In Vitro Digestibility of Ration Containing Different Level of Palm Oil Frond Fermented with Phanerochaetae Chrysosporium

Phanerochaete chrysosporium was widely used to delignify agricultural waste product and improve biodegradation of the substrate as animal feed. The experiment was carried out to increase the use of palm oil fronds as a substitute material for napier grass through biodegradation process with P. chysosporium. A completely randomized design with four treatments and four replications was used. The treatments were ration containing 60% napier grass (R1), ration containing 40% napier grass and 20% fermented palm oil frond (R2), ration containing 20% napier grass and 40% fermented palm oil frond (R3), ration containing 60% fermented palm oil frond (R4). Fourty percent concentrate was included in all treatment rations. Parameters measured were in vitro digestibilities of dry matter, organic matter, crude fiber, NDF, ADF, NH3, TVFA, and ruminal cellulolitic bacteria. Results showed that increasing level of fermented palm oil frond in the ration reduced (P<0.05) digestibilities of dry matter, organic matter, crude fiber, NDF, ADF, N-NH3, TVFA concentration and number of ruminal cellulolytic bacteria. It is concluded that fermentation of palm oil frondwith P. chysosporium decrease lignin content by 47.79%, but increasing the fermented palm oil frond in the ration reduces nutrient digestibilities, N-NH3 and TVFA concentrations and rumen cellulolytic bacteria counts. Fermented palm oil frond up to 40% could be used as a substitute for forages in ruminant rations.


INTRODUCTION
Oil palm fronds are an important co-product. However, the high neutral detergent fiber (NDF, 700 g/kg dry matter and lignin (205 g/kg contents are major constraints to OPF use as livestock feed (Zahari et al., 2003;Wan Rosli et al., 2004;Abdul Khalil et al., 2006) this is consistent with low OPF digestibility in cattle (400 g/kg DM (Kawamoto et al., 2001). Physical methods and chemical agents to disrupt the cell wall matrix in low quality forages and agriculture waste products have been investigated. Some methods are very effective in removing lignin and disrupting the cell wall matrix. Commercial application is plagued by disposal of chemical waste and hazards to persons applying treatments. One alternative is the use of biological agents like microbial for removal of lignin to increase digestibility of agriculture waste product as feedstuff for ruminants. The organisms predominantly responsible for lignocelluloses degradation are fungi, and the most rapid degraders in this group are basidiomycetes (Ten Have & Teunissen, 2001;Bennet et al. 2002;Rabinovich et al., 2004) Agriculture wastes like palm oil frond containing lignocellulose can be up grade by solid-state fermentation with white rot fungi. Moreover, lignin degradation is affected by the culture condition, among which aeration is highly important. Not even whiterot fungi are known to be capable of using lignin as a sole carbon and energy source, and it is generally believed that lignin break down is necessary to gain access to cellulose and hemicellulose. Although white-rot basidiomycetes have been shown to efficiently mineralize lignin, species differ gross morphological patterns of decay they cause Phanerochaetae chrysosporium strains simultaneously degrade cellulose, hemicellulose and lignin, whereas others such as Ceriporiopsis subvermispora tend to remove lignin in advance of cellulose and hemicelluloses (Sanchez, 2009).
The common pattern of attack on lignocellulose by white rot fungi is a simultaneous decay of polysaccharides and lignin, but preferential degradation of lignin may also occur. Much of the reported research has dealth with delignification of wood by white rot fungi, particularly P. chrysosporium. P. chrysosporium has been reported to liberate lignin from plant tissue, research has shown that lignin is oxidized and degraded by a ligninolytic system (Elisashvili et al., 2008;Rodrigues et al., 2008) composed by lignin peroxidase (LiP), manganese peroxidase (MnP) and Laccase (Panagiotou et al., 2007).
P. chrysosporium has recently been shown to produce an extracellular enzyme that catalyzes oxidative C α -C β cleavage in two important lignin substructures and thereby causes the partial depolymerization of natural lignin. Recent research reported about effect of temperature, pH, carbon and energy sources for P. chrysosporium activity, but little lignin degradation associated with decreased substrate digestibilty from waste agriculture product like palm oil frond has been reported.
The objective of this experiment was to study in vitro digestibility of ration containing different level of palm oil frond fermented by P. chrysosporium.

MATERIALS AND METHODS
This experiment was done in Dairy Nutrition Laboratory, Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University.

Stater Preparation
P. chrysosporium (obtained from Biotechnolgy Laboratory, Bandung Institute of Technology, Bandung) was grown in potato dextrose broth (PDB). PDB was prepared following the modification method of MacFaddin (1985). P. chrysosporium (10 7 cfu/mL) was inoculated into 50 mL PDB medium and incubated for three days.

Fermentation
Palm oil frond was removed its skin, chopped, dried and milled with a size 5 mm. Fifteen grams of milled palm oil frond was autoclaved for 15 min at 121 o C in Erlenmeyer. Then, palm oil frond was inoculated with stater P. chrysosporium and incubated at 27 o C for 10 d.

In Vitro Digestibility of Palm Oil Frond in Ruminant Ration
In vitro digestibility trial was conducted based on modification of Tilley & Terry (1963) method. Cattle rumen liquor from RPH was filtered with four layers cheese cloth. One part of rumen liquor (10 mL) was mixed with 4 parts medium (40 mL) i.e. buffer solution, macro and micro mineral solution, resazurine and reduction solution (Goering & Van Soest, 2002). One gram of sample was added into the 100 ml incubation tube containing 50 mL mixed solution. Before incubation tube was closed, CO 2 gas was purged for 30 sec and incubated for 24, 48, and 72 h. When each incubation time finished, 2 drops of HgCl 2 was added into the cultures. The incubation tubes were then centrifuged at 4000 rpm for 10 min. Supernatant was used for determination cellulolytic bacterial, NH 3 -N concentration and total VFA, then residue was added 50 mL pepsin-HCl 0.2% solution and incubation for 48 h. Residu filtered by whatman no 41, dried at 60 o C for 48 h. The dried sample was used for determination of in vitro digestibility of dry matter, organic matter, crude fiber, NDF, and ADF. Cellulolytic bacterial concentrations were estimated with the Hungate procedure (Ogimoto & Imai, 1981). NH 3 -N concentration in rumen fluid was determined by microdiffusion technique Conway. Total VFA (TVFA) concentration in rumen fluid was determined by Markhams distillation

Dry Matter and Organic Matter Digestibility
Digestibility of nutrient is one indicator to measure feed quality because it reflects the level of nutrients availability for livestock. The study showed that increasing level of fermented palm oil frond with P. chrysosporium in ruminant ration significantly reduced (P<0.05) digestibility of dry matter and organic matter. The average was 55.06%-71.27% for dry matter digestibility and 54.15%-69.66% for organic matter digestibility ( Table 2). The dry matter digestibility of ration with 20% fermented palm oil frond had 6.73% lower than that of ration containing 60% napier grass. Meanwhile, ration containing 40% and 60% fermented palm oil frond had 18.15% and 22.74% lower than that of ration containing 60% napier grass respectively. Organic matter digestibility of ration with 20% fermented palm oil frond had 7.00% lower than of ration containing 60% napier grass, meanwhile ration containing 40% and 60% fermented palm oil frond had 17.18% and 22.26% lower than that of ration containing 60% napier grass respectively. The differences in dry matter and organic matter digestibility were caused by crude fiber contents in each treatment (Table 1). Cherdthong et al. (2010) found that the digestibility of DM and organic matter was negatively correlated with NDF and ADF contents in the diet. Davidson et al. (2003) reported that high of crude fiber contributed to decreased DM and OM digestibility. Griswold et al. (2003) reported that increasing trend in organic matter digestibility related with dry matter digestibility because the difference was only in the ash content. Ration containing 60% napier grass had the highest organic matter digestibility.
Dry matter and organic matter digestibility of ration containing fermented palm oil frond were higher compared to that ration containing palm oil frond without fermentation. Suryadi et al. (2009) reported that use of 42% palm oil frond without fermentation of in ration obtained dry matter digestibility 30.77% and organic matter digestibility 30.91%, meanwhile Syarief (2010) obtained dry matter digestibility of ration containing 60% non fermented palm oil frond was 39.28%, and organic matter digestibility was 44.35%. According to Islam et al. (2000), used of 60% fresh palm oil frond and 40% concentrate gave in sacco dry matter digestibility of 30.85%, meanwhile in vivo dry matter, organic matter and ADF digestibility were 52%, 56% and 26%, respectively.

Fiber Digestibility
The increasing level of fermented palm oil frond with P. chrysosporium in ruminant ration significantly reduced (P<0.05) digestibility of NDF, ADF, and crude fiber. The average was 35.99%-55.90% for NDF digestibility, 41.02%-58.58% for ADF digestibility, and 32.87%-57.43% for crude fiber digestibility. The increased level of fermented palm oil frond in ration decreased NDF, ADF, and crude fiber digestibility. NDF, ADF, and crude fiber digestibility of ration containing 60% napier grass (R1) were higher than those rations containing 20% fermented palm oil frond (R2), 40% fermented palm oil frond (R3) and 60% fermented palm oil frond (R4), meanwhile the ration containing 60% fermented palm oil frond had the lowest NDF, ADF and crude fiber digestibility (Table 2). This indicated that although biodegradation of palm oil frond with P. crhysosporium capable to decrease lignin contents (  Biodegradation of palm oil frond with 10 7 cfu/mL P. crhysosporium and incubated for 10 d showed the best yield for lignin degradation. The lignin content of fermented palm oil frond was 13.27% while palm oil frond without biodegration has lignin content 25.42% (Table 3).
The result also showed that lignin content of fermented palm oil frond was higher than lignin of napier grass (11.42%). Lignin in the ration could decrease digestibility as reported by Lynd et al. (2002) that ration with lignocelulolytic contents generally have high structural carbohydrate by some level of crystalline and make stick to lignin so digested of ration is difficult. Digestion of ration with fiber content could be increased through that can disperse lignin and carbohydrater string (Annison et al., 2002). Rumen microbes can digest the nutrient in the ration easier as the content of lignin decreasing. Van Soest (2002) mentioned that lignin in the plant cell is important factor in limiting the digestibility of the nutrient. A positive relation between total lignin loss and in vitro DM degradability has been reported in a few studies, particularly for sugarcane bagasse colonized with L.
edodes and C. subvermispora for 16 wk (Okano et al., 2006). Similar positive relationships between lignin loss and in vitro DM degradability have been reported for other substrates and fungi, such as sugarcane bagasse colonized with P. eryngii (Okano et al., 2007) The negative relationship that seems to exist between lignin concentration and forage digestibility has been recorgnized for many years, because lignin as a component of the cell wall, the effect of lignin on forage digestibility is assumed to be a direct influence on wall digestibility rather than on digestibility of total forage organic matter . Nutrient digestibility was influenced with physical of fibrous component in the rations (Toharmat et al. 2006). Lignin seems to exert its negative effect on cell-wall polysaccharaide digestibility by shielding the polysaccharides from enzymatic hydrolysis (Jung & Deetz, 2003). This is apparently a steric hindrances such that the polysaccharides cannot align themselves properly with their substrate for hydrolysis to occur (Tripathi et al. 2008). In this experiment based on in vitro experiment, the value of dry matter, organic matter, crude fiber, NDF, ADF digestibility was negatively correlated to lignin content it seems in determination koefisien of correlation value. Determination koefisien (R) for correlation value of lignin content with dry matter digestibility= -0.97098 (y= -15.52x+177.3; R 2 = 0.942), organic matter digestibil-ity= -0.97351 (y= -14.26x+166.9; R 2 = 0.947), NDF digest-ibility= -0,99831 (y= -11.70x + 158.30; R 2 = 0.996), ADF digestibility= -0.97417 (y= -7.077x + 130.10; R 2 = 0.949) and crude fiber digestibility= -0.9032 (y= -19,38x+213.00; Fiber digestibility of ration with 20% fermented palm oil frond had 6.9% lower than ration with 60% napier grass. Meanwhile, ration with 40% and 60% fermented palm oil frond had fiber digestibility of 14.45% and 20.89%, respectively. Based on the result above, 60% of fiber in fermented palm oil frond could be digested by rumen microbes. This is the result of laccase and ligninase enzymes activity from P. crhysosoporium which capable of decreasing lignin content in palm oil frond up to 47.79%. Crude fiber digestibility of fermented palm oil frond in the ration was higher than the ration with palm oil frond without fermentation. This result support the experiment conducted by Syarief (2010) that NDF and ADF digestibility of ration with 42% palm oil frond without fermentation had 62.66% and 54.83%, respectively.

Concentrations of N-NH 3 , TVFA, and Cellulolytic Bacteria
The concentrations of N-NH 3 and TVFA and the number of cellulolytic bacteria were found to be decreasing with the increasing use of fermented palm oil frond in the rations. It was found that the average N-NH 3 concentration was about 6.5-8.71 mM, the average TVFA concentration was about 150.06-163.47 mM, and the number of cellulolytic bacteria was about 6.02-8.27 log cfu/ml. The figures of these three parameters were found to be higher in ration containing 60% napier grass (R1) than those in rations containing 20%, 40%, and 60% fermented palm oil frond (R2, R3, and R4). Ration containing 60% fermented palm oil frond was found to have the lowest N-NH 3 and TVFA concentrations and the number of cellulolytic bacteria (Table 4).
The decreasing N-NH 3 concentration with an increased use of fermented palm oil frond was in line with to that in R1 resulted in reducing N-NH 3 concentrations (3.09, 2.11, and 1.38 mg N/dl, respectively). This might 3.09, 2.11, and 1.38 mg N/dl, respectively). This might be explained by the notion that in ruminants, protein entering the rumen will be degraded to ammonia by proteolytic enzymes produced by rumen microbes. Ammonia production depends on feed protein solubility, feed protein content, feed retention in rumen, and rumen pH (Orskov, 1992). 92).
N-NH 3 concentration found in this study was considered adequate for optimum growth. Cherdthong et al. (2011) N-NH 3 concentration of rumen is 15.7 mg N/dl, meanwhile Tripathi et al. (2008) found  found N-NH NH 3 concentration 20.34 mg N/dl in rumen of diet containing mustard (Brassica compestris), and 10-15 mg N/dl rumen fluid (Alcaide et al. 2003) were required for optimum growth. Nagadi et al. (2000) found that NDF fermentation rate increased as N-NH NH 3 concentration increased. An N-NH increased. An N-NH 3 concentration of 8-15mg N/dl was found to be optimum 8-15mg N/dl was found to be optimum for NDF degradation and intake (Dermann, 2009) and 6.4 mg N/dl rumen fluid was found by Islam et al. (2000) in a ration containing 60% palm oil frond and 40% oil frond and 40% 40% concentrate.
In this study, the increasing level of fermented the increasing level of fermented palm oil frond with P. chrysosporium in ruminant ration significantly reduced (P<0.05) number of cellulolytic bacteria. Reducing number of cellulolytic bacteria was influenced by decreasing of N-NH 3 concentration. This was attributed to the fact that the resulted ammonia was the main source of nitrogen for rumen microbes. A dedeficiency of N-NH NH 3 concentration resulted in a reduction in rumen microbial population. Decreasing of N-NH Decreasing of N-NH 3 concentration could be reflection about sintetic rumen bacteria (Karabulut et al., 2007;Kongmun et al., 2010;Cutrignelli et al., 2010). Most ammonia (82%) is used by rumen microbes for multiplication, especially in their cell synthesis. The ammonia level needed for rumen microbe growth is about 5.9-9.4 mg N/dl  mg N/dl (Jalc et al. 2009).
In-vitro N-NH NH 3 measurement can be used to estimate protein degradation and utilization by rumen microbes.
A strong correlation appeared between the digestible organic matter intake and the microbial protein synthesis (Pathak, 2008). Volatile fatty acids (VFA) are the end products of carbohydrate metabolism by rumen microbes. TVFA concentration of 150.06-163.47mM in this study was 150.06-163.47mM in this study was found to be adequate for microbial growth as it was higher than the amount required (80-160 mM) for optimal rumen microbial growth (Sutardi, 1980). The decreasing VFA concentration as the amount of palm frond use increased was found to be correlated with the decreaing fiber digestibility. Lower fiber digetibility resulted in lower VFA concentration. NDF digestibilities were found to be lower by 16.68%, 9.88%, 16.68%, 9.88%, and 6.44% (in R4, R3, and R2, respectively) than that in R1. These reducing NDF digestibility figures were correspondingly accompanied by decreasing TVFA concentrations of 13.41, 10.05, and 3.65 mM in R4, R3, and R2, respectively. High lignification level in feed was known to limit rumen microorganisms in fermenting cellulose and hemicellulose to produce energy in the forms of VFAs (Tripathi et al., 2008). The improvement of access of cellulose and hemicellulose enzymes should be initiated by the breakdown of lignocellulose bonds of the cell wall. The degradation process in the rumen requires protein availability to ensure the optimum growth of microbes (Alcaide et al., 2003). TVFA concentration in this TVFA concentration in this study was found to be higher than that (22,13-91,95mM) resulted from fermented cocoa pods by fermented cocoa pods by P. chrysosporium (Nelson et al., 2011) CONCLUSION Fermentation of palm oil frond with P. crhysosporium decreases lignin content by 47.79%, but it can not improve nutrient digestibilities, N-NH 3 and TVFA concentrations, and rumen cellulolytic bacterial counts. Fermented palm oil frond can be used as a substitute for napier grass up to 40% in ruminant rations.