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.

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.