Improved organic matter biodegradation through pulsed H2 injections during in situ biomethanation.

During in situ biomethanation, microbial communities can convert complex Organic Matter (OM) and H2 into CH4. OM biodegradation was compared between Anaerobic Digestion (AD) and in situ biomethanation, in semi-continuous processes, using two inocula from the digester (D) and the post-digester (PoD) of an AD plant. The impact of H2 on OM degradation was assessed using a fractionation method. Operational parameters included 20 days of hydraulic retention time and 1.5 gVS.L-1.d-1 of organic loading rate. During in situ biomethanation, 485 NmL of H2 were injected for each feeding (3 times a week). Maximum organic COD removal was 0.6 gCOD in AD control and at least 1.6 gCOD for in situ biomethanation. Therefore, COD removal was 2.5 times higher with H2 injections. These results bring out the potential of H2 injections during AD, not only for CO2 consumption but also for better OM degradation.

Archaeal community composition as key driver of H2 consumption rates at the start-up of the biomethanation process.

The performance of hydrogen consumption by various inocula derived from mesophilic anaerobic digestion plants was evaluated under ex situ biomethanation. A panel of 11 mesophilic inocula was operated at a concentration of 15 gVS.L-1 at a temperature of 35 °C in batch system with two successive injections of H2:CO2 (4:1 mol:mol). Hydrogen consumption and methane production rates were monitored from 44 h to 72 h. Hydrogen consumption kinetics varies significantly based on the inoculum origin, with no accumulation of volatile fatty acids. Microbial community analyses revealed that microbial indicators such as the increase in Methanosarcina sp. abundance and the increase of the Archaea/Bacteria ratio were associated to high initial hydrogen consumption rates. The improvement in the hydrogen consumption rate between the two injections was correlated with the enrichment in hydrogenotrophic methanogens. This work provides new insights into the early response of microbial communities to hydrogen injection and on the microbial structures that may favor their adaptation to the biomethanation process.

Biomass acid pretreatment impacts on metabolic routes and bacterial composition of dark fermentation process.

Complex organic matter represents a suitable substrate to produce hydrogen through dark fermentation (DF) process. To increase H2 yields, pretreatment technology is often required. The main objective of the present work was to investigate thermo-acid pretreatment impact on sugar solubilization and biotic parameters of DF of sorghum or organic fraction of municipal solid waste (OFMSW). Biochemical hydrogen potential tests were carried out without inoculum using raw or thermo-acid pretreated substrates. Results showed an improvement in sugar solubilization after thermo-acid pretreatments. Pretreatments led to similar DF performances (H2 and total metabolite production) compared to raw biomasses. Nevertheless, they were responsible for bacterial shifts from Enterobacteriales towards Clostridiales and Bacillales as well as metabolic changes from acetate towards butyrate or ethanol. The metabolic changes were attributed to the biomass pretreatment impact on indigenous bacteria as no change in the metabolic profile was observed after performing thermo-acid pretreatments on irradiated OFMSW (inactivated indigenous bacteria and inoculum addition). Consequently, acid pretreatments were inefficient to improve DF performances but led to metabolic and bacterial community changes due to their impact on indigenous bacteria.

Microbial community redundance in biomethanation systems lead to faster recovery of methane production rates after starvation.

The Power-to-Gas concept corresponds to the use of the electric energy surplus to produce H2 by water electrolysis, that can be further converted to methane by biomethanation. However, the fluctuant production of renewable energy sources can lead to discontinuous H2 injections into the reactors, that may interfere with the adaptation of the microbial community to high H2 partial pressures. In this study, the response of the microbial community to H2 and organic feed starvation was evaluated in in-situ and ex-situ biomethanation. The fed-batch reactors were fed with acetate or glucose and H2, and one or four weeks of starvation periods were investigated. Methane productivity was mostly affected by the four-week starvation period. However, both in-situ and ex-situ biomethanation reactors recovered their methane production rate after starvation within approximately one-week of normal operation, while the anaerobic digestion (AD) reactors did not recover their performances even after 3 weeks of normal operation. The recovery failure of the AD reactors was probably related to a slow growth of the syntrophic and methanogen microorganisms, that led to a VFA accumulation. On the contrary, the faster recovery of both biomethanation reactors was related to the replacement of Methanoculleus sp. by Methanobacterium sp., restoring the methane production in the in-situ and ex-situ biomethanation reactors. This study has shown that biomethanation processes can respond favourably to the intermittent H2 addition without compromising their CH4 production performance.

Impact of microbial inoculum storage on dark fermentative H2 production.

Complex organic substrates represent an important and relevant feedstock for producing hydrogen by Dark Fermentation (DF). Usually, an external microbial inoculum originated from various natural environments is added to seed the DF reactors. However, H2 yields are significantly impacted by the inoculum origin and the storage conditions as microbial community composition can fluctuate. This study aims to determine how the type and time of inoculum storage can impact the DF performances. Biochemical Hydrogen Potential tests were carried out using three substrates (glucose, the organic fraction of municipal solid waste, and food waste), inocula of three different origins, different storage conditions (freezing or freeze-drying) and duration. As a result, H2 production from glucose with the differently stored inocula was significantly impacted (positively or negatively) and was inoculum-origin-dependent. For complex substrates, hydrogen yields with the stored inocula were not statistically different from the fresh inocula, offering the possibility to store an inoculum.

Role of indigenous bacteria in dark fermentation of organic substrates.

Hydrogen production by dark fermentation of complex organic substrates, such as biowaste, can naturally take place with indigenous bacteria or by adding an external microbial inoculum issued from various natural environments. This study aims to determine whether indigenous bacteria associated with thermal pretreatment could impact dark fermentation performances. Biochemical hydrogen potential tests were carried out on seven organic substrates. Results showed a strong influence of the indigenous bacteria which are as effective as thermally pretreated exogenous bacteria to produce H2 and metabolites. High abundance in Clostridiales and/or Enterobacteriales was associated with high H2 yield. This study shows that no inoculum nor pretreatment are required to achieve satisfactory dark fermentation performances from organic waste.

The hydraulic retention time influences the abundance of Enterobacter, Clostridium and Lactobacillus during the hydrogen production from food waste.

The influence of hydraulic retention time (HRT) on the microbial communities was evaluated in an anaerobic sequencing batch reactor (AnSBR) using organic waste from a restaurant as the substrate. The relationship among Lactobacillus, Clostridium and Bacillus as key micro-organisms on hydrogen production from organic solid waste was studied. The effect of the HRT (8-48 h) on the hydrogen production and the microbial community was evaluated. Quantitative PCR was applied to determine the abundance of bacteria (in particular, Enterobacter, Clostridium and Lactobacillus genera). An AnSBR fermentative reactor was operated for 111 cycles, with carbohydrate and organic matter removal efficiencies of 80 ± 15·42% and 22·1 ± 4·49% respectively. The highest percentage of hydrogen in the biogas (23·2 ± 11·1 %), and the specific production rate (0·42 ± 0·16 mmol H2 gVSadded -1 d-1 ) were obtained at an HRT of 48 h. The decrease in the HRT generated an increase in the hydrogen production rate but decreasing the content of the hydrogen in the gas. HRT significantly influence the abundance of Enterobacter, Clostridium and Lactobacillus during the hydrogen production from food waste leading the hydrogen production as well as the metabolic pathways. The microbial analysis revealed a direct relationship between the HRT and the presence of fermentative bacteria (Enterobacter, Clostridium and Lactobacillus genera). Clostridium sp. predominated at an HRT of 48 h, while Enterobacter and Lactobacillus predominated at HRTs between 8 and 24 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: It was demonstrated that hydrogen production using food waste was influenced by the hydraulic retention time (HRT), and closely related to changes in microbial communities together with differences in metabolic patterns (e.g. volatile fatty acids, lactate, etc.). The decrease in the HRT led to the dominance of lactic acid bacteria within the microbial community whereas the increase in HRT favoured the emergence of Clostridium bacteria and the increase in acetic and butyric acids. Statistical data analysis revealed a direct relationship existing between the HRT and the microbial community composition in fermentative bacteria. This study provides new insight into the relationship between the bioprocess operation and the microbial community to understand better and control the biohydrogen production.

Biohydrogen production at pH below 3.0: Is it possible?

Biological hydrogen production was investigated in continuous acidogenic reactors fed with sucrose at 30 °C without pH control. In the first experimental phase, three reactors were compared: a structured fixed-bed (FB), a granular UASB (UG) and a flocculent UASB (UF-1). They were run at 3.3 h HRT and 33 gCOD L-1d-1 OLR. Hydrogen production occurred throughout the experimental period with an average effluent pH of only 2.8. The FB, UG and UF-1 reactors presented volumetric hydrogen production rates (VHPR) of 95 ± 69, 45 ± 37 and 54 ± 32 mLH2 L-1h-1, respectively; and H2 yields (HY) of 1.5 ± 0.8, 0.8 ± 0.6 and 1.2 ± 0.7 molH2 mol-1 sucroseconsumed, respectively. The UF-1 reactor showed intermediate VHPR and HY, but no declining trend, contrary to what was observed in the FB reactor. Thus, aiming at continuous and long-term H2 production, a flocculent UASB was applied in the second experimental phase. In this phase, the HRT of the acidogenic reactor, which was named UF-2, was raised to 4.6 h, resulting in an OLR of 25 gCOD L-1d-1. The VHPR and the HY increased considerably to 175 ± 44 mLH2 L-1h-1 and 3.4 ± 0.7 molH2 mol-1 sucroseconsumed, respectively. These improvements were accompanied by greater sucrose removal, higher suspended biomass concentration, less production of lactate and more of acetate, and high ethanol concentration. Contradicting the current published literature data that reports strong inhibition of H2 production by dark fermentation at pH less than 4.0, the UF-2 reactor presented stable, long-term H2 production with satisfactory yields at pH 2.7 on average. 16 S rDNA sequencing revealed that two sequences assigned as Ethanoligenens and Clostridium accounted for over 70% of the microbiota in all the reactors. The non-necessity of adding alkalizing agents and the successful H2 production under very acid conditions, demonstrated in this study, open a new field of investigation in biological hydrogen production by dark fermentation towards a more sustainable and feasible technology.

Bioelectrochemical treatment of table olive brine processing wastewater for biogas production and phenolic compounds removal.

Industry of table olives is widely distributed over the Mediterranean countries and generates large volumes of processing wastewaters (TOPWs). TOPWs contain high levels of organic matter, salt, and phenolic compounds that are recalcitrant to microbial degradation. This work aims to evaluate the potential of bioelectrochemical systems to simultaneously treat real TOPWs and recover energy. The experiments were performed in potentiostatically-controlled single-chamber systems fed with real TOPW and using a moderate halophilic consortium as biocatalyst. In conventional anaerobic digestion (AD) treatment, ie. where no potential was applied, no CH4 was produced. In comparison, Bio-Electrochemical Systems (BES) showed a maximum CH4 yield of 701 ± 13 NmL CH4·LTOPW(-1) under a current density of 7.1 ± 0.4 A m(-2) and with a coulombic efficiency of 30%. Interestingly, up to 80% of the phenolic compounds found in the raw TOPW (i.e. hydroxytyrosol and tyrosol) were removed. A new theoretical degradation pathway was proposed after identification of the metabolic by-products. Consistently, microbial community analysis at the anode revealed a clear and specific enrichment in anode-respiring bacteria (ARB) from the genera Desulfuromonas and Geoalkalibacter, supporting the key role of these electroactive microorganisms. As a conclusion, bioelectrochemical systems represent a promising bioprocess alternative for the treatment and energy recovery of recalcitrant TOPWs.

Growth of Chlorella sorokiniana on a mixture of volatile fatty acids: The effects of light and temperature.

This study investigated the influence of light and temperature on Chlorella sorokiniana grown on a mixture of acetate and butyrate, two of the volatile fatty acids produced by dark fermentation. Exposure to light caused autotrophic biomass production (56% of the final biomass) and reduced the time to reach butyrate exhaustion to 7 days at 25°C from 10 days in the dark. For growth on acetate at the optimum temperature (35°C), the presence of butyrate reduced the growth rate (by 46%) and the carbon yield (by 36%). For successful microalgae growth on dark fermentation effluent, butyrate inhibition may be reduced by setting the temperature to 30°C and providing light.

Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production.

Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L(-1)), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H2 production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4 ± 0.1 molH2 mol(-1) glycerol consumed) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H2 production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.

Biomass hydrolysis inhibition at high hydrogen partial pressure in solid-state anaerobic digestion.

In solid-state anaerobic digestion, so-called ss-AD, biogas production is inhibited at high total solids contents. Such inhibition is likely caused by a slow diffusion of dissolved reaction intermediates that locally accumulate. In this study, we investigated the effect of H2 and CO2 partial pressure on ss-AD. Partial pressure of H2 and/or CO2 was artificially fixed, from 0 to 1 557mbars for H2 and from 0 to 427mbars for CO2. High partial pressure of H2 showed a significant effect on methanogenesis, while CO2 had no impact. At high [Formula: see text] , the overall substrate degradation decreased with no accumulation of metabolites from acidogenic bacteria, indicating that the hydrolytic activity was specifically impacted. Interestingly, such inhibition did not occur when CO2 was added with H2. This result suggests that CO2 gas transfer is probably a key factor in ss-AD from biomass.

Use of fermentative metabolites for heterotrophic microalgae growth: Yields and kinetics.

The growth of two lipid-producing Chlorella species on fermentative end-products acetate, butyrate and lactate, was investigated using a kinetic modeling approach. Chlorella sorokiniana and Auxenochlorella protothecoides were grown on synthetic media with various (acetate:butyrate:lactate) ratios. Both species assimilated efficiently acetate and butyrate with yields between 0.4 and 0.5g carbon of biomass/g carbon of substrate, but did not use lactate. The highest growth rate on acetate, 2.23d(-1), was observed for C. sorokiniana, and on butyrate, 0.22d(-1), for A. protothecoides. Butyrate removal started after complete acetate exhaustion (diauxic effect). However, butyrate consumption may be favored by the increase of biomass concentration induced by the initial use of acetate. A model combining Monod and Haldane functions was then built and fitted the experimental data well for both species. Butyrate concentration and (acetate:butyrate) ratios were identified as key parameters for heterotrophic growth of microalgae on fermentative metabolites.

Do furanic and phenolic compounds of lignocellulosic and algae biomass hydrolyzate inhibit anaerobic mixed cultures? A comprehensive review.

Nowadays there is a growing interest on the use of both lignocellulosic and algae biomass to produce biofuels (i.e. biohydrogen, ethanol and methane), as future alternatives to fossil fuels. In this purpose, thermal and thermo-chemical pretreatments have been widely investigated to overcome the natural physico-chemical barriers of such biomass and to enhance biofuel production from lignocellulosic residues and, more recently, marine biomass (i.e. macro and microalgae). However, the pretreatment technologies lead not only to the conversion of carbohydrate polymers (ie cellulose, hemicelluloses, starch, agar) to soluble monomeric sugar (ie glucose, xylose, arabinose, galactose), but also the generation of various by-products (i.e. furfural and 5-HMF). In the case of lignocellulosic residues, part of the lignin can also be degraded in lignin derived by-products, mainly composed of phenolic compounds. Although the negative impact of such by-products on ethanol production has been widely described in literature, studies on their impact on biohydrogen and methane production operated with mixed cultures are still very limited. This review aims to summarise and discuss literature data on the impact of pre-treatment by-products on H2-producing dark fermentation and anaerobic digestion processes when using mixed cultures as inoculum. As a summary, furanic (5-HMF, furfural) and phenolic compounds were found to be stronger inhibitors of the microbial dark fermentation than the full anaerobic digestion process. Such observations can be explained by differences in process parameters: anaerobic digestion is performed with more complex mixed cultures, lower substrate/inoculum and by-products/inoculum ratios and longer batch incubation times than dark fermentation. Finally, it has been reported that, during dark fermentation process, the presence of by-products could lead to a metabolic shift from H2-producing pathways (i.e. acetate and butyrate) to non-H2-producing pathways (i.e. lactate, ethanol and propionate) and whatever the metabolic route, metabolites can be all further converted into methane, but at different rates.

A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste.

AIMS: The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H(2)) producers from digested household solid wastes. METHODS AND RESULTS: A strict anaerobic extreme thermophilic H(2) producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70 degrees C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80 degrees C and an optimal pH 8.1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H(2) and carbon dioxide. Maximal H(2) production rate on glucose was 1.1 mmol l(-1) h(-1) with a maximum H(2) yield of 1.9 mole H(2) per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 +/- 5% and 13 +/- 5% for Bacillus and Clostridium, respectively. CONCLUSIONS: An extreme thermophilic, strict anaerobic, mixed microbial culture with H(2)-producing potential was enriched from digested household wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H(2) production from complex organic wastes.

Impact of anaerobic and aerobic processes on polychlorobiphenyl removal in contaminated sewage sludge.

Aerobic and anaerobic biodegradation of six priority PCBs was investigated in continuous stirred tank reactors fed with naturally contaminated sewage sludge. Anaerobic and aerobic abiotic losses were higher for the lightly chlorinated PCBs but remained for all PCBs below 20%. Under strict methanogenic conditions, PCB removals were about 40% whatever PCB molecular weight or their degree of chlorination. However, considering abiotic losses, the heaviest PCBs were more efficiently anaerobically biodegraded probably because of higher dechlorination rates. The aerating sludge process enhanced removal of the lightest chlorinated PCBs from 40% up to 100%, while removal rates of the heaviest PCBs remained around 40%. Although the mesophilic aerobic process exhibits better removal efficiencies because of operating conditions, the results suggest that PCB biodegradation was strongly limited by their bioavailability in naturally contaminated sludge, under both redox conditions. Indeed, since PCB removal was closely linked to the solid reduction rates, PCB bioavailability was likely the limiting factor for biodegradation. As a consequence, the raw PCB concentrations (in mg kg(-1) (dry weight)) which are concerned by legislative procedures did not decrease sufficiently in both processes to reach a limit value fulfilling the current French/European regulation about PCB contents in sewage sludge before spreading on agricultural land.

Enhancement of polycyclic aromatic hydrocarbons removal during anaerobic treatment of urban sludge.

Anaerobically stabilized sewage sludge has potential to partially substitute synthetic fertilizers. The main risk with the recycling of urban sludge on agricultural soils is the accumulation of unwanted products, such as trace metals and organic micropollutants. In this context, the polycyclic aromatic hydrocarbons (PAHs) are particularly monitored because of their toxic properties at low concentrations and their high resistance to biological degradation. The aim of the present study was to optimize PAHs removal during anaerobic digestion of contaminated sewage sludge. Thirteen PAHs were monitored in laboratory-scale anaerobic bioreactors under mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic conditions. Abiotic losses were statistically significant for the lightest PAHs, such as fluorene, phenanthrene and anthracene. It was shown that PAH removal was due to a specific biological activity. Biological PAHs removal was significantly enhanced by an increase of the temperature from 35 degrees C to 55 degrees C, especially for the heaviest PAHs. Bioaugmentation experiment was also performed by addition of a PAH-adapted bacterial consortium to a non-acclimated reactor. Significant enhancement of PAHs removal was observed. It was finally shown that PAH removal efficiencies and methanogenic performances were closely linked. The rate of biogas production may be used as an indicator of bacterial activity on PAH removal.