Guylaine Talbot

Bacterial community dynamics in an anaerobic plug-flow type bioreactor treating swine manure.

A plug-flow type anaerobic reactor consisting of eight sequential compartments was used to study shifts in a bacterial community adapted to degrade swine manure at 25 degrees C. The investigation was carried out during the first 6 months of reactor operation. The reactor successfully separated the hydrolysis/acidogenesis stage from the methanogenesis stage. Bacterial 16S rDNA- and rRNA-based fingerprints obtained through amplicon length heterogeneity PCR (LH-PCR) were analyzed with a view to characterizing the bacterial community structure and the metabolically active community, respectively. Multivariate statistical tools showed that the rDNA-based fingerprints described a more temporal than compartmentalized distribution of similar bacterial communities. By contrast, the rRNA-based multivariate analyses described a distribution that was linked more to reactor performance parameters, especially during short time periods. Diversity indices calculated from fingerprint data were used to assess overall diversity shifts. The decrease in rRNA-based diversity observed through the reactor compartments was greater than the decrease in rDNA-based diversity. This finding indicates that the analysis of metabolically active bacteria diversity was more discriminative than the analysis based on the mere presence of bacteria. The observed decrease in diversity suggests that the bacterial community became specialized in degrading less diversified substrates through the compartments. All these findings suggest that rRNA-based analyses are more appropriate for monitoring reactor performance.

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.

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

ABSTRACT 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-13C]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 13C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the 13C 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.

A novel fingerprint method to assess the diversity of methanogens in microbial systems

Understanding the ecology of methanogens in natural and engineered environments is a prerequisite to predicting or managing methane emissions. In this study, a novel high-throughput fingerprint method was developed for determining methanogen diversity and relative abundance within environmental samples. The method described here, designated amplicon length heterogeneity PCR of the mcrA gene (LH-mcrA), is based on the natural length variation in the mcrA gene. The mcrA gene encodes the alpha-subunit of the methyl-coenzyme M reductase, which is involved in the terminal step of methane production by methanogens. The methanogenic communities from stored swine and dairy manures were distinct from each other. To validate the method, methanogenic communities in a plug flow-type bioreactor (PFBR) treating swine manure were characterized using LH-mcrA method and correlated to mcrA gene clone libraries. The diversity and relative abundance of the methanogenic groups were assessed. Methanobrevibacter, Methanosarcinaceae, Methanoculleus, Methanogenium, Methanocorpusculum and one unidentified group were assigned to particular LH-mcrA amplicons. Particular phylotypes related to Methanoculleus were predominant in the last compartment of the PFBR where the bulk of methane was produced. LH-mcrA method was found to be a reliable, fast and cost-effective alternative for diversity assessment of methanogenic communities in microbial systems.

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.

Multivariate statistical analyses of rDNA and rRNA fingerprint data to differentiate microbial communities in swine manure

Fingerprint data from swine manure microbial community rRNAs and rRNA genes were treated by multivariate statistical and diversity analyses to differentiate swine manures. Microbial communities from finishing pig manure and from a mixture of manure slurries from maternity confinement and finishing pigs were characterized using a combination of amplicon length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP), using PCR primers targeting Bacteria and Archaea, respectively. Unweighted pair group method with arithmetic mean clustering, principal components analysis (PCA), indicator species analysis (ISA), and diversity analyses showed that rRNA-based fingerprinting methods [reverse transcription (RT)-LH-PCR and RT-T-RFLP] were more effective than rDNA-based fingerprinting methods for distinguishing the manure samples. Multiresponse permutation procedure from fingerprint data showed that all manure samples had distinct microbial communities. PCA and ISA showed that the major phylotypes differentiating the LH-PCR or the RT-LH-PCR profiles were distributed differently between manures, suggesting that the bacterial community structure was different from the metabolically active bacterial community. The detection of minor archaeal populations was greater using RT-T-RFLP instead of T-RFLP. The findings indicated that the analysis of microbial community rRNAs could differentiate each manure sample from the others and would be appropriate for the monitoring of metabolically active populations.

Spatial distribution of some microbial trophic groups in a plug-flow-type anaerobic bioreactor treating swine manure

Anaerobic digestion of swine manure is carried out by a consortium of microbial species, including volatile fatty acid (VFA) producers, VFA-degraders and methanogens. The distribution of five phylogenetic groups within a plug-flow-type anaerobic bioreactor consisting of eight serially-connected tanks was examined through the sequential digestion of swine manure. Quantification was carried out using reverse transcription real-time PCR (RT-Q-PCR) assays targeting the 16S rRNA of Clostridium (cluster XIVa), Peptostreptococcus, Syntrophomonas, Methanosaeta, and Methanosarcina spp. The VFA producers Peptostreptococcus spp. and Clostridium spp. were found predominantly in compartments where hydrolysis/acidogenesis took place. The spatial distribution of the aceticlastic methanogens, Methanosaeta and Methanosarcina, within the bioreactor was not correlated with methanogenic activity. In contrast the VFA-degrading genus Syntrophomonas spp. was more abundant in compartments with elevated methanogenic activity. Multivariate statistical analyses of the RT-Q-PCR data have provided new insights into our understanding of how the various trophic groups were distributed within this bioreactor system. While the distribution of clostridia, peptostreptococci and Syntrophomonas corresponded to their known metabolic functions, aceticlastic methanogens were not apparently linked to the methanogenesis stage occurring in latter compartments, suggesting that hydrogenotrophic methanogens were the primary methane generators in this bioreactor. However, aceticlastic methanogens could be involved in compartments related to the hydrolysis/acidogenesis stage.

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.

Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.

As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngμL(-1) to 100ngμL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.

Draft Genome Sequences of Three Arcobacter Strains of Pig and Dairy Cattle Manure Origin

ABSTRACT The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. Here, we announce the draft genome sequences of three strains of Arcobacter species cultured from pig and dairy cattle manure tanks. This information will assist in the characterization of features related to host specificities and identify potential pathogenic health risks to humans and animals.