Patterns of dark - Springer Link

Microb Ecol (1987) 13:249-259

MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1987

Patterns of Dark 14CO2 Incorporation by Natural Marine Phytoplankton Communities L. Ignatiades, M. Karydis,* and K. Pagou Nuclear Research Center "Democritos," Aghia Paraskevi, Attiki, Greece

Abstract. The rates of dark |4CO2fixation by natural phytoplankton communities growing in eutrophic and oligotrophic waters were studied with short-term in situ experiments. Three aspects were investigated: (1) the time course incorporation of t4CO2 in darkness, (2) the depth variability in dark ~4CO2 fixation, and (3) the variability in ~4CO2 fixation within a year. The highest dark ~4CO2 incorporation rates were observed during the first interval of incubation (20 min) after which they approached a constant rate with time. The observed differences in dark ~4CO2 fixation rates between populations from different depths were associated with differences in species composition as well as with physiological differences caused by exposure to different illumination conditions prior to their exposure to darkness. Autocorrelation coefficients were computed for the analysis of variability of dark ~4CO2 fixation rates within a year. It was suggested that dark ~4CO2 incorporation might be a periodic phenomenon depending mainly on the productive capacity of the phytoplankton community.

Introduction

It can be seen from a review of literature [10, 13, 15, 19, 22, 27, 36] that most information on the dark 14CO2 incorporation by phytoplankton has been derived from work with cultures and that it is related to algal physiology and biochemistry. The problem of dealing with plankton assemblages is more complex, so that, although carbon assimilation in the dark is of great ecological importance [23], there is only limited information from in situ experimental work [35]. Data indicate that dark t4CO2 fixation is influenced by cell density [20], availability of inorganic nutrients [21, 35], concentration of organic nutrients [29], and bacterial action [30]. The experiments reported in this paper were conducted in situ with three main objectives: (1) to investigate the time-course incorporation of t4CO2 in the dark by natural marine communities from different water types, (2) to examine the response of marine communities from different seawater depths to dark t4CO2 assimilation, and (3) to further examine the variability of dark * Present address: Hydrobiological Station and Public Aquarium of Rhodes, Rhodes, Greece.

250

L. Ignatiades et al.

14CO 2 fixation within a year in eutrophic and oligotrophic waters of the marine environment.

Methods The experimental work was done at six stations in the Saronicos Gulf, Aegean Sea. The experiments of dark z4CO2 fixation in relation to time and depth were performed at stations S~, $2, S~, and $4. These stations were located along a transect starting from the eutrophic inshore area of sewage outfall in W. Saronicos (S~) and following a gradient ($2, S~, $4) towards the offshore oligotrophic waters with a progressive decrease in phytoplankton standing stock. The experiments of variability of dark ~4CO2 fixation within a year were made at stations Ss and $6. Station $5 was located in the eutrophic inshore area of sewage outfall of W. Saronicos (near S~), and station $6 was in the inshore oligotrophic area ofE. Saronicos Gulf which was not influenced by the sewage effluents. The general procedure of the experimental work was as follows. Dark and light carbon fixation was measured by the ~4C-technique [34]. The samples were collected with a van Dorn sampler, dispensed to 100 ml dark and light glass bottles, injected with ~4C-NaHCO3, and incubated in situ. The samples were filtered immediately after the termination of each experiment under 100 m m Hg pressure differential. The filters (Millipore membrane filters, 0.8 #m pore size, 47 m m in diameter) were not treated with HCI solution since acidification can remove a large fraction of the incorporated carbon [3, 9], but they were washed thoroughly with filtered seawater [34]. Goldman and Dennett [11] demonstrated that by rinsing the filters with seawater, cell breakage might be caused. This possibility cannot be excluded but it is difficult to evaluate in the present investigation which has been performed with different experimental material and conditions. Filters of 0.8 #m pore size were chosen in order to avoid interference from the larger portion of heterotrophic virioplankton and bacterioplankton [31 ] as well as of photosynthetic ultraplanktonic species, whose identification, enumeration, and classification is a difficult task. Furthermore, their ecological behavior is not well understood [33]. Filters of 0.8 /~m pore size were used also for chlorophyll a determinations in order to obtain populations of comparable size to those treated with ~4CO2. Several attempts to estimate the inactive ~4CO2 binding by running dark blanks [18, 20, 35] gave nonreproducible results, and thus this parameter was not evaluated. This fraction was assumed to be constant with time [l 5]. Conditions specific for each experiment were as foUows: (1) The time-course incorporation of ~4CO2 in the dark was examined using populations sampled from 1 m depth of stations Sl, $2, $3, and $4. Nine sets of two dark bottles and one light bottle were simultaneously incubated at 1 m depth, and a set was removed after 20, 40, 60, 80, 100, 120, 140, 160, and 180 min. Four experiments were conducted during the period June 24-July 1, 1981. (2) Stations S~, S~, $3 and $4 were sampled also at l, 10, and 40 m depths for testing the dark J4CO2 fixation behavior of populations from different depths. Four dark and two light bottles were incubated at 1, 10, and 40 m depths for 3 hours. Four experiments were performed during the period June 22-June 29, 1981. (3) The examination of the variability of dark ~4CO2 fixation within a year was performed at station $5 (eutrophic) and station $6 (oligotrophic) during the period March 1982-May 1983. Sampling was monthly at 1 m depth, and duplicate dark and light bottles were incubated at this depth for 2 hours. All samples were analyzed for chlorophyll a [34], cell concentration (inverted microscopy), and ammonia [ 17]. Incident solar radiation was measured by 28A Gemware Star Pyranometer and the water transparency by the Secchi disc. Temperature was also recorded at each sampling depth. Serial correlation analysis or autocorrelation was used to determine the pattern of dark and light ~4CO2 fixation rates during the period of I year [ 16, 24, 26]. This method was used for a preliminary evaluation of the pattern of dark fixation within a year, although the number o f observations was not so large to assure statistically significant interpretations o f the results in all cases. The serial correlation coefficients between the dark (or light) ~4CO2 fixation rates at regular monthly intervals (t = 1, 2 . . . . n) were calculated from the equation described in Poole [26]:

Dark ~4CO2 Incorporation by Phytoplankton Table 1.

Date

251

Selected physical and biological parameters at the experimental stations Depth (m) Incident 1% radiaillu- Secchi tion Depth Temp. mina- disc gmcal/ Chl. a Stat. (m) (~ tion (m) cmVmin mg/m 3

% Taxa composition

Cells/liter

FlaDi- gelatoms lates Others

22.6.81

SI

1 10 40

23.8 22.0 19.2

8

3

0.72

9.67 1.74 1.05

2.5 • 106 5.5 • 105 9.1 x 104

29 50 80

70 49 20

1 1 --

23.6.81

$2

1 10 40

23.2 22.4 19.3

14

5

0.60

2.07 1.24 0.93

8.1 x 105 5.1 • 105 8.6 • 104

37 85 96

61 14 4

2 1 --

26.6.81

$3

1 I0 40

24.4 23.4 23.4

56

18

1.06

0.55 0.37 0.29

6.7 • lO s 3.5 x 105 1.8 x 104

73 72 91

26 27 7

1 1 2

29.6.81

$4

1 10 40

25.2 24.0 18.8

63

16

0.86

0.43 0.38 0.26

1.3 x 105 7.6 x 104 1.1 • 104

96 81 93

4 17 6

-2 1

n--t

n--I

n

rt =

Xi2 - n -~

Xi

:j

-]

i-I

where n is the number of observations from i = 1 to n (here n = 12), x~ is each of the observations and r, is the serial correlation or autocorrelation. The original data series was tested for tendency by the Kendall rank correlation test [16] and did not show any trend. The data were logarithmically transformed. A computer program [7] was used for the estimation of autocorrelation.

Results

Time Course Incorporation of 14C02 in the Dark The quantitative differences in phytoplankton concentration among the exp e r i m e n t a l s t a t i o n s Sl, $2, $3, a n d $4 a r e g i v e n i n T a b l e 1. D e c r e a s i n g o f p h y t o p l a n k t o n b i o m a s s a n d s t a n d i n g s t o c k (chl a, c e l l s / l i t e r ) f r o m s t a t i o n St t o s t a t i o n $4 is o b v i o u s . C h a n g e s i n t h e d a r k J4CO2 a c c u m u l a t i o n b y p h y t o p l a n k t o n w i t h l e n g t h o f i n c u b a t i o n p e r i o d a r e s h o w n in F i g . 1. T h e u p t a k e c u r v e s i n d i c a t e a s u b s t a n t i a l i n c r e a s e in t h e x4CO 2 f i x a t i o n l e v e l s a f t e r 180 m i n i n c u b a t i o n i n r e l a t i o n t o t h e i n i t i a l l e v e l s (20 m i n i n c u b a t i o n ) . T h i s i n c r e a s e w a s 1 7 3 % f o r s t a t i o n $ 1 , 6 8 % f o r s t a t i o n $2, 8 6 % f o r s t a t i o n $3, a n d 8 2 % f o r s t a t i o n $4. T h e t i m e c o u r s e o f t h e r a t e s o f d a r k 14CO 2 a s s i m i l a t i o n b y t h e p h y t o p l a n k t o n p o p u l a t i o n s f o r s t a t i o n s S~, $2, $3, a n d $4 a r e g i v e n i n F i g . 2. I t m a y b e s e e n

252

L. Ignatiadeset al.

E (,_)

f

~' z s

st. 51

Z o

D

IE 1.5

U

1.0 A

~

s

t

.

52

~st. 5 4

d~

st. 5a

vrr 0.5 0.0

I

I

I

2o ,o 6o do

12o' 1, o

IN 51TU I N C U B A T I O N

I

1;o

Fig. 1. Dark 14CO2 accumulation vs incubation period at the experimental stations.

(mln.)

that the shapes o f the curves derived from the four different stations are remarkably similar. The highest values of t4CO2 fixation rates were recorded at the 20 min incubation period after which the rates followed a slope approaching asymptotically a constant value with time. The initial m a x i m u m fixation rate at station St was higher (2.20 mg C/m3/hour) in relation to the initial rates o f stations $2, $3, and $4, which ranged from 0.78 to 1.55 mg C/ma/hour. Also, the level at which the rates became constant with time was higher for station St (0.72 mg C/m3/hour) in relation to the corresponding levels o f the other stations, which ranged from 0.22-0.35 mg C/m3/hour. Concerning the quantitative relationship between the '4CO2 uptake rates in darkness and light the results showed (Fig. 3) that the dark to light uptake ratio was higher in the first 20 min o f incubation (St, 8%; $2, 14%; $3, 37%; $4, 25%), and it decreased continuously to 3-5% after 180 min of incubation at all stations.

Dark 14C02Fixation in Relation to Depth The response o f algal populations grown in different light conditions to dark t4CO2 assimilation was examined by testing populations over a range o f depths. The samples were taken from 1, 10, and 40 m depths, and, because of the stability o f the water column and the light attenuation (Table 1), they represent populations from above or below the euphotic zone. At 1 m the phytoplankton biomass differed considerably at St (chl a 9.67 mg/m 3) and S 2 (chl a 2.07 mg/m3), but it was quantitatively similar at 10 and 40 m depth at the two stations (Table 1). Both stations had higher percentages o f flagellates (61-70%) over diatoms (29-37%) at 1 m depth. However, the percentage o f diatoms increased in the populations of 10 m depth, and at 40 m depth diatoms dominated (80-96%) over flagellates (4-20%). The highest 14CO2 fixation rates (Fig. 4) were recorded at 1 m depth (0.99-1.30 mg C/m3/

Dark J4CO2Incorporation by Phytoplankton

253

2.1

E 1.9 o',

.EE 1.7 Z

9 ~- 1.5 X Ix_

N 1.3 0 1.1 fY

\\~,+,,

O.9 U_

9

~

0,7

~

0

0

\ \

s t . 57

\ \

05 0.3 .....

0.1 I

I

I

I

20

4O

60

80

I

I

A I

I

100 120 140 160

~ s t . 5~ t

180

Fig. 2. Rates of dark t4CO2 fixation vs incubation period at the experimental stations.

IN 51TU INCUBATION (rain.)

hour) a n d declined considerably at 10 m (0.16--0.20 m g C / m 3 / h o u r ) a n d 40 m d e p t h (0.11--0.13 m g C/m3/hour). T h e s a m p l e s at 1 m d e p t h showed similar rates o f d a r k t4CO2 fixation in spite o f the great q u a n t i t a t i v e difference in p h y t o p l a n k t o n abundance. Stations $3 a n d $4 showed a small difference in p h y t o p l a n k t o n density (Table 1), h a v i n g at 1 m d e p t h chl a c o n c e n t r a t i o n 0.55 m g / m 3 a n d 0.43 m g / m 3 respectively. These quantities declined with depth, with similar degree o f decrease at b o t h stations. T h e analysis o f t a x a c o m p o s i t i o n s h o w e d t h a t all depths o f b o t h stations were d o m i n a t e d by d i a t o m s , which c o m p r i s e d 7 2 - 9 3 % o f the total population. T h e general trend o f d a r k t4CO2 fixation was similar at b o t h stations (Fig. 4), a n d the values were 0.39 a n d 0.28 m g C / m V h o u r at 1 m depth, 0.30 a n d 0.20 m g C / m 3 / h o u r at 10 m depth, a n d 0.25 a n d 0.17 m g (3/ m 3 / h o u r at 40 m d e p t h o f stations $3 a n d $4, respectively.

254

z

o

L. Ignatiades et al.

50

F
0.01 level of significance), but those between dark :4CO2 assimilation and cell density or the concentration of ammonia were nonsignificant in all instances.

Discussion

To explain '4CO2 uptake of a darkened phytoplankton population one should take into account the following reactions: (1) dark fixation of '4CO2, (2) dark loss of '4CO2, and (3) inactive fixation of '4COy

Dark t4CO2 Incorporation by Phytoplankton

255

1.4 st.52

7:: 1.2 m-' . E

st.51

1.o E z

0

0.8

5x

I

:1

kL

~ 0.6 9 _

2

cr.

I

st.53

0.4

b_ 0 0.2 Ld

-h

Fig. 4. Rates of dark ~"CO2 fixation vs depth at the experimental stations.

a: 0.0 DEPTH

(m;

The reactions by which carbon dioxide is incorporated in the dark have been discussed by Raven [28]. In most algae the major reaction of COz incorporation in the dark is/3-carboxylation, whose main function appears to be in the net synthesis of the four carbon acids and their derivatives. Another dark COz incorporation reaction is the synthesis of carbamoyl acid which is important in the biosynthesis of arginine and pyrimidines. Sugar phosphates are also products of dark CO2 fixation. Akoyunoglou and Calvin [2] and Akoyunoglou and Argyroudi-Akoyunoglou [1] have also reported the formation of COzaddition compounds with amino acids, peptides, protein amines, etc., which are in the carbamate form. Respiration appears to be a principal source of the dark 14CO2 loss by phytoplankton [5, 6, 8, 28]. Excretion of organic substances by algae in darkness might also contribute to the loss of the carbon fixed [14]. Respiration rates cannot be measured by the 14CO~ technique [12]. Inactive 14CO2 binding is associated with the isotopic technique for estimation of the carbon incorporation in the dark. It has been reported that the retention of ~4COz on algal cell walls and on membrane filters during filtration can cause a significant error in the evaluation of the dark fixation level, but the recommended methodology for avoiding these errors is controversial [9, 15, 18, 32, 37]. The time course experiments of the present investigation indicate that the dark 14CO2 fixation is a time-dependent reaction (Figs. 1 and 2). From the shapes of the curves (Fig. 2), it is evident that the highest dark ~4CO2 incorporation rates were observed during the first time interval of incubation (20 min), after which they followed a slope approximating an equilibrium with

256

L. Ignatiades et al. A

+1.0

P:I 0.05)

.--'"

+0.5 - - - X . . . . . . ; < i -

_~-0.5

~) -q51