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Published: 11 August 2015

Anaerobic microorganisms and bioremediation of organohalide pollution

Matthew Lee A , Chris Marquis A , Bat-Erdene Judger A and Mike Manefield A B

A School of Biotechnology and Biomolecular Sciences
University of New South Wales
Sydney, NSW 2052, Australia
Tel: +61 2 9385 1780

B Email: manefield@unsw.edu.au

Organohalide pollution of subsurface environments is ubiquitous across all industrialised countries. Fortunately, strictly anaerobic microorganisms exist that have evolved using naturally occurring organohalides as their terminal electron acceptor. These unusual organisms are now being utilised to clean anthropogenic organohalide pollution.

Subsurface water systems are a precious resource particularly in Australia where they are used for agricultural, industrial and domestic purposes including for drinking. However, a large range of organic and inorganic pollutants often compromise this precious resource, such as nitrates from overuse of fertilisers, heavy metals from the mining industry and BTEX compounds from petroleum spillages.

Bioremediation of organohalides has garnered a significant amount of research interest over the past 20 years1. Organohalides are a large family of compounds ranging from simple C1 and C2 aliphatics to complicated polyaromatic structures such as dioxins (Figure 1). The chemical and physical characteristics of these compounds have enabled them to be used in a large range of applications. Some of the more notable examples include: vinyl chloride (VC) in the manufacture of polyvinyl chloride (PVC), perchloroethene (PCE) as dry cleaning solvent and trichloromethane (chloroform; CF) as precursor chemical in the production of chlorofluorocarbon refrigerant gases. Because of their utility, these compounds have been synthesised in enormous quantities. For example PCE production in the USA peaked at 320 million kg per annum in the 1980s2. Industrial activity on such a large scale has resulted in the discharge of organohalides into the environment, where because of their high density and limited water solubility, tend to reside as solvent pools within subsurface water systems. These subsurface solvent pools are known as dense non-aqueous phase liquid (DNAPL) source zones. The DNAPL slowly dissolves into the surrounding water creating a toxic solvent plume radiating out from the source zone in the direction of the groundwater flow. To give some perspective on the extent of organohalide pollution, of the 1319 priority contaminated sites listed by the USA-EPA, 819 (61%) of them are polluted with PCE3.

Figure 1. Examples of organohalides that pollute Australian waterways. From left to right: perchloroethene (PCE); trichloromethane (chloroform; CF); and 2,3,7,8-tetrachlorobenzodioxin (TCDD).
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Fortunately, strictly anaerobic bacteria exist that have evolved to utilise organohalides as terminal electron acceptors. These organisms predate anthropogenic organohalide production and owe their existence to a wide range of naturally occurring organohalides in the environment4. Their metabolism results in the elimination of a halogen atom, which ultimately yields a much less toxic hydrocarbon. Organohalide respiring bacteria (ORB) perform the dehalogenation reaction by virtue of enzymes known as reductive dehalogenases that are constructed around a cobalt-containing cobalamin cofactor at the active site. The cobalt atom in its reduced form is a strong nucleophile that attacks the carbon atom in a carbon-halogen bond. In most ORB, the electrons that are ultimately transferred to the organohalide are acquired from the oxidation of hydrogen. The electrons are transferred via a series of membrane associated quinones and cytochrome-like proteins to the reductive dehalogenase (RdhA). The transfer of electrons generates a proton motive force and subsequently ATP5 (Figure 2).

Figure 2. A typical membrane associated electron transport system in ORB involved in the oxidation of molecular hydrogen and subsequent reduction of an organohalide. The proton motive force generated across the cytoplasmic membrane is used to synthesise ATP. MBH, membrane bound hydrogenase; MQ, menaquinone; RdhB, membrane anchor protein; RdhA, reductive dehalogenase (catalytic subunit).
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The first ORB reported about 30 years ago transformed 3-chlorobenzoic acid to benzoic acid6. However, the greatest advance in terms of organohalide bioremediation was the discovery of PCE-dechlorinating mixed cultures in the mid 1980s. These cultures produced various daughter compounds ranging from cis-dichloroethene and VC to ethane7,8. Isolation and characterisation of organisms capable of utilising PCE and trichloroethene (TCE) as terminal electron acceptors revealed the mechanism behind the dechlorination reactions observed in the mixed cultures. Strains from genera including Dehalospirillium, Desulfitobacterium, Desulfuromonas and Dehalobacter were shown to be capable of partial dechlorination of PCE and TCE to cis-DCE and the highly toxic carcinogen VC913 (Figure 3).

Figure 3. The transformation of the dry cleaning solvent (PCE) to the innocuous hydrocarbon ethene. Several genera of bacteria can capable of transforming PCE to cis-DCE or vinyl chloride; however, only Dehalococcoides strains are capable of complete dechlorination to ethene.
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For bioremediation, the production of partially chlorinated toxic daughter compounds is an undesirable outcome. Therefore, the discovery of Dehalococcoides mccartyii strain 19514, which could transform vinyl chloride to ethene, was extremely important, as this made bioremediation of chlorinated ethanes and ethenes a viable option. Dehalococcoides strains since that time have become synonymous with bioremediation because of their versatility. Dehalococcoides strains harbor up to 32 reductive dehalogenase homologs that are capable of dehalogenating not only halogenated C2 aliphatics, but also halogenated aromatic compounds such as hexachlorobenzene and chlorinated dioxins1517.

Dehalococcoides resides in the phylum Chloroflexi, to which the class Dehalococcoidetes was added to accommodate organohalide respiring members18. Dehalococcoides cells morphologically are unusual being disc shaped and only 0.3–1 µm in diameter and 0.2 µm thick. Another unusual feature of Dehalococcoides is the apparent lack of peptidoglycan layer in the cell-wall a feature typical of most bacteria18. Electron micrographs have revealed an unusual cell-wall S-layer resembling that found in archaea19. Dehalococcoides like most ORB and other strict anaerobes operate only at very low redox potentials (<110 mV)18. Therefore, minute traces of oxygen quickly curtail dehalogenating activity. This presents a difficulty in cultivating these types of organisms under laboratory conditions, where equipment and chemicals must be scrupulously deoxygenated in anaerobic chambers or with chemical reducing agents before they can be used.

Over the past 20 years numerous obligate and facultative ORB have been reported, and between them, these bacteria can use the vast majority of anthropogenic chlorinated ethanes and ethenes as sole electron acceptors (reviewed in Koenig et al.3). However, in reality, many organohalide-polluted sites contain more than one halogenated compound, which poses a challenge for ORB. The presence of chlorinated methanes in the solvent plume has been problematic, as they are strong inhibitors of most anaerobic microbial processes including organohalide respiration20,21. CF is particularly problematic as it is a widespread pollutant and highly recalcitrant, with a half-life of 3500 years in groundwater in the absence of appropriate microbes22. Until recently, the presence of CF has required site pretreatment for its removal, or the employment of abiotic strategies such as “pump and treat” or the installation of permeable reactive iron barriers23. However, recent discoveries of CF respiring Dehalobacter and Desulfitobacterium strains that transform CF to dichloromethane (DCM) have opened many CF-impacted sites to bioremediation strategies2426. Although DCM is not a favourable intermediate, recently Dehalobacter strains have been identified that can use DCM as a sole source of organic carbon and electrons, producing acetate, hydrogen and carbon dioxide. Both the CF respiring and DCM fermenting Dehalobacter strains have been shown to work together to effect complete dechlorination of CF26,27.

In conclusion, in subsurface environments where oxygen is in limited supply, microorganisms have evolved to use a multitude of alternative electrons acceptors for energy conservation. Organohalides are naturally occurring molecules in which the carbon halide bond is in a high oxidation state, hence bacteria have evolved to use these compounds as terminal electron acceptors in a process called organohalide respiration. These organisms have been exploited to remediate sub-surface environments that have been polluted with large quantities of anthropogenic organohalides. Research over the past 20 years has revealed numerous genera that are capable of organohalide respiration, with a large array of reductive dehalogenases that can tackle most organohalides found at polluted sites. However, knowledge gaps still exist around how ORB interact with other cohabiting microorganisms such as those supplying hydrogen and cobalamin cofactors.


Biographies

Matthew Lee is a Senior Research Associate at UNSW. His research interests include microbiology in subsurface environments, particularly chlorinated methane metabolising bacteria.

Chris Marquis is a bioprocess engineer who runs the UNSW Recombinant Products Facility at UNSW and has interests in applications of microbiology and protein.

Bat-Erdene Judger is a PhD student at UNSW. His research project is to heterologous production of functional reductive dehalogenases from Dehalobacter and Dehalococcoides species.

Mike Manefield is an Associate Professor at UNSW. His interests are in environmental microbiology and biotechnology development.

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