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OPEN ACCESS International Journal of Poultry Science ISSN: 1682-8356 DOI: 10.3923/ijps.2017.6.10 Research Article 
Palm Oil Sludge Fermented by Using Lignocellulolytic Fungi as Poultry Diet Nuraini, Ade Djulardi and Ade Trisna Faculty of Animal Science, Kampus Limau Manis, University of Andalas, Padang, Indonesia Abstract Background: Palm Oil Sludge (POS) represents an alternative to poultry feed but its utilization is limited due to the high content of crude fiber (lignin and cellulose) and low content of crude protein. Fermentation with lignocellulolytic fungi, which include Lentinus edodes, Pleurotus ostreatus and Phanerochaete chrysosporium, produce ligninase and cellulase enzyme have been used as an approach to increase POS nutrient levels and quality. This study evaluated the nutrient content and quality of POS after fermentation using different ligninocellulolytic fungi. Methodology: Two experiments were conducted in this study. Experiment 1 was performed as a 3×3 factorial arrangement in a completely randomized design with 3 replicates. The first factor was lignocellulolytic fungi (Lentinus edodes, Pleurotus ostreatus and Phanerochaete chrysosporium) and the second factor was incubation time (7, 11 and 15 days). Subsequently, Crude Fiber (CF), Crude Protein (CP), cellulose, lignin and cellulose enzyme activity were measured. Experiment 2 compared the nutrient content of fermented (the best treatment identified in experiment 1) and unfermented POS. In this experiment, the nitrogen retention, crude fiber digestion, Metabolizable Energy (ME) and amino acid profile of POS was measured. Results: In experiment 1, it was found that Pleurotus ostreatu significantly reduced CF, cellulose and lignin levels and increased CP content as well as cellulase enzyme activity after 11 days of incubation (p<0.01) Moreover, experiment 2 showed that nitrogen retention, crude fiber digestion and ME content levels in fermented POS were higher than in unfermented POS. Fermentation also improved the amino acid profile of POS. Conclusion: An 11 day fermentation with Pleurotus ostreatus was identified as the best condition for improving the nutrient content and quality of POS. Key words: Palm oil sludge, fermentation, lignocellulolytic fungi, nutrient content and quality, poultry feed Received: November 13, 2016 Accepted: November 30, 2016 Published: December 15, 2016 Citation: Nuraini, Ade Djulardi and Ade Trisna, 2017. Palm oil sludge fermented by using lignocellulolytic fungi as poultry diet. Int. J. Poult. Sci., 16: 6-10. Corresponding Author: Nuraini,Faculty of Animal Science, Kampus Limau Manis, University of Andalas, Padang, Indonesia Tel: +62 (751) 71464 Fax: +62 (751) 71464 Copyright: © 2017 Nuraini et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists. Data Availability: All relevant data are within the paper and its supporting information files. INTRODUCTION Palm Oil Sludge (POS) can be used as a non-conventional animal feed. Indonesia is currently the largest palm oil producer in the world, producing a total of 22.5 million tons per year. Moreover, 70% of the palm oil production originates from the island of Sumatra. During palm oil production, approximately 45-46% of the material results in waste, including POS and Palm Kernel Cake (PKC). The POS consists of approximately 11.30% crude protein, 10.43% fat, 25.80% crude fiber, 19.19% lignin, 16.15% cellulose and 1550 kcal kgG1 energy metabolism1. The utilization of POS as poultry feed is currently low with only 5% being used in broiler ration1. Poultry feed containing high content of crude fiber (lignin and cellulose) has low digestibility and therefore, utilization in the diet is also limited. Lignocellulose consists of three major components: Cellulose, hemicellulose and lignin2. Improving the nutrient quality of POS requires reducing the crude fiber levels through microorganism fermentation, which has been shown to increase the nutrient content, digestibility value and palatability of feed3 -7. It has been shown that fermentation using cellulolytic fungi can reduce the cellulose content of POS but lignin content remains high. Therefore, fermentation using lignocellulolytic fungi (Lentinula edodes, Pleorotus ostreatus and Phanerochaete chrysosporium) may reduce lignin levels due to the production of ligninase and cellulase enzymes. The ligninase enzyme produced by these fungi can degrade lignin as a substrate4-6,8-12. Previous studies have also shown these fungi produce cellulase enzymes4-13. The POS fermented with an 8% inoculum of Phanerochaete chrysosporium for 7 days decreased crude fiber levels (lignin and cellulose) and increased application in a broiler diet by 15%. Therefore, fermentation of POS with other types of lignocellulolytic fungi, including Lentinula edodes, Pleorotus ostreatus and Phanerochaete chrysosporium, would likely improve the nutrient content and quality of POS as well as reduce crude fiber, especially lignin and cellulose. In this study we assessed the effects of different types of lignocellulolytic fungi and incubation times on the nutrient quality of POS. MATERIALS AND METHODS The main objective of this study was to assess improvements in nutrient quality of POS through fermentation using several lignocellulolytic fungi and incubation times. Experiment 1: Palm Oil Sludge (POS) was dried under sunlight until the water content reach approximately 12-14%. This experiment was designed as a 3×3 factorial arrangement in a completely randomized design with 3 replicates per treatment. The first factor was the type of lignocellulolytic fungi (Lentinus edodes, Pleorotus ostreatus and Phanerochaete chrysosporium) assessed and the second factor was the incubation time (7, 11 and 15 days). In the first experiment, 100 g of substrate containing 80% POS and 20% rice bran mixture per experimental unit was added together with 100 mL of aquadest (moisture content 70%). The mixture was then sterilized in an autoclave (121EC for 15 min). After cooling to 27- 30EC, the sample was inoculated with 8% Lentinus edodes, Pleorotus ostreatus and Phanerochaete chrysosporium together with 100 ppm calcium and 40 ppm manganese. These mixtures were then incubated for 7, 11 and 15 days. All of the data obtained were statistically analyzed by analysis of variance. The differences among treatments were determined by using Duncan Multiple Range Test (DMRT)14. Experiment 2: The second experiment was designed to compare nutrient content between fermented (the best treatment obtained in the first experiment) and unfermented POS. Measured variables included the following: Nitrogen retention, crude fiber digestion, Metabolizable Energy (ME) and amino acid profile. The fermentation sample used in this experiment was based on the best conditions obtained in the first experiment. Amino acid analysis was performed by HPLC. All data obtained from the second experiment were compared numerically. RESULTS Experiment 1 Effect of lignocellulolytic fungi and incubation time on crude fiber, lignin, cellulose and cellulase enzyme activity: The effects of lignocellulolytic fungi and incubation time on crude fiber, lignin, cellulose content and cellulase enzyme activity of POS are shown in Table 1-4, respectively. We found that the combination of lignocellulolytic fungi and incubation time had a significant effect on crude fiber, lignin and cellulose content as well as cellulase enzyme activity of POS (p<0.05). Effect of lignocellulolytic fungi and incubation time on crude protein: The effect of lignocellulolytic fungi and incubation time on crude protein content of POS is shown in Table 5. It was found that the combination of the type of Table 1: Effect of lignocellulolytic fungi and incubation time on crude fiber (%) Treatments B1 (7 days) B2 (11 days) B3 (15 days) Averages A1 (Le) 18.36Aab 14.97Ba 14.59Ba A2 (Po) 17.96Ab 14.04Bb 14.12Bb A3 (Pc) 18.74Aa 14.91Ba 14.95Ba Averages 18.35 14.64 14.55 15.97 15.37 16.20 Table 2: Effect of lignocellulolytic fungi and incubation time on lignin (%) Treatments B1 (7 days) B2 (11 days) B3 (15 days) Averages A1 (Le) 16.17Aa 14.70Bb 14.78Ba A2 (Po) 15.14Ab 13.22Bb 13.39Bb A3 (Pc) 16.00Aa 14.49Ba 14.58Aa Averages 15.77 14.14 14.25 15.22 13.91 15.02 Table 3: Effect of lignocellulolytic fungi and incubation time on cellulose (%) Treatments B1 (7 days) B2 (11 days) B3 (15 days) Averages A1 (Le) 9.33Aab 7.81Bb 7.84Bb A2 (Po) 8.90Ab 7.68Bb 7.99Bb A3 (Pc) 9.98Aa 8.82Ba 8.50Aa Averages 9.40 9.10 8.11 8.33 8.19 9.10 Table 4: Effect of lignocellulolytic fungi and incubation time on cellulase enzyme activity (U mLG1) Treatments B1 (7 days) B2 (11 days) B3 (15 days) Averages A1 (Le) A2 (Po) A3 (Pc) Averages 4.83Bb 5.09Aa 5.03Aa 5.22Aa 4.84Ab 5.01Aa 4.90 5.11 4.87Bb 5.02Aa 4.80Ab 4.90 4.93 5.09 4.88 Table 5: Effect of lignocellulolytic fungi and incubation time on crude protein (%) Treatments B1 (7 days) B2 (11 days) B3 (15 days) Averages A1 (Le) 14.04Cc 20.11Ac 19.05Bc A2 (Po) 15.05Ca 21.86Aa 20.55Ba A3 (Pc) 14.51Bb 20.46Ab 20.40Ab Averages 14.53 20.81 20.00 17.73 19.15 18.46 lignocellulolytic fungi and incubation time had a strong significant effect on crude protein content of POS (p<0.01). Experiment 2 Comparison of nitrogen retention, crude fiber digestibility, metabolizable energy and amino acid profile between unfermented vs., fermented POS: Nitrogen retention, crude fiber digestion and metabolizable energy of POS under fermented and unfermented conditions are shown in Table 6. The amino acid profiles under both conditions are shown in Table 7. DISCUSSION The crude fiber, lignin and cellulose levels of POS were decreased after fermentation with Lentinus edodes, Pleorotus ostreatus and Phanerochaete chrysosporium, which was due to degradation by ligninase and cellulose enzyme produced in the fungi. The lowest crude fiber content of POS was Table 6: Nitrogen retention, crude fiber digestion and metabolizable energy of unfermented and fermented POS (A3B2) Compound and energy Unfermented Fermented Nitrogen retention (%) Crude fiber digestion (%) Metabolizable energy (kcal kgG1) 44.05 40.48 1540.09 69.02 60.93 2050.87 Table 7: Amino acid profile of fermented and unfermented POS (A3B2) Amino acids Unfermented Fermented ----------------------(DM%)----------------------- Aspartate 1.18 1.28 Glutamate 2.52 2.63 Serine 0.54 0.61 Histidine 0.49 0.57 Glycine 0.38 0.43 Threonine 0.51 0.58 Arginine 0.62 0.69 Alanine 0.98 1.14 Tyrosine 0.19 0.28 Methionine 0.50 0.58 Valine 0.43 0.52 Phenylalanine 0.52 0.59 Iso-leucine 0.39 0.47 Leucine 1.12 1.24 Lysine 1.06 1.21 Cysteine 0.51 0.61 Proline 0.27 0.35 observed in the fermentation reaction with Pleorotus ostreatus after 11 and 15 days of incubation (reduction of 31.04 and 30.64%, respectively). Bentil7 also found that fermentation of cocoa bean shells with Pleurotus ostreatus decreased the crude fiber content by 30.60 and 49.93% at the 2nd and 6th weeks of incubation, respectively. In addition, Akinfemi et al.4 also found that the crude fiber content of peanut husk fermented with Pleorotus ostreatus decreased from 26.20-16.9% (35.49% decrease) after 3 weeks of incubation. The lowest cellulose and lignin levels in POS were also observed after fermentation with Pleurotus ostreatus for 11 and 15 days (7.68 and 7.99% for cellulose and 13.22 and 13.39% for lignin, respectively). Cellulose degradation was likely facilitated by the hydrolytic enzymes (cellulases) secreted by the fungus during fermentation2,5,7. The cellulase enzyme activity in POS fermented with Pleorotus ostreatus also increased by the 11th and 15th days of incubation (5.22 and 5.02 U mLG1, respectively). Fungi are capable of producing a range of enzymes extracellularly and P. ostreatus is known to produce cellulases, hemicellulases and laccases that help degrade complex carbohydrates into soluble sugars9,4. Fermentation of POS with Lentinus edodes also exhibited reductions in crude fiber content at the 11th and 15th days of incubation by 26.76 and 26.57%, respectively. Elisashvili et al.8 previously reported that L. edodes IBB 123 reached maximal laccase activity on day 7 of cultivation but Pleurotus spp., achieved maximum acitivity after 10 days of cultivation. It was also observed a 26.76 and 26.57% reduction in crude fiber content in POS after 11 and 15 days of incubation, respectively, with Phanerochaete chrysosporium. Moreover, Nuraini and Nirwansyah13 reported that fermentation of cocoa pod with Phanerochaete chrysosporium and Monascus purpureus reduced the crude fiber, lignin and cellulose content. The highest crude protein levels in POS were observed after fermentation with Pleurotus ostreatus for 11 days. Fungi growth likely contributed to the increase in crude protein content in the POS, thus augmenting the crude protein content in the fermented product. The observed increase in protein levels could be due to bioconversion of some of the soluble carbohydrates in the colonized substrate into mycelia protein or Single Cell Protein (SCP) by the growing fungus5,6,13. The increase in crude protein content of POS after fermentation was associated with the process of protein enrichment, which was similar to the formation of a single cell protein and therefore could not be separated from proteins produced by the fungi. The increase in crude protein content of fermented POS was also related to the enzymes produced by the fungi (cellulase and ligninase). Nitrogen retention and metabolizable energy were enhanced when POS was fermented using Pleorotus ostreatus. In addition, all of the amino acids assessed were increased in fermented POS compared to unfermented POS. Importantly, the first limiting amino acids in a poultry diet (i.e., methionine and lysine) were slightly increased from 0.50, 1.06-0.58 and 1.21%, respectively, in the fermented POS compared to the unfermented POS. CONCLUSION Fermentation with Pleurotus ostreatus for 11 days was identified as the best condition for improving nutrient content (reducing crude fiber, lignin and cellulose levels as well as increasing crude protein and cellulose enzyme activity) and nutrient quality of POS. SIGNIFICANCE STATEMENT This study assessed the utilization of Palm Oil Sludge (POS) through fermentation using different lignocellulolytic fungi. The use of POS waste as an alternative poultry feed is still limited due to the crude fiber content and low protein content. This study found that fermenting POS using different lignocellulolytic fungi significantly reduced the crude fiber content. ACKNOWLEDGMENTS This experiment was supported by University of Andalas who provided funding for this study (SK No. 503/XIV/A/UNAND-2016, Hibah Penelitian Klaster Guru Besar), LPPM University of Andalas. REFERENCES 1. Nuraini, A. Djulardi and A. Trisna, 2016. Improving nutrient quality of palm oil sludge and palm kernel cake with lignocellulolityc fungi. Research Report, Hibah Kluster Guru Besar, University of Andalas, Indonesia. 2. Dashtban, M., H. Schraft, T.A. Syed and W. Qin, 2010. Fungal biodegradation and enzymatic modification of lignin. Int. J. Biochem. Mol. Biol., 1: 36-50. 3. Nuraini, S. and S.A. Latif, 2008. [Performances laying hen and egg quality feeding cassava waste fermented with Neurospora crassa]. J. Med. Peternakan, 31: 195-202. 4. Akinfemi, A., O.A. Adu and F. Doherty, 2010. Conversion of sorghum stover into animal feed with white-rot fungi: Pleurotus ostreatus and Pleurotus pulmonarius. Afr. J. Biotechnol., 9: 1706-1712. 5. Nuraini, A. Djulardi and M.E. 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