Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.     

Rhodococcus UKMP-5M, an endogenous lipase producing actinomycete from Peninsular Malaysia

Escalation in food industries unctuous wastes has led to serious anthropogenic problems to the environment. Parallel to “green strategy”, growing awareness in biological treatment emphasizes efficacy of enzymatic technology for bioremediation. Pertinently, researchers are in search for new lipase-lipid interaction for improved outcome. Rhodococcus species have documented inadequate evidences on lipase enzyme production. Consequent assessments on Rhodococcus isolates from Peninsular Malaysia have identified twelve promising strains as lipase producer. Interestingly, apart from usual lipolytic behaviour, Rhodococcus sp. exhibited significant level of lipase endogenously, while cryogenic grinding method effectively ruptured the cell. An isolate from petroleum-contaminated site, namely Rhodococcus UKMP-5M, projected the highest level of lipase specificity and has further been optimized. It was found out that the best specificity was apparent in acidic condition (pH 5) with 6% inoculum at 30°C for 72 hours of incubation. Due to high level of mycolic cell-surfactant developed in triacylglycerol supplements, cell lysis was employed with Triton X-100 detergent solubilisation. As a result, oil blend composed of various carbon-chain length fatty acids (composite 2) induces enzyme production extensively. Remarkably, R. UKMP-5M found to cater enzyme production without aid of inducer by nature, but additional carbon source like glucose represses lipase production. Further ability for biological treatment was revealed when the optimized R. UKMP-5M whole cell degraded waste cooking oil significantly by solubilizing fatty acids and commencing conversion into biomass. These qualities resemble practical new lipid-lipase biological lipid rich on-site treatment.     

Quantification of Gordona amarae Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes

Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems.     

Biodegradation and conversion of alkanes and crude oil by a marine Rhodococcus

A hydrocarbon degrader isolated from a chronically oil-polluted marine site was identified as Rhodococcus sp. on the basis of morphology, fatty acid methyl ester pattern, cell wall analysis, biochemical tests and G + C content of DNA. It degraded upto 50% of the aliphatic fraction of Assam crude oil, in seawater supplemented with 35 mM nitrogen as urea and 0.1 mM phosphorus as dipotassium hydrogen orthophosphate, after 72 h at 30 ° and 150 revolutions per minute. The relative percentage of intracellular fatty acid was higher in hydrocarbon-grown cells compared to fructose-grown cells. The fatty acids C₁₆ , C16_{16 :1} C₁₈ and C_{18 : 1} were constitutively present regardless of the growth substrate. In addition to these constitutive acids, other intracellular fatty acids varied in correlation to the hydrocarbon chain length supplied as a substrate. When grown on odd carbon number alkanes, the isolate released only monocarboxylic acids into the growth medium. On even carbon number alkanes only dicarboxylic acids were produced.     

Volatile Fatty Acid Requirements of Cellulolytic Rumen Bacteria

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 μmole of valeric per ml and 0.05 μmole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 μmole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 μmole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 μmole/ml, respectively. About 0.3 μmole/ml of each acid was required for satisfactory growth of C1a.     

Effect of Aromatic Compounds on Cellular Fatty Acid Composition of Rhodococcus opacus

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.     

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg²⁺, Ca²⁺, and Mn²⁺ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.     

Substrate Specificity of Regiospecific Desaturation of Aliphatic Compounds by a Mutant Rhodococcus Strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.     

Modified ferric hydroxamate spectrophotometry for assaying glycolic acid from the hydrolysis of glycolonitrile by Rhodococcus sp. CCZU10-1

We successfully modified a ferric hydroxamate spectrophotometry method for assaying glycolic acid. Comparable to the high-performance liquid chromatography (HPLC)-based method, ferric hydroxamate spectrophotometry can be used to accurately monitor the time course of glycolonitrile bioconversion. Glycolic acid was assayed simply and rapidly at room temperature (25 ～ 35°C). Optimum culture conditions were obtained using this method to assay the glycolonitrile-hydrolyzing activity of Rhodococcus sp. CCZU10-1. The preferred carbon and nitrogen sources and ideal inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (5 g/L), and ?-caprolactam (2 mmol/L), respectively. The optimal growth temperature and initial medium pH for Rhodococcus sp. CCZU10-1 glycolonitrile-hydrolyzing activity were 30°C and pH 7.0. Modified ferric hydroxamate spectrophotometry could potentially be employed to assay other carboxylic acids.     

Low-Temperature Microbial Degradation of Oil Products Differing in the Extent of Condensation

Out of the 30 strains capable of oil degradation at 4–6°C, four were selected by their ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol–benzene resins) at 4–6°C. The maximum degrees of degradation of masut and ethanol–benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). These strains provide a basis for developing biodegrader preparations applicable to the bioremediation of oil-polluted sites under the conditions of a cold climate.     

Characterization of a pyridine-degrading branched Gram-positive bacterium isolated from the anoxic zone of an oil shale column

Summary From the anoxic zone of an oil shale leachate column three pyridine-degrading bacterial strains were isolated. Two strains were Gram-negative facultative anaerobic rods and one strain was a branched Gram-positive bacterium. The branched Gram-positive strain had the best pyridine-degrading ability. This organism was aerobic, non-motile, catalase positive, oxidase negative, and had no flagellum. The G+C content of the DNA was 66.5 mol%. The major menaquinone was MK-8(H₂). The main cellular fatty acids were saturated and monounsaturated straight chains. This organism contained mycolic acid, meso-diaminopimelic acid, arabinogalactan and glycolyl residues in the cell wall. Due to morphological, physiological and chemotaxonomic characteristics this strain was placed in the genus Rhodococcus. The optimum culture conditions were as follows: temperature 32° C, pH 8.0 and 0.1% v/v of pyridine as sole carbon, energy and nitrogen source. Utilization of pyridine by a batch fermentor culture of Rhodococcus sp. was characterized by a specific growth rate of 0.13 h^–1, growth yield of 0.61 mg cell·mg pyridine^–1 and a doubling time of 5.3 h^–1. Offprint requests to: S.-T. Lee     

Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402

Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 μmol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system.     

Interaction of Operational and Physicochemical Factors Leading to Gordonia amarae-Like Foaming in an Incompletely Nitrifying Activated Sludge Plant

The overgrowth of Gordonia amarae-like bacteria in the mixed liquor of an incompletely nitrifying water reclamation plant was inversely correlated with temperature (r = −0.78; P < 0.005) and positively correlated with the solids retention time (SRT) obtained a week prior to sampling (r = 0.67; P < 0.005). Drops followed by spikes in the food-to-mass ratio (0.18 to 0.52) and biochemical oxygen demand concentrations in primary effluent (94 to 298 mg liter⁻¹) occurred at the initiation of G. amarae-like bacterial growth. The total bacterial concentration did not increase as concentrations of G. amarae-like cells increased, but total bacterial cell concentrations fluctuated in a manner similar to that of G. amarae-like bacteria in the pseudo-steady state. The ammonium ion removal rate (percent) was inversely related to G. amarae-like cell concentrations during accelerated growth and washout phases. The dissolved oxygen concentration decreased as the G. amarae-like cell concentration decreased. The concentrations of G. amarae-like cells peaked (2.47 × 10⁹ cells liter⁻¹) approximately 1.5 months prior to foaming. Foaming occurred during the late pseudo-steady-state phase, when temperature declines reversed. These findings suggested that temperature changes triggered operational and physicochemical changes favorable to the growth of G. amarae-like bacteria. Fine-scale quantitative PCR (qPCR) monitoring at weekly intervals allowed a better understanding of the factors affecting this organism and indicated that frequent sampling was required to obtain statistical significance with factors changing as the concentrations of this organism increased. Furthermore, the early identification of G. amarae-like cells when they are confined to mixed liquor (10⁷ cells liter⁻¹) allows management strategies to prevent foaming.     

Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native thraustochytrid strains

Liquid residues from beer (RB) and potato (RP) processing were evaluated as carbon sources for the production of docosahexaenoic acid (C22:6n-3, DHA) by two native Thraustochytriidae sp., M12-X1 and C41, in shaking flask experiments. Results were compared with those obtained in the fermentations of glucose, maltose, soluble starch and ethanol. Both strains produced the highest biomass concentration (2.3 g/L) in the fermentation of RB supplemented with nitrogen sources [yeast extract (YE) and monosodium glutamate (MSG)]. DHA content in the fatty acids produced by the native thraustochytrids was dependent on the fermented carbon source; the fatty acids from biomass grown on carbon sources that permitted a lower growth rate contained more DHA. The highest DHA productivity [55.1 mg/(day L)] was obtained in the fermentation of RB-YE-MSG by M12-X1 strain. In this medium, M12-X1 strain grew at a specific growth rate of 0.014 h^?1 and total fatty acid content in the biomass was 41.3%. Production of DHA by M12-X1 strain followed a non-growth rate associated pattern and DHA content in the biomass decreased significantly after growth ceased.     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.     

Effect of Growth Temperature on Fatty Acid Composition of Ten Thermus Strains

The fatty acid composition of Thermus spp., including T. aquaticus ATCC 25104, T. thermophilus DSM 579, T. flavus DSM 674, and seven wild strains was examined. Organisms were tested at a minimum of either 35, 40, or 45°C and at an optimum of 60 or 70°C. Total fatty acid content per dry weight of cells varied between 1.2 and 3.7%, and the quantity of fatty acids was higher at the high temperature range in the majority of strains. At the optimum temperature, strains could be assigned to three chemotaxonomic groups with reference to the ratio of iso C_15:0/iso C_17:0. In six of the strains the ratio of iso C_15:0/iso C_17:0 remained unchanged at the minimum temperature, whereas in four strains the ratio was reversed. The proportion of the C_15:0 and C_17:0 isobranched acids was decreased and the proportion of anteisobranched fatty acids, namely anteiso C_15:0, anteiso C_17:0, and anteiso C_17:1, was increased at the lower temperature range. Some changes were seen in the levels of the n-C_16:0 and iso C_16:0 acids, but these were strain specific.     

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175

Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of acidification activity of the cells after freezing, which differed according to the strain and the growth phase. Survival of B. longum R0175 was increased by 10% in cells with low membrane fluidity and high relative saturated fatty acid contents, without any change in acidification activity. Acidification activity was more degraded (70 min) in L. buchneri R1102 cells displaying low membrane fluidity and high saturated and cyclic fatty acid levels. Finally, this study showed that membrane modifications induced by the growth phase differed among bacterial strains in terms of composition. By lowering membrane fluidity, these modifications could be beneficial for survival of B. longum R0175 during the freezing process but detrimental for maintenance of acidification activity of L. buchneri R1102.