1. Mục đích và đối tượng nghiên cứu của luận án + Mục đích: Tuyển chọn được một số chủng VKTQH vừa có khả năng tạo tạo màng sinh học (MSH) vừa có khả năng phân hủy hydrocarbon dầu mỏ hiệu suất cao. Đánh giá hiệu suất phân hủy các hợp chất hydrocarbon dầu mỏ bởi MSH đơn chủng và đa chủng VKTQH trên các loại giá thể, từ đó đưa ra giải pháp xử lý ô nhiễm dầu ở điều kiện mô hình. + Đối tượng nghiên cứu: Vi khuẩn tía quang hợp, mẫu đất và nước thải nhiễm dầu, hợp chất hydrocarbon có trong dầu mỏ. 2. Các phương pháp nghiên cứu đã sử dụng 2.1. Các phương pháp phân tích vi sinh vật Phân lập các chủng VKTQH; Đánh giá khả năng sinh trưởng của các chủng VKTQH; Xác định mật độ tế bào VKTQH trên MSH; Đánh giá khả năng tạo MSH của các chủng đã lựa chọn; Xác định phổ hấp thụ Bacteriochlorophyll ở trong tế bào nguyên được xác định theo phương pháp quang phổ ở vùng 400 – 900 nm trên máy quang phổ; Xác định Gram của vi khuẩn; Đánh giá tính đối kháng của các chủng VKTQH; Đánh giá khả năng phân hủy hydrocarbon dầu mỏ bởi MSH của VKTQH bằng phương pháp sắc kí khối phổ (GCMS), sắc ký lỏng cao áp (HPLC). 2.2. Các phương pháp sinh học phân tử Tách chiết DNA tổng số, PCR, điện di DNA trên gel agarose. 2.3. Nhóm phương pháp phân tích hóa học Các thành phần hydrocarbon thơm có trong dịch nuôi cấy vi khuẩn được đem đi phân tích hóa học bằng sắc ký lỏng cao áp (HPLC); Hàm lượng dầu diesel được xác định bằng phương pháp phân tích khối lượng theo tiêu chuẩn TCVN 4582-88:100 ml. 3. Các kết quả chính và kết luận Từ 32 chủng, chúng tôi đã tuyển chọn được 3 chủng VKTQH là DQ41, DD4 và FO2 vừa có khả năng tạo MSH tốt vừa phân hủy thành phần hydrocarbon dầu mỏ hiệu suất cao. Các chủng này đã được định tên là Rhodopseudomonas sp. DD4 (LC318127.1), Rhodopseudomonas sp. DQ41 (LC318128.1) và Rhodopseudomonas sp. FO2 (LC318129.1). Khi tiến hành khảo sát ảnh hưởng của nhiệt độ, pH, nồng độ muối, chúng tôi thấy ba chủng VKTQH DQ41, DD4 và FO2 có khả năng sinh trưởng và tạo MSH tốt trong dải nhiệt độ 30-37oC, pH 5-9 và nồng độ NaCl 1 - 2%. Nhờ vậy, các chủng VKTQH đã được lựa chọn dễ dàng thích ứng với môi trường thực tế ngoài hiện trường. Sau 14 ngày nuôi cấy: MSH của ba chủng VKTQH đã lựa chọn có khả năng phân hủy 100% toluene (nồng độ ban đầu là 250 ppm), hiệu suất phân hủy naphthalene là 97,71; 97,23; 96,53% tương ứng với DQ41, DD4 và FO2 (nồng độ ban đầu 300 ppm) và hiệu suất phân hủy pyrene cũng rất tốt đều đạt trên 99% (nồng độ ban đầu là 300 ppm); MSH đa chủng VKTQH gắn trên xơ dừa cho hiệu suất phân hủy cao nhất các thành phần hydrocarbon trong dầu mỏ, cụ thể: hiệu suất phân hủy dầu diesel: 95% (nồng độ ban đầu là 17,2 g/l), n-alkane (C8-C16) trong dầu diesel: 80 - 94%, PAH: 91 - 96% (nồng độ ban đầu là 600ppm), dầu thô: 92% tổng số dầu thô (nồng độ ban đầu là 20g/l). Đây là những công bố đầu tiên về đánh giá hiệu suất phân hủy dầu thô, dầu diesel bởi MSH đơn chủng và đa chủng VKTQH trên giá thể (sỏi nhẹ, xơ dừa, mút xốp).
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 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 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 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 topic thesis: “Petroleum hydrocarbon degradation by several biofilm forming photosynthetic purple bacteria isolated in Vietnam” The research objectives of the thesis - Isolation and selection several well biofilm forming and highly degrading petroleum hydrocarbon photosynthetic purple bacteria from oil polluted samples taken in coastal zones in Vietnam - Estimation petroleum hydrocarbon degradation efficiency by single species biofilm and mixture biofilm immobilized on several carriers leading to the impression to treat oil pollution in laboratory modules The main research content of the thesis [1] Screening several well biofilm forming and highly degrading petroleum hydrocarbon photosynthetic purple bacteria from oil polluted samples taken in coastal zones in Vietnam; studying biological characteristics and identifying the selections [2] Studying several bio-physical and bio-chemical conditions such as pH, temperature, NaCl concentrations effected on the selections’ biofilm forming capacity [3] Estimation petroleum hydrocarbon degradation efficiency by single and mixture species biofilm immobilized on several carriers or without carriers in laboratory modules Content of the thesis: The thesis includes 98 pages (3 chapters): Abstract pages, chapter (Introduction) 27 pages, chapter (Materials and methods) 14 pages, chapter (Results and discussion) 56 pages, conclusion and request page, list of publications page, references 18 pages, appendix pages; the thesis has 34 figures, tables and 178 references CHAPTER OVERVIEW 1.1 Several mainly biological characteristics of photosynthetic purple bacteria PPB belong to aquatic bacteria which can grow in anaerobic condition by photosynthesizing without releasing oxygen because they not receive electrons from photolysis of water but from some substances such as hydrogen, simple organic acids, sulfur, hydrogen sulfide, simple sugars and alcohols PPB are usually pink to burgundy; their photosynthetic pigments contain bacteriochlorophyll (Bchl) and carotenoids This group of bacteria has flexible metabolic patterns depending on environmental conditions, so they are very widely distributed in nature According to Bergey's classification manual, PPB are divided into groups: (i) photosynthetic purple sulfur bacteria (PSB): has the ability to accumulate sulfur drops inside the cell; (ii) photosynthetic purple non-sulfur bacteria (PNSB): inability to accumulate sulfur droplets inside the cell 1.2 Application of photosynthetic purple bacteria in petroleum hydrocarbon degradation According to publications on oil pollution, the research and selection of safe and effective oil pollution treatment methods is an urgent issue today The physical and chemical methods give a quick, noticeable and immediate effect on oil cleansing However, they are often expensive and can lead to secondary pollution In contrast, natural methods, especially biodegradation, are considered green technologies, with high efficiency and friendly with the environment The only drawback of this method is to take a long time Therefore, the scientific combination of methods as well as the acceleration of biodegradation have been focused on recent research Isolation and selection of hydrocarbon-degrading microorganisms are important steps in bioremediation Aromatic compounds are difficult to decompose in nature because of the stable association of the benzene ring Some PNSBs are capable of using plant-derived aromatic compounds including various lignin monomers and some man-made compounds such as chlorobenzoates and toluene as carbon sources Rps palustris is capable of using benzoate as the sole carbon source for anaerobic photosynthesis Benzoate metabolism by Rps palustris; Rps fulvum; Rcy purpureus is the characteristic feature that distinguishes them from other species in the same genus Photosynthesis of aromatic compounds has also been detected in some other species such as: Rps acidophila, Rhodomicrobium (Rmi.) vannieli, Blastochloris (Blc.) sulfoviridis, Rubrivivax (Rvi.) benzoatilyticus Several studies have investigated the growth of Rps palustris on many different aromatic compounds showed that depending on the strain Rps palustris, beside benzoate, they could also use a number of other aromatic compounds such as 4-hydroxybenzoate, 4hydroxycinnamate, cinnamate, chorobenzoates, phenylacetate, phenol, methoxylated compounds, as well as aldehydes and aromatic alcohols as carbon and nitrogen sources for growth The ability to degrade lignin derivatives containing 3-methoxyl- or 3-hydroxylsubstituents is unique characteristic of Rps palustris Although chlorobenzoate compounds are often toxic to microorganisms, some strains of the species Rps palustris is likely to use these compounds as carbon sources In Vietnam, Dinh Thi Thu Hang et al (2007) have studied the biodegradation of cyclic hydrocarbon compounds by some PPB The study has detected strains of PPB that have strong ability to degrade 3-chlorobenzoate with co-substances of benzoate or 4hydroxybenzoate, in which 3-chlorobenzoate was used as the sole carbon source The badA gene encoding for the benzoate-CoA ligase enzyme in the benzoate degradation pathway in strain VKTQH MI1 is 93.3% similar to the corresponding gene of R palustris strain DCP3 which has been published on the International Gene Bank Recently, Sampaio et al (2017) isolated several strains of the genus Rhodovolum and the genus Rhodomicrobium capable of anaerobic degradation of petroleum hydrocarbons Venkidusamy and Megharaj (2021) isolated the strain Rps palustris RP2 degrades the n-alkane components in petroleum hydrocarbons 1.3 Petroleum hydrocarbon degrading and biofilm forming microorganisms Petroleum hydrocarbons including saturated hydrocarbons and aromatic hydrocarbons cause long-term pollution in nature and tends to spread into surface water and groundwater The polycyclic aromatic hydrocarbon (PAH) decomposition efficiency depends on its ability to survive in harsh environments Notably, the microflora in the biofilm is better able to resist the stress conditions of the environment, supports better metabolism and limits the competition of other microorganisms than planktonic organism Thereby organisms in biofilm show the potential to fight against environmental conditions They can overcome difficult conditions in the natural environments and can enhance the ability to degrade pollutants Kokoky et al (2017) isolated several strains of bacteria from petroleum-contaminated soil, including Acinetobacter sp PDB4 was selected to evaluate its ability to generate biofilm, degrade PAH and promote plant growth Shao et al (2015) also reported on Acinetobacter sp isolated from a coal mining area can use fluorene, phenanthrene and pyrene as its sole carbon source Research by Zhang et al (2015) showed that biofilm formed by strain Micrococcus sp PHE9 and Mycobacterium sp NJS-P is capable of degrading phenanthrene and pyrene, the decomposition rate in biofilm type is much higher than that of bacteria in planktonic one Wimpenny et al (2000) reported in a study that bioremediation by biofilm is a proficient approach and a safe choice because cells in biofilm have a better chance of survival than in suspended type Biofilm supports the growth of Pseudomonas sp and enhance the degradation of crude oil easily and extensively Biofilms formed in the presence of crude oil accumulated biomass were higher than those produced in the presence of glucose as the sole carbon source Research by Tribelli et al (2012) also showed that the strain Pseudomonas extremaustralis isolated from Antarctica is capable of generating biofilm In biofilm, the bacterial cells grew, produced surfactants and degraded diesel with higher efficiency than planktonic condition In biofilm type, this strain degraded long-chain and branched-chain alkanes, however, when cultured in a shaker, this strain degrades only medium-chain alkanes To investigate the potential of Pseudomonas aeruginosa NY3 for treatment of concentrated crude oil-contaminated water, strain NY3 was immobilized on the foam surface to evaluate the ability to generate biofilm and the ability to recover the used biofilms The results showed that the process of biofilm formation for strain NY3 took place quickly and easily Under optimal conditions, the biomass immobilized on the sponge surface could reach 488.32 mg dry cells/g dry sponge The results demonstrated that the average oil removal rate of g of crude oil/L in contaminated water was about 90% for 40 days Meanwhile, the biofilms can be recovered for reuse Stable, high oil recovery and removal rates have facilitated the application of biofilms to remove concentrated oils from wastewater Recently, Omarova et al (2019) also found that the biofilm of Alcanivorax borkumensis strain is capable of removing 40-50% of oil stains after days of culture and degraded up to 90% hexadecane after days of culture (while commercial dispersant Corexit 9500A only reduces 25 % of hexadecane) Le Thi Nhi Cong et al (2016) evaluated the ability to form biofilm of several oil-degrading bacterial and yeast strains isolated from oil-contaminated seawater samples in Thanh Hoa and Vung Tau The results showed that some Candida, Acinetobacter, Bacillus, Rhodococcus strains have been identified They were both biofilm-forming and well degrading and metabolizing petroleum component strains Moreover, after 24 h of culture, in biofilm type, they were able to degrade oil better than in the free cell form This showed the high potential of microorganisms that both generate biofilm and degrade/transform hydrocarbons in oil-contaminated soil and water samples in Vietnam Cung Thi Ngoc Mai (2019) selected bacterial strains and yeast strains including Rhodococcus sp BQN11, Paracoccus sp DG25, Ochrobactrum sp DGP2, Pseudomonas sp DGP4, Pseudomonas sp DGP8, Acinetobacter sp QND10, Rhodococcus sp VTPG5, C viswanathii TH1 and Candida sp TH4 from oil contaminated sites These strains with good biofilm formation at pH 5, 30oC were able to degrade up to 99.8% phenol, 76.07% pyrene, 65.4% anthracene and 35.7% isopentybenzene under laboratory culture conditions at initial concentrations head from 100-200 ppm Using carriers to immobilize microorganims has many advantages such as increasing the density of organisms bringing about treatment efficiency and stability Materials used as carriers are very diverse such as coconut fiber, light gravel, sponge cotton, dried water hyacinth, etc Currently, many wastewater treatment technologies using media for microorganisms have been applied for instance membrane bioreactor (MBR), moving bed biofilm bioreactor (MBBBR) and all bring high efficiency in the treatment process In 2016, Le Thi Nhi Cong et al studied the biofilm of a mixture of bacterial and yeast strains attached to coconut fiber which was both as an adsorbent and as a carrier The efficiency of degrading hydrocarbon components by the carrier attached by biofilm reached > 99% after day – incubation Do Van Tuan et al (2017) designed a biological treatment system for oily wastewater with a scale of 20,000 liters/batch at Do Xa petroleum depot, Thuong Tin, Hanoi using biofilm adherence on coconut fiber After days of treatment, 99.9% of hydrocarbon components in oily wastewater, 99.9% phenol and over 94.8% of polycyclic aromatic hydrocarbon (PAH) components were removed The use of anaerobic or microaerobic bacteria, yeasts capable of forming biofilm to degrade petroleum hydrocarbons is a solution to overcome oil pollution In particular, PPB are announced to have a flexible metabolism, using a variety of substrates including 11 - To determine the density of PPB cells on biofilm, the experiments were conducted according to the method of Allesandrello et al (2017) - The ability to form biofilm of selected strains was stained with crystal violet according to the method of O'Toole and Kolter (1998), Morikawa (2006) - Bacteriochlorophyll absorption spectrum in stem cells was determined by spectrophotometric method at 400 - 900 nm on Novaspec II spectrophotometer or UV-1650PC spectrometer - Determination of Gram of bacteria according to the method of Smith and Hussey (2005) - Antagonism of PPB strains was performed according to the description of Nguyen Lan Dung et al (1981) - Evaluation of the ability to degrade petroleum hydrocarbons by biofilm formed by selected PPB by means of mass spectrometry (GCMS), high pressure liquid chromatography (HPLC) 2.3.3 Molecular methods - The steps of total DNA extraction were conducted as described in the study of Ramana et al (2006) - PCR method with specific primers for 16S rRNA fragment was described as the method of Hillmer and Gest (1977) - The nucleotide sequence of the PCR product was determined by the ABI automatic DNA sequencer (USA) and analyzed using Bioedit, Clustal X and Mega4 bioinformatics software - DNA electrophoresis on agarose gel was carried out according to the method of Sambrook and Russel (2001) 2.4 Chemical analysis method: - The aromatic hydrocarbon components present in the bacterial culture were analyzed by high pressure liquid chromatography (HPLC) 12 - The total content of diesel oil is determined by the standard TCVN 4582-88: 100 ml 2.5 Statistical analysis The data were processed by biostatistical methods Rely on Excel software to calculate mean, standard deviation and plot Each experiment was repeated times to calculate the sample mean The recommended level of statistical significance is p < 0.05 Place to conduct the experiments: Environmental Biotechnology Department, Institute of Biotechnology, VAST CHAPTER RESULTS AND DISCUSSION 3.1 Isolation and selection biofilm forming and hydrocarbon degrading photosynthetic purple bacteria 3.1.1 Isolation photosynthetic purple bacteria from oil polluted water and sediment samples Fifteen PPB strains with different colors, sizes and shapes and very diverse were isolated from water and sediment samples collected from coastal zones in Vietnam Their colony colors are mainly pale red burgundy to dark red, light brown to dark brown, light yellow to dark yellow 3.1.2 Selection biofilm forming and hydrocarbon utilizing photosynthetic purple bacteria 3.1.2.1 Growth and biofilm formation of the isolates Fifteen isolated PPB strains were purified and combined with 17 PPB strains taken from the collection of Environmental Biotechnology Department, Institute of Biotechnology, VAST to investigate their growth and biofilm formation As the results, all 32 strains well grew on DSMZ27 and 10 of them were the best growth such as FO2, DQ52, DQ41, DQ42, DD3, DD4, DQ52, DQ82, FO1, DQ81 (∆OD800>1) 13 In parallel with the experiment to determine the growth ability of 32 strains of PPB, their biofilm formation capacity was conducted As the results, 32 strains of PPB were compared with positive control (strain P23), showing that 10 strains FO2, DQ52, DQ41, DQ42, DD3, DD4, DQ52, DQ82, FO1, DQ81 formed biofilm better than strain P23 The positive control had an OD590 value of 17.6 (Figures 3.3 and 3.4, page 50 full text) In which, four strains DQ41, DQ51, DD4 and DQ82 have strong ability to form biofilm, OD590 value from 20.6 to 22.4 and continue to increase even after days of culture Therefore, these 10 strains were selected for further studies 3.1.2.2 Utilizing hydrocarbon capacity of the selections The cultivations were supplemented with diesel, naphthalene, phenol, pyrene and toluene and the results were presented in Table 3.3 Table 3.3 Growth of selected PPB on different substrates Substrates Concentration The well growth strains (ppm) Toluene 200 DQ41, DD4, DQ52 FO2 250 DQ41, DD4 FO2 Phenol 150 DQ41, FO1, FO2, DD3 DD4 300 DQ41, FO2 DD4 Naphthalene 200 DQ41, DQ52, DQ81 DD4 250 DQ52, DQ81 DD4 Pyrene 200 DQ41, DQ51, FO2, DD3 DD4 250 DQ41, FO2 DD4 Dầu diesel 4-8% DQ41, DD4, FO2 Three strains including DQ41, DD4, FO2 were selected because they have both biofilm forming and hydrocarbon utilizing capacity Therefore, they were used for further investigation 3.2 Biological characteristics and identification selected PPB 3.2.1 Morphology characteristics 14 Gram staining results showed that all three strains of VKTQH are Gram (-) The colonies of strain DD4 are round, tiny, light red in color Its cells are rod-shaped, twisted, wrinkled, unicellular, about 700 x 2210 nm in size The colonies of strain DQ41 are also round, convex, glossy, non-nucleated, with an inner margin, dark red, 1.5 mm in diameter Its cells are rod-shaped, wrinkled, unicellular, about 540 x 1320 nm in size Colonies of strain FO2 are round, smooth, small and light red in color and covered with an opaque border, less than 0.5 mm in diameter Its cells are rod-shaped, slightly wrinkled, unicellular, about 515 x 1160 nm in size Observation under the electron microscope showed that all these strains were rod-shaped, with dimensions of 500 × 1100 ÷ 700 × 2200 nm Notably, strain FO2 has budding mode of reproduction Thus, the selected three strains have morphological characteristics similar to those of the genus Rhodopseudomonas 3.2.2 16S rRNA and identification of DQ41, DD4 và FO2 All three strains of PPB DD4, DQ41 and FO2 have 16S rRNA gene sequences that have the highest similarity to R palustris (>99%) and this similarity is lower than that of R faecalis (>98 %) Based on the morphological and sequence characteristics of the gene encoding 16S rRNA, the three selected strains were classified with high sequence homology and were named as Rhodopseudomonas sp DD4, Rhodopseudomonas sp DQ41 and Rhodopseudomonas sp FO2 (Figure 3.16) The 16S rRNA gene sequences of strains FO2, DQ41, DD4 have been registered in the Gene bank with codes LC318127, LC318128 and LC318129, respectively 15 Figure 3.16 Phylogenetic tree of strains DD4, DQ41, FO2 3.2.3 Biological characteristics 3.2.3.1 Characteristics of photosynthetic pigments The bacteriochlorophyll of all three strains have maxima in the region of 800 - 890 nm that are typical for bacteriochlorophyll a as described in the previous study Carotenoids in PPB are also very diverse, specific to each species and also different from this group of pigments in algae and higher plants in structure Strain DD4 has peaks at 591 nm, strain DQ41 has peaks at 406, 445, 464, 492, 591 nm, strain FO2 has peaks at 460, 491, 591 nm These absorption 16 peaks are consistent with the absorption spectrum of the spirilloxanthin family According to Plennig and Trueper (1992), the ability to synthesize pigments in PPB cells greatly depends on the growing conditions Species in the genus Rhodopseudomonas have the ability to synthesize pigments when grown under aerobic, dark conditions To determine the ability to synthesize pigments of selected strains, we cultured them on a petri dish containing DSMZ 27 medium supplemented with 2% agar and cultured under anaerobic, light and aerobic, and dark conditions The results of determining the influence of oxygen and light on the ability to synthesize pigments of the selected strains showed that they were all grown under aerobic and dark conditions, but the ability to synthesize pigments was partially inhibited Colony color becomes pale pink or discolored (Figure 3.18 page 61 full text) The obtained results confirmed that the three strains selected to synthesize photosynthetic pigments when exposed to light Although they can grow under aerobic and dark conditions, their ability to synthesize pigments is inhibited This is also one of the characteristic features of the genus Rhodopseudomonas 3.2.3.2 Utilizing replacing carbon sources by DQ41, DD4 and FO2 Comparing the ability to use some carbon sources of three strains DQ41, DD4 and FO2 with representatives of Rhodopseudomonas sp., the results in Table 3.4 page 62 full text show that all three strains DQ41, DD4 and FO2 are capable of using some carbon sources such as formate, acetate, succinate, citrate, benzoate, glycerol and malate They are not capable of growing on media containing methanol and tartate The ability to use carbon sources 17 such as acetate, succinate, malate and glycerol of the studied bacterial strains is similar to that of the species of the genus Rhodopseudomonas Among the species in this genus, there are two species, R palustris and R rhenobacensis and some strains of the species R acidophila, which are able to use formate as a carbon source for growth However, R acidophila only grow well in acidic environment and R rhenobacensis is able to use tartate as a carbon source In particular, all three strains of PPB DD4, FO2 and DQ41 were able to grow strongly on the medium containing glycerol as a carbon source According to a number of published studies, among the species of the genus Rhodopseudomonas, only R palustris has the ability to use benzoate as the sole carbon source for anaerobic growth in the light Thus, according to carbon nutritional characteristics, all three strains of PPB in this study are closer to R palustris Many of the morphological, physiological and nutritional carbon characteristics of these strains were similar to those of R palustris 3.3 Effect of several bio-physical on the biofilm formation The biofilm formation capacity of DQ41, DD4 and FO2 was investigated under the influence of temperature, pH, and salt concentration at the thresholds of 15, 30, 37, 40, 45oC; pH ranges from to 9; the salt concentration ranges from to 4% The results show that all strains DQ41, DD4 and FO2 have good growth ability at the test temperature thresholds, reaching the optimum at about 30 - 37oC; pH = (notably, all strains have the ability to grow and form biofilm well in the pH range from to 9); all strains form biofilm at different concentrations of NaCl, among the three strains, two strains DD4 and FO2 form the best biofilm at 1.5 to 2% 18 NaCl concentration while strain DQ41 produced the best biofilm at concentrations NaCl 1% However, in order to find the general conditions for the strains of PPB, the concentration of 1-2% was used for further studies This result also leads to the application of these microorganisms in freshwater, brackish and salt water environments 3.4 Petroleum hydrocarbon degradation efficiency by biofilm formed by selected strains 3.4.1 Petroleum hydrocarbon degradation efficiency by single species biofilm without carriers The results presented in Table 3.5 (page 67 in the full thesis) showed that the ability to degrade toluene, naphthalene and pyrene compounds of the biofilms formed by these bacterial strains was very good In particular, the toluene analysis showed that selected strains were capable of completely degrading toluene at an initial concentration of 250 ppm The pyrene degradation efficiency of the strains was also very good, reaching over 99% The FO2 strain had the highest pyrene degradation capacity with an efficiency of up to 99.37% corresponding to an initial concentration of 300 ppm The naphthalene degradation efficiency by DQ41, DD4 and FO2 was 97.71, 97.23; 96.53% respectively (initial substrate concentration was 300 ppm) The phenol degradation efficiency by DQ41, DD4 and FO2 was 76.48, 78.35, 86.53%, respectively (initial substrate concentration was 300 ppm) 3.4.2 Petroleum hydrocarbon degradation single/mixture species biofilm with/without carriers 3.4.2.1 Antagonism of selected PPB strains efficiency by 19 The purpose of the investigation was to estimate petroleum hydrocarbon degradation efficiency by mixture species biofilm with/without carriers and compare with the single biofilms And the suitable carriers were suggested after these experiments To culture all strains together in a medium, they were examined the antagonism by crossed cultivation in agar plates The results indicated that all strains did not restrict each other and could combine to form mixture biofilm (Figure 3.22, thesis full text) 3.4.2.2 Estimation biofilm formation on different carriers The biofilm formed by selected strain on different carriers including coconut fiber (CF), polyurethane foam (PUF) and cinder beads (CB) was observed under SEM The results showed that the porosity of all the carriers was suitable for the immobilization of bacteria onto them (Figure 3.24, thesis full text page 72) In which, CB has many spongy structures where bacteria can invade; CF has a fibrous structure formed by parallel fibers; PUF is not as porous as other media, but according to Alessandrello et al (2017), PUF is a potential material to attach bacteria and be applied in petroleum hydrocarbon degradation The ideal properties of such media are nontoxic, inexpensive, and highly porous, as well as having a fibrous structure 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 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 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% 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 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 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 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