Open ended lab

UNIVERSITI MALAYSIA PERLIS

Bioprocess EngineeringSem 2 2014/2015

ERT 314 Bioreactor System

Lab Report

Lab 5: Comparison of enzyme activity by using free and immobilized enzymes in Batch Culture

Group B4 Members:

Nur Amirah Binti Mahfod 121141662Nur Athirah Awatif Binti Abdul Rahman 121142810Pua Chee Wee 121142063Tan Siew Yen 121142363

Date of Experiment: 29 April 2015 (Wednesday)

Date of Submission: 6 May 2015 (Wednesday)

1.0 Objectives

1. To comparison enzymatic activity between immobilized and free cell reactor for

ethanol production in batch culture

2. To understand the principles of cell immobilization by using gel entrapment

method in alginate gel

3. To analyze the effect of immobilization on ethanol production

2.0 Introduction

A yeast cell-free enzyme system containing an intact fermentation assembly

and that is capable of bio-ethanol production at elevated temperatures in the absence of

living cells was developed to address the limitations associated with conventional

fermentation processes. Immobilized cells are cells attached to an inert, insoluble

material such as calcium alginate (produced by reaction mixture of sodium alginate

solution and cells with calcium chloride). This can provide increased resistance to

changes in conditions such as pH or temperature. Theoretically, cell immobilization in

calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of

the beads in several sequential fermentation cycles to be carried out with stable final

ethanol concentrations. In addition, there was no need to use antibiotics and no

contamination was observed. After several cycle, there was a significant rupture of the

beads making them inappropriate for reuse. It has also helped to prevent the

contamination of the substrate with enzyme or protein or other compounds, which

decreases purification costs. These benefits of immobilized cells have made them

highly applicable to a range of evolving biotechnologies.

Enzymes are biological catalysts that promote the transformation of chemical

species in living systems. Enzymes have the ability to catalyze reactions under very

mild conditions with a very high degree of substrate specificity, thus decreasing the

formation of by-products. Enzymes can catalyze reactions in different states: as

individual molecules in solution, in aggregates with other entities, and as attached to

surfaces. The attached or “immobilized” state has been of particular interest to those

wishing to exploit enzymes for technical purposes. The term “immobilized enzymes”

refers to “enzymes physically confined or localized in a certain defined region of space

with retention of their catalytic activities, and which can be used repeatedly and

continuously”. The introduction of immobilized catalysts has, in some cases, greatly

improved both the technical performance of the industrial processes and their

economy.

Entrapment is the physical enclosure of enzymes in a small space. When

immobilizing in a polymer matrix, enzyme solution is mixed with polymer solution

before polymerization takes place. Polymerized gel-containing enzyme is either

extruded or a template for shaping the particles from a liquid polymer-enzyme

mixture. Enzyme entrapment may have its inherent problems, such as enzyme leakage

into solution, significant diffusional limitations, reduced enzyme activity and stability,

and lack of control of microenvironmental conditions. Enzyme leakage can be

overcome by reducing the molecular weight cutoff of membranes or the pore size of

solid matrices. Diffusion limitations can be eliminated by reducing the particle size of

matrices and or capsules. Reduced enzyme activity and stability are due to unfavorable

microenvironmental conditions, which are difficult to control. However, by using

different matrices as chemical ingredients, by changing processing conditions, and by

reducing particle or capsule size, more favorable microenvironmental conditions can

be obtained. Diffusion barrier is usually less significant in mircocapsuled as compared

to gel beads.

This study was organised to understand the principles of cell immobilization;

how they works and what benefits will they provide, and analyzing their effect on the

ethanol in term of glucose production. These were determined using the gel

entrapment method.

3.0 Materials And Equipment

3.1 Malt Extract

3.2 Peptone

3.3 Glucose monohydrate (D-glucose)

3.4 Yeast Agar plate

3.4 1.0% and 3.0% (w/v) sodium alginate solution

3.6 0.2 M calcium chloride solution

3.7 20 g/L glucose solution

3.8 Distilled Water

3.9 Tap water

3.10 Ice

3.11 DNS

3.12 5 of 125 mL Erlenmeyer flasks

3.13 Pipette

3.14 Beaker

3.15 Autoclave

3.16 Incubator

3.17 Chemical balance

3.18 Spectrophotometer

3.19 5ml of measuring cylinder

3.20 Hot plate and magnetic stirrer

3.21 Retort Stand

3.22 Syringes

4.0 Procedures

4.1 Inoculation of yeast

1. 0.3% (w/v) malt extract, 0.5% (w/v) peptone and 1 % (w/v) glucose

monohydrate (D-glucose) is prepared for a working volume of 100ml.

2. (If want 500ml, times five times for each media)

3. This solution is put in a 100 ml Erlenmeyer flask and for glucose

monohydrate is separated from other media to prevent oxidization.

4. The flask is cotton-plugged and covered with aluminium foil.

5. The medium is sterilized in batch autoclave at 1210C for 15 minutes.

6. After sterilization, the media is let to cool down at room temperature

before inoculation.

7. 100ml of the medium is measured and poured it into a conical flask.

8. 2 loops of yeast is scraped from agar plate and is inoculated it into the

medium and are left in the incubator to be shaken overnight.

4.2 Preparation of immobilized enzyme beads

1. Mix 1 ml of yeast cell broth with 9 ml of 3% (w/v) sodium alginate

solution

2. Drip the polymer solution prepared in step 4.1.1 from a height of

approximately 20 cm into the stirred calcium chloride (CaCl2) solution

with a syringe at room temperature.

3. Leave the beads in the calcium solution to cure for 15minutes.

4. Filter the formed beads and wash thoroughly with distilled water.

5. Dry the beads using filter paper (Whatman no.1) followed by exposure to

the open air for 15 minutes before use.

4.3 Determination of ethanol produced from glucose in batch reactor for

immobilised and free cells

1. Transfer all of the gel beads prepared in Part 4.1 in a beaker

2. Add 50 ml of glucose solution in the beakers or column containing the

beads. The glucose concentration at this time is to be determined.

3. Shake the flasks at 150 rpm inside the incubator shaker.

4. Measure glucose concentration every 15 min by using DNS test.

5. For free cells, mix 1 ml of yeast cell broth and 10 ml of glucose solution in

conical flask and repeat steps 4.3.3 – 4.3.4.

4.4 DNS Measurement

1. 3 ml of DNS was added to the 3 ml of sample in the test tube and diluted

with distilled water.

2. The diluted solution was preheated for 5 min at 37°C in water bath.

3. Then, the diluted solution was heated from 5 to 15 min at 85°C until the

colour change from yellow to brownish.

4. Water tap was used to cool down the solution at room temperature.

5. The absorbance of solution was recorded at 540 nm.

5.0 Results

5.1 Glucose Standard Curve

In this experiment, standard curve of glucose is developed in order to calculate the

glucose concentration at different absorbance. The data and graph are shown below:

Glucose (g/L) Absorbance at 540 nm

0 0.000

0.2 0.133

0.4 0.318

0.6 0.474

0.8 0.557

1.0 0.753

5.2 Calculations of Molarity of Standard Solution

For glucose standard curve, 0.2 % glucose solution was used. To calculate the needed

concentration, the equation used is:

M1V1 = M2V2

M1 : Initial concentration of glusose solution

V1 : Initial volume of glucose solution

M2 : Final concentration of glucose concentration

V2 : Final volume of glucose solution

For standard 1,

( 0.2 g100 ml ) (0.1 ml )=(M 2)(1ml)

M2 =0.02 g100 ml

=0.02 g100 ml

x 1000 ml1L

= 0.2 g/L

Graph of Standard of Glucose and the equation is listed on the graph

0.000 0.200 0.400 0.600 0.800 1.000 1.2000.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

f(x) = 0.741857142857143 x + 0.00157142857142856R² = 0.992367302412008

Standard Curve of Glucose

Glucose Concentration (g/L)

Abso

rban

ce @

540n

m

5.3 Absorbance for Free and Immobilized Cells (Yeast)

Time (min)Absorbance at 540 nm

Free Cells Immobilized Cells

0 0.982 1.012

15 0.838 0.968

30 0.444 0.544

45 0.240 0.299

60 0.120 0.138

0 10 20 30 40 50 60 700

0.2

0.4

0.6

0.8

1

1.2

Graph of Absorbance vs Time

Free CellImmobilized Cell

Time, min

Absorbance, 540 nm

5.3 For Free and Immobilised Yeast Cells

Time (min)Absorbance at 540nm

Free cells Immobilised cells

0 0.982 1.012

15 0.838 0.968

30 0.444 0.544

45 0.240 0.299

60 0.120 0.138

*Note that all the absorbance for free and immobilised cells was carrying out at

dilution factor of 2 or 102.

From equation: y = 0.7419x + 0.0016

x= y−0.00160.7419 , y = Absorbance at 540nm; x = glucose concentration

(g/L)

Glucose concentration = [x] x [dilution factor (102)]

At 0 Minute:

i) For free cells, y = 0.982, x=0.982−0.0016

0.7419

x = 1.321 g/L x Dilution Factor

= 1.321 g/L x 102

Glucose concentration = 132.1 g/L

ii) For immobilised cells, y = 1.012, x=1.013−0.0016

0.7419


= 1.362 g/L x 102


At 15 Minute:


0.7419


= 0.596 g/L x 102



0.7419


= 0.731 g/L x 102


At 30 Minute:


0.7419


= 1.127 g/L x 102



0.7419


= 1.303 g/L x 102


At 45 Minute:


0.7419


= 0.321 g/L x 102



0.7419

= 0.401 g/L x Dilution Factor

= 0.410 g/L x 102


At 60 Minute:


0.7419


= 0.160 g/L x 102



0.7419

= 0.184 g/L x Dilution Factor

= 0.184 g/L x 102


6.0 Discussion

In this experiment, we are compared the using of free and immobilized enzyme on the

production of ethanol from glucose in batch culture in the time provided.

Before doing experiment, we have to do standard glucose solution graph by reading

the absorbance at every 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0 g/L. From the standard

glucose graph, we can determine the equation which is y = 0.7419x + 0.0016 .From

this equation, we can determine the concentration of glucose if we know the

absorbance value by substituting the absorbance values into equation.

The whole experiment has been done on our own, starting from preparing the yeast

culture media, inoculum the yeast into the suspension media, preparing calcium

chloride and sodium alginate for the immobilization. For the yeast culture media,

during the preparation, the carbon sources which come from glucose monohydrate are

separated from other nutrients to prevent oxidation. To inoculate the yeast into the

suspension media,4 loops are required in this experiment and the yeast suspension is

left in incubator to be shaken overnight at 150rpm.In this experiment, the whole yeast

culture method is conducted in the fume hood to prevent contamination.

Besides, to detect the glucose concentration, we have to use DNS test which is useful

for reducing sugars. This method tests for the presence of free carbonyl group (C=O),

the so-called reducing sugars. Reducing sugars has property, 3,5-dinitrosalicylic acid

(DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline condition. Since our

sample contains simple sugars like glucose, we can use Dinitro Salicylic Acid, (DNS)

method. Different reducing sugars generally yield different colour intensities, thus it

is necessary to calibrate for each sugar. For DNS test, the colour will changes from

yellowish to red brownish, which then indicated the presence of reducing sugars.

After detecting the colour changes, we use spectrophotometer to take the absorbance

reading. Before adding DNA reagent, the sample is diluted three times, so that the

spectrophotometer is able to read the sample. If too concentrated, the sample cannot

be read and therefore in this experiment, we are using two times of dilution factor.

From the result, the absorbance values for free enzyme are higher than the

immobilized enzyme in the time: 0minutes, 15 minutes, 30 minutes, 45minutes and 60

minutes. Therefore, by calculating the absorbance into the equation, we can determine

the concentration of the glucose.

From result, the concentrations of glucose at 0 minutes for free (132.1g/l) and

immobilized enzyme (136.2g/l) are the highest since there is no time for glucose to

become ethanol yet. As the time proceeds, there is more time for glucose to react with

the yeast to become ethanol, therefore the enzyme activity increase and the glucose

concentration depleted. From 15 minutes to 60 minutes, the glucose concentration is

decreased from 59.6g/L to 16 g/L for free enzyme, while for immobilized enzyme is

decreased from 73.1g/L to 18.4g/L. The glucose concentration level for free enzyme

is lower than the immobilized enzyme. This shows that it is better to use free enzyme

which the rate of conversion to ethanol from glucose is higher than using immobilized

enzyme. The free enzyme is better than immobilized because there are no mass

transfer limitation occurs in free enzyme. Since the glucose molecules take time to

pass through the membrane inside the immobilized enzyme, therefore it requires quite

much time to convert the glucose molecules to ethanol molecules.

While for free enzyme, the glucose molecules do not need to pass through the

membrane, time needed for reaction to occurs to occur is shorter and hence better

conversion is achievable. But even though, free enzyme is easier to be contaminated

since it is exposed to the environment. The immobilized enzymes are less to be

contaminated and are protected by the capsules or membrane outside it. There are

many factors like external diffusion, internal diffusion and so on which affect the rate

of conversion of ethanol.

Besides, for immobilized enzyme, the volume of glucose solution added is 50 ml,

which is larger than the free enzyme which only added 10ml of glucose solution. The

increase in glucose solution may cause the yeast to be insufficient to convert the

glucose to the ethanol which causing the concentration of glucose in the immobilized

enzyme is higher than the free enzyme.

7.0 Conclusion

In this experiment, we are comparing the free and immobilized enzyme activity. From

the result, we know that the absorbance reading value for free enzyme is lower than

the immobilized enzyme which mean that the concentration of glucose inside the free

enzyme is lower than the glucose concentration in immobilized enzyme. We can

conclude that the free enzyme activity is higher than immobilized enzyme since the

mass transfer limitation may occur inside the immobilized enzyme.

8.0 References

Asenjo, J. A. and Merchuk, J. A. (1995). Bioreactor System Design. Marcel

Dekker Inc, New York.

Barnfeld, P. (1995). Methods in Enzymmology 1, 149-158

Doran, P. M. (2006). Bioprocess Engineering Principles. Academic Press,

London

Shuler M. L. & Kargi F. (2014). Enzymes. Bioprocess engineering basic

concepts, 61- 66

Shuler M. L. & et.all. (2014). How cells grow. Bioprocess engineering basic

concepts, 155-174

Shuler, M. L. & et.all. (2001). Bioprocess Engineering: Basic Concepts.” 2nd

ed. Prentice Hall PTR, Upper Saddle River, NJ.

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