Linking Trace Organic Chemical Attenuation to Microbiome Metabolic

Downloaded by COLUMBIA UNIV on December 12, 2016 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1241.ch011

Chapter 11

Linking Trace Organic Chemical Attenuation to Microbiome Metabolic Capabilities: Insights from Laboratory- and Full-Scale Managed Aquifer Recharge Systems Julia Regnery,*,1 Dong Li,1,2 Simon Roberts,1,3 Christopher Higgins,1 Jonathan O. Sharp,1 and Jörg E. Drewes1,4 1Department

of Civil and Environmental Engineering, Colorado School of Mines, Golden, Colorado 80401, United States 2Bren School of Environmental Science & Management, University of California Santa Barbara, Santa Barbara, California 93106, United States 3Sciex LLC, Framingham, Massachusetts 01701, United States 4Chair of Urban Water Systems Engineering, Technical University of Munich, 85748 Garching, Germany *E-mail: [email protected].

Can trace organic chemical biodegradation in complex environments be explained by correlating the behavior of these water pollutants to the expression and abundance of specific enzymes related to their metabolism? In this study, we aim to link trace organic chemical attenuation to the metabolic capability of the microbiome by investigating intermediate xenobiotic transformation products catalyzed by specific enzymes. Water samples from both laboratory- and full-scale managed aquifer recharge systems were analyzed by suspected-target screening using liquid-chromatography time-of-flight mass spectrometry in ESI negative and positive mode. Identification of transformation products was carried out by accurate mass and MS/MS spectra. Out of 75 potential transformation products for 24 parent compounds, six metabolites for six parent compounds were tentatively identified in the analyzed source and groundwater samples. Identified transformation products were compared with the © 2016 American Chemical Society

Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


expression and abundance of functional genes quantified by 454 pyrosequencing. For the majority of functional genes involved in xenobiotic degradation pathways, no correlation with environmental parameters such as depth was observed. One important finding of our study is that technical guidelines for standardization of environmental ‘-omics’ research procedures are crucial and should cover amongst others sampling, technical data analysis, and interpretation of results, as well as the definition of cut off criteria, reference points, and normal values.

Introduction The metabolic versatility of certain microorganisms gives them the capacity to transform xenobiotic water pollutants. By definition, xenobiotics are compounds that are foreign to an ecological system, e.g., chemicals of anthropogenic origin such as trace organic chemicals that are introduced to the environment. This transformation process can be driven by co-metabolism, which is defined as the microbial transformation of a non-growth substrate in the presence of a growth substrate or another transformable substance (1). The emergence of ‘-omics’ technologies over the last decade offered new insights into the complex network of metabolic and regulatory interactions which frame biodegradation processes in microorganisms. Turnbaugh and Gordon (2) articulated an early scientific argument for uniting metagenomics with metabolomics to shed light on how microbial communities function in a variety of environments. Metagenomics seek to characterize the composition of microbial communities, their operations, and their dynamically coevolving relationships with the habitats they occupy without having to culture community members, whereas metabolomics characterize metabolites generated by one or more organisms in a given physiological and environmental context using analytical methods such as high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance spectroscopy (2). Since then, interest in using meta-omics associated studies to investigate trace organic chemical biotransformation has significantly increased. The microbial capacity to transform xenobiotics can be naturally enhanced by external factors such as the presence of essential nutrients, adequate electron acceptors, or appropriate environmental conditions (e.g., pH, redox potential, humidity, temperature) (3). The general strategy of assessing microbial community function is to characterize the complete set of genes, transcripts, or enzymes from in situ environmental microbial communities (e.g., by shotgun approaches) and use the abundances of particular ones to establish associations with the communities’ potential to biotransform specific trace organic chemicals (4). Recently, studies were performed to reveal the microbial community characteristics during simulated managed aquifer recharge (MAR) in soil column systems receiving different primary substrate composition and further link these to the biotransformation of trace organic chemicals (5). Li et al. (5) reported that the metabolic capabilities of the microbiome involved in xenobiotic biodegradation (e.g., cytochrome 164



P450 (CYP) genes) as well as the attenuation of trace organic chemicals were significantly promoted under carbon-limited conditions (i.e., lower biodegradable dissolved organic carbon (BDOC) and higher humic acid content in the feed water). However, occurrence and fate of transformation products were not monitored during that study. By collecting information on xenobiotic biotransformation reactions and pathways, the University of Minnesota pioneered a biocatalysis/biodegradation database (BBD), allowing the prediction of the most likely metabolic pathway of a given compound based on abstracted chemical reaction processes (6). At present this database is being further developed by the Swiss Federal Aquatic Research Institute in a pathway prediction system (Eawag-PPS) and currently comprises 250 biotransformation rules, 1,503 chemical reactions, 219 microbial degradation pathways, and 993 enzymes. Nevertheless, it should be noted that biotransformation pathways established by chemical reactions only provide little or no relation to the corresponding enzymes and proteins (3). Kern et al. (7) published a study on using high-resolution mass spectrometry to identify transformation products predicted by the Eawag-PPS (two generations, aerobic and anaerobic) as well as known environmental transformation products and human metabolites in natural water samples. Interestingly, only 19 transformation products out of 1,794 potential metabolites for 52 parent compounds were tentatively identified. In a different study by Card et al. (8), 14 biotransformation libraries encoded in eight software packages that predict metabolite structures were assessed for their sensitivity (proportion of reported metabolites that were predicted) and selectivity (proportion of predicted metabolites that were reported) toward reported mammalian and microbial metabolites for ten agrochemicals. No library averaged greater than 58% sensitivity or 69% selectivity. With an increasing number of predicted generations, sensitivity increased and selectivity decreased. Most often the information of biodegradation enzymes is only partially included in biodegradation databases, e.g., provided by enzyme names or the Enzyme Commission (EC) codes. The EC code is a numerical classification scheme for enzymes based on catalyzed chemical reactions. It is organized into four levels of detail, from more general (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases) to more specific (the substrate that transforms). Unfortunately, in many cases, the EC code is not sufficient to identify the protein complex responsible for the reaction, which is a consequence of the equivocality in the definition of the codes and the different criteria used by annotators during assignment (3). For example, only 36% of the 993 enzymes included in the Eawag-PPS (6) possess a complete EC number. Despite having a strong potential, it becomes apparent that the approaches currently used by biotransformation libraries are only able to identify the presence or absence of previously characterized enzymes and species in environmental samples. Although pure cultures of bacterial strains degrading individual trace organic chemicals have been isolated and reported (9–11), only limited results were published for complex mixtures (12). Another limitation is the relevance of laboratory cultivars to natural conditions as many yet unknown species contribute to biodegradation (3). 165


The present study was guided by the question of whether trace organic chemical biotransformation in complex environments such as soil-aquifer systems can be explained by comparing trace organic chemical attenuation with the expression and abundance of a targeted subset of putative biotransformation genes extracted from proximal soils. To shed light on this question, both metagenomics and HR-MS techniques were applied to corresponding soil and water samples from laboratory-scale soil column systems simulating MAR under controlled environmental conditions as well as pilot- and full-scale MAR operations in California and Colorado recharging impaired water via surface spreading.


Methodology Test Beds and Sample Collection Recharged source water and groundwater samples from two different MAR field sites in California and Colorado as well as two soil column systems were collected and analyzed according to Standard Methods (13) during sampling campaigns in 2012 and 2013 as described in Drewes et al. (14). Water samples for advanced chemical analysis were immediately preserved with sodium azide (1 g/L) to prevent further biodegradation. In addition, representative soil samples were collected from the soil column systems, the surface of the San Gabriel Spreading Grounds test basin in California at 1 cm and 5 cm depths, and over a 1 m depth profile from the central infiltration basin at the Prairie Waters Project aquifer recharge and recovery (ARR) facility in Colorado.

California The United States Geological Survey/Water Replenishment District of Southern California test basin at the Montebello Forebay was constructed in the early 90’s at the north end of the San Gabriel River Coastal Spreading Grounds located between Whittier Boulevard and Washington Boulevard in Pico Rivera, Los Angeles County, California. Tertiary treated reclaimed water (i.e., dual-media filtration followed by chlorination and dechlorination) is delivered from the water reclamation facilities to the Spreading Grounds through a culvert. A small percentage of the total flow can be diverted to the 2023 m2 large test basin through a pipeline using a submersible pump. The test basin is fully equipped with a multilevel sampler and monitoring wells and subsurface flow conditions are very well characterized (15–17). Prior to both sampling campaigns, the filling of the test basin with reclaimed water to activate/maintain the indigenous microbial community was scheduled as detailed in Drewes et al. (14). During this study, DOC, nitrate, and dissolved oxygen concentrations in the recharged tertiary treated wastewater effluent were in the range of 6.5 ± 1.0 mg/L, 4.3 ± 1.4 mg N/L, and 6.8 ± 0.6 mg/L, respectively. Due to the high amount of BDOC (approximately 4 mg/L), subsurface redox conditions at the test basin were characterized as oxic/suboxic in the upper aquifer and anoxic conditions in the 166


lower aquifer. Suboxic conditions were defined as nitrate reduction of less than 0.5 mg N/L and dissolved manganese concentrations of less than 0.05 mg/L.


Colorado In 2010, the city of Aurora launched the Prairie Waters Project North Campus to supplement its drinking water supply using surface water impaired by treated wastewater discharges. An MAR system consisting of riverbank filtration (RBF) galleries and an ARR facility as part of an advanced water treatment train was constructed along the South Platte River downstream of Denver, Colorado. The combined RBF filtrate is delivered via pipes into an array of surface spreading basins at the adjacent ARR facility. A continuous slurry wall along the entire perimeter of the ARR facility extending from the surface to the bedrock is used to isolate the recharged water from the surrounding native groundwater system. Recovery wells and monitoring wells are placed throughout the ARR site. During this study, DOC and nitrate concentrations in the recharged anoxic RBF filtrate were in the range of 3.3 ± 0.3 mg/L and 2.7 ± 1.2 mg N/L, respectively. Due to the low amount of BDOC in the RBF filtrate (approximately 0.4 mg/L) and re-aeration during surface spreading, subsurface redox conditions at the ARR site remained oxic to suboxic. A detailed description of the Prairie Waters Project North Campus is provided elsewhere (14, 18–20).

Soil Column Systems Two large-scale soil column systems (each 4.5 m in length, 0.15 m inner diameter), filled with a blend of 50:50 (v/v) technical sand and sandy soil from the Prairie Waters Project North Campus (grain size

Linking Trace Organic Chemical Attenuation to Microbiome Metabolic

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