Sustainable biohydrogen production by dark fermentation using carbon monoxide as the sole carbon and energy source

This study evaluated the kinetics of biomass growth, biohydrogen production and substrate utilization using carbon monoxide as the sole carbon and energy source. Experiments were conducted at different initial CO concentration in the range 1.8–5.12 mmol/L over a period of 144 h in order to assess the effect of CO concentration on biomass growth, substrate utilization and H₂ production. Complete utilization (100%) of CO was achieved up to an initial concentration of 3.8 mmol/L and it gradually decreased to 84.5% for 4.4 mmol/L and 83.7% for 5.12 mmol/L. The experimental results of CO utilization were fitted to substrate utilization kinetic models reported in the literature, and it followed a modified Gompertz model. A maximum yield of H₂ on CO was found to be 70.8% and a maximum H₂ production of 29.9 mmol/L was obtained for an initial CO concentration of 5.12 mmol/L. The experimental results on biohydrogen production matched well with the values predicted using the modified Gompertz model. Furthermore, the experimental data on specific growth rate of the ananerobic biomass at different H₂ concentration was fitted to different product inhibition models and the best fit was obtained with Aiba model. This study showed product inhibition on both specific growth rate of biomass and H₂ production due to H₂ accumulation in the gas phase. A very good correlation between the experimental specific growth rate and the Han-Levenspiel model predicted values were obtained with a high determination coefficient (R²) value of more than 0.96.     

Comparative evaluation of the biohydrogen production potential of thermophilic microorganisms isolated from hot springs located in Izmir

Biohydrogen production with the utilization of thermophilic microorganisms is a green process contributing to the mitigation of fossil fuel dependency. This study reports an improved water-gas shift reaction (WGSR) methodology with isolation of thermophilic and anaerobic microorganisms from 5 different closely-located hot springs, employing enrichment and characterization techniques to evaluate their biohydrogen production capacities with 100% CO in the headspace. Enriched mixed and pure cultures were screened for biohydrogen production. An isolate from Doğanbey hot spring reached the highest H₂ yield of 0.43 mmol H₂/mmol CO with a 43% CO conversion. Using growth profiling and morphologically with SEM imaging, this isolate was characterized biochemically, and was identified as an isolated bacilli shaped bacteria capable of producing hydrogen through WGSR. This is the first study to report potential thermophilic hydrogen producers in selected hot springs in the Izmir region.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

Biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures

Key factors affecting biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures were optimized using response surface methodology (RSM) with central composite design (CCD). Investigated parameters were waste glycerol concentration, sludge concentration, and the amount of Endo–nutrient addition. Concentrations of waste glycerol and sludge had a significant individual effect on hydrogen production rate (HPR) (p ≤ 0.05). The interactive effect on HPR (p ≤ 0.05) was found between waste glycerol concentration and sludge concentration. The optimal conditions for the maximum HPR were: waste glycerol concentration 22.19 g/L, sludge concentration 7.16 g-total solid (TS/L), and the amount of Endo–nutrient addition 2.89 mL/L in which the maximum HPR of 1.37 mmol H₂/L h was achieved. Using the optimal conditions, HPR from a co-digestion of waste glycerol and sludge (1.37 mmol H₂/L h) was two times greater than the control (waste glycerol without addition of sludge) (0.76 mmol H₂/L h), indicating a significant enhancement of HPR by sludge. Major metabolites of the fermentation process were ethanol, 1,3-propanediol (1,3-PD), lactate, and formate.     

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.     

Production of biohydrogen from waste wheat in continuously operated UPBR: The effect of influent substrate concentration

Utilization of waste materials is one of the most economical approaches to biohydrogen production. Continuous generation of biohydrogen in a bioreactor makes the process more economical with respect to the conventional physical and chemical method. The two main parameters that affect the biohydrogen production in a continuously operated bioreactor are hydraulic retention time (HRT) and influent substrate concentrations. The effect of influent substrate concentration on biohydrogen generation in an up-flow packed bed reactor (UPBR) at HRT = 3 h was investigated in this study. The substrate was waste wheat which was acid hydrolyzed in H₂SO₄ by adjusting the pH value to pH = 2, under high temperature as T = 90 °C in an autoclave to obtain fermentable sugar solution. A natural and porous support particle namely, aquarium biological sponge (ABS) was the microbial immobilization surface in the reactor. Total and hydrogen gas volumes, hydrogen percentage, influent and effluent substrate concentrations, VFA concentrations were monitored. The influent substrate concentration (TS_o) was varied between TS_o = 10 g/L and TS_o = 35 g/L. The process performance was evaluated as biohydrogen volume, percentages, rate and yield under varying operating conditions. The production volume (4275 ml/day) and the rate (3.05 L H₂/L day) were maximum at influent sugar concentration of TS_o = 25 g/L, but the yield reached to its maximum value as Y = 1.22 mol H₂/mol glucose at TS_o = 19 g/L. Substrate limitation and inhibitions were observed at influent concentrations of TS_o = 10 g/L and TS_o = 35 g/L, respectively. The results indicated that ABS could be suggested as a microbial support particle for hydrogen generation in immobilized systems.     

Hydrogen production and carbon dioxide enrichment using a catalytic membrane reactor with Ni metal catalyst and Pd-based membrane

We prepared a catalytic membrane reactor (CMR) by adopting a high-performance metal catalyst and Pd–Au membrane to investigate the possibility of hydrogen production concurrently with carbon dioxide enrichment (up to >80%) in a single-stage reactor from a simulated syngas of a coal gasification, via simultaneous WGS reaction and hydrogen separation process. The CO conversion was above 99% and the H₂ recovery was above 94% at del-P = 30 bar in a CMR. The best result for the concentration of the enriched CO₂ in the retentate side was 85.3% under the conditions of 350 °C, del-P = 30 bar and steam to carbon ratio of 2.0. These results show promise for a feasible simplified process able to achieve CO removal from a high-concentration CO mixture gas coming out of coal gasification via a water-gas shift reaction (WGS), to separate hydrogen and also to enrich CO₂ for pre-combustion capture and storage of CO₂ (CCS) in substitution for the conventional WGS and CO₂ separation stages in integrated gasification and combined cycle process integrated with CCS.     

Effect of silica-core gold-shell nanoparticles on the kinetics of biohydrogen production and pollutant hydrogenation via organic acid photofermentation over enhanced near-infrared illumination

A biological photoinduced fermentation process provides an alternative to traditional hydrogen productions. In this study, biohydrogen production was investigated at near IR region coupled to a near-field enhancement by silica-core gold-shell nanoparticles (NPs) over a range of acetate concentrations (5–40 mM) and light intensities (11–160 W/m²). The kinetic data were modeled using modified Monod equations containing light intensity effects. The yields of H₂ and CO₂ produced per acetate were determined as 2.31 mol-H₂/mol-Ac and 0.83 mol-CO₂/mol-Ac and increased to 4.38 mmol-H₂/mmol-Ma and 2.62 mmol-CO₂/mmol-Ma when malate was used. Maximum increases in H₂ and CO₂ productions by 115% and 113% were observed by adding NPs without affecting the bacterial growth rates (6.1–8.2 mg-DCM/L/hour) while the highest hydrogen production rate was determined as 0.81 mmol/L/hour. Model simulations showed that the energy conversion efficiency increased with NPs concentration but decreased with the intensity. Complete hydrogenation application was demonstrated with toxic 2-chlorobiphenyl using Pd catalysts.     

Biohydrogen production from palm oil mill effluent (POME) by two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of carbon sources in POME

The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H₂/mol-sugar and 8.54 mmol H₂/L·h respectively, while for the mesophilic stage were 1.19 mol H₂/mol-sugar and 1.47 mmolH₂/L·h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H₂/L·h to 10.34 mmol H₂/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 °C) and mesophilic (37.0 °C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique.     

Continuous biohydrogen production in immobilized biofilm system versus suspended cell culture

In this study, the performance of a new cell immobilization material, namely ceramic ball, was examined for continuous biohydrogen production in comparison to suspended cell culture system (CSTR). Production of biohydrogen in both systems was assessed under thermophilic conditions. Both systems were operated at varying hydraulic retention times (HRT) by shortening HRT values from 24 to 1.5 h at an influent sucrose concentration of 10 g/l. The immobilized bioreactor configuration outcompeted the CSTR bioreactor in terms of both volumetric hydrogen production (2.7 l H₂/l/day for immobilized system @ HRT = 3 h and 0.5 l H₂/l/day for CSTR @ HRT = 24 h) and resistance to cell-washout (CSTR reactor lost significant amount of biomass at short HRT values). It was concluded that immobilized bioreactor configuration is much more robust than CSTR against high organic loading rates and 5 fold more volumetric hydrogen production was achieved in 8 fold smaller immobilized bioreactor.     

Biohydrogen by Rhodobacter sphaeroides during photo-fermentation: Mixed vs. sole carbon sources enhance bacterial growth and H2 production

Mixed carbon sources fermentation by bacteria is a promising approach for biohydrogen (H₂) production biotechnology. In the present study, growth and Н₂ production by purple bacteria Rhodobacter sphaeroides MDC6521 during mixed carbon sources (succinate + acetate, succinate + malate, and malate + acetate) photo-fermentation was investigated. The growth rate of bacteria in mixed carbon sources containing medium was of ∼0.33 h⁻¹ which was considerably higher (1.3–1.7-fold) compared with sole carbon substrate containing one. Moreover, the H₂ production during photo-fermentation of succinate and acetate mixture was of ∼6.5 mmol H₂ g⁻¹ (dry weight of biomass) and significantly more (∼2–3-fold) than that with appropriate sole sources and higher (1.5-fold) than that with succinate and malate mixture. Probably, supplementation of the mixed carbon sources into bacterial culture alters the mode of metabolism, resulting in enhanced H₂ production, thus they can be preferable compared to the sole carbon source. The changed F_OF₁-ATPase activity of membrane vesicles suggested its important role in the increase of Н₂ production efficiency. The results showed that mixed carbon sources provide more H₂ than the sole carbon substrates and succinate with acetate mixture is better than succinate with malate.     

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H₂/g-substrate, hydrogen production rate of 39 mL/g h⁻¹ and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0-5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.     

Biohydrogen production in the two-stage process of anaerobic bioconversion of organic matter of liquid organic waste with recirculation of digister effluent

At present, hydrogen energy is gaining immense popularity in the world due to the problem of depletion of non-renewable energy sources, hydrocarbons, and environmental pollution caused by their growing consumption. Of particular interest is the dark process of producing hydrogen-containing biogas in the processing of organic waste under anaerobic conditions which allows to take advantage of both energy production and solving the problem of recycling organic waste. The article describes in detail an experimental plant for investigating a two-stage process of anaerobic bioconversion of organic matter of liquid organic waste and setting up an experiment to study the effect of recirculation of the methantenk effluent into an anaerobic bioreactor for the production of biohydrogen. Moreover, experimental studies were carried out in a continuous mode in reactors with increased volume. The average specific yield of biohydrogen (per kilogram of initial organic matter (OM)) during recirculation of the methantenk effluent increased by 4% (from 0.1046 to 0.1087 m³/(day 1 kg of OM_in)). In addition, recirculation of the methantenk effluent to the biohydrogen production reactor during two-stage anaerobic bioconversion allowed us to reduce fluctuations in the output of biohydrogen from the reactor. At the same time, there was no methanogenic activity in the anaerobic bioreactor for the production of biohydrogen. The self-stabilizing pH level in the anaerobic bioreactor for producing biohydrogen was less than 4.5 (3.94 without effluent recirculation and 3.88 with recirculation), however, there was no inhibition of hydrogen formation. Thus, the use of recirculation of the methantenk effluent into the anaerobic bioreactor for producing biohydrogen can enhance the efficiency of the two-stage anaerobic bioconversion of organic waste while maintaining the stability of the process.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Photo fermentative hydrogen production using dominant components (acetate, lactate, and butyrate) in dark fermentation effluents

Engineering strategies were applied to promote the phototrophic H₂ production of an indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5 using major components (i.e., acetate, butyrate, and lactate) of dark fermentation effluents as carbon sources. First, performance of cell growth and photo-H₂ production on each carbon source was examined individually. It appeared that acetate was the most effective carbon source for photo-H₂ production, giving an overall H₂ production rate and H₂ yield of 12.68 ml/h/l and 67.1%, respectively. Next, the effect of substrate concentration of each carbon source on photo-hydrogen production was investigated. Kinetic models were developed to describe the correlation between maximum specific growth rate/specific H₂ production rate and the substrate concentration. The results show that using acetate and lactate as the carbon source, the kinetics for the cell growth and photo-hydrogen production can be described by Monod-type and Michaelis–Menten models, respectively, whereas substrate inhibition occurred when using butyrate as the carbon source. The continuous cultures were also conducted at a hydraulic retention time of 96 h using synthetic dark fermentation soluble metabolites (with a 5 and 10 fold dilution) as the influent. The phototrophic H₂ production efficiency was stably maintained for over 30 days with an overall H₂ yield 10.30 and 11.97 mol H₂/mol sucrose, when using 5-fold and 10-fold diluted dark fermentation effluent, respectively, as the substrate for dark fermentation. This demonstrates the feasibility of using the sequential dark and photo fermentation for high-yield biohydrogen production.     

Optimization of conditions for hydrogen production from complex dairy wastewater by anaerobic sludge using desirability function approach

Present study deals with the multiple-response optimization for biohydrogen production using anaerobic sludge and outstanding approach to overcome the drawbacks of conventional response surface methodology (RSM). Dairy wastewater was used as source in batch fermentation was followed for this study. Response surface methodology (RSM), based on a three level, four variable Box–Behnken design, was employed to obtain the best possible combination of substrate concentration, pH, COD/N ratio and COD/P ratio for maximum H₂ yield (HY) and specific hydrogen production rate (SHPR). Experimental data were evaluated by applying RSM integrating a desirability function approach. The optimum H₂ yield and SHPR conditions were: substrate concentration 15.3 g COD/L, pH 5.5, COD/N ratio 100.5 and COD/P ratio 120 with maximum overall desirability D of 0.94. The confirmation experiment under these optimal condition showed a HY and SHPR of 13.54 mmol H₂/g COD and 29.91 mmol H₂/g-VSS.d, respectively. This was only 0.22% and 0.20%, respectively, different from the predicted values, suggesting that the desirability function approach with RSM was a useful technique to get the maximum H₂ yield and SHPR simultaneously.     

Biotechnological approach to generate green biohydrogen through the utilization of succinate-rich fermentation wastewater

Photofermentation seems to be an attractive mode of generating biohydrogen from fermentation effluent. Use of succinate fermentation effluent, however, has not been reported. Rhodobacter sphaeroides KKU-PS1 and Rhodopseudomonas palustris were acclimatised in succinate. It was determined that the KKU-PS1 was superior with respect to hydrogen productivity and was selected for further experiments. Photofermentation in succinate by the KKU-PS1 validated, generating 1217 mL H₂/L of cumulative hydrogen at a maximum rate of 6.7 mL H₂/L/h. Photofermentation from each single carbon sources that are components of effluent was performed and it was determined that acetate and succinate promoted the fastest growth of KKU-PS1 and hydrogen evolution, respectively. Photofermentation by the strain using mixed substrates mimicking diluted bio-succinate effluent produced yielded 1005 mL H₂/L cumulative hydrogen at a maximum rate of 4.1 mL H₂/L/h. The study highlighted potential of utilizing bio-succinate fermentation effluent for biohydrogen production, with further optimization required.     

Biohydrogen production using native carbon monoxide converting anaerobic microbial consortium predominantly Petrobacter sp.

In this study, anaerobic mixed microbial consortium isolated from a local sewage treatment plant in Guwahati, India, was used to convert carbon monoxide (CO) to hydrogen. The consortium was initially grown in acetate containing medium and later acclimatized to utilize CO as the sole carbon source for hydrogen production. By 16S rDNA analysis, the consortium was identified to be predominantly Petrobacter sp. Statistically designed experiments were then applied to optimize the CO conversion and hydrogen production by the anaerobic mixed consortium. To evaluate the significant factors that influenced the biohydrogen production, Plackett–Burman screening design of experiments was applied, which revealed that temperature and Fe²⁺ influenced the most on hydrogen production with P values less than 0.05 each. The effect due to pH and Ni²⁺ was less with P values 0.120 and 0.132, respectively. Concentration of Fe²⁺ and Ni²⁺ in the medium was then subsequently optimized by using Central Composite Design (CCD) of experiments followed by response surface methodology (RSM) which yielded the optimum value of 213 mg/L for Fe²⁺ and 2.2 mg/L for Ni²⁺. At these optimum conditions, 60.8 mol hydrogen production was achieved which was 8% higher than that observed from the screening experiment.     

Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values

An anaerobic fermentation of food waste was conducted in a 0.5 L bioreactor incubated at a thermophilic temperature of 55 °C to evaluate the effects of different controlled pH values (5.0, 5.5 and 6.0) on biohydrogen production. Effective biohydrogen production was found at controlled pH 5.5 and 6.0 corresponding to lower lactic acid production compared to pH 5.0. It was demonstrated that biohydrogen production from food waste was pH-dependent with hydrogen yields of 79, 76 and 23 mmol H₂/L-media/d for pH 5.5, 6.0 and 5.0, respectively. Specific microbial determination for Clostridium sp. and total bacteria quantification were carried out by the fluorescent in-situ hybridization (FISH) technique. The number of Clostridium sp. for acclimatized sludge, fermentation broth at pH 5.0, 5.5 and 6.0 were 2.9 × 10⁸, 3.6 × 10⁸, 7.8 × 10⁸ and 5.4 × 10⁸ cells/ml, respectively. The quantification analysis showed that 92% of the total bacteria belonged to Clostridium sp. from clusters I and XI from the sample at controlled pH 5.5. The denaturing gradient gel electrophoresis (DGGE) bands of the sample after heat-treatment, acclimatization and during fermentation indicated the presence of Bacteroidetes, Caloromator australicus sp. and Clostridium sp.