RESULTS AND DISCUSSION - Shodhgangashodhganga.inflibnet.ac.in › bitstream › 10603 › 145 › 18...
136
Transcript of RESULTS AND DISCUSSION - Shodhgangashodhganga.inflibnet.ac.in › bitstream › 10603 › 145 › 18...
4 RESULTS AND DISCUSSION
Part I SCREENING OF CELLUMLYTIC MICROORGANISMS
4.1 Isolation of cellulolytic bacteria and fungi
Cellulolytic microorganisms viz., fungi and bacteria
were isolated from various samples collected from paddy
field, timber saw mill dump, municipal garbage compost
pit, coconut husk retting spot, dung of elephant, goat,
rabbit, cow and horse as well as termite gut using
cellulose enriched modified Czapek's agar medium and Hans
agar medium for fungi and bacteria respectively. The
results are given in Table 4.1.
The population of different groups of cellulolytic
microorganisms differ in various substrates. Bacterial
population was maximum in cow dung followed by horse duns.
The population of cellulolytic fungi was more in coconut
husk retting spots followed by municipal garbage compost.
This might be due to the aerobic solid state fermentation
upon these substrates, leading to the development of
basidiomycetes, brown and white rot fungi. Dung of
animals recorded very low population of cellulolytic
fungi. Both paddy soil and timber saw mill dump recorded
same number of cellulolytic fungi. Only bacterial
population was observed in termite gut. The isolated
microorganisms were subjected to primary screening for the
selection of better strains.
Table 4.1. Population of cellulolytic microbes in different sources per gram of matter
Sources 3 Bacteria (10 )
4 Fungi (10 )
Paddy soil
Coconut husk rettiny pit
Saw mill compost
Municipal garbage compost
Cow duny
Elephant duny
Goat dung
Rabbit dung
Horse dung
Termite gut
4.1.1 Primary screening and selection
The ~rimary screening of the cellulolytic
microorganisms included a qualitative test of detecting
the ability to utilize cellulose waste as a sole carbon
(cellulose) source. These cellulolytic microorganisms
showed considerable variation in their capacity to degrade
cellulose waste because of the differences in the genetic
make up of microorganisms. Out of the 150 isolates of
bacteria, only eight isolates which showed maximum
degradation in 14 days were selected for secondary
screening. Among the 200 isolates of fungi, eleven showed
good cellulose degradation and they were selected for
further studies.
4.1.2 Identification of selected strains
The different isolates of bacteria selected after
primary screening were identified on the basis of their
morphological, physiological and bfochemical properties
and the fungi were identified on the basis of their growth
and morphological characters. The list of identified
microorganisms are given in Table 4.2.
4.1.3 Secondary screening
Cellulolytic microorganisms selected for secondary
screening are given in Table 4.2.
Cellulolytic activity was checked by measuring total
sugars and reducing sugars produced by the enzymatic
saccharification of tapioca waste and water hyacinth.
The sugars obtained from tapioca waste were more. Among
the different isolates of fungi, - M. verrucaria produced
the highest level of reducing sugar followed by
A. fumiqatus, C. comatus, P. florida, - - - Fusarium sp.,
T. harzianum, A. niqer, - - Penicillium sp., Mucor sp.,
P. citrinopileatus, and Poria sp. The level of total - - sugars was higher than the reducing sugars in all the
treatments and the variation was similar in tapioca stem
and water hyacinth.
Cellulomonas sp. produced the highest level of
reducing sugars followed by - B. subtilis. The same pattern
of changes in total sugars with higher values was recorded
for the enzyme from bacteria using both tapioca waste and
water hyacinth.
The growth of the organism was represented by
measuring the cell protein and the enzyme production by
estimating the protein content of cellulose enriched
liquid media. - M. verrucaria showed maximum growth and
enzyme production followed by - A . fumigatus, - - C. comatus,
P. florida, etc. Among the bacteria, Cellulomonas sp. was - found to be the most suitable organism for enzyme
production when compared to other strains. It was found
that fungal enzymes were more suitable for cellulose
degradation than bacterial enzymes.
On the basis of above data, the following four
microorganisms were selected for further studies.
1. Myrothecium verrucaria
2. Coprinus comatus
3. Pleurotus florida
4. Cellulomonas sp.
Even though, - A. fumigatus was good in enzyme
production, it is not included in the present study since
it is a pathogenic fungus.
4.2 Optimization of Hicrobial Growth and Enzyme Production
Studies on the effect of various factors on growth
and enzyme production of different isolates were carried
out by growing them in cellulose enriched modified
Czapek's and Hans broth medium for fungi and bacteria
respectively.
4.2.1 Effect of temperature
The effect of temperature on microbial growth and
enzyme production was studied and the results are given
in Figures 4.la, 4.lb and 4.1~.
TEMPERATURE (OC)
Figure 4.1 a. Effed of temperature on growth in terms of cell protein
0 10 20 30 40 50
TEMPERATURE ("C)
Figure 4.1 b. Effect on temperature on enzyme production in terms of filter paper activity
One unit will liberate one micromole of glucose per minute per mg protein
*M. verrucarm -KC. cornatus
* ' florida + Cellulomonas sp.
250
10 20 30 40 50
TEMPERATURE ("G)
Figure 4 . 1 ~ . Eftect d temperature on enzyme production in terns of 8-glucosidase activity
One unit will liberate one micromole of p nitm phenol per minute per mg protein
The rate of growth of verrucaria and Cellulomonas
0 sp. increased with increasing temperature from 10 to 30°c
and thereafter decreased. But comatus grew well at
40°c. On the other hand, & florida grew well at a low
temperature ( 20°c) and thereafter the growth decreased.
The enzyme production was tested bymeasuring the filter
paper activity and fi-glucosidase activity. Both filter
paper and fi-glucosidase activities were maximum for the
enzyme, secreted by - M. verrucaria and Cellulomonas sp. at
30°c, while for - P. florida and - C. comatus, the enzyme
activity was maximum, when these organisms were incubated
0 at 20 C and 40°c respectively. The enhanced cellulolytic
activity was observed by Tewari -- et While studying
with various isolates of Cellulomonas sp., Enriquez
observed optimum growth and enzyme production of this
o 117 organism in the temperature range 28-32 C . The
temperatures, for maximum growth and enzyme production for
P. florida and C. comatus were also found to be 20°c and - o 60,199 40 C
4 .2 .2 Effect of pH
The effect of pH on microbial growth and enzyme
production was studied and the results are given in
Figures 4.2a, 4.2b and 4.2~.
Figure 42a. Effect of pH on growth in terms of cell protein
Figure 4.2b. Effect of pH on enzyme production in terms of filter paper activity(FPA)
One unit will liberate one micromole of glucose per minute mg protein
*_M. verrucaria *C. comatus 4 Cellulomonas sp.
Figure 4 2 c Effect d pH on enzyme production in terms of &glucosidase activity
One unit will liberate one micromole of p. nitro phenol per minute per mg protein
The rate of growth increased with increasing pH from
3.0 to 6.0 for & verrucaria, 3.0 to 7.0 for florida
and Cellulomonas sp. and 3.0 to 8.0 for - C. comatus.
Even though M, verrucaria showed maximum growth at pH 6.0,
it could grow well in the pH range 4.0-7.0. Similarly
P. florida and Cellulomonas sp. grew well in the pH range - 6.0-7.0. Among the isolates tested only - C. comatus
showed the maximum growth at alkaline pH.
In the case of M, verrucaria, both the filter paper
and B-ylucosidase activities were maximum when the pH of
its culture broth was 6.0. Above and below this pH, the
enzyme activity gradually decreased. The enzyme secreted
by both P, florida and Cellulomonas sp. at pH 7.0 showed
maximum filter paper activity and 8-glucosidase activity.
On the other hand, the filter paper and a -glucosidase
activities of - C. comatus enzyme increased with increase in
the pH of the culture broth from 3.0 to 8.0 and then
decreased. 5 -glucosidase produced by Cellulomonas sp.
was comparatively low. In all the cases, the enzyme
production was directly proportional to the growth of
microorganisms.
4.2.3 Effect of substrate concentration
The effect of substrate concentration on microbial
growth and enzyme production of all the isolates at
various levels of substrate concentration ranging from
0.5% to 4 % was studied and the results are given in
Figures 4.3a, 4.3b and 4.3~.
The rate of growth increased with increase in the
substrate concentration from 0.5% to 4.0% for both the
fungi and bacteria. In all the cases, there was a
significant increase in growth (upto 2%) and thereafter
the increase in growth was not significant. Even though
the microbial growth increased with increasing the
substrate concentration, there was not a corresponding
increase in filter paper activity and P -glucosidase
activity for all the microbes. There was no significant
difference in enzyme activity when the substrate
concentration was increased from 1% to 3%. Similarly,
even though the microbial growth increased with increasing
the substrate concentration, the cell protein per gram of
the substrate decreased after 1% substrate concentration
for all fungi and bacteria. Substrate concentration had a
major role in the enzyme production. Mandels and Weber
also found that the enzyme production increased with
increasing the substrate concentration upto 1% and
6 1 thereafter there was not much difference for T. viride . -
* P florida 4 Cellulomonas sp.
Substrate Concentration (%)
Figure 4.3a. Effect of substrate concentration on growth in terms of cell protein
Substrate concentration (%)
Figure 4.3b. Effect of substrate concentration on enzyme production in terms of filter paper adivity(FPA)
One unit will liberate one micromole of glucose per minute per mg protein
4 3 0 0 r ~ M .wr.car. uc comatus
1 / + P florida + Cellulomonas sp.
? 0 - X -2oo- c .- a - e a
F . 150 2 - a z E g loo 0 3 - ?' a
Substrate concentration (%)
Figure 4 . 3 ~ Effect of substrate concentration on enzyme production in terms d B-glucosidase activity
One unit will liberate one micromole ol p. nitm phenol per minute per mg protein
4.2.4 Effect of incubation period
The effect of incubation period on microbial growth
and enzyme production of all the selected fungi and
bacteria was studied and the results are given in
Figure 4.4a, 4.4b and 4.4~.
The rate of growth of all the fungi and bacteria
increased with increasing the incubation period upto
11 days. But for M, verrucaria, the growth of the
organism increased upto 14 days and thereafter the growth
slightly decreased. This was because of the delay in
getting acclimatized to a medium containing cellulose
which is not easily degradable. Among the tested microbes
M. - verrucaria gave the maximum growth followed by
C. comatus, P, florida and Cellulomonas sp. and the same - trend was observed for filter paper and -glucosidase
activities.
The f i -glucosidase activity of comatus was found
to be slightly higher than - P. florida. All the isolates
except M, verrucaria showed same pattern of changes.
*t& verwcarla * C. cornatus
+ P florida Cellulomonas sp.
INCUBATION PERIOD (Days)
Figure 4.4a. Effect of incubation period on growth in terms of cell protein
1 *M vermcarla *s. cornatus 1 1 +iWmcla *Cellulomonas sp. I I
2 5 8 11 14 17 20 23
INCUBATION PERIOD (Days)
Figure 4.4b. Effect of incubation period on enzyme production in terms of filter paper act~ity(FPA)
One unit will liberate one micromole of glucose per minute per mg protein
0 2 5 0 0 8 11 14 17 20 23
INCUBATION PERIOD (Days)
Figure 4 . 4 ~ . Effect of incubation period on enzyme production in terms of Sglucosidase activity. One unii will liberate one micro mole of p. nitro phenol per minute per mg protein
4.2.5 Effect of agitation
The effect of agitation on microbial broth and enzyme
production was studied and the results are given in
Tables 4-3a and 4.3b.
The rate of growth of all the fungi and bacteria
increased with agitation. The activity of the enzyme
secreted by the organisms increased with increasing the
rate of agitation. Thus agitation gave maximum growth and
enzyme production of verrucaria, C, comatus, P, florida
and Cellulomonas sp. The trend of cell protein content
also corresponded with the trends of filter paper activity
and fi-glucosidase activity. The highest values for all
the parameters were obtained with the continuous shake
cultures followed by intermittent shake cultures for
M. - verrucaria followed by comatus, - P. florida and
Cellulomonas sp.
4.3.6 Effect of carbon sources
The effect of carbon sources on growth and enzyme
production by incorporating various carbon sources at 1%
concentration in the media was studied and the results are
given in Tables 4.4a and 4.4b.
-uyw ad ~ouaqdoqru-d jo aTowoJsTu auo aqeJaqrT TTTM ~~E~Tso~~T~-~Jo qyun auo -u~w ad uarqo~d 6w ad asosn~b 40 aTouoJsyu auo aqeJaqTT TTTM wdj JO ?Tun auo
- X~rnrqse ~aded ~aq~rj - vdj ~3s + ases qsea UT sanTen any4 40 abe~anw
- - OE'9 + OZV LL'O + OO'OL 89.T r OL'8ZT VZ'9 7 00'0~5 0Z'T 7 00.Z6 6'1.T T 08.6ET 6ur'levs snonurquo3
- TL'T 7 061 V5'0 + 00'9C 90.T r 81'9L ZT'C 7 CO'OVZ '18.0 + 00'CS 8S'T T OT.6L fJ~~yt?q~ ON
-
(u~a~o~d 6u/n) (aqelqsqns 6/6u) OT (uwo~d 6~) (aqeqsqns 6/6w) E - L - oor7e7rfie
All the carbon sources tested favoured the growth of
cellulolytic microorganisms. M, verrucaria showed maximum
growth with dextrin as carbon source. While - P. florida
and - C.' comatus showed maximum growth when glycogen was
added. But the presence of starch in the medium favoured
maximum growth of Cellulomonas sp. Among the various
carbon sources, the presence of cellulose in the media
gave the minimum growth of all the microorganisms.
When cellulose was used as the carbon source, both
filter paper and ~-glucosidase activities were found to
be high in all the organisms. Lactose and inulin were
found to induce cellulase production in all the cases.
Even though all the organisms grew well in starch,
glycogen, glucose and dextrin, the cellulase production
was decreased significantly. The enzyme production was
maximum in cellulose and carboxy methyl cellulose enriched
medium.
4.2.7 Effect of nitrogen sources
The effect of nitrogen sources on growth and enzyme
production by incorporating various nitrogen sources at
0.1% concentration into the media was studied and the
results are given in Tables 4.5a and 4.5b.
0
N
NJ
m
0
NI
?
43
ON
N
Jr
.0
0
0
3
+I
+I
+I
00
0
00
0
-3
m
aV
)a
fl +
I +
I 0
0
0
OO
N
N
0
0
nN
I?
4
-3
C
w
aJ JI
73
0
'3
LC
aJ LI
UU
o
~
m
3
TT
c -- a
U
ur
o
0
E
€1
3
3a
3
.3
C
0
Cc2 E
E
u
E
EL
I
=z
40
c 0
w
c
.3
w
UT
0 a
LI
0
a r
u
m .?
E
LI a
w a&
0
w
ro
w
0 3
u
0
3
E
4
0
3i LI u
L 3
0 E
0
w
3 c
6
go
LO
o
w
Flu
+I
.2 :
w
Ea
l
rn n
m
w .d
U
C
3
1
0 3
u
w3
m
u .-i
w ~
m +
U
LI
K'm w
a~
-.,>n ro
3 .r(
m
ro
ir
3D
0
U
.,
3
m-+
rn
3
30
m
r-., u
>m
33
a
-r w
m<
0
> aa
I
2 ,'-
9
WL
L
'4-U
0
0 0 3
'4
u u
w L .3
.rl
JI c
c
m1
33
LI
wu
m w
>
LL
c
C
<L
OO
Among the organic nitrogen sources, peptone followed
by yeast extract was found to be the best cellulase
inducer for M, verrucaria, comatus, P, florida and
Cellulomonas sp. Organic nitrogen sources favoured the
qrowth and enzyme production of all the microbes.
Among the inorganic nitrogen sources, the presence of
ammonium molybdate, followed by sodium nitrate, in the
media accelerated the growth and enzyme production of
C. comatus. Sodium nitrate, ammonium M. verrucaria and - molybdate and ammonium dihydrogen ortho phosphate were
found to be the inducers for - P. florida. Ammonium
dihydrogen orthophosphate and ammonium molybdate were
found to be the suitable inducer for Cellulomonas sp. The
presence of urea in the media was found to enhance the
growth and enzyme production of - C. comatus and II, florida.
Garcia-Martinez et al. found that urea was the most -- suitable nitrogen source for cellulase production by
2 2 5 Clostridum thermocellum . - M. verrucaria gave the highest
amount of enzyme in all the media containing different
nitrogen sources. Cellulomonas sp. gave the least amount
of enzyme in all the cases.
4.3 Growth curve
The growth curve of the microorganisms selected are
given in Figure 4.5.
: *g. ver~caria *s. comatus 1 +I? florida + Cellubmonas sp.
DAYS
Figure 4.5 The Growth curve
The growth curve of -- M. verrucaria showed that weight
of the mycelia was highest on the 14th day while for
C. comatus and & florida, the growth of the organisms was - maximum on 12th day. After the 14th day, there was a
slight decrease in the weight of the mycelia. In the case
of Cellulomonas sp. the growth of the organism was maximum
on the 14th day as indicated by the optical density.
As in the case of - C. comatus and P. florida, there was a 7
slight decrease in the weight of mycelia on the 20th day.
This may be due to the utilization of cellulose by the
microbes.
PART I1 COMPOSITION AND PRETREATMENT STUDIES OF WASTE SAMPLES
4.4 Composition
Cellulose, hemicellulose, lignin, starch, pectin,
proteins and total lipids present in the samples were
estimated and the results are given in Table 4.6.
Cellulose and lignin contents were found to be more
in tapioca stem. Hemicellulose was maximum in tapioca
petiole. But, there was not much difference in
hemicellulose content in tapioca stem and petiole. On the
other hand, tapioca leaves were rich in proteins and
lipids. Proteins and lipids were found to be less in
tapioca petiole. Tapioca stem contained maximum pectin
content followed by tapioca petiole, leaf and water
hyacinth. On the other hand, starch content was highest
in tapioca leaf followed by petiole, stem and water
hyacinth.
4.5 Pretreatment Studies
The effect of following physical and chemical
pretreatments of the cellulosic wastes om enzymatic
saccharification was studied.
4.5.1 Physical pretreatments
The effect of physical pretreatments by milling
and steaming the cellulosic wastes on enzymatic
saccharification was studied. The results are given in
Table 4.7.
The results of the effect of physical pretreatments
of cellulosic wastes showed that pretreatment of
cellulosic wastes by steaming made the samples more
suscepti-ble to enzymatic saccharification than milling.
Among the various cellulosic wastes, the maximum
saccharification was obtained for tapioca stem followed by
tapioca petiole, water hyacinth and tapioca leaf.
Among the various isolates, the enzyme secreted by
M. verrucaria gave the maximum saccharification rate for - different parts of the tapioca waste as well as water
hyacinth followed by - C. comatus, P, florida and the
bacteria Cellulomonas sp.. The results showed that water
hyacinth provided a better cellulose resource for
bioconversion than tapioca leaf. Both the wastes of
tapioca stem and petiole gave more or less same percentage
of saccharification, but slightly higher in tapioca stem.
The results indicate that the physical pretreatments had
some influence on the bioconversion.
Table 4.7 Effect o f physical pretreatrnents
Type o f Source o f enzyme p h y s i c a l Ce l l u l ose .................................................... pret reatment waste M. v . C. c . P. f . Cel l .
l ap ioca stem 7.75 + 0.06 5.75 + 0.04 5.00 + 0.06 3.50 + 0.07 MILLING Tepioca p e t i o l e 7.32 T - 0.01 5.32 ; 0.01 4.80 5 0.03 3.30 + 0.06
Tapioca l e a f 5.20 + 0.05 4.09 T 0 . 0 2 3 . 5 4 T 0 . 0 1 2 . 4 8 2 0 . 0 3 Water hyac in th 6.60 - ; 0.02 5.06 - ; 0.05 4.64 0.02 3.26 0.05
Tapioca stem 8.50 - + 0.10 6.06 + 0.13 5.36 + 0.10 3.90 + 0.05 STEAMING Tapioca p e t i o l e 7.50 - + 0.06 5.91 ; 0.10 5.10 0.08 3.75 T 0.06
Tapioca l e a f 5.34 + 0.04 4.30; 0.09 3.80 3 0.04 2.63 + 0.01 Water hyac in th 6.88 - 0.06 5.63 ; - 0.21 4.97 0.05 3.56 2 0.03
Average o f 5 values i n each case + SEM Values are expressed as mg o f r e d i c i n g sugars per 100 mg subs t ra te pe r mg p r o t e i n
M. v. - M. ve r ruca r i a C. c. - cornatus P. f. - - P. f l o r i d a C e l l . - Cellulomonas sp.
4.6 Chemical Pretreatments
4.6.1 Effect of sodium hydroxide
The effect of sodium hydroxide pretreatment using
1% to 10% sodium hydroxide solutions at two different
temperatures ( 2 7 O ~ and 121°c) was studied. The results
are given in Tables 4.8a, 4.8b and 4.8~.
The effect of sodium hydroxide pretreatment of the
cellulosic wastes showed that sodium hydroxide
pretreatment favoured the enzymatic hydrolysis of
cellulosic waste more than the physical pretreatments. As
in the case of physically pretreated samples, the maximum
percentage of saccharification was given by the enzyme
from M, verrucaria followed by - C. comatus, il, florida and
Cellulomonas sp.. The pretreatment with 1% sodium
hydroxide made tapioca stem more susceptible to enzyme
action. This was followed by tapioca petiole, tapioca
leaf and water hyacinth. On the other hand, the enzymatic
saccharification was the highest in tapioca stem followed
by tapioca leaf, tapioca petiole and water hyacinth, when
a 2% solution of sodium hydroxide was used. The
pretreatments carried out at higher temperatures favoured
the bioconversion of cellulosic wastes.
- TT-O 5 LB'E ST.O + zz.9 oc.0 7 ~6.9 00.0 LL.8 qqu~ae& ~aqe~ 60.0 + 2b.L T0.0 Z ZZ'OT BT'O Z Z5'OT LT'O - + LV'TT jea~ eso~del c0.0 + 6V.V 51'0 + 0T.6 01.0 + LZ'6 oT'O + LO'TT a~o~qad esordel 2 TZT 52'0 + 62'01 6T'O + 66'bT 5Z'O + 68'91 51.0 + ZO'OZ waqs esordel
- - 81.0 + 4b.V 710.0 5 8T'L 50.0 - + LZ.8 C0.0 7 Ofl'OT 47uT3'3L4 Ja?eM 6V.O + SL'9 90'0 - + S2.6 OT'O - + OZ'OT 61'0 ? 52'ZT ~ea~ eso~del 8r.0 + 07.8 60.0 2 OE'TT 50'0 2 ~8.~1 sz.0 5 zz.91 a~orqad eaoydel T TZT Ob'O + 68'8 51'0 + SE'ZT 51'0 + 96'ET OE'O + 9b'LT waqs eaordel
- 80.0 5 EL-E 60.0 1~0.9 ~T.O 5 ~13.9 50.0 + 25.8 vqur~eKq Jaw LO'O + 5E'L 50'0 - + ZT'OT 02'0 + 0T'OT 01'0 + 9S'TT jea~ eaoydel 10'0 Z TV'V OT'O + 00'6 EO'O + 91'6 90'0 - + SE'TT a~oyqad eaoydel Z LZ 82.0 + 6T'OT 12.0 + 56'bT 51.0 7 60.91 TT.0 + 88'6T waqs ea0~del
- - - vz.0 - + TS'V c0.0 + 80-L 10.0 + r0.8 20.0 7 V6.6 vqu~aeX4 JaJeM 1V'O + 19'9 50.0 + ZT.6 50.0 + iT.01 LO.0 7 - CT'ZT jea[ esordel OS-o + ZTY 90'0 7 - ~T-TT LO'O 7 65'ZT 61.0 2 01.91 a~orqad e3orde1 I L; ZS'O + 28.8 OT'O + 51.~1 ZT'O VL.ET OZ.O + 00.~1 waqs eaordel
C
.4
w
u
0
Li
a
m
E
L,
w
a
m
u
m Li
u
m 0
3
07
m
E
o
0
3
Li
w
n
10 r
m
01
3
10
x
m
WC
in .-I
0
+I$
ru
m
ZLi 0 'c
0
.c u rn
m
E
w m
c
m
-4
-0
6l w
m
ro J
m
3 w
m
r
> a
X
n
W
L
w
0 r
m
w
rn I0
m
m r
3
m-l
>
m
4 >
m r\ 0 ir\
0 0
3 0
....
0 0
0 0
+I +
I +I +
I r.Ir\N
a
nr.0
.
..
.
nrrr.a
nn
-in
3
N3
3
..
..
0 3
0 0
+I +
I +I +
I r
.w
no
0 J
r‘. rT
\ .
..
.
0r.o
r. 3
3
mn
mm
0 0
3 0
.
..
.
00
00
+I +
I +I +
I O
NJ
N
nr-r-n
.
..
.
N i‘. 3 m
3
3
n o
m N
N
3 3
3
..
..
0
00
0
+I +
I +I +
I N
0 L
n I?
N
On
N
..
..
?
-Or\O
\ 3 3 3
m
3
s
0
4J
E.
4L
C
wu
m-
4
uw
wo
m
a
3 m
X
m
m m
r
U 0
U
o
oo
r
.- .-I .i m
a
a au
m
mm
m
C t-- C
x
\O
i
N
3
N Y
\ O
I r.
00
00
.
..
.
00
00
+I +
I +I +
I r- m
3 o
c
\C
IL
nL
n
..
..
w
J r-
n
r.m
nr
-
33
30
.
..
.
00
00
+I +
I +I +
I PI r- n
o
NJ
nN
.
..
.
3m
ow
3
Po
em
3
N3
O
..
..
0 0
0 0
+I +
I +I +
I N
NP
\rr\
NO
NN
.
..
.
T\
O\
-I
h
3
3
mm
n3
3
00
3
..
..
0
00
0
+I +
I +I +
I' O
r-N
Ln
n
ma
n
..
..
C
OO
N^
3 4
3
m
3
s
0
u
E.
-!
L C
w
u m
.4
ur
nm
u
ro a
+
m
X
m m
mr
U
UU
o
o o
r4 .-I
.-! .-! 0
a
a nu
m m
m
m
-+
ex
J
-l
N
3
JU
33
n
00
30
d d d d
+I +
I +I +
I r.
N N
o
inaln
CI
..
..
n
nw
a
N~
\"-,J
3
33
3
..
..
3
00
0
fl +
I fl +
I m
N o
\O
"-I
\0
\O Ln
..
..
O
r-
Or
.
4
3
r-
r.m
n
0 0
0 0
.
..
.
00
00
+I +
I +I +
I Q
ON
3
am
r-
3
..
..
N
h3
m
3
3
ma
on
3 0
3 0
.
..
.
o o
o o
+I +
I +I +
I 0 N
m 0
N 4
N
CI
..
..
r-O
CIO
\ 3
3 3
m
3
s
0
u
E.
4L
C
mu
m.
4
u m
m u
ro a3 m
A
m
m m
s
u 0
0
o o
o r
.-I .-I .4
w
a a
nu
m
mm
m
c C
6 x
U3
r- N
"7 N
J N
0 0
0 0
.
..
.
00
00
+I +
I +I +
I n
o n a
NN
JJ
.
..
w
-3 r. <
nm
mn
i
0 0
0
..
..
0
00
0
+I +I +
I +I
P N
r. N
O
On
O
..
..
3
mo
a
"
3
r.
03
~3
0
30
0
..
..
0 0
0 0
+I +
I +I +
I Ln 0 P- r.
om
on
.
..
.
nm
i~
3 i
o fi, ~
.h
J
30
00
.
..
.
00
00
+I +
I +I +
I N
m N
r. O
Ln
*N
.
..
.
m0
~m
3 3
3
w
3
s
0
0
E.
+L
C
m
u
m-
+
uw
wo
ro
ah
m
X
m m
ms
U
0
L)
oo
o*
.-I
.- .-!
m
a a
au
m
mm
m
cc
cx
J
r. N
Y
1 rn P)
I,
2 P 4 'rl X 0 I,
T. C 9 1 li o
Y
8 (c
(c
w
i 4 P)
I
I
I
I I
I
I.
I - 1
4
I w
I
U
I
I I I
I
I I I.
I
L
0 I
El
.
xl
n
N
I C
I
m I
I
L
1 0
I
I
m I
0 1
UI
3
10
0
1 i
nl
.
I U
I I I
I I
I
I I 1 I
.
I
>
I I
.
I z
I
I
I
I
I
m
m
o
3
3
w
3 4
J
3 m
w
m
u 3
L
I
OC
u
o
c
c '4
w
ou
-
0 .-I
3 6
Q
Cu
d-
o
m o
,,m
I
Li
w w
n
u -
€3
u
I I
?$
O-
Table 4.812. Effect of d i m hydroxide pretreatment
Temper- Concent- ature ration of Source of enzyme
solution Cellulose ........................................................ ( OC! (%) waste M. v . C. c. P. f. Cell.
Tapioca stem 15.07 - + 0.20 11.72 - + 0.10 9.63 + 0.09 5.12 + 0.01 - - 27 8 Tapioca petiole 7.54 - + 0.09 6.52 - + 0.08 5.26 - + 0.03 2.87 + 0.02 -
lapioca leaf 15.17 - + 0.18 13.03 + 0.15 12.18 + 0.18 7.93 + 0.07 - Water hyacinth 8.72 - + 0.15 8.34 T - 0.10 6.83 T - 0.05 5.81 - + 0.05
Tapioca stem 12.37 + 0.20 8.85 + 0.10 8.71 2 0.20 4.30 - + 0.13 2 7 10 lapioca petiole 6.73 5 - 0.05 6.22 0.09 5.22 + 0.11 2.60 + 0.05
Tapioca leaf 16.98 - + 0.23 14.87 5 - 0.18 13.38 0.19 7.98 7 - 0.01 Water hyacinth 8.03 - + 0.07 6.54 - + 0.15 6.03 - + 0.05 5.00 - + 0.04
Tapioca stem 14.85 - + 0.25 11.70 + 0.09 9.39 + 0.01 5.01 + 0.05 121 8 Tapioca petiole 7.52+0.19 - 6.3550.07 5.12T0.05 2.75T0.03 -
Tapioca leaf 15.70+0.13 - 13.52T0.17 - 12.20;0.08 7.94+0.07 - Water hyacinth 8.20 - +.0.10 7.35 - + 0.20 6.72 - 0.07 5.04 - + 0.03
Tapioca stem 10.54 - + 0.05 8.03 - + 0.18 7.54 + 0.10 3.80 - + 0.10 121 10 Tapioca petiole 5.91 - + 0.09 4.26 + 0.05 4.50 ; 0.03 2.00 2 0.13
Tapioca leaf 17.03 - + 0.15 15.00 - 0.08 13.65 0.15 8.00 - + 0.05 Water hyacinth 8.29 - + 0.02 6.50 - + 0.07 5.54 - + 0.03 4.64 - + 0.09
Average of 5 values in each case 2 SEM Values are expressed as rng of reducing sugars per 100 mg substrate per mg protein
M. v. - - M. verrucaria C . c. - C. comatus P . f. - - florida Cell. - Cellulomonas sp.
The effect of sodium hydroxide pretreatment using 4%
and 6 % solutions showed that this pretreatment increased
the susceptibility of the cellulosic wastes to enzyme
action. As in the earlier cases, the maximum percentage
of saccharification was achieved by using the enzyme from
M. verrucaria followed by C, comatus, P. florida and - - Cellulomonas sp.. When the pretreatment was carried out
by using both 4% and 6% solution, tapioca stem gave the
highest saccharification rate followed by tapioca leaf,
tapioca petiole and water hyacinth. The results indicate
that as the concentration of the sodium hydroxide solution
was increased, the rate of saccharification and hence the
reducing sugars formed from tapioca leaf was found to
be increased. saccharification. was more when the
pretreatment was carried out at 121°c by using 4% sodium
hydroxide solution than in other cases. But the reverse
effect was found for tapioca petiole and water hyacinth,
when 6 % sodium hydroxide solution was used.
The effect of sodium hydroxide pretreatment of the
cellulosic wastes using 8% and 10% solution showed that as
the concentration of sodium hydroxide solution was
increased, the tendency of the cellulosic wastes to
undergo enzymatic saccharification was decreased except in
the case of tapioca leaf. The reducing sugars released
from the cellulosic wastes decreased in the order: tapioca
leaf > tapioca stem > water hyacinth > tapioca petiole.
The enzyme secreted by M, verrucaria gave the maximum
saccharification followed by - C. comatus, P, florida and
Cellulomonas.
4.6.2 Effect of sodium hydroxide-acetic acid
The effect of sodium hydroxide-acetic acid
pretreatment of the cellulosic wastes on enzymatic
saccharification was studied and the results are given in
Table 4.9.
The results showed that this pretreatment could make
water hyacinth to a much better substrate for enzymatic
saccharification than alkali treatment or physical
pretreatments. The results showed that tapioca stem waste
could serve as a good cellulose resource for
bioconversion. As in earlier cases, in this case also the
pretreatments carried out at higher temperatures favoured
enzymatic saccharification. This pretreatment favoured
water hyacinth for a better bioconversion than the other
pretreatments. Tapioca stem gave the highest percentage
of saccharification followed by tapioca leaf and tapioca
petiole. In the present study also the enzyme from
M. verrucaria gave the maximum bioconversion and - Cellulomonas sp. gave the least activity.
4.6.3 Effect of chloroform
The effect of chloroform pretreatment of the
cellulosic wastes on enzymatic saccharification was
studied and the results are given in Table 4.10.
The effect of chloroform pretreatment showed enhanced
bioconversion of cellulosic waste. Herealso the maximum
percentage of saccharification was given by the enzyme
from & verrucaria followed by comatus, P, florida and
Cellulomonas sp. The highest percentage of reducing
sugars was obtained by the enzymatic saccharification of
tapioca leaf followed by tapioca stem, tapioca petiole and
water hyacinth. This pretreatment was found to be better
for water hyacinth than the other pretreatments. The same
result was also found for tapioca leaf except with 10%
sodium hydroxide solution.
4.6.4 Effect of hydrochloric acid
The effect of hydrochloric acid pretreatment of
cellulose waste on enzymatic saccharification was studied
and the results are given in Table 4.11.
fable 4.10 Effect of chloroform p r e t r e a h n t
Source o f enzyme C e l l u l o s e ....................................................... waste M. v. C . c . P. f . C e l l .
l a p l o c a stem 13.94 - + 0.20 10.98 - + 0.25 9 .96 - + 0.10 4.78 - + 0.04
Tapioca p e t l o l e 1 2 . 5 3 - + 0 .18 lU.30 - + 0.17 9 .06 - + 0 .13 4 .61 - + 0 .03
l a p i o c a l e a f 15.62 - + 0 .11 13.29 - + 0.18 12.05 - + 0.19 7.97 - + 0 .07
Water h y a c i n t h 10 .80 - + 0.09 9 .66 - + 0.05 7.79 - + 0.04 5 .00 - + 0.01
Average of 5 v a l u e s i n each c a s e , SEM. Values a r e e x p r e s s e d a s mg o f r e d u c i n g s u g a r s p e r 100 mg s u b s t r a t e p e r my p r o t e i n .
M. v . - & v e r r u c a r i a C . c. - C. cornatus P. f . - - f l o r i d a C e l l . - Cel lulomonas s p .
Table 4.11 E f f e c t of hydrochlor ic a c i d pre t rea tment
Source of enzyme Cel lu lose ....................................................... waste M. v . C . c . P. f . Ce l l .
Tapioca stem 16.03 - + 0.25 13.09 - + 0.18 12.69 - + 0.15 7.83 - + 0.10
Tapioca p e t i o l e 10.62 - + 0.19 7.94 - + 0.08 7.70 - + 0.20 4.40 - + 0.01
Tapioca l e a f 11.09 - + 0.06 10.07 - + 0.21 9.92 - + 0.09 6.46 - + 0.09
Water hyacinth 12.43 - + 0.10 11.15 - + 0.03 10.17 - + 0.05 6.85 - + 0.13
Average o f 5 va lues i n each case -+ SEM. Values a r e expressed a s rng of reducing s u g a r s p e r 100 mg s u b s t r a t e per mg p r o t e i n .
M. v . - M. ve r ruca r i a C . c . - C. comatus P. f . - - f l o r i d a Ce l l . - Cellulomonas sp.
The results of the effect of hydrochloric acid
pretreatment of the cellulosic waste on enzymatic
saccharification showed that the maximum amount of
reducing sugars was obtained from tapioca stem followed
by water hyacinth, tapioca leaf and tapioca petiole.
In this case also, the maximum percentage of
saccharification was given by - M. verrucaria and the
minimum by Cellulomonas sp. This pretreatment was found
to be more suitable for water hyacinth when compared with
other cases.
4.6.5 Effect of sodium sulphite
The effect of sodium sulphite pretreatment of the
cellulosic wastes at two different temperatures on
enzymatic saccharification was studied and the results are
given in Table 4.12.
The action of sodium sulphite on cellulosic wastes at
room temperature showed no considerable improvement over
the physical pretreatments. On the other hand, the
pretreatment carried out at 121°c favoured slightly
the enzymatic hydrolysis. When the pretreatment was
carried out at room temperature, the percentage of
saccharification was more for tapioca stem than
for other cellulosic wastes. But the pretreatment
carried out at higher temperature made tapioca petiole
more susceptible to enzymatic saccharification.
Table 4.12 Effect o f sodiun sulphite pretreatment
Temper- Cel lu lose Source of enzyme a t u r e waste ........................................................
{ O C J M . v . C . c . P. f . Ce l l .
Tapioca stem 7.52 - + 0.19 5.86 - + 0.10 5.13 - + 0.04 3.55 - + 0.05 27 Tapioca p e t i o l e 7.38 - + 0.17 5.81 + 0.13 5.10 - + 0.03 3.53 + 0.10
Tapioca l e a f 4.97 - + 0.08 4.13 + 0.01 3.73 + 0.10 2.33 - + 0.01 Water hyacinth 6.63 - + 0.03 5.4U - + 0.04 P.63 5 - 0.11 3.31 - + 0.02
Taploca stem 8.52 - + 0.21 6.87 - + 0.03 6.07 - + 0.09 4.41 + 0.06 121 T a p i o c a p e t i o l e 8 . 4 0 t 0 . 1 5 6 . 7 2 + 0 . 0 2 - 6 . 0 1 + 0 . 1 0 - 4 . 3 7 5 0 . 0 1 -
Tapioca l e a f 6 . 0 6 ~ 0 . 1 1 1 4 . 8 9 i 0 . 1 0 4 . 3 2 + 0 . 0 3 3 . 2 4 + 0 . 0 7 - Water hyaclrith 7 .92 2 - 0.05 6.39 2 0.11 5 .66; - 0.06 4.09 - + 0.05
Averaye of 5 values l n each case 2 SEM. Values a r e expressed a s m g of reducing sugars per 100 mg s u b s t r a t e per mg p ro te in .
C . c . - C. comatus P. f . - P. f l o r i d a Ce l l . - ~ l l u l o m o n a s sp.
The maximum saccharification of cellulosic wastes
was given by the enzyme from M. verrucaria followed by
C. comatus, florida and Cellulomonas sp. The results - showed that sodium sulphite pretreatment was not effective
for the bioconversion of tapioca leaf.
4.6.6 Effect of peracetic acid
The effect of peracetic acid pretreatment of
cellulosic wastes on enzymatic hydrolysis was studied and
the results are given in Table 4.13.
Peracetic acid pretreatment of cellulosic wastes on
enzymatic saccharification showed that bioconversion was
most faciliated in water hyacinth when compared to
all other pretreatments. Water hyacinth gave the highest
percentage of reducing sugars followed by tapioca
petiole. - M. verrucaria gave the highest percentage of
saccharification followed by - C. comatus, - P. florida and
Cellulomonas sp.. The amount of sugars produced was found
to be almost the same in tapioca petiole and water
hyacinth.
4.6.7 Effect of butanol
Effect of butanol pretreatment on enzymatic
saccharification of the cellulosic wastes was studied and
the results are given in Table 4.14.
Table 4.U Effect of peracetic acid pretreatrent
Source of enzyme Cel lu lose .......................................................... waste M. v . C. c . P. f . Cel l .
Tapioca stem 15.04 + 0.19 12.56 - + 0.17 12.17 - + 0.10 5.03 - + 0.05
Tapioca p e t i o l e 18.22 - + 0.25 14.88 - + 0.15 13.17 - + 0.09 9.95 - + 0.09
Tapioca l e a f 16.68 - + 0.13 14.35 - + 0.19 10.88 - + 0.11 7.39 - + 0.06
Water hyacinth 18.54 - + 0.11 15.13 - + 0.18 13.62 - + 0.15 10.10 - + 0.09
Average of 5 va lues i n each case 2 SEM. Values a r e expressed a s rng of reducing sugars per 100 rng s u b s t r a t e per mg p ro te in .
M. v. - M, ver ruca r i a C. c . - C. cornatus P. f . - Florida - Cel l . - Cellulornonas sp.
Table 4.14 Effect o f butanol p r e t r e a b t
Source o f enzyme Cel lu lose ........................................................ waste M. v . C . c . P. f . Ce l l .
Tapioca stem 8.10 - + 0.10 6.52 + 0.04 6.11 - + 0.10 5.42 - + 0.20
Taploca p e t l o l e 7.04 - + 0.09 6.24 - + 0.09 5.54 - + 0.13 4.01 + 0.17
Tapioca l e a f 5.88 - + 0.05 4.53 - + 0.10 h.05 - + 0.05 2.93 - + 0.03
Water hyacinth 7.60 + 0.01 6.41 - + 0.01 5.60 - + 0.04 5.10 + 0.10
Average of 5 values i n each case + SEM. Values a r e expressed a s rng o f reducing sugars per 100 mg s u b s t r a t e per mg p ro te in .
M. v. - M. ver ruca r i a C. c . - comatus P. f. - - P. f l o r i d a Ce l l . - Cellulornonas sp .
Butanol pretreatment on enzymatic saccharification
showed that this pretreatment had only slight influence on
the cellulosic wastes for enzymatic saccharification.
The highest percentage of saccharification was given by
tapioca stem and hence the maximum amount of reducing
sugars was obtained from tapioca stem. M, verrucaria gave
the highest percentage of saccharification followed by
C. comatus, P, florida and Cellulomonas sp. -
4.6.7 Effect of hydrogen peroxide' and ferrous salt
The effect of hydrogen peroxide-ferrous salt
pretreatment on enzymatic saccharification was studied
and the results are given in Table 4.15.
Hydrogen peroxide-ferrous salt pretreatment on
enzymatic saccharification also had not much influence on
cellulosic wastes. Out of the cellulosic wastes, water
hyacinth gave the maximum reducing sugars followed by
tapioca petiole, tapioca stem and tapioca leaf. The
highest percentage of saccharification was given by the
enzyme from M, verrucaria followed by E, comatus. This
pretreatment was found to be not suitable for tapioca
leaf.
Table 4.15 Effect of hydrogen peroxide-ferrous salt pretreatment
Source of enzyme Cellulose ........................................................ waste M. v. C. c. P . f. Cell.
Tapioca stem 7.56 - + 0.10 6.22 - + 0.19 5.62 + 0.13 3.94 - + 0.09
lapioca petiole 7.60 - + 0.08 6.52 - + 0.15 5.74 + 0.14 4.29 - + 0.06
Tapioca leaf 5.92 - + 0.01 4.77 - + 0.08 4.46 - + 0.18 3.12 + 0.04
Water hyacinth 7.61 - + 0.05 6.57 - + 0.11 5.80 - + 0.06 4.47 - + 0.08
Average of 5 values in each case 2 SEM. Values are expressed as rng of reducing sugars per 100 mg substrate per rng protein.
M. v. - M. verrucaria C. c. - comatus P. f. - florida Cell. - Cellulomonas sp.
4.6.8 Effect of hydrogen peroxide and manganous salt
The effect of hydrogen peroxide-manganous salt
pretreatment on enzymatic saccharification was studied and
the results are given in Table 4.16.
M. verrucaria gave the highest percentage of - saccharification and Cellulomonas sp. gave the least.
Tapioca stem was found to be most susceptible to enzymatic
saccharification. The same trend of saccharification of
the cellulosic wastes was given by the enzyme from all the
four different isolates.
4.6.9 Effect of hydrochloric acid and zinc chloride
The effect of hydrochloric acid-zinc chloride
pretreatment on enzymatic saccharification was studied and
the results are given in Table 4.17.
Hydrochloric acid-zinc chloride pretreatment on
enzymatic saccharification showed that this pretreatment
could influence all the cellulosic wastes for enzymatic
saccharification. This pretreatment was found to be most
suitable for tapioca stem and tapioca leaf. The maximum
reducing sugars was obtained by the saccharification of
tapioca stem using cellulase enzyme from M, verrucaria and
the minimum from water hyacinth by Cellulomonas sp.
Table 4.16 Effect of hydrogen pemxide-rengamus salt pre treaht
Source of enzyme Cellulose ........................................................ waste M. v. C. c. P. f. Cell.
Tapioca stem 13.67 5 0.19 11.45 - + 0.15 10.23 - + 0.09 4.87 + 0.06
Tapioca petiole 8.52 - + 0.08 6.87 - + 0.17 6.05 - + 0.07 3.00 + 0.01 - Tapioca leaf 10.06 - + 0.11 9.07 - + 0.13 8.02 + 0.01 4.20 + 0.02 - -
Water hyacinth 9.94 - + 0.13 8.01 - + 0.09 7.08 - + 0.05 3.97 + 0.10 -
Average of 5 values in each case 2 SEM. Values are expressed as mg of reducing sugars per 100 mg substrate per mg protein.
M. v. - verrucaria C. c. - C. comatus P. f. - - florida Cell. - Cellulomonas sp.
Table 4.17 Effect of hydrochloric acid-zinc chloride pretreatment
Source of enzyme Cellulose ........................................................ waste M. v. C. c. P. f. Cell.
Tapioca stem 15.62 - + 0.19 12.59 - + 0.10 11.13 - + 0.16 7.18 - + 0.09
Tapioca petlole 9.86 - + 0.05 8.05 - + 0.20 7.17 - + 0.13 5.26 - + 0.01
Tapioca leaf 12.00 - + 0.10 10.52 - + 0.18 10.06 - + 0.10 5.62 - + 0.05
Water hyacinth 8.52 - + 0.08 6.91 - + 0.05 6.07 - + 0.06 3.64 - + 0.07
Average of > values in each case _r SEM. Values are expressed as mg of reducing sugars per 100 mg substrate per mg protein.
M. v . - M. verrucaria - C. c. - C. comatus P. f. - - florida Cell. - Cellulomonas sp.
4.6.10 Effect of acetic acid and hydrogen peroxide
The effect of acetic acid-hydrogen peroxide
pretreatment on enzymatic saccharification was studied and
the results are given in Table 4.18.
Effect of acetic acid-hydrogen peroxide pretreatment
on enzymatic saccharification showed that this
pretreatment was more suitable for water hyacinth.
Besides this, pretreatment could fascilitate tapioca stem
and petiole for enzymatic saccharification. Even though
the saccharification rate of acetic acid-hydrogen peroxide
pretreated tapioca leaf was found to be less, but this
pretreatment was better for leaf. The reducing sugars
released from the above mentioned pretreated cellulosic
wastes by enzymatic saccharification decreased in tapioca
stem followed by tapioca petiole. The highest percentage
of saccharification was achieved by using the
enzyme secreted by I?, verrucaria and the lowest by
Cellulomonas sp. The results showed that different
pretreatments have different action on different
cellulosic wastes.
The solid wastes of tapioca plant and water hyacinth
consist mainly cellulose and lignin in considerable
quantity. The physiochemical properties of
lignocellulosic wastes determine the rate of enzymatic
degradation of this cellulosic wastes. Both cellulose and
hemicellulose as such are highly susceptible to the action
of cellulolytic microbes. But in nature it exists as a
complex with lignin. Therefore liberation of cellulose
and hemicellulose moieties either by chemical or enzymic
reaction was reported to be essential for bioconversion.
For different cellulosic wastes, different methods of
pretreatments were suggested. Studies have shown that
both physical and chemical pretreatments had some
influence on saccharification rate.
The physical pretreatment, milling reduced the
particle size and provided more surface area for the
action of enzymes. The maximum saccharification in the
case of M, verrucaria might be due to the increase in
concentration of the enzyme. The difference in the
saccharification may also be due to the difference in the
various fractions of cellulase enzyme as indicated by
Srinivasan et al. -- 226 and ~ u n l a ~ ~ ~ ' . The enhanced rate of saccharification by enzymes of various microorganisms
after steaming may be due to the swelling of cellulosic
material.
The chemical pretreatments carried out by using
alkali, increased the saccharification rate of cellulosic
wastes. This might be due to the increase in the fibre
saturation point and the swelling capacity of
lignocellulosic materials. The increase in swelling
capacity results from the saponification of esters of
4-o-methyl glucuronic acid attached to xylan chains.
Table 4.18 Effect of acetic acid-hydrogefi peroxide pretreatment
Source of enzyme Cellulose ....................................................... waste M. v . C. c. P. f. Cell.
Tapioca stem 14.27 + 0.20 12.87 + 0.15 12.05 + 0.13 8.00 + 0.10
Tapioca petiole 13.49 - + 0.10 11.11 + 0.10 9.22 - + 0.15 7.07 - + 0.11
Tapioca leaf 6.39 + 0.09 5.26 - + 0.09 5.06 - + 0.20 3.72 - + 0.15
Water hyacinth 11.36 + 0.17 9.35 + 0.17 8.10 - + 0.21 6.02 - + 0.03
Average of 5 values in each case 2 SEM. Values are expressed as mg of reducing sugars per 100 my substrate per mg protein.
M. v . - M, verrucaria C. c. - comatus P. f. - - P. florida Cell. - Cellulomonas sp.
In the natural state, the esters act as crosslinks,
limiting the swelling or dispersion of polymer segments in
water. In the present study, pretreatment with alkali
was found to be the most suitable one for tapioca stem
and leaf. For water hyacinth and tapioca petiole,
pretreatment carried out by using peracetic acid was found
to be more suitable than alkali pretreatment. This might
be due to the difference in their composition and
structure.
PART XI1 PURIFICATION AND CEARACTWIZATION OF ENZYME
4.7 Purification
It would be advantageous if we suggest a cheap
culture medium, since industrial production of cellulase
is essential, because of its increasing demand for
saccharification purpose and protoplast fusion studies.
Hence based on the optimization of growth and enzyme
production studies, a medium was suggested for both fungi
and bacteria separatively. The composition is given in
Chapter I11 which was used for the studies. The organisms
were grown in the respective media for a period of
14 days. This was centrifuged and the filtrate was used
for purification as mentioned in Chapter 111.
The results of the purification steps for
cellulase from - M. verrucaria, C, comatus, li, florida and
Cellulomonas sp. are summarised in Tables 4.19a, 4.19b,
4.19~ and 4.19d.
LC. 0
- a,
'5
a, [D
3
n
ma
,
3
.3
o
m a,
3
i m
u
mu
I
3
0 -3
3
3
om
U
-3
3 0
3
30
D
1
51
3
C
w
14
m
m
-;f m
m
-
1
a, u
4.3
V1 3
I-
m
m
?< 2
0
u
3
3
01
a,
3
DO
u
151
CX
I
.
I m a,
XC
u .+ 9
I I
a! L
LY
0
ma,
00
Lo
m
4
u
mm
o
n .i
3 3
.3
C
33
ccn
2
43
3
E
3 .-,
a, ma,
DL
4
c
00
c
w 0
i
u
m,
*
w w
*a
LO 3
W 0
3 3 * m
m
f f\O
3u a I
w.3
w2
U
3 LOU
3
k
a, 3
a, LO
3
n
ma
, .-I
-3
n
m
w
4
rl m
u
LO
O
I
3
0 -4
3
3
UL
O
U
.rl
3 0
I
30
O
3
C
a,
13
m
V)
3 m
m
-1
a,
O
313
(D
.4
I
-
m
(0
q? 2
0
u
by
2
3
3
00
0
m
CX
I
.
I w
a,
x
c
I I
u .r
l =Q.
a, r
ru
0
al
w
00
LO
rn CI
u
mm
a
.-3
i 3
." C
3
3
cm
3
3
3
3E
i 3
a, a
m
a,*
C
UC
J
CW
0
I I
oa
X 3
* U
U
* *
Cellulase was purified by DEAE-sepharose CL-6B as
mentioned in Chapter 111. Endo- .8 -1,4-glucosidase,
exo- b -1,4-glucose (cx) and b-glucosidase components of
cellulase enzyme of - M. verrucaria, P, florida and
C. comatus were eluted with 0.4 M, 0.45 M and 0.5 M - ammonium acetate buffer (pH 5-0) respectively. In the
case of cellulomonas cellulase enzyme, the P-glucosidase
fraction was not recovered by gel filtretion while the
endo- and exo-P-1,4-glucosidase fractions were eluted
with 0.4 M and 0.45 M ammonium acetate buffer (pH 5.0).
M. verrucaria C -endo cellulase was purified to 16 - X
fold, C -exo-cellulase to 18 fold andfi-glucosidase to 15 1
fold. In the case of - C. comatus and - P. florida both
C - and C -cellulase were purified upto 16 fold and X 1
P-glucosidase to 12 fold. Almost the same fold of
purification was also obtained for the enzyme from
cellulomonas. The specific activity was found to be much
higher than that obtained from some species by several
workers. It is certain that better standardization may
yield greater recovery rate and high specific activity.
4 .8 Enzyme studies
4 . 8 . 1 Effect of pH on the activity of the enzyme
The effect of pH on activity of the enzyme was
studied and the results are given in Figures 4.Ga, 4.6b,
4 . 6 ~ and 4.Gd.
Figure 4.6~1. Effect of pH on activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca stem
50
- C .- a - g 4 0
E" . 0 .- 2 w 0 n 2 cD 30
E" 0 0 - . 2 ;; 20
5 S V)
a z 9 1 0 -
n W II:
0 3
*M verrucarla *C comahrs
*I? florlda -- Cellulomonas sp
-
.
8 4 2 4 6 5 5 4 5 8
E " ! 8 - . E" -
*&. verrucarla -*c C. comatus * Cellulomo~s sp.
Figure 4.6b. Effect of pH on activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca petiole
50
* I ? florida I- 4 Cellulomonas sp.
n 40
Figure 4. 6c. Effect of pH on activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca leaf
*c cornatus
Cellulornonas sp.
F~gure 4 .6d Ettect of pH on acttvlw of the enzvrne Reduclng sugar produced by the enzyrnatlc sacchar~f~cat~on of water hyac~nth
Effect of pH on the activity of the enzyme showed
that pH of the saccharification medium depended upon the
228 enzyme source and the nature of the cellulosic waste . The enzyme secreted by M, verrucaria gave the highest
saccharification rate at pH 4.6 for all the cellulosic
wastes studied. On the other hand, the enzyme from
C. comatus gave the highest percentage of reducing sugars - -- at pH 5.0 for all the cellulosic wastes, while the enzyme
from - P. florida and Cellulomonas sp. showed a pH optimum
at 4 .8 . Even though the pH optimum for M, verrucaria was
4.6 , it was active in the pH range 4.2-5.0. Similarly the
enzyme from C, comatus could give saccharification in the
pH range 4.6-5.2. P, florida showed a pH range of 4.4 to
5.0 for saccharification. The optimal pH for the growth
of - C. comatus was at an alkaline pH but the optimal pH
for saccharification was found to be at an acidic pH.
4.8.2 Effect of temperature on the activity of the enzyme
The effect of temperature on the activities of the
enzyme was studied and the results are given in
Figures 4.7a, 4.7b, 4 . 7 ~ and 4.7d.
*M. verrucarla *&. comatus
* P florida Cellulomonas sp.
TEMPERATURE ("C)
Figure 4.7a. Effect of temperature on activity of the enzyme. Reducing sugar produced by the saccharification of tapioca stem
TEMPERATURE ("C)
Figure 4.7b. Effect of temperature on the activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca petiole
TEMPERATURE ("C)
50
- C .- a, - g 4 0
Figure 4 . 7 ~ . Effect of temperature on the activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca leaf
-
* P florlda 4 Cellulornonas sp.
-
TEMPERATURE ("C)
Figure 4.7d Effect of temperature on the actlvlty of the enzyme. Reducing sugar produced by the enzymatic saccharlficatton of water hyacinth
The optimum temperature for bioconversion varied with
the enzyme source63. The enzyme secreted by - M. verrucaria
and P, florida released the maximum amount of reducing
0 sugars from the cellulosic samples at 40 C. On the other
hand, the highest percentage of saccharification of the
cellulosic wastes was given by the enzyme from C, comatus
at 50O~. While cellulase from Cellulomonas sp. gave the
0 maximum saccharification rate at 45 C. Above 50°c, the
saccharification rate decreased. Cellulase from - C. comatus
preferred a higher temperature for bioconversion while
M. verrucaria as well as 11, florida preferred lower - temperatures.
4.8.3 Effect of substrate concentration on the activity of the enzyme
The effect of substrate concentration on the activity
of the enzyme was studied and the results are given in
Figures 4.8a, 4.8b, 4 . 8 ~ and 4.8d.
As the concentration of the various cellulosic
samples increased, the yield of reducing sugars was found
to be increased, but the percentage of saccharification
was decreased correspondingly. The same trend in reducing
sugars as well as percentage of saccharification was
observed for all the enzymes from different sources. The
enzyme from all the microbes gave the highest percentage
of saccharification at 2% substrate concentration.
0 0.5 1 1.5 2 2 . 5 3 3 .5 4 4.5
SUBSTRATE CONCENTRATION (%)
1
Figure 4.88. Effect of substrate concentration on the activity of the enzyme. Reduang sugru produced by the enzymatic saccharificetion of tapioca stem
*M. verrucaria * C comatus
* P florida 4 Cellulomnas sp.
SUBSTRATE CONCENTRATION (%)
Figure 4 8 b Effect of substrate concentrat~on on the activity of the enzyme. Reduc~ng sugaf produced by the enzymatic saccharificalion of tapioca petiole
SUBSTRATE CONCENTRATION (%)
Figure 4 . 0 ~ . Effect of substrate concentration on the activity of the enzyme Reducing sugar produced by the enzymatic saccharification of tapioca leaf
SUBSTRATE CONCENTRATION (%)
Figure 4.84 Effect of substrate concentration on the activ~ty of the enzyme. Reducing sugar produced by the enzymatic saccharification of water hyacinth
Thereafter the percentage of saccharification gradually
decreased.
The percentage of saccharification gradually
increased from the substrate concentration 1.8% to 2%.
The highest percentage of saccharification and the maximum
amount of reducing sugars were given by the enzyme from
M. verrucaria. Of the various cellulosic samples, tapioca - stem gave the highest percentage of saccharification,
producing maximum yield of reducing sugars.
4.8.4 Effect of incubation period on the activity of the enzyme
The effect of incubation period on the activity of
the enzyme was studied and the results are given in
Figures 4..I)a, 4.9b, 4..9c and 4.9d.
When the incubation period was increased, the yield
of reducing sugars was also increased. The rate of
saccharification of all the cellulosic wastes increased
almost in a uniform manner upto 12 h of incubation
using the enzyme from various microbes. After 12 h of
incubation, even though the saccharification rate was
increased, the saccharification per unit time was
decreased. Tapioca stem gave the highest yield of
reducing sugars in 12 h of incubation followed by
tapioca petiole, water hyacinth and tapioca leaf.
INCUBATION PERIOD (h)
Figure 4.9a. Eftect ot Incubation period on the activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca stem
INCUBATION PERIOD (h)
F~gure 4 9b Effect of lncubatlon pertod on the acttvlty of the enzyme Reduc~ng sugar produced by the enzymattc sacchar~ftcatton of taploca petiole
* M verrucarla * C comatus 1 * - P florlda j_-
INCUBATION PERIOD (h)
Figure 4 . 9 ~ . Effect of Incubation period on the activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of tapioca leaf
* M. verrucarla *C. cornatus
+ Cellulornonas sp. I I
INCUBATION PERIOD (h)
Figure 4.9d. Effect of Incubation period on the activity of the enzyme. Reducing sugar produced by the enzymatic saccharification of water hyacinth
The enzyme secreted by 5 verrucaria gave the maximum
rate of saccharification during 1 2 h of incubation. This
was followed by - C. comatus, florida and Cellulom-
sp. The yield of reducing sugars was maximum for tapioca
stem, when the saccharification was carried out by the
enzyme from - M. verrucaria and minimum for tapioca leaf,
when the saccharification was carried out by the enzyme
from Cellulomonas sp.
4.8 .5 Effect of agitation on the activity of the enzyme
The effect of agitation on the activity of the enzyme
was studied and the results are given in Tables 4.20a and
4.20b.
Agitation of the reaction medium on the activity of
the enzyme resulted in an increased production of reducing
sugars. The enzyme from 5 verrucaria showed maximum
bioconversion of all the cellulosic wastes. The same
trend of saccharification rate was observed for all the
cellulosic wastes by the enzymes from different microbes.
PART IV BIOCONVERSION OF TAPIOCA WASTE AND WATER HYACINTH
4.9 Bioconversion
Bioconversion of tapioca waste and water hyacinth was
carried out by the three different methods.
4.9.1 Solid state fermentation by the action of microorganisms
The bioconversion of tapioca waste and water hyacinth
was carried out by solid state fermentation using
the fungi and bacteria. The results are given in
Tables 4.21a, 4.21b and 4.21~.
M. verrucaria was found to give maximum fermentation. - The efficiency of the microbes for bioconversion decreased
in the order of - M. verrucaria, comatus, P, florida and
Cellulomonas sp. Similarly, among the various cellulosic
wastes, tapioca stem gave the highest percentage of
fermentation. The ability of the cellulosic wastes to
undergo fermentation increased in the order tapioca
leaf < water hyacinth < tapioca petiole < tapioca stem.
The highest percentage of fermentation was achieved
by M, verrucaria on tapioca stem and the lowest by
Cellulomonas sp. on tapioca leaf.
Table 4.21 Sol id s t a t e fe-tatim o f ce l lu lose waste
( a ) Total sugars
Organism Percentage of s accha r i f i ca t ion Cellulose .................................................... ................................ waste M. v . C . c. P. f . Cel l . M . v . C . c. P . f . Cel l .
Tapioca stem 38.50 - + 0.34 31.90 - + 0.19 28.25 - + 0.14 17.40 - + 0.10 34.64 28.70 25.41 15.65
Tapioca p e t i o l e 33.05 - + 0.19 29.50 - + 0.30 26.96 - + 0.16 16.25 - + 0.07 29.73 26.54 24.25 14.62
Tapioca l ea f 31.50 - + 0.25 27.72 - + 0.16 24.35 - + 0.14 13.94 - + 0.20 28.34 24.94 21.90 12.54
Water hyacinth 32.90 - + 0.39 29.45 - + 0.23 26.55 - + 0.21 16.15 + 0.09 29.60 26.49 23.88 14.53 -
Average of 5 values i n each case + SEM. Values a re expressed as mg of t o t a l sugars per 100 mg s u b s t r a t e per rng p ro te in .
M . v . - - M . ve r ruca r i a C. c . - C . cornatus - -- P . f . - P. f l o r i d a Cel l . - Cellulomonas sp.
The yield of total sugars was higher than reducing
sugars and glucose in all cases. However, in the case of
fermentation carried out by fungi, the amount of reducing
sugars produced was almost equal to the amount of glucose.
But in the case of bacterial fermentation the reducing
sugars released from the cellulosic wastes were higher
than that of glucose.
4 .9 .2 Action of cellulolytic microbes and ligninolytic fungi
Solid state fermentation of cellulosic wastes was
carried out by the combined action of ligninolytic and
cellulolytic microbes and the results are given in
Tables 4.22a, 4.22b and 4.22~.
The combined action of cellulolytic and ligninolytic
organisms on tapioca wastes and water hyacinth showed
that the presence of ligninolytic organisms in the
fermentation medium favoured the rate of fermentation.
As in the earlier cases, here also, the highest percentage
of bioconversion was given by M. verrucaria followed by - C. comatus, P, florida and Cellulomonas sp. The yield
was found to be in the order of tapioca leaf < water
hyacinth < tapioca petiole < tapioca stem. The presence
of ligninolytic organisms in the fermentation medium
increased the release of total sugars, reducing sugars and
glucose in all cases.
4.9.3 Bioconversion of cellulose waste by enzyme
The enzymatic saccharification of the cellulosic
wastes was studied and the results are given in
Tables 4.23a, 4.23b and 4.23~.
Bioconversion of wastes by the enzyme showed that the
yield of total sugars, reducing sugars and glucose were
higher in the enzymatic conversion than in solid state
fermentation. On the other hand, the yield of glucose
produced by the enzymatic saccharification of cellulosic
wastes by Cellulomonas sp. was significantly low. The
yield of total sugars was much higher than reducing sugars
and glucose in all the cases. But the difference between
reducing sugars and glucose was not significant for the
enzymatic saccharification of cellulosic wastes by fungi.
The highest saccharification was given by the enzyme from
M. verrucaria followed by C. comatus. Tapioca stem gave - - the maximum yield of total sugars, reducing sugars and
glucose.
4.9.4 Bioconversion of cellulose waste using termite gut extract
Bioconversion of the wastes using termite gut extract
was studied and the results are given in Table 4.24.
X R m R r u u o 3 0 0 i . . , - 3 C ! a a a u m m m m ! - + - - =
C1 a, . a x
w a , m m +I g a,- LO Jl m u L
0 L u rn O E a!
LO c m .i
D m w
m 3 m 3 a, m i+ > a
X n a,
lable 4.24 Bioconversion of cellulose waste by termite gut extract
Cellulose Total Heduciny Glucose Total Reducing Glucose waste suyars sugars sugars sugars
:my/lUU mg substrate/mg p ro te ln ) ................................ Percentac]~ nf s a c c h a r ? f l c z t ~ o ! :
Tapioca stem 63.08 - + 0 .31 55.40 - + 0.66 >4.36 + 0.48 56.77 49.86 48.92 - lapluca petiole 61.83 - + U.5> 53.85 + U.48 52.64 - + 0.26 55.64 48.46 47.37
lapioca l e a f 56.97 - + 0 .68 50.56 - + 0.70 49.30 + 0.88 57.27 45. 50 44.39 - Water hyac~ri th 60.02 - + 0 . 4 8 52.60 - + 0 .94 50.60 + 0.85 54.00 47.34 45.54 -
Average of 5 values i n each case SEM.
Extracts of termite gut was most efficient for the
bioconversion of agricultural cellulosic wastes to sugars.
The percentage of saccharification was found to be in the
order: tapioca stem > tapioca petiole > water hyacinth >
tapioca leaf. There was also some difference in the yield
of total sugars and reducing sugars. But the difference
was not significant. The results showed that tapioca stem
could undergo saccharification more easily than other
wastes.
Solid state fermentation was the most efficient
method of bioconversion of lignocellulosic wastes under
optimum environmental conditions. Microorganisms secrete
a number of hydrolytic enzymes and attack a number of
complex compounds to simple compounds. Cellulase, being
the major hydrolytic enzyme, contributed a lot to the
solid state fermentation. The end product of this process
consist of a mixture of simple sugars like glucose.
In the present study both the amount of reducing sugars
and glucose are same, indicating that glucose is the only
reducing sugar released during solid state fermentation.
Solid state fermentation in the presence of the fungi
of P, chrysosporium was found to be good. Since lignin
in the reaction mixture was hydrolyzed, the cellulolytic
activity of the microbes was also enhanced. The
differential effect of microbial attack on different
substrate was mainly due to the biochemical composition
and structure of the substrates6*. Tapioca stem waste
contained maximum cellulose and hence showed higher level
of fermentation.
In the present study, it was observed that
fermentation rate was higher when pure enzyme was used.
All these studies indicated that tapioca stem waste
was superior to other substrates for enzymatic
saccharification.
Termites thrive on cellulose present in plant parts.
The cellulose entering into the gut of termite seems to be
broken down to simple sugars. A number of cellulolytic
bacteria were reported to be present in the gut of these
insects. The liquid collected from the gut showed
maximum saccharification which indicate that efficient
cellulolytic enzymes are present in the gut. In the
extract of termite gut, innumerable Trichonymphae were
observed. A study on the isolation of the microbes from
the gut may contribute a lot to the saccharification rate.
But in vitro, culture of the organism was found to be very
difficult and it could not be recommended for utilization
of wastes on a larger scale.
PART V ALCOHOL FERMENTATION
4.10 Fermentation Studies
Fermentation of the fermentable sugars present in the
fermentation medium of both tapioca waste and water
hyacinth was studied.
4.10.1 Effect of pH on alcohol fermentation
The effect of pH on alcohol fermentation of the
sugars obtained by the saccharification of the cellulosic
wastes using the enzyme secreted by the microbes was
studied and the results are given in Figures 4.10a, 4.10b,
4.10~ and 4.10d.
As the pH of the fermentation medium was increased,
the production of alcohol was also increased. The highest
yield was obtained by fermentation of sugars from tapioca
stem. The results indicated that at very low pH,
fermentation of sugars was very slow. As the pH was
increased, the yield of alcohol increased gradually.
There was only a slight difference in alcohol yield,
when the pH of the fermentation medium was 3.0 and 3.5.
Below pH 3.0, alcohol yield was found to be decreased
significantly. It was also found that the yield of
alcohol varied with the cellulosic source.
Figure 4.1 0a. Effect of pH on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca stem
*M. verrucarla * C. camatus
*I? florida * Cellulomonas sp,
Figure 4.1 0b Effect of pH on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca petiole
Figure 4 .10~ . Effect of pH on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca leaf
+ Cellulomonas sp.
Figure 4.10d. Effect of pH on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of water hyacinth
4.10.2 Effect of temperature
The effect of temperature on alcohol fermentation was
studied and the results are given in Figures 4.11a. 4.11b,
4 . 1 1 ~ and 4.11d.
Temperature has a good influence on alcohol
fermentation of sugars. When the temperature was increased
from 2o0c to 30°c, the fermentation of alcohol was also
increased. Thereafter tPe fermentation decreased. The
same trend was observed in all the cases. The maximum
amount of alcohol was obtained from tapioca stem, followed
by tasioca petiole.
4.10.3 Effect of incubation period
The effect of incubation period on alcohol
fermentation was studied and the results are given in
Figures 4.12a, 4.12b, 4 . 1 2 ~ and 4.12d.
When the incubation period of the fermentation was
increased, the rate of alcohol production was also
increased. The same observation was found for the
fermentation of all the cellulosic wastes. Even though
the yield of alcohol increased with the period of
incubation, the amount of alcohol produced per unit time
decreased after the first three days. The best incubation
period for fermentation was three days in all the cases.
The highest yield of alcohol was given by tapioca stem.
* M. verrucarla * C, comatus
* F? florida
4
TEMPERATURE ("C)
Figure 4.1 l a . Effect of temperature on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca stem
- 18 20 22 24 26 28 30 32 34 36
TEMPERATURE ("C)
.,
Figure 4.1 1 b. Effect of temperature on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca petiole
* M. verrucarii! * C. comatus
* I? tlorida Cellulomonas sp.
TEMPERATURE ("C)
Figure 4.1 1c. Effect of temperature on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca leaf
t-& verrucarla *s- cOmatuS
* P florida * Cellulomonas sp.
TEMPERATURE ("C)
Figure 4.1 Id. Effect of temperature on alcohol fermentation Alcohol produced from sugar obtained by the enzymatic saccarification of water hyacinth enzyme
Figure 4.12a Effect of incubation period on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca stem
INCUBATION PERIOD (days)
Figure 4.12b Effect of incubation period on alcohol fermentation Alcohd produced from sugar obtained by the enzymatic saccharification of tapioca petiole
INCUBATION PERIOD (days)
Figure 4 . 1 2 ~ . Effect of incubation period on alcohol fermentation Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca leaf
5
4
- 3 0 - z %
; - 3 - - 8 m2- % . m - 6 6 0, a 1
0 ' 0
+ M verrucarla * s cornatus - * P flor~da -- + Cellulomonas sp
- I
- --
5 1 1 5 2 2 5 3 3 5 4 4
INCUBATION PERIOD (days)
Figure 4.12d. Effect of incubation period on alcohol fermentation Alcohol produced from sugar obtained by the enzymatic saccarification of water hyacinth
4.10.4 Effect of substrate concentration
The effect of substrate concentration on alcohol
fermentation was studied and the results are given in
Figures 4.13a, 4.13b, 4.13~ and 4.13d.
As the concentration of the substrate was increased
upto 108, the yield of alcohol from the cellulosic wastes
also increased. The highest percentage of conversion was
given by a 10% solution of glucose. The same trend of
increase in alcohol yield was observed from tapioca stem,
tapioca petiole, tapioca leaf and water hyacinth. But the
alcohol yield per gram of the cellulosic waste was in the
order, tapioca stem > tapioca petiole > water hyacinth >
tapioca leaf.
4.10.5 Alcohol fennentati.on of the fermentable sugars
Alcohol fermentation of the fermentable sugars
obtained by acid hydrolysis was studied and the results
are given in Table 4.25.
SUBSTRATE CONCENTRATION (glucose percent)
Figure 4.13a. Effect of substrate concentration on alcohol fermentation Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca stem
SUBSTRATE CONCENTRATION (glucose percent)
Figure 4.13b. Effect of substrate concentraticn on alcohol fermentation. Alcohol produced from sugar obtained by the enzymatic saccharification of tapioca petiole
SUBSTRATE CONCENTRA-TION (glucose percent)
Figure 4 . 1 3 ~ . Effect of substrate concentration on alcohol Alcohol produced from sugar obtained by the enzymatic saccarification of tapioca leaf
SUBSTRATE CONCENTRATION (glucose percent)
F~gure 4 13d Effect of substrate concentrallon on alcohol fermentat~on Alcohol produced from sugar obtalned by the enzyrnatlc saccharlflcatlon of water hyac~nth
I 7J 1 .rl I 0 1 m I a:
I n L I . 0 ! r .
As the concentration of the acid was increased, the
amount of reducing sugars formed also increased, and hence
the yield of alcohol. So 'he highest amount of alcohol
was obtained from tapioca stem by the hydrolysis using 10%
acid solution. Tapioca stem gave maximum yield of
reducing sugars and alcohol. This was followed by tapioca
petiole, water hyacinth and tapioca leaf. It was also
found that when the cellulose source was varied, the
amount of reducing sugars and hence the alcohol produced
were also varied.
4.10.6 Alcohol fermentation of the fermentable sugars
Alcohol fermentation of the reducing sugars, obtained
by the enzymatic saccharification of cellulosic wastes was
studied and the results are given in Table 4.26.
Enzymatic saccharification was much superior to acid
hydrolysis of the cellulosic wastes. The maximum amount
of alcohol was obtained from tapioca stem followed by
tapioca petiole, water hyacinth and tapioca leaf. It was
also found that the enzyme from - M. verrucaria was most
suitable for saccharification of the cellulosic wastes.
By the combined action of the enzyme and yeast, tapioca
stem gave the maximum amount of alcohol. The results also
indicated that alcohol production depended upon the source
of cellulose and the source of enzyme.
A significant amount of ethanol was produced
by S, cerevisiae from the sugar obtained by the
saccharification of various substrates. The yield of
alcohol from tapioca stem was found to be maximum by yeast
at pH 4.0 and at a temperature between 28-30°c, when
substrate concentration was maintained at 10%. The
duration of the incubation was found to be 3 days.
The pH is an environmental factor affecting growth
and metabolic production. Yeast grow well in the pH range
of 3.5-5.0. Many reports are available indicating
different pH for maximum alcohol production. The optimum
229 In the pH in most cases was between 4.0 and 4.5 . present study the pH optima was found to be 4.0.
0 Normally, the fermentation was carried out at 29 C, while
the growth rate continues to increase upto a temperature
as high as 35O~. Maximum alcohol production was found to
be at a temperature of 2g0(:.
Alcohol production from sugars obtained by enzymes of
different organisms was found to be different. This may
be due to the difference in the level of sugars in the
reaction system. The fermentation of sugars to alcohol
started from the first day itself and reached at its peak
on the fourth day.
Substrates at the optimum level of 10% of glucose
(obtained by saccharification using different enzymes from
various sources) was found to favour the maximum
production of alcohol.
In both the cases, where saccharification was carried
out by chemical methods or by the action of enzyme,
glucose was the end produc:t. However, sugars obtained by
enzymatic saccharification enhanced the alcohol
production. This may be due to the absence of by-products
in enzymatic saccharification. During chemical hydrolysis
of cellulose waste, a lot of inhibitory compounds may be
formed which may inhibit fermentation. In addition to
this, the presence of excess chemicals may inhibit
fermentation.
PART VI FARMING OF OYSTER MUSHROOM
4.11 Farming of oyster mushroom
The cultivation of mushroom on tapioca stem and water
hyacinth was studied and the results are given in
Tables 4.27 and 4.28 and in plates (Plates la, lb, 2a
and 2b).
Mushroom production was more in tapioca stem than in
water hyacinth. The bioefficiency and spawn running time
in mushroom production in tapioca stem and water hyacinth
showed that among the three different methods of spawning,
thorough spawning gave the maximum bioefficiency followed
by triple layering and surface layering. The yield of
mushroom was considerably different in surface layering
and in thorough spawning methods. However there was not
much difference in mushroom yield between thorough
spawning and triple layering methods.
The morphological characters of the mushroom
developed on cassava waste showed that the mushroom had
umbrella shape with 7 cm in size and a weight of 16 g.
However, those developed from sides were oyster shaped.
The size of the mushroom was comparatively small in water
hyacinth. Minimum days were sufficient for the
development of mushroom by thorough spawning method. The
yields were found to be 400 g and 325 g per 500 g of
tapioca waste and water hyacinth respectively.
Plate la - P. florida mycelial growth on tapioca waste.
' Plate lb Oyster mushroom (L florida) on tapioca waste.
Table 4.27 Mushroom farming
Tapioca stem Water h y a c i n t h ..........................
Spawning Spawn Yie ld B i o e f f i - Y i e l d B i o e f f i - method runn ing iy/ky c i e n c y I g/kg c i e n c y
t ime o f t h e (?A) of t h e ($6) ( d a y s ) s u b s t r a t e s u b s t r a t e
S u r f a c e l a y e r i n g 10 100 1 0 7 5 7 . 5
Thorough spawning b 800 80 750 75.0
T r i p l e l a y e r i n g Y 751 7 5 700 70 .0
Table 4.28 Morphological characters of mushroam
Subs t r a t e s Shape
Tapioca stel11
Water hyacinth
Oysteriurnbrella
Oysteriurnbrella
P. florida was found to grow better in both tapioca - waste and water hyacinth. The cellulose and lignin
content of these substrates must be in the optimum level
for promoting the gr0wt.h of white rot fungi like
P. florida. Tapioca waste and water hyacinth were also - found to contain these pol.ymers in the proper ratio and
promoted the growth of mushroom fungi.
The growth and production of mushroom on tapioca
waste and water hyacinth were due to the capacity of
P. florida to grow on lignocellulosic wastes. In the - present study it was observed that this fungi produced
cellulase which led to the degradation of lignocellulosic
wastes and to mushroom production. The bioefficiency of
P. florida is 80% in tapioca waste and 75% in water - hyacinth.
Mushrooms contain not only high content of proteins
but also high amount of folic acid. Reports available
indicate that mushrooms are h y p ~ l i p i d e m i c ~ ~ ~ ' 231. Report
is also available on the anticancerous effects of
mushrooms232. Drugs isolated from mushrooms are found to
233 be good for the treatment of AIDS .
The work described in this thesis has the following
advantageous.
1. The work has local relevance: Tapioca waste and
water hyacinth are the major wastes causing pollution
problems, difficulties in agriculture and water
transportation in Kerala.
2. Nutritious products and useful chemicals could be
formed from very cheap raw materials (wastes).
3. Pharmacologically useful products for the treatments
of hyperlipidemia, cancer and AIDS could be produced
from the cheap raw materials--the wastes.
