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

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

Introduction

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

Introduction (Cont.)

• Current global concern

• Minimize cement use

• Enhancement

(Strength+Durability) by

biomineralization

Challenges

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.

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.

Objective

Physiology of B. Pasteurii

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.

Microbiologically Induced Calcium

Carbonate Precipitation (MICP)

MICP PROCESS (Cont.)

STEP 1

Mortar Cube

Secrete Urease Enzyme (Urea amino-hydrolase)

Control

Environment

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.

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

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↓

* 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.)

Culture of

Bacillus pasteurii(BP)

• 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

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

Mutation of Bacteria with

Ultra Violet Rays

Mutation of Bacteria (Cont.)

• Requirement of Mutation of bacteria

Bacteria optimum growth condition pH 9

Concrete Environment pH 12

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.

Bacteria Growth Comparison

Improvement of survivability of MBP at high pH 10.5

Experimental Design

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)

Sample Preparation

& Curing

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

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

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.

Compressive Strength

Test

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

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

• 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.)

Freeze Thaw

Test

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

Absorption

Test

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

XRD Analysis of Mortar

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°

SEM Analysis of

Mortar

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

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

Conclusion

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.

• 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/