APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June i986, p. 1186-1198 0099-2240/86/061186-13$02.00/0 Copyright C) 1986, American Society for Microbiology

Vol. 51, No. 6

Countermeasures to Microbiofotling in Simulated Ocean Thermal Energy Conversion Heat Exchangers with Surface and Deep Ocean Waters in Hawaii LESLIE RALPH BERGER* AND JOYCE A. BERGER

Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii 96822 Received 4 October 1985/Accepted 7 March 1986

Countermeasures to biofouling in simulated ocean thermal energy conversion heat exchangers have been studied in single-pass flow systemis, hsing cold deep and warm surface ocean waters off the island of Hawaii. Manual brushing of the loops after free fouling periods removed most of the biofouling material. However, over a 2-year period a tenacious film formed. Daily free passage of sponge rubber balls through the tubing only removed the loose surface biofouling layer and was inadequate as a countermeasure in both titanium and aluminum alloy tubes. Chlorination at 0.05, 0.07, and 0.10 mg liter-' for 1 h day'1 lowered biofouling rates. Only at 0.10 mg liter-' was chlorine adequate over a 1-year period to keep film formation and heat transfer resistance from rising above the maximum tolerated values. Lower chlotination regimens led to the buildup of uneven or patchy ifims which produced increased flow turbulence. The result was loWver heat transfer resistance values which did not correlate with the amount of biofouling. Strfaces which were let foill and then treated With intermittent or continuous chlorination at 0.10 mg of chlorine or less per liter were only partially of unevenly cleaned, although heat transfer measurements did not indicate that fact. It took cdntihubus chlorination at 0.25 mg liter-' to bring the heat transfer resistance to zero and eliminate the fouling layer. Biofouling in deep coid seawater was much slower than in the warm surface waters. Tubing in one stainless-steel loop had a barely detectable fouling layer after 1 year in flow. With aluminum alloys sufficient corrosion and biofouling material accumulated to require that some fouling coutermeasure be used in long-term operation of an ocean thermal energy conversion plant. The

thermal

and structures. In bacteria, in particular, exopolysaccharides (2) are secreted by the cells to form masses of fibers which have been termed "glycocalyx" (3). The bacterial cell surfaces and the adhering polymers are in turn suitable loci on which other bacteria and particulate materials may attach or on which inorganic precipitates may forrn. Such accumulations may be termed biofilms. A film only ca. 25 to 50 ,um thick may further reduce by 40 to 50% the heat transfer efficiency of the HXs, rendering the entire OTEC system economically impractical. This paper reports studies of microbiofouling and countermeasures to control or retard it. Long-term experiments are reported using open loops through which either surface warm water or deep cold sea-water flows year-round and continuously. The experiments have run for several years. They are part of an integrated program to determine the materials and conditions required for long-term OTEC operation.

conversion (OTEC) system ocean water and a potentially unlimited volume of deep cold ocean water with a heat engine to drive a conventional electricityproducing turbine generator. In a closed-cycle OTEC plant heat exchangers (HXs) extract the thermal energy from warm ocean waters. A small fraction of that energy is converted to electrical power and waste heat is rejected through a second HX to cold water pumped from the ocean depth. Solar energy absorbed by the ocean surface provides the heat source. The efficiency of the OTEC system is inherently low. The theoretical maximum based on the temperature difference between the water masses is only 6 to 7%. Thermal losses, the power requirements to pump large volumes of seawater and working fluid, power losses in turbines, generators, by microbiofouling (the attachment arid growth of microorganisms and their products on the HX surfaces), and corrosion of these HX surfaces by seawater are critical problems to be overcome if OTEC systems are to become a practical reality. All of these factors may reduce the net efficiency to as little as 1 or 2%. Unlike conventional power plants, however, the OTEC system uses an inexhaustible and virtually cost-free fuel which offsets the inherently low thermnodynah-iic efficiency and will make it a competitive alternative to energy generated from increasingly costly fossil and nuclear fuels. Many microorganisms adhere to surfaces, often with specificity and considerable firmrness. Chemical receptors on the surfaces of the cells or their appendages and in their extracellular secretions may bind the cell to other surfaces ocean

energy

connects a large heat source of warm surface

*

MATERIALS AND METHODS Site and experimental setups. The experimental site was at the Seacoast Test Facility at Ke-ahole Point on the western tip of the island of Hawaii. The surface (warm) water ranges between 24 and 280C and it is collected from about 100 m offshore. There, a large gyre of current brings in open ocean water. The shoreline is arid and mainly lava rock with relatively little vegetation. Most of the year, there is little rainfall and terrestrial runoff. The bottom drops off rapidly. The warm water is pumped through two 0.3-m-diameter polyvinyl chloride pipes to large header tanks from which the experiments are fed by gravity. The cold water intake is located at a depth of -580 m, -20 m above the sloping ocean floor some 1,400 m from the

Corresponding author. 1186

VOL . 5 1,

1986

1187

COUNTERMEASURES TO MICROBIOFOULING

TABLE 1. Operating conditions of the simulated HXs (open loops) Loop no.

Operating mode

Material

Warm surface seawater 1 2A

Titanium (2) Titanium (2)

2B

Aluminum (3004)

3 5

Titanium (2) Aluminum (3003) clad with aluminum Titanium (2)

6 7

Aluminum (3004) clad with aluminum Stainless steel (AL6X) Aluminum (5052)

10 11

Cold deep ocean water 13 16 17

Stainless steel (AL6X) Alclad (3003) Aluminum (5052)

Cycles of free-fouling and hand brushing Soft Amertap balls, 12 passes h-', 1 h day-', brush cleaned when required Hard Amertap balls, 12 passes h-', 1 h day-', brush cleaned when required Chlorinated at 0.05 mg liter-' for 1 h day-' Cycles of free-fouling and hand brushing Cycles of free-fouling and continuous chlorination at 0.05 mg liter-' or as indicated Chlorinated at 0.10 mg liter-' for 1 h day-' Chlorinated at 0.07 mg liter-' for I h day-' Cycles of free-fouling and chlorination at 0.10 mg liter-' for 1 h day-'

Free-fouling Free-fouling Free-fouling

shoreline. Details of these inlet lines have been fully described (J. Larsen-Basse, in Y. Mori and W.-J. Yang, ed., ASME/JSME Thermal Engineering Joint Conf. Proc. 2:285-289, 1982). The cold water is pumped directly into the laboratory building where it is diverted into the various test loops. Each test loop is fitted with a heat transfer monitor (HTM) plus sequential strings of corrosion and microbiofouling test coupons of the same test metal. Test coupons vary from 100 to 250 mm in length. They are 25.4 mm in outside diameter. Aluminum tubes are 22 Inm in inside diameter; titanium and stainless-steel tubes are 24 mm in inside diameter. Flow velocities were maintained at 1.8 m s-1. Table 1 summarizes the operating conditions of the simulated HXs discussed in this paper.

Heat transfer measurements. The HTMs were modified versions (T. M. Kuzay, G. N. Granneman, and A. P. Gavin, in W. L. Owens ed., ASME HTD 12:39-46, 1980) of the design by Fetkovich (J. G. Fetkovich, G. N. Granneman, L. M. Mahalingam, and D. L. Meier, Argonne National Laboratory Rep. OTEC/BCM-002, Argonne, Ill., p. 237-380, 1978) in which direct heating of the water is used transiently to determine the heat transfer resistance (HTR) of the test tubing. HTR values are cited in the text in Rf units, which are in meters square degree Celsius per watt x 105. The maximum HTR value tolerated in most of these experiments before antifouling measures were taken was approximately 9 Rf units. Three types of biofouling countermeasures were used: (i)

14

280

-j' E

12 31 E u n

I

LOOP 1, Titanium

240 200

o_

M

Z

8_ 6 _

120 80 1

0

Lr

u

4

Lo

40

2 _

0

o 21R

750

850

950

1050

1150

1250

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TIME IN FLOW (days) FIG. 1. HTR and biofouling levels in titanium loop 1. The loop surfaces were allowed to free-foul to about Rf 9 and then were cleaned with 10 manual passes of a bristle brush while seawater flowed slowly. Flow was resumed at 1.8 m s-' and the cycle was repeated. Only a portion of the experiment is shown.

1188

APPL. ENVIRON. MICROBIOL.

BERGER AND BERGER

TABLE 2. Percentage of total film removed by extraction for 15 min in loop 1l rli E u "I cn 3

Days In flow

z u L 0

81 323 610

1--

x U

Film DW

OC + TN

% 95 88 60

(%) 96 85 58

a Total film removed was estimated by extrapolation of the kinetic data curves to the point at which no further film would be removed on continued extraction.

.J' 31F cx 0

EXTRACTION TIME (min)

FIG. 2. Amount of biofouling removed from loop 1 plotted against extraction time. Biofouling samples were in the flow for different periods of time. Solid symbols are DW; open symbols are OC and TN.

manual brushing of the tubes, in which a bristle "test tube" brush was passed through each arm of a loop five times (10 strokes) for each "brush cleaning" while water flowed slowly; (ii) cleaning with sponge rubber balls (Amertap Co., Parkwest, Woodbury, N.Y.), in which one sponge rubber ball with a diameter of 25.5 or 26.5 mm (depending on the tubing used) was released every 5 min for 1 h once per 24-h period; and (iii) chlorination to prevent or retard microfouling or to remove already formed films, in which hypochlorite was generated electrochemically in situ immediately upstream of the HTM. Residual chlorine was determined by amperometric titration from samples taken immediately downstream of the HTM or at the end of the experimental loop 10 to 15 m downstream. Chlorination was done either for 1 h day-' or continuously following a period of free-fouling. In either case, chlorination was, done at nominal 0.05, 0.07, or 0.10 mg liter-1 except as noted. Sample preparation for scanning electron microscopy (SEM). Biofouling samples were removed from loops and

treated as follows. A piece of tubing about 2.5 cm was cut off from the sample (coupon), dipped in filtered seawater, and then fixed in 4% glutaraldehyde in filtered seawater, at pH 7.2 for 1 to 2 h at 15°C in the dark. The sections were dipped in filtered distilled water and then for 5 mmn each in 5% ethanol-distilled water (twice), 25% ethanol, 50% ethanol, and 75% ethanol. Iced samples were flown to Honolulu in tightly closed jars in 75% ethanol. The dehydration process was continued in 95% ethanol (twice), absolute ethanol (twice), and xylene (twice). Samples were then air dried and stored in a desiccator until they were cut and mounted on SEM stubs. They were then coated with carbon and gold before examination in the SEM (Cambridge Stereoscan model S4-10 or ISI model DS 130). For dry weight (DW), organic carbon (OC), and total nitrogen (TN) analyses, the other section of the tubing sample was rinsed in filtered distilled water, drained, bagged in polyethylene, and frozen in dry ice. The frozen samples were flown to Honolulu and kept frozen until analyzed. Extraction procedures. Sections of the frozen samples were cut in 2.6- to 3.3-cm lengths. One end of the cut piece was covered with Parafilm (American Can Co., Greenwich, Conn.) and a silastic cap. A 0.5-ml amount of acid-washed Ballotini-like microglass beads (Scotch Lite; 3M Co., St. Paul, Minn.) and 2 ml of glass double-distilled water were added. The open end was closed as above. The sample was

16

0 LOOP 5, Alclad 3003

14 :3

A

800

N

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400

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u

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700

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900

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_

600

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# 700

A

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900

1000

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TIME IN FLOW (days) FIG. 3. HTR and biofouling levels in Alclad (type 3003) loop 5. The tube surfaces were allowed to free-foul to about Rf 9 and then were cleaned with 10 passes of a bristle brush while seawater flowed slowly. Flow was resumed at 1.8 m s-' and the cycle was repeated. Only a portion of the experimental data is shown.

VOL. 51, 1986

COUNTERMEASURES TO MICROBIOFOULING

1189

I FIG. 4. SEM pictures of loop 5. (A) A complex film exists on the tube surfaces at Rf 9.3 before manual brushing (bar = 2 i±m). (B) Note areas free of film after brushing to Rf 2.3 (bar = 2 pLm).

placed in a model Vi2 vibrating ballistic disintegrator (RHO Scientific Co., Commack, N.Y.) and shaken at approximately 100 Hz for 5 min. After allowing the glass beads to settle, the supernatant fluid was removed. Two milliliters of glass double-distilled water was added to the sample, and the film removal procedure was repeated twice. The aqueous suspensions were pooled. For kinetic studies, fouled samples were progressively ballistically extracted with eight sequential 5-min treatments. The extracts were analyzed individually. Aliquots (1 ml) of the extracts were hydrolyzed in 4 N HCl at 121°C for 1 h. The hydrolysates were then dried over P205 and NaOH in vacuo at 55°C overnight. For each sample two vials of hydrolysate were used. Each hydrolysate was taken up in 0.1 ml of glass double-distilled water and pooled for analysis. A 20-,ul portion of pooled hydrolysate was added to

weighed tinfoil cups (two cups per sample) and dried in vacuo over P205 and NaOH at 55°C. A second aliquot was added to the tin cup and the sample was then dried overnight as above. Cups were closed and then weighed on a microbalance (model 21; Cahn Instrument Co., Cerritos, Calif). The length of tubing used for the extraction was measured to calculate the area of the inner surface. DWs (less carbonates) are reported in micrograms per square centimeter. OC and TN were determined on these samples in an elemental analyzer (Carlo Erba model 1106; Haak-Buchler Instruments, Saddle Brook, N.J.). Values are reported in micrograms per square centimeter. RESULTS Mechanical cleaning experiments. (i) Brushing as a fouling countermeasure. (a) Loop I (titanium, free-fouling and peri280

14

122

LOOP 2A, Titanium

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10 -

200 z

O* 160

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800

850

900

950

1000

105C

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TIME IN FLOW (days) FIG. 5. HTR and biofouling levels in titanium loop 2A. Twelve sponge rubber balls were passed through the sytem over 1 h, once daily to control fouling. When HTR reached about Rf 5, the tubes were cleaned with 10 passes of a bristle brush while water flowed slowly. The flow was then restarted at 1.8 m s-1, and the use of sponge rubber balls was resumed. Only a portion of the experimental data is shown. The asterisk indicates that four extractions were used instead of three.

1190

BERGER AND BERGER

odically brushed). Figure 1 shows part of the data from loop 1. The experiment ran for -3.5 years, although biofouling coupons were installed and replaced at various times. Early samples were undated. The loop was allowed to free-foul until the HTR reached Rf -9 (which took between 28 and 42 days). It was then brushed to bring the HTR to near 0. Microscopically, films were complex with filamentous organisms of various sizes and colonies of cocci, rods, and other forms all imbedded in a glycocalyx matrix. Macroscopically, the films appeared evenly dispersed and quite thin. The DW never exceeded 150 ,g cm-2 and the OC plus TN averaged -29 jxg cm-2, although the HTR was approximately at Rf 9. The recalcitrance of older films to removal was tested kinetically. Figure 2 shows the kinetics of film removal as functions of the time of extraction and the actual days in flow. Table 2 shows the percentage of the total film removed by extraction for 15 min. The data show that long periods of time in the flow and frequent brushings produced an increasing film tenacity. (b) Loop 5 (Alclad 3003, free-fouling and periodically brushed). Figure 3 summarizes some data from loop 5. The experiment had been running for -3.5 years. Biofouling samples were installed in the loop 376 and 667 days after the start of the experiment and additional replacement samples were added between days 700 and 900. None of these samples were labeled, although their positions in the loop were noted. Tubes were let free-foul from 26 to 43 days until the HTR reached Rf 9. They were then brushed back to Rf 1.5 to 2.5. The period between brushings was influenced by the condition of the bristle brushes and seasonal factors, in particular, water temperature. Film DW and OC-plus-TN analyses and SEM photographs (see Fig. 3 and 4B) demonstrated that there was always a fouling film residue after brushing. The bacterial flora also changed with time. Forms evident in Fig. 4A include prosthecate and spiral or colonial hat-shaped bacteria. The latter have not been previously described to our knowledge. Before brushing, the inner tube surfaces appeared smooth and evenly coated with a layer which varied from off-white to pale yellow. SEMs showed that the tube surfaces were covered with complex films similar in appearance to those found in (titanium) loop 1. After brushing the surface ap-

APPL. ENVIRON. MICROBIOL.

FIG. 6. SEM showing biofouling on the surface of titanium loop 2A through which sponge rubber balls had been passed as a fouling countermeasure. Note the combed appearance of the surface and the smaller diversity of microorganisms than in Fig. 4. Loose surface material is removed by the passage of the balls. Bar = 2 ,um.

peared streaked in the direction of the brushing. The DW of the extracted films just before brushing varied from 286 to 460 ,ug cm-2 which depended on the age of the sample, i.e., the number of free-fouling/brushing cycles and the efficiency of the brushing procedure itself. The OC-plus-TN values averaged 24 ,ug cm-2, values which were slightly lower than those in loop 1. HTR values before brushing varied from Rf 9 to 11. (ii) Sponge rubber balls as a fouling countermeasure. (a) Loop 2A, which ran for 34 months, using Amertap sponge rubber balls and titanium tubing. Only 6.5 months of this experiment is shown in Fig. 5. The HTR increased slowly compared with untreated free-fouling surfaces (Fig. 1). When the HTR approached Rf 5, the loop was brushed. HTR

1001 90

80 70

60

_

50
740 1-3 6.75 169 1-6 160.0 2-1 >740 135.0 >740 2-7 32.61 220 3-1 291.0 >740 3-2 435.4 >740 3-12 a HTR was Rf 2.9 when the loop was dismantled.

l_ 1

2

Days in flow

FIG. 12. Diagrammatic representation of loop 3, which is typical of the other loops. The electrolytic chlorinator (EC) was not present in all loops. The HTM was made of the same alloy as the sample coupons. Other parts of the loop were of polyvinyl chloride plastic pipe. Sample coupons were connected to each other by nylon or silastic couplings. The numbering system for the test samples is given. Each arm was approximately 3.5 m long. The numbers of test coupons in each arm and loop varied.

125.5 545.3 34.71 1,002 637.9 128.6 1,552 2,750

that, except near the HTM, the amount of biofouling is not dependent on the position of the sample in the loop. The lumpy and patchy coating on the HTM surface at the end of the 1,300-day experiment was not indicated by the low HTR values. (iii) Loop 10, (stainless steel [AL6X], chlorinated for 1 h day-' at 0.70 mg liter-'). Loop 10 was started in 1982. Twenty biocoupons were added sometime during that year. Over the 372 days of the experiment, HTR varied between Rf 0.6 and 2. The data indicate that chlorination at 0.07 mg liter-1 is sufficient to maintain good heat transfer. OC, TN, and DW analyses of the films showed that fouling occurred more slowly than that in the free-fouling cycle of loop 1. The biofouling parameters in loop 10, at Rf 1.8, howfver, were higher than those found in loop 1 at Rf 8.7. DWs were 128.5 and 97.21 ,ug cm2 and those for OC plus TN were 24.5 and 19.51 ,ug cm-2 in loops 10 and 1, respectively. (iv) Loop 7 (Alclad [3004], chlorinated at 0.10 mg liter-l for 1 h day-'). Loop 7 was run for about 15 months. The HTR gradually rose, reaching a maximum of Rf 2.8. Figure 14 summarizes data for 350 days of the experiment. The DW of the films varied between 196 and 432 ,ug cm2, while the OC plus TN was between 3.5 and 7.0 pug cm-2. After a few months of flow, the surfaces of the tubes were sparsely covered with a limited morphological range of bacteria imbedded in and on the inorganic/polysaccharide film matrix (Fig. 1SA). Four months later the film had become more complex (Fig. 15B), but the amounts of OC and TN had only fluctuated slightly. (v) Loop 6 (Ti, free-fouling, then cleaned by continuous chlorination at 0.050 mg liter-'). In the first experiment in

Fig. llB. The chlorination regimen slows film formation, but is does not prevent it. Figure 12 is a diagram of the setup for loop 3 and other loops showing the general positioning and the numbering system for biocoupons as used in this study. (ii) Biofouling and position of samples in the loop. There is evidence to indicate that with chlorination at low levels (e.g. 0.05 mg liter-') the hypochlorite generated was not uniformly in the bulk water as it flowed through the HTMs. Thus the HTM and those coupons closest to it were probably "cleaned" with more than 0.05 mg of hypochlorite liter-. Based on the appearance of the biocoupons, uniform mixing appears to have occurred by 1 m downstream of the chlorinator. Chlorination caused small pieces of the fouling layer to slough off periodically. The resulting surface roughness produced added turbulence which in the HTM results in lowered values of HTR. The level of residual chlorine was measured on several occasions to be 15 to 20% lower at the far end of the loop than just downstream of the HTM. Over a period of -3 years a 1- to 2-mm-thick biofouling layer

resulted. Figure 13 shows the effect of the time in flow on biofilm development. Only the data points for the sample closest to the HTM and the chlorinator (in the 1-1 position) are not on the curve. Additional data shown in Table 3 support the fact 600

N E

U

500 Oa m N Ln z

Ln) J

0

400

*

200 _

ILII

(1-5)

U

300

*

(3-1)

100

z

a

n

(1-1)

-

f00

(1-6) 200

300

400

500

600

700 ---

800

ACTUAL TIME IN FLOW (days)

FIG. 13. Loop 3. Film DW and OC plus TN in the fouling layer are plotted versus the time coupons were in the flowing system. Positions of the samples in the loop are indicated in parentheses. HTR varied between Rf 2.9 and 3.3.

1194

APPL. ENVIRON. MICROBIOL.

BERGER AND BERGER

500

16 I ,4 -LOOP 7, Alclad 3004 N

0 400

I2

0 C\j E

0

I

0~~

300

8

0

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4

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200

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100

150

200

250

300

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TIME IN FLOW (days) FIG. 14. HTR and biofouling levels in Alclad (type 3004) loop 7. Biofouling countermeasures consisted of chlorination at 0.10 for 1 h day-'.

loop 6, free-fouling followed by continuous chlorination at 0.05 mg liter-' invariably reduced HTR to Rf 0 or less, but not the film OC, TN, or DW. After 15 months of cyclic operation in this manner, when the continuous chlorination period was extended, Rf values dropped below 0 and stayed negative. To test whether increased turbulence was the cause, the chlorine level was increased to 0.1 mg liter-1 and run for an additional 50 days. HTR continued to drop but then started to rise. Concurrently, film DW, OC, and TN decreased. Increasing the chlorine to 0.25 mg liter-1 eventually brought the HTR up to Rf 0, at which point examination of the tube surfaces showed them to be free of all biofilm. Loop 6 was dismantled, acid cleaned, and restarted with

liter-'

new biofouling coupons. The regimen consisted of freefouling to Rf 9 followed by cleaning with continuous chlorination at 0.05 mg liter-'. The first four cycles of the experiment are shown in Fig. 16. Although each new cycle starts near Rf 0, there is a progressive increase in the amount of base-line fouling. (vi) Loop 11 (aluminum [5052], free-fouling, then chlorinated at 0.10 mg liter-' for 1 h day-'). This experiment was designed to test whether chlorination at 0.10 mg liter-' for 1 h day-' was sufficient to remove preformed biofouling layers on aluminum HX surfaces. Three cycles of fouling and cleaning were done. Figure 17 shows the biofouling data for the second and third cycles. Chlorination does not return the HTR to zero and each succeeding free-fouling cycle starts at

FIG. 15. SEMs of loop 7 which had been chlorinated daily for 1 h at 0.10 mg liter-'. (A) Fouling layer at exopolysaccharide slime (bar 1 ,um). (B) Sample taken 4 months later at Rf 2.0. Note the diversity of organisms (bar =

mg

Rf 1.8. Note the =

1

,um).

VOL. 51, 1986

1195

COUNTERMEASURES TO MICROBIOFOULING

700

c\j E u

600

.

500

IN :3:

z

C\M E u 0

Ur)

400

u

300

oD

200

I

4

0

(4-

100

U CY

w

:¢

-J La11

0 TIME IN FLOW (days) FIG. 16. Plot of first four cycles of data for free-fouling and cleaning by continuous chlorination at 0.05 mg liter-' of the cleaned and refurbished loop 6 titanium (second experiment).

a higher Rf value. Similar increases occur for OC, TN, and film DW. Table 4 shows that for the second and third fouling cycles, at comparable Rf values, the biofouling parameters are not the same; an uneven removal of the fouling film changes the heat transfer properties of the system. Similar observations were made for loop 6. Cold water loops (fouling with deep seawater). (i) Loop 13 (stainless steel [AL-6X]). During the 1-year period over which biocoupons were monitored, the HTR ranged from about Rf -1.0 to +3.3. Biofouling was not significant; SEM photographs showed mainly inorganic deposits. (ii) Loop 16 (Alclad [3003]). Biocoupons became completely corroded within 1 year and were useless for further analyses. Before corrosion began, however, biofouling was

observed in SEM photographs. A type of filamentous bacterium was imbedded and adhered to the hydrated aluminum oxide corrosion layer. The OC and TN comprised only about 2% of the total film weight and did not contribute significantly to the HTR. (iii) Loop 17 (aluminum [5052]). Data for approximately 2 years of flow are shown in Fig. 18. A second set of biocoupons was added to the system after -6 months of running. Some of the coupons tested are assumed to be from the second set. HTR increased nearly linearly during the entire period. After about 2 years the Rf reached 4.4, just 50% of the tolerable fouling level. The film mass accrued linearly during the first year and OC and TN also increased. The organic - 500

16 14 _ 3

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0

LOl C n4-

LOOP 11. Aluminum 5052

-

10

As0

k

0

0

8 _ 6

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0

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4 _

350

z

_ 300

.

250

4od

200

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_ 150

06 w LLi

_ 100

W

2 _ u

100

50 150

200

250

300

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N.

400

0

12 _

450

350

400

0

3 1-J

_

11

TIME IN FLOW (days) FIG. 17. HTR and biofouling levels in aluminum (type 5052) loop 11. Tubes were cleaned by chlorination at 0.10 mg liter-' for 1 h after HTR reached about Rf 9. Only part of the experiment is shown.

day-'

1196

BERGER AND BERGER

components of the film constituted only -3% of the total film weight. Only filamentous bacteria were observed in the films from the cold-water aluminum loops (Fig. 19).

DISCUSSION Studies of film formation in model laboratory systems have shown a direct correlation between nutrient concentration in the bulk phase and both the rates of film formation and film thickness so long as nutrient concentrations are not excessive (1, 8). At nutrient levels in which heterotrophic bacteria are commonly cultured in the laboratory, adhesion by bacteria to solid surfaces is generally weak. In these studies, we noted that the fouling rates increased during the summer and autumn months when the surface waters approached 30°C and the dissolved OC ranged from 0.76 to 1.14 versus 0.38 to 0.76 mg liter-' during winter and spring. The dissolved OC was consistently found to be -95% of the total OC. The experimental setup at the Seacoast Test Facility is unique in that some experiments have been maintained to date for well over 3 years, using single-pass (once-through) flow systems. The types of countermeasures which can be tested in these systems is more limited than in recirculating laboratory, pilot, or industrial reactors. Surfactants and high concentrations of other reagents may not be used for economic or environmental reasons. Chlorination is the method most used in industry for biofouling control. These studies provide a base line for the chlorination of seawater systems, using single-pass flow. Because of the huge surface areas and volumes and high flow velocities in OTEC HXs, countermeasures must be optimized. We have used low concentrations of chlorine, usually 0.05 to 0.10 mg liter-', because of the low organic content of the seawater used. For analysis, chlorine samples were taken downstream of the chlorinator either about 0.3 or 5 s after its generation. No measure of the actual chlorine consumed in those intervals is available as the water passed through the HTM or the entire loop. All chlorination treatments resulted in readily measured decreases of the biofouling rates. Use of 0.05 mg liter-' for 1 h day-' was inadequate as a fouling countermeasure. The minimum chlorine concentration which might be used under the field conditions tested is 0.1 mg liter-1 for 1 h day-'. Continuous chlorination at the same concentration was not adequate to clean prefouled tubes. Cleaning by chlorination led to changes in the smoothness of the biofilms. These, in turn, affected the HTR measurements, which led to the belief that the tubes were clean. In fact, the biofouling layers caused increased turbulence which lowered HTR below that expected from the other analytical data. The same observation was made by Tosteton et al. (7) in reference to mechanical cleaning regimes and in very early fouling (6). For OTEC-type model installations, Nosetani et al. (5) and Tipton (D. G. Tipton, Argonne National Laboratory Rep. ANL/OTEC-BCM-018, Argonne, Ill., part 2, p. 1-30, 1981) showed that abrasive-coated sponge rubber balls could be used effectively in titanium tubing to remove fouling layers without appreciably eroding the metal. The same type of countermeasure caused rapid wear of aluminum and copper alloys. Use of the plain sponge rubber balls retarded but did not control fouling film formation. They only removed the looser adhering surface layers. The resulting biofilm underlayers were dense and recalcitrant to removal in situ and in the

laboratory.

APPL. ENVIRON. MICROBIOL.

TABLE 4. Biofouling parameters during successive fouling and cleaning cycles in loop 11 m2 W'1) (105 °C W)Rf 101 oCm2

cm-2) (p.gDW

OC CM-2) + TN

Free fouling 2nd 3rd

5.98 5.33

217.1 372.9

22.89 54.76

Chlorinationa 2nd 3rd

1.85 1.83

119.8 172.6 at 0.10 mg liter-'.

15.48 20.93

Cycle Cycle

a Chlorination was limited to 1 h day-'

(ILg

We have noted seasonal and unexplained changes in the types of organisms which populate the biofilms. While a diverse range of microorganisms has been noted in the fouling layers, there is no visual evidence that any eucaryotic organisms colonize the loops. Entrapment of diatoms, whole or fragmentary, and portions of differentiated algae and other organisms have been noted in SEM preparations. The absence of light and the high velocity of the water in the tubing apparently prevents their colonization. Direct measurement for constituents specific to eucaryotes or procaryotes and other parameters of community structure by the techniques developed primarily by Nickels et al. (4) and by Uhlinger and White (9) and Berger et al. (L. R. Berger, W. F. McCoy, and J. A. Berger, in Proceedings of the OTEC Biofouling, Corrosion, and Materials Workshop, Argonne National Laboratory Publ. OTEC/BCM-002, Argonne, Ill., p. 38-55, 1979) has not been done. In these experiments, however, surfaces were cleaned whenever the HTR rose above Rf 9. For nonturbulent flow this HTR was reached when the wet film thickness was about 50 to 60 ,um. That some coupons were retrieved after several years in the warm-water loops having biofilms 20 to 40 times thicker demonstrates the need to use more than HTR alone to monitor the course of fouling in this type of tubular HX system. The OC/TN weight ratios in the fouling layers were not significantly different on the various metal surfaces and did not vary significantly (P = 0.001) between thick and thin films except on aluminum alloys. In loop 5 the OC/TN ratios before brushing averaged 3.90 ± 0.23 and 5.55 ± 1.85 after brushing. This suggests that most cellular material is removed from the hydrated oxide layer by brushing, leaving behind much of the base slime layer on or in the remaining underlying corrosion layer. In aerated seawater, aluminum oxidizes to form a hydrated aluminum oxide layer. The corrosion process goes rapidly at first but slows as the diffusion of oxygen becomes limited by the corrosion film itself (B. E. Liebert, in Proceedings of the OTEC Biofouling, Corrosion and Materials Workshop, Argonne National Laboratory Publ. OTEC/BCM-002, Argonne, Ill., p. 189-196, 1979). Independent of this process, biofouling proceeded much as it did on titanium and stainless-steel coupons in our experiments. The rate of early biofouling was shown to occur at approximately the same rate on all of these alloys whether or not corrosion also occurred [B. E. Liebert, L. R. Berger, H. J. White, J. Moore, W. E. McCoy, J. A. Berger, and J. Larsen-Basse, in G. L. Dugger, ed., Proceedings of the Sixth Ocean Thermal Energy Conversion (OTEC) Biofouling and Corrosion Symposium, 2:381-390, 1980). We observed that the biofilms accreted faster on aged

11

600

14 H e%

1197

COUNTERMEASURES TO MICROBIOFOULING

VOL. 51, 1986

LOOP 17, Aluminum 5052 Cold deep-sea water

cC\E

U On

IN

0 500

D3

12 F z

400 E

+ u

10-

0 -

0

8k

LO

6

0

0

300

co

0

0

200

C0 U-.

4 u 6-4

w

4k

_I

100

2

00L 0

I

I

I

100

200

300

w cm -J

400

500

0

600

701

6-

LL

0

TIME IN FLOW (days) FIG. 18. HTR and biofouling levels in cold water, aluminum (type 5052) loop 17. Two 6 months apart. Some of the samplings were from the second set.

aluminum alloys than on titanium. Unlike titanium, however, when a new piece of aluminum was placed in the flowing seawater, the HTR increased from Rf 1.7 to 3.5 as the early corrosion layer formed. Biofouling on top of this layer is responsible for most of the subsequent increase in HTR. A loose outer film of bacteria and organic matter extends into the flow and increases the width of the stagnant water layer; it is such a layer that relatively gentle brushing removes.

Studies in electrical impedance spectroscopy (P. K. Sullivan, Ph.D. thesis, University of Hawaii at Manoa, Manoa, 1985) recently showed that a fouled titanium surface can be resolved into three layers: (i) the loose outer layer mentioned above, (ii) a tougher older film which gradually develops under it and which is believed to contain mostly inorganic material accounting for much of the film DW, and (iii) the thin TiO2 protective barrier covering the bare metal.

sets

of biocoupons

were

installed in the flow about

Biofouling was greatly retarded in the cold-water loops. With aluminum (3003) clad with aluminum, corrosion was so extensive that it was not possible to follow biofilm formation. Essentially no fouling was observed on the stainlesssteel loop over the test period. However, biofouling did occur on aluminum 5052, although it was very slow. In terms of a closed-cycle OTEC plant, some sort of fouling countermeasure would be needed for the cold-water HXs, but its use would be infrequent. The deep-sea cold water used in these studies d 'ffers markedly from that used by Lewis (R. 0. Lewis, in Proceedings of the Eighth Ocean Energy Conference, Mar. Technol. Soc., 2:379-387, 1981), who compared fouling rates in Wrightsville Beach, N.C., waters between summer and winter months. While the fouling rates in the winter water were lower than those in summer, the differences were relatively small; extensive fouling occurred year-round. In contrast, we have compared two different water masses simultaneously over several years. The deep seawater is lower in dissolved organic matter and contains fewer heterotrophic bacteria than are found in the warm surface waters. In 2 years of examining aluminum loops in the cold seawater, we have only observed filamentous bacteria on the thin hydrated corrosion layers. ACKNOWLEDGMENTS Support for these studies came from the Ocean Energy Program of the U.S. Department of Energy through Argonne National Laboratory (ANL), Argonne, Ill. We acknowledge Tom Daniels, Director of the Natural Energy Laboratory of Hawaii (NELH), the staff of the NELH, and Ajay Bhargava for the measurements of HTR, C. Panchal and H. Stevens of ANL, and J. Larsen-Basse, Scientific Director of the project, for his advice, aid, and support throughout this work. We thank especially Barbara Lee for her meticulous attention to the many tasks which she performed for us at the test site, and we acknowledge Ted Walsch for the water quality data cited in this paper.

FIG.

19.

Biofouling of aluminum (type 5052), using deep cold

ocean water for about 20 months. Mostly filaments and bacillary forms are evident adhering to and imbedded in the hydrated aluminum

oxide-exopolysaccharide matrix. Bar

=

2

im.

LITERATURE CITED 1. Characklis, W. G., M. G. Trulear, J. D. Bryers, and N. Zelver 1982. Dynamics of biofilm processes: methods. Water Res.

16:1207-1216.

1198

BERGER AND BERGER

2. Corpe, W. A. 1980. Microbial surface components involved in adsorption of microorganisms onto surfaces, p. 105-144. In G. Bitton and K. C. Marshall (ed.), Adsorption of microorganisms to surfaces. John Wiley & Sons, Inc., New York. 3. Costerton, J. W., G. G. Geesey, and K. J. Cheng. 1978. How bacteria stick. Sci. Am. 238:86-95. 4. Nickels, J. S., R. J. Bobbie, D. F. Lott, R. F. Martz, P. H. Benson, and D. C. White. 1981. Effect of manual brush cleaning on biomass and community structure of microfouling film formed on aluminum and titanium surfaces exposed to rapidly flowing seawater. Appl. Environ. Microbiol. 41:1442-1453. 5. Nosetani, T., S. Sato, K. Onda, J. Kashiwada, and K. Kawaguchi. 1981. Effect of marine biofouling on the heat transfer performance of titanium condenser tubes, p. 345-353. In E. F. C. Somerscales and J. G. Knudsen (ed.), Fouling of heat transfer equipment. Hemisphere Publishing, New York.

APPL. ENVIRON. MICROBIOL. 6. Sasscer, D. S., T. 0. Morgan, C. Rivera, T. R. Tosteton, B. R. Zaidi, R. Revuelta, S. H. Imam, R. W. Axtmayer, D. DeVore, and D. L. Ballantine. 1981. OTEC biofouling, corrosion and materials study from a moored platform at Punta Tuna, Puerto Rico. I. Fouling resistance. Ocean Sci. Eng. 6:499-532. 7. Tosteton, T. R., B. R. Zaidi, R. Revuelta, S. H. Imam, R. W. Axtmayer, D. DeVore, D. L. Balantine, D. S. Sasscer, T. 0. Morgan, and D. Rivera. 1982. OTEC biofouling, corrosion and materials study from a moored platform at Punta Tuna, Puerto Rico. II. "Microbiofouling." Ocean Sci. Res. Eng. 7:21-73. 8. Trulear, M. G., and W. G. Characklis. 1982. Dynamics of biofilm

processes. J. Water Pollut. Control Fed. 54:1288-1301. 9. Uhlinger, D. J., and D. C. White. 1983. Relationship between physiological status and formation of extracellular polysaccharide glycocalyx in Pseudomonas atlantica. Appl. Environ. Microbiol. 45:64-70.