Optimizing the performance of microbially induced calcium carbonate precipitation (MICCP) through microbial engineering approaches

Seyed Ali Rahmaninezhad

doi:10.17918/00010665

Microbially Induced Calcium Carbonate Precipitation (MICCP) involves microorganisms producing calcium carbonate, presenting a cost-effective and efficient method that has garnered attention for concrete repair research since Cripps' seminal work in the previous century [1]. Despite advancements in bio self-repair of concrete, significant research gaps persist regarding the factors that impede the kinetics of calcium carbonate production during MICCP reactions. This dissertation addresses these gaps by investigating inhibiting factors and proposing solutions to mitigate their adverse effects on MICCP reactions, thereby enhancing calcium carbonate production kinetics. Drawing from an extensive literature review, we categorize impediments into two primary categories: "delaying factors" that prolong the onset of MICCP reactions, and "restricting factors" that diminish the overall yield of calcium carbonate production. To tackle these inhibiting factors, we structure MICCP reactions into three phases: the endospores-germination, lag, and log phases. We aim to expedite calcium carbonate production initiation, increase yield, and enhance production kinetics by addressing delaying and restricting factors through three corresponding approaches. Our first approach scrutinizes the challenging factors encountered during the endospores-germination phase. This phase spans from the inception of endospores' exposure to cement to their maturation into vegetative cells throughout the lifespan of concrete. Bio-concrete confronts a spectrum of rigorous environmental conditions, including extreme temperatures, variable pH levels, and desiccation. Literature underscores these conditions as dual impediments, both delaying and restricting factors, which impede the initiation of the logarithmic growth phase. Consequently, they diminish the efficiency of MICCP reactions by curtailing the successful transition of endospores into vegetative cells. Our hypothesis posits a correlation between endospores' vulnerability to these conditions and their morphology, marked by an insufficient protein-based protective layer around the core. Hence, we advocated refining the endosporulation process to bolster the protective layers, thus augmenting their resilience to adverse conditions. Our research reveals that the endosporulation ratio using the thermal shock (TS) method surpasses that of the commonly employed carbon starvation (CS) method. Furthermore, employing the TS method enhances endospore tolerance to saline environments, and freeze-thaw cycling results in a higher germination ratio, expediting the onset of the logarithmic growth phase, and amplifying calcium carbonate production yield. The second approach delves into the complex factors related to the lag phase, during which vegetative-phase bacteria acclimate to micro-environmental conditions preceding the exponential growth phase. Our research, aligning with existing literature, underscores that elevated initial urea concentrations impede bacterial growth, thus delaying the onset of the log phase and subsequent MICCP reactions. We posit that this inhibition stems from the time required for cells to produce urease, and exposure to higher urea concentrations further postpones urease production. To tackle this issue, we advocated for the introduction of pre-cultured urease-containing cells, which can alleviate bacterial growth inhibition by mitigating the initially high urea concentrations. Our findings demonstrated that incorporating pre-cultured urease-containing cells accelerates urease growth initiation, thereby expediting bacterial growth onset. Consequently, MICCP reactions are enhanced up to 2.6 times, leading to accelerated calcium carbonate production, and crack-filling rates. The third approach investigates the intricate factors influencing cell behavior during the transition from the lag phase to the logarithmic phase. Existing literature suggests that exposing bacterial cells to high concentrations of calcium ions adversely affects both bacterial growth rates and cell mobility. Addressing the research gap concerning the impact of calcium ion presence on MICCP performance, we proposed that reduced cell mobility may stem from cell encapsulation due to the formation of calcium carbonate crystal layers around the cells. These layers impede cell access to essential elements such as carbon, nutrients, oxygen, and urea, ultimately leading to the death phase. Furthermore, consuming carbonate ions during cell encapsulation diminishes the buffering capacity of the culture media. To address this, we proposed introducing exogenous carbonate ions to expedite bacterial growth by maintaining the microenvironment's buffering capacity, thereby providing more nucleation sites and mitigating full cell surface coverage. Our findings indicated that adding exogenous sodium carbonate enhances bacterial growth rates in calcium-rich culture media, accelerating the onset of MICCP reactions (up to 122-fold) and improving the kinetics of calcium carbonate production and crack-filling rates.

Optimizing the performance of microbially induced calcium carbonate precipitation (MICCP) through microbial engineering approaches

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Optimizing the performance of microbially induced calcium carbonate precipitation (MICCP) through microbial engineering approaches

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