Anaerobic microorganisms and bioremediation of organohalide pollution

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).

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).

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 ethane^7,8. 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 VC^9–13 (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.

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 dioxins^15–17.

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 respiration^20,21. 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 strategies^24–26. 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 CF^26,27.

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.