Alison M. Cupples

Detection of the antimicrobials triclocarban and triclosan in agricultural soils following land application of municipal biosolids

The occurrence of the antimicrobials triclocarban (TCC) and triclosan (TCS) was investigated in agricultural soils following land application of biosolids using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with negative ion multimode ionization. The method detection limits were 0.58 ng TCC/g soil, 3.08 ng TCC/g biosolids, 0.05 ng TCS/g soil and 0.11 ng TCS/g biosolids and the average recovery from all of the sample matrices was >95%. Antimicrobial concentrations in biosolids from three Michigan wastewater treatment plants (WWTPs) ranged from 4890 to 9280 ng/g, and from 90 to 7060 ng/g, for TCC and TCS respectively. Antimicrobial analysis of soil samples, collected over two years, from ten agricultural sites previously amended with biosolids, indicated TCC was present at higher concentrations (1.24-7.01 ng/g and 1.20-65.10 ng/g in 2007 and 2008) compared to TCS (0.16-1.02 ng/g and from the method detection limit, <0.05-0.28 ng/g in 2007 and 2008). Soil antimicrobial concentrations could not be correlated to any soil characteristic, or to the time of last biosolids application, which occurred in either 2003, 2004 or 2007. To our knowledge, our data represent the first report of TCC, and the first comparison of TCC and TCS concentrations, in biosolids-amended agricultural soils. Such information is important because approximately 50% of US biosolids are land applied, therefore, any downstream effects of either antimicrobial are likely to be widespread.

Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing

ABSTRACT Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [13C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.

Identification of a novel toluene-degrading bacterium from the candidate phylum TM7, as determined by DNA stable isotope probing.

ABSTRACT The dominant bacterium responsible for carbon uptake from toluene in an agricultural soil was identified by stable isotope probing. Samples were amended with unlabeled toluene or labeled [ring-13C6]toluene, and DNA was extracted over time. Sequencing indicated that the organism involved belongs to the candidate phylum TM7. Microorganisms in this candidate phylum are of particular interest because although they have been found in a variety of habitats, no stable culture of any species exists, so their general metabolic capabilities are largely unknown.

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (13C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for 13C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (13C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived 13C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).

Presence, diversity and enumeration of functional genes (bssA and bamA) relating to toluene degradation across a range of redox conditions and inoculum sources

The study investigates two functional genes for toluene degradation across three redox conditions (nitrate and sulfate amended and methanogenic). The genes targeted include benzylsuccinate synthase α-subunit (bssA) and a gene recently identified as being a strong indicator of anaerobic aromatic degradation, called 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase (bamA). In all, sixteen different anaerobic toluene degrading microcosms were investigated using several primers sets targeting bssA and one primer set targeting bamA. One bssA primer set (7772f/8546r) was the most successful in producing a strong amplicon (eight from sixteen) with the other bssA primers sets producing strong amplicons in six or less samples. In contrast, the bamA primer set (bam-sp9 and bam-asp1) produced a strong amplicon in DNA extracted from all except one microcosm. Partial bssA and bamA sequences were obtained for a number of samples and compared to those available in GenBank. The partial bssA sequences (from nitrate amended and methanogenic microcosms) were most similar to Thauera sp. DNT-1, Thauera aromatica, Aromatoleum aromaticum EbN1 and bssA clones from a study involving sulfate reducing toluene degradation. The bamA sequences obtained could be placed into five previously defined clades (bamA-clade 1, Georgfuchsia/Azoarcus, Magnetospirillum/ThaueraSyntrophus and Geobacter clades), with the placement generally depending on redox conditions. Gene numbers were also correlated with toluene degradation and the final gene number for both genes differed considerably between the range of redox conditions. The work is the first in depth investigation of bamA diversity over a range of redox conditions and inoculum sources.

The use of nucleic acid based stable isotope probing to identify the microorganisms responsible for anaerobic benzene and toluene biodegradation

The remediation of sites contaminated with gasoline has been limited by a lack of information on the microorganisms able to transform these chemicals under anaerobic conditions. To address this, researchers have recently adopted a molecular method, called stable isotope probing (SIP), to identify anaerobic toluene and benzene degraders from a number of environments across the globe. The approach involves incubation with (13)C labeled benzene or toluene, DNA or RNA extraction, ultracentrifugation and molecular analysis of the separated fractions to determine the organisms responsible for label uptake and therefore contaminant degradation. This manuscript reviews the methods and key results of the studies that have used SIP to specifically investigate anaerobic toluene and benzene degradation. These studies have examined toluene removal under sulfate reducing conditions and benzene degradation under methanogenic, sulfate reducing, iron reducing and nitrate reducing and aerobic conditions. The research to date indicates microorganisms affiliating with the Clostridia (genera Desulfosporosinus and Desulfotomaculum in the family Peptococcaceae) and Deltaproteobacteria (e.g. genera Desulfocapsa in the family Desulfobulbaceae and Desulfobacterium in the family Desulfobacteraceae) appear to be the active toluene degraders under sulfate reducing conditions. A greater variety of organisms were identified as anaerobic benzene degraders, likely because a range of anaerobic conditions were examined. However, several studies also linked anaerobic benzene degradation to the Deltaproteobacteria (e.g. Desulfobacteraceae, Desulfobulbacea) and the Clostridia (e.g. Peptococcaceae). In summary, these studies highlight the importance of SIP as a method for linking function (anaerobic toluene and benzene degradation) with identity for complex samples.

Direct Link between Toluene Degradation in Contaminated-Site Microcosms and a Polaromonas Strain

ABSTRACT Stable isotope probing (SIP) was used to identify the aerobic toluene-degrading microorganism in soil microcosms. Several approaches (terminal restriction fragment length polymorphism, 16S rRNA gene sequencing, and quantitative PCR) provided evidence that the microorganism responsible was a member of the genus Polaromonas and could grow on toluene. This microorganism also transformed benzene, but not m-xylene or cis-dichloroethene.

Dehalogenation of the Herbicides Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by Desulfitobacterium chlororespirans

ABSTRACT Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 μM) and DBHB (250 to 260 μM) were used as electron acceptors for growth. Doubling times for growth (means ± standard deviations for triplicate cultures) on bromoxynil (18.4 ± 5.2 h) and DBHB (11.9 ± 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.

Triclocarban and triclosan biodegradation at field concentrations and the resulting leaching potentials in three agricultural soils.

This study evaluated the leaching potential of the antimicrobials triclocarban (TCC) and triclosan (TCS) in three agricultural soils using a simple model based on biodegradation and adsorption. The antimicrobials were added to the soils at two moisture levels (10% or 15% w/w) to achieve initial concentrations of 0.05, 0.2, or 2 mg kg(-1). The low concentrations (0.05, 0.2 mg kg(-1)) are more representative of field concentrations, important because previous studies have typically focused on higher initial concentrations. After 100 d, significant residuals of both TCC and TCS occurred under all conditions and first-order degradation half-lives indicated TCC was more resistant to biodegradation. The estimated K(d) and K(oc) values were 193-296 L kg(-1) and 18175-33991 L kg(-1) for TCC and 33-55 L kg(-1) and 3968-6310 L kg(-1) for TCS. The resulting leaching models indicated these chemicals have a very low leaching potential and are thus unlikely to contaminate groundwater.

Anaerobic Methyl tert-Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing

ABSTRACT Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.