Sains Malaysiana 52(6)(2023): 1723-1735

http://doi.org/10.17576/jsm-2023-5206-09

Evaluation of Calcium Carbonate Precipitation by Bacillus spp. Isolated from Stingless Bee Produtcs

(Penilaian Kerpasan Kalsium Karbonat oleh Bacillus spp. yang Dipencilkan daripada Produk Lebah Kelulut)

NURUL ASYIQIN ADDENAN¹, MOHAMAD SYAZWAN NGALIMAT², RAJA NOOR ZALIHA RAJA ABD RAHMAN^1,2, RAKESH DONEPUDI³, NOOR AZLINE MOHD NASIR³, MOHD SALEH JAAFAR³ & SURIANA SABRI^1,2*

¹Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

²Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

³Department of Civil Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Received: 20 October 2022/Accepted: 12 June 2023

Abstract

Microbiologically Induced Calcium Carbonate Precipitation (MICCP) through urea hydrolysis is the most effective way to precipitate a high concentration of calcium carbonate (CaCO₃) within a short time. The MICCP process is used to remediate the micro-crack in the concrete. However, limited research has been conducted to determine CaCO₃ precipitation by bacteria, especially in Malaysia. Here, Bacillus spp. isolated from the Malaysian stingless bee products were evaluated for CaCO₃ precipitation. Bacillus spp. were selected for further study according to their ability to produce urease enzymes. The urease-positive Bacillus spp. were screened for CaCO₃ precipitation by culturing on both CaCO₃ precipitation agar and broth media. The survivability of the urease-positive Bacillus spp. in various temperatures, pH values, and NaCl concentrations were tested. Seven out of 11 Bacillus spp. were found as ureolytic bacteria. Among the ureolytic bacteria, bacteria belonging to the Bacillus subtilis species complex group showed the highest number of bacteria (36.4%) that are capable of precipitating CaCO₃. Bacillus stratosphericus PD6 and B. aryabhattai BD8 exhibited the largest CaCO₃ precipitation zones (15 mm). Bacillus stratosphericus PD6 also precipitated the highest amount of CaCO₃ (65 mg) and urease activity (0.197 U/mL). All the urease-positive Bacillus spp. were able to grow at 45 °C, pH (8 to 12), and 5% NaCl. Only B. subtilis BD3 can withstand high temperatures up to 55 °C and 15% NaCl concentration. In conclusion, Bacillus spp. isolated from stingless bee products showed the ability as the CaCO₃ precipitating bacteria; suggesting its potential application in self-healing concrete.

Keywords: Bacillus spp.; biomineralization; calcium carbonate precipitation; urease; ureolytic bacteria

Abstrak

Pemendakan Kalsium Karbonat Terinduksi Mikrobiologi (MICCP) melalui hidrolisis urea adalah cara paling berkesan untuk memendakkan kepekatan tinggi kalsium karbonat (CaCO₃) dalam masa yang singkat. Proses MICCP digunakan untuk memulihkan retakan mikro dalam konkrit. Walau bagaimanapun, kajian terhad telah dijalankan untuk menentukan pemendakan CaCO₃ oleh bakteria, terutamanya di Malaysia. Di sini, Bacillus spp. yang diasingkan daripada produk lebah Kelulut di Malaysia telah dinilai untuk pemendakan CaCO₃. Bacillus spp. telah dipilih untuk kajian lanjut mengikut kebolehan mereka menghasilkan enzim urease. Bacillus spp. yang positif urease telah disaring untuk pemendakan CaCO₃ dengan mengkultur pada media agar dan media kaldu pemendakan CaCO₃. Kebolehmandirian Bacillus spp. yang positif urease dalam pelbagai suhu, nilai pH dan kepekatan NaCl telah diuji. Tujuh daripada 11 Bacillus spp. ialah bakteria ureolitik. Antara bakteria ureolitik, bakteria yang tergolong dalam kumpulan kompleks spesies Bacillus subtilis menunjukkan bilangan bakteria tertinggi (36.4%) yang mampu memendakan CaCO₃. Bacillus stratosphericus PD6 dan B. aryabhattai BD8 didapati menunjukkan zon pemendakan CaCO₃ terbesar (15 mm). Bacillus stratosphericus PD6 juga memendakan jumlah tertinggi CaCO₃ (65 mg) dan aktiviti urease (0.197 U/mL). Semua Bacillus spp. yang positif urease mampu tumbuh pada suhu 45 °C, pH (8 hingga 12), dan 5% NaCl. Hanya B. subtilis BD3 boleh menahan suhu tinggi sehingga 55 °C dan kepekatan NaCl 15%. Kesimpulannya, Bacillus spp. diasingkan daripada produk lebah Kelulut menunjukkan keupayaan sebagai bakteria pemendakan CaCO₃; mencadangkan penggunaan potensinya dalam pemulihan diri konkrit.

Kata kunci: Bacillus spp.; bakteria ureolitik; biomineralisasi; pemendakan kalsium karbonat; urease

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*Corresponding author; email: suriana@upm.edu.my