Utilization of Gamma Irradiated Aspergillus niger to Improve Oil Palm by-Product Digestibility

1Field of Agriculture, Center for Application of Isotope and Radiation, National Nuclear Energy Agency of Indonesia, South Jakarta, 12070, Indonesia 2Field of Industry and Environment, Center for Application of Isotope and Radiation, National Nuclear Energy Agency of Indonesia , South Jakarta, 12070, Indonesia 3Faculty of Science and Technology, Syarif Hidayatullah State Islamic University, Banten, 15419, Indonesia


Introduction
Indonesia is a very massive growth country in palm oil plantations.In 2013, the area of large oil palm plantations (owned by the company) was 6,108,900 ha and increased to 6,404,400 ha in 2014.The area of smallholder palm oil plantations also increased from 4,356,090 ha in 2013 up to 4,551,850 in 20144,551,850 in (BPS, 2016)).Zahari et al. (2012) states that oil palm has a long economic value of about 20-25 years.This is a consideration that the oil palm by-products should be handled appropriately in order to provide a profitable added value.Proper by-products handling also supports environment-based management of intersectoral integration.
The oil palm by-products utilization as animal feed comes from two sources (direct byproducts and by-products from oil palm processing industry).Oil palm empty bunches and palm fronds are plantation by-products, while kernel shell is one of industrial by-products (Zahari et al., 2012).Islam et al. (2000) explains that the oil palm by-products needs to be investigated for digestibility because it is a potential source of ruminant feed.The constraints of oil palm byproducts utilization as ruminants feed are high fiber content and low digestibility (Antonius et al., 2015).Oil palm by-products including kernel shells also has a low palatability and characteristic of indigestible material (Chanjula et al., 2010).Oil palm empty bunches contain low quality fiber, high lignin content and resulting in low palatability performance (Akbar, 2007).Rahman et al. (2011) reported that the lignin content of palm fronds reaches 20% of dry biomass so that it is able to inhibit utilization as a fiber source.Various nutritional factors that inhibit the digestibility of feed in the rumen need to be processing first before being used as feed (Akbar, 2007).
Various strategies are done in overcoming these constraints so that oil palm by-products is able to be utilized as main feed or mixture in ruminant rations.In the previous study, Akbar (2007) used palm frond as a source of fiber, combined with protein by pass and defaunation treatment as sheep feed.Chanjula et al. (2010) utilize palm kernel cake (PKC) as a mixture in goat concentrate to accompany a Paspalum plicatulum based ration.Fariani et al. (2013) used oil palm frond fermentation using white root fungi (WRF) which is then used in complete feed block (CFB) formulation for beef cattle.The strategy of increasing the digestibility of oil palm by-products is by fermentation treatment to facilitate the solubility of fiber fraction.Mulyana et al. (2015) using Aspergilus niger fungi that has been irradiated by 500 Gy dose as a fermentation agent to improve rice straw digestibility.Furthermore, it is explained that the gamma irradiation treatment in A. niger inoculum is able to increase cellulase enzyme activity and glucose substrate production.Potential A. niger that has been irradiated gamma can be utilized as a fermentation agent that able to improve the digestibility of oil palm by-products.Based on the description, the purpose of this study was to determine the effect of palm oil fermentation using A. niger which has been irradiated to rumen fermentation characteristics in vitro.

Materials and Methods
The research was conducted at Animal Nutrition Laboratory, Livestock Production Group, Agriculture Field, Center for Application of Isotope and Radiation, BATAN, from July to October 2016.

Sample preparation
The kernel shells (CK), palm frond (PK) and oil palm empty bunches (TKKS) were dried at 60°C for three days then milled to a size of 5 mm.Non fermented and fermented feed samples then milled at 2 mm for further analysis of nutrient content.

Fermentation of oil palm by-products
The fermentation of oil palm by-products using three main components (inoculum A. niger, nutrient solution and oil palm by-products substrate).Preparation of nutrient solution was done by dissolving 28.8 g of PDB; 1.2 g (NH4) 2SO4; 0.6 g KH2PO4; 0.6 g of K2HPO4 and 0.24 g of MgSO4.7H20 in 1200 ml of distilled water and sterilized on autoclave for 15 minutes at 121°C.The inoculum used was A. niger which has been irradiated gamma using a 60 Co source irradiator at a dose of 500 Gy.A total of 200 ml of nutrient solution and 10 g of inoculum were dissolved into 200 ml of distilled water.The solution was mixed with 200 g of crude palm oil substrate which has been milled to a size of 5 mm then incubated into a 5 kg plastic bag and sealed with a cotton-clogged pipe.The substrate was incubated for 15 days.

In vitro incubation
A total of 380 mg samples were inserted into a 100 ml glass syringe volume (Fortuna model, Germany).Rumen liquids were collected from buffalo fistula before feeding in the morning.The feed ration for the buffalo consists of a combination of forage and concentrates (50:50) which was needed to maintain a basic life and production.The stage of the incubation process of the sample, the glass syringe containing the sample was incubated at 39°C before the rumen buffered fluid filled into the syringe.
The rumen fluid was filtered using a fourlayer cotton cloth.The incubation procedure uses Menke et al. (1979) which was modified by Blümmel et al. (1997).Incubation was carried out at 39°C for 48 hours.The sample was replicated as many as four replications.Observations of total gas production were performed at 0, 2, 4, 6, 8, 10, 12, 24 and 48 h incubation.Measurements of pH, ammonia (NH3), and total volatile acids (VFA) and protozoa populations were performed on the sample and rumen-buffered fluid that has been incubated for 48 hours.
Dry matter content (DM) and organic material (OM) (AOAC, 2010) were analyzed.Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were also analyzed using Goering and Van Soest (1970) procedures.The rumen fluid used in the study collected from fistulated buffalo.Total gas production, methane gas concentration measurements, CO2, and sample inoculum analysis were performed after 48 hours of incubation.Measurement of methane (CH4) and CO2 concentrations used MRU gas analyzer.Measurement of pH using Hanna instrument pH meter, NH3 measurement using Conway (1950) micro diffusion method and total VFA measurement based on AOAC (2010) method.

Data analysis
The gas kinetics variables were measured using the Ørskov and McDonald exponential models (1979) p=a+b(1-e -ct )."a" and "b" are soluble fractions and insoluble fractions but may also be degraded respectively.While c is the constant fraction solubility rate per t time unit.Calculation of fractions a, b and c used the Neway fit curve software.The data were analyzed using SPSS 16.0 followed by Duncan's Multiple Range Test (DMRT) test.This test is used to determine differences between treatments (Steel and Torrie, 1980).

Organic matter and fiber contents
The content of DM, OM, NDF and ADF of oil palm by-products before and after fermentation was presented in Table 1.The fermentation process decreased (P<0.05)DM content by The effect of fermentation on DM content was different between CK sample with PK, PKS and TKKS.This was caused by differences in the characteristics of CK, PKS and TKKS samples containing higher water content than CK.Sufficient water content would support the fungi to grow more, so the fermentation process utilizing the water content increased the percentage of DM content.In a previous study, Wajizah et al. (2015) reported that A. niger absorbed water for its growth.The condition of the substrate was drier along with the length of the fermentation process.The difference in the effect of fermentation was also due to the high lignin content in the kernel shell.Okoroigwe et al. (2014) reported that the lignin content of the kernel shell was 53.85%.According to Saka et al. (2008), the lignin content of the kernel shell was 49.9% while the lower palm oil was 21.2%.The decrease in the percentage of OM in palm oil was occur because of the use of organic compounds contained in substrates that were not offset by the resulting organic product.The fermentation process was able to increase the OM content if there was sufficient source of dissolved carbohydrate (Wajizah et al., 2015).It further mentioned that rice flour and sago starch acts as a soluble carbohydrate added in the fermentation process.
The NDF and ADF fraction of the kernel shell become higher after the fermentation process.This can be caused by the resistance of high lignin content.The fermentation process would involve dissolved carbohydrates in the sample and decrease the percentage thereby increasing the percentage of fractions containing lignin (NDF and ADF).The fermentation process using irradiated A. niger resulted in different effects on the NDF fraction and the ADF of oil palm empty bunches.The fermentation process proved that it has been able to decrease the content of both crude fiber fractions.In palm frond, fermentation was also to decrease the ADF fraction.It proves that the existence of fiberbreaking mechanism by cellulase enzyme produced by A. niger.Puastuti et al. (2014) explains that the fermentation process was able to convert more complex fractions into simpler fractions in order to be utilized by microbes.Mahesh and Mohini (2013) reported that the use of fungi as a fermentation agent was to decrease the cell wall component (NDF) so as to improve the digestibility of feed.

Total gas production
Total gas production in vitro of oil palm byproducts after incubation for 48 hours is presented in Table 2. Total gas production fluctuative greatly between treatments with incubation observation time from 2 to 48 hours.The difference was noticeable, occurring at the time of observation after 24 hours.At the time of incubation, TKKS and mix samples produced the highest total gas production (P<0.05)compared to the other six treatments.It was caused by the material characteristics of TKKS which were more easily fermented by rumen microbes.This affects the maximum gas production value (a + b), where TKKS produced the highest maximum of gas production (56.67 ml), and followed by mixed samples (37.81 ml).Different maximum gas production characteristics were shown between CK and PKS samples with TKKS and mix.In CK and PKS samples, the irradiated A. niger fermentation treatment had no significant effect on maximum in vitro total gas production.The fermentation treatment decreased (P<0.05) in TKKS and mix samples.The maximum total gas production was 52.92 and 35.60%, respectively.The "c" fraction (solubility rate fraction per t time unit) was not significantly different between treatments.The fastest rate of solubility of the fraction was in the TKKS sample.
Total gas production increased with increasing hours of observation.It represented a fermentation product by a microbe that turned into a gas form.The total gas produced came from direct substrate fermentation (CO2 and CH4) and came from indirect gas production through a buffering mechanism of VFA in the form of CO2 released from the bicarbonate buffer during the fermentation process (Getachew et al., 1998;Jayanegara et al., 2009).The dynamics of total gas production was influenced by differences in the fiber profile of the treatment substrate (Table 1).The data in Table 1 shows that there was a decrease in NDF percentage due to fermentation by A. niger.The decrease in the NDF fraction was in line with the decrease of maximum gas production on PKS, TKKS and mix substrate.Wahyono (2015) reported that the maximum gas production of incubation in vitro feed substrates was affected by fiber content and solubility rate.The maximum decrease in total gas production in TKKS samples appeared to be very massive (Table 2).This may be due to a decrease in the ADF fraction related to the fermentation treatment using A. niger (Table 1).In the fraction of the ADF was obtained component of cellulose which was the source of carbon element (C) in the fermentation process.
The efficiency of feed material utilization was not a result from the calculation of total gas production, therefore the production of gas in the form of CO2 and CH4 need to be measured so that the effectiveness of feed material utilization was able to be determined.The results of this study are in accordance with the Jahromi et al. (2010) who reported that although the fermentation process can decrease the content of lignocellulase in the feed substrate, it also has the effect of decreasing total gas production.Furthermore, it is explained that A. niger inoculum has antagonistic effect on rumen microbe so it can affect feed degradation process, and needs to be investigated further.The same results are also found in the study of Kaur et al. (2010) that reported if total gas production in vitro of rice straw decreased after fermentation process.

CH4 and CO2 gas production
The higher total gas production produced the higher CH4 and CO2 production.Therefore, the measurement of gas production based on the type of gas produced which determined from CH4 and CO2 gas concentration.The results of concentration of CH4, CO2 and CO2: CH4 ratio were showed in Figure 1.
CH4 concentrations of CKF and PKSF samples were 27.27 and 4.46% respectively lower than CK and PKS (P<0.05).Different things are shown on TKKSF samples which result in higher CH4 concentrations of 34.72% compared to TKKS samples (P<0.05).The fermentation process using A. niger had no significant effect on mixed samples.Fermentation using A. niger decreased CO2 concentration in CK, TKKS and mix samples (P<0.05).From the calculation of CO2 concentration ratio: CH4, the TKKS and mix samples decreased respectively by 49.69 and 25.63% after the fermentation treatment.
The effect of fermentation using irradiated A. niger differed greatly against the concentration of CH4 and CO2 on each substrate.This was influenced by differences in the structure of carbohydrates on the substrate.Increasing production of CH4 and CO2 gas because the production of excess CH4 gas that accompanied the release of CO2 also decreased feed efficiency.Baker (1999) reported that CH4 gas production indicated the amount of waste energy in the form of gas that indicated the low efficiency of feed utilization.CH4 gas was a fermentation product that was not utilized and contributed to environmental pollution.Mahesh and Mohini (2013) explain that enteric CH4 production came from microbial fermentation to the structural carbohydrate sources contained in the feeds of cellulose and hemicellulose.
The results of this study indicate that the ratio of CO2: CH4 gas in oil palm empty bunches and substrate mix became lower after the fermentation process using A. niger.This is different from the Mahesh (2012) study which states that the fermentation treatment using fungi is able to improve the quality of feed substrates that impact on the decline in CH4 production.This difference of results was thought to be caused by differences in fermented yields of partial VFA (Acetate, propionate, butyrate) each of which has a significant effect on CH4 production.Acetate and butyrate produced by carbohydrate fermentation feed substrate will provide H+ ions and methanogenic bacteria will utilize the H2 ion as a CH4 forming component (Wahyono et al., 2015).The mechanism of propionic acid formation will otherwise antagonistic with CH4 gas formation (Li et al., 2014).

Characteristics of rumen fermentation
In vitro rumen fermentation results including pH, NH3 and VFA are presented in Table 3.The lowest pH measurement results were TKKS samples (P<0.05),whereas differences were not seen in comparison among other treatments.The fermentation treatment using A. niger did not affect NH3 concentration.Total of VFA production in fermented kernel and palm kernel shells was higher (P<0.05)50 and 67.97%, respectively, than before fermentation.The fermentation treatment did not affect the total VFA production of the sample of empty bunches of palm oil and mixture.
The pH value of rumen fermentation tended to be the same between treatments except TKKS treatment.Nevertheless, the pH value of the eight treatments was still within the normal range so as not to interfere with the performance of rumen microbes especially cellulolytic bacteria.Martinez et al. (2010) reported that the rumen pH that continues to decrease, it will negatively affect population and activity of cellulolytic bacteria that assist the rumen fermentation process.Cardozo et al. (2000) identified that optimum and suboptimal pH values in the rumen were 6.4 and 5.5, respectively.The NH3 concentration of the eight treatments also corresponds to the normal range of NH3 produced in a closed culture.Wanapat et al. (2013) reported that optimal NH3 concentrations in in vitro cultures of closed systems were about 5 mg / 100 ml.
Characteristics of total VFA production of kernel shell substrate and palm oil frond increased after fermentation using A. niger.This is according to Mahesh and Mohini (2013) study which explains that the fermentation process can increase total VFA production.VFA production in oil palm by-products tended to be more toward acetate and butyrate.This can be proved by a considerable increase in CH4 gas (Figure 1).The process of producing acetate and butyrate produces H2 ions used in CH4 gas formation.Total VFA production of the eight treatments is still within the normal range, where the total ideal VFA production ranges from 80-160 mM (Van Soest, 1982).

Conclusion
Fermentation used irradiated A. niger affected the fiber profile in each of the oil palm byproducts.In the kernel shell, the fermentation increased the NDF and ADF fractions while in the empty palm oil bunch, the NDF and ADF fractions decreased after fermentation.The fermentation treatment resulted in the lowest total in vitro production of gas in the sample of oil palm empty bunches and a mixture of the three oil palm by products.Fermentation was able to increase total VFA production in kernel shell and oil palm frond samples.The fermentation process using irradiated A. niger tends to decrease CO2: CH4 ratio.This affected the decreased efficiency of energy consumption contained in the feed so that further research was needed.The best treatment was mixed treatment (CK + PKS + TKKS) because it produced the best CO2: CH4 ratio.

Table 1 .
Organic matter and fiber contents of treatments The concentration of NDF fraction decreased (P<0.05) on post-fermentation of PKS and TKKS samples (7.42 and 7.09%).Fermentation also decreased the ADF fraction in TKKS by 7.35% (P<0.05),whereas in CK samples increased NDF and ADF fractions by 6.92 and 11.38% (P<0.05).

Table 2 .
Total gas production in vitro of treatments