Biodegradability of unheated and laboratory heated dissolved organic matter.

Following wildfires, partially combusted biomass remains on the forest floor and erosion from the landscape can release dissolved pyrogenic organic matter (dPyOM) to surface waters. Therefore, post-fire alterations to dissolved organic matter (DOM) in aquatic systems may play a vital role in DOM stability and biogeochemical cycles. Dissolved PyOM biodegradation remains poorly understood and is expected to vary with combustion temperature and fuel source. In this study laboratory heating and leaching of forest floor materials (soil and litter) were used to compare the biodegradability of unheated, low (250 °C), and moderate (450 °C) temperature leachates. Inoculation experiments were performed with river microbes. Dissolved organic carbon (DOC) and nitrogen (DON), inorganic nitrogen, and DOM optical properties were monitored for 38 days. Inoculation experiments showed significantly greater DOC biodegradation of low and moderate temperature samples (64% and 71%, respectively) compared to unheated samples (32%). The greater DOC biodegradation may be explained by lower molecular weight DOM composition of heated leachates which was supported by higher initial E2/E3 ratios (absorbance at 250 nm/365 nm). Further, the observed decrease in the E2/E3 ratio after incubation suggests biodegradation of smaller compounds. This trend was greater for heated samples than unheated DOM. Specific ultraviolet absorbance increased after incubation, suggesting biodegradation of aliphatic compounds. Inoculated moderate temperature samples showed the greatest DON degradation (74%), followed by low temperature (58%) and unheated (51%) samples. Overall, results suggest that low and moderate temperature dPyOM was more biodegradable than unheated DOM, which may have implications for aquatic biogeochemical cycling, ecosystem function, and water quality in fire-impacted watersheds.

Fate and transport of biological microcontaminants bound to microplastics in the soil environment.

Microplastics, fragmented plastic particles with a maximum dimension <5 mm, are an emerging contaminant of concern that can also serve as a vector of other chemical and biological contaminants. Compared to chemical contaminants, the potential of microplastics to adsorb biological microcontaminants such as antibiotic resistance genes, small interference RNAs, and pathogenic viruses is not well understood. Many current microplastic studies are based in the aquatic environment (freshwater, seawater, and wastewater), even though the terrestrial environment is considered both an important sink and source of microplastics. Microplastics co-occur with biological microcontaminants in many terrestrial environments including agricultural soils, where biosolids containing both contaminants are often applied as a soil amendment. Recent research suggests that microplastics in these environments can increase gene persistence and flow, which could have unintended downstream consequences for environmental microbiome health and resilience. Antibiotic resistance genes and silencing RNAs bound to microplastics, for example, have the potential to increase resistance and alter gene expression in environmental bacteria, respectively. This review evaluates the sources and pathways of microplastics and biological microcontaminants in the terrestrial environment as well as potential sorption mechanisms that can encourage long-range transport and persistence. Novel sources of biological microcontaminants are considered, and the role of microplastics in promoting the persistence and flow of biological microcontaminants evaluated. Finally, future research directions are suggested to increase understanding of the mechanisms that drive the fate and transport of microplastic-biological microcontaminant complexes in the terrestrial environment and better inform risk management.

Evaluation of stormwater microbiomes for the potential biodegradation of tire wear particle contaminants.

AIM: Stormwater is a major source of many contaminants of emerging concern, which can be toxic to both aquatic and terrestrial organisms. This project aimed to identify novel biodegraders of toxic tire wear particle (TWP) contaminants associated with coho salmon mortality. METHODS AND RESULTS: This study has (i) characterized the prokaryotic communities of stormwater in both urban and rural settings; (ii) evaluated the ability of stormwater isolates to degrade two model TWP contaminants, hexa(methoxymethyl)melamine and 1,3-diphenylguanidine; and (iii) evaluated the toxicological impact of these model contaminants on the growth of six model bacteria. Rural stormwater possessed a diverse microbiome dominated by Oxalobacteraceae, Microbacteriaceae, Cellulomonadaceae, and Pseudomonadaceae taxa, while urban stormwater showed much less microbial diversity overall. Additionally, multiple stormwater isolates appeared capable of using model TWP contaminants as their sole carbon source. Each model contaminant was also found to alter growth patterns of model environmental bacteria including, with 1,3-DPG appearing more acutely toxic at high concentrations. CONCLUSION: This study identified several stormwater isolates that have the potential to be used as a sustainable solution to stormwater quality management.

Environmental fate and behaviour of antibiotic resistance genes and small interference RNAs released from genetically modified crops.

Rising global populations have amplified food scarcity across the world and ushered in the development of genetically modified (GM) crops to overcome these challenges. Cultivation of major crops such as corn and soy has favoured GM crops over conventional varieties to meet crop production and resilience needs. Modern GM crops containing small interference RNA molecules and antibiotic resistance genes have become increasingly common in the United States. However, the use of these crops remains controversial due to the uncertainty regarding the unintended release of its genetic material into the environment and possible downstream effects on human and environmental health. DNA or RNA transgenes may be exuded from crop tissues during cultivation or released during plant decomposition and adsorbed by soil. This can contribute to the persistence and bioavailability in soil or water environment and possible uptake by soil microbial communities and further passing of this information to neighbouring bacteria, disrupting microbial ecosystem services such as nutrient cycling and soil fertility. In this review, transgene mechanisms of action, uses in crops, and knowledge regarding their environmental fate and impact to microbes are evaluated. This aims to encapsulate the current knowledge and promote further research regarding unintended effects transgenes may cause.

Evaluation of developing maize microbiomes and associations among nitrogen cyclers and key fungal taxa.

More sustainable approaches to agriculture are urgently needed to protect existing resources and optimize crop yields and to provide food for a growing global human population. More sustainable agricultural practices that utilize plant-microbe relationships across cultivation are urgently needed. The main objectives of this study were to track the prokaryotic and fungal microbiomes associated with key growth stages of developing maize to evaluate the relationships among nitrogen cycling bacteria and major fungal genera including those known to contain arbuscular mycorrhizal fungi and other important taxa. Prokaryotic and fungal microbiomes associated with bulk soils, rhizosphere soils and tissues of developing maize were characterized using Illumina MiSeq sequencing. Similarities in microbiome diversity and abundance were compared to sample metadata to explore the influence of external factors on microbiome development. Correlations among target fungal taxa, bulk bacteria and nitrogen cycling bacteria were determined using non-parametric Spearman correlations. Important maize-associated fungal taxa were detected in all samples across growth stages, with Fusarium, Penicillium and Aspergillus fungi comprising up to 4.21, 4.26 and 0.28% of all fungal genera, respectively. Thirteen statistically significant correlations between nitrogen cycling genera and targeted fungal genera were also identified (rS≥0.70 or rS≤-0.70; P<0.05). This study is the first to note a strong positive association among several nitrifying bacteria and Fusarium (R=0.71; P=0.0046), Aspergillus (R=0.71; P=0.0055) and Cladosporium spcies (R=0.74; P=0.0038), suggesting the levels of soil nitrate, nitrite or nitrification intermediates may have large roles in the proliferation of important maize-associated fungi.

Loading, transport, and treatment of emerging chemical and biological contaminants of concern in stormwater.

Stormwater is a largely uncontrolled source of pollution in rural and urban environments across the United States. Concern regarding the growing diversity and abundance of pollutants in stormwater, as well as their impacts on water quality, has grown significantly over the past several decades. In addition to conventional contaminants like nutrients and heavy metals, stormwater is a well-documented source of many contaminants of emerging concern, which can be toxic to both aquatic and terrestrial organisms and remain a barrier to maintaining high quality water resources. Chemical pollutants like pharmaceuticals and personal care products, industrial pollutants such as per- and polyfluoroalkyl substances, and tire wear particles in stormwater are of great concern due to their toxic, genotoxic, mutagenic and carcinogenic properties. Emerging microbial contaminants such as pathogens and antibiotic resistance genes also represent significant threats to environmental water quality and human health. Knowledge regarding the transport, behavior, and the remediation capacity of these pollutants in runoff is key for addressing these pollutants in situ and minimizing ecosystem perturbations. To this end, this review paper will analyze current understanding of these contaminants in stormwater runoff in terms of their transport, behavior, and bioremediation potential.

A novel PCR-clamping assay reducing plant host DNA amplification significantly improves prokaryotic endo-microbiome community characterization.

Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers.

Examining the behavior of crop-derived antibiotic resistance genes in anaerobic sludge batch reactors under thermophilic conditions.

The consumption of transgenic crops and their by-products has become increasingly common in the United States. Yet, uncertainty remains regarding the fate and behavior of DNA within food matrices once it exits the digestive track and enters into wastewater treatment plants (WWTPs). Because many transgenic crops have historically contained antibiotic resistance genes as selection markers, understanding the behavior and uptake of these transgenes by environmental microbes is of critical importance. To investigate the behavior of free transgenic crop DNA, thermophilic anaerobic batch reactors were amended with varying concentrations of transgenic crop genes (i.e., LUG, nptII, and bla) and the persistence of those genes was monitored over 60 days using quantitative PCR. Significant levels of nptII and bla were detected in extracellular DNA (eDNA). Furthermore, LUG maize marker genes were also detected in the control reactors, suggesting that other crop-derived transgenes contained within digested transgenic foods may also enter WWTPs. Possible bacterial transformation events were detected within the highest dose treatments at Days 30 and 60 of incubation. These findings suggest that within the average conventional digester residence times in the United States (30 days), there is a potential for bacterial transformation events to occur with crop-derived transgenes found in eDNA.

A new framework for approaching precision bioremediation of PAH contaminated soils.

Bioremediation is a sustainable treatment strategy which remains challenging to implement especially in heterogeneous environments such as soil and sediment. Herein, we present a novel precision bioremediation framework that integrates amplicon based metagenomic analysis and chemical profiling. We applied this approach to samples obtained at a site contaminated with polycyclic aromatic hydrocarbons (PAHs). Geobacter spp. were identified as biostimulation targets because they were one of the most abundant genera and previously identified to carry relevant degradative genes. Mycobacterium and Sphingomonads spp. were identified as bioaugmentation and genetic bioaugmentation targets, respectively, due to their positive associations with PAHs and their high abundance and species diversity at all sampling locations. Overall, this case study suggests this framework can help identify bacterial targets for precision bioremediation. However, it is imperative that we continue to build our databases as the power of metagenomic based approaches remains limited to microorganisms currently in our databases.

A survey of crop-derived transgenes in activated and digester sludges in wastewater treatment plants in the United States.

The use of transgenic crops has become increasingly common in the United States over the last several decades. Increasing evidence suggests that DNA may be protected from enzymatic digestion and acid hydrolysis in the digestive tract, suggesting that crop-derived transgenes may enter into wastewater treatment plants (WWTPs) intact. Given the historical use of antibiotic resistance genes as selection markers in transgenic crop development, it is important to consider the fate of these transgenes. Herein we detected and quantified crop-derived transgenes in WWTPs. All viable US WWTP samples were found to contain multiple gene targets (p35, nos, bla and nptII) at significantly higher levels than control samples. Control wastewater samples obtained from France, where transgenic crops are not cultivated, contained significantly fewer copies of the nptII gene than US activated and digester sludges. No significant differences were measured for the bla antibiotic resistance gene (ARG). In addition, a nested PCR (polymerase chain reaction) assay was developed that targeted the bla ARG located in regions flanked by the p35 promoter and nos terminator. Overall this work suggests that transgenic crops may have provided an environmental source of nptII; however, follow-up studies are needed to ascertain the viability of these genes as they exit WWTPs.

Using laboratory-generated biosolids to evaluate the microbial ecotoxicity of triclosan in a simulated land application scenario.

Land application accounts for approximately 50% of wastewater solids disposal in the USA. Yet, little is known regarding the ecological impacts of many non-regulated chemicals found in biosolids. In most previous studies aimed at assessing ecological impacts, a model biosolid is generated by spiking high concentrations of the target chemical into a soil or biosolid. This approach does not account for the interaction of the chemical of interest with the solids throughout the biosolids production process (a.k.a., aging) which may impact the bioavailability and, thus, ultimate toxicity of the chemical. In the present study, using a lab-scale wastewater and digestion treatment system, we generated biosolids which contained aged triclosan and compared ecological impacts to that of spiked biosolids. Ecotoxicity was assessed based on functional and community structure changes to soil denitrifiers, microorganisms critical to nitrogen cycling. A decrease in denitrifier abundance and diversity was observed in the aged biosolids at concentrations of 17.9 ± 1.93 µg/kg while decreases in activity were observed at 26.9 ± 4.6 µg/kg. In the spiked biosolids treatment, lower denitrifier abundance, diversity, and activity were observed at triclosan (TCS) concentrations of 68.6 ± 26.9 µg/kg. This difference suggests a need to better understand TCS bioavailability dynamics.

Biochar and activated carbon act as promising amendments for promoting the microbial debromination of tetrabromobisphenol A.

The increasing occurrence of tetrabromobisphenol A (TBBPA) in the environment is raising questions about its potential environmental health impacts as it has been shown to cause various deleterious effects in humans. The fact that the highest concentrations of TBBPA have been reported in wastewater sludge is concerning as effluent discharge and biosolids land application are likely a route by which TBBPA can be further disbursed to the environment. Our objectives in this study were to evaluate the effect of biochar (BC) and activated carbon (AC) in promoting the biodegradation of TBBPA, and characterize the response of anaerobic sludge microbial communities following amendments. Both carbonaceous amendments were found to promote the reductive debromination of TBBPA. Nearly complete transformation of TBBPA to BPA was observed in the amended reactors ∼20 days earlier than in the control reactors. In particular, the transformation of diBBPA to monoBBPA, which appears to be the rate-limiting step, was accelerated in the presence of either amendment. Overall, microbial taxa responding to the amendments, i.e., 'sensitive responders', represented a small proportion of the community (i.e., 7.2%), and responded positively. However, although both amendments had a similar effect on TBBPA degradation, the taxonomic profile of the sensitive responders differed greatly from one amendment to the other. BC had a taxonomically broader and slightly more pronounced effect than AC. This work suggests that BC and AC show great potential to promote the biodegradation of TBBPA in anaerobic sludge, and their integration into wastewater treatment processes may be helpful for removing TBBPA and possibly other emerging hydrophobic contaminants.

Adsorption capacity of multiple DNA sources to clay minerals and environmental soil matrices less than previously estimated.

The cultivation and consumption of transgenic crops continues to be a widely debated topic, as the potential ecological impacts are not fully understood. In particular, because antibiotic resistance genes (ARGs) have historically been used as selectable markers in the genetic engineering of transgenic crops, it is important to determine if the genetic constructs found in decomposing transgenic crops persist long enough in the environment and if they can be transferred horizontally to indigenous microorganisms. In the present study, we address the question of persistence. Others have also estimated the DNA adsorption capacity of various clays, but have done so by manipulating the surface charge and size of particles tested which may overestimate sorption and underestimate the DNA available for horizontal transfer. In the present study, isotherms were generated using model Calf Thymus DNA and transgenic maize DNA without surface modification. Montmorillonite, kaolinite, and 3 soil mixtures with varying clay content were used in this study. The adsorption capacity of pure montmorillonite and kaolinite minerals was found to be one to two orders of magnitude less than previously estimated likely due to the distribution of clay particle sizes and heteroionic particle surface charge. However, it appears that a substantial amount of DNA is still able to adsorb onto these matrices (up to 200 mg DNA per gram of clay) suggesting the potential availability of free transgenic DNA in the environment may still be significant. Future studies should be conducted to determine the fate of these genes in agricultural soils.