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1. 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

2. Agenda
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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

3. Introduction

4. 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.
Bio calcite-Echinoderm
Synthetically
produced
calcite
* Source of the figure is “An Overview of Biomineralization Processes and the Problem of Vital Effect” by Steve Weiner and
Patricia M. Dove.

5. Introduction (Cont.)
• Current global concern
• Minimize cement use
• Enhancement
(Strength+Durability) by
biomineralization

6. Challenges

7. Challenges
 Survivability of microorganism (Cement environment
pH≅12).
 Fly ash can be used to lower the pH.
 Mutation of micro-organism
survivability in higher pH.
can
improve
its

8. Objective

9. Objective
• 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.

10. Physiology of B. Pasteurii

11. 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.

12. Microbiologically Induced Calcium
Carbonate Precipitation (MICP)

13. MICP PROCESS (Cont.)
STEP 1
Control
Environment
Mortar Cube
Secrete Urease Enzyme (Urea amino-hydrolase)

14. MICP PROCESS (Cont.)
STEP 2
Secrete Urease Enzyme
Break down Urea to NH3 & Dissolved
Inorganic Carbon
CO(NH2)2 + H2O → NH2COOH + NH3
NH2COOH + H2O → NH3 + H2CO3
2NH3 +2H2O ↔ 2NH4+* + 2OH−
2OH− + H2CO3 ↔ CO32-+ 2H2O
* Efflux of NH4+ via ATP synthesis cause proton to drive back into the cell due to increase in charge separation across the cell
membrane.

15. MICP PROCESS (Cont.)
STEP 3
In the presence of calcium ion in
Media, cell attract Ca2+ by surface
absorption
Ca2+
* Cell/ LPS which are anchored to the outer membrane and have lipophilic end, attract cation bindings in the presence of
phosphate and carboxylate group

16. MICP PROCESS (Cont.)
STEP 4
This Result is super-saturation of Ca2+ ion
in bacteria cell wall
Ca2+
Ca2+ + Cell → Cell-Ca2+
Cell-Ca2+ + CO32− → Cell-CaCO3↓

17. MICP PROCESS (Cont.)
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
* Source: Microbial carbonate precipitation in construction materials-A review by Muynck et al.,2010

18. Culture of
Bacillus pasteurii(BP)

19. Culture of B. pasteurii (Cont.)
• A vial was collected (ATCC* 11859)
• Tris-Buffer medium (ATCC 1376).
Ingredients
Yeast
Extract
* American Type Culture Collection
Ammonium
sulfate
Tris Buffer

20. pH
Temp.
Shake
Bacteria Stock
Add
BP
Control Environment
Medium Preparation
Culture of B. pasteurii (Cont.)
Sample
preparation
Frozen
stock

21. Mutation of Bacteria with
Ultra Violet Rays

22. Mutation of Bacteria (Cont.)
• Requirement of Mutation of bacteria
 Bacteria optimum growth condition pH 9
 Concrete Environment pH 12

23. 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.

24. Bacteria Growth Comparison
Improvement of survivability of MBP at high pH 10.5

25. Experimental Design

26. Experimental Design (Cont.)
• Mechanical Test: Compressive Strength Test (ASTM
C 109-08)
• Durability Test: Freeze thaw test (ASTM C 1645M09) and Absorption test (ASTM C 1585-11)

27. Sample Preparation
& Curing

28. 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

29. 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

30. 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.

31. Compressive Strength
Test

32. Compressive Strength Test
Compressive Strength (MPa)
40.0
3 Day Strength
7 Day Strength
28 Day Strength
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
CSW
CSP
CSF(5%)P CSF(10%)P CSF(20%)P CSF(30%)P CSF(40%)P
*C: Cement; S:Sand; F:Fly Ash; P:Sodium Phosphate Buffer; W: Water

33. Compressive Strength Test (Cont.)
Compressive Strength (MPa)
45.0
3 Day Strength
7 Day Strength
28 Day Strength
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
CSP
CSF(5%)P
CSF(5%)BP
CSMBP
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer; W: Water
CSF(5%)MBP

34. Compressive Strength Test (Cont.)
• 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

35. Freeze Thaw
Test

36. Freeze Thaw Test (Cont.)
BP & MB sample have less mortar loss
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer

37. Absorption
Test

38. Absorption Test (Cont.)
160
140
About 10% less Absorption
Initial Absorption Rate, x 10-4
mm/s1/2
180
120
100
80
60
40
20
0
CSP
CSF(5%)P
CSF(5%)BP
*C: Cement; S:Sand; F:Fly Ash; B:Wild Bacteria; MB: Mutated Bacteria; P:Sodium Phosphate Buffer
CSF(5%)MBP

39. XRD Analysis of Mortar

40. XRD of Mortar
C
60
Seconds
• Noticeable peak of
Gehlenite
(Ca2Al(AlSiO7))
was
found.
Strongest near 31.4°
50
Count Per
• Bacteria
treated
sample have more
calcite.
CSP
CSF(5%)P
CSF(5%)BP
CSF(5%)MBP
C:Calcite
40
G:Gehlenite
C
30
20
C
C
G
G
C
C
10
0
10 15
20 25
2-T 30 35 40
het
a,D 45 50
egr
ee
55
60
65
70
75
80

41. SEM Analysis of
Mortar

42. IMAGE ANALYSIS OF
MORTAR
SEM Image at 8000 Magnification of Different Samples
5 um
5 um
Normal Sample
Rhombohedral Calcite Crystal
Bacteria Treated Sample
Bacteria

43. EDS ANALYSIS OF
MORTAR
Elemental Analysis Result of Calcium for Different Samples
Calcium Amount (in %)
Sample Type
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

44. Conclusion

45. 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.

46. • 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/