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Microbiologically induced calcium carbonate precipitation to repair microcracks remaining after autogenous healing of mortars
Construction and Building Materials 141 (2017) 461–469
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Construction and Building Materials journal homepage: www.elsevier.com/locate/conbuildmat
Microbiologically induced calcium carbonate precipitation to repair microcracks remaining after autogenous healing of mortars C. Lors a,b,⇑, J. Ducasse-Lapeyrusse a,b,c, R. Gagné c, D. Damidot a,b a
Mines Douai, LGCgE-GCE, 941 rue Charles Bourseul, 59508 Douai, France Université de Lille, Cité Scientifique, 59650 Villeneuve-d’Ascq, France c Centre de recherche sur les infrastructures en béton (CRIB), Université de Sherbrooke, 2500 boulevard de l’Université, J1K2R1 Sherbrooke, Québec, Canada b
h i g h l i g h t s Biohealing can be efficient on cracks partially autogenously healed. Bacterial effects can be estimated with biotic and abiotic experiments. Biodegradable organic calcium compound is necessary to form calcite in autogenously healed mortar. Inorganic calcium salt improves the robustness if its anion reacts with cement paste.
a r t i c l e
i n f o
Article history: Received 28 August 2016 Received in revised form 27 February 2017 Accepted 4 March 2017
Keywords: Mortar Microcrack Biohealing Calcite Calcium lactate Calcium nitrate Bacillus pseudofirmus
a b s t r a c t Cracks remaining after autogenous healing of mortar can be further healed when subjected to a bacterial suspension of Bacillus pseudofirmus if it contains organic calcium compounds. The consumption of organic calcium compounds, such as calcium lactate, by bacteria is providing the necessary calcium to precipitate calcite. The addition of inorganic calcium salts is inefficient to promote the formation of calcite during bacterial growth experiments, but can be efficient during biohealing experiments if the anion of the inorganic calcium salt can react with the cement paste. The addition of both organic and inorganic calcium compounds improves the robustness of the biohealing. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Cracking is one of the most deleterious parameters for concrete durability. In addition, to decreasing mechanical performance, cracks promote various detrimental mechanisms, as they provide a preferential pathway for the penetration of aggressive substances. However, concrete can defend itself against small microcracks having a width between 50 and 100 lm, depending on its composition, through autogenous healing [1]. Autogenous healing involves both hydration of unreacted cement, leaching and carbonation of the cement paste. Nevertheless, larger more deleterious
⇑ Corresponding author at: Mines Douai, LGCgE-GCE, 941 rue Charles Bourseul, 59508 Douai, France. E-mail addresses: [email protected] (C. Lors), [email protected] (J. Ducasse-Lapeyrusse), [email protected] (R. Gagné), [email protected] (D. Damidot). http://dx.doi.org/10.1016/j.conbuildmat.2017.03.026 0950-0618/Ó 2017 Elsevier Ltd. All rights reserved.
cracks that remain unsealed after autogenous healing, need to be treated by other means, most often expensive, such as resin impregnation. Alternative methods based on the impregnation of chemical healing agent could also be used for medium sized microcracks (having a width lower than 300 lm). Additionally, this method could be extended to biological healing agents. Indeed, microorganisms can promote the precipitation of calcium carbonate in the crack [2,3]. Calcium carbonate is a highly compatible with concrete, and it presents itself as a sustainable and ecological repair solution to improve the durability of cementitious materials [4,5]. Microbiologically Induced Calcium Carbonate Precipitation (MICCP) uses microbial metabolism to generate CO2, leading to the formation of carbonate ions in the presence of water. Carbonate ions can react with available calcium ions to precipitate calcium carbonate if the suitable conditions are reached [6]. MICCP in a crack requires filling the crack with a bacterial suspension (mix of suitable bacteria, nutrients and possibly other compounds
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promoting calcium carbonate precipitation) in which bacteria can grow [7,8]. Nevertheless, the bacterial suspension should not contain chemical compounds harmful to concrete, such as chloride ions for concrete rebars. Additionally, when the cement paste is in contact with the bacterial suspension, the chemical elements leached from the cement paste have to be innocuous with respect to the microbial activity. Once cracks are formed, they are rapidly carbonated thanks to autogenous healing, leading to the formation of calcium carbonate layer on the wall of the crack [9]. Thanks to the formation of the calcite layer, carbonation reduces the pH at the surface of the crack that becomes lower than 10 compared to more than 13 for a fresh crack [10]. This drop of pH that can be measured by pH contact electrode [11], is beneficial to select suitable alkaliphilic bacteria for MICCP [12–17]. However, calcium carbonate layer restricts the diffusion process that is governing the release of calcium from the cement paste to the crack [18]. As a consequence, the amount of calcium carbonate that could be formed by MICCP in the already carbonated crack is limited. This calcium shortage needs to be compensated by the addition of calcium in the bacterial suspension in a chemical form and at concentrations that do not inhibit the bacterial growth. The complexity induced by all these constraints have led to propose a large variety of bacterial suspensions for MICCP in various materials and environments [3,4,13,19–27]. For example, ureabased bacteria are commonly used [4,6,12], despite the fact that they form ammonium ions (NH+4), which are deleterious for concrete [28]. Alternative nutriments, such as organic calcium compounds, may also appear to be an interesting source of nutrients in the presence of bacterial strains belonging to Bacillus genus. For example, calcium lactate ([CH3CH(OH)COO]2Ca) can be metabolized by different species of Bacillus, such as B. pseudofirmus or B. cohnii, in order to promote MICCP [13,27,29]. However, these strains were less studied than Sporosarcina pasteurii especially in the case of self-healed mortars. The aim of this paper is to perform MICCP on autogenously healed mortars by using a bacterial suspension of Bacillus pseudofirmus containing calcium lactate as the main calcium source. The first part of the study is devoted to better understanding the key parameters, in order to define a suitable growth medium leading to a good bacterial activity associated with the production of notable amounts of calcium carbonate. In the second part, the selected bacterial suspension was applied on autogenously healed mortar containing one incompletely healed radial crack. The efficiency of the biohealing process was estimated by the decrease in apparent opening of the crack measured by air permeability. The observation and the mineralogical characterization of the deposit in the crack were also performed. Finally, the effect of chemical reactions between the growth medium constituents and the mortar on the healing process was assessed by performing healing experiments carried out with the growth medium in abiotic conditions. This enabled us to differentiate the intensity of microbial activity relative to the chemical reactions induced by the growth medium, in order to produce calcium carbonate during the biohealing process.
2. Materials and methods 2.1. Bacterial strain The selected strain was Bacillus pseudofirmus DMS 2516, which was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. This strain was already used in a previous study for its abilities to grow in an alkaline environment and generate carbonate ions [12]. Bacterial stocks were stored at 80 °C in glycerol. This bacterial strain is an alkaliphilic strain able to survive from neutral to basic pH levels up to 11. Indeed, any significant bacterial activity was noticed at a pH value of 11.5 [30]. It is aerobic and heterotrophic,
i.e. in the presence of oxygen, it is able to consume organic compounds and produce carbon dioxide by cellular respiration according to Eq. (1) written for calcium lactate that was used:
½CH3 CHðOHÞCOO2 Ca þ 6 O2 ! CaCO3 ðsÞ þ 5 CO2 þ 5 H2 O
ð1Þ
2.2. Growth medium composition The studied culture media, named M1 and M2, were composed of yeast extract (3 g/L) and calcium lactate, [CH3CH(OH)COO]2Ca, (75 g/L corresponding to 0.34 mol/L). Calcium nitrate, Ca(NO3)2, (32.8 g/L corresponding to 0.2 mol/L) was only added to the medium called M2. The initial pH was not adjusted, and measured 6.5 and 6 for M1 and M2 media, respectively. Using a buffer solution [NaHCO3/Na2CO3] to increase the initial pH was not desired as it could induce side chemical reactions leading to form calcium carbonate that would not be induced by the bacterial metabolism. For example, a method to precipitate calcite is to mix a solution of Na2CO3 with another one containing CaCl2. The yeast extract and calcium lactate provide the nutrients necessary for the bacterial growth. Calcium lactate and calcium nitrate also provide calcium ions. It was observed that larger calcium nitrate concentrations than 0.2 mol/L inhibited the growth of Bacillus pseudofirmus [11]. Calcium nitrate was preferred over calcium chloride often found in media used for MICCP. Indeed, chloride ions are well-known catalysers for rebar corrosion and thus could be inadequate for a repair strategy. 2.3. Bacterial growth Bacteria were pre-cultivated from frozen inocula in the medium recommended by DMSZ, composed of 8 g/L of Nutrient Broth (NB) with a pH adjusted to 9.7 using a buffer solution (NaHCO3/Na2CO3) at 30 °C until reaching the exponential growth phase. Then, two Erlenmeyer flasks containing 250 mL of each liquid sterile media (M1 or M2) were inoculated with 250 mL of the pre-culture containing approximately 109 bacteria/mL. Erlenmeyer flasks were placed under stirring at a temperature of 30 °C and were regularly monitored over 35 days for pH measurements, lactate and calcium concentrations and bacterial density. After the filtration of the bacterial suspension at 0.2 mm, the lactate concentration was determined using ion chromatography (DIONEX ICS-3000, USA) and the calcium concentration was determined using inductively coupled plasma atomic emission spectroscopy (ICPAES) (VARIAN 720 ES). The bacterial concentration was evaluated using epifluorescence microscopy (LEICA DMLP), in order to carry out a direct enumeration of bacteria [31]. The bacterial cells were harvested on a 0.2 mm filter. The living bacteria can be quantified by microscopic counting using a fluorochrome (orange acridine) and an ultraviolet light. One control sample was made for each medium (M1 or M2). In this case, the Erlenmeyer flasks containing the media were not inoculated. Lactate and soluble calcium concentrations were measured at the beginning and the end of the experiment. Sterility in each control sample was evaluated by the direct bacterial enumeration in the course of the experiment. Additionally, for each media, three Erlenmeyer flasks were not sampled, but filtered (sieve of 0.45 mm) at the end of the bacterial growth test (35 days). The collected solid residue was weighed and analysed using thermogravimetric analysis (TGA) (NETZSCH STA 449 F3) and X-ray diffraction (XRD) (Brucker Advance D8) to determine the mineralogy of the solid residue. 2.4. Preparation of cracked mortars The mortar samples were made in accordance with ASTM C109-C109M [32]. This standard requires a water-to-cement mass ratio of 0.485 and a sand-tocement mass ratio of 2.75. The cement used was a Canadian GU Portland cement (General Use, similar to CEM I and ASTM Type 1). The cement composition (in mass%) was 60% C3S, 13% C2S, 7.5% C3A and 7% C4AF. The sand required by ASTM standard C788 [33] is a normalized Ottawa sand made of natural silica, having a particle size distribution between 150 and 600 mm, with a density of 2.65. The mortar samples were annular, with a diameter of 150 mm and a height of 50 mm. A central hole with a diameter of 55 mm allowed for the insertion of a steel expansive core, which was used to create one radial crack along the entire section of the mortar (Fig. 1). Thus, the crack had three openings accessible for healing: one opening at the top, one other at the bottom and the last one in the side surface of the ring. The surface of each crack wall was 50 47.5 mm2 and the apparent crack opening was computed from air-flow measurements [34]. The sample included an embedded steel ring to simulate a reinforcing bar that balances the internal stresses after cracking (Fig. 1). The ring was made from a hot-rolled steel rod having an internal diameter of 95.2 mm2 and a section diameter of 4.8 mm (reinforcement ratio of 0.72%) [34]. The mortars were cracked after 28 days of storage in an environmental chamber at 100% relative humidity and a temperature of 23 °C. The opening of each crack was more than 200 mm. After cracking, the mortar samples were stored for more than one year at 100% R.H. and a temperature of 23 °C. After this period, the autogenous healing process was almost complete and the walls of the cracks were carbonated at 23 °C under atmosphere of 4% of CO2 with 40% relative humidity. Thus,
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150 mm 55 mm
Crack
Expansive core 50 mm
Steel ring Mortar sample
Fig. 1. Schematic diagram of the annular mortar [34].
2.5. Biohealing of cracked mortars The mortar samples were immersed in contact either with M1 or M2 bacterial suspensions obtained by the culture of Bacillus pseudofirmus in M1 or M2 growth medium for seven days respectively. The cracked mortar samples with their expansive core were individually immersed in such a way that only the microcracked volume was in contact with the bacterial suspension. Thus, the bacterial suspension could enter the microcrack by its three openings. The immersion was done in a microbiological safety cabinet in which the air-flow was kept sterile, in order to avoid contamination by other microorganisms. For the control experiment, the mortar sample was immersed in the same way, but in a sterile culture medium. Its chemical composition was designed to be as close as possible to the bacterial suspension in which the cracked mortar was immersed. In order to do so, the control experiment was made after the biohealing experiment. This allowed us to determine both calcium and lactate concentrations in the bacterial suspension in which the mortar was immersed, as some calcium and lactate were consumed during seven days of the bacterial growth prior the immersion of the mortar. Thus, the lactate and calcium concentrations in the medium used for the control experiment carried out in abiotic conditions were identical to those of the biohealing experiment. Moreover, the biocide bronopol (0.1 g/L) was added in medium used for the control experiment, in order to avoid contamination by external microorganisms. For biotic and abiotic experiments, duplicate samples were made to assess the reproducibility. After the immersion, each mortar sample was placed for one month in its own initially sterile humidification system, which provided 100% R.H. at a temperature of 20 °C and avoided contamination. The totally saturated condition with respect to water vapour was required, in order to be sure that the bacterial activity continued during the period where biohealing was expected to occur. However, it also led water condensation on the surface of the cracked mortar and on the walls of the humidification system. Subsequently, some water accumulated at the bottom of the humidification system. The mortar samples were not in contact with the water at the bottom of the humidification system. Nevertheless, they were slightly leached by the water condensed at their surface, which was partly recovered at the bottom of the humidification system. The water accumulated at the bottom of the humidification system was analysed for the presence of microorganisms using epifluorescence microscopy in the case of the control assays, in order to check whether any contamination occurred. After one month spent in the humidification system, the mortar was removed from the system to measure the apparent crack opening using air-flow described in the next paragraph. As most of bacteria did not survive to drying at 40 °C during 24 h, the mortar was immersed again in a bacterial suspension to perform a new cycle of biohealing. This protocol was repeated over a four-months period, leading the cracked mortar to be subjected to four cycles of one day of immersion in the bacterial suspension, one month of storage in the humidification system, and then a 24 h drying at 40 °C followed by the measurement of the apparent crack opening. 2.6. Measurement of the apparent crack opening using air flow The evolution of the apparent crack opening was monitored every month before the next immersion in the bacterial suspension using air-flow measurements in a sterile air permeability apparatus [34]. The mortar samples were removed from the humidification systems and dried at 40 °C for 24 h. The samples were placed
in an air permeability cell specially designed to evaluate the apparent opening of the crack. Eq. (2) describes the relationship between Q (air flow in liter per minutes) and Wef (the effective crack opening in mm):
Q ¼ 1:57043:104 W2ef þ 1:40517:102 Wef
ð2Þ
This equation was obtained through a large number of assays on cracked mortar samples having the same geometry and cracked using the same device. In order to evaluate the biohealing process, the apparent reduction of the thickness of the crack at a given time (DWt) was calculated from the initial apparent crack opening (W0) and the apparent crack opening at a given time t (Wt) (Eq. (3)):
DWt ¼ W0 Wt
ð3Þ
It can be noticed that DWt does not depend on the initial thickness of the crack because of the planar geometry of the crack. Thus, the healing of cracks having different initial thickness can be compared. 2.7. Observations of the cracks During the biohealing experiment, observations of the opening of the crack at the mortar surface were made every month with a binocular magnifier (OLYMPUS, B061) on dried mortar just before airflow measurement. After 4 months, at the end of the biohealing assay, the mortar sample was cut to 1 cm on each side of the crack and the crack was split open by slight chisel strokes, enabling us to observe the deposit at the surface of the wall of the crack. Observations were made under a scanning electron microscope (SEM) (Hitachi SE 4300). Moreover, the products formed on the surfaces of the crack were scraped and investigated using XRD (Brucker Advance D8).
3. Results and discussion 3.1. Bacterial growth in the selected growth media Figs. 2 and 3 present the evolution of lactate and calcium concentrations, respectively, in the liquid phase recovered after filtration of the bacterial suspension during bacterial growth in the M1
0.8 0.7
[Lactate] (mol/L)
the measured pH at the surface of the crack wall was 9.2 in accordance with the formation of a layer of calcium carbonate on the wall. Using an already carbonated crack is important to have cracks that are representative of mortars or concretes in situ, considering biohealing treatment as a repair strategy. Since autogenous healing reduces the thickness of the microcrack, its opening was then adjusted using the expansive core to reach approximately 250 mm in thickness before the biohealing experiment. Additionally, mortars were sterilized by gamma ray irradiation (42 kGy) to remove microorganisms.
0.6 M1 (Rep 1)
0.5
M1 (Rep 2)
0.4
M1-control
0.3 0.2 0.1 0.0
0
5
10
15
20
25
30
35
Time (days) Fig. 2. Evolution of the lactate concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M1 medium.
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0.4
1.0E+10
(40.3%) (Fig. 6). In agreement with XRD results, calcite decarbonation was expected to occur during this major weight loss. Thus, calcite corresponded to 88% of the mass of the solid residue. The presence of a small quantity of amorphous calcium carbonate that contains water molecules could also be identified by the initial weight loss between 180 and 400 °C [35] corresponding to 9% of the mass of the solid residue. Thus, the removal of calcium in the bacterial suspension was mostly due to the formation of calcite. However, all calcium ions freed by lactate consumed due to bacterial activity were not used to precipitate calcium carbonate. A part of calcium unassociated to the lactate ions (0.54/2–0.17 = 0.10 mol/L), remained in the bacterial suspension under an undetermined form. Calcium ions may be complexed by some of organic metabolites produced by the bacterial degradation of yeast extract. We could also hypothesize that clusters of amorphous calcium carbonate were formed in the presence of bacteria [35,36] and remained in the liquid part of the bacterial suspension after filtration as they passed through the mesh of the filter (0.45 lm). Results obtained with the M2 growth medium showed similar evolution to experiments carried out with the M1 growth medium (Figs. 7–9). However, when using the M2 growth medium, the lactate was completely consumed by the bacteria (0.68 mol/L) (Fig. 7), leading to a living bacterial concentration of 2.1010 bacteria/mL, which was 10 times more than that of the M1 growth medium (Fig. 9). Moreover, the living bacterial concentration stated constant whereas it slightly decreases with M1 growth medium. Thus, the M2 growth medium appeared to be more efficient to promote the bacterial growth, perhaps with a positive impact of calcium and/or nitrate ions. The decrease in calcium concentration, 0.14 mol/L, was close to the value observed with M1 growth medium (Fig. 8). The recovered solid precipitated in the bacterial suspension was also composed of 91% ± 1% of calcite and also possibly small amounts of amorphous calcium carbonate. Thus, comparatively to M1 growth medium, a larger part of calcium was unassociated to lactate that was consumed by bacteria (0.68/2–0.14 = 0.20 mol/L). Consequently, the presence of Ca(NO3)2 in M2 growth medium did not appeared to contribute to the formation of additional calcium carbonate during bacterial growth experiments. Additional calcium ions provided by Ca(NO3)2 were not available to form calcium carbonate either during the bacterial growth experiment or the control experiment. Indeed, both concentrations of lactate and calcium remained constant during the control experiment (Figs. 7 and 8). Thus, even in the presence of carbonate ions generated by the bacterial activity or by the contact of the growth medium with air, calcium ions provided by Ca(NO3)2 cannot be used to precipitate calcium carbonate as nitrate ions cannot be counterbalanced by other cations than calcium ions (Eq. (4)):
1.0E+09
½CH3 CHðOHÞCOO2 Ca þ 6 O2 þ Ca2þ þ 2 NO3
[Calcium] (mol/L)
0.35 0.3 0.25 0.2 0.15 M1 (Rep 1)
0.1
M1 (Rep 2) M1-control
0.05 0 0
5
10
15
20
25
30
35
40
Time (days) Fig. 3. Evolution of the calcium concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M1 medium.
Living bacteria per mL
medium for two Erlenmeyer flasks. These figures demonstrate a good reproducibility of results and a fast growth occurred despite the lower initial value of the pH of the growth medium compared to the growth medium recommended by DMSZ. The pH of the bacterial suspension increased with time during the growth experiment to reach a final value close to 8.5. At 7 day, the exponential phase produced 2.109 bacteria/mL. Then, the number of live bacteria slightly dropped, but remained higher than 1.108 bacteria/mL indicating a good bacterial activity. The lactate and calcium concentrations both decreased rapidly during the first 20 days. Then, they decreased less markedly to become almost constant at 35 days. The decrease in lactate concentration, 0.54 mol/L, was due to its degradation by the bacteria (Eq. (1)) and thus was linked with the bacterial growth (Fig. 4). Indeed, any lactate was consumed in the control experiment (Fig. 2). A decrease in calcium concentration was also only observed in the presence of bacteria (Fig. 3). However, the consumption of calcium was less than the lactate one, equal to 0.17 mol/L. The dried residue obtained by filtration of the bacterial suspension was analysed using TGA and XRD. XRD pattern reported calcite as the only crystalized mineral (Fig. 5). TG reported a first weight loss between 180 and 400 °C (4.5%) followed by a smaller weight loss between 400 and 600 °C (1.2%). Then, the major weight loss appeared between 650 and 900 °C
! CaCO3 ðsÞ þ 5 CO2 þ 5 H2 O þ Ca2þ þ 2 NO3
ð4Þ
1.0E+08 M1 (Rep 1)
1.0E+07
M1 (Rep 2)
1.0E+06 1.0E+05 0
5
10
15
20
25
30
35
Time (days) Fig. 4. Evolution of the living bacteria concentration per mL of suspension during the bacterial growth in M1 medium.
On the other hand, if additional chemical elements would be present and could react or counterbalance nitrate ions, some calcium ions would become available to form calcium carbonate in the presence of carbonate ions. Consequently, in the reported growth experiments, only calcium initially provided by calcium lactate and then freed by the biodegradation of lactate ions, can react with carbonate ions, originated either by the bacterial metabolism or by the contact of growth medium with air, to precipitate calcium carbonate. This conclusion would have been different if other bacterial strains such as Diaphorobacter nitroreducens leading to denitrification would have been used for MICP [37].
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Fig. 5. XRD pattern of the dried residue obtained by filtration of the bacterial suspension. All the diffraction peaks are assigned to calcite.
Fig. 6. TGA pattern of the dried residue obtained by filtration of the bacterial suspension.
0.8
[Lactate] (mol/L)
0.7 0.6 M2 (Rep 1) M2 (Rep 2) M2-control
0.5 0.4 0.3 0.2 0.1 0 0
5
10
15
20
25
30
35
Time (days) Fig. 7. Evolution of the lactate concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M2 medium.
Fig. 8. Evolution of the calcium concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M2 medium.
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Fig. 9. Evolution of the living bacteria concentration per mL of suspension during the bacterial growth in M2 medium.
3.2. Biohealing of cracked mortar
sized that the apparent crack opening was constant in these control samples. This was also an additional proof that when the anion associated with calcium, lactate ions in this case, is not involved in some chemical reactions, calcium ions cannot be used to precipitate calcium carbonate. In the present case, only bacteria were able to degrade lactate, and thus in the control sample, calcium was not available to precipitate calcium carbonate that could heal the crack. A more robust biohealing was observed for the assays performed with the M2 bacterial suspension leading to DW values after 4 months of 179 and 155 lm for sample 1 and sample 2 respectively (Fig. 11). Moreover, the decrease of the apparent crack opening was more continuous during the duration of these experiments: DW values after 2 months of 82 and 54 lm for sample 1 and sample 2 respectively. This better efficiency could be attributed to the slightly higher bacterial activity in the M2 growth medium, as demonstrated previously, but also to the presence of Ca (NO3)2. Indeed, control experiments in M2 growth medium indicated a small reduction of the apparent crack opening. It continuously increased with time leading to DW values after 4 months of 16 and 48 lm for the two samples respectively (Fig. 11). As no bacterial activity was observed in the water collected at the bottom of the control experiments humidification system, the observed decrease of the apparent crack opening could only be attributed to chemical reactions that did not involve bacteria. These chemical reactions may form some additional calcium carbonate noted x in equation (5). Indeed, if 2x nitrate ions react with the cement paste of the mortar, x calcium ions may react with carbonate ions to form x additional calcium carbonate (Eq. (5)).
3.2.1. Evolution of the apparent crack opening Fig. 10 reports the evolution of the apparent crack opening of the mortar samples treated with the M1 bacterial suspension and their control experiments without bacteria. The two mortars in contact with the bacterial suspension showed a decrease of the apparent crack opening (DW), but at different rates. Sample 1 had an apparent reduction of the thickness of its crack at 2 months of 49 lm and then it increased to 167 lm at 4 months. For sample 2, DW reached 93 lm at 2 months but decreased to 69 lm at 4 months. A decrease instead of an increase of DW is surprising as it was the only mortar that had this behaviour. Perhaps, the crack of this sample had a kind of bottleneck made by some particles released during the cracking. First, these particles would lead to underestimate the apparent opening of the microcrack. Second, they would enhance the sealing effect as observed at 2 months comparatively to sample 1. Then, for some reasons, either during the drying of the sample before the permeability measurement or during the measurement itself, the particles were moved leading to measure the true and larger apparent crack opening that was subsequently more difficult to heal. Performing several experiments is thus a key point to check the reproducibility in such complex experiments. The control samples made with M1 growth medium proved that the bacterial activity was the only process involved in the reduction of the apparent crack opening. Indeed, considering experimental errors in air-flow measurements, it can be hypothe-
Amongst the likely chemical reactions, nitrate ions could react with cement paste, in order to form some calcium mononitroaluminate (3CaO.Al2O3.Ca(NO3)2.xH2O) [38] or to be adsorbed on calcium silicate hydrate (C-S-H). However, DW values for the control experiment were strongly lower than those obtained in the presence of bacteria: 179 and 155 lm compared to 16 and 48 lm respectively. This indicates that DW was mainly due to the effect of bacteria compared to the chemical reactions between mostly Ca(NO3)2 of M2 growth medium and the cement paste. Consequently, the addition of a soluble calcium salt to the growth medium may be an interesting possibility to form larger amounts of
Fig. 10. Effective crack opening evolution on biohealing experiments using a bacterial suspension of Bacillus pseudofirmus made with M1 growth medium and on control abiotic experiments.
Fig. 11. Effective crack opening evolution on biohealing experiments using a bacterial suspension of Bacillus pseudofirmus made with M2 growth medium and on control abiotic experiments.
½CH3 CHðOHÞCOO2 Ca þ 6 O2 þ Ca2þ þ 2 NO3 þ cement paste ! ð1 þ xÞCaCO3 ðsÞ þ ð5 xÞCO2 þ ð5 þ xÞH2 O þ ð1 xÞCa2þ þ ð2 2xÞNO3 þ cement paste containing NO3 ð5Þ
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Fig. 12. Surface of the mortar and opening of the crack before (left) and after (right) biohealing experiment.
Fig. 13. XRD pattern of the deposit on the wall of the crack that was scraped in order to be analysed.
calcium carbonate and thus to improve the robustness of the healing process. However, this statement will be only true if the anion of the soluble calcium salt can react with the cement paste to be removed from the aqueous phase. This kind of chemical reactions is expected to be more intense with the cement paste of a fresh crack that has not yet reacted to autogenous heals it. Indeed, self-healing experiments reported by Stuckrath et al. [39] on fresh cracks, lead to conclude that healing induced by the chemical reactions from the chemical compounds added was more intense that the biohealing. This result is not opposed to our as the intensity of biohealing with respect to the healing induced by chemical reactions will vary depending on the availability of calcium to form calcium carbonate. On the other hand, the efficiency of the healing process was comparable to our experiments lead to seal cracks between 80 and 220 lm. So, both strategies can be efficient depending on the purpose. Fig. 12 shows the opening of a crack at the surface of the mortar before treatment. The opening of the crack is well defined and its thickness is relatively similar all along the crack opening. The same crack but not exactly at the same localisation, is presented after the 4-months of biohealing using M2 bacterial suspension (Fig. 12). A deposit of beige colour was observed at the surface of the mortar and it almost covered the opening of the crack. This is in agreement
with the reduction of the apparent thickness of the crack. The deposit seemed to be composed of fibers engulfed into a mineral matrix. Larger magnification under SEM, reported the presence of a mixture of minerals and of biofilm. The mineral contained Ca, C and O as determined by EDS, pointing to the formation of calcium carbonate as expected. The deposit on the wall of the crack was scraped and analysed by XRD. Quartz coming from the sand grains and calcite were the major crystalized minerals (Fig. 13). The formation of layer of calcite is consistent with previous results using Bacillus strains in the presence of calcium lactate [27]. The remains of the biofilm were also observed but at a lesser extent than on the deposit at the mortar surface. The biofilm was sometimes developed in the volume of the crack, forming long and fine fibers, but also larger films (Fig. 14). These fibers and films were the consequence of the biological activity as they were not observed in the control mortars. Moreover, as calcium carbonate can be formed within the biofilm [35–42], the microbial induced calcium carbonate could be more efficient to heal the crack by forming some bottleneck than calcium carbonate precipitated from chemical reactions. Indeed, in the case of a crack with walls that have already been carbonated by autogenous healing, the further formation of calcium carbonate by a chemical reaction is expected to be mostly located on the walls of the crack that are already partially
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Fig. 14. Mix of calcite and biofilm at the surface of the wall of the crack (1 cm inside the mortar) after 4 months of the biohealing experiment using a bacterial suspension of Bacillus pseudofirmus made with M2 growth medium.
or completely covered by calcium carbonate. Here, heterogeneous nucleation of calcium carbonate on already formed calcium carbonate crystals will be favoured. Thus, calcium carbonate precipitated will mostly precipitate on the wall or at the opening the crack. In the case of biogenerated calcium carbonate precipitation, calcium carbonate crystals may also be precipitated on the wall of the crack but also in the volume of the crack where the biofilm has developed. 4. Conclusions Undertaking experiments in which mutual interactions between microorganisms and material take place is a complex process. First of all, a good reproducibility of the microbial activity is required. Second, the real impact of microorganisms has to be estimated by comparing results with similar abiotic experiments that are often difficult to perform. In the present study, the control experiments carried out in abiotic conditions were essential to differentiate the healing originated from the bacterial activity relative to chemical reactions between the chemical compounds contained the growth medium and the cement paste of the mortar. The reported biohealing experiments performed with Bacillus pseudofirmus demonstrate, that healing of already autogenous and carbonated microcracks by precipitating calcium carbonate can be very efficient thanks to an adapted bacterial suspension. Indeed, due to the calcite layer formed on the crack wall, the limiting component is not carbonate ions that can be generated through the bacterial activity or provided by air, but available calcium ions. Thus, before undertaking biohealing experiments, it is essential to perform some bacterial growth tests, in order to optimize the culture medium to obtain a good bacterial activity leading to suitable amounts of calcium carbonate precipitated. The key parameter is the choice of the chemical compound, in order to supply calcium that can thereafter be used to precipitate calcium carbonate. It is necessary to use an organic calcium compound for which the organic part can serve as a nutrient for the bacterial metabolism. The reported results indicated that significant amounts of calcium carbonate were formed when calcium lactate was consumed by Bacillus pseudofirmus. Inorganic calcium salts
can also be added to the growth medium at concentrations that do not inhibit the bacterial growth. These calcium salts will not provide additional calcium carbonate precipitation during the bacterial growth experiments, but may induce a better bacterial activity. Moreover, in the presence of cement paste and if the anion of the inorganic calcium salt can react with it, some additional calcium carbonate may be precipitated. This was the case in the reported experiment when using calcium nitrate in M2 growth medium. In the case of already autogenous and carbonated microcracks, the biohealing due to the bacterial activity was significantly larger than the healing induced by the reaction between calcium nitrate and the cement paste. This result may have been different in the case of a fresh crack leading to an important supply of calcium from the cement paste but also with different bacterial strains. Consequently, different strategies can be used to optimize calcium carbonate bioprecipitation relative to its precipitation by other chemical reactions, depending on the interactions between the bacterial suspension and the material, such as soil, stone or concrete. Acknowledgements The authors acknowledge the financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). References [1] M. De Rooij, K. Van Tittelboom, N. De Belie, E. Schlangen, Self-healing Phenomena in Cement-based Materials, State of Art Report of RILEM Technical Committee 221-SHC: Self-Healing Phenomena in Cement-Based Materials, Springer, 2013. [2] W. De Muynck, N. De Belie, W. Verstraete, Microbial carbonate precipitation in construction materials: a review, Ecol. Eng. 36 (2010) 118–136. [3] V. Achal, A. Mukherjee, D. Kumari, Q. Zhang, Biomineralization for sustainable construction – a review of processes and applications, Earth Sci. Rev. 148 (2015) 1–17. [4] K. Van Tittelboom, N. De Belie, W. De Muynck, W. Verstraete, Use of bacteria to repair cracks in concrete, Cem. Concr. Res. 40 (2010) 157–166. [5] W. De Muynck, D. Debrouwer, N. De Belie, W. Verstraete, Bacterial carbonate precipitation improves the durability of cementitious materials, Cem. Concr. Res. 38 (2008) 1005–1014.
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Contents lists available at ScienceDirect
Construction and Building Materials journal homepage: www.elsevier.com/locate/conbuildmat
Microbiologically induced calcium carbonate precipitation to repair microcracks remaining after autogenous healing of mortars C. Lors a,b,⇑, J. Ducasse-Lapeyrusse a,b,c, R. Gagné c, D. Damidot a,b a
Mines Douai, LGCgE-GCE, 941 rue Charles Bourseul, 59508 Douai, France Université de Lille, Cité Scientifique, 59650 Villeneuve-d’Ascq, France c Centre de recherche sur les infrastructures en béton (CRIB), Université de Sherbrooke, 2500 boulevard de l’Université, J1K2R1 Sherbrooke, Québec, Canada b
h i g h l i g h t s Biohealing can be efficient on cracks partially autogenously healed. Bacterial effects can be estimated with biotic and abiotic experiments. Biodegradable organic calcium compound is necessary to form calcite in autogenously healed mortar. Inorganic calcium salt improves the robustness if its anion reacts with cement paste.
a r t i c l e
i n f o
Article history: Received 28 August 2016 Received in revised form 27 February 2017 Accepted 4 March 2017
Keywords: Mortar Microcrack Biohealing Calcite Calcium lactate Calcium nitrate Bacillus pseudofirmus
a b s t r a c t Cracks remaining after autogenous healing of mortar can be further healed when subjected to a bacterial suspension of Bacillus pseudofirmus if it contains organic calcium compounds. The consumption of organic calcium compounds, such as calcium lactate, by bacteria is providing the necessary calcium to precipitate calcite. The addition of inorganic calcium salts is inefficient to promote the formation of calcite during bacterial growth experiments, but can be efficient during biohealing experiments if the anion of the inorganic calcium salt can react with the cement paste. The addition of both organic and inorganic calcium compounds improves the robustness of the biohealing. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Cracking is one of the most deleterious parameters for concrete durability. In addition, to decreasing mechanical performance, cracks promote various detrimental mechanisms, as they provide a preferential pathway for the penetration of aggressive substances. However, concrete can defend itself against small microcracks having a width between 50 and 100 lm, depending on its composition, through autogenous healing [1]. Autogenous healing involves both hydration of unreacted cement, leaching and carbonation of the cement paste. Nevertheless, larger more deleterious
⇑ Corresponding author at: Mines Douai, LGCgE-GCE, 941 rue Charles Bourseul, 59508 Douai, France. E-mail addresses: [email protected] (C. Lors), [email protected] (J. Ducasse-Lapeyrusse), [email protected] (R. Gagné), [email protected] (D. Damidot). http://dx.doi.org/10.1016/j.conbuildmat.2017.03.026 0950-0618/Ó 2017 Elsevier Ltd. All rights reserved.
cracks that remain unsealed after autogenous healing, need to be treated by other means, most often expensive, such as resin impregnation. Alternative methods based on the impregnation of chemical healing agent could also be used for medium sized microcracks (having a width lower than 300 lm). Additionally, this method could be extended to biological healing agents. Indeed, microorganisms can promote the precipitation of calcium carbonate in the crack [2,3]. Calcium carbonate is a highly compatible with concrete, and it presents itself as a sustainable and ecological repair solution to improve the durability of cementitious materials [4,5]. Microbiologically Induced Calcium Carbonate Precipitation (MICCP) uses microbial metabolism to generate CO2, leading to the formation of carbonate ions in the presence of water. Carbonate ions can react with available calcium ions to precipitate calcium carbonate if the suitable conditions are reached [6]. MICCP in a crack requires filling the crack with a bacterial suspension (mix of suitable bacteria, nutrients and possibly other compounds
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promoting calcium carbonate precipitation) in which bacteria can grow [7,8]. Nevertheless, the bacterial suspension should not contain chemical compounds harmful to concrete, such as chloride ions for concrete rebars. Additionally, when the cement paste is in contact with the bacterial suspension, the chemical elements leached from the cement paste have to be innocuous with respect to the microbial activity. Once cracks are formed, they are rapidly carbonated thanks to autogenous healing, leading to the formation of calcium carbonate layer on the wall of the crack [9]. Thanks to the formation of the calcite layer, carbonation reduces the pH at the surface of the crack that becomes lower than 10 compared to more than 13 for a fresh crack [10]. This drop of pH that can be measured by pH contact electrode [11], is beneficial to select suitable alkaliphilic bacteria for MICCP [12–17]. However, calcium carbonate layer restricts the diffusion process that is governing the release of calcium from the cement paste to the crack [18]. As a consequence, the amount of calcium carbonate that could be formed by MICCP in the already carbonated crack is limited. This calcium shortage needs to be compensated by the addition of calcium in the bacterial suspension in a chemical form and at concentrations that do not inhibit the bacterial growth. The complexity induced by all these constraints have led to propose a large variety of bacterial suspensions for MICCP in various materials and environments [3,4,13,19–27]. For example, ureabased bacteria are commonly used [4,6,12], despite the fact that they form ammonium ions (NH+4), which are deleterious for concrete [28]. Alternative nutriments, such as organic calcium compounds, may also appear to be an interesting source of nutrients in the presence of bacterial strains belonging to Bacillus genus. For example, calcium lactate ([CH3CH(OH)COO]2Ca) can be metabolized by different species of Bacillus, such as B. pseudofirmus or B. cohnii, in order to promote MICCP [13,27,29]. However, these strains were less studied than Sporosarcina pasteurii especially in the case of self-healed mortars. The aim of this paper is to perform MICCP on autogenously healed mortars by using a bacterial suspension of Bacillus pseudofirmus containing calcium lactate as the main calcium source. The first part of the study is devoted to better understanding the key parameters, in order to define a suitable growth medium leading to a good bacterial activity associated with the production of notable amounts of calcium carbonate. In the second part, the selected bacterial suspension was applied on autogenously healed mortar containing one incompletely healed radial crack. The efficiency of the biohealing process was estimated by the decrease in apparent opening of the crack measured by air permeability. The observation and the mineralogical characterization of the deposit in the crack were also performed. Finally, the effect of chemical reactions between the growth medium constituents and the mortar on the healing process was assessed by performing healing experiments carried out with the growth medium in abiotic conditions. This enabled us to differentiate the intensity of microbial activity relative to the chemical reactions induced by the growth medium, in order to produce calcium carbonate during the biohealing process.
2. Materials and methods 2.1. Bacterial strain The selected strain was Bacillus pseudofirmus DMS 2516, which was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. This strain was already used in a previous study for its abilities to grow in an alkaline environment and generate carbonate ions [12]. Bacterial stocks were stored at 80 °C in glycerol. This bacterial strain is an alkaliphilic strain able to survive from neutral to basic pH levels up to 11. Indeed, any significant bacterial activity was noticed at a pH value of 11.5 [30]. It is aerobic and heterotrophic,
i.e. in the presence of oxygen, it is able to consume organic compounds and produce carbon dioxide by cellular respiration according to Eq. (1) written for calcium lactate that was used:
½CH3 CHðOHÞCOO2 Ca þ 6 O2 ! CaCO3 ðsÞ þ 5 CO2 þ 5 H2 O
ð1Þ
2.2. Growth medium composition The studied culture media, named M1 and M2, were composed of yeast extract (3 g/L) and calcium lactate, [CH3CH(OH)COO]2Ca, (75 g/L corresponding to 0.34 mol/L). Calcium nitrate, Ca(NO3)2, (32.8 g/L corresponding to 0.2 mol/L) was only added to the medium called M2. The initial pH was not adjusted, and measured 6.5 and 6 for M1 and M2 media, respectively. Using a buffer solution [NaHCO3/Na2CO3] to increase the initial pH was not desired as it could induce side chemical reactions leading to form calcium carbonate that would not be induced by the bacterial metabolism. For example, a method to precipitate calcite is to mix a solution of Na2CO3 with another one containing CaCl2. The yeast extract and calcium lactate provide the nutrients necessary for the bacterial growth. Calcium lactate and calcium nitrate also provide calcium ions. It was observed that larger calcium nitrate concentrations than 0.2 mol/L inhibited the growth of Bacillus pseudofirmus [11]. Calcium nitrate was preferred over calcium chloride often found in media used for MICCP. Indeed, chloride ions are well-known catalysers for rebar corrosion and thus could be inadequate for a repair strategy. 2.3. Bacterial growth Bacteria were pre-cultivated from frozen inocula in the medium recommended by DMSZ, composed of 8 g/L of Nutrient Broth (NB) with a pH adjusted to 9.7 using a buffer solution (NaHCO3/Na2CO3) at 30 °C until reaching the exponential growth phase. Then, two Erlenmeyer flasks containing 250 mL of each liquid sterile media (M1 or M2) were inoculated with 250 mL of the pre-culture containing approximately 109 bacteria/mL. Erlenmeyer flasks were placed under stirring at a temperature of 30 °C and were regularly monitored over 35 days for pH measurements, lactate and calcium concentrations and bacterial density. After the filtration of the bacterial suspension at 0.2 mm, the lactate concentration was determined using ion chromatography (DIONEX ICS-3000, USA) and the calcium concentration was determined using inductively coupled plasma atomic emission spectroscopy (ICPAES) (VARIAN 720 ES). The bacterial concentration was evaluated using epifluorescence microscopy (LEICA DMLP), in order to carry out a direct enumeration of bacteria [31]. The bacterial cells were harvested on a 0.2 mm filter. The living bacteria can be quantified by microscopic counting using a fluorochrome (orange acridine) and an ultraviolet light. One control sample was made for each medium (M1 or M2). In this case, the Erlenmeyer flasks containing the media were not inoculated. Lactate and soluble calcium concentrations were measured at the beginning and the end of the experiment. Sterility in each control sample was evaluated by the direct bacterial enumeration in the course of the experiment. Additionally, for each media, three Erlenmeyer flasks were not sampled, but filtered (sieve of 0.45 mm) at the end of the bacterial growth test (35 days). The collected solid residue was weighed and analysed using thermogravimetric analysis (TGA) (NETZSCH STA 449 F3) and X-ray diffraction (XRD) (Brucker Advance D8) to determine the mineralogy of the solid residue. 2.4. Preparation of cracked mortars The mortar samples were made in accordance with ASTM C109-C109M [32]. This standard requires a water-to-cement mass ratio of 0.485 and a sand-tocement mass ratio of 2.75. The cement used was a Canadian GU Portland cement (General Use, similar to CEM I and ASTM Type 1). The cement composition (in mass%) was 60% C3S, 13% C2S, 7.5% C3A and 7% C4AF. The sand required by ASTM standard C788 [33] is a normalized Ottawa sand made of natural silica, having a particle size distribution between 150 and 600 mm, with a density of 2.65. The mortar samples were annular, with a diameter of 150 mm and a height of 50 mm. A central hole with a diameter of 55 mm allowed for the insertion of a steel expansive core, which was used to create one radial crack along the entire section of the mortar (Fig. 1). Thus, the crack had three openings accessible for healing: one opening at the top, one other at the bottom and the last one in the side surface of the ring. The surface of each crack wall was 50 47.5 mm2 and the apparent crack opening was computed from air-flow measurements [34]. The sample included an embedded steel ring to simulate a reinforcing bar that balances the internal stresses after cracking (Fig. 1). The ring was made from a hot-rolled steel rod having an internal diameter of 95.2 mm2 and a section diameter of 4.8 mm (reinforcement ratio of 0.72%) [34]. The mortars were cracked after 28 days of storage in an environmental chamber at 100% relative humidity and a temperature of 23 °C. The opening of each crack was more than 200 mm. After cracking, the mortar samples were stored for more than one year at 100% R.H. and a temperature of 23 °C. After this period, the autogenous healing process was almost complete and the walls of the cracks were carbonated at 23 °C under atmosphere of 4% of CO2 with 40% relative humidity. Thus,
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150 mm 55 mm
Crack
Expansive core 50 mm
Steel ring Mortar sample
Fig. 1. Schematic diagram of the annular mortar [34].
2.5. Biohealing of cracked mortars The mortar samples were immersed in contact either with M1 or M2 bacterial suspensions obtained by the culture of Bacillus pseudofirmus in M1 or M2 growth medium for seven days respectively. The cracked mortar samples with their expansive core were individually immersed in such a way that only the microcracked volume was in contact with the bacterial suspension. Thus, the bacterial suspension could enter the microcrack by its three openings. The immersion was done in a microbiological safety cabinet in which the air-flow was kept sterile, in order to avoid contamination by other microorganisms. For the control experiment, the mortar sample was immersed in the same way, but in a sterile culture medium. Its chemical composition was designed to be as close as possible to the bacterial suspension in which the cracked mortar was immersed. In order to do so, the control experiment was made after the biohealing experiment. This allowed us to determine both calcium and lactate concentrations in the bacterial suspension in which the mortar was immersed, as some calcium and lactate were consumed during seven days of the bacterial growth prior the immersion of the mortar. Thus, the lactate and calcium concentrations in the medium used for the control experiment carried out in abiotic conditions were identical to those of the biohealing experiment. Moreover, the biocide bronopol (0.1 g/L) was added in medium used for the control experiment, in order to avoid contamination by external microorganisms. For biotic and abiotic experiments, duplicate samples were made to assess the reproducibility. After the immersion, each mortar sample was placed for one month in its own initially sterile humidification system, which provided 100% R.H. at a temperature of 20 °C and avoided contamination. The totally saturated condition with respect to water vapour was required, in order to be sure that the bacterial activity continued during the period where biohealing was expected to occur. However, it also led water condensation on the surface of the cracked mortar and on the walls of the humidification system. Subsequently, some water accumulated at the bottom of the humidification system. The mortar samples were not in contact with the water at the bottom of the humidification system. Nevertheless, they were slightly leached by the water condensed at their surface, which was partly recovered at the bottom of the humidification system. The water accumulated at the bottom of the humidification system was analysed for the presence of microorganisms using epifluorescence microscopy in the case of the control assays, in order to check whether any contamination occurred. After one month spent in the humidification system, the mortar was removed from the system to measure the apparent crack opening using air-flow described in the next paragraph. As most of bacteria did not survive to drying at 40 °C during 24 h, the mortar was immersed again in a bacterial suspension to perform a new cycle of biohealing. This protocol was repeated over a four-months period, leading the cracked mortar to be subjected to four cycles of one day of immersion in the bacterial suspension, one month of storage in the humidification system, and then a 24 h drying at 40 °C followed by the measurement of the apparent crack opening. 2.6. Measurement of the apparent crack opening using air flow The evolution of the apparent crack opening was monitored every month before the next immersion in the bacterial suspension using air-flow measurements in a sterile air permeability apparatus [34]. The mortar samples were removed from the humidification systems and dried at 40 °C for 24 h. The samples were placed
in an air permeability cell specially designed to evaluate the apparent opening of the crack. Eq. (2) describes the relationship between Q (air flow in liter per minutes) and Wef (the effective crack opening in mm):
Q ¼ 1:57043:104 W2ef þ 1:40517:102 Wef
ð2Þ
This equation was obtained through a large number of assays on cracked mortar samples having the same geometry and cracked using the same device. In order to evaluate the biohealing process, the apparent reduction of the thickness of the crack at a given time (DWt) was calculated from the initial apparent crack opening (W0) and the apparent crack opening at a given time t (Wt) (Eq. (3)):
DWt ¼ W0 Wt
ð3Þ
It can be noticed that DWt does not depend on the initial thickness of the crack because of the planar geometry of the crack. Thus, the healing of cracks having different initial thickness can be compared. 2.7. Observations of the cracks During the biohealing experiment, observations of the opening of the crack at the mortar surface were made every month with a binocular magnifier (OLYMPUS, B061) on dried mortar just before airflow measurement. After 4 months, at the end of the biohealing assay, the mortar sample was cut to 1 cm on each side of the crack and the crack was split open by slight chisel strokes, enabling us to observe the deposit at the surface of the wall of the crack. Observations were made under a scanning electron microscope (SEM) (Hitachi SE 4300). Moreover, the products formed on the surfaces of the crack were scraped and investigated using XRD (Brucker Advance D8).
3. Results and discussion 3.1. Bacterial growth in the selected growth media Figs. 2 and 3 present the evolution of lactate and calcium concentrations, respectively, in the liquid phase recovered after filtration of the bacterial suspension during bacterial growth in the M1
0.8 0.7
[Lactate] (mol/L)
the measured pH at the surface of the crack wall was 9.2 in accordance with the formation of a layer of calcium carbonate on the wall. Using an already carbonated crack is important to have cracks that are representative of mortars or concretes in situ, considering biohealing treatment as a repair strategy. Since autogenous healing reduces the thickness of the microcrack, its opening was then adjusted using the expansive core to reach approximately 250 mm in thickness before the biohealing experiment. Additionally, mortars were sterilized by gamma ray irradiation (42 kGy) to remove microorganisms.
0.6 M1 (Rep 1)
0.5
M1 (Rep 2)
0.4
M1-control
0.3 0.2 0.1 0.0
0
5
10
15
20
25
30
35
Time (days) Fig. 2. Evolution of the lactate concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M1 medium.
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0.4
1.0E+10
(40.3%) (Fig. 6). In agreement with XRD results, calcite decarbonation was expected to occur during this major weight loss. Thus, calcite corresponded to 88% of the mass of the solid residue. The presence of a small quantity of amorphous calcium carbonate that contains water molecules could also be identified by the initial weight loss between 180 and 400 °C [35] corresponding to 9% of the mass of the solid residue. Thus, the removal of calcium in the bacterial suspension was mostly due to the formation of calcite. However, all calcium ions freed by lactate consumed due to bacterial activity were not used to precipitate calcium carbonate. A part of calcium unassociated to the lactate ions (0.54/2–0.17 = 0.10 mol/L), remained in the bacterial suspension under an undetermined form. Calcium ions may be complexed by some of organic metabolites produced by the bacterial degradation of yeast extract. We could also hypothesize that clusters of amorphous calcium carbonate were formed in the presence of bacteria [35,36] and remained in the liquid part of the bacterial suspension after filtration as they passed through the mesh of the filter (0.45 lm). Results obtained with the M2 growth medium showed similar evolution to experiments carried out with the M1 growth medium (Figs. 7–9). However, when using the M2 growth medium, the lactate was completely consumed by the bacteria (0.68 mol/L) (Fig. 7), leading to a living bacterial concentration of 2.1010 bacteria/mL, which was 10 times more than that of the M1 growth medium (Fig. 9). Moreover, the living bacterial concentration stated constant whereas it slightly decreases with M1 growth medium. Thus, the M2 growth medium appeared to be more efficient to promote the bacterial growth, perhaps with a positive impact of calcium and/or nitrate ions. The decrease in calcium concentration, 0.14 mol/L, was close to the value observed with M1 growth medium (Fig. 8). The recovered solid precipitated in the bacterial suspension was also composed of 91% ± 1% of calcite and also possibly small amounts of amorphous calcium carbonate. Thus, comparatively to M1 growth medium, a larger part of calcium was unassociated to lactate that was consumed by bacteria (0.68/2–0.14 = 0.20 mol/L). Consequently, the presence of Ca(NO3)2 in M2 growth medium did not appeared to contribute to the formation of additional calcium carbonate during bacterial growth experiments. Additional calcium ions provided by Ca(NO3)2 were not available to form calcium carbonate either during the bacterial growth experiment or the control experiment. Indeed, both concentrations of lactate and calcium remained constant during the control experiment (Figs. 7 and 8). Thus, even in the presence of carbonate ions generated by the bacterial activity or by the contact of the growth medium with air, calcium ions provided by Ca(NO3)2 cannot be used to precipitate calcium carbonate as nitrate ions cannot be counterbalanced by other cations than calcium ions (Eq. (4)):
1.0E+09
½CH3 CHðOHÞCOO2 Ca þ 6 O2 þ Ca2þ þ 2 NO3
[Calcium] (mol/L)
0.35 0.3 0.25 0.2 0.15 M1 (Rep 1)
0.1
M1 (Rep 2) M1-control
0.05 0 0
5
10
15
20
25
30
35
40
Time (days) Fig. 3. Evolution of the calcium concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M1 medium.
Living bacteria per mL
medium for two Erlenmeyer flasks. These figures demonstrate a good reproducibility of results and a fast growth occurred despite the lower initial value of the pH of the growth medium compared to the growth medium recommended by DMSZ. The pH of the bacterial suspension increased with time during the growth experiment to reach a final value close to 8.5. At 7 day, the exponential phase produced 2.109 bacteria/mL. Then, the number of live bacteria slightly dropped, but remained higher than 1.108 bacteria/mL indicating a good bacterial activity. The lactate and calcium concentrations both decreased rapidly during the first 20 days. Then, they decreased less markedly to become almost constant at 35 days. The decrease in lactate concentration, 0.54 mol/L, was due to its degradation by the bacteria (Eq. (1)) and thus was linked with the bacterial growth (Fig. 4). Indeed, any lactate was consumed in the control experiment (Fig. 2). A decrease in calcium concentration was also only observed in the presence of bacteria (Fig. 3). However, the consumption of calcium was less than the lactate one, equal to 0.17 mol/L. The dried residue obtained by filtration of the bacterial suspension was analysed using TGA and XRD. XRD pattern reported calcite as the only crystalized mineral (Fig. 5). TG reported a first weight loss between 180 and 400 °C (4.5%) followed by a smaller weight loss between 400 and 600 °C (1.2%). Then, the major weight loss appeared between 650 and 900 °C
! CaCO3 ðsÞ þ 5 CO2 þ 5 H2 O þ Ca2þ þ 2 NO3
ð4Þ
1.0E+08 M1 (Rep 1)
1.0E+07
M1 (Rep 2)
1.0E+06 1.0E+05 0
5
10
15
20
25
30
35
Time (days) Fig. 4. Evolution of the living bacteria concentration per mL of suspension during the bacterial growth in M1 medium.
On the other hand, if additional chemical elements would be present and could react or counterbalance nitrate ions, some calcium ions would become available to form calcium carbonate in the presence of carbonate ions. Consequently, in the reported growth experiments, only calcium initially provided by calcium lactate and then freed by the biodegradation of lactate ions, can react with carbonate ions, originated either by the bacterial metabolism or by the contact of growth medium with air, to precipitate calcium carbonate. This conclusion would have been different if other bacterial strains such as Diaphorobacter nitroreducens leading to denitrification would have been used for MICP [37].
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Fig. 5. XRD pattern of the dried residue obtained by filtration of the bacterial suspension. All the diffraction peaks are assigned to calcite.
Fig. 6. TGA pattern of the dried residue obtained by filtration of the bacterial suspension.
0.8
[Lactate] (mol/L)
0.7 0.6 M2 (Rep 1) M2 (Rep 2) M2-control
0.5 0.4 0.3 0.2 0.1 0 0
5
10
15
20
25
30
35
Time (days) Fig. 7. Evolution of the lactate concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M2 medium.
Fig. 8. Evolution of the calcium concentration of the filtered bacterial suspension during the growth of Bacillus pseudofirmus in M2 medium.
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Fig. 9. Evolution of the living bacteria concentration per mL of suspension during the bacterial growth in M2 medium.
3.2. Biohealing of cracked mortar
sized that the apparent crack opening was constant in these control samples. This was also an additional proof that when the anion associated with calcium, lactate ions in this case, is not involved in some chemical reactions, calcium ions cannot be used to precipitate calcium carbonate. In the present case, only bacteria were able to degrade lactate, and thus in the control sample, calcium was not available to precipitate calcium carbonate that could heal the crack. A more robust biohealing was observed for the assays performed with the M2 bacterial suspension leading to DW values after 4 months of 179 and 155 lm for sample 1 and sample 2 respectively (Fig. 11). Moreover, the decrease of the apparent crack opening was more continuous during the duration of these experiments: DW values after 2 months of 82 and 54 lm for sample 1 and sample 2 respectively. This better efficiency could be attributed to the slightly higher bacterial activity in the M2 growth medium, as demonstrated previously, but also to the presence of Ca (NO3)2. Indeed, control experiments in M2 growth medium indicated a small reduction of the apparent crack opening. It continuously increased with time leading to DW values after 4 months of 16 and 48 lm for the two samples respectively (Fig. 11). As no bacterial activity was observed in the water collected at the bottom of the control experiments humidification system, the observed decrease of the apparent crack opening could only be attributed to chemical reactions that did not involve bacteria. These chemical reactions may form some additional calcium carbonate noted x in equation (5). Indeed, if 2x nitrate ions react with the cement paste of the mortar, x calcium ions may react with carbonate ions to form x additional calcium carbonate (Eq. (5)).
3.2.1. Evolution of the apparent crack opening Fig. 10 reports the evolution of the apparent crack opening of the mortar samples treated with the M1 bacterial suspension and their control experiments without bacteria. The two mortars in contact with the bacterial suspension showed a decrease of the apparent crack opening (DW), but at different rates. Sample 1 had an apparent reduction of the thickness of its crack at 2 months of 49 lm and then it increased to 167 lm at 4 months. For sample 2, DW reached 93 lm at 2 months but decreased to 69 lm at 4 months. A decrease instead of an increase of DW is surprising as it was the only mortar that had this behaviour. Perhaps, the crack of this sample had a kind of bottleneck made by some particles released during the cracking. First, these particles would lead to underestimate the apparent opening of the microcrack. Second, they would enhance the sealing effect as observed at 2 months comparatively to sample 1. Then, for some reasons, either during the drying of the sample before the permeability measurement or during the measurement itself, the particles were moved leading to measure the true and larger apparent crack opening that was subsequently more difficult to heal. Performing several experiments is thus a key point to check the reproducibility in such complex experiments. The control samples made with M1 growth medium proved that the bacterial activity was the only process involved in the reduction of the apparent crack opening. Indeed, considering experimental errors in air-flow measurements, it can be hypothe-
Amongst the likely chemical reactions, nitrate ions could react with cement paste, in order to form some calcium mononitroaluminate (3CaO.Al2O3.Ca(NO3)2.xH2O) [38] or to be adsorbed on calcium silicate hydrate (C-S-H). However, DW values for the control experiment were strongly lower than those obtained in the presence of bacteria: 179 and 155 lm compared to 16 and 48 lm respectively. This indicates that DW was mainly due to the effect of bacteria compared to the chemical reactions between mostly Ca(NO3)2 of M2 growth medium and the cement paste. Consequently, the addition of a soluble calcium salt to the growth medium may be an interesting possibility to form larger amounts of
Fig. 10. Effective crack opening evolution on biohealing experiments using a bacterial suspension of Bacillus pseudofirmus made with M1 growth medium and on control abiotic experiments.
Fig. 11. Effective crack opening evolution on biohealing experiments using a bacterial suspension of Bacillus pseudofirmus made with M2 growth medium and on control abiotic experiments.
½CH3 CHðOHÞCOO2 Ca þ 6 O2 þ Ca2þ þ 2 NO3 þ cement paste ! ð1 þ xÞCaCO3 ðsÞ þ ð5 xÞCO2 þ ð5 þ xÞH2 O þ ð1 xÞCa2þ þ ð2 2xÞNO3 þ cement paste containing NO3 ð5Þ
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Fig. 12. Surface of the mortar and opening of the crack before (left) and after (right) biohealing experiment.
Fig. 13. XRD pattern of the deposit on the wall of the crack that was scraped in order to be analysed.
calcium carbonate and thus to improve the robustness of the healing process. However, this statement will be only true if the anion of the soluble calcium salt can react with the cement paste to be removed from the aqueous phase. This kind of chemical reactions is expected to be more intense with the cement paste of a fresh crack that has not yet reacted to autogenous heals it. Indeed, self-healing experiments reported by Stuckrath et al. [39] on fresh cracks, lead to conclude that healing induced by the chemical reactions from the chemical compounds added was more intense that the biohealing. This result is not opposed to our as the intensity of biohealing with respect to the healing induced by chemical reactions will vary depending on the availability of calcium to form calcium carbonate. On the other hand, the efficiency of the healing process was comparable to our experiments lead to seal cracks between 80 and 220 lm. So, both strategies can be efficient depending on the purpose. Fig. 12 shows the opening of a crack at the surface of the mortar before treatment. The opening of the crack is well defined and its thickness is relatively similar all along the crack opening. The same crack but not exactly at the same localisation, is presented after the 4-months of biohealing using M2 bacterial suspension (Fig. 12). A deposit of beige colour was observed at the surface of the mortar and it almost covered the opening of the crack. This is in agreement
with the reduction of the apparent thickness of the crack. The deposit seemed to be composed of fibers engulfed into a mineral matrix. Larger magnification under SEM, reported the presence of a mixture of minerals and of biofilm. The mineral contained Ca, C and O as determined by EDS, pointing to the formation of calcium carbonate as expected. The deposit on the wall of the crack was scraped and analysed by XRD. Quartz coming from the sand grains and calcite were the major crystalized minerals (Fig. 13). The formation of layer of calcite is consistent with previous results using Bacillus strains in the presence of calcium lactate [27]. The remains of the biofilm were also observed but at a lesser extent than on the deposit at the mortar surface. The biofilm was sometimes developed in the volume of the crack, forming long and fine fibers, but also larger films (Fig. 14). These fibers and films were the consequence of the biological activity as they were not observed in the control mortars. Moreover, as calcium carbonate can be formed within the biofilm [35–42], the microbial induced calcium carbonate could be more efficient to heal the crack by forming some bottleneck than calcium carbonate precipitated from chemical reactions. Indeed, in the case of a crack with walls that have already been carbonated by autogenous healing, the further formation of calcium carbonate by a chemical reaction is expected to be mostly located on the walls of the crack that are already partially
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Fig. 14. Mix of calcite and biofilm at the surface of the wall of the crack (1 cm inside the mortar) after 4 months of the biohealing experiment using a bacterial suspension of Bacillus pseudofirmus made with M2 growth medium.
or completely covered by calcium carbonate. Here, heterogeneous nucleation of calcium carbonate on already formed calcium carbonate crystals will be favoured. Thus, calcium carbonate precipitated will mostly precipitate on the wall or at the opening the crack. In the case of biogenerated calcium carbonate precipitation, calcium carbonate crystals may also be precipitated on the wall of the crack but also in the volume of the crack where the biofilm has developed. 4. Conclusions Undertaking experiments in which mutual interactions between microorganisms and material take place is a complex process. First of all, a good reproducibility of the microbial activity is required. Second, the real impact of microorganisms has to be estimated by comparing results with similar abiotic experiments that are often difficult to perform. In the present study, the control experiments carried out in abiotic conditions were essential to differentiate the healing originated from the bacterial activity relative to chemical reactions between the chemical compounds contained the growth medium and the cement paste of the mortar. The reported biohealing experiments performed with Bacillus pseudofirmus demonstrate, that healing of already autogenous and carbonated microcracks by precipitating calcium carbonate can be very efficient thanks to an adapted bacterial suspension. Indeed, due to the calcite layer formed on the crack wall, the limiting component is not carbonate ions that can be generated through the bacterial activity or provided by air, but available calcium ions. Thus, before undertaking biohealing experiments, it is essential to perform some bacterial growth tests, in order to optimize the culture medium to obtain a good bacterial activity leading to suitable amounts of calcium carbonate precipitated. The key parameter is the choice of the chemical compound, in order to supply calcium that can thereafter be used to precipitate calcium carbonate. It is necessary to use an organic calcium compound for which the organic part can serve as a nutrient for the bacterial metabolism. The reported results indicated that significant amounts of calcium carbonate were formed when calcium lactate was consumed by Bacillus pseudofirmus. Inorganic calcium salts
can also be added to the growth medium at concentrations that do not inhibit the bacterial growth. These calcium salts will not provide additional calcium carbonate precipitation during the bacterial growth experiments, but may induce a better bacterial activity. Moreover, in the presence of cement paste and if the anion of the inorganic calcium salt can react with it, some additional calcium carbonate may be precipitated. This was the case in the reported experiment when using calcium nitrate in M2 growth medium. In the case of already autogenous and carbonated microcracks, the biohealing due to the bacterial activity was significantly larger than the healing induced by the reaction between calcium nitrate and the cement paste. This result may have been different in the case of a fresh crack leading to an important supply of calcium from the cement paste but also with different bacterial strains. Consequently, different strategies can be used to optimize calcium carbonate bioprecipitation relative to its precipitation by other chemical reactions, depending on the interactions between the bacterial suspension and the material, such as soil, stone or concrete. Acknowledgements The authors acknowledge the financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). References [1] M. De Rooij, K. Van Tittelboom, N. De Belie, E. Schlangen, Self-healing Phenomena in Cement-based Materials, State of Art Report of RILEM Technical Committee 221-SHC: Self-Healing Phenomena in Cement-Based Materials, Springer, 2013. [2] W. De Muynck, N. De Belie, W. Verstraete, Microbial carbonate precipitation in construction materials: a review, Ecol. Eng. 36 (2010) 118–136. [3] V. Achal, A. Mukherjee, D. Kumari, Q. Zhang, Biomineralization for sustainable construction – a review of processes and applications, Earth Sci. Rev. 148 (2015) 1–17. [4] K. Van Tittelboom, N. De Belie, W. De Muynck, W. Verstraete, Use of bacteria to repair cracks in concrete, Cem. Concr. Res. 40 (2010) 157–166. [5] W. De Muynck, D. Debrouwer, N. De Belie, W. Verstraete, Bacterial carbonate precipitation improves the durability of cementitious materials, Cem. Concr. Res. 38 (2008) 1005–1014.
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