Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures.

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca2+ is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 °C) and cold acclimated (2 weeks at 12/4 °C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhanced the temperature at which half the cells survived freezing injury by 8 °C (LT50 =-20 °C), and reduced tissue permeability by 70-fold compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca2+ in the cell wall, and a lower number of visible cell wall pores compared with that observed in cold acclimated plants. Using cryo-microscopy, we observed that 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells, but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate that the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.

Aerobic biofilms grown from Athabasca watershed sediments are inhibited by increasing concentrations of bituminous compounds.

Sediments from the Athabasca River and its tributaries naturally contain bitumen at various concentrations, but the impacts of this variation on the ecology of the river are unknown. Here, we used controlled rotating biofilm reactors in which we recirculated diluted sediments containing various concentrations of bituminous compounds taken from the Athabasca River and three tributaries. Biofilms exposed to sediments having low and high concentrations of bituminous compounds were compared. The latter were 29% thinner, had a different extracellular polysaccharide composition, 67% less bacterial biomass per µm2, 68% less cyanobacterial biomass per µm2, 64% less algal biomass per µm2, 13% fewer protozoa per cm2, were 21% less productive, and had a 33% reduced content in chlorophyll a per mm2 and a 20% reduction in the expression of photosynthetic genes, but they had a 23% increase in the expression of aromatic hydrocarbon degradation genes. Within the Bacteria, differences in community composition were also observed, with relatively more Alphaproteobacteria and Betaproteobacteria and less Cyanobacteria, Bacteroidetes, and Firmicutes in biofilms exposed to high concentrations of bituminous compounds. Altogether, our results suggest that biofilms that develop in the presence of higher concentrations of bituminous compounds are less productive and have lower biomass, linked to a decrease in the activities and abundance of photosynthetic organisms likely due to inhibitory effects. However, within this general inhibition, some specific microbial taxa and functional genes are stimulated because they are less sensitive to the inhibitory effects of bituminous compounds or can degrade and utilize some bitumen-associated compounds.

Metatranscriptomic Insights Into the Response of River Biofilm Communities to Ionic and Nano-Zinc Oxide Exposures.

Manufactured Zn oxide nanoparticle (ZnO-NP) are extensively used world-wide in personal care and industrial products and are important contaminants of aquatic environments. To understand the overall impact of ZnO-NP contamination on aquatic ecosystems, investigation of their toxicity on aquatic biofilms is of particular consequence, given biofilms are known sinks for NP contaminants. In order to assess alterations in the functional activity of river microbial biofilm communities as a result of environmentally-relevant ZnO-NP exposure, biofilms were exposed to ionic zinc salt or ZnOPs that were uncoated (hydrophilic), coated with silane (hydrophobic) or stearic acid (lipophilic), at a total concentration of 188 µg l-1 Zn. ICP-MS analyses of biofilms indicated ZnO-NP concentrated in the biofilms, with hydrophilic, hydrophobic, and lipophilic treatments reaching 0.310, 0.250, and 0.220 µg Zn cm-2 of biofilm, respectively, while scanning transmission X-ray microspectroscopy (STXM) analyses of biofilms confirmed that Zn was extensively- and differentially-sorbed to biofilm material. Microbial community composition, based on taxonomic affiliation of mRNA sequences and enumeration of protozoa and micrometazoa, was not affected by these treatments, and the total transcriptional response of biofilms to all experimental exposures was not indicative of a global toxic-response, as cellular processes involved in general cell maintenance and housekeeping were abundantly transcribed. Transcripts related to major biological processes, including photosynthesis, energy metabolism, nitrogen metabolism, lipid metabolism, membrane transport, antibiotic resistance and xenobiotic degradation, were differentially expressed in Zn-exposures relative to controls. Notably, transcripts involved in nitrogen fixation and photosynthesis were decreased in abundance in response to Zn-exposure, while transcripts related to lipid degradation and motility-chemotaxis were increased, suggesting a potential role of Zn in biofilm dissolution. ZnO-NP and ionic Zn exposures elicited generally overlapping transcriptional responses, however hydrophilic and hydrophobic ZnO-NPs induced a more distinct effect than that of lipophilic ZnO-NPs, which had an effect similar to that of low ionic Zn exposure. While the physical coating of ZnO-NP may not induce specific toxicity observable at a community level, alteration of ecologically important processes of photosynthesis and nitrogen cycling are an important potential consequence of exposure to ionic Zn and Zn oxides.

Microscale and molecular analyses of river biofilm communities treated with microgram levels of cerium oxide nanoparticles indicate limited but significant effects.

Cerium oxide (CeO2) nanoparticles are used as in-fuel catalysts and in manufacturing processes, creating a potential for release to aquatic environments. Exposures at 1 and 10 µg/L CeO2-nanoparticles were made to assess effects during the development of river biofilm communities. Scanning transmission x-ray microscopy (STXM) indicated extensive sorption of nanoparticles to the community and co-localization with lipid moieties. Following 8 weeks of development, polycarbonate coupons were removed from the reactors and used for molecular analyses, denaturing gradient gel electrophoresis analysis (DGGE-16S rRNA) and 16S rRNA amplicon sequencing. Microscopic imaging of the biofilm communities (bacterial, photosynthetic biomass, exopolymer composition, thickness, protozoan numbers), as well as carbon substrate utilization fingerprinting was performed. There was a trend toward reduced photosynthetic biomass, but no significant effects of CeO2 exposure were found on photosynthetic and bacterial biomass or biofilm thickness. Sole carbon source utilization analyses indicated increased utilization of 10 carbon sources in the carbohydrate, carboxylic acid and amino acids categories related to CeO2 exposures; however, predominantly, no significant effects (p < 0.05) were detected. Measures of microbial diversity, lectin binding affinities of exopolymeric substances and results of DGGE analyses, indicated significant changes to community composition (p < 0.05) with CeO2 exposure. Increased binding of the lectin Canavalia ensiformis was observed, consistent with changes in bacterial-associated polymers. Whereas, no significant changes were observed in binding to residues associated with algal and cyanobacterial exopolymers. 16S rRNA amplicon sequencing of community DNA indicated changes in diversity and shifts in community composition; however, these did not trend with increasing CeO2 exposure. Counting of protozoans in the biofilm communities indicated no significant effects on this trophic level. Thus, based on biomass and functional measures, CeO2 nanoparticles did not appear to have significant effects; however, there was evidence of selection pressure resulting in significant changes in microbial community composition.

Visualization of the Sorption of Nickel within Exopolymer Microdomains of Bacterial Microcolonies Using Confocal and Scanning Electron Microscopy.

The sorption and distribution of nickel, a common metal contaminant in aquatic systems, were assessed in bacterial microcolonies using a combination of fluorescent staining with Newport Green and confocal laser scanning microscopy (CLSM) with confirmation by scanning electron microscopy (SEM) and X-ray microprobe analyses. CLSM with Newport Green, selected fluor-conjugated lectins, and DNA staining allowed for the discrimination of the microdomains present in the microcolony exopolymeric matrix and detection of bound nickel. This approach avoided the artefacts associated with drying and fixation required by analytical electron microscopy. The results obtained indicated that specific microcolonies within river biofilms sorbed nickel within limited microdomains present in the complex tripartite exopolymeric matrix surrounding bacterial cells. Sorption occurred such that nickel was concentrated within the exopolymeric matrix, but not directly associated with cells. These microdomains appeared to have neutral pH and be dominated by negatively charged residues favoring the sorption of nickel and other cations. These results also suggest an important role for specific community members in the sorption and concentration of metals in aquatic biofilm communities.

N,N-Diethyl-m-Toluamide Exposure at an Environmentally Relevant Concentration Influences River Microbial Community Development.

Studies of the South Saskatchewan River confirmed that N,N-diethyl-m-toluamide (DEET) is ubiquitous at 10 to 20 ng/L, whereas in effluent-dominated Wascana Creek, levels of 100 to 450 ng/L were observed. Effects of DEET exposure were assessed in microbial communities using a wide variety of measures. Communities developed in rotating annular reactors with either 100 or 500 ng/L DEET, verified using gas chromatography-mass spectrometry analyses. Microscale analyses indicated that both DEET concentrations resulted in significant (p < 0.05) declines in photosynthetic biomass, whereas bacterial biomass was unaffected. There was no detectable effect of DEET on the levels of chlorophyll a. However, pigment analyses indicated substantial shifts in algal-cyanobacterial community structure, with reductions of green algae and some cyanobacterial groups at 500 ng/L DEET. Protozoan/micrometazoan grazers increased in communities exposed to 500 ng/L, but not 100 ng/L, DEET. Based on thymidine incorporation or utilization of carbon sources, DEET had no significant effects on metabolic activities. Fluorescent lectin-binding analyses showed significant (p < 0.05) changes in glycoconjugate composition at both DEET concentrations, consistent with altered community structure. Principal component cluster analyses of denaturing gradient gel electrophoresis indicated that DEET exposure at either concentration significantly changed the bacterial community (p < 0.05). Analyses based on 16S ribosomal RNA of community composition confirmed changes with DEET exposure, increasing detectable beta-proteobacteria, whereas actinobacteria and acidimicrobia became undetectable. Further, cyanobacteria in the subclass Oscillatoriophycideae were similarly not detected. Thus, DEET can alter microbial community structure and function, supporting the need for further evaluation of its effects in aquatic habitats. Environ Toxicol Chem 2019;38:2414-2425. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Architectural adaptation and protein expression patterns of Salmonella enterica serovar Enteritidis biofilms under laminar flow conditions.

Salmonella enterica serovar Enteritidis is a significant biofilm-forming pathogen. The influence of a 10-fold difference in nutrient laminar flow velocity on the dynamics of Salmonella Enteritidis biofilm formation and protein expression profiles were compared in order to ascertain how flow velocity influenced biofilm structure and function. Low-flow (0.007 cm s(-1)) biofilms consisted of diffusely-arranged microcolonies which grew until merging by approximately 72 h. High-flow (0.07 cm s(-1)) biofilms were significantly thicker (36+/-3 microm (arithmetic mean+/-standard error; n=225) versus 16+/-2 microm for low-flow biofilms at 120 h) and consisted of large bacterial mounds interspersed by water channels. Lectin-binding analysis of biofilm exopolymers revealed a significantly higher (P<0.05) proportion of N-acetylgalactosamine (GalNAc) in low-flow biofilms (55.2%), relative to only 1.2% in high-flow biofilms. Alternatively, the proportions of alpha-L-fucose and N-acetylglucosamine (GlcNAc2)-N-acetylneuraminic acid (NeuNAc) polymer-conjugates were significantly higher (P<0.05) in high-flow biofilms (69.1% and 29.6%, respectively) than low-flow biofilms (33.1% and 11.7%, respectively). Despite an apparent flow rate-based physiologic effect on biofilm structure and exopolymer composition, no major shift in whole-cell protein expression patterns was seen between 168 h-old low-flow and high-flow biofilms, and notably did not include any response involving the stress response proteins, DnaK, SodB, and Tpx. Proteins involved in degradation and energy metabolism (PduA, GapA, GpmA, Pgk, and RpiA), RNA and protein biosynthesis (Tsf, TufA, and RpoZ), cell processes (Crr, MalE, and PtsH), and adaptation (GrcA), and some hypothetical proteins (YcbL and YnaF) became up-regulated in both biofilm systems relative to a 168 h-old planktonic cell control. Our results indicate that Salmonella Enteritidis biofilms altered their structure and extracellular glycoconjugate composition in response to flow and this response is suggested to be significant in the survival of this pathogen as biofilms.

Structural and functional responses of river biofilm communities to the nonsteroidal anti-inflammatory diclofenac.

Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) that has been detected widely in surface waters in North America and Europe. The impact of diclofenac on river biofilm communities was investigated at exposures of 10 and 100 microg L(-1) of diclofenac or its molar equivalent in carbon and nutrients. Experiments were carried out with river water during spring and summer using rotating annular reactors as model systems. Diclofenac or nutrients at 10 microg L(-1) were observed to have no significant effect on algal, bacterial, and cyanobacterial biomass in spring, whereas in the summer the nutrient equivalent reduced algal biomass and diclofenac reduced cyanobacterial biomass relative to control biofilms (p < 0.05). In contrast, at 100 microg L(-1) diclofenac or nutrients, the result was increased cyanobacterial and bacterial biomass, respectively, relative to control biofilms in spring. In summer, 100 microg L(-1) diclofenac significantly increased bacterial biomass and the nutrient treatment had no significant effect (p < 0.05); both treatments resulted in increased biofilm thickness. The glycoconjugate composition of the exopolysaccharide matrix was influenced differentially by the treatments in both seasons. Biolog assessments of carbon use indicated that 100 microg L(-1) diclofenac or nutrients resulted in significant depressions in the use of carbon sources in summer and significant increases in spring. Impacts on protozoan and micrometazoan populations also were assessed. Denaturing gradient gel electrophoresis analyses of community DNA and fluorescent in situ hybridization studies indicated that diclofenac had significant effects on the nature of the bacterial community in comparison with control and nutrient-treated river biofilm communities.

Quantitative mapping of chlorhexidine in natural river biofilms.

Soft X-ray scanning transmission X-ray microscopy has been applied to map chlorhexidine, a ubiquitous antimicrobial agent, relative to major biochemical components (proteins, lipids, polysaccharides, Ca2+, K+, CO3(2-)) in natural river biofilms. For the first time, bio-accumulation of chlorhexidine in diatoms has been observed unambiguously. The quantitative results show that chlorhexidine bioaccumulated extensively in lipid-rich regions of diatoms and bacteria. Confocal laser scanning microscopy was used to document changes in the biofilm community. The bioaccumulation provides a significant entry point for chlorhexidine into the aquatic food chain. It results in modification of the biofilm community and it impacts the photosynthetic and protozoan species in particular. X-ray microscopy mapping at high spatial resolution is shown to be a powerful tool for studies of antimicrobial agents in the environment.

Molecular and microscopic assessment of the effects of caffeine, acetaminophen, diclofenac, and their mixtures on river biofilm communities.

The authors examined effects of three common contaminants, caffeine (CF), acetaminophen (AC), and diclofenac (DF), as well as their mixtures on the development, functioning, and biodiversity of river biofilm communities. Biofilms were cultivated in rotating annular reactors. Treatments included AC, CF, DF, AC + CF, AC + DF, CF + DF, AC + CF + DF at 5 µg/L, and their molar equivalent as carbon and nutrients. Incubations using ¹4C-labeled AC, DF, and CF indicated that 90% of the CF, 80% of the AC, and less than 2% of the DF were converted to CO2. Digital imaging revealed a variety of effects on algal, cyanobacterial, and bacterial biomass. Algal biomass was unaffected by AC or CF in combination with DF but significantly reduced by all other treatments. Cyanobacterial biomass was influenced only by the AC + DF application. All treatments other than AC resulted in a significant decrease in bacterial biomass. Diclofenac or DF + CF and DF + AC resulted in increases in micrometazoan grazing. The denaturing gradient gel electrophoresis of Eubacterial community DNA, evaluated by principal component analysis and analysis of similarity, indicated that relative to the control, all treatments had effects on microbial community structure (r = 0.47, p < 0.001). However, the AC + CF + DF treatment was not significantly different from its molar equivalent carbon and nutrient additions. The Archaeal community differed significantly in its response to these exposures based on community analyses, confirming a need to integrate these organisms into ecotoxicological studies.

Morphological and biochemical changes in Pseudomonas fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial agents.

Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of biomacromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 microg/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.

Speciation and quantitative mapping of metal species in microbial biofilms using scanning transmission X-ray microscopy.

A scanning transmission X-ray microscope illuminated with synchrotron light was used to investigate the speciation and spatial distributions of metals in a microbial biofilm cultivated from river water. Metal 2p absorption edge signals were used to provide metal speciation (through shapes of the absorption spectra) and quantitative spatial distributions of the metal species. This paper presents sample data and describes methods for extracting quantitative maps of metal species from image sequences recorded in the region of the metal 2p edges. Comparisons were made with biochemical characterization of the same region using images recorded at the C 1s and O 1s edges. The method is applied to detailed quantitative analysis of ferrous and ferric iron in a river biofilm, in concert with mapping Ni(II) and Mn(II) species in the same region. The distributions of the metal species are discussed in the context of the biofilm structure. These results demonstrate that soft X-ray STXM measurements at the metal 2p absorption edges can be used to speciate metals and to provide quantitative spatial distribution maps for metal species in environmental samples with 50 nm spatial resolution.

Effects of selected pharmaceuticals on riverine biofilm communities.

Although pharmaceutical and therapeutic products are widely found in the natural environment, there is limited understanding of their ecological effects. Here we used rotating annular bioreactors to assess the impact of 10 microg.L(-1) of the selected pharmaceuticals ibuprofen, carbamazepine, furosemide, and caffeine on riverine biofilms. After 8 weeks of development, community structure was assessed using in situ microscopic analyses, fluor-conjugated lectin binding, standard plate counts, fluorescent in situ hybridization, carbon utilization spectra, and stable carbon isotope analyses. The biofilm communities varied markedly in architecture although only caffeine treated biofilms were significantly thicker. Cyanobacteria were suppressed by all 4 compounds, whereas the nitrogen containing caffeine, furosemide, and carbamazepine increased algal biomass. Ibuprofen and carbamazepine reduced bacterial biomass, while caffeine and furosemide increased it. Exopolymer content and composition of the biofilms was also influenced. Significant positive and negative effects were observed in carbon utilization spectra. In situ hybridization analyses indicated all treatments significantly decreased the gamma-proteobacterial populations and increased beta-proteobacteria. Ibuprofen in particular increased the alpha-proteobacteria, beta-proteobacteria, cytophaga-flavobacteria, and SRB385 probe positive populations. Caffeine and carbamazepine additions resulted in significant increases in the high GC354c and low GC69a probe positive cells. Live-dead analyses of the biofilms indicated that all treatments influenced the ratio of live-to-dead cells with controls having a ratio of 2.4, carbamazepine and ibuprofen being 3.2 and 3.5, respectively, and furosemide and caffeine being 1.9 and 1.7, respectively. Stable isotope analyses of the biofilms indicated delta 13C values shifted to more negative values relative to control biofilms. This shift may be consistent with proportional loss of cyanobacteria and relative increase in algal biomass rather than incorporation of pharmaceutical carbon into microbial biofilm. Thus, at 10 microg.L(-1) levels pharmaceuticals exhibit both nutrient-like and toxic effects on riverine microbial communities.

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.

Bacteria associated with cysts of the soybean cyst nematode (Heterodera glycines).

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 +/- 0.5) x 10(5) bacteria (mean +/- standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and STREPTOMYCES: A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.