High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor.

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of ∼ 100 % (HRT = 2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) analysis visually revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput sequencing analysis results further confirmed the biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest it is a potentially effective technique for high-toxic azo dyes removal from textile wastewater.

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments.

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60-86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils.

The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.

Substrate sources regulate spatial variation of metabolically active methanogens from two contrasting freshwater wetlands.

There is ample evidence that methane (CH4) emissions from natural wetlands exhibit large spatial variations at a field scale. However, little is known about the metabolically active methanogens mediating these differences. We explored the spatial patterns in active methanogens of summer inundated Calamagrostis angustifolia marsh with low CH4 emissions and permanently inundated Carex lasiocarpa marsh with high CH4 emissions in Sanjiang Plain, China. In C. angustifolia marsh, the addition of (13)C-acetate significantly increased the CH4 production rate, and Methanosarcinaceae methanogens were found to participate in the consumption of acetate. In C. lasiocarpa marsh, there was no apparent increase in the CH4 production rate and no methanogen species were labeled with (13)C. When (13)CO2-H2 was added, however, CH4 production was found to be due to Fen Cluster (Methanomicrobiales) in C. angustifolia marsh and Methanobacterium Cluster B (Methanobacteriaceae) together with Fen Cluster in C. lasiocarpa marsh. These results suggested that CH4 was produced primarily by hydrogenotrophic methanogens using substrates mainly derived from plant litter in C. lasiocarpa marsh and by both hydrogenotrophic and acetoclastic methanogens using substrates mainly derived from root exudate in C. angustifolia marsh. The significantly lower CH4 emissions measured in situ in C. angustifolia marsh was primarily due to a deficiency of substrates compared to C. lasiocarpa marsh. Therefore, we speculate that the substrate source regulates both the type of active methanogens and the CH4 production pathway and consequently contributes to the spatial variations in CH4 productions observed in these freshwater marshes.

Evaluation of system performance and microbial communities of a bioaugmented anaerobic membrane bioreactor treating pharmaceutical wastewater.

In this study, a control anaerobic membrane bioreactor (C-AnMBR) and a bioaugmented anaerobic membrane bioreactor (B-AnMBR) were operated for 210 d to treat pharmaceutical wastewater. Both the bioreactors were fed with the pharmaceutical wastewater containing TCOD of 16,249 ± 714 mg/L and total dissolved solids (TDS) of 29,450 ± 2209 mg/L with an organic loading rate (OLR) of 13.0 ± 0.6 kgCOD/m(3)d. Under steady-state condition, an average total chemical oxygen demand (TCOD) removal efficiency of 46.1 ± 2.9% and 60.3 ± 2.8% was achieved by the C-AnMBR and the B-AnMBR, respectively. The conventional anaerobes in the C-AnMBR cannot tolerate the hypersaline conditions well, resulting in lower TCOD removal efficiency, biogas production and methane yield than the B-AnMBR seeded from the coastal shore. Pyrosequencing analysis indicated that marine bacterial species (Oliephilus sp.) and halophilic bacterial species (Thermohalobacter sp.) were only present in the B-AnMBR; these species could possibly degrade complex and recalcitrant organic matter and withstand hypersaline environments. Two different dominant archaeal communities, genus Methanosaeta (43.4%) and Methanolobus (61.7%), were identified as the dominant methanogens in the C-AnMBR and the B-AnMBR, respectively. The species of genus Methanolobus was reported resistant to penicillin and required sodium and magnesium for growth, which could enable it to thrive in the hypersaline environment.

Retention and transport of an anaerobic trichloroethene dechlorinating microbial culture in anaerobic porous media.

The influence of solution chemistry on microbial transport was examined using the strictly anaerobic trichloroethene (TCE) bioaugmentation culture KB-1(®). A column was employed to determine transport behaviors and deposition kinetics of three distinct functional species in KB-1(®), Dehalococcoides, Geobacter, and Methanomethylovorans, over a range of ionic strengths under a well-controlled anaerobic condition. A quantitative polymerase chain reaction (qPCR) was utilized to enumerate cell concentration and complementary techniques were implemented to evaluate cell surface electrokinetic potentials. Solution chemistry was found to positively affect the deposition rates, which was consistent with calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energies. Retained microbial profiles showed spatially constant colloid deposition rate coefficients, in agreement with classical colloid filtration theory (CFT). It was interesting to note that the three KB-1(®) species displayed similar transport and retention behaviors under the defined experimental conditions despite their different cell electrokinetic properties. A deeper analysis of cell characteristics showed that factors, such as cell size and shape, concentration, and motility were involved in determining adhesion behavior.

Electrochemical nutrient recovery enables ammonia toxicity control and biogas desulfurization in anaerobic digestion.

Organic waste streams can be valorized and reduced in volume with anaerobic digestion (AD). An often-encountered key issue however is the high ammonium (NH4(+)) content of certain waste streams. Ammonia (NH3), in equilibrium with NH4(+), is a toxic compound to the methanogenic community, which limits the organic loading rate and endangers process stability. An electrochemical system (ES) linked to a digester could, besides recovering this nutrient, decrease NH3 toxicity through electrochemical extraction. Therefore, two digesters with and without ES attached in the recirculation loop were operated to test whether the ES could control NH3 toxicity. During periods of high ammonium loading rates, the methane (CH4) production of the ES-coupled reactor was up to 4.5 times higher compared to the control, which could be explained through simultaneous NH4(+) extraction and electrochemical pH control. A nitrogen flux of 47 g N m(­2) membrane d(­1) could be obtained in the ES-coupled reactor, resulting in a current and removal efficiency of 38 ± 5% and 28 ± 2%, respectively, at an electrochemical power input of 17 ± 2 kWh kg(­1) N. The anode also oxidized sulfide, resulting in a significantly lower H2S emission via the biogas. Lastly, limited methanogenic community dynamics pointed to a nonselective influence of the different operational conditions.

Glycine betaine as a direct substrate for methanogens (Methanococcoides spp.).

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics.

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part II: the effect of different methylated growth substrates.

Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part I: the effect of growth temperature.

The response of the cold-adapted (psychrophilic) methanogenic archaeon Methanococcoides burtonii to growth temperature was investigated using differential proteomics (postincorporation isobaric labeling) and tandem liquid chromatography-mass spectrometry (LC/LC-MS/MS). This is the first proteomic study of M. burtonii to include techniques that specifically enrich for both surface and membrane proteins and to assess the effects of growth temperature (4 vs 23 degrees C) and carbon source (trimethylamine vs methanol) on cellular protein levels. Numerous surface layer proteins were more abundant at 4 degrees C, indicating an extensive remodeling of the cell envelope in response to low temperature. Many of these surface proteins contain domains associated with cell adhesion. Within the cell, small proteins each composed of a single TRAM domain were recovered as important cold adaptation proteins and might serve as RNA chaperones, in an analogous manner to Csp proteins (absent from M. burtonii). Other proteins that had higher abundances at 4 degrees C can be similarly tied to relieving or resolving the adverse affects of cold growth temperature on translational capacity and correct protein folding. The proteome of M. burtonii grown at 23 degrees C was dominated by oxidative stress proteins, as well as a large number of integral membrane proteins of unknown function. This is the first truly global proteomic study of a psychrophilic archaeon and greatly expands knowledge of the cellular mechanisms underpinning cold adaptation in the Archaea.

Effect of antimicrobial compounds tylosin and chlortetracycline during batch anaerobic swine manure digestion.

Tylosin and chlortetracycline (CTC) are antimicrobial chemicals that are fed to >45% of the US swine herds at therapeutic and sub-therapeutic dosages to enhance growth rates and treat swine health problems. These compounds are poorly absorbed during digestion so that the bioactive compound or metabolites are excreted. This study investigated the degradation and stabilization of swine manure that contained no additives and compared the observed processes with those of manure containing either tylosin or CTC. The batch anaerobic incubation lasted 216 days. The breakdown of insoluble organic matter through anaerobic hydrolysis reactions was faster for manure containing CTC compared with tylosin or no-antimicrobial treatments. Volatile fatty acid (VFA) accumulation, including acetate, butyrate, and propionate, was greater for CTC-containing manure compared to tylosin and no-antimicrobial treatments. The relative abundance of two aceticlastic methanogens, Methanosaetaceae and Methanosarcinaceae spp., were less for CTC manure than manure with no-antimicrobial treatment. In addition, generation of methane and carbon dioxide was inhibited by 27.8% and 28.4%, respectively, due to the presence of CTC. Tylosin effects on manure degradation were limited, however the relative abundance of Methanosarcinaceae spp. was greater than found in the CTC or no-antimicrobial manures. These data suggest that acetate and other C-1 VFA compounds would be effectively utilized during methanogenesis in the presence of tylosin.

Methanolobus zinderi sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam.

A methanogenic organism from the domain Archaea (SD1(T)) was isolated from saline water released from a coal seam located 926 m below the surface via a methane-producing well near Monroe, Louisiana, USA. Growth and methanogenesis were supported with methanol, monomethylamine, dimethylamine or trimethylamine, but not with dimethylsulfide, formate, acetate or H(2)/CO(2). Cells grew in high-salt minimal medium but growth was stimulated with yeast extract or tryptone. Cells were single, non-motile, irregular coccoids 0.5-1.0 microm in diameter and the cell wall contained protein. Conditions for the maximum rate of growth were 40-50 degrees C, 0.2-0.6 M NaCl, 100->or=200 mM MgCl(2), and pH 7.0-8.0. The G+C content of the genomic DNA was 42+/-1mol %. A comparison of 16S rRNA gene sequences indicated that strain SD1(T) was most closely related to Methanolobus oregonensis DSM 5435(T) with 96 % gene sequence similarity. It is proposed that strain SD1(T) represents a novel species, Methanolobus zinderi sp. nov. The type strain is SD1(T) (=ATCC BAA-1601(T)=DSM 21339(T)).

Enrichment of hydrogenotrophic methanogens in coupling with methane production using an electrochemical bioreactor.

Anaerobic digestion sludge was cultivated in an electrochemical bioreactor (ECB) to enrich the hydrogenotrophic methanogens. A modified graphite felt cathode with neutral red (NR-cathode) was charged with electrochemical reducing power generated from a solar cell. The methane and carbon dioxide collected in a Teflon bag from the ECB were more than 80 ml/l of reactant/day and less than 20 ml/l of reactant/day, respectively, whereas the methane and carbon dioxide collected from a conventional bioreactor (CB) was around 40 ml/l of reactant/day, respectively. Moreover, the maximal volume ratios of methane to carbon dioxide (M/C ratio) collected in the Teflon bag from the ECB and CB were 7 and 1, respectively. The most predominant methanogens isolated from the CB on the 20th, 80th, and 150th days of incubation were hydrogenotrophs. The methanogenic diversity analyzed by temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region was higher in the ECB than in the CB. The DNA extracted from the TGGE bands was more than 95% homologous with hydrogenotrophic methanogens in the CB. In conclusion, the ECB was demonstrated as a useful system for enriching hydrogenotrophic methanogens and increasing the M/C ratio of the gas product.

Thermophilic degradation of phenolic compounds in lab scale hybrid up flow anaerobic sludge blanket reactors.

This Study describes the feasibility of anaerobic degradation of United States Environmental Protection Agency (USEPA) listed 4-chloro-2-nitrophenol (4C-2-NP), 2-chloro-4-nitrophenol (2C-4-NP), 2-chloro-5-methylphenol (2C-5-MP) from a simulated wastewater using four identical 7L bench scale hybrid up flow anaerobic sludge blankets (HUASBs) at five different hydraulic retention times (HRTs) under thermophilic condition (55+/-3 degrees C). The substrate to co-substrate ratios were maintained between 1:33.3 and 1:166.6. Continuous monitoring of parameters like pH, volatile fatty acids (VFAs) accumulation, oxidation reduction potential, chemical oxygen demand (COD), alkalinity, gas productions, methane percentages were carried out along with compound reduction to asses the efficiency of biodegradation. The compound reduction was estimated by using spectrophotometric methods and further validated with high-performance liquid chromatography (HPLC). Optimum HRT values were observed at 24h. Optimum ratio of substrate (phenolic compounds) to co-substrate (glucose) was 1:100. Scanning electron micrographs show that the granules were composed of thermophilic Methanobrevibacter and thermophilic Methanothrix like bacteria.

The competitive success of Methanomicrococcus blatticola, a dominant methylotrophic methanogen in the cockroach hindgut, is supported by high substrate affinities and favorable thermodynamics.

Methanomicrococcus blatticola is an obligately anaerobic methanogen that derives the energy for growth exclusively from the reduction of methylated compounds to methane with molecular hydrogen as energy source. Competition for methanol (concentration below 10 microM) and H(2) (concentration below 500 Pa), as well as oxidative stress due to the presence of oxygen are likely to occur in the peripheral region of the cockroach hindgut, the species' normal habitat. We investigated the ecophysiological properties of M. blatticola to explain how it can successfully compete for its methanogenic substrates. The organism showed affinities for methanol (K(m)=5 microM; threshold<1 microM) and hydrogen (K(m)=200 Pa; threshold <0.7 Pa) that are superior to other methylotrophic methanogens (Methanosphaera stadtmanae, Methanosarcina barkeri) investigated here. Thermodynamic considerations indicated that 'methanol respiration', i.e. the use of methanol as the terminal electron acceptor, represents an attractive mode of energy generation, especially at low hydrogen concentrations. Methanomicrococcus blatticola exploits the opportunities by specific growth rates (>0.2 h(-1)) and specific growth yields (up to 7 g of dry cells per mole of methane formed) that are particularly high within the realm of mesophilic methanogens. Upon oxygen exposure, part of the metabolic activity may be diverted into oxygen removal, thus establishing appropriate anaerobic conditions for survival and growth.

Degradation of methanethiol by methylotrophic methanogenic archaea in a lab-scale upflow anaerobic sludge blanket reactor.

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30 degrees C to H2S, CO2, and CH4. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter-1 day-1. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe.

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4+/-0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.

Growth kinetics and competition between Methanosarcina and Methanosaeta in mesophilic anaerobic digestion.

Methanosarcina species with a high maximum specific growth rate (mumax) and high half-saturation coefficient (KS) and Methanosaeta species with a low mumax and low KS are the only known aceticlastic methanogens. Because of Methanosaeta's low KS, the low acetate concentrations in conventional, mesophilic anaerobic digestion yield Methanosaeta dominance. However, Methanosarcina absorbs increases in acetate more efficiently and thus promotes more stable digestion. This paper tests the hypothesis that decreasing digester feeding frequencies can increase Methanosarcina predominance. Two acetate-fed reactors were established at a 17-day solids retention time. One reactor was fed hourly, and one was fed once daily. Microscopic and molecular methods were used to verify that the hourly fed reactor enriched for Methanosaeta, while the daily fed reactor enriched for Methanosarcina. Growth and substrate-use kinetics were measured for each reactor. A digester overload condition was simulated, and the Methanosarcina-enriched reactor was found to perform better than the Methanosaeta-enriched reactor. These findings indicate that Methanosarcina dominance can be achieved with infrequent feedings, leading to more stable digestion.