Tối ưu hóa điều kiện tách chiết và làm giàu axit béo không no omega6, 7, 9 từ sinh khối vi khuẩn tía quang hợp.

Tối ưu hóa điều kiện tách chiết và làm giàu axit béo không no omega6, 7, 9 từ sinh khối vi khuẩn tía quang hợp.Tối ưu hóa điều kiện tách chiết và làm giàu axit béo không no omega6, 7, 9 từ sinh khối vi khuẩn tía quang hợp.Tối ưu hóa điều kiện tách chiết và làm giàu axit béo không no omega6, 7, 9 từ sinh khối vi khuẩn tía quang hợp.Tối ưu hóa điều kiện tách chiết và làm giàu axit béo không no omega6, 7, 9 từ sinh khối vi khuẩn tía quang hợp.

BỘ GIÁO DỤC VIỆN HÀN LÂM KHOA HỌC VÀ ĐÀO TẠO VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Trần Thị Thu Quỳnh TỐI ƯU HÓA ĐIỀU KIỆN TÁCH CHIẾT VÀ LÀM GIÀU AXIT BÉO KHÔNG NO OMEGA-6, 7, TỪ SINH KHỐI VI KHUẨN TÍA QUANG HỢP LUẬN VĂN THẠC SĨ: SINH HỌC THỰC NGHIỆM Hà Nội - 2020 BỘ GIÁO DỤC VIỆN HÀN LÂM KHOA HỌC VÀ ĐÀO TẠO VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Trần Thị Thu Quỳnh TỐI ƯU HÓA ĐIỀU KIỆN TÁCH CHIẾT VÀ LÀM GIÀU AXIT BÉO KHÔNG NO OMEGA-6, 7, TỪ SINH KHỐI VI KHUẨN TÍA QUANG HỢP Chuyên ngành: Mã số: Sinh học thực nghiệm 8420114 LUẬN VĂN THẠC SĨ: SINH HỌC THỰC NGHIỆM NGƯỜI HƯỚNG DẪN KHOA HỌC: Cán hướng dẫn Cán hướng dẫn TS Hoàng Thị Yến GS TS Đặng Diễm Hồng Hà Nội - 2020 LỜI CAM ĐOAN Tôi xin cam đoan đề tài: “Tối ưu hóa điều kiện tách chiết làm giàu axit béo không no omega-6, 7, từ sinh khối vi khuẩn tía quang hợp” tơi trực tiếp thực hướng dẫn TS Hoàng Thị Yến GS TS Đặng Diễm Hồng Số liệu kết nghiên cứu luận văn hồn tồn xác, trung thực Mọi thông tin nội dung tham khảo báo cáo trích dẫn rõ ràng tên tác giả, tên cơng trình, thời gian, địa điểm nguồn gốc Tơi xin hồn tồn chịu trách nhiệm lời cam đoan này! Hà Nội, ngày tháng năm 2020 Học viên Trần Thị Thu Quỳnh LỜI CẢM ƠN Sau thời gian học tập nghiên cứu, để hoàn thành luận văn này, trước tiên em xin gửi lời cảm ơn chân thành sâu sắc tới TS Hoàng Thị Yến - cán Phịng Thí nghiệm trọng điểm Công nghệ gen GS TS Đặng Diễm Hồng - ngun trưởng Phịng Cơng nghệ tảo, Viện Cơng nghệ sinh học, Viện Hàn lâm khoa học Công nghệ Việt Nam, người thầy trực tiếp hướng dẫn, lên ý tưởng, định hướng nghiên cứu, tận tình bảo truyền đạt kinh nghiệm quý báu cho em suốt thời gian thực đề tài nghiên cứu Em xin bày tỏ lòng biết ơn chân thành đến Ths Lê Thị Thơm, Ths Lưu Thị Tâm - cán Phịng Cơng nghệ tảo, Viện Cơng nghệ sinh học, Viện Hàn lâm Khoa học Công nghệ Việt Nam hướng dẫn, nhiệt tình giúp đỡ em thí nghiệm gặp khó khăn truyền đạt cho em kinh nghiệm quý giá công tác nghiên cứu Sinh học Luận văn thực kinh phí Đề tài “Nghiên cứu quy trình cơng nghệ sản xuất omega 6, 7, từ vi khuẩn tía quang hợp ứng dụng cơng nghiệp thực phẩm dược phẩm” TS Hồng Thị Yến làm chủ nhiệm thuộc Đề án phát triển ứng dụng công nghệ sinh học lĩnh vực công nghiệp chế biến Bộ Công thương, năm 2017-2020 Em xin gửi lời cảm ơn tới Ban giám đốc, thầy, cô giáo thuộc Khoa Công nghệ sinh học Phòng Đào tạo, Quản lý Khoa học Hợp tác quốc tế Học viện Khoa học Công nghệ - Viện Hàn lâm Khoa học công nghệ Việt Nam tạo điều kiện, hướng dẫn, truyền đạt cho em nhiều kiến thức trình học tập Học viện Xin cảm ơn gia đình, bạn bè người thân động viên, hậu phương vững giúp em có động lực học tập Em xin chân thành cảm ơn! Học viên Trần Thị Thu Quỳnh DANH MỤC CHỮ VIẾT TẮT Từ viết tắt Tên đầy đủ Tên tiếng Việt VKTQH Vi khuẩn tía quang hợp Axit béo không bão hào MUFA Monounsaturated fatty acid PUFA Polyunsaturated fatty acid SFA Saturated fatty acid Axit béo bão hịa UFA Unsaturated fatty acid Axit béo khơng bão hòa OD Optical density Mật độ quang TFA Total fatty acid Axit béo tổng số nối đôi Axit béo khơng bão hịa đa nối đơi Sinh khối khơ SKK ω Omega Omega HUFA Highly unsaturated fatty acid Axit béo khơng bão hịa cao EPA Eicosapentaenoic acid Eicosapentaenoic acid DHA Docosahexaenoic acid Docosahexaenoic acid DPA Docosapetaenoic acid Docosapetaenoic acid ARA Arachidonic acid Arachidonic acid ACP Acyl carrier protein Protein mang acyl DANH MỤC BẢNG Trang Bảng 2.1 Phương pháp xác định hàm lượng kim loại nặng mẫu dầu sinh học 36 Bảng 2.2 Phương pháp xác định tiêu vi sinh vật có dầu sinh học 37 Bảng 3.1 Hàm lượng sinh khối khô, lipid omega-3, 6, 7, VKTQH nuôi bể quang sinh thể tích 1m3 38 Bảng 3.2 Thành phần axit béo hỗn hợp MUFAs PUFAs với tỷ lệ TFA: urê khác 46 Bảng 3.3 Hiệu suất thu hồi MUFAs, PUFAs số iot mẫu thu sau lần tạo phức với urê 50 Bảng 3.4 Phân tích tiêu cảm quan, hóa lý mẫu dầu sinh học omega-6, 7, 54 Bảng 3.5 Kết phân tích hàm lượng omega-6, 7, 55 Bảng 3.6 Kết phân tích dư lượng urê, kim loại nặng dầu sinh học giàu axit béo omega-6, 7, 56 Bảng 3.7 Kết phân tích tiêu vi sinh vật 56 DANH MỤC HÌNH Trang Hình 1.1 Tổng hợp axit béo 17 Hình 1.2 Phản ứng ester hóa dầu mỡ 21 Hình 3.1 Ảnh hưởng nhiệt độ lên hiệu suất tách chiết TFA từ sinh khối VKTQH 40 Hình 3.2 Ảnh hưởng chất xúc tác lên hiệu suất tách chiết TFA từ sinh khối VKTQH 41 Hình 3.3 Ảnh hưởng tỷ lệ sinh khối/ dung môi lên hiệu suất tách chiết TFA từ sinh khối VKTQH 42 Hình 3.4 Ảnh hưởng thời gian lên hiệu suất tách chiết TFA từ sinh khối VKTQH 43 Hình 3.5 Ảnh hưởng điều kiện khuấy trộn lên hiệu suất tách chiết TFA từ sinh khối VKTQH 43 Hình 3.6 Hiệu suất tách chiết SFAs (A) MUFAs, PUFAs (B) tỷ lệ TFA: urê khác 45 Hình 3.7 Hiệu suất tách chiết SFAs (A) MUFAs, PUFAs (B) tỷ lệ TFA: urê : methanol khác 47 Hình 3.8 Hiệu suất tách SFAs (A) MUFAs, PUFAs (B) nhiệt độ khác 49 Hình 3.9 Sơ đồ quy trình tách chiết làm giàu axit béo không no omega-6, 7, từ sinh khối VKTQH 51 Hình 3.10 Hình ảnh minh họa trình làm giàu hỗn hợp axit béo omega-6, 7, phương pháp tạo phức urê 53 MỤC LỤC Trang LỜI CAM ĐOAN LỜI CẢM ƠN DANH MỤC CHỮ VIẾT TẮT DANH MỤC BẢNG DANH MỤC HÌNH MỤC LỤC MỞ ĐẦU CHƯƠNG TỔNG QUAN TÀI LIỆU 1.1 TỔNG QUAN VỀ LIPID, AXIT BÉO KHÔNG NO MỘT NỐI ĐÔI (MUFAs) VÀ ĐA NỐI ĐÔI (PUFAs) (DẠNG OMEGA-6, 7, 9) 1.1.1 Giới thiệu lipid 1.1.2 Giới thiệu MUFAs PUFAs (dạng omega-6, 7, 9) 1.1.3 Các nguồn cung cấp omega-6, 7, 9 1.2 TỔNG QUAN VỀ VI KHUẨN TÍA QUANG HỢP (VKTQH) 13 1.2.1 Định nghĩa 13 1.2.2 Sinh thái học VKTQH 14 1.2.3 Ứng dụng VKTQH 14 1.2.4 Khả sinh tổng hợp axit béo không no VKTQH 17 1.3 CÁC PHƯƠNG PHÁP TÁCH CHIẾT VÀ TINH SẠCH DẦU 19 1.3.1 Các phương pháp tách chiết dầu 19 1.3.2 Các phương pháp làm giàu 23 1.4 TÌNH HÌNH NGHIÊN CỨU OMEGA-6, 7, TRÊN THẾ GIỚI VÀ Ở VIỆT NAM 25 1.4.1 Tình hình nghiên cứu giới 25 1.4.2 Tình hình nghiên cứu Việt nam 27 CHƯƠNG NGUYÊN VẬT LIỆU VÀ PHƯƠNG PHÁP NGHIÊN CỨU 29 2.1 ĐỊA ĐIỂM NGHIÊN CỨU 29 2.2 ĐỐI TƯỢNG VÀ VẬT LIỆU NGHIÊN CỨU 29 2.2.1 Đối tượng nghiên cứu 29 2.2.2 Vật liệu nghiên cứu 29 2.3 PHƯƠNG PHÁP NGHIÊN CỨU 30 2.3.1 Phương pháp chuyển vị ester trực tiếp từ sinh khối để tạo hỗn hợp axit béo dạng methyl ester 30 2.3.2 Tối ưu thông số trình tách chiết TFA 31 2.3.3 Làm giàu hỗn hợp axit béo omega-6, 7, dầu vi khuẩn tía phương pháp tạo phức với ure 32 2.3.4 Phương pháp phân tích thành phần axit béo dầu 33 2.3.5 Phương pháp xác định trạng thái cảm quan 34 2.3.6 Phương pháp xác định số axit 34 2.3.7 Phương pháp xác định số iot 35 2.3.8 Phương pháp xác định hàm lượng urê 35 2.3.9 Phương pháp xác định hàm lượng kim loại nặng dầu sinh học 36 2.3.10 Phương pháp xác định vi sinh vật dầu sinh học 37 2.3.11 Phương pháp xử lý thống kê 37 CHƯƠNG KẾT QUẢ VÀ THẢO LUẬN 38 3.1 HÀM LƯỢNG SINH KHỐI KHÔ, LIPID VÀ THÀNH PHẦN AXIT BÉO (OMEGA-6, 7, 9) CỦA SINH KHỐI HỖN HỢP CHỦNG VKTQH SẢN XUẤT TRONG BỂ QUANG SINH THỂ TÍCH 1M3 38 3.2 TỐI ƯU ĐIỀU KIỆN PHẢN ỨNG TÁCH CHIẾT TFA TỪ SINH KHỐI KHÔ VKTQH 39 3.2.1 Kết xác định ảnh hưởng nhiệt độ phản ứng 39 3.2.2 Kết xác định ảnh hưởng chất xúc tác 40 3.2.3 Kết xác định ảnh hưởng tỷ lệ nguyên liệu/ dung mơi (khối lượng/ thể tích) 41 3.2.4 Kết xác định ảnh hưởng thời gian phản ứng 42 3.2.5 Kết xác định ảnh hưởng điều kiện khuấy trộn 43 3.3 TỐI ƯU ĐIỀU KIỆN LÀM GIÀU OMEGA-6, 7, TỪ HỖN HỢP AXIT BÉO TỔNG SỐ THU ĐƯỢC BẰNG PHƯƠNG PHÁP TẠO PHỨC VỚI URÊ 44 3.3.1 Kết xác định ảnh hưởng tỷ lệ hỗn hợp TFA: urê trình làm giàu hỗn hợp axit béo 44 3.3.2 Kết xác định ảnh hưởng tỷ lệ TFA: urê: methanol trình làm giàu hỗn hợp axit béo 47 3.3.3 Kết xác định ảnh hưởng nhiệt độ kết tinh trình làm giàu hỗn hợp axit béo 48 3.3.4 Kết nghiên cứu tăng hiệu suất thu hồi omega-6, 7, việc tạo phức lần với urê 49 3.4 QUY TRÌNH TÁCH CHIẾT VÀ LÀM GIÀU AXIT BÉO KHÔNG NO OMEGA-6, 7, TỪ SINH KHỐI VKTQH 50 3.5 KẾT QUẢ KIỂM TRA CHẤT LƯỢNG DẦU SINH HỌC OMEGA6, 7, TÁCH CHIẾT ĐƯỢC SAU QUÁ TRÌNH LÀM GIÀU 54 3.5.1 Kết xác định tiêu cảm quan, hóa lý 54 3.5.2 Kết xác định thành phần axit béo 55 3.5.3 Kết xác định dư lượng urê, kim loại nặng 55 3.5.4 Kết tiêu vi sinh vật có dầu sinh học omega-6, 7, 56 CHƯƠNG KẾT LUẬN VÀ KIẾN NGHỊ 58 KẾT LUẬN 58 KIẾN NGHỊ 55 TÀI LIỆU THAM KHẢO 56 PHỤ LỤC PHỤ LỤC PHỤ LỤC CƠNG TRÌNH CƠNG BỐ LIÊN QUAN ĐẾN LUẬN VĂN Clean Technologies and Environmental Policy https://doi.org/10.1007/s10098-020-01966-0 Sustainable cultivation via waste soybean extract for higher vaccenic acid production by purple non‑sulfur bacteria ‑ Received: 11 July 2020 / Accepted: August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 The biomass production of Rhodovulum sulfidophilum HPB.6 was optimized via response surface methodology (RSM), and the optimal medium components such as waste soybean extract, yeast extract, and Mg2+ were determined using “one-singlefactor-at-one-time” approach RSM used a three-factor and central composite rotatable design consisting of 21 experimental runs conducted to optimize the final medium components The optimized conditions were as follows: 2.723 g/L waste soybean extract, g/L yeast extract, and 22 mg/L Mg2+ Under optimized conditions of Rhodovulum sulfidophilum HPB.6, the biomass production was 4.665 ± 0.326 g/L, which was 5.7-folds higher than that under non-optimized conditions Besides that, the total lipid production was 5.7 times higher corresponding to the increase in biomass productivity In addition, there was a change in total fatty acid composition with omega and omega which increased from 55.4 to 62.21 and from 3.4 to 9.41, respectively, while omega decreased from 9.79 to 4.54 and omega could not be detected This exploration of waste soybean under optimized conditions would be a significant impact for the higher biomass production from Rhodovulum sulfidophilum HPB.6 Graphic abstract Extended author information available on the last page of the article 13 T.Y Hoàng et al Keywords Rhodovulum sulfidophilum · Vaccenic acid · Validated model · Biomass production · Waste soybean extract Purple non-sulfur bacteria (PNSB) are a physiological group of bacteria distributed among either alpha or beta-proteobacteria that can carry out photosynthesis in the absence of oxygen production (Imhoff 1989) PNSB are the most diverse and most useful group of bacteria for various biotechnological applications like single-cell protein (SCP), bioactive compounds, and wastewater treatment and especially for functional food formation compounds (Sasikala and Ramana 1995) Functional food rich with unsaturated fatty acids (e.g., omega 3, 6, 7, 9) is beneficial to human health It is proven to be proficient of antioxidant, anti-inflammatory, and immune system modulator, and strengthens the cardiovascular system and mucous membrane tissue regenerator (Innes and Calder 2020; Koyande et al 2019) Many studies have positively correlated essential fatty acids with reduction of cardiovascular morbidity and mortality, infant development, cancer prevention, optimal brain and vision functioning, arthritis, hypertension, diabetes mellitus, and neurological/neuropsychiatric disorders (Kaur et al 2014; Kim et al 2013) PNSB are particularly rich in omega fatty acids also known as vaccenic acid (65–82% of total fatty acids) which has a crucial role in maintaining the health of skin and mucous membranes (Djoussé et al 2012) PNSB phototropic has been explored mainly for the production of bioenergy such as hydrogen and polyhydroxybutyrate (PHBT), along with wastewater treatment (Khatipov et al 1998; Kim et al 2006; Wu et al 2012) Previous study by Kim et al (2013) has shown the feasibility for microbial fatty acid production of photosynthetic bacteria, Rhodobacter sphaeroides KD131 cultivated in a continuous-flow, membrane-coupled bioreactor with lactate as carbon source with a cell productivity of 1.9 g dcw/L/d and fatty acid productivity of 665 mg FA/L/d, respectively The fatty acid produced was around 35% dcw and mainly consisted of vaccenic acid (Kim et al 2013) However, a previous study utilizes synthetic cultivation media which is not very economically viable The bacteria biomass production study can be performed via one-factor-at-a-time approach (OFAT) and response surface methodology (RSM) (Irfan et al 2014; Kong et al 2004) OFAT, also known as a single factor experiment, is a classical method in which only one factor is variable at one time while all others are kept constant This approach has several drawbacks: (i) it is time-consuming; (ii) there is an inability to evaluate the interaction between the variables; (iii) it is costly; and (iv) it is less effective than other methods (Khoo et al 2020; Torres-Acosta et al 2019) RSM is a statistical method which uses the data from experiments to 13 build and to solve multi-variable equations This approach overcomes the disadvantages of the OFAT method and has been applied in different kinds of research including microbiological biomass production In this study, we report the omega fatty acid production by Rhodovulum sulfidophilum HPB.6 in a culture medium with waste soybean extract as substrate Based on our previous study, waste soybean extract medium was selected to replace over synthetic cultivation medium, which resulted in rapid biomass growth and highest lipid accumulation specifically unsaturated fatty acid (i.e., omega 6, 7, 9) production by Rhodovulum sulfidophilum HPB.6 (Hoang et al 2019) In order to utilize Rhodovulum sulfidophilum HPB.6 for high biomass production, extraction of unsaturated fatty acid and other biotechnology applications, this study focused on the selection of suitable medium components via OFAT for the biomass production and the selected medium composition was further optimized using RSM to achieve the highest biomass production of Rhodovulum sulfidophilum HPB.6 for various biotechnology applications Rhodovulum sulfidophilum HPB.6 used throughout this study was isolated from the coastal area of Haiphong, Vietnam and identified as Rhodovulum sulfìdophilum by using morphological and physiological properties and 16S rRNA gene sequence analysis (Hoang et al 2019) It was maintained in DSMZ-27 medium at °C and lyophilized and kept at the Key Laboratory of Gene Technology, Biotechnology Institute, Vietnam Academy of Science and Technology (VAST) The DSMZ-27 medium (pH 7.0) was used for the cultivation of the bacterium DSMZ-27 medium composed the following components per liter of distilled water: 0.3 g yeast extract; 0.5 mL ethanol; g succinate; 0.5 g acetate; mL ferric citrate from 0.1% (w/v) stock; 0.5 g KH 2PO4; 0.4 g MgSO4·7H2O; 0.05 g CaCl2·2H2O; 0.4 g NH4Cl; 25 g NaCl; trace element solution SL6; and mL vitamin B 12 solution (filter sterilized) Trace element solution SL6 contains (l−1) 1.8 g FeCl2·4H2O; 0.25 g CoCl2·6H2O; 0.01 g NiCl·6H2O; 0.01 g CuCl2·5H2O, 0.07 g MnCl2·4H2O; 0.1 g ZnCl2; 0.5 g H3BO3; 0.01 g Na2SiO3·5H2O; 0.03 g Na2MoO4·2H2O Vitamin B12 solution was added after autoclaving stock solution Sustainable cultivation via waste soybean extract for higher vaccenic acid production by purple… and was used as Basal medium The stock solution was prepared by putting 10 g of waste soybean to cloth and tightly sealed, then the stock solution is put into 100 mL distilled water, sterilized at 121 °C for 30 The bacterium was cultivated in soybean extract concentration ranging from to g/L and added 25 g/L NaCl to evaluate the effect of stock solution concentration on the growth of Rhodovulum sulfidophilum HPB.6 The optimal concentration stock solution was added with carbon and nitrogen sources separately such as malic acid, acetate, yeast extract, and glutamate (2 g/L) to assess the effect of different carbon and nitrogen sources The yeast extract concentration (range from to g/L) and Mg2+ (8–22 mg/L) were examined by using OFAT to obtain the highest biomass production For each experiment, the culture was grown in a 13-mL penicillin tube composed of 10 mL of stock solution medium, along with the indicated sources and inoculated bacteria at an exponential phase with an initial concentration of 160 mg/L (Hoang et al 2019) Bacterium and culture medium were mixed uniformly by a shaker Response surface methodology (RSM) was performed using a three-factor and central composite rotatable design (CCRD) consisting of 21 experimental runs with (2 3) factorial points, (2 × 3) axial points (i.e., two axial points on the axis of each design variable at a distance of 1.68 from the design center, (23)1/3), and replicates at the center points, maximal and minimal factorial points The design variables were the waste soybean extract concentration (g/L; X1), the yeast extract concentration (g/L; X2), and the Mg2+ concentration (mg/L; X3) Each variable was coded at five levels − 1.68, − 1, 0, 1, and 1.68 The conversion of real values (Xi) to coded values (xi) was done by using the following equation xi = (Xi – X0)/ΔXi, where X0 is the real value of the independent variable i at the center point and ΔXi is the step change of Xi corresponding to a unit of variation In the present study, X0 and ΔXi were determined in the section of primary experiments The experimental data were fitted to the following second-order polynomial model: 3 , , , , Y= 0+ X+ X2 + XX ii i=1 ii i i=1 ij i j i=1 j=2 where Y is the response or dependant variable (∆OD800 of Rhodovulum sulfidophilum HPB.6 culture), β0, βi, βii, and βij are the regression coefficients for the intercept, linear, quadratic, and interactions terms, respectively, and Xi and Xj are the real values of variables For all runs, the biomass production was performed in a 13-mL penicillin tube containing 10 mL of medium and inoculated bacteria at an exponential phase with an initial concentration of 160 mg/L Bacterium and medium were mixed uniformly by a shaker After days of anaerobic incubation at a temperature within 30–32 °C and light intensity of lux (tungsten light bulbs), biomass production was estimated either by cell density at ∆OD800 or dried biomass (dcw) The optimal conditions for biomass production (∆OD800) were determined using the JMP 10 software The software was set to have the maximized desirability which was the highest biomass quantity Four experimental replicates were performed at the optimized conditions The experimental and predicted values were compared in order to validate the model The productivity of Rhodovulum sulfidophilum HPB.6 was determined by measuring the cell density at OD800 using UV–Vis spectrophotometer and by dry biomass quantity (g/L) (Cai et al 2012; Li et al 1995) Dry biomass (g/L) was determined as follows: after days of incubation, Rhodovulum sulfidophilum HPB.6 cells were harvested by the centrifugation method at 8000 rpm for 15 at °C The pellet was re-suspended in 10 mL distilled water and centrifuged again for washing The washed cells were then lyophilized until a constant weight is obtained The dry biomass was used to extract lipids and subjected to further analysis of fatty acid composition Determination of total lipid extraction Lipid extraction was performed using the modified methodology of Bligh and Dyer (1959) 100 g of dry biomass was mixed with 300 mL mixture of dichloromethane and methanol (2:1, v/v) was carried out for h integrated with ultrasonication for cell membrane disruption A further dilution was made with 100 mL of dichloromethane and 100 mL of water After the separation of the two layers, the upper aqueous layer containing methanol, water, and non-lipid compounds was discarded and the lower dichloromethane layer was filtered using a filter paper containing anhydrous sodium sulfate and collected in pre-weighed glass vials This procedure was repeated for the extraction of lipids remaining in the sample Both the organic phases containing the lipid extract was vacuum dried to remove the excess solvent until a constant weight was attained 13 T Y Hoàng et al Fatty acid analysis Fatty acid analysis of non-optimized biomass was conducted at Royal Research Laboratories, Secunderabad, India Biomass was methylated, separated, and identified according to the instructions for the Microbial Identification System (Sasser 1990) (Microbial ID; MIDI; version V 6.0-2007; RTSBA6 database; 6850 series II gas chromatograph, Olympus) Fatty acid analysis of the optimized biomass was determined by gas chromatography (GC) and a subsequent ISO draft standard method in Laboratory of Biochemistry, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology [Animal and Vegetable Fats and Oils—Preparation of Methyl Esters of Fatty Acids; Standard No 5509; ISO: Geneva, Switzerland, 1988] Approximately 10 mg of oil was dissolved in mL of petroleum ether, followed by the addition of 25 µL of M sodium methanolate methanol solution and the mixture was agitated vigorously for About 20 µL of water was added, and after centrifugation the aqueous phase was removed Then, 20 µL of methyl orange in 0.1 N HCl was added as a pH indicator The mixture was agitated carefully, and different derivatives were analyzed by a Hewlett–Packard Gas Chromatography Instrument Model 5890 Series II/5989 A80 equipped with a 0.25 mm ZB-1 fused-silica capillary column (30 m ì 0.25 àm i.d.; Phenomenex, Torrance, CA) The carrier gas is composed with helium at a flow rate of 1.0 mL/min The experimental results were analyzed using the SAS 9.1 software (SAS Institute, Cary, NC) In the primary experiments, the selection of suitable medium components was optimized using one-factor-at-a-time (OFAT) and the results Fig Effect of waste soybean extract concentration on the growth of HPB.6 13 were expressed as mean ± standard deviation One-way analysis of variance (ANOVA) and Duncan’s multiple range test were used to determine the differences among the means P-values (< 0.05) were considered to be significantly different In the RSM experiment, multiple linear regression analysis was performed using the software JMP 10 (SAS Institute, Cary, NC) Effect of soybean extract concentration The synthetic medium was used for isolation, studying the biological characteristics and microbial biomass production However, in considering the production at pilot scale, it is important to access an appropriate and cheap substrate to reduce the cost demanded Study conducted by He et al (2010) used purple non-sulfur bacteria (i.e., Rhodobacter sphaeroides) for the treatment of synthetic soybean wastewater with chemical oxygen demand concentration nearly 10,000 mg/L and achieved a recovery of g/L biomass in 96 h Our study showed that waste soybean extract was the best substrate compared to corn and rice extract for biomass production (Del Socorro et al 2013; Hoang et al 2019) This result indicated that waste soybean extract was a suitable substrate for PNSB in large-scale production In addition, we had analyzed the fatty acid composition when Rhodovulum sulfidophilum HPB.6 was grown in DSMZ-27 and waste soybean extract (2 g/L) The result showed that fatty acid composition was nearly similar (see SI-1 and SI-2) Therefore, waste soybean extract was chosen to study the effect of its concentration on biomass production Rhodovulum sulfidophilum HPB.6 strain was cultivated in soybean extract concentration ranging from to g/L (by diluted stock solution) and added 25 g/L NaCl The experiment was conducted as described in Sect 2.3 After cultivation for days, the results indicated that soybean extract concentration showed a significant effect on the growth of Rhodovulum sulfidophilum HPB.6 strain (P < 0.0001) Figure shows the effect of waste soybean extraction concentration on the growth of Rhodovulum sulfidophilum HDP.6 strain At g/L soybean extract, the biomass of HPB.6 strain revealed an absorbance of 1.207 On the other hand, with an increase in soybean extract concentration from to g/L, the biomass reaches about 111.4% and 116% compared to soybean extract concentration at and g/L, where the biomass was only 81.93% and 49.71%, respectively Therefore, soybean extract concentration of g/L was selected as the central Sustainable cultivation via waste soybean extract for higher vaccenic acid production by purple… value in the RMS experiment and was used in the subsequent parameter Effect of carbon and nitrogen sources PNSB can utilize various carbon and nitrogen sources for growth Malic acid (Ab Rahim et al 2020) acetate, yeast extract, and glutamate have been chosen as the most important components (Saejung and Thammaratana 2016) In order to achieve a high biomass production of Rhodovulum sulfidophilum HPB.6, the optimal concentration of waste soybean extract medium (3 g/L) was further added with some additives carbon and nitrogen sources such as malate (2 g/L), acetate (2 g/L), yeast extract (2 g/L), and glutamate (2 g/L) After days of incubation, the result was obtained, as shown in Fig The result showed that Rhodovulum sulfidophilum HPB.6 grew the best in soybean extract added with yeast extract In medium added with g/L of yeast extract and glutamate, the biomass increased to 170.24% and 151%, respectively, compared to the control experiment (soybean extract: g/L) In the waste soybean extract medium added with g/L acetate and malate, the absorbance increased from 121.03 to 135.24%, respectively Based on the result, the yeast extract was chosen as the optimal condition for the following parameters the medium containing g/L of soybean extract and yeast extract with concentration ranging from to g/L was investigated The experiment was conducted as an indication in the method After days incubation, the result in Fig showed a significant effect on the growth of HPB.6 strain (P < 0.0001) when yeast extract concentration was added in g/L soybean extract The absorbance increased from to with an increase in the yeast extract concentration and then remained constant At g/L of yeast extract, the biomass absorbance reaches about 1.986, increased by 161.84% compared to the control experiment (soybean extract: g/L) Therefore, the yeast extract concentration of g/L was selected for the next experiment Effect of Mg2+ concentration on the growth of HPB.6 Yeast extract is a common addition to media formulated for purple non-sulfur bacteria (Madigan and Jung 2009) It also stimulated the growth because of its assortment of organic compounds that can fuel photoheterotrophic growth Yeast extract is a source of B-vitamins, thiamine, nicotinic acid, biotin, and p-aminobenzoic acid as growth factor (Smith et al 1975; Stokes et al 1944) In this experiment, Wu et al (2014) investigated the enhancement of PNSB cell accumulation in soybean wastewater by implementing Mg2+ under the light anaerobic condition and the results showed that with Mg2+ at 10 mg/L, biomass production was improved by 70% and chemical oxygen demand removal reached 86% Therefore, in this experiment, Mg2+ concentration at a range within 5–25 mg/L was added into the optimized medium to evaluate the effect of Mg 2+ on the growth of HPB.6 strain The results shown in Fig indicated that an increase in Mg2+ concentration in medium played a significant role on the growth of Rhodovulum sulfidophilum HPB.6 strain (P < 0.0001) Indeed, the growth of biomass increased with an increase in the Mg2+ concentration from to 15 mg/L and then remained fairly constant At a concentration of 15 mg/L of Mg2+, the biomass reached an absorbance of 3.873 which significantly increase by 144.04% compared to the soybean extract without additional Mg2+ However, when the concentration of Mg2+ increased to 25 mg/L, the biomass decreased by 119.17% Based on this Fig Effect of carbon and nitrogen sources on the growth of HPB.6 Fig Effect of yeast extract concentration on the growth of HPB.6 Effect of YE concentration 13 T Y Hoàng et al result, the concentration of Mg2+ at 15 mg/L was chosen for the next parameter Biomass production from Rhodovulum sulfidophilum HPB.6 for the extraction of omega was further optimized through the RSM approach Based on the primary results, three factors namely soybean extract, yeast extract, and Mg2+ contents were considered as independent variables in the model Their ranges were determined in the primary experiments and are presented in Table The experimental design of a five-level, three-variable CCRD and the experimental results of the biomass (described through the optical density at OD800) are shown in Table By applying the multiple regression analysis, the relation between the tested independent variables and the response was explained by Eq 1, in which xi were the standardized or coded variables The equation of Rhodovulum sulfidophilum HPB.6 biomass production (Eq 2) was obtained by converting the coded values (xi) into real values (Xi) Fig Effect of Mg2+ concentration on the growth of HPB.6 Table Central value and variation of independent variable Independent variables Central values Variations Soybean extract (g/L) Yeast extract (g/L) Mg2+ (mg/L) 15 1 Table Rotatable central composite design setting in the coded form (x1, x2 and x3) and real values of the independent variables (X1, X2 and X3), and experimental results for the response variable (biomass production of Rhodovulum sulfidophilum HPB.6 described through the optical density ∆OD800) 13 Standard variables a00 −−− −−− −−− −−+ −+− −++ 0a0 00a 000 000 000 00A 0A0 +−− +−+ ++− +++ +++ +++ A00 ∆OD800 Real variables x1 x2 x3 Soybean extract Yeast extract Mg2+ − 1.68 −1 −1 −1 −1 −1 −1 0 0 0 1 1 1 1.681 −1 −1 −1 −1 1 − 1.681 0 0 1.681 −1 −1 1 1 0 −1 −1 −1 −1 − 1.681 0 1.681 −1 −1 1 1.318 2 2 2 3 3 3 4 4 4 4.681 1 1 3 0.318 2 2 3.681 1 3 3 15 8 22 22 15 3.227 15 15 15 26.772 15 22 22 22 22 15 4.494 4.545 4.204 4.202 4.280 5.342 5.778 4.348 5.379 5.412 5.379 5.546 5.194 5.754 5.158 4.899 4.707 5.502 5.599 5.648 5.047 Sustainable cultivation via waste soybean extract for higher vaccenic acid production by purple… Y = 5.445 + 0.128x1 + 0.389x2 + 0.066x3−0.286x1x2 + 0.027x1x3 + 0.2x2x3 − 0.239x2 − 0.14x2 − 0.057x2 (1) Y = 0.588 + 2.053X1 + 1.234X2 − 0.0146X3 − 0.286X1X2 + 0.0054X1X3 + 0.04X2X3 ± 0.239X2 − 0.14X2 − 0.00228X2 (2) To fit the response function and experimental data, the linear and quadratic effects of the independent variables, as well as their interactions in the response, were evaluated by analysis of variance (ANOVA), and regression coefficients are shown in Tables and The ANOVA of the regression model showed that the model was highly significant or useful due to a very low probability value (P < 0.0001) The fitness of the model was judged by the coefficient of determination (R2) In this study, the R2 value for the regression model of the biomass production was 0.9433, which was close to 1, suggesting that the predicted second-order polynomial model defined well the biomass production (∆OD800) and that 94.33% of variation for the biomass production was attributed to the three studied factors Besides, the lack of fit test is used to verify the adequacy of the model In our study, the absence of lack of fit (variation between average and predicted values at X) (P = 0.1401) meant that the total error Table Analysis of variance for the response surface quadratic model of biomass production Source DF Sum of squares Mean square F ratio Model Error Lack of fit Pure error C Total 11 20 5.4892806 0.3295647 0.2249000 0.1046647 5.8188452 0.609920 0.029960 0.044980 0.017444 20.3575 P < 0.0001* 2.5785 P = 0.1401 of the model was due to the pure error This strengthened the reliability of the model The effects of the concentration of soybean extract, yeast extract, and Mg2+ on the biomass production of Rhodovulum sulfidophilum HPB.6 are presented in Table and Fig As illustrated in Table 4, the soybean extract, yeast extract, and Mg2+ showed significant linear effects for the biomass production, respectively (P < 0.05) Biomass production (∆OD800) was mainly influenced by the soybean extract and yeast extract contents The soybean extract and yeast extract exerted significant individual and quadratic effects, respectively (P < 0.05) Mg2+, varying from to 22 mg/L, was not significant (P = 0.1534) The interactions between the three parameters soybean extract, yeast extract, and Mg2+ had a significant effect on the biomass production of Rhodovulum sulfidophilum HPB.6, except the interaction between soybean extract and Mg2+ contents (P = 0.6356) The negative quadratic coefficients of x1, x2, and x3 indicated that there was a maximum biomass production at a certain concentration of soybean extract, yeast extract, and Mg2+ The optimized medium composition for the HPB.6 biomass production was subjected using JMP10 software The software was set to search the optimum desirability for the response, meaning the maximum biomass production by Rhodovulum sulfidophilum HPB.6 By this way, the optimal medium composition was as follows: 2.723 g/L of soybean extract, g/L of yeast extract, and 22 mg/L of Mg2+, as shown in Fig In order to examine the validity of the model, biomass production (∆OD800) was completed with four replicates under the estimated optimum conditions generated by the software The measured values (5.852, 5.723, 5.754, and 5.801) were within a 95% mean confidence interval of the predicted value (5.698–6.146) The results confirmed the predictability of the model The second-order polynomial model can thus be effectively applied to predict the biomass production of Rhodovulum sulfidophilum HPB.6 *Indicate significant < 0.05 Table Parameter estimates of the predicted second-order model for the responses (biomass production of Rhodovulum sulfidophilum HPB.6) Term Intercept Soybean extract Yeast extract Mg2+ Soybean extract and yeast extract Soybean extract and Mg2+ Yeast extract and Mg2+ Soybean extract and Soybean extract Yeast extract and yeast extract Mg2+ and Mg2+ Estimate SE T ratio P > |t| 5.4458841 0.1286064 0.3893231 0.099725 0.043032 0.043032 54.61 2.99 9.05 < 0.0001* 0.0123* < 0.0001* 0.0659886 − 0.286474 0.027026 0.200776 − 0.239456 − 0.140285 − 0.057023 0.043032 0.055466 0.055466 0.055466 0.051231 0.051231 0.051231 1.53 − 5.16 0.49 3.62 − 4.67 − 2.74 − 1.11 0.1534 0.0003* 0.6356 0.0040* 0.0007* 0.0193* 0.2894 *Indicate significant < 0.05 13 T Y Hoàng et al Fig Response surface for the biomass production of Rhodovulum sulfidophilum HPB.6 in the function of the waste soybean extract, yeast extract, and Mg2+ Fig The concentrations of soybean extract (SE), yeast extract (YE), and Mg2+ for the maximal desirability After determining the optimal composition of the culture medium for biomass production in large scale, using the surface response model to determine the influence of factors on the ability to accumulate biomass Table compared the biomass production, lipid content, and fatty acid profile of Rhodovulum sulfidophilum HPB.6 growth in an optimized and non-optimized medium composition The composition of media for biomass production includes soybean extract: 2.723 g/L; yeast extract: g/L and Mg2+: 22 mg/L 13 Using this media to cultivate HPB.6 strain in a zip lock (1 L) and incubate at a temperature 30–32 °C, intensity illuminates at 4.000 lux After days of culturing (96 h), the biomass (DCW), lipid (w/v), and fatty acid (% total fatty acid) accumulation were determined and compared to control (before using RSM) According to Table 5, the results showed that after determining the composition and verifying via RSM approach to optimize the media composition, the biomass production of Rhodovulum sulfidophilum HPB.6 showed an increase of 5.7 times from 0.775 g/L in waste soybean extract medium to 4.665 g/L after using RSM The lipid content (% dcw) remains around 21.457 ± 2.272 and 20.324 ± 2.021, respectively, and the fatty acid (% of total Sustainable cultivation via waste soybean extract for higher vaccenic acid production by purple… Table Biomass production, lipid content, and fatty acid profile of Rhodovulum sulfidophilum HPB.6 grown in optimized and non-optimized medium composition Composition Rhodovulum sulfidophilum HPB.6 Non-optimized medium Dry biomass 0.775 ± 0.081 Lipid (% w/v) 21.457 ± 2.272 Fatty acid (% of total fatty acid) Omega 0.0 Omega 9.79 Omega 55.4 Omega 3.4 Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Optimized medium 4.665 ± 0.326 20.324 ± 2.021 1.04 4.54 62.21 9.41 fatty acid) had a little change in composition in which omega (mainly vaccenic acid) and omega increase (from 55.4 to 62.21 and from 3.4 to 9.41, respectively) and omega decreases (from 9.79 to 4.54) Here, we conclude that the response surface methodology has successfully optimized an optimal biomass production of Rhodovulum sulfidophilum HPB.6 The optimized parameter is composed of 2.723 g/L of waste soybean extract, g/L of yeast extract, and 22 mg/L of Mg2+ The optimized cultivation media to culture Rhodovulum sulfidophilum HPB.6 in a zip lock (1 L) at a temperature within 30–32 °C and light intensity of 4.000 lux, the biomass achieved an optimal growth of 4.665 ± 0.326 g/L, which was 5.7-fold higher than non-optimization medium The lipid content (% dcw) remains unchanged; however, the fatty acid (% of total fatty acid) had a slight reduction in composition where omega and omega increase from 55.4 to 62.21 and from 3.4 to 9.41, respectively, while omega decreases from 9.79 to 4.54 Nevertheless, the implementation of waste soybean extract as culture medium has shown the feasibility and reliability of this approach over synthetic culture medium for the enhancement, extraction of unsaturated fatty acid, and other biotechnology applications of Rhodovulum sulfidophilum HPB.6 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Thị Yến Hoàng hoangyen.ibt@gmail.com  Pau Loke Show showpauloke@gmail.com; PauLoke.Show@nottingham.edu.my Chyi-How Lay chlay0612@gmail.com Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Quỳnh Trần Thị Thu tranthithuquynh.k56@hus.edu.vn Faculty of Food Sciences and Technology, Vietnam National University of Agriculture, Gia Lam, Hanoi, Vietnam Tuyên Đỗ Thị dttuyen@ibt.ac.vn Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Bacterial Discovery Laboratory, Centre for Environment, IST, JNT University Hyderabad, Kukatpally, Hyderabad 500085, India Green Energy and Biotechnology Industry Development Research Center, Feng Chia University, Taichung City, Taiwan Kuan Shiong Khoo kuanshiong.khoo@hotmail.com Hà Lại Thị Ngọc hoangyen.ibt@gmail.com Hang Đinh Thị Thu dtthang@gust-edu.vast.vn Ha Chu Hoàng chuhoangha@ibt.ac.vn Sasikala Chinthalapati sasikala.ch@gmail.com 13 ... VKTQH làm giàu axit béo không no omega-6, 7, phương pháp tạo phức với urê  Nội dung nghiên cứu đề tài: - Tối ưu điều kiện phản ứng tách chiết TFA từ sinh khối VKTQH; - Tối ưu điều kiện làm giàu. .. Nội - 2020 LỜI CAM ĐOAN Tôi xin cam đoan đề tài: ? ?Tối ưu hóa điều kiện tách chiết làm giàu axit béo không no omega-6, 7, từ sinh khối vi khuẩn tía quang hợp” tơi trực tiếp thực hướng dẫn TS Hoàng... chiết TFA từ sinh khối VKTQH; làm giàu axit béo omega-6, 7, từ hỗn hợp TFA Để thu hiệu suất tách chiết axit béo không no omega-6, 7, cao cần tối ưu hiệu suất tách chiết giai đoạn Chính vậy, vi? ??c nghiên