Isolation and characterization of Rhodococcus sp. strains TMP2 and T12 that degrade 2,6,10,14-tetramethylpentadecane (pristane) at moderately low temperatures

Branched alkanes including 2,6,10,14-tetramethylpentadecane (pristane) are more resistant to biological degradation than straight-chain alkanes especially under low-temperature conditions, such as 10 degrees C. Two bacterial strains, TMP2 and T12, that are capable of degrading pristane at 10 degrees C were isolated and characterized. Both strains grew optimally at 30 degrees C and were identified as Rhodococcus sp. based on the 16S rRNA gene sequences. Strain T12 degraded comparable amounts of pristane in a range of temperatures from 10 to 30 degrees C and strain TMP2 degraded pristane similarly at 10 and 20 degrees C but did not degrade it at 30 degrees C. These data suggest that the strains have adapted their pristane degradation system to moderately low-temperature conditions.     

Microbial degradation of the multiply branched alkane 2,6,10,15,19, 23-hexamethyltetracosane (Squalane) by Mycobacterium fortuitum and Mycobacterium ratisbonense

Among several bacterial species belonging to the general Gordonia, Mycobacterium, Micromonospora, Pseudomonas, and Rhodococcus, only two mycobacterial isolates, Mycobacterium fortuitum strain NF4 and the new isolate Mycobacterium ratisbonense strain SD4, which was isolated from a sewage treatment plant, were capable of utilizing the multiply branched hydrocarbon squalane (2,6,10,15,19, 23-hexamethyltetracosane) and its analogous unsaturated hydrocarbon squalene as the sole carbon source for growth. Detailed degradation studies and high-pressure liquid chromatography analysis showed a clear decrease of the concentrations of squalane and squalene during biomass increase. These results were supported by resting-cell experiments using strain SD4 and squalane or squalene as the substrate. The degradation of acyclic isoprenoids and alkanes as well as of acids derived from these compounds was also investigated. Inhibition of squalane and squalene degradation by acrylic acid indicated the possible involvement of beta-oxidation in the degradation route. To our knowledge, this is the first report demonstrating the biodegradation of squalane by using defined axenic cultures.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

Degradation and bioconversion of aliphatic and aromatic hydrocarbons by Rhodococcus ruber 219

 A tetrahydrofuran-degrading bacterial strain, which had previously been tentatively assigned as Rhodococcus sp. strain 219, has now been identified as Rhodococcus ruber using physiological and chemotaxonomical tests. A comparison with the type strain DSM 43338 has revealed that the new strain differs in its ability to degrade or convert tetrahydrofuran and compounds of similar structure such as 2,5-dimethyltetrahydrofuran or tetrahydropyran. Tetrahydrofuran acts as an inducer for its degradation. When tetrahydrofuran-induced cells were incubated with 2,5-dimethyltetrahydrofuran two primary metabolites could be detected by gas chromatography, and 2-hydroxyhexane-5-one and hexane-2,5-dione were isolated and characterized by ¹H-NMR spectroscopy or as dinitrophenylhydrazones. The formation of these intermediates is consistent with an initial 2hydroxylation of the cyclic ether, which has not yet been described in microorganisms. Received: 19 July 1995/Received last revision: 31 October 1995/Accepted: 6 November 1995     

Identification of phenylalkane derivatives when Mycobacterium neoaurum and Rhodococcus erythropolis were cultured in the presence of various phenylalkanes

Phenylalkanes are ubiquitously found in nature as pollutants originating from oil, gas oil and petrol. Rising commercial demand for mineral oil fractions has led to the increased prevalence of environmental contamination, whereby these particular hydrocarbons are encountered by bacteria which have subsequently developed sophisticated metabolic routes for purposes of degradation. Herein a detailed analysis of these metabolic pathways in the degradation of phenylalkanes by Mycobacterium neoaurum and Rhodococcus erythropolis highlighted preponderance for the formation of certain metabolites of which 17 were identified and whereby striking differences were noticed depending specifically upon the length of the substrate’s alkyl chain. Although the degradation of even-numbered phenylalkane substrates was assumed to result in the generation of phenylacetic acid formed due to substrate terminal oxidation and subsequent β-oxidation, cultures of M. neoaurum and R. erythropolis were determined in an extracellular accumulation of odd-numbered acidic metabolites, suggesting a simultaneous presence of sub-terminal degradation mechanisms. However, results obtained from biotransformation assays containing even-chained phenylalkanoic acid intermediates as substrates revealed exclusive β-oxidative mechanisms and no generation of odd-numbered degradation products. R. erythropolis in contrast to M. neoaurum also proved viable for hydroxylation of the aromatic ring of metabolites. Interestingly, the generation of phenylacetic acid and subsequently 2-hydroxyphenyl acetic acid was monitored and entailed the presence of the lactone intermediate 2-coumaranone. These results enhance our understanding of the degradation of phenylalkanes and illustrate the potential application of such species in the bioremediation of these common environmental pollutants and in the strains’ diverse abilities to transform mineral oil compounds to new valuable products.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Identification of different alkane hydroxylase systems in Rhodococcus ruber strain SP2B,an hexane‐degrading actinomycete

Aims: To investigate the alkane‐hydroxylating system of isolate SP2B, closely related to Rhodococcus ruber DSM 43338^T and uncharacterized so far for its alkane degradation genes. Methods and Results: Although isolate SP2B and reference strain can grow on by‐products from hexane degradation, the type strain R. ruber was unable, unlike SP2B isolate, to use short‐chain alkanes, as assessed by gas chromatography. Using PCR with specific or degenerated primers, inverse PCR and Southern blot, two alkane hydroxylase encoding genes (alkB) were detected in both bacteria, which is in agreement with their alkane range. The first AlkB was related to Rhodococcus AlkB7 enzymes and contains a nonbulky residue at a specific position, suggesting it might be involved in medium‐ and long‐chain alkane oxidation. The second partial alkB gene potentially belongs to alkB5‐type, which was found in bacteria unable to use hexane. Moreover, a partial P450 cytochrome alkane hydroxylase, thought to be responsible for the hexane degradation, was detected only in the isolated strain. Conclusions: Rhodococcus ruber SP2B should prove to be a promising candidate for bioremediation studies of contaminated sites because of its large degradation range of alkanes. Significance and Impact of the Study: This is the first thorough study on R.ruber alkane degradation systems.     

Kinetics of the degradation of aliphatic hydrocarbons by the bacteria Rhodococcus ruber and Rhodococcus erythropolis

Consumption of aliphatic hydrocarbons by the bacteria Rhodococcus ruber Ac-1513-D and Rhodococcus erythropolis Ac-1514-D grown on mixed n-alkanes and diesel fuel was studied. Consumption of diesel fuel hydrocarbons by the strains was less intense in comparison with the n-alkane mixture. The strains showed differences in growth rate and consumption of the substrates, which suggests that they possess different mechanisms of hydrocarbon uptake.     

Kinetics of the degradation of aliphatic hydrocarbons by the bacteria Rhodococcus ruber and Rhodococcus erythropolis

Consumption of aliphatic hydrocarbons by the bacteria Rhodococcus ruber Ac-1513-D and Rhodococcus erythropolis Ac-1514-D grown on mixed n-alkanes and diesel fuel was studied. Consumption of diesel fuel hydrocarbons by the strains was less intense in comparison with the n-alkane mixture. The strains showed differences in growth rate and consumption of the substrates, which suggests that they possess different mechanisms of hydrocarbon uptake.     

新金色分枝杆菌ksdD基因的敲除及其对菌株AD(D)合成的影响

新金色分枝杆菌(Mycobacterium neoaurum)能将植物甾醇转化为药物中间体4-烯-雄甾-3,17-二酮(AD)和1,4-二烯-雄甾-3,17-二酮(ADD),其中3-甾酮-△1-脱氢酶(KSDD)是AD转化为ADD的关键酶.本实验室在筛菌过程中筛选到一株能将甾醇转化为AD(D)的菌株,经鉴定为M.neo...     

The uptake of pristane (2,6,10,14-tetramethylpentadecane) into phospholipid bilayers as assessed by NMR, DSC, and tritium labeling methods.

Unilamellar dioleoylphosphatidylcholine (DOPC) liposomes (250 microM) incorporated 2 mol% of [3H]pristane at 37 degrees C after addition of 50 microM pristane solubilized with beta-cyclodextrin. Conventional solubilization in dimethyl sulphoxide resulted in much lower uptake. Premixing of perdeuterated pristane with DOPC and dipalmitoylphosphatidylcholine (DPPC) prior to the formation of multilamellar liposomes resulted in homogeneous incorporation of up to 5 mol% pristane at 22 degrees C and 50 degrees C, respectively, as observed by 2H-NMR. Lipid order parameters measured by 31P and 2H-NMR remained unchanged after pristane uptake. Pristane induced the transformation of part of the dioleoylphosphatidylethanolamine (DOPE)/DOPC (3:1, mol/mol) liquid crystalline lamellar phase into an inverse hexagonal phase. 5 mol% pristane in DPPC bilayers decreased the midpoint of the main phase transition temperature of DPPC from 41.5 degrees C to 40.9 degrees C. Upon cooling in the temperature range from 41 degrees C to 36 degrees C, pristane was either displaced from the DPPC bilayer or the mode of incorporation changed. These results may aid in defining the mechanisms whereby pristane, an isoprenoid C19-isoalkane, induces plasmacytomagenesis in mice.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains.

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Abstract Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 μg/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 μg/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 μg/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Biocatalytic scrubbing of gaseous acrylonitrile using Rhodococcus ruber immobilized in synthetic silicone polymer (ImmobaSil) rings

Microbial detoxification of acrylonitrile vapor was performed using Rhodococcus ruber immobilized into rings of a synthetic silicone polymer (ImmobaSil ) sponge. The immobilization matrix was used as both the cell support material and also as the trickle-bed column packing. The elimination capacity of the biocatalytic scrubber was determined for different influent concentrations of gaseous acrylonitrile. The working life of the nongrowing biocatalyst was also examined under test conditions and was found to be at least 70 days before biocatalyst replacement was required. An elimination capacity of 4.0 kg m(-3) h(-1) was achieved with a 95% removal efficiency, and an elimination capacity of over 7.2 kg m(-3) h(-1) acrylonitrile was achieved with a removal efficiency of 90%. This elimination capacity is 10 times that previously published in the literature.     

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase. Received: 12 January 2000 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [¹⁴C]NDMA to ¹⁴CO₂, growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.N-Nitrosodimethylamine (NDMA) is a potent carcinogen that has recently been detected in groundwater, wastewater, and drinking water (1, 2, 17, 18). It forms as a disinfection byproduct in wastewater and drinking water treated with chloramine and other disinfectants (17, 18, 43). NDMA has also been found to be present in aquifers at several military sites that have used 1,1-dimethylhydrazine, a component of liquid rocket propellant that contained NDMA as an impurity (6, 9). Although there is presently no federal maximum contaminant level for NDMA in drinking water, a risk assessment conducted by the U.S. Environmental Protection Agency suggested that concentrations as low as 0.7 ng/liter can increase lifetime cancer risk by 1 × 10⁻⁶ (34). In addition, California currently has a 10 ng/liter notification level for NDMA concentrations in drinking water and has recently recommended an even lower public health goal of 3 ng/liter (3, 20). Thus, the presence of even trace concentrations of this chemical in drinking water represents a potential public health concern.The rates and extents of NDMA biodegradation in natural environments, including surface water, sludges, and soils, are highly variable. In some studies, the compound has been reported to be recalcitrant or only partially biodegraded (16, 30, 31); in others, fairly rapid and extensive biodegradation has been previously observed (2, 13, 22, 40). Few studies have been conducted to examine NDMA biodegradation in groundwater. However, the persistence of NDMA derived originally from 1,1-dimethylhydrazine-based rocket fuel over decades in some groundwater aquifers (e.g., Rocky Mountain Arsenal, CO; former Air Force Plant PJKS, CO; and Aerojet Superfund Site, CA) suggests that this molecule can be very recalcitrant (8, 9, 35). At sites where biodegradation has been observed, the organisms responsible and the microbial degradation pathways are largely unknown.The metabolism of NDMA and other nitrosamines by mammals has received extensive study. NDMA requires metabolic activation to the methyldiazonium ion (a strong alkylating agent) to exert its genotoxic effects (1, 19, 34). This activation reaction is catalyzed by specific isozymes of the cytochrome P-450-dependent mixed-function oxidase system and proceeds through an initial α-hydroxylation reaction. Alternately, NDMA can be oxidized by the P-450 system via a denitrosation route, which does not result in the formation of a highly carcinogenic intermediate (11, 28, 37).The bacterial transformation of NDMA has not been studied in significant detail. Several bacteria expressing broad-specificity monooxygenase enzymes have been reported to degrade NDMA via cometabolism. These include the propanotrophs Rhodococcus sp. strain RHA1 (25, 26) and Rhodococcus ruber ENV425 (29) as well as Mycobacterium vaccae JOB5 (25), the methanotroph Methylosinus trichosporium OB3b (42), and the toluene oxidizer Pseudomonas mendocina KR1 (7). We recently characterized the pathway of NDMA transformation used by P. mendocina KR1, a bacterium that utilizes the enzyme toluene-4-monooxygenase (T4MO) to cometabolically degrade NDMA and other anthropogenic pollutants (7, 38). The pathway of NDMA transformation by KR1 differs from the two pathways described for mammals. A majority of the NDMA metabolized by T4MO in this strain is oxidized to N-nitrodimethylamine (NTDMA) and then further to N-nitromethylamine (NTMA), which accumulates as a terminal product (7).In this report, we describe the pathway used by the propanotroph R. ruber ENV425 to catabolize NDMA. This strain was originally isolated from turf soil, where propane was used as the sole carbon source, and was previously reported to oxidize methyl tertiary-butyl ether and other gasoline oxygenates (27). Our data show that the pathway of NDMA degradation mediated by strain ENV425 differs from that mediated by P. mendocina KR1. Rather, the pathway used for transformation of NDMA by ENV425 appears to be similar to the denitrosation pathway catalyzed by various P-450 isozymes in mammals, resulting in the production of nitric oxide (NO), nitrite, nitrate, formaldehyde, formate, and methylamine (MA) (11, 12, 28, 39). A significant fraction of the carbon in the NDMA molecule was released as CO₂ by strain ENV425, although growth on NDMA could not be confirmed. However, the bacterium was observed to utilize NDMA as well as the NDMA-degradation intermediates MA and nitrate as sources of nitrogen during growth on propane as a sole carbon and energy source.