Constraints on CaCO3 precipitation in superabsorbent polymer by aerobic bacteria.

Microbially induced CaCO3 precipitation (MICP) can give concrete self-healing properties. MICP agents are typically bacterial endospores which are coated into shelled granules, infused into expanded clay, or embedded into superabsorbent polymer (SAP). When small cracks appear in the cured concrete, the encapsulation is broken and the metabolic CO2 production from the germinated bacteria causes healing of the cracks by precipitation of CaCO3. Such systems are being tested empirically at large scales, but survival of endospores through preparation and application, as well as germination and growth kinetics of the germinated vegetative cells, remains poorly resolved. We encapsulated endospores of Bacillus subtilis and Bacillus alkalinitrilicus in crosslinked acrylamide-based SAP and quantified their germination, growth, and, in the case of B. alkalinitrilicus, CaCO3 precipitation potential. The endospores survived crosslinking and desiccation inside the polymer matrix. Microcalorimetry and microscopy showed that ~ 80% of the encapsulated endospores of both strains readily germinated after rehydration of freeze-dried SAP. Germinated cells grew into dense colonies of cells inside the SAP, and those of B. alkalinitrilicus calcified with up to 0.3 g CaCO3 produced per g desiccated SAP when incubated aerobically. Measurements by planar optodes indicated that the precipitation rates were inherently oxygen limited due to diffusional constraints, rather than limited by electron donor or Ca2+ availability. Such oxygen limitation will limit MICP in all water-saturated and oxygen-dependent systems, and MICP agents based on anaerobic bacteria, e.g., nitrate reducers, should be developed to broaden the applicability of bioactive self-healing concretes to wet and waterlogged environments.

The Polyextremophilic Bacterium Clostridium paradoxum Attains Piezophilic Traits by Modulating Its Energy Metabolism and Cell Membrane Composition.

In polyextremophiles, i.e., microorganisms growing preferentially under multiple extremes, synergistic effects may allow growth when application of the same extremes alone would not. High hydrostatic pressure (HP) is rarely considered in studies of polyextremophiles, and its role in potentially enhancing tolerance to other extremes remains unclear. Here, we investigated the HP-temperature response in Clostridium paradoxum, a haloalkaliphilic moderately thermophilic endospore-forming bacterium, in the range of 50 to 70°C and 0.1 to 30 MPa. At ambient pressure, growth limits were extended from the previously reported 63°C to 70°C, defining C. paradoxum as an actual thermophile. Concomitant application of high HP and temperature compared to standard conditions (i.e., ambient pressure and 50°C) remarkably enhanced growth, with an optimum growth rate observed at 22 MPa and 60°C. HP distinctively defined C. paradoxum physiology, as at 22 MPa biomass, production increased by 75% and the release of fermentation products per cell decreased by >50% compared to ambient pressure. This metabolic modulation was apparently linked to an energy-preserving mechanism triggered by HP, involving a shift toward pyruvate as the preferred energy and carbon source. High HPs decreased cell damage, as determined by Syto9 and propidium iodide staining, despite no organic solute being accumulated intracellularly. A distinct reduction in carbon chain length of phospholipid fatty acids (PLFAs) and an increase in the amount of branched-chain PLFAs occurred at high HP. Our results describe a multifaceted, cause-and-effect relationship between HP and cell metabolism, stressing the importance of applying HP to define the boundaries for life under polyextreme conditions.IMPORTANCE Hydrostatic pressure (HP) is a fundamental parameter influencing biochemical reactions and cell physiology; however, it is less frequently applied than other factors, such as pH, temperature, and salinity, when studying polyextremophilic microorganisms. In particular, how HP affects microbial tolerance to other and multiple extremes remains unclear. Here, we show that under polyextreme conditions of high pH and temperature, Clostridium paradoxum demonstrates a moderately piezophilic nature as cultures grow to highest cell densities and most efficiently at a specific combination of temperature and HP. Our results highlight the importance of considering HP when exploring microbial physiology under extreme conditions and thus have implications for defining the limits for microbial life in nature and for optimizing industrial bioprocesses occurring under multiple extremes.

Optical Sensing of pH and O2 in the Evaluation of Bioactive Self-Healing Cement.

Leakage from cementitious structures with a retaining function can have devastating environmental consequences. Leaks can originate from cracks within the hardened cementitious material that is supposed to seal the structure off from the surrounding environment. Bioactive self-healing concretes containing bacteria capable of microbially inducing CaCO3 precipitation have been suggested to mitigate the healing of such cracks before leaking occurs. An important parameter determining the biocompatibility of concretes and cements is the pH environment. Therefore, a novel ratiometric pH optode imaging system based on an inexpensive single-lens reflex (SLR) camera was used to characterize the pH of porewater within cracks of submerged hydrated oil and gas well cement. This enabled the imaging of pH with a spatial distribution in high resolution (50 µm per pixel) and a gradient of 1.4 pH units per 1 mm. The effect of fly ash substitution and hydration time on the pH of the cement surface was evaluated by this approach. The results show that pH is significantly reduced from pH >11 to below 10 with increasing fly ash content as well as hydration time. The assessment of bioactivity in the cement was evaluated by introducing superabsorbent polymers with encapsulated Bacillus alkalinitrilicus endospores into the cracks. The bacterial activity was measured using oxygen optodes, which showed the highest bacterial activity with increasing amounts of fly ash substitution in the cement, correlating with the decrease in the pH. Overall, our results demonstrate that the pH of well cements can be reliably measured and modified to sustain the microbial activity.