msc final
TRANSCRIPT
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Chemical and Biological Engineering Department
MSc in Environmental and Energy Engineering
Algae Screening: Spirulina sp.
Name : Christine Ho Registration no : 130111904 Supervisor : Prof. Will Zimmerman Date : 27th August 2014
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Abstract The objective of this study was to evaluate the growth of blue-green algae Spirulina sp.
using the method airlift loop bioreactors to cultivate the algae. In airlift loop bioreactors, the
medium was supplied with CO2 nutrient by bubbling it to the medium at 30 minutes per day.
Its growth rate was compared without the presence of CO2 sparging. Besides that, the
Spirulina was tested with different concentrations of copper and acetaldehyde to determine
how well the Spirulina adapts in different growth conditions. Heavy metal copper is toxic to
microalgae but results shows that Spirulina could adapt in the tested concentration of 2 mg/L
and 5 mg/L over the span of 11 days. Spirulina adapts well with addition of acetaldehyde in
concentration between 100-150 over the span of 12 days. Therefore, it is suitable to
remove heavy metal of copper at and treat flue gas from industry emission that contains
acetaldehyde at the tested concentration range. Flask cultures were also used to compare
different culturing methods without CO2 bubbling. It shows that photosynthesis and growth
was inhibited when the same copper concentration was added in flask and resulted in cell
death. Spirulina added with acetaldehyde remains a linear growth and had a higher specific
growth rate when compared with ALB culture. It is concluded that, there is a difference on
how Spirulina cells react on different culturing methods and parameters such as pH will
affect the growth.
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Table of Contents Abstract .................................................................................................................................... 2
1. Introduction ....................................................................................................................... 5
2. Background ....................................................................................................................... 6
3. Project overview ............................................................................................................... 6
4. Objectives and Research Hypothesis ............................................................................... 7
5. Chemical Analysis Procedures ......................................................................................... 8 5.1 Chlorophyll concentration determination ....................................................................... 8 5.2 Protein determination .................................................................................................... 8 5.3 Dry weight determination .............................................................................................. 9
6. Biochemistry of CO2 Fixation ............................................................................................ 9
7. Effect on Spirulina Growth .............................................................................................. 11 7.1 Effect of pH ................................................................................................................. 11 7.2 Effect of Light Intensity ................................................................................................ 11 7.3 Effect of Mass Transfer ............................................................................................... 11 7.4 Effect of Mixing ............................................................................................................ 12 7.5 Effect of CO2 concentration ......................................................................................... 13 7.6 Effect of O2 accumulation ............................................................................................ 13
8. Mass Cultivation of Algae using ALB .............................................................................. 14
9. Metal Adsorption ............................................................................................................. 15
10. Copper Toxicity on Algae ............................................................................................ 15
11. Wastewater Treatment using Microalgae .................................................................... 16
12. Algal Biomass Harvest and Drying .............................................................................. 17
13. Experimental Methods ................................................................................................ 18 13.1 Materials ...................................................................................................................... 19
13.1.1 Microalgae ........................................................................................................... 19 13.1.2 Medium Recipe .................................................................................................... 20 13.1.3 Copper (II) Sulphate ............................................................................................. 21 13.1.4 Acetaldehyde ....................................................................................................... 21
13.2 Main Apparatus ........................................................................................................... 22 13.2.1 ALB ...................................................................................................................... 22 13.2.2 Spectrophotometer (DR2800) .............................................................................. 23
13.3 Experiment Settings .................................................................................................... 24 13.3.1 Experiment Setting for ALB .................................................................................. 24 13.3.2 Experimental Setting for Flasks ........................................................................... 24
14. Experiments ................................................................................................................ 25 14.1 Experiment I: Growth Rate of Spirulina (Flask Culture) .............................................. 25 14.2 Experiment II: Reaction of Spirulina with Acetaldehyde ............................................. 25
14.2.1 Flask Culture ........................................................................................................ 25 14.2.2 With ALB .............................................................................................................. 25
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14.3 Experiment III: Reaction with Heavy Metal, Copper (II) Sulphate ............................... 26 14.3.1 Flask Culture ........................................................................................................ 26 14.3.2 With ALB .............................................................................................................. 26 14.3.3 Preparation of Copper concentrations ................................................................. 26
14.4 Microflotation ............................................................................................................... 27 14.5 Preliminary Experiment for Metal Adsorption .............................................................. 27
15. Results and Discussions ............................................................................................. 29 15.1 Initial Observation ....................................................................................................... 29 15.2 Flask Culture compare with ALB ................................................................................. 29 15.3 ALBs Comparisons ..................................................................................................... 31
15.3.1 Control groups ..................................................................................................... 32 15.3.2 Spirulina with added acetaldehyde ...................................................................... 33 15.3.3 Spirulina with added CuSO4 ................................................................................ 33
15.4 Flask Cultures ............................................................................................................. 33 15.4.1 Spirulina with added CuSO4 ................................................................................ 33 15.4.2 Spirulina with added acetaldehyde ...................................................................... 35
15.5 Specific Growth Rate .................................................................................................. 37 15.6 Further Discussions .................................................................................................... 42
16. Limitations ................................................................................................................... 45
17. Conclusions ................................................................................................................. 45
18. Future works ............................................................................................................... 46 18.1 Determine the Protein content .................................................................................... 46 18.2 Reaction of acclimatised Spirulina with Acetaldehyde ................................................ 46 18.3 Microbubbles ............................................................................................................... 46
19. Acknowledgement ....................................................................................................... 47
20. Reference .................................................................................................................... 48
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1. Introduction The aim of this research is to investigate a sustainable and environmental friendly method to
treat wastewater using algal biomass. The algae species Spirulina has yet to be studied in
the Chemical and Biological Department, Sheffield. Therefore, there is an interest to
investigate more about this alga. Spirulina is an unbranched, helicoidal, filamentous
freshwater blue-green algal or also known as a cyanobacterium (Belay, et al., 1993). It is
commonly sold as a food supplement due to its rich protein content. Recently there is more
emphasis on Spirulina for its benefits to industrial applications.
The waste gases such as carbon dioxide and acetaldehyde are emitted from biological
processes and industry, these gases affects the environment adversely as acetaldehyde is a
toxic organic pollutant and carbon dioxide is a greenhouse gas. Algae are known to be able
to treat flue gasses. It is an ideal solution as carbon dioxide emitted will be used as
feedstock to algae growth and it can remove acetaldehyde and produce biomass for biofuels.
This project aims to screen and study Spirulina acclimation on acetaldehyde for
acetaldehyde removal in flue gas treatment.
There is a global concern regarding the release of heavy metals to the environment. Metals
such as cadmium, zinc, copper, lead and mercury are commonly detected in industrial
wastewaters. These metals are non-biodegradable and cause adverse effects to the aquatic
life. It is necessary to treat these wastewaters before discharging them.
There are chemical methods from aqueous solution such as precipitation, electrolysis, ionic
exchange, filtration and evaporation (Nalimova, et al., 2005). However, these methods are
uneconomical, low efficiency in heavy metal removal and require slag burial. Biological
methods are able to metal detoxify and remove heavy metals from the aqueous solution.
Adsorption process is known to be an effective method to remove heavy metal ion. Algae,
plant wastes, bagasse fly ash and recycled coal fly ash are known absorbents. (Hui, et al.,
2005, Wan Ngah & Hanafiah, 2008, Gupta & Ali, 2000). Microorganisms are able to
accumulate a wide range of heavy metal concentrations and convert it into inactive form.
From the technical review, it was reviewed that Spirulina is an effective adsorbent in heavy
metal ions removal. It is also easy to culture and an inexpensive method.
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2. Background The method chosen to cultivate Spirulina is a novel method that was introduced in the
University of Sheffield. There are advantages of using this method, which is to save energy
and to save cost for large scale of algae biomass production. So far, this method has not
been done with this algae species in this department. Therefore there is an interest to study
and conduct this experiment. This method uses an airlift loop bioreactor (ALB) which has
CO2 being bubbled from the bottom of the reactor via a ceramic diffuser. This method was
chosen because it is able to grow algae at a faster rate as to compare to conventional
methods such as open pond and tubular reactor. In theory, the circulation, mixing and mass
transfer that occurs in the airlift loop bioreactor is able to enhance the algae growth.
3. Project overview The aim of this project is to do algae screening for Spirulina algae. All the screenings were
done in ALB and flask culture to compare its effects on Spirulina growth and its
acclimatisation tendency.
The first screening is to study the effects of CO2 enriched bubbles on the growth of Spirulina
algae in an ALB and compare it with a controlled ALB without any enriched CO2 bubbles
being supplied to its growth medium.
The second screening is to test Spirulina with an organic contaminant. The organic
contaminant chosen for this experiment is acetaldehyde. The Department of Chemical and
Biological Engineering, Sheffield has conducted several experiments regarding the reaction
of acetaldehyde with different strains of algae. However, all these are marine algae strains
and a freshwater cyanobacterium was not studied before.
The third screening is to test Spirulina as biosorbent with a heavy metal. In this experiment,
different concentrations of copper (II) sulphate will be used. Spirulina is known to be a very
efficient biosorbent and different concentrations of copper are used to test its adsorption
efficiency.
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4. Objectives and Research Hypothesis Based on the literature review conducted, investigation on the points below was carried out.
• The growth Spirulina algae using ALB novel method
• The difference of Spirulina growth with and without enhanced CO2 supply
• The growth of Spirulina when added with copper (II) sulphate
• The acclimatation of Spirulina when added with acetaldehyde
• Compare the growth rate of Spirulina in different growth conditions
Hypothesis:
• The bubbling of CO2 in to the algae solution enhances its growth rate
• The copper (II) sulphate is adsorbed by the Spirulina
• Spirulina solution will acclimatized in the addition of acetaldehyde
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5. Chemical Analysis Procedures These are the methods and procedures mentioned by Vonshak (1997, p.214-215) to
analyse the Spirulina in further detail. The optimum growth conditions are at 35 ℃ and pH
9.8. The Spirulina cultures can be preserved for more than 6 moths on solidified medium
using 1.2-1.5 % of agar and kept at low light of 10-20 µμmol m!!s!! and 20℃. However, the
cultures must not be heavily contaminated by bacteria.
5.1 Chlorophyll concentration determination The chlorophyll content of Spirulina can be determined by take samples of 5 ml from the
algal suspension and centrifuges for 5 minutes at 3500 rpm and the supernatant is then
discarded while the pellet is kept. Alternatively, using a Whatman GF/C filter at 25 mm
diameter to filter it and re-suspend the sample in 5ml methanol and ground it in a glass
tissue homogenizer. The samples are then incubated in water at 70 ℃ for 2 minutes and
centrifuged; the clear supernatant is used for the chlorophyll measurements. The factor for
Spirulina is 13.9.
𝐶ℎ𝑙𝑜𝑟𝑜𝑝ℎ𝑦𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔𝑚𝑙
) = 𝑂𝐷!!" !"×13.9 (1)
5.2 Protein determination The pellet taken from chlorophyll measurements could be used to determine the protein
concentration by drying it with gentle stream of air or N2. The pellet is added with 2 ml of 0.5
N NaOH and incubated for 20 minutes at 100 ℃. The tubes were covered to prevent
evaporation. The supernatant is kept after centrifuged and 2 ml of hot 0.5 N NaOH at 70 ℃ is
added to the volume. The mixture is well mixed and centrifuged again and combines with
supernatant. For colour reaction, 0.1-0.5 ml supernatant is used and 0.5 N NaOH to a final
volume of 1 ml. BSA is used as a standard in the range of 50-200 mg.
Reagents preparations for colour reaction:
A : 2 % Na2CO3
B : 0.5% CuSO4.5H2O
C : 1% Na-tartarate
D : A (48 ml) + B (1 ml) + C (1 ml)
The reagents are well mixed and prepared fresh each time. D (4 ml) is added to the 1 ml
sample for 10 minutes before adding 1 ml of Folin-Ciocalteus reagent diluted with water at
1:1. The absorbance reading was taken at 660 nm after 30 minutes.
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5.3 Dry weight determination Sample of 25-50 ml from algal suspension is weighted and filtered through a Whatman GF/C
filter 47 mm in diameter was dried in an oven for 2 hours at 105 ℃ in a glass petri dish. 20 ml
of acidified water that pH 4 is used to wash the samples to remove the algae from insoluble
salts. After drying, the filter is cooled in a desiccator for 20 minutes before re-weighing.
6. Biochemistry of CO2 Fixation The overall reaction of photosynthesis is when CO2 is converted into glucose with the help of
ATP by carboxylase activity of enzyme RuBisCo in Calvin Cycle.
𝐶𝑂! + 𝐻!𝑂 + 𝐿𝑖𝑔ℎ𝑡 → (𝐶𝐻!𝑂)! + 𝑂! (2)
In cyanobacteria, the photosynthesis depends on RuBisCo; which has a low affinity for CO2
(Moroney & Somanchi, 1999). Microalgae are able to overcome the problem of CO2 diffusion
by accumulating HCO!!, which diffuses through the membrane slower than CO2. The enzyme
carbonic anhydrase is used to catalyse the CO2 for RuBisCo.
𝐶𝑂! + 𝐻!𝑂 → 𝐻𝐶𝑂!! + 𝐻! (3)
An increase in pH occurs due to CO2 uptake in photosynthesis, however when pH increases,
the CO!!! increases while HCO!! and CO2 decrease which inhibits photosynthesis due to lack
of CO2. Buffer solutions such as HEPES or acid addition are normally used in culture
medium to maintain pH at a certain level.
However, for ALB experiments neither buffer solution nor acid were added because the daily
CO2 dosing into ALB was able to neutralise the pH. The location of RuBisCo for
photosynthesis type in cyanobacteria is in the carboxysomes and it has the ability to
concentrate CO2 (Moroney & Somanchi, 1999).
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Figure 1: Carboxysomes and pyrenoids in different photosynthetic organisms. (A) Electron
micrograph of the cyanobacteria Anabaena; (B) green alga C. reinhardtii; (C) diatom
Amphora; (D), Immunogold labelling of the pyrenoid of C.reinhardtii with and anti-Rubisco
antibody. Bars = 0.5 𝜇m. Cs=Carboxyme; Py=pyrenoid (Moroney & Somanchi, 1999)
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7. Effect on Spirulina Growth There are a many parameters that will affect the algae growth such as light intensity,
temperature, pH, growth medium, cultivation methods, and contaminations and so on.
Microalgae cultures are susceptible to contamination by other species of microalgae, viruses,
bacteria, fungi and protozoa. The effects can alter the cell structure of microalgae and
reduces its yield. However, impurities in culture are normally acceptable if microalgae
biomass is used for biofuels, waste treatment, biofertilizers and CO2 fixation (Pandey, et al.,
2014).
7.1 Effect of pH For, pH, it could be a limiting factor which affects the metabolic rate of microalgae,
physiological growth and biomass production (Fagiri, et al., 2013). Spirulina is grown in huge
quantity is tropical and subtropical water source which have pH of up to 11. According to a
few journals, the optimum pH for Spirulina growth is between pH 9-10. With increased pH,
the environment will prevent auto inhibition effect on cell growth.
7.2 Effect of Light Intensity However, even though continuous supply of light promotes photosynthesis, prolonged light
exposure to high light conditions to green plant tissues will lead to photoinhibition of
Photosystem II (Bladier, et al., 1994).Thus, a decrease in yield and the rate photosynthesis
in light saturated conditions. Fluorescent lights are widely used, however new full-spectrum
fluorescent bulbs are able to close to natural light. According to Andersen (2005),
incandescent lighting should be avoided. By increasing light intensity does not means an
increase algae growth, it may be harmful to algal cells. Cultures are commonly illuminated at
30-60 𝜇𝑚𝑜𝑙 𝑚!!𝑠!! (Andersen, 2005).
7.3 Effect of Mass Transfer Hydrodynamics and mass transfer characteristics are important factors in factors in algae
cultivation including the overall mass transfer coefficient (kLa), mixing in reactor, gas bubble
velocity and gas holdup. The kLa depends on a few factors such as the type of sparger,
design of reactor, temperature and liquid viscosity of medium. Mass transfer coefficient for
liquid-gas film theory is presented in Figure 2. From the equation (4), mass transfer rate is
proportional to the difference between two concentrations at the interface and interfacial
area.
𝑁! = 𝐾!𝑎(𝐶! − 𝐶!) (4)
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Figure 2: Interfacial dynamics of mass transfer for gas exchange (Al-Mashhadani, et al., 2012)
7.4 Effect of Mixing The design of the reactor needs to have efficient mixing mechanism to retain algal cells in
suspension, ensure high cell concentration, evenly distribute nutrients, thermal stratification,
and lower probability of photoinhibition and improve gas exchange.
It was reported that mixing at induced turbulent flow in open pond system would result in
high yield of algal biomass when its nutrients and environmental condition are optimum
(Ugwu, et al., 2008). It is also known that algae productivity is higher in mixed culture
compare to unmixed under the same parameters. A proper mixing can prevent photo-
sharing in culture. Mixing can be done directly by bubbling air into the airlift system. In open
pond system, paddle wheels are used to induce turbulent flow, in some photobioreactors
have baffles for mixing in algae culture and in stirred tank, impellers were used.
Mixing can be improved by increasing aeration rate, however at high aeration rate; it could
also cause shear stress to algal cells. Therefore, fine spargers were used to produce smaller
bubbles and reduce shear stress and increase gas dispersion. However, poor mass transfer
rate can occur by reduction in contact surface when bubble coalesce during bubbling and
form interface between the liquid medium, gas and the wall of the reactor. When gas
flowrate increases, the bubble diameter and gas bubble velocity increases. The baffles are
installed inside reactors to increase gas dispersion.
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7.5 Effect of CO2 concentration Algal cells can only tolerate CO2 up to a certain concentration and once exceeded, CO2 is
detrimental to the algal growth. Environmental stress cause by high CO2 reduces its
capacity for algal cells for carbon sequestration and culture pH will decrease due to
formation of high amount of bicarbonate buffer (Kumar , et al., 2011). Biomass productivity
increases with increasing CO2 % (v/v), however, this is only applicable to certain percentage.
Table 1 shows that at lower CO2 % (v/v), higher CO2 is sequestered in a three-stage serial
tubular photobioreactor for Spirulina. In aqueous environment, the dissolved CO2 exist in
equilibrium with H2CO3, HCO! ! and CO! !! which concentration depends on pH and
temperature. According to Carvalho et al (2006), microalgal cells prefer the uptake of HCO! !
over CO2 despite being a poor source of carbon when compared to CO2.
Table 1: CO2 sequestration capabilities for Spirulina sp. (Adapted from Kumar, et al. (2011)
Algal species % CO2 at influent (% v/v) % CO2 sequestered
Spirulina sp 6 53.29
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7.6 Effect of O2 accumulation During photosynthesis, water is split to oxygen and hydrogen ions in photosystem II reaction
(Figure 3). Oxygen trapped in the liquid culture is known to reduce photosynthetic efficiency
and causes toxic effect such as photo-bleaching. Therefore, an effective method to strip
oxygen from accumulation is required in reactors with poor gas exchange system. One of
the main disadvantages of using tubular photobioreactors is its inefficiency to strip O2 due to
its long tubular structure (Ugwu, et al., 2008). Stripping O2 from algal cells is a challenge that
ALB design has manage to improve on. As O2 accumulation inhibits the algae growth.
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Figure 3: Photosystems in chloroplast (Pearson Education, 2005)
8. Mass Cultivation of Algae using ALB From the experiment, high algal growth was obtained when operated at laboratory scale. In
order to apply this method in industry and to mass produce algae, a scaled up ALB is need.
However, there are challenges in up scaling ALB to pilot scale such as difficulty in providing
light source evenly, maintaining optimum temperature, proper mixing and ensure good mass
transfers. With larger a reactor, the cost of building and maintaining will also increase.
There are other additional modifications that are needed such as thermal insulation to
maintain optimum temperature, additional light source surrounding the reactor and within the
reactor. The pressure drop for the diffuser will be higher due to a higher and bigger ALB and
it will reduce its efficiency to produce bubbles at high transfer rate. The mixing mechanism
may not be effective and consideration of adding impellers to ensure a high algal biomass
yield may be needed. As volume of reactor increases, the productivity of the algal biomass
yield decreases (Ugwu, et al., 2008).
One of the main concerns is also the availability of land mass area for cultivation sites. For
Spirulina cultivation, it is not ideal to culture in outdoors as the UK weather is below its
optimum growth rate temperature unless it is insulated to maintain a certain temperature.
The temperature ranges from 1℃ to 21 ℃ in the UK (Met Office, 2014).
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9. Metal Adsorption Metal ions can be immobilized by functional groups that belongs to the proteins, lipids and
carbohydrates on the cell wall of the organism (Fang, et al., 2011) Cyanobacterium are
known to be good biosorbents for heavy metals in bioremediation. Spirulina is an effective
biosorbent and its adsorption of copper ions was discussed.
Based on the experiment conducted in Fang et al, (2011), the amount of copper ion being
adsorbed by Spirulina could be determined by adding Potassium nitrate into medium as a
supporting electrolyte. And its samples were taken from the mixture and shake for 2 hours
before centrifuging it. The centrifugation occurs at 12,000 rpm for 10 minutes and the
supernatant was analysed by flame atomic absorption spectrometry.
Within this OD range it is shown that algal cells had sufficient time to adapt to copper.
Spirulina tolerance to Copper is related to the sorption by cell walls and secretion of metal
excess into the culturing medium and its conversion into the form inaccessible for cells.
(Nalimova et al, 2005).
As copper concentration in medium increases, so does the intracellular content. When
CuSO4 was added initially, its cell content increased rapidly and gradually decreases after a
few days. Copper accumulation in Spirulina cells had a biphasic character; firstly, Cu2+ was
absorbed by cell walls rapidly and binds within the cells; secondly was releasing as reduced
Copper, Cu+ in to the medium (Nalimova, et al., 2005).
10. Copper Toxicity on Algae Addition of copper affected the photosynthesis and growth rate. Copper is a micronutrient for
growth, metabolism and enzyme activities for cyanobacteria but not at high concentrations
(Cid, et al., 1995). The range of concentration depends on the microorganism tolerance to
heavy metals, pH of nutrient medium, presence of chelating agents and cell density
(Nalimova, et al., 2005). As the copper concentration increased in the medium, a decreased
in pH was observed. The toxic effect towards Spriulina is on its growth and cell death. The
cell walls of algae have functional groups such as aminic, carboxylic, thiolic, sulphydrylic and
phosphoric group that are potential for metal binding (Solisio, et al., 2006). The biosorption
intensity depends on ligands, its distribution on the cell wall and affinity for ions.
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There are studies conducted that shows that toxicity of metals such as copper for instance
decreases with decreasing pH (Franklin, et al., 2000). As pH increases in medium, the
number of negatively charged sites on the algal surfaces also increased (Crist, et al., 1988).
The interaction between metals and algal surfaces involves electrostatic bonding, which may
result in increased toxicity and metal adsorption. And in the author’s (Franklin, et al., 2000)
experiment, Copper was significantly more toxic to Chlorella sp. at pH 6.5 as to compare to
pH 5.7.
11. Wastewater Treatment using Microalgae Discharging wastewater to aquatic environment which contains high nutrient such as
nitrogen and phosphorus may cause eutrophication and phytoplankton blooms. It is a
serious environmental problem due to pollution and affects the marine life. For instance, the
beach in Qingdao, China was covered by thick layer of green algae due to industrial pollution
(Guardian, 2013). It suffocates marine life by blocking sunlight from entering the ocean and
absorbs oxygen in the water. Removal of nutrients and toxic metals from the wastewater to
an acceptable limit is needed. Microalgae are able to remove toxic metals, nitrogen and
phosphorus efficiently. In wastewater treatment microalgae utilizes the nutrients from
wastewaters reduce eutrophication.
Lau et al. (1995) did a lab scale batch experiment to remove nutrients from the primary
settled municipal sewage. The author used the microalgae Chlorella vulgaris to remove
nitrogen and phosphorus from wastewaters at inoculum sizes
between 1×10! cells ml!! to 1×10! cells ml!!. The results show that the higher algal density
is able to remove over 90 % of NH! ! and 80 % of PO! ! over the span of 10 days. However,
the residual concentrations show a negative correlation effect on cell numbers and
chlorophyll content of the cultures. The efficiency of nutrient removal from wastewater is
directly related to the physiological activity of algae growth.
Microalgae were grown as immobilized and free cells to compare its ability to remove
nitrogen and phosphorus in batch cultures in urban wastewaters. Results from Ruiz-Marin et
al. (2010) shows that immobilized systems are better as it could facilitate separation of
biomass from treated wastewater. However, in terms of nutritional value of biomass it does
not represent advantages over free-cell system.
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12. Algal Biomass Harvest and Drying The process of biomass harvest is by removing algae from growth medium. This process
can contribute to 20-30% of the total cost of biomass (Pandey, et al., 2014). Selecting the
harvesting methods depends of the properties of microalgae, its cell density, size and the
desired final product.
The common techniques to harvest algae biomass are sedimentation, flocculation, flotation,
filtration and electrophoresis. The main process in harvesting biomass is to separate
microalgae biomass from growth medium and remove excess medium and maintaining
biomass concentration. A way to reduce the total cost of biomass is by introducing the
microflotation method. This technique uses microbubbles for separation process and was
develop in the University of Sheffield.
For instance, it was observed during experiment that algae in ALB settled at the bottom after
a day due to gravity. If harvesting is required when algae is widely distributed in the reactor
the sedimentation method would not be time effective. The microflotation method is fast and
able to separate up to 99% of algae and medium within half an hour. This would reduce
production cost significantly.
Algal biomass is dried to produce desired products such as fuel, food, drug, feed, 𝛽-carotene
and polysaccharides. The drying process dehydrates the biomass and extends its shelf life.
The common methods to dry algal biomass are spray drying, drum drying, freeze dying and
sun drying (Pandey, et al., 2014).
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13. Experimental Methods For the extent of this experiment, emphasis on Spirulina specific growth rate and optical
density was carried out. The experiment was carried out for a month. Spirulina was grown in
closed-batch system in four ALBs. Each ALB has the capacity of 3 litres.
The experiments were carried out in Kroto lab in The University of Sheffield at room
temperature(22 ± 2℃). The growth mediums were added to the ALBs and mixed with
deionized water using 1:1 ratio. 10 ml of inoculated Spirulina were added into each ALB. The
experimental was set-up as shown in 13.3.1.
Each ALB consist of,
Growth medium : 1.25 litres
Ionize water : 1.25 litres
Algae inoculum : 10 ml
The ALBs were connect and supplied with enriched 5% CO2 supply. The CO2 supply was
bubbled in to the ALB for 30 minutes every day to provide feedstock for the algae. To study
the growth rate, the optical density of the mixed solutions was measure using a
spectrophotometer. The wavelength was set to 595 nm as this is the recommended
wavelength for algae.
The results obtained from the experiments were recorded and discussed in the later section
of this report. Along with the ALBs, Spirulina was also cultured in Erlenmeyer flasks to
compare the effects on growth rate in different methods of culturing algal. The CO2 dosing
was done at the same time of the day each time and its OD data was recorded for a more
consistent data.
While taking the OD reading, the cuvettes were wiped dry at the outer surface and gloves
were worn to ensure the surface is clear. This is to ensure that the light scattered through
the sample suspension is accurate. A new dropper was used to collect samples for each
ALD and flask each time to limit possibility of contamination. To prevent contamination
during experiment, microsol solution was used to wash and clean the ALBs thoroughly in
order to kill bacteria and ensure it is sterile before starting the experiment.
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Before taking the OD reading, thorough mixing was ensured in ALB culture mediums
through CO2 bubbling. For flask cultures, the flask was swirled thoroughly to ensure uniform
mixing.
Lab coat, safety goggles and gloves were worn throughout the experiment duration. When
acetaldehyde was added into ALB, safely mask was worn and the lab window was opened
for ventilation. As acetaldehyde may cause health hazards if inhaled for prolong period. At
the end of the experiments, the algae mediums from ALB were collected and stored in 1 litre
duren bottles for algal biomass harvesting using microflotation.
13.1 Materials
13.1.1 Microalgae
Brief description of microalgal species,
Domain : Bacteria
Phylum : Cyanobacteria
Class : Cyanophyceae
Order : Oscillatoriales
Genus : Spirulina
Figure 4: Light micrograph of Spriulina shows an open helical shape. Bar at the bottom left
represents 20 um (Vonshak, 1997)
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13.1.2 Medium Recipe
The media recipe used was BG 11 (Blue-Green Medium), which is for freshwater algae and
protozoa (Stanier, et al., 1971). The following medium was prepared in the Department of
Molecular Biology and Biotechnology, Sheffield. The medium was made up to 1 litre with
deionized water. The pH was adjusted to 7.1 with 1M NaOH and the medium was
autoclaved for 2 hours at 15 psi to sterilize the medium. Some algae died when inoculated
into full-strength culture. Therefore, is a good precautious to use a diluted for cultivation. In
this experiment a 1:1 ratio of medium to water was used.
Table 2 : Media recipe for BG11
Stocks per litre
(1) NaNO3 15.0 g
per 500ml
(2) K2HPO4 2.0 g
(3) MgSO4.7H2O 3.75 g
(4) CaCl2.2H2O 1.80 g
(5) Citric acid 0.30 g
(6) Ammonium ferric citrate green 0.30 g
(7) EDTANa2 0.05 g
(8) Na2CO3 1.00 g
(9) Trace metal solution: per litre
H3BO3 2.86 g
MnCl2.4H2O 1.81 g
ZnSO4.7H2O 0.22 g
Na2MoO4.2H2O 0.39 g
CuSO4.5H2O 0.08 g
Co(NO3)2.6H2O 0.05 g
Medium per litre
Stock solution 1 100.0 ml
Stock solution 2-8 10.0 ml each
Stock solution 9 1.0 ml
21
13.1.3 Copper (II) Sulphate
The copper used in this experiment is copper (II) sulphate pentahydrate (CuSO4.5H2O)
purchased from Sigma Life Science. It is in crystal salt form and needs to be dissolved prior
using it. It is over 98% purity.
Figure 5: Copper (II) sulphate pentahydrate
13.1.4 Acetaldehyde
According SEPA (2011), acetaldehyde is a reactive substance that is mainly used as an
intermediate in the synthesis of other chemicals. It has a fruity smell at low concentration but
an unpleasant pungent smell at high concentrations. It is a volatile organic compound (VOC)
that evaporates easily and is flammable. Acetaldehyde is also toxic when applied externally
for prolonged periods, an irritant, and a probable carcinogen (U.S EPA, 1994).It has the
chemical formula of CH3CHO. The acetaldehyde purchased for this experiment is from Fluka
Analytical at over 99.5% purity.
Figure 6: Acetaldehyde Lewis structure
22
13.2 Main Apparatus
13.2.1 ALB
Each ALB has a capacity of 3 L and by using the same ALB in Ying, et al. (2013), the
dimension of the ALB is 285 mm in height and 124 mm in diameter. Each ceramic diffuser
has a diameter of 78 mm and a pore size of 20 𝜇m. The gas draught tube is 170 mm in
height and diameter of 95 mm, it is hung 30 mm above the diffuser. The ceramic diffuser is
able to dose fine bubble of size 600 𝜇m. The bubble size produced is 30 times larger than its
pore size. The ALB (Figure 7) has gas draught tube inside its reactor to promote
recirculation for the bubbles and to increase mass transfer time and retention time. There is
a different design between the ceramic diffuser in ALB Control 1 and Control 2 as shown in
Figure 8. The rest of the ALB diffusers are the same as Control 2.
Figure 7: Structure of a 3 litre airlift loop bioreators (Ying , et al., 2013)
Figure 8: Ceramic diffuser of ALB : Control 1 (Left), Control 2 (Right)
23
13.2.2 Spectrophotometer (DR2800)
This spectrophotometer was used to measure OD (Abs) of the algae in the ALBs at
wavelength 595 nm to investigate the growth behaviour for the algae in different screenings.
This was done by taking samples (2 ml) and inserts it in cuvettes. The OD of a material is a
logarithmic ratio of the radiation falling upon a material, to the radiation transmitted through a
material.
𝐴! = − log!"𝐼!𝐼!
(5)
𝐴!= absorbance at certain wavelegth of light
𝐼!= Intensity of the radiatian (light) that pass through material
𝐼!= Intensity of radiation before passes through material
The purpose for measuring OD is to analyse the cell concentration. OD increases as
microalgae particles increases in suspension. The spectrophotometer measures the turbidity
of particles present in a suspension. In a spectrophotometer, a specific wavelength was
initially chosen and a cuvette filled with deionized water was used to calibrate the machine
so that the absorbance is calibrated to zero. During this process, a beam of light passes
through the water sample and the light intensity was transmitted and scattered as it passes
through the sample compartment where the cuvettes were inserted. While measuring the
algal suspension, the greater the scattered of light detected indicates that smaller light
intensity passes through the sample compartment. Thus, the sample is more turbid and
indicates the particle concentration in the suspension. The particles represent microalgal
cells.
Figure 9: Basic principle in a spectrophotometer in measuring optical density from
suspension
Light source Light intensity
Sample compartment
Light transmi4ed sca4ered Detector
24
13.3 Experiment Settings
13.3.1 Experiment Setting for ALB
As shown in Figure 10, the ALBs are connected to the CO2 supply. Each of the connecting
pipe has an adjustable valve to adjust the flowrate of the CO2 entering the ALB. The
fluorescent lamps are adjacent to the ALBs. The spectrophotometer is in between the CO2
gas cylinder and the ALBs. The flowrate of each ALB is maintained at about 0.7 L/min which
is within the optimum range for algal culturing.
Light source is needed as a nutrient supply to promote photosynthesis for the growth of
algae. Constant light source were supplied to the ALB by using two circular lamps (28 W
each) and a tubular lamp (33 W). The fluorescent lamps were switched on constantly to
promote a linear growth of algae through the process of photosynthesis.
CO2 gas cylinder contains 5% CO2 , 95% N2 at 200 bar pressure.
Figure 10: Experiment set up for ALB
13.3.2 Experimental Setting for Flasks
Erlenmeyer flasks were used ranging from 500 ml to 250 ml, depending on the amount of
medium used. The flasks were located beside a light source (florescent lamp) to gain
nutrient for cell growth. The OD was taken at the same time when OD from ALB cultures
was taken. Both of the experiments were running adjacently.
25
14. Experiments
14.1 Experiment I: Growth Rate of Spirulina (Flask Culture) In order to cultivate Spirulina algae, a growth medium was made using the medium recipe
(BG-11) described in 13.1.2. The medium was mixed with deionized water at 1:1 ratio.
Growth medium : 200 ml
Deionized water : 200 ml
Inoculated algal : 50 ml
These ingredients were mixed in a 500 ml Erlenmeyer flask. The OD was recorded by taking
samples (2 ml) and measured in spectrophotometer. The OD is recorded over the span of 12
days using the equation (1) and was plotted as OD against time. Its OD was compared with
ALB to determine the relation when fine bubbling dosing is added.
14.2 Experiment II: Reaction of Spirulina with Acetaldehyde
14.2.1 Flask Culture
The two Erlenmeyer flasks were used to test acetaldehyde with 150 ml of growth medium
added in each flask. One was made as a control and the other one was tested with
acetaldehyde with an initial concentration of 10 𝜇𝑙. The concentration was further increase to
20 𝜇𝑙 to observe the adaptability of Spirulina towards acetaldehyde. The amount of
acetaldehyde added was based on the scaled down amount of acetaldehyde added into
ALB.
For instance,
𝑉𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝐴𝐿𝐵𝑉𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝐹𝑙𝑎𝑠𝑘
=𝐴𝑐𝑒𝑡𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 𝑎𝑑𝑑𝑒𝑑 𝑖𝑛𝑡𝑜 𝐴𝐿𝐵 𝜇𝑙
𝑥 (6)
x= Amount of acetaldehyde needed to be added into flask culture (𝜇𝑙)
14.2.2 With ALB
The algae were grown in the ALB for 3 weeks before acetaldehyde was added into the ALB.
An initial amount of 100 𝜇𝑙 were added into the ALB every day for a span of 3 days and its
optical density was being monitored.
After noticing there was no significant decline in OD, the acetaldehyde amount was increase
to subsequently 150 𝜇𝑙 and 200 𝜇𝑙.
26
14.3 Experiment III: Reaction with Heavy Metal, Copper (II) Sulphate
14.3.1 Flask Culture
The algal was tested with different concentrations of copper (II) sulphate. Three 250 ml
Erlenmeyer flasks were used. Each flask consist of 150 ml of algae medium, One was set to
be a control with no copper (II) sulphate and the other three flask were tested with
concentration at 2 mg/L and 5 mg/L. The OD was measured and monitored for 7 days at
wavelength 595 nm. The Spirulina’s growth behaviour was studied and analysed.
14.3.2 With ALB
The 2.5 litre ALB was tested with 2 mg/L of copper concentration initially for 4 days and
subsequently increased to 5 mg/L. To determine the adaptability of the Spirulina as higher
concentration of copper were added. The concentration was increase when there was no
significant decrease in OD value.
14.3.3 Preparation of Copper concentrations
The preparation for Cu concentrations at 2 mg/L and 5 mg/L were done by using the
equations below for pre-experiment calculations.
𝐶!𝑉! = 𝐶!𝑉! (7)
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =𝑀𝑎𝑠𝑠𝑉𝑜𝑙𝑢𝑚𝑒
(8)
C1 = Concentration of CuSO4 at 1 g/L
C2 = Concentration of CuSO4 desired in ALB/flask
V1 = Volume of CuSO4 required to reach desired concentration
V2 = Volume of ALB/flask
An initial concentration of 1g/L was made by measuring 1 g of CuSO4.5H2O solid using a
highly sensitive weighing balance and mixed with 1 litre of deionized water until it is
dissolved. The Copper solution was then being customized further into different
concentrations by adding different volume of Copper in different flask and ALB. A 500-
5000 𝜇𝑙 pipette was used to obtained higher accuracy measurements.
27
Table 3: Amount of CuSO4 at 1 g/L added into ALB to achieve desired concentraton
Desired Concentration
(mg/l)
Amount inserted into 2.5
litre ALB (ml)
Amount inserted into 150
ml flask (ml)
2 5.0 0.3
5 12.5 0.75
14.4 Microflotation Microflotation is an important process in culturing algae. It is an economical and effective
way to harvest algae biomass. Metallic salts were used to coagulate the algae into flocs. The
microbubbles were produced using a fluidic oscillator and diffused into the ALB. After 30
minutes, there was a clear separation between the algae biomass and its growth medium.
The algae biomass is then harvested by draining the medium by opening a valve at the
bottom of the reactor.
14.5 Preliminary Experiment for Metal Adsorption The following experiment was carried out to determine the copper adsorption in Spirulina.
The aim was to determine the tolerance of cyanobacterium to copper for accumulation in this
heavy metal in its algal cells.
The experiment was carried out in a 250 ml Erlenmeyer flask. 150 ml of Spirulina with an
initial OD of 0.04 and 2 mg/L of copper was added to the flask. The absorbance for copper
used is 650 nm. The OD was monitored for 2 hours for any changes in OD with reading
taken every 15 minutes. The results showed that there were no changes in OD.
This method was proven to be not feasible due to failure to show the relation of copper being
adsorb in Spirulina. There are several factors why the experiment did not succeed such as
low density of Spirulina could not adsorb copper at that concentration. The suspension was
not separate and perhaps spectrophotometer was unable to detect its optimal wavelength.
An improvement would be to use a filter paper first to separate the algal biomass before
taking its OD or centrifuged the suspension beforehand and test it. A higher algae
concentration should be used as it has higher tolerance to copper toxicity.
The Langmuir adsorption model can be used to quantify the amount of adsorbate (copper)
adsorbed on an adsorbent (Spirulina) as a function of concentration at a specific
temperature. The Langmuir equation can be use is in linearized form as Ce/qe plotted against
Ce, a straight line shows that sorption is a monolayer.
28
The process of metal biosorption is fast and equilibrium could be reached within an hour.
The overall adsorption process is best described by pseudo-order kinetics (Keskinkan, et al.,
2004); the process was done using aquatic plants, which contains the process of biosorption
and bioaccumulation. The biosorption binds the metal and is initially fast and a reversible
process. The bioaccumulation is a slow, irreversible and ion-sequestration step. According to
Keskinkan et al. (2004), pH value at slightly below 6 is suitable for metal adsorption by C.
demersum at slight acidic environment and equilibrium was achieved within 20 minutes of
contact time. The kinetics of adsorption can be separated to stages of mass transfer,
sorption of ions onto sites and intraparticles diffusions.
Pseudo second-order equation (Keskinkan, et al., 2004):
𝑡𝑞!=
12×𝐾!×𝑞!! +
𝑡𝑞! (9)
qe =mass of metal adsorbed at equilibrium (mg/g)
qt =mass of metal adsorbed at time t (mg/g)
K’ =pseudo second-order rate constant of adsorption (g/mg min) (copper = 0.183 g/mg min)
29
15. Results and Discussions
15.1 Initial Observation Initially, the growth medium’s colour was transparent due to the minute amount of inoculum
(10 ml) being inserted into the ALB (2.5 L). After two weeks of CO2 bubbling, the medium
started to show visible Spirulina growth. It was deduced that only small amount of inoculum
was required to grow algae due to its fast growing tendency. The growth medium used was
suitable in this experiment as there was an increased in OD over time. Another possible
explanation could be also due to the CO2 supply had lowered the pH level, and affected the
growth rate initially. As the suitable pH to grow Spirulina is pH 9. The pH was tested after
CO2 bubbling and all four ALB medium had an average of pH 5.9. The acidic environment
due to the bubbling had inhibited the Spirulina growth. Therefore, by stopping dosing of
enriched CO2 for 4 days into the ALB, it enables the medium pH to increase to pH 9 which is
the ideal range to grow Spirulina and the green algal were visible in the ALB.
Even though same amount of inoculum were inserted into the ALBs, however the growth
rate differs. It is possible that the diffusers and pressure drop of each ALB had affected the
Spirulina growth rate. The cultivation of microalgae through bubbling enriched CO2 into the
ceramic diffusers in ALB has a gas transfer efficiency of only 13%-20% (Ying et al. 2013).
The CO2 supply enhanced the algal metabolism rate and acts as a buffer solution to
neutralize the increased pH due to Spirulina growth. As Spirulina OD increases, it is visual
that the colour of the culture became denser and dark green colour. The light source
became a limiting factor to the culture in ALB. As time passes and Spirulina continues to
grow, photo-shading will occur in the microalgal cells.
15.2 Flask Culture compare with ALB Based on the Experiment I results, it is deduced that the growth rate of Spirulina is faster in
the ALB as to compare to the cultivated Spirulina in a flask. Despite that the flask culture had
a higher OD initially, the ALB control group manage to surpasses in Spirulina growth as
shown in Figure 11. A linear growth was observed in all three cultures. This is due to the
continuous photosynthesis reaction from the 24 hours light source being supplied. The
enriched CO2 supply enhances the growth rate of Spirulina due to high mass transfer rate of
CO2 dosing. The flask result indicates that without the enhanced CO2 bubbling, microalgae is
still able to grow but at a slower rate. It can still receive nutrients from CO2 from the
atmosphere through simple gas diffusion from the medium surface.
30
Figure 11: Optical density of Spirulina compared between control groups of ALB and flask
Table 4: Optical density reading taken in the span of 12 days at absorbance 595 nm
Day Control 1 Control 2 Flask Culture
0 0.04 0.06 0.09
1 0.07 0.05 0.10
2 0.15 0.09 0.11
5 0.28 0.14 0.16
6 0.37 0.18 0.14
7 0.37 0.23 0.13
8 0.38 0.26 0.18
9 0.39 0.28 0.23
12 0.57 0.37 0.30
0
0.1
0.2
0.3
0.4
0.5
0.6
0 2 4 6 8 10 12
Op#
cal den
sity (A
bs) a
t 595 nm
Time (days)
Flask
Control 1
Control 2
31
15.3 ALBs Comparisons The Spirulina algae were cultured simultaneously and its reading was taken at the same
time to ensure a consistent data collection. Even though the flowrate the flowrate of CO2
being bubbled into the each ALB was different and may be the limiting factor of the
difference of Spirulina growth in each ALB.
Table 5: Optical density of Spirulina at 595 nm in ALB
Day Control 1 Acetaldehyde Copper Control 2
0 0.04 0.17 0.11 0.06
1 0.07 0.21 0.27 0.05
2 0.15 0.32 0.39 0.09
5 0.28 0.45 0.46 0.14
6 0.37 0.55 0.58 0.18
7 0.37 0.61 0.66 0.23
8 0.38 0.63 0.67 0.26
9 0.39 0.65 0.74 0.28
12 0.57 0.71 0.78 0.37
13 0.66 0.70 0.82 0.43
14 0.74 0.73 0.85 0.48
15 0.80 0.80 0.76 0.53
19 0.96 0.73 0.72 0.62
20 1.01 0.71 0.85 0.63
21 1.03 0.57 0.87 0.68
22 1.10 0.46 0.92 0.70
23 1.02 0.45 0.78 0.76
32
Figure 12: The growth comparisons between control groups, Spirulina added with
acetaldehyde and Spirulina added with CuSO4 over the span of 23 days in ALBs
15.3.1 Control groups
The growth of Spirulina in Control 1 exceeds Control 2 by approximately 25 %. Figure 12
shows that a linear growth on both controls. Two sets of control were used to provide
consistent data, however Spirulina in Control 1 has a significantly faster growth rate. There
are various factors that could contribute to a difference in growth such as CO2 mass transfer,
O2 accumulation and proper mixing in ALB. When difference of growth concentration is
higher, a higher mass transfer rate is deduced. It could be said that mass transfer rate in
Control 1 ALB is higher or diffusion of CO2 gas into medium was more efficient due to evenly
distributed bubble size.
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Op#
cal den
sity (A
bs) a
t 595nm
Time (Days)
Control 1
Acetaldehyde
Copper
Control 2
100 𝜇𝑙 150 𝜇𝑙
2 mg/L 5 mg/L
200 µμl
33
15.3.2 Spirulina with added acetaldehyde
A fixed amount 100 𝜇𝑙 of acetaldehyde was added daily from Day 7 to Day 12. After
monitoring that there were no significant changes in algal growth. The dosage was increased
to 150 𝜇𝑙 from Day 12 to Day 15. The growth rate decreased the most when 200 𝜇𝑙 were
added daily from Day 19 to Day 22 as shown in Figure 12.
Spirulina was able to acclimatise to the addition of 100 𝜇𝑙 and 150 𝜇𝑙 from Day 7 to Day 12.
However, when 200 𝜇𝑙 were added on Day 19, there was a rapid decreased in OD indicating
that the growth rate of Spirulina decreases. The high concentration of acetaldehyde may
have affected the photosynthesis rate and inhibited growth. It is shown that at this
acetaldehyde concentration, it is toxic to Spirulina.
15.3.3 Spirulina with added CuSO4
The ALB that was added with 2 mg/L from Day 12 to Day 15 and subsequently to 5 mg/L
from Day 19 to Day 22. When added with CuSO4, the growth of Spirulina was affected and
growth rate slowed down as some algae cell growth were inhibited. As presented in Table 5.
However, the OD did not reach a plateau as to compare with the flask culture (Figure 14)
The Spirulina OD was the highest initially but was surpassed by Control 1 at Day 15. As to
compare to Control 1, growth was reduced by about 20 %.
15.4 Flask Cultures
15.4.1 Spirulina with added CuSO4
After a day, it was noticed that the colour of the growth medium for 2 mg/L and 5 mg/L
turned from lime green to pale green colour in Figure 13. The sponges were used to prevent
the medium from contamination such as dusts and bacteria from contacting with the medium.
It shows that the OD decreased initially and remained plateau after that, which indicates that
the Spirulina’s growth was inhibited. A pH meter was used to measure the pH of the three
flasks and it was observed that the pH readings at Day 3 were presented in Table 6.
Table 6: The pH and colour obseration of the flask cultures when added with CuSO4
Spirulina with added CuSO4 pH Colour observation at Day 2
Control 9.2 Lime green
2 mg/L 6.7 Pale green
5 mg/L 6.3 Pale green
34
Figure 13: The colour difference of medium between the 3 flask cultures after a day
It shows that at pH was below the optimum growth range for Spirulina could not survive as
To determine whether the concentration of algae was a limiting factor, another set of
experiment was conducted at CuSO4 concentration of 2 mg/L in the flask. The starting OD
was at 0.57 abs, which was higher by more than two-folds compared to first experiement.
The culture medium colour changes from vibrate green to almost colourless over the span of
3 days. Even at a higher algal concentration, the algae growth was inhibited. This could be
explained that the Spirulina only can growth within certain range of pH and the pH was
proven to be too acidic for Spirulina to survive. On the contrary, in ALB Spirulina was able to
survive despite lower pH reading was measured.
Only the control group showed an increase in OD, the rest of the flasks that were added with
different concentrations of CuSO4 had inhibited Spirulina growth. The flask cultures could not
acclimatise at the selected CuSO4 concentrations. However, the same concentration was
added into the ALB and the results indicated that the Spirulina was able to acclimatise at the
given concentration and still increase in OD. Despite having a gradual growth, the growth
rate of Spirulina with added CuSO4 was still lower as to compare with the two ALB controls.
35
Table 7: Optical density for Spirulina at different CuSO4 concentrations
Day Optical density at 595 nm (Abs)
Control 2 mg/L 5 mg/L 2 mg/L
0 0.25 0.25 0.25 0.57
1 0.27 0.22 0.23 0.53
2 0.28 0.24 0.23 0.53
3 0.36 0.23 0.23 0.49
Figure 14: The effect of CuSO4 on Spirulina growth
15.4.2 Spirulina with added acetaldehyde
Both OD of Spirulina control and with acetaldehyde were recorded and presented in Figure
15 and Table 8 for over the span of 10 days. The results show that the Spirulina with added
acetaldehyde had a slightly slower growth rate than the control. The growth rate of Spirulina
was inhibited by an average of 15% when added with acetaldehyde.
10 𝜇𝑙 of acetaldehyde were added on Day 0 to Day 2. The amount was increased to 20 𝜇𝑙
and added on Day 6 till Day 10. The Spirulina was able to acclimatise despite the increased
amount of acetaldehyde. The pH was taken using a pH meter and reading shows that the
control was pH 9.5 and the Spirulina with added acetaldehyde was pH 9.3. Both pH reading
were within the optimum range for Spirulina growth.
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0 1 2 3
Op#
cal den
sity (A
bs) a
t 595 nm
Time (Days)
Control 5 mg/L 2 mg/L 2 mg/L
36
For a scaled down version of comparison between ALB and flask culture, the 20 𝜇𝑙
acetaldehyde in flask (150 ml) was a higher dose as to compare to 200 𝜇𝑙 (2.5 L) in ALB.
However, there were no observations on growth declining. The pH was the limitting factor in
this experiment.
Figure 15: Comparison between control and Spirulina added with acetaldehyde
Table 8: Optical density data of Control and Spirulina added with acetaldehyde
Day Control Acetaldehyde Acetaldehyde added
0 0.22 0.22 10 (𝜇𝑙)
1 0.23 0.22 10 (𝜇𝑙)
2 0.27 0.25 10 (𝜇𝑙)
6 0.45 0.42 20 (𝜇𝑙)
7 0.48 0.53 20 (𝜇𝑙)
8 0.56 0.52 20 (𝜇𝑙)
9 0.61 0.57 20 (𝜇𝑙)
10 0.65 0.60 20 (𝜇𝑙)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 1 2 3 4 5 6 7 8 9 10
Op#
cal den
sity (A
bs) a
t 595 nm
Time (Days)
Control
Acetaldehyde
10 μl
20 μl
37
15.5 Specific Growth Rate The growth rate of Spirulina is through simple cell division. Its specific growth rate (𝜇) is
calculated using the following equation (Vonshak, 1997):
𝜇 =ln 𝑥! − ln 𝑥!𝑡! − 𝑡!
(10)
Where 𝑥! and 𝑥! are biomass concentrations and at time intervals 𝑡!and 𝑡!.
The specific growth rate (𝜇) was plotted between the four ALBs and compares. It can be
seen that initially growth rate was high and it gradually decreases. It could be due to
photoinhibition when concentration of algal biomass increases. The negative values indicate
that the concentration measured had reduced and there were no algal growth during this
period.
Figure 16: The specific growth rate for the ALBs over the span of 23 days.
-‐0.3
-‐0.1
0.1
0.3
0.5
0.7
0.9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Specific grow
th ra
te (𝜇
)
Data frequency in 23 days
Control 1 Acetaldehyde Copper Control 2
38
Figure 17: The comparison between addition of CO2 dosing and without it across 12 days.
10 % error bar added.
Figure 18: Specific growth rates between ALBs over the span of 23 days. 10 % error bar
added.
0
0.05
0.1
0.15
0.2
0.25
0.3
ALB 1 ALB 2 Flask
Specific grow
th ra
te (𝜇
)
Control groups
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Control 1 Acetaldehyde Copper Control 2
Specific grow
th ra
te (𝜇
)
ALB
39
Figure 19: Specific growth rate between control and added CuSO4 in ALBs. 5 % eror bar
added.
Figure 20: Specific growth rate between control and added acetaldehyde in ALBs. 5% error
bar was added.
-‐0.05
0
0.05
0.1
0.15
0.2
0 2 5
Specific grow
th ra
te (𝜇)
Copper concentra#on in medium (mg/L)
Copper
Control 2
-‐0.8
-‐0.7
-‐0.6
-‐0.5
-‐0.4
-‐0.3
-‐0.2
-‐0.1
0
0.1
0.2
0 100 150 200
Specific grow
th ra
te (𝜇
)
Amount of acetaldehyde added (𝜇l)
Acetaldehyde
Control 2
40
Figure 21: Specific growth rate between control and added acetaldehyde in flasks. 5 % error
bar was added.
Figure 22: Specific growth rate between control and added CuSO4 in flasks. 10 % error bar
was added.
-‐0.05
0
0.05
0.1
0.15
0.2
0.25
10 10 20 20 20 20 20
Specific grow
th ra
te (𝜇
)
Acetaldehyde added into flask (𝜇l)
Control
Acetaldehyde
-‐0.1
-‐0.05
0
0.05
0.1
0.15
0 2 5 2
Specific grow
th ra
te (𝜇
)
Copper concentra#on in medium (mg/L)
41
Figure 17 clearly shows that with CO2 sparging, the specific growth rate is higher by about 2
and 1.5 folds. All three groups started at low OD of 0.04-0.09 Abs at 595 nm.
For Figure 18, each measurement, the initial and final values of algal concentration were
used over the span of the whole experiment duration. The overall specific growth rate of the
algae in ALBs with 10% error bars was inserted. This calculation method is least accurate
compare to calculating each specific growth rate per day and compare. Therefore, error bars
with a higher percentage were used. The specific growth rate is the highest in Control 1 and
followed by Control 2, Copper and Acetaldehyde. The growth rate of the control groups was
higher because it was not inhibited by toxic chemicals.
In Figure 19, the ALB Copper specific growth rate was compared with Control 2 to able to
see a clearer difference when Spriulina was added with heavy metal. It shows that despite a
gradual increase in OD the 𝜇 decreases upon addition of copper and have negative 𝜇. In
Figure 20 the ALB Acetaldehyde shows a positive growth rate with added of acetaldehyde
except when the dose was increase to 200 𝜇𝑙. Error bar of 5% was inserted.
In Figure 21, the acetaldehyde acclimation of when 𝜇 between ALB and flask were
compared, the flask has a higher specific growth rate than ALB. Which indicate that pH is the
limiting factor. The pH in flask was closer to the optimum range. Another explanation could
also be that the OD in ALB is 3 folds higher than flask, and with populated density, growth
rate decreases.
As seen in Figure 22, the OD value showed little or no increase when added with copper
concentration and the 𝜇 were all negative over the span of 3 days. The error bars at 10%
were added. At the same CuSO4 concentration (2 mg/L) added but with different initial OD, it
showed that specific growth rate with higher OD has a more negative 𝜇. The population
density is higher when higher OD is detected. A negative specific growth rate indicates that
there is a decline in growth rate algal cells.
To have a better comparison on 𝜇, Control 2 was chosen to compare the 𝜇 along with ALB
Copper and ALB Acetaldehyde instead of Control 1 because its initial 𝜇 value was closer to
the two ALBs 𝜇 value than Control 1. The average 𝜇 for both Control and Acetaldehyde in
flask were similar over the span of 10 days. The amount chosen at 10 𝜇𝑙 and 20 𝜇𝑙 to be
added in flask was because it is a scaled down value from the added acetaldehyde amount
in ALB.
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15.6 Further Discussions By connecting ALB with fine-bubbling CO2 (600 𝜇m), a high mass transfer rate of CO2
dissolution and O2 stripping was achieved. When photosynthesis occurs, O2 was produced
as a by-product in the algal cells and it inhibits the uptake of CO2. By stripping the O2,
Spirulina had a higher growth rate as to compare to the flask culture. Another reason for
having a higher growth rate in ALBs than flask cultures is because of its ability to have pH
control. After CO2 dosing, the ALBs were able to maintain at low pH at 5.9 due to the as CO2
is acidic. However, the next day the pH increased to pH 8 in the culture. This cycle ensured
that the pH was kept within a desirable range and not a limiting factor to the Spirulina growth.
In the flasks cultures, pH in the medium increases as Spirulina grows and may affect the
growth rate by getting slower if it increases beyond the optimum pH range.
In CuSO4 Spriulina experiment, the pH measured in the flasks (pH 6.7, pH 6.3) were higher
than the ALB (pH 5.9); the copper might be more toxic to Spirulina in the flasks. Copper may
interfere with cell permeability or the binding of essential metals, it may transport into the
chloroplast and react with –SH enzyme groups and free thiols, disrupting enzyme active
sites and cell division (Cid, et al., 1995). Therefore, the Spirulina did not survive in the flasks
culture as photosynthesis and growth were inhibited due to copper toxicity.
In the ALB, the concentration-respond curve for CuSO4 was flat over the range of 2 mg/L to
5 mg/L indicating that this is still within the threshold level for Spirulina as it had minute effect
on its growth rate. It is possible that this flat area of the concentration-response curve may
be due to detoxification of Copper by algal cells. In certain freshwater algal species such as
Chlorella fusca is able to produce organic substances that reduce the bioavailable copper
concentration if it is released extracellular in sufficient amounts (Franklin, et al., 2000).
Therefore, the Spirulina in ALB was able to reduce the copper concentration. The heavy
metal accumulation in its cells affects the Spirulina growth.
For flask culture control, there were absences of CO2 supply as nutrient and also an
accumulation of O2 over time which explains the slower growth rate compared to ALB control.
Before bubbling, the algal cells were settled down at the bottom of the ALB. The
accumulation of algal cells reduces its total surface area over volume and its exposure to
light source. During bubbling, the algal cells were thoroughly mixed and it minimized the
tendency of algal cells to accumulate and also increases its surface area.
43
As the optical density increases, it is observed that the colour of the culture became denser
and a dark green medium was observed. Spirulina concentration increases, the growth rate
slowed down towards Day X onwards as to compare to initial growth rate. As the
concentration increases, the algal cells did not received the same amount of light and CO2
source as initially due to its saturated environment. Algal growth increases proportionally
with light intensity when it is below saturation point, at above saturation point, photoinhibition
may occur.
Erlenmeyer flasks were used instead of ALB even though growth of Spirulina could be
carried out in ALB without any CO2 supply. However, Spirulina algal particles tend to settle
at the bottom of the ALB after a day; there were constraints in measuring the OD as the ALB
without gas bubbling was too bulky to ensure a thorough mixing manually before taking its
OD reading.
There are very few research has been done regarding methods of acetaldehyde removal
from flue gas using microalgae. The mechanism of how acetaldehyde reacts in microalgae
cells is still unclear. Similar experiment was done with a different algae strain Chlorella sp. in
the same department and it showed that the growth rate of Chlorella increases when added
with acetaldehyde. Different algal strains will have different reaction towards this toxic
pollutant. The decrease in OD for Spirulina at 200 𝜇𝑙 is due to acetaldehyde toxicity and
inhibition in photosynthesis.
It was suggested by Slatyer, et al. (1983) that acetaldehyde can be used as an inhibitor in
experiments designed to separate electron flow through the photosystems from the fixation
of CO2 and N2 in cyanobacterium Anabaena cylindria. In the author’s experiment,
acetaldehyde concentration of 50 mM prevented cell growth in the cyanobacterium and
resulted in death. There was no significant effect of acetaldehyde on CO2 fixation. A study of
acetaldehyde toxicity was conducted by Brank and Frank, (1998) on freshwater green algae
Chlamydomonas reinhardti. The lowest values of toxicity are 23 mg/L obtained as the 2-hr
EC5 in photosynthetic inhibition.
One of the theory may be that acetaldehyde is converted to acetate to provide as a nutrient
for Spirulina growth. Therefore, Spirulina was able to grow continuously. However, there is
no further research regarding this mechanism. It is known that at anaerobic conditions,
pyruvate degradation in green alga Chlorogonium elongatum forms acetate and ethanol
(Kreuzberg, 1985) as shown in Figure 23.
44
Figure 23: Scheme of the proposed formate dermentation pathway for anaerobic pyruvate
degradation in C. elongatum (Kreuzberg, 1985)
45
16. Limitations Ideally, this method is able to cultivate Spirulina successful. However to mass produce
Spirulina poses challenges such as scaled up ALB to pilot-scale for industry application. The
cost analysis needs to be conducted to determine if this method is cheaper and sustainable
compare to conventional culturing methods for large scale production. The microflotation
methods were unable to be carried out in the lab due damages in the reactor used for
microflotation. The parameter used was not the optimal values. Therefore, there is a need to
adjust it so that the culture medium in ALB is able to maintain at pH 9 and temperature of
35 ℃ to compare if growth rate would be significantly higher.
17. Conclusions
For ALB experiments, the result from this study demonstrated the feasibility of cultivating
Spirulina sp. in the three different growth conditions. Spirulina sp.could adapt well in all three
culture mediums (control, acetaldehyde and copper) in ALB with no lag phases observed
except when higher concentration of acetaldehyde was added. The high acetaldehyde
content could not support a productive algal growth by inhibiting photosynthesis system.
Algal growth was significantly enhanced in ALB because of its additional nutrient from CO2
bubbling and also a thoroughly mixing from bubbling enables high mass transfer rate of
dissolved CO2 to medium. The copper ions were able to be removed efficient by Spirulina
growth as there was no inhibited. The pH conditions and nutrients were able to sustain a
linear growth despite copper toxicity in cells occurs.
For flask cultures, the copper concentrations were proven to be toxic for Spirulina which
resulted cell death after one day. The acetaldehyde on the other hand, was shown to have
little effect on the growth for Spirulina.
The ALB method using fine bubbles was proven to be a successful method to cultivate
Spirulina for fast growth rate. It also shows that Spirulina is able to grow in this lab conditions
by using diluted growth medium and constant light exposure that promotes linear growth.
46
18. Future works
18.1 Determine the Protein content It is known that Spirulina is rich in protein source. There is an interest to study if the addition
of copper and acetaldehyde will affect the protein content. This experiment could be done
using the chemical procedure to determine protein mentioned in Vonshak (1997).
18.2 Reaction of acclimatised Spirulina with Acetaldehyde The experiment will be conducted in two ALBs with Spirulina that was previously cultured
with addition of acetaldehyde. One ALB will act as a control and the other one will be added
with acetaldehyde at 100 𝜇𝑙. In theory, both growth rates would be at the same. As the
Spirulina has already acclimatised in that particular concentration, it should not affect its
growth rate.
18.3 Microbubbles Another interest is to compare the growth rate when microbubbles (300 𝜇m) is used instead
of fine bubbles. The ALB will be connected with a fluidic oscillator to produce microbubbles.
The pH and temperature measured in ALB was lower than the optimum conditions.
Therefore, there is an interest to compare if there is a huge difference in the growth rate and
adaptability for copper and acetaldehyde in optimum conditions and the current condition.
47
19. Acknowledgement First and foremost, I would like to thank Prof. Will Zimmerman for giving me the opportunity
to be a part of this research group. Indeed it was a privilege, I have learnt so much for the
past year and I have developed a deeper understanding about this topic. I would like to also
thank Dr. James Hanotu for guiding me throughout this research project and pushing me
when I needed it. And to Mr. YuZhen Shi for helping me with my experiment by going
through the trouble of preparing the growth mediums, setting up and acquiring the Spirulina
algae. The little conversations that we had were very helpful towards my understanding
about algae and the culturing techniques. To Mr Tom Holmes, who was always in the lab
and provided me with advice and help on the spot. To my dear friends and course mates,
whom encouraged me to persevere and try my best during this period, I thank you very
much for the support. Lastly, thank you The University of Sheffield and especially the people
in Chemical and Biological Engineering department, thank you for making my time as a
student here nothing but wonderful.
48
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