The Nutritional Value Enhancement of Oil Palm Empty Fruit Bunches as Animal Feed Using the Fungus Coprinus Comatus, with Different Numbers of Inoculums and Incubation Times

The exploration of agricultural/plantation wastes is mainly conducted to address the limited availability of animal feed, including oil palm empty fruit branches. Furthermore, the Central Sulawesi Province has oil palm plantations covering 158,187 hectares with 316,781 tons of production [1]. The plantation produces biomass in empty fruit bunches, with about 3,680 kg fresh and 92.01% dry matter (3,386 kg dry matter). Oil palm empty fruit bunches (OPEFB) contain chemical compounds with the potential to be used as animal feed [2]. Ishola et al. [3] reported that OPEFB comprises 82.4% hemicellulose and 17.6% lignin. Also, the remaining fiber from palm fruit juice has the shape of a thread. Its chemical composition includes 4% and 36% crude protein and fiber (26% lignin), respectively [4].

However, the ability to digest and use these agricultural wastes has some limitations. These include their low lignin levels, which reduces their digestibility Furthermore, lignin as an antinutrient for livestock disturbs digestion and reduces the nutritional value of feed ingredients. Its content in oil palm empty bunches inhibits the conversion process of cellulose into simple sugars, which requires a process to release lignin [5]. One of the efforts to improve the digestibility of feed ingredients is through biological processing, which includes the use of lignocellulose-degrading fungi. Sarnklong et al. [6] in 2010, reported that the use of ligninolytic fungi including enzymes is a potential alternative with an environmentally friendly approach to change the nutritional value of rice straw. Several studies have also shown that white-rot fungi reduce lignin levels. Reddy et al. [7] reported that the use of Pleurotus sajor-caju in increasing the number of P.ostreatus and P. sajor-caju fungi will increase cellulolytic enzymes per dry weight of the material. Also, Belewu [8] used the Pleurotus sajor caju in sawdust media, and the remaining cotton was incubated for 60 days. The lignin content and the remaining cotton were reduced from 44.36% to 25.53% and 20% to 14.2%, respectively. Cellulose was found in small quantity, which is 31.99% to 30.89% for sawdust and 23.72% to 21.8% for the remaining cotton. Furthermore, Trametes Versicolor and Pleurotus ostreatus had the first and second-best activity in breaking down lignin holocellulose in rice straw [9]. According to Islamiyati et al. [10], corn straw treated with Trichoderma sp provides body weight gain. Also, crude protein and fiber fraction were better than those without treatment of Phanerocaeta crysosporium and Trichoderma sp. The highest was in the treatment of Trichoderma sp 5% with an incubation time of 2 weeks.

Mustabi [11] used a Coprinus comatus with an inoculum level of 5.0%, 7.5%, and 10.0% (w/w) at 15 and 30 days incubation period. The 10% inoculum level reduced lignin, crude fiber, and increase hemicellulose. Hermiati et al. [12] also found a weight loss of oil palm empty fruit bunches (OPEFB) fiber and an increase in the incubation time of OPF (oil palm fronds) until 3 weeks. However, there was no significant change in weight loss after. Fibers were found to be lower than OPF, which indicated that the mixed culture of P. crysosporium and T. Versicolor was more active in degrading OPF than OPEFB. Also, polysaccharides in the palm frond fibers are quickly degraded into glucose and xylose compared to empty fruit bunches (EFB).

This study, therefore, aims to determine the best white-rot fungi isolates from the five used in degrading lignin that produces low degradability of cellulose. Furthermore, the results were to obtain fungi that function optimally in degrading lignin in OPEFB media such that the great potential is useful as a forage substitute.

Selection of white rot fungi

The statistical analysis of the effect of five types of fungi on OPEFB lignin levels is shown in Table 1.

Table 1. Lignin and cellulose content of fermented OPEFB

Note: different subscribed values indicate a significant difference (p <0.05).

Based on Table 1, the five types of white-rot fungi applied to OPEFB have different abilities in degrading lignin at 20 days incubation period. However, the Trichoderma sp treatment was not statistically different from the control, it only decreased lignin levels on average. Furthermore, Coprinus comatus reduced lignin levels by 22.04%, which was the best in the degradation of OPEFB following Lentinus edodes (13.25%), Trametes Versicolor (9.86%), Pleurotus sajur-caju (9.62%), and Trichoderma sp (0.13). %). Jamila (2013) reported a 7.92% decrease in the application of Coprinus comatus on wood dust (11). D'Souza et al. [13] also revealed that white-rot fungi degrade all lignocellulosic material components, including lignin by secreting two types of ligninolytic enzymes, namely LiP and MnP. The lignin degradation of LiP is the main enzyme due to its ability to oxidize non-phenolic units.

The use of fungus for delignification has different results, which include the influence of several factors in the decrease in lignin levels, the type of fungus, and fermented material. Suprapti et al. [14], stated that the ability to decompose wood varies, this depends on its type and the nature of fungus work. Imsya et al. [15] showed that Phanerochaeta crysosporium produced a 47.79% reduction in lignin. Wang et al. [16], through a study on the Lenzites baeulinus, Trametes Orientalis, Trametes velutina, found that T.velutina provided a reduction in lignin levels of 58% after 12 weeks of processing. Also, white-rot fungi depolymerize and metabolize lignin to CO₂ and H₂O [17]. Srinivasan et al. [18] stated that Basydiomicetes white-rot generally synthesizes three kinds of enzymes, which are vital in the degradation of lignin, namely lignin peroxidase (LiPs), Manganese Peroxidase (MNPs), and Laccase. In general, treatment with the white-rot fungus reduces lignin levels due to the enzymes they produce.

The analysis showed differences in cellulose levels from the treatment of five types of fungi on OPEFB. Statistically, the highest cellulose content was Coprinus comatus (7.86%), which is different from the control, Pleurotus sajur-caju, and Trametes Versicolor. However, it is not significantly different from Lentinus edodes and Trichoderma sp. In this study, there were increased cellulose levels of about 16.67%, 13.13%, and 7.49%, in Coprinus Comatus, Lentinula edodes, Trichoderma sp., respectively. The most significant decrease occurred in Trametes Versicolor (14.05%) and Pleurotus sajur-caju (7.33%). Similarly, Donggeng [19] reported that the use of Colombian unidentified lipophilic hymenomycetes on palm fibers obtained higher cellulose levels than controls. Increased cellulose levels suggest that Coprinus comatus, Lentinula edodes, and Trichoderma sp. do not produce or use cellulose during fermentation. Furthermore, OPEFB cellulose was not degraded, however, a reasonable number of lignocellulose components has the potential to be broken down into lignin and cellulose. Therefore, a separate cellulose component from lignin needs to be added. Meanwhile, cellulose is thought to have originated from the fungal cell walls.

OPEFB lignin and cellulose levels on the different number of inoculum and incubation time

Based on the results, the levels of lignin produced after fermentation with the Coprinus comatus were different, as shown in Tables 2 and 3. Furthermore, the statistical tests on the number of inoculums and the incubation period of each treatment differed significantly. However, there was no interaction found between the two treatments as shown in Table 2.

Duncan's test on the levels of lignin levels for each treatment showed a very significant effect, however, there was no interaction. Table 2 shows that the different incubation periods and the number of inoculums are a result of the various lignin levels. The lowest lignin levels were inoculum of 0.5 ml (24.75) followed by control (26.37), an inoculum of 1.0 ml (27.25), and 0.75 ml (27.83). Furthermore, there was a decrease in lignin levels of 0.5ml inoculum by 6.17%. This has no significant difference compared to the control treatment, however, it differs significantly from 0.75 ml and 1.0 ml inoculums. Statistically, the incubation period between 20 and 30 days as well as 20 and 40 days was not significantly different. However, for the 40 days, the lowest lignin levels were obtained (24.40).

Table 2. Lignin levels at different number of inoculum and incubation periods

Table 3 shows that different amounts of inoculum had a very significant effect on cellulose levels. In addition, the best treatment was 0.5 ml, which yielded a cellulose content of 9.31, followed by 1.0 ml inoculum (8.33), control, and 0.75 ml inoculum (7.47) at different incubation periods (20, 30, and 40 days). It was also discovered that the cellulose level increased along with the incubation period. The increase in cellulose for the best incubation time was (40 days) was 18.19%. Furthermore, 0.5ml inoculum produced 9.31% cellulose of the dry matter and an increase of 18.54%.

Table 3. Cellulose levels at different number of inoculum and incubation periods

Note: different subscribed values indicate a significant difference (p <0.05)

The results showed that the fermentation with Coprinus comatus differs significantly in terms of the specific number of inoculums and incubation periods on the levels of lignin and cellulose of empty oil palm fruit bunches. However, there was no interaction between the two treatments. This study proved that the incubation period and the number of inoculums have a significant effect on delignification. Reddy et al. [7] reported that increasing the number of P. ostreatus and P. sajor caju fungi would increase cellulolytic enzymes per dry weight of the material. Furthermore, Belewu [8] used the Pleurotus sajor caju in sawdust and cotton residue with 60 days incubation period, the lignin content and the remaining cotton were reduced from 44.36% to 25.53%, and 20% to 14.2%, respectively. The reduction in cellulose also occurred at a lower rate from 31.99% to 30.89% for sawdust and 23.72 to 21.8% for the remaining cotton. This indicates that fungi are more likely to break down lignin than hemicellulose or cellulose.

Fadilah et al. [20] found that the most significant results with an 81.4% decrease rate occur at a 30days incubation period followed by 23% cellulose levels. Furthermore, Anita et al. [21] stated that the 4 weeks incubation period of a single culture P. ostreatus is more advantageous for bagasse pretreatment. This is due to the relatively high level of lignin degradation (17.95%). The degradation rate of cellulose was not too high (11.00%) compared to mixed culture. However, the optimum amount of inoculum at pretreatment T Versicolor was 15%, while P ostreatus was 10%. In addition, the white-rot fungi are the most efficient group due to their ability to degrade lignin compared to the brown and soft rot, which are limited to hemicellulose and cellulose degradation [22]. An increase in cellulose levels suggests that the Coprinus comatus uses only lignin as an energy source in fermentation. Therefore, there is an increase in cellulose, which emerges from the fungus itself. Donggeng [19] stated that the addition of cellulose comes from the cell walls of fungi. This is consistent with the statement of Kusumaningsih [23] that the components of the fungal cell wall include the chitin, beta-1,3 glucan, and the mannoprotein layer. 

This study illustrated that white-rot fungi such as Coprinus comatus tend to degrade lignin compared to cellulose. Furthermore, the decrease in lignin levels in empty oil palm fruit bunches is due to the active release of ligninolytic enzymes during fermentation. Coprinus comatus which was applied to empty fruit bunches contributed to the utilization of agro-industrial waste such as empty oil palm fruit bunches in reducing lignin content. Subsequently, it is utilized by livestock for growth. Also, Coprinus comatus, which is a part of the white-rot fungi group has the ability to degrade lignin as high as other fungi [11]. White rot mold increases the nutritional value of corn cobs used as animal feed and also depolymerizes oxidative by secreting several enzymes lignin peroxidase (LiP), manganese-peroxidase (MnP), and laccase [18, 24]. Furthermore, ligninolytic fungi use lignin as the only energy source and carbon for growth. as well as polysaccharides that are present in lignocellulosic substrates [25]. However, it is believed that the fermentation with Coprinus comatus does not secrete cellulase enzymes.

This study will contribute to the fermented empty oil palm fruit bunches by white-rot fungi as animal feed. The result showed that the higher the cellulose content of the ingredients, the better the animal feed. Furthermore, Haryanto [26] reported that cellulose and hemicellulose are polymers of glucose and xylose, respectively that are converted into free fatty acids by the activity of rumen microbial enzymes, which is a source of energy for livestock. According to Yanuartono et al. [27], the use of biological methods for utilizing fungi is able to improve the quality of feed from agricultural waste due to its ability to degrade lignin through the synthesis of ligninolytic enzymes. Furthermore, the use of fungi in biological methods is safe, environmentally friendly, easy to work with, and inexpensive.

These results were different from previous studies. The limitation is the lack of included laboratory analysis such as protein content, crude fiber, and other components that further explains the results.