Rulin Liu

Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir

The complete genome sequence of Geobacillus thermodenitrificans NG80-2, a thermophilic bacillus isolated from a deep oil reservoir in Northern China, consists of a 3,550,319-bp chromosome and a 57,693-bp plasmid. The genome reveals that NG80-2 is well equipped for adaptation into a wide variety of environmental niches, including oil reservoirs, by possessing genes for utilization of a broad range of energy sources, genes encoding various transporters for efficient nutrient uptake and detoxification, and genes for a flexible respiration system including an aerobic branch comprising five terminal oxidases and an anaerobic branch comprising a complete denitrification pathway for quick response to dissolved oxygen fluctuation. The identification of a nitrous oxide reductase gene has not been previously described in Gram-positive bacteria. The proteome further reveals the presence of a long-chain alkane degradation pathway; and the function of the key enzyme in the pathway, the long-chain alkane monooxygenase LadA, is confirmed by in vivo and in vitro experiments. The thermophilic soluble monomeric LadA is an ideal candidate for treatment of environmental oil pollutions and biosynthesis of complex molecules.

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes.

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.

Optimization of nutrient component for diesel oil degradation by Rhodococcus erythropolis.

A novel bacterium T7-2 was isolated from the oil-polluted sea-bed mud of Bohai Sea, northern China, which can degrade diesel oil at 15 degrees C. This bacterium was identified as a strain of Rhodococcus erythropolis according to its 16S rDNA gene. In order to enhance degradation efficiency, a five-level, three-factor central composite design was employed to optimize the nutrition supplied to artificial seawater. The results indicate that a supplement of 2.53 g (NH(4))(2)SO(4)L(-1), 2.75 g Na(2)HPO(4)L(-1) and 0.01 g yeast extract L(-1) to artificial seawater increases the degradation rate from 12.61% to 75% within 7d.

Genetic Rearrangement Strategy for Optimizing the Dibenzothiophene Biodesulfurization Pathway in Rhodococcus erythropolis

ABSTRACT Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by enzymes DszC, DszA, and DszB, which are encoded by the operon dszABC and contribute to the conversion in tandem. We investigated the expression characteristics of the dsz operon. Our results revealed that the levels of transcription and translation of dszA, dszB, and dszC decreased according to the positions of the genes in the dsz operon. Furthermore, the translation of dszB was repressed by an overlapping structure in the dsz operon. In order to get better and steady expression of the Dsz enzymes and optimize the metabolic flux of DBT, we rearranged the dsz operon according to the catalytic capabilities of the Dsz enzymes and expressed the rearranged dsz operon, dszBCA, in Rhodococcus erythropolis. After rearrangement, the ratio of dszA, dszB, and dszC mRNAs in the cells was changed, from 11:3.3:1 to 1:16:5. Western blot analysis revealed that the levels of expression of dszB and dszC had been enhanced but that the expression of dszA had decreased. The desulfurization activity of resting cells prepared from R. erythropolis DRB, which carried the rearranged dsz operon, was about 12-fold higher than that of resting cells of R. erythropolis DRA, which carried the original operon in a similarly constructed vector.

Sphingomonas sanxanigenens sp. nov., isolated from soil

Strain NX02(T), a Gram-negative, non-spore-forming, rod-shaped bacterium, was isolated from soil, and its taxonomic position was investigated using a polyphasic approach. Chemotaxonomic analysis revealed that strain NX02(T) possessed Q-10 as the predominant ubiquinone, sym-homospermidine as the major polyamine and C(18 : 1)omega7c, C(16 : 0) and C(14 : 0) 2-OH as the major fatty acids. The main polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and an unidentified glycolipid. The DNA G+C content was 66.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NX02(T) belongs to the alpha-4 subgroup of the Proteobacteria, exhibiting the highest sequence similarity with respect to Sphingomonas azotifigens NBRC 15497(T) (95.9 %), Sphingomonas pituitosa DSM 13101(T) (95.8 %) and Sphingomonas dokdonensis KCTC 12541(T) (95.8 %). On the basis of these results, strain NX02(T) represents a novel species of the genus Sphingomonas sensu stricto, for which the name Sphingomonas sanxanigenens sp. nov. is proposed. The type strain is NX02(T) (=DSM 19645(T) =CGMCC 1.6417(T)).

Monitoring exogenous and indigenous bacteria by PCR-DGGE technology during the process of microbial enhanced oil recovery

A field experiment was performed to monitor changes in exogenous bacteria and to investigate the diversity of indigenous bacteria during a field trial of microbial enhanced oil recovery (MEOR). Two wells (26–195 and 27–221) were injected with three exogenous strains and then closed to allow for microbial growth and metabolism. After a waiting period, the pumps were restarted and the samples were collected. The bacterial populations of these samples were analyzed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rRNA fragments. DGGE profiles indicated that the exogenous strains were retrieved in the production water samples and indigenous strains could also be detected. After the pumps were restarted, average oil yield increased to 1.58 and 4.52 tons per day in wells 26–195 and 27–221, respectively, compared with almost no oil output before the injection of exogenous bacteria. Exogenous bacteria and indigenous bacteria contributed together to the increased oil output. Sequence analysis of the DGGE bands revealed that Proteobacteria were a major component of the predominant bacteria in both wells. Changes in the bacteria population in the reservoirs during MEOR process were monitored by molecular analysis of the 16S rRNA gene sequence. DGGE analysis was a successful approach to investigate the changes in microorganisms used for enhancing oil recovery. The feasibility of MEOR technology in the petroleum industry was also demonstrated.