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THE SIGNIFICANCE OF MARINE BACTERIA IN THE
FOULING OF SUBMERGED SURFACES
CLAUDE E. ZOBELL AND ESTHER C. ALLEN
SCripp8 Institution of Oceanography of the University of California,
La Jolla, California
Received for publication July 24, 1934
The attachment and subsequent growth of the wildly promiscuous assemblage of visible plant and animal organisms on the hulls
of ships and other submerged marine structures is known as
fouling. Our present-day knowledge of the nature and extent of
fouling is discussed comprehensively by Visscher (1928b). The
vast economic loss resulting from fouling has instigated extensive
investigations of its cause and more particularly of practical
methods for its prevention. However, attention has been focused
mainly upon the habits, life histories and tolerances to poisonous
paints and metals of the macroscopic encroachers such as barnacles, mollusks, tunicates, hydroids, and bryozoans. Surprisingly, little, or no, attention has been devoted to the exact
sequence of events, especially during the initial stages, to the relationship of one group of organisms to another, and to the associated microscopic life. It is the purpose of this paper to report
the observations which have been made at the Scripps Institution of Oceanography at La Jolla during the last two years on the
attachment of bacteria and kindred microorganisms upon submerged surfaces,’ and to discuss their possible significance with
reference to fouling.
The studies of Wilson (1925) at the Scripps Institution on marine algal successions indicated that colonial diatoms were the first
1 Acknowledgment is here made to Captain A. H. Van Keuren of the Bureau
of Construction and Repair of the Navy Department for making available certain invaluable reports and otherwise encouraging the work, and to Professor
W. E. Allen of the Scripps Institution for generous assistance.
239
240
CLAUDE E. ZOBELL AND BETHER C. ALLEN
sessile organisms to appear on submerged plates; but his methods
of investigation made no provision for the observation of bacteria. Although he did not apply the procedure to the fouling
problem Naumann (1925) reported that, due to their tenacious
attachment, excellent preparations of iron bacteria can be prepared by the submergence of glass slides in iron-bearing waters.
Both Hentschel (1925) and Thomasson (1925) submerged slides
and later examined themimicroscopically to follow the distribution of diatoms and other minute sessile aquatic organisms, but
neither the fouling problem nor bacteria were considered. Using
a direct microscopic technique Henrici (1933) concluded from his
studies on fresh-water bacteria that “it is quite evident that for
the most part water bacteria are not free floating organisms, but
grow attached upon submerged surfaces.” He found that following the submergence of glass slides in lake water a deposit of
bacteria soon becomes apparent and increases progressively until
individual cells can be distinguished only with difficulty.
In preliminary observations on the nature and distribution of
marine bacteria and their r6le in the fouling of submerged surfaces, ZoBell and Allen (1933), using a procedure somewhat similar to that employed by Henrici, indicated that numerous bacteria soon attach themselves to glass slides submerged in sea
water and that bacteria, and to a lesser extent diatoms and actinomyces, usually precede the attachment of barnacles and other
fouling organisms. This report is a continuation of those studies.
METHODS OF INVISTIGATION
Standard micro-slides were submerged off the end of the Institution’s pier which extends one thousand feet seaward from shore.
The slides were tied to a carrier (fig. 1) which consists of a piece
of lead about 12 by 4 by 0.25 inches covered with wood, and the
whole coated with paraffin. The heavy lead gives anchorage
and stability, and the wood and paraffin keep the slides from
direct contact with the metal. Grooved wood strips and cord
string provide for fastening twelve to sixteen slides on the
faces of the carrier. The device was suspended by means of a
cotton rope in from six to twelve feet of water, depending upon
BACTERIA IN FOULING OF SUBMERGED SURFACES
241
the tide. Within these limits the depth of submergence did not
influence the results and neither did the tidal phase, the main
effect of the latter being merely to change the depth of the water,
because the pier virtually extends in the open ocean. Adherent
FIG. 1. WOOD COVERED LEAD CARRIER FOR HOLDING SUBMERGED GLASS SLIDES
growths were scraped from the carrier as often as required and
the latter was sterilized by dipping in hypochlorite solution.
Prior to submergence the slides were cleaned, wrapped in paper,
and heat sterilized. Aseptic precautions were exercised in their
242
CLAUDE E. ZOBELL
AND
ESTHER
C.
ALLEN
manipulation. They were exposed to the sea water for periods
varying from a few hours to a few days. They were examined in
the fresh condition and also after staining. Loeffler’s methylene
blue and Hucker’s ammonium-oxalate crystal-violet are useful
stains; but the most satisfactory results have been achieved with
Conn’s (1918) rose bengal consisting of 1.0 per cent of the dye and
0.02 per cent anhydrous calcium chloride in 5 per cent aqueous
phenol solution.
In order to obtain cultures of bacteria that form films or those
which attach to solid surfaces, some of the exposed slides were
plated directly in sea-water agar after rinsing them in sterile
water to dislodge all except the desired types. This procedure
was practicable only in the case of those slides which had been
submerged for a few hours, because later there were too many
bacteria on the slides. In other experiments bacteria from the
adherent films were brushed off with a cotton swab in sterile
sea water and appropriate dilutions thereof were plated. The
method had no quantitative significance but it furnished a means
of obtaining pure cultures of the film-formers.
RESULTS
The majority of the organisms which are found attached to
slides submerged in the sea for one to seven days are definitely
microscopic in size and most of these are bacteria. Table 1 illustrates the kind of record which was kept and shows the number of
bacteria, other microscopic forms, and larger organisms visible
to the naked eye, found on the 2- by 1-inch exposed area of the
slide after twenty-four hours’ submergence. All of the macroorganisms were enumerated, whereas the number of bacteria and
other microorganisms per slide were calculated by counting those
which appeared in a representative number of fields (magnification 980 X and 430 X, respectively) and multiplying by a factor.
Usually at least fifty fields were scrutinized.
Figure 2 shows graphically the number of bacteria by weekly
averages which were found attached to slides which had been
submerged for forty-eight hours during the first six months of
1933. There were millions of attached bacteria per 2 square-
243
BACTERIA IN FOULING OF SUBMERGED SURFACES
inches of slide while concomitant plate counts revealed only
hundreds of bacteria per cubic centimeter of water in which the
slides were submerged. The lack of relationship between the
number of marine bacteria found attached to slides and the number demonstrated by plating procedures is noteworthy.
Controlled laboratory experiments devised to simulate field
conditions show that there are several factors besides the number
of bacteria present in the sea water which influence the number
found attached to glass slides submerged therein. In the first
place, not all marine bacteria attach themselves to glass slides
even under favorable conditions. This was revealed by testing
TABLE 1
Number of bacteria, other microscopic organisms and macroscopic organisms
attached to S square inches of micro-slides submerged in the sea for 84 hours
OTHER
DATE OF SUBMERGENE
BACTERIA
MICROSCOPIC
ORGANISMS,
February 7, 1933.2,820,000
February 13, 1933 .1,860,000
February 14, 1933 .1,260,000
February 20, 1933 .600,000
February 27, 1933.
9,060,000
March 6, 1933 .7,800,000
March 9, 1933.
6,720,000
March 15, 1933.
3,240,000
March 20, 1933 .1,920,000
March 29, 1933 .4,260,000
3,500
500
8,900
500
1,000
10,300
12,000
400
300
600
MACROSCOPIC
ORGANISMS
0
0
0
0
0
1
0
1
0
0
73 pure cultures, differing physiologically or morphologically,
which had been isolated at random by planting sea water (ZoBell
and Feltham, 1934). These were cultivated in bottles of seawater broth into which sterile glass slides were inserted vertically.
After two days’ incubation at 250C. it was found by microscopic
examination of the slides that only one-third, or 24 of the 73
cultures, were firmly fixed to the solid surfaces. However, three
of the cultures were found attached only to solid surfaces, forming films of micro-colonies on the glass slides and the walls of the
bottles, while the broth itself was not perceptibly turbid.
Micro-colonies seldom appear on slides exposed to sea water
244
CLAUDE E. ZOBELL AND
ESTHER
C. ALLEN
for only a few hours; the cells for the most part occur singly or in
pairs, or more rarely, in long chains. The paucity of microcolonies is attributed to the lack of available nutrients in sea
water, because they appear abundantly on slides in sea water to
which nutrients have been added. Under the latter conditions
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Fit2s Brokeenlnd rpeent thes
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face of the slide and all stages of cell division”-cn be observed.
Ordinarily it requires two to four hours for appreciable numbers of
bacteria to become attached solidly to glass slides. Firm attachment after comig into contact with the surface is not immediate,
BACTERIA IN FOULING OF SUBMERGED SURFACES
245
as it seems to take several minutes for the bacteria to cement
themselves to the glass. Thus, the submergence of a slide in
sea water replete with bacteria, followed by its immediate removal, does not result in the attachment of bacteria, although a
few may adhere temporarily. But let the slide remain submerged
for an hour or two, and if the bacteria are of the attachment type
and are in the logarithmic phase of growth, they will be found
profusely, so firmly glued to the slide that running water will not
detach them.
Various sizes and shapes of bacteria appear. Ovoid organisms
with a diameter of less than 1 micron are most numerous. Such
coccobacilli comprise at least 60 per cent of the total number of
bacteria. Slender bacilli, 1js to 21 in length, are common. Larger
rods occur less frequently. True cocci and spirilla are seldom
seen. The majority of these organisms possess well-defined capsules which in some cases are twice or three times the size of the
enclosed bacterium. Nearly all of the bacteria are Gramnegative.
Assorted filamentous forms occasionally appear. These are of
two principal types: (a) actinomyces, consisting of small patches
of slender mycelial threads which appear both continuous and
fragmented, and (b) larger, straighter, branched filaments which
are probably Chlamydobacter. Leptothrix has been observed.
While it is impossible to identify species of bacteria from a
consideration of morphological features only, it is estimated from
the diversity of form, size and structure, that no less than forty
or fifty species of bacteria are represented regularly, and probably
more. Twenty-eight pure cultures have been isolated from
twenty-four-hour films for further study. Immediately following isolation the majority of these grow readily in sea-water
media, but not in a corresponding nutrient solution prepared
with fresh water, thereby indicating their halophilic specificity.
Several of these have been completely characterized according to
the methods of the Committee (1930) on the Pure Culture Study
of Bacteria, and they are found to be new species.
Three representative species which have been encountered
frequently attached to submerged surfaces are herewith described.
246
CLAUDE E. ZOBELL
AND
ESTHER C. ALLEN
No. 577. Achromobacter marinoglutinosus, n. sp. Short Gramnegative rods 0.7 to 1.0 by 1.8 to 2.4 microns with rounded ends. Methylene blue shows granular structure. Occur singly, in pairs, and in
clumps. Encapsulated. Motile by means of polar flagella.
Gelatin stab: Moderate filiform growth with slight napiform liquefaction. No pigment.
Agar slant: Moderate, filiform, flat growth. Butyrous consistency.
Agar colonies: Round with concentric circles and crinkled radial line,
1.5 to 5.0 mm. in diameter. No pigment.
Broth: Moderate clouding, marked ring, adherent film of growth on
test-tube wall, and flaky sediment. No growth in milk or on potato.
No indol.
Produces hydrogen sulphide and ammonia from Bacto-tryptone.
Reduces neither nitrate nor nitrite.
Does not ferment glucose, lactose, sucrose or mannitol.
Produces acid but no gas from xylose and dextrin.
Starch hydrolyzed.
Facultative aerobe.
Optimum temperature, 200 to 250C.
No. 580. Achromobader membranoformis, n. sp. Rods 0.9 to 1.2 by
3.5 to 4.8 microns, occurring singly and in pairs. Encapsulated. Motile by means of lophotrichous flagella.
Gelatin stab: Filiform growth, best at top with slow crateriform
liquefaction.
Agar slant: Moderate, beaded, raised growth. Membranous consistency. Becomes browned with age.
Agar colonies: Circular 1.0 to 2.5 mm. with crinkled surface.
Broth: Slight clouding, flocculent sediment, film of growth on walls
of test tube.
No growth in milk or on potato.
No indol or hydrogen sulphide.
Reduces neither nitrate nor nitrite.
Produces acid without gas from glucose, sucrose, dextrin, and mannitol. Lactose and xylose not fermented.
No diastatic action.
Aerobic.
Optimum temperature, 200 to 250C.
No. 588. Flavobacterium amocontactus, n. sp. Slender Gram-negative rods, 0.4 to 0.7 by 1.6 to 2.3 microns, with rounded ends. Stain
BACTERIA IN FOULING OF SUBMERGED SURFACES
247
very lightly. Occur singly or in irregular clumps. Possess well-defined
capsules. Actively motile by means of peritrichous flagella.
Gelatin stab: Good filiform growth with rapid saccate liquefaction.
Agar slant: Abundant, filiform, smooth, glistening bright yellow
growth having a butyrous consistency. Originally liquefied agar but
this property was lost following artificial cultivation.
Agar colonies: Circular 2.0 to 4.0 mm. in diameter, yellow.
Broth: Good growth in sea-water broth with ring at surface, strong
clouding and abundant viscid sediment. No odor.
No growth in milk.
No growth on ordinary potato but slight yellow growth on potato
dialyzed in sea water.
No indol.
Produces hydrogen sulphide.
Ammonia liberated from peptone.
Reduces both nitrate and nitrite.
Does not ferment glucose, lactose, sucrose, xylose or mannitol.
Starch not attacked.
Facultative aerobe.
Optimum temperature, 180 to 210C. Optimum reaction, pH. 8.0.
The microscopic organisms other than bacteria which become
attached to the submerged slides consist chiefly of diatoms, the
common genera being Grammatophora, Navicula, Liemophora,
Fragilaria, Striatella, and Nitzschia. Although the diatoms are
much less numerous than bacteria, they are always more abundant than the macroscopic organisms. The data summarized in
table 2 show that approximately a hundred times as many bacteria as of all other classes of organisms combined were attached
to the submerged slide during the primary stages. Furthermore,
it is of interest to note that the accumulation of bacteria and,
more particularly, of diatoms does not proceed in arithmetical
progression with twenty-four-hour intervals; or, in other words,
when the number of attached organisms is plotted against time,
a parabolic curve rather than a straight line results. This indicates either a multiplication of the attached organisms or a
favoring influence of the film-formers upon subsequent attachment. Both factors are probably operative.
As indicated by table 2, very few macroscopic organisms, or
248
CLAUDE E. ZOBELL AND ESTHER C.
ALLEN
those which could be located without the aid of a lens, were
attached to slides which had been submerged for only three days.
Their number continued to increase slowly but progressively
from the fourth to the seventh day, the longest submergence
period considered in these studies. Suctoria and hydroids were
most abundant, both appearing with regularity throughout the
year but in appreciable numbers only after five days. Cyprid
larvae of barnacles were found occasionally from March to
August, being common only in the last four months of that period. Most of the larvae were not firmly attached to the slides
in seven days and were readily washed off. Bryozoa appeared
very rarely. For more information concerning these macroscopic sedentary marine organisms in this vicinity the reader is
referred to Coe (1932).
TABLE 2
Average number of different classes of organisms, which were attached to 2 square
inches of slide after 24, 48, and 72 hours’ submergence during the first six
months of 1988
PERIOD OF
SUBMERGENCE
BACT
ERI
OTHER MICROORGANISMS
INCLUDING DIATOMS
MACROSCOPIC
ORGANISMS
2,560
10,840
28,310
0.3
1.2
1.9
hours
24
48
72
2,023,800
9,268,200
24,115,400
What is the relation of the primary bacterial film to the attachment of other forms? To elucidate this point further, filmcoated glass slides were submerged concurrently with sterile
slides as controls. The films were prepared in the laboratory by
placing slides in bottles of nutrient broth inoculated with cultures of attachment bacteria until a good film had formed. Following one to five day’s submergence in the sea the film-coated
and the originally sterile slides were examined for attached organisms. The film-covered slides had a noticeably greater number of attached organisms than did the slides which were originally sterile. Table 3 presents the average results.
Not only do bacteria play an important r6le as primary filmformers, but they are also found in abundance associated with the
249
BACTERIA IN FOULING OF SUBMERGED SURFACES
growths on fouled surfaces during the later stages of fouling. Several specimens of slimy film were scraped from the hull of the U.
S. N. Destroyer LAUB as it was being dry-docked. The vessel
had been in the water for thirty-eight months. Direct microscopic analyses by a modified Breed and Brew (1916) method of
this material properly diluted revealed that it contained from
a few million to several billion bacteria per gram of moist scrapings. From preliminary results which will be reported in greater
detail elsewhere it is estimated that, on an average, 8 to 9 per
cent by volume of the fouling cumulation on this particular
vessel consisted of bacteria. This plethora of bacteria is attributed to the abundance of organic matter on which they feed and
to their sedentary properties.
TABLE 3
Average number of microorganisms (excluding bacteria) and macroscopic organisms
attached to 2 square inches of sterile micro-slides and to slides coated with a
bacterial film under comparable conditions
OMICEO6GAMSMS
PERIOD OF
SUBMIERGENCE
MACRO6RGAMISM
Sterile slides
Film-coated slides
Sterile slides
Film-coated slides
15
23
98
852
42
89
276
1,257
0.3
1.1
5.4
7.2
18.6
43.8
hours
24
48
72
120
Hilen (1923) reported that the slime which forms on surfaces
in ocean water is “composed of a variety of bacteria as well as
yeasts and molds.” Corroborating these studies, Angst (1923)
found that the slime on ships’ bottoms is caused to a large extent
by bacteria, and, furthermore, he concluded that “the slime bears
a direct relation to the appearance of the barnacles.”
CONCLUSIONS
Our observations show quite conclusively that bacteria and, to
a lesser extent, other microorganisms are the primary filmformers on submerged glass slides, and that such films favor the
subsequent attachment of the larger and more inimical fouling
250
CLAUDE E. ZOBELL AND ESTHER
C.
ALLEN
organisms. The film of bacteria may promote the attachment of
macroscopic organisns in different ways. They may form a
mucilaginous surface to which the fouling organisms in the planktonic or free-swimming stage readily adhere until they can prepare their own holdfast. Again, the bacterial film together with
the particulate organic detritus which sticks to it, may furnish
the fouling organisms with a suitable source of food during their
infancy. Speaking of barnacle larvae in captivity, Visscher
(1928a) says, “These organisms have been observed to ‘walk’
for considerable distances, and have been seen to ‘test’ various
areas for a period of more than an hour before finally attaching.”
It is possible that at least a part of this delay in attaching may be
due to the influence of microbrganic films. Furthermore, while
it is still a matter of conjecture, it is entirely possible that in the
case of submerged surfaces which contain substances poisonous
to the fouling organisms, the bacterial film forms a protective
coating.
While it remains to be proved that barnacles, bryozoa, hydroids
and other macroscopic fouling organisms will not attach without
the aid of a primary bacterial or other microbrganic film, the
foregoing studies suggest that microorganisms merit considerable
attention in investigating the exact cause and ultimate prevention
of fouling.
REFERENCES
ANGST, E. C. 1923 The fouling of ship bottoms by bacteria. Report, Bureau
Construction and Repair, United States Navy Department, Washington.
BREED, R. S., AND BREW, J. D. 1916 Counting bacteria by means of the microscope. N. Y. Agric. Exp. Sta., Bull. 49.
COE, W. R. 1932 Bull. Scripps Inst. Oceanog., tech. series, 3, 37-86.
Committee 1930 Manual of methods for pure culture study of bacteria. Soc.
American Bact., Geneva, N. Y.
CONN, H. J. 1918 The microscopic study of bacteria and fungi in soil. N. Y.
Agric. Exper. Sta., Tech. Bull. 34.
HENRICI, A. T. 1933 Jour. Bact., 25, 277-286.
HENTSCHEL, E. 1925 “Anwasserbiologie” in Abderhalden’s Handb. der biol.
Arbeitsmethod, Abt. 9, 266.
HILEN, E. J. 1923 Report on a bacteriological study of ocean slime. Report
Bureau Construction and Repair, United States Navy Department,
Washington.
BACTERIA IN FOULING OF SUBMERGED SURFACES
251
NAumANN, E. 1925 “Wasserwerkbiologie” in Abderhalden’s Handb. der biol.
Arbeitsmethod, Abt. 9, 229.
THOMASSON, H. 1925 “Methoden zur Untersuchung der Mikrophyten, usw.”
In Abderhalden’s Handb. der biol. Arbeitsmethod, Abt. 9, 685.
VISSCHER, J. P. 1928a Biol. Bull., 54, 327-335.
VISSCHER, J. P. 1928b Bureau of Fisheries, Bull. 43, 193-252.
WILSON, 0. T. 1925 Ecology, 6, 303-311.
ZoBELL, C. E., AND ALLEN, ESTHER C. 1933 Proc. Soc. Exper. Biol. and Med.,
30, 1409-1411.
ZoBELL, C. E., AND FELTHAM, C. B. 1934 Preliminary studies on the distribution and characteristics of marine bacteria. Bull. Scripps Inst.
Oceanog., tech. series 3, 275-295.
clear concise fashion
2. (42)
Please read Zobell and Allen’s “the significance of marine bacteria in the fouling of
submerged surfaces?.” from 1934. The key passages to read are pages 239-243&249-250;
but since you’re a literary nerd, you may enjoy and benefit from reading the whole article.
ASSIN
b. (21) Critique the paper as it is (for its time period)
i. Forethought/ ingenuity
ii. Technique choices
iii. Mention at least one thing that could be improved (again, for its time period)
c. (21) Write a short summary (abstract) for this paper using modern day scientific
language. Only refer to what was reported to be done in this paper, but you can use
verbiage that has been developed since its publication.
iv. What was the problem?
V. What were the techniques used and in what ways?
vi. What was the primary finding and how novel/impactful is this finding?
osc
3. (18) The evaluation of a community and an ecosystem depends greatly on the definition of an
individual
a. (6) Define the following evaluations of an ecosystem:
i. Diversity
ii. Richness
iii. Evenness
b. (12) Calculate the following ecological indexes. Use the
following depiction of microbes and the equation.
i. diversity (4),
ii. richness (4),
iii. evenness (4)
H = Diversity Index
S = Species Count
P;= proportion of
made
the ith species
H–3p,In p.
EH = Pool Everness Ex =H/In S
I
osc