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Biohydrogen production from microalgae of Chlorella sp.

Imran Ali 1, Sudip K. Rakshit 2, Lakkhana Kanhayuwa 3

1,2 Food Engineering and Biotechnology, Asian Institute of Technology,P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand

3Fungal Diversity Laboratory, Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonbu-ri, 49 Thakam, Bangkhuntien, Bangkok, 10150, Thailand

E-mail: st106871@ait.ac.th

Abstract Three green algae of Chlorella sp were used to investigate their ability for biohydrogen production. Each strain was cultered normal BG-11 and sulfur-deprived BG-11 under anaerobic condition (at 28oC, 150 rpm for 48-72 hr) to monitor their growth. The results revealed that growth of all strains was suppressed in sulfur-deprived medium. Chlorella sp. TISTR 8262 yielded maximum hydrogen 13.03% of biogas produced, followed by C. ellipsoidea TISTR 8260 obtained at 3.05% of hydrogen, while there was no hydrogen production in case of C. vulgaris TISTR 8680. Keywords: biohydrogen/ biogas / Chlorella sp./ green algae.

1. Introduction Nowadays energy is an important component of daily life. About 80% of the world’s energy demands are fulfilled by fossil fuels. This utilization of fossil fuel will ultimately lead to the depletion of limited available fossil fuel resources [2]. Fossil fuels are also one of the contributing agents of global warming by the emission of pollutants. These pollutants also cause acid rain. Thus, a search for alternative energy source has become an urgent demand [1]. Currently, hydrogen comes as the best choice of fuel. Hydrogen has proven to be a very efficient fuel for number of applications. The most advantage factor of hydrogen is that it produces only water on combustion. Electricity can be generated by hydrogen fuel cells, as well as it can be directly used in engines for combustion. Different physical and chemical methods are widely used for hydrogen production such as steam cracking, partial oxidation of fossil fuels [2]. These processes are high energy intensive making hydrogen production expensive and also cause environmental pollution. However production of bio hydrogen provides a cost-effective, environmental friendly and can be produced under mild operating conditions. Mostly, biohydrogen production can be produced at ambient temperature and low pressure and do not require high energy input. In addition waste recycling can also be achieved by production process.

Biohydrogen production can be categorized from the process involved 4 mechanisms as 1) biophotolysis of water using algae and cyanobacteria, 2) Photodecomposition of organic compounds by photosynthetic bacteria, 3) Fermentation of organic compounds, and 4) Hybrid system of photosynthetic and fermentative bacteria [1]. Requirement and outcome are different in each process, depending upon raw material used and substrate consumption. Green algae can be preferred most for biohydrogen because only water is utilized for hydrogen production and no need much more for consideration about intermediate carbon metabolites [7]. This process is conducted via photosynthetic pathway. Photosynthesis is mainly based on two photosystems named as photosystem I and II. Absorption of light energy by these systems results in the decomposition of water in protons and oxygen. Protons are catalytically reduced in hydrogen at enzyme active sites. Required electrons are transferred from the photosystems to the enzyme using a transporters chain. These electrons are then processed at photosystem II in thylakoid membrane by ferredoxin, and release H2 via Fe-hydrogenase pathway [8]. The enzyme Fe-hydrogenase responsible for hydrogen production is present at stroma in chloroplast, after receiving the electrons from reduced ferredoxin, they donate them to protons [4], thereby producing molecular hydrogen. This seems to be a convenient and simple process but the enzyme Fe-hydrogenase is very sensitive to oxygen. Thus, anaerobic conditions are necessary for the expression and proper functioning of hydrogenase gene [3]. In this work we attempted to investigate how green microalgae, particularly Chlorella species produce biohydrogen under different cultivating conditions. 2. Methodology 2.1 Algal strains Chlorella species of Chlorella vulgaris TISTR 8680, C. ellipsoidea TISTR 8260 and C. sp. TISTR 8262 kept in N8 medium were obtained from Thailand Institute of Scientific and Technological Research, Thailand.

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2.2 Culture maintenance The cultures were transferred into 150 mL shake flask containing 100 mL BG-11 medium (28o C with 150 rpm) under fluorescent light in order to increase cell concentration as well as to preserve as stock cultures for further experiments.2.3 Growth monitor All cultures were grown in two different growth media named as BG-11 medium and sulfur-deprived BG-11 medium in which all sulfate salts were replaced by chloride salts. Cultures were cultivated in 50 mL shake flasks and incubated at 28o C, 150 rpm under fluorescent light. The cultures were withdrawn and were counted every 6 hr using haemacytometer (Boeco, Germany). The experiment was done in triplication.2.4 Pre-experiment apparatus design Lab scale bioreactors were designed for photo biochemical reaction as instructed by Janssen et al [5]. The graduated gas collection units were installed with the bioreactor. The collected gas was recorded by a water displacement system in the gas collection units.2.5 Biohydrogen production experiment The pure cultures from BG-11 medium at their log phase were centrifuged at 6000 rpm for 10 min. The cells then were washed twice with 0.1 M potassium phosphate buffer, pH 7.0. Each culture at final concentration of 4x107 cells/ mL was seperatley incubated in 500 mL of sulfur-deprived BG-11 medium in bioreactors under 28o C with 150 rpm. Fluorescent light was supplied for photolysis activity. An anaerobic condition was controlled by N2 flushing prior to experiment. Gas samples were taken and then was analyzed in gas chromatography (Model 14 A SHIMADZU Germany) using a thermal conductivity detector (TCD), with helium (He) as carrier gas. A 1.8 m -0.32 mm Alltech carbospher column was used. Column head pressure was maintained at 350 kPa. The temperatures of oven, injector port and detector were 100, 120 and 120 oC, respectively. The standard biogas was taken as a reference.

3. Results and Discussion3.1 Growth of Chlorella sp. Fig. 1 (a) shows comparative growth profiles of all Chlorella species under BG-11 medium. C. ellipsoidea TISTR 8260 gave the best growth behavior, followed by C. vulgaris TISTR 8680. Meanwhile, C. sp. TISTR 8262 seems to less acclimatize in the medium.

C. vulgaris TISTR 8680 and Chlorella sp. TISTR 8262 reached log phase after 6 hr, while C. vulgaris TISTR 8680 reached log phase in earlier. The maximum growth for all species obtained 48 hr of early stationary phase, while the

death phase was found after 66 hr. In case of Chlorella sp. TISTR 8262, the death phase seems to occur earlier than the other two strains Fig. 1 (b).

In sulfur-deprived BG-11 medium, the Chlorella sp. TISTR 8262 found as the best adaptable strain under unfavorable condition. C. vulgaris TISTR 8680 showed growth pattern similar to that of Chlorella sp. TISTR 8262. However, Chlorella vulgaris TISTR 8680 showed rapid decline when the cell reached the death phase compared to Chlorella sp. TISTR 8262. While, C. ellipsoidea TISTR 8260 showed the least adaptability to sulfur deprived conditions. The log and stationary phases have been found variable amongst all Chlorella species, but the death phase seems to start almost at 36 hrs in all Chlorella strains. Overall comparison of both growth profiles as in normal BG-11 and sulfur-deprived BG-11 medium. Chlorella species clearly shows that the sulfer-deprived condition affected their growth. It was clearly found that life time was reduced due to unavailability of sulfur content in the medium which was required for normal function of photosynthetic activity. For the deprived condition mentioned in literature reviews, the cells were forced to utilize the available starch. This activity cannot be prolonged for long time and the cells then reached death phase faster than the normal condition which was on depletion of all the starch stock available.

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a) b)

Fig. 1 Growth of Chlorella sp. (a) in normal BG-11 medium (b) in sulfur-deprived BG-11 medium.

Fig. 2 Chromatogram of Chlorella vulgaris TISTR 8680 (a), C. ellipsoidea TISTR 8260 (b), and C.sp. TISTR 8262 (c).

a)

b)

c)

Fig.1 Growth of Chlorella sp. (a) in normal BG-11 medium (b) in sulfru-deprived BG-11 medium

Fig.2 Chromatogram of Chlorella valgaris TISTR 8680 (a), C. ellipsoidea TISTR 8260 (b), and C.sp. TISTR 8286 (c).

a) b)

Fig. 1 Growth of Chlorella sp. (a) in normal BG-11 medium (b) in sulfur-deprived BG-11 medium.

Fig. 2 Chromatogram of Chlorella vulgaris TISTR 8680 (a), C. ellipsoidea TISTR 8260 (b), and C.sp. TISTR 8262 (c).

a)

b)

c)

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Physiological appearance of the cultures cultivated in 2 different media showed that highest yield was found under normal condition. Metabolic activity operated in the normal way was leading to accumulation of organic materials and polymers i.e. starch as a final product for cell development and reproduction. Under stress condition, like nutrient limitation, cell development is interfered. The deprivation of sulfur leads to the low deficiency of photosynthesis because sulfur is the main component to build up important structures such as chloroplast during photosynthesis. This mostly affects on partial inactivation of photosystem II and also makes anaerobic condition in the culture causes blocking starch production. This phenomena force cells to switch the mechanism to other pathway for maintaining their survival, leading to produce hydrogen [6].

3.2 Biohydrogen Production C. vulgaris TISTR 8680 has also been found to lack of activation of hydrogenases enzyme, which may be due to the lack of gene expression and activation even under optimal condition where other chlorella species are found to produce hydrogen. No hydrogen production was observed for C. vulgaris TISTR 8680. In total 53 mL biogas composts of 16.45% oxygen (8.7 mL) which shows a very high level of oxygen presence under which the hydrogenase enzyme is difficult to get activated because C. vulgaris has found to be vulnerable strain not to produce any hydrogen [8]. Fe-hydrogenase is extremely sensitive to O2 [9]. C. vulgaris TISTR 8680 has not been found active for hydrogen production metabolism. In case of C. ellipsoidea TISTR 8260 1.12 mL hydrogengas was observed along low level of oxygen concentration (1.5%). It was verified that anaerobic condition could be helped, in expression of Fe-hydrogenase enzyme which was responsible for hydrogen production. As high level of CO2 shows the utilization of available starch converted into energy. In sulfur-deprived BG-11 medium growth profile of C. ellipsoideaTISTR 8260 was least adaptable to the sulfur-deprived condition compared to others. This could be a reason for limited hydrogen production. Chlorella sp. TISTR 8262 has been found to be the most promising specie for bio hydrogen production amongst all the Chlorella species under investigation. Chlorella sp. TISTR 8262 exhibited the best adaptability in its growth profile of sulfur-deprived medium. The anaerobic conditions even did not much interrupted the metabolic functioning, leading for the switching over of normal photosynthesic mechanism to the hydrogen production. Starch stocks was utilized for the energy production at high level of CO2. The oxygen level showed was

higher than C. ellipsoidea TISTR 8260. It is implied that is more tolerant to oxygen than C. ellipsoidea TISTR 8260.

4. Conclusion It was concluded that sulfer-deprived medium suppressed normal growth for all Chlorella sp. However, compared among them, Chlorella sp. TISTR 8262 showed the best growth which was the maximum yield of optimum biohydrogen obtained from C. sp. TISTR 8262. C. vulgaris TISTR 8680 has also been found to survive under the sulfur-deprived conditions although there was no bio hydrogen produced by this strain. In case of C. ellipsoidea TISTR 8260, the sulfur-deprived condition is the most unfavorable for its growth compared to other under investigation. Biohydrogen produced from C. ellipsoidea TISTR 8260 has also been found that it was quite less in quantity.

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