Bacterial Mineralization Mechanism And Related Biomineral Organic Matter Analysis

Biomineral was ubiquitous in nature and had a characteristic structure and unique performance comparing with the inorganic minerals.Thus,the biomineral has broad application prospect in bioscience,geology,environment science and some other interdisciplinary subjects.The form of biominerals is the result of the environmental chemical change caused by the physiological activities of microorganisms.In long time scale,some organic matters secreted by microbes interacted with the inorganic ions,which resulted in the form of biomineral.For the biomineral,the existence of organic matters is a major feature,leading to the characteristic performance.Thus,the response of the organic matters secreted by microbes to the metal ions was an entry point for the biomineralization mechanisms research.However,some key scientific problems still need to be solved such as the compositions of organic matters,metabolic response mechanism and the roles of organic matters played in biomineralization and so on.In this paper,some selected model metal ions(Au³⁺/Ca²⁺/Mg²⁺)were mineralized in the presence of the typical bacteria(Bacillus subtilis and Paenibacillus mucilaginosus)by reduction mineralization,directed mineralization and weathering mineralization.During the process of biomineralization,the change of p H,the concentrations of metal ions,the amounts of the main organic matters(proteins or sugars)were detected.Combined with SEM-EDS,TEM-SAED,XRD,FTIR,Raman spectrum,Electrochemical analysis,FTICR MS and quantitative proteomics technology,the biomineralization and metabolic regulatory mechanism in biomineralization were investigated.Meanwhile,vapor diffusion crystallization experiments were also performed to further reveal the molecular mechanisms.The main research results are as following:1:P.mucilaginosus and Au³⁺were used as a model to performe reduction mineralization.During the process of Au³⁺mineralization,loose extracellular polymeric substances(L-EPS,the fraction which is not directly attached to the cell surface)and bound EPS(B-EPS,the inner fraction which is tightly linked via covalent or non-covalent associations)of the bacteria were separately extracted by cation exchange resin to examine the metabolic response to toxic Au³⁺ions.The experimental results indicated that B-EPS fractions were shown to contribute more significantly than L-EPS in reduction mineralization.When toxic metal ions attack cells,L-EPS contacted tightly with metal ions is the first barrier which hinders intracellular penetration of metal ions.Subsequently,with the penetration of metal ions,B-EPS could potentially act as the next barrier by protecting the cell wall and membranes to limit internalization of metal ions.Different fractions of EPS therefore play significant roles in cell defense against the attack of the toxic metal ions.2:In reduction mineralization,there is a higher sugar/protein ratio in L-EPS or B-EPS with increasing concentration of Au³⁺ions(0 ppm–180 ppm),indicating that sugars had a stronger response to the toxicity of Au³⁺.Thus,sugars contributed more significantly in reducing the toxicity of Au³⁺ions than proteins.Mass production of sugars in microbial EPS was shown not as a simple passive response to Au³⁺but a metabolically regulated stress response to metal toxicity.Meanwhile,FTIR analysis was performed to identify the major functional groups of the two EPS fractions before and after reacting with Au³⁺ions.For the pristine EPS,the band at 1246 cm^-1was assigned to the deformation vibration of the aldehyde group from sugars,whilst the band disappeared after reacting with Au³⁺ions,indicating that the aldehyde group of sugars may be used as the main reducing group in EPS that is responsible for the reduction Au³⁺ions.The FTIR result was also supported by an electrochemical titration result of aldehyde groups on EPS before and after reacting with Au³⁺ions.With an initial content of 3 mmol·g^-1,the final content of aldehyde groups decreased to 1.25 mmol·g^-1 after reacting with Au³⁺ions.3:B.subtilis and Ca²⁺were used as a model to performe directed mineralization.Biogenic vaterite was formed under the actions of organic matters according to the directed mineralization.FTICR MS was used to investigate the molecular compositions of organic matters in vaterite.The result indicated that the proteins played a vital role in the form of vaterite.Accoring to the Van Krevelen diagram of organic matters,it was confirmed that most of these key components were carboxylic acids and peptides.Meanwhile,the detailed molecular information also showed that the main components were O₂,NO₄and N₂O₆,and O₃,O₄,O₅ were also contained.In addition,the main carbon number is 15-20 and there are 1-3 unsaturation.By analyzing the structure of these key molecules,the stability and acid resistance of the vaterite was closely related to the compositions of these organic matters.4:B.subtilis and Mg²⁺were used as a model to performe weathering mineralization.The research results indicated that mineral carbonation involves dissolution of silicate minerals and subsequent precipitation of carbonate minerals.In the stages of serpentine weathering,B.subtilis regulated related metabolic pathways to up-regulate the expressions of oxidoreductases and then triggered organic acid metabolism,which promoted serpentine weathering and the release of magnesium to meet the needs of growth.In the formation of the secondary mineral,the growth of B.subtilis was gradually realized and amounts of nickel ions were also released with the release of magnesium from serpentine.Excessive Ni in the micro-environment drove N metabolism,resulting in elevation of the p H.A more alkaline p H provided optimal conditions for the formation of struvite and amorphous carbonate.In contrast,these alkaline minerals had a buffering effect on the bacterial growth because weakly alkaline conditions proved to be the optimal environment for B.subtilis.The self-regulating activities of microorganisms provided an optimal environment for serpentine weathering coupled with carbonation.5:FTICR MS was used to investigate the molecular compositions of organic matters in amorphous carbonate during the weathering mineralization.The research results indiacated that the amorphous carbonate minerals formed in the process of serpentine bio-weathering differed from traditional carbonate minerals owing to the microbial action.The minerals not only contained inorganic elements but also organic matter such as protein,carbohydrates,lignins,lipids,unsaturated hydrocarbons,and tannins secreted by microbes,and the relative amounts of proteins(44.65%)were higher than other organic matter.Proteins containing acidic amino acids such as Asp and Glu were found to be conducive to the improvement of stability in the formation of amorphous carbonate minerals.Thus,proteins and other organic matter were not only an important component of the secondary minerals,but also improved the stability of minerals owing to the form of organic-inorganic hybrid structures.The proteins acted like a sponge,accumulating Mg²⁺and CO₃^2-from solutions in the amorphous carbonate minerals,which were complexed by the carboxylic groups and amino groups of proteins.The interactions between mineralization ions and proteins showed a tendency to exist as a small ionic network in such amorphous carbonate minerals,thus stabilizing them,which would benefit CO₂ sequestration over geological timescales.In this paper,the compositions of organic matters,metabolic response mechanism and the roles of organic matters played in biomineralization were investigated by reduction mineralization,directed mineralization and weathering mineralization.These results further revealed the effects of organic matters in biomineralization,and have great value for the research of the biomineralization mechanism.It also provides basic data and theoretical basis for the further development and utilization of biominerals in related geology,life science,material science and environment science.