Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.     

Biohydrogen using leachate from an industrial waste landfill as inoculum

Since hydrogen is a renewable energy source, biohydrogen has been researched in recent years. However, there is little data on hydrogen fermentation by a leachate from a waste landfill as inoculum. We investigated hydrogen production using a leachate from an industrial waste landfill in Kanagawa prefecture. The results showed no methane gas production and the leachate was a suitable inoculum for hydrogen fermentation. The maximum H₂ yield was 2.67 mol of H₂ per mol of carbohydrate added, obtained at 30 °C and initial pH 7. The acetate and butyrate production was significant when the H₂ yield was higher. The oxidation–reduction potential analysis of the culture suggested that hydrogen-producing bacteria in the leachate were facultatively anaerobic. Scanning electron microscope observations revealed hydrogen-producing bacteria comprised bacilli of about 2 μm in length.     

Effects of inoculum and indigenous microflora on hydrogen production from the organic fraction of municipal solid waste

Biological production of hydrogen (H₂) by dark fermentation is an exciting scientific area for the conversion of low-cost residues and waste into biofuel. The main requirement for an efficient H₂ production process is the availability of efficient microbial consortia in which H₂-utilizing and non-H₂-producing bacteria are suppressed. This study was performed to evaluate the H₂ production potentials from the organic fraction of municipal solid waste (OFMSW) with and without addition of inoculum. The results showed that hydrogen productions from OFMSW without addition of inoculum were comparable to those obtained with inoculum but a latency phase of about 6 days occurred. On the contrary, addition of inoculum resulted in higher H₂ production potentials without any latency phase. The use of a properly pre-treated inoculum confirmed to be an interesting and improvable tool to obtain high H₂ yields from organic waste. However the indigenous OFMSW microbiota showed promising hydrogen yields especially toward the development of efficient hydrogen producing microbial inoculants.     

Electro-hydrolysis of cheese whey solution for hydrogen gas production and chemical oxygen demand (COD) removal using photo-voltaic cells (PVC)

Diluted cheese whey (CW) solution was used for hydrogen gas production by electro-hydrolysis using photo-voltaic cells (PVC) as source of electricity. Effects of initial chemical oxygen demand (COD) concentration on the rate and yield of hydrogen gas production were investigated using a completely mixed and sealed reactor with aluminum electrodes. Cumulative hydrogen gas formation (CHF) increased with increasing initial COD concentration. The highest cumulative hydrogen gas volume (26472 mL), hydrogen gas production rate (4553 mL d⁻¹), hydrogen yield (7004 mL H₂ g⁻¹ COD), and percent COD removal (21.5%) were obtained with initial COD of 35172 mg L⁻¹. H₂ gas formation from water control was only 5365 mL. pH of the CW solution increased with decreasing conductivities during the course of experiments. Gas phase contained more than 99% H₂ at the end of experiments. The highest energy efficiency (20.4%) was also obtained with the highest COD content. Nearly pure hydrogen gas formation by electro-hydrolysis of cheese whey using PVC panels was proven to be an effective method.     

Initial pH influences in-batch hydrogen production from scotta permeate

We studied the influence of initial pH on hydrogen (H₂) production using permeate from scotta (a partially deproteinized cheese whey from ricotta production) as substrate (51 g L^?1 lactose). Dark fermentation was carried out at 35 °C in laboratory batch reactors, in an unbuffered system. Hydrogen production and metabolite (volatile fatty acids, ethanol, and lactic acid) evolution during a 96-h period were monitored in reactors with initial pH varying in the range 4–10. In all reactors, H₂ production started only when pH fell below 6. However, it was much higher (+31%) in the reactors with initial alkaline pH. We conclude that H₂ production occurs only at acidic pH values, but initial alkaline pH values increase the overall H₂ production in dark fermentation of lactose-rich substrates.     

The effect of dilution and l-malic acid addition on bio-hydrogen production with Rhodopseudomonas palustris from effluent of an acidogenic anaerobic reactor

In this study, H₂ was produced from cheese whey wastewater in a two-stage biological process: i) first stage; thermophilic dark fermentation ii) second stage; the photo fermentation using Rhodopseudomonas palustris strain DSM 127 (R. palustris). The effect of both dilution and addition of l-malic acid on the hydrogen production was investigated. Among the dilution rates used, 1/5 dilution ratio was found to produce the best hydrogen production (349 ml H₂/g COD_fed). On the other hand, It was seen that the mixing the effluent with l-malic acid at increasing ratios had further positive effect and improved the hydrogen production significantly. It was concluded that dilution of the feeding helps to reduce the nitrogen content and the volatile fatty acid content that might be otherwise harmful to the photo-heterotrophic organisms. Overall hydrogen production yield (for dark + photo fermentation) was found to vary 2 and 10 mol H₂/mol lactose. Second conclusion is that cheese whey effluent should be mixed with a co-substrate containing l-malic acid such as apple juice processing effluents before fed into the photo fermentation reactor.     

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.     

Anaerobic solid-state fermentation of bio-hydrogen from microalgal Chlorella sp. biomass

A considerable amount of volatile solids (VS) contained in the biomass of microalgae makes it promising for use as feedstock in fermentation processes. In this study, a biomass of microalga Chlorella sp. was used as a sole substrate for hydrogen production in an anaerobic solid-state fermentation (ASSF). Optimization of the process was investigated on the selected critical variables, i.e., total solid (TS) content, initial pH, and feed to inoculum (F/I) ratio (on a VS basis) using response surface methodology (RSM) with central composite design (CCD). TS content and F/I ratio were found to have statistically significant effects on hydrogen production. Maximal hydrogen production of 165 ± 12 mL H₂, equivalent to 18.58 mL H₂/g VS and 0.28 L H₂/L reactor·d, was achieved under the optimal conditions of 38.83% TS, pH 6.03, and an F/I ratio of 4.33. Acetic and butyric acids were found to be main soluble microbial products (SMPs) in the fermented biomass. Based on the compositions of the biomass, an equation for theoretical bioconversion of Chlorella sp. biomass to hydrogen was proposed.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

Evaluation of biohydrogen production from glucose and protein at neutral initial pH

Organic wastes are considered as potential substances for economical biohydrogen production, because the carbohydrate and protein are main components. Previous investigations indicate that an optimum hydrogen production appear in acidic conditions to carbohydrates, or in alkali condition to protein. However, in practice, the treatment of these organic wastes by anaerobic fermentation usually carries out at neutral pH condition, in which biohydrogen production is only a middle process. So, the purpose of this paper is to evaluate the biohydrogen production at neutral pH condition from carbohydrates or protein. Batch tests were conducted to investigate the differences in biohydrogen production by anaerobic fermentation at neutral initial pH using carbohydrate and protein (glucose and peptone) as the sole carbon source. The experimental results showed that the maximal hydrogen yields of two substrates were about 0.14 ml H₂/mg glucose and 0.077 ml H₂/mg protein, respectively, at neutral initial pH. Although the hydrogen yields of glucose is far greater than that of protein at neutral pH, they were lower than previous results of hydrogen production in acidic condition to carbohydrate or in alkali condition to protein. This result shows that the neutral pH is not an optimal condition for biohydrogen production. In this experiment of biohydrogen production, a phenomenon has been observed that the hydrogen production and hydrogen consumption occurred simultaneously in the fermentation of protein, whereas the hydrogen production occurred only in the fermentation of glucose. Furthermore, the different evaluation of the main components of the organic liquid by-products produced by fermentation of each substrate implied that the biohydrogen production pathways of these two substrates were different. Molecular analysis indicated that the dominant microorganisms during the anaerobic fermentation of these two substrates are greatly different.     

An integrated biological hydrogen production process based on ethanol-type fermentation and bipolar membrane electrodialysis

An integrated bio-hydrogen production system involving fermentative hydrogen production and product separation is proposed. In this process, microorganisms conduct ethanol-type fermentation and generate H₂ gas in anaerobic bioreactor, and acetate is removed from fermentation broth by using a two chamber bipolar membrane electrodialysis as separation unit. A comparative study of fermentative hydrogen production of Ethanoligenens harbinese B49 in the integrated system with traditional fermentation process was carried out. Compared to traditional process, accumulated H₂ elevated 23%, glucose utilization ratio increased by 135% and cell growth increased by 27% in the integrated system. The specific hydrogen production rate reached 2.2 mol H₂/mol glucose, indicating that separation of acetate from fermentation system has a great role in promoting hydrogen producing capacity. Bipolar membrane electrodialysis showed high acetate separation efficiency and low glucose loss rate. In the integrated system, pH could be used to direct electrodialysis operation, since it has an exponential correlation with acetate concentration in fermentation broth. These results provide a new method for achieving efficient and stable H₂ production with simultaneous glucose recovery and acetate inhibition release.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

Optimization of hybrid inoculum development techniques for biohydrogen production and preliminary scale up

Inoculum pre-treatment is a crucial aspect of hydrogen fermentation processes to establish the required microbial community for hydrogen production. This paper models and optimizes two hybrid techniques of inoculum pre-treatment for fermentative hydrogen production: ¹pH and Autoclave (PHA); ²pH and heat shock (PHS) using Response Surface Methodology (RSM). Coefficients of determination (R²) of 0.93 and 0.90 were obtained for PHA and PHS respectively and the optimized pre-treatment conditions gave hydrogen yields up to 1.35 mol H₂/mol glucose and 0.75 mol H₂/mol glucose, thus a 37.75% and 15.38% improvement on model predictions for PHA and PHS respectively.     

Isolation and evaluation of a high H2-producing lab isolate from cow dung

Hydrogen producing bacterial strain was isolated from Indian cow dung and identified of the bacterial family Enterobacteriaceae. This lab isolate was differentiated from Citrobacter Y-19 at molecular level by using RAPD, PCR based technique, and OPO-03₄₆₀ and OPO-17₈₀₀ RAPD marker for this specific strain (lab isolate) was identified. Fermentative studies were investigated for important parameters, starting with pH of the culture, temperature, inoculum age and inoculum volume, initial substrate concentration and different substrates. Among different substrates, dextrose and sucrose were the preferred substrates for hydrogen production. The optimal starting pH of the culture was found to be 5.0. The H₂ production increased with increase in temperature up to 30 °C. The maximum value of H₂ production was recorded when inoculum volume was 12.5% of the culture broth and inoculum age was 14 h. Under batch fermentation conditions, the maximum hydrogen production rate and yield were 355.2 ml l⁻¹ h⁻¹ and 2.1 mol/mol glucose (conversion 35%), respectively. These results indicate that this lab isolate is an ideal hydrogen producer.     

A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process

A two-stage fermentation process combining hydrogen and methane production for the treatment of food waste was investigated in this paper. In hydrogen fermentation reactor, the indigenous mixed microbial cultures contained in food waste were used for hydrogen production. No foreign inoculum was used in the hydrogen fermentation stage, the traditional heat treatment of inoculum was not applied either in this bench scale experiment. The effects of the stepwise increased organic loading rate (OLR) and solid retention time (SRT) on integrated two-stage process were investigated. At steady state, the optimal OLR and SRT for the integrated two-stage process were found to be 22.65 kg VS/m³ d (160 h) for hydrogen fermentation reactor and 4.61 (26.67 d) for methane fermentation reactor, respectively. Under the optimum conditions, the maximum yields of hydrogen (0.065 m³ H₂/kg VS) and methane (0.546 m³ CH₄/kg VS) were achieved with the hydrogen and methane contents ranging from 29.42 to 30.86%, 64.33 to 71.48%, respectively. Biodegradability analysis showed that 5.78% of the influent COD was converted to the hydrogen in H₂-SCRD and 82.18% of the influent COD was converted to the methane in CH₄-SCSTR under the optimum conditions.     

Critical assessment of biofilm and suspended growth reactor configurations for acidogenic biohydrogen production using wastewater as a function of redox microenvironment

Comprehensive experiments were designed to study the relative function of two bioreactor configurations viz., biofilm and suspended growth for dark-fermentative hydrogen (H₂) production along with wastewater treatment at two varying feed pH conditions (6 and 7). Both the reactors were operated in sequencing batch mode using anaerobic inoculum after pretreatment (combined treatment: acid-shock, heat-shock and chemical-shock). Biofilm system showed efficient H₂ production over the corresponding suspended growth operation at feeding pH 6. VFA profiles visualized high acetate fraction supporting feasible microenvironment for higher H₂ production. Voltammogram profiles visualized significant variation in the bio-electrochemical behavior with the function of operating pH and reactor configuration. It can be inferred from this study that biofilm systems are efficient for H₂ production particularly at acidiophilic microenvironment.     

Biohydrogen production from pretreated sludge and synthetic and real biodiesel wastewater by dark fermentation

Different types of sludge pretreatments were tested, with thermal shock at 90°C to 95°C for 60 minutes plus a 6‐hour rest period achieving the best results for inhibition of methanogen microorganisms and inoculum enrichment with H₂‐producing bacteria, which produced a H₂‐rich biogas (up to 65% mol/mol) without the presence of CH₄. Wastewater from biodiesel production (WBP), containing mainly methanol (128 g/L) and glycerol (4 g/L), was evaluated as a potential substrate to produce H₂ through dark fermentation. Both methanol‐based solutions and methanol‐rich wastewater were not suitable for hydrogen production; however, these effluents showed a strong potential for CH₄‐rich biogas production. A fractional factorial design was employed to evaluate the effect of six substrate‐related variables (glycerol content, 25% and 75%; COD content, 4 and 50 g/L, COD:VSS ratio, 1:1 and 5:1; COD:N:P ratio, 350:0:0 and 350:5:1; NaCl content, 0.5 and 12.0 g/L; and pH, 4.0 and 5.5) on the H₂ production from glycerol‐methanol–based synthetic solutions (synthetic WBP). Some substrate‐related variables had a crucial impact on the hydrogen production potential from WBP, which was significantly affected by the COD and salinity content in the substrate. WBP containing high glycerol (representing until 75% of the COD) and salinity (up to 12 g/L as NaCl) content could be turned into a potential substrate for H₂ production through dark fermentation as long as specific fermentation conditions are maintained, such as pH 5.5 to 5.7 and a substrate COD content up to 50 g/L. Using this condition, glycerol conversion, H₂ productivity, and H₂ yield of 81.3 ± 8.9%, 102.8 ± 18.2 mL H₂/L.d, and 24.5 ± 4.4 mL H₂/g COD_applied, respectively, were obtained.     

Continuous biohydrogen production using cheese whey: Improving the hydrogen production rate

Due to the renewed interest in finding sustainable fuels or energy carriers, biohydrogen (Bio-H₂) from biomass is a promising alternative. Fermentative Bio-H₂ production was studied in a continuous stirred tank reactor (CSTR) operated during 65.6 d with cheese whey (CW) as substrate. Three hydraulic retention times (HRTs) were tested (10, 6 and 4 h) and the highest volumetric hydrogen production rate (VHPR) was attained with HRT of 6 h. Therefore, four organic loading rates (OLRs) at a fixed HRT of 6 h were tested thereafter, being: 92.4, 115.5, 138.6 and 184.4 g lactose/L/d. The highest VHPR (46.61 mmol H₂/L/h) and hydrogen molar yield (HMY) of 2.8 mol H₂/mol lactose were found at an OLR of 138.6 g lactose/L/d; a sharp fall in VHPR occurred at an OLR of 184.4 g lactose/L/d. Butyric, propionic and acetic acids were the main soluble metabolites found, with butyric-to-acetic ratios ranging from 1.0 to 2.4. Bacterial community was identified by partial sequence analysis of the 16S rRNA and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). The results showed that at HRT of 10 h and 6 h were dominated by the Clostridium genus. The VHPR attained in this study is the highest reported value for a CSTR system using CW as substrate with anaerobic sludge as inoculum and represents a 33-fold increase compared to a previous study. Thus, it was demonstrated that continuous fermentative Bio-H₂ production from CW can be significantly enhanced by an appropriate selection of parameters such as HRT and OLR. Enhancements in VHPR are significant because it is a critical parameter to determine the full-scale practical application of fermentation technologies that will be used for sustainable and clean energy generation.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.