m
TITI
INTRODUCTTON
Investigations have shown that that portion of the Coliform
group found in the gastrointestinal tract and feces of warm blooded
animals has the ability to ferment lactose with the production of gas
under suitable conditions involving restrictive media and elevated
temperatures. Since bacteria from other sources are, for the most
part, unable to produce gas under such conditions, this criterion has
been used to indicate the presence of fecal Coliforms in bodies of
water, and the presence of such bacteria has become a standard micro-
biological parameter indicating water contamination. The reliability
of this test for the marine environment has been questioned however,
since the number of Coliforms detectable often fluctuates unpredictaly
over short periods of time and may depend upon such factors as cloud
cover (1) and ocean currents. The microfauna associated with marine
sediments may not be subject to such fluctuations. The purpose of this
paper is to indicate a second biochemical parameter which may reflect
domestic sewage pollution, and which might be used to confirm or even
replace Coliform determinations in the marine environment. The rationaie
for this new criterion is related to the urease activity of the enteric
Proteus vulgaris, a common bacterium in the intestinal tract of man.
Amounts of the enzyme urease can be readily determined due to the ammonia
which is produced upon hydrolysis of urea by the enzyme. This study is
an attempt to correlate urease activity in marine beach sediments with
proximity to ocean sewage outfalls.
240
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M
p
MATERTALS AND METHODS
Marine Sediments
Sands were always obtained where they were barely awash.
Samples were taken at both low and high tide. The sediments were
collected in 500 ml. wide-mouthed, plastic, screw-capped jars along
with approximately 50 ml. of the ambient waters. In the laboratory,
the samples were stored at sea water temperature (13-15 C.) until they
could be tested. The time elapsed between collection and testing was
never more than two hours.
Urease Assay -
150 ml. of a test medium consisting of 30.0 grams Nacl and
50.0 grams urea CO(NH2)2 per liter of distilled water was filter ster-
ilized and placed in a small-mouthed, 250 ml. glass bottle. Recently
collected sand was dried for thirty seconds on a clean blotter, then
50.0 grams were weighed and placed in the test bottle. The ph was
adjusted to 9.5 with 0.1 N Naoh and the bottles were incubated at
37.0°C. (+ 0.5° C.) in a water bath. Air which had passed through a
cotton filter was bubbled through the test solution at the rate of
130 cc. per minute, and ammonia was trapped in 10 ml. of a saturate
boric acid solution containing a mixed indicator (2) held in a 125 ml.
Erlenmeyer flask. The amount of ammonia released from the test prep-
aration and trapped in the boric acid was determined by titration at
intervals of time with 0.01 N HCl. After each titration, a fresh boric
acid trap was attached.
a4
- 3 -
Cultures-
is and Escherechia coli used in
Cultures of Proteus vulge
these studies were obtained through the courtesy of Miss Adeline Larson
of the Department of Bacteriology, University of California at Berkeley.
Enu
eration of Proteus vulgaris and Escherechia coli-
The M.P.N. of E. coli was determined by standard methods (3).
Difco urea broth was used to test for the presence of P. vulga
is in
survival experiments and to detect the presence of the genus Proteus
in sea water. Plate counts (on Difco nutrient agar) were used to
enumerate P. vulgaris.
Enzyme Preparation -
Jack Bean urease was prepared from Matheson Coleman and Bell
tablets by grinding in distilled water. Urease preparations used in
subsequent tests were prepared using two 25.0 mg. tablets per ml. of
water. The urease was activated by the addition of 0.2 ml. of
14% sodium pyrophosphate solution.
ats
RESULT
Urease Activity of a Jack Bean Urease Preparation -
The action of several concentrations of Jack Bean urease upon
the test medium was investigated. So as to mimic conditions used in
tests upon ambient sands, 50 grams of sand sterilized for four hours
(250 C., 15p.s.i.) was included in the preparation. As can be seen
from Figure 1, the rate of ammonia evolution is directly related to
the enzyme concentration.
+47
Urease Acivit
of Proteus vulgaris-
The urease activity of various numbers of the urease containing
bacterium P. vulgaris was investigated. The results of this experiment
appear in Figure 2.
The rate of urea breakdown can be related to the density of
P. vulgaris. Although this relationship requires further quantitation,
Figure 3 shows a plot of the rate of the reaction vs. the number of
P. vulgaris, as revealed by this experiment.
Relationship Between Sediment Size and Urease Activity
The effect of sediment particle size on urease activity was
investigated using sand obtained 30 feet from the Pacific Grove sewage
outfall pipe (Station +10, Figure 6). This sediment was divided into
three fractions with the aid of Tyler screens: "coarse" : 3.9 - 2.9 mm.,
"medium" : 2.9 - 1.9 mm., and "fine" : 2 1.9 mm. Figure 4 presents the
results of this determination. A clear relationship between particle
size and urease activity in sand from this polluted area is indicated.
26
5-
Sands from the west beach of the Hopkins Marine Station (Station k7,
Figure 5), a relatively unpolluted area of Monterey Bay, were similariy
-r +
examined. Only a trace of urease activity was detected in any of these
sediment fractions. The slow rises before definitive slopes are
established in Figure 4 may be attributed to fluctuations in the air
flow, a condition which was corrected at 30 hours. In all subsequent
urease tests, the "fine" sediments were used and the air flow was
held constant.
Urease Activity in Marine Sediments
A study of beach sediments collected at various distances from
two marine outfalls was undertaken. The entire study area, comprising
some five miles of the southern shoreline of Monterey Bay is depicted
in Figure 5, with sampling stations 1 - 8 indicated. Figure 6 is an
enlargement of the Pacific Grove study area showing the positions of
stations 9 - 12 relative to the outfall pipe.
Sample stations 1 -6 were located in the vicinity of the
Monterey outfall, along a beach made up of homogenously fine (1.9 -
0.9 mm.) sand. This outfall is subtidal and 800 feet from shore.
Figure 7 indicates the amount of urease activity detectable on the day
these sediments were sampled. Current studies in this area indicate a
dominant onshore flow outside the surf zone in this area, and a net flow
to the west, in the direction of the Monterey Municipal Wharf No. 2 (4).
Urease activity reflects the current pattern as it is understood. Sample
station 6lies very close to the aforementioned wharf ; this area
receives an additional load of sewage from restaurants and moored boats.
p9
Sample stations 9 - 12 were located within a 200 yard radius
+
This outfall is intertidal, and clearly
of the Pacific Grove outfall.
visible at times of low tide. Station 9 (Figure 6) lies across a small
surge channel from the pipe and receives effluent only at high tide.
Station 8 (Figure 5) is a public bathing beach. Figure 8 shows the
amount of urease activity observed in sediments collected from these
areas.
The overlying waters at all stations were cultured for the
presence of urease producing bacteria. In all cases these tests were
negative.
Survival of Escherechia coli and Proteus vulgaris in Sea Water and Se
100 ml. of unfiltered Monterey Bay sea water was placed in a
250 ml. glass bottle. 1.0 ml. of a suspension of either bacterium
containing between 1X102 and 1X107 bacteria per mililiter was added, and
the preparation was aereated at 200 cc. per minute. The bottles were
incubated at sea water temperature (13 - 15° C.). At time intervals,
the bottles were vigorously shaken and 5.0 ml. aliquots of liquid were
removed for enumeration of surviving bacteria. Figure 9 shows the results
of this experiment. The viability of both organisms under these con-
ditions seems to be similar.
Survival in wet sand was tested by placing 5.0 grams of sterile
"fine" sand in 10 X 100 test tubes along with 0.5 ml. of sterile sea water.
The tubes were innoculated with a dose of organisms similar to that used
above. All tubes were incubated at sea water temperature and at time
intervals were tested for growth of urease producing or lactose fermenting
bacteria. Both species were able to survive for over a week in moist
vuls
tris survived for 7.5
sediments. In two separate experiments,

ents
5
- 7 -
and 8.5 days, while E. coli remained viable for over 9.0 days in both
tests.
prencer
DISCUSSION
The amount of urease activity in beach sediments along
Monterey Bay appears to be related to proximity to sewage outfalls
(from plants utilizing primary treatment processes only). Although
in this study no attempt was made to determine the source (s) of this
enzyme, the relationship between sediment particle size and urease
activity suggests the presence of microorganisms. Further, the urease
activity observed in local sediments can be mimicked through the use
of suspensions of Proteus vulgaris. This organism shows appreciable
survival in marine materials.
Urease activity is not a conspicuous property of marine
bacteria. Of 130 random bacterial isolates from the Monterey Bay
intertidal, only 4 showed the ability to decompose urea (5). Bergey'
Manual (6) lists only three marine organisms which show urease
activity : Sarcina ureae, Bacterium ammoniagenes, and Bacillus pasteurii.
All were originally isolated from sewage, feces, or decomposing urine,
but are thought to be able to exist in salt water. Members of the
genus Proteus, all of which show urease activity, have been isolated only
rarely from air, soil, or fresh water that has not been contaminated
with fecal material (7). Therefore, the measurement of urease activity
in marine sediments shows promise as a measure of the area contaminated
by sewage outfalls.
A lag of from 14 to 22 hours before ammonia can be detected
is evident in tests involving natural sediments and at all but the
highest concentration of P. vulgaris in innoculated sands. Yet only
a minimal lag appeared when Jack Bean urease was tested. Attempts to
remove this lag by pre-incubation for 24 hours in 3% NaCl before addition
of urea, increased air flow, and the addition of appropriate amounts
of ammonium chloride all met with failure. Growth of P. vulgaris in
nutrient broth containing 1% urea for 24 hours prior to harvest and
testing succeeded in lowering the time lag, but only to 8 hours.
Therefore, it seems unlikely that the observed lag is caused by a
purely adaptive enzyme system. Although natural sediments contain
some organic matter, growth must be minimal. No growth permitting
substances were included in the urease assay medium. The duration of
the lag did not seem to affect the rate of observed urease activity.
and this rate could be directly rélated to either the amount of
Jack Bean urease or the number of P. vulgaris added.
Further research is necessary to determine the source or
sources of urease present in the sediments studied. Also, an accurate.
simple assay must be developed before urease activity in marine
sediments can be used as a convenient test for sewage pollution.
This study indicates that such further research is warranted.
2
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SUMMAR!
1. A relationship between the amount of urease activity in marine
beach sediments and proximity to sewage outfalls has been
demonstrated.
2. The amount of urease activity detectable is directly related to
sediment particle size and can be related to the number of
Proteus vulgaris added to sterile sediments.
vulgaris, a typical urease producer and an inhabitant
3. Proteus
of the human gastrointestinal tract, shows substantial survival
in marine materials.
2
ADTTON
FIGURE CAPTIONS
-754
Figure 1. Urease Activity of Jack Bean Urease
Preparations.
A : 5.0 ml. urease; B : 1.0 ml. urease; C : 0.5 ml. urease;
d : 0.1 ml. urease.
Figure 2. Urease activity of Proteus vulgaris.
A : 1.6 X 1010 bacteria; B : 1.2 X 106 bacteria; C : 1.1 X 104 bacteria.
Figure 3. Relationship Between Number of Proteus vulgaris and the
Rate of Ammonia Evolution.
Figure 4. Relationship Between Sediment Size and Urease Activity.
A : "Coarse", 3.9 - 2.9 mm.; B : "Medium", 2,9 - 1.9 mm.;
0 : "Fine", 2 1.9 mm.
Figure 5. The Monterey
Bay Shorline Study Area.
Numbers indicate beach sediment sampling stations.
Figure 6. Enlargement of the Pacific Grove Outfall Study Area.
Numbers indicate beach sediment sampling stations.
+
Figure 7. Urease Activity in Marine Beach Sediments Collected in the
Vicinity of the Monte
Outfall.
+
Numbers correspond to sampling stations indicated in Figure 5.
e.
EIGURE CAPIIONS (Cont.)
Figure 8. Urease Activity in Marine Beach Sediments Collected in the
Vicinity of the Pacific Grove Outfall.
Numbers correspond to the sampling stations indicated in Figures 5 and 6.
).
and Escherechia coli in
Figure 9. Survival of Proteus vulgari
Sea Water.
Monterey Bay
28
102

L
—
L


68
51
34
17
10.2
O


Time: Hours
40
5
D
—

5.1.
3.4
1.7





40
Time: Hours
O
D
S

0.34
0.085
0.17
mg NH, Liberated per Hour
U
S
13.6
11.
10.2

+ 0.9
—
—
o
6.8
5.1
3.4
.7




20
Lime: Hours
a

12
38

scale:




— ++







Pacitic
8
Grove

41
Monterey



LIEI


51. 1
1 mile

t  5



Outfall
U
S
Pacific
Grove
Outfall
28
6
5
9 2.
—

10

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è
scale.

10 yards
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2
86

D

20




Time: Hours
5.1
3.
1.7.
+
8.
—

o
6.8
5.1
3.4
1.7


20

i



48
50
Time: Hours

o
S
D5
o
—

20


— Vulgaris
41
90
Survival Time: Hours
E. Cl
100
110
20
25
0
RTOT
ADIV
BIBLIOGRAFHI
Trumbauer, David S., "A Coliform Bacteria Survey of Monterey Bay off

Del Monte Beach", M.S. Thesis, U. S. Naval Posugraduate School, 1966.
Kabat, E. and Mayer, M., Experimental Immunochemistry, Charles C. Thomas,
Springfield, Illinois, 1961.
St
Standard Methods for the Examination of Water and Wastewater,
Ed.
12
American Public Health Association, Inc., 1965.
Stevenson, C.D., "A Study of Currents in southern Monterey Bay", M.S.
Thesis, U. S. Naval Postgraduate School, 1964.
Phillips, John H., June, 1970, Personal communication.

-+5
Bergey's Manual of Determinative Bacteriol
ogY, 6 Ed., Williams and
Wilkins Co., Baltimore, 1948.
Levine, M., 1942, "An Ecological Study of Proteus", J. Bact., 13: 34.
JKNC
LEDGEMEN
I thank Dr. John H. Phillips for encouragement and advice
in the course of this work, and Drs. C. B. Van Niel and W.B. Watt
for critically reading this manuscript. I further express my
gratitude to the faculty, students, and staff of the Hopkins Marine
m
Station for their enthusiastic support. Ihls work was supported in
part by the National Science Foundation Undergraduate Research
Program, Grant No. GY - 7244.
6