Microbially induced calcite precipitation (MICP) is a ground improvement approach that has advanced rapidly over the past two decades given its potential application in geotechnical engineering systems. In this process ureolytic MICP uses urea-hydrolyzing bacteria to precipitate calcium carbonate in the soil matrix to rapidly change improve the mechanical properties of the soil while purporting to have fewer environmental impacts than many traditional cement-based ground improvement techniques. The engineering performance gains of the process is relatively well-studied, but far less has been published about the biological processes and phenomena that enable MICP.
Ureolytic MICP has two main approaches: bioaugmentation, or the inoculation of the soil with, typically, the Gram-positive bacterium Sporosarcina pasteurii; and biostimulation, which uses sequential selective enrichments within the soil to generate a sufficiently ureolytic community. Presented herein is a conceptual synthesis of both methods from a microbiological perspective, with particular focus given to the current understanding of the factors that govern the effectiveness of biostimulation. A model for enrichment is proposed that includes the negative selectivity imposed by the synergistic effects of ammonia and elevated pH to suppress competitive growth as well as the positive selection based on energy-derived from the hydrolysis of urea to promote the targeted growth of important MICP species.
Biostimulated and bioaugmented MICP approaches yielded comparable precipitation in a 1.7 m diameter tank comparison, but the augmented S. pasteurii strain was seemingly outcompeted by native soil bacteria such that it was unable to be recovered one week after inoculation. Instead, other Sporosarcina species were cultured from both tanks with S. soli being the most abundant single species. These isolates manifested a wide range of whole-cell urease kinetics, but still demonstrated ureolytic rates that produce carbonate alkalinity as much as 100 times more rapidly than proposed biocementation processes dependent upon other types of bacterial energy metabolism.
The two methods were again compared using molecular approaches in small, less heterogeneous soil columns. Consistent with what was seen at the large-scale, the calcium carbonate precipitation was comparable between both methods. Moreover, the bioaugmented S. pasteurii strain, which was detected three days following the inoculation, was unable to be detected after seven, again an indication of its poor ability to compete with the native bacteria. The microbial communities in both columns were populated by Sporosarcina species (principally S. koreensis, S. luteola, S. ginsengisoli, S. soli, S. saromensis, S. pasteurii, and S. newyorkensis), reinforcing the importance of this genus for ureolytic MICP, though Lysinibacillus sphaericus and Lysinibacillus fusiformis came to prominence in the latter half of the treatments. The emergence of Lysinibacillus was not associated with a particular decrease in ureolytic activity. Instead, ureolytic activity continued to accrete, such that even generous estimates of ureolytic kinetics, when combined with measured suspended cell densities, explained only 10% of the observed ureolytic activity, implying analyses of the pore fluid neglect a substantial portion of the microbial participants.
Pure culture studies of the S. pasteurii strain typically used for MICP as well as MICP isolates demonstrate that some members of the genus Sporosarcina are able to generate energy from urea hydrolysis and the chemiosmotic gradients that result. These isolates appear to have the ability, upon repeated transfer of small inocula to fresh media, to indefinitely sustain urea-dependent multiplication under rigorously anoxic conditions. Furthermore, the energy yield of this metabolism is substantial, allowing the anaerobic growth and maintenance of cultures of 10⁷-10⁸ cfu/mL density, with an S. soli isolate having the potential to grow to even higher titers. The geotechnical importance of anaerobic, urea-fueled growth in this genus is emphasized by the demonstration that biostimulated soil columns experience anoxia for 80-90% of a typical treatment, an interval in which alternative modes of bacterial energy generation are ineffective. Modeling based on biomass yield did not exclude cytoplasmic alkalinization as a potential mechanism for energy generation, but ammonium transporters are predicted to be present to boost the efficiency in at least some species. The ability to grow with energy derived from urea could be an essential element to the function of biostimulation but may even play a more common role in urogenital pathogenesis.