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 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

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
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

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. .................................... 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

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 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

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
1. 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.

1


2. Purposes:

- Select the thermophilic fungi strains that grow best on the distillery
wastewater.
3. 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.

2


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; starchcontaining 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.

3


Table 1.1 . Wastewater generation in various operations.

Distillery operations

Average wastewater

Specific wastewater

generation

generation (L

(kLD/distillery)

wastewater/L alcohol

491.9

11.9

Fermenter cleaning

98.2

1.6

Fermenter cooling

355.1

2.0

Condenser cooling

864.4

7.9

Floor wash

30.8

0.5

Bottling plant

113.8

1.3

Others

141.6

1.2

Spent wash (from
distillation)

During the ethanol production process, after distillation, two groups of byproducts 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 2330%, 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
5


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 0.51.0% 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,
6


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 AcetoneButanol-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.
7


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
8


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

Zygomycetes,

a

heterogeneous

Ascomycetes,

physiological

Deuteromycetes

group

and

belonging

Mycelia

to

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
9


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

10


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 cellulose/wheatbran/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
11


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.

12


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:

13


Table 3.1: The instruments and equipment were used in the research

Equipment

Origin

Drying oven

Korea

Refrigerator

Vietnam

Incubator

China

Vortex shaker

Italia

Autoclave machine

China

Microwave

Vietnam

Freezer

Korea

pH titration machine

Switzerland

Shaker incubator

Korea

Centrifuge (5000rpm)

Korea

Refractometer

Japan

Precision balance 0.01g

Switzerland

Analytical balance 0.1mg

Switzerland

3.1.3 Chemistry
Table 3.2: Chemicals were used in the research

Chemicals

Origin

Yeast extract

Vietnam

Glucose

Vietnam

Agar
Ure
Viscozyme
Liquozyme SCDS

China

DAP
Glucoamylase
Thermosacc Dry

14


3.1.4 Medium

+ PDA medium:
Potato extract: Weigh 1 kg of peeled potatoes, then wash the thin slices,

then add 5 liters of RO water, boil for 1 hour, then filter off the pulp. The
solution extract is used as a PDA medium.
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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