Potency of Lignocellulose Degrading Bacteria Isolated From Buffalo and Horse Gastrointestinal Tract and Elephant Dung for Feed Fiber Degradation

Lignin is limiting factor for cellulose and hemicellulose degradation in rumen. Isolation andselection bacteria from buffalo and horse gastrointestinal tract and elephant dung could be foundbacteria that have superiority to degrade lignin, xylan, and cellulose. Those animals were chosenbecause they were herbivores that consume low quality crude fiber as their main energy sources.Lignocellulose degrading bacteria were isolated by Hungate selective media, by using lignin (tannicacid), xylan, and cellulose as selective substrates. The morphological identification used an enrichmentmedia by measuring color, colony size, diffusion zone, clear zone, and biochemical identification usingproduction of ligninase, xylanase, and cellulase enzymes. The best lignocellulose degrading bacteriathen was determined by the morphological and biochemical character. This study showed thatlignocellulose degrading bacteria could be found in gastrointestinal tract of buffalo and horse, andelephant dung. Highest number colony was found in samples from buffalo's colon (376), followed byhorse's cecum (203), elephant's dung (46), buffalo's cecum (23), buffalo's rumen (9) and horse's colon(7). The highest isolates activity of lignolytic, xylanolytic, and cellulolytic were reached by buffalo'scecum (7.64), horse's cecum (6.27), and buffalo's colon (2.48). Meanwhile the highest enzymesproductivities were: buffalo's cecum (0.0400 µmol), horse's cecum (1.3912 µmol) and buffalo's colon(0.1971 µmol). Based on morphologycal character and biochemical test, it could be concluded thatlignolytic from buffalo's cecum, xylanolytic from horse's cecum, and cellulolytic from buffalo's colonwere the superior isolates and they were 99% analyzed as Enterococcus casseliflavus/gallinarumspecies.


INTRODUCTION
Lignocellulose degrading bacteria has important role in energy supply for ruminants. Ruminants are able to convert low quality feed in rumen because role of the lignocellulolytic bacteria. Tropical buffalo can grow properly with low quality roughage, agricultural and industrial waste with basic structure high lignocellulose as main energy source (Wanapat, 2003). On the other hand, conversion of feed crude fiber to animal product was not optimum in intensive animal farming (especially dairy cattle). Only 10-35% energy from crude fiber can be used, while 20-70% undigested cellulose were carried out with feces (Varga dan Kovler, 1997). These were the prove that crude fiber degradation in rumen was not optimal and these crude fiber content in feces was still high and fermentable (Krause et al., 2003).
Lignocellulolytic bacteria existence in the rumen is influenced by feed, chemical substance, and environment stress factor. Using too much concentrate and chemical substance in intensive farming system would depress rumen lignocellulolytic bacteria population. In contrast, these microbes could grow rapidly in ruminants fed roughage, so that herbivores that consume high crude fiber would be good to be used as lignocellulolytic microbes source. The microbes perhaps could be found in cecum and colon due to their colonized with undigested crude fiber in rumen.
Consideration of low crude fiber degradation in dairy cattle, and potentiality of lignocellulose degrading microbes from cecum and colon herbivores, lead to conduct isolation for collecting lignocellulolytic bacteria that has high ability to improve crude fiber degradation.
Cellulolytic bacteria has been isolated often to get anaerobic bacteria that degrade cellulose and hemicellulose. Study of enzymes capability and fermentation media formulation also has been conducted oftenly. Isolation of lignin degrading bacteria (Martani, 2003) and lignocellulolytic fungi (Samingan, 1998) has been done, but both have been done in aerobic isolation.
Lignin degrading bacteria is presumed live both in rumen and colon, but has not been proven empirically. Further exploration about potency of lignocellulose degrading bacteria from cecum and colon, so far is limited. Lignocellulolytic from cecum and colon could be has better ability to degrade crude fiber because substrate in those location contain rumen undigested crude fiber. The coprophagy phenomenon in rabbit, and elephant fed their feces for the calf showed the more perfection process of feed fermentation and nutrient fiber usage by lignocellulolytic by non ruminants herbivore.
Besides as differ factor from the previous study, isolation of lignocellulose degrading bacteria from buffalo's cecum and colon, exploration of lignocellulose degrading bacteria from horse's cecum and colon and also elephant dung are the new issue added in this study. Isolation from various compartment from herbivores are expected for getting potential bacteria for probiotics or inoculum for fiber feed anaerobic degradation.
This study was conducted to isolate and identify lignocellulose degrading bacteria from herbivore's digestive tract anaerobically and then test their ability in synthetic medium to degrade feed fiber.

Isolate Sources
The bacteria were isolated from fresh sample of buffalo's rumen, cecum and colon, horse's' cecum and colon, and elephant's dung. Buffalo's sample were taken from Demak' slaughter house (abattoir), horse's sample from Imogiri, and Elephant's dung from Gembiraloka Zoo, Yogyakarta.

Qualitative Selection
The lignin degrader bacteria was selected qualitatively based on the diffusion zone diameter that formed around colony (Subbarao, 1993: Samingan, 1998: Martani, 2003. While xylan and cellulose degrading bacteria were selected by measured clear zone around colony (Ogimoto and Imai, 1981). Each isolate was inoculated by spot method on nutrient agar that contain 1% tannic acid (Subbarao, 1993). Cellulose and xylan degrader were isolated according clear zone around colonies on nutrient agar that contain 1% cellulose and 1% xylan respectively (modified Hungate method in Ogimoto and Imai, 1981). Diffusion and clear zone were measured after 7 days of anaerobic incubation. Ratio between diffusion or clear zone with colony size was used to determine the selected isolates.

Liquid Media
Isolates were grown in liquid media by Potency of Lignocellulose Degrading Bacteria (A. Wahyudi et al.) modified Hungate method (Bachruddin, 1985) which were mixed 150 ml mineral I solution, 150 ml mineral II solution, 1 ml rezasurin 0,1% (w/v) , 2,00g substrate, 400 ml rumen liquid extract, 2 g yeast extract as enrichment nutrient, and 250 ml aquadest in 1000 ml Erlenmeyer. Substrates that used were mixed lignin, xylan and cellulose, adjusted by each enzymes production test. All materials in Erlenmeyer then were heated 100 o C for 5 minutes for homogenized along with CO 2 gas. Temperature was sustained 45 o C in water bath. An aerobic condition was reached when red color was faded. Then, 32,3 ml sodium carbonate and 16,7 ml Cystein-HCl were added. Tube then was closed by rubber stopper and sealed then sterilized in 121 o C for 15 minutes. Each media (according to selective substrate) was divided for isolates number that would be grown in 50 ml serum bottle. Isolate from solid media was dissolved in dilute solution in 0,5 λ 600 absorbent, inoculated in bottle as much as 10%, incubated in 39 o C for 7 days. Growth culture media then was used as enzymes source.

Quantitative Selection
Enzyme extract was collected from centrifuged liquid media culture in 12.000 x g for 15 minutes in 4 o C. According to the substrate, extracts were tested in three kinds of substrates that contained 1% Whatman No.1 filter paper/xylan/lignin in 50 mM acetate buffer and pH 5,5. Each substrate liquid in buffer was taken 8 ml, added 1 ml enzymes source, and 1 ml aquadest. The mixture then were shaken by fortex, enzyme activity was measured in 60 minutes. Reduction sugar (glucose from filter paper, xylose from xylan), or vanillin from lignin produced from the reaction were the 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 vanilin: 1 ml of sample was added to 4 ml methanol, then measured the absorbent by spectrophotometer in λ 560 nm for glucose, 550 nm for xilosa and 335 nm for vanilin.

Research Design
The research was conducted based on qualitative and quantitative analysis. One way completely randomized design was used as statistically design. Six isolates sources were used as treatment with three replication and filter paperase, xylanase, and ligninase as parameters observed.

Isolation of Lignocellulolytic Bacteria
Samples were taken from lignocellulose material of buffalo's and horse's digestive compartment and also from elephant's dung. The compartments were consisted of buffalo's rumen, cecum and colon, horse's cecum and colon and elephant's dung (Table 1). Because of using lignin (tannic acid), xylan and cellulose as selected substrate in bacteria isolated media, the cultures were predicted containing lignolytic, xylanolytic and cellulolytic bacteria respectively. Every strain of bacteria needed a specific substrate as an energy source (Berra-Maillet et al., 2004).
Lignin, xylan, and cellulose degrading bacteria could be found from all samples. In 10 -5 dilution, the highest number of lignin degrading isolates was found from buffalo's colon (3), horse's cecum for xylan (188), and also buffalo's colon for cellulose (262). Buffalo's colon seem contain more lignocellulolytic bacteria than others. Puppo et al. (2002) and Wanapat (2003), stated that buffalo has higher fiber degrading bacteria than others animal farming, especially local cow.
Herbivores digestive tract, specifically ruminants in Indonesia, in general contain high lignocellulose feed, so that lignocellulose degrading bacteria was expected exist. This study proved that all compartment digestive tract of buffalo and horse, and also elephant dung possess lignin, xylan, and cellulose degrading bacteria. Several previous study showed that lignocellulose degrading 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). According to Table 1, it is proved that the lignocellulolytic microbes could be found from herbivores digestive tract.

Qualitative of Lignocellulolytic Activity
Qualitative selection (Table 2) showed that isolates from buffalo's cecum has highest activity in degrade lignin (7,64) followed by colon (3.98) and rumen (3.85) These data proved that lignolytic bacteria from buffalo has higher lignin degradation than others. This statement is strengthen by Wanapat (2003), Kennedy et al. (1992) and Hardjosubroto (2006) studies that buffalo could use fiber feed more efficient than other ruminants. This statement also support the hypothesis that fiber degrader bacteria from lower tract has higher ability than those from rumen.
The highest activity of xylan degrader isolate (Table 3) found from horse's cecum (6.27) and followed by buffalo's cecum (3.32) and then rumen (2.84) It still unclear explanation why horse bacteria has higher ability than others, even though buffalo's colon has also high number of this bacteria (Table 1) Xylan is main carbohydrate that form hemicellulose, consist of xylosa polymer and other sugar with β-1,4, bond and end side chain with α-1,2 or α-1,3 bonds (Peres et al., 2002). Differences of heteropolymerisity of sugar that form xylan in rumen and cecum or colon in this study are predicted as main reason why rumen and cecum xylanolytic bacteria have higher activity than colon xylanolytic bacteria .
Cellulolytic isolate from buffalo's colon has the highest activity (2.48) in cellulose degradation than others (Table 4). Buffalo's colon isolate seem has higher activity than isolate from horse's digestive tract and elephant's dung. This data strengthen the Puppo's et al. (2002) and Wanapat's (2003) statement that buffalo has higher fiber degrading bacteria than others animal farming. From buffalo point of view itself, cellulolytic isolate from colon is superior than bacteria from rumen (1,92) or cecum (1,40). This study also prove the hypothesis that lignocellulolytic from colon has higher activity in fiber degradation.

Quantitative Lignocellulolytic Activity
Several lignin, xylan, and cellulose degrading isolate were superior than the others (Table 5). It was concluded from quantitative analysis of enzymes productivity. The lignolytic isolates was from buffalo's cecum, xylanolytic was from horse's cecum, and cellulolytic was from buffalo's colon with enzyme productivities were 0.0400 µmol, 1.3912 µmol and 0.1971 µmol respectively. The data showed positive correlation with qualitative analysis based on each colonies activity ratio. Qualitatively, buffalo's cecum, horse's cecum and buffalo's colon were best selected among others isolates, with activity ratio 7,64; 6,27 and 2,48 (Table 2, 3 and 4).
Quantitative and qualitative test for ligninase, xylanase and cellulase enzymes activities showed that selected lignocellulolytic bacteria was found more frequently from cecum and colon both from buffalo and horse. Its strengthen the hypothesis above that lignocellulose degrading bacteria could be found from lower digestive tract of herbivores, including ruminants. This statement support Ullrey et al. (1997) study that cecum and colon monogastric herbivores have similar composition microbes with ruminants. Futhermore this study showed that the cecum and colon lignocellulolytic bacteria have higher ability to degrade lignocellulose than rumen bacteria.
The three selected bacteria then were tested for classification up to species. Result of morphological character and biochemical test were summarized in Table 6. Lignolytic isolate from bufallo's cecum, xylanolytic from horse's cecum and cellulolytic from horse's colon have a similarity.
According to microscopic, macroscopic, and biochemical analysis by Bergey's manual determinative bacteriology (Holt et al., 1994) (Ogimoto and Imai, 1981), the three isolates tend to be Ruminococcus sp genus, but based on biochemical analysis by BD Phoenix automated microbiology system for identification with 99% confidence value, the isolates were Enterococcus casseliflavus/gallinarum.

CONCLUSION
Lignocellulolytic isolates could be found from all samples in this study. Based on morphologycal character and biochemical test, it could be concluded that lignolytic isolate from buffalo's cecum, xylanolytic from horse's cecum, and cellulolytic from horse's colon were the superior isolates.