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Bioremediation of Halogenated Aliphatic Compounds

Paper Type: Free Essay Subject: Sciences
Wordcount: 6332 words Published: 8th Feb 2020

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A Halogenated compound is a chemical compound in which one or more hydrogen atoms have been replaced by a halogen atom such as - fluorine, chlorine, and bromine (ILO, 2011). Due to their excellent ability to dissolve oils, fast evaporation rate, and chemical stability, they have been widely used in industrial, commercial, and agricultural fields. They are used as pesticides, plasticizers, paint and printing-ink components, textile auxiliaries, refrigerants, and flame retardants (Chaudhry and Chapalamadugu, 1991). They are also widely used for cleaning purposes as dry cleaning fluid, degreasing solvent, and ink and paint strippers.

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Despite their efficacy, halogenated compounds require additional attention and cost to pretreat before they can be disposed. They are incompatible with other waste because they are toxic, mutagenic, and carcinogenic (Chaudhry and Chapalamadugu, 1991), thus, posing a threat to environment and causing human health problems. Humans are usually exposed to halogenated compounds by oral inhalation or dermal contact, as they are prevalent in drinking water and groundwater. Once exposed to humans, they cause not only cancer but also effect on nervous system, and injury of vital organ, particularly liver (Stellman, 1998). Due to the detrimental effects of halogenated compounds, their use has been forbidden worldwide. Even trace amounts of halogenated compounds present in effluents are of concern.

Therefore, renowned researchers have been doing studies to degrade halogenated compounds, and they have found efficient and effective methods to remediate sites contaminated by halogenated compounds by utilizing certain bacteria. In this context, this paper intends to provide background knowledge about halogenated compounds and a selected compound to aid better understanding of this paper. In addition, this paper introduces bioremediation pathways used in selected articles and discusses their approach and conclusion of the studies, and demonstrate writer’s own critical thinking on the papers.


Halogenated compounds are composed of two major groups: halogenated aromatic compounds and halogenated aliphatic compounds. Most halogenated aliphatic compounds are chemically stable, and lack a benzene ring typical in halogenated aromatic compounds (Flowers, 2017). This paper mainly focuses on halogenated aliphatic compound, and its biodegradation pathway. Therefore, the sections in this paper intend to provide general information about aliphatic compounds, and introduce proposed degradation pathways by utilizing certain bacteria on selected compounds from the articles. Furthermore, the following sections scrutinize the approach and conclusion on published papers in order to demonstrate depth of author’s understanding about dehalonization - process of replacing halogen atoms to hydrogen atoms - pathway. In the end of this paper, it reveals writer’s independent assessments on selected papers.

I.       Properties of Aliphatic Compounds

All of aliphatic compounds are divided into two categories; saturated aliphatic compounds and unsaturated aliphatic compounds. Saturated aliphatic compounds known as alkanes only contain single bonds. In contrast, unsaturated aliphatic compounds contains multiple double bonds, known as alkenes, and triple bonds, known as alkynes (Abozenadah et al., 2017). Both saturated and unsaturated aliphatic compounds are entirely linked carbon and hydrogen atoms, and contained straight-chain. Once hydrogen atoms are replaced by halogen atoms, it is called halogenated aliphatic compound. Moreover, unsaturated aliphatic compounds are linked less hydrogen atoms than saturated aliphatic compounds, therefore, this property makes alkenes and alkynes relatively reactive (Flowers, 2017).

Figure 1. Skeletal structure of Saturated Alkane and Unsaturated Alkene
(Abozenadah et al., 2017)

Aliphatic compounds will react differently with halogen atoms based on their physical feature, therefore, comprehending their physical feature is very important. The physical features of alkane and alkene are relatively similar. The boiling point of alkane and alkene increase with increasing molecule weight. They are insoluble in water, however, they are soluble in organic solvents.


Molecular Formula

Molecular Weight

Melting Point ()

Boiling Point ()




































Table 1. Physical Properties of Alkane Group (Mehendale, 2017)


Molecular Formula

Molecular Weight

Melting Point ()

Boiling Point ()































Table 2. Physical Properties of Alkene Group (Abozenadah et al., 2017)

II.    Selected Halogenated Aliphatic Compound; Chloroethenes

1.      Properties of Chloroethenes

Chloroethenes, unsaturated aliphatic compounds contained double bonds, are mainly used in the industrial fields as paint remover, solvent, and chemical intermediate for fibers and pesticides productions. They were produced about approximately 20 to 25 million tonnes/year in late 80s, and 90% of the total production was vinyl chloride (Agteren et al., 1998).



    Vinyl Chloride (VC)

     Trichloroethene (TCE)

Figure 3. Chemical structure of Chloroethene and selected Chloroethene compounds
(Callahan et al., 1979, Agteren et al., 1998)

They are transferred into atmospheric environment through volatilization, and once they are emitted into atmosphere, they are photolyzed with certain wavelength. World Health Organization (WHO) informs half-life, required time to be half of the substances, of vinyl chloride is about 20 hours, and they are the most unstable compound among chloroethene.


Molecular weight (g/mol)

Melting Point ()

Boiling Point ()

Density at 20

tetrachloroethene (PCE)





trichloroethene (TCE)





cis-1,2-dichloroethene (cis-1,2-DCE)





trans-1,2-dichloroethene (trans-1,2-DCE)





1,1-dicholoroethene (1,1-DCE)





vinyl chloride (VC)





Table 3. Physical Properties of Alkene Group (Agteren et al., 1998)

Tri- and perchloroethylene are the most prevailingly used in varied field. Chloroethene compound are persistence, therefore, they can be detected in groundwater under manufacture sites decades as a result even after introduction of those compounds have stopped (Agteren et al., 1998).

2.      Natural bioremediation pathway of Chloroethenes compound

Several steps of PCE dechlorination were taken happens naturally under anaerobic condition - oxygen absence and other terminal electron acceptor are presented condition, and this section discuss about natural bioremediation pathway of PCE compounds suggested by Agterne et al. (1998).

 The most significant step of chloroethene conversion is chloride atoms naturally replace to hydrogen. Tetrachloroethene (PCE) is reductively dehalorinated to trichloroethene (TCE) and 1,2-dichloroethene (DCE, mainly cis-1,2-DCE). In addition, 1,1-dicholoroethene (1,1-DCE) is reduced to vinyl chloride (VC). VC, which is end product proposed by Agrerne et al. (1998), is the most toxicity compound, and it is not naturally degraded to ethane or ethane. Therefore, it is easily accumulated at contaminated site.

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The reference covered anaerobic biodegradation is relatively outdated resource, nevertheless, it includes comprehensive as well as well-structured mechanisms about entire biodegradation pathway with providing chemical structure of all compounds on the pathway to aid better understanding. In addition, it clearly reveals limitation of natural bioremediation under anaerobic condition, which is end product under anaerobic condition with most toxic cannot achieve further degrade naturally. Though this contains constructive pathways of nature biodegradation, it does not proposed further research should take implementation in the future for dealing with VC residue under anaerobic condition. Furthermore, they were missed further VC biodegradation by utilizing certain bacteria proposed by Maymó-Gatell et al. (1997). Including further research to remediate residue VC compounds makes this reference more reliable resource to be foundation stone of PCE bioremediation.

3.      Bioremediation pathways of Chloroethene with certain microorganisms

This section includes three prominent research papers about Chloroethene bioremediation pathways in varied time (I was trying to describe first paper was published in 1997, second paper was published in 2008, and last paper was published in this year). It helps to understand how researchers develop their work to determine the overall pathway of Chloroethene degradation. In addition, all three papers used with different types of bacterium or bacteria consortia, therefore, it is easy to evaluate its effectiveness and reliability of their paper as well as their experimental design and conclusion of their paper.

3-1) Isolation of a Bacterium that Reductively Dechlorinates Tetrachloroethene to Ethene

 Maymó-Gatell et al. (1997) suggest that PCE can achieve complete dechlorination by natural microbial community and mixed microbial enrichment culture under anaerobic condition. They assumed isolated bacteria utilized hydrogen as electron donor and PCE as an electron acceptors for their growth. Therefore, when hydrogen is consumed, chloride supposed to be produced, and irregular coccus and a short rod bacteria are presented under the same condition, they can conclude those bacteria can dechlorination PCE products. They drove their result by stoichiometric comparison between hydrogen consumption and chlorine elimination. In order to avoid competition to consume hydrogen with methanogens and acetogen, they only put methanol-PCE and hydrogen with a mixture called ABSS [2 mM acetate, 0.05 mg of vitamin B12 per liter and 25% anaerobic digester sludge supernatant]. In addition, they only can observe the morphology of the stain under medium containing vancomycin (100mg/liter) or ampicillin (3g/liter), and those are the condition for PCE dechlorination. Under contained 20 mg/liter of tetracyline (a eubacterial protein-synthesis inhibitor) medium, PCE dechlorination wouldn’t take happened.

Figure 4. Proposed biodegradation pathway from PCE to ETH by strain 195
(Maymó-Gatell et al., 1997)

Due to strain 195 is classified into eubacteria, typically don’t have the cell wall, unlike Bacteria and Archaea do, certain nutrients - cholesterol and horse serum - require by mycoplasmas. In order to get quantitative information of bacterium, they measured the strain by direct microscopic cell counts and cell protein during metabolite pathway from PCE to VC and ethene. In addition, they found strain only grow about 5 days with 19.2 doubling times. However, after they cease to grow, they still can PCE dechlorination, and 90% of the PCE were used VC and ethane production. Furthermore, degradation pathway from VC to ethene or ethane only can initiate after PCE depletion, and the rate of conversion is faster than conversion rate from methanol-PCE to VC. Strain 195 only grow condition under hydrogen and PCE present. In order to determine morphology of the strain, they examined it through the electron microscopic method, and they found that the strain is irregular coccoid cells with the unusual cell wall, which resembled with S-layer protein typically Archaea have. In order to distinguish the presence of peptidoglycan cell wall, they tested it by fluorescently labeled wheat germ agglutinin. This binds to N-acetyl-glucosamine and N-acetylneuraminic acid, which gram-positive eubacterium Clostridium pasteurianum WF and gram-negative eubacterium E.coli DH5ɑ have.


Figure 5. Phylogenetic tree by 16S ribosomal DNA sequence and classification of strain 195 (Maymó-Gatell et al., 1997)

As a result, there is no binding on whole cell of stain, therefore, it is impossible to determine whether the existence of the cell wall with fluorescently labeled wheat germs agglutinin. To classify its phylogenetic position, it was tested by 16S ribosomal DNA sequence. Even strain didn’t cluster within any other phylogenetic lines, the branch of 195 strain is included cyanobacteria and planctomycetes. Moreover, strain shares high similarity with Clostridium butyricum with closer DNA distance. However, strain has few affiliations for other gram-positive branch. Therefore, they just classified strain 195 into eubacterial branches at that time.

Experimental design used in this research was not included uncertainty to compete for hydrogen with methanogen and acetogens. Even they knew the mechanism of microbial consortia, it cannot be assumed the correlation of coexistence between strain 195 and methanogens and acetogens through their research. In addition, there are other microbial group can utilize hydrogen as electron donor for their metabolite - sulfur oxidizer, nitrogen oxidizer, and iron oxidizer.

In order to get accurate information about rate of conversion, other microbial group should be included in their experiment design. With lacking of sufficient information on clone library, researchers couldn’t elucidate classification of strain 195 at that time, and they were put strain 195 into eubacterial branches based on DNA distance with vogue. Now we all know strain 195 is Dehalococcoides mccartyi strain 195, which contains multiple dehalogenase genes, therefore, it has the capacity to achieve complete dechlorination from PFC to VC. Furthermore, this study was used mixture called ABSS. However, this paper doesn’t reveal specific reason and role of being used ABSS in the experiment. It makes vague to me whether it was used as solvent for halogenated compound, or created a suitable environment for microbes for enrichment experiment.

3-2) Adaptation of aerobic, Ethene-Assimilating Mycobacterium Strains to Vinyl Chloride as a Growth substrate

Although researchers found several VC-assimilating bacteria, such as Dehalococcoides spp. under anaerobic condition, application have not been implemented in real world. In addition, microbes can easily enrich under aerobic - oxygen presents and plays a role as electron acceptor in reaction - condition, therefore, this study was conducted to test possibility of utilization VC as a growth substrate by currently isolated ethene-assimilating Mycobacterium strains. VC and ethene transform into VC epoxide (chlorooxirane) and epoxyethane (ethylene oxide) with alkene monooxygenase (AkMO) enzyme, therefore, all the microorganisms, which contained AkMO enzyme, can play a role as VC-assimilating bacteria. In addition, VC epoxide can reach further degradation, and epoxyethane can utilize as substrate into metabolite pathway with application of epoxyalkane: coenzyme M transferase (EaCoMT). Furthermore, ethene from anaerobic condition can be utilized as the only carbon and energy source by ethene-assimilating bacteria - Pseudomonas aeruginosa strain DL1 -  under the condition with exceed 30 mg/L in soil.

Currently isolated stains (Mycobacterium JS622, JS623, JS624 and JS625) contain genes shared similarities with EaCoMT gene. Moreover, after varied incubation periods, they observed VC depletion, and they assumed VC were utilized as their substrate to grow. To verify uncertainty on VC utilization as substrate, researchers tested strains growth with 20mM of acetate. In order to verify purity of the culture, experiment was used plate with 1/10-strength trypticase soy agar and 1% glucose (TSAG). Both samples of VC and ethene were analyzed by flame ionization detection, and empty space was evaluated by gas chromatography. A spectrophotometer was measured growth rate of the samples by optical density at 600 nano wavelength (OB600). In order to check purity of the strain, a bead-beating DNA extraction method were used with modification of the samples and it analyzed by PCR. (‘The Taq PCR Master Mix (Quiagen) kit was used for PCR with 16S rRNA gene primers while the Taq PCR Core Mix (Qiagen) was used for REP-PCR. Sequencing data were compared to 16S rRNA gene sequences deposited in Genbank. BioEdit and ClustalX were used for alignment and analysis of DNA sequences.- I think those idea should be included, but have no idea how to rephrase it :( Could you help me on modification of this sentence?). VC adaptation experiment when through with exthene-grown cultures, acetate-grown cultures, and TSAG-grown cultures, which harvested at mixexponential phase. For ethene-grown cultures, multiple strains were feed on MSM as well as initial VC concentration of 0.8-1.0mM and incubated at room temperature. For acetate-grown cultures, strains were grown on acetate, which one was VC provided and other one is not. For TSAG-grown cultures, they were incubated at 30℃. In addition, after estimating 7 to 14 days colonies were shown on the plate, and it successfully obtained one TSAG-cultures with sufficient biomass.

The researchers were observed that VC consumption with ethene-grown JS622, JS623, JS624 and JS625 cultures. They consumed VC as their substrate about 14 days. There is no further observation on VC degradation as well as stabilization of OB600 after VC depletion. Correlation between VC consumption and OB600 pattern indicated cometabolic VC biodegradation. TSAG-culture strains also were observed VC consumption with relatively slower growth rate than ethene-culture strains.

Unfortunately, both cultures didn’t possesed capability of utilization of VC as growth substrate. However, after varied VC adaptation period depending on types of culture, ethene-grown cultures initiated VC consumption with similar patterns for all cultures. TSAG-grown cultures also required varied VC adaptation period to initiate VC consumption. In addition, the correlation between VC consumption and OB600 pattern indicated that TSAG-grown cultures also consumed VC concentration. Moreover, higher VC concentration required longer incubation period than initial VC concentration.

Figure 6.Observed adaptation time in Mycobacterium strains (Yang & Mattes, 2008)

Due to the long incubation periods, this study validated purity of VC-adapted strains. They compared morphology of strains with VC-unadapted ethene-grown cultures. Most of the strains shown identical morphology with VC-unadapted ethene-grown cultures, however, VC-adapted JS623 shown different morphology and color even with 16S rRNA sequencing and REP-PCR method. The results with 16S rRNA sequencing shown VC-adapted and unadapted cultures have 100% identical gene, therefore, VC-adapted cultures used in experiment had purity.

The results of this paper shown that Mycobacterium stains have capability to degrade VC compounds, and could apply for remediating VC contaminated site as promising ethene-assilimiting bacterium. In addition, the requirement of varied VC adaptation period suggested that composition of fields microbial community would effect on period of VC adaptation. Moreover, this experiment was conducted with low concentration of oxygen; it may refers that ethene-assimilating strains can do VC degradation under groundwater condition with low oxygen concentration as well as high oxygen concentration. Additionally, they found large-plasmid in VC-assimilating bacteria, however, they didn’t cover the role of large-plasmid and didn’t put into their consideration to drive its result.

This paper clearly stated the objective on their experiment to reveal limitations of previous work from others - other VC-assimilating bacteria, such as Dehalococcoides spp, have limitation on VC degradation under aerobic condition. In addition, they constructively designed their experiment to compare VC-adapted and undapted cultures, and accurately understood mechanisms of VC degradation in terms of involved enzyme activity. However, they couldn’t clearly reveal morphology of VC-adapted JS623 strain with 16S rRNA sequencing and REP-PCR. I think amplified fragment length polymorphism (AFLP) can be utilized as alternative way to identify morphology of VC-adapted JS623. AFLP employs similar banding patterns as REP-PCR, but this is based digestion genomic DNA with larger number of bands. It may help to validate disparate morphology on VC-adapted JS623. Furthermore, the result didn’t include present of large-plasmid. Plasmid contains gene expression encode protein. If they accounted for present of large-plasmid in their consideration, they could determine reasons on morphology differences.

 3-3) Reductive dechlorination of high concentrations of chloroethenes by a Dehalococcoides Mccartyi strain 11G

A study conducted at the National University of Singapore and published on 18 October, 2018 was successfully able to show the reductive dechlorination of high concentrations of chlorethenes by a Dehalococcoides Mccartyi strain 11G. Reductive dechlorination has been established as a reliable in situ treatment for chloroethenes in anaerobic and anoxic conditions. Dehalococcoides is the most effective genus that can detoxify chloroethenes to the harmless compound ethene. This study isolated the Dehalococcoides Mccartyi strain 11G and found that it has tolerance to elevated chloroethene concentrations.

The authors of this study established a microcosm with mangrove sediment in defined DCB-1 medium and amended with acetate as a carbon source and and hydrogen as an electron donor exhibited complete dechlorination of TCE at high concentrations (up to 3.0 mM) to ethene, with the production of cis-DCE and trace amounts of VC as intermediates under strictly anaerobic conditions after 90 days’ incubation. To achieve complete dechlorination, 3 mM TCE rather than higher concentrations was spiked in the following consecutive sub-culturing, and a highly enriched culture which maintained TCE-to-ethene dechlorinating capability was obtained. This enrichment culture exhibited faster dechlorination (dechlorinating 3.0mM TCE within 40 days) than the original microcosm. Only three major populations were present in the enrichment, among which, Dehalococcoides, an obligate OHRB, became the dominant genus, accounting for 63.52% of the community, with Clostridium (17.95%) and Sulfurospirillum (12.94%) making up the rest.

Serial dilution-to-extinction was performed to separate potential TCE dechlorinating bacteria from the highly enriched culture. After five successive serial dilutions, complete conversion of TCE to ethene was repeatedly detected in 10−1–10−7 dilutions. Twenty randomly picked clones from a clone library analysis of a sample from the 10−7 dilution were identified as D. mccartyi and shared an identical sequence. After tests like Fluorescence microscopy revealed uniform disc-shaped morphology, which is similar to that of other Dehalococcoides strains (Löffler et al. 2013) and real-time quantitative PCR (Polymerase Chain Reaction) was applied to quantify the cell growth using bacteria- and Dehalococcoides-specific primer pairs, these results suggest that a pure D. mccartyi strain, designated D. mccartyi strain 11G, was obtained.

While the authors of the study did a great job of identifying and demonstrating the high tolerance to chloroethene of the 11G strain using practical experiments and existing literature to support their findings, the presentation of the paper was slightly complex and the writing and presentation of the paper was slightly flawed. The authors referred to figures that were not published in the paper and used acronyms that were not elaborated upon making the reading and understanding experience puzzling. 


 Chemical compounds are the double-edged swords. It helps us to live our life with enrichment and aids the reactions human cannot manipulate. However, as halogenated compounds, it also can detrimentally destroy environment, ecosystem, and corrupted water quality. Thus, I think this paper alters our attention on use of chemical compounds as well as appreciation on mechanisms of nature. Because microorganisms, even people are unknown, play a crucial role on remediate our nature, contaminated by human. Consequently, I think it is important to discovery mechanisms of their roles and pathways to aid their activity for achieving efficient result on bioremediation.

In this context, estimating and evaluating interaction in microbial community is imperative to perceive. Because it may help to be coexisted in same habitat as syntrophy, however, it can be treated to be living in same habitat. As mentioned in body paragraph, some microorganisms - methanogens and acetogens - compete for consuming hydrogen as electron acceptor with strains to degrade halogenated compounds. Therefore estimating total amount of required electron acceptor is essential step should take before evaluating effectiveness of remediation.

In addition, utilizing bacteria consortia can produce higher efficient result on degradation than employing single strain. Hence, based on current observation and understanding, it would be worth to construct bacteria consortia to remediate contaminated site by halogenated aliphatic compound.


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