- Email: [email protected]
Enhancing biohydrogen production of the alkalithermophile Thermobrachium celere
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 5 0 e5 5 5 8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Enhancing biohydrogen production of the alkalithermophile Thermobrachium celere Alessandro Ciranna*, Ville Santala, Matti Karp Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101 Tampere, Finland
article info
abstract
Article history:
In this study the effect of different buffering agents, pH control and N2 sparging on
Received 13 October 2011
biohydrogen production in Thermobrachium celere was investigated in batch cultivations.
Received in revised form
Among the tested buffers, none was able to prevent the medium acidification resulting in
21 November 2011
a premature interruption of the hydrogen production. Controlling the pH helped to sustain
Accepted 13 December 2011
the growth, the complete substrate consumption and the H2 production. However, in these
Available online 21 January 2012
conditions the increase of H2 partial pressure induced a partial metabolic shift towards ethanol production resulting in a decreased H2 yield. Analysis of formate accumulation
Keywords:
during growth suggests that this compound might play a relevant role in the anabolic
Anaerobic bacteria
routes in T. celere. When frequent N2 sparging was applied for H2 removal, together with pH
Hydrogen partial pressure
control, the H2 yield was remarkably enhanced from 2.26 to 3.53 mol H2/mol glucose, and
Dark fermentation
the maximum H2 production rate and specific H2 production rate reached 41.5 mmol H2/l/h
Volatile fatty acids
and 142.3 mmol H2/h/g, respectively. This result suggests that under proper conditions
pH control
T. celere is able to produce hydrogen at high yield and production rate.
Ethanol formation
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The depletion of fossil fuels and the negative impact of their combustion on the environment have led to exploration of alternative sources of energy. Hydrogen is considered an ideal and clean energy carrier for replacing fossil fuels in the future because of its high energy content per weight unit (122 kJ/g) and non-polluting properties [1]. Fermentation by anaerobic bacteria is a promising biological process for converting carbohydrate-rich substrates to hydrogen. Compared to photofermentation it has several advantages such as higher hydrogen production rate and capability to convert organic wastes to a more valuable energy source [2]. Hydrogen can be produced via dark fermentation by several mesophilic and thermophilic anaerobic bacteria. Thermophiles have shown great potential for H2 production
mainly because of thermodynamic advantages that allow to generate a lower variety of by-products and to reach the theoretical yield of 4 mol H2/mol glucose [3]. Moreover, thermophilic biohydrogen production is less sensitive to contamination of H2-consuming methanogens [4]. This poses a clear advantage when thermophiles are used for hydrogen production in an open system. However, the limited volumetric hydrogen productivity in thermophiles is one major drawback for industrial-scale application. In the attempt of optimizing the biohydrogenation process, a negative correlation has been observed between H2 yield and H2 production rate both in mesophiles and thermophiles [5,6]. At high H2 production rates the concentration of H2 in the liquid phase and in the headspace increases rapidly and H2 synthesis becomes thermodynamically unfavorable [7]. As a consequence, the disposal of
* Corresponding author. Tel.: þ358 40 1981 141; fax: þ358 3 3115 2869. E-mail address: [email protected] (A. Ciranna). 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.12.105
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 5 0 e5 5 5 8
accumulated reducing equivalents in the cell is mediated by a metabolic shift towards production of more reduced metabolites, such as lactate, ethanol, acetone, butanol, or alanine [2]. Despite their higher resistance towards high PH2 , thermophiles are still sensitive to the inhibition caused by the accumulation of hydrogen during the anaerobic fermentation [3,8]. Attempts to minimize the effect of hydrogen accumulation in small closed systems (e.g. serum bottles) have been made by either increasing the headspace/culture ratio [9,10] or removing hydrogen by gas sparging techniques [9,11,12]. The mantainence of an optimal culture pH is another important parameter to control for achieving an efficient H2 production [13]. During fermentation the hydrogen evolution is accompanied by accumulation of volatile fatty acids (VFAs) resulting in a drop of pH in the culture. Therefore, buffering agents such as phosphate, carbonate and HEPES are often used to maintain the pH at optimal level. Previous studies have shown that the anaerobic alkalithermophilic species Thermobrachium celere (soon to be reclassified as Caloramator celere (Bharat Patel, Personal communication)) produced hydrogen at high yields both in pure and mixed culture [10,14]. Considering these results, together with the great selective pressure provided by the optimal growth conditions (T ¼ 67 C and pH67 C ¼ 8.2) and the fast growth rate [15], T. celere is a promising strain for biohydrogen production in open (non-sterile) bioprocess system. A recent study showed that in T. celere H2 production is affected by the medium acidification caused by accumulation of VFAs and by the H2 build-up during the fermentation [10]. In batch conditions, without pH adjustments, the pH dropped below 6 which corresponded to a reduction of hydrogen production rate. Despite being able to produce H2 at PH2 as high as 223 kPa, the H2 yield in T. celere was severely influenced by the hydrogen accumulation decreasing from 3.36 to 1.02 mol H2/mol glucose with the decrease of the headspace/culture ratio [10]. In this study the effect of different buffering agents, pH control and N2 sparging was investigated to enhance H2 production during the glucose fermentation of the alkalithermophile T. celere.
autoclaving for 15 min at 121 C. The pH of the medium was adjusted after sterilization to 8.2 at 67 C with sterile and anaerobic 3 M NaOH. The serum bottles were inoculated with 1 ml (5% v/v) of a culture in the exponential phase and incubated at 67 C and 150 rpm for 7e12 h.
2.2.
Materials and methods
2.1.
Medium and culture conditions
T. celere (DSMZ 8682T from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) was grown under a nitrogen atmosphere in a modified version of the ATCC 2072 medium containing (g/l): KH2PO4 1.24; Na2HPO4 5.79; KCl 1; (NH4)2SO4 0.5; NH4Cl 0.5; MgCl2$6H20 0.1; CaCl2$6H20 0.11; Cystein-HCl 0.13; Na2S$9H20 0.13; yeast extract 2; tryptone 2; resazurin (redox indicator) 0.001. After sterilization glucose was added as substrate in concentration of 10 g/l (55 mM) as well as 10 ml/l of vitamin solution and 10 ml/l of trace element solution (ATCC medium No. 2072, American Type Culture Collection). All chemicals used were of analytical grade. The medium was made anaerobic by flushing with N2 for 20 min, dispensed as 20 ml aliquots into 120 ml serum bottles flushed with N2 and then sterilized by
Experimental procedures
All the experiments were performed in 120 ml serum bottles containing 20 ml of medium with 10 g/l of glucose as substrate. The effect of different buffering agents was studied by preparing media containing phosphate buffer, HEPES buffer, carbonate buffer or TriseHCl buffer to a final concentration of 50 mM. In the study the media were referred as PBM (phosphate-buffered medium), HBM (HEPES-buffered medium), CBM (carbonate-buffered medium) and TBM (Tris HCl-buffered medium). PBM was prepared as described in Section 2.1. HBM, CBM and TBM were prepared by omitting KH2PO4 and Na2HPO4 and by adding respectively 1 M solution of HEPES buffer, carbonate buffer and TriseHCl buffer to a final concentration of 50 mM. During the experiment, liquid (0.5 ml) and gas samples (0.3 ml) were taken every hour for the analyses with N2-flushed 1 ml syringes. PBM, HBM and CBM were used to test the effect of pH control during fermentation. In this experiment the pH of liquid samples withdrawn from the culture was measured hourly with a slim probe. When the pH reached the value of 6 or below it was manually adjusted by injecting in the bottles from 0.35 to 0.5 ml (depending on the buffer in the medium) of sterile and anaerobic 3 M NaOH with a N2-flushed 1 ml syringe. After the injection a liquid sample was taken to verify that the pH was in the range of the initial value (8.8e9). To evaluate the effect of N2 sparging on the H2 production, PBM was employed. The headspace was sparged with pure N2 gas (Oy AGA Ab, Espoo, Finland) every hour for 2 min to ensure complete removal of produced gases. Finally, the combined effect of pH control and N2 sparging was studied in PBM as described above. All the experiments were run in duplicate and included negative controls without added substrate.
2.3.
2.
5551
Analytical methods and calculations
Cell concentrations were determined by measuring the absorbance spectrophotometrically at 600 nm with an Ultrospec 500 Pro spectrophotometer (Amersham Biosciences, Munich, Germany). Cell dry weight (CDW) was determined by centrifuging the culture broths. For this, 20 ml aliquots of the culture were transferred into dried, pre-weighed 50 ml Falcon tubes and centrifuged at 5000 g for 15 min. Cell pellets were washed twice with saline solution (9 g/l NaCl) by centrifugation and dried at 70 C to a constant weight. The relation between OD and CDW of T. celere for growth on glucose was found to be CDW (g/l) ¼ 0.208 OD600 þ 0.071 (R2 ¼ 0.99). The pH of the cultures was measured with a pH330i pH meter (WTW, Weilheim, Germany) equipped with a Sentix 41 pH-electrode (WTW, Weilheim, Germany) or a Slimtrode pH-electrode (Hamilton, Bonaduz, Switzerland). For practical reasons the pH of the cultures was measured at room temperature. The amount of H2 and CO2 produced was analyzed according to Owen et al. [16]. The headspace was sampled by
5552
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 5 0 e5 5 5 8
using a gas-tight syringe (0.3 ml injection volume) and its composition analyzed with a GC-2014 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with Porapak N column (80/100 mesh) and a thermal conductivity detector (TCD). N2 was used as carrier gas and the temperatures of injector, column and detector were 110 C, 80 C and 110 C, respectively. Hydrogen production was calculated from the headspace gas sample and the total volume of gas produced at each time interval according to the mass balance equation (Eq. (1)): VH2 ;t ¼ VH2 ;t1 þ CH2 ;t Vg;t Vg;t1 þ VHead ðCH2 ;t CH2 ;t1 Þ
(1)
where VH2 ;t is the cumulative H2 gas volume at time t, VH2 ;t1 is the cumulative H2 gas volume at time t1, Vg,t is the cumulative total gas volume at time t, Vg,t1 is the cumulative total gas volume at time t1, VHead is the headspace volume in serum bottles, CH2 ;t is the H2 percentage of biogas in headspace
Fig. 1 e Time course of growth, sugar consumption, culture pH and metabolite formation by T. celere in PBM (A), HBM (B) and CBM (C). Optical density (;); glucose (C); pH (A); H2 (-); acetate (,); formate (6); ethanol (7). Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
at time t, and CH2 ;t1 is the H2 percentage of biogas in headspace at time t1. VHead was modified taking into account the change of headspace volume upon liquid withdrawal for sampling. Molar H2 and CO2 were calculated using the ideal gas law. Soluble end products (VFAs and ethanol) and glucose were analyzed by high liquid performance chromatography (HPLC) with an LC-20AC prominence liquid chromatograph equipped with an RID-10A refractive index detector, DGU20A5 prominence degasser and a CBM-20A prominence communications bus module (Shimadzu, Kyoto, Japan). The column was a 30-cm Rezex RHM Monosaccharide Hþ (8%) column (Phenomenex, Alleroed, Denmark). 0.01 N H2SO4 was used as mobile phase at a flow rate of 0.600 ml/min. Carbon balances were calculated from the total amount of carbon-containing products formed (in C-mol) and the amount of sugar consumed (in C-mol). Redox (electron) balances were calculated after multiplying the amount of each metabolite and the sugar by the corresponding degree of reduction (in mol electrons per C-mol) [17]. The chemical formula of biomass was assumed to be CH1.8O0.5N0.2. Metabolite yields as well as carbon and electron balances were calculated by subtracting
Fig. 2 e Time course of H2 production rate (A) and effect of change of culture pH during growth on H2 production rate (B) in PBM (-), HBM (C) and CBM (;). Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
5553
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 5 0 e5 5 5 8
Table 1 e Effect of different buffering agents on glucose degradation, yields of major fermentation products and biomass in T. celere. Data represents averages of the results of two replicate culture experiments ± standard deviation. Medium
Glucose degradation (%) 61.9 0.2 73.3 0.1 53.5 0.8
PBM HBM CBM
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
2.84 0.03 2.52 0.06 2.27 0.01
1.42 0.01 1.27 0.02 1.29 0.02
0.19 0.01 0.29 0.01 0.21 0.01
0.33 0.01 0.21 0.01 0.62 0.03
the background metabolite production in control cultivations (i.e. cultivation without the substrate) and the precultures’ carryover metabolites from the results.
3.
Results and discussion
3.1.
Effect of buffering agents on H2 production
The effect of four buffering agents (phosphate buffer, HEPES, carbonate buffer and TriseHCl) on hydrogen production by T. celere was evaluated during glucose fermentation. Among the tested media, TBM severely repressed the growth and consequently the hydrogen production (data not shown). Despite its optimal pKa of 8.06 for the growth of T. celere, TriseHCl is a polyamine that can have toxic effect on living cells by interacting with cellular components [18] and by inhibiting enzymatic activities [19]. In PBM and HBM the H2 accumulation ceased after 7 h of growth reaching 112.2 and 116.4 mmol H2/l culture, respectively (Fig. 1A, B), while in CBM 84.6 mmol H2/l culture were produced (Fig. 1C). In CBM H2 accumulation started immediately reaching an H2 production rate of 30.7 mmol H2/l/h after 2 h (Fig. 2A). Also the glucose consumption rate was higher compared to the other media (Fig. 1C). This indicates that carbonate buffer was able to maintain the pH near to the optimal value during the first hours of fermentation. However, a sudden drop in pH occurred between the 1st and the 3rd hour resulting in the inhibition of both growth and H2 production (Fig. 1C). In PBM and HBM the highest H2 production rate observed after 3 h was 26.9 and 30.6 mmol H2/l/h, respectively (Fig. 2A). As for carbonate, both buffering agents were not able to prevent the pH to drop at inhibiting value for T. celere [10] with consequent incomplete glucose consumption (Fig. 1A, B). In order to evaluate the effect of changes in culture pH on H2 evolution, in Fig. 2B the hydrogen production rate was plotted against the culture pH measured during the growth in the
CDW (g/l culture)
Carbon balance
Redox balance
0.44 0.01 0.34 0.01 0.44 0.01
1.00 0.01 0.93 0.01 1.16 0.01
0.93 0.01 0.87 0.01 0.89 0.01
different media (Fig. 1AeC). In T. celere the H2 production rate peaked at different pH depending on the buffer used in the medium. Interestingly, the pH at which the highest H2 production rate was observed correlates well with the pKa values of the different buffers. In fact, in CBM (pKa1 6.35, pKa2 10.25) the H2 production rate maintained high level from pH 7.5 to 5.7, while in HBM (pKa 7.55) and PBM (pKa 7.21) it peaked at 7.1 and 6.5, respectively. This implies that the dynamics of H2 production in T. celere is sensitive to the buffering system employed. For all the tested buffers a sudden decrease in the H2 production rate was observed below pH 6 confirming that acidification below this value results detrimental for the H2 production of T. celere. In all the tested conditions acetate was the most abundant soluble end product (Fig. 1AeC). Acetate yield ranged from 1.42 mol acetate/mol glucose in PBM to 1.27 mol acetate/mol glucose in HBM (Table 1). Formate and ethanol were detected in different amounts depending on the buffer used in the medium. Addition of carbonate buffer in the medium resulted in high production of formate yielding 0.62 mol formate/mol glucose (Table 1). This value was 2- and 3-fold higher than those observed in PBM and HBM, respectively. Members of genera closely related to Thermobrachium do not harbor genes encoding for a formate-hydrogen lyase complex to further convert formate to H2 and CO2 [20,21]. If the same applies to T. celere, the production of formate would divert the electron flow from H2 production decreasing the yield. In fact, H2 yield in CBM was 2.27 mol H2/mol glucose compared with 2.52 mol H2/mol glucose and 2.84 mol H2/mol glucose observed in HBM and PBM where the formate production was lower (Table 1). Also ethanol production negatively affects the H2 yield since it is used by the cells as an alternative pathway to dispose the excess of reducing equivalents generated during fermentation [2]. Ethanol production yield was highest in HBM (0.29 mol ethanol/mol glucose) and nearly similar in PBM and CBM (0.19 and 0.21 mol ethanol/mol glucose, respectively) (Table 1). Under the tested conditions in a non pH-controlled system the excessive medium acidification was the main limiting
Table 2 e Effect of different buffering agents combined with pH control on glucose degradation, H2 accumulation and production rate, yields of major fermentation products and biomass in T. celere. Data represents averages of the results of two replicate culture experiments ± standard deviation. Medium
PBM HBM CBM
H2 Glucose H2 degradation accumulation production (mmol/l rate (mmol/ (%) culture) l/h) > 99.0 > 99.0 > 99.0
142.9 5.7 137.6 0.2 133.9 2.2
26.8 3.4 32.0 0.8 32.7 1.2
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
2.26 0.07 1.27 0.03 0.40 0.01 0.31 0.02 2.28 0.03 1.24 0.04 0.48 0.01 0.16 0.01 2.19 0.09 1.29 0.04 0.47 0.02 0.56 0.01
CDW Carbon (g/l culture) balance
0.37 0.01 0.34 0.01 0.41 0.02
Redox balance
0.89 0.01 0.89 0.01 0.89 0.01 0.90 0.02 1.05 0.02 0.95 0.02
5554
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 5 0 e5 5 5 8
growth factor. In fact, when proper growth occurred none of the buffering agents was able to prevent the pH to reach inhibiting value for T. celere. Consequently, growth and hydrogen production ceased before the complete utilization of the substrate. Phosphate and carbonate are often used in media for biohydrogen production systems both in mixed and pure culture for their nutritional and buffering capacity [6,22]. Increase of phosphate buffer in the medium was found to have a positive effect on H2 production in T. celere up to 50 mM after which the growth was inhibited [10]. In the present study, the results showed that PBM was more effective in buffering the pH in the T. celere’s growing range and consequently allowed higher H2 accumulation and yield, while the use of carbonate buffer resulted in a premature inhibition of the H2 production due to a sharp drop in the pH. The better suitability of phosphate buffer for biohydrogenation process was also reported in other studies [22,23]. Recently, HEPES buffer has been successfully employed to enhance the H2 production from glycerol waste by Thermotoga neapolitana by maintaining the pH near the optimal value [12]. On the contrary, despite the slower acidification and a higher glucose consumption (Fig. 1B), H2 accumulation by T. celere in HBM was similar to the one observed in PBM resulting in a lower H2 yield.
3.2.
in PBM, 137.6 mmol H2/l culture in HBM and 133.9 mmol H2/l culture in CBM (Table 2). However, the H2 yield decreased significantly in PBM and HBM (by 20.4 and 9.5%, respectively) and only slightly in CBM. The increased H2 partial pressure was the most probable cause for the inhibition of H2 production and the consequent decrease of the H2 yield. T. celere partially shifts its metabolism to ethanol production as the H2 accumulates in the system during the fermentation [10]. The same behavior was observed during this study, where the
Effect of pH control on H2 production
The influence of three buffers on H2 production was tested under conditions where pH was not the limiting growth factor. Controlling the pH by adding NaOH was effective to prevent the pH value from decreasing below 6, thus maintaining H2 production by T. celere. Under these conditions, T. celere was able to completely utilize the substrate in all the tested media (Table 2). Cumulative H2 production improved with all the buffering agents reaching 142.9 mmol H2/l culture
Fig. 3 e Correlation between mmax and formate yield in T. celere in PBM without pH control (C), PBM with pH control (B), HBM without pH control (:), HBM with pH control (6), CBM without pH control (-) and CBM with pH control (,). Dashed lines represent the linear regression analysis. Data represents averages of the results of two replicate culture experiments.
Fig. 4 e Time course of H2 accumulation (A), H2 production rate (B) and, optical density (filled symbols) and culture pH (open symbols) (C) in T. celere grown in PBM without N2 sparging and with pH control (C, B), with N2 sparging and without pH control (-, ,), and with N2 sparging and with pH control (:, 6). When controlled, pH was adjusted to the initial value (8.8e9) at the 4th hour by NaOH addition. Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
5555
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 5 0 e5 5 5 8
decrease of H2 yields was accompanied by an increase of ethanol yields up to 2-fold (Table 2). In all media the formate yield did not vary significantly when compared with the non pH-controlled experiment. This is consistent with the fact that the formate production pathway is not involved in the process of reductant disposal and therefore was only minimally affected by the hydrogen partial pressure. Under pH-controlled fermentation the H2 production rate remains substantially unchanged ranging from 26.8 mmol H2/ l/h in PBM to 32.7 mmol H2/l/h in CBM (Table 2). Comparison with the results obtained in the experiment without pH control shows a decrease in biomass formation in PBM (from 0.44 to 0.37 g/l), while no significant change was observed in HBM and CBM. Given the performance and the economical convenience for future large-scale cultivations, phosphate buffer was used in the subsequent experiments.
3.3.
Role of formate during H2 production
During the glucose fermentation T. celere produced significant amount of formate which interfered with the H2 yield. The formate yield resulted particularly high in the CBM (Table 1, Table 2). Such high yield raises the question about the role of formate production during the anaerobic fermentation in T. celere. Formate was mainly synthesized in the first hours of growth during the exponential phase (Fig. 1AeC). In this phase the ratio between acetyl-CoA produced via pyruvate oxidoreductase (POR) and pyruvate formate-lyase (PFL) was estimated to be near 1:1 (data not shown), but at the transition to the stationary phase formate accumulation nearly ceased and further acetyl-CoA accumulation was catalyzed by POR. This suggests that the formation of formic acid might be strictly related to the exponential growth phase. As shown in Fig. 3, regression analysis showed linear correlation between formate yield and maximum specific growth rate observed in different media. In CBM mmax was the highest compared to those observed in other media and so was the formate yield. On the contrary, in HBM mmax was around 0.5 h1 and formate yield resulted reduced by 3-fold. The function of pyruvate formate-lyase in Escherichia coli is to mediate the conversion of pyruvate to formate and acetyl-CoA for ATP synthesis in catabolism [24], while the clostridial
pyruvate-formate lyase has been proposed to catalyze formate synthesis to supply the cells with C1 units in anabolism [25,26]. T. celere is phylogenetically related to clostridia and it could possibly possess a PFL with a similar function. This would be consistent with the higher formate synthesis observed at higher growth rates and with the formate production occurring mainly during the exponential phase when maximal cell anabolic activity is expected. However, further studies are needed in order to assess whether formate synthesis is essential for T. celere or whether formate production pathway could be a possible target for metabolic engineering to improve the H2 yield.
3.4.
Enhanced H2 production by N2 sparging
H2 production by T. celere in batch culture is affected by a feedback inhibition from H2 build-up. In order to prevent excessive accumulation of H2 in the system, N2 sparging was employed with and without pH control. Fig. 4A shows that N2 sparging was an efficient method to improve the H2 accumulation in small batch fermentation. When only N2 sparging was applied 184.4 mmol H2/l culture were produced, while combination of N2 sparging and pH control further increased the production to 266.3 mmol H2/l culture. The latter result marked an improvement of 86% and 44% respectively over the non-sparged culture and the sparged culture without pH correction. The H2 production rate improved significantly under sparged conditions reaching the highest value of 41.5 mmol H2/l/h (Fig. 4B). Without pH correction the H2 production rate decreased sharply after reaching the maximum (39.3 mmol H2/l/h) due to the inhibition caused by the drop of pH in the culture. When the pH was adjusted the H2 production was maintained at fairly high level ranging from 31.6 to 23.1 mmol H2/l/h in the 5 h following the addition of NaOH. The maximum H2 production rate of T. celere obtained during glucose fermentation was at least almost 2-fold higher than previously reported for other thermophilic bacteria, such as Caldicellulosiruptor saccharolyticus (23 mmol H2/l/h on sucrose) [27], Caldicellulosiruptor owensensis (19 mmol H2/l/h on xylose) [17] and T. neapolitana (2.1 mmol H2/l/h on glucose) [11] under N2 sparging. Also the maximum specific H2 production rate of 142.3 mmol H2/h/g (Table 3) observed in
Table 3 e Effect of N2 sparging and pH control on glucose consumption, yields of major fermentation products, specific H2 production rate and biomass in T. celere grown in PBM. Data represents averages of the results of two replicate culture experiments ± standard deviation. No N2 sparging & pH control Glucose degradation Yield H2 Acetate Ethanol Formate Specific H2 production rate CDW Carbon balance Redox balance
(%) (mol/mol glucose)
(mmol/h/g) (g/l culture)
>99.0 2.26 1.27 0.40 0.31 112.3 0.37 0.89 0.89
0.07 0.03 0.01 0.02 10.8 0.01 0.01 0.01
N2 sparging & no pH control
N2 sparging & pH control
68.6 0.12
>99.0
3.07 1.59 0.14 0.33 126.4 0.51 1.07 0.99
3.53 1.65 0.15 0.25 142.3 0.51 1.06 1.01
0.01 0.01 0.01 0.01 26.2 0.02 0.01 0.01
0.01 0.01 0.01 0.03 24.5 0.01 0.01 0.01
5556
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 5 0 e5 5 5 8
T. celere was significantly higher when compared to those of other thermophilic microorganisms: hyperthermophilic archaea Pyrococcus furiosus and Thermococcus kodakaraensis reached respectively 100 mmol H2/h/g on maltose [28] and 59.6 mmol H2/h/g on pyruvate [29], while C. saccharolyticus 30 mmol H2/h/g on glucose [6]. However, it must be noted that the reported results for other thermophiles were observed in larger culture volumes (from 0.6 to 7 l) and whether T. celere can achieve similar performance in the same conditions needs to be further investigated. The end product analysis showed that acetate accumulation and yield were maximized when both N2 sparging and pH correction were applied (Fig. 5A, Table 3), while ethanol production was significantly reduced (Fig. 5B, Table 3). As a consequence, H2 yield increased from 2.26 to 3.53 mol H2/ mol glucose with N2 sparging and pH control which corresponds to 88% of the theoretical maximum (i.e. 4 mol H2/mol glucose) (Table 3). As previously noticed, formate was mainly produced during the first hours of fermentation and under sparged condition its accumulation after the 4th hour was minimal (Fig. 5C). Interestingly, the comparison between the product yields in sparged condition without and with pH control shows that a minimal difference in the acetate yield (1.59 mol acetate/mol glucose vs. 1.65 mol acetate/mol glucose) corresponded to a much noticeable difference in H2 yield (3.07 mol H2/mol glucose vs. 3.53 mol H2/mol glucose) (Table 3). Kinetics analysis seems to suggest that under sparged conditions the disposal of reducing equivalents through H2 production is more prominent in the second half of the growth and therefore the drop of medium pH might have suddenly interrupted the process when the pH was not corrected lowering the H2 yield. This hypothesis is confirmed by the fact that with N2 sparging and pH control the H2 yield during the first 5 h of fermentation reached only 3.07 mol H2/mol glucose due to simultaneous production of formate and ethanol (Table 4). Later on the flux at the pyruvate node was mainly directed to acetate (1.91 mol acetate/mol glucose) and only minimal amounts of formate and ethanol were produced. As a consequence, in this interval of time the H2 yield reached 3.95 mol H2/mol glucose, corresponding to 98.75% of the theoretical yield (Table 4). This increase in H2 yield was observed when the culture entered in an exponential phase characterized by a decreased growth rate (Fig. 4C), in which probably most of the glucose was used for cell maintenance [6]. Similarly, an increase in the H2 yield during the late stage of growth was recently observed in T. neapolitana [11]. On the other hand, a different behavior was described for C. saccharolyticus in which the conversion of glucose to H2 was associated with the exponential phase of growth [7]. These differences in the redirection of electron flow during the microbial growth underline the complexity of the mechanisms regulating hydrogen production in different microorganisms and the necessity to thoroughly investigate their metabolism in order to improve the hydrogen production. The ability to yield almost stoichiometric conversion of glucose to H2 in the second half of the growth proves that T. celere is able to couple the complete reoxidation of both reduced ferredoxin and NADH with H2 production. However, the set of hydrogenases that catalyze H2 synthesis (either
a combination of ferredoxin- and NADH-dependent hydrogenases or a bifurcating hydrogenase) and the exact mechanism at molecular level involved in regulation of the metabolic shift from H2 to ethanol are not yet known in details. As shown by the H2 yields obtained with and without N2 sparging (Table 3), H2 concentration in the liquid and gaseous phases surely plays an important role, although
Fig. 5 e Time course of accumulation of acetate (A), ethanol (B) and formate (C) in T. celere grown in PBM without N2 sparging and with pH control (C), with N2 sparging and without pH control (-), and with N2 sparging and with pH control (:).When controlled, pH was adjusted to the initial value (8.8e9) at the 4th hour by NaOH addition. Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
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 5 0 e5 5 5 8
Table 4 e Yields of major fermentation products of T. celere at different growth phases under N2 sparging and pH control. Data represents averages of the results of two replicate culture experiments ± standard deviation. Growth phase (h) 0e5 5e11
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
3.07 0.08 3.95 0.05
1.44 0.02 1.91 0.02
0.17 0.01 0.07 0.01
0.38 0.02 0.03 0.01
electron flow redirection during fermentation can be modulated by other factors such as redox level and pool of energy carriers even at low PH2 [7]. Further investigations will aim to elucidate which metabolic and redox conditions can maximize the H2 synthesis in T. celere throughout the entire growth.
4.
Conclusions
The hydrogen production of T. celere in batch conditions is affected by the medium acidification and the H2 concentration in the system. When frequent N2 sparging was applied for H2 removal, together with pH control, the H2 yield was remarkably enhanced from 2.26 to 3.53 mol H2/mol glucose and the maximum H2 production rate and specific H2 production rate reached 41.5 mmol H2/l/h and 142.3 mmol H2/h/g, respectively. By far this is the highest H2 production rate observed in thermophilic species. Interestingly, the H2 yield achieved in the second half of the growth was near to the theoretical limit of 4 mol H2/mol glucose. These results suggest that under proper conditions T. celere is able to produce hydrogen at high yield and production rate. This performance can be achieved in alkalithermophilic anaerobic conditions which provide great selective pressure making T. celere an ideal candidate for biohydrogen production in open (non-sterile) bioprocess system.
Acknowledgements ¨ a¨rimikro The work was financed by the Academy of Finland (A project no.126974, BUSU project no.139830) and by the Maj and Tor Nessling Foundation (grant no. 620110). Dr. Simone Guglielmetti is kindly acknowledged for the valuable discussions and comments on the text.
references
[1] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzym Microb Tech 2006;38:569e82. [2] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29:173e85. [3] Verhaart MRA, Bielen AAM, Van Der Oost J, Stams AJM, Kengen SWM. Hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea: mechanisms for reductant disposal. Environ Technol 2010; 31:993e1003.
5557
[4] Chou CJ, Jenney Jr FE, Michael WW, Adams MWW, Kelly RM. Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels. Metab Eng 2008;10:394e404. [5] Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. Appl Environ Microbiol 2006;73:67e72. [6] de Vrije T, Mars AE, Budde MA, Lai MH, Dijkema C, de Waard P, et al. Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Microbiol Biotechnol 2007;74: 1358e67. [7] Willquist K, Zeidan AA, van Niel EWJ. Physiological characteristics of the extreme thermophile Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell factory. Microb Cell Fact 2010;9:89. [8] van Niel EWJ, Claassen PAM, Stams AJM. Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 2003;81:255e62. [9] Nguyen TAD, Han SJ, Kim JP, Kim MS, Sim SJ. Hydrogen production of the hyperthermophilic eubacterium, Thermotoga neapolitana under N2 sparging condition. Biores Technol 2010;101:38e41. [10] Ciranna A, Santala V, Karp M. Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study. Biores Technol 2011;102:8714e22. [11] d’Ippolito G, Dipasquale L, Vella FM, Romano I, Gambacorta A, Cutignano A, et al. Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana. Int J Hydrogen Energy 2010;35:2290e5. [12] Ngo TA, Kim MS, Sim SJ. High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana. Int J Hydrogen Energy 2011;36:5836e42. [13] Wang J, Wan W. Factors influencing fermentative hydrogen production: a review. Int J Hydrogen Energy 2009;34:799e811. [14] Koskinen PEP, Lay C, Puhakka JA, Lin P, Wu S, Orlygsson J, et al. High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring. Biotechnol Bioeng 2008;4:665e78. [15] Engle M, Li Y, Rainey F, DeBlois S, Mai V, Reichert A, et al. Thermobrachium celere gen. nov., sp. nov., a rapidly growing thermophilic, alkalitolerant, and proteolytic obligate anaerobe. Int J Syst Bacteriol 1996;46:1025e33. [16] Owen WF, Stuckey DC, Healyjr J, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e92. [17] Zeidan AA, van Niel EWJ. A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OLT. Int J Hydrogen Energy 2010;35:1128e37. [18] Irvin RT, MacAlister TJ, Costerton JW. Tris(hydroxymethyl) aminomethane buffer modification of Escherichia coli outer membrane permeability. J Bacteriol 1981;145:1397e403. [19] Desmarais WT, Bienvenue DL, Bzymek KP, Holz RC, Petsko GA, ˚ resolution crystal structure of the Ringe D. The 1.20 A aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition. Structure 2002;10:1063e72. [20] Rydzak T, Levin DB, Cicek N, Sparling R. Growth phasedependant enzyme profile of pyruvate catabolism and endproduct formation in Clostridium thermocellum ATCC 27405. J Biotechnol 2009;140:169e75. [21] Shaw AJ, Jenneyjr F, Adams MWW, Lynd LR. End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum. Enzym Microb Tech 2008;42:453e8. [22] Lin CY, Lay CH. Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic
5558
[23]
[24] [25]
[26]
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 5 0 e5 5 5 8
sewage sludge microflora. Int J Hydrogen Energy 2004;29: 275e81. Wang CH, Lin PJ, Chang JS. Fermentative conversion of sucrose and pineapple waste into hydrogen gas in phosphate-buffered culture seeded with municipal sewage sludge. Process Biochem 2006;41:1353e8. Sawers RG. Formate and its role in hydrogen production in Escherichia coli. Biochem Soc Trans 2005;33:42e6. Thauer RK, Kirschniawy FH, Jungermann KA. Properties and function of the pyruvate formate-lyase reaction in clostridiae. Eur J Biochem 1972;27:282e90. Weidner G, Sawers G. Molecular characterization of the genes encoding pyruvate formate-lyase and its activating
enzyme of Clostridium pasteurianum. J Bacteriol 1996;178: 2440e4. [27] Willquist K, Claassen PAM, Van Niel EWJ. Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus. Int J Hydrogen Energy 2009; 34:4718e26. [28] Schicho RN, Ma K, Adams MW, Kelly RM. Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 1993;175:1823e30. [29] Kanai T, Imanaka H, Nakajima A, Uwamori K, Omori Y, Fukui T, et al. Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. J Biotechnol 2005;116:271e82.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Enhancing biohydrogen production of the alkalithermophile Thermobrachium celere Alessandro Ciranna*, Ville Santala, Matti Karp Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101 Tampere, Finland
article info
abstract
Article history:
In this study the effect of different buffering agents, pH control and N2 sparging on
Received 13 October 2011
biohydrogen production in Thermobrachium celere was investigated in batch cultivations.
Received in revised form
Among the tested buffers, none was able to prevent the medium acidification resulting in
21 November 2011
a premature interruption of the hydrogen production. Controlling the pH helped to sustain
Accepted 13 December 2011
the growth, the complete substrate consumption and the H2 production. However, in these
Available online 21 January 2012
conditions the increase of H2 partial pressure induced a partial metabolic shift towards ethanol production resulting in a decreased H2 yield. Analysis of formate accumulation
Keywords:
during growth suggests that this compound might play a relevant role in the anabolic
Anaerobic bacteria
routes in T. celere. When frequent N2 sparging was applied for H2 removal, together with pH
Hydrogen partial pressure
control, the H2 yield was remarkably enhanced from 2.26 to 3.53 mol H2/mol glucose, and
Dark fermentation
the maximum H2 production rate and specific H2 production rate reached 41.5 mmol H2/l/h
Volatile fatty acids
and 142.3 mmol H2/h/g, respectively. This result suggests that under proper conditions
pH control
T. celere is able to produce hydrogen at high yield and production rate.
Ethanol formation
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The depletion of fossil fuels and the negative impact of their combustion on the environment have led to exploration of alternative sources of energy. Hydrogen is considered an ideal and clean energy carrier for replacing fossil fuels in the future because of its high energy content per weight unit (122 kJ/g) and non-polluting properties [1]. Fermentation by anaerobic bacteria is a promising biological process for converting carbohydrate-rich substrates to hydrogen. Compared to photofermentation it has several advantages such as higher hydrogen production rate and capability to convert organic wastes to a more valuable energy source [2]. Hydrogen can be produced via dark fermentation by several mesophilic and thermophilic anaerobic bacteria. Thermophiles have shown great potential for H2 production
mainly because of thermodynamic advantages that allow to generate a lower variety of by-products and to reach the theoretical yield of 4 mol H2/mol glucose [3]. Moreover, thermophilic biohydrogen production is less sensitive to contamination of H2-consuming methanogens [4]. This poses a clear advantage when thermophiles are used for hydrogen production in an open system. However, the limited volumetric hydrogen productivity in thermophiles is one major drawback for industrial-scale application. In the attempt of optimizing the biohydrogenation process, a negative correlation has been observed between H2 yield and H2 production rate both in mesophiles and thermophiles [5,6]. At high H2 production rates the concentration of H2 in the liquid phase and in the headspace increases rapidly and H2 synthesis becomes thermodynamically unfavorable [7]. As a consequence, the disposal of
* Corresponding author. Tel.: þ358 40 1981 141; fax: þ358 3 3115 2869. E-mail address: [email protected] (A. Ciranna). 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.12.105
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 5 0 e5 5 5 8
accumulated reducing equivalents in the cell is mediated by a metabolic shift towards production of more reduced metabolites, such as lactate, ethanol, acetone, butanol, or alanine [2]. Despite their higher resistance towards high PH2 , thermophiles are still sensitive to the inhibition caused by the accumulation of hydrogen during the anaerobic fermentation [3,8]. Attempts to minimize the effect of hydrogen accumulation in small closed systems (e.g. serum bottles) have been made by either increasing the headspace/culture ratio [9,10] or removing hydrogen by gas sparging techniques [9,11,12]. The mantainence of an optimal culture pH is another important parameter to control for achieving an efficient H2 production [13]. During fermentation the hydrogen evolution is accompanied by accumulation of volatile fatty acids (VFAs) resulting in a drop of pH in the culture. Therefore, buffering agents such as phosphate, carbonate and HEPES are often used to maintain the pH at optimal level. Previous studies have shown that the anaerobic alkalithermophilic species Thermobrachium celere (soon to be reclassified as Caloramator celere (Bharat Patel, Personal communication)) produced hydrogen at high yields both in pure and mixed culture [10,14]. Considering these results, together with the great selective pressure provided by the optimal growth conditions (T ¼ 67 C and pH67 C ¼ 8.2) and the fast growth rate [15], T. celere is a promising strain for biohydrogen production in open (non-sterile) bioprocess system. A recent study showed that in T. celere H2 production is affected by the medium acidification caused by accumulation of VFAs and by the H2 build-up during the fermentation [10]. In batch conditions, without pH adjustments, the pH dropped below 6 which corresponded to a reduction of hydrogen production rate. Despite being able to produce H2 at PH2 as high as 223 kPa, the H2 yield in T. celere was severely influenced by the hydrogen accumulation decreasing from 3.36 to 1.02 mol H2/mol glucose with the decrease of the headspace/culture ratio [10]. In this study the effect of different buffering agents, pH control and N2 sparging was investigated to enhance H2 production during the glucose fermentation of the alkalithermophile T. celere.
autoclaving for 15 min at 121 C. The pH of the medium was adjusted after sterilization to 8.2 at 67 C with sterile and anaerobic 3 M NaOH. The serum bottles were inoculated with 1 ml (5% v/v) of a culture in the exponential phase and incubated at 67 C and 150 rpm for 7e12 h.
2.2.
Materials and methods
2.1.
Medium and culture conditions
T. celere (DSMZ 8682T from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) was grown under a nitrogen atmosphere in a modified version of the ATCC 2072 medium containing (g/l): KH2PO4 1.24; Na2HPO4 5.79; KCl 1; (NH4)2SO4 0.5; NH4Cl 0.5; MgCl2$6H20 0.1; CaCl2$6H20 0.11; Cystein-HCl 0.13; Na2S$9H20 0.13; yeast extract 2; tryptone 2; resazurin (redox indicator) 0.001. After sterilization glucose was added as substrate in concentration of 10 g/l (55 mM) as well as 10 ml/l of vitamin solution and 10 ml/l of trace element solution (ATCC medium No. 2072, American Type Culture Collection). All chemicals used were of analytical grade. The medium was made anaerobic by flushing with N2 for 20 min, dispensed as 20 ml aliquots into 120 ml serum bottles flushed with N2 and then sterilized by
Experimental procedures
All the experiments were performed in 120 ml serum bottles containing 20 ml of medium with 10 g/l of glucose as substrate. The effect of different buffering agents was studied by preparing media containing phosphate buffer, HEPES buffer, carbonate buffer or TriseHCl buffer to a final concentration of 50 mM. In the study the media were referred as PBM (phosphate-buffered medium), HBM (HEPES-buffered medium), CBM (carbonate-buffered medium) and TBM (Tris HCl-buffered medium). PBM was prepared as described in Section 2.1. HBM, CBM and TBM were prepared by omitting KH2PO4 and Na2HPO4 and by adding respectively 1 M solution of HEPES buffer, carbonate buffer and TriseHCl buffer to a final concentration of 50 mM. During the experiment, liquid (0.5 ml) and gas samples (0.3 ml) were taken every hour for the analyses with N2-flushed 1 ml syringes. PBM, HBM and CBM were used to test the effect of pH control during fermentation. In this experiment the pH of liquid samples withdrawn from the culture was measured hourly with a slim probe. When the pH reached the value of 6 or below it was manually adjusted by injecting in the bottles from 0.35 to 0.5 ml (depending on the buffer in the medium) of sterile and anaerobic 3 M NaOH with a N2-flushed 1 ml syringe. After the injection a liquid sample was taken to verify that the pH was in the range of the initial value (8.8e9). To evaluate the effect of N2 sparging on the H2 production, PBM was employed. The headspace was sparged with pure N2 gas (Oy AGA Ab, Espoo, Finland) every hour for 2 min to ensure complete removal of produced gases. Finally, the combined effect of pH control and N2 sparging was studied in PBM as described above. All the experiments were run in duplicate and included negative controls without added substrate.
2.3.
2.
5551
Analytical methods and calculations
Cell concentrations were determined by measuring the absorbance spectrophotometrically at 600 nm with an Ultrospec 500 Pro spectrophotometer (Amersham Biosciences, Munich, Germany). Cell dry weight (CDW) was determined by centrifuging the culture broths. For this, 20 ml aliquots of the culture were transferred into dried, pre-weighed 50 ml Falcon tubes and centrifuged at 5000 g for 15 min. Cell pellets were washed twice with saline solution (9 g/l NaCl) by centrifugation and dried at 70 C to a constant weight. The relation between OD and CDW of T. celere for growth on glucose was found to be CDW (g/l) ¼ 0.208 OD600 þ 0.071 (R2 ¼ 0.99). The pH of the cultures was measured with a pH330i pH meter (WTW, Weilheim, Germany) equipped with a Sentix 41 pH-electrode (WTW, Weilheim, Germany) or a Slimtrode pH-electrode (Hamilton, Bonaduz, Switzerland). For practical reasons the pH of the cultures was measured at room temperature. The amount of H2 and CO2 produced was analyzed according to Owen et al. [16]. The headspace was sampled by
5552
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 5 0 e5 5 5 8
using a gas-tight syringe (0.3 ml injection volume) and its composition analyzed with a GC-2014 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with Porapak N column (80/100 mesh) and a thermal conductivity detector (TCD). N2 was used as carrier gas and the temperatures of injector, column and detector were 110 C, 80 C and 110 C, respectively. Hydrogen production was calculated from the headspace gas sample and the total volume of gas produced at each time interval according to the mass balance equation (Eq. (1)): VH2 ;t ¼ VH2 ;t1 þ CH2 ;t Vg;t Vg;t1 þ VHead ðCH2 ;t CH2 ;t1 Þ
(1)
where VH2 ;t is the cumulative H2 gas volume at time t, VH2 ;t1 is the cumulative H2 gas volume at time t1, Vg,t is the cumulative total gas volume at time t, Vg,t1 is the cumulative total gas volume at time t1, VHead is the headspace volume in serum bottles, CH2 ;t is the H2 percentage of biogas in headspace
Fig. 1 e Time course of growth, sugar consumption, culture pH and metabolite formation by T. celere in PBM (A), HBM (B) and CBM (C). Optical density (;); glucose (C); pH (A); H2 (-); acetate (,); formate (6); ethanol (7). Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
at time t, and CH2 ;t1 is the H2 percentage of biogas in headspace at time t1. VHead was modified taking into account the change of headspace volume upon liquid withdrawal for sampling. Molar H2 and CO2 were calculated using the ideal gas law. Soluble end products (VFAs and ethanol) and glucose were analyzed by high liquid performance chromatography (HPLC) with an LC-20AC prominence liquid chromatograph equipped with an RID-10A refractive index detector, DGU20A5 prominence degasser and a CBM-20A prominence communications bus module (Shimadzu, Kyoto, Japan). The column was a 30-cm Rezex RHM Monosaccharide Hþ (8%) column (Phenomenex, Alleroed, Denmark). 0.01 N H2SO4 was used as mobile phase at a flow rate of 0.600 ml/min. Carbon balances were calculated from the total amount of carbon-containing products formed (in C-mol) and the amount of sugar consumed (in C-mol). Redox (electron) balances were calculated after multiplying the amount of each metabolite and the sugar by the corresponding degree of reduction (in mol electrons per C-mol) [17]. The chemical formula of biomass was assumed to be CH1.8O0.5N0.2. Metabolite yields as well as carbon and electron balances were calculated by subtracting
Fig. 2 e Time course of H2 production rate (A) and effect of change of culture pH during growth on H2 production rate (B) in PBM (-), HBM (C) and CBM (;). Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
5553
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 5 0 e5 5 5 8
Table 1 e Effect of different buffering agents on glucose degradation, yields of major fermentation products and biomass in T. celere. Data represents averages of the results of two replicate culture experiments ± standard deviation. Medium
Glucose degradation (%) 61.9 0.2 73.3 0.1 53.5 0.8
PBM HBM CBM
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
2.84 0.03 2.52 0.06 2.27 0.01
1.42 0.01 1.27 0.02 1.29 0.02
0.19 0.01 0.29 0.01 0.21 0.01
0.33 0.01 0.21 0.01 0.62 0.03
the background metabolite production in control cultivations (i.e. cultivation without the substrate) and the precultures’ carryover metabolites from the results.
3.
Results and discussion
3.1.
Effect of buffering agents on H2 production
The effect of four buffering agents (phosphate buffer, HEPES, carbonate buffer and TriseHCl) on hydrogen production by T. celere was evaluated during glucose fermentation. Among the tested media, TBM severely repressed the growth and consequently the hydrogen production (data not shown). Despite its optimal pKa of 8.06 for the growth of T. celere, TriseHCl is a polyamine that can have toxic effect on living cells by interacting with cellular components [18] and by inhibiting enzymatic activities [19]. In PBM and HBM the H2 accumulation ceased after 7 h of growth reaching 112.2 and 116.4 mmol H2/l culture, respectively (Fig. 1A, B), while in CBM 84.6 mmol H2/l culture were produced (Fig. 1C). In CBM H2 accumulation started immediately reaching an H2 production rate of 30.7 mmol H2/l/h after 2 h (Fig. 2A). Also the glucose consumption rate was higher compared to the other media (Fig. 1C). This indicates that carbonate buffer was able to maintain the pH near to the optimal value during the first hours of fermentation. However, a sudden drop in pH occurred between the 1st and the 3rd hour resulting in the inhibition of both growth and H2 production (Fig. 1C). In PBM and HBM the highest H2 production rate observed after 3 h was 26.9 and 30.6 mmol H2/l/h, respectively (Fig. 2A). As for carbonate, both buffering agents were not able to prevent the pH to drop at inhibiting value for T. celere [10] with consequent incomplete glucose consumption (Fig. 1A, B). In order to evaluate the effect of changes in culture pH on H2 evolution, in Fig. 2B the hydrogen production rate was plotted against the culture pH measured during the growth in the
CDW (g/l culture)
Carbon balance
Redox balance
0.44 0.01 0.34 0.01 0.44 0.01
1.00 0.01 0.93 0.01 1.16 0.01
0.93 0.01 0.87 0.01 0.89 0.01
different media (Fig. 1AeC). In T. celere the H2 production rate peaked at different pH depending on the buffer used in the medium. Interestingly, the pH at which the highest H2 production rate was observed correlates well with the pKa values of the different buffers. In fact, in CBM (pKa1 6.35, pKa2 10.25) the H2 production rate maintained high level from pH 7.5 to 5.7, while in HBM (pKa 7.55) and PBM (pKa 7.21) it peaked at 7.1 and 6.5, respectively. This implies that the dynamics of H2 production in T. celere is sensitive to the buffering system employed. For all the tested buffers a sudden decrease in the H2 production rate was observed below pH 6 confirming that acidification below this value results detrimental for the H2 production of T. celere. In all the tested conditions acetate was the most abundant soluble end product (Fig. 1AeC). Acetate yield ranged from 1.42 mol acetate/mol glucose in PBM to 1.27 mol acetate/mol glucose in HBM (Table 1). Formate and ethanol were detected in different amounts depending on the buffer used in the medium. Addition of carbonate buffer in the medium resulted in high production of formate yielding 0.62 mol formate/mol glucose (Table 1). This value was 2- and 3-fold higher than those observed in PBM and HBM, respectively. Members of genera closely related to Thermobrachium do not harbor genes encoding for a formate-hydrogen lyase complex to further convert formate to H2 and CO2 [20,21]. If the same applies to T. celere, the production of formate would divert the electron flow from H2 production decreasing the yield. In fact, H2 yield in CBM was 2.27 mol H2/mol glucose compared with 2.52 mol H2/mol glucose and 2.84 mol H2/mol glucose observed in HBM and PBM where the formate production was lower (Table 1). Also ethanol production negatively affects the H2 yield since it is used by the cells as an alternative pathway to dispose the excess of reducing equivalents generated during fermentation [2]. Ethanol production yield was highest in HBM (0.29 mol ethanol/mol glucose) and nearly similar in PBM and CBM (0.19 and 0.21 mol ethanol/mol glucose, respectively) (Table 1). Under the tested conditions in a non pH-controlled system the excessive medium acidification was the main limiting
Table 2 e Effect of different buffering agents combined with pH control on glucose degradation, H2 accumulation and production rate, yields of major fermentation products and biomass in T. celere. Data represents averages of the results of two replicate culture experiments ± standard deviation. Medium
PBM HBM CBM
H2 Glucose H2 degradation accumulation production (mmol/l rate (mmol/ (%) culture) l/h) > 99.0 > 99.0 > 99.0
142.9 5.7 137.6 0.2 133.9 2.2
26.8 3.4 32.0 0.8 32.7 1.2
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
2.26 0.07 1.27 0.03 0.40 0.01 0.31 0.02 2.28 0.03 1.24 0.04 0.48 0.01 0.16 0.01 2.19 0.09 1.29 0.04 0.47 0.02 0.56 0.01
CDW Carbon (g/l culture) balance
0.37 0.01 0.34 0.01 0.41 0.02
Redox balance
0.89 0.01 0.89 0.01 0.89 0.01 0.90 0.02 1.05 0.02 0.95 0.02
5554
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 5 0 e5 5 5 8
growth factor. In fact, when proper growth occurred none of the buffering agents was able to prevent the pH to reach inhibiting value for T. celere. Consequently, growth and hydrogen production ceased before the complete utilization of the substrate. Phosphate and carbonate are often used in media for biohydrogen production systems both in mixed and pure culture for their nutritional and buffering capacity [6,22]. Increase of phosphate buffer in the medium was found to have a positive effect on H2 production in T. celere up to 50 mM after which the growth was inhibited [10]. In the present study, the results showed that PBM was more effective in buffering the pH in the T. celere’s growing range and consequently allowed higher H2 accumulation and yield, while the use of carbonate buffer resulted in a premature inhibition of the H2 production due to a sharp drop in the pH. The better suitability of phosphate buffer for biohydrogenation process was also reported in other studies [22,23]. Recently, HEPES buffer has been successfully employed to enhance the H2 production from glycerol waste by Thermotoga neapolitana by maintaining the pH near the optimal value [12]. On the contrary, despite the slower acidification and a higher glucose consumption (Fig. 1B), H2 accumulation by T. celere in HBM was similar to the one observed in PBM resulting in a lower H2 yield.
3.2.
in PBM, 137.6 mmol H2/l culture in HBM and 133.9 mmol H2/l culture in CBM (Table 2). However, the H2 yield decreased significantly in PBM and HBM (by 20.4 and 9.5%, respectively) and only slightly in CBM. The increased H2 partial pressure was the most probable cause for the inhibition of H2 production and the consequent decrease of the H2 yield. T. celere partially shifts its metabolism to ethanol production as the H2 accumulates in the system during the fermentation [10]. The same behavior was observed during this study, where the
Effect of pH control on H2 production
The influence of three buffers on H2 production was tested under conditions where pH was not the limiting growth factor. Controlling the pH by adding NaOH was effective to prevent the pH value from decreasing below 6, thus maintaining H2 production by T. celere. Under these conditions, T. celere was able to completely utilize the substrate in all the tested media (Table 2). Cumulative H2 production improved with all the buffering agents reaching 142.9 mmol H2/l culture
Fig. 3 e Correlation between mmax and formate yield in T. celere in PBM without pH control (C), PBM with pH control (B), HBM without pH control (:), HBM with pH control (6), CBM without pH control (-) and CBM with pH control (,). Dashed lines represent the linear regression analysis. Data represents averages of the results of two replicate culture experiments.
Fig. 4 e Time course of H2 accumulation (A), H2 production rate (B) and, optical density (filled symbols) and culture pH (open symbols) (C) in T. celere grown in PBM without N2 sparging and with pH control (C, B), with N2 sparging and without pH control (-, ,), and with N2 sparging and with pH control (:, 6). When controlled, pH was adjusted to the initial value (8.8e9) at the 4th hour by NaOH addition. Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
5555
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 5 0 e5 5 5 8
decrease of H2 yields was accompanied by an increase of ethanol yields up to 2-fold (Table 2). In all media the formate yield did not vary significantly when compared with the non pH-controlled experiment. This is consistent with the fact that the formate production pathway is not involved in the process of reductant disposal and therefore was only minimally affected by the hydrogen partial pressure. Under pH-controlled fermentation the H2 production rate remains substantially unchanged ranging from 26.8 mmol H2/ l/h in PBM to 32.7 mmol H2/l/h in CBM (Table 2). Comparison with the results obtained in the experiment without pH control shows a decrease in biomass formation in PBM (from 0.44 to 0.37 g/l), while no significant change was observed in HBM and CBM. Given the performance and the economical convenience for future large-scale cultivations, phosphate buffer was used in the subsequent experiments.
3.3.
Role of formate during H2 production
During the glucose fermentation T. celere produced significant amount of formate which interfered with the H2 yield. The formate yield resulted particularly high in the CBM (Table 1, Table 2). Such high yield raises the question about the role of formate production during the anaerobic fermentation in T. celere. Formate was mainly synthesized in the first hours of growth during the exponential phase (Fig. 1AeC). In this phase the ratio between acetyl-CoA produced via pyruvate oxidoreductase (POR) and pyruvate formate-lyase (PFL) was estimated to be near 1:1 (data not shown), but at the transition to the stationary phase formate accumulation nearly ceased and further acetyl-CoA accumulation was catalyzed by POR. This suggests that the formation of formic acid might be strictly related to the exponential growth phase. As shown in Fig. 3, regression analysis showed linear correlation between formate yield and maximum specific growth rate observed in different media. In CBM mmax was the highest compared to those observed in other media and so was the formate yield. On the contrary, in HBM mmax was around 0.5 h1 and formate yield resulted reduced by 3-fold. The function of pyruvate formate-lyase in Escherichia coli is to mediate the conversion of pyruvate to formate and acetyl-CoA for ATP synthesis in catabolism [24], while the clostridial
pyruvate-formate lyase has been proposed to catalyze formate synthesis to supply the cells with C1 units in anabolism [25,26]. T. celere is phylogenetically related to clostridia and it could possibly possess a PFL with a similar function. This would be consistent with the higher formate synthesis observed at higher growth rates and with the formate production occurring mainly during the exponential phase when maximal cell anabolic activity is expected. However, further studies are needed in order to assess whether formate synthesis is essential for T. celere or whether formate production pathway could be a possible target for metabolic engineering to improve the H2 yield.
3.4.
Enhanced H2 production by N2 sparging
H2 production by T. celere in batch culture is affected by a feedback inhibition from H2 build-up. In order to prevent excessive accumulation of H2 in the system, N2 sparging was employed with and without pH control. Fig. 4A shows that N2 sparging was an efficient method to improve the H2 accumulation in small batch fermentation. When only N2 sparging was applied 184.4 mmol H2/l culture were produced, while combination of N2 sparging and pH control further increased the production to 266.3 mmol H2/l culture. The latter result marked an improvement of 86% and 44% respectively over the non-sparged culture and the sparged culture without pH correction. The H2 production rate improved significantly under sparged conditions reaching the highest value of 41.5 mmol H2/l/h (Fig. 4B). Without pH correction the H2 production rate decreased sharply after reaching the maximum (39.3 mmol H2/l/h) due to the inhibition caused by the drop of pH in the culture. When the pH was adjusted the H2 production was maintained at fairly high level ranging from 31.6 to 23.1 mmol H2/l/h in the 5 h following the addition of NaOH. The maximum H2 production rate of T. celere obtained during glucose fermentation was at least almost 2-fold higher than previously reported for other thermophilic bacteria, such as Caldicellulosiruptor saccharolyticus (23 mmol H2/l/h on sucrose) [27], Caldicellulosiruptor owensensis (19 mmol H2/l/h on xylose) [17] and T. neapolitana (2.1 mmol H2/l/h on glucose) [11] under N2 sparging. Also the maximum specific H2 production rate of 142.3 mmol H2/h/g (Table 3) observed in
Table 3 e Effect of N2 sparging and pH control on glucose consumption, yields of major fermentation products, specific H2 production rate and biomass in T. celere grown in PBM. Data represents averages of the results of two replicate culture experiments ± standard deviation. No N2 sparging & pH control Glucose degradation Yield H2 Acetate Ethanol Formate Specific H2 production rate CDW Carbon balance Redox balance
(%) (mol/mol glucose)
(mmol/h/g) (g/l culture)
>99.0 2.26 1.27 0.40 0.31 112.3 0.37 0.89 0.89
0.07 0.03 0.01 0.02 10.8 0.01 0.01 0.01
N2 sparging & no pH control
N2 sparging & pH control
68.6 0.12
>99.0
3.07 1.59 0.14 0.33 126.4 0.51 1.07 0.99
3.53 1.65 0.15 0.25 142.3 0.51 1.06 1.01
0.01 0.01 0.01 0.01 26.2 0.02 0.01 0.01
0.01 0.01 0.01 0.03 24.5 0.01 0.01 0.01
5556
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 5 0 e5 5 5 8
T. celere was significantly higher when compared to those of other thermophilic microorganisms: hyperthermophilic archaea Pyrococcus furiosus and Thermococcus kodakaraensis reached respectively 100 mmol H2/h/g on maltose [28] and 59.6 mmol H2/h/g on pyruvate [29], while C. saccharolyticus 30 mmol H2/h/g on glucose [6]. However, it must be noted that the reported results for other thermophiles were observed in larger culture volumes (from 0.6 to 7 l) and whether T. celere can achieve similar performance in the same conditions needs to be further investigated. The end product analysis showed that acetate accumulation and yield were maximized when both N2 sparging and pH correction were applied (Fig. 5A, Table 3), while ethanol production was significantly reduced (Fig. 5B, Table 3). As a consequence, H2 yield increased from 2.26 to 3.53 mol H2/ mol glucose with N2 sparging and pH control which corresponds to 88% of the theoretical maximum (i.e. 4 mol H2/mol glucose) (Table 3). As previously noticed, formate was mainly produced during the first hours of fermentation and under sparged condition its accumulation after the 4th hour was minimal (Fig. 5C). Interestingly, the comparison between the product yields in sparged condition without and with pH control shows that a minimal difference in the acetate yield (1.59 mol acetate/mol glucose vs. 1.65 mol acetate/mol glucose) corresponded to a much noticeable difference in H2 yield (3.07 mol H2/mol glucose vs. 3.53 mol H2/mol glucose) (Table 3). Kinetics analysis seems to suggest that under sparged conditions the disposal of reducing equivalents through H2 production is more prominent in the second half of the growth and therefore the drop of medium pH might have suddenly interrupted the process when the pH was not corrected lowering the H2 yield. This hypothesis is confirmed by the fact that with N2 sparging and pH control the H2 yield during the first 5 h of fermentation reached only 3.07 mol H2/mol glucose due to simultaneous production of formate and ethanol (Table 4). Later on the flux at the pyruvate node was mainly directed to acetate (1.91 mol acetate/mol glucose) and only minimal amounts of formate and ethanol were produced. As a consequence, in this interval of time the H2 yield reached 3.95 mol H2/mol glucose, corresponding to 98.75% of the theoretical yield (Table 4). This increase in H2 yield was observed when the culture entered in an exponential phase characterized by a decreased growth rate (Fig. 4C), in which probably most of the glucose was used for cell maintenance [6]. Similarly, an increase in the H2 yield during the late stage of growth was recently observed in T. neapolitana [11]. On the other hand, a different behavior was described for C. saccharolyticus in which the conversion of glucose to H2 was associated with the exponential phase of growth [7]. These differences in the redirection of electron flow during the microbial growth underline the complexity of the mechanisms regulating hydrogen production in different microorganisms and the necessity to thoroughly investigate their metabolism in order to improve the hydrogen production. The ability to yield almost stoichiometric conversion of glucose to H2 in the second half of the growth proves that T. celere is able to couple the complete reoxidation of both reduced ferredoxin and NADH with H2 production. However, the set of hydrogenases that catalyze H2 synthesis (either
a combination of ferredoxin- and NADH-dependent hydrogenases or a bifurcating hydrogenase) and the exact mechanism at molecular level involved in regulation of the metabolic shift from H2 to ethanol are not yet known in details. As shown by the H2 yields obtained with and without N2 sparging (Table 3), H2 concentration in the liquid and gaseous phases surely plays an important role, although
Fig. 5 e Time course of accumulation of acetate (A), ethanol (B) and formate (C) in T. celere grown in PBM without N2 sparging and with pH control (C), with N2 sparging and without pH control (-), and with N2 sparging and with pH control (:).When controlled, pH was adjusted to the initial value (8.8e9) at the 4th hour by NaOH addition. Data represents averages of the results of two replicate culture experiments. Standard deviations are shown as error bars. In some cases the error bars are smaller than the symbols.
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 5 0 e5 5 5 8
Table 4 e Yields of major fermentation products of T. celere at different growth phases under N2 sparging and pH control. Data represents averages of the results of two replicate culture experiments ± standard deviation. Growth phase (h) 0e5 5e11
Yield (mol/mol glucose) H2
Acetate
Ethanol
Formate
3.07 0.08 3.95 0.05
1.44 0.02 1.91 0.02
0.17 0.01 0.07 0.01
0.38 0.02 0.03 0.01
electron flow redirection during fermentation can be modulated by other factors such as redox level and pool of energy carriers even at low PH2 [7]. Further investigations will aim to elucidate which metabolic and redox conditions can maximize the H2 synthesis in T. celere throughout the entire growth.
4.
Conclusions
The hydrogen production of T. celere in batch conditions is affected by the medium acidification and the H2 concentration in the system. When frequent N2 sparging was applied for H2 removal, together with pH control, the H2 yield was remarkably enhanced from 2.26 to 3.53 mol H2/mol glucose and the maximum H2 production rate and specific H2 production rate reached 41.5 mmol H2/l/h and 142.3 mmol H2/h/g, respectively. By far this is the highest H2 production rate observed in thermophilic species. Interestingly, the H2 yield achieved in the second half of the growth was near to the theoretical limit of 4 mol H2/mol glucose. These results suggest that under proper conditions T. celere is able to produce hydrogen at high yield and production rate. This performance can be achieved in alkalithermophilic anaerobic conditions which provide great selective pressure making T. celere an ideal candidate for biohydrogen production in open (non-sterile) bioprocess system.
Acknowledgements ¨ a¨rimikro The work was financed by the Academy of Finland (A project no.126974, BUSU project no.139830) and by the Maj and Tor Nessling Foundation (grant no. 620110). Dr. Simone Guglielmetti is kindly acknowledged for the valuable discussions and comments on the text.
references
[1] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzym Microb Tech 2006;38:569e82. [2] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29:173e85. [3] Verhaart MRA, Bielen AAM, Van Der Oost J, Stams AJM, Kengen SWM. Hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea: mechanisms for reductant disposal. Environ Technol 2010; 31:993e1003.
5557
[4] Chou CJ, Jenney Jr FE, Michael WW, Adams MWW, Kelly RM. Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels. Metab Eng 2008;10:394e404. [5] Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains. Appl Environ Microbiol 2006;73:67e72. [6] de Vrije T, Mars AE, Budde MA, Lai MH, Dijkema C, de Waard P, et al. Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Microbiol Biotechnol 2007;74: 1358e67. [7] Willquist K, Zeidan AA, van Niel EWJ. Physiological characteristics of the extreme thermophile Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell factory. Microb Cell Fact 2010;9:89. [8] van Niel EWJ, Claassen PAM, Stams AJM. Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus. Biotechnol Bioeng 2003;81:255e62. [9] Nguyen TAD, Han SJ, Kim JP, Kim MS, Sim SJ. Hydrogen production of the hyperthermophilic eubacterium, Thermotoga neapolitana under N2 sparging condition. Biores Technol 2010;101:38e41. [10] Ciranna A, Santala V, Karp M. Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study. Biores Technol 2011;102:8714e22. [11] d’Ippolito G, Dipasquale L, Vella FM, Romano I, Gambacorta A, Cutignano A, et al. Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana. Int J Hydrogen Energy 2010;35:2290e5. [12] Ngo TA, Kim MS, Sim SJ. High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana. Int J Hydrogen Energy 2011;36:5836e42. [13] Wang J, Wan W. Factors influencing fermentative hydrogen production: a review. Int J Hydrogen Energy 2009;34:799e811. [14] Koskinen PEP, Lay C, Puhakka JA, Lin P, Wu S, Orlygsson J, et al. High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring. Biotechnol Bioeng 2008;4:665e78. [15] Engle M, Li Y, Rainey F, DeBlois S, Mai V, Reichert A, et al. Thermobrachium celere gen. nov., sp. nov., a rapidly growing thermophilic, alkalitolerant, and proteolytic obligate anaerobe. Int J Syst Bacteriol 1996;46:1025e33. [16] Owen WF, Stuckey DC, Healyjr J, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e92. [17] Zeidan AA, van Niel EWJ. A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OLT. Int J Hydrogen Energy 2010;35:1128e37. [18] Irvin RT, MacAlister TJ, Costerton JW. Tris(hydroxymethyl) aminomethane buffer modification of Escherichia coli outer membrane permeability. J Bacteriol 1981;145:1397e403. [19] Desmarais WT, Bienvenue DL, Bzymek KP, Holz RC, Petsko GA, ˚ resolution crystal structure of the Ringe D. The 1.20 A aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition. Structure 2002;10:1063e72. [20] Rydzak T, Levin DB, Cicek N, Sparling R. Growth phasedependant enzyme profile of pyruvate catabolism and endproduct formation in Clostridium thermocellum ATCC 27405. J Biotechnol 2009;140:169e75. [21] Shaw AJ, Jenneyjr F, Adams MWW, Lynd LR. End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum. Enzym Microb Tech 2008;42:453e8. [22] Lin CY, Lay CH. Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic
5558
[23]
[24] [25]
[26]
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 5 0 e5 5 5 8
sewage sludge microflora. Int J Hydrogen Energy 2004;29: 275e81. Wang CH, Lin PJ, Chang JS. Fermentative conversion of sucrose and pineapple waste into hydrogen gas in phosphate-buffered culture seeded with municipal sewage sludge. Process Biochem 2006;41:1353e8. Sawers RG. Formate and its role in hydrogen production in Escherichia coli. Biochem Soc Trans 2005;33:42e6. Thauer RK, Kirschniawy FH, Jungermann KA. Properties and function of the pyruvate formate-lyase reaction in clostridiae. Eur J Biochem 1972;27:282e90. Weidner G, Sawers G. Molecular characterization of the genes encoding pyruvate formate-lyase and its activating
enzyme of Clostridium pasteurianum. J Bacteriol 1996;178: 2440e4. [27] Willquist K, Claassen PAM, Van Niel EWJ. Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus. Int J Hydrogen Energy 2009; 34:4718e26. [28] Schicho RN, Ma K, Adams MW, Kelly RM. Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 1993;175:1823e30. [29] Kanai T, Imanaka H, Nakajima A, Uwamori K, Omori Y, Fukui T, et al. Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. J Biotechnol 2005;116:271e82.