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

Diserahkan: 20 Oktober 2022/Diterima: 12 Jun 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

RUJUKAN

Aburawi, M.M. & Swamy, R.N. 2008. Influence of salt weathering on the properties of concrete. The Arabian Journal for Science and Engineering 33(1B): 105-116.

Alazhari, M., Sharma, T., Heath, A., Cooper, R. & Paine, K. 2018. Application of expanded perlite encapsulated bacteria and growth media for self-healing concrete. Construction and Building Materials 160: 610-619.

Algaifi, H.A., Bakar, S.A., Sam, A.R.M., Abidin, A.R.Z., Shahir, S. & Altowayti, W.A.H. 2018. Numerical modeling for crack self-healing concrete by microbial calcium carbonate. Construction and Building Materials 189: 816-824.

Algaifi, H.A., Bakar, S.A., Sam, A.R.M., Ismail, M., Abidin, A.R.Z., Shahir, S. & Altowayti, W.A.H. 2020. Insight into the role of microbial calcium carbonate and the factors involved in self-healing concrete. Construction and Building Materials 254: 119258.

Alshalif, A.F., Juki, M.I., Othman, N. & Al-gheethi, A.A. 2019. Improvement of mechanical properties of bio-concrete using Enterococcus faecalis and Bacillus cereus. Environmental Engineering Research 24(4): 630-637.

Amran, M., Onaizi, A.M., Fediuk, R., Vatin, N.I., Muhammad Rashid, R.S., Abdelgader, H. & Ozbakkaloglu, T. 2022. Self-healing concrete as a prospective construction material: A review. Materials 15(9): 3214.

Andalib, R., Abd Majid, M.Z., Hussin, M.W., Ponraj, M., Keyvanfar, A., Mirza, J. & Lee, H. 2016. Optimum concentration of Bacillus megaterium for strengthening structural concrete. Construction and Building Materials 118: 180-193.

Azmi, M.S., Lin, T.S., Tajaruddin, H.A., Mohd Zaini Makhtar, M. & Daud, Z. 2018. Characterisation of calcium carbonate formed by Bacillus sphaericus via fermentation of urea. International Journal of Integrated Engineering: Special Issue 10(9): 119-124.

Bachmeier, K.L., Williams, A.E., Warmington, J.R. & Bang, S.S. 2002. Urease activity in microbiologically-induced calcite precipitation. Journal of Biotechnology 93(2): 171-181.

Behnood, A., Tittelboom, K.V. & Belie, N.D. 2016. Methods for measuring pH in concrete: a review. Construction and Building Materials 105: 176-188.

Binici, H., Aksogan, O., Gorur, E.B., Kaplan, H. & Bodur, M.N. 2008. Performance of ground blast furnace slag and ground basaltic pumice concrete against seawater attack. Construction and Building Materials 22: 1515-1526.

Braissant, O., Decho, A.W., Dupraz, C., Glunk, C., Przekop, K.M. & Visscher, P.T. 2007. Exopolymeric substances of sulfate-reducing bacteria: Interactions with calcium at alkaline pH and implication for formation of carbonate minerals. Geobiology 5(4): 401-411.

Chahal, N., Rajor, A. & Siddique, R. 2011. Calcium carbonate precipitation by different bacterial strains. African Journal of Biotechnology 10(42): 8359-8372.

Chen, H., Peng, C., Tang, C. & Chen, Y. 2019. Self-healing concrete by biological substrate. Materials 12: 4099.

Damayanti, N.W., Sahlan, M., Lischer, K., Hermansyah, H., Kusumoputro, B. & Pratami, D.K. 2019. Comparison of original honey (Apis sp. and Tetragonula sp.) and fake honey compounds in Indonesia using gas chromatography-mass spectrometry (GC-MS). AIP Conference Proceedings 2193: 030018.

De Muynck, W., De Belie, N. & Verstraete, W. 2010. Microbial carbonate precipitation in construction materials: A review. Ecological Engineering Journal 36: 118-136.

Deng, W. & Wang, Y. 2018. Investigating the factors affecting the properties of coral sand treated with microbially induced calcite precipitation. Advances in Civil Engineering 2018: 1-7.

Dhami, N.K., Reddy, M.S. & Mukherjee, A. 2013. Biomineralization of calcium carbonates and their engineered applications: A review. Frontiers in Microbiology 4: 314.

Dhami, N.K., Reddy, M.S. & Mukherjee, A. 2014. Application of calcifying bacteria for remediation of stones and cultural heritages. Frontiers in Microbiology 5: 304.

Dupraz, C., Visscher, P.T., Baumgartner, L.K. & Reid, R.P. 2004. Microbe-mineral interactions: Early carbonate precipitation in a hypersaline lake (Eleuthera Island, Bahamas). Sedimentology 51(4): 745-765.

Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39(4): 783-791.

Fujita, Y., Ferris, F.G., Lawson, R.D., Colwell, F.S. & Smith, R.W. 2000. Subscribed content calcium carbonate precipitation by ureolytic subsurface bacteria. Geomicrobiology Journal 17(4): 305-318.

Golovkina, D.A., Zhurishkina, E.V., Ivanova, L.A., Baranchikov, A.E., Sokolov, A.Y., Bobrov, K.S., Masharsky, A.E., Tsvigun, N.V., Kopitsa, G.P. & Kulminskaya, A.A. 2020. Calcifying bacteria flexibility in induction of CaCO₃ mineralization. Life 10(12): 317.

Hammad, I.A., Talkhan, F.N. & Zoheir, A.E. 2013. Urease activity and induction of calcium carbonate precipitation by Sporosarcina pasteurii NCIMB 8841. Journal of Applied Sciences Research 9(3): 1525-1533.

Hammes, F., Boon, N., Villiers, J.D., Verstraete, W. & Siciliano, S.D. 2003. Strain-specific ureolytic microbial calcium carbonate precipitation. Applied and Enviromental Microbiology 69(8): 4901-4909.

Hammes, F. & Verstraete, W. 2002. Key roles of pH and calcium metabolism in microbial carbonate precipitation. Review in Enviromental Science and Biotechnology 1: 3-7.

Huang, F. & Zhou, S. 2022. A review of lightweight self-healing concrete. Materials 15(21): 7572.

Huynh, N.N.T., Imamoto, K. & Kiyohara, C. 2019. A study on biomineralization using Bacillus subtilis Natto for repeatability of self-healing concrete and strength improvement. Journal of Advanced Concrete Technology 17: 700-714.

Irwan, J.M. & Othman, N. 2013. An overview of bioconcrete for structural repair. Applied Mechanics and Materials 389: 36-39.

Jin, C., Yu, R. & Shui, Z. 2018. Fungi: A neglected candidate for the application of self-healing concrete. Frontiers in Built Environment 4: 62.

Joshi, K.A., Kumthekar, M.B. & Ghodake, V.P. 2016. Bacillus subtilis bacteria impregnation in concrete for enhancement in compressive strength. International Research Journal of Engineering and Technology 3(5): 1229-1234.

Khaliq, W. & Ehsan, M.B. 2019. Crack healing in concrete using various bio influenced self-healing techniques. Construction and Building Materials Journal 102: 349-357.

Krishnapriya, S., Venkatesh Babu, D.L. & Prince Arulraj, G. 2015. Isolation and identification of bacteria to improve the strength of concrete. Microbiological Research 174: 48-55.

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing plaforms. Molecular Biology and Evolution 35(6): 1547.

Luhar, S., Luhar, I. & Shaikh, F.U.A. 2022. A review on the performance evaluation of autonomous self-healing bacterial concrete: Mechanisms, strength, durability, and microstructural properties. Journal of Composites Science 6(1): 23.

Manikandan, A.T. & Padmavathi, A. 2015. An experimental investigation on improvement of concrete serviceability by using bacterial mineral precipitation. International Journal of Research and Scientific Innovation 2(3): 46-49.

Mitchell, A. & Grant Ferris, F. 2005. The coprecipitation of Sr into calcite precipitates induced by bacterial ureolysis in artificial groundwater: Temperature and kinetic dependence. Geochimica et Cosmochimica Acta 69(17): 4199-4210.

Mohammadizadeh, M., Ajalloeian, R., Nadi, B. & Soltani Nezhad, S. 2019. Investigating the shear strength of biologically improved soil using two types of bacteria. Geomicrobiology Journal 36(7): 581-590.

Natarajan, K.R. 1995. Kinetic study of the enzyme urease from Dolichos biflorus. Journal of Chemical Education 72(6): 556-557.

Nei, M. & Kumar, S. 2000. Molecular Evolution and Phylogenetics. Oxford: Oxford University Press.

Ngalimat, M.S., Raja Abd. Rahman, R.N.Z., Yusof, M.T., Syahir, A. & Sabri, S. 2019. Characterisation of bacteria isolated from the stingless bee, Heterotrigona itama, honey, bee bread and propolis. PeerJ 7: e7478.

Ni, M. & Ratner, B.D. 2008. Differentiation of calcium carbonate polymorphs by surface analysis techniques - An XPS and TOF-SIMS study. Surface and Interface Analysis: SIA 40(10): 1356-1361.

Nosouhian, F., Mostofinejad, D. & Hasheminejad, H. 2015. Influence of biodeposition treatment on concrete durability in a sulphate environment. Biosystems Engineering 133: 141-152.

Pachaivannan, P., Hariharasudhan, C., Mohanasundram, M. & Bhavani, M.A. 2020. Experimental anaylsis of self healing properties of bacterial concrete. Materials Today: Proceedings 33: 3148-3154.

Phillips, A.J., Gerlach, R., Lauchnor, E., Mitchell, A.C., Cunningham, A.B. & Spangler, L. 2013. Engineered applications of ureolytic biomineralization: A review. Biofouling 29(6): 715-733.

Reeburgh, W.S. 2007. Oceanic methane biogeochemistry. Chemical Reviews 107(2): 486-513.

Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chekroun, K. & Gonzalez-Muñoz, M.T. 2003. Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization. Applied and Environmental Microbiology 69(4): 2182-2193.

Saitou, N. & Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425.

Sarkar, M., Adak, D., Tamang, A., Chattopadhyay, B. & Mandal, S. 2015. Genetically-enriched microbe-facilitated self- healing concrete - A sustainable material for a new generation of construction technology. RSC Advances 5: 105363-105371.

Shahid, S., Aslam, M.A., Ali, S., Zameer, M. & Faisal, M. 2020. Self-healing of cracks in concrete using Bacillus strains encapsulated in sodium alginate beads. Chemistry Select 5: 312-323.

Siddique, R., Singh, K., Singh, M., Corinaldesi, V. & Rajor, A. 2016. Properties of bacterial rice husk ash concrete. Construction and Building Materials 121: 112-119.

Sierra-beltran, M.G., Jonkers, H.M. & Schlangen, E. 2014. Characterization of sustainable bio-based mortar for concrete repair. Construction and Building Materials 67: 344-352.

Van Paassen, L.A., Daza, C.M., Staal, M., Sorokin, D.Y., Van Der Zon, W. & Van Loosdrecht, M.C.M. 2010. Potential soil reinforcement by biological denitrification. Ecological Engineering 36(2): 168-175.

Vijay, K., Murmu, M. & Deo, S.V. 2017. Bacteria based self healing concrete - A review. Construction and Building Materials 152: 1008-1014.

Wang, J.Y., Soens, H., Verstraete, W. & De Belie, N. 2014. Self-healing concrete by use of microencapsulated bacterial spores. Cement and Concrete Research 56: 139-152.

Wegian, F.M. 2010. Effect of seawater for mixing and curing on structural concrete. The IES Journal Part A: Civil & Structural Engineering 3(4): 235-243.

Wiktor, V. & Jonkers, H.M. 2011. Quantification of crack-healing in novel bacteria-based self-healing concrete. Cement and Concrete Composites 33: 763-770.

Williams, P.D., Guilyardi, E., Madec, G., Gualdi, S. & Scoccimarro, E. 2010. The role of mean ocean salinity in climate. Dynamics of Atmospheres and Oceans 49: 108-123.

Zhang, Z., Weng, Y., Ding, Y. & Qian, S. 2019. Use of genetically modified bacteria to repair cracks in concrete. Materials 12(23): 3912.

*Pengarang untuk surat-menyurat; email: suriana@upm.edu.my