Degradation of Lignocellulosic Substrates By Bacteria Consortium of Bali Cattle Colon and Organic Waste.

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International Journal of Contemporary Applied Sciences Vol. 2, No. 11, November 2015

(ISSN: 2308-1365) www.ijcas.net

84

DEGRADATION OF LIGNOCELLULOSIC SUBSTRATES BY

BACTERIA CONSORTIUM OF BALI CATTLE COLON

AND ORGANIC WASTE

Mudita, I M1., I W. Wirawan2, A. A. P. P. Wibawa3, I G. L. O. Cakra1,I. B. G. Partama2 1

Laboratory of Feed and Animal Nutrition, 2Laboratory of Plant Tropical,

3

Laboratory of Biophysical Chemistry

Faculty of Animal Husbandry, Udayana University, Denpasar

Email:muditafapet_unud@yahoo.com

Abstract

A research has been carried out to evaluating potency of bacteria consortium inoculant

formulated by elected superior bacteria isolated of bali cattle colon and organic waste as

lignocellulosic substrates degrader. Evaluated based on degradation of lignocellulose

substrates measurement using diameter of clear/diffusion zone and lignocellulase enzyme

activity such as endoglucanase, exoglucanase. xylanase and lignase. This study showed that

inoculant of bacteria consortium produce clear zone diameter 0.30 – 0.72 cm, 0.27 – 0.62 cm,

0.43 – 0.78 cm, 0.21 – 0.48, 0.29 – 0.71 and 0.41 – 0.80 cmrespectively for carboxy methyle

cellulose/CMC, avicel micro crystalin, xylan, tannic acid, rice straw, and rice brand

substrates, while inoculant BW12C12 produced highest clear zone diameter on all

lignocellulose substrates evaluated. Meanwhile for enzym activity, bacteria consortium

inocullant coded BW12C12 has highest endoglucanase, exoglucanase, xylanase, and lignase

enzyme activity respectively for carboxy methyle cellulose/CMC, avicel micro crystalin,

xylan and tannic acid substrates. It was concluded that bacteria consortium inoculant coded

BW12C12 was highest potential as lignocellulosic substrates degrader.

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85

1. INTRODUCTION

The development of feeding system based on the local resources is the pillars supporting the development of sustainable animal production in Indonesian (Ginting, 2004). Utilization of agricultural by-product/waste as feed will reduce cost animal production.

However, research results Mudita et al. (2009 and 2012) and Putri et al. (2009) revealed the

use of local resources waste as fed without the application of processing technology will decrease the productivity of livestock and increase pollutant emissions due to high crude fiber content in particular lignocellulose compounds and very difficult to be utilized by animals.

Degradation of lignocellulosic substrates requires the cooperative action of family of lignocellulolytic enzyme that have been classified into three complex enzyme groups: i.e

lignolytic enzyme (lignase), cellulolytic enzim (cellulase) and hemicellulolytic enzyme

(hemicellulose). Lignocellulase complex enzyme can produce by certain microbe or corporative of lignocellulolytic bacteria consortium that work synergistically. Formulated the probiotic lignocelulolytic bacteria consortium used by selected bacteria isolate from the Bali

cattle colon and organic waste landfills based research result Mudita et al (2014) is a strategic

thing to be implemented. Besides that, the utilization of bacterial consortium will increase the overall digestibility, good digestibility in the rumen and post-rumen, improving health throughout the gastrointestinal tract as well as the balance of micro flora in the rumen and post-rumen.

The research results Mudita et al (2014) showed that from the bali cattle colon and

organic waste respectively selected two (2) superior probiotic lignocellulolytic bacteria

isolates with coded BCC4LC and BCC12.1LC (Bali Cattle Colon) and BW1LC and BW4LC (organic

waste of landfill) which has higher the enzyme activity and the ability of lignocellulosic substrates degradation. Formulation of selected bacteria isolates of bali cattle colon and organic waste of landfill as bacteria consortium inoculant is expected to produce a synergistic activity so that the process of degradation of lignocellulosic compounds will be more easily implemented.

The combination of two/any isolates from different sources is likely to produce bacteria consortium inoculant that are potentially capable to improving the productivity of ruminants

including Bali cattle fed agricultural waste.

2. MATERIALS AND METHODS

Isolates Bacteria

Bacterial isolates are utilized in the production of probiotic lignoselulolitik bacteria

consortium inoculant is elected superior isolates of the First Year of the research Mudita et al

(2014) coded BCC4LC and BCC12.1LC isolated from the bali cattle colon and code BW1LC

and BW4LC isolated from organic waste of landfill.

Production of bacterial cultures done by inoculating bacteria isolates of the pure stocks into fluid thioglicollate medium with 1 % lignocellulose substrates (combination of 50%

CMC, 30% Xylan and 20% tannic acid) at 0,1 600 nm. Going further cultures incubated

under anaerobic conditions for 5 days at 39oC. Culture that has grown further exploited in the

production inoculant.

Medium Inoculant

Medium inoculant is made from a combination of natural ingredients and chemical

materials such as Table 1. Mixing medium using vortex for 30 minutes at 80-100oC. Medium

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86

minutes T 121oC. After the medium inoculant begin cooling (T ± 40°C), medium ready to be

used for the production inoculant.

Tabel 1. Composition of Inoculant Medium (on 1 liter)

No Material Composition

1 Thioglicollate Medium (g) 0.1

2 Sugarcane (ml) 50

3 Urea (g) 1

4 Tannic Acid (g) 0,025

5 CMC (g) 0,025

6 Xylan (g) 0,025

7 Rice Straw (g) 0.25

8 Rice Brand (g) 0.25

9 Cassava 0.25

10 Multy vitamin-mineral!“Pignox”!(g) 0.15

11 Water until volume 1 liter

Bacteria Consortium Inoculant

Bacteria consortium inoculants were produced by inoculating 10% a combination of bacterial culture (according to treatment) on the inoculant medium aseptically under anaerobic conditions. The formulated inoculant are presented in Table 2.

Table 2. Formulated of bacteria consortium inoculant (on 1 liter)

No

Bacteria Consortium

Inoculant

Bacteria Isolate Culture from Landfill Waste (ml)

Bacteria Isolate Culture

fromBali Cattle Colon (ml) Medium Inoculant

(ml) BW 1 LC

(1)

BW4LC

(2)

BCC 12.1 LC

(1)

BCC4 LC

(2)

1 IW12 5 5 - - 990

2 IC12 - - 5 5 990

3 IW 1C1 5 - 5 - 990

4 IW 1C2 5 - - 5 990

5 IW 2C1 - 5 5 - 990

6 IW 2C2 - 5 - 5 990

7 IW 12C1 2.5 2.5 5 - 990

8 IW 12C2 2.5 2.5 - 5 990

9 IW 1C12 5 - 2.5 2.5 990

10 IW 2C12 - 5 2.5 2.5 990

11 IW 12C12 2.5 2.5 2.5 2.5 990

Evaluation of Lignocellulosic Substrates Degradation Ability

The ability of lignocellulosic substrates degradation is determined from clear or diffusion zone formed by bacteria isolates tested (Ogimoto and Imai, 1981). The substrate used: CMC for cellulose, xylan for hemicellulose, tannic acid for lignin and combination of 50% CMC, 30% Xylan and 20% tannic acid for lignocellulose substrate. Each pure bacteria

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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 4oC. Extracts enzyme were tested in three kinds of substrates that contained 1%

CMC powder/xylan/Tannic 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 cellulase/xylanase 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. Lignase/cellulase/xylanase enzyme activities was estimated by using vanillin/glucose/xylose calibration curve (Adney

and Baker, 2008; Ghose, 1987). One unit (U) of enzyme activity was! de#ned! as! 1! "mol!of

vanillin/glucose/xylose 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 (IW0C0) 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 IW0C0 0.30a1 0.27a 0.43a 0.21a 0.29a 0.41a

2 IW12 0.55b 0.43b 0.67bc 0.33ab 0.47b 0.70b

3 IC12 0.64b 0.55bc 0.69bcd 0.31ab 0.47b 0.72b

4 IW1C1 0.70b 0.62c 0.76de 0.44b 0.68d 0.79b

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6 IW2C1 0.69b 0.59c 0.76e 0.42b 0.67cd 0.77b

7 IW2C2 0.66b 0.55bc 0.70bcde 0.39ab 0.49b 0.72b

8 IW12C1 0.70b 0.62c 0.78e 0.44b 0.70d 0.78b

9 IW12C2 0.55b 0.53bc 0.65b 0.38ab 0.62bcd 0.71b

10 IW1C12 0.69b 0.61c 0.77de 0.43b 0.67cd 0.77b

11 IW2C12 0.68b 0.59c 0.75cde 0.42b 0.67cd 0.77b

12 IW12C12 0.72b 0.62c 0.78e 0.48b 0.71d 0.80b

SEM2 0,04 0,03 0,02 0,04 0,03 0,02

Notes: 1)Means in the same column with different letter differ significantly (P<0,05), 2)SEM = Standard error of the treatmens and means

In Table 3 it appears that inoculant coded IW12C12 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 IW12C12 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 (IW0C0) 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 and/or vanillin.

Table 4a Endo-Glukanase Enzyme Activity from Bacteria Consortium inoculant

No

Inoculant

Endo-Glukanase Enzyme Activity on Time Period (U)

10 minutes 20 minutes 30 minutes 60 minutes

1 IW0C0 0.0357a1 0.0574a 0.0546a 0.0337a

2 IW12 0.1025b 0.1133b 0.1053b 0.0598b

3 IC12 0.1046bc 0.1140b 0.1058b 0.0603b

4 IW1C1 0.1321def 0.1350def 0.1198b 0.0677c

5 IW1C2 0.1244de 0.1294cde 0.1131b 0.0644bc

6 IW2C1 0.1295def 0.1289cd 0.1206b 0.0682c

7 IW2C2 0.1182cd 0.1215bc 0.1108b 0.0628bc

8 IW12C1 0.1373ef 0.1415ef 0.1202b 0.0685c

9 IW12C2 0.1197d 0.1295cde 0.1161b 0.0660bc

10 IW1C12 0.1396f 0.1434f 0.1205b 0.0687c

11 IW2C12 0.1310def 0.1349def 0.1167b 0.0658bc

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SEM8 0,0028 0,0025 0,003 0,0015

Table 4b Exo-Glukanase Enzyme Activity from Bacteria Consortium Inoculant

No

Inokulan

Exo-Glukanase Enzyme Activity on Time Period (U)

10 minutes 10 minutes 10 minutes 10 minutes

1 IW0C0 0.0164a1 0.0304a 0.0353a 0.0267a

2 IW12 0.0847b 0.0902b 0.0907b 0.0527b

3 IC12 0.0857b 0.0922b 0.0925b 0.0537bc

4 IW1C1 0.1116cde 0.1136cd 0.1065cde 0.0642de

5 IW1C2 0.1066cd 0.1124c 0.1049cd 0.0605de

6 IW2C1 0.1030c 0.1121c 0.1021c 0.0584bcd

7 IW2C2 0.1003c 0.1124c 0.1024c 0.0593cde

8 IW12C1 0.1194de 0.1209cde 0.1123de 0.0646e

9 IW12C2 0.1142cde 0.1156cd 0.1102cde 0.0623de

10 IW1C12 0.1218e 0.1230de 0.1118de 0.0633de

11 IW2C12 0.1137cde 0.1156cd 0.1068cde 0.0621de

12 IW12C12 0.1236e 0.1252e 0.1152e 0.0673f

SEM8 0,0028 0,0019 0,0018 0,0012

Table 4c Xylanase Enzyme Activity from Bacteria Consortium Inoculant

No

Inokulan

Xylanase Enzyme Activity on Time Period (U)

1 IW0C0 4.1501a

1

9.2605a 12.9360a 9.7664a

2 IW12 7.7263ab 11.5342ab 19.3525b 15.1582bc

3 IC12 7.6159ab 11.9205b 19.2053b 14.9742b

4 IW1C1 12.3620cde 14.7903de 20.7138bcd 15.7837cde

5 IW1C2 9.9338bc 13.3554bcd 20.0515bc 15.4893bcd

6 IW2C1 12.1413cd 14.6799cde 20.5666bcd 15.7653cde

7 IW2C2 7.9470ab 12.3068bc 19.4628b 15.0846bc

8 IW12C1 15.4525de 16.5673e 21.6336cd 16.3907ef

9 IW12C2 12.5828cde 15.1876de 20.7873bcd 15.7653cde

10 IW1C12 14.6799de 16.1810e 21.4496cd 16.2804ef

11 IW2C12 13.5762cde 15.6843de 21.1185cd 16.1884def

12 IW12C12 16.1148e 16.8648e 22.1486d 16.7403f

SEM2 0,7457 0,4570 0,3110 0,1423

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Tabel 4d Lignase Enzyme Activity from Bacteria Consortium Inoculant

No

Bacteria Consortium

Inokulan

Lignase Enzyme Activity on Time Period (U)

1 IW0C0 0.0214a 0.0358a 0.0423a 0.0283a

2 IW12 0.1021b 0.1366b 0.0975b 0.0577b

3 IC12 0.1028b 0.1372b 0.0988bc 0.0582b

4 IW1C1 0.1404de 0.1408bc 0.1002bcd 0.0588bcd

5 IW1C2 0.1270cd 0.1375b 0.0987bc 0.0584bc

6 IW2C1 0.1292d 0.1390bc 0.0991bcd 0.0583bc

7 IW2C2 0.1125bc 0.1375b 0.0989bc 0.0578b

8 IW12C1 0.1542ef 0.1456cd 0.1018cd 0.0610d

9 IW12C2 0.1432def 0.1427bcd 0.1005bcd 0.0591bcd

10 IW1C12 0.15108ef 0.1447cd 0.1006bcd 0.0607cd

11 IW2C12 0.1470ef 0.1434bcd 0.1005bcd 0.0600bcd

12 IW12C12 0.1577f 0.1482d 0.1029d 0.0612d

SEM8 0032 0,0014 0,0008 0,0005

On Table 4a,b,c,d so showed that inoculant coded IW12C12 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 IW12C12

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 IW12C12 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

CMCase/endo-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! $-glucosidase/glukohydrolase. Olempska-Beer (2004) revealed that the

enzyme endo-1,4-$-xylanase! duty! hydrolyze! $-1,4 bond in the chain silane (a major

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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

1. Inoculant of bacteria consortium coded IW12C12 has produced highest diameter of clear

zone on lignocellulose substrates

2. Inoculant of bacteria consortium coded IW12C12 has produced highest lignocellulolytic

enzyme activity on all time period

3. The inoculant of bacteria consortium formulated from bali cattle colon and waste landfile

isolates most potential to be used as degrader of feedstuff rich in lignocellulosic substrates such as agriculture waste product.

ACNOWLEDGEMENTS

The authors acknowledge to the Directorate General of Higher Education, National Education Department, Republic of Indonesia and Udayana University through Competitive Research Grant 2015 for fund support in this experimentation. Thanks are also due to the Laboratory of feed and nutrition animal, Faculty of Animal Husbandry Udayana University and Analytic Laboratorium Udayana University for assistance in laboratory analysis.

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Degradation of Lignocellulosic Substrates By Bacteria Consortium of Bali Cattle Colon and Organic Waste.

International Journal of Contemporary Applied Sciences

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DEGRADATION OF LIGNOCELLULOSIC SUBSTRATES BY

Abstract

1. INTRODUCTION

2. MATERIALS AND METHODS

Isolates Bacteria

Medium Inoculant

Tabel 1. Composition of Inoculant Medium (on 1 liter)

Table 2. Formulated of bacteria consortium inoculant (on 1 liter)

Evaluation of Lignocellulolytic enzyme activity

3. RESULTS AND DISCUSSION

Ability of lignocellulosic compounds degradation

Table 3 Clear Zone Diameters produced by Bacteria Consortium Inoculant on Lignocellulose Substrates

Lignocellulase Enzyme Activity

Table 4a Endo-Glukanase Enzyme Activity from Bacteria Consortium inoculant

Table 4b Exo-Glukanase Enzyme Activity from Bacteria Consortium Inoculant

Table 4c Xylanase Enzyme Activity from Bacteria Consortium Inoculant

Bacteria Consortium

5. CONCLUSION

ACNOWLEDGEMENTS

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