Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil
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Abstract
This study reports the biodiversity of thermophilic cellulolytic bacterial strains that present in the north Malaysian mangrove ecosystem. Soil samples were collected at the four most northern state of Malaysia (Perak, Pulau Pinang, Kedah and Perlis). The samples obtained were first enriched in nutrient broth at 45°C and 55°C prior culturing in the carboxymethylcellulose (CMC) agar medium. Repeated streaking was performed on the CMC agar to obtain a pure culture of each isolate prior subjecting it to hydrolysis capacity testing. The isolates that showing the cellulolytic zone (halozone) were sent for 16S rRNA sequencing. Total seven isolates (two from Perak, three from Kedah, another two were from Perlis and Penang each) showed halozone. The isolate (KFX-40) from Kedah exhibited highest halozone of 3.42 ± 0.58, meanwhile, the one obtained from Perak (AFZ- 0) showed the lowest hydrolysis capacity (2.61 ± 0.10). Based on 16S rRNA sequencing results, 5 isolates (AFY-40, AFZ-0, KFX-40, RFY-20, and PFX-40) were determined to be Anoxybacillus sp. The other two isolates were identified as Bacillus subtilis (KFY-40) and Paenibacillus dendritiformis (KFX-0). Based on growth curve, doubling time of Anoxybacillus sp. UniMAP-KB06 was calculated to be 32.3 min. Optimal cellulose hydrolysis temperature and pH of this strain were determined to be 55°C and 6.0 respectively. Addition of Mg2+ and Ca2+ were found to enhance the cellulase activity while Fe3+ acted as an enzyme inhibitor.
Kajian ini melaporkan biodiversiti strain bakteria selulosa termofilik yang terdapat di ekosistem bakau utara Malaysia. Sampel tanah dikumpulkan dari empat negeri paling utara di Malaysia (Perak, Pulau Pinang, Kedah dan Perlis). Sampel yang diperoleh diperkaya terlebih dahulu dengan brot nutrien pada suhu 45°C dan 55°C sebelum dibiak dalam medium agar karboksimetilselulosa (CMC). Teknik corek digunakan untuk mendapatkan strain tulen setiap bakteria dan bakteria tersebut diuji untuk kapasiti hidrolisis. Strain bakteria yang menunjukkan zon selulosa (halozone) telah dihantar untuk penjujukan 16S rRNA. Secara keseluruhan, tujuh strain bakteria (dua dari Perak, tiga dari Kedah, satu dari Pulau Pinang dan satu lagi dari Perlis) menunjukkan zon selulosa. KFX-40 (strain dari Kedah) menunjukkan zon selulosa yang terbesar sebanyak 3.42 ± 0.58, manakala, AFZ-0 (strain dari Perak) menunjukkan zon selulosa yang terkecil sebanyak 2.61 ± 0.10. Hasil penjujukan 16S rRNA menunjukkan bahawa lima (AFY-40, AFZ-0, KFX-40, RFY-20, and PFX-40) daripada tujuh strain yang diperoleh adalah Anoxybacillus sp. Sementara itu, dua strain yang lain adalah Bacillus subtilis (KFY-40) dan Paenibacillus dendritiformis (KFX-0). Analisis keluk pertumbuhan menunjukkan masa ganda dua bagi bakteria Anoxybacillus sp. UniMAP-KB06 adalah 32.3 minit. Suhu dan pH optimum untuk strain tersebut adalah 55°C dan 6.0. Penambahan ion Mg2+ dan Ca2+ meningkatkan aktiviti selulase, manakala, Fe3+ membantut aktiviti selulase.
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