Nitrate reducing CaCO3 precipitating bacteria survive in mortar and inhibit steel corrosion
Introduction
Steel reinforcement plays an important role for durability of concrete structures, particularly under tensile loads. Yet, concrete is prone to cracking due to its heterogeneous matrix and brittle nature. Early age cracks in concrete mostly occur few days after casting and facilitate the migration of aggressive substances all the way to the steel rebar. Repair of these cracks, therefore, is essential to increase the durability of the concrete structures. In practice, extrinsic maintenance methods such as injection of various repair agents into concrete cracks are used [1]. Meanwhile researchers have been trying to implement self-healing technology into concrete to avoid manual labor and to repair the cracks immediately after crack initiation, i.e. starting from the micro-level [2]. Self-healing in concrete is the intrinsic repair of the cracks with the aid of rupture triggered chemical or biochemical processes and their reaction products. In order to achieve biochemical self-healing concrete, several types of axenic bacteria have been investigated [3], [4]. The main principle has been to close cracks by means of microbial induced CaCO3 precipitation. Previous results indicated that cracks up to 1 mm could be healed in 3 to 14 weeks depending on the type of the bacteria, the biochemical processes and the crack width [4], [5], [6]. Such self-healing processes however lack the preventive action to avoid exposure of the steel surface to corrosive substances during the healing period. It is possible to achieve simultaneous corrosion inhibition and crack healing by using NO3− reducing bacteria.
Biological NO3− reduction takes place during the microbial oxidation of organic matter by use of NO3− as an electron acceptor instead of O2 ((Reaction 1), (Reaction 2), (Reaction 3), (Reaction 4)). In the presence of calcium ions (Ca2 +) NO3− reduction induces CaCO3 precipitation (Reaction 5). Our works have revealed that through NO3− reduction, even enhanced CaCO3 precipitation performances could be achieved in nutrient-poor environments which makes the mechanism feasible for self-healing concrete [7]. Nitrate (NO3−) reduction is not only advantageous by inducing enhanced CaCO3 precipitation; it can also lead to the production of NO2− (Reaction 1) which is known as corrosion inhibitor [8], [9].
Studies on NO2− for corrosion inhibition have revealed that the optimum corrosion inhibition could be achieved when the [NO2−]:[Cl−] ratio was in the range of 0.34–1 [10], [11], [12]. Nitrite (NO2−) is an intermediate product in biological NO3− reduction (Reaction 1). In alkaline conditions (pH ~ 9), microbial NO2− reduction is mostly suppressed by high rate NO3− reduction causing NO2− to accumulate, which is called partial/incomplete denitrification [8]. It is possible to achieve NO2− concentrations up to 0.065 M in a few hours through partial denitrification [8]. To our knowledge, despite its significant potential, biological NO3− reduction has never been investigated either for self-healing concrete or for corrosion inhibition.
It is known that to be used in concrete applications, bacteria should be able to withstand (1) high shear stress during mixing, (2) high temperatures during cement hydration, (3) starvation, (4) highly alkaline pH (pH ~ 13 in concrete and pH ~ 10 in cracks), (5) dehydration stress, (6) cement hydration related shrinkage and pore sizes < 0.1 μm [3]. Previous studies revealed that regardless of their resilience, axenic cultures (spores or vegetative cells) require encapsulation or immobilization to withstand the harsh conditions, especially the high shear stress, the alkaline pH conditions and the crushing due to microstructure densification [5], [13]. We previously reported two NO3− reducing axenic cultures namely, Diaphorobacter nitroreducens and Pseudomonas aeruginosa for their resilience to heat, dehydration and starvation [7]. Therefore, it is possible that with protective carriers these two axenic cultures could become functional in the context of self-healing of concrete.
Besides, it is reported that bacteria are able to create self-organizing clusters, composed of compact self-immobilized cells to protect themselves from harsh conditions [14]. Moreover, our work revealed that such self-immobilized cultures are concrete compatible [15]. Therefore, a clustered microbial culture containing an activated compact denitrifying core (ACDC) could also be an option. The identified options (Diaphorobacter nitroreducens, Pseudomonas aeruginosa and ACDC) were tested for their potential to develop a corrosion resistant microbial self-healing concrete in three consecutive steps, (1) activity in alkaline pH environments (pH 9.5–10, pH ~ 13) (2) survival in mortar, (3) effect of biochemically produced NO2− on steel corrosion.
Section snippets
Production of axenic cultures
Axenic cultures were grown in nutrient media (NM) and harvested (1.25 g cell dry weight/L) by centrifuging at 6300 g for 7 min. Collected pellets were re-suspended in saline solution (0.15 M NaCl) prior to inoculation of test bottles.
Production of ACDC culture
The ACDC culture was cultivated in a cylindrical sequencing batch reactor (SBR) (effective h = 30 cm, Ø = 12.5 cm and 50% volume exchange ratio) by following a previously described procedure [16]. The SBR was operated with anoxic/aerobic period sequence (180 min
Effect of pH on bacterial activity
The NO3− reduction activities of unprotected axenic cultures at each pH condition (pH 7, pH 9.5 and pH 13) were used as a base to evaluate the effect of protection methods. The evolution of NOx-N (NO3-N and NO2-N) concentrations were used to compare the performances in each case. The NO3− reduction activities of Diaphorobacter nitroreducens with and without protection are given in Fig. 1. Same type of illustration was used in Fig. 2 to present NO3− reduction activities of Pseudomonas aeruginosa
Performance of unprotected bacteria at different pH environments
It is known that several environmental factors control the microbial NO3− reduction. Presence of NO3− reducing bacteria, available nutrients and absence of oxygen are the main controlling parameters of the microbial NO3− reduction [20]. Besides, pH and temperature influence the activities and thus the denitrification rate [21]. Microbial activity at different pH values is of importance for the development of microbial self-healing concrete. To be appropriate for concrete application, bacteria
Conclusion
It was shown that vegetative axenic NO3− reducing and CaCO3 precipitating bacteria, Pseudomonas aeruginosa and Diaphorobacter nitroreducens, could survive mortar environment if protected by either diatomaceous earth, expanded clay or granular activated carbon. The self-immobilized non-axenic ACDC culture did not require additional protection for mortar application and performed better than the protected axenic cultures in all the tested conditions.
The tested cultures tend to accumulate NO2− at
Acknowledgments
The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement no. 290308 (Marie Curie action SHeMat “Training Network for Self-Healing Materials: from Concepts to Market”).
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