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Winery effluents are agro-industrial wastes that can be treated to produce methane in anaerobic digestion systems. Understanding how the process configuration and operating conditions affect the microbial communities and their metabolism is essential to improve the performance of these systems. This work aimed to compare the taxonomic and functional profiles of four process configurations of an anaerobic digestion system treating winery effluents. Taxonomic profiles were obtained by sequencing the 16S rRNA gene, and the Tax4Fun2 package was used to predict the functional profiles. Different genera predominated under three configurations in the acidogenic reactor, Sporolactobacillus, Prevotella and Acetobacter, respectively; however, the analysis indicated high functional redundancy among configurations. Methane production by the acetoclastic pathway was mainly related to Methanosaeta in the single-stage configuration and related to five different archaea genera in the two-stage configuration. Syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis predominated under thermophilic conditions, where Methanothermobacter had up to 65% of the relative abundance. The two-stage configuration was more stable due to the higher microbial diversity and functional redundancy than the single-stage; the microbial community in mesophilic conditions also had higher microbial diversity and functional redundancy than in thermophilic conditions; finally, applying harsher operating conditions reduces the microbial diversity and the stability of the process. This analysis provided valuable knowledge to understand and improve methane production in anaerobic digestion systems.

The capacity of a heat-treated sludge (HTS) to produce hydrogen from the mono- and co-digestion of corn (NW, pH 13.1) and brewery (BW, pH 3.8) wastewater was evaluated. The co-digestion of NW and BW was conducted with ratios (NW/BW) from 40/60 to 80/20 (vol/vol) at pH 6 and under different initial pH values (from 5.8 to 12.3) according to the substrates mixtures. With the initial pH adjusted to 6, the highest production (302 mL) occurred for the mono-digestion of NW, but hydrogen was produced in all incubations. For incubations under variable pH values, the highest hydrogen production was obtained with the ratio 60/40 (270 mL), followed by the ratio 65/35 (260 mL) with pH values of 10.8 and 10.4, respectively. The initial pH influenced the kinetic parameters, especially on maximum production and lag phase. For the ratio 60/40, with an alkaline pH value (10.4), the lag phase was delayed up to 122 h, but the highest volume of hydrogen was obtained with this condition. The Clostridium genus, present in all samples, could be associated as the main responsible for hydrogen production. Besides, the presence of Burkholderia genus, previously related to hydrogen production, was identified as the main involved in the culture at pH 10.8.

The relationship between the taxonomic diversity and the function of microbial communities is complex. Specifically, the ecological mechanisms that drive the dynamics of microbial populations and the consequences of these dynamics on functional traits have remained elusive. Among the simplest but natural microbial communities are dark fermentation consortia, a subset of the more diverse and complex microbial communities, anaerobic digestion communities. Dark fermentation consortia have been of interest as they produce biofuels such as hydrogen and different alcohols that can be used as fossil fuels alternatives. However, these hydrogen-producing communities have unresolved instability and low yield issues. We have previously proposed that instability and low yields in dark fermentation communities could be due to reduced diversity that results from aggressive pretreatments of original anaerobic digestion communities. In this work, we used dark fermentation communities to examine experimentally the effect of diversity reduction in functional traits, including stability and microbial interactions. We established two types of treatment, (i) maintaining strict culture conditions that are known to induce hydrogen production and ii) applying a heat-shock treatment known for selecting hydrogen-producing bacteria, which resulted in two types of communities, high and low diversity. Each treatment consisted of 12 replicates that were transferred to fresh medium daily (during 28 days for the non-treated bioreactors and 61 days for the heat-shock treated bioreactors). Microbial communities of the two treatments were characterized in their function as well as resistance to invasion. Microbial composition was characterized by culture-independent 16S rRNA gene amplicon sequencing. We analyzed microbial community composition and function through time, establishing statistical relationships between bacterial groups and metabolite production. Also, we inferred the potential ecological interactions that might have been established. Results show that the replicate bioreactors for each treatment predictably shifted to a similar composition and increased and stable biogas production. The non-treated bioreactors showed less susceptibility to the invasion (with the invasive bacteria establishing only in one replicate in the non-treated bioreactors vs 6 invaded replicates of the heat-shock treated bioreactors). However, the effect observed in the non-treated bioreactor replicate where the invader bacteria established was more drastic since the invasive bacteria managed to become dominant.

Wine production has increased in recent years, especially in developing countries such as Mexico. This increase is followed by an increase of winery effluents that must be treated to avoid environmental risks. However, little information is available about the characteristics of these effluents and the possible treatments. This paper aimed to characterize the effluents and by-products generated by the Mexican winery industry and to evaluate the performance and stability of the anaerobic treatment using a single-stage and a two-stage process. Results showed that the winery effluents had a high content of biodegradable organic matter, with chemical oxygen demand (COD) values ranging from 221 to 436 g COD/L. The single-stage anaerobic process was able to treat an organic loading rate of 9.6 kg COD/(m3 d); however, it was unstable and highly dependent on the addition of bicarbonate alkalinity (0.31 g NaHCO3/g COD removed). The two-stage process was more stable working at a higher organic load (12.1 kg COD/(m3 d)) and was less dependent on the addition of bicarbonate (0.17 g NaHCO3/g COD removed). The results highlight the potential of the winery effluents to produce methane through anaerobic digestion in a two-stage process, making wine production more sustainable.

BACKGROUND: Fermentative hydrogen production has an inherent limitation caused by hydrogen‐consuming metabolic pathways such as homoacetogenesis related to high hydrogen partial pressures. In this study, a strategy based on recirculating the headspace gas was applied to increase the hydrogen release from the liquid to the gas phase in an upflow anaerobic sludge blanket(UASB) reactor fed with winery effluents. The influence of the gas upflow recirculation velocity on hydrogen production, hydrogen consumption by homoacetogenesis, and microbial community structure was evaluated. RESULTS: Under the control condition (only liquid recirculation), the hydrogen productivity was as much as 22 mL H₂ L⁻¹ h⁻¹. Conversely, the hydrogen productivity increased up to 62 mL H₂ L⁻¹ h⁻¹ when the reactor was operated with an upflow gas recirculation velocity of 28.6 m d⁻¹. The increase in mass transfer, due to the gas recirculation, produces a decrease (up to 70%) in the hydrogen consumption rate associated with homoacetogenesis. High‐throughput 16s rDNA sequencing characterization showed that the gas recirculation strategy promoted the development of hydrogen‐producing microorganisms related to Megasphaera elsdenii and decreased the abundance of hydrogen‐consuming bacteria related to Clostridium carboxidivorans and C. ljungdahlii. CONCLUSION: The results indicated that headspace recirculation at gas upflow velocities higher than 17.8 m d⁻¹ increased the hydrogen production rate concomitantly with the reduction of both the homoacetogenic activity and the abundance of H₂‐consuming bacteria. The study demonstrated that headspace recirculation could be a promising way to control homoacetogenesis, and therefore, to increase the biohydrogen productivity from complex substrates such as winery effluents. © 2019 Society of Chemical Industry

International audience; Hydrogen production by dark fermentation is an emerging technology of increasing interest due to its renewable feature. Recent scientific advances have well investigated the operational conditions to produce hydrogen through the valorization of several wastes or wastewaters. However, the development of standardized protocols to accurately assess the biohydrogen potential (BHP) is of crucial importance. This work is the first interlaboratory and international effort to validate a protocol estimating hydrogen potential using batch tests, using glucose as individual model substrate. The repeatability of the hydrogen potential (HP) increased with variations of the proposed protocol: reducing substrate concentration, increasing the buffer capacity, and using an automatic device. The interlaboratory variation of the HP was reduced from 32 to 12%, demonstrating the reproducibility and robustness of the proposed protocol. Recommendations to run BHP tests were formulated in terms of i) repeatability and reproducibility of results, ii) criteria for results validation and acceptance, iii) workload of the proposed protocols.

The heat-shock pretreatment (HST) is a useful method to select for H 2 -producing inocula when soluble substrates are employed. However, the HST has proven to have negative effects on the H 2 production performance from lignocellulosic substrates. We hypothesize that the negative effect of HST on H 2 production from lignocellulosic substrates is due to the loss of species involved in cellulose solubilization. In the present study, we tested this hypothesis by applying a heat-shock pretreatment (105 °C/24 h) on the microbial community for producing hydrogen from microcrystalline cellulose. Specifically, we compared a microbial community treated with 2-bromoethanesulfonate (BES-treated control) versus a heat-shock pretreated microbial community. For both experimental treatments, we determined the major fermentation products (hydrogen, acetic, butyric, propionic, and isovaleric acids), as well as the diversity of bacteria and fungi using Illumina MiSeq of amplicons in five sampling points. We found that HST immediately reduced alpha diversity of microbial communities, being fungi more affected than bacteria. We also found that the bacterial reduction in Comamonas , Ureibacillus , and Aneurinibacillus was related to a low hydrogen production in the heat-shock pretreated community. Strictly anaerobic fungi such as Orpinomyces , Cyllamyces , and Neocallimastix , which are recognized by their role in solubilization of fibrous materials, were unable to survive the HST. The reconstructed bacterial network predicted positive interactions between cellulase-producing and hydrogen-producing families. We conclude that the HST did not promote the high microbial diversity required for hydrogen production from cellulose.

The current pandemic COVID-19 caused by the coronavirus SARS-CoV-2, has generated different economic, social and public health problems. Moreover, wastewater-based epidemiology could be a predictor of the virus rate of spread to alert on new outbreaks. To assist in epidemiological surveillance, this work introduces a simple, low-cost and affordable electrochemical sensor to specifically detect N and ORF1ab genes of the SARS-CoV-2 genome. The proposed sensor works based on screen-printed electrodes acting as a disposable test strip, where the reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction takes place. Electrochemical detection relies upon methylene blue as a redox intercalator probe, to provide a diffusion-controlled current encoding the presence and concentration of RT-LAMP products, namely amplicons or double-stranded DNA. We test the performance of the sensor by testing real wastewater samples using end-point and time course measurements. Results show the ability of the electrochemical test strip to specifically detect and quantify RT-LAMP amplicons below to ~ 2.5 × 10

This work proves the feasibility of employing regular secondary activated sludge for the enrichment of a microbial community able to perform the anaerobic oxidation of methane coupled to nitrate reduction (N-AOM). After 96 days of activated sludge enrichment, a clear N-AOM activity was observed in the resulting microbial community. The methane removal potential of the enriched N-AOM culture was then studied in a stirred tank reactor (STR) operated in continuous mode for methane supply and semi-continuous mode for the liquid phase. The effect of applying nitrate loads of ∼22, 44, 66, and 88 g NO

Anaerobic digestion has been widely used in recent years for the treatment of agro-industrial or municipal wastes as carbon source to obtain alternative energy sources such as methane (CH4). Great efforts are currently being made to understand this phenomenon, generally as a first approximation in batch experiments, but due to the growing need to produce more environmentally friendly fuels, it is imperative to study the process as a continuous production. Starting from a three-state model and its parametric characterization in batch experiments, this paper analyzes the effect of the variation of two parameters of the mathematical model from continuous balances: the dilution rate (D) and the substrate feeding concentration (Si). The increase in the concentration of substrate in the inflow allows a greater production of methane, while small values allow reaching the trivial solution in the system. The choice of the dilution rate can be solved as an optimization problem since the production reaches a maximum at values close to 4 d_1.

To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor. One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8–2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2–2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions. More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

This work proves the feasibility of employing regular secondary activated sludge for the enrichment of a microbial community able to perform the anaerobic oxidation of methane coupled to nitrate reduction (N-AOM). After 96 days of activated sludge enrichment, a clear N-AOM activity was observed in the resulting microbial community. The methane removal potential of the enriched N-AOM culture was then studied in a stirred tank reactor (STR) operated in continuous mode for methane supply and semi-continuous mode for the liquid phase. The effect of applying nitrate loads of ∼22, 44, 66, and 88 g NO3− m−3 h−1 on (i) STR methane and nitrate removal performance, (ii) N2O emission, and (iii) microbial composition was investigated. Methane elimination capacities from 21 ± 13.3 to 55 ± 12 g CH4 m−3 h−1 were recorded, coupled to nitrate removal rates ranging from 6 ± 3.2 to 43 ± 14.9 g NO3− m−3 h−1. N2O production was not detected under the three nitrate loading rates applied for the assessment of potential N2O emission in the continuous N-AOM process (i.e. ∼22–66 g NO−3 m−3 h−1). The lack of N2O emissions during the process was attributed to the N2O reducing capacity of the bacterial taxa identified and the rigorous control of dissolved O2 and pH implemented (dissolved O2 values ≤ 0.07 g m−3 and pH of 7.6 ± 0.4). Microbial characterization showed that the N-AOM process was performed in absence of putative N-AOM archaea and bacteria (ANME-2d, M. oxyfera). Instead, microbial activity was driven by methane-oxidizing bacteria and denitrifying bacteria (Bacteroidetes, α-, and γ-proteobacteria).

Biogas production through anaerobic mesophilic digestion is the most straightforward biofuel production route integrated into microalgae-bacteria wastewater treatment plants. Improvement of this biofuel route without adding pretreatment units is possible through the temperature increase. This paper presents a comprehensive evaluation of the transitory effect of different temperatures (35 °C and 55 °C) and hydraulic retention times (HRT) of 15 and 30 d on the long-term methane production using non-pretreated microalgae-bacteria aggregates as a feedstock. The thermophilic transition from mesophilic inoculum adapted to microalgae-bacteria aggregate increased 1.7-fold the methane production (0.41 m3CH4 kgVS−1) at HRT of 30 d. A substantial decrease in the microbial community’s diversity present in the anaerobic reactor was observed when thermophilic conditions were applied, explaining the long adaptation period needed. The increase of the operative temperature condition promotes changes in the dominance pathway of methanogenesis from hydrogenotrophic to acetolactic. The energy balance assessment showed a positive net energy ratio when the digester was operated at an HRT of 30 d. A maximum net energy ratio of 1.5 was achieved at mesophilic temperature. This study demonstrated, based on experimental data, that microalgal digestion with an HRT of 30 d favors energy self-sustainability in microalgal wastewater treatment plants.

The use of ruminal fluid as a source of hydrolytic microorganisms for the pretreatment of a native consortium of microalgae (essentially Senedesmus ) was investigated. The hydrolytic enzyme activity of the ruminal culture was first enriched in a bioreactor. Then, using the enriched culture, the effect of the microalgae to the ruminal fluid ratio (S/X) on the hydrolysis and subsequent production of methane was investigated. An S/X ratio of 0.5 showed the best hydrolysis efficiency (29%) reaching in a second stage process a methane yield of 193 mL CH 4 g COD −1 . The processing time (pretreatment plus methanization) was only 7 days. The predominant ruminal hydrolytic bacteria selected in the enrichment were principally Clostridium , Proteocatella and Pseudomonas .

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.

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 H 2 /L-d), molar yield (1.29 mol H 2 /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).

Pretreatments to break down complex biopolymers in microalgae cells are a key process in the production of gaseous biofuels (methane and hydrogen) from such biomass. Biological pretreatment implies cell degradation by purified enzymes; enzymatic cocktails or by microorganisms with enzymatic activity capable of hydrolyzing the microalgae cell wall. This review presents relevant results using those methods that are less energy intensive and, in some cases, more specific than other strategies, such as chemical and physical pretreatments. Enzymatic pretreatments are specific and efficient, with cellulase, hemicellulase, pectinase, protease and amylase being the most explored enzymes. For biomass pretreatment, enzymatic cocktails have been more effective than single enzymes, as it is more feasible to obtain enzymatic extracts of one or more hydrolytic microorganisms than their purified enzymes. The potential use of hydrolytic cultures for cell disruption to breakdown complex biopolymers has been demonstrated. Their use is less specific than that of enzymatic extracts, but more cost-effective. Pure cultures of hydrolytic bacteria, most of which have carbohydrase activities, have increased the biofuel conversion efficiency from microalgae and from bacterial consortia. The use of natural microbial consortia with hydrolytic activities, such as ruminal microorganisms, represents a potential pretreatment for microalgae. In this review, common hydrolytic activities are highlighted and compared, and the use of ruminal microorganisms as a cell disruption strategy is discussed. Understanding the operational conditions applied to natural consortia, such as ruminal microorganisms, will favor a suitable system for microalgae cell disruption that may increase the biological hydrogen and methane recovery from microalgae.

A novel bioreactor, employing a silicone membrane for microaeration, was studied for partial sulfide oxidation to elemental sulfur. The objective of this study was to assess the feasibility of using an internal silicone membrane reactor (ISMR) to treat dissolved sulfide and to characterize its microbial community. The ISMR is an effective system to eliminate sulfide produced in anaerobic reactors. Sulfide removal efficiencies reached 96 % in a combined anaerobic/microaerobic reactor and significant sulfate production did not occur. The oxygen transfer was strongly influenced by air pressure and flow. Pyrosequencing analysis indicated various sulfide-oxidizing bacteria (SOB) affiliated to the species Acidithiobacillus thiooxidans, Sulfuricurvum kujiense and Pseudomonas stutzeri attached to the membrane and also indicated similarity between the biomass deposited on the membrane wall and the biomass drawn from the material support, supported the establishment of SOB in an anaerobic sludge under microaerobic conditions. Furthermore, these results showed that the reactor configuration can develop SOB under microaerobic conditions and can improve and reestablish the sulfide conversion to elemental sulfur.

Hydrogen production by fermentation with granular anaerobic biomass has been regarded as one alternative to obtain clean energy. However critical aspects such as process conditions to obtain hydrogenogenic granules have been scarcely investigated. In this work two inoculum pretreatments and different upward liquid velocities (ULV) in expanded granular sludge bed (EGSB) reactors were applied to determine the influence of these parameters on the formation, structure and specific hydrogenogenic activity (SHA) of the granules. Heat pretreated inoculum produced granules with greater manipulation resistance compared to those produced with wash-out pretreatment. Furthermore, the increase of ULV (2.5–4.5 m/h) generated bigger granules with a granule size distribution with a trend to a normal distribution and higher protein to carbohydrate ratios. Mass balances showed that propionate was the main metabolite, nonetheless, its production decreased substantially as the ULV applied increased, indicating a selective wash-out of propionate-producing bacteria. In the opposite way, SHA values were higher as the ULV applied increased as a result of selective enrichment and better mass transfer conditions. The whole results indicated that the inoculum pretreatment and hydrodynamic conditions play a key role in the formation, structure and in the biological properties of hydrogen-producing granules in EGSB reactors.

There are several parameters that affect the hydrogen (H2) production when using organic solid waste in a discontinuous process, such as the hydraulic retention time (HRT). To optimize the process, it is necessary to determine the HRT such that the H2 production is maximized. The objective of this study is to evaluate the effect of different HRTs on the hydrogen production in an anaerobic sequencing batch reactor (ASBR) using food waste as the substrate. The microbial community was analyzed and correlated with the reactor performance. The results showed that the highest percentage of H2 in biogas, maximum volumetric H2 production, and maximum H2 production rate was obtained with an HRT of 24 h. The percentage of H2 in biogas varied from 22 to 53%, which depended on the HRT. Acetic acid was the main volatile fatty acid obtained. The highest propionic acid production was observed with an HRT of 6 h, which was related to a decrease in H2 generation, where a low diversity and low evenness were obtained compared with those of the reactor samples obtained from different HRTs. The genus Megasphaera was found to be the dominant genus in the microbial community.

Agave bagasse is a lignocellulosic agroindustrial waste suitable for biofuel production. This study aimed to compare biological, enzymatic, chemical, and hydrothermal pretreatments applied to Agave bagasse in terms of solubilization of carbohydrates (CHO) and chemical oxygen demand (COD), as well as the biochemical methane potential (BMP) from hydrolyzates. Most pretreatments behave similarly, with an average yield of 0.16 ± 0.02 gCOD/g, only the chemical pretreatment overcame this yield by a factor of 2.6 with a CHO/COD ratio of 0.95. The concentrations of inhibitors – furfural, hydroxymethylfurfural, and phenols – were higher in the chemical hydrolyzates than in the biological and enzymatic hydrolyzates. BMP from most hydrolyzates was the same, on average 219 ± 15 mL/gCODin, hydrolyzates differentiate only in terms of lag phase and the methane production rates.

H2 production by dark fermentation using mixed cultures has been studied intensively during the last two decades, and its feasibility has been demonstrated. Different substrates, operational conditions, and reactor technologies have been widely studied and there is a general agreement that the use of non-sterile fermentable substrates is required to make the process feasible for scaling up. Nonetheless, stability problems during long term operation may hinder its application at large scale. This work, written by members of the Latin American Biohydrogen Network, analyse and discuss instability causes and possible solutions in the H2 production by dark fermentation. It is concluded that instability is mostly linked to the biotic aspects of the process (i.e., changes in the microbial community composition, presence of organisms that consume hydrogen and compete for the substrate, and accumulation of fermentation products); regardless of the reactor configuration. However, some problems like excessive growth of microorganisms and methanogens presence were mostly reported in fixed bed reactors and granular sludge reactors. The novelty of this work relies on the comprehensive revision of the main causes behind the unstable and low hydrogen production and how these causes are linked to the technology used. The strategies to overcome the problems as well as the potential implications are also analysed.

In the present work, the effect of anthraquinone-2-sulfonate (AQS) and its reduced form anthrahydroxyquinone-2-sulfonate (AH2QS), and different pH values were evaluated during a dark fermentation process using heat-treated anaerobic granular sludge for hydrogen production. The highest maximum hydrogen production (1.99 mmol) was obtained by adding the reduced electron shuttling compound (AH2QS) at pH 6. This experimental condition also reflected the highest substrate consumption efficiency and the lowest lag phase in relation to the AQS and the control (only sludge). In contrast, the lag phase and maximum hydrogen production were not impacted by AH2QS addition at initial pH of 6.4 and 7.8. Regardless of the initial pH value, the hydrogen production rate was increased after the addition of the electron shuttling compounds, which could be attributable to the performance of either the oxidized and reduced form of the electron shuttling compound. These findings demonstrate the feasibility of AH2QS addition during dark fermentation at pH 6.4 for improving the hydrogen production rate. Exoelectrogenic microorganisms identified in the inoculum could be responsible to improve the dark fermentation process in assays with electron shuttling compounds.

Graphical abstract, Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (Hmax) was defined to be

Winery wastewaters are acidic effluents with high content of organic matter and nutrients. Different initial values of chemical oxygen demand (COD), ranging from 4 to 50 g L−1, were tested in batch assays to evaluate the fermentative hydrogen production followed by a methane production step. The influence of adding a typical nutrient solution for hydrogen production was investigated. Nutrients include N–NH4, Mg, Fe, Co, Mn, I, Ni, and Zn. The best hydrogen production potential was obtained at a COD of 50 g L−1 without nutrient addition. This condition produced 528 mL H2 L−1. At a COD ≥ 35 g L−1, tests with only WW had a hydrogen potential 1.6 to 1.9 times higher than did tests where nutrients were added. The use of added nutrients reduced the hydrogen production by producing additional reduced acids, such as propionate and valerate. In a second stage, biomethane potential was evaluated using the effluent of a selected condition from hydrogen production tests. The methane production reached values of 207 ± 2.2 mL CH4 g−1 COD at 10 g COD L−1. The COD affected the specific methane production. The results of this study demonstrated the potential of winery effluents as a substrate for sequential hydrogen and methane production to increase the energy recovery from this effluent, with a maximum energetic yield and productivity of 7.15 kJ gCOD−1 and 11.51 kJ d−1.

Hydrogen production by dark fermentation is one of the most promising methods for obtaining clean energy. Inoculum pretreatments allow the selection of bacteria that have better performance in hydrogen production, because the selection of pretreatment limits the presence of some species while favoring others. In order to elucidate the inoculum pretreatment influence during the operation of two EGSB reactors, two pretreatments were assayed: heat shock and cell wash-out. Different organic loading rates (24–60 g glucose/L d) and hydraulic retention times (10–4 h) were applied to both reactors to determine population dynamics along 100 days of operation. Reactors exhibited differences in both volumetric hydrogen production rate and molar yield but with cell wash-out pretreatment showing better performance than heat shock pretreatment. Maximum molar yield (0.92 mol H 2 /mol hexose) and volumetric hydrogen production rate (4.23 L H 2 /L d) were obtained with organic loading rates of 36 g glucose/L d at HRT of 10 h in EGSB reactor inoculated with cell wash-out pretreated sludge. The microbial community of the reactors samples was analyzed by 16S rRNA genes profiles and the predominant bands were excised and their DNA sequence determined. Clostridium and representatives of Enterobacteriaceae were dominant, with a strong presence of Lactobacillus genus. The whole result indicates that the inoculum pretreatment has a strong initial effect during early stages of fermentation, after which the operating conditions have a greater impact on reactor performance.

Two biological methods for hydrogen production involving microalgae are revised. In one method, microalgae use the light energy to produce hydrogen from water. In the second one, bacteria capable of fermenting the carbohydrates (either structural or stored in the microalgal cell wall) are utilized to produce hydrogen. Different approaches for using direct and indirect biophotolysis and strategies for using microalgae as feedstock for dark fermentation are presented and discussed. Finally, a life cycle assessment on hydrogen production by these two methods is used to identify the technological bottlenecks, detecting the weaknesses and focusing the research efforts needed.

Methane occurrence is a common concern in hydrogen producing reactors. This study presents the analysis of the microbial community structure during the application of operational strategies to decrease methane production, in three different up-flow anaerobic sludge blanket hydrogen-producing reactors. Cloning and denaturing gradient gel electrophoresis approach were used to establish the presence of homoacetogens, methanogens and hydrogen producers. The results showed that homoacetogenic organisms related to Blautia hydrogenotrophica and Oscillibacter valericigenes, and the hydrogen producer Enterobacter aerogenes where favored during pH decreasing strategies (5.6 to 4.5). The increment of the organic loading rate from 20 to 30 g chemical oxygen demand/L-d, selected hydrogen producers similar to Clostridium tyrobutyricum, Citrobacter freundii and E. aerogenes ; further increments caused inhibition of hydrogen production due to the high undissociated acids concentration. Methane production was inhibited completely only when the biomass of the reactor was heat treated for a second time, this strategy selected hydrogen producers capable to sporulate, but homoacetogens were also favored. In all reactors the methanogenic activity was attributed to hydrogenotrophs related to the genera Methanobrevibacter and Methanobacterium .

One of the problems in fermentative hydrogen producing reactors, inoculated with pre-treated anaerobic granular sludge, is the eventual methane production by hydrogen-consuming methanogens. In this study, strategies such as reduction of pH and HRT, organic shock loads and repeated biomass heat treatment were applied to hydrogenogenic UASB reactors fed with cheese whey, that showed methane production after certain time of continuous operation (between 10 and 60 days). The reduction of pH to 4.5 not only decreased methane production but also hydrogen production. Organic shock load (from 20 to 30 g COD/L-d) was the more effective strategy to decrease the methane production rate (75%) and to increase the hydrogen production rate (172%), without stopping reactor operation. Repeated heat treatment of the granular sludge was the only strategy that inhibited completely methane production, leading to high volumetric hydrogen production rates (1.67 L H2/L-d), however this strategy required stopping reactor operation; in addition homoacetogenesis, another hydrogen-consuming pathway, was not completely inhibited. This work demonstrated that it was possible to control the methane activity in hydrogen producing reactors using operational strategies.

A native microalgae consortium treated under thermal-acidic hydrolysis was used to produce hydrogen and methane in a two-step sequential process. Different acid concentrations were tested, generating hydrogen and methane yields of up to 45 mL H 2 g VS −1 and 432 mL CH 4 g VS −1 , respectively. The hydrogen production step solubilized the particulate COD (chemical oxygen demand) up to 30%, creating considerable amounts of volatile fatty acids (up to 10 g COD L −1 ). It was observed that lower acid concentration presented higher hydrogen and methane production potential. The results revealed that thermal acid hydrolysis of a native microalgae consortium is a simple but effective strategy for producing hydrogen and methane in the sequential process. In addition to COD removal (50–70%), this method resulted in an energy recovery of up to 15.9 kJ per g of volatile solids of microalgae biomass, one of the highest reported.

This chapter serves as an introduction to the major types of microscopy that are applied to the characterization of lignocellulosic biomasses. The covered techniques include optical microscopies (light, Raman, and confocal microscopy), scanning probe microscopy, and electron microscopy. This chapter provides a general description of the principles, advantages and drawbacks, type of information that can be obtained using the different microscopic techniques, and includes a wide range of examples on the use of such techniques to characterize lignocellulosic biomass samples before and after pretreatments. Finally, some of the reviewed microscopic techniques were used to visualize samples of wheat straw nodes before and after acid and alkali pretreatments. This chapter is designed to help scientists select the best microscopic technique to study biomass feedstocks with recalcitrant natures.

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.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.

The feasibility of integrating biological hydrogen and methane production in a two-stage process using mixed cultures and cheese whey powder (CWP) as substrate was studied. The effect of operational parameters such as hydraulic retention time (HRT) and organic loading rate (OLR) on the volumetric hydrogen (VHPR) and methane (VMPR) production rates was assessed. The highest VHPR was 28 L H2/L/d, obtained during stable operation in a CSTR at HRT and OLR of 6 h and 142 g lactose/L/d, respectively. Moreover, hydrogen (13 L/L/d) was produced even at HRT as low as 3.5 h and OLR of 163 g lactose/L/d, nonetheless, the reactor operation was not stable. Regarding methane production in an UASB reactor, the acidified effluent from the hydrogen-producing bioreactor was efficiently treated obtaining COD removals above 90% at OLR and HRT of 20 g COD/L/d and 6 h, respectively. The two-stage process for continuous production of hydrogen and methane recovered over 70% of the energy present in the substrate. This study demonstrated that hydrogen production can be efficiently coupled to methane production in a two-stage system and that CWP is an adequate substrate for energy production.