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Remediation and Control Technologies

Bioremediation Strategies Aimed at Stimulating Chlorinated Solvent Dehalogenation can Lead to Microbially-Mediated Toluene Biogenesis William M Moe, Samuel J Reynolds, M Aaron Griffin, and J Bryan McReynolds Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02081 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Environmental Science & Technology

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Bioremediation Strategies Aimed at Stimulating

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Chlorinated Solvent Dehalogenation can Lead to

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Microbially-Mediated Toluene Biogenesis

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William M. Moe*,†, Samuel J. Reynolds†, M. Aaron Griffin†, J. Bryan McReynolds‡

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†

Louisiana State University, Department of Civil and Environmental Engineering, Baton Rouge, LA 70803, USA

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‡

Dayspring Group, LLC, Baton Rouge, LA 70808, USA

AUTHOR INFORMATION *Corresponding Author

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Address: Department of Civil and Environmental Engineering, Louisiana State University, 3255 Patrick F. Taylor Hall, Baton Rouge, LA 70803

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E-mail: [email protected]; Telephone: +1-225-578-9174

1 ACS Paragon Plus Environment


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ABSTRACT

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In situ bioremediation practices that include subsurface addition of fermentable electron donors

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to stimulate reductive dechlorination by anaerobic bacteria have become widely employed to

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combat chlorinated solvent contamination in groundwater. At a contaminated site located near

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Baton Rouge, Louisiana (USA), toluene was transiently observed in groundwater at

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concentrations that sometimes far exceeded the US drinking water maximum contaminant level

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(MCL) of 1 mg/L after a fermentable substrate (agricultural feed grade cane molasses) was

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injected into the subsurface with the intent of providing electron donors for reductive

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dechlorination. Here, we present data that demonstrate that indigenous microorganisms can

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biologically produce toluene by converting phenylacetic acid, phenylalanine, phenyllactate, and

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phenylpyruvate to toluene. When grown in defined medium with phenylacetic acid at

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concentrations ≤350 mg/L, the molar ratio between toluene accumulated and phenylacetic acid

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supplied was highly correlated (R2>0.96) with a toluene yield exceeding 0.9:1. Experiments

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conducted using

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resulted in production of toluene-α-13C, confirming that toluene was synthesized from these

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precursors by two independently developed enrichment cultures. Results presented here suggest

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that monitoring of aromatic hydrocarbons in warranted during enhanced bioremediation

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activities where electron donors are introduced to stimulate anaerobic biotransformation of

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chlorinated solvents.

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C labelled compounds (phenylacetic acid-2-13C and L-phenylalanine-3-13C)


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GRAPHIC FOR TABLE OF CONTENTS/ABSTRACT ART

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Phenylacetic acid-2- C

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L-Phenylalanine-3- C

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Toluene-α- C



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INTRODUCTION

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Chlorinated alkanes (e.g., 1,2-dichloroethane and 1,2-dichloropropane) and alkenes (e.g.,

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trichloroethene and vinyl chloride) are widespread soil and groundwater pollutants that pose a

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risk to human health on account of their toxicity and/or carcinogenicity.1 Fortunately, some

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bacterial genera possess the metabolic capacity to reductively dehalogenate chlorinated aliphatic

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hydrocarbons into environmentally benign final products. For example, Dehalococcoides

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mccartyi strains are able to transform the carcinogen vinyl chloride into ethene.2,3 Similarly,

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representatives from the genus Dehalogenimonas are able to dehalogenate 1,2-dichloroethane to

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ethene and 1,2-dichloropropane to propene.4-6 Because the chlorinated compounds serve as

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electron acceptors for organohalide respiring bacteria, a suitable electron donor must be available

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for both Dehalococcoides and Dehalogenimonas to transform the chlorinated substrates. The

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number of electron donors that may be utilized by some dechlorinating genera are quite limited,

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with H2 the only known electron donor for Dehalococcoides3 and H2 or formate the only known

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electron donors utilized by Dehalogenimonas.6

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A technique that has become widely implemented in recent years to enhance in-situ

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biotransformation of chlorinated compounds is to inject a fermentable substrate (e.g., sugars,

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lactate, molasses, emulsified vegetable oil, or poly-lactate esters) into the subsurface so that

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indigenous microbial populations can produce electron donors (e.g., H2) that can be utilized by

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dechlorinating bacteria.7-10 Fermentative bacteria able to produce H2 from various substrates

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include a variety of Clostridium species11 and other taxa12 that appear to be ubiquitous in the

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environment.13

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While bioremediation strategies involving electron donor additions have proven

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successful for chlorinated solvent remediation at many sites around the world, here we report on

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a potential unintended consequence of electron donor addition, the biologically mediated in-situ 4 ACS Paragon Plus Environment

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production of the aromatic hydrocarbon toluene, a pollutant conventionally thought to be

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associated with anthropogenic pollution sources. Data reported include a combination of field

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observations of toluene concentrations over a four year period as well as laboratory studies

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conducted to identify precursors that have the potential to form toluene.

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MATERIALS AND METHODS

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Field site and microcosm studies. Field monitoring was conducted at a Superfund Site located

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in southeast Louisiana (USA) where groundwater is contaminated with a variety of chlorinated

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alkanes (e.g., 1,2-dichloroethane and 1,2-dichloropropane) and alkenes (e.g., tetrachloroethene,

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vinyl chloride). In 2012, a series of groundwater wells (selected well locations provided in

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Supporting Information Table S1) was installed to allow the subsurface injection of amendments

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intended to facilitate establishment of anaerobic conditions and stimulate the biologically

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mediated reductive dechlorination of halogenated aliphatic compounds near the leading edge of

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the site’s contaminated groundwater plume. The subsurface injection of molasses in the study

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area was accomplished by pumping groundwater from adjacent wells, metering in agricultural

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feed grade cane molasses and in some cases bicarbonate buffer (injection quantities for selected

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wells shown in Supporting Information Table S2), and then re-injecting the amended

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groundwater. In the area reported on here, molasses amended groundwater was injected into each

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well on four separate occasions spaced at roughly one year intervals.

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Groundwater sampling was conducted prior to and after molasses addition to quantify

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groundwater concentrations of both contaminants and geochemical parameters. Concentrations

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of volatile organic compounds were measured via gas chromatography mass spectrometry (GC-

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MS) using EPA method 8260B. Dissolved ethene, ethane and methane were measured using

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method RSK 175. Nitrate and nitrite were measured using US EPA method 353.2. Chloride was 5 ACS Paragon Plus Environment


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measured using US EPA method 325.2. Sulfate was measured by ion chromatography using US

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EPA method 300.0. Sulfide was measured using US EPA method 376.2. Ferrous iron was

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measured using US EPA method 3500-Fe D. Total organic carbon (TOC) was measured using

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US EPA method 5310B. Detailed descriptions of the US EPA analytical methods referenced

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above are available elsewhere (National Environmental Index, http://www.nemi.gov/).

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Groundwater temperature, conductivity, turbidity, pH, and ORP were measured at the time of

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collection using probes and flow cell (In-Situ).

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A groundwater microcosm study was conducted using groundwater sampled from well

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ID SP024 70 days after the third subsurface injection of molasses in that well. For microcosms,

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groundwater was collected in sterile glass bottles filled leaving little or no gas headspace. After

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transport to the laboratory (approximately one hour), groundwater samples were transferred into

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an anaerobic chamber (Coy Laboratory Products), and 100 mL aliquots of groundwater were

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aseptically dispensed into sterile 160 mL glass serum bottles (Wheaton) that were capped with

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butyl rubber stoppers and aluminum crimp caps with no additional media supplements. Bottles

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were incubated in the dark, without mixing, at ambient laboratory temperature (21±2˚C),

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comparable to the 20.9°C site groundwater temperature on the day of collection (Table S4). At

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weekly intervals, samples from duplicate bottles were analyzed by gas chromatography to

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quantify toluene. Following analysis, microcosm replicates were stored at 4°C.

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Enrichment cultures. Two separate enrichment cultures were developed from groundwater

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collected from different wells on different dates. Groundwater sample locations (Supporting

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Information Table S1), contaminant concentrations (Supporting Information Table S3), and

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various geochemical concentrations at the time of initial groundwater sample collection

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(Supporting Information Table S4) are provided in Supporting Information. The enrichment


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cultures were prepared using strictly anaerobic protocols and aseptic techniques. All cultures

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were incubated at ambient laboratory temperature (21±2 °C) in the dark without mixing.

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The first enrichment culture, designated as AG, was established by propagating the

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microbial population from microcosms described above that were established using groundwater

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sampled from well ID SP024. Following storage at 4°C, the culture grown with only

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groundwater was subsequently inoculated (5% v/v) into anoxic TP medium14 but without

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Na2S·9H2O, phenylalanine added to a final concentration of 132 mg/L, and glucose added to a

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final concentration of 2.0 g/L. After incubation at ambient laboratory temperature for 6 weeks,

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the culture was subsequently transferred into anoxic modified TP medium prepared as described

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by Zargar et al.15 but with phenylacetic acid omitted and phenylalanine added to a concentration

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of 220 mg/L. The culture was serially transferred (0.1-5%, v/v) using sterile, disposable syringes

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and needles at 3-8 week intervals in the same medium but with either phenylalanine or

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phenylacetic acid supplied at concentrations in the range of 27 to 800 mg/L.

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A second enrichment culture, designated as SR, was established by inoculating

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groundwater (4% v/v) sampled from well ID SP022 at a time 222 days after the fourth injection

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of molasses-amended groundwater into anoxic modified TP medium as described previously15

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but with phenylacetic acid added to a final concentration of 350 mg/L, pH 7.6. The culture was

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serially transferred (0.1-10%, v/v) a total of 11 times at 2-8 week intervals in the same medium.

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Toluene production by enrichment cultures. To investigate the relationship between toluene

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production and the amount of phenylacetic acid supplied to the enrichment cultures as a potential

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precursor, we tested cultures supplied with phenylacetic acid (Aldrich P16621, 99% purity) over

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the concentration range of 0 to 800 mg/L. 25 mL glass serum bottles containing 15 mL liquid

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medium, sealed with PTFE-lined butyl rubber stoppers and aluminum crimp caps, were



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inoculated (0.1% v/v) with one of the two enrichment cultures (AG or SR). Serum bottles

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prepared with identical medium but without microbial inoculation served as abiotic negative

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controls. Bottles without phenylacetic acid addition but with microbial inoculation served as

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additional controls. For serum bottles containing 350 and 700 mg/L phenylacetic acid, triplicate

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bottles were sacrificed at regular (~3 day) intervals over an incubation period lasting 30 days.

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For other concentrations, triplicate bottles were sacrificed at a single time step after 30 days

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incubation.

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To further assess whether the enrichment cultures were able to transform phenylacetic 13

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acid to toluene, modified TP medium was prepared as described above but with

C-labelled

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phenylacetic acid (phenylacetic acid-2-13C, Aldrich 293849, 99 atom% 13C) replacing the regular

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(i.e., unlabeled) phenylacetic acid. The phenylacetic acid-2-13C was added to a final

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concentration of 250 mg/L. Replicate glass serum bottles sealed with PTFE-lined butyl rubber

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stoppers and aluminum crimp caps were inoculated 0.1% v/v with culture SR or AG. Abiotic

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negative controls were prepared in exactly the same manner but without inoculation. Triplicate

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bottles were sacrificed after 45 days incubation for analysis via GC-MS. Toluene-α-13C (Aldrich

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487082, 99 atom % 13C) dissolved in deionized water was included as a standard.

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Experiments conducted to investigate whether additional phenyl-containing compounds

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could serve as precursors for toluene production were conducted in 25 mL glass serum bottles

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containing 15 mL liquid medium, sealed with PTFE-lined butyl rubber stoppers and aluminum

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crimp caps. Bottles were inoculated (0.1% v/v) with one of the two enrichment cultures (AG or

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SR). L-Phenylalanine (Sigma P5482, assay 98.5-101.0%) was added to reach final aqueous-

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phase concentrations of 350 mg/L while sodium phenylpyruvate (≥95%, Aldrich P8001),

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phenylacetaldehyde (≥95%, Aldrich W287407), and L-(−)-3-phenyllactic acid (98%, Aldrich


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113069) were added to reach final aqueous-phase concentrations of 250 mg/L. Serum bottles

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prepared with identical medium but without microbial inoculation served as abiotic negative

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controls. Triplicate bottles were analyzed for toluene after incubation for 35-37 days. Bottles not

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exhibiting appreciable toluene accumulation were further incubated and reanalyzed at t=70 days.

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To fully confirm whether the enrichment cultures were able to transform phenylalanine to

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toluene, experiments described above were repeated but with 13C labelled L-phenylalanine -β-13C

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(Aldrich 490121, 99 atom%

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ambient laboratory temperature for 35 days prior to analysis by GC-MS.

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C) added to a concentration of 250 mg/L. Incubation was at

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For routine analyses, aqueous-phase toluene concentrations in laboratory enrichment

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cultures were measured using an Agilent Model 7820A Gas Chromatograph (GC) equipped with

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a flame ionization detector (FID) and a DB-624 capillary column (60 m × 0.32 mm × 1.80 µm).

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The GC thermal program included a five minute hold at 40°C, a 20°C/minute ramp to 260°C,

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and a 3-minute hold at 260°C. Aqueous-phase samples were introduced to the GC utilizing a

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Teledyne Tekmar AQUATek 100 autosampler in conjunction with a Teledyne Tekmar Purge and

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Trap. A subset of laboratory enrichment culture samples (n=13) were additionally analyzed via

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gas chromatography (GC) with mass spectrometry (MS) detection following EPA Method

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8260B using an Agilent model 6890N GC equipped with an Agilent 5975B detector and an Rtx-

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VMS fused silica column (30 m × 0.25 mm, Restek). Triplicate bottles from both cultures

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amended with

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temperatures were monitored using Traceable digital thermometers (Fisher Scientific).

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C-labelled precursors were analyzed by GC-MS. Laboratory incubation

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To facilitate mass balance calculations, the amount of toluene per serum bottle was

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calculated using experimentally measured aqueous-phase toluene concentrations in conjunction



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with an assumed toluene dimensionless Henry’s Law Constant of 0.209 [=0.00506

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atm·m3/mol].16

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Molasses analysis. Molasses from two bulk distributors of agricultural feed grade molasses

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(Table S6) were analyzed to determine total phenylalanine content (free amino acid +

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phenylalanine incorporated into proteins). Molasses aliquots were hydrolyzed in 6 N HCl for 24

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hours at 110 °C with N2 as the gas headspace, dried under vacuum, reconstituted in ultrapure

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water, and amino acids were quantified by reversed-phase HPLC with fluorescence detection

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after pre-column derivatization of with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

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(AQC) as described previously.17 Analysis employed AccQ-Tag chemistry (Waters) in

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conjunction with a Breeze 2 HPLC System equipped with a binary HPLC pump, a model 2475

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multi-λ fluorescence detector and a model 717plus autosampler injector (Waters). To determine

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the concentrations of phenyl-containing organic acids, aliquots of molasses were added to

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deionized water at a final concentration of 10 g/L, mixed on a magnetic stir plate for two hours,

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centrifuged (10 min, 10,000×g), and supernatants were analyzed via ion chromatography (IC) as

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described previously18 but with phenylacetic acid (Aldrich P16621, 99% purity), L-(−)-3-

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phenyllactic acid (98%, Aldrich 113069), and sodium phenylpyruvate (≥95%, Aldrich P8001)

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dissolved in deionized water as standards.

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RESULTS AND DISCUSSION

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Field-scale Observations and Microcosms. Prior to the first injection of molasses and

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bicarbonate buffer at the field site, groundwater collected from the two groundwater wells

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reported on here (SP022 and SP024) was moderately acidic (pH 5.5-6.0), aerobic (dissolved

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oxygen approximately 3 mg/L), contained low alkalinity (61-73 mg/L as CaCO3), moderate

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levels of sulfate (25.9 to 30.3 mg/L), and low TOC (

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