Ecotoxicological effects of copper oxide nanoparticles (nCuO) on the soil microbial community in a biosolids-amended soil.

This study represents a holistic approach in assessing the effects of copper oxide nanoparticles (nCuO) on microbial health and community structure in soil amended with municipal biosolids. The biosolids were amended with nCuO (<50 nm) and mixed into a sandy loam soil at measured Cu concentrations of 27, 54, 123, 265 and 627 mg Cu kg-1 soil. A suite of tests were used to assess the potential impact of nCuO on microbial growth, activity, and diversity. Microbial growth was determined by the heterotrophic plate count (HPC) method, while microbial diversity was assessed using both community level physiological profiling (CLPP) and 16S ribosomal DNA (rDNA) sequencing. Microbial activity was assessed by examining soil nitrification, organic matter decomposition, soil respiration (basal and substrate induced) and soil enzyme assays for dehydrogenase, phosphatase and ß-glucosidase activities. As a readily soluble positive control, copper sulfate (CuSO4) was used at measured Cu concentrations of 65, 140, 335 and 885 mg Cu kg-1 soil for select tests, and at the highest concentration for the remaining tests. Analysis on Cu bioavailability revealed that extractable Cu2+ was higher in CuSO4-spiked soils than nCuO-spiked soils. At a nCuO exposure concentration of ≤265 mg Cu kg-1 soil, stimulatory effects were observed in nitrification, ß-glucosidase and community level physiological profiling (CLPP) tests. nCuO showed no significant inhibitory effects on the soil microbial growth, activity or diversity at the highest concentration (i.e. 627 mg Cu kg-1 soil), with the exception of the dehydrogenase (i.e. ≥27 mg Cu kg-1 soil) and phosphatase (i.e. 627 mg Cu kg-1 soil) enzyme activities. In contrast, inhibition from CuSO4 at 885 mg Cu kg-1 soil was observed in all tests with the exception of ß-glucosidase enzyme activity. The growth of a Cu tolerant bacterium, Rhodanobacter sp., was observed at 885 mg Cu kg-1 soil (CuSO4).

Evaluation of denaturing gradient gel electrophoresis (DGGE) and next generation sequencing (NGS) in combination with enrichment culture techniques to identify bacteria in commercial microbial-based products.

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared. Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions. This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs.

Draft Genome Sequence of the Industrially Significant Bacterium Pseudomonas fluorescens ATCC 13525.

Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolic functions and potential industrial uses. We sequenced P. fluorescens strain ATCC 13525 with the goal of determining virulence factors and antibiotic resistance genes to predict the potential impacts on human and environmental health in the event of exposure.

Bacterial and fungal composition profiling of microbial based cleaning products.

Microbial based cleaning products (MBCPs) are a new generation of cleaning products that are gaining greater use in household, institutional, and industrial settings. Little is known about the exact microbial composition of these products because they are not identified in detail on product labels and formulations are often proprietary. To gain a better understanding of their microbial and fungal composition towards risk assessment, the cultivable microorganisms and rDNA was surveyed for microbial content in five different MBCPs manufactured and sold in North America. Individual bacterial and fungal colonies were identified by ribosequencing and fatty acid methyl ester (FAME) gas chromatography. Metagenomic DNA (mDNA) corresponding to each of the products was subjected to amplification and short read sequencing of seven of the variable regions of the bacterial 16S ribosomal DNA. Taken together, the cultivable microorganism and rDNA survey analyses showed that three of the products were simple mixtures of Bacillus species. The two other products featured a mixture of cultivable fungi with Bacilli, and by rDNA survey analysis, they featured greater microbial complexity. This study improves our understanding of the microbial composition of several MBCPs towards a more comprehensive risk assessment.

Development of a microbial test suite and data integration method for assessing microbial health of contaminated soil.

There is no standard methodology or guideline for assessing soil microbial health for the purposes of contaminant risk assessments. Here we propose a laboratory-based test suite and novel data integration method for evaluating soil microbial health using site-specific contaminated and reference soil. The test suite encompasses experiments for evaluating microbial biomass, activity, and diversity. The results from the tests are then integrated so that a Soil Microbial Health Score (SMHS) may be assigned. This test suite and data integration method was tested on soils from 3 different contaminated sites in Canada. The soil microbial health of a petroleum hydrocarbon (PHC) contaminated site was found to be 'Mildly Impacted' and 'Moderately Impacted' for two soil horizons at a boreal forest site. The soil microbial health of the mixed metal/PHC and mixed metal sites were both found to be 'Not Impacted'. Continued use of this test suite and data integration method will help create guidelines for assessing soil microbial health in ecological risk assessments.

Effect of silver nano-particles on soil microbial growth, activity and community diversity in a sandy loam soil.

Silver nano-particles (AgNPs) are widely used in a range of consumer products as a result of their antimicrobial properties. Given the broad spectrum of uses, AgNPs have the potential for being released to the environment. As a result, environmental risks associated with AgNPs need to be assessed to aid in the development of regulatory guidelines. Research was performed to assess the effects of AgNPs on soil microbial activity and diversity in a sandy loam soil with an emphasis on using a battery of microbial tests involving multiple endpoints. The test soil was spiked with PVP coated (0.3%) AgNPs at the following concentrations of 49, 124, 287, 723 and 1815 mg Ag kg-1 dry soil. Test controls included an un-amended soil; soil amended with PVP equivalent to the highest PVP concentration of the coated AgNP; and soil amended with humic acid, as 1.8% humic acid was used as a suspension agent for the AgNPs. The impact on soil microbial community was assessed using an array of tests including heterotrophic plate counting, microbial respiration, organic matter decomposition, soil enzyme activity, biological nitrification, community level physiological profiling (CLPP), Ion Torrent™ DNA sequencing and denaturing gradient gel electrophoresis (DGGE). An impact on microbial growth, activity and community diversity was evident from 49 to 1815 mg kg-1 with the median inhibitory concentrations (IC50) as low as 20-31 mg kg-1 depending on the test. AgNP showed a notable impact on microbial functional and genomic diversity. Emergence of a silver tolerant bacterium was observed at AgNP concentrations of 49-287 mg kg-1 after 14-28 days of incubation, but not detectable at 723 and 1815 mg kg-1. The bacterium was identified as Rhodanobacter sp. The study highlighted the effectiveness of using multiple microbial endpoints for inclusion to the environmental risk assessment of nanomaterials.

Comparative assessment of next-generation sequencing, denaturing gradient gel electrophoresis, clonal restriction fragment length polymorphism and cloning-sequencing as methods for characterizing commercial microbial consortia.

Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants.

Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms.

To augment the information on commercial microbial products, we investigated the persistence patterns of high-priority bacterial strains from the Canadian Domestic Substance List (DSL). Specific DNA markers for each of the 10 DSL bacterial strains were developed using the amplified fragment length polymorphism (AFLP) technique, and the fates of DSL strains introduced in soil were assessed by real-time quantitative PCR (qPCR). The results indicated that all DNA markers had high specificity at the functional strain level and that detection of the target microorganisms was sensitive at a detection limitation range from 1.3 × 10² to 3.25 × 105 CFU/g of dry soil. The results indicated that all introduced strains showed a trend toward a declining persistence in soil and could be categorized into three pattern types. The first type was long-term persistence exemplified by Pseudomonas stutzeri (ATCC 17587) and Pseudomonas denitrificans (ATCC 13867) strains. In the second pattern, represented by Bacillus subtilis (ATCC 6051) and Escherichia hermannii (ATCC 700368), the inoculated strain populations dropped dramatically below the detection threshold after 10 to 21 days, while in the third pattern there was a gradual decrease, with the population falling below the detectable level within the 180-day incubation period. These patterns indicate a selection effect of a microbial community related to the ecological function of microbial strains introduced in soil. As a key finding, the DSL strains can be quantitatively tracked in soil with high sensitivity and specificity at the functional strain level. This provides the basic evidence for further risk assessment of the priority DSL strains.

Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment.

Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines.

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

Fluorescent methods to study DNA, RNA, proteins and cytoplasmic membrane polarization in the pentachlorophenol-mineralizing bacterium Sphingomonas sp. UG30 during nutrient starvation in water.

The effect of sodium pentachlorophenolate (NaPCP) exposure on the nutrient-starved pentachlorophenol (PCP)-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescent methods to measure DNA, RNA, total cellular protein, and cytoplasmic membrane proteins. UG30 cells were inoculated into sterilized Speed River (Guelph, ON, Canada) water samples in the presence of 50, 100, and 250 ppm NaPCP. No marked changes were observed in the total cellular DNA, RNA or protein levels over 90 d, indicating the macromolecular composition of UG30 was not affected by both nutrient limitation and NaPCP. Total cell counts as determined by DAPI staining also did not change over 90 d. Over the same period, viable counts decreased with increasing concentrations of NaPCP. At 250 ppm NaPCP, viable cell counts decreased over 6 orders of magnitude after 1 hr exposure. Cell numbers partially recovered once NaPCP was degraded. The UG30 cytoplasmic membrane polarization ratio also decreased after NaPCP was depleted. The decreased polarization value at the end of the study period suggested the UG30 membrane was more fluid and that this increase in fluidity was due to nutrient starvation effects rather than exposure to NaPCP. These results indicated that UG30 is a robust organism that is able to degrade NaPCP even under adverse conditions and fluorescent methods are useful for determining macromolecular concentrations and cytoplasmic membrane polarization values.

Polychlorinated biphenyl (PCB) degradation and persistence of a gfp-marked Ralstonia eutropha H850 in PCB-contaminated soil.

Ralstonia eutropha H850 was labelled chromosomally with a gfp marker gene encoding for the green fluorescent protein, and designated R. eutropha H850g13. Visual observation of green fluorescent cells under an epifluorescence microscope, and PCR amplification products, confirmed that the bacterium was labelled with gfp. Southern blot hybridization products further confirmed the gfp was chromosomally labelled. Using resting cell assays, it was determined that insertion of the gfp gene decreased the microorganisms' ability to degrade biphenyl compared to the parent strain. However, this marker facilitated the identification and monitoring of R. eutropha H850g13 survival in soil microcosm experiments. Survival and polychlorinated biphenyl degradation by R. eutropha H850g13 was analysed in soil microcosms spiked with 2,2',5,5'-tetrachlorobiphenyl (TeCB). R. eutropha H850g13 was detected by viable plate counts and most-probable-number/PCR after 102 days in TeCB-contaminated soil microcosms, and was likely outcompeted by indigenous soil microorganisms in microcosms amended with oil and Daramend (an organic amendment, http://www.adventusremediation.com). R. eutropha H850g13 did not degrade TeCB in any of the soil microcosms. This research confirmed that gfp was useful as a marker to distinguish R. eutropha H850g13 from indigenous soil microorganisms over a 102 day period and that, under the experimental conditions used, R. eutropha H850g13 did not degrade TeCB.

Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes.

Membranes lipids are one of the most adaptable molecules in response to perturbations. Even subtle changes of the composition of acyl chains or head groups can alter the packing arrangements of lipids within the bilayer. This changes the balance between bilayer and nonbilayer lipids, serving to affect bilayer stability and fluidity, as well as altering lipid-protein interactions. External factors can also change membrane fluidity and lipid composition; including temperature, chemicals, ions, pressure, nutrients and the growth phase of the microbial culture. Various biophysical techniques have been used to monitor fluidity changes within the bacterial membrane. In this review, bacterial cytoplasmic membrane changes and related functional effects will be examined as well as the use of fluorescence polarization methods and examples of data obtained from research with bacteria.

Characterization of tetrachlorohydroquinone reductive dehalogenase from Sphingomonas sp. UG30.

Tetrachlorohydroquinone reductive dehalogenase (PcpC) is the second of three enzymes that catalyze the initial degradation of pentachlorophenol in Sphingomonas sp. UG30 and several other bacterial strains. The UG30 PcpC shares a high degree (94%) of primary sequence identity with the well-studied PcpC from Sphingobium chlorophenolicum ATCC 39723. Significant differences, however, were observed between the two PcpC enzymes in some of their functional and kinetic properties. The temperature optimum of the UG30 PcpC is 10 degrees C higher and the pH optimum is approximately 2 units higher than the S. chlorophenolicum PcpC. In addition, the S. chlorophenolicum PcpC is subject to inhibition by the substrate tetrachlorohydroquinone (TCHQ), and this has necessitated the use of a mutant enzyme, which was not inhibited by TCHQ, for kinetic studies. In contrast, the UG30 PcpC was not inhibited by TCHQ and this may allow detailed kinetic and mechanistic studies using the wild-type enzyme.

Survival of a GFP-Labeled Polychlorinated Biphenyl Degrading Psychrotolerant Pseudomonas spp. in 4 and 22 degrees C Soil Microcosms.

The green fluorescent protein gene (gfp) was inserted into the chromosome of Pseudomonas spp. Cam-1 and Sag-50G, two psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria. The gfp-transformed microorganisms, designated Cam-1-gfp1, Cam-1-gfp2, Sag-50G-gfp1, and Sag-50G-gfp2, exhibited green fluorescence under an epifluorescent microscope. The gfp was inserted into the chromosome of each psychrotolerant strain and was stable with no apparent adverse affects on the metabolism and growth of each organism. Activity of gfp-transformed microorganisms against biphenyl and 2,3-dichlorobiphenyl was determined by assaying for BphC activity and by resting cell assays. The patterns of BphC activity at two different growth temperatures in batch cultures were similar for each of the gfp-transformed microorganisms. Resting cell assays of both the parent strains (Cam-1, Sag-50G) and the gfp-transformed strains (Cam-1-gfp1, Cam-1-gfp2, Sag-50G-gfp1, Sag-50G-gfp2), grown on glycerol or glucose, exhibited BphC activity to a lesser extent and at a slower rate than those observed for biphenyl grown cells. In addition, all gfp-transformed microorganisms degraded 2,3-dichlorobiphenyl (2,3-DCB) in broth to the same extent as the parent strains. When Cam-1-gfp1 and Sag-50G-gfp1 were used as a bioremediation amendment in soil microcosms spiked with 2,3-DCB, both strains survived in high numbers (5.6 to 7.9 log cfu g?1 and 5.6 to 8.0 log cfu g?1) when inoculated into nonsterilized soil over 16 weeks at 22 degrees C and 18 weeks at 4 degrees C, respectively. Biodegradation of 2,3-DCB was enhanced with the microbial amendment; however, the addition of sunflower oil did not help the PCB degrading bacteria and may have enhanced the growth of the indigenous population, thereby decreasing the amended PCB-degrading population.

Low surfactant concentration increases fungal mineralization of a polychlorinated biphenyl congener but has no effect on overall metabolism.

Three white rot fungi were compared for their ability to attack polychlorinated biphenyl (PCB) congeners in the presence and absence of the non-ionic Triton X-100 or the anionic Dowfax 8390 surfactants at half their critical micelle concentrations. Neither surfactant affected PCB biodegradation monitored by gas chromatography but the release of 14CO2 from 2,4',5-[U-14C]trichlorobiphenyl by Trametes versicolor was stimulated 12% by Triton X-100. Since mineralization is the complete metabolism of the congener and biodegradation was measured as substrate disappearance, Triton X-100 is proposed to aid intracellular solubilization of 2,4',5-trichlorobiphenyl for complete oxidation by T. versicolor.

Comparison of gas chromatography and mineralization experiments for measuring loss of selected polychlorinated biphenyl congeners in cultures of white rot fungi.

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4'-dichlorobiphenyl, 2,4',5-trichlorobiphenyl (2,4',5-TCB), 2,2',4,4'-tetrachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl. The congener tested for mineralization was 2,4',5-[U-14C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower,