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Novel Remediation and Control Technologies
A Mixed Microbial Community for the Biodegradation of Chlorinated Ethenes and 1,4-Dioxane Alexandra Polasko, Alessandro Zulli, Phillip B Gedalanga, Peerapong Pornwongthong, and Shaily Mahendra Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00591 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018
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Environmental Science & Technology Letters
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A Mixed Microbial Community for the
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Biodegradation of Chlorinated Ethenes and 1,4-
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Dioxane
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Alexandra Polasko1, Alessandro Zulli1, Phillip B. Gedalanga2, Peerapong Pornwongthong3,
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Shaily Mahendra1*
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1
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Hall, Los Angeles, California 90095, USA
Department of Civil and Environmental Engineering, University of California, 5732 Boelter
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Room KHS-121, Fullerton, California 92834, USA
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Science, King Mongkut's University of Technology North Bangkok, 1518 Pracharat 1,
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Wongsawang, Bangsue, Bangkok 10800, Thailand
Department of Health Science, California State University, 800 North State College Blvd.,
Department of Agro-Industrial, Food and Environmental Technology, Faculty of Applied
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Running head: Sequential anaerobic-aerobic biodegradation of trichloroethylene and 1,4-dioxane
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*Corresponding author:
[email protected] 21 1 ACS Paragon Plus Environment
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Abstract
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We developed a microbial community capable of biodegrading mixtures of chlorinated
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ethenes and 1,4-dioxane under varying redox conditions. Achieving the removal of chlorinated
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ethenes as well as 1,4-dioxane to below health advisory levels in groundwater that has anaerobic
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and aerobic zones has proven to be challenging. However, our mixed community comprised of
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the anaerobic, chlorinated ethene-degrading consortium (KB-1) and aerobic bacterial strain,
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Pseudonocardia dioxanivorans CB1190, was able to reduce trichloroethene (TCE) in anaerobic
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environments and oxidize 1,4-dioxane after oxygen amendments. Aerobic biodegradation of cis-
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1,2-dichloroethene (cis-DCE) by CB1190 was also confirmed, thus decreasing the accumulation
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of TCE transformation products such as vinyl chloride. The results from this study demonstrate
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that the assembled microbial community not only survived significant redox changes, but
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sequentially biodegraded chlorinated ethenes and 1,4-dioxane. This approach towards degrading
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mixed contaminants with a consortium containing anaerobic and aerobic bacteria provides
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greater insight as to when and how to transition from active enhanced reductive dechlorination to
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enhanced aerobic attenuation of chlorinated ethenes and 1,4-dioxane.
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Supplemental Keywords: biotransformation, trichloroethylene, dioxane monooxygenase,
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reductive dechlorination
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Environmental Science & Technology Letters
Introduction
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Chlorinated ethenes and 1,4-dioxane are probable human carcinogens and commonly co-
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occurring groundwater contaminants.1-5 Biological treatment technologies for these compounds
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individually have become attractive remediation methods due to low implementation costs and
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minimal invasiveness.6, 7 Members of the bacterial genus, Dehalococcoides, are the most widely
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reported bacteria capable of metabolically degrading chlorinated ethenes, such as trichloroethene
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(TCE) via reductive dechlorination.8 However, a downside to this process is the formation and
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accumulation of intermediate transformation products, such as cis-1,2-dichoroethene (cDCE) and
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vinyl chloride (VC).9 Aerobic cometabolism of TCE can be carried out using substrates such as,
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methane, propane, methanol, toluene, phenol, and ammonia.6 However, cometabolism-based
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remediation requires additional amendments, such as electron donors for inducing appropriate
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biodegradative enzymes in microbial populations.10, 11
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A variety of microbial strains have been reported to metabolically or co-metabolically 1,4-dioxane
under
aerobic
conditions
only.7
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degrade
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monooxygenase responsible for initiating 1,4-dioxane degradation is the tetrahydrofuran
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(THF)/dioxane monooxygenase.12 The genes coding for this enzyme, thmADBC/dxmADBC,
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along with the aldehyde dehydrogenase, aldH, serve as biomarkers to verify 1,4-dioxane
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biodegradation.13 Some actinomycetes, especially, members of Pseudonocardia, are capable of
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mineralizing 1,4-dioxane via initial monooxygenase-catalyzed hydroxylation, followed by
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common cellular metabolic pathways.12-16 1,4-Dioxane-cometabolizing microorganisms are
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equipped with monooxygenases induced by multiple carbon sources such as methane, propane,
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phenol, tetrahydrofuran, and toluene.17,
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degradation may allow for the removal of lower concentrations of 1,4-dioxane 13, 19-24 but might
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A
bacterial
multicomponent
Decoupling cellular growth from 1,4-dioxane
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result in toxic products, such as 2-hydroxyethoxyacetic acid.20 Conversely, minimum substrate
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concentrations are required to sustain microbial growth
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expected to accumulate during 1,4-dioxane metabolizing processes.12
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but toxic intermediates are not
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Numerous in-situ bioremediation projects have been performed that use biostimulation
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along with bioaugmentation to promote biodegradation of chlorinated ethenes.26-29 Many of these
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projects have involved enhancing reductive dechlorination and have focused on promoting
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anaerobic conditions with very low redox environments (oxidation-reduction potential < -200
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millivolts). If complete reduction to ethene is not achieved, there is a risk of cDCE or VC
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migrating downgradient from source zones. As the concern of 1,4-dioxane rises, remedial
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technology selection process must consider approaches to remove chlorinated solvents as well as
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1,4-dioxane. Aerobic biodegradation of 1,4-dioxane is possible in the naturally changing redox
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conditions of plumes as they move downgradient from anaerobic source zones toward more
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aerobic environments.30 Few studies have evaluated the versatility and abilities of 1,4-dioxane
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degrading microorganisms in divergent redox environments. Opposing redox conditions favored
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by chlorinated ethene- and 1,4-dioxane-degrading bacteria pose a challenge for concurrent
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bioremediation of both contaminants.
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The objective of this study was to formulate a microbial community to simultaneously or
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sequentially degrade chlorinated ethenes and 1,4-dioxane under changing redox conditions.
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After various anaerobic incubation periods, the reactivation of the monooxygenase enzymes to
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aerobically biodegrade 1,4-dioxane and chlorinated solvents was also investigated.
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Environmental Science & Technology Letters
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Materials and Methods
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Chemicals
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1,4-Dioxane (99.8%, ACS grade), TCE (≥99.5%, ACS grade), cis-DCE (97%), sodium
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lactate (60% w/w), and titanium trichloride (TiCl3) (≥99.9%), were obtained from Sigma-
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Aldrich. Saturated chlorinated stocks were prepared as previously described.31
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Bacterial Cultures and Growth Conditions
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The widely used and resilient mixed consortium, KB-1, containing Dehalococcoides, was
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grown in a defined medium 31, with 20 mg/L TCE (aqueous) as electron acceptor and 60 mg/L
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lactate as carbon source.32 Sterile 160 mL serum bottles containing media were sparged for 3
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minutes with filtered (0.2 µm) N2 to achieve anaerobic conditions (Figure S1) and sealed with 20
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mm butyl rubber stoppers. TiCl3 was also used as reductant. CB1190 seed cultures were prepared
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using a 1% (v/v) transfer from an actively growing pure culture under aerobic conditions in the
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lactate-free medium previously used for growing KB-1. Experimental bottles were inoculated
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with stock cultures in their late exponential or early stationary phase after TCE (KB-1) or 1,4-
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dioxane (CB1190) was degraded to below 1,000 µg/L.
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Design of Batch Experiments
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The microcosms were prepared in sterile 500 mL glass bottles and maintained with 1:5
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liquid to headspace ratios with the following conditions: 1) KB-1 + CB1190 that transitioned
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from anaerobic to aerobic, 2) CB1190 only that transitioned from anaerobic to aerobic, and 3)
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CB1190 only that remained anaerobic. Experimental bottles were inoculated with 10% (v/v)
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CB1190 and 5% (v/v) KB-1. Once TCE was degraded to cis-DCE by KB-1, 60 mL of filtered,
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high purity oxygen was amended to transition anaerobic to aerobic conditions. All bottles were
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incubated upright at 30°C with 150 rpm shaking. TCE and 1,4-dioxane concentrations were
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amended as liquid concentrations. At each time point, 200 µL of liquid sample were collected
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and filtered for 1,4-dioxane quantification.
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Analytical Methods
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Chlorinated ethenes were measured by direct headspace injections (10 or 100 µL
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depending on concentration) using gas chromatography-mass spectrometry (GC-MS; Agilent
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6890 GC and 5973 MS). 1,4-Dioxane concentrations higher than 1,000 µg/L were measured
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using a GC equipped with a flame ionization detector in 2 µL aqueous samples, while 1,4-
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dioxane samples containing
