Background: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase—an enzyme with the capability to hydrolyse CO₂, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO₃ biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. Results: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO₂ and convert it to calcium carbonate. A significant decrease in CO₂ was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO₂ sequestration. Conclusion: Recombinantly produced carbonic anhydrase successfully sequestered CO₂ and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α‐helices, the α‐domain, and another predominantly composed of β‐sheets, the β‐domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate‐binding cleft between the α‐ and β‐domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X‐ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate‐binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site‐directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate‐binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases. Proteins 2011.

Bacterial cellulose (BC) has been explored for use in a range of applications including tissue engineering and textiles. BC can be produced from waste streams, but sustainable approaches are needed for functionalisation. To this end, BslA, a B. subtilis biofilm protein was produced recombinantly with and without a cellulose binding module (CBM) and the cell free extract was used to treat BC either ex-situ, through drip coating or in-situ, by incorporating during fermentation. The results showed that ex-situ modified BC increased the hydrophobicity and water contact angle reached 120°. In-situ experiments led to a BC film morphological change and mechanical testing demonstrated that addition of BslA with CBM resulted in a stronger, more elastic material. This study presents a nature inspired approach to functionalise BC using a biofilm hydrophobin, and we demonstrate that recombinant proteins could be effective and sustainable molecules for functionalisation of BC materials.

This study aimed to understand the morphological effects of (in)organic additives on microbially induced calcium carbonate precipitation (MICP). MICP was monitored in real-time in the presence of (in)organic additives: bovine serum albumin (BSA), biofilm surface layer protein A (BslA), magnesium chloride (MgCl₂) and poly-L-lysine. This monitoring was carried out using confocal microscopy to observe the formation of CaCO₃ from the point of nucleation, in comparison to conditions without additives. Complementary methodologies, namely scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction, were employed to assess the visual morphology, elemental composition, and crystalline structures of CaCO₃, respectively, following the crystals’ formation. The results demonstrated that in the presence of additives, more CaCO₃ crystals were produced at 100 minutes compared to the reaction without additives. The inclusion of BslA resulted in larger crystals than reactions containing other additives, including MgCl₂. BSA induced a significant number of crystals from the early stages of the reaction (20 minutes) but did not have a substantial impact on crystal size compared to conditions without additives. All additives led to a higher content of calcite compared to vaterite after a 24-hour reaction, with the exception of MgCl₂, which produced a substantial quantity of magnesium calcite. The work demonstrates the effect of several (in)organic additives on MICP and sets the stage for further research to understand additive effects on MICP to achieve controlled CaCO₃ precipitation.

Hydrostatic pressure is an important physical stimulus which can cause various responses in bacterial cells. The survival and cellular processes of Escherichia coli under hydrostatic pressures between 10 MPa and 110 MPa have been studied. However, understanding bacterial responses to moderately elevated pressure of up to 10 MPa is useful for a range of different applications including for example in smart and responsive materials. In this study, the genetic responses of E. coli K-12 MG1655 to 1 MPa pressure was examined using transcriptomic analysis by RNA-Seq. The results show that 101 genes were differentially expressed under 1 MPa pressure in E. coli cells, with 85 of them up-regulated. The analysis suggested that some genes were over expressed to adapt the increase of oxygen levels in our system, and several functional categories are involved including oxidative stress responses, Fe-S cluster assembly and iron acquisition. Two differentially expressed genes azuC and entC were further investigated using RT-qPCR, and GFP reported strains of those two genes were created, AG1319 (P_azuC azuC-msfgfp) and AG1321 (P_entC entC-msfgfp). A linear response of azuC expression was observed between 0 MPa to 1 MPa by monitoring the fluorescence signal of strain AG1319 (P_azuC azuC-msfgfp). This study is the first report to demonstrate the genetic response of bacterial cells under 1 MPa hydrostatic pressure, and provides preliminary data for creating pressure sensing bacterial strains for a wide range of applications.