Diversity of Sporosarcina-like Bacterial Strains Obtained from Meter

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Environmental Processes

Diversity of Sporosarcina-Like Bacterial Strains Obtained from MeterScale Augmented and Stimulated Bio-cementation Experiments Charles M.R. Graddy, Michael Gregory Gomez, Lindsay Marie Kline, Sydney Rose Morrill, Jason T. DeJong, and Douglas C. Nelson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04271 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Environmental Science & Technology

Diversity of Sporosarcina-Like Bacterial Strains Obtained from Meter-Scale Augmented and Stimulated Bio-cementation Experiments Charles M. R. Graddy1, Michael G. Gomez2, Lindsay M. Kline1, Sydney R. Morrill1, Jason T. DeJong2 and Douglas C. Nelson1*

University of California, Davis, CA, 95616, USA 1 2

Department of Microbiology and Molecular Genetics Department of Civil and Environmental Engineering

Corresponding Author: Douglas C. Nelson University of California, Department of Microbiology and Molecular Genetics; CA, 95616, USA, email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Microbially Induced Calcite Precipitation (MICP) is a bio-mediated soil cementation process that

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offers an environmentally conscious alternative to conventional geotechnical soil improvement

25

technologies. This study provides the first comparison of ureolytic bacteria isolated from sand

26

cemented in parallel, meter-scale, MICP experiments using either bio-stimulation or bio-

27

augmentation approaches, wherein colonies resembling the augmented strain (Sporosarcina

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pasteurii ATCC 11859) were interrogated. Over the 13-day experiment, 47 of the 57 isolates

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collected were strains of Sporosarcina and the diversity of these strains was high, with 20

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distinct strains belonging to 5 species identified. Although the S. pasteurii inoculant used

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for augmentation was recovered immediately after introduction in the augmented specimen, the

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strain was not recovered after 8 days in either augmented or stimulated soils, suggesting that it

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competes poorly with indigenous bacteria. Past studies on the physiological properties of

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Sporosarcina pasteurii ATCC 11859 suggest that close relatives may have selective advantages

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under the biogeochemical conditions employed during MICP; however, the extent to which these

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properties apply to isolates of the current study is unknown. Whole cell urease kinetic properties

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were investigated for representative isolates and suggest up to 100-fold higher rates of carbonate

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production when compared to other bio-mediated processes proposed for MICP.

39


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Introduction

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Worldwide demand for new and sustainable approaches to solve challenging geotechnical

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engineering problems has generated novel research opportunities in the emerging field of bio-

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mediated soil improvement.1 The most widely researched of these processes has been

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Microbially Induced Calcite Precipitation (MICP).2-4 In many of the first studies, bio-augmented

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MICP was accomplished by adding to a soil a high density of the constitutively ureolytic

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bacterium, Sporosarcina pasteurii.3 The amended soil was then supplemented with liquid

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medium containing calcium salts, urea, and sometimes growth-promoting organic compounds.

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Bacterial hydrolysis of one molecule of urea generates one molecule of carbonic acid (H2CO3)

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and two of ammonia (NH3). The resulting ammonia, being a weak base, equilibrates in water to

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form ammonium and hydroxide ions (pKa = 9.3).5 This shifts the H2CO3/HCO3-/CO32-

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equilibrium (pKa1 = 6.35; pKa2 = 10.33)5 toward carbonate, which will precipitate, in the

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presence of sufficient calcium, as calcium carbonate. Electron microscopy has shown that

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calcium carbonate deposition can occur in the immediate vicinity of these bacteria, thereby

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cementing adjacent soil particles, resulting in increases in soil strength and stiffness.1, 4 Bio-

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augmented MICP has shown promise for a variety of engineering applications2, 4, 6-12 including

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the mitigation of earthquake-induced soil liquefaction.13-15 The approach has also shown

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potential for co-precipitation of divalent radionuclides in artificial groundwater.16,

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Occasionally, other urease-positive bacterial species have also been employed for bio-augmented

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MICP.18

17

60 61

Researchers have also explored an alternate approach, termed bio-stimulated MICP, in which the

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proliferation of ureolytic bacteria naturally present in soils is promoted prior to the cementation



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phase, in lieu of adding a cultured strain. Early studies of bio-stimulation focused on ureolytic

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soil bacteria in aqueous microcosms, sometimes also demonstrating calcite precipitation.19-23

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More recently bio-stimulated MICP has been fully demonstrated in native sands24 with prospects

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for eliminating the financial costs and environmental impacts of propagating and transporting

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large quantities of bacteria and societal concerns over environmental release of non-native

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bacteria. Procedurally, bio-stimulation treats the target soil with a solution that lacks calcium

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salts, but contains urea and organic compounds that encourage the growth of ureolytic bacteria.

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When these bacteria have been sufficiently enriched, as judged by the rate of urea disappearance

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in the pore fluid, bio-cementation is enabled by the inclusion of calcium in subsequent

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applications of the treatment solution. A recent meter-scale side-by-side comparison of MICP

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completed by using bio-augmentation and bio-stimulation in two separate soil tanks suggested

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that both approaches were equally effective with respect to improving soil engineering

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properties.25 In column experiments completed on 14 different sandy soils from different

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depositional environments, including several samples obtained from natural deposits as deep as

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12 meters, bio-stimulated MICP was always successful.25-27 The achievement of bio-stimulated

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MICP in these experiments, involving diverse, nutrient poor sands, suggests that ureolytic

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bacteria capable of catalyzing this bio-geotechnical process may be widespread in natural soil

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systems.23, 24, 28

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Here we report on bacterial cultures isolated from the pore fluid of the bio-stimulation and bio-

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augmentation tanks in the aforementioned meter-scale experiment,25 with a focus on

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understanding the phylogenetic and functional diversity of the participating ureolytic bacteria.

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This study assesses ureolytic diversity at the end of bio-stimulated MICP, while focusing on


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colonies most resembling those of the type strain, to examine how the type strain of S. pasteurii,

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added at high density to the bio-augmentation tank, responds to competition from native bacteria

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present in the sand, and to determine whether contaminants of the type strain from the bio-

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augmentation tank could be detected in the bio-stimulation tank. The phylogenetic groupings of

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all cultured strains were assessed and for select strains urease kinetic properties (Vmax and KM)

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were evaluated. These data on the kinetic properties of intact native ureolytic bacteria allow for

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the comparison of their predicted efficiency in generating alkalinity (OH-) and CO2 relative to

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other microbial processes proposed for MICP.1, 29

94 95

Materials and Methods

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Large-scale Tank Experiment

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A detailed description of materials and methods for the meter-scale experiment are provided in

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Gomez, et al. 25, however, a summary of relevant details is presented here. Two 1.7 m diameter

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tanks containing 0.3 m thick layers of a poorly graded “Concrete Sand”26 (void ratio = 0.43)

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obtained from a local aggregate quarry (Woodland, CA) received treatments through three wells,

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which were slotted in the portion within the sand layer and placed in a triangular pattern (Figure

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S1). At the beginning of day 1, both tanks were saturated with deionized water from the bottom

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up. Thereafter, tanks received different treatments to bio-augment or bio-stimulate respective

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soils; however, following these treatments, identical injection schemes were used to cement soils

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in both tanks in a non-uniform spatial manner. Treatment details were as follows: For days 1 to

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5, the bio-stimulation tank received daily stimulation solution treatments (0.1 g/L yeast extract,

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12.5 mM ammonium chloride, 42.5 mM sodium acetate, 350 mM urea) in three sequential 0.5

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pore volume additions (107 L each) using a rotating injection scheme (e.g. injection at Well 1



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with overflow at Well 2, followed by injection from Well 2 to Well 3, etc.) to promote spatially

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uniform enrichment of native ureolytic microorganisms. On day 4, the bio-augmentation tank,

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which had remained saturated with deionized water from day 1, received three sequential 107 L

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volumes of S. pasteurii strain ATCC 11859 diluted to 3.5 x 107 cells/L in stimulation solution

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using the same rotating injection scheme for spatial uniformity. Inoculum density was based on a

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standard curve for the type strain of OD600 vs. total direct cell counts determined by the method

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of Hobbie, et al.30 Commencing on day 5, cementation solutions (pH 8.2), identical to the

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stimulation solution but containing calcium chloride at a concentration of 250 mM, were applied

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once daily to both tanks in a 161 L volumes from Well 1 to Well 2 for days 5 through 9 and from

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Well 2 to Well 3 for days 10 through 12. All injections (duration 25 to 45 minutes each) were

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under a constant gravimetric pressure head difference of 0.6 m between wells. The

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saturation state of the cementation solution was approximately 21, but maximum abiotic calcite

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precipitation occurring due to atmospheric CO2 was determined, using a PHREEQC equilibrium

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chemistry batch model,31 to consume only 0.17 mM Ca+2 (250 mM initial concentration)

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before calcite precipitation decreased pH and carbonate concentrations below values needed to

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maintain super-saturation. During treatments, pore fluid samples (60 ml each) were collected for

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chemical and biological analysis from the three slotted wells and ten aqueous sampling ports in

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each tank located at mid-depth within the sand layer. Samples were collected for chemical

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analysis on alternating treatment days, before and immediately after injections, and 2, 4, and 20

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hours after injections to monitor spatial and temporal changes in solution chemistry. Chemical

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samples were processed immediately for pH measurements or stored at -20°C for other analyses.

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All biological samples were obtained immediately prior to injections and were stored on ice prior

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to plating.


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Chemical Analysis

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After thawing, solution urea concentrations were quantified using a colorimetric assay similar to

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that presented in Knorst, et al.32 All pH measurements were completed using an Accumet AB15

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pH meter (Thermo Fisher Scientific, Waltham, MA).

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Isolate Collection

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Pore fluid samples from the bio-stimulation tank were sequentially diluted 10-fold in sterile 9

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g/L sodium chloride and 100 µL subsamples were spread on alkaline culturing agar. Colonies

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most closely matching the morphology of S. pasteurii ATCC 11859 were purified by a minimum

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of 4 sequential passages of isolated colonies. Following liquid propagation in alkaline culturing

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broth, cultures were preserved at -80°C with 20% (v/v) glycerol. Sporosarcina pasteurii ATCC

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11859 was obtained from the American Type Culture Collection and maintained as a frozen

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stock as detailed above.

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Culturing Media

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Alkaline culturing broth was modified from ATCC 1376 Bacillus pasteurii NH4-YE medium

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and contained 20 g/L yeast extract, 75 mM ammonium sulfate, 130 mM tris base in the broth,

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with the addition of 16 g/L agar for plating medium. All components were autoclaved separately

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and combined once cooled to 55°C. The final pH was approximately 8.5. Urease test slants were

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prepared based on the methods of Christensen.33 A solution of 1 g/L peptone, 1 g/L dextrose, 5

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g/L sodium chloride, 2 g/L potassium phosphate monobasic, 2.4 mL/L 0.5% phenol red, 15 g/L

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agar, was pH adjusted to pH 6.9, autoclaved, supplemented (after cooling to 55°C) to a final



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concentration of 333 mM urea from a filter-sterilized stock, and divided into 3 mL aliquots in

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angled screw cap 12 mm culture tubes. Isolated colonies from fresh plates were streaked onto the

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surface of slants, and urea hydrolysis was determined by the appearance of a pink color within

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48 hours at 30°C.

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Phylogenetic Characterization

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Isolate cultures were grown overnight at 30°C in alkaline culturing broth. DNA was extracted by

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re-suspending a small (≈50 µL) pellet of the overnight culture in an alkaline PEG solution (60 g

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200 molecular weight polyethylene glycol (PEG), 930 µL 2 M potassium hydroxide, 39 mL

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distilled water).34 The 16S rRNA gene was amplified with a high-fidelity DNA polymerase using

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the

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(GGTTACCTTGTTACGACTT) and sequenced with the same primers. Sequences were edited

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and assembled using CodonCode Aligner v 7.0.1 (CodonCode Corporation, Centerville, MA)

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and aligned and classified by most similar homologous strain using the Ribosomal Database

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Project’s online tools.35 Bayesian inference into the phylogeny of the cultured isolates was

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completed in MyBayes 3.236 using a general time reversible model with gamma substitution rate

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distributions and no invariable sites.

universal

bacterial

primers

8F

(AGAGTTTGATCCTGGCTCAG)

and

1492R

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Whole cell urease kinetics

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Urease kinetic properties were determined for the ATCC strain of Sporosarcina pasteurii and 6

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isolates chosen to represent a subset of the distinct lineages observed: an additional strain of S.

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pasteurii, three of S. soli and one each of S. aquimarina and Bacillus lentus. Increases in

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solution electrical conductivity during urea hydrolysis results from the production of charged


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species (HCO3-, CO32-, NH4+, OH-) from the uncharged molecule urea. Solution conductivity

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measurements can therefore indirectly monitor urea degradation, enabling the investigation of

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initial rate enzyme kinetics. Overnight cultures in alkaline medium with 75 mM urea were

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centrifuged and re-suspended twice in one volume and once in one-quarter volume of 30 mM

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HEPES pH 8.5 to wash and concentrate the cells. Between 500 and 1000 µL cell suspension,

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based on suspension activity, was added to 30 mM HEPES pH 8.5 and the conductivity was

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measured once every five seconds for 2 minutes using a YSI 3200 conductivity meter with a

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glass fill cell (YSI Incorporated, Yellow Springs, OH) to establish a baseline slope, which was

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generally less than 1% of the assayed rate. 5 M urea was added to solutions to final

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concentrations between 20 and 600 mM, bringing the total solution volume to 5 mL and

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measurements continued for 3 minutes. Conductivity measurements were compared to a

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conductivity standard curve created by adding 0.01 to 10 mM ammonium carbonate to 30 mM

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HEPES pH 8.5. Initial rates were typically determined, between 30 and 60 seconds after urea

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addition, by excluding as little data as possible to achieve random residuals. Total protein of the

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cell suspension was determined by incubating a 10-fold dilution in 10% trichloroacetic acid for

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an hour at 90°C, precipitating overnight at 4°C followed by centrifugation, and re-suspending the

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pellet in 0.1 M sodium hydroxide. This solution was quantified by a Bradford37 based

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colorimetric assay using an egg white albumin standard curve. Bulk rates, corrected for baseline

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slope, were standardized by protein concentration and used to determine specific rates for tested

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urea concentrations. A non-linear fit of these data using the Michaelis-Menten kinetic model was

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used to estimate whole cell urea hydrolysis half saturation (KM) and maximum rate (Vmax)

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parameters.

200



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Results

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Samples taken from the bio-stimulation tank on days 1 and 3 (bio-stimulation phase) and days 5,

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12 and 13 (cementation phase), yielded a total of 34 pure culture isolates (Figure 1). These

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represent at least 14 distinct phylogenetic lineages (Figures 1, 2, Table S1) and were obtained by

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plating 0.1 ml aliquots from dilutions ranging from 10-1 to 10-5, strongly suggesting that the most

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abundant strains were present at a minimum of 106 colony forming units (cfu) per ml. Day 1

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cultures were from pore fluid samples taken before addition of stimulation solutions; thus, the

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strains were initially present, including as spores, at roughly 103 cfu ml-1. The majority of these

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cultures (27 of 34) were judged by 16S rRNA sequence to belong to the genus Sporosarcina,

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with 6 different species represented (Figure 1, Table S1). The remaining 7 strains examined fell

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in closely related genera, Bacillus and Oceanobacillus. Strain diversity was highest prior to

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treatments (day 1) and at the end of the cementation phase (day 12/13), with only phylotypes of

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S. soli detected on days 3 and 5 (Figure 1). Single cultures of S. pasteurii, obtained on days 1 and

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12 from the bio-stimulation tank, were genetically distinct from ATCC 11859 at 20 out of 1389

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and 8 out of 1415 positions compared, respectively (see also Figure 2). Only two of the tabulated

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34 strains were urease negative: LS38 O. luteolus and LS47 S. aquimarina, both isolated from

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day 12 samples.

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For the bio-augmented tank, all 7 isolates obtained on day 5 (Figure 1) were genetically identical

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to each other, to the RDP sequence data for S. pasteurii ATCC 11859, and to our sequence data

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for this strain (Figure 2, Table S1), which had been added at approximately 1013 cells 15 hours

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earlier. Day 12 and 13 aggregate data (Figure 1) for 16 isolates show that 12 belonged to the

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genus Sporosarcina, with 10 of those being S. soli. There was also a single isolate each of S.


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aquimarina and S. pasteurii, with the latter differing from S. pasteurii ATCC 11859 by 22

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nucleotides. All but 3 of the total 23 bio-augmentation tank isolates were urease positive (Table

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S1). The aqueous samples that yielded the 14 (bio-stimulation) or 16 (bio-augmentation) isolates

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from day 12/13 were from locations (Table S1) that spanned most of the mid-height cross section

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of each tank, i.e. ports A, B, E, G and J for both plus Well 2 for stimulation and port D for

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augmentation. The physical location corresponding to each letter is shown in right most urea

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panels of Figure 4. Figure 1 significantly underrepresents the fine scale diversity of our isolates,

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binning the sequence data based on reference 16 S rRNA sequences only from pure cultures.

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This approach detects 2 clades/strains of S. soli and 3 of S. pasteurii in our aggregate data for 57

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isolates. An examination of the same sequence data, using grouping defined by posterior

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probability support values greater than 0.50 (Figure 2), identifies 11 distinct clades of S. soli and

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4 of S. pasteurii.

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Great kinetic diversity in whole-cell urease activity was found among the 7 tested strains (Figure

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3), with whole cell Vmax and KM values covering 120-fold and 15-fold ranges, respectively. The

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strain of S. pasteurii employed in the bio-augmentation tank had the highest Vmax (48 U mg prot-

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1

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strain (LS09) isolated from the bio-stimulation tank on day 1 were both significantly lower 12 U

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mg prot-1 and 150 mM urea. The three isolates of S. soli tested exhibited a wide range of kinetic

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parameters: strain LS28 (stimulation, day 5) showed the highest values both for KM and Vmax;

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strain LS63 (day 13, augmentation) showed the lowest values, and strain LS57 (day 12,

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augmentation) possessed intermediate values. Plots of hydrolysis rate versus urea concentration

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fit well with the Michaelis-Menten equation for five of the seven tested strains (Figure S2). For

) and an intermediate value of KM (301 mM). The corresponding parameters for an S. pasteurii



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the two strains with highest rates over the range of urea concentrations tested (S. pasteurii ATCC

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11859 and S. soil LS57), the data become quite erratic above 300 mM urea.

249 250

Measurements of solution pH and urea concentrations at various times following uniform

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stimulation on day 3 to 4 are presented in Figure 4 along with comparable measurements after a

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single cementation injection from Well 2 to Well 3 on day 12 to 13. It should be noted that the

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20- and 21-hour conditions are reflective of solution conditions immediately prior to the next

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treatment injection following precipitation and ureolysis reactions occurring during solution

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retention. Within 2 hours of stimulation on day 3, tank pH was uniformly high (9.35 to 9.65) and

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constant until the next injection. Although urea concentration was slightly higher near the last

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injection well (Well 2), it was uniformly distributed laterally after injection and declined over

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time in a similar manner across the entire tank, with detectable concentrations still present after

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hour 21 in some locations. In contrast, the cementation injection shown for day 12 to 13 (Figure

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4b) illustrated much more dynamic conditions both spatially and temporally. Immediately after

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injection, urea concentrations increased above 250 mM and solution pH decreased to near 7.5

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along the main flow path from Well 2 to Well 3 as the result of solution replacement. At this

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same time, a region near Well 1 remained nearly untreated with urea near 0 mM and solution pH

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near 8.5. Chemical contours show that the majority of urea hydrolysis was completed within a 2

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to 4 hour period, with most sampling locations showing urea near 0 mM and increases in

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solution pH to above 8.5.

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Discussion

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Isolate Diversity and Source


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This study represents the first comparison of bacteria cultured from bio-stimulated and bio-

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augmented ureolytic MICP treatments that were equally successful – as judged by similar

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engineering improvement and calcite precipitation yield between tanks – and performed in

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parallel, confined, meter-scale systems using identical native sands. Beginning 20 years ago2, the

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ATCC strain 11859 of Sporosarcina pasteurii (reassigned from the designation Bacillus

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pasteurii)38 gained primacy for driving bio-augmented MICP in an expanding array of

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geotechnical engineering applications.

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given the proliferation of Sporosarcina in the current bio-stimulation study. However, failure to

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culture the ATCC strain from day 12 or 13 of the bio-augmentation tank, in spite of introducing

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1013 cells 7 or 8 days earlier, strongly suggests that it is a poor competitor versus native strains in

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the sand. The array of enriched Sporosarcina strains characterized here, including specific

281

prevalence of S. soli strains, suggests that these are better suited to survive and proliferate in the

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conditions examined in this study.

283

A rationale for the isolation of indigenous ureolytic bacteria with colony morphologies like that

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of augmented S. pasteurii type strain ATCC 11859 was a concern that similar soil improvement

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and bio-cementation distributions observed in both tanks (Figure S1) might have reflected the

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activity of strain 11859 potentially resulting from accidental contamination of the bio-stimulation

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tank. This concern appears to have been unfounded, but the possibility should be considered that

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strain 11859 remained active in both tanks, without representation in pore water samples beyond

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day 5, due to steadfast attachment to soil particles and/or entombment in deposited calcite.

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Detection of non-augmented strains of Sporosarcina pasteurii from both tanks on days 12/13

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(Figure 1) renders this hypothesis unlikely. It is also important to consider the possibility that the

292

ureolytic bacteria cultured in this study were enriched, not from the sand, but as contaminants

At one level the initial species choice was prescient



293

from initially non-sterile surfaces in the tanks or the non-sterile solutions added daily. The

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strongest evidence that they were not contaminants comes from a recent demonstration27 wherein

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sands, from the same quarry as this experiment, achieved bio-stimulated MICP under conditions

296

where all components of the experiment, except the sand, were initially sterile. In that study,

297

where the sand was the only possible source of bacteria, 7 distinct strains of Sporosarcina spp.

298

were isolated, with 3 strain clusters being among the 9 identified in the current study (Graddy,

299

unpublished).

300

Bio-stimulated MICP Versus Classical Bacterial Enrichment

301

As a specific method, bio-stimulation to promote ureolytic MICP is an example of the much

302

broader “enrichment culture” approach, long practiced by microbiologists and producing much

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of our current understanding of the vast metabolic and physiological diversity of prokaryotes.39

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Passing an inoculum of soil through sequential liquid enrichment cultures typically results in

305

dominance by one or a few bacterial types best adapted to the “predetermined conditions,” i.e. to

306

the experimenter-selected sources/values of energy, organic carbon, electron acceptor, nitrogen,

307

pH, salinity, temperature, pressure, etc. Because the native sand in the bio-stimulation tank of the

308

current study was treated once daily for 12 days with an enrichment medium containing a modest

309

quantity of organic matter and an extremely high concentration of urea, it was reasonable to

310

expect dominance by one or a few strains of bacteria best adapted to those conditions. Thus, the

311

wide diversity of urease positive species recovered from the end of this experiment was

312

surprising. This variety may result from temporally dynamic lateral gradients in chemical

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conditions occurring spatially across tanks, which were especially pronounced during non-

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uniform cementation solution injections (Figure 4b). This heterogeneity leads to numerous

315

ecological niches in which bacteria can persist and is postulated to prevent domination by a


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single strain or species, contrary to the concept of a strict bacterial enrichment. The reduced

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diversity from samples obtained on days 3 and 5 (Figure 1) is postulated to reflect the treatment

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pattern during stimulation (Figure 4a), which resembles a more uniform classical enrichment

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procedure, with nutrients replaced daily. It is not yet known which of these isolates had the

320

largest contribution to bulk ureolysis in the bio-stimulation tank, or if different strains

321

contributed differentially at different times following daily injections and during different

322

treatment phases. There appears to be considerable functional redundancy among the enriched

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strains of Sporosarcina, Bacillus, and Oceanobacillus, as all but five are ureolytic and could

324

catalyze MICP in pure culture. The species diversity and ubiquity of ureolytic activity within the

325

enrichment supports the hypothesis that the observed bio-stimulated MICP was robust and not

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dependent on the presence of one or a few essential ureolytic bacterial strains in the selected

327

sand.

328

Because we were targeting colonies with morphology like that of S. pasteurii ATCC 11859 and

329

were not regularly sampling from colonies that arose from the highest successful plating

330

dilutions, few conclusions can be drawn from our data (Table S1) about absolute strain

331

abundance in sampled fluids or even relative abundances of different strains. Also unanswered

332

are questions about whether strains cultured on the medium of the current study are a fair

333

reflection of those suspended in the effluent and whether effluent populations accurately reflect

334

attached plus suspended populations in the treated, saturated sand layer. Nonetheless, cultures

335

analyzed in this study comprise a valuable reference collection for addressing these questions in

336

future studies using molecular approaches.

337

Urease Parameters: Kinetics and Comparison with Alternate MICP Strategies



338

Modeling whole cell ureolysis with apparent Michaelis-Menten parameters seems fully justified

339

both by the form of the rate vs. concentration data (Figure S2) and by cycling of urea

340

concentrations, from greater than 300 mM to near zero, within both tanks following each

341

treatment (Figure 4). In less dynamic systems, others have successfully modeled S. pasteurii

342

ureolysis using first-order kinetics.21,

343

parameters are sparse. A study of whole cells of S. pasteurii ATCC 11859 yielded KM values

344

comparable to ours (305 to 359 mM versus 301 mM);41 however, no comparisons were possible

345

between Vmax values, which were normalized to cell numbers measured by plate counts in that

346

study, and vary significantly for this strain based on choices of diluent and plate medium.

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Hammes et al.42 report kinetic parameters for ureolytic strains isolated from MICP treatments.

348

While their Vmax values for crude cell extracts from 4 S. pasteurii-like isolates match well the

349

value for our S. pasteurii LS09 isolate (Figure 3), their KM values are an order of magnitude

350

lower. This is to be expected, because the bacterial cell membrane – a diffusional barrier to urea

351

transport, which we believe is central to accurate modeling of MICP – was destroyed in their

352

sample preparation scheme.

40

Literature comparisons for our calculated kinetic

353 354

Urea hydrolysis by Sporosarcina pasteurii has been the biological process and organism most

355

fully explored to drive MICP. Nonetheless, other biological processes, namely sulfate reduction,

356

iron reduction, and denitrification have also been proposed.1, 29 The range of whole cell urease

357

kinetic properties reported here for the 6 strains isolated from both tanks represents the first

358

opportunity to compare their ability to produce carbonate species (in CO2 equivalents) and

359

alkalinity (essential for driving the inorganic carbon equilibrium toward carbonate to promote

360

precipitation) with available literature rates for denitrifying, iron-reducing and sulfate-reducing


Page 16 of 33

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361

pure cultures. Presented here (Figure 5, Table S2) are the ranges of production rates for

362

heterotrophic bacteria respiring anaerobically with these other electron acceptors. Data, including

363

stoichiometry for reduction of iron oxyhydroxide, are from literature studies43-46 and from rates

364

calculated for the 7 strains of this study using KM and Vmax data (Figure 3) at 350 mM urea, the

365

initial concentration of urea used in this experiment. The type strain of S. pasteurii, used in the

366

bio-augmentation tank, produces alkalinity and carbonate species at rates that are roughly 2

367

orders of magnitude greater than the most active of the other proposed biological processes.

368

Rates for the isolated ureolytic strains ranged from approximately 80% of the corresponding rate

369

for the S. pasteurii type strain to 2 orders of magnitude lower. Even the lowest measured rates

370

for the indigenous ureolytic isolates were on the same order as the highest known rates for the

371

other metabolic processes (Fig 5 Table S2). These data agree with the much slower rate of MICP

372

reported in denitrification soil column experiments8 wherein achieving a low calcite content

373

(near 2% by mass) required 100 days. In comparison, nearly twice this amount of bio-

374

cementation was achieved along the Well 1 to 2 path in both tanks after 8 days (Figure S1).

375 376

Potential field implementations of MICP are likely to impose practical time constraints on the

377

treatment frequency and overall duration of projects. To this end, much attention has been paid

378

to achieving the highest production rate of carbonate and alkalinity. However, it is unknown

379

what effect, if any, a lower rate of calcite precipitation may have on bio-cemented soil behavior.

380

This might be achieved, for example, by identifying treatment conditions that favor strains with

381

low Vmax values. At a minimum, lower production rates may present an opportunity to improve

382

the spatial uniformity of treatment by reducing the extent of hydrolysis and precipitation

383

reactions that occur during solution injection. With the hundred-fold range of specific activity



384

represented in urea hydrolytic MICP (Figure 5), stimulation protocol modifications may enable

385

increased selectivity for specific strains to achieve further control of process kinetics, increased

386

MICP treatment uniformity, and reductions in material consumption.

387

MICP-Adaptive Features of the S. pasteurii phenotype

388

All the strains isolated and characterized here exist within a single bacterial phylum, the

389

Firmicutes. Although bacterial representatives from other phyla are known to be urease

390

positive,47 the capacity to form true endospores, which is confined to the Firmicutes among all

391

bacterial phyla, is undoubtedly among the most decisive factors contributing to the presence and

392

survival of Sporosarcina and relatives in this low-organic quarried sand prior to enrichment for

393

ureolytic bacteria. However, other physiological properties of S. pasteurii appear to contribute to

394

the prevalence of this species and ureolytic relatives in bio-stimulated MICP enrichments.

395 396

Limited physiological studies on S. pasteurii offer additional possible explanations for

397

dominance of this group, including special tolerance for extremely high concentrations of

398

ammonia even in alkaline pH environments. Full hydrolysis of 350 mM urea in our bio-

399

stimulated MICP procedure can generate a total ammonia concentration (NH3 + NH4+) that

400

approaches 700 mM. Among 26 species of Firmicutes tested by Leejeerajumnean, et al.,48 it was

401

demonstrated that only S. pasteurii and one other species could tolerate an NH3 concentration of

402

500 mM (pH = 9, [NH3] + [NH4+] = 1431 mM), with the maximum concentration tolerated not

403

determined. As the environmental pH shifts from neutrality toward 9.3 during stimulation (pKa

404

for the ammonia/ammonium equilibrium) a greater fraction will be present as NH3. This non-

405

ionic form, unlike NH4+, is freely permeable across bacterial cell membranes. In bacterial

406

cytoplasm, this NH3 reaches a pH dependent equilibrium with NH4+ thereby generating


Page 18 of 33

Page 19 of 33


407

alkalinity. At a constant pH, as the external concentration of NH3 and NH4+ increases, or as the

408

external pH increases, NH3 flux across bacterial membranes and subsequent alkalization of

409

bacterial cytoplasm both increase, thereby raising the cytoplasmic pH. Most soil bacteria are

410

expected to be neutrophilic and therefore rely on abundant, energy-dependent, homeostatic

411

mechanisms to maintain their cytoplasmic pH between 7.5 and 8.0.49 Because the pKa of the

412

ammonia/ammonium equilibrium is significantly above the highest cytoplasmic pH tolerated by

413

neutrophilic bacteria, bio-stimulation of alkali-tolerant bacteria with high urease activity, e.g., S.

414

pasteurii and relatives, will generate an alkaline, ammonia-rich environment within which the

415

metabolism of a typical neutrophilic soil bacterium will be uncoupled. These bacteria will

416

therefore be forced to constantly expend energy on maintaining pH homeostasis against constant

417

NH3 influx, leaving limited energy for growth and other maintenance functions. By contrast, the

418

limited biophysical evidence available shows that S. pasteurii can tolerate an internal pH greater

419

than 9 and can grow in a complete medium at an external pH of 10 or higher.50-52 One strain each

420

of S. pasteurii and S. ureae were examined and found to express urease constitutively; thus, they

421

are always enzymatically prepared to hydrolyze urea.53, 54 As an adaptive strategy, this implies

422

that as soon as these bacteria encounter a urea-rich environment they rapidly render that

423

environment unsuitable for otherwise potentially competing neutrophilic bacteria enabling them

424

to dominate in bio-stimulation treatments.

425 426

The quantity of organic matter in bio-stimulation and bio-cementation solutions applied in this or

427

other studies is high enough to rapidly deplete the oxygen concentration in soil columns during

428

any stimulation or cementation treatment cycle. S. pasteurii is an obligate aerobe,55 which raises

429

questions about how well it survives and competes under anoxic conditions with other bacteria



430

capable of fermentation or anaerobic respiration. The principal organic carbon sources in our

431

stimulation solution, acetate and peptides in yeast extract, are anaerobically metabolized only

432

very slowly and under a restricted range of conditions by a narrow range of fermentative

433

bacteria. Additionally, applied solutions lack alternate electron acceptors. Collectively, these

434

observations suggest that the ability to survive during the extensive anoxic portion of each

435

stimulation- or cementation-cycle is significant for determining the bacteria best adapted to these

436

enrichment conditions. Importantly, the ability to generate a proton motive force and ATP,

437

fueled by urea hydrolysis, has been demonstrated in the laboratory for the type strain of S.

438

pasteurii.52 Experimental conditions that maximized energy production in this strain driven by

439

urea hydrolysis included: growth in the presence of high quantities of urea followed by washing

440

to greatly reduce the external ammonia concentration followed by re-suspension in fresh medium

441

containing urea. These are very similar conditions to those experienced daily by enriched, native

442

Sporosarcina relatives in bio-stimulated sands when a fresh volume of stimulation or

443

cementation solution is applied.

444 445

In total, an impressive suite of adaptations may account for the singular success of relatives of S.

446

pasteurii in the enrichment conditions employed in our bio-stimulated MICP treatments. These

447

include: possession of spores to enhance survival in dry and non-nutritive environments,

448

constitutive urease activity. production of an ammonia waste that will uncouple the energy

449

metabolism of competing neutrophilic bacteria. the ability to tolerate high cytoplasmic pH, and

450

the possibility of generating ATP during at least part of the anoxic portion of each stimulation or

451

cementation injection cycle. While ammonia- and alkali-tolerance and constitutive urease

452

activity are seemingly common among the isolates in this study, whether this full suite of


Page 20 of 33

Page 21 of 33


453

properties extends to some, most, or all of the ureolytic bacteria in bio-stimulated MICP, and

454

more specifically to the strains we isolated, remains to be determined. The superior competitive

455

ability of these isolates in stimulated native sands, when compared to the S. pasteurii type strain,

456

offers a reasonable prospect that they do.

457

Acknowledgements

458

This material is based upon work supported by the National Science Foundation grant CMMI-

459

1234367 and the Engineering Research Center Program of the National Science Foundation

460

under NSF Cooperative Agreement No. EEC‐1449501. Any opinions, findings and conclusions

461

or recommendations expressed in this material are those of the authors and do not necessarily

462

reflect the views of the National Science Foundation. The authors would also like to thank Collin

463

Anderson, Alana Erickson, Matthew Havey, and Jason Wong of the University of California,

464

Davis for their assistance with the project and Teichert Aggregates Woodland for providing sand

465

material.

466 467 468

Supporting Information

469

Supporting information is available free of charge and contains two figures and two tables.



470

References

471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

(1) DeJong, J.; Mortensen, B.; Martinez, B.; Nelson, D., Bio-mediated soil improvement. Ecological Engineering 2010, 36, (2), 197-210. (2) Ferris, F.; Stehmeier, L.; Kantzas, A.; Mourits, F., Bacteriogenic mineral plugging. Journal of Canadian Petroleum Technology 1997, 35, (08), 56-61. (3) Stocks-Fischer, S.; Galinat, J.; Bang, S., Microbiological precipitation of CaCO3. Soil Biology & Biochemistry 1999, 31, (11), 1563-1571. (4) DeJong, J.; Fritzges, M.; Nusslein, K., Microbially induced cementation to control sand response to undrained shear. Journal of Geotechnical and Geoenvironmental Engineering 2006, 132, (11), 1381-1392. (5) Stumm, W.; Morgan, J. J., Aquatic chemistry : chemical equilibria and rates in natural waters. 3rd ed.; Wiley: New York, 1996; p xvi, 1022 p. (6) Ramachandran, S.; Ramakrishnan, V.; Bang, S., Remediation of concrete using micro-organisms. Aci Materials Journal 2001, 98, (1), 3-9. (7) Whiffin, V.; van Paassen, L.; Harkes, M., Microbial carbonate precipitation as a soil improvement technique. Geomicrobiology Journal 2007, 24, (5), 417-423. (8) van Paassen, L. A.; Daza, C. M.; Staal, M.; Sorokin, D. Y.; van der Zon, W.; van Loosdrecht, M. C. M., Potential soil reinforcement by biological denitrification. Ecological Engineering 2010, 36, (2), 168-175. (9) Bang, S. M., SH; Bang, SS, Application of Microbially Induced Soil Stabilization Technique for Dust Suppression. International Journal of Geo-Engineering 2011, 3, (2), 27-37. (10) Cuthbert, M. O.; McMillan, L. A.; Handley-Sidhu, S.; Riley, M. S.; Tobler, D. J.; Phoenix, V. R., A Field and Modeling Study of Fractured Rock Permeability Reduction Using Microbially Induced Calcite Precipitation. Environmental Science & Technology 2013, 47, (23), 13637-13643. (11) Gomez, M. G.; Martinez, B. C.; DeJong, J. T.; Hunt, C. E.; deVlaming, L. A.; Major, D. W.; Dworatzek, S. M., Field-scale bio-cementation tests to improve sands. Proceedings of the Institution of Civil Engineers-Ground Improvement 2015, 168, (3), 206-216. (12) Montoya, B. M.; DeJong, J. T., Stress-Strain Behavior of Sands Cemented by Microbially Induced Calcite Precipitation. Journal of Geotechnical and Geoenvironmental Engineering 2015, 141, (6), 04015019. (13) Montoya, B. M.; Dejong, J. T.; Boulanger, R. W., Dynamic response of liquefiable sand improved by microbial-induced calcite precipitation. Geotechnique 2013, 63, (4), 302-312. (14) Simatupang, M.; Okamura, M., Liquefaction resistance of sand remediated with carbonate precipitation at different degrees of saturation during curing. Soils Found 2017, 57, (4), 619-631. (15) Zamani, A., Liquefaction Mitigation of Silty Sands with Microbial Induced Calcium Carbonate Precipitation. 2017, PhD Thesis, University of North Carolina, Raleigh, USA. (16) Mitchell, A.; Ferris, F., The coprecipitation of Sr into calcite precipitates induced by bacterial ureolysis in artificial groundwater: Temperature and kinetic dependence. Geochimica Et Cosmochimica Acta 2005, 69, (17), 4199-4210.


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(17) Mitchell, A.; Ferris, F., Effect of strontium contaminants upon the size and solubility of calcite crystals precipitated by the bacterial hydrolysis of urea. Environmental Science & Technology 2006, 40, (3), 1008-1014. (18) Cheng, L.; Cord-Ruwisch, R., In situ soil cementation with ureolytic bacteria by surface percolation. Ecological Engineering 2012, 42, 64-72. (19) Fujita, Y.; Ferris, E.; Lawson, R.; Colwell, F.; Smith, R., Calcium carbonate precipitation by ureolytic subsurface bacteria. Geomicrobiology Journal 2000, 17, (4), 305-318. (20) Fujita, Y.; Taylor, J.; Gresham, T.; Delwiche, M.; Colwell, F.; McLing, T.; Petzke, L.; Smith, R., Stimulation of microbial urea hydrolysis in groundwater to enhance calcite precipitation. Environmental Science & Technology 2008, 42, (8), 3025-3032. (21) Tobler, D. J.; Cuthbert, M. O.; Greswell, R. B.; Riley, M. S.; Renshaw, J. C.; Handley-Sidhu, S.; Phoenix, V. R., Comparison of rates of ureolysis between Sporosarcina pasteurii and an indigenous groundwater community under conditions required to precipitate large volumes of calcite. Geochimica Et Cosmochimica Acta 2011, 75, (11), 3290-3301. (22) Gat, D.; Tsesarsky, M.; Shamir, D.; Ronen, Z., Accelerated microbial-induced CaCO3 precipitation in a defined coculture of ureolytic and non-ureolytic bacteria. Biogeosciences 2014, 11, (10), 2561-2569. (23) Gat, D.; Ronen, Z.; Tsesarsky, M., Soil Bacteria Population Dynamics Following Stimulation for Ureolytic Microbial-Induced CaCO3 Precipitation. Environmental Science & Technology 2016, 50, (2), 616-624. (24) Burbank, M.; Weaver, T.; Green, T.; Williams, B.; Crawford, R., Precipitation of Calcite by Indigenous Microorganisms to Strengthen Liquefiable Soils. Geomicrobiology Journal 2011, 28, (4), 301-312. (25) Gomez, M.; Anderson, C.; Graddy, C.; DeJong, J.; Nelson, D.; Ginn, T., LargeScale Comparison of Bioaugmentation and Biostimulation Approaches for Biocementation of Sands. Journal of Geotechnical and Geoenvironmental Engineering 2017, 143, (5), 04016124. (26) Gomez, M.; Anderson, C.; DeJong, J.; Nelson, D.; Lau, X., Stimulating in-situ soil bacteria for bio-cementation of sands. In Geo-Congress 2014 Technical Papers, 2014; pp 1674-1682. (27) Gomez, M. G.; Graddy, C. M. R.; DeJong, J. T.; Nelson, D. C.; Tsesarsky, M., Stimulation of Native Microorganisms for Biocementation in Samples Recovered from Field-Scale Treatment Depths. Journal of Geotechnical and Geoenvironmental Engineering 2018, 144, (1), 04017098. (28) Burbank, M.; Weaver, T.; Lewis, R.; Williams, T.; Williams, B.; Crawford, R., Geotechnical Tests of Sands Following Bioinduced Calcite Precipitation Catalyzed by Indigenous Bacteria. Journal of Geotechnical and Geoenvironmental Engineering 2013, 139, (6), 928-936. (29) Castanier, S.; Le Metayer-Levrel, G.; Perthuisot, J. P., Ca-carbonates precipitation and limestone genesis - the microbiogeologist point of view. Sediment Geol 1999, 126, (1-4), 9-23. (30) Hobbie, J. E.; Daley, R. J.; Jasper, S., Use of Nuclepore Filters for Counting Bacteria by Fluorescence Microscopy. Applied and Environmental Microbiology 1977, 33, (5), 1225-1228.



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(31) Parkhurst, D. L.; Appelo, C. A. J., User's guide to PHREEQC (version 2) : a computer program for speciation, batch-reaction, one-dimensional transport, and inverse geochemical calculations. U.S. Department of the Interior, U.S. Geological Survey: Denver, Colo., 1999; p xiv, 312 p. (32) Knorst, M. T.; Neubert, R.; Wohlrab, W., Analytical methods for measuring urea in pharmaceutical formulations. J Pharmaceut Biomed 1997, 15, (11), 16271632. (33) Christensen, W. B., Urea Decomposition as a Means of Differentiating Proteus and Paracolon Cultures from Each Other and from Salmonella and Shigella Types. Journal of Bacteriology 1946, 52, (4), 461-466. (34) Chomczynski, P.; Rymaszewski, M., Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Biotechniques 2006, 40, (4), 454-456. (35) Cole, J. R.; Wang, Q.; Fish, J. A.; Chai, B. L.; McGarrell, D. M.; Sun, Y. N.; Brown, C. T.; Porras-Alfaro, A.; Kuske, C. R.; Tiedje, J. M., Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Research 2014, 42, (D1), D633-D642. (36) Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D. L.; Darling, A.; Hohna, S.; Larget, B.; Liu, L.; Suchard, M. A.; Huelsenbeck, J. P., MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012, 61, (3), 539-42. (37) Bradford, M. M., Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-dye Binding. Analytical Biochemistry 1976, 72, (1-2), 248-254. (38) Yoon, J. H.; Lee, K. C.; Weiss, N.; Kho, Y. H.; Kang, K. H.; Park, Y. H., Sporosarcina aquimarina sp nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb. nov and Sporosarcina pasteurii comb. nov., and emended description of the genus Sporosarcina. Int J Syst Evol Micr 2001, 51, 1079-1086. (39) van Niel, C. B., The "Delft School" and the Rise of General Microbiology. Bacteriological Reviews 1949, 13, (3), 161-174. (40) Ferris, F. G.; Phoenix, V.; Fujita, Y.; Smith, R. W., Kinetics of calcite precipitation induced by ureolytic bacteria at 10 to 20 degrees C in artificial groundwater. Geochimica Et Cosmochimica Acta 2004, 68, (8), 1701-1710. (41) Lauchnor, E. G.; Topp, D. M.; Parker, A. E.; Gerlach, R., Whole cell kinetics of ureolysis by Sporosarcina pasteurii. J Appl Microbiol 2015, 118, (6), 1321-1332. (42) Hammes, F.; Boon, N.; de Villiers, J.; Verstraete, W.; Siciliano, S. D., Strainspecific ureolytic microbial calcium carbonate precipitation. Applied and Environmental Microbiology 2003, 69, (8), 4901-4909. (43) Carlson, C. A.; Ingraham, J. L., Comparison of denitrification by pseudomonasstutzeri, pseudomonas-aeruginosa, and paracoccus-denitrificans. Applied and Environmental Microbiology 1983, 45, (4), 1247-1253.


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(44) Marschall, C.; Frenzel, P.; Cypionka, H., Influence of Oxygen on Sulfate Reduction and Growth of Sulfate-reducing Bacteria. Archives of Microbiology 1993, 159, (2), 168-173. (45) Nealson, K. H.; Saffarini, D., Iron and Manganese in Anaerobic Respiration Environmental Significance, Physiology, and Regulation. Annual Review of Microbiology 1994, 48, 311-343. (46) Canfield, D. E.; Kristensen, E.; Thamdrup, B., Aquatic geomicrobiology. Gulf Professional Publishing: 2005. (47) Mobley, H. L. T.; Hausinger, R. P., Microbial Ureases - Significance, Regulation, and Molecular Characterization. Microbiological Reviews 1989, 53, (1), 85-108. (48) Leejeerajumnean, A.; Ames, J. M.; Owens, J. D., Effect of ammonia on the growth of Bacillus species and some other bacteria. Letters in Applied Microbiology 2000, 30, (5), 385-389. (49) Booth, I. R., Regulation of Cytoplasmic pH in Bacteria. Microbiological Reviews 1985, 49, (4), 359-378. (50) Wiley, W. R.; Stokes, J. L., Requirement of an Alkaline pH and Ammonia for Substrate Oxidation by Bacillus pasteurii. Journal of Bacteriology 1962, 84, (4), 730734. (51) Hoddinott, M. H.; Reid, G. A.; Ingledew, W. J., The Respiratory Chain and Proton Electrochemical Gradient in the Alkalophile Bacillus pasteurii. Biochemical Society Transactions 1978, 6, 1295-1298. (52) Jahns, T., Ammonium urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii. Journal of Bacteriology 1996, 178, (2), 403-409. (53) Kaltwasser, H.; Conger, W. R.; Kramer, J., Control of urease formation in certain aerobic bacteria. Archiv Fur Mikrobiologie 1972, 81, (2), 178-196. (54) Morsdorf, G.; Kaltwasser, H., Ammonium Assimilation in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina urea. Archives of Microbiology 1989, 152, (2), 125-131. (55) Berkeley, R. C. W.; Ali, N., Classification and Identification of EndosporeForming Bacteria. Journal of Applied Bacteriology 1994, 76, S1-S8. (56) ASTM, Standard Test Method for Rapid Determination of Carbonate Content of Soils. In ASTM D4373-14, ASTM International: West Conshohocken, PA, 2014.



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646

Figure Legends

647

Figure 1. Strain assignments and abundances of isolates collected on days 1, 3, 5, and

648

12/13 (data combined) from post-treatment effluent of bio-stimulation tank (34 strains

649

total) and on days 5 and 12/13 from effluent of bio-augmentation tank (23 strains total).

650

Data from days 12 and 13 were combined to achieve approximately equal strains

651

numbers for both treatments at end of experiment, where, based on daily cementation

652

treatments for 7-8 days, steady state is expected. Data are grouped by closest homologous

653

pure culture strain represented in the RDP database. A finer-scale analysis of strain

654

diversity is presented in Figure 2.

655

Figure 2. Bayesian 16S rRNA phylogenetic reconstruction of MICP isolates, homolog

656

type strains, reference strains, and the augmented cell line of S. pasteurii ATCC 11859.

657

Nodes are labeled with posterior probability support; database reference strains and the

658

injected strain are presented in bold face; non-ureolytic isolates are indicated with an

659

asterisk (*). Taxon symbols indicate tank of origin for 57 strains cultured in this study.

660

Sporosarcina pasteurii HQ676600 is the database entry for the ATCC 11859 strain.

661

Figure 3. Whole cell maximum velocity (Vmax) and half saturation constant (KM) of urea

662

hydrolysis at pH 8.5 for select isolates and S. pasteurii type strain. LS28, LS57 and LS63

663

are S. soli isolates; LS09, LS35, and LS54 represent S. pasteurii, B. lentus, and S.

664

aquimarina, respectively.

665

Figure 4. Temporal and spatial changes in urea concentration and pH over the course of:

666

(a) 21 hours of uniform bio-stimulation treatment (day 3 to 4); (b) 20 hours of spatially

667

asymmetric cementation treatment (day 12 to 13; bio-stimulation tank, injection at Well 2

668

overflow at Well 3). To limit potential spatial bias, the starting injection well was rotated



669

daily during stimulation; thus, well 2 was the final injection well on day 3. Within each

670

panel: top row of 4 images show (left to right) contours of pore fluid pH at 0, 2, 4 and 20-

671

21 hours post injection; bottom row of images show urea concentration at corresponding

672

times. Contours were interpolated based on samples taken mid-depth within the sand

673

layer.

674

Figure 5. Rates, for individual strains and metabolically grouped strains, of whole-cell

675

production of CO2 and alkalinity (expressed as OH-), which are the drivers of carbonate

676

production, essential for MICP. Equations used to calculate production ratios for CO2 and

677

OH- are shown in Table S2, along with supporting references. Urea hydrolysis rates were

678

calculated for 350 mM urea, pH 8.5, based on Vmax and KM values from the current study.


Page 32 of 33

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Diversity of Sporosarcina-like Bacterial Strains Obtained from Meter

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