| With the wide exploitation, use and transportation of crude oil, the accidents of Crude oil pollution are frequent. By gravity, surface tension and capillary phenomenon the oil spill into the soil will spread to the surface and underground, affect the water permeability and air permeability, further influence soil fertility and underground water. Finally, harm to plant and animal life and bring three hazards "On human cancer, teratogenicity, mutagenic". Compared with crude oil pollution in the Marine environment, the crude oil pollution in soil in the hidden disadvantages sex is big, the incubation period is long, wide range, management more difficult.Commonly used methods for removing oil pollution has a physical method, chemical method and biological repair method. But physical method and chemical method has high cost, complex operation, high requirements for equipment, can produce secondary pollution, such as faults, and bioremediation method is to use microbial metabolism to break down pollutants itself, the end result is CO2, H2 O, etc, will not produce secondary pollution to the environment, is regarded as a green processing method, and using the strain can degrade some physical and chemical method is not easy to degradation of the material. So development of bioremediation technology will be greatly beneficial to oil pollution control work.But compared with interesting environment, the application of bioremediation technology in cold environment and more difficult, low temperature is an important limit condition of bioremediation technology. Low temperature will not only reduce the volatilization of low molecular weight alkanes, make big molecular weight of alkane stuck on the soil surface are difficult to degrade, will also reduce microbial cell SAP liquidity in the body, catalytic degradation of pollutants loss of enzyme activity are grown, microbial activity and chemical transport ability is abate, at the same time, low temperature can also affect the stability of DNA secondary structure so as to inhibit DNA replication, mRNA transcription and translation. If the temperature is lower than the cytoplasm freezing point, closed cell membrane channel, cytoplasm freezes, pollutants and nutrients cannot enter cells, the cells can destroy the cellular structure of ice crystal formation, the cells will stop life activities. So the research of crude oil at low temperature degradation bacteria the application of bioremediation technology in cold environment is a great supplement.The Qinghai-Tibet Plateau is a famous low temperature environment on the earth due to an average elevation of 4000 meters, all the year round in the low temperature, long sunshine environment. In recent years, due to the Tibetan engineering construction and operation of the corridor, and related to the development of tourism, has brought the special ecological environment of hydrocarbons, different levels of pollution, and the qinghai-tibet plateau, the sensitive and fragile ecological environment difficult to restore once they are damaged, so strengthening regional crude oil pollution prevention and control work is very important. Many previous studies indicated that there was a high diversity and richness of microbes in the soil of the Qinghai-Tibet Plateau. This study aimed to obtain microbial resources which can be used in crude oil degradation in cold environment. The degradation characteristics of the low temperature and high efficency crude oil degrading bacterialisolates were further studied to deepen the understanding of the bioremediation technology in oil-polluted cold environments. The main results of this study are listed as below.(1) Soil samples were collected along with Qinghai-Tibet highway and located at 10 sites, including Bangeqiao Bridge(BGQ), Tuotuohe River(TTH), Wudaoliang Ridge(WDL), Beiluhe River(BLH), Wuli(WL), Kaixinling(KXL), Yanshiping(YSP), the south side of Tanggula Mountain(TGL), Anduo(AD), and Dangxiong(DX). Under the condition of 10°C and crude oil used as the sole carbon source,a total of 53 crude oil degrading bacterial strains were isolated. The majority of them belong to Proteobacteria and Actinobacteria. Theoil degradation assay of single bacterial strain showed that two strains assigned asBGQ-6 and DX-7 had the capacity to degrade crude oil efficiently.Phylogenetic analyses based on 16 S rRNA gene sequence and BioLog analysis using GEN III MicroPlate indicated that strain BGQ-6 hasa 100% similarity with Rhodococcus qingshengii djl-6T(DQ090961) that belong to the phylum of Actinobacteria, and strain DX-7 has a 99.93% similarity with Acinetobacter calcoaceticus DSM 30006T(AIEC01000170) that belong to the phylum of Proteobacteria.(2) Strain BGQ-6 showed higherefficiency in crude oil degradingthan strain DX-7. Thus, we further determined the optimum conditionsof BGQ-6 for crude oil degrading. The results indicated that the optimum growth conditions were 20℃, p H 7.5, 2% of salinity, ammonium nitrate as nitrogen source, and potassium hydrogen phosphate and potassium dihydrogen phosphate as phosphorus source.The GC-MS method was used to determine the degradation rate after 15 days of the strains incubation at 10°C and 150 rpm. GC-MS detection showed that crude oil consists of 60 alkanes that belong to 5 groups, namely straight-chain alkanes, branched-alkanes, cycloalkanes, aromatic alkanes, and branched-olefin. The degradation rate of strain BGQ-6 for total crude oil was 74.14%;for straight-chain alkanes, branched-alkanes, cycloalkanes, aromatic alkanes, and branched-olefin, the degradation rate were 77.55%, 59.32%, 44.80%, 76.06%, and 93.69%, respectively. Furthermore, in the group of straight-chain alkanes, the degradation rates of short-chain alkane(C10), moderate long-chain alkanes(C10-C20), and long-chain alkanes(>C20) by strain BGQ-6 were 98.43%, 80%, and 60-80%, respectively; as to C28 and C29,the degradation rate was relatively low. This result indicates that the degrading rate decreases with the carbon chain lengthening. For the branched alkanes with carbon numbers ranged fromC12 to C26, the degradation rates of the strain BGQ-6 were about 60%.The degradation rate of the strain DX-7 for crude oil was 45.9%; for straight-chain alkanes, branched-alkanes, cycloalkanes, aromatic alkanes, and branched-olefin, they were 48.95%, 46.32%, 41.67%, 43.27%, and 49.75%, respectively. In the group of straight-chain alkanes, the degradation rates of strain DX-7 for short-chain alkanes was greater than 90%, andfor moderatelong-chain alkanes it was about 50%. For long-chain alkanes, the degradation ratewas higher than that of strain BGQ-6. For most of branched alkanes, the degradation rate was about 50% except 2-methyl-Octadecane, which was only 4.68%. Both strains DX-7 and BGQ-6 could degrade about 50% of tocycloalkanes and had a higher degrading rate of unsaturated alkanes.(3) To detect genes associated with crude oil degradation, several gene primers were designed to amplify the genes using PCR method from the genomes of the strainsBGQ-6 and DX-7. The results indicated that both two strain genomes contain alkB(encoding alkane hydroxylase) and almA(encoding flavin containing monooxygenase) genes, but have not P450(encoding cytochrome), ndoB(encoding naphthalene dioxygenase), and xylE(encoding 2, 3- catechol dioxygenase) genes. Forther detection indicated that the genome of strain BGQ-6 containfourmembers of alkB gene family(alkB1, alkB2, alkB3, and alkB4) and onecopy of almA gene. The gene sequences alkB1, alkB2, alkB3, alkB4, and almAhave been deposited in theGen Bank databaseunder the accession numbers HG530382, HG530385, HG530379, HG530384, and HG530364, respectively. Phylogenetic analysis indicated that alkB1, alkB2, and alkB4 are closely related to the three strains in same genus and have gene similarities greater than 90%. The gene alkB3 shows a similarity of 93% with thealkB gene of Geobacillus subterraneus. The gene almA of strain BGQ-6 shows only 73% gene similarity with the closestalmA gene in the database and the sequence coverage is lower than 50%. The genome ofstrain DX-7 contains only one copy ofalkB gene and one copy of almA gene and the GenBank accession numbers are HG530364 and KF802199, respectively.(4) In this study, we obtainedsix strains of Acinetobacter usingAcinetobacterspecific housekeeping genes rpo B and gyrB. Of these, two strains isolated from the Tuotuohe River and the Tanggula Mountain were assigned as TTH0-4 and TGL-Y2, which may be potential novel species. The analysis of DNA-DNA hybridization, Biolog GEN III systems with API ZYM and API 20 NE MicroPlates, and SEM observation confirmed that strain TTH0-4 is a novel species in the genus of Acinetobacter. |
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