An investigation of soil and groundwater metagenomes for genes encoding soluble and particulate methane monooxygenase, toluene-4-monoxygenase, propane monooxygenase and phenol hydroxylase.

Soil and groundwater were investigated for the genes encoding soluble and particulate methane monooxygenase/ammonia monooxygenase (sMMO, pMMO/AMO), toluene 4-monooxygenase (T4MO), propane monooxygenase (PMO) and phenol hydroxylase (PH). The objectives were (1) to determine which subunits were present, (2) to examine the diversity of the phylotypes associated with the biomarkers and (3) to identify which metagenome associated genomes (MAGs) contained these subunits. All T4MO and PH subunits were annotated in the groundwater metagenomes, while few were annotated in the soil metagenomes. The majority of the soil metagenomes included only four sMMO subunits. Only two groundwater metagenomes contained five sMMO subunits. Gene counts for the pMMO subunits varied between samples. The majority of the soil metagenomes were annotated for all four PMO subunits, while three out of eight groundwater metagenomes contained all four PMO subunits. A comparison of the blast alignments for the sMMO alpha chain (mmoX) indicated the phylotypes differed between the soil and groundwater metagenomes. For the pMMO/AMO alpha subunit (pmoA/amoA), Nitrosospira was important for the soil metagenomes, while Methylosinus and Methylocystis were dominant for the groundwater metagenomes. The majority of pmoA alignments from both metagenomes were from uncultured bacteria. High quality MAGs were obtained from the groundwater data. Four MAGs (Methylocella and Cypionkella) contained sMMO subunits. Another three MAGs, within the order Pseudomonadales, contained all three pMMO subunits. All PH subunits were detected in seven MAGs (Azonexus, Rhodoferax, Aquabacterium). In those seven, all contained catechol 2,3-dioxagenase, and Aquabacterium also contained catechol 1,2-dioxygenase. T4MO subunits were detected in eight MAGs (Azonexus, Rhodoferax, Siculibacillus) and all, except one, contained all six subunits. Four MAGs (Rhodoferax and Azonexus) contained all subunits for PH and T4MO, as well as catechol 2,3-dixoygenase. The detection of T4MO and PH in groundwater metagenomes and MAGs has important implications for the potential oxidation of groundwater contaminants.

Exploring structure-activity relationships for polymer biodegradability by microorganisms.

Research on the environmental biodegradation or microbial biodegradation of polymers has substantially increased recently due to growing demand for biodegradable polymers for certain applications. Environmental biodegradation of a polymer depends on the intrinsic biodegradability of the polymer and the characteristics of the receiving environment. The intrinsic biodegradability of a polymer is determined by the chemical structure and resulting physical properties (e.g., glass transition temperature, melting temperature, modulus of elasticity, crystallinity, and crystal structure) of the polymer. Quantitative structure-activity relationships (QSARs) on biodegradability have been well-established for discrete (non-polymeric) organic chemicals, but not for polymers due to the absence of adequate biodegradability data based on consistent and standardized biodegradation tests with appropriate characterization and reporting of the polymers tested. This review summarizes empirical structure-activity relationships (SARs) for biodegradability of polymers in laboratory studies involving various environmental matrices. In general, polyolefins with carbon-carbon chain are not biodegradable, while polymers containing labile bonds such as ester, ether, amide, or glycosidic bonds in their polymer chain may be favorable for biodegradation. Under a univariate scenario, polymers with higher molecular weight, higher crosslinking, lower water solubility, higher degree of substitution (i.e., higher average number of substituted functional groups per monomer unit), and higher crystallinity may result in reduced biodegradability. This review paper also highlights some of the challenges that hamper QSAR development for polymer biodegradability, stresses the need for better characterization of polymer structures used in biodegradation studies, and emphasizes the necessity for consistent testing conditions for the ease of cross-comparison and quantitative modeling analysis during future QSAR development.

Predicting the occurrence of monooxygenases and their associated phylotypes in soil microcosms.

Cometabolic oxidation involves the oxidation of chemicals often by monooxygenases or dioxygenases and can be a removal process for environmental contaminants such as trichloroethene (TCE) or 1,4-dioxane. Information on the occurrence of these genes and their associated microorganisms in environmental samples has the potential to enhance our understanding of contaminant removal. The overall aims were to 1) ascertain which genes encoding for monooxygenases (from methanotrophs, ammonia oxidizing bacteria and toluene/phenol oxidizers) and other key enzymes are present in soil microcosms and 2) determine which phylotypes are associated with those genes. The approach involved a predictive tool called PICRUSt2 and 16S rRNA gene amplicon datasets from two previous soil microcosm studies. The following targets from the KEGG database were examined: pmo/amo, mmo, dmp/pox/tomA, tmo/tbu/tou, bssABC (and downstream genes), tod, xylM, xylA, gst, dhaA, catE, dbfA1, dbfA2 and phenol 2-monooxygenase. A large number of phylotypes were associated with pmo/amo, while mmo was linked to only five. Several phylotypes were associated with both pmo/amo and mmo. The most dominant microorganism predicted for mmoX was Mycobacterium (also predicted for pmo/amo). A large number of phylotypes were associated with all six genes from the dmp/pox/tomA KEGG group. The taxonomic associations predicted for the tmo/tbu/tou KEGG group were more limited. In both datasets, Geobacter was a key phylotype for benzylsuccinate synthase. The dioxygenase-mediated toluene degradation pathway encoded by todC1C2BA was largely absent, as were the genes (xylM, xylA) encoding for xylene monooxygenase. All other genes investigated were predicted to be present and were associated with a number of microorganisms. Overall, the analysis predicted the genes encoding for sMMO (mmo), T3MO/T3MO/ToMO (tmo/tbu/tou) and benzylsuccinate synthase (bssABC) are present for a limited number of phylotypes compared to those encoding for pMMO/AMO (pmo/amo) and phenol monooxygenase/T2MO (dmp/poxA/tomA). These findings suggest in soils contaminant removal via pMMO/AMO or phenol monooxygenase/T2MO may be common because of the occurrence of these enzymes with a large number of phylotypes.

Carbamazepine, triclocarban and triclosan biodegradation and the phylotypes and functional genes associated with xenobiotic degradation in four agricultural soils.

Pharmaceuticals and personal care products (PPCPs) are released into the environment due to their poor removal during wastewater treatment. Agricultural soils subject to irrigation with wastewater effluent and biosolids application are possible reservoirs for these chemicals. This study examined the impact of the pharmaceutical carbamazepine (CBZ), and the antimicrobial agents triclocarban (TCC) and triclosan (TCS) on four soil microbial communities using shotgun sequencing (HiSeq Illumina) with the overall aim of determining possible degraders as well as the functional genes related to general xenobiotic degradation. The biodegradation of CBZ and TCC was slow, with ≤50% decrease during the 80-day incubation period. In contrast, TCS biodegradation was rapid, with ~80% removal in 25 days. For each chemical, when all four soils were considered together, between three and ten phylotypes (from multiple phyla) were more abundant in the soil samples compared to the live controls. The genera of a number of previously reported CBZ, TCC or TCS degrading isolates were present; Rhodococcus (CBZ), Streptomyces (CBZ), Pseudomonas (CBZ, TCC, TCS), Sphingomonas (TCC, TCS), Methylobacillus (TCS) and Stenotrophomonas (TCS) were among the most abundant (chemical previously reported to be degraded is shown in parenthesis). From the analysis of xenobiotic degrading pathways, genes from five KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology pathways were the most dominant, including those associated with aminobenzoate, benzoate (most common), chlorocyclohexane/chlorobenzene, dioxin and nitrotoluene biodegradation. Several phylotypes including Bradyrhizobium, Mycobacterium, Rhodopseudomonas, Pseudomonas, Cupriavidus, and Streptomyces were common genera associated with these pathways. Overall, the data suggest several phylotypes are likely involved in the biodegradation of these PPCPs with Pseudomonas being an important genus.

Diclofenac, carbamazepine and triclocarban biodegradation in agricultural soils and the microorganisms and metabolic pathways affected.

The incomplete elimination of pharmaceuticals and personal care products (PPCPs) during wastewater treatment has resulted in their detection in the environment. PPCP biodegradation is a potential removal mechanism; however, the microorganisms and pathways involved in soils are generally unknown. Here, the biodegradation of diclofenac (DCF), carbamazepine (CBZ) and triclocarban (TCC) in four agricultural soils at concentrations typically detected in soils and biosolids (50 ng g-1) was examined. Rapid DCF removal (<7 days) was observed under aerobic conditions, but only limited biodegradation was noted under other redox conditions. CBZ and TCC degradation under aerobic conditions was slow (half-lives of 128-241 days and 165-190 days for CBZ and TCC). Phylotypes in the Proteobacteria, Gemmatimonadales and Actinobacteria were significantly more abundant during DCF biodegradation compared to the controls (no DCF). For CBZ, those in the Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia were enriched compared to the controls. Actinobacteria and Proteobacteria were also enriched during TCC biodegradation. Such differences could indicate these microorganisms are associated with the biodegradation of these compounds, as they appear to be benefiting from their removal. The impact of these PPCPs on the KEGG pathways associated with metabolism was also examined. Four pathways were positively impacted during DCF biodegradation (propanoate, lysine, fatty acid & benzoate metabolism). These pathways are likely common in soils, explaining the rapid removal of DCF. There was limited impact of CBZ on the metabolic pathways. TCC removal was linked to genes associated with the degradation of simple and complex substrates. The results indicate even low concentrations of PPCPs significantly affect soil communities. The recalcitrant nature of TCC and CBZ suggests soils receiving biosolids could accumulate these chemicals, representing risks concerning crop uptake.

The identification of carbamazepine biodegrading phylotypes and phylotypes sensitive to carbamazepine exposure in two soil microbial communities.

Carbamazepine (CBZ), an antiepileptic drug, has been introduced into agricultural soils via irrigation with treated wastewater and biosolids application. Such contamination is problematic because CBZ is persistent and the risks to ecosystems or human health are unknown. The current study examined CBZ biodegradation in two agricultural soils (soil 1 and 2) and the effects on the soil microbial communities during CBZ exposure. The experimental design involved three CBZ concentrations (50, 500, 5000ng/g), under aerobic as well as anaerobic conditions. CBZ concentrations were determined using solid phase extraction and LC MS/MS. The effect of CBZ on the soil microbial community was investigated using high throughput sequencing and a computational approach to predict functional composition of the metagenomes (phylogenetic investigation of communities by reconstruction of unobserved states, PICRUSt). The most significant CBZ biodegradation occurred in soil 1 under aerobic conditions. In contrast, CBZ biodegradation was limited under anaerobic conditions in soil 1 and under both conditions in soil 2. For soil 1, several phylotypes were enriched following CBZ degradation compared to the controls, including unclassified Sphingomonadaceae, Xanthomonadaceae and Rhodobacteraceae, as well as Sphingomonas, Aquicella and Microvirga. These phylotypes are considered putative CBZ degraders as they appear to be benefiting from CBZ biodegradation. PICRUSt revealed that soil 1 contained a greater abundance of xenobiotic degrading genes compared to soil 2, and thus, this analysis method offers a potential valuable approach for predicting CBZ attenuation in soils. PICRUSt analysis also implicated Sphingomonadaceae and Xanthomonadaceae in drug metabolism. Interestingly, numerous phylotypes decreased in abundance following CBZ exposure and these varied with soil type, concentration, duration of exposure, and the availability of oxygen. For three phylotypes (Flavobacterium, 3 genus incertae sedis and unclassified Bacteroidetes), the relative abundance was reduced in both soils, indicating a notable sensitivity to CBZ for these microorganisms.