Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.
© Masset et al.; licensee BioMed Central Ltd. 2012
Received: 24 November 2011
Accepted: 22 May 2012
Published: 22 May 2012
Pure bacterial strains give better yields when producing H2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.
Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H2 production (sequence 1); and the H2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H2/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H2 at higher rates than the pure Clostridium cultures, without negatively affecting the H2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H2. In the second part of the study the co-culture (3) was used to produce H2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation.
The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H2 using glucose and starch as carbon sources. The artificial co-cultures produced H2 at higher rates than the pure strains, while the H2 yields were only slightly affected.
KeywordsClostridium spp. Fermentative H2 production qPCR
qPCR reaction efficiency
Glyceraldehyde 3-phosphate dehydrogenase
Quantitative real–time PCR
Sequencing batch reactor
Volatile fatty acids.
Hydrogen is regarded as a future energy vector for transportation and stationary power. However, its current production is heavily dependent on fossil fuels, yielding globally 40% of H2 from steam reforming of methane and another 48% from crude oil and coal. By contrast, biohydrogen production offers a sustainable alternative and by utilizing renewable carbon resources can be considered as a CO2 offset .
Hydrogen can be produced biologically by four different processes: direct and indirect biophotolysis, photofermentation and dark fermentation. Fermentative hydrogen production not only provides higher gas production rates compared to photosynthetic processes, but is also light independent and can utilize various carbon sources, including wastewaters .
Many agricultural and food-industry wastes and wastewaters contain large quantities of carbohydrates and proteins. However, their complex nature can adversely affect their digestibility for the fermentative organism(s) present in a H2 producing bioreactor . Naturally occurring microbial consortia derived from different sources, such as compost, anaerobic sludge or soil, are frequently used for H2 production. On one hand due to the metabolic flexibility, provided by the different members of the community, they are vital to the waste treatment systems, as they can tolerate different substrates and environmental conditions . On the other hand the same metabolic flexibility can undermine the H2 production efficiency by initiating and sustaining other metabolic pathways, e.g. increased lactate formation by lactic acid bacteria . Additionally it has been shown that within a complex and poorly defined population, bacterial successions can occur even within a batch culture, and that this can significantly impact H2 production [6, 7].
In comparison to the use of natural, mixed bacterial populations, pure strain cultures were shown to produce H2 at higher yields . However, the main drawback of the use of pure cultures is the necessity to maintain the fermentations under sterile conditions. In fact the autochthonous bacteria present in the waste materials can easily overgrow the pure strain used to produce H2 in a bioreactor. Therefore, the use of artificial microbial co-cultures and consortia, composed of pre-defined and well characterized species, has attracted particular interest in the developing biohydrogen industry. Co-cultures can perform complex functions, such as simultaneous pentose or hexose consumption, which generally cannot be performed by a single species . Being potentially more robust to changes in environmental conditions, microbial co-cultures can resist periods of nutrient limitation better; what is typically combined with the exchange of metabolites between the different bacteria . Another feature is biomass concentration with the formation of aggregates, which occurs more readily in co-cultures than with a single strain culture . However, until now, the creation of a stable artificial community composed of three or more members has been problematic due to the diverse rates of growth of the different members and the subsequent imbalance between the consumption and production of metabolites . To date, a stable coexistence of community members over a long period of time has only been reported for a few H2-producing artificial consortia and co-cultures [13–16].
Therefore this study has two goals. The first is to characterize H2 production by four Clostridium strains (C. beijerinckii DSM 1820, C. butyricum CWBI 1009, C. pasteurianum DSM 525 and C. felsineum DSM 794) grown as pure cultures with different substrates. The second goal is to develop stable H2 producing co-cultures composed of two different Clostridium spp. A simple three-step approach, consisting of three fermentation sequences, was used to first assess the optimal pH for H2 production for pure strains and for co-cultures with glucose (sequence 1). Then the potential of the pure cultures and co-cultures to stably produce H2 using different carbon sources was evaluated by performing glucose (sequence 2) and starch fermentations (sequence 3) at a controlled optimal pH. In order to assess the metabolic interactions between the different strains, the metabolites produced were monitored by HPLC. The potential of the co-culture, composed of C. butyricum and C. pasteurianum (co-culture 3), to grow consistently and produce H2 was also evaluated during a 13-day glucose fermentation in a sequencing batch bioreactor (SBR). The dynamics of the two strains was monitored throughout the fermentation using a previously optimized qPCR assay based on quantification of the recA and gyrA marker genes .
Results and Discussion
In the first part of this study a strategy of three-culture sequences was developed to characterize hydrogen production by four pure Clostridium strains (C. beijerinckii DSM 1820, C. butyricum CWBI 1009, C. pasteurianum DSM 525 and C. felsineum DSM 794), and three co-cultures composed of two closely related clostridia; i.e. co-culture (1) with C. pasteurianum and C. felsineum, co-culture (2) with C. butyricum and C. felsineum, and co-culture (3) with C. pasteurianum and C. butyricum. Two strains, namely C. butyricum and C. beijerinckii, have been widely investigated for H2-production in bioreactors and they were shown to ferment a variety of different carbon sources, including pure sugars (e.g. glucose, starch, sucrose, galactose [18–22] palm oil  yeast wastes  or sugar cane juice . C. pasteurianum has been often detected as a dominant strain in sludge-derived H2-producing mixed cultures . By contrast the application of C. felsineum for H2 production has not yet been reported.
The hydrogen production potential of the pure strains and co-cultures was evaluated in the same 20 L SBRs. The first sequence was conducted without pH control using glucose as a carbon source. By monitoring the hydrogen production rate for each pure strain and co-culture, the optimal pH for H2 production was determined. The second and the third sequences were carried out at optimal pH values, using either glucose or starch as fermentative substrates.
Additionally in the second part of the study, co-culture (3) with C. pasteurianum and C. butyricum, was used to produce hydrogen over a longer period (13 days) in a SBR, maintained at fixed pH using glucose as the carbon source.
Single batch fermentation to evaluate the optimal pH for H2 production (Sequence 1)
Optimal pH ranges for H 2 production by pure Clostridium strains and co-cultures determined during one-stage glucose fermentation (first sequence) at unregulated pH
C. beijerinckii DSM 1820
C. butyricum CWBI 1009
C. pasteurianum DSM 525
C. felsineum DSM 794
The optimal pH values defined for C. butyricum C. felsineum and C. pasteurianum were similar, on average 5.35 ±0.15. This pH value is similar to the optimal pH of 5.5 observed for the best H2 production by C. butyricum EB6 . By contrast the optimal pH for C. beijerinckii was pH 6.7 (Table 1). Furthermore, its H2 production was inhibited at pH 5.2, which overlaps with the optimal pH values for the three other Clostridium strains selected. Therefore, due to its very different pH requirements, C. beijerinckii was not used in any of the three two-strain co-cultures. Additionally, this species has been shown to inhibit the growth of other bacteria, including the Clostridium species analysed in this study (data not shown), probably by producing bacteriocin and circularin A .
To determine the optimal pH for each co-culture, the same single batch fermentation approach was used. For all three co-cultures the optimal pH for H2 production with glucose was 5.3 (Table 1). The inhibiting pH values for co-cultures (1) with C. pasteurianum and C. felsineum and (2) with C. butyricum and C. felsineum were found to be 4.7 and 4.6, respectively. However for co-culture (3) with C. butyricum and C. pasteurianum, the inhibiting pH of 5.0 was very close to the optimal one (5.3). Therefore, use of this co-culture in large bioreactors, in which significant pH variations occur, would require care to avoid inhibition of H2 production due to a small shift in pH . A narrow optimal pH range for H2 production had already been suggested in previous studies, which reported that even small pH variations caused a shift in the metabolic pathways leading to a reduction in the final H2 yield [21, 30].
Metabolites and H2 production during glucose batch fermentations with unregulated pH (Sequence 1)
Fermentation end products (molar concentrations) obtained during glucose fermentation at unregulated pH (first sequence)
H2yield (mol H2/mol glucose)
Biogas production rate (L biogas/ h)a
C. beijerinckii DSM 1820
C. butyricum CWBI 1009
C. pasteurianum DSM 525
C. felsineum DSM 794
Metabolites and H2 production during glucose batch fermentations at fixed optimal pH (Sequence 2)
Fermentation end products (molar concentrations) obtained during glucose fermentation performed at fixed optimal pH values (second sequence)
H2yield (mol H2/ mol glucose)
Biogas production rate (L biogas/ h)a
C. beijerinckii DSM 1820
C. butyricum CWBI 1009
C. pasteurianum DSM 525
C. felsineum DSM 794
Maintaining the culture at fixed optimal pH gave rise to a 10% increase in the H2 yield compared to unregulated pH conditions for co-culture (1) with C. pasteurianum and C. felsineum, and provided an increase of as much as 60% for co-cultures (2) and (3) (C. butyricum & C. felsineum and C. butyricum & C. pasteurianum, respectively) (Table 3). The 2.12 mol H2/mol glucose yield obtained by co-culture 3 was comparable to the results obtained for C. butyricum grown separately, but the co-culture 3 biogas production rate was 1.6 and 3.5 fold higher than that obtained with the pure C. pasteurianum and C. butyricum strains, respectively (Table 3).
Compared to the previous sequence, the composition of the fermentation end products changed radically for both the pure strains and for the three co-cultures, yielding on average a higher proportion of acetate and butyrate in the total soluble metabolites, when the pH was fixed at the optimal value (Table 3). For C. beijerinckii, C. butyricum and C. pasteurianum the production of lactate decreased significantly, while for C. felsineum it increased. The increased lactate production, observed for co-culture (1) with C. pasteurianum and C. felsineum during the previous batch glucose fermentation with unregulated pH, was no longer present and the three co-cultures produced similar amounts of lactate (4.49 ±0.23 mM) during this fermentation sequence. A decrease in formate production, ranging from 8 to 18 fold, was observed at fixed pH for co-cultures (3) with C. butyricum and C. pasteurianum, and (2) with C. butyricum and C. felsineum. Additionally, the increased H2 yield for the three co-cultures suggested that pyruvate was mainly metabolized to acetyl-CoA via PFOR (Figure 2).
Metabolites and H2 production during starch batch fermentations at fixed optimal pH (Sequence 3)
Simple sugars, e.g. glucose or lactose, are the preferred carbon sources for H2 producing fermentative bacteria, due to their fast biodegradability [20, 22, 41]. However, pure carbohydrates are far too expensive for use as feedstock and renewable waste material is therefore an attractive potential alternative for the sustainable production of hydrogen [3, 42]. Consequently, during the third fermentation sequence with removal/addition of 3 L of the MDT medium, the ability of the pure Clostridium strains and the co-cultures to grow on different carbon sources was further assessed using starch (5 g/ L), which is present at high levels in many food industry and agricultural wastes.
Pure C. butyricum and C. pasteurianum cultures consumed respectively 62.8% and 57.4% of the starch initially added to the medium (after 46.5 h ±1). These results seem to contradict the findings of Chen et al., where the authors had to employ a starch pre-treatment step in order to accelerate hydrogen production . In their work C. pasteurianum (different indigenous isolates from Taiwan) was reported to be unable to use starch, neither for the cell growth nor H2 production.
Of the other two pure strains, C. beijerinckii consumed merely 13.4% of the starch after 71 h of fermentation, and C. felsineum was unable to use starch as a fermentative carbon source. By contrast with these results, another strain of C. beijerinckii, namely C. beijerinckii AM21B isolated from termites, was reported to efficiently consume starch with concomitant production of H2.
By contrast with the pure strain cultures, starch was shown to be easily degraded by the three co-cultures and was entirely consumed after 47.5 h ±0.5 by co-culture (1) with C. pasteurianum and C. felsineum and by co-culture (3) with C. butyricum and C. pasteurianum, without the need for any pre-treatment such as acid or enzymatic hydrolysis. Co-culture (2) with C. butyricum and C. felsineum required 70 h of fermentation to consume all the starch, probably due to a lower hydrolysis rate.
Fermentation end products (molar concentrations) obtained during the starch fermentation performed at fixed optimal pH values (third sequence)
H2yield (mol H2/mol hexose)
Biogas production rate (L biogas/ h) a
C. beijerinckii DSM 1820
C. butyricum CWBI 1009
C. pasteurianum DSM525
C. felsineum DSM 794
E. coli and other Enterobacteriaceae can produce hydrogen from formate by the action of an enzymatic complex called formate:hydrogen lyase (FHL). However, none of the Clostridium strains studied has been shown to possess this type of enzyme. Surprisingly, another putative trimeric [FeFe] hydrogenase complex has been found in the genome of C. beijerinckii and it seems to be associated with a formate dehydrogenase-like protein . This could explain the ability of C. beijerinckii to utilise formate for H2 production. However the enzyme activity has never been studied in this strain. A similar enzymatic complex was reported for Eubacterium acidaminophilum, and indeed the bacterium was shown to couple formate oxidation to H2 production .
In case of the co-cultures, the highest H2 yield of 2.32 mol H2/ mol hexose was obtained for co-culture (3) with C. butyricum and C. pasteurianum. Interestingly, the yield obtained for C. butyricum grown as a pure strain culture on starch was higher (2.91 mol H2/ mol hexose), but the biogas production rate tripled when the strain was combined with C. pasteurianum in a co-culture (Table 4). A comparison of the H2 yields obtained during glucose and starch sequences showed that, for starch, the H2 yields were higher for co-culture (1) with C. pasteurianum and C. felsineum, and co-culture (3) with C. butyricum and C. pasteurianum, probably due to the lower biogas production rates and the resulting lower H2 partial pressure, as was shown for the pure strains. Indeed, the biogas production rates during the starch batch fermentations ranged from 0.95 (co-culture 1) to 1.3 (co-culture 2) L biogas/ h, and except for the co-culture (2) they were 30% lower compared to the previous glucose batch fermentation.
Recently Quéméneur et al. showed, by performing a series of H2-producing batch fermentations, that both the carbohydrate chain length and alpha- or beta-linkage have an impact on H2 production . In their work the H2 yields decreased from 1.84 (fructose fermentation) to 1.38 mol H2/ mol hexose (maltotriose fermentation) with the increasing chain length of the substrate. By contrast, the three co-cultures used in our study were characterized by equal or even higher H2 yields when grown on a polysaccharide (starch) compared to a monosaccharide (glucose). However different experimental conditions were applied in the two studies and the use of 500 ml glass bottles without pH control versus 20 L batch reactors operating at fixed pH may explain the differences in findings.
From the fermentation end-product analysis several observations can be made about the different strains’ and co-cultures’ metabolisms during the starch batch fermentation. A radical decrease in acetate production by a factor of 2.7 and 4.3 was found for pure strains of C. butyricum and C. pasteurianum, respectively (Table 4). Butyrate production was also shown to be lower for C. beijerinckii and C. butyricum, but it did not change significantly for C. pasteurianum. By contrast for the co-cultures fermenting starch a 1.3- and 2.1-fold increase in butyrate production was observed for co-cultures (2) with C. butyricum and C. felsineum, and (3) with C. butyricum and C. pasteurianum, compared to the previous batch sequence with glucose. At the same time the amount of acetate produced from starch decreased 2.5-fold and as much as 50-fold for co-cultures (2) with C. butyricum and C. felsineum and (1) with C. pasteurianum and C. felsineum, respectively. Interestingly the lactate produced during the former batch sequences with glucose was consumed in this step by the pure strains and by the three co-cultures. The highest rates of lactate consumption were observed for co-cultures (1) and (3), suggesting that C. pasteurianum may have been the main lactate consumer. Indeed this strain consumed the highest amounts of lactate during the starch fermentation stage when grown as a pure strain culture (Table 4).
Typical lactate consuming species, e.g. Clostridium propionicum, produces propionic and acetic acids together with CO2 during lactate fermentation, but no H2 is generated via this pathway . However the lactic acid present in a starch containing medium, undergoes a different, not completely understood metabolic pathway, resulting in H2 production together with the formation of butyrate as the major metabolite . Recently, Matsumoto and co-authors proposed a similar pathway, where one mol of acetate reacted with two moles of lactate giving in turn H2, CO2 and butyrate as the main products . Indeed a radical decrease in molar acetate concentration was characteristic for the pure strains and the three co-cultures grown on starch, compared to the previous glucose fermentation stage (Table 4).
Longer-term evaluation of H2 production using co-culture (3) with C. butyricum and C. pasteurianum and a glucose substrate in a SBR at fixed pH 5.3
As shown above, the Clostridium strains analysed in this study, when combined into two-strain artificial co-cultures, proved capable of producing H2 at significantly higher rates without negatively affecting the final H2 yield. Therefore, to further evaluate their H2 producing abilities, co-culture (3) was used as a model culture, since it had been shown to combine the best H2 yield and biogas production rate, during the glucose batch fermentation under controlled pH conditions. The co-culture was grown over a longer period of time (13 days), by extending the operating mode to 7 sequences. The fermentation was carried out at the fixed optimal pH value of 5.3, using glucose as a carbon source.
Although the maximal H2 yield obtained in this experiment represents less than 60% of the maximum theoretical yield that can be obtained by dark fermentation (4 mol H2/ mol glucose), only a few studies have reported higher yields, sometimes using much more sophisticated and expensive systems . For example, employing a two-stage process combining thermophilic starch hydrolysis and dark fermentation, Chen et al. obtained a yield of 2.4 mol H2/mol hexose (reported as 13.2 mmol H2/g glucose) for C. butyricum CGS2 . In another study a H2 yield of 2.79 mol H2/ mol sucrose was reported for an acclimated, mesophilic sewage sludge grown in an anaerobic SBR . Interestingly, when combining C. butyricum in a co-culture with immobilized cells of Rhodopseudomonas faecalis, a H2 yield of 4.134 mol H2/ mol glucose was reported . This indicates that the development of a two stage system, combining an artificial co-culture composed of Clostridium spp. with a photosynthetic H2-producing bacterium, could additionally increase the final H2 yield over the theoretical maximum of 4 mol H2/ mol glucose that can be reached by clostridia.
Species dynamics for co-culture (3) with C. butyricum and C. pasteurianum monitored by qPCR during the 13 days of glucose fermentation in the SBR
A sequenced or a continuous operational mode for H2 production is necessary for its future industrial application . However the behaviour and the genetic composition of a defined artificial co-culture can change over time, thus affecting the final H2 production.
In general, as shown by qPCR, both organisms coexisted in the co-culture and no species was completely displaced by the other, despite the fact that there was only one carbon source. A similar situation was previously observed for the two closely related Caldicellulospiruptor species, combined in a H2 producing co-culture and grown under nutrient limiting conditions . C. saccharolyticus which was in a minority during the batch start-up phase (less than 5% of the total population), overgrew C. kristjanssonii during the glucose and mixed sugar fermentations. Nevertheless none of the species was completely washed out from the co-culture. The occurrence of (complex) interspecies interactions between the microorganisms is hypothesized to be a key factor enhancing their stable co-existence in a population . It has been suggested that under nutrient limitation a minority species can become metabolically active, and that this activity can be crucial for the survival of the whole population . However further studies, including research on the species interactions and the metabolite trading between the bacterial members in a co-culture, are needed to better understand the behaviour of the different strains in a H2-producing artificial community.
Artificial co-cultures composed of different Clostridium species were shown to offer better performance in terms of efficient H2 production than the corresponding pure strain cultures. In contrast to the pure strain cultures, of which only C. butyricum and C. pasteurianum were able to partially hydrolyse starch, the three co-cultures completely consumed the starch without any need for pre-treatment. Surprisingly, all the Clostridium strains and co-cultures studied were shown to efficiently reconsume the lactate present in a starch-containing medium. Additionally, C. beijerinckii appeared to produce H2 by re-oxidizing the previously produced formate. The utilization of lactate and formate for H2 production by different Clostridium spp suggests the existence of novel pathways leading to H2 in this genus.
The hydrogen yields obtained in this study by mesophilic fermentation of glucose and starch were amongst the highest reported to date for Clostridium spp., cultured in a large lab-scale bioreactor (20 L). A comparison of the use of glucose or starch as a fermentation substrate resulted in contrasting data. Grown on starch the pure Clostridium cultures and the co-cultures provided higher yields compared to glucose fermentation. By contrast higher biogas production rates were reported when glucose was used as the carbon source.
Interestingly, co-culture (3), with C. butyricum and C. pasteurianum, produced H2 efficiently during 13 days of glucose fermentation in a SBR. Moreover both species co-existed stably in the co-culture during this time, despite the fact that there was only one carbon source available. Additionally, no species was completely displaced by the other, suggesting that positive interactions between the community members were at least as important as the simple competition for nutrients.
Future studies to improve substrate utilisation and metabolite trading should focus on the utilisation in H2 production of artificial co-cultures with increasing species complexity. In addition, the use of cellulose should be of prime interest since it would ensure the sustainability of the cheap carbon source. Therefore, the addition of a cellulose hydrolysing species, e.g. Clostridium cellulolyticum, to the co-culture should be considered.
Bacterial strains and culture conditions
The type strains of C. beijerinckii DSM 1820, C. pasteurianum DSM 525 and C. felsineum DSM 794 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). C. butyricum CWBI 1009 was previously isolated from a hydrogen-producing sludge . The strains were anaerobically maintained in MDT medium (5 g glucose, 5 g casein peptone, 0.5 g yeast extract, 1.2 g KH2PO4, 5.1 g Na2HPO4, 0.5 g MgSO4 x 7H2O and 0.5 g L-cysteine) as previously described . Before the bioreactor inoculation the strains were individually sub-cultured for 24 h at 30°C under nitrogen atmosphere in 5 L glass bottles filled with MDT medium and equipped with silicone tubing and gas filters for sterile liquid transfer and outlet gas sterilisation. To initiate a co-culture an equal volume (L) of the two respective strain cultures was used.
Bioreactor setup and sampling
All fermentations were carried out in MDT medium in a 20 L stainless steel tank bioreactor (Biolafite) equipped with a shaft with three Rushton impellers (four blades, diameter 100 mm, height 20 mm), 0.2 um gas filter, butyl septum and tubing for temperature regulation, gas inlet, gas outlet and medium removal/addition. After sterilization (120°C, 20 min) glucose or starch (rice starch, BENEO-Remy) were added sterilely and the medium was purged with nitrogen. The medium was continuously stirred at 100 rpm and the pH was maintained automatically at a given value using 3 N KOH (except for the unregulated pH fermentation). Finally 1 L of each (co-) culture was added to give a final working volume of 18 L. For the sequenced-batch mode after an initial growth in batch mode 2 L (3 L) of the medium were removed and the bioreactor was fed with 2 L (3 L) of fresh medium containing sufficient glucose to provide a final concentration of 5 g/ L in the bioreactor. Gas samples from the bioreactor headspace were regularly analysed. Culture samples were withdrawn to monitor the co-culture dynamics and analyse the metabolites. Cells were harvested by centrifugation at 13 000 x g for 1 min and the cell lysates were prepared as previously described . The supernatant was further filtrated through a 0.2 μm cellulose acetate filter (Minisart Sartorius) and analysed for sugar consumption and metabolite formation.
A three-step strategy was developed to characterize the hydrogen production of the pure strains and co-cultures with glucose and starch as alternative fermentative substrates. The first batch fermentation was carried out with unregulated pH and glucose at a final concentration of 5 g/ L. The initial pH was set at 7.3 and was allowed to decrease progressively as acidic metabolites accumulated. The optimal pH was defined as the value corresponding to the maximum biogas production rate. During the next sequence 3 L of the medium were replaced with the same volume of a fresh MDT medium containing glucose at a final concentration of 5 g/L. During this stage the pH was fixed at the optimal value by the automatic addition of sterile 1.5 N KOH. During the third sequence 3 L of the medium were replaced with the same volume of a fresh MDT medium containing starch at a final concentration of 5 g hexose/L. The pH was controlled during this stage. The determination of the optimal pH value by a multi-stage approach was performed as previously described .
The flow rate of the biogas produced in the bioreactor headspace was continuously measured with a wet flow meter (Ritter Gas meter Drum type TG01) connected to a computer running Rigamo data acquisition software (V1.30-K1). The proportion of hydrogen gas was determined using a gas chromatograph (GC) (Hewlett-Packard 5890 Series II) fitted with a thermal conductivity detector (TCD) and a 30 m x 0.32 mm GAS PRO GSC capillary column (Altech) in series with a 20 m × 0.25 mm CarboPLOT P7 column (Chrompak). The temperatures of the injection, TCD chambers and the oven were maintained at 90°C, 110°C and 55°C respectively. Nitrogen was used as the carrier gas in the column at a flow rate of 20 mL min-1. The HPLC analysis was carried out using an Agilent 1110 series (HP Chemstation software) as previously described .
qPCR specific primers used in this study
Primer name and sequence (5′ → 3′)
C. butyricum CWBI1009
The research was funded by an ARC project (Action de Recherche Concertée, ARC-07/12-04), granted by a French Community of Belgium. AW is a Research Associate of the FRS-FNRS. JM was the recipient of a FRIA fellowship (Fonds de la Communauté Française de Belgique pour la Formation à la Recherche dans l′Industrie et l′Agriculture). We would like to thank Laurent Beckers from the University of Liège for a critical review of the manuscript and Ian Hamilton for English correction.
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