Multiple roles of extracellular polymeric substances on resistance of biofilm and detached clusters.

In this study, multiple roles of biofilm EPS were assessed with respect to the resistance of biofilm and detached biofilm clusters to chlorine disinfection. Strains from an opportunistic pathogen, Pseudomonas aeruginosa (wild type, EPS- and EPS+) with altered extracellular polymeric substances (EPS) secretion capabilities were tested. The impact of biofilm EPS quantity on disinfection was evaluated by monitoring biofilm viability, biofilm structure, removal of dissolved organic matter (DOM), and viability of detached biofilm simultaneously during chlorine disinfection. The obtained results suggested that the presence of EPS increased biofilm and detached biofilm resistance to chlorine in both presence and absence of DOM. The quantity of EPS had an effect on biofilm structure and the structural characteristics were closely related to both overall biofilm viability and the spatial distribution of viable cells within the biofilm. Additionally, the increased amount of EPS influenced selective removal of DOM with polar functional groups. However the DOM removal did not have a significant impact on the viability of biofilm cells during chlorine disinfection. Meanwhile, the viability of detached biofilm clusters, particularly the EPS overproducing strain, was significantly increased in the presence of DOM. The combined results suggested that biofilm EPS played multiple roles toward influencing the resistance of both biofilm and detached biofilm to disinfectant.

Detection and quantification of Microcystis spp. and microcystin-LR in Western Lake Erie during the summer of 2007.

Microcystis spp. blooms have occurred annually in western Lake Erie since about 1995. Microcystis produce a group of toxins known as microcystins which can be harmful to livestock and to humans. In this study, surface water samples were collected from six sites during six sampling events from July to October in 2007. In situ environmental data (e.g. pH, temperature) and laboratory analyses (e.g. nutrients) were carried out to characterize the six sites. The Microcystis spp. density ranged from 10(2) to 10(7) cells/ml. Microcystin-LR concentration of 20 of all 36 samples were below the detection limit (0.15-5 ppb), while the microcystin-LR concentration in the 16 remaining samples ranged from 0.5 to 3 x 10(3) microg per gram dry weight. The aim of this research was to investigate the relationships between sampling location, environmental parameters, Microcystis spp. concentration, and microcystin-LR concentration. The results suggest that temperature, nutrient concentration, turbidity, and wind speed and direction (P<0.05) are factors which affected Microcystis spp. density. Sampling site 8M, located 13 m from the Maumee River, provided an advantage for Microcystis spp. growth, presumably due to intermediate water depth (5.5 m) combined with impact from the river. No relationship was found between Microcystis spp. density and microcystin-LR concentration. Temperature, nutrient concentration and DO (P<0.05) were associated with the production of microcystin-LR.

Activity assessment of microorganisms eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium chloride: a quantitative comparison of flow cytometry to epifluorescent microscopy.

Enhanced natural recovery may be successfully implemented at contaminated sediment sites, which are often characterized by large volumes of sediments with low to moderate levels of contamination to cost-effectively reduce human and ecological risks. In order to evaluate the potential for microbial contribution to remediation strategies, physiological assessment of indigenous microorganisms is essential. We report here a method for rapid and accurate assessment of metabolically (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]) active microorganisms eluted from sediment, based on flow cytometry (FCM). Microorganisms eluted from sediment and suspended in estuarine medium were stained with CTC and counterstained with the DNA stain Picogreen (PG). Optimal stain concentrations and incubation times were employed. FCM quantification of the dual-stained microorganisms was not statistically different (paired t test; alpha=0.05; df=10) from enumeration (total or active numbers) by an established method (fluorescent microscopy) over two orders of magnitude (approximately 10(4)-10(6)/ml). This research suggests that FCM, which allows the collection and analysis of multiple parameters (light scatter and fluorescence emission), is a good candidate for microbial characterization in complex environmental matrices, such as sediments, across a broad range of activity levels (approximately 2% to 84% of total). Potential applications for this FCM-based method include the rapid assessment of changes in sediment microbial activity in response to enhanced bioremediation strategies.

5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in a mesophilic anaerobic digester: measuring redox behavior, differentiating abiotic reduction, and comparing FISH response as an activity indicator.

The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been widely applied to assess microbiological activity in environmental samples. CTC reduction has previously been quantified in a variety of anaerobic systems (i.e., fermentative, nitrate reducing, sulfate reducing) using direct microscopy, solvent extraction, and flow cytometry. In this work, extracellular CTC reduction was observed and distinguished from its intercellular counterparts by the amorphous character and near uniform fluorescence of the resulting formazan precipitates (CTF). Fluorescence yielded by non-cellular-associated formazan precipitates bleached much more rapidly than CTF formed within cells under identical UV exposure (<2 min). Dehydrogenase activity assays and fluorescent in situ hybridization (FISH) were simultaneously carried out in microcosms containing active anaerobic digester biomass, propylene glycol, and settled sewage centrate for direct comparison. In substrate limited microcosms, quantitative FISH measurements remained well above their detection limit indicating sustained intercellular ribosomal RNA concentrations over a 5-day period, while dehydrogenase assays (CTC) decreased to background levels within 14 h of substrate limitation. Results from this work suggest that CTC reduction in cell-free samples may impede accurate enzyme activity measurements, particularly when quantification involves solvent extraction, flow cytometry, or software-aided counting. In addition, activity assessment in anaerobic digesters using FISH and CTC reduction assays may be comparable until substrate becomes limited.

Anaerobic digestion of aircraft deicing fluid wastes: interactions and toxicity of corrosion inhibitors and surfactants.

Corrosion inhibitors and surfactants are present in aircraft deicing fluids (ADFs) at significant concentrations (> 1% w/w). The purpose of this research was to study the interactions of a common nonionic surfactant with the commercially significant corrosion inhibitors used in modern ADF (4- and 5-methylbenzotriazole [MeBT]), and to determine the effects of their mixture on the conventional anaerobic digestion process. In mesophilic anaerobic microcosms codigesting wastewater solids, propylene glycol, and MeBT, increasing surfactant levels resulted in enhanced MeBT sorption on digester solids. As judged by anaerobic toxicity assays, responses from digesters containing surfactant concentrations below their critical micelle concentration (CMC) suggested that low nonionic surfactant concentrations could facilitate a reduction in the apparent toxicity of MeBT. In microcosms exposed to surfactant concentrations above their CMC, no increase in MeBT solubility was observed, and the anaerobic toxicity response corresponded to control systems not containing surfactant. Direct microscopic measurements of digesting biomass using fluorescent phylogenetic probes (fluorescent in situ hybridization) revealed that members of the domain Bacteria were more sensitive to MeBT in the presence of surfactant than were members of the domain Archaea.

A multiphasic characterization of the impact of the herbicide acetochlor on freshwater bacterial communities.

Acetochlor is the third most frequently detected herbicide in natural waters; however, it is unknown if exposure to environmentally relevant concentrations of acetochlor will impact bacterial community structure and function. This study examined the impact of acetochlor on freshwater heterotrophic bacteria number, and community structure and function using direct counting, community level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) analysis. Acetochlor concentration did not appear to correlate with the number of total (P=0.69) and viable (P=0.80) bacteria, even at concentrations up to 500 microg l(-1). However, CLPP indicated that acetochlor increased functional diversity as shown by (i) an increase in the number of carbon sources utilized by the microbial community, relative to nonexposed controls and (ii) increased functional evenness within the heterotrophic bacterial community. Conversely, DGGE fingerprints suggested that exposure to acetochlor generally decreased the community complexity, as the average number of DGGE bands in most treatments was significantly less than in the control treatment. Cluster analysis of DGGE fingerprints revealed three distinct, dose-dependent clusters (i) communities exposed to 0, 1 and 5 microg l(-1); (ii) 50 and 100 microg l(-1) and (iii) 500 microg l(-1), indicating a relationship between acetochlor concentration bacterial community changes. This study indicated that while exposure to environmentally relevant concentrations of acetochlor resulted in no significant impact to the number of freshwater bacteria, impacts to the function and structure of the community were revealed by adopting a multiphasic approach.

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids.

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.