Performance and microbial community composition dynamics of aerobic granular sludge from sequencing batch bubble column reactors operated at 20°C, 30°C, and 35°C

Two bubble column sequencing batch reactors fed with an artificial wastewater were operated at 20°C, 30°C, and 35°C. In a first stage, stable granules were obtained at 20°C, whereas fluffy structures were observed at 30°C. Molecular analysis revealed high abundance of the operational taxonomic unit 208 (OTU 208) affiliating with filamentous bacteria Leptothrix spp. at 30°C, an OTU much less abundant at 20°C. The granular sludge obtained at 20°C was used for the second stage during which one reactor was maintained at 20°C and the second operated at 30°C and 35°C after prior gradual increase of temperature. Aerobic granular sludge with similar physical properties developed in both reactors but it had different nutrient elimination performances and microbial communities. At 20°C, acetate was consumed during anaerobic feeding, and biological phosphorous removal was observed when Rhodocyclaceae-affiliating OTU 214 was present. At 30°C and 35°C, acetate was mainly consumed during aeration and phosphorous removal was insignificant. OTU 214 was almost absent but the Gammaproteobacteria-affiliating OTU 239 was more abundant than at 20°C. Aerobic granular sludge at all temperatures contained abundantly the OTUs 224 and 289 affiliating with Sphingomonadaceae indicating that this bacterial family played an important role in maintaining stable granular structure

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidate

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides"-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes" strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated

Identification of bacteria from wastewater for the oxidation of micropollutants

The term "micropollutants" includes xenobiotic compounds of various origins which can be found in aquatic environments at concentration levels of nano- to micrograms per liter and which present a negative effect on ecosystems. Many aromatic micropollutants present in municipal wastewater are due to human activities, such as pharmaceuticals or biocides, and are not easily removed by conventional biological treatments in wastewater treatment plants (WWTPs). Fungal laccases and other multicopper oxidases are known to efficiently oxidize a wide spectrum of aromatic compounds, while a few in vitro studies on bacterial enzymes have also suggested the potential to use bacterial oxidases to increase the removal efficiency of micropollutants in WWTPs. In the present study, we chose to focus on the oxidative activities displayed by bacteria present in WWTPs. One aim of this work was to develop a screening method to isolate microorganisms producing oxidases with a potential activity on aromatic micropollutants. The microorganisms were selected on solid rich medium for their ability to oxidize 2,6-dimethoxyphenol (DMP), a chromogenic compound which becomes brown upon oxidation. DMP was chosen among other chromogenic substrates as it was not affecting bacterial growth, is nicely oxidized at neutral pH and is mimicking the basic structure of aromatic micropollutants. Selected bacterial isolates were identified as members of different genera (Pseudomonas, Comamonas, Enterobacter, …) and their ability to oxidize DMP was confirmed in liquid cultures as well as using an in vitro enzymatic assay with crude extracts. Preliminary observations suggested that DMP oxidation is linked with the stationary phase of bacterial growth. Further characterization of the physiology and biochemistry of the oxidases is under investigation for a subset of isolates

An unusual tandem-domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell

Statistical Assessment of Variability of Terminal Restriction Fragment Length Polymorphism Analysis Applied to Complex Microbial Communities ▿ †

The variability of terminal restriction fragment polymorphism analysis applied to complex microbial communities was assessed statistically. Recent technological improvements were implemented in the successive steps of the procedure, resulting in a standardized procedure which provided a high level of reproducibility

Temporal evolution of bacterial communities associated with the in situ wetland-based remediation of a marine shore porphyry copper tailings deposit

Mine tailings are a serious threat to the environment and public health. Remediation of these residues can be carried out effectively by the activation of specific microbial processes. This article presents detailed information about temporal changes in bacterial community composition during the remediation of a section of porphyry copper tailings deposited on the Bahía de Ite shoreline (Peru). An experimental remediation cell was flooded and transformed into a wetland in order to prevent oxidation processes, immobilizing metals. Initially, the top oxidation zone of the tailings deposit displayed a low pH (3.1) and high concentrations of metals, sulfate, and chloride, in a sandy grain size geological matrix. This habitatwas dominated by sulfur- and iron-oxidizing bacteria, such as Leptospirillumspp., Acidithiobacillus spp., and Sulfobacillus spp., in a microbial community which structure resembled acid mine drainage environments. After wetland implementation, the cell was water-saturated, the acidity was consumed and metals dropped to a fraction of their initial respective concentrations. Bacterial communities analyzed by massive sequencing showed time-dependent changes both in composition and cell numbers. The final remediation stage was characterized by the highest bacterial diversity and evenness. Aside from classical sulfate reducers from the phyla δ-Proteobacteria and Firmicutes, community structure comprised taxa derived fromvery diverse habitats. The community was also characterized by an elevated proportion of rare phyla and unaffiliated sequences. Numerical ecology analysis confirmed that the temporal population evolution was driven by pH, redox, and K. Results of this study demonstrated the usefulness of a detailed follow-up of the remediation process, not only for the elucidation of the communities gradually switching from autotrophic, oxidizing to heterotrophic and reducing living conditions, but also for the long term management of the remediation wetlands

THE TWIN-ARGININE TRANSLOCATION PATHWAY IN THE OBLIGATE ANAEROBE DESULFITOBACTERIUM

The genus Desulfitobacterium belong to the low-GC gram-positive phylogenic group and is a versatile obligate anaerobe capable of reducing very diverse terminal electron acceptors such as sulfite, nitrate, fumarate, thiosulfate, and a few polluting chlorinated compounds in a environmental-friendly energy metabolism called dehalorespiration. Analysis of available desulfitobacteria genomes revealed on one hand a high number of predicted proteins that are translocated in a folded conformation to or across the cytoplasmic membrane by the twin-arginine translocation (Tat) pathway. On the other hand a striking redundancy in the pore-forming protein (TatA) of the Tat translocase was observed. It might reflect the strong need for the translocase, and/or some TatA specificity of the Tat-dependent translocated proteins. The presence of numerous Tat-dependent oxidoreductases, for most of which the substrate specificity is not yet known, correlates well with the high versatility of anaerobic respiration of this genus. Half of Tat-dependent proteins in Desulfitobacterium belong also to the class of lipoproteins that might keep them attached to the cytoplasmic membrane. The functional characterization of the Tat system in our model organism Desulfitobacterium hafniense strain TCE1 is under progress

PREDOMINANCE OF ZOOGLOEA SP. DURING AEROBIC GRANULAR SLUDGE BIOFILMS DEVELOPMENT FOR WASTEWATER TREATMENT

Aerobic granular sludge (AGS) biofilm process is a sustainable technology for full biological nutrients removal (BNR) from municipal and agro-industrial wastewater, and for secondary clarification in single sequencing batch reactors (SBR). Fundamental research on the structure and dynamics of microbial communities inside AGS biofilms remains to be performed in addition to full-scale implementation, in order to link process conditions and reactor efficiency, and to tailor metabolic activities for stable treatment performance. Ten bubble column SBRs were run during three successive campaigns for (i) cultivating AGS, (ii) optimizing the AGS biofilm stability, and (iii) investigating process conditions favouring full BNR. Molecular ecology analyses on eubacterial communities performed during each first month of start-up revealed that optimally dense and fast-settling AGS biofilms were composed of 60-90% of Zoogloea sp. of the β-proteobacterial order Rhodocyclales, and of less than 20% of undesired Burkholderiales filamentous bulking heterotrophic bacteria. Zoogloea sp. was favourably selected by combining optimal process parameters: inoculum from a full BNR plant, 20°C, 60min- slow plug-flow anaerobic influent feeding regime, 0.018 m s-1 air superficial velocity, and settling time lower than 5 min. The application of a short settling phase induced a washout dynamics between the times of SBR inoculation and AGS nucleation. This low initial sludge retention time selected for a fast-settling biomass, but hampered the growth of relevant phosphorus-accumulating organisms, nitrifiers and denitrifiers. Organic carbon was fully removed, while phosphorus and nitrogen were not treated during the first 70 d. Zoogloea-like organisms can store soluble volatile fatty acids into intracellular polyhydroxyalcanoates (PHA). PHA-accumulating bacteria are expected to be denser and to settle faster than other species, and can stay in the AGS-SBR system during the initial washout dynamics. Zoogloea sp. can produce in addition extracellular polysaccharide polymers which could help to form and stabilize the early AGS biofilm structure

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

BackgroundThe 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.ObjectivesThe 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 MethodsOverall, 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.ResultsThe 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 RelevanceThese 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