Screening for Urease-Producing Bacteria from Limestone Caves of Sarawak

  • Armstrong Ighodalo Omoregie
  • Nurnajwani Senian
  • Phua Ye Li
  • Ngu Lock Hei
  • Dominic Ong Ek Leong
  • Irine Runnie Henry Ginjom
  • Peter Morin Nissom
Keywords: Bacterial isolation, conductivity, enrichment culture, microbial induced calcite precipitation (MICP), Sporosarcina pasteurii, urease activity


Urease is a key enzyme in the chemical reaction of microorganism and has been found to be associated with
calcification, which is essential in microbially induced calcite precipitation (MICP) process. Three bacterial
isolates (designated as LPB19, TSB31 and TSB12) were among twenty-eight bacteria that were isolated from
samples collected from Sarawak limestone caves using the enrichment culture technique. Isolates LPB19, TSB31
and TSB12 were selected based on their quick urease production when compared to other isolates. Phenotypic
characteristics indicate all three bacterial strains are gram-positive, rod-shaped, motile, catalase and oxidase
positive. Urease activity of the bacterial isolates were measured through changes in conductivity in the absence of
calcium ions. The bacterial isolates (LPB19, TSB12 and TSB31) showed urease activity of 16.14, 12.45 and 11.41
mM urea hydrolysed/min respectively. The current work suggested that these isolates serves as constitutive
producers of urease, potentially useful in inducing calcite precipitates.


Achal, V. (2015). Production of bacteria for structural concrete. In T.F. Pacheco, J.A. Labrincha, M.V. Diamanti, C.P. Yu & H.K. Lee (Eds.), Biotechnologies and Biomimetics for Civil Engineering. Springer International Publishing. Pp 309-323.

Achal, V., Abhijit, M. & Reddy, S.M. (2010). Characterization of two urease-producing and calcifying Bacillus spp. isolated from cement. Journal of Microbiology and Biotechnology, 20: 1571-1576.

Achal, V., Mukherjee, A., Basu, P.C. & Reddy, M.S. (2009). Strain improvement of Sporosarcina pasteurii for enhanced urease and calcite production. Journal of Industrial Microbiology and Biotechnology, 36: 981-988.

Achal, V. & Pan, X. (2011). Characterization of urease and carbonic anhydrase producing bacteria and their role in calcite precipitation. Current Microbiology, 62: 894-902.

Al-Thawadi, S. & Cord-Ruwisch, R. (2012). Calcium carbonate crystals formation by ureolytic bacteria isolated from Australian soil and sludge. Journal of Advanced Science and Engineering Research, 2: 12-16.

Al-Thawadi, S.M. (2008). High strength in-situ biocementation of soil by calcite precipitating locally isolated ureolytic bacteria. (Doctoral thesis), Murdoch University, Perth, Australia.

Alves, P.D., Siqueira Fde, F., Facchin, S., Horta, C.C., Victoria, J.M. & Kalapothakis, E. (2014). Survey of microbial enzymes in soil, water, and plant microenvironments. The Open Microbiology Journal, 8: 25-31.

Ariyanti, D., Handayani, N.A. & Hadiyanto (2011). An overview of biocement production from microalgae. International Journal of Science and Engineering, 2: 30-33.

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

Banda, R.M., Gendang, R. & Ambun, A.V. (2004). Geology and geochemistry of limestone in Sarawak. The Sarawak Museum Journal, 6: 41-61.

Bang, S.S., Galinat, J.K. & Ramakrishnan, V. (2001). Calcite precipitation induced by polyurethane-immobilized Bacillus pasteurii. Enzyme and Microbial Technology, 28: 404-409.

Burbank, M.B., Weaver, T.J., Williams, B.C. & Crawford, R.L. (2012). Urease activity of ureolytic bacteria isolated from six soils in which calcite was precipitated by indigenous bacteria. Geomicrobiology Journal, 29: 389-395.

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

Cheng, L. & Cord-Ruwisch, R. (2013). Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture. Journal of Industrial Microbiology and Biotechnology, 40: 1095-104.

Chu, J., Stabnikov, V. & Ivanov, V. (2012). Microbially induced calcium carbonate precipitation on surface or in the bulk of soil. Geomicrobiology Journal, 29: 544-549.

Cole, L.E., Bhagwat, S.A. & Willis, K.J. (2015). Long-term disturbance dynamics and resilience of tropical peat swamp forests. Journal of Ecology, 103(1): 16-30.

Cuzman, O., Richter, K., Wittig, L. & Tiano, P. (2015a). Alternative nutrient sources for biotechnological use of Sporosarcina pasteurii. World Journal of Microbiology and Biotechnology, 31: 897-906.

Cuzman, O.A., Rescic, S., Richter, K., Wittig, L. & Tiano, P. (2015b). Sporosarcina pasteurii use in extreme alkaline conditions for recycling solid industrial wastes. Journal of Biotechnology, 214: 49-56.

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

Elmanama, A.A. & Alhour, M.T. (2013). Isolation, characterization and application of calcite producing bacteria from urea rich soils. Journal of Advanced Science and Engineering Research, 3: 377-399.

Gorski, L. (2012). Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths. PLoS ONE, 7(4): e34722.

Gueye, L., Samb, A., Ciss, M., Ndoye, O., Mbengue-Gaye, A. & Cisse, F. (2001). Ammonia-gas poisoning: respiratory troubles evaluated by functional exploration. Dakar Medical Journal, 46: 8-11.

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: 1525-1533.

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

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

Harkes, M.P., van Paassen, L.A., Booster, J. L., Whiffin, V.S. & van Loosdrecht, M.C.M. (2010). Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement. Ecological Engineering, 36: 112-117.

Ivanov, V. & Chu, J. (2008). Applications of microorganisms to geotechnical engineering for bioclogging and biocementation of soil in situ. Reviews in Environmental Science and Biotechnology, 7: 139-153.

Julaihi, L.C.J. (2004). Altitudinal analyses of limestone vegetation of Gunung Api, Gunung Mulu National Park, Miri, Sarawak. (Master of Science thesis), Universiti Malaysia Sarawak, Sarawak, Malaysia.

Kang, C.H., Choi, J.H., Noh, J., Kwak, D.Y., Han, S.H. & So, J.S. (2014). Microbially induced calcite precipitation-based sequestration of strontium by Sporosarcina pasteurii WJ-2. Applied Biochemistry and Biotechnology, 174: 2482-2491.

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

Kuek, F.W.I., Lim, L.F., Ngu, L.H., Mujahid, A., Lim, P.T., Leaw, C.P., & Müller, M. (2015). The potential roles of bacterial communities in coral defence: A case study at Talang-talang reef. Ocean Science Journal, 50: 1-14.

Lateef, A.A., Sepiah, M. & Bolhassan, M.H. (2014). Microfungi on green leaves and leaf litters from Santubong National Park, Sarawak. In M.T. Liong, R. Ahmad, S. Hena, W.N.W. Abdullah, C.K. Lee, N.A. Serri, H.A.B. Tajaruddin, J.S. Ong & S.Y.A. Lau (Eds.), Proceedings of the International Conference on Beneficial Microbes ICOBM 2014: Microbes for the Benefits of Mankind, Penang, Malaysia: Universiti Sains Malaysia. Pp 130-134.

Miyashita, N.T., Iwanaga, H., Charles, S., Diway, B., Sabang, J. & Chong, L. (2013). Soil bacterial community structure in five tropical forests in Malaysia and one temperate forest in Japan revealed by pyrosequencing analyses of 16S rRNA gene sequence variation. Genes and Genetic Systems, 88: 93-103.

Mohd, R.A.R., Roberta, C.T.T., Mohd, I.A., Noor, H.H. & Mohd, T.A. (2011). Bats of the Wind Cave Nature Reserve, Sarawak, Malaysian Borneo. Tropical Natural History, 11: 159-175.

Muniesa, M., Blanch, A.R., Lucena, F. & Jofre, J. (2005). Bacteriophages may bias outcome of bacterial enrichment cultures. Applied and Environmental Microbiology, 71: 4269-4275.

Omoregie, A.I., Senian, N., Li, P.Y., Hei, N.L., Leong, D.O.E., Ginjom, I.R.H. & Nissom, P.M. (2015). Isolation and characterisation of urease producing bacteria from Sarawak caves and their role in calcite precipitation. In K. Nadarajah, T.K. Lin, K.S. Kumar, D. Ibrahim, K. Philip, J. Santhanam, L.C. Weng, G. Krishnasamy, L.Y. Sze, M.J. Masarudin, C.L. Ching, B.C. Wei & S.K. Shin (Eds.), International Congress of the Malaysian Society for Microbiology, Penang, Malaysia. Malaysian Society for Microbiology. Pp 16- 21.

Rahman, M.R.A., Tingga, R.C.T., Noor Haliza, H., Sigit, W., Anang, S.A., Eileen, L., Besar, K., Huzal, I.H. & Abdullah, M.T. (2010). Diversity of bats in two protected limestone areas in Sarawak, Malaysia. Sarawak Museum Journal, 88: 209-246.

Sa'don, N.M., Karim, A.R.A., Jaol, W. & Lili, W.H.W. (2015). Sarawak peat characteristics and heat treatment. UNIMAS e-Journal of Civil Engineering, 5: 6-12.

Schabereiter-Gurtner, C., Saiz-Jimenez, C., Pinar, G., Lubitz, W. & Rolleke, S. (2004). Phylogenetic diversity of bacteria associated with paleolithic paintings and surrounding rock walls in two Spanish caves (Llonin and La Garma). FEMS Microbiology Ecology, 47: 235-247.

Stocks-Fischer, S., Galinat, J.K. & Bang, S.S. (1999). Microbiological precipitation of CaCO3. Soil Biology and Biochemistry, 31: 1563-1571.

Stabnikov, V., Jian, C., Ivanov, V. & Li, Y. (2013). Halotolerant, alkaliphilic ureaseproducing bacteria from different climate zones and their application for biocementation of sand. World Journal of Microbiology and Biotechnology, 29: 1453-1460.

Sugita, T., Kikuchi, K., Makimura, K., Urata, K., Someya, T., Kamei, K. & Uehara, Y. (2005). Trichosporon species isolated from guano samples obtained from bat-inhabited caves in Japan. Applied and Environmental Microbiology, 71: 7626-7629.

Tan, H.S., Nadiah, I. & Eryani, S.S. (2009). Tree flora of Sabah and Sarawak project - progress and future activities. Blumea - Biodiversity, Evolution and Biogeography of Plants, 54: 23-24.

Tomczyk-Żak, K. & Zielenkiewicz, U. (2015). Microbial diversity in caves. Geomicrobiology Journal, 33: 20-38.

Wei, S., Cui, H., Jiang, Z., Liu, H., He, H. & Fang, N. (2015). Biomineralization processes of calcite induced by bacteria isolated from marine sediments. Brazilian Journal of Microbiology, 46: 455-464.

Whiffin, V.S. (2004). Microbial CaCO3 Precipitation for the production of biocement. (Doctoral thesis), Murdoch University, Perth, Australia.

Whiffin, V.S., van Paassen, L.A. & Harkes, M.P. (2007). Microbial carbonate precipitation as a soil improvement technique. Geomicrobiology Journal, 24:417-423.

Woto-Gaye, G., Mendez, V., Boye, I.A. & Ndiaye, P.D. (1999). Death from ammonia poisoning: anatomo-pathologic features. Dakar Medical Journal, 44: 199-201.

Zhang, Y., Guo, H.X. & Cheng, X.H. (2015). Role of calcium sources in the strength and microstructure of microbial mortar. Construction and Building Materials, 77:160-167.

How to Cite
Omoregie, A. I., Senian, N., Ye Li, P., Hei, N. L., Leong, D. O. E., Henry Ginjom, I. R., & Nissom, P. M. (2016). Screening for Urease-Producing Bacteria from Limestone Caves of Sarawak. Borneo Journal of Resource Science and Technology, 6(1), 37-45.