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Editorial Board of IJAER

Editor in Chief Dr. Gautam Singh Rathore

MIT, India.

Board Members Prof. Muhamad Mat Noor, Mechanical Engineering, Malaysia

Prof. Dong-Chul Park, Myong Ji University, Korea

Prof. Anjaiah Devineni, Manipal University, India

Prof. Mohamed Ben Haj Frej, POST University, USA

Prof. Figueira, F. M. Monteiro, ULHT - Universidade Lusofona, Portugal

Mr.ROSHAN BABU OJHA, Soil Scientist,Nepal Agricultural Research Council.

Asso.Prof.L.K. Bhatiya,BHK collage of Engineering, India

Mr.BINOD GHIMIRE,Agriculture Extension Officer Ministry of Agricultural

Development,Government of Nepal, Nepal

Ass.Prof.H.A.Sayeswara,Department of Zoology, Sahyadri Science

College(Autonomous), Shivamogga-577203, Karnataka state,India

Dr.Mahadeva Swamy,Senior Research Fellow (SRF),Biopesticide

Laboratory(BPL),Division of Biotechnology,(IIHR), Hessarghatta lake post,

Bangalore, India

Ass.Prof. ARVIND SINGH TOMAR,Dept. of Irrigation & Drainage

Engineering,College of Technology,G.B. Pant Univ. of Agric. &

Technology,Pantnagar(Udham Singh Nagar)263145 Uttarakhand India

To Join our Editorial Board Email Your Resume to [email protected]

International journal of Agriculture and Environmental Research Promote your research with IJAER| www.ijaer.in

Volume 1 Issue 1 November 2015

#Article Title & Author name Page download

1

ANTIMICROBIAL AND CYTOTOXICITY OF Ag AND ZnO

NANOPARTICLES

Nabeel K.AL-Ani, Ayad,M.Ali,Sarah Ibrahim,Farooq

I.Mohammad,Salah M.Al-chalabi

01-11

2

POTENCY OF BACTERIA ISOLATED FROM BALI COLON

WASTE AS LIGNOCELLULOSE SUBSTRATES DEGRADER

Mudita, I M., I G. Mahardika, I N. Sujaya, I. B. G. Partama

12-23

3

NUTRITIVE EVALUATION OF SOME BROWSE TREES

FRUITS (SEED AND PODS) AS DRY SEASON

SUPPLEMENT FOR LIVESTOCK IN ARID AND SEMI-ARID

LANDS OF SUDAN

Izeldin A. Babiker, Mohamed S.A. Abdulla

24-31

4

THE EFFECT OF FEEDING THREE PEARL MILLET

CULTIVARS ON PERFORMANCE OF LAYING HENS AND

THYROID GLAND HISTOLOGY

Amal Eltayeb Mahmoud

32-41

5

GROWTH OF SESAME AS INFLUENCED BY VARIETIES

AND PLANT EXTRACTS ON THE CONTROL OF Cercospora

sp. IN ARDO-KOLA AND GASSOL, TARABA STATE,

NIGERIA

Tunwari, B.A., Nahunnaro, H.

42-64

International Journal of Agriculture and Environmental Research

Volume:01,Issue:01

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POTENCY OF BACTERIA ISOLATED FROM BALI COLON WASTE AS

LIGNOCELLULOSE SUBSTRATES DEGRADER

Mudita, I M., I G. Mahardika, I N. Sujaya and I. B. G. Partama

Faculty of Animal Husbandry, Udayana University, Denpasar

ABSTRACT

A research has been carried out to evaluate the potency of bacteria isolated from bali cattle colon

waste as lignocellulosic substrates degrader. Bacteria were isolated by Hungate selective media

using combination of tannic acid (lignin), xylan, and Carboxy Methyl Cellulose/CMC (cellulose)

as selective substrates. The potency of lignocellulolytic was identified based on the degradation

of lignocellulosic substrates measurement using clear/diffusion zone width and lignocellulolytic

enzyme activity. This study showed that lignocellulolytic bacteria isolated from bali cattle colon

produce clear/diffusion zones in the width of wide 0.078 – 3.440 cm2, 0.007 – 0.072 cm2, 0.172 –

4.497 cm2 and 0,392 – 5,864 cm2 respectively for lignocellulose, tannic acid, cellulose and xylan

substrates, while isolates coded BCC 7 LC, BCC 4 LC and BCC 12.1 LC produced higher

clear/diffusion zones. Meanwhile for lignocellulolytic enzyme activity, bacteria isolates coded

BCC 12.1 LC and BCC 6 LC has higher enzyme activity at tannic acid, CMC, and xylan

substrates. It was concluded that bacteria isolates coded BCC 12.1 LC is a lignocellulosic

substrates degrader.

Keywords: Bali Cattle Colon Waste, Clear Zone Width, Enzyme Activity, Lignocellulosic

Substrates

INTRODUCTION

Utilization of agricultural wastes as feed is one of the national policy in the development

of cattle livestock to achieve beef self-sufficiency. This step is more strategic for the livestock

sector in Bali in support of the Bali Green and Clean Provence by the Provincial Government of

Bali, Indonesian Country. Utilization of waste as feed will reduce the emission of pollutants in

the air (Hegarty, 2001). However, research results of Mudita et al. (2008-2012) and Putri et al.

(2009) revealed the use of waste as feed 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 livestock.


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Lignocellulose is composed of three polymers, including lignin, cellulose and

hemicellulose (Howard et al., 2003; Perez et al., 2002). Degradation of its polymer completely

will supply all potential nutrients contained in the agricultural waste feedstuffs. Lignocellulose

can only be degraded by certain microbes, such as lignocellulolytic bacteria. So the effort to

develop lignocellulolytic bacteria is very important in optimizing utility agricultural waste as

feedstuffs. Bali cattle colon contents waste is one of the source of lignocelluloses degrading

bacteria isolates.

The cattle colon contains many fiber degrading microbes (bacteria and fungi) such as

lignolytic, cellulolytic, hemycellulolytic, amylolytic and proteolytic and various probiotics

microbes (Wahyudi et al., 2010ab). Wahyudi et al. (2010) showed that the lignocellulolytic

bacteria of the colon and caecum have the ability to degrade crude fiber higher than the rumen

bacteria. The bacteria isolates capable of associating with fiber rich feed ingredients that are not

degraded in the rumen. Wahyudi et al. (2010) have isolated three lignolytic bacteria, bacterial

xylanolytic 111, and 262 of cellulolytic bacteria from the colon of buffalo with the highest

enzyme production of 0.1971 molecular weight (mol).Based on the potential of lignocellulosic

substrates degradation, bacteria isolated from bali cattle colon contents waste was made to

explore its potential as a source of inoculum agro-waste degrader on the development of

competitive and sustainable bali cattle livestock.

MATERIALS AND METHODS

Isolation of source and sample preparation

The bali cattle colon contents waste sample taken from cattle slaughter house in Kediri

Tabanan, Bali Provence-Indonesian Country. The colon content sample was obtained from gut

after bali cattle’s death by filtering into a prewarm (39oC) termos flask (Lowe, 1986). Anaerobic

conditions were created by inserting the bali cattle colon contents waste up to full and filling CO2

into the flask, and then covering it with asterile butyl rubber stopper. The sample was brought to

the laboratory for further processing as sources of bacteria isolates. Dilution of sample has been

conducted in series until 10-7foid dilution was obtained with dilution medium formula No 14

Bryant and Burkey (Ogimoto and Imai, 1981).

Solid Media and Isolation

Bacteria from samples were grown in selective solid media by Hungate method

(Ogimoto and Imai, 1981) countaining 0,02g Monopotassium phosphate (KH2PO4); 0,03g

Dipotassium phosphate (K2HPO4); 0,01g Magnesium Sulfate (MgSO4); 0,01g Calcium


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Tetrachloride (CaCl4); 0,10g Sodium Cloride (NaCl); 0,10g Ammonium Sulfate {(NH4)2SO4};

0,10ml Rezasurin 0,1% solution; 0,02g Cystein-HCl.H2O; 0,40g Sodium Carbonate (Na2CO3);

30,00 ml rumen liquid; 1,00g substrate; 70,00ml Aquadest and 1,8% Agar. Selective

substrate used were tannic acid (as lignin source), xylan (as hemicellulose source) and Carboxy

Methyl Cellulose/CMC (as cellulose source). All ingredients were mixed in erlenmeyer, pH was

determined 6.8 and heated until all ingredients dissolved. The flask then transferred aseptically

with oxygen-free CO2 gas displacing all air until red color faded was closed with rubber stopper,

sealed and then sterilized in the autoclave at 121oC for 15 minutes.

The dilution colon fluid in series 10-7in tube was transferred aseptically to a petri disc

with oxygen free CO2 gas displacing all air. Then medium in the enlenmeyer flask wasinoculated

into the petri disc and closed. The culture was incubated at 39oC for 5 – 7 days. The colonies

thus grown on this medium were selected for the isolation

Isolation of Colonies

From these inoculated tube containing the selection medium, the individual colonies of

lignocellulolytic bacteria were pricked. Bacteria isolates were carefully pricked using bent

platinum-irridium needle. The bacteria were then transferred to plate agar medium anaerobically

with gasses oxygen-free CO2. The plate was incubated at 39oC for 5 – 7 days. The bacteria

colonies that produces clear or diffusion zone were chosen for furification by repeated streaking.

Ability of Lignocellulosic Substrates Degradation

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 isolate (15μl) was

inoculated by spot method using paper disc blank (0.6 cm) and placed on 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.

Lignoselulase 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 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


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(lignin) produced from the reaction were the cellulase/xylanase or lignaseenzyme 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 (λ) 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 defined as 1 μmolof vanillin/glucose/xylose equivalent released per

minuteunder standard assay condition (Irfan et al., 2012; Lo et al., 2009).

RESULTS AND DISCUSSION

Ability of lignocellulosic compounds degradation

The results showed that the lignocellulolytic bacteria isolates were isolated from the bali

cattle colon waste has the ability to return high enough to degrade lignocellulosic compounds

shown with resultant wide clear zone large enough that 0.078 - 3.440 cm2, 0.007 -0.072 cm2,

0.172 - 4.497 cm2 and 0.392 - 5.864 cm2respectively for on lignocellulosic, tannic acid, CMC

and xylan substrates (Table 1). These showed its bacteria isolates as true lignocellulolytic

bacteria (lignocellulose degrading bacteria). These isolates were isolated using the combination

of lignin (tannic acid), cellulose (carboxy methyl cellulose/CMC) and xylan as selected

substrates in bacteria isolated media. Every strain of bacteria isolates needed a spesific substrate

as an energy source for growing (Howard et al., 2003; Perez et al., 2002). This suggests that in

the colon of Bali cattle are various types of bacteria that have the ability to degrade

lignocellulose compounds is high enough (Kamra, 2005) and capable of associating with fiber

rich feed ingredients that are not degraded in the rumen (Wahyudi et al., 2010). Kamra (2005)

stated that ruminants in tropic area such as Indonesia has higher fiber degrading bacteria than

other animal farming, especially local ruminants (bali cattle).

Table 1. Width of clear zone (cm2) obtained from 15 μl Bacteria isolateson variety

substrates

No Bacteria Isolates Lignocellulose Lignin CMC Xylan

1 BCC 1 LC 0,725ab1 0,007a 1,185b 2,178c

2 BCC 2 LC 2,803c 0,043abc 4,136cd 5,560ef

3 BCC 3 LC 0,885ab 0,027ab 1,335b 1,625b

4 BCC 4 LC 3,357c 0,045bc 4,206cd 5,864f


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5 BCC 5 LC 0,793ab 0,030ab 1,263b 2,253c

6 BCC 6 LC 0,978b 0,030ab 1,516b 2,339c

7 BCC 7 LC 3,440c 0,072c 4,497d 5,042d

8 BCC 11 LC 0,837ab 0,028ab 1,338b 1,858bc

9 BCC 12.1 LC 3,130c 0,044abc 3,901c 5,759f

10 BCC 13.1 LC 0,078a 0,030ab 0,172a 0,392a

SEM2 0,170 0,010 0,080 0,100 Notes: 1)Means in the same column with different letter differ significantly (P<0,05), 2)SEM = Standard

error of the treatmens and means

Herbivores digestive tract, specifically ruminants in Indonesia such as bali cattle, in

general contain high lignocellulose feed, so that lignocellulolytic bacteria isolates were expected

exist. This study proved that colon waste bali cattle possess lignocellulolytic bacteria. Several

previous study showed that lignocellulolytic bacteria was exist in rumen and goat’s feces has

used as rumen microbes replacement (Utomo et al., 2006). Another study showed that horse’s

cecum and colon, and elephant have microbes composition such as rumen. (Ulrey et al., 1997;

Wahyudi et al., 2010). According to Table 1, it is proved that the lignocellulolytic bacteria

isolates could be found from bali cattle colon waste.

Lignocellulose degrading bacteria (lignocellulolytic bacteria) isolates from bali cattle

colon waste has degrading lignocellulosic substrates shown of producing a wide clear zone of

0.078 to 3.440 cm2on lignocellulose substrates (mixture substrates) and individual substrates of

0.007 - 0.072 cm2, 0.172 – 4.497 cm2, 0.392 -5.864 cm2 respectively for lignin (tannic acid),

CMC (cellulose), and xylan (hemicellulose) substrates. The bacteria isolates coded BCC2LC,

BCC4LC, BCC7LC and BCC12.1LC with the higher clear zone wide and significantly different

(P<0.05) compared to the other isolates. This shows the great potential of these bacteria isolates

to degrade lignocellulosic compounds such as feed based on agricultural wasteand supply all

potential nutrients contained in the substrates/feed.

Bacteria isolates coded BCC 7 LC with the highest wide of clear zone on lignocellulose,

lignin, and cellulose substrates (3.440 cm2, 0.072 cm2 and 4.497 cm2) and significantly different

(P<0.05) compared to the other isolates. While on the xylan substrate, bacteria isolate coded

BCC 4 LC and BCC 12.1 LC with the highest wide of clear zone and significantly different

(P<0.05) compared to the other isolates (5.864 cm2 vs 1.685 – 5.560 cm2). These show its

bacteria isolates (isolates BCC7LC, BCC4 LC and/or BCC 12.1 LC) able to degrade lignocellulose,

lignin, cellulose and xylan to simple compounts and supply all potential nutrients contained in

the materials/feeds. Lignocellulose, lignin and cellulose will degrade to simples sugars, energy

(heat), CO2, H20 and the others compounts with action of thegroup lignocellulolytic enzyme such


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as lignase, cellulase and xylanase (Howard et al., 2003). At Table 2 also shows its bacteria

isolate have higher lignocellulolytic enzyme activity so wide of clear zone produced its isolate

are high. This shows the great potential of bacteria isolates to be applied as degrader of

lignocellulosic compounts such as agriculture waste and potential benefit in optimizing the

development of livestock based on agricultural waste

Lignocellulolytic Enzyme Activity

The results of study showed lignocellulose degrading bacteria from bali cattle colon

waste has high lignocellulolytic enzyme activities i.elignase, cellulase, and xylanase enzyme

activities (Table 2). These data proved that bacteria isolates has high lignocellulose degradation

to simple coumponent such as vanillin, glucose, and/or xylose. This statement is strengthen by

Wahyudi et al (2010) studies that the lignocellulolytic bacteria of the colon and/or caecum have

the ability fiber to degrade higher than the rumen bacteria. The bacteria isolates capable of

associating with fiber rich feed ingredients that are not degraded in the rumen.

The higher lignase enzyme activity of lignocellulolytic bacteria isolates from bali cattle

colon waste found from bacteria isolate coded BCC 12.1 LC, BCC 6 LC, BCC 4 LC The bacteria

isolates has high lignase activity 0.0045 – 0.0563 IU/ml, 0.0010 – 0.0427 IU/ml, 0.0050 – 0.0196

IU/ml and 0.0067 – 0.0105 IU/ml respectively after contact with the substrates for 30, 60, 180

and 360 minutes. The isolates coded BCC 12.1 LC and BCC 6 LC have shown higher (P<0.05)

lignase enzyme activity than the other isolates except observations at 30 min; of the other

bacteria isolates BCC 4 LC coded has the highest lignase enzyme activity similar to that of the

bacteria isolate coded with BCC 12.1 LC (Table 2A). This show that the bacteria isolates from

bali cattle colon waste capable produce lignase enzyme such as phenol oxidase/laccase, lignin

peroxidases/Li-P, and/or manganese peroxidade/Mn-P to breakdown of lignin compounds from

lignocellulose material (Howard et al., 2003). This statement is strengthen by Wahyudi et al

(2010) studies that the lignocellulolytic bacteria of the colon and/or caecum from herbivores

animal has produce lignase enzyme and has higher lignase enzyme activity compare with

bacteria rumen. In generally, lignase enzyme activities of bali cattle colon waste bacteria isolate

not too high. The enzyme activity this study similarly to the lignase enzyme activity of bacteria

isolates from Wahyudi et al (2010) research is 0.02 – 0.04 µmol for bacteria isolate from buffalo,

horse gastro intestinal tract and elephant dung. These numbers might be due lignin is the most

recalcitrant to degradation whereas cellulose or hemicellulose/xylanose, because of its highly

ordered crystalline structure and more resistant to hydrolysis (Howard et al., 2003).


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Table 2. Enzyme Activies from lignocellulolytic bacteria isolates from Bali Cattle colon

content waste

Bacteria Isolate Enzyme Activities (IU/ml)

t30 t60 t180 t360

A. Lignase enzyme activities1)

BCC 1 LC 0.0045a4 0.0289bc 0.0121c 0.0067a

BCC 2 LC 0.0435bc 0.0366bc 0.0163d 0.0085b

BCC 3 LC 0.0116a 0.0328bc 0.0050a 0.0087bc

BCC 4 LC 0.0563d 0.0401bc 0.0110c 0.0071a

BCC 5 LC 0.0130a 0.0305bc 0.0075b 0.0080ab

BCC 6 LC 0.0507cd 0.0439c 0.0196e 0.0105d

BCC 7 LC 0.0381b 0.0010a 0.0174d 0.0099cd

BCC 11 LC 0.0057a 0.0260b 0.0050a 0.0087bc

BCC 12.1 LC 0.0563d 0.0427c 0.0196e 0.0105d

BCC 13.1 LC 0.0130a 0.0051a 0.0073b 0.0074ab

SEM5 0,0021 0.0033 0.0004 0.003

B. Cellulase enzyme activities2)

BCC 1 LC 0.0493bc 0.0283bc 0,0108bcd 0.0127ab

BCC 2 LC 0.0448b 0.0238b 0.0093ab 0.0108a

BCC 3 LC 0.0503bc 0.0317cd 0.0105bc 0.0109a

BCC 4 LC 0.0682d 0.0309cd 0.0118cd 0.0138bcd

BCC 5 LC 0.0426b 0.0325cd 0.0123cde 0.0141bcd

BCC 6 LC 0.0720d 0.0398e 0.0141e 0.0154cd


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BCC 7 LC 0.0186a 0.0162a 0.0082a 0.0104a

BCC 11 LC 0.0214a 0.0140a 0.0087ab 0.0105a

BCC 12.1 LC 0.0716d 0.0398e 0.0142e 0.0157d

BCC 13.1 LC 0.0626cd 0.0359de 0.0129de 0.0133bc

SEM 0,0029 0,0014 0.0004 0.0005

C. Xylanase enzyme activities3)

BCC 1 LC 7.8734ab 9.7222abc 3.7282abc 1.9070a

BCC 2 LC 11.1111abc 11.5066bc 4.7216bcd 2.3485abcd

BCC 3 LC 8.0206ab 7.7134abc 2.5018a 1.9438a

BCC 4 LC 6.4018a 3.8355a 2.9065ab 2.0419ab

BCC 5 LC 11.6630abc 8.6553abc 4.2924abcd 2.3853abcd

BCC 6 LC 20.3826c 13.3830c 5.5923cd 2.9433d

BCC 7 LC 6.9904ab 6.3742ab 4.7952bcd 2.7349cd

BCC 11 LC 17.3657bc 8.7104abc 3.2009ab 1.9316a

BCC 12.1 LC 21.3392c 13.3462c 5.0711bcd 2.6613bcd

BCC 13.1 LC 16.9242abc 12.0438bc 6.4202d 2.2115abc

SEM 2.1088 1.2789 0.4389 0.1244

Notes: 1) Lignaseanalysis using tannic acid substrates, 2) Cellulase (Endo-glucanase)analysis using

carboxy methyl cellulose/CMC powder substrates, 3) Xylanaseanalysis using Xylanose substrates,

4)Mean in the same colom with different letter differ significantly (P<0,05).

On cellulase enzyme activity, the lignocellulolytic bacteria isolates has cellulase activities 0.0186

– 0.0720 IU/ml, 0.0140 – 0.0398 IU/ml, 0.0082 – 0.0142 IU/ml, and 0.0104 – 0.0157 IU/ml

respectively for 30, 60, 180 and 360 minutes after contact with the carboxy methyl cellulose

substrate. The bacteria isolates coded BCC 12.1 LC and BCC 6 LC have shown higher (P<0.05)

cellulase enzyme activity than the other isolates (Table 2B). This show that cellulases enzyme

from bali cattle colon bacteria isolates responsible for the hydrolysis of cellulose through the

breakdown of hydrogen bond in cellulose crystallin structure (α1,4 glukoside bond) from


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cellulose componds. CMC-ase (endoglucanase) are proposed to initiate attack randomly at

multiple internalsites in the amorphous regions of the cellulose fibre (Howard et al., 2003; Perez

et al., 2002).

Xylan is the most abundant hemicellulose, consist of xylosa polymer and other sugar

with ß-1,4 bond and end side chainwith a-1,2 or a-1,3 bonds (Peres et al., 2002).Xylanase

enzyme is one of the major hemicellulases which hydrolyse the β-1.4 bond in the xylan backbone

yielding short xylooligomers which are further hydrolysed intosingle xylose units by β-

xylosidase (Howard et al., 2003). Xylanase enzyme activities of lignocellulolytic bacteria

isolates from bali cattle colon waste showed at Table 2C. This study showed that isolate coded

BCC 6 LC, BCC 12.1 LC and BCC 13.1 LC has produce higher xylanase enzyme activity (P<0.05)

compared to the other isolates on all time durations. This show its bacteria isolates capable better

hydrolysis of xylanose to simple sugars ie xylose and the others sugars. Kamra (2005) stated that

ruminants in tropic area such as Indonesia has higher fiber degrading bacteria than other animal

farming.

In general, the activity of xylanase enzyme of bacterial isolates is quite high. This might

be due to xylan/hemicellulose has a structure that is relatively more easily degraded than the

other components of lignocellulose. The xylanase enzyme activity in this study is higher than the

xylanase enzyme activity of rumen bacteria isolates Bali cattle is 1.449 to 17.1264 U/ml

(Partama et al., 2012). This confirms that the isolated bacteria from the colon having enzyme

activity were higher than bacteria isolates of rumen cattle. This statement is strengthen by

Wahyudiet al (2010) studies that the lignocellulolytic bacteria of the colon and/or caecum have

the ability fiber to degrade higher than the rumen bacteria.

Based on the enzyme activities value obtained, the bacteria isolates coded BCC 12.1 LC

and BCC 6 LC have the higher quality and most potencial as lignocellulosic substrates degrader.

Lignoselulase high enzyme activity show a high ability of bacterial isolates to degrade

lignocellulose compounds as well as a variety of fiber-rich feedstuffs to simple

nutrients/compounds which easy to degrade or digestible for animal/livestock. It is feasible that

the isolates most potential to be used as fermentor/degrader of material/feed resources rich in

crude fiber and lignocellulose such as agriculture waste product.

CONCLUSION

1. Lignocellulolytic bacteria isolates coded BCC 7 LC, BCC 4 LC, BCC 12.1 LC, and BCC 2 LC

has produced higher width of clear zone


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2. Lignocellulolytic bacteria isolates coded BCC 12.1 LC and BCC 6 LC higher lignocellulolytic

enzyme activity

3. The lignocellulolytic bacteria isolates from bali cattle colon 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 2014 for fund support in this experimentation. Thanks are also due to the

Laboratory of feed and nutrition animal, Faculty of Animal Husbandry Udayana University for

assistance in isolation and characterization bacteria isolates analysis.

REFERENCES

Adney, B. and J. Baker. 2008. Measurement of Cellulase Activities. Laboratory Analytical

Procedure (LAP). Technical Report. National Renewable Energy Laboratory. 1617 Cole

Boulevard Golden, Colorado. 1-8

Efiok, B. J. S. 1996. Basic Calculation for Chemical and Biological Analysis.Gaithersburg. Md.;

AOAC International, Maryland, USA

Ghose, T. K. 1987. Measurement of Cellulase Activities. International Union of Pure and

Applied Chemistry. Pure and Appl. Chem., 59 (2); 257-268

Hegarty, R. .2001. Green House Gas Emission From The Australian Livestock Sector. What Do

We Know, What Can We Do. Australian Green House Office, Canberra ACT. ISBN: 1

876536 69 1. (accessed: December 24, 2007).

Howard, RL, Abotsi E., Rensburg JVEL., and Howard S. 2003. Lignocellulose Biotechnology:

Issues of Bioconversion and enzyme Production. African Journal of biotechnology 2:6002-

619. Available from: URL: Http://www.vtt.fi/inf/pdf [cited 2008, February 25]. 2(12): 602-

619

Irfan, M., Safdar A., Syed Q., Nadeem M. 2012. Isolation and screening of Cellulolytic Bacteria

from Soil and Optimization of Cellulase Production and Activity.TurkBiyokimyaDergisi

[Turkish Journal of Biochemistry] 2012: 37 (3); 287 – 293


Volume:01,Issue:01

www.ijaer.in


Kamra, D. N. 2005. Rumen Microbial Ecosystem. Special Section: Microbial Diversity. Current

Science. Vol. 89. No. 1. hal 124-135. [cited 2007 Decembre 20]. Available from: URL:

http://www.ias.ac.in/currsci/jul102005/124.pdf. 89 (1); 124-135

Lo, Y. C., G. D. Saratale, W. M. Chen, M. D. Bai, J. S. Chang. 2009. Isolation of cellulose-

hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen

production. Enzyme and Microbial Technology Journal 44; (6-7); 417 – 425.

Miller, G. L. 1959. Use of Dinitrosalisylic Acid Reagent for Determination of Reducing Sugar.

Anal. Chem. 31 (3): 426 - 428

Mudita, I M. 2008. Rumen Microbial Protein Synthesis Of Bali Cattle Given Complite Ration

Based On Ammoniated Rice Straw with Supplementation Of Multivitamins-Minerals.

Thesis. Graduate School (Magister), Udayana University, Denpasar. (in Indonesian with

abstract in English).140 p

Mudita, I M., AA. P.P. Wibawa, I W. Wirawan, N W. Siti and I G.L.O. Cakra. 2011.

Improvingthe Nutrition Quality of Complete Feeds Based on Local Waste with Fermentation

by Rumen Liquor and Enzyme Complex. Indonesian Journal of Nutrition and Feed Science 2

(1); 20-25,Bogor Indonesia

Mudita, I M., A.A.P.P. Wibawa, I W. Wirawan, and I G. N. Kayana. 2012.Use Bali Cattle

Rumen Liquor Waste and Termites on Produktion of Alternative Bioinocullant and Its

Application on Competitive and Sustainable Bali Cattle Livestock Development.

Competitive Research Report. Faculty of Animal Husbandry Udayana University, Denpasar

(In Indonesian with abstract in English)

Ogimoto, K. And Imai S. 1981. Atlas of Rumen Microbiology. Japan Scientific Societies Press,

Tokyo. 231 p

Partama, I. B. G. I M. Mudita, N. W. Siti, I W. Suberata, A.A. A. S. Trisnadewi. 2012. Isolation,

Identification, and Enzyme Activity of Lignocellulolytic Bacteria and Fungifrom Bali Cattle

Rumen Waste and Termites as Inocullant resources on bali cattle livestock development

based on Local Waste. Invention Research Grant. Udasyana University, Denpasar (In

Indonesian with abstract in English)

Perez, J., J. Munoz-Dorado, T. De la Rubia, and J. Martinez. 2002. Biodegradation and

Biological Treatment of Cellulose, Hemicellulose and Lignin; an overview. Int. Microbial, 5

(2): 53-62


Volume:01,Issue:01

www.ijaer.in


Putri, T. I., T. G. B. Yadnya, I M. Mudita, and Budi Rahayu T. P. 2009. Biofermentation of

Ration based on nonconventional local feed waste on development competitive and

sustainable bali cattle livestock. Competitive Research Grant under the national priorities.

Faculty of Animal Husbandry Udayana University, Denpasar (In Indonesian with abstract in

English)

SubhaRao, N.S. 1993. Biofertilizers in Agriculture and Forestry. 3rd ed. International Science

Publisher, New York. (Xi) p 242

Ullrey, D.E, S.D Crissey and H.F. Hintz. 1997. Elephants: Nutrition and DietaryHusbandry.

Michigan State University. EastLansing, Michigan 48824

Utomo, R., Z. Bachruddin, B.P. Widyobroto, M.Soejono and R. Antari. 2006. The use ofGoat’

feces as the microbial resources andmicrobial enzyme as a substitute for rumenliquor.

Bulletin Peternakan. 30 (3):106-114

Wahyudi, A., M. N. Cahyanto, M. Soejono, and Z. Bachruddin. 2010. Effect of Administration

of Lignocellulose-Degrading Fungi Isolated from Herbivore’s Gastrointestinal Tract for

Fiber Degradation. Journal of Animal Production 12 (1): 21-28

Wahyudi, A., M. N. Cahyanto, M. Soejono, and Z. Bachruddin. 2010. Potency of

lignocellulose degrading bacteria isolated from buffalo and horse gastrointestinal tract and

elephant dung for feed fiber degradation. Journal Indonesian Tropical Animal Agriculture 55(1):

34-41

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