Hydrogenotrophic Activity Research Articles

Recovery of anaerobic system treating sulfate-rich wastewater using zero-valent iron

The anaerobic digestion (AD) process often fails in treating wastewater containing high levels of sulfate because sulfate-reducing bacteria (SRB) can reduce sulfate to sulfide and compete with methane-producing archaea (MPA) for their common substrates. The sulfide produced from the sulfate reduction could inhibit both MPA and SRB. In this study, we investigated the application of zero-valent iron (ZVI) to recover the growth and activities of MPA and SRB in an AD process treating wastewater with a relatively low ratio of chemical oxygen demand (COD) to sulfate ratio (SO42-) (9,000 mg COD/L glucose and 4,500 mg/L SO42-). The one-time addition of 210 g ZVI increased the CH4 production rates from near zero to 0.54 ± 0.15 L/L·d. An electron flow analysis (in terms of COD) demonstrated that the CH4-COD was approximately eight times higher than the COD contributed by ZVI electrons. The results indicated that ZVI not only provided extra electrons for use in hydrogenotrophic methanogenesis, but also provided ferrous ions that could co-precipitate with sulfides and reduced the inhibition of acetoclastic methanogens from undissociated H2S. ZVI helped recover the acetoclastic activity of the biomass to approximately the same level as the activity of the inoculum before being inhibited by sulfides and improved the hydrogenotrophic activity by over 72 times. In addition, ZVI had different effects on the acetoclastic activities of the culture attached to ZVI and the suspended culture. The acetoclastic activity of the culture attached to ZVI (0.446 ± 0.003 g COD/g COD·d) was only approximately half of that of the suspended culture (1.056 ± 0.070 g COD/g COD·d).

Dynamic changes in anaerobic digester metabolic pathways and microbial populations during acclimatisation to increasing ammonium concentrations

Transitions in microbial community structure in response to increasing ammonia concentrations were determined by monitoring mesophilic anaerobic digesters seeded with a predominantly acetoclastic methanogenic community from a sewage sludge digester. Ammonia concentration was raised by switching the feed to source segregated domestic food waste and applying two organic loading rates (OLR) and hydraulic retention times (HRT) in paired digesters. One of each pair was dosed with trace elements (TE) known to be essential to the transition, with the other unsupplemented digester acting as a control. Samples taken during the trial were used to determine the metabolic pathway to methanogenesis using 14C labelled acetate. Partitioning of 14C between the product gases was interpreted via an equation to indicate the proportion produced by acetoclastic and hydrogenotrophic routes. Archaeal and selected bacterial groups were identified by 16S rRNA sequencing, to determine relative abundance and diversity. Acclimatisation for digesters with TE was relatively smooth, but OLR and HRT influenced both metabolic route and community structure. The 14C ratio could be used quantitatively and, when interpreted alongside archaeal community structure, showed that at longer HRT and lower loading Methanobacteriaceae were dominant and hydrogenotrophic activity accounted for 77% of methane production. At the higher OLR and shorter HRT, Methanosarcinaceae were dominant with the 14C ratio indicating simultaneous production of methane by acetoclastic and hydrogenotrophic pathways: the first reported observation of this in digestion under mesophilic conditions. Digesters without TE supplementation showed similar initial changes but, as expected failed to complete the transition to stable operation.

Hydrogenotrophic activity: A tool to evaluate the kinetics of methanogens

Anaerobic-digestion-based technology is key to achieving sustainable water management and resource recovery. It is essential to understand the material flux and kinetics involved in methanogenesis to optimize the organic matter removal and methane production. In this sense, specific methanogenic activity is a cost-effective tool to characterize the biological activity of anaerobic biosludge, to monitor the performance of reactors, and study the kinetics of acetate and H2 conversion to methane. Established protocols are applied for the acetoclastic activity test. However, hydrogenotrophic activity assay remains less widespread and is not standardized. In this work, the assay design for hydrogenotrophic activity is discussed and full calculation is presented, based on the kinetics for the H2/CO2 conversion to methane. An equation to calculate the inoculum size is proposed, suitable for a wide variety of types of biosludge: from a wastewater treatment plant to solid digesters, from a high-rate reactor to lagoons. The applied zero-order model fitted adequately to data for pilot-scale and full-scale anaerobic reactors: the p-values from the ANOVA F-test were below 1E-03; standard deviations for triplicate experiments were between 3 and 12%, coherent with the values found in the literature. Microbial growth during the test was negligible, below 1.2% of the biomass dosed in the vial. As a complement, acetoclastic activity was determined for each sample. The use of both acetoclastic and hydrogenotrophic activity is relevant for the study of the methanogenesis and gives a better characterization of the performance of the biosludge in anaerobic reactors rather than only using the specific acetoclastic methanogenic activity.

Reduced graphene oxide decorated with magnetite nanoparticles enhance biomethane enrichment

The addition of magnetite nanoparticles (MNPs), reduced graphene oxide (rGO), and reduced graphene oxide decorated with magnetite nanoparticles (rGO-MNPs) was evaluated during biomethane enrichment process. rGO-MNPs presented the highest beneficial impact on the hydrogenotrophic assays with an improvement of 47 % in CH4 production. The improvement was linked to the increase of the electron shuttling capacity (ESC) by rGO-MNPs addition, which boosted the hydrogenotrophic activity of microorganisms, to the rGO and rGO-MNPs, which served as reservoirs of hydrogen, improving H⁠2 transport from the gas to the liquid phase, and to the iron ions released, which acted as a dietary supply for microorganisms. Raman and XRD confirmed a greater disorder and lower crystallinity of rGO-MNPs after the hydrogenotrophic assays, with a lower effect at a nanoparticle concentration of 50 mg/L. Moreover, FTIR analysis indicated that rGO-MNPs were oxidized during the hydrogenotrophic tests. This study highlights the advantages of adding rGO-MNPs as a magnetic nanocomposite. Furthermore, rGO-MNPs can be easily recovered, minimizing their release to the environment.

Hydrogenotrophic activity of strain RWL1 similar to Methanothrix soehngenii

Methanogens are strictly anaerobic organisms that produce methane. Based on the nature of substrate utilization, they are classified into acetoclastic, hydrogenotrophic and methylotrophic methanogens. Methanothrix soehngenii is a methanogen believed to be acetoclastic. A strain RWL1, similar to M. soehngenii with hydrogenotrophic activity has been isolated, characterized and identified by 16S rRNA sequencing. The strain RWL1 utilizes H2+CO2 (4:1) and has recorded methane production of 74.82%, thereby opening a new gate for substrate diversity studies. This study reveals the hydrogenotrophic nature of strain RWL1 clustered with M. soehngenii..

Enhancing hydrogenotrophic activities by zero-valent iron addition as an effective method to improve sulfadiazine removal during anaerobic digestion of swine manure

In this study, the feasibility of Fe0 addition for driving sulfadiazine (SDZ) removal during anaerobic digestion of swine manure (SM) was tested. Compared with the Fe0-free digesters spiked with 200 mg/L SDZ (RSDZ), treatments with 5.0 g/L Fe0 (RSDZ/Fe0) significantly accelerated and optimized the acidification process by enriching Clostridia and Bacteroidia (key members responsible for VFAs/H2 production), providing more readily available substrates for methanogenesis. Furthermore, Fe0 increased the overall abundance of hydrogenotrophic methanogens, specifically toxicant resistant Methanoculleus and Methanosphaera spp. were selectively enriched, helping achieve a 36.9% higher CH4 yield and a 26.4% greater total solids removal efficiency. A positive correlation between the solid content and the SDZ concentration adsorbed in SM was observed. The addition of Fe0 increased the distribution of SDZ in liquid and facilitated its removal through the enhanced biodegradation and physicochemical processes. Overall, the total SDZ removal efficiency was improved by 86.8% with Fe0.

Methane Production and Conductive Materials: A Critical Review.

Conductive materials (CM) have been extensively reported to enhance methane production in anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in microbial communities, as an alternative or complementary to indirect electron transfer (via hydrogen or formate), is the main explanation given to justify the improvement of methane production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely does not explain all the reported observations. In fact, in methanogenic environments DIET was only unequivocally demonstrated in cocultures of Geobacter metallireducens with Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not detected in improved methane production driven systems. Furthermore, conductive carbon nanotubes were shown to accelerate the activity of methanogens growing in pure cultures, where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale anaerobic digesters treating for example brewery wastewaters, indicating that interspecies hydrogen transfer is an important electron transfer mechanism in those systems. This paper presents an overview of the effect of several iron-based and carbon-based CM in bioengineered systems, focusing on the improvement in methane production and in microbial communities' changes. Control assays, as fundamental elements to support major conclusions in reported experiments, are critically revised and discussed.

Compositional and functional dynamics of the bovine rumen methanogenic community across different developmental stages.

SummaryMethanogenic archaea in the bovine rumen are responsible for the reduction of carbon molecules to methane, using various electron donors and driving the electron flow across the microbial food webs. Thus, methanogens play a key role in sustaining rumen metabolism and function. Research of rumen methanogenic archaea typically focuses on their composition and function in mature animals, while studies of early colonization and functional establishment remain scarce. Here, we investigated the metabolic potential and taxonomic composition of the methanogenic communities across different rumen developmental stages. We discovered that the methanogenesis process changes with age and that the early methanogenic community is characterized by a high activity of methylotrophic methanogenesis, likely performed by members of the order Methanosarcinales, exclusively found in young rumen. In contrast, higher hydrogenotrophic activity was observed in the mature rumen, where a higher proportion of exclusively hydrogenotrophic taxa are found. These findings suggest that environmental filtering acts on the archaeal communities and select for different methanogenic lineages during different growth stages, affecting the functionality of this ecosystem. This study provides a better understanding of the compositional and metabolic changes that occur in the rumen microbiome from its initial stages of colonization and throughout the animals' life.

Inglés

This study identifed and quantifed microbial populations in cattle manure slurry, responsible for anaerobic degradation of kitchen waste using activity tests and Most Probable Number for key trophic groups in the process. Hydrolytic, acidogenic, acetogenic, specifc methanogenic acetoclastic and hydrogenotrophic activities were evaluated using as model substrates starch, glucose, propionate-butyrate mix, acetate and formate, respectively. Anaerobic digestion from kitchen waste was developed for 35 days in batch reactors of 50mL containing an inoculums/substrate ratio of 3. Samples were taken from digesters every 7 days to evaluate microbial populations by counting metabolic groups. This study demonstrates that there is an association between biomass activity and population of trophic groups related. Additionally, CMS is an inoculum with high quality to start-up the anaerobic process with kitchen waste. On the other side, MA/FB ratio and MA/SRB ratio are important microbial parameters to evaluate performance of reactor. Finally, AD from kitchen waste reached a yield coeffcient of 0.41m3 CH4/kgVS using cattle manure sludge as inoculum. Keywords: trophic groups, metabolic groups, kitchen waste, cattle manure slurry, MPN, microbial activities.

Formate‐derived H2, a driver of hydrogenotrophic processes in the root‐zone of a methane‐emitting fen

Wetlands are important sources of globally emitted methane. Plants mediate much of that emission by releasing root-derived organic carbon, including formate, a direct precursor of methane. Thus, the objective of this study was to resolve formate-driven processes potentially linked to methanogenesis in the fen root-zone. Although, formate was anticipated to directly trigger methanogenesis, the rapid anaerobic consumption of formate by Carex roots unexpectedly yielded H2 and CO2 via enzymes such as formate-H2 -lyase (FHL), and likewise appeared to enhance the utilization of organic carbon. Collectively, 57 [FeFe]- and [NiFe]-hydrogenase-containing family level phylotypes potentially linked to FHL activity were detected. Under anoxic conditions, root-derived fermentative Citrobacter and Hafnia isolates produced H2 from formate via FHL. Formate-derived H2 fueled methanogenesis and acetogenesis, and methanogenic (Methanoregula, Methanobacterium, Methanocella) and acetogenic (Acetonema, Clostridum, Sporomusa) genera potentially linked to these hydrogenotrophic activities were identified. The findings (i) provide novel insights on highly diverse root-associated FHL-containing taxa that can augment secondary hydrogenotrophic processes via the production of formate-derived H2 , (ii) demonstrate that formate can have a 'priming' effect on the utilization of organic carbon, and (iii) raise questions regarding the fate of formate-derived H2 when it diffuses away from the root-zone.

Microbial methanogenesis in the sulfate-reducing zone of surface sediments traversing the Peruvian margin

Abstract. We studied the concurrence of methanogenesis and sulfate reduction in surface sediments (0–25 cm below sea floor) at six stations (70, 145, 253, 407, 990 and 1024 m) along the Peruvian margin (12° S). This oceanographic region is characterized by high carbon export to the seafloor creating an extensive oxygen minimum zone (OMZ) on the shelf, both factors that could favor surface methanogenesis. Sediments sampled along the depth transect traversed areas of anoxic and oxic conditions in the bottom-near water. Net methane production (batch incubations) and sulfate reduction (35S-sulfate radiotracer incubation) were determined in the upper 0–25 cm b.s.f. of multiple cores from all stations, while deep hydrogenotrophic methanogenesis (> 30 cm b.s.f., 14C-bicarbonate radiotracer incubation) was determined in two gravity cores at selected sites (78 and 407 m). Furthermore, stimulation (methanol addition) and inhibition (molybdate addition) experiments were carried out to investigate the relationship between sulfate reduction and methanogenesis.Highest rates of methanogenesis and sulfate reduction in the surface sediments, integrated over 0–25 cm b.s.f., were observed on the shelf (70–253 m, 0.06–0.1 and 0.5-4.7 mmol m−2 d−1, respectively), while lowest rates were discovered at the deepest site (1024 m, 0.03 and 0.2 mmol m−2 d−1, respectively). The addition of methanol resulted in significantly higher surface methanogenesis activity, suggesting that the process was mostly based on non-competitive substrates – i.e., substrates not used by sulfate reducers. In the deeper sediment horizons, where competition was probably relieved due to the decrease of sulfate, the usage of competitive substrates was confirmed by the detection of hydrogenotrophic activity in the sulfate-depleted zone at the shallow shelf station (70 m).Surface methanogenesis appeared to be correlated to the availability of labile organic matter (C ∕ N ratio) and organic carbon degradation (DIC production), both of which support the supply of methanogenic substrates. A negative correlation between methanogenesis rates and dissolved oxygen in the bottom-near water was not obvious; however, anoxic conditions within the OMZ might be advantageous for methanogenic organisms at the sediment-water interface.Our results revealed a high relevance of surface methanogenesis on the shelf, where the ratio between surface to deep (below sulfate penetration) methanogenic activity ranged between 0.13 and 105. In addition, methane concentration profiles indicated a partial release of surface methane into the water column as well as consumption of methane by anaerobic methane oxidation (AOM) in the surface sediment. The present study suggests that surface methanogenesis might play a greater role in benthic methane budgeting than previously thought, especially for fueling AOM above the sulfate–methane transition zone.

Magnetite as a precursor for green rust through the hydrogenotrophic activity of the iron‐reducing bacteria Shewanella putrefaciens

Magnetite (Fe(II) Fe(III) 2 O4 ) is often considered as a stable end product of the bioreduction of Fe(III) minerals (e.g., ferrihydrite, lepidocrocite, hematite) or of the biological oxidation of Fe(II) compounds (e.g., siderite), with green rust (GR) as a mixed Fe(II) -Fe(III) hydroxide intermediate. Until now, the biotic transformation of magnetite to GR has not been evidenced. In this study, we investigated the capability of an iron-reducing bacterium, Shewanella putrefaciens, to reduce magnetite at circumneutral pH in the presence of dihydrogen as sole inorganic electron donor. During incubation, GR and/or siderite (Fe(II) CO3 ) formation occurred as secondary iron minerals, resulting from the precipitation of Fe(II) species produced via the bacterial reduction of Fe(III) species present in magnetite. Taking into account the exact nature of the secondary iron minerals and the electron donor source is necessary to understand the exergonic character of the biotic transformation of magnetite to GR, which had been considered to date as thermodynamically unfavorable at circumneutral pH. This finding reinforces the hypothesis that GR would be the cornerstone of the microbial transformations of iron-bearing minerals in the anoxic biogeochemical cycle of iron and opens up new possibilities for the interpretation of the evolution of Earth's history and for the understanding of biocorrosion processes in the field of applied science.

Hydrogenotrophic activity under increased H2/CO2 pressure: Effect on methane production and microbial community

Hydrogenotrophic activity under increased H2/CO2 pressure: Effect on methane production and microbial community

English

The influence of mixing H2/CO2 gas recirculation on the performance of hydrogenotrophic methanogens activity in continuous culture was studied at 37 and 20°C. Chemostat fermentation was used at laboratory scale to determine the bioconversion rate of H2/CO2 mixture gas to methane under different mixing rates. On comparison with continuous mixing, intermittent mixing at 45 min/h provided a better methane production, 1.94 ± 0.06 versus 1.83 ± 0.05 L for continuous mixing. When the temperature was progressively decreased to 20°C, the same configuration was observed. The methane production was lowered from 76.2, 80.8, 67.5, 61.2 to 27.9, 35.8, 29.6 and 26.7% for 60, 45, 30 and 15 min/h, respectively. The mixing at 45 min/h showed a stable methane production as compared to all proposed mixing duration especially at psychrophilic temperature. The results would facilitate an empiric model that could help to establish more economical biogas reactor model. Key words: Hydrogenotrophic methanogens, mixing, H2/CO2 gas, bioconversion, temperature fluctuation.

Improved Stability of Anaerobic Digestion through the Use of Selective Acidogenic Culture

Accumulation of volatile fatty acids (VFAs) under overloading conditions in anaerobic reactors is a common problem. There is a need to stop this accumulation to improve reactor stability. In this study, propionate-, butyrate-, and acetate-degrading cultures were enriched and used as inocula in a mesophilic two-stage reactor to improve system stability. The performance of a test reactor was compared with that of a control reactor that was not seeded with specific VFA-degrading inocula under normal and overloading conditions. The reactors were fed with simulated wastewater having a chemical oxygen demand (COD) of 10,000 mg L−1. The test reactor inoculated with VFA-degrading cultures showed better performance in terms of methane production, COD removal, and VFA degradation. Also, after two consecutive organic shocks (influent COD 20,000 mg L−1) the test reactor recovered within four days and low levels of VFAs, especially propionate (429 mg L−1), were observed. In contrast, in the control reactor the concentration of VFA did not decline to preshock levels, even after five days of two consecutive shocks. PCR-denaturing gradient gel electrophoresis analysis using 16S rRNA gene amplicons also indicated a significant difference in archaeal community structure in the control and test reactors. Methanosarcinaceae was found dominant in the test reactor whereas in the control reactor an equal abundance of both Methanosaetaceae and Methanosarcinaceae was observed. Specific methanogenic activity also suggested higher acetoclastic and hydrogenotrophic activity of the sludge in the test reactor. Overall, using enriched culture as inocula resulted in a more balanced and robust methanogenic consortium and in improved system stability, with efficient degradation of the high concentration of VFAs.

Thermophilic anaerobic co-digestion of sewage sludge with grease waste: Effect of long chain fatty acids in the methane yield and its dewatering properties

Thermophilic co-digestion of sewage sludge with three different doses of trapped grease waste (GW) from the pre-treatment of a WWTP has been assessed in a CSTR bench-scale reactor. After adding 12% and 27% of grease waste (on COD basis), the organic loading rate increased from 2.2 to 2.3 and 2.8kgCODm−3d−1 respectively, and the methane yield increased 1.2 and 2.2 times. Further GW increase (37% on COD basis) resulted in an unstable methane yield and in long chain fatty acids (LCFA) accumulation. Although this inestability, the presence of volatile fatty acids in the effluent was negligible, showing good adaptation to fats of the thermophilic biomass. Nevertheless, the presence of LCFA in the effluent worsens its dewatering properties. Specific methanogenic activity tests showed that the addition of grease waste ameliorates the acetoclastic activity in detriment of the hydrogenotrophic activity, and suggests that the tolerance to LCFA can be further enhanced by slowly increasing the addition of lipid-rich materials.

“Methano-compost”, a booster and restoring agent for thermophilic anaerobic digestion of energy crops

The influence of plant litter-compost of the hot rotten-phase as additional inoculum for anaerobic batch digestion of sugar beet silage (SBS) was studied. Four simultaneously driven batch-fermenters were inoculated with sewage sludge. Two of the fermenters were inoculated additionally with the same amount of organics by compost of the hot rotten-phase. Two of the fermenters were mesophilic (40 °C) and the other two were thermophilic (60 °C). The impact on the gas production rate and gas yield was observed to be boosted for thermophilic (60 °C) and only a minor effect of 6–13% for mesophilic (40 °C) digestion. The gas yield increased considerably up to 26.5% at 60 °C (batch). Also the methane content increased from 57.4% to 62.3% by adding compost (continuously run mesophilic digestion). Fluorescence In Situ Hybridization (FISH) indicated that a microbial effect was responsible for the observed stimulation of gas production rates, but not simply by increasing the bacterial counts. By analysing each fermenter for its mineral and trace element content a mineralic effect could be excluded. However, the bacterial counts by FISH of 10 different groups were somewhat ambiguous. But an effect on the presence of Chloroflexi could be demonstrated. They nearly doubled to 15–16% by supplementation with compost. Furthermore, under thermophilic conditions, the added compost induced a significant shift in the microbial composition towards hydrogenotrophic Methanobacteriales. The suggestive conclusion drawn is that this explicitly increase in hydrogenotrophic activity could alone or in combination with accompanying fermentative bacteria forces the microbial food chain towards stimulation of methane generation.

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m(-3) day(-1) (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.

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

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

Optimizing the electrode size and arrangement in a microbial electrolysis cell

This study investigates the influence of anode and cathode size and arrangement on hydrogen production in a membrane-less flat-plate microbial electrolysis cell (MEC). Protein measurements were used to evaluate microbial density in the carbon felt anode. The protein concentration was observed to significantly decrease with the increase in distance from the anode–cathode interface. Cathode placement on both sides of the carbon felt anode was found to increase the current, but also led to increased losses of hydrogen to hydrogenotrophic activity leading to methane production. Overall, the best performance was obtained in the flat-plate MEC with a two-layer 10mm thick carbon felt anode and a single gas-diffusion cathode sandwiched between the anode and the hydrogen collection compartments.