A bioelectrochemical reactor containing carbon fiber textiles enables efficient methane fermentation from garbage slurry

A packed-bed system includes supporting materials to retain microorganisms and a bioelectrochemical system influences the microbial metabolism. In our study, carbon fiber textiles (CFT) as a supporting material was attached onto a carbon working electrode in a bioelectrochemical reactor (BER) that degrades garbage slurry to methane, in order to investigate the effect of combining electrochemical regulation and packing CFT. The potential on the working electrode in the BER containing CFT was set to −1.0 V or −0.8 V (vs. Ag/AgCl). BERs containing CFT exhibited higher methane production, elimination of dichromate chemical oxygen demand, and the ratio of methanogens in the suspended fraction than reactors containing CFT without electrochemical regulation at an organic loading rate (OLR) of 27.8 gCODcr/L/day. In addition, BERs containing CFT exhibited higher reactor performances than BERs without CFT at this OLR. Our results revealed that the new design that combined electrochemical regulation and packing CFT was effective.     

Factors influencing the degradation of garbage in methanogenic bioreactors and impacts on biogas formation

Anaerobic digestion of garbage is attracting much attention because of its application in waste volume reduction and the recovery of biogas for use as an energy source. In this review, various factors influencing the degradation of garbage and the production of biogas are discussed. The surface hydrophobicity and porosity of supporting materials are important factors in retaining microorganisms such as aceticlastic methanogens and in attaining a higher degradation of garbage and a higher production of biogas. Ammonia concentration, changes in environmental parameters such as temperature and pH, and adaptation of microbial community to ammonia have been related to ammonia inhibition. The effects of drawing electrons from the methanogenic community and donating electrons into the methanogenic community on methane production have been shown in microbial fuel cells and bioelectrochemical reactors. The influences of trace elements, phase separation, and co-digestion are also summarized in this review.     

Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Organic loading rate and food-to-microorganism ratio shape prokaryotic diversity in a demo-scale up-flow anaerobic sludge blanket reactor treating domestic wastewater

We investigated the microbial community in an up-flow anaerobic sludge blanket (UASB) reactor treating domestic wastewater (DW) during two different periods of organic loading rate (OLR) and food-to-microorganism (F/M) ratio. 16S rDNA clone libraries were generated, and quantitative real-time PCR (qPCR) analyses were performed. Fluctuations in the OLR and F/M ratio affected the abundance and the composition of the UASB prokaryotic community, mainly at the species level, as well as the performance of the UASB reactor. The qPCR analysis suggested that there was a decrease in the bacterial cell number during the rainy season, when the OLR and F/M ratio were lower. However, the bacterial diversity was higher during this time, suggesting that the community degraded more diversified substrates. The diversity and the abundance of the archaeal community were higher when the F/M ratio was lower. Shifts in the methanogenic community composition might have influenced the route of methane production, with methane produced by acetotrophic methanogens (dry season), and by hydrogenotrophic, methylotrophic and acetotrophic methanogens (rainy season). This study revealed higher levels of bacterial diversity, metabolic specialization and chemical oxygen demand removal efficiency of the DW UASB reactor during the rainy season.     

Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process

The present study investigated a two-stage anaerobic hydrogen and methane process for increasing bioenergy production from organic wastes. A two-stage process with hydraulic retention time (HRT) 3 d for hydrogen reactor and 12 d for methane reactor, obtained 11% higher energy compared to a single-stage methanogenic process (HRT 15 d) under organic loading rate (OLR) 3 gVS/(L d). The two-stage process was still stable when the OLR was increased to 4.5 gVS/(L d), while the single-stage process failed. The study further revealed that by changing the HRT_hydrogen:HRT_methane ratio of the two-stage process from 3:12 to 1:14, 6.7%, more energy could be obtained. Microbial community analysis indicated that the dominant bacterial species were different in the hydrogen reactors (Thermoanaerobacterium thermosaccharolyticum-like species) and methane reactors (Clostridiumthermocellum-like species). The changes of substrates and HRT did not change the dominant species. The archaeal community structures in methane reactors were similar both in single- and two- stage reactors, with acetoclastic methanogens Methanosarcina acetivorans-like organisms as the dominant species.     

Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L⁻¹, the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L⁻¹. Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Microbial population in the biomass adhering to supporting material in a packed-bed reactor degrading organic solid waste

An anaerobic packed-bed reactor using carbon fiber textiles (CFT) as the supporting material was continuously operated using an artificial garbage slurry. 16S rRNA gene analysis showed that many bacteria in the biomass adhering to CFT were closely related to those observed from other anaerobic environments, although a wide variety of unidentified bacteria were also found. Dot blot hybridization results clarified that 16S rRNA levels of methanogens in the adhering biomass were higher than those in the effluent. Based on microscopic observation, the adhering biomass consisted of microorganisms, organic material, and void areas. Bacteria and Archaea detected by fluorescence in situ hybridization were distributed from the surface to the inner regions of the adhering biomass. Methanosarcina sp. tended to be more abundant in the inner part of the adhering biomass than at the surface. This is the first report to elucidate the structure of the microbial community on CFT in a packed-bed reactor.     

Microstructure and molecular microbiology of rapidly formed hydrogen‐ and methane‐producing granules in a phase‐separated anaerobic environment

Hydrogen and methane were simultaneously produced in a two‐phase reactor, operated to separate the reactions of hydrogen and methanogen production. Each reactor was inoculated with a seed enriched with different microbial consortia. The first phase was operated with a hydraulic retention time of 7 days and at an organic loading rate of 7.7 g VS L^?1 d^?1 that produced a stable pH of 5.5. This suppressed the growth of methanogens and as a result, the off gas contained up to 27% hydrogen. The second phase was operated with a hydraulic retention time of 12 days and at an organic loading rate of 3.6 g VS L^?1 d^?1. This permitted the growth of hydrogenotrophs and methanogens to produce methane at a concentration of 60%. Examination of the microbial population of the two reactors both microscopically and using PCR, showed an effective separation of hydrogen‐ and methane‐producing microbial communities. The study revealed that the suppression of hydrogentrophs and methanogens can be achieved by adopting rapid method that leads the growth of hydrogen‐ and methane‐producing granules in phase‐separated anaerobic environment.     

The influence of cell and substratum surface hydrophobicities on microbial attachment

This study investigated the role of hydrophobic/hydrophilic interaction between bacterial and support surfaces in microbial adhesion, and a model that correlates microbial adhesion and relative cell-hydrophobicity defined as the ratio of cell-support surface hydrophobicity over cell-support hydrophilicity was derived. This model quantitatively describes how cell hydrophobic and hydrophilic interactions affect microbial adhesion, and offers deep insights into the thermodynamic mechanisms of microbial adhesion. The proposed model was verified by literature data. It appears that a high cell-hydrophobicity strongly facilitates microbial adhesion on both hydrophobic and hydrophilic support surfaces.     

Granulation and Sludge Bed Stability in Upflow Anaerobic Sludge Bed Reactors in Relation to Surface Thermodynamics

Adhesion of bacteria involved in anaerobic consortia was investigated in upflow anaerobic sludge bed reactors and was related to surface thermodynamics. The adhesion of hydrophilic cells appeared to be enhanced at a low liquid surface tension ((gamma)(infLV)), while the adhesion of hydrophobic cells was favored at a high (gamma)(infLV). Growth in protein-rich growth media resulted in low granular biomass yields; addition of polycations, such as poly-l-lysine and chitosan, increased the (gamma)(infLV) and the granular biomass yield. On the basis of the results of activity tests and microbial counts with wash-out cells, we identified two types of structured granules that were related to the influence of (gamma)(infLV). In one type of granules, hydrophilic acidogens surrounded a more hydrophobic methanogenic association. These granules were selected at a low (gamma)(infLV) provided that carbohydrates were available as substrates. The other type of granules was selected at a high (gamma)(infLV); hydrophobic cells (i.e., methanogens) were predominant throughout these granules. The granules which had acidogens as solid-phase emulsifiers around a methanogenic association appeared to allow more stable reactor performance. Decreasing the (gamma)(infLV) in the reactor by adding trace amounts of a surfactant also increased reactor stability.     

Effects of aeration and organic loading rates on degradation of trichloroethylene in a methanogenic-methanotrophic coupled reactor

The effects of four aeration and four organic loading (OLR) rates on trichloroethylene (TCE) degradation in methanogenic-methanotrophic coupled reactors were studied using ethanol as the carbon source for the methanogens. Microcosm and PCR studies demonstrated that methanotrophs capable of mineralizing TCE and methanogens were present in the biomass throughout the study. The gene for the particulate form of methane monooxygenase (pMMO) was detected by PCR, but not that for the soluble form (sMMO). TCE mineralization by methanotrophs was therefore due primarily to pMMO activity. Low TCE concentrations were measured in effluent and off-gas samples in all cases. Volatilization losses were 0-5%. Dichloroethylene (DCE) was also observed, but vinyl chloride and ethylene were never detected. Changes in the aeration rate had no effect on TCE removal, but did influence DCE degradation. Reductive dechlorination of TCE to DCE was favored at low and no-aeration conditions, and DCE accumulation occurred due to slow DCE degradation. Low DCE levels were observed at the higher aeration rates, which indicated that conditions in these reactors were amenable to the aerobic co-metabolism of TCE and DCE. The OLR did have an effect on TCE removal. TCE and DCE removal were negatively affected when the OLR was increased. An OLR of 0.3 g COD l(rx)(-1)day(-1) or lower with an aeration rate of 3 l(O2 )l(rx)(-1)day(-1) and higher is the recommended operating condition of a coupled reactor for removal of TCE.     

Hydrolysis,acidification and methanogenesis during low-temperature anaerobic digestion of dilute dairy wastewater in an inverted fluidised bioreactor

The application of low-temperature (10 °C) anaerobic digestion (LtAD) for the treatment of complex dairy-based wastewater in an inverted fluidised bed (IFB) reactor was investigated. Inadequate mixing intensity provoked poor hydrolysis of the substrate (mostly protein), which resulted in low chemical oxygen demand (COD) removal efficiency throughout the trial, averaging ~69 % at the best operational period. Overgrowth of the attached biomass to the support particles (Extendospheres) induced bed stratification by provoking agglutination of the particles and supporting their washout by sedimentation, which contributed to unstable bioprocess performance at the organic loading rates (OLRs) between 0.5 and 5 kg COD m^?3 day^?1. An applied OLR above 2 kg COD m^?3 day^?1 additionally promoted acidification and strongly influenced the microbial composition and dynamics. Hydrogenotrophic methanogens appeared to be the mostly affected group by the Extendospheres particle washout as a decrease in their abundance was observed by quantitative PCR analysis towards the end of the trial, although the specific methanogenic activity and maximum substrate utilisation rate on H₂/CO₂ indicated high metabolic activity and preference towards hydrogenotrophic methanogenesis of the reactor biomass at this stage. The bacterial community in the bioreactor monitored via denaturing gradient gel electrophoresis (DGGE) also suggested an influence of OLR stress on bacterial community structure and population dynamics. The data presented in this work can provide useful information in future optimisation of fluidised reactors intended for digestion of complex industrial wastewaters during LtAD.     

Effect of temperature on performance and microbial diversity in hyperthermophilic digester system fed with kitchen garbage

The objective of this study was to evaluate the performances and microbial diversities for development of the effective hyperthermophilic digester system that consists of hyperthermophilic reactor and hyperthermophilic or thermophilic reactor in series. Lab-scale reactors were operated continuously fed with artificial kitchen garbage. The effect of temperature on the acidification step was firstly investigated. Results indicated that 43.1% of COD solubilization was achieved at 70 degrees C, while it was about 21% at 80 degrees C. The average protein solubilization reached to 31% at 80 degrees C. Methane conversion efficiency following the acidification was around 85% in average at 55 degrees C, but decreased with increasing temperature and methane gas was not produced over 73 degrees C. The upper temperature limits for growth of microbes were secondly observed and shown to be 73 degrees C for acetate oxidizers, 65 degrees C for propionate oxidizers, 70 degrees C for iso-butyrate oxidizers, 80 degrees C for lactate oxidizers and 65 degrees C for protein degrading bacteria in the methane fermenter. As well as, microbes affiliated with methanogens dominated the population below the 65 degrees C, while those affiliated with acidogens were predominant over the 73 degrees C. These results indicated that the hyperthermophilic processes have considerable benefits to treat wastewater or waste containing high concentration of protein.     

Immobilization patterns and dynamics of acetate-utilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and mumax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Performance and microbial communities of a continuous stirred tank anaerobic reactor treating two-phases olive mill solid wastes at low organic loading rates

A study of the performance and microbial communities of a continuous stirred tank reactor (CSTR) treating two-phases olive mill solid wastes (OMSW) was carried out at laboratory-scale. The reactor operated at a mesophilic temperature (35 degrees C) and an influent substrate concentration of 162 g total chemical oxygen demand (COD)L(-1) and 126 g volatile solids (VS)L(-1). The data analyzed in this work corresponded to a range of organic loading rates (OLR) of between 0.75 and 3.00 g CODL(-1)d(-1), getting removal efficiencies in the range of 97.0-95.6%. Methane production rate increased from 0.164 to 0.659 L CH(4)L(reactor)(-1)d(-1) when the OLR increased within the tested range. Methane yield coefficients were 0.225 L CH(4)g(-1) COD removed and 0.290 L CH(4)g(-1) VS removed and were virtually independent of the OLR applied. A molecular characterization of the microbial communities involved in the process was also accomplished. Molecular identification of microbial species was performed by PCR amplification of 16S ribosomal RNA genes, denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Among the predominant microorganisms in the bioreactor, the Firmicutes (mainly represented by Clostridiales) were the most abundant group, followed by the Chloroflexi and the Gamma-Proteobacteria (Pseudomonas species as the major representative). Other bacterial groups detected in the bioreactor were the Actinobacteria, Bacteroidetes and Deferribacteres. Among the Archaea, the methanogen Methanosaeta concilii was the most representative species.     

Hydrophobic interactions within biofilms of nitrifying and denitrifying bacteria in biofilters

Hydrophobicity of the solid surface and microbial cell surface is important factor for the development of biofilms applied in bioengineering systems. An adsorption of phenanthrene was used for analysis of the hydrophobicity of support fibers and bacterial cell surfaces within the biofilter of wastewater. The adsorption of phenanthrene was measured by synchronous fluorescence spectrometry. Cell surface hydrophobicity does not depend on the fixation procedure, pH of microbial suspension, and has no clear correlation with an adherence of the cells to hexadecane droplets. Notwithstanding high hydrophobicity of bacterial cells, the hydrophobicity of intact biofilm is determined by the hydrophobicity of the support fibers. New indexes were proposed to evaluate the reactor performance related with hydrophobic interactions within the biofilm. These indexes showed that significant share of hydrophobic sites within the nitrifying biofilm is protected from the hydrophobic interactions between the cells and environment.     

Illumination enhances methane production from thermophilic anaerobic digestion

Incandescent lamp illumination enhanced methane production from a thermophilic anaerobic digestion reactor (55°C) supplied with glucose. After 10 days of operation, the volume of methane produced from light reactors was approximately 2.5 times higher than that from dark reactors. A comparison of the carbon balance between light and dark conditions showed that methane produced from hydrogen and carbon dioxide in the light reactors was higher than that from the dark reactors. When hydrogen or acetate was fed into the reactors, methane production with added hydrogen was faster and higher under light conditions than under dark conditions. The use of blue light-emitting diodes also enhanced methane production over that under dark conditions. The 16S rRNA gene copy numbers for Methanothermobacter spp. in the light reactor and in the dark reactor were at the same level. The copy number for Methanosarcina spp. in the light reactors was approximately double than that in the dark reactors. These results suggest that blue light enhances the methanogenic activity of hydrogenotrophic methanogens.     

Improved biogas production from palm oil mill effluent by a scaled-down anaerobic treatment process

This is a scale-down study of a 500-m³ methane recovery test plant for anaerobic treatment of palm oil mill effluent (POME) where biomass washout has become one of the problems because of the continuous mixing of effluent during anaerobic treatment of POME. Therefore, in this study, anaerobic POME treatment using a scaled down 50-l bioreactor which mimicked the 500-m³ bioreactor was carried out to improve biogas production with and without biomass sedimentation. Three sets of experiments were conducted under different conditions in terms of biomass sedimentation applied to the system. The first experiment was operated under semi-continuous mode whereas the second and third experiments were operated based on mix and settle mode. As expected, biomass retention improved the anaerobic process as the POME treatment incorporated with mix and settle system were able to operate at an organic loading rate (OLR) of 3.5 and 6.0 kg COD/m³/day respectively, while the semi-continuous operated anaerobic treatment only achieved OLR of 3.0 kg COD/m³/day. The highest biogas and methane production rates achieved were 2.42 m³/m³ of reactor/day and 0.992 m³/m³ of reactor/day, respectively at OLR 6.0 kg COD/m³/day. The biomass or solids retention in the reactors was represented by the total solids measured in this study.