Enzymes in the Environment: Activity, Ecology and Applications - Chapter 12 ppsx

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
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(93) reported that a soil Pseudomonas sp. produced more exopolysaccharide and less pro-
tein 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 pro-
tecting the cells from desiccation. This suggests that biofilms are important for the survival
of microorganisms in the soil. Mucoid strains of Escherichia coli, Acinetobacter calcoace-
ticus, and Erwinia stewartii exhibited up to 35% greater survival under desiccating condi-
tions compared with isogenic nonmucoid mutants (97). The EPS matrices not only are
necessary for the microbes to survive low relative humidity and desiccation but may also
have a role in plant microbe symbiosis and plant pathogenesis (98). These polysaccharides
have also been shown to be virulence factors on plant pathogens such as Pseudomonas,
Erwinia, and Xanthomonas spp., however, they are also important in the symbiosis of

Rhizobia spp. (99). Both events depend on bacterial adhesion to surfaces and the formation
of a temporary biofilm.
VIII. ENZYMES IN BIOFILMS
A number of different processes that occur within a microbial biofilm contribute to the
creation of a heterogenous, dynamic environment. These processes, among many others,
include cycling and exchange of nutrients, plasmid transfer, communication via chemical
signals, and frequent deterioration of the surface (corrosion or degradation). The biofilm
microorganisms respond by having different physiological characteristics, metabolic activ-
ity, and growth rates from those of unattached organisms growing outside the biofilm
(e.g., soil aqueous phase, river water) (100).
Differences in the enzyme activities occurring within biofilms could account for
some of the differences between biofilm and planktonic cells. Although there have been
only a few reports on enzyme activities within biofilms, studies undertaken on samples
from natural environments suggest that many biofilm processes would be mediated by
enzymes. The bioremediation of pollutants by bacteria attached to soil particles, on the
surface of biodegradable materials, and on rocks in rivers or trickling filters exemplifies
enzymatic activity while bacteria are within biofilms. Potential substrates concentrated/
precipitated at the surface or diffusing into the biofilm are accessible to the attached micro-
organisms in a way that is not possible for planktonic bacteria. Vetter and Deming (101)
suggested that bacteria degrade both particulate and dissolved organic carbon by secreting
extracellular enzymes, as they are too large for direct uptake into the cell. In natural aquatic
environments it is difficult to imagine that these enzymes may have any substantial effects
except when localized near the carbon source, such as in adherent cells and biofilms (101).
This is supported by laboratory studies in which bacterial enzyme activity was important
in colloidal organic matter degradation by a biofilm community in a reactor (102). This
book identifies many processes in natural environments, and since biofilms are often found
in those environments, the enzymatic processes described would therefore be relevant
here.
Biofilms that accumulate on suspended particles in rivers, lakes, and marine systems
are considered beneficial to their environments as they play an essential role of purification

by removing suspended, settled, and dissolved organic material. Freeman et al. (103) de-
scribed river biofilms as a trophic link between dissolved nutrients in the water column
and the higher trophic levels of the ecosystem. Natural biofilms are able to biodegrade
organic compounds and transform inorganic compounds as part of their natural metabolic
Copyright © 2002 Marcel Dekker, Inc.
pathway. It is assumed that in a natural environment, such as the soil habitat, bacteria and
fungi do form biofilms and therefore the degradation of environmental pollutants occurs
in a biofilm state. The role of bacterial–fungal biofilms in the degradation of environmental
pollutants is not well understood. It is thought that the success of fungi in the role of
biodegradation is mainly attributable to the production of extracellular enzymes. Manga-
nese peroxidases and lignin peroxidases produced by Phanerochaete chrysosporium play
a key role in the detoxification and decolorization of pulp bleach plant effluent (104). A
1993 report has shown that enzymes produced by fungi and bacteria would act simulta-
neously in the biodegradation of pesticides under field conditions (105). Levanon (105)
described how the mineralization of alachlor and atrazine was mainly due to fungal activity
and that the mineralization of carbofuran and malathion was mainly due to bacterial activ-
ity. The bacterial enzyme identified to degrade malathion was a carboxylesterase. Fungi
release many 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 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 of toxic metals result
from enzymatic activity of microorganisms within biofilms. The degradation of mining
tailings by Thiobacillus ferrooxidans occurs when reduced iron and sulfur compounds in
ores or wastes are converted to sulfuric acid and iron (III) (106). The oxidation of these
metals is a chemical process enhanced by microbial biocatalysts, that is, enzymes that
encourage the flow of electrons (107,108). With Thiobacillus ferrooxidans, these enzymes
are believed to be located on the cell envelope; therefore, it is important that the minerals
are closely associated with the bacterium (109). Although the details of the bacterial–

mineral interaction are not fully understood, it is known that the bacterium colonizes the
mineral with the aid of its lipopolysaccharide (110). Studies have shown that even in
environments that solubilize metals, bacteria precipitate and accumulate metals within
their matrix (110). This is a natural system for cleaning the surrounding environment of
toxic metals so that other organisms and bacteria may survive.
Some surfaces act as both a colonization surface and a nutrient source. Often en-
zymes are required to degrade substances to a soluble and low-molecular-weight form that
can be utilized by the bacterium as a carbon and nitrogen source. Enzymatic degradation of
organic materials is often preceded by bacterial attachment and biofilm formation. A pri-
mary example is the hydrolysis of cellulose by anaerobic, thermophylic Clostridium ther-
mocellum (111). This organism produces an extracellular protein complex called a cellulo-
some that has a dual purpose; it both binds to and degrades cellulose. The cellulosome
produces catabolic enzymes that solubilize cellulose substances, primarily to the disaccha-
ride cellobiose, which then can be taken up by the bacterium as a nutrient source (111).
It is presumed that biofilm formation is required to position the bacterium close to the
substrate, thereby concentrating the enzyme for hydrolytic activity. Similar studies have
been undertaken on other cellulose-degrading microorganisms such as Fibrobacter succi-
nogenes and Ruminococcus albus (112).
In many cases the microorganism uses the degraded compounds as a nutrient source;
however, this process also liberates nutrients and ions for other bacteria in the biofilm
community as well as for higher organisms such as plants or animals. For example, bio-
films in the guts of ruminant animals help enzymatically degrade feed particles into sub-
stances that the animal is able to utilize (80). It is likely that each insoluble substance,
Copyright © 2002 Marcel Dekker, Inc.
such as cellulose, amylose, starch, and proteins, has its own biofilm population. The bacte-
ria produce the appropriate suite of extracellular degradative enzymes that convert the
substances into soluble nutrients available for the uptake by both bacteria and animal (80).
Many bacteria and fungi grow in association with a surface, and this process often
results in the enzymic deterioration of that surface. The surface can therefore act as a
substrate. For example, polyvinyl chloride (PVC) is largely composed of carbon, hydro-

gen, and chlorine, although these are unlikely to be available to a colonizing population;
the added UV stabilizers and colourant may well influence attachment. A 1999 report
(113) has shown that plasticizers increase the adhesion of the deteriogenic fungus Aureo-
basidium pallulans to PVC. The loss of plasticizers in PVC due to microbial degradation
results in brittleness, shrinkage, and ultimate failure of the PVC in its intended application
(113). The production of extracellular esterase by A. pallulans is thought to be instrumental
the biodeterioration of PVC (114). Stachybotrys chartarum (atra) is a saprophytic green–
black fungus that is found in many habitats and grows particularly well in high-cellulose
material, damp wallpaper, fiber board, lint, and dust. It produces potent macrocyclic thri-
chothecene toxins (satratoxins H, G, F; roridin E; verrucarin J; and trichoverrols A and
B), and is associated with chronic health problems, and is implicated in ‘‘sick building
syndrom.’’
Dental biofilms are highly complex consortia that cooperate via nutrient cycling. The
aerobic or facultative organisms often aid the survival of anaerobic bacteria by utilizing the
oxygen (115); thus the anaerobes are able to survive transient levels of 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 in-
clude β-galactosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, α- 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 nutrients released support a diverse range of organisms
with different nutrient requirements (116).
Microorganisms are attracted to an inert interface as a result of the increased concen-
tration of nutrients at that interface, including some ions and larger molecules such as
amino acids and glycoproteins, which may not be easily transported into the cell. Dissolved
organic matter, ions (e.g., NH

4
ϩ
) and metals that serve as energy sources for chemolitho-
trophs (e.g., Fe
2ϩ
,Mn
2ϩ
) may initially become adsorped at a surface and also by ionic and
physical entrapment within the exopolymer. Both these processes concentrate nutrients in
the proximity of the microorganisms. Extracellular enzymes produced by some microor-
ganisms hydrolyze and degrade organic ions into soluble products available for their or
other organisms’ use. For example, an exoprotease producing Pseudomonas sp. strain S9
was used to determine adhesion by taking advantage of its degradation of ribulose-1,5-
bisphosphate carboxylase adsorbed to the surface (117). In many cases, enzymes within
biofilms are one of the normal consequence of microbial metabolism. The microenviron-
ments within a biofilm may provide the conditions in which enzyme induction and secre-
tion are stimulated. The activity of some of these enzymes outside the biofilm may be
restricted as a result of the comparatively low concentrations of substrates, inducers, and
other effector molecules outside the biofilm.
Copyright © 2002 Marcel Dekker, Inc.
Biofilm maintenance or thickness may be controlled by specific enzymes produced
by the bacterium in response to environmental stimuli. Boyd and Chakrabarty (118) dis-
covered a P. aeruginosa strain that produces alginate lyase and degrades alginate. This
may be a mechanism through which bacteria are detached from a biofilm. Similarly, Strep-
tococcus mutans produces a protein called surface protein-releasing enzyme that catalyzes
the release of bacteria from biofilms (119). This may be one method whereby organisms
become dissociated from the biofilm environment when nutrients are depleted.
IX. CONCLUSIONS
Direct evidence for the production of specific enzymes within biofilms is sparse, possibly
because of the limitations of research techniques. However, there is no doubt that extracel-

lular 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 small population
responses to environmental stimuli is possible. A number of useful reviews (120,121) and
textbooks (122–124) have been published on current techniques to study microbial adhe-
sion 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 cells. It is assumed that biofilm cells produce
enzymes in response to environmental stimuli in order to maintain the integrity of the
overall structure, composition, and activity of the biofilm.
This chapter has shown biofilms to have three primary functions with respect to
enzyme production and activity. First, biofilm formation places the bacterium close to the
substrate or the substratum where it is concentrated for which an enzyme may be specific.
In this case, the bacterium may be producing enzymes still associated with the cell wall
(i.e., mural enzymes) requiring attachment to the substrate for catalysis to occur. Second,
a biofilm matrix may concentrate the enzyme so that it may reach concentrations at which
the activity becomes relevant. Third, the biofilm may create an environment that induces
the production of a specific enzyme.
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