In situ evidence for metabolic and chemical microdomains in the structured polymer matrix of bacterial microcolonies.

CLSM and fluorescent probes were applied to assess the structure, composition, metabolic activity and gradients within naturally occurring ß-proteobacteria microcolonies. Extracellular polymeric substances (EPS) as defined by lectin-binding analyses had three regions: (i) cell associated, (ii) intercellular and (iii) an outer layer covering the entire colony. We assessed structural, microenvironmental and metabolic implications of this complex EPS structure. Permeability studies indicated that the outer two layers were permeable to 20 nm beads, intercellular EPS to <40 nm beads and the outer layer was permeable to <100 nm beads. Phosphatase activity occurred at the cell surface and associated polymer. Glucose oxidase activity was only detected inside the cells and the cell-associated polymer. Rhodamine 123 suggested that activity was highest near the cell surface. The potential sensitive dye JC-1 concentrated within the outer EPS layer and the gradient was responsive to inhibition by KCN, dispersion using KCl and enhanced by addition of nutrients (nutrient broth). pH gradients occurred from the cell interior (pH 7) to the microcolony interior (pH 4+) with a gradient of increasing pH (pH 7+) to the colony exterior. The EPS provides a physical and chemical structuring mechanism forming microdomains that segregate extracellular activities at the microscale, possibly resulting in a microcolony with unitary structure and function.

Effects of erythromycin, trimethoprim and clindamycin on attached microbial communities from an effluent dominated prairie stream.

In this study, differing metrics were utilized to measure effects of erythromycin (ER), trimethoprim (TR) and clindamycin (CL) on the structure and function of attached Wascana Creek, SK microbial communities. All three test antibiotics, especially ER, affected community structure and function of biofilms grown in rotating annular reactors. Biofilm thickness, bacterial biomass, and lectin binding biovolume (exopolymeric substances) were consistently less in ER treated biofilms when compared to the control. As well negative effects on protozoan numbers, and carbon utilization were detected. Finally, PCA analyses of DGGE results indicated that bacterial community diversity in ER exposed biofilms was always different from the control. ER exhibited toxic effects even at lower concentrations. Observations on TR and CL exposed biofilms indicated that bacterial biomass, lectin binding biovolume and carbon utilization were negatively affected as well. In terms of bacterial community diversity, however, CL exposed biofilms tended to group with the control while TR grouped with nutrient additions suggesting both nutritive and toxic effects. This study results represent an important step in understanding antibiotic effects, especially ER, on aquatic microbial communities. And because ER is so ubiquitous in receiving water bodies worldwide, the Wascana study results suggest the possibility of ecosystem disturbance elsewhere. CAPSULE ABSTRACT: Erythromycin (ER) is ubiquitous in waterbodies receiving sewage effluent. Structure and function of microbial communities from an effluent dominated stream were negatively affected by ER, at realistic concentrations.

Microbial interactions with naturally occurring hydrophobic sediments: Influence on sediment and associated contaminant mobility.

The erosion, transport and fate of sediments and associated contaminants are known to be influenced by both particle characteristics and the flow dynamics imparted onto the sediment. The influential role of bitumen containing hydrophobic sediments and the microbial community on sediment dynamics are however less understood. This study links an experimental evaluation of sediment erosion with measured sediment-associated contaminant concentrations and microbial community analysis to provide an estimate of the potential for sediment to control the erosion, transport and fate of contaminants. Specifically the paper addresses the unique behaviour of hydrophobic sediments and the role that the microbial community associated with hydrophobic sediment may play in the transport of contaminated sediment. Results demonstrate that the hydrophobic cohesive sediment demonstrates unique transport and particle characteristics (poor settling and small floc size). Biofilms were observed to increase with consolidation/biostabilization times and generated a unique microbial consortium relative to the eroded flocs. Natural oil associated with the flocs appeared to be preferentially associated with microbial derived extracellular polymeric substances. While PAHs and naphthenic acid increased with increasing shear (indicative of increasing loads), they tended to decrease with consolidation/biostabilization (CB) time at similar shears suggesting a chemical and/or biological degradation. PAH and napthenic acid degrading microbes decreased with time as well, which may suggest that there was a reduced pool of PAHs and naphthenic acids available resulting in their die off. This study emphasizes the importance that any management strategies and operational assessments for the protection of human and aquatic health incorporate the sediment (suspended and bed sediment) and biological (biofilm) compartments and the energy dynamics within the system in order to better predict contaminant transport.

Effects of fullerene (C60), multi-wall carbon nanotubes (MWCNT), single wall carbon nanotubes (SWCNT) and hydroxyl and carboxyl modified single wall carbon nanotubes on riverine microbial communities.

Commercial production of nanoparticles (NP) has created a need for research to support regulation of nanotechnology. In the current study, microbial biofilm communities were developed in rotating annular reactors during continuous exposure to 500 µg L(-1) of each nanomaterial and subjected to multimetric analyses. Scanning transmission X-ray spectromicroscopy (STXM) was used to detect and estimate the presence of the carbon nanomaterials in the biofilm communities. Microscopy observations indicated that the communities were visibly different in appearance with changes in abundance of filamentous cyanobacteria in particular. Microscale analyses indicated that fullerene (C60) did not significantly (p < 0.05) impact algal, cyanobacterial or bacterial biomass. In contrast, MWCNT exposure resulted in a significant decline in algal and bacteria biomass. Interestingly, the presence of SWCNT products increased algal biomass, significantly in the case of SWCNT-COOH (p < 0.05) but had no significant impact on cyanobacterial or bacterial biomass. Thymidine incorporation indicated that bacterial production was significantly reduced (p < 0.05) by all nanomaterials with the exception of fullerene. Biolog assessment of carbon utilization revealed few significant effects with the exception of the utilization of carboxylic acids. PCA and ANOSIM analyses of denaturing gradient gel electrophoresis (DGGE) results indicated that the bacterial communities exposed to fullerene were not different from the control, the MWCNT and SWNT-OH differed from the control but not each other, whereas the SWCNT and SWCNT-COOH both differed from all other treatments and were significantly different from the control (p < 0.05). Fluorescent lectin binding analyses also indicated significant (p < 0.05) changes in the nature and quantities of exopolymer consistent with changes in microbial community structure during exposure to all nanomaterials. Enumeration of protozoan grazers showed declines in communities exposed to fullerene or MWCNT but a trend for increases in all SWCNT exposures. Observations indicated that at 500 µg L(-1), carbon nanomaterials significantly alter aspects of microbial community structure and function supporting the need for further evaluation of their effects in aquatic habitats.

Soft X-ray spectromicroscopy for speciation, quantitation and nano-eco-toxicology of nanomaterials.

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X-ray absorption properties, soft X-ray scanning transmission X-ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal-containing nanomaterials (Ti, Ni, Cu) and carbon-based nanomaterials (multiwalled carbon nanotubes, C60 fullerene). The benefits of X-ray fluorescence detection in soft X-ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.

Comparative responses of river biofilms at the community level to common organic solvent and herbicide exposure.

Residual pesticides applied to crops migrate from agricultural lands to surface and ground waters. River biofilms are the first aquatic non-target organisms which interact with pesticides. Therefore, ecotoxicological experiments were performed at laboratory scale under controlled conditions to investigate the community-level responses of river biofilms to a chloroacetanilide herbicide (alachlor) and organic solvent (methanol) exposure through the development referenced to control. Triplicate rotating annular bioreactors, inoculated with river water, were used to cultivate river biofilms under the influence of 1 and 10 µg L(-1) of alachlor and 25 mg L(-1) of methanol. For this purpose, functional (thymidine incorporation and carbon utilization spectra) and structural responses of microbial communities were assessed after 5 weeks of development. Structural aspects included biomass (chlorophyll a, confocal laser scanning microscopy) and composition (fluor-conjugated lectin binding, molecular fingerprinting, and diatom species composition). The addition of alachlor resulted in a significant reduction of bacterial biomass at 1 µg L(-1), whereas at 10 µg L(-1), it induced a significant reduction of exopolymer lectin binding, algal, bacterial, and cyanobacterial biomass. However, there were no changes in biofilm thickness or thymidine incorporation. No significant difference between the bacterial community structures of control and alachlor-treated biofilms was revealed by terminal restriction fragment length polymorphism (T-RFLP) analyses. However, the methanol-treated bacterial communities appeared different from control and alachlor-treated communities. Moreover, methanol treatment resulted in an increase of bacterial biomass and thymidine incorporation as well. Changes in dominant lectin binding suggested changes in the exopolymeric substances and community composition. Chlorophyll a and cyanobacterial biomass were also altered by methanol. This study suggested that the concentration-dependent effect of alachlor mainly remains limited to biomass and growth inhibition without apparent changes of structural and functional characteristics measured. Our work also establishes the potential toxic effects of organic solvents on river biofilm in ecotoxicological experiments. For the ecotoxicological experiments, the alternative of dissolution in organic solvent followed by its evaporation, depositing the chemical on a glass surface prior to dissolution in river water used here, appears to allow exposure while minimizing the effect of organic solvent.

Resilience and recovery: the effect of triclosan exposure timing during development, on the structure and function of river biofilm communities.

Triclosan (TCS) is a ubiquitous antibacterial agent found in soaps, scrubs, and consumer products. There is limited information on hazardous effects of TCS in the environment. Here, rotating annular reactors were used to cultivate river biofilm communities exposed to 1.8 µg l(-1) TCS with the timing and duration of exposure and recovery during development varied. Two major treatment regimens were employed: (i) biofilm development for 2, 4 or 6 weeks prior to TCS exposure and (ii) exposure of biofilms to TCS for 2, 4 or 6 weeks followed by recovery. Biofilms not exposed to TCS were used as a reference condition. Communities cultivated without and then exposed to TCS all exhibited reductions in algal biomass and significant (p<0.05) reductions in cyanobacterial biomass. No significant effects were observed on bacterial biomass. CLSM imaging of biofilms at 8 weeks revealed unique endpoints in terms of community architecture. Community composition was altered by any exposure to TCS, as indicated by significant shifts in denaturing gradient gel electrophoresis fingerprints and exopolymer composition relative to the reference. Bacterial, algal and cyanobacterial components initially exposed to TCS were significantly different from those TCS-free at time zero. Pigment analyses suggested that significant changes in composition of algal and cyanobacterial populations occurred with TCS exposure. Bacterial thymidine incorporation rates were reduced by TCS exposure and carbon utilization spectra shifted in terms substrate metabolism. Direct counts of protozoans indicated that TCS was suppressive, whereas micrometazoan populations were, in some instances, stimulated. These results indicate that even a relatively brief exposure of a river biofilm community to relatively low levels of TCS alters both the trajectory and final community structure. Although some evidence of recovery was observed, removal of TCS did not result in a return to the unexposed reference condition.

Biogeochemical activity of microbial biofilms in the water column overlying uranium mine tailings.

AIMS: To describe microbial diversity, biofilm composition and biogeochemical potential within biofilms in the water overlying uranium tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To estimate microbial diversity in biofilms formed in water columns overlying uranium mine tailings, culture-dependent and culture-independent methods were employed. High-throughput sequencing revealed the presence of 11 phyla; however, the majority of the sequences were affiliated with four major lineages (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) as confirmed by culture-based methods. Dominant phylotypes were closely related to methylotrophs (Methylobacterium) and bacterial groups able to utilize complex hydrocarbons (Aquabacterium and Dechloromonas). Microbial diversity in biofilms from the 13 m depth was significantly different that in biofilms from 1 to 41 m (P < 0·05). Phylotypes closely related to iron-reducing bacteria were identified at each depth; whereas sulphate-, thio-sulphate-, sulphite- and sulphur-reducing bacteria, at low abundance, were only detected at lower depths. Confocal scanning laser microscopy (CSLM) was used to investigate polymer quantity and composition of the biofilm components, and principal component analysis of the CLSM data revealed that the relative abundance of α-L-fucose and N-acetyl-glucosamine/lipopolysaccharide residues separated tailings-water interface biofilms from those from other depths. Reduced (ferrous) iron was detected within all the biofilm samples examined by scanning X-ray transmission microscopy. CONCLUSIONS: Microbial communities within the water column covering a highly alkaline uranium tailings body form biofilms with microenvironments where iron reduction takes place. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the biogeochemical potential of microbial biofilm communities in the water column covering an alkaline uranium tailings body; specifically, the nature of the bacterial groups detected (Aquabacterium, Dechloromonas) and the presence of reduced iron suggest that complex hydrocarbons are available for bacterial growth and geochemical change, such as iron reduction, can occur even though the system bulk phase is predominantly oxic.

Relationship between water quality parameters and bacterial indicators in a large prairie reservoir: Lake Diefenbaker, Saskatchewan, Canada.

Lake Diefenbaker (LD) is a large reservoir on the South Saskatchewan River used for agricultural irrigation, drinking water, and recreation. Our objectives were to determine the distribution and abundance of bacterial indicators in embayments and the main channel of LD and to relate these to environmental factors. Total coliforms (TCs), fecal coliforms (FCs), and fecal indicator bacteria (i.e., Escherichia coli) were measured concurrently with water quality parameters. Although TCs, FCs, and E. coli were present in LD, they rarely exceeded the TC and FC Canadian Council of Ministers of the Environment (CCME) water quality standards for agricultural use (1000 colony-forming units (CFU) per 100 mL and 100 CFU per 100 mL, respectively). The correlation between the bacterial indicators in the sediments and the water column indicates that higher embayment abundances may be related to sediment loading and (or) resuspension events in these frequently mixed embayments. With higher water temperatures and water levels, as well as higher microbial activity, CCME bacterial limits may be exceeded. The greatest contributor to bacterial indicator abundance was water temperature. We predict that water quality standards will be exceeded more frequently with climate warming.

Microbial communities in low permeability, high pH uranium mine tailings: characterization and potential effects.

AIMS: To describe the diversity and metabolic potential of microbial communities in uranium mine tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To assess microbial diversity and their potential to influence the geochemistry of uranium mine tailings using aerobic and anaerobic culture-based methods, in conjunction with next generation sequencing and clone library sequencing targeting two universal bacterial markers (the 16S rRNA and cpn60 genes). Growth assays revealed that 69% of the 59 distinct culturable isolates evaluated were multiple-metal resistant, with 15% exhibiting dual-metal hypertolerance. There was a moderately positive correlation coefficient (R = 0·43, P < 0·05) between multiple-metal resistance of the isolates and their enzyme expression profile. Of the isolates tested, 17 reduced amorphous iron, 22 reduced molybdate and seven oxidized arsenite. Based on next generation sequencing, tailings depth was shown to influence bacterial community composition, with the difference in the microbial diversity of the upper (0-20 m) and middle (20-40 m) tailings zones being highly significant (P < 0·01) from the lower zone (40-60 m) and the difference in diversity of the upper and middle tailings zone being significant (P < 0·05). Phylotypes closely related to well-known sulfate-reducing and iron-reducing bacteria were identified with low abundance, yet relatively high diversity. CONCLUSIONS: The presence of a population of metabolically-diverse, metal-resistant micro-organisms within the tailings environment, along with their demonstrated capacity for transforming metal elements, suggests that these organisms have the potential to influence the long-term geochemistry of the tailings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first investigation of the diversity and functional potential of micro-organisms present in low permeability, high pH uranium mine tailings.

Transient response of microbial communities in a water well field to application of an impressed current.

Deterioration of water wells due to clogging and corrosion over time is a common problem where solutions may be costly and ineffective. Pilot studies have suggested that impressed current or cathodic protection may be used to reduce microbially-induced declines in water well performance. Two water wells in an alluvial aquifer close to the North Saskatchewan River were selected to study the response of subsurface microbial communities to the application of an impressed current as an anti-fouling technology. The treated well was exposed to an impressed current while the untreated well was used as a reference site. Biofilms grown on in situ coupons under the influence of the impressed current were significantly (p < 0.05) thicker (mean thickness = 67.3 µm) when compared to the biofilms (mean thickness = 19.3 µm) grown outside the electric field. Quantitative PCR analyses showed significantly (p < 0.05) higher numbers of total bacteria, iron- and nitrate-reducers in the electrified zone. Molecular analysis revealed that the predominant bacteria present in biofilms grown under the influence of the impressed current belonged to Rhodobacter spp., Sediminibacterium spp. and Geobacter spp. In addition to favouring the growth of biofilms, direct microscopic and ICP-AES analyses revealed that the impressed current also caused the deposition of iron and manganese on, and in the vicinity of, the well screen. Together, these factors contributed to rapid clogging leading to reduced specific pumping capacities of the treated well. The study revealed that the impressed current system was not effective as an anti-fouling technology but actually promoted both microbial growth and physical clogging in this aquifer.

Spatial variation in microbial community structure, richness, and diversity in an alluvial aquifer.

Relatively little is known regarding the spatial variability of microbial communities in aquifers where well fouling is an issue. In this study 2 water wells were installed in an alluvial aquifer located adjacent to the North Saskatchewan River and an associated piezometer network developed to facilitate the study of microbial community structure, richness, and diversity. Carbon utilization data analysis revealed reduced microbial activity in waters collected close to the wells. Functional PCR and quantitative PCR analysis indicated spatial variability in the potential for iron-, sulphate-, and nitrate-reducing activity at all locations in the aquifer. Denaturing gradient gel electrophoresis analysis of aquifer water samples using principal components analyses indicated that the microbial community composition was spatially variable, and denaturing gradient gel electrophoresis sequence analysis revealed that bacteria belonging to the genera Acidovorax , Rhodobacter , and Sulfuricurvum were common throughout the aquifer. Shannon's richness (H') and Pielou's evenness (J') indices revealed a varied microbial diversity (H' = 1.488-2.274) and an even distribution of microbial communities within the aquifer (J' = 0.811-0.917). Overall, these analyses revealed that the aquifer's microbial community varied spatially in terms of composition, richness, and metabolic activity. Such information may facilitate the diagnosis, prevention, and management of fouling.

Effect of pumping on the spatio-temporal distribution of microbial communities in a water well field.

A water well field adjacent to the North Saskatchewan River (City of North Battleford, Saskatchewan, Canada) with a history of rapid deterioration of both well water quality and yield was selected to study the spatial and temporal distribution of subsurface microbial communities and their response to water pumping. A range of conventional cultural, microscopic and molecular techniques, including confocal laser scanning microscopy (CLSM), Biolog, qPCR and Denaturing Gradient Gel Electrophoresis (DGGE), was used during this study. Redox data and water and sediment chemistry showed that the aquifer was anoxic and harbored substantial amounts of Fe and Mn. CLSM analyses of incubated coupons indicated extensive biofilm growth in the zone immediately surrounding the well and was coincident with reduced water well yield. PCR screening and qPCR analyses showed that the potential for iron- and sulfate-reducing activity increased with proximity to the well. Bacterial communities inhabiting the zone closest to the well showed the greatest changes and differences in metabolic activities and composition as revealed by PCA (Principal Components Analysis) of the Biolog and DGGE data. The sequence analysis of all the samples revealed that Sulfuricurvum spp., Methylobacter spp., Geobacter spp. and Rhodobacter spp. were most commonly detected in this aquifer. Overall the findings demonstrated that the microbial numbers, metabolic activities and the community composition changed in response to water pumping but effects did not extend beyond 1-2m zone from the well.

Controls and rates of acid production in commercial-scale sulfur blocks.

Acidic drainage (pH 0.4-1.0) from oxidizing elemental sulfur (S(0)) blocks is an environmental concern in regions where S(0) is stockpiled. In this study, the locations, controls, and rates of H(2)SO(4) production in commercial-scale S(0) blocks ( approximately 1-2 x 10(6) m(3)) in northern Alberta, Canada, were estimated. In situ modeling of O(2) concentrations ([O(2)]) suggest that 70 to >97% of the annual H(2)SO(4) production occurs in the upper 1 m of the blocks where temperatures increase to >15 degrees C during the summer. Laboratory experiments show that S(0) oxidation rates are sensitive to temperature (Q(10) = 4.3) and dependent on the activity of autotrophic S(0)-oxidizing microbes. The annual efflux of SO(4) in drainage water from a S(0) block (5.5 x 10(5) kg) was within the estimated range of SO(4) production within the block (2.7 x 10(5) to 1.2 x 10(6) kg), suggesting that H(2)SO(4) production and removal rates were approximately equal during the study period. The low mean relative humidity within the block (68%; SD = 17%; n = 21) was attributed to osmotic suction from elevated H(2)SO(4) concentrations and suggests a mean in situ pH of approximately -2.1. The low pH of drainage waters was attributed to the mixing of fresh infiltrating water and low-pH in situ water. Heat generation during S(0) oxidation was an important factor in maintaining elevated temperatures (mean, 11.1 degrees C) within the block. The implications of this research are relevant globally because construction methods and the physical properties of S(0) blocks are similar worldwide.

The role of rpoS on the survival of a p-nitrophenol degrading Pseudomonas putida strain in planktonic and biofilm phases.

The survival of and interactions between a Pseudomonas putida strain labelled with a red fluorescent protein gene (WT-rfp) and its green fluorescent protein gene-labelled rpoS(-) mutant (KO-gfp) were examined. The generation times of the planktonic WT-rfp and KO-gfp in trypticase soy broth were not significantly different (i.e., p > 0.05) from each other at 30 degrees C. However, the biovolume of the KO-gfp biofilm was about 7 times larger than its WT-rfp counterpart after 48 h of growth. Furthermore, the presence of WT-rfp suppressed the biofilm development of KO-gfp significantly in co-culture biofilms. In planktonic conditions, the pre-carbon-starved WT-rfp achieved a 3-fold greater survival than the pre-carbon-starved KO-gfp in 0.85% saline after a 13-day incubation. In a 1:1 ratio co-culture, the pre-carbon-starved WT-rfp outcompeted the pre-carbon-starved KO-gfp by 20-fold. However, the survival of WT-rfp and KO-gfp were not significantly different from each other in biofilm conditions. Additionally, 11.4% and 61.2% of the WT-rfp and KO-gfp biofilms, respectively, remained intact after washing in 0.2% SDS for 60 min. In conclusion, the rpoS had a significant impact on survival and competitiveness of planktonic P. putida, and on biofilm development, being implicated in competitive suppression of biofilm development in co-culture biofilms and decreased biofilm cohesiveness.

Soft X-ray spectromicroscopy of nickel sorption in a natural river biofilm.

Scanning transmission X-ray microscopy (STXM) at the C 1s, O 1s, Ni 2p, Ca 2p, Mn 2p, Fe 2p, Mg 1s, Al 1s and Si 1s edges was used to study Ni sorption in a complex natural river biofilm. The 10-week grown river biofilm was exposed to 10 mg L(-1) Ni(2+) (as NiCl(2)) for 24 h. The region of the biofilm examined was dominated by filamentous structures, which were interpreted as the discarded sheaths of filamentous bacteria, as well as a sparse distribution of rod-shaped bacteria. The region also contained discrete particles with spectra similar to those of muscovite, SiO(2) and CaCO(3). The Ni(II) ions were selectively adsorbed by the sheaths of the filamentous bacteria. The sheaths were observed to be metal rich with significant amounts of Ca, Fe and Mn, along with the Ni. In addition, the sheaths had a large silicate content but little organic material. The metal content of the rod-shaped bacterial cells was much lower. The Fe on the sheath was mainly in the Fe(III) oxidation state. Mn was found in II, III and IV oxidation states. The Ni was likely sorbed to Mn-Fe minerals on the sheath. These STXM results have probed nano-scale biogeochemistry associated with bacterial species in a complex, natural biofilm community. They have implications for selective Ni contamination of the food chain and for developing bioremediation strategies.

Comparative microscale analysis of the effects of triclosan and triclocarban on the structure and function of river biofilm communities.

The broad spectrum antimicrobials triclosan (TCS) and triclocarban (TCC) are commonly detected in the environment. However, there is very limited understanding of the aquatic ecological implications of these agents. During this study, river biofilms were cultivated using 10 microg l(-1) of TCS or TCC and the equivalent in nutrients (carbon, nitrogen) over a developmental period of 8 weeks. Confocal laser microscopy showed that the biofilm communities developing under the influence of TCS and TCC had community architecture and composition different from either control or nutrient exposed communities. Microscale analyses of biofilm community structure indicated a significant reduction in algal biomass (p<0.05) as a result of exposure to either TCS or TCC. Thymidine incorporation did not detect significant differences between control and treated communities. The use of carbon utilization assays based on growth indicated that, in general, TCS and TCC suppressed utilization. The community was altered from one dominated by autotrophic processes to one dominated by heterotrophic processes. Both TCS and TCC treatments resulted in significant (p<0.05) alterations in the composition of the EPS matrix of the communities, suggesting significant changes in community composition. Denaturing gradient gel electrophoresis and PCA-ANOSIM analyses indicated a significant change occurred in the bacterial community as a consequence of TCS treatments. Enumeration of micrometazoa and protozoa revealed an increase in micrometazoan numbers over control values, whereas no clear impact on protozoa was detected in any treatment. This study indicated significant effects of 10 microg l(-1) TCS and TCC on microbial community composition, algal biomass, architecture and activity.

Community-level assessment of the effects of the broad-spectrum antimicrobial chlorhexidine on the outcome of river microbial biofilm development.

Chlorhexidine is a common-use antibacterial agent found in a range of personal-care products. We used rotating annular reactors to cultivate river biofilms under the influence of chlorhexidine or its molar equivalent in nutrients. Studies of the degradation of [(14)C]chlorhexidine demonstrated that no mineralization of the compound occurred. During studies with 100 microg liter(-1) chlorhexidine, significant changes were observed in the protozoan and micrometazoan populations, the algal and cyanobacterial biomass, the bacterial biomass, and carbon utilization. Denaturing gradient gel electrophoresis (DGGE) in combination with statistical analyses showed that the communities developing under control and 100 microg liter(-1) chlorhexidine were significantly different. At 10 microg liter(-1) chlorhexidine, there was significantly increased algal and cyanobacterial biomass while the bacterial biomass was not significantly affected (P < 0.05). No significant effects on protozoan or metazoan grazing were detected at the 10-microg liter(-1) chlorhexidine level. Fluorescent in situ hybridization indicated a significant reduction in the abundance of betaproteobacteria and gammaproteobacteria (P < 0.05). Archaeal cell counts were significantly reduced by both chlorhexidine and nutrient treatments. DGGE and statistical analyses indicated that 10 microg liter(-1) chlorhexidine and molar equivalent nutrient treatments were significantly different from control communities. In contrast to community level observations, toxicological testing with a panel of cyanobacteria, algae, and protozoa indicated no detectable effects at 10, 50, and 100 microg liter(-1) chlorhexidine. Thus, community level assessment indicated a risk of low levels of chlorhexidine in aquatic habitats while conventional approaches did not.

In situ evidence for microdomains in the polymer matrix of bacterial microcolonies.

Confocal laser scanning microscopy and fluorescent lectin-binding analyses (FLBA) were used to study the form, arrangement, and composition of exopolymeric substances (EPS) surrounding naturally occurring microcolonies in biofilms. FLBA, using multiple lectin staining and multichannel imaging, indicated that the EPS of many microcolonies exhibit distinct multiple binding regions. A common pattern in the microcolonies is a three zone arrangement with cell-associated, intercellular, and an outer layer of EPS covering the exterior of the colony. Differential binding of lectins suggests that there are differences in the glycoconjugate composition or their arrangement in the EPS of microcolonies. The combination of FLBA with fluorescent in situ hybridization (FISH) indicates that the colonies consist of the major groups, alpha- and beta-Proteobacteria. It is suggested that the EPS arrangement observed provides a physical structuring mechanism that can segregate extracellular activities at the microscale.

Influence of nutrient inputs, hexadecane, and temporal variations on denitrification and community composition of river biofilms.

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P < or = 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.