Marine Georesources & Geotechnology, 33: 403–407

Copyright # 2015 Taylor & Francis Group, LLC

ISSN: 1064-119X print/1521-0618 online
DOI: 10.1080/1064119X.2014.909912

Bacteria-Induced Cementation Process in Loose Sand

Medium
IBRAHIM SEL1, HACER BILIR OZHAN1, RECEP CIBIK2, and ESRA BUYUKCANGAZ3
1
Department of Civil Engineering, Bursa Orhangazi University, Bursa, Turkey
2
Department of Food Hygiene and Technology, Uludag University, Bursa, Turkey
3
Department of Microbiology, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey

Received 3 August 2013, Accepted 12 March 2014

Several conventional methods for treatment of soil are available for engineers. The most widely used method includes solution=
grout, but they alter the ground pH level and could be toxic. In order to avoid the potential environmental harms and make
use of naturally occurring microorganisms, biological methods to improve the engineering properties of soils have been intensely
studied. This article presents the results of a research project in which Sporosarcina pasteurii (formerly known as Bacillus pasteurii)
was used to induce biological cementation in loose sand medium. The bacteria was injected into sand, which led to the development
of cementation (calcium carbonate crystal) between sand grains. The calcification was observed using a scanning electron
microscope (SEM).
Keywords: bacteria-induced cementation, biological method, calcification, loose sand

Introduction on kinetic details indicated that calcite precipitation is a

function of cell concentration, ionic strength, and pH in
Harnessing biological processes in soils promises to be the the medium. In addition, micro-organisms whose net cell
next transformative practice in geotechnical engineering. In surface charge is negative also draw cations from the
fact, many chemical processes are enabled or mediated by environment, including Ca2þ, to deposit on the cell surface.
biological processes. Though ignored for centuries with In this study, which is the first phase of a long-term pro-
respect to geotechnical behavior, bacteria are omnipresent ject focused on the development of biotechnological meth-
in soils at surprisingly high concentrations, almost regardless ods for large-scale geotechnical and=or civil engineering
of saturation, mineralogy, pH, and other environmental applications, the results of biocementation induced by
factors (DeJong et al. 2013). bacteria are presented.
Successful implementation of a biological process lies
in understanding the basic chemical mechanism involved.
Calcium and carbonate, to form calcium-carbonate crystal- Literature Review
line by bonding, should first dissolve in water. Bacteria first Below is a brief summary of what has been done in the
force them to melt and then tightly compact the resulting field of biomediation and its potential full-scale engineering
natural cement, leading to a concrete-type material. The applications.
formation of calcium carbonate is expressed as follows: Use of microbiological processes can be especially useful
Ca2þ þ CO3 2 $ CaCO3 # ð1Þ near or beneath existing structures, where the application
of traditional soil improvement techniques is limited because
The micro-biological interaction or calcite precipitation of the ground deformations and=or high cost associated with
was expressed by Ramakrishnan et al. (2001) as: alternative techniques. The successful application of micro-
Ca2þ þ HCO3  þ OH ! CaCO3 # þH2 O ð2Þ biological processes to soil improvement will likely depend
on a variety of factors, including the type of microbial
Ramakrishnan et al. (2001) further stated that microbial metabolism desired, interactions with other microbes present
CaCO3 precipitation has a complex mechanism. Their study in the environment, soil type, available nutrients, implemen-
tation, and reduced impact to the environment (Karatas
Address correspondence to Ibrahim Sel, Bursa Orhangazi 2008). The microbial-induced calcium carbonate precipi-
University, Mimar Sinan Mahallesi Mimar Sinan Bulvari, No: tation also known as the MICP treatment process (DeJong
177 Yildirim, Bursa 16310, Turkey. E-mail: [email protected] et al. 2006; Fritzges 2005) may be conceptualized by a zone
Color versions of one or more of the figures in the article of loose, saturated sand susceptible to geotechnical loading
can be found online at www.tandfonline.com/umgt. (e.g., shaking). Biological activity is initiated within the
404 I. Sel et al.

target treatment zone through augmentation of microbes et al. (2011) have performed a MICP experiment in artificial
into the subsurface, followed by discrete injections of nutri- ground water medium, inoculated with two model bacteria,
ents and chemical components to catalyze the precipitation S. pasteurii and B. subtilis, and supplemented with Nutrient
of calcite. The bacterial strain S. pasteurii utilizes the urease Broth and 7 mM of urea. Control was inoculated with S.
enzyme to convert urea to carbonate ions, which are marked pasteurii. Study results show that dissolved calcium was
by an increase in pH. Carbonate ions will precipitate with depleted at a higher rate in the presence of non-ureolytic
supplied calcium onto the bacterial cell wall to produce cal- bacteria, despite lower pH and CO32 concentration.
cium carbonate (Stocks-Fischer et al. 1999). S. pasteurii is Martinez et al. (2009) have presented an overview of bio-
chosen for laboratory experiments due to its highly active mediated soil improvement systems in the context of
urease enzyme, which catalyzes the reaction network toward bio-mediated calcite precipitation of sands. Micro-scale
precipitation of calcite (Fujita et al. 2000). and macro-scale investigations of MICP identify funda-
From an engineering perspective, it is desirable to link the mental material properties and mechanical characteristics
bio-geochemical processes occurring locally in soil structure of biocementation. Comparison of load tests on the footing
with changes in the bulk soil properties as they relate to the for MICP-treated and untreated experiments reveals up to a
performance of geosystems supported by bio-improved five-fold reduction in foundation settlement.
soils. The upscaling of micro-scale cementation to macro- For MICP to be effective, a microorganism must be selec-
scale system response is the objective herein. Advanced ted that is capable of CO2 production paralleled by a pH rise
microscopy techniques, including scanning electron in the surrounding environment to an alkaline level that
microscopy (SEM) and energy dispersive X-ray spec- induces precipitation of calcium carbonate. Aerobic micro-
troscopy (EDS), are used to visualize the reaction network organisms capable of consuming urea as an energy source
at the microscale (Han et al. 2009). A scaled, shallow are particularly good candidates because they provide two
foundation model treated with MICP is constructed and sources of CO2: respiration by the cell and decomposition
analyzed for settlement reduction of a footing underlain by of urea. B. pasteurii, a common bacterium naturally occur-
loose sands. A number of investigators have investigated ring in the subsurface, is such an aerobic microorganism.
the impact of biopolymers on saturated hydraulic con- In addition, cells of B. pasteurii do not aggregate; this
ductivity with respect to the potential for forming ensures a high cell surface to volume ratio, a condition that
hydraulic barriers, or biobarriers, to contaminant transport. is essential for efficient cementation initiation (DeJong et al.
Khachatoorian et al. (2003) performed a series of per- 2006).
meability tests to evaluate the ‘‘plugging’’ of fine sand by
biopolymer slurry impregnation using five different biopoly-
mers. Results of these tests demonstrated a permeability Material and Method
decrease of up to 14 orders of magnitude in fine sand in less
Culture
than two weeks. Kim et al. (2011) present a basic study on
the development of construction materials in an eco-friendly The reference strain S. pasteurii DSfM 33 (formerly known
manner by using biomineralization of effective micro- as B. pasteurii) was obtained from DSMZ (Leibniz Institute
organisms. The results of this experiment showed formation DSMZ-German Collection of Microorganisms and Cell
of calcite and hardening of mortar surface. SEM verified Cultures). The culture was activated in 5 ml of Tryptone
formation with precipitated calcite forming bonds at Soya Broth (Oxoid1 CM0129B) supplemented with 40%
particle-particle contacts. X-ray diffraction (XRD) analysis Urea solution (Oxoid1 SR0020K with) to have a final
confirmed that the observed calcite was composed of concentration of 3% urea (Figures 1 and 2). All reagents
calcium carbonate. were prepared according to DeJong et al. (2006). Briefly,
Hata et al. (2011) have proposed several soil improvement the pH of the medium was adjusted to 6.0 with 6 N HCl
techniques based on microbial functions. These techniques prior to autoclaving. Final concentration of CaCl2 was
can control soil strength and permeability. The ureolytic 25.2 mM. The culture was incubated for 48 hours in the
bacterium B. pasteurii was used to accelerate the precipi- humidified atmosphere at 30 C. DEN-1 Mac Farland
tation of calcium carbonate (CaCO3). They attempted to Densitometer MF-units (Biosan1) was used for adjusting
explain the scheme of these methods and illustrate the results the density of the bacterial suspension that was used for
of each experiment. Each technique controls the physical improvement of the stock culture. A bacterial culture that
property of soil and can be beneficial in reducing construc- was adjusted to 4 Mac Farland (60 109) was used as a work-
tion costs and lowering environmental impact. Inagaki ing culture and suspended in 25 ml Tryptic Soy Broth (TSB)
et al. (2011) have focused on how the amount of calcium car- media supplemented with CaCO3 þ 3% urea. Then the
bonate precipitation is affected by the injection conditions of suspension was added onto the 25 ml sterile standard sand
the microorganism and nutrient salt, such as the number of previously replaced in a 50 ml sterile vial having very thin
injections and the soil type. The results indicate that the holes in the bottom part, allowing the evacuation of used
amount of precipitation is affected by injection conditions media over the experimental incubation period. The
and soil type, suggesting that, in order for soil improvement bacteria–sand mixture was incubated at 22 C for three
by MCP to be effective, it is necessary to set injection con- weeks. Every three days, 25 ml of TSB containing
ditions that are in accordance with the soil conditions. Gat CaCO3 þ 3% urea suspension was added onto the mixture.
Bacteria-Induced Cementation in Loose Sand Medium 405

structure characteristics, namely the pore throats (DeJong

et al. 2006). Thus, this study employed DSM 33 S. pasteurii
and Gemlik Sand, whose properties are tabulated in Table 1.

Results and Discussion

The presence of CaCO3 on the face of sand grains and pores
of sand was detected by SEM. The results are given in
Figures 3–5 for
. Untreated sand,
. Sand with CaCO3, and
. Biologically treated sand medium.
As expected no bonding between sand grains exists in
Figure 3. The medium is totally loose. When some CaCO3
Fig. 1. Remarkable usease activitiy of DSM 33 in urea medium. was added into this medium, it was observed that the sand
DSM 33 inoculated culture is shown in the left image (pink). grains were covered with CaCO3 (please note the white color
around the grains) (Figure 4). However there is no bonding

Fig. 2. Gram stain was applied to Sporosarcina pasteurii (100x

magnification).
Fig. 3. Untreated sand.

One control group without bacteria was also tested. Each

experiment was performed in two replicates.
The properties of sand used in the present study are
tabulated in Table 1.

Scanning Electron Microscopy

The influence of microbial cementation on granular beha-
vior is dependent on the ability of microbes to freely move
either by locomotion or injection throughout the pore space
and on sufficient particle-particle contacts per unit volume at
which cementation will occur. These conditions require a
balanced relationship between the microbe size and the pore

Table 1. Properties of gemlik sand

D50 (mm) Cu Cc Gs emin emax Shape

0.35 2.5 1.09 2.60 0.52 0.60 rounded Fig. 4. Sand and CaCO3.
406 I. Sel et al.

evidence that these parts are nitrogen-rich regions. This

would result from the high urease activity of S. pasteurii,
which consumes urea and produces ammonia. These prelimi-
nary results suggest that the strain used has the potential to
produce bonds between sand grains in convenient con-
ditions, and the large presence of nitrogen in these regions
indicates the role of urea in these formations. Detailed stu-
dies are required to better understand the biocementation
processes in large-scale systems to be able to use this
technique in practical applications.

Conclusions
Biocementation using S. pasteurii (formerly known as B.
pasteurii) was achieved in this study. The bacteria were
injected into sand and led to the development of cemen-
tation (calcium carbonate crystal) between sand grains.
The calcification was observed using SEM. It was clearly
observed that new bonds between loose sand particles
developed as a result of the calcification process. This study
is the first phase of a long-term project that aims to develop
biotechnological methods for various large-scale applica-
tions. In order to achieve that goal, the improvement of
the mechanical properties of soil due to biocementation will
be studied.

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