Effects of nicotinamide and riboflavin on the biodesulfurization activity of dibenzothiophene by Rhodococcus erythropolis USTB-03

Rhodococcus erythropolis USTB-03 is a promising bacterial strain for the biodesulfurization of dibenzothiophene （DBT） via a sulfurspecific pathway in which DBT is converted to 2-hydroxybiphenyl （2HBP） as an end product. The effects of nicotinamide and riboflavin on the sulfur specific activity （SA） of DBT biodesulfurization by R. erythropolis USTB-03 were investigated. Both nicotinamide and riboflavin were found to enhance the expression of SA, which was not previously reported. When R. erythropolis USTB-03 was grown on a medium containing nicotinamide of 10.0 mmol or riboflavin of 50.0 μmol, SA was raised from 68.0 or so to more than 130 mmol 2HBP/（kg dry cells.h）. When R. erythropolis USTB-03 was grown in the presence of both nicotinamide of 5.0 mmol and riboflavin of 25.0 μmol, SA was further increased to 159.0 mmol 2HBP/（kg dry cells.h）. It is suggested that the biological synthesis of reduced form of flavin mononucleotide （FMNH2）, an essential coenzyme for the activities of biodesulfurization enzyme Dsz C and A, might be enhanced by nicotinamide and riboflavin, which was responsible for the increased SA of R. erythropolis USTB-03.

Effect of Iron Nanoparticles Synthesized by a Sol-Gel Process on Rhodococcus erythropolis T902.1 for Biphenyl Degradation

Nanoparticles (NPS) are considered as a new generation of compounds to improve environmental remediation and biological processes. The aim of this study is to investigate the effect of iron NPS encapsulated in porous silica (SiO2) on the biphenyl biodegradation by Rhodococcus erythropolis T902.1 (RT902.1). The iron NPS (major iron oxide FexOy form) were dispersed in the porosity of a SiO2 support synthesized by sol-gel process. These Fe/SiO2 NPS offer a stimulating effect on the biodegradation rate of biphenyl, an organic pollutant that is very stable and water-insoluble. This positive impact of NPS on the microbial biodegradation was found to be dependent on the NPS concentration ranging from 10-6 M to 10-4 M. After 18 days of incubation the cultures containing NPS at a concentration of 10-4 M of iron improved RT902.1 growth and degraded 35% more biphenyl than those without NPS (positive control) or with the sole SiO2 particles. Though the microorganism could not interact directly with the insoluble iron NPS, the results show that about 10% and 35% of the initial 10-4 M iron NPS encapsulated in the SiO2 matrix would be incorporated inside or adsorbed on the cell surface respectively and 35% would be released in the supernatant. These results suggest that RT902.1 would produce siderophore-like molecules to attract iron from the porous silica matrix.

Sulfur-Selective Desulfurization of Dibenzothiophene and Diesel Oil by Newly Isolated Rhodococcus erythropolis NCC-1

A dibenzothiophene （DBT）-desulfurizing bacteria strain was isolated from oil-contaminated soils and identified as Rhodococcus erythropolis NCC-1. Strain NCC-1 was found to convert DBT to hydroxybiphenyl （2-HBP） via the 4S pathway and also be able to use organic sulfur compounds other than DBT as a sole sulfur source. The strain could desulfurize 4,6-dimethyldibenzothiophene （4,6-DMDBT）, which is one of the most recalcitrant dibenzothiophene derivatives to hydrodesulfurization. When two type of oils, a model oil [n-hexadecane （n-C16） containing DBT] and a hydrodesulfurized diesel oil with various organic sulfur compounds, were treated with Rhodococcus erythropolis NCC-1 cells, the total sulfur content significantly decreased, from 150 to 20 mg/L for n-C16 and from 554 to 274 mg/L for diesel oil. The newly isolated strain NCC-1 is considered to have good potential for application in the biodesulfurization of fossil fuels.

Physiological cellular responses and adaptations of Rhodococcus erythropolis IBB_(Po1) to toxic organic solvents

A new Gram-positive bacterium, Rhodococcus erythropolis IBBPo1（KF059972.1） was isolated from a crude oil-contaminated soil sample by enrichment culture method. R. erythropolis IBBPo1 was able to tolerate a wide range of toxic compounds, such as antibiotics（800–1000 μg/mL）,synthetic surfactants（50–200 μg/mL）, and organic solvents（40%–100%）. R. erythropolis IBBPo1 showed good tolerance to both alkanes（cyclohexane, n-hexane, n-decane） and aromatics（toluene, styrene, ethylbenzene） with logPOW（logarithm of the partition coefficient of the solvent in octanol–water mixture） values between 2.64 and 5.98. However, alkanes were less toxic for R. erythropolis IBBPo1 cells, compared with aromatics. The high organic solvent tolerance of R. erythropolis IBBPo1 could be due to the presence in their large genome of some catabolic（alkB, alkB1, todC1, todM, xylM）, transporter（HAE1） and trehalose-6-phosphate synthase（otsA1, KF059973.1） genes. Numerous and complex physiological cellular responses and adaptations involved in organic solvent tolerance were revealed in R. erythropolis IBBPo1 cells exposed 1 and 24 hr to 1% organic solvents. R. erythropolis IBBPo1 cells adapt to 1% organic solvents by changing surface hydrophobicity, morphology and their metabolic fingerprinting.Considerable modifications in otsA1 gene sequence were also observed in cells exposed to organic solvents（except ethylbenzene）.

Evaluation of the function of a luciferase-like monooxygenase homologue in 4,4´-dithiodibutyric acid catabolism in Rhodococcus erythropolis MI2

The bacterium Rhodococcus erythropolis MI2 uses 4,4´-dithiodibutyric acid(DTDB)as carbon source to synthesize polythioesters(PTE).The first step for the production of PTE using DTDB is catalyzed by an NADH:flavin oxidoreductase(nox)as it was previously shown in our laboratory,and the second step is catabolized by a putative luciferase-like monooxygenase(Llm).In the current study,experiments were carried out to identify the function of Llm.Hence,the llm gene,which encodes for the Llm protein,was amplified from the genomic DNA of MI2 using polymerase chain reaction and expressed in Escherichia coli BL21 cells.Protein purification was done using His Spin Trap affinity columns.Enzyme assay was carried out using the purified protein and p-coumaric acid as substrate giving a specific activity of 1.6 U/mg protein for the purified Llm.The responsible gene(llm)was deleted in the genome of MI2,and a single deletion mutant was subsequently generated.Finally,growth of the wild-type strain(MI2)and the mutant strain(MI2Δllm)were compared using DTDB or succinate as carbon sources.Whereas the wild type was successfully grown with DTDB or succinate,the llm-negative mutant exhibited low grow with DTDB although it grows very well with succinate.

Isolation and preliminary characterization of a 3-chlorobenzoate degrading bacteria

A study was conducted to compare the diversity of 2-, 3-, and 4-chlorobenzoate degraders in two pristine soils and one contaminated sewage sludge. These samples contained strikingly different populations of mono-chlorobenzoate degraders. Although fewer cultures were isolated in the uncontaminated soils than contaminated one, the ability of microbial populations to mineralize chlorobenzoate was widespread. The 3- and 4-chlorobenzoate degraders were more diverse than the 2-chlorobenzoate degraders. One of the strains isolated from the sewage sludge was obtained. Based on its phenotype, chemotaxonomic properties and 16S rRNA gene, the organism S-7 was classified as Rhodococcus erythropolis. The strain can grow at temperature from 4 to 37℃. It can utilize several （halo）aromatic compounds. Moreover, strain S-7 can grow and use 3-chlorobenzoate as sole carbon source in a temperatures range of 10-30℃ with stoichiometric release of chloride ions. The psychrotolerant ability was significant for bioremediation in low temperature regions. Catechol and chlorocatechol 1,2-dioxygenase activities were present in cell free extracts of the strain, but no （chloro）catechol 2,3- dioxygenase activities was detected. Spectral conversion assays with extracts from R. erythropolis S-7 showed accumulation of a compound with a similar UV spectrum as chloro-cis,cis-muconate from 3-chlorobenzoate. On the basis of these results, we proposed that S-7 degraded 3-chlorobenzoate through the modified ortho-cleave pathway.