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

16 3.2.3 Evaluate growth ability on thin stillage agar, dried distiller grains, liquid stillage: .... 22 4.2 Evaluate the ability of growth and development of thermophilic fungi strains

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

ALCOHOL PRODUCTION

Student : Vu Thi Thu Trang Faculty : Biotechnology Supervisor : Nguyen Thanh Hao, PhD

Ha Noi, 02/2021

Hanoi, January 2021

Student

Vu Thi Thu Trang

ii

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 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

iii

CONTENT

COMMITMENT i

ACKNOWLEDGEMENTS ii

CONTENT iii

LIST OF TABLE iv

LIST OF FIGURE v

ABBREVIATION LIST vi

ABSTRACT viii

Part I INTRODUCTION 1

1 Subject 1

2 Purposes: 2

3 Requirements: 2

Part II LITERATURE REVIEW 3

2.1 Overview of distillery wastewater 3

2.1.1 Bioethanol production and distillery wastewater 3

2.1.2 Characteristics and composition of distillery wastewater 3

2.1.3 Direction of application to improve the value of distillery wastewater 7

2.2 Thermophilic fungi strains 8

2.2.1 General characteristics of thermophilic fungi strains 8

2.2.2 Some thermophilic fungi strains 9

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

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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

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LIST OF TABLE

Table 1.1 Wastewater generation in various operations 4

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

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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 4 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

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ABBREVIATION LIST

DDGS Distillers dried grains with solubles

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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

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

- 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

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Part II LITERATURE REVIEW

2.1 Overview of distillery wastewater

2.1.1 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

2.1.2 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 1.1

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Table 1.1 Wastewater generation in various operations

Distillery operations Average wastewater

generation (kLD/distillery)

Specific wastewater generation (L wastewater/L alcohol Spent wash (from

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

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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 30%, fat 2.9 - 12.8%, neutral detergent fiber (NDF) from 28.8 to 40.3%, acid fiber (ADF) from 10.3 to 18.1%, ash from 3.4 - 7.3%, lysine from 0.43 - 0.89%, methionine (met) from 0.44 - 0.55%, threonine (thr) from 0.89-1.16%, tryptophan (trp) from 0.16 - 0.23%, calcium is 0.06%, phosphorus 0.89% Among the minerals in DDGS, sodium is the metal with the largest variation from 0.09 - 0.44% [3]

DDGS does not provide many vitamins, trace minerals (thiamine, riboflavin and other vitamins) but contains many bioactive substances such as nucleotides, mannooligosaccharides, beta-1.3 / 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 0.85%, ranging from 0.72 to 1.02% Lysine was found to be the most variable out of the 10 amino acids measured, with average CV = 17.3% Methionine values range from 0.49 to 0.69% The average tryptophan and threonine values were 0.25 and 1.13%, respectively The mean values for arginine, histidine, phenylalanine, isoleucine, leucine and valine were 1.20, 0.76, 1.47, 1.12, 3.55 and 1.50%, respectively 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 0.48 to 0.76% 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

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reaction Almeida et al (2013) have also shown that the ratio between the lysine content and crude protein has an effect on the digestible amino acid content [11] The main minerals in DDGS are Ca, P, K, Mg, S and Na The average content ranged from 0.05% for Ca to 1.15% for K (dry matter) The auxiliary minerals in DDGS include Zn, Mn, Cu, Fe, Al and Se [3] Their concentrations ranged from 6 ppm for Cu and 149 ppm for Fe, among studies The variation in mineral composition is much greater than that of other nutrients For some minerals such as S, Na, and Ca, CV values may be abnormally high in a single study Exogenous supplementation of some minerals during processing could be

an explanation For example, ethanol plants can use sodium hydroxide to disinfect equipment and machinery They can also use it, along with sulfuric acid, to adjust the pH of the mixture for optimum enzyme activity during liquefaction and or to meet yeast requirements during fermentation The high concentration and high variability of the minerals affect the value and end use of DDGS as animal feed High content ratios can lead not only to excessive nutritional disturbances, while high variability in mineral content makes it difficult to formulate the correct diet because synthetic concentrations are present may differ from actual concentration Out of all the minerals in DDGS, phosphorus (P) is of the greatest concern for all foods as it is the third most expensive nutrient in the diet and has important implications not only nutrition for animals but also for the environment The concentration range is about 0.5-1.0% so the range is much higher than that in conventional grains and exceeds the requirements of most ruminants Hence, the P concentration in DDGS has become an emerging problem [10] When ruminants consume a diet containing elevated concentrations of P, such as a diet containing high DDGS, the amount

of P excreted in the waste is increased

Lipids: The main lipids in DG are triglycerides and auxiliary substances including phytosterols, tocopherols, tocotrienols and carotenoids However,

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unlike the starting material, DG was found to contain abnormally high amounts

of free fatty acids (6-8 vs 1-2% in corn, based on weight of the oil extracted) Oils extracted from solutes are also found to contain higher content of free fatty acids (7.92-12.18 vs 2.28%, oil mass basis) The oil content in DG is about 10% [15]

Carbohydrates and low mass molecules: During the dry milling process, the starch is converted to simple sugars, which are then fermented into ethanol and carbon dioxide However, other carbohydrates, such as the cell wall, remain chemically relatively unchanged DDGS also contains low molecular weight organic compounds that are either in the starting material or produced in the process Because starch conversion cannot be completed under normal processing conditions, there is also some remaining starch and sugars in the product Low-mass molecules with corn-based DDGS include lactic acid (10.40

g / L), glycerol (5.8 g / L) and alanine (free amino acid, 4.08 g / L), as well as small amounts of ethanol other types of nitrogen and nitrogen acids, polyhydroxy alcohols, sugars, and glucosides [8]

2.1.3 Direction of application to improve the value of distillery wastewater

Currently, there are many research directions to increase the value of distillery wastewater such as biofuel production: Bioethanol and Acetone-Butanol-Ethanol (ABE), methane gas, hydrogen gas Substrate hydrolysis and gaseous biofuel generation; organic acid production: short-chain volatile fatty acids, citric acid, lactic acid, succinic acid; production of surfactants or creating other value-added products such as polysaccharide, flavoring production, and bio-fertilizers

In particular, the current trend is towards researching DDGS to become animal feed With a rather large content of crude protein, amino acids, phosphorus and nutrients, it is suitable as a feed ingredient for animals In some ruminants they can easily digest foods high in fiber, such as forage and DDGS

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In a number of studies that have examined proteins, amino acids, and nutrients that are quite good for rumen function (including pH and volatile fatty acid concentrations), other studies have identified digestibility and the decomposition potential of different chemical components Overall, studies have agreed that DDGS actually works as a protein supplement when used with a grain-based diet that partially replaces cornstarch and soybeans with no effects are negative

in growth, increase productivity and do not lead to acidosis or flatulence Application of DDGS as feed has been studied for many different types of livestock

Topic: "Research to utilize by-products of alcohol factories to produce feed ingredients." chaired by Assoc.Prof Dr Chu Ky Son The research results have evaluated the volume and use of by-products of our country's food alcohol and fuel alcohol factories The replacement of maize with rice wine residue and cassava up to 5-10% did not affect the growth and digestion of experimental pigs The study also assesses the potential of using this by-product in the production of DDGS, which is a raw material for animal feed production, which

is currently completely imported

Worldwide, according to Nitayavardhana et al., 2013, the fungus Rhizopus oligosporus is capable of growing at 37°C in an environment containing 75%

post-distillation fluid (v/v), supplemented with inorganic minerals and up to 80 types % COD, the cumulative mold biomass contains up to 50% protein and contains essential amino acids comparable to commercial protein sources from fishmeal or soybean meal products

2.2 Thermophilic fungi strains

2.2.1 General characteristics of thermophilic fungi strains

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature

of growth extending up to 60 to 62°C As the only representatives of eukaryotic

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organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60°C to 62°C Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests

Thermophilic fungi strains are present in soils and within the decomposition of plants including: compost, hay heap, stored grain, nesting material of birds and animals, garbage of Cities and other organic substances that accumulate in warm, humid and aerobic environments provide the basic physiological conditions for the growth of thermophilic fungi strains Thermophilic fungi strains are more common in acidic heat than in neutral or alkaline environments

They form a heterogeneous physiological group belonging to Zygomycetes, Ascomycetes, Deuteromycetes and Mycelia Sterilia Thermophilic fungi strains have received little research interest Only approximately 30 species out of about 50,000 fungal species grow within the temperature range of 45-62°C [12]

2.2.2 Some thermophilic fungi strains

Rhizomucor miehei (also: Mucor miehei) is a species of fungus It is

commercially used to produce enzymes which can be used to produce a microbial rennet to curd milk and produce cheese Under experimental conditions, this species grows particularly well at temperatures between 24 and 55°C, and their growth becomes negligible below 21°C or above 57°C.It is also

used to produce lipases for interesterification of fats

Rhizomucor pusillus is a species of Rhizomucor It can cause disease in humans R pusillus is a grey mycelium fungi most commonly found in compost

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piles Yellow-brown spores grow on a stalk to reproduce more fungal cells

Rhizomucor pusillus is a thermophilic fungus that lives in hot environments Its growth optimum is between 50 and 70°C Celsius R pusillus cells have stolons,

rhizoids, and branched sporangiophores Because of the high temperatures required for this microorganism, it is difficult to study in laboratory environments Thermophiles reproduce both sexually and asexually.[clarification needed] Most common reproduction is asexually, through mitosis Thermophiles reproduce asexually, when a male spore and a female spore come in contact with each other.[clarification needed] Different

strains of R pusillus segregate into two subclusters at very high levels causing

different EST and G6D patterns

Rhizopus microsporus is a fungal plant pathogen infecting maize,

sunflower, and rice This fungus is most commonly found in soil, plant debris, and foodstuffs.[8] It is a pathogen of many crops and therefore is found in many

diverse environments R microsporus is generally found in soils with a neutral

pH These soil levels usually have lower salinity for optimum growth

conditions The growth range of R microsporus ranges from 25℃ to 55℃ with

an optimal temperature of 28℃

Thermomyces lanuginosus is a species of thermophilic fungus that

belongs to Thermomyces, a genus of hemicellulose degraders It is classified as

a deuteromycete and no sexual form has ever been observed It is the dominant fungus of compost heaps, due to its ability to withstand high temperatures and

use complex carbon sources for energy T lanuginosus is classified as a

thermophile, and experiences rapid growth at high temperatures In the lab, colonies can be cultured in a glucose-salt liquid medium fortified with peptone Colonies are white and velvety at first, generally less than 1 mm high, but soon turn grey or green-ish grey, starting from the center Mature colonies are dull

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dark brown to black, often with pink or vinaceous diffusing pigment secreted

from the colony The optimal growth temperature for T lanuginosus is 45-50°C Thermomyces dupontii, a widely distributed thermophilic fungus, is an

ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments The species is commercially used for the production of various enzymes

Rasamsonia is a genus of fungi in the family Trichocomaceae It is characterized from other genera of the Trichocomaceae by the following

combination of features: species are thermotolerant or thermophilic; their conidiophores have distinctly rough-walled stripes; conidia are olive brown; and

ascomata, if present, have minimal covering Rasamsonia phenotypically resembles Paecilomyces, in that both have thermotolerant species, produce

olive-brown conidia, and form ascomata with no or scarce ascometal covering;

Rasamsonia, however, differs from Paecilomyces in having more regularly

branched conidiophores with distinct rough-walled structures

Malbranchea cinnamomea is a thermophilic fungus belonging to the order

Onygenales This ascomycete fungi is often isolated from higher-temperature environments It is naturally found in composting soil and has the capability of

degrading plant biomass M cinnamomea has biochemical relevance, as it produces

a quinone antibiotic (6-(1-methylethyl)-2-methoxy-2,5-cyclohexadiene-1,4-dione) named malbranicin, as well as thermostable enzymes, such as alpha-glucosidases, xylanases, alpha-amylases, and glucanases

Myco Thermus thermophilus (Syn Scytalidium thermophilum/Humicola

insolens), a thermophilic fungus, is being reported to produce appreciable titers

of cellulases and hemicellulases during shake flask culturing on bran/rice straw based production medium

Myceliophthora thermophila is an ascomycete fungus that grows

optimally at 45–50 °C (113–122 °F) but not above 60 °C It efficiently degrades

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cellulose and is of interest in the production of biofuel Myceliophthora thermophila colonies have been commonly isolated from composts, where they

generate high temperatures from cellular activities Moist, sun-heated soils and

hay provide ideal places for M thermophila growth because they do not easily dissipate heat and help insulate the colony Colonies of M thermophila initially

appear cottony-pink, but rapidly turn cinnamon-brown and granular in texture Microscopic examination reveals septate hyphae with several obovoid to pyriform conidia arising singly or in small groups from conidiogenous cells

Thielavia is a genus of fungi in the family Chaetomiaceae Circumscribed

by German botanist Friedrich Wilhelm Zopf in 1876, Thielavia is a teleomorph

of Myceliophthora Collectively, the genus is widely distributed, and according

to a 2008 estimate, contains 31 species

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Part III MATERIALS AND METHODS OF RESEARCH

3.1 Materials and research equipments

3.1.1 Materials

- Thermophilic fungi strains have been isolated in Vietnam and are currently stored in the strains collection of the Center for Industrial Microbiology, Food Industries Research Institute

- Distillery wastewater from the alcohol fermentation process from Tung Lam's limited liability company

Figure 3.1: Distillery wastewater 3.1.2 Equipment

The instruments and equipment were used in the Center for Industrial Microbiology, Food Industries Research Institute:

pH titration machine Switzerland

Liquozyme SCDS

DAP Glucoamylase Thermosacc Dry

PDA medium: 2% glucose and 1.7% agar per liter of potato extract Autoclave 121 ° C for 15 minutes, then pour about 20 ml into Petri dishes

+ Thin stillage agar:

Distillery wastewater is centrifuged at 4000v / 30 minutes / 4 ° C The

liquid after centrifugation is added with 2% agar Then, bring the mixture to a

steam bath at 118 ° C / 10 minutes and then pour about 20 ml into Petri dishes

+ Dried distiller grains:

Distillery wastewater is centrifuged at 4000v / 30 minutes / 4 ° C to obtain the dried distiller grains Dry the pulp with absorbent paper to a moisture content

of 70% Then, take the residue and steam it in a water bath at 118 ° C / 15 minutes Next, place a quantity of the medium on the Petri dishes, equivalent to

about 25 g

+ Liquid stillage:

Distillery wastewater is centrifuged at 4000v / 30 minutes / 4 ° C The liquid after centrifuging is steamed in a water bath at 118 ° C / 15 minutes Using a clean steam measuring cup (falcon tube) transfer 50 ml of the liquid medium to each clean steamed 100 ml flask

3.1.5 Location and time:

Location: This subject was conducted in the Center for Industrial Microbiology, Food Industries Research Institute

Time: 8/2020 – 2/2021

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3.2 Research methods

3.2.1 Dry cassava fermentation and post-fermentation distillery wastewater treatment in the laboratory

Raw materials: Dried cassava, moisture sampling

Lakeization: Transfer cassava into a pot of liquefied chemicals with

available water with a ratio of 1/3 cassava/water Turn on the paddle motor to mix the cassava with water Then adjust pH 5.0 (about 4.5 - 5.5 Add 0.3% Viscozyme Cassava C and heat at 50°C, for 60 minutes, stir continuously at 160 rpm After one hour, adjust pH 6.0 (range 5.5 - 6.0) Add 0.05% Liquozyme SCDS, divided into two supplements: 1st time: 0.01% at 95°C for 30 minutes 2nd: 0.04% at 85°C for 120 minutes, then add water to give Measure Bx, pH, Cool to 60°C Add 0.7 g/L urea and 0.04 g / L DAP Adjust pH 5.0 (range 4.5 - 5.0) Add 0.06% ( dw) Glucoamylase (Spirizyme Fuel)

Saccharification: saccharification at 60°C for 60 minutes Then test iodine,

measure Bx, pH

Fermentation: Supplement 0.25g/L dry yeast (Thermosacc Dry) Before

adding, dissolve dry yeast (3.5%) in water and cultivate at 37°C, 30 minutes Or use thermosacc enzyme enrichment in the saccharification fluid Fermented at 32°C for a period of 5-7 days

Distillation: The fermented fluid is boiled to remove alcohol, and the

distillery wastewater residue is obtained

Distillery wastewater: Centrifuge 4000 rpm for 30 minutes at 25°C

Separation of the liquid and the distillery wastewater residue (absorb the water from the distillery wastewater residue using a desiccant until the lowest possible moisture content)

3.2.2 Clean and store strains

Take a seed of thermophilic fungi strains to be cleaned and inoculated on Petri dishes containing PDA medium, then incubate at 45°C for 2-3 days When

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you see that the mold has grown and no strange colonies appear, select a separate colony and inoculate it to the PDA agar tube used for future experiments

3.2.3 Evaluate growth ability on thin stillage agar, dried distiller grains, liquid stillage:

Prepare strains: Inoculate the fungus strains inoculated on PDA plates

cultured at 45°C for 2-3 days to activate the seeds, let the seeds grow on the agar surface, Carry out additional seed for culture to determine growth and thrive in hematoma environments

3.2.3.1 Evaluate growth ability on thin stillage agar

Thin stillage agar: Take the seed quantity on a PDA dish (in a circle of

1cm diameter) inoculate in the center surface of thin stillage agar dish Incubate

at 45°C for 4 days Take pictures and observe to record growth over 2 and 4 days

Figure 3.2.3.1: Culture thermophilic fungi strains on thin stillage agar 3.2.3.2 Evaluate growth ability on dried distiller grains

Dried distiller grains: Take the seed quantity on a PDA dish (in a circle

of 1cm diameter) inoculated in the center surface of the dried distiller grains

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dish Incubate at 45°C for 4 days Take pictures and observe to record growth

over 2 and 4 days

Figure 3.2.3.2: Culture thermophilic fungi strains on dried distiller grains 3.2.3.3 Evaluate growth ability on liquid stillage

Liquid stillage: Take the seed quantity on a PDA dish (in a circle of 1cm

diameter) inoculated in the culture flask Use the implant to mix well after adding the seeds Shake culture at 45°C / 150v for 4 days

Figure 3.2.3.3: Culture thermophilic fungi strains on liquid stillage

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Weigh the culture vessel of each strain after adding the seeds before incubation, and record the parameters After 5 days of incubation weigh the weight of the culture flask again

Samples with post-culture growth were filtered to obtain the mold culture biomass Biomass collection process: Filter paper has been dried and kept moisture in a glass desiccant Take 1 piece of filter paper, numbered according

to No Pour the culture solution into the filter, filter it, then the biomass is retained on the filter paper and put into the oven Wet biomass was dried at 105 degrees to constant weight After drying, put in the desiccator immediately to allow temperature equilibrium Weighing biomass after drying

Figure 3.2.3.4: Dry biomass of some thermophilic fungi strains after drying

Cultures were collected in Falcon tubes and stored in a freezer for reduction sugar analysis, Brix, pH

3.2.4 Estimation of Reducing Sugars by the Dinitro Salicylic Acid (DNS) Method

Principle: This method is based on the reduction reaction of the

dinitrosalicylic acid reagent (DNS) The color intensity of the reaction mixture is directly proportional to the reducing sugar concentration in a given range Color comparison is carried out at 540nm Based on the standard curve graph of pure

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glucose with DNS reagent will calculate the reducing sugar content of the sample

DNS reagent composition: 708 g distilled water, 5.3 g 3.5 dinitrosalicylic

acid (DNS), 9.9 g sodium hydroxide (NaOH) Dissolution and addition: 153 g of rochelle salt (sodium potassium tartrate), 3.8 ml of crystalline phenol (to dissolve at 50°C prior to use, 4.15 g of sodium metabisulfite (Na2S2O5)

Re-check the pH of the DNS reagent solution: Take 3 ml of DNS solution

just mixed, add phenolphthalein in the solution above, the solution turns red Small each 1ml 0.1N HCl If you have added 3 ml of 0.1N HCl solution and the solution does not change color, then the DNS solution is just mixed If less than

3 ml of 0.1N HCl used causes the solution to turn yellow, less than 1 ml of 0.1N HCl should be added to the solution per 2 g of NaOH

Blank sample: distilled water instead of liquid and proceed in the same

way as the test sample

Calculation: C (mg/mL) = (y-b) d / a

C: number of mg / mL reducing sugar

y: measuring value OD 540nm

d: number of dilutions of the sample

a, b: coefficients of equations of standard curves

3.2.5 Measure sweetness

Principle: Brix is a unit of measurement for the evaluation of any soluble

solids in plant juices (fruits and vegetables) These solids include amino acids, proteins, minerals, vitamins, and fructose and sucrose The Brix value, expressed in degrees Brix (°Bx), is the number of grams of sucrose per 100 grams of liquid

Brix values are based on "refractive index" and "specific gravity" It uses purified water as its benchmark as the zero point When other substances dissolve in water, the specific gravity increases and the value increases above 0

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Therefore, the higher the concentration of dissolved solids in the water, the higher the Brix value

Use the Brix scale: The Brix scale is a popular scale that comes from a

solution's refractive index at 20 degrees C (68 Fahrenheit) This simple scale has

been used since the 19th century because quickly and accurately