1. MICROBIAL CALCIFICATION IN SUBSURFACE ENVIRONMENTS Sookie S. Bang Department of Chemistry and Chemical Engineering South Dakota School of Mines and Technology

2. Microbial Calcification • Microorganisms • Soil bacteria (Urease-positive) • Phototrophs • Occurs in • Terrestrial environments: alkaline soil e.g., plugging of porous media • Aquatic environments: marine and freshwaters e.g., whitings, calcareous mats

3. Calcification Ca2+ + HCO3- CaCO3 + H+

4. Microbial Urease • Intracellular Enzyme • Urea hydrolysis UREASE NH2-CO-NH2 + H2O —— 2NH3 + CO2 NH3 + H+ NH4+ (pH ) • Microorganisms: Eubacteria-Bacillus pasteurii, Proteus vulgaris, Pseudomonas spp., etc.

5. CaCO3 Precipitation Experiments • Microorganism: Bacillus pasteurii ATCC11859 • Medium: 3 g Nutrient broth, 20 g Urea, 2.8 g CaCl2, and 2.12 g NaHCO3, pH 7.8 – 8.0

11. Microbiologically InducedCalcite Precipitation (MICP) At higher pH : in medium containing Urea, CaCl2 and NaHCO3 Ca2+ + Cell  Cell–Ca2+ Cl- + HCO3- + NH3 NH4Cl + CO32- Cell–Ca2+ + CO32- Cell-CaCO3

12. Calcification in Aquatic Environments • Photosynthetic microorganisms: Ca2+ + HCO3- CaCO3 + H+ H+ + HCO3-  CH2O + O2 • Ureolytic microorganisms: Ca2+ + HCO3- CaCO3 + H+ NH3 + H+ NH4+

16. Potential Applications of MICP • Microbial plugging in porous media: (NSF/CMS-9412942) • Remediation of cracks and fissures in granite and concrete • Subsurface stabilization in highways with urease enzyme • Dust control for surface soils • Carbon sink in ecosystems

18. Potential Applications of MICP • Microbial plugging in porous media • Remediation of cracks and fissures in granite and concrete: (NSF/CMS-9412942; CMS-9802127 ) • Subsurface stabilization in highways with urease enzyme • Dust control for surface soils • Carbon sink in ecosystems

21. Potential Applications of MICP • Microbial plugging in porous media • Remediation of cracks and fissures in granite and concrete • Subsurface stabilization in highways with urease enzyme: (NSF/INT-0002608) • Dust control for surface soils • Carbon sink in ecosystems

22. Potential Applications of MICP • Microbial plugging in porous media • Remediation of cracks and fissures in granite and concrete • Subsurface stabilization in highways with urease enzyme • Dust control for surface soils • Carbon sink in ecosystems

23. Potential Applications of MICP • Microbial plugging in porous media • Remediation of cracks and fissures in granite and concrete • Subsurface stabilization in highways with urease enzyme • Dust control for surface soils • Carbon sink in ecosystems

24. Proposed Research Experiments at NeSS • Identification of diversity in microorganisms that participate in CaCO3 precipitation: • DNA extraction / PCR amplification / phylogenetic analysis • MICP in subsurface environments: • Effects of pressure, temperature, and CO2 concentration on CaCO3 precipitation kinetics • Measurement of CO2 sequestration rates: • CO2 flux using the eddy covariance methods

25. Hypotheses/Possibilities • CaCO3 at Homestake has percolated from the surface. • Surface soil microbial populations may have been introduced to the subsurface. • Ecological interactions among microbes in the subsurface result in phyogenetic diversity. • Subsurface environmental factors will influence kinetics of CaCO3precipitation and CO2 flux.

26. Significance of Proposed Research • Phylogenetic diversity of microbial communities involved in subsurface calcification • Effects of MICP on subsurface hydrology • Application of MICP in subsurface bioremediation • Evaluation of the range of carbon sequestration in deep subsurface