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 do 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. 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. iii LIST OF TABLE. iv LIST OF FIGURE.
Overview of distillery wastewater. Bioethanol production and distillery wastewater. Characteristics and composition of distillery wastewater. Direction of application to improve the value of distillery wastewater.
Thermophilic fungi strains. General characteristics of thermophilic fungi strains. Some thermophilic fungi strains. MATERIALS AND METHODS OF RESEARCH.
Materials and research equipments. Location and time: .1 Dry cassava fermentation and post-fermentation distillery wastewater treatment in the laboratory .2 Clean and store strains .3 Evaluate growth ability on thin stillage agar, dried distiller grains, liquid stillage: .4 Estimation of Reducing Sugars by the Dinitro Salicylic Acid (DNS) Method. 20 Part IV: RESULTS AND DISCUSSION .1 Dry cassava fermentation and the treatment of distillery wastewater after fermentation in the laboratory .2 Evaluate the ability of growth and development of thermophilic fungi strains on dried distiller grains and thin stillage agar .3 Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage .1 Biomass average of thermophilic fungi species on liquid stillage.2 Brix (%) of thermophilic fungi species on liquid stillage .3 DNS of thermophilic fungi species on liquid stillage .5 pH of thermophilic fungi species on liquid stillage. 49 Part V: CONCLUSIONS AND PROPOSALS.
51 iv LIST OF TABLE Table 1. Wastewater generation in various operations.1: The instruments and equipment were used in the research .2: Chemicals were used in the research. Parameters of the post-fermentation solution. Comparing samples of factory distillery wastewater and laboratory distillery wastewater samples .2: Growth of thermophilic fungi strains on dried distiller grains and thin stillage agar .1: Biomass, Bx, pH of thermophilic fungi strains and reducing sugar concentration on liquid stillage.
Biomass average of thermophilic fungi species on liquid stillage. 45 v LIST OF FIGURE Figure 3.1: Culture thermophilic fungi strains on thin stillage agar .2: Culture thermophilic fungi strains on dried distiller grains .3: Culture thermophilic fungi strains on liquid stillage .4: Dry biomass of some thermophilic fungi strains after drying .1 Processing of steps distillery wastewater after fermentation.2: Laboratory distillery wastewater .3: Laboratory dried distiller grains .4: Laboratory liquid stillage.2: Growth of thermophilic fungi strains on dried distiller grains and thin stillage agar .1: Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage after 4 days of incubation. Biomass average of thermophilic fungi species on liquid stillage .3: Brix (%) of thermophilic fungi species on liquid stillage. DNS of thermophilic fungi species on liquid stillage.
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. 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.
Purposes: - Select the thermophilic fungi strains that grow best on the distillery wastewater. Requirements: - Dry cassava fermentation and post-fermentation distillery wastewater treatment in the laboratory. - Evaluate the ability of thermophilic fungi strains to grow at 45°C on thin stillage agar. - Evaluate the ability of thermophilic fungi strains to grow at 45°C on dried distiller grains.
- Evaluate the ability of thermophilic fungi strains to grow in the liquid stillage. Overview of distillery wastewater 2. Bioethanol production and distillery wastewater Materials for bioethanol production can be divided into 3 main categories: sugar-containing materials such as sugar beets, sugarcane, molasses; starch- containing materials such as corn, rice, cassava, wheat; Lignocellulose-rich materials such as straw and agricultural and forestry residues. The fermented ingredients are converted to ethanol and carbon dioxide, and the rest of the raw materials contain proteins, lipids, fiber, minerals and vitamins, which chemically change relatively little.
The bioethanol production process in Vietnam mainly consists of the following steps: Raw materials are cleaned and crushed using wet or dry crushing technology. After that, the gelatinized (liquefied) materials are made using the products of glycemic enzymes and liquefied with the dry matter content of about 20%. After that, the solution was cooled to add dry yeast, urea and fermented at 30°C for 3 - 5 days. Proceed to distillation to collect ethanol.
The raw ethanol is then anhydrous, the CO₂ generated can be recovered for dry ice production or cleaned for carbonated beverages. The liquid after distillation is the distillery wastewater, usually separated from the residue for livestock or as fertilizer. The fluid and wastewater are put into fermentation anaerobic tanks to collect biogas as fuel burning materials. Characteristics and composition of distillery wastewater Alcohol distilleries are highly water intensive units generating large volumes of high strength wastewater which pose a serious environmental concern.
The quantum and characteristics of wastewater generated at various stages in the manufacturing process is provided in table 1. Wastewater generation in various operations. Distillery operations Average wastewater Specific wastewater generation generation (L (kLD/distillery) wastewater/L alcohol Spent wash (from 491.9 distillation) Fermenter cleaning 98.2 During the ethanol production process, after distillation, two groups of by- products are formed: solid portion and soluble portion. The solid residue consists of elements from non-fermentable ingredients that can be used as animal feed in its original form (Distillers Wet Grains-DWG), or dried (Distillers Dried Grains-DDG ).
The clarified residue has a low dry matter content. For animal feed, this fermentation is concentrated and then dried to obtain a dry soluble distillation by-product (Distillers Dried Solubles-DDS). Dry liquor residue (DDGS) is a blend between two components of solid phase (DDG) and liquid phase (DDS), with different chemical composition and nutritional value depending on the source of raw materials and the mixing ratio, especially when examining physical properties [1]. Most of the ingredients used to make bio-alcohol are low in protein.
Since most of the starch has been converted to alcohol, the byproducts (DDGS) are quite high in crude protein compared to the original material. The source of dry wine residue from rice alcohol factories contains a very high protein content (over 70% of dry matter), while for cassava, the fiber content in the wine pulp is 4 obtained 15-30%[7]. DDGS has a 2 to 3 times increase in the remaining nutrient content of starch compared to pre-fermented cereals. Due to its high nutrient content of protein, amino acids, phosphorus and other nutrients, DDGS is used as feed or as a feed ingredient for livestock.
Normally, crude protein is from 23- 30%, fat 2.8%, neutral detergent fiber (NDF) from 28.3%, acid fiber (ADF) from 10. Among the minerals in DDGS, sodium is the metal with the largest variation from 0. DDGS does not provide many vitamins, trace minerals (thiamine, riboflavin and other vitamins) but contains many bioactive substances such as nucleotides, mannooligosaccharides, beta-1.6-glucan, inositol, glutamine and nucleic acids. These compounds help boost immunity and health for animals.
Amino acids: In the study of Spiehs et al. (2002), 119 DDGS samples were analyzed for 10 essential amino acids. On a dry basis, mean lysine content was found to be 0. Lysine was found to be the most variable out of the 10 amino acids measured, with average CV = 17.
Methionine values range from 0. The average tryptophan and threonine values were 0. The mean values for arginine, histidine, phenylalanine, isoleucine, leucine and valine were 1. Due to being very sensitive to high temperatures, the content and especially digestibility of lysine in DDGS samples is also very variable.
Lysine tends to be the lowest concentration in darkest color DDGS and highest in lightest color DDGS, Lysine ranges from 0. The digestibility of amino acids in maize-produced DDGS in poultry was lower than that of maize due to the effect of temperature, increasing the Maillard 5 reaction. Almeida et al. The main minerals in DDGS are Ca, P, K, Mg, S and Na.