Evaluating putative ecological drivers of microcystin spatiotemporal dynamics using metabarcoding and environmental data.

Microcystin is a cyanobacterial hepatotoxin of global concern. Understanding the environmental factors that cause high concentrations of microcystin is crucial to the development of lake management strategies that minimize harmful exposures. While the literature is replete with studies linking cyanobacterial production of microcystin to changes in various nutrients, abiotic stressors, grazers, and competitors, no single biotic or abiotic factor has been shown to be reliably predictive of microcystin concentrations in complex ecosystems. We performed random forest regression analyses with 16S and 18S rRNA gene sequencing data and environmental data to determine which putative ecological drivers best explained spatiotemporal variation in total microcystin and several individual congeners in a eutrophic freshwater reservoir. Model performance was best for predicting concentrations of the congener MC-LR, with ca. 88% of spatiotemporal variance explained. Most of the variance was associated with changes in the relative abundance of the cyanobacterial genus Microcystis. Follow-up RF regression analyses revealed that factors that were the most important in predicting MC-LR were also the most important in predicting Microcystis population dynamics. We discuss how these results relate to prevailing ecological hypotheses regarding the function of microcystin.

Draft Genome Sequences of Two Bacillus sp. Strains and Four Cellulomonas sp. Strains Isolated from Heavy-Metal-Contaminated Soil.

We present the draft genome sequence for Bacillus sp. strain PF3, Bacillus sp. strain K6W, Cellulomonas sp. strain B12, Cellulomonas sp. strain K38, Cellulomonas sp. strain K39, and Cellulomonas sp. strain K42B. These bacteria were isolated from contaminated soils, and their genomes contain genes related to chromate transport and reduction.

Bacterial community changes in copper and PEX drinking water pipeline biofilms under extra disinfection and magnetic water treatment.

AIMS: To study the stability of biofilms and water quality in pilot scale drinking water copper and PEX pipes in changing conditions (extra disinfection, magnetic water treatment, MWT). METHODS AND RESULTS: Next-generation sequencing (NGS) of 16S ribosomal RNA genes (rDNA) to describe total bacterial community and ribosomal RNA (rRNA) to describe active bacterial members in addition to traditional microbiological methods were applied. Biofilms from control copper and PEX pipes shared same most abundant bacteria (Methylobacterium spp., Sphingomonas spp., Zymomonas spp.) and average species diversities (Shannon 3·8-4·2) in rDNA and rRNA libraries, whereas few of the taxa differed by their abundance such as lower total Mycobacterium spp. occurrence in copper (<0·02%) to PEX (<0·2%) pipes. Extra disinfection (total chlorine increase from c. 0·5 to 1 mg l-1 ) affected total and active population in biofilms seen as decrease in many bacterial species and diversity (Shannon 2·7, P < 0·01, rRNA) and increase in Sphingomonas spp. as compared to control samples. Furthermore, extra-disinfected copper and PEX samples formed separate clusters in unweighted non-metric multidimensional scaling plot (rRNA) similarly to MWT-treated biofilms of copper (but not PEX) pipes that instead showed higher species diversity (Shannon 4·8, P < 0·05 interaction). CONCLUSIONS: Minor chlorine dose addition increased selection pressure and many species were sensitive to chlorination. Pipe material seemed to affect mycobacteria occurrence, and bacterial communities with MWT in copper but not in PEX pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study using rRNA showed that chlorination affects especially active fraction of bacterial communities. Copper and PEX differed by the occurrence of some bacterial members despite similar community profiles.

Inhibitory effect of cyanide on wastewater nitrification determined using SOUR and RNA-based gene-specific assays.

UNLABELLED: The effect of cyanide (CN(-) ) on nitrification was examined with samples from nitrifying bacterial enrichments using two different approaches: by measuring substrate (ammonia) specific oxygen uptake rates (SOUR), and by using RT-qPCR to quantify the transcripts of functional genes involved in nitrification. The nitrifying bioreactor was operated as a continuous reactor with a 24 h hydraulic retention time. The samples were exposed in batch vessels to cyanide for a period of 12 h. The concentrations of CN(-) used in the batch assays were 0·03, 0·06, 0·1 and 1·0 mg l(-1) . There was considerable decrease in SOUR with increasing dosages of CN(-) . A decrease of more than 50% in nitrification activity was observed at 0·1 mg l(-1) CN(-) . Based on the RT-qPCR data, there was notable reduction in the transcript levels of amoA and hao for increasing CN(-) dosage, which corresponded well with the ammonia oxidation activity measured via SOUR. The inhibitory effect of cyanide may be attributed to the affinity of cyanide to bind ferric haeme proteins, which disrupt protein structure and function. The correspondence between the relative expression of functional genes and SOUR shown in this study demonstrates the efficacy of RNA-based function-specific assays for better understanding of the effect of toxic compounds on nitrification activity in wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of cyanide on nitrifying bacteria was characterized by measuring physiological and transcriptional response. Cyanide was inhibitory to nitrification at concentrations that may be found in industrial waste. The RNA-based function-specific assays represent a mechanistic approach for better understanding the effect of toxic compounds on nitrification activity in wastewater. Moreover, the relative abundance of RNA transcripts can be used to closely track in situ nitrifying bacterial activity which can be used to predict inhibition events, thereby providing a metric to potentially improve performance of wastewater nitrifying systems.

Changes in bacterial composition of biofilm in a metropolitan drinking water distribution system.

AIMS: This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e. groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. METHODS AND RESULTS: The biofilm community was characterized using 16S rRNA gene clone libraries and functional potential analysis, generated from total DNA extracted from coupons in biofilm annular reactors fed with onsite drinking water for up to 18 months. Differences in the bacterial community structure were observed between GW and SW. Representatives that explained the dissimilarity were associated with the classes Betaproteobacteria, Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Firmicutes. After 9 months the biofilm bacterial community from both GW and SW were dominated by Mycobacterium species. The distribution of the dominant operational taxonomic unit (OTU) (Mycobacterium) positively correlated with the drinking water distribution system (DWDS) temperature. CONCLUSIONS: In this study, the biofilm community structure observed between GW and SW were dissimilar, while communities from different locations receiving SW did not show significant differences. The results suggest that source water and/or the water quality shaped by their respective treatment processes may play an important role in shaping the bacterial communities in the distribution system. In addition, several bacterial groups were present in all samples, suggesting that they are an integral part of the core microbiota of this DWDS. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide an ecological insight into biofilm bacterial structure in chlorine-treated drinking water influenced by different water sources and their respective treatment processes.

Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

AIMS: Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. METHODS AND RESULTS: The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. CONCLUSIONS: Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods.

Molecular characterization of bacteria inhabiting a water distribution system simulator.

The objective of this study was to monitor the impact of chlorination and chloramination treatments on heterotrophic bacteria (HB) and ammonia-oxidizing bacteria (AOB) inhabiting a water distribution system simulator. HB densities decreased while AOB densities increased when chloramine was added. AOB densities decreased below detection limits after the disinfection treatment was switched back to chlorination. The presence of AOB was confirmed using a group-specific 16S rDNA-PCR method. 16S rDNA sequence analysis showed that most bacterial isolates from feed water, discharge water, and biofilm samples were alpha-Proteobacteria or beta-Proteobacteria. The latter bacterial groups were also numerically dominant among the sequences recovered from water and biofilm 16S rDNA clone libraries. The relative frequency of each culturable bacterial group was different for each sample examined. Denaturing gradient gel electrophoresis analysis of total community 16S rDNA genes showed notable differences between the microbial community structure of biofilm samples and feed water. The results of this study suggest that disinfection treatments could influence the type of bacterial community inhabiting water distribution systems.

Survival of Salmonella species in river water.

The survival of four Salmonella strains in river water microcosms was monitored by culturing techniques, direct counts, whole-cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytometry. Plate counts of bacteria resuspended in filtered and untreated river water decreased several orders of magnitude within the first week of incubation, while they did not decrease as rapidly in autoclaved water. In situ hybridization studies suggested a rapid decrease in ribosomal content, as determined by the drastic decrease in the number of detectable cells after 72 h. In contrast, direct counts remained relatively constant during 45 days in all microcosoms. Although the culturable counts of two bacterial strains in filtered water after 31 days represented approximately 0.001% of the total counts, direct viable counts and resuscitation studies with a dilution series suggested that the number of viable bacteria was at least four orders of magnitude higher. Additionally, notable changes in forward scatter and in nucleic acid content were observed only after 4 h of nutrient amendments by flow cytometry. However, cells from the resuscitation experiments did not grow on solid media unless cell-free supernatant from viable cultures was added during the resuscitation period. The results in this study suggest the presence of a not immediately culturable status in Salmonella.

Microbiology of spent nuclear fuel storage basins.

Microbiological studies of spent nuclear fuel storage basins at Savannah River Site (SRS) were performed as a preliminary step to elucidate the potential for microbial-influenced corrosion (MIC) in these facilities. Total direct counts and culturable counts performed during a 2-year period indicated microbial densities of 10(4) to 10(7) cells/ml in water samples and on submerged metal coupons collected from these basins. Bacterial communities present in the basin transformed between 15% and 89% of the compounds present in Biologtrade mark plates. Additionally, the presence of several biocorrosion-relevant microbial groups (i.e., sulfate-reducing bacteria and acid-producing bacteria) was detected with commercially available test kits. Scanning electron microscopy and X-ray spectra analysis of osmium tetroxide-stained coupons demonstrated the development of microbial biofilm communities on some metal coupons submerged for 3 weeks in storage basins. After 12 months, coupons were fully covered by biofilms, with some deterioration of the coupon surface evident at the microscopical level. These results suggest that, despite the oligotrophic and radiological environment of the SRS storage basins and the active water deionization treatments commonly applied to prevent electrochemical corrosion in these facilities, these conditions do not prevent microbial colonization and survival. Such microbial densities and wide diversity of carbon source utilization reflect the ability of the microbial populations to adapt to these environments. The presumptive presence of sulfate-reducing bacteria and acid-producing bacteria and the development of biofilms on submerged coupons indicated that an environment for MIC of metal components in the storage basins may occur. However, to date, there has been no indication or evidence of MIC in the basins. Basin chemistry control and corrosion surveillance programs instituted several years ago have substantially abated all corrosion mechanisms.

Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes.

Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The Bacteroides-Prevotella probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets.

Survival and activity of Streptococcus faecalis and Escherichia coli in petroleum-contaminated tropical marine waters.

The in situ survival and activity of Streptococcus faecalis and Escherichia coli were studied using membrane diffusion chambers in tropical marine waters receiving oil refinery effluents. Protein synthesis, DNA synthesis, respiration or fermentation, INT reduced per cell, and ATP per cell were used to measure physiological activity. Cell densities decreased significantly over time at both sites for both S. faecalis and E. coli; however, no significant differences in survival pattern were observed between S. faecalis and E. coli. Differences in protein synthesis between the two were only observed at a study site which was not heavily oiled. E. coli was more active in protein synthesis and respiration than S. faecalis at both oiled and unoiled sites, and the percentage of the E. coli population that was respiring was significantly higher than S. faecalis fermenting cells at both sites. However, S. faecalis cells were more active in DNA synthesis and higher in ATP content than E. coli cells at both sites. Although fecal streptococci have been suggested as a better indicator of fecal contamination than fecal coliforms in marine waters, in this study both E. coli and S. faecalis survived and remained physiologically active for extended periods of time. These results suggest that the fecal streptococci group is not a better indicator of fecal contamination in tropical marine waters than the fecal coliform group, especially when that environment is high in long-chained hydrocarbons.