Effect of Anolyte & Catholyte on Biofilms: Adhesion to surfaces is a common and well known behaviour of microorganisms in oligotrophic habitats (Zobell, 1943). This adhesion and subsequent metabolism lead to the formation of biofilms (39). Bacterial biofilms promote increased biomass deposition (54), resulting in fluid flow resistance, loss of heat exchange and microbial induced corrosion in industrial water cooling systems (13). Industries control unwanted biofilms, with varying degrees of success, by using biocides (13). The use of biocides, especially chlorine, in water reticulation and heat exchange systems is effective only if the biofilm is removed manually. Chlorination of a mature biofilm is usually unsuccessful because the biocide only reacts with the outer portion of the biofilm, leaving a healthy and substantial bacterial community on the surface that rapidly regrows (10). Bacteria within biofilms develop increasing resistance to the biocide on repeated dosing (13). BrÃ¶zel and Cloete (10, 11, 12) found that biocides also induced cross-resistance to other biocides.
Microbial biofilms are problematic in a range of industrial environments where large areas of submerged surfaces are exposed to relatively high nutrient fluxes, providing niches for the formation of copious surface-associated growth (13, 18). Bacterial colonisation of surfaces in an aqueous environment is a basic strategem for survival in nature as nutrients are more available at the solid – liquid interface (27, 32). The resulting aggregates form microcolonies which develop into biofilms (39). Biofilms increase fluid frictional resistance (39) and decrease the rate of heat energy transfer, collectively termed biofouling. These biofilms also promote corrosion of ferrous and other metals by the concerted metabolic activity of a number of biofilm-associated bacterial types (38), a process collectively termed microbially influenced corrosion (MIC). MIC encompasses a number of specific mechanisms relating either directly or indirectly to the metabolic activity of a variety of microorganisms, notably the action of sulphidogenic bacteria (19, 33). As the costs attributable to MIC and biofouling are high, effective control of bacterial numbers in industrial aqueous environments is essential. A range of bactericidal substances, commonly termed biocides or microbicides, are available, all of which are claimed by their producers to kill bacteria occurring in aqueous systems quantitatively. Biocides target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material (47). However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins (9), or to the development of resistance, either by adaptation or by genetic exchange (11). Bactericides should therefore be evaluated against the organisms which they are chosen to control, i.e. the dominant ones in the system to be treated. The composition of microbial populations in systems varies with the type of water used, and changes considerably following treatment with various biocides by selection for resistant strains (10). Bacteria growing as biofilms are also significantly more resistant to most antimicrobial agents known currently, so that methods for their control pose an ongoing challenge (14, 18) Successful biofouling control depends on rationally developed treatment strategies which are based on information of the specific system. The primary target should always be the biofilm-associated flora as it is the catalyst for MIC and impacts negatively on system operation.
Five approaches are currently available and may be used in combination:
A novel way of the electro chemical activation of water was recently introduced in South Africa. During Electro Chemical Activation (ECA) of water, a dilute saline solution is “activated” by passing through acylindrical electrolytic cell in which the anodic and cathodicchambers are separated by a permeable membrane. During the process of electrochemical activation three broad classes of product are produced:
Stable products – these are acids (in the Anolyte) and bases (in the Catholyte) which influence the pH of the solution in question, as well as other active species.
Highly active unstable products – these include free radicals and other active ion species with a typical lifetime of less than 48 hours. Included here would be electrically and chemically active micro bubbles of electrolytic gas 0,2 – 0,5 micrometer in diameter and with concentrations up to 107 ml-1, distributed uniformly through the solution. All these species serve to enhance the oxidation-reduction potentials (ORP) of the Anolyte, which is reducing, resulting in anomalous ORP values for both.
Quasi-stable structures – these are structures formed at or near the electrode surface as a consequence of the very high voltage drop (107 V cm-1) in those regions. These are free structural complexes of hydrated membranes around ions, molecules, radicals and atoms. The size of these water clusters is reduced to approximately 5-6 molecules per cluster.
All these features enhance the diffusion, catalytic and biocatalytic properties of the water. The chemical composition of ECA solutions may be altered by utilizing various hydraulic arrangements linking electrolytic cell modules, together with other supplementary devices, in order to optimally address the requirements of specific areas of application. Some other variables are flow rate; hydraulic pressure; current density; voltage on the electrodes. Two separate streams of activated water are produced: Anolyte with a pH range of 2-9 and an oxidation- reduction potential (ORP) of +400 mV to +1200 mV. Anolyte is an oxidizing agent due to a mixture of Free Radicals and has an antimicrobial effect. Catholyte with pH of 12 to 13 and an ORP of about -900mV. It has reducing and surfactant properties and is an antioxidant. The aim of this study was to use DAPI staining and scanning electron microscopy (SEM) evaluate the biofilm removal efficiency of anolyte and a combination of anolyte and catholyte.
Bactericides are antimicrobial agents employed in various spheres of human activity to prevent, inhibit or eliminate microbial growth. They can be divided into two groups; those derived from naturally occurring antimicrobial agents (termed antibiotics), and those not occurring readily in nature (termed antiseptics, disinfectants, biocides, bactericides, sanitisers and preservatives). Members of the second group are classified, depending either on their chemical nature, but more often on their specific field of application. The use of biocides to control biofouling in water systems is still an accepted practice although higher levels of environmental awareness and tighter legislation have placed increased pressure on the water treatment industry to seek alternative means of control (6). Nevertheless biocides are still indispensable components for effective control of biofouling as they remain the core technology for decimating viable numbers whereas all other techniques merely aid in their efficacy.
The modes of action of a plethora of antibiotics have been investigated in detail. Much less is known about the mechanisms of action of the many biocides available for biofouling control. As antibiotics, biocides for water treatment target a range of specific cellular components and functions, from membrane permeability and electron transport to enzyme function. Bactericides attack functional cell components, placing the bacterium under stress (52). At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations (58). Targets of bactericide action are components of the cytoplasmic membrane or of the cytoplasm (47). For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Therefore different bacteria react differently to bactericides due to differing permeabilities of their cell wall properties (40). Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high biocide concentration will kill bacteria with enhanced penetration barriers (47). Water treatment bactericides fall into two categories, oxidising (eg. chlorine and hydrogen peroxide) and non-oxidising. Non-oxidising bactericides can be divided into five groups based on their chemical nature or mode of action, and these will be discussed below.
Oxidising biocides are general chemical oxidants. They are not selective for living organisms, but react with any oxidisable matter. However, they are bactericidal because certain bacterial cell components can react readily with them, having a higher oxidation potential than most other chemicals present in water. Three classes of oxidising biocides are available for bactericidal applications; oxidising halogens, peroxides and ozone.
These include a variety of organic chemical compounds which have antimicrobial activity. Their modes of action differ vastly, and their only common denominator is that they are non-oxidising organic molecules. Most currently used biocides fall into five distinct categories, although a number of miscellaneous compounds are also usefull.
Alternative approaches of chemical control
Factors affecting efficacy of treatment programmes
The antibacterial activity of bactericides is determined by their chemical reactivity with certain organic groups. Bactericides do not select between free and cell-bound groups. Therefore oxidising bactericides react with any readily oxidisable organic compound, and not only with live cells. Bactericide activity is influenced by the chemistry of the surrounding where it is employed (53) . Factors affecting bactericide effectivity are the following:
These factors affect different bactericides to different degrees. Some bactericides are not very stable in concentrated form and undergo changes. Formaldehyde polymerises when exposed to polar compounds (acids or alkalis) or high temperature and oxidises to formic acid when exposed to air (53). Isothiazolones are unstable at temperatures above 40°C and chlorhexidine is unstable above 70°C (53). A decrease in the efficacy of a bactericide treatment programme can be due to a decrease in bactericide activity, or due to inactivation by adverse conditions, and does not always indicate bacterial resistance (13).
Chemistry of the water
Chemicals inhibiting scaling and corrosion are also added to industrial water systems, and some interact with certain biocides. Chromates are used to inhibit corrosion, but also suppress microbial growth, acting synergistically with the biocide used. Glycolic acid secreted by algae can, however, reduce chromate, rendering it inactive. Dithiocarbamates reduce chromate, so the two substances are incompatible (34). QAC’s form insoluble chromate precipitates at high concentrations, so the two should not be added simultaneously to water. Careless application of chloride lowers the pH to a point where the protective chromate film is solubilised. Na-2- mercaptobenzothiazole is a corrosion inhibitor which is oxidised by chlorine dioxide. Methylene bis-thiocyanate is hydrolysed under slightly alkaline conditions (pH 7,5). Where the chlorine demand of the water is high, the large quantity of chlorine added leads to a high chloride level which increases the corrosion potential of the water. Chlorine and quaternary ammonium compounds increase the corrosion rate of copper alloys.
Bactericide treatment regimes for cooling water systems often fail, posing the question of bacterial resistance to the bactericide. Certain authors have argued that failure of treatment programmes was due to selection for resistant strains. We have however shown that susceptible bacterial isolates do acquire increased tolerance to bactericides following serial transfer in subinhibitory concentrations. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and / or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they were not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tollerance is a genetic change, and where the biochemical basis is known. Whereas the basis of bacterial resistance to antibiotics is well known, that of resistance to antiseptics, disinfectants and food preservatives is less well understood. The basis of bacterial development of resistance to water treatment bactericides is little known. As biocides are selective in their action, application of any one could result in selection for resistant bacteria. As cells in biofilms and planktonic communities are in continuous exchange, death of cells in the planktonic phase would influence the equilibrium and shifts would occur in both the planktonic and the sessile populations. Biocides attack targets of cell function, placing the bacterium under stress. It is well recognized that communities under stress have a lower species diversity and select for fitter species. Therefore a more resistant community could develop. The concentration of a biocide is not related linearly to its activity; a concentration exponent is involved in the relationship. In many cases a small decrease in concentration will result in a notable decrease in activity. In an in situ biocide evaluation study we found the dominant planktonic survivor after 48h was a species most effectively killed by the relevant biocide under laboratory pure culture conditions. An example is dichlorophen which killed 99.94% ofPseudomonas stutreri at 50 ppm and yet left this species the dominant isolate after 48h (43%) in the cooling system. Also, thiocarbamate killed 99.87% of P. stutzeri at l74ppm and left it the dominant planktonic survivor (62.5%) in the system treated. QAC-tin killed 100% of Acinetobacter calcoaceticus and left Acinetobacter spp. ominant (40%)in the system. Although the surviving strains could be different ones, the correlation is striking.
Two reasons exist why the efficacy of bactericide treatment programmes can decrease at times. The one is a decrease in the activity of the bactericide, and the other is a decrease in the bacterial susceptibility towards the bactericide. Three mechanisms of resistance have been reported in the field of antibiotic study:
Bactericides are less specific in their action than some antibiotics, so that the alteration of a reactive site or the substitution of an amino acid in a protein will not render bacterial cells resistant. Therefore inaccessibility and inactivation are the two classes of possible mechanisms of resistance. Additionally, active removal of biocide is a third class of resistance.
Bacteria in biofilms are much more protected from bactericidal action than are planktonic bacteria. In a recent study, biofilm bacteria were found 150 to 3000-fold more resistant to hypochlorous acid and 2 to 100 times more to monochloramine than were unattached cells. Pseudomonas aeruginosa growing as a biofilm has been found 20 times as resistant to tobramycin as are planktonic cells. Three reasons for the increased biocide resistance of biofilm bacteria have been put forward. These do not adequately explain the phenomenon of biofilm resistance, but they are listed below:
The first reason is questionable because the reason why bacteria grow in biofilins is because the EPS acts as a nutrient sink, attracting organic compounds from the surrounding water. Recent work has supported the theory that organic material is somehow attracted to the EPS, and associates favourably with it. At least some of these molecules must diffuse to and into the microorganisms embedded in the EPS to facilitate the observed growth. Non-oxidising biocides, being organic molecules of small to intermadiate size would also associate favourably with the EPS. At least some would diffuse to and into the microorganisms embedded in theEPS and exert their antibav\cterial activity. The mechanism of increased resistance must be related to alterred surface properties of cells growing in the biofilm environment. Reasons 2 and 3 are possibly correct.
Mutagenicity of biocides
Some bactericides, being toxic substances, have a broader spectrum of toxicity than merely bacteria. Some are mutagens or even carcinogenic. Table 4 summarises the LD50 values available for bactericides, as well as mutagenicity data where available. Mutagens are substances which induce mutations in DNA of any living organisms. Furthermore many mutagens are carcinogens. Mutation in bacteria from water cooling systems will increase the rate of development of resistance to biocide, and necessitate the addition of higher levels of biocide. Such treated water, when released into the environment will cary with it large volumes of mutagenic and possibly carcinogenic biocides. This will affect ground water sources, rivers, dams and by irrigation agricultural soils. It could also reach drinking water sources. The release of such substances into nature is dangerous to human life. Formaldehyde itself is carcinogenic. A second mutagenic compound used in biocides is 5-chloro- N-methylisothiazolone (CMIT). The related isothiazolones benzisothiazolone (BIT) and Nmethylisothiazolone (MIT) are not mutagenic. Thiocarbamates are not toxic to humans as they are degradation products of certain pharmaceuticals used in the treatment of alcoholism.
Materials and Methods
Organism used: Pseudomonas aeruginosa isolated from a cooling water system was used for all the experiments
Biocide used: Anolyte and Catholyte was supplied by Radical waters. In the first experiment anolyte was used in a 1:10 dilution. In the second experiment a combination of anolyte and catholyte at a ratio of 2:1 was used in a 1:10 dilution.
Experimental procedures: A continuous flow – through system Pedersen device (39) was used to determine the biofilm removal of Ps. aeruginosa on a stainless steel surface and on glass.
DAPI-staining: DAPI staining was done as described in a previous study (Wolfaardt et al., 1996). Qantification of attached bacteria using 4,6-diamidino-z-phenylidole (DAPI). The 75 x 27 x 1mm coupons, were removed from the Pedersen device and rinsed with sterile water as described for the SEM studies of biofilm formation and stained with DAPI for epifluorescence microscopy (Wolfaardt et al., 1991). Attached bacteria were observed under oil immersion using Epifluorescence microscopy. Ten randomly chosen microscope fields were counted under the 800 x magnification.
Scanning Electron Microscopy (SEM): Coupons (25 x 27 x 1mm) were removed, in duplicate, from the modified Pedersen device at 4, 8, 24, 32, 48 and 56 h, with a sterile forceps and replaced with a sterile coupon, in order to keep the flow constant. After removal the coupons were rinsed with sterile distilled water for 30 s to remove any unattached cells and then fixed for SEM by the following series of treatments: 2% gluteraldehyde (1 h); 0.175M Phosphate- buffer (3 x 15min); 50% ethanol (1 x 15min); 70% ethanol (1 x 15min); 90% ethanol (1 x 15 min) and 100% ethanol (3 x 15min). The coupons were thereafter dried in a critical point dryer, mounted on studs and coated with gold plasma and examined using the Hitachi S-450 scanning electron microscope.
Biofilm removal: To study biofilm removal, the bacteria were allowed to adhere to the surface of the 3CR12
Viable bacteria counts: The total number of viable bacteria in the planktonic phase was determined before biocide addition and again after 6h. Plate counts were done on R2A agar and incubation at ambient temperature to simulate the experimental conditions.
Results and Discussion
The anolyte solution (1:10 dilution) effectively removed a mature P.aeruginosa biofilm within 6h (Fig 1). The anolyte also reduced the planktonic bacteria numbers from 2,41 x 107 cfu ml-1 to <10 cfu ml-1 during the same period (Table 1). The anolyte killed the bacteria in the biofilm within 1h indicated by the fading of the DAPI stain. The system was oprated for a further 72 h to determine whether biofilm regrowth would occur. Regrowth of the biofilm was observed 24h after treatment (Fig 2). Regrowth of the planktonic bacteria occurred as reflected by the increase in cfu to 1,33 x 106 cfu ml-1 after 72 h (Table 1). These results are in agreement with BrÃ¶zel and Cloete (10) who indicated that regrowth normally occurs within 48 h after biocide treatment. Regrowth can be attributed to mainly two factors: firstly, in some instances, a microbial population shift may occur to organisms resistant to the biocide, or secondly, the biocide is “consumed” by organic matter allowing the regrowth of the surviving bacteria.
The anolyte/catholyte (2:1 ratio) solution added at a 1:10 ratio also effectively removed the mature P.aeruginosa biofilm. The anolyte/catholyte solution effectively removed the biofilm within 3-4h (Fig 2). Noticeable is the dispersion of the biofilm structure (after 1h) before removal occurs (Fig 2). Regrowth of the biofilm started taking place 24h after treatment (Fig 2). Regrowth of the planktonic bacteria occurred after 72h (Table 2).
Fig’s 3-9 are representative scanning electron micrographs of the biofilm behaviour before and after biocide treatment. Figure 3 and 4 represent the biofilm before and after 1h of treatment. Surface colonization can clearly be seen by numerous microcolonies. Also noticeable is the dehydrated glycocalyx structure (biofilm). These micrcolonies are still visible after 2h (Fig 5) and 3h (Fig 6) of treatment. The microcolonies are nevertheless fewer in number and smaller in size than at 0h and 1h. After 4h of treatment (Fig 7) very few microcolonies were observed and the glycolacyx (biofilm) was no longer noticeable. After 24h of treatment the situation remained unchanged (Fig 8). Neverthless, DAPI staining indicated regrowth of the biofilm (Fig 2). This difference was attributed to the difference in the method of preparation for DAPI and SEM, where the preparation of slides for DAPI is less harsh than for SEM.