Growth Rate Determinations of the Macrophyte Ulva in Continuous Culture Thomas D. Waite,’ Lloyd A. Spielman, and Ralph Mitchell2 Division of Engineering and Applied Physics, Laboratory of Applied Microbiology, Harvard University, Cambridge, Mass. 02138
Continuous culture experiments were run with the benthic macrophyte Ulcn iactuca. Using oxygen evolution as a monitor of photosynthesis and dry weight determinations for biomass synthesis, growth rates and stoichiometric growth constants were evaluated. The data showed that the ratio of oxygen production to algal mass synthesis is relatively independent of nutrient concentration and growth rate, but is affected by light intensity. The data also showed that the amount of oxygen evolved per unit of algal material was almost a factor of 10 higher than is predicted from carbohydrate synthesis. It appears that Uloa is capable of synthesizing compounds with carbon oxidation states of 1 or +2, thus estimates of biomass synthesis may be in error when the average algal material is assumed to be carbohydrate.
ganism (cells ml/min), Rid = volumetric decay rate of microorganisms (cells/ml/min), and V = volume of reactor (ml). Chemostats or algastats are run at steady state as cells are washed out, thus dc‘ldt = 0. In most cases the influent flow does not contain microorganisms, therefore = 0, and with the introduction of first-order rate experiments with specific growth rate constant, p , and specific decay rate constant, ud,Equation 1 becomes:
ecent evidence has shown that benthic algae may contribute substantially to the productivity of coastal waters (Waite and Mitchell, 1971); however, very little is known about the kinetics of macrophyte growth. The reason for our poor understanding of the growth kinetics of this important group of algae has been the difficulty of accurately measuring productivity responses to changes in nutrient concentration. Continuous-flow culture assays using chemostats or algastats have recently become popular for growth rate determinations. Biological populations can be kept at steady state while their growth rates are varied over a wide range. It is also possible to determine the maximum cell yield in a given hydraulic situation using a continuous-flow culture system. Microbiologists have long utilized the chemostat for heterotrophic growth kinetics, and the theory and mathematics have been reviewed by Mtilek and Fencl (1966). The use of continuous culture methods for algal growth kinetics was introduced by Pipes and Koutsoyannis (1962), and a review of algastat theory is given by Retovsky (1966) and Pearson (1969). The most used type of chemostat or algastat is a flowthrough, mixed reactor. A cellular material balance can be written around the reactor to define the growth of any unicellular microorganism. (input - output)
+ (growth - decay) = change
or
=
0
(2)
or
+
R
+ ~ c ’ V- u ~ c ’ V
-Qc‘
p
-
ud =
Q/V
=
l / ~
(3)
where p and ud are in min-I and T = hydraulic residence time, min . Equation 3 is the basic re!ationship describing an algastat operating at steady state, and has proved useful for evaluating phytoplankton growth kinetics as a function of nutrient enrichment. The data derived from such experiments have helped elucidate the response of phytoplankton in systems fertilized by domestic sewage. The problem of productivity stimulation owing to nutrient enrichment is not limited to the phytoplankton, however, as recent data have shown that benthic macrophytes are also stimulated by nutrient fertilization (Waite and Gregory, 1969). It has also been shown that benthic plants may have a higher productivity per unit area than the phytoplankton (Waite and Mitchell, 1971). Methods for measuring primary production of aquatic plants are still inaccurate and poorly developed. Several methods have been proposed for in situ determinations, including oxygen evolution, 14C02uptake, and biomass changes (Kanwisher, 1966; Wetzel, 1964; Westlake, 1965). Because of the size and growth complexity of the plants, laboratory culturing is difficult ; thus, reliable growth rate relationships have not been well developed. We have investigated the possibility of utilizing a continuous-flow stirred reactor for growth rate determinations of benthic macrophytes. This is a novel case as there is no washout of cells, thus Vdc’ldt in Equation 1 does not equal zero. The purpose of this study was to derive a simple relationship that would reliably model the growth response of benthic plants in a reactor using oxygen production. It has been found that in addition to obtaining the growth rate, the stoichiometric ratio of oxygen produced to biomass produced can also be evaluated provided measurable increases in biomass occur over the duration of an experiment, even though only the final biomass is measured. Theory
where Q = flow (mljmin), e’ = concentration of cellular material (cellsjml), R’, = volumetric growth rate of microor-
A mass balance of oxygen can be written for the reactor in the same manner as a cellular balance was written in Equation 1. Qco - Qc
Present address, Dept . of Civil Engineering, Universiry of Miami, Coral Gables, Fla. 33124. 2 To whom correspondence should be addressed. 1096 Environmental Science & Technology
+ R,m
- Rdm = Vdcjdt
(4)
where Q = flow-through reactor (mlihr), c = concentration of dissolved oxygen in reactor (mg O,jml), R, = specific algal growth rate in the light reactor (mg 02/mg/hr), Rd = specific
algal decay rate (respiration) in the dark reactor (mg Os/mg/hr), rn = algal mass (mg), and v = volume of reactor (ml). The plant material was fastened in the reactor, thus algal material accumulated as the plant grew during the experiment. Figure 1 shows the conceptual difference between a reactor running at steady state-Le., washout of material, and one with an increasing biomass. It can be seen that the slope foI the nonsteady reactor data should be proportional to the rate of biomass increase in the reactor. Initially it was assumed that the increase in biomass in the light reactor would follow a first-order relationship : dmldt
=
( k L R , - kdRd)m
(5)
where k L = stoichiometric growth constant (mg/mg 02), and li,, = stoichiometric decay constant (mgimg 02) thus
Realizing the oxygen increase in the light reactor represents the net productivity, Equation 4 may be rewritten as: Qc,
- Qc + ( R , - RJm,e (kgEo - kdRid)t
Taking c
=
-
- Vdcjdr
(7)
c, at t = 0, this equation can be solved to yield:
If we assume there will be only a small amount of growth in the reactor-Le. (kLR,r - kf(RfIT)t/r
