[27] Evaluation and quantification of bacterial attachment, microbial activity, and biocide efficacy by microcalorimetry

[271

MICROCALORIMETRY

361

Furthermore, the standard deviation on cell density determined by conductance measurements was decreased compared to traditional plate counts. The described method was particularly useful for the evaluation of enzymatic degradation of biofilm, as cells left on the substratum after enzyme treatment was more detachable than untreated biofilm cells. The enzyme treatment can thereby interfere with the traditional evaluation methods involving biofilm removal followed by bacterial enumeration by colony formation, resulting in an underestimation of the enzyme efficacy. The use of a calibration curve for determinating the number of biofilm cells involves the assumption that, in the Malthus tubes, the carbon dioxide metabolism of cells from the biofilm and cells from the planktonic suspension is comparable. This assumption was found to be acceptable, despite the indications of differences in the metabolism of biofilm and planktonic cells, respectively.17 This can be explained by the fact that biofilm cells do not continue to grow as biofilm cells when transferred to Malthus tubes and thereby the actual detection will be on the outgrowth of planktonic cells in the Malthus medium. 17 S. M¢ller, C. Kristensen, L. K. Poulsen, J. M. Carstensen, and S. Molin, Appl. Environ. Microbiol. 61, 741 (1995).

[27] E v a l u a t i o n a n d Q u a n t i f i c a t i o n o f B a c t e r i a l Attachment, Microbial Activity, and Biocide Efficacy by Microcalorimetry

By W O L F G A N G

SAND a n d H E N R Y VON RIDGE

Introduction In the field of biofilm research, the interactions between microorganisms and the animate or inanimate surface are only partially understood. The primary process, which finally leads to a firm attachment of a microorganism to a substratum, is the focus of much attention. Up until now it remains impossible to predict which strain of a bacterial species will attach to a specific substratum/material. Furthermore, once a cell has attached to a surface, the direct and continuous monitoring of its growth, multiplication, metabolic activity, and so on is very difficult. Most techniques allow only measurements at certain points (ATP, fatty acids, etc.), are destructive (electron microscopy, many electro-

METHODS IN ENZYMOLOGY, VOL. 310

Copyright © 1999by AcademicPress All rights of reproductionin any form reserved. 0076-6879/99 $30.00

362

PHYSICALMETHODS

[27]

chemical techniques, etc.), or are only applicable for aerobic samples (02 electrode). In addition, special test rigs are required to expose sample coupons for consecutive detection and analyses. Thus, there is a need for an on-line test system that allows for undisturbed, continuous measurements under aerobic as well as under anaerobic conditions. In addition, the test system should allow one to scrutinize the relevant microorganisms with respect to the relevant materials, to determine possible interactions such as attachment, and, in the case of unwanted effects such as biocorrosion, the optimization of countermeasures including biocide dosage. To achieve these goals, the technique of microcalorimetry is the most suitable one. It is a nondestructive, highly sensitive technique that allows the detection on-line of a minimum of 104 metabolically active cells 1 without the need to disturb the system for/by sampling. D e s c r i p t i o n of Microcalorimetric M e a s u r e m e n t S y s t e m Calorimetry has been applied as early as 1780 by Lavoisier for a determination of metabolic rates of mammals. From this first ice calorimeter it has been a long way to the development of present instruments. 2 Whereas the ice calorimeter used the amount of water, resulting from molten ice, as a measure of the heat evolved, present-day instruments are based on thermocouples, allowing for an accurate determination of the power output. Modern calorimeters, and here only the isothermal type of equipment will be referred to, allow the detection of thermal power quantities as low as 50 nW. Measuring Principle

The principle on which m o d e r n instruments are based is given in Fig. 1. The measuring cup is the center of the system. It can take up closed ampoules of between 4 and 25 ml volume, microreactors of the same volume (open ampoules), and/or be surrounded by a coil of gold tubing, allowing for continuous flow experiments. The three systems are shown in Fig. 2. The flow system is available as a flow-through or as a flow-mix system. The latter allows the addition of a compound to the system under supervision immediately prior to the passage of a sample liquid through the thermocoupies. Using this assembly short time reactions become detectable. All metabolic reactions are accompanied by heat production or absorption (exergonic or endergonic reactions). In the microcalorimetric measure1j. p. Belaich, in "Biological Microcalorimetry" (A. E. Beezer, ed.). p. 1. Academic Press, London, 1980. 2j. Suurkuusk and I. Wads0, Chem. Scr. 20, 150 (1982).

[271

MICROCALORIMETRY

Heat sink/'/(

Measuring

cup

&

363

~Heat sink \

J t flow

~hermopil(

Heat

Thermopile ~

FIG. 1. Measuring unit of the microcalorirneter.

ment unit this heat is completely exchanged with a large, surrounding heat sink. This sink is a large water bath, accurately maintained at a constant temperature. When a thermal energy change occurs in a sample as a consequence of bacterial metabolism, a small temperature difference arises (relative to the heat sink), which forces a heat flow, until equilibrium is obtained again. This temperature difference is directly proportional to the heat flow. Highly sensitive thermopiles, located around the reaction vessel, are used to detect and quantify the temperature difference. The potential, generated

Flow system Insertion ampoules Flow mix Flowthrough Closed Open

FIG. 2. Measuring systems of the microcalorimeter.

364

PHYSICALMETHODS

[271

Ampoule lifter

Heat exchanger coil (for

Jflow

mode)

Ampoule

Sample ~measuring cup

Reference measuring cup

-- Thermopilar arrays

Heat sink

Calibration heater

\ Intermediate

heat sink

FIG. 3. View of a calorimetric unit.

by the thermopiles, is amplified and recorded as heat flow. Figure 3 gives the details of a calorimetric unit, displaying the spacial arrangement of the measuring elements. This unit allows for ampoule and/or flow-through experiments.

Measuring Units Three general types of measuring units are available: (1) the 4-ml twinampoule/microreaction system unit, (2) the 20/25-ml twin-ampoule/micro-

[2 7]

MICROCALORIMETRY

365

reaction system, and (3) the combined twin-ampoule/microreaction system plus flow or flow-mix installation unit. Ampoule units are equipped with a twin detector for measuring and a reference cup. The ampoules are available with 4-, 20-, or 25-ml volumes. In all instances, either closed or open insertion ampoules (microreaction systems) may be used. The sensitivity of the system amounts to 50 nW (0.1 /xW) for the 4-ml ampoules (microreaction system) or to 0.2/zW (0.4/zW) for the 20-ml units, respectively. Closed ampoules are generally used for stability or compatibility tests? More specifically, they may be used for a quantification of biocide efficacy against biofilm samples on coupons or determinations of the microbial activity of cell suspensions with dissolved and/or particulate compounds.4 Microreaction systems are equipped with a stirrer and gas/liquid inlets and outlets. Thus, experimental conditions can be manipulated during a run. Oxygen, nitrogen, or hydrogen gas may be added. In addition, nutrients and/or trace elements as well as toxic compounds such as biocidal substances can be included in the experiment. These ampoules allow the study of ligand interactions, enzyme kinetics, binding, and conformational changes and, with living microorganisms, interactions between solids and liquids occurring as in biocorrosion systems. If test coupons of metallic or other materials are used, which contain a biofilm, material- and/or microorganism(s)-specific tests become possible. The response of the biofilm community to an addition of growth-enhancing or growth-inhibiting substances may be tested using planktonic, planktonic and attached, or only attached bacteria. Thus, effective measures for antifouling strategies with regard to a specific material can be developed or the course of biocorrosion be followed (see later). Combined calorimetric units allow one to perform all of the previously described experiments plus continuous culture tests in the bypass mode. The solution or suspension under investigation may originate from an external fermenter and is pumped through the measuring coil for the quantification of the heat flow. The volume, available for a registration of the metabolic reactions, amounts to 0.6 ml. The whole system of a flow unit contains 1.8 (flow unit) or 2.1 (flow-mix unit) ml. The sensitivity of the system amounts to 0.1/zW for both types. Typical experiments, run in a flow unit, are assays involving the tests of drug effects on cell cultures or fermentation processes. Regarding biofilm, these flow units allow the detection of pure and mixed cultures, whether comprised of defined or 3 K. Bystr6m and A, B. Draco, "Thermometric Application note 22,004." Thermometric, Broma, Sweden. 4 H. yon R6ge and W. Sand, J. Microbiol. Methods 33, 227 (1998).

366

PHYSICALMZTHODS

[27]

unknown (enrichment) microorganisms, the capability to attach to the surface of the tubing (in the measurement position). Because gold, an inert material, is used for the tubing, adhesion may be studied without any interfering effects resulting from corrosion. However, it needs to be pointed out that material-specific adhesion experiments are not yet possible. The advantage of the flow unit is that those microorganisms may be detected that attach readily to the surface of the tubing. They may even be detected in mixed cultures, allowing identification of the relevant ones for biofilm formation. In the case of pathogenic bacteria, to become established in potable water system biofilms, this possibility is of considerable importance. Up until now, no other test system is available that unequivocally allows for the detection of adhesion on line and, in addition, the test of countermeasures. The latter is achievable by adding antimicrobials before entering the measuring unit, i.e., outside of the instrument, to the pumped solution/ suspension in the case of the flow-type unit or by using the flow-mix type unit (mixing occurs inside the instrument) immediately prior to the measuring position with the pumped solution. The first possibility is used to measure long-term effects on pure cultures, attached, and/or planktonic ones, as well as on mixed cultures. The second possibility allows for tests of rapid reactions occurring immediately after the addition of toxic compounds. Other experiments that are possible using the flow-mix unit are useful for determinations of thermodynamic properties of reactions, such as the heat of dilution or ligand binding. Practical Applications of Microcalorimetry Following the previous explanation of the microcalorimetric system, specific experimental possibilities are described for an evaluation of adhesion, microbial activity of biofilms on materials, surfaces, and biocide efficacy. Adhesion to Surfaces

Two examples are described: determining the possibility to detect the ability of bacteria to attach to a surface (of gold) and determining the amount of energy used for attachment and for biofilm formation. Chemoorganotrophic bacteria of the species Bacillus subtilis were isolated from the river Elbe in Germany near Hamburg. The water samples originated from the surface and from deep water (0 to 12 m). From the water phase and from flocs found in the samples, strains of B. subtilis were isolated and subcultured. These were analyzed for their heat output and growth characteristics using flow-through measurement units (2277 type

[2 7]

80-

MICROCALORIMETRY

367

1 ~,

heat output P [~tW]

sessile strain -*-planktonic strain

60

40

20

0

5

10

20

15

25

30

time [h] FIG. 4. Heat output of different strains of Bacillus subtilis. Arrows indicate the change from the culture suspension with bacteria to the sterile nutrient solution (M. Rudert, unpublished results).

T A M of Thermometric, Sweden, or C-3 Technik, Germany). The strains were cultivated aerobically and pumped through the flow-through unit with peristaltic pumps. Intermittently, air and solution at a rate of 20 ml each were pumped. The resulting power-time curves were recorded and registered. It has been shown previously that these power-time curves are strain specific.5 Figure 4 gives a typical example of such a power-time curve. For evaluation, whether these strains had attached to the surface of the tubing, the flow from the external culture was interrupted and the solution was replaced by a fresh, sterile one. The consequences are shown in Fig. 4. In case of a strain able to attach, after the exchange of the nutrient solution, a measurable, constant heat output remained detectable. If growth occurs under these conditions, the heat output would increase. In contrast, in the experiment with the nonattaching strain the heat output decreased to almost the baseline after solution replacement. Obviously, all cells were washed out by the sterile solution, rendering the measuring position virtually free of bacterial cells. The ability for attachment was correlated with the origin of the strains. The attaching ones originated from flocs, whereas the planktonic ones did not attach. As a consequence, the calorimetric test allows an 5 A. W. Schr6ter and W. Sand, F E M S Microbiol. Rev. 11, 79 (1993).

35

368

PHYSICALMETHODS

[27]

300

~ 250- |

~

/j~.%~,~

I-- 200:D ::D150

0

LU 1 0 0

-r

!

0

1

0

....

10

20

30

40

50

60

70

TIME [h] FIG. 5. Influence of adhering cells on the heat output of Thiomonas intermedia growing on thiosulfate. A, power-time curve without interruption by cleaning; B, power-time curve with cleaning intervals. Arrow, Start of cleaning procedure; circle, change from steep to moderate increase of heat output. Reprinted from S. Wentzien et aL, Arch. Microbiol. 161, 116 (1994), with permission. explanation why some strains occurred in floes and others only as planktonically growing ones. In addition to c h e m o o r g a n o t r o p h i c bacteria, autotrophic species are able to attach to surfaces and to f o r m a biofilm. The following experiments were conducted with T h i o m o n a s intermedia (previously Thiobacillus intermedius 6) strain K12, which originally had b e e n isolated f r o m corroded sites of a concrete pipe in the H a m b u r g sewer system 7 and Thiobacillus versutus strain D S M 582. Both strains were cultured, using their respective growth media with thiosulfate as an energy source. 8 Microcalorimetric experiments were p e r f o r m e d using a flow-through unit and a flow rate of 20 ml nutrient solution plus 20 ml air per hour each, achieved by means of a peristaltic pump. First growth characteristics were determined for b o t h strains. The resulting p o w e r - t i m e curves of typical experiments are given in Fig, 5. In the course of substrate degradation, T. interrnedia exhibited a p o w e r - t i m e curve with two peaks, accompanied by a large heat output. 6 D. Moreira and R. Amils, Int. 3. Syst. Bacteriol. 47, 522 (1997). 7 K. Milde, W. Sand, W. Wolff, and E. Bock, J. Gen. Microbiol. 129, 1327 (1983). 8 S. Wentzien, W. Sand, A. Albertsen, and R. Steudel, Arch. MicrobioL 161, 116 (1994).

[271

MICROCALORIMETRY

369

After exhaustion of the substrate, the heat output decreased rapidly to the baseline. In the experiment with a culture of T. versutus, the power-time curve increased gradually in the course of the experiment. After substrate consumption, the curve returned rapidly to baseline. In contrast to the experiment with T. intermedia, only a comparably small area, indicating a small total heat output, was registered for T. versutus in the course of substrate degradation. To demonstrate that the large heat output registered with T. intermedia resulted from attachment and biofilm formation, the experiment was repeated in the following way. From an external culture of T. intermedia, a suspension with growing, thiosulfate-oxidizing cells was split in two, each measured with an independent measuring unit of the microcalorimeter. In one unit the course of substrate degradation was recorded, as has been described previously. The same power-time curve was consequently recorded. In the other measuring unit, however, which was run under identical conditions, the flow of the culture solution was interrupted regularly and replaced by a cleaning solution [0.1 M NaOH/ 0.1% sodium dodecyl sulfate (SDS)] that had been shown previously to kill all attached bacteria and to remove most of the cell debris. 5After circulating this latter solution for about 20 min, a thorough washing with distilled water followed to remove all traces of the cleaning solution. Afterward the culture solution was again pumped through this measuring unit. The resulting power-time curve is shown in Fig. 5. As a consequence of the application of the cleaning solution, the heat output decreased after each application almost to baseline. After reswitching to the culture solution, a rapid increase was registered. The heat output soon reached a point where it leveled off to a slight increase. By connecting these points, a power-time curve resulted, which resembled the one obtained with cells of T. versutus. As a consequence, by applying the cleaning solution the attached cells of T. intermedia were removed from the system, rendering it free from their heat output. The curve, resulting from the connected points, reflects the heat output of the planktonic population. The latter was almost identical to the curve of the planktonic cells of T. versutus (not shown). In addition, the area below the curve allows the determination of the amount of energy consumed by attachment and excretion, and biofilm formation as well. In the present case, about 85% of the total heat evolved had been used for attachment, and so on, whereas only 15% resulted from substrate oxidation. Quantification of Microbial Activity Most techniques require the removal of a biofilm from the sample surface for determinations of microbial activity. Because this alters the environmental conditions for biofilm microorganisms, a microcalorimetric

370

PHYSICALMETHODS

[27]

procedure was developed that allows one to nondestructively determine microbial activity in biofilm samples. As a consequence, a realistic testing of a biocide treatment becomes possible. Furthermore, screening and optimization of other countermeasures against microbiologically influenced corrosion (MIC) and biofouling also become possible. In the case of a biofilm on metal surfaces, biological and chemical processes such as corrosion produce a measurable heat. To determine the biological and the chemical contribution to the heat output, mild and stainless steel (AISI 304) coupons were incubated in a miniplant. 9,1° A mixed culture consisting of the biocorrosion-relevant bacteria Desulfovibrio vulgaris (NCIMB 8457), T. intermedia, and the chemoorganotrophic biofilm isolate Ochrobactrum anthropi were used as inocula (108/ml each). Medium 9 containing these bacteria was pumped continuously through ring columns of the miniplant to produce a biofilm on the metal surfaces. A continuous change between anaerobic and aerobic conditions within the columns was produced by alternatively gassing with nitrogen or compressed air during the incubation. As a result, high cell counts of all of these bacterial species were achieved. After 6 weeks of incubation, biofilm samples were withdrawn and analyzed for cell counts, for mass loss, and for microbial (biological) as well as chemical activity (heat output). Microcalorimetric measurements were performed with a 25-ml stainless steel ampoule (TAMcylinder 2277-205). After sampling, the coupons (with biofilms) were put into the ampoule, together with 15 ml of sterile medium. 9 After stabilization of the heat output about 2 hr later, the value was registered. To differentiate between biological and chemical contributions to the heat output of biofilm samples, consecutive to the first measurement, a 24-hr incubation in 5% (w/v) formaldehyde was enclosed to kill all biofllm microorganisms. Afterward, the remaining (chemical) heat output was determined. The difference between the two values can be attributed to microbial activity. The total heat output in this example, originating from biofilms on mild steel coupons, was 30 /xW/cm 2. After formaldehyde incubation, a heat output of 14 /zW/cm 2 remained, indicating that biological and chemical reactions contributed to about 50% each to the total value (Fig. 6). Cell counts in the biofilm samples were reduced by the formaldehyde treatment to 102 cells/cm 2 (not shown). Because this cell number is below the microcalorimetric limit of detectability, it was proven that the remaining output 9H. von R6ge and W. Sand, in "Biodeterioration and Biodegradation, DECHEMA Monographs Vol. 133" (G. Kreysa and W. Sand, eds.), p. 325. VCH, Weinheirn, Germany, 1996. 10U. Eul, H. von R6ge, E. Heitz, and W. Sand, in "Microbially Influenced Corrosion of Materials" (E. Heitz, W. Sand, and H.-C.Flemming, eds.), p. 188. Springer-Verlag, Berlin, 1996.

[2 7]

MICROCALORIMETRY

mass loss rate x 10 [mg/cm2 x d cell count [log N/cm~ 7

heat output P [pW/cmO 20

371

/ l b i o l o g i c a l heat output

15

: chemical heat output

6

E:]total cell count

5

F~mass loss rate

4 10

3 2 1

/o

/

z

mild steel

stainless steel

FIG. 6. Biological and chemical heat output, cell counts, and mass loss of mild or stainless steel coupons with biofilms consisting of Thiomonas intermedia, Desulfovibrio vulgaris, and Ochrobactrum anthropi. Coupons were incubated for 6 weeks under alternating aerobic or anaerobic conditions in a miniplant.

resulted from chemical activity only (possibly from the chemical oxidation of reduced sulfur compounds or from the iron compound oxidation). The microbial heat output of stainless steel coupons with biofilms was 4.4/zW/ cm 2. Chemical heat output, measured after formaldehyde incubation, was only 0.6/.~W/cm z (Fig. 6). Also, in this case, cell numbers in the biofilm after formaldehyde treatment were too low (not shown) to produce a detectable heat output. Thus, in contrast to the experiment with mild steel, the biological activity a m o u n t e d to 86%. Biofilm samples on mild or stainless steel coupons incubated under fully aerobic or fully anaerobic conditions exhibited similar results (not shown). In the case of a biofilm on a mild steel coupon, the chemical activity generally contributed m o r e to the total activity (due to corrosion, as indicated by the high mass loss observed) than in the case of a biofilm on a stainless steel coupon (without corrosion) where microbial activity was the main source for the heat output. Summarizing, this technique allows a rapid detection and quantification of the ongoing processes in a biofilm on a steel surface.

Biocide Efficacy Testing The effects of chemical agents, biocides, or cleaning procedures are easily quantifiable too, as has been shown earlier for the formaldehyde

372

pI-IYSICALM~THODS

[271 70 6O

8

5O O

40 6

30

4

~

lO

0

I control

I Dilurit

I TM

I FA

I DHA

FIG. 7. Influence of four different biocides on microbial activity and cell counts of biofilm samples on mild steel. Coupons were incubated for 10 weeks with a mixed culture consisting of SRB and COT. Batch experiments with biocides lasted for 24 hr at 30°. Dilurit (500 mg/liter); TM (tetramethylammonium hydroxide, 500 mg/liter); D H A (1,8-dihydroxyanthraquinone, 4.8 mg/liter); F A (formaldehyde, 50,000 mg/liter).

treatment. Biocide application, to fight undesired biofilms, is still the most common practice in industry. Effective monitoring methods for an evaluation of biocide efficacy are lacking. Microcalorimetry offers a potential to fill this gap. For a demonstration, the efficacy of biocides against biofilm microorganisms on mild steel coupons was scrutinized. Coupons were incubated anaerobically in the presence of a mixed culture containing suffate-reducing bacteria (SRB) and chemoorganotrophic bacteria (COT): After 10 weeks, identical coupons with biofilms were treated with several biocides to screen for the most effective one. The biocides were commercial products with glutaraldehyde as the active compound (Dilurit, BK Ladenburg), a quaternary ammonium compound (tetramethylammonium hydroxide), 1,8-dihydroxyanthraquinone (an ancoupler of ATP synthesis for SRB11), and formaldehyde. The latter compound was added in a high concentration in order to reduce the cell counts of living microorganisms in the biofilm samples 11 F. B. Cooling, C. L. Maloney, E. Nagel, J. Tabinowski, and J. M. Odom. Appl. Environ. MicrobioL 62, 2999 (1996).

[2 7]

MICROCALORIMETRY

373

to such a low amount that chemical contribution to the heat output could be determined. As a result of a biocide application with a contact time of 24 hr, the microbial activity was in general reduced considerably (Fig. 7). The share of the chemical heat output only amounted to about 10% of the total heat output. Obviously, the biological activity was the dominating one. Cell counts of SRB remained stable in three of four experiments, despite biocide application, whereas those of C O T were reduced slightly. Only in the case of the formaldehyde application were the cell counts of both groups reduced strongly. After an exchange of the medium against a biocide free one and 7 days of further incubation, regrowth occurred, but microbial activity did not reach the initial level (not shown). Microcalorimetry, consequently, enables differentiation between killing or inhibition of the microbiota as a result of a biocide application. Thus, a direct monitoring of biocide action for determining the time, when a further application becomes necessary, is possible. In industries where biocide use is unavoidable, as in paper and pulp manufacturing, this possibility is of considerable advantage. On-line measurements of biocide action against biofilms by the flowthrough technique offer additional possibilities. A biofilm established on the inner surface of the gold tubing (in the flow-through cylinder, described earlier) allows screening for the optimal dosage of a biocide. This was demonstrated by experiments with Vibrio natriegens (DSM 759). The bacteheat output P [~tW]

1 20

110 100

addition of biocide ~"

-7

80 70 ~ heat output 60 ~frombi°filmcell:

40

~

~ -I-25 mg/liter biocide -o- 1O0 mg/liter biocide -~ 500 mg/liter biocide

50

30 20 10

0E 0

117

t 100

200

[

300

•

[

400

~?

j

-/

500

time [min] FIG. 8. Influence of biocide concentration on a Vibrio natriegens biofllm in continuous culture. Reprinted from H. von R6ge and W. Sand, J. Microbiol. Methods 33, 227 (1998).

600

374

PHYSICALMETHODS

1271

rium formed a biofilm inside the gold tubing. After a stable heat output signal had been obtained, only sterile nutrient solution 4 was further pumped (20 ml/hr), followed by an addition of 25 rag/liter biocide to the medium. At this concentration only a slight and transient decrease of microbial activity was measured (Fig. 8). A concentration of 100 mg/liter had a more pronounced effect (see Fig. 8), although a considerable amount of microbial activity was still detectable. Only with a biocide concentration of 500 rag/ liter was microbial activity almost totally abolished, remaining negligible until the end of the experiment. However, by replacing the biocide-containing medium against a biocide-free one (see earlier discussion), microbial activity increased within a 10-hr period. Obviously, some bacteria survived in the depth of the biofilm and were able to regrow. This technique allowed elaboration for this biocide. Furthermore, data demonstrated that some microorganisms remained alive within the (protecting) biofilm, inaccessible even to high biocide dosages. No other technique is known that allows such accurate determinations within such a short time. If it becomes possible to exchange the gold tubing by other materials, selective tests for adhesion and biocorrosion will allow for more insight into the interfacial processes governing biofilm formation and stability. S u m m a r y and Outlook Microcalorimetry is not yet a widely established technique in the field of biofilm research. This will change with time because of the potential the technique has to offer. Up until now, no other technique allows an on-line measurement of an attachment, which is also material specifc. Furthermore, the possibility of measuring aerobic and/or anaerobic metabolism and differentiating between biotic and abiotic reactions is also unique. The comparably short times needed for an experiment are also a strong argument in favor of microcalorimetry, in addition to the simple handling required.