Fungal carbonatogenesis process mediates zinc and chromium removal via statistically optimized carbonic anhydrase enzyme.

INTRODUCTION: With rapid elevation in population, urbanization and industrialization, the environment is exposed to uncontrolled discharge of effluents filled with broad-spectrum toxicity, persistence and long-distance transmission anthropogenic compounds, among them heavy metals. That put our ecosystem on the verge or at a stake of drastic ecological deterioration, which eventually adversely influence on public health. Therefore, this study employed marine fungal strain Rhodotorula sp. MZ312369 for Zn2+ and Cr6+ remediation using the promising calcium carbonate (CaCO3) bioprecipitation technique, for the first time. RESULTS: Initially, Plackett-Burman design followed by central composite design were applied to optimize carbonic anhydrase enzyme (CA), which succeeded in enhancing its activity to 154 U/mL with 1.8-fold increase comparing to the basal conditions. The potentiality of our biofactory in remediating Zn2+ (50 ppm) and Cr6+ (400 ppm) was monitored through dynamic study of several parameters including microbial count, CA activity, CaCO3 weight, pH fluctuation, changing the soluble concentrations of Ca2+ along with Zn2+ and Cr6+. The results revealed that 9.23 × 107 ± 2.1 × 106 CFU/mL and 10.88 × 107 ± 2.5 × 106 CFU/mL of cells exhibited their maximum CA activity by 124.84 ± 1.24 and 140 ± 2.5 U/mL at 132 h for Zn2+ and Cr6+, respectively. Simultaneously, with pH increase to 9.5 ± 0.2, a complete removal for both metals was observed at 168 h; Ca2+ removal percentages recorded 78.99% and 85.06% for Zn2+ and Cr6+ remediating experiments, respectively. Further, the identity, elemental composition, functional structure and morphology of bioremediated precipitates were also examined via mineralogical analysis. EDX pattern showed the typical signals of C, O and Ca accompanying with Zn2+ and Cr6+ peaks. SEM micrographs depicted spindle, spherical and cubic shape bioliths with size range of 1.3 ± 0.5-23.7 ± 3.1 µm. Meanwhile, XRD difractigrams unveiled the prevalence of vaterite phase in remediated samples. Besides, FTIR profiles emphasized the presence of vaterite spectral peaks along with metals wavenumbers. CONCLUSION: CA enzyme mediated Zn2+ and Cr6+ immobilization and encapsulation inside potent vaterite trap through microbial biomineralization process, which deemed as surrogate ecofriendly solution to mitigate heavy metals toxicity and restrict their mobility in soil and wastewater.

Preparation of an environment-friendly microbial limestone dust suppressant and its dust suppression mechanism.

The development of an efficient and environmentally friendly dust suppressant is crucial to address the issue of dust pollution in limestone mines. Leveraging the synergistic microbial-induced calcium carbonate precipitation (MICP) technology involving NaHCO3 and dodecyl glucoside (APG), the optimal ratio of the dust suppressant was determined through single-factor and response surface tests. The dust suppression efficacy and mechanisms were analyzed through performance testing and microscopic imaging techniques, indicating that the optimal ratio of the new microbial dust suppressant was 20% mineralized bacteria cultured for 72 h, 0.647 mol L-1 cementing solution, 3.142% NaHCO3, and 0.149% APG. Under these conditions, the yield of calcium carbonate increased by 24.89% as compared to when no NaHCO3 was added. The dust suppressant demonstrated excellent wind, moisture, and rain resistance, as well as curing ability. More calcite was formed in the dust samples after treatment, and the stable form of the dust suppressant contributed to consolidating the limestone dust into a cohesive mass. These findings indicate that the synergistic effect of NaHCO3 and APG significantly enhanced the dust suppression capabilities of the designed microbial dust suppressant.

Application of urease-producing microbial community in seawater to dust suppression in desert.

In order to solve the dust problem caused by sandstorms, this paper aims to propose a new method of enriching urease-producing microbial communities in seawater in a non-sterile environment. Besides, the difference of dust suppression performance of enriched microorganisms under different pH conditions was also explored to adapt the dust. The Fourier-transform infrared spectrometry (FTIR) and Scanning electron microscopy (SEM) confirmed the formation of CaCO3. The X-ray diffraction (XRD) further showed that the crystal forms of CaCO3 were calcite and vaterite. When urease activity was equivalent, the alkaline environment was conducive to the transformation of CaCO3 to more stable calcite. The mineralization rate at pH = 10 reached the maximum value on the 7th day, which was 97.49 ± 1.73%. Moreover, microbial community analysis results showed that the relative abundance of microbial community structure was different under different pH enrichment. Besides, the relative abundance of Sporosarcina, a representative genus of urease-producing microbial community, increased with the increase of pH under culture conditions, which consistent with the mineralization performance results. In addition, the genus level species network diagram also showed that in the microbial community, Sporosarcina was negatively correlated with another urease-producing genus Bacillus, and had a reciprocal relationship with Atopostipes, which means that the urease-producing microbial community was structurally stable. The enrichment of urease-producing microbial communities in seawater will provide empirical support for the large-scale engineering application of MICP technology in preventing and controlling sandstorms in deserts.

Sugarecane molasse and vinasse added as microbial growth substrates increase calcium carbonate content, surface stability and resistance against wind erosion of desert soils.

Wind erosion is one of the main factors of soil degradation and air pollution in arid and semi-arid regions. In this study we evaluated microbial-induced carbonate precipitation (MICP) as an alternative soil conservation method against wind erosion using sugar cane molasse and vinasse as growth substrates in comparison to tryptic soy broth (TSB). The three substrates were applied in laboratory tests with and without addition of MICP cementing solution (1 M urea plus calcium chloride) to two sandy soils differing in calcium carbonate content. The performance of MICP solution inoculated with a cultured urease-producing strain of Sporosarcina pasteurii was compared to that of an autoclaved MICP solution. For control we also performed a blank treatment without substrate, MICP solution and inoculation. In addition to lab tests in which we determined the effects of treatments on soil pH, electrical conductivity (EC), calcium carbonate (CaCO3) content and surface penetration resistance, we performed wind tunnel experiments to determine soil loss by deflation under different wind velocities. Applying vinasse and molasse strongly increased soil CaCO3 content and penetration resistance, with and without addition of inoculated or non-inoculated MICP solution. Vinasse generally had stronger effects than molasse, while TSB was less effective, especially on penetration resistance. The addition of MICP solution in most treatments did not enhance but rather decrease the substrate effects. In the treatments with vinasse and molasse, increase in penetration resistance translated into substantially decreased soil loss in the wind tunnel tests, down to around one third of the loss in the blank treatment. In contrast, soil loss substantially increased in the treatments with TSB, probably due to the high input of sodium with this substrate. Our results show that molasse and, even more, vinasse can have a strong soil stabilization effect against wind erosion, which is primarily related to the formation of CaCO3 content and does not depend on additional amendments. Thus, these substrates have a great potential to be used on their own as environmentally friendly and cost-effective amendments to control wind erosion of bare sandy soils in arid environments.

Comparison of calcium carbonate production by bacterial isolates from recycled aggregates.

The new technology of microbially induced calcium carbonate precipitation (MICP) has been applied in construction materials as a strategy to enhance their properties. In pursuit of solutions that are more localized and tailored to the study's target, this work focused on isolating and selecting bacteria capable of producing CaCO3 for posterior application in concrete aggregates. First, eleven bacterial isolates were obtained from aggregates and identified as genera Bacillus, Lysinibacillus, Exiguobacterium, and Micrococcus. Then, the strains were compared based on the quantity and nature of calcium carbonate they produced using thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy with energy dispersive spectroscopy. Bacillus sp. dominated the cultured isolates and, along with Lysinibacillus sp., exhibited the highest CaCO3 conversion (up to 80%). On the other hand, Exiguobacterium and Micrococcus genera showed the poor ability to MICP (21.3 and 20.3%, respectively). Calcite and vaterite were the dominant carbonate polymorphs, with varying proportions. Concrete aggregates have proven to be a source of microorganisms capable of producing stable calcium carbonates with a high conversion rate. This indicates the feasibility of using microorganisms derived from local sources for application in construction materials as a sustainable way to enhance their characteristics.

Biotrapping Ureolytic Bacteria on Sand to Improve the Efficiency of Biocementation.

Microbially induced calcium carbonate precipitation (MICP) has emerged as a novel technology with the potential to produce building materials through lower-temperature processes. The formation of calcium carbonate bridges in MICP allows the biocementation of aggregate particles to produce biobricks. Current approaches require several pulses of microbes and mineralization media to increase the quantity of calcium carbonate minerals and improve the strength of the material, thus leading to a reduction in sustainability. One potential technique to improve the efficiency of strength development involves trapping the bacteria on the aggregate surfaces using silane coupling agents such as positively charged 3-aminopropyl-methyl-diethoxysilane (APMDES). This treatment traps bacteria on sand through electrostatic interactions that attract negatively charged walls of bacteria to positively charged amine groups. The APMDES treatment promoted an abundant and immediate association of bacteria with sand, increasing the spatial density of ureolytic microbes on sand and promoting efficient initial calcium carbonate precipitation. Though microbial viability was compromised by treatment, urea hydrolysis was minimally affected. Strength was gained much more rapidly for the APMDES-treated sand than for the untreated sand. Three injections of bacteria and biomineralization media using APMDES-treated sand led to the same strength gain as seven injections using untreated sand. The higher strength with APMDES treatment was not explained by increased calcium carbonate accrual in the structure and may be influenced by additional factors such as differences in the microstructure of calcium carbonate bridges between sand particles. Overall, incorporating pretreatment methods, such as amine silane coupling agents, opens a new avenue in biomineralization research by producing materials with an improved efficiency and sustainability.

Isolation and characterization of a resistance Bacillus subtilis for soil stabilization and dust alleviation purposes.

Dust poses environmental, geological, health, and economic concerns, and microorganisms can help mitigate these adverse consequences by improving soil properties. Microbial calcium carbonate precipitation (MICP) has been found to be an efficient strategy for increasing soil strength, reducing soil porosity, and preventing erosion; however, severe environmental conditions such as pH and high temperatures may impede this process. To identify the best strain for MICP, 60 bacteria strains were obtained from arid soils using the enrichment culture technique. They were tested for the capacity of calcium carbonate deposition and biocement synthesis in stress environments. Phenotypic characterization indicated that the majority of the bacterial isolates were gram-positive and rod-shaped, with strong catalase and oxidase enzyme activity. Furthermore, MALDI-TOF MS identification revealed that the isolates were from the Bacillus and Pseudomonas genera. Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD) were used to analyze the microstructures and composition of bacterial cement. The results represented that B. subtilis isolate S56 has a higher production yield and forms distinctive calcite crystals as a result of fast urease synthesis. B. subtilis isolate S56 can be applied in situ to reduce soil erosion and dust pollution. This study reveals the potential of the B. subtilis S56 strain for soil consolidation and dust prevention in harsh environments and has the prospect of promoting its application in desertification control and ecological restoration.

Overcoming the inhibitory effects of urea to improve the kinetics of microbial-induced calcium carbonate precipitation (MICCP) by Lysinibacillus sphaericus strain MB284.

Among different microbial-induced calcium carbonate precipitation (MICCP) mechanisms utilized for biomineralization, ureolysis leads to the greatest yields of calcium carbonate. Unfortunately, it is reported that urea-induced growth inhibition can delay urea hydrolysis but it is not clear how this affects MICCP kinetics. This study investigated the impact of urea addition on the MICCP performance of Lysinibacillus sphaericus MB284 not previously grown on urea (thereafter named bio-agents), compared with those previously cultured in urea-rich media (20 g/L) (hereafter named bio-agents+ or bio-agents-plus). While it was discovered that initial urea concentrations exceeding 3 g/L temporarily hindered cell growth and MICCP reactions for bio-agents, employing bio-agents+ accelerated the initiation of bacterial growth by 33% and led to a 1.46-fold increase in the initial yield of calcium carbonate in media containing 20 g/L of urea. The improved tolerance of bio-agents+ to urea is attributed to the presence of pre-produced endogenous urease, which serves to reduce the initial urea concentration, alleviate growth inhibition, and expedite biomineralization. Notably, elevating the initial concentration of bio-agents+ from OD600 of 0.01 to 1, housing a higher content of endogenous urease, accelerated the initiation of MICCP reactions and boosted the ultimate yield of biomineralization by 2.6 times while the media was supplemented with 20 g/L of urea. These results elucidate the advantages of employing bio-agents+ with higher initial cell concentrations to successfully mitigate the temporary inhibitory effects of urea on biomineralization kinetics, offering a promising strategy for accelerating the production of calcium carbonate for applications like bio self-healing of concrete.

Biomineralization Process of CaCO3 Precipitation Induced by Bacillus mucilaginous and Its Potential Application in Microbial Self-healing Concrete.

Microbial induced calcium carbonate precipitation (MICP) is widely common in nature, which belongs to biomineralization and has been explored carefully in recent decades. The paper studied the effect of temperature, initial pH value and Ca2+ concentration on bacterial growth and carbonic anhydrase activity, and then revealed the biomineralization process through the changes of Ca2+ concentration and calcification rate in alkali environment. Meanwhile, microbial healing agent containing spores and calcium nitrate was prepared and used for the early age concrete cracks repair. The self-healing efficiency was assessed by crack closure rate and water permeability repair rate. The experimental results showed that when the optimal temperature was 30 °C, the pH was 8.0-11.0, and the optimal Ca2+ concentration was 0-90 mM, the bacteria could grow better and the carbonic anhydrase activity was higher. Compared with reference, the crack closure rate with the crack width up to 0.339 mm could reach 95.62% and the water permeability repair rate was 87.54% after 28 d healing time of dry-wet cycles. XRD analysis showed that the precipitates at the crack mouth were calcite CaCO3. Meanwhile, the self-healing mechanism of mortar cracks was discussed in detail. In particular, there is no other pollution in the whole mineralization process, and the self-healing system is environmentally friendly, which provides a novel idea and method for the application of microbial self-healing concrete.

[Advances of enzymes related to microbial cement].

Microbial induced calcium carbonate precipitation (MICP) refers to the natural biological process of calcium carbonate precipitation induced by microbial metabolism in its surrounding environment. Based on the principles of MICP, microbial cement has been developed and has received widespread attention in the field of biology, civil engineering, and environment owing to the merits of environmental friendliness and economic competence. Urease and carbonic anhydrase are the key enzymes closely related to microbial cement. This review summarizes the genes, protein structures, regulatory mechanisms, engineering strains and mutual synergistic relationship of these two enzymes. The application of bioinformatics and synthetic biology is expected to develop biocement with a wide range of environmental adaptability and high performance, and will bring the MICP research to a new height.

Biomineralization of Plastic Waste to Improve the Strength of Plastic-Reinforced Cement Mortar.

The development of methods to reuse large volumes of plastic waste is essential to curb the environmental impact of plastic pollution. Plastic-reinforced cementitious materials (PRCs), such as plastic-reinforced mortar (PRM), may be potential avenues to productively use large quantities of low-value plastic waste. However, poor bonding between the plastic and cement matrix reduces the strength of PRCs, limiting its viable applications. In this study, calcium carbonate biomineralization techniques were applied to coat plastic waste and improved the compressive strength of PRM. Two biomineralization treatments were examined: enzymatically induced calcium carbonate precipitation (EICP) and microbially induced calcium carbonate precipitation (MICP). MICP treatment of polyethylene terephthalate (PET) resulted in PRMs with compressive strengths similar to that of plastic-free mortar and higher than the compressive strengths of PRMs with untreated or EICP-treated PET. Based on the results of this study, MICP was used to treat hard-to-recycle types 3-7 plastic waste. No plastics investigated in this study inhibited the MICP process. PRM samples with 5% MICP-treated polyvinyl chloride (PVC) and mixed type 3-7 plastic had compressive strengths similar to plastic-free mortar. These results indicate that MICP treatment can improve PRM strength and that MICP-treated PRM shows promise as a method to reuse plastic waste.