Spatial Patterns of Carbonate Biomineralization in Biofilms

Abstract

dMicrobially catalyzed precipitation of carbonate minerals is an important process in diverse biological, geological, and engineered systems. However, the processes that regulate carbonate biomineralization and their impacts on biofilms are largely unexplored, mainly because of the inability of current methods to directly observe biomineralization within biofilms. Here, we present a method for in situ, real-time imaging of biomineralization in biofilms and use it to show that Pseudomonas aeruginosa biofilms produce morphologically distinct carbonate deposits that substantially modify biofilm structures. The patterns of carbonate biomineralization produced in situ were substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation should occur at the biofilm surface, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, detaching biofilm-resident cells and deforming the biofilm morphology. These findings indicate that biomineralization is a general regulator of biofilm architecture and properties. Microbially induced carbonate precipitation represents an essential pathway for sequestration of large amounts of carbon in ancient and modern environments (1-5). Rock records reveal that Precambrian stromatolites formed as a consequence of trapping, binding, and precipitation of calcium carbonate by the growth and metabolism of microorganisms (2-4). Modern carbonate microbialites are found in a wide range of environments, including freshwater, oceans, hypersaline lakes, and soils (6, 7). Clinically, prolonged bacterial infection of indwelling urinary catheters leads to the formation of mineralized biofilms that can occlude the catheter lumen and cause serious complications (8-10). Some persistent infections of the lungs, particularly those associated with the genetic disorder primary ciliary dyskinesia, involve precipitation of calcium-rich stones or coatings (11)(12)(13)(14). In engineered systems, microbially induced scale formation decreases the performance of a wide variety of processes, including membrane separations in water treatment and heat exchange efficiency in cooling towers (15, 16). More recently, calcium carbonate biomineralization has also been explored as a novel biotechnology for the purpose of bioremediation and stabilization of porous structures, including soils, sediments, and construction materials (17-21).The microorganisms involved in carbonate biomineralization cover nearly all classes, including bacteria, algae, and fungi (22-27). It has been reported that more than 200 soil bacteria, including Pseudomonas spp., Bacillus subtilis, and Azotobacter spp., are capable of inducing calcium carbonate precipitation (5). Diverse microbial metabolisms, such as photosynthesis, sulfate reduction, and urea hydrolysis, can induce carbonate precipitation by significantly changing the saturation state of calcium carbonate (28,29). Microbial respiration p...

In Situ Biomineralization and Particle Deposition Distinctively Mediate Biofilm Susceptibility to Chlorine

“…Calcium carbonate biomineralization was induced by treating 3-day-old PAO1 biofilms with a supersaturated calcium carbonate medium composed of 15 mM (each) CaCl 2 and NaHCO 3 in 1% TSB at a flow rate of 10 ml/h for 12 h. The medium has a pH of 7.6 and a calcium carbonate saturation index with a log⍀ value of 1.88 at room temperature (ϳ22°C) under atmospheric CO 2 concentrations, yielding the biomineralization of calcium carbonate within the biofilms. We previously showed that this medium is nontoxic to PAO1 biofilms and planktonic cells (29).…”

“…The biofilms were imaged through the visualization of constitutively expressed green fluorescent protein (GFP). Mineral deposits in the biofilms were imaged by collecting the confocal laser reflectance signal following methods described previously (29).…”

“…We found that in situ carbonate biomineralization produced unexpected spatial patterns, in that it primarily occurred from the base of the biofilm architecture rather than at the biofilm surface. The biomineralized deposits grew over time, deformed the biofilm structure, and detached cells from the biofilm (29). Based on these previous findings, we hypothesized that in situ biomineralization should enhance internal solute transport within biofilms and therefore increase biofilm killing by biocides.…”

“…In our previous investigations, we used Pseudomonas aeruginosa as a model organism to study calcium carbonate biomineralization in biofilms (29). P. aeruginosa is a common pathogen in aquatic systems and building water systems, as well as an organism well studied as a model for biofilm formation (30,31).…”

Detection Techniques for Extracellular Polymeric Substances in Biofilms: A Review

“…Virdis et al (2012) demonstrated that CRM allowed monitoring of biofilm development at different growth stages, without impacting its structural or metabolic activity. Li et al (2015) presented CRM for in situ, real-time imaging of the biomineralization in biofilms, through which it was shown that Pseudomonas aeruginosa biofilms could produce morphologically distinct carbonate deposits that substantially modified biofilm structures.…”