Effects of Calcium, Phosphorus and Manganese Supplementation During Oil Palm Frond Fermentation by Phanerochaete chrysosporium on Laccase Activity and in vitro Digestibility

MATERIALS AND METHODS

Oil palm frond fermentation: Oil Palm Frond (OPF) substrates used in this study were cut, dried and finely milled. Calcium (Ca) was obtained from CaSO₄, P was obtained from KHPO₄ and Mn was obtained from MnSO₄.H₂O. Phanerochaete chrysosporium was maintained on Potato Dextrose Agar (PDA) slants at 4°C, transferred to PDA plates at 37°C for 6 days and subsequently grown on OPFs mixed with rice bran. The ratio of the leaves and stems of OPFs was 1:1, the mineral solution from Brook et al.¹⁸ was then added. The fermentation process was initiated by adding water to the OPFs until the water level reached 70%, either Ca, P or Mn was added, depending on the treatment. Observations were made every 120 h for 20 days. After 20 days, samples were taken for proximate analysis and fiber fraction determination. The proximate components were determined as described by AOAC¹⁹. The predominant fiber residues (hemicellulose, cellulose and lignin) were determined according to the method of Van Soest et al.²⁰.

Laccase activity: Enzyme extraction was initiated with the centrifuge method²¹; about 1 g of the sample was mixed with 50 mM phosphate buffer (pH 6.5) at a ratio of 10/1 (v/w) and stirred at a speed of 150 rpm for 1 h. The samples were then centrifuged at a speed of 7000 g for 20 min. The resulting supernatant (liquid) was separated and used for analysis of enzyme activity.

Laccase enzyme activity was determined by the oxidation of 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). The reaction mixture consisted of 0.1 mL ABTS, acetate buffer (0.5 mL, pH 5) at a concentration of 0.5 M and 0.4 mL of filtrate enzymes, the total volume was 1 mL. The cuvettes were shaken to ensure that all the ingredients were mixed. The enzyme activity reaction was carried out at a temperature of 20±1°C. Absorbance was measured at 0 and 30 min at a wavelength of 420 nm using a spectrophotometer. Enzyme activity can be calculated by following formula:

where, ε max is molar absorptivity ABTS (36000 M^–1cm^–1), Vol. Is volume and d is wave length.

In vitro digestibility assay: In vitro digestibility was analyzed by Tilley and Terry²². Fistula cow rumen fluid was diluted using McDougal Buffer (1:4) and dispensed into a 1 g substrate-prepared incubation tube, which was purged with CO₂ to maintain an aerobic condition. The tubes were incubated in a water bath at 39°C for 24 h. After fermentation, the erlenmeyer tube containing the sample was inserted into ice water to stop the fermentation. All samples were then centrifuged at 1,200 rpm for 15 min. The pH, NH₃-N and total VFA of the supernatants were then recorded. The NH₃-N concentration was measured using the micro-diffusion conway method and the total VFA concentration was measured using the steam distillation method. The previously incubated samples were vacuum filtered (Whatman No. 41) and dried at 60°C in an oven. The dried samples were used to analyze the NDF, ADF and cellulose in the in vitro digestibility assay.

Experimental design and statistical analysis: The study was carried out using a randomized block design (3×5) with five replications (Addition of Ca, P and Mn as treatments). Differences between treatment means were analyzed using Duncan's multiple range test.

The following treatments were carried out:

T1=Ca 2000 ppm+P 1000 ppm+Mn 150 ppm
T2=Ca 2000 ppm+P 1500 ppm+Mn 150 ppm
T3=Ca 2000 ppm+P 2000 ppm+Mn 150 ppm

RESULTS AND DISCUSSION

The analysis of the data for the laccase activity and digestibility of NDF, ADF, cellulose and hemicellulose due to the addition of Ca, P and Mn in the fermentation process of OPFs by Phanerochaete chrysosporium is presented in Table 1.

Laccase enzyme is one of the ligninase enzymes produced by Phanerochayte chrisosporium. Treatment T3 shows the significant effect (p<0.05) against laccase enzyme activity, which indicates that the treatment is able to improve the activity of the laccase enzyme. Laccase enzyme activity was lowest for T1 (1.18 U mL^–1).

Table 1:	Laccase activity and digestibility of NDF, ADF, cellulose and hemicellulose due to the addition of minerals in the fermentation of OPFs by Phanerochaete chrysosporium
Means in the same row with different a,bletters are significant at p<0.05, NDF: Neutral detergent fiber, ADF: Acid detergent fiber

It is suspected that the T1 treatment combination was not optimal for the mineral to extend to the fungi mycelia and was not optimal for the growth of molds; therefore, the resulting laccase enzyme activity was low. With increasing doses of P, the enzyme activity also increased due to laccase, in which P plays an important role in the metabolism of molds²³. Laccase activity was highest in the T3 treatment (2.88 U mL^–1). The high activity of laccase in T3 indicates that fungus grows better and the fungi mycelia are longer; therefore, more enzymes are secreted. The high activity of laccase indicates that the degradation of lignocellulose and lignohemicellulose also increased, making it easier to utilize the rumen microbial cellulose and hemicellulose, which in turn increases digestibility. The laccase activity obtained in this study is higher than in previous study, in which Apriyani²⁴ was obtaining laccase activity of 2.002 U mL^–1 in palm fronds fermented by Pleurotus ostreatus. However, the present results were lower than previous study which reported by Perdana²⁵, in which the laccase activity was 3.1 U mL^–1 in fermented bagasse by Ganoderma lucidum.

The addition of mineral Ca, P and Mn significantly (p<0.05) affects the NDF digestibility of OPF fermentation. The NDF digestibility was lowest at 65.32% in the T1 treatment. Low NDF digestibility and the resulting mycelium growth of mold and production of enzymes for degrading the ligninolytic substrate was not optimal, which was evident from the low laccase enzyme activity (1:18 U mL^–1). The lower enzyme activity was a result of the limit of the lignin degradation process. The lignification process limits the microbes that break down cellulose and hemicellulose as a source of energy for growth it²⁶. The combination in the T3 mineral treatment produces the highest NDF digestibility. The increased digestibility of NDFs in the treatment with T3 was due to the combination of these minerals triggering an extension of mycelial fungi and ligninolytic enzymes with the resulting mold Phanerochayte chrysosporium. This optimized condition can loosen the bonds of lignocellulose and lignohemicellulose, which causes the cellulose and hemicellulose to be utilized by microbes. This is supported by the study of Febrina et al.¹², in which Phanerochayte chrysosporium could decompose lignin in the substrate, penetrating the cellulose and hemicellulose attached to the lignin matrix. Release of the lignocellulosic bonds and lignohemicellulose result in cellulose and hemicellulose that can be used by fungi to grow and develop so that the process of fermentation in the rumen can better function. This is evident by the high digestibility of NDFs in this treatment. The biodegradation of lignin is a key process for lignocellulosic waste composting, in which the increased use of carbon increases the activity of micro-organisms that cause the enzyme activity of ligninolytics, thus increasing the degradation of lignin²⁷. Increased doses of P also greatly aid the growth of microbes because P is instrumental in the process of glycolysis. Glycolysis produces chemical energy in the form of ATP compounds. Compounds of ATP (AP~P~P) contain a high-energy phosphate group that plays an important role in the metabolic process of phosphorylation via the radical transfer of phosphate groups. The cell also contains phosphoproteins, phospholipids and nucleic acids. The NDF digestibility obtained in this study is higher than the 66.429% reported by Febrina et al.¹² that only added Ca and Mn without P, in the fermentation of OPFs by Phanerochayte chrysosporium.

The treatments had a significant effect (p<0.05) on the ADF digestibility of OPFs. Treatment T1 provided the lowest ADF digestibility at 48.50%. The low activity of the laccase enzyme in this treatment shows that it is not optimal for fungi to degrade lignocellulose and lignohemicellulose, causing difficulty for the rumen microbes to degrade the feed to the low digestibility of ADF. The ADF digestibility was highest at 59.97% in the T3 treatment. The high digestibility in this treatment is associated with the higher activity of the enzyme, with optimal conditions for laccase in the lignin degradation process. The optimal level of lignin degradation causes the release of lignin from cellulose and hemicellulose. Sanchhez²⁸ noted that Phanerochayte chrysosporium is a white rot fungi that can degrade the lignocellulose components simultaneously. The ADF digestibility value in this treatment is higher than the results obtained by Pulungan²⁹, in which fermented oil palm trunks using Phanerochayte chrysosporium with 0.3 g urea achieved 36.50%.

Treatment T3 significantly affects (p<0.05) the digestibility of cellulose and hemicellulose. The treatment T3 causes the release of the lignocellulosic bonds and lignohemicellulose that cellulose and hemicellulose can use as an energy source with the rumen microbes that ultimately improve rumen microbial populations. The increasing population and cellulolytic microbe activity increased NDF, ADF and cellulose digestibility³⁰. The addition of minerals into the substrate is still within normal levels, as seen from the digestibility of cellulose. Dias et al.³¹ reported Ca, P, Mg and Mo at normal levels in the rumen microbes increase activity in digesting cellulose fibers. The digestibility of cellulose and hemicellulose was lowest at 57.05 and 72.57% in treatment T1, low digestibility showed low activity and the number of rumen microbes in the remodeled lignocellulose and lignohemicellulose resulted in still bound lignin with cellulose and hemicellulose. This is in accordance with Zain et al.³² and Genther and Hansen³³, who noted that the digestibility of feed in ruminants was closely related to the amount and activity of rumen microbes. The digestibility of cellulose and hemicellulose increases with increasing laccase activity. High enzyme activity would increase the level of degradation of lignin so that the cellulose and hemicellulose were more widely used by the rumen microbes. The combination of minerals in T3 optimized the digestibility of cellulose and hemicellulose. Mineral supplementation was sufficient and appropriate microbial activity was indispensable in fulfilling normal functions to stimulate the proliferation and activity of fermentation³². The digestibility rates of cellulose and hemicellulose in this study were higher than in Febrina et al.¹⁷, who added P, S and Mg in the fermentation of OPFs by Phanerochayte chrysosporium, at 55.97 and 78.63%. Hemicellulose digestibility is higher than the digestibility of cellulose because hemicellulose has a smaller molecular weight than cellulose due to the short chain branch that consists of different sugars, thus making it easier to be hydrolyzed³⁴.

CONCLUSION

Supplementation of fermented OPFs by Phanerochayte chrysosporium with treatments of Ca (2000 ppm), P (2000 ppm) and Mn (150 ppm) (T3) provided the highest laccase activity and in vitro digestibility.

ACKNOWLEDGMENTS

This study was funded by the Andalas University Professor Grant 2016 and part of the Ph.D. dissertation of Roni Pazla in Animal Nutrition, Faculty of Animal Science, Andalas University.