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.     

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.     

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.     

Sustainable fermentation approach for biogenic hydrogen productivity from delignified sugarcane bagasse

Improper lignocellulosic wastes management causes severe environmental pollution and health damage. Conversion of such wastes particularly sugarcane bagasse (SCB) onto bioenergy is a sustainable approach due to a continuous depletion of conventional biofuels. The delignification of SCB is necessary to proceed for bio-genic H₂ productivity by anaerobic bacteria. The effect of autoclaving, pre-acidification/autoclaving and pre-alkalization/autoclaving of SCB on glucose recovery and subsequently H₂ productivity by dark fermentation was comprehensively investigated. Pre-acidified SCB with 1% H₂SO₄ (v/v) provided H₂ productivity of 8.5 ± 0.14 L/kg SCB and maximum H₂ production rate (R_m) of 105.9 ± 8.3 mL/h. Those values were dropped to 2.7 ± 0.13 L/kg SCB and 58.3 ± 12.9 mL/h for fermentation of delignified SCB with 2% H₂SO₄. This was linked to high levels of total phenolic compounds (1775.3 ± 212 mg/L) in the feedstock. Better H₂ productivity of 13.9 ± 0.58 L/kg SCB and R_m of 133.9 ± 3.6 mL/h was achieved from fermentation of pre-alkalized SCB with 1%KOH (v/v). 256.8 ± 9.8 U/100 mL of α-amylase, 165.7 ± 7.6 U/100 mL of xylanase, 232.8 ± 6.1 U/100 mL of CM-Cellulase, 176.5 ± 5.0 U/100 mL of polyglacturanase and 0.702 ± 0.013 mg M B. reduced/min. of hydrogenase enzyme was accounted for the batches supplied with delignified SCB by KOH. The Clostridium and Bacillus spp. was dominance and prevalence resulting a higher H₂ productivity and yield. A novel strain of Archea and alpha proteobacterium were also identified and detected.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

Enhancement of biohydrogen production in industrial wastewaters with vinasse pond consortium using lignin-mediated iron nanoparticles

The aim of this work was the enhancement of biohydrogen production by an anaerobic bacterial consortium with incorporation of lignin-mediated iron nanoparticles in the fermentation medium. Lignin magnetic nanoparticles (LMNP), identified as magnetite by XRD, exhibited spherical shape and average particle size of 8.6 nm, while lignin non-magnetic nanoparticles (LNMNP) exhibited high agglomeration and an amorphous nature in TEM and XRD, respectively. The fermentation medium (pH 7) was composed of 88% soft drink wastewater (SDW) and 12% corn steep liquor (CSL) and supplemented with NaHCO₃ (1.0 g/L) and cysteine-HCl (0.5 g/L). Under optimal conditions, BioH₂ production was 17.67 ± 0.54 mL, after 48 h of fermentation at 37 °C. Addition of LMNP and LNMNP increased BioH₂ production in 91.0 and 74.3%, respectively. Additionally, 200 mg/L of LMNP and LNMNP in the fermentation medium improved the BioH₂ yields (mL H₂/g COD_removed) in 2.8- and 2.3-fold, respectively.     

Synergistic strategy for the enhancement of biohydrogen production from molasses through coculture of Lactobacillus brevis and Clostridium saccharobutylicum

This study aimed to evaluate the capacity of different inoculum sources and their bacterial diversity to generate hydrogen (H₂). The highest Simpson (0.7901) and Shannon (1.581) diversity indexes for H₂-producing bacterial isolates were estimated for sewage inocula. The maximum cumulative H₂ production (H_max) was 639.6 ± 5.49 mL/L recorded for the sewage inoculum (SS30) after 72 h. The highest H₂-producing isolates were recovered from SS30 and identified as Clostridium saccharobutylicum MH206 and Lactobacillus brevis MH223. The H_max of C. saccharobutylicum, L. brevis, and synergistic coculture was 415.00 ± 24.68, 491.67 ± 15.90, and 617.67 ± 3.93 mL/L, respectively. The optimization process showed that the H_max (1571.66 ± 33.71 mL/L) with a production rate of 58.02 mL/L/h and lag phase of 19.33 h was achieved by the synergistic coculture grown on 3% molasses at 40 °C, pH 7, and an inoculum size of 25% (v/v). This study revealed the economic feasibility of the synergistic effects of coculture on waste management and biohydrogen production technology.     

Evaluation of process performance on biohydrogen production in continuous fixed bed reactor (C-FBR) using acid algae hydrolysate (AAH) as feedstock

In this study, a novel inoculation method to mitigate the inhibition of 5-hydroxymethylfurfural (5-HMF) is proposed. Acid algae hydrolysate containing 1.5 g 5-HMF/L and 15 g hexose/L hexose was fed to a continuous fixed bed reactor (C-FBR) partially packed with hybrid-immobilized beads. The inoculation method enabled a high rate of H₂ production, due to the reduction of 5-HMF inhibition and enhanced biofilm formation. Maximum hydrogen production was achieved at a hydraulic retention time of 6 h with a hydrogen production rate (HPR) of 20.0 ± 3.3 L H₂/L-d and a hydrogen yield (HY) of 2.3 ± 0.4 mol H₂/mol hexose _added. Butyrate and acetate were the major soluble metabolic products released during fermentation. Quantitative real-time polymerase chain reaction analysis revealed that Clostridium butyricum comprised 94.3% of the total bacteria, which was attributed to the high rate of biohydrogen production.     

Sequential hydrogen and methane production using the residual biocatalyst of biodiesel synthesis as raw material

Residual Fermented Solid (RFS) is the used biocatalyst obtained after enzymatic biodiesel production carried out applying the fermented solid (FS) with lipase activity. Approximately 350 g of RFS are generated for each liter of biodiesel produced from palm residues fermented solid. In this study, this residue was used for the first time as a raw material for biological hydrogen production through dark fermentation and sequential application of the hydrogen production liquid waste (HPLW) for methane obtainment via anaerobic digestion. The RFS was composed mostly of oils and fats (60% wt.%), and carbohydrates, such as mannose, glucose, and xylose. Hydrogen yield reached 239 ± 44 mL H₂/L after 24 h of fermentation using 31 g_RFS/L at the beginning of the process. Additionally, 204 ± 13 mL CH₄/g COD were produced through the anaerobic digestion of HPLW, which represented 61% of efficiency.     

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.     

Influence of alkaline peroxide assisted and hydrothermal pretreatment on biodegradability and bio-hydrogen formation from citrus peel waste

The effect of pretreatments by hydrothermolysis (180 °C; 15 min) and alkaline delignification (NaOH 5M; H₂O₂ 1%; 24 h) in citrus peel waste (CPW) was evaluated, as well as the effect on H₂, organic acids and alcohols production, in addition to characterization of the microbial community involved in fermentation. Batch reactors at 37 °C were operated with 3 gTVS/L of CPW with allochthonous consortium (UASB reactor sludge; 2 gTVS/L) and autochthonous of CPW (1.5 gTVS/L) as inocula. H₂ production was higher in reactors with in natura CPW (13.31 mmol/L) compared to hydrothermolysis (8.19 mmol/L) and alkaline delignification (7.27 mmol/L). The acetogenic pathway was predominant in the in natura CPW (4,355 mg/L acetic acid). The most abundant genera in the in natura CPW and after hydrothermolysis were Clostridium (18.97 and 12.90%, respectively) and Ruminiclostridium (16.65 and 1.04%, respectively) commonly related to cellulolytic bacteria and/or H₂ production.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Coproduction of hydrogen,ethanol and 2,3-butanediol from agro-industrial residues by the Antarctic psychrophilic GA0F bacterium

In this study, the simultaneous production of hydrogen, ethanol, and 2,3-butanediol was assessed using three agro-industrial residues: cheese whey powder (CWP), wheat straw hydrolysate (WSH) and sugarcane molasses (SCM), by the Antarctic psychrophilic GA0F bacterium [EU636050], which is closely related to Pseudomonas antarctica [KX186936.1]. The main soluble metabolites produced in all the fermentations were ethanol and 2,3-butanediol. CWP demonstrated to be the most effective carbon source, since fermentation of this substrate resulted in the highest yields of H₂ (73.5 ± 10 cm³ g⁻¹), ethanol (0.24 ± 0.03 g g⁻¹) and 2,3-butanediol (0.42 ± 0.04 g g⁻¹), followed by the use of SCM, whereas WSH showed to have an inhibitory effect during the fermentation process, showing the lowest production values. Our results demonstrated the ability of the Antarctic psychrophilic GA0F bacterium to produce valuable products using low-cost substrates at room temperature conditions.     

Influence of pretreatment and pH on the enhancement of hydrogen and volatile fatty acids production from food waste in the semi-continuously running reactor

In this study, FW effluent was used as a substrate for both hydrogen and volatile fatty acids (VFAs) production by a lab scale set up of the semi-continuously running reactor system with a mesophilic fermentation to examine the influence of pH and pretreatment. Repeated measurement analysis showed that the factors (pH and Pretreatment) significantly influenced H₂, VFAs concentration, VFA/soluble chemical oxygen demand (SCOD), and H₂/SCOD traits (P < 0.0001). Duncan comparisons showed that both concentration and yield of H₂ were the highest in the chemical treatment (CT) at pH 7, which were (280.82 ± 5.72) ml/L, and (4.44 ± 0.10) ml/g SCOD, respectively. While concentration and yield of the VFAs were the highest in the chemical treatment (CT) at pH 6, which were (55.44 ± 2.39) g/L, and (926.21 ± 42.27) mg/g SCOD, respectively. The butyrate and acetate for the optimal blend (pH 6, CT pretreatment) counted for 62.43% of the total VFAs.     

Hydrogen production by sequential dark and photofermentation using wet biomass hydrolysate of Spirulina platensis: Response surface methodological approach

Microalgae and cyanobacteria can be used as a potential biomass to produce hydrogen from stored glycogen and starch through fermentation and photofermentation. In this study, the potential of algal biomass i.e. Spirulina platensis hydrolysate as a substrate for sequential fermentative (I-stage) and photo-fermentative (II-stage) biohydrogen production was evaluated. Response Surface Methodology (RSM) was employed to find the optimum photofermentation conditions. From the preliminary optimization experiments, it was found that the significantly affecting factors for H₂ production were pH, dilution fold (D.F.) of fermentate and Fe(II) sulfate concentration during photofermentation (second stage). In the present study, 1% (w/v) Spirulina platensis hydrolyzate produced 23.06 ± 3.63 mmol of H₂ with yield of 1.92 ± 0.20 mmol H₂/g COD reduced. In the second stage experiment 1510 ± 35 mL/l hydrogen was produced using inoculum volume-20.0% (v/v) and inoculum age-48 h of co-culture of Rhodobacter sphaeroides NMBL-01 and Bacillus firmus NMBL-03 under conditions pH-5.95, D.F. of dark fermentate-20.30 folds, Fe(II) sulfate concentration-0.412 μM, temperature-32±2 °C and light intensity-2.5 klux.     

Optimization of key factors affecting hydrogen production from coffee waste using factorial design and metagenomic analysis of the microbial community

The objective of this study was to evaluate the fermentation conditions that led to the optimization of H₂ production from coffee waste (wastewater, pulp and husk) and the taxonomic and functional characterization of autochthonous microorganisms. Assays in batch reactors with microbial consortium bioaugmentation (bacteria and fungi) evaluated the pH (4.82–8.18), pulp and husk concentration (6.95–17.05 g/L) and headspace factor (33.18–66.82%) by means of rotational central composite design and response surface. Operating conditions in the reactor optimized for 3.04 LH₂/Ld were at pH 7.0, 7 g/L pulp and husk and 30% headspace. The main metabolites observed were butyric acid (3838 mg/L), isobutyric acid (506 mg/L), methanol (226 mg/L) and butanol (156 mg/L). Clostridium sp. (87.9%), Lactobacillus sp. (1.7%), Kazachstania sp. (18.6%) and Saccharomyces sp. (16.3%) were the main genera identified in the optimized reactor, which had functional gene diversity for H₂ production, alcoholic fermentation, cellulose degradation, lignin, hemicellulose and phenol.     

High-solid dark fermentation of cassava pulp and cassava processing wastewater for hydrogen production

Dark fermentation (DF) is a promising biological process for hydrogen production from biomass. However, low hydrogen yield (HY) is a major hurdle impeding its use at large-scale operation. A potential way to mitigate this problem is to increase the concentration of substrate in the process to increase hydrogen production. The present study investigated the possibility of using high-solid DF to produce hydrogen from cassava processing wastes, i.e., cassava pulp (CP) and cassava processing wastewater (CPW). CP was suspended in CPW and hydrolyzed enzymatically under optimum conditions of 150 g-CP/L, 29 U/g of α-amylase, 47 U/g of glucoamylase, and 60 FPU/g of cellulase. The hydrolysis performed at 50 °C for 24 h yielded a reducing sugar concentration of 117.7 ± 1.8 g/L, equivalent to 0.78 g-reducing-sugar/g-CP. Subsequent DF of CP-CPW enzymatic slurry, which contained 12.1% water insoluble solids, resulted in a cumulative production of 13.72 ± 0.22 L-H₂, equivalent to 225.2 ± 3.7 mL-H₂/g-VS. This was 83.1% of a maximum stoichiometric HY, based on carbohydrate content of CP and soluble metabolites production. The present study shows clearly the applicability of high-solid DF in the production of hydrogen from cassava processing wastes.     

Bio-hydrogen production from tequila vinasses: Effect of detoxification with activated charcoal on dark fermentation performance

Hydrogen production from dark fermentation is a potential source of sustainable fuel when it is generated from waste. This study compared hydrogen production resulting from fermentation using raw and detoxified tequila vinasse. Vinasse was detoxified with granular activated charcoal, which was used to adsorb compounds that could inhibit the production of hydrogen by dark fermentation. In batch cultures detoxification of vinasse led to up to 20% higher maximum velocities of hydrogen production, a 5.4 h reduction in the lag phase and an 11% higher molar yield, compared to results obtained with raw vinasse. Losses of sugars after detoxification provoked that the specific hydrogen volumetric yields obtained with detoxified vinasse were 30–40% lower with 5 g COD/L and 15 g COD/L initial concentrations, compared to the ones obtained with raw vinasse. For an initial 30 g COD/L no differences in specific hydrogen yields were observed between raw or detoxified vinasse in batch fermentation. Continuous culture fermentation of vinasse showed hydrogen production rates between 1.32 ± 0.07 to 1.39 ± 0.14 NL H₂/L-d when extra nutrients were added, while a stable production of hydrogen through fermentation of detoxified vinasse could not be maintained despite nutrient addition. Production of hydrogen from vinasse diluted with water with no additional nutrients was assessed and rates close to 0.42 ± 0.02 NL H₂/L-d and hydrogen content close to 37% were obtained. Accumulation of lactic acid and a predominant production of butyric acid over acetic acid suggested that the fermentation dynamics of vinasse with no supplementary nutrients were especially susceptible to high substrate loading rates and prolonged hydraulic retention times.     

Optimization of high-solid waste activated sludge concentration for hydrogen production in microbial electrolysis cells and microbial community diversity analysis

To enhance hydrogen recovery from high-solid waste activated sludge (WAS), microbial electrolysis cells (MECs) were used as an efficient device. The effects of WAS concentrations were firstly investigated. Optimal concentration for hydrogen production was 7.6 g VSS/L. Maximum hydrogen yields reached to 4.66 ± 1.90 mg-H₂/g VSS and 11.42 ± 2.43 mg-H₂/g VSS for MECs fed with raw WAS (R-WAS) and alkaline-pretreated WAS (A-WAS) respectively, which was much higher than that obtained traditional anaerobic digestion. Moreover, no propionic acid accumulation was achieved at the optimal concentration. Effective sludge reduction was also achieved in MECs feeding with A-WAS. 52.9 ± 1.3% TCOD were removed in A-WAS MECs, meanwhile, protein degradation were 50.4 ± 0.8%. The 454 pyrosequencing analysis of 16S rRNA gene revealed the syntrophic interactions were existed between exoelectrogen Geobacter and fermentative bacteria Petrimonas, which apparently drove the efficient performance of MECs fed with WAS.