Temperature and Inoculum Origin Influence the Performance of Ex-Situ Biological Hydrogen Methanation.

The conversion of H2 into methane can be carried out by microorganisms in a process so-called biomethanation. In ex-situ biomethanation H2 and CO2 gas are exogenous to the system. One of the main limitations of the biomethanation process is the low gas-liquid transfer rate and solubility of H2 which are strongly influenced by the temperature. Hydrogenotrophic methanogens that are responsible for the biomethanation reaction are also very sensitive to temperature variations. The aim of this work was to evaluate the impact of temperature on batch biomethanation process in mixed culture. The performances of mesophilic and thermophilic inocula were assessed at 4 temperatures (24, 35, 55 and 65 °C). A negative impact of the low temperature (24 °C) was observed on microbial kinetics. Although methane production rate was higher at 55 and 65 °C (respectively 290 ± 55 and 309 ± 109 mL CH4/L.day for the mesophilic inoculum) than at 24 and 35 °C (respectively 156 ± 41 and 253 ± 51 mL CH4/L.day), the instability of the system substantially increased, likely because of a strong dominance of only Methanothermobacter species. Considering the maximal methane production rates and their stability all along the experiments, an optimal temperature range of 35 °C or 55 °C is recommended to operate ex-situ biomethanation process.

Biomethanation processes: new insights on the effect of a high H2 partial pressure on microbial communities.

BACKGROUND: Biomethanation is a promising solution to upgrade the CH4 content in biogas. This process consists in the injection of H2 into an anaerobic digester, using the capacity of indigenous hydrogenotrophic methanogens for converting the injected H2 and the CO2 generated from the anaerobic digestion process into CH4. However, the injection of H2 could cause process disturbances by impacting the microbial communities of the anaerobic digester. Better understanding on how the indigenous microbial community can adapt to high H2 partial pressures is therefore required. RESULTS: Seven microbial inocula issued from industrial bioprocesses treating different types of waste were exposed to a high H2 partial pressure in semi-continuous reactors. After 12 days of operation, even though both CH4 and volatile fatty acids (VFA) were produced as end products, one of them was the main product. Acetate was the most abundant VFA, representing up to 94% of the total VFA production. VFA accumulation strongly anti-correlated with CH4 production according to the source of inoculum. Three clusters of inocula were distinguished: (1) inocula leading to CH4 production, (2) inocula leading to the production of methane and VFA in a low proportion, and (3) inocula leading to the accumulation of mostly VFA, mainly acetate. Interestingly, VFA accumulation was highly correlated to a low proportion of archaea in the inocula, a higher amount of homoacetogens than hydrogenotrophic methanogens and, the absence or the very low abundance in members from the Methanosarcinales order. The best methanogenic performances were obtained when hydrogenotrophic methanogens and Methanosarcina sp. co-dominated all along the operation. CONCLUSIONS: New insights on the microbial community response to high H2 partial pressure are provided in this work. H2 injection in semi-continuous reactors showed a significant impact on microbial communities and their associated metabolic patterns. Hydrogenotrophic methanogens, Methanobacterium sp. or Methanoculleus sp. were highly selected in the reactors, but the presence of co-dominant Methanosarcinales related species were required to produce higher amounts of CH4 than VFA.

Soft Microwave Pretreatment to Extract P-Hydroxycinnamic Acids from Grass Stalks.

The aim of this article is to provide an analysis of microwave effects on ferulic and coumaric acids (FA and CA, respectively) extraction from grass biomass (corn stalks and miscanthus). Microwave pretreatment using various solvents was first compared to conventional heating on corn stalks. Then, microwave operational conditions were extended in terms of incident power and treatment duration. Optimal conditions were chosen to increase p-hydroxycinnamic acids release. Finally, these optimal conditions determined on corn stalks were tested on miscanthus stalks to underlie the substrate incidence on p-hydroxycinnamic acids release yields. The optimal conditions-a treatment duration of 405 s under 1000 W-allowed extracting 1.38% FA and 1.97% CA in corn stalks and 0.58% FA and 3.89% CA in miscanthus stalks. The different bioaccessibility of these two molecules can explain the higher or lower yields between corn and miscanthus stalks.

Addition of granular activated carbon and trace elements to favor volatile fatty acid consumption during anaerobic digestion of food waste.

The effect of supplementing granular activated carbon and trace elements on the anaerobic digestion performance of consecutive batch reactors treating food waste was investigated. The results from the first batch suggest that addition of activated carbon favored biomass acclimation, improving acetic acid consumption and enhancing methane production. Adding trace elements allowed a faster consumption of propionic acid. A second batch proved that a synergy existed when activated carbon and trace elements were supplemented simultaneously. The degradation kinetics of propionate oxidation were particularly improved, reducing significantly the batch duration and improving the average methane productivities. Addition of activated carbon favored the growth of archaea and syntrophic bacteria, suggesting that interactions between these microorganisms were enhanced. Interestingly, microbial analyses showed that hydrogenotrophic methanogens were predominant. This study shows for the first time that addition of granular activated carbon and trace elements may be a feasible solution to stabilize food waste anaerobic digestion.

Methanosarcina plays a main role during methanogenesis of high-solids food waste and cardboard.

Anaerobic digestion of food waste is a complex process often hindered by high concentrations of volatile fatty acids and ammonia. Methanogenic archaea are more sensitive to these inhibitors than bacteria and thus the structure of their community is critical to avoid reactor acidification. In this study, the performances of three different inocula were compared using batch digestion tests of food waste and cardboard mixtures. Particular attention was paid to the archaeal communities in the inocula and after digestion. While the tests started with inocula rich in Methanosarcina led to efficient methane production, VFAs accumulated in the reactors where inocula initially were poor in this archaea and no methane was produced. In addition, higher substrate loads were tolerated when greater proportions of Methanosarcina were initially present in the inoculum. Independently of the inoculum origin, Methanosarcina were the dominant methanogens in the digestates from the experiments that efficiently produced methane. These results suggest that the initial archaeal composition of the inoculum is crucial during reactor start-up to achieve stable anaerobic digestion at high concentrations of ammonia and organic acids.

Cardboard proportions and total solids contents as driving factors in dry co-fermentation of food waste.

This study evaluated the influence of the co-substrate proportions (0-60% of cardboard in dry basis) and the initial total solid contents (20-40%) on the batch fermentation performance. Maximum hydrogen yields were obtained when mono-fermenting food waste at high solids contents (89mlH2·gVS-1). The hydrogen yields were lower when increasing the proportions of cardboard. The lower hydrogen yields at higher proportions of cardboard were translated into higher yields of caproic acid (up to 70.1gCOD·kgCODbio-1), produced by consumption of acetic acid and hydrogen. The highest substrate conversions were achieved at low proportions of cardboard, indicating a stabilization effect due to higher buffering capacities in co-fermentation. Clostridiales were predominant in all operational conditions. This study opens up new possibilities for using the cardboard proportions for controlling the production of high added-value products in dry co-fermentation of food waste.

Accumulation of propionic acid during consecutive batch anaerobic digestion of commercial food waste.

The objective of this study was to test three different alternatives to mitigate the destabilizing effect of accumulation of ammonia and volatile fatty acids during food waste anaerobic digestion. The three options tested (low temperature, co-digestion with paper waste and trace elements addition) were compared using consecutive batch reactors. Although methane was produced efficiently (∼500ml CH4gVS-1; 16l CH4lreactor-1), the concentrations of propionic acid increased gradually (up to 21.6gl-1). This caused lag phases in the methane production and eventually led to acidification at high substrate loads. The addition of trace elements improved the kinetics and allowed higher substrate loads, but could not avoid propionate accumulation. Here, it is shown for the first time that addition of activated carbon, trace elements and dilution can favor propionic acid consumption after its accumulation. These promising options should be optimized to prevent propionate accumulation.

Kinetic study of dry anaerobic co-digestion of food waste and cardboard for methane production.

Dry anaerobic digestion is a promising option for food waste treatment and valorization. However, accumulation of ammonia and volatile fatty acids often occurs, leading to inefficient processes and digestion failure. Co-digestion with cardboard may be a solution to overcome this problem. The effect of the initial substrate to inoculum ratio (0.25 to 1gVS·gVS-1) and the initial total solids contents (20-30%) on the kinetics and performance of dry food waste mono-digestion and co-digestion with cardboard was investigated in batch tests. All the conditions produced methane efficiently (71-93% of the biochemical methane potential). However, due to lack of methanogenic activity, volatile fatty acids accumulated at the beginning of the digestion and lag phases in the methane production were observed. At increasing substrate to inoculum ratios, the initial acid accumulation was more pronounced and lower cumulative methane yields were obtained. Higher amounts of soluble organic matter remained undegraded at higher substrate loads. Although causing slightly longer lag phases, high initial total solids contents did not jeopardize the methane yields. Cardboard addition reduced acid accumulation and the decline in the yields at increasing substrate loads. However, cardboard addition also caused higher concentrations of propionic acid, which appeared as the most last acid to be degraded. Nevertheless, dry co-digestion of food waste and cardboard in urban areas is demonstrated asan interesting feasible valorization option.

Dry anaerobic digestion of food waste and cardboard at different substrate loads, solid contents and co-digestion proportions.

The increasing food waste production calls for developing efficient technologies for its treatment. Anaerobic processes provide an effective waste valorization. The influence of the initial substrate load on the performance of batch dry anaerobic co-digestion reactors treating food waste and cardboard was investigated. The load was varied by modifying the substrate to inoculum ratio (S/X), the total solids content and the co-digestion proportions. The results showed that the S/X was a crucial parameter. Within the tested values (0.25, 1 and 4gVS·gVS-1), only the reactors working at 0.25 produced methane. Methanosarcina was the main archaea, indicating its importance for efficient methanogenesis. Acidogenic fermentation was predominant at higher S/X, producing hydrogen and other metabolites. Higher substrate conversions (≤48%) and hydrogen yields (≤62mL·gVS-1) were achieved at low loads. This study suggests that different value-added compounds can be produced in dry conditions, with the initial substrate load as easy-to-control operational parameter.

Total solid content drives hydrogen production through microbial selection during thermophilic fermentation.

In this study, the effect of total solid content (TS) on thermophilic hydrogen production from wheat straw was investigated. Six TS contents ranging from wet to dry conditions (10-34%TS) were tested in batch tests. A decrease of H2 yields was observed and three statistical groups were distinguished according to the TS content: wet conditions (10% and 14%TS) with 15.3 ± 1.6 NmlH2 gTS(-1), intermediate conditions (19%TS) with 6.4 ± 1.0 NmlH2 gTS(-1) and dry conditions (25-34%TS) with 3.4 ± 0.8 NmlH2 gTS(-1). Such a decrease in biohydrogen yields was related to a metabolic shift with an accumulation of lactic acid under dry conditions. Concomitantly, a microbial population shift was observed with a dominance of species related to the class Clostridia under wet conditions, and a co-dominance of members of Bacilli, Clostridia classes and Bacteroidetes phylum under dry conditions.

Single-phase and two-phase anaerobic digestion of fruit and vegetable waste: comparison of start-up, reactor stability and process performance.

Single-phase and two-phase digestion of fruit and vegetable waste were studied to compare reactor start-up, reactor stability and performance (methane yield, volatile solids reduction and energy yield). The single-phase reactor (SPR) was a conventional reactor operated at a low loading rate (maximum of 3.5 kgVS/m3 d), while the two-phase system consisted of an acidification reactor (TPAR) and a methanogenic reactor (TPMR). The TPAR was inoculated with methanogenic sludge similar to the SPR, but was operated with step-wise increase in the loading rate and with total recirculation of reactor solids to convert it into acidification sludge. Before each feeding, part of the sludge from TPAR was centrifuged, the centrifuge liquid (solubilized products) was fed to the TPMR and centrifuged solids were recycled back to the reactor. Single-phase digestion produced a methane yield of 0.45 m3 CH4/kg VS fed and VS removal of 83%. The TPAR shifted to acidification mode at an OLR of 10.0 kgVS/m3 d and then achieved stable performance at 7.0 kgVS/m3 d and pH 5.5-6.2, with very high substrate solubilization rate and a methane yield of 0.30 m3 CH4/kg COD fed. The two-phase process was capable of high VS reduction, but material and energy balance showed that the single-phase process was superior in terms of volumetric methane production and energy yield by 33%. The lower energy yield of the two-phase system was due to the loss of energy during hydrolysis in the TPAR and the deficit in methane production in the TPMR attributed to COD loss due to biomass synthesis and adsorption of hard COD onto the flocs. These results including the complicated operational procedure of the two-phase process and the economic factors suggested that the single-phase process could be the preferred system for FVW.

Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion.

BACKGROUND: In solid-state anaerobic digestion (AD) bioprocesses, hydrolytic and acidogenic microbial metabolisms have not yet been clarified. Since these stages are particularly important for the establishment of the biological reaction, better knowledge could optimize the process performances by process parameters adjustment. RESULTS: This study demonstrated the effect of total solids (TS) content on microbial fermentation of wheat straw with six different TS contents ranging from wet to dry conditions (10 to 33% TS). Three groups of metabolic behaviors were distinguished based on wheat straw conversion rates with 2,200, 1,600, and 1,400 mmol.kgVS-1 of fermentative products under wet (10 and 14% TS), dry (19 to 28% TS), and highly dry (28 to 33% TS) conditions, respectively. Furthermore, both wet and dry fermentations showed acetic and butyric acid metabolisms, whereas a mainly butyric acid metabolism occurred in highly dry fermentation. CONCLUSION: Substrate conversion was reduced with no changes of the metabolic pathways until a clear limit at 28% TS content, which corresponded to the threshold value of free water content of wheat straw. This study suggested that metabolic pathways present a limit of TS content for high-solid AD.

Microbial community signature of high-solid content methanogenic ecosystems.

In this study, changes in bacterial and archaeal communities involved in anaerobic digestion processes operated with high solid contents were investigated. Batch tests were performed within a range of total solids (TS) of 10-35%. Between 10% and 25% TS, high methanogenic activity was observed and no overall specific structure of active bacterial communities was found. At 30% and 35%, methanogenesis was inhibited as a consequence of volatile fatty acids accumulation. Here, a specific bacterial signature was observed with three main dominant bacteria related to Clostridium sp., known for their ability to grow at low pH. Additionally, archaeal community was gradually impacted by TS content. Three archaeal community structures were observed with a gradual shift from Methanobacterium sp. to Methanosarcina sp., according to the TS content. Overall, several species were identified as biomarkers of methanogenesis inhibition, since bacterial and archaeal communities were highly specific at high TS contents.

Anaerobic co-digestion of solid waste: Effect of increasing organic loading rates and characterization of the solubilised organic matter.

The impact of stepwise increase in OLR (up to 7.5kgVS/m(3)d) on methane production, reactor performance and solubilised organic matter production in a high-loading reactor were investigated. A reference reactor operated at low OLR (<2.0kgVS/m(3)d) was used solely to observe the methane potential of the feed substrate. Specific methane yield was 0.33lCH(4)/gVS at the lowest OLR and dropped by about 20% at the maximum OLR, while volumetric methane production increased from 0.35 to 1.38m(3)CH(4)/m(3)d. At higher loadings, solids hydrolysis was affected, with consequent transfer of poorly-degraded organic material into the drain solids. Biodegradability and size-fractionation of the solubilised COD were characterized to evaluate the possibility of a second stage liquid reactor. Only 18% of the organics were truly soluble (<1kD). The rest were in colloidal and very fine particulate form which originated from grass and cow manure and were non-biodegradable.

Chemical and toxicological assessment of a full-scale biosolid compost.

The impact of a full-scale biosolid composting plant on the fate of a broad range of priority organic pollutants was investigated. Chemical analysis was performed at different steps of the process during two seasons. Simultaneously, the toxicological quality was assessed using estrogen α-, dioxin-, and pregnane X-receptor reporter cell lines. Mass-balance calculation highlighted the removal of easily degradable pollutants during composting. The important variations observed for each compound and for the two seasons might be explained by pollutant-fate dependency on process parameters like temperature. The final compost displayed low pregnane X activity but high estrogenic activity. The dioxin-like activity stayed constant through the process. The chemical and toxicological results highlight the importance of combining both approaches to accurately assess the compost quality. Such compilation of data on full-scale processes may be also very helpful for the environmental risk assessment of new organic waste disposal practices.

Total solids content drives high solid anaerobic digestion via mass transfer limitation.

The role of the total solids (TS) content on anaerobic digestion was investigated in batch reactors. A range of TS contents from 10% to 35% was evaluated, four replicates were performed. The total methane production slightly decreased with TS concentrations increasing from 10% to 25% TS. Two behaviors were observed at 30% TS: two replicates had similar performances to that at 25% TS; for the two other replicates, the methane production was inhibited as observed at 35% TS. This difference suggested that 30% TS content corresponded to a threshold of the solids content, above which methanogenesis was strongly inhibited. The Anaerobic Digestion Model No. 1 (ADM1) was used to describe the experimental data. The effects of hydrolysis step and liquid/gas mass transfer were particularly investigated. The simulations showed that mass transfer limitation could explain the low methane production at high TS, and that hydrolysis rate constants slightly decreased with increasing TS.

Control of start-up and operation of anaerobic biofilm reactors: an overview of 15 years of research.

Anaerobic biofilm reactors have to be operated in a way that optimizes on one hand the start-up period by a quick growth of an active biofilm, on the other hand the regular operation by an active control of the biofilm to avoid diffusion limitations and clogging. This article is an overview of the research carried out at INRA-LBE for the last 15 years. The start-up of anaerobic biofilm reactors may be considerably shortened by applying a short inoculation period (i.e. contact between the inoculum and the support media). Then, the increase of the organic loading rate should be operated at a short hydraulic retention time and low hydrodynamic constraints in order to favor biofilm growth. After the start-up period, biofilm growth should be controlled to maintain a high specific activity and prevent clogging. This can be done in particulate biofilm systems by using hydrodynamics to increase or decrease shear forces and attrition but is much more difficult in anaerobic fixed bed reactors.

Occurrence of estrogens in sewage sludge and their fate during plant-scale anaerobic digestion.

Estrogens, which contribute greatly to the endocrine-disrupting activity in sewage, are partially sorbed onto particulate matter during sewage treatment. We thus investigated the occurrence of estrogens in different kinds of sludge and throughout a plant-scale anaerobic digestion process. The analytical method was first validated when sorption interaction between spiked estrogens and sludge could occur. Hence, the recovery ratio of estrone (E1), 17beta-estradiol (E2), estriol (E3) and 17alpha-ethinylestradiol (EE2) were determined when added to liquid sludge and mixed under various conditions. We show that minor non-extractable residues were formed (5-10%), suggesting that the sorption interaction established with sludge did not limit estrogen extraction. Estrogen concentrations measured in collected samples varied with sludge type. Secondary sludge showed higher E1 contents than primary sludge: respectively, 43 and 8 ng g(-1) dry weight (dw). Two pathways of E1 production during secondary treatment are proposed to explain such a result. Higher estrogen concentrations were found in secondary sludge from a conventional plant (55 ng g(-1)dw) compared to those from an advanced plant (13 ng g(-1)dw). Based on estimated estrogen concentrations in sewage, we conclude that operating parameters play a role in the sorption of estrogens during secondary treatment. Also, the hydrophobic properties of the estrogens influenced the individual adsorption of each molecule. Thus, E3 showed the highest estimated concentrations in sewage but very low concentrations in sludge. Finally, plant-scale anaerobic digestion showed low efficiency (<40%) for removing estrogens and, regarding the final dewatering process, concentrations increased for E2 and EE2.

Selective microbial aerosolization in biogas demonstrated by quantitative PCR.

Aerosolization of Bacteria, Archaea, Synergistes, Staphylococcus spp. and Propionibacterium acnes was investigated in situ with quantitative real-time PCR of DNA isolated from sludge and biogases of anaerobic digesters. The data revealed that in biogas, Staphylococcus spp. and P. acnes were, respectively, aerosolized 30 and 220 times more and Archaea and Synergistes, respectively, 8 and 20 times less aerosolized than Bacteria. This is the first demonstration of selective microbial aerosolization for anaerobic digestors microorganisms. This study illustrates the fact that some microbial groups, such as opportunistic pathogens, are more susceptible to be aerosolized, since they use air as a dissemination vector, and that this has to be taken in account when up-grading biogas into natural gas networks.

Molecular and kinetic characterization of mixed cultures degrading natural and synthetic estrogens.

The biodegradation of estradiol (E2), estrone (E1), and ethinylestradiol (EE2) was investigated using mixed bacterial cultures enriched from activated sludge. Enrichments were carried out on E2 or EE2 in batch conditions with acetonitrile as additional carbon source. Degradation experiments were performed both using hormones as sole carbon source or with an additional source. The hormones were completely degraded by these cultures. Estradiol was rapidly converted to E1 within 24 h. Thereafter, E1 degradation began, displaying a lag phase ranging from 3 to 4 days. Estrone depletion took from 48 h to more than 6 days, depending on the culture conditions. For EE2 degradation, when it was the sole carbon source, the lag phase and the time required for its complete removal (7 and 15 days, respectively) were shorter that in cultures with a supplementary carbon source. The specific degradation rates observed for E2 both with and without an additional carbon source were similar. By contrast, the specific degradation rates for E1 and EE2 were, respectively, seven and 20 times faster when these hormones were supplied as the sole carbon source. The bacterial community structure of each culture was characterized by molecular and cultural methods. The mixed cultures were made up of species belonging to Alcaligenes faecalis, Pusillimonas sp., Denitrobacter sp., and Brevundimonas diminuta or related to uncultured Bacteroidetes. The isolated strain B. diminuta achieved the conversion of E2 to E1.