Life Cycle of Biofilm?

Each number corresponds with a stage in biofilm development. The photomicrographs are of an actual Pseudomonas aeruginosa biofilm.

Biofilm Basics

Contrary to common belief, most bacteria are not “free living” – instead, they live in a self-sustaining community where they cooperate with other bacteria and are largely protected from outside influences. This is properly known as a “Biofilm” although most people would recognise it as “slime”. This makes up a large proportion of the brown deposits you see on the pipework above. Among many other bacteria, Biofilm harbours the dangerous pathogens responsible for poor growth reduced liveability and poor food conversation to mention but a few.

Biofilm removal is the most important part of any drinking water treatment system. If the sanitation product used does not have the ability to remove biofilm it is impossible to have a safe pathogen free environment.  Many products – even down to the level of citrus juices – have the ability to kill free-living bacteria, but only the best have the ability to penetrate and destroy biofilm.

In the early days since recognition of this problem in the Poultry industry, application of basic industrial water treatment products such as occasional addition of chlorine tablets have shown some benefit over doing nothing, however due to their inability to tackle the complex problem of biofilm forming throughout the water pipe-work over a long growth cycle, these approaches have been found to be not necessarily protective or economic.

Since the early 90’s vast improvements have been made in biocide dosing technology and chemical monitoring equipment, which when combined with new microbiological analysis techniques have proven to secure the viability of treatment programs. Currently, there is a number of “new generation” water treatment approaches being used, ranging from professional ozone and chlorine dioxide dosing units, through to the addition of Peracetic acid, pH adjusters and eveso-calleded “organic bio-flavonoids”.

The Microbiology of Biofilms

Bacteria are initially attracted to pipe surfaces for lots of reasons.  They may be attracted to the positive charge on some inorganic surface because they often have a negatively charged outer envelope themselves, or they may arrive and settle out due to gravity or water flows.  There is evidence that biofilm formation is much more than simple random physical force.  Many surfaces attract and concentrate nutrients, and many bacteria have the skill to detect and follow such high concentrations (an ability called chemotaxis).

Some cells produce lots of polysaccharides which act as mucus layers, gaining a foothold and gluing others to the selected host surface.  These are called the primary colonisers.  This external slime gives a helping hand to other passing bacteria who add another tier of inhabitants called the secondary colonisers who live and feed on the waste produced by the first colonisers.  Before long a thriving complex microbial community has been established inside the polysaccharide slime and this is called the biofilm.

• Protection from antibiotics and biocides. Much higher doses of antibiotics & biocides are required to kill bacteria in biofilms compared with their freewheeling relatives.  At first, it was thought that the biofilm provided a complete physical barrier to efforts at destruction but there is now evidence that the  very nature of the colonies themselves provides protection.  By growing in colonies, the late arrivals protect the inner cells from penetration, leaving the latter free to grow and multiply.

• Concentration of nutrients As negative charges are often associated with the biofilm matrix, many nutrients are drawn to settle and nutrients with negative charges can exchange with ions on the surface.  This activity provides an attractive source of nourishment compared to the surrounding water.

• Community feeling No bacterium likes to be alone for long and nearly all live with other micro-organisms for energy, carbon and other nutrients.  We see a classic example in the degradation of cellulose; celluolytic microbes break the cellulose into sugar monomers which fermenting bacteria can use, giving off smaller organic acids, carbon-dioxide and hydrogen gas, which methanogens or sulfate reducers use for their carbon and energy.  Finally, genetic material can be easily exchanged within the close confines of the biofilm.  This increases the potential for the successful emergence of better adapted and stronger strains of bacteria.

Water Treatment Approaches

Chlorine Disinfection of Water

Chlorine disinfection can be achieved either by the use of chlorine gas or by using sodium or calcium hypochlorite. Chlorine gas can be produced by generation using salt plus acid or it be purchased as a bottled gas. When free chlorine gas is dissolved in water, a mixture of hydrochloric and hypochlorous acids is formed which will react with water by dissociation to an extent determined by pH. At pH 7 approximately 80% remains effective as hypochlorous acid. As pH increases this figure falls dramatically so that at pH 8 only around 20% remains active. Optimum use of chlorine requires pH control.

When sodium or calcium hypochlorite are used, the chemical reactions involved also produce sodium and calcium hydroxide which increases the pH thereby limiting the efficacy of the active acids. Chlorine as hypochlorous acid readily reacts with both organic and non organic material such as sulphides and ammonia.  Initially the chlorine will be used up in these reactions and testing may show that none remains as a residual. in the water. As further hypochlorous acid is added ammonia and some organics will react to form to produce chloramines and chloro-organic compounds resulting in taste and odour problems. Additional hypochlorous acid will be used in oxidising the compounds just formed and only after that has happened will there be an available residual in the water.

In water with a high organic load chlorine is used up in complex multiple reactions. These reactions are temperature, and pH related. Chlorine will start to dissociate or “gas off” from water at about 40oC.

Although chlorine will oxidise as a tertiary reaction, its primary reaction is chlorination and many of the chloro-organics formed will not be re-oxidised. These residuals include the group trihalomethanes, which are known carcinogens and difficult and costly to remove, especially once they have contaminated the ground water.

Free chlorine gas is toxic and corrosive when in solution. Biocidal effectiveness is limited to simpler organisms and viruses in operating conditions of pH6.5 – 8 and below 40oC. Chlorine has limited effectiveness against biofouling and will not kill all of the more complex organisms such as cysts and protozoa. Research has shown amoebic cysts infected with hundreds of legionella bacteria surviving in well chlorinated systems. When the host dies the legionella bacteria are released and infect the bases of calorifiers, silt traps and re-seed themselves in the biofouling. Because chlorine readily combines with both organic and inorganic material to form both toxic and carcinogenic by-products its use as a potable water disinfectant is rapidly declining and will continue to do so.

Ultra Violet and Ozone Disinfection of Water

The growth of biofilms was studied using a biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. The biofilms were also tested for the presence of Legionella spp, a typical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the UV disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection.

Conclusions: The results indicated that bacteria are able to regenerate and proliferate more effectively after UV irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water.

Significance and Impact of the Study: As far as the application of UV disinfection is used for conditioning of critical water sources for drinking water, the efficiency of UV irradiation in natural systems should reach a high standard to avoid adverse impacts on human health.

Since UV cannot provide residual disinfection capability, it can only be utilized to inactivate bacteria and Cryptosporidium at the raw or finish water treatment points in the plant. Therefore, chlorine, chloramines and combined disinfectants (chlorine dioxide and chlorine or chloramines) will likely always be required to provide water plant and distribution system disinfection capabilities. Gordon Finch, a Canadian Researcher in the late 90s, determined that combined disinfectants such as chlorine dioxide and chlorine or chloramines showed a synergistic capability to inactivate Cryptosporidium on an equal basis compared to UV alone.

Chlorine Dioxide Disinfection of Water

Chlorine dioxide is a powerful oxidising biocide and has been successfully used as a water treatment disinfectant for several decades in many countries. Rapid progress has been made in the technology for generation of the product and knowledge of its reactivity has increased with improved analytical techniques. Chlorine dioxide is a relatively stable radical molecule. It is highly soluble in water, has a boiling point of 110C, absorbs light and breaks down into ClO3- and Cl–. Because of its oxidising properties chlorine dioxide acts on Fe2, Mn2+ and NO2- but does not act on Cl, NH4+ and Br– when not exposed to light. These ions are generally part of the chemical composition of natural water.

Because of its radical structure, chlorine dioxide has a particular reactivity – totally different from that of chlorine or ozone. The latter behave as electron acceptors or are electrophilic, while chlorine dioxide has a free electron for a homopolar bond based on one of its oxygens. The electrophilic nature of chlorine or hypochloric acid can lead, through reaction of addition or substitution, to the formation of organic species while the radical reactivity of chlorine dioxide mainly results in oxycarbonyls.

The oxidising properties and the radical nature of chlorine dioxide make it an excellent virucidal and bactericidal agent in a large pH range. The most probable explanation is that in the alkaline media the permeability of living cell walls to gaseous chlorine dioxide radicals seems to be increased allowing an easier access to vital molecules. The reaction of chlorine dioxide with vital amino acids is one of the dominant processes of its action on bacteria and viruses.

Chlorine dioxide is efficient against viruses, bacteria and protoza clumps usually found in raw water. A rise in pH level further increase its action against f2 bacteriophages, amoebic clumps, polioviruses and anterovirus. It is efficient against Giardia and has an excellent biocidal effect against Cryptosporidium which are resistant to chlorine and chloromines.  It has been demonstrated that ClO2 has greater persistence than chlorine. In a recent report for dosages 3 times lower than those of chlorine at the station outlet, the residual of ClO2 used alone was always higher than that of Cl2 which also required 3 extra injections of chlorine in the distribution system.