enhanced methane and hydrogen production in reverse...
TRANSCRIPT
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Thesis for the Degree of Doctor of Philosophy
Enhanced Methane and Hydrogen production in Reverse Membrane
Bioreactors via Syngas Fermentation
Konstantinos Chandolias
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Copyright © Konstantinos Chandolias, 2019
Swedish Centre for Resource Recovery
University of Borås
SE-50190 Borås, Sweden
Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-21740
ISBN 978-91-88838-45-2 (printed)
ISBN 978-91-88838-46-9 (PDF)
ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 99
Cover page photo by Bruno Ramos Lara on Unsplash
Borås 2019
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Abstract
The increase in the waste production and the energy demand worldwide stimulates the
development of waste treatment processes, such as the anaerobic digestion. This biochemical
process converts organic substrates into biogas, with anaerobic microorganisms. However, some
types of substrates have low bio-degradability due to its recalcitrance or the presence of
inhibitors. This can be solved by the coupling of anaerobic digestion with gasification, a
thermochemical process that can convert organic substrates into syngas (H2, CO, and CO2)
regardless of the substrate´s degradability. Consequently, syngas can be converted into biogas
and other fermentative products via anaerobic digestion, in a process known as syngas
fermentation. In comparison to the catalytic conversion of syngas, syngas fermentation has
several advantages such as lower sensitivity to CO/H2/CO2 ratio and to syngas contaminants as
well as higher product specificity.
The main goal of this thesis was to improve the syngas conversion rate into CH4 and H2 by
addressing the cell washout, the cell inhibition by syngas contaminants, and the low gas-to-liquid
mass transfer, which are major challenges in syngas fermentation. For this purpose, a reverse
membrane bioreactor, containing a mixed culture encased in membranes, was used in various set
ups. The membranes were used in order to retain the cells inside the bioreactors, to protect the
cells against inhibitors, and to improve the gas holdup and gas-to-cell contact by decreasing the
rise velocity of syngas bubbles. As evident from the results, the cell washout was successfully
tackled during a continuous experiment that lasted 154 days. In addition, membrane bioreactors
fed with the syngas contaminants, toluene and naphthalene, achieved approximately 92% and
15% higher CH4 production rate, respectively, compared with the free cell bioreactors. In order to
improve the gas holdup and consequently the gas-to-liquid mass transfer of syngas, a floating
membrane bed bioreactor was set up. This bioreactor contained membrane sachets, filled with
inoculum that formed a packed floating membrane bed and achieved an increase of 38% and 28%
for the conversion rate of H2 and CO, respectively. Furthermore, the addition of a mixture of
heavy metals improved the production rates and yields during the syngas conversion into
fermentative H2.
Keywords: syngas fermentation; CH4; H2; cell washout; inhibitors; mass transfer
SVANENMÄRKET
Trycksak3041 0234
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PUBLICATIONS INCLUDED IN THE THESIS
I. Youngsukkasem, S., Chandolias, K., Taherzadeh, M. J. 2015. Rapid bio-methanation of
syngas in a reverse membrane bioreactor: Membrane encased microorganisms. Bioresource
Technology, 178, 334-340.
II. Westman, Y.S., Chandolias, K., Taherzadeh, M. J. 2016. Syngas Biomethanation in a Semi-
Continuous Reverse Membrane Bioreactor (RMBR). Fermentation, 2(8), 1-12.
III. Chandolias, K., Wainaina, S., Niklasson, C., Taherzadeh, M.J. 2018. Effects of heavy
metals and pH on the conversion of biomass to hydrogen via syngas fermentation.
Bioresources, 13(2), 4455-4469.
IV. Chandolias, K., Alan L., Izazi, N., Ylitervo, P., Wikandari, R., Milati, R., Taherzadeh, M., J.
2019. Protective effect of RMBR against syngas impurities. [submitted manuscript]
V. Chandolias, K., Pekgenc, E., Taherzadeh, M.J. 2019. Floating Membrane Bioreactors with
High Gas Hold-Up for Syngas-to-Biomethane Conversion. Energies, 12(6), 1046.
STATEMENT OF CONTRIBUTION
I. Responsible for the experimental work and partially responsible for the data analysis.
II. Responsible for the experimental work and partially responsible for the data analysis and
writing of the manuscript.
III. Conception of the idea and design of the experimental work. Partially responsible for the
experimental work, responsible for the data analysis and writing of the manuscript.
IV. Conception of the idea and design of the experimental work. Partially responsible for the
experimental work, responsible for the data analysis and writing of the manuscript together with
the co-authors.
V. Conception of the idea and design of the experimental work. Partially responsible for the
experimental work, responsible for the data analysis and writing of the manuscript.
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PUBLICATIONS NOT INCLUDED IN THE
THESIS
VI. Chandolias, K., Pardaev, S., Taherzadeh, M.J. 2016. Biohydrogen and carboxylic acids
production from wheat straw hydrolysate. Bioresource Technology, 216, 1093-1097.
VII. Patinvoh, R.J., Osadolor, O.A., Chandolias, K., Sárvári Horváth, I., Taherzadeh, M.J. 2017.
Innovative pretreatment strategies for biogas production. Bioresource Technology, 224, 13-
24.
VIII. Chandolias, K., Richards, T., Taherzadeh, M.J. 2018. Chapter 5 - Combined
Gasification-Fermentation Process in Waste Biorefinery, in Waste Biorefinery, (Eds.) T.
Bhaskar, A. Pandey, S.V. Mohan, D.-J. Lee, S.K. Khanal, Elsevier, pp. 157-200.
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To Magda and Angelos, my sunshine
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PREFACE
This thesis is a part of the requirements for completing the Ph.D. program in Resource
Recovery at the University of Borås. The scientific results and articles that are presented were
produced under the supervision of Professor Mohammad Taherzadeh, Dr. Supansa Westman,
Professor Tobias Richards, and Professor Claes Niklasson.
Global issues, such as climate change, environmental deterioration, extinction of flora and
fauna, overconsumption, and dramatic increase in waste generation have been the main focus of
intensified scientific research projects in recent years. Syngas fermentation has several
advantages, such as rapid waste treatment, product variety, and improvement of the environment
as well as new job opportunities. In addition, the concept of coupling thermochemical and
biochemical processes can be a part of the sustainable solution to the increasing energy demand
and waste generation. This work focused on the syngas fermentation, which is the biochemical
conversion of syngas into fermentative biofuels and value-added chemicals. Several experiments
were conducted using different bioreactor designs. The background, the main results, and
conclusions are discussed in the following chapters.
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RESEARCH JOURNEY
Ever since I can remember, I have always been fascinated by chemistry and biology. My
father worked in the textile Industry, and I used to spend many summer days in the chemistry
room. While I was studying chemical engineering at the Aristotle University in Thessaloniki, the
issue of global pollution and climate change was becoming more popular. I thought that this will
be the major global issue in the future, we should make a difference, and change things for the
better; therefore, I started reading about renewable resources, while I did an internship in
environmental pollution and waste treatment. When I was accepted at the master’s program of
resource recovery-industrial biotechnology at the University in Borås, I was excited! After 2
years, I got the chance to start my research and teaching in the same area!
Good things come at a cost and my research journey was no exception. During my Ph.D.
studies, I tried to prioritize my tasks although as any Ph.D. understands, this is quite tricky.
Literature reviews, courses, experiments, conferences, teaching, administrative work, and
machines that broke down too often. At the same time, I was involved in several activities, such
as the working environment committee and the study visits from high school students, which
were very interesting experiences, although they required extra time and energy. However, it was
also an interesting period, as I tested my limits and interacted with many hard-working and smart
people. A main challenge at the beginning of my studies was to prove that I could use syngas, a
toxic and explosive gas, in a safe way; therefore, I put a lot of effort into writing risk declarations
and building a safe set up for the gas feeding inside a fume hood. Luckily, no accidents have
occurred during my experiments.
In the first year, I failed to finalize two experimental projects that lasted almost the whole
year. However, I had also gathered material from my previous experiments on the cell washout
from the bioreactors during repeated batch and continuous syngas fermentation. To achieve this, I
used a reverse membrane bioreactor, with my supervisor, Dr. Supansa Westman, who had come
up with this type of bioreactor. In this bioreactor, anaerobic cells are retained inside the
membrane sachets, which are immersed inside the liquid medium of the membrane bioreactor.
The main challenge in this work was to avoid membrane clogging because there was no way to
do a backwash in order to clean the membranes during the experiment. However, after trial and
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error with different set ups and because the produced biogas was pushing outwards through the
membrane pores, the membranes did not clog! The results were promising, and we managed to
publish the first two articles (paper I and paper II).
At the beginning of the second year, after discussions with my main supervisor, Professor
Mohammad Taherzadeh, we planned a new strategy for my research. Meanwhile, I started
writing a book chapter on the coupling of fermentation and gasification (paper VIII). This gave
me the chance to do a literature review in my area of studies, something that is difficult to do
when experiments are ongoing. During that year, the gas chromatograph, for which I was
responsible, stopped working and was fixed after 4 months. Then, I ran an experiment on the
conversion of straw hydrolysate into H2 and carboxylic acids. The results were not so promising;
therefore, the experiment was repeated in the third year, when it was finally published (paper
VI).
During the third year, I worked on the concept of using waste from gasification in order to
improve the fermentation yields and reduce the footprint of gasification. The results showed that
heavy metals found in gasification ashes could improve the H2 production, during syngas
fermentation (paper III). In addition, I was involved in the writing of a review paper on
innovative processes for biogas production (paper VII).
In the fourth year, I focused on the improvement of the low gas-to-liquid mass transfer,
which is a major bottleneck in syngas fermentation. I was trying to come up with an interesting
suggestion on how to use the reverse membrane bioreactor in order to overcome this limitation.
After several discussions with my supervisor and trials, I noticed that in a bioreactor that contains
enough membrane sachets and nourishment for the cells, a floating membrane bed is formed,
which could block the rising velocity of syngas and therefore increase the gas hold up. The idea
of passing syngas through several reverse membrane bioreactors, connected in line, failed.
However, syngas conversion rates were improved during a single pass through a reverse
membrane bioreactor (paper V).
In the fifth year, I tried to address the issue of the gas-cleaning requirement for raw syngas,
prior to fermentation. If gas cleaning is not required, then the economics of the overall process
can be improved. The idea was to use hydrophilic membranes in a reverse membrane bioreactor
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to block the contact of hydrophobic syngas contaminants with the cells. The main challenge was
which contaminants to choose and how they would react with the membranes. A long literature
review resulted in selecting some contaminants, which were tested. Finally, two of them, toluene
and naphthalene were picked for the experiment. The results proved the protective effect of the
membranes towards high concentrations of the studied syngas contaminants (paper IV).
During these 5 years, the failures were too many; however, they made the success-moments
even greater. The most valuable lessons were to learn to take criticism, turn failure into success,
and to collaborate with people from different cultures. I enjoyed being a Ph.D. student, and I feel
blessed for getting to experience this in Sweden. It also feels good to think that your work can
have an impact, and I hope that others will be helped and inspired by this study as I was from
other works.
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ACKNOWLEDGMENTS
I am grateful to the Swedish Research Council for the funding of this work.
I would like to express my gratitude to my main supervisor, Professor Mohammad
Taherzadeh for his guidance, availability, and for the opportunity to start with this Ph.D.
program. I also wish to thank Dr. Supansa Westman for her support and guidance during my first
year as a Ph.D. student and before that during my M.Sc. studies. I also thank Professor Tobias
Richards and Professor Claes Niklasson for their constructive discussions. Special thanks to
Assistant Professor Ilona Sárvári Horváth, for the encouragement, support, and warm welcome.
To the laboratory staff: Tomas, Kristina, Marlén, Haike, Faranak, and Jonas and to the
section staff especially to Peter, Patrick, Päivi, Jorge, Tomas, Solveig, Kamran, Magnus, Louise,
Jonas, Camilla, Tatiana, Jonas, Akram, and Dan; thank you for all your help and for creating a
nice working environment.
During my studies, I had the privilege to work with many bachelor’s, master’s, and Ph.D.
students from all around the world. I have learned a lot from you all, and I hope we will meet
again in the future! Thank you Karthik, Foluke, Wikan, Martin, Ram, Regina, Alex, Julius,
Mostafa, Maryam, Adib, Veronika, Farzad, Abas, Kehinde, Solmaz, Behnaz, Sunil, Amir, Luki,
Andreas, Rebecca, Sindor, Gürlu, Mohsen, Sofie, Madumita, Steven, Anette, Danh, Mukesh,
Pedro, Sajjad, Taner, Moein, Tuba, Hanieh, Babak, Eboh, Supri, David, and the rest of the Ph.D.
students that worked in the Swedish Centre for Resource Recovery. I also want to express my
gratitude to my master’s and bachelor’s students, Björn, Sara, Müge, Enise, Sara, and Karolin;
thank you for your hard work and efforts.
I feel very blessed to have my friends in Borås, and I feel that they have also helped me in
their own way in order to finish this thesis. Thank you Stamatis, Katerina, Argyro, Martha,
Iordanis, Vasiliki, Vicki, Thomas, Nelly, Spiros, Vaya, Stratos, Thanassis, Stavros, and Vasso.
My thoughts are always with my friends in Greece, especially to my best friend George;
thank you for the nice summer moments, I really needed them between my studies.
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To my lovely parents, Angelos and Rodi, my beloved brother Lampis, and my grandmother
Nikki. You have been a role model for me; thank you for your love, patience, and kindness. I
wish we could see each other more often.
To my wonderful wife Magda and my son Angelos. Without you, I could not make it.
Thank you for your love, and I hope that we will have more time together now. I love you!
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Contents Abstract ......................................................................................................................................................... iii
PUBLICATIONS INCLUDED IN THE THESIS ...................................................................................................... iv
STATEMENT OF CONTRIBUTION ................................................................................................................... iv
PUBLICATIONS NOT INCLUDED IN THE THESIS ............................................................................................... v
PREFACE ....................................................................................................................................................... vii
RESEARCH JOURNEY .................................................................................................................................... viii
ACKNOWLEDGMENTS ................................................................................................................................... xi
NOMENCLATURE .......................................................................................................................................... xv
LIST OF FIGURES .......................................................................................................................................... xvi
LIST OF TABLES ........................................................................................................................................... xvii
Chapter 1 ....................................................................................................................................................... 1
Introduction ................................................................................................................................................... 1
1.1 Aim of this work .................................................................................................................................. 2
1.2 Thesis structure ................................................................................................................................... 3
1.3 Socioeconomic and ethical reflections ................................................................................................ 3
Chapter 2 ....................................................................................................................................................... 7
Anaerobic digestion ................................................................................................................................... 7
2.1 Biogas: an overview ............................................................................................................................. 7
2.2 Bio-H2: a renewable and sustainable product ................................................................................... 10
2.3 Basic principles of anaerobic digestion ................................................................................... .......... 11
2.3.1 Hydrolysis ................................................................................................................................... 12
2.3.2 Acidogenesis ............................................................................................................................... 12
2.3.3 Acetogenesis............................................................................................................................... 13
2.3.4 Methanogenesis ......................................................................................................................... 13
2.5 Waste with low degradability ............................................................................................................ 15
Chapter 3 ..................................................................................................................................................... 17
Thermochemical treatment for methane and hydrogen production ......................................................... 17
3.1 The gasification process .................................................................................................................... 18
3. 2 Types of gasifiers .............................................................................................................................. 21
3.3 Raw gas cleanup ................................................................................................................................ 22
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3.4 Catalytic conversion of syngas .......................................................................................................... 26
3.4.1 Hydrogen production ..................................................................................................... ............ 26
3.4.2 Methane production .................................................................................................................. 27
Chapter 4 ..................................................................................................................................................... 29
Coupling of thermochemical and biochemical process .............................................................................. 29
4.1 Metabolic activity for hydrogen and methane production ............................................................... 30
4.4 Operational parameters .................................................................................................................... 38
4.4.1 pH ............................................................................................................................................... 38
4.4.2 Temperature ............................................................................................................................... 38
4.4.3 Pressure ...................................................................................................................................... 39
4.4.4 Partial pressure........................................................................................................................... 39
4.5 Fermentation systems for hydrogen and methane production ........................................................ 40
4.5.1 Methane production .................................................................................................................. 40
4.5.2 Hydrogen production ..................................................................................................... ............ 43
4.6 The reverse membrane bioreactor ................................................................................................... 44
Chapter 5 ..................................................................................................................................................... 47
Improving syngas fermentation .................................................................................................................. 47
5.1 Cell washout ...................................................................................................................................... 47
5.2 Inhibitors ........................................................................................................................................... 50
5.2.1 Effect of heavy metals in anaerobic digestion ........................................................................... 50
5.2.2 Protective effect of rMBR against syngas contaminants ........................................................... 53
5.3 Gas-to-liquid mass transfer ............................................................................................... ................ 56
Chapter 6 ..................................................................................................................................................... 61
Conclusions and future work ....................................................................................................................... 61
6.1 Major experimental results and conclusions ................................................................................ .... 61
6.2 Future work ....................................................................................................................................... 62
6.2.1 Membrane material ................................................................................................................... 62
6.2.2 Bioreactor´s design ..................................................................................................................... 62
6.2.3 Effect of syngas contaminants and heavy metals ...................................................................... 62
References ................................................................................................................................................... 63
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NOMENCLATURE
ADP : Adenosine diphosphate
ATP : Adenosine triphosphate
CODH : Carbon monoxide dehydrogenase
ECH : Energy-conserving hydrogenase
ETP : Electron transport phosphorylation
FBEB : Flavin-based electron bifurcation
FCBR : Free cell bioreactor
Fd : Ferredoxin
ISR : Inoculum to syngas ratio
GHG : Greenhouse gas
SLP : Membrane bioreactor
NAD : Nicotinamide adenine dinucleotide
PBR : Packed bioreactor
PHA : Polyhydroxyalkanoates
PHB : Polyhydrobutyrate
PVDF : Polyvinylidene difluoride
RMBR : Reverse membrane bioreactor
SRB : Sulphate-reducing bacteria
SLP : Substrate level phosphorylation
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LIST OF FIGURES
Figure 2.1 Global biogas production in 2012 and its trend to 2022 9 Figure 2.2 Simplified scheme of the anaerobic stages 11 Figure 4.1 Schematic illustration of CO-oxidation, electron transfer, H2-formation and proton
transfer through the cell-membrane. CODH: CO dehydrogenase; Fd: ferredoxin; ECH: energy-conserving hydrogenase 33
Figure 4.2 The Wood-Ljungdahl pathway. H4F: tetrahydropholate; CoFeSP: corrinoid iron-sulphur protein 35
Figure 4.3 Metabolic pathway of aceticlastic methanogenesis. CoA: coenzyme A; H4SPT: tetrahydrosarcinapterin; HS-CoM: sulfhydryl coenzyme M; HS-CoB: sulfhydryl coenzyme B; CoM-S-S-CoB: heterodisulfide coenzymes CoM and CoB 36
Figure 4.4 Metabolic pathway of hydrogenotrophic methanogenesis. MFR: methanofuran; H4MPT: tetrahydromethanopterin; HS-CoM: sulfhydryl coenzyme M; HS-CoB: sulfhydryl coenzyme B; CoM-S-S-CoB: heterodisulfide coenzymes CoM and CoB 37
Figure 4.5 Schematic illustration of the reverse membrane bioreactor (RMBR) in the semi-continuous biomethanation process of syngas and organic substances. (a) Digesting sludge encased in PVDF membrane, (b) Organic and nutrient medium, (c) Syngas, (d) Peristaltic pump, (e) Gas outlet, (f) Warm water bath, (g) Effluent, (h) Flow meter, (i) Data analysis 45
Figure 5.1 Elimination of cell washout in a RMBR 48 Figure 5.2 Comparison of the CH4 production in RMBR and FCBR, during the semi-continuous
biomethanation of syngas and organic substances 49 Figure 5.3 Utilization of heavy metals from gasification ash during syngas fermentation 51 Figure 5.4 Relationships between H2 production activity, Ah (%) = (mol H2/mol H2 control) 100%
(a), H2 yield, YH = mol H2 prod./mol CO fed (b), initial medium pH and heavy metal concentrations (mg L-1) 52
Figure 5.5 Protective effect of membranes against syngas contaminants 54 Figure 5.6 The effect of naphthalene on the (a) CH4 production rate, mmol CH4 L-1 d-1; (b) CH4
production activity, (mol mol-1control) 100%; and (c) the theoretical naphthalene concentration, g L-1; in the FCBR and the RMBR 55
Figure 5.7 (a) Main resistances during the mass transfer of syngas from the gas phase to the site of reaction in the cells. Movement 1) in the gas bubble; 2) across the gas-liquid interfacial; 3) through the liquid film surrounding the gas bubble; 4) in the liquid bulk; 5) through the membrane pores; 6) inside the membrane; 7) across the liquid film surrounding the microbial cell; 8) through the cell membrane; 9) through the cell and end up in the site of reaction. (b) Movement of A through the interfacial boundary. CAL: concentration of A in the liquid phase; CAli concentration of A in the liquid boundary; CAGi concentration of A in the gas boundary and CAG concentration of A in the gas phase 57
Figure 5.8 Improvement of gas-to-liquid mass transfer rates in a floating MBR 59 Figure 5.9 Syngas biomethanation in a floating MBR, floating MBR/FCBR, PBR, and FCBR during
continuous syngas feeding. Consumption of (a) H2 and (b) CO and production of (c) CO2 and (d) CH4 in mmol·L-1·d-1) 60
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LIST OF TABLES
Table 3.1 Principal reactions in gasification 19 Table 3.2 Composition of bottom and fly ashes from gasification of wood pellets, expressed as
mg kg-1 of the dry substance 20 Table 3.3 Composition of dry gas in oxygen and steam gasification 22 Table 3.4 Examples of feedstock contaminant levels 23 Table 3.5 Analysis of tar content in syngas derived from downdraft gasification with wood
pellets 24 Table 3.6 Analysis of syngas trace contaminants collected on filters after gasification of rice
hulls and wood chips. All concentrations are expressed in μg m-3syngas 25 Table 3.7 Gas cleaning requirements for different syngas applications 26 Table 4.1 Syngas fermenting bacteria 31 Table 4.2 Syngas fermenting methanogens 32
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Chapter 1
Introduction
Due to the increasing global waste generation, the climbing energy demand, and the
environmental deterioration from fossil fuels, there is an urgent need for renewable and
sustainable solutions. The anaerobic conversion of waste streams into biofuels, biochemicals, and
other bioproducts is a sustainable answer to the above challenges. During anaerobic digestion,
organic molecules, such as monosaccharides and amino acids, are broken down by
microorganisms, in the absence of oxygen. The main product of anaerobic digestion is biogas, a
mixture of CH4 and CO2, while other valuable products such as H2, acids, and bioplastics can also
be generated [1-3]. Although there are organic substrates that can be easily degradable by the
anaerobic cells, there are several types of potential substrate with low degradability, such as
lignocellulose. These substrates can be treated with chemical (e.g., acid) or biological (e.g.,
enzymatic) processes, which have a high cost.
Another more efficient method for the treatment of substrates with low degradability is
gasification. In this method, the feedstock is thermally degraded in the presence of an oxidizing
agent, usually air, oxygen, or steam. Gasification encompasses several advantages such as
feedstock flexibility and high conversion rates [4, 5]. The main product of gasification is syngas,
a gaseous mixture of mainly CO, H2, and CO2 with several applications, such as heat and power,
fuels (ethanol, H2, CH4), value-added chemicals (ammonia), and bioplastics [6]. During the past
few years, the anaerobic fermentation of syngas for the generation of biofuels and other value-
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added products, has gained broad interest in scientific, social, and industrial fields [7]. This
process has several advantages in comparison with the traditional catalytic processes, which
valorize inorganic/metal catalysts. More specifically, syngas fermentation is less sensitive to
syngas contaminants and to the H2/CO ratio; it does not require high temperature and pressure, it
is more product-specific, and no hazardous components are formed [8].
The coupling of gasification and fermentation is a relatively new technology, and several
challenges need to be tackled so that the efficacy of the overall process will be improved. Low
cell-density bioreactors are a common challenge in continuous anaerobic processes, where
productive bacteria are washed out of the bioreactors during the exchange of fresh and old liquid
medium. Another bottleneck is the high concentration of heavy metals inside the gasification-
derived ash, which poses an environmental and health hazard. Moreover, extended exposure to
high concentrations of syngas contaminants may be toxic for the anaerobic cells. Finally, a main
concern during syngas fermentation is the low gas-to-liquid mass transfer rates of syngas
components. The above challenges were the main focus of this thesis.
1.1 Aim of this work
The aim of this thesis was to overcome important challenges in syngas fermentation, such
as to eliminate the cell-wash out, to investigate the use of gasification-derived heavy metals in
fermentation, to study and address the cell sensitivity in syngas contaminants, and to improve the
low gas-to-liquid mass transfer of syngas components in bioreactors. For this purpose, a reverse
membrane bioreactor (RMBR) was employed and developed. The focus of the thesis can be
summarized in five steps:
1. To study syngas biomethanation in a RMBR, with high cell density, in batch conditions
(Paper I)
2. To operate a RMBR in continuous syngas biomethanation and report the effects of cell
retention (paper II)
3. To investigate the effect of different concentrations of heavy metals in syngas
fermentation for H2 production (paper III)
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4. To report the beneficial and inhibitory concentrations of two syngas contaminants,
toluene and naphthalene, and the possible protective effect of the RMBR in batch and
continuous anaerobic digestion (paper IV)
5. To increase syngas holdup for higher gas-to-liquid mass transfer rates and thus higher
syngas conversion rates in a floating MBR with high gas holdup (paper V)
1.2 Thesis structure
This thesis is divided into 6 Chapters. In Chapter 1, the background, the aim, as well as the
socioeconomic and ethical reflections of this work are presented. The anaerobic digestion,
including the microbiology, digestion steps, and microorganisms are discussed in Chapter 2.
Moreover, the challenge of substrates with low bio-degradability is introduced. In Chapter 3,
gasification is presented as a sustainable treatment process for substrates with low bio-
degradability. Chapter 4 presents the syngas fermentation process, including biochemical
reactions, pathways, and bioreactors. The main challenges of syngas fermentation, which were
the focus of this thesis, including the cell washout, the utilization of heavy metals derived from
the gasification ashes, the effect of contaminants in the raw syngas, and the low gas-to-liquid
mass transfer rates of syngas components, are described in Chapter 5. Finally, the main
conclusions of this thesis and future work are discussed in Chapter 6.
1.3 Socioeconomic and ethical reflections
The ultimate goal in research is to create better living conditions for humanity. The way to
do that is by asking questions and answering with scientific data. During my research journey, I
have tried to correlate the importance and the effect of my studies to society´s well-being. At the
end of this work, I believe that my research can have a positive influence in society.
Although technology has rapidly evolved and improved our life standards, the dramatic
increase of waste and the rising energy demand create concerning environmental and social
challenges. Fossil fuels are the traditional resources used for energy; however, their use generates
serious pollution problems that are considered a main reason for our planet’s pollution and
climate change. The concept of circular economy with its three principles, to reduce, reuse, and
recycle materials, is becoming increasingly popular. Therefore, the use of abundant resources and
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the simultaneous reduction of waste are incredibly important for the present and future
generations.
This thesis is based on the waste-to-energy concept, with the combination of gasification
and fermentation for waste reduction and biofuel production. During the last few decades, there
has been an increasing interest toward biofuels, with global and regional policies. The production
of fermentative products such as biofuels and value-added chemicals can lead to a more
sustainable alternative to fossil fuels and petrochemicals. However, the use of first-generation
biofuels creates the ethical dilemma of food-to-energy conversion. The use of arable land, water,
and pesticides contradicts the concept of sustainability and raises global concerns. On the other
hand, the gasification-fermentation process uses renewable, abundant feedstock, such as forest
residues and municipal waste, for the production of second-generation biofuels. Moreover,
gasification and fermentation are two established industrial processes; thus, the infrastructure
exists. Moreover, intensification of the two processes will lead to more job opportunities in both
research and industrial projects.
Gasification offers rapid waste reduction with important social advantages. This creates
better living conditions, reduces the health effect and environmental pollution, and leaves more
space for human activities such as agriculture and farming. The reduction of waste affects
relatively large populations that live near landfills, especially in developing countries. These
populations depend on recycling or retrieving of valuable compounds from the waste, but they
are exposed to dangerous emissions and even explosions that take place in landfills because of
toxic and explosive gases formed inside the waste piles. The reduction of landfill waste also
decreases the threat of underground water pollution by leaching and the greenhouse gas (GHG)
effect by carbon dioxide and methane emissions. In addition, in many parts of the world, cooking
and waste reduction are still done by burning waste in the backyards, thus, creating
environmental and health threats. The development of a technology that converts various types of
waste, in a controlled and efficient way, into energy can dramatically minimize the uncontrolled
waste burning.
Syngas fermentation via anaerobic microorganisms can generate different products in the
concept of a biorefinery, thus, creating better economic opportunities. This work aimed to
achieve higher H2 and CH4 yields during syngas fermentation. These gases have numerous
5
applications, such as vehicle fuels, electricity generation, and production of chemicals. The
improvement of the yields could give a push on the biofuel market with higher revenues and thus
greater opportunities for new projects and jobs. In addition, except for syngas, other industrial
off-gases, with similar composition, could be used as a substrate. The reduction of CO and CO2
emissions could significantly reduce the industrial pollution in industrial areas. In addition, no
pathogens or other dangerous microorganisms are generated during fermentation. During this
work, only naturally occurring microorganisms, present in sewage sludge, were used as
inoculum. Therefore, there were no considerations about genetically modified cells and the
danger of them escaping into the environment.
This work was funded by public means; therefore, there has been a significant effort to
make the findings of the experiments publicly available by Open Access publications. In
addition, ethical norms, such as reliability, honesty, and respect for intellectual property were
followed in order to reassure the high publication standards.
6
6
7
Chapter 2
Anaerobic digestion
Currently, the world is experiencing increasing rates of development, which aim for better
living conditions for societies. However, the drawbacks of this development are the
intensification in the use of fossil fuels and the increasing waste generation. This contributes
greatly to the global pollution and climate change. In order to address the above challenges,
renewable processes, such as anaerobic digestion, have gained considerable interest in the last
few decades. During anaerobic digestion, organic substrate is converted into biogas, by
microorganisms, under anoxic conditions. In addition, other intermediate products, such as
fermentative H2 can be obtained during anaerobic digestion.
2.1 Biogas: an overview
Biogas production technology is diverse, as it combines small and large scale plants and a
variety of possible substrates. In developing countries, biogas is commonly produced in small
domestic digesters for cooking or lighting, with food residues being the main substrate. In
developed countries, biogas production mainly takes place in larger scale industrial digesters for
electricity and heat production. In this case, wastewater sludge, food waste, manure, industrial
wastes, and agricultural residues are common microbial substrates. Biogas is usually produced in
digesters with operating temperature that ranges from 30 40 °C (mesophilic) to 50 60 °C
(thermophilic). Different anaerobic microbes dominate in different temperatures. In general,
8
8
thermophilic conditions offer a faster degradation process and greater pathogen kill. The digestate
from thermophilic digesters can be used for land application with no restrictions, according to the
US Environmental Protection Agency (EPA) [9]. However, mesophilic conditions may be less
costly and easier to operate and maintain [9]. Wet substrate, containing less than 15% solids
content, is a more common substrate than dry feedstock, due to the fact that the wet feedstock can
be easily pumped in and out of the digester and be homogenized inside the digesters. Modern
digesters are equipped with other facilities that can deliver a broad range of products, from heat
generation to upgraded CH4 for vehicle fuel use. Other digesters are utilized for the treatment of
animal manure and human wastewater, and they are thus placed near farms and cities.
Biogas, consisting mainly of CH4 and CO2, has numerous applications, such as electricity
generation, combined heat and power plants (CHP), direct burning for energy generation and
cooking, injection into the natural gas pipeline, vehicle fuel, and fuel cells. In Europe, biogas is
largely used for generation of electricity, heat, or heat and power. The heat produced can be used
for the local facility demands or external users. In the case of biogas injection in the natural gas
network, biogas upgrade with removal of trace gases such as H2S, water, and CO2 is required
[10]. Biogas production from wastewater and landfill gas recovery, as well as biogas upgrading
into CH4 for vehicle fuel use or injection to the natural gas grid are gaining attention in several
countries [10].
Except from energy and fuel generation, biogas production results in environmental
benefits, such as decrease in water, soil, and air pollution [10]. Manure has traditionally been
used as a fertilizer in agriculture. However, this can cause environmental pollution due to
pathogen growth and CH4 and CO2 release in the atmosphere [11, 12]. In anaerobic digestion,
manure and other substrates are degraded in a controlled environment, resulting in a reduction of
odor and removal of pathogens that can pose health risks for humans and animals, while CH4 and
CO2 are used as the main products. In other words, the anaerobic digestion process results in the
reduction of Green House Gas (GHG) emissions. In addition, digestate from biogas production
can still be used as a biofertilizer containing similar nutrients to manure. This results in additional
economic benefits, while it reduces the use of chemical fertilizers, the nutrient runoff, and CH4
emissions [11-13].
9
Figure 2.1 Global biogas production in 2012 and its trend to 2022 [14]
The biogas market is expected to grow significantly in the following decade [15, 16] as
shown in Figure 2.1. The status of biogas market depends greatly on the price and availability of
fossil fuels. For example, the global oil crisis of 1973 led to an increased interest in biogas
production [17]. Although the oil prices decreased in 1985, and since 2015, there is still interest
in anaerobic digestion, due to environmental considerations [18]. Among the developed
countries, Germany is the leading country in biogas generation, which in 2010, generated 61% of
the total electricity produced from biogas in Europe [19]. The European commission legislates
incentives that aim for the production of biofuels and specifically biogas and biofertilizer. A new
European legislation includes a legally-binding EU-wide target of 32% for renewable energy by
2030 [20]. In 2015, biogas production in Europe represented half of the global biogas production
[10]. Furthermore, Europe is the world´s leading producer of bio-CH4 with 459 plants in 2015,
while most of the bio-CH4 production plants are in Germany (185 plants), UK (80 plants), and
Sweden (61 plants), in the same year [10]. In Asia, China and India are the leading countries in
biogas generation. China has the highest amount of domestic biogas plants, which in 2011
reached 41.68 million [21], while the large scale agricultural and industrial organic waste biogas
installations were 4,700 and 1,600, respectively [22].
9
Figure 2.1 Global biogas production in 2012 and its trend to 2022 [14]
The biogas market is expected to grow significantly in the following decade [15, 16] as
shown in Figure 2.1. The status of biogas market depends greatly on the price and availability of
fossil fuels. For example, the global oil crisis of 1973 led to an increased interest in biogas
production [17]. Although the oil prices decreased in 1985, and since 2015, there is still interest
in anaerobic digestion, due to environmental considerations [18]. Among the developed
countries, Germany is the leading country in biogas generation, which in 2010, generated 61% of
the total electricity produced from biogas in Europe [19]. The European commission legislates
incentives that aim for the production of biofuels and specifically biogas and biofertilizer. A new
European legislation includes a legally-binding EU-wide target of 32% for renewable energy by
2030 [20]. In 2015, biogas production in Europe represented half of the global biogas production
[10]. Furthermore, Europe is the world´s leading producer of bio-CH4 with 459 plants in 2015,
while most of the bio-CH4 production plants are in Germany (185 plants), UK (80 plants), and
Sweden (61 plants), in the same year [10]. In Asia, China and India are the leading countries in
biogas generation. China has the highest amount of domestic biogas plants, which in 2011
reached 41.68 million [21], while the large scale agricultural and industrial organic waste biogas
installations were 4,700 and 1,600, respectively [22].
10
10
2.2 Bio-H2: a renewable and sustainable product
The current global H2 generation market is promising, and it was estimated to be larger than
100 billion USD in 2017 [23]. Between 2003 and 2008, there were reports of approximately 6%
yearly increase in the H2 sales [24]. Until 2026, the H2 market is expected to increase by more
than 2 times [23]. During the same period, Asia Pacific is expected to dominate the global H2
market [23]. The increase of the H2 market depends greatly on global trends for renewable
policies, on the status of technologies for producing/consuming H2, and the price of H2 and its
rivals, such as fossil fuels [25].
H2 is one of the most abundant components with a high value, several production processes,
applications, and properties. It has a high-energy content of 120 MJ/kg to 142 MJ/kg that is more
than 2.5 times higher than that of hydrocarbon fuels and releases only water during combustion
[26, 27]. The global H2 production is mainly used for ammonia manufacturing, with
approximately 50% contribution [24, 28]. Other typical applications of H2 are chemical
processes, lamps, vehicle fuel, laboratories, power-to-gas storage, and redox reactions (reductive
agent). The H2 use as a combustible fuel in stationary and transportation sector, such as in
internal combustion engines, rockets, fuel cell electric vehicles, and high-temperature industrial
furnaces is considered very promising [29, 30]. Moreover, H2 can be used as a blend with other
fuels, such as ethanol, in internal combustion engines [31].
H2 can be generated via several processes based on fossil fuels, such as reforming and
pyrolysis, or renewable sources. The main fraction of global H2 production is generated by CH4
reforming of natural gas, which utilizes fossil fuels [32]. Renewable processes for H2 production
include biomass treatment via biological or biochemical routes and water splitting. Water
electrolysis is a water splitting method with less environmental footprint, higher costs, and small
scale availability [27]. One biological process includes special bioreactors that use biocatalysts,
such as microalgae and phototrophic bacteria, for H2 production via photofermentation, which
have been studied [33]. In addition, fermentative H2 production by anaerobic digestion is
considered as a less expensive, less energy-demanding, and more eco-friendly process than other
production methods [34, 35]. Bio-H2 is an intermediate product in anaerobic digestion, in contrast
to biogas that is the end product of the process. The production of H2 and biogas is discussed in
more details in the following section.
11
2.3 Basic principles of anaerobic digestion
Figure 2.2 Simplified scheme of the anaerobic stages (adapted from [36])
Anaerobic digestion is a microbial process in which organic carbon is converted via
successive redox biochemical reactions into its most oxidized state (CO2), and to its most reduced
form (CH4) [18]. Anaerobic consortia contain a mixture of diverse microorganisms. In nature,
these cells thrive in different environments such as hot springs, wetlands, manure, and forest
sediments [37-40]. The anaerobic and facultative anaerobic microorganisms that take part in
anaerobic digestion have evolved a special symbiotic collaboration, called syntrophism due to the
small amount of energy available in CH4 generation. This means that different groups of bacteria
rely on each other for their metabolic activity, while together these bacterial groups show a
metabolic activity that no group could carry out on their own. The anaerobic digestion process
can be divided into four main stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis
as schematically presented in Figure 2.2.
11
2.3 Basic principles of anaerobic digestion
Figure 2.2 Simplified scheme of the anaerobic stages (adapted from [36])
Anaerobic digestion is a microbial process in which organic carbon is converted via
successive redox biochemical reactions into its most oxidized state (CO2), and to its most reduced
form (CH4) [18]. Anaerobic consortia contain a mixture of diverse microorganisms. In nature,
these cells thrive in different environments such as hot springs, wetlands, manure, and forest
sediments [37-40]. The anaerobic and facultative anaerobic microorganisms that take part in
anaerobic digestion have evolved a special symbiotic collaboration, called syntrophism due to the
small amount of energy available in CH4 generation. This means that different groups of bacteria
rely on each other for their metabolic activity, while together these bacterial groups show a
metabolic activity that no group could carry out on their own. The anaerobic digestion process
can be divided into four main stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis
as schematically presented in Figure 2.2.
12
12
2.3.1 Hydrolysis
Hydrolysis is the first stage of anaerobic digestion where undissolved compounds, such as
carbohydrates, proteins, and fats are cracked down into water-soluble compounds. This
conversion is catalyzed by hydrolytic extracellular enzymes, which are excreted by hydrolytic
facultative and obligatorily anaerobic bacteria. Thermoanaerobium brockii is a representative
thermophilic, hydrolytic, bacterium. The presence of different hydrolytic bacteria depends on the
type of different substrates, and their task is to solubilize complex compounds, which will be
consequently transferred into the cells and further degraded by endoenzymes. The rate of this
stage depends on parameters, such as the production of enzymes, substrate structure, and
adsorption of enzymes on the substrate surface [41]. Hydrolysis can be the limiting stage of
anaerobic digestion when the solid and complex substrates are fed into the digester [42, 43].
Anaerobic bacteria, such as Clostridium and Bacteroides, pose an extra cellular multi-
enzyme complex, called cellulosome, for the decomposition of substrates such as carbohydrates
[44]. The typical structure of a cellulosome consists of large non-catalytic scaffoldin protein that
contains a carbohydrate-binding-module (CBM), surface layer homology (SLH), and several
cohesin domains [45]. Various enzymatic subunits are bound to the scaffoldin subunit via cohesin
and dockerin interactions. The SLH can bind on the bacterial cell wall, regardless of whether the
CBM binds on the substrate surface and the enzymatic subunits hydrolyze the substrate.
2.3.2 Acidogenesis
In acidogenesis, the products of hydrolysis are degraded by different facultative and
obligatorily anaerobic bacteria into compounds of one to five carbon units. Typical products of
acidogenesis are volatile fatty acids (VFAs), such as propionic, butyric, and valeric acid, and two
examples of acetogenic bacteria are the Butyribacterium methylotrophicum and the Clostridium
ragsdalei. Depending on the parameters, such as the type of substrate, the anaerobic conditions
and microbial consortia, others products, such as H2, CO2, alcohols, and ammonia can be
generated in this stage. Imbalances in this stage can affect the CH4 production as well. For
example, in case of high acid production (acidogenesis), the bioreactor may be overloaded and a
sudden drop in the pH may occur (souring). This can inhibit the methanogenic activity and
endanger the overall digestion process.
13
2.3.3 Acetogenesis
The main role of acetogenesis is the conversion of the products of acidogenesis, such as
volatile fatty acids and alcohols into H2, CO2, and acetate. Some examples of acetogenic bacteria
are Acetobacterium bakii, Acetoanaerobium noterae, and Acetitomaculum ruminis. The
production of excess H2 may result in the increase of H2 partial pressure. If this partial pressure
exceeds a limit, then the acetogenesis is not thermodynamically feasible. Therefore, a symbiotic
interaction between the H2 producing bacteria (acetogens) and the H2 consuming bacteria
(hydrogenotrophic methanogens) is vital for the digestion process [46]. In the presence of
sulphates, sulphate-reducing bacteria (SRB), such as bacteria of the genus Desulfobacter,
Desulfosarcina and Desulfovibrio, thrive in anaerobic digesters. These bacteria use H2 and
acetate and reduce sulphate into H2 sulphide. This leads to a concurrence of H2 use with the
hydrogenotrophic methanogens. In the presence of low acetate amounts and high sulphate
concentrations, the sulphate reducing bacteria obtain H2 and acetate more easily than the
methanogens.
2.3.4 Methanogenesis
In the last stage, CH4-forming microorganisms convert the products of acidogenesis and
acetogenesis, mostly acetate, H2, and CO2, into CH4. CH4 can also be formed by other organic
compounds, such as CO. Therefore, it is crucial that all substrate compounds have been
transformed to a form that can be treated by methanogens. This can be the slowest stage of
anaerobic digestion mainly because the methanogenic cells are more sensitive in comparison to
the microorganisms in the previous stages, they have lower growth rates, and they can consume
only a limited type of substrates. This means that for easily hydrolyzed substrates,
methanogenesis may be the limiting stage of the anaerobic digestion [47].
Methanogens can be classified according to their substrate utilization into three main
groups: the hydrogenotrophic, the acetotrophic, and the methylotrophic methanogens. The
hydrogenotrophic methanogens convert CO2 and H2 into CH4 and water. By this mechanism, they
maintain a low H2 pressure that is vital for the acetogenic and the acetotrophic bacteria. The
acetotrophic methanogens can convert acetate into CO2 and H2. The CO2 produced can be further
converted into CH4 by the hydrogenotrophic methanogens. Moreover, some acetotrophic
methanogens can convert CO and water into CH4 and CO2. The acetotrophic methanogens
14
14
reproduce more slowly than the hydrogenotrophic methanogens and are negatively affected by
the high H2 partial pressure [47]. Therefore, the maintenance of low H2 pressure is vital for both
acetate and CH4 production. The methylotrophic methanogens convert water and substrates
containing the methyl group (CH3-), such as methanol and methylamine, into CH4, CO2, and
NH3.
2.4 Wastes-substrates for anaerobic digestion
The volumes of waste generated by human activity have been exponentially increasing after
the industrial revolution. According to the World Bank, the global waste generation will increase
by 70% on current levels by 2050. The waste generation per capita is affected by the national
gross domestic product (GDP); therefore, wealthier countries tend to produce more waste per
capita than poor countries [48]. However, as the GDP of giant countries such as India and China
is improving, the global waste generation is increasing.
Wastes can be categorized into solid, liquid, and gas streams including food, agricultural,
and forest residues, construction and electronic waste, municipal waste, and emissions from
transportation and industries. The composition of wastes can include glass, paper, metal, plastic,
lignocellulosic biomass, hair, and animal and human feces. Globally, efforts are made in order to
achieve a proper waste treatment, where the waste generation is reduced, reused, and recycled
(3Rs). The waste reduction can be achieved via a reduced consumption, less use of packaging
material and reuse of wastes, such as old textiles. Components, such as glass, paper, plastic, and
metal, can be recycled and reused. However, several waste streams are not recyclable and can be
treated for the generation of energy and other products, via other processes, such as anaerobic
digestion. The conversion of waste for the production of energy and value-added chemicals can
favor the concept of circular economy toward zero-waste societies, and lead to less dependency
on fossil fuels.
Various types of wastes including agricultural and forest residues, animal manure,
slaughterhouse waste, and municipal waste (e.g., household and sewage waste) can be used as a
substrate in anaerobic digestion. Historically, liquid waste streams, such as sewage sludge and
industrial wastewater have been fed in digesters. In the last few decades, the digestion of
municipal solid wastes and agricultural residues is gaining attention. The yield and composition
15
of fermentative products can vary greatly due to the content of carbohydrates, proteins, and lipids
in feedstocks. The elemental composition and the Buswell formula (Eq. 2.1) can be used in
practice in order to calculate the theoretical CH4 potential of each substrate. The theoretical CH4
potential of carbohydrates, proteins, and lipids is 0.42, 0.50, and 1.01 Nm3 CH4 kg-1 VS,
respectively [12]. Therefore, the theoretical methane potential of municipal solid waste, food
waste, and manure can be calculated at 180 350, 400 800, and 100 300 Nm3 CH4 ton-1 VS,
respectively [49], depending on their content in carbohydrates, lipids, and proteins.
CcHhOoNnSs + yH2O → xCH4 + nNH3 + sH2S + (c-x)CO2 (2.1)
Where: x = 1/8 (4c+h-2o-3n-2s)
For liquid substrates, with low solid content, the theoretical CH4 potential can be calculated
by the chemical oxygen demand (COD). In Eq. 2.2, one mole of CH4 needs two moles of oxygen
to oxidize CH4 to CO2 and water. Therefore, 0.35 L CH4 is produced per g COD substrate.
CH4 + 2O2 → CO2 + 2H2O (2.2)
A basic assumption for the above equations is that the substrate is completely degraded and that
the substrate utilization for biomass (microbial) growth is negligible [12, 50, 51]. The
calculations were made with the ideal gas formula, at standard conditions.
2.5 Waste with low degradability
Several waste streams, such as lignocellulosic material, textile residues, and keratin-rich
waste have relatively low degradability rates in anaerobic digestion. The protective structural
mechanism of these materials that prevents their degradation is called recalcitrance. One example
of recalcitrance is that the structure of some waste does not allow for efficient contact of the
substrate with enzymes and therefore, the digestion is inhibited. For instance, although
lignocellulose has high carbohydrate content, it cannot be easily degraded by anaerobic
microorganisms due to its lignin content [52]. Chicken feather and wool, with a high content of
protein, have low degradation rates because of their high content in keratin [53]. Moreover, there
are wastes that cannot be completely degraded or their degradation requires considerable amount
of time, for example, mixed landfill waste containing plastic, lignocellulose, food residues, etc.
16
16
These materials contain degradable components; however, their diversity makes them
challenging substrates for anaerobic digestion. Another reason for the low degradability rate of
some wastes is their content of components that have antimicrobial activity. For example, the
fruit flavors, such as D-limonene in citrus waste can inhibit the anaerobic digestion process [54].
In order to convert wastes with low degradability into degradable substrates for anaerobic
digestion, various pretreatment methods can be employed. The available methods can be
categorized into mechanical, biological, chemical, and thermochemical methods. The mechanical
pretreatment includes substrate size reduction by grinding or milling and has a high-energy
demand. Enzymatic hydrolysis is a biological pretreatment that has low energy and cost-input
and long hydrolysis rates [53]. Alkalis, acids, oxidizing agents, and organic solvents are used in
chemical pretreatments. The efficiency of this type of pretreatment depends on the lignin content
and may lead to cellulose and hemicellulose losses, corrosion, cell inhibition, and loss of highly
volatile chemicals [53]. Another method to treat recalcitrant waste and waste containing
microbial inhibitors is via thermochemical treatment, such as combustion and gasification.
Especially in gasification, a large variety of wastes can be converted, at high temperatures and in
the presence of an oxidation agent, into a gas product that can consequently be converted into
fermentative products via anaerobic digestion. The process of gasification and the digestion of
the gaseous product are discussed in the following chapters.
17
Chapter 3
Thermochemical treatment for methane and
hydrogen production
Currently, thermochemical treatments, such as incineration (combustion), pyrolysis, and
gasification, of waste streams are applied in large scale. Incineration is commonly used for the
burning of feedstock with oxygen for heat and power generation [55]. Pyrolysis converts the
feedstock in the absence of oxygen into char and bio-oil [55]. Gasification is the conversion of
feedstock into a gas product. The thermochemical treatment of wastes offers advantages in
comparison to other types of waste treatment [5], such as the high conversion rates of solid waste
[4] and thus, the reduction of eventual uncontrolled CO2 and CH4 emissions from landfills [56].
Moreover, the thermochemical processes have wide feedstock flexibility, including waste with
low degradability rate, such as forest residues. Another advantage is the destruction of pathogenic
microbes because of high operational temperatures [57]. The main advantage of gasification in
comparison to other thermochemical processes is the generation of the gaseous product that has a
high value and numerous applications. In addition, several operating conditions of the
gasification can be altered upon demand, creating a flexible process [58, 59]. This chapter
focuses on the process of gasification, equipment, product applications, and challenges, such as
the toxicity of residuals, the contaminants in raw syngas, and the catalytic conversion of syngas
into CH4 and H2.
18
18
3.1 The gasification process
During gasification, low-value feedstock is converted into high value products at high
temperatures and the presence of oxidizing media. At temperatures below 1000 °C, a gas blend of
mainly CO, H2, CH4, CxHy aliphatic hydrocarbons, tars, CO2, and water is produced [60]. At
temperatures above 1200 °C, syngas is produced, which consists mainly of CO, H2, CO2, together
with a small amount of CH4, nitrogen, and contaminants, depending on the operating conditions
[60].
Gasification consists of four main steps: drying of feedstock, pyrolysis, oxidation, and
reduction. The order of these steps depends on the gasifier set-up. Drying takes place in order to
remove moisture, at temperatures above 100 °C. Pyrolysis is the thermal decomposition of
feedstock in the absence of an oxidation agent, and occurs at 150 700 °C. The feedstock
moisture ranges between 5–35% and generates steam when evaporated. Pyrolysis gases, such as
CO2, CO, H2, CH4, and water vapor together with char, tar, and volatile compounds are released
in this stage. The use of oxygen as an oxidation agent, although it is more expensive than air, is
often preferred in order to avoid high amounts of nitrogen [61]. Table 3.1 shows important
reactions that take place during gasification. In the oxidation zone, the oxygen can react with
solid carbonized fuel, producing CO or CO2 as shown in Eq. 3.1 and Eq. 3.2. In Eq. 3.3, the
water vapor introduced with air or produced by drying or pyrolysis reacts with the hot carbon,
according to the reversible heterogeneous water gas reaction. In the reduction zone, several
reactions take place in the absence of oxygen. Principal reduction reactions are the water, gas and
Boudouard reaction (Eq. 3.3 3.4). In addition, other important reduction reactions are the water
shift (Eq. 3.5) and the methanation reaction (Eq. 3.6). The water shift reaction is exothermic
when water is in surplus. When the reactions in Eq. 3.7 and Eq. 3.8 occur, heat is produced;
however, the heat value is reduced.
19
Table 3.1 Principal reactions in gasification [62]
Reaction ΔH, kJ mol-1
(25 °C, 1 atm)
Equation
C + 0.5O2 → CO -123.1 (3.1)
C + O2 → CO2 -393.8 (3.2)
C + H2O ↔ CO + H2 +118.5 (3.3)
CO2 + C 2CO +159.9 (3.4)
CO + H2O CO2 + H2 -40.9 (3.5)
C + 2H2 CH4 -87.5 (3.6)
CO + 0.5O2 → CO2 -283.9 (3.7)
H2 + 0.5O2 → H2O -285.9 (3.8)
After gasification, a relatively small amount of feedstock that has not been converted into the gas
phase is left in the gasifier in the form of ash. The elemental composition of ash that can contain
carbon, minerals, and metals can include Ca, K, P, Cu, Zn, Mn, Fe, and Mg [63] (Figure 3.2).
The residual phase is classified according to its carbon content, to char (high content) and ash,
which contains high concentrations of metals and minerals [63]. The char is commonly used in
combustion for heat generation, while the ash can be used as a construction material after
pretreatment [63]. For example, a study that investigated alternative uses of gasification ash
stated that the ash could be used as a component in bricks without pretreatment [64]. However,
there is a lack of research on the efficient uses of gasification-derived ashes in the literature. A
main challenge with incineration ashes used as construction material in roads is leaching, which
depends greatly on the weather conditions [65]. Another potential use of these ashes is the
anaerobic digestion. Components of the ashes, such as the heavy metals, can improve the yields
of fermentative products [66]. This was the focus of a part of this thesis (paper III), and it is
discussed in more details in chapter 5.
20
20
Table 3.2 Composition of bottom and fly ashes from the gasification of wood pellets, expressed as mg kg-
1 of the dry substance (adapted from [67])
Bottom ash Fly ash
Element 550 °C 800 °C 550 °C 800 °C
Ca 308.0 365.3 309.2 354.7
K 108.0 47.1 48.3 33.8
Mg 32.6 37.8 34.3 35.4
Fe 17.8 23.1 33.3 51.4
Al 11.7 19.6 16.0 17.5
Na 7159.0 8929.0 6212.0 5932.0
Mn 3625.0 4362.0 4979.0 5055.0
Phosphate 36.7 44.7 41.0 44.3
Sulphate 17.1 19.9 18.5 21.6
Si 73.7 n.d. 82.9 n.d.
Zn 466.0 283.0 1416.0 1700.0
B 397.0 464.0 546.0 538.0
Cu 159.0 353.0 150.0 178.0
Cr 111.0 234.0 252.0 455.0
Ni 92.0 104.0 143.0 165.0
Pb 7.0 8.0 47.0 53.0
Cd 0.8 1.1 6.3 6.3
Co 9.0 14.1 n.d. n.d.
21
3. 2 Types of gasifiers
There is a wide variety of commercial gasifiers for waste gasification, with the majority of
them employed for heat and power generation. The main difference in these gasifiers is the
altered parameters, such as design and the operating conditions. The feedstock entry can be done
from the lower side, bottom, or upper side, and the oxidizing agent is usually oxygen, air or
steam. Typical gasifier designs are downdraft and updraft gasifier, bubble, circulating and dual
fluidized bed gasifier, and plasma gasifier [68]. The downdraft gasifier is commonly used for
small or medium applications, with the feedstock added at the top of the gasifier and landing on a
grate. In the updraft gasifier, the feedstock is added at the top of the gasifier, more diverse
feedstock can be treated, and the gasifier has high-energy efficiency. A main difference in these
gasifiers is that the oxidizing media are fed at the bottom of the updraft gasifier. In entrained flow
gasifiers, the feedstock and the oxidation agent are moving in the same direction. The main
feature of the fluidized bed gasifiers is that the feedstock and bed material are levitated by the air
stream that is supplied at the bottom of the gasifier. Finally, in plasma gasification, the feedstock
is gasified by electrically generated plasma at high temperatures (1500 5000 °C) and
atmospheric pressure, in the presence of oxidizing media for the production of high quality
syngas. Autothermal gasifiers provide the heat required for the gasification reactions, in means of
partial oxidation inside the gasifier. This is the main advantage of this type of gasifiers in
comparison to the allothermal gasifiers, where the heat production and heat consumption are
done in separate facilities [69].
Another categorization of gasifiers is by the type of oxidizing media, such as the oxygen
blown and the steam blown gasifiers. The composition of the gas compounds differs greatly when
oxygen or steam is used as an oxidation agent during gasification (Table 3.3). During oxygen
gasification, the CO2 and water produced during the combustion, take part in the chemical
reactions. On the other hand, higher amounts of H2, which derives from the steam, are obtained in
the steam gasification. The tar content is highly dependent on the operating temperature. For
example, in entrained gasification with operating temperature above 1000 °C, a low amount of tar
is produced.
22
22
Table 3.3 Composition of dry gas in oxygen and steam gasification (adapted from [69])
Compound (content) Oxygen gasification
(entrained flow)
Oxygen gasification
(fluidized bed)
Steam gasification
CO (vol %) 40–60 20–30 20–25
CO2 (vol %) 10–15 25–40 20–25
H2 (vol %) 15–20 20–30 30–45
CH4 (vol %) 0–1 5–10 6–12
N2 (vol %) 0–1 0–1 0–1
LHV (MJ Nm-3) 10–12 10–12 10–14
Tar content (g Nm-3) <0.1 1–20 1–10
3.3 Raw gas cleanup
Typical gas contaminants consist of char, tars, nitrogen (NH3, HCN, etc.), sulfur (H2S,
COS, etc.), H2 (HCl, HF, etc.), trace metals (Na, K, etc.) S, alkali, Cl, and particulate matter [70].
The composition of these contaminants varies greatly and is significantly influenced by the
feedstock contaminants and the gasification operating conditions [70]. Examples of feedstock
contaminant composition, tar content in syngas, and trace syngas contaminants are shown in
Table 3.4, Table 3.5, and Table 3.6, respectively.
23
Table 3.4 Examples of feedstock contaminant levels [71-73], (adapted from [70])
Contaminant Wood Wheat straw Coal
Percent by mass
Sulfur 0.01 0.2 0.1–5.0
Nitrogen 0.25 0.7 1.5
Chlorine 0.03 0.5 0.12
Ash (Major Components) 1.33 7.8 9.5
K2O 0.04 2.2 1.5
SiO2 0.08 3.4 2.3
Cl 0.001 0.5 0.1
P2O5 0.02 0.2 0.1
The cleanup of raw syngas is essential prior to the syngas application in downstream
processes [74]. Table 3.7 shows examples of gas cleaning requirements for different syngas
applications. The gas cleanup processes can be classified according to the temperature range into:
hot gas cleanup (HGC), warm gas cleanup (WGC), and cold gas cleanup (CGC) [70]. Cold gas
cleanup takes place at ambient temperatures, where water spray is used and water condenses in
the outlet. Contaminants are either washed away with the condensed water or act as condensation
sites for water. Hot gas cleanup occurs at temperatures as low as 400 °C, while only few hot
cleanup processes manage to operate at temperatures higher than 600 °C. During this type of gas
cleanup, syngas is purified by contaminants, such as alkali compounds [75]. Warm gas cleanup
takes place at temperature ranges between the water boiling point and 300 °C, which allows
contaminants, such as ammonium chloride, to condensate. In addition, at high temperature gas
cleanup processes, candle filters for removing solid contaminants and sorbents for removing fluid
contaminants are employed [76]. An alternative way to treat syngas containing contaminants, to
avoid syngas cleaning requirements, was investigated in this thesis. The protective effect of
membrane encasement of cells against syngas contaminants (tars) was studied in a RMBR
24
24
(Paper IV). As shown in Figure 3.4, a long list of tars can be found in syngas in various
concentrations; however, in this work, due to time and resource restrictions, two common syngas
contaminants, toluene and naphthalene, were investigated [77]. The results of the experimental
work are discussed in more details in chapter 5.
Table 3.5 Analysis of tar content in syngas derived from downdraft gasification with wood pellets (adapted from [77])
Compound Concentration,
mg Nm-3
Compound Concentration,
mg Nm-3
Toluene 76.8 198.3 2-Vinylnaphthalene 0.4 6.7
o/p-Xylene 9.3 111.6 Furfural 0.0 4.0
Naphthalene 62.3 126.1 Naphthalene, 1,8-dimethyl- 0.6 3.6
Phenol 6.9 67.2 Naphthalene, 1,5-dimethyl- 0.0 3.6
Styrene 21.0 65.1 Dibenzofuran 0.4 3.4
Indene 15.7 55.8 alpha-Methylstyrene 1.5 3.1
Ethylbenzene 2.5 25.0 2-Ethyltoluene 0.6 3.0
Phenol, 3-methyl- 1.3 25.4 Benzene, 1,2,3-trimethyl- 1.4 2.4
Benzofuran 8.5 24.9 Phenol, 2,4-dimethyl- 0.0 2.4
Biphenylene 7.1 22.2 Acenaphthene 0.3 2.1
Benzofuran, 2-methyl- 0.0 23.8 Phenol, 3,5-dimethyl- 0.0 1.9
m-Methylstyrene 6.6 18.8 Naphthalene, 2,3-dimethyl- 0.0 1.4
Naphthalene, 2-methyl- 5.1 16.2 Phenol, 3-ethyl- 0.0 1.3
Naphthalene, 1-methyl- 5.9 14.6 Phenol, 4-ethyl- 0.0 1.0
Biphenyl 2.6 10.1 Naphthalene, 1,8-dimethyl- 0.0 0.8
Phenol, 2-methyl- 0.5 8.9 Total tar 340 680
25
Table 3.6 Analysis of syngas trace contaminants collected on filters after gasification of rice hulls and wood chips (adapted from [78]). All concentrations are expressed in μg m-3syngas.
Feedstock Feedstock
Compound Rice hulls Wood chips Compound Rice hulls Wood chips
PM2.5 12.74 11.88 Na 1.45 1.9 Organic carbon
18.43 21.69 Mg 0.09 0.33
Elemental carbon
0.12 1.57 Al 0.87 0.18
Cl- <0.01 <0.01 Si 0.12 0.02
NO3- 0.87 0.55 S <0.01 <0.01
SO4-2 0.40 0.35 Cl 0.37 0.07
NH4+ 0.93 0.90 K 0.44 0.05
Na+ 0.54 <0.01 Ca 0.04 0.06
K+ 0.67 0.12 Fe 0.01 0.08
NH3 35.35 24.17 Zn 0.01 0.07
HCl n.d. n.d. Br <0.01 0.03
HNO3 2.62 3.08 Pb <0.01 0.01
SO2 5.74 <0.01 Others (sum) 1.22 1.80
H2S 0.53 <0.01
26
26
Table 3.7 Gas cleaning requirements for different syngas applications [79-82] (adapted from [70])
Contaminant Application
Internal combustion engine
Gas turbine Methanol synthesis
Fischer-Tropsch synthesis
Particulate <50 mg m−3 <30 mg m−3 <0.02 mg m−3 n.d.*
Tars (condensable) <100 mg m−3 <0.1 mg m−3
Sulfur <20 μL L−1 <1 mg m−3 <0.01 μL L−1
(H2S, COS)
Nitrogen <50 μL L−1 <0.1 mg m−3 <0.02 μL L−1
(NH3, HCN)
Alkali <0.024 μL L−1 <0.01 μL L−1
Halides (primarily HCl)
1 μL L−1 <0.1 mg m−3 <0.01 μL L−1
* n.d. = not detectable
3.4 Catalytic conversion of syngas
The main applications of syngas include the production of ammonia (50%), H2 (25%),
methanol, and Fischer-Tropsch products [83]. In the last decade, R&D processes have mainly
focused on the production of transportation fuels [69]. The production processes of H2 and CH4
from syngas via catalytic conversion are further discussed in the following paragraphs.
3.4.1 Hydrogen production
Syngas can be converted into H2 via the water gas shift (Eq. 3.4) and steam reforming (Eq.
3.5) reactions [61]. Steam reforming is a chemical synthesis process for the production of H2 and
CO from hydrocarbons, at a high temperature and pressure and the presence of a catalyst. Most of
the global H2 production is generated via the steam reforming of natural gas [84]. The CO can be
converted by catalysts, such as iron and chromium, at high temperatures (400 500 °C) [85].
Relatively pure H2 can also be produced by the steam reforming process followed by water gas
27
shift reaction [85]. The CO content can decrease to approximately 2% and 0.2% after the water
gas shift reaction at high and low temperature, respectively [85].
Another way to produce H2 from natural gas is the partial oxidation, where CH4 and other
hydrocarbons react with a limited amount of oxygen, to produce H2, CO, and heat (Eq. 3.9). In
comparison to steam reforming, this process is faster, requires a smaller reactor and gives off
heat. On the other side, more H2 per unit of fuel is produced in steam reforming. The CO
produced can consequently be removed by the water gas shift reaction (Eq. 3.5).
CH4 + 0.5O2 ↔ CO + 2H2 + (heat) (3.9)
During the water gas shift reaction, metals and metal oxides are utilized as catalysts.
Copper-based catalysts are preferred at lower temperatures, while iron oxide-based catalysts are
used at high temperatures [86]. These catalysts are sensitive to impurities. For example, the high
temperature catalyst Fe/Cr (350–500 °C) can be deactivated at sulphur concentrations higher than
200–250 ppm, and the low temperature catalyst Cu/Zn (150–300 °C) can be deactivated at
sulphur concentrations as low as 1–10 ppm [87, 88]. Other catalysts based on cobalt and
molybdenum sulphides are tolerant to sulphur [85]. The CO2 after the water gas shift reaction can
be removed by water absorption [89].
3.4.2 Methane production
CH4 is the main component of synthetic natural gas (SNG), a gas generated by coal or
biomass that can substitute natural gas [90]. The methanation takes place after H2 and CO react in
the presence of catalysts, such as Ni, according to the reaction below:
CO + 3H2 → CH4 + H2O (3.10)
The reaction can occur even at atmospheric pressure, although higher pressure is preferred.
A H2/CO ratio of approximately 3.0 is required, which can be achieved by the water gas shift
reaction. In some types of reactors, such as fluidized beds, methanation can occur in parallel with
the water gas shift reaction; thus, there is no need for additional adjustment of the H2/CO ratio
[61]. An important limitation of this process is that the Ni catalysts are sensitive to sulphur
poisoning. In syngas methanation, removal of CH4 from syngas is not required. This is an
28
28
important advantage for the production of bio-SNG. Both fixed and fluidized bed reactors have
been employed for syngas methanation [90]. A main concern of this process is the heat removal,
which can be addressed by a series of fixed bed reactors with gas cooling [90]. On the other hand,
the use of fluidized bed offers better heat transfer and removal capabilities [91]. However, low
rates and product selectivity in fluidized beds, due to gas and solids backmixing, are important
challenges [91].
An alternative route to the catalytic conversion of syngas to H2 and CH4 is the syngas
fermentation, where microorganisms are utilized as biocatalysts. This combination of
thermochemical and biochemical processes is considered to become more cost effective and more
robust. The technology of syngas fermentation and its advantages in comparison to the catalytic
conversion processes are discussed in the next chapter.
29
Chapter 4
Coupling of thermochemical and biochemical process
The biochemical process of anaerobic digestion can convert organic feedstock into H2 and
CH4. However, the anaerobic digestion cannot efficiently treat a large fraction of potential
feedstock with low degradability, such as lignocellulosic material. This can be overcome by
combining thermochemical and biochemical treatments. Gasification can treat feedstocks
regardless of its low degradability, and the syngas produced can be further anaerobically digested
via syngas fermentation. The coupling of thermochemical and biochemical processes for the
production of fermentative products is a promising strategy with several advantages [92, 93].
Gasification offers high conversion efficiency and a broad range of feedstock, regardless of their
biodegradability, while the biomethanation of syngas is a flexible multipurpose method that can
be used for converting biomass-derived syngas and industrial off-gases, rich in CO and CO2 [94].
In addition, an in-situ biogas upgrading can take place by coupling of an external H2 supply to the
syngas biomethanation bioreactor [95]. In comparison to the combined thermochemical and
catalytic conversion, syngas fermentation has a higher biocatalyst specificity, lower energy costs,
lower sensitivity of biocatalysts to impurities and to the H2/CO ratio, while ambient pressures and
temperatures are required [96, 97]. Moreover, the biological catalysts in the syngas fermentation
can generate a larger variety of products, and the biochemical reactions are irreversible.
Syngas fermentation was the main focus of this thesis. During this process, H2 and CO can
be converted into fermentative products by anaerobic microorganisms that use syngas as a carbon
30
30
and energy source. Some of the main products that have been investigated in recent studies are
ethanol, acetone, butanol, 2.3-butanediol, and polymers, with ethanol being the most popular [8,
96, 98-100]. The effect of parameters, such as pH, temperature, syngas contaminants, and gas
partial pressure has been investigated [101-105]. In addition, several works studied the effect of
bioreactor design in syngas fermentation [94, 106]. The slow gas-to-liquid mass transfer of
syngas components is a main challenge; thus, it has been investigated in different operating
conditions, bioreactor designs, and cultures. For example, a study reported that the CO mass
transfer improved by using an external hollow fiber membrane for gas diffusion [107], while
another study achieved improved mass transfer of syngas in a monolithic biofilm reactor [108]. In
addition, the effect of some syngas contaminants on the microbial activity has been studied [104]
in correlation with the use of pure and mixed cultures. Research has shown that the use of a
mixed culture could be more beneficial in large-scale syngas fermentation due to no requirements
for sterile conditions and due to the higher tolerance and adaptation to syngas contaminants [8,
109, 110].
The technology of syngas fermentation for H2 and CH4 production is still not commercial
because of the high sale prices that are required to support it [110, 111]. The scaling-up of the
process can be achieved by actions such as the coupling of syngas fermentation with already
active gasification activities and achieving higher productivity yields in syngas fermentation
[110]. One of the most important steps toward the commercialization of syngas fermentation is
the local and global policies that aim to replace fossil fuel resources though sustainable processes.
4.1 Metabolic activity for hydrogen and methane production
Syngas can be converted into biofuels and chemicals by several anaerobic microorganisms
with diverse sizes, shapes, and inhabitants. Syngas fermenting microorganisms also have diverse
catabolic metabolisms and can generate different products, such as carboxylic acids, alcohols, H2,
CH4, and CO2. Several of the known strains that have been isolated are gram-positive, and it is
mainly carboxydotrophic hydrogenogens that grow chemolithotrophically during the conversion
of CO and water into H2 and CO2, at 55–80°C [26]. Table 4.1 and Table 4.2 show known
acetogens and methanogens that can ferment syngas components. The vast majority of the known
syngas-fermenting microorganisms are acetogens.
31
Table 4.1 Syngas fermenting bacteria (adapted from [68])
Microorganisms pH T,
°C Substrate Products Reference
Acetobacterium
woodii (wild) 7 30
CO2-H2,
PH2 = 1700 mbar Acetate [112]
Butyribacterium
Methylotrophicum
(adapted on CO)
7.3 37 CO:CO2 =
70:30
Acetic &
butyric
acid
[113]
Clostridium
aceticum (wild) 7.1–8.9 30
CO:H2:Ar =
78:4:18 Acetate [114]
Clostridium
ljungdahlii (wild) 4–6 37 CO:CO2:H2:Ar=
55:10:20:15
Acetate,
ethanol [115]
Clostridium
ragsdalei, P11
(wild)
5–6 37 CO:CO2:H2:N2=
20:15:5:60
Ethanol,
acetic
acid, 1-
butanol
[116]
Carboxydothermus
hydrogenoformans
(wild)
6.8–7 70 CO, 100% Acetate,
methanol,
ethanol
[117]
Citrobacter sp Y19
(wild) 7 30
CO: air+organics
=
2.5:97.5 (v/v)
H2 [118]
Clostridium drakei
(wild) 6–7 25
30 H2/CO2, CO/CO2 Acetate [119]
32
32
Table 4.2 Syngas fermenting methanogens (adapted from [68])
Microorganisms pH T,
°C Substrate Products Reference
Methanosarcina
barkeri (adapted on
CO)
7.4 37 CO, 100% CH4 [120]
Methanosarcina
acetivorans strain
C2A (adapted on CO)
7 37 CO, 100% Acetate,
formate,
CH4
[121]
Methanothermobacter 7.4 65 CO: H2/CO2= CH4 [122]
Thermococcus
onnurineus NA1 6.1–6.2 80 CO, 100% H2 [123]
The production of H2 and CH4 can take place via different biochemical reactions [124],
depending on the syngas composition, existence of co-substrates, operating conditions, and type
of consortia. The conversion of CO can be done by carboxydotrophic hydrogenogens via the
biological water gas shift reaction (Eq. 3.1), by carboxydotrophic acetogens (Eq. 3.2) via the
Wood Ljundgahl metabolic pathway, and by carboxydotrophic methanogens (Eq. 3.3). However,
the CH4 production via carboxydotrophic methanogenesis is considered rare in syngas
biomethanation [110]. The conversion of H2/CO2 is done by homoacetogens (Eq. 3.4) via the
Wood Ljundgahl metabolic pathway by hydrogenotrophic (Eq. 3.5) and aceticlastic (Eq. 3.6)
methanogens. During syngas fermentation, ATP can be produced through different ways, such as
substrate level phosphorylation (SLP), electron transport phosphorylation (ETP), and electron
bifurcation [125-127]. The biochemical reactions, the pathways, and energy conservation are
further discussed in the following paragraphs.
The biochemical reactions are presented with the standard change of Gibbs free energy
[110, 124].
CO + H2O → CO2 + H2, ΔG0 = -20 kj/mol (3.1)
33
4CO + 2H2O → CH3COOH + 2CO2, ΔG0 = -165.4 kj/mol (3.2)
4CO + 2H2O → 3CO2 + CH4, ΔG0 = -210.9 kj/mol (3.3)
2CO2 + 4H2 → CH3COOH + 2H2O, ΔG0 = -104.6 kj/mol (3.4)
CO2 + 4H2 → CH4 + 2H2O, ΔG0 = -135.6 kj/mol (3.5)
CH3COOH → CH4 + CO2, ΔG0 = -31 kj/mol (3.6)
The carboxydotrophic hydrogenogens conserve energy via the H2 formation (Figure 4.1)
during the biological water gas shift reaction. The main enzyme that catalyzes this reaction is
CO-dehydrogenase (CODH), which oxidizes CO into CO2. An iron-sulfur protein called
ferredoxin (Fd) transfers the released electrons to an energy-conserving hydrogenase (ECH),
where they are bound with protons and form molecular H2 [128, 129]. A fraction of the CO2 is
used as cell components and biomass, and the rest is released from the cells along with H2.
Another important function of ECH enzyme is the transfer of protons or sodium ions through the
cellular membrane creating an electrochemical proton gradient. This mechanism generates energy
through the ATP-synthesis that is initiated by the ATP-synthase [128, 129].
Figure 4.1 Schematic illustration of CO-oxidation, electron transfer, H2-formation, and proton transfer
through the cell-membrane. CODH: CO dehydrogenase; Fd: ferredoxin; ECH: energy-conserving
hydrogenase (adapted from [26, 128])
34
34
During syngas fermentation, CO can be converted into CO2 (Eq. 3.1), and acetate can be
produced by acetogens via the reductive Acetyl-CoA or Wood-Ljungdahl pathway (Eq. 3.2 and
Eq. 3.4) as seen in Figure 4.2. The reductive Acetyl-CoA is an important pathway utilized by
microorganisms in syngas fermentation in order to achieve autotrophic CO2 fixation. This means
that carbon from an inorganic source is used by the cells as the sole carbon and energy source. In
syngas fermentation, several microorganisms follow the Acetyl-CoA pathway [68]. The Acetyl-
CoA synthase/CO-dehydrogenase complex (ACS/CODH) binds to a carbonyl and a methyl group
[130, 131], while CO-dehydrogenase enzyme reduces the CO2 to CO, which serves as a carbonyl
group [26]. The methyl group is formed after successive reductions of CO2 to several
intermediates, which are bound to a pterin coenzyme. The intermediate product Acetyl-CoA is
used as a cellular precursor and as an energy source. During the Acetyl-CoA pathway, H2 is the
electron donor in the presence of the hydrogenase enzyme. If H2 is absent, CO becomes the
electron donor, in the presence of CO-dehydrogenase (CODH). Therefore, the supply of adequate
H2 or CO during syngas fermentation is crucial [132]. Energy (1 mol ATP) and electrons are
required in order to reduce the CO2 to Acetyl-CoA, and the same amount of energy is recovered
when the acetyl-synthase catalyzes the Acetyl-CoA and CO reaction for acetate formation. In the
Acetyl-CoA pathway, 1 mol ATP is consumed while the CO2-pterin is reduced to methyl-pterin
[133], and 1 mol ATP is generated when acetate is formed. This energy conservation is done via
the substrate level phosphorylation.
35
Figure 4.2 The Wood-Ljungdahl pathway. H4F: tetrahydropholate; CoFeSP: corrinoid iron-sulphur protein
(adapted from [134]).
Acetate can be converted into CH4 and CO2 via aceticlastic methanogenesis as shown in
Figure 4.3. Initially, acetate is converted into acetyl-CoA, and then the methyl group is attached
to the tetrahydrosarcinapterin, and consequently to coenzyme M. Finally, the methyl-SCoM is
reduced to CH4 with coenzyme B as the electron donor [135]. In this pathway, 1 mol ATP is
generated for 1 mol of CH4 produced [136]. All acetotrophic methanogens gain energy by the
36
36
electron transfer from ferredoxin to CoM-S-S-CoB with the creation of a proton gradient, as
shown in Figure 4.1 [135].
Figure 4.3 Metabolic pathway of aceticlastic methanogenesis. CoA: coenzyme A; H4SPT:
tetrahydrosarcinapterin; HS-CoM: sulfhydryl coenzyme M; HS-CoB: sulfhydryl coenzyme B; CoM-S-S-CoB:
heterodisulfide coenzymes CoM and CoB (adapted from [135]).
In hydrogenotrophic methanogenesis, the CO2 is converted into CH4 by the use of H2.
Therefore, this pathway controls the H2 to low levels and therefore assists the acetogenesis
reaction. Figure 4.4 shows the so-called Wolfe cycle of hydrogenotrophic methanogenesis [137].
The pathway has been described in literature [138] and starts with the reduction of CO2 to formyl
methanofuran with ferredoxin as the electron donor. Thereafter, the formyl group is attached to
the tetrahydromethanopterin (electron carrier) and then converted into methenyl-
tetrahydromethanopterin. Consequently, the electron donor reduces the electron carrier F420 to
F420H2 with hydrogenases and dehydrogenases. F420 catalyzes the reduction of methylene-
37
H4MPT to methyl-H4MPT. Afterwards, the methyl group is transported to the coenzyme M and
reduced to CH4. Energy is conserved by the electron bifurcation that occurs, heterodisulfide
reductase complex, when the CoM-S-S-CoB heterosulphide is converted into HS-CoM and HS-
CoB. This reaction generates the energy required for the formyl-MFR formation reaction [137,
139, 140]. Hydrogenotrophic methanogens can also use H2 in order to oxidize CO to CO2, which
is thereafter reduced to CH4 by utilizing H2 [141].
Figure 4.4 Metabolic pathway of hydrogenotrophic methanogenesis. MFR: methanofuran; H4MPT:
tetrahydromethanopterin; HS-CoM: sulfhydryl coenzyme M; HS-CoB: sulfhydryl coenzyme B; CoM-S-S-
CoB: heterodisulfide coenzymes CoM and CoB (adapted from [138]).
38
38
4.4 Operational parameters
4.4.1 pH
The pH can affect the microbial activity during syngas fermentation. More specifically, it
can influence the cellular metabolism, by altering the intracellular pH and the electrochemical
gradient across the membrane [110]. The methanogens grow mostly at a neutral pH at a range of
6.8 8.5 [142]. Thus, most of the syngas fermentation studies are operated at pHs close to 7.0
[110]. The acetogenic bacteria are possibly the most tolerant group in various pH values,
including both alkaline and acidic environments [143]. These microorganisms are known to
produce acids at a higher pH and alcohols at a lower pH [144]. In addition, the hydrogenogens
mostly thrive at a neutral pH [145]. In a study on the mesophilic syngas biomethanation in
different pH and syngas pressures, the highest CH4 production of 0.89 mol CH4/mol syngas was
achieved for pH 5.8 and syngas pressure 1atm [146]. In this thesis, the effect of different pH
values (5, 6, and 7) was investigated during the addition of various concentrations of three heavy
metals (Paper III). According to the results, at high metal concentrations of 0.625 mg Cu/L, 3.75
mg Zn/L, and 17.5 mg Mn/L, the microbial activity was stimulated at pH 5 [66].
4.4.2 Temperature
The temperature in the bioreactors is of vital importance for the microbial activity in syngas
fermentation. The range of temperature affects the metabolic pathways used by the
microorganisms [110]. For example, acetate is considered the main CH4 precursor at mesophilic
conditions, while H2 is considered the main CH4 precursor in thermophilic conditions probably
because of the higher diversity of carboxydotrophic hydrogenogens in thermophilic conditions
[110, 147]. Another possible reason could be that the H2 formation from CO is
thermodynamically favored at thermophilic conditions [148]. On the other hand, even the
hydrogenotrophic methanogenesis is enhanced at thermophilic conditions in comparison to
mesophilic conditions. A study on the enrichment of mesophilic and thermophilic mixed culture
showed that CH4 productivity was approximately 18 times higher in mesophilic conditions and
that the thermophilic metabolism was dominated by carboxydotrophic hydrogenogens and
hydrogenotrophic methanogens [149]. Similarly, another study reported that the CO conversion
rates to CH4 were increased along with higher fermentation temperatures [147]. In this thesis,
(paper I), higher CH4 yields were observed during the conversion of artificial syngas (H2, CO,
39
CO2) by mixed culture, at 55 °C in comparison to 35 °C [150]. Another study reported that
various types of sludge had higher CO conversion to CH4, at thermophilic conditions, even if
they had been previously incubated at mesophilic conditions [147]. In general, higher syngas
conversion rates and CH4 production rates have been observed in thermophilic conditions;
however, higher energy input is also required and lower gas solubility can occur, in comparison
to mesophilic fermentation. The coupling of gasification and syngas fermentation could be an
advantage due to the fact that less effort is needed for the cooling of the syngas to thermophilic
levels than to mesophilic. Moreover, the lower gas solubility at a higher temperature can be
balanced by the increased gas diffusivity. For example, a study on gas diffusivity in water
showed that the CO diffusivity doubled from 35 °C to 60 °C [151].
4.4.3 Pressure
The pressure can affect the mass transfer rates of syngas components and consequently the
syngas conversion rates. An increase in the bioreactor´s operating pressure can increase the gas
solubility and achieve higher conversion rates without the need for bigger bioreactors [106, 152,
153]. The conversion of H2/CO2 mixture into CH4 has been studied in different pressures. In a
study on the influence of pressure in trickle bed bioreactors, the CH4 content increased by 34%
by increasing the pressure from 1.5 to 9 bar [153]. However, based on the author´s knowledge,
there are no studies on the effect of pressure during syngas conversion to H2 and CH4.
Theoretically, high CO pressures can cause inhibitory effects on the microbial activity. The effect
of the partial pressure of syngas components is discussed below.
4.4.4 Partial pressure
The partial pressure of each syngas component affects the mass transfer of this component
from the gas to the liquid phase inside the bioreactor´s broth. H2 and CO have low solubility in
liquids; thus, their partial pressure is important for efficient syngas conversion. On the other
hand, high CO partial pressure can lead to cellular inhibition. During syngas fermentation, the
most sensitive microorganisms to CO are carboxydotrophic methanogens and sulphate reducers,
which can usually tolerate PCO of 0.5 1.0 atm and 0.2 0.5 atm, respectively [121, 122, 154]. The
anaerobic culture can be more tolerant to high PCO after adaptation. A study reported that
although methanogenesis was totally inhibited at PCO ≥1 atm, higher methanogenic potential was
obtained after acclimatization to a 100% CO atmosphere [155]. This adaptation favored the
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40
domination of hydrogenotrophic methanogens, which were able to take over acetoclastic
methanogens, whereas syntrophic acetate oxidizing (SAO) bacteria converted acetate into CO2
and H2. Another study also reported the shift in microbial community from the addition of CO
during H2/CO2 fermentation. More specifically, the addition of CO resulted in the enrichment of
the microbial community with bacteria from the genera Thermincola and Thermoanaerobacter
[156]. The PH2 is considered to have less negative effect, in comparison to PCO, on the microbial
community. A study reported that the hydrogenase activity improved, along with the PH2, until
inhibition occurred probably due to saturation of the hydrogenase [157]. Another work reported
that increasing the initial pressure of H2/CO2 from 1 to 5 bar led to an increase in the CH4 rate
production from 0.035 ± 0.014 mmol h-1 to 0.072 ± 0.019 mmol h-1 [158].
4.5 Fermentation systems for hydrogen and methane production
The aim of the different bioreactor designs is to address the limitations of the syngas
fermentation process. Various bioreactor types have different advantages and disadvantages. For
example, the continuous stirred tank bioreactor (CSTR) has high mass transfer rates although it
requires high energy input [101, 159]. Trickle bed bioreactors have a lower energy demand than
CSTR and good gas-to-cell contact; however, gas channeling is a potential challenge [96, 160].
Membrane bioreactors (MBR), such as hollow fiber BR, offer high mass transfer rates, although
there is the challenge of clogging due to microbial growth on the fibers [161].
4.5.1 Methane production
Several studies have investigated the production for CH4 via syngas fermentation. The
conversion of CO into CH4 was studied in a continuous CSTR at both mesophilic and
thermophilic conditions with a mixed culture [162]. The H2:CO2 was fed in the bioreactor with a
ratio of 4:1, according to the stoichiometric requirements for hydrogenotrophic methanogenesis
(Eq. 3.5), and the cells were recycled in order to achieve high cell density. According to the
results, the highest CH4 productivity of 446 mmol LR-1 h-1 was achieved at thermophilic
conditions. Syngas biomethanation, with different types of anaerobic sludge, was also studied in
an upflow anaerobic sludge bed (UASB) bioreactor, where a maximum methanogenic activity of
1 mmol CH4 g-1 VSS d-1 was obtained at a PCO of 0.2 atm [155]. Higher CO biomethanation rates
were reported in a CSTR, by the use of a thermophilic co-culture of Carboxydothermus
41
hydrogenoformans and Methanothermobacter thermoautotrophicus in comparison with the
Methanothermobacter thermoautotrophicus monoculture [163]. The maximum CO conversion
efficiency and CH4 production rate achieved in that study were 93% and 17.6 mmol LR-1 d-1,
respectively.
A mixed culture was also used for the biomethanation of syngas. More specifically, syngas
was converted into CH4 by a triculture, consisting of the photosynthetic bacterium R. rubrumand
the methanogens M. formicicum, and M. barkeri [164]. The bacterium was chosen for the
conversion of CO into CO2 and H2 by the water gas shift reaction and the methanogens for the
conversion of CO2 and H2 into CH4. Two types of bioreactors were utilized, a packed bubble
column and a trickle bed bioreactor. The bubble column bioreactor was filled with glass Raching
rings (6mm × 6 mm) and the liquid medium was fed at the top of the bioreactor, whereas the
syngas was fed at the bottom of the vessel. The trickle bed bioreactor had a column height of 515
mm, with an inner diameter of 51 mm. The packing material was 6.35-mm Intalox saddles; the
total volume of the packed section was 1052 mL, and the gas and liquid flowed downward
through the packing material. The results showed CO conversion rates of 100% and 79% in the
trickle bed and bubble column bioreactor, respectively and mass transfer coefficients of 780 h-1
and 3.5 h-1 in the trickle bed and the bubble column bioreactor, respectively. The same triculture
was later used for the comparison of syngas fermentation in 5-cm and 16.5-cm diameter trickle
bed bioreactors [165]. The results showed a yield of 0.20 mol CH4 mol-1 H2, which is comparable
to the theoretical yield of 0.20 mol CH4 mol-1 H2. However, the larger bioreactor showed poor
performance probably due to low cell density or because of poor distribution of the liquid through
the column.
A new type of anaerobic trickle bed bioreactor was used for the biomethanation of H2 and
CO2 [160]. The 61-L pilot scale bioreactor was operated as a plug flow, at mesophilic conditions
with immobilized culture on a biofilm without gas-recirculation. The results showed an
impressive 98% CH4 concentration and a CH4 productivity of 2.77 mmol LR-1 h-1 at a gas
retention time of 4 h. A 30 L gas-lift bioreactor was used in another work for the biomethanation
of syngas with granular sludge [166]. The industrial granular sludge was first used for mesophilic
batch carboxydotrophic methanogenesis; thereafter, it was introduced in a gas-lift bioreactor and
fed with CO. The highest CO-conversion efficiency achieved was 75% at a PCO of 0.6 atm and a
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42
gas recirculation ratio of 20:1. A 5-fold higher CO-conversion potential was achieved at
thermophilic conditions. The highest CH4 specific productivity obtained was 0.126 mmol·g-
1VSS·h-1 (95% of the theoretical CH4 yield), at a gas recirculation rate of 600 mL min-1 and gas
retention time of 8.6 d. In a similar work, a stirred tank bioreactor with 400 mL working volume
was operated at 55 °C, pH 7 for the CO biomethanation with sewage sludge as the inoculum
[167]. The gaseous substrate was fed to the bioreactor via a hollow fiber membrane (HFM), and a
high CH4 productivity of 3.7 mmol·LR-1·h-1was reported although the CH4 percentage in the
gaseous product was only 19.2%. This was probably a result of the poor CO solubility in the
medium and the high CO flow rate of 6.6 mL·min-1·LR-1. The main archaeal species involved in
the CO conversion were Methanosarcina barkeri and Methanothermobacter thermautotrophicus,
while the bacterial species could not be identified.
The direct biomethanation of H2 and CO2 was achieved in an anaerobic bioreactor, with
Methanobacterium thermoautotrophicum cells immobilized on a cellulose acetate membrane or
inside a porous silica-alumina ceramic support [168]. The ceramic bioreactor was cylindrical (30
mm × 70 mm), with an average pore size and porosity of 100 μ and of 79.7%, respectively. In the
membrane bioreactor, a CH4 production rate of 0.75 ml CH4 per cm2 contact area per h was
achieved, while the initial fixed-cell mass increased from 0.2 mg dry cell cm-2 of contact area to 1
mg cm-2 after 12 h of cultivation. On the other hand, in the ceramic bioreactor, a CH4 production
rate of 6 L CH4 Lceramic-1 LR
-1 was achieved while the methanogen growth was homogeneous
inside the ceramic up to 7 cm depth, with a cell density between 20 and 30 mg dry cell cm-3
ceramic.
In this thesis (paper II), a reverse membrane bioreactor was operated at thermophilic
conditions for the biomethanation of artificial syngas in continuous mode. The bioreactor was a
serum glass bottle with 600-mL working volume, closed with rubber caps. The inoculum was a
mixed culture from a food digester enclosed inside hydrophilic PVDF flat sheet membranes that
formed 6 cm × 3 cm rectangles by heat sealing. Each sachet contained 3 g of inoculum, and each
bioreactor contained 15 sachets, while it was filled with 475 mL of synthetic medium. Syngas
was fed at the bottom of the bioreactor, and it was continuously recirculated. Although this type
of bubble column bioreactor has moderate mass transfer rates, it demands lower power
consumption, maintenance, and operational costs in comparison to other bioreactor types [159,
43
169]. The results showed that the reverse membrane bioreactor retained successfully high cell
density by eliminating the cell washout for 154 days. The maximum CH4 productivity achieved
was 8.3 mmol·L-1·h-1.
4.5.2 Hydrogen production
The syngas bioconversion to H2 has not been investigated so extensively as the syngas
biomethanation. Nevertheless, several studies with various bioreactor designs have been
conducted. Two studies reported the use of a mesophilic continuous stirred tank reactor for the
production of H2 from syngas (20% H2, 15% Ar, 55% CO, and 10% CO2), with the anaerobic
photosynthetic bacterium Rhodopirillum rubrum [170, 171]. The highest H2 production activity
and productivity achieved were approximately 16 mmol gcell-1 h-1 and 24 mmol L-1 h-1,
respectively, at pH 6.5, agitation of 500 rpm, syngas flow rate of 14 mL min-1 and the use of
acetate for cell growth [170]. In another work, the isolated strain Rhodopseudomonas palustris
PT was used in batch mesophilic experiments for the conversion of CO-rich gas into H2 [172].
The maximum H2 production in response to the CO consumption (86%) and the highest H2
concentration of 33.5 mmol L-1 was achieved at a concentration of 1.5 g L-1 sodium acetate.
The performance of a hollow fiber membrane bioreactor was studied for the CO conversion
to H2, with Carboxydothermus hydrogenoformans [173]. The study reported maximum CO
conversion ratio and H2 production rate of 97.6% and 0.46 mol d-1 at a gas loading rate of 0.22
mol d-1 and a liquid recirculation speed of 1.5 L min-1. The same bioreactor and strain were used
for the bioconversion of CO into H2 under various operational conditions [174]. In this work, a
biofilm was formed on the hollow membrane surface inside the liquid medium. This allowed for
the highest CO conversion activity of 0.44 mol CO g VSS d-1 and a maximum H2 productivity of
125 mmol L-1 h-1, at 70°C, with a PCO of 2 atm and liquid velocity of 65 m·h−1. The same
bioconversion was operated by the same pure strain in an 35 L gas-lift bioreactor, with
continuous gas supply for three months, at 70 °C [117]. The results showed highest conversion
efficiency of up to 90%, with a CO conversion rate of 2.7 mol g-1VSS d-1 and a H2 productivity of
6.7 mmol L-1 d-1. This performance was limited due to low cell density of the bioreactor.
A continuous stirred bioreactor with a working volume of 5 L was operated at mesophilic
conditions for the CO conversion to H2 [175]. At the beginning of this experiment, sucrose was
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44
supplied to the cells as a carbon and energy source and thereafter, the substrate was switched to
CO under anaerobic conditions. The maximum H2 productivity obtained was 41 mmol L-1 d-1 and
the CO conversion efficiency was 61%, at gas retention of 5 min, pH 7, and agitation of 700 rpm.
Another study employed a similar strategy of sucrose feeding before switching to CO feeding in a
CSTR with a working volume of 3 L [118]. The innovation of this study was the use of an
isolated bacterium strain, Citrobacter sp. Y19, which grows on aerobically organic carbon source
and converts CO and water into H2 in anaerobic environments. The study reported a maximum
H2 productivity of 5.7 mmol L-1 d-1, at pH 7, 30 °C and 500 rpm agitation, and three times higher
H2 production activity (27.1 mmol gcell-1 h-1) in comparison to that of Rhodospirillum rubrum.
The use of a genetically modified Thermococcus onnurineus NA1 for CO-dependent H2
production was investigated in a CSTR, with a working volume of 2 L, at 80 °C, pH 6.1–6.2, and
agitation of 300 rpm [123]. The mutant strain MC01 had a 30-fold higher transcription of the
mRNA encoding CODH and hydrogenase. This resulted in a maximum H2 productivity of 123.5
mmol L-1 d-1 that was 3.8-fold higher than that of the wild strain. The catabolic activity of the
mixed culture can be affected by components that favor the H2-formation while they inhibit
methanogenesis. For example, during a study on the effects of heavy metals in syngas
fermentation with a mixed culture (paper III), the addition of HNO3 inhibited the production of
CH4 and favored the H2 production [66]. The experiments were conducted in repeated batch
conditions with serum glass bottle bioreactors, at 55 °C. The results of this work are further
discussed in the following chapter.
4.6 The reverse membrane bioreactor
The concept of reverse membrane bioreactors (RMBR) is relatively new, and it has been
initiated and studied in the University of Borås since 2012 [176, 177]. In order to prepare the
membrane sachets, a flat sheet polyvinylidine difluoride (PVDF) membrane was cut into a
square, with dimensions 6 × 6 cm. Then, the membrane was folded into half, and the long side
and one of the smaller ends were heat-sealed, forming an envelope. Thereafter, the inoculum was
placed inside the envelope, and the last side was finally heat-sealed. The membrane sachet
containing the anaerobic cells was then immersed into the liquid medium of the bioreactor.
Liquid substrate passed though the membrane pores by passive diffusion, and gaseous products
exited the membrane when the required pressure was built inside the sachets. Figure 4.5 shows a
45
schematic illustration of the RMBR system that was operated continuously for 154 days (paper
II).
Figure 4.5 Schematic illustration of the reverse membrane bioreactor (RMBR) in the semi-continuous
biomethanation process of syngas and organic substances. (a) Digesting sludge encased in PVDF
membrane, (b) Organic and nutrient medium, (c) Syngas, (d) Peristaltic pump, (e) Gas outlet, (f) Warm
water bath, (g) Effluent, (h) Flow meter, (i) Data analysis [169].
Syngas fermentation for H2 and CH4 production has been investigated in several works, and
important progress has been achieved. However, there are main challenges that still need to be
overcome prior to the commercialization of the process. The main challenges, such as cell
washout, toxic effect of inhibitors on the microbial activity, and low gas-to-liquid mass transfer
rates, are discussed in the following chapter.
46
46
47
Chapter 5
Improving syngas fermentation
Syngas fermentation for CH4 and H2 production has several advantages in comparison to
the catalytic conversion processes, as discussed in the previous chapter. However, this is a
relatively new technology; thus, there are still challenges that need to be addressed. For example,
the low gas-to-liquid mass transfer rates of syngas components and the systematic cell washout in
continuous processes in combination with the low growth rates of microorganisms, especially
methanogens, cause low fermentation yields. The presence of toxic syngas contaminants can
inhibit the microbial activity and lead to bioreactor failure. Furthermore, the unsustainable use of
gasification ashes, containing heavy metals, poses high health risks and leads to environmental
pollution. The main task of this thesis was to address the challenges mentioned above.
5.1 Cell washout
In continuous fermentation processes, the cell washout from bioreactors has been a
bottleneck that causes low cell density and lower capacity. Especially when CH4 is the desirable
product of fermentation, cell-washout is more important because of the low growth rate of
methanogens. For example, during a continuous syngas fermentation experiment with mixed
suspended culture, the CH4 production rate started to decrease after 40 days of fermentation
because of systematic cell-washout [169]. In another study, low yields were observed for cell-
dilution rates greater than 0.55 d-1 [159]. Different strategies have been investigated in order to
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48
minimize the cell-washout. Cell immobilization in tubes or packing material with cavities can
provide high cell-density, better gas-to-cell contact, and no need for mechanical agitation [102,
106]. The concept of immobilized sludge was investigated in a horizontal-flow anaerobic
immobilized sludge (HAIS) bioreactor [178]. In another work, a horizontal packed-bed
bioreactor, fed with glucose-based synthetic wastewater, was used for the conversion of low
contents of organic matter into organic acids and H2 [179]. However, the gas channeling caused
by the cell overgrowth on the tubes and cavities and the limiting column dimensions are
considered to be the main drawbacks of this method [102, 106]. In a study that aimed to address
the improvement of the biogas production rates, the anaerobic cells were encapsulated in natural
membranes that were made with alginate, using chitosan or Ca2+ as counter-ions, together with
the addition of carboxymethylcellulose (CMC) [176]. The results of that study showed that cell
encapsulation is a promising technique in order to achieve high cell density in bioreactors.
Figure 5.1 Elimination of cell washout in a RMBR
MBRs have been also used in order to increase the cell density and stop cell washout. Both
submerged and side stream membrane setups have been investigated. The submerged modules do
not require recirculation; however, the membrane area in contact with the liquid broth is limited,
and concentration polarization may occur [180]. Cell recycling in a continuous MBR allowed for
higher yields at higher dilution rates during syngas fermentation [181]. In another study, high
49
COD removal and CH4 yield with no VFA accumulation was achieved in an anaerobic biofilm
MBR for wastewater treatment [182]. The concept of the use of membranes in RMBR for high
cell density is schematically illustrated in Figure 5.1. The preparation and setup of the RMBR
was explained in section 4.5. This type of bioreactor has been successfully used in a previous
study for improving the biogas production rates during anaerobic digestion [176]. In another
similar set up, the cells were encased in multiple membrane horizontal layers, placed inside a
packed MBR [183]. However, the membrane encasement concept had never been used in syngas
fermentation.
In this thesis, a RMBR was used in order to address cell washout in syngas fermentation.
The membrane-encased cells were retained successfully inside the bioreactor during syngas
fermentation in both batch and continuous experiments [150, 169]. In the repeated batch
experiment (Paper I), the RMBR was successfully operated for the first time in syngas
biomethanation. Higher CH4 production was observed at 55 °C in comparison to 35 °C, and the
addition of a synthetic organic medium improved the CH4 production. In the case of the
continuous experiment (Paper II), a RMBR was successfully operated for 154 days without any
visual cell leaking from the membranes.
Figure 5.2 Comparison of the CH4 production rate in RMBR and FCBR, during the biomethanation of
syngas and organic substances [169]
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50
Figure 5.2 shows the comparison of CH4 production rate, in a RMBR and a free cell
bioreactor (FCBR) during continuous syngas fermentation. The addition of a liquid organic
medium after period III and the systematic cell washout caused a decrease in the CH4 production
in the FCBR, while the CH4 production in the RMBR was significantly higher. This result
showed that the high cell density was crucial for the higher CH4 production rate inside the RMBR
and that the membranes can be used for a long period without failing.
5.2 Inhibitors
In a syngas fermentation process, several contaminants may act as inhibitors of the
microbial activity, especially that of the methanogens. However, the reported effect of inhibitors
differs in the various studies due to the fact that the inhibition levels depend on several
parameters, such as the inhibitor concentration, the composition of the inoculum, the microbial
acclimatization to inhibitors as well as the environmental and operational conditions [184].
Moreover, synergistic and antagonistic effects between anaerobic microorganisms can influence
their tolerance in inhibiting components [184]. At low concentrations, some inhibitors may not
affect the microbial activity or they can be used as substrates. Two common types of
contaminants that are formed or released during the gasification process are tars and heavy
metals. Tars are formed during gasification, and they are contaminants of raw syngas. Syngas
contaminants need to be removed after gasification by costly processes. High concentration of
heavy metals are contained inside gasification ashes, which can leak into the environment. The
effect of three heavy metals, in syngas fermentation, was studied in this thesis. Furthermore, the
use of the RMBR in order to protect the anaerobic cells against high concentrations of two
common tars was investigated.
5.2.1 Effect of heavy metals in anaerobic digestion
Heavy metals are important for the metabolism of the anaerobic consortium because many
of them are part of enzymes that catalyze anaerobic reactions [185]. However, high
concentrations of heavy metals may lead to toxic conditions for the cells. In anaerobic digesters
fed with waste, such as municipal sewage and sludge and industrial waste, heavy metals can be
present in high concentrations. Heavy metals can disrupt the enzymatic activity and structure by
binding on enzyme prosthetic groups or by binding with thiol on proteins [186, 187]. Unlike
51
other components, the microbial community cannot degrade heavy metals, which can instead
accumulate to toxic concentrations [187] and lead to heavy metal toxicity and failure of the
anaerobic digester.
The stimulatory or inhibitory effect of heavy metals depends on the type of
microorganisms, the metal concentration in the digester, the chemical form of metals, and other
parameters, such as the pH and redox potential [188-190]. Studies that observed 50% inhibition
of methanogenesis reported that metal toxicity decreased in the order of Cu > Zn > Ni [190-192].
Another study on anaerobic digestion of sewage sludge reported that metal toxicity decreased in
the order of Cr > Ni > Cu > Zn [193]. In addition, the toxicity of some metals can be either
synergistic or antagonistic. More specifically, most mixed heavy metals, such as Cr–Cd–Pb, and
Zn–Cu–Ni were found to act synergistically [192], although Ahring and Westermann [194]
reported that the presence of Ni decreased the toxicity of Cd and Zn. A review of the effects of
heavy metals in anaerobic digestion reported that metals, such as Cu, Zn, Ni, Cd, Cr, and Pb have
been overwhelmingly reported to have inhibitory activity, depending on their concentrations
[195]. On the other side, the same review reported that metals, such as iron can have stimulatory
effect [195]. For example, a study reported that the conversion of acetate, propionate, and
methanol, by granulated sludge, was stimulated by the continuous addition of trace metals in the
influent [185]. Higher H2 yields were also achieved after the addition of Cu and Cr ions, during
anaerobic fermentation [35].
Figure 5.3 Utilization of heavy metals from gasification ash during syngas fermentation
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52
Figure 5.4 Relationships between H2 production activity, Ah (%) = (mol H2 mol-1 H2 control) 100 (a), H2
yield, YH = mol H2 mol-1 CO fed (b), initial medium pH and heavy metal concentrations (mg L-1) [66]
Heavy metals are found in large concentrations in the ash of the gasifier, at the end of the
gasification process. The ash is rich in heavy metals in various forms, such as metal oxides and
metal chlorides [67, 196, 197]. The use of these ashes in road and other constructions raise
53
significant concerns about the environment and public health because of leaching [198]. Heavy
metals are considered a threat because they are classified as possible carcinogens [199].
Therefore, the addition of heavy metals deriving from gasification ashes (Figure 5.3) could
reduce the footprint of gasification and improve the yields in syngas fermentation.
In this thesis (paper III), the effect of a metal mixture of Cu, Zn, and Mn as well as the
effect of pH were investigated during the syngas conversion into fermentative H2 [66]. The heavy
metals were mixed in various concentrations, and the results showed that the utilization of these
components improved the fermentative H2 production. Moreover, the beneficial and inhibitory
concentrations were reported. Figure 5.4 shows the H2 production activity in
(mmol/mmolcontrol) 100% and H2 yield, in mol H2 mol-1 CO fed, at different mixtures of Cu, Zn,
and Mn at a pH of 5, 6, and 7. According to the results, heavy metal concentrations of up to 0.1
mg Cu L-1, 0.67 mg Zn L-1 and 2.8 mg Mn L-1 were beneficial at all pH values. On the contrary, at
a higher heavy metal concentration of 0.625 mg Cu L-1, 3.75 mg Zn L-1 and 17.5 mg Mn L-1,
stimulation was observed only at pH 5. This shift in the preference of lower pH at higher metal
concentrations could be connected to the types of different thriving anaerobic strains in the
culture [66].
5.2.2 Protective effect of rMBR against syngas contaminants
Raw syngas can contain contaminants, such as particulate matter, tars, NOx, S, N2, and
alkali [70], which can inhibit the cellular performance during syngas fermentation [110]. Tar is
one of the most common types of contaminants; thus, it has been the main focus of several gas
cleaning technologies and studies [200]. Tars can be classified into: 1) >7 carbon ring
compounds; 2) heterocyclic, such as phenol; 3) light aromatic, such as toluene, styrene, and
xylene; 4) light polyaromatic, such as naphthalene and anthracene; and 5) heavy polyaromatic,
such as fluoroanthracene, pyrene, and chrysene [201]. Tars are condensable organic compounds
that are created mainly at lower temperatures during gasification. Toluene and naphthalene are
two common contaminants in raw syngas [202], and they can be toxic at high concentrations
[203-205]. The presence of tars and the tar content in raw syngas depends highly on the
gasification operating conditions and the type of feedstock. For example, benzene and toluene
can be found in syngas from gasification of switchgrass [206]. Although there is a high content of
tars in raw syngas, there is insufficient knowledge on the inhibitory effect of different syngas
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54
contaminants and on the minimum gas-cleaning requirements prior to fermentation [110]. From a
review in the literature, different inhibition levels of tars, such as toluene and naphthalene, are
reported in different studies [203, 207, 208]. The microbial tolerance toward these compounds
depends greatly on the composition of the microbial culture, as several bacteria can degrade
toluene and naphthalene [204, 209]. Microbial adaptation is also an important factor for the
toluene and naphthalene effect [203]. Furthermore, studies have shown that the presence of other
compounds can influence the effect of tar presence in digesters. For instance, a study reported
that the presence of toluene inhibited the anaerobic digestion, whereas the addition of toluene and
methanol did not cause any inhibition effect on the microbial activity [207]. Toluene and
naphthalene in raw syngas can be removed during costly gas cleaning processes as discussed in
Chapter 3. However, if this cleaning process could be avoided, the overall cost of the syngas
fermentation process could drop considerably.
Figure 5.5 Protective effect of membranes against syngas contaminants
An alternative and cost-effective solution to the syngas cleaning methods is to use raw
syngas as substrate and cover the cells in the bioreactor with a protective membrane. Figure 5.5
illustrates the concept of membrane protection against syngas contaminants. This concept is
based on the hydrophilicity of the membrane surface and the hydrophobic nature of syngas
55
contaminants. Similar MBRs have been successfully used in a previous study that showed their
protective effect against D-limonene, an inhibitor from citrus pills [210].
Figure 5.6 The effect of naphthalene on the (A) CH4 production rate, mmol CH4 L-1 d-1; (B) CH4 production
activity, (mol mol-1control) 100%; and (C) the naphthalene concentration, g L-1; in the FCBR and the RMBR
[submitted manuscript]
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In this thesis (paper IV), a RMBR was used in order to protect the encased cells during
their exposure to high toluene and naphthalene concentrations. The experiment was operated in
continuous mode, at 55 °C while the bioreactors were continuously shaken inside a shaking water
batch. According to the results, the CH4 production rate was higher in the RMBR in comparison
to the FCBR. More specifically, at the highest toluene and naphthalene concentration of 6.44 g L-
1 and 2.47 g L-1, the CH4 production rate in the RMBR was approximately 92% and 15%,
respectively, higher than in the FCBR. Figure 5.6 shows the effect of naphthalene´s increasing
concentration on the CH4 production rate, in mmol CH4 L-1 d-1 and CH4 production activity in
(mol mol-1control) 100% in the FCBR and in the RMBR. It can be concluded that CH4 production
was significantly higher in the RMBR than in the FCBR, from day 81 until day 120 for a
naphthalene concentration of 1.8 g L-1. A similar effect was observed from the increasing
concentration of toluene.
5.3 Gas-to-liquid mass transfer
The low gas-to-liquid mass transfer of syngas components and especially of CO and H2 is a
main challenge toward a more efficient syngas fermentation process. Several approaches such as
different bioreactor design, feeding method for syngas, cell immobilization, and bioreactor
conditions have been investigated. Studies reported that the utilization of packed bed and MBRs
led to higher mass transfer rates in comparison to CSTRs and bubble column bioreactors [94].
Furthermore, the syngas feeding with microspargers and hollow fibers increased the gas-to-liquid
mass transfer rates [96] and created better gas-to-cell contact through the biofilm attached on the
hollow fibers [161]. Packed material has also been used in order to increase the mass transfer
rates of syngas for the production of biogas. For example, an anaerobic trickle bed bioreactor
filled with packed material achieved a high CH4 concentration of 98%, from H2 and CO2
conversion [160].
During syngas fermentation, the gas components are consumed by microorganisms;
therefore, the rate of gas-to-liquid mass transfer is crucial for the process. The mass transfer rate
is often evaluated by the calculation of the mass transfer coefficient. Syngas components face
several resistances inside the bioreactor until they reach the cells (Figure 5.7-a). In the case of
cell encasement inside the membranes, there is a potential extra mass transfer resistance, which
57
can be considered negligible, however [150]. The main mass transfer resistance of syngas
compounds is considered to be the liquid film surrounding the cells (Figure 5.7-b), while all
other resistances can be considered as negligible [211].
Figure 5.7 (a) Main resistances during the mass transfer of syngas from the gas phase to the site of
reaction in the cells. Movement 1) in the gas bubble, 2) across the gas-liquid interfacial, 3) through the
liquid film surrounding the gas bubble, 4) in the liquid bulk, 5) through the membrane pores, 6) inside
the membrane, 7) across the liquid film surrounding the microbial cell, 8) through the cell membrane, 9)
through the cell, and ending up in the site of the reaction. (b) Movement of A through the interfacial
boundary. CAL: concentration of A in the liquid phase; CAli concentration of A in the liquid boundary; CAGi
concentration of A in the gas boundary; and CAG concentration of A in the gas phase [211]
The rate of mass transfer of component A though the gas (NAG) and the liquid (NAL)
boundary phase (in gmol m-3 s-1) can be calculated by the following equations:
NAG = kG α (CAG – CAGi) (5.1)
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can be considered negligible, however [150]. The main mass transfer resistance of syngas
compounds is considered to be the liquid film surrounding the cells (Figure 5.7-b), while all
other resistances can be considered as negligible [211].
Figure 5.7 (a) Main resistances during the mass transfer of syngas from the gas phase to the site of
reaction in the cells. Movement 1) in the gas bubble, 2) across the gas-liquid interfacial, 3) through the
liquid film surrounding the gas bubble, 4) in the liquid bulk, 5) through the membrane pores, 6) inside
the membrane, 7) across the liquid film surrounding the microbial cell, 8) through the cell membrane, 9)
through the cell, and ending up in the site of the reaction. (b) Movement of A through the interfacial
boundary. CAL: concentration of A in the liquid phase; CAli concentration of A in the liquid boundary; CAGi
concentration of A in the gas boundary; and CAG concentration of A in the gas phase [211]
The rate of mass transfer of component A though the gas (NAG) and the liquid (NAL)
boundary phase (in gmol m-3 s-1) can be calculated by the following equations:
NAG = kG α (CAG – CAGi) (5.1)
57
can be considered negligible, however [150]. The main mass transfer resistance of syngas
compounds is considered to be the liquid film surrounding the cells (Figure 5.7-b), while all
other resistances can be considered as negligible [211].
Figure 5.7 (a) Main resistances during the mass transfer of syngas from the gas phase to the site of
reaction in the cells. Movement 1) in the gas bubble, 2) across the gas-liquid interfacial, 3) through the
liquid film surrounding the gas bubble, 4) in the liquid bulk, 5) through the membrane pores, 6) inside
the membrane, 7) across the liquid film surrounding the microbial cell, 8) through the cell membrane, 9)
through the cell, and ending up in the site of the reaction. (b) Movement of A through the interfacial
boundary. CAL: concentration of A in the liquid phase; CAli concentration of A in the liquid boundary; CAGi
concentration of A in the gas boundary; and CAG concentration of A in the gas phase [211]
The rate of mass transfer of component A though the gas (NAG) and the liquid (NAL)
boundary phase (in gmolm-3s-1) can be calculated by the following equations:
NAG = kGα(CAG – CAGi) (5.1)
58
58
NAL = kL α (CALi – CAL) (5.2)
Where kG and kL is the gas-phase and liquid-phase mass transfer coefficient (m s-1),
respectively; and α is the total interfacial area for the mass transfer (m-1); CAL is the concentration
of A in the liquid bulk; and CAli is the concentration of A in the liquid boundary; CAG is the
concentration of A in the gas phase; and CAGi is the concentration of A in the gas boundary
(gmol m-3).
The Eq. 5.1 and Eq. 5.2 can be simplified to Eq. 5.3 and Eq. 5.4, respectively [211].
NAG = kG α (CAG – C*AG) (5.3)
NAL = kL α (C*AL – CAL) (5.4)
Where mCAL = C*AG is the gas-phase concentration of A in equilibrium with CAL; CAG/m =
C*AL is the liquid-phase concentration of A in equilibrium with CAG, and m is the distribution
factor. The last equations are when the main mass transfer resistance is the gas-phase film
resistance or the liquid-phase film resistance. Therefore, the overall mass transfer coefficients
KGα and KLα can be replaced by kGα and kLα [211]. In syngas fermentation, H2 and CO have low
solubility in the aqueous solution; thus, the kGα is significantly larger than kLα, and Eq. 5.3 is the
main mass transfer equation of the system.
Higher mas transfer rates can be achieved with higher gas holdup. In this thesis (paper V),
a packed floating MBR was used (Figure 5.8) in order to increase the gas holdup of syngas
bubbles. The membrane barrier in the floating MBR blocked the free anodic movement of the
syngas bubbles that rose from the bottom of the bioreactor. This caused an increase in the gas
holdup and the gas-to-cell contact, which consequently resulted in higher syngas mass transfer
and conversion rates. The FCBR had a poor gas-to-cell contact because of the distribution of the
cells that were mainly concentrated at the bottom of the FCBR. According to the results [212],
the H2 and CO conversion rates in the floating MBR were approximately 38% and 28% higher in
comparison to the FCBR. Moreover, the increase in the thickness of the membrane bed led to
even higher syngas conversion rates.
59
Figure 5.8 Improvement of gas-to-liquid mass transfer rates in a floating MBR
Figure 5.9 shows a comparison of the H2 and CO consumption rates and CH4 and CO2
production rates in a floating MBR, a bioreactor containing floating membranes and free cells
(floating MBR/FCBR), a bioreactor with free cells growing on packed material (PBR) and a
FCBR. According to the results, the highest consumption and production rates were observed in
the floating MBR. More specifically, the highest H2 and CO consumption rates obtained were
18.47 and 39.67 mmol L-1 d-1, respectively. In the same bioreactor, the highest CH4 production
and the highest CH4 yield of 21.33 mmol CH4 L-1 d-1 and 0.36 mol CH4 mol-1 (H2 + CO),
respectively, were reported.
60
60
Figure 5.9 Syngas biomethanation in floating MBR, floating MBR/FCBR, PBR, and FCBR during continuous
syngas feeding. Consumption of (a) H2 and (b) CO and production of (c) CO2 and (d) CH4 in mmol·L-1·d-1)
[212]
The results from this chapter showed that the efficiency of syngas fermentation for CH4 and
H2 production was improved by addressing the main challenges of the process. The use and
development of the rMBR technology was a main tool in this research. This work focused on a
part of the challenges and their parameters in syngas fermentation. It is clear that more research is
needed in order to achieve a holistic approach and address all the main bottlenecks. In the
following chapter, the main conclusions are presented, and future work is suggested.
61
Chapter 6
Conclusions and future work
6.1 Major experimental results and conclusions
Syngas fermentation is a flexible process with several advantages in comparison to other
processes for waste treatment. However, there are still challenges, such as the cell washout, the
high heavy metal content inside the remaining ashes after gasification, the toxic effect of syngas
impurities on the cells, and the low gas-to-liquid mass transfer rates that have to be tackled. This
work aimed at the improvement of syngas fermentation by focusing on the above challenges. The
main results from the experimental work are the following:
1. The cell washout was successfully eliminated by the membrane encasement in a RMBR.
The membrane system was still efficient after 154 days of syngas biomethanation.
2. The syngas conversion rates into H2 were improved by the addition of specific amounts of
heavy metals.
3. The use of a RMBR had a protective effect on the cells against high concentrations of
toluene and naphthalene.
4. The use of a floating MBR increased the gas holdup and thus the syngas conversion rates
in syngas fermentation.
62
62
6.2 Future work
This work addressed the use of RMBR for the improvement of syngas fermentation by
focusing on specific challenges mentioned in the previous section. However, several other factors
still need to be investigated.
6.2.1 Membrane material
One important consideration in MBRs is the cost and durability of membranes. A
commercial PVDF membrane was utilized in this thesis. The cost of this membrane is considered
high for large-scale MBRs. However, in our lab, a composite membrane that contains textile
waste was also prepared. The results that are not presented in this thesis showed similar syngas
conversion rates with both types of membranes (composite and PVDF), which indicates that
membranes from reused material with lower cost can be efficient alternatives to commercial
membranes. More research on alternative materials, based on sustainability, cost, and durability
should be conducted in the future.
6.2.2 Bioreactor´s design
One of the most important factors in syngas fermentation is the bioreactor´s design due to
the limited mass transfer rates of syngas components in the liquid medium. The high efficiency of
the floating MBR showed that the RMBR can be improved by internal adjustments. Several
features such as the height to diameter ratio and the use of a microsparger should be studied for
the further improvement of the rMBR.
6.2.3 Effect of syngas contaminants and heavy metals
This work focused on the effect of specific syngas contaminants and heavy metals
contained in the gasification ash, during syngas fermentation. However, a wider range of
contaminants and heavy metals as well as their antagonistic and synergistic effects should be
investigated in order to reach higher efficiencies in syngas fermentation.
63
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Paper I
Rapid bio-methanation of syngas in a reverse membrane bioreactor: Membrane encased
microorganisms
Rapid bio-methanation of syngas in a reverse membrane bioreactor:Membrane encased microorganisms
Supansa Youngsukkasem ⇑, Konstantinos Chandolias, Mohammad J. TaherzadehSwedish Centre for Resource Recovery, University of Borås, 50190 Borås, Sweden
h i g h l i g h t s
� A reverse membrane bioreactor (RMBR) was applied for syngas bio-methanation.� The cells encased in PVDF membrane could convert syngas and produce CH4 in 1 day.� Encased cells in the RMBR performed better at 55 �C compared to 35 �C.� Addition of organic waste with syngas improved the methane productivity.
a r t i c l e i n f o
Article history:Received 3 June 2014Received in revised form 18 July 2014Accepted 19 July 2014Available online 1 August 2014
Keywords:Syngas fermentationCo-digestionMethaneCell retentionMembrane bioreactor
a b s t r a c t
The performance of a novel reverse membrane bioreactor (RMBR) with encased microorganisms for syn-gas bio-methanation as well as a co-digestion process of syngas and organic substances was examined.The sachets were placed in the reactors and examined in repeated batch mode. Different temperaturesand short retention time were studied. The digesting sludge encased in the PVDF membranes was ableto convert syngas into methane at a retention time of 1 day and displayed a similar performance asthe free cells in batch fermentation. The co-digestion of syngas and organic substances by the RMBR(the encased cells) showed a good performance without any observed negative effects. At thermophilicconditions, there was a higher conversion of pure syngas and co-digestion using the encased cells com-pared to at mesophilic conditions.
� 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The global energy demand has constantly increased for severaldecades, and it has triggered the need for research and develop-ment on renewable energy sources. The potential to create renew-able energy from waste material, including municipal solid waste(MSW), industrial waste, agricultural waste, andwaste by-productshas been developed (Fatih et al., 2011). Biogas or bio-methane is arenewable energy source with several applications in e.g., car fuel,heating, cooking, or electricity production. Biogas mainly consistsof methane and carbon dioxide but may also contain minor impu-rities of other components (Deublein and Steinhauser, 2008).
There are different types of wastes used for methane production,which can be classified as easily degradablewastes, hard degradablewastes, and non-degradable wastes. In general, to obtain methanefrom easily degradable wastes such as food wastes, a biochemical
approach called an anaerobic digestion process has been employed.On the other hand, the recalcitrance of the hardly degradable mate-rials, such as crystalline cellulose and non-degradable materialssuch as lignin and plastic wastes cannot be decomposed by themicroorganisms in an anaerobic digestion process (Nizami et al.,2009). Thermochemical processes however have the potential toconvert this kind of wastes as well as non-degradable residues fromthe digestion process intomethane. In this approach, feedstocks canbe thermally gasified into intermediate gases, called syngas,through a partial oxidation process, at a relatively high temperature.Syngas or synthesis gas primarily contains carbon monoxide (CO),hydrogen (H2), and carbon dioxide (CO2). Raw syngas can be synthe-sized intomethane by the use of metal catalysts, first introduced byFranz Fischer and Hans Tropsch. However, the high manufacturingcost and challenges of the toxic impurities for the catalysts limitits economic feasibility (Fatih, 2009; Fatih et al., 2011).
Anaerobic microorganisms, primarily acetogens, carboxytrophs,and methanogens are able to use CO and/or CO2 sources as carbonsources and use H2 as an energy source for their metabolism andproduce different bio-products (Daniell et al., 2012). Thus, the
http://dx.doi.org/10.1016/j.biortech.2014.07.0710960-8524/� 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +46 33 435 4608; fax: +46 33 435 4008.E-mail address: [email protected] (S. Youngsukkasem).
Bioresource Technology 178 (2015) 334–340
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combination of thermal and biological processes for the conversionof feedstocks into biofuel has been explored. This alternativemethod can generate a variety of products including methane froma wide variety of materials (Daniels et al., 1977; Kimmel et al.,1991; Klasson et al., 1990, 1992). Using the fermentation processesand microorganisms offers several benefits over the catalytic pro-cess, such as being a more specific process, resulting in higheryields, having a lower energy consumption, being environmentallyfriendly, and having better robustness (Mohammadi et al., 2011;Munasinghe and Khanal, 2010).
The bio-methanation of CO, H2, and CO2 in the syngas by themicroorganisms can be performed in two pathways (Kimmelet al., 1991). The first one utilizes an acetate pathway as a methaneprecursor. Thereafter, methanogenic microorganisms convert ace-tate into methane. The other pathway utilizes the H2/CO2 pathway.CO can be converted into CO2 by the microorganisms (Henstraet al., 2007). The H2 and CO2 produced and initially present inthe syngas are converted into methane by some methanogenicmicroorganisms (Kimmel et al., 1991). However, this microbialflora including methanogens requires a long retention time, sincetheir growth rate is very low. Methanogenic microorganisms arealso very sensitive to the process conditions; hence, the cells areeasily washed out from the digester at high dilution rates. For thesereasons, the population size of the microbial cells is easily reducedresulting in a decreased methane production. Moreover, fermenta-tion processes with a low cell density need long start-up periods,and larger digesters are required for proper function, which meansthat the capital cost is high. To achieve a high process efficiency inconverting syngas into methane using microorganisms in a fer-mentation process, retaining the microbial cells inside a compactreactor might be a solution to overcome these problems (Klassonet al., 1992). Immobilized cell technology, meaning the confine-ment of cells in a specific region or matrix, has been widely usedin a variety of laboratory experiments and industrial applications(Raymond et al., 2004). These systems have been used to solveproblems encountered in conventional bioreactors using sus-pended cell culture, including low biomass concentrations, lowbiomass productivity and product formation, inefficiency in con-tinuous production, low stability to sudden fluctuation, and limiteddilution rate in the case of continuous operation (Chinnayelka andMcShane, 2004).
A membrane bioreactor (MBR) is the combination of a mem-brane process such as microfiltration with a suspended growthbioreactor (Judd and Judd, 2011). MBRs have been widely usedfor cell retention, especially in the municipal and industrial waste-water treatment. For example, MBR was studied by Badani et al.(2005) to treat textile wastewater, and it was found to performperfectly compared to the conventional system. Kanai et al.(2010) employed a submerged anaerobic membrane bioreactorprocess in the anaerobic digestion of food wastes. They found thatMBR enhanced the degradation of wastes, the rejection of toxics,and the reactor volumes could be scaled down compared to theconventional system. This technology has several benefits overthe conventional systems such as having low energy consumption,being environmentally friendly, and being easily implemented.Using MBR to retain the microbial cells for the enhancement ofthe bio-methanation of syngas could be an interesting system.However, currently available commercial MBR processes employedin biological process are designed to filter the liquid through themembranes, retaining the cells in the reactor to obtain a clarifiedand disinfected product. It is this type of MBR (the biomass rejec-tion MBR), which is in primary focus of the most research in thisfield (Judd and Judd, 2011). In order to increase the conversionefficacy and bio-productivity, a new technique for retaining thecells inside the bioreactor is probably necessary for efficient bio-methanation systems of syngas.
The current work focuses on applying the reverse membranebioreactor (RMBR) to retain the cells for the bio-conversion processof synthesis gas into methane. RMBR used in this work, the liquidpermeate is not actively passes through the membrane, but thesubstrate diffuse through the membrane and the metabolic prod-ucts diffuse back to the medium. It is a novel technique that usesmembranes (PVDF) to enclose the microbial cells completelyfollowed by immersion into a bioreactor in order to retain a highcell density in the bioreactor and increase the methane productiv-ity, without the risk of a clogged outlet. In this work, the efficiencyof the RMBR contained the encased cells and the resistance of usingthe membrane as a barrier for the syngas fermentation was testedcompared to the free cells, in repeated batch mode with a short-ened retention time and different temperatures. Moreover, thepossibility of enhancing the methane productivity with additionof organic substances as a co-substrate with syngas using theRMBR was also examined.
2. Methods
2.1. Anaerobic culture, medium, and syngas
An anaerobic culture was obtained from a 3000 m3 municipalsolid waste digester, operating under thermophilic (55 �C) condi-tions (Borås Energy and Environment AB, Sweden). The inoculumwas incubated at 55 �C for 3 days to keep the bacteria active whileconsuming the carbon source provided by the inoculum. Afterincubation, the inoculum was filtered through a sieve (1 mm poresize) to remove the large particles. The digesting sludge was thencentrifuged at 31,000�g for 15 min to separate the solid inoculum(85% TS), the methane-producing microorganisms, which wereloaded into the membrane sachets. Two kinds of synthetic mediumsolutions for two different experiments were prepared. One wasfor the co-digestion; the synthetic organic medium contains ace-tate, propionate, butyrate, and vitamins solution (basal medium)with a ratio of 3:1:1:1 (Osuna et al., 2003). The other was thevitamin solution, that is, the basal medium for the pure syngas fer-mentation. Medium solutions were buffered to pH 7.0 ± 0.2 withNaHCO3 (Isci and Demirer, 2007). Synthetic syngas containing CO(55% mole), H2 (20% mole), and CO2 (10% mole) (Kimmel et al.,1991) was purchased from AGA gas (Borås, Sweden).
2.2. Membrane sachet preparations and cell containment procedure
The cell entrapment in the membranes was performed follow-ing a previously described method (Youngsukkasem et al., 2012,2013). Flat plain PVDF (polyvinylidene fluoride, Durapore�) mem-branes (Thermo Fisher Scientific Inc., Sweden) were used as syn-thetic membranes. The PVDF membrane filters were hydrophilewith the pore size, thickness and diameter of 0.1 lm, 125 lmand 90 mm, respectively. The membranes were cut into rectangu-lar shapes of 6 � 6 cm and folded to create membrane pockets of3 � 6 cm2. They were then heat-sealed (HPL 450 AS, Hawo,Germany) on two sides with heating and cooling times of 4.5 and4.5 s, leaving one side open for the insertion of the inoculum. Solidsludge inoculum (3 g per sachet) was then injected carefully intothe synthetic membrane sachets, and the fourth side sealed. Thesachets containing the inoculum were used immediately for thebio-methanation of the syngas and the co-digestion process.
2.3. Repeated batch fermentation process
The repeated batch fermentation processes (with cell reuse andmedium replacement) were performed in order to preliminarilyexamine the performance of the RMBR (microorganisms encased
S. Youngsukkasem et al. / Bioresource Technology 178 (2015) 334–340 335
in the synthetic membrane) for the syngas bio-methanation andthe co-digestion of syngas and organic medium at different reten-tion times. The schematic diagram of an experiment shows inFig. 1. In the experiment with the syngas bio-methanation, eachreactor contained one sachet of inoculum and 30 mL of basalmedium. For the experiment with the co-digestion process, eachdigester contained one sachet of inoculum and 30 mL of syntheticorganic medium. The reactors used were serum glass bottles with118 mL working volume, closed with butyl rubber seals and alumi-num caps. The headspace of each bottle was flushed with syngas toobtain a sufficient amount of gas substrate at 1 atm. Reactors wereinclined and carried out in a water bath with constant agitation of100 rpm in order to provide good gas–liquid mass transfer. Anaer-obic fermentations at temperatures of 35 �C and 55 �C were exam-ined. The retention time was gradually shortened: 4, 4, 2, 2, and1 day, respectively. The encased cells were reused; the syngaswas exchanged with fresh syngas at 1 atm, and necessary nutrientswere replaced prior to the start of every new batch. Anaerobicfermentations of the free cells with syngas were performed for9 days of retention time in parallel in order to examine theefficiency of the encased cells for the bio-methanation of syngas,especially, the membrane resistance, under identical conditionsas the reference.
2.4. Analytical methods
Methane, hydrogen, carbon monoxide, and carbon dioxide weremeasured regularly, using a gas chromatograph (Perkin-Elmer,U.S.A.), equipped with a packed column (CarboxenTM 1000, SUPE-LCO, 60 � 1.800 OD, 60/80 Mesh, U.S.A.) and a thermal conductivitydetector (Perkin-Elmer, U.S.A.) with an injection temperature of200 �C. The carrier gas was nitrogen, with a flow rate of30 mL/min at 75 �C. A 250 lL gastight syringe (VICI, PrecisionSampling Inc., U.S.A.) was used for the gas sampling. The obtainedpeak area was compared with a standard gas analyzed at the samecondition (STP, 273.15 K and 101.325 kPa). The volatile fatty acids(VFA) were analyzed using a gas chromatograph (Auto System,Perkin-Elmer, U.S.A.) equipped with a capillary column (ZebronZB-WAX plus, Polyethylene glycol (PEG), 30 m � 0.25 mm �0.25 lm, U.S.A.) and a flame ionized detector (Perkin-Elmer,U.S.A.) with an injection and detection temperature of 250 �C and300 �C, respectively. The carrier gas was nitrogen, with a flow rateof 2 mL/min at a pressure of 20 psi. The experiment was performed
in triplicate and the results were presented as mean ± standarddeviation.
3. Results and discussion
3.1. The performance of the RMBR (digesting microorganisms encasedin the PVDF membranes) compared to the free cells for the bio-methanation of syngas
During the anaerobic bio-methanation process of syngas, it wasobserved that the encased microorganisms performed smoothly.Sachets containing the cells stayed intact and no leakage of cellsoccurred throughout the anaerobic conversion process.
Fig. 2 shows the performance of the encased cells compared tothe free cells in the anaerobic bio-methanation of syngas. Theencased microbial cells had a similar accumulated methane levelcompared to the free cells in the thermophilic fermentation inthe 9-day batch. Methane was rapidly accumulated in the reactorswith both the encased and the free cells at thermophilic conditions(55 �C), from the first day until the 3rd day of fermentation. There-after, the trend of methane production was stable until the last daydue to slow production (Fig. 2a). The accumulated methane pro-duced on the 9th day for the encased cells and the free cells at55 �C and 35 �C were 0.72, 0.41, 0.51, and 0 mmol, respectively.The amount of H2 and CO decreased dramatically during the first3 days. Under the thermophilic conditions, the free cells showedbetter performance in rapid assimilation of the syngas comparedto the encased cells. CO and H2 were completely consumed bythe free cells already in the 2nd day of bio-methanation. The anaer-obic digesting sludge used as the inoculum contained differentspecies of microorganisms, and used for biogas production, as anactive source of methane-producing microorganisms. In addition,it has been found to be a potential source of microorganisms forthe syngas fermentation in different bioprocess systems (Sipmaet al., 2003). The individual microbial cells (free cells) most likelyhave better contact to the medium with dissolved gases in the fer-mentation system compared to the compact cells encased in themembranes. However, cells encased in the PVDF membranes con-verted all H2 into products in 4 days while CO required a longertime to be completely used up by the encased cells. The trend ofCO2 consumption was stable for all treatments (Fig. 2d).
This experiment revealed that microorganisms encased in thePVDF membrane displayed a similar performance compared tothe free cells in syngas bio-methanation, but the conversion rateof syngas was found to be a bit slower than for the free cells with
A reverse membrane bioreactor (RMB)
(Encased cells)Free microbial cells
Pure syngas fermenta�on
Co-digestion of syngas and organic
substances
Conditions:Shortened retention times and different
temperatures
Analyzation
CO, H2, CO2, CH4and VFAs
Fig. 1. The schematic diagram of an experiment.
Fig. 2. Anaerobic bio-methanation of syngas using the RMBR (encased cells)compared to free cells. (a) Methane, (b) Hydrogen, (c) Carbon monoxide, and (d)Carbon dioxide. Symbols: Encased cells at 35 �C ( ), Free cells at 35 �C ( ),Encased cells at 55 �C ( ), and Free cells at 55 �C ( ).
336 S. Youngsukkasem et al. / Bioresource Technology 178 (2015) 334–340
the same process conditions. However, the results indicate thatusing the PVDF membrane to retain microbial cells for bio-methanation of syngas is feasible. Hence, it would be interestingto investigate this novel technique in a long-term process.
3.2. The efficiency of the RMBR (encased digesting microorganisms) inrepeated batch process for bio-methanation of syngas
3.2.1. Effect of temperaturesFig. 3 shows the performance of the encased cells in the anaer-
obic bio-methanation of syngas at different retention times andtemperatures compared to the control treatments with no syngas,under the same conditions. The result shows that at the firstretention time of 9 days, methane was produced continuouslywhile the syngas concentration decreased. The remarkable resultshows that methane was produced immediately from the firstday of fermentation in all conditions. In addition, the interestingresult from this experiment was that the encased cells, at bothtemperature conditions, produced more methane than the controlswith no syngas at the same conditions. Furthermore, it wasobserved that under thermophilic conditions of 55 �C, the encasedcells had the highest methane production of 1.53 mmol on the 9thday compared to the mesophilic conditions at 35 �C and the con-trols at both temperatures (0.78, 0.81, and 0.37 mmol, respec-tively). Kundiyana et al. (2011) studied the syngas fermentationusing microbial cells at different temperatures and found that amesophilic temperature (35 �C) was the best condition for enhanc-ing the gas solubility in the liquid medium and consequentlyenhancing the bio-productivity. However, in the current experi-ment, under mesophilic conditions (35 �C), the encased cells pro-duced less methane than those at thermophilic conditions. Thismeans that the gas solubility may not be the main obstacle in
increasing the process efficiency of syngas fermentation. The nat-ure of the fermenting microorganisms, which might work betterat thermophilic conditions, also plays an important role for thisprocess. In this experiment, the active inoculum obtained from athermophilic biogas plant is the likely reason for the improvedbio-conversion of syngas. Thermophilic conditions generally stim-ulate the microbial metabolism compared to lower temperatures.Thus, faster bio-methanation of syngas occurred at thermophilicconditions with the community of microbial cells originating fromthe thermophilic biogas plant.
Both H2 and CO have a lower solubility in the liquid phase com-pared to CO2, but the amount of syngas, including H2 and CO(Fig. 3b and c), decreased continuously from the first day, at thesame time as methane was produced (Fig. 3a). The H2 concentra-tion of 0.7 mmol at 55 �C anaerobic condition at the first day haddecreased to 0.1 mmol at the 5th day and thereafter, was verylow during the remainder of the fermentation period (Fig. 3b). At35 �C, the encased cells had a lower conversion efficiency of H2
than at thermophilic condition. Here, the same amount of H2
(0.7 mmol) decreased slowly to 0.06 mmol on the 9th day offermentation. However, the encased cells had no problems to takeup H2 for methane formation. H2 is usually contained in syngas atdifferent concentrations depending on the composition of the bio-mass used for the thermal gasification process and also on the pro-cess parameters (Fatih, 2009). To produce methane, methanogenicmicroorganisms such as Methanothermobacter thermoautotrophicus(Sipma et al., 2003) utilize H2 together with CO2. However, precur-sors for methane-producing microorganisms, such as acetate, canalso be formed by microbial cells such as Acetoanaerobium noteraeusing H2.
With carbon monoxide (Fig. 3c), no problems regarding theinhibitory effects on the cells were observed during the fermenta-tion process. The encased cells performing at thermophilic condi-tions were able to also consume CO faster than the cells atmesophilic conditions. The CO (1.8 mmol) was completely assimi-lated on the 7th day at 55 �C. At 35 �C, the same amount of CO wascompletely used up by the encased cells by the 9th day. In order tomimic the raw syngas, CO2 was added as one of the main gases forthis experiment. The result shows that the production trends ofCO2 by the encased cells using syngas (35 and 55 �C) were quitestable (Fig. 3d). It may be because of CO2, even as a metabolicproduct (cf. controls on Fig. 2d). The amount of CO2 at the condi-tions of 35 �C and 55 �C were in the ranges of 0.25–0.50 and0.33–0.59 mmol, respectively. To formmethane, the microbial cellsprobably used CO2 from their metabolisms, so they did not needthe CO2 supplied from the syngas. However, CO2 accumulationhad no negative effect on the bio-methanation of syngas in thisexperiment.
3.2.2. Effect of short retention timesThese results reveal that anaerobic microorganisms encased in
the PVDF membrane were able to convert syngas into methaneat the retention time of 9 days. However, to investigate the effi-ciency of the encased cells in shorter periods of bio-methanation,the experiment was thereafter operated in repeated batch mode,in which the encased cells were reused and new syngas wasreplaced at the beginning of every new batch. The batch timewas gradually shortened to 4, 4, 2, 1, and 1 day, respectively. Thecompositions of the syngas and methane were analyzed regularly.
The results showed that the encased cells still performedefficiently, and no negative effect was observed during the bio-methanation of the syngas. In addition, the encased microbial cellshad better conversion efficiency of the syngas at anaerobic thermo-philic conditions than at mesophilic conditions throughout allbatches (Fig. 3). Under thermophilic anaerobic conditions, COwas completely used up by the encased cells on the 3rd day of
Fig. 3. Performance of the RMBR in anaerobic repeated batch bio-methanation ofsyngas. (a) Methane production, (b) Hydrogen concentration, (c) Carbon monoxideconcentration, and (d) Carbon dioxide concentration. Symbols: Syngas at 35 �C( ), Blank at 35 �C ( ), Syngas at 55 �C ( ), and Blank at 55 �C ( ).
S. Youngsukkasem et al. / Bioresource Technology 178 (2015) 334–340 337
the batch. When the retention time was gradually shortened to2 days, CO was still completely used up. With a retention time of1 day, the encased cells used CO, but it was not enough time forthe encased cells to completely use all CO (Fig. 3c). For H2, theamount decreased continuously from the beginning of the batches,especially, at the retention time of 4 days. Nevertheless, the reten-tion times of 4, 2, and 1 day were not enough for the encased cell toentirely convert the H2 into products. The trend for the CO2 levelwas found to be the same throughout the process, even whenthe batch time was shortened. The accumulated methane produc-tion of the encased cells in thermophilic bio-methanation withbatch times of 4, 4, 2, 1, and 1 day were 0.90, 0.88, 0.73, 0.35,and 0.35 mmol, respectively. The most interesting point from thisexperiment was that the reused encased cells performed well,and were still able to convert syngas into methane continuouslywhen the retention time was shortened. The accumulated methaneproduction was lower compared to the first period of 9 days. How-ever, a decrease was expected due to the used up methane poten-tial of the inoculum.
All these results indicate that the digesting sludge retained inthe hydrophilic PVDF membrane was able to convert syngas intomethane. Furthermore, the encased cells were easy to handle whenreused for the repeated batch fermentation process. HydrophilicDurapore� PVDF membrane allowed all gases, including syngas(substrate) and methane (product) together with the necessaryliquid nutrients for the microbial cells to diffuse through the mem-brane layer. Consequently, the microbial cells encased in the PVDFmembranes were able to take up syngas and produce methane.Thermophilic anaerobic conditions were found to enhance the con-version efficiency of syngas and facilitate faster methane produc-tion compared to at mesophilic conditions.
3.3. Enhancing the methane productivity using the encasedmicroorganisms in simultaneous bio-methanation of syngas andorganic substances
In anaerobic digestion processing of biomass, co-digestion hasbeen proven to be a powerful process due to its advantages in theimprovement of productivity and process efficiency. In this system,different kinds of biomass are put together in the same digesterand digested simultaneously in an often synergistic manner(Abouelenien et al., 2014; Long et al., 2012). CO, H2, and CO2 in syn-gas, the intermediate gas, can be used for the metabolism of themicroorganisms. However, syngas has a low energy, which maynot be sufficient for the microbial cells to produce methane, thus,to increase the efficacy of the encased cells in bio-methanation ofsyngas, a simultaneous fermentation process for the syngas andorganic substance to produce methane using the encased cellswas studied. In this experiment, the digesting sludge was encasedin the PVDF membranes and used as an inoculum. A syntheticorganic medium containing: acetate, propionate, and butyrate,and syngas, containing CO, H2 and CO2, were used as a co-substrate.Mesophilic and thermophilic anaerobic conditions were examinedand performed in repeated batch at different retention times. Syn-gas concentrations, total volatile fatty acids (VFA), and methaneproduction were analyzed regularly. Syngas bio-methanation bythe encased cells in the same condition was used for comparison.
The results show that the encased cells performed efficiently inthe co-biomethanation of syngas and organic substances. Thesachets got swollen during the anaerobic fermentation process,due to the high pressure of the methane produced. This means thatthe methanogenic microbes encased in the PVDF membranes hadobtained sufficient substrates for product formation. Here, thethermophilic anaerobic conditions were found to be advantageouscompared to the mesophilic conditions. Digesting sludge encasedin the hydrophilic PVDF membranes was able to perform in the
co-digestion process without any observed negative effects. Inthe first period of 9 days, the trends of methane production bythe encased cells in the co-digestion and in the pure syngasfermentation were similar. The highest accumulated methane pro-duction was obtained by the encased cells in the co-digestion andpure syngas fermentation (0.72 and 0.79 mmol) at thermophilicconditions (Fig. 4a). Looking at the gas amounts in the same period,H2 and CO were completely consumed in both processes by theencased cells on the 9th day. CO2 was consumed a bit at the begin-ning and was thereafter stable.
Total volatile fatty acids (VFA) in the liquid nutrients were alsomeasured on the last day of the different batches. After the firstbatch, with retention time of 9 days, the VFA levels decreased from0.97 g/L of pure syngas fermentation and 8.05 g/L of co-digestionunder thermophilic conditions at the first day to 0.08 and 0.05 g/L(Table 1). Hence, the VFA utilizations were 92% and 99%,respectively. These results reveal that the encased cells canconsume syngas simultaneously with the digestion of organicsubstrates. Nevertheless, the performance of the encased cells inthe co-digestion process did not show that the co-digestionresulted in a higher methane production during the first batchperiod (Fig. 4a). Thus, repeated anaerobic batch digestions withshorter retention times were performed.
When the encased cells were reused, the substrates (syngas andorganic solution) were exchanged with new substrates, and theretention time was shortened to 4, 4, 2, and 1 days, interestingresults were observed. It was found that the methane producedfrom the thermophilic co-digestion (55 �C) showed an increasingtrend (Fig. 4a), even as the retention time was shortened. Themicrobial cells encased in the hydrophilic PVDF membranes
Fig. 4. Comparison of syngas bio-methanation and co-digestion process of syngasand organic substances by the RMBR. (a) Methane, (b) Hydrogen, (c) Carbonmonoxide, and (d) Carbon dioxide. Symbols: Pure syngas at 35 �C ( ), Pure syngasat 55 �C ( ), Syngas + organic substances at 35 �C ( ), and Syngas + organicsubstances at 55 �C ( ).
338 S. Youngsukkasem et al. / Bioresource Technology 178 (2015) 334–340
produced the highest amount of methane from 4, 4, and 2 days of0.42, 1.41, and 1.92 mmol in thermophilic co-digestion comparedto the others. The encased microbial cells themselves may alreadyhave adapted from the first batch, so that they could perform bet-ter and produce methane faster in the following batch periods.However, the methane level was lower when the retention timewas shortened to 1 day (1.14 and 1.59 mmol for the two batches,respectively) due to a too limited process time. In contrast, themethane production from the encased cells using only syngas asa substrate was lower than in the co-digestion process, and theproduced amounts were similar in all batches except for the short-est batches, where the production rate was obviously too slow.
After 9 days of fermentation, a new batch was started and theretention time was shortened, then H2 was completely consumedby the encased cells in 4 days of two batches for thermophilicco-digestion process (Fig. 4b). The encased cells were still able touse H2 for their metabolism at the shorter retention time, but H2
was not completely used up in the two and one day batches. COwas found to be consumed more rapidly by the encased cells inthe thermophilic pure syngas fermentation compared to in theco-digestion (Fig. 4c). It seems that the microorganisms encasedin the PVDF membranes preferred to use the other sources of sub-strates compared to CO. The encased microorganisms managed tocompletely convert the CO that was present in 2 days. For the CO2
values in this experiment (Fig. 4d), it was shown to have an inter-esting trend. After the first period of 9 days, the CO2 was consumedrapidly by the encased cells under both mesophilic and thermo-philic conditions of the co-digestion processes, but not in the sys-tems with only syngas fermentation. It is presumed that theencased digesting sludge probably contains specific microorgan-isms such as M. thermoautotrophicus, which are able to utilizeCO2 together with gases such as H2 to form precursors (acetate)or methane. Furthermore, the co-digestion process may providesuitable conditions for this microbial group, which was not thecase with only syngas fermentation.
According to the methane production and syngas consumption,the VFA concentration in the effluents from the co-digestion pro-cess was found to decrease (Table 1). The lower VFA concentrationsin the system with the encased cells in thermophilic co-digestioncompared to mesophilic conditions show that the encased cellshad spent more organic substances for their metabolism, and ithas been shown that the hydrophilic PVDF membrane allowedliquid organic substrates to diffuse through the membrane layerfor the metabolism of the microorganism in the digestion process(Youngsukkasem et al., 2013). However, the cells in co-digestionwere probably inhibited by the VFA in the first 2 and 3 batchesand thereafter, they adapted and could quickly utilize the VFA aswell as the syngas (Table 1).
The results presented here reveal that the microbial cellsencased in the hydrophilic PVDF membranes were capable of
simultaneous fermentation of the syngas and organic substratesfor methane production, without any detected negative effects.These successful results show the feasibility of applying this noveltechnique in combined anaerobic organic digestion with syngasfermentation, in order to increase the methane productivity. Inaddition, the PVDF membranes turned out to be an interestingsupporting material in this experiment. It was able to retain themicrobial cells in the bioreactors, and at the same time it was ableto allow both organic and gas substrates to pass through, whichcan provide the necessary nutrients for the metabolism of themicrobial cells to produce methane.
4. Conclusion
Retaining microorganisms in the reverse membrane bioreactor(RMBR) using the PVDF membranes was a successful approachfor bio-methanation of syngas, as well as simultaneous fermenta-tion of syngas and organic substances. The PVDF membranesallowed the liquid and gas diffusion through the membrane sur-face. The encased cells in RMBR could convert CO, H2, and CO2
and produce methane in 1 day. Thermophilic conditions (55 �C)enhanced the syngas fermentation, and the co-digestion usingthe encased cells improved the methane productivity. However,to develop RMBR system for the industrial scale, RMBR performsunder continuous bio-methanation of syngas should be furtherdeveloped.
Acknowledgement
This work was financially supported by Swedish ResearchCouncil.
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Table 1The concentrations of total volatile fatty acids (VFAs) from the effluent of each reactorat the start-up and at the end of the different batches.
Retention time (Days) Total volatile fatty acids (g/L)
Pure syngasfermentation
Co-digestionprocess
35 �C 55 �C 35 �C 55 �C
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Paper II
Syngas Biomethanation in a Semi-Continuous Reverse Membrane Bioreactor (RMBR)
fermentation
Article
Syngas Biomethanation in a Semi-ContinuousReverse Membrane Bioreactor (RMBR)Supansa Y. Westman *, Konstantinos Chandolias and Mohammad J. Taherzadeh
Swedish Centre for Resource Recovery, University of Borås, Borås 50190, Sweden;[email protected] (K.C.); [email protected] (M.J.T.)* Correspondence: [email protected]; Tel.: +46-70-487-7100
Academic Editor: Thaddeus EzejiReceived: 22 January 2016; Accepted: 21 March 2016; Published: 25 March 2016
Abstract: Syngas biomethanation is a potent bio-conversion route, utilizing microorganisms toassimilate intermediate gases to produce methane. However, since methanogens have a long doublingtime, the reactor works best at a low dilution rate; otherwise, the cells can be washed out during thecontinuous fermentation process. In this study, the performance of a practical reverse membranebioreactor (RMBR) with high cell density for rapid syngas biomethanation as well as a co-substrate ofsyngas and organic substances was examined in a long-term fermentation process of 154 days andcompared with the reactors of the free cells (FCBR). The RMBR reached maximum capacities of H2, CO,and CO2 conversion of 7.0, 15.2, and 4.0 mmol/Lreactor.day, respectively, at the organic loading rate of3.40 gCOD/L.day. The highest methane production rate from the RMBR was 186.0 mL/Lreactor.dayon the 147th day, compared to the highest rate in the FCBR, 106.3 mL/Lreactor.day, on the 58th day.The RMBR had the ability to maintain a high methanation capacity by retaining the microbial cells,which were at a high risk for cell wash out. Consequently, the system was able to convert moresyngas simultaneously with the organic compounds into methane compared to the FCBR.
Keywords: syngas fermentation; methane production; semi-continuous process; reverse membranebioreactor; co-substrate; cell retention
1. Introduction
Biogas is a renewable energy source with several applications in heating, cooking, or electricityproduction. Biogas mainly consists of methane and carbon dioxide but may also contain minorimpurities of other components [1]. It can also be upgraded to about 97% biomethane, which is usedas a popular renewable form of energy such as car fuel.
Biogas or biomethane can be produced from waste resources that can be classified as easilydegradable, difficult to degrade, and non-degradable wastes. To obtain biogas from easily degradablewastes such as food wastes, a biochemical approach called anaerobic digestion process is traditionallyemployed. In this process, complex polymers of organic substances are degraded easily bymicroorganisms through the different steps of the anaerobic fermentation process [2]. In the sameway, difficult to degrade materials such as lignocelluloses from crop residues and agricultural residuescan also be converted into methane by the performance of microorganisms in the anaerobic digestionprocess. However, the recalcitrance of these difficult to degrade biomass sources, such as the coverageof lignin or the crystallinity of cellulose, limits the access of the microorganisms and enzymes forefficient digestion [3]. The hydrolysis rate can be accelerated by pretreating the substrate with chemical,physical, or biological processes before feeding the substrate to the digester [4]. Nevertheless, thepre-treatment process has been found to be an expensive and rate limiting factor [5]. Thus, thebiochemical process may face challenges in converting difficult to degrade wastes into methane.Non-degradable materials such as lignin or plastic wastes cannot be decomposed by microorganisms
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in an anaerobic digestion process. Consequently, it is common to incinerate this category of wasteto energy.
On the other hand, it is possible to convert non-degradable wastes and lignocelluloses to biofuelsvia gasification to syngas and the fermentation routes. The biomass is first gasified into a gas mixturecalled syngas or synthesis gas. Syngas contains primarily CO, H2, and CO2. The syngas can beupgraded by methanization, either by using chemical catalysts (e.g., Fischer-Tropsch Synthesis) orthrough fermentation with microbial catalysts. The fermentation process offers several benefits over thechemical catalytic process, such as a more specific process, higher yields, lower energy consumption,being environmentally friendly, and having better robustness [6,7]. Syngas fermentation for methaneproduction, i.e., syngas biomethanation, is a relatively new technology and a good alternative to thefirst-generation biofuel.
The biomethanation of the syngas by microorganisms can be performed in two pathways [8]. Thefirst one utilizes an acetate pathway as a methane precursor (Reactions 1 and 2). Acetobacterium woodiiand Enbacterium limosum, for example, are microbial cells that perform this reaction. Thereafter,methanogenic microorganisms such as Methanosarcina barkeri convert acetate into methane (Reaction 3):
2CO2 ` 4H2 Ñ CH3COOH ` 2H2O Reaction 1
4CO2 ` H2O Ñ CH3COOH ` 2CO2 Reaction 2
CH3COOH Ñ CH4 ` CO2 Reaction 3
The other pathway utilizes the H2/CO2 pathway. CO can be converted into CO2
(Reaction 4) by microorganisms, for example, Methanothermobacter thermoautotrophicus andClostridium thermoaceticum [9]. The H2 and CO2 produced by reaction 4 and initially present inthe syngas are converted into methane by some microorganisms such as Methanosarcina formicicum(Reaction 5) [8]:
CO ` H2O Ñ CO2 ` H2 Reaction 4
4H2 ` CO2 Ñ CH4 ` H2O Reaction 5
However, these microbial groups, especially, methane-producing microorganisms, require a longretention time in the digester, since their growth rate is very low. In addition, they are very sensitive toharsh conditions; hence, the cells can easily be washed out from the digester at high dilution rates ina continuous process. For these reasons, the population size of the microbial cells is easily reduced,resulting in a decreased methane production and low process efficiency. Moreover, fermentationprocesses with low cell density need long start-up periods, and larger digesters are required for properfunction, which means that the capital cost is high. Retaining the microbial cells inside a compactbioreactor with a low chance of bio-fouling and abraded microorganisms might be a solution toovercome these problems.
Different bioreactor designs have been developed in order to enhance the process efficiency ofthe syngas fermentation [10–15]. For instance, continuous stirred tank reactors (CSTR) have beenfrequently employed for syngas fermentation. A high speed of agitation in the CSTR can be used toenhance the mass transfer efficiency. However, it has been observed to result in high shear stress onthe cells and high-energy consumption [16,17].
In another study, a bioreactor for syngas fermentation was connected to a hollow fiber membranemodule [16]. The system enhanced the gas-liquid mass transfer as well as allowed the microbial cellsto be attached on the membrane layer [16]. However, a long start-up period for cell attachment to theouter membrane layer was crucial. The same authors also developed a novel bioreactor configuration,called a monolithic biofilm reactor, for the same purpose. The results revealed that the performanceof the syngas fermentation process depended not only on the mass transfer efficiency, but the lowefficiency was also related to bio-fouling and abrading of the microbial cells attached to the channelof the monolithic wall [18]. Liu et al. [19] used the membrane module for the cell retention system in
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continuous syngas fermentation for ethanol production. However, cell wash out still occurred, whichwas one of the reasons for low product formation.
From these results and the nature of methane-producing microorganisms, retaining a highmicrobial cell density in the bioreactor and preventing cell washout during the syngas continuousfermentation processes are important aspects to keep the process stable and increase the methaneproductivity [15].
The reverse membrane bioreactor (RMBR) is a configuration of a bioreactor for methaneproduction. The RMBR system can be defined as a process that employs a fixed microbial growthsystem with the use of membranes. This bioreactor system was designed to enclose the cells completelyin the permeable membrane filter, while substrates in the surrounding liquid are able to diffuse throughthe membrane to the cells. The fermentation products can thereafter diffuse out to the surroundingsolution. Additional equipment for the cell recycle system is not necessary for this kind of process.This technique provided a high cell density; consequently, the process efficiency and productivity ofthe anaerobic digestion process for methane production were improved [20–23].
The current work focuses on applying the RMBR for syngas biomethanation under anaerobicsemi-continuous mode in order to retain a high cell loading and consequently, improve the processperformance. The simultaneous fermentation of syngas and organic compounds was also investigated.The performance of the system was evaluated under thermophilic anaerobic conditions and comparedto the conventional system of free cells bioreactor (FCBR) under the same conditions.
2. Materials and Methods
2.1. Anaerobic Culture, Medium, and Raw Syngas
An anaerobic culture was obtained from a 3000-m3 municipal solid waste digester, operatingunder thermophilic (55 ˝C) conditions (Borås Energy and Environment AB, Borås, Sweden). Theinoculum was incubated at 55 ˝C for 3 days to keep the bacteria active while consuming the carbonsource provided by the inoculum. After incubation, the inoculum was filtered through a sieve(1-mm pore size) to remove the large particles. The digesting sludge was then centrifuged at 31,000ˆ gfor 15 min to separate the solid inoculum (85 % Total solid (TS)) and the microorganisms, whichwere loaded into membrane sachets (Section 2.2). The synthetic medium solution contained acetate,propionate, butyrate with COD strength of 6.72 gCOD/L, and a vitamin solution (basal medium)with COD strength of 3.34 gCOD/L at a ratio of 3:1:1:1 [24]. The medium solution was buffered topH 7.0 ˘ 0.2 with NaHCO3 [25]. The artificial syngas containing CO (55% mol), H2 (20% mol), andCO2 (10% mol) [11] was provided by AGA gas AB (Borås, Sweden).
2.2. Cell Containment Procedure for the RMBR Configuration
The microbial cell encasement was performed following a previously described method [23].Flat plain hydrophilic PVDF (polyvinylidene fluoride, Durapore®) membranes (Merch Millipore Ltd.,Cork, Ireland) with a pore size, thickness, and diameter of 0.1 μm, 125 μm, and 90 mm, respectively,were used as supporting materials. The main physicochemical characteristics of the membranes are: airflow rate of 0.15 L/min.cm2, 0.5% gravimetric extractables, 70% porosity, and water flow rate greaterthan 0.33 mL/min.cm2. The membranes were cut into rectangular shapes of 6 ˆ 6 cm2 and folded tocreate membrane pockets of 3 ˆ 6 cm2. They were then heat-sealed (HPL 450 AS, Hawo, Obrigheim,Germany) on three sides with heating and cooling times of 4.5 and 4.5 s, leaving one side open for theinsertion of the inoculum. Solid sludge inoculum (3 g per sachet) was then injected carefully into thesynthetic membrane pockets, and the fourth side was subsequently sealed. The sachets containing theinoculum were used immediately for biomethanation of syngas.
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2.3. Reactor Set up for Semi-Continuous Operation
The schematic illustration of the RMBR system is shown in Figure 1. The reactors used wereserum glass bottles with 600-mL working volume, closed with rubber seals and an outlet for the biogas.The RMBR contained 15 sachets of encased inoculum (a total of 45 g) and 475 mL of synthetic medium.In parallel, the same amount of solid inoculum and medium was used in a reference reactor of freecells (FCBR) and performed under the same process conditions. The thermophilic temperature wasmaintained at 55 ˘ 1 ˝C throughout the process by placing the reactors in a water bath. The syngasamount was gradually increased by flushing fresh syngas at different gas pressures. Simultaneously,the OLR was gradually increased by increasing the concentrations of the medium fed (Table 1). Duringthe fermentation process, the syngas was continuously bubbled through the reactors, from the bottomrising up to the top of the reactors, from where it was circulated to the bottom at different flow rates.Each co-substrate loading rate and gas circulation rate was maintained for different experiments(different periods of time), giving a total digestion process of 154 days.
Figure 1. Schematic illustration of the reverse membrane bioreactor (RMBR) in the semi-continuousbiomethanation process of syngas and organic substances. (a) Digesting sludge encased in PVDFmembrane; (b) Organic and nutrient inlet; (c) Syngas inlet; (d) Peristaltic pump; (e) Product outlet;(f) Warm water bath; (g) Effluent outlet; (h) Gas sampling; and (i) Data analysis.
Table 1. Parameter profiles during the simultaneous semi-continuous biomethanation process.
Periods (Days) Gas CirculationRate (mL/min)
Organic Loading(gCOD/Lreactor.day)
Syngas Amounts (mmol/Lreactor.day)
H2 CO CO2
I (0–16) 100 0.02 1.1 3.1 1.6II (17–36) 200 0.02 2.2 6.1 1.7III (37–58) 200 0.02 2.8 7.7 1.6IV (59–87) 300 0.43 1.5 4.0 0.7V (88–126) 300 0.86 1.7 4.7 1.0
VI (127–147) 300 1.70 3.8 9.4 1.9VII (148–154) 300 3.40 7.0 15.2 4.0
The parameter profiles are shown in Table 1. At the beginning of the digestion (periods I,II, and III), the syngas amount added was gradually increased. The OLR was kept relatively low,according to a very low organic content in the basal medium that was used as the vitamins and
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minerals solution during these periods. This was done in order to allow the anaerobic culture to adaptto the environment with a low risk of cell washout and to investigate the ability of both systems toresult in pure syngas fermentation. Then, from period IV to VII, the organic compounds containingacetate, propionate, and butyrate were added simultaneously with syngas. The OLR was doubled inorder to investigate the efficiency of the systems under the harsh conditions at a shorter retention time.
For the adjustment of the syngas compounds inside the reactor, syngas was placed in a 1-L gassampling bag and then the gas was fed to the reactor by a peristaltic pump. Inside the sampling bag,there was a known syngas volume that could be adjusted, and the content of each compound wasanalysed. Then, the gas was placed inside the reactor (with a known headspace), and the percentageof each syngas component was measured again. The ideal gas law was used for calculating themole number:
PV = nRT, where:P: 1 atm, adjusted with a needle after the syngas feeding; V: the headspace of the reactor (stable);
R: the ideal gas law constant; T: the temperature of the reactor; n: the unknown mole value thatwas calculated.
2.4. Analytical Methods
The composition of the gas samples (methane, hydrogen, carbon monoxide, and carbon dioxide)was measured regularly, using a gas chromatograph (Perkin-Elmer, Norwalk, CT, USA), equippedwith a packed column (CarboxenTM 1000, SUPELCO, 6’ ˆ 1.8” OD, 60/80 Mesh, Shelton, CT, USA)and a thermal conductivity detector (Perkin-Elmer) with an injection temperature of 200 ˝C. Thecarrier gas was nitrogen, with a flow rate of 30 mL/min at 75 ˝C. A 250-μL gas tight syringe (VICI,Precision Sampling Inc., Baton Rouge, LA, USA) was used for the gas sampling. The obtained peakarea was compared with a standard gas, analyzed under the same conditions (STP, 273.15˝K and101.325 kPa). The volatile fatty acids (VFAs) were analyzed using a gas chromatograph (Auto System,Perkin-Elmer) equipped with a capillary column (Zebron ZB-WAXplus, Polyethylen glycol (PEG),30 m ˆ 0.25 mm ˆ 0.25 μm, Shelton, CT, USA) and a flame ionized detector (Perkin-Elmer) with aninjection and detection temperature of 250 and 300 ˝C, respectively. The carrier gas was nitrogen, witha flow rate of 2 mL/min at a pressure of 20 psi. The experiment was performed in duplicate, and theresults were presented as the average ˘ standard deviation.
3. Results and Discussion
Syngas biomethanation is a potent bio-conversion route, utilizing microorganisms to assimilateintermediate gases to produce methane. However, the cell density in continuous bioreactor operationsplays an important role in the process efficiency. In this work, a novel application of the RMBR wasinvestigated: rapid syngas biomethanation in a semi-continuous operation at high cell loading. Theperformance of the RMBR was compared to the performance of a reactor with free cells (FCBR) underotherwise identical conditions.
During the semi-continuous biomethanation process of 154 days, the performance of the RMBRand the FCBR was investigated under seven experimental conditions (seven periods). The conditionsof each period are presented in Table 1.
3.1. Methane Production
Figure 2 and Table 2 show the trend of the methane production throughout the anaerobic process.Methane was produced from mainly pure syngas at the beginning of the process (Periods I–III),to thereafter be produced also from the co-substrates until the last day. The methane formed fromthese substrates was likely made by the acetic cleavage of acetate and the reduction of carbon dioxide.The microorganisms assimilating these substrates are mainly hydrogenotrophic methanogens andacetotrophic methanogens. It was observed that the methane production increased with the increasingconcentration of gaseous and organic substrates fed, until the maximum conversion capacity was
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reached and the production decreased. This shows that the metabolism of the microorganisms occurredas expected.
Figure 2. Comparison of the methane production in the reverse membrane bioreactor (RMBR)system and the free cell bioreactor (FCBR) during the semi-continuous biomethanation of syngasand organic substances.
Table 2. Investigated parameters during the semi-continuous biomethanation process ofthe co-substrates.
PeriodsMethane Production (mL/Lreactor.day) Total VFA (g/L)
RMBR * FCBR ** RMBR * FCBR **
I 39.1 ˘ 0.4 31.4 ˘ 0.5 0.4 ˘ 0.0 0.5 ˘ 0.0II 38.0 ˘ 0.7 46.4 ˘ 0.5 0.4 ˘ 0.0 0.5 ˘ 0.0III 108.9 ˘ 0.2 106.3 ˘ 0.2 0.4 ˘ 0.0 0.5 ˘ 0.0IV 92.5 ˘ 1.5 84.4 ˘ 1.2 0.8 ˘ 0.3 1.0 ˘ 0.3V 160.8 ˘ 1.1 74.5 ˘ 1.7 0.4 ˘ 0.3 1.2 ˘ 0.7VI 186.0 ˘ 1.3 46.0 ˘ 0.6 4.4 ˘ 3.5 4.0 ˘ 3.0VII 176.5 ˘ 0.3 36.2 ˘ 0.1 7.8 ˘ 0.8 9.4 ˘ 0.5
* The reverse membrane bioreactor. ** The free cells bioreactor.
In the beginning of the experiment, a total pure syngas amount of 5.8 mmol/L.day was added tothe reactors. The amount was thereafter gradually increased throughout the operation of the reactors(Table 1). The basal medium with an organic content was fed at a low rate of 0.02 gCOD/L.dayduring the periods I, II, and III, and a similar methane production was observed in both reactors.The methane amount produced in the RMBR and the FCBR during these periods ranged between38.0–108.9 mL/L.day and 31.4–106.3 mL/L.day, respectively. These results show that under a low riskof cell wash-out condition, the encased microorganisms in the RMBR displayed a similar methaneproduction performance from syngas as the free microorganisms in the FCBR. The results also indicatethat using PVDF membranes as a cell supporting material in the current reactor setup was not a majorissue for the continuous syngas fermentation when it came to diffusion limitations. In addition, due tothe practical design of the RMBR, the problematic issues of cell clogging and cell abrading are likelyless severe.
During the periods IV, V, and VI, the organic compounds were introduced to both the reactorswhile the syngas amount added to the systems was decreased slightly in order to maintain a mildcondition for microbial adaptation. From these experiments, the microorganisms retained in theRMBR could assimilate the co-substrate perfectly with maintained efficient performance, resulting in acorresponding increase of the methane production (Table 2 and Figure 2). The maximum methaneproductivity of the RMBR during these periods was 92.5, 160.8, and 186.0 mL/L.day, respectively.On the other hand, the wash-out of the microbial cells from the FCBR was clearly occurring during
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these periods, resulting in a substantially lower methane production from the FCBR than the RMBRduring the periods V and VI (74.5 and 46.0 mL/L.day). In the RMBR, the microbial cells were stillenclosed completely in the membrane sachets, and cell abrading was not visually occurring.
A better cell retention performance of the RMBR for syngas biomethanation in a semi-continuousprocess was clearly shown during period VII, when a higher syngas amount (26.2 mmol/L.day) was fedto both the reactors, while the organic loading was doubled in comparison to period VI. The methaneproduction in the RMBR was higher than the production in the FCBR (176.5 and 36.2 mL/L.day,respectively). These results indicate that the RMBR had the ability to maintain a high methanationcapacity, by retaining the microbial cells under the high risk of cell-wash out condition. Consequently,the system was able to convert more substrates into methane than the one with the free cells. Thisresult stresses the importance of cell-retention inside anaerobic digestion as shown in other studiesin which biofilm was used for high cell-density. However, with the method of cell-enclosure in themembranes, there is no danger of cell-detachment in contrast with the use of biofilm [26].
In the last period, the higher co-substrate concentration was fed to the systems in order toinvestigate if the product profiles changed. The results showed that the methane production in boththe reactors was maintained at a low level, observed as a lower utilization of H2 and CO (Figure 3a,b)and higher concentration of VFAs (Table 2). The co-substrate was not completely converted by themicrobial cells in this period. This result indicates that the maximum conversion capacity of thesystems had been reached. The lower methane production was probably because of the inhibitoryeffect of a high hydrogen partial pressure from the syngas, as well as the accumulation of the volatilefatty acids from the organic substances [2]. However, a higher amount of methane was still formed inthe RMBR compared to the FCBR. This is an effect of the higher amount of microbial cells retained inthe RMBR, still able to consume the co-substrates and convert them into methane.
Figure 3. Comparison of the performance of the reverse membrane bioreactor (RMBR) system andthe free cell bioreactor (FCBR) during the semi-continuous biomethanation of syngas and organicsubstances. The utilization of (a) Hydrogen; (b) Carbon monoxide; and (c) Carbon dioxide.
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The above results indicate that during 154 days of syngas biomethanation, the RMBR systemwas an efficient and practical process in retaining the microbial cells, resulting in a high methaneproduction under a semi-continuous process condition.
3.2. Syngas Utilization Efficiency
Periods I, II, and III were designed to allow the microbial cells to adapt to the environmentalconditions in the beginning of the experiment. The artificial syngas fed was gradually increased, whilethe organic loading rate was controlled at a very low level (0.02 gCOD/L.day). The medium usedonly contained nutrients and vitamins during these periods. The flow rate of the gas recirculationwas 100 mL/min in the first experiment, and thereafter increased to 200 mL/min during periods IIand III in order to test the performances of the systems with shortened gas retention time. Figure 3and Table 3 present the percentage of gas utilization during the semi-continuous syngas fermentationprocess of 154 days.
Table 3. Investigated parameters during the semi-continuous biomethanation process ofthe co-substrates.
PeriodsSyngas Component Utilization (%) RMBR * Syngas Component Utilization (%) FCBR **
H2 CO CO2 H2 CO CO2
I 88.9 ˘ 0.0 80.8 ˘ 0.4 ´0.0 ˘ 0.5 96.5 ˘ 0.0 87.2 ˘ 0.2 ´1.7 ˘ 0.0II 100.0 ˘ 0.0 98.7 ˘ 0.0 ´16.6 ˘ 0.1 100.0 ˘ 0.0 99.5 ˘ 0.0 ´21.7 ˘ 0.0III 100.0 ˘ 0.0 100.0 ˘ 0.0 ´11.4 ˘ 0.2 100.0 ˘ 0.0 100.0 ˘ 0.0 ´12.2 ˘ 0.1IV 100.0 ˘ 0.0 98.2 ˘ 0.1 ´39.3 ˘ 0.1 100.0 ˘ 0.0 99.8 ˘ 0.0 ´36.3 ˘ 0.0V 99.7 ˘ 0.0 100.0 ˘ 0.0 ´17.6 ˘ 0.2 99.5 ˘ 0.0 100.0 ˘ 0.0 ´23.8 ˘ 0.2VI 94.7 ˘ 0.2 88.4 ˘ 0.7 ´40.3 ˘ 0.1 59.3 ˘ 0.6 42.3 ˘ 3.3 ´22.3 ˘ 0.1VII 56.6 ˘ 0.2 46.8 ˘ 4.9 25.6 ˘ 0.7 16.4 ˘ 0.3 38.9 ˘ 5.7 0.8 ˘ 0.7
* The reverse membrane bioreactor. ** The free cells bioreactor.
The amount of H2 in both the reactors remained at a very low level even when the H2 (syngas)fed was increased from 1.1 to 2.8 mmol/L.day, and the gas circulation rate was increased from 100 to200 mL/min. The percentage of H2 consumed in the RMBR and the FCBR was close to 100 percent.This shows that the microbial cells in both the RMBR and the FCBR could convert the H2 easily within1 day. Similar to the conversion of H2 in both the reactors during these periods, the microorganismsalso converted all the CO, without any observed negative effects. The CO in the gas fed was increasedfrom 3.1 to 7.7 mmol/L.day (Table 1), and it was utilized by the microbial cells in both the systemsat almost the same rate (about 100 percent). The CO2 amounts showed a different trend from theother gases. It increased during the first 3 days, and then stayed stable throughout periods I–III.It was observed that the CO2 amounts in both the reactors increased rather than decreased duringthese periods (a CO2 amount of 1.6–1.7 mmol/L.day was present in the syngas fed). This trendindicated that the microbial cells in both the reactors had performed as expected, with the CO2 beingboth produced and consumed during the fermentations. Thus, even though CO2 was supplementedtogether with the other compounds in the syngas, CO2 was also produced, leading to a stable amountof CO2 (Figure 3c). The most interesting conclusion that can be drawn from the results of these periodsis that the gases transfer through the PVDF membrane used, as the cell encasing material was not alimiting factor during the semi-continuous fermentation process, since the amount of the differentcomponents of the syngas were at similar levels as in the free cell system. The encased cells in theRMBR could assimilate the syngas continuously for product formation.
During periods IV and V, the gas circulation rate was increased to 300 mL/min, reducing the gasretention time in the systems. The syngas amount was decreased slightly while higher organic loadingrates with a medium containing acetic, propionic, and butyric acid was introduced. The results showedthat the syngas conversion performance of both the reactors was maintained during the simultaneous
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co-substrate fermentation. The microorganisms in both of the systems utilized almost 100 percent ofthe H2 and CO. However, CO2 started accumulating during these periods, indicating that the microbialcells decomposed the organic acids.
During the last two periods, VI and VII, a higher concentration of co-substrates was providedto the reactors. The results showed that the syngas utilization showed a decreasing trend in both thereactors, despite a higher concentration being added to the reactors during period VII (Table 1). Thehigher gas amounts, especially of H2, in the systems probably increased the partial pressure, therebyleading to a higher pH of the liquid solution (Figure 4b). Furthermore, additional organic substances,which resulted in the accumulated VFA, were present during these periods (Figure 4a). This resultedin a decreased performance of the microorganisms in the reactors. However, the RMBR still showed abetter ability to utilize the high concentration of co-substrates than the FCBR during these periods,shown by a higher percentage of H2 and CO utilization (Table 2). The percentage of H2 utilizationin the RMBR and the FCBR from period VI was 94.7 and 59.3, respectively. The CO utilization inthe RMBR and the FCBR was 88.4 and 42.3 percent, respectively. CO2 was accumulated, resultingin negative utilization values of ´40.3 and ´22.3 percent, respectively. These results revealed thatthe RMBR had a better capacity than the conventional system of the FCBR to convert higher syngasand co-substrate concentrations in a semi-continuous fermentation process. However, a positive CO2
utilization in both the reactors was observed in period VII. This means that CO2 was consumed ata higher rate than it was produced. In general, CO2 is produced during the anaerobic digestion oforganic compounds. In period VII, the VFAs were accumulated, meaning that the organic substanceswere not much used. This leads to less CO2 production in both the RMBR and FCBR. Although ahigher amount of CO2 was fed to the reactors (Table 1), CO2 was likely utilized by the microbial cells,thus resulting in a positive CO2 utilization. It can be concluded that retaining the microorganismsin the RMBR was a successful technique for improving the syngas biomethanation in the long-termfermentation process. Encased microbial cells in the RMBR showed a similar performance to assimilatethe syngas in a semi-continuous biomethanation process as the system with the free cells at a loworganic loading rate. Moreover, when higher syngas and organic loading rates were applied, a higherprocess efficiency of the RMBR, compared to the FCBR, was observed.
Figure 4. Accumulation of organic acids and pH during the semi-continuous biomethanation ofco-substrates. (a) Volatile fatty acids; (b) pH.
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3.3. Volatile Fatty Acids and pH Profile
In this work, the performance of the bioreactors with cells degrading a mixture of two substratesin a semi-continuous process was investigated. Organic acids, including acetic, propionic, and butyricacid, were used as co-substrates to the syngas in the fermentations. The organic loading rate (OLR),in the form of the organic acids fed, was gradually increased in both the systems. The data oftotal volatile fatty acids (VFAs) in the effluents represented the organic acids utilization during thesimultaneous fermentation process. The VFA value is also an indicator for monitoring the stability ofthe anaerobic digestion process [4]. In a previous work, repeated batch syngas biomethanation wasexamined [27]. All of the medium was removed, and the encased cells were reused in a new batchexperiment. In that setup, the inhibiting substances probably disappeared, which is different from thecontinuous process of the present work, where most of the substrates remained in the reactors. Thus,the trend of the VFA content and the pH values were investigated in this experiment.
Figure 4 represents the total VFA concentration (a) and the pH values (b) in the effluents duringthe semi-continuous fermentation processes in the two investigated cell systems. The VFA amountsin both the reactors were close to zero during the periods I, II, III, and IV, even when the organicloading was increased from 0.02 to 0.43 g COD/L.day. This indicates that the microorganisms in boththe reactors were able to convert the organic substances simultaneously with syngas, without anyobserved negative effect. However, the pH value decreased slightly when the organic acids wereintroduced to the system during these periods. The pH was in the range of 7.0–7.8, and the changeswere not observed to affect the stability of the processes.
A low amount of VFAs in the reactors shows that the microorganisms are coping wellwith the process conditions. In the anaerobic digestion process, VFAs can be consumed by themethane-producing microorganisms. This reduction of VFAs increases the alkalinity of the surroundingmedium. The pH in the anaerobic digester is significantly affected by the production of VFAs and theCO2 content of the biogas.
In period V (days 88–126), there were substantially lower VFA concentrations measured in theeffluent from the RMBR (around 0.2 g/L) compared to the free cell system FCBR (around 0.6 g/L),as the OLR was increased to 0.86 gCOD/L.day. However, the VFAs concentration still remained at alow level during this period. The VFAs concentration in the RMBR was totally consumed. The reasonfor this fact was probably the high active microbial cell density retained in the RMBR, while the cellwash out was visually occurring from the FCBR. During this experiment, the pH value of both thereactors started to ramp up slightly. This probably happened because of the high conversion rate ofthe VFAs to biogas (methane and CO2), as well as the additional CO2 fed contained in the syngas(Figure 4b). In particular, the value from the RMBR had lower residual VFAs content than the FCBR.The pH increased to 8.7 and 8.1 in the RMBR and the FCBR, respectively.
When the concentration of the organic acids and syngas were increased in the last period, theaccumulation of the VFAs was even higher, while the pH dropped slightly. The VFA accumulation inthe FCBR increased, reaching a maximum amount of 9.4 g/L, while the total VFAs in the RMBR weredetected at 7.8 g/L, indicating that the cell encasement in the RMBR helped to maintain a higher abilityto degrade the VFAs compared to the FCBR in a semi-continuous fermentation process. However, therecommended VFA concentrations during the anaerobic digestion, without negatively affecting themethane-producing microorganisms in the process, are under 4 g/L [1,2]. At the observed levels in thereactors, it was thus clear that the maximum process capacity had been reached in this condition.
These results indicated that the RMBR system performed efficiently during the semi-continuoussyngas biomethanation with the addition of organic substances as a co-substrate. Organic acids weredegraded simultaneously with the reduction of the syngas until too high amounts of substrates werefed to the systems.
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4. Conclusions
The syngas biomethanation under simultaneous semi-continuous fermentation could be enhancedby retaining a high microbial cell density in a reverse membrane bioreactor (RMBR). No cell washoutwas visually observed from the RMBR during the process, running for 154 days. The performanceof the RMBR for the simultaneous fermentation of syngas and the organic compounds was betterthan the conventional system of free cells (FCBR). The microorganisms in the RMBR were able toconvert the syngas simultaneously with organic acids to methane at higher substrate concentrationsand productivity than the FCBR system. The RMBR reached maximum capacities of H2, CO,and CO2 conversion of 7.0, 15.2, and 4.0 mmol/L.day, respectively, at the organic loading rate of3.40 gCOD/L.day. The highest methane production rate from the RMBR was 186.0 mL/Lreactor.day onthe 147th day, compared to the highest rate in the FCBR, 106.3 mL/Lreactor.day, on the 58th day.
Acknowledgments: This work was financially supported by the Swedish Research Council. The authors wouldlike to thank Johan Westman for the useful discussions.
Author Contributions: All authors contributed to the conception and design of experiments; Supansa Y. Westmanand Konstantinos Chandolias performed the experiments and analyzed the data; all authors contributed to writingthe paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Paper III
Effects of heavy metals and pH on the conversion of biomass to hydrogen via syngas
fermentation
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Chandolias et al. (2018). “Biomass to hydrogen,” BioResources 13(2), 4455-4469. 4455
Effects of Heavy Metals and pH on the Conversion of Biomass to Hydrogen via Syngas Fermentation Konstantinos Chandolias,a,* Steven Wainaina,a Claes Niklasson,b and Mohammad J. Taherzadeh a
The effects of three heavy metals on hydrogen production via syngas fermentation were investigated within a metal concentration range of 0 to 1.5 mg Cu/L, 0 to 9 mg Zn/L, 0 to 42 mg Mn/L, in media with initial pH of 5, 6, and 7, at 55 °C. The results showed that at lower metal concentration, pH 6 was optimum while at higher metal concentrations, pH 5 stimulated the process. More specifically, the highest hydrogen production activity recorded was 155% ± 12% at a metal concentration of 0.04 mg Cu/L, 0.25 mg Zn/L, and 1.06 mg Mn/L and an initial medium pH of 6. At higher metal concentration (0.625 mg Cu/L, 3.75 mg Zn/L, and 17.5 mg Mn/L), only pH 5 was stimulating for the cells. The results showed that the addition of heavy metals, contained in gasification-derived ash, can improve the production rate and yield of fermentative hydrogen. This could lead to lower costs in gasification process and fermentative hydrogen production and less demand for syngas cleaning before syngas fermentation.
Keywords: Gasification; Syngas; Fermentative hydrogen; Heavy metals; pH Contact information: a: Swedish Centre for Resource Recovery, University of Borås, S-50190 Borås, Sweden; b: Department of Chemistry and Chemical Engineering, Chalmers University of Technology, S-412 96 Gothenburg, Sweden; *Corresponding author: [email protected] INTRODUCTION
To face the increasing global waste generation and energy demand, sustainable strategies for the conversion of waste into green energy are required. Anaerobic fermentation is a relatively simple and cost-effective method that converts organic wastes, such as food residuals, sewage waste, and manure, into biofuels. However, there is a big fraction of solid waste, such as forest residues and mixed landfill wastes, which are difficult or impossible to degrade biologically because of their complex structure. An effective way to treat this recalcitrant biomass is with the combination of gasification and anaerobic fermentation processes. During this two-stage process, the feedstock is converted into syngas, a gas consisting mainly of hydrogen, carbon monoxide, and carbon dioxide. Thereafter, anaerobic bacteria digest the syngas and create value-added chemicals, such as volatile fatty acids (VFA), and biofuels such as hydrogen.
Hydrogen is a valuable gas with a high-energy content of 120 MJ/kg to 142 MJ/kg, which is higher than that of hydrocarbon fuels by a factor of approximately 2.75 and releases only water during combustion (Henstra et al. 2007; Tufa et al. 2016). This gas has numerous applications in chemical processes, lamps, balloons, vehicle fuels, laboratories, as a reductive agent (redox reactions), etc. Hydrogen is also a product of several processes. The largest fraction of the global hydrogen production (80% to 85%) is obtained by the steam methane reforming (SMR) process of natural gas (Fan et al. 2016), which uses fossil
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Chandolias et al. (2018). “Biomass to hydrogen,” BioResources 13(2), 4455-4469. 4456
fuels as the main substrate. In addition, electricity can be stored in hydrogen form through the power-to-gas process. Another process for hydrogen generation, with less environmental drawbacks but higher costs, is the water electrolysis method, although it currently can only be used in small-scale applications (Tufa et al. 2016). Biocatalysts, such as microalgae and phototrophic bacteria, have been employed in special-designed photobioreactors for hydrogen production (Skjånes et al. 2016). Another biological process is the dark fermentation of biomass such as food residues, manure, and straw. The main advantage of the fermentation process for hydrogen production is that it is less expensive, less energy-demanding, and more eco-friendly than the other conventional processes (Levin et al. 2004; Lin and Shei 2008). All the above processes can lead to a substantial production of hydrogen to be used for energy and as material resources.
The anaerobic syngas conversion into hydrogen can occur via a biological water gas shift process. In this process, carbon monoxide acts as an electron donor and the CO-dehydrogenase (CODH) enzyme provides electrons and protons for the carbon monoxide. Thereafter, the electrons released by the oxidation reaction are transferred to another enzyme called energy-converting hydrogenase (ECH) (Henstra et al. 2007), which catalyzes the reduction of electrons for the production of molecular hydrogen. Fatty acids are by-products that can be produced through the H2/CO2 or CO consumption pathway (Grimalt-Alemany et al. 2017), shown in Eq. 1,
CO + H2O → CO2 + H2, ΔG0 = -20 KJ/mol (1)
At the end of the gasification process, inorganic components, such as heavy metals, still remain in the residual ashes (Dong et al. 2015). These metals can be present in different forms such as metal oxides or metal chlorides (Liao et al. 2007; Tafur-Marinos et al. 2014), and their composition depends on the feedstock and gasification conditions (Dong et al. 2015). Despite the fact that the gasification ashes have been used as building materials for roads and other constructions, they can leach and pose a threat to the environment and to the public’s health (James et al. 2012). The main threat derives from the heavy metals, which are toxic and classified as known or possible carcinogens for humans, according to the U.S. Environmental Protection Agency and the International Agency for Research on Cancer (Tchounwou et al. 2012). In contrast, heavy metals have been shown to be beneficial for the growth of fermentative microbes (Osuna et al. 2003). For instance, a study on fermentative hydrogen production reported that the addition of Cu and Cr ions improved the hydrogen yield (Lin and Shei 2008). Therefore, the use of heavy metals during syngas fermentation could reduce the environmental footprint of gasification while enhancing the fermentation process.
Although several studies have been conducted on the effects of heavy metals on anaerobic methane production (Fang and Chan 1997; Lin and Chen 1999; Abdel-Shafy and Mansour 2014), there are only a handful of studies on the effects of heavy metals on the fermentative hydrogen production (Li and Fang 2007; Lin and Shei 2008). According to the authors’ best knowledge, this is the first study that investigates the impact of heavy metals in syngas fermentation. The aim of this work is to provide valuable data on the beneficial and inhibitory effects of heavy metals and pH during syngas fermentation and suggest strategies for improving the efficacy of this process. This knowledge can be used in order to reduce the environmental impact of gasification and reduce the costs of syngas cleaning and fermentative hydrogen production.
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EXPERIMENTAL Materials Liquid medium
The liquid medium inside the reactors consisted of micronutrients in trace amounts, macronutrients, and the three heavy metals that were under investigation (except the control reactors).
The concentration of the inorganic macronutrients in the used liquid medium was 280 mg NH4Cl/L, 330 mg K2HPO4•3H2O/L, 100 mg MgSO4•7H2O/L, and 10 mg CaCl2•2H2O/L (Osuna et al. 2003). Moreover, 1 mL of the trace element stock solution was contained in 1 L of the liquid medium. The concentration of the trace element stock solution included 2000 mg FeCl2•4H2O/L, 50 mg H3BO3/L, 50 mg ZnCl2/L, 500 mg MnCl2•4H2O/L, 38 mg CuCl2•2H2O/L, 50 mg (NH4)6Mo7O24•4H2O/L, 2000 mg CoCl2•6H2O/L, 142 mg NiCl2•6H2O/L, and 164 mg Na2SeO3•5H2O/L (Osuna et al. 2003).
The metal ions used were in the form of metal oxides (ZnO, CuO) and salt (MnCl2•4H2O) powder. Initially, 0.25 g of each metal powder was dissolved in 20 mL of 2% nitric acid (HNO3), and deionized water was added to achieve a total volume of 500 mL. The metal stock solutions were dissolved to achieve a metal ratio (Cu:Zn:Mn) of approximately 1:6:28. This metal ratio is similar to the heavy metal composition that was found in the fly and bottom ashes of a wood pellet pyro-gasification facility (Tafur-Marinos et al. 2014).
Syngas composition
A commercial syngas mixture containing carbon monoxide (55 mol%), hydrogen (20 mol%), and carbon dioxide (10 mol%) (Klasson et al. 1990) was provided by AGA Gas AB (Gothenburg, Sweden). The overpressure in the gas cylinder containing the syngas was built by nitrogen gas. Inoculum
The mixed anaerobic consortium was obtained from a local thermophilic 3000 m3 anaerobic digester that typically digested the organic fraction of municipal solid waste (Borås Energy and Environment, Borås, Sweden). A typical pH value in the digester was 8 to 8.2 and the main fraction of the substrate was food waste. The total solids (% TS) and volatile solids (% VS) of the inoculum were 14.23% ± 0.27% and 14.15% ± 0.27%, respectively. Methods Reactor characteristics, inoculation, and start-up
The reactors were serum glass bottles with plastic caps, rubber sealing, and a total volume of 118 mL (Bioprocess Control AB, Lund, Sweden). The anaerobic culture was incubated at 55 °C for 5 days to consume all the nutrients prior to the experiment. After incubation, the excess water from the inoculum was removed by centrifugation at 4300 ×g for 10 min. Then, each reactor was loaded with 3 g of inoculum and filled with 40 mL of liquid medium. The headspace of each reactor was purged with syngas for 3 min with a flow rate of 50 mL/min in order to remove air and feed the reactor with the gaseous substrate. After the syngas-purging, the pressure inside the headspace was adjusted at 1
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atm with a needle that removed the excess gas. The reactors were continuously shaken (100 rpm) in a warm water bath (55 °C ± 1 °C) and placed at a 45° angle. The gaseous samples were collected from the top of the reactors through the rubber sealing regularly, and the liquid samples were collected at the end of the experiment from the liquid medium.
The inoculum was acclimated to syngas as the sole carbon and energy source for 15 days before the experimental results were obtained. The experiment was conducted in batch mode and in triplicates, and the results are presented as the mean values ± standard deviation. The duration of the experiment was 96 h. Gas analysis
Gas samples were collected from the headspace of the bioreactors every 12 h with a 0.25-mL gas-tight syringe (VICI, Precision Sampling Inc., Baton Rouge, LA, USA). The gas components were analyzed by using gas chromatography (GC). For the analysis of hydrogen and carbon dioxide, a Perkin-Elmer gas chromatograph (Clarus 500; Norwalk, CT, USA) was used, equipped with a packed column (CarboxenTM 1000, 6’ × 1.8” OD, 60/80 Mesh, Supelco, Shelton, CT, USA) using a thermal conductivity detector (Perkin-Elmer, Norwalk, CT, USA) with an injection temperature of 200 °C. The levels of the carbon monoxide were also analyzed by another gas chromatograph (Clarus 400; Perkin-Elmer, Norwalk, CT, USA), equipped with a packed column (CarboxenTM 1000, 6’ × 1.8” OD, 60/80 Mesh, Supelco, Shelton, CT, USA) and a thermal conductivity detector (Perkin-Elmer, Norwalk, CT, USA) with an injection temperature of 200 °C. Both of the gas chromatographs used nitrogen as a carrier gas with a flow rate of 30 mL/min at 75 °C.
Liquid analysis
The VFA content in the liquid samples was analyzed using a Waters 2695 high-performance liquid chromatograph (HPLC; Waters Corporation, Milford, CT, USA) with a hydrogen-based column (Aminex HPX87-H; BioRad Laboratories, München, Germany) at 60 °C and 0.6 mL/min (5 mM H2SO4 eluent) equipped with a refractive index (RI) detector (Waters 2410, Waters Corporation, Milford, CT, USA). The metal concentrations of the liquid samples were measured with a microwave plasma-atomic emission spectrometer (MP-AES 4200; Agilent Technologies, Santa Clara, CA, USA) equipped with an inlet air filter kit - 4107 nitrogen generator (Agilent Technologies, Santa Clara, CA, USA) and an SPS 3 Autosampler (Agilent Technologies, Santa Clara, CA, USA). RESULTS AND DISCUSSION
The coupling of thermochemical and biochemical processes, such as gasification and dark fermentation, can convert recalcitrant biomass into hydrogen. The use of heavy metals derived from the ashes of thermochemical processes can reduce the environmental footprint of these processes and boost the efficacy of biochemical processes.
This work focused on the biochemical conversion of syngas into hydrogen through dark fermentation. The goal was to investigate the effect of heavy metals and pH and to report the optimum, inhibiting, and toxic metal concentrations and pH values. Hydrogen production activity (Eq. 2), hydrogen yield (Eq. 3), and total VFA yield (Eq. 4) were employed to monitor the extent of heavy metal and pH effects on the syngas fermentation
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process. The heavy metal uptake (%), was also obtained. Equation 2 shows the hydrogen production activity (%),
AH (%) = (Ai / Ac) × 100 (2)
where Ac is the hydrogen amount (mmol) produced by the control reactors, and Ai is the hydrogen amount (mmol) produced by the metal-dosed reactors after 96 h of anaerobic fermentation. The yield values were calculated as follows,
YH = H2 produced / CO fed (3) YVFA = VFA concentration / CO fed (4)
where YH is the hydrogen yield (mmol H2/mmol CO fed) and YVFA is the VFA yield (g VFA/L/mmol CO fed) obtained after 96 h of anaerobic fermentation. The Effect of HNO3
The addition of HNO3 in the reactors had two main purposes. First, the acid dissolved the heavy metal compounds inside the liquid medium and, second, the addition of HNO3 shifted the biological activity from methane to hydrogen production. Thus, no inoculum pretreatment that favored hydrogen production and the inhibition of methanogens was necessary. Figure 1 (panels a through d) shows how the consumption/production of gas components was affected by the addition of HNO3. The HNO3 concentration that inhibited methane production was 0.08%. This nitrate concentration was relatively low in comparison to the concentration used in other studies. For example, in a work that investigated the effect of ammonia and nitrate on biogas production from food waste, no inhibitory effect on methane production was reported at total ammonia nitrate addition levels below 1.1 g/L (Sheng et al. 2013). However, these inhibitory levels depend also on the anaerobic species of the inoculum.
Several mechanisms have been proposed to explain the inhibition of methanogens by HNO3. One possible mechanism is that nitrate raises the redox potential, Eh, of the medium (Jones 1972). Another reason may be the toxic effects of nitrogen intermediates such as nitrite and nitrous oxide (Iwamoto et al. 1999; Ungerfeld 2015). Recent studies suggest that nitrate can be used as an alternative hydrogen sink so that it can compete with available hydrogen, which is one of the main methane substrates together with carbon dioxide (Yang et al. 2016). However, in this work, hydrogen production was favored, and therefore it can be assumed that the inhibiting mechanism was based on the toxicity of nitrate on methanogens.
The Effect of Heavy Metals and pH
Syngas fermentation for hydrogen production took place in bioreactors containing media with heavy metal concentrations between 0 mg Cu/L, 0 mg Zn/L, 0 mg Mn/L, and 1.5 mg Cu/L, 9 mg Zn/L, 42 mg Mn/L and the different initial medium pHs that were investigated were 7, 6, and 5. The metals were added in a specific ratio in the medium to mimic the real metal composition in the ash of a wood pellet gasification plant as described previously.
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Fig. 1. Effect of the addition of HNO3 during syngas fermentation
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Figure 2 shows the relationships between the accumulated hydrogen production, heavy metal concentrations, and initial pH of the medium.
Fig. 2. Relationships between accumulative hydrogen production, initial medium pH of 7 (a), 6 (b), and 7 (c), and heavy metal concentrations (mg/L)
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The accumulative hydrogen amount was calculated by the total biogas production and hydrogen gas content. Figure 2 also shows the time (h) that was required for each bioreactor to reach its maximum hydrogen production in 96 h of fermentation. The results showed that the different heavy metal loadings and the initial pHs of the medium influenced the hydrogen production.
The role of heavy metals in anaerobic fermentation is essential, as many of them are part of the enzymes as metallic co-factors. More specifically, Mn stabilizes the enzyme methyltransferase (MPB) and affects redox reactions (Fisher et al. 1973; Perry and Silver 1982), Cu affects the superoxide dismutase (SODM) and hydrogenase in MPB in Clostridia and facultative anaerobes (Jones et al. 1987; Kirby et al. 1981), and Zn affects the activity of hydrogenase in MPB, the sulfate-reducing bacteria (SRB), and formate dehydrogenase (FDH) (Adams et al. 1986; Kirby et al. 1981). A total lack of or overdose of heavy metals may cause the inhibition or loss of enzyme functions. Usually, at high concentrations, metals act as nonspecific, reversible inhibitors and do not compete with the substrate. In this type of inhibition, the metals bind to enzymes and form the enzyme-inhibitor (EI) or enzyme-substrate-inhibitor (ESI) complexes of EI or ESI type (Wood and Wang 1983). However, some heavy metals, such as Zn and Cd, may cause competitive inhibition, meaning that they may compete with the substrate. Possible microbial inhibition mechanisms include substitution of the metallic enzyme co-factors, combining with the sulfhydryl group (-SH), and tight binding to acid groups in the polypeptide chains (Wood and Wang 1983).
The hydrogen production activity (AH) and hydrogen yield (YH) are shown in Fig. 3. The reactors dosed with low heavy metal concentrations showed a higher hydrogen production in comparison to the control reactors. In particular, the metal concentrations of 0.04 mg Cu/L, 0.25 mg Zn/L, and 1.06 mg Mn/L and 0.1 mg Cu/L, 0.67 mg Zn/L, and 2.8 mg Mn/L stimulated the hydrogen production activity (Ah > 100%). The highest hydrogen production activity recorded was 155% ± 12% at a metal concentration of 0.04 mg Cu/L, 0.25 mg Zn/L, and 1.06 mg Mn/L and initial medium pH 6. Another study reported that a metal concentration of 3 mg Cu/L resulted in a 10% to 20% increase in hydrogen production (Lin and Shei 2008). Other studies on fermentative hydrogen production also have shown that the highest hydrogen production was achieved in mediums with a pH of 6 and that lowering the pH inhibited hydrogen formation (Dareioti et al. 2014). This phenomenon depends on the type of the dominant bacterial species inside the anaerobic microbial community of the inoculum. For example, Clostridia, which are usually dominant in this process, function within a pH range of 6.0 to 6.7. The pH is a very crucial parameter for the microbial growth of anaerobic communities and it directly affects the fermentative hydrogen production. The pH of the medium changes the intracellular pH and the electrochemical gradient across the microbial membrane and thus influences the regulation of metabolism and the bioenergetics of microorganisms.
Metal concentrations higher than 0.1 mg Cu/L, 0.67 mg Zn/L, and 2.8 mg Mn/L were demonstrated to be inhibitory for the cells in media with pH 6 and 7 (Fig. 3). More specifically, media containing metal mixtures of 1.25 mg Cu/L, 7.57 mg Zn/L, and 35 mg Mn/L and 1.5 mg Cu/L, 9 mg Zn/L, and 42 mg Mn/L were shown to be inhibitory and reduced the hydrogen production activity to values as low as 35%. Another study reported a 50% reduction in the hydrogen production activity for mixed inoculum that was dosed with media containing 4.5 mg Zn/L and 6.5 mg Cu/L (Lin and Shei 2008). These
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concentrations are higher but comparable to the inhibiting metal concentrations found in this study. However, the metal concentrations of 0.625 mg Cu/L, 3.75 mg Zn/L, and 17.5 mg Mn/L stimulated the hydrogen production activity in mediums with a pH of 5. Therefore, for substrates with higher metal concentrations, pH 5 is recommended. This shift in the preference of pH at higher metal concentrations is possibly connected to the types of different strains of the microbial community and their optimum conditions of growth.
Fig. 3. Relationships between hydrogen production activity, Ah (%) = (mol H2/mol H2 control)*100 (a), hydrogen yield, YH = mol H2 prod./mol CO fed (b), initial medium pH and heavy metal concentrations (mg/L)
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The hydrogen yield showed a similar result to the hydrogen production activity (Fig. 3). The hydrogen yields were stimulated at lower metal concentrations and inhibited at higher metal concentrations. The highest hydrogen yield achieved was 1.32 mol ± 0.02 mol H2 prod./mol CO fed in bioreactors containing 0.1 mg Cu/L, 0.67 mg Zn/L, and 2.8 mg Mn/L and medium with initial pH 7.
Volatile fatty acid production was detected only in the control reactors (Fig. 4). This meant that the presence of heavy metals inhibited complete acidogenesis even though hydrogen formation was favored at low metal concentrations (Fig. 3). The VFA formation increased as pH decreased and the highest VFA yield reported was 1.086 g VFA/L per mmol of carbon monoxide fed, at a pH of 5.
Fig. 4. Relationships between VFA yield, YVFA = gVFA/L/mmol CO fed, initial medium pH, and heavy metal concentrations (mg/L) Heavy Metal Uptake (%)
During anaerobic fermentation, the heavy metals can end up inside the cells or they can be absorbed or precipitated with other inorganics (Hayes and Theis 1978; Howgrave-Graham and Wallis 1991). The availability of heavy metals as nutrients or as inhibitors is affected by the concentration of the metals as well as other parameters such as the pH of the liquid medium (Oleszkiewicz and Sharma 1990). However, even if there is a full availability of the metals as nutrients, this does not mean that the microorganisms utilize the metals. The metal uptake may be hindered by the presence of other metals, the lack of carrier molecules inside the cells, high excretion of metal ions by the cells, and the failure of the energy driven system (Wood and Wang 1983). In the case of the opposite conditions, excessive metal uptake may take place. In general, the uptake of heavy metals and all other nutrients indicate the growth rate of the cells (Oleszkiewicz and Sharma 1990).
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Fig. 5. Relationships between metal uptake (%) of Zn (a) and Mn (b), initial medium pH, and initial heavy metal concentrations (mg/L)
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In this work, the metal uptake was calculated from the initial and final metal concentrations inside the liquid medium of the reactors (Fig. 5). The results showed that the uptake of Zn and Mn was approximately 100% up to a metal concentration of 0.1 mg Cu/L, 0.67 mg Zn/L, and 2.8 mg Mn/L. At a metal concentration of 0.625 mg Cu/L, 3.75 mg Zn/L, and 17.5 mg Mn/L the metal uptake decreased to 90 80%, while at a higher metal concentration of 1.25 mg Cu/L, 7.57 mg Zn/L, and 35 mg Mn/L the Zn and Mn uptake dropped especially in media with lower pHs. This result was similar to the findings of another study that investigated the uptake of several heavy metals, at different pHs, during anaerobic digestion (Wang et al. 2007). That study also concluded that heavy metal uptake increased at higher pH values. In addition, several studies have shown that low pHs may hinder the heavy metal uptake (Cheng et al. 1975; Wang et al. 1999) because of the pH value’s influence on the binding of metals on the microbial cell wall (Wang et al. 2007). However, the pH inhibition level also depended on the type of each metal. In studies performed with activated sludge, it was reported that the main binding sites of the metals on the sludge sites were amino acids on the cell wall. Some metals, such as Cu and Pb, exhibit a strong affinity for anaerobic sludge sites (Artola et al. 1997), while other metals, such as Zn and Ni, show a relatively low affinity (Leighton and Forster 1997; Artola et al. 2000).
The above results showed that the addition of heavy metals during syngas fermentation improved hydrogen production. In addition, the appropriate combination of metal concentrations and initial pH of the liquid medium increased the efficacy of the process. CONCLUSIONS 1. The addition of Cu, Zn, and Mn improved hydrogen production during syngas
fermentation.
2. The highest hydrogen production activity achieved was 155% ± 12% at a metal concentration of 0.04 mg Cu/L, 0.25 mg Zn/L, and 1.06 mg Mn/L and a pH of 6.
3. At a higher metal concentration of 0.625 mg Cu/L, 3.75 mg Zn/L, and 17.5 mg Mn/L, pH 5 was optimum.
4. The uptake of Zn and Mn decreased at higher metal doses and lower pHs. ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the Swedish Research Council.
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Article submitted: January 9, 2018; Peer review completed: April 9, 2018; Revised version received: April 19, 2018; Accepted: April 25, 2018; Published: April 30, 2018. DOI: 10.15376/biores.13.2.4455-4469
77
Paper IV
Protective effect of RMBR against syngas impurities
1
Protective Effect of a Reverse Membrane Bioreactor against
Toluene and Naphthalene in Anaerobic Digestion
Konstantinos Chandoliasa, Laurenz Alan Ricardo Sugiantob, Nurina Izazib, Ria Millatib*,
Rachma Wikandarib, Päivi Ylitervoa, Claes Niklassonc, Mohammad Taherzadeha
a Swedish Center for Resource Recovery, University of Borås, 50190 Borås, Sweden,
bDepartment of Food and Agricultural Product Technology, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia,
cDepartment of Chemistry and Chemical Engineering, Chalmers University of Technology,
41296 Gothenburg, Sweden
*corresponding author: [email protected]
2
Abstract
A combined route for biogas production is thermochemical conversion of wastes to syngas
(H2, CO and CO2) followed by their biological conversion into CH4 during anaerobic
digestion. However, raw syngas contains tar contaminants that can be toxic for the cells.
This work investigated the effect of two common tar contaminants, toluene and
naphthalene, at a concentration range of 0 6.44 g/L and 0 1.28 g/L, respectively, on
methane production, with a mixed culture. Free cell bioreactors and reverse membrane
bioreactors were operated in parallel, during batch and continuous thermophilic anaerobic
digestion. The results from the continuous digestion showed inhibition at a toluene and
naphthalene concentration of 3.14 g/L and 0.63 g/L, respectively in the free cell
bioreactors. At a toluene and naphthalene concentration of 4.80 g/L and 0.96 g/L, the CH4
production rate in membrane bioreactors was approximately 47% and 160% higher than in
the free cell bioreactors.
Keywords: Anaerobic Digestion; Syngas Contaminants; Reverse Membrane Bioreactor;
Naphthalene; Toluene; Protective Effect.
3
1. Introduction
Biofuel production is one of the main strategies of international policies, such as
Agenda 21 and Kyoto Protocol, in order to meet the increasing energy demand, the waste
generation and the environmental pollution caused by the use of fossil fuels (Weiland,
2010). Biogas, containing mainly CH4 and CO2, is produced during anaerobic digestion of
organic material and it is considered as a sustainable alternative to fossil fuels. The use of
upgraded biogas (>90% methane), in transportation sector can cause a significant
reduction in greenhouse gas emissions, in comparison to gasoline (Sahota et al., 2018).
Biogas production creates also decentralized energy production sites. This valuable gas
product is used for heat and electricity production, as vehicle fuel, cooking or it can be
injected to the natural gas grid (Pöschl et al., 2010). However, there are limitations to the
type of substrate that the anaerobic microorganisms can degrade.
Poorly degradable and non-degradable substrates are recalcitrant and require a
pretreatment prior to their anaerobic digestion. One such pretreatment is gasification, a
thermochemical process in which the feedstock is thermally degraded at high temperature
and at the presence of a controlled amount of an oxidizing agent, such as O2, air or steam.
In comparison to other pretreatment processes, such as enzymatic and acid hydrolysis,
gasification gathers advantages, such as feedstock flexibility, high conversion rates and
rapid mass and volume waste reduction (Consonni et al., 2005; Gershman, 2013). The
product of gasification of carbonaceous feedstock is syngas, a mixture of mainly CO, H2
and CO2. This versatile gas has numerous applications, such as electricity generation and
production of methanol, ammonia, mixed alcohols, acids, waxes, olefins, and biofuels
(Spath & Dayton, 2003).
4
The Fischer-Tropsch process is widely used in industrial application for syngas
conversion into biofuels, via the use of metal catalysts. Prior to this process, syngas has to
be upgraded, by removing contaminants. Typical syngas contaminants are particulate
matters, tars, NOx, S, N2, and alkali (Woolcock & Brown, 2013). Tars are condensable
organic compounds, which are mainly formed due to the operating parameters of
gasification. The type of tar contaminants varies from primary to heavier oxygenated
hydrocarbons and polycyclic aromatic hydrocarbons (Stevens, 2001). Several syngas-
cleaning methods are available, such as hot gas clean-up (HGC), inertial separation
(cyclons) and barrier filtration (Woolcock & Brown, 2013), however, these methods have
a relatively high cost (Patinvoh et al., 2017).
During the past years, the biological conversion of syngas into methane and value-
added chemicals via anaerobic fermentation has gained increasing interest in scientific,
social, and industrial fields (Bengelsdorf et al., 2018). In comparison to catalytic syngas
conversion, such as Fischer-Tropsch process, syngas fermentation is more product
specific, less sensitive to CO/H2/CO ratio, generates no hazardous components and does
not require high temperature and pressure (Drzyzga et al., 2015). Furthermore, syngas can
be co-fermented with other carbonaceous substrates, such as food waste, in order to
increase the fermentation efficiency. In a study of syngas fermentation, the addition of
synthetic organic waste containing acetic, butyric and propionic acid, caused an increase
in CH4 production (Youngsukkasem et al., 2015).
One of the main challenges in syngas biomethanation is the low gas-to-liquid mass
transfer, which is greatly affected by the low gas hold up in the bioreactors. A recent study
5
reported higher syngas conversion rates in a reverse membrane bioreactor (RMBR) due to
a higher gas hold up (Chandolias et al., 2019). This type of membrane bioreactor contains
anaerobic cells enclosed inside polyvinylidene difluoride (PVDF) membrane sachets that
float inside the medium of the bioreactor. The same type of bioreactor was used in order to
protect the anaerobic cells against fruit flavours, such as D-limonene in another study
(Wikandari et al., 2014). The hydrophilic surface of the membranes blocked successfully
the hydrophobic inhibitor, while volatile fatty acids were able to completely permeate the
membranes after 30 min. The RMBR has also been used in order to avoid the cell washout
and achieve high cell-density during continuous syngas fermentation (Westman
Youngsukkasem et al., 2016).
The RMBR could also be used in order to protect the anaerobic cells from the
contaminants present in the raw syngas. This would eliminate the costly requirement for
syngas cleaning prior to syngas fermentation. Syngas contaminants can inhibit the
microbial performance or change other parameters such as pH and redox potential
(Grimalt-Alemany et al., 2017). Toluene and naphthalene are two common chemical
compounds present in tars that can be found in syngas (Lopes et al., 2018). These
compounds can be used as a microbial substrate at low amounts (Maillacheruvu & Pathan,
2009), however, high concentrations could be toxic for the cells (Edwards & Grbić-Galić,
1994; Foght, 2008; Marozava et al., 2018). Although studies have shown that low
amounts of syngas contaminants do not affect the anaerobic syngas conversion, there is no
sufficient knowledge on the minimum gas-cleaning requirements before biomethanation.
Moreover, different anaerobic microorganisms have different tolerance towards syngas
contaminants. Therefore, further investigation is required in order to study the inhibitory
6
effect of syngas contaminants in the conversion of syngas into biogas (Grimalt-Alemany
et al., 2017). The most sensitive anaerobic microbes are the methanogens and therefore a
strong and robust methanogen community is required. This can be achieved by feeding the
cells with adequate carbon and energy source, such as a synthetic organic medium
(Youngsukkasem et al., 2015).
In this work, the effect of toluene and naphthalene was investigated during anaerobic
fermentation of synthetic organic medium by a mixed culture, at thermophilic conditions.
In addition, a RMBR was employed in order to study a possible protective effect of the
hydrophilic membranes against high concentrations of the hydrophobic toluene and
naphthalene. The experiments were operated in both batch and continuous conditions and
the RMBRs were operated in parallel with free cell bioreactors (FCBR).
2. Materials and Methods
2.1 Inoculum and membrane-encasement
The inoculum used was a mixed culture collected from a recirculating stream of a local
3000-m3 anaerobic digester (Borås Energy and Environment AB, Borås, Sweden), which
operated at thermophilic conditions and was fed with food residues. Prior to the
experiment, the culture was incubated for 3 days at 55 °C so that all nutrients would be
consumed. After incubation, large particles and other debris were trapped by a 1-mm sieve
and the excess water was removed by centrifugation at 9000×g for 3 min (Hereaus
Megafuge 8, Thermo Scientific, Germany). The inoculum used had a total solid (TS) and
volatile solid (VS) content of 71.16% and 18.63%, respectively. The inoculum was placed
inside the reactors either as free (suspended) cells or as membrane-encased cells.
7
The membrane used was a hydrophilic polyvinylidene fluoride flat sheet membrane
with a pore size of 0.1μm (Durapore, Thermo Fisher Scientific Inc., Sweden). The cell-
encasement process has been described in detail in a previous study (Youngsukkasem et
al., 2012).
2.2 Liquid medium
The liquid medium used in this work was a mixture stock solution. The stock solution
contained basal medium (macronutrients and micronutrients) and organic acids (Osuna et
al., 2003). Toluene and naphthalene dissolved in methanol were mixed with the stock
solution before the feeding of the bioreactors. Methanol was selected as a solvent because
at low concentrations, it has no inhibitory effect for the cells and it is a good solvent of
toluene and naphthalene, which are not easily dissolved in water solutions (Dickhut et al.,
1989; Du et al., 2016). In the stock solution, the concentration of the macronutrients was
28 mg/L NH4Cl, 33 mg/L K2HPO43H2O, 10 mg/L MgSO47H2O, and 1 mg/L
CaCl22H2O. The concentration of the micronutrients was 2 mg/L FeCl24H2O, 0.5 mg/L
H3BO3, 0.5 mg/L ZnCl2, 5 mg/L MnCl24H2O, 0.038 mg/L CuCl22H2O, 0.05 mg/L
(NH4)6Mo7O244H2O, 2 mg/L CoCl26H2O, 0.142 mg/L NiCl26H2O, and 0.164 mg/L
Na2SeO35H2O. The concentration of the acids was 10 g/L acetic acid, 3.33 g/L propionic
acid and 3.33 g/L butyric acid. The toluene and naphthalene solutions had a concentration
of 234.4 g/L and 46.88 g/L, respectively. Different volumes of these solutions were added
into the bioreactors in order to achieve toluene and naphthalene concentrations of 0 6.44
g/L and 0 2.46 g/L, respectively. The methanol concentration in the feed was ≤ 0.034%.
The pH of the medium fed to the bioreactors was adjusted to 7.0 ± 0.2 with 5M NaOH.
8
2.3 Reactor characteristics, seeding and experimental setup
The batch bioreactors were serum glass bottles (Bioprocess control AB, Lund, Sweden)
with a total volume of 118 mL. Each bioreactor was loaded with 23.34 mL liquid medium
and seeded with 3 g of suspended cells, in FCBRs or a membrane sachet containing 3 g or
encased cells, in RMBRs. The batch digestion was run in duplicates and the batch
bioreactors were continuously shaken in a water batch (55 °C), at an inclination of
approximately 45 ° and a shaking frequency of 100 rpm.
The continuous bioreactors were glass bottles (Bioprocess control AB, Lund, Sweden)
with a total volume of 600 mL. Each bioreactor was loaded with 350 mL liquid medium
and seeded with 45 g of suspended cells, in FCBRs, or equal amount of encased cells in
RMBRs (3 g/membrane). The continuous bioreactors were placed in a shaking water bath,
at 55 °C and 45 rpm, without inclination.
After seeding, the bioreactors were sealed with butyl caps and then purged with N2, in
order to remove oxygen from the bioreactor´s headspace. The ratio of mass of inoculum to
volume of liquid substrate was approximately 0.13 g/mL in both batch and continuous
bioreactors. This ratio has an direct effect on the product yields as it has been reported in a
previous study (Chandolias et al., 2019).
2.4 Analytical methods
During the experiment, the composition of the gas headspace of the bioreactors and the
liquid medium were regularly analysed, in order to ensure the good operation of the
bioreactors. In the continuous experiments, the outlet of the bioreactors was connected to
an Automatic Methane Potential Test System (AMPTS II, Bioprocess control AB, Lund,
Sweden), which measured the volume of the daily gas production. Gas samples were
9
collected from the headspace of the bioreactors and analysed with gas chromatography
(GC). The gas samples (100 μL) were collected with a 0.25-mL gas-tight syringe (VICI,
Precision Sampling Inc., Baton Rouge, LA, USA) and injected into a Varian 450 Gas
Chromatograph (Palo Alto, CA, USA). The GC was equipped with a wall coated open
tubular (WCOT) capillary column (WCOT, J&W Scientific GC-Gas Pro, bonded silica
based 30 m × 0.32 mm, Agilent Technologies, Santa Clara, CA, USA) and a thermal
conductivity detector (TCD, Varian, Palo Alto, CA, USA) and Galaxie Chromatography
Data System Single Instrument (v.1.9, Varian, Walnut Creek, CA, USA) as the analysis
software. The GC was connected to a computer using Galaxie Chromatography Data
System v.1.9 Single Instrument as the data recording software. N2 was used as carrier gas
with a flow rate of 2.0 mL/min, through the column and 30 mL/min, through the detector.
The temperatures of injector, oven, detector, heater, and detector filament were set to 75
°C, 60 °C, 120 °C, and 200 °C, respectively, and the injection split ratio was set to 5.
The amount of toluene and naphthalene in the bioreactors was measured with a gas
chromatograph with mass spectroscopy detector, GC-MS (GC-MS Trace GC Ultra,
Themoscientific, Waltham, MA, USA) equipped with a capillary column (Zebron ZB-5ms
fused silica, 30 m×0.25 mm×0.25 μm, Torrance, CA, USA). The injection had a split of
50:1, temperature of 230 °C and 1 μL of sample. Helium was used as carrier gas at a
constant flow of 0.7 mL/min and the oven was set to 40 °C for 2 min and 210 °C for 3 min
with a ramp of 17 °C/min, while the MS detector was set to 250 °C. The preparation of the
liquid samples was done with and without solvent extraction. For the solvent extraction, 1
mL of sample was mixed with 1 mL of diethyl ether and then shaken overnight, at room
temperature. Sonication of 5 min was used in order to disrupt the cells.
10
3. Results and Discussion
Syngas components are good substrate for CH4 production during anaerobic
fermentation. However, syngas contaminants may have an inhibitory effect on anaerobic
microorganisms. Thus, cleaning of raw syngas prior to syngas fermentation is required.
However, the cost of this gas-cleaning is significant in comparison to the overall operating
cost of the process. In addition, few information is available on the inhibitory effect of
various syngas contaminants.
This work investigated the effect of two common tar contaminants in raw syngas,
toluene and naphthalene, during the anaerobic digestion of synthetic organic substrate and
the cell-encasement into membrane sachets inside a RMBR as a protective mean. Each
RMBR was operated in parallel with a FCBR, containing free/suspended cells. The
experiments took place in both batch and continuous conditions while the accumulative
CH4 production, CH4 production rate, and CH4 production activity were used as indicators
of the bioreactors` efficacy. The results showed good tolerance of the cells against high
concentrations of toluene and naphthalene compared to previous studies and improvement
of the CH4 production rate in RMBRs, in which toluene and naphthalene were added.
3.1 Investigation of toluene and naphthalene effect in batch anaerobic digestion
A batch anaerobic digestion took place for 34 days and duplicate bioreactors were
operated with a synthetic organic medium with a COD content of 42 g COD/L. Prior to
the experiment, the mixed culture was acclimatized in synthetic organic medium for 35
days. The range of toluene and naphthalene concentrations investigated was 0 1.0 g/L and
0 0.2 g/L, respectively. These concentrations were higher than other inhibitory
concentrations found in the literature (Akyol et al., 2015; Dou et al., 2009). Toluene and
11
naphthalene are aromatic hydrocarbons, labelled as highly toxic to living organisms.
Toluene consists of one benzene ring and one methyl group, while naphthalene consists of
two benzene rings. The presence of toluene and naphthalene in anaerobic environments
can inhibit the cellular metabolism. Naphthalene can disrupt the membrane of anaerobic
cells and produce toxic metabolites. More specifically, the lipophilic nature of naphthalene
can alter the fluidity of the cell membrane, cause swelling of the lipid bilayer and affect
the intracellular energy transduction. The produced toxic metabolites can be more harmful
to the cells because of the disrupted cellular membrane, the production of reactive oxygen,
and the disturbance in DNA and protein formation (Pumphrey & Madsen, 2007). The
tolerance of anaerobic microorganisms to these compounds depends mainly on the type of
culture, compound concentrations, and type of substrate (Akyol et al., 2015; Dou et al.,
2009; Govind et al., 1991; Shimp & Pfaender, 1985). Studies in anaerobic digestion have
shown inhibitory effect of toluene and naphthalene at concentrations above 0.09 g/L and
0.01 g/L, respectively (Edwards & Grbić-Galić, 1994; Ince et al., 2009; Pumphrey &
Madsen, 2007).
Fig. 1 presents the total amount of CH4 obtained in bioreactors fed with 0.5 g/L and 1.0
g/L of toluene and 0.1 g/L and 0.2 g/L of naphthalene. The results showed no inhibitory
effect of toluene and naphthalene at the investigated concentrations in FCBRs. On the
contrary, CH4 production was increased at higher contaminant concentrations. More
specifically, the increase in CH4 production at a high toluene concentration of 1 g/L,
suggests that the cells were able to utilize this compound as a substrate (Fig 1A). Another
study investigated the effect of toluene on acetyl-CoA synthetase expression level of
acetoclastic methanogen, Methanosaeta concilii. The results showed complete inhibition
12
after the 3rd exposure at toluene concentrations higher than 0.046 g/L, or 0.023 g toluene
per g VS inoculum (Akyol et al., 2015). In the current work, at toluene concentrations of
up to 1.0 g/L or at a ratio of 0.06 g toluene per g VS inoculum, no inhibition effect was
observed, however, the cells were not exposed to toluene multiple times.
Fig. 1B showed also a tendency for higher CH4 production at a high concentration of
0.2 g/L naphthalene or 0.012 g naphthalene per g VS inoculum. Another study on the
anaerobic degradation of naphthalene by mixed bacteria under nitrate reducing conditions,
showed also no inhibition and complete degradation of naphthalene within a period of 25
days incubation when the initial concentration was below 0.030 g/L (Dou et al., 2009).
Moreover, higher degradation rates of naphthalene were recorded in bioreactors with
higher initial concentrations of naphthalene (Dou et al., 2009). However, another work,
reported inhibition at lower naphthalene concentration. More specifically, a study on the
naphthalene metabolism and growth inhibition by naphthalene in Polaromonas
naphthalenivorans strain CJ2 showed inhibition for naphthalene concentrations above
0.01 g/L (Pumphrey & Madsen, 2007). In addition, naphthalene has been reportedly toxic
to the archetypal naphthalene degraders P. putida G7 and P. putida NCIB 9816-4 under
nitrogen or oxygen-limiting conditions (Ahn et al., 1998) or at high concentration of 2 g/L
of naphthalene crystals (Park et al., 2004).
The results shown in Fig. 1 did not indicate any inhibitory effect while CH4 production
was higher at higher contaminant concentration. The yields obtained were approximately
40 50% of the theoretical yield. The use of mixed consortia and the incubation with
organic acids prior to the experiment possibly made the mixed culture more tolerant to
toluene and naphthalene in comparison to other works. Previous studies have reported a
13
relation between the degradation of easily degradable substrates such as organic acids and
the effect of phenolic compounds during anaerobic digestion. For example, the cellular
adaptation to increasing concentrations of amino acids, carbohydrates, or fatty acids
improved the ability of aquatic bacteria to tolerate and degrade phenols (Shimp &
Pfaender, 1985). A mixed culture can be tolerant towards inhibitors due to the diversity of
microorganisms and the domination of the strongest cells according to the type of
substrate and growth conditions. Moreover, the tolerance at high concentrations of toluene
and naphthalene in this work could be a result of the relatively high amount of g VS
inoculum seeded in the bioreactors.
3.2 Effect of toluene and naphthalene in continuous anaerobic digestion
The results from batch experiment suggested that in order to observe inhibition in CH4
production, higher toluene and naphthalene concentrations should be investigated.
However, from the literature, different studies suggest different inhibiting concentrations
for these compounds depending on the type of culture, substrate, medium and other
parameters (Akyol et al., 2015; Edwards & Grbić-Galić, 1994; Ince et al., 2009).
Therefore, the contaminant concentrations were increased daily until inhibition on CH4
production was observed. A synthetic organic medium was used as a carbon and energy
source throughout the experiment; with an OLR of 1.24 g COD/Ld and a hydraulic
retention time of 17.5 days. During the feeding of FCBRs, cell washout was avoided by
centrifugation of the digestate and recycling of the pelleted cells into the bioreactors. Until
day-35, only synthetic organic medium was fed into the bioreactors in order to create a
strong and robust anaerobic culture. From day-35 until day-75, the concentrations of
14
contaminants in the bioreactors were increased daily until inhibition was observed. From
day-75 until day-80, no contaminants were added into the bioreactors and from day-80
until day-120 the concentration of contaminants into the bioreactors was stable. More
specifically, from day-35 until day-55, 0.035 g of toluene and 0.007 g of naphthalene were
added daily into the bioreactors. From day-55 until day-75, 0.1755 g of toluene and 0.035
g of naphthalene were fed into the bioreactors daily. Between day-75 and day-80, no
contaminants were added into the bioreactors and from day-80 until day-120, 0.096 g of
toluene and 0.019 g of naphthalene were fed into the bioreactors daily. Fig. 2-4 present the
CH4 production rate, CH4 production activity, and the theoretical concentration of
contaminants inside the bioreactors, with the assumption that they were not consumed,
during the continuous experiment. The CH4 yield obtained in this study, in the bioreactor
with 0 g/L of contaminants was close to the theoretical yield (result not shown), which
indicated favorable conditions for CH4 production, during the anaerobic digestion. The
theoretical yield in the above calculations is based on the assumption that all organic
compounds were converted into CH4.
Fig. 2 shows the effect of different toluene concentrations on CH4 production rate and
production activity. The increase in CH4 production rate in FCBR, from day-35 until day-
55, shows that the anaerobic cells were able to grow and used toluene as a substrate.
Toluene is in general the most readily degraded compound of the monoaromatic BTEX
(benzene, toluene, ethylbenzene and xylene isomers) components. In comparison to
naphthalene, toluene can be degraded by a larger variety of anaerobic bacteria, which
belong mostly either to the Azoarcus or Thauera genus (Foght, 2008; Weelink et al.,
2010). Moreover, toluene degradation takes place in the presence of various terminal
15
electron acceptors such as nitrate, iron (III), sulfate and CO2 (Edwards & Grbić-Galić,
1994). In the presence of CO2, toluene can be directly converted into CH4 while
naphthalene can be converted into acetate and H2 (Christensen et al., 2004; Edwards &
Grbić-Galić, 1994). A study on anaerobic degradation by methanogenic consortium
showed that, after a long adaptation period of 100 days, the cultures were able to degrade
toluene but not naphthalene and that toluene degradation was inhibited by the presence of
acetate, propionate and methanol (Edwards & Grbić-Galić, 1994). However, in this
current work, the addition of toluene had a positive effect on the CH4 production rate.
Another study, on the acetyl-coA synthetase expression level of acetoclastic methanogen
Methanosaeta concilii showed positive effect during the co-digestion of toluene and
methanol. More specifically, the co-digestion of methanol and toluene did not cause
combined inhibition, however, singular toluene-exposed cells were inhibited (Akyol et al.,
2015). Therefore, the small amount of methanol used for dissolving naphthalene and
toluene in this work could even have had a positive effect on the degradation of these
compounds.
The CH4 production rate started to decrease on day-57 at a toluene concentration of
2.05 g/L (Fig. 2A) in the FCBR, while toluene inhibition was observed on day-60 at a
toluene concentration of 3.14 g/L when the CH4 production activity was lower than 100%
(Fig. 2B). The FCBR system showed signs of recovery during the no-feeding period and
during the last period with a stable toluene concentration of 4.8 g/L. The average CH4
production activity, in the last period, was approximately 100%. The inhibiting toluene
concentration obtained in the current work is quite high in comparison to the reports from
other similar studies. For example, during an anaerobic digestion with aquifer-derived
16
mixed consortium, the observed inhibiting toluene concentration was higher than 0.092
g/L, after 100 120 days of adaptation (Edwards & Grbić-Galić, 1994). Another study with
anaerobic sludge in an up flow anaerobic sludge blanket bioreactor (UASB) reported a
62% inhibition on potential methane production (PMP) at a toluene concentration of 0.091
g/L (Ince et al., 2009). The system recovery phenomenon has been observed in other
studies with toluene and other aromatic hydrocarbons as substrates. Warthmann et al.
(2004) observed that xylene degradation could be inhibited by adding toluene to an
anaerobic sludge digester and it could be recovered again when toluene was not added.
Moreover, Datar et al. (2004) observed also that cells remained in the dormant state after
exposure to syngas contaminants and cells would start growing again after the
contaminant feeding was omitted.
The use of RMBRs could have a protective effect against syngas contaminants. RMBRs
have been used in previous studies in order to improve the gas hold up and consequently
the gas-to-liquid mass transfer in syngas fermentation (Chandolias et al., 2019) and as
protective barriers against fruit flavors during anaerobic digestion (Wikandari et al.,
2014). The membrane blocked the hydrophobic inhibitors because of its hydrophilic
surface while all substrate and products were free to pass through its pores. The RMBR
could also block the passage of hydrophobic inhibitors, such as tars, NOx, and particles,
which are present in the raw syngas after gasification.
In order to improve the cellular tolerance to substrates containing high concentrations of
toluene, a RMBR was operated in parallel with the FCBR. The increase in CH4 production
rate (RMBR) after toluene feeding on day-36 indicates that an amount of toluene could
pass through the membrane pores and that it was used as substrate by the cells. This result
17
agrees with the results from batch anaerobic digestion in the previous section. In contrast
to the FCBR, the CH4 production rate in RMBR increased from day-36 until day-67. This
proves that the membranes protected the cells from high toluene concentration while at the
same time; a fraction of toluene could enter the membranes and was converted to CH4. On
the other side, the free cells were inhibited due to direct exposure to the extreme toluene
concentrations. The stimulation of CH4 production in RMBR can be also verified by its
CH4 production activity, which was above 100%, during these days. After day-68, at a
toluene concentration of 5.25 g/L, the CH4 production rate decreased although the CH4
production activity remained above 100%. This indicates that the concentration of toluene
in the medium was too high even for the encased cells. The lowest CH4 production
activity observed in the RMBR was approximately 106%, at a toluene concentration of 5.4
g/L, on day-78, while the highest CH4 production activity was approximately 144% and it
was achieved at a stable toluene concentration of 4.8 g/L. This value was significantly
higher compared to the CH4 production activity in the FCBR of approximately 99% at the
same toluene concentration.
Naphthalene is considered as a more toxic component to the cells in comparison to
toluene, mainly due to the toxic metabolites that are generated during its degradation. In
comparison to toluene degrading microorganisms, fewer cells can degrade naphthalene. In
addition, the cells can use fewer electron acceptors in the presence of naphthalene
compared to toluene metabolism while both toluene and naphthalene degradation can
occur with nitrate as electron acceptor (Cunningham et al., 2001; Edwards & Grbic-Galic,
1992; Heider et al., 1998; Rockne & Strand, 2001). In a study, where an anaerobic culture
was enriched with 1-methylnaphthalene as a sole carbon source and Fe(OH)3 as electron
18
acceptor, the gram-positive Thermoanaerobacteraceae could degrade polycyclic aromatic
hydrocarbons while the Desulfobacterales were mainly responsible for Fe(III) reduction
(Marozava et al., 2018). Because of the high toxicity of naphthalene, lower amount of this
inhibitor was added into the bioreactors in comparison to toluene.
Fig. 3 shows the effect of different naphthalene concentrations on the CH4 production
rate and production activity. The CH4 production rate in FCBR increased until day-57. On
day-60, inhibition was observed at a naphthalene concentration of 0.63 g/L. The methane
production increased when naphthalene feeding was paused between day-76 and day-80.
However, the CH4 production activity dropped from approximately 114% on day-80 to
41% on day-120, which indicates inhibition at a stable naphthalene concentration of 0.96
g/L. In another study on naphthalene inhibition during anaerobic digestion, the inhibitory
naphthalene concentration was approximately 0.01 g/L (Pumphrey & Madsen, 2007),
which is approximately 100-fold lower compared to the current results in this work. The
CH4 production in RMBR followed a similar trend with the FCBR until day-80. Between
day-80 until day-120, the highest CH4 production rate and CH4 production activity of
approximately 56.7 mmol/(Ld) and 175%, respectively, were achieved in the RMBR. In
addition, the CH4 production rate was rather stable during this period. This was probably a
result of cell adaptation at high naphthalene concentrations and proved the protective
effect of the cell encasement towards high naphthalene concentrations.
The effect of simultaneous toluene and naphthalene addition was examined and the
results are shown in Fig. 4. In the FCBR, inhibition occurred on day-60, at a toluene and
naphthalene concentration of 3.14 g/L and 0.63 g/L, respectively. After a short increase in
19
CH4 production rate between day-75 and day-80, the CH4 production activity dropped
from approximately 141% (day-80) to 28% (day-120), indicating inhibition. Interestingly
enough, no inhibition was observed in the RMBR at the highest toluene and naphthalene
concentrations of 6.44 g/L and 1.26 g/L, respectively. However, similar results were
obtained in both FCBR and RMBR between day-80 and day-120 due to severe cell
leaking from the membrane sachets in RMBR. From the comparison of Fig. 2 4, between
day-55 and day-75, it can be concluded that the presence of toluene had a positive effect
on anaerobic digestion with naphthalene also present in RMBRs. This was probably a
result of the greater diversity of microorganisms that thrived in the bioreactors containing
both toluene and naphthalene and created a more robust culture (Liang & Ramesh, 2012).
Some amount of the contaminants could have been accumulated inside the cells. This
explanation is verified by the result of liquid analysis that showed low amounts of
contaminants detected after cell disruption. The complete degradation of toluene and
naphthalene is considered not possible because of the high contaminant concentration.
Furthermore, the yields were low, approximately 40% of the theoretical yield in the
RMBR fed with naphthalene and less than 20% of the theoretical yield in all other
bioreactors fed with contaminants (after day-80). The inhibiting effect of the contaminants
was also depicted on the consumption of the organic medium since there was higher
accumulation of organic acids in the FCBRs especially from day-80 until day-120 in
comparison to the RMBRs (results not shown).
4. Conclusions
Batch and continuous anaerobic digestion took place in this study, for the investigation
of CH4 production in the presence of two syngas contaminants, toluene and naphthalene.
20
The results showed no inhibition on CH4 production at low concentrations of toluene and
naphthalene. However, at higher concentrations of these contaminants, the CH4 production
was inhibited. In addition, significantly higher CH4 production rate and stimulation were
observed in RMBRs even at high concentrations of contaminants, in comparison to the
FCBRs.
Acknowledgements
The authors would like to express sincere gratitude to the Swedish Research Council and
Swedish Council for Higher Education for the financial support.
The authors declare no conflict of interest.
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38. Westman Youngsukkasem, S., Chandolias, K., Taherzadeh, J.M. 2016. Syngas Biomethanation in a Semi-Continuous Reverse Membrane Bioreactor (RMBR). Fermentation, 2(8), 1-12, doi:10.3390/fermentation2020008.
39. Wikandari, R., Youngsukkasem, S., Millati, R., Taherzadeh, M.J. 2014. Performance of semi-continuous membrane bioreactor in biogas production from toxic feedstock containing D-Limonene. Bioresour Technol, 170, 350-5, doi:10.1016/j.biortech.2014.07.102.
40. Woolcock, P.J., Brown, R.C. 2013. A review of cleaning technologies for biomass-derived syngas. Biomass Bioenerg, 52, 54-84, doi:10.1016/j.biombioe.2013.02.036.
41. Youngsukkasem, S., Chandolias, K., Taherzadeh, M., J. 2015. Rapid bio-methanation of syngas in a reverse membrane bioreactor: Membrane encased microorganisms. Bioresour Technol, 178, 334-340, doi:10.1016/j.biortech.2014.07.071.
42. Youngsukkasem, S., Rakshit, S.K., Taherzadeh, M.J. 2012. Biogas production by encapsulated methane-producing bacteria. Bioresources, 7(1), 56-65.
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Figure captions
Fig. 1. Accumulative CH4 production, mmol; at (A) toluene concentrations of 0.5 g/L and 1.0 g/L and at (B) naphthalene concentrations of 0.1 g/L and 0.2 g/L; in RMBR and FCBR, during batch anaerobic digestion
Fig. 2 Effect of toluene on the (A) CH4 production rate, mmol CH4/(Ld); (B) CH4 production activity, (mol/molcontrol)100%; and (C) the theoretical toluene concentration, g/L; in FCBR and RMBR, during continuous experiment
Fig. 3 Effect of naphthalene on the (A) CH4 production rate, mmol CH4/(Ld); (B) CH4 production activity, (mol/molcontrol)100%; and (C) the theoretical naphthalene concentration, g/L; in FCBR and RMBR, during continuous anaerobic digestion
Fig. 4 Effect of toluene and naphthalene on the (A) CH4 production rate, mmol CH4/(Ld); (B) CH4 production activity, (mol/molcontrol)100%; and (C) the theoretical toluene and naphthalene concentration, g/L; in FCBR and RMBR, during continuous anaerobic digestion
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Paper V
Floating Membrane Bioreactors with High Gas Hold-Up for Syngas-to-Biomethane
Conversion
energies
Article
Floating Membrane Bioreactors with High GasHold-Up for Syngas-to-Biomethane Conversion
Konstantinos Chandolias 1,* , Enise Pekgenc 2 and Mohammad J. Taherzadeh 1
1 Swedish Centre for Resource Recovery (SCRR), Faculty of Textiles engineering and Business, University ofBorås, Allégatan 1, 501 90 Borås, Sweden; [email protected]
2 Department of Environmental Engineering, Istanbul Technical University, Maslak,34467 Sarıyer/Istanbul, Turkey; [email protected]
* Correspondence: [email protected]
Received: 20 February 2019; Accepted: 14 March 2019; Published: 18 March 2019����������������
Abstract: The low gas-to-liquid mass transfer rate is one of the main challenges in syngasbiomethanation. In this work, a new concept of the floating membrane system with high gas hold-upwas introduced in order to enhance the mass transfer rate of the process. In addition, the effectof the inoculum-to-syngas ratio was investigated. The experiments were conducted at 55 ◦C withan anaerobic mixed culture in both batch and continuous modes. According to the results fromthe continuous experiments, the H2 and CO conversion rates in the floating membrane bioreactorwere approximately 38% and 28% higher in comparison to the free (suspended) cell bioreactors.The doubling of the thickness of the membrane bed resulted in an increase of the conversion rates ofH2 and CO by approximately 6% and 12%, respectively. The highest H2 and CO consumption ratesand CH4 production rate recorded were approximately 22 mmol/(L·d), 50 mmol/(L·d), and 34.41mmol/(L·d), respectively, obtained at the highest inoculum-to-syngas ratio of 0.2 g/mL. To conclude,the use of the floating membrane system enhanced the syngas biomethanation rates, while a thickermembrane bed resulted in even higher syngas conversion rates. Moreover, the increase of theinoculum-to-syngas ratio of up to 0.2 g/mL favored the syngas conversion.
Keywords: floating MBR; syngas-to-biomethane conversion; high gas hold-up; inoculum-to-syngas ratio
1. Introduction
Gasification is a thermochemical process which converts biomass into a gaseous mixture,called syngas (mostly CO, H2, and CO2). This gas can be employed for the production of electricity,energy, and transport fuels. Currently, approximately 50% of the generated syngas is converted intoNH3, 25% into H2, and the remaining fraction is used for the production of Fischer Tropsch fuels,methanol, and other valuable chemicals [1,2]. Syngas is also considered as a promising vector for heatand power generation [3]. Another promising application of syngas and other industrial off-gases isanaerobic fermentation and conversion into biofuels, alcohols, bioplastics, and value-added chemicals.Syngas biomethanation, in particular, is considered as a sustainable alternative to the applicationsmentioned above [4].
An important limitation of syngas fermentation is the low syngas conversion rate due to poorgas-to-liquid mass transfer rates. Different approaches, in order to improve the mass transfer,focus mainly on the bioreactor’s design and the syngas feed. More specifically, different bioreactortypes such as stirred tank, bubble, packed bed, airlift, and trickle bed bioreactors have been studied.Packed bed and membrane bioreactors allowed higher mass transfer and cell-density than thetraditional bubble column and stirred tank reactors during syngas fermentation [5]. In addition,
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Energies 2019, 12, 1046 2 of 14
the use of microbubble sparging reduced the power-to-energy demand [6]. Hollow fibre membraneswere also employed for the syngas feed and led to high mass transfer efficiency [7]. Another effect ofthe hollow fibre membranes was the creation of a biofilm on the surface of the membranes for bettergas-cell contact [8].
Another way to increase the gas-to-liquid mass transfer without using costly andenergy-demanding methods, such as agitation, is to increase the gas hold-up. Higher gas hold-upallows for better diffusion of the gas inside the bulk medium and better contact with the cells. This canbe achieved by altering the bioreactor´s design [4], such as by increasing the height and decreasingthe diameter of the bioreactor or by blocking the anodic path of the rising gas bubbles inside theliquid medium.
The main novelty in this work is in the function of polymeric membranes which were employedin order to decelerate the anodic bubble-rise velocity and thus increase the gas hold-up of syngas in afloating membrane bioreactor (floating MBR). Initially, the membranes were shaped into rectangularsachets which were seeded with anaerobic cells and then heat-sealed and placed inside the medium ofthe floating MBR. The membrane-floating effect was caused by the biogas that was produced insidethe membranes. The swollen membranes formed a packed membrane bed which floated under thesurface of the liquid medium with the help of a plastic net. In other words, the floating MBR systemis actually a reverse membrane bioreactor (reverse MBR) [9] in which the membranes form a packedfloating bed inside the medium of the reactor.
Another factor that was studied in this work and can affect the syngas conversion rate is theinoculum-to-syngas ratio (ISR). Although the effect of ISR on the methane potential of organic wastehas been investigated in other studies [10,11], there is no previous study on the effect of the ISR insyngas biomethanation. This parameter could be essential for the improvement of the process asa low ratio could lead to toxic syngas concentrations for the cells, while a high ratio could causecell-starvation. For this purpose, several ISRs were tested in both batch and continuous experiments.
The aim of this work was to investigate the effect of a MBR with a floating membrane bed on thesyngas conversion rate and the effect of increasing ISR during syngas biomethanation. According tothe authors’ best knowledge, there are no similar studies in the literature.
2. Materials and Methods
2.1. Inoculum, Syngas, and Liquid Medium
The inoculum was a mixed culture collected from a local thermophilic 3000 m3 anaerobic digesteroperating on organic fraction of food solid waste (Borås Energy and Environment AB, Borås, Sweden).Prior to the experiment, the inoculum was incubated for four days at 55 ◦C in order to consume all thenutrients. The pH of the inoculum was 8.0 and the total solid (% TS) and volatile solid (% VS) contentwas 15.01 ± 0.17% and 65.11 ± 0.44%, respectively.
The gaseous substrate was syngas, containing 20% H2, 55% CO, and 10% CO2, and was providedby AGA gas AB (Gothenburg, Sweden). The gas cylinder, containing the syngas, was pressurized withnitrogen gas at 1500 psi.
The liquid medium consisted of basal medium (micronutrients and macronutrients) and aceticacid. The basal medium recipe was obtained from the literature [12]. The macronutrients concentrationin the liquid medium was 280 mg NH4Cl/L, 330 mg K2HPO4·3H2O/L, 100 mg MgSO4·7H2O/L,and 10 mg CaCl2·2H2O/L. In addition, 2 mL of trace elements solution was added per 1 L of liquidmedium. The composition of the trace elements solution was 2000 mg FeCl2·4H2O/L, 50 mg H3BO3/L,50 mg ZnCl2/L, 500 mg MnCl2·4H2O/L, 38 mg CuCl2·2H2O/L, 50 mg (NH4)6Mo7O24·4H2O/L, 2000mg CoCl2·6H2O/L, 142 mg NiCl2·6H2O/L, and 164 mg Na2SeO3·5H2O/L.
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2.2. Bioreactor Characteristics, Seeding, and Start Up
The excess water of the inoculum was removed by centrifugation at 9000× g for 3 min (HereausMegafuge 8, Thermo Scientific, Bremen, Germany) prior to seeding. Both batch and continuousexperiments were conducted. The batch bioreactors were serum glass bottles with a rubber sealed capand a total volume of 118 mL (Bioprocess control AB, Lund, Sweden). Each bioreactor was loadedwith 40 mL of liquid medium and different amounts of inoculum in order to investigate the effect ofdifferent ISRs of 0.01, 0.04, 0.07, and 0.1 g/mL. After seeding, the bioreactors were placed inside ashaking water bath at 100 rpm, at an inclination of approximately 45◦, and a temperature of 55 ◦C.
The bioreactors that were operated in the continuous mode were glass bubble column vesselswith rubber sealing caps and a total volume of 2.2 L. These bioreactors were placed inside a stationarywater bath at 55 ◦C. Each bubble column reactor contained 1.7 L of liquid medium and was seededwith 45 g of the pelleted inoculum. The continuous experiments were conducted in order to comparethe floating MBR with other types of free cell bioreactors and the effect of thicker membrane bed andhigher ISR. More specifically, the bioreactor designs that were used were a floating MBR; a bioreactorwith both floating membrane bed and free cells (floating MBR/FCBR); a bioreactor with free cells,filled with packing material (PBR); and a free cell bioreactor (FCBR). The FCBR had an ISR of 0.1 g/mLand was operated in parallel with another free cell bioreactor (FCBR.2), which was loaded with 90 ginoculum (ISR = 0.2 g/mL). The Floating MBR contained 15 membranes filled with 3 g of inoculumeach. In the floating MBR/FCBR, a part of the inoculum (24 g) was encased inside eight membranesand another part (26 g) was suspended in the liquid medium of the reactor. The PBR and FCBRcontained free cells with the difference that the PBR was filled with 1.5 L of packed material, designedfor cell-attach growth, with a diameter of 15 mm, made from PVC (HR 15–7, Rauschert, Hannover,Germany). Finally, another bioreactor, the floating MBR.2, was loaded with 30 membrane sachets (1.5g inoculum/membrane), forming a floating membrane bed with a thickness of approximately 6 cm,while the thickness of the membrane bed in the floating MBR was approximately 3 cm.
The membranes were hydrophilic polyvinylidene difluoride (PVDF) flat sheets (Merck MilliporeLtd., Cork, Ireland) with a pore size and thickness of 0.1 μm and 125 μm, respectively, while themethod of cell-encasement was described in a previous work [13].
2.3. Gas and Liquid Feeding
Figure 1 shows a scheme of the gas and liquid feeding process. Syngas was fed in the bioreactorswith a flow meter control (11–110 mL/min, Swagelok, Sollentuna, Sweden) at a flow rate of 15 mL/min.The gaseous substrate was introduced to the bottom of the bioreactors with a sparger (Air diffuser2 × 3 cm, Zalux, Zaragoza, Spain). There was no gas-recirculation and the conversion rate of syngaswas reported after one pass through the medium broth. The gas composition was analysed at theinlet and outlet of the reactors, while the gas flow rate at the outlet was recorded by a data acquisitionsystem (AMPTS II, Bioprocess control, Sweden AB, Sweden). The feeding of the liquid medium tookplace with a plastic 50 mL syringe though a tube that was immersed inside the medium. Regularcontrols with gas and liquid sampling ensured the stable operation of the bioreactors.
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Figure 1. Schematic of gas and liquid feeding in the floating MBR (membrane bioreactor).
2.4. Analytical Methods
The gas components were detected with Gas Chromatography. The CO levels were obtained bya Gas Chromatograph (Perkin-Elmer 480, Norwalk, CT, USA) with a packed column (CarboxenTM1000, SUPELCO, 6� × 1.8” OD, 60/80 Mesh, Shelton, CT, USA) and a thermal conductivity detector(Perkin-Elmer) with an injection temperature of 200 ◦C. The H2, CO2, and CH4 levels were analysedwith a Gas Chromatograph (Perkin-Elmer 590, Norwalk, CT, USA), equipped with a packed column(CarboxenTM 1000, SUPELCO, 6� × 1.8” OD, 60/80 Mesh, Shelton, CT, USA), using a thermalconductivity detector (Perkin-Elmer, Norwalk, CT, USA) with an injection temperature of 200 ◦C.The gas sampling was performed with a 0.25 mL gas-tight syringe (VICI, Precision Sampling Inc., BatonRouge, LA, USA) and the carrier gas in the chromatographs was N2 with a flow rate of 30 mL/min at75 ◦C.
For the analysis of the liquid samples, a High-Performance Liquid Chromatography (Waters 2695,Waters Corporation, Milford, CT, USA) with a hydrogen-based column (Aminex HPX87-H, BioRadLaboratories, München, Germany), equipped with a refractive index (RI) detector (Waters 2410, WatersCorporation, Milford, CT, USA), was operated at 60 ◦C and 0.6 mL/min (5 mM H2SO4 eluent).
The gas analysis of the continuous experiment took place every five days starting from the 50thday and ending on the 90th day of fermentation, while the batch experiment was run in triplicatesfor a period of seven days and gas samples were analysed every 24 h. The results are presented asaverage value ± standard deviation.
3. Results and Discussion
A main challenge during syngas fermentation is the low gas-to-liquid mass transfer, which can beimproved by increasing the gas hold-up inside the liquid medium of bioreactors. For this purpose,anaerobic cells were encased in membrane sachets which were then heat-sealed and immersed insidea bubble column bioreactor. During the fermentation process, syngas was introduced to the bottomof the bioreactor with a sparger and the bubble ascent of syngas components was delayed because
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of the membrane bed, which operated as a mechanical barrier. This delay increased the gas hold-upin the liquid phase. Consequently, the syngas components diffused in the liquid phase and throughthe 0.1 μm membrane pores. The total interfacial area for gas mass transfer (α) increases with highergas hold-up and smaller bubble size. Thus, higher mass transfer rates are achieved in systems withhigher gas hold-up and smaller bubble size [14]. In addition, the position of the membrane bed underthe liquid surface and the gas passing through the membrane pores and into the cell-contained areafavoured the gas-to-cell contact without agitation.
The membranes were hydrophilic and, therefore, accessible to dissolved syngas componentsand organic acids in the liquid phase. These components diffused through the membrane pores andthereafter were converted into biogas. The produced biogas built a pressure inside the membraneswhich increased until it reached the membrane bubble point. Then, biogas exited the inner membranearea by blowing the membrane pores dry. Consequently, the inner pressure of the membrane droppedagain, and fresh liquid diffused through the membrane. The fact that the membrane sachets werecontinuously swollen led also to the conclusion that there was probably minimum gas exchangefrom the outside to the inside of the sachets. The membranes are accessible to gas when they aredry, however once they are wet, the differential pressure has to be greater than the bubble point for agas exchange to occur. This means that the membranes worked as conductors of the diffused liquidphase and the produced gas phase. From previous studies, it was observed that the floating effect wasdirectly stirred by the organic loading rate (OLR) of the bioreactors [15] because the biogas productionrate inside the membranes had to be high in order to create the floating effect. The experiments inthis work showed that a minimum OLR of approximately 1 g COD/(L·d) was required in order toinitiate the membrane floating phenomenon. The undissolved syngas bubbles ascended between themembrane sachets to the surface of the liquid.
Figure 2 illustrates the different bioreactor designs that were investigated. The floating MBRwas operated in parallel with a free cell bioreactor (FCBR) containing free suspended cells, a packedbioreactor (PBR) containing free cells growing on packed material, and a hybrid bioreactor (floatingMBR/FCBR) containing both membrane encased cells and free cells. These bioreactors are describedin more detail in the Materials and Methods section. The effect of a thicker membrane bed was alsoinvestigated as well as the effect of a higher inoculum-to-syngas ratio (ISR). The H2 and CO conversionrates and the CO2 and CH4 production rates in mmol/(L·d) were used as indicators of the reactors’efficacy. The results showed that the use of the membrane bed resulted in higher syngas conversionrates. The increase of the thickness of the membrane bed led to higher conversion rates. In addition,higher ISRs increased the conversion rates in both batch and continuous experiments.
During the starting up period (first 30 days) of the continuous experiment, the bioreactors werefed with basal medium which contained micro- and macro-nutrients and syngas as the sole carbonand energy source. From the 31st day and until the 90th day, acetic acid was also introduced into theliquid medium with an OLR of 1.21 g COD/(L·d). The HRT was 34 d during the experiment.
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Figure 2. Syngas biomethanation in bioreactors containing (a) membrane-encased cells (floating MBR),membrane-encased and free cells (floating MBR/FCBR), free cells growing on packed material (PBR),and free (suspended) cells (FCBR); (b) membrane bed thickness of 3 cm (floating MBR) and 6 cm(floating MBR.2), and (c) inoculum to syngas ratio (ISR) of 0.1 g/mL (FCBR) and 0.2 g/mL (FCBR.2).
3.1. Efficacy of the Membrane Floating Bed System
The inoculum used in this work consisted of mixed cells which take up syngas from the liquidphase. Therefore, the rate of the mass transfer and, thus, the mass transfer coefficient from thegas-to-liquid phase is of vital importance. The syngas components face several resistances during theirjourney from the gas phase to the cells (Figure 3a). In the case of floating MBR, there is an extra masstransfer resistance of the membrane surface, however previous studies proved that this resistance isnegligible [15]. The liquid film surrounding the cells (Figure 3b) is considered to be the main resistanceof the gas-to-liquid mass transfer inside the investigated bioreactors, while the rest of the resistancesare considered negligible [14]. The following equations show the rate of mass transfer of component Athough the gas (NAG) and the liquid (NAL) boundary in gmol/(m3·s):
NAG = kGα(CAG − CAGi), (1)
NAL = kLα(CALi − CAL), (2)
where kG and kL is the gas-phase and liquid-phase mass transfer coefficient, m/s, respectively, and α
is the total interfacial area for mass transfer, 1/m. The concentration of A in the liquid bulk is CAL and
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in the liquid boundary is CAli, while the concentration of A in the gas phase is CAG and in the gasboundary is CAGi, in gmol/m3.
Figure 3. (a) The resistances during the mass transfer of syngas from the gas phase to the site ofreaction in the cells. 1. Travel in the gas bubble; 2. Move across the gas-liquid interfacial; 3. Travelthrough the liquid film surrounding the gas bubble; 4. Travel in the liquid bulk; 5. Enter the interiorof the membrane through its pores; 6. Move inside the membrane; 7. Travel across the liquid filmsurrounding the microbial cell; 8. Pass through the cell membrane; 9. Move through the cell and endup in the site of reaction; (b) Movement of A through the interfacial boundary. CAL: concentration of Ain the liquid phase; CAli concentration of A in the liquid boundary; CAGi concentration of A in the gasboundary; and CAG concentration of A in the gas phase [14].
Equations (1) and (2) can be simplified to Equations (3) and (4) [14], respectively, where mCAL
= C*AG is the gas-phase concentration of A in equilibrium with CAL and CAG/m = C*
AL is theliquid-phase concentration of A in equilibrium with CAG, and m is the distribution factor.
NAG = kGα(CAG − C*AG), (3)
NAL = kLα(C*AL − CAL), (4)
Equations (3) and (4) are valid for systems in which the main mass transfer resistance is either thegas-phase film resistance or the liquid-phase film resistance. Thus, the overall mass transfer coefficientsKGα and KLα were replaced by kGα and kLα [14]. In the case of syngas fermentation, H2 and COare poorly soluble to the aqueous solution, which means that kGα is significantly larger than kLα
and, therefore, Equation (3) is the main equation that can describe the limiting mass transfer rate inthe system.
The consumption rates of H2 and CO and the production rates of CH4 and CO2 in floating MBR,floating MBR/FCBR, PBR, and FCBR are presented in Figure 4. The highest consumption rates ofH2 and CO obtained were 18.47 ± 1.25 and 39.67 ± 1.45 mmol/(L·d), respectively, in floating MBR.In the same bioreactor, the highest CH4 production of 21.33 mmol CH4/(L·d) was also achieved
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and the highest CH4 yield of 0.36 mol CH4/mol (H2 + CO). The pH of the effluent was 8.2 ± 2.0in all bioreactors during the experiment. In previous studies, a triculture (R. rubrum, M. barkeri, M.formicicum) converted syngas of a similar composition into CH4 in a trickle bed bioreactor with a CH4
production rate of 48–72 mmol CH4/(L·d) and yields of 0.2–0.214 mol CH4/mol (H2 + CO) [16,17].Moreover, the same triculture converted syngas in a packed bed bioreactor with a CH4 productionof 4.8–7.2 mmol/(L·d) and a yield of 0.214 mol CH4/mol (H2 + CO) [17]. Higher yields of 0.6–0.8mol CH4/mol (H2 + CO) and a CH4 production of 73 mmol/(L·d) were obtained in a multi-orificeoscillatory baffled bioreactor where granular sludge converted syngas with a gas recirculation rate of600 mL/min [18].
Figure 4. Syngas biomethanation in floating MBR, floating MBR/FCBR, PBR, and FCBR duringcontinuous syngas feeding. Consumption of (a) H2 and (b) CO and production of (c) CO2 and(d) CH4in mmol/(L·d).
The lowest syngas biomethanation rates were observed in the PBR and the FCBR. This resultcontradicts with other studies, which reported that the use of packed material in the PBR significantlyimproved the syngas conversion rates and CH4 production rate. For instance, Burkhardt, et al. [19]reported a CH4 concentration of higher than 98% in their final biogas product, achieved in a trickle bedbioreactor filled with packing material. Trickle bed bioreactors with packed material are consideredeffective in syngas biomethanation because of higher mass transfer coefficients and lower cost thanthe continuous stirred bioreactors (CSTR) [16]. The liquid recirculation, which may be co-current orcounter-current with the gas flow, is one of the main reasons for high mass transfer in the trickle bedbioreactors. However, in the present study, there was no liquid recirculation and, therefore, the lowersyngas conversion rates in PBR are likely a result of inadequate substrate distribution inside thebioreactor, which is probably the reason for the lower acetic acid consumption (Table 1) in comparisonto other bioreactors.
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Table 1. Consumption of acetic acid in the bioreactors.
Bioreactor Type Acetic Acid Consumed, g/(L·d)
floating MBR (membr. bed = 3 cm) 1.40 ± 0.42floating MBR/FCBR 1.01 ± 0.41
PBR 0.78 ± 0.51FCBR (ISR = 0.1 g/mL) 0.92 ± 0.52
floating MBR.2 (membr. bed = 6 cm) 1.15 ± 0.35FCBR.2 (ISR = 0.2 g/mL) 1.79 ± 0.62
The floating MBR/FCBR system proved to be less efficient in terms of syngas conversion incomparison to the floating MBR system. The reason for this was probably the better gas-to-cell contactin floating MBR due to the fact that the suspended inoculum was mostly concentrated at the bottom ofthe floating MBR/FCBR. Likewise, in the FCBR and the PBR, the inoculum was mostly concentratedat the bottom of the bioreactors. In bubble column bioreactors with free cells, when syngas feed isintroduced at the bottom of the bioreactor, the H2 and CO levels decrease as the gas flows up thecolumn because of cellular consumption. This causes spatial difference of dissolved syngas inside thebubble column which can affect the cellular growth and the product profile [20]. Therefore, the aim isto establish favourable syngas concentration profiles in the liquid medium according to the desirableproduct [20]. However, in the floating MBR, the cells were assembled in packed formation at the upperparts of the liquid medium so that the gas bubbles stayed longer in the liquid and were dispersedthough the membrane pores. This cell-placement was probably a main factor which caused bettergas-to-cell contact and, therefore, faster syngas biomethanation in the floating MBR.
The floating MBR resembles the concept of a trickle bed or a packed bed bioreactor where thepacked material is replaced by membranes. Trickle bed bioreactors are preferred to other bioreactortypes, such as continuous stirred bioreactors, because of their enhanced gas-to-liquid mass transfermechanisms. A mass transfer analysis on various bioreactors claimed that the mass transfer wasenhanced over three times in a trickle bed bioreactor in comparison to continuously stirred tankreactor [21]. In addition, the optimum CO mass transfer coefficient of stirred tank bioreactors can besubstantially improved in bubble column bioreactors because of higher mass transfer driving forces,which are a result of gas composition spatial profiles and longer gas hold-up [20].
3.2. The Effect of Membrane Bed Thickness
The floating MBR, containing a floating membrane bed, presented the highest syngas conversionefficacy in comparison to other bioreactor types in the previous section. In order to study the effectof membrane bed thickness, another bioreactor was operated in parallel with the floating MBR. Thissecond bioreactor (floating MBR.2) consisted of a higher amount of membranes (30) that was twotimes the amount of membranes in the floating MBR. The floating membrane bed in floating MBR.2was approximately 6 cm thick in comparison with the membrane bed in floating MBR which had athickness of approximately 3 cm. The results in Figure 5 show that the reinforcement of the membranebed increases the conversion rates of H2 and CO by approximately 6% and 11%, respectively, in floatingMBR.2. This result proved that bioreactors with a thicker membrane bed had higher conversion rates,although the improvement was not proportional to the magnitude of the increase of the thickness ofthe membrane bed, which was approximately 100%.
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Figure 5. Syngas biomethanation in floating MBR and floating MBR.2 with a membrane bed thicknessof 3 cm and 6 cm, respectively, during continuous syngas feeding. Consumption of (a) H2 and (b) COand production of (c) CO2 and (d) CH4 in mmol/(L·d).
3.3. Impact of Inoculum-to-Syngas Ratio (ISR)
In order to study the effect of different ISRs, both batch and continuous bioreactors were operated.During the batch experiment, syngas consumption and biogas production was investigated at differentISRs (0.01, 0.04, 0.07, and 0.1 g/mL), and the results are presented in Figure 6. According to thefigure, the increase of ISR led to faster consumption of H2 and CO. More specifically, the completeconsumption of H2 and CO took one day in bioreactors with an ISR of 0.1 g/mL, while in bioreactorswith 10 times less inoculum (ISR = 0.01 g/mL), the complete consumption of H2 and CO took fourand five days, respectively. The CH4 production followed the same trend. The CH4 production inthe bioreactor with an ISR of 0.1 g/mL was stabilized on the third day, while the CH4 productionin the bioreactor with an ISR of 0.01 g/mL reached its highest CH4 production on the sixth day offermentation. In bioreactors with an ISR of 0.04 g/mL, the complete conversion of H2 and CO tookthree and five days, respectively. These conversion rates were lower in comparison with a previousstudy with a similar system (ISR = 0.38 g/mL) during which H2 and CO were totally consumed intwo days [22]. However, in this work, the initial amount of H2 and CO content in the bioreactors wasapproximately 50% and 24% higher than in the previous study. The highest CH4 yield (at an ISR of0.1 g/mL) obtained in the current work was 0.39 mol CH4/mol (H2 + CO2), which is comparativelyhigher than in other similar studies. For example, the CH4 yield achieved during the conversion ofH2/CO2 and CO in a batch bubble column bioreactor with an ISR of 0.0037 g/mL was 0.22–0.26 molCH4/mol (H2 + CO2) and 0.25 mol CH4/mol CO, respectively [23]. The lower yields in the abovework could have been caused by the lower ISR and the different syngas composition in comparison tothe current work.
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Figure 6. Syngas biomethanation in batch bioreactors with an ISR of 0.01, 0.04, 0.07, and 0.1 (g/mL).Consumption of (a) H2 and (b) CO and production of (c) CO2 and (d) CH4 in mmol.
During the continuous experiment, the FCBR that had an ISR of 0.1 g/mL was operated inparallel with FCBR.2 with an ISR of 0.2 g/mL. The aim was to investigate the effect of doubling theISR and the operation in continuous mode. The results (Figure 7) showed an increase in the H2 andCO consumption rate of approximately 66% and 61%, respectively, and an increase of approximately94% in CH4 production rate. The CH4 production rate in the bioreactor with an ISR of 0.2 g/mLwas 34.41 mmol/(L·d), the highest achieved in this work. This production rate is comparable toresults from other similar studies. A CH4 production rate of 72 mmol/(L·d) was achieved duringbiomethanation of syngas with a similar composition at a gas flow rate of 70 mL/min. The syngas wasconverted in a trickle bed bioreactor by a triculture of R. rubrum, M. barkeri, and M. formicicum [4,16].Another study was conducted in a packed bed bioreactor with the same triculture, similar syngascomposition, and gas flow rate of 80 mL/min. In that work, a lower CH4 production rate between 4.8and 7.2 mmol/(L·d) was reported [17]. In the literature, there are previous studies that have reportedthat higher ISRs can improve the CH4 potential yields. These studies have mainly focused on theinvestigation of using different ISRs based on g VSadded, with mixed anaerobic sludge. For example, astudy on the effect of ISR on the CH4 potential of microcrystalline cellulose production wastewaterreported that the fastest CH4 production rate and highest kinetic constant were achieved at the highestISR of 2.0 [10]. However, extremely high ISRs may be inhibiting for the anaerobic process. Lim andFox [24] studied three different ISRs (1, 0.33, and 0.125) and reported that the highest CH4 productionrate was obtained at the ratio of 0.33, whereas the minimum production rate was obtained at an ISRof 0.125. The low ISR probably caused low substrate concentration and the high ISR caused highconcentration of volatile fatty acids and thus, low pH [24].
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Figure 7. Syngas biomethanation in FCBR (ISR = 0.1 g/mL) and FCBR.2 (ISR = 0.2 g/mL) duringcontinuous syngas feeding. Consumption of (a) H2 and (b) CO and production of (c) CO2 and (d) CH4
in mmol/(L·d).
4. Conclusions
The new concept of floating MBR was successfully applied during continuous syngasbiomethanation with a mixed culture and thermophilic conditions. The floating MBR was operatedin parallel with a hybrid bioreactor containing both membrane encased and free cells, a bioreactorwith free cells growing on packed material, and a free cell bioreactor. The results showed thatthe use of the floating MBR improved the gas hold-up and accelerated the syngas conversionrate. Thus, syngas conversion rates were higher in the floating MBR in comparison to the otherbioreactors. The increase of the thickness of the membrane bed by twofold resulted in higher syngasconversion rates of 6% and 11% for H2 and CO consumption, respectively. In addition, differentinoculum-to-syngas ratios (ISR) were tested during both batch and continuous experiments and thehighest syngas conversion rates were achieved at the highest ISR of 0.2 g/mL during continuousbiomethanation. The study of the limiting effect of ISR in continuous syngas biomethanation isconsidered an interesting future step.
Author Contributions: Conceptualization, K.C.; methodology, K.C. and E.P.; validation, K.C.; formal analysis,K.C.; investigation, E.P. and K.C.; resources, E.P. and K.C.; data curation, E.P. and K.C.; writing—original draftpreparation, K.C.; writing—review and editing, K.C. and M.J.T.; visualization, K.C. and E.P.; supervision, M.J.T.;project administration, K.C. and M.J.T.; funding acquisition, M.J.T.
Funding: This research was funded by the Swedish Research Council.
Acknowledgments: The authors would like to thank Jean-Francois Charlot from Merck, Millipore S.A.S., Franceand Amir Mahboubi from University of Borås, Sweden for their technical support on membranes.
Conflicts of Interest: The authors declare no conflict of interest.
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