Vol. 43, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1982, p. 6-130099-2240/82/010006-08$02.00/0

Method for Studying Microbial Biofilms in Flowing-WaterSystems

KARSTEN PEDERSEN

Department ofMarine Microbiology, University of Goteborg, Carl Skottsbergs Gata 22, S413 19 Goteborg,Sweden

Received 6 July 1981/Accepted 21 September 1981

A method for the study of microbial biofilms in flowing-water systems wasdeveloped with special reference to the flow conditions in electrochemicalconcentration cells. Seawater was circulated in a semiclosed flow system throughbiofilmn reactors (3 cm s-1) with microscope cover slips arranged in lamellar pilesparallel with the flow. At fixed time intervals cover slips with their biofilm wereremoved from the pile, stained with crystal violet, and mounted on microscopeslides. The absorbances of the slides were measured at 590 nm and plotted againsttime to give microbial biofilm development. From calibration experiments astaining time of 1 min and a rinse time of 10 min in a tap water flow (3 cm s 1) wereconsidered sufficient. When an analysis of variance was performed on biofilmdevelopment data, 78% of the total variance was found to be due to randomnatural effects; the rest could be explained by experimental effects. The absor-bance values correlated well with protein N, dry weight, and organic weight intwo biofilm experiments, one with a biofilm with a high (75%) and one with a low(-25%, normal) inorganic content. Comparisons of regression lines revealed thatthe absorbance of the stained biofilms was an estimate closely related to biofilmdry weight.

The development of microbial biofilms inflowing-water systems has been the subject ofmany investigations, usually to elucidate a largenumber of undesirable effects concomitant withgrowing biofilms. Increased flow resistance, in-duction and acceleration of corrosion processes,decreasing heat exchanger capacity, and clog-ging of filters are examples of problems causedby biofilms.

All biofilm studies require more or less com-plicated devices both for the biofilm cultivationand for the study of effects caused by microbialbiofilms. Methods to determine biofilm mass asa function of time and regulating factors are alsorequired. The design of these devices and meth-ods depends very much upon the aim of thebiofilm study. Those described in this paperhave been designed for the study of factorsregulating biofillm development under flow con-ditions as in electrochemical concentration cells.A concentration cell consists of a number of

anion and cation-exchange membranes. Themembranes are alternately arranged in lamellarpiles between an anode and a cathode with saltwater and freshwater alternately flowing overthe membranes. By use of such cells naturalseawater and freshwater can be utilized for theproduction of electrical power (14).The availability of ion-exchange membranes

with the efficiency required for a useful concen-

tration cell is very limited, so I constructedlamellary glass piles that imitate the flow condi-tions apparent in a concentration cell, and I usednatural seawater to develop the method.

MATERIALS AND METHODSBioffim reactors. Microscope cover slips (60 by 24

by 0.15 mm) were fitted into acrylic plastic holdersforming two parallel test piles, each with room for 19slips (Fig. 1). The distance between the slips was 1mm.To get even and comparable surface energies

throughout manufactured batches of slips, they wereheated in a muffle furnace at 550°C for 6 h, resulting inglass surfaces on which water drops spread complete-ly. The test piles were placed in flow cells, which,together with the diffusors and stabilizers describedbelow, are denoted as biofilm reactors. At the flowvelocity used, 3 cm s- , the flow is laminar. Toseparate the flow at the inlet of the reactor, threediffusors with different hole patterns were used (Fig.1). Finally, a laminar flow between the slips wasestablished with flow stabilizers that, except for theirlength of 32 mm, were identical to the test pile (60mm). The hydrodynamic considerations are adoptedfrom Vennard and Street (18). Since the flow directionthrough the reactor was alternated (see circulatingwater system), diffusors and stabilizers were placed onboth sides of the test pile. The flow pattern through thecell was visualized by pouring Difco agar into thewater pumped through the cell and observing withperpendicular illumination. No inequalities in the flowwere seen. To avoid sedimentation effects and air

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FIG. 1. The biofilm reactor, made of acrylic plastic.

bubbles on the slips, they were placed in parallel withthe gravitational force during the experiments.

Sampling. At fixed times a desired number of slipswere taken out for biomass determinations. Normally,one sample consisted of two slips, one from each ofthe two parallel piles. The sampled slips were replacedwith new ones so that the flow conditions through thepile were maintained.

Circulating water system. Seawater was collected byboat from a depth of 2 m in the outer coast line ofGothenburg (57°39.0' N, 11°36.5' E) every 7 to 10days. A 200-liter tank was continuously fed with 30liters of the water a day, and a corresponding waterquantity was expelled by brim drainage. From the tanka 316 stainless steel Teflon impellar pump (LiquifloSeries 86 pump) circulated the water in the watersystem constructed of polypropylene tubes (0 10-mmEastman impolen tubing; ITE), nylon tube fittings(Jaco), and stainless steel valves (Fig. 2). Water circu-lation in the tank was maintained by the return flowfrom the system. Before entering the reactors, thewater was passed through one of two parallel 125-,urmstainless steel ifiters, otherwise the edges of the flowstabilizers became covered with fibrous material dis-turbing the flow. The ifiters were automatically re-flushed every 12 h. The flow velocity through thereactors, 3 cm s-1, was regulated by valves and pumpspeed and controlled by flow meters (Rota-meterseries 2000, ±4%). The tank and the pump were kept

in a cold-storage room at 8°C; the rest of the systemwas placed in the laboratory. The resulting watertemperature in the system was 17.3 ± 0.°C. Thebioffilm reactors were placed in series with the circulat-ing system (Fig. 2). To avoid transport gradientswithin or between the reactors, the flow directionthrough the reactor series was alternated every 12 h.

Biofilms growing on the slips decrease the distancebetween the slips. It could be suspected that above acertain biofilm thickness, the hydraulic performancethrough the reactors may be impaired. The system wasinvestigated for such effects in the following way.Manometers were connected to the inlet and outlet ofthe circulating water system indicating an inlet pres-sure of 86.66 kPa and an outlet pressure of 38.66 kPa;the resulting pressure difference of 48 kPa did notchange significantly during any of the growth periods.During one of the growth experiments a piece of iron

tubing was placed in the system. The rust that devel-oped and circulated in the system resulted in a biofilmwith a high inorganic content. In this manner theanalytical methods could be tested on a biofilm with ahigh (75%) inorganic content and on a biofilm with alow (25%, normal) inorganic content.

Blofilm experiments. Four growth experiments werecompleted. The first involved calibration of a stainingprocedure, developed as a method for biofilm massdetermination. In the second growth experiment ananalysis of variance was performed, and the variance

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FIG. 2. Schematic diagram of the circulating water system.

components were calculated. To accelerate the biofilmdevelopment in these two growth experiments the feedwater was enriched with 10 ,ug of glucose per ml and10 ,ul of nutrient broth (Difco) per ml, resulting inmature biofilms covering the slip surfaces after 10days.The last two growth experiments were used to

correlate growth, measured as stained and unstainedbiofilm on the slips, with the same growth measured as

micrograms of protein N per square centimeter of slipand micrograms of dry weight and micrograms oforganic weight per square centimeter of slip undergrowth conditions without nutrient enrichments. Pilotexperiments revealed that a growth time of about 25days then was needed to acquire a mature biofilm.

Calibration of the staining procedure. According tothe calibration experiments described below, the fol-lowing staining procedure was developed. The sam-

FIG. 3. Cover slip rinse, made of acrylic plastic.

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MICROBIAL BIOFILMS IN FLOWING-WATER SYSTEMS 9

pled slips with biofilm were stained for 1 min in acuvette with Huckers crystal violet, a basic dye withmaximum absorbance at 589 to 593 nm (3). The excessstain was rinsed off in a specially designed cover sliprinse (Fig. 3) at a flow of 3 cm s-' for 10 min.Subsequently, each slip was dipped in distilled water,air dried (20°C), and mounted with Permount mountingmedium between a microscope slide and a clean coverslip. Finally, the absorbances of the slides were mea-sured at 590 nm on five fixed points of each slip(Beckman DU spectrophotometer completed withMultilog 311 and Multiblank 171, both from OptilabAB, Sweden).Four reactors were used for the calibration. After 10

days, a series of 9 or 10 slips from reactors 1 and 2were stained at different times and rinsed for 10 min; astaining time of 1 min was judged sufficient (Fig. 4).Also, a series of 9 or 10 slips from reactors 3 and 4were stained for 1 min and rinsed at different times.The absorbance decreased exponentially with time; 10min was considered as a sufficient rinsing time, enoughfor handling 18 slips with maintained accuracy (Fig. 4).

Analysis of variance experiment. After 10 days ofgrowth all slips in five reactors were sampled andstained. A hypothesis of effects that could have affect-ed the absorbance measurement data is summarizedin the following analysis of variance model: Aijkl = m+ Rj + Pji + Sijk + Eijkl where Aijkl is the Ithmeasurement on the kth slip in the jth height positionin the ith reactor; m is the overall mean; Ri is the effectfrom the ith reactor; Pji is the effect from the jth heightposition in the ith reactor; Suk is the effect from the kthslip in the jth height position in the ith reactor; and Ejjklis the residual. It is assumed that all effects are random(16). An analysis of variance and an estimation of thevariance-components were executed by using the sta-

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Days

FIG. 4. Absorbance of biofilm slips stained fordifferent times and rinsed for 10 min (0) or stained for1 min and rinsed for different times (0). The barsindicate standard deviations for 9 or 10 slips.

tistical analysis system (8) on the absorbance dataobtained.

Correlation experiments. The two correlation experi-ments lasted for 21 and 25 days. Seven reactors wereused. During the first experiment an iron tube wasinserted, and the developed biofilm was denoted ironwater biofilm (IWB). The biofilm developed during thesecond experiment was denoted normal water biofilm(NWB). From each reactor, pairs of slips were sam-pled on each sample occasion and treated as in thestaining procedure, but without the staining step.Subsequently the following treatments were per-formed. Biofilms from reactors 1 and 2 were analyzedfor protein N content, reactors 3 and 4 were used forweight determinations, slips from reactors 5 and 6were stained and mounted, and finally, slips fromreactor 7 were mounted but without stain.

Protein assay. A heated biuret-Folin assay (5) wasperformed at the end of the growth periods. The slips(stored at -20°C) were thawed and crushed into thereaction tubes. To ensure that the biofilm protein cameoff the slips into the solution, 30 g of sodium deoxy-cholate (8) per liter was added to the reagent solutionin the protein assay.Weight determinations. The slips in question were

weighed (Mettler ME 30 ± 1 ,ug) before being put in thepiles. The sampled slips were dried at 70°C for 6 h andweighed to give the dry weight of the biofilm. Re-weighing after 12 h in a muffle fumace at 450°C gavethe ash weight. The difference between dry weight andash weight was taken as the organic weight.The correlation calculations included not only the

correlation of the total absorbance with protein N, dryweight, and organic weight, but also the correlationbetween the total absorbance minus the absorbance ofunstained biofilm and protein N, dry weight, andorganic weight. This was done because part of theabsorbance was due to the stain and the rest to thebiofilm itself.

RESULTSCharacters of the biofilms. At the end of the

growth experiments the four biofilms developedwere studied in an inteiference contrast micro-scope. Their thickness was approximated byfocusing the top and bottom of the biofilm; thedifference read on the fine-adjustment knob gavethe thickness. The characters for each biofilmare summarized below.

(i) Growth experiment 1. At the bottom therewere bacteria and some protozoa evenly distrib-uted and firmly linked with the surface. At thetop a network of a filamentous bluegreen bacte-rium was spread. The thickness of the film was20 to 40 ,um.

(ii) Growth experiment 2. A dense cover ofbacteria in aggregates was firmly linked with thesurface and many protozoa, but few species.The thickness of the film was 50 to 70 ,um.

(iii) Growth experiment 3. There were fewbacteria, much deposited undifferentiated mate-rial in aggregates loosely linked with the surface,and very few protozoa. The thickness of the filmwas 30 to 50 ,um.

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TABLE 1. Results from analysis of variance with pertaining variance component estimationa

ource rees Sum of Mean F-testc F pr> F Expected mean Variancefreedom squares square square model component

R 4 0.052026 0.013007 MS (R) 1Var(residual) + Var(R) 0.000057MS [S(RP)] 5.0000 5 var[S(RP)]

+ 190 var(R)p 18 0.047176 0.002621 MS (P) 1.19 0.2744 Var(residual) + Var(P) 0.000008

MS (S(RP)] 1.9024 var[S(RP)]+ 50 var(P)

S(RP) 167 0.368349 0.002206 MS [S(RP)| Var(residual) + Var[S(RP)] 0.000235MS(esidal)2.14 0.0001 5 var[S(RP)]

Residual 760 0.782367 0.001029M (eiul

Var(residual) Var(residual) 0.001029

a The total number of observations was 950, distributed over 190 slips in 19 height positions in five reactors.The overall mean was 0.410. Var, Variance.

b R, Effect from reactor; P, effect from height position; S(RP), effect from slips, nested within R and P.c MS, Mean square.d Probability of getting an F-value smaller than that obtained.

(iv) Growth experiment 4. The highest diversi-ty of the four biofilms appeared, with bacteria inaggregates, solitary, stalked, and filamentousbacteria, firmly linked with the surface andmany protozoa of several species. The thicknessof the film was 40 to 60 ,um.

All of the biofilms had at the bottom, on theslip surface, a thin film of a transparent gelati-nous material easy to observe when the filmswere scratched. No algae were observed, exceptisolated algae that had stuck from the over-flowing water.

Variance study experiment. The results fromthe analysis of variance are presented in Table 1.The effects of R and S(RP) were significantlydifferent from the residual effect, whereas theeffect of P was not. In the variance componentestimation 4 and 18% of the total variance weredue to effects from the reactors and slips, re-spectively, whereas the rest was due to naturalrandom effects contained in the residual.For interpretation of the variance analysis

results it is necessary to expand the classifica-tion descriptions. The slips were stained inbatches of 19 slips. Variations in batch treat-

TABLE 2. Mean of each of the five bioffim reactorsused in the variance study experiment

Reactor no. Mean na Groupingb5 0.414 190 A6 0.405 190 C7 0.422 190 B8 0.403 190 C9 0.404 190 C

a Number of observations on which the mean isbased.

b Means with the same letter are not significantlydifferent. The grouping was obtained by Duncan'smultiple-range test.

ments will affect the reactor mean and show upas a reactor effect. Variations in rinse flowvelocity due to pressure differences in the tapwater system, not always notified and compen-sated for, may then explain the small but signifi-

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FIG. 5. A, Ash weights as percentage of the bio-films in the correlation experiments. The bars indicatestandard deviations for four biofilm slips. B, Growthcurves from the correlation experiments, measured asabsorbance with and without the staining step. Thebars indicate standard deviations from 10 to 20 absor-bance value measurements.

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MICROBIAL BIOFILMS IN FLOWING-WATER SYSTEMS 11

TABLE 3. Linear regression functions with pertaining correlation coefficients (r) for the correlation betweenmicrograms of protein N per square centimeter (P) and absorbance (A) with IWB and NWB in the correlation

experiments (n = 18)Correlation Function r

IWB P - total A A = 1.58P - 0.015 0.965IWB P - total A - A off unstained biofilm A = 0.824P - 0.015 0.953NWB P - total A A = 0.343P - 0.004 0.997NWB P - total A - A off unstained biofilm A = 0.323P - 0.006 0.996

cant effect of R. When a Duncan multiple-rangetest was made on the biofilm reactor data,reactors 5 and 7 differed from the others (Table2).

All treatment effects connected to separateslips, such as position in the rinse and mountingeffects, are included within S(RP). The majorpart of S(RP) may be explained by an unevenflow pattern in the rinse observed when dyetrace experiments were executed. By perform-ing Duncan's multiple-range test (2), the connec-tion between a somewhat higher absorbancevalue and a somewhat lower rinse column flowvelocity was confirmed. A later improvement ofthe rinse with insertion of flow diffusers in thebottom of the rinse smoothed out the unevenflow velocities between the rinse columns.

Correlation experiments. The biofilm develop-ments, measured as absorbance of stained andunstained biofilms, are presented in Fig. 5. Theunstained NWB had a low absorbance through-

out the experiment; at most all absorbance of thestained NWB was due to the stain. The IWB hada brown to yellow color caused by an ironprecipitation on the slips responsible for about50% of the absorbance of the stained IWB (Fig.5B). The iron precipitation also constituted aconsiderable part of the ash weight (Fig. 5A).There was a strong correlation between absor-

bance and protein (Table 3) and between absor-bance and organic weight (Fig. 6) in both experi-ments, which means that the relationshipbetween protein and organic weight was con-stant throughout the experimental periods. As-suming it is mainly the organic material that isstained, the slope of the regression line (x2, Y2) inFig. 6 should coincide with the slope of the (X4,y4) line in the same figure. This is not the case.However, when repeating the comparison forthe absorbance-dry weight regression, theslopes of the lines (x2, Y2) and (x4, y4) (Fig. 7)approach each other. This indicates that the

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00 10 20 30 40 50

pg organic weight cm-2FIG. 6. Correlation of absorbance (A) and organic weight for IWB and normal water biofilm NWB. Symbols:

A, IWB (xl, Yi) = (micrograms of organic weight per square centimeter, total A); A, IWB (x2, Y2) = (microgramsof organic weight per square centimeter, total A minus A of unstained biofilm); 0, NWB (X3, yO) = (microgramsof organic weight per square centimeter, total A); 0, NWB (X4, y4) = (micrograms of organic weight per squarecentimeter, total A minus A of unstained biofilm).

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go

* .y3 = C.00675X3*0.0043Y3 r ~~~~~~~~=0.996

N .18

o -o y4 =0.00634X4.0.002r =0.994

X2i2 ~~~~~~N.180 10 20 3 0 40 50

|1g dry weight cm2FIG. 7. Correlation of absorbance (A) and dry weight for IWB and NWB. Symbols: A, IWB (xl, Yl) =

(micrograms of dry weight per square centimeter, total A); A, IWB (x2, Y2) = (micrograms of dry weight persquare centimeter, total A minus A of unstained biofilm); 0, NWB (X3, y3) = (micrograms of dry weight persquare centimeter, total A); 0, NWB (X4, y4) = (micrograms of dry weight per square centimeter, total A minusA of unstained biofilm).

absorbance difference between a stained and anunstained biofilm is a measure closely related toits dry weight.

DISCUSSIONDevelopment of various biofilm types. The na-

ture of biofilms that develop in flowing watersystems depends on characteristics of the flowsystem and the flowing water itself. The maturebiofilms differed from each other when studiedunder a microscope. The biofilm obtained withnormal seawater resembled biofilms obtainedwith continuously flowing seawater (unpub-lished data). The IWB and the accelerated bio-films differed from natural seawater biofilms. Inthe study of factors regulating microbial bio-films, it is important to use water identical withthe water present in the water system that thestudy is related to. Otherwise, the results ob-tained will be less applicable. When the purposeof a biofilm study is of a methodological charac-ter, then modulations of the water may giveadvantages such as less complicated biofilmsand faster growth.Assay of bioflim components. In the study of

biofilms the purpose of the study indicateswhich measurement should be made. When bio-film activity is sought, an assay of ATP concen-tration offers an acceptable procedure for ob-taining an answer (11). If the effect of biofilmformation is of interest, then the investigator hasto find ways for measuring the effect as a func-

tion of biofilm mass, decreasing heat exchangecapacity (12), increased flow resistance (15), andcorrosion problems (10).

Biofilm mass can be assayed by various meth-ods, i.e., by measuring thickness (9), weight, orthe content of some biochemical components ofthe biofilm such as organic nitrogen, organiccarbon and chlorophyll (1), or lipopolysaccha-rides (4).Dry weight and ash weight determinations

were chosen as ways to assay the total biofilmmass and its organic content. The protein assayreflects an organic part of the biofilm, excludingpolysaccharides, which sometimes constitute aconsiderable part of biofilms (17). The stainingprocedure was designed to give a fast anduncomplicated method with high accuracy forthe measurement of bioflm mass. Performanceof protein and weight determinations at the endof a biofilm growth experiment measured by thestaining procedure will make it possible to calcu-late protein content, dry weight, and organicweight for absorbance values within the range ofgood correlation.

Biofilm study equipment. In the design ofbiofilm study equipment two main aspects of theproblem require special consideration, namely,the hydrodynamics of the equipment and thecharacter of the surface.A biofilm development depends completely

on the transport of biofilm components to thesurface. Oxygen, nutrients, particles, orga-

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nisms, and, in fact, everything have to be trans-ported to the surface in some form where theywill be adsorbed, metabolized or metabolizing.When the biofilm becomes mature, reentranceof material into the water will be significant. Inflowing water systems the water movement ef-fects the major transport to the immediate vicini-ty of the surfaces. The transport of biofilmcomponents over the last few micrometers to,and for some components such as oxygen andnutrients into, the biofilm (and vice versa) iscompleted by a variety of processes (6, 13).Because of this transport, the flow patternthrough biofilm study equipment should be ana-lyzed for inequalities which increase the vari-ance or even give misleading results.When the flow pattern through the reactors

was visualized by pouring particles into thewater, no flow inequalities could be observed.This observation was confirmed by the analysisof variance in which the position effect could notbe shown to be significant. Transport gradientsthat appeared in spite of the alternation of theflow direction should range the means of thereactors in one way or another. When the Dun-can multiple-range test was performed, reactors5 and 7 differed a little, but significantly, fromthe others (Table 2). The distribution of the fivereactor means cannot be explained by transportgradients. Some other random effects, probablythe rinse effect suggested in the variance study,must be responsible.

If a time and a treatment effect are added tothe variance analysis model, the method de-scribed above allows detection of small treat-ment effects on the development of biofilms.Biofilm development series with different treat-ments can be compared, and even small treat-ment effects will be significant.

If the surface can react with the liquid in thesystem (corrosion), if it is toxic, or if microorga-nisms can utilize the surface as a substrate oraffect it by their activity, the interpretations ofthe results will be more complicated as com-pared with biofilm studies on inert surfaces.The membranes in an electrochemical concen-

tration cell are hydrophilic and charged, and atleast the anion exchange membrane with highnitrogen content can theoretically be utilized bymicroorganisms. In addition, charge-charge in-teractions between the biofilm components andthe membranes may have a significant influenceon the biofilm formation.To facilitate the further study of bioffilms in

concentration cells used with natural water, theinvestigation has been split into two parts. Thefirst part is a study of how factors not related tothe surface regulate biofilm development (sub-mitted for publication). The second part will beconcentrated on biofilm development on single

membranes in simplified concentration cells. Byputting these results together predictions aboutmicrobial bioflm problems in a salt gradientpower plant can be made.

ACKNOWLEDGMENTS

This research was supported by the Swedish NationalBoard for Energy Source Development, grant 5565 060.

I am grateful to the late Kaare Gundersen for, among manyother things, his never-ceasing encouragement. I am gratefulto Birgitta Norkrans for many valuable discussions, to LarsBrandstrom for his excellent construction of the equipment, toBj6rn Rosander for help with the statistics, and to the crew ofr/v Falsterbo.

LITERATURE CITED1. Aftring, R. P., and B. T. Taylor. 1979. Assessment of

microbial fouling in an ocean thermal energy conversionexperiment. Appl. Environ. Microbiol. 38:734-739.

2. Alder, H. L., and E. B. Roessler. 1977. Introduction toprobability and statistics, 6th ed. W. H. Freeman and Co.,San Francisco.

3. Conn, H. J. 1953. Biological stains, 6th ed. W. F. Hum-phrey Press Inc., Geneva.

4. Dexter, S. C., J. D. Sullvan, Jr., J. William III, andS. W. Watson. 1975. Influence of substrata wettability onthe attachment of marine bacteria to various surfaces.Appl. Microbiol. 30:298-308.

5. Dorsey, T. E., P. W. McDonald, and 0. A. Roels. 1977. Aheated Biuret-Folin protein assay which gives equal ab-sorbance with different proteins. Anal. Biochem. 78:156-164.

6. Harremoes, P. 1978. Biofilm kinetics, p. 71-109. In R.Mitchell (ed.), Water pollution microbiology 2. John Wi-ley & Sons, Inc., New York.

7. Helenius, A., and K. Simons. 1975. Solubilization ofmembranes by detergents. Biochim. Biophys. Acta415:29-79.

8. Helwig, J. T., and K. A. Council. 1979. SAS users guide,1979 ed. SAS Institute Inc., Cary, N.C.

9. Hoehn, R. C., and A. R. Ray. 1973. Effects of thicknesson bacterial film. J. Water Pollut. Control. Fed. 45:2302-2320.

10. Iverson, W. P. 1974. Microbial corrosion of iron, p. 476-513. In J. B. Neilands (ed.), Microbial iron metabolism.Academic Press, Inc., New York.

11. La Motta, E. J. 1976. Kinetics of growth and substrateuptake in a biological film system. Appl. Environ. Micro-biol. 31:286-293.

12. Lkebert, B. E., L. R. Berger, H. J. White, J. Moore, Wm.McCoy, J. A. Berger, and J. Larsen-Basse. 1979. Theeffect of biofouling and corrosion on heat transfer mea-surements, p. 7A-1, 1-10. In G. Dugger (ed.), Proceedingsof 6th OTEC Conference, "Ocean thermal energy for the80's," Washington D.C. National Technical InformationService, Springfield, Va.

13. Marshall, K. C. 1976. Interfaces in microbial ecology.Harvard University Press, Cambridge.

14. Pattle, R. E. 1955. Electricity from fresh and salt water-without fuel. Chem. Process. Eng. (Bombay) 36:351-354.

15. Plcologlou, B. F., N. Zelver, and W. G. Charackls. 1980.Biofllm growth and hydraulic performance. J. Hydraul.Div. Am. Soc. Civ. Eng. 106:733-746.

16. Scheffe, H. 1964. The analysis of variance. John Wiley &Sons, Inc., New York.

17. Sutherland, I. W. 1980. Polysaccharides in the adhesionof marine and fresh-water bacteria. In R. C. W. Berkeley,J. M. Lynch, J. Melling, P. R. Ruttner, and B. Vincent(ed.), Microbial adhesion to surfaces. Society of chemicalindustry, London. Ellis Horwood Ltd. Publishers, Lon-don.

18. Vennard, J. K., and R. L. Street. 1976. Elementary fluidmechanics. John Wiley & Sons, Inc., New York.

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