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Use of Mutated Microorganism to
Produce Sustainable Mortar
By
Bijoy Krishna Halder
Graduate Student
Department of Civil Engineering
The University of Texas at El Paso
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Agenda
Introduction
Challenges
Objective
Physiology of Bacillus pasteurii
MICP Process
Culture of B. pasteurii
Mutation of Bacteria
Experimental Design
Compressive Strength Test
Freeze Thaw Test
Absorption Test
Micro Scale Analysis
Conclusion
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Introduction
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Introduction
• Microbial technology is a new branch.
• The aim is to improve the properties of civil engineering material using
biomineralization process.
• Biomineral refers not only to a mineral produced by micro-organism, but
also to the fact that almost all of these mineralized products are composite
material comprised both mineral & organic components & formed under
Bcontrol conditions.
* Source of the figure is “An Overview of Biomineralization Processes and the Problem of Vital Effect” by Steve Weiner and
Patricia M. Dove.
Bio calcite-Echinoderm Synthetically
produced
calcite
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Introduction (Cont.)
• Current global concern
• Minimize cement use
• Enhancement
(Strength+Durability) by
biomineralization
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Challenges
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Challenges
Survivability of microorganism (Cement environment
pH≅12).
Fly ash can be used to lower the pH.
Mutation of micro-organism can improve its
survivability in higher pH.
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Objective
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• Evaluation of the mechanical and durability
properties due to microorganism (Bacillus Pasteurii)
application in cement mortar.
• Mutation of B. Pasteurii to improve its endurance in
higher pH.
• Micro level tests.
Objective
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Physiology of B. Pasteurii
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Physiology of B. Pasteurii (BP)
• Rod shaped
• Non pathogenic
• Aerobic Bacteria.
• Size 1-4 µm.
• Optimum growth temperature 30 ̊ C and pH
9.0.
• Precipitate biomineral calcite.
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Microbiologically Induced Calcium
Carbonate Precipitation (MICP)
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MICP PROCESS (Cont.)
STEP 1
Mortar Cube
Secrete Urease Enzyme (Urea amino-hydrolase)
Control
Environment
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STEP 2 Secrete Urease Enzyme
MICP PROCESS (Cont.)
CO(NH2)2 + H2O → NH2COOH + NH3
NH2COOH + H2O → NH3 + H2CO3
2NH3 +2H2O ↔ 2NH4+* + 2OH−
2OH− + H2CO3 ↔ CO32-+ 2H2O
Break down Urea to NH3 & Dissolved
Inorganic Carbon
* Efflux of NH4+ via ATP synthesis cause proton to drive back into the cell due to increase in charge separation across the cell
membrane.
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STEP 3 In the presence of calcium ion in
Media, cell attract Ca2+ by surface
absorption
Ca2+
MICP PROCESS (Cont.)
* Cell/ LPS which are anchored to the outer membrane and have lipophilic end, attract cation bindings in the presence of
phosphate and carboxylate group
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STEP 4 This Result is super-saturation of Ca2+ ion
in bacteria cell wall
Ca2+
MICP PROCESS (Cont.)
Ca2+ + Cell → Cell-Ca2+
Cell-Ca2+ + CO32− → Cell-CaCO3↓
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* Source: Microbial carbonate precipitation in construction materials-A review by Muynck et al.,2010
STEP 5
• NH3 produced in step 1 increase pH
of bacterial micro-environment.
• Favors heterogeneous precipitation
of calcium carbonate.*
• After a while, whole cell becomes
encapsulated. CaCO3
MICP PROCESS (Cont.)
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Culture of
Bacillus pasteurii(BP)
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• A vial was collected (ATCC* 11859)
• Tris-Buffer medium (ATCC 1376).
* American Type Culture Collection
Culture of B. pasteurii (Cont.)
Ingredients
Yeast Extract
Ammonium sulfate
Tris Buffer
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Culture of B. pasteurii (Cont.) M
ediu
m P
rep
ara
tio
n
Add
BP
Co
ntr
ol
En
vir
on
men
t
pH
Temp.
Shake Bact
eria
Sto
ck
Sample preparation
Frozen stock
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Mutation of Bacteria with
Ultra Violet Rays
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Mutation of Bacteria (Cont.)
• Requirement of Mutation of bacteria
Bacteria optimum growth condition pH 9
Concrete Environment pH 12
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Mutation of Bacteria (Cont.)
• Achal Mukherjee et al. (2009) investigation found the
UV irradiation effect on BP (Grow at high pH and ↑
urease activity).
• Mutated bacteria was culture several times in culture
media (pH 10.5) before stocking.
*UV irradiation damage a part of DNA (by binding adjacent thymine bases to form dimers that cant function in protein synthesis ),
but to survive cell able to repair that part. An enzyme first excise damaged part of DNA . The excise part then replace by DNA
polymerase and DNA ligase forms the final phosphodiester bond.
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Bacteria Growth Comparison
Improvement of survivability of MBP at high pH 10.5
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Experimental Design
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Experimental Design (Cont.)
• Mechanical Test: Compressive Strength Test (ASTM
C 109-08)
• Durability Test: Freeze thaw test (ASTM C 1645M-
09) and Absorption test (ASTM C 1585-11)
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Sample Preparation
& Curing
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Sample Preparation (Cont.)
ASTM* C-109 (2008)
Cement: Sand: Mixing Liquid= 1:2.75:0.49
Samples were prepared in 2 × 2 × 2 in..
*American Society of Testing material
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Sample Preparation (Cont.)
Bacteria is cultured.
Centrifuged at 4,000 rpm to get cell pellet.
Washed with Sodium phosphate buffer.
Bacteria OD*600=0.6 was adjusted by
spectrophotometer.
OD : Optical density
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Curing Process
• Tap Water (For standard samples)
• Urea-Calcium Chloride Medium (For bacteria treated
sample)*
*Park, Sung-Jin; Yu-Mi, Park; Young Chun, Woo; Jung Kim, Wha; and Youl Ghim, Sa. “Calcite-Forming Bacteria for Compressive
Strength Improvement in Mortar.” J. Microbiol. Biotechnol, 2009.
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Compressive Strength
Test
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Compressive Strength Test
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
CSW CSP CSF(5%)P CSF(10%)P CSF(20%)P CSF(30%)P CSF(40%)P
Co
mp
ress
ive
Str
en
gth
(M
Pa)
3 Day Strength 7 Day Strength 28 Day Strength
*C: Cement; S:Sand; F:Fly Ash; P:Sodium Phosphate Buffer; W: Water
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Compressive Strength Test (Cont.)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
CSP CSF(5%)P CSF(5%)BP CSMBP CSF(5%)MBP
Co
mp
ress
ive
Str
en
gth
(M
Pa)
3 Day Strength 7 Day Strength 28 Day Strength
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer; W: Water
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• Summary
– Early strength gain of CSP is mainly due to slight higher pH environment of
buffer solution.
– CSF(5%)P & CSF(40%)P have 5 & 25 percent lower strength respectively.1
– So only 5% fly ash replacement was used.
– CSF(5%)BP/CSF(5%)MBP have 10,14,20% more comp. strength than
CSMBP, CSP & CSF(5%)P samples respectively.
– Improvement reason (primarily): biocalcite precipitation, CSH/CSAH gel
formation, gehlenite (new gluey mineral).
1. http://www.flyash.info/2013/064-Tandon-2013.pdf
Compressive Strength Test (Cont.)
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Freeze Thaw
Test
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Freeze Thaw Test (Cont.)
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer
BP & MB sample have less mortar loss
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Absorption
Test
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Absorption Test (Cont.)
0
20
40
60
80
100
120
140
160
180
CSP CSF(5%)P CSF(5%)BP CSF(5%)MBP
Init
ial A
bso
rpti
on
Rate
, x 1
0-4
mm
/s1/2
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer
Ab
ou
t 10
% less A
bso
rptio
n
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XRD Analysis of Mortar
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XRD of Mortar
10152025 3035404550
5560
6570
7580
0
10
20
30
40
50
60
CC
C
CSP
CSF(5%)P
CSF(5%)BP
CSF(5%)MBP
Count P
er
Seco
nds
2-Theta,Degree
C
C
GG
C
C:Calcite
G:Gehlenite
• Bacteria treated
sample have more
calcite.
• Noticeable peak of
Gehlenite
(Ca2Al(AlSiO7))
was found.
Strongest near 31.4°
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SEM Analysis of
Mortar
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SEM Image at 8000 Magnification of Different Samples
Normal Sample Bacteria Treated Sample
IMAGE ANALYSIS OF
MORTAR
5 um 5 um
Bacteria Rhombohedral Calcite Crystal
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Elemental Analysis Result of Calcium for Different Samples
EDS ANALYSIS OF
MORTAR
Sample Type
Calcium Amount (in %)
By weight By Atomic weight
CSP 36.46 17.83
CSF(5%)P 33.99 16.01
CSF(5%)BP 48.22 25.35
CSF(5%)MBP 67.19 44.30
MB showed 20% more calcite precipitation than BP in surface analysis
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Conclusion
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Conclusion
• The mortar samples prepared with mutated Bacillus pasteurii and wild B. pasteurii gained the
highest 28-day compressive strength. This strength development is due to precipitation of
calcite over the surface and plugging of pores due to microbial activity.
• Mutated bacteria and fly ash-treated samples exhibited better resistance against freezing and
thawing. The samples prepared with mutated bacteria and fly ash have about 63 percent less
mortar disintegration than conventional mortar specimens.
• Bacteria and fly ash-treated samples had the lowest absorption rate due to plugging of pores
by calcite and CSH gel, indicating better durability.
• XRD analysis displayed larger and intense calcite peak and new mineral gehlenite.
• SEM investigation indicated full growth of calcite crystals and presence of more calcium in
bacteria treated sample.
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• This presentation is given at International Concrete Sustainability Conference, May 6-8, 2013, San Francisco,
USA (http://www.concretesustainabilityconference.org/sanfrancisco/speakers.asp).
• Paper is accepted in ACI and under publication (Use of Mutated Micro-Organism to Produce Sustainable Mortar,
Manuscript ID M-2012-381).
• For more information: http://digitalcommons.utep.edu/dissertations/AAI1518200/
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