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What are biofilms?Biofilms are a collection ofmicroorganisms 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 ofthe 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 ifchlorine use in animal drinking wateris prohibited? Or what if water qualityrequirements become even morestringent with the use of new

specialized 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

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Steps in biofilmdevelopment1. Surface conditioningAlmost immediately after a cleanpipe contacts water, organic moleculesadhere to the surface. These organicsneutralize the surface charge whichmay repel approaching bacteria.

2. Adhesion of “pioneer” bacteriaPlanktonic (free-floating) bacteriafirst attach themselves by electrostaticattraction and physical forces. Someof these cells will permanently adhereto the surface with their extracellularpolymeric substances, or stickypolymers.

3. “Slime” formationThe extracellular polymers consist ofcharged 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 ofbacteria 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 andprotectionThe 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.

NutrientsPotable 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?

• Trace organics will concentrateon surfaces.

• Extracellular polymers will furtherconcentrate trace nutrients fromthe bulk water.

• Secondary colonizers use thewaste products from theirneighbors.

• By pooling their biochemicalresources, several species ofbacteria, each armed withdifferent enzymes, can breakdown food supplies that no singlespecies could digest alone.

Protection against disinfectantsBiofilm 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.

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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 colonizersBesides 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 biofilmThe mature, fully functioning biofilmis like a living tissue on the pipe surface.It is a complex, metabolicallycooperative community made up ofdifferent 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 discoveriesIn the past, microbiologists assumed thatbiofilms contained disorderly clumps ofbacteria located in no particular structureor pattern. New techniques to magnifybiofilms without destroying the gel-likestructures have enabled researchers todiscover the complex structure ofbiofilms 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% ofcell 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 evolutionBacteria have evolved the means tofind and attach to surfaces in order toincrease the chances of encounteringnutrients. Motile bacteria likePseudomonas 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 factorsSurface materialSurface material has little or no effecton biofilm development. Microbeswill adhere to stainless steel or plasticswith nearly equal enthusiasm.

Surface areaSurface area is one primary factorinfluencing biofilm development.Plumbing systems, unlike mostnatural environments (lakes andrivers), offer a tremendous amount ofsurface area. RO membranes, DIresins, storage tanks, cartridge filters,and piping systems all providesurfaces suitable for bacterialattachment and growth.

SmoothnessAlthough smoother surfaces delay theinitial buildup of attached bacteria,smoothness does not significantlyaffect the total amount of biofilm ona surface after several days.

Flow velocityHigh 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 nutrientsLike 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 systemsTo visualize biofilm in an automatedwatering system, it helps to compare thescale of cell size, biofilm thickness andmicroroughness on the pipe surface.

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Figure 5.Biofilm extending outside the depth of the laminar layer will be sheared off duringflushing. 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 to

the 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 ofPseudomonas 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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depth of the oxygen gradient into thebiofilm will vary, but one sourceindicates that oxygen can be depletedwithin 30-40 microns of the water/biofilm interface. Anaerobic zones aremost likely to occur in crevices at o-ring pipe joints and threaded fittings.

Microbiologicallyinfluenced corrosionThe physical presence of microbialcells on a metal surface, as well astheir metabolic activities, can causeMicrobiologically InfluencedCorrosion or biocorrosion.

Nonuniform (patchy) colonies ofbiofilm result in the formation ofdifferential aeration cells where areasunder respiring colonies are depletedof oxygen relative to surroundingnoncolonized areas. Having differentoxygen concentrations at twolocations on a metal causes adifference in electrical potential andconsequently corrosion currents.

Stainless steel relies on a stable oxidefilm to provide corrosion resistance.When the oxide film is damaged oroxygen is kept from the metal surface,corrosion can occur.

Some biofilm bacteria producecorrosive chemicals like acids andhydrogen gas during their metabolism.Anaerobic zones in biofilm can havesulfate-reducing bacteria (SRBs). Thisgroup of bacteria reduce sulfate tohydrogen sulfide which corrodesmetals. SRBs can grow in watertrapped in stagnant areas, such as deadlegs of piping. One way to limit SRBactivity is to reduce the concentrationof their essential nutrients:phosphorus, nitrogen, and sulfate.Thus, purified (RO or DI) water wouldsupport less SRBs.

Any practices which minimize biofilmthickness (flushing, sanitizing,

eliminating dead-end crevices) willminimize the anaerobic areas whichcan cause biocorrosion.

Sanitizing biofilmsBiofilm can be removed and/ordestroyed by chemical and physicaltreatments. Chemical biocides can bedivided into two major groups:oxidizing and nonoxidizing. Physicaltreatments include mechanicalscrubbing and hot water.

Oxidizing biocidesChlorineProbably the most effective and leastexpensive of all chemical biocides,chlorine not only kills free-floatingand biofilm bacteria, but it alsodestroys the polysaccharide web andits attachments to the surface. Bydestroying the extracellular polymers,chlorine breaks up the physicalintegrity of the biofilm.

Higher concentrations of chlorine areneeded to disinfect biofilm than to killfree-floating bacteria. As chlorinediffuses into a biofilm, it is used up

reacting with bacteria cells and slime.At low chlorine levels, biofilmbacteria shield themselves byproducing slime faster than chlorinecan diffuse through. By increasing theconcentration, chlorine will diffusefarther into the biofilm. When itcomes to disinfection of biofilms,high chlorine concentration for shortdurations is more effective than lowconcentration for long durations.However, since it is corrosive tostainless steel, there is a limit on therecommended chlorine concentrationfor sanitization.

Chlorine dioxideChlorine dioxide has biocidal activitiessimilar to those of chlorine. Because itis unstable, it must be mixed andprepared on-site. Like chlorine, chlorinedioxide is corrosive to metals and mustbe handled with care.

OzoneAs an oxidizer, ozone is approximatelytwice as powerful as chlorine at thesame concentrations. However,because of its limited solubility, it isdifficult to produce high concentrations

Figure 6.Nonuniform colonization by bacteria results in differential aeration cells. This schematicshows pit initiation due to oxygen depletion under a biofilm. (Borenstein 1994)

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of ozone in water. Chlorine can beused in higher sanitizingconcentrations with equal disinfectingstrength. Like chlorine dioxide, ozonemust be generated on-site because ofits high reactivity and relativeinstability. Systems must be designedwith appropriate ozone resistantmaterials.

PeroxideHydrogen peroxide (10% by volumesolution) is used as a biocide inmicroelectronic-grade water systemsbecause it rapidly degrades to waterand oxygen. It is less effective andmore expensive than chlorine.

Non-oxidizing biocidesQuatsIn addition to their biocidal activity,quaternary ammonium compoundsare effective surfactants which helpremove biofilm from surfaces.However, it requires exhaustiverinsing to flush them out of watersystems.

FormaldehydeFormaldehyde has been used tosanitize pharmaceutical-grade watersystems. It is relatively noncorrosiveto stainless steel, but its effectivenessagainst biofilm is questionable and itis a toxic carcinogen.

Physical TreatmentsHeatPharmaceutical Water-for-Injectionsystems use continuously recirculatinghot water loops (greater than 80oC) tokill bacteria. Periodic hot watersanitization can also be used todestroy biofilm, but since this requiresa temperature of 95oC for 100 minutes,it’s usually not practical in an animaldrinking water system.

Detecting and countingRoutine monitoring of bacterial levelsis an essential part of monitoring thequality of laboratory animal drinkingwater. The classic way to enumeratebacteria in water is to do a plate countwhich is to spread a known volumeof sample on the surface of alaboratory medium and count thenumber of visible colonies thatdevelop after a period of time.However, plate counts mayunderestimate the total number ofbacteria present in a watering system.

Water samples only collect free-floating bacteria which are eithersloughed off of the biofilm or passthrough from the incoming water

supply. A low plate count doesn’tmean that bacteria are not presentbecause more than 99% of bacteria inwater systems are attached to pipesurfaces. If the integrity of a maturebiofilm hasn’t been disrupted byrecent flushing or sanitization, it maynot slough off many cells into thedrinking water, but it is still there.

Plate counts are based on the abilityof bacteria in a sample to grow on adefined nutrient medium and formdistinct colonies. Theoretically, acolony is derived from a singlebacteria cell, but some underestimationis caused by clumps of bacteria thatform only one colony. In purifiedwater samples, plate counts may be

Because they shield themselves inslime, bacteria associated withbiofilms are much more difficult tokill than free-floating organisms. Itis common to observe a rapidregrowth of biofilm immediatelyfollowing chlorine sanitization asshown in Figure 7. Incompleteremoval of the biofilm will allow itto quickly return to its equilibriumstate, causing a rebound in totalplate counts following sanitization.

Slime-producing organisms likePseudomonas are less susceptible tobiocides and may accumulateselectively in distribution systemsthat are sanitized.

Perhaps if sanitization could beautomated, a watering systemcould be easily re-sanitized everyfew days before biofilm fullyrecovers.

Figure 7.Example of sanitization followed by biofilm recovery. Bacteria count samples weretaken on a daily basis. (Mittelman 1986)

Biofilm recovery (regrowth)

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...and how to fight back.Purify anyway!

It will limit nutrients somewhat, especially nutrients for microbes likesulfate-reducing bacteria which cause corrosion problems. Nutrient-poor RO water will support less bacteria than tap water. This meansa thinner biofilm. Besides, the animals will be getting better qualitydrinking water.

Flush anyway!

Periodic flushing will minimize the thickness of the biofilm. Thinnerbiofilm have less anaerobic zones and sanitizing chemicals will have ashorter distance to diffuse through to reach the pipe surface.

Minimize crevices anyway!

Maybe surface finish doesn’t matter much as far as total biofilmaccumulation, but eliminating large crevices (like o-ring joints) willeliminate deep pockets of biofilm which are harder to sanitize and aremore corrosive. Also, electropolishing will aid in resisting corrosion.

Sanitize anyway!

If biofilm recovers within 3 days after sanitization, knock it backdown by sanitizing every 1-2 days. This could be done by automatingchlorine or ozone sanitization.

low because the media is too rich togrow bacteria in a starved state.

Equilibrium biofilm thickness isdependent on water velocity andnutrients. As biofilms grow, singlecells or “rafts” of cells are sloughedoff during flushing. This results inrandom ‘particle showers’ of bacteriawhich can explain day-to-dayfluctuations and occasional highbacteria count results.

What you can doBacteria constitute a very successfullife form. In their evolution, they havedeveloped successful strategies forsurvival which include attachment tosurfaces and development ofprotective biofilms where they behavevery differently than free-floatingbacteria. Their successful strategiesmake it difficult to control biofilmgrowth in automated wateringsystems.

There is no “silver bullet” forcontrolling bacterial growth inautomated watering systems. Unlessa continuous chlorine level is allowed,it will take a combination strategy. Buta multi-pronged strategy should resultin a bacterial water quality which willsatisfy the needs of animal research.

Questions orComments

If you have any questions orcomments on biofilms or ifyou’d like a copy of thecomplete Biofilm report,including references, pleasecontact:Edstrom Industries, Inc.(800)-558-5913 e-mail: lab@edstrom.com

The biofilm assault...

We purify water to remove nutrients and ask “How could anythinglive in it?"

Biofilm bacteria use their polymer web to concentrate nutrients. Theycan live on nutrient levels we can’t even measure.

We flush water lines trying to scour them off the pipes.

They cement themselves to surfaces with their sticky polymers underthe laminar layer where shear forces are too weak to remove them.

We smooth the inside surfaces of fittings so they can’t take shelter increvices and crannies.

It doesn’t matter. They will attach themselves speedily and inevitablyanyway.

We sanitize piping with chlorine. They shield themselves in slime.