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87 the selective medium solid growth medium containing 1 substrate test for experimentation
Subbarao, 1993. Diffusion and clear zone wide were measured after 24 hours of anaerobic incubation.
Evaluation of Lignocellulolytic enzyme activity
Enzyme extract was collected from centrifuged liquid media culture in 12.000 rpm for 15 minutes at 4
o
C. Extracts enzyme were tested in three kinds of substrates that contained 1 CMC powderxylanTannic Acid as source of lignin in 50 mM acetate buffer pH 5.5. Each
substrate liquid in buffer was taken 8 ml, added 1 ml enzymes source, and 1ml aquadest. The mixture then were shaken by shaking bath, enzyme activity was measured in 30, 60, 180
and 360 minutes durations. Reduction sugar glucose from CMC and xylose from xylan, or vanillin from tannic acid lignin produced from the reaction were the cellulasexylanase or
lignase enzyme activities Efiok, 1996. For sugar reduction:1 ml of sample was added to 3 ml DNS reagent and 1 ml aquadest Miller, 1959, for vanillin: 1ml of sample was added to
4 ml methanol, then measured the absorbent by spectrophotometer in wavelength 508,5 nm for glucose, 509 nm for xilosa and 279 nm for vanillin. Lignasecellulasexylanase
enzyme activities was estimated by using vanillinglucosexylose calibration curve Adney and Baker, 2008; Ghose, 1987. One unit U of enzyme activity was dened as 1 mol of
vanillinglucosexylose equivalent released per minute under standard assay condition Irfan et al., 2012; Lo et al., 2009.
3. RESULTS AND DISCUSSION Ability of lignocellulosic compounds degradation
The results showed that the probiotic lignocellulolytic bacteria consortium inoculant were formulated from combination the 1st and 2nd superior isolates from bali cattle colon and
landfill waste has the ability to return high enough to degrade lignocellulosic compounds shown with resultant diameter clear zone higher than inoculant without consortium bacteria
isolates IW
C that 0.055 – 0.72 cm
Vs 0.30 cm, 0.43 - 0.62 cm Vs 0.27 cm, 0.67 – 0.78 cm 0.43 cm, 0.31 – 0.48 cm Vs 0.21 cm, 0.47 – 0.71 cm Vs 0.29 cm, and 0.70 – 0.80 cm
Vs 0.41 cm respectively for on CMC, Avicel, Xylan, Tannic Acid, Rice Straw, and Rice Brand substrates Table 3. These showed its probiotic lignocellulolytic bacteria consortium
inoculants as true lignocellulolytic bacteria consortium which work sinergitically. Every strain of bacteria isolates needed a spesific substrate as an energy source for growing and can
used output product produce by the others bacteria isolates Howard et al., 2003; Perez et al., 2002.
Table 3 Clear Zone Diameters produced by Bacteria Consortium Inoculant on Lignocellulose Substrates
No Bacteria
Consortium Inoculant
Clear Zone Diameter on Lignocellulose Substrates
CMC Avicel
Xylan Tannic Acid
Rice Straw Rice Brand
cm cm
cm cm
cm cm
1 IW
C 0.30a
1
0.27a 0.43a
0.21a 0.29a
0.41a 2
IW
12
0.55b 0.43b 0.67bc
0.33ab 0.47b
0.70b 3
IC
12
0.64b 0.55bc 0.69bcd 0.31ab
0.47b 0.72b
4 IW
1
C
1
0.70b 0.62c 0.76de
0.44b 0.68d
0.79b 5
IW
1
C
2
0.69b 0.58c 0.70bcde
0.39ab 0.52bc
0.72b
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88 6
IW
2
C
1
0.69b 0.59c 0.76e
0.42b 0.67cd
0.77b 7
IW
2
C
2
0.66b 0.55bc 0.70bcde 0.39ab
0.49b 0.72b
8 IW
12
C
1
0.70b 0.62c 0.78e
0.44b 0.70d
0.78b 9
IW
12
C
2
0.55b 0.53bc 0.65b 0.38ab
0.62bcd 0.71b
10 IW
1
C
12
0.69b 0.61c 0.77de
0.43b 0.67cd
0.77b 11
IW
2
C
12
0.68b 0.59c 0.75cde
0.42b 0.67cd
0.77b 12
IW
12
C
12
0.72b 0.62c 0.78e
0.48b 0.71d
0.80b SEM
2
0,04 0,03
0,02 0,04
0,03 0,02
Notes:
1
Means in the same column with different letter differ significantly P0,05,
2
SEM = Standard error of the treatmens and means
In Table 3 it appears that inoculant coded IW
12
C
1
2 have highest substrates degradation ability with a clear zone diameter of 0.72 cm; 0.62 cm; 0.78 cm; 0.48 cm, 0.71 cm, and 0.80
cm respectively on the substrate CMC, Avicel, Xylan, Tannic Acid, rice straw and rice bran. Based on these data can be predicted bioinokulan will be able to be a good starter in the
fermentation process to degraded crude fiber-rich feed ingredients such as agricultural waste. Moreover, based on Table 4 also shows inoculant coded IW
12
C
1
2 has produce highest lignocellulolytic enzyme activity, thus the potential for inoculant superior high quality.
Lignocellulase Enzyme Activity
The results of study showed probiotic lignocellulolytic bacteria consortium inoculant formulated by bacteria isolated from bali cattle colon waste and waste landfill has higher
lignocellulolytic enzyme activities i.e endo glucanase, exo-glucanase, xylanase and lignase enzyme activities than inoculant without bacteria isolate IW
C on all times period Table
4a,b,c,d. These data proved that formulated bacteria consortium inoculant by bacteria isolates from bali cattle colon and waste landfill can produce good quality inoculant with
synergistically work which has high ability of lignocelluloses degradation to simple component such as glucose, xylose andor vanillin.
Table 4a Endo-Glukanase Enzyme Activity from Bacteria Consortium inoculant No
Bacteria Consortium
Inoculant Endo-Glukanase Enzyme Activity on Time Period U
10 minutes 20 minutes 30 minutes 60 minutes
1 IW
C 0.0357a
1
0.0574a 0.0546a
0.0337a 2
IW
12
0.1025b 0.1133b
0.1053b 0.0598b
3 IC
12
0.1046bc 0.1140b
0.1058b 0.0603b
4 IW
1
C
1
0.1321def 0.1350def
0.1198b 0.0677c
5 IW
1
C
2
0.1244de 0.1294cde
0.1131b 0.0644bc
6 IW
2
C
1
0.1295def 0.1289cd
0.1206b 0.0682c
7 IW
2
C
2
0.1182cd 0.1215bc
0.1108b 0.0628bc
8 IW
12
C
1
0.1373ef 0.1415ef
0.1202b 0.0685c
9 IW
12
C
2
0.1197d 0.1295cde
0.1161b 0.0660bc
10 IW
1
C
12
0.1396f 0.1434f
0.1205b 0.0687c
11 IW
2
C
12
0.1310def 0.1349def
0.1167b 0.0658bc
12 IW
12
C
12
0.1414f 0.1442f
0.1209b 0.0699c
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89 SEM
8
0,0028 0,0025
0,003 0,0015
Notes:
1
Means in the same column with different letter differ significantly P0,05,
2
SEM = Standard error of the treatmens and means
Table 4b Exo-Glukanase Enzyme Activity from Bacteria Consortium Inoculant No
Bacteria Consortium
Inokulan Exo-Glukanase Enzyme Activity on Time Period U
10 minutes 10 minutes
10 minutes 10 minutes
1 IW
C 0.0164a
1
0.0304a 0.0353a
0.0267a 2
IW
12
0.0847b 0.0902b
0.0907b 0.0527b
3 IC
12
0.0857b 0.0922b
0.0925b 0.0537bc
4 IW
1
C
1
0.1116cde 0.1136cd
0.1065cde 0.0642de
5 IW
1
C
2
0.1066cd 0.1124c
0.1049cd 0.0605de
6 IW
2
C
1
0.1030c 0.1121c
0.1021c 0.0584bcd
7 IW
2
C
2
0.1003c 0.1124c
0.1024c 0.0593cde
8 IW
12
C
1
0.1194de 0.1209cde
0.1123de 0.0646e
9 IW
12
C
2
0.1142cde 0.1156cd
0.1102cde 0.0623de
10 IW
1
C
12
0.1218e 0.1230de
0.1118de 0.0633de
11 IW
2
C
12
0.1137cde 0.1156cd
0.1068cde 0.0621de
12 IW
12
C
12
0.1236e 0.1252e
0.1152e 0.0673f
SEM
8
0,0028 0,0019
0,0018 0,0012
Notes:
1
Means in the same column with different letter differ significantly P0,05,
2
SEM = Standard error of the treatmens and means
Table 4c Xylanase Enzyme Activity from Bacteria Consortium Inoculant No
Bacteria Consortium
Inokulan Xylanase Enzyme Activity on Time Period U
10 minutes 10 minutes
10 minutes 10 minutes
1 IW
C 4.1501a
1
9.2605a 12.9360a
9.7664a 2
IW
12
7.7263ab 11.5342ab
19.3525b 15.1582bc
3 IC
12
7.6159ab 11.9205b
19.2053b 14.9742b
4 IW
1
C
1
12.3620cde 14.7903de
20.7138bcd 15.7837cde
5 IW
1
C
2
9.9338bc 13.3554bcd
20.0515bc 15.4893bcd
6 IW
2
C
1
12.1413cd 14.6799cde
20.5666bcd 15.7653cde
7 IW
2
C
2
7.9470ab 12.3068bc
19.4628b 15.0846bc
8 IW
12
C
1
15.4525de 16.5673e
21.6336cd 16.3907ef
9 IW
12
C
2
12.5828cde 15.1876de
20.7873bcd 15.7653cde
10 IW
1
C
12
14.6799de 16.1810e
21.4496cd 16.2804ef
11 IW
2
C
12
13.5762cde 15.6843de
21.1185cd 16.1884def
12 IW
12
C
12
16.1148e 16.8648e
22.1486d 16.7403f
SEM
2
0,7457 0,4570
0,3110 0,1423
Notes:
1
Means in the same column with different letter differ significantly P0,05,
2
SEM = Standard error of the treatmens and means
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90
Tabel 4d Lignase Enzyme Activity from Bacteria Consortium Inoculant No
Bacteria Consortium
Inokulan Lignase Enzyme Activity on Time Period U
10 minutes 10 minutes
10 minutes 10 minutes
1 IW
C 0.0214a
0.0358a 0.0423a
0.0283a 2
IW
12
0.1021b 0.1366b
0.0975b 0.0577b
3 IC
12
0.1028b 0.1372b
0.0988bc 0.0582b
4 IW
1
C
1
0.1404de 0.1408bc
0.1002bcd 0.0588bcd
5 IW
1
C
2
0.1270cd 0.1375b
0.0987bc 0.0584bc
6 IW
2
C
1
0.1292d 0.1390bc
0.0991bcd 0.0583bc
7 IW
2
C
2
0.1125bc 0.1375b
0.0989bc 0.0578b
8 IW
12
C
1
0.1542ef 0.1456cd
0.1018cd 0.0610d
9 IW
12
C
2
0.1432def 0.1427bcd
0.1005bcd 0.0591bcd
10 IW
1
C
12
0.15108ef 0.1447cd
0.1006bcd 0.0607cd
11 IW
2
C
12
0.1470ef 0.1434bcd
0.1005bcd 0.0600bcd
12 IW
12
C
12
0.1577f 0.1482d
0.1029d 0.0612d
SEM
8
0032 0,0014
0,0008 0,0005
Notes:
1
Means in the same column with different letter differ significantly P0,05,
2
SEM = Standard error of the treatmens and means
On Table 4a,b,c,d so showed that inoculant coded IW
12
C
12
has highest endo- glucanase enzyme activity 0.141 U, 0.144 U, 0.121 U and 0.070 U respectively after 10
minutes, 20 minutes, 30 minutes and 60 minutes time period of contact with the substrates CMC, exo glucanase produces the highest enzyme activity that is equal to U 0124, U 0125, U
0115 and U 0067 in the period of time of 10 minutes, 20 minutes, 30 minutes and 60 minutes after contact with the substrate avicel micro-crystalline cellulose, Inoculant coded IW
12
C
12
also able to produce xylanase enzyme activity of 16 115 U, U 16 865, 22 149 and 16 740 U U in the period of 10 minutes, 20 minutes, 30 minutes and 60 minutes after contact with the
substrate xylanosa and produce lignase enzyme activity of 0158 U, 0148 U , U 0103 and U 0061 in the period of time of 10 minutes, 20 minutes, 30 minutes and 60 minutes after contact
with the substrate tannic acid.
The high enzyme activity was particularly on Inoculant coded IW
12
C
12
will as that inoculant a good starterfor fermentation process, including for coarse fiber-rich feed materials
such as agricultural waste. Lignocelluloses enzyme activity CMC ase, exo glucanase, xylanase or lignase will be able to degrade lignocellulose compounds into simpler organic
compounds and can be utilized by livestock as a source of energy Perez et al., 2002. Beauchemin et al. 2003 and Lynd et al 2002 states that the enzyme CMCaseendo-
glucanase is the first enzyme that will degrade cellulose polymers randomly by breaking the hydrogen bonds in the cellulose crystalline structure internal bond -1,4-glucoside thus
forming chains of individual cellulose oligodekstrin. Furthermore exo-glucanase enzyme will outline the cellulose from the reducing and non-reducing end through cutting the ends of
the individual chains of cellulose the reducing end and a non-reducing resulting disaccharide eg, cellobiose. The third stage final is the stage of decomposition of cellobiose to glucose
by the enzyme -glucosidaseglukohydrolase. Olempska-Beer 2004 revealed that the enzyme endo-1,4--xylanase duty hydrolyze -1,4 bond in the chain silane a major
component of hemicellulose produce short xylooligomer which would then be hydrolyzed
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91 into xylose units single by -xylosidase. While enzymes lignase will describe bonding
lignocellulose compounds in the lignin into simple components. Lignocelluloses enzyme activity at all bacteria consortium inoculant produced will
make all inoculant produced has a high quality especially in degrading crude fiber-rich feedstuffs such as agricultural waste. The high population of bacteria and nutrient content of
inoculant will support growth and microbial activity inoculant degrade lignocellulosic substrates.
5. CONCLUSION
