Optimisation and Scale-up of a Biotechnological Process
for Production of L(+)-Lactic Acid from Waste Potato
Starch by Rhizopus arrhizus
Zhanying Zhang
The University of Adelaide
May 2008
Optimisation and Scale-up of a Biotechnological Process for
Production of L(+)-Lactic Acid from Waste Potato Starch by
Rhizopus arrhizus
Zhanying Zhang
B.Eng. (Biochemical Engineering)
M.Eng. (Biochemical Engineering)
Thesis Submitted for the Degree of
Doctor of Philosophy
School of Chemical Engineering
The University of Adelaide
May 2008
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page I
TABLE OF CONTENTS
Table of Contents ...................................................................................................................... I
Abstract................................................................................................................................... VI
Declaration............................................................................................................................VIII
Acknowledgements ..................................................................................................................X
Preface..................................................................................................................................... XI
Chapter 1. Introduction............................................................................................................1
1. L(+)-Lactic Acid and Its Application .................................................................................2
2. Waste Potato Starch and Environmental Concerns ............................................................2
3. Biological Production of L(+)-Lactic Acid.........................................................................3
4. Technical and Economic Chanllenges Associated with L-Lactic Acid Production
by Rhizopus fungi ...............................................................................................................4
5. Aim of This Project.............................................................................................................6
6. References...........................................................................................................................7
Chapter 2. Literature Review: Production of Lactic Acid from Renewable Materials
by Rhizopus Fungi ..................................................................................................9
Statement of Authorship .......................................................................................................10
Abstract .................................................................................................................................11
Contents ................................................................................................................................11
1. Introduction.......................................................................................................................12
2. Substrates for Lactic Acid Production .............................................................................12
2.1 Starch ..........................................................................................................................13
2.2 Lignocellulose.............................................................................................................13
3. Simultaneous Saccharification and Fermentation (SSF) ..................................................13
4. Bioprocess Parameters in Lactic Acid Production............................................................14
4.1. Nutrients.....................................................................................................................14
4.1.1. Nitrogen ..............................................................................................................14
4.1.2. Inorganic Salts ....................................................................................................14
4.2. Morphology................................................................................................................15
4.3. Immobilization...........................................................................................................15
4.4. pH...............................................................................................................................16
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page II
4.4.1. Effect of pH.........................................................................................................16
4.4.2. Neutralizing Agents ............................................................................................16
4.5. Oxygen Supply...........................................................................................................16
4.6. Temperature ...............................................................................................................17
5. Bioreactor System and Scale-up .......................................................................................17
6. Process Modelling.............................................................................................................18
7. Economic Evaluation of Fermentation Processes.............................................................18
8. Mechanisms of Lactic Acid Production by Rhizopus species ..........................................19
8.1 Lactate Dehydrogenase Enzymes ...............................................................................19
8.2 Molecualr Genetic Strategies to Increase Lactic Acid Production .............................19
8.3 Metabolic Flux Analysis .............................................................................................20
9. Conclusions.......................................................................................................................20
References.............................................................................................................................20
Chapter 3. Experimental Materials and Methods ...............................................................24
1. Microorganism and Media................................................................................................25
1.1 Microorganism............................................................................................................25
1.2 Preculture Medium......................................................................................................25
1.3 Production Medium ....................................................................................................25
2. Bioreactor Systems ...........................................................................................................26
3. Cultivation Conditions ......................................................................................................28
3.1 Precultures...................................................................................................................28
3.2 Experimental Set Up and Operation ...........................................................................28
4. Measurement and Analysis ...............................................................................................28
4.1 Measurement of Initial kLa..........................................................................................28
4.2 Analysis of Morphology and Biomass........................................................................29
4.3 Analysis of Samples by HPLC ..................................................................................29
4.4 Calculation of L(+)-Lactic Acid Yield and Productivity............................................30
4.5 Data Analysis ..............................................................................................................31
5. References.........................................................................................................................31
Chapter 4. Production of Lactic Acid and Byproducts from Waste Potato Starch
by Rhizopus arrhizus: Role of Nitrogen Sources ............................................32
Statement of Authorship .......................................................................................................33
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page III
Summary ...............................................................................................................................34
Introduction...........................................................................................................................34
Materials and Methods..........................................................................................................35
Microorganism...................................................................................................................35
Culture Media and Cultivation Methods ............................................................................35
Sample Preparation and Analytical Methods .....................................................................36
Results and Discussion ..........................................................................................................36
Effect of Nitrogen Sources.................................................................................................36
Kinetics of the Formation of Lactic Acid, Fumaric Acid and Ethanol ...............................39
Conclusion.............................................................................................................................41
References .............................................................................................................................41
Chapter 5. Production of Lactic Acid Using Acid-Adapted Precultures of
Rhizopus arrhizus in a Stired Tank Reactor ......................................................42
Statement of Authorship ........................................................................................................43
Abstract .................................................................................................................................44
Introduction...........................................................................................................................44
Materials and Methods..........................................................................................................45
Microorganism...................................................................................................................45
Preculture Medium ............................................................................................................46
Production Medium ...........................................................................................................46
Preparation of Acid-Adapted Precultures...........................................................................46
Lactic Acid Production in the STR ....................................................................................46
Analytical Methods............................................................................................................46
Results ...................................................................................................................................47
Effect of pH on Precultures................................................................................................47
Effect of Acid-Adapted Precultures on the Morphology of R. arrhizus in the STR ...........48
Kinetics of Lactic Acid Production in the STR..................................................................50
Summary of Production of Lactic Acid and By-Products in the STR ................................50
Discussion .............................................................................................................................53
References .............................................................................................................................55
Chapter 6. Enhancement of Lactic Acid Production Using Acid-Adapted Precultures
of Rhizopus arrhizus in a Bubble Column Reactor ..........................................56
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page IV
Statement of Authorship ........................................................................................................57
Abstract .................................................................................................................................58
1. Introduction.......................................................................................................................59
2. Materials and Methods......................................................................................................60
2.1 Microorganism and Media ...........................................................................................60
2.2 Acid-Adapted Precultures ............................................................................................62
2.3 Lactic Acid Production in the BCR..............................................................................62
2.4 Simulation of Scale-up Processes in the BCR..............................................................63
2.5 Analytical Methods......................................................................................................63
3. Results ...............................................................................................................................64
3.1 Effect of Precultures on the Morphology of R. arrhizus...............................................64
3.2 Effects of Precultues on Dissolved Oxygen (DO) Level, Lactic Acid Production
and Starch Consumption ..............................................................................................66
3.3 Summary of Production of Lactic Acid, Fumaric Acid and Ethanol ............................69
3.4 Simulation of Scale-up Processes for Lactic Acid Production .....................................73
4. Discussion..........................................................................................................................75
References .............................................................................................................................77
Chapter 7. Effect of Cultivation Parameters on the Morphology of Rhizopus arrhizus
and the Lactic Acid Production in a Bubble Column Reactor..........................81
Statement of Authorship ........................................................................................................82
(Abstract) ..............................................................................................................................83
1 Introduction........................................................................................................................83
2 Materials and Methods.......................................................................................................84
2.1 Microorganism and Media ...........................................................................................84
2.2 Cultivation Conditions .................................................................................................84
2.3 Analytical Methods......................................................................................................84
3 Results and Discussion .......................................................................................................84
3.1 Glucose and Waste Potato Starch.................................................................................84
3.2 pH Value......................................................................................................................86
3.3 Starch Concentration....................................................................................................87
3.4 Spargers and Aeration Rate..........................................................................................87
4 Conclusions ........................................................................................................................88
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page V
References .............................................................................................................................88
Chapter 8. Conclusions and Future Direction. ......................................................................90
1. Conclusions .......................................................................................................................91
1.1 A Brief Introduction........................................................................................................91
1.2 Major Achievements.......................................................................................................91
1.3 Summary .........................................................................................................................93
2. Future Direction.................................................................................................................93
2.1 Enhancement of Lactic Acid Yield and Productivity .................................................93
2.2 Scale-up of Bench-Scale Process to Pilot Plant..........................................................95
3. References .........................................................................................................................95
Appendix. Production of Fungal Biomass Protein Using Microfungi from Winery
Wastewater Treatment................................................................................................97
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VI
ABSTRACT
L(+)-Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use
in food and food-related industries, and its potential for the production of biodegradable and
biocompatible polylactate polymers.
The aim of this study was to optimize and scale-up a biotechnological process of L(+)-lactic
acid production by suspended cells of R. arrhizus DAR 36017 with waste potato starch as the
substrate. Commonly used inorganic and organic nitrogen sources, including ammonium
sulphate, ammonium nitrate, urea, yeast extract and peptone, were assessed in conjunction
with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio
enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio led to
production of more fumaric acid as a by-product. The use of organic nitrogen sources (yeast
extract, peptone and urea) resulted in a significant reduction of lactic acid yields by 15 % - 34
% with a decrease of C:N from 168 to 28. The use of inorganic nitrogen sources (ammonium
nitrate and ammonium sulphate) led to a high lactic acid yield of 84 % - 91 % at a C:N below
168. Therefore, ammonium nitrate and ammonium sulphate were considered to be better
nitrogen sources for lactic acid production.
Small pellets are the favoured morphological form for many fermentation processes by
filamentous fungi. However, to control filamentous Rhizopus sp in the pellet form in a
submerged fermentation system is difficult due to its filamentous characteristics. An acid-
adapted preculture technique was developed to induce the formation of the pellet form in
bioreactors. Using the acid-adapted precultures, the fungal biomass can be controlled in small
dispersed pellets as a dominant morphological form. With these small pellets, a lactic acid
yield of 86-89%, corresponding to a concentration of 86-89g/L, was obtained in a laboratory
scale process using a stirred tank reactor (STR) and a bubble column reactor (BCR). A batch
bioprocess for lactic acid production was successfully scaled-up from shake flasks to
laboratory scale bioreactors. Results from a simulated scale-up process revealed that the
concentration and productivity of lactic acid decreased with the increase of the scale-up steps
because of increased pellet size. This suggested that a one-step scale-up process using the
acid-adapted preculture may be feasible in an industrial-scale bioreactor system.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VII
A comprehensive investigation of the impact of cultivation parameters on the morphology of
R. arrhizus and lactic acid production was carried out in the BCR. The results showed that the
fungal morphology was significantly influenced by carbon sources, pH, starch concentrations,
sparger designs and aeration rates. The favoured morphology for lactic acid production was
freely dispersed small pellets, which could be retained as a dominant morphology under
operation conditions at pH 5.0 – 6.0, starch concentrations of 60 – 120 g/L and aeration rates
of 0.2 – 0.8 vvm, using a sintered stainless steel disc sparger. The optimal cultivation
conditions at pH 6.0 and aeration rate of 0.4 vvm resulted in the formation of the freely
dispersed small pellets and production of 103.8 g/L lactic acid, with a yield of 87 %, from 120
g/L liquefied potato starch in 48 h.
This study shows a technically feasible and economically promising process for the production
of lactic acid from waste potato starch. The use of waste potato starch instead of pure glucose
or starch as substrate can significantly reduce the production cost, making this technology
environmentally and economically attractive.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VIII
DECLARATION
This work contains no material which has been accepted for the award of any other degree or
diploma in any university or other tertiary institution and, to the best of my knowledge and
belief, contains no material previously published or written by another person, except where
due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being made
available for loan and photocopying, subject to the provisions of the Copyright Act 1968.
The author acknowledges that copyright of published works contained within this thesis (as
listed below*) resides with the copyright holder(s) of those works.
*List of publications contained in this thesis and copyright holder(s):
1. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Production of lactic acid from renewable materials
by Rhizopus fungi (review). Biochemical Engineering Journal 35:251-263. Copyright for this
paper belongs to Elsevier B.V..
2. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Production of lactic acid and byproducts from waste
potato starch by Rhizopus arrhizus: role of nitrogen sources. World Journal of Microbiology
and Biotechnology 23:229-236. Copyright for this paper belongs to Springer Science &
Business Media.
3. Z.Y. Zhang, B. Jin, J.M. Kelly. 2008. Production of L(+)-lactic acid using acid-adapted
precultures of Rhizopus arrhizus in a stirred tank reactor. Applied Biochemistry and
Biotechnology. D.O.I., 10.1007/s12010-007-8126-7. Copyright for this paper belongs to
Humana Press.
4. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Effects of cultivation parameters on the morphology
of Rhizopus arrhizus and the lactic acid production in a bubble column reactor. Engineering in
Life Sciences 7:490-496. Copyright for this paper belongs to WILEY-VCH Verlag GmbH &
Co. KGaA, Weinheim.
An additional publication which employs similar research methodologies, not included in this
thesis directly but presented as an appendix of this thesis,
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page IX
Z.Y. Zhang, B. Jin, Z.H. Bai, X.Y. Wang. 2008. Production of fungal biomass protein using
microfungi from winery wastewater treatment. Bioresource Technology 99: 3871-3876.
Copyright for this paper belongs to Elsevier Ltd.
Zhanying Zhang
Signed………………………………………
Date…………………………………………
ACKNOWLEDGEMENTS
I would like to express my gratitude to all those who accompanied and supported me to
complete this thesis. Without their kind help, it would have been impossible for me to finish
the research.
Firstly, my greatest appreciations are given to my principal supervisor, Associate Professor Bo
Jin, who gave me the opportunity to study in Australia. During my PhD study, Bo has
impressed me deeply by his enthusiasm for research and insight into science. His
encouragement and personal guidance have provided outstanding support for the present thesis.
Besides being an excellent supervisor, Bo is also a close friend to me and provides me with a
lot of help beyond work.
I would like to warmly thank my co-supervisor Dr. Joan M. Kelly as well for her important
support throughout the project, especially for her detailed and constructive advice on this
project. I am also grateful to my labmates in the Water Environment Biotechnology
Laboratory: Dr. Richard Haas, Dr. Zhihui Bai, and PhD candidates Adrian Hunter, Xiaoyi
Wang, Meng Nan Gabriel Chong, Vipasiri Vimonses and Florian Zepf for their help on this
project.
I also wish to thank the University of South Australia and the University of Adelaide, in which
I have done my PhD project and from which I have received scholarships. I would like to
acknowledge the Australia Research Council, which contributed to this project substantially
by funding the project.
Finally, I would like to give my special thanks to my wife Shuang Zhou for her love and
understanding and to my parents and parents-in-law for their encouragement and support from
China during my PhD study.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page X
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page XI
PREFACE
This thesis contains eight chapters, of which five chapters (Chapter 2, 4, 5, 6 and 7) comprise
the main body. In Chapter 1, a general introduction to this project and thesis is outlined.
Chapter 2 contains a comprehensive literature review, which has been published. Chapter 3
contains a general introduction to the experimental materials and methods used in this study.
Specific details of the methods are given in the relevant chapters. This project aimed to
optimize and scale-up the lactic acid production process using waste potato starch by Rhizopus
arrhizus. The research outcomes and findings are presented thoroughly in Chapters 4 to 7. In
Chapter 4, the focus is on identification of the role of nitrogen sources, and optimization of the
ratio of C:N for lactic acid production in shake flasks. In Chapter 5, a newly developed
inoculation strategy using acid-adapted precultures as the inoculum is described. With this
strategy, the lactic acid production process was successfully scaled up from shake flasks to a
stirred tank reactor (STR). Furthermore, the newly developed inoculation strategy was applied
to a self-designed bubble column reactor (BCR) resulting in successful process scale-up,
described in Chapter 6. In addition, the scale-up between reactors was simulated in this BCR
to determine the ideal scale-up steps for lactic acid production. The results from Chapter 5
and Chapter 6 also proved that the BCR is more suitable for lactic acid production in terms of
yield and productivity. Therefore, in Chapter 7, the results of further comprehensive
optimization of cultivation conditions carried out in the BCR are presented. Chapter 8 draws
the conclusions from each individual published paper and discusses the possible prospects of
this project.
Chapters of 2, 4, 5 and 7 have been published or accepted for publication in refereed academic
journals. Chapter 6 will be submitted to a refereed academic journal. All the papers are closely
related to the research field of this work.
CHAPTER 1
INTRODUCTION
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 1
1. L(+)-lactic acid and its application
Lactic acid (2-hydroxypropionic acid, CH3CHOHCOOH) is the most widely occurring
hydroxycarboxylic acid. It exists naturally in two optical isomers: D(-)-lactic acid and L(+)-
lactic acid. As humans have only L-lactate dehydrogenase that metabolizes L(+) lactic acid,
L(+)-lactic acid is the preferred isomer in the food and pharmaceutical industries and elevated
levels of D(-)-lactic acid are harmful to humans (Expert Committee on Food Additives, 1967).
Lactic acid is the most important multifunctional organic acid due to its versatile applications
in food, pharmaceutical, textile, leather, and chemical industries (Vickroy, 1985). A review
published in 1995 stated that 85% of lactic acid in the USA was used in food and food-related
applications (Datta et al., 1995). An emerging application of lactic acid is its use for
production of biodegradable and biocompatible polylactate polymers, which provide an envi-
ronmentally friendly alternative to biodegradable plastics derived from petrochemical materials
The growth of lactic acid demand is expected to come from the development of new, large-
volume uses, particularly as a feedstock for biodegradable polymers, ‘green’ solvents and
oxygenated chemicals (Datta & Henry, 2006). With the development and commercialization of
biodegradable polymers, their use has increased considerably, and 20–30% of the 120,000
tonnes global production of lactic acid was estimated to be used in these new applications in
2005 (Datta and Henry, 2006). Lactic acid global demand is expected to shoot up to 200,000
tonnes world wide by the end of year 2011 (Ramesh, 2001).
2. Waste potato starch and environmental concerns
Potatoes are the world's fourth largest crop after wheat, rice and maize. The world’s
production of potato is 314.37 million tonnes with the world's largest producer, China,
producing 70.34 million tonnes in 2006 (Food and Agricultural Organisation, 2008).
Internationally, Australia ranks 35th of the list of potato growing countries, with potato
production totalling over 1,200,000 tonnes since 2001 (Australian Bureau of Statistics, 2008).
The data from the Australian Bureau of Statistics show that the gross value of potatoes was
$513.7 million in 2006-2007, accounting for 3.2 % of the total principal agricultural
commodities. The potato processing industry consumes millions of tonnes of potato while
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 2
generating large amounts of potato wastewater which contains high starch content. This
wastewater can significantly pollute the environment if it is discharged directly into an
ecosystem without treatment. This practice results in large losses of valuable nutrient
resources. With increased environmental awareness, people are increasingly concerned about
the disposal of wastes.
Waste potato starch collected from wastewater in a potato processing industry can be used as
animal feed or supplied as a substrate for alcohol production by fermentation conventionally,
as the main component is a biodegradable material — starch (Natu et al., 1991). In addition,
the amounts of protein, vitamins, minerals and other nutrients in the potato starch may be
sufficient to meet the growth requirements for many microorganisms, especially for fungi.
Therefore, the biotechnological process converting waste potato starch to other valuable end-
products, eg. microbial biomass protein (MBP) and organic acids, by fungi only needs the
addition of a small amount of other nutrients such as nitrogen and inorganic salts (Collen &
Kenneth, 1987; Jin et al., 1999; Tsao et al., 1999). Therefore, reuse of the waste potato starch
not only can reduce and eliminate potential damage to the environment, but also may lower
the production cost.
3. Biotechnological production of L(+)-lactic acid
Lactic acid can be produced by either chemical synthesis or a biotechnological process, such
as fermentation. Chemical synthetic production relies on using expensive chemicals from non-
renewable raw materials, such as petroleum, via a high energy-cost process, and results in a
racemic mixture of the two isomers. Biotechnological production has an ability to yield an
optically pure form of lactic acid alone or a racemate, depending on the microorganisms and
culture conditions used (Ǻkerberg et al., 1998; Huang et al., 2003; Yin et al., 1997). In
addition, fermentative production of lactic acid can use cheap, renewable resources, such as
whey, molasses, corn and potato, as well as starch wastes from starch processing plants
(Aristidous & Penttila, 2000; Huang et al., 2003; Jin et al., 2003; Richter & Berthold, 1998;
Tsao et al., 1999).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 3
Lactic acid can be produced using bacteria and fungi. Lactic acid producing bacteria (LAB)
have received wide interest because of their high growth rate and product yield. However,
LAB have complex nutrient requirements because of their limited ability to synthesize B-
vitamins and amino acids (Chopin, 1993), making supplementation of sufficient nutrients such
as yeast extracts to production media necessary. In addition, the difficulty in separating
fermentation broth containing lactic acid from bacterial biomass increases the overall cost of
production process.
Fungal Rhizopus species have attracted a great attention in recent decades, and have been
recognized as suitable candidates for lactic acid production. Unlike the LAB, lactic acid
producing Rhizopus strains generate L-lactic acid as a sole isomer of lactic acid (Yin et al., 1997;
Soccol et al., 1994; Yu & Hang, 1989). Rhizopus strains grow better under nitrogen-limited
environments than the LAB (Rosenberg & Krištofícová, 2003). When a starch-based material
is used as the substrate, only small amounts of inorganic salts and inorganic nitrogen are
needed for the lactic acid production using Rhizopus fungi. Separation of the fungal biomass
from the fermentation broth is easy because of their filamentous or pellet forms, leading to a
simple and cheap downstream process. Furthermore, as a by-product from the lactic acid
production, the fungal biomass from Rhizopus strains can be used in bioadsorption processes for
purification of contaminated effluents (Fourest, 1994), for fungal chitosan production
(Pochanavanich et al., 2002; Yoshihara et al., 2003) and as an additive in animal feeds to
improve the feed quality (Kusumaningtyas et al., 2006).
4. Technical and economic challenges associated with lactic acid production by Rhizopus
fungi
Although Rhizopus fungi have been widely investigated for lactic acid production, there are
some significant technical challenges associated with the biotechnological process for the
production of lactic acid:
(1) The cost of lactic acid production is high. It has been estimated that substrate cost is one of
the major operational costs, representing 30–40% of total production costs (Saito et al.,
2003). Commercial glucose and starch from agricultural products (wheat, barley, corn, etc.)
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 4
were the main carbon sources used for lactic acid production by either lactic acid bacteria
or Rhizopus sp. in most previous investigations and industrial processes. Therefore, there is
no doubt that the use of low cost substrate, such as starch wastes, can lower the total
production cost significantly. However, there are only a few studies focusing on the studies
of lactic acid production using starch wastes (Huang et al., 2003; Jin et al., 2003).
(2) Lactic acid yield by Rhizopus sp. is low. A lactic acid yield less than 80 % with suspended
cells of Rhizopus species was reported commonly in the literature. The low yield of lactic
acid is due to the production of by-products, such as ethanol and other organic acids, which
competitively consume the substrate. Among these by-products, ethanol is an anaerobic
metabolite and can be accumulated in large amounts if the production process is limited by
oxygen supply. Other by-products are mainly organic acids, such as fumaric acid, malic
acid and succinic acid, which are aerobic metabolites present in small amounts compared to
lactic acid, even under good oxygen supply conditions. Therefore, it is critical to improve
aeration efficiency in the submerged fermentation process. However, the improvement of
oxygen supply and mixing is frustrated because of the difficulty in controlling the
morphology of Rhizopus in the desirable form.
(3) Morphology control is a crucial and difficult task for a submerged fermentation system
using filamentous fungi. The filamentous fungi can easily grow in “filamentous” mycelial
forms. The growth of filamentous fungi in submerged cultures as filamentous mycelia
results in increasing viscosity of the fermentation broth. The highly viscous fermentation
broth makes the bioreactor into a heterogeneous system, and therefore, can significantly
reduce the gas–liquid mass transfer efficiency in the reactor. The filamentous mycelial
biomass can possibly block aeration nozzles, cling to impellers and baffles, and form large
cakes, leading to poor mixing and mass transfer performance in the bioreactor system.
Immobilization has been recognised as an alternative technique to improve the oxygen and
mass transfer efficiency in the submerged fermentation system. However, the use of an
immobilization system results in an expensive and technically complex process. The study
on scale-up of an immobilization fermentation process is missing, which may limit its
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 5
application in industry. Therefore, it is worth studying how to enhance the lactic acid yield
using suspended cultures of Rhizopus cells.
It has been proven that growth of filamentous fungi in pellet forms can overcome the
problems caused by filamentous mycelial growth. Enormous efforts have been given to
control the morphological form of fungal cells to pellets, especially to small uniform pellets.
This is because the large pellets may limit internal mass transfer, resulting in a decrease in
the production rate. Therefore, control of the formation of small uniform pellets is a
prerequisite for industrial applications to ensure adequate mass and heat transfer, and
metabolite production. Methods to control the formation of pellets for the lactic acid
production by Rhizopus sp. mainly focus on the control of inoculum size, culture medium,
addition of calcium, cultivation time or a combination of these operational conditions. The
control strategies reported in the literature are either too complex or inefficient for lactic
acid production. The lactic acid yields with these methods are low, less than 80 %.
5. Aim of this project
The aim of this research was to develop a cost-effective biotechnological process for the
production of L(+)-lactic acid using a cheap substrate, waste potato starch, by filamentous
fungus R. arrhizus strain DAR 36017. Research has focused on the technical challenges to
control fungal morphology, to minimize the accumulation of by-products, such as ethanol and
fumaric acid, and consequently to maximize the yield of lactic acid. The specific objectives of
this study was to
(1) investigate the role of nitrogen source in the fermentation of waste potato starch by R.
arrhizus for lactic acid production,
(2) develop a preculture technique and control strategy to obtain desirable morphological
forms of R. arrhizus for lactic acid production in reactors,
(3) scale-up the lactic acid production process from shake flask culture to a bioreactor process,
and select a suitable bioreactor for further optimization, and
(4) optimize lactic acid production processes in the selected bioreactor system
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 6
6. References
Ǻkerberg, C., Hofvendahl, K., Zacchi, G., Hahn-Hägerdal, B., 1998. Modelling the influence
of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid
production by Lactococcus lactis ssp Lactis ATCC 19435 in whole-wheat flour. Appl.
Microbiol. Biotechnol. 49, 682–690.
Aristidous, A., Penttila, M., 2000. Metabolic engineering applications to renewable resource
utilization. Curr. Opin. Biotechnol. 11, 187-198.
Australian Bureau of Statistics, 2008. Year Book Australia. pp 497-500.
Chopin, A., 1993. Organization and regulation of genes for amino acid biosynthesis in lactic
acid bacteria. FEMS Microbiol. Rev. 12, 21–38.
Collen, S.A., Kenneth, F.G., 1987. Production of microbial biomass protein from potato
process waste by Cephalosporim eichhoriae. Appl. Environ. Microbiol. 53, 824-829.
Datta, R., Tsai, S.P., Bonsignor, P., Moon, S., Frank, J., 1995. Technological and economic
potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol. Rev. 16, 221-
231.
Datta, R., Henry, M., 2006. Lactic acid: recent advances in products, processes and
technologies- a review. J. Chem. Technol. Biotechnol. 81, 1119–1129.
Expert Committee on Food Additives., 1967. Lactic acid. WHO Food Addit. Ser. 29, 144–148.
Food and Agricultural Organisation., 2008. International Year of the Potato,
http://www.potato2008.org/en/world/index.html.
Fourest, E., Canal, C., Roux, J., 1994. Improvement of heavy metal biosorption by mycelial
dead biomasses (Rhizopus arrhizus, Mucor miehei and Penicillium chrysogenum): pH
control and cationic activation. FEMS Microbiol. Rev. 14, 325–332.
Huang, L.P., Jin, B., Lant, P., Zhou, J., 2003. Biotechnological production of lactic acid
integrated with potato wastewater treatment by Rhizopus arrhizus. J. Chem. Technol.
Biotechnol. 78, 899-906.
Jin, B., Huang, L.P., Lant, P., 2003. Rhizopus arrhizus-a producer for simultaneous
saccharification and fermentation of starch waste materials to L(+)-lactic acid. Biotechnol.
Lett. 25, 1983-1987.
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Jin, B., van Leeuwen, H.J., Patel, B., Doelle, H.W., Yu, Q., 1999. Production of fungal protein
and glucoamylase by Rhizopus oligosporus from starch processing wastewater. Process
Biochem. 34, 59-65.
Kusumaningtyas, E., Widiastuti, R., Maryam, R., 2006. Reduction of aflatoxin B 1 in chicken
feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination.
Myopathology 162, 307–311.
Natu, R.B., Mazza, G., Jadhav, S.J., 1991. Potato: production, processing, and products, in
Waste Utilizaiton, Edited by Salunkhe, D.K., Kadam, S.S., Jadhav, S.J., CRC Press, Boca
Raton, FL, pp175-201.
Pochanavanich, P., Suntornsuk, W., 2002. Fungal chitosan production and its characterization.
Lett. Appl. Microbiol. 35, 17–21.
Ramesh, M.V., 2001. A wonder chemical that will help make biodegradable plastic, why India
needs to milk the full potential of lactic acid. India markets empowering business. April 2.
Richter, K., Berthold, C., 1998. Biotechnological conversion of sugar and starchy crops into
lactic acid. J. Agri. Eng. Res. 71, 181-191.
Rosenberg, M., Krištofícová, L., 1995. Physiological restriction of the l-lactic acid production
by Rhizopus arrhizus. Acta Biotechnol. 15, 367–374.
Saito, K., Kawamura, Y., Oda, Y., 2003. Role of the pectinolytic enzyme in the lactic acid
fermentation of potato pulp by Rhizopus oryzae. J. Ind. Microbiol. Biotechnol. 30, 440–444.
Soccol C.R., Marin, B., Raimbault, M., Lebeault, J.M., 1994. Potential of solid state
fermentation for production of L(+)-lactic acid by Rhizopus oryzae. Appl. Microbiol.
Biotechnol. 41, 286–290.
Tsao, G.T., Cao, N.J., Du, J., Gong, C.S., 1999. Production of multifunctional organic acids
from renewable resources. Adv. Biochem. Eng./Biotechnol. 65, 243-279.
Vickroy, T.B., 1985. Comprehensive biotechnology. Dic Pergamon, Toronto.
Yin, P.M., Nishina, N., Kosakai, Y., Yahiro, K., Park, Y., Okabe, M., 1997. Enhanced
production of L(+)-lactic acid from corn starch in a culture of Rhizopus oryzae using an
air-lift bioreactor. J. Ferment. Bioeng. 84, 249-253.
Yoshihara, K., Shinohara, Y., Hirotsu, T., Izumori, K., 2003. Chitosan productivity
enhancement in Rhizopus oryzae YPF-61A by D-psicose. J. Biosci. Bioeng. 95, 293–297.
Yu, R.C., Hang, Y.D., 1989. Kinetics of direct fermentation of agricultural commodities to L(+)
lactic acid by Rhizopus oryzae. Biotechnol. Lett. 11, 597–600.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 8
CHAPTER 2
LITERATURE REVIEW
PRODUCTION OF LACTIC ACID FROM RENEWABLE MATERIALS BY
RHIZOPUS FUNGI
Z.Y. Zhang a, B. Jin a,b, J. M. Kelly c
a SA Water Centre for Water Sciences and Systems, The University of South Australia,
Mawson Lakes, SA 5095, Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia
c School of Molecular and Biomedical Science, The University of Adelaide,
SA 5005, Australia
Biochemical Engineering Journal 2007, 35: 251-263.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 9
STATEMENT OF AUTHORSHIP
PRODUCTION OF LACTIC ACID FROM RENEWABLE MATERIALS BY
RHIZOPUS FUNGI
Biochemical Engineering Journal 2007, 35: 251-263.
Zhang, Z.Y. (Candidate) Performed literature review; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date……………………….. Jin, B.
Supervision of manuscript preparation; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..
Kelly, J. M.
Supervision of manuscript preparation; manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 10
Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Production of lactic acid from renewable materials by Rhizopus fungi Biochemical Engineering Journal, v.35 (3), pp. 251-263, August 2007
NOTE: This publication is included on pages 9 – 31 in the print
copy of the thesis held in the University of Adelaide Library.
It is also available online to authorised users at: http://dx.doi.org/10.1016/j.bej.2007.01.028
CHAPTER 3
EXPERIEMNTAL MATERIALS AND METHODS
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 24
1. Microorganism and media
1.1 Microorganism
R. arrhizus DAR 36017, purchased from Orange Agricultural Institute, Sydney, Australia, was
used for lactic acid production in this project. R. arrhizus WEBL 0401 in Chapter 4 was the
same strain as R. arrhizus DAR 36017, but was named after the abbreviation of our laboratory
name (WEBL, Water Environment Biotechnology Laboratory). Previous research revealed
that this strain could produced L(+)-lactic acid as the sole isomer (Jin et al., 2003). The strain
was maintained and grown for spore production on potato dextrose agar slants at 30 oC for 7
days and stored at 4 oC. Spores were harvested using an inoculation loop, suspended in
sterilized water, and counted under a microscope (BA 400, Mitoc, USA).
1.2 Preculture medium
The preculture medium was prepared based on the medium described by Huang et al. (2003),
and contained (g/L): soluble starch, 10; peptone, 5.0; yeast extract, 5.0; KH2PO4, 0.2;
MgSO4·7H2O, 0.2. The preculture medium had a pH around 6.8. For acid-adapted precultures,
the pH of the preculture medium was adjusted according to the experimental requirements
(Chapter 5 and Chapter 6).
1.3 Production medium
Production medium consisted of waste potato starch or glucose, nitrogen source, KH2PO4
(0.25 g/L), MgSO4·7H2O (0.15 g/L), ZnSO4·7H2O (0.04 g/L). Waste potato starch was
collected from Smith’s Chips Ltd. In the shake flask experiments in Chapter 4, 40 g/L waste
potato starch was used with the addition of 20 g/L CaCO3 as a neutralizing agent. The
production medium for shake flask cultures was gelatinized and sterilized at 100 oC for 40
minutes in an autoclave (HV-110, HIRAYAMA, Japan). For reactor use, 600 – 700 g/L waste
potato starch was liquefied at 95 – 100 oC for 4 h with the addition of 0.05 % α-amylase
(Termamyl® Classic, Novoenzymes, Denmark). The liquefied waste potato starch was stored
in a freezer for later use. When the liquefied starch was used in the reactor, frozen starch was
thawed first and diluted to required concentrations with the addition of other medium
components, followed by sterilization at 121 oC for 20 min in the autoclave.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 25
2. Bioreactor systems
A 3.3 L standard stirred tank reactor (STR) with a working volume of 2.5 L (Bioflo III, New
Brunswick, USA) and a 11.5 L bubble column reactor (BCR) with a working volume of 7.5 L
were used in this study. The STR is shown in Figure 1. The BCR was designed by ourselves
and made by Stainless Steel Welding Services Pty. Ltd (Australia). The BCR system and
design scheme are shown in Figure 2.
Figure 1 Stirred tank reactor (Bioflo III, New Brunswick, USA).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 26
A B
Sterile air Sampling port
580 mm
405 mm
Ø=155 mm
Ø=195 mm
Probes
Air sparger
Figure 2 Bubble column reactor. (A) photograph of the BCR system; (B) design scheme.
The STR was equipped with two conventional 6-flat-bladed turbine impellers and an annular
stainless steel sparger (Ø = 55 mm) with five evenly distributed pores (Ø = 0.5 mm). The
aeration for the BCR was provided by either an annular stainless steel sparger (Ø = 70 mm)
with eight evenly distributed pores (Ø = 0.5 mm) or a sintered stainless steel disc sparger (Ø =
75 mm; pore size, 50 – 70 μm). The BCR was equipped with a biocontrol system (LabVIEW
7.0, National Instruments, USA) to control and monitor the experiment parameters (pH,
dissolved oxygen level and temperature). The electrodes of pH, dissolved oxygen (DO) and
temperature used in the both STR and BCR were purchased from METTLER TOLEDO
(Switzerland).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 27
3. Cultivation conditions
3.1 Precultures
For preparation of the inocula (except acid-adapted precultures), a 5 mL spore suspension was
transferred into a 250 mL shake flask with 100 mL preculture medium under aseptic
conditions. The seed cutlures were incubated for 18 h before inoculation for production use.
The detailed procedures for preparation of the acid-adapted precultures are described in
Chapter 5. Precultures were conducted in an incubator (OM15, Orbital incubator, Ratek,
Australia) at 150 rpm and 30 °C.
3.2 Experimental set up and operation
For shake flask experiments, 5 mL preculture was transferred into a 250 mL shake flask with
100 mL of production medium. The cultivation conditions for shake flask experiments were
set up in the same way as those for the precultures described at 3.3.1.
In the reactor experiments, the inoculum size was 5 % (v/v) for both STR and BCR (except the
scale-up simulation experiments in the BCR). For example, 0.125 L preculture was inoculated
to the STR with 2.375 L production medium (total glucose of 250 g). All the experiments in
the reactors were carried out at 30 °C. The agitation speed and aeration rate for operating the
STR were set at 300 rpm and 1.0 vvm, respectively, through the experiments. The aeration
rate of the BCR was set up at 0.1 – 1.0 vvm, depending on the experimental design. 10 M
NaOH was pumped to neutralize the organic acids produced in reactors during cultivations.
Operation pH was controlled at pH 6.0 in the STR, while the growth pH was controlled
according to the requirements in the BCR.
4. Measurement and analysis
4.1 Measurement of initial kLa
The initial volumetric oxygen transfer coefficient (kLa) in the BCR was determined by the
dynamic gassing-out method (Bandyopadhyay et al., 1996). The measurement of initial kLa
was carried out in water phase at 30 oC under a series of aeration rates. During the
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 28
measurement, a DO probe was used to record the change of DO level. The reactor was firstly
aerated with nitrogen. When the DO level was below 5 %, the addition of nitrogen was
stopped, and aeration started at a required flow rate. The DO change was recorded at an
interval of 5 – 15 sec. The initial kLa was calculated using the following equation:
ln C* − CL
C*= −kLa (1)
4.2 Analysis of morphology and biomass
The morphology of R. arrhizus DAR 36017 was observed and recorded using a camera
(PowerShot A95, Canon, Japan). A series of stainless steel sieves (Mining grade, Labtech Essa,
Australia) with an aperture of 1.0 mm, 1.4 mm, 1.7 mm, 2.0 mm and 2.8 mm were used to
collect the pellets with different diameters. Biomass weights were determined after drying at
60 oC for 72 h.
4.3 Analysis of samples by HPLC
High performance liquid chromatography (HPLC) was used to analyse the concentrations of
organic acids, ethanol and residual sugar in the fermentation broth. The HPLC system (Model
350, Varian, Australia; Fig. 3) was equipped with a Rezex ROA-Organic Acid analysis
column (300 × 7.8 mm, Phenomenex, Australia), a refractive index detector (Model 350,
Varian, Australia) and an autosampler (Model 400, Varian, USA). Mobile phase was 4 mM
H SO2 4 solution. Water used for mobile phase was from a water purifying machine
(NANOpure DiamondTM, Barnstead, USA). Fermentation broth samples were centrifuged at
10,000 rpm by a centrifuge (5415R, Eppendorf, USA). 0.5 mL supernatant from centrifuged
sample was diluted 10 times and mixed with 37 % HCl at a ratio of 10:1 (Tay & Yang, 2002).
The mixture was autoclaved at 121 oC for 1 h, and filtered for glucose analysis. Another 0.5
mL supernatant was diluted 25 times and filtered for analysis by HPLC.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 29
Figure 3 High performance liquid chromatography system.
4.4 Calculation of L(+)-lactic acid yield and productivity
The yield of lactic acid was calculated according to the following equation.
Lactic acid concentration
Initial starch concentration (as glucose)Lactic acid yield = (2)
Lactic acid productivity =
Lactic acid concentration
Cultivation time (3)
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 30
4.5 Data analysis
The results presented in this thesis were the means of triplicate experiments in shake flasks
and at least duplicate experiments in the STR and BCR. To evaluate the experiment errors, the
standard deviation (SD) was calculated. The calculation of SD was conducted automatically
using the function of STDEV included in Microsoft Office software, Excel.
5. References
Bandyopadhyay, B., Humphrey, A.E., Taguchi, H., 1996. Dynamic measurement of the
volumetric oxygen transfer coefficient in fermentation systems. Biotechnol. Bioeng. 51,
511-519.
Huang, L.P., Jin, B., Lant, P., Zhou, J., 2003. Biotechnological production of lactic acid
integrated with potato wastewater treatment by Rhizopus arrhizus. J. Chem. Technol.
Biotechnol. 78, 899-906.
Jin, B., Huang, L.P., Lant, P., 2003. Rhizopus arrhizus-a producer for simultaneous
saccharification and fermentation of starch waste materials to L(+)-lactic acid. Biotechnol.
Lett. 25, 1983-1987.
Tay, A., Yang, S.T., 2002. Production of L(+)-lactic acid from glucose and starch by
immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor, Biotechnol.
Bioeng. 80, 1–12.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 31
CHAPTER 4
PRODUCTION OF LACTIC ACID AND BYPRODUCTS FROM WASTE POTATO
STARCH BY RHIZOPUS ARRHIZUS: ROLE OF NITROGEN SOURCES
Z.Y. Zhang a, B. Jin a,b, J. M. Kelly c
a SA Water Centre for Water Sciences and Systems, The University of South Australia,
Mawson Lakes, SA 5095, Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia
c School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,
Australia
World Journal of Microbiology and Biotechnology 2007, 23: 229-236.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 32
STATEMENT OF AUTHORSHIP
PRODUCTION OF LACTIC ACID AND BYPRODUCTS FROM WASTE POTATO
STARCH BY RHIZOPUS ARRHIZUS: ROLE OF NITROGEN SOURCES
World Journal of Microbiology and Biotechnology 2007, 23: 229-236. Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date………………………..
Jin, B.
Interpreted data; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..
Kelly, J. M.
Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 33
Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Production of lactic acid from and byproducts from waste potato starch by Rhizopus arrhizus: role of nitrogen sources. World Journal of Microbiology and Biotechnology, v.23 (2), pp. 229-236, February 2007
NOTE: This publication is included on pages 34 - 41 in the print
copy of the thesis held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1007/s11274-006-9218-1
CHAPTER 5
PRODUCTION OF L(+)-LACTIC ACID USING ACID-ADAPTED PRECULTURES
OF RHIZOPUS ARRHIZUS IN A STIRRED TANK REACTOR
Z.Y. Zhang a, b, B. Jin a, b, c, J. M. Kelly d
a School of Earth and Environmental Sciences, The University of Adelaide, Australia
b School of Chemical Engineering, The University of Adelaide, Australia c Australian Water Quality Centre, Bolivar, SA 5095, Australia
d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,
Australia
Applied Biochemistry and Biotechnology 2008, D.O.I.: 10.1007/s12010-007-8126-7.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 42
STATEMENT OF AUTHORSHIP
PRODUCTION OF L(+)-LACTIC ACID USING ACID-ADAPTED PRECULTURES
OF RHIZOPUS ARRHIZUS IN A STIRRED TANK REACTOR
Applied Biochemistry and Biotechnology 2008, D.O.I.: 10.1007/s12010-007-8126-7. Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date…………………….…
Jin, B.
Interpreted data; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date…………………….…
Kelly, J. M.
Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 43
Zhang, Z.Y., Jin, B. and Kelly, J.M. (2008) Production of L(+)-lactic acid using acid-adapted precultures of Rhizopus arrhizus in a stirred tank receptor. Applied Biochemistry and Biotechnology, v. 149 (3), pp. 265-276, June 2008
NOTE: This publication is included on pages 44 - 55 in the print
copy of the thesis held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1007/s12010-007-8126-7
CHAPTER 6
ENHANCEMENT OF L(+)-LACTIC ACID PRODUCTION USING ACID-ADAPTED
PRECULTURES OF RHIZOPUS ARRHIZUS IN A BUBBLE COLUMN REACTOR
Z.Y. Zhang a, b, B. Jin a, b, c, J. M. Kelly d
a School of Earth and Environmental Sciences, The University of Adelaide, Australia
b School of Chemical Engineering, The University of Adelaide, Australia c Australian Water Quality Centre, Bolivar, SA 5095, Australia
d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,
Australia
Manuscript to be submitted.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 56
STATEMENT OF AUTHORSHIP
ENHANCEMENT OF L(+)-LACTIC ACID PRODUCTION USING ACID-ADAPTED
PRECULTURES OF RHIZOPUS ARRHIZUS IN A BUBBLE COLUMN REACTOR
Manuscript to be submitted.
Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date………………….……
Jin, B.
Manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……
Kelly, J. M.
Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 57
Enhancement of L(+)-lactic acid production using acid-adapted precultures
of Rhizopus arrhizus in a bubble column reactor
Zhan Ying Zhang a,b, Bo Jin* a, b, c , Joan M. Kelly d
a School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA
5005, Australia b School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia
c Australian Water Quality Centre, Bolivar, SA 5095, Australia d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,
Australia * To whom correspondence should be addressed.
Tel.: +61 8 8303 5996. Fax: +61 8 8303 4308. Email: [email protected].
Abstract
Controlling the morphology of filamentous fungi in certain forms in submerged cultures is a
prerequisite for the successful production of metabolites. In this study, an acid-adapted
preculture method was successfully developed to manipulate the morphology of Rhizopus
arrhizus in a bubble column reactor (BCR). The morphology of R. arrhizus in the BCR varied
from fluffy mycelia to freely dispersed small loose pellets and big compact pellets, depending
on the acid-adapted precultures inoculated. With the formation of freely dispersed small loose
pellets, a high concentration of 87.9 – 88.7 g/L lactic acid from 100 g/L potato starch in the
BCR was achieved in 42 h fermentation. Results from a simulated scale-up process revealed
that the concentration and productivity of lactic acid decreased with an increased number of
scale-up steps because pellet size increased, indicating that a one-step scale-up process using
the acid-adapted preculture appears to be feasible in an industrial-scale bioreactor system.
Keywords
Lactic acid; Rhizopus arrhizus; morphology; acid-adapted precultures; bubble column reactor;
scale-up
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 58
1. Introduction
Filamentous fungi are morphologically complex microorganisms. The cultivations of
filamentous fungi in submerged cultures for the production of biomass protein and metabolites
such as organic acids, antibiotics and enzymes have been investigated and applied widely over
the past several decades. The successful production of a metabolite often requires a certain
morphological form of the fungus used. However, it is not easy to control the morphology of
filamentous fungi, which can vary from a filamentous form to a pellet form, depending on the
species and cultivation conditions [1].
Strains of filamentous fungus, Rhizopus arrhizus (syn. Rhizopus oryzae), have attracted great
interest because of their capacity to produce pure L(+)-lactic acid, which is a widely used
multifunctional organic acid in the food, pharmaceutical and chemical industries [2], as well
as their amylolytic ability and low nutrient requirements [2-6]. However, due to low lactic acid
yield and productivity, the application of R. arrhizus in industrial production is still in
question. One of the main reasons causing low yield and productivity is the difficulty in
controlling the morphology of R. arrhizus in bioreactors [7-9]. Although immobilization is
considered to be an effective approach to produce lactic acid [9-11], extra operational and
material costs limit its application in an industrial process. Therefore, exploration of
economical and stable operational techniques to control the morphology of R. arrhizus in a
submerged fermentation system is a challenge in creating an industrial process for efficient
production of lactic acid.
It has been demonstrated that pellets are a promising morphological form in industrial
fermentation processes. The pellet biomass results in a low viscosity of the fermentation broth,
and consequently a simple downstream process required to separate the fungal biomass from
the fermentation broth [1, 12, 13]. However, big pellets may limit nutrient and oxygen transfer
to the pellet interior, leading to a low production yield [12, 14]. Therefore, the development of
an engineering strategy to control the fungal morphology in a small pellet form to ensure
adequate mass and heat transfer, and efficient metabolite production, is a prerequisite for
industrial applications [12]. To achieve a pellet form of R. arrhizus, most research has focused
on controlling inoculum size [15-18]. Yang and co-workers obtained small pellets in a stirred
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 59
tank reactor (STR) using precultures growing on xylose [19]. However, in most previous
studies, either lactic acid yield or productivity was low.
In a previous study, we have developed an acid-adapted preculture method to control the
morphology of R. arrhizus in pellet forms successfully in a STR [20]. The yield of lactic acid
in the STR reached over 86 % with a productivity of 1.6 g/L/h (Table 1). In this study, the
application of an acid-adapted preculture method to control the morphology of R. arrhizus
DAR 36017, and consequently to enhance the lactic acid yield and productivity was further
investigated in a self-designed BCR. The BCR can generate considerably less shear stress than
conventional STRs since mechanical agitation is not introduced, and therefore it is more
suitable for cultivation of filamentous fungi sensitive to shear force. A simulation experiment
of scale-up process was also carried out to investigate the effects of scale-up steps on the
morphology of R. arrhizus and lactic acid production.
2. Materials and methods
2.1 Microorganism and media
R. arrhizus DAR 36017, obtained from the Orange Agricultural Institute, Sydney, Australia,
was used in this research. This strain was maintained and grown for spore production on
potato dextrose agar slants at 30 oC for 7 days and stored at 4 oC.
The preculture medium was prepared based on the medium described by Huang et al. [21],
and contained (g/L): soluble starch, 10; peptone, 5.0; yeast extract, 5.0; KH2PO4, 0.2;
MgSO4·7H2O, 0.2. The pH of preculture medium was adjusted to an initial value ranged from
2.5 to 5.5, as required, before sterilization. pH in the control preculture medium was 6.8
(unadjusted).The production medium used in the BCR consisted of 100 g/L waste potato
starch, 3.0 g/L (NH4)2SO4, 0.25 g/L KH2PO4, 0.15 g/L MgSO4·7H2O and 0.04 g/L
ZnSO4·7H2O, which was same as that used previously in a STR [20]. Waste potato starch used
in this study was from Smiths Chips Ltd (Australia), where potato starch was separated from
wastewater. Approximately 600 – 700 g/L waste potato starch was liquefied at 95 – 100 oC for
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 60
Table 1 Summary of lactic acid production with Rhizopus species in batch fermentations.
Productivity ConcentrationReactor Morphology Substrate
Yield
(%) (g/L/h) (g/L) Reference
89 2.1 88.7 This study BCR
Small pellets (acid-adapted precultures)
Waste potato starch
86
1.6
85.7
20
STR 86 1.7 103.6 7
ALR
Cotton-like flocs via
immobilization
Glucose
87
1.8
104.6
8
STR
Small pellets (precultures growing on
xylose)
Glucose 41 0.6 33 19
STR
Small pellets (precultures by
control of inoculum size and addition
time of CaCO3)
Glucose 76 — 76.1 15
ALR 85 1.4 102.3 18
ALR
Small pellets (precultures by
control of inoculum size)
Corn starch
77 1.9 92 17
ALR
Small pellets (precultures by
control of inoculum size)
Glucose 80 2.2 95 16
Shake flask
Immobilization on polymer
support
Glucose
65
–
–
11
Glucose 94 5.0 112.7 Shake flask
Immobilization on poly(vinyl
alcohol)-cryogel
Starch 52 1.8 56.7 10
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 61
4 h by the addition of 0.05 % α-amylase (Termamyl® Classic, Novoenzymes, Denmark).
When production medium was prepared, the concentration of the liquefied starch solution was
adjusted to 100 g/L. The potato starch concentration was calculated and expressed as glucose
in this paper. The preculture and production media were autoclaved at 121 oC for 20 min.
2.2 Acid-Adapted precultures
The precultures were prepared in a 250 mL shake flask containing 100 mL precutlure medium.
The procedures for preparation of the acid-adapted precultures were described previously [20].
Spores were harvested from slants using a platinum loop and suspended in sterilized water.
The precultures were acidified at an initial pH from 2.5 to 5.5 with an inoculum size of 105
spores/mL. The 1st precultures with spores were incubated for 18 h at a designated pH of 2.5 –
5.5. The 2nd precultures at a designated pH were prepared using 5 mL of the 1st preculture as
the inoculum and grown for 12 h. In this paper, pH of the precultures refers to the initial pH
unless otherwise stated. The control preculture was inoculated with the same amount of spores
as acid-adapted precultures. All the precultures were cultivated at 30 oC in an orbital shaker at
150 rpm.
2.3 Lactic acid production in the BCR
Production of lactic acid was carried out in a self-designed 11.5 L stainless steel bubble
column reactor with a 7.5 L working volume. The BCR was equipped with a sintered stainless
steel sparger (pore size, 50 – 70 µm) at the bottom. The inoculation was conducted by
transferring 375 mL of acid-adapted preculture into reactors with 7.125 L production medium
(total glucose of 750 g). The temperature and aeration rate in the BCR were maintained at 30
oC and 0.4 vvm throughout the experiments. The cultivation pH was controlled at pH 6.0 with
the addition of 10 M NaOH solution. 0.1 % antifoam (v/v) (Dow Corning® 1510, BDH, UK)
was added to the reactor before sterilization. A few drops of antifoam were added if necessary
during the cultivations. All the cultivations were stopped at 60 h.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 62
2.4 Simulation of scale-up processes in the BCR
The BCR was used for simulation of one-step and multi-step batch processes for lactic acid
production. One-step scale-up cultivation is a process which was scaled-up from shake flask to
reactor. To simulate the two-step scale-up, about 750 mL 18 h culture in the BCR remained
under aeration (so that pellets did not sediment) following the addition of 6.75 L of new
production medium. The cultivation for the two-step scale-up was further carried out for 60 h.
This process was serially repeated for the three-step scale-up. Aeration rate, cultivation
temperature and pH were controlled described in section above.
2.5 Analytical methods
30 to 50 mL samples were taken from the BCR at 6 h to 12 h intervals. The morphology of R.
arrhizus DAR 36017 was recorded in a 9-cm petri-dish using a digital camera (PowerShot
A95, Canon, Japan). For pellet distribution analysis, a series of sieves with an aperture of 1.0
mm, 1.4 mm, 1.7 mm, 2.0 mm and 2.8 mm were used to collect the pellets with a diameter less
than 1.0 mm, between 1.0 mm and 1.4 mm, between 1.4 mm and 2.0 mm, and above 2.8 mm.
Biomass was harvested after filtration and washed three times using tap water. Biomass
weight was determined after drying at 60 oC for 72 h. A Rezex ROA-Organic Acid analysis
column (300×7.8 mm, Phenomenex, Australia) and a refractive index detector (Model 350,
Varian, Australia) were used to analyze all organic compounds, including glucose, lactic acid,
fumaric acid and ethanol. The mobile phase for HPLC was 4 mM H2SO4 solution with a flow
rate of 0.6 mL/min. The column temperature was maintained at 70 oC. 0.5 mL of supernatant
from the centrifuged sample was diluted 10 times and mixed with 37 % HCl at a ratio of 10:1
[9]. The mixture was autoclaved at 121 oC for 1 h, and filtered for glucose analysis by HPLC.
Another 0.5 mL of supernatant was diluted 25 times and filtered for analysis of lactic acid,
fumaric acid and ethanol by HPLC. All the experiments were carried out at least twice and the
results presented were the means of at least duplicate experiments.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 63
3. Results
3.1 Effect of precultures on the morphology of R. arrhizus
Fig. 1 shows the representative morphological forms of R. arrhizus in the BCR using different
precultures. It was observed that inoculation of different precultures significantly affected the
morphology of the fungal biomass. When the control preculture was used, big and irregular
pellets were formed, leading to a range of pellet sizes between 5 and 15 mm (Fig. 1d). In
contrast, the morphology of R. arrhizus changed from coalesced fluffy mycelia to freely
dispersed small pellets in the BCR inoculated with the 1st precultures. The fluffy mycelia
(Figs. 1b1. and 1c1) were formed in the reactor which was inoculated with the 1st precultures
adapted at pH 3.5 – 5.5 (pH referred to the initial pH of precultures in shake flasks unless
otherwise stated, and the cultivation pH in the BCR was 6.0 in the BCR in all cases), whereas
the freely dispersed small pellets appeared in the reactor using the 1st precultures at pH 2.5 and
3.0 (Figs. 1a1). The use of the 2nd precultures at pH 4.0 – 5.5 resulted in forming freely
dispersed small pellets as the dominant morphological form in the BCR (Fig. 1c2). Freely
dispersed compact and big pellets (Fig. 1a2) were formed with the 2nd precultures at pH 2.5
and 3.0 while fluffy mycelia were observed in the reactor using the 2nd preculture at pH 3.5
(Fig. 1b2). It was found that the freely dispersed small pellets were loose and “light” while the
big pellets were compact and “heavy”.
Analysis of pellet distribution of dispersed pellets at 48 h shows that when the 1st preculture at
pH 2.5 was used, the total pellets smaller than 2.0 mm made up 93 % of the total biomass
(w/w) while pellets over 2.0 mm were only 7.0 % (Fig. 2). The use of the 2nd preculture at pH
2.5 and pH 3.0 resulted in a significant reduction in the percentage of the small pellets and an
increase in the numbers of the big pellets. For instance, only 4 % small pellets (< 2.0 mm) but
96 % big pellets ( > 2.0 mm) were formed in the reactor using the 2nd preculture at pH 2.5
(Fig. 2). About 90 % small pellets (< 2.0 mm) were also formed in the BCR using the 2nd
precutlures at pH 4.5 to 5.5. It was found that the fluffy mycelia dominated in the BCR with
the 1st preculture at pH 5.0 (Fig. 2). Pellets smaller than 1.0 mm were only 17 % with the 2nd
preculture at pH 5.0, much less than 42 % with the 1st preculture at pH 2.5. Pellets between 1.4
mm and 2.0 mm were 40 % with the 2nd preculture at pH 5.0, higher than 16 % with the 1st
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 64
stpreculture at pH 2.5 (Fig. 2). In general, the use of either the 1 precultures pH 2.5 and 3.0 or
the 2nd precultures at pH 4.5 to 5.5 led to the formation of small pellets whereas fluffy mycelia
were found in the BCR which was inoculated with other 1st nd and 2 precultures.
Figure 1 Effect of precultures on the morphology of R. arrhizus in the BCR. The precultures
used were the 1st precultures adapted at pH 2.5 (a1), pH 3.5 (b1), pH 5.0 (c1), the
2nd precultures adapted at pH 2.5 (a2), pH 3.5 (b2) and pH 5.0 (c2), and the control
preculture (d). Photos were taken at 48 h from a 9.0 cm petri-dish.
d
10 mm
a1
a2 c2
b1 c1
b2
d
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 65
0
15
30
45
60
<1.0 1.0-1.4 1.4-2.0 2.0-2.8 >2.8Pellet size (mm)
Pelle
t dist
ribut
ion
(%
Figure 2 Effect of precultures on pellet distribution (48 h). The precultures used were the 1st
preculture adapted at pH 2.5 (grey bar), and the 2nd precultures at pH 2.5 (dark bar)
and pH 5.0 (blank bar).
3.2 Effects of precultures on dissolved oxygen (DO) level, lactic acid production and
starch consumption
Fig. 3 and Fig. 4 display the representative kinetic profiles of DO level, lactic acid production
and starch consumption in the BCR using the acid-adapted precultures. Starch consumption
was expressed as reduction in residual sugar (Fig. 4). Obviously, the change of morphology of
R. arrhizus significantly affected the DO level, lactic acid production and starch consumption
under the operation conditions. The lowest DO level (<30%) was measured in the broth which
had coalesced fluffy mycelia, while the broth with freely dispersed small pellets corresponded
to a DO level above 45 % (Fig. 3). Approximately 85 % DO level was obtained in the broth
dominated by big pellets (Fig. 3b).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 66
0
20
40
60
80
100
0 12 24 36 48 60 72Time (h)
DO
(%)
0
20
40
60
80
100
0 12 24 36 48 60 72Time (h)
DO
(%)
a
b
Figure 3 Effect of precultures on process DO. The precultures inoculated were the 1st (a) and
the 2nd (b) precultures adapted at pH 2.5 ( ), pH 3.5 ( ) and pH 5.0
( ).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 67
0
20
40
60
80
100
0 12 24 36 48 60 72Time (h)
Lact
ic a
cid
and
resid
ual s
ugar
(g/L
0
20
40
60
80
100
0 12 24 36 48 60 72Time (h)
Lact
ic a
cid
and
resid
ual s
ugar
(g/L
a
b
Figure 4 Kinetics of lactic acid production and starch consumption using different precultures.
The precultures inoculated were the 1st (a) and 2nd (b) precultures at pH 2.5 (square),
pH 3.5 (triangle) and pH 5.0 (circle); open symbols, lactic acid production; dark
symbols refer to starch consumption.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 68
The kinetics of lactic acid production was affected by the morphology and DO level in the
BCR (Fig. 4). Using the 1st preculture at pH 2.5 and 2nd preculture at pH 5.0 as inoculum,
lactic acid concentration reached 88.7 g/L and 88.1 g/L at 42 h associated with the formation
of freely dispersed small pellets. The formation of big pellets with the 2nd preculture at pH 2.5
produced 82.3 g/L lactic acid at 48 h. The lactic acid concentration dropped significantly with
the 1st and 2nd precultures at pH 3.5, and 1st preculture at pH 5.0, which resulted in the
formation of coalesced fluffy mycelia (Fig. 1 and Fig. 4). A low starch consumption rate
corresponded to high residual sugar left in the reactor (Fig. 4). This reveals that low DO
caused by fluffy mycelia inhibited the consumption of sugar by R. arrhizus and, consequently,
slowed down the lactic acid production.
3.3 Summary of production of lactic acid, fumaric acid and ethanol
Fig. 5 shows a summary of the maximum lactic acid concentration and productivity in the
BCR using the 1st and 2nd acid-adapted precultures at pH 2.5 – 5.5. Only 45.9 g/L lactic acid
was produced in the BCR inoculated with the 1st preculture at pH 5.5, corresponding to a
productivity of 0.8 g/L/h. The lactic acid concentration in the BCR was enhanced significantly
from 45.9 g/L to 88.7 g/L with the reduction of the adaptation pH of the 1st precultures from
5.5 to 2.5. The use of 2nd precultures at pH 4.0 – 5.5 produced 87.9 g/L to 88.5 g/L lactic
acid. The lactic acid productivity reached 2.1 g/L/h with the 1st precultures at pH 2.5 and pH
3.0, and 2nd precultures at pH 4.5 – 5.5, higher than others. The high lactic acid concentration
and productivity were associated with the formation of freely dispersed small pellets, whereas
low lactic acid concentration and productivity were accompanied with the growth of compact
big pellets or fluffy mycelia. When the BCR was inoculated with the control preculture, less
lactic acid (74.1 g/L) but more ethanol (10.7 g/L) was accumulated due to the formation of big
pellets (Fig. 1d).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 69
0
20
40
60
80
100
2.5 3.0 3.5 4.0 4.5 5.0 5.5Adapted pH
Lact
ic a
cid
conc
entra
tion
(g/L
0.0
0.5
1.0
1.5
2.0
2.5
Lact
ic a
cid
prod
uctiv
ity (g
/L/h
Figure 5 Concentration and productivity of lactic acid. The precultures inoculated were the 1st
(grey) and the 2nd (dark) precultures; Symbols: lactic acid concentration (bar), lactic
acid productivity (square).
The fumaric acid concentration varied between 1.5 g/L and 3.2 g/L when freely dispersed
small pellets were formed but it was below 1.0 g/L when other forms of biomass appeared
(Fig. 1 and Fig. 6a). More ethanol was produced in the fermentation with fluffy mycelia than
with freely dispersed small pellets (Fig. 6b). When the 2nd precultures adapted at pH 2.5 and
pH 3.0 were used as the inoculum, 2.8 – 3.5 g/L ethanol was produced, which was comparably
higher than that measured in the BCR inoculated with the 1st precultures at pH 2.5 and pH 3.0
and the 2nd precultures at pH 4.5 – 5.5 (Fig. 5b). However, the DO level in the bulk broth of
the BCR with the 2nd precutlure at pH 2.5 and pH 3.0 was much higher than that with the 1st
preculture at pH 2.5 and pH 3.0, and the 2nd precultures at pH 4.5 – 5.5 (Fig. 3), indicating that
the accumulation of more ethanol was attributed to the increase in pellet size because of the
poor oxygen transfer inside the big pellets.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 70
0
1
2
3
4
2.5 3.0 3.5 4.0 4.5 5.0 5.5Adapted pH
Fum
aric
aci
d (g
/L)
0
1
2
3
4
5
2.5 3.0 3.5 4.0 4.5 5.0 5.5
Adapted pH
Etha
nol (
g/L)
a
b
Figure 6 Maximal concentration of fumaric acid and ethanol. a, fumaric concentration; b,
ethanol concentration; The precultures inoculated were the 1st (grey bar) and 2nd
(dark bar) precultures.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 71
3.4 Simulation of scale-up processes for lactic acid production
The 7.5 L bench-scale reactor was used to simulate scale-up processes to determine the effect
of the dilution of seed culture on lactic acid production, and the appropriate scale-up
approaches. The 2nd preculture adapted at pH 5.0 was used as the inoculum in one-step scale-
up process from shake flasks to the 7.5 L reactor.
Fig. 7 shows the biomass production during the simulation processes and pellet distribution at
48 h. After the cultivation was scaled up from shake flask to reactor (one-step), the biomass
increased from 0.1 g/L to 2.3 g/L at 18 h. 10 % of culture in the BCR, which was still in
exponential phase, was used for the next scale-up step. The seed cultures were diluted in the
two-step scale-up process. The biomass growth rate slowed down from 0.14 g/L/h to 0.09
g/L/h in 24 h fermentation (Fig. 7a). Three-step scale-up resulted in the further decrease of
biomass growth rate to 0.06 g/L/h in 24 h fermentation (Fig. 7a). In addition, the final biomass
concentration also reduced from 4.6 g/L (one-step) to 3.0 g/L (two-step) and 2.6 g/L (three-
step).
Analysis of pellet distribution at 48 h showed that the increase in the scale-up steps promoted
the growth of pellet size (Fig. 7b). 90 % of the pellets were smaller than 2.0 mm after one-step
scale-up while two-step and three-step scale-up processes resulted in only approximately 39 %
and 9 % small pellets (<2.0 mm), respectively. These results showed that the dilution of seeds
associated with the scale-up processes corresponded to a decrease in biomass production, but
an increase in the pellet size. Furthermore, pellets became loose and light after one-step scale-
up, but compact and heavy after two- and three-step scale-up. As expected, the decrease of
biomass concentration and increase of pellet size resulted in a reduction of the sugar
consumption rate and the lactic acid production rate (Fig. 8a). The production rate of lactic
acid after one-step scale-up was 3.3 g/L/h in 12 – 36 h. However, it was only 2.6 g/L/h and 1.9
g/L/h after two- and three-step scale-up, respectively. The lactic acid concentration after one-
step scale-up was 88.1 g/L, which was higher than 85.1 g/L after two-step scale-up. Further
scale-up (three-step) lead to a prolonged fermentation time over 60 h to achieve a maximum
lactic acid concentration of 81.4 g/L. The increase of scale-up steps promoted ethanol
production (Fig. 8b). The highest ethanol concentration was only 1.6 g/L (42 h) after one-step
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 72
scale-up, but it reached 2.2 g/L (42 h) and 4.1 g/L (60 h) after two- and three-step scale-up,
respectively. 1.9 g/L furmaic acid was produced in the one-step scale-up process. The two-
and three-step scale-up processes resulted in producing less than 1.0 g/L fumaric acid.
0
1
2
3
4
5
0 12 24 36 48 60 72
Time (h)
Bio
mas
s (g/
L)
0
15
30
45
60
<1.0 1.0-1.4 1.4-2.0 2.0-2.8 >2.8Pellet size (mm)
Pelle
t dist
ribut
ion
(%
a
b
Figure 7 Effects of scale-up processes on biomass production and pellet distribution (48 h). a,
biomass production in one-step (square), two-step (triangle) and three-step (circle)
scale-up; b, pellet distribution; Symbols: one-step (grey bar), two-step (dark bar) and
three-step (blank bar).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 73
0
20
40
60
80
100
0 12 24 36 48 60 72
Time (h)
Lact
ic a
cid
and
resid
ual s
ugar
(g/L
0
1
2
3
4
5
0 12 24 36 48 60 72Time (h)
Etha
nol (
g/L)
a
b
Figure 8 Kinetics of lactic acid production, starch consumption and ethanol production in
different scale-up processes. a, lactic acid production and starch consumption; b,
ethanol production; Symbols: one-step (square), two-step (triangle) and three-step
(circle).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 74
The DO profiles of three scale-up processes are presented in Fig. 9. After one-step scale-up,
the lowest DO level was 46 %. The DO levels were approximately 64 % and 79 % in the two-
and three-step scale-up processes. It is well known that production of lactic acid and fumaric
acid by Rhizopus is an aerobic process and accumulation of ethanol by Rhizopus takes place in
an anaerobic process. The high DO level was accompanied by low yields of lactic acid and
fumaric acid but high yield of ethanol, indicating that the internal oxygen transfer of pellets
was hampered due to the increased pellet size.
0
20
40
60
80
100
0 12 24 36 48 60 72
Time (h)
DO
(%)
Figure 9 Effect of scale-up processes on DO. Symbols: one-step ( ), two-step ( )
and three-step ( ) scale-up.
4. Discussion
It has been demonstrated that fungal morphology affects nutrient and oxygen uptake rate in
submerged cultures due to the change of viscosity of the fermentation broth [22, 23]. Fluffy
mycelia lead to an increase in the viscosity of the fermentation broth, causing a reduction in
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 75
efficiency of mixing and oxygen supply [23]. This can explain why the DO level in the BCR
with fluffy mycelia and coalesced fluffy pellets was low. Growth of filamentous fungi in
freely dispersed pellets decreases the viscosity and improves mixing and mass transfer
capacity [23], which was also demonstrated by our observation.
Fumaric acid and ethanol are the main by-products produced during lactic acid production by
Rhizopus fungi [3, 9, 20]. Synthesis of L-lactic acid, fumaric acid and ethanol occurs in the
cytosol, with pyruvate at the crossroad [24]. Both fumaric acid and lactic acid are produced in
aerobic processes by Rhizopus fungi [24]. In our study, the highest lactic acid yield of 89 %
was achieved with a highest fumaric acid yield of 3.2 % when the freely dispersed small
pellets were formed. A fumaric acid yield of 3.2 % equated to consumption of less than 3.5 %
of the glucose, considering that a maximum of 0.93 g fumaric acid is produced from 1.0 g
glucose [25]. In contrast, high ethanol yields of 2.8 – 4.1 % produced with big pellets and
fluffy mycelia of R. arrhizus could consume 5.5 – 8.0 % of the glucose (theoretical ethanol
yield is 0.51 g/g glucose consumed). Therefore, controlling of the morphology of R. arrhizus
in small loose pellets instead of big compact pellets or fluffy mycelia is favourable for
converting more glucose to lactic acid. 87.9 – 88.7 g/L lactic acid was produced from 100 g/L
starch within a shorter fermentation time (42 h), with less than 1.8 g/L ethanol accumulated in
the presence of freely dispersed small loose pellets. In contrast, less lactic acid and more
ethanol was produced with big compact pellets when the 2nd precultures at pH 2.5 and pH 3.0,
and two- or three-step scale-up were applied.
Although the lactic acid yield of 89 % in this study is slightly higher than that obtained in the
STR using the same inoculation method, the productivity was enhanced by 30 % (Table 1).
The significant increase in the productivity was attributed to the faster biomass growth rate in
the BCR (data not shown). The enhanced biomass growth may be due to the lower shear stress
generated in the BCR. Using waste potato starch as substrate, the lactic acid yield and
productivity achieved in this study were also higher than those in most previous studies using
commercial starch materials and glucose as substrates with the control of inoculum size and
with immobilized Rhizopus cells in batch fermentations (Table 1).
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 76
Freely dispersed loose small pellets formed with the inoculation of acid-adapted precultures
made the oxygen transfer easier and required a low aeration rate with sintered stainless steel
sparger. It was found that the aeration rate of 0.4 vvm was enough to maintain the DO level
above 45 % (above 3.4 ppm, 30 oC) in the BCR. The ethanol yield was no more than 1.8 g/L
based on 100 g/L potato starch. In contrast, high oxygen supply conditions were always
required by fermentations with immobilized cells of Rhizopus. Tay and Yang aerated over 1.0
vvm of the mixture of air and pure oxygen (5:1) to maintain a DO level at 90 % [9]. However,
an ethanol yield was as high as 5.9 % because of the difficulty of oxygen diffusion crossing
the mycelial layer. A high ethanol concentration of 5 g/L was also detected with 120 g/L
glucose in the immobilized fermentation with an aeration rate from 0.5 vvm to 2.0 vvm (to
maintain 3 – 5 ppm DO level) by Park et al., despite a lactic acid concentration of 104.6 g/L
lactic acid [8].
The results achieved in this study reveal that the application of acid-adapted precultures is a
promising and economical engineering approach to control R. arrhizus in pellet forms for
lactic acid production. The use of BCR instead of STR results in a higher lactic acid yield and
productivity, indicating that the BCR is more suitable for cultivation of filamentous R.
arrhizus. The results from a simulation experiment of scale-up process reveal that a one-step
scale-up process may be preferred in terms of lactic acid yield and productivity.
Acknowledgement
We greatly acknowledge the research fund from Australian Research Council Discovery grant
(DP0452516).
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D.O.I.: 10.1007/s12010-007-8126-7
[21] Huang L P, Jin B, Lant P, Zhou J (2003) Biotechnological production of lactic acid
integrated with potato wastewater treatment by Rhizopus arrhizus. J Chem Technol
Biotechnol 78:899 – 906
[22] Gibbs P A, Seviour R J, Schimid F (2000) Growth of filamentous fungi in submerged
culture: problems and possible solutions. Crit Rev Biotechnol 20:17 – 48
[23] Papagianni M (2004) Fungal morphology and metabolite production in submerged
mycelia processes. Biotechnol Adv 22:189 – 259
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 79
[24] Wright, B E, Longacre, A, Reimers, J, (1996) Models of metabolism in Rhizopus oryzae J
Theor Biol 182:453–457
[25] Tsao, G. T., Cao, N. J., Du, J. and Gong, C. S., (1999) Production of multifunctional
organic acids from renewable resources. Adv Biochem Eng Biotechnol 65:243–279
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 80
CHAPTER 7
EFFECT OF CULTIVATION PARAMETERS ON THE MORPHOLOGY OF
RHIZOPUS ARRHIZUS AND THE LACTIC ACID PRODUCTION IN A BUBBLE
COLUMN REACTOR
Z.Y. Zhang a, b, B. Jin a, b, c, J. M. Kelly d
a School of Earth and Environmental Sciences, The University of Adelaide, Australia
b School of Chemical Engineering, The University of Adelaide, Australia c Australian Water Quality Centre, Bolivar, SA 5095, Australia
d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,
Australia
Engineering in Life Sciences 2007, 7: 1-8.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 81
STATEMENT OF AUTHORSHIP
EFFECT OF CULTIVATION PARAMETERS ON THE MORPHOLOGY OF
RHIZOPUS ARRHIZUS AND THE LACTIC ACID PRODUCTION IN A BUBBLE
COLUMN REACTOR
Engineering in Life Sciences, 7: 1-8.
Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date…………………….…
Jin, B.
Manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..
Kelly, J. M.
Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 82
Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor. Engineering in Life Sciences, v. 7 (5), pp. 490-496, October 2007
NOTE: This publication is included on pages 83 - 89 in the print
copy of the thesis held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1002/elsc.200700002
CHAPTER 8
CONCLUSIONS AND FUTURE DIRECTION
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 90
1. Conclusions
1.1 A brief introduction
High production cost and low lactic acid yield are the key issues which limit lactic acid
production by Rhizopus sp in an industrial process. One of the major production costs is the
substrates used, which can be approximately a quarter of the total production cost if wheat-
flour is used (Åkerberg & Zacchi, 2000). In this study, waste potato starch from a local food
company was used to produce L(+)-lactic acid, which can significantly reduce the costs for
lactic acid production. The low lactic acid yield produced by the filamentous Rhizopus species
is mainly attributed to the formation of ethanol, a major by-product during lactic acid
production. The accumulation of ethanol is caused by poor mass transfer performance,
especially poor oxygen supply, due to the formation of viscous filamentous mycelia, or big
biomass aggregates and pellets during the fermentation processes. These forms of fungal
biomass can significantly reduce the biochemical reaction rate, resulting in low lactic acid
productivity. Therefore, the aim of this thesis study was to develop an efficient and cost-
effective biotechnological process for the production of lactic acid by R. arrhizus using waste
potato starch. The research focused on the optimization and scale-up of the lactic acid
production process.
1.2 Major achievements
Determination of suitable nitrogen sources
To enhance the lactic acid production by R. arrhizus DAR 36017, five commonly used organic
and inorganic nitrogen sources, ammonium sulphate, ammonium nitrate, urea, yeast extract
and peptone, were tested in terms of C:N ratio in order to select a technically and
economically suitable nitrogen source for lactic acid production. It has been found that the
lactic acid concentrations produced from waste potato starch using inorganic nitrogen sources
(ammonium nitrate and ammonium sulphate) were high and stable compared to organic
nitrogen sources (urea, yeast extract and peptone). As an important process operation
parameter, nitrogen sources were studied in a number of investigations. This investigation was
one of few studies which employed C:N ratio, rather than total nitrogen amount in lactic acid
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 91
production media. The results reveal that the C:N is a precise nitrogen-related parameter,
showing precisely the role of nitrogen in a fermentation process.
Development of a new inoculation method
In suspended cultures of R. arrhizus small pellets are the preferred morphological form for
lactic acid production in submerged reactor systems. However, the formation of pellets of
filamentous R. arrhizus depends upon the inoculation strategies and cultivation conditions,
which make the control of morphology complicated and difficult. The major achievement of
this research was the development of a new inoculation strategy, namely, using acid-adapted
precultures of R. arrhizus DAR 36017 as inoculum for lactic acid production. This inoculation
method is effective and an easy technique to control the morphology of R. arrhizus in a
desirable form, such as small pellets, in the reactors.
Scale-up of the lactic acid production process to reactor systems
The use of the acid-adapted precultures resulted in the success of scale-up processes from
shake flask cultures to bioreactor systems. This was one of few studies comparing the physical
morphologies and metabolic activities in lactic acid fermentation systems employing two
commonly used bioreactors, namely, a stirred tank reactor and a bubble column reactor. In the
both reactor processes, over 90% fungal biomass was formed as small pellets. The success in
controlling the fungal morphology led to the lactic acid yields of 86 - 89 % based on 100 g/L
waste potato starch. The accumulation of ethanol, a major by-product during lactic acid
production by Rhizopus strains, was minimized due to the formation of small pellets. The
results also showed that the BCR was a more suitable bioreactor for the cultivation of
filamentous Rhizopus for lactic acid production than the STR in terms of the lactic acid yield
and productivity. The better performance of the BCR was possibly attributed to the simpler
reactor structure and lower shear stress environment generated in the BCR, which favoured the
cultivation of Rhizopus. Further study on a simulated scale-up process revealed that the
increase of the scale-up steps resulted in the growth of big and compact pellets, and
consequently, resulted in low lactic acid yield. Therefore, a one-step scale-up process may be
feasible for an industrial-scale bioreactor system.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 92
Comprehensive optimization of cultivation conditions
The optimization of the cultivation conditions using the newly developed inoculation method
was carried out in the laboratory scale BCR process. The cultivation parameters, including
carbon sources, waste potato starch concentration, growth pH, sparger design and oxygen
supply were optimized comprehensively in the BCR. The results further proved the
relationship between the morphological form, and the lactic acid yield and productivity. A
high lactic acid concentration of 103.8 g/L, with a yield of 87 %, was achieved in 48 h
cultivation with the morphological form growing as uniform small pellets.
1.3 Summary
This project successfully developed an inoculation strategy, optimized the cultivation
conditions, and scaled-up the lactic acid production process from shake flask cultures to two
different bioreactor systems. The lactic acid yield achieved in this study is comparable to those
reported processes, which either used complicated and expensive immobilization systems or
other low efficient morphology control strategies by Rhizopus fermentations. It is worth noting
that waste potato starch was used as the substrate for lactic acid production in this study,
which will significantly reduce the production cost and make the process more competitive in
an industrial process.
2. Future direction
2.1 Enhancement of lactic acid yield and productivity
Firstly, the high lactic acid yield of 94 %, with a productivity of 5.0 g/L·h, achieved using a
immobilized Rhizopus strain (Efremenko et al., 2006) indicates that it is possible to further
enhance the lactic acid yield and productivity via bioprocess optimization, including
optimization of cultivation parameters such as inoculum size, agitation speed, impeller design
in the STR and improvement of pellet characteristics in the STR and BCR.
The research on optimization of cultivation parameters is recommended to combine the
quantitative study on the characterisation of pellet formation of R. arrhizus and of the
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 93
rheology of the fermentation broth. Uniform small pellets are preferred for lactic acid
production by Rhizopus strains. The pellet’s characteristics such as size and porosity have
been proved to be important for the performance of fermentation broth (Cui et al., 1998). In
the literature, intensive studies have been performed on quantitative analysis of the
morphological variation of filamentous Aspergillus fungi, which are widely used for
production of citric acid, amylase and other enzymes (Papagianni, 2004). Studies on the
formation of pellets of the filamentous fungus, R. arrhizus, may improve the characteristics of
the fermentation broth rheology, which influences significantly the mixing, mass and heat
transfer capacity in a reactor. The research results can provide more precise information on
how to efficiently control the morphology of R. arrhizus in desirable forms and maintain the
rheology of the fermentation broth in a good condition benefiting lactic acid production.
Secondly, the lactic acid yield may be improved by using genetically modified Rhizopus
strains. Yields of lactic acid are compromised primarily because Rhizopus also produces
ethanol with fermentative growth. The production of lactic acid from pyruvate is catalysed by
lactate dehydrogenase (LDH) under aerobic conditions. On the other hand, pyruvate
decarboxylase (PDC) catalyses the conversion of pyruvate to acetaldehyde, which is
subsequently reduced to ethanol by alcohol dehydrogenase (ADH) under anaerobically
stressed growth conditions that may result from inadequate aeration or mycelial clumping.
Therefore, the production of lactic acid competes for available pyruvate with ethanol
fermentation. Consequently, it is expected that lactic acid fermentation could become more
effective if the LDH activity is increased, and activities of ADH and PDC are decreased at the
same time.
Skory and his coworkers (1998, 2004) have developed several mutants with modified ADH
activity and transformants with modified LDH in order to enhance lactic acid yield. Some of
these modified strains did produce more lactic acid than the parent strains. However, the lactic
acid yields achieved using genetically modified Rhizopus were lower than those obtained by
controlling the morphology of Rhizopus. Therefore, research on using genetically modified
strains of Rhizopus should be combined with research on morphology control and
characterisation in order to maximise the yield and productivity of lactic acid.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 94
2.2 Scale-up of bench-scale process to pilot plant
The scale up of the lactic acid production process into a pilot plant is an essential step towards
a commercial process. So far, research on scale up of lactic acid production between reactors
was only reported by Miura and his coworkers (2003, 2006). The results from this project
have shown that a one-step scale-up process is optimal because high lactic acid yield can be
achieved. However, it may not be industrially feasible to scale-up lactic acid production from
shake flask cultures to reactors directly. A promising approach may be to prepare the
precultures in bench-scale or pilot-scale reactors, or avoid pellet formation in precultures until
precultures are inoculated into industrial reactors. Therefore, further study on scale-up of the
lactic acid production process by R. arrhizus from bench-scale to pilot-scale is necessary,
which needs a better understanding of the mechanism of pellet formation as well.
3. References
Åkerberg, C., Zacchi, G., 2000. An economic evaluation of the fermentative production of
lactic acid from wheat flour. Biores. Technol. 75: 119-126.
Cui, Y.Q., van der Lans, R.G.J.M., Luyben, K.Ch.A.M., 1998. Effects of dissolved oxygen
tension and mechanical forces on fungal morphology in submerged fermentation.
Biotechnol. Bioeng. 57: 409-419.
Efremenko, E.N., Spiricheva, O.V., Veremeenko, D.V., Baibak, A.V., Lozinsky, V.I., 2006.
L(+)-Lactic acid production using poly(vinyl alcohol)-cryogelentrapped Rhizopus oryzae
fungal cells. J. Chem. Technol. Biotechnol. 81, 519–522.
Liu, T., Miura, S., Yaguchi, M., Arimura, T., Park, E. Y., Okabe, M., 2006. Scale-up of L-
lactic acid production by mutant strain Rhizopus sp. MK-96-1196 from 0.003 m3 to 5 m3
in airlift bioreactors. J. Biosci. Bioeng. 101, 9–12.
Miura, S., Arimura, T., Hoshino, M., Kojima, M., Dwiarti, L., Okabe, M., 2003. Optimization
and scale-up of L-lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in
airlift bioreactors. J. Biosci. Bioeng. 96, 65–69.
Papagianni, M., 2004. Fungal morphology and metabolite production in submerged mycelia
processes. Biotechnol Adv. 22, 189–259.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 95
Skory, C.D., 2004. Lactic acid production by Rhizopus oryzae transformants with modified
lactate dehydrogenase activity. Appl. Microbiol. Biotechnol. 64, 237–242.
Skory, C.D., Freer, S.N., Bothast, R.J., 1998. Production of l-lactic acid by Rhizopus oryzae
under oxygen limiting conditions. Biotechnol. Lett. 20, 191–194.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 96
APPENDIX
PRODUCTION OF FUNGAL BIOMASS PROTEIN USING MICROFUNGI FROM
WINERY WASTEWATER TREATMENT
Zhan Ying Zhang a, Bo Jin a,b, Zhi Hui Bai a,c, Xiao Yi Wang a
a School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA 5005,
Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia
c Research Centre for Eco-Environmental Sciences, Beijing 100085, China
Bioresource Technology 2008, 99: 3871-3876.
Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 97
Zhang, Z.Y., Jin, B. and Kelly, J.M. (2008) Production of fungal biomass protein using microfungi from winery wastewater treatment. Bioresource Technology, v. 99 (9), pp. 3871-3976, June 2008
NOTE: This publication is included on pages 97 – 103 in the print
copy of the thesis held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1016/j.biortech.2006.10.047