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.     

Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.     

Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Role of beta 1-4 linked polymers in the biofilm structure of marine Pseudomonas sp. CE-2 on 304 stainless steel coupons

Pseudomonas sp CE-2 cells attach and form biofilms on 304-stainless steel (SS) coupons. A series of experiments were carried out in order to understand the role of exopolysaccharides (EPS) in the formation and maintenance of CE-2 biofilms on SS coupons. The biofilm density and EPS concentration increased over the period of incubation and the highest values for both were recorded after 72 h. Calcofluor and the lectin concanavalin A (Con A) showed a positive interaction with 72-h old biofilms, indicating the presence of beta 1-4 linked polymers, and alpha-d-glucose and alpha-d-mannose in the biofilm matrix of CE-2. When the CE-2 cells were grown in the presence of calcofluor (200 microg ml(-1)), biofilm formation was significantly reduced (approximately 85%). Conversely, the lectins Con A or WGA did not influence the CE-2 biofilms on the SS coupons. Furthermore, treatment with cellulase, an enzyme specific for the degradation of beta 1-4 linked polymers, removed substantial amounts of CE-2 biofilm from SS coupons. These results strongly suggest the involvement of beta 1-4 linked polymers in the formation and maintenance of Pseudomonas sp. CE-2 biofilms on SS coupons.     

Characterisation of conditioning layers formed by exopolymeric substances of Pseudomonas NCIMB 2021 on surfaces of AISI 316 stainless steel

This investigation aimed to characterise conditioning layers formed on AISI 316 stainless steel by different types of extracellular polymeric substances (EPS), i.e. biofilm, planktonic and capsular exopolymers, isolated from continuous cultures of marine Pseudomonas received from the National Collection of Industrial and Marine Bacteria (strain NCIMB 2021). Colorimetric assays and gas chromatography‐mass spectrometry analysis confirmed previously obtained results based on a FTIR and SDS‐PAGE study of Pseudomonas NCIMB 2021 EPS demonstrating the presence of protein, neutral and amino sugars and uronic acids. The content and the ratio of these macromolecules differed depending on the type of EPS. X‐ray photoelectron spectroscopy revealed that conditioning layers formed upon exposure of steel to EPS solutions were chemically dissimilar. It is proposed that the observed difference in the chemistry of conditioning layers is the likely reason for reported differences in attachment of Pseudomonas cells to EPS‐conditioned steel surfaces.     

IR study of self-assembly of capsular exopolymers from Pseudomonas sp. NCIMB 2021 on hydrophilic and hydrophobic surfaces

Capsular exopolymers (EPS) of the bacterium Pseudomonas sp. NCIMB 2021 are allowed to self-assemble on hydrophilic and hydrophobic gold surfaces. Tapping mode atomic force microscopy confirms the differences in the surface topography between EPS adsorbed on both surfaces. Fourier-transform IR spectroscopy indicates that the EPS surface coverage is much greater on the hydrophobic surface. Furthermore, an increased contribution is observed from hydrophobic (i.e., methyl and tyrosyl residues) and electrostatic (i.e., carboxylate residues) groups at the hydrophobic surface, but there is relatively less neutral polymer compared to the hydrophilic surface. The behavior of this EPS is in agreement with the behavior of cells of Pseudomonas sp. NCIMB 2021 at hydrophilic and hydrophobic surfaces.     

Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms

To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria–Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera. Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold. The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion. Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls. Pseudomonas mendocina KR1 reduced corrosion the most in VNSS. It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm. Received 09 December 1996/ Accepted in revised form 19 March 1997     

Disinfection of bacterial biofilms in pilot-scale cooling tower systems

The impact of continuous chlorination and periodic glutaraldehyde treatment on planktonic and biofilm microbial communities was evaluated in pilot-scale cooling towers operated continuously for 3 months. The system was operated at a flow rate of 10,080 l day(-1). Experiments were performed with a well-defined microbial consortium containing three heterotrophic bacteria: Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. The persistence of each species was monitored in the recirculating cooling water loop and in biofilms on steel and PVC coupons in the cooling tower basin. The observed bacterial colonization in cooling towers did not follow trends in growth rates observed under batch conditions and, instead, reflected differences in the ability of each organism to remain attached and form biofilms under the high-through flow conditions in cooling towers. Flavobacterium was the dominant organism in the community, while P. aeruginosa and K. pneumoniae did not attach well to either PVC or steel coupons in cooling towers and were not able to persist in biofilms. As a result, the much greater ability of Flavobacterium to adhere to surfaces protected it from disinfection, whereas P. aeruginosa and K. pneumoniae were subject to rapid disinfection in the planktonic state.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.     

A green biocide enhancer for the treatment of sulfate-reducing bacteria (SRB) biofilms on carbon steel surfaces using glutaraldehyde

Generally speaking, a much higher concentration of biocide is needed to treat biofilms compared to the dosage used to for planktonic bacteria. With increasing restrictions of environmental regulations and safety concerns on large-scale biocide uses such as oil field applications, it is highly desirable to make more effective use of biocides. In this paper a green biocide enhancer ethylenediaminedisuccinate (EDDS) that is a biodegradable chelator, was found to enhance the efficacy of glutaraldehyde in its treatment of sulfate-reducing bacteria (SRB) biofilms. Experiments were carried out in 100 ml anaerobic vials with carbon steel coupons. The ATCC 14563 strain of Desulfovibrio desulfuricans was used. Biofilms on coupon surfaces were visualized using scanning electron microscopy (SEM). Experimental results showed that EDDS reduced the glutaraldehyde dosages considerably in the inhibition of SRB biofilm establishment and the treatment of established biofilms on carbon steel coupon surfaces.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.     

Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers.

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Comparison of the Efficacy of Free Residual Chlorine and Monochloramine against Biofilms in Model and Full Scale Cooling Towers

The presence of microbial cells on surfaces results in the formation of biofilms, which may also give rise to microbiologically influenced corrosion. Biofilms accumulate on all submerged industrial and environmental surfaces. The efficacy of disinfectants is usually evaluated using planktonic cultures, which often leads to an underestimate of the concentration required to control a biofilm. The aim of this study was to investigate the efficacy of monochloramine on biofilms developed in a cooling tower. The disinfectants selected for the study were commercial formulations recommended for controlling microbial growth in cooling towers. A cooling tower and a laboratory model recirculating water system were used as biofilm reactors. Although previous studies have evaluated the efficacy of free chlorine and monochloramine for controlling biofilm growth, there is a lack of published data concerning the use monochloramine in cooling towers. Stainless steel coupons were inserted in each tower basin for a period of 30 d before removal. Monochloramine and free chlorine were tested under identical conditions on mixed biofilms which had been allowed to grow on coupons. Monochloramine was found to be significantly more effective than free chlorine against cooling tower biofilms.     

Effect of biofouling on anodized and sol-gel treated titanium surfaces: a comparative study

Anodization and sol-gel treatments of titanium (Ti) were evaluated as biofilm control measures on surfaces exposed to seawater exposed to ultraviolet light. Anodized and sol-gel treated specimens were characterized using Raman spectroscopy to confirm the presence of TiO₂. The single anatase phase was observed at the anodized surfaces whereas the anatase/rutile mixed phase was detected on the sol-gel coated surfaces. After exposure of the specimens to seawater, biofilms were characterized by total viable counts, and epifluorescence and Raman microscopy. These techniques confirmed the reduction in biofilm formation on both the anodized and sol-gel coated Ti specimens compared to the untreated specimens. Biofilm control by anodization was found to be more effective than by sol-gel treatment of the specimens. The higher particle size and the inhomogeneity at the sol-gel coated surfaces produced less effective biofilm control.     

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech     

The influence of the chemical composition of drinking water on cuprosolvency by biofilm bacteria

AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.     

Biofilm adaptation to iron availability in the presence of biotite and consequences for chemical weathering

Bacteria in nature often live within biofilms, exopolymeric matrices that provide a favorable environment that can differ markedly from their surroundings. Biofilms have been found growing on mineral surfaces and are expected to play a role in weathering those surfaces, but a clear understanding of how environmental factors, such as trace‐nutrient limitation, influence this role is lacking. Here, we examine biofilm development by Pseudomonas putida in media either deficient or sufficient in Fe during growth on biotite, an Fe rich mineral, or on glass. We hypothesized that the bacteria would respond to Fe deficiency by enhancing biotite dissolution and by the formation of binding sites to inhibit Fe leaching from the system. Glass coupons acted as a no‐Fe control to investigate whether biofilm response depended on the presence of Fe in the supporting solid. Biofilms grown on biotite, as compared to glass, had significantly greater biofilm biomass, specific numbers of viable cells (SNVC), and biofilm cation concentrations of K, Mg, and Fe, and these differences were greater when Fe was deficient in the medium. Scanning electron microscopy (SEM) confirmed that biofilm growth altered the biotite surface, smoothing the rough, jagged edges of channels scratched by hand on the biotite, and dissolving away small, easy‐to‐access particles scattered across the planar surface. High‐resolution magic angle spinning proton nuclear magnetic resonance (HRMAS ¹H NMR) spectroscopy showed that, in the Fe‐deficient medium, the relative amount of polysaccharide nearly doubled relative to that in biofilms grown in the medium amended with Fe. The results imply that the bacteria responded to the Fe deficiency by obtaining Fe from biotite and used the biofilm matrix to enhance weathering and as a sink for released cation nutrients. These results demonstrate one mechanism by which biofilms may help soil microbes overcome nutrient deficiencies in oligotrophic systems.