The Potential of Cellulolytic Yeast Pichia manshurica UNJCC Y-123, Saccharomyces cerevisiae UNCC Y-84, and Saccharomyces cerevisiae UNJCC Y-83 to Produce Cellulase Enzyme by Using Substrate Skin Delignification of Cocoa (Theobroma cocoa)
Authors: Sukmawati Dalia , Balqis Mutia , Adisyahputra Adisyahputra , Nurjayadi Muktiningsih , Annisyah Sheyla , Ichsanty Famira , Supiyani Atin , Widowati Retno , Ali El Enshasy Hesham , Sulistiani Sulistiani , Yusuf Dandy , Roswita Dewi Fera , Anshory Lutfi , Bimo Setiarto Raden Haryo 
Published: August 2023

Exploring the possibility of using agricultural waste as a substrate for the synthesis of cellulase enzymes for fuel and renewable energy is the main goal of this study, which is in line with the notions of sustainable development and environmental stewardship. The product of delignification of cocoa peel can be used as a substrate for cellulase enzymes producing by yeast isolated from Balinese palm wine. Cellulase enzymes made from yeast from Balinese palm wine can be produced using the delignified cocoa peel as a substrate. This study aims to analyze the cellulase enzyme activity of yeast from Bali's palm wine on delignified cocoa bark substrates. The tests carried out were delignification of cocoa bark (solvent variations: HCl, H2SO4 and NaOH; concentrations: 1, 1.5 and 2 %; biomass 1:15 and 1:20 (w/v)); screening of cellulolytic yeast from Balinese palm wine; molecular identification and morphological characteristics of yeast; and cellulase enzyme activity (variation of yeast isolates and fermentation time of 48, 72 and 96 h). The results of delignification of cocoa shells showed that the use of 1.5 % NaOH solvent with cocoa shell powder biomass of 1:15 (9.59 ± 0.11) significantly differed from the value of reducing sugar content. Based on the screening results of 6 yeast isolates, 3 yeast isolates with the highest cellulolytic index values were selected, namely UNJCC Y-83 (0.29 ± 0.01 mm), UNJCC Y-123 (0.24 ± 0.01 mm) and UNJCC Y-84 (0.23 ± 0.01 mm). For testing cellulase enzyme activity. S. cerevisiae UNJCC Y-83 (4.11 ± 0.41 U/mL); S. cerevisiae UNJCC Y-84 (4.11 ± 0.33 U/mL) and P. manshurica UNJCC Y-123 (4.06 ± 0.12 U/mL) at 96 h of fermentation had significantly different cellulase enzyme activity. The results of identification of yeast rDNA in the D1/D2 region with NL1/NL4 primers obtained the identity of Saccharomyces cerevisiae UNJCC Y-83; Saccharomyces cerevisiae UNJCC Y-84 (99.66 % homology); and Pichia manshurica (100 % homology). Theobroma cocoa rind can be used as a yeast substrate to produce cellulase enzymes for fuel and renewable energy.
HIGHLIGHTS

Screening of 6 yeast isolates, 3 yeast isolates with the highest cellulolytic index values were selected, namely UNJCC Y-83 (0.29 ± 0.01 mm), UNJCC Y-123 (0.24 ± 0.01 mm) and UNJCC Y-84 (0.23 ± 0.01 mm) for testing molecular identification and cellulase enzyme activity
The result of rDNA sequence analysis in the D1/D2 region showed that the yeast isolates UNJCC Y-83 and UNJCC Y-84 had a homology level of 99.66 % as Saccharomyces cerevisiae with a bootstrap value of 92 %. UNJCC Y-123 yeast isolate has a homology level of 100 % as Pichia manshurica with a bootstrap value of 100 %
cerevisiae UNJCC Y-83 (4.11 ± 0.41 U/mL); S. cerevisiae UNJCC Y-84 (4.11 ± 0.33 U/mL); and P. manshurica UNJCC Y-123 (4.06 ± 0.12 U/mL) at 96 h of fermentation had significantly different cellulase enzyme activity
Theobroma cocoa rind can be used as a yeast substrate to produce cellulase enzymes for fuel and renewable energy

GRAPHICAL ABSTRACT