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.

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.

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.

Bacterial contamination of tile drainage water and shallow groundwater under different application methods of liquid swine manure.

A 2 year field experiment evaluated liquid manure application methods on the movement of manure-borne pathogens (Salmonella sp.) and indicator bacteria (Escherichia coli and Clostridium perfringens) to subsurface water. A combination of application methods including surface application, pre-application tillage, and post-application incorporation were applied in a randomized complete block design on an instrumented field site in spring 2007 and 2008. Tile and shallow groundwater were sampled immediately after manure application and after rainfall events. Bacterial enumeration from water samples showed that the surface-applied manure resulted in the highest concentration of E. coli in tile drainage water. Pre-tillage significantly (p < 0.05) reduced the movement of manure-based E. coli and C. perfringens to tile water and to shallow groundwater within 3 days after manure application (DAM) in 2008 and within 10 DAM in 2007. Pre-tillage also decreased the occurrence of Salmonella sp. in tile water samples. Indicator bacteria and pathogens reached nondetectable levels within 50 DAM. The results suggest that tillage before application of liquid swine manure can minimize the movement of bacteria to tile and groundwater, but is effective only for the drainage events immediately after manure application or initial rainfall-associated drainage flows. Furthermore, the study highlights the strong association between bacterial concentrations in subsurface waters and rainfall timing and volume after manure application.

Evaluation of Petrifilm EC method for enumeration of E. coli from soil.

AIMS: To evaluate the suitability of commercially available Petrifilm EC plates for enumeration of Escherichia coli from soil. METHODS AND RESULTS: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10(2), 10(3), 10(5) CFU g(-1) of soil. The efficiency of recovery on Petrifilm EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m-FC basal medium supplemented with 3-bromo-4-chloro-5-indoyl-beta-D-glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) broth. Petrifilm EC and m-FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0.05) were observed between Petrifilm EC, m-FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure-applied field soil samples showed a significant difference (P < 0.05) between the Petrifilm EC method and the m-FC method in enumerating E. coli possibly as a result of false positives on m-FC. CONCLUSION: The Petrifilm EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g(-1) soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The commercially available Petrifilm EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.