APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, p. 1629-16350099-2240/92/051629-07$02.00/0

Measurements of the Distribution of Adenylate Concentrations andAdenylate Energy Charge across Pseudomonas aeruginosa Biofilms

S. L. KINNIMENT* AND J. W. T. WIMPENNYSchool ofPure and Applied Biology, University of Wales College Cardiff,

Cardiff CFI 3TL, United KingdomReceived 16 December 1991/Accepted 27 February 1992

Adenine nucleotide pools and adenylate energy charge distributions were determined by using a laboratory-generated quasi-steady-state Pseudomonas aeruginosa biofilm. The method used involved freezing andsectioning of the intact biofilm, followed by extraction and assay of the adenylates in the sectioned material.Results indicated an increase in adenylate energy charge of about 0.2 units from the bottom to the surface ofthe biofilm. However, energy charge values were generally low throughout the biofilm, reaching a maximumof only 0.6 units. Of the adenylates measured, AMP was the predominant nucleotide, especially in the deeperparts of the biofilm profile.

Biofilm is the general term that applies to microbialcommunities forming coherent layers on solid surfaces.These structures are ubiquitous and can lead to seriouseconomic loss to industry, besides posing health risks in thedental and medical spheres. These problems are exacerbatedbecause biofilms are generally less sensitive to antimicrobialagents than are freely suspended organisms (22).As a spatially heterogeneous community, very little is

known about the structure and physiology of biofilm. Anumber of experimental models which allow the investiga-tion of biofilm throughout the growth cycle exist (10, 24, 28).The constant-depth film fermentor developed by Coombeand colleagues (5) and by Peters and Wimpenny (23) gener-ates quasi-steady-state biofilm. This is defined on the basisthat total protein and viable count remain constant over aperiod of time. Biofilm can be of any chosen thickness up toabout 500 p,m.One indicator of the energetic status of living cells is the

adenylate pool (1). Sensitive assays which are capable ofmeasuring femtomole quantities of ATP exist (27). It was feltthat this level of sensitivity would allow us to measureadenylates in 12-p,m-thick cryostat sections of biofilm.Adenine nucleotides, especially ATP, play a central role

as intermediate carriers of chemical energy linking catabo-lism and biosynthesis. In vitro studies have revealed that theactivities of certain enzymes are affected by the concentra-tion of ATP; others are affected by ADP or AMP. Theadenylate energy charge (ECA) is a measure of the effect ofthe ratios of adenosine phosphate concentrations on the rateof cellular metabolism (12) and is defined as follows: ECA =

([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]). The ECA iSa linear measure of the total amount of potential energymomentarily stored in the adenine nucleotide pool. It is adimensionless number which ranges from 0 to 1 (2).

Since there is claimed to be a positive correlation betweenthe growth potential or functional capacity of a cell and itsECA (4), these measurements provide a framework forestimating metabolic potentials of naturally occurring micro-bial populations.

This article describes a method for freezing and sectioningbiofilm generated in the constant-depth film fermentor. By

* Corresponding author.

using this technique, adenylate pools and energy chargevalues have been determined as a function of depth througha Pseudomonas aeruginosa biofilm.

MATERIALS AND METHODS

Organisms. A metalworking fluid isolate, identified as P.aeruginosa (14), was used to inoculate the fermentors. Thismicroorganism was selected because it emerged as thepredominant bacterium in the development of a stable bio-film derived from contaminated metalworking fluids. Themicroorganism was stored by freezing on glass beads at-70-C (11).Media. The amine-carboxylate medium used was a simpli-

fied simulated cutting fluid based on a mixture of fatty acids(predominantly oleic acid) and triethanolamine supple-mented with mineral salts and vitamins. The medium con-tained, per liter of distilled water, an 0.085% (vol/vol)mixture of cutting fluid components consisting, by weight, of60% triethanolamine and 40% fatty acids (of which 66% wasoleic acid) and (in grams) the mineral salts FeSO4. 7H20(1.5 x 10-2), MgSO4. 7H20 (0.2), ZnSO4- 7H20 (1.75 x10-3), MnSO4 * 4H20 (1 X 10-4), CUS04 5H20 (1 X 10-4),NH4Cl (1.0), K2HPO4 3H20 (1.2), and CaCl2- 6H20 (1 x

10-2) and the vitamins inositol (1 x 10-2), calcium panto-thenate (2.0 x 10-3), and biotin (1.0 x 10-5). For platecultures, the same medium was used, except that the cuttingfluid components were increased to 2% (vol/vol) and 2%(wt/vol) agar (Difco) was added. Cutting fluid componentswere supplied by Castrol Research Laboratories (Whitch-urch Hill, Pangbourne, Reading, United Kingdom). Otherchemicals were AnalaR grade (BDH) unless otherwisestated.

Biofilm growth. Biofilm formation. The constant-depth filmfermentor used by Kinniment and Wimpenny (14) was usedto generate a reproducible biofilm. The constant-depth filmfermentor consists of an enclosed rotating stainless steel discin which are located 15 removable biofilm pans. Each biofilmpan is drilled with a central threaded hole, and around theedge of this are arranged six biofilm plugs. The latter arerecessed to the selected depth up to 500 p.m by using a

specially machined tool. Biofilm develops on the flat surfaceof the plug in the recessed space. The steel disc rotatesbeneath angled polytetrafluoroethylene (PTFE) scraper

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1630 KINNIMENT AND WIMPENNY

blades which remove excess growth, maintaining the biofilmat a constant depth. Medium drips onto the steel disc directlyin front of one scraper blade. The latter distributes it over thebiofilm pans, and any excess is wiped away. Samples ofbiofilm can be taken by replacing pans at selected timeintervals by using two sterilizable stainless steel tools. Athreaded extractor tool is used to remove and replace thepans, while a flat tamping tool is used to push replacementpans down flush within the turntable. The fermentor is heatsterilizable, the design allowing aseptic removal and replace-ment of biofilm pans. In addition, the system is operatedunder controlled gas phase and temperature conditions.The inoculum for the fermentor was prepared by asepti-

cally flooding a 48-h-streaked amine-carboxylate plate of P.aeruginosa with 2 ml of sterile Tris-HCl buffer, pH 8.8. Onemilliliter of the homogenized mixture was then removed andresuspended in 4 ml of sterile buffer.

This inoculum was allowed to recirculate through thefermentor in 500 ml of medium for 12 h. The main 10-literbatch of sterile medium was then aseptically connected tothe fermentor. At the same time, the waste outlet port wasconnected to a large sterile waste receiver.The fermentor was operated with a medium flow rate of 1

ml min-1, an air flow rate of 250 ml min-1, a disc rotationspeed of 3 rpm, and a constant temperature of 30°C. Samplepans were taken from the fermentor at selected time inter-vals. Each pan contained six PTFE sample plugs recessed toa depth of 300 p.m. The diameter of the plug surface was 4.75mm, giving a surface area of approximately 17.72 mm2 and abiofilm volume, if the available space was completely full, ofabout 5.32 mm3.The fermentor was run to quasi-steady state before sam-

ples were removed for sectioning. The fermentor was closeddown after 10 liters of medium had passed through thesystem.

Estimation of total protein accumulation. Protein analysisbased on a modified Lowry method was carried out (18). Asample pan was removed and four plugs were carefullypushed out of the pan, with the biofilm still intact on thesurface. Protein on each plug was assayed separately bybeing boiled in 0.5 ml of 1 M NaOH for 5 min. These wereeach diluted fourfold in 1 M NaOH, and 0.5 ml (each) of thediluted mixtures was then used in the standard Folin proce-dure. Protein standards containing 5 to 150 p.g of protein(bovine albumin fraction five; BDH) were prepared eachtime the assay was performed. The absorbance values forthe biofilm samples were then read against these standardcurves.Viable count. The two remaining sample plugs from each

pan were removed with flame-sterilized forceps. To dispersethe biofilm, the plugs were transferred to 10 ml of steriledeflocculant solution in a universal container. The defloccu-lant consisted of 0.01% (wt/vol) Cirrasol (ICI Organics) in0.01% (wt/vol) sodium pyrophosphate as described by Gay-ford and Richards (8). Approximately 250 2.5- to 3.5-mm-diameter glass beads (BDH) had been added to this solution.Sample plugs and biofilm were then agitated by vortexmixing for 90 s. After treatment, the sample was seriallydiluted in Tris-HCl buffer at pH 8.8 and 0.1 ml of eachdilution was plated out in triplicate onto amine-carboxylateplates. The plates were incubated at 30°C for 24 to 48 h, andCFU were determined.Method for sectioning sampled biofilm. Before sectioning, a

method for freezing and mounting the biofilm to a cryostatsample holder was developed, and it is described below.Agar (2% [wt/vol]) (Oxoid) plates were poured aseptically

to a depth of about 5 mm. Agar plugs were cut from theseplates with a sterile no. 15-size cork borer. The plug of agarwas removed from the petri dish with a sterile spatula,inverted, and placed on the upper surface of the agarremaining in the petri dish. A circle of lens tissue was placedon top of the agar plug, and a drop of water was placed on itto moisten it. With forceps, a cryostat sample holder wastouched firmly to the surface of the damp lens tissue. Thewhole assembly was inverted and the sample holder, withthe agar layer attached, was then lifted away from thesurface of the agar plate and placed in a polystyrene con-tainer.Sample pans containing quasi-steady-state biofilm were

removed from the fermentor at 55 and 105 h. The PTFEsample plugs were carefully removed from the sample pans.Sample plugs, biofilm upper surface down, were positionedbetween a pair of forceps which were then placed in the jawsof a micromanipulator. The whole assembly was placeddirectly above the sample holder, and 1 drop of water wasquickly placed onto the surface of the agar. By using themicromanipulator, the PTFE sample plug with biofilm at-tached was lowered onto the drop of water so that the biofilmsurface was just submerged. Liquid nitrogen was thenpoured into the polystyrene container to the depth of theagar plug, and the agar and biofilm were left to freeze. Oncethe biofilm was frozen, the forceps were removed from themicromanipulator and the PTFE sample plug was pulledaway from the frozen biofilm.A modification of this method was used for one set of

replicate samples; the main difference was that the sampleholder was chilled to -37°C prior to being mounted onto theagar plug. The biofilm, previously frozen in liquid nitrogen,was then stuck onto the frozen agar surface with a drop ofwater, and once frozen the PTFE sample plug was removed.The time between taking samples of and freezing the

biofilm was approximately 2 to 3 min for the first method and1 to 2 min for the second. The sample holder was fixed to themicrotome in a Starlet 2212 cryostat (Bright InstrumentCompany Ltd., Huntingdon, United Kingdom), and theknife blade was adjusted to cut 12-p.m, entire, horizontalbiofilm sections. Sections were removed with a clean sterilemicrospatula. In the first method, sections were removed inthrees; in the second method, the first section was discardedand subsequent sections were then removed in pairs.A total of six biofilm profiles were sectioned, three 55-h

and three 105-h samples.Method of adenylate extraction of sectioned biofilm. The

method described below was adapted from that used byLundin and Thore (19) and Scourfield (25). Each set of twoor three sections was transferred to 0.5 ml of 2.3 M perchlo-ric acid containing 6.7 mM EDTA. The mixture was stirredconstantly for 15 min on ice and centrifuged for 3 min in anEppendorf bench top centrifuge at approximately 13,000 xg, and the supernatant was neutralized with 2 M KOH-0.5 Mtriethanolamine buffer. The mixture was left on ice for 5 minuntil all the potassium perchlorate had precipitated and thenwas centrifuged for a further 2 min. The supernatant wasfinally frozen at -70°C until needed.Adenylate and energy charge analyses of sectioned biofilm.

ATP, ADP, and AMP were assayed in triplicate by standardmethods (19, 20) by using a purified firefly lantern extract(Bio-Orbit Oy, Turku, Finland). An LKB 1251 computer-interfaced luminometer (Bio-Orbit Oy) was used for thebioluminescence assay. One hundred twenty-five microlitersof the assay mixture was placed in a cuvette in the sampleholder of the luminometer. Once the sample had been loaded

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ADENYLATE MEASUREMENTS THROUGH BIOFILM 1631

1000

10 I I _

0 25 50 75 100 125 150 17'

Time (h)

.0I

lo 9

X. 10 8.

8 1 70I

52

10 6.

0I I .

25 50 75 100

Time (h)

I I

125 150

'5

175

FIG. 1. (a) Typical growth curve (each value expressed as mi-crograms of protein per plug) of the biofilm. Error bars showstandard deviations based on four sample plugs. (b) Viable count,expressed as CFU per plug (average for two sample plugs).

into the detection chamber, 100 ,ul of ATP monitoringreagent (Bio-Orbit Oy) was rapidly injected, the sample andreagent were pulse-mixed, and the 10-s integral of lightemitted from each assay was recorded immediately. Allassays were performed at 25°C.ATP was measured directly; ADP was determined after

conversion to ATP by using pyruvate kinase (Sigma) andphospho(enol)pyruvate (Sigma); AMP was measured bybeing converted to ADP and then to ATP by using adenylatekinase (Sigma) together with the pyruvate kinase and phos-pho(enol)pyruvate. Internal standards were assayed to de-tect possible inhibition of the bioluminescence reaction, andan ATP calibration curve was also constructed.

RESULTS

Biofilm growth. The P. aeruginosa biofilm was grown inamine-carboxylate medium for a period of 150 h. Quasi-steady-state conditions were reached after about 45 h. Qua-si-steady-state protein values plateaued at about 250 jig perplug (Fig. la). The viable count was about 2 x 108 CFU perplug. The slope of the log of viable count versus time

suggests a doubling time of approximately 3.5 h (P.max = 0.2h-1) (Fig. lb).

Analysis of adenylate concentration through the biofilm.The results of adenylate measurements showed randomfluctuations (Fig. 2a and b); therefore, the data were

smoothed by taking a three-point moving average. Althoughthe first and last datum points are lost by using this ap-proach, trends in adenylate levels and ECA values are easierto discern.

Errors for the moving average adenylate concentrationsand the ECA values were estimated by calculating the rootmean square deviation of the original data from the movingaverage data for the former and then the ratios of randomvariables for the latter (13). An estimate of the probability ofany significant difference in ECA between the base andsurface points was calculated by comparing two samples,and then standard Student's t tables were consulted.Adenylate concentrations for two of the six biofilm pro-

files are shown in Fig. 2, as examples. For the remainingreplicate samples, the total adenylates and the percentagesof ATP, ADP, and AMP in relation to the total adenylatesare presented in Table 1. The results for total adenylates andindividual adenylates and the fraction of each expressed as a

percentage of the total adenylates through the biofilm profileshow similar trends for all six profiles (Fig. 2c to f and Table1). Different biofilms were used for each profile, so somevariation is to be expected.

In the 55-h biofilms, concentrations of total adenylates,AMP, and ATP rose from the base and reached a maximumnear the middle of the biofilm. By 105 h these peaks hadmoved toward the surface. ADP stays relatively constant,showing a small peak at the center of the 55-h biofilms andnear the surface of the 105-h biofilms. All adenylate levelsfall very near the surface of the biofilm in both sets ofsamples (Fig. 2c and d and Table 1).

In the 55-h biofilms, the percentage of AMP in relation tothe total adenylates drops toward the surface of the biofilm,that of ATP increases, and that of ADP stays relativelyconstant. For the 105-h biofilms, ATP and AMP follow asimilar course but ADP either increases or decreasesthrough the center before levelling off again near the surface.The dominant adenylate appeared to be AMP in all samples(Fig. 2e and f and Table 1). ATP levels were generally low.Values ranged from about 1 to 30 pmol per section. While the55-h biofilms appeared to have reached a quasi-steady state,judging by the protein and CFU values, the number ofsections taken from the 55-h samples was generally smallerthan that of the 105-h samples. (Fig. 1 to 3).

Examination of ECA through the biofilm. Energy chargevalues showed a consistent increase across both sets ofbiofilm from base to surface. While the range was not large,it represented a value of about 0.2, from values of 0.22 to0.28 near the base to 0.4 to 0.45 near the surface of the 55-hbiofilms (Fig. 3a) and 0.17 to 0.35 near the base to 0.37 to 0.6at the surface of the 105-h biofilms (Fig. 3b). The probabilityof a significant difference between the points at the biofilmbase and the surface ranged from 70 to 99.9%, with an

average value of 90%.

DISCUSSION

A number of different sectioning procedures were testedbefore the methods described in this article were selected. Itwas not always easy to produce complete sections, andoccasionally material was lost during the transfer of sectionsfrom the microtome blade to the sample container. The raw

(a)

1-1

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(b)

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1632 KINNIMENT AND WIMPENNY

55 h orginal data(c)

55 h moving average data(e)

C)

CuI0

55 h moving average dataau -

70 base surface

60 4+ +50

40

30 =

20 -

10

0 I0 10 20 30 0 10 20 30 0 10 20

Section number Section number Section number30

(D105 h original data

Section number

70-

0-

c-% 50-

1 40-V 30-o 20-

10 -

0Section number

105 h moving average data

I n10 20Section number

FIG. 2. Graphical examples of adenylate concentrations through the biofilm. (a and b) Concentration of adenylates in each section (originaldata) (error bars show standard deviations of triplicate samples). (c and d) Concentration of adenylates in each section (moving average data)(error bars show root mean square deviations of the original data from the moving average data). (e and f) Adenylates expressed aspercentages of the total. Symbols: E], ATP; *, ADP; *, AMP; O, total adenylates.

data therefore show considerable fluctuations. However,some of these fluctuations may also be due to variations inadenylate extraction. Some of the variability in the resultshas been removed by calculating a three-point movingaverage .of the data. The deviation of the original data fromthe moving average data was then calculated. Although thisprocedure is statistically more complicated, especially withregard to the calculation of the ECA deviation, these proce-dures enable any trends to be clearly visualized and anystatistical significances in these trends to be estimated.

All of the adenylates were present throughout the biofilm,suggesting that a considerable proportion of the bacteriawere viable throughout the structure. The observation thattotal adenylates peak at some position across the biofilmwhich depends on biofilm age is interesting. The observedmaximum is near the middle at 55 h but approaches thesurface of the biofilm in the older samples.

It is possible that this position corresponds to a peak in"healthy" microbial biomass. This suggestion has also beenmade by Kornegay and Andrews (16), who showed that oncebiofilm growth had reached a critical thickness of 70 pum, anyincrease did not lead to an increase in the rate of bothdissolved oxygen and substrate removal. This indicated thatthe biofilm had a limited "active" layer. Differences inmicrobial biomass and in cell density have been observed inelectron and light microscope studies and more recently byusing sectioning techniques with cryoprotected biofilm todetermine the number of viable cells through the biofilm

profile (15). The decrease in adenylates near the surfacecould be explained by irregular surface layers which produceincomplete sections.As a proportion of the total adenylates, the AMP level is

higher in the lower depths of the biofilm and decreasestoward the surface. There is an increase in the proportion ofATP toward the surface, while that of ADP remains fairlyconstant.The change in ECA values from base to surface, although

relatively small (roughly 0.2 units), could suggest that thedeeper cells in the biofilm become diffusion limited fornutrient and/or oxygen. The predominantly low ECA valuesuggests that the cell population has a low energy status. Itis unlikely that this is due to freezing the biofilm, since a lowenergy charge value for unfrozen, immediately extractedentire biofilm has previously been observed (15). Addition-ally, Dobbs and LaRock (7) reported that rapid freezing ofmarine sediment, in liquid nitrogen, did not lead to asignificant loss of ATP relative to that found on immediateextraction of unfrozen samples. While the turnover in ade-nylates is rapid, it was considered that the sampling time of1 to 3 min before freezing would have little effect on theproportions of adenylates through the biofilm. This wasbecause the biofilm is effectively a buffered system by virtueof the relatively slow rates of solute exchange due todiffusion processes.Low ECA values have been noted before with spores,

which may have ECA values of less than 0.1 (26). Chapman

(a)1

* .2

5)

5).r2,

(b)120

rA100.n0800° 80

10cAf4

'e 53

base surface

4 44

30

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ADENYLATE MEASUREMENTS THROUGH BIOFILM 1633

TABLE 1. Additional data for replicate samples

Replicate no. and Section no. Total adenylates RMSDa % ATP % ADP % AMPsample time (pmol/section) (pmol/section)

Replicate 1, 55 h

Replicate 2, 55 h

Replicate 1, 105 h

Replicate 2, 105 h

58

111417202326

19.1824.5921.3125.1127.7828.7522.4318.00

4.56.58.5

10.512.514.516.518.520.5

58

111417202326293235

4.56.58.5

10.512.514.516.518.520.522.524.526.528.530.532.5

78.5475.08

102.92113.72123.17105.6291.3373.2153.62

6.147.04

11.5321.4032.7344.6657.6272.2880.0870.7153.05

31.1940.5950.1656.4861.0265.2866.6973.0580.0789.99107.37106.15105.4586.3880.74

7.915.902.802.522.742.841.961.60

12.7715.8220.0419.2314.615.175.594.763.91

2.893.855.457.76

11.2010.149.858.318.136.975.87

6.716.265.314.576.927.116.765.147.449.209.208.377.457.967.61

8.658.178.88

10.1211.5014.3119.9825.34

12.2214.2517.2920.9624.3926.5430.5431.3632.71

11.9410.419.264.677.748.13

17.5520.1622.6920.7520.65

13.4217.4418.4518.3020.3722.8830.8429.6230.8526.9328.8329.2031.3633.6334.55

25.6327.8734.4136.7035.2533.6737.2640.02

65.7363.9756.7153.1853.2552.0142.7534.63

16.7317.8214.1812.2810.7711.9012.8013.6713.76

71.0567.9368.5366.7664.8461.5656.6654.9753.53

13.6013.5628.9838.8041.8642.9738.2233.6231.5130.8432.97

74.4676.0361.7756.5250.3948.9044.2346.2245.8148.4046.38

37.8131.7226.0723.6722.7322.0020.6418.3218.5517.7817.7816.6019.1121.2822.94

48.7650.8455.4858.0356.9155.1248.5252.0750.6155.2953.3954.2149.5345.0942.51

a RMSD, root mean square deviation of original data from moving average data.

and colleagues (4) showed that exhaustion of glucose fromthe growth medium caused the intracellular ECA of Esche-nchia coli to decrease from about 0.8 to a value of 0.6 to 0.5.The lower value and viability were then maintained for 60 to80 h. Low ATP values have also been reported for cellsunder starvation conditions which recovered rapidly uponreplenishment of the nutrient supply (6). Whole biofilm oforal bacteria investigated by Wimpenny and colleagues (29)showed low steady-state film ECA values similar to thoseobserved in this study. In their work, by using a similarperchloric acid extraction method, high ECA values could beinduced in young biofilm exposed to high glucose concen-trations.The biofilm generated in this study was produced by P.

aeruginosa under the special circumstances of low nutrientconcentration, and it is possible that there could be large

numbers of dead cells trapped within the biofilm, especiallyin the basal regions. It is clear, however, that the biofilm wasgrowing and that it retained a large population of viable cells(ca. 2 x 108 per plug). Marshall (21) showed that starvedbacteria adhering to surfaces were able to grow to normalsize and to complete a number of cycles of cell division andconcluded that if the limiting substrate were continuallyreplenished, as in natural, flowing systems, rapid biofilmdevelopment would be expected.These observations raise the interesting question of what

is normal for energy charge values in a structured, nutrient-limited habitat. There is some evidence for a wide range inECA values in other structured ecosystems. Thus, Witzel(30) examined 356 water samples from several deep lakes inGermany. Witzel's results indicated that the ECA valuesranged from 0.16 to 0.97. The highest ECA values were

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1634 KINNIMENT AND WIMPENNY

55 h moving average data

I I , ,

0 10 20 30

(b)

1.0

0.9

c 0.8

4, 0.7

u 0.6

0 0.50

2 0.4

0 0.3

0.2

0.1

0.0

105 h moving average data

0 10 20 30 40

Section number Section number

FIG. 3. ECA values across the biofilm. Error bars show root mean square deviations of original data from moving average data, which werecalculated by using the ratios of random variables (13). Symbols: *, biofilm sample 1; El, biofilm sample 2; a, biofilm sample 3.

measured in samples collected in the metabolically activeepilimnion. In deep water samples, values were generallylower than 0.6.

It has been proposed that bacteria associated with slowgrowth rate and nutrient deprivation have a lower sensitivityto antibacterial compounds (3). Since biofilms often form inlow-nutrient conditions (9, 17), the low ECA values observedin the biofilms reported here and those of the oral bacteriamay support this theory.The methods described in this communication for freeze

sectioning, coupled with the constant-depth film fermentor,have been shown to be useful tools for the study of thephysiology of biofilms.

ACKNOWLEDGMENTS

We thank K. Slater for helpful advice, T. Iles for statisticalguidance, and T. Bater and Bio-Orbit Oy for the loan of the 1251LKB luminometer system. We particularly appreciate the help andadvice given to us by P. Ruane and C. Townsley on behalf of theInstitute of Petroleum.

This research was supported by a contract from the Institute ofPetroleum, which we gratefully acknowledge.

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15. Kinniment, S. L., and J. W. T. Wimpenny. Unpublished data.16. Kornegay, B. H., and J. F. Andrews. 1968. Kinetics of fixed-film

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