International University School of Biotechnology

Viet Nam National University – HCMC Department of Biochemistry

FERMENTATION TECHNOLOGY

LABORATORY REPORT
Instructor: MSc. Le Tran Hong Ngoc

Date of submission: 13th, January , 2021

Group 3’s members:

Pham Thi Linh Chi BTBCIU17061

Nguyen Huu Duyen BTBCIU17063

Nguyen Thanh Hang BTBCIU17050

Score: ____________
 I. Abstract

The focus of this fermentation laboratory course was to investigate Trichoderma reesei which is

recognized as having high potential value for the industry. Trichoderma reesei was fermented at

laboratory level, which began with the preparation of cultivation medium, following by sub-culture,

morphology observation and screening for cellulose produced by the fungus. In the Trichoderma

reesei culture and observation experiment, the fungus was cultured on PDA agar plates for a week

and then using techniques such as staining, wet mount to observe under a microscope. As a result,

T.reesei was an aerobic organism due to its development on the slant agar surface and its morphology

was also clearly observed under a microscope. In the cellulose activity experiment, the ability of

fungus reduce sugar and produce protein was assessed through the DNS method and Bradford assay.

From there, the result of calculating enzyme activity was 0.54 based on the measured amount of

glucose and protein.

II. Introduction

 Fermentation is a process of utilizing microorganisms to produce commercial products. In

biochemistry, it refers to the metabolic pathway conducted by microorganisms in which

carbohydrates are converted to alcohol and carbon dioxide or other acids under anaerobic condition.

 Aseptic technique is a fundamental and essential laboratory competency in microbiology field.

Utilizing proper aseptic technique will help reduce the possibility of contamination, which promotes

the growth and preservation of pure culture. In this laboratory, aseptic technique involves in a number

of steps including inoculation of culture, isolation of pure cultures and microbiological testing. [1]

 The Bradford protein assay is used to measure the concentration of total protein in a sample. Bradford
method states negatively-charged Coomassie brilliant blue dye binds to positively-charged proteins.
When it binds to basic amino acids in the protein, it changes from brown to blue color and absorbs at
595 nm. The absorption in the sample can then be compared to a standard curve. [3]
  Trichoderma reesei is a mesophilic and filamentous fungus. It can secrete large amounts of
cellulolytic enzymes (cellulases and hemicellulases). Microbial cellulases have industrial application
in the conversion of cellulose, a major component of plant biomass, into glucose.[2]


Figure 1: Morphology of Trichoderma reesei. First row: colonies on potato dextrose agar. Second
row: colonies on V8 agar. Third row: colonies on corn meal agar. Fourth row: conidiophores and
conidia. Chlamydospores of T. reesei (H) (scale bars: D, H = 20 µm).
III. Materials and methods

A. Medium preparation - Aseptic technique and culture preparation

Materials:

The equipment were 2 petri dishes, 2 test tubes, 2 Erlenmeyer flasks, test tube rack, plastic wrap,

inoculating loop, scalpel blades and 2 alcohol burners for the Aseptic technique and culture

preparation. The chemicals used for this experiment were PDA, cellulose-yeast extract medium and a

cultured dish of Trichoderma reesei which were prepared by TA.

Cellulose-yeast extract medium includes the chemicals follow the table 1 below

Chemicals Amount
Yeast extract 10g/L

Glucose 10g/L
 (NH4)2SO4 1.4g/L

KH2PO4 2g/L

CaCl2.2H2O 0.4g/L

MgSO4.7H2O 0.3g/L

FeSO4.7H2O 0.005g/L

CoCl2.6H2O 0.0037g/L

MnSO4.H2O 0.0016g/L

ZnSO4.7H2O 0.014g/L

Distilled water 1 liter

Table 1: The chemicals were used for cellulose-yeast extract broth

Method:

The sub-culturing of Trichoderma reesei was conducted using aseptic technique. It means the

working area must be disinfected using 70% alcohol before conducting the experiments and during

performance, everything should be put near the flame to avoid contaminants.

In the inoculation of fungal spore from stock to solid media preparation, three piece of its stock

culture were cut like a small diamond and transferred to two fresh PDA plates by using a scalpel

blade. The PDA plates were sealed with before being incubated at 30 0C and the result was observed

after seven days. And in the inoculation of the fungal spore into slant agar, a metal loop was used to

pick up some of its mycelia and streaked on the surface of agar and submerging into the deep inside

of agar. Then, they were incubated at room temperature in seven days for growth and the results were

obtained. The purpose of slant agar culture is to test whether the fungus is anaerobic or aerobic or

both.
 For the inoculation of the fungal spore into liquid media part, an inoculating loop was used to take a

piece of fungus grown in the PDA plate or slant agar and transferred to Erlenmeyer flasks containing

cellulose-yeast extract broth. These flasks were covered with cotton-gauze plugs, aluminum foil and

stretch film, then incubated in one week to carry out glucose and protein related assays.

B. Observation of fungal morphology

Materials
The equipment used were microscope, two glass slides and cover slips, inoculating loop, and alcohol
burner were prepared for the observation process. The chemical used in this experiment were actively
growing culture of Trichoderma reesei in plate, distilled water, congo red dye, CMC plated.
Methods
Before doing the experiment, alcohol 70% used to clean our hands, and area working for limit
contamination and bacteria.
For heat fix method: Inoculating loop used to take small piece of Trichoderma reesei in CMC plate
and transfer into glass slide and spread out. After that, this glass slide contained cells was heat near
alcohol lamp for fixing cells. Adding 2-3 drops of congo-red for dye and then heat this slide by
alcohol burner until the liquid evaporated completely. And then, added congo red fulfill all the
surface of the plate in 30s, this slide was washed by ethanol 70% to release congo red. Finally,
observing the morphology of cell under microscope.
For wet mount method: It was simpler step than heat fix, a piece of culture is placed on a glass slide,

covered by coverslip. The coverslip and drop are then inverted over the well of a depression slide.

Lastly, observing the morphology of cell under microscope.

C. Measurement of cellulase activity

Materials
The equipment used were 9 test tubes, 5ml glass pipette, water bath. The chemicals were falcon
supernatant, phosphate citrate buffer, DNS reagent, Bradford reagent.
Methods
Chemical preparation: Reagents Dinitrosalicyclic acid, Sample has been prepared by TA advance.
Dilute 10 times of 1ml sample by 9mL of Buffer solution (Phosphate citrate buffer)
 For sugar analysis, we have three test tubes. For the control, added 2mL of control, putting 2mL of
sample in the second test tube, and then added 2mL of Phosphate citrate buffer to the last test tube.
After that, 4mL of DNS reagent is added to each tube and boiled at 100 oC for 5- 10 minutes. Finally,
the solutions were poured into the cuvette and measured in the Spectrophotometer at 540 nm.
For protein determination, we also have three test tubes. For the control, added 1mL of control,
putting 1mL of sample in the second test tube, and then added 1mL of Phosphate citrate buffer to the
last tube. After that, 2.5mL of Bradford reagent is added to each tube and incubated at room
temperature for 10- 45 minutes. Finally, the solutions were poured into the cuvette and measured in
the Spectrophotometer at 595 nm.
D. Application. (making bread)
Materials
The used equipment was a basket, a plastic tub, a set of five knives, a set of planes, an oven, different
types of grill, a spatula, two sets of tablespoon and teaspoon. 
The materials of yeast bread included one half cup of whole wheat flour and one fourth cu of white
flour, one half teaspoon of yeast, one fourth teaspoon of salt, and a 90 mL of warm water at 55oC. 
The materials of soda bread included of one-half cup of white flour, one-half of white meal flour,
third eight teaspoon of baking soda, one fourth teaspoon of salt and one-half of butter milk. 
The 1 cup of butter milk was prepared by 1 cup of milk mixed one table spoon of vinegar. 
Methods
There are two types of yeast used to make bread: instant dried yeast and butter milk. The method in
this experiment was combined and kneaded all the ingredients. With instant dried yeast, each group
performed at least 30 minutes for incubation’s step. With butter milk, the incubation’s time was not
counted, but the butter milk should be poured to the mixing just before baking time. All the breads
were baked at 180oC within 30 minutes.  
IV. Results

A. Medium preparation - Aseptic technique and culture preparation

Figure 2: Culture of Trichoderma reesei in PDA plate after incubating one week at 30 0C

Figure 3: The stock of T.reesei was grown in two agar slant after 1 week
 Figure 4: Submerged culture of T.reesei in cellulose-yeast extract broth after 1 week

B. Observation of fungal morphology

Figure 5: Trichoderma reesei observed on heat fix under microscope (10x)

 Figure 6: Trichoderma reesei observed on wet mount under microscope (40x)

C. Measurement of cellulase activity

Figure 7: Glucose sample Figure 8: Protein sample

 Figure 9: Absorbance value of sugar Figure 10: Absorbance value of protein

Table 2: The absorbance of sugar

Absorbance 1 Absorbance 2 Absorbance 3 Average of
absorbance
Control 0.015 0.016 0.016 0.016
Sample 0.045 0.045 0.045 0.045
Net absorbance of glucose = absorbance sample – absorbance control = 0.0293

D-Glu std
0.5 0.45

0.4 f(x) = 0.05 x − 0.12

R² = 0.99 0.31
Absorbance

0.3

0.19
0.2

0.09
0.1
0.01
0
2 3 4 5 6 7 8 9 10 11
Concentration (mg/ml)

Figure 11: Standard curve of DNS assay

The equation from the standard curve of D-Glucose is:

y = 0.0548x - 0.1166

 x = (y+0.1166) / 0.0548

 x = (0.0293+0.1166) / 0.0548
 x = 2.66

Thus, the concentration of the diluted sample is 2.66 mg/ml

The dilution factor is 1:10

 The concentration of the sample is 2.66 x 10 = 26.6 mg/ml.

Table 3: The absorbance of protein

Absorbance 1 Absorbance 2 Absorbance 3 Average of
absorbance
Control 0.003 0.003 0.003 0.003
Sample 0.075 0.075 0.075 0.075
 Net absorbance of protein = absorbance sample – absorbance control = 0.072

Protein - BSA
1
0.9 f(x) = 0.53 x − 0.01
0.8 R² = 0.97
0.7
Absorbance

0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Concentration (mg/ml)

Figure 12: The standard curve of protein

The equation from the standard curve of protein is:

y = 0.5292x – 0.0148

 x = (y+0.0148) / 0.5292

 x = (0.072+0.0148) / 0.5292

 x = 0.164
 Thus, the concentration of the diluted sample is 0.164 mg/ml

The dilution factor is 1:10

 The concentration of the protein sample is 0.164 x 10 = 1.64 mg/ml.

Table 4: Mass of cell pellet

Group Initial weight (gram) Final weight (gram)
3 28.7510 1.4018

amount of glucose liberated (%)

Enzyme activity = :time incubation =
%Protein

mg
concentration of Glucose ( )
ml
:time incubation
mg
concentration of Protein ( )
ml
With:
mg
concentration of Glucose ( )
 Glucose content = %Glucose = ml
x 100
DCW (mg)
mg
Concentration of Protein ( )
 Protein content = %Protein = ml
x 100
DCW (mg)

Therefore:
26.6
Enzyme activity = = 0.54 enzyme unit.
1.64 x 30
D. Making bread (application): Making bread following two recipes 

Using instant dried yeast: The product got a crispy crust, sweet taste, good smell, and softer than the
soda bread. 
 Figure 13: Instant yeast dried bread.
Using baking soda: The bread tastes was soapy, salty but was tougher. The crust was too dark with
dry in texture and came denser than the yeast one.  

Figure 14: Soda banking bread.

V. Discussion

A. Medium preparation - Aseptic technique and culture preparation

 The culture medium is of vital importance to the growth of microorganisms in the laboratory,

providing a source of carbon, nutrients, aeration, and pH conditions for microbial growth. [4] The

preparation of agar on Petri dishes requires a high degree of aseptic environmental conditions, as

small acts of detail can lead to contamination inside the agar plates that make them unusable. This

will significantly affect the overall speed of the process as well as less likely to provide accurate and

reproducible microbiological test results. The results of Trichoderma reesei cultured on the PDA plate

showed that the fungus grew well after 1 week. However, colonies also appeared beside the fungus,

which indicates that the culture plate was contaminated. This contamination could be caused by

improperly performed aseptic procedures as well as the skill of the performers. In order to obtain the

best results, closing all doors including windows and main doors aid to the

prevention of flown air from outside environment which can bring undesired contaminants to the

dishes; working in a well-prepared workbench sterilized with alcohol, using alcohol lamps can also

prevent any nearby microorganism possible of entering the plates. The most ideal location is to work

in Biological Safety Cabinet. In addition, perform precise laboratory techniques such as opening the

lid in a minimized way in the direction near the alcohol lamp, and working at maximum speed. In

addition, the performers should perform precise laboratory techniques such as opening the lid in a

minimized direction towards the alcohol lamp, working at maximum speed but still follow procedures

and techniques correctly.

Compared with PDA dishes, culture on slant agar had better results. The smooth surface of the fungus

indicated that they were well-populated and not contaminated. Therefore, it was the desirable colony

to be inoculated with the cellulose yeast extract broth. In addition, the fungus grew on the surface of

slant agar where has the presence of oxygen, thus, the Trichoderma reesei was an aerobic one.

B. Observation of fungal morphology

 Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the

sample to more readily take up stains. Allow the smear to air dry. Congo-red was used to qualitative

detected these microorganisms or activity of fungal were still active or not. Congo red tendency to

bind with cellulose then removed by ethanol at the place where did not contain cellulose. Cellulases

used as an enzyme for breakdown beta-1,4-glycosidic linkage to convert cellulose into monomeric or

dimeric structure hence carboxymethyl cellulose (CMC) and used it as a carbon source which is a

soluble form of cellulose.

C. Measurement of cellulase activity

In DNS assay for reducing sugar estimation; 3,5-Dinitrosalicylic acid is an aromatic compound that

reacts with reducing sugars to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540

nm. This will result in a solution that has red brown color whose color intensity depends on the

amount of 3-amino-5-nitro salicylic acid is formed. The color intensity of a sample then can be

inferred by measuring its absorbance, and from which the concentration of reducing sugar can be

calculated throughout the standard curve. In our group’s experiments, we noticed the red brown color

in our sample test tube, which can be a little hard to differentiated from the test tube containing blank

and control solutions. Since the standard curve calculated by the TA is excellent with the R 2 = 0.987

and consistency of the absorbance over three samples, the concentration of sugar can be inferred with

confidence to be 26.6 mg/mL.

In Bradford assay for total protein quantification, the purpose of this task is to determine the

concentration of unknown protein throughout Bradford assay. The reagent (Coomassie Brilliant Blue

G-250) in the Bradford assay associates with proteins changes color from red to blue. This reaction

caused the amino acids to change blue in color, thus, the reagent-protein complex can be analyzed

through the spectrophotometer as it is within the wavelength of the visible light spectrum. There are
 20 amino acids in general but Coomassie dye tends to bind with aromatic groups (phenylalanine,

tyrosine, tryptophan) and basic side chains (lysine, arginine, and histidine). CBB G exists in 3 forms

with 3 colors respectively: anionic (blue), neutral (green), and cationic (red). [5] The red form of the

dye is converted into blue color when negatively charged of the dye binds to the positive amine

groups of the protein under acidic conditions. If there is no protein to bind, the solution will remain

brown. The standard curve also was provided by TA with a good R2 value (0.974).

According to the data and standard curve, we can calculate the concentration of unknown sample and

the result was 1.64 mg/ml.

The enzyme activity was defined as moles of substrate converted per unit time. The recorded result of

enzyme activity was 0.54 enzyme unit.

D. Application (making bread)

Baking soda is a simple base and therefore needs to be combined with an acid (in our experiment,

vinegar was used) in order to become activated. The mixing of soda bread cannot wait too much due

to the rapid nature of the gas bubbles that are produced through the reaction of acid plus sodium

bicarbonate. That is the reason why the buttermilk should be poured just before baking time.  

The product got the taste soapy, salty, bitter, and the crust is too dark, the texture is dry due to some

problems. First, our group might have added too much baking soda and salt. Secondly, our group

might have spent too much time to waiting for the dough after buttermilk was poured. Baking soda

starts to react and release its gas as soon as it comes into contact with the sour milk. The gas will

escape before the bread is baked then lead to dry bread in result when taking too long. [6]

Yeast differs from baking soda, mainly because it is a live organism and takes substantially longer to

leaven the dough. So, in the experiment, the mixing needs to incubated at least 30 minutes. Unlike

baking soda, yeast leavens dough through a biological process and results in fermentation. Through
 fermentation, yeast can affect the taste associated with dough through residual alcohol, making it a

great option for bread. That's why there is a bit of sweetness in the taste of the product. 

In conclusion, our product proposed that yeast is better than baking soda. 

VI. Conclusion

T. reesei is an important commercial and industrial micro-organism due to its cellulase production

ability. [7] The focus of this fermentation laboratory course was to investigate Trichoderma sinense

which was fermented at laboratory level, which began with the preparation of cultivation medium,

following by sub-culture, morphology observation and screening for cellulase produced by the

fungus. Medium preparation and subculture were performed well using aseptic technique. However,

the result was not as expected due to the long culture time undergoing Covid 19. The sample then re-

prepared for all the group by teaching assistant and enzyme activity of cellulase was estimated 0.54

enzyme unit. In addition, an application of yeast was performed in making bread. Type of yeasts and

incubation time is the main points that would change the taste and scent of bread.

VII. References
[1] Bykowski, T., & Stevenson, B. (2008). Aseptic technique. Current protocols in
microbiology, 11(1), A-4D.
[2] Reczey, K., Szengyel, Z. S., Eklund, R., & Zacchi, G. (1996). Cellulase production by T.
reesei. Bioresource Technology, 57(1), 25-30.
[3] Kruger, N. J. (2009). The Bradford method for protein quantitation. In The protein protocols
handbook (pp. 17-24). Humana Press, Totowa, NJ.
[4] Cohen, D. (1994). The culture medium. Environmental Effects and their Control in Plant Tissue
Culture 393, 15-24.
[5] Anal. Chem. 2017, 89, 7, 4314–4319 Publication Date:March 15, 2017
[6] Healthy Living, Baking 101, Learning Center on February 2, 2018 by Bob's Red Mill
[7] Seiboth, Bernhard; Ivanova, Christa; Seidl-Seiboth, Verena (September 15, 2011). "Chapter 13:
Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels". In Dos Santos Bernardes,
Marco Aurélio (ed.). Biofuel Production-Recent Developments and Prospects. InTech. p. 321.