Biodegradation of Lignin by White Rot Fungi (Pleurotus ostreatus) to Decrease the Fibre Components in the Palm Midrib

MATERIALS AND METHODS

The raw materials was used in this study include palm midrib and the white rot fungus Pleurotus ostreatus as an inoculant. The palm midrib was already chopped and dried and the fungus was cultured on PDA medium and then inoculated on immobilization medium in a 1:1:1 mixture of media sawdust, fine bran and corn flour.

Implementation of research: Pleurotus ostreatus that had been cultivated on the immobilization medium was inoculated using an inoculant dose that was further fermented in 1 kg of palm fronds in a cylindrical bag, hence, forth called a log-shaped bag. The duration of fermentation depended on the treatment (1, 2 or 3 months).

Parameters evaluated: The parameters evaluated in this study were components of the fibre fraction of palm fronds, neutral detergent fibre (NDF), acid detergent fibre (ADF), hemicellulose, cellulose and lignin. Each component was measured both before and after fermentation and the results were analysed using the method of Goering and Van Soest¹⁰.

Statistical analysis: This study used a completely randomized factorial design with three replications per combination. The treatments in this study were as follows: P. ostreatus inoculum (3, 5 and 7%) and long fermentation (1, 2 and 3 months). Further tests were conducted using Duncan's new multiple range test (DNMRT) at the 5% level of significance.

RESULTS AND DISCUSSION

NDF content: Statistical analysis showed that there was no interaction between the P. ostreatus dose and the fermentation time. However, a long duration of fermentation had a significant (p<0.05) effect on the NDF content of the palm fronds. Table 1 shows that before fermentation, the NDF content of the palm midrib was 77.349% and this value continuously decreased at months 1-3 during fermentation, with values of 74.960, 70.43 and 67.099%, respectively (Table 2).

Table 1:	Fibre fraction contents of palm midrib before bio-fermentation with P. ostreatus
*Laboratory analysis results Ruminant Basic Nutrients, Faculty of Animal Husbandry, Andalas University in Padang, based on 100% dry matter

Table 2:	NDF contents of palm midrib after bio-fermentation with P. ostreatus
The numbers in the same column followed by the same small letters are not significantly different at the 5% based on DNMRT DF analysis results after treatment

Table 3:	ADF contents of palm midrib after bio-fermentation with P. ostreatus
The numbers in the same column followed by the same small letters are not significantly different at the 5% level based on DNMRT DF analysis results after treatment

Table 4:	Hemicellulose contents of palm midrib after bio-fermentation with P. ostreatus
The numbers in the same column followed by the same small letters are not significantly different at the 5% level based on DNMRT DF analysis results after treatment

While the dose of P. ostreatus used was not statistically significant, the higher dose resulted in a reduction in the content of NDF.

The decline in the NDF content confirms that the lignocellulases produced by P. ostreatus can hydrolyse the lignin and hemicellulose bonds so effectively that they were decomposed. The decrease in the NDF content results in a decrease in the content of substrate that then affects the composition of the fibres.

In this study, the NDF content of the palm midrib decreased because the white rot fungus disrupted the lignocellulose bonds during fermentation. The disruption of those bonds was also accompanied by the development of microbial activity¹¹.

ADF content: Statistical analysis showed no significant interaction between the dose of P. ostreatus and the fermentation time. However, long-term fermentation significantly decreased (p<0.05) the ADF content of palm midrib. Before fermentation, the content of ADF in the palm midribs was 66.193% and over the 3 months period, that value decreased to 57.296% (Table 3). The amount of fungus applied was non-significant, but on average, the dose of 0.7% resulted in a lower ADF content due to P. ostreatus activity.

The decrease in the ADF content in the palm midrib is due to the P. ostreatus enzyme that can disrupt the cellulose bonds. Thus, optimization of the fermentation process, resulting in the optimal production of cellulase, caused the disruption of previously strong bonds.

Studies that use different microbes with the same substrate can reduce the ADF content of the untreated substrate by 50.32%. In this study, fermentation for 30 days decreased the ADF content by 42.69%. The success of fermentation is strongly influenced by both the dose of inoculum and the fermentation time¹¹.

Hemicellulose content: Statistical analysis showed no significant interactions between the dose of P. ostreatus and the fermentation time in terms of the hemicellulose content. However, the duration of fermentation significantly affected (p<0.05) the hemicellulose content of the palm midribs. The hemicellulose content of the palm midribs before fermentation was 11.156% and it decreased to 9.803% after 3 months of fermentation with P. ostreatus (Table 4). Although the dose was not statistically significant, on average, it appears that the higher doses of P. ostreatus resulted in lower levels of hemicellulose.

The decrease in the hemicellulose content was correlated with the decrease in the lignin content over the course of the fermentation. Since the isolates of P. ostreatus degrade cellulose, the hemicelluloses are also degraded. The degradation of the lignin results in access to the cellulose and hemicelluloses, enabling alteration of their bonds. Reshuffling the components of cellulose will produce simple sugars. Degradation of cellulose limits the production of most enzymes that degrade hemicellulose. This process is performed by the white rot fungus.

Table 5:	Cellulose contents of palm midrib after bio-fermentation with P. ostreatus
The values in this table show no significant differences at the 5% level based on the F-test

Table 6:	Lignin contents of palm midrib after bio-fermentation with P. ostreatus
The numbers in the same column followed by the same small letters are not significantly different at the 5% level based on DNMRT DF analysis results after treatment

A further explanation is that during the degradation of hemicellulose, the hemicelluloses were attacked first by endoenzymes (mannanase and xylanase), which resulted in the intensive hydrolysis of short bonds to simple sugars by various enzymes (mannosidase, xylosidase and glucosidase). As with the cellulases, the production of simple sugars limits the production of most of the enzymes of the white rot fungi that degrade hemicellulose. Cellulose is thought to be the source of the carbon that accelerates the formation of the fungal enzymes that degrade hemicellulose¹².

Cellulose content: Statistical analysis showed that the cellulose content of the palm midribs was not significantly affected by the P. ostreatus dose, the fermentation time or the interaction of those factors, although there were fluctuations in the average cellulose content. Before fermentation, the palm midribs contained 37.196% cellulose, this decreased to 31.743, 31.356 and 31.456% after 1, 2 and 3 months, respectively (Table 5). Although, the dose of P. ostreatus was not statistically significant, on average, it appears that the higher P. ostreatus dose resulted in a higher content of cellulose.

The cellulose content has a tendency to decrease with a longer incubation period because the fungus continues to degrade cellulose to generate substrates for its growth. Pleurotus ostreatus has been shown to degrade cellulose to compounds that it can more effectively use as substrates for growth¹³.

The cellulose degradation peaked when P. ostreatus formed fruiting bodies, because at that time, different environmental conditions induce changes in fungal metabolism. Decreased levels of cellulase due to the increasing thickening of the mycelia can restructure the cellulose¹⁴.

Long incubation times increase the concentration of the mycelium in the substrate. The optimal concentration of the mycelium produces cellulase more effectively, decreasing the content of cellulose. When the mycelia have grown thick and evenly cover the entire surface of the substrate, the concentration of the enzyme is high. These results in the degradation of the cell wall components, especially those of the fibres that then have their cellulose degraded¹⁵.

Lignin content: Statistical analysis showed no significant interaction effect of the dose of P. ostreatus and the fermentation time on the lignin content. However, the fermentation time significantly (p<0.05) decreased the lignin content of the palm midribs. The lignin content of the palm midribs was 22.936% before fermentation but decreased to 17.458% after 3 months (Table 6). Although, the doses of P. ostreatus did not differ significantly, on average, it appears that the higher doses of P. ostreatus resulted in lower amounts of lignin present.

The levels of lignin in the bag log with P. ostreatus decreased because the fungus used lignin for growth. After 2 months of incubation with P. ostreatus, the fermentation has begun and the first harvest has occurred. This observation agrees with a previous report¹⁶ which indicated that P. ostreatus belongs to a group of microorganisms that can thoroughly break down lignin to carbon dioxide and water. This group of fungi produces a family of enzymes that are directly involved in the restructuring of lignin, including the phenoloxidase laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP). Pleurotus ostreatus includes spoilage white rot fungi that can degrade lignin and improve the digestibility of dry matter and organic ingredients such as rice straw¹⁷.

Reduced lignin levels lead to the increased availability of nutrients such as dry matter and organic matter, allowing the palm midribs to be used as feed for ruminants. However, the bag log composed of palm midrib has a low nutrient content. Thus, more research is needed to produce technological breakthroughs that will increase its nutritional value.

CONCLUSION

The results showed that the dosage of P. ostreatus and the fermentation time did not interact significantly. However, the fermentation time of 3 months significantly (p<0.05) decreased the contents of NDF, ADF, hemicellulose and lignin palm midribs. It can therefore be used to improve the quality of animal feed ingredients, especially feed used for ruminants.

ACKNOWLEDGMENT

This study was funded by the Directorate General of Higher Education of Indonesia (DIKTI), Ministry of National Education, Ref. No. 48 / KONTRAK-PENELITIAN / 010.KM / 2016 (Dipa Kopertis Wil X).