Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

The potential contribution of aquatic wildlife to antibiotic resistance dissemination in freshwater ecosystems: A review.

Antibiotic resistance (AR) is one of the major health threats of our time. The presence of antibiotics in the environment and their continuous release from sewage treatment plants, chemical manufacturing plants and animal husbandry, agriculture and aquaculture, result in constant selection pressure on microbial organisms. This presence leads to the emergence, mobilization, horizontal gene transfer and a selection of antibiotic resistance genes, resistant bacteria and mobile genetic elements. Under these circumstances, aquatic wildlife is impacted in all compartments, including freshwater organisms with partially impermeable microbiota. In this narrative review, recent advancements in terms of occurrence of antibiotics and antibiotic resistance genes in sewage treatment plant effluents source compared to freshwater have been examined, occurrence of antibiotic resistance in wildlife, as well as experiments on antibiotic exposure. Based on this current state of knowledge, we propose the hypothesis that freshwater aquatic wildlife may play a crucial role in the dissemination of antibiotic resistance within the environment. Specifically, we suggest that organisms with high bacterial density tissues, which are partially isolated from the external environment, such as fishes and amphibians, could potentially be reservoirs and amplifiers of antibiotic resistance in the environment, potentially favoring the increase of the abundance of antibiotic resistance genes and resistant bacteria. Potential avenues for further research (trophic transfer, innovative exposure experiment) and action (biodiversity eco-engineering) are finally proposed.

Biogeography of microbial communities in high-latitude ecosystems: Contrasting drivers for methanogens, methanotrophs and global prokaryotes.

Methane-cycling is becoming more important in high-latitude ecosystems as global warming makes permafrost organic carbon increasingly available. We explored 387 samples from three high-latitudes regions (Siberia, Alaska and Patagonia) focusing on mineral/organic soils (wetlands, peatlands, forest), lake/pond sediment and water. Physicochemical, climatic and geographic variables were integrated with 16S rDNA amplicon sequences to determine the structure of the overall microbial communities and of specific methanogenic and methanotrophic guilds. Physicochemistry (especially pH) explained the largest proportion of variation in guild composition, confirming species sorting (i.e., environmental filtering) as a key mechanism in microbial assembly. Geographic distance impacted more strongly beta diversity for (i) methanogens and methanotrophs than the overall prokaryotes and, (ii) the sediment habitat, suggesting that dispersal limitation contributed to shape the communities of methane-cycling microorganisms. Bioindicator taxa characterising different ecological niches (i.e., specific combinations of geographic, climatic and physicochemical variables) were identified, highlighting the importance of Methanoregula as generalist methanogens. Methylocystis and Methylocapsa were key methanotrophs in low pH niches while Methylobacter and Methylomonadaceae in neutral environments. This work gives insight into the present and projected distribution of methane-cycling microbes at high latitudes under climate change predictions, which is crucial for constraining their impact on greenhouse gas budgets.

Gut microbiota impairment following graphene oxide exposure is associated to physiological alterations in Xenopus laevis tadpoles.

Graphene-based nanomaterials such as graphene oxide (GO) possess unique properties triggering high expectations for the development of technological applications. Thus, GO is likely to be released in aquatic ecosystems. It is essential to evaluate its ecotoxicological potential to ensure a safe use of these nanomaterials. In amphibians, previous studies highlighted X. laevis tadpole growth inhibitions together with metabolic disturbances and genotoxic effects following GO exposure. As GO is known to exert bactericidal effects whereas the gut microbiota constitutes a compartment involved in host homeostasis regulation, it is important to determine if this microbial compartment constitutes a toxicological pathway involved in known GO-induced host physiological impairments. This study investigates the potential link between gut microbial communities and host physiological alterations. For this purpose, X. laevis tadpoles were exposed during 12 days to GO. Growth rate was monitored every 2 days and genotoxicity was assessed through enumeration of micronucleated erythrocytes. Genomic DNA was also extracted from the whole intestine to quantify gut bacteria and to analyze the community composition. GO exposure led to a dose dependent growth inhibition and genotoxic effects were detected following exposure to low doses. A transient decrease of the total bacteria was noticed with a persistent shift in the gut microbiota structure in exposed animals. Genotoxic effects were associated to gut microbiota remodeling characterized by an increase of the relative abundance of Bacteroides fragilis. The growth inhibitory effects would be associated to a shift in the Firmicutes/Bacteroidetes ratio while metagenome inference suggested changes in metabolic pathways and upregulation of detoxification processes. This work indicates that the gut microbiota compartment is a biological compartment of interest as it is integrative of host physiological alterations and should be considered for ecotoxicological studies as structural or functional impairments could lead to later life host fitness loss.

A combined microbial and biogeochemical dataset from high-latitude ecosystems with respect to methane cycle.

High latitudes are experiencing intense ecosystem changes with climate warming. The underlying methane (CH4) cycling dynamics remain unresolved, despite its crucial climatic feedback. Atmospheric CH4 emissions are heterogeneous, resulting from local geochemical drivers, global climatic factors, and microbial production/consumption balance. Holistic studies are mandatory to capture CH4 cycling complexity. Here, we report a large set of integrated microbial and biogeochemical data from 387 samples, using a concerted sampling strategy and experimental protocols. The study followed international standards to ensure inter-comparisons of data amongst three high-latitude regions: Alaska, Siberia, and Patagonia. The dataset encompasses different representative environmental features (e.g. lake, wetland, tundra, forest soil) of these high-latitude sites and their respective heterogeneity (e.g. characteristic microtopographic patterns). The data included physicochemical parameters, greenhouse gas concentrations and emissions, organic matter characterization, trace elements and nutrients, isotopes, microbial quantification and composition. This dataset addresses the need for a robust physicochemical framework to conduct and contextualize future research on the interactions between climate change, biogeochemical cycles and microbial communities at high-latitudes.

Combining sequential extraction and 3D fluorescence to investigate the behavior of antibiotic and polycyclic aromatic hydrocarbons during solar drying of sewage sludge.

Solar drying and liming are commonly used for sludge treatment, but little is known about their efficiency on antibiotics and Polycyclic Aromatic Hydrocarbons (PAHs) removal. This study aimed to investigate the removal of antibiotics and PAHs during solar drying of Limed Sludge (LS) and Non-Limed Sludge (NLS). Thus, organic matter fractionation and 3D fluorescence were used to assess the accessibility and the complexity of organic matter. 2 experiments have been conducted using LS and NLS for 45 days of drying in a pilot scale tunnel. Physicochemical results indicated significant decrease of water content (90%) for both sludge samples within 15 days of drying. For both treatments, the removal of total organic carbon and total nitrogen was low and similar for both treatments. Through this study, it has been confirmed that liming and drying contributed to a strong modification of the organic matter quality with an increase of its accessibility. On the other hand, drying alone increased the less accessible compartments, while the presence of lime affected the interconnexion between the organic matter pools. 3D fluorescence confirmed the obtained results and indicated that LS leads to obtaining more simple molecules in the most accessible compartments, while NLS leads to obtaining more complex molecules in the less accessible compartments. In addition, solar radiations and leaching may contribute to the significant removal (p < 0.01) of roxithromycin, benzo(a)anthracene, chrysene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo(g, h, i) perylene in the presence of lime. Furthermore, the evolution of organic matter pools in terms of accessibility and complexity may drive the bioavailability of these pollutants, leading to their significant removal.

Graphene-Based Nanomaterials Modulate Internal Biofilm Interactions and Microbial Diversity.

Graphene-based nanomaterials (GBMs), such as graphene oxide (GO) and reduced graphene oxide (rGO), possess unique properties triggering high expectations for the development of new technological applications and are forecasted to be produced at industrial-scale. This raises the question of potential adverse outcomes on living organisms and especially toward microorganisms constituting the basis of the trophic chain in ecosystems. However, investigations on GBMs toxicity were performed on various microorganisms using single species that are helpful to determine toxicity mechanisms but fail to predict the consequences of the observed effects at a larger organization scale. Thus, this study focuses on the ecotoxicological assessment of GO and rGO toward a biofilm composed of the diatom Nitzschia palea associated to a bacterial consortium. After 48 and 144 h of exposure to these GBMs at 0, 0.1, 1, and 10 mg.L-1, their effects on the diatom physiology, the structure, and the metabolism of bacterial communities were measured through the use of flow cytometry, 16S amplicon sequencing, and Biolog ecoplates, respectively. The exposure to both of these GBMs stimulated the diatom growth. Besides, GO exerted strong bacterial growth inhibition as from 1 mg.L-1, influenced the taxonomic composition of diatom-associated bacterial consortium, and increased transiently the bacterial activity related to carbon cycling, with weak toxicity toward the diatom. On the contrary, rGO was shown to exert a weaker toxicity toward the bacterial consortium, whereas it influenced more strongly the diatom physiology. When compared to the results from the literature using single species tests, our study suggests that diatoms benefited from diatom-bacteria interactions and that the biofilm was able to maintain or recover its carbon-related metabolic activities when exposed to GBMs.

Temperature differently affected methanogenic pathways and microbial communities in sub-Antarctic freshwater ecosystems.

Freshwater ecosystems are responsible for an important part of the methane (CH4) emissions which are likely to change with global warming. This study aims to evaluate temperature-induced (from 5 to 20 °C) changes on microbial community structure and methanogenic pathways in five sub-Antarctic lake sediments from Magallanes strait to Cape Horn, Chile. We combined in situ CH4 flux measurements, CH4 production rates (MPRs), gene abundance quantification and microbial community structure analysis (metabarcoding of the 16S rRNA gene). Under unamended conditions, a temperature increase of 5 °C doubled MPR while microbial community structure was not affected. Stimulation of methanogenesis by methanogenic precursors as acetate and H2/CO2, resulted in an increase of MPRs up to 127-fold and 19-fold, respectively, as well as an enrichment of mcrA-carriers strikingly stronger under acetate amendment. At low temperatures, H2/CO2-derived MPRs were considerably lower (down to 160-fold lower) than the acetate-derived MPRs, but the contribution of hydrogenotrophic methanogenesis increased with temperature. Temperature dependence of MPRs was significantly higher in incubations spiked with H2/CO2 (c. 1.9 eV) compared to incubations spiked with acetate or unamended (c. 0.8 eV). Temperature was not found to shape the total microbial community structure, that rather exhibited a site-specific variability among the studied lakes. However, the methanogenic archaeal community structure was driven by amended methanogenic precursors with a dominance of Methanobacterium in H2/CO2-based incubations and Methanosarcina in acetate-based incubations. We also suggested the importance of acetogenic H2-production outcompeting hydrogenotrohic methanogenesis especially at low temperatures, further supported by homoacetogen proportion in the microcosm communities. The combination of in situ-, and laboratory-based measurements and molecular approaches indicates that the hydrogenotrophic pathway may become more important with increasing temperatures than the acetoclastic pathway. In a continuously warming environment driven by climate change, such issues are crucial and may receive more attention.

Ecotoxicological assessment of commercial boron nitride nanotubes toward Xenopus laevis tadpoles and host-associated gut microbiota.

Despite the growing interest for boron nitride nanotubes (BNNT) due to their unique properties, data on the evaluation of the environmental risk potential of this emerging engineered nanomaterial are currently lacking. Therefore, the ecotoxicity of a commercial form of BNNT (containing tubes, hexagonal-boron nitride, and boron) was assessed in vivo toward larvae of the amphibian Xenopus laevis. Following the exposure, multiple endpoints were measured in the tadpoles as well as in bacterial communities associated to the host gut. Exposure to BNNT led to boron accumulation in host tissues and was not associated to genotoxic effects. However, the growth of the tadpoles increased due to BNNT exposure. This parameter was associated to remodeling of gut microbiome, benefiting to taxa from the phylum Bacteroidetes. Changes in relative abundance of this phylum were positively correlated to larval growth. The obtained results support the finding that BNNT are biocompatible as indicated by the absence of toxic effect from the tested nanomaterials. In addition, byproducts, especially free boron present in the tested product, were overall beneficial for the metabolism of the tadpoles.

Anaerobic oxidation of methane and associated microbiome in anoxic water of Northwestern Siberian lakes.

Arctic lakes emit methane (CH4) to the atmosphere. The magnitude of this flux could increase with permafrost thaw but might also be mitigated by microbial CH4 oxidation. Methane oxidation in oxic water has been extensively studied, while the contribution of anaerobic oxidation of methane (AOM) to CH4 mitigation is not fully understood. We have investigated four Northern Siberian stratified lakes in an area of discontinuous permafrost nearby Igarka, Russia. Analyses of CH4 concentrations in the water column demonstrated that 60 to 100% of upward diffusing CH4 was oxidized in the anoxic layers of the four lakes. A combination of pmoA and mcrA gene qPCR and 16S rRNA gene metabarcoding showed that the same taxa, all within Methylomonadaceae and including the predominant genus Methylobacter as well as Crenothrix, could be the major methane-oxidizing bacteria (MOB) in the anoxic water of the four lakes. Correlation between Methylomonadaceae and OTUs within Methylotenera, Geothrix and Geobacter genera indicated that AOM might occur in an interaction between MOB, denitrifiers and iron-cycling partners. We conclude that MOB within Methylomonadaceae could have a crucial impact on CH4 cycling in these Siberian Arctic lakes by mitigating the majority of produced CH4 before it leaves the anoxic zone. This finding emphasizes the importance of AOM by Methylomonadaceae and extends our knowledge about CH4 cycle in lakes, a crucial component of the global CH4 cycle.

Sub-oxycline methane oxidation can fully uptake CH4 produced in sediments: case study of a lake in Siberia.

It is commonly assumed that methane (CH4) released by lakes into the atmosphere is mainly produced in anoxic sediment and transported by diffusion or ebullition through the water column to the surface of the lake. In contrast to that prevailing idea, it has been gradually established that the epilimnetic CH4 does not originate exclusively from sediments but is also locally produced or laterally transported from the littoral zone. Therefore, CH4 cycling in the epilimnion and the hypolimnion might not be as closely linked as previously thought. We utilized a high-resolution method used to determine dissolved CH4 concentration to analyze a Siberian lake in which epilimnetic and hypolimnetic CH4 cycles were fully segregated by a section of the water column where CH4 was not detected. This layer, with no detected CH4, was well below the oxycline and the photic zone and thus assumed to be anaerobic. However, on the basis of a diffusion-reaction model, molecular biology, and stable isotope analyses, we determined that this layer takes up all the CH4 produced in the sediments and the deepest section of the hypolimnion. We concluded that there was no CH4 exchange between the hypolimnion (dominated by methanotrophy and methanogenesis) and the epilimnion (dominated by methane lateral transport and/or oxic production), resulting in a vertically segregated lake internal CH4 cycle.

Gut microbiota of aquatic organisms: A key endpoint for ecotoxicological studies.

Gut microbial communities constitute a compartment of crucial importance in regulation of homeostasis of multiple host physiological functions as well as in resistance towards environmental pollutants. Many chemical contaminants were shown to constitute a major threat for gut bacteria. Changes in gut microbiome could lead to alteration of host health. The access to high-throughput sequencing platforms permitted a great expansion of this discipline in human health while data from ecotoxicological studies are scarce and particularly those related to aquatic pollution. The main purpose of this review is to summarize recent body of literature providing data obtained from microbial community surveys using high-throughput 16S rRNA sequencing technology applied to aquatic ecotoxicity. Effects of pesticides, PCBs, PBDEs, heavy metals, nanoparticles, PPCPs, microplastics and endocrine disruptors on gut microbial communities are presented and discussed. We pointed out difficulties and limits provided by actual methodologies. We also proposed ways to improve understanding of links between changes in gut bacterial communities and host fitness loss, along with further applications for this emerging discipline.

Transfer and Ecotoxicity of Titanium Dioxide Nanoparticles in Terrestrial and Aquatic Ecosystems: A Microcosm Study.

With the advancement in nanotechnology, particularly the use of TiO2 nanoparticles (NPs), there is a need to study their release into the environment and assess the related risk in an environmentally relevant contamination scenario. In the present study, the transfer and toxicity of TiO2 NPs in microcosms mimicking terrestrial and aquatic ecosystems were evaluated. The contaminated soil was prepared by spiking natural soils, with these then used as the basis for all exposure systems including preparation of soil leachates for amphibian exposure. Results demonstrated significant reductions in bacterial (-45%) and archaeal (-36%) nitrifier abundance; significant translocation of Ti to M. truncatula leaves (+422%); significant reductions in plant height (-17%), number of leaves (-29%), and aboveground biomass (-53%); nonsignificant Ti uptake in snail foot and viscera, and excretion in feces; and genotoxicity to X. laevis larvae (+119% micronuclei). Our study highlights a possible risk of engineered TiO2 NPs in the environment in terms of trophic transfer and toxicity in both terrestrial and aquatic environments.

A pressurized liquid extraction approach followed by standard addition method and UPLC-MS/MS for a fast multiclass determination of antibiotics in a complex matrix.

In this work a fast analytical method for the determination of macrolides, tetracyclines and fluoroquinolones in a compost originating from a mixture of sewage sludge, palm waste and grass was developed by ultra-high performance liquid chromatography coupled to mass spectrometry (U-HPLC/MS). Antibiotics were extracted from compost by using the accelerated solvent extraction (ASE). The chromatographic separation was carried out on a T3 Cortecs C18 column using a mobile phase gradient mixture of water acidified with 1% of formic acid and acetonitrile. Recoveries of 24-30%, 53-93%, 33-57%, 69-135% and 100-171% were obtained for roxithromycin (ROX), chlortetracycline (CTC), oxytetracycline (OTC), enrofloxacin (ENR) and ciprofloxacin (CIP), respectively. As the most part of antibiotics showed significant matrix effect (ME), the method was validated using the standard addition method (SAM) to correct the observed ME. Instrumental variation, of LC/MS system, showed that 93.75% of the relative standard deviation (RSD %) are below 15%, although the organic load of extracts. This analytical method was applied to assess the fate of antibiotics during composting. Two composting experiments were conducted separately after spiking sludge at 2 different concentrations levels. The resulting elimination rates were of 52-76, 69-100, 100 and 24-50% for ROX, CTC, OTC and CIP, respectively. These results suggest that composting process contributes to the removal of residuals concentrations of macrolides and tetracyclines while the fluoroquinolones persist in the final compost product.

Human and veterinary antibiotics during composting of sludge or manure: Global perspectives on persistence, degradation, and resistance genes.

Wastewater treatment plant effluent, sludge and manure are the main sources of contamination by antibiotics in the whole environment compartments (soil, sediment, surface and underground water). One of the major consequences of the antibiotics discharge into the environment could be the prevalence of a bacterial resistance to antibiotic. In this review, four groups of antibiotics (Tetracyclines, Fluoroquinolones, Macrolides and Sulfonamides) were focused for the background on their wide spread occurrence in sludge and manure and for their effects on several target and non-target species. The antibiotics concentrations range between 1 and 136,000 µg kg-1 of dry matter in sludge and manure, representing a potential risk for the human health and the environment. Composting of sludge or manure is a well-known and used organic matter stabilization technology, which could be effective in reducing the antibiotics levels as well as the antibiotic resistance genes. During sludge or manure composting, the antibiotics removals range between 17-100%. The deduced calculated half-lives range between 1-105 days for most of the studied antibiotics. Nevertheless, these removals are often based on the measurement of concentration without considering the matter removal (lack of matter balance) and very few studies are emphasized on the removal mechanisms (biotic/abiotic, bound residues formation) and the potential presence of more or less hazardous transformation products. The results from the few studies on the fate of the antibiotic resistance genes during sludge or manure composting are still inconsistent showing either decrease or increase of their concentration in the final product. Whether for antibiotic or antibiotic resistance genes, additional researches are needed, gathering chemical, microbiological and toxicological data to better understand the implied removal mechanisms (chemical, physical and biological), the interactions between both components and the environmental matrices (organic, inorganic bearing phases) and how composting process could be optimized to reduce the discharge of antibiotics and antibiotic resistance genes into the environment.

Evaluation of the antibiotics effects on the physical and chemical parameters during the co-composting of sewage sludge with palm wastes in a bioreactor.

The objective of this study was to investigate thermal and physicochemical parameters of sewage sludge-palm waste mixtures contaminated by different families of antibiotics (tetracyclines, macrolides and fluoroquinolones) during co-composting. Sludge was spiked with chlortetracycline (CTC), oxytetracycline (OTC), roxithromycin (RXY), enrofloxacin (ENR) and ciprofloxacin (CIP). Antibiotics were spiked at a low level, medium level, high level and a control without antibiotics was conducted. The results showed that the organic matter degradation was delayed and the carbon/nitrogen (C/N) ratio was affected by an increase of the antibiotics concentration. The presence of antibiotics, especially at high level, delayed the coming of the temperature maxima, and disturbed the thermophilic phase. The calorific model showed that the heat release is affected during the thermophilic phase when high antibiotics concentrations were used. In addition, the microbiological approach showed that the adsorption of antibiotics on the rachis could be probably responsible of the fungi inhibition especially during the maturation phase. Therefore, the medium and high levels of antibiotics affected the thermal, physical and chemical parameters as well as the compost quality.

Phylogenetic identification of methanogens assimilating acetate-derived carbon in dairy and swine manures.

In order to develop approaches for reducing the carbon footprint of the swine and dairy industries, it is important first to identify the methanogenic communities that drive methane emissions from stored manure. In this study, the metabolically active methanogens in substrate-starved manure samples taken from two dairy and one swine manure storage tanks were identified using [(13)C]-acetate and DNA stable-isotope probing (DNA-SIP). Molecular analysis of recovered genomic [(13)C]-DNA revealed that two distinct clusters of unclassified methanogen populations affiliated with the Methanoculleus genus, and the populations affiliated with Methanoculleus chikugoensis assimilated acetate-derived carbon (acetate-C) in swine and dairy starved manure samples, respectively. Furthermore, carbon flow calculations indicated that these populations were the primary contributors to methane emissions during these anoxic SIP incubations. Comparative analysis of mcrA gene abundance (coding for a key enzyme of methanogenesis) for Methanoculleus spp. in fresh feces and a wider range of stored dairy or swine manure samples, by real-time quantitative PCR using newly designed specific primers, demonstrated that the abundance of this genus significantly increased during storage. The findings supported the involvement of these particular methanogen populations as methane emitters from swine and dairy manure storage tanks. The study revealed that the ability to assimilate acetate-C for growth in manure differed within the Methanoculleus genus.

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Physico-chemical characteristics and methanogen communities in swine and dairy manure storage tanks: spatio-temporal variations and impact on methanogenic activity.

Greenhouse gas emissions represent a major environmental problem associated with the management of manure from the livestock industry. Methane is the primary GHG emitted during manure outdoor storage. In this paper, the variability of two swine and two dairy manure storage tanks was surveyed, in terms of physico-chemical and microbiological parameters. The impact of the inter-tank and spatio-temporal variations of these parameters on the methanogenic activity of manure was ascertained. A Partial Least Square regression was carried out, which demonstrated that physico-chemical as well as microbiological parameters had a major influence on the methanogenic activity. Among the 19 parameters included in the regression, the concentrations of VFAs had the strongest negative influence on the methane emission rate of manure, resulting from their well-known inhibitory effect. The relative abundance of two amplicons in archaeal fingerprints was found to positively influence the methanogenic activity, suggesting that Methanoculleus spp. and possibly Methanosarcina spp. are major contributors to methanogenesis in storage tanks. This work gave insights into the mechanisms, which drive methanogenesis in swine and dairy manure storage tanks.

Methanoculleus spp. as a biomarker of methanogenic activity in swine manure storage tanks.

Greenhouse gas emissions represent a major problem associated with manure management in the livestock industry. A prerequisite to mitigate methane emissions occurring during manure storage is a clearer understanding of how the microbial consortia involved in methanogenesis function. Here, we have examined manure stored in outdoor tanks from two different farms, at different locations and depths. Physico-chemical and microbiological characterization of these samples indicated differences between each tank, as well as differences within each tank dependent on the depth of sampling. The dynamics of both the bacterial and archaeal communities within these samples were monitored over a 150-day period of anaerobic incubation to identify and track emerging microorganisms, which may be temporally important in the methanogenesis process. Analyses based on DNA fingerprinting of microbial communities identified trends common among all samples as well as trends specific to certain samples. All archaeal communities became enriched with Methanoculleus spp. over time, indicating that the hydrogenotrophic pathway of methanogenesis predominated. Although the emerging species differed in samples obtained from shallow depths compared to deep samples, the temporal enrichment of Methanoculleus suggests that this genus may represent a relevant indicator of methanogenic activity in swine manure storage tanks.