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Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well
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Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well H.S. Jayasinghearachchi, Priyangshu M. Sarma, Banwari Lal* Environmental and Industrial Biotechnology Division, The Energy and Resource Institute, Darbari Seth Block, Habitat Place, Lordhi Road, New Delhi 110003, India
article info
abstract
Article history:
Hydrogen producing novel bacterial strain was isolated from formation water from oil
Received 5 October 2011
producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene
Received in revised form
sequencing. Hydrogen production by novel strain was pH and substrate dependent and
20 December 2011
favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0e9.0 for glucose fermen-
Accepted 27 December 2011
tation at 70 C. The highest H2 yield was 2.64 0.40 mol H2 mol glucose at 10 g/L,
Available online 25 January 2012
5.36 0.41 mol H2 mol e sucrose at 10 g/L, 17.91 0.16 mmol H2 g e starch at 5 g/L and 2.09 0.21 mol H2 mol xylose at 5 g/L. The maximum specific hydrogen production rates
Keywords:
6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type
Biological hydrogen production
fermentation pathway (approximately 97%) was found to be dominant in strain A3N,
Soluble starch
whereas butyrate formation was found in sucrose and xylose fermentation. Lactate
Extremely thermophilic
production increased with high xylose concentrations above 10 g/L.
Thermoanaerobacter mathranii
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Hydrogen is considered as one of the most promising alternative energy carrier to fossil fuel in the future. It is clean and environmentally friendly fuel that produces only water when combusted with oxygen. It is a high energy fuel (122 KJ/g) than hydrocarbon fuels [1]. Approximately 95% of commercially produced hydrogen comes from carboncontaining raw materials, primarily fossil in origin [2]. Due to the depletion of fossil fuel and emission of greenhouse gas (CO2) during conventional hydrogen production process, biological hydrogen production from biomass has been recognized as an eco-friendly, and less energy intensive process to produce hydrogen compared to photosynthetic or chemical processes [3].
Thermophilic anaerobic fermentation processes hold tremendous potential for the forthcoming generations as well as commercial production of hydrogen fuel [4]. The energy required for heating is the main drawback of dark fermentation at high temperatures. However, the biohydrogen fermentation at extremely thermophilic temperatures (over 70 C) has many outstanding advantages over lower temperature conditions, which could compensate the energy expenses from temperature increased such as higher yields [5], stable continuous production [3], higher production rate [6], better pathogenic destructions [7], higher rate of hydrolysis of complex materials such as household solid waste or manure [8,9]. Further, the extreme thermophilic microorganisms are known to generate low cell densities, which result in rather moderate hydrogen productivities [10].
* Corresponding author. Tel.: þ91 11 24682100; fax: þ91 11 24682144. E-mail addresses: [email protected], [email protected] (B. Lal). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.12.145
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Efficient extreme thermophilic hydrogen producing bacteria can be applied for fermentation of high temperature substrates such as hydrolyzates obtained from hydrothermal pretreatment of lignocelluloses or high temperature industrial effluents where the temperature is already high. Thus, additional heating would not be required [11,6]. Moreover, it has been proposed that combination of hydrogen fermentation with methane to produce hythane, which is a mixture of hydrogen and methane [6]. Thermoanaerobacter species might be the efficient dark fermentative bacteria [12]. The dark fermentative hydrogen production is affected by several environmental factors such as initial pH, substrate concentration and fermentation temperature [11,13,14]. It has been observed that the initial cultivation pH affects the hydrogenase enzyme activity while substrate concentration affects the ability of hydrogen producing bacteria to produce hydrogen during dark fermentation [15].The research on dark fermentative hydrogen production at extreme thermophilic temperatures is scanty and yet to be investigated. Further, to the best of our knowledge, Thermoanaerobacter mathranii have not been investigated before in detail for the dark fermentative hydrogen production at extremely thermophilic temperatures. In view of the above, this study investigates the optimum conditions for fermentative hydrogen production by T. mathranii strain A3N using different substrates such as starch, sucrose, glucose and xylose in batch operation under extremely thermophilic condition.
2.
Materials and methods
2.1.
Sample collection and isolation of the strain A3N
Oil water mixture samples (97% water, 3% oil) were collected from oil producing well (well No KD 269) in Nawagam, Ahamadabad, India into 50 mL of anaerobic sterilized serum bottles containing 1 mL of 2% Na2S during April, 2010. The reservoir temperature was 87 C and salinity of formation water was 15 g/L. Reservoir pressure was 700 psi.
2.2.
Isolation of the strain A3N
Samples (0.1 mL) were inoculated in liquid anaerobic basal medium consisting of (g/L): NH4Cl 0.5; Yeast extract 5; K2HPO4 0.25; KCl 0.002; MgCl26H2O 0.125; NH4HCO3 0.4; Peptone 1; NH4H2PO4 0.4; NaH2PO4 0.5. trace element 1 mL, vitamin solution 1 mL [16]. Sucrose (10 g/L) was used as sole carbon and energy source. All the culture bottles were incubated at 70 C for 3e5 days and sub cultured in same medium after 3 days of incubation. Serial dilution technique was used to obtain pure cultures. In order to be sure to obtain a pure isolates, serial dilution steps were repeated several times. All the sub cultures and diluted cultures were incubated at 70 C under atmospheric pressure. Cells were observed under a light microscope (Olympus, Japan) and pure isolate A3N was routinely cultivated in basal medium.
2.3. Identification of strain using 16S rRNA gene sequence Genomic DNA from the pure isolate A3N was extracted as reported [17]. The 16S rRNA gene sequence was amplified by PCR using MicroSec full Gene Kit (Applied Biosystems, UK) as per the manufactures instructions. Amplified product was sequenced using the DyeDeoxy Terminator Cycle sequencing Kit (Applied Biosystems, UK) as directed in the manufacturer’s protocol with an automatic Genetic analyser (Model 300; Applied Biosystems, USA). The nucleotide sequence of 16S rRNA gene of the isolate A3N was compared with other related sequences available in GenBank using BLAST programme [18]. Further, the nucleotide sequence of isolate A3N was aligned with closely related sequences found in GenBank, using CLUSTAL W, and pair wise evolutionary distances were computed using JukeseCanter Model [19]. Phylogenetic analysis was performed using MEGA version 4. Confidence in the tree topologies was evaluated by re-sampling 100 bootstrap trees [20].
2.4.
Study on the growth properties of the strain A3N
Effect of different temperatures (40, 45, 55, 65, 70 and 80 C), NaCl concentrations (0e7%) (w/v) and different carbon sources (5 g/L glucose, fructose, sucrose, xylose, xylan, ribose, raffinose, mannose, arabinose, xylan, rhamnose, galactose, cellobiose, dextrose, lactose, maltose, sorbitol, molasses, starch and cellulose) on the growth of the isolate A3N were studied in anaerobic liquid basal medium. All the growth experiments were performed in triplicate, using 60 mL serum bottles containing 30 mL of anaerobic basal medium.
2.5. Optimization of hydrogen production in batch fermentation Batch dark fermentation studies were performed in 125 mL anaerobic bottles with working volume of 10 mL of liquid anaerobic basal medium aforementioned. The inoculum acquired during exponential growth phase was added at 0.5% (v/v) by using disposable syringe. The inoculum was adapted to the each substrate by subculturing several cycles in batch cultures. Unless noted otherwise as a variable, initial starch, sucrose, glucose and xylose concentrations were kept at 10 g/L. Influence of different initial pH levels for the hydrogen production on different substrates (starch, glucose, xylose and sucrose) was studied. The initial pH levels ranging from 5.5 to 9.5 with 0.5 increments were selected for this study. The pH of the medium was adjusted with 1 M HCl or 1 M NaOH prior to dispensing the medium into the 125 mL serum bottles. Further, effect of initial substrate concentration on hydrogen production was investigated in batch experiments as described above. Optimal pH levels obtained for the maximum hydrogen production from each individual substrate were used as initial culture pH. Initial substrate concentrations tested were 5, 10, 15, 20 and 25 g/L for each individual substrate. The obtained optimal initial cultivation pH and initial substrate concentration which gave the maximum hydrogen
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production and yield for each individual substrate was further used to study the hydrogen production performance at two different temperatures (60 and 70 C). The batch experiments were conducted in 1 L anaerobic bottles with a working volume of 300 mL. The effect of headspace volume on hydrogen production, substrate degradation and biomass accumulation was also studied using starch as substrate under optimum initial cultivation pH and temperature. The experiment was conducted in a 1350 mL anaerobic glass bottles with different fermentation volumes ranging from 150 to 750 mL with 150 mL increment. Total pressure in the headspace was measured with mechanical pressure gauge directly connected to the anaerobic bottle. All experiments were repeated thrice, and hydrogen production was quantified at an interval of 24 h.
2.6.
Analytical methods
The headspace gas composition in each batch experiment was analysed by the gas chromatography (GC 6890N, Agilent, USA) equipped with a thermal conductivity detector (TCD) and HP PLOTQ column (15 m 530 mm 40 mm film thickness) as described in our previous study [16,21]. Volatile fatty acids (VFA) composition in the liquid phase i.e. acetate, butyrate, isobutyrate, valerate, isovalerate and propionate were analysed by gas chromatography (GC 6890N, Agilent, USA) equipped with a flame ionization detector (FID) and DB-WAXetr column (30 m 530 mm 1 mm film thickness) [16]. The concentrations of sugar and ethanol in the culture supernatant was analysed by High Performance Liquid Chromatography (HPLC, Agilent 1100 series, USA) equipped with Sugar-PAK.1 column (Water Research, USA) and refractive index detector (RID) [16]. AminexR HPX-87H, (300 mm 7.8 mm) column (BIO RAD, CA, USA) was used to quantify lactate, succinate and formate using water as the mobile phase at a flow rate of 0.6 mL/min. The column temperature was maintained at 35 C. Residual starch concentration was estimated by Anthrone-sulfuric method [22]. The percentage substrate degradation was estimated by dividing the amount of carbohydrate consumed by the amount of initial carbohydrate. The bacterial growth in each batch experiment was directly monitored by measuring the increase in optical density at 600 nm using UV visible spectrophotometer (UV2450, Shimadzu, Japan). The biomass concentrations were determined by standard method [23]. In brief, the samples (10 mL) were filtered through pre-weight 0.45 mm filter paper, dried at 105 C until constant weight. All measures were performed in triplicate. The rate of hydrogen production (mmol H2/L/h) in each experiment was calculated from total hydrogen production (mmol/L) divided by time duration (h) for which H2 evolution occurred. The specific rate of H2 production was calculated from rate of H2 production (mmol/L/h) divided by dry cell weight (DCW) (g/L). Hydrogen yield was calculated as total molaric amount of H2 divided by molaric amount of consumed total sugar (mol H2/mol total sugar consumed) [24]. In the case of starch, H2 yield was calculated by molaric amount of H2 divided by gram of starch consumed (mmol H2/g starch).
3.
Results and discussion
3.1.
General properties of the strain A3N
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The novel strain A3N is found to be extremely thermophilic, rod-shaped, Gram negative, fermentative anaerobe with an optimum growth at 74 C. The growth was weak at less than 40 to above 85 C. The strain A3N is moderately alkaliphilic, and able to grow at wide range of pH from 5.0 to 9.5 at 74 C. There was no growth observed at pH 4.0 or below. Further, pH in the range of 7.0e9.0 was found to be a favorable pH range for optimum biomass accumulation at 74 C. Similar growth pattern was observed at 60 C. However, total biomass accumulation was comparatively low at 60 C. It was able to grow up to 4% NaCl concentration (w/v) with an optimum of 0.5e0.75% NaCl (w/v) in the presence of sucrose as a sole carbon and energy source, and could utilize glucose, sucrose, fructose, arabinose, dextrose, lactose, sorbitol, xylan, xylose, starch and ribose, raffinose, rhamnose, molasses and maltose.
3.2. Identification of the strain A3N based on the 16S rRNA gene sequence analysis The 16S rRNA gene sequence of isolate A3N was compared with others available in GenBank database. The sequence of A3N had 99% identity to that of T. mathranii (Y11279), and the two sequences grouped together in a phylogenetic tree (Fig. 1). The 16S DNA sequence reported in this paper has been deposited in GenBank under the accession number HM179101.
3.3. Effect of initial culture pH on growth and hydrogen production from different substrates 3.3.1.
Hydrogen production from starch
T. mathranii A3N grew well in soluble starch medium under extremely thermophilic condition (Table 1). Cell growth was affected by initial culture pH affected the cell growth and poor cell growth was observed at initial cultivation pH between 5.0 and 5.5. High initial cultivation pH enhanced the bacterial cell growth, and the highest cell growth was observed at initial cultivation pH 8.0 at 70 C. The strain preferred high temperature (70 C) for optimum cell growth and the cell growth was slightly low at 60 C. The hydrogen production in the starch medium was initial culture pH dependent and the hydrogen production occurred within the wide range of pH (5.0e9.5). The hydrogen evolution was low at pH between 5.0 and 6.0. When the initial pH increased from 6.5 to 7.0, there was a significant increase in total hydrogen production (Table 1). Total hydrogen production of 108e110 mmol H2/L at 70 C was observed at initial pH between 8.0 and 9.0 suggesting that the initial pH between 8.0 and 9.0 was favorable for the cells to utilize starch for growth and hydrogen production. Almost similar hydrogen production performance was observed at 60 C within the same pH range.
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Fig. 1 e Phylogenetic dendrogram based on 16S rRNA sequences indicating the position of strain A3N among closely related members. The topology shown is an unrooted tree obtained with a neighbor joining algorithm (JukeseCantor corrections) with bootstrap values expressed as percentages of 100 replications.
3.3.2.
Hydrogen production from glucose, sucrose and xylose
T. mathranii A3N grew well on glucose, sucrose and xylose under extremely thermophilic condition (70 C). High cell densities were observed at alkaline range of pH, and optimum cell growth in glucose medium was observed at pH between
9.0 and 9.5 (Table 2). In sucrose medium, the highest cell growth was observed at pH 8.5 whereas in xylose medium the highest cell growth was at pH 9.0 (Table 3). Hydrogen production from glucose, sucrose and xylose fermentation was significantly influenced by initial culture pH
Table 1 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during soluble starch fermentation at different initial culture pH and at temperature 60 and 70 C. Initial pH
Fermentation temperature 60 C H2 (mmol/L)
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
47.57 53.19 61.09 68.10 82.86 90.65 98.49 103.40 109.25 97.22
0.34 0,36 0.97 0.67 0.72 0.82 0.96 0.56 0.42 0.64
AA (mmol/L) 14.25 16.96 17.98 28.55 27.30 29.30 34.88 39.92 35.33 31.75
0.18 10.12 0.42 0.21 0.24 0.62 0.42 0.30 0.56 0.64
70 C Cell growth (OD600 nm) 0.48 0.011 0.57 0.009 0.74 0.013 1.11 0.015 1.50 0.026 1.520.013 1.71 0.011 1.70 0.066 1.40 0.013 1.11 0.011
AA-acetate; Each experiment is representative of three independent replicates.
H2 (mmol/L) 42.57 50.67 62.09 69.20 103.46 100.30 108.60 107.60 110.00 99.52
0.49 0.76 0.11 0.68 0.12 0.25 0.33 0.25 0.41 0.31
AA (mmol/L) 19.26 21.73 25.14 28.02 41.89 40.61 43.97 43.56 46.15 38.29
0.27 0.16 0.32 0.19 0.11 0.21 0.33 0.32 0.19 0.12
Cell growth (OD600 nm) 0.41 0.63 1.01 1.41 1.67 1.73 1.79 1.72 1.78 1.31
0.016 0.008 0.015 0.011 0.009 0.012 0.005 0.006 0.012 0.008
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Table 2 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during sucrose and glucose fermentation at different initial culture pH and at temperature 70 C. Initial pH
Substrate Sucrose
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Glucose
H2 (mmol/L)
AA (mmol/L)
Cell growth (OD600 nm)
H2 (mmol/L)
AA (mmol/L)
Cell growth (OD600 nm)
e 25.62 0.25 31.13 0.45 58.90 0.31 73.09 0.29 84.18 0.46 82.25 0.12 79.22 0.45 73.67 0.25 68.93 0.43
e 10.25 0.45 12.45 0.67 23.56 0.27 29.24 0.25 33.67 0.21 32.90 0.25 31.69 0.23 29.47 0.41 26.36 0.13
e 0.37 0.005 0.42 0.012 0.54 0.008 0.75 0.010 1.03 0.006 1.07 0.013 1.34 0.011 1.13 0.008 1.03 0.011
21.54 0.34 32.94 0.21 58.25 0.42 55.52 0.21 68.12 0.34 72.68 0.71 72.96 0.23 73.18 0.15 91.14 0.41 76.49 0.35
11.32 0.17 13.78 0.11 22.90 0.24 24.57 0.11 22.40 0.23 28.95 0.41 25.93 0.34 27.10 0.26 27.20 0.62 27.88 0.21
0.51 0.002 0.68 0.004 1.30 0.001 1.36 0.003 1.62 0.011 1.62 0.008 1.87 0.006 1.87 0.011 1.92 0.009 1.94 0.003
AA-acetate; Each experiment is representative of three independent replicates.
(Tables 2 and 3). Significantly low hydrogen evolution was observed in glucose and sucrose medium at low initial culture pH (below pH 6.0). Further, no growth and hydrogen production occurred in sucrose medium at initial cultivation pH 5.0 (Table 2). Increase in initial cultivation pH led to an increase in total hydrogen production. Optimum initial culture pH for maximum hydrogen production (84.18 mmol/L) in sucrose medium was observed at initial culture pH 7.5. Whereas the initial culture pH 9.0 was observed as optimum pH for maximum hydrogen production (91 mmol/L) in glucose medium. The initial cultivation pH 7.5 was found to be the optimum pH for maximum hydrogen production in xylose medium.
3.4.
Effect of initial substrate concentration
Batch fermentations using starch, glucose, sucrose and xylose at different concentrations ranging from 5 to 35 g/L were followed until hydrogen production in each bioreactor has stopped. Nearly complete substrate degradation (approximately w98.6%) was observed in medium supplemented with 5 g/L of each substrate. Further, the substrate degradation was drastically reduced while increasing initial substrate concentration. Residual substrate concentration was high in cultures
supplemented with high initial substrates and similar to all substrates used. Maximum hydrogen yield of 17.91 mmol/g starch (398 mL/g starch) was obtained at the concentration of starch 7.5 g/L, pH levels 7.5e9.0 and temperature at 70 C (Table 4). This hydrogen is nearly 78e80% of the theoretically derived maximum value of 553 mL of hydrogen per gram of starch [25]. The result of this study is comparable with study reported by Hasyim [4] using Thermoanaerobacterium dominant mixed culture. Further, the hydrogen yield achieved in this study is comparatively higher as compared to the some previous reports [13,26,27]. With increase in starch concentration, the hydrogen yield and substrate degradation tended to decrease, and at initial starch concentration of 35 g/L, substrate removal was approximately 32.8% (Table 4). Zhang et al. [25] has also reported that the variation tendency was also different from thermophilic hydrogen fermentation of starch in which H2 yield decrease with increasing starch concentration. High initial starch concentration up to 30 g/L enhanced the biomass accumulation, and further increased in starch concentration inhibited the biomass formation in batch experiments. At the end of the fermentation, it was found that high concentrations of reducing sugar accumulate in fermentation broth in which the high initial starch concentration was added. This
Table 3 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during xylose fermentation at different initial culture pH and at temperature 70 C. Initial pH 5.0 5.5 6.0 7.0 7.5 8.0 8.5 9.0 9.5
H2 (mmol/L) 57.60 56.15 56.20 57.47 66.84 61.27 61.69 50.14 52.99
0.12 0.10 0.21 0.16 0.24 0.09 0.07 0.15 0.08
AA (mmol/L) 14.44 12.86 14.20 13.93 16.33 12.86 14.43 14.15 18.34
0.14 0.34 0.02 0.24 0.31 0.13 0.17 0.87 0.69
Cell growth (OD600nm) 1.66 1.80 1.76 1.98 2.05 2.04 2.20 2.32 1.84
AA-acetate; Each experiment is representative of three independent replicates.
0.024 0.040 0.013 0.024 0.013 0.017 0.016 0.019 0.023
BA (mmol/L) 1.24 1.09 1.12 1.13 1.29 1.28 1.23 1.44 0.32
0.012 0.034 0.016 0.013 0.014 0.015 0.016 0.012 0.003
LA (mmol/L) 0.036 0.048 0.029 0.026 0.014 0.026 0.021 0.022 0.022
0.013 0.0012 0.012 0.004 0.006 0.013 0.005 0.002 0.014
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Table 4 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N and substrate degradation at optimum initial culture pH of 8.0 and temperature of 70 C under different initial soluble starch concentrations. Soluble starch (g/L) 5 7.5 10 15 20 25 30 35
H2 yield (mol/g starch) 15.95 0.43 17.91 0.16 17.03 0.42 14.79 0.45 14.17 0.17 13.82 0.67 12.26 0.57 12.79 0.11
Biomass (g/L) 0.71 0.78 0.93 1.03 1.08 1.06 1.19 1.09
0.004 0.011 0.03 0.09 0.012 0.09 0.01 0.04
HPR (mmol/L/h) 2.39 3.40 3.65 3.42 3.37 3.49 3.27 3.11
0.013 0.071 0.092 0.031 0.072 0.061 0.045 0.037
SHPR (mmol/g cell/h)
TSM (mmol/L)
% Substrate degradation
3.40 0.021 4.25 0.033 3.93 0.062 3.32 0.014 2.86 0.011 3.29 0.071 2.75 0.046 2.86 0.023
22.03 0.18 21.34 0.25 23.41 0.41 24.98 0.27 22.24 0.33 19.56 0.19 17.49 0.28 21.45 0.61
97.2 94.53 62.80 58.34 51.90 42.48 36.97 32.85
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, lactate and ethanol); Each experiment is representative of three independent replicates.
indicates the strong amylolitic enzyme activity of the strain A3N, and showed the high potential to apply for biohydrogen production from starch. Few of the studies have emphasized the importance of amylase secreting dark fermentative hydrogen producing bacteria [28,29]. The hydrogen evolution was totally inhibited due to low pH and accumulation of intermediate products. The low or no hydrogen production at low pH (between 4.5 and 5.0), could be partially attributing to strong decrease in hydrogenase activity. Similar observation has also been reported from continuous fermentation, that at high starch loading rates, hydrogen formation is inhibited due to high starch accumulation [30]. The best hydrogen production conditions (i.e. initial cultivation at pH 7.5, temperature at 70 C and sucrose concentration at10 g/L) gave the maximum hydrogen yield of 5.36 mol of H2 mol of sucrose (Table 5). High initial substrate concentration has led to accumulation of high biomass and organic metabolites, which probably has resulted in unfavorable conditions for further hydrogen evolution in the present batch experimental conditions. Meantime, low hydrogen yield at 5 g/L sucrose could be possibly due to main part of the organic substrates under low substrate concentration could go to maintain the cell growth resulting in low hydrogen production per substrate amount [31]. Further, the concentration of residual sucrose increased with the initial sucrose concentration. Thus, it seems that substrate inhibition occurred if the sucrose concentration was higher than 10 g/L.
Nearly complete substrate removal was observed at a concentration of 5 g/L of xylose (Table 6). The substrate degradation decrease with an increase in the concentration of substrate, and hydrogen production yield was lower than that obtained at low concentrations. The upper limit of substrate concentration for xylose without marked decrease in molar hydrogen yield was 5 g/L. The maximum hydrogen yield observed in this study was 2.09 0.21 mol H2/mol xylose, which is higher than that of reported previously using mixed culture fermentation [6]. Theoretically xylose can be converted to hydrogen with a maximum yield of 3.33 mol-H2/mol xylose when the acetate is produced as the fermentation by product [6]. Further it has been reported that thermophiles typically generate higher hydrogen yield from xylose (1.7e2.4 mol H2/mol xylose) than that of mesophiles (commonly, 1 mol H2/mol xylose) [13]. Lower hydrogen yields from xylose in our experiment compared to the theoretical yield could be explained with some conversion of substrate in to biomass, formation of other intermediate products such as ethanol, lactate and pH drop during fermentation. Few studies have been carried out on hydrogen production under extreme thermophilic conditions [6]. The maximum hydrogen yield 2.64 mol/mol glucose was observed at initial glucose concentration of 10 g/L (Table 7). As explained above, high initial glucose concentration yielded low hydrogen yield. Thus initial glucose concentration (10 g/L) is likely to be the most suitable for hydrogen production by strain A3N.
Table 5 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 7.5 and temperature of 70 C under different initial sucrose concentrations. Sucrose (g/L) 5 10 15 20 25 30 35
H2 yield (mol/mol sucrose) 4.21 5.36 5.20 4.19 3.92 3.69 3.69
0.19 0.41 0.27 0.18 0.31 0.42 0.38
Biomass (g/L) 0.66 0.73 0.81 0.82 0.89 0.94 1.17
0.006 0.002 0.011 0.003 0.008 0.010 0.012
HPR (mmol/L/h) 2.08 4.48 5.07 4.16 4.79 4.13 4.25
0.054 0.061 0.037 0.051 0.047 0.026 0.029
SHPR (mmol/g cell/h) 3.15 6.13 6.24 5.05 5.38 5.49 5.52
0.066 0.043 0.038 0.029 0.033 0.015 0.061
Sucrose consumed (g/L)
TSM (mmol/L)
22.57 40.39 41.56 36.57 33.84 39.32 42.57
4.26 6.12 7.23 7.14 7.92 9.01 9.45
0.062 0.029 0.017 0.041 0.035 0.016 0.036
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM-total soluble metabolites - (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
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Table 6 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 7.5 and temperature of 70 C under different initial xylose concentrations. Xylose (g/L) 5 10 15 20 25 30 35
H2 yield(mol/mol xylose) 2.09 1.73 1.45 1.19 0.79 0.65 0.55
0.21 0.13 0.26 0.11 0.06 0.08 0.02
Biomass (g/L) 0.63 0.81 0.93 0.98 1.01 1.06 1.08
0.003 0.001 0.009 0.012 0.007 0.003 0.010
HPR (mmol/h/L) 3.33 3.97 4.02 3.75 2.30 2.47 2.49
0.021 0.018 0.042 0.017 0.052 0.021 0.027
SHPR (mmol/g cell/h) 5.74 4.90 4.31 3.82 2.25 2.33 2.31
0.032 0.015 0.029 0.031 0.036 0.027 0.018
Xylose consumed mmol/L
TSM (mmol/L)
25.86 24.01 25.42 24.08 25.28 25.63 29.60
4.95 5.78 6.14 7.02 7.32 8.75 9.37
0.076 0.043 0.091 0.037 0.062 0.087 0.093
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
The growth and hydrogen production at two different temperatures (60 and 70 C) is shown in Table 8. The best fermentation temperature for maximum H2 evolution was found to be at 70 C. However, H2 production at 60 C was also found to be considerably high. Formation of ethanol and lactate was found at higher levels in 60 C incubated cultures that that of at 70 C. The growth, substrate consumption and hydrogen production performance of strain A3N with fermentation time are shown in Figs. 2 and 3. The fermentation behavior of the strain A3N on different substrates has been found to be same. The maximum specific hydrogen rate was observed at late exponential growth phase of the cultures. Nevertheless, some significant differences in metabolite formation, and consequently in hydrogen production were observed. The maximum specific hydrogen production rates in starch and sucrose media was 6.29 and 9.34 mmol/g cell/h respectively (Fig. 2A). Maximum specific hydrogen production rate of 5.76 mmol/g cell/h was observed in xylose medium whereas maximum specific hydrogen production rate in glucose medium was 4.89 mmol/g cell/h (Fig. 2B). With the progress of the fermentation, pH value in the fermentation broths dropped due to the release of organic acids. At the early stationary phase of the culture growth of the cultures (approximately 42 h), the pH in the fermentation broth was between 4.2 and 4.5 irrespective of the substrate used. Thus no hydrogen evolution was found after 42 h of fermentation. Nevertheless, the residual substrates in the fermentation broth in all batch experiments were observed.
In this study, under pH uncontrolled batch experiments, the best H2 yield from the strain T. mathranii A3N was 2.64 0.40 mol H2/mol e glucose, 5.36 0.41 mol H2/mol e sucrose, 17.91 0.16 mmol H2/g e starch and 2.09 0.21 mol H2/mol e xylose. In another study in the literature, it has been reported that under pH controlled conditions the best H2 yield from Thermotoga neapolitana at extreme thermophilic condition was 2.22 0.11/mol H2 mol e xylose, 3.2 0.16 mol/H2mol e glucose and 4.95 .0.25 mol H2/mol sucrose [24].
3.5.
Distribution of intermediate metabolites
Formation of VFAs and other metabolic products during fermentative hydrogen process under extremely thermophilic conditions are scanty and yet to be investigated. In the literature, it has been reported that the hydrogen yields can be improved by increasing acetate formation, and decreasing butyrate formation by using a high-temperature fermentation process with thermophiles or extreme thermophiles, operating at temperature higher than 60 C [32]. In this study an attempt was made to understand fermentation pathways during hydrogen production under extremely thermophilic condition with different carbon sources. Acetate-type fermentation pathway was found to be the major fermentation type in xylose fermentation by T. mathranii A3N. Butyrate formation was found in sucrose and xylose medium during fermentation. In other studies, acetate and butyrate formation associated with xylose fermentation by Thermoanaerobacterium thermosaccharolyticum W16 [33],
Table 7 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 9.0 and temperature of 70 C under different initial glucose concentrations. Glucose (g/L) 5 10 15 20 25 30 35
H2 yield (mol/mol glucose) 1.93 2.64 2.18 2.02 1.62 1.69 1.40
0.11 0.40 0.32 0.46 0.62 0.27 0.056
Biomass (g/L) 0.62 0.72 0.85 0.92 0.88 0.91 1.04
0.004 0.0071 0.002 0.0041 0.009 0.0051 0.0031
HPR (mmol/L/h) 2.77 4.49 4.64 5.06 4.13 4.07 4.09
0.032 0.024 0.051 0.072 0.036 0.057 0.081
SHPR (mmol/g cell/h) 4,47 6.23 5.46 5.50 4.69 4.48 3.93
0.044 0.051 0.018 0.064 0.091 0.062 0.052
Glucose consumed (g/L)
TSM (mmol/L)
24.66 29.58 33.67 36.31 28.75 29.35 27.62
4.92 6.43 7.29 7.62 8.69 8.23 8.95
0.057 0.066 0.052 0.047 0.026 0.038 0.059
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
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Table 8 e Comparison of hydrogen production performance of Thermoanaerobacter mathranii strain A3N at two different temperatures (60 and 70 C) under optimum pH and substrate concentrations obtained for maximum hydrogen production in this study. Substrate Fermentation (g/L) temperature ( C) Glucose Sucrose Starch Xylose
70 60 70 60 70 60 70 60
H2 yield (mol/mol substrate) 2.64 2.11 5.36 4.67 17.91 14.21 2.09 1.92
HPR (mmol/L/h)
0.40 0.28 0.41 0.35 0.16 (mmol/g starch) 0.29 (mmol/g starch 0.21 0.18
4.49 3.74 4.48 4.11 3.4 3.56 3.33 2.86
0.023 0.029 0.061 0.042 0.071 0.052 0.032 0.041
Biomass (g/L) 0.78 0.69 0.73 0.68 0.63 0.60 0.72 0.62
0.0071 0.0042 0.002 0.001 0.011 0.002 0.003 0.001
TSM (mmol/L) % Substrate degradation 34.78 32.97 40.39 38.42 45.26 42.31 33.97 38.26
64.3 62.3 61.2 57.4 98.6 80.72 99 97
HPR-hydrogen production rate; TSM-total soluble metabolites (acetate, butyrate, lactate and ethanol).
acetate as a metabolic intermediate followed by ethanol and lactate by mixed culture [6] were found in xylose fermentation at extreme thermophilic temperatures. In this study, a significant increase in lactate production was found in cultures supplemented with high xylose concentrations above 10 g/L resulting low hydrogen yield as there is no hydrogen generation with lactic acid formation [34,35]. Further, we found that mixed type fermentation occurred in sucrose fermentation by strain A3N. Further, during early
A
exponential phase, acetate formation was found to be the dominant fermentation type, whereas when the cultures entered early stationary phase, equal amount of acetate and butyrate was observed. In contrast, acetate and lactate were found as the main intermediate products during sucrose fermentation by Caldicellulosiruptor saccharolyticus at 70 C [36]. The metabolic changes observed could be due to the expression levels of various acid forming enzymes such as phospho-transbutyrylase (PTB), which is responsible for the
A
B
B
Fig. 2 e A&B. Time course of growth, substrate consumption and hydrogen production by Thermoanaerobacter mathranii A3N in (A) starch medium; (B) Sucrose medium, under optimal conditions. Each experiment is representative of three independent replicates.
Fig. 3 e A&B. Time course of growth, substrate consumption and hydrogen production by Thermoanaerobacter mathranii A3N in (A) xylose medium; (B) Glucose medium under optimal conditions. Each experiment is representative of three independent replicates.
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butyrate formation had high expression levels at pH 6.3 [37]. Less quantities of formate, succinate and lactate were detected when initial culture pH is below 6.5 during sucrose fermentation. Acetate was the major VFA observed (approximately 97%) during soluble starch fermentation and butyrate formation was not detected. Small quantities of iso-butyrate (1e3 mg/L), succinate, formate (2e3 mg/L) and lactate (5e7 mg/L) were detected at all pH levels tested. Similar metabolic profile was also observed in xylose fermentation. In addition, trace amount of butyrate was observed (Table 3).
4.
Conclusion
In this study, we have isolated extremely thermophilic hydrogen producing bacterial strain from oil water mixtures (formation water) from oil well, Ahamadabad, India. It was identified as T. mathranii by 16S rRNA gene sequencing. Hydrogen production by the strain A3N was optimized in batch fermentation at 70 C. The highest H2 yields were 2.64 0.40 mol H2 mol glucose at 10 g/L, 5.36 0.41 mol H2 mol e sucrose at 10 g/L, 17.91 0.16 mmol H2 g e starch at 5 g/L and 2.09 0.21 mol H2 mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was dominant in strain A3N. Butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L. Results of this study demonstrates the high potential of the novel bacterium T. mathranii strain A3N for dark fermentative hydrogen production at 70 C.
Acknowledgments The authors are indebted to the Department of Biotechnology, Govt. of India for financial support. The authors are thankful to Dr. R. K. Pachauri, DG, TERI, New Delhi, for providing infrastructure facility to carry out the present study. We thank all technical staff in the laboratory for providing their assistance to complete this study successfully.
references
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[4] Hasyim R, et al. Biological production from sago starch in wastewater using enriched thermophilic mixed culture from hot spring. Int Journal Hydrogen Energy 2011. doi:10.1016/jijhydene.2011.04.211. [5] Cakur A, Ozmihci S. KargiF.Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation. Int J Hydrogen Energy 2010; 35:13214e8. [6] Kongjan P, Min B, Angelidaki I. Biohydrogen production from xylose at extreme thermophilic temperatures (70 C) by mixed culture fermentation. Water Res 2009;43:1414e24. [7] Li S, Lai C, Cai Y, Yang X, Yang S, Zhu M, et al. High efficiency hydrogen production from glucose/xylose by the Idh-deleted Thermoanaerobacerium strain. Bioresources Technol 2010;101: 8718e24. [8] Hartmann H, Ahring BK. A novel process configuration for anaerobic digestion of source-sorted household waste using hyper-thermophilic post-treatment. Biotechnol Bioeng 2005; 90:830e7. [9] Lu J, Gavala H, Skiadas I, Mladenovska Z, Ahring B. Improving anaerobic sewage sludge digestion by implementation of a hyper-thermophilic prehydrolysis step. J Environ Manage 2008;88(4):881e9. [10] Peintner C, Zeidan AA, Schnitahofer W. Bioreactor systems for thermophilic fermentative hydrogen production: evaluation and comparison of appropriate systems. J Cleaner Production 2010;18:515e22. [11] Wang J, Wan W. Effect of temperature on fermentative hydrogen production by mixed cultures. Int J Hydrogen Energy 2008;33:5392e7. [12] Hniman A, O-Thong S, Prasertsan P. Developing a thermophilic hydrogen-producing microbial consortia from geothermal spring for efficient utilization of xylose and glucose mixed substrates and oil palm trunk hydrolyzate. Int J Hydrogen Energy 2011;36:8785e93. [13] Ren N, Cao G, Wang A, Lee DJ, Guo W, Zhu Y. Dark fermentation of xylose and glucose mix using isolated Thermoanaerobacterium thermosaccharolyticumW16. Int J Hydrogen Energy 2008;33:6124e32. [14] Abreu AA, Denko AS, Costa JC, Ferreira EC, Alves MM. Inoculum type response to different pHs on biohydrogen production from L-arabinose, a component of hemicellulosic biopolymer. Int J Hydrogen Energy 2009;34:1744e51. [15] Gilroyed BH, Chang O, Chu A, Hao X. Effect of temperature on anaerobic fermentative hydrogen gas production from feed lot cattle manure using mixed microflora. Int J Hydrogen Energy 2008;33:4301e8. [16] Jayasinghearachchi HS, Sarma PM, Singh S, Aginihotri A, Mandal AK, Lal B. Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea-buried crude oil pipelines. Int J Hydrogen Energy 2009;34:7197e207. [17] Sambrook J, Fritsch EF, Maniatis T. Molecular cloninig: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: ColdSpring Harbor Laboratory; 1989. [18] Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programme. Nucleic Acids Res 1997;25:3389e402. [19] Jukes TH, Cantor CR. Evolution of protein molecules. In: Munro HN, editor. Mammalian protein metabolism. New York: Academic Press; 1969. p. 211e32. [20] Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1995;30:783e91. [21] Jayasinghearachchi HS, Singh S, Sarma PM, Aginihotri A, Lal B. Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crudeoil pipeline. Int J Hydrogen Energy 2010;35:6665e73.
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[22] Dubois M, Gilles K, Hmilton J, Rebers P, Smith F. Colourimetric method for determination of sugars and related substances. Anal Chem 1956;28:350e6. [23] Greenberg AE, Clesceri LS, Eaton AD, editors. Standard methods for the examination of water and waste water. 21st ed. Washington DC, USA: America Public Health Association (APHA); 2005. [24] Ngo TA, et al. Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture. Int J Hydrogen Energy 2011;36:14014e23. [25] Zhang T, Liu H, Fang HHP. Biohydrogen production from starch in wastewater under thermophilic condition. J Environ Mang 2003;69:149e56. [26] Nguyen TAD, Kim JP, Kim MS, Oh YK, Sim SJ. Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation. Int J Hydrogen Energy 2008;33:1483e8. [27] van Niel EWJ, Classen PAM, Stams AIM. Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldocellulosiruptorsaccharalyticus. Biotechnol Bioeng 2002;81:255e62. [28] Wang Y-H, Li S-L, Chen I-C, Cheng S-S. Starch hydrolysis characteristics of hydrogen producing sludge in thermophilic hydrogen fermenter fed with kitchen waste. Int J Hydrogen Energy 2009;34:7435e40. [29] Higuchi Y, Ohashi A, Imachi H, Harada H. Hydrolytic activity of alpha-amylase in anaerobic digested sludge. Water Sci Technol 2005;52:259e66. [30] Ozmihci S, Kargi F. Effects of starch loading rate on performance of combined fed-batch fermentation of ground
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Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well H.S. Jayasinghearachchi, Priyangshu M. Sarma, Banwari Lal* Environmental and Industrial Biotechnology Division, The Energy and Resource Institute, Darbari Seth Block, Habitat Place, Lordhi Road, New Delhi 110003, India
article info
abstract
Article history:
Hydrogen producing novel bacterial strain was isolated from formation water from oil
Received 5 October 2011
producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene
Received in revised form
sequencing. Hydrogen production by novel strain was pH and substrate dependent and
20 December 2011
favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0e9.0 for glucose fermen-
Accepted 27 December 2011
tation at 70 C. The highest H2 yield was 2.64 0.40 mol H2 mol glucose at 10 g/L,
Available online 25 January 2012
5.36 0.41 mol H2 mol e sucrose at 10 g/L, 17.91 0.16 mmol H2 g e starch at 5 g/L and 2.09 0.21 mol H2 mol xylose at 5 g/L. The maximum specific hydrogen production rates
Keywords:
6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type
Biological hydrogen production
fermentation pathway (approximately 97%) was found to be dominant in strain A3N,
Soluble starch
whereas butyrate formation was found in sucrose and xylose fermentation. Lactate
Extremely thermophilic
production increased with high xylose concentrations above 10 g/L.
Thermoanaerobacter mathranii
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Hydrogen is considered as one of the most promising alternative energy carrier to fossil fuel in the future. It is clean and environmentally friendly fuel that produces only water when combusted with oxygen. It is a high energy fuel (122 KJ/g) than hydrocarbon fuels [1]. Approximately 95% of commercially produced hydrogen comes from carboncontaining raw materials, primarily fossil in origin [2]. Due to the depletion of fossil fuel and emission of greenhouse gas (CO2) during conventional hydrogen production process, biological hydrogen production from biomass has been recognized as an eco-friendly, and less energy intensive process to produce hydrogen compared to photosynthetic or chemical processes [3].
Thermophilic anaerobic fermentation processes hold tremendous potential for the forthcoming generations as well as commercial production of hydrogen fuel [4]. The energy required for heating is the main drawback of dark fermentation at high temperatures. However, the biohydrogen fermentation at extremely thermophilic temperatures (over 70 C) has many outstanding advantages over lower temperature conditions, which could compensate the energy expenses from temperature increased such as higher yields [5], stable continuous production [3], higher production rate [6], better pathogenic destructions [7], higher rate of hydrolysis of complex materials such as household solid waste or manure [8,9]. Further, the extreme thermophilic microorganisms are known to generate low cell densities, which result in rather moderate hydrogen productivities [10].
* Corresponding author. Tel.: þ91 11 24682100; fax: þ91 11 24682144. E-mail addresses: [email protected], [email protected] (B. Lal). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.12.145
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Efficient extreme thermophilic hydrogen producing bacteria can be applied for fermentation of high temperature substrates such as hydrolyzates obtained from hydrothermal pretreatment of lignocelluloses or high temperature industrial effluents where the temperature is already high. Thus, additional heating would not be required [11,6]. Moreover, it has been proposed that combination of hydrogen fermentation with methane to produce hythane, which is a mixture of hydrogen and methane [6]. Thermoanaerobacter species might be the efficient dark fermentative bacteria [12]. The dark fermentative hydrogen production is affected by several environmental factors such as initial pH, substrate concentration and fermentation temperature [11,13,14]. It has been observed that the initial cultivation pH affects the hydrogenase enzyme activity while substrate concentration affects the ability of hydrogen producing bacteria to produce hydrogen during dark fermentation [15].The research on dark fermentative hydrogen production at extreme thermophilic temperatures is scanty and yet to be investigated. Further, to the best of our knowledge, Thermoanaerobacter mathranii have not been investigated before in detail for the dark fermentative hydrogen production at extremely thermophilic temperatures. In view of the above, this study investigates the optimum conditions for fermentative hydrogen production by T. mathranii strain A3N using different substrates such as starch, sucrose, glucose and xylose in batch operation under extremely thermophilic condition.
2.
Materials and methods
2.1.
Sample collection and isolation of the strain A3N
Oil water mixture samples (97% water, 3% oil) were collected from oil producing well (well No KD 269) in Nawagam, Ahamadabad, India into 50 mL of anaerobic sterilized serum bottles containing 1 mL of 2% Na2S during April, 2010. The reservoir temperature was 87 C and salinity of formation water was 15 g/L. Reservoir pressure was 700 psi.
2.2.
Isolation of the strain A3N
Samples (0.1 mL) were inoculated in liquid anaerobic basal medium consisting of (g/L): NH4Cl 0.5; Yeast extract 5; K2HPO4 0.25; KCl 0.002; MgCl26H2O 0.125; NH4HCO3 0.4; Peptone 1; NH4H2PO4 0.4; NaH2PO4 0.5. trace element 1 mL, vitamin solution 1 mL [16]. Sucrose (10 g/L) was used as sole carbon and energy source. All the culture bottles were incubated at 70 C for 3e5 days and sub cultured in same medium after 3 days of incubation. Serial dilution technique was used to obtain pure cultures. In order to be sure to obtain a pure isolates, serial dilution steps were repeated several times. All the sub cultures and diluted cultures were incubated at 70 C under atmospheric pressure. Cells were observed under a light microscope (Olympus, Japan) and pure isolate A3N was routinely cultivated in basal medium.
2.3. Identification of strain using 16S rRNA gene sequence Genomic DNA from the pure isolate A3N was extracted as reported [17]. The 16S rRNA gene sequence was amplified by PCR using MicroSec full Gene Kit (Applied Biosystems, UK) as per the manufactures instructions. Amplified product was sequenced using the DyeDeoxy Terminator Cycle sequencing Kit (Applied Biosystems, UK) as directed in the manufacturer’s protocol with an automatic Genetic analyser (Model 300; Applied Biosystems, USA). The nucleotide sequence of 16S rRNA gene of the isolate A3N was compared with other related sequences available in GenBank using BLAST programme [18]. Further, the nucleotide sequence of isolate A3N was aligned with closely related sequences found in GenBank, using CLUSTAL W, and pair wise evolutionary distances were computed using JukeseCanter Model [19]. Phylogenetic analysis was performed using MEGA version 4. Confidence in the tree topologies was evaluated by re-sampling 100 bootstrap trees [20].
2.4.
Study on the growth properties of the strain A3N
Effect of different temperatures (40, 45, 55, 65, 70 and 80 C), NaCl concentrations (0e7%) (w/v) and different carbon sources (5 g/L glucose, fructose, sucrose, xylose, xylan, ribose, raffinose, mannose, arabinose, xylan, rhamnose, galactose, cellobiose, dextrose, lactose, maltose, sorbitol, molasses, starch and cellulose) on the growth of the isolate A3N were studied in anaerobic liquid basal medium. All the growth experiments were performed in triplicate, using 60 mL serum bottles containing 30 mL of anaerobic basal medium.
2.5. Optimization of hydrogen production in batch fermentation Batch dark fermentation studies were performed in 125 mL anaerobic bottles with working volume of 10 mL of liquid anaerobic basal medium aforementioned. The inoculum acquired during exponential growth phase was added at 0.5% (v/v) by using disposable syringe. The inoculum was adapted to the each substrate by subculturing several cycles in batch cultures. Unless noted otherwise as a variable, initial starch, sucrose, glucose and xylose concentrations were kept at 10 g/L. Influence of different initial pH levels for the hydrogen production on different substrates (starch, glucose, xylose and sucrose) was studied. The initial pH levels ranging from 5.5 to 9.5 with 0.5 increments were selected for this study. The pH of the medium was adjusted with 1 M HCl or 1 M NaOH prior to dispensing the medium into the 125 mL serum bottles. Further, effect of initial substrate concentration on hydrogen production was investigated in batch experiments as described above. Optimal pH levels obtained for the maximum hydrogen production from each individual substrate were used as initial culture pH. Initial substrate concentrations tested were 5, 10, 15, 20 and 25 g/L for each individual substrate. The obtained optimal initial cultivation pH and initial substrate concentration which gave the maximum hydrogen
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production and yield for each individual substrate was further used to study the hydrogen production performance at two different temperatures (60 and 70 C). The batch experiments were conducted in 1 L anaerobic bottles with a working volume of 300 mL. The effect of headspace volume on hydrogen production, substrate degradation and biomass accumulation was also studied using starch as substrate under optimum initial cultivation pH and temperature. The experiment was conducted in a 1350 mL anaerobic glass bottles with different fermentation volumes ranging from 150 to 750 mL with 150 mL increment. Total pressure in the headspace was measured with mechanical pressure gauge directly connected to the anaerobic bottle. All experiments were repeated thrice, and hydrogen production was quantified at an interval of 24 h.
2.6.
Analytical methods
The headspace gas composition in each batch experiment was analysed by the gas chromatography (GC 6890N, Agilent, USA) equipped with a thermal conductivity detector (TCD) and HP PLOTQ column (15 m 530 mm 40 mm film thickness) as described in our previous study [16,21]. Volatile fatty acids (VFA) composition in the liquid phase i.e. acetate, butyrate, isobutyrate, valerate, isovalerate and propionate were analysed by gas chromatography (GC 6890N, Agilent, USA) equipped with a flame ionization detector (FID) and DB-WAXetr column (30 m 530 mm 1 mm film thickness) [16]. The concentrations of sugar and ethanol in the culture supernatant was analysed by High Performance Liquid Chromatography (HPLC, Agilent 1100 series, USA) equipped with Sugar-PAK.1 column (Water Research, USA) and refractive index detector (RID) [16]. AminexR HPX-87H, (300 mm 7.8 mm) column (BIO RAD, CA, USA) was used to quantify lactate, succinate and formate using water as the mobile phase at a flow rate of 0.6 mL/min. The column temperature was maintained at 35 C. Residual starch concentration was estimated by Anthrone-sulfuric method [22]. The percentage substrate degradation was estimated by dividing the amount of carbohydrate consumed by the amount of initial carbohydrate. The bacterial growth in each batch experiment was directly monitored by measuring the increase in optical density at 600 nm using UV visible spectrophotometer (UV2450, Shimadzu, Japan). The biomass concentrations were determined by standard method [23]. In brief, the samples (10 mL) were filtered through pre-weight 0.45 mm filter paper, dried at 105 C until constant weight. All measures were performed in triplicate. The rate of hydrogen production (mmol H2/L/h) in each experiment was calculated from total hydrogen production (mmol/L) divided by time duration (h) for which H2 evolution occurred. The specific rate of H2 production was calculated from rate of H2 production (mmol/L/h) divided by dry cell weight (DCW) (g/L). Hydrogen yield was calculated as total molaric amount of H2 divided by molaric amount of consumed total sugar (mol H2/mol total sugar consumed) [24]. In the case of starch, H2 yield was calculated by molaric amount of H2 divided by gram of starch consumed (mmol H2/g starch).
3.
Results and discussion
3.1.
General properties of the strain A3N
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The novel strain A3N is found to be extremely thermophilic, rod-shaped, Gram negative, fermentative anaerobe with an optimum growth at 74 C. The growth was weak at less than 40 to above 85 C. The strain A3N is moderately alkaliphilic, and able to grow at wide range of pH from 5.0 to 9.5 at 74 C. There was no growth observed at pH 4.0 or below. Further, pH in the range of 7.0e9.0 was found to be a favorable pH range for optimum biomass accumulation at 74 C. Similar growth pattern was observed at 60 C. However, total biomass accumulation was comparatively low at 60 C. It was able to grow up to 4% NaCl concentration (w/v) with an optimum of 0.5e0.75% NaCl (w/v) in the presence of sucrose as a sole carbon and energy source, and could utilize glucose, sucrose, fructose, arabinose, dextrose, lactose, sorbitol, xylan, xylose, starch and ribose, raffinose, rhamnose, molasses and maltose.
3.2. Identification of the strain A3N based on the 16S rRNA gene sequence analysis The 16S rRNA gene sequence of isolate A3N was compared with others available in GenBank database. The sequence of A3N had 99% identity to that of T. mathranii (Y11279), and the two sequences grouped together in a phylogenetic tree (Fig. 1). The 16S DNA sequence reported in this paper has been deposited in GenBank under the accession number HM179101.
3.3. Effect of initial culture pH on growth and hydrogen production from different substrates 3.3.1.
Hydrogen production from starch
T. mathranii A3N grew well in soluble starch medium under extremely thermophilic condition (Table 1). Cell growth was affected by initial culture pH affected the cell growth and poor cell growth was observed at initial cultivation pH between 5.0 and 5.5. High initial cultivation pH enhanced the bacterial cell growth, and the highest cell growth was observed at initial cultivation pH 8.0 at 70 C. The strain preferred high temperature (70 C) for optimum cell growth and the cell growth was slightly low at 60 C. The hydrogen production in the starch medium was initial culture pH dependent and the hydrogen production occurred within the wide range of pH (5.0e9.5). The hydrogen evolution was low at pH between 5.0 and 6.0. When the initial pH increased from 6.5 to 7.0, there was a significant increase in total hydrogen production (Table 1). Total hydrogen production of 108e110 mmol H2/L at 70 C was observed at initial pH between 8.0 and 9.0 suggesting that the initial pH between 8.0 and 9.0 was favorable for the cells to utilize starch for growth and hydrogen production. Almost similar hydrogen production performance was observed at 60 C within the same pH range.
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Fig. 1 e Phylogenetic dendrogram based on 16S rRNA sequences indicating the position of strain A3N among closely related members. The topology shown is an unrooted tree obtained with a neighbor joining algorithm (JukeseCantor corrections) with bootstrap values expressed as percentages of 100 replications.
3.3.2.
Hydrogen production from glucose, sucrose and xylose
T. mathranii A3N grew well on glucose, sucrose and xylose under extremely thermophilic condition (70 C). High cell densities were observed at alkaline range of pH, and optimum cell growth in glucose medium was observed at pH between
9.0 and 9.5 (Table 2). In sucrose medium, the highest cell growth was observed at pH 8.5 whereas in xylose medium the highest cell growth was at pH 9.0 (Table 3). Hydrogen production from glucose, sucrose and xylose fermentation was significantly influenced by initial culture pH
Table 1 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during soluble starch fermentation at different initial culture pH and at temperature 60 and 70 C. Initial pH
Fermentation temperature 60 C H2 (mmol/L)
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
47.57 53.19 61.09 68.10 82.86 90.65 98.49 103.40 109.25 97.22
0.34 0,36 0.97 0.67 0.72 0.82 0.96 0.56 0.42 0.64
AA (mmol/L) 14.25 16.96 17.98 28.55 27.30 29.30 34.88 39.92 35.33 31.75
0.18 10.12 0.42 0.21 0.24 0.62 0.42 0.30 0.56 0.64
70 C Cell growth (OD600 nm) 0.48 0.011 0.57 0.009 0.74 0.013 1.11 0.015 1.50 0.026 1.520.013 1.71 0.011 1.70 0.066 1.40 0.013 1.11 0.011
AA-acetate; Each experiment is representative of three independent replicates.
H2 (mmol/L) 42.57 50.67 62.09 69.20 103.46 100.30 108.60 107.60 110.00 99.52
0.49 0.76 0.11 0.68 0.12 0.25 0.33 0.25 0.41 0.31
AA (mmol/L) 19.26 21.73 25.14 28.02 41.89 40.61 43.97 43.56 46.15 38.29
0.27 0.16 0.32 0.19 0.11 0.21 0.33 0.32 0.19 0.12
Cell growth (OD600 nm) 0.41 0.63 1.01 1.41 1.67 1.73 1.79 1.72 1.78 1.31
0.016 0.008 0.015 0.011 0.009 0.012 0.005 0.006 0.012 0.008
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Table 2 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during sucrose and glucose fermentation at different initial culture pH and at temperature 70 C. Initial pH
Substrate Sucrose
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Glucose
H2 (mmol/L)
AA (mmol/L)
Cell growth (OD600 nm)
H2 (mmol/L)
AA (mmol/L)
Cell growth (OD600 nm)
e 25.62 0.25 31.13 0.45 58.90 0.31 73.09 0.29 84.18 0.46 82.25 0.12 79.22 0.45 73.67 0.25 68.93 0.43
e 10.25 0.45 12.45 0.67 23.56 0.27 29.24 0.25 33.67 0.21 32.90 0.25 31.69 0.23 29.47 0.41 26.36 0.13
e 0.37 0.005 0.42 0.012 0.54 0.008 0.75 0.010 1.03 0.006 1.07 0.013 1.34 0.011 1.13 0.008 1.03 0.011
21.54 0.34 32.94 0.21 58.25 0.42 55.52 0.21 68.12 0.34 72.68 0.71 72.96 0.23 73.18 0.15 91.14 0.41 76.49 0.35
11.32 0.17 13.78 0.11 22.90 0.24 24.57 0.11 22.40 0.23 28.95 0.41 25.93 0.34 27.10 0.26 27.20 0.62 27.88 0.21
0.51 0.002 0.68 0.004 1.30 0.001 1.36 0.003 1.62 0.011 1.62 0.008 1.87 0.006 1.87 0.011 1.92 0.009 1.94 0.003
AA-acetate; Each experiment is representative of three independent replicates.
(Tables 2 and 3). Significantly low hydrogen evolution was observed in glucose and sucrose medium at low initial culture pH (below pH 6.0). Further, no growth and hydrogen production occurred in sucrose medium at initial cultivation pH 5.0 (Table 2). Increase in initial cultivation pH led to an increase in total hydrogen production. Optimum initial culture pH for maximum hydrogen production (84.18 mmol/L) in sucrose medium was observed at initial culture pH 7.5. Whereas the initial culture pH 9.0 was observed as optimum pH for maximum hydrogen production (91 mmol/L) in glucose medium. The initial cultivation pH 7.5 was found to be the optimum pH for maximum hydrogen production in xylose medium.
3.4.
Effect of initial substrate concentration
Batch fermentations using starch, glucose, sucrose and xylose at different concentrations ranging from 5 to 35 g/L were followed until hydrogen production in each bioreactor has stopped. Nearly complete substrate degradation (approximately w98.6%) was observed in medium supplemented with 5 g/L of each substrate. Further, the substrate degradation was drastically reduced while increasing initial substrate concentration. Residual substrate concentration was high in cultures
supplemented with high initial substrates and similar to all substrates used. Maximum hydrogen yield of 17.91 mmol/g starch (398 mL/g starch) was obtained at the concentration of starch 7.5 g/L, pH levels 7.5e9.0 and temperature at 70 C (Table 4). This hydrogen is nearly 78e80% of the theoretically derived maximum value of 553 mL of hydrogen per gram of starch [25]. The result of this study is comparable with study reported by Hasyim [4] using Thermoanaerobacterium dominant mixed culture. Further, the hydrogen yield achieved in this study is comparatively higher as compared to the some previous reports [13,26,27]. With increase in starch concentration, the hydrogen yield and substrate degradation tended to decrease, and at initial starch concentration of 35 g/L, substrate removal was approximately 32.8% (Table 4). Zhang et al. [25] has also reported that the variation tendency was also different from thermophilic hydrogen fermentation of starch in which H2 yield decrease with increasing starch concentration. High initial starch concentration up to 30 g/L enhanced the biomass accumulation, and further increased in starch concentration inhibited the biomass formation in batch experiments. At the end of the fermentation, it was found that high concentrations of reducing sugar accumulate in fermentation broth in which the high initial starch concentration was added. This
Table 3 e Hydrogen production performance and cell growth of Thermoanaerobacter mathranii strain A3N during xylose fermentation at different initial culture pH and at temperature 70 C. Initial pH 5.0 5.5 6.0 7.0 7.5 8.0 8.5 9.0 9.5
H2 (mmol/L) 57.60 56.15 56.20 57.47 66.84 61.27 61.69 50.14 52.99
0.12 0.10 0.21 0.16 0.24 0.09 0.07 0.15 0.08
AA (mmol/L) 14.44 12.86 14.20 13.93 16.33 12.86 14.43 14.15 18.34
0.14 0.34 0.02 0.24 0.31 0.13 0.17 0.87 0.69
Cell growth (OD600nm) 1.66 1.80 1.76 1.98 2.05 2.04 2.20 2.32 1.84
AA-acetate; Each experiment is representative of three independent replicates.
0.024 0.040 0.013 0.024 0.013 0.017 0.016 0.019 0.023
BA (mmol/L) 1.24 1.09 1.12 1.13 1.29 1.28 1.23 1.44 0.32
0.012 0.034 0.016 0.013 0.014 0.015 0.016 0.012 0.003
LA (mmol/L) 0.036 0.048 0.029 0.026 0.014 0.026 0.021 0.022 0.022
0.013 0.0012 0.012 0.004 0.006 0.013 0.005 0.002 0.014
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Table 4 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N and substrate degradation at optimum initial culture pH of 8.0 and temperature of 70 C under different initial soluble starch concentrations. Soluble starch (g/L) 5 7.5 10 15 20 25 30 35
H2 yield (mol/g starch) 15.95 0.43 17.91 0.16 17.03 0.42 14.79 0.45 14.17 0.17 13.82 0.67 12.26 0.57 12.79 0.11
Biomass (g/L) 0.71 0.78 0.93 1.03 1.08 1.06 1.19 1.09
0.004 0.011 0.03 0.09 0.012 0.09 0.01 0.04
HPR (mmol/L/h) 2.39 3.40 3.65 3.42 3.37 3.49 3.27 3.11
0.013 0.071 0.092 0.031 0.072 0.061 0.045 0.037
SHPR (mmol/g cell/h)
TSM (mmol/L)
% Substrate degradation
3.40 0.021 4.25 0.033 3.93 0.062 3.32 0.014 2.86 0.011 3.29 0.071 2.75 0.046 2.86 0.023
22.03 0.18 21.34 0.25 23.41 0.41 24.98 0.27 22.24 0.33 19.56 0.19 17.49 0.28 21.45 0.61
97.2 94.53 62.80 58.34 51.90 42.48 36.97 32.85
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, lactate and ethanol); Each experiment is representative of three independent replicates.
indicates the strong amylolitic enzyme activity of the strain A3N, and showed the high potential to apply for biohydrogen production from starch. Few of the studies have emphasized the importance of amylase secreting dark fermentative hydrogen producing bacteria [28,29]. The hydrogen evolution was totally inhibited due to low pH and accumulation of intermediate products. The low or no hydrogen production at low pH (between 4.5 and 5.0), could be partially attributing to strong decrease in hydrogenase activity. Similar observation has also been reported from continuous fermentation, that at high starch loading rates, hydrogen formation is inhibited due to high starch accumulation [30]. The best hydrogen production conditions (i.e. initial cultivation at pH 7.5, temperature at 70 C and sucrose concentration at10 g/L) gave the maximum hydrogen yield of 5.36 mol of H2 mol of sucrose (Table 5). High initial substrate concentration has led to accumulation of high biomass and organic metabolites, which probably has resulted in unfavorable conditions for further hydrogen evolution in the present batch experimental conditions. Meantime, low hydrogen yield at 5 g/L sucrose could be possibly due to main part of the organic substrates under low substrate concentration could go to maintain the cell growth resulting in low hydrogen production per substrate amount [31]. Further, the concentration of residual sucrose increased with the initial sucrose concentration. Thus, it seems that substrate inhibition occurred if the sucrose concentration was higher than 10 g/L.
Nearly complete substrate removal was observed at a concentration of 5 g/L of xylose (Table 6). The substrate degradation decrease with an increase in the concentration of substrate, and hydrogen production yield was lower than that obtained at low concentrations. The upper limit of substrate concentration for xylose without marked decrease in molar hydrogen yield was 5 g/L. The maximum hydrogen yield observed in this study was 2.09 0.21 mol H2/mol xylose, which is higher than that of reported previously using mixed culture fermentation [6]. Theoretically xylose can be converted to hydrogen with a maximum yield of 3.33 mol-H2/mol xylose when the acetate is produced as the fermentation by product [6]. Further it has been reported that thermophiles typically generate higher hydrogen yield from xylose (1.7e2.4 mol H2/mol xylose) than that of mesophiles (commonly, 1 mol H2/mol xylose) [13]. Lower hydrogen yields from xylose in our experiment compared to the theoretical yield could be explained with some conversion of substrate in to biomass, formation of other intermediate products such as ethanol, lactate and pH drop during fermentation. Few studies have been carried out on hydrogen production under extreme thermophilic conditions [6]. The maximum hydrogen yield 2.64 mol/mol glucose was observed at initial glucose concentration of 10 g/L (Table 7). As explained above, high initial glucose concentration yielded low hydrogen yield. Thus initial glucose concentration (10 g/L) is likely to be the most suitable for hydrogen production by strain A3N.
Table 5 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 7.5 and temperature of 70 C under different initial sucrose concentrations. Sucrose (g/L) 5 10 15 20 25 30 35
H2 yield (mol/mol sucrose) 4.21 5.36 5.20 4.19 3.92 3.69 3.69
0.19 0.41 0.27 0.18 0.31 0.42 0.38
Biomass (g/L) 0.66 0.73 0.81 0.82 0.89 0.94 1.17
0.006 0.002 0.011 0.003 0.008 0.010 0.012
HPR (mmol/L/h) 2.08 4.48 5.07 4.16 4.79 4.13 4.25
0.054 0.061 0.037 0.051 0.047 0.026 0.029
SHPR (mmol/g cell/h) 3.15 6.13 6.24 5.05 5.38 5.49 5.52
0.066 0.043 0.038 0.029 0.033 0.015 0.061
Sucrose consumed (g/L)
TSM (mmol/L)
22.57 40.39 41.56 36.57 33.84 39.32 42.57
4.26 6.12 7.23 7.14 7.92 9.01 9.45
0.062 0.029 0.017 0.041 0.035 0.016 0.036
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM-total soluble metabolites - (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
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Table 6 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 7.5 and temperature of 70 C under different initial xylose concentrations. Xylose (g/L) 5 10 15 20 25 30 35
H2 yield(mol/mol xylose) 2.09 1.73 1.45 1.19 0.79 0.65 0.55
0.21 0.13 0.26 0.11 0.06 0.08 0.02
Biomass (g/L) 0.63 0.81 0.93 0.98 1.01 1.06 1.08
0.003 0.001 0.009 0.012 0.007 0.003 0.010
HPR (mmol/h/L) 3.33 3.97 4.02 3.75 2.30 2.47 2.49
0.021 0.018 0.042 0.017 0.052 0.021 0.027
SHPR (mmol/g cell/h) 5.74 4.90 4.31 3.82 2.25 2.33 2.31
0.032 0.015 0.029 0.031 0.036 0.027 0.018
Xylose consumed mmol/L
TSM (mmol/L)
25.86 24.01 25.42 24.08 25.28 25.63 29.60
4.95 5.78 6.14 7.02 7.32 8.75 9.37
0.076 0.043 0.091 0.037 0.062 0.087 0.093
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
The growth and hydrogen production at two different temperatures (60 and 70 C) is shown in Table 8. The best fermentation temperature for maximum H2 evolution was found to be at 70 C. However, H2 production at 60 C was also found to be considerably high. Formation of ethanol and lactate was found at higher levels in 60 C incubated cultures that that of at 70 C. The growth, substrate consumption and hydrogen production performance of strain A3N with fermentation time are shown in Figs. 2 and 3. The fermentation behavior of the strain A3N on different substrates has been found to be same. The maximum specific hydrogen rate was observed at late exponential growth phase of the cultures. Nevertheless, some significant differences in metabolite formation, and consequently in hydrogen production were observed. The maximum specific hydrogen production rates in starch and sucrose media was 6.29 and 9.34 mmol/g cell/h respectively (Fig. 2A). Maximum specific hydrogen production rate of 5.76 mmol/g cell/h was observed in xylose medium whereas maximum specific hydrogen production rate in glucose medium was 4.89 mmol/g cell/h (Fig. 2B). With the progress of the fermentation, pH value in the fermentation broths dropped due to the release of organic acids. At the early stationary phase of the culture growth of the cultures (approximately 42 h), the pH in the fermentation broth was between 4.2 and 4.5 irrespective of the substrate used. Thus no hydrogen evolution was found after 42 h of fermentation. Nevertheless, the residual substrates in the fermentation broth in all batch experiments were observed.
In this study, under pH uncontrolled batch experiments, the best H2 yield from the strain T. mathranii A3N was 2.64 0.40 mol H2/mol e glucose, 5.36 0.41 mol H2/mol e sucrose, 17.91 0.16 mmol H2/g e starch and 2.09 0.21 mol H2/mol e xylose. In another study in the literature, it has been reported that under pH controlled conditions the best H2 yield from Thermotoga neapolitana at extreme thermophilic condition was 2.22 0.11/mol H2 mol e xylose, 3.2 0.16 mol/H2mol e glucose and 4.95 .0.25 mol H2/mol sucrose [24].
3.5.
Distribution of intermediate metabolites
Formation of VFAs and other metabolic products during fermentative hydrogen process under extremely thermophilic conditions are scanty and yet to be investigated. In the literature, it has been reported that the hydrogen yields can be improved by increasing acetate formation, and decreasing butyrate formation by using a high-temperature fermentation process with thermophiles or extreme thermophiles, operating at temperature higher than 60 C [32]. In this study an attempt was made to understand fermentation pathways during hydrogen production under extremely thermophilic condition with different carbon sources. Acetate-type fermentation pathway was found to be the major fermentation type in xylose fermentation by T. mathranii A3N. Butyrate formation was found in sucrose and xylose medium during fermentation. In other studies, acetate and butyrate formation associated with xylose fermentation by Thermoanaerobacterium thermosaccharolyticum W16 [33],
Table 7 e Hydrogen production performance of Thermoanaerobacter mathranii strain A3N at optimum initial culture pH of 9.0 and temperature of 70 C under different initial glucose concentrations. Glucose (g/L) 5 10 15 20 25 30 35
H2 yield (mol/mol glucose) 1.93 2.64 2.18 2.02 1.62 1.69 1.40
0.11 0.40 0.32 0.46 0.62 0.27 0.056
Biomass (g/L) 0.62 0.72 0.85 0.92 0.88 0.91 1.04
0.004 0.0071 0.002 0.0041 0.009 0.0051 0.0031
HPR (mmol/L/h) 2.77 4.49 4.64 5.06 4.13 4.07 4.09
0.032 0.024 0.051 0.072 0.036 0.057 0.081
SHPR (mmol/g cell/h) 4,47 6.23 5.46 5.50 4.69 4.48 3.93
0.044 0.051 0.018 0.064 0.091 0.062 0.052
Glucose consumed (g/L)
TSM (mmol/L)
24.66 29.58 33.67 36.31 28.75 29.35 27.62
4.92 6.43 7.29 7.62 8.69 8.23 8.95
0.057 0.066 0.052 0.047 0.026 0.038 0.059
HPR-hydrogen production rate; SHPR- specific hydrogen production rate; DCW-dry cell weight; TSM- total soluble metabolites (acetate, butyrate, lactate and ethanol); Each experiment is representative of three independent replicates.
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Table 8 e Comparison of hydrogen production performance of Thermoanaerobacter mathranii strain A3N at two different temperatures (60 and 70 C) under optimum pH and substrate concentrations obtained for maximum hydrogen production in this study. Substrate Fermentation (g/L) temperature ( C) Glucose Sucrose Starch Xylose
70 60 70 60 70 60 70 60
H2 yield (mol/mol substrate) 2.64 2.11 5.36 4.67 17.91 14.21 2.09 1.92
HPR (mmol/L/h)
0.40 0.28 0.41 0.35 0.16 (mmol/g starch) 0.29 (mmol/g starch 0.21 0.18
4.49 3.74 4.48 4.11 3.4 3.56 3.33 2.86
0.023 0.029 0.061 0.042 0.071 0.052 0.032 0.041
Biomass (g/L) 0.78 0.69 0.73 0.68 0.63 0.60 0.72 0.62
0.0071 0.0042 0.002 0.001 0.011 0.002 0.003 0.001
TSM (mmol/L) % Substrate degradation 34.78 32.97 40.39 38.42 45.26 42.31 33.97 38.26
64.3 62.3 61.2 57.4 98.6 80.72 99 97
HPR-hydrogen production rate; TSM-total soluble metabolites (acetate, butyrate, lactate and ethanol).
acetate as a metabolic intermediate followed by ethanol and lactate by mixed culture [6] were found in xylose fermentation at extreme thermophilic temperatures. In this study, a significant increase in lactate production was found in cultures supplemented with high xylose concentrations above 10 g/L resulting low hydrogen yield as there is no hydrogen generation with lactic acid formation [34,35]. Further, we found that mixed type fermentation occurred in sucrose fermentation by strain A3N. Further, during early
A
exponential phase, acetate formation was found to be the dominant fermentation type, whereas when the cultures entered early stationary phase, equal amount of acetate and butyrate was observed. In contrast, acetate and lactate were found as the main intermediate products during sucrose fermentation by Caldicellulosiruptor saccharolyticus at 70 C [36]. The metabolic changes observed could be due to the expression levels of various acid forming enzymes such as phospho-transbutyrylase (PTB), which is responsible for the
A
B
B
Fig. 2 e A&B. Time course of growth, substrate consumption and hydrogen production by Thermoanaerobacter mathranii A3N in (A) starch medium; (B) Sucrose medium, under optimal conditions. Each experiment is representative of three independent replicates.
Fig. 3 e A&B. Time course of growth, substrate consumption and hydrogen production by Thermoanaerobacter mathranii A3N in (A) xylose medium; (B) Glucose medium under optimal conditions. Each experiment is representative of three independent replicates.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 5 5 6 9 e5 5 7 8
butyrate formation had high expression levels at pH 6.3 [37]. Less quantities of formate, succinate and lactate were detected when initial culture pH is below 6.5 during sucrose fermentation. Acetate was the major VFA observed (approximately 97%) during soluble starch fermentation and butyrate formation was not detected. Small quantities of iso-butyrate (1e3 mg/L), succinate, formate (2e3 mg/L) and lactate (5e7 mg/L) were detected at all pH levels tested. Similar metabolic profile was also observed in xylose fermentation. In addition, trace amount of butyrate was observed (Table 3).
4.
Conclusion
In this study, we have isolated extremely thermophilic hydrogen producing bacterial strain from oil water mixtures (formation water) from oil well, Ahamadabad, India. It was identified as T. mathranii by 16S rRNA gene sequencing. Hydrogen production by the strain A3N was optimized in batch fermentation at 70 C. The highest H2 yields were 2.64 0.40 mol H2 mol glucose at 10 g/L, 5.36 0.41 mol H2 mol e sucrose at 10 g/L, 17.91 0.16 mmol H2 g e starch at 5 g/L and 2.09 0.21 mol H2 mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was dominant in strain A3N. Butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L. Results of this study demonstrates the high potential of the novel bacterium T. mathranii strain A3N for dark fermentative hydrogen production at 70 C.
Acknowledgments The authors are indebted to the Department of Biotechnology, Govt. of India for financial support. The authors are thankful to Dr. R. K. Pachauri, DG, TERI, New Delhi, for providing infrastructure facility to carry out the present study. We thank all technical staff in the laboratory for providing their assistance to complete this study successfully.
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