Advances in Biomineralized Building Materials 

Wil V. Srubar III, PhD, Associate Professor 

Department of Civil, Environmental, and Architectural Engineering 

Materials Science and Engineering Program 

University of Colorado Boulder 
My group engineers low-carbon, biomineralized, living materials for the built environment. 

Question Convention 
Carbonate Mineralization 

1 kg CaCO3 = -0.44 kg CO2
 

 

There are multiple mechanisms of microbial biomineralization*. 

Urea Hydrolysis Photosynthesis Sulfate Reduction 

or 

Denitrification 

Organic Compound Conversion 

Beatty, Williams, and Srubar (2022). In press. *Calcium carbonate precipitation; mechanisms for biomineralization of other minerals (e.g., SiO2) also exist.
 

Biomineralization can be biologically induced or biologically controlled. 

Beatty, Williams, and Srubar (2022). In press. 
 

 

 

 

Example.

Biomineralization can be biologically induced or biologically controlled. 

Biologically Induced CaCO3 Precipitation // Urea Hydrolysis & Photosynthesis Biologically Controlled CaCO3 Precipitation // Photosynthesis 

10 μm 1 μm 

Heveran, et al. (2019). SEM Photograph by S. Gschmeissner 
Microbial metabolisms can affect crystal structure and properties. 

www.nature.com/scientificreports 

OPEN 

Received: 29 March 2019 

Accepted: 25 September 2019 

Engineered Ureolytic 

Microorganisms Can Tailor the 

Morphology and Nanomechanical 

Properties of Microbial-Precipitated 

Calcium Carbonate 

Chelsea M. Heveran1, Liya Liang2, Aparna Nagarajan2, Mija H. Hubler1, Ryan Gill2,3, 

Jefrey C. Cameron2,4,5, Sherri M. Cook 1 & Wil 1,6 V. Srubar III 

We demonstrate forthe frsttime thatthe morphology and nanomechanical properties of calcium

carbonate (CaCO3) can be tailored by modulating the precipitation kinetics of ureolytic microorganisms 

through genetic engineering. Many engineering applications employ microorganisms to produce 

CaCO3. However, control over bacterial calcite morphology and material properties has not been 

demonstrated. We hypothesized that microorganisms genetically engineered for low urease activity 

would achieve larger calcite crystals with higher moduli. We compared precipitation kinetics, 

morphology, and nanomechanical properties for biogenic CaCO3 produced by two Escherichia coli (E. 

coli) strains that were engineered to display either high or low urease activity and the native producer 

Sporosarcina pasteurii. While all three microorganisms produced calcite, lower urease activity was 

associated with both slower initial calcium depletion rate and increased average calcite crystal size. 

Both calcite crystal size and nanoindentation moduli were also signifcantly higher forthe low-urease

activity E. coli compared with the high-urease activity E. coli. The relative resistance to inelastic 

deformation, measured via the ratio of nanoindentation hardness to modulus, was similar across 

microorganisms. These fndings may enable design of novel advanced engineering materials where

modulus is tailored to the application while resistance to irreversible deformation is not compromised. 

Microbially induced calcium carbonate (CaCO3) precipitation (MICP) is ubiquitous in nature and is responsible 

for CaCO3 formations in terrestrial and marine environments1–3

. MICP has been widely used for a variety of 

applications, including soil stabilization4

, in situ cement repair5,6, oil and gas well fracture-sealing7, bioremediation of metals8,9

, and sealing subsurface fractures to mitigate leakage from geologically sequestered CO2

10. 

Biogenic CaCO3 mineralization is instigated by changes to solution chemistry local to microorganisms4,5

. 

Microorganisms such as the soil bacterium Sporosarcina pasteurii (S. pasteurii, previously known as Bacillus pasteurii) produce the enzyme urease. !is enzyme hydrolyzes urea to form ammonia and carbamic acid, which then 

spontaneously hydrolyzes to ammonia and carbonic acid. Near the bacterial cell, pH increases with the generation 

of hydroxide ions, and shi"s solution equilibria towards the availability of bicarbonate and carbonate ions. When 

calcium (Ca2+) ions are available, CaCO3 is formed5,11,12. !e negatively-charged bacterial surface o"en serves as 

a nucleation center for CaCO3 precipitation, leading to the formation of crystals with bacterial imprints11. 

1

Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, ECOT 441 UCB 

428, Boulder, Colorado, 80309-0428, USA. 2Renewable and Sustainable Energy Institute, University of Colorado

Boulder, 027UCB SuiteN321, Boulder,Colorado, 80309,USA. 3

Department of Chemical and Biological Engineering, 

University of Colorado Boulder, 596 UCB, Boulder, Colorado, 80309, USA. 4

Department of Biochemistry, University 

of Colorado Boulder, 596 UCB, Boulder, Colorado, 80309, USA. 5National Renewable Energy Laboratory, 15013

Denver West Parkway, Golden, Colorado, 80401, USA. 6Materials Science and Engineering Program, 027 UCB,

Boulder, Colorado, 80303, USA. Correspondence and requests for materials should be addressed to W.V.S. (email:

wsrubar@colorado.edu) 

E

Biologically Induced CaCO3 Precipitation for Biocement Applications // Urea Hydrolysis 

A C

B D F

 

 

 

 

                         

                           

                       

 

 

 

 

 

 

 

 

 

 

Published: xx xx xxxx

SCIENTIFIC REPORTS | (2019) 9:14721 | https://doi.org/10.1038/s41598-019-51133-9 1 
 

 

 

 

                         

                           

                       

 

 

 

 

 

 

 

 

 

 

Published: xx xx xxxx

Microbial metabolisms directly affect crystal structure and properties. 

www.nature.com/scientificreports 

A B 

45 

40 

30 

OPEN Engineered Ureolytic 

Microorganisms Can Tailor the 

Morphology and Nanomechanical Received: 29 March 2019 

Accepted: 25 September 2019 Properties of Microbial-Precipitated 

Calcium Carbonate 

Chelsea M. Heveran1, Liya Liang2, Aparna Nagarajan2, Mija H. Hubler1, Ryan Gill2,3, 1,6 Jefrey C. Cameron2,4,5, Sherri M. Cook 1 & WilV. Srubar III 

We demonstrate forthe frsttime thatthe morphology and nanomechanical properties of calcium

carbonate (CaCO3) can be tailored by modulating the precipitation kinetics of ureolytic microorganisms 

through genetic engineering. Many engineering applications employ microorganisms to produce 

CaCO3. However, control over bacterial calcite morphology and material properties has not been 

demonstrated. We hypothesized that microorganisms genetically engineered for low urease activity 

would achieve larger calcite crystals with higher moduli. We compared precipitation kinetics, 

morphology, and nanomechanical properties for biogenic CaCO3 produced by two Escherichia coli (E. 

coli) strains that were engineered to display either high or low urease activity and the native producer 

Sporosarcina pasteurii. While all three microorganisms produced calcite, lower urease activity was 

associated with both slower initial calcium depletion rate and increased average calcite crystal size. 

Both calcite crystal size and nanoindentation moduli were also signifcantly higher forthe low-urease

activity E. coli compared with the high-urease activity E. coli. The relative resistance to inelastic 

deformation, measured via the ratio of nanoindentation hardness to modulus, was similar across 

microorganisms. These fndings may enable design of novel advanced engineering materials where

modulus is tailored to the application while resistance to irreversible deformation is not compromised. 

Microbially induced calcium carbonate (CaCO3) precipitation (MICP) is ubiquitous in nature and is responsible 

for CaCO3 formations in terrestrial and marine environments1–3

. MICP has been widely used for a variety of 

applications, including soil stabilization4

, in situ cement repair5,6, oil and gas well fracture-sealing7, bioremediation of metals8,9

, and sealing subsurface fractures to mitigate leakage from geologically sequestered CO2

10. 

Biogenic CaCO3 mineralization is instigated by changes to solution chemistry local to microorganisms4,5

. 

Microorganisms such as the soil bacterium Sporosarcina pasteurii (S. pasteurii, previously known as Bacillus pasteurii) produce the enzyme urease. !is enzyme hydrolyzes urea to form ammonia and carbamic acid, which then 

spontaneously hydrolyzes to ammonia and carbonic acid. Near the bacterial cell, pH increases with the generation 

of hydroxide ions, and shi"s solution equilibria towards the availability of bicarbonate and carbonate ions. When 

calcium (Ca2+) ions are available, CaCO3 is formed5,11,12. !e negatively-charged bacterial surface o"en serves as 

a nucleation center for CaCO3 precipitation, leading to the formation of crystals with bacterial imprints11. 

Nanoindentation modulus (GPa) 

* 

4

GPa)

3 

2

ntation hardness (

0 

0 20 30 40 50 60 

Nanoindentation modulus (GPa) 

Nanoinde

20 

10 

0 

Count Count Count 

20 30 40 50 60 35 30 

20 30 40 50 60 

20 

10 

0 

30 

0 

30 

20 

10 

20 30 40 50 60 E. 0 coli HB101 / E. coli HB101 / pBU11 1

Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, ECOT 441 UCB 

428, Boulder, Colorado, 80309-0428, USA. 2Renewable and Sustainable Energy Institute, University of Colorado

Boulder, 027UCB SuiteN321, Boulder,Colorado, 80309,USA. 3

Department of Chemical and Biological Engineering, 

University of Colorado Boulder, 596 UCB, Boulder, Colorado, 80309, USA. 4

Department of Biochemistry, University ure-integration S. pasteurii of Colorado Boulder, 596 UCB, Boulder, Colorado, 80309, USA. 5National Renewable Energy Laboratory, 15013

Denver West Parkway, Golden, Colorado, 80401, USA. 6Materials Science and Engineering Program, 027 UCB,

Boulder, Colorado, 80303, USA. Correspondence and requests for materials should be addressed to W.V.S. (email:

wsrubar@colorado.edu) 

Nanoindentation modulus (GPa) 

SCIENTIFIC REPORTS | (2019) 9:14721 | https://doi.org/10.1038/s41598-019-51133-9 1 
 

 



 

 

 

 

Microbial biomineralization is used widely in construction applications. 

1. Biodeposition for Historic Preservation Conservation of monumental stones 

by bacterial biomineralization 

Brunella Perito & Giorgio Mastromei 

Application of living cultures of selected calcinogenic 

bacteria on limestone has been shown to induce calcite 

precipitation inside stone porosity, but it might generate 

other problems. In fact, chemical reactions due to 

metabolic by-products and growth of fungi, resulting 

from the application of organic nutrients for bacterial 

development, may have negative effects on the stone 

itself. For this reason, development of a stone treatment 

without viable cells seems a better biotechnological tool. 

To achieve this, it is necessary to understand the 

molecular mechanisms by which bacteria foster CaCO3 

precipitation, since this phenomenon is poorly 

understood both at molecular and genetic levels. 

Work with Bacillus subtilis 

In our laboratory we are studying calcite crystal 

formation in Bacillus subtilis in order to identify bacterial 

genes and cell structures involved in the biomineralization process. This work is part of the ‘Bioreinforce’ 

EC project (http://www.ub.es/rpat/bioreinforce/ 

bioreinforce.htm) directed to develop a biomediation 

calcite precipitation method for conservation treatment 

of monumental stones. 

B. subtilis produces calcite crystals when grown on 

an appropriate medium (Fig. 2). We isolated several 

B. subtilis mutants impaired in calcite crystal 

Monumental stone decay is a consequence of 

the weathering action of physical, chemical 

and biological factors, which induce a 

progressive dissolution of the mineral matrix (Fig. 1). 

Attempts to slow down monument deterioration have 

used conservation treatments with inorganic or organic 

products, but their use presents several drawbacks. A 

new approach to conservation treatment of calcareous 

stones exploits bacterial biomineralization. 

Calcium carbonate (CaCO3) precipitation is a major 

biogeochemical process very common to microbes living 

in different environments. Several studies have pointed 

out the complexity of the phenomenon, which is 

influenced by the environmental physico-chemical 

conditions, and is correlated both with metabolic 

activity and cell-surface structures. 

(a)

(b)

Microbes are 

often responsible 

for the decay of 

stonework, but 

Brunello Perito and 

Giorgio Mastromei 

have found a way 

of using bacteria 

to conserve 

monuments 

through the 

technique of 

biomineralization. 

TOP LEFT: 

Fig. 1. Marble Statue (1700), 

on the balustrade of the Villa 

Dosi-Dolfini, Pontremoli, Italy. The 

presence of old biological patina 

and new lichen encrustation can 

be seen. 

COURTESY B. PERITO & G. MASTROMEI 

LEFT: 

Fig. 2. Calcite crystal production 

by B. subtilis. (a) Crystal formation 

on the surface of B. subtilis cells. 

(b) Crystals produced by B. subtilis 

observed by optical microscopy. 

COURTESY B. PERITO & G. MASTROMEI 

MICROBIOLOGYTODAYVOL30/AUG03 113
 











Microbial biomineralization is used widely in construction applications. 

1. Biodeposition for Historic Preservation

2. Soil Stabilization and Biogeotechnics

Downloaded from ascelibrary.org by CASA Institution Identity on 06/06/22. Copyright ASCE. For personal use only; all rights reserved. 

State-of-the-Art Review

State of the Art Review of Emerging and Biogeotechnical

Methods for Liquefaction Mitigation in Sands

Meghna Sharma1; Neelima Satyam2; and Krishna R. Reddy3

Abstract: Earthquake-induced liquefaction causes soil to exhibit uidlike behavior due to a sudden increase in pore water pressure and a

concurrent decrease in effective stress. The liquefaction can destroy or damage existing substructures and superstructures that results in

considerable economic and human losses. Hence, there is a need for ground improvement in lique able soils for liquefaction hazard mitigation. Various conventional methods, such as soil replacement, densi cation, and grouting have been used for liquefaction mitigation

historically. However, these methods are carbon-intensive, uneconomic, and environmentally unfriendly. Recently, some researchers

have demonstrated new techniques that can signi cantly mitigate liquefaction and achieve cost-effectiveness, are ecologically friendly,

and have less associated disturbances. The objective of this review is to provide an overview and the associated challenges of emerging

techniques that increase the liquefaction resistance of sandy soils. Initially, the advantages and disadvantages of conventional methods

are discussed to justify the requirement for advanced methods. The rapid evolution of novel materials and techniques, as well as multidisciplinary collaborations, has led to new and innovative advanced methods for effective mitigation of liquefaction. Among these methods, the biogeotechnological methods that have received great attention recently are discussed in detail. Many studies have reported the effects of biotreatment

on soil properties and liquefaction resistance, factors affecting the biocementation process, and various challenges associated with the biocementation methods. Finally, additional research directions needed for biogeotechnical methods to be effective, sustainable, and resilient for liquefaction mitigation in actual eld applications are presented. DOI: 10.1061/(ASCE)HZ.2153-5515.0000557. © 2020 American Society of Civil

Engineers.

Author keywords: Biocementation; Ground improvement; Liquefaction phenomenon; Microbially induced calcite precipitation.

Introduction vibrational energy from underground layers to surface structures.

However, liquefaction leads to serious hazards when permanent

The term soil liquefaction can be elucidated as the transformation of ground movement occurs during earthquakes due to quicksand

saturated cohesionless soil from the solid phase to the liquid phase conditions, landslides with nite displacement, and ow landslide

due to the consequences of excess pore water pressure increases dur- failures (Seed 1968). The history of highly intense earthquakes ining an earthquake. Seed and Idriss (1971) demonstrated that satu- cludes major soil liquefaction events and associated damages,

rated cohesionless soils, such as uniformly (or poorly) graded such selected cases are summarized in the Appendix.

sands, are highly vulnerable to liquefaction. Earthquake-induced liq- The calamity of liquefaction can be reduced by adopting a suituefaction in this type of saturated sand creates sand boils, mud vol- able ground improvement technique to increase the liquefaction recano eruptions, and extensive ooding of discharged water onto the sistance of the potentially lique able soils. The conventional

ground. The settlement of buildings and structures can be generated techniques, including cement and chemical grouting, deep compacdue to the underlying foundation soil liquefaction. In addition, the tion, relief wells construction, and soil reinforcement, have been

damage is associated with underground structures, such as water used for many decades. However, the grouting methods are either

mains, storage, septic tanks, manholes and sewage conduits, and carbon-intensive or environmentally unfriendly, because the chemdeep foundations (piles) that have drifted up to the ground surface icals can create soil and groundwater pollution (Benhelal et al.

after the earthquake (Ambraseys and Sarma 1969). According to 2013). Densi cation by compaction is challenging at greater

Seed (1968) and Ambraseys (1973), liquefaction itself does not depths, and it will affect the stability of nearby structures and buildpose any particular hazard. The lique ed strata at depth act as an iso- ings (Wang et al. 2017). The addition of supplementary cementilator during an earthquake, which hinders the transport of seismic tious material, such as y ash, rice husk ash, ground granulated

blast furnace slag, and silica fume, is not practically possible for 1

Doctoral Student, Discipline of Civil Engineering, Indian Institute of eld application at greater depths.

Technology Indore 453552, Madhya Pradesh, India. ORCID: https://orcid Various advanced methods have been developed recently to

.org/0000-0002-0351-1637. Email: phd1801204002@iiti.ac.in deal with the challenges of conventional ground improvement 2

Associate Professor, Discipline of Civil Engineering, Indian Institute techniques. These advanced methods include the use of novel ma- of Technology Indore 453552, Madhya Pradesh, India (corresponding au- terials, such as nanomaterials, synthetic bers, recycled materials, thor). Email: neelima.satyam@iiti.ac.in biopolymers, and biomaterials (bacteria and enzymes). Recently, 3

Professor, Dept. of Civil and Materials Engineering, Univ. of Illinois at bioremediation has gathered attention as a novel, ecologically Chicago, Chicago, IL 60607. ORCID: https://orcid.org/0000-0002-6577 sound, economic, and sustainable approach. The active bacterial -1151. Email: kreddy@uic.edu

Note. This manuscript was submitted on April 21, 2020; approved on phase of soil is considered as a nucleation site for calcium carbonate precipitation between granular soil particles that improves the

open until January 28, 2021; separate discussions must be submitted for in- strength and reduces the hydraulic conductivity of the soil leading

dividual papers. This paper is part of the Journal of Hazardous, Toxic, and to effective liquefaction mitigation (DeJong et al. 2006, 2010,

Radioactive Waste, © ASCE, ISSN 2153-5493. 2011; Ferris et al. 1996; Fritzges et al. 2006).

June 10, 2020; published online on August 28, 2020. Discussion period

© ASCE 03120002-1 J. Hazard. Toxic Radioact. Waste

 J. Hazard. Toxic Radioact. Waste, 2021, 25(1): 03120002 
Microbial biomineralization is used widely in construction applications. 

1. Biodeposition for Historic Preservation 

2. Soil Stabilization and Biogeotechnics 

3. Self-Healing Concrete 
Microbial biomineralization is used widely in construction applications. 

1. Biodeposition for Historic Preservation 

2. Soil Stabilization and Biogeotechnics 

3. Self-Healing Concrete 

4. Living Building Materials 

Article

Biomineralization and Successive

Regeneration of Engineered Living Building

Materials

Living building materials (LBMs) were engineered using photosynthetic

cyanobacteria and an inert sand-gelatin scaffold. Microorganisms biomineralized

LBMs with calcium carbonate, which imparted higher fracture toughness

compared with no-cell controls. The microorganisms maintained relatively high

viability in LBMs as long as sufficient humidity conditions were provided. The

microorganisms were capable of on-demand exponential regeneration in

response to temperature and humidity switches. Looking forward, LBMs represent

a new class of structural materials that can be engineered to exhibit multiple

biological functionalities.

Chelsea M. Heveran, Sarah L.

Williams, Jishen Qiu, ..., Sherri

M. Cook, Jeffrey C. Cameron,

Wil V. Srubar III

wsrubar@colorado.edu

HIGHLIGHTS

Living building materials (LBMs)

were grown and regrown using

physical switches

Cyanobacteria biomineralized

hydrogel-sand scaffolds

Biomineralization increased the

fracture toughness of LBMs

Three child generations of LBMs

were grown from one parent

generation

Microbial viability in the living

building materials was maintained

through 30 days

Heveran et al., Matter 2, 1–14

February 5, 2020 a 2019 The Authors. Published

by Elsevier Inc.

https://doi.org/10.1016/j.matt.2019.11.016
 

 

Living bacteria 

We use biomineralizing photosynthetic microorganisms to “grow” strong, tough biological concretes. 

Regeneration 

Ability 

Sand + Media 

+ Cells @ 37°C Microbial Inoculation 

Growth Precipitation Gelation 

Physical Crosslink 

CaCO3 Formation Formation

Post-Gelation 

Living 

Bacteria 

Decreasing Humidity

Post-Gelation-Desiccation 

Structural 

Integrity 

Heveran, et al. 2020; Qiu, et al. 2021 
 

 

Living bacteria 

We use biomineralization to “grow” strong, tough biological concretes. 

Regeneration 

Ability 

Sand + Media 

+ Cells @ 37°C Microbial Inoculation 

Growth Precipitation Gelation 

Physical Crosslink 

CaCO3 Formation Formation 

Post-Gelation 

Living 

Bacteria 

Decreasing Humidity

Post-Gelation-Desiccation 

Structural 

Integrity 

Heveran, et al. 2020; Qiu, et al. 2021 
 

 

 

 

 

 

CO2-storing 3D Printed Earth

Materials and Structures

Bioinspired CO2-storing Portland

Cement Technologies

Embodied Carbon Benchmarking of

Residential and Commercial Buildings

Minus

Materials

Goal: Define kgCO2e/m2

for DOE Reference Buildings

Prometheus Materials is commercializing our biomineralized concrete technology. 
 

 

Microbial biomineralization is used widely in construction applications. 

1. Biodeposition for Historic Preservation 

2. Soil Stabilization and Biogeotechnics 

3. Self-Healing Concrete 

4. Living Building Materials 

5. Biogenic Limestone for PLC and Clinker Production 
We are growing CO2-storing limestone quarries using photosynthesis. 

CO2 + • CaCO3 CaCO3 • CaO + CO2

60%

40%

Courtesy of Algal Resources Collection 

Applications:

+ Carbonation Emissions-Free 

Clinker Production 

+ CO2-storing PLC Fillers 

 

 

 
 

 

 

 

What are the metrology & measurement science needs for biomineralized materials? 

Challenges

• In situ polymorph characterization (i.e., calcite, vaterite, aragonite) 

• Standard viability measurements within concrete and other biomineralized building materials 

• Characterization of the type and quantity of embedded in situ biominerals 

• Standards for characterization and durability testing of biomineralized building materials 

• Prototyping and standardized demonstration testbeds 

• Scale-up production of biological organisms for large-scale testbed applications 

• Linking genomics with process-structure-property relationships of minerals and materials 

Srubar III, WV, Trends in Biotechnology, 2021. 
 

Thank you! 

Wil V. Srubar III, PhD, Associate Professor 

wsrubar@colorado.edu