Hydrobiologia 164: 271-286 (1988)© Kluwer Academic Publishers. 271

Pelagic food web processes in an oligotrophic lake

Morten Sondergaard Ma, Bo Riemann2 , Lars Moller Jensen 3, Niels O. G. Jrgensen4 , Peter K. Bj0rnsen 2,

Michael Olesen2 , Jens B. Larsen 2 , Ole Geertz-Hensen 2 , Jan Hansen2 , Kirsten Christoffersen 2 , Anne-Mette

Jespersen 2 , Flemming Andersen2 & Suzanne Bosselmann 2

1Corresponding author: Morten S0ndergaard, Botanical Institute, University of Aarhus, 68 Nordlandsvej,DK-8240 Risskov, Denmark; aPresent address: Institute of Biology and Chemistry, University of Roskilde,P Box 260, DK-4000, Roskilde, Denmark; 2Freshwater Biological Laboratory, University of Copenhagen,

51 Helsingorsgade, DK-3400 Hillero0d, Denmark; 3International Agency for 14 C Determination, 15 Agern

Alle, DK-2970 H0rsholm, Denmark; 4 Department of Microbiology, Royal Veterinary and Agricultural

University, 21 Rolighedsvej, DK-1958 Frederiksberg C, Denmark

Received 17 November 1986; in revised form 19 August 1987; Accepted 20 October 1987

Key words: primary production, extracellular release, bacterial production, pelagic carbon budgets

Abstract

Major pelagic carbon pathways, including primary production, release of extracellular products (EOC), bac-

terial production and zooplankton grazing were measured in oligotrophic Lake Almind (Denmark) and in en-

closures (7 m3) subjected to artificial eutrophication. Simultaneous measurements at three days interval of

carbon exchange rates and pools allowed the construction of carbon flow scenarios over a nineteen day ex-

perimental period.The flow of organic carbon was dominated by phytoplankton EOC release, which amounted from 44 to

58% of the net fixation of inorganic carbon. Gross bacterial production accounted for 33 to 75% of the primaryproduction. The lower values of EOC release (44%7o) and bacterial production (33%) were found in the en-

closures with added nutrients. The release of recently fixed photosynthetic products was the most important

source of organic carbon to the bacterioplankton. Uptake of dissolved free amino acids was responsible for52 to 62% of the gross bacterial production. Thus, amino acids constituted a significant proportion of theEOC. Zooplankton (< 50 /xm) grazing on algae and bacteria accounted only for a minor proportion of the

particulate production in May. Circumstantial evidence is presented that suggests the chrysophycean algaDinobryon was the most important bacterial remover.

The results clearly demonstrated EOC release and bacterial metabolism to be key processes in pelagic carboncycling in this oligotrophic lake.

Introduction tion (Fuhrman & Azam, 1982, Riemann & Sonder-gaard, 1984) has enabled a better description and un-

The introduction of new methods to measure carbon derstanding of some important pelagic food webflux from phytoplankton to bacterioplankton via ex- processes. The quantitative, and to some extent thetracellular organic carbon (EOC) (Cole et al., 1982; qualitative, description of the pelagic marine scenar-Larsson & Hagstr6m, 1982; Bell & Kuparinen, 1984; io forwarded by Williams (1981) is currently under-Jensen & Sondergaard, 1985) and bacterial produc- going modification. Thus, biological interactions

272

and control mechanism at various trophic levels and,especially, the role of microheterotrophic organismsand sources of dissolved organic carbon for bacterialgrowth are presently major areas of research (e.g.Azam et al., 1983; Hobbie & Cole, 1984).

The activity of bacteria is related to primaryproduction and algal biomass (Hobbie & Rublee,1977). However, there is current some discussion ofthe potential importance of different carbon path-ways from algae to bacteria. Contrary to the resultsof Hobbie & Cole (1984), Fuhrman et al. (1980)found a better correlation between bacterial activityand algal biomass than algal primary production.The activity of zooplankton was considered to be thedominating DOC generating process in this study.Recently, Fuhrman et al. (1985) found evidence fora close coupling between light-forced (photosynthe-sis) and microheterotrophic processes on the shelfoff Southern California. Other investigations haveforwarded a combination of algal cell lysis and EOC(i.e. phytoplankton photosynthesis) to be of primaryimportance in the production of dissolved organiccarbon (DOC) (Cole et al., 1982; Larsson & Hag-strom, 1982; Riemann & Sondergaard, 1986a).Specific situations where EOC dominates totallyhave also been reported (Bell & Kuparinen, 1984;Lancelot & Billen, 1984; Sondergaard et al., 1985).

The carbon uptake by planktonic bacteria for avariety of waters range from about 20 to 80% of thephytoplankton primary production (Cole et al.,1982; Larsson & Hagstr6m, 1982; Riemann, 1983,Hobble & Cole, 1984; Lancelot & Billen, 1984; Fuhr-man et al., 1985; Riemann & Sondergaard, 1986a).The high carbon demand of planktonic bacteria em-phasizes the need to gain knowledge about thesources and pathways of DOC to bacteria.

At present, there is little evidence on which, if any,specific pathway dominates carbon flow in anyspecific environment; marine or freshwater, oligo-trophic or eutrophic. Riemann & Sndergaard(1986a) argued for a partly structural dependentpathway dominance in two eutrophic lakes. In agrowing algal population not subjected to any phys-iological stress, EOC dominated the bacterial sub-strates, whereas a dominance of zooplankton medi-ated DOC flow was observed in situations wherethere was grazer control of the phytoplankton bi-

omass (clear water phase). The total picture was,however, complex as all DOC generating processesare interacting, and further experiments are requiredbefore any generalizations can be made.

The purpose of this investigation was to study thebiological structure and the pelagic carbon processesin an oligotrophic lake. To allow continued samplingfrom a well defined water body and to study the ef-fects of artificial eutrophication, we used large en-closures, containing lake water manipulated with re-spect to fish content and nutrients.

Study site

The investigation was carried out in oligotrophicLake Almind, Denmark from 3 to 21 May, 1983. Theexperiments were partly repeated in September 1983.Lake Almind is a clear-water lake of moderate alka-linity (-0.5 meq 1-l). It is situated in mid-Jutlandin the outskirts of Silkeborg, does not receivewastewater and has a low nutrient content.Phytoplankton primary production is about70-90 g C m -2 year-'.

Information on lake morphology, nutrients, lightclimate and submerged macrophytes has beenpresented by Sand-Jensen & Sondergaard (1981).

Material and methods

Physico-chemical and biological variables weremeasured every three or four days both in the lakeand in six experimental enclosures fixed to a pon-toon bridge. The enclosures (1.5 m in diameter and4.2 m deep) consisted of 0.1 mm clear plastic bagsand contained about 7 m3 water each. The ex-perimental design, filling procedure and details ofthe manipulations are described elsewhere (Rie-mann & Sndergaard, 1986a). Briefly, bag no. 1served as a control, no. 2 was supplemented with in-organic phosphate and nitrate corresponding to anestimated consumption during a spring period, andno. 3 was supplemented with twice this amount ofnutrients (see Fig. 1). About 24 fish (50 g wet weighttotal of 1 + Perca fluviatilis (Perch)) were added toall enclosures in a parallel set-up in order to induce

273

e.a-

Mz

0

Z+D

&

May 1983

Fig. 1. Concentrations of phosphate and nitrate. Bars indicate+SD, n = 2.

predation on the macrozooplankton.The addition of fish did not change the growth

patterns of the phytoplankton or bacteria. This isprobably due to the low density of macro-zooplankton at the start of the experiment. In addi-tion the two nutrient treatments gave similarresponses. Consequently, the results of the experi-ments were treated in two sets i.e. with and withoutthe addition of nutrients.

All analyses and experiments were carried out us-ing water mixed from subsamples taken at 0, 1.5 and3 m depth. Unless otherwise stated, water was col-lected shortly after sunrise. Stratification did not de-velop in the enclosures during the investigation. Sur-

face quantum flux densities, transparency, nutrients,pH and total inorganic carbon were determined ac-cording to conventional standard methods.

Enumeration of bacteria was made by epifluores-cence microscopy (Hobbie et al., 1977). Cell numberswere converted to carbon biomass by multiplying theaverage cell volume of 0.09 lgm 3 (Riemann, 1983)and a carbon content of 0.35 pg C tm- 3 (Bratbak,1985; Bjornsen, 1986a).

Bacterial heterotrophic production was estimatedfrom 3 H-thymidine incorporation into TCAprecipitate (Riemann, 1984). An average conversionfactor of 1.1 x 1018 cells per mole was applied tocalculate production (Riemann et al., 1987). Thebacterial production was measured in three diel sam-ples. The total bacterial carbon uptake was estimat-ed by assuming thymidine incorporation to repre-sent the net biomass production and using thegrowth yield of 0.25 recently found for natural bac-terial assemblages (Newell et al., 1981; Bell & Kupari-nen, 1984; Linley & Newell, 1984; Bjornsen, 1986b).

Bacterial uptake and metabolism of dissolved freeamino acids (DFAA) followed the procedure out-lined in Jorgensen et al. (1983).

The phytoplankton biomass was measured aschlorophyll a extracted in methanol for 12 h withouthomogenization (Riemann & Ernst, 1982). Thecomposition and succession of phytoplankton weredetermined on fixed samples using an invertedmicroscope.

Particulate organic carbon (POC) was measuredby means of dry combustion of particles retained onGF/C filters followed by detection of CO2 by infrared gas analysis.

Phytoplankton primary production, extracellularrelease and bacterial EOC assimilation were meas-ured with the '4C-method and particle size separa-tion using 1.0 and 0.2 Itm pore size Nuclepore filters(Sondergaard et al., 1985). Presence ofpicophytoplankton passing the 1.0 Jtm filters waschecked under the microscope (Caron et al., 1985).No such organisms were observed in any of the sam-ples. The in situ incubations at the surface and at 1/2Secchi-depth were from sunrise to sunset. To estab-lish a depth-profile, two incubations including fivedepths were performed in the lake. All filtrationswere undertaken within two hours after incubation.

274

Prior to filtration the samples were stored cold indarkness whereafter they were filtered in randomorder. Dark values were subtracted from all fractionsbefore calculations.

The distribution of bacterial activity in size frac-tions > 1.0 um and < 1.0 > 0.2 tm was measured oneach occasion using several radioactive organic sub-strates, including glucose, fructose, amino acids andEOC from Microcystis aeruginosa (Sondergaard etal., 1985). We used the size distributions obtainedfrom the glucose experiments to complete calcula-tions of total bacterial net EOC uptake, Bn (Jensen& Sondergaard, 1985). Glucose was recommendedby Sondergaard et al. (1985).

Apparent release of EOC was estimated by adopt-ing a bacterial respiration (Br)' of 50% (Jensen,1985a); thus, apparent release is Bn + Br + EOCn,where EOCn is the amount in the dissolved fraction.The transport of EOC to bacteria is the ratioBn + Br/Bn + Br + EOCn, which reflects the labilityof the released products and the bacterial activity.Unless otherwise stated all results are presented perarea integrated over a 4.2 m deep water column.

Macro-zooplankton (> 140 lam) biomass was cal-culated from numbers and the mean dry weight ofthe dominating species. The dry weight (60 C, 24 h)was determined by weighing 100 individuals on anelectrobalance. The carbon content is assumed to be40% of the dry weight.

Zooplankton ingestion of algae and bacteria wasestimated using radiolabelled food sources. The in-gestion of bacteria was measured by means of 3 H-thymidine labelled bacteria using two size fractionsof the zooplankton; >140 tam and 50-140 tm(Bjornsen et al., 1986).

Macro-zooplankton (>140 m) ingestion ofsmall phytoplankters (< 50 m) was determined us-ing 4C-labelled algae collected in situ (Hansen,1985).

Results

The time-courses of most physico-chemical varia-bles were closely related to the addition of nutrientsand the enhanced biological processes (Figs. 1 and2). The water temperature increased from 8.5 at the

0

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4

0

2

4

0

2

3 6 9 12 15 18 21

May 198325

9

8

7

9

8

7

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Fig. 2. Time-courses of transparency and pH. Bars indicate+ SD, n = 2 and 4 for enclosures without and with addednutrients, respectively.

start to 13 °C on May 21. Stratifications did not de-velop and temperature differences between the lakeand enclosures could not be recorded.

With respect to nutrients, pH and transparency,the lake and the enclosures without added nutrientsshowed similar fluctuations around rather constantlevels (Figs. 1 and 2). Phosphate concentrations werelow (0-5 tag PO4-P 1-'), and were probably themost important factor limiting the biomass produc-tion of the phytoplankton. The addition of nutrientscreated a fast response in metabolic activity reflectedin the uptake of nutrients, an increase in pH and adecrease in transparency (Figs. 1 and 2).

The development of the physico-chemical varia-bles was tightly coupled to the development of theconcentrations of chlorophyll and particular organ-ic carbon (POC) (Fig. 3). The rather constant levelsin the lake and in the enclosures without addednutrients were contrasted by a five fold increase ofboth variables in the nutrient enriched enclosures.From May 3 to 18, the algal biomass increased from6 to 38 g chl. a 1-1 and then reached a plateau.The plateau level was probably controlled by silicatedepletion, as diatoms dominated the phytoplank-

Lake Almind

O O ° ° o --- "'- ° ~ o pH

Transparency

Enclosures without nutrients

o, ° ° -' o{ ppH

'"~ * · /t'--'-'-? jTransparency

Enclosures added nutrients - " pH

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275

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00

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May 1983

18 21 25

Fig. 3. Time-courses of particulate organic carbon (POC)chlorophyll a. Bars indicate + SD, n = 2, 4 and 8 for POC meurements in the lake, enclosures without and with adnutrients, respectively; n = 4 and 8 for chlorophyll a measiments.

ton. Unfortunately, silicate was not measured.The composition of the phytoplankton popu

tion in the lake (and all enclosures) was initially raler diverse with Fragillaria and Dinobryon domining both numerically and in biomaStephanodiscus, Phagus and Mallomonas were a]important species.

During the experiment, the numbersDinobryon increased from about 0.93.0 x 106 1- ' in the lake and all enclosures and came the dominant (based on biomass) species in tlake and enclosures without added nutrients. Fraglaria became the dominant species in the enclosewith a nutrient addition and its number increas5 fold from about 2 to 10 x 106 cells 1-1. Fragilla,was responsible for 60- 70% of the total biomass

the nutrient enriched enclosures on May 18.At the start of the experimental period, the size

20 distribution of '4 C-fixation in light was dominatedby particles between 3 and 8 m, and they accounted

10 for about 50% of the total fixation (Fig. 4). This sizedistribution remained constant in the lake and the

0 enclosures without added nutrients throughout thestudy. The artificial eutrophication started aprogressive development towards dominance by

20 larger particles. On May 21 about 50% of the carbonfixation took place in particles > 40 Jim in the nutri-

10 ent enriched enclosures (Fig. 4).e2 No pattern could be determined in the develop-> Z ment of the bacterial biomass (Fig. 5). The cell

counts were performed to the 95% confidence level,so statistically significant differences were obtained.However, the only constant trend with time was adoubling of the biomass in the nutrient enriched en-

40 closures (the lower level) where fish were present(Fig. 5).

20o The biomass of the macro-zooplankton was ini-tially low (35 tjg C 1-1) but increased with time

0 (Fig. 6). From the difference between the biomass inthe lake and the enclosures, it is clear that we did not

and succeed in getting the macro-zooplankton popula-aas- tion in the lake into the enclosures. The absence ofeas-ded any measurable effects on the algae by the additionure- of fish is most likely explained by the low density of

macro-zooplankton and, consequently, low grazingpressure on the algae. Any significant changes inzooplankton biomass would take longer to developthan the time covered by the experiment. Selective

la-th-I.t_ 80 r I AD mi n Ms a h1

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Iso

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`Z

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>40 <40>20 <20>8 <8>3 <3>1 <1 0.2Pore size (ym )

Fig. 4. Particle size distribution of DI' 4C-fixation. Bars indicate

+ SD, n = 4, for the period May 3 to 21. The measurement in theenclosure added nutrients was on May 21.

Lake Almind

0 0- 0 ° 00 °'oChlorophyUll

Enclosures without nutrients

. _._.od+ POC

°.... , ~ , l _, ~ Chlor ophyll

Enclosures added nutrients

rophyll

Lwe tmlno, veruage nay -Z1 I

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May 1983

Fig. 5. Bacterial biomass in Lake Almind and all enclosures.

predation by fish on the larger zooplankton specieswas, however, recorded (data not shown).

The development of the phytoplankton biomassis a function of the primary production and loss

E

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a

10

3 6 9 12 15 18 21May 1983

Fig. 6. Macro-zooplankton biomass (>140 /rm). Control =enclosure without treatment. Nutrients = enclosure with added2 x (N + P).

May 1983

Fig. 7. Incident quantum flux and phytoplankton net primaryproduction ( 4 C-fixation in algae + Bn + EOCn). Bars indicate

+ SD, n = 2 and 4 for enclosures without and with nutrients,respectively.

rates. The daily net carbon fixation(particles + EOCn) is shown in Fig. 7. The minorfluctuations in the lake and in enclosures withoutadded nutrients were predictable from the constantalgal biomass and light data. In the enriched en-closures, primary production peaked aroundMay 12-15, about six days before the biomass peak.The net fixation then rapidly decreased. The carbonfixation per jg chlorophyll and light was about 0.3on May 21 compared with values around 1 at all oth-er dates. The photosynthetic potential of the popu-lation, thus, clearly dropped.

In all measurements of phytoplankton primaryproduction, the release of EOC could account for asubstantial fraction of the carbon fixation and theEOC release rates showed distinct patterns related tothe experimental treatments. Initially, EOCn and

Lake Almind

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o Fish

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Enclosures added 2(NP)

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277

apparent release (Bn + Br + EOCn) were about 20and 45% of the net fixation, respectively (Fig. 8A).In the lake and enclosures without nutrients, bothvariables increased with time and reached 40 and60%. The opposite pattern was found where therewas nutrient enrichment. Here EOC n and apparentrelease decreased steadily to about 10 and 25 %7o of thetotal carbon fixation (Fig. 8A), although the actualvalues were still higher than in the unenriched sam-ples. Differences in percent EOC release as a func-tion of incubation depth were not observed.

Lake water enrichment and the addition of fish in-duced a relatively lower apparent EOC release in theSeptember experiment (Fig. 8B). In the control en-closure not subjected to any treatment, apparent

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EOC release fluctuated between 40 and 58%. Con-trary to the May experiments, the addition of fish inthis series of experiments was immediately succeed-ed by an accumulation of algal biomass suggestingthat nutrient release by the fish acted as a controllingfactor. As in May, algal biomass accumulation andthe decrease in the percent EOC release occurredsimultaneously.

The transport of EOC to bacteria exhibited twodifferent patterns. In the enriched enclosures, trans-port remained constant around 50%, whereas itdecreased to 20-30% in the two other systems(Fig. 9). These differences in development of bac-terial EOC uptake were paralleled by a change in themolecular weight distribution of EOCn. In the lake

B1uU

80

S 600

O 40

20' 20M:

3 6 9 12 15September 1983

19 22

May 1983

Fig. 8. Phytoplankton release of EOC expressed as percentages of carbon fixation and calculated as outlined in the text. A: experimentsin May, bars indicate SD, n = 2 and 4 for enclosures without and with nutrients, respectively. B: Experiments in September, where Controlis an enclosure without any treatment.

Enclosures in Lake Almind

** Control

x ' °~ +Fish

N+P

U I. v

.

278

(and enclosures without nutrients), the contributionof small molecules (< 700 Daltons) increased from52 to 62% of the total and the intermediate sizedmolecules (700 to 5 000 D) decreased from 32 to 19%of the total over the course of the experiment. In theenriched enclosures the distribution of moleculesdid not change with time and remained similar tothat presented in Table 1. We interpret the relative in-crease of small molecules in the EOC pool to be dueto a lower bacterial EOC uptake as seen in thedecreasing transport values in the latter half of thestudy period (Fig. 9).

The net bacterial production (Bp) estimated bymeans of 3 H-thymidine incorporation showed aconstant pattern with nearly similar values under allexperimental conditions (Fig. 10). From a low of10 mg C m - 2 24 h-' on May 3, Bp peaked at about40 mg C m - 2 24 h- l on May 15. After May 15, Bpagain decreased in all the enclosures, but remainedat a higher level in the lake.

Net bacterial uptake of EOC (Bn) is plotted alongwith Bp in Fig. 10. Although the time-courses of Bnwere not as distinct as those for Bp, Bn and Bp wereof similar magnitude in the lake and in enclosureswithout added nutrients. The enrichment created asituation which separated the values of Bn and Bpdramatically (Fig. 10). In these enclosures Bn

peaked about three days before Bp and with valuestwice as high. Only on May 18 and 21 did the twomethods give similar results. B was only weakly

Table 1. Molecular weight composition' of EOCn in LakeAlmind and enclosures. Incubation from sunrise to sunset. May1983.

Site Distribution (7o)

Date > 100.00 Intermediate <700Daltons Daltons

Lake May 3 16 32 52Lake 12 14 29 57Lake 28 19 19 62Enclosure with

nutrients May 122 18 32 50

Sephadex G-50 (Sondergaard & Schierup 1982);.2 Identical distributions were found May 3 and 21.

2

o

gt

-0C

2

Enclosures added nutrients60 -

60

03 6 9 12 15 18 21

May 1983

Fig. 9. Transport of EOC to bacteria. Bars as in Fig. 8.

correlated with Bn (r = 0.34) and primary produc-tion (r <0.3).

Contrary to the pattern with Bn, the time coursesof net DFAA uptake closely paralleled those of Bpwith a peak on May 15 (Fig. 11). It is, however, in-teresting that with the exception of the enriched en-closures, bacterial net carbon uptake from aminoacids was about twice as high as the estimated Bp's.There were no significant differences in DFAA as-similation between the lake and the enclosures.

The ingestion of smaller algae (< 50 /m) by themacro-zooplankton basically followed the develop-ment in zooplankton biomass (see Fig. 6). Themeasured ingestion of bacteria followed alsozooplankton biomass development. Bacterial graz-ing was due mainly to the macro-zooplankton andaccounted for 70 to 80% of the ingestion by thezooplankton size fraction > 50 Am. However, the in-gestion of bacteria by flagellates (including

60 Lake Almind

20

Enclosures without nutrients

4F~~~~~t-*+\~~~~,

20~~~ *-**

6

4

4

279

a

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EE

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ia

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11C)

May 1983

Fig. 10. Net bacterial production (Bp) and net bacterial uptakeof EOC (B,). Bars as in Fig. 8.

E

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b Y 17 l l LI

May 1983

Fig. 11. Net bacterial uptake of dissolved free amino acids. Barsas in Fig. 8.

Dinobryon) and other smaller organisms (< 50 Atm)was not measured and a major part of the bacteriagrazing is, thus, probably not accounted for.

The above results give some key values for a quan-titative presentation of the processes involved in theorganic carbon pathways. To produce a comprehen-sive picture of these pathways the rate measurementswere integrated into single values covering the ex-perimental period of 19 days (Table 2). The develop-

Table 2. Comparison of primary production, extracellular release and bacterial activity in Lake Almind and experimental enclosures.Values integrated for the period May 3 to 21, 1983 and a 4.2 m deep water column. Bn = bacterial net uptake of EOC; EOC, =EOC in the dissolved fraction; Bp = net bacterial production; DFAA = net bacterial uptake of amino acids.

Site Net carbon Bn EOC, Bp DFAA Apparent EOCfixation

carbon fixationmg C m- 2 070

Lake 3542 612 750 662 900 56Enclosures without nutrients 2 3032 ± 300 442 ± 67 872 ± 49 500 +± 145 720 + 145 58Enclosures added nutrients3 7141±770 1081±65 1015±98 590+ 65 953+100 44

Bacterial respiration of EOC included (50°70 respiration).2 Means SD, n=2.3 Means SD, n=4.

-A 2

280

ment of carbon budgets for the lake and the en-closures requires a set of assumptions and will befully treated in the discussion section.

An apparent major discrepancy emerged from themeasured rates of bacterial activity and production.The net production (Bp) was lower than the bacteri-al net uptake of both EOC and DFAA (Table 2). Inthe discussion we will show that this discrepancy isprobably due to the methods employed.

Discussion

As expected, the addition of nutrients created eu-trophication with an algal bloom and profound ef-fects on physico-chemical variables. Due to the lowconcentration of macro-zooplankton in the en-closures, the effects of the fish additions on thezooplankton were insignificant. However eutrophi-cation did change the physiological behaviour of thephytoplankton with respect to the release of extracel-lular organic carbon and the experiments created anopportunity to construct carbon budgets based onsimultaneous measurements of pelagic processes. Inthe discussion below, we have concentrated on theenclosure experiments.

The major pathway of organic carbon was the

detrital food web. The build-up of phytoplanktonbiomass in the enriched enclosures would ultimatelydecompose. The carbon-14 experiments and theresults from differential filtration showed that theapparent release of EOC comprised from 44 to 56%of the daily carbon fixation (Table 2). The inducedchanges with eutrophication were followed by a rela-tive decrease of EOC release to about 25% of the to-tal carbon fixation in both May and September, butwith higher absolute values in the enriched en-closures (Fig. 8). There was a shift towards Fragillar-ia and other larger species at the end of the studyperiod, although Fragillaria and Dinobryonpredominated during the entire experiment. Thechanges in species dominance were probably physio-logical responses to nutrient availability. A similarresponse of single species in relation to nutrient con-centrations has been found by Lancelot (1983) in astudy of marine environments.

The high relative release of EOC in oligotrophic

environments is a dominating feature of thephytoplankton (Cole et al., 1982; Chrost, 1983; Bell& Kuparinen, 1984) compared with most resultsfrom eutrophic lakes (Riemann et al., 1982; Sonder-gaard et al., 1985; Sondergaard & Jensen, 1986). Inoligotrophic environments the application of tradi-tional primary production measurements which in-clude only 14C-fixation into particles, would un-derestimate primary production from 30 to 50%,whereas less than a 10% underestimation would oc-cur in most eutrophic environments.

The biological processes in the pelagic were domi-nated in this study by photosynthesis and bacterialmetabolism of DOC. An evaluation of sources andcomposition of DOC must include accuratemethods to measure bacterial production and car-bon uptake. In addition, all methods should be com-parable. The apparent discrepancies between bac-terial activity measured as Bp, Bn and net DFAAuptake might be due to errors inherent in each meth-od, but also to the fact that each method emphasizesspecific bacterial characteristics. Thus, direct com-parison of results from the different methods asshown in Table 2 might be misleading due to differ-ences between the methods.

The reliability of differential filtration and the'4C-method as a tool in the assessment of a directcarbon flow from photosynthesizing algae to thebacterioplankton has recently been discussed byCole et al. (1982), Jensen & Sondergaard (1985) andS0ndergaard et al. (1985). Results from both oligo-trophic (Cole et al., 1982; Bell & Kuparinen, 1984)and eutrophic lakes (Riemann & Sndergaard,1986a) have not disproven the reliability of differen-tial filtration. However, the specific activity of EOCis still unknown and calculations based on '4 C incu-bations shorter than the time required for four celldivisions are likely to underestimate the true releaserates (Jensen, 1985b; Jensen et al., 1985). The magni-tude of error is largely unknown, but is most proba-bly low when EOC is dominated by small molecules(Jensen et al., 1985), as was found in this investiga-tion (Table 1).

Release of EOC is mainly, but not only, a light-dependent process as release of radiolabelled organ-ics fixed in a previous light period can be detectedin a following dark period (Mague et al., 1980;

281

Sondergaard et al., 1985). Previous diel experimentsin Lake Almind showed a continuous release in dark-ness, although at a lower rate (S0ndergaard et al.,1985), so the presented values of apparent EOC areminimum estimates.

The presence of picophytoplankton passing the1.0 tm filters would lead to an overestimation of Bnand consequently Br. We found no chlorophyll con-taining organisms in the 0.2-1.0 Am fraction, butpicophytoplankton just below 1.0 tm in diameterhas been recorded in Lake Almind in June, July andAugust 1985 (Sondergaard, unpubl. results) and inother lakes (e.g. Craig, 1984; Caron et al., 1985). Theinfluence of picophytoplankton on results obtainedwith differential filtration might vary seasonally andshould always be monitored.

The apparent release of EOC includes the dis-solved fraction of EOCn which is not utilized by thebacteria during an incubation. It has been shownthat about 75% of EOCn is metabolised within afew days (Herbland, 1975; Iturriaga & Zsolnay, 1983;Watanabe, 1984). Thus, the carbon source for bac-terial uptake should include EOCn as in the studiesby Larsson & Hagstr6m (1982), Kato & Stabel (1984)and Lancelot & Billen (1984). The theoretical esti-mate of the bacterial carbon supply from EOC isnow: Bn + B, + (EOCn x 0.75). These values arepresented in Table 3.

To convert bacterial net production (Bp) to totalbacterial carbon demand (Bg), we have used the re-cently established growth yield factor of 0.25 for nat-ural populations. The use of higher values (0.4 - 0.6)in earlier investigations does not seem appropriateas they are found on specific substances in short-term incubations. However, the respiration of EOCand DFAA are set at 50% as evidenced by recentstudies of their assimilation and metabolism(Watanabe, 1984; Jensen, 1985a; Jorgensen, 1986,1987). The use of these growth yield values andEOCn availability to calculate bacterial carbon up-take and sources gives ecologically 'reasonable'results in that - except for EOC in the enclosureswith added nutrients - overall bacterial carbon up-take (Bg) is higher than or equal to the estimatedEOC and measured DFAA uptakes (Table 3).

The estimated values of total bacterial carbon de-mand are sensitive to variations in bacterial cell vol-

Table 3. Theoretical values of total bacterial carbon uptakeand organic carbon available as EOC and DFAA, calculated forthe period May 3 to 21 and a 4.2 m deep water column.

Site B: 0.25 (Bn:0.5) + DFAA:0.5(EOC n x 0.75)

mg C m- 2

Lake 2648 1780 1800Enclosures without

nutrients 2000 1540 1440Enclosures added

nutrients 2360 2920 1906

ume (biomass) and the 3 H-thymidine conversionfactor. The observed average cell volume is in thelower range of published values (Riemann & Sonder-gaard, 1986b) and the conversion factors in fresh-water are often higher (1.5-2.2 x 1018) than the1.1 x 1018 cells per mol used here (e.g. Bell & Kupar-inen, 1984). Consequently, the reported Bp and Bgmost likely represent minimum values. However,current knowledge concerning bacterial biomass,growth yield and production is rather volatile withnew suggestions of correction factors frequently ap-pearing. An upward adjustment of Bp might provereasonable in the near future, but seems prematureat the moment.

In a series of diel studies considering carbon flowfrom algae to bacteria and bacterial activity, Sonder-gaard et al. (1985) also observed that bacterial netproduction estimated by 3 H-thymidine incorpora-tion on several occasions was lower than Bn andDFAA net uptake. The incomparability of overallbacterial growth yield and the respiration rates ob-tained in short-term radiotracer studies may offer anexplanation for this difference. The similar time-course patterns of B and DFAA assimilation(Figs. 10 and 11) revealed a close relationship be-tween these two variables. Amino acids were a DOCsource involved in the control of bacterial activity,the DFAA source being either EOC or phytoplank-ton decomposition (Jorgensen, 1986, 1987).

The carbon ingested by the zooplankton is dis-tributed in biomass production, respiration, fecalmaterial and excretion, the latter including 'sloppyfeeding'. We have used the ratios 0.32, 0.38, 0.2 and

282

Fig. 12. Constructed carbon flux scenarios based on assumptions outlined in text. All values in g C 1-1 19 days-' averaged for a 4.2 mdeep water column. Values inserted in different pools represent measured or calculated net accumulations. Broken lines are tentative path-ways; for the algal population it is cell lysis, leaching from zooplankton feces (F), macro-zooplankton and algal grazing of bacteria, andbacterial uptake of zooplankton generated DOC.

0.1, respectively to describe these pathways (see Lam-pert, 1978; Riemann et al., 1982). The value for netbiomass production was verified by the biomass in-crease in the enclosures (Fig. 6).

From the available values for primary production,bacterial production, ingestion of algae and bacteriaby zooplankton and the application of the above as-sumptions concerning EOC utilization, bacterialgrowth yield and zooplankton metabolism make itpossible to construct crude carbon flow budgets.The resulting scenarios, recalculated to average

values per litre for the experimental period of19 days are presented in Fig. 12. Comparative valuesof carbon sources and sinks are summarized in Ta-ble 4.The constructed budgets are balanced with respectto bacterial carbon demand. Thus, in the lake andthe unenriched enclosures where the supply of car-bon from EOC and zooplankton activity does notmeet the bacterial demands, a hypothetical carbonsource from algal lysis was inserted. The carbon me-tabolism of the bacteria accounted for from 66 to

Table 4. Comparison of carbon fixation, bacterial activity and zooplankton ingestion. Based on values from Tables 2 and 3.

Site Bg I BEOC2 DFAAg 3 Ingestion of algae Ingestion of bacteria

Carbon fixation Bg Bg Carbon fixation Bp

Lake 75 67 68 22 30Enclosures without nutrients 66 77 72 10 13Enclosures added nutrients 33 124 81 8 9

I Bacterial gross production = Bp:0.25.2 Bacterial carbon from EOC, see Table 3.3 DFAA gross uptake.

ENCLOSURES WITHOUT NUTRIENTS ENCLOSURES ADDED NUTRIENTSLAKE ALMINDlDC

283

75% of the daily carbon fixation in the lake and theunenriched enclosures. The basic carbon budgets inthese two cases were almost balanced. The apparent'steady state' of algal biomass (chlorophyll) andPOC was verified by the rate measurements. Only aminor part of the particulate production would sedi-ment during this period. In the enriched enclosures,the detrital foodweb and zooplankton grazing ac-counted only for 33 and 8% of the carbon fixation,respectively (Table 4). This is in accordance with themajor build up of algal biomass and POC. However,the measured POC increase of about 1500 ig C 1-

(Fig. 3) was about twice the value emerging from the'4C measurements (Fig. 12). The 4 C-method ap-parently underestimated the net primary produc-tion, but at present we cannot forward a comprehen-sive explanation for such a dramatic differencebetween the enriched and the unenriched condi-tions.

Extracellular organic carbon released by thephytoplankton provided the basis for the major partof the bacterial carbon uptake (67 to 124%) (Ta-ble 4). Values above 100% are, of course, theoretical-ly impossible. However, considering the many as-sumptions applied to the estimates of both bacterialproduction and EOC uptake, the values seemreasonable in an ecological context.

The results obtained in September were similar tothose in May and emphasize the reliability of the ob-servations concerning precision but not necessarilyaccuracy. In September, the carbon demand of thebacterioplankton ranged from 20 to 55% of the au-

Table 5. Comparison of carbon fixation, bacterial activity andEOC flux in enclosure experiments in Lake Almind, September3 - 22, 1983. Abbreviations and assumptions as in Table 4.

Site Carbon fixation Bg Bn BEOCmg C m- 2

Carbon Bp Bgfixation

070

Untreated control 1863 55 137 90Fish 5816 44 91 68Nutrients 6392 29 102 80Fish + nutrients 7456 20 178 126

totrophic fixation (Thble 5). The lower values werefound in situations with increasing phytoplanktonbiomass, as in the May study. The importance ofEOC for the bacteria was likewise high in Sept. (68to 126%). The one value significantly above 100%was, as in May, found in the enclosure (fish andnutrients) where the algal population increased andremained at a high level (30 /Ag chl. a 1-1) through-out the study. Separate additions of fish andnutrients also increased the biomass to about 30 Agchl. a I-, but during the last week of the three weekstudy, the biomass dropped by about 66%. Thesimilarity of both series of experiments could berelated to the same unknown systematic error. TheBEoc:Bg values above 100% suggest that one orseveral of the assumptions are incorrect. But thisshould not influence the relative differences betweenbacterial carbon utilization and phytoplanktonproduction, which are related to the biological struc-ture and nutrient availability. It must, however, beemphasized that all presented results are based onstatic assumptions concerning bacterial cell volume,growth yield, thymidine conversion factor and EOCutilization. These values might vary as a function ofgrowth rates and nutrient regime.

The contributions of EOC to bacterial carbon re-quirements found in this study are all at the upperend of the range from 13 to about 100% previouslyrecorded in freshwater and the sea and in both oligo-trophic and eutrophic situations (Cole et al., 1982;Larsson & Hagstr6m, 1982; Bell & Kuparinen, 1984;Brock & Clyne, 1984; Lancelot & Billen, 1984; Rie-mann & Sondergaard, 1986a). Although the empiri-cal data are scarce, there seems to be a trend forgreater EOC importance in oligotrophic than in eu-trophic areas (Cole et al., 1982; Bell & Kuparinen,1984). However, seasonal variations related to thedevelopment in biological structure e.g. the impactfrom zooplankton and algal lysis can be expectedand could change the ecological importance ofEOC. Thus, Riemann & Sondergaard (1986a) foundthree different situations, in which EOC, algal lysisand zooplankton activity, respectively, were the sin-gle most important contributor of carbon to the bac-terioplankton. In eutrophic Lake Hylke the impor-tance of EOC peaked (60%), when the algal biomasswas increasing and there was no major impact from

284

zooplankton grazing (Riemann & Sondergaard,1986a). The results from this oligotrophic lake sup-port the earlier findings.

The bacterial uptake of amino acids was almostequally as important as EOC (Table 4). Consequent-ly, a significant fraction of the released productsmust have been amino acids. Previous investigationshave shown that amino acids can comprise from 5to 32070 of EOC in natural waters (Mague et at, 1980;Jorgensen et al., 1983; Jorgensen, 1986b; Sonder-gaard & Jorgensen, unpubl. results). Taking into ac-count the high bacterial affinity for amino acids(Jorgensen & Sondergaard, 1984) most of thereleased amino acids are probably metabolized dur-ing an incubation and are recovered in the fractionsBn and Br where no chemical identification is possi-ble.

The net bacterial production has to either ac-cumulate as biomass or be lost through grazing andsedimentation. The grazing of bacteria by zooplank-ton larger than 50 Atm accounted for only about 9 to14% of the net production in the enclosures andabout 30% in the lake (Table 4). As the bacterial bi-omass did not increase (Fig. 5) grazing by small or-ganisms (e.g. flagellates) was probably important.Evaluation from microscopic examinations using adouble-counting technique (Davis & Sieburth, 1982)showed the number of heterotrophic flagellates to beinsignificant (J. B. Larsen and J. J. Cole, unpubl.results). Bacterial grazing by Dinobryon has recentlybeen reported by Bird & Kalff (1986) and Dinobryoncan be suggested as the most important bacteriaremover. Bird & Kalff (1986) found that an algal cellconsumed about 3 bacteria every 5 minutes or in-gested 30% of its weight in bacteria per day. Basedon this information, the population of Dinobryoncould ingest between 190 and 414 Atg C 1-1 duringthe experimental period. The higher value occurswhen 3 bacteria per 5 min is used to estimate con-sumption. We suggest, therefore, that the surplus ofbacterial production of 77 to 102 tag C -1' (Fig. 12),which does not accumulate as biomass (Fig. 5), isutilized by Dinobryon.

During this investigation, the detrital foodwebdominated the organic carbon flow in the lake andin the unenriched enclosures, where bacterial metab-olism accounted for some 50-70% of the photosyn-

thetic carbon input (Tables 4 and 5). Hobbie & Cole(1984) found that 31% of the primary productionwas metabolised by the bacterioplankton in seawaterenclosures. This value did not change with increasedphytoplankton production. The bacterial carbonuptake was calculated with a 50%7o growth yield. Inthe nutrient enriched enclosures of our study, thebacterial production did not immediately respond toa higher primary production and only 20 to 30% ofthe current primary production was channeleddirectly to the bacteria (Tables 4 and 5). However,viewed over a longer period, the phytoplankton bi-omass would decompose and create a major inputof DOC available to bacteria (Cole et al., 1984; Han-sen et al., 1986) triggering a higher bacterial produc-tion.

In the present investigation, bacterial activitymediated by phytoplankton release of EOC was themain carbon pathway.

Acknowledgements

We appreciate the suggestions offered by J. J. Cole,C. Howard-Williams, W. Vincent, and K. Richard-son. The valuable technical assistance byB. Philkjer, J. Bargholz, W. Martinsen and R. Ar-thur is acknowledged. This study was supported bythe Danish Natural Science Research Council andthe Freshwater Laboratory, National Agency of En-vironmental Protection. Nutrient analyses werekindly carried out by the Freshwater Laboratory.

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