Isolation of alkaliphilic calcifying bacteria and their feasibility for enhanced CaCO3 precipitation in bio-based cementitious composites

Abstract

Microbially induced calcite precipitation (MICP), secreted through biological metabolic activity, secured an imperative position in remedial measures within the construction industry subsequent to ecological, environmental and economical returns. However, this contemporary recurrent healing system is susceptible to microbial depletion in the highly alkaline cementitious environment. Therefore, researchers are probing for alkali resistant calcifying microbes. In the present study, alkaliphilic microbes were isolated from different soil sources and screened for probable CaCO₃ precipitation. Non-ureolytic pathway (oxidation of organic carbon) was adopted for calcite precipitation to eliminate the production of toxic ammonia. For this purpose, calcium lactate Ca(C₃ H₅ O₃ )₂ and calcium acetate Ca(CH₃ COO)₂ were used as CaCO₃ precipitation precursors. The quantification protocol for precipitated CaCO₃ was established to select potent microbial species for implementation in the alkaline cementitious systems as more than 50% of isolates were able to precipitate CaCO₃ . Results suggested 80% of potent calcifying strains isolated in this study, portrayed higher calcite precipitation at pH 10 when compared to pH 7. Ten superlative morphologically distinct isolates capable of CaCO₃ production were identified by 16SrRNA sequencing. Sequenced microbes were identified as species of Bacillus, Arthrobacter, Planococcus, Chryseomicrobium and Corynebacterium. Further, microstructure of precipitated CaCO₃ was inspected through scanning electron microscopy (SEM), X-ray diffraction (XRD) and thermal gravimetric (TG) analysis. Then, the selected microbes were investigated in the cementitious mortar to rule out any detrimental effects on mechanical properties. These strains showed maximum of 36% increase in compressive strength and 96% increase in flexural strength. Bacillus, Arthrobacter, Corynebacterium and Planococcus genera have been reported as CaCO₃ producers but isolated strains have not yet been investigated in conjunction with cementitious mortar. Moreover, species of Chryseomicrobium and Glutamicibacter were reported first time as calcifying strains.

Fig. 1

Process of identification of calcifying bacteria. (i) White halos represent the secreted calcite around the inoculation of calcifying bacteria in CPM plate. (ii) Red circle pointing the precipitated calcite at the bottom of falcon tube.

Fig. 2

Quantification of calcite precipitation by calcifying bacterial strains under different calcium sources, i.e. calcium lactate and calcium acetate at the incubation age of 7 and 14 days.

Fig. 3

Calcite precipitation of selected calcifying strains at pH 7 and pH 10 with calcium lactate as calcium source.

Fig. 4

The evolutionary history was inferred using the neighbour‐joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.59515366 is shown. The evolutionary distances were computed using the Tamura–Nei method (Tamura and Nei, 1993) and are in the units of the number of base substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). This analysis involved 38 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1501 positions in the final data set. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Fig. 5

Effect of calcium source on calcite morphology; (A) calcite precipitates of calcium lactate source are mostly spherical vaterite (A.1) and rhombohedral calcite crystals (A.2); (B) calcite precipitates of calcium acetate source are mainly spherical oval vaterite (B.2) and few are rhombohedral (B.1) and amorphous CaCO₃ (B.3).

Fig. 6

EDS of precipitated calcite.

Fig. 7

SEM analysis of precipitated calcite by (A) B. safensis, (B) C. imtechense, (C) A. luteolus and (D) G. mysorens using calcium lactate source.

Fig. 8

XRD pattern of precipitated calcite using different calcium sources.

Fig. 9

Thermal degradation curve of precipitated CaCO₃ powder showing weight loss in the range of 600 to 800ºC.

Fig. 10

Mechanical properties of calcifying bacterial formulations. A. Flexural strength and (B) compressive strength.