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.     

Advances towards the understanding of microbial communities in dark fermentation of enzymatic hydrolysates: Diversity,structure and hydrogen production performance

Microbial communities involved in hydrogen (H₂) production from enzymatic hydrolysates of agave bagasse were analyzed through 16S rRNA sequencing. Two types of reactor configurations and four different enzymatic hydrolysates were evaluated. Trickling bed reactors led to highly-diverse microbial communities, but low volumetric H₂ production rates (VHPR, maximum: 5.8 L H₂/L-d). On the contrary, well-controlled environments of continuous stirred-tank reactors favored the establishment of low diverse microbial communities composed by Clostridium-Sporolactobacillus leading to high-performance H₂-production (VHPR maximum: 13 L H₂/L-d). Cellulase-Viscozyme and Celluclast-Viscozyme hydrolysates led to the co-dominance of Clostridium and Sporolactobacillus, possibly due to the presence of xylose and hemicellulose-derived carbohydrates. Cellulase hydrolysates were linked to communities dominated by Clostridium, while maintaining low abundance of Sporolactobacillus. Stonezyme hydrolysates favored microbial communities co-dominated by Clostridium-Lachnoclostridium-Leuconostoc. Moreover, contrary to the prevailing theory, it was demonstrated that H₂ production performance was inversely related to microbial diversity.     

Effects of pretreatment method of natural bacteria source on microbial community and bio-hydrogen production by dark fermentation

The effects of pretreatment method of cow dung compost, which was employed as natural hydrogen bacteria source, on the microbial community, population distribution of microbes and hydrogen production potential were investigated in the batch tests. The maximum hydrogen yield of 290.8 mL/L-culture appeared in the pretreated method A (infrared drying) by dark fermentation. The pretreated method of compost significantly affected microbial succession, population distribution of microbes. Both Clostridium sp. and Enterobacter sp. were found to be two species of preponderant hydrogen-producing bacteria, the next best was Bacteroides sp. and Veillonella sp., the last was Lactobacillus sp. and Streptococcus sp., which were also essential. The results showed that the mutualism and symbiosis relations of the mixed bacteria played a critical role in hydrogen fermentation process.     

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.     

Impact of the microbial inoculum source on pre-treatment efficiency for fermentative H2 production from glycerol

Hydrogen (H₂) production by dark fermentation can be performed from a wide variety of microbial inoculum sources, which are generally pre-treated to eliminate the activity of H₂-consuming species and/or enrich the microbial community with H₂-producing bacteria. This paper aims to study the impact of the microbial inoculum source on pre-treatment behavior, with a special focus on microbial community changes. Two inocula (aerobic and anaerobic sludge) and two pre-treatments (aeration and heat shock) were investigated using glycerol as substrate during a continuous operation. Our results show that the inoculum source significantly affected the pre-treatment efficiency. In aerobic sludge no pre-treatment is necessary, while in anaerobic sludge the heat pre-treatment increased H₂ production but aeration caused unstable H₂ production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and unstable H₂ production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H₂ production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8–3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H₂ mol⁻¹ glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H₂ mol⁻¹ glucose. The biogas produced was composed of H₂ and CO₂, and the H₂ content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H₂ fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1–53.3% and 37.7–44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs.     

Influence of seed sludge and pretreatment method on hydrogen production in packed-bed anaerobic reactors

Several studies have reported on the effects of inoculums source and pretreatment on biological hydrogen production. However, there have been few studies on continuous reactors. This paper investigated the influence of different seed sludge sources and pretreatment methods on biohydrogen production in up-flow anaerobic fixed-bed reactors fed with sucrose. The following inoculum sources were included in the study: (1) anaerobic sludge from an up-flow anaerobic sludge blanket (UASB) reactor used to treat poultry slaughterhouse wastewater (Sl), (2) anaerobic sludge from a UASB reactor used to treat swine wastewater (Sw) and (3) autofermentation (A). Heat (He) and acid (Ac) shock were used to increase hydrogen production and suppress hydrogen consumption. The average hydrogen yields (HY) in the experiment were 2.1 (A), 2.0 (SlHe), 2.0 (SlAc), 1.0 (Sl), 1.0 (SwAc), 0.7 (SwHe) and 0.7 (Sw) mol H₂ mol⁻¹ sucrose. Although heat shock produced the maximum HY value (SlHe), acid pretreatment (SlAc) resulted in more stable hydrogen production with the largest average value, which could be an advantage of using pH shock. The autofermentation process presented HY values similar to those produced with SlAc and SlHe, making it a suitable seed sludge for biohydrogen production because pretreatment was not required.     

A meta-analysis of research trends on hydrogen production via dark fermentation

This work provides a meta-analysis of the state-of-the-art research on H₂ and value-added products production from biomass, via Dark Fermentation (DF) between 2015 and 2019. The meta-analysis data clusters are created considering inputs (i.e., feedstocks, and microorganisms used in DF), process conditions (i.e., feedstock pretreatments, and temperature, pH, working volume, substrate concentration in DF), yield and productivity of H₂ and the most common by-products (i.e., acetic, lactic, butyric, propionic acids and ethanol). Agricultural and green residues were the most common feedstock (36.5%), followed by Aquatic biomass (29.8%). Pretreated feedstocks and mixed cultures were employed in 72% and 79% of the studies, respectively. The meta-analysis relates H₂ high productivity to 6 ≤ pH ≤ 6.8 and 35 °C ≤ T ≤ 37 °C and H₂ high yield to 5.5 ≤ pH ≤ 7.5 under mesophilic conditions. The paper elaborates on the production strategies tested at the laboratory scale for each of the DF-products mentioned above, highlighting the pros and cons towards improving yield and productivity and discussing what are the challenges to integrating DF in large-scale biorefining schemes for industrial production of H₂ and value-added products.     

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.     

Ultrasonic pretreatment of palm oil mill effluent: Impact on biohydrogen production,bioelectricity generation,and underlying microbial communities

Substrate bioavailabity is one of the critical factors that determine the relative biohydrogen (bioH₂) yield in fermentative hydrogen production and bioelectricity output in a microbial fuel cell (MFC). In the present undertaking, batch bioH₂ production and MFC-based biolectricity generation from ultrasonically pretreated palm oil mill effluent (POME) were investigated using heat-pretreated anaerobic sludge as seed inoculum. Maximum bioH₂ production (0.7 mmol H₂/g COD) and COD removal (65%) was achieved at pH 7, for POME which was ultrasonically pretreated at a dose of 195 J/mL. Maximum value for bioH₂ productivity and COD removal at this sonication dose was higher by 38% and 20%, respectively, than unsonicated treatments. In batch MFC experiments, the same ultrasound dose led to reduced lag-time in bioelectricity generation with concomitant 25% increase in bioelectricity output (18.3 W/m³) and an increase of COD removal from 30% to 54%, as compared to controls. Quantitative polymerase chain reaction (qPCR) tests on sludge samples from batch bioH₂ production reflected an abundance of gene fragments coding for both clostridial and thermoanaerobacterial [FeFe]-hydrogenase. Fluorescence in situ hybridization (FISH) tests on sludge from MFC experiments showed Clostridium spp. and Thermoanaerobacterium spp. as the dominant microflora. Results suggest the potential of ultrasonicated POME as sustainable feedstock for dark fermentation-based bioH₂ production and MFC-based bioelectricity generation.     

Effects of pretreatment in a vortex layer apparatus on the properties of confectionery wastewater and its dark fermentation

Pretreatment prior to anaerobic digestion is an effective option for increasing the biodegradability of organic waste. Vortex layer apparatus (VLA) is considered one of the promising types of equipment for pretreatment. In this work, confectionery wastewater (CW) was pretreated in VLA for 1 and 3 min before dark fermentative hydrogen production in anaerobic upflow biofilters. The pretreatment resulted in a slight increase in soluble chemical oxygen demand (COD), soluble sugars and acetic acid, and a decrease in the concentration of propionic, butyric and caproic acids. Due to the abrasion of steel needles in VLA, the concentration of iron in the pretreated CW increased by 2.57 times. Hydraulic retention time in anaerobic upflow biofilters was gradually reduced from 5.6 to 1.8 and 1.3 days, which corresponded to organic loading rate of 2.0, 6.3 and 8.8 kg COD/(m³ day). Although the highest hydrogen yield (96.2 ± 8.1 ml/g COD) was obtained for non-pretreated CW, the pretreatment contributed to a significant increase in methane yield (39.2 ± 2.5 ml/g COD), possibly due to higher iron content (1.8 ± 0.3 mg/L). The highest energy production rate (4407 J/(L day)) was achieved after 3 min CW pretreatment. Thus, pretreatment in VLA can be a promising method for improving the biohythane production process.     

Hydrogen production performance from food waste using piggery anaerobic digested residues inoculum in long-term systems

The objective of this study was to investigate the hydrogen production performance from food waste using piggery anaerobic digested residues (PADRs) inoculum. Multiple parameters were evaluated such as organic load rate (OLR), pH, and hydraulic retention time (HRT), over a wide range of values in long-term dark fermentation systems. Results showed that a value of 126.50 mL/gVS·d hydrogen yield was achieved at OLR 6 g VS/L·d under thermophilic condition. A relatively stable structural composition dominated by Thermoanaerobacterium was maintained even suffering from OLR and acid shock. On the contrary, mesophilic fermentation performed acetic acids accumulation and an average hydrogen yield of less than 80 mL/gVS·d. High OLR and low pH (range of 5.0–5.5) led to the establishment of Lactobacillus. Beyond this range, the relative abundance of Olsenella, Streptococcus, and other bacteria showed a significant difference under different operating conditions, which caused weak resistance to external shocks during mesophilic fermentation. It showed that PADRs was capable of obtaining optimal hydrogen production performance under thermophilic condition from food waste with a stable microbial community structure.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l⁻¹ d⁻¹ gave a maximum hydrogen yield of 0.27 l H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ l⁻¹ d⁻¹ (16.9 mmol l⁻¹ h⁻¹). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l⁻¹ d⁻¹) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Thermophilic versus mesophilic dark fermentation in xylose-fed fluidised bed reactors: Biohydrogen production and active microbial community

Dark fermentative biohydrogen production in a thermophilic, xylose-fed (50 mM) fluidised bed reactor (FBR) was evaluated in the temperature range 55–70 °C with 5-degree increments and compared with a mesophilic FBR operated constantly at 37 °C. A significantly higher (p = 0.05) H₂ yield was obtained in the thermophilic FBR, which stabilised at about 1.2 mol H₂ mol^?1 xylose (36% of the theoretical maximum) at 55 and 70 °C, and at 0.8 mol H₂ mol^?1 xylose at 60 and 65 °C, compared to the mesophilic FBR (0.5 mol H₂ mol^?1 xylose). High-throughput sequencing of the reverse-transcribed 16S rRNA, done for the first time on biohydrogen producing reactors, indicated that Thermoanaerobacterium was the prevalent active microorganism in the thermophilic FBR, regardless of the operating temperature. The active microbial community in the mesophilic FBR was mainly composed of Clostridium and Ruminiclostridium at 37 °C. Thermophilic dark fermentation was shown to be suitable for treatment of high temperature, xylose-containing wastewaters, as it resulted in a higher energy output compared to the mesophilic counterpart.     

Initial pH influences microbial communities composition in dark fermentation of scotta permeate

This study addresses for the first time the influence of initial pH on the evolution of microbial consortia in dark fermentation of scotta permeate, using a high-throughput sequencing approach. Three fermentation phases could be detected: 1) a lag phase with no substantial differences in microbial composition at different initial pH values; 2) an exponential H₂ production phase, accompanied by a general increase of Clostridium genus components and higher incidence of Trichococcus genus at neutral and alkaline pH; 3) a final stationary phase, characterized by a general increase of Bifidobacterium and Lactobacillus genera in all reactors. The initial pH value influenced the relative abundance of Trichococcus at 16–48 h of incubation. The metabolic activity of this genus increased the amount of metabolic precursors of H₂ so that, when pH lowered to 5.4, clostridia in the reactors with initial alkaline pH become more active H₂-producers than those in the others.     

Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

Effects of architectural changes and inoculum type on internal resistance of a microbial fuel cell designed for the treatment of leachates from the dark hydrogenogenic fermentation of organic solid wastes

A new design of a single chamber MFC-A based on extended electrode surface (larger σ, specific surface or surface area of electrode to cell volume) and the assemblage or ‘sandwich’ arrangement of the anode-proton exchange membrane-cathode (AMC arrangement) and a standard single chamber MFC-B with separated electrodes were tested with several inocula (sulphate-reducing, SR-In; methanogenic, M-In, and aerobic, Ab-In) in order to determine the effects on the internal resistance R_int and other electrical characteristics of the cells. In general, the R_int of the new design cell MFC-A was consistently lower than that of the standard MFC-B, for all inocula used in this work. Resistances followed the order R_int,SR-In < R_int,M-In ? R_int,Ab-In.These results were consistent with reports on reduction of ohmic resistance of cells by decreasing inter-electrode distance. Also, the volumetric power P_V output was higher for the MFC-A than for MFC-B; this was congruent with doubling the σ in the MFC-A compared to MFC-B. Yet, power density P_An delivered was higher for MFC-A only when operated with SR-In and Ab-In, but not with M-In. The MFC-A loaded with SR-In showed a substantial improvement in P_V (ca. 13-fold, probably due to the combined effects of increased σ and decreased of R_int) and a 6.4-fold jump in P_An compared to MFC-B. The improvement was higher than the expected improvement factors (or algebraic factors; 6.5 improvement expected for P_V due to combined effects of increase of σ and lowering the R_int; 3.25 improvement expected for P_An due to lowering the R_int).Our results point out to continuing work using the two-set, sandwich-electrode MFC and sulphate-reducing inoculum as a departing model for further studies on effects of inoculum enrichment and electrode material substitution on cell performance. Also, the MFC-A model seems to hold promise for future studies of bioelectricity generation and pollution abatement processing leachates produced during biohydrogen generation in dark fermentation processes of organic solid wastes.