Research on the growth and development of thermophilic fungi strains on distillery wastewater in alcohol production (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: RESEARCH ON THE GROWTH AND DEVELOPMENT OF THERMOPHILIC FUNGI STRAINS ON DISTILLERY WASTEWATER IN ALCOHOL PRODUCTION Student : Vu Thi Thu Trang Faculty : Biotechnology Supervisor : Nguyen Thanh Hao, PhD Ha Noi, 02/2021 COMMITMENT I hereby declare that all the information and data provided in this study are true, accurate, and not used in any other reports I assure all of the information in my study is referred to at the end of my thesis and I have already given all of my respect and appreciation to every person helping me complete my thesis Hanoi, January 2021 Student Vu Thi Thu Trang i ACKNOWLEDGEMENTS Firstly, I would like to express my gratitude to my supervisor PhD Nguyen Thanh Hao for providing me an opportunity to the final work in Vietnam National University of Agriculture and giving me all support, which made me complete the project Secondly, I owe my deep gratitude to Prof PhD Vu Nguyen Thanh, Institute of Food Industry enthusiastically instructed and imparted specialized knowledge to me, inspired me to research ideas and facilitated me to complete the thesis during the time I intern at the Center of Industrial Microbiology Finally, I would like to thank the brothers and sisters at the Center for Industrial Microbiology who have always enthusiastically guided, helped and created all the conditions for me to complete my work well during the experiment in the center It also gives my thankfulness to my family, to all of my friends, for sharing my difficulties, and giving me various used advice during the process of learning and studying Thank you very much! Hanoi, January 2021 Student Vu Thi Thu Trang ii CONTENT COMMITMENT i ACKNOWLEDGEMENTS ii CONTENT iii LIST OF TABLE iv LIST OF FIGURE v ABBREVIATION LIST vi ABSTRACT viii Part I INTRODUCTION 1 Subject Purposes: Requirements: Part II LITERATURE REVIEW 2.1 Overview of distillery wastewater 2.1.1 Bioethanol production and distillery wastewater 2.1.2 Characteristics and composition of distillery wastewater 2.1.3 Direction of application to improve the value of distillery wastewater 2.2 Thermophilic fungi strains 2.2.1 General characteristics of thermophilic fungi strains 2.2.2 Some thermophilic fungi strains Part III MATERIALS AND METHODS OF RESEARCH 13 3.1 Materials and research equipments 13 3.1.1 Materials 13 3.1.2 Equipment 13 3.1.3 Chemistry 14 3.1.4 Medium 15 3.1.5 Location and time: 15 3.2 Research methods 16 iii 3.2.1 Dry cassava fermentation and post-fermentation distillery wastewater treatment in the laboratory 16 3.2.2 Clean and store strains 16 3.2.3 Evaluate growth ability on thin stillage agar, dried distiller grains, liquid stillage: 17 3.2.4 Estimation of Reducing Sugars by the Dinitro Salicylic Acid (DNS) Method 19 3.2.5 Measure sweetness 20 Part IV: RESULTS AND DISCUSSION 22 4.1 Dry cassava fermentation and the treatment of distillery wastewater after fermentation in the laboratory 22 4.2 Evaluate the ability of growth and development of thermophilic fungi strains on dried distiller grains and thin stillage agar 25 4.3 Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage 36 4.3.1 Biomass average of thermophilic fungi species on liquid stillage 45 4.3.2 Brix (%) of thermophilic fungi species on liquid stillage 47 4.3.3 DNS of thermophilic fungi species on liquid stillage 48 4.3.5 pH of thermophilic fungi species on liquid stillage 49 Part V: CONCLUSIONS AND PROPOSALS 50 5.1 Conclusion 50 5.2 Proposals 50 REFERENCES 51 iv LIST OF TABLE Table 1.1 Wastewater generation in various operations Table 3.1: The instruments and equipment were used in the research 14 Table 3.2: Chemicals were used in the research 14 Table 4.1.1 Parameters of the post-fermentation solution 22 Table 4.1.2 Comparing samples of factory distillery wastewater and laboratory distillery wastewater samples 23 Table 4.2: Growth of thermophilic fungi strains on dried distiller grains and thin stillage agar 27 Table 4.3.1: Biomass, Bx, pH of thermophilic fungi strains and reducing sugar concentration on liquid stillage 44 Table 4.3.2 Biomass average of thermophilic fungi species on liquid stillage 45 v LIST OF FIGURE Figure 3.1: Distillery wastewater 13 Figure 3.2.3.1: Culture thermophilic fungi strains on thin stillage agar 17 Figure 3.2.3.2: Culture thermophilic fungi strains on dried distiller grains 18 Figure 3.2.3.3: Culture thermophilic fungi strains on liquid stillage 18 Figure 3.2.3.4: Dry biomass of some thermophilic fungi strains after drying 19 Figure 4.1.1 Processing of steps distillery wastewater after fermentation 23 Figure 4.1.2: Laboratory distillery wastewater 24 Figure 4.1.3: Laboratory dried distiller grains 24 Figure 4.1.4: Laboratory liquid stillage 24 Figure 4.2: Growth of thermophilic fungi strains on dried distiller grains and thin stillage agar 35 Figure 4.3.1: Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage after days of incubation 43 Figure 4.3.2 Biomass average of thermophilic fungi species on liquid stillage 46 Figure 4.3.3: Brix (%) of thermophilic fungi species on liquid stillage 47 Figure 4.3.4 DNS of thermophilic fungi species on liquid stillage 48 Figure 4.3.4 pH of thermophilic fungi species on liquid stillage 49 vi ABBREVIATION LIST PDA Potato dextrose agar DDS Distillers Dried Solubles DWG Distillers Wet Grains DDG Distillers Dried Grains DDGS Distillers dried grains with solubles DNS 3,5-Dinitrosalicylic acid Ppm Parts per million vii ABSTRACT The alcohol distilleries are growing extensively worldwide due to widespread industrial applications of alcohol such as in chemicals, pharmaceuticals, cosmetics, beverages, food and perfumery industry, etc The industrial production of ethanol by fermentation results in the discharge of large quantities of high-strength liquid wastes Distillery wastewater is one of the most polluted waste products to dispose of because of the low pH, high temperature, dark brown colour, high ash content and high percentage of dissolved organic and inorganic matter with high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) values One of the research directions currently of interest is the use of thermophilic fungi strains to increase the protein content in the distillery wastewater and reduce the organic matter content in the distillery wastewater Thereby, increasing the nutritional value of animal feed This study discusses screen the strain of thermophilic fungi strains is able to develop on the distillery wastewater for application in fermentation to create microbial biomass for livestock viii Part I INTRODUCTION Subject Natural resources are important to the development of every country in the world However, along with the development of the economy, social resources are facing the risk of exhaustion The application of bioethanol to replace fossil resources is getting more and more attention Bioethanol is mainly produced by fermentation technology Currently, bioethanol in Vietnam is often produced from raw materials for cassava chips and cassava roots, or corn The remaining product of the material after distillation is also known as distillery wastewater During the production of ethanol will produce a very large amount of wort with composition that varies depending on the quality of raw materials In addition, sanitary water and other residues in production also contribute to increased production waste With such a large amount, if not thoroughly handling the environmental consequences will be very serious The high nutritional value of corn residue products is often used for livestock, while cassava residue has low nutritional value and contains a lot of fiber, so the efficiency in livestock is not high A research direction that is currently interesting is to use the fungus strains capable of generating hydrolyzed enzymes to make use of residual nutrients to create protein-rich biomass for use in animal feed and reduce the waste after alcohol fermentation With the title "Research on the growth and development of thermophilic fungi strains on distillery wastewater in alcohol production", this thesis aims to screen the strain of fungi Thermophilic fungi strains is able to develop on the distillery wastewater for application in fermentation to create microbial biomass for livestock FCH116.3 FCH5.7 FCH20.1 LPH176 LPHT228 LPH180 38 LPH084 LPH156 LPH143 BMM933 BMM723 BMM313 39 BMM413 FCH130.2 FCH10.4 LPHT246 LPHT227 LPH158 40 LPHT234 LPH074 LPH067 LPH130 FCH10.5 FCH5.3 41 LPHT231 FCH5.4 FCH23.1 FCH102.1 FCH112.2 LPH172 42 LPH212 DC Figure 4.3.1: Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage after days of incubation 43 Table 4.3.1: Biomass, Bx, pH of thermophilic fungi strains and reducing sugar concentration on liquid stillage No 10 11 12 13 14 15 16 17 18 19 20 21 Symb ol strain s LPH 122 FCH 5.2 LPH 080 LPHT 224 LPH 131 LPH 123 FCH 116.3 FCH 5.7 FCH 20.1 LPH 176 LPHT 228 LPH 180 LPH 084 LPH 156 LPH 143 BMM 933 BMM 723 BMM 313 BMM 413 FCH 130.2 FCH 10.4 22 LPHT 246 23 LPHT 227 LPH 158 24 Species ( Latin name) Initial volum e Biomass(g/ L) Bx 0h Bx 96 h pH 0h PH 96 h DNS (mg/m L) 0h DNS (mg/m L) 96h 0,213 8,4 20,202 8,4 7,5 16,478 19,379 0,195 8,5 7,1 17,533 20,000 0,184 8,5 7,1 17,626 19,659 0,144 8,5 7,5 17,642 22,389 0,188 8,5 7,1 14,229 21,132 0,273 8,5 16,106 16,246 0,261 8,5 6,6 5,7 6,0 5,5 5,8 5,5 5,6 5,5 5,2 17,782 0,175 4,9 4,8 4,8 4,9 4,8 4,9 17,006 17,285 0,253 8,4 8,5 6,7 17,875 16,882 0,207 8,5 6,9 17,533 17,363 0,14 8,5 18,107 5,960 0,148 8,5 5,1 16,882 5,092 0,149 8,5 4,9 17,068 5,371 0,159 8,5 4,8 13,686 5,123 0,148 8,5 5,7 15,191 14,477 0,197 8,5 5,3 17,146 7,325 0,133 8,5 6,6 17,177 14,400 0,158 8,5 6,2 16,665 14,369 50,05 45,34 0,254 9,5 22,637 27,338 50,61 47,95 0,157 10, 10 5,1 5,2 5,0 5,0 5,0 5,0 5,4 4,7 4,8 4,7 4,4 5,1 16,696 0,274 29,215 47,53 0,101 10, 10 4,9 5,0 23,940 50,73 4,9 4,7 4,9 5,0 4,9 4,8 4,9 4,9 4,9 4,7 4,9 4,5 4,4 4,4 16,975 51,26 Mass after cultur e 46,54 46,40 47,81 47,04 46,64 47,51 47,34 47,14 46,68 46,63 46,60 46,50 46,16 46,21 45,91 45,58 46,45 47,40 51,26 23,630 27,276 49,65 46,26 0,241 9,6 0,282 4,9 5,1 22,823 45,84 4,4 4,4 24,794 50,09 10, 10, 23,754 20,171 50,09 50,42 50,58 Rhizomucor miehei 50,38 50,09 50,72 50,29 50,54 50,1 Rhizomucor pusillus 49,98 50,03 49,96 52,04 49,85 Rhizopus microsporus 50,28 50,36 51,02 51,24 Thermomyces lanuginosus Thermomyces dupontii Rasamsonia byssochlamydoi des Rasamsonia emersonii 44 9,9 8,8 4,8 4,9 25 26 27 28 29 30 31 32 33 34 35 36 37 38 LPHT 234 LPH 074 LPH 067 LPH 130 FCH 10.5 FCH 5.3 LPHT 231 FCH 5.4 FCH 23.1 FCH 102.1 FCH 112.2 LPH 172 LPH 212 Đối chứng Malbranchea cinnamomea Mycothermus thermophilus Myceliophthora fergusii Myceliophthora heterothallica Thielavia sp Thielavia sp 11 49,91 46,49 0,286 50,05 47,61 0,22 50,05 47,48 0,21 50,12 45,8 0,229 50,01 46,2 0,228 50,11 46,5 0,026 49,57 46,48 0,132 50,49 44,6 0,05 49,75 45,72 0,313 49,8 46,12 0,41 50,65 46 0,451 50,46 46,86 0,276 49,93 45,6 0,02 50,41 46,75 0,003 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, 8,1 8,2 9,6 9,7 9,6 10, 10, 10, 9,2 8,7 8,5 7,2 9,8 10, 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 4,4 5,1 5,0 5,1 23,677 23,428 24,421 19,410 23,118 21,427 5,3 24,235 24,871 5,1 4,5 4,5 23,428 22,948 23,987 23,754 22,684 24,747 4,6 5,4 7,1 7,5 5,0 4,9 4,5 25,569 20,869 23,909 25,818 24,608 21,893 22,032 20,248 23,894 22,296 22,979 17,580 24,313 23,506 4.3.1 Biomass average of thermophilic fungi species on liquid stillage Table 4.3.2 Biomass average of thermophilic fungi species on liquid stillage Thermophilic fungi species Rhizomucor miehei Rhizomucor pusillus Rhizopus microspores Thermomyces lanuginosus Thermomyces dupontii Rasamsonia byssochlamydoides Rasamsonia emersonii Malbranchea cinnamomea Mycothermus thermophiles Myceliophthora fergusii Myceliophthora heterothallica Thielavia sp Thielavia sp 11 Biomass average (g/L) 0.196 0.249 0.154 0.254 0.157 0.101 0.2447 0.228 0.079 0.005 0.391 0.276 0.002 45 0.391 0.254 0.249 0.24470.228 0.196 0.157 0.154 0.101 0.079 0.276 0.05 0.002 Figure 4.3.2 Biomass average of thermophilic fungi species on liquid stillage Of all the strains groups, Myceliophthora heterothallica was the strain group that produced the most dry biomass (0.391g) It was followed by the Thielavia sp (0.276g), Thermomyces lanuginosus (0.254g), Rhizomucor pusillus (0.249g), Rasamsonia emersonii (0.2447g), Malbranchea cinnamomea (0.228g), Rhizomucor miehei (0.196g), Rhizopus microspores (0.154g), Rasamsonia byssochlamydoides (0.101g), Mycothermus thermophiles (0.079), Myceliophthora fergusii (0.005)… In contrast, the strain group Thielavia sp 11 was the strain group that produced the least dry biomass (0.002g) Furthermore, in the Myceliophthora heterothallica strain group, FCH112.2 produced the most biomass (0.451g), the strain that produces the lowest biomass is FCH 23.1 (0.313g) Moreover, in the Rhizomucor miehei, FCH116.3 and LPH122 are two strains that produce more biomass than the other strains Besides, LPH158 (0.282g) and LPHT234 (0.286g) are the two species that produce the most biomass in the Rasamsonia emersonii strain group About Rhizomucor pusillus strain, LPH176 (0.274g) produced the driest biomass In Thielavia sp 11, LPH212 produces the least biomass 46 The biomass of the fungus strains can be very dependent on the biological characteristics and structure of the strains themselves 4.3.2 Brix (%) of thermophilic fungi species on liquid stillage Figure 4.3.3: Brix (%) of thermophilic fungi species on liquid stillage Brix degree (° Bx) after days of culture of the strains groups almost decreased, except for the Myceliophthora fergusii and Mycothermus thermophilus strains This can be assumed that those strains of mold used sugar to generate biomass, while strains Myceliophthora fergusii and Mycothermus thermophilus during growth could produce sugars 47 4.3.3 DNS of thermophilic fungi species on liquid stillage Figure 4.3.4 DNS of thermophilic fungi species on liquid stillage The strains group Rhizomucor miehei, Thermomyces lanuginosus, Thermomyces dupontii, Rasamsonia byssochlamydoides, Myceliophthora heterothallica are the strains that had reduced sugar concentration after days of culture In which strain of Rhizomucor miehei LPH123 had the highest concentration of reducing sugar (increased 6.9mg / L) Among the strains group with the remaining reduced sugar concentration, the Rhizopus microsporus strain group was the group with the strongest reduction in sugar concentration 48 4.3.5 pH of thermophilic fungi species on liquid stillage Figure 4.3.4 pH of thermophilic fungi species on liquid stillage After culture, most of the strain groups had pH increased However, strains of Rhizopus microsporus BMM933, Rhizopus microsporus BMM723 and Rhizopus microsporus 413 had a decrease in pH Myceliophthora thermophila strain group was the group of strains with the highest increase in pH (FCH102.1 increased from 4.46 to 7.12 and FCH112.2 increased from 4.45 to 7.53) Control samples, Rhizomucor pusillus LPHT228, Rhizopus microsporus LPH180, Rhizopus microsporus BMM 313, Mycothermus thermophilus FCH5.3, Mycothermus thermophilus LPHT231 were strains groups with slightly increased pH Followed by: 0.07, 0.06, 0.03, 0.03, 0.07, 0.04 49 Part V: CONCLUSIONS AND PROPOSALS 5.1 Conclusion Successfully fermented dry cassava Treatment of distillery wastewater after fermentation in laboratories obtain dried distiller grains, thin stillage agar, liquid stillage Surveyed 37 thermophilic fungi strains on different culture media: dried distiller grains, thin stillage agar, liquid stillage On both thin stillage agar and dried distiller grains, the Rhizomucor mieheinosus, Rhizomucor pusillus, Rhizopus microsporus, Rasamsonia byssochlamydoides, Rasamsonia emersonii are the strains group that grow well and produce the most dry biomass Beside, on liquid stillage, Myceliophthora heterothallica, Thielavia sp 8, Thermomyces lanuginosus, Rhizomucor pusillus, Rasamsonia emersonii produce the most dry biomass 5.2 Proposals Continue screening from strains that have developed well on the distillery wastewater Finally, selecting the best strains helps to increase the protein content in the distillery wastewater and reduce the organic matter content in the distillery wastewater 50 REFERENCES Chu-Ky S., T.-H.H Pham, K.-L.T.L.T Bui, T.-T.T.-H.H Nguyen, K.-D.D Pham, H.-D.T.D.T Nguyen, H.-N.N Luong, V.-P.P Tu, T.-T.T.-H.H Nguyen, P.-H.H Ho, T.-M.M Le, Simultaneous liquefaction, saccharification and fermentation at very high gravity of rice at pilot scale for potable ethanol production and distillers dried grains composition, Food Bioprod Process 98 (2016) 79–85 Canevascini, L M.-R Coudray, R J G Southgate and H Meier (1978), "Induction and catabolite repression of cellulase synthesis in the thermophilic fungus Sporotrichum thermophile" J Gen Microbiol, 110, pp 91-303 Cromwell, G L.; Herkelman, K L.; Stahly, T S Physical, Nchemical, and nutritional characteristics of distillers dried grains with solubles for chicks and pigs J Anim Sci 1993, 71, 679–686 Gilbert M, Breuil C, Yaguchi M, Saddler JN (1992), "Purification and characterization of a xylanase from the thermophilic ascomycete Thielavia terrestris 255B", Appl Biochem Biotechnol, pp 247-59 Gilbert M, Yaguchi M, Watson DC, Wong KK, Breuil C, Saddler JN (1993), “A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus", Appl Microbiol Biotechnol, 40, pp.508-14 Kuiper, L., Ekmekci, B., Hamelinck, C., Hettinga, W., Meyer, S., & Koop, K (2007) Bio-ethanol from cassava Ecofys Netherlands BV, 1-38 KeShun Liu, Chemical Composition of Distillers Grains and review, journal of Agricultural and food chemistry Kim, Y.; Mosier, N S.; Hendrickson, R.; Ezeji, T.; Blaschek, H Dien, B.; Cotta, M.; Dale, B.; Ladisch, M Composition of corn dry-grind ethanol byproducts: DDGS, wet cake, and thin stillage Bioresour Technol 2008, 995165– 5176 Liu., K S Effects of particle size distribution, compositional and color properties of ground corn on quality of distillers dried grains with solubles (DDGS) Bioresour Technol 2009, 100, 4433–4440 51 10 Rausch, K D.; Belyea, R L The future of co-products from corn processing Appl Biochem Biotechnol 2006, 128(4), 47–86 11 Spiehs, M J.; Whitney, M H.; Shurson, G C Nutrient database for distiller’s dried grains with solubles produced from new ethanol plants in Minnesota and South Dakota J Anim Sci 2002, 80(3), 2639– 2645 12 Thanh V.N., Thuy N.T., Huong H.T.T., et al (2019) Surveying of acidtolerant thermophilic lignocellulolytic fungi in Vietnam reveals surprisingly high genetic diversity Sci Rep, 9, 3674 13 Taranu I., T.-T Nguyen, P Dang, M Gras, G Pistol, D Marin, M.-C Rotar, M.Habeanu, P.-H Ho, M Thanh, T.T.-H Bui, D.-V Mai, C Son, Rice and Cassava Distillers Dried Grains in Vietnam: Nutritional Values and Effects of Their Dietary Inclusion on Blood Chemical Parameters and Immune Responses of Growing Pigs,Waste and Biomass Valorization (2018) 14 Tuohy MG, Puls J, Claeyssens M, Vrsanská M, Coughlan MP (1993), "The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans", Biochem J., 290, pp 515-523 15 Wang, H.; Wang, T.; Johnson, L A.; Pometto, A L Effect of the corn breaking method on oil distribution between stillage phases of dry-grind corn ethanol production J Agric Food Chem 2008, 56(5), 975–9980 16 Winkler, J K.; Rennick, K A.; Eller, F J.; Vaughn, S F Phytosterol and tocopherol components in extracts of corn distiller’s dried grain J Agric Food Chem 2007, 55, 6482–6486 17 R Belyea Fl, S Eckhoff, M Wallig & M Tumbleson, Variability in the nutritional quality of distillers solubles Bioresource Technol 94, 293–298 18 Luận văn thạc sĩ: Hàn T.T.H (2014) Phân lập nghiên cứu đặc tính nấm mốc chịu nhiệt có khả thủy phân Lignocellulose 52