Inoculum pretreatment differentially affects the active microbial community performing mesophilic and thermophilic dark fermentation of xylose

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H₂ production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H₂ mol^?1 xylose. The H₂ and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H₂ production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H₂ mol^?1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum.     

Biohydrogen production from biomass and industrial wastes by dark fermentation

Hydrogen is a clean energy carrier which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. This article summarizes the dark fermentative biohydrogen production from biomass. Types of potential biomass that could be the source for biohydrogen generation such as food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol are discussed in this article. Moreover, the microorganisms, factors affecting biohydrogen production such as undissociated acid, hydrogen partial pressure and metal ions are also discussed.     

Comparison of thermophilic and hyperthermophilic dark fermentation with subsequent mesophilic methanogenesis in expanded granular sludge bed reactors

Herein, dark fermentation (DF, V = 5.5 L) and subsequent mesophilic methanogenesis (V = 43.5 L) are run as expanded granular sludge bed reactors (EGSB) at thermophilic (υDF = 60 °C) and hyperthermophilic (υDF = 80 °C) temperatures. A synthetic glucose wastewater is run with a 22.5 g/L chemical oxygen demand (COD) and 48–9 h hydraulic retention times (HRTs), giving organic loading rates (OLRs) of 11–60 g COD/L/d for DF. The maximum hydrogen production rate (HPR) is HPR = 3.0 m³/m³/d for HRT = 9 h with a 50 L/kg COD hydrogen yield (HY) and 40 vol% H₂. Methane production rate (MPR) reaches MPR = 2.6 m³/m³/d with 70 vol% CH₄ at HRT = 2.8 d. The highest H₂ yields are HY = 180 L/kg COD with 53 vol% H₂ (thermophilic, HRT = 48 h). Hyperthermophilic temperatures led to lower HPRs (0.7 m³/m³/d) and MPRs (1.6 m³/m³/d). 53% of Thermoanaerobacterium thermosaccharolyticum as an H₂ producer are found. Discoloration of granular sludge from black to white and granule stability was observed in DF.     

Effect of inoculum pretreatment on the microbial community structure and its performance during dark fermentation using anaerobic fluidized-bed reactors

The effect of two different inoculum pretreatments, thermal and cell wash-out (A1 and A2, respectively) on the performance of anaerobic fluidized bed reactors for hydrogen production was determined. The reactors were operated for 112 days under the same operational conditions using glucose as substrate at increasing organic loading rates and decreasing hydraulic retention times. Both treatments were effective avoiding methanogenesis. Reactor A2 showed better performance and stability than reactor A1 in each one of the different operational conditions. Cell wash-out treatment produced higher hydrogen volumetric production rates and yields than thermal treatment (7 L H₂/L-d, 3.5 mol H₂/mol hexose, respectively). DGGE analysis revealed that the microbial communities developed were affected by the inoculum treatment. Organisms from the genera Clostridium and Lactobacillus predominated in both reactors, with their relative abundances linked to hydrogen production. Resilience was observed in both reactors after a period of starvation.     

Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H₂ yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H₂ generation potential (171.3 NL H₂/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.     

Changes in microbial community structure during dark fermentative hydrogen production

Microbial community structure plays a significant role in the efficiency of dark fermentative hydrogen production using mixed culture. However, the detailed evolutions in microbial community structure during dark fermentation process are still unclear. This study investigated the detailed evolution patterns of microbial community structure during dark fermentation process by high-throughput pyrosequencing. Results showed that microbial community structure changed significantly over time in dark fermentation. Microbial diversity showed a constant decreasing trend during the fermentation process. The analysis of microbial community composition showed that Clostridium sensu stricto 1, Paraclostridium, Romboutsia and Paeniclostridium, which were all rarely existed in the inoculum, dramatically became dominant genera in the system after 6 h fermentation, with total relative abundance of more than 99%. This interesting result revealed that how quickly hydrogen-producing genera overwhelmed the microbial community in dark fermentation. Spearman correlation analysis showed that Clostridium sensu stricto 1 contributed the most to hydrogen fermentation performances.     

Biohydrogen production by dark fermentation: Experiences of continuous operation in large lab scale

Hydrogen is a clean energy carrier which can be used as fuel in fuel cells. Today, hydrogen is produced mainly by steam reforming of fossil fuels like natural gas or oil. But only hydrogen produced by renewable sources can be called clean energy production. One possibility for hydrogen production is the biological fermentation of biogenous wastes by hydrogen producing bacteria. For the experimental setup four 30-L-working-volume reactors were constructed for continuous biohydrogen production. As inoculum, heat-treated sludge of a wastewater treatment plant was used. Different hydraulic retention times (HRT) were tested and an organic loading rate (OLR) of 2–14 kg VS/m³*d. As starting substrate, waste sugar medium was used. The pH and other parameters were observed to find boundary conditions for a stable continuous process with a minimum of online-control measurements. The high concentration of organic acids in the reactor led to a very low pH, which was controlled manually and online > 4 up to 5.5, otherwise the biohydrogen production decreased rapidly. The gas amount varied with the different OLRs, but could be stabilised on a high level as well as the hydrogen concentration in the gas with 44–52%. No methane was detected in the gas. It turned out, that continuous biohydrogen production with stable gas amounts and qualities could be achieved at different operation conditions. The results showed, that the operation of a continuous biohydrogen reactor has to be observed very carefully to ensure a constant gas production, and that pH-control is necessary to ensure stable operation conditions.     

Selective adaptation of an anaerobic microbial community: Biohydrogen production by co-digestion of cheese whey and vegetables fruit waste

The co-digestion process of crude cheese whey (CCW) with fruit vegetable waste (FVW) for biohydrogen production was investigated in this study. Five different C/N ratios (7, 17, 21, 31, and 46) were tested in 2 L batch systems at a pH of 5.5 and 37 °C. The highest specific biohydrogen production rate of 10.68 mmol H₂/Lh and biohydrogen yield of 449.84 mL H₂/g COD were determined at a C/N ratio of 21. A pyrosequencing analysis showed that the main microbial population at the initial stage of the co-digestion consisted of Bifidobacterium, with 85.4% of predominance. Hydrogen producing bacteria such as Klebsiella (9.1%), Lactobacillus (0.97%), Citrobacter (0.21%), Enterobacter (0.27%), and Clostridium (0.18%) were less abundant at this culture period. The microbial population structure was correlated with the lactate, acetate, and butyrate profiles obtained. Results demonstrated that the co-digestion of CCW with FVW improves biohydrogen production due to a better nutrient balance and improvement of the system's buffering capacity.     

Bio-hydrogen production from cheese whey powder (CWP) solution: Comparison of thermophilic and mesophilic dark fermentations

Cheese whey powder (CWP) solution was used as the raw material for hydrogen gas production by mesophilic (35 °C) and thermophilic (55 °C) dark fermentations at constant initial total sugar and bacteria concentrations. Thermophilic fermentation yielded higher cumulative hydrogen formation (CHF = 171 mL), higher hydrogen yield (111 mL H₂ g⁻¹ total sugar), and higher hydrogen formation rate (3.46 mL H₂ L⁻¹ h⁻¹) as compared to mesophilic fermentation. CHF in both cases were correlated with the Gompertz equation and the constants were determined. Despite the longer lag phase, thermophilic fermentation yielded higher specific H₂ formation rate (2.10 mL H₂ g⁻¹cells h⁻¹). Favorable results obtained in thermophilic fermentation were probably due to elimination of H₂ consuming bacteria at high temperatures and selection of fast hydrogen gas producers.     

Lactate wastewater dark fermentation: The effect of temperature and initial pH on biohydrogen production and microbial community

Biohydrogen production using dark fermentation (hydrolysis and acidogenesis) is one of the ways to recover energy from lactate wastewater from the food-processing industry, which has high organic matter. Dark fermentation can be affected by the temperature, pH and the microbial community structure. This study investigated the effects of temperature and initial pH on the biohydrogen production and the microbial community from a lactate wastewater using dark fermentation. Biohydrogen production was successful only at lower temperature levels (35 and 45 °C) and initial pH 6.5, 7.5 and 8.5. The highest hydrogen yield (0.85 mol H₂/mol lactate consumed) was achieved at 45 °C and initial pH 8.5. The COD reduction achieved by fermenting the lactate wastewater at 35 °C ranged between 21 and 30% with the maximum COD reduction at pH 8.5, and at 45 °C, the COD reduction ranged between 12 and 21%, with the maximum at pH 7.5. At 35 °C, the lactate degradation ranged between 54 and 95%, while at 45 °C, it ranged between 77 and 99.8%. 16S rRNA sequencing revealed that at 35 °C, bacteria from the Clostridium genera were the most abundant at the end of the fermentation in the reactors that produced hydrogen, while at 45 °C Sporanaerobacter, Clostridium and Pseudomonas were the most abundant.     

Biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated packed bed reactor: The effect of hydraulic retention time

The aim of the study is biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated up-flow packed bed reactor. For this purpose, the effect of hydraulic retention time (HRT) on the rate (R_H2) and yield (Y_H2) of hydrogen gas formation were investigated. In order to determine the most suitable hydraulic retention time yielding the highest hydrogen formation, the reactor was operated between HRT = 1 h and 8 h. The substrate was the acid hydrolyzed wheat powder (AHWP). Waste wheat was sieved down to 70 μm size (less than 200 mesh) and acid hydrolyzed at pH = 2 and 90 °C in an autoclave for 15 min. The sugar solution obtained from hydrolysis of waste wheat was used as substrate at the constant concentration of 15 g/L after neutralization and nutrient addition for biohydrogen production by dark fermentation. The microbial growth support particle was aquarium biological sponge (ABS). Heat-treated anaerobic sludge was used as inoculum. Total gas volume and hydrogen percentage in total gas, hydrogen gas volume, total sugar and total volatile fatty acid concentrations in the feed and in the effluent of the system were monitored daily throughout the experiments. The highest yield and rate of productions were obtained as Y_H2 = 645.7 mL/g TS and R_H2 = 2.51 L H₂/L d at HRT = 3 h, respectively.     

Biohydrogen production in anaerobic fluidized bed reactors: Effect of support material and hydraulic retention time

This study evaluated two different support materials (polystyrene and expanded clay) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBRs contained either polystyrene (R1) or expanded clay (R2) as support materials were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C and a pH of approximately 5.5. The AFBRs were operated with a range of hydraulic retention times (HRTs) between 1 and 8 h. For R1 with an HRT of 2 h, the maximum hydrogen yield (HY) was 1.90 mol H₂ mol⁻¹ glucose, with 0.805 mg of biomass (as total volatile solids, or TVS) attached to each g of polystyrene. For R2 operated at an HRT of 2 h, the maximum HY was 2.59 mol H₂ mol⁻¹ glucose, with 1.100 mg of attached biomass (as TVS) g⁻¹ expanded clay. The highest hydrogen production rates (HPR) were 0.95 and 1.21 L h⁻¹ L⁻¹ for R1 and R2, respectively, using an HRT of 1 h. The H₂ content increased from 16–47% for R1 and from 22–51% for R2. No methane was detected in the biogas produced throughout the period of AFBR operation. These results show that the values of HY, HPR, H₂ content, and g of attached biomass g⁻¹ support material were all higher for AFBRs containing expanded clay than for reactors containing polystyrene.     

Biohydrogen production,sludge granulation,and microbial community in an anaerobic inner cycle biohydrogen production (AICHP) reactor at different hydraulic retention times

This study investigated the continuous biohydrogen production in an anaerobic inner cycle biohydrogen production (AICHP) reactor fed with synthetic molasses wastewater as the model substrate under mesophilic conditions (37 ± 1 °C). The hydraulic retention times (HRTs) were set as 6.12, 4.90, 4.08, 3.50, and 3.06 h. Both maximum hydrogen production rate (HPR) (8.08 ± 0.48 L/L/d) and maximum granule formation were achieved at the HRT of 3.50 h. Acetic acid and butyric acid were the dominant metabolites in all tested HRTs throughout the experiment. Microbial community analysis showed that shortening the HRT promoted hydrogen production. This was mainly achieved by enhancing the growth of acetogenic bacteria in the AICHP reactor, rather than the growth of hydrogen-producing bacteria.     

High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition

Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis was investigated under thermophilic condition. The optimum chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g·L⁻¹ and 6.5 with a hydrogen yield of 73 mL-H₂·gCOD⁻¹. The dark fermentation effluent consisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was 0.7 V with a hydrogen yield of 163 mL-H₂·gCOD⁻¹. The hydrogen yield of continuous two-stage dark fermentation and microbial electrolysis was 236 mL-H₂·gCOD⁻¹ with a hydrogen production rate of 7.81 L·L⁻¹·d⁻¹. The hydrogen yield was 3 times increased when compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the microbial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under thermophilic condition is a highly promising option to maximize the conversion of palm oil mill effluent into biohydrogen.     

Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation

Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were compared in batch dark fermentations under mesophilic (37 °C) and thermophilic (55 °C) conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 °C and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H₂ h⁻¹) was obtained with the acid-hydrolyzed and the lowest (1.12 ml H₂ h⁻¹) with the boiled wheat at 55 °C. The highest hydrogen gas yield (333 ml H₂ g⁻¹ total sugar or 2.40 mol H₂ mol⁻¹ glucose) and final total volatile fatty acid (TVFA) concentration (10.08 g L⁻¹) were also obtained with the acid-hydrolyzed wheat under thermophilic conditions (55 °C). Dark fermentation of acid-hydrolyzed ground wheat under thermophilic conditions (55 °C) was proven to be more beneficial as compared to mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.     

Early warning indicators and microbial community dynamics during unstable stages of continuous hydrogen production from food wastes by thermophilic dark fermentation

Early warning indicators (EWIs) and microbial community dynamics during thermophilic dark fermentation-based hydrogen production (TFHP) from food wastes were investigated during stable and fluctuating operations in continuous systems. Difference and redundancy analyses revealed the linkage between dominant genera, metabolites and process parameters and characterized the EWIs and mechanism of instability of the TFHP. Besides biogas production rate (GPR), hydrogen proportion and volatile fatty acids (VFAs) concentrations, the potential warning function of the microbial community were observed. Thermoanaerobacterium was the dominant genus which decreased during the unstable stages. Bacillus is another EWI in unstable stages when the value of pH is lower. Lactobacillus and Weissella appeared as OLR increased to 9 g VS L⁻¹ d⁻¹ in the OLR-9 unstable stage. This study demonstrates the key EWIs for monitoring and controlling continuous hydrogen production and its stability.     

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

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

Biohydrogen production from 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.     

Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities

Hydrogen (H₂) production from simulated cheese processing wastewater via anaerobic fermentation was conducted using mixed microbial communities under mesophilic conditions. In batch H₂ fermentation experiments H₂ yields of 8 and 10 mM/g COD fed were achieved at food-to-microorganism (F/M)$(F/M)$ ratios of 1.0 and 1.5, respectively. Butyric, acetic, propionic, and valeric acids were the major volatile fatty acids (VFA) produced in the fermentation process. Continuous H₂ fermentation experiments were also performed using a completely mixed reactor (CSTR). The pH of the bioreactor was controlled in a range of 4.0–5.0 by addition of carbonate in the feed material. Maximum H₂ yields were between 1.8 and 2.3 mM/g COD fed for the loading rates (LRs) tested with a hydraulic retention time (HRT) of 24 h. Occasionally CH₄ was produced in the biogas with concurrent reductions in H₂ production; however, continuous H₂ production was achieved for over 3 weeks at each LR. The 16S rDNA analysis of DNA extracted from the bioreactors during periods of high H₂ production revealed that more than 50% of the bacteria present were members of the genus Lactobacillus and about 5% were Clostridia. When H₂ production in the bioreactors decreased concurrent reductions in the genus Lactobacillus were also observed. Therefore, the microbial populations in the bioreactors were closely related to the conditions and performance of the bioreactors.     

Biohydrogen production by a novel integration of dark fermentation and mixotrophic microalgae cultivation

Biohydrogen is usually produced via dark fermentation, which generates CO₂ emissions and produces soluble metabolites (e.g., volatile fatty acids) with high chemical oxygen demand (COD) as the by-products, which require further treatments. In this study, mixotrophic culture of an isolated microalga (Chlorella vulgaris ESP6) was utilized to simultaneously consume CO₂ and COD by-products from dark fermentation, converting them to valuable microalgae biomass. Light intensity and food to microorganism (F/M) ratio were adjusted to 150 μmol m⁻² s⁻¹ and F/M ratio, 4.5, respectively, to improve the efficiency of assimilating the soluble metabolites. The mixotrophic microalgae culture could reduce the CO₂ content of dark fermentation effluent from 34% to 5% with nearly 100% consumption of soluble metabolites (mainly butyrate and acetate) in 9 days. The obtained microalgal biomass was hydrolyzed with 1.5% HCl and subsequently used as the substrate for bioH₂ production with Clostridium butyricum CGS5, giving a cumulative H₂ production of 1276 ml/L, a H₂ production rate of 240 ml/L/h, and a H₂ yield of 0.94 mol/mol sugar.