J Ind Microbiol Biotechnol (2016) 43:567575

DOI 10.1007/s10295-015-1726-2

NATURAL PRODUCTS

Optimization ofgrowth medium forSporosarcina pasteurii

inbiobased cement pastes tomitigate delay inhydration kinetics
SarahL.Williams1 MaryJoKirisits1 RaissaDouglasFerron1

Received: 7 July 2015 / Accepted: 19 December 2015 / Published online: 21 January 2016
Society for Industrial Microbiology and Biotechnology 2016

Abstract Microbial-induced calcium carbonate precipi- Introduction

tation has been identified as a novel method to improve
durability and remediate cracks in concrete. One way to
introduce microorganisms to concrete is by replacing the
mixing water with a bacterial culture in nutrient medium.
In the literature, yeast extract often has been used as a
carbon source for this application; however, severe retar-
dation of hydration kinetics has been observed when
yeast extract is added to cement. This study investigates
the suitability of alternative carbon sources to replace
yeast extract for microbial-induced calcium carbonate
precipitation in cement-based materials. A combination
of meat extract and sodium acetate was identified as a
suitable replacement in growth medium for Sporosarcina
pasteurii; this alternative growth medium reduced retar-
dation by 75% (as compared to yeast extract) without
compromising bacterial growth, urea hydrolysis, cell
zeta potential, and ability to promote calcium carbonate
formation.
Keywords Biomineralization Hydration Calcium
carbonate Meat extract Yeast extract
Recently, interest has focused on microbial-induced cal-
cium carbonate precipitation (MICCP) as a technique to
improve properties of concrete and other porous construc-
tion materials. Ureolytic microorganisms can catalyze
the conversion of urea into ammonia and carbon dioxide
(Reaction 1), and these products can participate in acid
base reactions (Reactions24). These reactions trigger an
increase in pH that promotes calcium carbonate precipita-
tion if sufficient dissolved calcium is present (Reaction5)
[24, 29, 31]. Sporosarcina pasteurii is one of the most com-
mon microorganisms studied for biomineralization applica-
tions due to its urease activity and its alkaliphilic nature,
which gives it the ability to tolerate the alkaline conditions
of cement paste.
CO(NH2 )2 + H2 O 2NH3 + CO2 (1)
NH+
4 NH3 + H
+
pKa = 9.3 (2)
H2 CO3 HCO
3 + H
+
pKa = 6.3 (3)

2
HCO
3 CO3 + H
+
pKa = 10.3 (4)

* Raissa Douglas Ferron CaCO3 (s) CO2

3 + Ca
2+
Ksp = 3.8 x 109 (5)
rferron@mail.utexas.edu
Sarah L. Williams Studies utilizing MICCP as an external treatment for con-
sarah.l.williams@usace.army.mil crete, by applying microbial cultures as a surface treatment
Mary Jo Kirisits [1214] or crack remediation agent [1, 34], have demon-
kirisits@utexas.edu strated improvements in durability by reducing the permea-
1
Department ofCivil, Architectural, andEnvironmental
bility of the concrete. These applications are classified as
Engineering, University ofTexas atAustin, 301 East Dean external because microorganisms are not incorporated inside
Keeton Street, Stop C1700, Austin, TX 78712, USA the bulk material. However, substantial interest exists

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568
regarding the possibility of creating a self-healing concrete,
where internal cracks could be healed. This could be achieved
by incorporating microorganisms and their nutrients into the
cement paste matrix during the mixing process, with the
intention that these embedded microorganisms could then
trigger the MICCP process to heal cracks. This approach has
shown promise in initial studies [6, 42], but concerns remain
that microorganisms might not survive for an extended period
of time inside concrete due to unfavorable environmental
conditions (e.g., high pH, increase in temperature due to heat
generated by cement hydration, nutrient depletion). To
address these concerns, researchers have explored adding
bacterial cells in endospore form (i.e., dormant state) because
endospores are more tolerant of harsh conditions than are
vegetative cells (i.e., cells that are in a metabolically active
state) [7, 27]; some researchers have suggested providing fur-
ther protection by encapsulating or immobilizing endospores
in a protective carrier [5, 20, 28, 35, 36]. However, this
approach might not be economically feasible based on pre-
sent-day costs. Silva etal. [30] estimated that the cost of con-
crete at an industrial scale could be increased from 60 to
75 /m3 (approximately $5062/yd3)1 for ordinary concrete
to 5760 /m3 (approximately $4787/yd3) (see footnote 1) for
concrete mixed with encapsulated endospores. This sharp
increase in cost was attributed primarily to the need for asep-
tic laboratory conditions, labor, and the cost of encapsulating
endospores [30]. As such, one of the main conclusions of that
study was that a more cost-effective bio-based additive is
needed for MICCP to be implemented at the field-scale [30].
One way to achieve this goal is to use unencapsulated vegeta-
tive cells in microbial concrete, which avoids costs associated
with endospore formation and synthetic encapsulation.
Although it has been shown that the viability and metabolic
activity of vegetative S. pasteurii are impacted by elevated
temperature and high pH [39], a previous study of the vegeta-
tive-inoculation approach in our group showed that viable
S. pasteurii were still detected in hardened mortar samples at
330days, where 48% of those viable cells were in a vegeta-
tive state [6].
Another issue that might impact cost is the severe retar-
dation of cement hydration kinetics that has been observed
when microorganisms in their growth medium are incorpo-
rated into the cement paste matrix [6]. Specifically, the addi-
tion of yeast extract to cement paste has been shown to retard
cement hydration [9, 36]. Microbial media are designed to
provide the chemical requirements for cell growth including
free water, essential minerals, growth factors, and sources of
carbon, nitrogen, and phosphorus [10]. The optimal medium
recommended by the American Type Culture Collection
(ATCC) for 6453 S. pasteurii contains 0.13M Tris base (pH
1
Exchange rate: 1 USD=0.92 . determine the mass composition of oxides (Table1).
13
J Ind Microbiol Biotechnol (2016) 43:567575
9), 20g/L yeast extract (2%), and 10g/L ammonium sul-
fate (1%). Urea can be substituted for ammonium sulfate to
produce urease activity for MICCP (Reaction1). Tris base
controls the pH while yeast extract and ammonium sulfate
(or urea) serve as the primary carbon and nitrogen sources
for bacterial growth, respectively. While Tris base and urea
have been shown to have no adverse effect on cement hydra-
tion, a substantial extension of the induction period during
hydration has been observed when yeast extract is added to
cement paste [6, 9, 36]. In general, extension of the induc-
tion period corresponds to longer setting times, which could
result in longer construction times and increased costs [32].
As such, this is a significant barrier that must be overcome
for microbial concrete to become feasible.
Previous studies have addressed identifying alternative
nutrient sources for bacteria in microbial concrete [2, 3], but
none of these studies have examined the effects of alternative
nutrient sources on hydration kinetics. Instead, attention has
focused primarily on the use of waste materials as nutrients
[2, 3]. That approach is advantageous from an environmental
standpoint, and it also has been proposed that the use of waste
materials could alleviate the substantial costs associated with
using commercial nutrients [2, 3]; however, further cost anal-
ysis must be carried out to support this claim. While using
waste materials for bacterial growth media might reduce the
material costs for microbial concrete, other drawbacks associ-
ated with the use of waste materials must be considered. For
instance, the chemical composition and physical properties
of waste materials tend to vary considerably due to irregu-
larities and variations in processing [41]. As such, extensive
quality control and/or processing of waste materials might be
required prior to use in microbial concrete because microbial
growth and fresh-state properties of the concrete could be
heavily influenced by variation in the waste materials.
In the current study, the influence of alternative carbon
sources on cement hydration was explored for the first
time, and the chemical constituents of the growth medium
were optimized to reduce the retardation of cement hydra-
tion. Results were used to develop an improved medium for
S. pasteurii that has less impact on hydration (as compared
to medium containing yeast extract) while maintaining
similar bacterial growth, cell zeta potential, and ability to
promote calcium carbonate formation.
Microorganism andcement
ATCC 6453 S. pasteurii was used in this study; cells were
grown aerobically at 30C with shaking. An ASTM Type I/II
portland cement (Buda, TX, United States) was used for all
cement paste mixtures. X-ray Fluorescence was performed
using a Bruker S4 Explorer (Madison, WI, United States) to
J Ind Microbiol Biotechnol (2016) 43:567575 569

Table1Type I/II portland cement oxide composition; LOI: Loss on those conditions, S. pasteurii grew substantially in growth
ignition medium containing yeast extract, meat extract, corn steep
Oxides % (w/w) Composition liquor, or lactose mother liquor (Fig.1).
CaO 63.63
Isothermal calorimetry
SiO2 20.14
Al2O3 5.42
To examine the effects of the alternative carbon sources on
Fe2O3 2.47
hydration, isothermal calorimetry was performed on neat
MgO 1.32 paste and carbon source pastes. Neat paste refers to a mix-
SO3 3.09 ture that was prepared with distilled water and cement; the
Na2O 0.17 carbon source pastes were prepared with a carbon source
K 2O 0.95 solution (20g/L of carbon source dissolved in water) and
LOI 2.82 cement. Pastes were prepared by mixing 16g of cement
with either 8g of distilled water (for neat pastes) or 8g
of the carbon source solution by hand for 2min. Thus,
Carbon sources the solution-to-cement ratio (s/c) for all pastes was 0.5.
Immediately after a paste sample was prepared, 20g of
In addition to yeast extract (Sigma-Aldrich, St. Louis, MO, the sample were placed in a Thermometric AB 3114/3236
United States), five alternative carbon sources were exam- TAM Air Isothermal Calorimeter (Sweden) for 45h. The
ined: lactose mother liquor (Brewster Dairy, Inc., Brewster, temperature was maintained at 23C during testing. Dupli-
OH, United States), corn steep liquor (Sigma-Aldrich, St. cate specimens were prepared for each type of paste. At the
Louis, MO, United States), meat extract (Sigma-Aldrich, initial tested carbon source concentration of 20g/L (2%
St. Louis, MO, United States), glucose (Fisher Scien- (w/w)), cement pastes containing lactose mother liquor or
tific, Pittsburgh, PA, United States), and sodium acetate glucose exhibited a constant rate of heat generation during
(AMRESCO, Solon, OH, United States). This group the 45-h testing period, which indicated that hydration was
of potential carbon sources was selected to provide vari- severely retarded by these substances. As such, isothermal
ety, including both undefined and defined carbon sources. calorimetry was repeated with a lower concentration of lac-
Lactose mother liquor, corn steep liquor, and meat extract tose mother liquor and glucose (1% (w/w)) to produce data
are undefined carbon sources, meaning that their exact useful for comparative purposes.
chemical composition is unknown; glucose (C6H12O6) and The calorimetry results for all the pastes are pre-
sodium acetate (NaCH3COO) are defined carbon sources. sented in Fig.2, and the hydration kinetics varied

Evaluation ofcarbon sources

Growth tests

ATCC recommends the following standard liquid growth

medium for S. pasteurii (6453): 20g/L yeast extract, 10g/L
ammonium sulfate, and 15.75g/L Tris base (pH 9); urea
can be substituted for ammonium sulfate to induce urea
hydrolysis (Reaction1). The effects of the five alternative
carbon sources on microbial growth were examined by sub-
stituting each alternative carbon source for yeast extract in
the standard S. pasteurii growth medium on an equal mass
basis. Therefore, each medium contained 20g/L of the car-
bon source, 10g/L of urea, and 15.75g/L of Tris base (pH
9). S. pasteurii was grown in 200L of each medium in a
Fig.1Growth of S. pasteurii (averaged from five replicates) at 30C
96-well plate at 30C with shaking. Five wells were inocu-
in media containing 15.75g/L Tris base (pH 9), 10g/L urea (U), and
lated for each medium, and absorbance at 600nm (OD600) 20g/L of a carbon source: yeast extract (YE), meat extract (ME),
was recorded hourly using a BIO-TEK Synergy HT spec- corn steep liquor (CSL), lactose mother liquor (LML), sodium acetate
trophotometer (Winooski, VT, United States). Under (SA), or glucose (Glu)

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Fig.2Rate of heat evolution (averaged from duplicate samples) for
neat and carbon source pastes with an s/c of 0.5; yeast extract (YE),
meat extract (ME), corn steep liquor (CSL), lactose mother liquor
(LML), sodium acetate (SA), glucose (Glu); LML and Glu were
tested at a reduced concentration (1% (w/w)) as compared to the
other carbon sources (2% (w/w))
considerably among the carbon sources. The cement
paste containing sodium acetate did not exhibit any sub-
stantial retardation as compared to neat paste. However,
the addition of glucose caused severe retardation even at
a 1% (w/w) concentration, which was expected because
sugars are known to be effective retarders [33]. Due to
the poor performance of glucose with respect to hydra-
tion, it was removed from further consideration. Each of
the undefined carbon sources caused substantial retar-
dation at the tested concentrations, particularly lactose
mother liquor.
Chemical oxygen demand (COD)
COD was measured to determine the electron-donating
capacity of each undefined carbon source (Table2). The
reactor digestion method [4] was used, with potassium
hydrogen phthalate (KHP) as the standard. Both corn steep
liquor and lactose mother liquor exhibited a lower COD per
unit mass than did yeast extract; thus, on an electron-donat-
ing basis, a higher mass concentration of corn steep liquor
or lactose mother liquor is needed to yield an equivalent
COD to 2% (w/w) yeast extract. Corn steep liquor and lac-
tose mother liquor were removed from further considera-
tion because increasing the concentration of either of these
carbon sources in the growth medium would likely exac-
erbate the already substantial retardation demonstrated at a
2% (w/w) concentration (Fig.2). On the other hand, meat
extract contains 13% more COD per unit mass as com-
pared to yeast extract, such that 17.7g/L meat extract pro-
vides an equivalent COD to that in the 20g/L yeast extract
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Table2COD per unit mass of yeast extract, meat extract, corn steep
liquor, and lactose mother liquor; error represents the standard devia-
tion of triplicate measurements
Carbon source COD (mg O2/mg substance)
Yeast extract 0.5840.005
Meat extract 0.6600.010
Corn steep liquor 0.2740.005
Lactose mother liquor 0.2000.022
specified in the standard medium recommended by ATCC
for 6453 S. pasteurii.
Growth media selection
Based on results from the growth tests, isothermal calo-
rimetry, and COD measurements, two alternative media
were developed to compare with Urea-Yeast Extract
(UYE) medium. UYE medium is a modified version of the
medium recommended for S. pasteurii by ATCC, where
ammonium sulfate is replaced with urea on an equal mass
basis. Urea-meat extract (UME) medium was developed to
provide COD equal to that of the UYE medium, where the
yeast extract was replaced with 17.7g/L meat extract. A
hybrid medium, urea-meat extract-sodium acetate (UME-
SA) medium, was developed from UME medium by reduc-
ing meat extract content by half (to 8.85g/L) and adding
hydrated sodium acetate such that the total COD was equal
to that of UYE medium.
All media (i.e., UYE, UME and UME-SA) were pre-
pared by dissolving Tris base in 1 liter of distilled, deion-
ized (DDI) water and adjusting the pH to 9 with hydro-
chloric acid. The Tris base solution was then divided into
two equal volumes. The carbon source (yeast extract, meat
extract, or meat extract and sodium acetate) was added to
one volume, and urea was added to the other volume; the
solutions were autoclaved separately. After sterilization,
the urea solution and the carbon source solution were com-
bined after cooling.
Methods toexamine performance ofgrowth media
The bacterial growth media were examined for their
impact on bacterial growth, ammonia production via
urea hydrolysis (to infer carbonate production), and zeta
potential. Growth curves were obtained to verify growth
in each medium; ammonia production and zeta potential
were examined to gain insight about the metabolic activ-
ity and surface charge, respectively, of the cells in each
medium. The media mixed with cement were interrogated
J Ind Microbiol Biotechnol (2016) 43:567575 571
by isothermal calorimetry and quantitative X-ray diffrac-
tion (XRD). Isothermal calorimetry was employed to deter-
mine how each medium would impact cement hydration.
XRD was carried out on neat cement paste, nutrient cement
pastes, and bacterial cement pastes to assess the morphol-
ogy of the precipitated calcium carbonate and to quantify
the amount of calcite in each paste.
Isothermal calorimetry
Isothermal calorimetry was performed according to the
aforementioned procedure; neat paste and nutrient pastes
were examined. Nutrient paste refers to a mixture that was
prepared with bacterial growth medium and cement. The
solution-to-cement ratio (s/c) for all pastes was 0.5, and
triplicate specimens were prepared for each sample type.
Growth curves
To examine the growth of S. pasteurii in each medium,
OD600 for triplicate 100-mL bacterial cultures in 250-mL
flasks at 30C was recorded for up to 24h.
Urea hydrolysis
S. pasteurii cells grown in each medium were used to exam-
ine urea hydrolysis to ammonia and carbon dioxide (Reac-
tion 1). Triplicate 100-mL cultures in 250-mL flasks were
grown at 30C with shaking in each medium for 24h.
The cells were harvested by centrifugation at 7500g for
10min, resuspended in fresh medium, and incubated again
at 30C with shaking. Then, ammonia (NH3) concentra-
tions were measured using a Thermo Scientific Orion
High-Performance Ammonia Electrode (Waltham, MA,
United States) over 10h. To correct for the small amount of
NH3 present in fresh media at t=0, the NH3 concentration
was measured immediately after cell resuspension (t=0),
and this value was subtracted from all subsequent measure-
ments. The cumulative percentage of urea consumed was
calculated according to the stoichiometry of Reaction1,
which shows that 1mol of urea is hydrolyzed to form two
moles of NH3 and one mole of carbon dioxide. Because
each medium contains 10g/L urea, or 167mM urea, the
maximum amount of NH3 (denoted as NH3 (max) in Eq.6),
that can be produced in each culture is 333mM. The amount
of NH3 present at t=0 (denoted as NH3 (0)) was subtracted
from both the NH3 concentration at time x (denoted as NH3
(x)) and the maximum possible ammonia concentration, and
the percent urea consumed was calculated using Eq.6:
[NH3(x) ] [NH3(0) ]
% Urea consumed(x) = 100 %
[NH3(max) ] [NH3(0) ]
(6)
Electrophoretic mobility andzeta potential
Electrokinetic potential in a colloidal dispersion can be
determined from zeta potential measurements, and zeta
potential often is used to approximate the surface charge of
bacterial cells [40]. S. pasteurii cells grown in each medium
were harvested by centrifugation at 4000g for 6min. The
cells were then washed and resuspended in sterile 20mM
Tris buffer at pH 9 for testing. The isoelectric point of
bacterial cells has been reported as ranging from pH 2 to
pH 4, and the bacterial cells typically possess a net posi-
tive charge in medium with a lower pH than the isoelectric
point and a net negative charge in medium with a higher pH
than the isoelectric point [17]. Therefore, the zeta potential
of the bacterial cells in this pH 9 medium depicts the nega-
tive charge that the cells will likely possess when subjected
to the high pH environment of concrete. To compare zeta
potential of the bacterial cells to that of cement particles, a
small amount of cement was suspended in pH 9 Tris buffer
and analyzed. A Malvern Zetasizer Nano ZS (Malvern,
Worcestershire, United Kingdom) was used to measure
electrophoretic mobility, and the Henry equation (Eq.7)
was used to calculate zeta potential [26]:
2zf (Ka)
UE = (7)
3
where UE is the electrophoretic mobility, is the dielectric
constant, z is the zeta potential, f(Ka) is Henrys function,
and is viscosity. For Henrys function, f(Ka)=1.5, or the
Smoluchowski approximation, was used.
XRD
Sample preparation andoperating procedure
XRD was conducted using a Siemens Bruker X-ray Dif-
fractometer (Madison, WI, United States) to examine the
morphology of the calcium carbonate precipitate and to
quantify the amount of calcite in each sample. Three types
of pastes were prepared: neat paste, nutrient paste, and bac-
terial paste. It was expected that mainly calcite would be
precipitated in neat, nutrient, and bacterial paste samples
because calcite is the most common calcium carbonate
polymorph observed in ordinary cement paste and in bacte-
rial cement paste [9, 12, 38]. Neat and nutrient pastes were
prepared by mixing cement with distilled water or nutrient
medium, respectively. Bacterial paste was prepared by mix-
ing cement with S. pasteurii cultures in the mid-exponen-
tial growth phase (OD600 =0.6) grown in each medium.
Samples were prepared by mixing 40g of cement with 20g
of the appropriate aqueous component by hand for 2min.
Pastes were then cast into 33cm cylindrical molds and
cured at 100% relative humidity for 24h. At this time, the
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572 J Ind Microbiol Biotechnol (2016) 43:567575

specimens were removed from the molds, crushed with a media are more desirable than is UYE medium with respect
mortar and pestle, and passed through a No. 325 sieve to the criterion of hydration.
such that the particles were finer than 45m. The pulver-
ized material was then ground with ethanol to arrest hydra- Growth curves
tion [43], and 10% zincite was added to each sample as
an internal standard. The samples were stored in a vacuum Figure4 shows that S. pasteurii grew substantially in UYE,
desiccator until the time of testing. In general, the analysis UME, and UME-SA media.
was conducted from 2540 2 with 6s dwell time. The
diffractometer was operated at 40keV and 30mA, at a step Urea hydrolysis
size of 0.02 2. Triplicate specimens were prepared for
each paste. NH3 production by S. pasteurii in UYE, UME, and UME-
SA media are presented in Fig.5a. Figure5b shows the
Reference intensity ratio (RIR) procedure percent of urea consumed in each medium, as calculated
from the NH3 profiles.
Calcite was quantified in each paste using the RIR method
[19]. A ratio was established between intensities of the pri-
mary zincite (internal standard) and calcite peaks by cre-
ating samples with known mass proportions of calcite to
zincite and plotting the results on a standard curve. Calcite
content was varied from 2% (w/w) to 18% (w/w) while
zincite was kept at 10% (w/w); NaCl was selected as a
filler material. Calcite was quantified in each paste sample
according to the standard curve.

Statistical analysis

A two-sample t test with one-tailed distribution [11] was

used to determine whether the mean value of calcite con-
tent in bacterial pastes was greater than the mean value of
calcite content in nutrient pastes. An alpha of 0.01 was used
for all t tests, and critical values of t were obtained from
Fig.3Rate of heat evolution (averaged from triplicate samples) for
standard tables [25].
neat, UYE nutrient, UME nutrient, and UME-SA nutrient pastes with
an s/c of 0.5; dark circles represent the end of the induction period for
each paste
Results anddiscussion

Isothermal calorimetry

Results of isothermal calorimetry for neat cement paste,

UYE nutrient paste, UME nutrient paste, and UME-SA
nutrient paste are presented in Fig.3. The beginning of the
acceleratory period (or similarly, the end of the induction
period) occurred at approximately 2h for neat paste, 4.5h
for UME-SA nutrient paste, 7h for UME nutrient paste,
and 12h for UYE nutrient paste. Therefore, as compared
to neat paste, the induction periods for UME-SA, UME,
and UYE nutrient pastes were extended by 2.5, 5, and
10h, respectively. The use of UME-SA medium in nutri-
ent paste reduced the extension of the induction period by
75% as compared to standard UYE medium, and the use
of UME medium in nutrient paste reduced the extension Fig.4Growth of S. pasteurii at 30C in UME-SA, UYE and UME
of the induction period by 50% as compared to standard media; error bars represent the standard deviation for triplicate cul-
UYE medium. These data indicate that UME-SA and UME tures

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J Ind Microbiol Biotechnol (2016) 43:567575 573
Fig.5NHs concentration (a) and percent urea consumed (b) by S.
pasteurii at 30C in UYE, UME, and UME-SA media; error bars
represent the standard deviation for triplicate cultures
The substantial hydrolysis of urea (with concomitant
production of ammonia and carbonates) suggests that all
three media allow production of the carbonates needed
for biomineralization applications. However, the ammo-
nia concentration (due to urea hydrolysis) increased more
rapidly in UYE and UME-SA cultures as compared to the
UME culture (Fig.5a).
Electrophoretic mobility andzeta potential
It has been assumed in several studies that S. pasteurii has
a negative net surface charge because most bacterial cells
possess a negative zeta potential in neutral and basic envi-
ronments [8, 15, 21, 22, 37]. Further, it has been suggested
that this charge can attract positively charged calcium
ions and induce CaCO3 precipitation through heterogene-
ous nucleation at the bacterial surface [2, 16, 24, 31, 34].
Results of electrophoretic mobility and zeta potential meas-
urements are presented in Table3. S. pasteurii exhibited a
similar zeta potential in UYE, UME, and UME-SA media.
S. pasteurii cells exhibited a substantially more negative
Table3Electrophoretic mobility and zeta potential for S. pasteurii
grown in UYE, UME, or UME-SA medium and cement particles
suspended in 20mM Tris buffer (pH 9); error represents the standard
deviation for triplicate samples
Sample type Electrophoretic Zeta potential (mV)
mobility (m-cm/V-s)
S. pasteurii grown in 2.790.22 35.62.8
UYE
S. pasteurii grown in 2.560.30 32.73.8
UME
S. pasteurii grown in 2.550.11 32.51.3
UME-SA
Cement 0.080.02 1.00.3
Fig.6Mass percentages of calcite at 1day; error bars represent the
standard deviation of triplicate samples; Neat paste (Neat), Nutrient
paste: UME (Nut-UME), Nutrient paste: UYE (Nut-UYE), Nutrient
paste: UME-SA (Nut-UME-SA), Bacterial paste: S. pasteurii in UME
(Bac-UME), Bacterial paste: S. pasteurii in UYE (Bac-UYE), Bacte-
rial paste: S. pasteurii in UME-SA (Bac-UME-SA)
zeta potential as compared to cement particles, which sup-
ports the idea that bacterial cells are good sites for hetero-
geneous nucleation of CaCO3. Another factor that might
influence the nucleation process is the small size of the bac-
teria cells. Rod-shaped S. pasteurii cells have been reported
as ranging from 1.3 to 4.0m in length and 0.51.2m in
diameter [7]. In general, the heterogeneous nucleation pro-
cess is accelerated as particle size decreases since there is
greater surface area available for attachment [18, 23].
XRD andstatistical analysis
Calcite was the only calcium carbonate polymorph detected
in each of the pastes, and Fig.6 displays the calculated
mass percentages of calcite. For all samples, the primary
calcite and zincite peaks were observed within the range of
29.429.8 2 and 36.236.6 2, respectively.
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574
From Fig.6, calcite content was substantially higher in
all three of the bacterial pastes as compared to neat and
nutrient pastes at 1day. In fact, the bacterial pastes with
UME, UYE, and UME-SA media had average calcite con-
tents that were 58.6, 70.4, and 49.3%, respectively, higher
than their corresponding nutrient pastes at 1day. A t test
(alpha 0.01) showed that the mean mass% calcite for all
bacterial pastes was significantly greater than the mean
mass% calcite for all nutrient pastes. Taken together, the
data from the growth curves, ammonia production via urea
hydrolysis, zeta potential, and XRD experiments suggest
that S. pasteurii can utilize the nutrients provided in UYE,
UME, and UME-SA media to produce carbonates and
induce calcite precipitation.
Conclusions
Chemical composition of the bacterial growth media has
the potential to impact microbial concrete in several ways,
with the retardation of cement hydration kinetics being
one of the most critical. UYE medium is commonly used
with S. pasteurii in microbial concrete research, but UYE
medium severely delays hydration due to the presence of
yeast extract. To mitigate this delayed hydration, five alter-
native carbon sources were explored in this study to replace
yeast extract in S. pasteurii growth medium. Following
analysis of these carbon sources, two alternative media
were developed using meat extract and sodium acetate as
replacements for yeast extract, and these media caused
substantially less retardation of cement hydration than did
UYE medium. Mixing cement with UME-SA medium
reduced the extension in the induction period by 75%,
or approximately 7.5h, as compared to mixing cement
with UYE medium. Similarly, mixing cement with UME
medium reduced the extension in the induction period by
50%, or approximately 5h, as compared to mixing cement
with UYE medium. Bacterial growth, ammonia produc-
tion via urea hydrolysis, and zeta potential of S. pasteurii
cells grown in UME-SA and UME medium were similar to
those of cells grown in standard UYE medium. XRD analy-
sis demonstrated that bacterial pastes (containing cement
and S. pasteurii grown in UYE, UME, or UME-SAnutri-
ent medium) had significantly greater calcite content at
1day as compared to nutrient pastes (containing cement
and sterile UYE, UME, or UME-SA nutrient medium).
Therefore, meat extract or a combination of meat extract
and sodium acetate is a good replacement for yeast extract
in S. pasteurii growth medium to mitigate retardation in
cement hydration kinetics. Because of the inherent rela-
tionship between hydration kinetics and setting time, this
strongly suggests that microbial concrete made with S. pas-
teurii grown in UME-SA or UME medium will set more
13
J Ind Microbiol Biotechnol (2016) 43:567575
quickly than will concrete mixed with S. pasteurii grown in
UYE medium, which is beneficial in practice. This is a sig-
nificant improvement and a step towards making microbial
concrete a more practical construction material; however,
further work is needed to examine the influence of UME
and UME-SA growth media on other concrete properties
including cost, strength, shrinkage, corrosion resistance,
and workability.
Acknowledgments The authors gratefully acknowledge Brewster
Dairy, Inc. for donating the lactose mother liquor used in this study.
Stephanie Chu is acknowledged for her help with the calorimetry
experiments.
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