ABSTRACT Aminobacter niigataensis MSH1 is a candidate for bioaugmentation of sand filters in drinking water treatment plants (DWTP), as it mineralizes the ubiquitous groundwater micropollutant 2,6-dichlorobenzamide (BAM). The DWTP sand filter isolate Piscinibacter sp. K169 improves BAM mineralization by MSH1 in an apparent accidental mutual cooperation, and co-inoculation of the organism was proposed to assist bioaugmentation with MSH1. In this study, we questioned whether this accidental mutual positive interaction extends to four other pesticide catabolic bacterial strains of the same or a different genus of MSH1, and examined the longevity of the cooperation. Negative interactions were never observed in either direction. As observed for BAM mineralization by MSH1, K169 stimulated BAM mineralization by A. niigataensis LG1 and 2,4-D mineralization by Cupriavidus pinatubonensis JMP134 without affecting the cell density of the catabolic strains. Linuron mineralization by Variovorax sp. SRS16 and carbofuran mineralization by Novosphingobium sp. KN65.2 were not affected. In the other direction, growth of K169 was stimulated by all pesticide catabolic strains except JMP134, indicating a common underlying mechanism. After 2 weeks, the beneficial effects of K169 on MSH1, LG1, and JMP134 functionality diminished or even reversed, likely because of organic carbon depletion. In contrast, cell densities of K169 in all dual-species systems remained higher than in the K169 monoculture system. This study extends our knowledge on accidental interactions and the beneficial effect of a sand filter isolate toward other pesticide degraders, opening doors for Piscinibacter sp. K169-assisted bioaugmentation of other/multiple pesticide degraders in DWTPs. IMPORTANCE Sand‑filter bioaugmentation with the BAM‑catabolic Aminobacter niigataensis MSH1 represents an advanced strategy for removing BAM from groundwater in drinking water treatment; however, prior studies indicate that efficacy lasts only for 1–2 weeks. Piscinibacter sp. K169, an isolate from drinking‑water sand filters, supports mineralization of BAM by MSH1 through accidental mutual cooperation, and co‑inoculation with K169 was suggested as an innovation to improve MSH1 bioaugmentation. We show that K169 promotes mineralization of OMPs by other bacteria and, hence, that the K169-degrader cooperation can be extended to support removal of other or even multiple OMPs. Benefits declined over time, likely due to nutrient depletion, making nutrient management a requirement for maintaining the cooperation. To the best of our knowledge, this is the first study to examine specificity in accidental microbial cooperation, especially in a bioaugmentation context of water treatment. It is relevant both to a fundamental understanding of accidental microbial interactions and to applications in water treatment.

Organic micropollutants (OMPs) compose a group of emerging contaminants that occur in environmental waters at trace concentrations (μg/L to ng/L) with suspected adverse effects on ecosystems and human health. Conventional wastewater treatment plants (WWTPs) are not designed to eliminate the more recalcitrant OMPs. As such, WWTP-effluents are a major source of OMPs in the aquatic environment, and sustainable advanced treatment options are required. One option concerns the biodegradation of OMPs, but several studies show that it might be constrained by the residual low energy content in effluent waters. Since molecular hydrogen (H2) has been identified as a universally available energy source utilised by various bacteria in oligotrophic environments, supporting mixotrophic growth, we examined the hypothesis that H2 can enhance OMP-removal and concomitant ecotoxicity from domestic wastewater effluent. To this end, a lab-scale biological trickling filter supplemented with H2 (1.5% in the aeration with ambient air) was operated in continuous mode for treating a field-collected domestic WWTP-effluent. In total, 51 OMPs were detected in the WWTP-effluent, of which azithromycin and clarithromycin contributed to 76.5% of the total ecotoxic effects, as demonstrated in a cyanobacterial growth inhibition assay. The H2-supplemented trickling filter enhanced the removal of azithromycin and clarithromycin by (69.5 ± 1.5)% and (46.0% ±2.9)%, respectively, and increased the reduction of total OMP-associated ecotoxic effects by 56.5%, compared to the non-H2-supplemented control. Moreover, H2 supplementation improved PO43- and NO3- removal by a factor of 2.3 and 1.5, respectively. These findings demonstrate that H2 supplementation may support microbial processes involved in OMP-removal from domestic wastewater in a microbial treatment system, thereby reducing concomitant ecotoxicity.

Organic micropollutants (OMPs) occur in natural aquatic environments at trace concentrations with suspected adverse effects on the ecosystem and human health. Microbial biodegradation plays a crucial role in OMP-elimination from drinking water resources. However, long-term OMP-biodegradation remains challenging since the metabolic activity of degrading strains is restricted by energy-limited conditions in treatment systems. Molecular hydrogen (H2) has been identified as a universally available energy source utilized by various bacteria under nutrient-starved conditions, and it can be hypothesized that H2 might also support OMP-degrading microbes when other energy carriers are scarce. The potential of H2 as a supporting energy source for OMP-degradation was tested by examining its effect on the biodegradation of 2,6-dichlorobenzamide (BAM) by Aminobacter niigataensis MSH1 and on the physiological status of the MSH1 cells during both nongrowth-linked (500 μg BAM/L) and growth-linked (10,000 μg BAM/L) regimes. MSH1 cells used as inoculum were either not or pre-exposed to H2 and were harvested at different growth phases. During the nongrowth-linked BAM biodegradation, MSH1 pre-exposed to H2 exhibited a 1.2 to 1.5-fold higher initial specific BAM biodegradation rate, resulting in more rapid BAM removal, likely due to the retention of more metabolically active cells, as suggested by a cell vitality assay. During the growth-linked biodegradation, MSH1 pre-exposed to H2 demonstrated accelerated growth with a 1.5-fold higher maximum specific growth rate, which coincided with an improved BAM removal. The positive effects of H2 were only evident for MSH1 cells harvested either at the stationary and/or starvation phase. Evidence of H2 metabolism was supported by H₂ consumption measurements. Collectively, this study reveals that microbial H2 metabolism enables OMP-degrading bacteria to sustain metabolic activity under starvation conditions, offering a novel strategy to enhance long-term OMP-biodegradation.

Aminobacter niigataensis MSH1 mineralizes the groundwater micropollutant 2,6-dichlorobenzamide (BAM) and is a candidate for bioaugmentation of sand filters in drinking water treatment plants (DWTP) to avert BAM-contamination. Piscinibacter sp. K169 is a sand filter isolate that improves MSH1-mediated BAM mineralization through a cooperative interaction, and co-inoculation of MSH1 with K169 is proposed as a strategy to support bioaugmentation with MSH1. In this study, boundaries regarding the initial population size and the supply of organic carbon resources determining the interaction between MSH1 and K169 in sand filter microcosms were explored. The cooperative interaction was only disturbed when initial cell densities of one of the two partners were 104 cells/mL or lower. Supplying acetate as a carbon source appeared redundant for supporting BAM mineralization. Instead, the organic carbon present on the sand drove the cooperative interaction between K169 and MSH1 as the effect of K169 on BAM mineralization disappeared, and none of the two strains showed growth in sand devoid from organic carbon. These findings highlight the feasibility of K169-assisted bioaugmentation with MSH1 under realistic field conditions, as it requires no supplementary organic carbon and remains effective, even at relatively low inoculum densities, thereby addressing key challenges in bioaugmentation strategies.IMPORTANCEBioaugmentation of sand filters exploited in drinking water treatment, with the BAM catabolic strain Aminobacter niigataensis MSH1, has previously been successful during the first 1-2 weeks, where after BAM degradation deteriorated together with the loss of MSH1 cell density and cell activity. Bacterial isolates obtained from sand filters can support BAM degradation activity by MSH1 involving mutualistic interactions which resulted in the proposition of a novel bioaugmentation approach involving the co-inoculation of "support" bacteria that are adapted to the target environment. This paper focuses on understanding the boundary conditions required for sustaining the mutualistic interaction between MSH1 and such a "supportive" sand filter isolate in sand microcosm, showing that the interaction could be maintained when using relatively low cell densities and with no additional carbon supplemented. To the best of our knowledge, this paper is the first study to examine the boundary conditions of a bacterial mutualistic interaction, particularly in a bioaugmentation context of water treatment.

Microplastic pollution in aquatic environments is a growing global concern. Microplastics, defined as plastic fragments smaller than 5 mm, accumulate in freshwater reservoirs, especially in urban areas, impacting resident biota. This study examined the effects of microplastics (MP) on the performance and microbiome of Daphnia, a keystone organism in freshwater ecosystems, through both in situ sampling of freshwater ponds and a controlled 23-day in vitro exposure experiment. Using bacterial 16S ribosomal RNA gene amplicon sequencing and whole-genome shotgun sequencing, we analyzed the microbiome's composition and functional capacity in relation to microplastic pollution levels. Urban ponds contained higher microplastic concentrations in water and sediment than natural ponds, with distinct differences in plastic composition. Bacterioplankton communities, defined as bacterial assemblages in the water column, were more diverse and richer than Daphnia-associated microbiomes. Overall, the in situ study showed that the composition of the Daphnia-associated community was influenced by many factors including microplastic levels but also temperature and redox potential. Functional analysis showed increased relative abundances of polyethylene terephthalate degradation enzymes and antibiotic resistance genes in microbiomes from high-microplastic ponds. In the in vitro experiment, the bacterioplankton inoculum source significantly influenced Daphnia survival and microbiome composition. Network analysis identified specific taxa associated with MP within the Daphnia microbiome. Our findings highlight that urbanization leads to higher microplastic and antibiotic resistance gene burdens, influencing host-associated microbiomes through taxonomic shifts, functional enrichment, and survival outcomes, with potential implications for the resilience of aquatic ecosystems.

Insertion sequences (IS) are drivers of bacterial diversification by facilitating recruitment and horizontal transfer of adaptive genes involving composite transposon structures, but their evolutionary role at the community level is rarely addressed. This study explores the dynamics of IS1071 and the cargo of IS1071-associated putative composite transposons in the establishment of a pesticide-degrading microbiome in an on-farm biopurification system (BPS)-which treats pesticide-contaminated wastewater and is considered a hotspot of microbial evolution-during the crucial start-up phase. Pesticide mineralisation assays and quantitative PCR targeting pesticide catabolic genes showed that the microbial community, upon feeding on the pesticide-contaminated wastewater, rapidly evolved into a pesticide-degrading microbiome. Concomitantly, an increase in the relative abundances of several mobile genetic elements, including IS1071, was observed, as well as a striking enrichment of xenobiotic catabolic genes in the cargo of putative IS1071-flanked composite transposons. The IS1071 cargo catabolic genes diversified over time and were mainly of Betaproteobacterial origin. Clear changes in community composition were observed both in the total bacterial community and the Betaproteobacterial community. We conclude that IS1071 supports the rapid establishment of pesticide catabolism in the BPS microbiome, highlighting the contribution of IS elements to microbial community adaptation to environmental changes.

Bioaugmentation of sand filters is an alternative process for eliminating organic micropollutants in drinking water treatment. Bioaugmentation resembles an invasion process and niche availability is a prime determinant for successful invasion. This is particularly relevant for bioaugmentation of oligotrophic environments where organic micropollutants (OMPs) hardly provide a selective C-source and exploitative competition for the scarce intrinsic organic carbon exists between inoculated OMP-degraders and resident microbiota. Building on microbial invasion theories, we tested the hypothesis that the success of bioaugmentation and associated OMP degradation can be enhanced through niche creation by supplying a selective carbon source for the introduced degrader. Sand filter microbiota reduced growth of the 2,6-dichlorobenzamide degrading strain Aminobacter niigataensis MSH1 and 2,6-dichlorobenzamide degradation in different natural waters. This was counteracted by adding benzamide as a selective C-source for MSH1 resulting in a 3-fold faster 2,6-dichlorobenzamide biodegradation and a 6-fold increase in MSH1 growth. An additive biokinetic model underpredicted growth of MSH1 in the presence of sand filter microbiota suggesting that the community, despite its overall negative effect, supported MSH1 growth. Moreover, benzamide retarded 2,6-dichlorobenzamide degradation likely due to enzyme competitive inhibition. The results demonstrate the use of deliberately creating dedicated niches selective for the inoculum and the successful translation of ecological invasion theories into microbial community management, for improved bioaugmentation of complex communities.

The negative diversity-invasion relationship observed in microbial invasion studies is commonly explained by competition between the invader and resident populations. However, whether this relationship is affected by invader-resident cooperative interactions is unknown. Using ecological and mathematical approaches, we examined the survival and functionality of Aminobacter niigataensis MSH1 to mineralize 2,6-dichlorobenzamide (BAM), a groundwater micropollutant affecting drinking water production, in sand microcosms when inoculated together with synthetic assemblies of resident bacteria. The assemblies varied in richness and in strains that interacted pairwise with MSH1, including cooperative and competitive interactions. While overall, the negative diversity-invasion relationship was retained, residents engaging in cooperative interactions with the invader had a positive impact on MSH1 survival and functionality, highlighting the dependency of invasion success on community composition. No correlation existed between community richness and the delay in BAM mineralization by MSH1. The findings suggest that the presence of cooperative residents can alleviate the negative diversity-invasion relationship. This paper explores whether pair-wise interactions between an invader and resident bacteria affect the commonly observed negative diversity-invasion relationship in microbial invasion studies. To this end, using ecological and mathematical approaches, we examined the survival and functionality of Aminobacter niigataensis MSH1 to mineralize 2,6-dichlorobenzamide (BAM), a groundwater micropollutant affecting drinking water production, in sand microcosms when inoculated together with synthetic assemblies of resident bacteria with known pair-wise interactions with MSH1. While overall, the negative diversity-invasion relationship was retained, residents engaging in cooperative interactions with the invader had a positive impact on MSH1 survival and functionality. Our findings highlight the dependency of invasion success on community composition and suggest that the presence of cooperative residents can alleviate the negative diversity-invasion relationship.image

This study aimed for a better understanding of the niche specification of bacteria carrying the tfd-genes for aerobic 2,4-dichlorphenoxyacetic acid (2,4-D) degradation in the rice paddy ecosystem. To achieve this, a dedicated microcosm experiment was set up to mimic the rice paddy system, with and without 2,4-D addition, allowing spatial sampling of the different rice paddy compartments and niches, i.e., the main anaerobic bulk soil and the aerobic surface water, surface soil, root surface and rhizosphere compartments. No effect of 2,4-D on the growth and morphology of the rice plant was noted. 2,4-D removal was faster in the upper soil layers compared to the deeper layers and was more rapid after the second 2,4-D addition compared to the first. Moreover, higher relative abundances of the 2,4-D catabolic gene tfdA and of the mobile genetic elements IncP-1 and IS1071 reported to carry the tfd-genes, were observed in surface water and surface soil when 2,4-D was added. tfdA was also detected in the root surface and rhizosphere compartment but without response to 2,4-D addition. While analysis of the bacterial community composition using high-throughput 16S rRNA gene amplicon sequencing did not reveal expected tfd-carrying taxa, subtle community changes linked with 2,4-D treatment and the presence of the plant were observed. These findings suggest (i) that the surface soil and surface water are the primary and most favorable compartements/niches for tfd-mediated aerobic 2,4-D biodegradation and (ii) that the community structure in the 2,4-D treated rice paddy ecosystem is determined by a niche-dependent complex interplay between the effects of the plant and of 2,4-D.

2,6-Dichlorobenzamide (BAM) is an omnipresent micropollutant in European groundwaters. Aminobacter niigataensis MSH1 is a prime candidate for biologically treating BAM-contaminated groundwater since this organism is capable of utilizing BAM as a carbon and energy source. However, detailed information on the BAM degradation kinetics by MSH1 at trace concentrations is lacking, while this knowledge is required for predicting and optimizing the degradation process. Contaminating assimilable organic carbon (AOC) in media makes the biodegradation experiment a mixed-substrate assay and hampers exploration of pollutant degradation at trace concentrations. In this study, we examined how the BAM concentration affects MSH1 growth and BAM substrate utilization kinetics in a AOC-restricted background to avoid mixed-substrate conditions. Conventional Monod kinetic models were unable to predict kinetic parameters at low concentrations from kinetics determined at high concentrations. Growth yields on BAM were concentration-dependent and decreased substantially at trace concentrations; i.e., growth of MSH1 diminished until undetectable levels at BAM concentrations below 217 μg-C/L. Nevertheless, BAM degradation continued. Decreasing growth yields at lower BAM concentrations might relate to physiological adaptations to low substrate availability or decreased expression of downstream steps of the BAM catabolic pathway beyond 2,6-dichlorobenzoic acid (2,6-DCBA) that ultimately leads to Krebs cycle intermediates for growth and energy conservation.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the environment pose serious threats to environmental security and public health. There is an urgent need for methods to specifically and effectively control environmental pollution or pathogen infection associated with ARGs and ARB. This review aims to provide an overview of methods abating the prevalence and spread of ARGs and ARB from species to community level. At the species level, species-specific technologies, such as nanoparticle-, photosensitizer-, CRISPR-Cas-, and phage-related technology can be utilized to clear a particular class of ARGs or ARB, and in combination with low-dose antibiotics, a higher removal efficiency can be achieved. Moreover, the combination of antibiotics can be used to reverse microbial resistance and treat recurrent antibiotic resistant pathogen infections. At the community level, community-specific strategies, such as biochar, hyperthermophilic compost, and fecal microbiota transplantation can eradicate most types of ARGs or ARB in one shot, reducing the probability of resistance development. Though some progress has been made to eliminate ARGs and ARB in disease treatment or decontamination scenarios, further research is still needed to elucidate their mechanisms of action and scopes of application, and efforts should be made to explore novel strategies to counter the prevalence of antibiotic resistance.

Strain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae, being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae. Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LMG 32615T=NCCB 100883T).

A thermophilic chain elongating bacterium, strain MDTJ8, was isolated from a thermophilic acidogenic anaerobic digestor producing n-caproate from human waste and grows optimally at 55°C and pH 6.5. 16S rRNA gene analysis suggests MDTJ8 represents a new species/genus within a group of recently identified mesophilic chain elongating bacteria of the Oscillospiraceae family. Functional genome analysis showed that strain MDTJ8 contains homologues of genes encoding for chain elongation and energy conservation but also indicates the production of n-caproate from carbohydrates including polymeric substances. This was confirmed by culturing experiments in which MDTJ8 converted at 55°C mono-, di- and polymeric carbohydrates (starch and hemicellulose) to n-caproate, n-butyrate, acetate, formate and lactate. MDTJ8 is the first axenic organism that thermophilically performs chain elongation. Its availability as an axenic culturable strain opens doors to understand and intensify thermophilic bioprocesses targeting the utilization of organic waste towards the production of the high-value platform chemical n-caproate.

Bioaugmentation often involves an invasion process requiring the establishment and activity of a foreign microbe in the resident community of the target environment. Interactions with resident micro-organisms, either antagonistic or cooperative, are believed to impact invasion. However, few studies have examined the variability of interactions between an invader and resident species of its target environment, and none of them considered a bioremediation context. Aminobacter sp. MSH1 mineralizing the groundwater micropollutant 2,6-dichlorobenzamide (BAM), is proposed for bioaugmentation of sand filters used in drinking water production to avert BAM contamination. We examined the nature of the interactions between MSH1 and 13 sand filter resident bacteria in dual and triple species assemblies in sand microcosms. The residents affected MSH1-mediated BAM mineralization without always impacting MSH1 cell densities, indicating effects on cell physiology rather than on cell number. Exploitative competition explained most of the effects (70%), but indications of interference competition were also found. Two residents improved BAM mineralization in dual species assemblies, apparently in a mutual cooperation, and overruled negative effects by others in triple species systems. The results suggest that sand filter communities contain species that increase MSH1 fitness. This opens doors for assisting bioaugmentation through co-inoculation with "helper" bacteria originating from and adapted to the target environment.

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC, a main insertion site of adaptive trait determinants, is amplified and its content analyzed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, genes with catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long-range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main obvious selective trait imposed on that community. Instead, these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

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 frequent exposure of agricultural soils to pesticides often leads to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify the in situ linuron degrading bacteria. DNA-SIP unambiguously linked Variovorax and its linuron catabolic genes to linuron dissipation, likely through synergistic cooperation between two species. Additionally, two linuron mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as the in situ degrader of linuron in the studied agricultural field and corroborate the genus as key in the maintenance of a robust genetic memory regarding linuron degradation functionality in the examined field.

Lipids are one of the main organic components in industrial and municipal wastewaters. Lipid hydrolysis is the first step in the biological conversion process and requires a close contact between lipid emulsion droplets and microbial hydrolytic enzymes. Adequate lipid hydrolysis monitoring is crucial to obtain mechanistic knowledge on lipid hydrolysis in response to changes in the process conditions and to improve the overall lipid conversion efficiency in aerobic and anaerobic wastewater treatment systems. We set out to develop a high-throughput lipid hydrolysis monitoring method based on vegetable oil model substrates and fluorescent quantification of product formation by exploiting the interaction with Rhodamine B. Olive oil and soybean oil emulsions were prepared with a high interfacial area and acceptable emulsion stability. The method was easy to apply and allowed to obtain detailed kinetic data over a time course of several hours for up to 16 samples in parallel. A proof-of-concept was obtained with a commercial enzyme, Amano lipase, but remains to be provided for wastewater treatment sludge samples. The findings of this study pave the way for further method development in lipid hydrolysis monitoring.

The formation of estrogenic intermediates, i.e. nonylphenol diethoxylate (NP2EO), nonylphenol monoethoxylate (NP1EO), and nonylphenol (NP), following nonylphenol ethoxylates (NPEOs) biodegradation in textile wastewater raises concerns about its endocrine disruptive activity, but the estrogenicity changes of textile wastewater throughout biological treatment processes remain unknown. In the present study, the estrogenicity of textile wastewater sampled from 10 wastewater treatment plants (WWTPs) were investigated using the reporter gene-based T47D-KBluc bioassay. Results showed that the estrogenicity of the textile wastewater significantly increased after either anaerobic or aerobic treatment in all WWTPs, with an average fold change of 3.21, although traditional pollutants were effectively removed. The estradiol equivalents of the effluent (ranging from 1.50 to 4.12 ng-E2/L) were generally higher than published effect based trigger values, indicating an increased risk for the receiving waters. Removal efficiency was high (84.46%) for NPEOs, but was low for NP2EO and NP1EO in the biological treatment processes. Nevertheless, NP had increased concentrations after the treatment. Bioanalytical equivalent concentration of the textile wastewater and that of NP2EO, NP1EO, and NP showed a good linear correlation, of which NP alone contributed more than 70% to the observed estrogenicity. Extending hydraulic retention time was found effective in reducing the estrogenicity as it allows relatively complete degradation of NP, which was further confirmed by running lab-scale A/O reactors fed with NP10EO. The results may extend our knowledge regarding the estrogenicity of textile wastewater and its reduction technologies used in WWTPs.