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

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.. This is reflec

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

However, it is important to be aware that in many instances, particularly in natural environ-ments, biofilms consist not only of bacteria but of fungi (9,10), yeasts (11), algae (12), and protozoa (13) Nonetheless, much of the published literature concentrates on bacterial biofilms, although with the increased emergence of infections (11) and problem associated with fungal biofilms (10) the importance of studying more complex mixed biofilms con-taining both prokaryotes and eukaryotes has recently been realized (14)

III BIOFILM LIFE CYCLE

It is often easier to understand what a biofilm is in terms of the events that lead to its formation Most studies of biofilms in natural environments have concentrated on events

at solid–liquid interfaces, the colonization of a submerged abiotic surface is depicted in Fig 1 Upon immersion of a nonbiological material, such as glass or silica, the surface becomes coated rapidly with a layer of proteinacous material called a conditioning layer (15–17) Ions and other nutrient sources accumulate at the interface, giving rise to higher microenvironment concentrations that will attract microorganisms from the nutrient- and energy-starved liquid phase to the surface (18) Bacteria, which are often the first coloniz-ers, begin to synthesize copious amounts of exopolysaccharide (EPS) material initiated

Figure 1 A schematic illustrating the life cycle of a biofilm

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

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 asγ-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-N-(3-oxododecanoyl)-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

rhlI or just lasI gene products were able to adhere to a glass surface but were not able

to differentiate into thick multilayered biofilms This system also regulates the expression

of other factors (59), such as type IV pili in P aeruginosa (twitching motion), which have

also been found to influence the differentiation of adherent monolayers to thick biofilm structures (60) An increasing number of bacteria are being found to be associated with new density-dependent communication molecules, both in the laboratory and in situ (53,61) For example, the presence of AHLs was detected in naturally occuring aquatic

biofilms on stones by introducing Agrobacterium tumefaciens A136 with a lacZ fusion

as an indicator organism (61) However, it would be nai¨ve to assume that adhesion and biofilm formation rest solely on the production of chemical signals (52,62) Other research has shown that although AHLs play an important role in the accumulation of cells on the surface and the formation of biofilms, the overall structure of biofilms growing in aqueous environments during the early stages of colonization is determined largely by the flow conditions (37,63)

There are two main delimiting factors that influence the structure of a biofilm in aqueous environments: flow rate and nutrient availability Flow can be categorized as laminar or turbulent Laminar flow is the smooth flow of fluid through a pipe or duct In contrast, when flow becomes erratic and irregular it is described as turbulent Lewandow-ski and Walser (64) found that the thickness of a mixed culture biofilm was at a maximum near the transition between laminar and turbulent flows However, many different biofilm structures have been observed, often explained by examining the mass transfer properties

of the bulk liquid In a turbulent system there is good mixing of nutrients, and the bulk liquid comes into contact with large proportions of the biofilm where uptake of nutrients can take place In comparison, under laminar flow conditions there is poor mixing of nutrients in the bulk liquid, limiting nutrient availability Indeed, Lewandowski and Walser (64) hypothesized that there was an optimal flow rate below which biofilm accumulation was limited by mass transfer and above which biofilm accumulation was limited by contin-ual cell detachment Many of the recent confocal microscope studies have shown that a biofilm consists of microcolonies of bacteria in a dense EPS matrix with less dense intersti-tial voids or water channels (38,65,66) Using microelectrodes (50) it has been demon-strated that these interstitial voids contain greater concentrations of nutrients than the microcolonies and thus can act as transport channels for nutrients and the removal of by-products, making them an essential structure in any biofilm (66) Others (67) have shown that there were fewer channels, which were less defined in a maturing biofilm Reduction

of these channels would decrease the mass transport characteristics within the bulk liquid phase, thereby controlling growth rate of the microbes within the biofilm due to reduced nutrient and, possibly, oxygen availability (68) In the laboratory under nutrient-rich con-ditions, bacterial monocultures may form thin layer biofilms; however, even these bio-films are not uniform in their structure For example, thin layered biobio-films produced

by P aeruginosa often contain bacteria distributed over a surface interdispersed with

uncolonized regions (Figs 2 and3), and these spaces are as important to a biofilm as 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)

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

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 100µm

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⫺1, there was a parallel increase in biofilm thickness from

30 to 130µm 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

of rocks (trickling filter) or associated with a rotating biological contactor (biodisk) are used in the removal of the organic carbon during sewage treatment (72) Wastewater

is passed over the surface containing the adherent microbial communities, formed of

primarily slime producing Zooglea ramigera and other bacteria (72) The thick EPS

matrix can retain a large number of other organisms to produce a consortium that is able

to absorb and utilize the dissolved organic carbon present in the water Similar systems have been used for biodegradation and remediation of industrial wastewaters In the envi-ronment, natural selection favors microbial communities that can survive and grow by utilizing the waste as nutrients However, this is often a slow process Studies are under way into methods for increasing the population of biodegrading organisms at contam-inated sites by enrichment techniques and immobilization of the organisms to substrata Using immobilized communities in biofilms is more advantageous because higher con-centrations of toxic compounds can be applied and they are less susceptible to washout under high flow or loading (73) In drinking water purification systems, sand filters con-taining microbial communities are used to remove potential pathogens by trapping them within the EPS matrix of the biofilm (72) In most instances, however, considerable prob-lems are associated with biofilm growth or biofouling in industrial processes and cost industry a significant amount of money to develop control regimens (74) In water distribu-tion systems, biofilms cause corrosion and degrade the quality of the water through micro-bial by-products Biofilms may also harbor pathogens that put consumers and workers at risk In the food and drink industry, biofilms cause contamination and spoilage of the product

One of the most common occurrences of a biofilm community is dental plaque, which has been studied for nearly 300 years (75) Over 500 different microbial species have been identified in dental plaque (76) Whereas normal microbial flora can exist in the mouth without causing any problems, when pathogens are present there is a potential for periodontal disease This biofilm exemplifies cooperation and coexistence in a complex microbial consortium in response to continual environmental changes One example of

this within the dental biofilm is the presence of the obligate anaerobe Fusobacterium nucleatum, which aggregates with both aerobes and anaerobes within a microbial

popula-tion The presence of this organism aids in the survival of obligate anaerobes by promoting aggregation in association with aerobes that remove oxygen from the immediate environ-ment, thereby creating a localized anaerobic region Bradshaw and associates (76) found

that without F nucleatum present in the microbial consortium, the anaerobic population

was significantly decreased Therefore, within this particular microbial community, bacte-ria interact with each other to create suitable microenvironments that support the growth

of a diverse microbial population that often would not survive as monocultures in the same environment (76)

Other frequently studied biofilms are those found in aquatic habitats, including fresh-water, groundfresh-water, and marine environments, where the microorganisms are attached to abiotic or biotic surfaces (Fig 5) These biofilms include a large number of bacteria and 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

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)

Microbial mats are examples of thickly layered biofilms of photosynthetic micro-organisms attached to rocks and sediment particles in aqueous habitats (25) They are often found under extreme environmental conditions For example, in the vicinity of deep sea hydrothermal vents, microorganisms within biofilms survive extreme temperatures (86,87) Hot springs are another extreme habitat where both high temperatures and sulfide concentrations harbor mats containing layers primarily composed of Archaea, including

sulfate-reducing purple bacteria (e.g., Chloroflexis spp., Chromatium spp., Thiopedia ro-seopersicinia) in association with cyanobacteria (25) Additional extreme environments

where microbial mats may be found include hypersaline lakes (88), terrestrial deserts with cyclical drought and desiccation, soda lakes and acid thermal waters containing extreme

pH conditions, and regions with high levels of ultraviolet (UV) irradiation (88) The micro-bial species that are found in these extreme environments are limited to primarily

cyano-bacteria (e.g., Oscillatoria and Spirulina spp.) and others such as Desulfovibrio spp., Beg-giatoa spp., and Thiovulum spp., with differing and varying degrees of tolerance (89).

Although mats are primarily composed of prokaryotes, other organisms, such as the

eukar-yotic Cyanidium sp., have been found at pH levels below 4.5 (89) Studies have shown that

most of the organisms within a mat are often not physiologically adapted to the extreme environment but growth within layers of a thick biofilm helps them survive and find a suitable microniche (89) Microbial mats are a good example of the protective nature of biofilm growth and the method with which stratification can encourage nutrient availability and cycling (90)

Biofilms have been observed at other aquatic interfaces besides those at a solid– liquid interface For example, in stagnant waters, biofilms are sometimes found at the air– liquid interface and are often seen as brown or green layers composed of algae and other aquatic microorganisms Another example is the waxy type biofilm at the air–liquid

inter-face formed from the rugose phenotype of Vibrio cholerae isolated from starvation

me-dium (91) The interface between jet fuels and water can also harbor biofilm growth, such

as the fungus Cladosporium resinae (92).

VII NONAQUATIC ENVIRONMENTS

Although biofilms have often been studied in aquatic environments, more recent studies have shown that microorganisms within thick EPS matrices or biofilms are also found in nonaquatic environments such as the rhizosphere (Chapter 4), soil, and subsurface environ-ments (93,94) One of the more complex environenviron-ments is the soil ecosystem, with its many different particles and pore spaces (95) Microorganisms in the soil adhere to surfaces such

as inorganic solid particles, humic matter, plant material (roots), and microfauna Plants provide large amounts of carbon and other nutrients to encourage microbial growth in the vicinity of the roots, and, in turn, the microorganisms fix nitrogen, assist the plant in adsorption of nutrients from the soil, and protect the roots against pathogens Another example of a nonaquatic biofilm is the colonization of the leaves of plants—the phyllo-sphere (96;Chapter 6) These biofilms consist of a diverse population of microorganisms, 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