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MINISTRY OF EDUCATION VIETNAM ACADEMY MINISTRY OF EDUCATION VIETNAM ACADEMY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY - NGUYEN THI MINH NGUYET PETROLEUM HYDROCARBON DEGRADATION BY SEVERAL BIOFILM FORMING PHOTOSYNTHETIC PURPLE BACTERIA ISOLATED IN VIETNAM Major: Microorganism Code: 42 01 07 SUMMARY OF BIOLOGICAL DOCTORAL THESIS HA NOI, 2022 download by : skknchat@gmail.com The trình thesisđược washồn accomplished Institute of Biotechnology, Công thành tại: Họcatviện Khoa học Công nghệ Viện Công nghệ sinh học Vietnam Academy of Science and Technology HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ; VIỆN CÔNG NGHỆ SINH HỌC, VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM The science 1: Dr Thi Cong Người advisor hướng dẫn khoaLe học 1: Nhi TS Lê Thị Nhi Công The science advisor 2: Asscio Prof.sinh Dr.học Dong Van Quyen - Viện Công nghệ Người hướng dẫn khoa học 2: PGS.TS Đồng Văn Quyền Reviewer 1: Reviewer 2: Reviewer 3: - Viện Công nghệ sinh học Phản biện 1: …………………………………… Phản biện 2: …………………………………… Phản biện 3: …………………………………… The thesis will be defended at the Graduate University of Science and Technology, Vietnam Academy of Science and Luận án đượcby bảo Institutes vệ trước HộiCouncil đồng đánhfor giá luận án tiến sĩThesis cấp Technology Doctoral Học viện, họpattại……… , Học viện………… , Khoa học 202 Công nghệ - Viện Hàn lâm Evaluation Khoa học Công nghệ Việt Nam vào hồi … ….’, ngày … tháng … năm 2020 Có search thể tìm in: hiểu luận án tại: The Thesis could by - The Graduate University ofKhoa Science Technology Library - Thư viện Học viện học and Công nghệ - The Vietnam National Library - Thư viện Quốc gia Việt Nam - Thư viện Học- viện vàsinh Công nghệ ViệnKhoa Cônghọc nghệ học - Thư viện Quốc gia Việt Nam - Viện Công nghệ sinh học download by : skknchat@gmail.com INTRODUCTION The urgency of the thesis Petroleum has been used for a long time and has become increasingly important in society, especially in economy, politics and technology Besides economic benefits, petroleum and its products cause serious sources of environmental pollution They are released from the extraction, use and processing of oil Petroleum contains many toxic compounds those are difficult to decompose in nature, and can cause serious consequences for the ecological environment In particular, aromatic compounds such as benzene, toluene, naphthalene, pyrene, phenol have high solubility in water and are poisonous to many living organisms Remediation of petroleum pollution can be carried out by mechanical (physical), chemical and biological methods In particular, physical and chemical methods are often used to treat petroleum hydrocarbon pollution at high concentrations, with high costs Biological treatment using microorganisms to degrade petroleum hydrocarbons is an effective measure in the petroleum hydrocarbon pollution treatment at low concentrations, which are beyond the capabilities of mechanical/chemical treatment The application of petroleum hydrocarbon decomposing and biofilm forming microorganisms could increase the efficiency of biological treatment Photosynthetic purple bacteria (PPB) are anaerobic organisms which are reported to be metabolically flexible and use a variety of substrates including petroleum hydrocarbons Several PPB have the ability to form biofilms that may play an important role in the transformation/degradation of petroleum hydrocarbon compounds PPB are widely distributed in nature then they have high potential for download by : skknchat@gmail.com application in the treatment of petroleum hydrocarbon pollution in situ and ex situ According to theory and the fact of petroleum pollution, we proposed the toptructure for the attachment of growing cells PPB was observed to have a rod shape with a size of about x 0.3 (nm), creating a stable biofilm structure The highest thicknesses of biofilm formed on CB, CF and PUF carriers were determined to be 13, 20 and 12 nm, respectively As reported by Levison et al (1994), substrate and oxygen can easily diffuse for metabolic activities in biofilm with thickness from 10–20 mm In addition, the number of PPB mounted on CF was higher than that of CB and PUF (Figure download by : skknchat@gmail.com 20 3.23, thesis full text page 71) Thus, it can be concluded that CF is the most suitable substrate for PPB adhesion 3.4.2.3 Diesel degradation efficiency by biofilms The GC analysis results (Figure 3.25, page 74 thesis full text) showed that the biofilm formed by each strain DD4, DQ41 and FO2 degraded diesel oil with an efficiency of 56, 60 and 58%, respectively The diesel oil degradation efficiency by mixture strain biofilm without carriers reached 78% Figures 3.25a and 3.25b showed that the increase in the number of bacteria cells was only evident after days of culture, indicating that after days of culture, the diesel degradation efficiency increased rapidly After days, the number of cells tended to decrease because the bacterial strains were in the dying phase and the environmental nutrients were exhausted From the obtained results, it can be inferred that to produce biomass, cells require the extraction of diesel as the sole source of carbon and energy Figure 3.26 (page 75 of the full text) demonstrated the ability of diesel oil to be adsorbed by three types of CB, PUF and CF without bacteria at 16, 12 and 15%, respectively, while those of mixture species biofilm formed on CB, PUF and CF degraded diesel up to 90, 91 and 95%, respectively The results (Figure 3.25 and Figure 3.26) indicate that the biofilm formed by the bacteria on carriers has a diesel oil degradation efficiency > 90%, especially the biofilm on CF 95% (p < 0.05), much higher than biofilms formed withour carriers (56 - 78%) In our study, the main components of diesel oil are n-alkane with carbon number from to 16 Therefore, we conduct experiments to evaluate the degradation efficiency of different n-alkanes in diesel oil download by : skknchat@gmail.com 21 (controls were carriers without bacteria and mixture species biofilms without carriers) The degradation efficiency of n-alkane components in diesel oil by mixture species biofilm after 14 days was shown in Figure 3.27 (page 76 thesis full text) The obtained results show that the carriers can only adsorb a small part of n-alkane, the degradation efficiency by mixture species biofilm attached on the carriers was much higher than that of mixture species biofilm without carriers, specifically more than 77% of hydrocarbon components were degraded mixture species biofilm on three types of carriers, the highest yield of nalkane degradation of mixture species biofilm attached on CF (ranged from 80 to 94%), while that of biofilm on CB and PUF degradation capacity was lower than the on adherence on CF and there was no major difference between CB and PUF The obtained results show that CF is the best carrier to remove diesel oil 3.4.2.4 PAH degradation efficiency by biofilms PAHs are recalcitrant compounds with high hydrophobicity Therefore, substances such as anthracene, naphthalene, phenanthrene and pyrene were selected to evaluate the degradation efficiency The results were illustrated in Figure 3.28 (page 81 thesis full text) The anthracene degradation efficiency by single species biofilm formed by DD4, DQ41, FO2 and mixture species biofilm after 14 days of culture was 78.3, 75.4, 74.1 and 83.2%, respectively (initial PAH concentration was 600 ppm) Meanwhile, mixture species biofilm formed on CB, CF and PUF could degrade anthracene with yield of 89.2, 95.3 and 87.2%, respectivel The percentage of anthracene that was absorbed by the controls was only 12.2-15.1% download by : skknchat@gmail.com 22 Similarly, the naphthalene degradation efficiency was 62.2, 67.4, 64.3 by single species biofilm formed by DD4, DQ41, FO2 and 74.6, 92.3, 94.2, 88.3% by mixture species biofilm without and with each kind of carriers, respectively The absorbance of naphthalene of the carriers ranged from 10.1 to 11.4 % The degradation of phenanthrene and pyrene also occurs similarly with anthracene and naphthalene The highest degradation rate of phenanthrene and pyrene was caused by mixture species biofilm on CF (about 91.4 and 96.2 %, respectively) These results are also in agreement with those of Lamichhane et al (2016) on activated carbon substrates that can adsorb PAH 3.4.2.4 Crude oil degradation efficiency by mixture species biofilms GC analysis results shown in Figure 3.29, 3.30, 3.31, 3.32 (pages 85 - 89 thesis full text) after 14 days with the initial concentration of 20 g/l crude oil presented that crude oil degradation efficiency by species biofilm formed by DD4, DQ41, FO2 about 61.3, 63.7 and 62.9%, respectively; about 67.5% of the total crude oil was degraded, by mixture species biofilm without carrier; crude oil degradation efficiency by mixture species biofilm attached on CB, CF and PUF is about 84.5, 92.6 and 89.3%, respectively Among these experiments, mixture species biofilm formed on CF showed the highest crude oil removal capacity The carriers could only adsorb a small amount of crude oil Analysis of the remaining crude oil fractions including alkane, aromatics, resin and bitumen showed that these components were significantly degraded, especially alkane The decomposition download by : skknchat@gmail.com 23 efficiency of alkane, aromatic, resin and bitumen fractions is described in Table 3.6 (page 90 full text) The degradation efficiency of lower alkanes such as C8, C9 and C10 was 34% after 14 days of culture; but the decomposition efficiency of higher carbon differs by biofilm attached on different carriers (Figures 3.32, 3.33, page 89, 91 full text, Appendix 5, Appendix 6) The results in Figure 3.34 and Appendix showed that the mixture species biofilm formed on CB, CF and PUF carriers has the ability to degrade saturated hydrocarbons about 77.25 - 89.71%, 85.57 – 92.38%, 32.21 – 99.05%, respectively Thus, mixture species biofilm formed on CF and CB carriers are capable of degrading about 90% of saturated hydrocarbons, mixture species biofilm formed on PUF is capable of degrading up to 99.05% saturated hydrocarbons Meanwhile, mixture species biofilm without carriers and single species biofilm formed by DD4, DQ41, FO2 only had the highest degradation capacity of about 86.95% When evaluating the degradation efficiency of aromatic hydrocarbons in crude oil, the obtained results also showed that the degradation efficiency by mixture species biofilm formed on CF (about 83-86%) was also higher than that of mixture species biofilm formed on CB and PUF and much higher than mixture species biofilm without carriers and single species biofilm formed by DD4, DQ41, FO2 (Table 3.7 page 89 full text) CONCLUSION Three highly biofilm forming and well petroleum hydrocarbon degrading strains including DD4, DQ41, FO2 were selected from 32 PPB strains They were identified as Rhodopseudomonas sp DD4 download by : skknchat@gmail.com 24 (LC318127.1), Rhodopseudomonas sp DQ41 (LC318128.1) and Rhodopseudomonas sp FO2 (LC318129.1) These selected strains well grew and formed biofilm at 30-37oC, pH 5-9 and with concentration of NaCl 1-2% Biofilm formed by each strain could degrade 100 % of toluene (initial concentration of 250 ppm); 97.71, 97.32 and 96.53 % of naphthalene in parallel with DQ41, DD4 and FO2 (initial concentration of 300 ppm) and 99 % of pyrene (initial concentration of 300 ppm) were degrade/transform after 14 days of cultivation Among carriers such as coconut fiber (CF), polyurethane foam (PUF) and cinder beads (CB), CF was the best carrier to attach bacteria Mixture species biofilm formed on coconut fiber had the highest degradation efficiency of oil components such as diesel: 95 % (initial concentration of 17.2 g/L), n-alkane (C8-C16) in diesel: 80 94%, PAH: 91 - 96% (initial concentration of 600ppm) and crude oil: 92 % (initial concentration of 20 g/L) FURTHER REQUEST Conduct further investigation to understand the predicted petroleum hydrocarbon degradation/transformation by biofilm formed by these PPB on carriers Study on bio-product from these PPB attached on carriers to apply in oil polluted water THE NEW CONTRIBUITION OF THE THESIS - Selected 03 highly biofilm forming and well petroleum hydrocarbon degrading PPB strains - To our knowledge, it is the first time to estimate crude and diesel oil degradation by single and mixture species of PPB on different carriers including coconut fiber, polyurethane foam and cinder beads download by : skknchat@gmail.com 25 LIST OF THE PUBLISHED WORKS Nguyen Thi Minh Nguyet, Do Thi Lien, Cung Thi Ngoc Mai, Hoang Phuong Ha, Le Thi Nhi Cong, Toluen Degradation ò Biofilm Formed by Photosynthetic Purple Bacteria Isolated from Oil Polluted Water Samples Taken at Cau Da Port, Khanh Hoa, VNU Journal of Science: Natural Sciences and Technology, 2017, 33 (2S), 71 – 76 Le Thi Nhi Cong, Nguyen Thi Minh Nguyet, Vu Ngoc Huy, Nguyen Binh Hieu, Dong Văn Quyen, Phenol degradation of several biofilm-forming photosynthetic purple bacterial strains isolated in Vietnam, The 5th Academic Conference on Natural Science for Young Scientists, Master and PhD Student form Asean Countrie 4-7 October, 2017, Dalat, Vietnam, 204-211 Le Thi Nhi-Cong, Do Thi Lien, Bhaskar Sen Gupta, Cung Thi Ngoc Mai, Hoang Phuong Ha, Nguyen Thi Minh Nguyet, Tran Hoa Duan, Dong Van Quyen, Hayyiratul Fatimah Mohd Zaid, Revathy Sankaran, Pau Loke Show, Enhanced Degradation of Diesel Oil by Using Biofilms Formed by Indigenous Purple Photosynthetic Bacteria from Oil - Contaminated Coasts of Vietnam on Different Carriers, Applied Biochemistry and Biotechnology, 2019 Le Thi Nhi-Cong, Do Thi Lien, Cung Thi Ngoc Mai, Nguyen Quang Lich, Nguyen Thi Minh Nguyet, Hoang Phuong Ha, Dong Van Quyen, Show Pau Loke, Biodegradation of crude oil polluted seawater by a purple phototrophic bacterial community isolated from oil-contaminated coastal zones in Vietnam immobilized in agricultural waste-based biocarriers, Water, Air, & Soil Pollution, 2020 Nguyen Thi Minh Nguyet, Hoang Phuong Ha, Dong Van Quyen, Nguyen Ngoc Huong Tra, Le Thi Nhi Cong, Degradation of naphthalene and pyrene by several biofilm - forming photosynthesis purple bacterial strains, Vietnam Journal of Biotechnology, 2020, 18 (3), 561-570 download by : skknchat@gmail.com ... Thư vi? ??n Học vi? ??n học and Công nghệ - The Vietnam National Library - Thư vi? ??n Quốc gia Vi? ??t Nam - Thư vi? ??n Học- vi? ??n v? ?sinh Công nghệ Vi? ??nKhoa Cônghọc nghệ học - Thư vi? ??n Quốc gia Vi? ??t Nam - Vi? ??n... Cơng thành tại: Họcatviện Khoa học Công nghệ Vi? ??n Công nghệ sinh học Vietnam Academy of Science and Technology HỌC VI? ??N KHOA HỌC VÀ CÔNG NGHỆ; VI? ??N CÔNG NGHỆ SINH HỌC, VI? ??N HÀN LÂM KHOA HỌC VÀ CÔNG... Technology, Vietnam Academy of Science and Luận án đượcby bảo Institutes vệ trước HộiCouncil đồng đánhfor giá luận án tiến sĩThesis cấp Technology Doctoral Học vi? ??n, họpattại……… , Học vi? ??n………… , Khoa học
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