Environment rather than breed or body site shapes the skin bacterial community of healthy sheep as revealed by metabarcoding.

BACKGROUND: The skin is inhabited by a variety of micro-organisms, with bacteria representing the predominant taxon of the skin microbiome. In sheep, the skin bacterial community of healthy animals has been addressed in few studies, only with culture-based methods or sequencing of cloned amplicons. OBJECTIVES: The objectives of this study were to determine the sheep skin bacterial community composition by using metabarcoding for a detailed characterisation and to determine the effect of body part, breed and environment. MATERIALS AND METHODS: Overall, 267 samples were taken from 89 adult female sheep, belonging to three different breeds and kept on nine different farms in Switzerland. From every individual, one sample each was taken from belly, left ear and left leg and metabarcoding of the 16S rRNA V3-V4 hypervariable region was performed. RESULTS: The main phyla identified were Actinobacteriota, Firmicutes, Proteobacteria and Bacteriodota. The alpha diversity as determined by Shannon's diversity index was significantly different between sheep from different farms. Beta diversity analysis by principal coordinate analysis (PCoA) showed clustering of the samples by farm and body site, while breed had only a marginal influence. A sparse partial least squares discriminant analysis (sPLS-DA) revealed seven main groups of operational taxonomic units (OTUs) of which groups of OTUs were specific for some farms. CONCLUSIONS AND CLINICAL RELEVANCE: These findings indicate that environment has a larger influence on skin microbial variability than breed, although the sampled breeds, the most abundant ones in Switzerland, are phenotypically similar. Future studies on the sheep skin microbiome may lead to novel insights in skin diseases and prevention.

Disentangle genus microdiversity within a complex microbial community by using a multi-distance long-read binning method: example of Candidatus Accumulibacter.

Complete genomes can be recovered from metagenomes by assembling and binning DNA sequences into metagenome assembled genomes (MAGs). Yet, the presence of microdiversity can hamper the assembly and binning processes, possibly yielding chimeric, highly fragmented and incomplete genomes. Here, the metagenomes of four samples of aerobic granular sludge bioreactors containing Candidatus (Ca.) Accumulibacter, a phosphate-accumulating organism of interest for wastewater treatment, were sequenced with both PacBio and Illumina. Different strategies of genome assembly and binning were investigated, including published protocols and a binning procedure adapted to the binning of long contigs (MuLoBiSC). Multiple criteria were considered to select the best strategy for Ca. Accumulibacter, whose multiple strains in every sample represent a challenging microdiversity. In this case, the best strategy relies on long-read only assembly and a custom binning procedure including MuLoBiSC in metaWRAP. Several high-quality Ca. Accumulibacter MAGs, including a novel species, were obtained independently from different samples. Comparative genomic analysis showed that MAGs retrieved in different samples harbour genomic rearrangements in addition to accumulation of point mutations. The microdiversity of Ca. Accumulibacter, likely driven by mobile genetic elements, causes major difficulties in recovering MAGs, but it is also a hallmark of the panmictic lifestyle of these bacteria.

Towards a standardization of biomethane potential tests: a commentary.

Inter-laboratory reproducibility of biomethane potential (BMP) is dismal, with differences in BMP values for the same sample exceeding a factor of two in some cases. A large group of BMP researchers directly addressed this problem during a workshop held in Leysin, Switzerland, in June 2015. The workshop resulted in a new set of guidelines for BMP tests published in 2016, which is the subject of the present commentary. The work has continued with two international inter-laboratory studies and one additional workshop held in Freising, Germany, in 2018. The dataset generated by the two inter-laboratory studies were used to refine the validation criteria for BMP tests. Based on these new results an update to the original guidelines is proposed here.

Modeling hydraulic transport and anaerobic uptake by PAOs and GAOs during wastewater feeding in EBPR granular sludge reactors.

New-generation bioprocesses using granular sludge aim for a high-rate removal of nutrients from wastewater with low footprint. Achieving enhanced biological phosphorus removal (EBPR) relies on the design of sludge beds and wastewater feeding conditions to optimally load the biomass and to select for polyphosphate- (PAOs) over glycogen-accumulating organisms (GAOs) and over other heterotrophs. A hydraulic-metabolic mathematical model was developed to elucidate the impact of hydraulic transport patterns and environmental conditions on the PAO/GAO competition during up-flow feeding through an EBPR granular sludge bed. Tracer experiments highlighted plug-flow regimes with dispersion under both rapid (9 m h-1 , Rebed = 1.6, Pez = 7.2, Pet = 4.6) and slow (0.9 m h-1 , Rebed = 0.2, Pez = 21.3, Pet = 3.4) feeding. Non-turbulent regimes (Rebed << 103 ) promote a safe implementation of simultaneous fill/draw. Feeding time, pH, and temperature significantly impacted bacterial competition for carbon uptake under anaerobic slow feeding. Feeding duration should be designed to avoid full depletion of intracellular storage polymers within static granules. PAOs bear twice longer feeding than GAOs by using both polyphosphate and glycogen hydrolysis to sustain anaerobic C-uptake. Alkaline conditions (pH 7.25-8.0) by, e.g., dosing lime in the feed select for PAOs independently of temperature (10-30°C). A twice higher bed is required for full anaerobic conversions at 10 rather than 20°C. Biosystem responses for anaerobic C-uptake can be anticipated using the model toward designing robust anaerobic selectors to manage the microbial resource in EBPR granular sludge. Biotechnol. Bioeng. 2017;114: 1688-1702. © 2017 Wiley Periodicals, Inc.

Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation.

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and ß-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of ß- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of ß- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.

The Swiss Society of Microbiology: Small Bugs, Big Questions and Cool Answers.

The Swiss Society for Microbiology (SSM) represents around 700 scientists working in the fields of medical (human and veterinary), microbial biotechnology as well as fundamental, environmental, and food microbiology. Five sections: Clinical Microbiology, Environmental Microbiology, Mycology, Prokaryotic Biology, and Virology reflects the main interests of the membership.

Role of ammonia-oxidizing bacteria in micropollutant removal from wastewater with aerobic granular sludge.

Nitrifying wastewater treatment plants (WWTPs) are more efficient than non-nitrifying WWTPs to remove several micropollutants such as pharmaceuticals and pesticides. This may be related to the activity of nitrifying organisms, such as ammonia-oxidizing bacteria (AOBs), which could possibly co-metabolically oxidize micropollutants with their ammonia monooxygenase (AMO). The role of AOBs in micropollutant removal was investigated with aerobic granular sludge (AGS), a promising technology for municipal WWTPs. Two identical laboratory-scale AGS sequencing batch reactors (AGS-SBRs) were operated with or without nitrification (inhibition of AMOs) to assess their potential for micropollutant removal. Of the 36 micropollutants studied at 1 µg l(-1) in synthetic wastewater, nine were over 80% removed, but 17 were eliminated by less than 20%. Five substances (bisphenol A, naproxen, irgarol, terbutryn and iohexol) were removed better in the reactor with nitrification, probably due to co-oxidation catalysed by AMOs. However, for the removal of all other micropollutants, AOBs did not seem to play a significant role. Many compounds were better removed in aerobic condition, suggesting that aerobic heterotrophic organisms were involved in the degradation. As the AGS-SBRs did not favour the growth of such organisms, their potential for micropollutant removal appeared to be lower than that of conventional nitrifying WWTPs.

Towards a standardization of biomethane potential tests.

Production of biogas from different organic materials is a most interesting source of renewable energy. The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.

Divergent PCB organohalide-respiring consortia enriched from the efflux channel of a former Delor manufacturer in Eastern Europe.

Polychlorinated biphenyl (PCB) organohalide-respiring communities from the efflux channel of a former Delor manufacturer in Eastern Slovakia were assessed using metagenomic, statistical and cultivation-adapted approaches. Multivariate analysis of environmental factors together with terminal restriction fragment length polymorphisms of the bacterial communities in the primary sediments revealed both temporal and spatial heterogeneity in the distribution of microbial populations, which reflects the dynamic pattern of contamination and altered conditions for biodegradation activity along the channel. Anaerobic microcosms were developed from eight sediments sampled along the channel, where high concentrations of PCBs - from 6.6 to 136mg/kg dry weight, were measured. PCB dehalorespiring activity, congruent with changes in the microbial composition in all microcosms, was detected. After 10 months of cultivation, the divergently evolved consortia achieved up to 35.9 percent reduction of the total PCB concentration. Phylogenetic-analysis of the active Chloroflexi-related organohalide-respiring bacteria by partial sequencing of 16S rRNA genes in cDNA from microcosms with the highest PCB dechlorination activity revealed diverse and unique complexity of the populations. The predominant organohalide respirers were either affiliated with Dehalococcoides sp. and Dehalococcoides-like group (DLG) organisms or were composed of currently unknown distant clades of DLG bacteria. The present study should encourage researchers to explore the full potential of the indigenous PCB dechlorinating populations to develop effective bioremediation approaches that can perform the complete mineralization of PCBs in polluted environments.

Aerobic granules: microbial landscape and architecture, stages, and practical implications.

For the successful application of aerobic granules in wastewater treatment, granules containing an appropriate microbial assembly able to remove contaminants should be retained and propagated within the reactor. To manipulate and/or optimize this process, a good understanding of the formation and dynamic architecture of the granules is desirable. Models of granules often assume a spherical shape with an outer layer and an inner core, but limited information is available regarding the extent of deviations from such assumptions. We report on new imaging approaches to gain detailed insights into the structural characteristics of aerobic granules. Our approach stained all components of the granule to obtain a high quality contrast in the images; hence limitations due to thresholding in the image analysis were overcome. A three-dimensional reconstruction of the granular structure was obtained that revealed the mesoscopic impression of the cavernlike interior of the structure, showing channels and dead-end paths in detail. In "old" granules, large cavities allowed for the irrigation and growth of dense microbial colonies along the path of the channels. Hence, in some areas, paradoxically higher biomass content was observed in the inner part of the granule compared to the outer part. Microbial clusters "rooting" from the interior of the mature granule structure indicate that granules mainly grow via biomass outgrowth and not by aggregation of small particles. We identify and discuss phenomena contributing to the life cycle of aerobic granules. With our approach, volumetric tetrahedral grids are generated that may be used to validate complex models of granule formation.

Use of silicate minerals for pH control during reductive dechlorination of chloroethenes in batch cultures of different microbial consortia.

In chloroethene-contaminated sites undergoing in situ bioremediation, groundwater acidification is a frequent problem in the source zone, and buffering strategies have to be implemented to maintain the pH in the neutral range. An alternative to conventional soluble buffers is silicate mineral particles as a long-term source of alkalinity. In previous studies, the buffering potentials of these minerals have been evaluated based on abiotic dissolution tests and geochemical modeling. In the present study, the buffering potentials of four silicate minerals (andradite, diopside, fayalite, and forsterite) were tested in batch cultures amended with tetrachloroethene (PCE) and inoculated with different organohalide-respiring consortia. Another objective of this study was to determine the influence of pH on the different steps of PCE dechlorination. The consortia showed significant differences in sensitivities toward acidic pH for the different dechlorination steps. Molecular analysis indicated that Dehalococcoides spp. that were present in all consortia were the most pH-sensitive organohalide-respiring guild members compared to Sulfurospirillum spp. and Dehalobacter spp. In batch cultures with silicate mineral particles as pH-buffering agents, all four minerals tested were able to maintain the pH in the appropriate range for reductive dechlorination of chloroethenes. However, complete dechlorination to ethene was observed only with forsterite, diopside, and fayalite. Dissolution of andradite increased the redox potential and did not allow dechlorination. With forsterite, diopside, and fayalite, dechlorination to ethene was observed but at much lower rates for the last two dechlorination steps than with the positive control. This indicated an inhibition effect of silicate minerals and/or their dissolution products on reductive dechlorination of cis-dichloroethene and vinyl chloride. Hence, despite the proven pH-buffering potential of silicate minerals, compatibility with the bacterial community involved in in situ bioremediation has to be carefully evaluated prior to their use for pH control at a specific site.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Multiple dual C-Cl isotope patterns associated with reductive dechlorination of tetrachloroethene.

Dual isotope slopes are increasingly used to identify transformation pathways of contaminants. We investigated if reductive dechlorination of tetrachloroethene (PCE) by consortia containing bacteria with different reductive dehalogenases (rdhA) genes can lead to variable dual C-Cl isotope slopes and if different slopes also occur in the field. Two bacterial enrichments harboring Sulfurospirillum spp. but different rdhA genes yielded two distinct δ(13)C to δ(37)Cl slopes of 2.7 ± 0.3 and 0.7 ± 0.2 despite a high similarity in gene sequences. This suggests that PCE reductive dechlorination could be catalyzed according to at least two distinct reaction mechanisms or that rate-limiting steps might vary. At two field sites, two distinct dual isotope slopes of 0.7 ± 0.3 and 3.5 ± 1.6 were obtained, each of which fits one of the laboratory slopes within the range of uncertainty. This study hence provides additional insight into multiple reaction mechanisms underlying PCE reductive dechlorination. It also demonstrates that caution is necessary if a dual isotope approach is used to differentiate between transformation pathways of chlorinated ethenes.

Conditional essentiality of the 11-subunit complex I-like enzyme in strict anaerobes: the case of Desulfitobacterium hafniense strain DCB-2.

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

Microbial genome collection of aerobic granular sludge cultivated in sequencing batch reactors using different carbon source mixtures.

Aerobic granular sludge (AGS) consists of a microbial consortium that has an important role in wastewater treatment. This study investigates AGS microorganisms cultivated in a laboratory-scale sequencing batch reactor. Metagenomic sequencing was conducted using PacBio and Illumina, resulting in 759 metagenome-assembled genomes, 331 of which remained after dereplication.

MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

Diversity of cobalamin riboswitches in the corrinoid-producing organohalide respirer Desulfitobacterium hafniense.

The strategic adaptation of prokaryotes in polluted niches involves the efficient regulation of their metabolism. The obligate anaerobe and metabolically versatile Desulfitobacterium hafniense reductively dechlorinates halogenated organic compounds (so-called organohalides). Some D. hafniense strains carry out organohalide respiration (OHR), a process which requires the use of corrinoid as a cofactor in reductive dehalogenases, the key enzymes in OHR. We report here the diversity of the cobalamin riboswitches that possibly regulate the corrinoid metabolism for D. hafniense. The analysis of available D. hafniense genomes indicates the presence of 18 cobalamin riboswitches located upstream of genes whose products are mainly involved in corrinoid biosynthesis and transport. To obtain insight into their function, the secondary structures of three of these RNA elements were predicted by Mfold, as well as analyzed by in-line probing. These RNA elements both display diversity in their structural elements and exhibit various affinities toward adenosylcobalamin that possibly relates to their role in the regulation of corrinoid metabolism. Furthermore, adenosylcobalamin-induced in vivo repression of RNA synthesis of the downstream located genes indicates that the corrinoid transporters and biosynthetic enzymes in D. hafniense strain TCE1 are regulated at the transcriptional level. Taken together, the riboswitch-mediated regulation of the complex corrinoid metabolism in D. hafniense could be of crucial significance in environments polluted with organohalides both to monitor their intracellular corrinoid level and to coexist with corrinoid-auxotroph OHR bacteria.

Metabolomics of hexachlorocyclohexane (HCH) transformation: ratio of LinA to LinB determines metabolic fate of HCH isomers.

Although the production and use of technical hexachlorocyclohexane (HCH) and lindane (the purified insecticidal isomer γ-HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ-HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α-, ß-, γ-, δ-, ε-HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ-HCH degradation, a dehydrochlorinase (LinA) and a haloalkane dehalogenase (LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ-HCH and that under these conditions other HCH-isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side-products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated.

Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.