Surface biomineralization with Pythium aphanidermatum in cracked cement pastes: Limitations and perspectives

ABSTRACT

This study investigates the limitations of biomineralization for the surface treatment of cracks in cement pastes. The proposed strategy involves the application of Pythium aphanidermatum spores on pre-carbonated cement matrices with induced cracks. The pastes were reinforced with polypropylene fibers, cracked via diametral compression, and subsequently subjected to a controlled carbonation process. Three treatment conditions were evaluated: water (Ref) and two biological solutions (T1 and T2) containing calcium acetate, Potato Dextrose Broth (PDB), and Pythium spores; T2 also included urea as an additional nutrient source. Treatment performance was assessed through load recovery and crack width closure. Additionally, SEM analysis was performed to detect microbial colonization along crack surfaces. The results showed limited mechanical improvement, with slightly better performance in T1 and T2. However, no measurable crack width healing (CWH ≈ 0%) was detected, and no microbial growth was observed, likely due to high alkalinity, low surface porosity, and poor nutrient retention in the treated zone. Despite the modest outcomes, the study introduces an innovative approach that combines accelerated carbonation and surface biomineralization using a non-bacterial microorganism. For future studies, it is recommended to investigate multiple treatment applications, encapsulation systems for spore delivery, surface modification to enhance microbial adhesion, and local pH monitoring to ensure optimal conditions for microbial growth and activity.

Keywords:
Biomineralization; Cracks; Cement pastes; Pythium aphanidermatum; Crack-healing

1. INTRODUCTION

Cement-based materials are characterized by high compressive strength, durability, and workability [¹, ²]. However, their brittle nature makes them susceptible to crack formation [³], which necessitates maintenance and repair interventions [⁴]. To address this issue, the development of self-healing cementitious matrices has been explored. The literature suggests that autogenous healing mechanisms may occur through different pathways: chemical reactions, where unreacted cement particles can hydrate, or through carbonation processes, where CaCO₃ is formed in the presence of CO₂; physical mechanisms, such as the expansion of certain materials; and mechanical effects, where particles may bridge or block fissures. In any case, the presence of water is essential for these processes. Nevertheless, this phenomenon is limited to ideal conditions, such as the availability of moisture, and to crack widths below 0.3 mm [⁵].

To seal wider cracks through CaCO₃ precipitation, autonomous healing systems have been developed, which involve the incorporation of additional materials into the conventional cementitious matrix [²]. Among the most studied approaches are the use of fibers, chemical additives, and microorganisms [⁶, ⁷], with microorganisms gaining particular interest due to their lower environmental impact compared to other products [⁸]. In fact, microbial-mediated mineralization has demonstrated potential in diverse applications, ranging from environmental remediation and non-reactive carriers to industrial and biomedical systems [⁹, ¹⁰]. Compared with chemical inducers, microorganisms can provide localized nucleation sites and sustainable precipitation of CaCO₃, although their performance is more dependent on environmental conditions.

The use of microorganisms for the self-healing of cementitious matrices is based on the biomineralization process [¹¹], where these organisms act as nucleation sites for CaCO₃ crystal formation. However, the efficiency of this process is influenced by several variables, including medium pH, availability of a calcium source, and the presence of CO₂ [¹²]. Although biomineralization has been widely studied in bacteria, fungi, and some algae [¹³,¹⁴,¹⁵], the survival of these microorganisms in highly alkaline environments remains a major challenge. To overcome this issue, research has explored strategies such as microorganism encapsulation to protect them from the hostile cementitious environment [⁶, ¹⁶], and the identification of species with greater tolerance to alkalinity [⁸]. Although certain microbial species have been identified as capable of surviving under alkaline conditions, many cannot tolerate pH levels above 11 [¹⁷], which limits their effectiveness in cementitious matrices with an initially high pH (11–13) [¹⁸].

In this context, a promising alternative is the reduction of matrix alkalinity through accelerated carbonation [¹⁹], a process that simulates the natural aging of structures exposed to outdoor conditions, such as buildings, bridges, and dams. Furthermore, previous studies have demonstrated the effect of surface treatment of concrete using bacteria; for example, the company Basilisk has developed a liquid system applied at real scale for maintenance of aged concrete structures [²⁰].

Although bacterial systems, particularly those based on Bacillus subtilis, have shown promising results in self-healing concrete through MICP, their mechanism relies mainly on localized CaCO₃ precipitation within or around bacterial cells, which restricts mineral formation to specific microzones where metabolic activity remains viable [²¹,²²,²³]. In contrast, the selection of a microorganism such as Pythium aphanidermatum, which exhibits a three-dimensional hyphal network rich in cellulose, capable of providing a larger and more widely distributed number of nucleation sites for CaCO₃ precipitation, represents a promising alternative. Furthermore, this oomycete tolerates alkaline conditions (pH < 11), supporting its survival and activity within carbonated cementitious matrices [²⁴,²⁵,²⁶,²⁷]. With this motivation, the objective of the present work is to evaluate the crack-healing effect in carbonated cement pastes through the surface application of microorganisms as the basis of the biomineralization process.

Unlike previous studies focused on incorporating microorganisms into the bulk of fresh cementitious mixes, the present work proposes an alternative strategy based on the surface application of biomineralization on pre-carbonated cement pastes. This combination takes advantage of the pH reduction induced by carbonation to enhance microbial viability without altering the internal composition of the matrix. Moreover, the use of the oomycete Pythium aphanidermatum, a rarely explored organism in cementitious applications, introduces a biologically distinct approach from the conventional use of bacteria, offering a novel perspective in the pursuit of sustainable solutions for crack repair.

2. MATERIALS AND METHODS

2.1. Cement matrix preparation

To prepare the test specimens, methods reported in the literature were adapted [²⁸]. Cement (OPC Type V) pastes were formulated with a water-to-cement (w/c) ratio of 0.4 and reinforced with polypropylene fibers (relative density 0.91 g/cm³, diameter 12 μm, and length 6 mm) at 0.4% by weight of cement. Table 1 presents the mix design used in this study. Cylindrical specimens measuring 50 mm in diameter and 25 mm in height were cast. These specimens were cured in a fog room for 28 days.

Table 1
Mix design of the cement paste specimens.

COMPONENT	PROPORTION	QUANTITY PER 1 m3	DESCRIPTION/DETAILS
Cement (OPC)	100% (reference)	1429 (kg)	Brazilian Type V Portland Cement
Water	w/c = 0.40	571 (kg)	Water-to-cement ratio
Polypropylene fibers	0.4%	5.7 (kg)	ρ = 0.91 g/cm3; ϕ = 12 μm; L = 6 mm

After the curing period, cracks were induced in the diametral section using a Shimadzu UHX testing machine (1000 kN) at a loading rate of 0.5 µm/s until a crack width of approximately 200 µm was achieved. A clip gauge was used to control the crack size. Additionally, crack behavior was monitored using the Digital Image Correlation (DIC) technique, as recommended in the literature [²⁹,³⁰,³¹] (Figure 1).

Figure 1
Set-up for mechanical tests and DIC.

After the crack generation process, the specimens were placed in a carbonation chamber to reduce the alkalinity of the medium and ensure the viability of the microorganism Pythium aphanidermatum (oomycete). This process lasted for 21 days, following recommendations from the literature: a constant CO₂ flow of 4%, 40% relative humidity, and a temperature of 30 ± 5 °C [³²].

2.2. Evaluation of the repairing effect

To evaluate the repair effect, treatment combinations were prepared as shown in Table 2. The solutions used for the biomineralization process (T1 and T2) were prepared following a previous study [²⁷]: 100 mM calcium acetate concentration, 2% urea, and Pythium spores at 10⁵ cells/mL in Potato Dextrose Broth (PDB). Spore concentration was quantified using a Neubauer counting chamber. Microbial viability was confirmed through optical microscopy by observing active hyphal growth and cytoplasmic integrity. A representative micrograph (Figure 2a, b) was included to illustrate the morphological confirmation of viability prior to surface application. A single application of 2.5 mL/cm (a value similar to the ER7 product by Basilisk) was applied to each specimen. After the biological treatment was applied, the specimens were placed inside sterile Petri dishes and sealed with plastic film to maintain a humid microenvironment around the sample. The sealed specimens were stored at ambient laboratory temperature (approximately 25 °C) for 28 days, without external CO₂ exposure, in order to simulate a realistic surface repair scenario without additional environmental intervention.

Table 2
Types of treatment.

CODE	DESCRIPTION
Ref	Water treatment
T1	Treatment with water, acetate, PDB and Pythium
T2	Treatment with water, acetate, PDB, urea and Pythium

Figure 2
Optical micrographs of Pythium aphanidermatum (a) Spores, and, (b) Germination and hyphal formation.

The inclusion of a reference group without microorganisms (Ref) and two biological formulations (T1 and T2) allows for the evaluation of the specific contribution of microorganisms and urea to the biomineralization process. Treatment T2 includes urea as an additional source of carbon and nitrogen, aiming to enhance the microorganism’s metabolic activity and, consequently, the formation of CaCO₃ crystals.

After the treatment, the repair capacity was assessed by quantifying the load recovery percentage, using the equation proposed by RILEM 221-SHC [³³] Equation (1).

(1) $$C=\frac{{\sigma }_2-{\sigma }_0}{{\sigma }_1-{\sigma }_0}\times 100$$

Where σ₀ is the stress at unloading during the first tensile test, σ₁ is the tensile strength in the first test, and σ₂ is the tensile strength after repair.

Regarding the crack closure percentage, the procedure reported in the literature was adapted [²⁹]. Measurements were taken at four marked points (C1–C4) on the application face of the specimen, where the healing solutions were applied. For crack width monitoring, these points were positioned consecutively at 1 cm intervals along the visible crack, starting from the upper edge. This setup allowed evaluating the healing effect at specific sections of the fissure, ensuring consistency in the measurements and avoiding variations when applying the crack width healing (CWH) calculation Equation (2).

(2) $$CWH=\frac{W_i-W_f}{W_i}\times 100\%$$

Where wᵢ is the initial crack width and w_f is the crack width after treatment.

The potential for crack repair was evaluated qualitatively through visual observation under an Olympus SZ11 stereoscopic microscope with a digital camera. The samples were scanned using a computer-controlled motorized stage, field images were acquired, and then extended images, completely covering the cracks, were stitched as described by [³⁴].

To verify the presence and potential development of Pythium aphanidermatum within the treated cracks, the internal microstructure was analyzed by scanning electron microscopy (SEM), using a HITACHI TM 3000 microscope. Observations were carried out on cross-sectional slices extracted from the specimens after treatment, specifically focusing on the internal crack walls.

3. RESULTS AND DISCUSSION

Regarding the T1 and T2 treatments, no improvements in strength were observed. This result can be attributed to the lack of microorganism development, which is necessary for nucleation and subsequent calcium carbonate (CaCO₃) precipitation [¹²]. Despite both solutions containing calcium acetate at a concentration of 100 mM, no positive effect on mechanical behavior was observed, which is consistent with findings reported by [³⁵].

Table 3 summarizes the diametral compression tensile strength values (σ₀, σ₁, σ₂) and the corresponding strength recovery rate (c) for each treatment. In this table, values below 0 indicate material degradation, values between 0 and 100% indicate slight recovery, and values above 100% represent significant load recovery. The results confirm that the Ref specimens exhibited negative recovery, while T1 and T2 presented slight recovery, though still within a non-significant range.

Table 3
Diametral compression tensile strength (σ) and strength recovery rate (c) for reference and treated specimens (mean ± SD).

CODE	DIAMETRAL COMPRESSION TENSILE STRENGTH (MPa)			STRENGTH RECOVERY RATE c(%)
	σ 1	σ 2	σ 0
Ref	0.72±0.02	0.51±0.04	0.57±0.03	-40.20
T1	0.61±0.04	0.45±0.04	0.42±0.10	18.85
T2	0.75±0.02	0.34±0.06	0.31±0.05	7.10

Figure 3a shows the crack development behavior obtained through DIC analysis. A 27.81% difference was observed compared to the values obtained with the clip gauge, with higher crack widths recorded by the DIC method. This difference arises because the crack generated on the front side of the specimen (monitored with DIC) differs from the crack on the back side (monitored with the clip gauge). The presence of polypropylene fibers also influenced the crack width evolution. In this study, the 0.4% polypropylene fiber dosage did not promote the formation of secondary microcracks; instead, it contributed to stabilizing a single dominant crack during the diagonal compression process. The fibers acted mainly through crack-bridging and pull-out mechanisms, which limited crack opening and enabled the control of the target crack width to approximately 200 µm. This controlled localization is consistent with previous studies using low-volume polypropylene fibers in cementitious matrices [²⁸]. As a result, the DIC strain fields primarily captured the progressive opening of the main crack rather than the development of a distributed microcracking pattern. This behavior should be interpreted within the context of paste-based matrices, where fiber efficiency in crack-width stabilization may differ from that observed in mortars or concretes with higher aggregate content.

Figure 3
Crack analysis, stress-strain behavior, and load recovery (a) Crack width: DIC vs. clip gauge comparison, (b) Mechanical behavior – Ref, (c) Mechanical behavior – T1, and, (d) Mechanical behavior – T2.

In the stress strain analysis, only the specimens that exhibited a typical mechanical behavior, characterized by an initial resting phase, peak load, and progressive crack opening, were considered. Specimens that did not display this behavior were excluded. Figures 3b, 3c, and 3d show the stress–strain response before (purple line) and after (green line) the treatment. None of the treatments showed a post-treatment stress value that exceeded the unloading stress from the first test, indicating that a single application of any of the tested solutions does not produce a significant effect on load recovery. Although the literature reports that water treatment (Ref) can induce self-healing in cracks smaller than 0.3 mm [⁵], this process relies on the continuous presence of moisture to promote the hydration of unreacted cement particles and subsequent CaCO₃ formation. In the present study, such conditions were not met, as only a single superficial application of 2.5 mL/cm was performed, which was insufficient to sustain the chemical reactions required for effective autogenous healing.

Additionally, the analysis of the mechanical response after treatment shows that Ref (Figure 3b) provides a stiffer behavior, which correlates with literature reporting that fibers such as PP and HPVA present lower deformation capacity [³⁶]. However, this effect was more variable in T1 and T2 (Figure 3c and d), which showed a more elastic response. It is believed that this behavior can be attributed to the glucose present in the PDB solution.

These results suggest that a single application is insufficient to achieve effective structural recovery. Furthermore, they highlight the importance of monitoring the development of the applied biological agents. Even in partially carbonated matrices, limiting factors may persist, for example, the smooth surface of cracks can hinder spore adhesion, while reduced porosity restricts CO₂ penetration and maintains high pH values (11–13), which are unfavorable for microbial growth.

Figure 4 presents the qualitative visual analysis of section recovery, evaluating the crack at four control points (C1–C4) located at 1 cm intervals along the application face. After the treatment period, there was no visual evidence of partial or complete closure in any of the analyzed sections. The recorded crack openings ranged from 213 µm (C1) to 130 µm (C4), indicating the persistence of the crack along its entire length. Accordingly, the crack width healing (CWH) values calculated with Equation (2) remained at 0%, confirming the absence of measurable visual closure. These results suggest that the microorganism Pythium aphanidermatum did not develop effectively throughout the crack. It is likely that the spores were activated in highly localized areas, possibly in deeper zones or favorable microenvironments, but their proliferation was insufficient to induce visible recovery in section and mechanical behavior. Similar observations have been reported in the literature, where limited results are noted when complementary strategies such as encapsulation, continuous nutrient supply, or multiple applications of the biological agent are not implemented [³⁷]. Although a proposed solution has been the recurrent application of spores along with calcium sources, this approach is not very feasible in real-world structural maintenance conditions. Additionally, the characteristics of the crack, such as its smooth surface and variable geometry, may have hindered spore adhesion and retention, which, together with potential residual alkalinity, limited the success of the surface biomineralization process.

Figure 4
Visual assessment of crack closure at four control points (C1–C4) marked at 1 cm intervals along the application face after the treatment period (T2).

In order to verify the development of the microorganism Pythium aphanidermatum within the cementitious matrix, SEM analysis was performed on the internal walls of the treated cracks. As shown in Figure 5, no structures compatible with spores or evidence of microbial growth were identified in any of the analyzed areas. The examined surfaces displayed a relatively smooth and compact texture, with no signs of biomineralization or accumulation of extracellular materials. It is presumed that the spores, approximately 13 µm in size, were unable to adhere or develop on this surface due to several possible limitations: a) low surface roughness, b) poor retention of moisture and nutrients, and c) high residual alkalinity, despite the carbonation process.

Figure 5
SEM analysis of crack surfaces after treatment with Pythium aphanidermatum, shown at different magnifications: (a) x300, b) x800, and, (c) x1000.

It is worth noting that no microbial activity was observed in other regions within the test environment, which reinforces the hypothesis that the physicochemical conditions were not suitable for the viability of the oomycete. This observation is consistent with previous studies emphasizing the need for optimal pH, texture, and nutrient availability for effective surface biomineralization processes in cementitious matrices [¹²].

To confirm the conditioning of the specimens, phenolphthalein testing was conducted at different exposure times (7, 14, and 21 days). As shown in Figure 6, carbonation of the cement paste was only partial, with non-carbonated regions persisting even after 21 days. This finding supports the assumption that residual alkalinity remained high in localized areas of the matrix, limiting the establishment of microbial growth. Compared to mortar matrices, which exhibit higher porosity and more uniform carbonation, the compact microstructure of cement paste hindered CO₂ penetration. In fact, in a previous study using mortar specimens, effective hyphal growth of Pythium aphanidermatum and CaCO₃ deposition were observed under similar treatment conditions, although with less control over crack formation [²⁷]. The contrast between both studies highlights the critical role of porosity and matrix characteristics in determining the success of microbial biomineralization.

Figure 6
Carbonation depth of cement paste specimens at 7, 14, and 21 days, assessed with phenolphthalein.

Although treatments T1 and T2 showed slight apparent recovery in load compared to the reference specimens, the overall results of the study indicate that the surface biomineralization process under the tested conditions was not effective in restoring either crack closure or mechanical performance. This behavior may be related to a combination of limiting factors: first, the low roughness of the internal crack surface, which hinders spore adhesion; second, the reduced porosity of the cementitious matrix, which may have limited CO₂ ­penetration during carbonation, maintaining a high pH (11–13) unfavorable for microbial viability; and finally, the single application of 2.5 mL/cm, which was possibly insufficient to induce effective colonization along the entire crack.

These findings are consistent with studies that highlight the need for more controlled conditions for successful biomineralization, such as the use of encapsulated microorganisms, environments with moderate pH, or the repeated application of the biological solution along with nutrients and calcium sources [³⁸]. Nevertheless, this study introduces a novel approach by combining the use of a pre-carbonated cementitious matrix with the surface application of Pythium aphanidermatum spores, a microorganism rarely explored in cementitious environments. Although limited in its effectiveness under current conditions, this strategy provides an experimental foundation for future research seeking sustainable and non-invasive crack repair solutions.

4. CONCLUSIONS

This study evaluated the effectiveness of the surface biomineralization process through the application of Pythium aphanidermatum oospore suspensions on pre-carbonated cement pastes with induced cracks. Based on the results obtained, the following conclusions can be drawn:

A single application of 2.5 mL/cm of the biological solution was not sufficient to produce a significant recovery in the load-bearing capacity of the cracked samples, even under previously optimized carbonation conditions.

Treatments with solutions T1 and T2 showed slight apparent recovery in the strength recovery rate (c); however, neither treatment induced effective crack closure (CWH = 0%) nor significant improvements in the stress–strain behavior. Therefore, the biomineralization approach evaluated in this study did not achieve the desired effects under the specific experimental conditions applied, particularly considering the surface characteristics of carbonated cement paste and the limitations in microbial adhesion.

SEM analysis and visual observations confirmed the absence of microbial development on the internal crack surfaces, which is attributed to factors such as the smooth surface texture, possibly elevated post-carbonation pH, and limited spore retention.

Despite the limited results, this research introduces an innovative strategy based on the combination of accelerated carbonation and surface biomineralization using an alternative microorganism to the traditional use of bacteria. For future studies, it is recommended to explore the effectiveness of multiple applications, the use of encapsulated systems, surface modification of cracks to enhance microbial adhesion, and the assessment of local pH in the treated area to ensure optimal conditions for microbial growth and activity.

5. ACKNOWLEDGMENTS

The authors would like to acknowledge CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPEMIG (Research Support Foundation of Minas Gerais) and Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support.

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Publication Dates

• Publication in this collection
  02 Feb 2026
• Date of issue
  2026

History

• Received
  27 June 2025
• Accepted
  05 Jan 2026