Luận án Nghiên cứu khả năng phân hủy hợp chất vòng thơm của các chủng vi sinh vật tạo màng sinh học phân lập tại một số địa điểm ô nhiễm dầu ở Việt Nam nghiên cứu khả năng phân hủy hợp chất vòng thơm của các chủng vi sinh vật tạo màng sinh học phân lập tạ

ntylbenzene bởi chủng nấm men Candida viswanathii TH1. 101 NHỮNG CÔNG TRÌNH CỦA TÁC GIẢ ĐÃ CÔNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN 1. Cung Thị Ngọc Mai, Lê Thị Nhi Công, Nghiêm Ngọc Minh (2013). Degradation of naphthalene by biofilm forming Rhodococcus sp. BQN11 isolated from petroleum-polluted water samples in Quang Ninh coastal zone, Vietnam. Proceedings of VAST-IRD symposium on marine science, Hai Phong, Vietnam: 125-133. 2. Cung Thị Ngọc Mai, Lê Thị Nhi Công, Nghiêm Ngọc Minh (2013). Khả năng phân hủy các hợp chất hydrocarbon có trong dầu diesel bởi màng sinh học của chủng Rhodococcus sp. BN5 phân lập từ nước thải của bể chứa kho xăng dầu Đỗ Xá, Thường Tín, Hà Nội. Báo cáo khoa học hội nghị khoa học công nghệ sinh học toàn quốc 2013: 355-359. 3. Cung Thị Ngọc Mai, Lê Thị Nhi Công, Lê Thành Công, Nghiêm Ngọc Minh (2014). Khả năng chuyển hóa và phân hủy phenol do màng sinh học tạo thành từ các chủng vi khuẩn phân lập tại kho xăng dầu Đức Giang, Gia Lâm, Hà Nội. Tạp chí Công nghệ sinh học 12(2): 381-386. 4. Le Thi Nhi Cong, Cung Thi Ngoc Mai, Masaaki Morikawa, Nghiem Ngoc Minh (2014). Transformation of iso-pentylbenzene by a biofilm - forming strain of Candida viswanathii TH1 isolated from oil-polluted sediments collected in coastal zones in Vietnam. Journal of Environmental Science and Health, part A 49: 777-786. 5. Le Thi Nhi Cong, Cung Thi Ngoc Mai, Vu Thi Thanh, Le Phi Nga and Nghiem Ngoc Minh (2014). Application of a biofilm formed by a mixture of yeasts isolated in Vietnam to degrade aromatic hydrocarbon polluted wastewater collected from petroleum storage. Water Science & Technology 70(2): 329-336. 6. Le Thi Nhi Cong, Cung Thi Ngoc Mai, Vu Thi Thanh, Nghiem Ngoc Minh, Hoang Phuong Ha, Do Thi Lien, Do Thi To Uyen (2014). Pyrene degradation of biofilm-forming Paracoccus sp. DG25 isolated from oil polluted samples collected in petroleum storage Duc Giang, Hanoi. Journal of Vietnamese environment, special issue 6(2): 178-183. 7. Cung Thị Ngọc Mai, Vũ Thị Thanh, Nghiêm Ngọc Minh, Lê Thị Nhi Công (2015). Hiệu suất phân hủy dầu diesel của chủng vi khuẩn có khả năng tạo màng tốt phân lập từ mẫu nước ô nhiễm dầu ở Quảng Ngãi. Tạp chí Khoa học ĐHQGHN: Khoa học Tự nhiên và Công nghệ 31(4S): 214-219. 102 SUMMARY BIODEGRADATION OF AROMATIC HYDROCARBONS BY BIOFILM FORMING MICROORGARNISMS ISOLATED FROM SEVERAL OIL- POLLUTED AREAS IN VIET NAM Aromatic hydrocarbons such as naphthalene, anthracene, etc (named polycylic aromatic hydrocarbons - PAHs) and phenol are persistant compounds in natural. They are available in oil; synthetic resin of resin, pesticides, herbicide, textile dyeing, TNT, detergens or in coffee production industry, additive of other industries. These compounds are insoluble in water but soluble in grease, therefore they are easy accumulated in soil, sand and sediment. Hence, they cause many diseases to health of human and ecosystem. Therefore, removal of these organic components in polluted-areas is necessary. To treat these compounds, many approaches such as physics, physical chemistry are used and has obtained positive results. These methods have fast processing; but the process is not thoroughly and create many secondary byproducts that affect the surrounding environment. Therefore, using biological agenets in bioremediation by microorganisms will overcome above mentioned disadvantages. Recently, scientists have proposed a method using biofilm formed by microorganisms to treat persistent organic compounds in oil polluted samples. A biofilm is any group of microorganisms in which cells are attached in any substrate that maining polysaccharide and protein. These characteristics help the microorganisms cell resistant to extreme environment conditions such as the decline of nutrition and changing of physical factors (pH, temperature, absorption or water loss of cells, etc) thereby improving the efficiency polluted-compound degradations in the field. However, in Vietnam, there are not many researches on biofilm forming microorgamisms to treat aromatic hydrocarbon compounds. From this fact, we have conducted investigation on thesis: “Study on biodegradation of aromatic 103 hydrocarbons by biofilm forming microorganisms isolated from several oil- polluted areas in Vietnam”. MAIN OBJECT Detection of microorganisms are not only forming biofilm but also degrading aromatic hydrocarbon compounds for application in environmental pollution treatment. RESEARCH CONTENT: 1. Enrich and isolate microorganisms on medium supplemented with aromatic hydrocarbons as a source and energy; 2. Screen the possibility of biofilm formation on the possibility of aromatic compound utilization; 3. Study on some biological characteristics, classification and identification of some represent microorganisms; 4. Estimate antagonistic and safe of selected microorganisms; 5. Study on some factors affecting on biofilm formation of some represent microorganisms; 6. Study on degradation of several aromatic hydrocarbons of some selected microorganisms; 7. Evaluate the effectiveness treatment of aromatic organic compounds in oil polluted water of forming biofilm microorganisms. NEW CONTRIBUTIONS OF THESIS: (1) This is the systematic study of the isolation, screening the strain of bacteria, yeasts with high abilitiesof biofilm formation and degradation of aromatic compounds. (2) It is the first time to establish in Vietnam a transformation pathway of iso- pentylbenzene by Candida viswanathii TH1. RESULTS In this study, some oil-polluted sediment and water samples were collected in Hanoi, Quang Ninh, Thanh Hoa, Quang Ngai and Vung Tau. After three-time 104 enrichment, 25 bacterial and 10 yeast strains were isolated. The colonies of these strainswere observed. As a result, the colonies of thesebacterial strainsoften have round shape, convex, white or orange colour with 0.3-3 mm in diameter. And yeast strains have round, rough colonies with 3-5 mm in diameter. There are seven bacterial strains (BQN11, VTPG5, ĐGP2, ĐGP4, ĐGP8, DG25, QND10) and two yeast strains (TH1, TH4) could form biofilm and grow on all tested aromatic hydrocarbons (such as anthracene, iso-pentylbenzene, naphthalene, phenol, pyrene). BQN11, VTPG5 strains were Gram positive and the others were Gram negative. Under Scanning Electron Microscope (SEM) with amplification of 5,000-10,000 times, these bacterial cells have short- rod shape and these yeasts have spherical cells. By analysis of 16S rRNA and ITS1-5.8S rRNA- ITS2 region sequence, these bacterial strains were named and registered at NCBI. They are Rhodococcus sp. BQN11 (KC151262), Rhodococcus sp. VTPG5 (LC057207), Ochrobactrum sp. DGP2 (KJ700308), Pseudomonas sp. DGP4 (KJ748401), Pseudomonas sp. DGP8 (KJ700307), Paracoccus sp. DG25 (KJ608354), Acinetobacter sp. QND10 (LC033904); two yeast strains were Candida sp. TH1 (JX129175) and Candida sp. TH4 (JX129176). Several factors affecting the biofilm formation such as pH values, temperature, NaCl concentrations, carbon and nitrogen sources were investigated. The results showed that six bacterial strains could form biofilm at pH 7 except the strain of BQN11; the yeast strain of TH1 at pH 5 and TH4 at pH 4. Two strains of BQN11 and QND10 form biofilm at 30 o C, the other bacterial strains and two yeast strains are at 37 o C. The strains of VTPG5, QND10, TH1 and TH4 form biofilm at 1.5% of NaCl and the others at 1%. Saccharose is a carbon source for the most effective biofilm forming of ĐGP8 and DG25 strains; maltose is for QND10 strain and for other strains are glucose. Beside, five bacterial and two yeast strains using KNO3 asnitrogen source, except VTPG5 strain uses (NH4)2SO4 and QND10 strain uses peptone. When using aromatic cyclic hydrocarbons, BQN11 could degrade 61.1% of 105 naphthalene (with initial concentration of 200 ppm); three strains of VTPG5, ĐGP2, ĐGP4 and ĐGP8 degraded phenol with 99.8% (with initial concentration of 200 ppm), 78.5%, 85.2% and 77.1%, respectively (with initial concentration of 100 ppm). And two strains of DG25 and TH4 could use pyrene up to 76.1% (with initial concentration of 300 ppm) and 64.2% (with initial concentration of 100 ppm). Moreover, QND10 strain could degrade 65.4% anthracene (with initial concentration of 100 ppm). Beside we studied the transformation pathway of iso- pentylbenzene by biofilm formed by TH1 strain. As a result, 7 intermediate products were identified by using gas chromatography/mass spectra and high performance liquid chromatography analysis. They were phenylacetic acid, benzoic acid, 2-methyl-4-phenyl-butan-1-ol, iso-valerophenone, succinic acid, 2-hydroxy phenylacetic acid and 2-methyl-4-phenylbutyric acid. In order to form a biofilm from a group of bacteria and a biofilm from many yeasts, the general biofilm-forming conditions for them were chosen. Specically, the conditions for bacteria are pH 7, 37 o C, 1% NaCl, saccharose, KNO3; and for yeasts are pH 5, 30 o C, 1.5% NaCl, saccharose, KNO3. Next, seven bacterial and two yeast strains were estimated their safe and antagonistic among them. As a result, all bacterial strains and two yeast strains were safe and not antagonistic between each others. Under Scanning Electron Microscope (SEM), cells in biofilm adhered more closely to each others than planktonic. Moreover, cell density of bacteria after treatment decreases from 14±0,1 x10 9 to 9±0,2 x 10 6 CFU/ml; and yeast cell decreases from 4,1±0,1 x10 8 to 2,1±0,1 x 10 5 by CFU method after 14 days at incubation. In addition, using PCR-DGGE technique, the number of bacteria strains after 7 and 14 days fluctuate slightly when compere with initial samples. Three represent bands were cut, purified and sequenced. The result showed that all these bands were similar 100% with strains Rhodococcus sp. BQN11, Acinetobactersp. QND10 and Ochrobactrum sp. DGP2. Moreover, mixed-species biofilm formed by bacteria and mixed-species biofilm formed by yeasts degraded 99% and 87% phenol (wih initial concentration of 202 mg/l phenol) after 7 days incubation. 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Website tham khảo: 123. 1 PHẦN PHỤ LỤC Phụ lục 1: KHẢ NĂNG TẠO BIOFILM CỦA CÁC CHỦNG VI SINH VẬT Hình 1. Khả năng bắt giữ tím tinh thể của biofilm do các chủng vi khuẩn tạo thành (K: đối chứng không có vsv) Hình 2. Khả năng bắt giữ tím tinh thể của biofilm do các chủng nấm men tạo thành (K: đối chứng không có vsv) 2 Phụ lục 2: PHÂN LOẠI VI SINH VẬT BẰNG KIT CHUẨN SINH HÓA Bảng 1. Kết quả phân loại bằng Kit chuẩn sinh hóa API 20E của các chủng vi khuẩn Gram âm TT Kít thử DGP2 DGP4 DG25 DGP8 QND10 1 ONPG - + - + - 2 ADH + - + - - 3 LDC - + - - + 4 ODC - - - - - 5 CIT + - + - + 6 H2S - - - + - 7 URE - - - - - 8 TDA - - - + - 9 IND - + - + + 10 VP + + + + - 11 GEL - - - - - 12 GLU + + + + + 13 MAN - + - - - 14 INO - - + - + 15 SOR - - - + - 16 RHA - - - - - 17 SAC - - + - + 18 MEL - + - - + 19 AMY - - - - - 20 ARA + + - - - 21 OX - - - - - (-, âm tính; +, dương tính) Bảng 2. Kết quả phân loại bằng Kit chuẩn sinh hóa API 20NE của các chủng vi khuẩn Gram âm TT Kít thử DGP2 DGP4 DG25 DGP8 QND10 1 NO3 - + + + - 2 TRP + + + - - 3 3 GLU + + + + + 4 ADH - + + + - 5 URE - - - - - 6 ESC - + + + - 7 GEL + + + + - 8 PNPG + + - + + 9 GLU + + + - - 10 ARA - + + + - 11 MNE - + + + - 12 MAN - + - + - 13 NAG + + + - - 14 MAL + + + + - 15 GNT + + + + - 16 CAP + + + + + 17 ADI - - - - - 18 MLT + + + + + 19 CIT - - - - + 20 PAC - - - - + 21 OX + + + + + (-, âm tính; +, dương tính) Bảng 3. Kết quả phân loại bằng Kit chuẩn sinh hóa API 50CH của các chủng vi khuẩn Gram dương TT Kit thử VTPG5 BQN11 TT Kit thử VTPG5 BQN11 0 0 - - 25 ESC - - 1 GLY - + 26 SAL + + 2 ERY - - 27 CEL - - 3 DARA - + 28 MAL - - 4 LARA + + 29 LAC - + 5 RIB - - 30 MEL - + 6 DXYL - + 31 SAC - + 7 LXYL - - 32 TRE - + 8 ADO - - 33 INU - - 4 TT Kit thử VTPG5 BQN11 TT Kit thử VTPG5 BQN11 9 MDX - + 34 MLZ - - 10 GAL - + 35 RAF - - 11 GLU - + 36 AMD + + 12 FRU - - 37 GLYG - + 13 MNE - + 38 XLT - + 14 SBE - + 39 GENE - + 15 RHA - - 40 TUR - + 16 DUL - - 41 LYX - - 17 INO + + 42 TAG + + 18 MAN - - 43 DFUC + - 19 SOR - - 44 LFUC + - 20 MDM - - 45 DARL - + 21 MDG - + 46 LARL - + 22 NAG - + 47 GNT - + 23 AMY - + 48 2KG - + 24 ARB + - 49 5KG - + (-, âm tính; +, dương tính) Bảng 4. Kết quả phân loại bằng Kit chuẩn sinh hóa API 20 C AUX của các chủng nấm men TT Kít thử B1 TH1 TH4 TT Kít thử B1 TH1 TH4 1 O - - - 12 MDG - + + 2 GLU + + + 13 NAG - + + 3 GLY -+ + + 14 CEL - + + 4 2KG + + + 15 LAL - + + 5 ARA - + - 16 MAL + + + 6 XYL - + + 17 SAC + + + 7 ADO - + + 18 TRE + + + 8 XLT - - - 19 MLZ + + + 9 GAL +- + + 20 RAF + - + 10 INO - - - 21 H/PH + - - - 11 SOR + + + (-, âm tính; -+, đổi màu khoảng 15-25%; +-, đổi màu khoảng 25-35%; +, dương tính) 5 Phụ lục 3: KHẢ NĂNG SINH TRƢỞNG CỦA CÁC CHỦNG VI SINH VẬT TRÊN CÁC NGUỒN HYDROCARBON KHÁC NHAU Hình 3(A).Khả năng sinh trưởng của chủng BQN11 trong MT có bổ sung naphthalene ở các nồng độ khác nhau Hình 3(B).Khả năng sinh trưởng của chủng VTPG5 trong MT có bổ sung phenol ở các nồng độ khác nhau Hình 3(C).Khả năng sinh trưởng của chủng DGP2 trong MT có bổ sung phenol ở các nồng độ khác nhau Hình 3(D).Khả năng sinh trưởng của chủng DGP4 trong MT có bổ sung phenol ở các nồng độ khác nhau 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 100 ppm 200 ppm 300 ppm 0 1 2 3 4 5 6 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 100 ppm 150 ppm 200 ppm 250 ppm 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 100 ppm 150 ppm 200 ppm 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 100 ppm 150 ppm 200 ppm 6 Hình 3(E). Khả năng sinh trưởng của chủng DGP8 trong MT có bổ sung phenol ở các nồng độ khác nhau Hình 3(F).Khả năng sinh trưởng của chủng DG25 trong môi trường có bổ sung pyrene ở các nồng độ khác nhau Hình 3(G).Khả năng sinh trưởng của chủng TH4 trong MT có bổ sung pyrene ở các nồng độ khác nhau Hình 3(H).Khả năng sinh trưởng của chủng QND10 trong MT có bổ sung anthracene ở các nồng độ khác nhau 0 0.5 1 1.5 2 2.5 3 3.5 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 100 ppm 150 ppm 200 ppm 0 0.5 1 1.5 2 2.5 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 100 ppm 200 ppm 300 ppm 400 ppm 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 100 ppm 150 ppm 0 0.5 1 1.5 2 2.5 3 0 24 48 72 96 120 144 168 O D 6 0 0 n m Thời gian (giờ) 50 ppm 150 ppm 100 ppm 7 Phụ lục 4: KẾT QUẢ PHÂN TÍCH GCMS DO BIOFILM ĐA CHỦNG TẠO THÀNH (A) (B) (C) Hình 4. Sắc ký đồ các thành phần hydrocarbon có trong các mẫu thí nghiệm do biofilm đa chủng vi sinh vật tạo thành; (A)- đối chứng; (B) - thí nghiệm biofilm đa chủng vi khuẩn; (C)- thí nghiệm biofilm đa chủng nấm men sau 14 ngày 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 2 2 .0 0 2 4 .0 0 2 6 .0 0 2 8 .0 0 3 0 .0 0 3 2 .0 0 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 5 0 0 0 0 0 6 0 0 0 0 0 7 0 0 0 0 0 8 0 0 0 0 0 9 0 0 0 0 0 1 0 0 0 0 0 0 T im e --> A b u n d a n c e T IC : N T -1 7 8 -1 .D 1 6 .6 0 1 8 .0 3 2 0 .1 9 2 3 .8 4 2 4 .7 2 2 6 .1 4 2 7 .0 6 2 7 .1 8 2 8 .1 1 2 8 .9 2 2 9 .7 3 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 0 5000 10000 15000 20000 25000 T ime--> Abundanc e T IC: N T 193-12-M 1.D 11.32 11.43 11.70 13.21 13.27 13.48 13.70 13.80 14.09 15.02 15.26 15.58 15.70 15.95 16.04 16.52 16.74 16.85 17.16 17.50 18.36 18. 1 18.76 19.92 20.29 21.40 21.55 21.65 21.89 21.97 22.81 23.44 24.16 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 2 2 .0 0 2 4 .0 0 2 6 .0 0 2 8 .0 0 3 0 .0 0 3 2 .0 0 3 4 .0 0 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 T im e --> A b u n d a n c e T IC : N T 1 9 3 -1 2 -M 3 .D 9 .8 3 1 0 .9 0 1 1 .2 4 1 1 .8 1 1 1 .8 9 1 2 .1 8 1 3 .0 3 1 3 .2 4 1 3 .7 0 1 3 .9 8 1 4 .2 0 1 4 .2 8 1 4 .5 8 1 5 .5 1 1 5 .5 9 1 5 .7 5 1 6 .0 8 1 6 .1 9 1 6 .4 3 1 6 .5 4 1 6 .7 4 1 7 .2 3 1 7 .3 4 1 7 .6 4 1 8 .0 0 1 8 .8 7 1 9 .1 3 1 9 .5 2 2 0 .3 8 2 0 .4 2 2 0 .7 9 2 1 .4 5 2 1 .7 3 2 1 .9 0 2 2 .0 3 2 2 .1 3 2 2 .3 9 2 2 .4 7 2 3 .3 1 2 3 .6 0 2 3 .6 9 2 3 .9 3 2 4 .0 3 2 4 .6 6 2 5 .1 4 2 5 .3 9 2 5 .5 0 2 5 .9 5 2 6 .6 5 2 7 .2 0 2 8 .2 7 2 8 .4 5 2 9 .9 1 3 1 .7 2 3 4 .0 2 8 Bảng 5. Kết quả phân tích GCMS của mẫu đối chứng P K RT Area Pct Library/ID Ref CAS Qual 1 3.8045 0.6612 p-Dimethylbenzene 10754 000106-42-3 95 2 4.1377 0.3531 Benzene, 1,3-dimethyl- 10741 000108-38-3 93 3 4.1694 0.4409 n-Nonane 25155 000111-84-2 95 4 5.2324 1.0395 Benzene, 1-ethyl-3-methyl- 18927 000620-14-4 93 5 5.2906 0.3828 Benzene, 1-ethyl-2-methyl- 18922 000611-14-3 90 6 5.3752 0.5199 Benzene, 1,2,3-trimethyl- 18949 000526-73-8 95 7 5.5445 0.4305 1-Methyl-2-ethylbenzene 18919 000611-14-3 94 8 5.8353 1.4916 Benzene, 1,2,4-trimethyl- 18964 000095-63-6 94 9 5.8724 1.1898 Decane 38801 000124-18-5 93 10 6.2849 0.3323 4-Methyldecane 56256 002847-72-5 74 11 6.3536 0.5914 Benzene, 1,2,3-trimethyl- 18949 000526-73-8 95 12 6.6286 0.6889 1-Phenyl-2-propene 18044 000300-57-2 86 13 6.8349 0.3778 1,2-Diethylbenzene 29901 000135-01-3 81 14 6.9036 0.5733 Benzene, (1-methylpropyl)- 29853 000135-98-8 83 15 7.02 1.3973 Benzene, 4-ethyl-1,2-dimethyl- 29941 000934-80-5 93 16 7.1099 0.5334 Decane, 2-methyl- 56250 006975-98-0 94 17 7.2315 0.4433 Decane, 3-methyl- 56253 013151-34-3 97 18 7.3849 0.5739 1-Methyl-3-isopropylbenzene 29878 000535-77-3 95 19 7.4484 0.6059 1,3-Dimethyl-4-ethylbenzene 29930 000874-41-9 91 20 7.57 1.2029 1-Ethyl-2,4-dimethylbenzene 29930 000874-41-9 94 21 7.6176 0.7666 1-ethenyl-3-ethyl-Benzene 28328 055319-72-7 87 22 7.8239 1.6116 n-Undecane 56237 001120-21-4 95 23 7.9614 0.2841 1,3-Dimethyl-4-ethylbenzene 29930 000874-41-9 94 24 8.2152 0.5048 1,2,3,4-Tetramethylbenzene 29955 000488-23-3 94 25 8.284 0.761 Benzene, 1,2,4,5-tetramethyl- 29967 000095-93-2 95 26 8.5748 0.5385 Benzene, 1-methyl-4-(1- methylpropyl)- 44859 001595-16-0 76 27 8.6542 0.6662 1-Methyl-3,5-diethylbenzene 44749 002050-24-0 83 28 8.7124 1.5659 1H-Indene, 2,3-dihydro-5- methyl- 28364 000874-35-1 81 29 8.8869 1.5618 1H-Indene, 2,3-dihydro-4- methyl- 28361 000824-22-6 91 30 8.9186 1.7691 Benzene, 1,2,4,5-tetramethyl- 29963 000095-93-2 90 31 9.0032 0.6161 Undecane, 4-methyl- 74420 002980-69-0 89 32 9.0614 0.9075 trans-1-methyl-2-indanol 44525 102330-13-2 68 33 9.109 0.5921 Undecane, 2-methyl- 74411 007045-71-8 89 34 9.1672 0.7209 Benzene, 1-methyl-4-(1- methylpropyl)- 44859 001595-16-0 68 35 9.2359 0.7002 5-Ethyldecane 74465 017302-36-2 52 9 P K RT Area Pct Library/ID Ref CAS Qual 36 9.3629 0.5394 Benzene, 1-methyl-4-(1- methylpropyl)- 44859 001595-16-0 90 37 9.5956 0.6248 Benzene, (1-methyl-1-butenyl)- 42930 053172-84-2 92 38 9.6802 3.6543 Naphthalene 25268 000091-20-3 76 39 9.7331 0.8703 1-Cyclopropyl-1-methylspiro 42943 000000-00-0 83 40 9.8336 4.5694 n-Dodecane 74405 000112-40-3 64 41 10.0239 0.4522 Benzene, ethyl-1,2,4-trimethyl- 44754 054120-62-6 87 42 10.0874 0.7175 2,6-Dimethylundecene 93403 017301-23-4 87 43 10.3413 0.7044 4-Cyclopentylphenol 62534 001518-83-8 72 44 10.5528 1.1952 Bicyclo[4.2.1]nona-2,4,7-triene, 7-ethyl- 42920 000000-00-0 76 45 10.8966 1.7468 1H-Indene, 2,3-dihydro-4,7- dimethyl- 42832 006682-71-9 96 46 11.1028 0.5913 Nonadecane, 9-methyl- 229004 013287-24-6 46 47 11.1398 1.069 1H-Indene, 2,3-dihydro-4,7- dimethyl- 42832 006682-71-9 93 48 11.2509 1.7751 3-p-Tolylprop-2-enal 42713 000000-00-0 42 49 11.4202 0.6083 Naphthalene, 1,2,3,4-tetrahydro- 6,7-dimethyl- 60409 001076-61-5 64 50 11.4572 0.3766 1-Isopropyl-3,5- dimethylbenzene 44748 004706-90-5 62 51 11.5153 0.4104 1H-Indene, 2,3-dihydro-4,7- dimethyl- 42832 006682-71-9 96 52 11.6846 0.3318 Benzene, 4-(2-butenyl)-1,2- dimethyl-, (E)- 60335 054340-86-2 86 53 11.7375 0.9212 1H-Indene, 2,3-dihydro-1,1,3- trimethyl- 60371 002613-76-5 64 54 11.8115 1.5277 n-Tridecane 93376 000629-50-5 97 55 11.8803 5.23 1-Methylnaphthalene 38907 000090-12-0 93 56 12.1235 0.4861 Benzo[b]thiophene, 4-methyl- 44168 014315-11-8 53 57 12.1764 2.8148 1-methyl naphthalene 38907 000090-12-0 93 58 13.0279 0.9302 n-Tetratriacontane 363627 014167-59-0 46 59 13.1548 0.5149 Nonadecane, 9-methyl- 229004 013287-24-6 43 60 13.2447 0.5186 5-ethyl-1,3-dimethylindan 79301 116384-72-6 80 61 13.6149 0.9572 Benzene, 4-(2-butenyl)-1,2- dimethyl- 60335 054340-86-2 91 62 13.7154 3.6954 n-Tetradecane 113303 000629-59-4 96 63 13.9587 1.7551 Naphthalene, 1,6-dimethyl- 56327 000575-43-9 95 64 13.9799 2.602 Naphthalene, 2,7-dimethyl- 56360 000582-16-1 95 65 14.1967 3.2892 Naphthalene, 1,6-dimethyl- 56327 000575-43-9 97 66 14.2813 3.0812 Naphthalene, 1,6-dimethyl- 56327 000575-43-9 96 67 14.588 1.3848 Naphthalene, 1,4-dimethyl- 56318 000571-58-4 96 10 P K RT Area Pct Library/ID Ref CAS Qual 68 14.6462 0.5819 1,6-Dimethylnaphthalene 56328 000575-43-9 90 69 14.8419 1.1114 1,4-Dimethylnapthalene 56317 000571-58-4 96 70 15.3813 0.5146 Pyrene 71665 001855-47-6 95 71 15.4554 0.511 Naphthalene, 1-propyl- 74519 002765-18-6 46 72 15.5189 1.6761 Pentadecane 134012 000629-62-9 96 73 15.741 2.0611 Naphthalene, 1,4,6-trimethyl- 74494 002131-42-2 91 74 16.0689 1.3449 Naphthalene, 1,6,7-trimethyl- 74497 002245-38-7 97 75 16.1958 1.1564 Naphthalene, 2,3,6-trimethyl- 74500 000829-26-5 97 76 16.4338 0.8456 Naphthalene, 1,6,7-trimethyl- 74497 002245-38-7 98 77 16.5343 0.9724 Naphthalene, 1,6,7-trimethyl- 74497 002245-38-7 96 78 16.6352 0.7768 Phenanthrene 74503 000829-26-5 97 79 17.2324 0.8345 Hexadecane 154901 000544-76-3 97 80 17.3276 1.2146 Naphthalene, 1-(2-propenyl)- 71647 002489-86-3 64 81 17.4175 0.6661 2-Methyl-1,1'-biphenyl 71617 000643-58-3 89 82 17.9992 0.4212 1-Acetyl-3-methylazulene 93351 000000-00-0 50 83 18.0386 0.8314 Fluorene 174552 000629-78-7 98 84 20.1998 0.746 Anthracene 87532 002523-37-7 95 85 20.3632 0.4319 Naphthalene, 2,3,6-trimethyl- 84482 000085-01-8 81 86 20.4161 0.7515 n-Octadecane 193185 000593-45-3 98 87 21.897 0.6009 Nonadecane 211481 000629-92-5 98 88 22.0345 0.6055 1H- Cyclopropa[l]phenanthrene,1a,9 b-dihydro- 103941 000949-41-7 96 89 22.1191 0.7698 Phenanthrene, 2-methyl- 103929 002531-84-2 96 90 23.3143 0.6147 Eicosane 228992 000112-95-8 98 91 23.8484 0.5192 Naphthalene 125000 001576-69-8 94 92 24.6629 0.5644 Acenaphthalene 134048 000629-62-9 95 93 25.9586 0.3558 Hexadecane 154904 000544-76-3 97 94 27.1962 0.3928 Tricosane 275686 000638-67-5 98 95 28.9243 0.7859 Benzo(k)fluoranthene 154914 000544-76-3 95 Bảng 6. Kết quả phân tích GCMS của biofilm đa chủng vi khuẩn tạo thành P K RT Area Pct Library/ID Ref CAS Qual 1 13.48 5.5839 Naphthalene 38929 000091-57-6 96 2 13.7101 5.0129 Acenaphthylene 113301 000629-59-4 94 3 13.9904 11.4563 1,5-Dimethylnaphthalene 56324 000571-61-9 90 4 14.2072 11.3701 Naphthalene, 1,7-dimethyl- 56332 000575-37-1 96 5 14.2866 7.4606 2,7-Dimethylnaphthalene 56361 000582-16-1 87 6 15.5135 5.4875 n-Pentadecane 134011 000629-62-9 74 11 P K RT Area Pct Library/ID Ref CAS Qual 7 15.7515 8.4094 Anthracene 74506 018242-86-9 87 8 16.0088 4.8038 Naphthalene, 2,3,6-trimethyl- 74500 000829-26-5 91 9 16.0485 5.3377 Fluorene 74500 000829-26-5 91 10 17.227 5.8372 n-Tridecane 93372 000629-50-5 68 11 19.9251 5.5728 Benzo(k)fluoranthene 90602 055836-29-8 93 12 20.2914 7.8182 Pyrene 244391 004292-19-7 95 13 21.945 5.1168 Phenanthrene 103905 007151-64-6 98 14 22.1243 5.1788 Anthracene, 2-methyl- 103918 000613-12-7 80 15 23.9436 5.5539 2,7-Dimethylphenanthrene 124999 001576-69-8 80 Bảng 7. Kết quả phân tích GCMS của biofilm đa chủng nấm men tạo thành P K RT Area Pct Library/ID Ref CAS Qual 1 9.8334 1.8119 Dodecane 74406 000112-40-3 47 2 10.8964 1.0733 1H-Indene, 2,3-dihydro-4,7- dimethyl- 42830 006682-71-9 76 3 11.8113 1.8647 Phenanthrene 93366 000629-50-5 95 4 11.8907 5.1625 Naphthalene 38933 000091-57-6 97 5 12.1869 3.4642 1-Methylnaphthalene 38918 000090-12-0 91 6 13.2446 0.6147 Hexacosane 311166 000630-01-3 43 7 13.71 2.7969 Tetradecane 113289 000629-59-4 95 8 13.7476 2.0884 Anthracene 56296 000939-27-5 93 9 13.9903 6.3202 Naphthalene, 1,7-dimethyl- 56332 000575-37-1 95 10 14.2018 5.3006 Benzo(k)fluoranthene 56315 000571-58-4 96 11 14.2864 5.3188 2,7-Dimethylnaphthalene 56361 000582-16-1 96 12 14.7038 0.7186 Fluorene 56314 000571-58-4 94 13 15.5187 3.0978 Pentadecane 134049 000629-62-9 94 14 15.7567 3.9871 1,4,6-Trimethylnaphthalene 74495 002131-42-2 93 15 16.074 3.2925 2,3,6-Trimethylnaphthalene 74504 000829-26-5 97 16 16.1956 3.2339 Naphthalene, 1,6,7-trimethyl- 74497 002245-38-7 97 17 16.4336 1.8333 4,6,8-Trimethylazulene 74479 000941-81-1 91 18 16.5394 1.7511 2,3,6-Trimethylnaphthalene 74501 000829-26-5 91 19 16.7351 0.6124 2,3,6-Trimethylnaphthalene 74501 000829-26-5 87 20 17.2322 1.7423 Hexadecane 154908 000544-76-3 93 21 17.3433 2.7081 1-Isopropenylnaphthalene 71665 001855-47-6 45 22 17.4279 2.061 2-Methylbiphenyl 71617 000643-58-3 50 23 18.8664 1.8798 Heptadecane 174548 000629-78-7 93 24 19.1256 2.4333 4,4'-Dimethylbiphenyl 90542 000613-33-2 43 25 21.8968 1.5435 n-Tetradecane 113297 000629-59-4 86 26 22.0396 2.0087 METHYL-ANTHRACENE 103909 000610-48-0 58 12 P K RT Area Pct Library/ID Ref CAS Qual 27 22.1348 2.9698 Anthracene, 9-methyl- 103921 000779-02-2 81 28 23.3142 1.2577 n-Eicosane 228993 000112-95-8 94 29 23.9329 3.2792 Pyrene 125000 001576-69-8 95 30 24.0334 2.0898 3,6-Dimethylphenanthrene 125004 001576-67-6 76 31 24.6628 2.5805 n-Hexadecane 154914 000544-76-3 90 32 25.144 1.8888 11-Methylene-2-oxa-4,4,7- trimethyltricyclo(1,6)]dodecane 145347 000000-00-0 59 33 25.5089 1.4153 Phenanthrene, 2,3,5-trimethyl- 145448 003674-73-5 91 34 25.9532 1.1889 n-Docosane 261311 000629-97-0 95 35 27.196 1.3854 n-Octadecane 193184 000593-45-3 83 36 28.2749 5.7557 Acenaphthylene 313828 000123-79-5 98 37 28.4442 3.406 Hexadecane 154901 000544-76-3 38 38 29.9144 1.7846 Eicosane 228988 000112-95-8 38