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.
Trang 1AND TRAINING 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
Trang 2Vietnam 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
The Thesis could by search in:
- The Graduate University of Science and Technology Library
- The Vietnam National Library
- Thư viện Học viện Khoa học và Công nghệ
- Thư viện Quốc gia Việt Nam
- Viện Công nghệ sinh học
Trang 3INTRODUCTION
1 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 oil-contaminated 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
Trang 4Therefore, 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
2 Thesis objectives
Selecting the biofilm-forming microbial strains those are concurrently capable of degrading hydrocarbon components in oil-contaminated wastewater; and finding the suitable carrier materials for microbial attachment to evaluate their degradation efficiency of hydrocarbon components in oily wastewater
3 Thesis contents
Major contents of the research include:
(1) Selection of microbial strains that are both capable of forming and utilizing hydrocarbon components in oily wastewater (2) Selection of the inexpensive available carriers in Vietnam those are suitable for microbial immobilization
biofilm-(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
Trang 5wastewater in Do Xa petroleum tanks, Hanoi
(6) Study on the sec-hexylbenzene degradation pathway by the yeast strain Trichosporon sp B1
CHAPTER 1 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 oil-contaminated wastewater The sources of this pollution could be divided into 3 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
Trang 6contaminated 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
Trang 71.2.3 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
Trang 8The 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
diesel-contaminated 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 2 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 contaminated water and ssediment samples in Vietnam were kindly provided
oil-by the Department of Environmental Biotechnology, Institute of
Biotechnology The Acinetobacter calcoaceticus P23 strain with high
biofilm-forming 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)
Trang 9The 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)
Trang 10- 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 forming unit method (CFU)
colony 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
2.2.3 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
Trang 11range of 15,23 to 27,4 The two strains HY1 and QNB3 were the lowest biofilm-forming ability among the experimental strains Seven microbial strains including four bacterial strains (QN1, B8, BN5, DX3) and three yeast strains (QNN1, QN5, B1) were selected
for further experiments to evaluate their antagonism characteristic
Figure 3.1 Biofilm formation by microorganisms
3.2 Antagonism of microbial strains
The experimental results proved that the 6 microbial strains including QN1, B8, BN5, DX3, QNN1, and QN5 have high biofilm-forming ability and have no mutual antagonism, which is suitable for further studies of co-culturing them in one medium
Figure 3.2 Antagonism of selected microbial strains
(1: QN1; 2: B8; 3: BN5; 4: DX3; 5: QNN1; 6: QN5; 7: B1)
9.58 12.06
26.83 15.23 27.4 26.35
4.31 1.59
19.71 11.04
Trang 123.3 The biofilm formation ability of microbial strains on the carrier material
Microbial strains were cultured statically on the 50-liter bioreactors those filled with the different carrier material After 18,
24, and 36 h, samples of carriers were collected and the attached microbial density was determined by counting the number of microbial colonies, the results were presented in Table 3.1
Table 3.1 The biofilm formation capacity of the mixture microorganisms on the carriers
In the biological treatment method, apart from the ability of microorganisms to degrade pollutants, and physical conditions such
Trang 13as 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 7 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/cm3after 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 7 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 7 days of treatment