Global survey of hgcA-carrying genomes in marine and freshwater sediments: Insights into mercury methylation processes.

Mercury (Hg) methylation is a microbially mediated process that produces methylmercury (MeHg), a bioaccumulative neurotoxin. A highly conserved gene pair, hgcAB, is required for Hg methylation, which provides a basis for identifying Hg methylators and evaluating their genomic composition. In this study, we conducted a large-scale omics analysis in which 281 metagenomic freshwater and marine sediment samples from 46 geographic locations across the globe were queried. Specific objectives were to examine the prevalence of Hg methylators, to identify horizontal gene transfer (HGT) events involving hgcAB within Hg methylator communities, and to identify associations between hgcAB and microbial biochemical functions/genes. Hg methylators from the phyla Desulfobacterota and Bacteroidota were dominant in both freshwater and marine sediments while Firmicutes and methanogens belonging to Euryarchaeota were identified only in freshwater sediments. Novel Hg methylators were found in the Phycisphaerae and Planctomycetia classes within the phylum Planctomycetota, including potential hgcA-carrying anammox metagenome-assembled genomes (MAGs) from Candidatus Brocadiia. HGT of hgcA and hgcB were identified in both freshwater and marine methylator communities. Spearman's correlation analysis of methylator genomes suggested that in addition to sulfide, thiosulfate, sulfite, and ammonia may be important parameters for Hg methylation processes in sediments. Overall, our results indicated that the biochemical drivers of Hg methylation vary between marine and freshwater sites, lending insight into the influence of environmental perturbances, such as a changing climate, on Hg methylation processes.

Continuous co-treatment of mine drainage with municipal wastewater.

Acid mine drainage (AMD) and municipal wastewater (MWW) are commonly co-occurring waste streams in mining regions. Co-treating AMD at existing wastewater facilities represents an innovative solution for simultaneous AMD reclamation and improved MWW treatment. However, unknowns related to biological processes and continuous treatment performance block full-scale use. The overarching goal of this work was to address questions related to efficacy and performance of continuous processing of AMD in a biological MWW treatment system. Synthetic AMD was co-treated with synthetic MWW in a continuously-operating bench-scale sequencing batch reactor (SBR). SBRs treated MWW with two strengths of AMD (91 and 720 mg/L as CaCO3 Acidity) to capture the variations of coal AMD chemistry and strength observed in the field. Each co-treatment phases lasted 40+ days, during which clarified effluent and settled sludge quality was routinely monitored to determine impacts of co-treatment relative to conventional MWW treatment performance. Co-treatment produced effluent that met key standards for secondary treatment including biochemical oxygen demand (BOD) < 5 mg/L, total suspended solids (TSS) < 20 mg/L, and pH ∼7.0. Addition of AMD also improved treatment performance, increasing Phosphate (PO4) removal by >60% and pathogen removal by an order of magnitude. Furthermore, AMD co-treatment did not exhibit any major impacts on the overall diversity of the wastewater microbial community. Co-treatment sludge had slightly higher settleability and a lower bound water content, but notable changes in sludge morphology was observed. This study demonstrates co-treatment allows for continuous mitigation of AMD without adversely impacting MWW treatment performance in conventional biological MWW processes.

Non-mercury methylating microbial taxa are integral to understanding links between mercury methylation and elemental cycles in marine and freshwater sediments.

The goal of this study was to explore the role of non-mercury (Hg) methylating taxa in mercury methylation and to identify potential links between elemental cycles and Hg methylation. Statistical approaches were utilized to investigate the microbial community and biochemical functions in relation to methylmercury (MeHg) concentrations in marine and freshwater sediments. Sediments were collected from the methylation zone (top 15 cm) in four Hg-contaminated sites. Both abiotic (e.g., sulfate, sulfide, iron, salinity, total organic matter, etc.) and biotic factors (e.g., hgcA, abundances of methylating and non-methylating taxa) were quantified. Random forest and stepwise regression were performed to assess whether non-methylating taxa were significantly associated with MeHg concentration. Co-occurrence and functional network analyses were constructed to explore associations between taxa by examining microbial community structure, composition, and biochemical functions across sites. Regression analysis showed that approximately 80% of the variability in sediment MeHg concentration was predicted by total mercury concentration, the abundances of Hg methylating taxa, and the abundances of the non-Hg methylating taxa. The co-occurrence networks identified Paludibacteraceae and Syntrophorhabdaceae as keystone non Hg methylating taxa in multiple sites, indicating the potential for syntrophic interactions with Hg methylators. Strong associations were also observed between methanogens and sulfate-reducing bacteria, which were likely symbiotic associations. The functional network results suggested that non-Hg methylating taxa play important roles in sulfur respiration, nitrogen respiration, and the carbon metabolism-related functions methylotrophy, methanotrophy, and chemoheterotrophy. Interestingly, keystone functions varied by site and did not involve carbon- and sulfur-related functions only. Our findings highlight associations between methylating and non-methylating taxa and sulfur, carbon, and nitrogen cycles in sediment methylation zones, with implications for predicting and understanding the impact of climate and land/sea use changes on Hg methylation.

Three dimensional printed biofilms: Fabrication, design and future biomedical and environmental applications.

Three dimensional printing has emerged as a widely acceptable strategy for the fabrication of mammalian cell laden constructs with complex microenvironments for tissue engineering and regenerative medicine. More recently 3D printed living materials containing microorganisms have been developed and matured into living biofilms. The potential for engineered 3D biofilms as in vitro models for biomedical applications, such as antimicrobial susceptibility testing, and environmental applications, such as bioleaching, bioremediation, and wastewater purification, is extensive but the need for an in-depth understanding of the structure-function relationship between the complex construct and the microorganism response still exists. This review discusses 3D printing fabrication methods for engineered biofilms with specific structural features. Next, it highlights the importance of bioink compositions and 3D bioarchitecture design. Finally, a brief overview of current and potential applications of 3D printed biofilms in environmental and biomedical fields is discussed.

One-Week-Ahead Prediction of Cyanobacterial Harmful Algal Blooms in Iowa Lakes.

Cyanobacterial harmful algal blooms (CyanoHABs) pose serious risks to inland water resources. Despite advancements in our understanding of associated environmental factors and modeling efforts, predicting CyanoHABs remains challenging. Leveraging an integrated water quality data collection effort in Iowa lakes, this study aimed to identify factors associated with hazardous microcystin levels and develop one-week-ahead predictive classification models. Using water samples from 38 Iowa lakes collected between 2018 and 2021, feature selection was conducted considering both linear and nonlinear properties. Subsequently, we developed three model types (Neural Network, XGBoost, and Logistic Regression) with different sampling strategies using the nine selected variables (mcyA_M, TKN, % hay/pasture, pH, mcyA_M:16S, % developed, DOC, dewpoint temperature, and ortho-P). Evaluation metrics demonstrated the strong performance of the Neural Network with oversampling (ROC-AUC 0.940, accuracy 0.861, sensitivity 0.857, specificity 0.857, LR+ 5.993, and 1/LR- 5.993), as well as the XGBoost with downsampling (ROC-AUC 0.944, accuracy 0.831, sensitivity 0.928, specificity 0.833, LR+ 5.557, and 1/LR- 11.569). This study exhibited the intricacies of modeling with limited data and class imbalances, underscoring the importance of continuous monitoring and data collection to improve predictive accuracy. Also, the methodologies employed can serve as meaningful references for researchers tackling similar challenges in diverse environments.

Systematic Performance Evaluation of Reinforcement Learning Algorithms Applied to Wastewater Treatment Control Optimization.

Treatment of wastewater using activated sludge relies on several complex, nonlinear processes. While activated sludge systems can provide high levels of treatment, including nutrient removal, operating these systems is often challenging and energy intensive. Significant research investment has been made in recent years into improving control optimization of such systems, through both domain knowledge and, more recently, machine learning. This study leverages a novel interface between a common process modeling software and a Python reinforcement learning environment to evaluate four common reinforcement learning algorithms for their ability to minimize treatment energy use while maintaining effluent compliance within the Benchmark Simulation Model No. 1 (BSM1) simulation. Three of the algorithms tested, deep Q-learning, proximal policy optimization, and synchronous advantage actor critic, generally performed poorly over the scenarios tested in this study. In contrast, the twin delayed deep deterministic policy gradient (TD3) algorithm consistently produced a high level of control optimization while maintaining the treatment requirements. Under the best selection of state observation features, TD3 control optimization reduced aeration and pumping energy requirements by 14.3% compared to the BSM1 benchmark control, outperforming the advanced domain-based control strategy of ammonia-based aeration control, although future work is necessary to improve robustness of RL implementation.

Biological phosphorus removal and its microbial community in a modified full-scale activated sludge system under dry and wet weather dynamics.

Enhanced biological phosphorus removal (EBPR) performance and microbial community dynamics during dry and wet-weather conditions of a full-scale treatment plant was evaluated by converting a section of activated sludge basins using low-cost operational modifications into an anoxic/anaerobic zone to promote EBPR. Two trains of the activated sludge system at the Des Moines, Iowa Metropolitan Wastewater Reclamation Facility were used for the study with one train modified for EBPR, and the other remained as nitrification-only for comparison. In addition to measuring the modification effectiveness for phosphorus removal, performance was compared during dry and wet weather conditions over the course of two summer seasons to improve understanding of wet and dry weather dynamics for EBPR. DNA sequencing and qPCR tests were conducted to develop an understanding of microbial population changes between control and modified basins and wet and dry weather conditions. Basin hydraulic retention times varied from 2.6 to 12.7 hours with an average of 8.9 hours. EBPR activity was successfully established in the modified basins with average phosphorus content of the return activated sludge 0.032 ± 0.002 compared to 0.016 ± 0.001 mg TP/mg TSS (95% confidence) in the control basins. Phosphorus removal was significantly decreased by prolonged wet weather conditions, particularly in year two of the study, however the modified basin maximum removal of 96% and average of 43.7 ± 5.3% remained significantly higher than the maximum of 46% and average 12.6 ± 2.4% removal in the control basins. DNA sequencing showed a significant increase in relative abundance of phyla Chloroflexi, Nitrospirae, and Verrucomicrobia in the modified basins, but no correlation to EBPR performance. qPCR indicated significant increase in relative quantity of Accumulibacter, but not for Actinetobacter-like phosphorus accumulating organisms (PAOs), which includes the PAO Tetrasphaera. Significant abundance of some Accumulibacter clades found within the modified basins was contrary to previous literature which focused on small-scale and batch studies. A higher than expected dominance of clade I and increased relative quantities of clades IIB and IIC during extended wet weather was observed which may have contributed to rapid recovery of phosphorus removal when dry weather resumed. The abundance of PAOs did not significantly correlate with changes in phosphorous removal performance, contrary to reports from previous small-scale and batch studies.

Sulfite-activated ferrate for water reuse applications.

Ferrate is a promising, emerging water treatment technology. However, there has been limited research on the application of ferrate in a water reuse paradigm. Recent literature has shown that ferrate oxidation of target contaminants could be improved by "activation" with the addition of reductants or acid. This study examined the impact of sulfite-activated ferrate in laboratory water matrix and spiked municipal wastewater effluents with the goal of transforming organic contaminants of concern (e.g., 1,4-dioxane) and inactivating pathogenic organisms. Additionally, the formation of brominated disinfection byproducts by activated ferrate were examined and a proposed reaction pathway for byproduct formation is presented. In particular, the relative importance of reaction intermediates is discussed. This represents the first activated ferrate study to examine 1,4-dioxane transformation, disinfection, and brominated byproduct formation. Results presented show that the sub-stoichiometric ([Sulfite]:[Ferrate] = 0.5) activated ferrate treatment approach can oxidize recalcitrant contaminants by >50%, achieve >4-log inactivation of pathogens, and have relatively limited generation of brominated byproducts. However, stoichiometrically excessive ([Sulfite]:[Ferrate] = 4.0) activation showed decreased performance with decreased disinfection and increased risk of by-product formation. In general, our results indicate that sub-stoichiometric sulfite-activated ferrate seems a viable alternative technology for various modes of water reuse treatment.

Effect of ferrate and monochloramine disinfection on the physiological and transcriptomic response of Escherichia coli at late stationary phase.

Biological mechanisms of disinfection not only vary by disinfectant but also remain not well understood. We investigated the physiological and transcriptomic response of Escherichia coli at late stationary phase to ferrate and monochloramine in amended lake water. Although ferrate and monochloramine treatments similarly reduced culturable cell concentrations by 3-log10, 64% and 11% of treated cells were viable following monochloramine and ferrate treatment, respectively. This observed induction of viable but non-culturable (VBNC) state following monochloramine treatment but not ferrate is attributed to slower monochloramine disinfection kinetics (by 2.8 times) compared to ferrate. Transcriptomic analysis of E. coli at 15 min of exposure revealed that 3 times as many genes related to translation and transcription were downregulated by monochloramine compared to ferrate, suggesting that monochloramine treatment may be inducing VBNC through reduced protein synthesis and metabolism. Downregulation of universal stress response genes (rpoS, uspA) was attributed to growth-related physiological stressors during late stationary phase which may have contributed to the elevated expression levels of general stress responses pre-disinfection and, subsequently, their significant downregulation post-disinfection. Both disinfectants upregulated oxidative stress response genes (trxC, grxA, soxS), although levels of upregulation were time sensitive. This work shows that bacterial inactivation responses to disinfectants is mediated by complex molecular and growth-related responses.

Improved detection of mcyA genes and their phylogenetic origins in harmful algal blooms.

Microcystins, a group of cyanotoxins produced by cyanobacterial strains, have become a significant microbial hazard to human and animal health due to increases in the frequency and intensity of cyanobacterial harmful algal blooms (CyanoHABs). Many studies have explored the correlation between microcystin concentrations and abundances of toxin-producing genes (e.g., mcyA genes) measured using quantitative PCR, and discrepancies between toxin concentrations and gene abundances are often observed. In this study, the results show that these discrepancies are at least partially due to primer sets that do not capture the phylogenetic diversity of naturally present toxin-producers. We designed three novel primer gene probes based on known mcyA genes to improve the detection and quantification of these genes in environmental samples. These primers were shown to improve the identification of mcyA genes compared to previously published primers in freshwater metagenomes, cyanobacterial isolates, and lake water samples. Unlike previously published primers, our primer sets could selectively amplify and resolve Microcystis, Anabaena, and Planktothrix mcyA genes. In lake water samples, abundance estimations of mcyA genes were found to correlate strongly with microcystin concentrations. Based on our results, these primers offer significant improvements over previously published probes to accurately identify and quantify mcyA genes in the environment. There is an increasing need to develop models based on microbial information and environmental factors to predict CyanoHABs, and improved primers will play an important role in aiding monitoring efforts to collect reliable and consistent data on toxicity risks.

Feasibility test of autotrophic denitrification of industrial wastewater in sequencing batch and static granular bed reactors.

In order to evaluate the efficacy of using reduced sulfur species in lieu of conventional substrates, a sequencing batch reactor (SBR) was used to develop an autotrophic denitrifying culture which in turn was used to seed a static granular bed reactor (SGBR) for continuous flow treatment. Both bioreactors were able to quickly acclimate to the anoxic environment and achieve stable autotrophic denitrification within several weeks of being placed in operation. The seed for the SBR was obtained from operating basins at the Cedar Rapids plant. MiSeq analysis showed the presence of the autotrophic denitrifier Thiobacillus in the seed from the sulfur oxidation basin; however, Shinella and Sulfurovum became the dominant autotrophic denitrifiers in the SBR. Both the SBR and SGBR achieved excellent nitrate removal (i.e., >95%) with stoichiometric amounts of thiosulfate added to the synthetic influent. The results of this feasibility study suggest that anaerobic granules from the UASB at the plant serve as good seed biomass for autotrophic denitrification when augmented by sulfur oxidation basin and sulfide scrubber biomass, and that reduced sulfur species at the plant (or augmented with an external sulfur source) can serve as electron donors for nearly complete denitrification. PRACTITIONER POINTS: Autotrophic denitrification of industrial wastewater was investigated to evaluate reduced sulfur species as electron donor for nitrogen removal. An autotrophic denitrifying culture was cultivated in an SBR, and continuous autotrophic denitrification was accomplished in an SGBR. No increase in head loss was observed in the SGBR, and it was able to operate without the need for backwashing in more than 200 days of operation. Reduced sulfur was demonstrated to be a sufficient electron donor for nearly complete denitrification. MiSeq analysis resolved primary species responsible for autotrophic denitrification in this study.

White blood cell DNA methylation and risk of breast cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO).

BACKGROUND: Several studies have suggested that global DNA methylation in circulating white blood cells (WBC) is associated with breast cancer risk. METHODS: To address conflicting results and concerns that the findings for WBC DNA methylation in some prior studies may reflect disease effects, we evaluated the relationship between global levels of WBC DNA methylation in white blood cells and breast cancer risk in a case-control study nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) cohort. A total of 428 invasive breast cancer cases and 419 controls, frequency matched on age at entry (55-59, 60-64, 65-69, ≥70 years), year of entry (on/before September 30, 1997, on/after October 1, 1997) and period of DNA extraction (previously extracted, newly extracted) were included. The ratio of 5-methyl-2' deoxycytidine [5-mdC] to 2'-deoxyguanine [dG], assuming [dG] = [5-mdC] + [2'-deoxycytidine [dC]] (%5-mdC), was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry, an especially accurate method for assessing total genomic DNA methylation. RESULTS: Odds ratio (OR) estimates and 95% confidence intervals (CI) for breast cancer risk adjusted for age at entry, year of entry, and period of DNA extraction, were 1.0 (referent), 0.89 (95% CI, 0.6-1.3), 0.88 (95% CI, 0.6-1.3), and 0.84 (95% CI, 0.6-1.2) for women in the highest compared to lowest quartile levels of %5md-C (p for trend = .39). Effects did not meaningfully vary by time elapsed from WBC collection to diagnosis. DISCUSSION: These results do not support the hypothesis that global DNA hypomethylation in WBC DNA is associated with increased breast cancer risk prior to the appearance of clinical disease.

Impacts of coal ash on methylmercury production and the methylating microbial community in anaerobic sediment slurries.

Mercury (Hg) associated with coal ash is an environmental concern, particularly if the release of coal ash to the environment is associated with the conversion of inorganic Hg to methylmercury (MeHg), a bioaccumulative form of Hg that is produced by anaerobic microorganisms. In this study, sediment slurry microcosm experiments were performed to understand how spilled coal ash might influence MeHg production in anaerobic sediments of an aquatic ecosystem. Two coal ash types were used: (1) a weathered coal ash; and (2) a freshly collected, unweathered fly ash that was relatively enriched in sulfate and Hg compared to the weathered ash. These ash samples were added to anaerobic sediment slurries constructed with a relatively pristine sediment (containing 0.03 mg kg-1 Hg) and a Hg-contaminated sediment (containing 0.29 mg kg-1 Hg). The results of these experiments showed negligible net production of MeHg in microcosms with no ash and in microcosms amended with the low sulfate/low Hg ash. In contrast, slurry microcosms amended with high sulfate/high Hg ash showed increases in total MeHg content that was 2 to 3 times greater than control microcosms without ash (p < 0.001). 16S amplicon sequencing of microbial communities in the slurries indicated that the coal ash addition generally increased the relative abundance of the methylating microbial community, including sulfate-reducing bacteria and iron-reducing bacteria species that are known to be efficient methylators of Hg. The stimulation of these microorganisms was likely caused by the release of substrates (sulfate and Fe) originating from the ash. Overall, the results highlight the need to incorporate both environmental parameters and coal ash characteristics into risk assessments that guide coal ash management and disposal.

Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment.

When nanoparticles meet biofilms-interactions guiding the environmental fate and accumulation of nanoparticles.

Bacteria are essential components of all natural and many engineered systems. The most active fractions of bacteria are now recognized to occur as biofilms, where cells are attached and surrounded by a secreted matrix of "sticky" extracellular polymeric substances. Recent investigations have established that significant accumulation of nanoparticles (NPs) occurs in aquatic biofilms. These studies point to the emerging roles of biofilms for influencing partitioning and possibly transformations of NPs in both natural and engineered systems. While attached biofilms are efficient "sponges" for NPs, efforts to elucidate the fundamental mechanisms guiding interactions between NPs and biofilms have just begun. In this mini review, special attention is focused on NP-biofilm interactions within the aquatic environment. We highlight key physical, chemical, and biological processes that affect interactions and accumulation of NPs by bacterial biofilms. We posit that these biofilm processes present the likely possibility for unique biological and chemical transformations of NPs. Ultimately, the environmental fate of NPs is influenced by biofilms, and therefore requires a more in-depth understanding of their fundamental properties.

Aging of fullerene C60 nanoparticle suspensions in the presence of microbes.

Despite the growing use of carbon nanomaterials in commercial applications, very little is known about the fate of these nanomaterials once they are released into the environment. The carbon-carbon bonding of spherical sp(2) hybridized fullerene (C60) forms a strong and resilient material that resists biodegradation. Moreover, C60 is widely reported to be bactericidal. Here however, we observe the changing properties of fullerene nanoparticle aggregates aged in the presence of microbes. C60 aggregates were observed to decrease in size with aging, while hydroxylation and photosensitized reactivity measured by the production of reactive oxygen species (ROS) increased, suggesting that chemically and/or biologically-mediated activity is capable of partially transforming fullerene structure and reactivity in the environment. However, stable-isotope-labeling C60 aggregates incubated with microbial cultures from aged suspensions for 203 days did not produce significant labeled carbon dioxide, despite significant reduction in aggregate radius for biological samples. These results suggest that either the rate of biodegradation of these particles is too slow to quantify or that the biologically-enhanced transformation of these particles does not occur through microbial biodegradation to carbon dioxide.

Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations.

As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a "side-on" or an "end-on" conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NP-protein interactions.

Functionality of the TOL plasmid under varying environmental conditions following conjugal transfer.

Conjugation of catabolic plasmids in contaminated environments is a naturally occurring horizontal gene transfer phenomenon, which could be utilized in genetic bioaugmentation. The potentially important parameters for genetic bioaugmentation include gene regulation of transferred catabolic plasmids that may be controlled by the genetic characteristics of transconjugants as well as environmental conditions that may alter the expression of the contaminant-degrading phenotype. This study showed that both genomic guanine-cytosine contents and phylogenetic characteristics of transconjugants were important in controlling the phenotype functionality of the TOL plasmid. These genetic characteristics had no apparent impact on the stability of the TOL plasmid, which was observed to be highly variable among strains. Within the environmental conditions tested, the addition of glucose resulted in the largest enhancement of the activities of enzymes encoded by the TOL plasmid in all transconjugant strains. Glucose (1 g/L) enhanced the phenotype functionality by up to 16.4 (±2.22), 30.8 (±7.03), and 90.8 (±4.56)-fold in toluene degradation rates, catechol 2,3-dioxygenase enzymatic activities, and xylE gene expression, respectively. These results suggest that genetic limitations of the expression of horizontally acquired genes may be overcome by the presence of alternate carbon substrates. Such observations may be utilized in improving the effectiveness of genetic bioaugmentation.

Impacts of organic carbon availability and recipient bacteria characteristics on the potential for TOL plasmid genetic bioaugmentation in soil slurries.

The effectiveness of genetic bioaugmentation relies on efficient plasmid transfer between donor and recipient cells as well as the plasmid's phenotype in the recipient cell. In the present study, the effects of varying organic carbon substrates, initial recipient-to-donor cell density ratios, and mixtures of known recipient bacterial strains on the conjugation and function of a TOL plasmid were tested in sterile soil slurry batch reactors. The presence of soil organic carbon was sufficient in ensuring TOL plasmid transconjugant occurrence (up to 2.1±0.5%) for most recipient strains in soil slurry batch mating experiments. The addition of glucose had limited effects on transconjugant occurrence; however, glucose amendment increased the specific toluene degradation rates of some Enterobacteriaceae transconjugants in soil slurry. Initial cell density ratios and mixtures of recipient strains had smaller impacts on plasmid conjugation and resulting phenotype functionality. These observations suggest that genetic bioaugmentation may be improved by minimal altering of environmental conditions.

Genetic bioaugmentation as an effective method for in situ bioremediation: functionality of catabolic plasmids following conjugal transfers.

Genetic bioaugmentation is an in situ bioremediation method that stimulates horizontal transfer of catabolic plasmids between exogenous donor cells and indigenous bacteria to increase the biodegradation potential of contaminants. A critical outcome of genetic bioaugmentation is the expression of an active catabolic phenotype upon plasmid conjugation. Using a pWW0-derivative TOL plasmid, we showed that certain genetic characteristics of the recipient bacteria, including genomic guanine-cytosine (G + C) content and phylogeny, may limit the expression of the transferred catabolic pathway. However, such genetic limitations observed in transconjugants could be overcome by the presence of an additional carbon source. Glucose and Luria-Bertani broth were shown to enhance the toluene degradation rates of transconjugants; these enhancement effects were dependent on transconjugant genomic G + C contents. Based on these observations, thorough genetic characterization of the indigenous microbial community in the contaminated environment of interest may provide a predictive tool for assessing the success of genetic bioaugmentation.