George D. W. Swerhone

Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

ABSTRACT Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.

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.

Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms

Abstract Techniques are required for the simultaneous or sequential determination of multiple parameters within microbial biofilms. Confocal scanning laser microscopy in combination with a range of fluorescent probes and markers offers an approach to quantitatively defining many aspects of biofilm communities. By applying multispectral imaging in conjunction with nucleic acid stains, fluor conjugated lectins, and autofluorescence we have developed a simple approach to evaluate biofilm community composition. Biofilms were treated with the fluorescent nucleic acid stain SYTO 9 to allow quantification of bacterial biomass and fluor conjugated lectins (i.e., Triticum vulgaris lectin) to identify and allow quantification of exopolymeric substances. Far red autofluorescence was imaged to quantify algal biomass. Digital image analysis of the CSLM optical thin sections in each of the channels was used to determine such parameters as biofilm depth, bacterial cell area (biomass), exopolymer area and algal biomass at various depths and locations. In addition, three colour red–green–blue projections of the biofilms were computed. The method proved simple and effective for determining treatment effects such as grazing by invertebrates.

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.

A simple rotating annular reactor for replicated biofilm studies

The performance of two types of rotating annular reactors for the cultivation of river biofilms was compared qualitatively and quantitatively. One reactor was a commercially available system with a rotating inner solid cylinder and polycarbonate slides in the outer fixed cylinder. The other, a non-commercial system manufactured in the laboratory, had the polycarbonate slides positioned on a machined, rotating inner cylinder. Microscale comparison of the biofilms was carried out using confocal laser scanning microscopy techniques including, fluorescent nucleic acid staining, fluor conjugated lectins and autofluorescence imaging. The results obtained indicated that the reactors were similar in terms of biofilm development pattern, thickness, bacterial biomass, and exopolymer production. Significant differences were found in terms of photosynthetic biomass with the glass bodied non-commercial reactor providing more favourable conditions for algal growth than the opaque polycarbonate outer cylinder of the commercial reactor. The study indicated that a simple inexpensive reactor constructed from available components and materials, produced river biofilms similar to those obtained using a commercial system but at substantially lower cost. The availability of such inexpensive annular reactors should facilitate much needed replicated studies of biofilm development.

Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines)

ABSTRACT 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) × 105 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.

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.

Galvanic sulphide oxidation as a metal-leaching mechanism and its environmental implications

Following a brief review of the theoretical aspects of galvanic sulphide oxidation, the significance of galvanic interaction in the oxidation of natural mixed sulphide assemblages is demonstrated using test materials prepared from sulphide-containing rocks in a series of chemical and microbial weathering experiments. The test results indicate that: (1) metal leaching can effectively proceed even in a near-neutral environment; and (2) the occurrence of acid mine drainage can be delayed due to cathodic protection of an acid-generating sulphide such as pyrite from oxidative dissolution. While microbial mediation may enhance the weathering of sulphides with a high electrode potential, the competing galvanic processes diminish the dominating role of the microbes in effecting the oxidation of sulphides with a low electrode potential when the sulphides occur in a mixed assemblage. In-situ potential measurements on sulphide surfaces with micro-electrodes demonstrated the occurrence of a significant potential difference between a contacting sulphide pair sufficient to sustain galvanic interaction for the duration of the experiments. It is also shown that galvanic sulphide oxidation and hence metal leaching can occur even under an oxygenated water cover. Therefore, subaqueous disposal may not be the best management option for all reactive mine wastes, especially those containing metals or trace elements that remain mobile under near-neutral pH conditions.

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.

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.