VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM VIỆN CÔNG NGHỆ SINH HỌC VƯƠNG THỊ NGA NGHIÊN CỨU VÀ ỨNG DỤNG VI KHUẨN SỬ DỤNG HYDROCARBON TRONG XỬ LÝ Ô NHIỄM DẦU VEN BIỂN LUẬN ÁN TIẾN SĨ SINH HỌC HÀ NỘI, 2016 VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM VIỆN CÔNG NGHỆ SINH HỌC Vương Thị Nga NGHIÊN CỨU VÀ ỨNG DỤNG VI KHUẨN SỬ DỤNG HYDROCARBON TRONG XỬ LÝ Ô NHIỄM DẦU VEN BIỂN Chuyên ngành: Vi sinh vật Mã số: 62 42 01 07 LUẬN ÁN TIẾN SĨ SINH HỌC NGƯỜI HƯỚNG DẪN KHOA HỌC: PGS TS Trần Đình Mấn Viện Công nghệ sinh học i LỜI CẢM ƠN Tôi xin gửi lời biết ơn chân thành sâu sắc tới PGS TS NCVCC Lại Thúy Hiền, phòng Vi sinh vật dầu mỏ, Viện Công nghệ sinh học, Viện Hàn Lâm Khoa học Công nghệ Việt Nam, hướng dẫn, giúp đỡ tận tình, cặn kẽ chuyên môn dìu dắt, bảo ban từ bước nhỏ sống Tôi xin bày tỏ lòng cảm ơn sâu sắc đến PGS TS NCVC Trần Đình Mấn, nguyên Viện phó Viện Công nghệ sinh học, phòng Vật liệu sinh học, đồng hướng dẫn nghiên cứu tôi, bảo cặn kẽ bổ sung kiến thức quý báu, giúp hoàn thành tốt luận án Tôi xin gửi lời cảm ơn sâu sắc tới TS Kiều Thị Quỳnh Hoa anh chị, bạn đồng nghiệp phòng Vi sinh vật dầu mỏ, Viện Công nghệ sinh học, tập thể vững mạnh, đoàn kết mà vô yêu quý Các anh chị, bạn phối hợp chặt chẽ, giúp đỡ tận tình, chia sẻ với niềm vui, nỗi buồn, vượt qua khó khăn, giúp hoàn thành tốt luận án Tôi vô biết ơn người thân gia đình, đặc biệt người bạn đời, mang đến cho nhiều niềm vui, hạnh phúc, bên để động viên, quan tâm, giúp đỡ lúc khó khăn Tôi xin trân trọng gửi lời cảm ơn tới Ban lãnh đạo Viện Công nghệ sinh học ThS Bùi Thị Hải Hà giúp đỡ, hợp tác tạo điều kiện thuận lợi để hoàn thành luận án Hà Nội, ngày tháng Tác giả Vương Thị Nga năm 2015 ii LỜI CAM ĐOAN Tôi xin cam đoan: Đây công trình nghiên cứu số kết cộng tác với cộng khác; Các số liệu kết trình bày luận án trung thực, phần công bố Tạp chí Khoa học chuyên ngành với đồng ý cho phép đồng tác giả; Phần lại chưa công bố công trình khác Hà Nội, ngày tháng Tác giả năm 2015 iii MỤC LỤC Lời cảm ơn i Lời cam đoan ii Mục lục iii Danh mục ký hiệu, chữ viết tắt .vii Danh mục bảng viii Danh mục hình x MỞ ĐẦU CHƢƠNG TỔNG QUAN VẤN ĐỀ NGHIÊN CỨU 1.1 Tình hình ô nhiễm hydrocarbon dầu mỏ giới Việt Nam 1.1.1 Tình hình ô nhiễm hydrocarbon dầu mỏ giới 1.1.2 Tình hình ô nhiễm hydrocarbon dầu mỏ Việt Nam 1.2 Một số đặc điểm, tính chất dầu mỏ 1.2.1 Thành phần hóa học dầu mỏ 1.2.2 Các tính chất vật lý quan trọng dầu mỏ 10 1.3 Quá trình phân hủy hydrocarbon dầu mỏ nhờ vi sinh vật 11 1.3.1 Quá trình oxy hóa hydrocarbon chất nhận điện tử oxygen 11 1.3.2 Quá trình oxy hóa hydrocarbon chất nhận điện tử khác 18 1.4 Những nét chung chất hoạt hóa bề mặt sinh học .20 1.4.1 Vai trò chất hoạt hóa bề mặt sinh học vi sinh vật phân hủy dầu 20 1.4.2 Các loại chất hoạt hóa bề mặt sinh học từ vi khuẩn .21 1.4.3 Tính chất chất hoạt hóa bề mặt sinh học 24 1.5 Vi sinh vật có khả sử dụng hydrocarbon dầu mỏ tạo chất hoạt hóa bề mặt sinh học .26 1.5.1 Tình hình nghiên cứu vi sinh vật sử dụng hydrocarbon dầu mỏ tạo chất hoạt hóa bề mặt sinh học giới 26 1.5.2 Tình hình nghiên cứu vi sinh vật sử dụng hydrocarbon dầu mỏ tạo chất hoạt hóa bề mặt sinh học Việt Nam 30 iv 1.6 Ứng dụng vi sinh vật sử dụng hydrocarbon dầu mỏ tạo chất hoạt hóa bề mặt sinh học xử lý ô nhiễm dầu ven biển 33 CHƢƠNG VẬT LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU 35 2.1 Vật liệu nghiên cứu 35 2.1.1 Vật liệu 35 2.1.2 Thiết kế mô hình thực nghiệm xử lý ô nhiễm dầu ven biển 35 2.1.3 Máy móc thiết bị 36 2.1.4 Hóa chất 36 2.1.5 Môi trường nuôi cấy .36 2.2 Phƣơng pháp .37 2.2.1 Phương pháp thu mẫu 37 2.2.2 Xác định số lượng vi sinh vật theo phương pháp pha loãng tới hạn Most Probable Number (Man, 1983) 37 2.2.3 Phân lập vi khuẩn sử dụng hydrocarbon .38 2.2.4 Quan sát hình thái tế bào vi khuẩn kính hiển vi điện tử quét .38 2.2.5 Đặc điểm sinh hóa vi khuẩn kít chuẩn sinh hóa 38 2.2.6 Phân loại vi khuẩn phân tích trình tự 16S rRNA 38 2.2.7 Phân tích quần xã vi sinh vật Metagenomics .40 2.2.8 Phương pháp, tiêu chí đánh giá số lượng taxa (giới, ngành, lớp, chi loài) 41 2.2.9 Nghiên cứu ảnh hưởng nhiệt độ, nguồn carbon, nitrogen, pH nồng độ NaCl lên sinh trưởng tổng hợp chất hoạt hóa bề mặt sinh học vi khuẩn 41 2.2.10 Khả tạo chất hoạt hóa bề mặt sinh học số nhũ hóa E24 42 2.2.11 Hiệu nhũ hóa chất hoạt hóa bề mặt sinh học phương pháp đẩy màng dầu loang Oil spreading 42 2.2.12 Hoạt tính nhũ hóa chất hoạt hóa bề mặt sinh học số hydrocarbon độ ổn định nhũ hóa tác động nhiệt độ, nồng độ muối pH thay đổi 42 v 2.2.13 Tối ưu hóa môi trường tạo chất hoạt hóa bề mặt sinh học phương pháp đáp ứng bề mặt 43 2.2.14 Tách chiết thô chất hoạt hóa bề mặt sinh học .43 2.2.15 Xác định cấu trúc chất hoạt hóa bề mặt sinh học sắc ký khối phổ (GC-MS) 43 2.2.16 Xác định dầu tổng số cân trọng lượng .44 2.2.17 Xác định thành phần dầu sắc ký khí khối phổ (GC-MS) 44 2.2.18 Xử lý số liệu 44 CHƢƠNG KẾT QUẢ NGHIÊN CỨU 45 3.1 Số lƣợng thành phần loài vi khuẩn sử dụng hydrocarbon nƣớc trầm tích số cảng, vịnh ven biển Việt Nam 46 3.1.1 Phân tích số lượng vi khuẩn sử dụng hydrocarbon 46 3.1.2 Đặc điểm phân loại vi khuẩn sử dụng hydrocarbon .50 3.2 Khả tạo chất hoạt hóa bề mặt sinh học vi khuẩn sử dụng hydrocarbon 56 3.2.1 Sàng lọc chủng vi khuẩn tạo chất hoạt hóa bề mặt sinh học cao 56 3.2.2 Động thái sinh trưởng khả tạo chất hoạt hóa bề mặt sinh học chủng A soli H1, A calcoaceticus H3 R ruber TD2 .57 3.2.3 Đặc điểm chất hoạt hóa bề mặt sinh học chủng A soli H1, A calcoaceticus H3 R ruber TD2tạo 58 3.2.4 Ảnh hưởng yếu tố môi trường đến sinh trưởng tạo chất hoạt hóa bề mặt sinh học chủng A soli H1, A calcoaceticus H3 R ruber TD2 60 3.3 Khả phân hủy dầu diesel dầu thô chủng A soli H1, A calcoaceticus H3 R ruber TD2 .68 3.4 Cấu trúc hóa học chất hoạt hóa bề mặt sinh học chủng R ruber TD2 tạo 71 vi 3.5 Tối ƣu hóa khả tạo chất hoạt hóa bề mặt sinh học chủng R ruber TD2 phƣơng pháp đáp ứng bề mặt 73 3.6 Ứng dụng vi khuẩn sử dụng hydrocarbon tạo chất hoạt hóa bề mặt sinh học xử lý ô nhiễm dầu ven biển 77 3.6.1 Tạo chế phẩm sinh học từ chủng R ruber TD2 77 3.6.2 Biến động số lượng vi khuẩn sử dụng hydrocarbon trình xử lý .79 3.6.3 Biến động loài vi khuẩn sử dụng hydrocarbon chiếm ưu trình xử lý .81 3.6.4 Xác định quần xã vi sinh vật mô hình xử lý phân tích Metagenomics 86 3.6.5 Hiệu xử lý ô nhiễm dầu ven biển 88 CHƢƠNG BÀN LUẬN KẾT QUẢ NGHIÊN CỨU 94 * Số lượng thành phần loài vi khuẩn sử dụng hydrocarbon nước trầm tích số cảng, vịnh ven biển Việt Nam 94 * Khả tạo chất hoạt hóa bề mặt sinh học đặc điểm chất hoạt hóa bề mặt sinh học vi khuẩn sử dụng hydrocarbon tạo 96 * Khả phân hủy dầu diesel dầu thô số chủng vi khuẩn sử dụng hydrocarbon 101 * Ứng dụng vi khuẩn sử dụng hydrocarbon tạo chất hoạt hóa bề mặt sinh học xử lý ô nhiễm dầu ven biển 103 KẾT LUẬN 111 KIẾN NGHỊ 111 DANH MỤC CÁC CÔNG TRÌNH CÔNG BỐ 113 TÀI LIỆU THAM KHẢO 114 TÓM TẮT TIẾNG ANH 125 vii DANH MỤC CÁC KÝ HIỆU, CÁC CHỮ VIẾT TẮT BLAST Basic local alignment search tool = Công cụ tìm kiếm xếp CHHBMSH Chất hoạt hóa bề mặt sinh học DNA Deoxyribonucleic acid = axit deoxyribonucleic EDTA Ethylene diamine tetra acetic acid = axit etilen điamin tetra axetic dNTP Deoxyribonucleotide triphosphate FO Fuel oil = dầu nhiên liệu DT Dầu thô DO Diesel oil = dầu diesel GC-MS Gas chromatography-Mass spectrometry = sắc ký khối phổ GHCP Giới hạn cho phép PAH Polycyclic aromatic hydrocarbon = hydrocarbon thơm đa vòng MPN Most probable number = số lượng tế bào có mẫu NCBI National Center for Biotechnology Information = Trung tâm Quốc gia Tin sinh học OD Optical density = mật độ quang học OS Oil spreading = màng dầu loang PBS Phosphate buffered saline = muối đệm photphat PCI phenol choloroform isoamylalcohol PCR Polymerase chain reaction = phản ứng trùng hợp chuỗi SDS Sodium dodecyl sulfate = muối natri dodexil sunphat VSV Vi sinh vật VK Vi khuẩn VK SDHC Vi khuẩn sử dụng hydrocarbon VK HK Vi khuẩn hiếu khí STT Số thứ tự viii DANH MỤC CÁC BẢNG Bảng 1.1 Các hydrocarbon riêng lẻ xác định loại dầu mỏ Bảng 1.2 Các chi vi sinh vật có khả phân hủy hydrocarbon no 15 Bảng 1.3 Các chi vi khuẩn có khả phân hủy hydrocarbon thơm 18 Bảng 1.4 Các nhóm vi khuẩn có khả oxy hóa hydrocarbon điều kiện kị khí 19 Bảng 1.5 Chất hoạt hóa bề mặt sinh học tách từ vi khuẩn 21 Bảng 2.1 Các điều kiện nuôi cấy khảo sát 41 Bảng 2.2 Giá trị mã hóa yếu tố thực nghiệm 43 Bảng 3.1 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích vịnh Lan Hạ 46 Bảng 3.2 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích ven biển Đồ Sơn 47 Bảng 3.3 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích cảng Nghi Sơn 47 Bảng 3.4 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích cảng, ven biển Quy Nhơn, Huế, Vũng Tàu 48 Bảng 3.5 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích ven biển Bình Thuận 49 Bảng 3.6 Số lượng vi khuẩn sử dụng hydrocarbon hiếu khí mẫu nước trầm tích ven biển Bến Tre 49 Bảng 3.7 Đặc điểm hình thái vi khuẩn sử dụng hydrocarbon Gram âm phân lập số cảng, vịnh ven biển Việt Nam 50 Bảng 3.8 Đặc điểm hình thái vi khuẩn sử dụng 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BACTERIA AND THEIR APPLICATION IN OIL POLLUTION REMEDIATION IN VIETNAM COASTAL ZONE Vietnam has a long coast zone and a developing petrochemical industry From 1986 up to now, 200 million tons of crude oil have been exploited, the highest annual amount is 20 tons and turn-over is 8,8 billion USD Although petrochemical industry provides huge income source, untreated wastes of this industry results in the environmental pollution Annually, hundred thousands tons of crude oil seepage have caused the serious environmental pollution in coastal zones from North to South of Vietnam Soil and water contamination with hydrocarbon causes extensive damage of local system and affects negatively to aquaculture in coastal zones Thus, the selection of the reasonable technologies for clean-up of the oil is required Recently, the bioremediation technology using microorganisms (especially bacteria) to remove oil contamination is an attractive alternative over other methods because of cost-effectiveness, complete mineralization of pollutants and environment-friendliness Petroleum bioremediation is carried out by natural population microorganisms capable of utilizing hydrocarbons as a source of energy and carbon There microorganisms are capable of degrading the various types of hydrocarbon including short-chain, long-chain and aromatic compounds However, all these compounds have low solubility in water It make difficult for bacteria to come in direct contact with the hydrocarbon substrates for degrading Therefore, bacteria growing on petroleum hydrocarbon usually produce potent emulsifierscalled biosurfactant Due to their amphiphilic structure, biosurfactants increase the surface area of hydrophobic water-insoluble substances and increase their bioavailability, thereby enhancing the growth of bacteria and the rate of bioremediation In comparison to their chemically synthesized equivalents they have many advantages such as: environmentally friendly, biodegradable, less toxic and non-hazardous Moreover, they are active at extreme temperatures, pH and 126 salinity as well, and can be produced from industrial wastes and from by-products Because of their potential advantages, biosurfactant-producing microorganisms are widely used in many industries such as agriculture, chemistry, cosmetics, pharmaceutics, especially in removal of oil contamination In Vietnam, over the decade years, diverse bacteria capable of degrading petroleum hydrocarbons have been isolated and characterized, including the genera Pseudomonas, Aeromonas, Rhodococcus, Bacillus, Shigella, Acinetobacter etc However, there have been only few researches on biosurfactant-producing bacteria, especially studies on their application in bioremediation of oil contamination were still lacking Thus, this thesis studied on the hydrocarbon-degrading bacteria isolated from Vietnam coastal zones and their capability of biosurfactant production Then, they were of use as probiotics for removal of oil pollution in coastal zones From 112 water and sediment samples taken from some of ports, gulfs and coastal zones of Do Son, Cat Ba, Nghi Son, Hue, Quy Nhon, Vung Tau, Ben Tre and Binh Thuan, the enumeration and isolation of hydrocarbon-degrading bacteria were investigated The results have shown that all samples were present hydrocarbon-degrading bacteria with the number of 1.1x102-7.5x106 MPN/ml(g) Among, the number of hydrocarbon-degrading bacteria in sediment samples were higher 5-100 fold in water samples Cultivate on Gost 9023-74 media, 124 hydrocarbon-degrading bacterial strains were isolated Based on physiological, biochemical properties (API 50 CHB, 50 CHL and 20 NE kit) and 16S-rDNA sequence analysis, they are belong to 16 genera and phylums, including: Rhodococcus, Janibacter (Actinobacteria); Aeromonas, Acinetobacter, Burkholderia, Brevundimonas, Chryceomonas, Ochrobactrum, Enterobacter, Pseudomonas, Serratia, Morganella (Proteobacteria); Bacillus, Leuconostoc, Lactobacillus and Paenibacillus (Firmicutes) Among the genera, Acinetobacter, Rhodococcus, Pseudomonas and Bacillus were dominant in some of ports, gulfs and coastal zones of Vietnam 127 For efficient bioremediation of oil contamination, we screened the capability of biosurfactant production of the selected strains The results shown that, all strains are capable of biosurfactant production with the emulsification activity (E24) of 35±1.2-75±1% Three of them (Acinetobacter soli H1, Acinetobacter calcoaceticus H3 and Rhodococcus ruber TD2) showed high biosurfactant-producing capacity with the E24 of 67.5±1.2, 70.2±1.1 and 75±1%, respectively In addition, the result of the oil spreading test (Morikawa et al., 2000) on the production of biosurfactants from these strains is 100% In order to their application in oil polluted treatment, we determined characterization of biosurfactant, the optimal growth conditions for biosurfactantproducing capacity and oil biodegradation by strains H1, H3 and TD2 The data indicated that the maximum biomass concentration of three strains were achieved after days of growth At the same time, the emulsification activity of biosusurfactant produced by strains H1, H3 and TD2 were highest with the E24 of 70.3±1.2, 74±1 and 80±1%, respectively Moreover, the surface tension of the their culture broth reduced from 68.6 to 29.7-36.5mN/m Emulsification activity of biosurfactant with some hydrocarbons (xylene, n-hexane, DO and JetA1) and effects of temperature, pH and sodium chloride on biosurfactant stability were investigated The data indicated that biosurfactants produced by strains H1, H3 and TD2 emulsified effectively with all hydrocarbons Moreover, the their biosurfactant were stable during exposure to temperatures of 4-121oC for 20 min, within a wide pH range (6-12 by H1, 4-12 by H3 and 2-12 by TD2) and salinity of 0-10% NaCl Environmental conditions such as temperature, nutrients, pH and salinity affected the bacterial growth and their biosurfactant-producing capacity The best environmental conditions for H1, H3 and TD2 growing and producing biosurfactant were 30oC, 5% (v/v) DO, 2gL-1 (NH4)2HPO4, pH 7.5, 2% (w/v) NaCl; 30oC, 5% (v/v) DO, 2gL-1 (NH4)2SO4, pH7, 1% (w/v) NaCl and 30oC, 5% (v/v) DO, 2gL-1 NaNO3, pH8, 2% (w/v) NaCl, respectively In these condition, strains H1, H3 and TD2 produced crude biosurfactant of 12.9±0.3, 13.57±0.4 and 13.7±0.45 gL-1, 128 respectively The biosurfactant production by Rhodococcus ruber TD2 were optimized by using response surface methodology (RSM) based on changes of medium component: diesel oil, NaNO3 and pH RSM analysis showed that the highest biosurfactant production was obtained in medium containing 5.7 % (v/v) DO, 3.3 gL-1 NaNO3 and pH 8.3 In this medium, strain TD2 produced the maximum yield of crude biosurfactant of 30.05 gL-1 after days It was 2.2-fold higher than before optimization Base on GC-MS analyses, the biosurfactant produced by strains H1 and H3 consist of hydrophobic (-CH3) and hydrophilic (COOH) groups in chemical structure C16H22O4 (1,2 benzendicarboxylic acid, bis 2methyl propyl ester) The biosurfactant of Rhodococcus ruber TD2 was ester of Hexadecenoic acid (C16H30O2) and Hexanedioic acid bis 2-ethylhexyl (C22H42O4) containing of many –OH and C=O groups in structural chemicals Degradation of diesel and crude oil of three selected strains were estimated Analysis of total petroleum hydrocarbon (TPH) in diesel oil (DO) indicated that the TPH degradation was contributed by 98.44, 98.68 and 99.27% by H1, H3 and TD2 during days of incubation, respectively As evident from GC-MS chromatogram of hydrocarbon components of DO, H1, H3 and TD2 could degrade C10-C35 by 90.73-99.47%, 91.68-99.45% and 97.11-97.11%, respectively Similarly, the degradation of THP and its fractions in crude oil (CO) by these strains was also observed The results indicated that the degradation rate of THP and components (C11-C36) were 81% and 39.21-100% by H1, 85.2% and 60.27-100% by H3, 90.07% and 70.76-100% by TD2 after days of incubation, respectively According to the above analyses, strain Rhodococcus ruber TD2 showed the high capability of biosurfactant production and degradation of petroleum hydrocarbon Hence, it was of use as the main components of probiotics for removal of oil contamination in coastal zones To evaluate the effectiveness of probiotics, four experimental microcosms were applied in Do Son beach (each measuring by 6mx3m) Four treatments consisted of a diesel oil control, a diesel oil plus probiotics, a crude oil control and a crude oil 129 plus probiotics Diesel oil or Bach Ho crude oil were applied in each microcosm, resulting in a calculated diesel and crude oil contamination level of approximately and 10 g/kg of sand, respectively Probiotic was applied, resulting in a calculated number of bacteria of approximately 103 CFU/g of sand Once a days, mineral nutrients (7g of NH4NO3 and 3g of Ca3(PO4)2) predissolved in about 10 liters of seawater were applied in each microcosm via a sprinkler Biodegrading processes was experimented during 28 days in winter (12/2009) and summer (6/2010) In order to assess the effect of probiotics, microbial population changes and amount of total and hydrocarbon components of petroleum during bioremediation were examined The results showed that in treated microcosms, the number of useful bacteria (aerobic, fermentative and hydrocarbon-degrading bacteria) was increased during the biodegrading process while the number of sulfate-reducing bacteria, fungi and streptomyces was decreased In the highest hydrocarbon degradation stage (the 21st day of experiment in winter and the 14th day of experiment in summer), the hydrocarbon-degrading bacteria were dominant with 50 and 65-70% of total aerobic bacteria in the microcosms supported probiotics, respectively Based on 16S rDNA sequence analysis and standard API kit indicated that dominant hydrocarbon-degrading bacteria in processes are belonging to genera: Acinetobacter, Bacillus, Brevundimonas, Ochrobactrum, Pseudomonas, Pasteurella, Strenotromonas, Staphylococcus and Rhodococcus Four genera Bacillus, Pseudomonas, Pasteurella and Bacillus were dominant in control microcosms, while seven genera Rhodococcus, Acinetobacter, Brevundimonas, Ochrobactrum, Pseudomonas, Strenotromonas and Staphylococus were dominant in probiotic microcosms Though, the number of each dominant genera was changed during the different periods of treatment, they are demonstrated to play an significant role in degrading process Using non-cultivation method (metagenomics) for investigation the biodiversity of microorganisms in probiotics microcosms revealed that the dominant genera are belonging to phylum: Actinobacteria (Rhodococcus, Nocardioides, 130 Mycobacterium ), Proteobacteria (Acinetobacter, Pseudomonas, Ochrobactrum ), Firmicutes (Bacillus, Staphylococcus, Leuconostoc ) and Bacteroidetes (Flavobacterium, Shingobacterium ) with ratio of 97,17% Among genera, two genera Rhodococcus and Acinetobacter are dominant in both analysis of cultivation and non-cultivation methods The obtained result indicated that Rhodococcus ruber and other microorganisms play a key role in oil-degrading process in Do Son beach Besides, the decrease in total and hydrocarbon components of the petroleum could also serve as a proof for the effectiveness of probiotic in oil degradation Thus, approximately 99.92-99.89% and 41.16-74.65 of total petroleum, 98.35-100% and 14,56-92,71 hydrocarbon components in treated and control microcosms were removed, respectively The data obtained in this study support the application of biosurfactant-producing bacteria in the treatment of oil pollution along coastlines in Vietnam