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Thermophilic fermentative hydrogen production by the newly isolated Thermoanaerobacterium thermosaccharolyticum PSU-2
Sompong O-Thonga, Poonsuk Prasertsanb, Dimitar Karakasheva, Irini Angelidakia,
a b

Institute of Environment and Resources, Technical University of Denmark, DK-2800 Lyngby, Denmark Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat-Yai, Songkhla 90120, Thailand

ar t ic l e i n f o
Article history: Received 29 June 2007 Received in revised form 22 October 2007 Accepted 8 December 2007 Available online 1 February 2008 Keywords: Thermophilic fermentation Thermoanaerobacterium thermosaccharolyticum PSU-2 Hydrogen production

abs tra ct
A thermophilic H2 -producing bacterial strain was isolated from a biohydrogen reactor fed with palm oil mill efuent (POME) and identied as Thermoanaerobacterium thermosaccharolyticum using 16S rRNA gene analysis. The isolated bacterium, designated as T. thermosaccharolyticum PSU-2, showed a high yield and production rate of H2 . Temperature optimum, pH optimum and substrate utilization for H2 production were investigated in batch conditions. All of tested substrate was utilized for H2 production, while sucrose, xylose and starch were the preferred substrates. The strain produced H2 within a wide range of pH (4.58) and temperature (4570  C), with the optimal temperature 60  C and optimal initial pH about 6.25. Maximum of H2 production rate was registered from hour 8 to hour 16 in late exponential phase. The H2 production was drastically reduced in a prolonged fermentation (24 h) and stopped at pH 4.5 due to the accumulation of organic acids. The maximum H2 production yield and rate at sucrose concentration of 20 g l1 , pH 6.25 and temperature 60  C were 2:53 mol H2 mol1 hexose and 12:12 mmol H2 l1 h1 , respectively. Organic nitrogen amended medium improved the H2 production with 68% compared to inorganic nitrogen amended medium. The strain performed ethanolacetate type fermentation in inorganic nitrogen amended medium, while it performed butyrateacetate type fermentation in organic nitrogen amended medium. & 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Hydrogen H2 is a clean and promising fuel when it is ultimately derived from renewable energy sources. It is also efcient and environmentally friendly, since it has high energy content, and water is the sole end product after combustion [1,2]. H2 can be produced sustainably by anaerobic bacteria degrading carbohydrate-rich substrates to organic acids, H2 and CO2 . Dark-fermentation of carbohydrate-rich substrates as biomass presents a promising route
Corresponding author. Tel.: +45 4525 1429; fax: +45 4593 2850.

of biological H2 production, compared with photosynthetic routes [3]. For example, fermentative H2 process produces H2 at higher rate (0.565:0 l H2 l1 d1 ) than light driven process (0.044:3 l H2 l1 d1 ) [4]. In addition, the major advantages are rapid bacterial growth rates, low energy demands, minimal pollution generation, operation without light sources, no oxygen limitation problems and low capital costs for at least small-scale production facilities (1001000 l H2 h1 ) [4,5]. The isolation and identication of fermentative H2 producers with a high yield and high production rate of H2 are

E-mail address: ria@er.dtu.dk (I. Angelidaki). 0360-3199/$ - see front matter & 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.12.015

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important for development of commercial sustainable biohydrogen production process. Various H2 -producing strains including species of Enterobacter spp. [6], Clostridium spp. [7] and Bacillus spp. [8] have been identied and studied. Among the H2 producers, mesophilic bacteria have been studied most extensively. Normally, it is observed that H2 production yield of 12 mol H2 mol1 hexose are obtained with mesophiles, while thermophiles display a yield higher than 2 mol H2 mol1 hexose [9]. H2 yields can be improved by increasing H2 production through acetate end product reaction, and decreasing or preventing butyrate, ethanol and propionate product reaction. One way to accomplish this is through fermentation process with thermophiles or extreme thermophiles, operating at temperatures higher than 60  C [9,10]. Thus, a higher temperature is more feasible for the conversion reaction toward H2 due to favorable thermodynamics conditions. Thermophilic bacteria are therefore considered as most promising microorganisms than mesophilic bacteria for H2 production [11]. Maximum theoretically H2 production yield could be obtained by thermophilic (5055  C) bacteria or extreme thermophilic (5580  C) bacteria [9]. Thermophilic bacteria are able to utilize a wide range of organic wastes [12]. Thermophilic mixed and pure cultures have been examined for their potential as H2 producers [1315]. High H2 production rate and less variety of fermentation end products were observed under thermophilic conditions compared to mesophilic ones [16]. These properties make application of thermophiles for H2 production economically and technical feasible. However, a limited number of bacteria can convert carbohydrate into H2 with a satisfactory yield and productivity. The aim of the present study is to isolate, characterize and identify a thermophilic fermentative bacterium from a sequencing batch reactor digesting palm oil mill efuent for continuous H2 production. Operational conditions for optimal H2 production process in terms of carbon and nitrogen sources, temperature and pH were investigated. Metabolic pathways and kinetic parameters for hydrogen production from glucose were identied.

series were inoculated to fresh liquid medium and analyzed further for H2 production. The strain with the highest level of H2 production was named PSU-2. In this study, the characterization of PSU-2 isolate and its H2 production properties were investigated.

2.2.

Microscopic examination and biochemical tests

Characterization and morphology of the strain PSU-2 isolate were determined according to Bergeys manual of determinative bacteriology [18]. Morphological studies were performed with light microscope (Eclipse E400; Nikon) equipped with digital camera (Nikon) and a transmission electron microscope, JEM-2010, JEOL. Gram staining was performed by Hucker method [19]. For biochemical characterization, substrate utilization and fermentation end products proles were investigated. Arabinose, fructose, galactose, glucose, mannose, rhamnose, xylose, cellobiose, lactose, maltose, sucrose, trehalose, rafnose, inulin, pectin, starch, xylan, cellulose, mannitol and glycerol were tested for hydrogen production at concentration of 10 g l1 .

2.3.

Strain identication

Total genomic DNA was extracted from strain PSU-2 isolate using the Ultraclean Soil DNA Kit (MoBio Laboratory Inc., USA). The region of the 16S rRNA genes corresponding to position 271492 in the 16S rRNA of Escherichia coli was PCR-amplied using 16SF (50 -GAGTTTGATCCTGGCTCAG-30 ) and 16SR (50 -GAAAGGAGGTGATCCAGCC-30 ), as described by Dahle and Birkeland [20]. The double stranded PCR products were sequenced using an ABI PRISM Big Terminator Cycle Sequencing Kit version 3.1 (Applied Biosystems, USA) in accordance with the manufacturers instructions. Closest matches for partial 16S rRNA gene sequences were identied by database searches in GenBank using BLAST [21]. Alignments for phylogenetic analysis were made by using CLUSTAL X [22]. Phylogenetic trees were then constructed using the neighborjoining method with MEGA 2.1 [23]. Bootstrapings analysis [24] for 1000 replicates was performed to estimate the condence of tree topologies. The DNA base composition of strain PSU-2 was determined by HPLC as described elsewhere [25].

2.
2.1.

Materials and methods

2.4. Isolation of the bacterial strain
In batch fermentation H2 production, 10 ml of microbial suspension at exponential growth phase (OD660 0:5) was transferred into 90 ml basic anaerobic (BA) medium supplemented with sucrose as carbon source to nal concentration of 10 g l1 . Medium was dispensed anaerobically in 250 ml serum bottle capped with rubber stopper and closed with aluminum caps. The headspace was replaced with nitrogen gas. To determine the effects of operating parameters (temperature, initial pH, type and concentration of substrate) on H2 production, pH value, type and concentration of substrates, temperature and nitrogen source were alternately varied. The pH was set to various levels (in the range of 4.09.0 with increments of 0.5) in serum bottle using 2 N HCl and 2 N NaOH. The incubation temperature was varied from 35 to 80  C with increments of 5  C. The concentrations of all components in medium except

H2 production in batch

Anaerobic sludge was obtained from a H2 -producing anaerobic sequencing batch reactor (ASBR) running at steady state conditions for approximately 3 months. ASBR was operated at 60  C with a hydraulic retention time of 2 d and organic loading rate of 60 g COD l1 d1 in continuous mode. The palm oil mill efuent (POME) was fed into the reactor as substrate. The sludge sample was transferred anaerobically (under N2:CO2 80:20) into 50 ml BA medium prepared as previously described [17]. The medium was further supplemented with sucrose 10 g l1 and incubated at 60  C for 24 h at strict anaerobic conditions. The culture was then serially diluted in the same medium solidied by 0.3% (w/v) Gelrite (Kelco) and incubated anaerobically at the same temperature as that used for the primary enrichment. Colonies from each dilution

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for sucrose concentration were xed in experiment to study the effect of substrate concentration. Sucrose concentrations in the medium varied from 5 to 50 g l1 with increments of 5 g l1 . All experiments were done in triplicates. Liquid samples from the serum bottles were taken for the analysis of pH, substrate utilization and fermentation end products. The volumetric H2 production rate ml H2 l1 h1 was calculated from the total gas production rate and the concentration of H2 in the headspace. The molar H2 production rate mmol H2 l1 h1 ) was calculated using the ideal gas law as molar H2 production rate mmol H2 l1 h1 volumetric H2 production rate ml H2 l1 h1 =RT, where R 8:31447215 J K1 mol1 equal to R 0:08205784 L atm K1 mol1 , and T 333 K [26]. Hydrogen production yield was calculated as total molaric amount of H2 divided by molaric amount of consumed sucrose mmol H2 =mmol sucrose. Cell growth was initially investigated based on simple Monod equation [27]. Linearization of Monod equation was done to nd out the kinetics parameters with the help of LineweaverBurk plot. Regression analysis was used to nd the best t for a straight line on a plot of 1=m vs 1/S to predict values of mmax and KS , where m is specic growth rate h1 , S is substrate concentration g l1 , mmax is maximum specic growth rate h1 and KS is half saturation constant g l1 . The effect of nitrogen source on the hydrogen production yield and growth of strain PSU-2 was observed with modied BA medium where inorganic nitrogen source NH4 Cl was replaced by organic nitrogen source (peptone) to nal concentration of 1 g l1 . The effect of nitrogen source was studied with 10 g l1 of glucose, sucrose and starch as carbon sources. The C:N ratios in inorganic nitrogen medium of glucose, sucrose and starch were 38:1, 75:1 and 770:1, respectively. The C:N ratios in organic nitrogen medium of glucose, sucrose and starch were 35:1, 71:1 and 714:1, respectively.

absorbance is equal to 0:45 g dry cell l1 . The volume of gas produced was recorded daily using a gas meter with water displacement method. The H2 concentration in the gas phase was measured by using a gas chromatograph (MicroLab, Arhus, Denmark) equipped with a thermal conductivity detector (TCD) and a s-m stainless column packed with Porapak Q (50/80 mesh). Fermentation liquid end products (volatile fatty acids and ethanol) were determined by gas chromatograph (HP 5890 series II) equipped with a ame ionization detector (FID) and HP FFAP column (dimensions 30 m 0:53 mm 1:0 mm). The GC-TCD and GC-FID conditions were set according to Liu et al. [28]. Lactate and formate were determined by suppressed ion chromatography, which consisted of HPLC pump (HP 1100, Waldbronn, Germany). Sucrose was analyzed by phenolsulfuric acid method [29] and volatile suspended solids (VSS) were measured according to the standard methods [30].

3.
3.1.

Results and discussion

Identication and phenotypic characterization

2.5.

Analytical methods

The cell concentration was determined by measuring the absorbance at 660 nm using spectrophotometer. One unit of

Isolated H2 -producing bacteria have a rod shape, with a length from 0.3 to 2:3 mm. The cells had a rounded ends and spore forming. They occurred singly or in pairs, though rarely in chains (Fig. 1). They exhibited gram-positive staining in lag growth phase. 16S rRNA analysis revealed that PSU-2 is a member of the genus Thermoanaerobacterium. The closet phylogenetic relative was Thermoanaerobacterium thermosaccharolyticum DSM571 (formerly Clostridium thermosaccharolyticum), where the recorded similarity of the 16S rRNA genes was 98% of 1478 bp. The 16S rRNA accession number at GenBank and EMBL databases is AM408569. A phylogenetic tree was constructed (Fig. 2). The phenotypic characteristics properties of isolate PSU-2 were also consistent with those of the Thermoanaerobacterium spp. and most of them have been reported to produce H2 [3133]. Several species of the genus Thermoanaerobacterium are known for their H2 production characteristics, including T. thermosaccharolyticum,

Fig. 1 Transmission electron micrograph of T. thermosaccharolyticum PSU-2.

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Thermoanaerobacterium thermosulfurigenes (L09171) 97 Thermoanaerobacterium xylanolyticum (L09172)

Thermoanaerobium lactoethylicum (L09170) 46 83 Thermoanaerobacterium saccharolyticum (L09169)

Thermoanaerobacterium islandicum strain AK17 (EF088330) 76 99 Clostridium thermoamylolyticum (X76743)

100

Thermoanaerobacterium aotearoense (X93359)

99

Thermoanaerobacterium bryantii (AY140670)

100

Thermoanaerobacterium thermosaccharolyticum PSU-2

Thermoanaerobacterium thermosaccharolyticum strain DSM571 (M59119)

Thermoanaerobacterium polysaccharolyticum (U40229) 100 Thermoanaerobacterium zeae (U75993)

Clostridium thermocellum (L09173) 0.01

Fig. 2 Neighbor-joining tree showing the phylogenetic position of isolated strain, Thermoanaerobacterium thermosaccharolyticum PSU-2, based on the 16S rRNA sequences of the genus Thermoanaerobacterium. The numbers at the nodes indicate the levels of bootstrap support percentages based on the neighbor-joining of 1000 replicates. Bar 0.01 nucleotide substitutions per site. GenBank accession numbers are given in parentheses.

T. polysaccharolyticum, T. zeae, T. lactoethylicum and T. aotearoense. All these organisms have optimal growth conditions at 5570  C and at pH 5.27.8 [34]. The PSU-2 isolate showed growth with various type of carbohydrate as the sole carbon and energy source under thermophilic condition (Table 1). As shown in Table 1, the substrate spectrum of strain PSU-2 comprises a variety of macromolecules such as starch, dextran and xylan. The substrate spectrum of PSU-2 except rhamnose and dextran was identical to the one of the T. thermosaccharolyticum type strain, DSM571, while except peptone was identical to T. thermosaccharolyticum strain FH1. Thus we concluded on the basis of the 16S rRNA gene analysis and the substrate spectrum that PSU-2 represents a new strain within the species T. thermosaccharolyticum. Effective fermentative bacteria should use various type of substrates especially complex carbohydrate [10,33]. Therefore, the level of H2 production of T. thermosaccharolyticum PSU-2 from various types of carbohydrates was examined. The concentration of each substrate was xed at 10 g l1 .

T. thermosaccharolyticum PSU-2 was able to produce H2 from various types of carbohydrate (Fig. 3). The highest level of H2 production, around 250 ml H2 g1 substrate, was obtained from starch, xylose and sucrose. However, H2 production from xylan and cellulose was quite low (54 and 44 ml H2 g1 substrate, respectively). The low hydrogen production from xylan and cellulose could be explained with end fermentation products prole. Propionate was the main end product from xylan fermentation, while ethanol was the main end product from cellulose fermentation (data not shown). Formation of reduced metabolites as propionate and ethanol was already shown to disfavor hydrogen production [5]. There have been many reports indicating that Thermoanaerobacterium spp. possess the capability to utilize complex carbohydrate such as starch, xylan, cracked corn, cellulose and simple sugar accompanied with H2 and acid production [15,17,32,33]. The ability of the bacterium to utilize complex carbohydrate demonstrates its capability to produce H2 from renewable resources such as biomass [34]. Thus strain PSU-2 could be a

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Table 1 Carbon sources utilization of T. thermosaccharolyticum strain PSU-2, T. thermosaccharolyticum strain DSM571 and T. thermosaccharolyticum strain FH1 Carbon sources
References Arabinose Fructose Galactose Glucose Mannose Rhamnose Xylose Cellobiose Lactose Maltose Sucrose Trehalose Rafnose Dextrin (Dextran) Glycogen Inulin Pectin Starch Xylan Cellulose Yeast extract Chitin Peptone Manitol Glycerol G+C contain ND not determined.

Strain DSM571
[30] ; ND ND ND ND 32

Strain FH1
[37] ND ND ND ND ND ND ND

Strain PSU-2
This study ND ND ND 33.2

promising candidate for the thermophilic fermentative H2 production with possible application for H2 production from organic wastewater that usually contains a mixture of sugars.

3.2.

Effect of operational parameters on H2 production

respectively. Therefore, the results obtained showed that a pH 6.25 is optimal for the growth and H2 production of PSU-2. The growth of stain PSU-2 was observed at temperatures from 45 to 65  C with optimal temperature of 60  C. This is in a range previously reported for the genus Thermoanaerobacterium [37]. Cell yield, H2 production yield and rate of strain T. thermosaccharolyticum PSU-2, a moderate thermophile, decreased when temperature was elevated above 60  C (Fig. 4B). Dropped of cell yield, H2 production yield and rate at temperatures higher than 60  C could be attributed to protein deactivation [38]. The initial substrate concentration usually plays an important role in H2 production [39]. The isolate PSU-2 was used to investigate the effect of substrate concentration on cell yield and H2 production. Sucrose with different concentrations (550 g l1 ) at pH 6.5 was tested in batch cultures. It was observed that the H2 production rate and cells yield of PSU-2 increased positively with sucrose concentration up to 20 g l1 and then H2 production rate decreased gradually along with increasing sucrose concentration (Fig. 4C). Cell yield and rate of H2 production decreased when sucrose concentration increases above 20 g l1 . The most probable reason is substrate inhibition [8], end products inhibition [13] or combination between both types of inhibition. The end product inhibition may be due to accumulation of acetate, it being a weak acid, and therefore impairing the growth and also increasing osmolality. The increase of sucrose concentration also contributes to the osmolality [40]. When the concentration of sucrose was 20 g l1 , the maximum H2 production rate of 12:12 mmol l1 h1 was achieved. In contrast H2 production yield decreased with increasing sucrose concentration, decreased from 5:45 mol H2 mol1 sucrose 5 g l1 to 0:87 mol H2 mol 1 sucrose 50 g l1 . This implies that hydrogen production by T. thermosaccharolyticum PSU-2 is a compromise between technical efciency based on H2 yield and economic efciency based onH2 production rate when substrate concentration is increased. Cell growth and substrate consumption kinetics of strain PSU-2 were investigated with Monod model. LineweaverBurk plots were considered to predict the values of maximum specic growth rate mmax and half-saturation constant KS (Fig. 5). The values of mmax and KS of strain PSU-2 were 0:31 h1 and 1:47 g l1 , respectively, using sucrose as a substrate.

The H2 production of strain PSU-2 was highly dependent on fermentation conditions such as pH, temperature and substrate concentration (Fig. 4). The PSU-2 isolate was strict anaerobe and favored the growth in weak acidic environment. The maximum growth rate was observed at pH 6.25, while no or poor growth was observed below pH 4. At pH 5.5 and 6.5, growth was favored compared to higher pH values, showing the acid tolerance nature of PSU-2 isolate, which was in consensus with reported values [35]. Low pH (pH 4) facilitates formation of acidic metabolites, which may destabilize the cells ability to maintain internal pH, resulting in the lowering of intracellular level of ATP and inhibiting substrate uptake [36]. Higher pH (7.09.0) restricted the growth of bacterium and also the H2 production [7]. Fig. 4A shows the H2 yields of PSU-2 isolate at different pH levels. At pH 6.25, the maximum cell yield, H2 production rate and yield were 0:267 g cell g1 sucrose, 5:73 mmol H2 l1 h1 and 4:91 mol H2 mol1 sucrose,

3.3. H2 production and soluble metabolites production characteristics

Fig. 6A shows the pH and biomass concentration during fermentation of sucrose when the initial pH was not controlled. The pH decreased from 6.0 to 4.2 within 24 h. The decrease of pH was attributed to the accumulation of organic acids. As fermentation progresses, various organic acids (mainly acetate and butyrate) accumulate resulting in a signicant decrease in pH in the culture medium. This decrease in pH caused a signicant decrease in H2 production. H2 production began when cell growth entered the early exponential phase (4 h) and the rate of H2 production reached a maximum in the late exponential phase (Fig. 6B). Most of H2 was produced in the late exponential phase and early stationary phase. H2 production was accompanied with the

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350 Hydrogen production (ml H2g-1 substrate) 300 250 200 150 100 50 0 25 acetate Metabolite concentration (mM) 20 butyrate butanol 15 propionate ethanol

10

0 Soluble starch Potato starch Xylan Cellulose Cellobiose Glucose soluble starch and sucrose sucrose cellulose Xylose and sucrose

Fig. 3 H2 production with T. thermosaccharolyticum PSU-2 from different carbon sources at 10 g l1 under anaerobic conditions, 60  C, initial pH 6.5 and without pH control: (A) hydrogen production and (B) metabolite concentration. Error bars represent standard deviation from a triplicate analysis.

production of VFA and alcohols. T. thermosaccharolyticum PSU2 produced H2 through acetate and butyrate as main fermentation end products. Low amounts of propionic acid and lactic acid were produced by this strain (Fig. 6C). Butyric acid and acetic acid constituted more than 80% of total end products. In general, production of these two organic acids favors the production of H2 , according to Eqs. (1) and (2). Theoretically, 4 mol of H2 are produced from 1 mol of glucose in acetate C2 H4 O2 type fermentation: C6 H12 O6 2H2 O ! 4H2 2CO2 2C2 H4 O2 , (1)

with only 2 mol of H2 produced when butyrate C4 H8 O2 is the main fermentation product: C6 H12 O6 ! 2H2 2CO2 C4 H8 O2 . (2)

It has been reported that butyric acid to acetic acid (B/A) ratios are directly proportional to H2 yields [41]. The B/A ratios observed in batch culture ranged from 0.9 to 1.1 on a molar basis. Therefore, in this study, Eq. (3) was used to characterize the stoichiometric reaction for the production of H2 from glucose using a B/A ratio of 1.0. 3C6 H12 O6 2H2 O ! 8H2 6CO2 2C2 H4 O2 2C4 H8 O2 . (3)

recorded in our experiments was obtained with sucrose at initial concentrations of 5 g L1 producing 2:53 mol H2 mol1 hexose. However, this value is still lower than the theoretical maximal value, but this could be explained by the conversion of some of the substrate into microbial biomass. Kim et al. [41] reported that approximately 11% of the substrate, sucrose, is converted to microbial biomass. The maximum H2 production rate and yield of strain PSU-2 were 12:12 mmol H2 l1 h1 and 2:53 mol H2 mol1 hexose, respectively. Maximum H2 production yields from different reported strains were compared with that of PSU-2 isolate. Thermotoga eli [42], Calidicellulosiruptor saccharolyticus [9], C. thermocellum [43], C. thermolacticum [44], C. thermobutyricum [45] and C. thermosaccharolyticum [46] are known to posses H2 producing abilities under thermophilic and hyperthermophilic conditions, corresponding to 2.7, 3.3, 1.95, 1.5, 1.9 and 1:72 mol H2 mol1 hexose, respectively. Strain PSU-2 had H2 yield nearly equivalent to those of T. eli and C. saccharolyticus.

3.4.

Effect of nitrogen source on growth of strain PSU-2

This would result in a theoretical H2 yield of 2:67 mol H2 mol1 hexose. Experimental H2 yield of 5:06 mol H2 mol1 sucrose

The volume of produced hydrogen ml l1 , H2 yield mol H2 mol1 substrate), cell yield (g cell g1 substrate) and concentrations of fermentation by-products during 2 d cultivation are summarized in Table 2. During 2 d of batch

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0.3
Cell yield (g cell g-1 sucrose)

7 6 5

0.25 0.2

4 0.15 3 0.1 0.05 0 4 4.5 5 5.5 6 6.5 pH 7 7.5 8 8.5 9 2 1 0

0.35 0.3
Cell yield (g cell g-1 sucrose)

8 7 6 5

0.25 0.2

4 0.15 3 0.1 0.05 0 35 40 45 50 55 60 65 Temperature (C) 70 75 80 2 1 0

0.35 0.3
Cell yield (g cell g-1 sucrose)

14 12 10 8 6 4 2 0 5 10 15 20 30 40 50 Sucrose concentration (g l-1)

0.25 0.2 0.15 0.1 0.05 0

Fig. 4 Comparison of cell concentration &, H2 yield D and H2 production rate  under different conditions; effect of pH (A), effect of temperature (B), effect of initial sucrose concentration (C). Error bars represent standard deviation from a triplicate analysis.

Hydrogen production rate (mmol l-1 h-1) Hydrogen production yield (mol H2 mol-1 sucrose)

Hydrogen production rate (mmol l-1 h-1) Hydrogen production yield (mol H2 mol-1 sucrose)

Hydrogen production rate (mmol l-1 h-1) Hydrogen production yield (mol H2 mol-1 sucrose)

0.35

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10 9 8 7 6 5 4 3 2 1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1/substrate concentration (l g-1)

Fig. 5 LineweaverBurk plot for prediction of growth kinetic parameters at pH 6.25 and temperature of 60  C.

cultivation, PSU-2 produced 2195 ml of H2 gas per liter of inorganic nitrogen amended medium, and 6762 ml of gas per liter of organic nitrogen amended medium using sucrose as substrate. Organic nitrogen amendment medium improved the H2 production with 68% compared to inorganic nitrogen amended medium. Source of nitrogen, organic or inorganic, resulted in different metabolic proles of hydrogen production. The productivity of ethanol in inorganic nitrogen amended medium was increased 3 times (10.510.9 mM) when compared with organic nitrogen amended medium (2.42.9 mM). Acetate production in inorganic nitrogen amended medium was in the same levels in both medium when using glucose and starch a carbon sources, while it increased 2 times (17.8 mM) compared to organic nitrogen

1/specific growth rate (h)

1.6 1.4 Cell concentration (g l-1) 1.2 1 0.8 0.6 0.4 0.2 0 30 Hydrogen production rate (ml l-1h-1) 25 20 15 H2 rate H2 accumulation cell pH

6.5 6 5.5 5 4.5 4 350 300 250 200 150 Hydrogen production accumulation (ml g-1 sucrose) pH 100 50 0
acetate butyrate propionate ethanol butanol lactate

10 5 0 16

Metabolite concentration (mM)

14 12 10 8 6 4 2 0 0

12 16 Culture time (h)

20

24

Fig. 6 Physiological characterization of T. thermosaccharolyticum PSU-2 grown on sucrose 10 g l1 medium inoculated with 10 ml of cells in exponential growth phase OD660 0:5. (A) Growth rate and pH variation, (B) H2 production rate and H2 accumulation and (C) fermentation metabolites. Error bars represent standard deviation from a triplicate analysis.

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Table 2 Effects organic nitrogen sources on relative hydrogen production and fermentation metabolites of T. thermosaccharolyticum PSU-2

IN inorganic nitrogen NH4 Cl; ON organic nitrogen (peptone); SD represents standard deviation from a triplicate analysis.

H2 yield SD (mol H2 mol1 substrate)

amended medium (8.1 mM) when using sucrose as carbon source. In contrast, butyrate production in organic nitrogen amended medium was increased 10 times (611.6 mM) compared to inorganic nitrogen amended medium (0.51.4 mM). Propionate was not produced in presence of either organic or inorganic nitrogen source. Strain PSU-2 tends to grow and produced more H2 when medium contains organic nitrogen source. C:N ratios of inorganic nitrogen amended medium (38:1) and organic nitrogen amended medium (35:1) were almost the same, and the increase of bacterial biomass and hydrogen production was most probably due to peptone, a source of amino acids which can be essential growth factors for the bacteria. Strain PSU-2 performs ethanolacetate type fermentation in inorganic nitrogen amended medium and butyrateacetate type fermentation in organic nitrogen amended medium. The nitrogen source in the medium affected the substrates fermentation of T. thermosaccharolyticum PSU-2, caused a change in H2 yield. H2 production yield (2:53 mol H2 mol1 hexose) of butyrateacetate type fermentation was high, whereas H2 production yield (0:75 mol H2 mol1 hexose) of ethanolacetate type fermentation was low (Fig. 7). H2 yield was about 3 times higher than the maximum H2 yield quantied when strain PSU-2 was cultivated in inorganic nitrogen amended medium. The different fermentation yields in the medium used were due to the difference in the nitrogen source. The presence of peptone as organic nitrogen source enhanced production of H2 by strain PSU-2. The cell yield in presence of organic nitrogen amended medium increased 2 times compared to inorganic nitrogen amended medium. The utilization of such organic nitrogen sources could be energetically more advantageous from aspect of cell material synthesis [15,33]. Results obtained showed that presence of organic nitrogen resulted in a complete shift in the metabolism of the cell, production of butyrate, which was accompanied by further production of ATP and additional reducing power. H2 can be produced if surplus electrons are formed in the reaction, and then protons are reduced by hydrogenase [47]. Decient organic nitrogen in medium prevented the H2 production by the bacteria and modied metabolic pattern toward the production of reduced end product such as ethanol [33]. Therefore, it appears that the organic nitrogen amendment could be responsible for increased H2 production. The fact that ethanol was obtained in similar concentrations as acetate for all cultivations, where inorganic nitrogen amended media were used, explains the decrease of H2 yield to less than 2 mol H2 mol1 hexose. In case of H2 production associated with ethanol production, mixed acetateethanol type fermentation could be described with following equation: C6 H12 O6 H2 O ! 2H2 2CO2 C2 H4 O2 C2 H6 O. (4)

Concentration of fermentation metabolites SD (mM)

Produced H2 SD (ml l1 )

Cell yield SD (g cell g1 substrate)

0.7570.02 1.7570.05 1.370.03 2.4370.04 5.0670.16 2.870.09

0.3070.01 0.3270.01 0.2570.01 0.170.01 0.5170.05 0.6470.02 0.4570.01

107073 219576 193075 24070.5 342076 6762715 4137712

2.8370.08 10.5270.32 10.9170.33 0.6070.02 2.9370.1 2.4070.12 2.51 70.11

Ethanol

0.0270.01 0.00 0.00 0.00 0.00 0.3070.01 0.00

Butanol

6.7370.2 17.7870.5 11.6870.4 2.0070.05 7.3670.2 8.1370.21 7.0270.25

Acetate

0.3170.08 0.00 0.3170.05 0.00 0.2970.06 0.00 0.00

Isobutyrate

1.4670.04 0.6770.02 0.5970.02 1.7570.03 6.0970.2 8.4170.25 11.6070.35

Butyrate

0.5970.02 0.6470.02 0.9170.03 0.9070.02 0.3470.01 0.00 0.00

Lactate

Therefore, in case of H2 production associated with butyrate, the fermentation pathway could be described with Eq. (3). It was proposed that shift of ethanolacetate type fermentation to butyrateacetate type fermentation is due to organic nitrogen amendment. The suggested metabolic pathway deduced for carbohydrate fermentation by strain PSU-2 was shown in Fig. 7. Organic nitrogen stimulation for butyrate acetate type fermentation achieved maximum H2 production

Medium components

IN Glucose IN Sucrose IN Starch IN ON ON Glucose ON Sucrose ON Starch

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Propionic 2 CH3CH2COOH 2 NADH 2 CH3CHOHCOOH 2CO2 Lactate Butyrate-acetate type fermentation (organic nitrogen amended medium)

C6H12O6 Glucose 2 ADP 2 ATP 2 CH3COCOOH Pyruvate

2 Fd 2 NADH 2 CO2 H2ase 2FdH2 2 CH3CH2OH Ethanol 2 NADH 2 Fd H2ase 2 H2 2 H2

2 CH3COSCoA Acetyl-CoA

2FdH2 Ethanol-acetate type fermentation (inorganic nitrogen amended medium)

CH3CH2CH2COOH Butyrate
3C6H12O6 + 2H2O 8H2 + 6CO2 + 2C2H4O2 + 2C4H8O2

2 CH3COOH Acetate
2C6H12O6 + 2H2O

4H2 + 4CO2 + 2CH3COOH + CH3CH2OH

Fig. 7 Suggested metabolic pathway deduced for carbohydrate fermentation by T. thermosaccharolyticum PSU-2 under organic nitrogen added medium and organic nitrogen decient medium.

yield of 2:67 mol H2 mol1 hexose. Inorganic nitrogen stimulation for ethanolacetate type fermentation achieved maximum H2 production yield of 1:0 mol H2 mol1 hexose.

4.

Conclusions

A strict anaerobic thermophilic bacterial strain with high production rate and yield of H2 , named T. thermosaccharolyticum PSU-2, was isolated. T. thermosaccharolyticum PSU-2 appeared a suitable candidate for the thermophilic fermentative H2 production due to its capacity to use various types of carbon source, acid tolerance and high H2 production rate. The level of fermentative H2 production by this strain was inuenced by the medium pH and the initial sucrose concentration. The optimum initial sucrose concentration, pH and temperature were 20 g l1 , 6.25 and 60  C, respectively, which resulted in a respective maximum H2 productivity of 12:12 mmol H2 l1 h1 . This strain might be applicable to the treatment plant of organic wastewater that usually contains a mixture of sugar.

Acknowledgments
This work was support by the Thailand Research Fund (TRF). Additionally the authors wish to acknowledge the nancial support of Danish Research Council STVF Project No. 2058-030020. Mr. O-Thong thanks Hector Garcia for technical support.
R E F E R E N C E S

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