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.     

Discontinuous biomass recycling as a successful strategy to enhance continuous hydrogen production at high organic loading rates

A novel strategy to discontinuously increase the biomass concentration in a continuous stirred-tank reactor was evaluated to enhance the performance of dark fermentation. Different concentrations of biomass were evaluated at organic loading rates (OLR) ranging from 90 to 160 g lactose/L-d with a hydraulic retention time (HRT) of 6 h. The study revealed that the discontinuous increase of biomass enhanced the hydrogen (H₂) production rates and carboxylic acids concentrations by 19–25% and 8–23%, respectively. In particular, a maximum H₂ production rate of 30.8 L H₂/L-d with carboxylic acids concentration of 20 g/L was reached at an OLR of 138 g lactose/L-d with a biomass concentration of 15 g volatile suspended solids/L. The analysis of microbial communities showed the co-dominance of Clostridium and lactic acid bacteria. Overall, the discontinuous increase of biomass was an effective strategy to improve the performance of suspended-biomass reactors operated at high OLR and low HRT.     

Cell wash-out enrichment increases the stability and performance of biohydrogen producing packed-bed reactors and the community transition along the operation time

Most of the fermentative hydrogen production studies based on mixed cultures have shown enrichment of the microbial community by means of a heat treatment. This heat treatment enrichment strategy selects for Clostridum spp., an efficient hydrogen producer; however, other bacteria that may contribute to the systems performance could be excluded. Another enrichment strategy based on high dilution rates selects different taxonomic groups, which may affect hydrogen production and the system stability. In this work, two enrichment strategies were evaluated, heat shock and cell wash-out, for hydrogen production and the system stability in continuous stirred reactors. The enriched communities were then inoculated in packed bed reactors and operated up to 70 days. Both strategies selected hydrogen producing bacteria, mainly Clostridium spp. The highest hydrogen production rate (6.01 L H₂/L-d), molar yield (1.29 mol H₂/mol glucose_consumed), and stability were achieved by the wash-out procedure; this high performance was attributed to facultative bacteria like Lactobacillus and Lactococcus. Furthermore, there was a transition within the community (along the operation time in the reactor with cell wash-out inoculum) and a selection for methanogenic activity (due to the long solids retention time).     

Plant-associated microbial communities converge in fermentative hydrogen production and form a core microbiome

This study presents the taxonomic characterization and fermentative activity of bacteria and fungi present in four plant-associated microbial communities: corn stover (CS), wheat straw (WS), sugarcane bagasse (SCB), and agave bagasse (AB). Fiber soaking, fermentation number, and organic loading rate (OLR) were studied in semicontinuous reactors fed with untreated substrates under a consolidated bioprocessing approach. WS, SCB, and AB communities converged to a core microbiome with the predominance of Lactobacillus, Clostridium, Enterobacter, and the fungus Pichia. After the fourth fermentation, hydrogen productivity became stable and similar among microbial communities (1.62 L-H₂/kg-day), except for the CS community. The OLR increase promoted the hydrogen production from lactate and acetate. Lactobacillus positively correlated with H₂ productivity and polysaccharide degradation, and Clostridium positively correlated only with polysaccharide degradation. Results demonstrated that Lactobacillus played a key role in producing high hydrogen productivities from lignocellulosic substrates. While the OLR increase promoted metabolic pathways that favored fermentative hydrogen production.     

Microbial ecology of a lactate-driven dark fermentation process producing hydrogen under carbohydrate-limiting conditions

Despite the high prevalence of lactic acid bacteria in dark fermentation (DF) processes, their ecological role is not yet completely elucidated, preventing their systematic use as “helpers” for hydrogen production. The aim of this study was to investigate the microbial community structure of a lactate-driven DF process that successfully produced hydrogen under carbohydrate-limiting conditions using tequila vinasse as a substrate. Microbial responses to stepwise decreases in hydraulic retention time (HRT) from 24 to 4 h were assessed by using Illumina MiSeq sequencing. HRTs above 12 h and below 6 h led to a lower hydrogen production rate (HPR; 0.2–3.3 L/L-d) and process stability (HPR variations within 25–65%), which were associated with the presence of Acetobacter lovaniensis, Clostridium luticellari, Blautia coccoides, and the high abundance of propionate and lactate. Interestingly, transient conditions from unsteady-to-steady state occurred at an HRT of 12 h, where species richness and evenness decreased remarkably. Accordingly, HRTs between 12 and 6 h resulted in higher HPRs of up to 11.7 ± 0.7 L/L-d with HPR variations of less than 10%, which closely matched with the dominance of Clostridium sp., and butyrate and acetate as the main aqueous products. Overall, the results indicate that the successfulness of exploiting the ‘unwanted’ LAB proliferation through lactate-driven DF processes requires the enrichment of lactate-consuming and hydrogen-producing bacteria, which entails the selection of proper biocatalysts and operating conditions/strategies such as the operation of DF reactors under carbohydrate-limiting conditions and low HRTs.     

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.     

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.     

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.     

Hydrogen production from acid and enzymatic oat straw hydrolysates in an anaerobic sequencing batch reactor: Performance and microbial population analysis

Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H₂/mol sugar consumed) and hydrogen production rate (HPR, 278 mL H₂/L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H₂/mol sugar consumed and 29.6 mL H₂/L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.     

Reactor start-up strategy as key for high and stable hydrogen production from cheese whey thermophilic dark fermentation

Using the right start-up strategy can be vital for successful hydrogen production from thermophilic dark fermentation (55 °C), but it needs to be affordable. Hence, three start-up strategies modifying only influent concentration and temperature were assessed in a reactor fed with cheese whey: (i) high temperature (55 °C) and a high organic loading rate (OLR_A - 15 kgCOD m^?3 d^?1) right at the beginning of the operation; (ii) slowly increasing temperature up to 55 °C using a high OLR_A and (iii) slowly increasing temperature and OLR_A up to the desired condition. Strategy (iii) increased hydrogen productivity in 39% compared to the others. The combination of high temperature and low pH thermodynamically favored H₂ producing routes. Synergy between Thermoanaerobacterium and Clostridium might have boosted hydrogen production. Three reactors of 41 m³ each would be needed to treat 3.4 × 10³ m³ year^?1 of whey (small-size dairy industry) and the energy produced could reach 14 MWh month^?1.     

Performance and composition of bacterial communities in anaerobic fluidized bed reactors for hydrogen production: Effects of organic loading rate and alkalinity

This study evaluated the effects of the organic loading rate (OLR) and pH buffer addition on hydrogen production in two anaerobic fluidized bed reactors (AFBRs) operated simultaneously. The AFBRs were fed with glucose, and expanded clay was used as support material. The reactors were operated at a temperature of 30 °C, without the addition of a buffer (AFBR1) and with the addition of a pH buffer (AFBR2, sodium bicarbonate) for OLRs ranging from 19.0 to 140.6 kg COD m⁻³ d⁻¹ (COD: chemical oxygen demand). The maximum hydrogen yields for AFBR1 and AFBR2 were 2.45 and 1.90 mol H₂ mol⁻¹ glucose (OLR of 84.3 kg COD m⁻³ d⁻¹), respectively. The highest hydrogen production rates were 0.95 and 0.76 L h⁻¹ L⁻¹ for AFBR1 and AFBR2 (OLR of 140.6 kg COD m⁻³ d⁻¹), respectively. The operating conditions in AFBR1 favored the presence of such bacteria as Clostridium, while the bacteria in AFBR2 included Clostridium, Enterobacter, Klebsiella, Veillonellaceae, Chryseobacterium, Sporolactobacillus, and Burkholderiaceae.     

Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis

A two-stage dark-fermentation and electrohydrogenesis process was used to convert the recalcitrant lignocellulosic materials into hydrogen gas at high yields and rates. Fermentation using Clostridium thermocellum produced 1.67 mol H₂/mol-glucose at a rate of 0.25 L H₂/L-d with a corn stover lignocellulose feed, and 1.64 mol H₂/mol-glucose and 1.65 L H₂/L-d with a cellobiose feed. The lignocelluose and cellobiose fermentation effluent consisted primarily of: acetic, lactic, succinic, and formic acids and ethanol. An additional 800 ± 290 mL H₂/g-COD was produced from a synthetic effluent with a wastewater inoculum (fermentation effluent inoculum; FEI) by electrohydrogensis using microbial electrolysis cells (MECs). Hydrogen yields were increased to 980 ± 110 mL H₂/g-COD with the synthetic effluent by combining in the inoculum samples from multiple microbial fuel cells (MFCs) each pre-acclimated to a single substrate (single substrate inocula; SSI). Hydrogen yields and production rates with SSI and the actual fermentation effluents were 980 ± 110 mL/g-COD and 1.11 ± 0.13 L/L-d (synthetic); 900 ± 140 mL/g-COD and 0.96 ± 0.16 L/L-d (cellobiose); and 750 ± 180 mL/g-COD and 1.00 ± 0.19 L/L-d (lignocellulose). A maximum hydrogen production rate of 1.11 ± 0.13 L H₂/L reactor/d was produced with synthetic effluent. Energy efficiencies based on electricity needed for the MEC using SSI were 270 ± 20% for the synthetic effluent, 230 ± 50% for lignocellulose effluent and 220 ± 30% for the cellobiose effluent. COD removals were ∼90% for the synthetic effluents, and 70–85% based on VFA removal (65% COD removal) with the cellobiose and lignocellulose effluent. The overall hydrogen yield was 9.95 mol-H₂/mol-glucose for the cellobiose. These results show that pre-acclimation of MFCs to single substrates improves performance with a complex mixture of substrates, and that high hydrogen yields and gas production rates can be achieved using a two-stage fermentation and MEC process.     

Co-fermentation of glucose,starch, and cellulose for mesophilic biohydrogen production

The aim of this study was to assess the synergistic effects of co-fermentation of glucose, starch, and cellulose using anaerobic digester sludge (ADS) on the biohydrogen (H₂) production and the associated microbial communities. Yields of H₂ were 1.22, 1.00, and 0.15 mol H₂/mol hexose-added in fermentation reactions containing glucose, starch, and cellulose as mono-substrates. The H₂ yields were greater by an average of 27 ± 4% than expected in all the different co-substrate conditions, which affirmed that co-fermentation of different substrates improved the H₂ potential. Glucose addition to starch and/or cellulose favored acetate synthesis, while cellulose degradation was associated with the propionate synthesis pathway. Illumina sequencing technology and bioinformatics analyses revealed operational taxonomic units (OTUs) in the Phyla Bacteroides, Chloroflexi, Firmicutes, Proteobacteria, Spirochaetes, Synergistes, and Thermotogae were common in mono- and co-substrate batches. However, OTUs in the Phyla Acidobacteria, Actinobacteria, and Bacteroidetesee were unique to only the co-substrate conditions.     

Different start-up strategies to enhance biohydrogen production from cheese whey in UASB reactors

The effect of different operational strategies and inoculum structure (granules and disaggregated granules) during the start-up of four up-flow anaerobic sludge blanket hydrogenogenic reactors was investigated. The more stable volumetric hydrogen production rates obtained were 0.38 and 0.36 L H₂/L-d, in reactors operated with a constant organic loading rate (OLR) with both inoculum structures, whereas in reactors operated with an increasing OLR methane started to be produced earliest in time. Specific hydrogenogenic activity results proved that the disaggregated inoculum produced a more active biomass than the granular one, but not granule formation was evident. The methane hydrogenotrophic activity was the main limitation of the systems evaluated. In the reactors inoculated with disaggregated sludge the start-up strategy did not influence the bacterial DGGE fingerprint, in contrast to the reactors started-up with granular sludge; members of the Clostridium genus were always present. The results demonstrated that operational conditions during the start-up period are crucial for the production of hydrogenogenic biomass.     

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.     

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.     

Glucose electro-fermentation as main driver for efficient H2-producing bacteria selection in mixed cultures

Electro-fermentation has been recently proposed as a new operational mode of bioprocess control using polarized electrodes. This paper aims to evaluate how polarized electrodes are affecting microbial metabolic fermentative pathways, with a special focus on how the bacterial populations are affected during hydrogen production by dark fermentation. Four different potentials were applied on the working electrode in batch electro-fermentation tests operated with mixed culture and using glucose as a substrate. Two different metabolic behaviours for H₂ production were observed in electro-fermentation. The first one led to a higher H₂ production compared to conventional fermentation with a strong selection of Clostridium sp. The second behaviour led to lower H₂ production along with ethanol, and strongly correlated with the selection of Escherichia and Enterobacter genera. However, the effect of the applied potential on population selection was mostly non-linear and no simple relationship was found between these two parameters. Overall, electro-fermentation process has shown its potential as a new type of control for mixed-culture bioprocesses with significant effects of polarized electrodes on glucose fermentation.     

Mitigating the variability of hydrogen production in mixed culture through bioaugmentation with exogenous pure strains

Process stability is a key operational issue when operating dark fermentation with mixed microbial cultures for hydrogen production. This study aimed at mitigating the instability of hydrogen production by separately adding exogenous pure strains suspected to have key roles in fermentative cultures. Among them, Clostridium acetobutylicum, Clostridium pasteurianum and Lactobacillus bulgaris which became predominant within the mixed culture strongly reduced the spectrum of produced metabolites and H₂ production variability. Interestingly, Escherichia coli and Cupriavidus necator, which remained in minor abundance, maintained a high and stable H₂ production while lowering the metabolic variability. 16S rRNA revealed that this could correlate to a simplification of the microbial diversity and the non-emergence of spore-forming competitors such as Sporolactobacillus sp. These results illustrate the potential beneficial role of minor OTUs as keystone species on H_2-producing complex ecosystem and support the possibility of using them to engineer the ecosystem and maintain high and stable performances.     

Continuous H2 production during fermentation of the carbon components of lignocellulose hydrolysates: Insight into the influence of pH conditions on the conversion efficiency of individual sugars

Sugars released from lignocellulose biomass are a promising substrate for biohydrogen production. This study evaluates the effect of pH controlled between 4.0 and 7.5 on continuous dark-fermentative H₂ production from the mixture of cellobiose, xylose and arabinose. High hydrogen production rate was obtained for pH values between 6.0 and 7.0 with a maximum of 7.41 ± 0.16 L/L-d at pH 7.0. On the other hand, the highest H₂ yields of around 1.74 ± 0.02 mol/mol_consumed were obtained at pH 4.5, 5.0 and 6.0. Cellobiose was completely utilized in nearly the entire pH range, while the highest consumption of xylose and arabinose was obtained at pH 6.0 and 7.0, respectively. It shows the challenges in selecting optimum pH for fermentation of mixed sugars. Significant impact of pH conditions on the microbial structure was observed. Between pH 4.0 and 7.0 Clostridium genus dominated the consortium, while above pH 7.0 relative abundance of Bacillus genus increased significantly.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.