Mục đích và đối tượng nghiên cứu của luận án + Mục đích: Chọn lọc các chủng vi sinh vật tạo có khả năng phân hủy mạnh các thành phần hydrocarbon có trong nước thải nhiễm dầu và tìm được vật liệu mang phù hợp để gắn các chủng đó nhằm đánh giá được hiệu quả phân hủy một số thành phần hydrocarbon có trong nước thải nhiễm dầu. + Đối tượng nghiên cứu: Vi sinh vật phân hủy dầu, mẫu nước thải nhiễm dầu, một số thành phần hợp chất hydrocarbon có trong dầu mỏ, một số loại vật liệu mang vi sinh. 2. Các phương pháp nghiên cứu đã sử dụng 2.1. Các phương pháp phân tích vi sinh vật Đánh giá khả năng tạo màng sinh học (biofilm) của vi sinh vật; Kiểm tra tính đối kháng của các chủng vi sinh vật; Đánh giá mật độ tế bào vi sinh vật; Phân loại và định tên nấm men bằng phương pháp so sánh trình tự đoạn ITS1 với các chủng nấm men khác trên ngân hàng gene (NCBI). 2.2. Nhóm phương pháp phân tích hóa học Lượng dầu tổng số trong nước thải được xác định bằng phương pháp phân tích khối lượng theo tiêu chuẩn TCVN 5070:1995; Xác định hàm lượng các thành phần hydrocarbon trong nước thải nhiễm dầu bằng sắc ký khí (GC), sắc ký khí kết hợp khối phổ (GCMS) và sắc ký lỏng hiệu năng (HPLC); Nghiên cứu con đường chuyển hóa sec-hexylbenzene của chủng B1 bằng phương pháp phân tích các sản phẩm tạo thành sắc ký khí kết hợp khối phổ (GCMS) và sắc ký lỏng hiệu năng (HPLC). 3. Các kết quả chính và kết luận Từ 9 chủng vi sinh vật được phân lập từ các mẫu nước và đất ô nhiễm dầu tại Việt Nam, chúng tôi đã tuyển chọn được 6 chủng vi sinh vật bao gồm QN1, B8, BN5, DX3, QNN1 và QN5 có khả năng tạo biofilm tốt và không có tính đối kháng lẫn nhau. Hỗn hợp các chủng này cho thấy khả năng hình thành biofilm tốt nhất trên vật liệu mang xơ dừa với mật độ vi sinh đạt 3,9*1012 CFU/cm3, trên các vật liệu sỏi nhẹ, cellulose và mút xốp mật độ vi sinh lần lượt đạt 2,1*1012, 4,25*109 và 1,65*1010 CFU/cm3 sau 36h. Khi tiến hành thử nghiệm khả năng phân hủy các thành phần hydrocarbon trong nước thải nhiễm dầu, biofilm vi sinh vật trên vật liệu mang xơ dừa có hiệu quả phân hủy tốt nhất so với các vật liệu sỏi nhẹ, cellulose và mút xốp, đạt 99,8% lượng dầu tổng số, 85,56% phenol và trên 96% các thành phần PAH sau 7 ngày ở mô hình 50 lít. Từ kết quả trên chúng tôi lựa chọn vật liệu mang xơ dừa để tiếp tục đánh giá hiệu quả xử lý của biofim vi sinh vật ở quy mô thử nghiệm 300 lít và 20m3/mẻ. Trên hệ thống xử lý 300 lít, biofilm vi sinh vật cho hiệu quả xử lý tốt các thành phần hydrocarbon trong nước thải nhiễm dầu và ở hệ thống xử lý 20 m3/mẻ, biofilm vi sinh vật cho hiệu quả xử lý đạt 99,94% hàm lượng dầu tổng số, 99,97% phenol và trên 94% các thành phần PAH, kết quả nước thải đầu ra đạt QCVN 40:2011/BTNMT tiêu chuẩn B. Chúng tôi cũng đã xác định được con đường giả định về sự phân hủy sec-hexylbenzene của chủng nấm men Trichosporon asahii B1 thông qua các sản phẩm trung gian bao gồm benzoic acid, 2-phenylpropionic acid, 3-phenylbutyric acid, 5-phenylhexanoic acid, ß-methylcinnamic acid, acephenone và 2,3-dihydroxybenzoic. Đây là những công bố đầu tiên tại Việt Nam về khả năng tạo biofilm của các chủng vi sinh vật phân hủy dầu trên các vật liệu mang và ứng dụng trong xử lý nước thải nhiễm dầu, cũng như đề xuất được con đường giải định về sự phân hủy sec-hexylbenzene của chủng nấm men Trichosporon asahii B1 phân lập tại Việt Nam.
MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY Do Van Tuan DEGRADATION OF HYDROCARBON COMPONENTS IN OIL POLLUTED WASTWATER BY BIOFILM FORMING MICROORGANISMS IMMOBILIZED ON CARRIERS Major: Microbiology Code: 9420107 SUMMARY OF BIOLOGICAL DOCTORAL THESIS HA NOI, 2022 The thesis was accomplished at Institute of Biotechnology, 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 advisor 1: Dr Le Thi Nhi Cong The science advisor 2: Asscio Prof Dr Dong Van Quyen Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the Graduate University of Science and Technology, Vietnam Academy of Science and Technology by Institutes Council for Doctoral Thesis Evaluation at ……… , ………… , 202 The Thesis could by search in: - The Graduate University of Science and Technology Library - The Vietnam National Library - Thư viện Học viện Khoa học Công nghệ - Thư viện Quốc gia Việt Nam - Viện Công nghệ sinh học INTRODUCTION Relevance of the research topic Currently, besides the economic benefits of the industry development, the expansion of this economic sector also leads to an increase of environmental pollution issues to an alarming level One of the common pollution sources is oil-contaminated wastewater that not only formed from the oil extraction and transportation activities but also due to the washing of engines, oil leaks, and spills The oilcontaminated water was changed the physicochemical properties of water such asincreasing its viscosity, reducing dissolved oxygen, and seriously affecting the environment and living organisms There are many effective treatment approaches for oily wastewater based on chemical and physical methods, but most of them have high costs and complicated operations which are not suitable to apply in low-income countries Moreover, these processes also could lead to secondary contamination that was negative impact on the environment and the ecosystem Recently, there have been more and more reports on the ability of microorganisms to degrade persistent organic compounds in oily wastewater, especially groups of microorganisms capable of biofilm formation The positive results and low cost of applying biofilms in wastewater treatment, including oil-contaminated wastewater treatment have gained the attention of researchers around the world However, in Vietnam, the study and application of the microbial biofilm-forming ability as well as evaluation their potential in hydrocarbon degradation in oil-polluted water is still limited Therefore, we proposed to conduct a study on the topic "Degradation of hydrocarbon components in oil polluted wastewater by biofilm forming microorganisms immobilized on carriers" to select microbial strains capable of highly degrading hydrocarbon components in oily wastewater and evaluate the treatment ability of their biofilm formed on suitable carriers Thesis objectives Selecting the biofilm-forming microbial strains those are concurrently capable of degrading hydrocarbon components in oilcontaminated wastewater; and finding the suitable carrier materials for microbial attachment to evaluate their degradation efficiency of hydrocarbon components in oily wastewater Thesis contents Major contents of the research include: (1) Selection of microbial strains that are both capable of biofilmforming and utilizing hydrocarbon components in oily wastewater (2) Selection of the inexpensive available carriers in Vietnam those are suitable for microbial immobilization (3) Evaluation the most suitable carrierfor microbial immobilization based on their oily wastewater treatment efficiency in a 50-liter bioreactor (4) Estimation the degradation ability of pollutants containing in oily wastewater by the microorganisms immobilized on suitable carrier in a 300-liter bioreactor (5) Application and evaluation of the efficiency of the selected organisms immobilized on carrier to treat oil-contaminated wastewater in Do Xa petroleum tanks, Hanoi (6) Study on the sec-hexylbenzene degradation pathway by the yeast strain Trichosporon sp B1 CHAPTER OVERVIEW 1.1 Overview of oil-contaminated wastewater The oil-polluted water is a common phenonmenom in many countries Besides the natural oil spills, human production activities also regularly discharge into the environment a large amount of oilcontaminated wastewater The sources of this pollution could be divided into main reasons including oil extraction process and the leaking of oil-contaminated wastewater at petrol storage due to rainwater, incident in oil transportation process as well as machinery operation and production activities using oil products The main component of oil-contaminated wastewater are hydrocarbons, additional garbage, sediment, etc Depending on the source, the diversity of composition as well as the pollutants content in the oil-contaminated wastewater is different significantly Oil-contaminated wastewater not only causes serious harm to the environment but also has a strong impact on the ecosystem, human health as well as the economy and society Oil-contaminated wastewater was changed its physicochemical properties The oil layer floating on the water reduces dissolved oxygen, leading to a decrease in the self-cleaning ability of the water The oil layer also prevents water evaporation, reducing rainfall, affecting the climate of the area Oil polluted sediment is the cause of soil pollution Moreover, oil contaminated water is the cause of economic losses, especially in the mining, aquaculture, seafood, and tourism industries 1.2 Approaches applied in oil-contaminated wastewater treatment 1.2.1 Flotation techniques Flotation technology is widely used in the treatment of oily wastewater in factories around the world Reports showed that flotation technology was capable of removing more than 90% of oil emulsion components using pretreatment with aluminum sulfate and ferric sulfate Flotation technology has a good ability to remove free oil components, oil in emulsion form but is less effective in removing oil components in soluble form and emulsion form with very small particle size Moreover, the use of coagulants to ensure effective treatment leads to the risk of secondary pollution because these substance residues remain in the wastewater after treatment 1.2.2 Membrane filtration technology Membrane filtration technology uses specially structured, highly porous materials capable of capturing pollutant particles to remove them from wastewater Currently, membrane filtration technology is investigatedand applied in oil-contaminated wastewater treatment at oil refineries with high efficiency, with the removal of over 98% of the oil content in the wastewater However, membrane filtration technology is not capable of removing soluble oil components in the treatment of oily wastewater, the membrane system is easied to clogging by hydrophobic components in wastewater and has low treatment throughput 1.2.3 Advanced oxidation process Advanced oxidation process (AOPs) is a chemical wastewater treatment method that utilizes the reaction of hydroxyl radicals (OH-) with pollutants to convert them into small inorganic molecules The hydroxyl radicals can be generated from one or more oxidants such as ozone, hydrogen peroxide, or energy sources such as ultraviolet, electrochemistry AOPs technology is commonly used in the treatment of industrial, domestic, and oily wastewater Numerous reports showed the removal efficiency of over 99% for oil components in a very short time AOP approaches have high treatment efficiency and is friendly to the environment However, the method has not yet been perfected to be widely applied in oil-contaminated wastewater treatment Additionally, its high costs, as well as complicated technical requirements for operation, are the must overcome the obstacle before the technology can be applied around the world 1.3 Biofilm and its application in oil pollution treatment Biofilm is a complex structure that is formed when microorganisms come into contact with a solid or liquid surface A biofilm consists of two main components: the cellular component and the network of extracellular EPS compounds that surround the cells, creating a characteristic structure for the biofilm Biofilm is applied worldwide in oil pollution treatment with high efficiency, research results show that biofilm has many important properties that help increase the efficiency of pollutant treatment while still being environmentally friendly Studies also reported that the use of biofilm-forming microorganismsmuch higher treatment efficiency than the suspended ones The biofilm contained Candida tropicalis on gravel-bearing material could quickly and effectively decompose components of diesel oil, the treatment efficiency reaches 98% after 10 days, which was much higher than that of the free form (80%) Chavan & Mukherji suggested that the use of the formed biofilm by a single strain Burkholderia cepacia in dieselcontaminated wastewater treatment achieved a removal efficiency of over 95% for the n-alkanes components from C9-C20 after 15 days In Vietnam, several studies carried out in the last few years showed the high potential of applying biofilm-forming microorganisms those are concurrently capable of degrading and metabolizing hydrocarbons in oil-contaminated water and sediment samples However, most of the studies are still on the application of biofilm in oil-contaminated wastewater treatment are still limited at the laboratory scale CHAPTER MATERIALS AND METHODS 2.1 Materials Oil contaminated water and sediment samples were taken at Do Xa petroleum depot, Thuong Tin district, Hanoi city Oil-degrading microorganisms those were previously isolated from oilcontaminated water and ssediment samples in Vietnam were kindly provided by the Department of Environmental Biotechnology, Institute of Biotechnology The Acinetobacter calcoaceticus P23 strain with high biofilmforming ability was provided by the research team of Prof Dr Masaaki Morikawa, Hokkaido University, Japan was used as a positive control The culture medium: MPA, HKTS (for bacteria), and Hansen medium (for yeast) The carrier including polyurethane foam, zeolite, cellulose, and coconut fiber those used in this study was selected based on the characteristics of large surface area, rough surface, inexpensive and available in Vietnam Equipments used in the experiments belongs to Environmental Biotechnology Department, Institute of Biotechnology; Key Laboratory of Gene Technology, Institute of Biotechnology; Institute of Industrial Chemistry, Ministry of Industry and Trade; Department of Microbial Ecology, Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi 2.2 Methods 2.2.1 Experimental procedue The experimental steps of the thesis were conducted as follows: (1) Screening for microbial strains those have high biofilm-forming capacity (2) Testing for antagonism of microbial strains (3) Evaluating the biofilm-forming ability of microbial strains on carrier materials (4) Estimating the ability of hydrocarbon component degradation by the mixed strain biofilm attached on carriers in a 50-liter bioreactor (5) Validating the ability of hydrocarbon component degradation by the mixed strain biofilm attached on carriers in a 300-liter bioreactor (6) Estimating the ability of hydrocarbon component degradation by the mixed strain biofilm attached on carrier materials in a 20 m3 bioreactor scale (7) Identifying and studying of the sec-hexyl benzene metabolism pathway by the Trichosporon sp B1 strain 2.2.1 Microbiological methods - The ability to form biofilm of microbial strains was conducted according to the method of O'Toole et al (2006) - Antagonism of microbial strains was performed according to the description of Nguyen Lan Dung et al (1981) - The microbial cells density was determined by the colonyforming unit method (CFU) - The yeast strain Trichosporon sp B1 was preliminarily classified by morphology and by the API 20 C AUX Biochemistry Standard Kit as described by Maria et al (1997) Then, the ITS1, 5.8S rRNA, ITS2 gene fragments were sequenced and blasted into the gene bank by the BLAST tool 2.2.2 Chemical methods - The amount of total oil in the wastewater is determined as described in the National standard TCVN 5070:1995 - The hydrocarbon components in oil-contaminated wastewater were analyzed by gas chromatography (GC), gas chromatography-mass spectrometry (GCMS), and high-performance liquid chromatography (HPLC) 2.2.3 Data processing methods The data were processed by a biological statistical method using Microsoft excel 2013 III RESULTS AND DISCUSSION 3.1 Biofilm formation capacity of microbial strains Strains QN1 and B8 showed good biofilm formation ability after 24 h of culture, ∆OD570 index reached from 9,58 to 12,06 in comparision with strain P23 as a positive control (∆OD570 reached 11,04), Especially, four strains, including BN5, DX3, QNN1, and B1 have the excellent biofilm-forming ability with ∆OD570 in the 11 as temperature, light, etc., the density of microorganisms plays an important role in affecting the treatment process The higher the microbial concentration, the higher treatment efficiency and the shorter treatment time can be achieved 3.4 Degradation of diesel oil (DO) and aromatic hydrocarbons of the microbial biofilms attached to various carrier materials 3.4.1 Degradation of DO and aromatic hydrocarbons of microbial biofilms attached to polyurethane foam carrier The immobilized microbial biofilm on polyurethane foam has good ability to degrade DO with a decomposition efficiency of 90,85% after days In addition, the phenol and aromatic hydrocarbon component removal efficiency reached 87,36% of phenol, 55,59% of naphthalene, 68,35% of anthracene, 95,77% of pyrene, and 72,51% of fluorene, in comparison to their initial concentrations 3.4.2 Degradation of DO and aromatic hydrocarbons of microbial biofilms attached to cellulose Compared with polyurethane foam, cellulose carriers had a lower biofilm formation ability with a microbial density of 109 CFU/cm3 after 36 hours Therefore, a significant reduction in aromatic hydrocarbon contents was seen The decomposition efficiency was 89,12% for phenol, 57,11% for naphthalene, 70,32% for anthracene, 97,33% for pyrene, and 76,61% fluorene after days of treatment However, the DO degradation efficiency remained as higher as 79,54% for DO content on the 5th day, and the latter reached 93,41% after days of treatment 12 3.4.3 Degradability of DO and aromatic hydrocarbons of microbial biofilms attached on coconut fiber On coconut fiber, microbial biofilm gave the best formation ability with microbial density reached to 1012 CFU/cm3 After days of treatment, biofilm immobilized coconut fiber had an excellent DO decomposition efficiency of 99,98% Concurrently, the removal of aromatic hydrocarbons components was increased to 98,23% for phenol and 100% for both pyrene and fluorene after days of treatment 3.4.4 Degradation of DO and aromatic hydrocarbons of microbial biofilms attached on zeolite Similar to the experimental results gained with coconut fiber, the zeolite had a very high microbial density of up to 1012 CFU/cm3 After days of treatment, the degradation efficiency of the attached biofilm for DO was 99,95% The phenolic components and aromatic hydrocarbons were also well decomposed with an efficiency of 92,32% for phenol and over 59% for the naphthalene, anthracene, pyrene, and fluorene components 3.5 Degradation of hydrocarbon components in oily wastewater by mixute species biofilm attached on carrier material 3.5.1 Quality of oil-contaminated wastewater Samples from the wastewater storage tank at Do Xa petroleum depot, Thuong Tin, Hanoi, were collected at different locations wherein the oily scum and a pungent odor were observed The wastewater samples were then analyzed for the several criteria parameters at the Institute of Chemistry, Vietnam Academy of Science and Technology, and the Institute of Industrial Chemistry, the analysis results are presented in Table 3.3 13 Table 3.3 Analysis results of wastewater at Do Xa petroleum depot, Thuong Tin, Hanoi National Technical No Parameters Unit Initial Regulation content “QCVN 40:2011/BTNMT (Column B)” pH 8,5 5,5-9 SS (Suspended solid) o mg/l 374 100 BOD5 (20 C) mg/l 13345 50 COD mg/l 26686 150 N (Total nitrogen) mg/l 694,1 40 P (Total phosphorus) mg/l 20,22 Tổng dầu mỡ khoáng mg/l 675560 10 Phenol mg/l 910 0,5 PAH mg/l - Acenaphthylene 1159 - Fluorene 2005 - Phenanthrene 802 - Anthracene 57 - Fluoranthene 1070 - Pyrene 47,8 -Benzo(k)fluoranthene 16,8 Oil-contaminated wastewater at the Do Xa petroleum depot which has not been treated, had all critical parameters exceeding the National Technical Regulation “QCVN 40:2011/BTNMT” (Column B), especially the total mineral oil and grease content exceeds 67.556 times, and the phenol content was 1.820 times 14 higher than accepted concentrations With high oil content in wastewater, especially high levels of phenol and toxic PAHs, oily wastewater should be treated to avoid adverse impacts on the surrounding environment Oil-contaminated wastewater was diluted 10 times to match the threshold of adaptation and growth of microorganisms before conducting tests to evaluate the ability of attached biofilm on carriers to degrade hydrocarbon components The tests were carried out in the 50 liters and 300-liter bioreactor systems 3.5.2 Degradation of hydrocarbon components in oily wastewater by mixture species biofilm attached on carriers at a scale of 50 liters After the biofilm-forming process that lasted for 36 hours, the bioreactor was filled with a volume of 50 liters of oil-contaminated wastewater (10 times diluted) and operated under aerobic and continuous mixing conditions The bioreactor contained carrier without microbial attachment was concurrently set up as negative control The wastewater samples were regularly collected on days 3rd, 5th, and 7th of the experiment All four tested carrier materials, including polyurethane foam, cellulose, zeolite, and coconut fiber have a good removal efficiency of about 90% for total oil content after days of operation The phenolic components achieved treatment efficiency of over 70% and over 69% for PAH contents (Figure 3.16, 3.17) Amongst, the microbial biofilm immobilized on the coconut fiber carrier had the highest hydrocarbon components decomposition efficiency of 99,8% for total oil, 85,56% for phenol, and over 96% of PAH components after days of treatment These results were consistent with the results of microbial density on the 15 material The higher the attached microbial density, the better higher Decomposition efficiency (%) treatment efficiency could be achieved 100 99.80 86.45 93.16 84.34 91.26 82.71 95.54 85.09 80 60 40 42.09 39.18 37.54 40.60 days days 20 days Polyurethane Cellulose coconut fiber foam zeolite Carrier types Figure 3.16 Total oil degradation ability of attached microbial Decomposition efficiency (%) biofilms on various carrier materials 100 80 72.77 76.6 Polyurethane foam Cellulose 85.56 78.68 60 40 20 coconut fiber zeolite Carrier types Figure 3.17 The phenol degradability after days of attached microbial biofilm on carrier materials The obtained results indicated that the best hydrocarbon components decomposition efficiency belonged to the attached biofilm coconut fiber 16 Decomposition efficiency (%) carrier material Therefore, we continued to design a 300-liter system to evaluate biofilm's ability to treat oily wastewater on a larger scale to evaluate the applicability of this carrier in practical oil-contaminated wastewater treatment at the field 3.5.3 Degradation of hydrocarbon components in oily wastewater of attached by mixture species biofilm attached on coconut fiber in a 300 liters system 3.5.3.1 Degradation of hydrocarbon components in oily wastewater by mixture species biofilm attached on coconut fiber at a system of 300 liters/batch After 36 hours of the biofilm formation process, a microbial density of 3,9*1012 CFU/cm3 was detected on the carrier Then, the system was filled with 300 liters of oily wastewater and operated at Co Nhue Bio-Experimental Station (Co Nhue, Ha Noi) The analyzed results showed that the microbial biofilm on the carrier material was capable of degrading 85,36% and 99,76% of the total oil content after and 14 days of treatment, respectively Moreover, the phenol and PAH components were degraded up to 85,89%, and 97% after 14 days, respectively 100.00 99.76 85.36 80.00 60.00 40.00 Biofilm 40.38 Control 20.00 0.00 days days 14 days Figure 3.18 Total oil degradation ability of microbial biofilms on coir material in a 300 liters batch-bioreactor 17 Table 3.12 Degradation of phenols and PAHs in oily wastewater by mixture species biofilm attached on coconut fiber in a 300 liters batch-bioreactor Concentration (mg/l) Parameter th Decomposition efficiency (%) Initial On 14 90,7+1,53 12,8+1,1 85,89+1,43 Acenaphthylene 115,47+0,67 100 Fluorene 200,6+1,75 6,01+0,87 97+0,44 Phenanthrene 80,44+0,74 100 Anthracene 5,76+0,12 100 107,14+1,07 100 Pyrene 4,8+0,04 100 Benzo(k)fluoranthene 1,62+0,02 100 Phenol Fluoranthene 3.5.3.2 Degradation efficiency of hydrocarbon components in oily wastewater by mixture species biofilm attached on coconut fiber at a flow rate of 300 liter/day The pre-treated coconut fiber was fixed to the holding frame to form complete modules with dimensions of 85*85*15cm Modules were installed in tanks and the biofilm formation was carried out in 36 hours Afterward, the microbial density was estimated at 3,9*1012 CFU/cm3 Next, the treatment system is operated with a flow rate of 300 liters/day The GC chromatography analysis results of wastewater samples revealed that the 300 liter/day system was capable of removing 100% of n-alkane components C9, C12, C13, C19 and C20 after 14 days Decomposition efficiency (%) 18 100 90 80 70 60 50 40 30 20 10 Control after 14 days days days 14 days C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 n-alkane Figure 3.21 The removal efficiency of n-alkane components by microbial biofilm attached to coir material in a system with a flow rate of 300 liters/day Components C10, C11, C14-18 were treated with a removal efficiency of over 97% compared to the original concentration Biphenyl was used as a standard component to evaluate the content of these n-alkane components at different time points The GCMS analyzed results showed that, after 14 days of treatment, the mixed strain biofilm on coconut fiber was capable of degrading up to 98,9% of saturated hydrocarbon components, 95,03% of aromatic hydrocarbons and 95% of resin and asphaltene 3.5.4 Degradation efficiency of hydrocarbon components in oily wastewater by mixture species biofilm attached on coconut fiber in a 20m3 scale system Compared with the initial oil content of wastewater that used in the 50 liter and 300 liter bioreactor, the initial oil content in the present experiment had a slight increase while the total oil content, phenol, and PAH components did not change significantly However, the 19 tested wastewater parameters remained much higher than National Techincal Regulation “QCVN 40:2011/BTNMT (Column B)” To facilitate the adaptation of oil-degrading microorganisms, the wastewater is pre-treated with a DAF pressure flotation system, the treatment lasted 24 hours After preliminary treatment, wastewater was re-analyzed and the results were presented in Table 3.14 Table 3.14 Quality of wastewater after preliminary treatment No Parameter Unit Concentration pH SS (Suspended solid) mg/l 154,83+1,06 BOD5 (20oC) mg/l 5872,4+4,85 COD mg/l 15239,5+3,98 N (Total nitrogen) mg/l 342,5+0,7 P (Total phosphorus) mg/l 98,2+0,95 Total mineral oil grease mg/l 31950+1,53 Phenol mg/l 723+6,7 PAH mg/l 7,9+0,02 - Acenaphthylene 559,2+6,21 - Fluorene 205,5+4,79 - Phenanthrene 402,1+2,16 - Anthracene 57,3+1,23 - Fluoranthene 107,7+1,57 - Pyrene 48,3+0,63 - Benzo(k)fluoranthene 16,8+0,25 After 36 hours of culture, biofilm was formed on coconut fiber with a microbial density of 2,1*1012 CFU/cm3 The carrier modules were then installed in the treatment tank that filled with 20 m3 of wastewater The bioreactor was mixed and aerated throughout the 20 treatment process After 14 days of treatment, the total oil content Decomposition efficiency (%) removal efficiency was as high as 99,94% 120 99.94 100 80 62.92 Biofilm 60 40 Control 20.33 20 0.65 1.18 2.11 days days 14 days Figure 3.26 Total oil degradation ability of mixed strains biofilm attached on carrier material at a scale of 20m3 During the treatment process of the 20 m3 system, the biofilm on the coir material maintained a high microbial density The scanning electron microscope (SEM) images showed that the biofilm was thick, and had a uniform structure on the carrier material with high microbial density cells adhering to the EPS substrate (Figure 3.27) (a) (b) Figure 3.27 SEM images of the structure of the carrier material before (a) and after (b) the biofilm formation in the 20 m3 bioreactor During the treatment process, the content of phenol and PAH components in the wastewater was also determined regularly After 14 days of treatment, 21 microorganisms on the carrier material are capable of removing 100% of anthracene, fluoranthene, pyrene, and benzo(k)fluoranthene components and over 94% of phenol, acenaphthylene, fluorene, and phenanthrene components Table 3.16 Treatment efficiency of phenol and PAH components in the 20m3 bioreactor system Decomposition after 14 days th th of treatment On day On 14 day (%) 272+1,77 0,2+0,01 99,97 325+5,14 5,4+0,15 99, 03 164+2,31 10,7+0,12 94,79 288+5,13 0,1+0,01 99,98 21,3+0,9 100 47+0,32 100 13,6+0,31 100 6,8+0,23 100 Concentration (mg/l) Substrate 0h Phenol Acenaphthylene Fluorene Phenanthrene Anthracene Flouranthene Pyrene Benzo(k) flouranthene 723+6,7 559,2+6,21 205,5+4,79 402,1+2,16 57,3+1,23 107,7+1,57 48,3+0,63 16,8+0,25 The analyzed results of output wastewater quality after 14 days of treatment (Table 3.17) showed that all test parameters were within the accepted range of National Technical Regulation “QCVN 40:2011/BTNMT (column B)” Especially, the total mineral oil grease and phenol in wastewater before treatment was 3.195 times and 1.446 times higher than the standard, respectively, however, these contaminant concentrations reduced to the safety level of 3,4mg/l and 0,2mg/l after 14 days of treatment Table 3.17 Output wastewater quality No Parameter pH SS (Suspended solid) Unit mg/l Conc after treatment National Technical Regulation “QCVN 40:2011/BTNMT (Column B)” 7,4 5,5-9 6,3 100 22 BOD5 (20oC) mg/l 41 50 COD mg/l 110 150 N (Total nitrogen) mg/l 39,7 40 P (Total phosphorus) mg/l 1,2 Total mineral oil mg/l 3,4 10 Phenol mg/l 0,2 0,5 PAH mg/l - Acenaphthylene 5,4 - Fluorene 10,7 - Phenanthrene 0,1 - Anthracene - Fluoranthene - Pyrene Benzo(k)fluoranthene 3.6 The ability to convert sec-hexylbenzene of Trichosporon asahii B1 The ITS1, 5.8S rRNA, ITS2 gene fragments of strain B1 were sequenced with the size of 492bp and registered on Gene bank with an accession number of KC139404 The gene sequences were then compared on the gene bank by the BLAST tool, showing that strain B1 was closely related to Trichosporon asahii with 98.39% similarity From the HPLC and GCMS analysis results, the names of the intermediate products of sec-hexyl benzene that were degraded by strain B1 were identified as A, B, C, D, and E, corresponding to benzoic acid, 2-phenyl propionic acid, 3-phenyl butyric acid, 5phenylhexanoic acid, and ß-methyl cinnamic acid, respectively Based on these formation products and published studies, we have constructed a putative pathway for the metabolism of sec-hexyl benzene compounds by the yeast strain Trichosporon asahii B1 23 Figure 3.33 Putative degradation pathway of sec-hexylbenzene by Trichosporon asahii B1 CONCLUSION The study has achieved the following major results: Mixed strains of microorganisms formed the best biofilm on coconut fiber with the highest microbial density of 3,9*1012 CFU/cm3 compared to other carriers such as zeolite, cellulose, and polyurethane foam that had a density of 2,1*1012, 4,25*109, and 1,65*1010 CFU/cm3, respectively, after 36 hours Microbial biofilm on coconut fiber has the highest ability to degrade 99,8% of total oil, 85,56% of phenol, and over 96% of PAH components after days of treatment in a 50-liter bioreactor, compared to the zeolite, cellulose, and polyurethane foam carriers On a treatment system of 300 liters batch-bioreactor, microbial biofilm could effectively treat 99,76% of total oil content and over 85% of phenol and PAH components after 14 days of treatment On a continuous treatment system of 300 liters/day, the formed biofilm have a treatment efficiency of over 97% for n-alkane 24 components C10, C11, C14-18, 100% for C9, C12, C13, C19 and C20 components, 100% for phenol and PAH components, 98,9% for saturated hydrocarbons, 95,03% for aromatic hydrocarbons, and 95% resin and asphaltene On the treatment system scale of 20 m3, the microbial biofilm showed a treatment efficiency of 99,94% of total oil, 99,97% of phenol, and over 94% of PAH components Output wastewater results meet the National Technical Regulation “QCVN 40:2011/BTNMT column B” Putative pathway of sec-hexyl benzene degradation of Trichosporon asahii B1 was identified The intermediate products of the pathway included benzoic acid, 2-phenyl propionic acid, 3-phenyl butyric acid, 5-phenylhexanoic acid, ß- methyl cinnamic acid, acephenone and 2,3-dihydroxybenzoic FURTHER REQUEST: Further research for evaluating the ability to degrade hydrocarbon components in oil-contaminated wastewater by microbial biofilm at other gasoline depots in Vietnam is recommended NEW CONTRIBUTIONS OF THE DISSERTATION This research has brought the following contributions: This is the first study in Vietnam to evaluate the ability of oildegrading microbial biofilm attached to various carrier materials such as coconut fiber, polyurethane foam, zeolite, cellulose in treating oil-contaminated wastewater treatment This is the first time, the postulated pathway for sec-hexyl benzene degradation by the yeast strain Trichosporon asahii B1 isolated in Vietnam was proposed LIST OF RELATED PUBLICATIONS Le Thi Nhi Cong, Vu Thi Thanh, Cung Thi Ngoc Mai, Nghiem Ngoc Minh, Do Thi Lien, Hoang Phuong Ha, Do Van Tuan, Do Thi To Uyen, Proving diesel oil degradation of biofilm formed by microorganisms on cellulose material at 50 litre module, Journal of Biotechnology 2015, 13(2A), 703-708 Le Thi Nhi Cong, Cung Thi Ngoc Mai, Nghiem Ngoc Minh, Hoang Phuong Ha, Do Thi Lien, Do Van Tuan, Dong Van Quyen, Michihiko Ike, Do Thi To Uyen, Degradation of sec-hexylbenzene and its metabolites by a biofilm-forming yeast Trichosporon asahii B1 isolated from oilcontaminated sediments in Quangninh Coastal Zone, Vietnam, Journal of environmental science and health 2016, part A 51(3), 267-275 Do Van Tuan, Do Thi To Uyen, Dong Van Quyen, Le Thi Nhi Cong, Hydrocarbon degradation in oily wastewater by microbial biofilm attached on polyurethane foam carriers, Proceeding at the 4th Academic conference on natural science for Young Scientists, Master and PhD Student from Asean countries 15-18 December, 2015, Bangkok, Thailand: O16, 40-45 Do Van Tuan, Le Thi Nhi Cong, Do Thi Lien & Dong Van Quyen, Degradation of hydrocarbon components contaminated in oily waste-water collected in Doxa petroleum storage, Hanoi by microbial biofilm attached on coconut fiber, VNU Journal of Science 2017, 33 (2S), 274-279 Do Van Tuan, Le Thi Nhi Cong, Vu Ngoc Huy, Hoang Phuong Ha, Hydrocarbon degradation in oily waste water by microbial biofilm attached on zeolite carriers, Journal of Biotechnology 2017, 15(4A), 291-297 Do Van Tuan, Le Thi Nhi Cong, Vu Ngoc Huy, Phi Quyet Tien & Hoang Phuong Ha, Assessment of oil contaminated wastewater treatment by microbial biofilm attached on coconut fiber in 20,000 liter system, Proceeding at the 5th Academic conference on natural science for Young Scientists, Master and PhD Student from Asean countries 4-7 October, 2017, Da Lat, Vietnam, 170-176 ... Ecology, Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi 2.2 Methods 2.2 .1 Experimental procedue The experimental steps of the thesis were conducted as follows:... Identifying and studying of the sec-hexyl benzene metabolism pathway by the Trichosporon sp B1 strain 2.2 .1 Microbiological methods - The ability to form biofilm of microbial strains was conducted according... 5.8S rRNA, ITS2 gene fragments were sequenced and blasted into the gene bank by the BLAST tool 2.2 .2 Chemical methods - The amount of total oil in the wastewater is determined as described in