00144'7
MttlttlMlll轟 )
POTENTIAL OF AGRICULTURAL WASTES FOR GLUCOSE PRODUCT10N
BY
UKIS
卜
PORNRAKSADEE
‐
THE FACULTY OF ENVIROMENtt AND RESOuRCE SttUDIES
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
(丁
ECHNOLOGY OF ENVIRONMENTAL MANAGEMENT)
IN THE
FACULTY OF GRADUATE STUDIES
OF
MAHIDOL
UNIVERSITY
1986
Copyright by Mahidol University
The Thesis Supervisory Corrnittee
UKIS
for
PORNRAIGADEE
unanimously approve the
thesis entiiled
POTENTIAL OF AGRICULTUML I,IASTES FOR GLUCOSE PRODUCTION
and recormend the candidate
to
subnrit the thesis
to the
Faculty of Graduate Studies, Mahidol University for the
MASTER
0F SCIENCE degree
on
19r
[,t,iylg66
Ukis
. .$.-,
Pornraksadee
.W:::...... .....
Rungjarat Hutacharoen,
M.Sc.
Major Advisor
....?...
Thanakorn Uan-0n, Ph.D.
Coadvisor
.
!i o lr
Pisit
"
.
"{:l:1
.n!:lYl:.T:
;
Sukreeyapong, M.Sc.
Coadvisor
Monthree Chulasamaya,
M.D.
Dean
Faculty of Graduate Studies
Mahidol University
Copyright by Mahidol University
The Thesis Defense Cormittee
UKIS
for
PORT{RAKSADEE
unanimous'ly approve
the thesis entiiled
P0TEI'ITIAL 0F AGRICULTURAL IIASTES FOR GLUCOSE PRODUCTION
and agree
that he has satisfactorily
and be awarded the degree
of
defended
his thesis
of Master of Science (Technology
Environtnental Management) from litahidol University on
31 0:11980
(L,**
Thanakorn Uan-On, Ph.D.
Cha irman
,,4. A*Ut^.vma^,a.
""'d.'...
l'lonthree Chulasamaya, l,l.D., ph.D.
$1ipy"l.--., O[ ). ^*-V--x\ ".:Sirichai Chinatangul , Ph.D.
Member
Member
..{t.
J*::ly;:=. :............
RungJarat Hutacharoen, M.Sc.
M
ember
..t(.(fum6u.."...
Monthree Chulasamaya, M.D., ph.D.
D
ean
..1ilu"un*,*.-.-Thanakorn Uan-0n, Ph.D.
Dean
Facultjr of Graduate Studies
Mahidol University
Faculty of
Envi ronment
and Resource Studi es,
f'lahi do
l
University
Copyright by Mahidol University
BIOGRAPHY
NAME:
UKIS, PORNRAKSADEE
DAttE OF BIRTH:
octOber 3, 1945
PLACE OF BIRTH:
Saraburi, Thailand
EDUCAT10NAL INSTITUT10N ATTENDED:
Taweethapisek Schoo19 Bangkok
March9 1962 : Cert. of Mathayom VI
March, 1964 : Certo of Mathayomsuksa v
(Self Study)
College of Education Bangsaen
March, 1968 : B.Ed.
Copyright by Mahidol University
￨
ACKNOt^lLEDGEMENTS
to express his sincere gratitude to
Uan-On, Dean of the Faculty of Environment and Resource
The author wishes
Dr.
Thanakorn
studies, Mahidol university, and Assistant professor Rungjarat
for his valuable guidance and encouragement through out
the study of this research.
He is grateful to Assistant professor Pisit Sukreeyapong,
Hutacharoen,
Mr. Somchart Roong-In from Pulp and Paper Laboratory Department of
Science Service, Mp. Niyom Petchpud from Department
tions Faculty of Forestry, Sublieutenant
of Forest Produc-
Sombut Tuengwiwat from
Pathology Departrent, Police General Hospital, and Dr. Sirichai
Chinatangul
for their valuable help in
is also grateful to Mrs. Emma Jalin
help in English comments.
He
valuable
comments.
Suitisang
for
her
to thank the Fermentation Technology Laboratory
and Bangkok MIRCEN for providing the micro-organism I.oirile Q.M.9414
Finally he wishes to express his gratitude to Dr- Nart
He wishes
Tuntawiroon and
orther teachers for their unselfish efforts
and
inspirations given.
Copyright by Mahidol University
Thesis
Title
Potenti
al of Agricutural Wastes for
Glucose
Production
Ukis
Narne
Thesis
Advisor
Pornraksadee
Thanakorn Uan-0n
Rungi
arat
Pisit
Academic
Year
Hutacharoen
SukreeYaPong
1986
ABSTRACT
This study
is to i nvesti gate production of glucose from
cellulosebyusingenzyneextractedfroml.ltitideQ.M.9414byinduction rcthod, This study use agricultural wastes which are corn stover,
rice straw,
sugarcane bagasse and cotton seed
sodium hydroxide
The
hulls as substrates
and
solution as the delignifying agent.
first
step in the experiment
is the
production
by growing the inoculum on gyratory shaker (120 rpm)
of
enzyme
at 28'C,for
5 days. The enzyme should hydrolyze substrates such as corn stover'
rice straw, sugarcane bagasse and cotton seed hulls at pH 5 and
i ncubated
at 50'C for 8 hours. Before the different substrates
hydrolyzed, they are pretreated by using
in order to
separate
ls
be
sodium hydroxide solution
lignin from the substrated. After the substrates
are being hydrolyze<!, glucose
is
produced. Ilhen corn stover, rice
straw, sugar cane bagasse and cotton seed hulls are used as substrates,
glucose
order.
iS
produced o.oI7%, 0.0t78%. 0.0134%, 0.0267%
in chronological
Copyright by Mahidol University
economic
In addition, this study is in connection with quality and
patential of agricultural wastes (corn stover, rice straw,
sugarcane bagasse and cotton seed
hulls),
of
of quality potential of the agricut-
glucose production. The study
tural wastes, it is
found
cellulose content with
that cotton
and study
seed
hulls
of
estimated costs
has the highest
rice straw having the least cellulose
content with 33%. As to the aspect of economic potential, rice straw
59%
has the highest economic
easy
and
potential due to
collection, cotton seed hulls
has
its
abundancy, low
price
and
the lowest economic potential
of its few quantity and high price. In the analysis of
costs of glucose production from the agricultural wastes, it was found
because
out that the cost
stover),
1236
is very high which is
tiath/kg (from rice straw), 1570 Bath/kg (from
bagasse), 97A Bath/kg (from cottonseed
glucose
1341 Bath/kg (from corn
hulls).
is only about 14 Bath per kg, thereby
The market
showing
profitable to do glucose production from cellulose.
development
this
in
technology
that
sugarcane
price of
it is not
If in the future
is achieved, reducing the cost of production,
study may contribute greatly to economic efficiency.
Copyright by Mahidol University
TABLE OF CONTENTS
PAGE
CHAPTER
l
INTRODUCT10N ―‐“―――…――¨――――‐‐‐―――――――――――――=― ―‐-
1
― ――――¨¨――――――――‐――1. GENERAL SITUAT10N ―――――
・
1
¨―¨――――――――――¨―‐-
4
2. SIGNIFICANCE OF THE STUDY
¨―‐―‐――――¨――――――――‐‐‐
3. THE PURPOSES OF SttUDY
――――――――中――――¨‐―ニーー‐____
4
BACKGROUND ――――――――――――‐―‐――¨¨―――‐―――――¨“――――――-
8
1。
CELLULOSE ―…―――‐――――――¨¨‐―――――……――‐―――――――-
8
2。
SOURCES OF CELLULOSE
4。
CHAPTER
2
4
SCOPE OF THE STUDY
――“‐―――――‐―‐―‐―‐―‐‐―……
2。
l Forest production ¨―――――………――‐‐‐¨‐‐―――-
10
2。
2 Agricultural production
――――…―――――――――-
11
2.3 Weed ―――――¨――――¨――‐――――――――――――‐―¨‐――……
2.4 Solid waste of industries
2.5 Municipal solid waste
3. NATURE OF CELLULASES
“―‐―――……――――-
―¨―¨¨―――――………――……
¨――“――¨‐――――…―‐―――――‐-
3.l Cellulytic enzymes
―‐―¨“―‐――――――――‐―¨―‐
――‐――‐―‐―‐‐―‐-
3。
2 Properties of cellulases
3。
3 Mbde of action of cellulases
3.4 Activity determination
13
13
15
―――…―‐―¨―‐――――……
20
l Strain development ――………――¨―‐―‥‐――――‐‐―
5。
2 Mbdia and cultural condition for
5.3 rン づ ο′aコ蘭α cellulase ―“―――‐――――‐―‐―‐―
６ ７
２ ２
――――――――――‐‐――…
４ ４
２ ２
‐―“……―――‐――
5。
`乃
13
17
VRIDE Q.M. 9414 -―
production of cellulase
13
――““‐‐―――‐
MICROBIAL SOURCES OF CELLULASE ――‐……………―――¨
5. rRIαπθDtt
12
２
２
4。
10
Copyright by Mahidol University
PAGE
6.1 PretreaflEnt
6.2 Enzymatic hydrolysis __― ―___¨ ―――――‐――‐‐‐¨
CHAPTER
3
MATERIAL AND METHOD
９ ９ ２
２ ２ ３
6. HYDROLYSIS OF CELLULASE
35
1. SUBSTRATES
35
of substrates --------------1.2 Pretreatment of the substrates
35
1.2.1 Physical pretreatment -----------__
.|.2.2 Chemical pretreatment
-------------
35
1.1 Preparation
2. CELLULASE ENZYM[ PRODuCT10N
35
35
‐―――‐―――‐―――¨‐――-
36
2.l Micro ―organism __… ……____― ―――‐―――¨――‐――-
36
2.2 Cultural medium ―¨__¨ _― ――――¨‐―――‐―――‐――¨-
36
2.3 Culture conditiOn and innoculum
preparation ………………―__― ―――‐―‐‐――――‐‐――¨―‐-
36
3. ENZYMATIC HYDROLYSIS OF CELLULOSIC MATERIAL
(ENZYME SACCHARIFICAT10N)― ‐‐――‐――――――‐――‐―‐… 37
4.
ANALYSIS METHOD ――――――――――――――¨¨‐―――‐――¨――‐¨-
37
_α
37
4.l Growth ofコ ンづ
`λ
aご
υ うごο Q.M. 9414 ¨=′
4.2 Residual dry weight determinatiOn
4.3 Cellulase assays
‐___¨ ――――‐―‐――――――‐――¨¨-
4.4 Determination of reducing sugar
CHAPTER
4
――――……‐
‐―¨___¨ _-
RESULT
37
37
38
39
1. POTENTIAL OF RAW MATERIALS
1.l Qualitative potential
――¨¨―‐――_‐ ‐‐‐―¨‐――
39
―二¨―___― ¨―――――‐――――
39
1.2 Economic Potential ―__¨ ¨____¨ ――――¨¨――¨¨――
41
Copyright by Mahidol University
PAGE
２ ３
ENZYME PRODUCT10N
41
PRETREATMENT
42
3.l Physical pretreatment _… ______‐ ‐―‐¨――――‐―
42
3.2 Chemical pretreatment _____― ―――――‐¨―‐‐―――
43
4. MOISTURE OF AGRICULTURAL WASTES ―――――‐___‐ ‐――
44
5。
ENZYMATIC HYDROLYSIS OF CELLULOSE
(SACCHARI FICAT10N)― ――¨――‐―――――――――‐¨――――¨‐――
CHAPTER
5
45
COST ESTIMAT10N OF GLUCOSE PRODUCT10N FROM THE
AGRICULTURAL WASTES
――――――――――――¨―――‐――‐―――‐‐―――‐‐
47
1. DIAGRAM OF THE PROCESS OF GLuCOSE
PRODUCT10N ―――――‐――――――¨―‐―――‐‐――¨‐―‐――‐‐―――-
47
2. UTILITY UNIT COST ――――――___― ――――‐―¨――¨―――¨―‐‐
48
3. CHEMICAL F9R GLuCOSE PRODuCT10N
49
―‐___‐ ‐―――‐―-
4. HYDROLYSATE AND PERCENT OF CELLULOSE
CONVERS10N ――――――――――――――――……――‐――――――‐――――‐‐
50
MATERIAL BALANCE FLOW DIAGRAM _1__‐ ¨___― ¨‐‐―¨
51
6. COST ESTIMAT10N ‐¨―――――――――――――――――‐‐――¨――¨――
55
l Chemicals fOr enzyme productiOn cost _― ――
55
6.2 Glucose production cost _____― ‐¨―――¨‐――――
56
DISCuSsloN ―‐――‐―――――‐―¨―――‐―――――‐―――‐――‐――¨―――‐――
61
5。
6。
CHAPTER
6
1. OVERALL RESuLT ‐‐―――――__― ――‐―――‐―‐‐―――‐
61
2. THE COMPARISON BETWEEN COST oF GLUCOSE
PRODUCT10N FROM THE AGRICULTURAL WASTES ―――¨_
62
Copyright by Mahidol University
Ｅ
Ｇ
Ａ
Ｐ
7
CONCLUS10N AND RECOMMENDATION
５
６
CHAPTER
1. CONCLUS10N
2。
RECOMMENDAT10N FOR FURTHER STUDY
65
‐‐―¨_― ――――――――
BIBL10GRAPHY
66
68
APPENDIX I
CALCULAT10N 中―‐_¨ ――――¨――‐―――――“―――――――――――………―――
74
APPENDIX II
BUFFER USED IN THIS sTUDY
78
APPENDIX III
EFFECT OF MILLING ――――‐――――‐―――――――¨―¨―――‥―‐――――
79
APPENDIX IV
MESH SIZE ¨‐―¨―¨‐¨¨"― ―‐――¨―¨―¨――¨―‐――――¨―……――――¨
80
APPENDIX V
EFFECT OF pH DURING HYDROLYSIS __― ____― ―――――‐―――
81
APPENDIX VI
丁HE PRODUCT10N AND HARVESTED AREA OF AGRICUL‐
丁URAL PRODUCTS IN VEAR 1984/85 -― ―__― ―――――――――――
APPENDIX VII
CONVERS10N FAC丁 ORS FOR UNITS usED IN THIS
APPENDIX VIII
PILOT PLANT ―――――____― ――――‐¨―‐――――¨‐―――‐―――――………
APPENDIX IX
COS丁
APPENDIX X
GLUCOSE PRODUCT10N COST (INCLUDE COST oF
OF PRINCIPAL EQUIPMENT
ENERGY)― ――¨――――――――"― ――¨中――¨―¨――――¨―――――――――――-
４ ５ ６
８ ８ ８
STUDY ―――¨……――――――――――――――‐¨―――――――¨―――‐――――――――
88
Copyright by Mahidol University
LIST OF TABLES
TABLES
PAGE
l.
Cellulose content 9f CrOp plants and residues
2.
Some
――__― ――――‐ 12
properti es of cellulolytic enzyme isolated frOm
Tticlpdanna oitide
3.
16
Relative cellulase activities of the component of
!,lonhgii
cellulase alone and in
for
combi
nation __-__-____ lg
degradation
4.
A mechanism
5。
lbtblpdqna strains in Natick col lection
6.
r.ofuide
7.
lGthods
8.
Compari son
medium
for
enzymatic cellulose
for cellulase
pretreatment
of physical
25
production
of cellulose
Zl
------____
and chemical pretreatrEnt
ZO
Zg
of
cellulose
32
9.
The chemical composition
10.
The production
11.
Price of agricultural wastes
of
some
of agricultural
agricultural wastes __-
wastes (1984/85)
40
------
12. Relationship betrieen yield of milling
39
40
substrates
and
42
13. Delignification of cellulosic material with sodium
‐―
¨¨―‐¨――‐――――――― ‐―――¨‐――‐―¨‐―
４ ５
Moisture of agricultural wastes
…
―‐――___― ‐―――――¨‐¨――‐¨‐――
３ ４
４ ４
hydroxlde ―‐――__― ――■―‐
Hydrolysis of delignified cellulosic material with
enzyme of r.υ ′rぅ′ο ―――‐―――――――‐――‐‐――¨―‐¨――‐――‐――‐‐――‐‐― 45
Copyright by Mahidol University
TABLES
PAGE
16.
17.
18.
Utility unit cost
48
------Hydrolysate of enzyne hydrolysis
50
conversion
50
Chemicals and chemical
of cellulose
price
49
19.
Percent
20.
Chemicals
21.
Glucose production
cost (from corn stover)
22.
Glucose production
cost (from rice
23.
Glucose production
cost (from sugarcane bagasse)
24.
Glucose production
cost (from cotton seed hulls) ________ 59
25。
Estimated glucose production cost
26.
Mesh
27.
The production and harvesled area
for
enzynre
production cost
________
______________ 56
straw)
Sl
--------
(
5g
63
size
prcducts
55
gO
of agricultural
1984/i985)
83
of principal equipment
28.
Cost
29.
Glucose production
cost (from corn stover) _________-____
30.
Glucose production
cost (from rice
31.
Glucose production
cost (from sugarcane bagasse)
32.
Glucose production
cost (from cotton seed hulls)
86
straw)
8g
g9
--------
90
9t
Copyright by Mahidol University
LIST OF FIGURES
FIGURES
l.
PAGE
Possible routes to petrcchemicals from cellulosic
wastes.
From Brenner
2.
0peration plan
3.
Flow chart
of
of
et al. (1977)
research
experiment
tork
________
process
4. Cellulose
5.
3
6
-_-_______
-------------
Structure of cellulose
in
biomass
naterials
g
:
(a) cellulose, (b) cellulose crystallite (c) cellulose
crystallites in microfibril
6.
Tzblodqna otuide slrain imprcvement by mutation
ultraviolet; N.A., not analyzed.
Hydrolysis
8.
Diagram
9.
Material balance flow diagram for integrated
____
46
production
47
of the
process
of
of
glucose
glucose production from corn
10. Material balance flow diagram for
processing scheme
of
integrated
glucose production from rice
-------
52
11. Material balance flow diagram for
processing scheme
bagasse
26
of delignified agricultural wastes
processing scheme
straw
9
UV,
From Ghose (1978)
7.
7
of
i ntegrated
glucose production from sugarcane
53
Copyright by Mahidol University
FIGURES
PAGE
12. Material balance flow
processing scheme
of
diagram
for
integrated
glucose production from cotton
seed hulls
13.
54
cost of glucose production from the agricurturar
wastes
14.
64
Effect of milling on the susceptibility Of Solka Floc
fraction passing
sw40
400 mesh
but not passing 500 mesh,
ball milled fraction passing 400 mesh but not passing
500 mesh
15- Effect of
79
pH on
cellulases.
the activity of wiclod,anna oi.ride
Formation
solution from a
of
glucose with a
dilute
enzyme
of solka Floc within
1 (r-r), 2 (H); and 3 (o-o) days of hydrolysis ---
16. Effect of
pH on
cellulases.
2%
suspension
g1
the activity of
Formation
of
glucose with a
dilute
enzyme
solution from a 4.6% suspension of furfural process
waste
days
within I (o*o)., 2 (e-.r), 3 (+o) and 4 (o--o)
of hydrolysis -------
_--___---__--
gz
17.PitotplantprocesSofglucoseproduction-.--.
Copyright by Mahidol University
CHAPTER l
lNTRODUCT10N
1.
GENERAL SITUAT10N
The food and energy shortages and
related to the
economi
c
change
of
many
oil crisis
countries, especially the
loping countries which do not have their own sources
So, besides trying to explore their own sources
kinds
of
renewable sources
of
have severely
energy have
to
of
of
peteroleum.
petrcIeum,
be searched
are also emphasized on the use of various kinds
of
deve_
for.
all
Interests
renewable sources,
solar, water, wind, etc., as well as energy from the bioconversion of organic matters. The source of energy which is one more
such as
attracti ve for the agricultural developing countries, is cellulose.
Cellulose, a carbohydrate polymer occurs in the primary and
secondary cell walls of plants. unlike the limited reserves of fossil
reduction of c0,
in photosynthesis. The sunright used in the photosynthetic production of cellulose is free, but the fossil sources of
energy are grcwing scarcer and dearer. Because
ways
to utilize the sunlight
conserved
of this,
we seek new
in the cellulose skeleton of
pl ants .
is the most abundant organic material that can be
of food, fuels, and chemicals. The net world wide
Cellulose
used as a source
production
of cellulose is estimated at I00 billion tons per year
(Spano, 1976). This is approximatly 56 kg (l5O Ib) of cellulose per
day for each and every one of the earth's 3.9 billion people. The
energy to produce this vast quantity of cellulose comes from the sun
and
is fixed by photosynthesis.
The utilization
of this annually
Copyright
by Mahidol
University
replenishabre resource
is greaily simprified if it is first hydroryzed
into glucose. From glucose, various sources of food consumabre
by man
derived. In addition, it can be used as a feedstock
to make solvents plastics, and other chemicals now made from petroleum;
and animal can be
it
can be converted microbially
into single cell protein or it can be
fennented to a clean burning fuel, such as ethanol. The various
routes for the utirization of cellurose and'the possible routes
to
petrochemicals from cellulose are shown
in Fig I. This i nterest in the
earthrs [Dst abundant renewable resource is due to a combination of
several factor the energy crisis, food and feed protein shortages and
envi roumental pol
lution.
Copyright by Mahidol University
︵
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.2
、
日
‐
O C
ミ
=
lll:
ξ
=‐
=0
H
・
O
C
2
●
0
ヽ
‐
・
■J
ヽ
‐
O
=C
0
8 _0
も
0
0
0
0
Copyright by Mahidol University
4
2.
SIGNIFICANCE OF THE STUDY
Thailand
is
an agricultural country
that
has produced large
of agriculture products and left a lot of agricultural wastes
in each year. The agricultural wastes includes crop wastes,
amount
such as
rice straw,
bagasse, cotton seed
hulls, corn stover, etc.; forestry
residues, such as saudust i and animal wastes (manures).
ihe agricul_
tural wastes have high percentage of cellulose. If they are produced
to glucose, their value will be added.
food fuel and other chemical products.
cheap
it
changed
From glucose we can change
Because
to
agricurtural wastes
are
the production cost of glucose which can be
This kind of fuel may be used instead of fossil fuel
may reduce
to fuel.
in the future.
In order to find
some
apprcpriate alternatives, develope
alternative sources of energy in form of grucose frrm ceuurose and
encouragement of the maximized utirization of the agricurtural
wastes,
re ought to have a primary research of the potential of agricultural
wastes as cellulosic raw material for glucose production.
3.
THE PURPOSES OF STUDY
3.1
To analyze and evaluate the potenti
wastes (Cellulosic materials)
3.2
agricultural
4.
for
al of
agri
cu
ltura I
glucose production.
To analyze estimated cost
of gluiose production
form
wastes.
SCOPE OF THE STUDY
4.I
rice straw,
The study
of celluiose from agricultural wastes, such
and cornUniversity
Copyright by Mahidol
stover
sugarcane bagasse, cotton seed hu1ls,
:4
4.2
4.3
The study
of
enzyme production
The study
of
glucose production from cellulose by hydro-
lysis of tuinhodmna oinide
4.4
4.5
from
ryiehod,enna oi,ride
enzyme.
of potential of agricultural wastes
The study of estimated cost of glucose production
The study
the agricultural
from
wastes.
In order to achieve the above
posed purposes, steps
of
work
plan. This operation plan is
show in Figure 2 and the experimental process of research work is
designed as demonstrated in Figure 3.
are divided as shown in the operation
Copyright by Mahidol University
DATA COLLECT10N
FIELD SURVEY
EXPERIMENTAT10N
DATA ANALYSIS
UTILIZAT10N
EVALUAT10N
CONCLuS10N AND RECOMMENDAT10N
Fig。
2
Operation plan
of
research work.
Copyright by Mahidol University
A survey
of agricultural
Utilization
wastes
Selected raw materials
High percent
of cel lulose
Selected substrates
Cel
lulose
Hydro
Hydrolysis
lysi
of
analysi
s
Cel
lulose
Determi nation
of
s
sugar
Final analysis
Fig.
3
Flow chart
of experiment
process.
Copyright by Mahidol University
CHAPTER 2
BACKGROUND
1.
CELLULOSE
Biomass
materials in general consist of cellulose,
hemicellu―
lignin in the ratio of approximately 4 : 4 : 3 (1).
cel I u lose is linear polymer of D-anhydro glucopyranose units
lose
and
The
linked by
9-1-4 - glucosidic bond, as shown below.
T
9H
ズ k:
「_。 ハH
りH
Fig 4, Cellulose
of xylan, a polymer of B - 1,4 - liked
xylose units, having arabi nofurano sy I ,4 - o - methyl glucuronosyi,
hemicellulose consists mainly
acetyl and substituted cinnamyl substituents (Wilkie, 1979). Lignin
is a polyphenolic
polymer containing phenylpropane
and has been reviewed
and Ludwing,
earlier
(Freudenberg and Neish, 1958, Sarkanen
1971). As well as these major constituents the
material also contain small amouts of
aci
structural units
ds, and ash.
biomass
silica, pectin, protein,
uronic
Copyright by Mahidol University
Ce:lu:ose― 'Jo branchOs
G・ G・ G・ G・ G‐ G‐
G
Gし
C‐ 6‐ 6‐ G・ G・ G‐
G
G‐ G‐ G・ G・ G‐ G・
●、
6・ G・ 6‐ 6・ 6・ G・
G‐ G・ G・ G・ G‐ G・ G・ 6・
G
こ
β
q
・6・
G・
・
‐
6‐ C‐ G・ G‐ G‐ G
p_G・
G G
G‐
6‐ G‐
・G・ G‐ 6・ G
●
G・ G‐ G‐ G‐ G・ G・ G・
6‐ 6‐ G・ G・ G・ G‐ G‐ G‐ G・ 6‐ G・ G・ G・ 6‐ G・ G・ G‐
G.
G
G
.
Square
crOss section
Folded『 ibbons
︵︵︵︵︵＾︵
︵︵︵︵︵︵
慾
Longiludinal
seclion
,,h,,n^,]
Cellulosc
crvslalli!e
il.],fl
●
120A
30A
Fig.5. Structure 6f cellulose in
biomass
materials; (a) cellulose,
(b) cellulose crystallite, (c) cellulose crystallites in
mi
crofi bri
I.
Copyright by Mahidol University
l0
The number
of
glucose units per rclecule (degreg
of
polymeri_
zation) range from as Iitile as 15 or less to, as high as
10,000 _
14,000 (2). Both starch and cellulose could be
thought of as poly_
glucose. Starch has a structure which is readily hydrolyzed:
Incomparision, Cellulose (Fig 5 a) form a crystalline structure
(Fig 5 b) which protects the internal bond from
hydrolysis (3). Also,
cellulose in plants is protected by lignin. This potyphenolic
material forms a seal around the cellulose
protection against hydrolysis (4).
2.
(fig
S
.) for further
SOURCES OF CELLULOSE
is our rost abundant natural product. The crude
cellulose content of agricurtural crops varies four fold from about
20% 6f dry weight for bluegrass to 91S for cotton
fiber (5) on tne
Cellulose
other hand, the ahpha
from about 40
-
cellulose content of
to slighily
Major sources
rnore than 50%
wood ranges narrowly
of dry weight (5).
of cellulose are
l)
forest production
2) Agricultural production 3) weed 4) sotid waste of industries.
5) Municipal solid waste.
2.1
Forcst production
It is not suitable to use forest wood as a source of
cellulose,
The
because Thai
forest
was destroyed by people
aerial photo survey in 196r showed that
area
of the country or
However
area
17.l
million rai
the satenite-photo survey
in
53%
at high rate.
of the totar
were covered by
1973 showed
land
forest (66).
that the forest
in Thailand was 43.20% of the country,s total land area, and
Copyright by Mahidol University
1t
of the country,s total land area in 1g7g (67). At present
(1982) the forest area in Thailand is
estimated at 30.% (66).
34.15S
However,
there
is
another way
by raising quick growing trees
in
to
produce cellulose from rcod
vacant areas. In the selection of
of tree to be planted, the following should be considered :
1) lle have to choose fast growi ng trees with their maximum
lenght of
gncwth for 5 years. lle have to consider y.ears
5
the maximum to
species
economi
that
ze funds and save
interest. 2) l,le have to
be aware
of
insects
attack the tree planted; the endurance of tree against
diseases. 3) The output vre can get per rai. 4) Tne best way of
reproduci ng plants should be studied. 5) l,le should
consider also
may
the
of the plants to the climate changes, etc. There are many
of trees which the Forestry Division suggests people grow such
endurance
species
as:
Leucaena leucocephala
Acacia auri cul i formi
s
Casuarina equi seti fol i a
Casuarina j unghuhni ana
Eucalyptus spp.
Ayadi
rachta
i ndi ca
The quick growing trees mentioned above are rnore
suitable for
produci ng
cellulose than another kinds of the trees because of their
duration in growing is shorter.
2.2 Agricultural products.
cultural products
such
},Ie can
as, cotton, kenaf
and
get cellulose from agri -
agricultural
resi dues
(wastes ) .
Copyright by Mahidol University
12
Table
1.
Cellulose content
Crop
of
crop plants and resi dues
Cellulose content ( % )a
Component
Peanuts
hul ls
49
Ri ce
hulls
42
straw
32
hay
51
huI Is
31
hay
52
hul ls
42
mature pl ants
48
bagasse
62
wheat
straw
50
Corn
stover
36,
Cotton
seed hul ls
60
f,i ber
91
grai
n
1B
straw
40
hulls
51
Sorghum
Soybeans
Sugarcane
0ats
aBased
Source
:
on oven dry wight.
C.R. Hilke "CeIIuIose as a Chemical
Net,l
2.3
43
and
Energy Resource"
York, ,lohn I'liley & Sons, 1975, p 29.
Weeds. l,Ie can get cellulose from many kinds
of
weeds
as water hyacinth, cogon grass, kajonjob grass, etc.
Copyright by Mahidol University
t3
2.4 Industrial
SoIid wastes such as waste paper pulp, waste
texti Ie materials etc.
2.5
Municipal solid waste, such as waste newspaper, refuse
paper etc.
3.
NATURE OF CELLULASES
3.1 Cellulolytic
enzymes
of crystalline cellulose is a complex
process' requiring the participation of many enzymes. It
is not., t{ell
established that there are at least three di fferent types of celrulolytic activity : exo- B-1 , 4-glucanase, endo-B_,l, 4-glucanase and
The degradation
-glucosldase- A strong synergistic effect has been observed
exo-and endo-glucanases hydrolyzing
crystalline
cel
but not
chain cello
-
when
cellobiose and short
glucose, but have no effect.on
-
cellulose.
aryl-B-glucosides, but not cellobiose
concerning the nature
forward by Reese et
of
enzymati
aI. (g).
(7).
lulose (Avicel ),
i gosacchari
des to
some B-glucosidases
attack
The
or
first
hypothesis
c hydrolysis of cellulose
They reported
between
luas put
the existence of
a
nonhydrolytic enzyne, C,, which initiated the hydrolysis of native
cellulose by breaking hydrogen bonds between cellulose chains. This
first step was a prerequisite for hydrolysis by hydrolytic enzyme,
Cx (9). It was also bel ieved that those microorganisms unable to
grow on native cellulose
did not subsequently been extensively
ques_
tioned. In particular, the nature of the initial step in the
hydrolysis of native celrurose is stiil obscure. At present the most
generally accepted view is that C,, -enzyme is an exo-B-1, 4-glucanase.
Copyright by Mahidol University
14
In the case of tutbhodana oiride
and
?ptbhadana koningi.i purif ied
c1
to be a cellobiohy-drolase (10). Clearly we should now
reconsider the theory of Reese and corcrkers and redefi ne the mechanisms
of cellulase action in the light of new understanding of the properties
has been shown
of cellulase.
Accordi ng
to the present view,
cooperati ve action
of
- and exogrucanases hydrolyzes crystalline cerlurose to solubre
cello - o I i gosacchari des mainly cellobiose, which is released by
endo
exoglucanases (6).
3.1.1
.
Exo
Wood
produced by wicltodqna
components were
-
and Endoglucanases
and McCrae separated the cellulase complex
koniqi.i into
pure components. These
exo-B-l, 4-glucanase (C,),
endo_p_1, 4-glucanases (C*)
and B-glucosidases. The complex thus contains many isoenzymes. The
splitting off cellobiose from the non-reducing end of
exoglucanase was
the cellulose chain
(r1).
it
Thus,
may be
systematicaily designated
B-1, 4-glucan cellobiohydrolase. The endoglucanases hydrolyze B_,l,
4-glucan
in a random fashion
and are systematically called B_1,
4-glucan glucanohydrolases. The endogrucanases can be differentiated
by the randomness
by the rate
of their attack
of sorubirization of
on carbosymethyl cellulose (CMC)
phosphoric acid-swolIen cellulose (1r).
Pettersson fractionated the cellulase complex
oiride into
of
lyi,chodesna
pure components. The components were endoglucanases,
exoglucanase, and cellobiase. The exoglucanase was shown
ce I
and
lobi ohydro I ase
,
which was inhibited by
cellobiose.
to
be
a
Consequently
g-glucosidase greatly accelerates the action
of exoglucanase on
micnccrystalline cellulose by renovi ng cellobiose. It there for
seems
Copyright by Mahidol University
t5
clear that fungi produce at least five different endo_B_1, 4_glucanases,
the old Cr-components, varyi ng in degree of randomness of hydrolytic
action.
So
for only
one exo-E-|, 4_glucanase has been
purified
and
fully characteri zed. It
has, however, been shown clearly that all
known organisms hydroryzing native ceilulose are able
to produce at reast
one exo-B-glucanase. In the case of ?.oi,ride, I.koni.ngii and
S.pttlo*ttZenhtn, this enzyme
It
is
B-1
has also been claimed
,
4-glucan cellobiohydrolase.
that fungiproduce a B-1,
4-glucan
glucosylhydrolase, but none of these enzymes has been isolated
in a
pure state. Preparations releasing glucose from cellulosic
substrates
have been isolated from culture media
nig
of r.oiride (12)
a (13). tbwever, thes preparations
completly exclude the present
3.1.2
is
illue
were not pure enough to
of cellobiose.
B-Glucosidases
The
cellulose
and Aeperg
third activity involved in the
breakdown
or cellobiose, which hydrolyzes
B-glucosidase
of
mainly
cellobiose' but also higher cellodex-trins to glucose. These enzymes
accelerate the hydrolysis of crystalline cellulose by removing cello_
is an inhibitor of exo-p-g r ucanase. g-Grucosidases are
widespread in fungi. Bucht and Eriksson (7) isolated both
B-glucosidase
and aryl-g-glucosidase fron stq,an eoquinolelhat" L koningii produces
biose' which
two B-glucosidases
3.2
(
l4).
Properties
of
cel lulase
The molecular weights
28300 and 37500
have also been
(i5).
found.
of
endoglucanases vary between
Small differences
The
in the
amrno-acid composetion
isoelectric points vary between 4.20
and
Copyright by Mahidol University
16
5.32 making possible their separatiOn by isOelectric fOcusingo
with
the exception of One compOnent, all endoglucanases are glycOproteins。
In Table 2 some properties of cellulases isOlated from
summarized according to PetterssOn (16).
exo‐ and
endoglucanases of r.υ
=′
ιづ
レれι
z″ ∫
ο′ι
ο―
Paれ
ぅ′夕,
r。
.υ
『
ι′ σ are
`′
The molecular weights Of the
た
。4.4g`づ , Fレ ,α ′ι″η`ο ι
αηιand
lie in the reglon 40,000 .... 75,000, with the
`ο `ι
exception of the low‐ molecular― weight compOnents frOm r.た ο′じ′ jt and
ク
r.υ ι
′ぅdι .
T8Ы ●
2
These have a mOlecular weight of 12,500 。... 13,000 (16)。
+
+
+
―
●
+
―
一
properties
5,71
＋
Thermostabi
47000
一 ●
,-Glucorid.ic
０
ll
42000 379
12500 460
50000 339
２
Endo.r.t,4.tluc.n.tc
I
C bo. CMC Micro. R.prcci. Ccllo.
hydr.tc
Cryrt!|. Dit.tcd trt.
contcnt
lina
aallulo!. ,.oic
(p., enl)
ccllulotc
１
c. nrsc
ndo-r.t ,4.tluc.n.t.
Aclivity tow.rd diff.rent tub3lralct
９
Exer-l J -!lu
価
山
﹂
恥
咄
山
Typc ofcnzymc
E
c en2ym● S idated iom 77た 力
ο
d″ π″ッ
″ldg(16)
somcP=openLs oFcenddy●
+
lity is one of the most important
of cellulases, since the hydrolysis of
faster at higher temperatures.
technical
cel lulose proceeds
Endoglucanases are more
exoglucanases. Endoglucanases are quite stable
6o'c ano pH 5.0 B-Grucosidase and exogrucan
ase
for
up
stable than
to 4 hrs at
of r. kDningz:i resemble
in their heat stability at 60'c : they roose about g0% of
their original activity at 60.C and pH S.0 in 4 hrs (9). In the
present of cotton the cellulases of ?.l<oningii and F.eoZoti
are
one another
remarkably stable, showing no ross
of activity
when incubated
for
4
Copyright
by Mahidol University
weeks at 37'C and pH S.0
(17).
17
3.3
t'bde
of action of
cellulases
It is now weH estabrished that ceilurase is a murticomponent enzyme system- using various
chnrmatographic and electrophoretic
techniqueS, the comprex has been resolved into
its
it
component
part
and
has become apparent
that there are at least three different types of
enzyne in the complex. These are as follows (11).
1- Ct component. This feature
considered
fi
is
so named because
to initiate attack on crystalrini cellulose (e.g.,
it
was
cotton
ber) .
2- B-1, 4-glucanases.
These are
also called c,
enzymes.
C, enzlmes can hydrolyze soluble derivatives of cellulose
or swollen
and
partlally
degraded
ceiluroses.
cMc
is
normally used for the assay
of C, activity.
3- B-glucosidases. These hydrolyze cellobiose and short
chain cello-oligosaccharides.
The
to
glucose but have no
syergistic properties of the
complex enzyme were studied by lr'lood and McCrae
effect on cellulose.
component
(17).
parts of the
They could
reconsitute the cellulase complex from the fraction because
the
recoveries of the enzymes and protein were very high, over g0% (14).
Table 3 shows some of the reconstitution results.
Copyright by Mahidol University
18
Table
3.
Relative cel lulase activities of the
component
of I. koning ii
cellulase alone and in combination ( 14).
Enzyme
Relative cel lul
c1
<,1
cx(t)
<l
<l
cx(2)
0-GIucosi dase
ase
l;
0
0-Glucosidase,r,
0
Ct+cx(t) + cx(z)
24
C,
l
+B-Glucosi dase
(i
(%)
5
*2 )
Ci+Cx(t+2) + 6-9lucosidase
( t+2)
103
original culture fi ltrate
100
All of the original cellulase activity
all
activity
was reconstituted when
the components, Ct + Cx(t+Z) + B-glucosidase(t*Z), were
recombi ned
in their original proportions. The rnost potent synergistic effect
,.as
found between exoglucanase (Cr) and the endoglucanase component (C*(Z))
when
cotton
was used as
cellulose, but the
gistic effect at
substrate. Cx11; and Cl act synergistically
low-mo lecu
alr.
lar-r'ei ght
low:8%
component showed no syner-
Grucose and ceilobiose uere
when exo-and endoglucanases were
glucose was
c, -
when
on
the main products
combined. Ho,rever, the proportion of
the combination Cl + Cx11;was used, and 14%
for cl + cx(2).
Copyright by Mahidol University
19
also observed other differences in the hydrolytic
capaci-
Wood
lies of ?.1<oniilg?:?: cellulases. The endoglucanase, Cx(1) _
component,
hydrolyzed 29% of phosphoric acid swolren
cellulose in 4 h, while the
Ｃ
，
︲
ｔ
ｉ
cx(t)'
Ｓ
endoglucanase, Cx(Z), hydrolyzed B3S, and
exoglucanase,
The endoglucanase, cx(Z) - component,
is more random in
32? (14',).
action than
obviously, therefore, the combination of exoglucanase
with the
endoglucanase, Cx(2) - Component, hydrolyzes
cotton cellulose ncre
efficiently ;
endoglucanase, Cx(2), opens more and groups
for
the
action of exoglucanase. This finding supports the theory
that
glucanases initiate the attack on native
cellulose.
endo_
Exo-g-gl ucanase f rom S. pu.hs ew,Lanhan showed no
vi scosi ty_
activity toward CMC (lg). The mainproduct, cellobiose, is
released in the c-configuration (6). Eriksson and pettersson (,l5)
found that the u,eight ratio of exoglucanase protein
to endoglucanase
decreasing
protei n was
l:1.
They also'found
a strong synergistic action
between
exo-and endogrucanases when hydrolyzing
when hydrolyzing phosphori
crystalrine cellulose, but not
c-aci d-src I l en cellulose (6). Endoglucanase
pretreatment arso increased the production
of celrobiose from cotton
cellulose by exoglucanase. This also supports the theory
that the
endroglucanases open chain ends
for exoglucanases. This evidence
strongry supports the nrechani sm for enzymati c degradation
of ceuurose
as described by petterson (16) (Table. 4).
In the
first
reaction, free ends for exoglucanase ane released
at the sites of non-crystanine regions of the celrurose. Thts kind of
mechanism was
nases have di
a variety
of
first
suggested by Eriksson
(19). Different endogluca_
fferent substrate specificities
and therefore, can attack
Copyright by Mahidol University
substrates.
20
Table
4.
A mechanism
for
enzymati
1. Nati ve cel
c cellulose degradation (.l6).
lulose
Endoglucanase- Cel lulosea
--_..,-----_..->
Native cel I ulosea
2。
3. Native cel lulose
aFormed
canase on
-....-..-------_
Exoglucanase-
CeI
B-glucosidase^
- Gl ucose
-...-.---.--_...
from native cellulose by the action
non-crystalline regions of the cellulose
Iu
lose
of the endoglu-
fiber.
Free chain
ends are created.
The
initiation
not yet been completely
unknown enzymes
mechani sm
of the degradation of cellulose
clarified. It is still
has
possible that hither to
are involved in the degradation. The existence of
such enzyre was demonstrated by Eriksson and couorkers
(20).
one
The
quantitative purification of both exo-and endoglucanases from culture
filtrate
it
possible
the culture solution using purified
enzymes.
of
S. WLo
a,Lilqran
3.4 Activity
3.4.
I
measurement
tive
CMC
of
for
them
to reconsti tute
determination
Endo-p-Gl ucanase
Endo-B-l
4-Iinkages in
nade
,
4-glucanases randomly attack B_,|,
or srcllen cellulose.
endo-B-gl ucanase
The best substrate
for
the
activity is a soluble cellulose deriva-
such as CMC. This substrate has been employed by many workers,
either the decrease in viscosity (2t) or the production of
reduci ng sugars (22). Measurement of the decrease in viscosity is a
very sensitive technique, slnce even a fel{ breaks in a chain cause a
who measured
Copyright by Mahidol University
21
"
marked decrease
in the
.
reducing sugars
is
rength.
ress sensitive and
of other cellulolytic
(Table
average chain
enzymes
J,reasurement
is also influenced
rather than
endo_B_g l
by the presence
ucanase (16)
2), but the cellobiose forned is, of course,
!-glucosidase.
of the
hydrolyzed by
tuted celluroses, for example cMc, are not
the ideal substrates for viscometric assays. Difficulties are caused
Ioni c-substi
fact that the viscosity of ionic substrates is dependent on pH,
ionic strength, and polyvalent cations tor itris reason, non_ionic
by the
substituted celluloses, such as hydroxyethyl cellulose (HEC), are
prefemed for the determination of low endo_B_gl ucanase activities (23).
3.4.2
Exo_B_Glucanase
Exo-g-glucanase
.
breakdown
of insolubre
is the first
enzyme
involved in the
ce1lulose preparations. consequentry these are
suitabre substrates for the determination of exo-B-g r ucanase. Usually
cotton is consi dered the best substrate (22), but mi cro_crysta I l i ne
cellulose is also suitable. Microcrystalline cellulose is hydrolyzed
only by exo-B-glucanase (Tabre 2), Since the enzyme produces celrobiose as the reaction product, the measurement
,
formed
of the reducing sugars
is
infruenced by the presence of.B-grucosidase. An the producing
mre substrate for the exo-B-g r ucanase. If the reducting sugars
produced from microcrystalrine cerlulose
[Easurement
is a true
detenni nati on
of
of cotton are estimated,
the
exo-g-grucanase only when other
cel lulyticenzyme are absent.
3
-4.3
B_Gl
ucosi dase
FehEst problems
arise in the activity determinations
for the enzyme hydrolyzing cellobiose. This enzyme hydrolyzes both
Copyright by Mahidol University
22
cellobiose and B-1,
4-o I i gosacchori
des (Table 2)
be determi ned using cellobiose as a substrate
can also uti
lize a pseudosubstrate ,
to glucose. It
(24).
B_Glucosidase
p_ni tropheny I _B _gl ucos i
de (25),
the use of which provided a rapid and convenient
determination
4.
can
method.
MICROBIAL SOURCES OF CELLULASE
Only a fetr fungi have been reported to produce
cellulases
degrading native cellulose. Such fungi are i r.oi{id.e,
r, Li,gnorwn and
I. loning i,i, S pto tr iclaan puh: aul etzturt, p azi c i.LL t\nt
funieulo swt and
polgpmte
P. ixieneie,
ahtekts, Myrotheeiun o arucati.a., Eueati.uttt
solani
and
Claetaniwt thanophiT.e uat. diesiAnn, pellieulari,a
filonentosa (Tanaka,
i977). Itpea Lactarc
(Kawai,
et al.,
197g).
fungi and bacteria produce cellulases which degrade
pretreated cellulose of carboxymethyl
cellutose (CUC), 5r1 not crystal_
line cellulose (26). Ce I I u tose-degradi ng fonns are found
among
Many nnre
the
gliding bacteria,
annng Gram-negati ve and Gram_positive
and among acti nomycetes
(27). Cellulolylic ability is
true bacteria,
also found
among
obligate aerobes (gliding bacteria, peanilomonae), facultative
anaerobes (Baci.Llue c ellulqnonaa ) and obligate anaerobes (Clostridiun).
In recent therrmphiric
organisms have also been
studied. The
ascomycete, Chadorn*m thanophile oat dieei.twn,
is a typical thermo_
philic fungus able to produce a cerulorytic system
decomposing native
cel
lulose
(27).
Claetaniun thamophile, .sprottbVan themophilit*n,
and ?hannoascue antqntia,cus gnow on and decompose cellulose
very
rapidly' but the ceuulase activities of the curture filtrates
are row
(28), fnqnonono stwe cun:ata hasbeen observed
to produce both endo_and
Copyright by Mahidol University
23
exoglucanases when grow on
by
this
cellulose (29).
organism caused less than
cating that
1%
The enzyme system secreted
hydrolysis of cotton
fiber, indi-
it is unable to decompose native cellulose.
Interest in therrxrphilic organisms has been stimulated by the
search
for
cellulases.
thernrostable
However,
cellulases from therrn-
philes are not necessarily more heat-stable than cellulases from mesophiles (241. Mandels (29) compared the cell.ulase systems produced by
rhavnoactinonyees and rbip?oderrna
tible
oinid,e. In short assayi on suscep-
substrates both cellulases showed higher
at 50'c. In a 24 hrs
activity at 65.c than
assay on cotton the r.oit,id,e ce\lulase
was
inactivated at 60'C, but rhqrnoaetinomyees cellulase was found to
deficient in
exoglucanase
activity,
so
further
be
comparison could not
be
made.
Although much work has been done with other organisms,
r,tsirid'e
still
cellulases.
seems
to
be tire most convenient source
of extracellular
possibility of attaining high over all cellulolytic
activities lies in mixing the enzymes produced by different of organims.
One
the mechanisms of cellulose decomposition may be different in
different microbes. Not all endoglucanases act synergistically with
However
all
exoglucanases
in the.from of
(27).
For synergism, two enzymes must work together
aloose complex, which cannot be formed between
all
exo-and endoglucanases
Copyright by Mahidol University
24
5.
¨
コ鴨俎 げ RIDE
・
5。
l
QM 9414
strain development
During the last ten years, studies carried Out by the
toxonomlsts at u.s.
Army Natic LaboratOrles, Nati ck, Massachusetts,
U.SoAc involved cOllecting and identifying the Organisns active in
cellu10se degradatiOno
This resulted in the QM COllectiOn of Over
14,000 fungi active in degradatiOn of material as w。
cellu10se, and Other polysaccharideso
。1, leather,
This c011ectiOn is nOw hOused
at the Unlverslty Of Massachusetts ln AmherstD U●
S●
A.
Among thOse
collected fungi9 r.υ arぅ ご。 is regarded as One of the most potent
strain of cellulolytic fungi producer (GhoSe, 1977; Herr. et.al.,
1978; Mandels and stermberg,1976).
The strain has been treated by
irradiatiOn and chemical mutagens tO induce mutation, Details Of type
and cellulase activity of r.υ
arぅ αo
are shOwn table 5 (Mandels and
Sternberg, 1976)strains Of QM 9123 and QM 9414 (Mandels and sternberg,
1976).
strains Of QM 9123 and QM 9414 (Mandels et al., 1971)have
been selected that yield higher levels Of cellulase.
丁he specific
activity of enzyme and the propOrtion of the CompOnents Of the complex
are similar tO the wild strain.
Copyright by Mahidol University
25
Table 5e
QM No.
Fン づむ乃a′ aη α strains in Natick c011ection (38).
“
A T C c No.
Type
Cellulase Activity
(PP units/ml)
a
13631
Wild
strain
0.5 - 0.7
9123
24449
cellulase
mutant derived from
QM 6 a
1.0 _ 1.2
6
9414
26921
Enhanced
cellulase
mutant derived from
Enhanced
QM
The strai n
9123
of r.oinid,e
was
further
1.s _ 2.0
developed on
the basis of
mutation by mutagens which detailed studies concerned the effects of
strain and substrate on production of cellulase were reported by
Kosaric (1980). Figure 5 shows the improvement of the fungus strain.
Copyright by Mahidol University
26
TRI範の コ]“
『
ェRDE
rr.E圏 弼EIリ
Linear accelerator
UV
￨
￨
QM 9123
1969
回
Linear lccelerator
Nitro,° guanldi ne
1%
山
"ん
￨
NG 14
1977
ｎ ｉ
ｄ ￨
・
UV
７
７
９
￨
I C 3σ l 1978
７
７
９
Fig.
6
tbinhodqntaoiril,e strain
improvement by mutation UV,
ultraviolet; N.A., not analyzed. From Ghose (197g).
5-2
l{edia and cultural condition
The media
standard mineral
for
60
production of the enzyme consisted
salts with 0.75-l% cellulose,
0.2% Tween 80 (Table
121'c
for
for production of cellulase
min.
6).
initial
pH
the
o.o7s% proteose peptone,
Both vessels and media were
The medium
of
sterilized at
5.5-6.0. This medium
was
inoculated with a spore suspension and incubated at 30.C on shaker for
five
days.
Copyright by Mahidol University
27
Table
6.
r.uieide
medium
for cellulase productiona (52)
g/1iter
mg/ I i
(NH4)2 So4
1.4
Fe S04 ・ 7 H20
5.0
KH2P04
2.0
MnS04 ・ H20
1.5
urea
0.3
ZnS94
1.4
CaC12
0.3
C° C12
MgS04
0.3
. 7 H20
2.0
acellulose, 0.75% proteose peptone,
;
initial
pH
i
Tween
g0,
0.2%
Cel lu lase
5.3.1 Cellulase
produced
O.O7S%
5.5-6.0.
5.3 Lyie?pdmru
is
ter
only
when
is
the fungus
an induced enzyme
is
grown on
in rz,b?adenna
and
cellulose or on other
glucosides containing 9-1 , 4-linkages, such as cellobiose, lactose, or
sepharose. This means that for partical purpose the fungus must be
grown 0n
cellulose, which is a slowly rnetabolized. insoluble substrate.
This creates challenging fermentation problems.
5.3.2 Cellulose and insoluble impurities, such as lignin,
absorb cellulase, thus reducing the amount of cellulase avai lable for
hydrolysi
s.
5.3.3 The synthesi s of
enzyme
is
strongly repressed
by
soluble sugar or other easily metabolizable substrates.
Copyright by Mahidol University
28
5.3.4 control of pH is
critical. If
pH
is not controlled,
will be inactivated by severely acidic conditions.
the yield of cellulase is reduced at higher pH.
enzymes
However,
5.3.5 Cellulase has a low specific activity on crystalline
cellulose, which
is
because
of its insolubility
several alternative routes are proposed
:
and recalcitrance.
(a) increase of cellulose
concentration, (b) addition of glucose to the cellulose media,
(c) use of continuous curtures, (d) use of two stage fermentation,
and (e) reeveling of cells. Among these alternatives, Mandels and
Andreotti (1978) considered cell recycling as the more promising area
for further
research.
5-3.6
Because
concentrations result
of
low bulk density
of ceilulose,
in thick shumies, which leads to
higher
severe
foami ng.
-
5.3.7 Temperature profiling and
inputs for productivity enhancement.
5。 3。
ttma
8 ■ンづοttοごθ
rθ θ
sθ tt
pH
cycling are essential
QM 9414 cellulase i
s
complex
cellulase contains three types Of cOmponents;
1)endo-3-1, 4¨ glucanase (cx)
2)Ce1lobiohydrolase (CBH)
3)3-Glucosidase (ce1lobi ase)
Copyright by Mahidol University
29
6.
HYDROLYSIS OF CELLULOSE
6.1
Pretreatment
In order to enhance the susceptibility of cellulose for
enzymatic hydrolysis, various techniques to pretreat the cellulosic
material have been developed. Table 7 gives a
summary
of the pretreat-
ment processes.
Table
7.
Methods
for
pretreament
Physi cal
Ball milling
Hammer
of celtutosea(3g)
Chemical
(liquid)
(gas )
NOi and
Ammonia
Weatheri ng
turunoni
Boiling
Hydrochloric acid
High-pressure steam
Acetic acid
Electron irradiation
sulfuric acid
Photooxidation
Sodium
Wetti ng
Sulfur dioxide
-
Iradiation
a
nation
ball milling
NaOH and ball milling
Sodium hydroxide
milling
Combi
Hot
irradiation
sulfide
Nitrogen dioxide
Potassium hydroxide
Phosphoric acid
oFrom
Dunlap
et al.
(1976)
Copyright by Mahidol University
30
Ball milling an_irradiation are amcng the more
effecti
ve
physical methods' whereas sodium hydroxide
treatment and sulfur dioxide
gas treatment are the viable chemical
methods.
6.1.1 Ball m.illing
popular rnethod
Milling celluloslc material is an often used and
for increasing ce,ulose digestibility. The materiar
t0 be treated is simple praced on a ba, mill
and compressive forces generated by the
time.
and subjected
to
shearing
milt for a specific period of
The rnost obvious changes
in cellulose physical properties
include a reduction in crystallinity, a reduction
in mean degree of
polymerization, an increase in the fraction
of the material that is
water soluble, and a marked decrease in particle
size. It is reported
that the dlgestibility of the milled cellulose is dlrecily
related to
the time of milling.
6.1.2 B-Irradi ation
g_Irradiation of cellulosic materials has provided
increased digestibilities resulting in a lower degree
of polymeriza_
tion'
rower
crystalrinity,
and higher npisture absorption capacity.
Studies have shown that a level
and various hard$/oods
is
of
about 5
needed before
x
107 rad on wheat straw
a significant increase in diges-
tibility is observed (Dunlap et al. 1976). It is know that addition
of certain nitrate
imadiation
seems
nitrite
sarts of celrulosic materials prior to
to aid in catalyzing the degradation reactions.
and
6.1.3
Sodi um
Hydroxide Treatrnent
Sodium hydroxide treatment
the oldest and best
of cellulose is
probaly
of increasing
Copyright
by Mahidol
University
digestibility.
The
known method
う０
application of the alkali results in disruption of lignin structure,
hydration and swelling of the cellulose, and decrease in ceilulose
crystallinity.
As long as the treated cellulose remains moist these
modifications persist, but drying often irreversibly negates them.
Maximum
digestibility
between
0.1 and 0.15
was found
gm NaOH
to be reached at an alkali lever
per gram solids.
6.1.4 Sulfur Dioxide Treatmeni,
Sulfur dioxide gas treatment of cellulose is pro_
bably the most recent innovation in ceuulose treatment techniques
and may be one
of the
most
promising. Early
Products Laboratory has shown
r,rork
that reaction of
at the U.S.
gaseous
Forest
sulfur dioxide
tiith moist cellulosic materials at 120'c for about 2-3 hours resulted
in an increase of severar times in digesttbility. The possibility of
using a gaseous agent to treat cellulose
several reasons
:
after treatment
and presumably recovered
is
the gast could be easily
most interesting
removed from
for
the cellulose
for total reuse; the treated
material would be in a solid form and solubilized materials could
removed
it
desired by a small volume
of
wash
liquid;
be
and penetration
of
the agent into the cellulose structure rvould be nnre rapid and complete
with a small molecule the size of sulfur solution.
A comparison
chemical pretreatment methods
is
of
some
features
in
Table g.
given
of physical
and
Copyright by Mahidol University
32
Table
8.
Compari son
of p,hysical
and chemical
p
retreatment of
cellulosea (40)
Phys i ca
Bulk
density
Reactor
space
I
Chemical
Increases (8-30%) but at
Decreases Considerably;
increased cost (10_15% even 7_10% slurry
slurry can be handled becomes difficult to
easi ly)
hand le
Increased
reactor
volume requi red
of lignin
of
hydrolysis
Extent
Waste
outflow
aFrom
6.2
because
less reactor volume is
requi red
Nearly 50X when
cellulose is
With detignification
l0%
hydrolyzed
None
Nearly the same extent
as in mechanical trea_
tment Causes problems
for u,ash water which
can be recyc led.
Ghose (1978)
Enzymatic hydrolysis
Factors affecti ng the hydrolysis
include : type of substrate, pretreatment,
of cellulosic materials
characteristics of the
preparation, temperature, time, pH, substrate concentration,
reuse of enzyme, and type of reactor. Many of these factors
are inter_
enzyme
dependent, making the whole process
mind the stringent demands
of
quite complicated. Bearing in
economic
feasibility, there are many
Copyright by Mahidol University
001447
33
CENTRAL LIBRARY
limitations on the process design
」袢Uゝ 鴨ぎR9ヽ fbNIVERSITY
F these limitatiOns are
mutually incompatible. For example, the cost of enzyme does not allow
high enzyme concentrations, but slow reaction caused by low enzyme
concentration does not bear out the cost
Simi
larly, the
reuse
of
enzyrne does
not
of
i nvestment
permi
t
in the process.
a high reaction tenpera-
ture, but the reaction is too slow at low temperatures.
In an ideal
leading
of
case
the reaction rrould be rapid and complete,
to a high glucose concentration without
enzJme, which could
of cellulose
any substantial loss
therefore be reused. At present, the hydrolysis
this ideal situation, but the results
achieved are at least promising enough to encourage further development
and optimization of the process.
does not apprpach
0n a small scale hydrolyzates with 30t
of
glucose have
been
achieved using high enzyme and substrate concentrations and a long
reaction time
(30).
fne rest economical substrate concentration is
near 10% (3,l ). Adsorption or
r€use
of
enzyme
(32).
ultrafiltration
has been proposed
for
Cellulose adsorbs cellulase efficienily at
pH
to 5 and at a temperature of 25 to So.C, i.e. at the optimal range
for enzyre activity. Adsorption is rapid at the beginning of hydroly4
hydrolysis. During hydrolysis the enzyne is Iiberated and bound again
to new substrate. Adsorption depends on the concentration and particle
size of cellulose and on the
(33). Reports on the
desorption of cellulase are contradictory (33). Ultrafiltration for
enzyme component
of enzyrc has been studied only with pure cellulose as the
substrate. In thls cases no problems with lignin and other compounds
remaining in the reactor are encountered.
reuse
Copyright by Mahidol University
110311'ら 1
34
An increase
in
temperature from 45
to 55-c enhances the
hydrolysis, but impairs the possibilities of reusing
the enzyme (3+1.
During the first hours the rate of hydrolysis
is higher at 55.c, but
after 10 h 45'c is advantageous. At a temperature 0f 45.c,
the
usual reaction time has been ronger than one day,
for example 40 h,
efficient mixing acedlerates the hydrolysis, but may arso inactivate
the
the shear stress effect. High substrate concentrations cause technical problems in mixing (35). The pH optimum
is
enzyme through
slightly
5.0, but higher than 4.0 since the pH optimum
range is relatively narrow, pH control during hydrolysis
may be
lower than
necessary- The pH tends to decrease during the hydrolysis (36).
Copyright by Mahidol University
CHAPTER 3
MATERIAL AND METHOD
1.
SUBSTRATES
1.1
Preparation
of
substrates
This study used cellulosic ralv material in form
cultural crop residues
such as
rice straw,
of
agri_
.sugarcane bagasse, corn stover,
cotton seed hulls, as the substrates. These cellulosic raw material
were washed with clean water, and rvere dried in the sun all day long,
and kept
in hot air
1.2
oven
at
Pretreatment
105.C over
of the
night until dry.
substrates
1.2.1 Physical pretreatment
tere pretreated by milling to
in pariticle size. They were milled untill they could pass
The substrates
decrease
through a sieve
is
used
for
of
100 mesh
size. Milling cellulosic material
increasing cellulose
method
digestibility.
1.2.2 Chemical pretreatment
After milling, the cellulosic material
brought
to pretreat by chemical
reagent which
is
were
sodium hydroxide
concentrated 1%. The milling cellulosic materials were treated with
1%
sodium hydroxide
claving at 120'C
centrifuge,
in
at ratio of
1%
l:7.
They u/ere
sodium hydroxide
delignified by auto_
for I hour.
Then separated by
and was thoroughly washed
40'C, kept in hot air oven at
105.C
with water and air-drying at
over night.
Copyright by Mahidol University
36
2.
cELLULASE ENZYME PRODUCTIoN
2.l
Mi crO_Organism
The micro― Organism used tO produce cellulase enzyme are
ぅ
。み
。
αanttα υι
′う
ごοQM 9414. These fungus were supplied by Thailand
lnstitute of scientific and Techno10gical
Research.
rン
These fungus were malntalned On potatO_dextrose‐
agar
slant at 30° c as stOck culture and was subcilture monthly。
2.2
Cultivation medium
The medium for production of the enzyme consitsted Of
(NH4)2S041・ 49;KH2P04 2.Og:urea o.3g;caC12 0・
39;MgS04・ 7H20
0.3g;FeS04・ 7H20 5 mg;MnSo4・ H201・ 6 mg;znS04・
7H201・
4 mg;cOc12
2.O mg; proteose peptone 1 9; tween 80 1 ml
l litreo
cellulose pOwder were added at l%.
and distilled water
The vessel (cOni cal
flasks plugged with cOttOn wり ol)cOntained the medium but nO tween 80
was added, bOth vessels and medium were sterized at 121・
C fOr 60 min.
After sterilization of the mediun the pH was 5.2.
2.3
Culture conditiOn and innoculum preparatiOn
Culture of rン
`cヵ
οαοコ物α υ′′ぅα′QM 9414 were maintained on
the stOck culture slant, and were transfered to
plate, and kept fOr 5 days at 30・
c.
pOtato―
dextrose_agar
The spOres On the plate were
washed by steriled distilled water and the anount Of the spOres were
adjusted t0 175 ng dry weight (39,64).
This spore suspension was used
as an inoculun that cOuld be transfered
intO a shake flast (1000 ml.
conicalflasks plugged with cottOn wool)and cOntainin9 500 ml. Of
Copyright by Mahidol University
37
medium were used
to
grow
the
inoculum on a gyratory shaker (120 rpm)
at 28'C for 5 days. Cellulase enzyme yield as a supernatant
}Jas
separated by Centrifuge.
3.
ENZYMETIC HYDROLYSIS OF CELLULOSIC MATERIAL
(
Enayme sacchari
fi cati on )
of cellulosic. materials is a method to
cellulosic materials. The enzymeti c hydrolysis in
Enzymetic hydrolysis
produce sugar from
this study used milling delignified cellurosic materials mixed with
tuiclodesna oinide CeLlulase solution at a 5f, substrate concentration
pH 5 (cellulosic material 200 mg + acetatebuffer pH 5 2
ml + enzyme
2 ml), and incubated at 50.C
4.
for 2-g hr.
ANALYSIS METHOD
4.1
Growth
of tutblodana ufuide
Growth
of the fungus during cultivation
QM 9114
lras examined by
observing the growth on potato-dextrose-agar plate and by microscopy.
4.2
Mycelium
dry r,reight detemination
Taking the mycelium which were kept
for 5 days from
potato-dextrose-agar plate to wash with steried disti lled water, and
dried over night at 105'C and weighted.
4.3
Cellulase assays
Filter
paper degradati ng
determined by mixed 0.5
(l'lhatman
ml
activity (Cl activity)
. of enzyme solution
and
filter
No. 1) 50 mg. with acetate buffer pH 5 0.5
ml
,
were
paper
strip
incubate at
Copyright by Mahidol University
38
50'c
for t hr.
and determined reducing
sugar.
The reducing sugar
was
determined by o-toluedine method.
4.4
Determination
The amount
glucose
of
of
reducing sugar from enzymetic hydrolysis
reducing sugar, consisting mainly of
in the sugar solution
produced
after enzymetic hydrolysis
was
determined by o-toluedine method.
Copyright by Mahidol University
CHAPTER 4
RESULT
1.
POTENTIAL OF RA‖ MATERIALS
Corn stover,
hulls
rice straw,
were the selected materials
sugarcane bagasse, and cotton seed
for
glucose production,
in
considera-
tion of the potential of these agricultural wastes. The potential of
these agricultural wastes have tro things to be considered, one is the
qualitative potenti al
and
1.1 Qualitative
The
the other is
economi
c potential.
potential
qualitative poential of agricultural
wastes were
of agricultural wastes. The
agricultural wastes were mainly compri sed of cellulose, hemicellulose
considered from chemical composition
and
lignin.
rice straw
compared
From
consideration, the corn stover, cotten seed hulls,
and sugarcane bagasse have highly
to other materiali
lignin, as shown in
Table
9.
qualitative potential
because they have much
as
cellulose and few
Table 9.
The chemical composition
of
some
agricultural wastes (% of
dry }teight)
Agricultural
waste
Li gni n
８
4.3
７
３ ３
１ １
Sugarcane bagasse
Cottonseed hul Is
５ ６ ９ ５
１ ２ ２ １
Rice straw
６ ３ ０ ９
３ ３ ４ ５
Corn stover
Cellulose Hemicellulose
Reference
l'lilke et al
( 1981 )
Sharma (1974)
Sharma ( 1974 )
Johnson and
Peyo (1975)
Copyright by Mahidol University
40
Table '10. The production
of agricurtural
wastes (1984/85)
Quantity (1000 tons)
Materi a I
Total
Used in pa
i ndustry
Used as
fuel in
Waste
i ndustry
Corn stover
295.215
Rice straw
24074。 4
Sugarcane bagasse
Price
Materi a I
225(63)
5.558
aself-generated (Shown
11.
30('3)
2756.05
Cottonseed hulls
Table
295.215
in
24044。 4
2480.4
50.65
5.558
appendix
I)
of agricultural wastes
'li ot
rnoi
sture
Pri ce
Prlce
sture)
Bath/ton
Cel
(dry)
Content
(rnoi
Bath/ton
(%
lulose
of
wt. )
Corn stover
10。 4
4004
446.4
36.1
Rice straw
9。
8
400b
443.5
33
Sugarcane bagasse
7.2
3ooc
323.3
40
6
1000d
1144。 2
59
Cottonseed hutls
12。
dry
dFrom
interviewing farmer.
bFro, Bangpra-in paper
Mill.
cF.o, Singburi Cane Mill.
dF.o, Industrial
Viwath
Company.
Copyright by Mahidol University
41
1.2
cri teri
as
Economic potenti at
The economic potenti
al
1) price of materi
s,
was determi ned by
the following
:
al
2) quantity of materials,
3) source of materials and collection,
4) alternative of useful of raw materials,
5) substituation of raw materials.
From
l0-ll
table
and
corn stover, rice straw, and sugarcane
quantity and lo', price or can get free, because they
are agricultural wastes,their price get along with collection and
bagasse have much
transportation. Cotton seed hulls
is
also agricultural waste. but
their cost higher than the others, because the cotton seed hulls can
be brought to make fuel and.to grow mushnoom in high quantity, and Ieft
few quantity. Hot,ever the cotton seed hulls are i riterested, because
they have cellulose
59%.
considering economic potenti
a
r
aspects can concruse
that rice
is the highest potential follolled by, corn stover, sugarcane
bagasse and cotton seed hulls is the lowest.
straw
2.
ENZYME PRODUCTION
The enzyme
of
tyiclodqrna
hydrolyzing agent because
oirile
of its effecti
QM
9414 was selected as the
veness against a
variety of
cellulosic materials (37).
Copyright by Mahidol University
42
In the exeperiment
QM
9414 by induction
we used enzyme
from tuieladarm. oiyid.e
lhe wiertoitqna oiei.dee[
9414
with cellulose
at 28'C for 5 day on gyratory shaker (.l20 rpm). Filter
ting (FPD) activity of enzyme = 0.0183 Unit/ml .
3.
1%
paper degrada-
PRETREATMENT
In this experirEnt physical pretreatnent and chemical
pretreatment was used.
3.,l
Physical pretreatment
The physical pretreatment t.',as
milling method. The subdi_
vision of substrates to a very small particle size yield a product
remarkably susceptible
to enzymatic hydrolysis attack.
case' substrates were milled
used time
to
r00 mesh
size.
Thus
Each kind
of
in this
substrates
to mill into small particle size not equal as shown in the
table
12.
Table
12-
Relationship between yield
of milling substrates
and time
of vibratory milling.
Substrate
Time of
milling
(mi nute)
Weight
weight after Yield of
b:f:IRg(g)T::ユ
l::hi8) :llll:: 188-
meSh(%)
2
Sugarcane bagasse
Cottonseed hul Is
2
2
2
０ ０ ０ ０
２ ２ ２ ２
Corn stover
Rice straw
8.30
41.5
7.80
39.0
7.70
38.5
8.65
43.25
Copyright by Mahidol University
43
3.2
Chemical pretreatment
Effect of delignifying agent on cellulosic materials.
Air-dried cerrulosic materiars were treated with chemicar derignifying
agents' at a ratio
of 1:7. Mirting rice straw,, s ugarcane
bagasse, corn stover,
cotton seed hulls, and sawdust were delignified by autoclaving
2' I
and 0.5% sodium hydroxide solution
Table
13
1%
sodium hydroxide
for the delignification of
Table
solution was.considered
these
Rice straw
Sugarcane bagasse
Corn stover
NaOH
15, lO,
120.c as shown in
to
be
sufficient
cellulosic material.
13. Delignification of cellurosic
Substrate
for t hr. at
in
sol!
(%)
materiar !,ith sodium hydroxide
Delignified ppduct(%)
0.5
73.75
1
67.50
2
52.50
10
40.00
15
35.00
0.5
89.25
1
68。 20
2
58.75
10
50.25
15
47.50
0.5
72.5
1
60.0
2
51.25
10
48。
15
47.5
10
Copyright by Mahidol University
44
Table 13.(con
Substrate
Na0H
Cotton seed hulls
4.
sot!
Delignified product (%)
(%)
0.5
72.5
1
67。 5
2
63.75
10
54。 5
15
42。 5
MOISTURE OF AGRICULTURAL WASttES
the milled agricultural
listed in table
Table
14.
wastes
ｆ ｔ
Ｏ ａ
Find out the rnoisture
agricultural
wastes by incubating
105'C overnight,
the result
are
14.
Moisture
Milled material
of agricultural wastes
before drying
(gm)
after drying
%
of
moisture
(gm)
Corn stover
25
22.40
10。 4
Rice straw
25
22。 55
9.8
Sugarcane bagasse
25
23.20
7.2
Cottonseed hulls
25
21.85
12.6
Copyright by Mahidol University
45
5.
(Sacchari
fi cation )
Cellulase enzyme hydrolyse the delignified cellulosic material
at 50'c pH 5 for 2,4 and B hours in incubator. The result are shown
in Table 15 and Figure
Table
15.
7.
of delignified cellulosic material with enzyme
of I,uiride
Hydrolysis
After 2 hrs of After 4 hrs of After g hrs of
i ncubati.ng sugar i ncubat-i.ng sugar i niunaii ng-slgur"
--
Substrate
(mg
%)
(mg
%)-
(;s-*i"
Corn stover
9.3
15.0
17.0
Rice straw
I
15.3
17.8
5
12.8
r3.4
25.s
26.7
Sugarcane
bagasse
Cotton seed hulls
13.7
Copyright by Mahidol University
46
﹂”
¨
一
“
一
寵¨
”
¨
一
¨
中
一
﹁
Ｌ
一
洲
︲
＝
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＝
ノ﹁ゴ
ノ
ン
一
﹁コ
■
＝“
＝
︲
︰
︲
峰´
■静
１ １ ■
■＝ 甘 ＝
．
．
﹃
¨
一
﹁
‘
一
‘
・
一＝＝
劇
噺列
■ ”ヽ 一 ，Ｌ＝ ︱
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ｒ一
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＝ロ ー
ｌ
劃﹁
︱︱
山 踊 中︱ ︱ 一
︲︱
十一
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・．
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而撃 ＝ 一
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︲〓﹁
‘
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一
¨
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再 ′
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■ ”
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轟 ＝ｌ
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名
!1
::
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︱ 〓 ■ ■ 一︱ ︱ ¨‘
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”一¨一 ¨
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ー
０ ＝Ｌ ト ト
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0ざ
． ¨ ¨
﹂‘
¨﹂一
1hr)
i:
，
％ И
１り 乙
¨ ．︱ “
￨`:
:l'
!:i
￨￨
︲
■＝〓¨
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︰
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．”
¨〓
■︲・
ソ な
た■
Ⅳた
¨ ︰﹁
:
︲
一﹁ 〓 ８
:￨:Tl品
﹁ コ 引﹁
一¨一¨一
¨
一
¨”¨
¨¨
﹁
γ声
一／
７ ■
︰
一！
ｒ〒
︰
︱
円︱
︱
﹂
ｒ劇沖喘一
︱ “１
︲ ︰︲ 一．
一
¨
１ ０
，
I
1111
:!￨￨':4:￨￨
::
JI
ぎ ，
¨
二¨・■
＝〓
i
ｉ
ｌ 十一
＝︸
ｉｉ
︲
︱〓 ＝ ４ １
:￨[
::!
1
出ギ が 一
＝﹁
“
Ｌ １
︲ １
:
●
ll
日」
￨
国
￨:
￨￨
￨11
￨li
ill
￨￨
ll
i「
=ト
::
+
!:
::
￨￨
‖
‖
r
illl
l
」
::
1:
L
硼
￨lil
!
:
l
‖
:ヽ
d 01 ::Lili:五 lagii
li昌
Fig 7:H
l:
i:
J::
鯉
‖
:!:
「
「
￨￨
!:￨:::￨
臨土
!
:▲
欄
::
I
:コ
￨
，Ｆ
ｒｌ
il
6■ 1
!
￨::￨:￨￨:
￨￨
「
::
l
!:
I
l:
:
,J
:
131 p・
囲
....t'.
!:
:1
L
百
Ы
111
::!
1・
1
￨■
:lill
:::
Copyright by Mahidol University
.
CHAPTER 5
COST ESTIMAT10N OF CLUCOSE PRODuCT10N FROM THE AGRICULTURAL wASTES
The pncduction costs are principall.y estimated
scale.
These estimated
capital cost,
in
laboratory
costs do not include chemical recovery, fixed
energy consumption costrlabour
cost, and pollution
contro I .
1.
DIAGRAM OF THE PROCESS IN CONSIDERAT10N OF GLuCOSE PRODUCT10N
Diagram
of the
process
of glucose pylduction is
shown
in
Figure 8.
Ｓ
ｔ
ｎ
ｅ
ｒ
ｔ
ｕ←
Ｎ
Agricultural
J
Feed
Feed
preparation
steri
―
Cel
Ii
zation
I
waste
ulose
pretreatment
↓
Enzyme
CeI
production
hydro lysi s
Enzyme
Gl ucose
lulose
Separation
J
Funga
I
Materi a I
Fig 8
Di
agram
ati on
J
Glucose
SoI
ution
of the process of glucose producti
on .
Copyright by Mahidol University
48
2。
uTILIttY UNItt COST
Utility unit cOst are listed in ttable 16.
Utility unit cOst
Table 16。
Item
Unit
Unit cOst
(Bath)
ｈ
Ｗ ３
ｋ Ｍ
Power
}'later
1.434
9.504
ｌ
Fuel oi I
3.80b
aFrom
Pubtic
Utility of Thailand. (1gg5)
bF.or 0ffice
of Nationat Energy. (1gg5)
Copyright by Mahidol University
49
3。
fHEMICALS FOR CLUCOSE PRODUC丁 10N
chemicals and price
Table
17.
of
ls
Bath/kg
Bath/1iter
２
４
(NH4 )2504
２ ７
KH2P04
Urea
４ ４
CaC12
MoSO*.7H20
４
FeS04・ 7H20
ｂ ｂ ｂ
４ ９ ６ ¨ ７ ２ ０ ５
１ ４ １ １ １
H20
ZnS0O.7Hr0
C°
listed in Table 17.
Chemicals and chemical pricea
Chemi ca
MNS04・
chemicals are
C12
Proteose peptone
Tween 80
Cellulose
CH3C00H
CH3C00Na
NaOH
aFrom
Department
of
80b
Business Economics Ministry
bF.o, chemical
stores
(CHEI,IIKIT
C0.,
of
corunerce.
LTD.)
Copyright by Mahidol University
4.
HYDROLYSATE AND PERCENT OF CELLULOSE CONVERS10N
Hydrolysate and percent
of cellulose
conversion are
listed in
Table 18 and Table i9.
Table
18.
Materi
a
of
HydrcIysate
enzyme hydrolysis
Glucose solut 10n
I
口g%
Corn stover
17.0
%
lysate
kg/i of solh
Hydro
g/1
0.017
0。
17
1
/5882.35
Rice straw
17.8
0.0178
0.178
1/5617 -98
Sugarcane bagasse
13.4
0.0134
0.134
1/7462.69
Cotton seedhulls
26.7
0,0267
0.267
Table
19.
percent
Materi al
of cellulose
/3475.32
conversion
Substrate
(Delignified
materi al)
(kg)
1
Cell ulose
% in
delig。
:￨￨:lly―
f-*
￨lil!:}:li:。 se
materi al
Corn stover
Rice straw
294。 12
53.48
157.29
1
0.636
280.90
48.39
135。
96
1
0。
1
0.402
Sugarcane
bagasse
373.13
66.67
248.81
Cotton seedhulls
187.13
87.41
163.71
736
0.611
Copyright by Mahidol University
51
5。
MATERIAL BALANCE IN FLOW DIAGRAM
Material balance (including energy in
shown
in
appro<imate) are
Figure 9-12.
490.20 oven
dry corn
Milling
stover
stover
corn
Alkali
treatment
water 3431.4
:
1_
l晏
!。
:
!
ー ー ー ー ー ー ー ー ー
ＯＮ ｒい。︺
l
'l
T.b:高
ＮＯ一 一Ｎ
・
TT
water 3431.4
←
・
・
ＯＯ︼い０つい﹁い００ い０︼い０
〓”一０﹁ ＮＯ一一。Ｎ
!
ト
Pentose and Iignin
―
←
Power
and
:
:
K:
0:
=￨
Fuel
⊥
l,leutrients
Enzyme
Residual solid
water 5882.4
ｇ
ｍ ・
ｕ
ｌ ｒ ｓ
ｅ ｅ ｒ
ｌ ｎ
一 ・
ｌ ａ
。
ｒ
ｒ
4rig 9.
Hydro Iysi s
water 2941.2
ｅ
鴨ｌ
ｅｔ
ａ
ｂ 詢 Ｗ
units:
ｔ
ｉ
胤Ｆ 帥︲
¨
・
water 2941.2
soI!
solid
Material balance flow diagram
Glucose so11 0.017%
Hydroly sate 1 9
for
of glucose production form corn
i
ntegrated processing
stover;
(-)
scheme
material;
(-..-)
Fuel oil; (… …‐―――――¨‐) power.
Copyright by Mahidol University
●‘
ｒυ
416.15 oven
dry rice
Mi I
straw
ling ri ce
Pentose and Iignin
straw
water 2913.05
一
Power
and
FueI
。
∞一や Ｃ
い一寸．︸
Enzyme
sol!
water 2809
MyceIi um
I
↑
．
︻
ＸＷ︼ぃ一
︼一
︵︶
ｏ
一
の０ い０一﹄︵
Ｎ∞
０コぃ﹁︼
・
〓口一Φヽ Ｎ∞００
l+--.l
I
+-.._.._
0.0303
Hydro
lysi
s
Residual solid
solid
water 5618
Glucose
sol!
o.ot78
Hydrolysate 1 g
Units:
- kl.Ih
Fuel oil - liter
Power
l'later and others -
Fig
10.
s
Material balance flow diagram
processing
mateial;
for
i
ntegrateci
Copyright
by Mahidol
University
of glucose productlon
form rice straw; (_-)
scheme
(-.,-)
Fuel oi
l; (-----)
power.
53
547.12 oven
dry sugarcane bagasse
547.12
Mi
sugarcane
bagasse
----.._-_-'
lling
Al kal
PI
\1
Power
and
-r
ｌ ψ
Neutrients
0.04079
ωＮω。一
口０︼い０つい﹁い００ ∽０︻い０一
ω
０一０．一
ll
ωＮω一。
ω
〓ｏ一ｏヽ 一
︺︸卜．Φ
k-lt
Water 3829.84
Fue
I
I
I
Enzyme
Enzyme
sol!
Hydrolysi
water
3731.3 pnoduction
----_.--'
Residual solid
s
Water 3731.3
Mycelium solid
water 7462.5
Gucose
sol!
Hydrplysate
Units:
︱ １１． １ １１ ｏ︰
﹃
．
．
， １， ，，
treatment
一
Water 3829.84
Pentose and lingnin
i
o.ot34g
I
g
- k}.th
Fuel oil - liter
Power
Irater and others _
g
Fig 11. Material balance flow diagram for i ntegrated
processing scheme of glucose prcduction
form Sugarcane
bagasse; (_)
material; (_.._) Fuel oil; (_____) power.
Copyright by Mahidol University
54
377.43 oven
dry cotton
seed hul ls
Mi I
ling cotton
seed
Pentose and lingnin
hulls
water 1942.01
-----}
water 1942.01
1
ｒ
ｄ
ｌ
腱 ｎ ｅ
Ｏ ａ ｕ
Ｐ Ｆ
ａ
︹
︺
Ч。
ヽ
一
﹃
口
︼
い
い
﹁
”
﹂
い
５
︵
Ｘ
∽
０︻
０
一９
〓一
一Φ∞∞
。
〓●一ｏヽ 一一∞ЧＮ。ヽ
一
一
一
８瑠ド ー ︲ｋ ︲︲︲︲ｂ娼ド
，
．
0:
0.02068
V
Enzyme
Nutri ents
ン
water 1872.7
solution
production
water 1872.7
Residual sOlid
４
ｎ 一
● 一
ｌ ，
５ ０ ４ Ｓ ７ ・ ３ ｅ ｒ Ｓ ｌ
ｅ ∞ Ю
ｔ ｕ
ａＧ
︲ 町ｄ
ｗ
ｕ
ｍ
ｅ
Ｃ
Ｌド←
Enzyme
soli d
ｅ
ｔ
ａ
Ｓ
ｙ
Units:
19
- kl'lh
Fuel oil - liter
Power
water and others
Fig 12.
0.0267%
-
g
al
baiance flow di agi"am for i ntegrated
processing scheme of glucose production form cotton seed
hults;
material r (*
Fuel oil; (----- ) power.
t''lateri
(-)
-..-)
Copyright by Mahidol University
55
6.
COST ESTIMAT10N
.
Cost analysis
of
glucose production
is
subdivided
into
2
catagories;
(1) Chemical for
enzyme production cost
(2) Glucose production cost
6.1
Chemicals
for
enzyme productlon cost
Chemicals
for
enzyme production
listed in Table
Table
20.
(NH4)2S04
KH2P04
for
enzyre production cost
Bath/kg
gath/g
g/1iter of
enzyme
Bath/1iter of
enzyme
2
0.002
1。
4
0.004
2.0
0.008
urea
.2
0.002
0.3
0.0006
CaC12
7
0.007
0。
3
0.0021
MgS0o.7Hr0
4
0.004
0.3
0.0012
FeS0O.7Hr0
4
0.004
0.005
0.00002
MnS04.H20
4
0.004
0.0016
0.0000064
2nS04.7H20
4
0.004
0.0014
0.0000056
CoClz
19
0.019
0.002
0.000038
Proteose peptone
CeI I ulose
46
0.046
0。
75
0.03450
17
0.017
7.5
Tween 80
and
20.
Chemicals
Chemicals
cost are concluded
4
20 3ath/1iter
0.0028
0.1275
0.04(2CC/1iter
of enzyme)
Total Chemicals Cost
0.217
Copyright by Mahidol University
56
6.2
Glucose production cost
Glucose was produced by hydrolysing
delignified cellulo_
sic material (corn stover, rice straw, sugarcane bagasse, cotton seed
hulls) at 50'C pH 5. Cost of glucose production are listed in
Table 20-23.
Table 20.
Glucose productiOn cost (from
Unit/g of
Item
gI
Raw materi
corn stover)
a
Bath/Unit
Bath/g of
glucose
ucose
I
Corn stover
(dry,
kg)
0.4902
0.4464
0.219
Tota1 0.219
Chemi
cals
NaOH (g)
34.314
CH3C00H (g)
5。
CH3C00Na (g)
2741
16.979
0.005
0.172
0.012
0.063
0.010
0.170
0.217
0.638
Chemicals for enzyme
production(liter Of enzyme)
2.9412
Tota1 1.043
Uti lity
Water(M3)
0.009314
8.5
0.079
Tota1 0.079
TotaI producti
Remark.
The actual prcduction cost excluded cost
on cost
of
l.341
energy which is
2.302 Bath/S, because the energy consurtion fixed by
apparatus
in lab sca le.
Copyright by Mahidol University
57
Table
21.
Glucose production cost (frpm
Unit/g of
Item
rice straw)
Bath/Unit
Bath/9 of
91ucose
glucose
Raw materi
a
I
Rice straw (dry, kg)
0。
41615
0.4435
0.185
Tota1 0.185
Chemicals
NaOH (g)
29.1305
0.005
0。
CH3C00H (g)
5.3699
0.0,2
0.060
CH3C00Na (g)
16.2158
0.010
0.612
2.809
0.217
0.610
146
Chemicals enzyme production
(liter of enzyme)
Tota1 0.978
Utiliti es
Water (M3)
0.008531
8.50
0.073
Tota1 0.073
Total production
Remark.
The actual production cost excluded cost
cost
of energy which is
2.089 Bath/g, because the energy consumtion fixed
apparatus
1.236
by_
in lab scale.
Copyright by Mahidol University
58
Table
22.
Glucose production cost (from sugarane bagasse
Item
Raw materi
a
)
Bath/g of
glucose
Bath/Unit
Bath/g of
glucose
0.54712
0.3233
0.177
I
Sugarcne bagasse
(dry,
kg)
Tota1 0.177
Chemicals
NaOH (g)
38.2984
0.005
0。
CH3C00H (9)
6.6908
0.012
0.080
CH3C00Na (g)
21.5400
0.01
0.2154
0.217
0。
Chemicals for enzyme
productiOn(liter of enzyme)
3.7313
1915
810
Tota1 1.297
Utilities
Water (M3)
0.0112924
8.5
o.o96
Tota1 0.096
Total production
Remark. The actual prcduction cost excluded cost
cost
of
1.57
energy which is
4.226 Bath/g. because the energy consumption fixed by
apparatus in lab scale.
Copyright by Mahidol University
59
Table
23.
Glucose production
cost (from cotton seed hulls)
Unit/g of
gluCOse
Item
Raw
Bath/9 of
glucose
Bath/Unl t
material
Cotton seed hulls (dry, kg)
0.27743
1。
1442
0.317
丁ota1 0.317
Chemi ca
NaOH
ls
(g)
19。 4201
0.005
0。
097
CH3C00H (g)
3.3581
0。
012
0.040
CH3C00Na (g)
10.8107
0。
010
0。
108
0.217
0。
406
Chemical for enzyme
production( liter of enzyme)
1.8727
Tota1 0.611
Uti lities
Water (M3)
0。
0056874
8.5
0。
048
Tota1 0。 048
Total productionl
Remark.
cost
O.976
The actual production cost excluded cost of energy which is
2.184 Bath/g, because the energy consumption fixed by
apparatus
in lab scale.
Copyright by Mahidol University
60
TabIe 20'23 show
stover
or
that the production cost of glucose from
is 1.341 Bath/g or l34l Bath/kg;
from
rice straw is
1236 Bath/kg3 from sugarcane bagasse 1."57 Bathlg
from cotton seed hulls
is
0.976 Bath/g
or
976
or
corn
1.236 Bath/g
1570 Bath/kg;
Bath/kg. These
costs
are estimated cost ln laboratory scale, and these costs do not include
fixed capital cost, energy comsumption cost labour cost, chemical
recovery, pollution control, purifying and by-product
these costs are
stlll
credit.
However,
being very high when compare with the market
price of glucose (14 Bath/kg).
purlfication of glucose has to be done before
the application through precipitation. Heat is the major resource to
be used in the precipitation method. so the production cost in addiMoreover, The
tion with the purification cost remain higher than as followed;
Bath/g
for cornstover,
sugarcane bagasse,
1.341
for rice straw, 1.57 Bath /g for
and 0.976Bathlg for cotton seed hulls.
L.236 Bath/g
Copyright by Mahidol University
CHAPTER 6
DISCuSsloN
Overall result and compari son betueen production cost of
glucose from corn stover,
seed
1.
rice straw,
sugarcane bagasse, and cotton
hulls, are discussed in this chapter.
OVERALL RESULT
From chapter 4
hulls
has the highest
the result of the study
shows
quality potential containing cellulose up to
59S, sugarcone baggasse the second, corn stover the
straw whlch containing
The economic
potenti
trial
just
al
because
33%
of cellulose
potentidl
of its
sector, Iow price
burn the
that cotton seed
mass
shows
third
and rice
having the lowest.
that rice straw
availability,
and may minimize
rice straw dovn the farm.
has the highest
low demand
in the indus-
air pollution rather
than
we
Corn stover comes second to
in the industrial sector is lower than
that of the sugarcane bagasse and is available at about 295,000 tons.
Sugarcane bagasse places third because demand in the industrial sector
is higher than that of corn stover and the availability left is only
the highest because the
about 50,000
tons.
demand
Cotton seed hulls ranks the last because of
its
least availability due to the fact that the industrial sector has
thus agricultural waste for fuel
Another use
of it is in the
until
growing
of
used
consumed alnrost completely.
mushrooms
thereby leaving almost
for further processing of other by-products. The high demand
of them in the industrial sector makes their price higher.
nothing
Copyright by Mahidol University
62
These
agricultural
These
four agricultural
differ in quality
potenti a I and economi c potential are still considered to be
the important source of cel lu lose.
combination
wastes though they
wastes being studied are a good
in the production of glucose
alternatively
if
one
is not available
The production
of
because they can be used
due
to their
seasonal character.
glucose from ihe agricultural wastes through
hydrolization with enzyne from r.oi"ide are in conditioned with the
following r (l) Use of enzyme fron r.oiri,.de which have (FpD) activity
of
.
enzyme = 0.0183 unit/ml
wastes
to
be pretreated by
(2)
Use substrate from
milling until
the agricultural
to 100 mesh and
delignify with 1S of Sodium Hydroxide solution at 120.C for t hour.
(3) Hydnclysis at 50'C pH 5. for g hours. The hydrolysis of agricultural
wastes resulted out
reduced
that cotton seed hulls has the best result,
followed by rice straw, corri stover and the louest
bagasse as shown in the table .l5. and figure
is
sugarcane
7.
2.
THE COMPARISON BETWEEN COST OF GLUCOSE PRODuCT10N FROM THE AGRICUL―
TURAL WASTES.
This study showed that production cost of glucose in chapter
found out that glucose production costs is higher as compared to its
market
price (14 Bath/kg).
agricultural
The production cost
wastes are shown
of
5
glucose from the
in Table 25.
Copyright by Mahidol University
63
Table
25.
Estimated glucose production cost
CENTRAL LIBRARY
MAHIDOL UNIVERSITY
Glucose production cost
Bath/g
Bath/kg
Substrate
Corn stover
1.341
1341
Rice straw
1.236
1236
Sugarcane bagasse
1.570
1570
Cotton seed hulls
0。
976
976
From Table
25, and Figure 13,
it is shown that the production
cost from sugarcane bagasse has the highest cost which
or
1570 Bath/kg,
is
,|.57 Bath/S
followed by the production cost from corn stover, the
production cost from rice straw, and the production cost from cotton
seed
hulls
has
the tonest cost which is 0.976 Bath/g or
In the production cost, cost of chemicals are highest
976 Bath/kg.
and cost
of
is lowest. The production cost exclude fixed capital cost
(sueh "as^fermehtor, hydrolyzer, etc.), labour cost, chemical recovery,
water
purification, by-product credit, pollution control
and energy consump-
tion cost. Additional energy cost in the production cost making the
cost higher is shown in the appendix X. The reason (that was found)
causing the high cost
of
production
From Table 29-32 appendix X
Costs
of
is
is
shown
due
to the cost of
energy.
the total glucose production costs.
energy are 2.302 Bath/g from corn stover, 2.184 Bath/g from
rice steraw, 4.226
Bailh/g from sugarcane bagasse, and 2.089 Bath/g
from cotton seed hulls.
Copyright by Mahidol University
64
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Copyright by Mahidol University
崎
ト
0
０一０一
一゛０● 一
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1国
CHAPTER 7
CONCLuS10N AND RECOMMENDAT10N
1.
CONCLuS10N
The study
found out
of the quality potential of agricultural
that cotton
seed
hulls has the highest cellulose which is
folloned by sugarcane bagasse with
straw with 36.lX and
33%
The economic
following rating
40%
59%
cellulose, corn stover and rice
respectively.
potential of the agricultural wastes has the
: rice straw being the first follow
sugarcane bagasse being the
However, these
wastes
agricultural
third
and cotton seed
wastes are
still
i
by corn stover,
hulls being the last.
mportant sources of
cel lulose.
The
out put of hydrolysis of the agricultural wastes are of
different levels
even though the method used
in the hydrolysis is
the
same. Cotton seed hulls afier being hydrolyzed has the best output.
Corn stover
follows, rice straw
cones
third
and Sugarcane bagasse the
lowest.
This study has found out that the estimated glucose production
cost of the different agricultural wastes are as
follows.
The estima-
ted production cost of cotton seed hulls is 0.976 Bath/g, rice straw
is 1.236 Bath/g, corn stover is 1.341 gath/g, and the highest is
sugarcane bagasse which
is
1.57
Bath/g.
The glucose production costs
stated in this study as compared to the maket price are higher.
The
findings indicated that
substrates, the prcduction cost
if
cotton seed hulls be used
is the lowest followed by rice
as
straw,
Copyright by Mahidol University
66
corn stover being the third and Sugarcane bagasse being the highest.
It is concluded that from the potential quality and cost of
production, the ncst suitable substrate in the production of glucose
is
rice straw, corn stover follows, sugarcane bagasse the third anci the last
being the cotton seed
hulls.
is used as substrate in
the production of grucose the production cost is the rowest among the
four substates. But the availability of cotton seed hulls is rare that
it may lead to the shortage of cotton seed hulls it production is to be
cotton seed hulls
If all these four agricultural wastes are used in the
productlon of glucose at the same time it may save the problem of
storage, avoiding foilage or fire. Since these agricultural wastes are
made
widely.
seasonal they can be used
2.
alternatively one after another.
RECOI,TMENDATION FOR FURTHER STUDY
Recormendation
agricultural
1.
for.further study on glucose production
from
wastes are the followings.
Achievement
of
mi
croorgani sm
in
producing high
activity
widely studied. There also should be study of
reused enzyme for reduction production cost.
enzyrned should be
2.
necessary
Improvement
of
for further study
methodology
of
such as; making up enzyme, reducing the
substrate loner than i00 mesh, and the use
chemical
for
glucose production is
of suitable kind of
pretreatment.
Copyright by Mahidol University
67
3.
scale.
At present, glucose
llowever,
in the future
of energy crisis, glucose }{ill
will
be used as
4.
if
to
be
for the
in
making alcohol which
machines.
mre stuOies
made
in using the
products such as lignin hemicellulose and mycelium. By
cost
of
business
production may be possible because
be more used
fuel for various
There ought
can not be produced
this
by-
way, the
production may be reduced.
Copyright by Mahidol University
68
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Biotechnol. Bioengo symp. No. 6, 35 (1976).
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57
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Copyright by Mahidol University
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Copyright by Mahidol University
74
APPENDIX I
CALCULAT10N
Calculation of power consumption
for
warming water up to
50'c.
Water
I
kg want heat
to raise
up temperature more beginning
25'c.
.
I
heat
Heat consumption
Power requi rerent
Water
kg, using
kcal
power consumption
1
x 103 cal.
25 x i03 cal.
25 x 103 cal.
25 kcal.
1.1622 x tO-3 kwh
25 x 1.1622 x lO-3 fWn
25
=
=
=
=
=
of water 25.C
Raise up temperature of water I kg to
Beginning temperaturet
using
por,rer
l,later 5.8824 kg,,using
=
power =
50.C,
x
x to-3 run
5.g824 x 25 x t.1622 x
25
to-3
=
lrlater 5.618 kg, using porver =
5.61g
x 25 x
7.4626
klrJh
x 25 x t.1622
x
rwn
0.216826
3.1454
to-3
1.622 x
twn
0.163231
to-3
=
Water 3.7454 kg, using power =
khJh
0. 170913 kl'lh
to-3
=
Water 7.462 kg, using power =
1.1622
khlh
x 25 x
kwn
I
.1622 x
Copyright by Mahidol University
=
0.t08823
k}Jh
75
Calculation of fuel consumption for heating water up to
120°
C.
Water 1 kg want heat
to raise up temperature more begining
95° C.
Water
I
kg, using
heat
95
=
95
kg
=
95
kcal
=
9500a
kca1/kg
=
_95
9500
kg
・
1:0
Fuel requi rement
Heat value of fuel oil
.
Fuel oll cOnsumption
Fuel oll l tOn
. .
= 0・
cal
01 kg
=
6.7a
barel
=
6.7 x 42
gal
=
6.7 x 42 x 3.785
=
1065.099
Fuel oll l kg
Fuel oll cOnsumption
x .|03
=
=
=
=
1
1
1065.099 = 1.o651
1000
o.01 x l.0651 1
0.010651
1
1
Ralse up temperature of water
l kg to 120・ C; using fuel oil
=
Water 3.4314 kg, using fue1 011
1
=
3.4314 x O.010651 1
=
0.03655
Water 2.91305 kg, using fue1 011 =
=
Water 3.82984 kg, using fue1 0il =
1
2.91305 x O.010651
0.03103
Water l.94201 kg, using fuel oil =
1
0.04079 1
1.94201 x o.01651
=
1
1
3.82984 x O.01651
・
aFrom
o.olo651
0.02068
1
1
Copyright by Mahidol University
0ffice of National Energy.
76
Agricultural
wastes.
Corn stover
In crop year 1984/85, corn products
Ratio
of corn product: corn straw
一
一
〓
Corn straw
Ratio of cOrn
straw: corn stover
In year 1984/85, there
)
4,226,ooo(55
(63)
I .1
ton,
4,226,000
2863 : 2oo
(
tons
63 )
are
= 4,226,000 x
〇 ３
０ 一
６
２ ８
２
corn stover
= 295,215
tons
tons
Rice straw
In year 1984/85, Harvested area of rice
There are
rice straw in harvested area
In year 1984/85, there are rice straw
60,186,ooo(65) rai s
1
rai
o.4a
tons
60,186,000 x 0.4 tons
19,905,000
tons
aFrom Rice Research lnstitute.
Copyright by Mahidol University
77
Sugarcane bagasse
Sugarcane bagasse t1% (63)
一
一
〓
In year 1984/85, sugarcane products
25,055,000(65)
tons
25,055,000 x ll
tOns
100
2,755,050
fuel in
sugar
mills
90% (63)
2,756,050 x
〇 ０
９ 一０
︲
Used as
tons
tons
2,480,445
tons
97,400(65)
もons
Cotton seed hulls
In year 1984/85, cotton products
Cotton
in cotton seeds
12.5%(63)
Total seeds and cotton
97,400 x 10o
12.5
635,200
Seeds
nutts tX(53) of
seed
of cotton
tons
635,200-97,400
tOns
555,800
tOns
= 555,800 x
= 5558
０
︲ 一０
︲
cottonseed
of cotton
tons
tons
tons
Copyright by Mahidol University
78
APPEDIX II
BUFFER USED IN THIS sTUDY
Acetate buffera
Stock solutions
A
:
0.2
M
solution of acetic acid
},,as prepared by mixing
of acetic acid (sp gr. 1.049) in distilled
water and made to 1000 ml with distilled water.
B : 0.2 M solution of sodium aceiate was prepared by diluting
16.4 sm of C2H302Na or 27.2 gm of C2H3O2Na.3Hr0 in
.1000
ml of distilled water.
11.55 ml
l,trorki
ng
so I uti
on
Mix X ml
of
distilled
A and y ml
of
B and
dilute total of
100
ml with
water.
X
osourcer WaIpole, G.S.,
Y
pH
46、 3
3.7
3.6
44.0
6.0
3.8
41.0
9.0
4.0
36.8
13.2
4.2
30.5
19.5
4。
25.5
24.5
4.6
20.0
30.0
4.8
14.8
35.2
5.0
10.5
39.5
5。
8.8
4.8
41.2
5.4
45.2
5.6
1941, J. Chemo sOc. 105,
4
2
1501 (1914).
Copyright by Mahidol University
＾ソ
﹁′
APPENDIX lll
EFFECT OF MILLING
The samples tested
for effect of milling
were Solka Floc.
Hydrolysis was carried out using the 400 mesh fraction
samples. The Iower curve represents
previously lrdrrer
the
same
rni
Iled floc
this fraction
Sll40 (40 rnesh
size fraction obtai ned after
tion passing
of the tvJo
obtained from
size).
St,l40 had been
The upper curve
aOO
in figure
14-
‘０〓 ●●〓 一﹄０‘０００● ド
Ｃ●ヽ ９´
﹂ ０い０●３〓︶
一
-t
---P'!ot
t"'-
is
ball milled frac-
400 mesh but not passing 500 mesh, as shown
'
a
h..n-to?,
Fig 14. Effect of milling on the susceptibitity of Solka Floc SH40
fraction passing 400 mesh but not passing 500 mesh, ball
milled fraction passing 400 resh but not passing 500
Source:
mesh.
John Nystrom, Biotechnol. Bioeng. Symp.
No.5,223(1975).
Copyright by Mahidol University
80
APPENDIX IV
MESH SIZE
Mesh size is listed in Table 26.
Table 26.
Mesh size.
Mesh
size
Inches
Micrpns
40
0.0165
420
80
0.007
117
100
0.0059
149
200
0.0029
74
270
0.0021
53
Source: 」ohn Nystrom,31otechnol.Bloengo symp。
No.5,224(1975).
Copyright by Mahidol University
_
ｎ０
APPENDIX V
EFFECT OF pH DURING HYDROLYSIS (41)
is slightly lower than 5.0,
but higher than 4.0. Since the pH optimum range is relatively narrow,
During hydrolysis., the pH optimum
pH
control during hydrolysis
may be
necessary. The
during the hydrolysis. Hhen Solka Floc
distinct
The
pH optima can be seen, one
situation is similar
is
us.ed
pH tends
to
decrease
as a substrate, two
at 4.25 and another at 4.75 (Fig t5l.
when using waste from
the furfural
manufac-
turing process as a substrate, although the optimum range is sharper
and the lower optimum less clear (Fig 16). The fact that there are tho
ootima may result from the presence
cel
lulase
compl
of different
components
in
the
ex.
一Ｅゝ Ｅ 一●も じコΦ
Fig 15.
EffeCt of pH On the activity Ofル を ん。dar″ α υづ
2・
`da Cellulases.
Formation of glucose with a dilute enzyme solution from a 2%
suspension of Solka Floe within l(0- )2 (―
), and
Copyright by Mahidol University
3 (0-0 )days of hydrolysis (41)
82
︵
〓δ二 ３８ あ
，
F19 16.
Effect of
the activity of 1\.bl1odens. u i:ride cellulases.
Foination of giucose with a dllute enzyme sr.riution frorrr a
pH on
of furfural process waste within I (o--{),
2 (c------o ), 3 ( H)
and 4 ( H
) days of hydrolysis (4t)
4.6% suspension
Copyright by Mahidol University
83
APPENDIX VI
丁HE PRODUCttION AND HARVESttED AREA OF AGRICULTURAL PRODUCTS IN
VEAR 1984/1985
Table 27.
The production and harvested area of agricultaral prOducts
(1984/1985).
.
I
area
rais)
Harvested
production
(1000 tons)
Corn
10,866
4,226
Rice
60,186
19,905
3,319
25,055
Item
Sugarcane
Cotton
(1000
437
79。 4
Source: Center for Agricultural Statistics,
0ffice of Agricultural
Economics,
Minitry of Agriculture and Co-operatives,
Bangkok, Thailand, .l985.
Copyright by Mahidol University
84
APPENDIX VII
CONVERSION FOR UNIttS uSED IN THIs sTUDY
l ton
1000
ks
l lb
0.44
kg
lg
10-6
l lt
1000
.ml
l ga1lon
3.785
tt
l barel
42 gallon (5.6146 cuft)
l kcal
l Gcal
103
109
l Btu
0.2644 kcal
l Btu
778 ft-tb
l hp
745.7 watts
l inch
2.54
kWh
3.6 x to6
cal
4.184 J
g
cal
cal
cm
.l
Copyright by Mahidol University
85
APPENDIX VIII
PILOT PLANT
PILOT PLANT PROCESS FOR CELLULAS[ PRODuCT10N
←
STERILE AIR_
STERILE AIR
INOCULUM VESSEL
一
PRODUCT■ ON VESSEL
FILTER
RECYCLE _
FORPROTEIN SCURCE
CAKE
FILTRATE
TO HYDROLYSIS REACTOR
PILOT PLANT PROCESS FOR CELLULOSIC‖ ASTE HYDROLYSIS
BUFFER
SOLUT10N
FILTER
SOLID
IPASTE
Fig 17.
lot plant process of glucose production [Adapt from the pilot
plant of glucose production
Copyright
by Mahidol
University
froni newsgaper
by Natic Development
Pi
Center (NDC) (381
86
APPENDIX Ix
COST OF PRINCIPAL EQUIPMENT
The
principal
estimated as shown
Table
28.
Cost
in
equi pment
mirr
are
equipmenta
Description
each
ammer
pilot prant
Table 29.
of principal
Item
costs which used in
unit
of
unit
fi::',i:::i ::[:i::-
cost
Unit costc
process
480'000 833'208
Mirring
Bath
1974 satn
ii85 ;;;ii;;
90 hp, 3600 rpm motor,
air system cyclone,
capacity 5,000-8,000
I
bs/hr.
tank
for alkali
agitated carbon steel l,2B0,OO0 2,221 ,BB7 Alkali_
mixing vessel, volume
treatrnent
treatnrent(A) 18.5 x 104 gal.
Mixing
'
Agitator
unit rating
100
hp
70,000
trptor couple
Solids
filter
-
vacuum
drum filter, fil_
industrial
i
,086,000
i2t,509
I ,g85,132
tration area
1380 ft2
Inoculum
vessel (Bl )
volunre
4.0gx104
gal, agitaled stai_
less steel construc_
tion, 30 hp agitator
rnotor.
446,000
774,189
Enzyme
produc_
tion
Copyright by Mahidol University
87
Item
Description
each
Induction
votume
unit
of
Unit
Bath
cost
Unit costc process
1974 Bath 1985 seiiion
4.08xt05gal 1,780,000 3,0g9,81t
fer.mentor(82) agitated, stainless
(Enzyme produc- steel Constructi on
vessel )
gitator
motor unit rating 200 hp
70,000
121,509
coupled with B,
-
Mycelium
fi lter
Enayme
filter
pressure
'27Z,OOO
filter,
fi ltration
7$ f*
472,151
area
plate and frame
366,000
635,32i
fi lter,
fi ltration area
t01o ft2
pressure
'
Refrigration
volume
t.32lxl04
gal
780,764b 1,355,288
storage tank
sorid
filter
firtration area
t38o
Hydrolyzer
ftz
concrete
1,086,000 1,985,132 Hydrolysis
digestor,
4,440,000
7.107,169.s
agitated place below
:
-
ground level , volume
5.54x10" gal.
Holding
tank
volure 1.585x104
gal
234,22gb
406,586
Source: aBiotechnol. Bioeng.
一 Symp. No. 5,262-263 (1gll)
bBiotechnol. Bioeng.
Symp. No. 6, 73 (1976)
cCost in
1985 = 6s51 in 1974 )( llholesa'le price index in 1985
.
llholesale price index in l9l4
，
Copyright by Mahidol University
88
APPENDIX X
GLuCOSE PRODuCT10N COST (INCLUDE COsT oF ENERGY)
Glucose was produced by hydrolysing
delignified cellulosic
material (corn stover, rice straw, sugarcane bagasse, cotton seed
huus)
at 50'c' pH 5- cost of glucose pncduction are listed in Table 29-32.
Table
29.
Glucose production
Item
cost (from
Unit/g of
glucose
corn stover)
Bathノ unit
Bath/g of
glucose
Raw material
Corn stover(dり ,k9)
0。
4902
0。
4464
0.219
Tota1 0.219
Chemicals
NaCH (g)
34.314
CH3C00h (g)
5.2741
CH3C00Na (g)
16.979
0.005
0.172
0.012
0.063
0.010
0。
Chemicals fOr enzyme
productiOn(liter of enzyme)
2.9412
0。
217
170
0.638
Total l.o43
Uti lity
3.80
∫
0.03655
１
1.43
Fuel oll (1)
９
８
３
︲
1.5126
３
６
︲
8.5
９
７
０
0.0093,4
Power(kWh)
2.302
１
８
３
ａ
ｔ
Ｏ
Ｔ
０ ２ ０ ２
Water (M3)
Total production cost
Copyright by Mahidol University
89
Table
30,. Glucose production cost (from rice straw)
Uni tノ g of
glucose
Item
Raw
Bath/unit
Bath/g of
glucose
material
Rice straw (dr.y, kg)
0.41615
0.4435
0。
185
丁ota1 0.185
Chemicals
NaOh (g)
29。
CH3C00H (g)
5。
CH3C00Na (g)
Chemi
1305
0.005
0。
146
03699
0。
012
0。
060
0.010
0。
162
0.217
0.610
16。 2158
cal s-enzyme
production(
liter
of enzyme) 2。 809
Tota1 0.978
Utili ties
Water (M3)
06008531
8.50
Power(kWh)
Fuel oil(1)
1.445
1。
0。
031027
0.073
43
::￨::12.184
3.80
Tota1 2.257
Total production cost
3.347
Copyright by Mahidol University
90
Table
31
-
Glucose production
cost (from sugarcane bagasse)
Item
Raw
unit/g of
glucose
Bath/Unit
Bath/g of
glucose
0.54112
0.3233
0.177
material
二
Sugarcane bagasse (dr:y,
kg)
、
．
Tota1 0。 177
Chemicals
NaOH (g)
38。 2984
CH3C00H (g)
Chemicals
for
0。
1915
6908
0。
012
0.080
21。 5400
0。
01
0。
6。
CH3C00Na (g)
0.005
2154
enzyme
production(liter of
enzyme)
3。
7313
0.217
0.810
Tota1 1.297
Utili ti es
Water (M3)
0。
Power(kWh)
2.847
Fuel oil (1)
0。
01,2924
04079
8.5
0。
096
1.43
3.80
226
::￨:11}4。
Tota1 4.322
Total production cost
5。
796
ノ ．
ヽ
Copyright by Mahidol University
001447
91
ｆ¨ １
CENTRAL LIBRARY 1
MAHIDOL UNTVERSTTy/
Table
32.
Glucose production
Item
cost (from cotton seed hulls)
Unit/g of
Bath/Unit
Bath/g of
glucose
Raw
material
Cotton seed hulls (dry, kg)
〕
gl ucose
0.27743
1.1442
0。
Tota1
317
0。 317
Chemicals
NaOH (g)
4201
0。
005
0.097
3.3581
0。
012
0.040
19。
CH3C00H (g)
■
CH3C00Na (g)
10。 8107
、
Chemical for enzyme
producti6n(liter of enzyme)
1.8727
0.010
0。
108
217
0。
406
0。
Tota1
0.611
Utilities
Water (M3)
0。
Power (kWh)
1.3927 .
Fuel oll (1)
0.02068
0056874
8。 5
0.048
1.43
1.992)2.089
3。 80
0.079」
■ ¨ 一■ Ｆ ヽ ´
丁ota1 2。 137
Total production cost
3.065
Ｒｔ ”
Copyright by Mahidol University