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Proteins, an important fraction of the organic matter in wastewater, typically enter a treatment facility as high molecular weight components. These components are degraded by extracellular protein hydrolytic enzymes, denoted as proteases. Adequate protein hydrolysis monitoring is crucial, since protein hydrolysis is often a rate-limiting step in wastewater treatment. However, current monitoring tools lack a high sample throughput and reliable quantification. Here, we present an improved assay for high-throughput protein hydrolysis rate measurements in wastewater treatment applications. A BODIPY FL casein model substrate was implemented in a microplate format for continuous fluorescent quantification. Case studies on a conventional and a high-rate aerobic municipal wastewater treatment plant and a lab-scale, two-stage, anaerobic reactor provided proof-of-concept. The assay presented in this study can help to obtain monitoring-based process insights, which will in turn allow improving biological performance of wastewater treatment installations in the future.Key points• Protein hydrolysis is a crucial step in biological wastewater treatment.• Quantification of the protein hydrolysis rate enables in-depth process knowledge.• BODIPY FL casein is a suitable model substrate for a protein hydrolysis assay.• High sample throughput was obtained with fluorescent hydrolysis quantification.

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1–3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi . In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP.

Marine microbial consortium UBF, enriched from a beach polluted by the Prestige oil spill and highly efficient in degrading this heavy fuel, was subcultured in pyrene minimal medium. The pyrene-degrading subpopulation (UBF-Py) mineralized 31 % of pyrene without accumulation of partially oxidized intermediates indicating the cooperation of different microbial components in substrate mineralization. The microbial community composition was characterized by culture dependent and PCR based methods (PCR-DGGE and clone libraries). Molecular analyses showed a highly stable community composed by Alphaproteobacteria (84 %, Breoghania , Thalassospira , Paracoccus, and Martelella ) and Actinobacteria (16 %, Gordonia ). The members of Thalasosspira and Gordonia were not recovered as pure cultures, but five additional strains, not detected in the molecular analysis, that classified within the genera Novosphingobium, Sphingopyxis, Aurantimonas ( Alphaproteobacteria ) , Alcanivorax ( Gammaproteobacteria ) and Micrococcus ( Actinobacteria ), were isolated. None of the isolates degraded pyrene or other PAHs in pure culture. PCR amplification of Gram-positive and Gram-negative dioxygenase genes did not produce results with any of the cultured strains. However, sequences related to the NidA3 pyrene dioxygenase present in mycobacterial strains were detected in UBF-Py consortium, suggesting the representative of Gordonia as the key pyrene degrader, which is consistent with a preeminent role of actinobacteria in pyrene removal in coastal environments affected by marine oil spills.

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are \"hot spots\" of plasmids potentially carrying catabolic genes.

The impact of four electron acceptors on hydrocarbon-induced methanogenesis was studied. Methanogenesis from residual hydrocarbons may enhance the exploitation of oil reservoirs and may improve bioremediation. The conditions to drive the rate-limiting first hydrocarbon-oxidizing steps for the conversion of hydrocarbons into methanogenic substrates are crucial. Thus, the electron acceptors ferrihydrite, manganese dioxide, nitrate or sulfate were added to sediment microcosms acquired from two brackish water locations. Hexadecane, ethylbenzene or 1-¹³C-naphthalene were used as model hydrocarbons. Methane was released most rapidly from incubations amended with ferrihydrite and hexadecane. Ferrihydrite enhanced only hexadecane-dependent methanogensis. The rates of methanogenesis were negatively affected by sulfate and nitrate at concentrations of more than 5 and 1 mM, respectively. Metal-reducing Geobacteraceae and potential sulfate reducers as well as Methanosarcina were present in situ and in vitro. Ferrihydrite addition triggered the growth of Methanosarcina-related methanogens. Additionally, methane was removed concomitantly by anaerobic methanotrophy. ANME-1 and -2 methyl coenzyme M reductase genes were detected, indicating anaerobic methanotrophy as an accompanying process [Correction added 16 December after online publication: ‘methyl coenzyme A' changed to ‘methyl coenzyme M' in this sentence]. The experiments presented here demonstrate the feasibility of enhancing methanogenic alkane degradation by ferrihydrite or sulfate addition in different geological settings.

Abstract tmoA and related genes encode the α-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

Anaerobic digestion (AD) is a biological process that is acquiring increasing attention for both solid waste and wastewater treatment, as well as for the production of valuable chemicals. Despite the importance of the inoculum, the relationship between inoculum community composition, reactor performance, and reactor community composition remains vague. To understand the impact of the starting community on the composition and functioning of the AD microbiome, we studied three sets of biologically replicated AD reactors inoculated with different communities, but operated identically, targeting both total and active community compositions. All reactors performed highly similar regarding volatile fatty acid and methane production. The community analyses showed reproducible total and active community compositions in replicate reactors, indicating that particularly deterministic factors shaped the AD community. Moreover, strong variation in community composition between the differently seeded reactors was observed, indicating the role of inoculum composition in community shaping. In all three reactor sets, especially species that were low abundant or even not detected in the inoculum contributed to the reactor communities, supporting the importance of functional redundancy and high diversity in inocula used for AD seeding. The careful start-up of the AD process using initially low organic loading rates likely contributed to the successful assembly of initial low-abundance/rare species into a new cooperative AD community in the reactors.

We report here the draft genome sequence of Aeromonas sp. strain EERV15 isolated from sand filter. The organism most closely related to Aeromonas sp. EERV15 is Aeromonas veronii B565, with an average 83% amino acid sequence similarity of putatively encoded protein open reading frames.

In recent years, the application of pesticide biodegradation in remediation of pesticide-contaminated matrices moved from remediating bulk soil to remediating and mitigating pesticide pollution of groundwater and surface water bodies. Specialized pesticide-degrading microbial populations are used, which can be endogenous to the ecosystem of interest or introduced by means of bioaugmentation. It involves (semi-)natural ecosystems like agricultural fields, vegetated filter strips, and riparian wetlands and man-made ecosystems like on-farm biopurification systems, groundwater treatment systems, and dedicated modules in drinking water treatment. Those ecosystems and applications impose challenges which are often different from those associated with bulk soil remediation. These include high or extreme low pesticide concentrations, mixed contamination, the presence of alternative carbon sources, specific hydraulic conditions, and spatial and temporal variation. Moreover, for various indicated ecosystems, limited knowledge exists about the microbiota present and their physiology and about the in situ degradation kinetics. This review reports on the current knowledge on applications of biodegradation in mitigating and remediating freshwater pesticide contamination. Attention is paid to the challenges involved and current knowledge gaps for improving those applications.