Encapsulation of fungal spores for fungi-mediated self-healing concrete

. Although concrete is a prominent building material in nearly all construction applications, it is also known for its reinforcement corrosion and thus material degradation due to crack formation. These severe durability issues ignited the use of microorganisms to self-heal concrete cracks in a biological way by promoting the precipitation of CaCO 3 on their cell walls. Filamentous fungi have recently emerged as high-potential self-healing agents because of their ability to grow in large mycelial networks providing abundant nucleation sites for CaCO 3 precipitation. Based on the extensive research already conducted on bacteria-based self-healing concrete, protection of the microbial spores in the concrete mix is key to the survival of the microorganism. This research therefore applied a natural encapsulation technique derived from bacteria-based literature on fungal spores. The fungus Trichoderma reesei , already known in the field of self-healing concrete, was used to prepare the capsules. First results showed that the fungus was able to withstand the encapsulation process, yet could not survive when embedded in cement due to its harsh conditions. The possibilities to optimize the procedure are however discussed in the paper and give rise to a broad range of research opportunities.


Introduction
Concrete is an essential building material in the construction sector, it is used in nearly all construction cases and the global production of cement was estimated at 4.4 billion tons in 2021 (1). The material is however prone to the formation of cracks caused by its shrinkage during hardening, limited tensile strength and brittle behaviour. These cracks allow water, oxygen, CO2 and harmful particles to enter the material and consequently cause reinforcement corrosion and material deterioration (2,3). To tackle these durability issues in a sustainable way, research on microbial induced calcite precipitation (MICP) has emerged (4)(5)(6)(7). Calcite or calcium carbonate (CaCO3) is thanks to its natural presence, and therefore its high compatibility with concrete, a very suitable self-healing product (8). Certain bacterial and fungal species have the native ability to precipitate CaCO3 on their cell walls through biomineralization (7)(8)(9)(10)(11)(12)(13)(14)(15). Whereas vast research on bacteria-based self-healing concrete has been conducted, the use of fungi has recently gained interest. Their extensive three-dimensional network-like hyphal growth could potentially allow for the self-healing of cracks larger than 1 mm, which is currently the limit achieved with bacteria (16). Pioneering studies have identified several potential fungal candidates for the * Corresponding author: aurelie.van.wylick@vub.be application, a recent review has compiled these candidates and their characteristics (15). This research focusses on the fungus Trichoderma reesei, as research has demonstrated its survival and growth on concrete and its capability of CaCO3 biomineralization (9,14). The MICP pathway in this research is however ureolytic, which relies on the generation of carbonate ions (CO3 2-) from the hydrolysis of urea (17,18). This approach is novel for T. reesei. Based on the research conducted on bacteria-based self-healing concrete, protection of the microbial spores in the concrete mix is crucial to the survival of the microorganism and to avoid adverse effects on the concrete's mechanical properties (7). This research therefore investigates a natural encapsulation technique derived from bacteria-based literature using sodium alginate and calcium lactate (7). Alginate is a hydrophilic and biodegradable polymer extracted from brown algae (7). A calcium-alginate gelatinous matrix can be formed by combining alginate with organic calcium salts, such as calcium lactate, resulting into cross-linking of alginate fibres (7). This research aims to explore the encapsulation of Trichoderma reesei spores, to assess the viability of the spores and to investigate the functionality of the capsules. Simultaneously, growth experiments are conducted, the amount of grown biomass is measured quantitatively and the precipitation of CaCO3 is verified.

Encapsulation
The encapsulation method was based on the method used by Fahimizadeh et al. (7). The media were adapted to the fungi and fungal spores instead of bacterial spores were used. Media contained 2 % sodium alginate, 2 % urea and 1.5 % nutrient source (type A). Two different nutrient sources were considered: corn steep liquor (type A1) and malt extract (type A2). The use of corn steep liquor was based on a study about bacterial MICP (19). Nutrients and sodium alginate were autoclaved separately for 15 min at 121 °C, urea was added filter sterile afterwards. The pH of the nutrient solutions was set at 5. Media were inoculated by means of cylindrical disks made with a 200 µl pipette in the 7 days-old cultures. One disk per 10 mL of medium was considered. Once the fungal spores were added, the media were put aside for 10 min and shaken manually at several time intervals. The aim was to spread the fungal spores homogenously throughout the media. The media were then added dropwise to a 200 mL sterile 0.20 M calcium lactate solution using a 20 mL sterile syringe. The calcium lactate solution was continuously stirred by using a magnetic stirrer for 20 minutes. Afterwards, the capsules were retrieved with a sterile spoon and spread over multiple sterile 90 mm Petri dishes. The capsules were dried for 48 h at 30 °C. Once dried, capsules were inspected on their viability. For both A1 and A2, capsules were placed on malt extract agar plates. Two conditions were considered: closed (taken directly from the oven) versus open (capsules were cut in two after drying). Additionally, the functionality of the capsules of both type A1 and A2 was investigated by embedding the capsules in cement paste. Cement (CEM III/B 42,5 N-LH/SR LA) was obtained from Holcim (20). Cement and water were autoclaved separately for 15 min at 121°C. The cement paste was prepared with w/c factor 0.4 using 30 g of sterile cement and 12 mL of sterile water per sample in a laminar flow cabinet. The cement paste was poured in sterile 90 mm Petri dishes and capsules were either added manually spread over the surface and pushed in the cement paste or mixed with the cement paste. One capsule per 1 g of cement was considered. After 7 days of curing, samples were broken into pieces. The pieces were either laid on a MEA plate or kept in an empty Petri dish. Additionally, capsules were taken out of the cement paste, cut open and placed on a MEA plate. All plates were incubated at 30 °C with 12 h light/dark cycles for 7 days. All these experiments were conducted in sterile conditions.

Growth
Fungal growth was assessed in parallel with the encapsulation experiments. Two sub-types of media were considered: the same (type A1-1 and A2-1) versus the addition of 2 mM calcium lactate to the media (type A1-2 and A2-2). Calcium lactate was added as a representation of the 0.20 M calcium lactate solution. Only 2 mM was considered because of the instant reaction with sodium alginate when a higher concentration was added. Additionally, type B, type C and type D were considered as well. Type B contained 1.5 % of the previously mentioned nutrients. To type C and D, 2 % urea and respectively 2 mM and 25 mM calcium lactate were added. Type D was only considered for CSL. All media were sterilized by means of autoclaving for 15 min at 121 °C. Urea and calcium lactate were added filter sterile. The pH of the nutrient solutions was set at 5. Erlenmeyer flasks with a 100 mL volume were filled with 20 mL media. Media were inoculated with cylindrical disks, as previously explained, each flask contained 1 disk. The samples were incubated in shaking conditions at 30 °C and 120 rpm for 7 days. After 7 days the pH of all media was measured, and fungal biomass was retrieved by using a vacuum pump. Biomass was killed with 70% ethanol, dried in the oven and then weighed. Scanning electron microscopy (SEM) combined with energy dispersive Xray spectroscopy (EDX) were used to visualize the morphology of fungal precipitates (calcium carbonates) and to characterize its composition. The samples were first sputter coated with a 1.3 nm layer of gold. To determine whether and how much calcium carbonate crystals were precipitated on the fungal hyphae, the fungal biomass was immersed in a 15 mL 2 M HCl solution for 15 min. Samples were then rinsed with distilled water and the biomass was retrieved with a vacuum pump. Samples were dried for 48h in an oven at 40 °C and weighed again to determine the weight of the biomass and the CaCO3 crystals. An overview of all media is given in Table 1. All these experiments were conducted in sterile conditions.

Encapsulation
Capsules were successfully made for both CSL-and Malt-based media, although optimal size and shape were not yet obtained. Freshly made capsules have an average length of 7.5 mm and an average width of 4 mm. Dried capsules have an average length of 5 mm and an average width of 1.8 mm. Fresh and dried capsules can be seen in Fig. 1a- Fig. 2a-b. Capsules were better embedded in the cured cement when dried for 72 h, while the 48 h capsules were much looser and fell out of the cement paste more easily because they still shrunk after mixing with the cement. Fungal spores in capsule-containing cement paste pieces were not able to grow on MEA plates (Fig. 2c), nor in the empty Petri dishes. The capsules that were removed from the cement paste also showed no growth. Additionally, 72 h dried Malt-based capsules were mixed with the cement paste, following a ratio of 2 capsules per 1 g of cement to ensure that sufficient capsules would be broken. Again, no growth was witnessed after breaking the cement paste into pieces. Although the spores can survive the encapsulation procedure, these experiments show that the capsules can't protect the spores from the cement paste's harsh environment.

Growth
Fungal growth was witnessed in all types of media, the biomass for both CSL-and Malt-based media is shown in Fig. 3. Fig. 3. Fungal biomass grown in CSL-and Malt-based media after 1 week of incubation. The labels refer to the types of media: A1-1 contains 2% sodium alginate, 2% urea and 1.5% CSL, to A1-2 2mM calcium lactate was added, B1 only contains 1.5% CSL, C1 and D1 are composed of 2% urea, 1.5% CSL and respectively 2mM and 25mM calcium lactate. The Malt-based samples (A2-1, A2-2, B2 and C2) follow the same compositions with malt extract instead of CSL. Scale bar: 15 mm The highest amount of fungal biomass for the CSL-and Malt-based media was obtained for respectively medium type B1 and type B2, with an average amount of 0.13 g and 0.12 g. These media only contained nutrients. By adding sodium alginate, calcium lactate and/or urea, fungal growth was reduced. This can be due to the increase in pH caused by the breakdown of urea, or stress conditions. The pH value at the beginning (T0) and end (T1) of the experiments, as well as the dry weight of the biomass before (T1) and after (T2) HCl treatment are shown in Fig. 4. Although pH values were similar for both types of media (within 1 type), less biomass was measured for Malt-based media. After HCl treatment, little to no difference in weight was observed for all types except for D1, indicating the absence of CaCO3 crystals. From SEM-EDX analyses, the same conclusion could be drawn. The EDX analysis showed the presence of both C, O and Ca on fungal hyphae for some samples, however almost no crystals were observed. Precipitation of CaCO3 was expected for media containing urea and calcium lactate. The added amount of 2 mM calcium lactate was not enough to promote the precipitation of CaCO3, whereas 25 mM significantly enhanced the precipitation. Half of the dry weight at T1 was coming from the CaCO3 crystals. This demonstrates that a certain concentration of calcium is required to promote the formation of CaCO3 crystals. However, as the CaCO3 precipitation also strongly depends on the pH value, the initial pH of 5 should be considered as well. Complementary experiments with more alkaline conditions should therefore be performed to determine the balance between the initial pH value and calcium concentration to reach optimal growth and CaCO3 precipitation.

Conclusion
Trichoderma reesei is able to endure the encapsulation process yet suffers severely when added to cement paste. Future research will involve an exploration to improve the survivability of the fungal spores, in parallel with defining the optimal medium for CaCO3 precipitation. Different aspects could be investigated, such as the required concentrations of the different elements to obtain both abundant fungal growth and CaCO3 precipitation, other encapsulation techniques that might better protect the fungal spores from the cement paste and fine-tuning of the currently used protocol (add more spores, add an additional protective layer, use a different calcium source, explore different nutrients, etc.).