Bio Film

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Biofilm Key to understanding and controlling bacterial growth in Automated Drinking Water Systems What are biofilms? Biofilms are a collection of microorganisms surrounded by the slime they secrete, attached to either an inert or living surface. You are already familiar with some biofilms: the plaque on your teeth, the slippery slime on river stones, and the gel-like film on the inside of a vase which held flowers for a week. Biofilm exists wherever surfaces contact water. Why learn about bio
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  1 What are biofilms? Biofilms are a collection of microorganisms surrounded by theslime they secrete, attached to eitheran inert or living surface. You arealready familiar with some biofilms:the plaque on your teeth, the slipperyslime on river stones, and the gel-likefilm on the inside of a vase which heldflowers for a week. Biofilm existswherever surfaces contact water. Why learn about biofilms? As in any water system, 99 per cent of the bacteria in an automated wateringsystem is likely to be in biofilmsattached to internal surfaces. Biofilmsare the source of much of the free-floating bacteria in drinking water,some of which can cause infectionand disease in laboratory animals.One common biofilm bacteria, Pseudomonas aeruginosa , is anopportunistic pathogen which caninfect animals that have suppressedimmune systems. Besides being areservoir of bacteria which can affectanimal health, biofilms can alsocause corrosion in stainless steelpiping systems. Key to understanding and controlling bacterial growthin Automated Drinking Water Systems Figure 1 .Wild bacteria are “hairy” cells with extracellular polymers which stick to surfaces,concentrate nutrients, and protect bacteria from disinfectants. Understanding bacteria in biofilms isone step toward preparing for thefuture. Animal facilities can meet themost demanding water qualityrequirements by supplying chlorinatedreverse osmosis water and bymaintaining water quality throughflushing and sanitization. But what if chlorine use in animal drinking wateris prohibited? Or what if water qualityrequirements become even morestringent with the use of newspecialized animals? In order todesign and operate automatedwatering systems that deliver thecleanest water possible we shouldunderstand how biofilms develop,some of the problems they can cause,and how biofilm growth can becontrolled. This issue of UPDATE isdevoted to understanding biofilms,since this information will directenhancements to automated watering   Biofilm  2 Steps in biofilmdevelopment 1. Surface conditioning Almost immediately after a cleanpipe contacts water, organic moleculesadhere to the surface. These organicsneutralize the surface charge whichmay repel approaching bacteria.2. Adhesion of “pioneer” bacteria Planktonic (free-floating) bacteriafirst attach themselves by electrostaticattraction and physical forces. Someof these cells will permanently adhereto the surface with their extracellular  polymeric substances , or stickypolymers. 3. “Slime” formation The extracellular polymers consist of charged and neutral polysaccharidegroups that not only cement the cellto the pipe wall, but also act as an ion-exchange system for trapping andconcentrating trace nutrients from thewater. As nutrients accumulate, thepioneer cells reproduce. The daughtercells then produce their ownexopolymers, greatly increasing thevolume of ion exchange surface.Pretty soon a thriving colony of bacteria is established. In a mature  Figure 2 . Bacteria in biofilms bind together in a sticky web of tangled polysaccharide fiberswhich anchor them to surfaces and to each other.  Figure 3 .Conceptual model of the architecture of a single-species biofilm based on direct observationsusing a confocal microscope. (Costerton 1995) Benefits tobacteria: food andprotection The association of bacteria with asurface and the development of abiofilm can be viewed as a survivalmechanism. Bacteria benefit bycapturing nutrients from the waterand developing protection againstdisinfectants. Nutrients Potable water, especially high-purity water systems, are nutrient-limited environments, but evennutrient concentrations too low tomeasure are sufficient for microbialgrowth and reproduction. Howdoes life in a biofilm help bacteriaacquire nutrients?ãTrace organics will concentrateon surfaces.ãExtracellular polymers will furtherconcentrate trace nutrients fromthe bulk water.ãSecondary colonizers use thewaste products from theirneighbors.ãBy pooling their biochemicalresources, several species of bacteria, each armed withdifferent enzymes, can break down food supplies that no singlespecies could digest alone. Protection against disinfectants Biofilm bacteria may be 150-3000times more resistant to free chlorinethan free-floating bacteria. In orderto destroy the cell responsible forforming the biofilm, the disinfectantmust first react with the surroundingpolysaccharide network. The cellsthemselves are not actually moreresistant, rather they have surroundedthemselves with a protective shield.The disinfectant’s oxidizing powercan be used up before it reaches thecell. In fact, biofilm bacteria oftenproduce more exopolymers afterbiocide treatment to further protectthemselves.  3 biofilm, most of the volume (75-95%)is occupied by the loosely organizedpolysaccharide matrix filled with water.This watery slime is what makesbiofilm-covered surfaces gelatinous andslippery. 4. Secondary colonizers Besides trapping nutrient molecules, theexopolymer web also snares other typesof microbial cells through physicalrestraint and electrostatic interaction.These secondary colonizers metabolizewastes from the primary colonizers aswell as produce their own waste whichother cells then use. 5. Fully functioning biofilm The mature, fully functioning biofilmis like a living tissue on the pipe surface.It is a complex, metabolicallycooperative community made up of different species each living in acustomized microniche. An anaerobiclayer may develop underneath theaerobic biofilm. As the film grows to athickness that allows it to extendthrough the quiescent zone at the pipewall into zones of more turbulent flow,some cells will be sloughed off. Thesereleased cells can then colonizedownstream piping.A mature biofilm may take severalhours to several weeks to develop,depending on the system. Pseudomonasaeruginosa is a common pioneer bacteria which can adhere to stainlesssteel, even to electropolished surfaces,within 30 seconds of exposure. New discoveries In the past, microbiologists assumed thatbiofilms contained disorderly clumps of bacteria located in no particular structureor pattern. New techniques to magnifybiofilms without destroying the gel-likestructures have enabled researchers todiscover the complex structure of biofilms as if viewing a city from asatellite. See Figure 3. Past researchers assumed that biofilmbacteria behaved much like solitary,free-floating microorganisms. Nowthey are discovering that while biofilmbacteria have the exact same geneticmakeup as their free-floating cousins,their biochemistry is very differentbecause they switch on a different setof genes. For example, up to 40% of cell wall proteins differ betweenbiofilm and free-floating bacteria. Inmedicine, this makes biofilm bacteriadifficult to kill because some of thetargets for antibiotics are no longerthere. Bacterial evolution Bacteria have evolved the means tofind and attach to surfaces in order toincrease the chances of encounteringnutrients. Motile bacteria like Pseudomonas aeruginosa can swimalong a chemical concentrationgradient towards higher nutrientconcentrations at the pipe wall. Manyorganisms will alter their cell wall toincrease their affinity for surfaces. Thecell wall becomes hydrophobic . Onceat the surface, bacteria cells anchorthemselves with their sticky polymers. Biofilm development factors Surface material Surface material has little or no effecton biofilm development. Microbeswill adhere to stainless steel or plasticswith nearly equal enthusiasm. Surface area Surface area is one primary factorinfluencing biofilm development.Plumbing systems, unlike mostnatural environments (lakes andrivers), offer a tremendous amount of surface area. RO membranes, DIresins, storage tanks, cartridge filters,and piping systems all providesurfaces suitable for bacterialattachment and growth. Smoothness Although smoother surfaces delay theinitial buildup of attached bacteria,smoothness does not significantlyaffect the total amount of biofilm ona surface after several days. Flow velocity High flow will not prevent bacteriaattachment nor completely removeexisting biofilm, but it will limitbiofilm thickness. Regardless of thewater velocity, it flows slowest in thezone adjacent to pipe surfaces. Evenwhen water flow in the center of thepipe is turbulent, the flow velocityfalls to zero at the pipe wall. Thedistance out from the pipe wall inwhich the flow rate is not turbulent iscalled the laminar sublayer  . Thisdistance can be considered equal tothe maximum biofilm thickness. Limited nutrients Like other living creatures, bacteriarequire certain nutrients for growthand reproduction. Limiting thesenutrients will limit bacteria growth,but even minute amounts of organicmatter will support many bacteria.Theoretically, just 1 ppb of organicmatter in water is enough to produce9,500 bacteria/ml! Current technologycannot reduce nutrient levelscompletely, so total control of bacteriais not achievable by simplycontrolling nutrients. Similarly, verysmall quantities of oxygen willadequately support luxurious bacterialgrowth. Although it won’t eliminatebacteria, nutrient-poor reverseosmosis water will support lessbiofilm than regular tap watersupplies. Biofilm in automatedwatering systems To visualize biofilm in an automatedwatering system, it helps to compare thescale of cell size, biofilm thickness andmicroroughness on the pipe surface.  4  Figure 5 . Biofilm extending outside the depth of the laminar layer will be sheared off during flushing. Higher flow velocity results in less biofilm. Maximum biofilm thickness inside½ stainless steel pipe compared to the crevice of an o-ring joint. Assumes biofilmthickness only limited by flushing, not by nutrients or sanitization.  Figure 4 .Compare the size of  Pseudomonas cells to the profile of a 180 grit (32 microinch RA)stainless steel surface. available nutrients, so it could bethinner in pure water.You can see that biofilm can grow toover 100 layers of individual bacterialcells. Also notice that irregularities insurface finish are small compared tothe maximum biofilm thickness. Thisillustrates why surface smoothnesshas little impact on the total amountof biofilm on a pipe surface.Figure 5 also shows that biofilm is athin layer in a flushed pipe. Incomparison, a much deeper biofilmcould fill the crevice of an o-ring joint.These areas of deeper biofilm growthcan have more corrosion problems andare harder to sanitize.  Aerobic bacteria near the outer surfaceof a biofilm consume oxygen. If biofilmis thick enough, oxygen will be depletedat the pipe surface creating an anaerobiczone. These zones inside a stainlesssteel pipe can cause corrosion.Could the biofilm in automatedwatering systems be thick enough tohave anaerobic zones? Possibly. The FLOW The inside surface of stainless steeltubing used in room piping andmanifolds is a rolled finish. Althoughnot quantitatively defined, we canassume it is no smoother than a 32microinch RA or 180 grit finish(which is considered sanitary fordairy, food, and pharmaceutical uses).Figure 4 shows that the surfaceirregularities in such a finish are largeenough to harbor several layers of  Pseudomonas aeruginosa .Biofilm will reach a certain equilibriumthickness depending on flow velocityand nutrient levels. Assuming nutrientsaren’t limiting, biofilm thickness willbe approximately the same as the depthof the laminar layer for a particularflushing flow rate. In current automatedwatering systems, piping is flushed atabout 2 ft/sec. At 2 ft/sec, biofilmthickness is limited to approximately125 microns. The goal for the futureis to increase the flushing flow rate to2.3 gpm which is an average velocityof 5 ft/sec. At 5 ft/sec, biofilmthickness should be limited toapproximately 50 microns.Figure 5 shows the maximum biofilmthickness for 2 and 5 ft/sec flushingvelocities in Edstrom Industries’stainless steel RDS piping. Rememberthat biofilm is also limited by the
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