STRAIN ENCHANCEMENT OF

DIFFERENT SPORE FORMING BACTERIA

FOR THEIR CALCIUM CARBONATE
PRECIPITATION PROPERTIES IN THE
OPTIMIZATION OF BIO-CEMENT

By : Adriel Braganza and C. Tamil Selvan

Department of Plant Biology and Biotechnology
Loyola College, Chennai.
Components of Cement
• lime (60-65%)
• Silica (17- 25%)
• Alumina (1-3%)
• Magnesia (1-3%)
• Iron oxide (0.5-6%)
• Calcium sulphate (0.1-0.5%)
• Sulfur trioxide (1-3%)
• Alkaline (0-1%)
Left side shows process of concrete making and right side shows GHG emissions from concrete, of
which 90% are from cement and 10% from aggregates and other inputs as well as quarrying,
transport, and preparation of raw materials. (Source: Dr. Veena Singla, Cut carbon and toxic pollution, make
cement clean and green 2022)
Cement manufacturing emissions in India have experienced a steep climb in recent decades. In 2021, figures
reached a high of 149 million metric tons of carbon dioxide (MtCO₂), after experiencing a drop in the previous
year due to the implications of the coronavirus pandemic. India is the second-largest polluter from cement
manufacturing across the globe.
(Source: Tiseo, India: Cement co₂ emissions 1960-2021, 2023)
Bacteria based Self-Healing Concrete
(BBSHC)

Calcium carbonates formation on bacterial cell wall.

(Source :Kunamineni Vijay et al., Bacteria based Self Healing Concrete – a review 2017)
Current process development for obtaining BBSHC. (1) The first step is to select a bacterial strain capable of CaCO3 precipitation,
appropriate for the application considered. (2) The second step is to characterize the selected strain and its carbonate precipitation
efficiency. (3) Step three is to select a growth medium and to produce spores. (4) In step four there is the selection of the method for
including the biological material in concrete, followed in step five (5) by testing the healing of cracked lab samples. The mechanical
properties of the healed samples are evaluated in step six (6). At the end of the process and based on a rigorous economic analysis,
large-scale applications are considered (7), but very few are reported to date. ( Source: Manpreet Bagga a et al., Advancements in
bacteria based self-healing concrete and the promise of modelling 2022)
Microbiologically Induced Calcium
Carbonate Precipitation (MICP)
• autotrophic (e.g., photosynthesis or methane oxidation)
• heterotrophic (e.g., sulphate reduction, organic acid oxidation or
nitrogen cycle)

The Nitrogen cycle, and in particular Ureolysis, are the most studied
pathways for MICP.
The precipitation of the mineral takes place on the cell surface and on
the extracellular polymeric substances surrounding the cells, which
provide nucleation sites
The Mechanism

Schematic of microbially
induced calcite precipitation
(MICP) catalyzed by S.
pasteurii cells.
(Source : Wu et al., Biomineralization
induced by cells of sporosarcina
pasteurii: Mechanisms, applications and
challenges 2021)
The Mechanism
• One mole of urea is hydrolyzed intracellularly to 1 mol of ammonia
and 1 mol of carbon.
• This spontaneously hydrolyzes to form an additional 1 mol of
ammonia and carbonic acid.
• These products subsequently equilibrate in water to form
bicarbonate and 2 mol of ammonium and hydroxide ions.
• . The latter give rise to a pH increase, which in turn can shift the
bicarbonate equilibrium, resulting in the formation of carbonate
ions.
• Which in the presence of soluble calcium ions precipitate as CaCO3.
Use of Organic Calcium Source.

Whey Water Egg Shells

Bacteria used for the study
• Sporosarcina pasteurii
• Bacillus subtilis
• Bacillus sphaericus
Sporosarcina pasteurii

A) Single colonies of S. pasteurii on the agar plate B) S. pasteurii with density adjusted
to Mc Farland 3 standard tube (approx. cell density of 9 ×10 8 Colony-forming unit/ml)
C) Microphotograph of Sporosarcina pasteurii (Gram-positive) strain.
(Source: TANYILDIZI et al., Comparison of bacteria-based self-healing methods in metakaolin
geopolymer mortars, 2022)
Bacillus subtilis

Intracellular calcium levels of bacterial cells stained

with calcein-AM, a calcium-specific fluorescent dye.
Cells were isolated from B. subtilis biofilm colonies
grown with calcium
(Source: Paz et al., The roles of intracellular and extracellular calcium in
bacillus subtilis biofilms 2022)

Calcium-rich deposits associated with a membrane from the

outside of the cell (Indicated by Arrows)
(Source: Paz et al., The roles of intracellular and extracellular calcium in bacillus subtilis
biofilms 2022)
Bacillus sphaericus

SEM image for Bacillus Sphaericus

(Source: Krishnakumar, Comparitive study on
durability properties of bacterial concrete 2016)
Work Done

Screening of starch hydrolysing bacteria on Starch Agar from soil

sample.
(Arrows indicating Zone of clearance due to starch hydrolysis)
Work Done

Gram Positive Rod shaped bacteria Observed under

40X magnification.
 Future Workplan
Phase 1
1. Obtain pure culture of
Sporosarcina pasteurii,
Bacillus subtilis and Bacillus
sphaericus.
2. Grow them on nutrient agar.
3. Culture till optimum density.
Phase 2 Phase 3
1. Make 3 triplicates of 1. Check calcium content
nutrient broth. before and after its
2. Introduce powdered egg utilization by the organism.
shells, whey water and 2. Using complexometric
calcium chloride in each titration.
of the triplicates 3. Compare and infer result.
respectively.
3. Organism with fixed
concentration of cell
density added.
 References
• Castro-Alonso, M. J., Montañez-Hernandez, L. E., Sanchez-Muñoz, M. A., Macias Franco, M. R., Narayanasamy, R., & Balagurusamy, N. (2019, May 14).
Microbially induced calcium carbonate precipitation (MICP) and its potential in bioconcrete: Microbiological and molecular concepts . Frontiers.
https://www.frontiersin.org/articles/10.3389/fmats.2019.00126/full
• Dr. Veena Singla, S. S. (2022, January 18). Cut carbon and toxic pollution, make cement clean and green. Be a Force for the Future.
https://www.nrdc.org/bio/veena-singla/cut-carbon-and-toxic-pollution-make-cement-clean-and-green#:~:text=Making%20cement%20also%20emits%20a,
(NOx)%20and%20carbon%20monoxide.
• Krishnakumar, K. (2016). Comparitive study on durability properties of bacterial concrete. Research Gate.
https://www.researchgate.net/publication/313029996_COMPARITIVE_STUDY_ON_DURABILITY_PROPERTIES_OF_BACTERIAL_CONCRETE.
• Kunamineni Vijay, Khaliq, W., Luo, M., Hung, C. C., Tziviloglou, E., Muynck, W. D., Siddique, R., Andalib, R., Wang, J., Wang, J. Y., Chahal, N., Tittelboom,
K. V., Qiu, J., Achal, V., Pei, R., Zhang, Y., & Wiktor, V. (2017, July 15). Bacteria based Self Healing Concrete – a review. Construction and Building Materials.
https://www.sciencedirect.com/science/article/pii/S0950061817313752
• Manpreet Bagga . (2022, October 19). Advancements in bacteria based self-healing concrete and the promise of modelling. Construction and Building
Materials. https://www.sciencedirect.com/science/article/pii/S0950061822030689
• Paz, A. K., Maan, H., Karunker, I., & Olender, T. (2022, April 27). The roles of intracellular and extracellular calcium in bacillus subtilis biofilms. iScience.
https://www.sciencedirect.com/science/article/pii/S258900422200579X
• TANYILDIZI, H., Ziada, M., Uysal, M., Güngör, N. D., & Coşkun, A. (2022). Comparison of bacteria-based self-healing methods in metakaolin geopolymer
mortars,. science direct. https://www.sciencedirect.com/science/article/pii/S2214509522000274
• TANYILDIZI, H., Ziada, M., Uysal, M., Güngör, N. D., & Coşkun, A. (2022). Comparison of bacteria-based self-healing methods in metakaolin geopolymer
mortars,. science direct. https://www.sciencedirect.com/science/article/pii/S2214509522000274.
• Tiseo, I. (2023, February 6). India: Cement co₂ emissions 1960-2021 . Statista. https://www.statista.com/statistics/1198693/carbon-dioxide-emissions-cement-
manufacturing-china/
• Wu, Y., Li, H., & Li, Y. (2021, November 21). Biomineralization induced by cells of sporosarcina pasteurii: Mechanisms, applications and challenges . MDPI.
https://www.mdpi.com/2076-2607/9/11/2396
Thank You!