Abstract— Bio-photolysis to produce the H2 gas carried out

using algal strain Chlorella vulagaris. In this study the effect of various parameters affecting on the hydrogen production were investigated to find the optimum conditions that maximize the hydrogen production using standard procedures for experiment design. These parameters include; initial substrate concentration, initial pH and total of nitrogen and phosphate content for the Bold's Basal culture (BBC) media concentration. Glucose as substrate was studied at varied concentrations (5- 40) g/l and the high production of hydrogen was observed at 10 g/l. A set of tests in this study was 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 were observed at pH 8.0 ± 0.2. Total of nitrogen and phosphate content for the (BBC) media concentration were increased by mole (10%, 20% and 30%). Results showed that the maximum production of H2 with increasing both of components nitrogen and phosphate about 10% concentration.

Keywords— Hydrogen production; green microalgae; anaerobic process; renewable energy.

I. INTRODUCTION YDROGEN is one of the alternative fuels to meet our energy requirements and its consumption as a fuel is completely deprived of carbon dioxide emissions as

compared to conventional fuels. Hydrogen gas is thought to be the ideal fuel for a world in which air pollution has been alleviated, global warming has been arrested, and the environment has been protected in an economically sustainable manner. Hydrogen can be used as a clean transportation fuel as well as for electricity production via fuel cells. Hydrogen and electricity could team to provide attractive options in transportation and power generation. Hydrogen gas can be produced by some chemical process but produce hydrogen from biomass has been declared as innovative and promising biotechnology [1, 2]. One way that hydrogen can be produced biologically is by microalgae exposed to anaerobic conditions [3, 4, 5]. Microalgae produce hydrogen by adopting a two-stage process, in stage 1, CO2 is fixed in the presence of sunlight through photosynthesis and allowing O2 production. In stage 2, hydrogen is produced by the degradation of stored organic compounds via bioreactor under sulfur deprivation Walid M. Alalayah, Yahia A. Alhamed, Abdulrahim Al-Zahrani and Gaber Edris, are with Chemical and Materials Engineering Department College of Engineering, King Abdulaziz University KAU, P.O. Box 80204, Jeddah 21589 Saudi Arabia

[6,7]. Sulfur deprivation in green algae causes a reversible inhibition of oxygenic photosynthesis due to impeded protein biosynthesis; consequently the algae cannot perform the required turnover of photosystem II [8]. After sulfur deprivation, nitrogen is purged to drive off remaining oxygen present in the culture medium. This study focus on the investigatory studies of some key environmental factors influencing the hydrogen production during biophotolysis, that including initial substrate concentration, initial pH, phosphate and nitrogen content of the culture media concentration.

II. MATERIALS AND METHODS

A. Algal strain, culture media and growth conditions Chlorella vulgaris, stored on agar, obtained from Carolina Biological Supply (Burlington, North Carolina, Catalogue No. 15-2075) used in this study. The strain was grown on Bold's Basal Medium [9,10] with the composition (mgL-1 of deionized water) shown in the following Table 1 :

TABLE 1

THE COMPOSITION OF BOLD'S BASAL MEDIUM

Stock solution No formula Weight (g) Distillate water

(ml)

1 K2HPO4 1.875 250

2 KH2PO4 4.375 250 3 MgSO4.7H2O 1.875 250 4 NaNO3 6.250 250 5 CaCl2 .4H2O 0.625 250 6 NaCl 0.625 250 7 Na4 EDTA 5.000 100 8 KOH 3.100 100 9 FeSO4.7H2O 0.498 100 10 H3BO3 1.142 25 11 MnCl2.4H2O 0.058 25 12 ZnSO4. 7H2O 0.353 25 13 Co(NO)2.6H2O 0.020 25 14 Na2MoO4. 2H2O 0.048 25

From the above Table it can prepare the stock of BBM medium in 1L to active the cell biomass through mixing the medium as following: 10 ml of each solutions 1-6 1 ml of each solutions 7-9 0.1ml of each solutions 10-14. After stirring, the pH of the medium is measured and it is adjusted at 6.9±0.2. The growth experiments will performed in 1000 mL Erlenmeyer flasks containing 500 of Bold's Basal Medium .The operation conditions are defined as following:

Experimental Investigation Parameters of Hydrogen Production by Algae

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

H

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

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Temperature set at 30 °C and air containing 0.04% CO2 was bubbled through fritted glass bubbles at a flow rate of 200 mLmin-1. Eight fluorescent lamps of 20 W were fitted [11].

B. Experimental set- up Duran bottle as a bioreactor was provided and used in this study for bio-hydrogen production with an inlet for nitrogen gas flushing and an outlet collecting the gas evolved by the culture. Microalgae was transferred to anaerobic process using the sulfur-deficient medium [12]. The hydrogen was produced by the degradation of stored organic compounds (glucose) via bioreactor; the evolved gas were allowed to pass through 5M 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 Diagnostics corporation [14].

III. RESULTS AND DISCUSSION

A. Effect of initial glucose concentration The initial glucose concentration plays an important role in the size and yield of hydrogen production during photosynthesis/ fermentation at relatively low initial glucose levels. The rate of photosynthesis was low according to the law of mass action as reported in the literature [15, 16]. Results shows that the highest production of hydrogen on varying glucose concentration with time was observed when initial glucose concentration was 10 g/l, as displayed in Figure 1. Then the production decreased with increasing glucose concentration. This result is in agreement with the results reported by reference [14, 16], which indicated when higher initial glucose concentrations were associated, the volume of hydrogen produced is decreased.

Fig. 1 hydrogen production at varying initial glucose concentrations.

B. Effect of initial medium pH Solution pH is an important controlling factor of anaerobic photolysis/ fermentation processes. Many researchers have reported that pH could affect the metabolic pathway in pure culture [17, 18, 19, 20]. pH is a critical factor in hydrogen production process. Algae can grow in a pH range of 4 –10. A set of tests in this study was performed by varying the pH from 6.0 ± 0.2 to 9.0 ± 0.2. Our result shows that the maximum rate of hydrogen production was measured at pH 8.0 ± 0.2, while the minimum rate of hydrogen production was recorded at pH 6.0 ± 0.2 as presented in Figure 2.

Fig. 2. Effect of initial medium pH on hydrogen production.

C. Effect of nitrogen and phosphate content of the culture media In hydrogen production fermentation/photosynthesis processes several researcher reported on the effect of Nitrogen and phosphate on the algal metabolism. Nitrate, ammonia, organic urea, and nitrite are the nitrogen forms utilized by most algae, ammonia or urea require the least energy to metabolize. Most media cultures contain nitrates or ammonium as the N source [20]. Table 2 Effect of addition of nitrogen and phosphate content on hydrogen production by C. Vularis

Parameters Bold's Basal Medium

addition 10% by (mole)

addition 20% (mole)

addition 30% (mole)

Initial pH 8 8 8 8

Final pH 6.5 7.1 6.8 6.9 Specific growth rate, h-1

0.0159 0.0162 0.0169 0.0173

InitialOD660nm 0.052 0.052 0.052 0.052

Final OD660nm 0.81 0.892 1.01 1.031 Duration Production, h

174 174 174 174

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 (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

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

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(BBC) media in anaerobic process as listed in Table (2). The volume of hydrogen production was increased by added 10% more than BBC media then decreased when higher addition of nitrogen and phosphate content more than 10 %. All experimental runs carried out at room temperature.

IV CONCLUSION The main aim in this study was investigated photosynthesis hydrogen production using algal C. Vulgaris in a bio reactor. It was found that some of the environmental parameters studied affected production, including; initial substrate concentration, initial pH and concentrations of nitrogen and phosphate content for (BBC) media. The optimum glucose concentration was investigated, and the highest yield of hydrogen was 2.85 mol (mol glucose)-1 when an initial glucose concentration of 10 g/l as a substrate. Increasing the initial substrate concentration above 10g/l decreased the hydrogen production rate. Another set of tests in this study was performed by varying pHs from 6.0± 0.2 to 9.0± 0.2. The maximum rate of hydrogen production was at pH 8.0± 0.2, while the minimum rate of hydrogen production was seen at pH 6.0± 0.2. The effect of addition nitrogen and phosphate content of the (BBC) media concentration on hydrogen production was also studied, and the best production was measured when increased 10% by mole. The hydrogen yield (YP/S) was about 2.85 mole (mole glucose)-1 at the optimum conditions (10 g, 8±0.2, 10%).

ACKNOWLEDGMENT This project was funded by Saudi Arabia Basic Industries Corroboration (SABIC) and deanship research (DSR), King Abdulaziz University. Jeddah, under grand No.( 15/472/1434). The authors, therefore, acknowledge with thanks SABIC and DSR technical and financial support.

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