Micro-Scale Analysis of Microbial-Induced Calcite Precipitation in Sandy Soil through SEM/FIB Imaging


Kejun Wen,1


37209-1561 3


Lin Li,2 * Rong Zhang,3 Yang Li,1 and Farshad Amini1


1Department of Civil and Environmental Engineering, Jackson State University, 1400 J.R. Lynch St., Jackson, MS, 39217 2Department of Civil and Architectural Engineering, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN


Electron Microscopy Core Laboratory, Jackson State University, 1400 J.R. Lynch St., Jackson, MS, 39217 *lli1@tnstate.edu


Abstract: Microbial-induced calcite precipitation (MICP) has gained much attention in soil improvement studies, where it can enhance the physical properties of sandy soil. Small-scale sand cylinder tests were conducted to investigate the formation and failure of calcium carbonate precipitation bonding between individual sand particles. Bonding formation by precipitation was examined by scanning elec- tron microscopy. Energy-dispersive X-ray spectroscopy analysis veri- fied the existence of calcium, carbon, and oxygen, which could form CaCO3


after MICP-treatment. Focused-ion-beam milling was applied


to study the interior structure of calcium carbonate precipitation dur- ing the MICP process.


Keywords: Microbial-induced calcite precipitation (MICP), CaCO3 bonding, scanning electron microscopy (SEM), focused ion beam


(FIB) milling, X-ray microanalysis


Introduction An emerging soil improvement technique, microbial-


induced calcite precipitation (MICP), can bond soil grains together and improve the engineering properties of soil [1]. A biological reaction network requiring the presence of ureo- lytic bacteria (or urease), urea, and calcium-rich solution can develop cementation between soil particles and improve soil mechanical properties. Te strength improvement of MICP- treated sand mainly relies on bonding formed by calcium car- bonate precipitation. Bacteria producing the urease enzyme can catalyze urea to carbonate and, in the presence of Ca2+ at elevated pH, result in CaCO3


precipitation. Te formation


of calcium carbonate precipitation around the bacteria limits the availability of oxygen and nutrients for the bacteria, thus reducing the efficiency of bacteria in producing urease enzyme. Moreover, the MICP process faces the challenges of non-uni- formity of calcite precipitation and limited penetration depth in the treated soil, which may limit the application of this inno- vative soil improvement technology [2,3]. Qabany et al. [4] found that when the cementation media


concentration was as high as 1 M Ca, the calcium carbonate precipitation was less uniform and contained larger crystal sizes, whereas the use of lower Ca concentrations over a larger number of injections resulted in a more homogeneous calcium carbonate precipitation. In addition, Zhao et al. [5] developed full contact flexible molds for the MICP process and achieved more uniform specimens. Te MICP process relies on molecular-level chemical and


biological processes that must be better understood for large- scale implementation. Crystal size and shape were found to influence the particle-bond failure mechanism in the form of either a sand-particle-bond interface failure or an internal fail- ure of the carbonate crystals along a suture [6]. Te strength


24 doi:10.1017/S1551929518001293


improvement of MICP-treated sand mainly relies on the pre- cipitation bond, and the unconfined compression strength


of MICP-treated samples increases with increasing CaCO3 content [5]. Scanning electron microscopy (SEM) has proven to be one


of the most useful tools for analysis of these materials, provid- ing the ability to non-invasively visualize, differentiate, and quantify the various components. Tis method has been used to examine the formation of MICP and biofilms on the sur- face of the soil matrix [5–7]. Focused-ion-beam (FIB) milling has been used by a number of researchers to serially section and visualize subsurface microstructure. Tis technique pro- vides a tremendous advance in our ability to view the true 3D microstructure of materials that have complex microstructural morphology and crystallography [8]. However, there is limited research on the formation and failure of calcium carbonate pre- cipitation bonding in MICP treatment at the scale of the pores between sand particles. Tis article provides a description of mineral formation in the interior of MICP-treated sandy soil as revealed by focused ion beam/scanning electron microscopy (FIB/SEM).


Materials and Methods Materials. Uniform, clean, Ottawa silica sand was used


in the experiments. Te average particle diameter of the sand (D50


) was 0.54mm, and no fines were included. Bacteria Spo- nutrient broth (3 g/L). Te urea-Ca2+ ·H2 O (73.5 g/L), NH4


rosarcina Pasteurii was used because of its highly active urease enzyme. A cementation medium was used to provide chemi- cals that induce calcium carbonate precipitation: urea (30 g/L), CaCl2


Cl (10 g/L), NaHCO3 (2.12 g/L), and molar ratio was fixed at 1:1. Batch preparation. Te specimens were prepared in full-


contact flexible molds as described in Zhao et al. [5]. Tese molds were made of non-woven geotextile. Te fibrous struc- ture of the geotextile mold increased the penetration of chemi- cals into sand pores and maintained suitable precipitation, which caused the MICP-treated soil samples to be more homo- geneous. All samples were prepared in a continuously stirred tank reactor, as shown in Figure 1a. With a bacteria concentra- tion (OD600


) of 0.6 and a cementation medium containing 0.5


M Ca, each treatment in the reactor lasted for 7 days. Up to four MICP treatment cycles were conducted on each sample to increase calcium carbonate precipitation and to improve the strength of MICP-treated sample. For each MICP treat- ment cycle, the specimen was taken out from the old flexible molds and mixed with new bacteria in a new geotextile mold to treat for another 7 days with new cementation media. Figure


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