Heterotrophic Glucose Assimilation in Lake Ontario

J. Great Lakes Res. 14(2):157-163 Internal. Assoc. Great Lakes Res., 1988

HETEROTROPHIC GLUCOSE ASSIMILATION IN LAKE ONTARIO

G. D. Haffner, M. L. Yallop, and P. D. N. Hebert Great Lakes Institute Department of Biology University of Windsor Windsor, Ontario N9B 3P4 ABSTRACT. Heterotrophic glucose uptake was measured during 1980 in Lake Ontario plankton populations. Uptake rates of glucose were generally low (0.003 - 0.457 mg C'm-3'h- I ) compared with corresponding photosynthetic carbon uptake (8 - 32 mg c-m-3'h- I ). Particle size fractionation studies confirmed that a majority of heterotrophic carbon uptake was in the 1 - 8 p.m diameter size class. Although heterotrophic carbon utilization by algae and bacteria was small when directly compared with photosynthetic production, heterotrophy might contribute significantly to the carbon flux within a water column over a 24-hour period. ADDITIONAL INDEX WORDS: Phytoplankton, productivity, bacteria.

HETEROTROPHIC CARBON ASSIMILATION IN LAKE ONTARIO

compounds can be utilized after their incorporation into algal cells (Palmer and Togaski 1971). Vincent and Goldman (1980) suggested that ultraplankton chlorophycean isolates had high affinities for organic compounds such as acetate and could metabolize acetate to support cell growth. Similar observations have suggested algal heterotrophy could play an important role at least in terms of population maintenance (Vincent 1978). In the Great Lakes, Moll and Brahce (1986) concluded that algae were responsible for a significant portion of dark heterotrophy measured in Lake Michigan. Of particular interest in the Lake Michigan study was evidence of potential temporal and spatial resource partitioning by bacteria and algae. This evidence of resource partitioning as opposed to direct resource competition can significantly alter the role significance of heterotrophy in regulating the composition of algal populations. Sole dependence on photosynthetic carbon assimilation could require phytoplankton cells to be able to track short-term variability in resources such as light, nutrients, and inorganic carbon. The availability and utilization of these resources in Lake Ontario are often associated with random events resulting in assemblages of opportunistic species (Haffner et al. 1984, Harris et al. 1980). The use of alternative energy and carbon sources that are available at low but consistent levels could permit populations to remain viable until more

Heterotrophic carbon assimilation is often considered to be a minor contribution to a phytoplankton cell's carbon budget (Pechlaner 1971). Although phytoplankton species have been shown to have both passive (Hobbie and Wright 1965) and active (Hellebust 1971) transport mechanisms for assimilating organic carbon, it has been generally concluded that heterotrophy in algal populations is insignificant when compared with bacterial populations (Wright and Hobbie 1966). Measurements of heterotrophic uptake of carbon by algae in aquatic systems have generally suggested uptake rates of 0.001 to 0.024 mg glucose C·m-3·h- 1 commonly observed in the Great Lakes (Vollenweider et al. 1974).

When comparing photosynthetic and heterotrophic carbon uptake rates, however, it is important to remember that heterotrophy can occur over a 24-hour period and throughout the entire water column. On a diel basis, Overbeck (1975) observed glucose uptake to range 2.3-12.3070 of the rate of primary productivity, suggesting that the ecological importance of heterotrophic uptake might have been previously underestimated, especially when considered in terms of resource competition among phytoplankton species. Conversely, there is still the question if organic

157

HAFFNER et al.

158

adequate environmental conditions for metabolism were encountered. In the Great Lakes, where physical processes can alter environmental conditions in very short time periods (Blanton 1974), a process such as heterotrophic carbon assimilation would provide a selective advantage during physical transition periods when light and nutrient levels are highly variable, or when populations are mixed out of the euphotic zone for extended periods of time. In order to assess the rate of heterotrophic uptake of glucose in the Great Lakes, a series of in situ and laboratory measurements were made. The experiments were designed to compare primary productivity measurements with glucose uptake, and to determine which size fraction of the plankton biocoenosis was most active in glucose uptake. These observations allowed some insight to the nature of the glucose uptake process, and an assessment of the ecological role of heterotrophy in the Great Lakes.

MATERIALS AND METHODS Sample Collection and Analysis Samples were collected along a transect 5 km east of Toronto Harbour. Three stations of 6, 20, and 50 m depth (see Haffner et al. 1984) were selected for determining carbon uptake rates. Primary productivity and heterotrophy were jointly measured from 17 July 1980 until 5 November 1980. Water samples for size fractionation were kept cool and transported immediately to the laboratory, where experiments were conducted the same day. For all field and laboratory experiments, subsamples of water were removed for chlorophyll a determination and phytoplankton identification and enumeration as described in Haffner et al. (1984). Chlorophyll a determinations were made by extraction of the pigments in 900/0 acetone for 20 hours at 5°C. A 1% suspension of magnesium carbonate was added to maintain alkaline conditions during extraction. The extract was then centrifuged and the absorbance of the sample measured at 750 and 665 nm on a Spectronic 20. The extract was then acidified by addition of 2 drops of 2N HCI and re~measured after a 10 minute period in the dark, thereby correcting for phaeophytin.

Field Analysis During 1980, 11 cruises were conducted to measure autotrophic and heterotrophic carbon uptake. Autotrophic carbon uptake was measured using the 14C method. Five /lCi of NaH14C03 (New England Nuclear) were added to 125 mL water samples and incubated throughout the water column in both light and dark bottles for 2-3 hours over the midday period (Vollenweider 1969). Glucose uptake was determined by addition of 1 /lCi of a mixture of labelled D (U_ 14C) glucose (Amersham) of specific activity 255 /lCilmmol and unlabelled glucose to a 125 mL water sample. This yielded a final concentration of 8 x 10-4 mM glucose/L (144 /lg/L). It was assumed that natural glucose concentrations would be minor compared with the added concentrations. Samples were incubated in situ in light and dark bottles for approximately 4 hours. Control bottles were prepared by the addition of formalin to make a 4% solution. All samples were filtered under low vacuum (15 cm Hg) onto 0.45 /lm Millipore HA filters and washed with 20 mL de-ionized water. The filters were placed in vials containing 15 mL of Aquasol-2 fluor. Radioactivity of the samples was determined on a Beckman 3150 P Liquid Scintillation Counter. Quench curves were prepared by the External Standard Channels Ratio method to determine counting efficiency. Size Fractionation Water samples collected from the 50-m station during July, August, and September were filtered through Nuclepore polycarbonate membrane filters of 142 mm diameter using reverse filtration techniques. The filters employed had pore sizes of 8.0, 5.0, 3.0, 1.0, and 0.45 /lm. Nuclepore filters were preferable as they act as sieves. Aliquots of each size fraction were taken and fixed in Lugol's iodine for microscopic examination. Size fractionated samples were then injected with 1 /lCi radio labelled glucose as previously described and incubated for 4 hours in 125-mL bottles in triplicate in the light and dark. Incubations were carried out in temperature controlled cabinets at 15°C and lighting was provided by means of a bank of CoolWhite fluorescent tubes providing a light intensity of 37 /lE.m-2·sec· 1 • Uptake Kinetics Raw water samples were incubated in situ over a range of radioisotope additions yielding final glu-

HETEROTROPHIC GLUCOSE ASSIMILATION

cose concentrations of 28.8 to 288 ",g.L-l. Light and dark uptake measurements were made along with formaldehyde controls. The following formula was applied for calculation of uptake rates, V

=

c(S + A) Co u ° t

(where V = uptake velocity (mg uptake ° L-lohr 1), S = natural substrate concentration (assumed to be zero), A = added substrate concentration, c = radioactivity of filtered organisms (cpm) , C = cpm for 1 ",Ci of isotope in counting assembly used, u = activity in ",Ci, t = time in hours). RESULTS

Field Observations

Primary productivity in Lake Ontario illustrated typical surface inhibition. Pmax was frequently observed between the 1.5 and 5 m depth range (Fig. 1), and varied between 2 to 32 mg Com-3oh- l during 1980. The higher values of Pmax were often associated with the deep offshore station. Integral productivity during this period ranged 28.4-112 mg Com-2oh- l for the 6 m nearshore station; 83.2-213 mg Com-2 oh- 1 for the 20 m station, and 25.2-164 mg Com-2 oh- 1 for the 50 m offshore station. Measurements of heterotrophy were very low compared with the above estimates of primary productivity and uptake rates varied little with depth. Table 1 summarizes the observed rates of glucose uptake at the three stations for both light and dark bottle experiments. Using a one way analysis of variance there was no significant difference in measured uptake rates and station depth (p > 0.10) and therefore there was no evidence of an offshore-nearshore gradient in glucose uptake kinetics. The glucose uptake rates observed in light bottles tended to be depressed as compared with the dark bottle uptake rates. This might have been a function of photolytic damage to bacterial and perhaps algal cells, and possibly accounts for there not being a vertical gradient of in situ glucose uptake rates. Hourly volume uptake rates of glucose carbon by both algae and bacteria were one to two orders of magnitude less than those those observed for primary productivity. Glucose uptake rates were observed between a relatively broad range of 0.003 to 0.457 mg Com-3oh. There was no correlation of either light or dark uptake rates of glucose with the concentration of chlorophyll a (P

159

> 0.10) although a relationship of heterotrophic uptake rate and biomass might be more species dependent than would primary productivity. A more complete description of the algal community during the time of the productivityheterotrophy study is given in Haffner et af. (1984). Generally, the summer populations were complex assemblages or chlorophytes, cryptophytes, and bacillariophytes with no one group dominating the others. Many of the species such as Pediastrum and Microcystis maintained slow growing populations, and diatoms such as Tabellaria and Stephanodiscus were common during the summer months. This composition and general lack of community structure might reflect the high turbulence regimes and temporal variability of the epilimnion of Lake Ontario (Haffner et af. 1984). Size Fractionation and Uptake Kinetics

On three occasions during 1980, samples from the 50 m water column were transported to the laboratory for determining glucose uptake rates in the various size fractions of the phytoplankton assemblage. Figure 2 illustrates that a majority of the glucose uptake occurred in the particle size fractions greater than 1.0 ",m diameter, and that particles less than 8.0 ",m diameter accounted for over 80010 of the heterotrophic uptake. Again, note that dark uptake rates generally exceeded light uptake rates, particularly during August 1980, when approximately 82% of the uptake in the light occurred in organisms greater than 8.0 ",m. The general depression of light uptake rates might be a function of the low light intensities used for incubating the samples. The uptake kinetics of glucose in natural Lake Ontario plankton populations were determined in October and December 1980, under both light and dark conditions. It was found that glucose uptake followed first order kinetics relative to substrate concentration. Half saturation concentrations (Ks) and the maximum uptake rates (Vmax) were determined and the results are summarized in Table 2. It is important to note that these results provide values which represent the kinetics of the phytoplankton assemblage and are not characteristic of individual species. It is assumed that the kinetics of substrate uptake of this mixed assemblage conform to kinetic equations applied to single species (Williams 1970). Uptake kinetics for light and dark incubated samples were similar on both occasions. Vmax

160

HAFFNER et af. P(mg C.m~3hr.-I)

8

P

P

16

8

24

16

P

P

24 32

16

8

8

24

16

8

32

16

24

2 0 E

4

4

P

A

T

H 6 (m)

4

B

6 8

8

8

10

14/7/80

14/7/80

13/8/80

29/7/80

12

I

P

8

0 E

16

24

P

8

16

32

16

8

B

P

24 32

C

T H (m)

10

50

P

24

13/8/80

P

8

16

P

24 32

8

2

2

4

4

6

6

8

8 5/11/80

10

12

12

14

14

16

50

50

16

24

C

5/11/80

50

FIG. 1. Primary productivity profiles in Lake Ontario, Toronto area, 1980 (A-Nearshore Station, B-Midshore Station, C - Offshore Station).

rates were lower for light uptake than for 'dark uptake on both sampling days. If, as size factionation studies indicate, the majority of the glucose uptake occurs in the nanoplankton component of

TABLE 1. Glucose uptake rates (mg C . m-] . h-l) in Lake Ontario between July and November 1980 (Mean

±

1 S.D.).

Uptake Station

Station Depth(m)

Light

Dark

n

1550 1552 1554

6 20 50

0.05 ± 0.05 0.06 ± 0.06 0.07 ± 0.08

0.12 ± 0.07 0.11 ± 0.04 0.09 ± 0.10

20 19 19

the seston, differences between light and dark uptake may be a result of changes in uptake responses under varying light conditions. As these measurements were made in the late autumn under relatively low light conditions, this might account for the little differences between the light and dark glucose uptake kinetics reported in Table 2. DISCUSSION Heterotrophic glucose uptake in Lake Ontario was small compared with the resident algal population's carbon requirements. During this present study, the largest observed heterotrophic uptake rate was 0.4 mg C·m- 3·h- 1 , approximately 2070 of the concurrent photosynthetic rate. The low rates of heterotrophic carbon assimilation in Lake Ontario

HETEROTROPHIC GLUCOSE ASSIMILATION

~ 100

A

u

Fraction

:::J

aI

80

Species

0.45

None

}'

1.0

Rhodomonas minuta

'0 60

3.0

R. minuta

lD

o

40

~

20

~

<;e

Light Uptake 0 Dark Uptake •

5.0

R. min uta, C. ovata

8.0

C. erosa, C. ovata

> 8.0

A. formosa

12345678 Pore Diameter(lJm)

lD

8u 100

B

Fraction

Species

:::J

~

0.45

80

}'

'0 60 & 40

51l/I

~

No cells

1.0

R. minuta

3.0

R. minuta

5.0

C. erosa, R. minuta

8.0

C. erosa, C. ovata

> 8.0

20

Stephanodiscus hantzschii, C. ovata

<;e LQ,--........--r-T'--r-"T"""1rl.f-r-

I 2 3 4 5 6 7 8 Total Pore Diameter (IJm)

§ 100

c

:::J

a I

Fraction

80

:P

'0 60

t

~

<;e

Species

0.45

No cells

1.0

R. min uta

3.0

R. minuta

5.0

R. minuta

40

8.0

R. minuta

20

> 8.0

S. hantzschii

L..-r-'-T"""""'--r--r--r-r-I/-r--

I 2 3 4 5 6 7 8 Total Pore Diameter (IJm)

FIG. 2. Size fraction uptake of glucose by Lake Ontario phytoplankton assemblages of the offshore station (A-July, B-August, C-September).

would probably not meet the respiratory carbon loss of phytoplankton cells. Consequently, the ecological advantage of heterotrophy might be confined to shorter time scale perturbations such as when cells are temporally mixed out of the euphotic zone, as opposed to maintaining cell viability for prolonged periods such as those cells resting in the surficial sediments or suspended in the water column under ice. For shorter time periods, however, algal species with the potential for hetero-

161

trophic uptake might maintain a physiological advantage compared with those without such a capacity, and therefore be able to seed later populations when more favorable conditions for growth return. The glucose uptake rates observed in this study involved uptake by both algal and bacterial populations, and it is not possible to asses the relative contribution of each group to the carbon flux measured. What is important to note here is that glucose uptake by both groups is small when directly compared with the autotrophic process. Although it could be argued that the bacterial carbon pool was smaller and therefore was being recycled much faster, these rates would have had to be at least ten times faster than those measured in Lake Ontario during this study for bacterial carbon flux to be equal to phytoplankton carbon flux in the euphotic zone. However, if one was to consider carbon turnover throughout the entire water column for a diurnal period, the minimum dark uptake rates of glucose observed in this study of 0.02 mg C'm- 3'h- l can result in a column rate of 8.4 mg C·m-z·d- 1 • Although this is only a small portion of the algal productivity (119-2,003 mg C'm-z'd- 1 estimated for Lake Ontario by Stadlemann et al. (1974», it is possible that autotrophic production is in the same order of magnitude of heterotrophic production in deep water columns. Scavia et al. (1986) have suggested that such a process might be important in the Lake Michigan food web. Flint (1986) did not account for such a process in his description of the dynamics of the Lake Ontario food web, and observations from this present study would indicate that in deeper water columns, heterotrophy might become more important in food web dynamics. Kinetics of glucose uptake observed for Lake Ontario plankton assemblages yielded Ks values of 0.5 x 10-4 to 0.63 X 10-3 mM, somewhat higher than the range Schneider and Wiley (1971) reported for bacteria (1 x 10-5 to 4 X lO- z mM). As natural levels of glucose in Lake Ontario are well below these values, direct competition for the resources would favor bacterial populations. Considering, however, that the total dissolved organic levels for Lake Ontario range between 1-4 mg/L, it is possible there is a large potential organic resource base. Combined with the observations of Moll and Brahce (1986) on resource partioning, the differences in Ks values between algal and bacterial populations might not result in one group having a distinct advantage over the other.

162 TABLE 2.

Date 9/10/80

HAFFNER et al. Uptake kinetics of glucose (Ks and Vmax) for Lake Ontario phytoplankton assemblages.

Light or Dark Light Dark

6/12/80

Light Dark

Ks for glucose (Standard error) 0.115 mg'L-l (0.03) (0.63 x 10-3mM) 0.105 mg'L-l (0.01) (0.58 x 1O-3mM) 0.083 mg'L-l (0.02) (0.46) x 1O-4 mM) 0.09 mg'L-l (0.01) (0.5 x 1O-4mM)

The size fractionation experiments indicated much of the glucose uptake was associated with cells between 1 to 8 pm diameter. It is possible that aggregates containing bacteria were in this size category, and there is no direct evidence as to which compartment, algal or bacterial, was more important. This size class of plankton biomass, because of its association with autotrophic and heterotrophic carbon assimilation, is probably the basis of the Lake Ontario foodweb as noted by Flint (1986). Unfortunately, the results of this study do not resolve the contribution of algal and bacterial components within this size class, but do suggest that heterotrophy might play a more important role than originally thought. Although heterotrophy is not a major factor in the production of Great Lakes phytoplankton populations, it might be of considerable ecological importance when viewed with respect to phytoplankton population composition and maintenance and the potential driving of the microbial loop within deep water columns. It is probable that the mechanism described by Scavia et al. (1986) for bacterial carbon requirements in Lake Michigan also operates in Lake Ontario. In a deep column (> 100 m) bacterial carbon demands could exceed autrotrophic production.

ACKNOWLEDGMENTS The authors wish to thank Dr. N. Billington for his review of the manuscript, and the Ontario Ministry of the Environment (Ms. M. Griffiths) for supporting this research project. The authors (G.D.H. and P.D.N.H.) acknowledge financial assistance of respective grants from N.S.E.R.C.

Vmax. (mg glucose L-l) (Standard error) 0.55 x 10-4 (0.12) 0.31 x 10-3 (0.09)

0.59

X

10-4 (0.14)

0.48

X

10-3 (0.08)

REFERENCES Blanton, J. O. 1974. Some characteristics of nearshore currents along the north shore of Lake Ontario. J. Phys. Oceanogr. 4:415-424. Flint, R. W. 1986. Hypotherized carbon flow through the deepwater lake Ontario food web. J. Great Lakes Res. 12:344-354. Haffner, G. D., Giffiths, M., and Hebert, P. D. N. 1984. Phytoplankton Community structure and distribution in the nearshore zone on Lake Ontario. Hydrobiologia 114:51-66. Harris, G. P., Haffner, G. D., and Piccinin, B. B. 1980. Physical variability and phytoplankton communities: II. Primary productivity by phytoplankton in a physically variable environment. Arch. Hydrobiol. 88:393-425.

Hellebust, J. A. 1971. Kinetics of glucose transport and growth of eye/otella cryptica. J. Phycol. 1:1-14. Hobbie, J. E., and Wright, R. T, 1965. Competition between planktonic bacteria and algae for organic substrates. Mem. 1st. Ital. Idrobiol. 18 (Supp!.): 175-185.

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Overbeck, J. 1975. Distribution pattern of uptake kinetic responses in a stratified eutrophic lake (PlurBsee Ecosystem Study IV). Mitt. Internat. Verein. Limnol. 19:2600-2615. Palmer, E. G., and Togaski, R. K. 1971. Acetate metabolism by an obligate phototrophic strain of Pandorina morum. J. Protozool. 18:640-644. Pechlaner, R. 1971. Factors that control the production rate and biomass of phytoplankton in high mountain lakes. Mitt. Internat. Verein. Limnol. 19:125-145. Scavia, D., Laird, G. A., and Fahnenstiel, G. C. 1986. Production of planktonic bacteria in Lake Michigan. Limnol. Oceanogr. 31 :612-626.

HETEROTROPHIC GLUCOSE ASSIMILATION Schneider, R. P., and Wiley, W. R. 1971. Regulation of sugar transport in Neurospora crassa. J. Bacteriol. 106:487-492. Stadlemann, P., Moore, J. E., and Pickett, E. 1974. Primary production in relation to temperature structure, light condition and biomass at an inshore and offshore station in Lake Ontario. J. Fish. Res. Board Can. 31:1215-1232. Vaccaro, R. F., Hicks, H. E., Jannasch, J. H., and Carey, F. G. 1968. The occurrence and role of glucose in sea water. Limnol. Oceanogr. 13:356-360. Vincent, W. F. 1978. Survival of aphotic phytoplankton in Lake Tahoe throughout prolonged stratification. Int. Ver. Theor. Angew. Limnol. Verh. 20:401-406. _ _ _ _ , and Goldman, C. R. 1980. Evidence for algal heterotrophy in Lake Tahoe, CaliforniaNevada. Limnol. Oceanogr. 25:89-99.

163

Vollenweider, R. A. 1969. Ed. Primary Production in Aquatic Environments. IBP Handbook No 12. Blackwell Scientific Publications. _ _ _ _ ,Munawar, M., and Stadlemann, P. 1974. A comparative review of phytoplankton and primary production in the Laurentian Great Lakes. J. Fish Res. Board Can. 31 :739-762. Williams, P. J. 1970. Heterotrophic utilization of dissolved organic compounds in the sea. I. Size distribution of populations and relationship between respiration and incorporation of growth substrates. J. mar. bioi. Ass. U.K. 50:859-870. Wright, R. T., and Hobbie, J. E. 1966. Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology 47:447-464.