Tartrate fermentation with H2 production by a new member of Sporomusaceae enriched from rice paddy soil.

In rice paddies, soil and plant-derived organic matter are degraded anaerobically to methane (CH4), a powerful greenhouse gas. The highest rate of methane emission occurs during the reproductive stage of the plant when mostly dicarboxylic acids are exudated by the roots. The emission of methane at this stage depends largely on the cooperative interaction between dicarboxylic acid-fermenting bacteria and methanogenic archaea in the rhizosphere. The fermentation of tartrate, one of the major acids exudated, has been scarcely explored in rice paddy soils. In this work, we characterized an anaerobic consortium from rice paddy soil composed of four bacterial strains, whose principal member (LT8) can ferment tartrate, producing H2 and acetate. Tartrate fermentation was accelerated by co-inoculation with a hydrogenotrophic methanogen. The assembled genome of LT8 possesses a Na+-dependent oxaloacetate decarboxylase and shows that this bacterium likely invests part of the H2 produced to reduce NAD(P)+ to assimilate C from tartrate. The phylogenetic analysis of the 16S rRNA gene, the genome-based classification as well as the average amino acid identity (AAI) indicated that LT8 belongs to a new genus within the Sporomusaceae family. LT8 shares a few common features with its closest relatives, for which tartrate degradation has not been described. LT8 is limited to a few environments but is more common in rice paddy soils, where it might contribute to methane emissions from root exudates.IMPORTANCEThis is the first report of the metabolic characterization of a new anaerobic bacterium able to degrade tartrate, a compound frequently associated with plants, but rare as a microbial metabolite. Tartrate fermentation by this bacterium can be coupled to methanogenesis in the rice rhizosphere where tartrate is mainly produced at the reproductive stage of the plant, when the maximum methane rate emission occurs. The interaction between secondary fermentative bacteria, such as LT8, and methanogens could represent a fundamental step in exploring mitigation strategies for methane emissions from rice fields. Possible strategies could include controlling the activity of these secondary fermentative bacteria or selecting plants whose exudates are more difficult to ferment.

Genome-centric metagenomic insights into the role of Chloroflexi in anammox, activated sludge and methanogenic reactors.

BACKGROUND: The phylum Chloroflexi is highly abundant in a wide variety of wastewater treatment bioreactors. It has been suggested that they play relevant roles in these ecosystems, particularly in degrading carbon compounds and on structuring flocs or granules. Nevertheless, their function is not yet well understood as most species have not been isolated in axenic cultures. Here we used a metagenomic approach to investigate Chloroflexi diversity and their metabolic potential in three environmentally different bioreactors: a methanogenic full-scale reactor, a full-scale activated sludge reactor and a lab scale anammox reactor. RESULTS: Differential coverage binning approach was used to assemble the genomes of 17 new Chloroflexi species, two of which are proposed as new Candidatus genus. In addition, we recovered the first representative genome belonging to the genus 'Ca. Villigracilis'. Even though samples analyzed were collected from bioreactors operating under different environmental conditions, the assembled genomes share several metabolic features: anaerobic metabolism, fermentative pathways and several genes coding for hydrolytic enzymes. Interestingly, genome analysis from the anammox reactor indicated a putative role of Chloroflexi in nitrogen conversion. Genes related to adhesiveness and exopolysaccharides production were also detected. Complementing sequencing analysis, filamentous morphology was detected by Fluorescent in situ hybridization. CONCLUSION: Our results suggest that Chloroflexi participate in organic matter degradation, nitrogen removal and biofilm aggregation, playing different roles according to the environmental conditions.

Extracellular hydrolytic potential drives microbiome shifts during anaerobic co-digestion of sewage sludge and food waste.

Bacterial community structure and dynamics in anaerobic digesters are primarily influenced by feedstock composition. It is therefore important to unveil microbial traits that explain microbiome variations in response to substrate changes. Here, gene and genome-centric metagenomics were used to examine microbiome dynamics in four laboratory-scale reactors, in which sewage sludge was co-digested with increasing amounts of food waste. A co-occurrence network revealed microbiome shifts in response to changes in substrate composition and concentration. Food waste concentration correlated with extracellular enzymes and metagenome-assembled genomes (MAGs) involved in the degradation of complex carbohydrates commonly found in fruits and plant cell walls as well as with the abundance of hydrolytic MAGs. A key role was attributed to Proteiniphillum for being the only bacteria that encoded the complete pectin degradation pathway. These results suggest that changes of feedstock composition establish new microbial niches for bacteria with the capacity to degrade newly added substrates.

Long-run bacteria-phage coexistence dynamics under natural habitat conditions in an environmental biotechnology system.

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage-host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage-host interactions have been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction between Gordonia and their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Coevolution was followed by monitoring changes over time in the CRISPR loci of Gordonia metagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability of Gordonia, and almost clonal conservation of the trailer end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. The host population carrying a shorter CRISPR locus that contain only ancestral spacers, which has not acquired newer spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and viral genomes revealed how the complexity at the population level has important consequences for bacteria-phage coexistence.

Time Series Genome-Centric Analysis Unveils Bacterial Response to Operational Disturbance in Activated Sludge.

Understanding ecosystem response to disturbances and identifying the most critical traits for the maintenance of ecosystem functioning are important goals for microbial community ecology. In this study, we used 16S rRNA amplicon sequencing and metagenomics to investigate the assembly of bacterial populations in a full-scale municipal activated sludge wastewater treatment plant over a period of 3 years, including a 9-month period of disturbance characterized by short-term plant shutdowns. Following the reconstruction of 173 metagenome-assembled genomes, we assessed the functional potential, the number of rRNA gene operons, and the in situ growth rate of microorganisms present throughout the time series. Operational disturbances caused a significant decrease in bacteria with a single copy of the rRNA (rrn) operon. Despite moderate differences in resource availability, replication rates were distributed uniformly throughout time, with no differences between disturbed and stable periods. We suggest that the length of the growth lag phase, rather than the growth rate, is the primary driver of selection under disturbed conditions. Thus, the system could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions.IMPORTANCE Disturbance is a key determinant of community assembly and dynamics in natural and engineered ecosystems. Microbiome response to disturbance is thought to be influenced by bacterial growth traits and life history strategies. In this time series observational study, the response to disturbance of microbial communities in a full-scale activated sludge wastewater treatment plant was assessed by computing specific cellular traits of genomes retrieved from metagenomes. It was found that the genomes observed in disturbed periods have more copies of the rRNA operon than genomes observed in stable periods, whereas the in situ mean relative growth rates of bacteria present during stable and disturbed periods were indistinguishable. From these intriguing observations, we infer that the length of the lag phase might be a growth trait that affects the microbial response to disturbance. Further exploration of this hypothesis could contribute to better understanding of the adaptive response of microbiomes to unsteady environmental conditions.

Crop monoculture rather than agriculture reduces the spatial turnover of soil bacterial communities at a regional scale.

The goal of this study was to investigate the spatial turnover of soil bacterial communities in response to environmental changes introduced by the practices of soybean monoculture or crop rotations, relative to grassland soils. Amplicon sequencing of the 16S rRNA gene was used to analyse bacterial diversity in producer fields through three successive cropping cycles within one and a half years, across a regional scale of the Argentinean Pampas. Unlike local diversity, which was not significantly affected by land use type, agricultural management had a strong influence on ß-diversity patterns. Distributions of pairwise distances between all soils samples under soybean monoculture had significantly lower ß-diversity and narrower breadth compared with distributions of pairwise distances between soils managed with crop rotation. Interestingly, good agricultural practices had similar degree of ß-diversity as natural grasslands. The higher phylogenetic relatedness of bacterial communities in soils under monoculture across the region was likely determined by the observed loss of endemic species, and affected mostly to phyla with low regional diversity, such as Acidobacteria, Verrucomicrobia and the candidates phyla SPAM and WS3. These results suggest that the implementation of good agricultural practices, including crop rotation, may be critical for the long-term conservation of soil biodiversity.

Bacterial indicator of agricultural management for soil under no-till crop production.

The rise in the world demand for food poses a challenge to our ability to sustain soil fertility and sustainability. The increasing use of no-till agriculture, adopted in many areas of the world as an alternative to conventional farming, may contribute to reduce the erosion of soils and the increase in the soil carbon pool. However, the advantages of no-till agriculture are jeopardized when its use is linked to the expansion of crop monoculture. The aim of this study was to survey bacterial communities to find indicators of soil quality related to contrasting agriculture management in soils under no-till farming. Four sites in production agriculture, with different soil properties, situated across a west-east transect in the most productive region in the Argentinean pampas, were taken as the basis for replication. Working definitions of Good no-till Agricultural Practices (GAP) and Poor no-till Agricultural Practices (PAP) were adopted for two distinct scenarios in terms of crop rotation, fertilization, agrochemicals use and pest control. Non-cultivated soils nearby the agricultural sites were taken as additional control treatments. Tag-encoded pyrosequencing was used to deeply sample the 16S rRNA gene from bacteria residing in soils corresponding to the three treatments at the four locations. Although bacterial communities as a whole appeared to be structured chiefly by a marked biogeographic provincialism, the distribution of a few taxa was shaped as well by environmental conditions related to agricultural management practices. A statistically supported approach was used to define candidates for management-indicator organisms, subsequently validated using quantitative PCR. We suggest that the ratio between the normalized abundance of a selected group of bacteria within the GP1 group of the phylum Acidobacteria and the genus Rubellimicrobium of the Alphaproteobacteria may serve as a potential management-indicator to discriminate between sustainable vs. non-sustainable agricultural practices in the Pampa region.

[Impact of the recent advances in the analysis of microbial communities on the control of the wastewater treatment process]. / Impacto de los recientes avances en el análisis de comunidades microbianas sobre el control del proceso de tratamiento de efluentes.

One of the main functions of environmental biotechnology is to address the study of microbial communities that provide essential services to society. Beyond the similarities with industrial and agricultural microbiology, the unique features exhibited by environmental biotechnology, such as process objectives, biomass characteristics and type and mode of feeding (substrates), allow a clear distinction from the other related disciplines. Recent advances in microbial ecology, ecophysiology, genomics and process engineering are herein reviewed to illustrate how the integration of the new knowledge can help overcome the shortcomings of classic microbiological analyses to understand, predict and optimize the performance of wastewater treatment.

Impacto de los recientes avances en el análisis de comunidades microbianas sobre el control del proceso de tratamiento de efluentes / Impact of the recent advances in the analysis of microbial communities on the control of the wastewater treatment process

Una de las funciones principales de la biotecnología ambiental es ocuparse del estudio de comunidades microbianas que proveen servicios esenciales para la sociedad. Más allá de las similitudes que presenta con la microbiología industrial y la agrícola, la biotecnología ambiental presenta peculiaridades, tales como los objetivos de proceso, las características de la biomasa y el tipo y modo de alimentación (sustratos), que la distinguen claramente de las otras disciplinas relacionadas. En este artículo se reseñan recientes avances en la ecología microbiana, la ecofisiología, la genómica y la ingeniería de procesos, para ilustrar cómo la integración de los nuevos conocimientos permite superar las limitaciones del análisis microbiológico clásico para entender, predecir y optimizar el funcionamiento de los procesos de tratamiento de efluentes.

One of the main functions of environmental biotechnology is to address the study of microbial communities that provide essential services to society. Beyond the similarities with industrial and agricultural microbiology, the unique features exhibited by environmental biotechnology, such as process objectives, biomass characteristics and type and mode of feeding (substrates), allow a clear distinction from the other related disciplines. Recent advances in microbial ecology, ecophysiology, genomics and process engineering are herein reviewed to illustrate how the integration of the new knowledge can help overcome the shortcomings of classic microbiological analyses to understand, predict and optimize the performance of wastewater treatment.

Nonrandom assembly of bacterial populations in activated sludge flocs.

The aim of this work was to investigate the dynamics of assembly of bacterial populations in activated sludge flocs. We approached this question by following the development of active bacterial populations during floc development in four replicated lab-scale activated sludge reactors, in which solid retention time (SRT) was set at 4 days. The null hypothesis was that the similarities in community composition could be accounted for by the probability that the same organisms occur in more than one replicated reactor. Microscopic imaging showed that the size of flocs in reactors with biomass retention increased during the first few days until a steady-state size was reached. The diversity and community structure of the sludge in all reactors were analyzed during a period of up to ten SRT, using denaturing gradient gel electrophoresis (DGGE) of reverse-transcription polymerase chain reaction-amplified 16S rRNA. High rates of change in DGGE profiles from consecutive sampling points suggested a high level of dynamics in all reactors. This conclusion was confirmed by the application of the Raup and Crick probability-based similarity index (S(RC)) for the comparison of rRNA-based fingerprinting patterns, which indicated that bacterial communities within reactors were not significantly similar after three SRT (0.05 < S(RC) < 0.95) and became significantly dissimilar after five SRT (S(RC) < 0.05). More importantly, significant similarity between replicate reactors was observed at all times analyzed (S(RC) > 0.95). The fact that the patterns between replicates were more reproducible than expected by chance under highly dynamic conditions allowed us to reject the null hypothesis that activated sludge floc communities assemble randomly from the available source pool of bacteria. We suggest that communities progressively recruit from the available pool of bacterial species, each with particular ecological requirements that determine their time of emergence into the community.

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination. RESULTS: Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus. CONCLUSION: These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.