Isolasi dan Identifikasi Bacillus Penghasil Enzim Hidrolitik dari Sampah Padat
Abstract
Berbagai upaya untuk mengatasi persoalan sampah telah dilakukan di antaranya melalui pengomposan, biodigester, biodrying, hingga pembuatan pelet atau pakan ternak. Namun demikian, sejumlah upaya tersebut seringkali masih menemui kendala di antaranya waktu yang diperlukan cukup lama, biaya operasional yang tinggi, dan kualitas produk yang dirasa masih rendah. Bioaugmentasi dan biostimulasi mikrob potensial dekomposer menjadi alternatif solusi yang efisien dan efektif, serta aman bagi lingkungan jika diaplikasikan. Oleh karenanya pencarian mikrob potensial dekomposer hingga kini masih terus dilakukan salah satunya dari kelompok Bacillus. Penelitian ini bertujuan mengisolasi dan mengidentifikasi Bacillus yang mampu menghasilkan enzim hidrolitik (selulolitik, amilolitik, dan proteolitik) dari sampah padat.
Penelitian diawali oleh tahapan isolasi Bacillus yang dilakukan dengan memberi perlakuan heat-shock (dipanaskan selama 80 °C selama 10 menit lalu didinginkan) pada sampel, kemudian dibuat pengenceran serial, dilanjutkan dengan pemurnian, dan diuji potensi hemolisisnya pada media agar-agar darah, sedangkan uji potensi selulolitik, amilolitik, dan proteolitik dilakukan dengan menumbuhkan isolat pada media agar-agar carboxyl methyl cellulose (CMC), pati, dan skim. Isolat dengan ketiga kemampuan yang baik berdasarkan indeks hidrolisis diuji antagonis. Isolat yang tidak saling antagonis diidentifikasi berdasarkan fenotipe (morfologi koloni dan pewarnaan Gram) dan genotipe (gen 16S rRNA). Karakterisasi isolat berdasarkan aktivitas ketiga enzim hidrolitik (amilase, protease, dan selulase) yang dimiliki dilakukan terhadap salah satu isolat terpilih secara kuantitatif.
Hasil seleksi menunjukkan empat belas isolat diduga Bacillus sp. yang diketahui memiliki kemampuan selulolitik, amilolitik dan proteolitik. Empat isolat terpilih berdasarkan indeks hidrolitik terbaik, di antaranya yakni isolat 2.5 dan 2.7 memiliki kemiripan dengan Bacillus stercoris, sedangkan dua isolat lainnya yakni 5,5 dan 5,9R masing-masing memiliki kemiripan dengan Calidifontibacillus erzurumensis dan Bacillus amyloliquefaciens. Aktivitas spesifik enzim amilase dari isolat 2.7 sebagai isolat terpillih lebih tinggi, dibandingkan protease dan selulase yakni 3,522 Unit/mg pada jam ke-27. Karakterisasi enzim diperlukan untuk optimasi dengan tujuan produksi dan pemanfaatan enzim secara berkelanjutan. Pengujian isolat dalam mendegradasi sampah tetap perlu dilakukan begitupula analisis whole genome sequencing (WGS) untuk memastikan hasil identifikasi yang lebih akurat dan deteksi gen hidrolitik yang dimiliki. Various efforts have been made to overcome the waste problem, including composting, biodigesters, biodrying, and making pellets or animal feed. However, some of these efforts still need help, including the time required, which is quite long, high operational costs, and the product quality still needs to be considered high. Bioaugmentation and biostimulation of potential decomposer microbes are alternative solutions that are efficient, effective, and safe for the environment. Therefore, searching for potential decomposer microbes, one of which is from the Bacillus group, is ongoing. This study aims to isolate and identify Bacillus that can produce hydrolytic enzymes (cellulolytic, amylolytic, and proteolytic) from solid waste.
The study began with the Bacillus isolation stage, which was carried out by giving heat-shock treatment (heated at 80 °C for 10 minutes, then cooled) to the sample. Serial dilutions were made, continued with purification, and tested for hemolysis potential on blood agar media, while cellulolytic, amylolytic, and proteolytic potential tests were carried out by growing the isolate on carboxyl methyl cellulose (CMC), starch, and skim agar media. Isolates with all three good abilities based on the hydrolysis index were tested antagonistic. Isolates that were not antagonistic to each other were identified based on phenotype (colony morphology and Gram staining) and genotype (16S rRNA gene). Isolate characterization based on the activity of the three hydrolytic enzymes (amylase, protease, and cellulase) they had was carried out on one of the selected isolates quantitatively.
The selection results showed fourteen isolates suspected of being Bacillus sp. known to have cellulolytic, amylolytic, and proteolytic abilities. Four isolates were selected based on the best hydrolytic index, including isolates 2.5 and 2.7, similar to Bacillus stercoris. In comparison, the other two isolates, namely 5.5 and 5.9R, are similar to Calidifontibacillus erzurumensis and Bacillus amyloliquefaciens, respectively. The specific activity of the amylase enzyme from isolate 2.7 as the selected isolate was higher, compared to protease and cellulase, namely 3.522 Units/mg at the 27th hour. Enzyme characterization is needed for optimization for sustainable enzyme production and utilization. Testing of isolates in degrading waste still needs to be done, as well as whole genome sequencing (WGS) analysis to ensure more accurate identification results and detection of the hydrolytic genes they have.