Hydrogel encapsulated bacterial spores for self-healing concrete: Proof of concept

HYDROGEL ENCAPSULATED BACTERIAL SPORES FOR

SELF-HEALING CONCRETE: PROOF OF CONCEPT

J.Y. Wang 1,2, S. Van Vlierberghe 3, P. Dubruel 3, W. Verstraete 2 and N. De Belie 1

1

Magnel Laboratory for Concrete Research, Ghent University, Technologiepark-Zwijnaarde 904, 9052 Ghent, Belgium – e-mail: jianyun.wang@ugent.be; nele.debelie@ugent.be

2

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium – e-mail: willy.verstraete@ugent.be

3

Polymer Chemistry and Biomaterials Group, Ghent University, Krijgslaan 281,S4-bis, 9000 Ghent, Belgium – e-mail: sandra.vanvlierberghe@ugent.be; peter.dubruel@ugent.be

Keywords: self-healing, concrete, hydrogel, bacterial spores, microbial CaCO3

ABSTRACT

Self-healing concrete is regarded as a promising solution to reduce the high maintenance and repair cost of concrete infrastructure. Due to the limited autogenous healing capacity of concrete as such, additives are needed to enhance its self-healing properties. Among various strategies, microbial-based self-healing has gained increasing attention because of its distinct features including environmental friendliness, long-term viability and low cost. Within the framework of this strategy, bacteria and the relevant bio-reagents are pre-added into the concrete during the casting and are expected to play their role (heal cracks) when cracking occurs. Due to the high alkalinity and small pore sizes of concrete, bacteria cannot be added directly, and hence an immobilization process is required prior to incorporation into concrete. In the present work, a bio-compatible hydrogel was evaluated as the carrier to encapsulate an efficient carbonate precipitating bacteria, Bacillus sphaericus, which was selected based on previous research. As proof of concept, the activity of bacterial spores after immobilization, the carbonatogenesity of the hydrogel encapsulated spores, the influence of the bio-agents on the hydrogel swelling properties, and the crack healing efficiency were investigated. Interestingly, no significant viability loss was observed after the immobilization process. The precipitation of CaCO3 in/on the

hydrogel matrix by the encapsulated spores was demonstrated by thermogravimetric analysis (TGA). The swelling capacity of the hydrogel was slightly increased after incorporation of the bio-agents. In addition, the specimens combined with the bio-hydrogels showed an obvious superiority in crack healing efficiency, both with respect to the healing rate as well as the maximum healed crack width. A maximum crack width of about 0.5mm can be healed in the specimens containing bio-hydrogels within 7d, while no crack healing was observed in the reference specimens.

The feasibility of using hydrogel immobilized bacteria for self-healing concrete is therefore demonstrated.

1. INTRODUCTION

Hydrogels are hydrophilic gels which have high water absorption capacity and can retain large amount of water or aqueous solution in the network without dissolving. The water absorbed would be gradually released to the exposing environments. There would be three benefits to use hydrogel for encapsulation of bacterial spores for

self-healing: 1) Hydrogel can protect bacterial spores during the mixing and hydration stage; 2) Swollen hydrogel can be used as the water reservoir for spores germination and bacterial activity when cracking occurs; 3) The precipitated microbial CaCO3 can

restrain the re-opening of the cracks due to long exposure to dry environment. 2. MATERIALS

Bacillus sphaericus LMG 22557 (Belgian coordinated collection of microorganisms,

Ghent) was used in this study. MBS medium [1] was used to cultivate B. sphaericus spores. The hydrogel used was synthesized based on the commercial Pluronic®F-127 (Sigma Aldrich) which is a tri-block copolymer and has a polymer chain of polyethylene oxide ― polypropylene oxide ― polyethylene oxide ((PEO)-(PPO)-(PEO)) units.

3. METHODS

Bacterial spores suspension (109 cells/mL) and/or the bio-reagents (yeast extract, urea, etc) were first mixed with a 20% w/w polymer solution (modified Pluronic®F-127). Then the initiator (Irgacure 2959) was also added to the solution. The mixture was degassed and mixed for 5min, which was subsequently subjected to UV radiation for 1h and a gel sheet was formed. The hydrogel sheet was then subjected to freeze grinding (IKA Yellowline A10 Analytical Grinder) and freeze drying (Christ Alpha 2-4 LSC, Germany) to obtain the dry powders.

The viability was evaluated by the amount of urea decomposed by the hydrogel immobilized spores, which was tested by TAN method [2]. Both pure (H) and blended hydrogels (with different combinations of spores (S), urea (U) and yeast extract (Y)) were tested. Thermogravimetric analysis (TGA) was used to demonstrate the formation of CaCO3 in/on the hydrogels.

Five  kinds  of  mortar  prisms  (30  mm  x  30  mm  x  360  mm)  with  a  reinforcement  (Φ  =  6   mm, L = 660 mm) in the center were made. The composition is shown in Table 1. The amount shown in the table is for one batch of the specimens, which also need 450g cement (OPC CEM I 52.5N) and 1350g sand (DIN EN 196-1 Standard sand). The last column shows whether bacteria were present (Y) or not (N). Urea and Ca-nitrate in the third and fourth column were added to the mortar matrix.

Multiple cracks were created in the prisms after 28d by means of a uniaxial tensile test. The cracked specimens were then subjected to the wet – dry cycles (1h in water and 11h exposure to 60%RH). The crack healing efficiency was evaluated by the decrease of crack widths monitored by light microscope (Leica S8 APO, Switzerland).

Table 1: Composition of the specimens

Type Water (g) Urea (g) Ca(NO3)2.4H2O (g) Yeast extract (g) Hydrogel(g) Spores

R 225 0 0 0 0 N N 214 18 36 3.84 0 N m-H 214 18 36 3.84 9 (H) N m-HYUC 214 18 36 3.84 22.5(HYUC) N m-HSYUC 214 18 36 3.84 22.5(HSYUC) Y ICSHM2013_________________________________________________________________________________ 607

4. RESULTS

After encapsulation and the subsequent treatments (grinding and drying), spores were still viable and kept high ureolytic activity. As shown in Fig.1, only limited amount (about 1~2g/L) of urea was decomposed after 1d and 2d in the media containing the hydrogels without spores inside. While in the media with bio-hydrogels, around 14~16g/L urea was decomposed in the first 24 hours. This was lower than that of free spores (about 18g/L urea decomposed in 1d). Yet, after 2 days, urea in the media with free or immobilized spores, was all completely decomposed. The subsequent grinding and drying had very limited negative effect on the immobilized spores.

Figure 1: Urea decomposed in the media with the addition of different hydrogels (with or without encapsulated spores)

TGA results demonstrated the presence of CaCO3 precipitation in the bio-hydrogels.

CaCO3 decomposes into CaO and CO2 in the temperature range of 650~800°C and

this causes weight loss. As shown in Fig.2, the samples from pure hydrogel (H, data not shown) and the ones with bio-reagents (HYUC) had no weight loss at 650~800°C; while the hydrogels with encapsulated bacterial spores (HSYUC) had distinct weight loss at the range of 650~800°C. The weight loss percentage was about 26%.

The swelling capacity of the hydrogel was slightly increased after incorporation of the bio-agents (Fig.3). No significant difference in swelling properties was observed in de-ionized water or filtered cement slurry. The swelling and re-swelling properties of the hydrogels (pure and blended) were also equivalent.

Figure 2: TGA graphs of samples from the hydrogels (encapsulated with only bio-agents (HYUC), and with both bio-agents and spores (HSYUC)) after immersion

in water 0 5 10 15 20 25

S H HY HU HYU HS HYS HUS HYUS

U re a de co m po se d (g /L )

After UV After UV+FG After UV+FG+FD urea decomposed after 48h

urea decomposed after 24h urea decomposed after

48h by free spores urea decomposed after 24h by free spores -0.01 0 0.01 0.02 0.03 0 20 40 60 80 100 0 200 400 600 800 1000 1/ ℃ W ei gh t (% ) Temperature (℃) -0.01 0 0.01 0.02 0.03 0 20 40 60 80 100 0 200 400 600 800 1000 1/ ℃ W ei gh t (% ) Temperature (℃) a: HYUC b: HSYUC ICSHM2013_________________________________________________________________________________ 608

Figure. 3: Swelling properties of hydrogels Figure 5 Healed crack (within 7d) in the in de-ionized water and filtered cement specimens with bio-hydrogels

slurry

The specimens with the bio-hydrogels showed an obvious superiority in crack healing efficiency, both with respect to the healing ratio (Fig.4) and the maximum healed crack width (data not shown). A maximum crack width of about 0.5mm can be healed in the specimens containing bio-hydrogels within 7d (Fig.5), while no crack healing was observed in the reference specimens.

Figure 4: Average crack healing ratio in different ranges of crack widths of the specimens

5. CONCLUSIONS

It was demonstrated that hydrogel encapsulated spores have high viability and have great potential to be used for enhancing the self-healing capacity of the concrete. ACKNOWLEDGEMENTS

Financial support from the Research Foundation Flanders (FWO-Vlaanderen) (Project No. G.0157.08) and Ghent University (a BOF grant) is gratefully acknowledged

REFERENCES

[1] Kalfon, A., et al. (1983) Growth, sporulation and larvicidal activity of bacillus-sphaericus, European Journal of Applied Microbiology and Biotechnology, 18, 168-173.

[2] Ivanov, V.M., et al. (2005) Chromaticity characteristics of NH2Hg2I3 and I2:

Molecular iodine as a test form alternative to Nessler's reagent, Journal of Analytical Chemistry, 60, 629-632. 0 10 20 30 40 H HYUC HSYUC Wa te r a bs or be d (g /g ) 1st in water 2nd in water 3rd in water 1st in filtered cement slurry 2nd in filtered cement slurry 3rd in filtered cement slurry 0 20 40 60 80 100 He al in g ra ti o (% ) R N H HYUC HSYUC 0-50 50-100 100-150 150-200 200-250 250-300 300-350 350-400 400-700 Initial crack width ranges (μm)