Exploring freshwater stream bacterial communities as indicators of land use intensity.

BACKGROUND: Stream ecosystems comprise complex interactions among biological communities and their physicochemical surroundings, contributing to their overall ecological health. Despite this, many monitoring programs ignore changes in the bacterial communities that are the base of food webs in streams, often focusing on stream physicochemical assessments or macroinvertebrate community diversity instead. We used 16S rRNA gene sequencing to assess bacterial community compositions within 600 New Zealand stream biofilm samples from 204 sites within a 6-week period (February-March 2010). Sites were either dominated by indigenous forests, exotic plantation forests, horticulture, or pastoral grasslands in the upstream catchment. We sought to predict each site's catchment land use and environmental conditions based on the composition of the stream bacterial communities. RESULTS: Random forest modelling allowed us to use bacterial community composition to predict upstream catchment land use with 65% accuracy; urban sites were correctly assigned 90% of the time. Despite the variation inherent when sampling across a ~ 1000-km distance, bacterial community data could correctly differentiate undisturbed sites, grouped by their dominant environmental properties, with 75% accuracy. The positive correlations between actual values and those predicted by the models built using the stream biofilm bacterial data ranged from weak (average log N concentration in the stream water, R2 = 0.02) to strong (annual mean air temperature, R2 = 0.69). CONCLUSIONS: Freshwater bacterial community data provide useful insights into land use impacts on stream ecosystems; they may be used as an additional measure to screen stream catchment attributes.

Surface-modified activated carbon for N-nitrosodimethylamine removal in the continuous flow biological activated carbon columns.

The carcinogenic nitrogenous disinfection by-product, N-nitrosodimethylamine (NDMA), is challenging to adsorb due to its high polarity and solubility. Our previous research demonstrated that the adsorptive removal of NDMA can be improved using surface-modified activated carbon (AC800). The current study evaluated the efficacy of AC800 in removing NDMA in a continuous-flow column over 75 days, using both granular activated carbon (GAC) and biologically activated carbon (BAC) columns. The AC800 GAC column demonstrated extended breakthrough and exhaustion times of 10 days and 22 days, respectively, compared to the conventional GAC column at 4 days and 10.5 days. The surface modification effect persisted for 25 days before the removal trends became indistinguishable. The AC800 BAC column outperformed the conventional BAC column with a longer breakthrough time of 11.3 days compared to 7.4 days. BAC columns consistently showed greater NDMA removal, emphasizing the role of biodegradation in NDMA removal on carbon. The higher NDMA removal in the inoculated columns was attributed to increased microbial diversity and the dominance of six specific genera, Methylobacterium, Phyllobacterium, Curvibacter, Acidovorax, Variovorax, and Rhodoferax. This study provides new insights into using modified activated carbon as GAC and BAC media in a real-world continuous-flow setup.

Time after time: detecting annual patterns in stream bacterial biofilm communities.

To quantify the major environmental drivers of stream bacterial population dynamics, we modelled temporal differences in stream bacterial communities to quantify community shifts, including those relating to cyclical seasonal variation and more sporadic bloom events. We applied Illumina MiSeq 16S rRNA bacterial gene sequencing of 892 stream biofilm samples, collected monthly for 36-months from six streams. The streams were located a maximum of 118 km apart and drained three different catchment types (forest, urban and rural land uses). We identified repeatable seasonal patterns among bacterial taxa, allowing their separation into three ecological groupings, those following linear, bloom/trough and repeated, seasonal trends. Various physicochemical parameters (light, water and air temperature, pH, dissolved oxygen, nutrients) were linked to temporal community changes. Our models indicate that bloom events and seasonal episodes modify biofilm bacterial populations, suggesting that distinct microbial taxa thrive during these events including non-cyanobacterial community members. These models could aid in determining how temporal environmental changes affect community assembly and guide the selection of appropriate statistical models to capture future community responses to environmental change.

Identifying congruence in stream assemblage thresholds in response to nutrient and sediment gradients for limit setting.

The setting of numeric instream objectives (effects-based criteria) and catchment limits for major agricultural stressors, such as nutrients and fine sediment, is a promising policy instrument to prevent or reduce degradation of stream ecosystem health. We explored the suitability of assemblage thresholds, defined as a point at which a small increase in a stressor will result in a disproportionally large change in assemblage structure relative to other points across the stressor gradient, to inform instream nutrient and sediment objectives. Identification and comparison of thresholds for macroinvertebrate, periphyton, and bacterial assemblages aimed at making the setting of objectives more robust and may further provide a better understanding of the underlying mechanisms of nutrient and fine sediment effects. Gradient forest, a novel approach to assemblage threshold identification based on regression-tree-based random forest models for individual taxa, allowed inclusion of multiple predictors to strengthen the evidence of cause and effect between stressors and multispecies responses. The most prominent macroinvertebrate and periphyton assemblage threshold across the nitrogen (N) gradient was located at very low levels and mainly attributed to declines of multiple taxa. This provided strong evidence for stream assemblages being significantly affected when N concentrations exceed reference conditions and for effects cascading through the ecosystem. The most prominent macroinvertebrate assemblage threshold across a gradient of suspended fine sediment was also located at very low levels and attributed to declines of multiple taxa. However, this threshold did not correspond with periphyton assemblage thresholds, suggesting that the sensitivity of macroinvertebrate assemblages is unrelated to sediment effects on periphyton assemblages. Overall, the spectrum of N concentrations and fine sediment levels within which these stream assemblages changed most dramatically were relatively narrow given the wide gradients tested. We conclude that assemblage thresholds can inform the setting of generic instream nutrient and sediment objectives for stream ecosystem health. For example, the most stringent objective for instream N concentration should be set at values similar to reference concentrations for full protection of sensitive taxa or overall stream biodiversity. To avoid severe degradation of stream biodiversity, the least stringent N objective should stay well below the point where significant turnover subsided.

Functional gene composition, diversity and redundancy in microbial stream biofilm communities.

We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.

Inactivation of bacteria and yeast using high-frequency ultrasound treatment.

High-frequency (850 kHz) ultrasound was used to inactivate bacteria and yeast at different growth phases under controlled temperature conditions. Three species of bacteria, Enterobacter aerogenes, Bacillus subtilis and Staphylococcus epidermidis as well as a yeast, Aureobasidium pullulans were considered. The study shows that high-frequency ultrasound is highly efficient in inactivating the bacteria in both their exponential and stationary growth phases, and inactivation rates of more than 99% were achieved. TEM observation suggests that the mechanism of bacteria inactivation is mainly due to acoustic cavitation generated free radicals and H2O2. The rod-shaped bacterium B. subtilis was also found to be sensitive to the mechanical effects of acoustic cavitation. The study showed that the inactivation process continued even after ultrasonic processing cessed due to the presence of H2O2, generated during acoustic cavitation. Compared to bacteria, the yeast A. pullulans was found to be more resistant to high-frequency ultrasound treatment.

Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound.

The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20kHz) and high-frequency (850kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50W for 60min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the κ-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of α-lactalbumin and ß-lactoglobulin slightly decreasing.

Using biofilm as a novel approach to assess stormwater treatment efficacy.

Contaminants associated with stormwater are among the leading causes of water quality impairment in urban streams. Multiple device treatment systems are commonly installed with the aim of reducing contaminant loads within stormwater discharge. However, the in situ performance of such systems remains poorly understood. We investigated the efficacy of an advanced stormwater treatment system by monitoring biofilm associated metals and biofilm bacterial community composition at multiple locations through the treatment system (which included rain gardens, grassy swales, a stormwater filter and a wetland) and in the receiving stream above and below the stormwater discharge. Changes in bacterial community composition were assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and concentrations of biofilm associated metals monitored by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Significant differences in bacterial community composition were detected throughout the stormwater network. Bacterial communities gradually changed towards a community more similar to that within the receiving stream and the discharge of treated stormwater had little effect on the composition of bacterial communities in the receiving stream, suggesting the effective conditioning of water quality by the treatment system. Concentrations of some biofilm-associated metals declined following sequential treatment, for example copper (73% reduction), zinc (48% reduction) and lead (46% reduction). In contrast, levels of arsenic, cadmium, chromium and nickel were not reduced by the treatment system. We demonstrate that biofilm bacterial community composition is a sensitive indicator of environmental changes within freshwater ecosystems and an efficient indicator to monitor water quality in enclosed stormwater networks where traditional biological indicators are not available.

Inactivation of microorganisms by low-frequency high-power ultrasound: 1. Effect of growth phase and capsule properties of the bacteria.

The aim of this study was to determine the effects of high-intensity low-frequency (20 kHz) ultrasound treatment on the viability of bacteria suspension. More specifically, we have investigated the relationship between the deactivation efficiency and the physical (size, hydrophobicity) and biological (gram-status, growth phase) properties of the microbes. Enterobacter aerogenes, Bacillus subtilis, Staphylococcus epidermidis, S. epidermidis SK and Staphylococcus pseudintermedius were chosen for this study owing to their varying physical and biological properties. The survival ratio of the bacteria suspension was measured as a function of the ultrasound power (up to 13 W) for a constant sonication time of 20 min. Transmission electron microscopy was used to evaluate the ultrasound-induced damages to the microbes. Ultrasound treatment resulted in lethal damage to E. aerogenes and B. subtilis (up to 4.5-log reduction), whereas Staphylococcus spp. were not affected noticeably. Further, E. aerogenes suspensions were more sensitive to ultrasonication in exponential growth phase than when they were in stationary phase. The results of this study demonstrate that the main reason for bacterial resistance to ultrasonic deactivation is due to the properties of the bacterial capsule. Microbes with a thicker and "soft" capsule are highly resistant to ultrasonic deactivation process.

Inactivation of microorganisms by low-frequency high-power ultrasound: 2. A simple model for the inactivation mechanism.

A simple theoretical model based on shear forces generated by the collapse of the ultrasound cavities near the surface of a microorganism is proposed. This model requires two parameters which take into account the number of acoustic cavitation bubbles, and the resistance of the cell wall of the microorganism to the shear forces generated by bubble collapse. To validate the model, high-power low frequency (20 kHz) ultrasound was used to inactivate two microorganisms with very different sizes, viz., a bacterium, Enterobacter aerogenes and a yeast, Aureobasidium pullulans. The inactivation ratio was experimentally measured as a function of sonication time for different ultrasound power and for different initial cell numbers. For both E. aerogenes and A. pullulans the Log of the inactivation ratio decreased linearly with sonication time, and the rate of inactivation increased (D-value decreased) with the increase in sonication power. The rate of inactivation was also found, for both microorganisms, to increase with a decrease in the initial cell number. The fits, obtained using the proposed model, are in very good agreement with the experimental data.

Evaluation of human adenovirus and human polyomavirus as indicators of human sewage contamination in the aquatic environment.

Discharge of inadequately treated human wastewater into surface waters used for recreation, drinking water, irrigation and shellfish cultivation may present a public health hazard due to the potential shedding of high concentrations of pathogenic viruses from the human gastrointestinal tract. Human adenovirus (HAdV) and human polyomavirus (HPyV) are ubiquitous in humans and have excellent survival characteristics in the environment, so are potential candidates for indicators of human sewage contamination. Using qPCR assays, the prevalence and quantity of HAdV and HPyV JC and BK were determined in influent and effluent wastewater and receiving waters (river, urban stream, estuarine), then compared with norovirus (NoV) presence, a significant human pathogen which is not necessarily ubiquitously excreted into the environment. HAdV and HPyV were frequently detected in high concentrations in wastewater and wastewater-contaminated waters confirming their use as potential indicators for the presence of human sewage. Overall, there was a correlation between the presence of HAdV and HPyV with NoV but there were some notable exceptions including the higher frequency of NoV compared to HAdV and HPyV in estuarine waters impacted by wastewater overflows. We found that HAdV and HPyV detection by qPCR was a suitable tool for evaluating water quality and that their detection can aid in determining pollution sources, thus providing useful information for health risk assessments.

Understanding the combined influence of fine sediment and glyphosate herbicide on stream periphyton communities.

Pesticides and deposited fine sediment have independently been associated with changes in relative abundance and species richness in aquatic ecosystems, but the interplay between these two stressors in agricultural streams is poorly understood. A 28-day experiment in outdoor stream mesocosms examined the effects of four levels each of fine sediment coverage (0, 25, 75, 100%) and glyphosate-based herbicide concentration (0, 50, 200, 370 µg/L) on periphyton communities (algae and bacteria) in a fully factorial, repeated-measures design. Our aims were to determine whether (i) increased levels of sediment and glyphosate had individual and/or additive effects, (ii) increased sediment reduced the toxicity of glyphosate (antagonistic multiple stressor interaction), or (iii) sediment-adsorbed glyphosate prolonged the effects of exposure (synergistic interaction). We also assigned all algal taxa to three ecological guilds (low-profile, high-profile and motile growth forms) and separately determined their responses to the treatments. As individual stressors, sediment addition affected all algal community-level metrics, whereas glyphosate addition only affected algal community evenness. Bacterial taxon richness was unaffected by either stressor. In combination, however, significant overall sediment by glyphosate interactions were detected, demonstrating synergistic (algal evenness, high-profile and motile guilds) or antagonistic effects (low-profile guild). Our experiment underscores the importance of considering both structural and functional indicators, including algal guild representation, when assessing the mechanisms by which periphyton communities respond to multiple stressors.

Naturalized Escherichia coli from New Zealand wetland and stream environments.

This research investigates the presence of a naturalized clade of Escherichia coli in wetland and stream biofilms. Escherichia coli is used as a faecal indicator in water quality monitoring programmes worldwide, with the assumption that this bacterium is exclusively a commensal of the vertebrate gut. However, recent findings indicate growth and multiplication of E. coli in water and soils. This study seeks to clarify the relationships between environmental and commensal E. coli strains retrieved from New Zealand streams by evaluating fundamental genetic differences using the multilocus sequence typing (MLST) method. Environmental and commensal strains showed a high diversity of MLST profiles. Genetic analyses of linkage disequilibrium, index of association and rates of synonymous and nonsynonymous substitutions were used to investigate sequence variability and nature of change. Phylogenetic trees based on the concatenated sequences of the seven MLST housekeeping genes displayed distinct clustering of environmental strains. Comparison of the New Zealand sequences with worldwide E. coli strains retrieved from the Shigatox MLST database online did not allow the identification of a clear environmental genotype. However, some New Zealand aquatic E. coli isolates showed close relationships with strains from human and bovine origins, suggesting that environmental isolates were originally derived from subpopulations of commensal E. coli from these sources.

Metal concentrations in stream biofilm and sediments and their potential to explain biofilm microbial community structure.

Concentrations of metals associated with sediments have traditionally been analysed to assess the extent of heavy metal contamination in freshwater environments. Stream biofilms present an alternative medium for this assessment which may be more relevant to the risk incurred by stream ecosystems as they are intensively grazed by aquatic organisms at a higher trophic level. Therefore, we investigated zinc, copper and lead concentrations in biofilms and sediments of 23 stream sites variously impacted by urbanisation. Simultaneously, biofilm bacterial and ciliate protozoan community structure was analysed by Automated Ribosomal Intergenic Spacer Analysis and Terminal Restriction Fragment Length Polymorphism, respectively. Statistical analysis revealed that biofilm associated metals explained a greater proportion of the variations observed in bacterial and ciliate communities than did sediment associated-metals. This study suggests that the analysis of metal concentrations in biofilms provide a good assessment of detrimental effects of metal contaminants on aquatic biota.

Influence of wastewater treatment process and the population size on human virus profiles in wastewater.

Human adenovirus (AdV and AdV species F), enterovirus (EV) and norovirus (NoV) concentrations entering wastewater treatment plants (WWTP) serving different-sized communities, and effectiveness of different treatment processes in reducing concentrations were established. Data was combined to create a characteristic and unique descriptor of the individual viral composition and termed as the sample virus profile. Virus profiles were generally independent of population size and treatment process (moving bed biofilm reactors, activated sludge, waste stabilisation ponds). AdV and EV concentrations in wastewater were more variable in small (<4000) and medium-sized (10,000-64,000) WWTP than in large-sized (>130,000 inhabitants) plants. AdV and EV concentrations were detected in influent of most WWTP (AdV range 1.00-4.08 log(10) infectious units (IU)/L, 3.25-8.62 log(10) genome copies/L; EV range 0.7-3.52 log(10) plaque forming units (PFU)/L; 2.84-6.67 log(10) genome copies/L) with a reduced median concentration in effluent (AdV range 0.70-3.26 log(10) IU/L, 2.97-6.95 log(10) genome copies/L; EV range 0.7-2.15 log(10)PFU/L, 1.54-5.28 log(10) genome copies/L). Highest culturable AdV and EV concentrations in effluent were from a medium-sized WWTP. NoV was sporadic in all WWTP with GI and GII concentrations being similar in influent (range 2.11-4.64 and 2.19-5.46 log(10) genome copies/L) as in effluent (range 2.18-5.06 and 2.88-5.46 log(10) genome copies/L). Effective management of WWTP requires recognition that virus concentration in influent will vary - particularly in small and medium plants. Irrespective of treatment type, culturable viruses and NoV are likely to be present in non-disinfected effluent, with associated human health risks dependent on concentration and receiving water usage.

Three common metal contaminants of urban runoff (Zn, Cu & Pb) accumulate in freshwater biofilm and modify embedded bacterial communities.

We investigated the absorption rates of zinc, copper and lead in freshwater biofilm and assessed whether biofilm bacterial populations are affected by exposure to environmentally relevant concentrations of these metals in flow chamber microcosms. Metals were rapidly accumulated by the biofilm and then retained for at least 14 days after transfer to uncontaminated water. Changes in bacterial populations were assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and 16S rRNA gene clone libraries. Significant differences in bacterial community structure occurred within only three days of exposure to metals and remained detectable at least 14 days after transfer to uncontaminated water. The rapid uptake of stormwater-associated metals and their retention in the biofilm highlight the potential role of biofilms in the transfer of metals to organisms at higher trophic levels. The sensitivity of stream biofilm bacterial populations to metal exposure supports their use as an indicator of stream ecological health.

Spatial and temporal heterogeneity of the bacterial communities in stream epilithic biofilms.

The spatial and temporal variability in bacterial communities within freshwater systems is poorly understood. The bacterial composition of stream epilithic biofilms across a range of different spatial and temporal scales both within and between streams and across the profile of individual stream rocks was characterised using a community DNA-fingerprinting technique (Automated Ribosomal Intergenic Spacer Analysis, ARISA). The differences in bacterial community structure between two different streams were found to be greater than the spatial variability within each stream site, and were larger than the weekly temporal variation measured over a 10-week study period. Greater variations in bacterial community profiles were detected on different faces of individual stream rocks than between whole rocks sampled within a 9-m stream section. Stream temperature was found to be the most important determinant of bacterial community variability using distance-based redundancy analysis (dbRDA) of ARISA data, which may have broad implications for riparian zone management and ecological change as a consequence of global warming. The combination of ARISA with multivariate statistical methods and ordination, such as multidimensional scaling (MDS), permutational manova and RDA, provided rapid and effective methods for quantifying and visualising variation in bacterial community structure, and to identify potential drivers of ecological change.

A heteroduplex method for detection of targeted sub-populations of bacterial communities.

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.