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.     

Auto-selection of microorganisms of sewage sludge used as an inoculum for fermentative hydrogen production from different substrates

When mixed organic waste is used for hydrogen production by dark fermentation, the microbial community which is most adapted to the actual biopolymer composition of the substrate is auto-selected. In this research, six substrates simulating different biopolymers (proteins, fats, carbohydrates) and their mixtures were used to enrich hydrogen-producing bacteria adapted to these substrates from non-pretreated sewage sludge. Phylum Firmicutes dominated in the microbial community (67–100%) regardless of the substrate used, as was shown by high-throughput sequencing. Microbial diversity was low when using carbohydrate-rich substrates and the microbial community was mainly represented by Ruminococcus (26–90%) and Thermoanaerobacterium (6–67%). Dark fermentation of fats and proteins was characterized by higher microbial diversity. Thermoanaerobacterium (21%), Thermobrachium (19%), Tepidiphilus (16%) and Acetomicrobium (14%) dominated when using fats, while Thermobrachium (34%), Acetomicrobium (16%) and Clostridium sensu stricto 7 (12%) dominated when using proteins, as substrate. Different microbial communities and substrates resulted in diverse process performance and metabolic pathways. Dark fermentation of starch achieved the maximum hydrogen yield of 138 mL/g volatile solids with 60.4% hydrogen content in biogas. The dominance of genus Ruminococcus was thought to be responsible for the highest hydrogen production. Minor quantities of methane from proteins and fats were produced by Methanothermobacter and Methanosarcina. Based upon the stable ¹³C isotope analysis, the hydrogenotrophic pathway was a slightly more predominant methane formation route than the others considered.     

Coupling dark fermentation and microbial electrolysis to enhance bio-hydrogen production from agro-industrial wastewaters and by-products in a bio-refinery framework

The aim of this work is to evaluate biohydrogen production from agro-industrial wastewaters and by-products, by combining dark fermentation and microbial electrolysis in a two-step cascade process. Such coupling of both technologies constitutes a technological building block within a concept of environmental biorefinery where sustainable production of renewable energy is expected.Six different wastewaters and industrial by-products coming from cheese, fruit juice, paper, sugar, fruit processing and spirits factories were evaluated for the feasibility of hydrogen production in a two-step process. The overall hydrogen production when coupling dark fermentation and microbial electrolysis was increased up to 13 times when compared to fermentation alone, achieving a maximum overall hydrogen yield of 1608.6 ± 266.2 mLH₂/gCOD_consumed and a maximum of 78.5 ± 5.7% of COD removal.These results show that dark fermentation coupled with microbial electrolysis is a highly promising option to maximize the conversion of agro-industrial wastewaters and by-products into bio-hydrogen.     

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.     

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.     

Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge

This paper is a comprehensive review of H₂ consumption during anaerobic mixed culture H₂ dark fermentation with a focus on homoacetogenesis. Homoacetogenesis consumed from 11% to 43% of the H₂ yield in single and repeated batch fermentations, respectively. However, its quantification and extent during continuous fermentation are still not well understood. Models incorporating thermodynamic and kinetic controls are required to provide insight into the dynamic of homoacetogenesis during H₂ dark fermentation. Currently, no adequate method exists to eliminate homoacetogenesis because it does not depend on the culture's source, pre-treatment, substrate, type of reactor, or operation conditions. Controlling CO₂ concentrations during dark fermentation needs further investigation as a potential strategy towards controlling homoacetogenesis. Incorporating radioactive labeling technique in H₂ fermentation research could provide information on simultaneous production and consumption of H₂ during dark fermentation. Genetic studies investigating blocking H₂ consuming pathways and enhancing H₂ evolving hydrogenases are suggested towards controlling homoacetogenesis during dark fermentation.     

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.     

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.     

Enhanced hydrogen production in a UASB reactor by retaining microbial consortium onto carbon nanotubes (CNTs)

Biohydrogen and subsequent biomethane generation from biomass is a promising strategy for renewable energy supply, because this combination can lead to higher energy recovery efficiency and faster fermentation than single methane fermentation. Microbial consortium control by retaining hydrogen-producers through the addition of microbial carriers is an alternative to constructing hydrogen-producing reactors. Here we report the use of carbon nanotubes (CNTs) as microbial carriers to enhance microbial retention and the production of biohydrogen. Laboratory-scale upflow anaerobic sludge blanket (UASB) reactors with CNTs at 100 mg/L achieved a maximal hydrogen production rate of 5.55 L/L/d and a maximal hydrogen yield of 2.45 mol/mol glucose. Compared to frequently used activated carbon (AC) particles, CNTs resulted in quicker startup and better performance of hydrogen fermentation in UASB reactors. Scanning electron microscopy (SEM) and pyrosequencing results revealed that the reactor with CNTs led to a high proportion of hydrogen-producing bacteria among the microbial consortium, which endowed the microbes with strong flocculation capacity and hydrogen productivity.     

Silage as source of bacteria and electrons for dark fermentative hydrogen production

In this study, grass silage was used both as a source of bacteria and as a substrate for dark fermentative hydrogen production. Silage is produced by lactic acid fermentation controlled by end point pH (<4.0). In this study, the fermentation of silage was successfully continued and directed to hydrogen production by neutralizing the pH. Highest hydrogen yield of 37.8 ± 5.8 mL H₂/g silage was obtained at 25 g/L of silage. The main soluble metabolites were acetate and butyrate with the final concentrations of 1.5 ± 0.2 and 0.5 ± 0.0 g/L, respectively. Bacteria present (at 25 g silage/L) included Ruminobacillus xylanolyticum, Acetanaerobacterium elongatum and Clostridium populeti and were involved in silage fermentation to hydrogen. In summary, this work demonstrates that grass silage becomes amenable to hydrogen fermentation by indigenous silage bacteria through pH neutralization.     

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.     

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.     

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.     

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.     

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.     

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 productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate

Hydrogen productivities of different photosynthetic bacteria have been searched on real thermophilic dark fermentation effluents (DFE). The results obtained with potato steam peels hydrolysate (PSP) DFE were compared to glucose DFE. Photobiological hydrogen production has been carried out in indoor, batch photobioreactors using several strains of purple non-sulfur (PNS) bacteria such as Rhodobacter capsulatus (DSM1710), Rhodobacter capsulatus hup- (YO3), Rhodobacter sphaeroides O.U.001 (DSM5864), Rb. sphaeroides O.U.001 hup- and Rhodopseudomonas palustris.The efficiency of photofermentation depends highly on the composition of the effluent and the PNS bacterial strain used. Rb. sphaeroides produced the highest amount of hydrogen on glucose DFE. Rb. capsulatus gave better results on PSP DFE. This study demonstrates that photobiological hydrogen production with high efficiency and productivity is possible on thermophilic dark fermentation effluents. Consequently, a sequential operation of dark fermentation and photofermentation is a promising route to produce hydrogen, and it provides a higher hydrogen yield compared to single step processes.     

Predictive functional profiling of microbial communities in fermentative hydrogen production system using PICRUSt

PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) is based on 16S rRNA sequencing data, which can analyze the microbial functions in fermentation system. In this study, PICRUSt was adopted to figure out the direct evidence of functions of the microbial community in biohydrogen production system. PICRUSt analysis demonstrated that metabolic flux shifted from acid-producing pathway to hydrogen-producing pathway, and the hydrogen-consuming homoacetogenic pathway was eliminated by ionizing radiation pretreatment at 5 kGy. KEGG (Kyoto Encyclopedia of Genes and Genomes) based functional genes analysis showed enriched energy metabolism and diminished homoaceto gene, which enhanced the hydrogen production. However, the diminished carbohydrate metabolism indicated that the pretreatment reduced the activity of microbial consortia to degrade various substrates. This study suggested that PICRUSt is an effective approach to analyze the functional profiling of microbial community in fermentative hydrogen production system.     

Improvement of biohydrogen production from glycerol in micro-oxidative environment

Glycerol is a highly available by-product generated in the biodiesel industry. It can be converted into higher value products such as hydrogen using biological processes. The aim of this study was to optimize a continuous dark fermenter producing hydrogen from glycerol, by using micro-aerobic conditions to promote facultative anaerobes. For that, hydrogen peroxide (H₂O₂) was continuously added at low but constant flow rate (0.252 mL/min) with three different inlet concentrations (0.2, 0.4, and 0.6% w/w). A mixture of aerobic and anaerobic sludge was used as inoculum. Results showed that micro-oxidative environment significantly enhanced the overall hydrogen production. The maximum H₂ yield (403.6 ± 94.7 mmolH₂/molGly_consumed) was reached at a H₂O₂ concentration of 0.6% (w/w), through the formate, ethanol and butyrate metabolic pathways. The addition of H₂O₂ promoted the development of facultative anaerobic microorganisms such as Klebsiella, Escherichia-Shigella and Enterococcus sp., likely by consuming oxygen traces in the medium and also producing hydrogen. Despite the micro-oxidative environment, strict anaerobes (Clostridium sp.) were still dominant in the microbial community and were probably the main hydrogen producing species. In conclusion, such micro-oxidative environment can improve hydrogen production by selecting specific microbial community structures with efficient metabolic pathways.