Alicycliphilus denitrificans

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Figure 1: Portion of a phylogenetic tree determined from 16S rRNA sequencing. This figure was derived from Weelink et al. (2008) [9].

Higher Order Taxa

Bacteria (Kingdom), Proteobacteria (Phylum), Betaproteobacteria (Class), Burkholderiales (Order), Comamonadaceae(Family), Alicycliphilus(Genus)


Alicycliphilus denitrificans

Description and Significance

Figure 2: A primary settling tank from Benfleet sewage treatment plant. Image attributed to John Rostron.

Alicycliphilus denitrificans is a gram-negative, motile, and non-spore-forming facultative anaerobe. It is approximately 0.6μm wide, 1-2μm long, and rod-shaped [9]. Alicycliphilus denitrificans is the first species to be isolated from the genus Alicycliphilus [1].

Alicycliphilus denitrificans has been isolated from wastewater treatment and sewage plants in the water and soil [3]. K601 and BC are the two best known strains and have different methods for their metabolism. Alicycliphilus denitrificans are capable of degrading hydrocarbons in the environment under both oxic and anoxic conditions [5]. Alicycliphilus denitrificans are capable of degrading alicyclic or aromatic compounds. The anaerobic degradation of these compounds is less well understood than their aerobic degradation [5]. This uncommon anaerobic alicyclic hydrocarbon degradation feature is one reason that Alicycliphilus denitrificans is a novel bacterium that has generated some interest and research.

Genome Sequence

The genome of Alicycliphilus denitrificans is comprised of plasmids and a circular chromosome [7]. The use of 16S rDNA sequencing was able to determine the phylogenetic relationship of Alicycliphilus denitrificans as a lineage of the family Comamonadaceae [5].

The genomes of both Alicycliphilus denitrificans strains (K601 and BC) have been determined. The size of the two strains were 4.84Mb for the BC genome and 5.07Mb for the K601 genome, both had a G+C content of approximately 68% [7].

The K601 and BC strain of Alicycliphilus denitrificans both contain the same gene cluster that encodes for the enzyme benzene and phenol monooxygenase and catechol 2,3-dioxygenase that are used for degradation of hydrocarbons [7]. However, the genome of the BC strain of Alicycliphilus denitrificans contains genes for the expression of chlorate reductase and chlorite dismutase enzymes that are not found in the K601 strain [9]. The genes for the two enzymes are located in a plasmid of the BC strain, which supports the theory that the unique ability of the BC strain to undergo chlorate respiration is obtained by horizontal gene transfer [7]. The K601 strain has a gene that encodes a bifunctional hydratase and alcohol dehydrogenase enzyme. This enzyme is not found in the BC strain and is thought to be responsible for the ability to degrade cyclohexanol in the K601 strain [4][7].


Alicycliphilus denitrificans can degrade hydrocarbons as a carbon source. They are significant for their capability of coupling oxidation of cyclic compounds to the degradation of nitrate or chlorate [5][7]. Both strains can use nitrate, oxygen, and nitrite as an electron acceptor; only the strain BC can use chlorate as an electron acceptor [9]. Alicycliphilus denitrificans has a NosZ gene that encodes a nitrous oxide reductase that is used to reduce N2O for denitrification activity when it uses nitrate or nitrite as electron acceptors [3]. Alicycliphilus denitrificans are facultative anaerobes that have metabolism that must be oxidative [5].

Figure 3: The reduction pathway of chlorate to produce oxygen in Alicycliphilus denitrificans strain BC with specific enzymes. This diagram was derived from the scheme described by Jin et al. (2011) [9].

The Alicycliphilus denitrificans strain BC has a strict oxidative metabolism, but it is still able to degrade compounds such as benzene in anaerobic conditions [7]. This is due to the specific enzymes that are found in its genome. Nitrate cannot be used as an electron acceptor if the benzene is used as the energy source due to the oxidative metabolism requirement, but chlorate can still be used as an electron acceptor even under anoxic condition [9]. The main reason is due to the reduction of chlorate, by the enzyme chlorate reductase, to chlorite that then provides oxygen and chlorine to the organism through the action of chlorite dismutase [9]. The oxygen is used with oxygenase to attack benzene or other organic material [9]. The strain BC is incapable of degrading cyclohexanol or phenol since they lack the enzyme for their degradation, thus they are often impossible intermediate in the BC strain's organic compound degradation such as benzene degradation [10].

Figure 4: The possible cyclohexanol degradation pathway with bifunctional MhyADH enzyme. This diagram was derived from the scheme presented by Jin et al. (2011) [9].

Alicycliphilus denitrificans strain K601 is the currently the only identified strain of the genus Alicycliphilus that is capable of degrading cyclohexanol and phenol as a carbon source [5]. In aerobic conditions, the K601 strain can use lactate, acetate, and many other types of hydrocarbons coupled to oxygen as terminal electron acceptor [5]. Under denitrifying conditions, the strain K601 can still grow using nitrate and nitrite as an electron acceptor with alicyclic compounds as carbon sources such as cyclohexanol and cyclohexone [4]. This Alicycliphilus denitrificans contains enzymes such as carboxylase that will allow for anaerobic growth from ketone molecules with nitrate [2]. A proposed mechanism for the metabolic pathway of degrading cyclohexanol involves an initial oxidation of a cyclohexanol into a ketone that is then followed by the formation of a carbon-carbon double bond forming a structure known as a vinylketone [4]. The vinylketone then undergoes an oxidative addition of H2O to become a 1,3-cyclohexodione, which is followed by a hydrolytic cleavage [4]. This overall process would explain growth in anaerobic conditions as the H2O is used as the source of oxygen in this model [4]. A study performed by Jin et al. showed that this process of an oxidative addition of H2O is performed by a single enzyme called MhyADH that is both a hydratase and a dehydrogenase as opposed to two separate enzymes [4].


Alicycliphilus denitrificans can have denitrifying or chlorate-reducing effects to its environments. These bacteria are often isolated from wastewater treatment plants [6]. The overall environmental and ecological effects of their metabolism have not yet been well studied [8]. The ability of Alicycliphilus denitrificans to degrade many different organic compounds that could be pollutants in water, air, or soil indicates that it may be a useful organism in the bio-degradation of these compounds in both aerobic and anaerobic environments [6].


(1) Cervantes, A., García, A., López, N., Nuñez, S., Gómez, M., Wacher, C., and Tavera, H. “Characterization of the Polyurethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N-Methylpyrrolidone.” Applied and Environmental Microbiology. 2007. Volume 73. p.6214–6223.

(2) Dullis, C., Chen, C., and Schink, B. “Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes.” Applied and Environmental Microbiology. 2011. Volume 77. p.6821–6825.

(3) Hou, J., Li, L., Zhang, S., Wang, P., and Wang, C. “Diversity of NosZ gene in three municipal wastewater treatment plants located in different geographic regions.” African Journal of Microbiology Research. 2012. Volume 6. p.3574–3581.

(4) Jin, J., Straathof, A., Pinkse, M., and Hanefeld, U. “Purification, characterization, and cloning of a bifunctionalmolybdoenzyme with hydratase and alcohol dehydrogenase activity.” Applied Microbiology and Biotechnology. 2011. Volume 89. p.1831–1840.

(5) Mechichi, T., Stackebrandt, E., and Fuchs, G. “Alicycliphilus denitrificans gen. nov., sp. Nov., a cyclohexanol-degrading, nitrate-reducing betaproteobacterium.” International Journal of Systematic and Evolutionary Microbiology. 2012. Volume 55. p.147–152.

(6) Okunishi, S., Morita, Y., Higuchi, T., Maeda, H., and Nishi, K. “Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE.” Journal of the Air & Waste Management Association. 2012. Volume 62. p.748–757.

(7) Oosterkamp, M., Veuskens, T., Plugge, C., Langenhoff, A., Gerritse, J., van Berkel, W., Pieper, D., Junca H, Goodwin, L., Daligault, H., Bruce, D., Detter, J., Tapia, R., Hans, C., Land, M., Hauser, L., Smidt, H., and Stams, A. “Genome Sequences of Alicycliphilus denitrificans Strains BC and K601T.” Journal of Bacteriology. 2011. Volume 193. p.5028–5029.

(8) Schwarz, A., Urrutia, H., Vidal, J., and Pérez, N. “Chlorate reduction capacity and characterisation of chlorate reducing bacteria communities in sediments of the rio Cruces wetland in southern Chile.” Water Research. 2012. Volume 46. p.3283–3292.

(9) Weelink, S., Tan, N., Broeke, H., van den Kieboom, C., van Doesburg, W., Langenhoff, A., Gerritse, J., Junca, H., and Stams, A. “Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor.” Applied and Environmental Microbiology. 2008. Volume 74. p.6672–6681.

(10) Weelink, S., Tan, N., Broeke, H., van Doesburg, W., Langenhoff, A., Gerritse, J., Junca, H., and Stams, A. “Physiological and phylogenetic characterization of a stable benzene-degrading, chlorate-reducingmicrobial community.” Federation of European Microbiological Societies. 2007. Volume 60. p.312–321.