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12 Microbes and Enzymes in Biofilms Jana Jass Umea ˚ University, Umea ˚ , Sweden Sara K. Roberts University of Illinois–Chicago, Chicago, Illinois Hilary M. Lappin-Scott Exeter University, Exeter, England I. INTRODUCTION Microbial enzymes and their activities have been studied primarily in pure liquid cultures under laboratory conditions. However, in natural environments microorganisms grow at interfaces as attached (sessile) mixed communities rather than as suspended planktonic populations (1). Studies of microbial enzymes in soil go some way to recognizing this, but data interpretation has often been difficult because the methodologies do not easily differentiate between enzymes associated with surface-attached populations and those loosely attached or free in the liquid phase. It is the aim of this chapter to discuss the biological characteristics of these sessile microbial populations with particular reference to their enzyme activities. II. WHAT IS A BIOFILM? Microorganisms attached to a surface are collectively referred to as a biofilm and are of current interest because they are different in their phenotype and physiological characteris- tics from the planktonic populations. Early research on biofilms was conducted in the 1920s and 1930s, primarily by Claude ZoBell (2–4), who was one of the first people to note that bacteria existed as what he termed attached films. Three of ZoBell’s many observations (3,4) include that bacteria attach rapidly to surfaces; planktonic bacteria are not covered in ‘‘sticky’’ material, but sessile bacteria are; and these organisms, once asso- ciated with a surface, secrete a ‘‘cementing’’ substance. It is significant that many of the problems encountered by biofilm researchers today are the same as those from as long as 60 years ago; they include understanding the mechanisms underlying attachment, detach- ment (3), microbial interactions, population diversity, biofilm structure, and growth (5). There have been many proposed definitions of a biofilm over the years (6,7), the most useful is given by Costerton and associates (8), who defined the biofilm as bacteria attached to surfaces and aggregated in a hydrated polymeric matrix of their own synthesis. Copyright © 2002 Marcel Dekker, Inc. However,itisimportanttobeawarethatinmanyinstances,particularlyinnaturalenviron- ments,biofilmsconsistnotonlyofbacteriabutoffungi(9,10),yeasts(11),algae(12), andprotozoa(13).Nonetheless,muchofthepublishedliteratureconcentratesonbacterial biofilms,althoughwiththeincreasedemergenceofinfections(11)andproblemassociated withfungalbiofilms(10)theimportanceofstudyingmorecomplexmixedbiofilmscon- tainingbothprokaryotesandeukaryoteshasrecentlybeenrealized(14). III.BIOFILMLIFECYCLE Itisofteneasiertounderstandwhatabiofilmisintermsoftheeventsthatleadtoits formation.Moststudiesofbiofilmsinnaturalenvironmentshaveconcentratedonevents atsolid–liquidinterfaces,thecolonizationofasubmergedabioticsurfaceisdepictedin Fig.1.Uponimmersionofanonbiologicalmaterial,suchasglassorsilica,thesurface becomescoatedrapidlywithalayerofproteinacousmaterialcalledaconditioninglayer (15–17).Ionsandothernutrientsourcesaccumulateattheinterface,givingrisetohigher microenvironmentconcentrationsthatwillattractmicroorganismsfromthenutrient-and energy-starvedliquidphasetothesurface(18).Bacteria,whichareoftenthefirstcoloniz- ers,begintosynthesizecopiousamountsofexopolysaccharide(EPS)materialinitiated Figure1Aschematicillustratingthelifecycleofabiofilm. Copyright © 2002 Marcel Dekker, Inc. upon contact with a surface (19,20). The microbial cells become embedded within this matrix, grow, and divide to form microcolonies. Other microorganisms present in the surrounding environment are recruited into the biofilm at all stages of biofilm development to form complex functioning communities (14,21,22). Bryers and Characklis (23) pro- posed a three-step colonization process that is widely accepted by many authors: initial biofilm formation, exponential accumulation of cells and biomass, and steady state. This pattern of colonization dictates a typical sigmoidal growth curve. Steady state is reached when attachment of cells is equal to detachment of cells as a consequence of such processes as predation, sloughing, and erosion. Although biofilms are complex and dynamic and differ from environment to envi- ronment, they all have three primary common features. First, a biofilm is associated with an interface at which the cells accumulate. The solid–liquid interface is most frequently studied and well characterized, however, biofilms may also form at air–liquid (24,25), solid–air (26), and, in some cases; when a phase separation occurs, liquid–liquid inter- faces. Second, a biofilm contains a number of microbial cells of one or more species at an interface. A single attached microorganism does not constitute a biofilm although opin- ions differ as to how dense the attached organisms must be to constitute, a biofilm (27). Third, the sessile microorganisms produce an extracellular polymer matrix within which they are embedded. This matrix, often composed of EPS synthesized by the bacteria, may contain materials and components trapped from their surrounding environment. For example, biofilms in natural water habitats contain particles of sediments and plant mate- rial trapped within the matrix. In addition, the EPS matrix is believed to be important in a variety of biofilm functions, which are discussed in the following sections. Studies have also shown that biofilm bacteria are more resistant to antimicrobial regimes than their planktonic counterparts (8). The exopolymer matrix may contribute to the increased resis- tance to antimicrobial agents by either ionically binding the compounds or physically reducing penetration of the agent through the structure, although other factors may be involved (28–30). IV. FUNCTION OF BIOFILM STRUCTURE AND ARCHITECTURE It would be naive to assume that a biofilm community is simply defined as microorganisms residing and growing at an interface. Microbes are, in fact, components of complex com- munities continuously responding to both their immediate microenvironment and their surrounding habitat. This is reflected in the range of biofilm structures: from thin layers of attached cells, as seen with monocultures of some Pseudomonads or smooth colony variants of Vibrio cholerae (31), to more complex forms of attached communities con- taining multiple species interacting with each other (22,32). Biofilm structure (three-dimensional) and architecture (microbial organization) are strongly connected to the functions and survival of the microorganisms within. Research has shown that there are many conditions that contribute to biofilm architecture; these can be categorized as physical factors (i.e., flow rates, hydrodynamic forces, and viscosity), chemical factors (i.e., nutrient availability and EPS composition), and biological factors (i.e., competition and predation) (33). In practice it is difficult to separate the influences of these categories, as there is overlap between them. The combination of species specific- ity and physical, chemical, and biological factors influence biofilm structure in such a manner that it is virtually impossible (and probably unrealistic) to agree on a standard Copyright © 2002 Marcel Dekker, Inc. Table 1 Factors That Influence Biofilm Structure Factor Examples of variables Reference Surface Hydrophobicity Bos et al. (15) Roughness Lewandowski et al. (34) Electrochemical properties Geesey et al. (35) Hydrodynamics Mass transfer Lewandowski et al. (36) Flow rate/velocity Stoodley et al. (37,38) Nutrients Concentration Stoodley et al. (37,38) Mass transfer Xu et al. (39) Availability deBeer and Stoodley (40) Møller et al. (41) Exopolymeric matrix Exopolysaccharide Sutherland (42, 43) production Skillman et al. (44) Ecology Consortia Stoodley et al. (37) Predation Caron (45) Rogers et al. (46) Cell signaling Davies et al. (47) Source: Adapted From Ref. 13. biofilm model. In practice, different models are available for different growth conditions, based on a consensus of variables that influence biofilm architecture (Table 1). With ad- vances in imaging technology, such as confocal scanning laser microscopy (48), real- time image capture (49), and fluorescent staining (41,50,51), our understanding of biofilm structure is increasing rapidly. Some researchers believe that biofilm structure and in- creased resistance to antimicrobial regimes are attributable to the production of chemical signals (52). An increasing number of microorganisms, including bacteria and fungi, are found to produce a range of molecules that regulate their population density; these are called quorum sensing or cell–cell communication molecules (53). Many gram-negative bacteria produce N-acylhomoserine lactones (AHL-s) as sensor molecules (54); however, other substances have been implicated in signaling including 3-hydroxypalmatic acid methyl ester produced by the plant pathogen Ralstonia solanacearum (55). Gram-positive organ- isms (e.g. streptomyces spp.) produce different signal molecules such as small posttransla- tionally modified peptides or other compounds related to AHLs such as γ-buytrolactones (56). These small diffusible molecules accumulate at high cell densities within the biofilm and, at a critical concentration, activate a genetic response in the microorganisms. The response is not always restricted to the same species producing the sensing molecules; other bacterial species or even eukaryotic cells (fungi, plant, or animal cell cultures) may respond to these chemical signals (57,58). Davies et al. (47) reported that the quorum sensing system of Pseudomonas aeruginosa that affects biofilm formation is composed of a two-gene cascade systems, lasR-lasI and rhlR-rhlI. The lasI and rhlI gene products are involved in the synthesis of two different AHL molecules, N-(3-oxododecanoyl)-l- homoserine lactone and N-buytryl-l-homoserine lactone, respectively (47). The AHL mol- ecules are required to activate the transcriptional regulators (products of lasR and rhlR) in a sequential order, where the gene product of lasR activates the rhlR-rhlI system and a number of virulence factors and secondary metabolites. Mutants lacking both lasI and Copyright © 2002 Marcel Dekker, Inc. rhlIorjustlasIgeneproductswereabletoadheretoaglasssurfacebutwerenotable todifferentiateintothickmultilayeredbiofilms.Thissystemalsoregulatestheexpression ofotherfactors(59),suchastypeIVpiliinP.aeruginosa(twitchingmotion),whichhave alsobeenfoundtoinfluencethedifferentiationofadherentmonolayerstothickbiofilm structures(60).Anincreasingnumberofbacteriaarebeingfoundtobeassociatedwith newdensity-dependentcommunicationmolecules,bothinthelaboratoryandinsitu (53,61).Forexample,thepresenceofAHLswasdetectedinnaturallyoccuringaquatic biofilmsonstonesbyintroducingAgrobacteriumtumefaciensA136withalacZfusion asanindicatororganism(61).However,itwouldbenai ¨ vetoassumethatadhesionand biofilmformationrestsolelyontheproductionofchemicalsignals(52,62).Otherresearch hasshownthatalthoughAHLsplayanimportantroleintheaccumulationofcellsonthe surfaceandtheformationofbiofilms,theoverallstructureofbiofilmsgrowinginaqueous environmentsduringtheearlystagesofcolonizationisdeterminedlargelybytheflow conditions(37,63). Therearetwomaindelimitingfactorsthatinfluencethestructureofabiofilmin aqueousenvironments:flowrateandnutrientavailability.Flowcanbecategorizedas laminarorturbulent.Laminarflowisthesmoothflowoffluidthroughapipeorduct.In contrast,whenflowbecomeserraticandirregularitisdescribedasturbulent.Lewandow- skiandWalser(64)foundthatthethicknessofamixedculturebiofilmwasatamaximum nearthetransitionbetweenlaminarandturbulentflows.However,manydifferentbiofilm structureshavebeenobserved,oftenexplainedbyexaminingthemasstransferproperties ofthebulkliquid.Inaturbulentsystemthereisgoodmixingofnutrients,andthebulk liquidcomesintocontactwithlargeproportionsofthebiofilmwhereuptakeofnutrients cantakeplace.Incomparison,underlaminarflowconditionsthereispoormixingof nutrientsinthebulkliquid,limitingnutrientavailability.Indeed,LewandowskiandWalser (64)hypothesizedthattherewasanoptimalflowratebelowwhichbiofilmaccumulation waslimitedbymasstransferandabovewhichbiofilmaccumulationwaslimitedbycontin- ualcelldetachment.Manyoftherecentconfocalmicroscopestudieshaveshownthata biofilmconsistsofmicrocoloniesofbacteriainadenseEPSmatrixwithlessdenseintersti- tialvoidsorwaterchannels(38,65,66).Usingmicroelectrodes(50)ithasbeendemon- stratedthattheseinterstitialvoidscontaingreaterconcentrationsofnutrientsthanthe microcoloniesandthuscanactastransportchannelsfornutrientsandtheremovalofby- products,makingthemanessentialstructureinanybiofilm(66).Others(67)haveshown thattherewerefewerchannels,whichwerelessdefinedinamaturingbiofilm.Reduction ofthesechannelswoulddecreasethemasstransportcharacteristicswithinthebulkliquid phase,therebycontrollinggrowthrateofthemicrobeswithinthebiofilmduetoreduced nutrientand,possibly,oxygenavailability(68).Inthelaboratoryundernutrient-richcon- ditions,bacterialmonoculturesmayformthinlayerbiofilms;however,eventhesebio- filmsarenotuniformintheirstructure.Forexample,thinlayeredbiofilmsproduced byP.aeruginosaoftencontainbacteriadistributedoverasurfaceinterdispersedwith uncolonizedregions(Figs.2and3),andthesespacesareasimportanttoabiofilmas the regions containing the bacteria. Dalton et al. (69) showed that a marine bacterium, Psychrobacter sp, SW5, produced a tightly packed multilayered biofilm on a hydrophobic surface (silanized glass). In contrast, the biofilm formed on a hydrophilic surface (glass) was composed of multicellular chains arranged in a more open architecture with greater distances between the chains of bacteria. The more open biofilm structure may improve nutrient flux and availability; however, it may have a negative effect on other processes such as plasmid transfer, nutrient exchange, and effects of signaling molecules (70). Copyright © 2002 Marcel Dekker, Inc. Figure 2 A scanning electron micrograph of a P. aeruginosa biofilm formed on a silastic surface over 48 h. This biofilm has thick regions visible here and areas that are only sparsely covered with cells. Figure 3 A transmission electron micrograph of a cross section of a P. aeruginosa biofilm on a silastic surface demonstrating the cell distribution and biofilm thickness. Copyright © 2002 Marcel Dekker, Inc. Figure 4 A schematic illustrating some of the variability in biofilm formation under different flow conditions. In aerial view: B, biofilm clusters, shading, biofilm thickness; S, streamer structures; R, ripples; dashed arrows, oscillation with flow; bold arrows, direction of flow around the channels and biofilm clusters. (Based on Ref. 38.) Stoodley et al. (38) showed that a mixed culture biofilm grown under laminar flow conditions was ‘‘patchy’’ in that it consisted of rounded clusters of cells up to 100 µm in diameter separated by interstitial voids containing only a thin dispersion of single cells on the surface. Biofilms grown in turbulent flow conditions were also patchy but consisted of migratory ripple-like patches and elongated tapered colonies termed streamers, which oscillated in the direction of flow (38). Figure 4 is a schematic representation of the differ- ent structures under these flow conditions. In addition to flow dynamics, the biofilm struc- ture was affected by changing nutrient conditions. When the glucose concentration was increased from 40 to 400 mg L Ϫ1 , there was a parallel increase in biofilm thickness from 30 to 130 µm over a 2-day period (38). However, 10 hours after the addition of glucose, migratory ripple-like structures had disappeared and the streamers became rounded to form larger porous structures. When the glucose concentration was reduced to the original concentration, the migratory ripple formation was again observed after 2 days. This may be indicative of the biofilm responding to a decrease in nutrient availability, thereby in- creasing its surface area and thus contact with the bulk fluid. V. WHERE ARE BIOFILMS FOUND? Biofilms are ubiquitous and may be beneficial or detrimental, depending on where they are found. Beneficial biofilms are those actively employed in processes such as wastewater and drinking water treatment (71). Slimy adherent microbial populations on the surface Copyright © 2002 Marcel Dekker, Inc. ofrocks(tricklingfilter)orassociatedwitharotatingbiologicalcontactor(biodisk) areusedintheremovaloftheorganiccarbonduringsewagetreatment(72).Wastewater ispassedoverthesurfacecontainingtheadherentmicrobialcommunities,formedof primarilyslimeproducingZooglearamigeraandotherbacteria(72).ThethickEPS matrixcanretainalargenumberofotherorganismstoproduceaconsortiumthatisable toabsorbandutilizethedissolvedorganiccarbonpresentinthewater.Similarsystems havebeenusedforbiodegradationandremediationofindustrialwastewaters.Intheenvi- ronment,naturalselectionfavorsmicrobialcommunitiesthatcansurviveandgrowby utilizingthewasteasnutrients.However,thisisoftenaslowprocess.Studiesareunder wayintomethodsforincreasingthepopulationofbiodegradingorganismsatcontam- inatedsitesbyenrichmenttechniquesandimmobilizationoftheorganismstosubstrata. Usingimmobilizedcommunitiesinbiofilmsismoreadvantageousbecausehighercon- centrationsoftoxiccompoundscanbeappliedandtheyarelesssusceptibletowashout underhighfloworloading(73).Indrinkingwaterpurificationsystems,sandfilterscon- tainingmicrobialcommunitiesareusedtoremovepotentialpathogensbytrappingthem withintheEPSmatrixofthebiofilm(72).Inmostinstances,however,considerableprob- lemsareassociatedwithbiofilmgrowthorbiofoulinginindustrialprocessesandcost industryasignificantamountofmoneytodevelopcontrolregimens(74).Inwaterdistribu- tionsystems,biofilmscausecorrosionanddegradethequalityofthewaterthroughmicro- bialby-products.Biofilmsmayalsoharborpathogensthatputconsumersandworkersat risk.Inthefoodanddrinkindustry,biofilmscausecontaminationandspoilageofthe product. Oneofthemostcommonoccurrencesofabiofilmcommunityisdentalplaque, whichhasbeenstudiedfornearly300years(75).Over500differentmicrobialspecies havebeenidentifiedindentalplaque(76).Whereasnormalmicrobialfloracanexistin themouthwithoutcausinganyproblems,whenpathogensarepresentthereisapotential forperiodontaldisease.Thisbiofilmexemplifiescooperationandcoexistenceinacomplex microbialconsortiuminresponsetocontinualenvironmentalchanges.Oneexampleof thiswithinthedentalbiofilmisthepresenceoftheobligateanaerobeFusobacterium nucleatum,whichaggregateswithbothaerobesandanaerobeswithinamicrobialpopula- tion.Thepresenceofthisorganismaidsinthesurvivalofobligateanaerobesbypromoting aggregationinassociationwithaerobesthatremoveoxygenfromtheimmediateenviron- ment,therebycreatingalocalizedanaerobicregion.Bradshawandassociates(76)found thatwithoutF.nucleatumpresentinthemicrobialconsortium,theanaerobicpopulation wassignificantlydecreased.Therefore,withinthisparticularmicrobialcommunity,bacte- riainteractwitheachothertocreatesuitablemicroenvironmentsthatsupportthegrowth ofadiversemicrobialpopulationthatoftenwouldnotsurviveasmonoculturesinthe sameenvironment(76). Otherfrequentlystudiedbiofilmsarethosefoundinaquatichabitats,includingfresh- water,groundwater,andmarineenvironments,wherethemicroorganismsareattachedto abioticorbioticsurfaces(Fig.5).Thesebiofilmsincludealargenumberofbacteriaand unicellular marine organisms. However, there are many other habitats that are currently being investigated with respect to microbial adhesion and biofilms, including soil particles (77,78), plant surfaces (25,79), and animal guts (80). Microbial adhesion and physiological processes are much more difficult to investigate in these habitats because of their diversity and range in conditions. These habitats are divided into aquatic and nonaquatic environ- ments and are discussed separately. Copyright © 2002 Marcel Dekker, Inc. Figure 5 A scanning electron micrograph of a biofilm formed on a glass slide immersed in pond water. This multispecies biofilm demonstrates the diverse population, variable structure, and debris present within a natural biofilm. VI. AQUATIC ENVIRONMENTS In fresh alpine rivers, there are nearly 1000 times more bacteria attached to surfaces (square centimeters) than are present as planktonic cells (ml) (1,81). Biofilms composed of bacteria and algae have been found on sediments and rock surfaces in both freshwater and marine ecosystems. The organisms synthesize large amounts of exopolymer material, creating a complex matrix that aids in sediment cohesion and stability in intertidal sedi- ments (82). In other instances, when the river is polluted and has high organic matter content, these biofilms may become so thick that they clog the river beds, creating drainage problems and stagnation (78). Microorganisms in aquatic environments adhere to inorganic rocks and clay particles as well as biological/organic surfaces. Although at times biofilms are also found on living marine animals (83) and plants (84), their surfaces have mechanisms that resist microbial adhesion and often remain free of biofilms. In some cases, however, a biofilm on a plant or animal surface is in a symbiotic relationship whereby the microorganisms enhance the growth of the higher organisms. In the highly integrated rhizobia–legume symbiosis, bio- film formation is preceded by recognition and attachment of the microorganisms to the root surface. Root colonization is often multifunctional in that the organisms aid in nutrient acquisition and also provide a protective environment for the plant. For example, the colonization of mangrove roots is believed not only to help with nitrogen fixation and solublization of phosphorus but also to protect mangroves growing in saline or brackish waters (85). Copyright © 2002 Marcel Dekker, Inc. Microbialmatsareexamplesofthicklylayeredbiofilmsofphotosyntheticmicro- organismsattachedtorocksandsedimentparticlesinaqueoushabitats(25).Theyare oftenfoundunderextremeenvironmentalconditions.Forexample,inthevicinityofdeep seahydrothermalvents,microorganismswithinbiofilmssurviveextremetemperatures (86,87).Hotspringsareanotherextremehabitatwherebothhightemperaturesandsulfide concentrationsharbormatscontaininglayersprimarilycomposedofArchaea,including sulfate-reducingpurplebacteria(e.g.,Chloroflexisspp.,Chromatiumspp.,Thiopediaro- seopersicinia)inassociationwithcyanobacteria(25).Additionalextremeenvironments wheremicrobialmatsmaybefoundincludehypersalinelakes(88),terrestrialdesertswith cyclicaldroughtanddesiccation,sodalakesandacidthermalwaterscontainingextreme pHconditions,andregionswithhighlevelsofultraviolet(UV)irradiation(88).Themicro- bialspeciesthatarefoundintheseextremeenvironmentsarelimitedtoprimarilycyano- bacteria(e.g.,OscillatoriaandSpirulinaspp.)andotherssuchasDesulfovibriospp.,Beg- giatoaspp.,andThiovulumspp.,withdifferingandvaryingdegreesoftolerance(89). Althoughmatsareprimarilycomposedofprokaryotes,otherorganisms,suchastheeukar- yoticCyanidiumsp.,havebeenfoundatpHlevelsbelow4.5(89).Studieshaveshownthat mostoftheorganismswithinamatareoftennotphysiologicallyadaptedtotheextreme environmentbutgrowthwithinlayersofathickbiofilmhelpsthemsurviveandfinda suitablemicroniche(89).Microbialmatsareagoodexampleoftheprotectivenatureof biofilmgrowthandthemethodwithwhichstratificationcanencouragenutrientavailability andcycling(90). Biofilmshavebeenobservedatotheraquaticinterfacesbesidesthoseatasolid– liquidinterface.Forexample,instagnantwaters,biofilmsaresometimesfoundattheair– liquidinterfaceandareoftenseenasbrownorgreenlayerscomposedofalgaeandother aquaticmicroorganisms.Anotherexampleisthewaxytypebiofilmattheair–liquidinter- faceformedfromtherugosephenotypeofVibriocholeraeisolatedfromstarvationme- dium(91).Theinterfacebetweenjetfuelsandwatercanalsoharborbiofilmgrowth,such asthefungusCladosporiumresinae(92). VII.NONAQUATICENVIRONMENTS Althoughbiofilmshaveoftenbeenstudiedinaquaticenvironments,morerecentstudies haveshownthatmicroorganismswithinthickEPSmatricesorbiofilmsarealsofoundin nonaquaticenvironmentssuchastherhizosphere(Chapter4),soil,andsubsurfaceenviron- ments(93,94).Oneofthemorecomplexenvironmentsisthesoilecosystem,withitsmany differentparticlesandporespaces(95).Microorganismsinthesoiladheretosurfacessuch asinorganicsolidparticles,humicmatter,plantmaterial(roots),andmicrofauna.Plants providelargeamountsofcarbonandothernutrientstoencouragemicrobialgrowthinthe vicinityoftheroots,and,inturn,themicroorganismsfixnitrogen,assisttheplantin adsorptionofnutrientsfromthesoil,andprotecttherootsagainstpathogens.Another exampleofanonaquaticbiofilmisthecolonizationoftheleavesofplants—thephyllo- sphere(96;Chapter6).Thesebiofilmsconsistofadiversepopulationofmicroorganisms, including gram-positive and gram-negative bacteria, yeasts, and filamentous fungi, sup- ported within extensive exopolymer matrices (96,25). The primary component of biofilms is the EPS matrix produced by the bacteria. In nonaquatic environments, the EPS matrix is of primary importance for microbial survival since they experience intermittent flux of nutrients and water. Roberson and Firestone Copyright © 2002 Marcel Dekker, Inc. [...]... β-N-acetylgalactosaminidase, - and β-mannosidase, and α-fucosidase (116) Other bacteria then produce proteolytic enzymes, such as glyprodiamino peptidase, which catalyzes the degradation of proteins into peptides and amino acids The degradation of these complex molecules requires cooperative and synergistic enzymic interactions within the microbial community and, inevitably, bacterial diversity In turn, the resultant... hydrolytic enzymes into the soil, and these enzymes are capable of hydrolyzing pesticides Degradation by fungal enzymes may be due to less specific enzymes, as in the case of lignin-degrading enzyme systems However, the ability of fungi to degrade a wide range of pesticides is believed to be related to the structural similarity of lignin to the pesticides The degradation of inorganic minerals and the precipitation... exopolysaccharide and less protein under low-water conditions than when growing in a water-rich environment The polysaccharide adsorbs large amounts of water, thus reducing the rate of drying and protecting the cells from desiccation This suggests that biofilms are important for the survival of microorganisms in the soil Mucoid strains of Escherichia coli, Acinetobacter calcoaceticus, and Erwinia stewartii... Williams Investigation of quorum sensing in Aeromonas hydrophilia biofilms formed on stainless steel In: J Wimpenny, P Gilbert, J Walker, M Brading, R Bayston, eds Biofilms: The Good, the Bad and the Ugly Cardiff: BioLine, 1999, pp 209–222 63 MG Brading, J Boyle, HM Lappin-Scott Biofilm formation in laminar flow using Pseudomonas fluorescens EX101 J Ind Microbiol 15:297–304, 1995 64 Z Lewandowski, G Walser In uence... iron-oxidising protein Appl Environ Microbiol 58:450–454, 1992 108 G Southam, FG Ferris, TJ Beveridge Mineralized bacterial biofilms in sulphide tailings and in acid mine drainage systems In: HM Lappin-Scott, JW Costerton, eds Microbial Biofilms Cambridge: Cambridge University Press, 1995, pp 148–170 109 G Southam, TJ Beveridge Enumeration of Thiobacilli within pH-neutral and acidic mine tailings and their... oxygen They also produce degrading enzymes that can catalyze the degradation of polysaccharides, glycoproteins, and complex macromolecules encountered in the oral cavity to smaller compounds usable by the microflora For example, some of the enzymes known to be produced by dental microorganisms for the hydrolysis of polysaccharide components include β-galactosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase,... Watson, K Schilling The effect of the conditioning films on adhesion In: J Wimpenny, P Handley, P Gilbert, HM Lappin-Scott, eds The Life and Death of Biofilm Cardiff: BioLine, 1995, pp 47–52 18 PC Griffith, M Fletcher Hydrolysis of protein and model dipeptide substrates by attached and non-attached marine Pseudomonas sp Strain NCIMB 2021 Appl Environ Microbiol 57: 2186–2191, 1991 19 P Vandevivere, DL Kirchman... In: J Wimpenny, P Handley, P Gilbert, HM Lappin-Scott, M Jones, eds Biofilms Community Interactions and Control Cardiff: BioLine, 1997, pp 133–141 22 LC Skillman, IW Sutherland, MV Jones Co-operative biofilm formation between two species of Enterobacteriaceae In: J Wimpenny, P Handley, P Gilbert, HM Lappin-Scott, M Jones, eds Biofilms Community Interactions and Control Cardiff: BioLine, 1997, pp 119 127 ... extracellular enzymes, either secreted or arising from dead and lysing cells, are found in the biofilm matrix Biofilms represent a dynamic and heterogeneous environment; therefore, localized (and low) concentrations of enzymes have been difficult to detect With the current development of more sensitive microscopy methods, reporter gene technology, molecular biology, and nanotechnology, investigating single-cell and. .. reviews (120 ,121 ) and textbooks (122 124 ) have been published on current techniques to study microbial adhesion and biofilm formation in natural environments This has provided a large resource for future studies into enzymes within biofilms Indirect evidence suggests that there are a number of different processes that involve microbial enzymes and that they occur within biofilms but do not occur in planktonic . systems, lasR-lasI and rhlR-rhlI. The lasI and rhlI gene products are involved in the synthesis of two different AHL molecules, N-(3-oxododecanoyl)-l- homoserine lactone and N-buytryl-l-homoserine lactone,. Inc. ofrocks(tricklingfilter)orassociatedwitharotatingbiologicalcontactor(biodisk) areusedintheremovaloftheorganiccarbonduringsewagetreatment(72).Wastewater ispassedoverthesurfacecontainingtheadherentmicrobialcommunities,formedof primarilyslimeproducingZooglearamigeraandotherbacteria(72).ThethickEPS matrixcanretainalargenumberofotherorganismstoproduceaconsortiumthatisable toabsorbandutilizethedissolvedorganiccarbonpresentinthewater.Similarsystems havebeenusedforbiodegradationandremediationofindustrialwastewaters.Intheenvi- ronment,naturalselectionfavorsmicrobialcommunitiesthatcansurviveandgrowby utilizingthewasteasnutrients.However,thisisoftenaslowprocess.Studiesareunder wayintomethodsforincreasingthepopulationofbiodegradingorganismsatcontam- inatedsitesbyenrichmenttechniquesandimmobilizationoftheorganismstosubstrata. Usingimmobilizedcommunitiesinbiofilmsismoreadvantageousbecausehighercon- centrationsoftoxiccompoundscanbeappliedandtheyarelesssusceptibletowashout underhighfloworloading(73).Indrinkingwaterpurificationsystems,sandfilterscon- tainingmicrobialcommunitiesareusedtoremovepotentialpathogensbytrappingthem withintheEPSmatrixofthebiofilm(72).Inmostinstances,however,considerableprob- lemsareassociatedwithbiofilmgrowthorbiofoulinginindustrialprocessesandcost industryasignificantamountofmoneytodevelopcontrolregimens(74).Inwaterdistribu- tionsystems,biofilmscausecorrosionanddegradethequalityofthewaterthroughmicro- bialby-products.Biofilmsmayalsoharborpathogensthatputconsumersandworkersat risk.Inthefoodanddrinkindustry,biofilmscausecontaminationandspoilageofthe product. Oneofthemostcommonoccurrencesofabiofilmcommunityisdentalplaque, whichhasbeenstudiedfornearly300years(75).Over500differentmicrobialspecies havebeenidentifiedindentalplaque(76).Whereasnormalmicrobialfloracanexistin themouthwithoutcausinganyproblems,whenpathogensarepresentthereisapotential forperiodontaldisease.Thisbiofilmexemplifiescooperationandcoexistenceinacomplex microbialconsortiuminresponsetocontinualenvironmentalchanges.Oneexampleof thiswithinthedentalbiofilmisthepresenceoftheobligateanaerobeFusobacterium nucleatum,whichaggregateswithbothaerobesandanaerobeswithinamicrobialpopula- tion.Thepresenceofthisorganismaidsinthesurvivalofobligateanaerobesbypromoting aggregationinassociationwithaerobesthatremoveoxygenfromtheimmediateenviron- ment,therebycreatingalocalizedanaerobicregion.Bradshawandassociates(76)found thatwithoutF.nucleatumpresentinthemicrobialconsortium,theanaerobicpopulation wassignificantlydecreased.Therefore,withinthisparticularmicrobialcommunity,bacte- riainteractwitheachothertocreatesuitablemicroenvironmentsthatsupportthegrowth ofadiversemicrobialpopulationthatoftenwouldnotsurviveasmonoculturesinthe sameenvironment(76). Otherfrequentlystudiedbiofilmsarethosefoundinaquatichabitats,includingfresh- water,groundwater,andmarineenvironments,wherethemicroorganismsareattachedto abioticorbioticsurfaces(Fig.5).Thesebiofilmsincludealargenumberofbacteriaand unicellular. hydrolytic enzymes into the soil, and these enzymes are capable of hydrolyz- ing pesticides. Degradation by fungal enzymes may be due to less specific enzymes, as in the case of lignin-degrading enzyme

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  • Enzymes in the Environment: Activity, Ecology and Applications

    • Table of Contents

      • Chapter 12: Microbes and Enzymes in Biofilms

        • I. INTRODUCTION

        • II. WHAT IS A BIOFILM?

        • III. BIOFILM LIFE CYCLE

        • IV. FUNCTION OF BIOFILM STRUCTURE AND ARCHITECTURE

        • V. WHERE ARE BIOFILMS FOUND?

        • VI. AQUATIC ENVIRONMENTS

        • VII. NONAQUATIC ENVIRONMENTS

        • VIII. ENZYMES IN BIOFILMS

        • IX. CONCLUSIONS

        • REFERENCES

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