Bio-generated metal binding polysaccharides as catalysts for synthetic applications and organic pollutant transformations

Iron and palladium binding an exopolysaccharide (EPS) were obtained and purified from cultures of bacterial cells of Klebsiella oxytoca BAS-10. The strain BAS-10 was able to grow under anaerobic conditions with Fe(III)-citrate as energy and carbon source, producing Fe(III)-EPS that was extracted and used as catalyst in the oxidation reaction of phenol with H(2)O(2). The same bacterial strain was cultivated anaerobically with Na-citrate and Pd(2)(NO)(3) was added during the exponential growth to afford a Pd-EPS, named Bio-Pd (A), that, after isolation and purification, was used as catalyst in the reductive dehalogenation of chlorobenzene as model reaction. For comparison other two palladium binding polysaccharides were prepared: (a) a second type Pd-EPS, named Bio-Pd (B), was obtained by an exchange reaction with Pd acetate starting from an iron-free EPS produced by strain BAS-10 growing on Na-citrate medium; (b) a third type of palladium, named Bio-Pd (C), bound to a different polysaccharide, was recovered after the same exchange reaction applied on glycolipid emulsan obtained from an aerobic culture of Acinetobacter venetianus RAG 1. The superiority of Bio-Pd (A), as catalyst, vs Bio-Pd (B) and (C) was demonstrated. This approach to use microorganisms to prepare metal bound polysaccharides is novel and permits to prepare metal species, sequestrated in aqueous phase that can be useful either as catalysts for synthetic applications or to support the microbial biotransformation of pollutants.     

XAS analysis of a nanostructured iron polysaccharide produced anaerobically by a strain of Klebsiella oxytoca

A strain of Klebsiella oxytoca, isolated from acid pyrite-mine drainage, characteristically produces a ferric hydrogel, consisting of branched heptasaccharide repeating units exopolysaccharide (EPS), with metal content of 36 wt%. The high content of iron in the EPS matrix cannot be explained by a simple ferric ion bond to the sugar skeleton. The bio-generated Fe-EPS is investigated by X-ray absorption spectroscopy. Fe K-edge XANES analysis shows that iron is mostly in trivalent form, with a non-negligible amount of Fe(2+) in the structure. The Fe EXAFS results indicate that iron in the sample is in a mineralized form, prevalently in the form of nano-sized particles of iron oxides/hydroxides, most probably a mixture of different nano-crystalline forms. TEM shows that these nanoparticles are located in the interior of the EPS matrix, as in ferritin. The strain produces Fe-EPS to modulate Fe-ions uptake from the cytoplasm to avoid iron toxicity under anaerobic conditions. This microbial material is potentially applicable as iron regulator.     

Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17

Aims:  To isolate an iron-reducing bacterium and examine its ability of Fe(III) oxide reduction and dechlorination.
Methods and Results:  A fermentative facultative anaerobe, strain L17 isolated from subterranean sediment, can reduce Fe(III) oxides and carbon tetrachloride (CT). It was identified as Klebsiella pneumoniae by 16S rRNA sequence analysis. Strain L17 can metabolize fermentable substrates such as citrate, glycerol, glucose and sucrose coupled with the reduction of hydrous ferric oxide, goethite, lepidocrocite and hematite. Fe(III) reduction was influenced by crystal structure of Fe(III) oxide, type of fermentable substrate, metabolic status of the strain, and significantly enhanced by addition of anthraquinone-2,6-disulfonate (AQDS). Strain L17 could dechlorinate CT to chloroform, and the rate was accelerated in the presence of Fe(III) oxide and AQDS. Biotic dechlorination by strain L17 and abiotic dechlorination by sorbed Fe(II) were proposed as the two main mechanisms. AQDS might accelerate the dechlorination by transferring electrons from strain L17 to Fe(III) oxide and CT.
Conclusions:  K. pneumoniae L17 can reduce Fe(III) oxides and CT. The two reductions can occur simultaneously, and be significantly promoted by AQDS.
Significance and Impact of the Study:  This is the first report of a strain of K. pneumoniae capable of reducing Fe(III) oxides and CT. As a strain of environmental origin, strain L17 may have the potential for bioremediation of chlorinated compound-contaminated sites.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

Ferric iron reduction-linked growth yields of Shewanella putrefaciens MR-1

The anaerobic reduction of ferric citrate by Shewanella putrefaciens MR-1 cells was inhibited markedly by p -chloromercuriphenylsulphonate, moderately by potassium cyanide, and to a small extent by 2-heptyl-4-hydroxyquinolone- N -oxide. Iron reduction was accompanied by increases in total cellular protein, with values of 0.33-7.54 g cell protein produced per mol Fe(III) reduced. The growth yields were dependent upon the growth conditions of the inoculum and the initial concentration of Fe(III) citrate in the medium. Specifically, maximum growth yields were obtained when the inoculum was pregrown anaerobically and when the initial Fe(III) citrate concentrations were 5–10 mmol l^-1. Lower growth yields were obtained with initial Fe(III) citrate concentrations of 20–30 mmol l^-1, suggesting that cell growth was partially inhibited by higher concentrations of Fe(III) or Fe(II). Maximal growth yields were also observed early (6–24 h), after which continued increases in cell protein were minimal.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Efficient method to isolate and purify viruses of bacteria from marine environments

Aim:  To isolate viruses of specific heterotrophic bacterial strains from marine environments using a host addition/virus amplification protocol (HAVAP) for use in phage/host systems.
Methods and Results:  Bacteria-free seawater samples containing natural viruses assemblages were inoculated with a single laboratory grown bacterial host of interest in a nutrient-enriched [peptone, Fe(III) and yeast extract] seawater suspension. These conditions enhanced the replication of only those virus(s) capable of infecting the host bacterium. After incubation, free viruses were recovered at concentrations ranging 10⁵–10¹⁰ infectious virus particles per ml of seawater. Using this approach, 15 viruses were isolated and represented 12 unique phage/host systems. Two of the hosts tested were infected by more than one virus.
Conclusions:  Isolation of high concentrations of specific viruses is possible even if their initial concentrations in native waters are low. This approach allows the recovery of phage/host systems that may not be numerically dominant.
Significance and Impact of the Study:  This host enrichment protocol for virus detection and isolation is well-suited for aquatic viral ecology studies that require phage/host systems.     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system

Aims:  To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied.
Methods and Results:  An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and β-galactosidase activities of the EPS⁺ strains were higher than those of the EPS⁻. Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected.
Conclusions:  The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH.
Significance and Impact of the Study:  The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.     

Shewanella putrefaciens produces an Fe(III)-solubilizing organic ligand during anaerobic respiration on insoluble Fe(III) oxides

The mechanism of Fe(III) reduction was investigated using voltammetric techniques in anaerobic incubations of Shewanella putrefaciens strain 200 supplemented with Fe(III) citrate or a suite of Fe(III) oxides as terminal electron acceptor. Results indicate that organic complexes of Fe(III) are produced during the reduction of Fe(III) at rates that correlate with the reactivity of the Fe(III) phase and bacterial cell density. Anaerobic Fe(III) solubilization activity is detected with either Fe(III) oxides or Fe(III) citrate, suggesting that the organic ligand produced is strong enough to destabilize Fe(III) from soluble or solid Fe(III) substrates. Results also demonstrate that Fe(III) oxide dissolution is not controlled by the intrinsic chemical reactivity of the Fe(III) oxides. Instead, the chemical reaction between the endogenous organic ligand is only affected by the number of reactive surface sites available to S. putrefaciens. This report describes the first application of voltammetric techniques to demonstrate production of soluble organic-Fe(III) complexes by any Fe(III)-reducing microorganism and is the first report of a Fe(III)-solubilizing ligand generated by a metal-reducing member of the genus Shewanella.     

Phosphorus transformations in the epilimnion of humic lakes: abiotic interactions between dissolved humic materials and phosphate

SUMMARY. 1. Sephadex gel filtration of filtered water from small, Finnish forest lakes demonstrated abiotic movement of ³³P from added PO₄ to two higher molecular weight fractions. This movement was most pronounced in waters with high humic content which also had high iron content. The two fractions which took up ¹³P had nominal molecular weights of > 100,000 and 10,000-20,000.
2. An equilibrium existed between free PO₄ and the two fractions. However, one fraction, at least, appeared to exist in two phases, with one phase in rapid equilibrium with free PO₄ but the other in only slow equilibrium.
3. Additions of ferric iron up to 1 mg Fe l⁻¹ to the filtered lake water stimulated movement from free PO₄, provided high concentrations of humic materials were present. In the absence of humic materials even 0.1 mg Fe 1⁻¹ would precipitate all added ³³PO₄.
4. The high molecular weight P was only partially reactive with standard molybdate reagents. Exposure of the high molecular weight P to sunlight caused a small release of PO₄ under the experimental conditions employed.
5. Possible implications for biological phosphorus demand of such sequestration of free PO₄ by humic materials in combination with iron are discussed.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Optimization of a new heteropolysaccharide production by a native isolate of Leuconostoc sp. CFR-2181

Aim:  This work is aimed at optimizing the production of a new heteropolysaccharide (HePS) of Leuconostoc sp. CFR-2181 by standardizing the fermentation conditions in a low cost semi-synthetic medium.
Methods and Results:  Both nutritional and cultural parameters, such as carbon source and its concentration, initial pH of the exopolysaccharide (EPS) medium, fermentation temperature and fermentation period were optimized. Fermentation of the EPS medium (pH 6·7), containing sucrose at 5% (w/v) and 5% (v/v) inoculum, at 25 ° C resulted in maximum production of HePS (18·38 g l⁻¹) by the isolate in 4 h of fermentation.
Conclusions:  The isolate was found to produce good amount of HePS in just 4 h in a low cost semi-synthetic EPS medium.
Significance and Impact of the Study:  This is the first report on rapid production of HePS by any lactic culture, which can significantly reduce the cost of the EPS.     

Ecophysiology and the energetic benefit of mixotrophic Fe(II) oxidation by various strains of nitrate-reducing bacteria

In order to assess the importance of nitrate-dependent Fe(II) oxidation and its impact on the growth physiology of dominant Fe oxidizers, we counted these bacteria in freshwater lake sediments and studied their growth physiology. Most probable number counts of nitrate-reducing Fe(II)-oxidizing bacteria in the sediment of Lake Constance, a freshwater lake in Southern Germany, yielded about 10⁵ cells mL⁻¹ of the total heterotrophic nitrate-reducing bacteria, with about 1% (10³ cells mL⁻¹) of nitrate-reducing Fe(II) oxidizers. We investigated the growth physiology of Acidovorax sp. strain BoFeN1, a dominant nitrate-reducing mixotrophic Fe(II) oxidizer isolated from this sediment. Strain BoFeN1 uses several organic compounds (but no sugars) as substrates for nitrate reduction. It also reduces nitrite, dinitrogen monoxide, and O₂, but cannot reduce Fe(III). Growth experiments with cultures amended either with acetate plus Fe(II) or with acetate alone demonstrated that the simultaneous oxidation of Fe(II) and acetate enhanced growth yields with acetate alone (12.5 g dry mass mol⁻¹ acetate) by about 1.4 g dry mass mol⁻¹ Fe(II). Also, pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans strains can oxidize Fe(II) with nitrate, whereas Pseudomonas fluorescens and Thiobacillus denitrificans strains did not. Our study demonstrates that nitrate-dependent Fe(II) oxidation contributes to the energy metabolism of these bacteria, and that nitrate-dependent Fe(II) oxidation can essentially contribute to anaerobic iron cycling.     

Stable isotope analysis of macroinvertebrates and their food sources in a glacier stream

1. Food sources and trophic structure of the macroinvertebrate community along a longitudinal gradient were examined in a glacier stream of the Swiss Alps (Val Roseg). Analysis of multiple stable isotopes (δ¹³C and δ¹⁵N) and measurement of C : N ratios were used to differentiate between allochthonous and autochthonous organic matter.
2. Although isotopic signatures of algae varied widely among sites and dates, it was possible to discriminate between allochthonous and autochthonous food sources using a site-specific approach.
3. Dominant food sources of herbivorous invertebrates in all main channel sites were epilithic diatoms and the filamentous gold alga Hydrurus foetidus . Allochthonous organic matter was of some importance only in a groundwater-fed stream close to the floodplain margin.
4. Seasonal changes in the δ¹³C signature of the macroinvertebrates corresponded with seasonal changes in δ¹³C of the gold alga H. foetidus . This indicated that the energy base remains autochthonous throughout the year.
5. Because of limited food sources, feeding plasticity of the invertebrate community was high. Both grazers and shredders fed predominantly on algae, whereas gatherer-collectors seemed to be omnivorous.
6. The overall enrichment of δ¹⁵N was 2.25‰ ( r ²=0.99) per trophic level. On a gradient from the glacier site to a downstream forested site trophic enrichment was constant but variation in δ¹⁵N within trophic levels decreased.     

Robust experimental design for optimizing the microbial inhibitor test for penicillin detection in milk

Aims:  To use experimental design techniques and a multiple logistic regression model to optimize a microbiological inhibition test with dichotomous response for the detection of Penicillin G in milk.
Methods and Results:  A 2³ × 2² robust experimental design with two replications was used. The effects of three control factors (V: culture medium volume, S: spore concentration of Geobacillus stearothermophilus , I: indicator concentration), two noise factors (Dt: diffusion time, Ip: incubation period) and their interactions were studied. The V, S, Dt, Ip factors and V × S, V × Ip, S × Ip interactions showed significant effects.
Conclusions:  The use of 100  μ l culture medium volume, 2 × 10⁵ spores ml⁻¹, 60 min diffusion time and 3 h incubation period is recommended. In these elaboration conditions, the penicillin detection limit was of 3·9  μ g l⁻¹, similar to the maximum residue limit (MRL). Of the two noise factors studied, the incubation period can be controlled by means of the culture medium volume and spore concentration.
Significance and Impact of the Study:  We were able to optimize bioassays of dichotomous response using an experimental design and logistic regression model for the detection of residues at the level of MRL, aiding in the avoidance of health problems in the consumer.     

Effect of Lactobacillus gasseri BNR17 on blood glucose levels and body weight in a mouse model of type 2 diabetes

Aims:  To investigate the effect of Lactobacillus gasseri BNR17 isolated from human breast milk on blood glucose and body weight in type 2 diabetic animals.
Methods and results:  db/db mice were divided into one control group and five sample groups; the sample groups received BNR17 (10⁷, 10⁸, 10⁹ and 10¹⁰ CFU) or rosiglitazone (8 mg kg⁻¹) orally twice a day for 12 weeks. BNR17 groups had a dose-dependent reduction in food, water intake and amount of excrement. Body weight loss was not seen in the BNR17 groups. Fasting and postprandial 2 h blood glucose levels were significantly lower in the BNR17 (10¹⁰ CFU) group compared with the control group. HbA1c decreased in the BNR17 group, although it was not statistically significant. During the oral glucose tolerance test, the BNR17 groups exhibited dose-dependent improvement in glucose sensitivity.
Conclusions:  Lactobacillus gasseri BNR17 has a suppressing effect on blood glucose levels and improved diabetic symptoms in db/db mice.
Significance and Impact of the Study:  Blood glucose-lowering lactic acid bacteria are expected to be useful as a therapeutic for treating type 2 diabetes in humans.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.