Renewable Energy 37 (2012) 174e179 Contents lists available at ScienceDirect Renewable Energy journal homepage: www.elsevier.com/locate/renene Thermophilic fermentative hydrogen production from xylose by Thermotoga neapolitana DSM 4359 Tien Anh Ngo a, b, *, Tra Huong Nguyen b, Ha Thi Viet Bui b a b Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan Department of Microbiology, Hanoi University of Science, Hanoi, Viet Nam a r t i c l e i n f o a b s t r a c t Article history: Received September 2010 Accepted 11 June 2011 Available online July 2011 Biohydrogen production from xylose by Thermotoga neapolitana was investigated in batch culture using serum bottles and a continuously stirred anaerobic bioreactor (CSABR) The effect of various xylose concentrations on growth and H2 production were studied in small batch culture for highly efficient H2 production The highest hydrogen production of 32.1 Ỉ 1.6 mmol-H2/L and maximum biomass concentration of 959.63 Ỉ 47.9 mg/L were obtained at initial xylose concentration of 5.0 g/L To develop a large-scale biohydrogen production system as well as overcome the problems in small batch culture, a continuously stirred anaerobic bioreactor was tested on T neapolitana in both pH-uncontrolled batch culture and pH-controlled batch culture The results showed that the production level of H2 from fermentation in a pH-controlled batch culture was much higher than those from a pH-uncontrolled batch culture for H2 production from xylose The H2 yield in a pH-controlled batch culture on xylose substrate was 2.22 Ỉ 0.11 mol-H2 molÀ1 xyloseconsumed, which was nearly 1.2-fold higher than pH-uncontrolled batch cultures In order to study the precise effect of a stable pH on hydrogen production, and metabolite pathway involved, cultures was conducted with pH-controlled at different levels ranging from 6.5 to 7.5 The maximum H2 yield of 2.8 Ỉ 0.14 mol-H2 molÀ1 xyloseconsumed was measured while the pH was maintained at 7.0 The acetic acid and lactic acid production were 2.98 Ỉ 0.15 g/L and 0.36 Ỉ 0.02 g/L, respectively Ó 2011 Elsevier Ltd All rights reserved Keywords: Batch culture Biohydrogen Thermotoga neapolitana Xylose CSABR Introduction Biohydrogen is a green energy with the greatest potential to replace fossil fuels in the future, given its high energy content, lack of CO2 emissions, and readily available production sources from various renewable feedstocks [1e3] Carbohydrate rich wastes such as lignocellulosic agricultural residues are promising feedstocks with huge biofuel potential [4] Pentose sugar (xylose) accounts for up to 35e45% of the total sugars in the lignocellulosic hydrolysate derived from wood, agricultural by products or crops [5] To convert cellulosic feedstock to high value hydrogen, full fermentation of pentose (xylose) or hexoses (glucose, sucrose) in the hydrolysates of cellulose and hemicelluloses is very important [6] The hyperthermophilic bacteria Thermotoga neapolitana has garnered increasing interest for potential biohydrogen production with high yield from a wide range of carbohydrates, such as * Corresponding author Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan E-mail address: tiennabio@gmail.com (T.A Ngo) 0960-1481/$ e see front matter Ó 2011 Elsevier Ltd All rights reserved doi:10.1016/j.renene.2011.06.015 glucose, sucrose [7,8], and especially direct fermentation of starch, hemicelluloses, cellulose [9,10] and glycerol wastes in our previous research [11] Moreover, the biohydrogen fermentation of T neapolitana at an ambient 75 C makes its H2 fermentation less sensitive to contamination from methanogenic archaea, with a higher rate of hydrolysis, and H2 yield [12e14,25] Most studies on biological hydrogen production in Thermotogales are based on small batch fermentation in serum bottle and few of works have used glucose as carbon source in pH-controlled bioreactors [26,27] Investigations of xylose fermentation to biohydrogen were mainly concentrated on fermentation using anaerobic mix culture at mesophilic temperatures [15e17] However, only limited study has been carried out on the possibility of xylose utilisation for hydrogen production by T neapolitana [8] Because of the main products produced from the metabolic activities of T neapolitana from strictly anaerobic fermentation, including organic acids (such as lactic and acetic acid), carbon dioxide (CO2) and H2 [10,18], growth and H2 production by T neapolitana in small batch cultures were reported to be limited by a rapid decrease in pH and the effect from high hydrogen partial pressure [8,12,19] To overcome this problem, an N2 sparging method and appropriate buffering [7,8,11,18] were T.A Ngo et al / Renewable Energy 37 (2012) 174e179 175 successfully applied to enhance anaerobic H2 fermentation T neapolitana in a small To develop a large-scale biohydrogen production system and overcome the problems in small batch culture, T neapolitana was studied in pH-controlled batch culture using a continuously stirred anaerobic bioreactor In this work, we describe the fermentative production of biohydrogen from xylose substrate by the hyperthermophile T neapolitana in serum bottle cultures and a L-CSABR with pHuncontrolled/controlled batch cultures and a continuous pure N2 gas flow The optimization of initial substrate concentration for growth and H2 production were determined in serum bottles Investigation of pH values was carried out in a bioreactor with pH control Materials and methods 2.1 Strain and cultivation medium The T neapolitana strain DSM 4359 was obtained from Deutsche Sammlung von Mikroorganismen und Zelhulturen, Germany The cultures were grown in 120 mL serum bottles containing 40 mL culture medium of modified Thermotoga maritima basal medium (TMB) at 75 C and pH 7.5, with 10% (v/v) inoculation [12] The medium used for H2 fermentation consisted of (amounts are in grams per litre of deionised water): 1.5 g KH2PO4; 4.2 g Na2HPO4$12H2O (22 mM PO3À ); 0.5 g NH4Cl; 0.2 g MgCl2$6H2O (1 mM); 20.0 g NaCl; 2.0 g yeast extract; 5.0 g carbon source (glucose, sucrose, xylose); 15.0 mL of the trace element solution (DSM-TES, see DSMZ medium 141); and 1.0 mg resazurin, which was used as a redox indicator The anaerobic conditions for growth were created by adding 1.1 g cysteine hydrochloride as a reducing agent and flushing the headspace of the serum bottles with pure N2 within A batch culture using a L bioreactor (Biotron, Korea), charged with 900 mL of fresh medium and a 100 mL inoculum of T neapolitana, was performed under the controlled of temperature, pH and agitation at 75 C, 7.5, and 300 rpm, respectively, using a Biotron controller system The pH was kept constant by the addition of 2.0 N NaOH Temperature was kept at 75 C using a heating coil wrapped around the bioreactor The gas headspace was sparged with a continuous and pure N2 gas flow; the gas outlet from the reactor was connected to a condenser The flow and partial pressure of the gas headspace in the outlet gas was monitored by a gas meter The complete setup is illustrated on Fig 2.2 Sampling and analyses The biomass was monitored by dry cell weight (DCW) and optical density (OD600), with sterile medium as the control The H2 gas in the headspace was sampled by a gas-tight syringe (100 mL injection volume, Hamilton, USA) and determined by gas chromatograph (GC, Hewlett Packard 5890 Series II, USA) employing a thermal conductivity detector (TCD) and a 2-m stainless column packed with Carboxen 1000, 50/80 mesh (Supelco) The operational temperatures of the injection port, the oven, and the detector were 120, 70, and 120 C, respectively Nitrogen was used as the gas carrier at the flow rate of 55 mL per The moles of produced H2 gas (nH2, mol) and the partial pressure of H2 (pH2, atm) in the batch culture using serum bottles were calculated from the mole percentage of H2, which was determined by GC analysis The hydrogen yield (YH2) is the molar amount of H2 produced from the consumed substrate It was calculated using Eq (1): YH2 ¼ nH2 nS (1) Fig Scheme of the anaerobic fermentation bioreactor (where nH2 is the moles of hydrogen produced, and nS is the moles of substrate consumed.) The volumetric gas flow rate (F, mL minÀ1) from the bioreactor and pH2 were used to calculate the molar flow rate of H2 (Q H2 , mmol.hÀ1) using Eq (2): Q H2 ¼ FpH2 60 RT h (2) (where R is the gas constant for ideal gas (0.08206 L atm molÀ1 KÀ1), and T is 298 K.) The amount of H2 (nH2 , mole) produced at the time (t) of inoculation (to ¼ 0) was calculated by numerical integration of the molar H2 flow rate, Q H2 ;t , with respect to time (Eq (3)): NH2 ;tiỵ1 ẳ X Q H2 ;ti ỵ Q H2 ;tiỵ1 tiỵ1 ti Þ (3) The organic acid concentration was quantified using an HPLC system equipped with a reflective index detector (Agilent 1100, USA): 50 mL of 0.2 mm filtered-culture supernatant was separated on a Rezex ROA-Organic acid Hỵ (8%) 300 Â 7.80 mm column (Phenomenex, USA) and eluted with 0.5 mL minÀ1 of 0.005 M H2SO4 at room temperature The residual xylose concentration in the culture supernatant was quantified using HPLC: 50 mL of 0.2 mm filtered-culture supernatant was injected and analysed on a Rezex RCM-Monosaccharide column (Phenomenex, USA) and eluted with 0.5 mL minÀ1 water at 60 C Detection was performed with a refractive index detector (Agilent 1100, USA) Results and discustion 3.1 Investigation of H2 production of T neapolitana from xylose T neapolitana were grown in serum bottles for application of converting xylose into biohydrogen When T neapolitana was grown anaerobically on xylose, a mixture of acetic and lactic acid was produced (data not shown) H2 accumulated in the headspace and accumulation of cells increased dry cell weight (DCW) from 0.08 to 0.73 Ỉ 0.03 g/L within 20 h and almost maintained from 25 to 55 h of cultivation (Fig 2) H2 production was observed soon after h upon initially entering into the log phase of growth, achieving 35 1.0 H2 content Cell growth A 0.8 30 25 0.6 20 0.4 15 10 0.2 Cell growth (g DCW/L) H content (%, v/v headspace) 40 36 54 32 48 28 42 24 36 20 30 16 24 12 18 H2 content 12 H2 production H2 production (mmol/L) T.A Ngo et al / Renewable Energy 37 (2012) 174e179 H content (% v/v, headspace) 176 0.0 10 20 30 40 50 0 Cultivation time (hour) Xylose concentration (g/L) Fig Investigation of H2 production by T neapolitana from xylose in serum bottle without pH control All data points are averages of three replicate bottles B 1.4 3.2 Fermentative H2 production in batch anaerobic bioreactor In T neapolitana fermentations there was a rapid decrease in pH, resulting in increasing pH stress In some cultures, the process stopped before all of substrate was consumed This has previously been observed also in cultures of T neapolitana [7,10,12] and T neapolitana [9] from glucose substrate In this study, T neapolitana was investigated in both pH-uncontrolled and pHcontrolled batch cultures from xylose substrate using a L-CSABR system at an initial pH of 7.5, and at temperature of 75 C A mixture of acetic and lactic acid was also produced when T neapolitana was 100 0.8 0.6 0.4 Cell growth 0.2 80 60 40 Xylose utilization (%) 1.0 Residual xylose (g/L) 1.2 Cell growth (g DCW/L) high level of H2 from 20 h to the end of the cultivation, with approximately 30% of H2 content in the headspace (Fig 2) This result indicated that there were no significant differences in the H2 production released in xylose fermentation and glucose fermentation which was studied in the previous research [12,7] To determine the sole effect of the xylose concentration on H2 production and substrate utilisation of T neapolitana, a complex medium containing a fixed yeast extract concentration 2.0 g/L supplemented with different initial xylose concentrations was applied to the small batch cultures Fig shows the effect of the different initial xylose concentrations versus H2 production and xylose utilisation The cumulative H2 production increases with the rising of xylose concentration in the range of 2.0e5.0 g/L, the maximum H2 production of 32 Ỉ 1.6 mmol-H2/L culture and maximum H2 content of 30% occurring at a xylose concentration of 5.0 g/L Then, H2 production gradually decreased as the xylose concentration increased (Fig 3A) At an initial xylose concentration of 5.0 g/L, the biomass of T neapolitana reached to the highest value of 0.96 Ỉ 0.05 g/L culture after 24 h of cultivation (Fig 3B) However, at the initial xylose concentration of 5.0 g/L, converted H2 yeild from xylose of 1.1 Ỉ 0.05 mol-H2 molÀ1 xylose, was lower than the initial xylose concentration of 2.0 g/L, with the highest yield of 1.7 Ỉ 0.08 mol-H2 molÀ1 xylose Accordingly, a high concentration of xylose was not favorable for T neapolitana growth and H2 production The results herein indicate that the change in xylose concentration remarkably affected H2 production and substrate utilisation Obviously, hydrogen production specific to the amount of xylose added depends both on the degree of substrate conversion, as well as the metabolic conversion pathway, giving information about the potential of substrate to release a specific amount of H2 Expression of H2 production per amount of substrate added is often used to describe H2 production efficiency [20] 20 xylose utilization Residual xylose 0.0 Xylose concentration (g/L) Fig Growth of T neapolitana from batch cultures in serum bottle at different initial xylose concentrations: (A) H2 content in biogas and molar amount H2 production; (B) biomass concentration as DCW and xylose initialization All data points are averages of three replicate bottles grown on xylose substrate H2 production process started at around h and the pH rapidly decreased from 7.5 to below 5.5 after 18 h of cultivation (Fig 4A and B) As shown in Fig 4A, in the first 24 h of cultivation, the cellular metabolism of T neapolitana was robust With xylose as the sole carbon source, the biomass concentration reached the maximum value of 1.4 Ỉ 0.07 g DCW/L and culture pH almost unchanged after dropping to 5.17 at 30 h of cultivation After fermenting for 10 h, the CSABR produce biogas with a H2 content of over 30% Steady-state operation was achieved from 10 to 17 h and the maximum of H2 production rate and H2 yield were respectively 3.1 Ỉ 0.1 mmol-H2 hÀ1 and 1.8 Ỉ 0.1 mol-H2 molÀ1 xylose at 15 h, (Fig 4B) The increase in H2 production was accompanied with a proportional increase in xylose utilisation and acetic acid production Fig 4B shows that approximately 86% of the xylose was consumed within the first 24 h of cultivation The acetic acid production began at h, while the lactic acid production started at around 12 h from medium containing xylose At the end of fermentation, 2.1 Ỉ 0.1 g/L acetic acid, 0.34 Ỉ 0.02 g/L lactic acid was obtained from xylose (Table 1) This result shows that the obtained acetic acid production in medium was consistently shown to be above ten fold higher than lactic acid production It implies the H2acetic acid production pathway predominated over the main compete lactic acid production pathway In this study, the pH T.A Ngo et al / Renewable Energy 37 (2012) 174e179 Cell growth Culture pH 1.0 7.5 7.0 6.5 0.5 6.0 Culture pH Cell growth (g DCW/L) B 8.0 5.5 0.0 5.0 12 18 24 30 36 42 Xylose, H , Lactic acid, acetic acid (mmol/L) 1.5 Cultivation time (hour) 70 4.0 H2 production rate Residual xylose Accumulated H production 60 3.5 Lactic acid Acetic acid 50 3.0 2.5 40 2.0 30 1.5 20 1.0 10 H production rate (mmol/h) A 177 0.5 0.0 10 20 30 40 Cultivation time (hour) 1.0 7.0 6.5 0.5 6.0 Cell growth Culture pH 0.0 Culture pH Cell growth (g DCW/L) 7.5 5.5 5.0 12 18 24 30 36 42 80 4.0 H2 production rate Residual xylose Accumulated H production 70 3.5 Lactic acid Acetic acid 60 3.0 50 2.5 40 2.0 30 1.5 20 1.0 10 0.5 H production rate (mmol/h) D 8.0 Xylose, H , Lactic acid, acetic acid (mmol/L) C 1.5 0.0 10 Cultivation time (hour) 20 30 40 50 Cultivation time (hour) Fig Growth of T neapolitana from xylose as the main substrate in the batch culture using a L-CSABR: (A) Cell growth (DCW) and pH in batch culture without pH control; (B) Metabolites (xylose, H2, lactic acid, acetic acid) in batch culture without pH control; (C) Cell growth (DCW) and pH in batch culture with pH control; Metabolites (xylose, H2, lactic acid, acetic acid) in batch culture with pH control decreased in the culture supernatants, resulting in cessation of the H2 fermentation process prior to complete consumption of the substrate At the end of fermentation, approximately 89.4% of xylose was consumed pH was initially 7.5 (Ỉ0.01) in all experiments In the pHcontrolled batch culture, pH was kept constant by the addition of 2.0 N NaOH during cultivation A very strong metabolism occurred in bacterial cells within the initial 15 h-cultivation with xylose as the Table Performance and metabolite analysis of T neapolitana fermentation Parameters Working volume of 1000 mL of culture in a 3L CSABRa system Without pH control With pH controlb 31.9 Ỉ 1.6 3.1 Ỉ 0.1 1.84 Ỉ 0.09 2.1 Ỉ 0.1 0.34 Ỉ 0.02 89.4 Ỉ 4.5 5.17 38.3 3.76 2.71 2.98 0.21 97.8 6.5 6.5 Maximum H2 content (%) Maximum H2 production rate (mmol-H2hÀ1) H2 yieldc Final [acetic acid] (g/L) Final [lactic acid] (g/L) Xylose consumption (%) Final pH Ỉ Ỉ Ỉ Ỉ Ỉ Ỉ 1.9 0.18 0.14 0.15 0.01 0.8 7.0 7.5 39.6 Ỉ 1.98 3.89 Ỉ 0.19 2.8 Ỉ 0.1 3.02 Ỉ 0.15 0.16 Æ 0.01 98.8 Æ 1.1 7.0 33.5 Æ 1.7 3.3 Æ 0.16 2.2 Æ 0.11 2.5 Æ 0.12 0.15 Æ 0.01 95.4 Ỉ 4.4 7.5 Each measurement was repeated three times and averaged Error indicates one standard deviation of uncertainty a CSABR ¼ continuously stirred anaerobic bioreactor b The culture was conducted with the pH-controlled at three different levels ranging from 6.5 to 7.5 with standard error in measurement of (ặ0.01) c H2 yield ẳ (amount (mol) of H2 formed)/(amount (mol) of substrate consumed) 178 T.A Ngo et al / Renewable Energy 37 (2012) 174e179 main substrate (Fig 4C) The cell growth significantly increased and reached the highest concentration of 1.42 Ỉ 0.07 g DCW/L at 15 h and was nearly maintained from 15 to 33 h of cultivation The residual xylose concentration in medium was 0.6 Ỉ 0.03 g/L, that means about 90% of xylose was consumed only at the first 24 h (Fig 4D) These results indicated that growth and xylose utilisation of T neapolitana in the pH-controlled batch cultures greater than that in pHuncontrolled batch culture As shown in Fig 4D, the H2 production rate achieved the best value of 3.3 Ỉ 0.16 mmol-H2 hÀ1 responding to H2 content in the mixture gas was 33.5 Ỉ 1.7% at 13 h After that, the H2 production rate was gradually decreased over time and negligible after 33 h Therefore, a decrease in H2 production of T neapolitana in the pH-controlled cultures was due to exhaustion of the growthlimiting substrate which was xylose in this case The acetic acid production also observed after h, while the lactic acid production started at 42 h from xylose (Fig 4D) Similar to the H2 production and xylose utilisation results, the levels of acetic acid from fermentation with pH control were much higher than those of the fermentation without pH control In contrast to acetic acid production, lactic acid production was the lowest under conditions of pH control In other words, the acetic acid concentration was much higher than the lactic acid concentration This result implies that the batch culture with pH control was found for highly efficient hydrogen production The observed correlation between the acetic acid/lactic acid ratio and the H2 yield implies a metabolic association between acetic acid and H2, as is well-described in prokaryotic fermentation such as the Clostridia and Enteric bacteria Results from Table show that maximum H2 yield, acetic acid production, and xylose utilisation in pHcontrolled culture was higher than those in pH-uncontrolled culture The best of H2 yield of T neapolitana obtained in the pHcontrolled culture was 2.2 Ỉ 0.11 mol-H2 molÀ1 xylose 3.3 Determination of optimal pH for H2 production from xylose using a L-CSABR system The effect of pH on fermentative H2 production by T neapolitana strain was investigated in a L-CSABR system with pH-controlled at three different levels ranging from 6.5 to 7.5 while keeping other operating conditions constant (stirring, temperature, pressure, and initial culture medium) In Table 1, the maximum H2 production yields are indicated for different pH conditions, with the related final acetic and lactic acid concentrations, and xylose utilisation The maximum H2 production yield and H2 production rate, 2.8 Ỉ 0.1 mol-H2 molÀ1 xylose and 3.89 Ỉ 0.19 mmol-H2 hÀ1 respectively, were obtained at a pH of 7.0 These optimum pH and yields were in accordance with previous studies with the T neapolitana culture [12] Since at this pH level utilisation of xylose and organic acid concentration also peaked, overall performance for the process was at a maximum (Table 1) Subsequently, the H2 production rate increased exponentially reaching the maximum level in comparing with those of T neapolitana cultured at other pH levels until substrate depletion (data not shown) The H2 yield of 2.8 Ỉ 0.1 mol-H2molÀ1 xylose in this study was substantially higher than in other studies using xylose as carbon source from pure cultures of bacteria (0.14 mol-H2molÀ1 xylose by Thermoanaerobacter finnii [21], 0.77 mol-H2molÀ1 xylose by Clostridium tyrobutyricum ATCC 25755 [22], and 0.73 mol-H2molÀ1 xylose by Clostridium butyricum CGS5 [16]) and mixed culture fermentation (1.36 mol-H2molÀ1 xylose [23], 1.84 mol-H2molÀ1 xylose [24]) The successful investigation in this study may be a potential culture technique for hydrogen production systems from T neapolitana Conclusions H2 production obtained from xylose fermentation of T neapolitana in the batch culture During the cultivations at different initial xylose concentrations, the optimal xylose concentration for T neapolitana growth and H2 production were defined in the medium at an initial xylose concentration of 5.0 g/L We successfully conducted biohydrogen fermentation by T neapolitana from xylose in a continuously stirred anaerobic bioreactor system The results indicated that the H2 yield and H2 production rate in pH-controlled batch culture was much higher than those from pH-uncontrolled batch culture The optimal condition of pH 7.0 obtained from the L reactor by considering H2 yield The maximum 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Cultivation time (hour) Xylose concentration (g/L) Fig Investigation of H2 production by T neapolitana from xylose in serum bottle without pH control All data points are averages of three replicate... efficiency [20] 20 xylose utilization Residual xylose 0.0 Xylose concentration (g/L) Fig Growth of T neapolitana from batch cultures in serum bottle at different initial xylose concentrations: (A) H2... fermentation of T neapolitana in the batch culture During the cultivations at different initial xylose concentrations, the optimal xylose concentration for T neapolitana growth and H2 production