MARINE ECOLOGY - PROGRESS SERIES Published March 21 Mar. Ecol. Prog. Ser. l

Bacteria as a food source for microzooplankton in the and Baltic Sea with special reference to ciliates*

V. Gast

Institut fiir Meereskunde an der Universitat Kiel. D-2300 Kiel, Diisternbrooker Weg 20. Federal Republic of Germany

ABSTRACT: In situ investigations revealed that number and biomass of microzooplankton increased with eutrophication along the length of the Schlei Fjord (FRG).The same observation was made for total bacterial numbers and biomass. Microzooplankton of the Schlei and total bacterial numbers showed a minimum in winter and major periods of development in late summer/autumn and spring. Usually the microzooplankton biomass in the Schlei was greater than the bacterial biomass. In contrast. the bacterial biomass for 5 of 6 stations in the Baltic Sea surpassed that of the microzooplankton during summer. Number and biornass of microzooplankton in both bodies of water can mostly be attributed to protozoans of the 3 to 30 pm fraction. Determined with the aid of radioactively labelled bacterial cultures, the filtration rate of 'natural' microzooplankton populations exhibited a distinct dependency on microzooplanktonic biomass and water temperature. In March 1982 microzooplankton populations in the eutrophic Schlei Fjord filtered 5 to 58 % of the water per day. In the central Baltic Sea in August 1982 the rate was 70 % d-' during the late stage of a decaying blue-green algae bloom. Laboratory experiments with Uronema marinurn clearly showed that bacteria concentrations exert a considerable influence on the development of ciliates. Only when a limiting concentration of about 1 X 106 bacteria ml-' is attained does a proliferation of ciliates commence. Hence, bacteria can represent an important food source for microzooplankton, especially in biotopes with a high bacterial number and biomass.

INTRODUCTION Consumption of bacteria is primarily attributed to microzooplanktonic organisms. The term 'microzoo- Along with particulate organic material, hetero- plankton' refers to organisms less than 200 pm in size. trophic aquatic bacteria chiefly utilize dissolved It includes small metazoans, but for the most part organic compounds for cellular energy requirements comprises heterotrophic protozoans such as flagellates as well as for the formation of bacterial biomass. This and ciliates (Beers & Stewart 1971, Takahashi & Hos- bacterial biomass can be utilized by filtering organ- kins 1978, Heinbokel & Beers 1979). The microzoo- isms as a food source. Through this process dissolved plankton filters particles in the pico- and nanoplank- organic material is introduced into the food web. ton range especially effectively (Capriulo & Carpenter The major fraction of pelagic bacteria in the western 1980), while for larger metazooplankton, bacterial Baltic Sea is free-living; attached bacteria generally nourishment is rather unimportant (Marshal1 1973, account for less than 10 % of the total bacterial number Nival & Nival 1976). (Zimmermann 1977). An enormous concentration of Numerous studies have shown that ciliates and bacteria may, however, develop in microbiotopes such flagellates can be grown in cultures with different as the mucous sheaths of blue-green algae or in decay- bacteria as nutrient (Fenchel 196813, Hamilton & Pres- ing phytoplankton blooms (Rieper 1976, Hoppe 1981). lan 1969, Lighthart 1969, Tezuka 1974, Ashby 1976, Berk et al. 1976, Haas & Webb 1979, Fenchel 1982a, Kracht 1982). Excerpts from the thesis of the same title were completed Laboratory experiments on the decomposition of under the supervision counselling of Professor Dr. G. Rheinheimer, Institut fiir Meereskunde an der Universitat plant material and detritus led to a typical succession Kiel, Federal Republic of Germany of organisms, whereby protozoan development com-

O Inter-Research/Printed in F. R. Germany 108 Mar. Ecol. Prog. Ser. 22: 107-120, 1985

mences following a strong proliferation of bacteria. This effects a considerable reduction of the bacteria (Harrison & Mann 1975, Fenchel & Jsrgensen 1977, Linley & Newel1 1981, Stuart et al. 1981, Robertson et al. 1982). These experiments suggest a special impor- tance of protozoans as consumers of bacteria also under natural conditions. Hence, it is necessary to obtain more information concerning the transfer of bacterial biomass to primary consumers (Rheinheimer 1981). In light of these considerations, data on the abun- dance and distribution of microzooplanktonic organ- isms, especially ciliate fauna, were recorded and their biomass estimated. Concentrations and biomass of bacteria were calculated, and the filtration rates of 'natural' microzooplankton populations measured Fig. l. Location of stations in Schlei Fjord. l Schleimiinde: N 54",40,l'; E loo,3,4'; 2 : N 54",39,6'; E go,56,2'; 3 using a radioactively labelled bacterial culture. These Lindaunis: N 54", 35,O';E go,49.2'; 4 Missunde: N 54", 31.7'; investigations were conducted from June 1981 to E go, 43.1'; 5 GroDe Breite: N 54O, 31.2';E go,40,2'; 6 Kleine October 1982 in the strongly eutrophic Schlei, a Baltic Breite: N 54', 30,s';E go,34,7' fjord polluted by municipal and rural sewage sources. A cruise to the central Baltic undertaken in August ature, only slightly in the Bornholm Basin and south of 1982 supplied data from an unpolluted brackish water the Gotland Deep, and remained almost constant with area. The influence of bacterial concentrations on cili- depth in the Gotland Deep proper. In the central Baltic ate growth was studied in laboratory experiments with region, horizontal and vertical salinity differences Uronema marinurn. remained relatively small (7.3 to 8.1 %). In contrast, the

salinity in the Fehmarn Belt at 2 m depth was 18.4 %O and increased to 24.3 %o at 20 m depth. MATERIALS AND METHODS ZoBell water samplers or 2 1 containers constructed for sterile water sampling were employed for mi- Area of investigation and sampling. The Schlei is one crobiological studies. Nansen samplers were used for of 4 of the Kiel Bight. A narrow, man-made other analyses. Samples were taken at a depth of 1 m in opening of the Schlei permits the only exchange of the Schlei and at 2, 10 and 20 m in the Baltic. The water with the Baltic Sea. The fjord measures about 40 qualitative assessment of the phytoplankton standing km from its outlet to the town of Schleswig. The salin- stock was made with 20 pm and 50 pm plankton nets, ity of the Schlei shows a sharp decline from the mouth trawled for several minutes. Usually, sampled mate-

region (14 to 20 %) to Schleswig (2 to 5 9/00).Eutrophica- rials were processed on board immediately following tion steadily increases from the fjord's mouth towards their retrieval. In a few cases, water samples obtained Schleswig (Rheinheimer 1970). High inorganic nut- from a small pier were transported in insulated con- rient content leads to considerable phytoplankton tainers to the laboratory and processed there within 3 h. blooms. In the period from summer to autumn the Bacteriological parameters. Saprophyte counts were bloom is dominated by the mass development of Mi- determined with the pour plate method using 2 mod- crocystis. Schlei Fjord studies were conducted at 5 ified ZoBell nutrient media 2216 E (Oppenheimer & stations between Kappeln and Schleswig and an addi- ZoBell 1952) consisting of 0.5 % peptone, 0.1 % yeast tional station directly off the Schlei outlet (Fig. 1). extract and 1.5 % agar in tapwater (0 %o = ZL) and in Eleven samplings of the fjord were undertaken from brackish water (8 %o = ZB). The fluorescence micro- June 1981 to October 1982. scopical assessment of the total bacterial number The studies in the Baltic Sea were conducted from 16 (TBN) was conducted according to Zimmermann et al. to 21 August 1982 during a cruise by the Department of (1978). Bacterial biomass (BBM) was deduced from size Marine Microbiology at the Institute of Marine Sci- categories and cell volumes as described by ences in Kiel (Fig. 2). At the time of investigation warm Rheinheimer (1977). The volume of bacteria with a summer temperatures of 15.6 to 17.9 'C were recorded specific weight of ca. 1 can be equated with mass (= in the upper 10 m for the Baltic stations. However, in wet weight). The dry weight of bacterial cells is the Fehmarn Belt and Arkona Basin a temperature assumed to be 20 % of the wet weight, and the carbon reduction of 7 C" was recorded at 20 m depth. The high content comprises about 50 % of the dry weight temperature decreased, relative to the surface temper- (Schlegel 1981). Gast: Bacteria as a food source for microzooplankton 109

Microzooplankton. Living specimens of ciliates and Matthes & Wenzel (1978) and Corliss (1979). Non- heterotrophic flagellates were used. Qualitative inves- identifiable organisms were assigned to 2 size tigations were designed to provide information on the categories: (1.) 30 to 60 pn long ciliates; (2.)3 to 30 pm major forms present. Quantitative investigations pro- size category containing ciliates and heterotrophic vided biomass estimates. A quick survey of the mi- (fluorescence microscopically lacking chlorophyll) crozooplankton in a water sample was afforded by flagellates. 20 *m and 55 pm plankton net samples. Due to the Biomass was determined volumetrically and ex- relatively high ciliate population density in the Schlei, pressed in pg C (after Strathmann 1967). Calculated counts were always made from unconcentrated water from measurements of over 250 individual cells, aver- samples. Of each sample 80 m1 were transferred to 100 age biomass was 138 X 106 pg C individual -' for the 3 m1 bottles and maintained at 4 "C for a maximum of 4 h to 30 pm fraction and 865 X 106 pg C indiv. -' for the before preparation for analysis. In order to study less 30 to 60 pm fraction. Biomass for the 3 to 30 pm fraction frequent larger forms, as well as the smallest more from the Baltic averaged 60 X 10d pg C indiv. -'. commonly occurring forms, volumes of 10 ml, lml and Filtration rate of 'natural' microzooplankton popula- 100 p1 to 10 p1 were sorted out with a stereomicroscope tion~.Filtration rate, i. e. the quantity of water effec- (Wild M8) equipped with bright- and darkfield light- tively cleared of food particles via filtering by a popu- ing. With the exception of the smallest volumes count- lation per unit time, was determined with the aid of ing was carried out by removal of individual cells with radioactively labelled bacteria. A 24 h old culture of a Pasteur pipette. The number of individuals in the Vibrio sp. grown at 20 'C in 300 m1 of ZS-medium subsamples from 10 m1 to 100 p1 varied between (same as ZB but without agar and with 24 %) was used 1 and 150 organisms. Five parallel assessments result- for labelling. Following centrifugation (l0 min, 5000 g) ing in a total of 50 to 100 individuals were conducted and resuspension in 100 m1 of isotonic, particle-free (= for the smallest volumes. Taxonomic identifications 0.2 pm filtered) water (the process was repeated twice), were made on the basis of Jsrgensen (1927), Kahl 200 pCi (methyl-l', 2'-3H)-thymidine (110 Ci mMol -l) (1934), Bick (1972), Borror (1973), Curds (1975), were added to the bacterial suspension. This was con-

Fig. 2. Location of stations in the Baltic Sea. A Fehmarn Belt: N 54", 32,3'; E 1l0,20'; B Arcona Basin: N 55', 00'; E 13", 18'; C Bomholm Basin: N 55", 15'; E 15",59'; CN:North of Bornholm: N 55",41,5'; E 16", 01'; D: South of Gotland Deep: N 56",05'; E 19", 10'; E Gotland Deep: N 57", 20'; E 20". 03' 110 Mar. Ecol. Prog. Ser.

tinuously shaken and incubated for 24 h. Repeated centrifugation removed unincorporated 3H-thymidine from the solution. When necessary, a gradual adjust- where t = duration of experiment (h). C indicates ment to the station's salinity in steps of 5 %O S was average bacterial number in the receptacle over the made at this point. Finally, labelled bacteria were time interval elapsed. Reduction in number of bacteria transfemed to isotonic water and filtered twice through during the feeding experiment is considered to be 3.0 pm polycarbonate filters (Nuclepore) in order to linear, and the mean bacterial concentration at time t/2 exclude bacterial agglomerates and to ensure distinct is the basis for filtration-rate calculations. separation of the bacteria (<3.0 pm) from the micro- Laboratory experiments with Uronema marinum and zooplankton (>3.0 pm) fraction in the subsequent Vibrio sp. For these studies a 4 d-old Uronema feeding experiments. 100 m1 bottles filled with 25 m1 of marinum culture fed with bacteria (Vibrio sp.) on the the prefiltered (150 pm net) water sample and 200 p1 of first and third day was employed. Bacterial food sus- the 3H-bacterial suspension served as experimental pension was prepared by growing Vibrio in a ZS- receptacles. The concentration of the radioactively medium followed by repeated washing via centrifuga- labelled bacteria was about 40 X 106 ml-l, and 6 X 106 tion. The cultures were maintained under sterile con- ml-I for investigations of the Schlei and Baltic samples, ditions in 1 1 Erlenmeyer flasks with cotton stoppers. respectively. Each of 3 parallel samples was incubated The flasks were kept in the dark and filled with 300 m1 in the dark at in situ temperature. Immediately after particle-free, aged North Sea water (30 %), 10 m1 cili- the start of the experiment and after 24 h (48 h for the ate culture and varying quantities of food organisms. station at Schleimiinde) the samples were fixed with The final concentrations were 0.60, 1.39, 6.40, 11.39, 500 p1 of 37 % formalin. Control samples without mi- 58.65 and 89.09 X 106 bacteria ml-l. Control samples crozooplankton were treated similarly with particle- without food or without ciliates were also made. Ten free, filtered water from the respective station. Subse- m1 subsamples were taken immediately following the quent to fixation, all samples underwent filter-fraction- start of experiment and at intervals during several ation with a 3.0 pm polycarbonate filter (microzoo- days. The subsamples were fixed with 0.2 m1 37 % plankton fraction) followed by a 0.2 pm membrane formalin. The bacterial number was determined with filter (bacterial fraction). Filters (25 mm in diameter) fluorescence microscopy. Ciliates were counted on were transferred to scintillation vials containing 10 m1 Unipore filters of 0.4 pn pore size after staining in of scintillator (120 g naphthalene, 5.5 g Permablend 111 erythrosine. filled to 1 1 with Dioxan). Radioactive microzooplank- ton excretion products and radioactive water resulting from bacterial respiration were measured in the <0.2 pm filtrate. Aquasol served as the scintillator for this RESULTS procedure. Results were computed from dprn as cor- rected by the quench factor (established employing a Schlei Fjord investigations 3H-toluene standard). For further details see Gast (summer 1981 to autumn 1982) (1983). (1) Uptake (U,) of radioactive bacteria by microzoo- Development of saprophyte counts and total bacte- planktonic organisms present in the water sample (25 rial numbers along the length of the Schlei are pre- ml) during the time period, t: sented in Fig. 3. Bacterial numbers in the Schlei outlet are always lowest and increase from the outer to inner U, = (dprn 3 pm + dprn filtrate) - (dpm 3 pm + dpm filtrate) (1) l l l I strongly eutrophicated fjord regions. This increase is water sample with control without microzooplankton microzooplankton especially evident from Schleimunde to Missunde. The high bacterial counts in the inner Schlei may be in part where dprn = disintegrations min-l; dprn 3 pm = due to an allochthonous input. A considerable input of radioactivity incorporated by microzooplankton; dprn organic nutrients, which promote autochthonous filtrate = excreted or released radioactivity. (2) Calcu- bacterial growth, may be the primary cause for the lation of the number (X,) of 3H-bacteria filtered by the water's high content on bacteria. microzooplankton present in the water sample (25 ml) The saprophyte counts disclose a population change during the time period, t parallel to falling salinity, whereby the component of marine and halophilic brackish water bacteria decreases in favour of halotolerant brackish water and where B = dprn (3H-bacterium)-1. (3) Determination of freshwater forms (see also Rheinheimer 1970 and filtration rate (F) of microzooplanktonic organisms pre- Rieper 1976). In this investigation bacterial numbers sent in the water sample (25 ml) within the Schlei varied as follows: Gast: Bacteria as a food source for microzooplankton 111

=106 TBN rnl-' 50

\

- 25.11.81

40 Fig. 3. Horizontal distribu- - tion of bacterial numbers in the Schlei. Saprophyte counts on ZB- (8%0) and ZL- (0%) media (left) and - 20 total bacterial numbers (right). Schleimiinde (Smd); Kappeln (Ka); Lin- daunis (Lin) ; Missunde (Miss); GroDe Breite (Gr. I I B); Kleine Breite (H. B.) Srnd Ka Lln Miss GrB KI B. Srnd Ka Lln MISSGcB K1.B. Mar. Ecol. hog. Ser. 22: 107-120, 1985

Saprophytes on ZL: 0.5 to 150 X 103 ml-' consuming ciliates play a significant role in the Schlei. Saprophytes on ZB: 20 to 320 X 103 ml-I The number of bacteriovorous ciliate species is espe- Total bacterial numbers: 5 to 50 X 106 ml-l. cially large in the inner Schlei. Often encountered are Distinct seasonal changes in bacteriological parame- Tintinnidium fluvia tile and Vorticella octava. Both ters were also observed. Following breakdown of phy- appear simultaneously during the Microcystis bloom. toplankton blooms in spring and autumn, saprophyte The mucous sheaths of these blue-green algae are counts were high. They dropped in winter and exhi- usually well populated by bacteria. Live material re- bited a summer minimum. Total bacterial numbers veals how numerous motile bacteria concentrate were very high in late summer, remained at this level around Microcystis aggregates, penetrate the sheath in autumn, and exhibited lowest values during the cold and subsequently disappear, indicating that feeding months. conditions for bacteriovorous ciliates are especially Conditions for bacteria-consuming microzooplank- favourable in the immediate environment of this mi- ton with respect to food sources become more favour- crobiotope. Microcystis itself is apparently not used as able with increasing removal from the Schlei mouth a food source. towards the inner fjord. Freely suspended bacteria and bacteria attached to The distribution of microzooplanktonic biomass detritus particles represent a plentiful food source for (MZP-BM) along the length of the Schlei compared to consumers of bacteria throughout the year. Diatom- bacterial biomass (BBM) is presented in Fig. 4. Shown consuming Strombidium and Strombilidium are pre- together with total MZP-BM is the biomass of larger sent all year round in the entire area of investigation. ciliates (>30 pm). The progression of total MZP-BM The outer Schlei and the Kiel Bight are the major zones distinctly parallels that of BBM. However, MZP-BM is of distribution of tintinnids (Stenosomella, Helicos- generally greater than BBM. The lowest biomass val- tomella, Tintinnopsis and Coxliella). Along with ues in both cases were observed in the Schlei-mouth diatoms, armoured dinoflagellates, flagellates and sampling station. The biomass of the microzooplank- bacteria can serve as a supplemental food source for ton increases rapidly with distance from the mouth and these ciliates. In a mixed diet the preference of a shows a slow increase as one moves further inwards. particular source depends on the respective concentra- Investigations from 10 September 1981 present an tion and biomass of the individual components (Webb exception. In this case a large increase in total MZP- 1956). Most Schlei ciliate species are either bac- BM from the middle to inner Schlei is observed due to teriophagous, herbivorous or exploit both types of food. the strong development of larger ciliates (>30 pm; Carnivorous ciliates are only represented by isolated 366 pg C 1-I of which Tintinnidium fluviatile comprises specimens of Litonotus sp., Lacrymaria sp. and 303 pg C 1-l). The bulk of the microzooplankton is Didinium sp. Consumers of Schlei ciliates predomi- composed of smaller protozoans (3 to 30 pm), which on nantly consist of rotifers. the average accounted for 92 % of total MZP-BM. In late summer/autumn and spring the Schlei MZP-BM attained high values. A minimum was observed in Baltic Sea investigations (August 1982) winter. In July 1982 the MZP-BM was likewise rela- tively low. Similar to MZP-BM, the BBM was signifi- Total bacterial numbers ranging between 2.6 X 106 cantly smaller in winter than in other seasons. MZP- and 6.4 X 106 ml-I were obtained at 0 to 20 m depths in BM in the Schlei ranged from 19 to 1162 pg C 1-I and the area investigated. Bacterial biomass was 40 to BBM from 48 to 436 yg C 1-l. 140 yg C 1-l. Regional and vertical fluctuations in total Ciliates longer than 30 km constitute only a minor bacterial number and bacterial biomass were rela- component of the total number of microzooplankton. tively small (Fig. 5), whereas saprophyte counts The numbers of Protozoa in the 3 to 30 pm fraction are demonstrated larger variations (297 to 1817 colony commonly 2 to 3 orders of magnitude greater than the forming units ml-l). The salinity of the ZB-medium sum of the larger ciliates. Within the Schlei the popula- (8 %) corresponds roughly to the surface water salinity tion density of the 3 to 30 pm fraction ranged from 6500 of the Bornholm Basin. Hence, this medium offers to 84 000 cells (10 m1)-l. The number of ciliates over better possibilities for the development of saprophytic 30 pm in length lay between a few specimens and bacteria from the Bornholm station than those of the 1400 cells (10 m1)-l. Nevertheless, the latter also play more highly saline water of the Fehmam Belt station. an important role as consumers of bacteria. More than During summer, blue-green algae blooms can be half of the species listed in Table 1 can at least par- observed frequently in the Baltic Sea. Even when a tially cover their nutritional requirements from bac- mass development of blue-greens was not encountered teria. A review of the comprehensive list of species during our expedition, microscopical studies of the compiled by Bock (1960) also reveals that bacteria- complex microbiotope of blue-green flocks at the sta-

114 Mar Ecol. Prog. Ser. 22: 107-120, 1985

Table 1. Ciliates found in the Schlei

Ciliates Place of occurrence Season Frequency Food source'

hostomatida Prorodon sp. Kleine Breite + Protophytes, histophages (2) Coleps sp. Kleine Breite + Small algae, ciliates, detr., histophages (4, 5. 6) Tiarina sp. Schleimiinde. Kleine Breite a + Bacteria, flagellates (7) Haptorida Didinium sp. Kappeln, Missunde. S a + Ciliates (1,5, 6) Kleine Breite Mesodinium pulex Total Schlei S su a W + + + Bacteria, small algae, detritus (5, 6) Lscrymaria sp. large Schleimunde, Lindaunis. ) S su a + + Lacrymaria sp, small Missunde, Kleine Breite Cll~ates,detritus (1, 4, 5, 6, 9) Pleurostomatida Litonotus sp. Missunde su + Protozoa (1, 4, 5, 6) Scuticociliatida Cyclidium sp. Missunde su + Bacteria (5) Peritrichida (Sessilia) Epistylis sp. Vorticella octa va Lindaunis, Missunde, su a + ++ Bacteria (5.9) GroDe Breite. Kleine Breite Vorticella patellina Kleine Breite S + Bacteria (9) Zoothamnium sp. Kappeln. Lindaunis. S GroDe Breite. Kleine Breite Heterotrichida Stentor roeseli GroDe Breite a + Bacteria, flagellates, algae, ciliates (1, 5) Spirostomum feres Kappeln. Missunde, S su a + + Bacteria, diatoms, algae, flagellates (4, 5) Kleine Breite Strom bilidium sp Total Schlei S su a W + + + Diatoms, detritus (l, 4, 8) Strombidium sp. Total Schlei S su a W + + + D~atoms,detritus, bacteria, flagellates (1, 4, 7, 8) Oligotrichida Stenosomella ventricosa Schleimunde. Kappeln a W Stenosomella nucula Schleim~de,Kappeln, a W + )Bacteria, flagellates Lindaunis Helicostomella subulata Schleimunde a W Tintinnopsis beroidea Schleimunde, Kappeln, a W Diatoms, armoured dinoflagellates (3) Lindaunls Coxliella helix Schleimunde a W + I Tintinnidium fl uvla tile (Kappeln), Lindaunis, S su a ++ + Bacteria, diatoms, flagellates (5, 9) Missunde. GroDe Breite, Kleine Breite Hypotrichida Oxitrichidae Missunde. Kleine Breite Euplotes sp. large Schleimiinde, Kappeln. S su a + + Bacteria, diatoms, detritus, algae (4, 5, 9) Euplotes sp. small GroDe Breite. Kleine Breite Diophrys sp. Kleine Breite su + Bacteria. diatoms, detritus, algae (4, 7, 9) Aspidisca sp Kappeln. Missunde, su a + + Bacteria, detritus (1, 2, 4, 5, 9) GroDe Breite, Kleine Breite

s Spring, su summer, a autumn, W winter + Isolated. + +some, + ++ numerous After (1) Aumann (1952), (2)Webb (1956), (3) Zeitzschel (1967), (4) Fenchel (1968a), (5) Bick (1972), (6) Matthes & Wenzel (1978), (7) Klekowski & Tumantseva (1981), (8) Smetacek (1981), (9) own observations tions B - E could be undertaken employing the concen- Diatoms (e. g. Chaetoceros species) were present in trated net samples. Among the dominating cyano- lesser numbers. Along with some extended strands of phytes were Nodularia spumigena, Aphanizomenon Nodularia without attached bacteria occurred numer- flos-aquae and some specimens of Anabaena baltica. ous spiral-shaped Nodulana filaments, whose mucous Gast: Bacteria as a food source for microzooplankton

sheaths were heavily covered with bacteria. Many ally in unconcentrated water samples. The microzoo- bacteria-consuming groups of Protozoa could be plankton count was dominated by protozoans of the observed in these agglomerates: Stentor, Vorticella, 3 to 30 pm fraction, whose density ranged from 1900 to flagellates, tintinnids and naked free-swimming cili- 3800 cells (10 m1)-I with the exception of the station ates. Considerable numbers of rotifers (Keratella quad- north of Bornholm where a maximum of 12 000 cells rata and K. cochlearis) and suctorians, as well as iso- (10 m1)-I was demonstrated. Apart from the fact that lated crustaceans were also present. Primarily dino- the microzooplankton count at 20 m depth was smaller flagellates (species of Ceratium and Prorocentrum) than that at 10 or 2 m, no distinct tendency was recog- were found in 55 pm net hauls in the Fehmarn belt. nizable in the vertical distribution of microzooplank- Ciliates over 30 pm in length (species of Vorticella, ton at these depths (Fig. 6). The stations at Fehmarn Strombidium, Strombilidium, etc.) appeared occasion- Belt, Arkona Basin, Bornholm Basin, south of the Got- land Deep and the Gotland Deep provided MZP-BM values between 11.4 and 25.1 pg C 1-I with an average , saprophytes (ZB) value of 17.1 pg C lkl; these values lay considerably l i.rwI under that of the corresponding BBM with a value of

~ 1zmyil il! 81.2 pg C 1-l. The situation was quite different north of Bornhom, where a maximal MZP-BM value of 73.8 pg C 1-I coincided with a remarkably low BBM of 39.4 bg C I-'.

Filtration rate of 'natural' microzooplankton populations

,I.''': TEN With the aid of a radioactively labelled bacterial culture, the filtration rate of 'natural' microzooplank- ton populations could be determined in spring for the Schlei (Schleimunde, Kappeln, Missunde, Kleine thG, :c 1 BBM Breite) and in summer for the Baltic (north of Born- holm). Filtration rates of 25 m1 samples of microzoo- plankton amounted to 0.055 to 0.600 m1 h-' at 5 "C in the Schlei and to 0.730 m1 h-' north of Bornholm at 18 "C; they were clearly dependent on the microzoo- planktonic biomass (Table 2). Low filtration rates in Flg. 5. Saprophyte count, total bacterial number (TBN) and bacterial biomass (BBM)at depths of 2, 10 and 20 m in August samples from Schleimunde and Kappeln stations coin- 1982, Baltic Sea cided with low microzooplanktonic biomass, and the surge in MZP-BM in the middle and inner Schlei resulted in a large rise in filtration rate. A comparison of these rates from microzooplankton populations of the Schlei and Baltic stations clearly indicates the influence of water temperature.

Laboratory experiments with 70 WC1'' Uronema marinum 1. 60: MZP BM The influence of bacterial concentrations on the development of Uronema marinum is presented in Fig. 7. In the control without bacteria the number of ciliates remains fairly constant. Ciliates nourished on bacteria produce a characteristic growth curve. For all but the smallest bacterial concentrations (0.6 X 106 bacterial O' Fehrna!r; Arkona Bornholm r~orrh roult> Gnr- tior1al.c Bell ~as~11 Ba5,n R'huiill I,4nd &ED Drrp cells ml-l), the exponential (log) phase commences following a ca. h initial (lag) phase. The duration of Fig. 6. Number (MZP) and biomass (MZP-BM) of the mi- 8 crozooplankton at depths of 2. 10 and 20 m in August 1982, the log phase depends on bacterial concentration, i. e. Baltic Sea the greater the food source, the shorter the duration. Mar. Ecol. Prog. Ser. 22: 107-120, 1985

The stationary phase and a more or less distinct death and biomass were dominated by small protozoans, i. e. phase complete the sequence. ciliates and flagellates, 3 to 30 pm in length. The ratios Uronemageneration time is clearly shorter (g = 2.1 of microzooplankton to bacterial biomasses in the to 2.7 h) with high concentrations of bacteria (IV, V, VI) Schlei were clearly different from those found in the than with low concentrations (11, 111) (g = 3.8 to 5.1 h). Baltic proper. Whereas the microzooplankton biomass At the lowest bacterial concentration (0.6 X 106 cells ml-l), no ciliate proliferation was observed within the Uronerna rnartnurn first 24 h of experimentation. As demonstrated by the progression of bacterial counts (I Fig. ?), the bacterial number, however, increases within this time period to about 1 X 106 cells ml-l. Apparently a proliferation of ciliates necessitates a preceding rise in Vibrionum- bers. The bacterial count in arrangements I11 to V1 distinctly sinks during the feeding experiments, whereby a concentration of 1 X 106 cells ml-' is the lower limit. In the receptacle lacking ciliates, the bacterial number remains fairly constant, about the initial value of 15 X 106 cells ml-l. No multiplication or lysis of bacterial cells was detectable. In contrast, with an initial concentration of 0.6 X 106 cells ml-' there is an increase in bacterial cell number. Initial concentra- tions of 1.3 X 106 cells ml-I remain relatively un- changed. However, since an obvious ciliate develop- ment occurs in this case, proliferation of bacteria is probably counterbalanced by grazing. This experiment clearly showed that ciliate produc- tion depends on the available amount of bacteria, i. e. the greater the food supply, the higher the ciliate yield.

DISCUSSION

In order to obtain a better understanding of the role of bacteria as food source for primary consumers, quan- titative and qualitative studies of microzooplankton with special reference to ciliate fauna were undertak- - en along with assessments of bacteriological parame- :I B >d 31 40 2 t) 161 ters. The investigations were conducted in the Schlei Fig. 7. Influence of bacterial concentration (Vibrio sp.) on the development of Uronerna marinurn. Above: development of Fjord from June 1981 to October 1982 and on a cruise ciliate numbers; below: development of bacterial numbers. in the Baltic in August 1982. Dotted lines: control samples without bacteria (above), with- In both bodies of water, microzooplankton number out ciliates (below)

Table 2. Filtration rate and feeding rate of 'natural' microzooplankton populations. BBM: Bacterial biomass; MZP-BM: Microzooplankton biomass

Station Date Temp. Filtration Filtered Feeding rate MZP-BM BBM ("c) rate' vol. d-l (pg C BBM (m1 h-') ("/.l 1-1 d-l 1 (W c 1-l) c 1-'1

Schleimdnde 23. 3. '82 5 0.084 8.1 0.7 59.8 8.5 Kappeln 23. 3. '82 5 0.055 5.3 3.0 57.6 56.3 Missunde 23. 3. '82 5 0.465 44.6 42.4 334.8 95.1 Kleine Bre~te 23. 3 '82 5 0.600 57.6 51.3 446.4 89.0 North of Bornholm 18. 8. '82 18 0.730 70.1 27.6 73.8 39.4

Of mlcrozooplankton (< 150 pm in length) in 25 m1 Gast: Bacteria as a food source for microzooplankton 117

in 5 of 6 stations from the Baltic was less than the 100 to 3500 pg bacterial carbon 1-l) for bacteriovorous bacterial biomass, with few exceptions the fjord's ciliates. biomass of the microzooplankton distinctly surpassed In the light of these considerations, a sufficiently that of bacteria. large bacterial biomass permitting the maintenance or The most important factor amongst parameters development of planktonic ciliate populations can only influencing ciliate populations is their food source, be expected in strongly eutrophic coastal regions or in especially since ciliates possess a broad spectrum of microbiotopes with high bacterial activity. Such mi- tolerance to physical and chemical conditions (e. g. crobiotopes are provided by the summer blue-green Noland 1925, Noland & Gojdics 1967, Fenchel 1968a). algae blooms occurring in the central and northeastern According to Faure-Fremiet (1967), the influence of Baltic. According to a calculation by Hoppe (1981), a abiotic factors is more likely to be indirect in view of Nodularia flock of 1 cm in length and 0.5 cm in diame- the stronger dependency of food organisms on physical ter contains at least 7.5 X 108 bacteria, which presents and chemical environmental conditions. an enormous enrichment of bacteria as compared with Rieper & Flotow (1981) carried out some feeding the surrounding water mass. Thus, it is not surprising experiments with Uronerna sp. and different species of that together with other microzooplanktonic organ- bacteria. They showed that Uronerna grew better with isms, numerous ciliates are encountered in blue-green 'List 7' and a mixture of oil-degrading bacteria than algae agglomerates. A frequent bacteriovorous ciliate with Serratia sp. 'Lst 7', identified as Vibrio sp. by in this microbiotope is Vorticella (see also Bursa 19631, Rieper (pers, comm.), was used in the present investi- which apparently requires both a substrate for attach- gations. ment and sufficient bacterial biomass. Coinciding with Laboratory studies with Uronema marinum disclosed the Schlei Microcystis bloom from summer to autumn that the concentration of the bacterial food source has a are Tintinnidium fluvia tile and Vorticella octava. The significant effect on ciliate development. Hence, the Microcystis colonies possess a mucous sheath heavily proliferation of ciliates required a minimal concentra- colonized by bacteria and this offers favourable feed- tion of about 1 X 106 bacteria ml-l. During feeding ing conditions for bacteria-consuming ciliates. experiments the bacterial concentration sank occa- In conjunction with the discussed limiting concen- sionally slightly below this limiting value. Tezuka trations of bacterial number and biomass for the nutri- (1974) reported that the survival minimum for the tion of ciliates, the question arises: To what degree do much larger Pararnecium caudatum corresponded to ciliates depend on bacteria as an exclusive food 1 X 107 bacteria ml-l. A critical bacterial density of 5 source? Table 1 demonstrates that most forms exploit a 106 - 107 bacteria ml-l was calculated for U. nigricans fairly broad spectrum of nutritional sources; they can by Berk et al. (1976), below which growth of the cili- consume flagellates and small algae as well as bac- ate~no longer occurred. Taylor (1978) found minimal teria. The sum of the biomass of available foods is concentrations of 7 X 106 bacteria ml-' for Colpidium decisive for ciliate development under conditions of campylum and 4 X 106 for C. colpoda. These investiga- supplemental bacterial nourishment. Some species, tions were based on experiments utilizing culture bac- however, apparently feed only on bacteria. Few teria, whose cell volumes greatly exceeded those of species are carnivorous and independent of bacteria as 'natural' bacteria. For example, the median volume of food sources. cultured Vibrio sp. is 1.15 pm3, while the median Ciliate production was dependent on the amount of volume for bacteria commonly occurring in the Baltic bacteria available; the larger the food source (Vibrio and Schlei is ca. 0.1 to 0.2 pm3. sp.), the greater the yield (Uronema marinum). This The limiting concentration of 1 X 106 bacteria ml-I experimental result agrees fundamentally with in situ using Vibrio sp. corresponds to a biomass of 115 kg observations along the profile of the Schlei. Hence, the C 1-l. A comparison of these results with in situ condi- biomass of microzooplankters accordingly rose with tions shows that although the bacterial concentration increasing bacterial biomass from Schleimiinde to of 1 X 106 bacteria ml-I was surpassed throughout the Schleswig. With respect to seasonal aspects, however, whole period of investigation in the Schlei (1.4 to 49.8 the data for bacterial and microzooplanktonic biomass X 106 bacteria ml-l), values greater than 100 pg C 1-I did not clearly run parallel to one another. For exam- were only detected in the central and inner Schlei ple, in September, October and November a rise in (maximum: 436 kg C 1-l) and in the Fehmarn Belt. In microzooplanktonic biomass at Missunde was coupled general, a bacterial biomass of 20 pg C 1-I is seldom with a reduction in bacterial biomass. Quite possibly, surpassed in the Baltic proper (Zimmermann 1977, the grazing effect by the bacteriovorous microzoo- Meyer-Reil et al. 1979, Hoppe 1981). Fenchel (1980) plankton outweighed bacterial production at this time. calculated a required minimal content on organic Experimental evidence for this assumption was given material of 0.2 to 7 mg dry weight 1-I (corresponding to by v. d. Ende (1973) and Ashby (1976): in studies with Mar. Ecol. Prog. Ser 22: 107-120, 1985

continuous cultures of ciliates and bacteria a steadily plankton feeding rate of the same water sample. declining amplitude of countercurrent oscillations for Bauerfeind (1983) calculated from turnover times for both components was observed. glucose and natural substrate concentrations bacterial Food uptake apparently influences the generation production for the central Baltic in May 1982 ranging time of ciliates. The generation time of Uronema from 5.4 to 54 pg C l-Id-'. Employing data on the marinum is distinctly shorter (g = 2.1 to 2.7 h) when frequency of dividing cells, Hagstrom et al. (1979) coupled with high bacterial concentrations (11 to 89 X obtained a daily Baltic bacterial production of 10 pg 106 ml-l) than with low bacterial concentrations (>l to C 1-l. A smaller production of 1.0 to 5.7 pg C l-Id-' in 6 X 106 ml-l; g = 3.8 to 5.1 h). Kracht (1982) reported the Kiel Bight from July to October was determined by that the generation time of Colpoda cucullusincreases Meyer-Reil (1977) with a 'flow system'. Azam et al. when the bacterial concentration is less than 4 X 108 (1983) assume a bacterial production of 2 to 250 pg C Escherichia coli ml-l. According to Banse (1982), cili- l-Id-' for coastal waters. These references show that ate~can only take advantage of their high reproductive there is a relatively wide range in bacterial production potential in patches with high nutrient concentrations. for different aquatic sites and seasons. Accordingly, In situ studies and laboratory experiments revealed microzooplankton production will also fluctuate within that bacterial populations can exert a significant influ- a certain range. ence on the development of microzooplanktonic In terms of number and biomass the major compo- organisms and, in turn, bacteria are held in a state of nent of microzooplankton populations comprised small physiological equilibrium by grazing (Johannes 1965, protozoans, including 3 to 30 pm long ciliates and Barsdate et al. 1974, Harrison & Mann 1975, Fenchel& flagellates. A bacteriovorous mode of feeding is also Jsrgensen 1977). An additional mechanism of regula- well known for flagellates (Lighthart 1969, Haas & tion was indicated by Legner (1973), who found that Webb 1979). Applying laboratory data from flagellate the metabolities of cillates may stimulate bacterial cultures and data of naturally occurring microflagel- proliferation. late abundances, Fenchel (1982b) calculated filtration For the study of the interaction between bacteria and rates of 12 to 67 % of the Limfjord water per day. This bacteriovorous microzooplankters it is interesting to value lies somewhat below that of the summer filtra- calculate the microzooplankton-induced reduction of tion rate of Baltic microzooplankton (70 % d-l) deter- bacteria and compare this with data on bacterial pro- mined in this paper. Fenchel (1982b) presumes that duction. If the filtration rate of the 25 m1 microzoo- heterotrophic microflagellates are the major consum- plankton population is known, then the percent water ers of bacteria in the marine pelagic system. It remains volume per day filtered to a particle-free state by the to be clarified, what proportion of the 3 to 30 p long microzooplankton can be calculated. Providing the protozoan fraction is actually represented by micro- standing stock of bacterial biomass has been deter- flagellates. mined, one can further calculate feeding rates in pg In areas of higher bacterial concentration, e, g. in the bacterial carbon l-Id-'. One must, of course, assume strongly eutrophic Schlei and in microbiotopes of blue- that 'natural' bacteria are filtered in the same manner green algae agglomerates, ciliates of over 30 pm in as are tritium-labelled cultured bacteria. Spring feed- length can play an important role as consumers of ing rates of 0.7 to 51.3 pg bacterial carbon l-Id-' were bacteria. measured in the Schlei. During the summer in the Our investigations have demonstrated that bacteria Baltic a reduction of 27.6 yg C l-Id-' in bacterial can represent an important food source for microzoo- biomass occurred as a result of microzooplankton graz- plankters. Moreover, due to their high reproductive ing. Schlei bacterial production measured by Hoppe potential, microzooplankton organisms can respond (unpubl.) according to the method of Fuhrma.n & Azam almost immediately to favourable environmental con- (1980) was as follows: ditions and, thus, effect a rapid introduction of bacte- Schleimiinde: 0.03 to 0.18 pg C l-Id-' rial biomass to the food web. Kappeln: 1.21 to 7.88 yg C l-Id-' Missunde: 5.11 to 33.21 pg C l-Id-' Acknowledgements. Sincere thanks are extended to my Even though a direct comparison of Schlei bacterial supervisor Professor Dr. G Rheinheirner for constructive sug- gestions and guidance during the preparation and completion production and reduction by microzooplankton graz- of this work, and for the Baltic Sea data placed at my disposal. ing is not possible due to seasonal dissimilarities in I thank the members of our research team, Dr. S. Bauerfeind, data, both parameters obviously are of the same order Prof. Dr. H.-G. Hoppe and Dr U. Horstmann, for helpful of magnitude. discussions. I further thank Dr E. Hartwig (Hamburg) for aid in the identification of ciliates and Dr. G. Uhlig (Helgoland) Hoppe (unpubl.) measured a bacterial production of for isolating and identifying Uronerna marinurn. This work 0.47 to 3.04 pg C l-Id-' for the station lying north of was supported by the Federal Ministry for Research and Bornholm. This value lies far below the microzoo- Technology, Bonn, BMFT Grant no. V 9. Gast: Bacteria as a food source for microzooplankton 119

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This paper was submitted to the editor; it was accepted for printing on November 26, 1984