Bacteria; Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Rhodanobacter; denitrificans .
2APBS1 and 116-2 .
Rhodanobacter denitrificans is a yellow-pigmented, gram-negative, non-sporulating, motile,slow-growing, and facultatively anaerobic rod-shaped bacterium with the ability to perform complete denitrification . For Rhodanobacter species, denitrification was not a characteristic until the discovery of R. denitrificans . Denitrification is the conversion of nitrate to N2 through nitrite, nitric and nitrous oxide intermediates catalyzed by microbial enzymes . Only two species of Rhodanobacter, one of them being Rhodanobacter denitrificans, is capable of this process . In the absence of oxygen, growth is fueled by the use of nitrate, nitrite, and nitrous oxide as electron acceptors . Rhodanobacter denitrificans has a salt tolerance up to 2% NaCl . Growth was observed at pH 4-8 and 10-35ºC, optimally at pH 6.5 and 30ºC . The two strains of R. denitrificans, 2APBS1 and 116-2, were found at Oak Ridge Integrated Field Research Challenge (ORIFRC) . ORIFRC is a model nuclear legacy site with high levels of nitrate and acidity, with uranium and other heavy metal contaminants . At ORIFRC, 2APBS1 was isolated from Area 2 (pH 6-7 and nitrate level <2 mM) and 116-2 was isolated from Area 3 (pH 3-4 and nitrate level 10s to 100s mM) . The two strains are highly abundant and active in acidic, nitrate rich subsurface environments with high metal (e.g. uranium) concentrations . The characteristic of survival in these harsh environments allow R. denitrificans to conduct bioremediation of the contamination sites by denitrification .
Ecology and Metabolism
Microbes can reduce contaminants in the subsurface ecosystem, such as in ORIFRC . Nitrate concentration is inversely proportional to pH . High levels of nitrate inhibits most microbes, but R. denitrificans can thrive in a nitrate rich environment conducting denitrification, which is important in subsurface ecology . Low pH, high nitrate, and other co-contaminants cause strong selective pressure on the bacterial community . Selection minimizes microbial diversity and leads to the survival of dominant species such as R. denitrificans . pH is the strongest determination factor of abundance for Rhodanobacter . Rhodanobacter thrives in conditions of high nitrate and uranium, and low pH . Also, electron donors are the limiting factors of denitrification; nitrate levels exceed the number of electron donors . The deaths of other native bacteria in the acidic subsurface led to favoring of horizontal gene transfers, giving complete denitrification capability and acid tolerance to R. denitrificans . R. denitrificans also have a flexible metabolism and able to grow on many organic substrates . They grow extremely well on acetate (great carbon source and electron donor), used to stimulate denitrification and neutralize liquid waste at ORIFRC . All in all, low pH groundwater and high nitrate levels created a specific niche for Rhodanobacter denitrificans .
Significance in Bioremediation
Bioremediation is a cost effective process that microbes use to remove pollutants from the environment by enzymatically transforming the contaminants . It is a natural solution to health and ecological impacts, and leads to nontoxic end-products such as N2 and precipitation of metals contaminants . Contamination of aquifers is a major problem because clean water is an important resource for agriculture and industry . For many countries, groundwater is a major source of drinking water . Nitrate is one of the most widespread groundwater contaminants . In ORIFRC, nitrate contamination is accompanied with uranium, which has no function biologically and is toxic to most bacteria and humans . It has two oxidation states, one being the soluble U(VI) uranyl ion and the other is extremely insoluble U(IV) precipitate . Uranium contamination is associated with the production of nuclear weapons, and is widespread across the world . The growth of R. denitrificans at low pH is important because uranyl ion exists in the most toxic form at low pH . Reductive immobilization of U(VI) is a currently favoured bioremediation process . U(VI) contaminates aquifers and drinking water rapidly . Nitrate is a competitive electron acceptor in the subsurface so U(VI) reduction is inhibited by its presence, and the ORIFRC research operation is used to test this correlation . Therefore, uranium precipitation is most efficient when nitrate is depleted . Rhodanobacter denitrificans effectively reduces nitrate in environments with low pH and high levels of nitrate and heavy metals. In ORIFRC, bioremediation of aquifers co-contaminated with nitrate and uranium can be stimulated by adding organic electron donors such as acetate . Acetate facilitates the removal of U(VI) from contaminated aquifers , by U(VI) reduction which is strictly a biological reaction by microbes . R. denitrificans are also able to reduce the emission of N2O from low pH soils, which is a strong greenhouse gas and a denitrification intermediate that depletes ozone and causes global warming . This is due to their capability to undergo complete denitrification, therefore reducing nitrate and other harmful intermediates to N2 which is released into air .
The complete genome of R. denitrificans 2APBS1 is 4.23 Mb and 116-2 is 3.98 Mb . 2APBS1 strain contains nitrate, nitrite, nitric oxide, and nitrous oxide reductase genes . Comparing 16S rRNA gene sequences, the two strains have high sequence similarity to R. thiooxydans LCS2; 99.2% match for 2APBS1 and 98.7% match for 116-2 . LCS2 is a thiosulfate-oxidizing bacterium with almost the same phenotype as Rhodanobacter denitrificans . However, nitrate is reduced, but nitrite is not for this bacterium . The highest rRNA sequence similarity is between the two strains, with a 99.4% match . Using DNA-DNA hybridization, results show that 2APBS1 has the highest DNA similarity of 86% to 116-2, whereas the next two closest only have 56.7% (R. soli) and 41.1% (R. thiooxydans) similarity . Overall, knowing that the DNA-DNA hybridization threshold is 70% for determining species, and with the genus-unique capability of denitrification, the two strains represent a novel species of Rhodanobacter with the proposed name Rhodanobacter denitrificans .
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