Anaerobic Enrichment Research Articles

Description and genome analysis of a novel archaeon isolated from a syntrophic pyrite-forming enrichment culture and reclassification of Methanospirillum hungatei strains GP1 and SK as Methanospirillum purgamenti sp. nov.

The archaeal isolate J.3.6.1-F.2.7.3T was obtained from an anaerobic enrichment culture, where it may play an important role in methane production during pyrite formation. The new isolate formed a species-level clade with Methanospirillum hungatei strains GP1 and SK, which is separate from the type strain JF-1T. Cultivation-independent surveys indicate the occurrence of this phylogenetic group in sediments and anaerobic digesters. The abundance of this clade appears to be negatively affected by high nitrogen loads, indicating a sensitivity to certain nitrogen compounds that is not known in M. hungatei JF-1T. The relatively large core genome of this Methanospirillum clade is indicative of niche specialization and efficient control of horizontal gene transfer. Genes for nitrogenase and F420-dependent secondary alcohol dehydrogenase contribute to the metabolic versatility of this lineage. Characteristics of the new isolate such as the ability to utilize 2-propanol as an electron donor or the requirement for acetate as a carbon source are found also in the strains GP1 and SK, but not in the type strain M. hungatei JF-1T. Based on the genomic differences to related species, a new species within the genus Methanospirillum is proposed with the name M. purgamenti sp. nov. The determined phenotypic characteristics support this proposal and indicate a metabolic adaptation to a separate ecological niche.

Enrichment of anaerobic and facultative anaerobic bacteria from a graphite mine sample on graphene oxide

Graphene oxide (GO) is progressively synthesized and applied in various industrial fields, such as healthcare, medicine, gas transport, electric power industry, etc. Thus, its distribution in the environment increases, which leads to changes in various microbial communities. Mechanisms of interaction between bacterial communities and novel carbon-based nanomaterials, such as GO, are still to be elucidated. We developed stable consortia in order to identify bacteria with the ability to thrive in the presence of GO. Here, we show the results of metagenomic analyses of the graphite mine sample, a consortium with GO as a sole source of carbon and a consortium with fumarate as an additional carbon source. The aim of this study was to establish anaerobic enrichment cultures from a graphite mine sample and GO, and to identify their diversity/community composition.

Genome-Centric Metatranscriptomic Characterization of a Humin-Facilitated Anaerobic Tetrabromobisphenol A-Dehalogenating Consortium.

Tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant in electronics manufacturing, has caused global contamination due to improper e-waste disposal. Its persistence, bioaccumulation, and potential carcinogenicity drive studies of its transformation and underlying (a)biotic interactions. This study achieved an anaerobic enrichment culture capable of reductively dehalogenating TBBPA to the more bioavailable bisphenol A. 16S rRNA gene amplicon sequencing and quantitative PCR confirmed that successive dehalogenation of four bromide ions from TBBPA was coupled with the growth of both Dehalobacter sp. and Dehalococcoides sp. with growth yields of 5.0 ± 0.4 × 108 and 8.6 ± 4.6 × 108 cells per μmol Br- released (N = 3), respectively. TBBPA dehalogenation was facilitated by solid humin and reduced humin, which possessed the highest organic radical signal intensity and reducing groups -NH2, and maintained the highest dehalogenation rate and dehalogenator copies. Genome-centric metatranscriptomic analyses revealed upregulated putative TBBPA-dehalogenating rdhA (reductive dehalogenase) genes with humin amendment, cprA-like Dhb_rdhA1 gene in Dehalobacter species, and Dhc_rdhA1/Dhc_rdhA2 genes in Dehalococcoides species. The upregulated genes of lactate fermentation, de novo corrinoid biosynthesis, and extracellular electron transport in the humin amended treatment also stimulated TBBPA dehalogenation. This study provided a comprehensive understanding of humin-facilitated organohalide respiration.

Identification of acacia gum fermenting bacteria from pooled human feces using anaerobic enrichment culture.

Commercial acacia gum (AG) used in this study is a premium-grade free-flowing powder. It is a gummy exudate composed of arabinogalactan branched polysaccharide, a biopolymer of arabinose and galactose. Also known as food additive, acacia gum (E414), which is presently marketed as a functional dietary fiber to improve overall human gut health. The health effects may be related to the luminal pH regulation from the short-chain fatty acids (SCFA) production. Studies suggested that amylolytic and butyrogenic pathways are the major factors determining the SCFA outcome of AG in the lower gut. However, the primary bacteria involved in the fermentation have not been studied. This study aimed to investigate the putative primary degraders of acacia gum in the gut ecosystem. Isolation and identification of gum-fermenting bacteria were performed through enrichment culture fermentation. The experiment was conducted in an anaerobic chamber for 144 h in three stages. The study was conducted in triplicate using an anaerobic chamber system. This culture system allows specific responses to support only bacteria that are responsible for gum fermentation among the gut microbiota. Five bacterial strains were isolated and found to be gum-fermenting bacteria. Based on the 16s RNA sequence, the isolates matched to butyrate-producing Escherichia fergusonii, ATCC 35469.

New dechlorination products and mechanisms of tris(2-chloroethyl) phosphate by an anaerobic enrichment culture from a vehicle dismantling site

End-of-life vehicles (ELVs) dismantling sites are the notorious hotspots of chlorinated organophosphate esters (Cl-OPEs). However, the microbial-mediated dechlorination of Cl-OPEs at such sites has not yet been explored. Herein, the dechlorination products, pathways and mechanisms of tris(2-chloroethyl) phosphate (TCEP, a representative Cl-OPE) by an anaerobic enrichment culture (ZNE) from an ELVs dismantling plant were investigated. Our results showed that dechlorination of TCEP can be triggered by reductive transformation to form bis(2-chloroethyl) phosphate (BCEP), mono-chloroethyl phosphate (MCEP) and by hydrolytic dechlorination to form bis(2-chloroethyl) 2-hydroxyethyl phosphate (TCEP-OH), 2-chloroethyl bis(2-hydroxyethyl) phosphate (TCEP-2OH), 2-chloroethyl (2-hydroxyethyl) hydrogen phosphate (BCEP-OH). The combination of 16S rRNA gene amplicon sequencing, quantitative real-time PCR (qPCR) and metagenomics revealed that the Dehalococcoides played an important role in the reductive transformation of TCEP to BCEP and MCEP. A high-quality metagenome-assembled genome (completeness >99% and contamination <1%) of Dehalococcoides was obtained. The sulfate-reducing bacteria harboring haloacid dehalogenase genes (had) may be responsible for the hydrolytic dechlorination of TCEP. These findings provide insights into microbial-mediated anaerobic transformation products and mechanisms of TCEP at ELVs dismantling sites, having implications for the environmental fate and risk assessment of Cl-OPEs at those sites.

Biodegradation of the emerging contaminant 3-nitro-1,2,4-triazol-5-one and its product 3-amino-1,2,4-triazol-5-one in perlite/soil columns

3-Nitro-1,2,4-triazol-5-one (NTO) is an ingredient of new safer-to-handle military insensitive munitions formulations. NTO can be microbially reduced to 3-amino-1,2,4-triazol-5-one (ATO) under anaerobic conditions if an electron donor is available. Conversely, ATO can undergo aerobic biodegradation. Previously, our research group developed an anaerobic enrichment culture that reduces NTO to ATO. A second culture could aerobically mineralize ATO. This study aimed to combine anaerobic/aerobic conditions within a down-flow perlite/soil column for simultaneous NTO reduction and ATO oxidation. Acetate biostimulation was investigated to promote oxygen depletion and create anaerobic micro-niches for NTO reduction, whereas perlite increased soil porosity and oxygen convection, allowing ATO oxidation. Two columns packed with a perlite/soil mixture (70:30, wet wt.%) or 100% perlite were operated aerobically and inoculated with the NTO- and ATO-degrading cultures. Initially, the influent consisted of ∼280 μM ATO, and after 30 days, the feeding was switched to ∼260 μM NTO and ∼250 μM acetate. By progressively increasing acetate from 250 to 4000 μM, the NTO removal gradually improved in both columns. The perlite/soil column reached a 100% NTO removal after 4000 μM acetate was supplemented. Additionally, there was no ATO accumulation, and inorganic nitrogen was produced, indicating ATO mineralization. Although NH4+ was produced following ATO oxidation, most nitrogen was recovered as NO3− likely via nitrification reactions. Microbial community analysis revealed that phylotypes hosted in the enrichment cultures specialized in NTO reduction (e.g., Geobacter) and ATO oxidation (e.g., Hydrogenophaga, Ramlibacter, Terrimonas, and Pseudomonas) were established in the columns. Besides, the predominant genera (Azohydromonas, Zoogloea, and Azospirillum) are linked to nitrogen cycling by performing nitrogen fixation, NO3− reduction, and nitroaromatics degradation. This study applied a bulking agent (perlite) and acetate biostimulation to achieve simultaneous NTO reduction and ATO oxidation in a single column. Such a strategy can assist with real-world applications of NTO and ATO biodegradation mechanisms.

Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Phylogeny and Physiological Diversity of Cold-adapted Anaerobic Bacteria Isolated from Rice Field Soil in Japan.

Cold-adapted or psychrotrophic fermentative anaerobic bacteria were isolated from rice field soil in a temperate area in Japan using anaerobic enrichment cultures incubated at 5°C. Most isolates were obligately anaerobic, spore-forming rods and affiliated with different lineages of the genus Clostridium based on 16S rRNA gene sequences. The growth temperature ranges and physiological properties of three representative clostridial isolates (C5S7, C5S11T, and C5S18) were examined. Strain C5S7 grew at 0°C, but not at 20°C, and was identified as Clostridium estertheticum, a psychrophile isolated from spoiled, vacuum-packed, chilled meat (blown pack spoilage, BPS). Strain C5S7 produced butyrate, n-butanol, and abundant gases (H2 and CO2) as major fermentation products from the carbohydrates utilized. Strain C5S11T, which was recently described as Clostridium gelidum sp. nov., possessed psychrotrophic properties and grew at temperatures between 0 and 25°C. Strain C5S11T was saccharolytic, decomposed polysaccharides, such as inulin, pectin, and xylan, and produced acetate, butyrate, and gases. Strain C5S18 also grew at 0°C and the optimum growth temperature was 15°C. Strain C5S18 did not ferment carbohydrates and grew in a manner that was dependent on proteinaceous substrates. This strain was identified as the psychrotolerant species, Clostridium tagluense, originally isolated from a permafrost sample. Collectively, the present results indicate that psychrotrophic anaerobic bacteria with different physiological properties actively degrade organic matter in rice field soil, even in midwinter, in a cooperative manner using different substrates. Furthermore, different psychrotrophic species of the genus Clostridium with the ability to cause BPS inhabit cultivated soil in Japan.

Elucidating the mechanisms associated with the anaerobic biotransformation of the emerging contaminant nitroguanidine

Nitroguanidine (NQ) is a constituent of gas generators for automobile airbags, smokeless pyrotechnics, insecticides, propellants, and new insensitive munitions formulations applied by the military. During its manufacture and use, NQ can easily spread in soils, ground, and surface waters due to its high aqueous solubility. Very little is known about the microbial biotransformation of NQ. This study aimed to elucidate important mechanisms operating during NQ anaerobic biotransformation. To achieve this goal, we developed an anaerobic enrichment culture able to reduce NQ to nitrosoguanidine (NsoQ), which was further abiotically transformed to cyanamide. Effective electron donors for NQ biotransformation were lactate and, to a lesser extent, pyruvate. The results demonstrate that the enrichment process selected a sulfate-reducing culture that utilized lactate as its electron donor and sulfate as its electron acceptor while competing with NQ as an electron sink. A unique property of the culture was its requirement for exogenous nitrogen (e.g., from yeast extract or NH4Cl) for NQ biotransformation since NQ itself did not serve as a nitrogen source. The main phylogenetic groups associated with the NQ-reducing culture were sulfate-reducing and fermentative bacteria, namely Cupidesulfovibrio oxamicus (63.1% relative abundance), Dendrosporobacter spp. (12.0%), and Raoultibacter massiliens (10.9%). The molecular ecology results corresponded to measurable physiological properties of the most abundant members. The results establish the conditions for NQ anaerobic biotransformation and the microbial community associated with the process, improving our present comprehension of NQ environmental fate and assisting the development of NQ remediation strategies.

Composition of Prokaryotic Communities in Anaerobic Enrichment Cultures of Crude Oil from Romashkino Oilfield (Russia)

Despite the many papers published on oilfield ecosystems, there is a lack of data about microbial community structure and functioning in many crude oils or oilfields and wells. One of such poorly studied oilfields is supergiant Romashkino situated in Tatarstan (Russia). In the present study, prokaryotic community composition and abundance of functional genes encoding sulfate reduction and rhamnolipid production, was investigated in enrichment cultures of four samples from Romashkino oilfield production wells with different oil/water ratios. The enrichment was conducted under anaerobic conditions, sulfate and nitrate were used as electron acceptors, and acetate – as an electron donor. In enrichment culture A and enrichment culture B, bacteria of the Pseudomonadaceae family predominated with 99% and 93% relative abundance, respectively. In enrichment culture C (obtained on the basis of initial sample contained oil with medium water content) and D (obtained on the basis of initial sample contained oil with very high water content), Pseudomonadaceae were one of the two predominating families, along with Micrococcaceae (enrichment culture C) and Dietziaceae (enrichment culture D). The proportion of dsrA and apsA genes encoding sulfate reduction to 16S rRNA gene copy numbers was quite low and ranged between 0.003% (enrichment cultures A, B) and 0.027% (enrichment culture D). The proportion of rhlB gene encoding rhamnolipid production from the total 16S rRNA gene copy number was 1.10% in sample A and 0.06% in sample B. In enrichment cultures C and D the rhlB gene was not found.

Organohalide Respiration with Diclofenac by Dehalogenimonas.

Diclofenac (DCF) is a pharmaceutically active contaminant frequently found in aquatic ecosystems. The transformation pathways and microbiology involved in the biodegradation of DCF, particularly under anoxic conditions, remain poorly understood. Here, we demonstrated microbially mediated reductive dechlorination of DCF in anaerobic enrichment culture derived from contaminated river sediment. Over 90% of the initial 76.7 ± 3.6 μM DCF was dechlorinated at a maximum rate of 1.8 ± 0.3 μM day-1 during a 160 days' incubation. Mass spectrometric analysis confirmed that 2-(2-((2-chlorophenyl)amino)phenyl)acetic acid (2-CPA) and 2-anilinophenylacetic acid (2-APA) were formed as the monochlorinated and nonchlorinated DCF transformation products, respectively. A survey of microbial composition and Sanger sequencing revealed the enrichment and dominance of a new Dehalogenimonas population, designated as Dehalogenimonas sp. strain DCF, in the DCF-dechlorinating community. Following the stoichiometric conversion of DCF to 2-CPA (76.0 ± 2.1 μM) and 2-APA (3.7 ± 0.8 μM), strain DCF cell densities increased by 24.4 ± 4.4-fold with a growth yield of 9.0 ± 0.1 × 108 cells per μmol chloride released. Our findings expand the metabolic capability in the genus Dehalogenimonas and highlight the relevant roles of organohalide-respiring bacteria for the natural attenuation of halogenated contaminants of emerging concerns (e.g., DCF).

Genomic characterization of denitrifying methylotrophic Pseudomonas aeruginosa strain AAK/M5 isolated from municipal solid waste landfill soil.

Municipal landfills are known for methane production and a source of nitrate pollution leading to various environmental issues. Therefore, this niche was selected for the isolation of one-carbon (C1) utilizing bacteria with denitrifying capacities using anaerobic enrichment on nitrate mineral salt medium supplemented with methanol as carbon source. Eight axenic cultures were isolated of which, isolate AAK/M5 demonstrated thehighest methanol removal (73.28%) in terms of solublechemical oxygen demand and methane removal (41.27%) at the expense of total nitrate removal of 100% and 33% respectively. The whole genome characterization with phylogenomic approach suggested that the strain AAK/M5 could be assigned to Pseudomonas aeruginosa with close neighbours as type strains DVT779, AES1M, W60856, and LES400. The circular genome annotation showed the presence of complete set of genes essential for methanol utilization and complete denitrification process. The study demonstrates the potential of P. aeruginosa strain AAK/M5 in catalysing methane oxidation thus serving as a methane sink vis-à-vis utilization of nitrate. Considering the existence of such bacteria at landfill site, the study highlights the need to develop strategies for their enrichment and designing of efficient catabolic activity for such environments.

Trickle-Bed Bioreactors for Acetogenic H2/CO2 Conversion

Acetic acid is an essential industrial building block and can be produced by acetogenic bacteria from molecular hydrogen (H2) and carbon dioxide (CO2). When gasses are supplied as substrates, bioreactor design plays an important role for their availability. Trickle-bed bioreactors (TBs) have an enhanced gas-to-liquid mass transfer and cells remain in the system by forming a biofilm on the carriers. So far, TBs have been investigated extensively for bio-methanation processes, whereas studies for their use in acetic acid production are rare. In this study, we evaluated the reproducibility of two parallel TBs for acetic acid production from H2:CO2(= 70:30) by a mixed culture with a gas flow rate of 3.8 mL min−1and a medium flow rate of 10 mL min−1. Additionally, the effect of glucose addition during the starting phase on the resulting products and microbial composition was investigated by setting up a third TB2. Partial medium exchanges to decrease the internal acetic acid concentration (AAC) combined with recycling of withdrawn cells had a positive impact on acetic acid production rates with maxima of around 1 g L−1d−1even at high AACs of 19–25 g L−1. Initial glucose addition resulted in the accumulation of unwanted butyric acid up to concentrations of 2.60 ± 0.64 g L−1. The maximum AAC of 40.84 g L−1was obtained without initial glucose addition. The main families identified in the acetogenic TBs were Peptococcaceae, Ruminococcaceae, Planococcaceae, Enterobacteriaceae, Clostridiaceae, Lachnospiraceae, Dysgonomonadaceae and Tannerellaceae. We conclude that a TB is a viable solution for conversion of H2/CO2to acetate using an anaerobic enrichment culture.

Relevance of using both aerobic and anaerobic enrichment vials for optimizing rapid diagnosis of osteoarticular infections

The performance of a pair of blood culture vials (BACTEC® Plus Aerobic/F, and Anaerobic Lytic/F) were analyzed in 496 osteoarticular specimens (246 synovial fluids and 250 crushed bone samples), obtained in patients during routine diagnostic procedure at the Teaching Hospital of Rennes (France). The positive detection times were recorded for a 14 day-incubation period, and compared between both vials and with agar cultures. For samples from infected patients, the positive detection time was significantly shortened when vials were used compared to agar plates (p < 0.001). Median positive detection time was later with the Anaerobic Lytic/F vials (15.0 h) compared to the Plus Aerobic/F (13.0 h). Positivity rate was similar for Anaerobic Lytic/F vials (80.4%) and Plus Aerobic/F vials (83.2%) (p = 0.25). Some microorganisms were only identified from aerobic vials (15.5%) or from anaerobic vials (12.7%). The use of both atmosphere conditions for optimal positive detection time is therefore critical.

Anaerobic carboxydotrophy in sulfur-respiring haloarchaea from hypersaline lakes.

Anaerobic carboxydotrophy is a widespread catabolic trait in bacteria, with two dominant pathways: hydrogenogenic and acetogenic. The marginal mode by direct oxidation to CO2 using an external e-acceptor has only a few examples. Use of sulfidic sediments from two types of hypersaline lakes in anaerobic enrichments with CO as an e-donor and elemental sulfur as an e-acceptor led to isolation of two pure cultures of anaerobic carboxydotrophs belonging to two genera of sulfur-reducing haloarchaea: Halanaeroarchaeum sp. HSR-CO from salt lakes and Halalkaliarchaeum sp. AArc-CO from soda lakes. Anaerobic growth of extremely halophilic archaea with CO was obligatory depended on the presence of elemental sulfur as the electron acceptor and yeast extract as the carbon source. CO served as a direct electron donor and H2 was not generated from CO when cells were incubated with or without sulfur. The genomes of the isolates encode a catalytic Ni,Fe-CODH subunit CooS (distantly related to bacterial homologs) and its Ni-incorporating chaperone CooC (related to methanogenic homologs) within a single genomic locus. Similar loci were also present in a genome of the type species of Halalkaliarchaeum closely related to AArc-CO, and the ability for anaerobic sulfur-dependent carboxydotrophy was confirmed for three different strains of this genus. Moreover, similar proteins are encoded in three of the four genomes of recently described carbohydrate-utilizing sulfur-reducing haloarchaea belonging to the genus Halapricum and in two yet undescribed haloarchaeal species. Overall, this work demonstrated for the first time the potential for anaerobic sulfur-dependent carboxydotrophy in extremely halophilic archaea.

Conversion of Waste Cooking Oil to Rhamnolipid by a Newly Oleophylic Pseudomonas aeruginosa WO2.

The components of waste cooking oil (WCO) are complex and contain toxic substances, which are difficult to treat biologically. Pseudomonas aeruginosa WO2 was isolated from oily sludge by an anaerobic enrichment–aerobic screening method, which could efficiently utilize WCO and produce rhamnolipid. The effects of nutrients and culture conditions on bacterial growth and lipase activity were investigated to optimize the fermentation of WCO. The results showed that strain WO2 utilized 92.25% of WCO and produced 3.03 g/L of rhamnolipid at 120 h. Compared with inorganic sources, the organic nitrogen source stabilized the pH of fermentation medium, improved lipase activity (up to 19.98 U/mL), and promoted the utilization of WCO. Furthermore, the WO2 strain exhibited inferior utilization ability of the soluble starch contained in food waste, but superior salt stress up to 60 g/L. These unique characteristics demonstrate the potential of Pseudomonas aeruginosa WO2 for the utilization of high-salinity oily organic waste or wastewater.

Metagenomic Investigation of a Low Diversity, High Salinity Offshore Oil Reservoir.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.

Detection of methanotrophic microorganisms in sludge and sediment samples from sewage treatment systems

Abstract This study investigated various groups of methanotrophic microorganisms, including the anaerobic methane-oxidizing archaea (ANME) group, aerobic methanotrophic bacteria, and bacteria of the phylum NC10 (Candidatus Methylomirabilis oxyfera). The polymerase chain reaction (PCR) technique was employed with specific primers for the pmoA gene from the aerobic methanotrophic and M. Oxyfera methanotrophic denitrifying (NC10 phylum) and for the anaerobic methanotrophic archaea 16S rRNA gene (ANME-1 and ANME-2). Sludge and sediment samples were analyzed from different sewage treatment systems installed at the Sanitation Research and Training Center (CePTS) at the Arrudas wastewater treatment plant (Arrudas WWTP), in Belo Horizonte, Brazil. ANME was shown to be present in sludges from the upflow anaerobic sludge blanket (UASB) reactor and activated sludge system, and in sediments from the polishing pond and wetlands. On the other hand, aerobic methanotrophic bacteria were detected only in the polishing pond samples, while no methanotrophic denitrifiers of the M. oxyfera genre were detected at all. Besides investigating and detecting different groups of methanotrophs in the treatment systems, the study provided support for another in which samples from the systems analyzed were selected for use as inoculum for methane anaerobic oxidation potential experiments and methanotrophic microorganism enrichment.

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N2O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB’s, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB’s were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

Identification of mucin degraders of the human gut microbiota

Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.