Influence of surface copper content on Stenotrophomonas maltophilia biofilm control using chlorine and mechanical stress

Abstract

This work aimed to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. Stenotrophomonas maltophilia isolated from drinking water was used as a model microorganism and biofilms were developed in a rotating cylinder reactor using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10?mg l^?1 of free chlorine for 10?min, an increased shear stress (a fluid velocity of 1.5?m s^?1 for 30s), and a combination of both treatments. These shock treatments were not effective in biofilm control. The benefits from the use of copper surfaces was found essentially in reducing the numbers of non-damaged cells. Copper materials demonstrated better performance in biofilm prevention than chlorine. In general, copper alloys may have a positive public health impact by reducing the number of non-damaged cells in the water delivered after chlorine exposure.     

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.     

Direct measurement of chlorine penetration into biofilms during disinfection.

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.     

Influence of pipe materials and VBNC cells on culturable bacteria in a chlorinated drinking water model system

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Measurement of chlorine dioxide penetration in dairy process pipe biofilms during disinfection

Biofilms are considered a significant health risk in the food and dairy industries because they can harbor pathogens, and direct contact with them can lead to food contamination. Biofilm control is often performed using strong oxidizing agents like chlorine and peracetic acid. Although chlorine dioxide (ClO₂) is being used increasingly to control microbiological growth in a number of different industries, not much is known about disinfection in biofilms using chlorine dioxide. In this study, a microelectrode originally made for chlorine detection was modified to measure the profiles of chlorine dioxide in biofilm as a function of depth into the biofilm. In addition, discarded microelectrodes proved useful for in situ direct measurement of biofilm thicknesses. The chlorine dioxide microelectrode had a linear response when calibrated up to a ClO₂ concentration of 0.4 mM. ClO₂ profiles showed depletion of disinfectant at 100 μm in the biofilm depth, indicating that ClO₂ may not reach bacteria in a biofilm thicker than this using a 25 mg/l solution.     

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.     

Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Investigation of platelet margination phenomena at elevated shear stress

Thrombosis is a common complication following the surgical implantation of blood contacting artificial organs. Platelet transport, which is an important process of thrombosis and strongly modulated by flow dynamics, has not been investigated under the shear stress level associated with these devices, which may range from tens to several hundred Pascal.The current research investigated platelet transport within blood under supra-physiological shear stress conditions through a micro flow visualization approach. Images of platelet-sized fluorescent particles in the blood flow were recorded within microchannels (2 cm x 100 microm x 100 microm). The results successfully demonstrated the occurrence of platelet-sized particle margination under shear stresses up to 193 Pa, revealing a platelet near-wall excess up to 8.7 near the wall (within 15 microm) at the highest shear stress. The concentration of red blood cells was found to influence the stream-wise development of platelet margination which was clearly observed in the 20% Ht sample but not the 40% Ht sample. Shear stress had a less dramatic effect on the margination phenomenon than did hematocrit. The results imply that cell-cell collision is an important factor for platelet transport under supra-physiologic shear stress conditions. It is anticipated that these results will contribute to the future design and optimization of artificial organs.     

Effect of wall shear rate on biofilm deposition and grazing in drinking water flow chambers

The effect of four-wall shear rates (34.9, 74.8, 142.5, and 194.5 s(-1)) on bacterial deposition on glass slides in drinking water flow chambers was studied. Biofilm image acquisition was performed over a 50-day period. Bacterial accumulation and surface coverage curves were obtained. Microscopic observations allowed us to obtain information about the dynamics and spatial distribution of the biofilm. During the first stage of biofilm formation (210-518 h), bacterial accumulation was a function of the wall shear rate: the higher the wall shear rate, the faster the bacterial deposition (1.1 and 1.9 x 10(4) bacterial cells . cm(-2) for wall shear rates of 34.9 and 142.5 s(-1), respectively). A new similarity relationship characteristic of a non-dimensional time and function of the wall shear rate was proposed to describe initial bacterial deposition. After 50 days of exposure to drinking water, surface coverage was more or less identical under the entire wall shear rates (7.44 +/- 0.9%), suggesting that biofilm bacterial density cannot be controlled using hydrodynamics. However, the spatial distribution of the biofilm was clearly different. Under low wall shear rate, aggregates were composed of bacterial cells able to "vibrate" independently on the surface, whereas, under a high wall shear rate, aggregates were more cohesive. Therefore, susceptibility to the hydraulic discontinuities occurring in drinking water system may not be similar. In all the flow chambers, significant decreases in bacterial biomass (up to 77%) were associated with the presence of amoebae. This grazing preferentially targeted small, isolated cells.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Biomass evolution in porous media and its effects on permeability under starvation conditions

The purpose of this study was to understand bacteria profile modification and its applications in subsurface biological operations such as biobarrier formation, in situ bioremediation, and microbial-enhanced oil recovery. Biomass accumulation and evolution in porous media were investigated both experimentally and theoretically. To study both nutrient-rich and carbon-source-depleted conditions, Leuconostoc mesenteroides was chosen because of its rapid growth rate and exopolymer production rate. Porous micromodels were used to study the effects of biomass evolution on the permeability of a porous medium. Bacterial starvation was initiated by switching the feed from a nutrient solution to a buffer solution in order to examine biofilm stability under nutrient-poor conditions. Four different evolution patterns were identified during the nutrient-rich and nutrient-depleted conditions used in the micromodel experiments. In phase I, the permeability of the porous micromodel decreased as a result of biomass accumulation in pore bodies and pore throats. In phase II, starvation conditions were initiated. The depletion of nutrient in the phase II resulted in slower growth of the biofilm causing the permeability to reach a minimum as all the remaining nutrients were consumed. In phase III, permeability began to increase due to biofilm sloughing caused by shear stress. In phase IV, shear stress remained below the critical shear stress for sloughing and the biofilm remained stable for long periods of time during starvation. The critical shear stress for biofilm sloughing provided an indication of biofilm strength. Shear removal of biofilms occurred when shear stress exceeded critical shear stress. A network model was used to describe the biofilm formation phenomenon and the existence of a critical shear stress. Simulations were in qualitative agreement with the experimental results, and demonstrate the existence of a critical shear stress.     

96-well microtiter plates for biofouling simulation in biomedical settings

Microtiter plates with 96 wells are routinely used in biofilm research mainly because they enable high-throughput assays. These platforms are used in a variety of conditions ranging from static to dynamic operation using different shaking frequencies and orbital diameters. The main goals of this work were to assess the influence of nutrient concentration and flow conditions on biofilm formation by Escherichia coli in microtiter plates and to define the operational conditions to be used in order to simulate relevant biomedical scenarios. Assays were performed in static mode and in incubators with distinct orbital diameters using different concentrations of glucose, peptone and yeast extract. Computational fluid dynamics (CFD) was used to simulate the flow inside the wells for shaking frequencies ranging from 50 to 200?rpm and orbital diameters from 25 to 100?mm. Higher glucose concentrations enhanced adhesion of E. coli in the first 24?h, but variation in peptone and yeast extract concentration had no significant impact on biofilm formation. Numerical simulations indicate that 96-well microtiter plates can be used to simulate a variety of biomedical scenarios if the operating conditions are carefully set.     

Disinfection of bacteria in water systems by using electrolytically generated copper:silver and reduced levels of free chlorine

As an alternative disinfectant to chlorination, electrolytically generated copper:silver (400 and 40 micrograms/L copper and silver, respectively) with and without free chlorine (0.3 mg/L) was evaluated over a period of 4 weeks in indoor and outdoor water systems (100 L tap water with natural body flora and urine). Numbers of total coliform, pseudomonas, and staphylococci were all less than drinking water standards in systems treated with copper:silver and free chlorine and systems treated with free chlorine alone (1.0 mg/L). No significant differences (p less than or equal to 0.05) in bacterial numbers were observed between systems with copper:silver and free chlorine and those with free chlorine alone. Overall, free-chlorine treatments (0.3 or 1.0 mg/L) showed significantly lower heterotrophic plate numbers than those without free chlorine. When challenged with a natural Staphylococcus sp. isolate, water with copper:silver and free chlorine had a 2.4 log10 reduction in bacterial numbers within 2 min, while free chlorine alone or copper:silver alone showed 1.5 and 0.03 log10 reductions, respectively. Addition of copper:silver to water systems may allow the concentration of free chlorine to be reduced while still providing comparable sanitary quality of the water.     

Modulation of growth media influences aggregation and biofilm formation between <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> and <Emphasis Type="Italic">Trichoderma viride</Emphasis>

The effect of growth media manipulation on the in vitro biofilm forming ability of Azotobacter chroococcum MTCC 25045 and Trichoderma viride ITCC 2211, both as individual cultures and co-culture was evaluated for 16 days. Growth curves (Bioscreen C lab system) and type of microbial population (planktonic and biofilm) helped to validate the aggregation and biofilm data. Modulation of combinations of routine growth media—Jensen’s broth (J) and potato dextrose broth (P) by changing their ratios (100, 75: 25, 50: 50, and 25: 75) was undertaken. In individual bacterial or fungal inoculation, the growth media–J25: P75 and P100 caused significantly (p < 0.01) higher growth, aggregation, and biofilm formation. In co-culture, J25: P75 medium showed enhanced planktonic as well as biofilm population, aggregation, and biofilm formation followed by J50: P50 and J75: P25 media. This is a first report on interrelationships among growth, aggregation and biofilm formation in relation to medium optimization for fungal-bacterial biofilm development.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Alumina surfaces with nanoscale topography reduce attachment and biofilm formation by Escherichia coli and Listeria spp.

This work reports on a simple, robust and scientifically sound method to develop surfaces able to reduce microbial attachment and biofilm development, with possible applications in medicine, dentistry, food processing, or water treatment. Anodic surfaces with cylindrical nanopores 15 to 100 nm in diameter were manufactured and incubated with Escherichia coli ATCC 25922 and Listeria innocua. Surfaces with 15 and 25 nm pore diameters significantly repressed attachment and biofilm formation. Surface–bacteria interaction forces calculated using the extended Derjaguin Landau Verwey-Overbeek (XDLVO) theory indicate that reduction in attachment and biofilm formation is due to a synergy between electrostatic repulsion and surface effective free energy. An attachment study using E. coli K12 strains unable to express appendages also suggests that the small-pore surfaces may inhibit flagella-dependent attachment. These results can have immediate, far-reaching implications and commercial applications, with substantial benefits for human health and life.     

Effectiveness of chlorine washing disinfection and effects on the appearance of artichoke and borage

AIM: Optimal conditions for chlorine application to obtain a reasonable decrease in the microbial counts without damaging the appearance of artichoke and borage have been established. METHODS AND RESULTS: The influence of chlorine concentration (0-200 mg l(-1)), pH, addition of organic acids, contact time and presence of protective structures on the microflora and vegetal appearance were studied. When pH was not controlled the effect of chlorine depended on its concentration until the pH increase caused by addition of chlorine reached 8.8. Any further increase in chlorine concentration was nullified by the pH increase. When pH was adjusted to 4.5 with acetic acid, the effectiveness increased with concentration. However, the use of citric acid to control pH caused a sharp decrease in effectiveness at concentration about 250 mg l(-1). The higher effectiveness of chlorine on homogenized plant extracts compared with the whole plant showed the impact of the vegetal structures on the resistance of the microorganisms. For artichoke, a relationship between the effectiveness of chlorine disinfection and its structures was also found. Extended washing times did not affect the total counts. However, in both vegetables, the appearance was affected by the extended contact times. CONCLUSIONS: The solutions rendering the highest microbial reduction with minimum damages were: 50 mg l(-1) free chlorine without pH control for artichoke and 100 mg l(-1) free chlorine at pH 7.0 for borage. SIGNIFICANCE AND IMPACT OF THE STUDY: Specific conditions for chlorine disinfection of artichoke and borage were determined to reduce the microorganisms in minimally processed artichoke and borage without damaging their appearance.