SKKB 3121

BIOPROCESS ENGINEERING 1
LABORATORY REPORT

TITLE: CELLULOSE DEGRADATION &

GLUCOSE ANALYSIS (E5)

NAME MATRIC NO
1) VICTOR LAI WEI XIANG A16KT0445
2) SOO MING HUEY A16KT0397
3) KOAY WENG MAY A16KT0143
4) CHONG FUNG CHUN A16KT0065
5) LIAU TSAE LING A16KT0162

SECTION : 03
GROUP NO. : Group 2
DATE OF EXPERIMENT : 18th OCTOBER 2018
DATE OF SUBMISSION : 1st NOVEMBER 2018
LECTURER’S NAME : DR. ROSNANI HASHAM @ HISAM
SUPERVISORS : 1) YAAKOP SABUDIN
2) MOHD HAFZAN BIN SAIDIN

DEPARTMENT OF BIOPROCESS ENGINEERING

FACULTY OF CHEMICAL ENGINEERING
UNIVERSITI TEKNOLOGI MALAYSIA
 TABLE OF CONTENTS

PARTICULARS PAGES

Task Division

1.0 Abstract

2.0 Introduction

3.0 Literature review

4.0 Methodology

5.0 Results

6.0 Discussion and questions

7.0 Conclusion

8.0 References

9.0 Appendix

TASK DIVISION

No. Content Person in charge (PIC)

1. Abstract Victor
2. Introduction Victor
3. Literature Review Tsae Ling
4. Methodology Weng May
5. Results Ming Huey & Fung Chun
6. Discussion Ming Huey & Fung Chun
7. Conclusion Weng May
8. References Weng May
 5.0- RESULTS

1) Standard curve for glucose analysis

Figure 1: Turbidity indicated by colour appearance of D-glucose

solutions at different concentrations

Samples Concentration Volume of Volume of Total Absorbance

label of D-glucose D-glucose buffer Volume Reading
solution (g/ml) solution (ml) added (ml)
(ml)

1 0.001 1 9 10 mL 0.198
2 0.003 3 7 10 mL 0.659
3 0.005 5 5 10 mL 1.087
4 0.007 7 3 10 mL 1.479
Table 1: Absorbance Readings for different concentrations of D-Glucose solution
 Absorbance against Concentration of D-glucose
Solution (g/ml)
1.6 1.479

1.4

1.2 1.087
Absorbance

1
y = 213.7x + 0.0008
0.8
0.659 R² = 0.9992
0.6

0.4
0.198
0.2
0
0
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008
Concentration of D-glucose solution (g/ml)

Graph 1: Standard curve for absorbance of glucose content analysis using D-glucose

Basic Calculations for dilution of D-glucose solution

Using glucose standard curve equation: y = 2137x + 0.0008 substitute value of absorbance at y
to calculate the reducing sugar content in every sample (concentration).

Sample 1

0.198 − 0.0008 𝑔
= 𝑥 = 9.23 𝑥 10−4 (≈ 0.001 𝑔/𝑚𝑙)
213.7 𝑚𝑙

Sample 2

0.659 − 0.0008 𝑔
= 𝑥 = 3.08 𝑥 10−3 (≈ 0.003 𝑔/𝑚𝑙)
213.7 𝑚𝑙

Sample 3

1.087 − 0.0008 𝑔
= 𝑥 = 5.08 𝑥 10−3 (≈ 0.005 𝑔/𝑚𝑙)
213.7 𝑚𝑙

Sample 4

1.479 − 0.0008 𝑔
= 𝑥 = 6.92 𝑥 10−3 (≈ 0.007 𝑔/𝑚𝑙)
213.7 𝑚𝑙
Then, using dilution equation to calculate the volume of D-glucose added (ml) by substituting
the concentration obtained above:

A) For 0.001 g/ml concentration of D-glucose:

M1V1 = M2V2
(0.01 g/mL) (V1) = (0.001 g/ml) (10ml)
𝑔
0.001 (10𝑚𝑙)
𝑚𝑙
V1 = 𝑔
0.01
𝑚𝑙
V1 = 1 ml

Volume of distilled water added: (10 – 1) ml = 9 ml

B) For 0.003 g/ml concentration of D-glucose:

M1V1 = M2V2
(1 g/L) (V1) = (0.003 g/ml) (10ml)
𝑔
0.003 (10𝑚𝑙)
𝑚𝑙
V1 = 𝑔
0.01
𝑚𝑙
V1 = 3 ml

Volume of distilled water added: (10 – 3) ml =7 ml

C) For 0.005 g/ml concentration of D-glucose:

M1V1 = M2V2
(1 g/L) (V1) = (0.005 g/ml) (10ml)
𝑔
0.005 (10𝑚𝑙)
𝑚𝑙
V1 = 𝑔
0.01
𝑚𝑙
V1 = 5 ml

Volume of distilled water added: (10 – 5) ml = 5 ml

D) For 0.007 g/ml concentration of D-glucose:

M1V1 = M2V2
(1 g/L) (V1) = (0.007 g/ml) (1ml)
𝑔
0.007 (10𝑚𝑙)
𝑚𝑙
V1 = 𝑔
0.01
𝑚𝑙
V1 = 7 ml

Volume of distilled water added: (10 – 7) ml = 3 ml

2) Cellulose Degradation

Figure 2: Turbidity indicated by colour appearance of D-glucose solution

for with and without shake

Cellulose Degradation, Absorbance

mg/mL

Shake 0.765
Without shake 0.083
QUESTIONS
Question 1: Give one example of other methods for analysing glucose and explain briefly the
principle of the method.

 A rapid and inexpensive plasma glucose estimation by two-point kinetic method based
on glucose oxidase and peroxidase (GOD-POD) enzymes has been developed. It takes
only 1.5 minutes of time and validity of the method. Enzymatic methods by using the
highly specific enzyme glucose oxidase or “notatin” were developed for the routine
determination of blood glucose level since it does not react with blood saccharides.
Glucose oxidase is a is a FAD coferment containing mutarotase enzyme extracted from
the growth medium of Aspergillus niger or Penicilium glaucum molds and is measured
in an immobilized glucose oxidase (E.C 1.1.3.4) column. For the principle of this method,
the dimeric enzyme of glucose oxidase catalyses the oxidation of -D- glucose present in
the plasma to D-glucono-1,5-actone with the formation of hydrogen peroxide as part of
the pentone phosphate pathway. The lactone is then slowly hydrolysed to D-gluconic acid.
Meanwhile, stoichiometric amount of H2O2 is also formed in the reaction. With the use
of a third enzyme, peroxidase (E.C l.ll.l.7), in a coupled reaction, the H2O2 is transformed
into water, H2O while the necessary hydrogens are removed from an organic substrate
molecule by reacting with an oxygen acceptor such as ortho toluidine or either ortho-
dianisidine. Besides, the produced H2O2 can also be transformed into a conjugated
compound in the presence of 4-aminoantipyrin and phenol. The oxidized form of ortho-
dianisidine is a coloured compound and its amount can be determined colorimetrically
and spectrophotometrically. As shown in the reaction scheme below, the advantage of
this enzymatic method will not respond to any other physiologic constituent except
glucose.

[O] + phenol + 4–aminoantipyrine → Quinoneimine dye (Reddish colour)

Schematic reaction: Principle of glucose oxidase-peroxidase (GOD-POD) method
 Rate of intensity of reddish colour formation is directly proportional to the glucose
concentration in the sample and change of absorbance per minute (Δ A/min) was
measured at 500 nm. The change in absorbance per minute of unknown plasma samples
were compared with that of standard glucose solution (100mg/dL) to find out plasma
glucose concentrations. In short, the major use of glucose oxidase has been in the
quantitative determination of free glucose in body fluids, in foodstuffs such as baking
agents, diet beer and dietetic foods, as well as in pharmaceuticals, cosmetics and
biological samples. (Basak,2007)

Question 2: Give examples of other material that contains cellulose and find out some
information on the research of degradation of this material.

First and foremost, one of the examples illustrated is cellulosic bioethanol from
generation of lignocellulosic biomass. Bioethanol is considered as an important sustainable and
renewable fuel to partly replace fossil-derived fuels that can reduce both the consumption of
crude oil and environmental pollution. Fermentation of sugar-based raw materials is referred to
as “first generation” bioethanol, whereas the use of lignocellulose raw materials is commonly
called “second generation” bioethanol. The development of this technology could deal with a
number of cellulose-containing agricultural by-products, such as wheat straws, wood trimmings,
sawdust, bamboo, and others. In this context, bioethanol contains lignocellulose, the principal
component of the plant cell walls, is mainly composed of cellulose (40–60% of the total dry
weight), hemicellulose (20–40%), and lignin (10–25%). Lignin is the principal impediment in
the production of biofuel from different lignocellulosic sources through hydrolysis,
saccharification and fermentation route. (Qian Kang et al.,2014)

 For production of bioethanol from cellulosic biomass, a pre-treatment process is used to

reduce the sample size, followed by lignin degradation that breaks down the
hemicelluloses to sugars, and open up the structure of the cellulose component. The
released cellulose portion is then hydrolyzed by acids or enzymes into glucose sugar that
is fermented to bioethanol. The conversion of cellulose and hemicellulose can be
expressed by the reaction of glucan (for hexoses) and xylan (for pentose) with water:
  There are two main types of treatment for lignin separation from the other main
constituent plant materials, hemicellulose and cellulose which are biodegradation and
the chemical degradation including base‐catalyzed, acid‐catalyzed, ionic liquids‐assisted
lignin depolymerization. Severe reaction conditions, such as high pressure, high
temperature and extreme pH, are being in practice for chemical degradation and result in
the requirement of specially designed reactors. This leads to high costs of facility and
handling along with the inadvertent generation of toxins. Biological degradation seems
to be widespread and cost effective if enzymes degrading lignin could be produced by
isolation of fungi. Bacterial strains could also be prepared that produce the lignin‐
splitting enzyme laccase.

2 stages of lignocellulose degradation:

 In the stage of natural lignocellulose degradation, many archaea, bacteria, and fungi, as
well as some protozoa, nematodes, insects, crustaceans, mollusks, and herbivores (aided
by symbiotic microbes) can be used to break down the biomass with elaborate enzymatic
machineries. Cellulose and hemicellulose are degraded directly by dilute acid, concentrated
acid, or hydrolytic enzymes (preferably such as cellulase, hemicellulase, esterase and lyase)
which may be extracellular and freely dissociated, (i) as in the case of cellulolytic aerobic
fungi or cell-bound and supramolecularly assembled into cellulosomes, (ii) as in the case
of many cellulolytic anaerobic bacteria or a combination of intracellular and extracellular
ones, (iii) as in the case of cellulolytic aerobic bacteria. The glycoside hydrolase (GHs) and
lyases cleave various backbone or side chain glycosidic bonds, while esterases cleave
various ester substituents in hemicellulose. Cellulose may also be degraded by nonspecific
attacks from radicals, exemplified by the action of brown rot fungi which use peroxidase
of H2O2-generating enzymes. In addition, natural lignocellulose degradations often involve
contributors other than the cellulolytic or ligninolytic enzymes aforementioned. For
instance, several kinds of noncatalytic “accessory proteins,” including CBM, swollenin,
and expansin, may assist biomass-active enzymes by disrupting H-bonds in cellulose.
 In the stage of industrial lignocellulose degradations (especially for those commercial
pulping and cellulosic bioethanol production) are, in general, very different from their
natural counterparts, in terms of substrate’s form, substrate-to-enzyme stoichiometry,
water content, reaction time, pH, temperature, presence of deactivators, and so on. Some
cellulolytic and ligninolytic enzymes may be applied without major structural changes,
while others may need protein engineering to make them more active under selected, non-
native conditions. Being either wild type or genetically engineered, enzymes active in
degrading lignocellulose need to be thoroughly investigated so that their potential as
 industrial biocatalysts for biomass conversion can be assessed, explored, and developed.
In this context, genetically engineered fungi that produce large volumes of cellulase,
xylanase, and hemicellulase enzymes are under investigation with regard to their structure,
activity, specificity, and stability, as well as their interdependence on or cooperation with
other enzymes, as these could convert agricultural residues (e.g., corn stover, straw, and
sugar cane bagasse) and energy crops (e.g., switchgrass) into fermentable sugars.
Additional researches also tried to find microorganisms which can effectively ferment both
types of sugars into ethanol with Escherichia coli, Klebsiella oxytoca, and Zymomonas
mobilis as promising candidates. (Feng Xu, 2011)

Moreover, another material that contains cellulose is cellulose acetate (CA) polymer in a
variety of consumer products such as textiles, plastic films, and cigarette filters. Besides
biological degradation, photo degradation is an important route for decomposing cellulose
acetate materials in the environment. There are different ways to induce photo degradation,
which have in common the formation of radicals initiated by the absorption of light. Cellulose
acetate is made from raw materials with a high content of α-cellulose, but still contains some
impurities which might be responsible for the absorption of light in the far UV light region
with wavelengths shorter than 280 nm range. Cellulose acetate has an absorption at
approximately 260 nm which is attributed to ketonic carbonyl groups. In consideration of the
fact, that the sunlight reaching earth’s surface after being filtered by the atmosphere has a lower
cut-off of approximately 300 nm, one might conclude that cellulose acetate does not
significantly photo degrade in a natural environment. This is only valid if other factors like air,
water and contaminations are neglected. Secondary mechanisms are important in the photo
degradation of cellulose acetate, which comprise other substances absorbing light and
generating radicals for reacting with the cellulose acetate structure, these include photocatalytic
oxidation or photosensitized degradation. The two latter routes can enhance the photo
degradation of cellulose acetate significantly if suitable additives are applied. The degradation
of pure cellulose acetate by irradiation with light in the far UV-range (λ < 300 nm) and the
regarding studies are helpful in obtaining an understanding of the general photo degradation
mechanism. (Puls & Wilson, 2011)
 8.0-REFERENCES

1) Juergen Puls, Steven A. Wilson (March 2011). Degradation of Cellulose Acetate-Based

Materials: A Review. Volume 19, Issue 1, pp 152–165
Retrieved from: https://link.springer.com/article/10.1007/s10924-010-0258-0

2) Qian Kang et al. (2014). Bioethanol from Lignocellulosic Biomass: Current Findings
Determine Research Priorities. Volume 2014, Article ID 298153, 13 pages
Retrieved from: https://www.hindawi.com/journals/tswj/2014/298153/

3) Anuradha Mukherjee, Tamal Mandal, Amit Ganguly, Pradip K. Chatterjee (April,2016).

Lignin Degradation in the Production of Bioethanol – A Review. Volume3, Issue2.
Pages 86-96.
Retrieved from: https://onlinelibrary.wiley.com/doi/abs/10.1002/cben.201500016

4) Anjan Basak (2007). Development of a Rapid and Inexpensive Plasma Glucose Estimation
by Two-Point Kinetic Method Based On Glucose Oxidase-Peroxidase Enzymes.
Volume22, Issue1. pp 156-160.
Retrieved from: http://medind.nic.in/iaf/t07/i1/iaft07i1p156.pdf

5) Feng Xu (12 April 2011). Enzymatic Degradation of Lignocellulosic Biomass. Published by

John Wiley & Sons, Ltd. Pages 361-366. Retrieved from:
https://onlinelibrary.wiley.com/doi/pdf/10.1002/9781118028308.ch14

6) Estimation of blood glucose by Glucose oxidase method.

Retrieved from: http://vlab.amrita.edu/?sub=3&brch=63&sim=1343&cnt=1

7) Determination of blood glucose level. Retrieved from:

http://semmelweis.hu/biokemia/files/2014/01/gy_EN_glucose-
tolerance_or_BK_20150227.pdf