A M . ZOOLOGIST. 8:95-106 (1968).
Dissolved Organic Matter as a Potential Source of Nutrition for
Marine Organisms
GROVER C. STEPHENS
Department of Organismic Biology, University of California,
Irvine, California 92664
SYNOPSIS. The general features of accumulation of amino acids by marine invertebrates are
outlined. The wide distribution of this ability is noted. Data from observations of this process
for periods of several days are presented for the polychaete worm, Doruillea articulata. Measurements of the constituents in the pool of free amino acids of this same organism are presented
together with data concerning the rate of appearance of radioactivity dn the medium after permitting animals to accumulate amino acids. These data permit tentative estimates of rates at
which amino acids are lost to the medium. These apparent "leakage" rates are low when compared with rates of uptake. The thermodynamic work necessary to move material against the
concentration differences involved is calculated. The fraction of metabolic energy needed is
small. The possible biological significance of uptake of small organic compounds is discussed.
It is concluded that uptake of amino acids occurs sufficiently generally and at such rates that
it should be included in any analysis of the pathways whereby material is acquired from the
environment by marine organisms.
The possibility that dissolved organic
compounds may contribute to the nutrition
of aquatic organisms is not a new idea. It
is usually associated with the name of Putter (1909). A very large early literature
was brilliantly reviewed by Krogh (1931).
The subsequent literature in limited areas
of the field has been summarized in several
places. The present paper is not intended
as a comprehensive review but rather as a
discussion of some selected aspects of the
problem.
Darnell (1967) defines detritus broadly
to include small organic molecules as well
as larger particles. He classifies the material on the basis of size, denning particles
smaller than one micron as subparticulate
detritus. He notes that there will be contributions of material from one size class
to another so that some brief notice of the
possible use of particles of colloidal size is
in order in this paper, even though most
of our discussion will be concerned with
small organic compounds.
I wish to acknowledge the help of Dr. Mary Clark
and Lyle Wong in obtaining the data on Dorvillea.
I should also like to express my debt to Dr. Clark
for discussion and clarification of many of the
points in the present manuscript. New work reported here has been supported by USPHS Grant,
GM 12889, and by ONR Contract, N00014-67-A03230001.
95
There are certainly organisms which can
utilize organic detritus of colloidal dimensions and these particles are no doubt present in aquatic habitats. Presumably some
of the detritus in this size range arises as
a result of the mechanical, chemical, and
biological breakdown of larger particles.
Also, there are physical processes operating
which increase the size of particles or,
phrased alternatively, which make available
small molecules to organisms which would
otherwise be unable to obtain them. Fox,
el al. (1952, 1953) have discussed the role
of leptopel in this regard. Inorganic particles of colloidal dimensions provide a
surface on which small organic molecules
can be concentrated by adsorption. Filterfeeding organisms may then utilize the resulting micelles. Cheesman (1956) notes
that organisms may crop protein at the
surface of a body of water where it has
been concentrated and denatured at the
air-water interface. Baylor and Sutcliffe
(1963) and Riley (1963) extend this idea
to the iormation of organic aggregates at
the air-water interface of bubbles.
All of these colloidal or suspended forms
of "subparticulate organic matter" occur,
although the quantitative contribution they
make to the nutrition of aquatic animals
under natural conditions is just beginning
to be analyzed (Johannes, 1967). Clearly,
96
GROVER C. STEPHENS
such pathways for utilization of organic
material in watery habitats may be very
important. The failure of further discussion of this aspect of the subject here is
not intended to minimize its potential imjjortance. The emphasis of this paper
merely reflects my interest in the process
of accumulation of small organic compounds from solution by marine organisms.
UPTAKE OF DISSOLVED ORGANIC COMPOUNDS
Our laboratory has published a series of
papers (Stephens, 1960, 1962, 1963, 1964,
1967; Stephens and Schinske, 1961; Stephens, et al., 1965; Stephens and Virkar,
1966; North and Stephens, 1967; Virkar,
1966) concerning the phenomenon of uptake of organic compounds by marine organisms. Ferguson (1967) has independently reported accumulation of small organic
compounds by starfishes, and the uptake
of acetate and glucose by Antarctic euphausid shrimp has been reported by
McWhinnie and Johanneck (1966). Table
1 lists phyla in which at least some representatives accumulate amino acids from
dilute solution.
It is apparent that the capacity for removing small organic compounds from
dilute solution is very broadly distributed.
In our experience, essentially all softbodied marine invertebrates are capable of
such accumulation though rates vary. With
respect to marine algae, some genera in
each of the phyla listed remove glycine
from sea water very rapidly while other
genera either fail to do so or pick it up so
slowly as to appear negative when subjected to our standard procedures. The
only vertebrate we have found which is
TABLE 1. Phyla which have been shown to be
capable of removing amino acids from dilute solutions in ambient sea water.
Chlorophyta
Phaeophyta
Rhodophyta
Cnidaria
Platj'helminthes
Rhynchocoela
Ectoprocta
Ajinelida
Sipunculoidea
Mollusca
Arthropoda
Echinodermata
Hemichordata
Chorda ta
TJrochordata
Cephalochordata
Vertebrata
capable of removing amino acids from
ambient solution is the hagfish, Eptatretus,
although a number of other animals has
been tried.
Most fresh water organisms seem incapable of removing small organic molecules from solution at anything like the
rate which characterizes marine forms.
Stephens (1964) studied two species of
nereid polychaetes which can be kept at
various salinities. At salinities below a welldefined threshold, the ability of these
worms to accumulate glycine decreased
dramatically. This threshold is also the
salinity at which regulation of chloride
and of osmotic concentration becomes apparent. On the basis of this as well as the
results of attempts to measure uptake of
aniino acids in a number of genera of fresh
water invertebrates with no strongly positive cases, it was suggested that the uptake
of amino acids and the process of osmoregulation were incompatible in metazoans.
Several protists are capable of removing
amino acids from dilute solution very rapidly even at low ambient osmotic concentrations (Stephens and Kerr, 1962; Yaden,
1965; North and Stephens, 1967). On the
other hand, Wright and Hobbie (1966)
find that fresh water algae (e.g., Chlamydomonas) take up glucose or acetate from
dilute solution at natural concentrations
very slowly. Sloan and Strickland (1966)
report this is also the case for several marine phytoplankters. Most of our discussion will be concerned with marine invertebrates.
Despite variations in rates and in the
details of the process, it is convenient to
describe the accumulation of an amino
acid in a typical marine invertebrate as
seen in our usual screening procedures.
Uniformly labelled glycine-C14 is supplied
in the ambient sea water at 10~7 to 10-°
moles/liter (about 20 ^c/liter). Accumulation begins as soon as the organism is
placed in the solution with no demonstrable lag period. Figure 1 is redrawn
from Stephens (1963) and is typical. It is
likely that accumulation occurs directly
across the body wall. The rate of accumu-
NUTRITIONAL ROLE OF ORGANIC SOLUTES
1
2
3
4
TIME (MINUTES)
S
FTC. 1. Uptake of glycine-C" by Clymenella torfjiinla with time. Units are arbitrary. The bars
represent means ± one s.d. The dashed line is the
calculated regression line for the data. (Redrawn
from Stephens, 1963.)
lation at low concentrations is such that
many invertebrates concentrate amino
acids supplied in the environment by a
factor of the order of 100 to 1 within an
hour. It is not reasonable to think of a
mechanism involving the passage of this
much water through the digestive tract of
a worm such as Nereis or Clymenella or a
mollusc such as Mytilus or Mercenaria.
Aside from this a priori argument, it has
been demonstrated in several cases that
ligation of the mouth and anus of the animals does not significantly modify the
measured rate of accumulation.
As the ambient concentration of glycine
or whatever compound is being used is
progressively increased, the physiological
system which mediates accumulation is
eventually saturated, and further increases
in concentration do not influence the rate
of accumulation. There are exceptions to
this statement (e.g., Stephens and Virkar,
1966). The general relation between concentration and rate seems to be adaptively
related to the low ambient concentrations
of organic material typical in the natural
habitat of inshore marine animals. Thus
the maximum rate at which material can
be accumulated is quite low, typically of
the order of 10"7 to 10-° moles/g/hr.
However, the concentration (Km) at which
uptake occurs at half the maximal rate is
also low; often between 10~4 and 10~6
97
Moles/liter. Thus the system has a low
capacity but functions well at very low
concentrations.
The reports now in the literature are
based on comparatively short-term observations of this process. Exposure of animals to labeled solutions has usually been
continued for an hour or two with most
ot the data being provided by observations
of 15-30 minutes. Longer experiments have
been conducted in such a way as to fail to
clarify whether the process of accumulation is continuing or whether a steadystate concentration ratio between the animal and the environment is simply established and then maintained. We have recently extended our observations in time
and will report some preliminary results
here.
Glycine-C14 was supplied to a subtidal
polychaete, Dorvillea articulata, at a concentration of approximately 2 X 10~8
moles/liter and at a rate of approximately
12 ml/hr using a peristaltic pump. The
animals were confined in a chamber of
approximately 9 ml volume by sintered
glass discs at the ends of the chamber. The
sea water supplied to the worms was sterilized by filtration into a sterile container
and contained 1000 units/ml of penicillin
to suppress bacterial growth. The worms
were starved for three days prior to use
and were kept in a solution of penicillin
in sea water which was changed daily.
Samples of the solution which passed
through the chamber containing the worms
were collected in a fraction-collector and
radioactivity determined with a scintillation counter. Daily samples of the stock
solution were also counted to verify that
initial levels of radioactivity were maintained. The observations continued for
periods of 72-84 hr.
Figure 2 shows the results of one such
experiment. The lower of the two lines is
a plot of the radioactivity remaining in
the sea water after passing through the
chamber containing eleven worms weighing 44.3 mg. The upper line plots radioactivity remaining in sea water in a control experiment done at the same time.
The same initial solution was pumped
98
GROVER C.
36
48
TIME (HOURS)
FIG. 2. The dashed line indicates radioactivity remaining in a solution of glycine-C14 after passage
through a chamber containing 11 worms {Dorvillea
articidata). The solid line shows radioactivity remaining in such a solution after passage through
a control chamber. The arrow indicates radioactivity of the stock solution. See text for details.
through a comparable chamber containing
eleven worms weighing 40.6 mg which had
been killed by exposure to 0.5 M KC1. It
is clear that no significant loss in radioactivity occurs in the controls. Table 2
further validates this impression by listing
the recovery of radioactivity from various
fractions of the two systems. The conclusion seems clear that the uptake can continue for considerable periods under the
conditions described. The slight decrease
in rate of uptake with time is not a constant feature of our observations.
When a labeled amino acid such as glycine is accumulated by a marine invertebrate, it presumably enters all of the usual
synthetic and oxidative pathways. The following qualitative account is typical. The
fraction of radioactivity found in the cold
TCA-soluble (5% trichloracetic acid) fraction steadily decreases. A steady increase
in activity in residual protein accounts for
much but not all of this decrease. A small
but steadily increasing fraction of the activity can be found in the hot TCA-soluble
fraction and the same is true of the poorly
chaiacterized residue after digestion of protein in 6N HC1. Finally, C14O2 is evolved
indicating that a fraction of the material
obtained is entering oxidative pathways.
When the cold TCA-extractable material
is chromatographed after exposure to gly-
STEPHENS
cine-C14, the bulk of the radioactivity is
still in the chemical form in which it was
supplied for as long as 24 hr after exposure
to labeled material. In the case of other
amino acids, conversion to other alcoholsoluble compounds may occur.
Our experience with marine algae has
been somewhat different. We find that
when glycine-C14 is supplied to Ulva and
Enteromorpha, the radioactivity is rather
quickly incorporated into alcohol-insoluble
forms. Further, when alcoholic extracts of
the green alga, Platymonas, are chromatographed, it is apparent that the glycine
which was supplied has been converted into
several other compounds although it can
be shown that the alcohol-soluble material
is largely in the form of glycine for brief
periods after exposure (North and Stephens, 1967).
We have reported accumulation of labeled carbon into various fractions of several marine organisms. We have also repeatedly reported concomitant decrease in
the level of ambient radioactivity in the
medium bathing the organisms and have
established a balance between the two
processes as in Table 2. This kind of data
has been supplemented by showing that
more concentrated ambient solutions of
amino acid in sea water (of the order of
10~3 to 10~4 moles/liter) decrease rapidly
in the presence of these marine organisms.
The last observations demonstrate a substantial net movement of material into the
organism since they are based on chemical
determinations of the amino acids remaining in the medium. However, it must be
noted that the concentrations employed
even in the most careful attempts to do
TABLE 2. Recovery of radioactivity in long-term
observations of uptake of glycine-C" by Dorvillea
articulata. Numbers are total cpm in various
fractions.
Alive (44 mg) Dead (41 mg)
Removed by animals
In chamber and tubing
Remaining in effluent
301,100
7,600
1,390,800
12,600
6,300
1,730,400
Total
Input
Difference
Recovery
1,699,500
1,742,200
42,700
97.5%
1,749,300
1,703,000
46,300
102.7%
NUTRITIONAL ROLE OF ORGANIC SOLUTES
this (2 x 10- 5 moles/liter; Stephens and
Schinske, 1961) are higher than any that
could reasonably be expected in the normal
environment of the organisms.
RATES OF LEAKAGE OF AMINO ACIDS
Although we have attempted in the past
to measure radioactivity which might return to the ambient sea water after an
organism has accumulated amino acids labeled with C14, the relevant data have not
been formally presented nor have apparent
rates of leakage been calculated. We shall
do these things here in a preliminary way.
The total free amino acids were determined colorimetrically (Clark, 1964) in
an 80% alcoholic extract of ten individuals
of Dorvillea articulata. Aliquots containing 0.1-0.5 /xmoles total amino nitrogen
were chromatographed on a commercial
TLC plate prepared from cellulose powder
(Brinkman Instruments, Inc., MN-Polygram Cel 300, 20 x 20 cm) using isopropanol: formic acid: water (40:2:10), and
£er£-butanol: methyl-ethyl-ketone :NH 4 OH:
water (25:15:5:5) as the solvent systems
(Jones and Heathcote, 1966). The resulting chromatogram was developed with ninhydrin in the dark for 24 hr. Spots were
eluted into 2.0 ml of 50% n-propanol and
optical density read at 570 in/*. Concentrations were estimated from standard curves
for each amino acid. The total ninhydrinpositive material in the ethanol extract
was 108.4 |imoles/g wet weight. Table 3
lists the amino acids and their concentrations as determined by the above procedure. The sum of the concentrations of the
individual amino acids is 103.9 /jnoles/g
wet weight which represents a recovery of
approximately 95.8% of the total ninhydrin-positive material.
Apparent rates of leakage of five amino
acids were measured in the following way.
A group of worms was exposed to solutions
of the C14-labeled amino acid involved.
Concentrations of the order of 1 to 3 X
10~6 moles/liter were used. Radioactivity
was determined in alcoholic extracts of the
worms made after an exposure of one hour,
and the radioactivity in the alcohol-insoluble fraction of the animals was also esti-
99
mated. One group of animals was rinsed
twice in sea water for 15 min and then
placed in 30 ml of sea water. Another
group was rinsed and placed in sea water
to which the same amino acid, unlabeled,
had been added at a concentration of 10~4
moles/liter. Samples of the sea water and
of the sea water with cold carrier amino
acid added were taken at 30-min intervals.
Radioactivity was determined directly in
one portion from each group. Another portion was acidified with HC1 and the radioactivity determined after 24 hr. The worms
from each group were sacrificed and the
radioactivity determined in the alcoholsoluble and alcohol-insoluble fractions.
The rate at which radioactivity appears
in the medium can be interpreted as a
measure of the rate at which the amino
acid concerned leaks from the organism.
It is clear that several assumptions are necessary. First, acidification removes considerable amounts of the activity of the rinsing medium in the case of some of the
amino acids. One must assume that the
24-hr period is sufficient to permit any
labeled CO2 to escape from solution. Second, one must assume that the residual
label after acidification is in the same
chemical form supplied in the experiment.
In general, we have no evidence that this is
so and some evidence that it may not be.
Thus, (he fact that at least some amino
acids are converted to others in the pool
of free amino acids does not support the
assumption. Third, we must assume that
the rate of release of material is not modified by the comparatively crowded and
artificial conditions of the experiment. In
at least some cases in other animals, crowding is a crucial factor (Corner and Newell,
1967).
If we are willing to make all of these
assumptions, we can combine the information concerning the rate of appearance of
radioactivity with the information in
Table 3 concerning the concentration of
each amino acid in the pool and calculate
an apparent rate of leakage. Table 4 lists
such rates for five amino acids. The rate
listed is the average of the apparent rate
for the second and the third 30-min sam-
100
GROVER C.
TABLE 3. Free amino acids in Dorvillea artieulata
expressed as ji.moles/gram wet weight. In collecting
these data, taurine was not identified and separated
from glycine.
Alanine
Arginine
Aspartic aeid
Cysteine
Glutamic acid
Glycine
Histi dine
Isoleuoine
Leueine
Lysine
Methionine
Phenylalaiiine
Serine
Threoiiine
Tryptophano
Tyrosine
Valine
2.3
2.2
4.0
0.9
1.1
33.6
2.8
0.2
1.3
1.1
trace
0.2
49.5
1.7
trace!
3.0
trace
103.9
Total
pies. The two values listed for glycine are
the result of two independent experiments.
The addition of amino acids to the rinsing
solution made no consistent difference in
apparent leakage rates.
It is apparent that little weight can be
placed on these estimates in view of the
assumptions which are necessary to derive
the figures. To draw more precise conclusions, it is particularly necessary to have
more information about the chemical nature of the labeled material which is lost.
We are currently attempting to get such
information. However, we can say that at
ambient levels in the range of one ^mole/
liter, rates of uptake of individual amino
acids exceed the apparent rates of leakage,
often by a large margin.
It is quite unlikely that a simple model
or hypothesis based on accumulation from
solution and passive diffusion back into
solution can account for our present information. As we have pointed out above,
the material accumulated enters a great
TABLE 4. Apparent leakage rates of the listed
amino acids, expressed as moles/g/hr.
Alanine
Glutamie acid
Glycine
Phenylalanine
Valine
8.0
9.7
9.85
4.15
4.3
1.07
X 10-"
X -io
X l O " 83
X IOX -io
X -io
10
10
10
STEPHENS
variety of synthetic as well as oxidative
pathways. If accumulation and diffusion
were the only factors of importance, a
steady state would be achieved with time,
and the rate of disappearance of radioactivity would be expected to decline. We
may thus conclude that the long maintained constant rate of removal of material from very low ambient concentrations
(Fig. 2) implies a more complicated interaction between the organism and the surrounding medium.
We must add that the "leakiness" of the
organisms under study should be determined in each particular case. Hellebust
(1967) has summarized work concerning
small organic molecules liberated into the
medium by marine phytoplankters. There
are early data in the literature of elimination of specific compounds by particular
animals: for example, citric acid in oysters
(Creac'h, 1955). Amino acids are given as
excretory products for marine invertebrates
in a number of early publications (summarized in Prosser-Brown, 1961, pp. 140141).
More recently, Johannes and Webb
(1965) reported that substantial amounts
of amino acid were lost by zooplankters.
Since the amount lost did not seem to be
related to the species present in their sample, they felt such losses might be characteristic of zooplankton generally. Corner
and Newell (1967) investigated the form
in which nitrogen was excreted in Calanus
helgolandicus and concluded that release
of amino acids in this copepod might be a
result of crowding. Hammen, et al. (1966)
report the loss of about one ^.mole/100 g
wet weight/hr of amino acid in the common oyster. Other work on excretion of
nitrogen in molluscs is reviewed by Potts
(1967). Although some of the earlier workers reported substantial release of amino
acids, we must be aware that their experiments lasted for long periods and they do
not report any precautions to retard bacterial growth. Staddon (1959) states that
as much as 80% of uric acid excreted by
dragonfly nymphs may be converted to
other forms during a 24-hr collecting period
in distilled water. Duerr (1965) makes the
NUTRITIONAL ROLE OF ORGANIC SOLUTES
same point in studying the form in which
nitrogen is excreted in the gastropod,
Lymnaea. In any case, we need additional
data concerning normal levels of loss of
amino acids in marine organisms.
ENERGY REQUIREMENTS FOR TRANSPORT
OF SOLUTES
We can interpret the data presented to
this point as supporting the idea that
amino acids are accumulated from extremely dilute solution in the medium and
transferred into the "free amino acid" pool
(FAA). The FAA in many marine invertebrate tissues is quite high and may represent a substantial fraction of their total
osmotic concentration (see Awapara, 1962,
and Kittredge, et al, 1962). Consequently,
it is necessary to discuss the energy requirements of the process of transfer of compounds against the differences in concentration which are involved. We are accustomed to thinking of concentration-gradients of 10:1 or even as much as 100:1 in
biological systems. We may be less happy
with the concept of transfer of material
occurring across concentration-gradients of
10°-107 to 1. This is a consequence of a
process which is apparently capable of removing amino acids from a concentration
of 10 nanomoles/liter and transferring them
to concentrations of the order of 0.1 mole/
liter.
Accumulation of a solute against a concentration-gradient represents a change in
free energy which can be interpreted as the
work necessary to accomplish the movement. The relevant formula is:
AF = R T l n —-
V Q>
Substituting for the variables and the gas
constant one obtains roughly 1400 cal/
mole solute transferred against a ten-fold
concentration-gradient. Since the logarithm
of the ratio of concentration is involved,
the work or change in free energy involved
in transferring solutes proves to be very
modest even when the gradient is very
large. Our data indicate that glycine can
be removed from concentrations of the
101
order of 10~8 moles/liter in the ambient
sea water and appears in a free glycine
pool of the order of 10- 1 moles/kg cell
water. An overall gradient of approximately 10 million-fold implies a change in free
energy of about 10 kcal/mole of solute
accumulated. For a compound such as
glycine, this represents only a small fraction of the energy derivable by oxidation
of the molecule.
It is clear that the actual energy expended in pumping a mole of solute will
be greater than the figure obtained from
this thermodynamic calculation by some
unknown factor. Despite this uncertainty,
we can apply a simple test to our postulated accumulation-system. The organism
must have sufficient metabolic energy available to meet the energy cost of the observed
rate at which solutes appear to be transported. If this condition is not met, our
interpretation of the phenomenon must be
revised. Stephens (1967) published Km and
Vmax data for the accumulation of several
organic compounds by several genera of
marine invertebrates. The most rapid
accumulation against the largest gradient
occurs in Clymenella torquata and in Nereis succinea with glycine as a substrate. The
calories required for uptake (i.e., A F)
from an ambient concentration (Km) at
which half the maximum velocity is
achieved can be calculated from the available data and amount to 22.2 and 3.7 cal/
kg/worm/hr for the two organisms mentioned. The metabolic energy can be derived from the oxygen consumption and is
360 and 104 cal/kg/hr, respectively. Thus
6.2% and 3.6% of the available metabolic
energy would supply the thermodynamic
energy necessary for the observed accumulation. A more realistic set of circumstances can be analyzed using earlier data
(Stephens, 1963) to calculate the energy requirements of accumulation from a mixture of amino acids at concentrations observed in the normal environment of Clymenella. When a comparable estimate is
made of the change in free energy involved
in this situation, one finds that 1.5% of
the available metabolic energy suffices to
account for the necessary work.
102
GROVER C. STEPHENS
An even less satisfactory argument is the
following: Since so many organisms can
accumulate small organic compounds, surely this capacity must provide something of
importance. This may or may not be amino
acids or glucose. These molecules are simply convenient to study and do not adequately represent the range of compounds
which are undoubtedly present in marine
sediments. Not only is this argument teleological, but it is not testable empirically
since it depends on the impossible requirement of a complete inventory of the compounds in the environment. No help is to
be obtained from generalized arguments of
this type, although it is well to be aware
of their existence so that they do not inadvertently influence the discussion.
There is a very large literature in which
investigators have attempted to establish
the biological importance of dissolved organic material by simply adding compounds to the medium and observing
whether animals or plants survived longer
or grew more rapidly in their presence.
BIOLOGICAL SIGNIFICANCE OF ACCUMULATING
Such
experiments are extremely difficult to
SOLUTES
interpret. Most have used very high conWe may now consider the more difficult centrations of material compared to those
question of the extent to which accumula- normally present, and generally have not
tion of small organic molecules is a bio- been performed axenically.
logically significant process. There are a
It is possible to design procedures which
priori arguments which are vague and un- are informative btit extremely difficult to
convincing but should be noted briefly. carry out. Recent workers have become inFirst is the fact that a system mediating the creasingly aware of problems presented by
rapid accumulation of solutes from the en- high concentrations and microbial activity.
vironment represents a form of differenti- Gillespie and coworkers (1964, 1966) atation. There is presumably selective pres- tempted to control bacterial multiplication
sure favoring its maintenance. Otherwise, and are appropriately cautious in their
it is difficult to explain the persistence of conclusions. They found that glucose added
such a transport system in such a wide di- at a level of 5 mg/liter (about 3 X 10~5
versity of organisms. If the accumulation moles/liter) increased longevity, maindid not serve some critical function in the tained general condition, and supported
economy of these forms, surely there would slight growth and the laying down of glycobe a selective advantage accruing to the gen in the oyster, Crassostrea virginica.
forms which dropped the excess baggage Despite the value and intrinsic interest of
of the functionless operons coding the pro- such studies, I believe that the analysis of
teins involved. This argument is unpro- the normal role of uptake of small organic
ductive since it does not focus on any par- compounds in marine organisms must be
ticular function. It may be that accumu- considered in more detail and tied more
lation is epiphenomenal with respect to closely to natural conditions.
some other process which has escaped us in
A possible biological role for this procthe present context.
ess would be a contribution to the nutriThe preceding argument is analogous to
that of Keynes and Maisel (1954) with respect to the energy required for extrusion
of sodium by frog skeletal muscle. The
calculated change in free energy is the
minimum required for the process. It does
not, of course, take into account the efficiency with which metabolic energy can be
used by organisms to accomplish the accumulation of compounds against a gradient.
It is not likely that the transfer of solute
will occur at an efficiency greater than
about 50%. We must also note that any
leakage to the exterior by diffusion or any
other process would need to be considered.
However, this is probably small. When we
double the estimate of the fraction of metabolic energy required to account for the
observed accumulation, we still obtain a
maximum figure of 12-13% and a more
likely figure of 3-4% under natural conditions. Thus the energy demands for accumulating amino acids are not excessive,
and the process is energetically possible.
NUTRITIONAL ROLE OF ORGANIC SOLUTES
tion of the organism. The classical approach in considering the nutrition of terrestrial organisms is to compare the caloric
value of food-intake with the heat production per unit time (Lavoisier and Laplace,
1780). Typically, the latter is estimated by
measuring gaseous exchange (CO2-production or O2-consumption). This general approach was used by Stephens (1963) in discussing the possible contribution of amino
acids to the nutrition of the polychaete,
Clymenella. An inventory of the amino
acids present in the intertidal muds where
the animals were living was compared with
the rate of accumulation of amino acid
measured in laboratory experiments. This
permitted an estimate of the rate at which
amino acids might be supplied by this
process of accumulation. A figure of 13.5
^g amino acid/100 mg worm/hr resulted.
Oxygen consumed by these worms was 0.09
ml/g/hr, a figure which agrees reasonably
well with that reported by Mangum (1963).
A simple calculation shows that the rate at
which amino acid can be obtained represented roughly 150% of the reduced carbon
necessary to support the observed consumption of oxygen by the animals.
Two comments should be made at this
point. First, there is no intention to suggest that all the amino acid accumulated
by an animal is funneled into oxidative
pathways. We have noted earlier that this
is not the case. This is no more than a
convenient way to estimate the turnover of
organic material in the animal. To the
extent that reduced carbon is supplied by
accumulation, the organism is spared the
necessity of obtaining it in other ways (e.g.,
feeding on large particles, photosynthesis,
etc.). Second, it should be stressed again
that ". . . the uptake of amino acids (and
possibly other dissolved compounds) is not
being urged as a complete account of the
feeding of Clymenella but rather as a supplement to other feeding methods which
are well established." The quotation is
from Stephens (1963) and deserves repetition as a caution against the natural desire
for an all-or-none acceptance or rejection
of a nutritional role of subparticulate organic matter.
103
The estimated concentration of amino
acids in the interstitial water of the mud
flat which was used in this calculation
agrees reasonably well with the determinations reported by Belser (1959, 1963). It is
substantially higher than other estimates
of free amino acids in sea water (Degens,
et ah, 1964: Chau and Riley, 1966; Webb
and Wood, 1967). Most of these estimates
refer to concentrations free in inshore
waters and are of the order of 10~6 moles/
liter total rather than 10~6 as measured by
Stephens and by Belser. It may be that the
higher concentrations are characteristic of
interstitial water. However, Webb has recently obtained concentrations of the order
of a few micromoles per liter total FAA in
interstitial water (data kindly supplied in
a personal communication). A calculation
based on such levels reduces the contribution of FAA to 5-10% of the reduced carbon necessary to support oxygen consumption.
The level of available FAA in the microhabitat of a particular organism is extremely difficult to determine but we can hope
for better information as analytical techniques continue to improve. When this
level is established, it will make it possible
to assess the extent of the contribution of
this category of compounds as supplementary nutrients and will also permit us to
make an informed estimate of the significance of leakage of amino acids to the environment in that particular case.
A related but conceptually distinct approach to an analysis of the potential contribution of accumulation of organic compounds was undertaken by North and
Stephens (1967). After measuring the rate
of uptake of several amino acids by the
green alga, Platymonas, they calculated the
contribution which such accumulation
would make to the nitrogen necessary for
a doubling of cell mass. Choosing an arbitrary environmental concentration of 1
//.mole/liter as consistent with the lower of
the reports cited above, they calculated
that the nitrogen obtained by uptake of
glycine via this pathway would supply approximately 10% of the nitrogen necessary
for log phase growth under optimal condi-
104
GROVER C. STEPHENS
tions. Since it is known that rates of division decline under less favorable conditions and cell nitrogen falls to 10 or 15%
of the value measured in the log phase,
this can be argued to be a very conservative
estimate of the possible contribution under
normal conditions.
If one makes an estimate of the potential
contribution of a particular organic compound from the environment either on the
basis of its quantitative relation to some
measure of oxidative metabolism or to the
needs for amino nitrogen in growth or
some other organismic feature, the fundamental question can be raised of the extent
to which the measure is a reliable one. Can
one defend oxygen consumption or doubling time as a measure of energy needs or
needs for reduced carbon for organisms in
general? The answer must certainly be
negative. Oxygen consumption is a nonsensical measure for an anaerobe and unreliable for animals capable of survival at
minimal oxygen tensions. In the same way
an animal may invest none of its food-intake in growth or may indeed undergo
negative growth under some circumstances.
Clearly no single measure will be valid for
all cases.
We may feel somewhat more confident
when dealing with a particular animal or
a particular plant. Yet here, too, caution
is required. A polychaete like Glycera or
Nereis seems clearly to be differentiated as
a predator, a large-particle feeder. Yet the
gut contents of Glycera do not validate this
suggestion, and Nereis has been described
as a filter-feeder (Harley, 1950). Both animals accumulate amino acids, Glycera
rather slowly and some species of Nereis
quite rapidly. To take another example,
surely Viva in direct sunlight is an autotroph. One can watch the bubbles of gas
appear. Yet Ulva accumulates glycine at
as high a rate as any organism we have examined.
We can attempt to diagram some of the
major inputs and outputs of material in a
generalized organism (Fig. 3). The categories are greatly simplified and omit such
things as particular inorganic materials
which must be regulated and organic com-
INPUTS
LARGE AND SMALL PREY, DETRITUS PARTICLES;
FINE SUSPENDED MATERIAL: LEPTOPEL. MUCUS;
SMALL MOLECULES: AMINO ACIDS, SUGARS. LIPIDS;
CO.
n
MM
[ORGANISM!
TTTT
OUTPUTS
LARVAE, GAMETES, FECAL PELLETS, CELL DEBRIS;
FINE SUSPENDED MATERIAL: PROTEIN, MUCUS;
AMINO ACIDS, SUGARS, LIPIDS, GLYCOLATE, UREA;
CO,
FIG. 3. Diagram o£ interaction between the organism and the aquatic environment as discussed
in the text. Inputs and outputs are listed in order
of decreasing size. The multiple arrows from the
inputs to the organism and the organism to the
outputs are intended to represent movement oE material by several pathways. The dashed arrows to
the left are intended to indicate that the distinction between inputs and outputs may not always
be meaningful since some materials may play both
roles for the same organism.
pounds needed in minute amounts such as
vitamins or pheromones. Also, no attempt
has been made to include the various forms
of nitrogen in a systematic way. Inputs
and outputs are listed in order of decreasing size of particles. It is clear that inputs
can occur of compounds which are either
reduced or oxidized, paniculate or subparticulate. The several input arrows serve
to emphasize the variety of pathways of
carbon dioxide fixation, nitrogen fixation,
feeding, and accumulation of organic material. Similarly, outputs may be of reduced
or oxidized compounds, cover a comparable
range of particle size, and arise by a great
variety of pathways.
When we attempt to interpret this diagram in terms of a particular organism,
some materials are clearly acquired from
the environment and belong on the input
side. Others are equally clearly excretory
and belong on the output side. Further-
NUTRITIONAL ROLE OF ORGANIC SOLUTES
more, materials which are outputs for one
organism may very well be inputs for another. Indeed, the same material may be
input and output for the same organism.
These remarks apply both to paniculate
material and to subparticulate compounds.
A familiar example is carbon dioxide
which arises as a product o£ energy metabolism and may contribute to the input side
of the same or another organism by a variety of fixation-pathways. An example at
the paniculate level is the important process of autocoprophagy as noted by Newell
(1965) and Johannes and Satomi (1966)
although it must be admitted that the fecal
pellet which is produced cannot really be
considered to be the same as that which is
consumed in a strict sense. The dotted arrows at the left of the diagram in Figure 3
are intended to represent this lack of a
meaningful distinction between input and
output for some materials.
The burden of the present paper is that
reduced organic compounds such as amino
acids and sugars represent inputs to a wide
variety of organisms. These occur at rates
which make it necessary to evaluate their
contribution to the total economy of particular plants and animals and to the marine biota in general.
The temptation is great to elaborate on
the various pathways suggested and to
multiply examples, but any interested
reader can do this for himself. Perhaps the
most general conclusion that can be drawn
from the discussion is that the time has
passed when it is possible to talk meaningfully of "dissolved organic material" or
any synonymous phrase as though it represented some single entity. We must focus
upon particular compounds and upon particular organisms. Our estimate of the significance of the various input pathways for
a particular organism amounts to an informed guess which we must then refine
by a continuing process of direct and indirect measurement of the various inputs
and outputs under circumstances as close
to normal as we can obtain. The obvious
pitfalls to avoid are too simplistic an interpretation of the relation of morphology
and function, and the natural desire for
105
simplification and generalization when it
may be premature.
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