Download A rapid method of quantifying algal carbon uptake kinetics

NOTES
A rapid method
of quantifying
algal carbon
ABSTRACT
A rapid and inexpensive
method of obtaining and assembling kinetic data from algal
cultures is presented which allows evaluation
of the interactions
between limits imposed on
algal carbon uptake kinetics by nutrient
or
physical factors and those imposed by carbon
availability.
Traditionally
studies of algal growth
kinetics are conducted in chemostats under
steady-state conditions,
IIowever maintenance of constant pH, alkalinity, and inorganic carbon supply while alga1 kinetic
response is related to single nutrients or
factors ignores interactions between these
parameters and the variable under study.
In natural waters algal fixation of carbon
from the system results in a variety of
changes including alterations in the relative
ability of algae to compete (King 1970,
1972). Verduin ( 1951, 1957)) Beyers and
Odum ( 1959)) Beyers et al. ( 1963)) Beyers
(1963, 1965), Park et al. ( 1958), and many
others have used measured changes in pH
and the alkalinity system to calculate algal
carbon fixation. Park ( 1969) reviewed the
various calculation methods involved. These
changes in the inorganic carbon budget
associated with algal photosynthesis can
be used to relate algal carbon fixation
kinetics rapidly to any of several parameters, while allowing the alkalinity system
of the culture to shift as it does in nature.
Our method involves growing algal cultures in sterile inorganic growth media in
l,OOO-ml erlenmeyer flasks designed to
minimize atmospheric recarbonation but to
permit atmospheric pressure equalization.
Stoppers for each flask are drilled with two
holes: a large median hole of 13-mm diameter, and a smaller one, 3.5 mm in diameter,
about 15 mm from the stopper center. The
small hole is fitted with a 50-mm length of
4-mm-OD glass tubing to provide pressure
equalization
during growth.
The larger
hole is fitted with a serum cap to permit
LIMNOLOGY
AND
OCEANOGRAPHY
uptake
kinetics
sample removal with a syringe without
exposing the flask contents to the atmosphere. Samples are removed for pH measurement by swirling the flask contents to
obtain a homogeneous suspension and then
penetrating the serum cap septum with a
syringe and withdrawing
the required
volume.
To accurately determine the pH of such
samples without interference from atmospheric recarbonation, we use a 50-ml beaker
fitted with a rubber stopper ( No. 9%)
drilled with a 14- and a 7-mm-diameter
hole. The larger hole is just large enough
to permit insertion of a combination pH
electrode and is capped with a No. 0 rubber
stopper when not in use. The smaller hole
is fitted with a 45-mm length of 6.35-mmOD glass tubing and capped with a small
serum cap. Before sample injection, the
apparatus is flushed completely of air and
COn with nitrogen gas and rapidly sealed
to preserve a nitrogen atmosphere. A 25-ml
sample is then withdrawn from the culture
flask with a syringe and injected into the
nitrogen atmosphere, with care to limit turbulence and prohibit
direct air contact.
Finally, the pH probe is quickly inserted
and pH determined.
Culture pH as a function of time, from
initial seeding until continued carbon uptake ceased, is illustrated in Fig. 1 for Anacystis nidulans. Calculated values of carbon
fixed by the algae can be obtained at any
point or over any interval of such curves by
applying the following equation.
where M = biomass in terms of organic
carbon, M; At = t - to = time increment, hr;
t, to = boundary parameters of time interval, hr; and
&x3, = total molar sum of inorganic
978
NOVEMBER
1973,
carbon
V.
18(6)
979
NOTES
o-280
-I -
tux
0
"
IO
0
PH
me
9
8. I
100
200
300
ELAPSED
400
TIME
500
I
600
1
(HOURS)
Fig. 1. Culture pH as a function of time during
photosynthesis
for several cultures
of Anacystis
niduhs
grown under light intensities
ranging
from 280 to 915 lux (Sylvania Gro-Lux).
species, M; a = carbonate-bicarbonate
alkalinity, eq liter-l; H = hydrogen ion activity
as determined by a pH meter, M; K1, K2 =
first and second dissociation constants of
carbonic acid. Values for K1 and K2 are
from Buch (1951) as listed by Harvey
( 1957) for various water temperatures.
Although it is difficult to evaluate the
precision of this method, comparison of calculated biomass estimates with direct measures of organic carbon with a carbon
analyzer (Beckman IR-315) yielded a coefficient of correlation between these estimates of 0.986. However, the standard
error of 20.998 mg C liter-l incurred by
estimating carbon fixed from calculated
values appears to be more a function of the
precision of the carbon analyzer than a
reflection of the precision of the calculation
method.
After the curves representing pH as a
function of time, as in Fig. 1, are transformed with equation 1 to curves of carbon
fixed as a function of time, estimates of the
specific growth rate, defined as follows, can
be obtained.
dM,‘dt
p=M)
(2)
where ,u = specific growth rate, hr-r; M =
carbon fixed as biomass, M; t = time, hr.
Specific growth rate as used here is the
instantaneous time rate of change of biomass (inorganic
carbon fixed) per unit
average biomass over the time interval in
-4
-3
log
I
0
-I
-2
CO,
(JI moles
I
I
liter-‘)
Fig. 2. Specific growth rate (P) of Anacystis
niduhs
as a function of the free CC& concentration
under two light intensities.
The curves represent
calculations
based on kinetic
constants derived
from the data, and photosynthetic
minima indicate
equilibrium
CO, concentrations
at which
continued carbon uptake ceased.
question,
becomes
t-b
The specific
AM/At
= -=
m
(Mt
growth
- Mt,)/(t
rate then
- to),
(3)
(Mt + wow
where pAt = specific growth rate during
the time increment, hr-l; AM = biomass
increment
( inorganic carbon fixed), M;
t, to = boundary parameters of time increment, hr; At= t- to = time increment, hr;
m = average standing crop biomass during
the time increment; M.
We chose to use the average standing
crop biomass rather than the biomass at the
beginning of the period to approximate
more closely the integral rate over the
period considered. This will yield a slightly
lower specific growth rate than using the
initial value, but we feel that it more nearly
approximates the true rate over the changing conditions during a given interval. For
the same reason we plot the resulting specific growth rate (p) against the mean
substrate (free CO,) concentration
over
that period.
Such specific growth rates for A. nidulans
are plotted in Fig. 2 against the average
free CO2 concentrations over the time intervals for which the specific growth rates
were determined. Free CO2 concentrations
980
NOTES
were calculated from pH and alkalinity data
according to Park ( 1969). The two curves
drawn through the data were calculated
by the empirical
Monod
formulation
(Monod 1949). Kinetic constants for these
curves were calculated by a regression
analysis of experimental data using V/S
vs. V linear transform of the parent growth
rate equation ( Dowd and Riggs 1965).
The logarithmic
axes used in this plot
allow illustration of the different minimum
attainable external free CO, concentrations
and the change in specific growth rates at
given free COa concentrations when other
parameters, in this case light intensity, are
varied. Figure 2 shows that a reduction in
light intensity caused a large reduction in
specific growth rate at all concentrations
considered as well as a reduction in excess
of an order of magnitude in the minimum
attainable external free CO2 concentration. Although the curves based on empirical
formulation drawn through the data in Fig.
2 would predict photosynthesis to continue
to zero free CO2 concentration, this clearly
is not the case, as is indicated by the points
at which additional carbon fixation ceased.
This indicates a marked interaction
between light intensity, carbon availability,
and both rate and extent of photosynthetic
carbon fixation by A. nidulans.
We have used this method successfully
to evaluate algal carbon uptake kinetics as
a function of available nitrogen and phosphorus over a range of free CO2 concentrations by withdrawing
samples from the
cultures at routine intervals for measurement of nitrogen or phosphorus uptake by
the algae. Such additional measurements
from a series of flask microcosms in which
the amount of nitrogen, phosphorus, light,
or temperature is varied sequentially allow
evaluation of interactions between limits
imposed by such parameters with those
imposed by carbon availability,
as well as
determinations of C: P and C:N ratios of
algae under various conditions of stress.
This method allows rapid accumulation
of carbon uptake kinetic data and can be
used to illustrate interaction between various limiting factors, but there are some
cautions. The carbon supply to the algae
is comprised solely of carbonate-bicarbonate alkalinity since sterile inorganic media
are used and the net flux of oxygen out of
the flask through the small pressure equalization vent minimizes recarbonation from
the atmosphere. Calculations are based on
the assumptions that all carbon fixed by the
algae comes from the alkalinity system and
that all carbon removed from the alkalinity
is fixed by the algae. Carbonate precipitates would be counted as algal carbon
uptake and therefore the growth medium
used must be dominated by monovalent
cations. The sheer volume of measurement
and calculation involved in estimating carbonate precipitation
in media dominated
by divalent cations limits the usefulness of
this method in such waters.
THOMAS C. YOUNG
DARRELL L. KING
Department of Civil Engineering
University of Missouri-Columbia
Columbia 65201
REFERENCES
R. J. 1963. The metabolism of twelve
aquatic
laboratory
microecosystems.
Ecol.
Monogr. 33: 281-306.
1965. The pattern
of photosynthesis
-.
and respiration in laboratory
microecosystems,
p. 61-74. In C. R. Goldman [ea.], Primary
productivity
in aquatic environments.
Mem.
1st. Ital. Idrobiol. 18( suppl.), also Univ. Calif.
1966.
J, LARIMER, H. T. ODUM, R. B. PARKER,
N. E. AFLMSTRONG. 1963. Directions
AA
for the determination
of changes in carbon
dioxide concentration
from changes in pH.
Publ. Inst. Mar. Sci. (Texas)
9: 454-489.
AND H. T. ODUM.
1959. The use of
caibon dioxide to construct
pH curves for
the measurement
of productivity.
Limnol.
Oceanogr. 4 : 499-502.
BUCH, K. 1951. Das Kohlensare Gleichgewichtssystem im Meerwasser.
Havsforskningsinst.
Skr. Helsingfors
151. 18 p.
Down, J. E., AND D. S. RIGGS. 19fXL A comof estimates
of Michaelis-Menten
parison
kinetic
constants from various linear transformations.
J. Biol. Chem. 240: 863-869.
1957. Chemistry
and fertility
HARVIZY,
H. W.
of sea water, 2nd ed. Cambridge.
KING, D. L.
1970. The role of carbon in eutrophication.
J. Water Pollut. Control Fed. 42:
2035-2051,
BEYERS,
981
NOTES
-.
1972.
Carbon limitation
in sewage
lagoons, p. 9S-105.
In G. E. Likens [ed.],
Nutrients
and eutrophication.
Amer.
Sot.
Limnol. Oceanogr. Spec. Symp. 1.
MONOD,
J. 1949. The growth of bacterial
cultures. Annu. Rev. Microbial.
3: 371-388.
PARK, K.
1969. Oceanic COz system: An evaluation of ten methods of investigation.
Limnol.
Oceanogr. 14: 179-186.
1958.
-,
D. W. HOOD, AND H. T. ODUM.
Diurnal pH variation
in Texas bays, and its
application
to primary production
estimation.
Publ. Inst. Mar. Sci. (Texas) 5: 47-64.
VERDUIN,
J. 1951. Photosynthesis
in naturally
reared aquatic
communities.
Plant Physiol.
26: 45-49.
1957.
Daytime
variation
in phyto-.
plankton
photosynthesis.
Limnol.
Oceanogr.
2 : 333-336.
Submitted:
Accepted:
20 March 1973
17 August 1973
Trends in drogue design1v2
ABSTRACT
Drogues in use today in the Lagrangian
measurement
of currents are in general inferior in behavior to those in use a century
or more ago. This results from the historical
tendency
toward
smaller drogues.
For optimum measurements,
one should select a
drogue design that maximizes ease in handling
and then scale it up to the largest size
practicable.
In conducting a general review of devices
used in the Lagrangian measurement of
current, we considered in particular that
category of drogue, drag, or sea anchor
whose motion is determined by tracking a
surface buoy from which the submerged
device is suspended at a selected depth.
The various types of drogue are illustrated
in Fig. I3 Each design is assigned to one
of four classes based on geometrical considerations. Certain trends in drogue usage
can be seen that limit the quality of the
measurements obtained with the modern
drogues.
BASIC
DROGUE
MECHANICS
A drogue-buoy pair undergoes only very
slow changes in speed or direction of
motion, i.e. very slight accelerations. It is
1 Study supported in part by the University
of
Michigan
Sea Grant
Program,
maintained
by
NOAA, US. Dept. of Commerce.
a Publication
No. 213 from the Department
of
Atmospheric
and Oceanic Science, The University
of Michigan.
a A copy of this figure, with a complete list of
bibliographic
citations, is available from the University of Michigan Sea Grant Program (Monahan
and Monahan, in prep. ).
thus appropriate to assume at any instant
that a drogue-buoy
system has no net
external force acting on it. In the unique
instance where the horizontal flows at the
surface and at the depth of the drogue are
the same, then the drogue-buoy system has
no horizontal forces acting on it, and the
drogue and the buoy, taken individually,
have no horizontal external forces acting
on them. (In this example, and in the one
to follow, it is assumed that the force
exerted by the wind on the exposed portions
of the buoy can be neglected. This is a
reasonable assumption if the buoy is almost
awash and has only a thin radio antenna or
a small radar transponder element protruding upward, but if the buoy has considerable freeboard, a flag, a radar reflector,
or several such features, then for winds
greater than a few meters per second, the
wind force will be significant and must be
included as an external force on the droguebuoy system. )
In the situation typically encountered,
where the surface current is greater than
the current at the depth of the drogue, the
drogue-buoy system has several external
forces acting on it which sum to zero. In
those cases where the wire by which the
drogue is suspended from the buoy is not
of excessive length, these horizontal external forces are two in number and equal
in magnitude: the drag force resulting from
the motion of the surface water relative to
the buoy and the drag force due to the
motion of the drogue relative to the current
at its depth. The drag force on the buoy is
essentially proportional
to the square of