International Conference on Chemical, Environment & Biological Sciences (CEBS-2014) Sept. 17-18, 2014 Kuala Lumpur (Malaysia)

Experimental Investigation Parameters of Hydrogen Production by Algae Vulgaris

Walid M. Alalayah, Yahia A. Alhamed, Abdulrahim Al-Zahrani and Gaber Edris

[6,7]. Sulfur deprivation in green algae causes a reversible

Abstract— Bio-photolysis to produce the H2 gas carried out inhibition of oxygenic photosynthesis due to impeded using algal strain Chlorella vulagaris. In this study the effect of biosynthesis; consequently the algae cannot perform the various parameters affecting on the hydrogen production were required turnover of photosystem II [8]. After sulfur investigated to find the optimum conditions that maximize the deprivation, nitrogen is purged to drive off remaining oxygen hydrogen production using standard procedures for experiment present in the culture medium. This study focus on the design. These parameters include; initial substrate concentration, investigatory studies of some key environmental factors initial pH and total of nitrogen and phosphate content for the Bold's Basal culture (BBC) media concentration. Glucose as substrate was influencing the hydrogen production during biophotolysis, that studied at varied concentrations (5- 40) g/l and the high production of including initial substrate concentration, initial pH, phosphate hydrogen was observed at 10 g/l. A set of tests in this study was and nitrogen content of the culture media concentration. performed by varying the initial pH from 6.0 ± 0.2 to 9.0 ± 0.2, The most suitable conditions for hydrogen production in a bio-reactor II. MATERIALS AND METHODS were observed at pH 8.0 ± 0.2. Total of nitrogen and phosphate content for the (BBC) media concentration were increased by mole A. Algal strain, culture media and growth conditions (10%, 20% and 30%). Results showed that the maximum production Chlorella vulgaris, stored on agar, obtained from Carolina of H2 with increasing both of components nitrogen and phosphate Biological Supply (Burlington, North Carolina, Catalogue No. about 10% concentration. 15-2075) used in this study. The strain was grown on Bold's Basal Medium [9,10] with the composition (mgL-1 of Keywords— Hydrogen production; green ; anaerobic deionized water) shown in the following Table 1 : process; renewable energy. TABLE 1 I. INTRODUCTION THE COMPOSITION OF BOLD'S BASAL MEDIUM Stock Distillate water YDROGEN is one of the alternative fuels to meet our formula Weight (g) Henergy requirements and its consumption as a fuel is solution No (ml) completely deprived of carbon dioxide emissions as compared to conventional fuels. Hydrogen gas is thought to be 1 K2HPO4 1.875 250 the ideal fuel for a world in which air pollution has been 2 KH2PO4 4.375 250 alleviated, global warming has been arrested, and the 3 MgSO4.7H2O 1.875 250 environment has been protected in an economically 4 NaNO3 6.250 250 5 CaCl2 .4H2O 0.625 250 sustainable manner. Hydrogen can be used as a clean 6 NaCl 0.625 250 transportation fuel as well as for electricity production via fuel 7 Na4 EDTA 5.000 100 cells. Hydrogen and electricity could team to provide 8 KOH 3.100 100 attractive options in transportation and power generation. 9 FeSO4.7H2O 0.498 100 10 H3BO3 1.142 25 Hydrogen gas can be produced by some chemical process but 11 MnCl2.4H2O 0.058 25 produce hydrogen from biomass has been declared as 12 ZnSO4. 7H2O 0.353 25 innovative and promising biotechnology [1, 2]. One way that 13 Co(NO)2.6H2O 0.020 25 hydrogen can be produced biologically is by microalgae 14 Na2MoO4. 2H2O 0.048 25 exposed to anaerobic conditions [3, 4, 5]. Microalgae produce From the above Table it can prepare the stock of BBM hydrogen by adopting a two-stage process, in stage 1, CO2 is fixed in the presence of sunlight through photosynthesis and medium in 1L to active the cell biomass through mixing the allowing O production. In stage 2, hydrogen is produced by medium as following: 2 10 ml of each solutions 1-6 the degradation of stored organic compounds via bioreactor 1 ml of each solutions 7-9 under sulfur deprivation 0.1ml of each solutions 10-14. Walid M. Alalayah, Yahia A. Alhamed, Abdulrahim Al-Zahrani and Gaber After stirring, the pH of the medium is measured and it is Edris, are with Chemical and Materials Engineering Department College of adjusted at 6.9±0.2. The growth experiments will performed Engineering, King Abdulaziz University KAU, P.O. Box 80204, Jeddah in 1000 mL Erlenmeyer flasks containing 500 of Bold's Basal 21589 Saudi Arabia Medium .The operation conditions are defined as following:

http://dx.doi.org/10.15242/IICBE.C914010 41 International Conference on Chemical, Environment & Biological Sciences (CEBS-2014) Sept. 17-18, 2014 Kuala Lumpur (Malaysia)

Temperature set at 30 °C and air containing 0.04% CO2 was B. Effect of initial medium pH bubbled through fritted glass bubbles at a flow rate of 200 Solution pH is an important controlling factor of anaerobic mLmin-1. Eight fluorescent lamps of 20 W were fitted [11]. photolysis/ fermentation processes. Many researchers have B. Experimental set- up reported that pH could affect the metabolic pathway in pure Duran bottle as a bioreactor was provided and used in this culture [17, 18, 19, 20]. pH is a critical factor in hydrogen study for bio-hydrogen production with an inlet for nitrogen production process. Algae can grow in a pH range of 4 –10. A gas flushing and an outlet collecting the gas evolved by the set of tests in this study was performed by varying the pH culture. Microalgae was transferred to anaerobic process using from 6.0 ± 0.2 to 9.0 ± 0.2. Our result shows that the the sulfur-deficient medium [12]. The hydrogen was produced maximum rate of hydrogen production was measured at pH by the degradation of stored organic compounds (glucose) via 8.0 ± 0.2, while the minimum rate of hydrogen production was bioreactor; the evolved gas were allowed to pass through 5M recorded at pH 6.0 ± 0.2 as presented in Figure 2. KOH solution to absorb any of carbon dioxide gas and then subjected to analysis by gas chromatography (GC) [13]. C. Analysis methods The composition of the evolved gas was analyzed using gas chromatography. (GC-17A, Shimazdu) with a column packed and molecular sieve 5A column (Alltech), using argon as a carrier gas. The packed column was maintained at 80°C and the thermal conductivity detector was set at 120°C. The cell biomass concentration was estimated by measuring the optical density at the 450 nm wavelength at the initial time and the end of experiment. The concentration of glucose in the medium during the experiment was determined by blood- glucose analyzer at the initial time and the end of experiment“ ACUU- CHECK ACTIVE” which manufacturing by Roche Fig. 2. Effect of initial medium pH on hydrogen production. Diagnostics corporation [14]. C. Effect of nitrogen and phosphate content of the culture III. RESULTS AND DISCUSSION media A. Effect of initial glucose concentration In hydrogen production fermentation/photosynthesis processes several researcher reported on the effect of Nitrogen The initial glucose concentration plays an important role in and phosphate on the algal metabolism. Nitrate, ammonia, the size and yield of hydrogen production during organic urea, and nitrite are the nitrogen forms utilized by photosynthesis/ fermentation at relatively low initial glucose levels. The rate of photosynthesis was low according to the most algae, ammonia or urea require the least energy to law of mass action as reported in the literature [15, 16]. metabolize. Most media cultures contain nitrates or Results shows that the highest production of hydrogen on ammonium as the N source [20]. Table 2 Effect of addition of varying glucose concentration with time was observed when nitrogen and phosphate content on hydrogen production by C. initial glucose concentration was 10 g/l, as displayed in Figure Vularis 1. Then the production decreased with increasing glucose Bold's addition addition addition concentration. This result is in agreement with the results Parameters Basal 10% by 20% 30% reported by reference [14, 16], which indicated when higher Medium (mole) (mole) (mole) initial glucose concentrations were associated, the volume of Initial pH 8 8 8 8 hydrogen produced is decreased. Final pH 6.5 7.1 6.8 6.9 Specific growth 0.0159 0.0169 0.0162 0.0173 rate, h-1

InitialOD660nm 0.052 0.052 0.052 0.052

Final OD660nm 0.81 0.892 1.01 1.031 Duration 174 174 174 174 Production, h

The addition of nitrogen and phosphate have some effects on growth and the rate of production as literature by [21, 22]. The effect of increasing nitrogen and phosphate content of the (BBC) media concentration was investigated in this work with

Fig. 1 hydrogen production at varying initial glucose concentrations. (10%, 20% and 30%) by mole respectively. The results found enhanced production of hydrogen, growth of biomass and it was observed that the highest hydrogen yield enhanced by adding nitrogen and phosphate content 10% by mole up to

http://dx.doi.org/10.15242/IICBE.C914010 42 International Conference on Chemical, Environment & Biological Sciences (CEBS-2014) Sept. 17-18, 2014 Kuala Lumpur (Malaysia)

(BBC) media in anaerobic process as listed in Table (2). The [9] Bischoff, H.W. & Bold, H.C. (1963): Phycological studies. IV. Some volume of hydrogen production was increased by added 10% soil algae from Enchanted Rock and related algal species. University of Texas Publications 6318: 1-95. more than BBC media then decreased when higher addition of [10] Bold, H. C. (1949). The morphology of Chlamydomonas chlamydogama nitrogen and phosphate content more than 10 %. All sp. Bull. Torrey Bot. Club, 76:101-108. experimental runs carried out at room temperature. http://dx.doi.org/10.2307/2482218 [11] Bischoff, H. W., & Bold, H. C. (1963). Some soil algae from Enchanted Rock and related algal species. Phycological Studies IV. , 1-95. IV CONCLUSION [12] He, L., Xu, Y.Q. & Zhang, X.H. 2008. Medium factors optimization and The main aim in this study was investigated photosynthesis fermentation kinetics for phenazine-1-carboxylic acid production by Pseudomonas sp. M18G. Biotechnology and Bioengineering 100:250–9. hydrogen production using algal C. Vulgaris in a bio reactor. http://dx.doi.org/10.1002/bit.21767 It was found that some of the environmental parameters [13] Wooshin P, Seung HH, Sang, EO, Bruce EL, Ins K. Removal of studied affected production, including; initial substrate Headspace CO2 Increases Biological Hydrogen Production. concentration, initial pH and concentrations of nitrogen and Environmental Science & Technology, American Chemical Society 2006 vol: 39 No 12. phosphate content for (BBC) media. The optimum glucose [14] Rashid, N., Lee, K., Mahmood, Q., 2011. Bio-hydrogen production by concentration was investigated, and the highest yield of Chlorella vulgaris under diverse photoperiods. Bioresource Technology, hydrogen was 2.85 mol (mol glucose)-1 when an initial 102 (2) 2101–2104. glucose concentration of 10 g/l as a substrate. Increasing the http://dx.doi.org/10.1016/j.biortech.2010.08.032 [15] Ahmad, I., & Hellebust, J.. Nitrogen metabolism of the marine initial substrate concentration above 10g/l decreased the microalgae , Plant Physiology, 76 (1984) 658- hydrogen production rate. Another set of tests in this study 663. was performed by varying pHs from 6.0± 0.2 to 9.0± 0.2. The http://dx.doi.org/10.1104/pp.76.3.658 maximum rate of hydrogen production was at pH 8.0± 0.2, [16] Aslan, S., & Kapdan, I. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecological Engineering, 28 while the minimum rate of hydrogen production was seen at (2006) 64-70. pH 6.0± 0.2. The effect of addition nitrogen and phosphate http://dx.doi.org/10.1016/j.ecoleng.2006.04.003 content of the (BBC) media concentration on hydrogen [17] Ji, Y., & Sherrell, R. Differential effects of phosphorus limitation on production was also studied, and the best production was cellularmetals in Chlorella and Microcystis. Limnology and Oceanography, 53(5) (2008) 1790-1804. measured when increased 10% by mole. The hydrogen yield http://dx.doi.org/10.4319/lo.2008.53.5.1790 -1 (YP/S) was about 2.85 mole (mole glucose) at the optimum [18] Wood, A., Everroad, R., & Wingard, L. (2005). Measuring growth rates conditions (10 g, 8±0.2, 10%). in microalgal cultures. In R. A. Anderson (Ed.), Algal Culturing Techniques (pp. 269-285). Burlington, MA: Academic Press. [19] MacIntyre, H., & Cullen, J. (2005). Using cultures to investigate the ACKNOWLEDGMENT physiological ecology of microalgae. In R. A. Anderson (Ed.), Algal This project was funded by Saudi Arabia Basic Industries Culturing Techniques. (pp. 287-326). Burlington, MA: Academic Press. [20] Kaushik N, Anish K, Debabrata D. Effect of some environmental Corroboration (SABIC) and deanship research (DSR), King parameters on fermentive hydrogen production. Canadian J of Abdulaziz University. Jeddah, under grand No.( 15/472/1434). Microbiology 2006; 52:525-535. The authors, therefore, acknowledge with thanks SABIC and http://dx.doi.org/10.1139/w06-005 DSR technical and financial support. [21] Kumer N, Das D. Emhancement of hydrogen production by Enterobacter cloacae IIT-BT 08 Process Biochem 2000; 35:589-593. http://dx.doi.org/10.1016/S0032-9592(99)00109-0 REFERENCES [22] Khanal SK, Chen WH, Li L, Sung S. Biohydrogen production in continuous flow reactor using mixed microbial culture. Int J Water [1] Alalayah W M, Kalil M S, Kadhum A H, Jahim J M & Alaug N M, Environ. Res 2004; 78(2):110-117. International J Hydrogen Energy 33 (2008) 7392. http://dx.doi.org/10.2175/106143005X89562 http://dx.doi.org/10.1016/j.ijhydene.2008.09.066 [2] Melis, A and Happe T, Hydrogen Production. Green Algae as a Source of Energy, American Society of Plant Biologists, 127(2011) 740–748. [3] Vijayaraghavan, K., Trends in hydrogen production- a review. 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