Biological saccharification by Clostridium thermocellum and two-stage hydrogen and methane production from hydrogen peroxide-acetic acid pretreated sugarcane bagasse

The present work aimed at establishing an efficient degradation and energy recovery system form sugarcane bagasse (SCB) through hydrogen peroxide-acetic acid (HPAC) pretreatment, thermophilic hydrogen production and mesophilic methane production. The degradation ratio of HPAC pretreated SCB (HPAC-SCB, 2%, w/v) exceeded 90% under the biological hydrolysis of C. thermocellum without enzyme addition. The hydrogen yield in the co-culture fermentation of T. thermosaccharolyticum and C. thermocellum from HPAC-SCB (2%, w/v) reached 226 mL/g substrate. The long-term hydrogen fermentation was successfully established with 1.59 L/(L·d), 0.159 L/g substrate for average hydrogen productivity and yield, respectively. Methane production of 0.341 L/g COD (chemical oxygen demand)_added was recovered by semi-continuous methane fermentation from hydrogen-producing effluent at 12 days of hydraulic retention time (HRT). Average energy recovery of 8.79 MJ/kg SCB was obtained under the optimal conditions. The present work indicated the promising application of the established process in valorization of lignocellulosic bio-waste.     

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.     

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.     

Dilution strategy to obtain H2 in the 2nd acidogenic phase from enzymatically pre-treated sugarcane bagasse

Sugarcane bagasse (SCB) pre-treated with laccase was used as substrate in two acidogenic phases (1 P and 2 P). In 1 P, the pretreatment conditions were statistically optimized (pH 4.0, 37 °C, 0.4 U of laccase/mL, 24.0 g SCB/L and 120 min of reaction), which allowed obtaining 84% more H₂ (166.8 mL/L) than SCB in natura (90.4 mL/L). In 2 P, the liquid fraction of the 1 P reactors was diluted (90%) and more 108.5 ml H₂/L was obtained. Furthermore, the quantification of enzymes genes responsible for the hydrolysis (Cel) and H₂ production (Hyd) was carried out. An increase in the expression of both genes was observed from the phase of highest H₂ production rates in P1. In 2 P, the 1 P low genic expression was indicative of the autochthonous bacteria activity. The 2nd acidogenic phase strategy proved to be a promising novelty for the use of pre-hydrolyzed substrates with concomitant production of more value-added products.     

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.     

Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration

Pre-heated elephant dung was used as inoculum to produce hydrogen from sugarcane bagasse (SCB) hydrolysate. SCB was hydrolyzed by H₂SO₄ or NaOH at various concentrations (0.25-5% volume) and reaction time of 60 min at 121 °C, 1.5 kg/cm² in the autoclave. The optimal condition for the pretreatment was obtained when SCB was hydrolyzed by H₂SO₄ at 1% volume which yielded 11.28 g/L of total sugar (1.46 g glucose/L; 9.10 g xylose/L; 0.72 g arabinose/L). The maximum hydrogen yield of 0.84 mol H₂/mol total sugar and the hydrogen production rate of 109.55 mL H₂/L day were obtained at the initial pH 6.5 and initial total sugar concentration 10 g/L. Hydrogen-producing bacterium (Clostridium pasteurianum) and non hydrogen-producing bacterium (Flavobacterium sp.) were dominating species in the elephant dung and in hydrogen fermentation broth. Sporolactobacillus sp. was found to be responsible for a low hydrogen yield obtained.     

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.     

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.     

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.     

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

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

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization

This work evaluated the effects of individual alkaline, sodium carbonate (Na₂CO₃ denoted as; NaC), sodium sulfide (Na₂SO₃ denoted as; NaS) and combination of NaC + NaS pretreatment for the saccharification of sugarcane bagasse (SCB). The effects of different pretreatments on chemical composition and structural complexity of SCB in relation with its saccharification were investigated. For enzymatic hydrolysis of pretreated SCB we have utilized the produced crude enzymes by Streptomyces sp. MDS to make the process more cost effective. A enzyme dose of 30 filter paperase (FPU) produced a maximum reducing sugar (RS) 592 mg/g with 80.2% hydrolysis yield from NaC + NaS pretreated SCB under optimized conditions. The resulted enzymatic hydrolysates of each pretreated SCB were applied for hydrogen production using Clostridium beijerinckii KCTC1785. NaC + NaS pretreated SCB hydrolysates exhibited maximum H₂ production relative to other pretreatment methods. Effects of temperature, initial pH of culture media and increasing NaC + NaS pretreated SCB enzymatic hydrolysates concentration (2.5–15 g/L) on bioH₂ production were investigated. Under the optimized conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1485 mL/L, 61.87 mL/L/h and 1.24 mmol H₂/mol of RS (0.733 mmol H₂/g of SCB), respectively. The efficient conversion of the SCB hydrolysate to H₂ without detoxification proves the viability of process for cost-effective hydrogen production.     

Evaluating biohydrogen production by Clostridium hydrogenum sp. nov. strain CUEA01 isolated from mangrove sediments in Thailand

The hydrogen (H₂) fermentative Clostridium hydrogenum sp. nov. strain CUEA01 was isolated from a mangrove sediment in Thailand. Genome sequencing and analysis revealed a genome size of 5,501,482 bp that encoded for 3,292 predicted protein coding genes with annotated functional assignments and many genes associated with carbon utilization and H₂ evolution. The H₂ production performance was evaluated in batch fermentation, and revealed that this strain can grow and produce H₂ at a broad range of temperatures (15–40 °C), pH (4–10), and initial glucose concentrations (5–60 g/L). The maximum H₂ yield (3.11 mol_H2/mol_glucose) was obtained at 37 °C, pH 8, and an initial glucose concentration of 10 g/L. Furthermore, this strain could utilize various carbon sources, including xylose, xylan, starch, mannose, glycerol, and avicel cellulose, amongst others. Additionally, CUEA01 was compatible with agro-industrial wastes and could achieve a maximum CHP of 4639 mL/L and 4024 mL/L from sugarcane molasses and cassava pulp, respectively. This demonstrates that CUEA01 has a potential for H₂ fermentation from complex organic wastes since it can secrete enzyme cocktails that consolidate the fermentation process.     

Comparison of suspended and granular cell anaerobic bioreactors for hydrogen production from acid agave bagasse hydrolyzates

Acid agave bagasse hydrolyzates have been used as a substrate for hydrogen production, however, bioreactors are unstable and with poor performance. Granular biomass could be more successful in producing hydrogen from acid agave bagasse hydrolyzates in comparison with suspended biomass. Thus, this study aimed to evaluate the effect of increasing concentrations of acid agave hydrolyzates on hydrogen production, to compare the hydrogen productivity and stability of granular biomass in an expanded granular sludge bed (EGSB) reactor and suspended biomass in an anaerobic sequencing batch reactor (AnSBR) fed with acid hydrolyzates, and finally to determine the variation of microbial communities established in both bioreactor configurations. In batch tests, the heat-treated inoculum produced hydrogen from acid agave hydrolyzates without observing inhibition at 6.3 g/L of carbohydrates (_CHO). This hydrolyzate concentration was used to start up the AnBSR, which reached a productivity of 226 ± 53 mL H₂/L⋅d at organic loading rates (OLR) from 3.2 to 4.5 g_CHO/L⋅d. The hydrogen production stability index decreased from 0.8 to 0.6 at increasing OLR, and the AnSBR failed at the highest OLR of 5.7 g/L⋅d. The EGSB reactor reached the highest productivity of 361 ± 130 mL H₂/L⋅d at an OLR of 7.4 g_CHO/L⋅d, but with a low stability index of 0.6. Independently of the bioreactor configuration, microbial communities associated with the production of acetate/lactate were successfully established in both configurations with the prevalence of Lactobacillus spp. A low abundance of typical H₂ producers like Clostridium was always observed over the whole period of operation (<10% of the total abundance). In sum, the hydrogen productivity from acid agave hydrolyzates was higher for the EGSB reactor than for the AnSBR, but with much lower stability. The evidence provided by this study suggests the establishment of metabolic pathways for hydrogen production from organic acids.     

Optimization of batch dilute-acid hydrolysis for biohydrogen production from red algal biomass

Marine algae are promising alternative sources for bioenergy including hydrogen. Their polymeric structure, however, requires a pretreatment such as dilute-acid hydrolysis prior to fermentation. This study aimed to optimize the control variables of batch dilute-acid hydrolysis for dark hydrogen fermentation of algal biomass. The powder of Gelidium amansii was hydrolyzed at temperatures of 120–180 °C, solid/liquid (S/L) ratios of 5–15% (w/v), and H₂SO₄ concentrations of 0.5–1.5% (w/w), and then fed to batch hydrogen fermentation. Among the three control variables, hydrolysis temperature was the most significant for hydrogen production as well as for hydrolysis efficiency. The maximum hydrogen production performance of 0.51 L H₂/L/hr and 37.0 mL H₂/g dry biomass was found at 161–164 °C hydrolysis temperature, 12.7–14.1% S/L ratio, and 0.50% H₂SO₄. The optimized dilute-acid hydrolysis would enhance the feasibility of the red algal biomass as a suitable substrate for hydrogen fermentation.     

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.     

Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).