Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Pilot-scale hydrogen fermentation system start-up performance

A high-rate hydrogen production process able to produce H₂ at a maximum rate of 15 L/L/h was successfully developed by the Feng Chia University (FCU) biohydrogen research team. This highly efficient hydrogen fermentation system includes a 400 L pilot-scale system constructed for determining scale-up operation parameters for commercializing the bioH₂ production technology. The pilot-scale system is composed of a feedstock tank, mixing system, fermentor, gas/liquid separator and automatic control system. The fermentor is fed with sucrose (20 g COD/L) and operated at 35 °C. A batch strategy is used for system start-up. The fermentor was first operated in a batch mode for two days and then switched to a continuous-feeding mode (HRT 12 h) for one month. During the continuous operation, pH notably affected H₂ production efficiency and bacterial community. For the first 14-day operation, the H₂ production rate increased from 0.017 to 0.256 L/L/h with a pH variation from 5.0 to 7.0. The DGGE results indicate the presence of two Clostridium species (namely, Clostridium butyricum and Clostridium pasteurianum) in the fermenter. Stable hydrogen production rate was obtained at pH 5.5–6.0 when C. pasteurianum became dominant in the mixed culture.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

Quantitative analysis of microorganism composition in a pilot-scale fermentative biohydrogen production system

Five specific real-time polymerase chain reaction primers targeting the 16S rRNA gene of Clostridium spp., Klebsiella spp., Streptococcus spp., Pseudomonas spp., and Bifidobacterium spp., and two primer sets targeting the hydrogenase genes of hydrogen-producing Clostridium pasteurianum and Clostridium butyricum were designed and tested in the present study to quantify the microorganisms in fermentative biohydrogen production systems. The former primers revealed the composition of all coexisting microorganisms, whereas the latter ones provided information on which clostridia were responsible for the biohydrogen production in various operational conditions. When sucrose was selected as the feeding substrate, the biogas production and hydrogen production rate (HPR) of the system increased as the percentage of Clostridium spp. (especially C. pasteurianum) increased. The cell count of C. pasteurianum increased up to 90% of the total cell population when the system approached its maximum hydrogen production. C. butyricum was identified as the main hydrogen-producing clostridium in the condensed molasses soluble wastewater feeding system, but there was no significant correlation between system HPR and C. butyricum cell count. At the same time, other microorganisms, such as Bifidobacterium spp. and Klebsiella spp., were the predominant ones throughout the whole operation and possibly caused the unsatisfied biohydrogen production. The composition of microorganisms is the principal factor affecting biohydrogen production. Aside from the well-known hydrogen-producing Clostridium spp., several other microorganisms not only coexist but can also significantly affect system performance. The monitoring method established in the present study provides a fast quantification procedure to help operators understand how the system works and therefore quickly respond in operations.     

Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria

Glycerol is an inevitable by-product from biodiesel synthesis process and could be a promising feedstock for fermentative hydrogen production. In this study, the feasibility of using crude glycerol from biodiesel industry for biohydrogen production was evaluated using seven isolated hydrogen-producing bacterial strains (Clostridium butyricum, Clostridium pasteurianum, and Klebsiella sp.). Among the strains examined, C. pasteurianum CH4 exhibited the best biohydrogen-producing performance under the optimal conditions of: temperature, 35 °C; initial pH, 7.0; agitation rate, 200 rpm; glycerol concentration, 10 g/l. When using pure glycerol as carbon source for continuous hydrogen fermentation, the average H₂ production rate and H₂ yield were 103.1 ± 8.1 ml/h/l and 0.50 ± 0.02 mol H₂/mol glycerol, respectively. In contrast, when using crude glycerol as the carbon source, the H₂ production rate and H₂ yield was improved to 166.0 ± 8.7 ml/h/l and 0.77 ± 0.05 mol H₂/mol glycerol, respectively. This work demonstrated the high potential of using biodiesel by-product, glycerol, for cost-effective biohydrogen production.     

Bioproduction of hydrogen by simultaneous saccharification and fermentation of cassava starch with 2-deoxyglucose-resistant mutant strains of Clostridium tyrobutyricum

Laboratory mutagenesis of microorganisms offers the possibility of relating acquired mutations to improve the quality of microbial cultures. In the present study, a mutant strain, Clostridium tyrobutyricum ATCC 25755 DG-8, with significantly elevated α-amylase activity as well as resistant to the non-metabolizable and toxic glucose analog 2-deoxyglucose (2-DG) was obtained by implanting the low-energy nitrogen ion beam. DG-8 was further developed to produce hydrogen by simultaneous saccharification and fermentation (SSF) directly form cassava starch in batch fermentation mode, which to our knowledge is at the first attempt in genus Clostridium. Our results demonstrated that the increased activity of α-amylase would be attributed to the hydrogen over-producing. Higher hydrogen yield (3.2 mol/mol glucose) was achieved with the volumetric productivity of 0.41 L/h/L when the initial total sugar concentration of cassava starch rise up to 100 g/L. The present work will help to decrease the cost of hydrogen fermentation process and stimulate its industrial application in the near future.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum

Clostridium acetobutylicum is an attractive industrial microorganism for biochemical production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, metabolically engineered C. acetobutylicum carrying glucose-6-phosphate dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, than the wild type. Furthermore, when pH and glucose concentration were optimized for the CA-hydA strain, enhanced hydrogen productivity of 25.8% was achieved in 7 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production.     

Co-producing hydrogen and methane from higher-concentration of corn stalk by combining hydrogen fermentation and anaerobic digestion

In this paper, the high concentration of corn stalk (60 g/L) was employed as feedstock to produce bio-hydrogen and methane by combining hydrogen fermentation and anaerobic digestion. In the first stage of hydrogen fermentation, the effects of several key parameters, such as strain enhancement technique, cetyl trimethyl ammonium bromide (CTAB), NH₄HCO₃ on hydrogen production from cornstalk were investigated and optimized. The maximum hydrogen yield of 79.8 ± 1.5 ml H₂/g-TS and hydrogen production rate of 3.78 ml/g-cornstalk h was observed at fixed acidizing cornstalk of 60 g/L, strains Bacillus sp. FS2011 dosage of 10%(v/v), CTAB of 30 mg/L, NH₄HCO₃ of 1.2 g/L and initial pH of 7.5 ± 0.5 at 36 ± 1 °C, respectively. In the second stage of anaerobic digestion, the effluent from hydrogen production bio-reactor was further employed as the feedstock to produce methane by methanogenic bacteria, the maximum methane yield of 227 ± 2.5 ml CH₄/g-COD and COD removal rate of 95  ± 1% was recorded. The interesting observations were that the total amount of the organic wastewater produced in a higher substrate concentration (60 g/l) by hydrogen fermentation was reduced by about two-thirds compared with that of traditional low substrate concentration (≤20 g/l).     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.     

Different feedback effects of aqueous end products on hydrogen production of Clostridium tyrobutyricum

Feedback inhibition is one of the main challenges of fermentative hydrogen production. In this study, the effects of butyrate and acetate on hydrogen production of Clostridium tyrobutyricum were investigated. Substrate consumption and hydrogen production were accelerated when acetate ≤15 g/L was fed. Exogenous acetate induced acetate assimilation and increased the metabolic flux of butyrate synthesis. Exogenous butyrate significantly decreased biomass formation, and slowed substrate consumption and hydrogen production. Metabolic and gene expression analyses showed that butyrate impaired glycolysis and acetate production pathway. The increased butyrate/acetate molar ratio was deemed as a strategy for cells to alleviate pH decrease and reduce the inhibition of undissociated butyric acid. Inhibition model analyses indicated butyrate was the main inhibitor in butyrate-type hydrogen production. This study demonstrates the different feedback effects of acetate and butyrate on hydrogen production of C. tyrobutyricum and provides strategies to relieve the feedback inhibition for efficient hydrogen production.     

Anaerobic fermentative system based scheme for green energy sustainable houses

The green energy sustainable house based on bio-hydrogen and bio-methane energy technologies proposed in this study employs dark fermentation technology to complete a scheme for green energy sustainable house that includes energy production, storage, distribution control, load applications, recycling, waste treatment, and reuse. In order to resolve the problem of wastewater discharge from hydrogen production in green energy sustainable houses, this study proposes wastewater chemical oxygen demand (COD) treatment research, and suggests the use of two-stage anaerobic treatment to produce two types of bio-energy i.e. hydrogen and methane, while simultaneously reducing COD levels.Methane production employed a condensed molasses fermentation solubles (CMS) and hydrogen fermentation tank effluent as a substrate to test the COD reducing efficiency and overall efficiency of methane production. It was found that if CMS is used during the hydrolysis and acidogenesis stages, the maximum carbohydrate degradation rate will be approximately 70% (F/M ratio of 1.9-2.3), and the COD removal rate will increase from 15 to 20% (F/M ratio of 1.9-2.3) to 68% (F/M ratio of 0.5). This study showed that the total gas (H₂ and CH₄) production yield from effluent of hydrogen fermentation tank (56.2 KJ/mol substrate) is greater than the value for CMS.In this study, a 3.2 m³ anaerobic hydrogen reactor is evaluated to provide a family with 3-4 kW of power. When acclimatization is performed under conditions of 20 g COD/L substrate and hydraulic retention time (HRT) of 8 h, the COD removal rate can reach approximately 50%. If a methane-generating reactor with a 95% COD removal rate is used to degrade effluent from the hydrogen reaction tank, it will be possible to reduce the COD of organic effluent to under 500 mg/L. Since this water quality is not far from that of ordinary untreated household wastewater (approximately 300-500 mg COD/L), the effluent can be discharged into a community sewer system and treated in a community sewage treatment facility.     

Enhanced biohydrogen production from date seeds by Clostridium thermocellum ATCC 27405

Biohydrogen production from waste lignocellulosic biomass serves the dual purpose of converting waste into valuable products and alleviates waste disposal issues. In this study, waste date seeds were valorized for biohydrogen production via consolidated bioprocessing by Clostridium thermocellum ATCC 27405. Effect of various surfactants (PEG1000, surfactin, Triton X-100) and sodium carbonate (buffering agent) on biohydrogen production from the acid pre-treated substrate was examined. Among the various surfactants, addition of Triton X-100 resulted in the maximum biohydrogen yield of 103.97 mmol/L at an optimal dosage of 0.75% w/v. Triton X-100 supplementation favoured the production of ethanol and acetate as co-metabolites than butyrate. Addition of Na₂CO₃ to date seed fermentation medium at a concentration of 15 mM enhanced the biohydrogen production by 33.16%. Also, Na₂CO₃ buffering supported the glycolytic pathway and subsequent ethanol production than acetate/butyrate formation. Combined effect of the optimal dosages of Triton X-100 and Na₂CO₃ resulted in high hydrogen productivity up to 72 h (0.443 mmol/g h of H₂) with a total increase in hydrogen yield of 40.6% at the end of 168 h, as compared to fermentation supplemented with Triton X-100 alone. Further analysis revealed that the combined effects of the additives resulted in better substrate degradation, favourable pH window and cell growth promotion which ensured enhanced hydrogen productivity and yield. Thus, the study highlights a novel stimulatory approach for enhanced biohydrogen production from a new substrate.     

Evaluation of hydrogen producing cultures using pretreated food waste

In order to enhance bio-hydrogen production from food waste, pretreatment methods are widely used. The influence of the initial pH and autoclaving were investigated in batch experiments. Fermentative studies showed that pure cultures like Clostridium beijerinckii could directly utilize raw food waste to produce hydrogen, while other cultures (Clostridium butyricum and Clostridium pasteurianum) could produce hydrogen only after pH adjustment. In this case, the optimal starting pH of the culture was found to be 7. Autoclaving could further enhance hydrogen yields due to increased hydrolysis of food waste. The maximum hydrogen yield was achieved by C. butyricum (38.9 mL-H₂/g-VS_added) after autoclaving food waste with pH adjustment at 7. In addition, the ratio acetic to butyric acid was decreased by autoclaving pretreatment, because butyrate metabolic pathway was favored in the fermentation process. However, suitable pH for bacteria growth and the low ammonia production could be achieved from autoclaving food waste.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.     

Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis

A two-stage dark-fermentation and electrohydrogenesis process was used to convert the recalcitrant lignocellulosic materials into hydrogen gas at high yields and rates. Fermentation using Clostridium thermocellum produced 1.67 mol H₂/mol-glucose at a rate of 0.25 L H₂/L-d with a corn stover lignocellulose feed, and 1.64 mol H₂/mol-glucose and 1.65 L H₂/L-d with a cellobiose feed. The lignocelluose and cellobiose fermentation effluent consisted primarily of: acetic, lactic, succinic, and formic acids and ethanol. An additional 800 ± 290 mL H₂/g-COD was produced from a synthetic effluent with a wastewater inoculum (fermentation effluent inoculum; FEI) by electrohydrogensis using microbial electrolysis cells (MECs). Hydrogen yields were increased to 980 ± 110 mL H₂/g-COD with the synthetic effluent by combining in the inoculum samples from multiple microbial fuel cells (MFCs) each pre-acclimated to a single substrate (single substrate inocula; SSI). Hydrogen yields and production rates with SSI and the actual fermentation effluents were 980 ± 110 mL/g-COD and 1.11 ± 0.13 L/L-d (synthetic); 900 ± 140 mL/g-COD and 0.96 ± 0.16 L/L-d (cellobiose); and 750 ± 180 mL/g-COD and 1.00 ± 0.19 L/L-d (lignocellulose). A maximum hydrogen production rate of 1.11 ± 0.13 L H₂/L reactor/d was produced with synthetic effluent. Energy efficiencies based on electricity needed for the MEC using SSI were 270 ± 20% for the synthetic effluent, 230 ± 50% for lignocellulose effluent and 220 ± 30% for the cellobiose effluent. COD removals were ∼90% for the synthetic effluents, and 70–85% based on VFA removal (65% COD removal) with the cellobiose and lignocellulose effluent. The overall hydrogen yield was 9.95 mol-H₂/mol-glucose for the cellobiose. These results show that pre-acclimation of MFCs to single substrates improves performance with a complex mixture of substrates, and that high hydrogen yields and gas production rates can be achieved using a two-stage fermentation and MEC process.