Topraktan İzole Edilen Bacillus Türlerinin Biyokalsifikasyon Özelliklerinin Belirlenmesi

Bu çalışma, üreaz aktivitesine sahip Bacillus türlerinin topraktan izolasyonunu, morfolojik ve moleküler tanımlamalarını ve biyokalsifikasyon özelliklerini belirlemeyi amaçlamıştır. Toprak örneklerinden Bacillus türüne ait toplam 11 bakteri izole edilmiştir. İzolatların tanımlanması, 16S rRNA dizileme analizi ile gerçekleştirilmiştir. İzolatların üreaz aktiviteleri fenol-hipoklorit yöntemine göre gerçekleştirilmiştir. Sıvı ortamda CaCO3 birikimi belirlenmiştir. CaCO3 kristalleri, taramalı elektron mikroskobunda (SEM) analiz edilmiştir. İzolatların üreaz enzim aktiviteleri kontrol grubu Sporosarcina pasteurii ile karşılaştırılmıştır. Sonuçlara göre F10 (Bacillus mycoides) spesifik üreaz aktivitesi 8.50±0.00 U/mg ile en yüksek aktiviteye sahip tür olarak belirlendi. En düşük spesifik aktiviteye sahip bakteri, 2,75±0,04 U/mg spesifik üreaz aktivitesi ile F14 (Bacillus thuringiensis) olarak belirlendi. Üre ve kalsiyum klorür katkılı sıvı ortamda S. pasteurii'de maksimum CaCO3 birikimi 0,53 g olarak belirlendi. Kontrol suşundan sonra maksimum CaCO3 birikimi F9, F13 ve F8 izolatlarında sırasıyla 0.20g, 0.20g ve 0.18g olarak ölçülmüştür. SEM incelemelerinin sonucuna göre izolatların kalsit birikimini gerçekleştirdiği belirlenmiştir.

Anahtar Kelimeler:

Biyokalsifikasyon, Bacillus, 16S rRNA, Ureaz aktivitesi

Determination of Biocalcification Properties of Bacillus Species Isolated from Soil

This study aimed to determine isolation of Bacillus species having urease activityfrom soil, morphological and molecular identification and on biocalcification features. Total 11 bacteria belong to Bacillus species were isolated from soilsamples. Identification of the isolates was carried out with 16S rRNA sequencing analysis. Urease activity of the isolates was carried out according to the phenol-hypochlorite method. CaCO3 accumulation was determined and liquid media. CaCO3 crystals were analyzed in the scanning electron microscope (SEM). Urease enzyme activities of the isolates were compared to with control group Sporosarcina pasteurii. According to results, F10 (Bacillus mycoides) was determined as species having the highest activity with specific urease activity 8.50±0.00 U/mg. The bacterium having the lowest specific activity was determined F14 (Bacillus thuringiensis) with specific urease activity 2.75±0.04 U/mg value. In liquid medium supplemented with urea and calcium chloride, the maximum CaCO3 accumulation was determined to be 0.53 g in S. pasteurii. After the control strain, the maximum CaCO3 accumulation was measured as 0.20g, 0.20 g and 0.18 g in F9, F13 and F8 isolates, respectively. According to the results of SEM examinations, it was determined that the isolates carried out calcite accumulation.

Keywords:

Biocalcification, Bacillus, 16S rRNA, Urease activity,

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[1] Ramachandran, S. K., Ramakrishnan, V., Bang, S. S. 2001. Remediation of concrete using microorganisms. ACI Materials Journals. 98 (2011), 3–9.
[2] Sarda, D., Choonia, H. S., Sarode, D. D., Lele, S. S. 2009. Biocalcification by Bacillus pasteurii urease: a novel application). Journal of Industrial Microbiology and Biotechnology. 36(2009), 1111–1115.
[3] Castanier, S., Metayer-Levrel, G. Le., Perthuisot, J. P. 1999. Ca-carbonates precipitation and limestone genesis–the microbiol geologist point of view. Sedimentary Geology. 126(1999), 9-23.
[4] Braissant, O., Verrecchia, E. P. And Aragno, M. 2002. Is the contribution of bacteria to terrestrial carbon budget greatly under estimated. Naturwissenschaften, 89(2002), 366-370.
[5] Dick, J., Windt, W. De., Graef, B. D., Saveyn, H., Meeran, P. V., Belie, N., Verstraete W. 2006. Biodeposition of a calcium carbonate layer on degraded limestone by Bacillus species. Biodegradation. 17(2006), 357- 67.
[6] 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(2013), 1525-1533.
[7] Maniatis, T., Fristsh, E. F., Sambrook, J. 1989. In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York,
[8] Giffel, M. C., Beumer, R. R., Granum, P. E., Rombouts, F. M. 1997. Isolation and characterization of Bacillus cereus from pasteurised milk in house hold refrigerators in the Netherland. International Journal of Food Microbiolgy. 34(1997), 307-318.
[9] Prescott, H., 2002. Laboratory Exercise in Microbiology, Fifth edition. The Mc GrawHill.
[10] Mobley, H., Hausinger, R. 1989. Microbial ureases: Significance, regulation, and molecular characterization. Microbiological Reviews. 53(1989), 85–108.
[11] Stocks-Fischer, S., Galinat, J.K., Bang, S.S. 1999. Microbiological precipitation of CaCO3. Soil Biology and Biochemistry. 31(1999), 1563-1571.
[12] Hammes, F., Boon, N., Villiers, J. de.,Verstraete,W., Siciliano, S.D. (2003a). Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation. Applied and Environmental Microbiology. 69(2003a), 4901-4909.
[13] Whiffin, V.S. 2004. Microbial CaCO3 Precipitation for the Production of Biocement. PhD thesis. Murdoch University, Western Australia.
[14] Al-Thawadi, S.M, 2008. High Strength In-Situ Biocementation of Soil by Calcite Precipitating Locally Isolated Ureolytic Bacteria. PhD thesis. Murdoch University, Western Australia.