Screening of Fungi for the Application of Self-Healing Concrete

Rakenth R. Menon1, Jing Luo2, Xiaobo Chen3, Hui Zhou3, Zhiyong Liu4, Guangwen Zhou1,3, Ning Zhang2,5*, Congrui Jin1,3*

1 Department of Mechanical Engineering, Binghamton University, NY 13902, USA

2 Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA

3 Materials Science and Engineering Program, Binghamton University, NY 13902, USA

4 Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at
Birmingham, Birmingham, AL 35294, USA

5 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ 08901, USA

*Corresponding author: [email protected]; [email protected]

Abstract

Concrete is susceptible to cracking owing to drying shrinkage, freeze-thaw cycles, delayed ettringite
formation, reinforcement corrosion, creep and fatigue, etc. Continuous inspection and maintenance of
concrete infrastructure require onerous labor and high costs. If the damaging cracks can heal by
themselves without any human interference or intervention, that could be of great attraction. In this study,
a novel self-healing approach is investigated, in which fungi are applied to heal cracks in concrete by
promoting calcium carbonate precipitation. The goal of this investigation is to discover the most
appropriate species of fungi for the application of biogenic crack repair. Our results showed that, despite
the significant pH increase owing to the leaching of calcium hydroxide from concrete, Aspergillus
nidulans (MAD1445), a pH regulatory mutant, could grow on concrete plates and promote calcium
carbonate precipitation.

1. Introduction

Sustainable infrastructure is the key to creating a sustainable community, due to its significant impact on
energy consumption, land use, and global economy. However, many countries are facing the downfall of
progressively aging infrastructure that needs rehabilitation. In particular, concrete infrastructure suffers
from serious deterioration owing to the effect of various physical and chemical phenomena, such as
drying shrinkage, freeze-thaw cycles, reinforcement corrosion, creep and fatigue, and delayed ettringite
formation, all of which could lead to concrete cracking.

Cracks themselves may not significantly reduce the load-carrying capacity of concrete in the short run,
but they considerably weaken the durability of concrete structures, as they channel in water, oxygen, and
carbon dioxide, which could potentially corrode the steel reinforcement. Moreover, cracking may
promote severe degradation of the non-mechanical properties of concrete, such as the radiation-shielding
properties of concrete elements used in nuclear applications. Nowadays, concrete has been the key
construction material for reactor containment and biological shielding structures, which are essential
components of the nuclear reactors in service worldwide for power generation. In addition, cementitious
grouts, mortars, and concrete are also often used to provide shielding and encapsulation of various

1
radioactive waste materials from military, research, and power generation applications. Some waste
isotopes as well as their decay products will become a serious radiation hazard for hundreds of thousands
of years, which requests exceptionally durable storage.

In view of the remarkable social significance of concrete infrastructures and their exceptional service
demands, the maintenance and inspection for concrete structures have come into focus. However,
continuous inspection and maintenance usually require onerous labor and high investments, which
presents a huge and costly challenge. Fortunately, inspired by the amazing capability of the human body
to fix broken bones through mineralization, researchers have conducted numerous investigations to equip
concrete structures with self-healing properties and generated many innovative solutions1-23.

So far, concrete can fix its own cracks mainly through the following three mechanisms: autogenous
healing, embedment of polymeric material, and bacteria-mediated CaCO3 precipitation, which have been
summarized in a comprehensive review provided by Seifan et al.20 Among them, the biotechnological
approach by using mineral-producing microorganisms is most desirable. It has been demonstrated that in
the environment of concrete, CaCO3 can be precipitated by certain species of bacteria through
biologically induced mineralization processes1-21. Owing to its sufficient compatibility with concrete
compositions, CaCO3 is often regarded as the most appropriate filler for concrete cracks.

Bacteria can precipitate CaCO3 mainly through three different pathways, as summarized below. (1)
Certain species of ureolytic bacteria have been explored based on their ability to indirectly cause the
formation of calcium precipitates by increasing the local pH via catalyzing the hydrolysis of urea to
ammonium1-5. (2) Microbiologists at Delft University of Technology in the Netherlands11-13 tested the
second pathway, in which aerobic oxidation of organic acids results in production of CO2, leading to
CaCO3 production in the environment of concrete. However, in the case of low oxygen concentration,
such as for most underground structures, the efficiency of this approach can be limited. (3) The third
pathway is known as dissimilatory nitrate reduction16. This approach is less efficient than the other two in
terms of the amount of produced CaCO3, but it can be applied in anaerobic zones.

2 Fungi-Mediated Self-Healing Concrete

Although the research on bacteria-mediated self-healing concrete indeed achieved a certain level of
success, it still suffers from serious limitations. So far, the viability of bacterial spores embedded in
concrete is generally less than six months13, which is far from being practical considering the fact that the
lifetime of concrete infrastructure can easily be fifty years or even a century. The detrimental environment
of concrete, such as very high pH values, tiny pores, serious moisture deficit, varied temperatures, and
limited nutrient availability, dramatically influences the microbial metabolic activities and makes bacteria
and their spores susceptible to death. In addition, owing to the limited ability of bacteria to produce large
amounts of CaCO3, bacteria can only heal small cracks with crack widths less than 0.8 mm10-20.

To address the above-mentioned problems, further investigation on alternative microorganisms becomes

potentially essential. In fact, microorganisms are very diverse, and besides bacteria, they also include
Archaea, protists, and fungi. Among them, fungi form a species-rich group of eukaryotic organisms,
which exhibits vast biodiversity with more than 100 K described species and approximately 1.5 M
unknown species. Common fungi include yeasts, lichen-forming fungi, molds, and mushrooms, etc.

2
Recent studies in geomycology demonstrated that certain species of fungi can play a central role in
CaCO3 precipitation24,25, but those species have never been tested in the environment of concrete.
Currently, the investigation of fungi has been mostly focusing on their importance in the degradation of
organic matter, and their connection with inorganic constituents is limited to acquisition of mineral
nutrition by mycorrhizal fungi as well as mineral weathering of lichens and ectomycorrhizae.

The cells of filamentous fungi grow as threadlike structures called hyphae, which possess multiple nuclei
and grow apically with new apices emerging from the formation of lateral branches, creating an
intertwined 3D network called mycelium. In fact, the existing literature suggests that filamentous fungi
have unique features to be used in various applications of biomineralization-based technologies26-30. For
example, compared with other microbial groups, filamentous fungi exhibit higher surface-to-volume
ratios, and therefore possess a larger fraction of organic substrates available for mineral precipitation.

The calcification of fungal filaments is a complex process, and it remains incompletely understood29-36.
However, it has been concluded that there are two critical factors determining the amount of CaCO3
production, i.e., carbonate alkalinity and Ca2+ concentration24. The metabolic activities of filamentous
fungi that can increase carbonate alkalinity typically include water consumption, physicochemical
degassing of fungal respired CO2, oxidation of organic acids, nitrate assimilation, and urea
mineralization24. In addition to carbonate alkalinity, fungal metabolic activities can also affect calcium
concentration. Ca2+ concentration within fungal cells should be strictly controlled, i.e., most of the
calcium ions need to be gathered at the apex for apical growth and sharply reduced in subapical regions24.
To maintain this sharp gradient, fungi need to effectively regulate Ca2+. Ca2+ in the cytoplasm is kept at
sufficiently low concentrations by deliberately pumping it out of the cell or by binding it onto
cytoplasmic proteins24.

In addition, fungi can also influence calcium concentration out of self-protection. Metals that are required
for fungal growth and metabolism may become toxic if their concentrations are too high. However, unlike
other microbial group, many species of fungi can survive and prosper in regions that are seriously
polluted by metals. Precipitation of metal minerals onto hyphae was considered as one of the most
important mechanisms to explain the superior metal tolerance in fungi. Concrete is a calcium-rich
environment, which represents a stress for fungal cells owing to calcium cytotoxicity and subsequent
osmotic pressure24. The production of calcium oxalates has been regarded as a method to decrease their
internal Ca2+ concentration24. Precipitation of CaCO3 may be due to a similar passive mechanism to
immobilize excessive Ca2+. Excessive alkalinity could also pose a source of stress and precipitation of
CaCO3 may be due to intracellular protection24.

While bacteria promote mineral precipitation only through induced biomineralization processes, fungi can
do it through both induced biomineralization and organomineralization processes. There exists a
substance called chitin in fungal cell walls, which is a long carbohydrate polymer forming a substrate that
could considerably reduce the required activation energies for nuclei formation so that the interfacial
energy between the fungus and the mineral crystal becomes significantly lower than the one between the
mineral crystal and the solution32,33. Thus, both living and dead fungal biomass can bind ions onto their
cell walls, resulting in nucleation and deposition of mineral phases31. Bound Ca2+ can then interact with
the dissolved carbonate, resulting in CaCO3 precipitation on the fungal hyphae.

3
Therefore, thanks to their ability to directly and indirectly promote CaCO3 production, fungi could be
used as self-healing agents. The goal of this investigation is to discover the most appropriate species of
fungi for the application of biogenic crack repair. To make fungi-mediated self-healing concrete, the
fungal spores alongside their nutrients, will be blended into the concrete before the curing process starts.
When cracks appear and water trickles into the concrete, the dormant fungal spores will wake up, grow,
consume the nutrient soup, and promote CaCO3 precipitates to fix the cracks in situ. After the cracks are
finally healed, the bacteria or fungi will make spores and go dormant once more – ready to start a new
cycle of self-healing when cracks form again. For existing concrete infrastructures with cracks, the fungal
spores and their nutrients can be injected or sprayed into the cracks.

Besides wild-type fungal strains, genetically engineered fungi are also important candidates for self-
healing concrete. Since the extremely high pH of the concrete environment can be handled by only a few
fungi, pH regulatory mutants were the focus here. It is well known that many microorganisms that are
capable of growing over a relatively wide pH range can adjust their gene expression according to the
ambient pH levels37,38. Fungi make responses to the extracellular pH by means of activation of a dedicated
transcription factor, PacC39-44. Mutants in the pacC regulatory gene are extremely heterogeneous in
phenotype37. A major class of mutations are gain-of-function pacCc created by truncating the C-terminal
region38. The pacCc mutations bypassing the need for the extracellular pH signal result in permanent
activation of alkaline genes and superrepression of acidic genes, which leads to alkalinity mimicry41.
Irrespective of ambient pH, fungi exhibiting alkalinity-mimicking mutations believe that they are always
at alkaline pH and trigger a gene expression pattern similar to that of the wild-type grown at high pH
values, which is exactly what is needed for self-healing concrete.

3. Materials and Methods

In our previous investigation45, we have found that the spores of a wild-type Trichoderma reesei
(ATCC13631) germinated into hyphal mycelium on concrete plates and grew well. However, there exist
only a few studies on the pH regulation of T. reesei. In filamentous fungi, the best characterized member
for gene regulation by ambient pH is Aspergillus nidulans38. Therefore, gene manipulations are easily
achievable in A. nidulans. In this study, three different types of alkalinity-mimicking mutants of A.
nidulans, i.e., MAD1445, MAD0305, and MAD0306, will be tested.

The following five different wild-type strains were purchased from American Type Culture Collection
(ATCC): Rhizopus oryzae (ATCC22961), Phanerochaete chrysosporium (ATCC24725), A. nidulans
(ATCC38163), A. terreus (ATCC1012), and A. oryzae (ATCC1011). The genomes of these fungal strains
have been sequenced and annotated and are publicly available46-54. In addition, these species do not
exhibit any toxicity and belong to Biosafety Level 1 (BSL-1), which is the lowest risk level.

The above-mentioned fungi are all filamentous fungi. In addition to filamentous fungi, there are also
single-celled fungi, i.e., yeasts, that do not form hyphae. One of the most well-known species of yeast is
Saccharomyces cerevisiae, also called baker’s yeast, as it has been instrumental to winemaking, baking,
and brewing since ancient times. Recently, a series of publications were released on the success of
designing synthetic set of S. cerevisiae chromosomes, which could allow S. cerevisiae to rapidly evolve
and rearrange its genome, optimizing itself for certain applications55-57. Thus, in this study, S. cerevisiae
will also be tested.

4
To characterize the fungal precipitates, X-ray diffraction (XRD), scanning electron microscope (SEM),
and transmission electron microscope (TEM) were applied in this study. XRD is an established technique
to identify unknown crystalline phases58,59. SEM has been used to visualize fungal precipitates60,61, and
TEM has been used to study fungal-biotite interfaces and weathered alkali feldspars62,63. SEM will be
applied to analyze the morphology and composition of the fungal precipitates, which will complement the
very local characterization by TEM.

In this section, the experimental procedures of gene replacement to obtain alkalinity-mimicking mutations,
survival test of fungi on concrete plates, as well as phase identification and microscopic characterization
of fungal precipitates will be presented. The procedures of the yeast strain identification and preparation
of concrete specimens are described in the Supplementary Information.

3.1 Gene Replacement Procedure to Obtain Alkalinity-Mimicking Mutations

The full genotypes of the strains investigated in the current work are given in Table 1. The detailed about
the parental strains and the selection procedures of isolating the pacC mutations were previously
published64-68.

Table 1. The full genotypes of the strains.

Strain Genotype Reference64-68

MAD1445 yA2 pabaA1 pacC14900 Penas et al. 2007; Hervas-Aguilar et al. 2007
MAD0305 pabaA1 pacC14 Caddick et al., 1986; Tilburn et al. 1995; Mingot et al. 1999
MAD0306 pabaA1 pacC202 Tilburn et al. 1995; Mingot et al. 1999

Briefly, the gene replacement procedure to obtain alleles pacC14 and pacC202 can be described as
follows. The parental strains with genotype pabaA1 gatA2 palF15 fwA1 (p-aminobenzoate-requiring,
lacking  -amino acid transaminase, mimicking growth at acidic pH, and having fawn conidial color)
were mutagenized with ultraviolet treatment followed by growth selection on media supplemented with 5
mM γ-amino-n-butyrate in presence of 10 mM ammonium tartrate as nitrogen source69-71. The pacC14
mutant strain was selected as it mimics the growth at alkaline pH values, downregulates the γ-amino-n-
butyrate permease, decreases the γ-amino-n-butyrate level in cells, and thus relieves the γ-amino-n-
butyrate-induced cytotoxicity. The pacC202 strain was a spontaneous mutant strain derived from the
parental strains with genotype pabaA1 sasA60 (p-aminobenzoate-requiring and semialdehyde sensitive),
selected as it relieves the toxicity of 10 mM γ-amino-n-butyrate. The pacC genomic sequences in pacC14
and pacC202 strains were then obtained by Polymerase Chain Reaction (PCR) amplification.

The procedure to obtain MYC3-tagged wild-type pacC allele, designated as pacC900, as well as the
engineered allele pacC14900 can be described as follows. A null allele of pacC was first constructed by
transforming the orthologous pyr4 (orotidine 5’-phosphate decarboxylase) gene of Neurospora crassa
flanked by 750 bp of upstream and 700 bp of downstream pacC genomic sequences into a pyrG89
(resulting in pyrimidine auxotrophy) pacC14 strain. Transformation was carried out using linearized
fragments containing the pacC moiety of the plasmid. The long flanking sequence ensures high efficiency
of homologous recombination-mediated gene replacement. The transformants displaying pyrimidine
prototrophy were then selected and tested by Southern blot analyses to confirm that pacC14 was replaced

5
by pyr4. The pacC null strain was then used as recipient strain and transformed with linearized MYC3-
tagged pacC carrying C2437A mutation flanked with genomic sequence 825 bp upstream of pacC start
codon and 925 bp downstream of stop codon72. Transformants displaying pacC colony morphology were
tested for pyrimidine auxotrophy. The gene replacement was again confirmed using Southern blot
analyses. The genotypes in the resulting pacC14900 strain were further confirmed by PCR sequencing.
The protein expression was confirmed by Western blot analyses using anti-MYC antibody.

3.2 Survival Test of Fungi on Concrete Plates

In this study, potato dextrose agar (PDA) was chosen as growth medium. To investigate the impact of the
extracellular pH on fungal growth, the inert pH buffer 3-(N-morpholino)propanesulfonic acid (MOPS,
20mM, pH 7.0) was added to some of the growth media. Sterilization of concrete plate was carried out in
a steam autoclave at 121 °C under 15 psi of pressure for 30 min. A 5 mm diameter mycelial disc of each
fungal strain removed from 7-day-old cultures was aseptically placed at the center of each 60 mm Petri
dish containing 10 ml growth medium. In some Petri dishes, there exists a concrete plate of 3 mm
thickness at the bottom. Sterile PDA plugs were used as the negative inoculum control. The Petri dishes
were incubated at 22 oC and 30 oC, respectively, for 21 days.

To summarize, as shown in Fig. S1 in the Supplementary Information, for each type of fungal strain, eight
different cases were investigated including PDA30, PDA22, MPDA30, MPDA22, CPDA30, CPDA22,
CMPDA30, and CMPDA22. All the cases were investigated independently in triplicates.

Radial growth was measured based on two perpendicular diameters. The fungal samples prepared by the
tape touch method73 were studied using an optical microscope. pH values were recorded after 21 days of
incubation by five independent measurements using an Orion 9110DJWP double junction pH electrode,
which is a semi-micro combination pH electrode.

3.3. Phase Identification of Fungal Precipitates

Phase identification of the fungal precipitates were performed on a Siemens-Bruker D5000 powder
diffractometer with Cu-Kα radiation in the θ/θ configuration operating at 40 kV and 30 mA. The samples
were analyzed over the range of 10° to 80° 2θ at a scan rate of 1°/min with an increment of 0.02° 2θ.
Before the measurements, the solid precipitates were isolated by dissolving the fungal sample in a vat of
dilute bleach (NaOCl less than 3% by weight) at room temperature and washing it repeatedly in methanol
following the published protocol59.

3.4 Microscopic Characterization of Fungal Precipitates

3.4.1 Scanning Electron Microscope (SEM)

The fungal precipitates were studied by a Zeiss Supra 55 VP Field Emission SEM with an EDAX Genesis
energy-dispersive X-ray spectrometer (EDS) at accelerating voltages of 5 kV to 20 kV. The collected
solids were placed in an oven at 50 oC for two days, and then were sputter-coated with carbon by an
Emitech K950X Carbon Vacuum Evaporator in order to ensure electrical conductivity. The chemical
microanalysis technique EDS was applied in conjunction with the SEM analysis.

6
3.4.2 Transmission Electron Microscope (TEM)

TEM analysis was performed by a JEOL JEM-2100F TEM operating at 200 kV. The precipitates were
collected by centrifugation (6000×g, 10 min) and washed multiple times in methanol to get rid of the
growth medium. The particles were dried at room temperature then ground using a set of agate mortar and
pestle into fine powders. The powders were then collected on copper grids overlain with a carbon-coated
collodion film prior to the examination. Bright-field imaging (BFI), selected area electron diffraction
(SAED), and high-resolution transmission electron microscopy (HRTEM) were applied to analyze the
calcite phases.

3.5 Pore Size Distribution Inside Cement Paste Specimen

High-resolution X-ray computed microtomography (μCT) was applied to characterize the pore size
distribution inside the cement paste specimen. Images were acquired using UltraTom X-ray μ-CT system
equipped with a LaB6 X-ray source and a CCD camera with resolution of 4000 × 2624 pixels. Small
cubic samples of 0.4 cm × 0.4 cm × 0.4 cm were used, for which the voxel size of the obtained images
will be about 1.2 μm. Samples were firmly fixed on a cylindrical holder mounted on a rotating table using
an adhesive paste. Acquisition parameters were set as follows: source 230 kV, voltage 90 kV, intensity
200-300 μA, filament LaB6, frame rate 0.4, and 8 averaging images. To impose small angular increments
to cover 360° along the rotation axis, a total of 1664 phase contrast images were collected. The
acquisition duration was about 30 min for each sample. Based on the raw data measured by μCT, the
reconstruction of inner microstructure was carried out by using the Octopus Imaging software74. Images
were post-processed by using the ImageJ software75.

4. Results and Discussion

4.1 Mutant Sequence Changes and Resulting Proteins

The mutant sequence changes in pacC and the resulting proteins are summarized in Table 2. Note that c
denotes constitutive phenotype, which bypasses the requirement for the pH signal and mimics alkaline
growth conditions; and an asterisk denotes a stop codon. It can be seen that the pacC14 genotype carries a
C2437A allele, causing an early stop codon that results in a C-terminal truncation of PacC protein at 492
amino acid. The pacC202 genotype has a deletion from 2353 bp to 2555 bp, resulting in an open reading
frame shift after amino acid 464 and an early stop codon. The resulting pacC14900 strain has a MYC3-
tagged pacC carrying the pacC14 mutation at the pacC locus.

Table 2. Mutant sequence changes in pacC and resulting proteins.

Genotype Phenotype Mutation Mutant protein Reference65,66

pacC14 c C2437A 1→492* Tilburn et al. 1995
pacC202 c Δ2353→2555 1→464 IDRPGSPFRISGRG* Tilburn et al. 1995
pacC14900 c C2437A 1→492* Hervas-Aguilar et al. 2007

4.2 Identification of the Yeast Strain

7
 The yeast strain has 99% ITS sequence similarity to the S. cerevisiae ex-type culture CBS 1171
(NR_111007) and has the same morphological features of S. cerevisiae. Therefore, it is confirmed as the
baker's yeast, i.e., S. cerevisiae.

4.3 Fungal Growth on Concrete Plates

The measurement results show that, because of the leaching of calcium hydroxide from concrete, the pH
values increased significantly from 6.5 to 13.0 (Table 3). On the concrete plates, only one type of pH
regulatory mutants of A. nidulans, i.e., MAD1445, has been found to be able to grow well (Table 4). At
30 oC, its growth rate was 3.2 mm/day in the case of CMPDA30. Plentiful conidia were found on the
concrete plates, which possess similar morphology with those found in the Petri dishes without concrete
(Fig. 1). However, A. nidulans (MAD1445) did not grow in the cases of CPDA22 or CPDA30. The other
eight species grew well in all the cases without concrete but did not grow on any concrete plate. The
fungal growth in each case and the associated optical microscopic images are shown in Fig. S1, Fig. S2,
and Fig. S3 in the Supplementary Information.

Note that of all these three strains, which carry pacC constitutive mutations and otherwise possess very
similar phenotypes, only MAD1445 grows well on the concrete plates in the presence of MOPS. This
could be due to the fact that other unknown genetic factors could lead to increased survival against high
pH values. The three strains were obtained through either mutagenesis or homologous recombination.
Although the pacC locus was sequence-verified, it is possible that mutations happened at other undesired
loci of the MAD1445 strain resulted in increased survival in the alkaline environment.

Table 3. pH measurement results after 12 days of inoculation.

PDA MPDA CPDA CMPDA

Control 6.13±0.12 6.83±0.14 13.14±0.14 12.02±0.11
Aspergillus nidulans (ATCC38163) 6.50±0.12 7.22±0.12 13.04±0.13 11.60±0.10
Aspergillus nidulans (MAD1445) 6.42±0.09 7.12±0.15 13.22±0.16 11.21±0.10
Aspergillus nidulans (MAD0305) 6.31±0.12 7.08±0.13 13.01±0.10 12.11±0.10
Aspergillus nidulans (MAD0306) 6.19±0.19 7.11±0.19 13.11±0.06 11.32±0.08
Aspergillus oryzae (ATCC1011) 6.34±0.13 7.14±0.20 13.63±0.13 12.19±0.19
Aspergillus terreus (ATCC1012) 6.33±0.22 7.03±0.15 13.14±0.20 13.04±0.08
Rhizopus oryzae (ATCC22961) 6.71±0.20 7.02±0.06 13.24±0.19 12.84±0.12
Phanerochaete chrysosporium (ATCC24725) 6.81±0.20 7.00±0.05 13.31±0.13 12.11±0.13
Saccharomyces cerevisiae (Yeast) 6.25±0.13 7.04±0.19 13.01±0.06 12.81±0.15

Table 4. Average growth rates (mm/day) after 21 days of inoculation (n = 6). Note that [mean ± standard
deviation] followed by different letters are remarkably different according to Tukey’s HSD test (p = 0.05).

PDA30 PDA22 MPDA30 MPDA22 CPDA30 CPDA22 CMPDA30 CMPDA22

Aspergillus 2.6±0. 2.6±0. 0.5±0.12b 0.7±0.23b 0 0 0 0

8
nidulans 07a 25a
(ATCC38163)
Aspergillus
3.9±0. 1.9±0.
nidulans 1.2±0.15d 1.2±0.17d 0 0 3.2±0.52b 0.9±0.23e
18a 14c
(MAD1445)
Aspergillus
3.2±0. 2.1±0.
nidulans 1.6±0.08c 1.5±0.21c 0 0 0 0
17a 06b
(MAD0305)
Aspergillus
2.1±0. 1.4±0.
nidulans 0.3±0.06c 0.3±0.12c 0 0 0 0
20a 33b
(MAD0306)
Aspergillus oryzae 4.3±0. 2.8±0.
3.3±0.09b 2.3±0.13d 0 0 0 0
(ATCC1011) 06a 14c
Aspergillus terreus 4.6±0. 2.8±0.
1.8±0.06c 1.7±0.22c 0 0 0 0
(ATCC1012) 46a 35b
Rhizopus oryzae 7.8±0. 7.8±0.
7.8±0.06a 7.8±0.12a 0 0 0 0
(ATCC22961) 07a 15a
Phanerochaete
7.8±0. 7.8±0.
chrysosporium 7.8±0.15a 4.2±0.07b 0 0 0 0
06a 07a
(ATCC24725)
Saccharomyces
1.9±0. 1.9±0.
cerevisiae 1.9±0.29a 1.5±0.11b 0 0 0 0
09a 23a
(Yeast)

Figure 1. For the case of A. nidulans (MAD1445), plentiful conidia were found in the case of CMPDA 30.
The diameter of A. nidulans spores is typically larger than 3 μm. More images are shown in Fig. S3 in the
Supplementary Information.

4.4 Characteristics of the Fungal Precipitates

The XRD analysis shows that the precipitates promoted by fungi are indeed CaCO3 crystals (Fig. 2). The
sharp peak at approximately 30o 2θ indicates highly crystalline phases of calcite. In comparison, the
mortar specimens obtained from the control medium were composed of both quartz and calcite.

9
Figure 2. XRD results for the solids collected from A. nidulans (MAD1445)-inoculated media and fungus-
free media.

Fig. 3(a) shows massive mineral crystals in the A. nidulans (MAD1445)-inoculated media: CMPDA22
and CMPDA30. The particles had regular shapes with well-defined crystalline faces, indicating high
crystallinity of the precipitates. The mineral crystals exhibited a range of crystalline morphologies from a
single pure bulk crystal to plate-like crystals stacked upon each other. Besides, the crystals confirmed
fungal involvement, as many cylindrical holes were found in the crystals, which probably occurred in the
space inhabited by fungi. The holes also suggested that fungal filaments functioned as nucleation sites
during the biomineralization process. As shown in Fig. 3(b), EDS results show that the crystals are
composed of calcium, carbon, and oxygen and their atomic percentage approximately matches that of
CaCO3. Note that EDS data may carry error if the sample is not homogeneous or flat. In our test, EDS
data are obtained from several relatively flat sample areas, and the results are consistent. In comparison,
the amount of calcium precipitation in the control medium was significantly reduced, as evidenced by the
absence of faceted particles (Fig. 3(c)).

10
Figure 3. SEM and EDS results of the collected solids: (a) A. nidulans (MAD1445)-inoculated media; (b)
EDS spectra; and (c) fungus-free medium.

TEM analyses, as shown in Fig. 4, further confirm the crystalline nature of the CaCO3. Fig. 4(a) shows a
bright-field TEM image of the fragments. Fig. 4(b) is a SAED pattern from the collection of the particles
shown in Fig. 4(a), which demonstrates the crystalline nature of the precipitated particles, as evidenced by
the presence of well resolved diffraction rings of (104), (110), (11-6), (122), and (1010) planes, matching
well with the calcite phase. Fig. 4(c) is a higher magnification TEM image of the precipitate particle,
which shows the presence of crystalline lattice (moiré fringes are also visible due to the overlapping of
particles). Fig. 4(d) is a HRTEM image obtained from the area as marked with the black dashed square in
Fig. 4(c). As shown in Fig 4(e), the interplanar spacing of the crystalline planes in Fig. 4(d) is measured
to be 0.308 nm, which matches with the interplanar spacing of (104) of the hexagonal crystal of calcite
with the lattice parameters of a=b=4.989 Å, c=17.062 Å, == 90, and =120.

11
Figure 4. TEM analysis of the fragments of precipitated particles: (a) Bright-field TEM image of the
fragments of crushed precipitated calcite; (b) SAED obtained from the crushed precipitate shown in (a),
the indexing of the diffraction rings matches well with the crystal structure of the calcite phase; (c)
Higher-magnification TEM image of the precipitated particles, showing the presence of crystalline lattice
fringes; (d) HRTEM image from the area marked with the black dashed square in (c); (e) Measurement of
the interplanar spacing by averaging ten lattice fringes marked in (d).

The experiments presented in this study have been repeated three times, confirming that the results are
reproducible. However, it is still not clear what are the optimal conditions for fungal growth and CaCO3
precipitation. Along with our previous work45, we found CaCO3 precipitates in both A. nidulans
(MAD1445)-inoculated and T. reesei (ATCC13631)-inoculated media, but the amount of mineral crystals
was rather limited. As our future endeavors, the effects of the multiple factors promoting fungal growth
and CaCO3 precipitation will be tested, such as concentrations of calcium ions and dissolved inorganic
carbon, temperature, exposure to ultraviolet light, growth medium composition, fungal spore
concentration, and availability of nucleation sites, etc. For examples, so far, our tests have confirmed that
adding 4.0 µg/ml aminobenzoic acid and 0.02 µg/ml biotin to the growth medium significantly promotes
the growth of A. nidulans (MAD1445); and adding 5.0 mM CaCl2 to the growth medium can promote
calcium carbonate precipitation. In addition, a systematic investigation will be conducted to assess the

12
fungal spore germination. According to Wang et al., whether or not the fungal spores in/on the concrete
crack germinate can be measured by the oxygen consumption on the crack surface76. Furthermore, we are
also testing urease-positive fungi Neurospora crassa77,78 and Myrothecium gramineum79 for this specific
application.

4.5 Embedment of Fungal Healing Agents in Concrete

If the fungal spores are larger than the pore sizes in concrete, if they are directly put into cement paste,
most spores will be crushed and lost viability during the hydration process. Our results on μCT
characterization of concrete microstructure have been shown in Fig. 5. The 3D renderings, as shown in
Fig. 5(a) to Fig. 5(c), are particularly useful for qualitative assessment of phase distribution, which is
complementary to higher resolution 2D microscopy imaging. From a typical 2D slice as shown in Fig.
5(d), it shows that the matrix pore sizes after 28 days of curing are much smaller than the typical
diameters of fungal spores, which are usually larger than 3 μm (Fig. 1). The matrix pore sizes in air-
entrained specimens after 28 days of curing are shown in Fig. 5(e). It shows that air-entraining agents
could be applied to generate plentiful additional tiny air bubbles in concrete matrix to provide the housing
for the healing agents. Although the specimen without air-entraining agents may also contain a small
number of inhomogeneous air voids, the specimen containing air-entraining agents usually shows a large
number of homogeneous air voids with appropriate size, which is more beneficial for both workability
and durability of concrete.

Figure 5. A 3D rendering of a grayscale volume is shown in (a), the corresponding high-density particles
and pores are shown in (b) and (c), respectively. (d) Pore size distribution inside cement paste specimens
after 28 days of curing measured by μCT. (e) Effect of air-entraining agent on pore size distribution
measured by μCT.

5. Concluding Remarks

13
In this study, a novel self-healing approach is investigated, in which fungi are used to heal concrete cracks
by promoting CaCO3 precipitation. The experimental results showed that, despite the significant pH rise
caused by the leaching of calcium hydroxide from concrete, one type of pH regulatory mutants of A.
nidulans (MAD1445) could grow on concrete plates. In comparison, A. nidulans (ATCC38163), A.
nidulans (MAD0305), A. nidulans (MAD0306), R. oryzae (ATCC22961), P. chrysosporium
(ATCC24725), A. terreus (ATCC1012), and A. oryzae (ATCC1011) could not grow on concrete. The
characterization by XRD, SEM, and TEM confirmed that the precipitates promoted by fungal activities
were mainly composed of calcite.

It is important to note that according to the existing literature, A. nidulans is usually considered relatively
harmless to healthy human beings. However, according to the investigation conducted by Bignnell et al.80
on the role of the PacC-mediated pH adaptation in the pathogenesis of pulmonary aspergillosis,
elimination of the PacC protein increases virulence in a murine neutropenic model, indicating that
activities under PacC control may be crucial for A. nidulans pathogenicity. Therefore, a complete
assessment needs to be performed to examine the possible short-term and long-term adverse outcomes of
pacCc mutation of A. nidulans on both environment and human beings before it is used in concrete
structures.

The research findings from this study will also be useful to many other applications, such as removal of
radionuclides, bio-consolidation of soil and sand, and removal of calcium ions from industrial wastewater.
(1) The discharge of radionuclide wastewater leads to serious contamination in the environment81.
Traditionally, chemical reactions, ion exchange, as well as membrane technologies are used to handle this
challenge, but these methods are ineffective or costly. In contrast, microbial CaCO3-based coprecipitation
offers a cost-effective solution82. The current study will shed light on how fungi could effectively promote
formation of radionuclide carbonate minerals. (2) Bio-consolidation is often involved in stabilization of
erosion and increasing slope stability83. Microbe-mediated CaCO3 precipitation offers a reliable and low-
cost method to bind sand particles together, which can significantly influence the important mechanical
properties of the sand, including permeability, compressibility, and shear strength84. The current study
will help us understand how fungi could be used in such applications. (3) Large amounts of calcium ions
in industrial wastewater often clog the pipelines, boilers, and heat exchangers85. Microbially induced
CaCO3 precipitation presents an effective and eco-friendly solution to remove inorganic contaminants
from the environment. The current study will advance our knowledge in how fungi could applied to get
rid of calcium ions from industrial wastewater.

Acknowledgement

Congrui Jin was funded by the Research Foundation for the State University of New York (TAE-
16083068). Congrui Jin was also supported by the Small Scale Systems Integration and Packaging (S3IP)
Center of Excellence. Dr. Miguel Penalva at the Biological Research Center of the Spanish National
Research Council was thanked for kindly providing three different alkalinity-mimicking mutants of A.
nidulans, i.e., MAD1445, MAD0306, and MAD0305. Dr. Joan Tilburn at Department of Microbiology of
Imperial College London, Dr. Daniel Lucena-Agell at the Biological Research Center of the Spanish
National Research Council, and Dr. America Hervas-Aguilar at the Division of Biomedical Sciences of
University of Warwick were thanked for useful discussion.

14
Author Contributions Statement

CJ and NZ conceived and initiated the project. RRM, JL, HZ, and XC designed and performed the
experimental work. GZ, NZ, and CJ supervised the research tasks. RRM and CJ wrote the initial draft of
the manuscript. RRM, JL, XC, ZL, GZ, NZ, and CJ co-edited the manuscript. All authors reviewed the
manuscript before submission.

Competing Interests

The authors declare that they have no competing interests.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the
corresponding author on reasonable request.

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