The vast beds of the giant kelp Macrocystis in
Monterey Bay provide a major source of food for beach
fauna in the form of wrack. A major component poly
saccharide in Macrocystis, alginic acid, has been
shown to be digestible by a number of invertebrates
(Meeuse & Fleugel, 1958; Eppley & Lasker, 1959; Oshima
1931; Galli & Giese, 1959; Fraussen & Jeuniaux, 1965;
Favorov & Vaskovsky, 1971). This polysaccharide con-
stitutes up to 30% of the dry weight of Phaeophytes (Haug
& Larsen,1965). However, all of those shown to digest
alginate are subtidal or very low intertidal dwellers;
there has been reported no direct use of this polysaccharide
by fauna of the sandy beach. A number of bacteria capable
of hydrolyzing alginate have been described (Kooiman. 1954;
Waksman, 1934). Working with the abalone Haliotis, Nakada
& Sweeney (1967) have isolated two different alginases;
alginases I rapidly reduces the viscosity of an alginate
broth, apparently by cleaving internal ø1,4, glycosidic
linkages. Alginase II shows almost no activity when assayed
by viscosimetry, but spectrophotometric assays suggest that
it catalyzes an eliminase reaction, attacking the ends of
the polymer and releasing diuronide units. Though alginase
producing bacteria have been found, the nature of their
decomposition of alginate is unclear. Furthermore, no
attempt has been made to correllate their activity with
the import of food to the beach community. Thus, this
study of the decomposition of alginic acid by bacteria of
Macrocystis was undertaken.
Materials and Methods
Large samples of M. pyrifera and M. integrefolia
were cut from the beds off the Hopkins Marine Station
and placed on the beach above the high tide mark. Samples
from these piles were taken from moist fronds that lay
immediately below the dried, crusty surface layer.
Enzyme assays: One disk (diameter 2.4 cm) of alga
was cut and shaken in 10 ml distilled water with a vortex
mixer for 30 seconds. One ml of this suspension was
added to 5 ml 0.25% Na alginate (Keltone, supplied by the
Kelco Co., Los Angeles). Viscosity was determined after
a variable incubation period by adding 3 ml of the test
solution to an Ostwald viscosimeter at 25°C. Standards
of distilled water were tested with each sample. Glucuronic
acid and 0.25% Na alginate were used as reference materials.
Culture media: Alginase-producing bacteria were
isolated from decomposing Macrocystis by inoculating
alginate-agar plates (2% agar; 0.5% Ca alginate; 0.1% KH2PO4:
0.02% KpHPOp: 0.1% yeast extract; in 3.5% artificial sea
water) with suspensions derived from shaking one algal
disk in 10 ml sterile artificial sea water. Colonies
thus isolated were grown in alginate liquid medium (as
above, except Na alginate was used in place of Ca alginate)
for 48 hours at 25°C. Two aliquots of this medium were cen
trifuged at 6000 g for 10 minutes; two aliquots were heated
in a boiling water bath for 1 hour. Samples of the medium,
the supernatant, the pellet, and the heated preparations were
assayed for alginase activity by viscosimetry. Samples were
also tested for alginase activity spectrophotometrically by
the method of Nakada (1960).
Results
In figure 1, alginase activity, as measured by
viscosimetry, in algae undergoing decomposition on the
beach is shown to increase for the first six days.
Two forms of bacteria showing marked differences in
colonial morphology were isolated; a white colonial form,
found at every sampling and comprised of actively motile,
rod-shaped organisms 2x.5 microns; and a brown colonial
form, found only on algae that had been on the beach for at
least two days and comprised of motile, rod-shaped bacteria,
1.5x.5 microns. Both the brown and the white colonial
forms showed alginase activity in their ability to liquify
an alginate gel within 48 hours. Culture supernatants
prpared using alginate liquid medium contained alginase
activity (figure 2). Little activity remained with the
cells themselves. Boiling moderately reduced alginase activity.
Adding additional enzyme after 2 hours of digestion had
no further effect on viscosity as compared with dilution
with an equal volume of water. In all preparations tested,
80-90% of the decrease in viscosity evident after the
addition of enzyme suspension occurredwithin the first
5 minutes. No difference in the specificity of the two
bacterial enzymes could be demonstrated by a study of mixtures.
The spectrophotometric assay of Nakada & Sweeney (1967) did not
indicate that the bacterial enzymes function as eliminases.
Discussion
Alginase activity of decomposing Macrocystis is maximal
after six days of decomposition. Two forms of bacteria
capable of using alginate as a sole carbon source were
—

e

13
12
DAYS ON BEACH
(



—
1.

—
TIME (HRS
DIGESTION
Captions
Figure 1. Change in viscosity as a measure of alginase
Macrocystis pyrifera
in decomposing Macrocystis. H-
-  Macrocystis integrefolia
Viscosity is given in viscosity coefficients; viscosity-d.
dot.
where d-density of water, t- time of outflow of water,
dodensity of test medium, to-time of outflow of test medium.
Figure 2. Change in viscosity as a measure of activity of
White culture supernatant,
various enzyme preparations.
Brown culture supernatant, +- - —+ Heat-treated
white culture supernatant, G-- — Heat-treated brown culture
White culture pellet,
supernatant,
Brown culture pellet, o....... Na alginate
standard.
isolated from this decomposing material. Enzyme preparations
from both forms behave very similarly. No synergistic
effect could be demonstrated when the enzymes were tested
in combination. This enzyme acts extra-cellularly and
is relatively heat-stable, suffering only 30% inactivation
after 1 hour at 100°C. No eliminase activity like that
described by Nakada and Sweeney (1967) was observed in the
cultures or culture supernatants. These experiments
suggest that the rate of decomposition of alginate is
directly a function of the amount of hydrolytic alginase
present and active. The use of viscosimetry for the
assay of alginase activity has proved to be an accurate
means for measuring this enzyme in a variety of materials.
Alginate as an energy-rich food source to the sandy beach
community appears to be made available to non-alginase
producing organisms through microbial activity.
10.
11.
Literature cited
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2.
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