Microbial exopolymers provide a mechanism for bioaccumulation of contaminants

Scanning confocal laser microscopy was used to directly visualize accumulation of the herbicide diclofop methyl and its breakdown products by a degradative biofilm community, cultivated in continuous-flow cell cultures. Some bacterial cells accumulated these compounds. However, most accumulation occurred in cell capsules and certain regions of the exopolymer matrix. Mass spectroscopic analysis of the biofilm material confirmed accumulation of the parent compound and its breakdown products in the biofilms. Lower molecular weight degradation products were found in the effluent, indicating mineralization of diclofop by the flow cell cultures. Grazing protozoa feeding on the biofilms nonselectively ingested cell capsules and exopolymers, suggesting direct transfer and accumulation of the contaminants in protozoa. These findings demonstrated that microbial exopolymers can play an important role in the bioaccumulation of contaminants in natural systems. Correspondence to: J.R. Lawrence.     

The effects of exopolymers on cell morphology and culturability of Leuconostoc mesenteroides during starvation

Biofilm formation by bacterial cells can be used to modify the subsurface permeability for the purpose of microbial enhanced oil recovery, bio-barrier formation, and in situ bioremediation. Once injected into the subsurface, the bacteria undergo starvation due to a decrease in nutrient supply and diffusion limitations in biofilms. To help understand the starvation response of bacteria in biofilms, the relationship between exopolymer formation and cell culturability was examined in a batch culture. The average cell diameter was observed to decrease from 0.8 μm to 0.35 μm 3 days after starvation began. Cell chain fragmentation was also observed during starvation. Cells that underwent starvation in the presence of insoluble exopolymers showed a slower rate of decrease in cell diameter and in cell chain length than cells without insoluble exopolymers. The rate of decrease in the average cell diameter and cell chain length were determined using a first order decay model. Cells starved in the presence of exopolymers showed greater culturability than cells starved without exopolymers. After 200 days starvation, 2.5 × 10⁻³% cells were culturable, but no increase in cell number was observed. During starvation, the exopolymer concentration remained constant, an indication that the exopolymer was not consumed by the starving bacteria as an alternative carbon or energy source. Received: 8 April 1999 / Received revision: 16 July 1999 / Accepted: 6 August 1999     

Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation

Biofilms are communities of microbial cells that are encased in a self-produced, polymeric matrix and are adherent to a surface. For several species of bacteria, an enhanced ability to form biofilms has been linked with an increased capability to produce exopolymers. To identify exopolymers of Bacillus subtilis that can contribute to biofilm formation, we transferred the genetic determinants that control exopolymer production from a wild, exopolymer-positive strain to a domesticated, exopolymer-negative strain. Mapping these genetic determinants led to the identification of gamma-poly-dl-glutamic acid (gamma-PGA) as an exopolymer that increases biofilm formation, possibly through enhancing cell-surface interactions. Production of gamma-PGA by Bacillus subtilis was known to be dependent on the two-component regulator ComPA; this study highlighted the additional dependence on the DegS-DegU, DegQ and SwrA regulator proteins. The inability of the domestic strain of B. subtilis to produce gamma-PGA was mapped to two base pairs; a single base pair change in the promoter region of degQ and a single base pair insertion in the coding region of swrA. Introduction of alleles of degQ and swrA from the wild strain into the domestic strain was sufficient to allow gamma-PGA production. In addition to controlling gamma-PGA production, ComPA and DegSU were also shown to activate biofilm formation through an as yet undefined pathway. The identification of these regulators as affecting gamma-PGA production and biofilm formation suggests that these processes are regulated by osmolarity, high cell density and phase variation.     

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C₆) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C₈) fatty acyl chain.     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gasliquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition of exopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Monosaccharide composition of exopolymer complex in Thiobacillus thioparus and Stenotrophomonas maltophilia

The bacteria of Thiobacillus thioparus and Stenotrophomonas maltophilia are capable to form a thick biofilm complicated as to its structure on the surface of low-carbon steel. This biofilm formation on any surface occurs under the adhesion of the cells of the plankton growth model with the help of synthesis of muciferous exopolymers with adhesive properties. Hence the monosaccharide composition of the exopolymer complex in the form of polyol acetates was studied by chromate-mass-spectrum method. A significant difference in the composition of exopolymer monosaccharides with the presence of steel model in the medium and without it was established; a change in the monosaccharide composition of mono- and binary culture in the conditions of plankton and biofilm growth was also observed.     

Terminal electron acceptors influence the quantity and chemical composition of capsular exopolymers produced by anaerobically growing Shewanella spp

Bacterial exopolymers perform various roles, including acting as a carbon sink, a protective layer against desiccation or antimicrobial agents, or a structural matrix in biofilms. Despite such varied roles, little is known about the heterogeneity of bacterial exopolymer production under varying growth conditions. Here we describe experiments designed to characterize the quantity and quality of exopolymers produced by two commonly studied members of the widely distributed genus Shewanella. Electrokinetic, spectroscopic, and electron microscopic techniques were employed to demonstrate that cell surfaces of Shewanella oneidensis MR-1 (electrophoretic softness, lambda(-1), range from 0.4 to 2.6 nm) are associated with less extracellular polymeric material than surfaces of Shewanella putrefaciens 200R (lambda(-1) range from 1.6 to 3.0 nm). Both species exhibit similar responses to changes in electron acceptor with nitrate- and fumarate-grown cells producing relatively little exopolymer compared to trimethylamine N-oxide (TMAO)-grown cells. In S. oneidensis, the increase in exopolymers has no apparent effect upon cell-surface fixed charge density (-7.7 to -8.7 mM), but for S. putrefaciens a significant drop in fixed charge density is observed between fumarate/nitrate-grown cells (-43 mM) and TMAO-grown cells (-20.8 mM). For both species, exopolymers produced during growth on TMAO have significant amide functionality, increasing from approximately 20-25% of C-containing moieties in nitrate-grown cells to over 30% for TMAO-grown cells (determined from X-ray photoelectron spectroscopy). The increased exopolymer layer associated with TMAO-grown cells appears as a continuous, convoluted layer covering the entire cell surface when viewed by low-temperature, high-resolution scanning electron microscopy. Such significant changes in cell-surface architecture, dependent upon the electron acceptor used for growth, are likely to influence a variety of cell interactions, including aggregation and attachment to surfaces, and the binding of aqueous metal species.     

Development and Structure of Eukaryotic Biofilms in an Extreme Acidic Environment,Río Tinto (SW,Spain)

An in situ colonization assay was performed to study the early stages of biofilm formation in Río Tinto (SW, Spain), an extremely acidic environment (pH ca. 2). Eukaryotic assemblages were monitored at monthly intervals for 1 year. Diversity, colonization rates, and seasonal variations were analyzed. Structural features of naturally grown biofilms were explored by light and scanning electron microscopy in backscattered electron mode. A total of 14 taxa were recognized as constituents of the eukaryotic assemblages. The eukaryotic communities were dissimilar at the different sampling sites. The lowest diversity was found at the most extreme locations, in terms of pH and heavy metal concentrations. The biofilms were mainly formed by species from the genera Dunaliella and Cyanidium. Two genera of filamentous algae, Zygnemopsis and Klebsormidium, were principally responsible for the variability in the cell number throughout the year. These species appear in June to decrease almost completely between October and November. In contrast, the number of heterotrophic flagellates and ciliates remained constant throughout the year. The microcolonization sequence showed an initial accumulation of amorphous particles composed of bacteria and inorganic grains of minerals. By the end of the second month, the organic matrix was also populated by fungi, bacteria, and a few eukaryotic heterotrophs such as amoebae and small flagellates. Diatoms only showed significant colonization in regions where mycelial matrices were first established. Flagellated green algae such as Dunaliella or Chlamydomonas as well as Euglena were also present at the very beginning of the biofilm development, although in low numbers (<100 cells cm^–2). After the flagellated cells, sessile species of algae such Chlorella or Cyanidium appeared. Filamentous algae were the last species to colonize the biofilms. Most of the naturally grown biofilms were found to be structures composed of different species organized in different layers separated, probably by extracellular polymeric substances, although more analysis should be done in this regard. The possible implications of the biofilm structure in the adaptation to this extreme habitat are discussed.     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

Abundance of the Multiheme c-Type Cytochrome OmcB Increases in Outer Biofilm Layers of Electrode-Grown Geobacter sulfurreducens

When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.     

Microscale evaluation of the effects of grazing by invertebrates with contrasting feeding modes on river biofilm architecture and composition

River biofilms are a valuable food resource for many invertebrates. In the present study biofilms were cultivated in a rotating annular bioreactor with river water as sole source of inoculum. The resulting biofilms were then presented to starved snails, ostracods, and mayflies as sole food source. The biofilms were then removed and microscopically examined to determine areas that had been grazed. The grazed and ungrazed areas were marked and analyzed for the effects of grazing using confocal laser scanning microscopy and image analyses. Samples were treated with fluorescent probes for nucleic acids to quantify bacterial biomass and fluor-conjugated lectins to quantify exopolymer, and far red autofluorescence was imaged to quantify algal or photosynthetic biomass. Grazing by snails significantly reduced algal biomass (1.1 +/- 0.6 micro m 3 micro m 2 to 0.02 +/- 0.04 micro m 3 micro m 2), exopolymer (5.3 +/- 3.4 micro m 3 micro m 2 to 0.18 +/- 0.18 micro m 3 micro m 2), and biofilm thickness (154 micro m +/- 50 to 11 micro m +/- 5.2; ANOVA, p < or= 0.05). Although bacterial biomass was influenced by grazing snails the impact was not statistically significant (p 相似文献   

Bioaccumulation of the Herbicide Diclofop in Extracellular Polymers and Its Utilization by a Biofilm Community during Starvation

Continuous-flow cell systems were used to cultivate a degradative biofilm community with the herbicide diclofop methyl as the sole carbon and energy source. The aromatic character of this compound and its breakdown products enabled direct visualization of their accumulation in the biofilm matrix. This accumulation could be inhibited by addition of a more labile carbon source to the culture medium or by inhibition of cell activity. The fluorescence of diclofop-grown biofilms remained constant after 14 to 21 days but decreased with time when diclofop was omitted from the irrigation solution. However, this decrease was inhibited by cyanide, indicating either utilization or release of accumulated diclofop when the cells were viable. Subsequent experiments with [(sup14)C]diclofop also indicated that decreased fluorescence in the absence of an exogenous carbon source resulted from degradation of adsorbed diclofop and its breakdown products by the biofilm bacteria. These results demonstrate that biofilm exopolymers can facilitate storage of nutrients for subsequent mineralization during periods of carbon limitation.     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gas-liquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition ofexopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water‐replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water‐limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water‐limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water‐replete and solute stress conditions. There was greater spatial stratification of dead cells under water‐limiting than water‐replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water‐limiting conditions, ROS accumulation is greater in an ΔalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration‐mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water‐replete and water‐limiting conditions.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

The effect of extracellular polymeric substances on the attachment of Pseudomonas NCIMB 2021 to AISI 304 and 316 stainless steel

Surfaces of AISI 304 and 316 stainless steels were pre-treated with three different types of extracellular polymeric substances, viz. (i) exopolymers released into the culture medium ("free"; or planktonic exopolymers), (ii) capsular exopolymers, and (iii) biofilm exopolymers, produced by continuous cultures of marine Pseudomonas NCIMB 2021. The initial attachment of Pseudomonas cells to exopolymer-conditioned steel surfaces varied with the exopolymer type and concentration. Results gained from wettability studies of exopolymer-treated steel using contact angle measurements, as well as from the surface roughness measurements conducted employing atomic force microscopy analysis, could not account for the observed, statistically significant differences (p < 0.1) in the level of bacterial surface colonisation. It is therefore proposed that neither surface hydrophobicity nor roughness play an important part in the early attachment of Pseudomonas NCIMB 2021 to the conditioned steel surfaces and that a difference in the chemistry of the exopolymers is most likely a key parameter influencing initial cell adhesion to pre-treated steel.     

Photosynthesis,Respiration and Exopolymer Calcium-Binding in Biofilm Calcification (Westerhöfer and Deinschwanger Creek,Germany)

The impact of microbial activity on biofilm calcification in aquatic environments is still a matter of debate, especially in settings where ambient water has high CaCO₃ mineral supersaturation. In this study, biofilms of two CO₂-degassing karst-water creeks in Germany, which attain high calcite supersaturation during their course downstream, were investigated with regard to water chemistry of the biofilm microenvironment. The biofilms mainly consisted of filamentous cyanobacteria (Phormidium morphotype) and heterotrophic bacteria (including sulfate-reducing bacteria), which affect the microenvironment and produce acidic exopolymers. In situ and ex situ microelectrode measurements showed that a strong pH increase, coupled with Ca^{2 +} consumption, occurred in light conditions at the biofilm surface, while the opposite occurred in the dark. Calcite supersaturation at the biofilm surface, calculated from ex situ Ca^{2 +} and CO₃ ^2? microelectrode measurements, showed that photosynthesis resulted in high omega values during illumination, while respiration slightly lowered supersaturation values in the dark, compared to values in the water column. Dissociation calculation demonstrated that the potential amount of Ca^{2 +} binding by exopolymers would be insufficient to explain the Ca^{2 +} loss observed, although Ca^{2 +} complexation to exopolymers might be crucial for calcite nucleation. No spontaneous precipitation occurred on biofilm-free limestone substrates under the same condition, regardless of high supersaturation. These facts indicate that photosynthesis is a crucial mechanism to overcome the kinetic barrier for CaCO₃ precipitation, even in highly supersaturated settings.     

The role of exopolymers in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.     

Calcium dynamics in microbialite‐forming exopolymer‐rich mats on the atoll of Kiritimati,Republic of Kiribati,Central Pacific

Microbialite‐forming microbial mats in a hypersaline lake on the atoll of Kiritimati were investigated with respect to microgradients, bulk water chemistry, and microbial community composition. O₂, H₂S, and pH microgradients show patterns as commonly observed for phototrophic mats with cyanobacteria‐dominated primary production in upper layers, an intermediate purple layer with sulfide oxidation, and anaerobic bottom layers with sulfate reduction. Ca²⁺ profiles, however, measured in daylight showed an increase of Ca²⁺ with depth in the oxic zone, followed by a sharp decline and low concentrations in anaerobic mat layers. In contrast, dark measurements show a constant Ca²⁺ concentration throughout the entire measured depth. This is explained by an oxygen‐dependent heterotrophic decomposition of Ca²⁺‐binding exopolymers. Strikingly, the daylight maximum in Ca²⁺ and subsequent drop coincides with a major zone of aragonite and gypsum precipitation at the transition from the cyanobacterial layer to the purple sulfur bacterial layer. Therefore, we suggest that Ca²⁺ binding exopolymers function as Ca²⁺ shuttle by their passive downward transport through compression, triggering aragonite precipitation in the mats upon their aerobic microbial decomposition and secondary Ca²⁺ release. This precipitation is mediated by phototrophic sulfide oxidizers whose action additionally leads to the precipitation of part of the available Ca²⁺ as gypsum.     

Cathodic protection and microbially induced corrosion

Cathodic protection, using sacrificial anodes or impressed current, has been recognized for a long time as an effective way to prevent marine corrosion. Cathodic polarization leads to the formation of a protective calcareous layer on the surfaces. It is well documented that the attachment of bacteria to metal surfaces and subsequent biofilm formation changes some physical and chemical parameters at the interface and influences the corrosion process. The objective of this study was to determine whether there is a relationship between cathodic polarization and development of biofilms on surfaces exposed to both synthetic and natural seawater. Experiments were conducted on clean surfaces, biofilmed surfaces, in natural or synthetic seawater using bacterial monocultures and cocultures. In marine sediments, cathodically produced hydrogen encouraged growth of hydrogenase-containing sulphate-reducing bacteria while in aerated seawater biofilms competed with the magnesium and calcium deposition. Both low pH induced by bacterial metabolism and exopolymers affect the deposition process and the stability of the calcareous layer.