Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH₄⁺, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H₂ photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L⁻¹), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H₂ production. The influence of various fermentation end products at different concentrations (up to 30 g L⁻¹) on dark, H₂ production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L⁻¹) resulted in 5.7 L H₂ L⁻¹ of culture (1.6 mol H₂ per mole of hexose) with the co-production mainly of butyrate and acetate.     

Bio-hydrogen production from ground wheat starch by continuous combined fermentation using annular-hybrid bioreactor

Continuous combined fermentation of ground wheat starch was realized in an annular-hybrid bioreactor (AHB) for hydrogen gas production. A mixture of pure cultures of Clostridium beijerinkii (DSMZ-791) and Rhodobacter sphaeroides-RV were used as seed cultures in combined fermentation. The feed contained 5 g L⁻¹ ground wheat with some nutrient supplementation. Effects of hydraulic residence time (HRT) on the rate and yield of hydrogen gas formation were investigated. Steady-state daily hydrogen production decreased but, hydrogen yield increased with increasing HRT. The highest hydrogen yield was 90 ml g⁻¹ starch at HRT of 6 days. Hydrolysis of starch and fermentation of glucose to volatile fatty acids (VFA) were readily realized at all HRTs. However, slow conversion of VFAs to H₂ and CO₂ by photo-fermentation caused accumulation of VFAs in the medium. Specific and volumetric rates of hydrogen formation also decreased with increasing HRT. High hydrogen yields obtained at high HRTs are due to partial fermentation of VFAs by Rhodobacter sp. The system should be operated at HRTs longer than 5 days for effective hydrogen gas formation by the dark and photo-fermentation bacteria.     

Effects of dark/light bacteria ratio on bio-hydrogen production by combined fed-batch fermentation of ground wheat starch

Bio-hydrogen production by combined dark and light fermentation of ground wheat starch was investigated using fed-batch operation. Serum bottles containing heat-treated anaerobic sludge and a mixture of Rhodobacter sp. was fed with a medium containing 20 g dm^?3 wheat powder (WP) at a constant flow rate. The system was operated at different initial dark/light biomass ratios (D/L). The optimum D/L ratio was 1/2 yielding the highest cumulative hydrogen (1548 cm³), yield (65.2 cm³ g^?1 starch), and specific hydrogen production rate (5.18 cm³ g^?1 h^?1). Light fermentation alone yielded higher hydrogen production than dark fermentation due to fermentation of volatile fatty acids (VFAs) to H₂ and CO₂. The lowest hydrogen formation was obtained with D/L ratio of 1/1 due to accumulation of VFAs in the medium.     

Fermentative hydrogen production from cheese whey with in-line,concentration gradient-driven butyric acid extraction

Hydrogen (H₂) generation from cheese whey with simultaneous production and extraction of volatile fatty acids (VFAs) was studied in UASB reactors at two temperatures (20 and 35 °C) and pH values (5.0 and 4.5). The extraction module, installed through a recirculation loop, was a silicone tube coil submerged in water, which allows concentration-driven extraction of undissociated VFAs. Operating conditions were selected as a compromise for the recovery of both H₂ and VFAs. Batch experiments showed a higher yield (0.9 mol H₂ mol⁻¹ glucose_eq.) at 35 °C and pH 5.0, regardless of the presence of the extraction module, whereas lower yields were obtained at pH 4.5 and 20 °C (0.5 and 0.3 mol H₂ mol⁻¹ glucose_eq., respectively). VFAs crossed the silicone membrane, with a strong preference for butyric over propionic and acetic acid due to its higher hydrophobicity. Sugars, lactic acid and nutrients were retained, resulting in an extracted solution of up to 2.5 g L⁻¹ butyric acid with more than 90% purity. Continuous experiment confirmed those results, with production rates up to 2.0 L H₂ L⁻¹ d⁻¹ and butyric acid extraction both in-line (from the UASB recirculation) and off-line (from the UASB effluent). In-line VFA extraction can reduce the operating costs of fermentation, facilitating downstream processing for the recovery of marketable VFAs without affecting the H₂ production.     

Effects of starch loading rate on performance of combined fed-batch fermentation of ground wheat for bio-hydrogen production

Ground wheat powder solution (10 g L⁻¹) was subjected to combined dark and light fermentations for bio-hydrogen production by fed-batch operation. A mixture of heat treated anaerobic sludge (AN) and Rhodobacter sphaeroides-NRRL (RS-NRRL) were used as the mixed culture of dark and light fermentation bacteria with an initial dark/light biomass ratio of 1/2. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. The highest cumulative hydrogen formation (CHF = 3460 ml), hydrogen yield (201 ml H₂ g⁻¹ starch) and formation rate (18.1 ml h⁻¹) were obtained with a starch loading rate of 80.4 mg S h⁻¹. Complete starch hydrolysis and glucose fermentation were achieved within 96 h of fed-batch operation producing volatile fatty acids (VFA) and H₂. Fermentation of VFAs by photo-fermentation for bio-hydrogen production was most effective at starch loading rate of 80.4 mg S h⁻¹. Hydrogen formation by combined fermentation took place by a fast dark fermentation followed by a rather slow light fermentation after a lag period.     

Sequencing batch reactor enhances bacterial hydrolysis of starch promoting continuous bio-hydrogen production from starch feedstock

Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H₂ fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H₂ from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 °C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H₂ production with the starch hydrolysate, the highest H₂ production rate and yield was 0.52 L/h/L and 13.2 mmol H₂/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH₄HCO₃) concentration was 2.62 g/L, attaining a good H₂ production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H₂ fermentation while using dark fermentation effluents as the substrate.     

New tolerant strains of purple nonsulfur bacteria for hydrogen production in a two-stage integrated system

In this study we described the isolation of eight new strains of purple non-sulfur bacteria resistant to salinity ≥30 g L⁻¹ and high concentration of VFAs (200 mM). These strains were characterized by their general physiological properties and the occurrence of hupSL genes. Some correlation was observed between the rate of H₂ photoproduction, the absence of hupSL genes and hydrogenase activity. Two fast-growing strains without hupSL genes showed high nitrogenase activity and hydrogen accumulation during growth on Ormerod medium. These strains were capable of H₂ photoproduction using non-treated dark culture (75% in water) after dark fermentation of starch at 30 g L⁻¹, unlike control strains, Rhodobacter capsulatus B10 and Rb. sphaeroides GL. New N7 and 13 strains identified as Rb. sphaeroides can be recommended for application in a two-stage H₂ production system.     

Inhibitory effects of substrate and soluble end products on biohydrogen production of the alkalithermophile Caloramator celer: Kinetic,metabolic and transcription analyses

In this study the tolerance of the alkalithermophile Caloramator celer towards substrate (glucose) and soluble end product (acetate, formate and ethanol) inhibition was assessed employing nonlinear inhibition models. In addition, the effects of subinhibitory concentrations of end products on fermentative metabolism and regulation of 12 key genes involved in pyruvate catabolism were studied. Optimal growth and H₂ production were found at 50 mM of glucose and the critical substrate concentration was observed at 290–360 mM. Two inhibition models revealed that ethanol had a higher inhibitory effect on growth rate, whereas H₂ production kinetics was more sensitive towards increasing concentrations of acetate and formate. Acetate, the main soluble metabolite of the fermentation, inhibited the H₂ production by increasing the ionic strength in the medium. Subinhibitory concentrations of soluble end products induced changes in the metabolite profile of C. celer, specifically exogenous acetate (80 mM) and ethanol (40 mM) slightly increased the H₂ yield by 4 and 7%, respectively. However, despite the observed metabolic shifts, gene regulation was minimal and not always in agreement with the measured product yields. Overall, the results suggest that further optimization of the H₂ production process from C. celer should focus on methods to evolve adapted osmotolerant strains and/or remove soluble metabolites, especially acetate, from the culture.     

Evaluation of sludge liquors from acidogenic fermentation and thermal hydrolysis process as feedstock for microbial electrolysis cells

In this study, mesophilic acidogenic fermentation, thermophilic acidogenic fermentation, and thermal hydrolysis process (THP) were compared to generate sludge liquors for bioenergy recovery with microbial electrolysis cells (MECs). The results showed that THP at 170 °C was the most effective for hydrolysis of particulate organics in sewage sludge, while fermentation under thermophilic temperature led to the highest accumulation of volatile fatty acids (VFAs) in sludge liquor. However, THP yielded the highest percentage of acetate in VFAs, which resulted in superior MEC performance compared to fermented sludge liquors in terms of current density (2.7 vs. ~1.3 A/m²), coulombic efficiency (50% vs. 31–34%), bio-H₂ potential (1114 vs. 839–881 mL), and H₂ production rate (50.3 mL/d vs. 28–32 mL/d). The utilization sequence of the VFAs was found to be acetate > butyrate > propionate. Overall, our results show that generating sludge liquors through THP could provide a feasible solution to produce bio-H₂ from sewage sludge; however, coulombic efficiencies should be further improved before practical application.     

Integrating waste activated sludge (WAS) acidification with denitrification by adding nitrite (NO2−)

Adding nitrite (NO₂⁻) to waste activated sludge (WAS) fermentation systems is an efficient approach to integrate fermentation with denitrification, and utilize volatile fatty acids (VFAs) to achieve a high nitrogen removal rate even at low C/N ratios. In this study, the effect of nitrite on the integrated WAS fermentation and denitrification (termed as WASFD) was investigated under acidic and alkaline conditions. The results indicated that adding nitrite achieved a most reduction of nitrite to N₂ and improved the acidification of WAS with high VFAs production. Under acidic condition (pH = 5), the maximum VFAs produced with nitrite addition was 3.3 times that without nitrite addition. Higher concentration of free nitrous acid (FNA) at the pH of 5 increased WAS particulates, improved the hydrolysis of organic substrates, and finally promoted VFAs yields. Under alkaline condition (pH = 9), adding nitrite only increased the VFAs production by 1.5 times, indicating that acidic condition was preferable for acidification than alkaline condition.     

Improving hydrogen production from cassava starch by combination of dark and photo fermentation

The combination of dark and photo fermentation was studied with cassava starch as the substrate to increase the hydrogen yield and alleviate the environmental pollution. The different raw cassava starch concentrations of 10–25 g/l give different hydrogen yields in the dark fermentation inoculated with the mixed hydrogen-producing bacteria derived from the preheated activated sludge. The maximum hydrogen yield (HY) of 240.4 ml H₂/g starch is obtained at the starch concentration of 10 g/l and the maximum hydrogen production rate (HPR) of 84.4 ml H₂/l/h is obtained at the starch concentration of 25 g/l. When the cassava starch, which is gelatinized by heating or hydrolyzed with α-amylase and glucoamylase, is used as the substrate to produce hydrogen, the maximum HY respectively increases to 258.5 and 276.1 ml H₂/g starch, and the maximum HPR respectively increases to 172 and 262.4 ml H₂/l/h. Meanwhile, the lag time (λ) for hydrogen production decreases from 11 h to 8 h and 5 h respectively, and the fermentation duration decreases from 75–110 h to 44–68 h. The metabolite byproducts in the dark fermentation, which are mainly acetate and butyrate, are reused as the substrates in the photo fermentation inoculated with the Rhodopseudomonas palustris bacteria. The maximum HY and HPR are respectively 131.9 ml H₂/g starch and 16.4 ml H₂/l/h in the photo fermentation, and the highest utilization ratios of acetate and butyrate are respectively 89.3% and 98.5%. The maximum HY dramatically increases from 240.4 ml H₂/g starch only in the dark fermentation to 402.3 ml H₂/g starch in the combined dark and photo fermentation, while the energy conversion efficiency increases from 17.5–18.6% to 26.4–27.1% if only the heat value of cassava starch is considered as the input energy. When the input light energy in the photo fermentation is also taken into account, the whole energy conversion efficiency is 4.46–6.04%.     

Sequential dark–photo fermentation and autotrophic microalgal growth for high-yield and CO2-free biohydrogen production

Dark fermentation, photo fermentation, and autotrophic microalgae cultivation were integrated to establish a high-yield and CO₂-free biohydrogen production system by using different feedstock. Among the four carbon sources examined, sucrose was the most effective for the sequential dark (with Clostridium butyricum CGS5) and photo (with Rhodopseudomonas palutris WP3-5) fermentation process. The sequential dark–photo fermentation was stably operated for nearly 80 days, giving a maximum H₂ yield of 11.61 mol H₂/mol sucrose and a H₂ production rate of 673.93 ml/h/l. The biogas produced from the sequential dark–photo fermentation (containing ca. 40.0% CO₂) was directly fed into a microalga culture (Chlorella vulgaris C–C) cultivated at 30 °C under 60 μmol/m²/s illumination. The CO₂ produced from the fermentation processes was completely consumed during the autotrophic growth of C. vulgaris C–C, resulting in a microalgal biomass concentration of 1999 mg/l composed mainly of 48.0% protein, 23.0% carbohydrate and 12.3% lipid.     

Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation

A two-step process of sequential anaerobic (dark) and photo-heterotrophic fermentation was employed to produce hydrogen from cassava and food waste. In dark fermentation, the average yield of hydrogen was approximately 199 ml H₂ g⁻¹ cassava and 220 ml H₂ g⁻¹ food waste. In subsequent photo-fermentation, the average yield of hydrogen from the effluent of dark fermentation was approximately 611 ml H₂ g⁻¹ cassava and 451 ml H₂ g⁻¹ food waste. The total hydrogen yield in the two-step process was estimated as 810 ml H₂ g⁻¹ cassava and 671 ml H₂ g⁻¹ food waste. Meanwhile, the COD decreased greatly with a removal efficiency of 84.3% in cassava batch and 80.2% in food waste batch. These results demonstrate that cassava and food waste could be ideal substrates for bio-hydrogen production. And a two-step process combining dark fermentation and photo-fermentation was highly improving both bio-hydrogen production and removal of substrates and fatty acids.     

Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: An unexpected deviation from the dark fermentation model

The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of organic substrates. The process is referred to as dark fermentation and is typically complemented by production of acetic acid. Here we show that synthesis of products derived by reductive metabolism of pyruvate, mainly lactic acid, occurs to the detriment of acetic acid fermentation when the cultures of the thermophilic bacterium are flushed by saturating level of CO₂. Sodium bicarbonate in a very narrow range of concentrations (∼14 mM) also causes the same metabolic shift. The capnophilic (CO₂-requiring) re-orientation of the fermentative process toward lactic acid does not affect hydrogen productivity thus challenging the currently accepted dark fermentation model that predicts reduction of this gas when glucose is converted into organic products different from acetate.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

H2-rich biogas recirculation prevents hydrogen supersaturation and enhances hydrogen production by Thermotoga neapolitana cf. capnolactica

This study focused on the supersaturation of hydrogen in the liquid phase (H_2aq) and its inhibitory effect on dark fermentation by Thermotoga neapolitana cf. capnolactica by increasing the agitation (from 100 to 500 rpm) and recirculating H₂-rich biogas (GaR). At low cell concentrations, both 500 rpm and GaR reduced the H_2aq from 30.1 (±4.4) mL/L to the lowest values of 7.4 (±0.7) mL/L and 7.2 (±1.2) mL/L, respectively. However, at high cell concentrations (0.79 g CDW/L), the addition of GaR at 300 rpm was more efficient and increased the hydrogen production rate by 271%, compared to a 136% increase when raising the agitation to 500 rpm instead. While H_2aq primarily affected the dark fermentation rate, GaR concomitantly increased the hydrogen yield up to 3.5 mol H₂/mol glucose. Hence, H_2aq supersaturation highly depends on the systems gas-liquid mass transfer and strongly inhibits dark fermentation.     

Fermentative hydrogen production from hydrolyzed cellulosic feedstock prepared with a thermophilic anaerobic bacterial isolate

Hydrogen gas was produced via dark fermentation from natural cellulosic materials and α-cellulose via a two-step process, in which the cellulosic substrates were first hydrolyzed by an isolated cellulolytic bacterium Clostridium strain TCW1, and the resulting hydrolysates were then used as substrate for fermentative H₂ production. The TCW1 strain was able to hydrolyze all the cellulosic materials examined to produce reducing sugars (RS), attaining the best reducing sugar production yield of 0.65 g reducing sugar/g substrate from hydrolysis of α-cellulose. The hydrolysates of those cellulosic materials were successfully converted to H₂ via dark fermentation using seven H₂-producing bacterial isolates. The bioH₂ production performance was highly dependent on the type of cellulosic feedstock used, the initial reducing sugar concentration (C_RS,o) (ranging from 0.7 to 4.5 mg/l), as well as the composition of sugar and soluble metabolites present in the cellulosic hydrolysates. It was found that Clostridium butyricum CGS5 displayed the highest H₂-producing efficiency with a cumulative H₂ production of 270 ml/l from α-cellulose hydrolysate (C_RS,o = 4.52 mg/l) and a H₂ yield of 7.40 mmol/g RS (or 6.66 mmol/g substrate) from napier grass hydrolysate (C_RS,o = 1.22 g/l).     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Dark fermentative biohydrogen production by Acinetobacter junii-AH4 utilizing various industry wastewaters

Hydrogen (H₂) is one of the most promising renewable energy sources, anaerobic bacterial H₂ fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H₂ is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H₂ production was achieved by using the bacteria Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H₂ production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H₂ and Acinetobacter junii-AH4 has shown the highest cumulative H₂ of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H₂/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H₂ production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H₂.