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Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment
Bioresource Technology 102 (2011) 194–199
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Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment K. Bala Amutha, A.G. Murugesan * Manonmaniam Sundaranar University, Sri Paramakalyani Centre of Excellence in Environmental Sciences, Alwarkurichi 627 412, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 20 February 2010 Received in revised form 1 June 2010 Accepted 2 June 2010
Keywords: Algal biomass Chlorella vulgaris MSU 01 strain Carbon source Optimization conditions Kinetic parameters
a b s t r a c t Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, L-alanine, L-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6–19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (Ps) of 7.784 mL and production yield of (Pr) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R2 0.9988. The final pH of the broth was increased from 7.0 to 8.5–8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H2 evolution rate and higher butyrate concentration in the fermentate. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction The biological hydrogen production phenomena have been observed for more than hundred years, related R & D is of growing interest since the 1970s. Attention was first paid to photosynthetic process during the last 30 years and a great number of publications dealing with biological hydrogen production were published, but only few progresses are in practical applications. Photo biological production of H2 by eukaryotic algae is of interest because it holds the promise of generating a renewable fuel from abundantly available light and water (Kosourov et al., 2003; Hallenbeck, 2004). An alternative approach of photo producing H2 is based on the concept of indirect bio photolysis, in which metabolite accumulation acts as an intermediate step between photosynthetic H2O oxidation and H2 production (Melis et al., 2000). Unicellular microalgae hold the attention of commercial exploitation for hydrogen production and biomass production (Oncel and Sukan, 2009). Photosynthetic H2 production by microalgae is a promising process due to its minimal nutritional requirements. Electrons released upon the oxidation of water are transported to Fe-S protein ferredoxin on the reducing side of photo system I (Ghirardi et al., 2002). The O2 evolution on the oxidizing side of photo system II and H2 production
* Corresponding author. Tel.: +91 4634 283883; fax: +91 4634 283270. E-mail address: [email protected] (A.G. Murugesan).
on the reducing side of PS I have the ratio of H2:O2 = 2:1 has not yet been achieved. This is because of the hydrogenase which is sensitive to O2 (Ghirardi et al., 2004 Melis et al., 2000). In micro algal production system, the achievable photosynthetic productivity and light utilization efficiency of the algae are the most important factors in cost determination (Lindblad, 2004; Polle et al., 2002). The algae also have the ability to operate two distinct environments, namely aerobic and anaerobic (Melis, 2007). Alteration of the photosynthesis–respiration relationship leads to a continuous H2 photo production process that sustained for many days (Ghirardi et al., 2000). The processes of oxygenic photosynthesis, mitochondrial respirations, catabolism of endogenous substrates, and electron transport via, Fe–hydrogenase pathway lead to H2 production (Melis and Happe, 2001). According to Melis and Happe (2003), electrons for the Fe– hydrogenase contribute to both oxygenic photosynthesis (primary source) and photon fermentation (secondary source) with notable catabolism of starch and protein. The evolution of H2 in light/dark cycle was the early method for hydrogen production by green algae (Polle et al., 2002). It was based on the fact that algal cells accumulate starch during their growth in light. The expression of the Fe– hydrogenase is elicited in the light, leading to H2 production by the algae (Melis et al., 2000). In S-deprivation dependent process, the enzymatic production of H2 is the last step of complex cellular metabolism, which entail unknown metabolic, regulatory and electron-transport reactions (Melis and Happe, 2003).
0960-8524/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.008
K. Bala Amutha, A.G. Murugesan / Bioresource Technology 102 (2011) 194–199
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So in this present study, different sources like mannitol, glucose, citric acid, aspartic acid, L-alanine, L-cystine, sodium succinate and sodium pyruvate were used for algal media optimization to enhance the biomass production which intern favor hydrogen production. Corn stalk, from 1 to 5 g/L was also tested for its effective algal growth and hydrogen production. Modified Logistic model and modified Gompertz model were used to fit the experimental data in this study.
where H (mL) was the cumulative hydrogen production at the reaction time t (h), P (mL) was the hydrogen production potential, Rm (mL/h) was the maximum hydrogen production rate and k (h) was the lag time. In this study, the modified logistic model was used to fit the cumulative hydrogen production data obtained from each batch test to obtain H, Rm and k. Once the three parameters were obtained, Eq. (2) was used to calculate the hydrogen production rate in each batch test (Lay, 2001).
2. Methods
Rate ðmL=hÞ ¼ P=k þ P=Rm
2.1. Identification and production of algal biomass The sediment from the pond was taken from Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi, Tirunelveli. Modified BG-11 medium that contained (g/L): NaNO3, 1.5; K2HPO4.3H2O, 0.04; MgSO4.7H2O, 0.075; CaCl2.2H2O, 0.036; citric acid, 0.006; ferric ammonium citrate, 0.006; Na2EDTA, 0.001; Na2CO3, 0.02 and trace metal solution 1 mL (including H3BO3, 2.86 g; MnCl2.4H2O, 1.81 g;ZnSO4.7H2O, 0.222 g; Na2MoO4.2H2O, 0.390 g; CuSO4.5H2O, 79 mg; and Co(NO3)2.6H2O, 49.4 mg per liter) at pH 7.4 and MJ medium (Modified Jorgensen’s media) contained (g/ L): NaNO3, 15; KH2PO4, 2; EDTA, 10 mg; Na2SiO3.H2O, 22.7 mg; Vitamin B12, 1 mg; and trace element solution, 1 mL (containing H3BO3, 2.86 g; MnCl2.4H2O, 1.81 g; CuSO4.5H2O, 80 mg; ZnSO4.7H2O, 220 mg; Na2MoO4, 210 mg; and 1 drop of conc. H2SO4 per liter) were used as the media for the isolation of the algae. Both the media were used for the isolation of microalgae from fresh water (Rippka et al., 1979). The broth culture was smeared on different solid media and cultivated at 30 °C ± 2. Colonies were picked and transferred to the same media for purification in 250 mL Erlenmeyer culture. The broth culture medium was mixed constantly by shaker at 60 rpm. Microalgae were identified as described by Kessler (1985). The dry weight of the algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (Lee et al., 2009). The cell density was determined by measuring the chlorophyll concentration using ultraviolet/visible absorption spectroscopy (Kosourov et al., 2002). 2.2. Optimization of parameters for hydrogen production The Chlorella vulgaris (MSU 01 strain of 10 mL) was cultivated in various conditions in cultural media to obtain the optimal growth and high constitutive hydrogen production. Hydrogen was accumulated in the micro algal culture during period of environmental stress (i.e., anaerobic conditions). Different sources like mannitol, Glucose, alanine, citric acid, aspartic acid, L-alanine, L-cysteine, sodium succinate and sodium pyruvate were used for algal growth optimization. Corn stalk, from 1 to 5 g/L was also used for the analysis of effective algal growth and hydrogen production. It was mixed with an agitator to ensure the uniform mixing and illumination of 8 klux (2 nos.) was provided by using halogen lamps. 2.3. Analysis Hydrogen gas in the head space was analyzed by a gas chromatograph equipped with molecular sieve 5Å column using a thermal conductivity detector. The concentration of organic acids was measured using HPLC fitted with an Aminex HPX-87H organic acid analysis column. The modified Logistic model (Eq. (1)) was used to describe the progress of cumulative hydrogen production in the batch tests (Chandrashekar et al., 1999).
HðtÞ ¼ P: exp expRm e =Pðk tÞ þ 1
ð1Þ
ð2Þ
The hydrogen yield was calculated by dividing the hydrogen production potential by the amount of glucose consumed in each batch test. The substrate degradation efficiency was estimated by dividing the amount of glucose consumed by the amount of initial glucose (Lay, 2001).
3. Results and discussion 3.1. Identification and production of algal biomass Modified BG-11 and BJ medium were used to isolate the fresh water algal species from pond sediment and identified as C. vulgaris MSU 01 strain. Cells cultured under 8 klux (2 nos.) were used as the inoculum for 500 mL bioreactor. At first, growth profile of C. vulgaris MSU 01 strain was measured by spectrometric method using different substrates (wavelength 660 nm) (Fig. 1). Obviously C. vulgaris MSU 01 strain had a growth pattern without notable lag phase and a longer log phase. Initial biomass concentration of 0.13 g dry cell weight (DCW)/L was increased to 1.18 g DCW/L after 72 h and then decreased with time (Kim et al., 2003). Similar increased biomass concentration of 2.35 g/L and 2.6 g DCW/L were noted for sodium pyruvate and glucose supplemented medium at 120 h respectively. The estimated chlorophyll content was also high for sodium pyruvate and glucose medium with 6.14% and 6.79% respectively. The photosynthetic O2 evolution activity fell below the limit under sulfur deprivation which initiated anaerobic respiration (Kosourov et al., 2002; Kosourov et al., 2005; Zhang and Melis, 2002; Laurinavichene et al., 2002). C. vulgaris MSU 01 strain grown under 8 klux illumination was attained by two halogen lamps and corn stalk was added as the carbon source from 1 to 5 g/L. CO2 was provided by bubbling the growth medium with CO2. The growth profile of the isolated C. vulgaris MSU 01 strain at different amount of corn stalk supplemented medium was given in Fig. 2. In the present study, the initial biomass concentration was increased from 1.6 g/L to 19 g dry cell weight (DCW)/L at 10th day. The algal biomass concentration (OD – 660 nm) was found to increase from initial 0.373 to 1.33 till 6th day, and further decreased. The chlorophyll content was found to be high (8.03%) in corn stalk supplemented medium. The chlorophyll content of the Chlamydomonas cells cultured in TAP medium under synchronized illumination showed the maximum level (19.8 mg/L cell density) at late exponential phase (Sim et al., 2005). The maximum amount of hydrogen production (159 mL H2/g cells) was obtained at late exponential phase with 0.96 g/L cell density (Kim et al., 2003). In this study, the final protein content was found to be 26–32 g/L. High chlorophyll content induced large amount of hydrogen production under anaerobic condition (Kosourov et al., 2002). But according to Chinnasamy et al. (2010), biomass obtained from algal consortium grown in carpet industry wastewater medium was rich in proteins (53.8%) and low in carbohydrates (15.7%), lipids (5.3%) and chlorophyll (0.9%). From these results, it was explained that the high chlorophyll content produced in late exponential phase brought about increased water oxidation activity in PS II, which intern increased the level of electrons for hydrogen production.
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3.5
Algal biomass (OD660nm)
3
Mannitol
Glucose
S.Pyruvate
Aspartic acid
Citric acid
Alanine
Cysteine
2.5 2 1.5 1 0.5 0 1
2
3
4
5
6
7 8 Days
9
10
11
12
13
14
Fig. 1. Growth profile of the algae C. vulgaris MSU 01 strain (OD – 660 nm) using different substrates.
16 Algal biomass concentration (OD660nm)
1g/L
2 g/L
3 g/L
4 g/L
5 g/L
14 12 10 8 6 4 2 0 1
2
3
4
5
6
Days Fig. 2. Estimation of growth profile of the algae C. vulgaris MSU 01 strain using the corn stalk (g/L) as the carbon source.
3.2. H2 production from algal biomass There were four stages for algal hydrogen production: aerobic, oxygen consumption, anaerobic and hydrogen production stage (Kosourov et al., 2000). It was found that oxygen consumption rate increased with the increased cell density, which resulted in quick conversion of anaerobic environment. Hydrogen production from corn stalk supplemented medium (g/L) using the algae C. vulgaris MSU 01 strain was widely investigated in the study, because they rapidly assimilate various carbon sources under anaerobic conditions. H2 production rate was almost higher (26 mL/L/day) than the results of other studies even though the complex corn stalk medium was used as the substrate for breakdown. In this study, the total hydrogen production was 220 mL/L at the end of the fermentation period. The increased algal biomass (19 g/L) also
increased the hydrogen production rate. But 12,128 m3 of bio methane/ha/year was produced from the carpet industry wastewater medium (Chinnasamy et al., 2010). The anaerobic digestion by Taihu blue algae showed that the COD, biogas accumulation and hydrogen content was found to reach 26 mg/L, 500 mL and 37.2% (Yan et al., 2010). H2 evolution was enhanced to 30 and 50% at optimum 25 mM acetate and 37.5 mM glucose medium (Guan et al., 2004). The production of biogas and bio methane was noticed in higher amount than the hydrogen production from both carpet industry wastewater medium and anaerobic wastewater digestion by Taihu blue algae (Yan et al., 2010). But the hydrogen production in this study was found to be 26% using 4 g/L corn stalk complex medium at pH 7. A 14-fold increase in H2 production was obtained when the pH of the medium was increased from 5 to 8; with a sharp decline at
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od without initial delay (Tsygankov et al., 1999). It was also reported that the fructose at high concentration inhibited the hydrogen production of algal culture (Lee and Lee, 2002). Hydrogen production was catalyzed by nitrogenase in filamentous cyanobacteria and it was assumed that sugars were applied as hydrogen donors, the source of reductants and ATP energy (Oncel and Sukan, 2009). The kinetic parameters of the algal biomass involved in hydrogen production at different corn stalk supplemented medium (g/L) using modified logistic gas equation was found to be 0.968 mol and the shortest k phase was found to be 12 h (4 g/L) respectively (Table. 1). The ability of Anabaena strain CH 3 that utilize sugars as the substrate for hydrogen production showed high potential usage of the above strain in waste water treatment (Shah et al., 2001). In this study, amount of hydrogen produced from the substrate corn stalk medium (220 mL/L) was higher than the substrate used by other studies. The obtained hydrogen production rate was approximately 1.55 times higher than the TAP medium under sulfur deficiency (Park et al., 2006). Most preferred substrates for hydrogen production by Anabaena sp. strain CH 3 were noted as fructose and sucrose (Lee et al., 2009). The rate of 2.0 to 2.5 mL H2/L/h was observed in the sulfur deprived medium at 24–70 h (Melis and Happe 2001). Shortest log phase of biomass (6 days) was noted for the ammonium chloride supplemented medium which also increased the hydrogen production rate (Lee and Lee, 2002). The maximum hydrogen production was 2.152 mL H2 from 10 mL algal culture with the density of 6 106 cells mL/L at 96 h under conditions of NHþ 4 9.20 mM, PO3 2.09 mM, and pH 7.00 (Park et al., 2006). The average rate 4 of hydrogen production was 4 ± 0.9 mL/mg/h during 2–30 days of incubation. The composition of organic acids, especially butyric acid content in the fermentate by anaerobic algal biomass fermentation was substantially different at different concentration of corn stalk supplemented medium. Acetic acid content was found to be in trace amount only. Butyric acid content was found to increase from 12.23 to 32 mM at the end of catabolic breakdown of the substrate corn stalk that ranges from 1 to 4 g/L, then decrease further at 5 g/L corn stalk medium (Fig. 1). This was due to the final pH around 8 found at the end of the fermentation. The accumulation of butyric acid, acetic acid and hydrogen reached 1.7, 1.4 and 3.8 times for the pretreated blue green algae under alkali condition (Yan et al., 2010). In conclusion, the optimization of hydrogen
Hydrogen production potential (mL); substrate consumption rate (g/L)
hydrogen production was due to the utilization of complex corn stalk medium by the isolated C. vulgaris MSU 01 strain. The kinetic parameters involved in hydrogen production at 4 g/ L corn stalk medium using the algal inoculums (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential of (Ps) 7.784 mL; production yield (Pr) of 15.534 mL, respectively. The lag phase was found to be 48 h. The final pH of the corn stalk media broth was increased from 7.0 to 8.5–8.7. It was reported that the heterocystous cyanobacteria produced hydrogen through degradation of stored glycogen or additional organic substrates (Yoon et al., 2002). An optimal anaerobic incubation period of 32 h gave the maximum H2 evolution in the absence of sulfur in the medium (Guan et al., 2004). The lag phase obtained for the hydrogen production using 4 g/L corn stalk medium was noted to be 12 h. At 5 g/L corn stalk medium, the lag phase was found to be 60 h. This increased level of lag phase was due to the substrate inhibition by the isolated strain. When 1000 ppm fructose was added to the medium, only little hydrogen was produced by log phase Anabaena CH 3 strain under the light intensity of 65 mmol/m2/s (Yoon et al., 2002). The growth profile of algal biomass at optimized corn stalk medium (4 g/L) using the algal inoculum of 50 mL in the bioreactor (500 mL) expressed the logistic model with best fit R2. The optimized hydrogen production profile of the algal biomass using the logistic model was found to be with R2, 0.99 and the Rmax was 16.46 mL respectively. Correlation of substrate degradation (g/L corn stalk) and product formation (hydrogen) profile of algal biomass concentration by Luedeking–Piret model was noted with R2, 0.88. This study also confirmed that the substrate degradation and product formation by the isolated C. vulgaris MSU 01 strain was correlated regarding hydrogen production and substrate degradation. But the hydrogen production of 0.6 mmol (40 mL head space) was obtained at 100 h by algal culture in acetate medium (Lee and Lee, 2002). It was suggested that nitrogenase enzyme operate more proton to produce hydrogen than nitrogen reduction in such sub stage of cells. It has been known that A. variabilis used fructose as the substrate to produce hydrogen under anaerobic condition (Kosourov et al., 2000). Correlation profile of hydrogen production potential (mL/ L) and substrate consumption rate (g/L corn stalk) using algal biomass on different days was given in Fig. 3. The fructose medium raised the hydrogen yield and maintained longer production peri-
9 8 7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
7
Days Fig. 3. Correlation profile of hydrogen production potential (mL) and substrate consumption rate (corn stalk g/L) using the algae C. vulgaris MSU 01 strain on different days; algal inoculums – 50 mL; Bioreactor volume – 500 mL.
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Table 1 Kinetic parameters of algal biomass involved in hydrogen production at different amount of corn stalk (g/l) using modified logistic gas equation; algal inoculums – 50 mL; Bioreactor volume – 500 mL.
a b c d e
S.no
Concentration (g/L)
ka
H (mL)b
Rm (mL)c
Pr (mL)d
Ps (mL/h)e
Butyrate concentration (mM)
1 2 3 4 5
1 2 3 4 5
36 24 36 12 60
12 18 20 26 19
0.760 0.859 0.961 0.968 0.440
0.717 0.8107 0.907 0.913 0.415
0.231 0.400 0.352 0.669 0.184
12.23 19 23 32 31
k, Lag phase. Hmax, Cumulative hydrogen production. Rmax, Maximum hydrogen production rate. Pr, Specific hydrogen production yield. Ps, Specific hydrogen production rate.
production in this study was achieved by controlling several parameters like sulfur deprived medium, creating anaerobic environment, the use of complex substance corn stalk medium, which had the highest increase of chlorophyll concentration, needed to increase the electrons for hydrogen production. From these results, it was concluded that the most preferred substrate for hydrogen production by C. vulgaris MSU 01 strain isolated from the pond sediment of Sri Paramakalyani Centre for Environmental Sciences was in capable of utilizing the complex corn stalk medium ranges from 1 to 4 g/L. Thus the optimized 4 g/L corn stalk supplemented medium was suitable for increased hydrogen production. We expect that the results presented from this study would be useful in the system design for algal hydrogen production using then agro waste corn stalk medium. 4. Conclusion Growth of the freshwater green algae C. vulgaris MSU 01 strain was high when various carbon sources including corn stalk was supplied by bubbling CO2 in the medium. But the addition of corn stalks (1–5 g/L) as the carbon source increased the cell growth at the optimized 4 g/L corn stalk and yielded high hydrogen than the other studies which used simple carbon sources as the substrates. Since protein (%) and chlorophyll content (%) were increased by algal biomass utilizing the complex substrate corn stalk medium (4 g/L), the above mentioned amount of corn stalk medium seems to be good substrate for H2 production. Accumulation of high content of butyrate in the anaerobic fermentate of algal biomass, rich in fat and protein contents were explained by known pathways. Thus we succeeded in the fermentative conversion of corn stalk medium by algal biomass to organic acids by anaerobic fermentation using C. vulgaris MSU 01 strain. The composition of organic acids, especially butyric acid content in the fermentate by anaerobic fermentation of algal biomass was substantially different for different amount of added corn stalk. We will further investigate the degradation of other biomass, protein, fats and identify the other metabolites from the fermentate. The anaerobic fermentation by C. vulgaris MSU 01 strain in the conversion process of complex carbon source has some advantages in increased H2 evolution rate and the presents of butyrate concentration in the fermentate. References Chandrashekar, K., Arthur, F.P., Panda, T., 1999. Optimization of temperature and initial pH and kinetic analysis of tartaric acid production by Gluconobacter suboxydans. Bioprocess. Eng. 20, 203–207. Chinnasamy, S., Bhatnagar, A., Claxton, R., Das, K.C., 2010. Biomass and bioenergy production potential of microalgae consortium in open and closed bioreactors using untreated carpet industry effluent as growth medium. Bioresour. Technol. 101 (17), 6751–6760.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment K. Bala Amutha, A.G. Murugesan * Manonmaniam Sundaranar University, Sri Paramakalyani Centre of Excellence in Environmental Sciences, Alwarkurichi 627 412, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 20 February 2010 Received in revised form 1 June 2010 Accepted 2 June 2010
Keywords: Algal biomass Chlorella vulgaris MSU 01 strain Carbon source Optimization conditions Kinetic parameters
a b s t r a c t Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, L-alanine, L-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6–19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (Ps) of 7.784 mL and production yield of (Pr) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R2 0.9988. The final pH of the broth was increased from 7.0 to 8.5–8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H2 evolution rate and higher butyrate concentration in the fermentate. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction The biological hydrogen production phenomena have been observed for more than hundred years, related R & D is of growing interest since the 1970s. Attention was first paid to photosynthetic process during the last 30 years and a great number of publications dealing with biological hydrogen production were published, but only few progresses are in practical applications. Photo biological production of H2 by eukaryotic algae is of interest because it holds the promise of generating a renewable fuel from abundantly available light and water (Kosourov et al., 2003; Hallenbeck, 2004). An alternative approach of photo producing H2 is based on the concept of indirect bio photolysis, in which metabolite accumulation acts as an intermediate step between photosynthetic H2O oxidation and H2 production (Melis et al., 2000). Unicellular microalgae hold the attention of commercial exploitation for hydrogen production and biomass production (Oncel and Sukan, 2009). Photosynthetic H2 production by microalgae is a promising process due to its minimal nutritional requirements. Electrons released upon the oxidation of water are transported to Fe-S protein ferredoxin on the reducing side of photo system I (Ghirardi et al., 2002). The O2 evolution on the oxidizing side of photo system II and H2 production
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on the reducing side of PS I have the ratio of H2:O2 = 2:1 has not yet been achieved. This is because of the hydrogenase which is sensitive to O2 (Ghirardi et al., 2004 Melis et al., 2000). In micro algal production system, the achievable photosynthetic productivity and light utilization efficiency of the algae are the most important factors in cost determination (Lindblad, 2004; Polle et al., 2002). The algae also have the ability to operate two distinct environments, namely aerobic and anaerobic (Melis, 2007). Alteration of the photosynthesis–respiration relationship leads to a continuous H2 photo production process that sustained for many days (Ghirardi et al., 2000). The processes of oxygenic photosynthesis, mitochondrial respirations, catabolism of endogenous substrates, and electron transport via, Fe–hydrogenase pathway lead to H2 production (Melis and Happe, 2001). According to Melis and Happe (2003), electrons for the Fe– hydrogenase contribute to both oxygenic photosynthesis (primary source) and photon fermentation (secondary source) with notable catabolism of starch and protein. The evolution of H2 in light/dark cycle was the early method for hydrogen production by green algae (Polle et al., 2002). It was based on the fact that algal cells accumulate starch during their growth in light. The expression of the Fe– hydrogenase is elicited in the light, leading to H2 production by the algae (Melis et al., 2000). In S-deprivation dependent process, the enzymatic production of H2 is the last step of complex cellular metabolism, which entail unknown metabolic, regulatory and electron-transport reactions (Melis and Happe, 2003).
0960-8524/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.008
K. Bala Amutha, A.G. Murugesan / Bioresource Technology 102 (2011) 194–199
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So in this present study, different sources like mannitol, glucose, citric acid, aspartic acid, L-alanine, L-cystine, sodium succinate and sodium pyruvate were used for algal media optimization to enhance the biomass production which intern favor hydrogen production. Corn stalk, from 1 to 5 g/L was also tested for its effective algal growth and hydrogen production. Modified Logistic model and modified Gompertz model were used to fit the experimental data in this study.
where H (mL) was the cumulative hydrogen production at the reaction time t (h), P (mL) was the hydrogen production potential, Rm (mL/h) was the maximum hydrogen production rate and k (h) was the lag time. In this study, the modified logistic model was used to fit the cumulative hydrogen production data obtained from each batch test to obtain H, Rm and k. Once the three parameters were obtained, Eq. (2) was used to calculate the hydrogen production rate in each batch test (Lay, 2001).
2. Methods
Rate ðmL=hÞ ¼ P=k þ P=Rm
2.1. Identification and production of algal biomass The sediment from the pond was taken from Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi, Tirunelveli. Modified BG-11 medium that contained (g/L): NaNO3, 1.5; K2HPO4.3H2O, 0.04; MgSO4.7H2O, 0.075; CaCl2.2H2O, 0.036; citric acid, 0.006; ferric ammonium citrate, 0.006; Na2EDTA, 0.001; Na2CO3, 0.02 and trace metal solution 1 mL (including H3BO3, 2.86 g; MnCl2.4H2O, 1.81 g;ZnSO4.7H2O, 0.222 g; Na2MoO4.2H2O, 0.390 g; CuSO4.5H2O, 79 mg; and Co(NO3)2.6H2O, 49.4 mg per liter) at pH 7.4 and MJ medium (Modified Jorgensen’s media) contained (g/ L): NaNO3, 15; KH2PO4, 2; EDTA, 10 mg; Na2SiO3.H2O, 22.7 mg; Vitamin B12, 1 mg; and trace element solution, 1 mL (containing H3BO3, 2.86 g; MnCl2.4H2O, 1.81 g; CuSO4.5H2O, 80 mg; ZnSO4.7H2O, 220 mg; Na2MoO4, 210 mg; and 1 drop of conc. H2SO4 per liter) were used as the media for the isolation of the algae. Both the media were used for the isolation of microalgae from fresh water (Rippka et al., 1979). The broth culture was smeared on different solid media and cultivated at 30 °C ± 2. Colonies were picked and transferred to the same media for purification in 250 mL Erlenmeyer culture. The broth culture medium was mixed constantly by shaker at 60 rpm. Microalgae were identified as described by Kessler (1985). The dry weight of the algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (Lee et al., 2009). The cell density was determined by measuring the chlorophyll concentration using ultraviolet/visible absorption spectroscopy (Kosourov et al., 2002). 2.2. Optimization of parameters for hydrogen production The Chlorella vulgaris (MSU 01 strain of 10 mL) was cultivated in various conditions in cultural media to obtain the optimal growth and high constitutive hydrogen production. Hydrogen was accumulated in the micro algal culture during period of environmental stress (i.e., anaerobic conditions). Different sources like mannitol, Glucose, alanine, citric acid, aspartic acid, L-alanine, L-cysteine, sodium succinate and sodium pyruvate were used for algal growth optimization. Corn stalk, from 1 to 5 g/L was also used for the analysis of effective algal growth and hydrogen production. It was mixed with an agitator to ensure the uniform mixing and illumination of 8 klux (2 nos.) was provided by using halogen lamps. 2.3. Analysis Hydrogen gas in the head space was analyzed by a gas chromatograph equipped with molecular sieve 5Å column using a thermal conductivity detector. The concentration of organic acids was measured using HPLC fitted with an Aminex HPX-87H organic acid analysis column. The modified Logistic model (Eq. (1)) was used to describe the progress of cumulative hydrogen production in the batch tests (Chandrashekar et al., 1999).
HðtÞ ¼ P: exp expRm e =Pðk tÞ þ 1
ð1Þ
ð2Þ
The hydrogen yield was calculated by dividing the hydrogen production potential by the amount of glucose consumed in each batch test. The substrate degradation efficiency was estimated by dividing the amount of glucose consumed by the amount of initial glucose (Lay, 2001).
3. Results and discussion 3.1. Identification and production of algal biomass Modified BG-11 and BJ medium were used to isolate the fresh water algal species from pond sediment and identified as C. vulgaris MSU 01 strain. Cells cultured under 8 klux (2 nos.) were used as the inoculum for 500 mL bioreactor. At first, growth profile of C. vulgaris MSU 01 strain was measured by spectrometric method using different substrates (wavelength 660 nm) (Fig. 1). Obviously C. vulgaris MSU 01 strain had a growth pattern without notable lag phase and a longer log phase. Initial biomass concentration of 0.13 g dry cell weight (DCW)/L was increased to 1.18 g DCW/L after 72 h and then decreased with time (Kim et al., 2003). Similar increased biomass concentration of 2.35 g/L and 2.6 g DCW/L were noted for sodium pyruvate and glucose supplemented medium at 120 h respectively. The estimated chlorophyll content was also high for sodium pyruvate and glucose medium with 6.14% and 6.79% respectively. The photosynthetic O2 evolution activity fell below the limit under sulfur deprivation which initiated anaerobic respiration (Kosourov et al., 2002; Kosourov et al., 2005; Zhang and Melis, 2002; Laurinavichene et al., 2002). C. vulgaris MSU 01 strain grown under 8 klux illumination was attained by two halogen lamps and corn stalk was added as the carbon source from 1 to 5 g/L. CO2 was provided by bubbling the growth medium with CO2. The growth profile of the isolated C. vulgaris MSU 01 strain at different amount of corn stalk supplemented medium was given in Fig. 2. In the present study, the initial biomass concentration was increased from 1.6 g/L to 19 g dry cell weight (DCW)/L at 10th day. The algal biomass concentration (OD – 660 nm) was found to increase from initial 0.373 to 1.33 till 6th day, and further decreased. The chlorophyll content was found to be high (8.03%) in corn stalk supplemented medium. The chlorophyll content of the Chlamydomonas cells cultured in TAP medium under synchronized illumination showed the maximum level (19.8 mg/L cell density) at late exponential phase (Sim et al., 2005). The maximum amount of hydrogen production (159 mL H2/g cells) was obtained at late exponential phase with 0.96 g/L cell density (Kim et al., 2003). In this study, the final protein content was found to be 26–32 g/L. High chlorophyll content induced large amount of hydrogen production under anaerobic condition (Kosourov et al., 2002). But according to Chinnasamy et al. (2010), biomass obtained from algal consortium grown in carpet industry wastewater medium was rich in proteins (53.8%) and low in carbohydrates (15.7%), lipids (5.3%) and chlorophyll (0.9%). From these results, it was explained that the high chlorophyll content produced in late exponential phase brought about increased water oxidation activity in PS II, which intern increased the level of electrons for hydrogen production.
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3.5
Algal biomass (OD660nm)
3
Mannitol
Glucose
S.Pyruvate
Aspartic acid
Citric acid
Alanine
Cysteine
2.5 2 1.5 1 0.5 0 1
2
3
4
5
6
7 8 Days
9
10
11
12
13
14
Fig. 1. Growth profile of the algae C. vulgaris MSU 01 strain (OD – 660 nm) using different substrates.
16 Algal biomass concentration (OD660nm)
1g/L
2 g/L
3 g/L
4 g/L
5 g/L
14 12 10 8 6 4 2 0 1
2
3
4
5
6
Days Fig. 2. Estimation of growth profile of the algae C. vulgaris MSU 01 strain using the corn stalk (g/L) as the carbon source.
3.2. H2 production from algal biomass There were four stages for algal hydrogen production: aerobic, oxygen consumption, anaerobic and hydrogen production stage (Kosourov et al., 2000). It was found that oxygen consumption rate increased with the increased cell density, which resulted in quick conversion of anaerobic environment. Hydrogen production from corn stalk supplemented medium (g/L) using the algae C. vulgaris MSU 01 strain was widely investigated in the study, because they rapidly assimilate various carbon sources under anaerobic conditions. H2 production rate was almost higher (26 mL/L/day) than the results of other studies even though the complex corn stalk medium was used as the substrate for breakdown. In this study, the total hydrogen production was 220 mL/L at the end of the fermentation period. The increased algal biomass (19 g/L) also
increased the hydrogen production rate. But 12,128 m3 of bio methane/ha/year was produced from the carpet industry wastewater medium (Chinnasamy et al., 2010). The anaerobic digestion by Taihu blue algae showed that the COD, biogas accumulation and hydrogen content was found to reach 26 mg/L, 500 mL and 37.2% (Yan et al., 2010). H2 evolution was enhanced to 30 and 50% at optimum 25 mM acetate and 37.5 mM glucose medium (Guan et al., 2004). The production of biogas and bio methane was noticed in higher amount than the hydrogen production from both carpet industry wastewater medium and anaerobic wastewater digestion by Taihu blue algae (Yan et al., 2010). But the hydrogen production in this study was found to be 26% using 4 g/L corn stalk complex medium at pH 7. A 14-fold increase in H2 production was obtained when the pH of the medium was increased from 5 to 8; with a sharp decline at
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od without initial delay (Tsygankov et al., 1999). It was also reported that the fructose at high concentration inhibited the hydrogen production of algal culture (Lee and Lee, 2002). Hydrogen production was catalyzed by nitrogenase in filamentous cyanobacteria and it was assumed that sugars were applied as hydrogen donors, the source of reductants and ATP energy (Oncel and Sukan, 2009). The kinetic parameters of the algal biomass involved in hydrogen production at different corn stalk supplemented medium (g/L) using modified logistic gas equation was found to be 0.968 mol and the shortest k phase was found to be 12 h (4 g/L) respectively (Table. 1). The ability of Anabaena strain CH 3 that utilize sugars as the substrate for hydrogen production showed high potential usage of the above strain in waste water treatment (Shah et al., 2001). In this study, amount of hydrogen produced from the substrate corn stalk medium (220 mL/L) was higher than the substrate used by other studies. The obtained hydrogen production rate was approximately 1.55 times higher than the TAP medium under sulfur deficiency (Park et al., 2006). Most preferred substrates for hydrogen production by Anabaena sp. strain CH 3 were noted as fructose and sucrose (Lee et al., 2009). The rate of 2.0 to 2.5 mL H2/L/h was observed in the sulfur deprived medium at 24–70 h (Melis and Happe 2001). Shortest log phase of biomass (6 days) was noted for the ammonium chloride supplemented medium which also increased the hydrogen production rate (Lee and Lee, 2002). The maximum hydrogen production was 2.152 mL H2 from 10 mL algal culture with the density of 6 106 cells mL/L at 96 h under conditions of NHþ 4 9.20 mM, PO3 2.09 mM, and pH 7.00 (Park et al., 2006). The average rate 4 of hydrogen production was 4 ± 0.9 mL/mg/h during 2–30 days of incubation. The composition of organic acids, especially butyric acid content in the fermentate by anaerobic algal biomass fermentation was substantially different at different concentration of corn stalk supplemented medium. Acetic acid content was found to be in trace amount only. Butyric acid content was found to increase from 12.23 to 32 mM at the end of catabolic breakdown of the substrate corn stalk that ranges from 1 to 4 g/L, then decrease further at 5 g/L corn stalk medium (Fig. 1). This was due to the final pH around 8 found at the end of the fermentation. The accumulation of butyric acid, acetic acid and hydrogen reached 1.7, 1.4 and 3.8 times for the pretreated blue green algae under alkali condition (Yan et al., 2010). In conclusion, the optimization of hydrogen
Hydrogen production potential (mL); substrate consumption rate (g/L)
hydrogen production was due to the utilization of complex corn stalk medium by the isolated C. vulgaris MSU 01 strain. The kinetic parameters involved in hydrogen production at 4 g/ L corn stalk medium using the algal inoculums (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential of (Ps) 7.784 mL; production yield (Pr) of 15.534 mL, respectively. The lag phase was found to be 48 h. The final pH of the corn stalk media broth was increased from 7.0 to 8.5–8.7. It was reported that the heterocystous cyanobacteria produced hydrogen through degradation of stored glycogen or additional organic substrates (Yoon et al., 2002). An optimal anaerobic incubation period of 32 h gave the maximum H2 evolution in the absence of sulfur in the medium (Guan et al., 2004). The lag phase obtained for the hydrogen production using 4 g/L corn stalk medium was noted to be 12 h. At 5 g/L corn stalk medium, the lag phase was found to be 60 h. This increased level of lag phase was due to the substrate inhibition by the isolated strain. When 1000 ppm fructose was added to the medium, only little hydrogen was produced by log phase Anabaena CH 3 strain under the light intensity of 65 mmol/m2/s (Yoon et al., 2002). The growth profile of algal biomass at optimized corn stalk medium (4 g/L) using the algal inoculum of 50 mL in the bioreactor (500 mL) expressed the logistic model with best fit R2. The optimized hydrogen production profile of the algal biomass using the logistic model was found to be with R2, 0.99 and the Rmax was 16.46 mL respectively. Correlation of substrate degradation (g/L corn stalk) and product formation (hydrogen) profile of algal biomass concentration by Luedeking–Piret model was noted with R2, 0.88. This study also confirmed that the substrate degradation and product formation by the isolated C. vulgaris MSU 01 strain was correlated regarding hydrogen production and substrate degradation. But the hydrogen production of 0.6 mmol (40 mL head space) was obtained at 100 h by algal culture in acetate medium (Lee and Lee, 2002). It was suggested that nitrogenase enzyme operate more proton to produce hydrogen than nitrogen reduction in such sub stage of cells. It has been known that A. variabilis used fructose as the substrate to produce hydrogen under anaerobic condition (Kosourov et al., 2000). Correlation profile of hydrogen production potential (mL/ L) and substrate consumption rate (g/L corn stalk) using algal biomass on different days was given in Fig. 3. The fructose medium raised the hydrogen yield and maintained longer production peri-
9 8 7 6 5 4 3 2 1 0 0
1
2
3
4
5
6
7
Days Fig. 3. Correlation profile of hydrogen production potential (mL) and substrate consumption rate (corn stalk g/L) using the algae C. vulgaris MSU 01 strain on different days; algal inoculums – 50 mL; Bioreactor volume – 500 mL.
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Table 1 Kinetic parameters of algal biomass involved in hydrogen production at different amount of corn stalk (g/l) using modified logistic gas equation; algal inoculums – 50 mL; Bioreactor volume – 500 mL.
a b c d e
S.no
Concentration (g/L)
ka
H (mL)b
Rm (mL)c
Pr (mL)d
Ps (mL/h)e
Butyrate concentration (mM)
1 2 3 4 5
1 2 3 4 5
36 24 36 12 60
12 18 20 26 19
0.760 0.859 0.961 0.968 0.440
0.717 0.8107 0.907 0.913 0.415
0.231 0.400 0.352 0.669 0.184
12.23 19 23 32 31
k, Lag phase. Hmax, Cumulative hydrogen production. Rmax, Maximum hydrogen production rate. Pr, Specific hydrogen production yield. Ps, Specific hydrogen production rate.
production in this study was achieved by controlling several parameters like sulfur deprived medium, creating anaerobic environment, the use of complex substance corn stalk medium, which had the highest increase of chlorophyll concentration, needed to increase the electrons for hydrogen production. From these results, it was concluded that the most preferred substrate for hydrogen production by C. vulgaris MSU 01 strain isolated from the pond sediment of Sri Paramakalyani Centre for Environmental Sciences was in capable of utilizing the complex corn stalk medium ranges from 1 to 4 g/L. Thus the optimized 4 g/L corn stalk supplemented medium was suitable for increased hydrogen production. We expect that the results presented from this study would be useful in the system design for algal hydrogen production using then agro waste corn stalk medium. 4. Conclusion Growth of the freshwater green algae C. vulgaris MSU 01 strain was high when various carbon sources including corn stalk was supplied by bubbling CO2 in the medium. But the addition of corn stalks (1–5 g/L) as the carbon source increased the cell growth at the optimized 4 g/L corn stalk and yielded high hydrogen than the other studies which used simple carbon sources as the substrates. Since protein (%) and chlorophyll content (%) were increased by algal biomass utilizing the complex substrate corn stalk medium (4 g/L), the above mentioned amount of corn stalk medium seems to be good substrate for H2 production. Accumulation of high content of butyrate in the anaerobic fermentate of algal biomass, rich in fat and protein contents were explained by known pathways. Thus we succeeded in the fermentative conversion of corn stalk medium by algal biomass to organic acids by anaerobic fermentation using C. vulgaris MSU 01 strain. The composition of organic acids, especially butyric acid content in the fermentate by anaerobic fermentation of algal biomass was substantially different for different amount of added corn stalk. We will further investigate the degradation of other biomass, protein, fats and identify the other metabolites from the fermentate. The anaerobic fermentation by C. vulgaris MSU 01 strain in the conversion process of complex carbon source has some advantages in increased H2 evolution rate and the presents of butyrate concentration in the fermentate. References Chandrashekar, K., Arthur, F.P., Panda, T., 1999. Optimization of temperature and initial pH and kinetic analysis of tartaric acid production by Gluconobacter suboxydans. Bioprocess. Eng. 20, 203–207. Chinnasamy, S., Bhatnagar, A., Claxton, R., Das, K.C., 2010. Biomass and bioenergy production potential of microalgae consortium in open and closed bioreactors using untreated carpet industry effluent as growth medium. Bioresour. Technol. 101 (17), 6751–6760.
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