I

I. INTRODUCTION

Biofilms are abundant in a multitude of aquatic environments in which

they cover all kinds of inorganic and organic solid surfaces. Biofilms can be

defined as microorganisms attached to a surface and embedded in an extracellular

gellike matrix of polymeric substances. This matrix is excreted by

the microorganisms themselves and may be enriched by molecules adsorbed

from the surrounding water.

Aquatic Ecosystems: Interactivity of Dissolved Organic Matter

286 Helmut Fischer

Biofilms are important in many medical and industrial fields (e.g.,

Flemming, 1987; Blenkinsopp and Costerton, 1991; Marsh, 1995; Costerton

et al., 1999; Mittelman, 1999; Flemming and Wingender, 2001a). They are

major foci of the self-purification capacity of streams and rivers (Wuhrmann,

1974; Cazelles et al., 1991; Grischek et al., 1998; Pusch et al., 1998) and are

made use of in technical waste water treatment (e.g., Bryers and Characklis,

1990). A differentiation between various types of biofilms covering solid

surfaces and freely floating organic flocs and colloids is somewhat arbitrary.

In a broad sense, particles such as organic aggregates inhabited by bacteria

(e.g., marine snow) can be seen as motile biofilms and many of the properties

of biofilms on solid surfaces are also valid for these organic particles.

Research from the beginning of the twentieth century showed that the

addition of sterile soils or colloidal substances had stimulating effects on

various microbial processes in culture media (S6hngen, 1913). Intense research

on biofilm bacteria was conducted in the 1930s and 1940s when

interest was drawn toward natural systems (e.g., Henrici, 1933), and enhanced

growth of bacteria in small vessels compared with natural aquatic

environments was observed (ZoBell and Anderson, 1936; Heukelekian and

Heller, 1940). It was found then that "surfaces enable bacteria to develop in

substrates otherwise too dilute for growth" (Heukelekian and Heller, 1940).

ZoBell (1943) speculated that this ability of bacteria depends on the role

of surfaces in concentrating nutrients by adsorption, in providing resting

places for sessile bacteria, and in retarding the diffusion of exoenzymes and

hydrolyzates away from the cell. Despite this early interest, the pure culture

of bacterial isolates in suspensions has been the focus of most microbiological

research. Geesey et al. (1977, 1978) were the first to closely examine bacterial

biofilms in mountain streams and to state the numerical dominance of

biofilm bacteria in these systems. Lock et al. (1984) developed a structuralfunctional

model describing the role of river biofilms in the adsorption and

transformation of nutrients and placing these biofilms into the focus of ecological

research in running waters. More recently, a shift in paradigms can

be observed. The biofilm mode of living (in mixed microbial communities)

is now seen as the mode generally preferred for bacterial growth, and it was

stressed that these sessile populations predominate in almost all natural

ecosystems (Costerton et al., 1995; Costerton and Lappin-Scott, 1995). It is

therefore necessary to determine how bacteria function as successful members

of these interacting communities and as components in the environment

(Caldwell, 1995).

The development of biofilms is a succession of processes that can be

described in four steps (e.g., Characklis, 1990; Marshall, 1996):

1. Development of a conditioning film. This process should depend on

surface properties of the substratum and on the charge and polarity of the

sorbed matter. It is a relatively fast [half-saturation times of 5-72 s (Armstrong

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 287

and B/irlocher, 1989)] physical and chemical process, the kinetics of which

can be described by so-called sorption isotherms. By sorbing onto a surface,

large macromolecules may significantly alter their quarternary structure

[reviewed by Norde (1986)], which may also change their susceptibility to

enzymatic attack (Quiquampoix, 2000).

2. Attachment of bacteria. At low ionic strength of the medium ~ as in

many freshwaters~bacteria-surface interactions are controlled by the

effects of van der Waals attraction and electrostatic repulsion. At high ionic

strength ~ as in seawater ~ steric interactions between the outer cell surface

macromolecules and the substratum gain in importance (van Loosdrecht et

al., 1989; Rijnaarts et al., 1999). Additionally, flagellar and twitching motility

of bacteria was found to be essential in the process of attachment by bacteria

onto surfaces (Pratt and Kolter, 1998; O'Toole and Kolter, 1998). It seems

that extracellular polysaccharides of bacteria are not involved in the adhesion

process itself. However, bacterial extracellular polysaccharides are necessary

for the development of a biofilm and for the formation of microcolonies

(Allison and Sutherland, 1987; Hoyle et al., 1993).

3. Development of a polysaccharide matrix. Adhered bacteria are able to

produce polysaccharides that are not synthesized when they are in the pelagic

mode of growth. In the process of adhesion of Pseudomonas aeruginosa, the

algC and algD genes that control important enzymes in the alginate synthesis

pathway, are up-regulated (Davies et al., 1993; Hoyle et al., 1993).

For the same species it was also found that the formation of type IV pili

seems to be necessary to facilitate movement of bacteria along an abiotic

surface and to form microcolonies within a developing biofilm (O'Toole and

Kolter, 1998). The planktonic-biofilm transformation of several phenotypic

expressions of the bacterial genome are controlled by o factors of the RNA

polymerase that initiate transcription (Deretic et al., 1994; discussed in

Costerton et al., 1995).

4. Growth and detachment of bacteria. A highly dynamic equilibrium is

reached during biofilm maturation. In a flowing system, the hydrodynamic

drag will control the biofilm shape (Stoodley et al., 1999c) and eventually

lead to active detachment of bacteria or passive sloughing of parts of the

biofilm. Bacteria in biofilms are often larger and more active than their freeliving

counterparts. This is true not only for cell-specific activity, but also for

biomass specific activity, so that biofilm bacteria in their natural environment

also may exhibit shorter doubling times (Kjelleberg et al., 1982; Fletcher,

1986; Fischer and Pusch, 1999, 2001).

Much of this research was performed on monospecific biofilms in

medical research; it is probable, but not yet substantiated, that the results

obtained in these genetic studies can be transferred to natural multispecies

biofilms in aquatic environments. In aquatic systems, researchers mostly

deal with existing biofilms and with their relationships to the surrounding

288 Helmut Fischer

water. The focus of this chapter will therefore be on the sorption of

dissolved organic matter (DOM) onto and the transformations of DOM in

these preexisting biofilms.

II. BIOFILM STRUCTURE

Biofilms exhibit different morphologies depending on the environmental

conditions (Lock, 1993; van Loosdrecht et al., 1995; Wimpenny and

Colasanti, 1997; Stoodley et al., 1999a, b, c). In general, laboratory biofilms

with one or a few species of bacteria are thicker and less dense in nutrientrich

media than in nutrient-poor media, and they are thinner and denser under

high flow velocities than under low flow velocities (e.g., van Loosdrecht et

al., 1995; Beyenal and Lewandowski, 2000). Mixed species river biofilms

growing under high flow velocities of 52-59 cm/sec in an annular biofilm

reactor developed a ridged structure (Neu and Lawrence, 1997). The ridges

were orientated parallel to the direction of flow. These biofilms were highly

heterogeneous; the ridges consisted of individual bacteria and microcolonies

embedded in a complex colloidal matrix of polymers. The heterogeneous

morphological structure of biofilms is reflected by their diffusive characteristics,

as will be discussed in Section IIIB. Recently, it was hypothesized

that cell-cell signaling would also play an important role in determining

structural complexity of biofilms (Stoodley et al., 1999a) and a signaling

molecule (acyl homoserine lactone) that is instrumental in the formation of

biofilms of P. aeruginosa was identified (Davies et al., 1998).

A structural and functional model of a biofilm on an inert substratum

in an aquatic system (e.g., on sediment particles) is shown in Figure 1. This

biofilm exhibits an enlarged surface area with interstitial voids and lacunae

(deBeer et al., 1994). The formation of biofilm streamers and the flattening

of the biofilm under higher flow velocities (Stoodley et al., 1999c) are indicated

by the arrows at the left margin and on the top of the figure. Regions

of varying biofilm thickness are alternating. Regions of the biofilm composed

of extracellular polymeric substances (EPS) of different density are symbolized

by different shadings. Growth and detachment of bacteria and flocs of EPS

are depicted. DOM from the water column is sorbed, and additional DOM

is exuded by algae and released by particulate organic matter entrapped in

the biofilm. Organic matter is cleaved by extracellular enzymes, some of

which are deactivated as a function of time after production or in regions

close to the substratum. Bacteria and algae are interacting, as well as colonies

of syntrophic or competing bacteria.

In natural streambed biofilms, the biomass of colloidal carbohydrates

was found to be 5 times greater than the biomass of bacteria inhabiting the

biofilm (Hall and Meyer, 1998). The biofilm volume/bacterial volume ratio is

much higher, because the fraction of the biofilm volume actually occupied

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 289

Eucaryotic alga

Flow direction ~:~ Decaying euca~otic alga, POM

0 • Active bacteria

@ o Dormant bacteria

w~ DOC

~, il bl

-,~Z. . . . . . .of .a b.lo.film. .str.ea mer ~ ~v~ 6 Extracellular enzyme

e6;epe_.~.. .... Length ~ ~, Inactivated extracellular enzyme

.....~. o% OO 0 | "., ,

g ~ 0 0 0 ;

h~ o o°o 0 0 0 o, 0

FIGURE I Diagrammatic representation of the structure and function of bacterial biofilms.

Dissolved organic matter (DOM) is sorbed onto the biofilm (1), additional DOM is released

from algae and organic particles (2). The organic matter is cleaved by extracellular enzymes

(3). Interactions can occur between clones of syntrophic or competing bacteria (4). See Section

II for details.

with solids may be very small (Flemming and Wingender, 2001b). The

relatively low biomass therefore shows the gellike structure of the biofilm,

whose volume is to a large extent occupied by water.

III. SUPPLY OF DISSOLVED ORGANIC MATTER TO BIOFILM BACTERIA

The retention of DOM in microbial biofilms involves several processes:

(A) sorption of a DOM molecule to the biofilm, (B) diffusion into the biofilm,

organic matter), and (D) uptake and microbial utilization of the

DOM molecule.

A. Sorption of Dissolved Organic Matter onto Biofilms

To measure sorption of a dissolved substance (sorbate) to a solid surface

(sorbent), the dissolved substance is generally incubated with the solid for

a short time (seconds to minutes) at low temperatures. These conditions are

chosen to minimize biotic uptake of the dissolved compound (e.g., Henrichs

and Sugai, 1993). The partition coefficient

% adsorbed/gram of solid

Kp = % dissolved/milliliter of solution

then gives information about the proportion of the dissolved compound

that is adsorbed to the surface. Kp (often referred to as Ka, the distribution

290 HelmuFt ischer

coefficient) not only depends on the properties of the surface (e.g., organic

carbon content) and the dissolved compound (charge and polarity) but also

on the concentration of the dissolved compound in the solution and the

amount of the dissolved compound compared with the amount of solids

(Stumm, 1996). From the results of multiple adsorption experiments with

different concentrations of the sorbate, typical adsorption isotherms can be

calculated. These adsorption isotherms are linear in many environmental

situations because in sufficiently diluted systems solute-solute interactions

can be ignored in both the sorbent and the sorbate (Karickhoff, 1984). In

these cases, Kp is constant and the sorbed phase (S) can be calculated from

the concentration in solution (C):

S=Kp×C.

If solute-solute interactions occur (e.g., because of the presence of a limited

number of sorption sites), then sorption can be modeled by fitting the experimentally

derived isotherm to theoretical equations, the Freundlich isotherm

and the Langmuir isotherm

S=KxC n

S ._.

Q°xKxC

I+KxC'

where n is another constant, Q0 is the maximum sorptive capacity for

the sorbent, and K is a specific partition coefficient reflecting the extent of

sorption (Podoll and Mabey, 1987; Domenico and Schwartz, 1998). The

sorption characteristics can additionally be influenced by cations (e.g., Ca 2+)

in solution, which may compete with positively charged sorbates for adsorption

sites or which can enhance sorption of negatively charged sorbates due

to bridging effects with the sorbent (Tipping and Cooke, 1982; Armstrong

and B~irlocher, 1989; Henrichs and Sugai, 1993).

Working with the multitude of possible solid surfaces in a natural environment

is somewhat simplified by the similar physicochemical properties

of the microbial biofilm. In general, these biofilms exhibit a neutral or

negative surface charge, the latter being caused by the uronic acids that are

common in extracellular polysaccharides (Christensen, 1989). The EPS may

further include proteins, nucleic acids, amphiphilic polymeric compounds

such as (phospho)lipids, and humic substances (Neu, 1996; Wingender et al.,

1999, Flemming and Wingender, 2001b). The chemical heterogeneity of the

EPS has recently been made visible by staining the biofilm with fluorescently

labeled lectins that bind to specific simple sugars of the biofilm (Wolfaardt

et al., 1998; Neu and Lawrence, 1999; Neu, 2000). In general, sorption of

DOM to biofilms was found to be higher than that to the clean surface with

the biofilm removed (e.g., Armstrong and B~irlocher, 1989). The EPS thus

represent a major structural and functional component of biofilms.

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 291

For two general reasons it is difficult to distinguish abiotic sorption of

DOM onto biofilms from biotic uptake:

1. A comparison of DOM uptake by living and killed biofilms could

be accomplished using carbon-free poisons. Sodium azide (N3Na) and mercury

chloride (HgCI2) are the most promising substances for this purpose

(Tuominen et al., 1994; Ribas et al., 1995). These substances can be used in

continuous flow systems such as those described in Table II. However, I found

that with a HgC12 concentration of 100 mg/L in perfused sediment cores

leucine uptake by bacteria in lower layers of the cores was not completely

suppressed, suggesting that HgC12 was readily adsorbed within the uppermost

layers. Additionally, killing the biofilm during experimentation leads

to a release of DOM from the biofilm, which masks the actual sorption

(Dahm, 1981; Tuominen et al., 1994; personal observations). The application

of short-term sorption experiments as described earlier in this section therefore

seems to be a more useful approach to measure abiotic sorption. However,

it should be taken into account that in most of these experiments

natural stratification of sediments would be disrupted and existing redox

gradients destroyed.

2. In aquatic systems, a physicochemical equilibrium between overlying

water and the biofilm is quickly accomplished. By taking up substances,

bacteria work against that equilibrium. Thus, the biofilm providing the

primary sorption sites for DOM and bacteria using this DOM form an

entity. Little is known about the process of formation and decomposition of

EPS in living biofilms and whether a "mature" biofilm is a constant, fixed

structure or produced and decomposed in a highly dynamic equilibrium. A

powerful alternative to measuring sorption onto biofilms is to use labeled

model substances. To do so, one can use radioactively labeled substances

and determine their fate and distribution in several compartments of the

biofilm (biofilm matrix and bacterial biomass), the overlying water (CO2),

or the food web (e.g., Dahm, 1981; Fiebig, 1997; Hall and Meyer, 1998).

In this case, sorption to the biofilm and bacterial turnover of the substances

can be documented.

In a biofilm, the transport rate of particles and solutes is further

influenced by the biofilm matrix itself. It has been calculated that, compared

to a clean surface, biofilms may increase convective mass transport near the

surface because their compliant nature can increase hydraulic roughness

(Bouwer, 1987). Roughness of the biofilm surface in some instances increased

eddy diffusion and external mass transfer rate into the biofilm (Siegrist and

Gujer, 1985). The viscoelasticity of biofilms has been experimentally demonstrated

by changing the hydrodynamic conditions (shear stress and flow

velocity) in a flow cell and measuring the structural deformations of biofilms

caused by these changes (Stoodley et al., 1999c). It was found that biofilms

were compliant and readily deformed by changes in shear stress. A decrease

292 HelmutF ischer

of biofilm thickness by 25% was measured when the shear stress was

increased from 0 through 10 N/m 2. This deformation was reversible to some

extent~ the biofilm returned to its original shape when changes in shear

stress were applied below the shear stress at which they were grown. Mass

transfer will certainly be influenced by this biofilm deformation. Biofilms

also can provide shelter from shear forces and increase surface area for

attachment (Bouwer, 1987).

Sorption experiments have been conducted under various experimental

conditions, and their results vary depending on whether clean or biofilmcoated

sorbents have been used and whether the biofilm was living or had

been killed. Armstrong and B~irlocher (1989) demonstrated that sorption on

clean natural sediments was higher for positively charged and for uncharged

polar amino acids than for negatively charged amino acids. Nonpolar amino

acids exhibited intermediate sorption in their study (Table I). Henrichs and

Sugai (1993) in their experiments used lower, near natural, amino acid

concentrations in seawater sediments covered by a natural biofilm. They

also found the highest sorption for the positively charged amino acid lysine;

however, they found higher sorption for the negatively charged glutamic

acid than for leucine, which is in contrast with the sorption characteristics

on clean surfaces (Table I). This finding may be attributed to reactive

functional groups in the biofilm, which alter the sorption capacities of the

clean surface and provide a greater spatial heterogeneity in the sorption

characteristics. Much of the sorption in these experiments was reversible

with use of ammonium chloride and/or cesium chloride, demonstrating the

functional role of biofilms as cation exchangers as stressed by Freeman et al.

(1995). However, substantial proportions of the amino acids sorbed onto

the biofilm were not extractable by ion exchange. By performing sorption

experiments with different amino acid concentrations, Henrichs and Sugai

(1993) also found that the sites for this type of irreversible binding were

limited. Thus, a chemical equilibrium develops between the sorbate and the

TABLE ! Sorption of Amino Acids onto Clean Stream Sediment Surfaces (SiO2)

with the Biofilm Removed

Sorbate Properties of Sorption

(DOM-Fraction) the sorbate (Kp)

Arginine, lysine

Aspartic acid, glutamic acid

Glycine, serine, threonine,

glutamine, asparagine

Alanine, valine, leucine, isoleucine,

phenylalanine

Positively charged High

Negatively charged Low

Uncharged, but polar High

Nonpolar or hydrophobic

From Armstrong, S., and E B~irlocher, 1989.

Intermediate

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 293

sorbent, and further retention of DOM in long-term experiments can be

attributed mostly to microbial processes (Section IIIC and Table II).

B. Diffusion of Dissolved Organic Matter into Bi0films

In a flow system with turbulent flow, a laminar boundary layer is formed

between the turbulent flow and the biofilm. Mass transfer in the turbulent

flow is accomplished by convection and within the biofilm possibly by

diffusion. Mass transfer in the boundary layer is accomplished by a mix of

diffusion and convective (micro)flow (deBeer et al., 1996; Mildenberger,

1999). Mathematical models of biofilm diffusivity assume the existence of

a thin diffusive boundary layer above the biofilm. However, some empirical

evidence contradicts this assumption. Measurements using nuclear magnetic

resonance imaging and particle velocimetry combined with confocal laser

scanning microscopy demonstrated that convective flow can be found even

within biofilm structures (deBeer et al., 1994, Lewandowski et al., 1995).

Using an oxygen microelectrode Lewandowski (1998) demonstrated that

there was only a slight decrease of the mass transfer coefficient above and

within a biofilm and a significant reduction only in thick cell clusters. He

therefore concluded that biofilms present much less resistance to mass transport

than traditionally believed.

The gellike biofilm matrix may be quite unimportant in impeding diffusion

in certain circumstances because of the detailed consequences of the

diffusion law in special geometries (Koch, 1991). The diffusion coefficient

of oxygen in the biofilm of nitrifying trickling filters was calculated to be

40-80% of the value in pure water, with thin biofilms exhibiting higher

diffusion resistance than thick biofilms. This was attributed to a higher

density of the thin biofilms (Siegrist and Gujer, 1985, 1987). Beyenal and

Lewandowski (2000) measured local effective diffusivities of glucose in a

three-species artificial biofilm using cathodically polarized microelectrodes,

and they spatially integrated the local measurements to give surface averaged

effective diffusivities. They found that relative effective diffusivities

increased with an increase in glucose concentration and with a decrease in

flow velocities. This means that the density of biotilms growing under low

nutrient concentrations and high flow velocities ~ as, for example, in streams

and rivers ~ is probably higher than the density of biofilms in eutrophic

lakes or in wastewater treatment plants.

Diffusion into a biofilm may be seen as directed into one dimension.

Diffusion time (t) can therefore be calculated as

t = -~-~" ~

where s is the diffusion distance (in centimeters) and D is the diffusion coefficient

of a chemical solute (in square centimeters per second) (Berg, 1983).

. o,,., .~

I::I

. o..,. ,~

r,~

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I::I

..,o.~

Ora,~

~r,.)

I:::I

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° .,..~

,.,... ~

o o--.t

r.~

I~-~

• 1,,,.

r,...3

I,,,,..

.,,,~

0",,

~'u

, ~=.

.~o~

I o ~

. ~eq

J oo ~~

¢:::1

I 0

• V

I'.-

Ox,

x,--I

.,...d

t-.: ,-4~

.~ ~ o

~~ ~-~

0,,.~

t"q o oo

~. oo .,¢-

r--, t--:

e,,i t,'q

o ~ ~ - ~ ~ ~ ~

• . ~

0 ~-"~ ~ " ' - '

294

eq -'--

0 O'~

e,l 0",,

¢;

o4 ~ o4

..~ .~ .~

U: U:

-~ ~.,~

.,,~

• -- 0

:Eo :Bo

.,.P.~' --~ .~P " 0

_ o - ~

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o z~

• ~, ~r,p

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• ,..,-..4 .~ eq

-~o ~ -~ =o,~= ~~!~

- _ ,.~~ N-s

...~

.,.~

-a

-k

295

296 Helmut Fischer

If the biofilm is seen simply as a two-dimensional structure on an inert

surface, the diffusion time within the biofilm would be

$ 2

and in three dimensions, as, for example, in organic particles or thick

biofilms, diffusion time would be

$ 2

(Berg, 1983). The diffusion coefficient for a small molecule in water at

room temperature is about 10 -5 cm2/s. Because diffusion time increases with

the square of diffusion distance, the consequence of the above equations is

that molecular diffusion is very fast on a small scale and very slow at a large

scale (Table III). Given a diffusivity in biofilms of about 50% of that in

water, diffusion is probably not the rate-limiting process in thin biofilms,

but is certainly a limiting factor in thick biofilms and microbial mats (e.g.,

Revsbech, 1989). Diffusion limitation can lead to experimental artifacts in

laboratory incubations with labeled tracer molecules, as often used for

measurements of bacterial production or substrate uptake. In this context,

Ploug and Grossart (1999) have measured the thymidine and leucine uptake

by bacteria on riverine aggregates incubated individually in a threedimensional

diffusion field versus similar aggregates pooled on the bottom

of vials. They found that thymidine and leucine incorporation rates were

5.6- to 5.3-fold lower, respectively, when aggregates were pooled. They

attributed this effect to limited (one-dimensional) diffusion of the tracer

molecules in vials as opposed to fast (three-dimensional) diffusion into freely

floating aggregates.

TABLE Ill Diffusion Times for One- and Three-Dimensional Diffusion of Leucine

and Oxygen Molecules in Water at Room Temperature

Diffusion time

Diffusion distance

Leucine (D = 0.73 x 10 -s) Oxygen (D = 2.24 x 10 -s)

One- Three- One- Threedimensional

dimensional dimensional dimensional

1 ~tm 0.68 ms 0.23 ms 0.22 ms 0.074 ms

10 ~tm 68 ms 23 ms 22 ms 7.4 ms

100 lam 6.8 s 2.3 s 2.2 s 0.74 s

1 mm 11.4 min 3.8 min 3.7 min 1.2 min

1 cm 19.0 h 6.3 h 6.2 h 2.1 h

*Calculated after Berg (1983).

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 297

(3. Cleavage by Extracellular Enzymes and Microbial Uptake

and Utilization

Small molecules diffuse through the biofilm and are taken up by bacteria

via membrane permeases or via diffusion through porins (Nikaido and

Vaara, 1985). According to Fick's first law, the driving forces for molecular

diffusion are the coefficient of diffusion and the concentration gradient.

Various strategies can further enhance the availability and uptake of dissolved

organic carbon (DOC) by bacteria. Voids and channels can occur in the biofilm

and provide ways for convective transport to the deeper biofilm layers

(deBeer et al., 1994; Lewandowski et al., 1995). It may therefore be hypothesized

that biofilm bacteria actively communicate to organize biofilm structures

that allow continuous nutrient supply via these interstitial voids (cf.

Shapiro, 1998). Uptake of DOC can further be enhanced by an increased

surface area of the bacterial cell. For example, the anaerobic cellulolytic

bacterium Clostridium thermocellum bears intricate multienzyme complexes

(the cellulosomes) within specialized cell surface organelles. In addition to

its enzymatic function, the cellulosome contains functional domains that

adhere the bacterium to its particulate cellulosic substrate, so that the enzyme

complex can be efficiently used in close proximity to the substrate (Bayer et

al., 1996). Another way to increase DOC uptake is to quickly remove the

organic compound from the internal pool. In this case, the substrate reacts

with a phosphorylated enzyme via the phosphoenol phosphotransferase

system within the membrane, and a phosphate ester of the substrate is formed

and released into the cytoplasm (Gottschalk, 1986). The substrate then

appears inside the cell in a chemically modified form, thus not steepening

the concentration gradient.

Section IIIA described how amino acids are adsorbed onto the biofilm.

However, in natural systems physical sorption and chemical interactions of

the sorbed molecules with the biofilm constituents are quickly superseded

by microbial activity. Experiments lasting several hours, days, or months

have been used to discriminate microbial effects from sorption effects that

occur over a time span of a few minutes. In these studies it became clear that,

if applied at near natural concentrations, more than 70% of amino acids

added to perfused sediment cores were retained in river biofilms (Fiebig,

1992; Fiebig and Marxsen, 1992; Volk et al., 1997). However, it may take

much longer for this material to be turned over, mineralized, and exported

from the cores. Only 14-36% of the sorbed amino acids were mineralized

during the first 4 h, the time of the incubation (Fiebig, 1992; Fiebig and

Marxsen, 1992). In a long-term experiment using amino acids and hyporheic

sediments it took 28 weeks for 88% of the retained carbon to be exported

as CO2 (Fiebig, 1997). The initial rate of immobilization exceeded the rate

of export by a factor of 710 in this study. These data demonstrate that biofilms

serve as a medium for DOM storage and may accumulate sufficient

298 Helmut Fischer

nutrients to negate the effects of short-term changes in carbon supply as

proposed by Freeman and Lock (1995).

Bacteria living in a biofilm are more or less fixed to a certain position.

To sustain their metabolic needs, they therefore depend on a constant flux

of organic matter toward their cells. In a recent study, Thompson and

Sinsabaugh (2000) have separated biofilms into a polysaccharide matrix

fraction and a fraction containing microbial cells and other particulate

material. They measured enzyme activity and kinetics of alkaline phosphatase

and leucine aminopeptidase in both fractions for both lightexposed

and shaded biofilms. They found an average of about 25% of the

total activity in the cell free matrix fraction, suggesting that the biofilm

matrix was retaining enzymes. They also found that for both enzymes Km

values were significantly higher and Vmax values were significantly lower in

the biofilm matrix than in the cell plus particulate fraction. The authors

discuss this phenomenon as an artifact of diagenesis: enzymes released into

the environment are conformationally constrained by reactions with the

exopolysaccharides, humic substances, or other molecules that reduce their

substrate binding capability. However, the matric enzyme activity can

comprise a significant fraction of total biofilm activity. Matric enzymes may

therefore be regarded as a community resource, uncoupled from the

metabolism of individual cells (Thompson and Sinsabaugh, 2000), a function

that will be further discussed in the next paragraphs.

By modeling bacterial foraging by means of freely released extracellular

enzymes, Vetter et al. (1998) defined a "foraging distance" within which

90% of the diffusion current of hydrolysate to the cell is produced. This

foraging distance was conservatively modeled to be about 10 gm and would

exceed 50 ~tm in large aggregates and in sediments under conditions of low

enzyme inactivation rates (Vetter et al., 1998). These calculated distances

may have implications for the spatial structures of biofilm communities. If

the biofilm is sparsely colonized by single bacteria in distances of more than

10 ~tm from each other, these bacteria would depend on hydrolysates

produced by their own extracellular enzymes. A relatively high proportion

of the extracellular enzymes produced may be lost in this case because of

inactivation or diffusion into the overlying water. The growth of bacteria in

clones forming microcolonies would support a more efficient utilization of

extracellular enzymes. Enzymes diffusing farther away from a hypothetical

bacterium in the center of the microcolony may then still be used by the

clone, because bacteria from the outer margins of the microcolony may

benefit from the hydrolysates. On the other hand, bacteria may have developed

strategies to avoid excessive production of extracellular enzymes and

seek to benefit from enzymes produced by different clones. To do so, they

would have to intrude into cell clusters of the other clone and refrain from

the production of their own extracellular enzymes. In this scenario, it would

not be surprising if the "host" bacteria would have developed defense

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 299

mechanisms, such as production of allelochemicals, to hinder the intrusion

into their cell clusters. These scenarios would afford extensive interactions

between bacteria of the same clone as well as between bacteria of different

clones or species. Indeed, cell-to-cell signaling to sense and control cell

density ("quorum sensing") is known from bacteria in suspension cultures

(Fuqua et al., 1994; Dunny and Leonard, 1997) and was recently shown to

exist in biofilms as well (Davies et al., 1998). Combining the modeling

approach (Vetter et al., 1998) with the hypotheses of intrabiofilm cell-cell

signaling (Davies et al., 1998) opens a new perspective for the understanding

of the spatial community structure in biofilms.

Gradients of nutrients and oxygen in biofilms additionally promote high

diversity, which may ultimately result in functional differences of the bacterial

community in biofilms compared with free-floating bacteria. Additionally,

increased species diversity may provide spatial and temporal niches not

available within monocultures or may create microenvironments within the

biofilm (Gieseke et al., 2001; Whiteley et al., 2001). These thoughts reinforce

the need for community-level biofilm studies as opposed to monocultures.

IV. EFFECTS OF THE BIOFILM ON MICROBIAL ACTIVITY

Bacteria afford extracellular enzymes to degrade and effectively use

large molecules. Whether biofilm bacteria on inert surfaces develop enzyme

patterns different from those of free-living bacteria has rarely been tested

(Arnosti, 2000; see Chapter 13). If there was a frequent turnover of the bacterial

extracellular polysaccharides in biofilms, the activity of polysaccharases

needed for synthesis and degradation of this material can be assumed to be

higher in biofilm bacteria than in free-living bacteria. These enzymes can have

a marked effect on the structure and the integrity of biofilms (Sutherland,

1999). However, enzymes adsorbed to particle surfaces mostly undergo

changes in their confirmation and thus in their kinetics. The optimal catalytic

activity shifts to higher pH values when enzymes are adsorbed on negatively

charged surfaces (Quiquampoix, 2000). Probably the electrostatic interactions

between the positively charged enzymes and negatively charged surfaces

lead to an unfolding of the enzymes at pH values below the isoelectric

point of the enzymes. Usually, Vma x decreases and Km increases in these

cases (Quiquampoix, 2000). These restrictions to enzymatic activity on

particle surfaces need not necessarily apply to fully hydrated biofilms, but

they probably occur in those regions of the biofilms that are located close

to a negatively charged substratum. Thus, the often-observed higher activity

of biofilm bacteria compared with that of free-living bacteria contrasts with

physical and physiological constraints (possible diffusion limitation and deactivation

of enzymes) exerted on the bacteria. Whereas in most cases the

advantages for growth in a biofilm predominate, they can be offset to some

300 Helmut Fischer

extent by disadvantages (Fletcher, 1991). Bacteria living close to the substratum

should be more strongly affected by those disadvantages than

bacteria from outer biofilm layers. Indeed, it has often been found that microorganisms

from outer biofilm layers develop higher activities (Paul and Duthie,

1989; Burkholder et al., 1990; Fischer et al., 1996; Okabe et al., 1996).

The type of the biofilm may thereby significantly influence the activity

of attached bacteria. In epilithic biofilms, mineral nutrients (Fe, Si, and

trace elements) can leach from the substratum and be used by the attached

microflora (Wetzel, 1993). However, if the underlying solid substratum can

be used as an additional source of carbon and nutrients, bacteria generally

exhibit higher activity than those living on a mineral surface. In this context,

Sinsabaugh et al. (1991) found that ATP content and a suite of extracellular

enzyme activities were higher in epixylic biofilms of a boreal river than in

epilithic biofilms under similar environmental conditions. Those epixylic

biofilms are inhabited by groups of bacteria specialized in the degradation

of complex polysaccharides such as cellulose (Reichenbach, 1992; Bayer

et al., 1996) and polyphenolics such as lignin (Vicufia, 1988; Hendel and

Marxsen, 2000). Detrital particles in the hyporheic zone of a prealpine river

were important for bacteria as colonizable surface areas and as a source of

nutrition and thus had a higher power in predicting total bacterial abundance

and production than inorganic particles (Brunke and Fischer, 1999). In

pelagic aggregates, intense activity of several ectohydrolases has been

revealed (e.g., Smith et al., 1992; Grossart and Simon, 1998; but see Miiller-

Niklas et al., 1994; see also Chapter 13). Largely overlooked, these aggregates

can contain considerable amounts of interstitial DOC (Alldredge,

2000), which may serve as a carbon source in addition to the particulate

fraction. In conclusion, the adverse effects for bacteria living in a biofilm

mostly predominate in regions close to the substratum, whereas bacteria in

the outer regions of the biofilm or on freely floating organic flocs may

benefit from the advantages of continuous substrate supply via sorption to

the biofilm.

V. EFFECTS OF DISSOLVED ORGANIC MATTER QUALITY AND

QUANTITY ON THE ACTIVITY OF BIOFILM BACTERIA

DOC, whatever its form or origin, either directly or indirectly represents

the ultimate source of organic carbon for sustaining the metabolism of

heterotrophic bacteria. The metabolic activity of biofilm bacteria can therefore

be influenced by the ambient concentration and composition of DOC

(e.g. Kaplan and Bott, 1989; Baker et al., 1999; Fischer et al., 2002; see

Chapter 15). Biofilms are able to retain inorganic and organic solutes (e.g.,

Bencala et al., 1984; Mc Dowell, 1985; Fiebig, 1992). As shown in Section

IIIC, they can buffer the supply of organic substrates so that short-term

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 301

changes in the quality and quantity of DOC need not have an immediate

effect on biofilm metabolism (Freeman and Lock, 1995; Fiebig, 1997).

In Table II it is shown that DOM fractions are differentially retained in

perfused cores with multispecies biofilms during long-term incubations.

This microbially mediated retention is high for low-molecular-weight

organic matter, most notably amino acids and mono- and disaccharides,

and for the polysaccharide fraction of high-molecular-weight organic matter.

It is relatively low for the intermediate-molecular-weight substances predominantly

consisting of humic substances. The retention of freshly extracted

DOC (Battin et al., 1999; Table II) is particularly high, probably because of

a high proportion of labile substances in these extracts. Figure 2A depicts

an example for DOC retention in a flowthrough incubation of riverine

sediments derived from the sixth-order lowland River Spree, Germany

(see Table II for details). It can be seen that humic substances are retained

to a much lesser extent than the high-molecular-weight compounds (polysaccharides)

and the low-molecular-weight compounds (mono- and

disaccharides and amino acids). However, the humic substances form the

largest group of DOC and can therefore make up a substantial proportion

of the total organic matter retained in the cores (Fig. 2A; Volk et al., 1997;

Fischer et al., 2002).

The quality of the DOC available obviously influences bacterial activity

in the biofilm. The composition of DOC in natural river water retained in

sediment cores has significant effects on bacterial production, whereas changes

in the bulk quantity of DOC alters the activity of biofilm bacteria to a lesser

extent (Fig. 3A-C; see Chapter 15). Under light conditions, autotrophic

algae in the biofilm are a possible source of labile compounds that may be

used by biofilm bacteria living in close proximity to the algae. Therefore,

bacterial activity often correlates with algal biomass and production in the

biofilm (Haack and McFeters, 1982; Chappell and Goulder, 1994; Sobczak,

1996). In a Piedmont stream, biofilm bacteria kept in the light grew twice

as fast than those kept in the dark. This was attributed to biofilm-internal

DOC cycling mediated by epilithic algae, which had a 5-fold higher biomass

in the light than in the dark (Kaplan and Bott, 1989). In a Mediterranean

stream, bacterial density and 13-glucosidase activity were higher in lightgrown

biofilms than in dark-grown biofilms. Algal biomass increased with

the use of cellobiosic as opposed to xylobiosic polysaccharides, probably

because of the presence of high-quality algal exudates (Romani and Sabater,

1999). In the same study, bacteria in dark-grown biofilms responded rapidly

to algal activity, although a greater increment in chlorophyll density was

necessary to obtain a similar increase in enzymatic activity in light-grown

biofilms. This resilience of bacteria in light-grown biofilm is probably related

to algal exudates stored in the biofilm. Wetzel (1993) hypothesized that

the high productivity of attached microcommunities of algae and bacteria

is due to efficient internal recycling of carbon and nutrients. Whereas

r~ _.e

o~,~

II II II

x~- I,O ¢0

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-ac

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302

t--

,,?,

r 2 = 0.36

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n=67

• d'

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• .... -"

• ..- • • .Jm ;

• ~ ,-,-'"" ql~ • g o

o •

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• .'-" •

00 0 100 200

Retention of total DOC [pg C / core]

300

400

o')

.-.i

o L...

63 .R

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(1)

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p < 0.0001

n=67

• oag~.. ~- i

• ~ O O . ..,-" "" O djlJo~l D • •

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-20 0 20 40 60

Retention of polysaccharides [pg C / core]

80 100

r 2 = 0.09 • C

p < 0.05 • •

n=67 •

o • •

• f •

"°° , , , , x°

"o •

00 -50 0 50 100 150 200

Retention of humic substances [IJg C / core]

FIGURE 3 Relationships between DOC retention and bacterial production in sediments of

the River Spree, Germany, February-November 1998 at temperatures ranging from 7 to 22°C

(Fischer et al., 2002). (A) Total DOC, (B) polysaccharidesi and (C) hurnic substances.

303

304 Helmut Fischer

heterotrophic bacterial biofilms would be largely dependent upon external

importation of DOC, the mixed community of auto- and heterotrophs decreases

this dependency upon importation from the ambient environment.

VI. ECOSYSTEM CONSEQUENCES

The previous sections of this chapter dealt with biofilm structure and

function in a microscopic scale; in Section V the view was extended toward

a mesocosm scale. In these mesocosms built as flowthrough reactors, significant

retention and/or transformation of DOC was found when inflow and

outflow were compared (Ribas et al., 1995; Fiebig, 1997; Volk et al., 1997;

Fischer et al., 2002.; Table II). The same condition applied to larger systems

built as artificial streambeds (Gantzer et al., 1988, 1991). These studies

imply that biofilms have the potential to play an important role in the

retention and transformation of organic matter from the water column on

an ecosystem scale. The relevant processes m adsorption to the biofilm and

subsequent bacterial utilization- may occur in natural systems similarly as

in laboratory incubations. Processes such as extracellular decomposition of

organic macromolecules (e.g., Marxsen and Fiebig, 1993) and selective

utilization of specific DOC fractions may thus enable biofilm bacteria to

exert a considerable influence on quantity and quality of DOC in natural

waters (e.g., Fiebig and Marxsen, 1992; Findlay et al., 1993; Findlay and

Sobczak, 1996).

Therefore, it seems worthwhile to transfer the ideas gained by mesocosm

experiments to larger systems as, for example, to stream sections or to

whole rivers. In running waters, sediments are the major sites of bacterial

metabolism (Pusch et al., 1998; Fischer and Pusch, 2001; see Chapter 4),

especially in those rivers with a high proportion of discharge through the

hyporheic zone (Findlay, 1995). Their large internal surface area promotes

colonization of these sediments by bacterial biofilms (Lock, 1993; Fischer

et al., 1996; Brunke and Fischer, 1999). These biofilms are supplied with

nutrients and oxygen by flowing interstitial water, which originates either

from the overlying water column being forced into the sediment interstices

by physical processes or from groundwater exfiltration (Hendricks, 1993;

Brunke and Gonser, 1997; Pusch et al., 1998).

When whole river systems are studied, it is difficult to separate the bacterial

influence on longitudinal changes in DOC amount and composition

from various other influences such as allochthonous inputs, algal exudation,

or photochemical cleavage. However, attempts have been made to investigate

the amount and composition of DOC and its availability to bacteria in ecosystem

studies performed along the Ogeechee River continuum (Left and

Meyer, 1991; Sabater et al., 1993; Sun et al., 1997). At low flow situations,

the relative availability of DOC to bacteria decreased along the continuum,

12. Biofilms in Uptake and Transformation of Dissolved Organic Matter 305

which was also reflected by a relative increase in the more refractory DOC

fractions and by an increase in total DOC. At high discharges, channel

processes were superimposed by inputs of allochthonous DOC from surrounding

swamps and the longitudinal patterns of DOC were less clear

(Sabater et al., 1993).

Changes in the amount and composition of DOC from the River Spree

after an algal bloom are shown in Figure 2B. There is a striking similarity

between the sequence of elution diagrams obtained in the laboratory incubations

before and after sediment core perfusion (Fig. 2A) and those obtained

directly from the river water during and after the algal bloom (Fig. 2B).

During the algal bloom, a considerable amount of labile DOC, especially

polysaccharides, was released into the river water. In the middle of May, the

chlorophyll- a content, as an indicator of algal biomass, decreased dramatically

from 45 to 3 Bg/L (data not shown). Concomitantly, the amount of

labile substances decreased strongly in the river water. It is concluded that

this change in riverine DOC composition was caused mainly by biofilm

bacteria, analogous to the bacterial decomposition of DOC in the laboratory

incubation. Therefore, in a number of ways biofilms exert a strong influence

on the carbon biogeochemistry of aquatic ecosystems.

In streams and rivers the surface-bound bacterial activity greatly

exceeds the activity of free-living bacteria. The share of the metabolism that

can be attributed to biofilm-coated surfaces has usually been calculated for

only small (first- and second-order) streams. In these streams, carbon turnover

is highest in the hyporheic zone, and less than 1% of the community

respiration occurred in the water column (Fuss and Smock, 1996; Naegeli

and Uehlinger, 1997). However, biofilm-bound activity can also dominate

the heterotrophic metabolism of larger rivers and lakes. It was shown that

in the sixth-order section of the blackwater Ogeechee River benthic (biofilm)

bacteria accounted for >90% of the system metabolism (Edwards et al.,

1990). In the Spree, a sixth-order lowland river with sandy sediments and

a mean depth of I m, the upper 2 cm of sediment had an areal bacterial

production 17- to 35-fold higher than that in the water column (Fischer and

Pusch, 2001). Even if the deep sediment layers were not hydrologically

connected with the overlying water, biofilm bacteria may exert a major

influence on the biogeochemistry of the water column. This was found for

shallow Danish lakes, in which the production of bacteria in the biofilm on

aquatic plants (epiphyton) was measured. On an areal basis, bacterial

production in this epiphytic biofilm was up to 7 times higher than the

activity of free-living bacteria (Theil-Nielsen and Sondergaard, 1999). From

an ecosystem perspective it seems that in many aquatic systems the water

column is the medium that transports carbon and nutrients to the foci of

heterotrophic metabolism. These foci are located in the biofilm of the

sediments and the epiphyton and serve as important sinks of organic matter

in these ecosystems.

306 HelmuFt ischer

VII. CONCLUSIONS

Biofilms enhance bacteria-DOM interactions by several means. Their

spatial and chemical heterogeneity provides additional sorption sites for

DOM compared with clean surfaces. Their loose architecture with interstitial

voids and channels increases diffusivity and to some extent allows

convective flow within biofilm structures. Because bacteria metabolize

organic matter sorbed to the biofilm, a diffusion flux from the free water to

the biofilm is maintained. Large proportions of organic matter sorbed to the

biofilm are not instantly turned over but remain in the biofilm as a reservoir,

which buffers direct effects of DOM depletion in the water column.

For bacteria, there may be some adverse effects of living in a biofilm,

especially in regions close to the substratum; there, bacteria are to some

extent cut off from the flux of DOM from the surrounding water.

Additionally, bacterial enzymes can be inactivated if they are sorbed to

surfaces. On the other hand, fixed positions of bacteria in biofilms support

interactions between bacteria either within bacterial clones or between

different bacterial species. They take advantage of growing in clones by

saving energy spent for the production of extracellular enzymes, and they

mutually interact between species in several ways.

Because of the high area of solid surfaces covered with biofilms, these

biofilms dominate the heterotrophic metabolism in many aquatic ecosystems.

In streams, rivers, and shallow lakes, bacterial activity in epilithic

and epiphytic biofilms may be several times higher on an areal basis than

the activity of free living bacteria. By the differential use of specific DOM

fractions, biofilm bacteria influence the biogeochemical composition of

DOM in these ecosystems. Biofilms thus can control biogeochemical fluxes

of DOM and are important sinks of organic matter.

ACKNOWLEDGMENTS

I thank Hans-Curt Flemming and Thomas Griebe for helpful comments

on an earlier draft of this contribution.

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