The 14C method: Patterns of dark CO, fixation and DCMU correction to replace the dark bottle1y2

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1 Limnol. Oceanogr., 28(5), 1983, 1983, by the American Society of Limnology and Oceanography, 111~. The 14C method: Patterns of dark CO, fixation and DCMU correction to replace the dark bottle1y2 Louis Legendre GIROQ, Ddpartement de biologic, Universitk Laval, Qu&ec, Qu&ec GlK 7P4 Serge Demers Centre Champlain des Sciences de la Mcr, Minis&e des P&hes et dcs O&am, B.P , Qu&x, Qu&ec GlK 7Y7 Clurice M. Yentsch und Churles S. Yentsch Bigelow L&oratory for Ocean Sciences, McKown Point, West Boothbuy IIarbor, Maine Abstmct Subtracting dark 14C fixation from light 14C fixation can lead to serious underestimates of phytoplankton production in aquatic environments. Light, temperature, and nutrient timecourse experiments show that there is an active and an inactive incorporation component of C in the dark. The active incorporation con be prevented by adding DCMU (dissolved in water) to either a light or a dark bottle, thus estimating the inactive component. It is therefore recommended that a DCMU treatment be subtracted from the standard light carbon dioxide fixation for accurate estimation of phytoplankton production. Since Steemann Nielsen (1952) first published the 14C method for estimation of primary productivity, environmental physiologists and aquatic ecologists have pumped vast quantities of 14C into light and dark bottles in an attempt to estimate phytoplankton productivity in the world s freshwaters and oceans. It is unsettling that there are no satisfactory explanations for the methodology or the interpretirtion of the results. The consequence is 30 years of contradiction and controversy. Estimates of productivity vary by over 100% and extreme problems are witnessed under conditions of low nutrients, high light, and low ratios of photosynthesis to respiration. As pointed out by Peterson (1980), one major point of departure from agreement concerns the treatment of the results from L Research was supported in part by: Champlain Centre for Marine Science and Sllrveys (Fisheries and Oceans) (S-D.), The National Sciences and Engineering Hescarch Council of Canada (L.L.), the National Science Foundation and the State of Maine (C.S.Y. and C.M.Y.). 2 This is Bigelow Laboratory contribution and a contrilmtion to the program of GIROQ (Groupe interuniversitaire de recherches o&anographiques du Qu&ec). the dark bottle. Although most aquatic scientists carry out the exercise of incubating a dark bottle, values for carbon uptake in the dark bottle may be subtracted from those in the light bottle, recorded separately, omitted, or a standard fudge correction factor may be applied. We agree with Peterson that the most severe shortcoming of the r4c method is that, unlike the oxygen method where oxygen uptake in the dark bottle does represent respiration, the 14C uptake in the dark bottle does not. It measures two components: nonphotosynthetic incorporation of 14C and inactive fixation. Dark incubations always indicate a positive carbon uptake. We present here evidence for the two components (active and inactive) of dark carbon dioxide fixation and, for the first time, a rational approach to the treatment of 14C uptake in the dark bottle. In- 996 stead of subtracting the dark carbon dioxide fixation, some portion of the dark carbon dioxide fixation, or a zero time correction, we suggest a DCMU treatment as the standard corrective technique. Our several approaches for these stud- ies are to assess what fraction of dark carbon dioxide fixation can be explained

2 14C method: DCMU correction 997 Experiment Effect of light intensity before darkness Effect of temperature during 14C incubation Effect of nutrients (ammonium) added at beginning of 14C incubation Effect of cell density Effect of DCMU added * 24-h light. Organism Light* (PEinst.,a. Temp Nutri- T s-1) 03 ents incubation Fig. Phaeodactylum F/l 100 PG.250 ml- 1,2 tricornutum P. tricornu turn F/l 100 &i a250 ml- 3 P. tricornu turn F/l 20 &i. 130 ml-l 4 P. tricornutum F/l 20 PCi * 130 ml- l 5 Dunaliella F/2 100 j&i * liter-l 6,7,&g primolecta metabolically. The obvious experiments include time-course studies, with varying light history, temperature effects, and nutrient effects. We present these results as a background to demonstrate the degree to which dark fixation rate can be modified by factors which regulate carbon fixation. These studies are followed by experiments using DCMU. We thank J. Hellehust for suggestions on metabolic pathways in darkness with DCMU. Experimental assistance is acknowledged by C.-A. Boudreuu, L. Leveille, D. A. Horton, and L. Strube. W. Balch, R. Eppley, I. Morris, and D. Redalje gave helpful comments on a previous draft of the manuscript. The figures were prepared by J. Rollins and L. Corriveau. Methods Specifics of incubation conditions and periods, and 14C activity and volumes are given in Table 1. Experiments were run in one of three ways: a standard incubation in separate containers with zero time and final values measured; time-course experiments in large samples with subsamples taken approximately every hour; and a 2-liter light-tight dispenser flask with a magnetic stirrer. In the last the culture or natural population was placed in the flask with only the plunger and sy- ringe for 14C entry exposed; at time zero, the 14C was injected into the culture and samples plunged off as often as every 30 s; enough material was pumped to clear the plunger between samples. For studies with DCMU, batch cultures of Dunaliellu primolectu (Butcher) were grown under constant illumination at 16 C in F/2 medium (Guillard and Ryther 1962). For each cell density, three experiments were done: standard incubation in dark bottle; incubation in dark bottle with 10B5 mol. liter- DCMU added; and incubation in light bottle with 10m5 mol. liter- 1 DCMU added (omitted for the third cell density). For all experiments, 0.5 N HCl was added to the scintillation vials (Lean and Burnison 1979). The DCMU was dissolved in water, not in acetone or alcohol. For each experiment, 2 liters of diluted culture (1 liter in the third series of experiments) were inoculated with 100 PCi. liter-l NaH14C09 and incubated in dispenser flasks (light or dark). During incubation, the temperature of the flasks was kept constant and the medium was continuously mixed with a magnetic stirrer. In all experiments, 5.0 ml were pipetted from the incubated sample each minute for the first 30 min, and each 10 min afterwards up to 220 min of incubation. These 5.0 ml were filtered on Nuclepore 0.4 pm, and the filters were

3 998 Legendre et al. immediately put into a scintillation vial, The scintillation liquid was 11 toluene : 11 ethylene glycol monoethyl ether : 1 Scintiprep 2, and the samples were counted on a LKB scintillation counter (Pugh 1973) * Results The effect of light intensity on the subsequent rate of dark 14C0, uptake during the 2 h before cells were put in the dark is shown in Figs. 1 and 2. These results show how dark 14C0, fixation is affected by the light history of the organisms. Most of the uptake is limited to the first 30 min of incubation and is reflected in different dark 14C fixation for several hours thereafter. The effects of temperature of incubation on dark fixation (Fig. 3) are not dramatic, but there is a difference in the slope of the active fixation as well as in the total cumulative dark r4c fixation. Nutrient addition during incubation shows the most dramatic effects. Results of ammonium enhancement in cultures (Fig. 4) are similar to those found in natural pop- P-J 03 X :I T 4 v 1 I I I I I I PERCENT LIGHT INTENSITY PREDARKNESS Fig. 1. Four-hour l*c incorporation in darkness as a function of light intensity during the 2 h before darkness. Note analogous plot to P:Z curve. Phneodactylurn tricornutum cultures. 100% corresponds to 100 PEinst * mp2* s-l. ulations (Morris et al. 1971b). These light, temperature, and nutrient effects cannot always be demonstrated. For example, where a single culture was diluted to var- 75 % 5:; 100% 430% 22% 7 O/o I I I 1, PREDARKNESS 0 I TIME (hours) Fig. 2. Time-course of l*c incorporation in darkness as a function of light intensity during the before darkness in Phaeodactylum tricomutum culture. 100% corresponds to 100 ~Einst*m-2*s-1. 2h

4 14C method: DCMU correction % dllutlon L -- - I I.5 TIME (hours) Fig. 3. Time-course of *C incorporation in darkness as a function of temperature during incubation. Growth temperature was 18 C for Phaeodactylum tricornutum. ious cell densities, three different cases can be demonstrated (Fig. 5). Our DCMU experiments show that dark 14C fixation is affected by DCMU treatment, as has been shown for light fixation (Kimmcl and White 1979). Figure 6 shows results for the difference between standard dark bottles and dark bottles with DCMU added at three cell concentrations. The high frequency variability in each data series was smoothed out by using a moving average on three successive values. The difference between dark 14C l-.--l-i 1 I TIME (hours) Fig. 5. Time-course of 14C incorporation in darkness as a function of Phaeodactylum tricornutum cell density for dilutions of 43, 22, and 4% with sterilized filtered seawater. Dark l*c uptake as measured for the total samples. fixation in standard incubations and with DCMU added was computed from the smoothed series; consequently, there are no values at 0 and 220 min. The three uptake curves reach a maximum after about min of incubation, with subsequent slow decrease. The slopes of r p 200 g e -t 160 oa- STANDARD DARK --.--I _-I _- L-II I TIME (hours I Fig. 4. Time-course of 14C incorporation in darkness as a function of NH*+ addition (10 mmol added at the start of 14C incubation). The nitrogen-deficient cells are Phaeoductylum tricornutum (adapted from Morris et al. 1971b) I TIMEfhours) 2~10~ CELLS.J- 3 Fig. 6. Time-course of 14C incorporation in darkness at different DunieZZu primolectu cell densities plotted as difference between standard dark bottles and dark bottles with DCMU added at three ccl1 densities.

5 1000 Legendre et al. 240 t DARK BOTTLES i OCMU ADDED / &A-_L 1 1 I 1 I 0 I 2 3 TIME (hours) Fig. 7. Time-course of 14C incorporation in darkness as a function of Dunieh primolecta cells with DCMU added at three cell densities. the initial portion of the three curves increase linearly with the cell concentration (see Fig. S), the overall initial uptake rate being about 55 dpm. ( lo8 cells - liter-r)-l. h-l. M aximum uptake also increases with the cell concentration (see Fig. 8: plateau), but we cannot assess the linearity of this relationship. Figure 7 shows the 14C fixation in dark bottles with DCMU added at the three cell concentrations (after smoothing). The fixation increases linearly, no asymptote being reached during the experiments. There is a linear relationship between the slopes of 14C incorporation for the three experiments and the cell concentration (Fig. 8): the fixation uptake rate is about 30 dpm. ( lo8 cells. liter-r)-l * h-l. Light bottles with added DCMU (Fig. 9) show results similar to the dark bottles with DCMU added (Fig. 7). Again, 14C fixation increases linearly from the lreginning to the end of the experiment at both cell concentrations. In a field experiment on natural populations, the 14C incorporation with DCMU added was about 14% of the r4c incorporation under saturating light conditions. Discussion Since the introduction of the 14C method, most practitioners have been aware that bulk dark fixation is comprised of uptake mediated through metabolic processes of the cell and of uptake not obviously related to physiological processes. For the sake of simplifying this discus- 107 CELL!+& Fig. 8. Slopes of 14C incorporation in darkness as a function of cell density. Slope computed from Figs. 6 and 7; plateau from Fig. 6. sion we will refer to the two features of dark fixation as active and inactive. Our approach was first to demonstrate that active dark fixation responds to external factors which influence cellular metabolism by experiments mostly of the type where fixation is followed at intervals over a period of several hours. Fig TIMEfhoursl Fig. 9. Time-course of 14C incorporation in light as a function of Duniella primolecta cells with DCMU added at two cell densities.

6 14C method: DCMU correction 1001 ures 1 and 2 show what happens when cultures that have been grown at one light intensity are placed in darkness after having been exposed to various light intensities for 2 h before darkness. Dark carbon dioxide uptake can be described by a hyperbolic curve. The initial rapid fixation of carbon dioxide occurs in the first 30 min of darkness, after which slower fixation continues at a relatively constant rate. Both the initial rapid uptake and the maximum depend on the light intensity to which the algae were exposed for 2 h before darkness. When replotted as a function of light (Fig. l), the uptake rate produces a curve which strongly resembles a conventional photosynthesis : light curve. This suggests that the level of dark fixation was closely coupled with either the rate of photosynthesis or the amount of accumulated photosynthate before darkening. Since the process of dark carbon fixation is at least partially under the influence of enzymes, the dark fixation rate can be expected to be influenced by temperature. In this case (Fig. 3), the timecourse was again hyperbolic with the maximum reached after 30 min of darkness, after which the rate of uptake remained constant for at least 1 h. The effect of lowering the temperature from 17 C to 12 C lowers the initial uptake slope by about 20% so that the level of maximum uptake is reached somewhat later at 12 C than at 17 C. Morris et al. (1971a) observed that the dark fixation rate could be changed markedly by the addition of nitrogen to nitrogen-deficient algae. Figure 4 shows the effects of adding ammonium to nitrogendeficient cultures on dark carbon fixation, In the cultures without ammonium added, the uptake changes little. With the addition of ammonium, the uptake rate is markedly accelerated and the resulting pattern of dark uptake for several hours resembles the hyperbolic curves shown in Figs. 2 and 3. To summarize, knowing nothing more than what has been demonstrated in the foregoing experiments, one would conclude that a close coupling exists be- To TIME CASE I Fig. 10. Schematic representation of time-course generalized cases of 14C incorporation in darkness. Case I has final value >T, (value measured initially). Case II has final value about equal to T,. Case III has final value <TO. Y-axes-time when 14C added; T&irst measurement. tween the light-driven reactions of carbon fixation and the subsequent dark fixation of carbon dioxide. Further, the field observer could under the proper conditions use the time-course techniques as a way of assessing the degree of environmental stress. However, precautions should be taken to separate batterial from algal 14C uptake. Careful time-course experiments reveal that there are at least two and most probably three patterns of dark 14C fixation (Fig. 10). We present here evidence for a substantial (> 100% increase) active component in case I, rcfiected by the initial slope of the timc-course, followed by a plateaulequilil~riuln. Case II and case III patterns have a nearly insignificant active component. Light, temperature,

7 1002 Legendre et al. and nutrient effects can be demonstrated in all case I situations. These effects are not shown in case II and case III situations. We are as yet uncertain of the physiological and biochemical processes distinguishing case I from cases II or III. One might be tempted to postulate that populations from oligotrophic waters will fall into one category of cases and populations from eutrophic waters into another; that cultures of healthy physiological state will fall into one category, those of unhealthy physiological state into another. Our results to date do not support this, The same population can be case I, case II, and case III (Fig. 5): 43% dilution is case I, 22% dilution is case II, and 4% dilution is case III. In a companion experitnent, the same dilutions had no significant effect on the light j4c fixation. The total dark 14C fixation of the population is altered, but even more extreme is the dark 14C fixation per cell. We have no explanation for this. Perhaps the cells in a new environment are prone to extreme excretion or osmotic shock following the initial r4c intake. Our experiments demonstrate that dark fixation is far more sensitive than light fixation in this regard: this is seen most clearly in the category of case I. The addition of DCMU changes the uptake kinetics of 14C in both the dark and in the light bottles. With DCMU added (Figs. 7 and S), the 14C uptake is lower than in standard dark incubations (Figs. 6 and 8). The differences between the two series of incubations (Fig. 6) suggest an active 14C assimilation, since the curves rapidly saturate (within min) and then slowly decrease. The cell density influences the kinetics (Fig. 8): the initial slope is a linear function of cell density. The maximum incorporation also increases with cell density (Fig. S), but the relationship is perhaps not linear (a loglog plot suggests a better fit). Therefore, the curves of the differences between standard incubations and those with DCMU added (Fig. 6) may represent the kinetics of the active dark 14C incorporation by the phytoplankton. Obviously, active dark fixation processes (Fig. 6) are not the same as those observed with added DCMU (Fig. 7). Other experiments have shown that part of the fixation in the dark is caused by the Wood-Werkman reaction (Syrett I962), a part of the Krebs respiratory cycle. This reaction is not related to photosynthesis, since Krebs cycle respiration is known to be inhibited in the light (Holm-Hansen 1962). Through the Wood-Werkman reaction, pyruvate is carboxylated into oxaloacetate. The energy for this reaction is supplied by mitochondrial ATP (Lehningcr 1975). When the algae are photosynthesizing, DCMU may act as an inhibitor of photosystem II, through its action on cytochrome Q and plastoquinone (Duysens and Sweers 1963). Plastoquinone is involved in the electron transport from organic substances to oxygen. In the dark, DCMU may act on ubiquinone (or coenzyme Q), an electron transport coenzyme analogous to plastoquinone. If DCMU does inhibit the activity of coenzyme Q, it would thus inhibit the Wood-Werkman reaction. Therefore, the linear 14C increase observed in cultures with added DCMU (Fig. 7) would be the result of an inactive mechanism, either the inactive transport of r4c through the cell membranes, or the adsorption of 14C onto the cell walls (which seems unlikely after the HCl treatment: Lean and Burnison 1979). Standard dark incubations and those with DCMU added show the two mechanisms simultaneously involved in dark 14C fixation: an active uptake, perhaps related to the Wood-Werkman reaction, and an inactive 14C uptake by the cells perhaps combined with some adsorption. The inactive dark 14C fixation (Fig. 8: with DCMU added) is slower than the active r4c incorporation (Fig. 8: standard minus DCMU added) and was linear with both time and cell number in all the experiments (Figs. 7 and 8). The two linear relationships in Fig. 8 suggest that, as the cell concentration decreases, the initial uptake of the two mechanisms becomes very similar. This could explain the drastic change in the slope of the relationship between the ratio of light : dark fixation

8 14C method: DCMU correction 1003 and cell density, found by Morris et al. (1971a: fig. 2) at around lo7 cells-liter-i. As to the practical use of dark bottles in measuring primary production, Morris et al. (1971a,h) came to the conclusion that subtracting dark from light bottles could lead to serious underestimates of phytoplankton production. It is obvious that dark bottles per se do not provide proper values to be subtracted from light bottles, since dark 14C fixation includes the effect of the Wood-Werkman reaction, which does not occur in the light bottles. However, it is debatable whether dark 14C fixation via the dark Wood-Werkman pathway should be considered net primary production in the dark period. On the other hand, 14C dark incorporation with added DCMU is a good candidate to be subtracted from the light bottles, since it represents only the inactive 14C incorportition, uncontaminated by the active dark uptake. The same is true of i4c light incorporation with DCMU (Fig. 9). How these results relate to field studies needs to bc assessed under various conditions. At low algal concentrations, Figs. 6 and 8 suggest that no significant difference can be demonstrated between standard dark incubations and those with DCMU added. This could lead to the conclusion that at low cell concentrations standard dark bottles are at times equivalent to those with DCMU. However, in any case when a difference may be anticipated then the dark bottle with DCMU added is the only one interpretable. The evidence presented here is informative in two ways. First, it indicates that the more complete data set from timccourse experiments permit new interpretations of dark 14C fixation. Second, it demonstrates that the best correction factor for primary productivity studies is not any dark fixation or zero time treatment, but a dark or a light bottle plus DCMU in a water solution. The carryover of a dark bottle from early oxygen method measurements is unmerited, as has been suggested repeatedly in the literature (e.g. Peterson 1980) and as is demonstrated by our experiments. Because DCIMU treatmcnts of the light and of the dark bottles result in similar estimates of the passive 14C fixation, we suggest that for productivity studies one should use a standard light bottle and one with added DCMU. References DuYsENs,L.N.,ANDII.E. SWEERS Mechanism of two photochemical reactions in algae as studied by means of fluorescence, p In N. Shokubutsu and S. Gakkai [eds.], Studies on microalgae and photosynthetic bacteria. Univ. Tokyo. GUILLARD, R.R., AND J. H. RYTHER Studits on marine planktonic diatoms. Can. J. Microbiol. 8: HOLM-HANSEN, Assimilation of carbon dioxide, p Zn R. A. Lewin red.], Physiology and biochemistry of algae. Academic. KIMMEL, B. L., AND M. M. WHITE DCMU enhanced chlorophyll fluorescence as an indicator of the physiological status of reservoir phytoplankton: An initial evaluation. U.S. Army Corps of Eng. Waterways Exp. Sta. Vicksburg, Miss. Tech. Rep. TC-3265, p LEAN, D. R., AND B. K. BURNISON An evaluation of errors in the 14C method of primary production measurement. Limnol. Oceanogr. 24: LEEININGER, A. L Biochemistry, the molecular basis of cell structure and function. Worth. MORRIS, I., C. M. YENTSCH, AND C. S. YENTSCII. 1971n. Relationship between light carbon dioxide fixation and dark carbon dioxide fixation by marine algae. Limnol. Oceanogr. 16: ,-, AND lh. The physiological state with respect to nitrogen of phytoplankton from low nutrient subtropical waters as measured by the effect of ammonium ion on dark carbon dioxide fixation. Limnol. Oceanogr. 16: PETERSON, P. J Aquatic primary productivity and the 14C-CO, method: A history of the productivity problem. Annu. Rev. Ecol. Syst. 11: PUGII, P. R An evaluation of liquid scintillation counting techniques for use in aquatic primary production studies. Limnol. Oceanogr. 18: STEE~MANN NIELSEN, E The USC ofradioactive carbon (CL4) for measuring organic production in the sea. J. Cons. Cons. Int. Explor. Mer 18: SYRETT, P. J Nitrogen assimilation, p Zn R. A. Lewin led.], Physiology and biochemistry of algae. Academic. Submitted: 7 June 1982 Accepted: 17 January 1983