Influence of combined abiotic/biotic factors on decay of P. aeruginosa and E. coli in Rhine River water.

Understanding the dynamic change in abundance of both fecal and opportunistic waterborne pathogens in urban surface water under different abiotic and biotic factors helps the prediction of microbiological water quality and protection of public health during recreational activities, such as swimming. However, a comprehensive understanding of the interaction among various factors on pathogen behavior in surface water is missing. In this study, the effect of salinity, light, and temperature and the presence of indigenous microbiota, on the decay/persistence of Escherichia coli and Pseudomonas aeruginosa in Rhine River water were tested during 7 days of incubation with varying salinity (0.4, 5.4, 9.4, and 15.4 ppt), with light under a light/dark regime (light/dark) and without light (dark), temperature (3, 12, and 20 °C), and presence/absence of indigenous microbiota. The results demonstrated that light, indigenous microbiota, and temperature significantly impacted the decay of E. coli. Moreover, a significant (p<0.01) four-factor interactive impact of these four environmental conditions on E. coli decay was observed. However, for P. aeruginosa, temperature and indigenous microbiota were two determinate factors on the decay or growth. A significant three-factor interactive impact between indigenous microbiota, temperature, and salinity (p<0.01); indigenous microbiota, light, and temperature (p<0.01); and light, temperature, and salinity (p<0.05) on the decay of P. aeruginosa was found. Due to these interactive effects, caution should be taken when predicting decay/persistence of E. coli and P. aeruginosa in surface water based on a single environmental condition. In addition, the different response of E. coli and P. aeruginosa to the environmental conditions highlights that E. coli monitoring alone underestimates health risks of surface water by non-fecal opportunistic pathogens, such as P. aeruginosa. KEY POINTS: Abiotic and biotic factors interactively affect decay of E. coli and P. aeruginosa E.coli and P.aeruginosa behave significantly different under the given conditions Only E. coli as an indicator underestimates the microbiological water quality.

Identifying Eukaryotes and Factors Influencing Their Biogeography in Drinking Water Metagenomes.

The biogeography of eukaryotes in drinking water systems is poorly understood relative to that of prokaryotes or viruses, limiting the understanding of their role and management. A challenge with studying complex eukaryotic communities is that metagenomic analysis workflows are currently not as mature as those that focus on prokaryotes or viruses. In this study, we benchmarked different strategies to recover eukaryotic sequences and genomes from metagenomic data and applied the best-performing workflow to explore the factors affecting the relative abundance and diversity of eukaryotic communities in drinking water distribution systems (DWDSs). We developed an ensemble approach exploiting k-mer- and reference-based strategies to improve eukaryotic sequence identification and identified MetaBAT2 as the best-performing binning approach for their clustering. Applying this workflow to the DWDS metagenomes showed that eukaryotic sequences typically constituted small proportions (i.e., <1%) of the overall metagenomic data with higher relative abundances in surface water-fed or chlorinated systems with high residuals. The α and ß diversities of eukaryotes were correlated with those of prokaryotic and viral communities, highlighting the common role of environmental/management factors. Finally, a co-occurrence analysis highlighted clusters of eukaryotes whose members' presence and abundance in DWDSs were affected by disinfection strategies, climate conditions, and source water types.

Aeromonas species from non-chlorinated distribution systems and their competitive planktonic growth in drinking water.

Aeromonas is included in the Dutch Drinking Water Decree as an indicator for elevated microbial regrowth in non-chlorinated drinking water distribution systems (DWDS). The temporal and spatial diversity of Aeromonas species in ten DWDS and their planktonic growth characteristics for different carbon sources was investigated. Genotyping of the gyrB gene of isolates showed a non-systematic temporal and spatial variable prevalence of seven different Aeromonas species in these DWDS and no correlation with AOC-P17/NOX and Aeromonas concentrations. Pure cultures of these seven species showed a high affinity to low concentrations (µg/L) of individual amino acids and fatty acids, compounds associated with biomass. Growth occurred at 0.5 µg-C/L of an amino acid mixture. Growth of a mixed community of A. rivuli, A. salmonicida, A. sobria and A. veronii in drinking water occurred in pasteurized samples, however, no growth and decay occurred in competition with the autochthonous bacteria (non-pasteurized samples). This community also failed to grow in non-pasteurized distribution samples from a location with clear increase in planktonic Aeromonas concentrations in the transported drinking water. For competitive planktonic growth of Aeromonas an amino acid concentration of ≥5 µg-C/L is required. AOC-P17/NOX concentrations showed that such concentrations are not expected in Dutch drinking water. Therefore, we suspect that competitive planktonic growth is not the major cause of the observed non-compliance with the Aeromonas standard in non-chlorinated DWSD.Importance The occurrence of the bacterial genus Aeromonas in non-chlorinated drinking water in the Netherlands is regarded as an indication for elevated microbial regrowth in the distribution system. Identification of the prevalent species in ten distribution systems by genotyping yielded seven different species, with A. rivuli, A. veronii and A. sobria as the most dominant ones. Planktonic growth experiments of pure cultures confirmed former published affinity of Aeromonas for certain biomass compounds (amino and fatty acids). In competition with the autochthonous microflora, however, planktonic growth was not observed, only after addition of a threshold amino acid concentration of 5 µg-C/L. Based on our results and further observations we deduced that planktonic growth of Aeromonas in the DWDS is not very likely. Benthic growth in loose deposits and planktonic release is a more plausible explanation for the observed planktonic increase of Aeromonas.

Effects of cold recovery technology on the microbial drinking water quality in unchlorinated distribution systems.

Drinking water distribution systems (DWDSs) are used to supply hygienically safe and biologically stable water for human consumption. The potential of thermal energy recovery from drinking water has been explored recently to provide cooling for buildings. Yet, the effects of increased water temperature induced by this "cold recovery" on the water quality in DWDSs are not known. The objective of this study was to investigate the impact of cold recovery from DWDSs on the microbiological quality of drinking water. For this purpose, three pilot distribution systems were operated in parallel for 38 weeks. System 1 has an operational heat exchanger, mimicking the cold recovery system by maintaining the water temperature at 25 °C; system 2 operated with a non-operational heat exchanger and system 3 run without heat exchanger. The results showed no significant effects on drinking water quality: cell numbers and ATP concentrations remained around 3.5 × 105 cells/ml and 4 ng ATP/l, comparable observed operational taxonomic units (OTUs) (~470-490) and similar Shannon indices (7.7-8.9). In the system with cold recovery, a higher relative abundance of Pseudomonas spp. and Chryseobacterium spp. was observed in the drinking water microbial community, but only when the cold recovery induced temperature difference (ΔT) was higher than 9 °C. In the 38 weeks' old biofilm, higher ATP concentration (475 vs. 89 pg/cm2), lower diversity (observed OTUs: 88 vs. ≥200) and a different bacterial community composition (e.g. higher relative abundance of Novosphingobium spp.) were detected, which did not influence water quality. No impacts were observed for the selected opportunisitic pathogens after introducing cold recovery. It is concluded that cold recovery does not affect bacterial water quality. Further investigation for a longer period is commended to understand the dynamic responses of biofilm to the increased temperature caused by cold recovery.

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms.

UNLABELLED: Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm(-2); BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. IMPORTANCE: The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely.

Microbial biogeography of drinking water: patterns in phylogenetic diversity across space and time.

In this study, we collected water from different locations in 32 drinking water distribution networks in the Netherlands and analysed the spatial and temporal variation in microbial community composition by high-throughput sequencing of 16S rRNA gene amplicons. We observed that microbial community compositions of raw source and processed water were very different for each distribution network sampled. In each network, major differences in community compositions were observed between raw and processed water, although community structures of processed water did not differ substantially from end-point tap water. End-point water samples within the same distribution network revealed very similar community structures. Network-specific communities were shown to be surprisingly stable in time. Biofilm communities sampled from domestic water metres varied distinctly between households and showed no resemblance to planktonic communities within the same distribution networks. Our findings demonstrate that high-throughput sequencing provides a powerful and sensitive tool to probe microbial community composition in drinking water distribution systems. Furthermore, this approach can be used to quantitatively compare the microbial communities to match end-point water samples to specific distribution networks. Insight in the ecology of drinking water distribution systems will facilitate the development of effective control strategies that will ensure safe and high-quality drinking water.

Polysaccharides and proteins added to flowing drinking water at microgram-per-liter levels promote the formation of biofilms predominated by bacteroidetes and proteobacteria.

Biopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 µg C liter(-1) in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 µg C liter(-1) per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm(-2) day(-1)), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm(-2) day(-1)). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells. Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew during polysaccharide addition, and Alpha-, Beta-, and Gammaproteobacteria, Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might impair the biological stability of distributed drinking water.

Bioremediation of rapid sand filters for removal of organic micropollutants during drinking water production.

Rapid sand filtration (RSF) is used during drinking water production for removal of particles, possible harmful microorganisms, organic material and inorganic compounds such as iron, manganese, ammonium and methane. However, RSF can also be used for removal of certain organic micropollutants (OMPs). In this study, it was investigated if OMP removal in columns packed with sand from full scale RSFs could be stimulated by bioaugmentation (i.e. inoculating RSFs with sand from another RSF) and/or biostimulation (i.e. addition of nutrients, vitamins and trace-elements that stimulate microbial growth). The results showed that removal of PFOA, carbamazepine, 1-H benzotriazole, amidotrizoate and iopamidol in the columns was low (< 20 %). Propranolol and diclofenac removal was higher (50-60 %) and propranolol removal likely occurred via sorption processes, whereas for diclofenac it was unclear if removal was a combination of physical-chemical and biological processes. Moreover, bioaugmentation and biostimulation resulted in 99 % removal of gabapentin and metoprolol after 38 days and 99 % removal of acesulfame after 52 days of incubation. The bioaugmented column without biostimulation showed 99 % removal for gabapentin and metoprolol after 52 days, and for acesulfame after 80 days. In contrast, the non-bioaugmented column did not remove gabapentin, removed < 40 % metoprolol and showed 99 % removal of acesulfame only after 80 days of incubation. Removal of these OMPs was negatively correlated with ammonium oxidation and the absolute abundance of ammonia-oxidizing bacteria. 16S rRNA gene sequencing showed that OMP removal of acesulfame, gabapentin and metoprolol was positively correlated to the relative abundance of specific bacterial genera that harbor species with a heterotrophic and aerobic or denitrifying metabolism. These results show that bioaugmentation of RSF can be successful for OMP removal, where biostimulation can accelerate this removal.

Nontuberculous mycobacteria, fungi, and opportunistic pathogens in unchlorinated drinking water in The Netherlands.

The multiplication of opportunistic pathogens in drinking water supplies might pose a threat to public health. In this study, distributed unchlorinated drinking water from eight treatment plants in the Netherlands was sampled and analyzed for fungi, nontuberculous mycobacteria (NTM), and several opportunistic pathogens by using selective quantitative PCR methods. Fungi and NTM were detected in all drinking water samples, whereas Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Aspergillus fumigatus were sporadically observed. Mycobacterium avium complex and Acanthamoeba spp. were not detected. Season had no influence on the occurrence of these organisms, except for NTM and S. maltophilia, which were present in higher numbers in the summer. Opportunistic pathogens were more often observed in premise plumbing water samples than in samples from the distribution system. The lowest number of these organisms was observed in the finished water at the plant. Thus, fungi, NTM, and some of the studied opportunistic pathogens can multiply in the distribution and premise plumbing systems. Assimilable organic carbon (AOC) and/or total organic carbon (TOC) had no clear effects on fungal and NTM numbers or on P. aeruginosa- and S. maltophilia-positive samples. However, L. pneumophila was detected more often in water with AOC concentrations above 10 µg C liter(-1) than in water with AOC levels below 5 µg C liter(-1). Finally, samples that contained L. pneumophila, P. aeruginosa, or S. maltophilia were more frequently positive for a second opportunistic pathogen, which shows that certain drinking water types and/or sampling locations promote the growth of multiple opportunistic pathogens.

Pyrosequence analysis of the hsp65 genes of nontuberculous mycobacterium communities in unchlorinated drinking water in the Netherlands.

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.

Impacts of shallow geothermal energy production on redox processes and microbial communities.

Shallow geothermal systems are increasingly being used to store or harvest thermal energy for heating or cooling purposes. This technology causes temperature perturbations exceeding the natural variations in aquifers, which may impact groundwater quality. Here, we report the results of laboratory experiments on the effect of temperature variations (5-80 °C) on redox processes and associated microbial communities in anoxic unconsolidated subsurface sediments. Both hydrochemical and microbiological data showed that a temperature increase from 11 °C (in situ) to 25 °C caused a shift from iron-reducing to sulfate-reducing and methanogenic conditions. Bioenergetic calculations could explain this shift. A further temperature increase (>45 °C) resulted in the emergence of a thermophilic microbial community specialized in fermentation and sulfate reduction. Two distinct maxima in sulfate reduction rates, of similar orders of magnitude (5 × 10(-10) M s(-1)), were observed at 40 and 70 °C. Thermophilic sulfate reduction, however, had a higher activation energy (100-160 kJ mol(-1)) than mesophilic sulfate reduction (30-60 kJ mol(-1)), which might be due to a trade-off between enzyme stability and activity with thermostable enzymes being less efficient catalysts that require higher activation energies. These results reveal that while sulfate-reducing functionality can withstand a substantial temperature rise, other key biochemical processes appear more temperature sensitive.

Initiating guidance values for novel biological stability parameters in drinking water to control regrowth in the distribution system.

Nine novel biological stability parameters for drinking water have been developed recently. Here, we report data for these nine parameters in treated water from 34 treatment plants in the Netherlands to deduce guidance values for these parameters. Most parameters did not show a strong correlation with another biological stability parameter in the same sample, demonstrating that most parameters hold different information on the biological stability of drinking water. Furthermore, the novel biological stability parameters in treated water varied considerably between plants and five parameters in treated water were significantly lower for drinking water produced from groundwater than surface water. The maximum biomass concentration (MBC7), cumulative biomass potential (CBP14) from the biomass production potential test (BPP-W) and the total organic carbon concentration in treated water from groundwater were predictive parameters for HPC22 and Aeromonas regrowth in the distribution system. Guidance values of 8.6 ng ATP L-1, 110 d·ng ATP L-1 and 4.1 mg C L-1 were deduced for these parameters, under which the HPC22 and Aeromonas numbers remain at regulatory level. The maximum biomass growth (MBG7) from the BPP-W test, the particulate and/or high molecular organic carbon and the iron accumulation rate in treated water from surface water were predictive parameters for HPC22 and Aeromonas regrowth in the distribution system. Deduced guidance values for these biological stability parameters were 4.5 ng ATP L-1, 47 µg C L-1 and 0.34 mg Fe m-2 day-1, respectively. We conclude from our study that a multiple parameter assessment is required to reliable describe the biological stability of drinking water, that the biological stability of drinking water produced from groundwater is described with other parameters than the biological stability of drinking water produced from surface water, and that guidance values for predictive biological stability parameters were inferred under which HPC22 and Aeromonas regrowth is under control.

Influence of Temperature on Growth of Four Different Opportunistic Pathogens in Drinking Water Biofilms.

High drinking water temperatures occur due to climate change and could enhance the growth of opportunistic pathogens in drinking water systems. We investigated the influence of drinking water temperatures on the growth of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Mycobacterium kansasii and Aspergillus fumigatus in drinking water biofilms with an autochthonous microflora. Our results reveal that the growth of P. aeruginosa and S. maltophilia in the biofilm already occurred at 15.0 °C, whereas M. kansasii and A. fumigatus were able to grow when temperatures were above 20.0 °C and 25.0 °C, respectively. Moreover, the maximum growth yield of P. aeruginosa, M. kansasii and A. fumigatus increased with increasing temperatures up to 30 °C, whereas an effect of temperature on the yield of S. maltophilia could not be established. In contrast, the maximum ATP concentration of the biofilm decreased with increasing temperatures. We conclude from these results that high drinking water temperatures caused by, e.g., climate change can result in high numbers of P. aeruginosa, M. kansasii and A. fumigatus in drinking water systems, which poses a possible risk to public health. Consequently, it is recommended for countries with a more moderate climate to use or maintain a drinking water maximum standard temperature of 25 °C.

Influence of filter age on Fe, Mn and NH4+ removal in dual media rapid sand filters used for drinking water production.

Rapid sand filtration is a common method for removal of iron (Fe), manganese (Mn) and ammonium (NH4+) from anoxic groundwaters used for drinking water production. In this study, we combine geochemical and microbiological data to assess how filter age influences Fe, Mn and NH4+ removal in dual media filters, consisting of anthracite overlying quartz sand, that have been in operation for between ∼2 months and ∼11 years. We show that the depth where dissolved Fe and Mn removal occurs is reflected in the filter medium coatings, with ferrihydrite forming in the anthracite in the top of the filters (< 1 m), while birnessite-type Mn oxides are mostly formed in the sand (> 1 m). Removal of NH4+ occurs through nitrification in both the anthracite and sand and is the key driver of oxygen loss. Removal of Fe is independent of filter age and is always efficient (> 97% removal). In contrast, for Mn, the removal efficiency varies with filter age, ranging from 9 to 28% at ∼2-3 months after filter replacement to 100% after 8 months. After 11 years, removal reduces to 60-80%. The lack of Mn removal in the youngest filters (at 2-3 months) is likely the result of a relatively low abundance of mineral coatings that adsorb Mn2+ and provide surfaces for the establishment of a microbial community. 16S rRNA gene amplicon sequencing shows that Gallionella, which are known Fe2+ oxidizers, are present after 2 months, yet Fe2+ removal is mostly chemical. Efficient NH4+ removal (> 90%) establishes within 3 months of operation but leakage occurs upon high NH4+loading (> 160 µM). Two-step nitrification by Nitrosomonas and Candidatus Nitrotoga is likely the most important NH4+ removal mechanism in younger filters during ripening (2 months), after which complete ammonia oxidation by Nitrospira and canonical two-step nitrification occur simultaneously in older filters. Our results highlight the strong effect of filter age on especially Mn2+but also NH4+ removal. We show that ageing of filter medium leads to the development of thick coatings, which we hypothesize leads to preferential flow, and breakthrough of Mn2+. Use of age-specific flow rates may increase the contact time with the filter medium in older filters and improve Mn2+ and NH4+ removal.

Stratified prokaryote network in the oxic-anoxic transition of a deep-sea halocline.

The chemical composition of the Bannock basin has been studied in some detail. We recently showed that unusual microbial populations, including a new division of Archaea (MSBL1), inhabit the NaCl-rich hypersaline brine. High salinities tend to reduce biodiversity, but when brines come into contact with fresher water the natural haloclines formed frequently contain gradients of other chemicals, including permutations of electron donors and acceptors, that may enhance microbial diversity, activity and biogeochemical cycling. Here we report a 2.5-m-thick chemocline with a steep NaCl gradient at 3.3 km within the water column betweeen Bannock anoxic hypersaline brine and overlying sea water. The chemocline supports some of the most biomass-rich and active microbial communities in the deep sea, dominated by Bacteria rather than Archaea, and including four major new divisions of Bacteria. Significantly higher metabolic activities were measured in the chemocline than in the overlying sea water and underlying brine; functional analyses indicate that a range of biological processes is likely to occur in the chemocline. Many prokaryotic taxa, including the phylogenetically new groups, were confined to defined salinities, and collectively formed a diverse, sharply stratified, deep-sea ecosystem with sufficient biomass to potentially contribute to organic geological deposits.

Sulfur cycling and methanogenesis primarily drive microbial colonization of the highly sulfidic Urania deep hypersaline basin.

Urania basin in the deep Mediterranean Sea houses a lake that is >100 m deep, devoid of oxygen, 6 times more saline than seawater, and has very high levels of methane and particularly sulfide (up to 16 mM), making it among the most sulfidic water bodies on Earth. Along the depth profile there are 2 chemoclines, a steep one with the overlying oxic seawater, and another between anoxic brines of different density, where gradients of salinity, electron donors and acceptors occur. To identify and differentiate the microbes and processes contributing to the turnover of organic matter and sulfide along the water column, these chemoclines were sampled at a high resolution. Bacterial cell numbers increased up to a hundredfold in the chemoclines as a consequence of elevated nutrient availability, with higher numbers in the upper interface where redox gradient was steeper. Bacterial and archaeal communities, analyzed by DNA fingerprinting, 16S rRNA gene libraries, activity measurements, and cultivation, were highly stratified and metabolically more active along the chemoclines compared with seawater or the uniformly hypersaline brines. Detailed analysis of 16S rRNA gene sequences revealed that in both chemoclines delta- and epsilon-Proteobacteria, predominantly sulfate reducers and sulfur oxidizers, respectively, were the dominant bacteria. In the deepest layers of the basin MSBL1, putatively responsible for methanogenesis, dominated among archaea. The data suggest that the complex microbial community is adapted to the basin's extreme chemistry, and the elevated biomass is driven largely by sulfur cycling and methanogenesis.

Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments.

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at µg liter(-1) levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 µg C liter(-1) in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10(-2) liter·µg(-1) of C·h(-1)) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10(-2) liter·µg(-1) of C·h(-1)). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell(-1) level was achieved at ≤10 µg C liter(-1) than at >10 µg C liter(-1) with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at µg liter(-1) levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.

Riverine microplastic and microbial community compositions: A field study in the Netherlands.

Plastic pollution in aquatic environments, particularly microplastics (<5 mm), is an emerging health threat. The buoyancy, hydrophobic hard surfaces, novel polymer carbon sources and long-distance transport make microplastics a unique substrate for biofilms, potentially harbouring pathogens and enabling antimicrobial resistance (AMR) gene exchange. Microplastic concentrations, their polymer types and the associated microbial communities were determined in paired, contemporaneous samples from the Dutch portion of the river Rhine. Microplastics were collected through a cascade of 500/100/10 µm sieves; filtrates and surface water were also analysed. Microplastics were characterized with infrared spectroscopy. Microbial communities and selected virulence and AMR genes were determined with 16S rRNA-sequencing and qPCR. Average microplastic concentration was 213,147 particles/m3; polyamide and polyvinylchloride were the most abundant polymers. Microbial composition on 100-500 µm samples differed significantly from surface water and 10-100 µm or smaller samples, with lower microbial diversity compared to surface water. An increasingly 'water-like' microbial community was observed as particles became smaller. Associations amongst specific microbial taxa, polymer types and particle sizes, as well as seasonal and methodological effects, were also observed. Known biofilm-forming and plastic-degrading taxa (e.g. Pseudomonas) and taxa harbouring potential pathogens (Pseudomonas, Acinetobacter, Arcobacter) were enriched in certain sample types, and other risk-conferring signatures like the sul1 and erm(B) AMR genes were almost ubiquitous. Results were generally compatible with the existence of taxon-selecting mechanisms and reduced microbial diversity in the biofilms of plastic substrates, varying over seasons, polymer types and particle sizes. This study provided updated field data and insights on microplastic pollution in a major riverine environment.

Unsuitability of quantitative Bacteroidales 16S rRNA gene assays for discerning fecal contamination of drinking water.

Bacteroidales species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for Bacteroidales 16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.

Turning Negatives into Positives for Pet Trading and Keeping: A Review of Positive Lists.

The trading and keeping of exotic pets are associated with animal welfare, conservation, environmental protection, agricultural animal health, and public health concerns and present serious regulatory challenges to legislators and enforcers. Most legislation concerning exotic pet trading and keeping involves restricting or banning problematic species, a practice known as "negative listing". However, an alternative approach adopted by some governments permits only the keeping of animals that meet certain scientifically proven criteria as suitable in respect of species, environmental, and public health and safety protections. We conducted an evaluation of positive lists for the regulation of pet trading and keeping within the context of the more prevalent system of restricting or prohibiting species via negative lists. Our examination of international, national, and regional regulations in Europe, the United States, and Canada found that criteria used for the development of both negative and positive lists were inconsistent or non-specific. Our online surveys of governments received limited responses, although telephone interviews with officials from governments either considering or developing positive lists provided useful insights into their attitudes and motivations towards adopting positive lists. We discuss key issues raised by civil servants including perceived advantages of positive lists and anticipated challenges when developing lists of suitable species. In addition, we compare functions of negative and positive lists, and recommend key principles that we hope will be helpful to governments concerning development and implementation of regulations based on positive lists.