Comparative Microbial Profile of Eco-enzymes Produced from Orange (Citrus sinensis) and Pineapple (Ananas comosus) Peels using Metabarcoding of the 16S and ITS Regions

Alifya Laras Ardana Pudijanto, Dr. Miftahul Ilmi, S.Si., M.Si.

2026 | Skripsi | BIOLOGI

Eco-enzyme (EE) merupakan zat organik antibakteri yang dimanfaatkan sebagai pembersih ramah lingkungan. Namun, meskipun studi mengenai penggunaan praktisnya telah banyak dilakukan, identifikasi keragaman mikroba melalui metabarcoding 16S dan ITS masih sangat terbatas. Penelitian ini bertujuan untuk mengarakterisasi biodiversitas organisme bakteri dan jamur pada EE yang berasal dari limbah kulit jeruk dan nanas menggunakan metode metabarcoding pada daerah 16S (untuk bakteri) dan ITS (untuk jamur). Sampel EE disiapkan dengan rasio 1:3:10 (molase:bahan organik:air), kemudian difermentasi selama tiga bulan sebelum dilakukan ekstraksi DNA dan amplifikasi PCR menggunakan primer 16S serta ITS. Hasil amplikon PCR divisualisasikan melalui elektroforesis gel, sementara sekuensing DNA dilakukan oleh Integrated Genomic Factory (IGF) menggunakan Oxford Nanopore Technology (ONT) dengan analisis data lanjutan di RStudio. Temuan penelitian menunjukkan bahwa profil bakteri pada EE nanas berhasil mengidentifikasi 10 spesies berbeda, sedangkan pada EE jeruk hanya ditemukan 9 spesies dalam kategori 10 besar. Spesies yang teridentifikasikan pada EE nanas adalah: Lentilactobacillus buchneri (166,579), Komagataeibacter saccharivorans (51,730), Lentilactobacillus parafarraginis (15,691), Lentilactobacillus parabuchneri (2550), Lentilactobacillus fungorum (1873), Lentilactobacillus farraginis (1135), Lentilactobacillus kefiri (1223), Lentilactobacillus parakefiri (550), Lentilactobacillus otakiensis (741), dan Ligilactobacillus aviaries (466). Sedangkan, profil jamur pada EE jeruk menunjukkan keberagaman spesies yang lebih tinggi dibandingkan dengan EE nanas yang hanya mengidentifikasi 4 spesies. Spesies yang ditemukan pada jamur adalah: Zygosaccharomyces bailii (126, 832), Wickerhamiella qilinensis (203), Starmerella stellata (70,513), Starmerella dacenportii (235), Pichia manshurica (339,000), Pichia kudriavzevii (180), Phaeococcomyces rothmanniae (1,774), dan Lamelloclavaria petersenii (351). Penelitian ini dilakukan sebanyak satu kali repetisi; Maka itu, studi lebih lanjut sangat direkomendasikan untuk mencakup analisis profil enzim dan pengujian aplikasi praktis guna memahami potensi penuh EE, serta perlunya eksplorasi lebih mendalam terhadap metabarcoding wilayah ITS pada produk-produk eco-enzyme.

Eco-enzyme (EE) is an organic, antibacterial substance used as an eco-friendly cleaner. Although there are many studies on the practical use of EE, the identification of microbial diversity through 16S and ITS metabarcoding remains limited. This study aims to characterise the biodiversity of bacterial and fungal organisms in orange (Citrus sinensis) and pineapple (Ananas comosus) peel-derived EE using metabarcoding of the 16S (bacteria) and ITS (fungi) regions. The EE samples were prepared using a 1:3:10 (molasses:organic waste:water) ratio and fermented for 3 months. DNA extraction and PCR amplification using the 16S and ITS primers were performed. PCR amplicons were visualised using gel electrophoresis. DNA sequencing was conducted by the Integrated Genomic Factory (IGF) using Oxford Nanopore Technology (ONT). Data analysis was performed in RStudio, with the provided data based solely on the ONT results. The results reveal that the bacterial profile of the pineapple EE identified 10 different species, while orange EE identified only 9 species within the top 10. The identified bacterial species in the pineapple EE are: Lentilactobacillus buchneri (166,579), Komagataeibacter saccharivorans (51,730), Lentilactobacillus parafarraginis (15,691), Lentilactobacillus parabuchneri (2550), Lentilactobacillus fungorum (1873), Lentilactobacillus farraginis (1135), Lentilactobacillus kefiri (1223), Lentilactobacillus parakefiri (550), Lentilactobacillus otakiensis (741), and Ligilactobacillus aviaries (466). On the other hand, the fungal profile of the orange EE identified more species than that of the pineapple EE, which identified only 4. The fungal species found in the orange EE include: Zygosaccharomyces bailii (126, 832), Wickerhamiella qilinensis (203), Starmerella stellata (70,513), Starmerella dacenportii (235), Pichia manshurica (339,000), Pichia kudriavzevii (180), Phaeococcomyces rothmanniae (1,774), and Lamelloclavaria petersenii (351). This study was carried out with a single repetition; Therefore, further studies are recommended to include a variety of tests, such as enzyme profiling and analysis, and conducting practical use to better understand the potential applications of EEs. Furthermore, metabarcoding of the ITS regions in EEs should be further explored.

Kata Kunci : 16S and ITS Regions, Eco-enzyme, Metabarcoding, Orange, Oxford Nanopore Technology, Pineapple