The effect of molybdenum on biofilm development

Little is known about the formation and effects of biofilms on stainless steel pipes in freshwater environments, particularly as they are considered as a direct replacement for copper pipes for ‘problem’ water. There is some cause for concern especially as stainless steel cannot claim the inherent biocidal potential of copper. As molybdenum is known to be leached out of stainless steel grade 316, in very small amounts, a study was set up to see if molybdenum could retard the development of biofilms. When a comparison of biofilm viable and total cell counts was made between pure molybdenum metal and stainless steel grade 304, it was found that cell counts were significantly higher (P < 0.05) on grade 304 stainless steel after 5 weeks exposure to flowing water (0.64 m s⁻¹). Molybdenum (above a concentration of 1 g L⁻¹) affected the growth rate of Acinetobacter sp, a pioneering bacterium of biofilms in potable water. Received 18 February 1998/ Accepted in revised form 17 May 1999     

The effect of turbulent flow and surface roughness on biofilm formation in drinking water

There is considerable interest in both Europe and the USA in the effects of microbiological fouling on stainless steels in potable water. However, little is known about the formation and effects of biofilms, on stainless steel in potable water environments, particularly in turbulent flow regimes. Results are presented on the development of biofilms on stainless steel grades 304 and 316 after exposure to potable water at velocities of 0.32, 0.96 and 1.75 m s⁻¹. Cell counts on slides of stainless steel grades 304 and 316 with both 2B (smooth) and 2D (rough) finishes showed viable and total cell counts were higher at the higher flow rates of 0.96 and 1.75 m s⁻¹, compared to a flow rate of 0.32 m s⁻¹. Extracellular polysaccharide levels were not significantly different (P< 0.05) between each flow rate on all stainless steel surfaces studied. higher levels were found at the higher water velocities. the biofilm attached to stainless steel was comprised of a mixed bacterial flora including Acinetobacter sp, Pseudomonas spp, Methylobacterium sp, and Corynebacterium/Arthrobacter spp. Epifluorescence microscopy provided evidence of rod-shaped bacteria and the formation of stands, possibly of extracellular material attached to stainless steel at high flow rates but not at low flow rates. Received 04 February 1998/ Accepted in revised form 12 February 1999     

Biofilm Colonization Dynamics and Its Influence on the Corrosion Resistance of Austenitic UNS S31603 Stainless Steel Exposed to Gulf of Mexico Seawater

Viable bacterial counts, chemical markers, phospholipid fatty acid analysis (PLFA), and Fourier-transformed infrared spectroscopy (FTIR), together with electrochemical methods, were used to study biofilm dynamics and its impact on the corrosion resistance of UNS S31603 stainless steels exposed to the Gulf of Mexico seawater. Biofilms progressively accumulated, peaking on day 20, but finally detached. The extracellular polysaccharide (EPS)/cellular biomass ratio remained low most of the time, but reached its highest level (4.2 ± 1.9) also on day 20. Viable bacterial cells reached their highest abundance earlier (∼800 CFU/cm²), on day 15. Biofilms were seen covering the stainless steel surfaces heterogeneously and were composed mainly of gram-negative rods, presumably EPS-producing bacteria. Despite the different levels of biofilm biomass and attachment state, field-exposed steel coupons ennobled significantly and showed more active pitting potentials (∼+500 mV_SCE) than on the abiotic control (+650 mV_SCE), where no significant ennoblement occurred. These results suggest that the heterogeneous distribution of biofilms, as opposed to the quantity of surface-associated biomass, promotes formation of differential aeration cells, and that this in turn contributes to the ennoblement of these steels.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

The Effect of a Cationic Porphyrin on Pseudomonas aeruginosa Biofilms

Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen (¹O₂) that damages biomolecules associated with cells and biofilms, e.g., proteins, polysaccharides, and DNA. The effect of a cationic porphryin on P. aeruginosa PAO1 biofilms was assessed by exposing static biofilms to 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) followed by irradiation. Biofilms were visualized using confocal laser scanning microscopy (CLSM) and cell viability determined using the LIVE/DEAD BacLight viability assay and standard plate counts. At a concentration of 100 μM TMP, there was substantial killing of P. aeruginosa PAO1 wild-type and pqsA mutant biofilms with little disruption of the biofilm matrix or structure. Exposure to 225 μM TMP resulted in almost complete killing as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Standard plate counts of cells recovered from attached biofilms revealed a 4.1-log₁₀ and a 3.9-log₁₀ reduction in viable cells of wild-type PAO1 and pqsA mutant strains, respectively. Our results suggest that the action of photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

The growth of Bacillus stearothermophilus on stainless steel

AIMS: To determine the potential for Bacillus stearothermophilus cells to form biofilms of significance in dairy manufacture. METHODS AND RESULTS: The ability of isolates of B. stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces was demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology. Spores attached more readily than vegetative cells. The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel. The growth of B. stearothermophilus as a biofilm on stainless steel surfaces was determined using a continuously flowing experimental reactor. Vegetative cells were released in greater numbers than spores from biofilms of most strains studied. Biofilms of one strain (B11) were studied in detail. Biofilms of > 106 cells cm-2 formed in the reactor and released approximately 106 cells ml-1 into milk passing over the biofilm. A doubling time of 25 min was calculated for this organism grown as a biofilm. CONCLUSION: The formation of biofilms of thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products. Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms of B. stearothermophilus growing in dairy manufacturing plants can explain the contamination of dairy products with these bacteria.     

Development of a CrN/Cu nanocomposite coating on titanium-modified stainless steel for antibacterial activity against Pseudomonas aeruginosa

A relatively simple method was developed to fabricate CrN/Cu nanocomposite coatings using pulsed DC magnetron sputtering for application in antibacterial activity. These nanocomposite coatings were applied on titanium (Ti)-modified stainless steel substrata (D-9 alloy) and the antibacterial activity of these coating with respect to the Gram-negative bacterium Pseudomonas aeruginosa was investigated qualitatively and quantitatively. Scanning electron microscopy, epifluorescence microscope analyses, and total viable counts confirmed that inclusion of copper in the CrN/Cu nanocomposite coatings provided antibacterial activity against P. aeruginosa. The quantitative examination of the bacterial activity of P. aeruginosa was estimated by the survival ratio as calculated from the number of viable cells which formed colonies on nutrient agar plates.     

Optimization of conditions for in vitro development of <Emphasis Type="Italic">Trichoderma viride</Emphasis>-based biofilms as potential inoculants

Biofilms represent mixed communities present in a diverse range of environments; however, their utility as inoculants is less investigated. Our investigation was aimed towards in vitro development of biofilms using fungal mycelia (Trichoderma viride) as matrices and nitrogen-fixing and P-solubilizing bacteria as partners, as a prelude to their use as biofertilizers (biofilmed biofertilizers, BBs) and biocontrol agents for different crops. The most suitable media in terms of population counts, fresh mass and dry biomass for Trichoderma and Bacillus subtilis/Pseudomonas fluorescens was found to be Pikovskaya broth ± 1 % CaCO_3, while for Trichoderma and Azotobacter chroococcum, Jensen’s medium was most optimal. The respective media were then used for optimization of the inoculation rate of the partners in terms of sequence of addition of partners, fresh/dry mass of biofilms and population counts of partners for efficient film formation. Microscopic observations revealed significant differences in the progress of growth of biofilms and dual cultures. In the biofilms, the bacteria were observed growing intermingled within the fungal mycelia mat. Further, biofilm formation was compared under static and shaking conditions and the fresh mass of biofilms was higher in the former. Such biofilms are being further characterized under in vitro conditions, before using them as inoculants with crops.     

Characterization of Adhesive Exopolysaccharide (EPS) Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Under Starvation Conditions

Pseudomonas aeruginosa synthesizes large quantities of exopolysaccharide (EPS), making it an excellent model organism for the study of EPS-mediated adhesion. The purpose of this investigation was to evaluate the influence of limited nutrients availability in the culture medium on the composition of EPS produced by P. aeruginosa. The relationship between the EPS production and the adhesion process of the P. aeruginosa cells to stainless steel surface (type 316 L) under starvation conditions were also examined. In all experimental variants P. aeruginosa produced more EPS with an increase of incubation period upon starvation conditions. Under limited nutrients condition, glucose dominated in the EPS materials. After 6 days of the process, only glucosyl units were detected in the extracellular matrix produced by nutrient-deprived P. aeruginosa cells. These extracellular molecules promoted more advanced stages of P. aeruginosa biofilm formation on the surface of stainless steel.     

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     

Evaluation of biocathodes in freshwater and brackish sediment microbial fuel cells

Biofilms on biocathodes can catalyze the cathodic oxygen reduction and accordingly guarantee high cathode redox potentials. The present research assessed the use of biocathodes in full-sediment microbial fuel cells. Carbon felt-based biocathodes were evaluated in freshwater systems, and an extension of their application to brackish systems and/or stainless steel webs as base material was considered. Efficient biocathodes could be developed within days through inoculation with active microorganisms. Carbon felt was found most suited for the biocathodes in freshwater with increased performance at salinities around 80–250 mM. Maximum long-term performance reached 12.3 μW cm⁻² cathode. The relative benefit of stainless steel seemed to increase with increasing salinity. A combination of stainless steel cathodes with biofilms could, however, also result in decreased electrical performance. In an efficiently catalyzing cathodic biofilm, an enrichment with an uncultured Proteobacterium—previously correlated with steel waste—was observed.     

Enhancement of Glutaraldehyde Biocidal Efficacy by the Application of an Electric Field. Effect on Sessile Cells and on Cells Released by the Biofilm

Summary The aim of this paper was to evaluate the possible enhancement of the biocidal efficacy of glutaraldehyde against Pseudomonas fluorescens biofilms by the application of an electric field. The behaviour of sessile cells and cells released by the biofilms was assed. Biofilms were formed on thin stainless steel coupons immersed in culture media inoculated with Pseudomonas fluorescens. Treatments using glutaraldehyde (TGA) and both glutaraldehyde and electric field application (TGAEF) were carried out with the samples with biofilms. TGA: samples with biofilms were immersed in glass cells containing a buffer solution with different glutaraldehyde concentrations in the 25–500 ppm range. TGAEF: samples with biofilms were immersed in an electrochemical cell containing glutaraldehyde solution where a direct electric current (4 × 10⁻⁴ A cm⁻²) was delivered to the chamber. The evolution of biofilms was observed through optical microscopy at real time. Results show that the electric field enhanced glutaraldehyde efficacy reducing the number of surviving cells in the range of one to four orders with respect to those with TGA treatment. The sensitivity of the cells to the treatments decreased in the following order: planktonic cells > cells released by the biofilm > sessile cells.     

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

Whereas the transfer of Listeria from surfaces to foods and vice versa has been well documented, little is known about the mechanism of bacterial transfer. The objective of this work is to gain a better understanding of the forces involved in listerial biofilms adhesion using atomic force microscopy (AFM). L. monocytogenes Scott A was grown as biofilms on stainless steel surfaces by inoculating stainless steel coupons with Listeria and incubating the coupons for 48 h at 32 °C with a diluted 1:20 tryptic soy broth. After growth, biofilms were equilibrated over saturated salt solutions at a constant relative humidity (%RH) before measurement of adhesion forces using AFM. The effects of contact time, loading force, and biofilm relative humidity (%RH) suggested that neither contact time, loading force nor biofilm %RH had a significant effect on biofilm adhesiveness at a cellular level (P > 0.05). In a second set of experiments, the influence of material type on biofilm adhesiveness was evaluated using two different colloidal probes (SiO₂ and polyethylene). Results showed that the maximum pull-off force and retraction work needed to retract the cantilever for glass (−85.42 nN and 1.610⁻¹⁵ J, respectively) were significantly lower than those of polyethylene (−113.38 nN and 2.7 × 10^–15 J, respectively; P < 0.001). The results of this study suggest that Listeria biofilms adhere more strongly to hydrophobic surfaces than hydrophilic surfaces when measured at a cellular level. These results provide important insights that could lead to new ways to remediate and avoid listerial biofilm formation in the food industry.     

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air–liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid–liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air–liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.     

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.     

Development and characterization of biofilms on stainless steel and titanium in spent nuclear fuel pools

The aim of the present research was to study the biofilms developed in a Spanish nuclear power plant and their ability to entrap radionuclides. In order to carry this out, a bioreactor, which was then submerged in a spent nuclear fuel pool, was designed. To characterise the biofilm on two different metallic materials (stainless steel and titanium), standard culture microbiological methods and molecular biology tools, as well as epifluorescence and scanning electron microscopy were used. The bacterial composition of the biofilm belongs to several phylogenetic groups (α, β, and γ-Proteobacteria, Actinobacteridae, and Firmicutes). The radioactivity of the biofilms was measured by gamma-ray spectrometry. Biofilms were able to retain radionuclides from radioactive water, especially ⁶⁰Co. The potential use of these biofilms in bioremediation of radioactive water is discussed.     

Biofilm material properties as related to shear-induced deformation and detachment phenomena

Biofilms of various Pseudomonas aeruginosa strains were grown in glass flow cells under laminar and turbulent flows. By relating the physical deformation of biofilms to variations in fluid shear, we found that the biofilms were viscoelastic fluids which behaved like elastic solids over periods of a few seconds but like linear viscous fluids over longer times. These data can be explained using concepts of associated polymeric systems, suggesting that the extracellular polymeric slime matrix determines the cohesive strength. Biofilms grown under high shear tended to form filamentous streamers while those grown under low shear formed an isotropic pattern of mound-shaped microcolonies. In some cases, sustained creep and necking in response to elevated shear resulted in a time-dependent fracture failure of the “tail” of the streamer from the attached upstream “head.” In addition to structural differences, our data suggest that biofilms grown under higher shear were more strongly attached and were cohesively stronger than those grown under lower shears. Received 06 February 2002/ Accepted in revised form 13 June 2002     

Development of cyanobacterium-based biofilms and their in vitro evaluation for agriculturally useful traits

The ability of cyanobacteria to be useful as matrices for agriculturally important bacteria was evaluated. Biofilms were generated with the selected strain Anabaena torulosa after co-culturing with Azotobacter chroococcum, Pseudomonas striata, Serratia marcescens, and Mesorhizobium ciceri. The biochemical attributes were compared with individual bacterial and cyanobacterial cultures. The biofilms were characterized in terms of proteins, chlorophyll, IAA production, acetylene-reducing activity, phosphate solubilization, and antagonism towards selected phytopathogenic fungi. An enhancement in the population counts was recorded in A. torulosa–S. marcescens and A. torulosa–P. striata biofilms. The A. torulosa–A. chroococcum and A. torulosa–M. ciceri biofilms were also able to utilize new saccharides as compared to the individual cultures. Such novel biofilms with agriculturally useful traits can provide additional advantages including the broader spectrum of activity and the presence or formation of biologically active compounds; they also suggest the way to effective inoculants for sustainable and environment friendly agriculture.