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Microbial diversity and activity in a Danish Fjord withanoxic deep waterTom Fenchel a , Catherine Bernard a , Genoveva Esteban b , Bland J. Finlay b , Per JuelHansen a & Niels Iversen ca Marine Biological Laboratory, University of Copenhagen, DK-3000, Helsingør, Denmarkb Windermere Laboratory, Institute of Freshwater Ecology, Ambleside, Cumbria, LA22 OLP,U.K.c Laboratory of Environmental Engineering, Aalborg University Center, Sohngaardsholmsvej57, DK-9000, Aalborg, Denmark

To cite this article: Tom Fenchel , Catherine Bernard , Genoveva Esteban , Bland J. Finlay , Per Juel Hansen & Niels Iversen(1995): Microbial diversity and activity in a Danish Fjord with anoxic deep water, Ophelia, 43:1, 45-100

To link to this article: http://dx.doi.org/10.1080/00785326.1995.10430576

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OPHELIA 43 (1): 45-100 (September 1995)

MICROBIAL DIVERSITY AND ACTIVITYIN A DANISH FJORD

WITH ANOXIC DEEP WATER

10m Fenchel! ~ Catherine Bernardl, Genooeoa Estebanl, Bland] Finlay2,PerJuel Hansen! & Niels Ioersenl

lMarine Biological Laboratory, University ofCopenhagen, DK-3000 Helsinger, Denmark

2Institute ofFreshwater Ecology, Windermere Laboratory, Ambleside, Cumbria LA22 OLP, U.K.

3Laboratory of Environmental Engineering, Aalborg University Center,Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark

"Corresponding author

ABSTRACT

Microbial diversity and activity were studied in a stratified basin ofMariager Fjord, Denmark inAugust 1994. The basin is about 30 m deep and the lower half of the water column is anoxie andsulphidic. The hydrographieal and biological features ofthe system are described. Based on chemi­cal gradient profiles and measurements of process rates, we found that the relative importance ofsulphate reduction, denitrification and methanogenesis in terms of anaerobic terminal mineralisa­tion was about 5:1:0.4. It is possible, however, that methanogenesis is underestimated because anunknown fraction ofthe methane production escaped by ebullition. It was estimated that 10-15 %of the net primary production is mineralised anaerobically. The mean residence time of methane,sulphide and ammonia beneath the chemocline is within the range 1.6-2.3 yrs. Chernolithotrophicproduction in the chemocline (sulphide oxidation and nitrification) accounted for about 3% ofthenet primary production of the system. Methane was oxidised (anaerobically or aerobically)throughout a large part of the water column, but most escaped to the atmosphere.

The fjord has an impoverished zooplankton. This may be due to the sulphidic deep water whichwill be lethal to sedimenting eggs. The protozoan biota were studied quantitatively and qualitative­ly. Eighty-one species of protozoa were identified in the water column; ofthese, 37 were ciliates andthe remainder were flagellates or rhizopods. Only one new species of ciliate was found. All flagel­lates from the aerobic zone could be assigned a generic name. In contrast, the microaerobic and es­pecially the anaerobic parts ofthe water column revealed about ten undescribed flagellates (threeofwhich are formally described), suggesting that the sm aller protists of anaerobic habitats are stillpoorly known. Three relatively distinct protistan assemblages could be identified, being associatedwith the oxic water column, the microaerobic zone around the chemocline, and the anoxie zone,respectively.

Keyuiords: Anoxie basins, an aerobic microbial processes, chemocline, protozoa.

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46 ruM FENCHEL ET AL.

INTRODUCTION

The existence of marine stratified basins with an anoxic water column is knownfrom many parts of the world (Grasshoff 1975). These include the Black Sea(Deuser 1975, Sorokin 1972), the Cariaco Basin (Richards 1975), the large deepbasins ofthe Baltic Sea (Rheinheimer et al. 1989, Detmer et al. 1993), Norwegianfjords with a sill (Indrebe et al. 1979, Skei 1983) and various shallower fjords andestuaries with a permanent or transient anoxic water column (e.g. Fenchel et al.1990, Kemp et al. 1992, Sieburth & Danaghay 1993). Different biogeochemicalaspects of such systems have previously been studied, especially with respect togradients of sulphide and ammonia, vertical fluxes of oxygen and sulphide andsulphide oxidation and nitrification at the chemocline (e.g. Dyrssen 1986; severalof the above mentioned papers). Environmental aspects, including the role of eu­trophication and the effect on the fauna (e.g. J0rgensen 1980, Kemp et al. 1992)have also drawn attention. However, regarding the protozoan biota ofthe anoxiczone and of the chemocline there are only few studies with a limited scope (Fen­chel et al. 1990, Setälä 1991, Zubkov et al. 1992, Detmer et al. 1993). A compre­hensive study ofsuch systems which includes quantification ofthe major microbi­al processes in the anaerobic layer and at the chemocline, together with a qualita­tive and quantitative study of microbial populations, has not previously beenpublished.

Here we present a detailed study of the anaerobic zone and the chemocline ofthe central basin in Mariager Fjord (east coast ofJutIand, Denmark) with specialreference to the hydrographical and biological properties which maintain ananoxic water column, the quantitative role of the major microbial processes andthe protozoan biota. An earlier study ofMariager Fjord which was carried out in1986 (Fenchel et al. 1990) concentrated mainly on the vertical distribution ofcili­ates and on protozoan and bacterial biomasses in relation to the position of thechemocline.

We are gratefuI to captain and crew ofRV 'Ophelia' and to Ms Kirsten Maagaard for technicaI as­sistance. We thank Mr. Finn Andersen M.Sc. for permission to use hydrographical data coIIectedby the Office ofEnvironment, County ofNorthernJutland and Drs M. Olesen and C. Lundsgaardfor obtaining additional water sampies in Iate August 1994. The studies were supported by grantsfrom the EU MAST2-programme, contract no ct93-00S8, from the Danish Natural ScienceResearch CounciI (11-0088-1)to TF, from Consejo Superior de Investigaciones Cientificas (Spain)to GE, from the Natural Environment Research CounciI (U.K.) to BJF and from the EU Environ­ment Programme, contract EVSV-CT94-0499 to NI.

MATERIAL AND METHODS

Sampling. The material was collected in the period from August 8 to August 12,1994 from RV 'Ophelia', Most water column sampies were taken at astation

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MICROBIAL LIFE IN AN ANOXIC FJORD 47

56°39'80"N, 9°58'69"E elose to the center ofthe deep basin and with a depth ofabout 28 m (Fig.1). Additional samples were taken 1-2 km further east and sam­pIes of sediment and sediment cores were collected on the northern slope of thebasin at depths of 16-17 and 20 m. Data on temperature, salinity, fluorescence(chlorophyll) and oxygen tension were collected two or three times daily with aCTD-probe. H ydrographic data (salinity, temperature, oxygen, chlorophyll) forsome previous periods (ineluding 1979-81, 1991 and the periodJanuary-August1994) were obtained from the Office of Environment, County of Northern Jut­land (Anon. 1983 and unpublished data); some data collected in 1986 (Fenchel etal. 1990) are also ineluded.

We collected water with a 5 I water sampler, On three occasions samples weretaken at 1 m intervals from 0 to 9 m, at 0.5 m intervals from 9 to 16 m and at 2m intervals from 17 to 25 m. These series were used for chemical analysis and forquantifying and identifying organisms. Additional series were taken at 0.5 m in­tervals at the chemocline (9-16 m) for more ac cu rate 02-determinations and forincubations with HC-bicarbonate.

Sediment cores (25 cm long) were collected with a HAPS-bottom corer (Kan­neworff & Nicolaisen 1973). Subcores for measuring CH4-oxidation rates, porewater chemistry and sediment porosity were sampled with plexiglass tubes fromthe HAPS-sample and kept cool until they could be analysed. Samples ofthe su­perficial sediment layer were also obtained with a detritus sIedge. Since the sedi­ment of the main sampling station is very loose and flocculent, samples had to betaken along the slope ofthe basin where sediments are somewhat more consoli­dated.

Chemicalmethods. Oxygen was determined in situ with an oxygen electrode (YSI)mounted on the CTD-probe and in water samples using the Winkler method.Ammonia, nitrite and nitrate concentrations were determined in 5 ml filteredGF/F water samples using a Teenion TRAACS 800 autoanalyser (Bran andLuebbe Inc., Elmersford, NY); the samples were kept frozen until they could beanalysed. Sulphide was determined with the methylene blue method of Cline(1969). Water samples for methane determination were collected from the watersampIer with a 20 ml syringe which was flushed three times and transferred to a58 ml serum bottle prefilled with 2 mI2.5% NaOH (to terminate bacterial activi­ty). The vials were closed with a thick butyl rubber stopper (Apodan, Denmark)and headspace gas was later analysed by gas chromatography (Chrompack) anda FID-detector.

Measurements offluxes and rates 01microbialprocesses. For determination of methaneproduction, three replicate 20 ml samples were collected with a syringe and trans­ferred to 50 ml serum bottles which were closed with a butyl rubber stopper andsealed with aluminum crimps. Samples from oxic strata were flushed with at­mospheric air (using a 50 ml syringe and a needle as vent) and sarnples from

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48 10M FENCHEL ET AL.

anoxic strata were flushed with N 2. The samples were stored in a cooler and,within three hours, placed in an incubator (11°C) and left overnight. HeadspaceCH4-concentrations were then followed for four days.

Sampies for measuring CH4-oxidation rates were collected in 26 ml serumbottles (three at each sampling depth). The bottles were filled by a tube attachedto the bottom of the water sampler and water was allowed to overflow (approxi­mately three times the bottle volume) before closure with a butyl stopper fittedwith a syringe needle to avoid air bubbles. The vials were kept in a cool box untilthey could be processed (within three hours after sampling). The rate ofmethaneoxidation was determined by the HC-tracer technique of Iversen & Blackburn(1981). After four hours in an incubator (11°C) 50 /LI HCH4-tracer (10 /LCimll)were injected. The tracer was prepared by diluting labelled methane (88 mCimmol-1; Amersham) with N 2 and it was cleaned and checked as described else­where (Iversen et al. 1987). The incubations were stopped after 20 h. Ten ml werewithdrawn with a syringe from each vial, displacing the volume by injection ofN 2. The syringe content was transferred to a 21 ml serum bottle containing 2 ml5% NaOH in order to terrninate activity and to bind CO2. Two ml NaOH werealso added to the original serum bottle. Methane concentration and specific ac­tivity was measured for each vial, The gases were separated with a Hyacep Qcolumn (2 m x 2 mm) and detected with a flame ionisation gas chromatographwith nitrogen as carrier (30 ml minI}. The HCH4 was combusted in the detectorand collected as HC02 at the detector vent as described in Iversen &J0rgensen(1985). The two sampIes were then pooled and HCOTactivity was measured asdescribed by Iversen & Blackburn (1981). Filter sterilised sampIes (from 9.5 and14.5 m) served as controls. Parallel samples containing dimethyl ether (1 mM)were also treated as described above. Dimethyl ether inhibits aerobic methane ox­idation (Oremland & Culbertson 1992), but does not affect anaerobic methaneoxidation (Iversen, unpublished). Recovery ofthe added label was> 70% at alldepths..

Methane flux across the water surface to the atmosphere was estimated bymeasuring the increase of methane during 1 h in the headspace of a floatingchamber. This was constructed from a PVC barrel (5 mm wall thickness, 50 cmtall and with an internal diameter of 45 cm). A float was attached to the chamber20 cm from the top, which had a hole for fitting a serum stopper. The stopper wasplaced in the hole after the chamber had been placed in the water. Ten ml air sam-

.....Fig. 1.Manager Fjord, Denmark and an enlarged presentation ofits eentral part. The shaded por­tion of the longitudinal depth profile (> 16 m depth) represents the anoxie part of the watereolumn. The loeation ofthe main sampling station offMariager is also indieated (+). The longitu­dinal seetion follows the rnid-Iine between the north and south coasts for the entire length of the

fjord.

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MARIAGER FJORD

'""~"E

MICROBIAL LIFE IN AN ANOXIC FJORD 49

N

A. I ~I~ r ~KATTEGAT

IMARIAGER

10 km

o

10

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30 ....' _

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50 10M FENCHEL ET AL.

ples were collected by syringe and transferred to 8 ml serum bottles prefilled withwater so that the samples complete1y displaced the water through a venting nee­dle. The flux eh amber was used twice on a calm day (wind speed < 2 m s·l) andfour times on a more windy day (8 m sol).

Measurements ofpotential nitrification were initiated within six hours ofsarn­pling. For each sampling depth, 100 ml water was added to each of four Erlen­meyer flasks and 100/LI of a solution of (NH.4,hS04 (final concentration 2mM)were added. In two of the flasks NaCI03 (final concentration 10 mM) was addedto inhibit the last step in the nitrification process. The flasks were covered withparafilm and incubated in a rotary shaker (200 rpm) at 12°C. For each ofthe fol­lowing three days, 10 ml samples were filtered through a G F/F filter and analysedfor N02- and NOr .

Denitrification was studied by the acetylene blocking technique (Flett et al.1976). Water samples (25 ml in 58 ml serum bottles and with N2 as headspacegas) were placed on a rotary shaker (200 rpm) at 12°C). After 4- h, 1.5 ml C2H2were added to the headspace. Produced N20 was measured by gas chromatogra­phy fit ted with a Hyacep Q column with Ar/CH4 as carrier gas (flow rate 20 mlmin-1) and an electron capture detector.

Incubation untb. HG-bicarbonate. In order to quantify chemotrophic CO2-assimila­tion in the chemocline we used a modification ofmethods previously used by Det­mer et al. (1993) and Sorokin (1972). On two occasions, water samples (4 ml) col­lected at 0.5 m intervals between 8 and 16 m and between 9.5 and 15 m, weredrawn into 5 ml syringes. One ml 14C-bicarbonate (4/LCi; Danish C-14 Agency)was added and the syringes were incubated on the boat for one hour at in situ tem­perature ("" 15°C) in the dark. Blanks (with 0.5 ml formalin) were also incubat­ed. After the incubation the sampies were transferred to scintillation vials, and0.5 ml 4% formalin and 200 /LI N HCI were added. The sampies were left openon the deck for 24 h (when all dissolved 14C02 was assumed to have equilibratedwith the air), closed and later counted with a liquid scintillation counter. Ambientbicarbonate concentrations were estimated from salinity.

Studies 01thebiota. Living protozoa were collected by picking them up in capillarypipettes from freshly collected sampies or after concentrating cells by centrifuga­tion of 10 or 50 ml sampies. Ciliates were fixed and silver impregnated as soon aspossible after sampling (Esteban et al. 1995). Qualitative studies were also basedon formalin-fixed material. In addition to the sampies taken during the cruise,two freshly collected samples, taken at 2 and 20 m depth on August 20, were usedto supplement the qualitative studies of protozoa.

Enrichment cultures were prepared immediately after sampling. For anaero­bic incubations, 2 ml samples were injected into stoppered 20 ml serum vials with15 ml sterile, anoxie seawater and a boiled wheat grain and an headspace withN 2. Aerobic and 'microaerobic' incubations were made in tissue culture flasks

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MICROBIAL LIFE IN AN ANOXIC FJORD 51

(Nunc, Denmark) with sterile seawater and one or two boiled wheat grains. In thecase of aerobic incubations the water constituted only about Y3 of the volume ofthe flasks, whereas the 'rnicroaerobic' flasks were filled almost completely. Theseincubations were monitored regularly during the following weeks and the de­veloping biota and some individual species were subcultured at regular intervals.

For quantitative studies, two sets ofLugol-fixed and two sets of formalin-fixed(final concentration 1%) sampIes were collected. The former were used for quan­tifying and identifying tintinnids and larger flagellates with an inverted rnicro­scope, after sedimentation in settling chambers with volumes of 10-50 ml accord­ing to the expected cell density. The formalin-fixed sarnples were used for quan­tifying bacteria and quantifying and identifying representatives of other pro­tozoan groups (except rhizopods). Fifteen ml samples and a drop of acridineorange solution were filtered through black 0.2 JLm nuclepore filters. The filterswere mounted between slides and coverslips with a drop of paraffin oil andstudied with an epifluorescence microscope and blue light. The preparationswere also studied with green excitation to quantify eyanobaeteria and eryp­tomonads.

The volume of each species of protozoon (or ofdifferent size classes in the caseofflagellates) were estimated from linear dimensions. In the ease ofbaeteria, weused previous data (Fenchel et al. 1990) for the average volume of baeteria fromdifferent depths. The average volume ofbacteria from the anoxie zone is signifi­cantly higher than from the oxic zone.

RESULTS AND DISCUSSION

General properties oJMariager Fjord

The following two seetions serve as a general introduetion to the fjord. Specialreferenee is given to particular hydrographieal properties and to primary produe­tivity ofthe system. These aspects represent a necessary background for the studyand their treatment is based mainly on unpublished data.

Hydrography and deuelopment oJan anoxiewatercolumn. Mariager Fjord is about 40 kmlong, but almost everywhere less than 2 km wide (Fig.1). The fjord has a sill: theouterpart is shallow( < 2 m) exeeptfor a 6-7 m deep ehannel which is maintainedto allow ships to enter the fjord. The eentral part of the fjord elose to the town ofMariager consists ofa deep (max. 30 m) basin. Towards the innermost part of thefjord the bottom slopes upwards again. The volume ofthe central part of the fjordis about 1.37 x 108 m>. In the summer the water column is anoxie beneath about16 m depth and the anoxie and sulphidic water then constitutes about 2.6 x 107

m 3 or 19% of the total volume of this part of the fjord.The water exchange of Mariager Fjord is relatively small, the average resi­

dence time ofwater in the central part (above the chemocline at about 16m) being

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52 '!DM FENCHEL ET AL.

10

~ 12

E 14

16

18

20

22

24

26

20

30

4050 70 100 120

re mpe ra tu re ('C)

<2°2

0

30

100

70

50

AugustJulyJuneMayAprilMarchFebruaryIIo

January

18 -___________ ......

20 - - - - - - - - - - - - --...

21 _

19

14.5

----

, ,, ,,, ,,,,, ,

-,,,

15

16

17

salinity (pp t )

10

20

22

16

18

24

~ 12

~ 14

26 20

January Febru a ry March April May June July August

Fig. 2. Isopleths of temperature and salinity in the central part of Mariager Fjord for the period]anuary-August, 1994 (broken line indicates absence of data).

about 8 months (Anon. 1983). The circulation pattern is in many respects similarto other shallow fjords, lagoons and estuaries (Officer 1976). Due to an input offresh water from terrestrial runoff and rivulets, the surface salinity is diluted andthere is a net surface outflow to the sea. During prevailing westerly winds the sur­face outflow increases and this is compensated by an inflow along the bottom ofwater frorn the Kattegat (which, at the mouth of the fjord, has a surface salinity

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Po,

MICROBIAL LIFE IN AN ANOXIC FJORD 53

1S I 0•

12 I 21 - - 16

--. - 6• I 4

• ..2 14 IS '. I 6

\ I ~ 8

~ 10 ~ ! 1018 "

\" 20•

~" 22,.~

I 18I

S

IS 16 17 18 19 0 50 100

ppt

Fig. 3. A salinity:temperature plot (numbers are depth in metres) and the 02-profile on Octoher9, 1991 showing an intrusion ofwarmer and oxygen-rich water at a depth of 14-15m. In a two-layersystem with mixing a plot oftemperature against salinity yields a straight line whereas two line seg­ments indicate an intrusion layer (Pedersen 1986). Salinity-temperature data often, hut not always,yield such segmented plots showing that intrusions are typically situated between 12 and 16 m.

of ca 25 ppt). In the innermost parts of the fjord this inflowing water is subjectto upwelling and mixing with the surface water which flows Out of the fjord. Asthe deeper inflowing water undergoes some turbulent mixing with the outflowingsurface water along the entire length ofthe fjord, the salinity differential betweenthese opposing currents is usually low (1-2 ppt) at the entrance to the central partof the fjord.

A characteristic feature of the deep basin is that at depths exceeding 18-20 mthe water has a salinity (::::::: 20 ppt) which is higher than that ofthe water whichusually enters the central part of the fjord (Fig. 2). An intrusion of water with asufficiently high salinity and ofsufficient magnitude to flush the bottom ofthe ba­sin and render it oxic occurs only at intervals of years and probably only underextreme weather conditions. It has not occurred since 1992. There is evidencethat it did happen during the winter of 1985-86, as the water column was oxicalmost to the bottom in spring 1986; at that time the temperature at 21 m was0.9°C, increasing to only 2°C in October (Fenchel et al. 1990 and unpublisheddata). The deepest part of the water column is therefore relatively stable andhomogeneous during long periods. Inflowing water normally has an intermedi­ate salinity and it forms an intrusion layer between the deep, permanently cold

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54 'IDM FENCHEL ET AL.

o I 1

cxygc n te nsion (% atmospheric saru rano n )

10

~ 12 r 100

E 14

"I 50

18 20

1020

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24

26

LJanuary February March April May

JOD (> IDO)

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16

18

20

22

24

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2 5

chlorophyll (!'g e ')

<2

EEJ 20-30

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JO 10 10

January February March April May June July August

Fig. 4. Isopleths of O, and of chlorophyll during the periodJanuary-August, 1994.

« 4°C) and relatively saline layer, and the more brackish surface layer. Whenthe inflowing current is strong (during strong winds from the west) the rniddlepart of the fjord becomes a three layer system with an intrusion layer which istypically located at a depth of 13-15m. Sometimes this results in an oxygen maxi­mum at this depth (Fig. 3). On some occasions, intrusions are also reflected indeeper chlorophyll maxima (Fig. 4). The fact that the deepest (5-10 m) part of thewater column is generally stable and mixes only very slowly with the overlying

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MICROBIAL LIFE IN AN ANOXIC FJORD

P02 (% atm. sat.)

55

Vl(l)...........(l)

S

o

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4

6

8

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16

18

20

22

24

26

o 50

March 16

100 0 50

August 9

100

8 10 12 14 16 18 8 10 12 14 16 18

o (p - 1000 kg rn")

Fig. 5. Examples of density and 02-profiles in late winter and in surnrner, 1994-.

water is a eharaeteristie feature ofthe middle part ofthe fjord whieh explains thepresenee of anoxia in the water column. Intrusions of seawater may disturb theehemoc1ine during summer when the anoxie layer migrates higher in the water

eolumn.

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56 ruM FENCHEL ET AL.

'"<U........<U

S

o

5

10

15

20

25

-0.2 o 0.2 0.4 0.6 0.8 1.2

so m'Fig. 6. The density gradient on August 9, 1994. The upper peak of the gradient is mainly a resultof the thermocline (and is found only in surnrner) while the deeper peak is due mainly to the salinity

gradient. Below ca 21 m the water column is relatively homogeneous.

During the winter and early spring the water is mixed down to 12-14m and themaximum gradient ofthe pycnoc1ine (which at this time is almost exc1usively dueto the salinity gradient) occurs at 14-16 rn depth. As the water warms up duringspring and early summer, a thermoeline develops closer to the surface; the pyc­nodine is then more complex and the water column is already stratified at a depthof 7-8 m (Figs 5-6). As a result (probably in conjunction with an increasedsedimentation of organic material) the chemocline migrates upwards duringspring and summer. In 1994 the anoxie zone rose from about 5 m above the bot­tom in]anuary to about 15m above the bottom in mid-August. In late August theoxyeline moved downwards again as a result of a weakened thermodine and morewindy weather (Fig. 4).

Signs of a weak intrusion between 12 and 14 m were evident on the first day ofour investigation (August 8), but the effect in terms of the discontinuity of the sa­linity:temperature plot disappeared during the following days.

Phytoplankton andprimary productivity. Mariager Fjord is highly productive; basedon 14C-bicarbonate uptake, production has been estimated to be 860-1100 gCm-2yr1, corresponding to 2.3-3.1 gC m-2day-l during the period 1991-93 (unpub­lished data, County of Northern Jutland; Fig. 7). However, these figuresrepresent an overestimate of the net production of particulate organic material

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MICROBIAL LIFE IN AN ANOXIC FJORD

12

10 I-

8 Ir:;>,~-e

N

6 r's

~~U0.0

4 I-

57

oJanuary 1993

2 I-

\ v~

January 1994Fig. 7. Primary (14C-uptake) production in the central part ofthe fjord during 1993.

which would be available to grazers or to sedimentation. The measurements arebased on short incubation times. Also, thernixed zone penetrates several metresbelow the photic zone during most of the spring and summer, resulting in a con­siderable (but unmeasured) algal respiration. A crude estimate ofthe net produc­tion would be about half of the above mentioned figures.

During the summer, chlorophyll concentrations may re ach > 50 /Lg 1-1 (Fig. 4)and Secchi disk readings are often < 2m. From mid April until the beginning ofSeptember, about five distinct blooms develop, interrupted by periods with lowerchlorophyll concentrations and lower productivity. The high productivity of thefjord is supported by mineral nutrients wh ich drain from the surrounding farmland. In the central part of the fjord, average winter concentrations ofnitrate andphosphate are about 70 and 3 /LM respectively, but during blooms, nutrient con­centrations reach limiting levels in the photic zone suggesting that production isthen based on rapid re-mineralization (the input rate of nutrients from land be-

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58 '!DM FENCHEL ET AL.

350:\00

\•II

250200pM

15010050

C1I,

o ./", 2 ,-

o r:" .----- ..\ ..I•I

r\,·0,. 0

'0I .~ '0t 111,,,.0,4 <:D. 11-", ...... 0

D. ....

L::. o~6 0;--......... 11• 0 "<,

\ 0 ---------------~ ~ 111D. 0 S' --

\ 0Nil,' 0

<,

oo

2

4

6

8

10

12E

..c 14ä.d.)

"0 16

18

20

22

24

26

28

Fig. 8. Profiles of oxygen, sulphide, ammonia and methane on August 8, 1994.

ing far too low to explain the production rate). The oscillatory pattern ofalgal bi­omass and of productivity is probably generated by protozoan grazing as indicat­ed by the periodically very high densities of algivorous protozoa (see later sec­tions). The possibility of spatial heterogeneity is ruled out by the lang residencetime of the water. Sediment trap studies (County of Northern ]utland, unpub­lished) indicated that mass sedimentation ofdiatoms does not take place and thatonly a small fraction (about 3%) of the production (as measured by HC-bicar­bonate) reaches the bottom. This surprisingly low value is, however, not inconsis­tent with our quantification ofbenthic anaerobic mineralization (as discussed inmore detail below), in particular when it is considered that the production rates(with which the sedimentation is compared) overestimate the net production.

The quantitatively most important phytoplankton organisms are the diatomsSkeletonema costatum and Stephanodiscus hantzschiiwhich alternate as the dominatingspecies. Other important primary producers are various diatoms, dinoflagellatessuch as Heterocapsa and Katodinium, cryptomonads, a variety of phototrophicnanoflagellates and the phototrophic ciliate Mesodinium rubrum.

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MICROBIAL LIFE IN AN ANOXIC FJORD 59

<,

Nil,

40:\020

/IM

10o4030

~M

2010

~.-­/" 0,

I

o .I \I "\ ~

\,NO, •6. )\\68 I

NO,\-,. 6.::,.. r»:y'. 4;', "I ~\ ~ .6 '

~ .~ i\ .6 I\ s'

6 eH. . B ......

\ .

10

E

-" 14'ii"-o

16

18

20

Fig. 9. Profiles of oxygen, sulphide, ammonia, nitrate, nitrite and methane at the chemocline onAugust 8, 1994.

The density and diversity ofmetazoan plankton is low. I t is dominated by cope­pods (Eurytemora hirundoides, Pseudocalanus minuius and Acartiaand Centropages spp.)and rotifers (Synchaeta sp.). Meroplankton (mollusc and polychaete larvae) aremore numerous. The poor zooplankton may in part be explained by the sulphidicdeep water which will be lethal to sedimenting eggs.

Chemical gradients and microbial processes

Gradients 0]oxygen, inorganic N-compounds, totalsulphideand methane. The vertical dis­tributions of dissolved CH4, total S2-, NH4 + and O 2 are shown in Fig. 8; Fig. 9shows the gradients around the chemocline in more detail and also includes verti­cal profiles of N03- and N02-. The oxygen tension decreased from roughly at­mospheric saturation to 5-10% atm. sat. between 7 and 9 m; below that, 02-ten­sion decreased more gradually and O 2was undetectable at 16 m. Measurementsmade during the aftemoon showed supersaturation (up to 120% atm. sat.) be­tween 2 and 5 m. Sulphide became detectable at 14.5-15 m and increased to300-400 /LMbelow 25 m. Methane concentrations exceeded 30 /LM in the deepestwater and decreased to about 1 JLM at the oxycline; above the oxycline CH4-con­

centrations were within the range 0.5-0.9 /LM. Ammonia decreased from > 200/LM at the bottom to 13-15 /LM at the oxycline and then decreased further to 2-3/LM in the oxic layer. Nitrite and nitrate showed a clear maximum at 13 m depth.

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60 TOM FENCHEL ET AL.

Table 1. Potential nitrification rates measured at 11 °C.

Depth (m)

9.510.511.012.013.014.0

/Lmol J-1 day'!

0.470.180.080.071. 710.00

SD

0.020.010.200.410.820.00

These two compounds were undetectable at greater depths; in the oxic zone con­centrations ofN02- and N03- were about 0.1 and 1-3 p.M, respectively. It is elearthat sulphide oxidation took place mainlybetween 14.5 and 16 m and that thebulk of nitrification took place around 13 m depth (see also Table 1).

With the exception of a small peak in NH4 + -concentration around 11 m (Fig.9) which we are unable to explain, the general picture resembles the chemical zo­nation patterns recorded in other anoxie basins (e.g. Rheinheimer et al. 1989 forthe Baltic Sea).

Rates0]nitrification and denitrification. Nitrification was measured as potential rates(Table 1) and thus at ammonia concentrations exceeding in situ levels. A widerange of Km-values have been reported for nitrification (Henriksen & Kemp1988) and there seems to be a tendency for Km-values to be elose to in situ ammo­nia concentrations. Very low Km-values (0.7-1.5 p.M) have been reported foropen oceanic waters. It is reasonable to assurne that the Km ofnitrifiers in Mari­ager Fjord is also elose to the measured ammonia concentrations and that themeasured potential rates yield a reasonable estimate of actual rates. They will beused as such in the following calculations concerning the carbon budget of thefjord.

Table 1 shows (in accordance with Fig. 9) that nitrification took piace mainlyaround 13 m. The nitrate profile also suggests that denitrification took place inthe water column immediately below the chemoeline. Attempts to quantify thisdirectly failed, however, because rates were lower than the detection limit of themethod (<=::: 1 p.M d-l) at all depths. Indirectly we have estimated denitrificationto be about 1 mmol m-2 d-l (see below); if denitrification took place over morethan 1 m ofthe water column then the true rate (per unit volume) would be belowthe detection limit.

The methane budget. Methane production could not be detected in the watercolumn (detection limit 0.1 nM d-l). Since ciliates with endosymbiotic methano­genic bacteria were present in the anaerobic water column some methanogenesismust have taken place. Such species may produce up to 8 pmol cell-l h-l (Fenchel& Finlay 1992), but their relatively low numbers « 1 mI-!) could not haveproduced CH4 at a detectable rate.

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MICROBIAL LIFE IN AN ANOXIC FJORD

mM CH 4

61

~

Su

'-"

..c+->c,(1)

Cl

o

5

10

o

<,

1

<,

TI

2

<,<,

<,

Q

\Q

Iq

-,Q.

<,o

3

15 Methane saturationat in-situ presslIre

Methane xuturätionut STP

20Fig. 10. Sediment methane profiles of two cores; also shown are the saturation limit at at­

mospheric (STP) and at in situ pressure.

Sinee methanogenesis eould not be deteeted in the water eolumn the methanein the water eolumn must be supplied from the sediment. Two sediment concen­tration profiles (Fig. 10) were obtained. Applieation of Fiek's first law, using theslope of the (almost) linear part of the profile elose to the surfaee, and a diffusioneoeffieient value for CH4- of 5.8 x 10-6 crn? s-l (Iversen &J0rgensen 1993) yieldedfluxes of 0.3 and 1.5 mmol m-2 d-I , respeetively. Sinee only these two eores wereanalysed we ean only offer this erude estimate of sediment methanogenesis.

Methane oxidation was observed in the oxie (below 5 m) as weIl as in the anoxiepart ofthe water column (Fig. 11),the highest rates being observed at the oxyeline;

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62 'IQM FENCHEL ET AL.

nmol CH4e .1d- 1

o 1 2 3 4 5 6 7

o

5

10.-E

'-"..c

15

c<......Cl.

tvQ

20

+DME--,25

30F'ig. 11. Methane oxidation rates in the water column, measured with and without the ad­

dition of dimethyl ether on August 9, 1994.

relatively low values were observed below the oxycline. Addition of dimethyl ethercompletely inhibited methane oxidation above the oxycline and significantly in­hibited methane oxidation down to 16m. Below 17 m dimethyl ether had no effecton CH4-oxidation dernonstrating anaerobic oxidation below that depth.Anaerobic methane oxidation could also be measured in the upper 3 cm of thesediment, where it amounted to 52 /Lmol m-2 d-i.

The depth integrated methane oxidation for the entire water column was 0.05mmol m-2 d-I of which about 33% is anaerobic. This result differs from thatfound in other stratified marine basins (Reeburgh 1980, Lidstrom 1983, Wardet al. 1987, Reeburgh et al.1992) and in merornictic lakes (Iversen et al. 1987)where in general< 1% of the methane oxidation took place in the oxic part of thewater column. On the other hand our aerobic rates are 10-10000 times higherthan aerobic rates measured elsewhere. Our measured anaerobic rates are

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MICROBIAL LIFE IN AN ANOXIC FJORD

Table 2. Methane flux from water to atmosphere.

Date mmol m? day' wind speed (m S-l)

10 Aug. 0.19 < 10.30

17 Aug. 4.32 -84.524.174.98

63

comparable to the average rate of anaerobic methane oxidation in the watercolumn ofthe Black Sea, but about 1000 times lower than reported from a strati­fied fjord (Lidstrom 1983) and 25 times lower than in a meromictic lake (Iversenet al. 1987). One reason for this may be the relatively short residence time formethane (1-2 yrs) in Mariager Fjord (see below) as compared to 3.6-73 yrs fortheBlack Sea (Reeburgh et al. 1992) or 1.5-2.9 yrs for Big Soda lake (Iversen et al.1987).

The methane flux from the water column to the atmosphere was measured un­der two different wind conditions (Table 2). The measuredfluxes were comparedto the predictions of an eddy diffusion model (Harman & Hammond 1985). Themodel predicts that the flux equals K I (C m - Ce) where K I is a gas transfer coeffi­eient, and C m and Ce are the measured CH4-concentration, and a concentrationin equilibrium with the atmosphere (1.7 ppm), respectively (Yamamoto et al.1976). The result corresponded to a supersaturation of about 100 times. Thevalue of K, depends on wind speed, and an empirical relation was proposed bySebacher et al. (1983). Using this model with our data, we predict a CH4-flux tothe atmosphere of 0.21 and 2.24 mmol m-2 d-I for the calm and the windy day,respectively. The former figure corresponds weIl with our data while the latter isonly about half the measured value. In fact, the employment of a closed chambershould have minimised the effect ofwind, but wind driven pumping action ofthefloating chamber may have increased the flux. Finally, it is possible that some ofthe flux results from ebullition from the sediment. This process is likely to be het­erogeneous in time and space and to occur especially during periods of fallingbarometrie pressure (which again correlates with high wind speeds). Gas bubblesbreaking the water surface have been observed by scientists of the Office of En­vironment, County of Northern Jutland (personal communication) and we ob­served that bubbles form at around 15 cm depth in sediment cores. Our sedimentmethane profiles resembled those observed by Chanton et al. (1989) in the WhiteOak Estuary at Cape Lookout Bight (North Carolina) and there about 50% ofthe methane was released as bubbles.

The water column was about 100 times supersaturated with CH4 which ismuch higher than the 30% supersaturation reported for the oceans in general(Ehalt 1974). The global estimate for methane emission from the sea is 10 Tg yr l

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64 TOM FENCHEL ET AL.

Table 3. Methane budget for Mariager Fjord, Aug. 1994. The total integrated methane pool in thewater column was 175 mmol rrr".

mmol m·2d· j

Sourees:production water columnflux sediment/water column

Sinks:flux water surface/atmosphereoxidation in water columnoxidation in sedimentflux in water columnI

Ibased on concentration gradients.

low estimate

o0.3

0.24±O.080.050.0520.43

high estimate

o1.5

4.50±O.350.050.0520.43

(Cicerone & Oremland 1988) and the open oceans represent the major part ofthesurface of the sea. However, our results indicate that fjords and other coastal ma­rine waters may constitute a relatively important source of atmospheric methaneas compared to the open sea. The surface area of the stratified part of MariagerFjord is about 3.8 km2 and our measurements suggest a yearly methane ernis­sion ofO.5 to 1x10-5 Tg yr1 which corresponds to 0.0005 to 0.001 0/ 00 of the globalestimate ofmethane flux from the sea to the atmosphere. Table 3 summarises themethane budget of Mariager Fjord.

Dark uptake of Ne-bicarbonate in the chemocline. The result of one of the incubationexperiments is shown in Fig. 12. A distinct peak is evident at 14-15 m depth anda less distinct one at 8-9 m. The former can possibly be assigned to sulphide oxi­dation. The integrated C-assimilation amounts to about 10 mmol C m-2 day-l.This is probably an overestimate of chemolithotrophic produetion beeause anunknown fraetion must be assigned to CO2-assimilation by heterotrophie bae­teria (Sorokin 1972).

The other incubation experiment yielded a lower value (3.2 mmol C m-2

day-l); in this case, however, ineubations were made only with water sarnplesdown to 15 m and only partially included the lower peak ofbiearbonate assimila­tion.

The carbon budget and therelative role ofterminalanaerobic mineralisation processes. I t is ap­parent from Fig. 8 that the gradients of sulphide, methane and ammonia are ap­proximately linear below 18-19 m and down to 25 m, suggesting a relatively cons­tant vertical mixing coeffieient at these depths. Provided that the three com­pounds are eonservative in the anoxie water eolumn, the slopes of the gradientsshould be proportional to the vertical fluxes (Table 4) and they can therefore beused for calculating the relative importance of methanogenesis and sulphatereduction in the sediment, and the relative role ofmethane, sulphide and ammo-

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MICROBIAL LIFE IN AN ANOXIC FJORD 65

8

9

10

11

~

s'--'

-5 120..(1)

-0

13

14

15

16

o 0.1

zzrno! C EI h'

0.2 0.3 0.4

Fig. 12. CO2-assimilation in the chemocline based on 14C-labelled bicarbonate incubations onAugust 9, 1994.

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66 ruM FENCHEL ET AL.

nia oxidation in the chemocline. The absolute rates of the individual processesand the total anaerobic mineralisation of organic C could also be estimatedprovided that the absolute rate of at least one of these processes is known.However, the assumption that the three compounds are totally conservative in theanaerobic water column is at best an approximation. Some anaerobic degrada­tion (fermentation) and sulphate reduction is likely to take place in the watercolumn and this may lead to a net release of ammonia. Sulphate reduction below25 m was indicated since the sulphide profile was convex upwards beneath thisdepth (not shown). Indrebe et al. (1979) found for a similar Norwegian stratifiedestuary that the largest fraction of sulphate reduction took place in the anoxiewater column rather than in the sediment. Anaerobic methane oxidation(through sulphate reduction) was evident, but its rate was low relative to themethane flux. It is more important that some methane is released through ebulli­tion from the sediment. As discussed in more detail below, this mechanism willlead to an underestimation oftotal anaerobic C-mineralisation and ofthe role ofmethanogenesis when the gradients (Table 4) are used for such calculations.

Under anaerobic conditions sedimenting organic material is in part minera­lised through nitrate reduction. The bulk of the material will, however, bedegraded by fermenting bacteria leading to low molecular weight fatty acids(principally acetate), H 2 and CO2; organic N will be released as NHi + throughdeamination. Hydrogen and volatile fatty acids will then be completely minera­lised through sulphate reduction or, when sulphate has become depleted (in thesediment), be transformed into CHi by methanogenic bacteria. The terminalanaerobic mineralisation of 1 mol C will yield either Y2 mol CHi or Y2 mol S2­(Fenchel & Finlay 1995).

If we assurne that the C:N ratio of sedimenting organic material is 6, then arelative vertical NHi + -flux of unity (Table 4) corresponds to the anaerobicdegradation of6 units ofC. Ignoring denitrification for the moment we would ex­pect that the sum ofthe vertical fluxes of CH, arid S2-equals Y2 ofthe total rateof anaerobic C-mineralisation. Table 4 shows that the fluxes (relative to that ofammonia) are 2.3 and 0.21 for S2- and CHi, respectively. This result,2 x [2.3 +0.21] = 5, is therefore consistent with the theoretical ratio betweenNHi + flux and the sum of CHi and S2- fluxes, in particular since the missing[sulphide + methane] equivalents may be accounted for by denitrificationand/or an underestimation of methane production due to ebullition.

The nitrification rate (= vertical flux of ammonia) was estimated to be 2.16mmol m-2 day! (Table 1). Based on the relative fluxes of NHi +, S2- and CHi(Table 4) it is therefore possible to estimate the quantitative rates of methanogen­esis and sulphate reduction to be 0.42 and 4.97 mmol m-2 day-l, respectively. Wemay further estimate that about halfofthe nitrate produced in the chemocline ( ""1 mmol m-2 day·l) diffused downwards to be denitrified in the upper part oftheanaerobic zone. One mole of nitrate can oxidise 5/4 mole C. The total rate of

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MICROBIAL LIFE IN AN ANOXIC FJORD 67

Table 4. Concentration gradients', and vertical flux of sulphide, ammonia and methane in theanoxie water eolumn of Mariager Fjord.

Gradient Relative flux Flux?mmol m" mmol m-2day'

S2- 43.7 2.3 4.97NH: 19.0 1.0 2.16CH4 4.0 0.2 3 0.43 3

"The gradients are calculated for 18-25 m depth. The S2--gradient represents the mean of threeprofiles measured August 8, 9 and 10, respectively; the NH; and CH4-gradients are based on pro­files measured on August 8. 2Calculated with the assumption that the measured nitrification rateequals the vertical NH; -flux. 3The CH4-flux applies only to dissolved methane and ignores ebul-

lition.

anaerobic C-mineralisation can therefore be calculated to be 2 X [4.97 +0.42] +5/4x1 = 12.03 mmol C m-2 day-1 (144 mg C m- 2 dayl}.

There are several uncertainties in these calculations, some of which have al­ready been discussed. To these we may add that it is possible that sulphide consti­tutes a substantial part of the substrate for denitrification in which case the C­mineralisation is overestimated. Conversely, if a large fraction of the methaneproduced in the sediment escaped through ebullition (cf. Tables 3-4) the rate ofanaerobic degradation is underestimated. If we accept the highest measuredCH4--flux from the water to the atmosphere (Table 3) of 1=:: 4.5 mmol m-2 day! asan average value (viz. that methanogenesis almost equals sulphate reduction inimpartance), then the above estimate of anaerobic C-mineralisation should beincreased by about 60%, but this is rather unrealistic.

It is unknown to wh at extent these estimates represent annual averages orwhether they are representative only for the period of investigation. Sedimenta­tion of organic material is confined mainly to the summer months with highprimary production. It is therefore of some interest to calculate the mean resi­dence time in the anoxie zone. In the deepest part of the water column (> 18 m)the vertical (mixing) diffusion coefficient can be estimated as [flux/gradient] to beabout 0.12 m 2 day! (0.013 cm? s·l). The average transport time of a moleeulemoving from the sediment to the chemocline can therefore be calculated to beabout 2 yrs. Another way to estimate residence time is to divide for each com­pound the depth integrated concentration by flux. For sulphide this again yieldsabout 2 yrs, for ammonia 2.3 yrs, whereas methane yields 1.6 yrs. If the highwater column/atmosphere flux rate far methane is used in the calculations a resi­dence time of only 0.1 yr is obtained. This is unrealistically low and suggests thatthe high waterlatmosphere flux measured on one occasion reflects an episodicrelease of gas bubbles from the sediment. The low estimate of methane flux istherefore the most realistic one as it renders consistent the figures in Table 3. Resi­dence times varying between 1.5 and 78 yrs have been found for other stratifiedwater bodies (Scranton & Brewer 1978, Iversen et al. 1987, Reeburgh et al. 1992).

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68 IDM FENCHEL ET AL.

Short term fluctuations in the primary production of the surface layers willtherefore not be reflected in pulses of sulphide arriving at the chemoeline.However, it is still possible that the vertical fluxes ofsulphide, ammonia and (dis­solved) methane show seasonal changes and lag months behind the primaryproduction rather than reflecting an annual average. The fact that thechemoeline migrates about 5 m downwards during the winter rnay also indicatethat the gradients never reach a steady state. A methane profile measured in Sep­tember 1991 (L. Dueholm, H. Kloch & P.K. Kristensen; unpublished mastersthesis) is almost identical to our methane profiles suggesting some reproducibilityfrom year to year. But we have no data which can resolve whether the estimatesof fluxes really represent an annual average of process rates.

Ifwe accept that the estimate of anaerobic C-mineralisation is valid as an an­nual average it may be compared to the annual primary production. As discussedin the introductory section, the annual average net production of particulate or­ganic material in the photic zone is probably 1-1.5 g C m-2 dayJ. If 144 mg C m-2

day'! is accepted as a reliable average value for anaerobic degradation this meansthat 10-15% of the production sinks down to be degraded in the anaerobic zone.To this should be added the accumulation of organic non-degradable materialand ofpyrite in the sediment. Measurements ofthe downward flux ofparticulatematerial in sediment traps (M. Olesen & C. Lundsgaard, in preparation) duringalgal blooms support the result that the largest part of the primary production ismineralised in the oxic zone.

There are few estimates ofrates ofmicrobial anaerobic processes in other waterbodies with an anaerobic water column with which to compare our results.Results from studies of sulphate reduction rates or sulphide fluxes in the watercolumn of some Norwegian fjords (Indrebe et al. 1979, Dyrssen 1986), however,are very elose to those found in the present study. Kemp et al. (1992) reported ver­tical fluxes of ammonia and of sulphide in anoxie bottom water of ChesapeakeBay which were about 3 times higher than those found in the present study.

Processes at the chemocline. The high chlorophyll content of the oxic zone and theresulting attenuation of light in the upper metres of the water column preeludephototrophic oxidation of sulphide in Mariager Fjord. Reoxidation of thereduced metabolites of anaerobic mineralisation is therefore oxidative and takesplace in the chemocIine. Nitrification and methane oxidation were measured tobe 2.16 and 0.05 mmol substrate m-2 day-l , respectively. We may further assurnethat the re oxidation of sulphide is complete and equals the sulphide flux estimat­ed to be 4.97 mmol m-2 dayl , Values ofmolar yield of chemolithotrophic bacter­ia (moles of C fixed per mole substrate oxidised) are somewhat variable in theliterature, but mean values can be taken to be 0.35,0.2 and 0.35 for complete sul­phide, ammonia and methane oxidation, respectively (Schlegel & Bowien 1989).

U sing these values the assimilation of inorganic C in the chemoeline due to

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MICROBIAL LIFE IN AN ANOXIC FJORD 69

chemolithotrophic bacteria can be estimated to be about 2.3 mmol C m-2 day!(=:; 28 mg C m·2 day'!) which represents about 19% of the organic C which isdegraded anaerobically or =:; 3 % of the primary production in the photic zone.This estimate ofC-assimilation is considerably lower than that derived from the14C-incubation experiment (=:; 10 mmol C m? day'!).

Heterotrophie flagellates and rhizopods

The study ofthe diversity and distribution of these organisms was less straightfor­ward than in the case of the ciliates. Only a few large flagellates could be identifiedto species level with certainty in Lugol-fixed sampies or with the fluorescencemicroscope (the remaining species being lumped together in groups such as'nanoflagellates', 'dinoflagellates') . The vertical range ofmost species was deter­mined on the basis of presence in enrichment cultures (incubated under aerobic,microaerobic or anaerobic conditions) and on their presence in centrifuged for­malin-fixed sarnples and in centrifuged unfixed water samples from 2 and 20 mdepth. Many ofthe aerobic forms were found only in the unfixed 2 m sampie, butit is assumed that these were distributed throughout the mixed (0-7 m) zone.Some species which are considered to belong to the oxic zone were also recordedin low numbers in fixed sampies from the anoxie zone, or they appeared in aero­bic cultures based on water from the anaerobic and mieroaerobie zones. Thesespecies were predominantly attaehed or semi-attaehed forms (some ehoano­flagellates, bieosoecids, the amoeba Jiznella) which, it may be assumed, hadreached the deep parts of the water column together with sinking detritus parti­eIes. These species did not grow in anaerobie ineubations (except for one popula­tion of Bicosoeca sp. which maintained itselffor some days). Otherwise there wasgenerally a elose correspondence between the speeies obtained from anaerobieenriehment eultures, and those which oeeurred in fixed or living samples from thechemoeline and downwards.

Altogether at least 44 speeies of heterotrophie flagellates and rhizopods werefound in the water column. Among them, six seemed to be restrieted to themicroaerobic zone and nine to the anoxie zone while the remaining 29 specieswere exelusively or predominantly restricted to the oxic zone. Figure 13 providesan almost eomplete list of the recorded species and a presentation of vertical dis­tribution patterns. The figure also suggests the existence of characteristic aero­bic, microaerobie and anaerobie assemblages offlagellates and rhizopods. Figure14 shows the quantitative distribution ofthree species whieh could be identified;these species represent the three flagellate communities.

During the last decade, understanding ofheterotrophie flagellate diversity hasadvanced considerably (Larsen & Patterson 1990, Patterson & Larsen 1991 andpapers cited therein) although it has not yet been possible to assign all describedspecies to the major recognised taxonomie groups. Also, comprehensive descrip-

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70 lDM FENCHEL ET AL.

t:: .~

~ ~ ~

':I ~t::.., .., - t)o

~ ::: :.:: t:: 0. t::l"" • '- - "' ...e:: 0. ec t::I ...

-s: .., <n t:::- t:: e::::- ~ ~: E ..,?~ Ec Cl c.. 0.:: '::: '- Cl '-

E E ~ ~ .~ ~ ~ E~~ .s ;; ~ :; E 'E ~ ~

~ t ~~'E ~ ~.~ ~~ ~ ~ ~ ~ ... ~ t:: Clf-Q..~;;::r:t--.:Z::~Q..

<'Ici." ~

. ~ "-: ~ c: ~.~ ~~~~tiEOu <U 4J - ..., E__ .. :::"_ t::I

~ <;1 ro t::..s: co

~~~~~.~

-=.:~~a~

E'" ~

.., . ~ .~ ~ ~ E ~::: ~~ ~ ~ ~ s ~ ci..~._ ~ t3 ~ .~~ c, V1 ... ~ ~ ~ ~. e VI E .~ ~.:: ~ ...~'" .~ ~~ '~'-.~~t::~~~~ ... EiEO().~ ~ e .:: ~ ~.;; E ~ "'l'~ :: e ~ ~ \.J ~ -e .:; _~~o.t::I~~o::;~~E:~t::~O~ !E~'~ .~.~ ~ ~ ~ (; t:: ~ t:: ~ t:: ~ ~ 0 g,::: Cl ~.~ .'~::: ti ~ ~ ~ ~~ ~.~ ~} ~ ~.~ E E E ~ ~ .~:~ ~ ~ ~ ~~:§ ~ ~ e ~ ~

-e - ~ .s Cl. ~ ~ ~ ,0 ~ ~ CL"t;s t::~ ~ ~.~ Vl ~"'t:l ~ ~~ ~ ~~~~~~~~~~~~~·~·:t::I~:::E~Q.::~~~~.9

Q~~~~~~~~~~c~~&~~~~~Q~~~~]

4

10

16

12

14

18

26

20

24

22

""E

I,•,I•,

~ !t t t;e.I I I

I I II

I I 1I

• ., I

I II

I I1

, +I, +I I

Fig. 13. All heterotrophie flagellates and rhizopods eneountered in Mariager Fjord and their verti­cal distribution in the water eolumn. (Some insuffieiently observed forms have been excluded, and

three speeies of Bodohave been lumped together as 'bodonids').

tions ofplanktonic flagellate and rhizopod diversity have appeared only recently(Thomsen 1992, Vers 1992 and papers cited therein), These have made it possibleto identify practically all forms encountered in the oxic surface layers to species,or at least genus level. These species will therefore not be discussed in detail be­yond the species list provided in Fig. 13. However, it should be mentioned thatrelative to the usual planktonic assemblage offorms such as the choanoflagellates,Paraphysomonas spp., Pteridomonas sp., the bodonids, Pseudobodo tremulans Griess­mann, Cafeteria roenbergensis Fenchel & Patterson, and Bicosoeca sp. (Griessmann1914, Fenchel 1982, Vars 1992), algal-feeding species (Quadricilia rotundata (Skuja)Vers, Leucocryptos remigera Vers, an unidentified heliozoan and probably several ofthe heterotrophie dinoflagellates) played an unusually large quantitative role.This reflects the fact that the samples were taken towards the end of an algalbloom.

In contrast to the flagellates in the surface layers, about % ofthose encountered

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MICROBIAL LIFE IN AN ANOXIC FJORD 71

Postgaardi m ar i ag er e n si s Oxyrrhis m ar i n a

o 10 20 o 100 200 . . .0

2

4

6

8

10

'"'" 12...'"E

14

16

18

20

22

24

26

cells rnl'

Fig. 14. The vertical distribution ofthree flagellate species in the water column on August 8,1994.

in the microaerobic and anaerobic layers seemed to be undescribed, reflectingthefact that these forms are still rather neglected in the literature. Anaerobic andmicroaerobic habitats harbour several species of flagellates whieh (at least at thelight microscopicallevel) are difficult or impossible to assign to major recognisedtaxonomie groups. Some of these organisms combine traits which are charac­teristic of species belonging to Heterolobosea, Retortamonadida or to the generaJakoba or Cryptaulax (see Patterson & Larsen 1991), but most differ to a larger orsmaller extent from anything previously described. We will here provide abriefdescription ofsuch forms (flagellate spp. 1-5) found in the Mariager Fjord materi­al, but we believe it is not useful to give names to this complex ofspecies until theirtaxonomy has been further clarified.

Nine species occurred exclusively or almost so in the anaerobie zone beneath15 m depth. All were observed alive in water samples and most of them also infixed material. All except two eould be grown anaerobically.

Tetradimorpha marina n.sp. (Figs 15,a and 16,d-f). This heliozoan-Iike organismis almost spherical and most cells measured between 15 and 20 Jlm in diameter.The spherical nucleus is situated towards one side adjacent to a depression in the

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72 TOM FENCHEL ET AL.

cell surface, from the bottom of which either two or four flagella arise. Cells withfour flagella might represent early division stages, but there was no correlationbetween the number of flagella and cell diameter. About twenty axopodia withextrusomes radiate from the cell. The cells are packed with spherical food vacu­oles containing bacteria and the surrounding cytoplasm is filled with rod-shaped("'" 1 pm) organelles or endosymbiotic bacteria. The cells are capable of absorb­ing the axopodia and oftransforming into a slug-like flagellated form (Figs 15,aand 16,f). The organism resembles in many ways the helioflagellate Ciliophrys ma­rina (see Davidson 1982) which, however, has only one flagellum which is immo­bile in the heliozoan stage. In most respects, however, it re sembles more eloselythe centrohelidean Tetradimorpha (see Brugerolle & Mignot 1983,1984) althoughthis genus has not previously been reported from seawater. Only studies of thefine structure will reveal the true taxonomic position of the organism from Mari­ager Fjord, but due to its elose resemblance at the light microscopical level we ten­tatively assign it to the genus Tetradimorpha. The species was exdusively foundgrowing in an anaerobic enrichment culture from a deep sample which containedsome sedimentary material and it is possible that it is exclusively a benthic form.

Postgaardi mariagerensis n.gen.n.sp. (Figs 15,c and 16,a-c) was found throughoutthe anaerobic water column with a distinct maximum in abundance between 18and 22 m depth (Fig. 14). Its shape is ovoid and the cell is rigid. Two almost equal­ly sized flagella emerge fromthe bottom of a reservoir at the anterior end of thecell; the posteriorly directed flagellum is acronematic. The ovoid nucleus is situ­ated in the anterior halfof the cell. The most characteristic feature is that the cellis covered by 50-100, "'" 7 p.m long rod-shaped bacteria, which in fixed specimenstend to peel off(Fig. 16,b). Most features ofthis organism suggest that it is an eu­glenoid flagellate and that it is related to the genus Anisonema, although it differsin that the reservoir is not situated on the left, that the flagella are ofroughly equalsize and that the trailing flagellum lacks the characteristic thick basal part. Thespecies was observed alive in freshly collected water sampIes, butfailed to grow incultures. The presence of ectosymbiotic bacteria is reminiscent of many marineanaerobic ciliates such as in the anaerobic Cristigera spp. (see the ciliate section ofthe present paper). The nature of the symbionts of Postgaardi is unknown.

A colourless dinoflagellate (Fig. 15,b) also occurred throughout the watercolumn below 15 m depth. The cell is roundish-ovoid, flattened dorso-ventrallyand measures about 17p.m in length. The epicone and hypocone are more or lessequal in size and they are separated by a girdle which runs only on the left halfof the cell, The sulcus of the hypocone widens towards the apex. The cytoplasmcontains numerous inclusions which may be bacteria. It has a delicate theca andit undoubtedly belongs to the genus Hemidinium. It is possibly identical to H sali­num Anissornova which has previously been recorded from a saline lake (Anis­somova 1926, Javornicky 1962). A related species (H thiophilum Konrad) hasbeen recorded from sulphidic waters in a Belgian salt marsh 0 avornicky 1962).

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b

MICROBIAL LIFE IN AN ANOXIC FJORD 73

Fig. 15. Anaerobic flagellates from Mariager Fjord. a: Tetradimorpha marina n. sp.; b: Hemidinium sp.;c: Postgaardi managerensis n.gen., n. sp.; d: flagellate sp. 5. All scale bars: 5 p.m.

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74 TDM FENCHEL ET AL.

Two species belonging to the Diplomonadida, Hexamita inflata Dujardin andTiepomonas agilisDujardin (Fig. 17,c and d) are known to inhabit a variety ofanox­ic habitats (Brugerolle 1991, Mylnikov 1991) and they also occurred throughoutthe anoxie water eolumn in Mariager Fjord (a few Hexamita eells were also foundin a forrnalin-fixed sample from 10 m depth). Both species could be grown inanaerobic cultures.

The pelobiont Mastigamoeba sp. 1 (Figs 16, g-h and 17,b) was found in anaerobicenriehment cultures at all depths below 19 m and could be maintained in purecultures. The oblong eell measures about 20 p-m and the anteriorly directedflagellum is 40-45 p-m lang. The cell is smooth and the cytoplasm seems almostempty and featureless. Typically only a few small pseudopodia form in theposterior end, but the cell can change in to an amoeboid form with pseudopodiaforming in several directions. The species can also eneyst. It differs from all previ­ously described species in that the nucleus, which is situated at the anterior endofthe cell, is divided into an anterior and a posterior part (Fig. 16,h). It has previ­ously been isolated from anaerobic shallow water sediments (Fenchel & Patter­son, unpublished observations),

A flagellate which appeared in anoxic incubations and in fixed samples fromdepths exeeeding 20 m is identical to Percolomonas cuspidata described by Larsen &Patterson (1990). These authors, who found it in sulphidic sarnples, considered itto be a heterolobosean, but it has also been suggested that it is a Chilemastix sp. be­longing to the retortamonads (Brugerolle 1991). It is shown in Figs 16, i and 17,f.The cells measure around 25 p-m and are drop-shaped, ending posteriorly in acup-shaped tail which may attach to a substratum. It has four flagella ofroughlyequallength. Two of the flagella are typically found in the groove whieh has astrongly developed right margin; bacteria are brought into the groove with watercurrents generated by the flagella and the bacteria are ingestedin the groove.

Flagellate sp. 4 (Fig. 17,e) grew in anaerobic cultures and was found in fixedsamples at all depths below 15 m. It is bean-shaped and measures about 12 p.m.

It carries two aeronematic flagella of which the posterior one is slightly langer.The flagella are inserted behind the anterior tip of the cell. The posterior flagel­lum is normally situated in a groove; in the posterior end the groove narrows intoan open channel in which the posterior flagellum is normally situated. The or­ganism resembles (and may be identical to) a species described as Cryptobia bialataby Ruinen (1938) although she found that the posterior flagellum is attached tothe bottom of the groove by a membranaus structure.

Flagellate sp. 5 (Fig. 15, d) occurred in anaerobic enrichment cultures incubat-

~

Fig. 16. Anaerobic and microaerobic flagellates from Mariager Fjord. a-c: Postgaardi mariagerensis;d-f: Taradimorphemarina; g-h: Mastigamoebasp. 1; i: Percolomonas cuspidata;j: flagellate sp. 1; k: flagel­

late sp. 3. All scale bars: 5 usu.

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QP::o~o§z<z-cz-

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76

b

TOM FENCHEL ET AL.

uFig. 17. Anaerobic and microaerobic flagellatcs from Mariager Fjord. a: Mastigamoeba sp. 2; b:Mastigamoeba sp. 1; c: Tiepomonas agilis; d: Hexamita inflata; e: flagellate sp. 4; f: Percolomonas cuspidata.

All scale bars: 5 p.m.

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MICROBIAL LIFE IN AN ANOXIC FJORD

c

b

77

Fig. 18. Microaerobic flagellates frorn Mariager Fjord. a: flagellate sp. 1;b. flagellate sp. 2; c: flagel­late sp. 3; d: Cryptobia sp. All scale bars: 5/lID.

ed with water from 16 and 20 m depth. It measures 6-8 p.m in length. It has aweakly-developed groove near the anterior end, in which two flagella are insert­ed. The trailing flagellum is acronematic and it is much longer ("'" 34 p.m) thanthe anterior flagellum. The periphery ofthe cell is uneven due to vacuoles situat­ed beneath the cell membrane. It was not possible to find any described formwhich closely resembles this species.

Six species offlagellates seemed to be confined to the chemocline. Among themthe well-known heterotrophie dinoflagellate Oxyrrhis marina Dujardin occurred ata higher level in the water column, with a distinct peak of abundance at a depthof9.5 m (Fig. 14) where oxygen tensions of about 20% atm. sat. were measured.The five other species occurred deeper, with P02 values ranging between 0 and10% atm. sat. They all appeared exclusively in enrichment cultures underanaerobic or microaerobic conditions incubated with water within the depthrange 12-16 m (two species also turned up in incubations from 21 m depth).

Flagellate sp. 1 (Figs 16, j and 18, a) is ovoid and measures 10-15p.m. It has fourequally sized flagella which are inserted in the anterior part of a groove which ex­tends posteriorly for about % ofthe cell. The groove has a distinct right margin.Two or three ofthe flagella are usually found in the groove in the living cell. Thecell surface is covered by what looks like extrusomes. Superficially it does resem-

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78 TDM FENCHEL ET AL.

ble Percolomonas (see Patterson & Larsen 1991), but the equally sized flagella, theextrusomes and motile behaviour do not suggest a elose relationship. It was foundexelusively in incubations from 15 and 16 m depth.

Flagellate sp. 2 (Fig. 18, b) measures 8-12 p.m; it is flattened and, seen from theside, has an almost semicircular shape. There is a groove which extends alongalmost the entire ventral side and two flagella of unequal size are inserted at theanterior end of the groove. The organism elosely resembles, and may be identicalto, a flagellate which Ruinen (1938) found in sulphidic seawater samples anddescribed as Bodo edax. Our organism, however, does not seem to have akinetoplast so that it is probably not a bodonid. It occurred regularly in incuba­tions with water from between 12.5 and 15.5 m depth.

Flagellate sp. 3 (Figs 16,k and 18,c) is drop-shaped, the posterior end beingdrawn into a thin tail which sometimes seems to form fine pseudopodia withwhich it attaches to the coverslip. It measures about 8 p.m in length. Two flagellaof unequallength are inserted in the anterior end of the cell, The Ion ger trailingflagellum follows a narrow groove which spirals posteriorly along the cell. The an­terior flagellum is often held in a hook-like position in front of the cell (Fig. 16,k).The spherical nueleus is found in the anterior end of the cell. The two flagella, ofwhich the anterior is held like a hook, and the spiral groove, are reminiscent ofJakoba (Cryptobia) libera described by Ruinen (1938); see also Larsen & Patterson1990, but it is not identical to this species. It could also be a Retortamonas species(see Brugerolle 1991).It was found in enrichment cultures based on water sampIesfrom 12-16m depth, but it was also observed in a water sample from 21 m and itgrows weIl in strictly anaerobic cultures.

Cryptaulaxsp. (Fig. 18, d) has a cylindrical slightly curved cell which measuresabout 10p.min length. Two flagella are inserted elose to the anterior end. The long(about twice the length ofthe cell) trailing flagellum follows a groove which con­tinues almost to the posterior end of the cell. The shorter, anterior flagellum isacronematic. Given the elose resemblance to Cryptaulaxas described in Patterson& Larsen (1991) we have assigned the organism to this genus. It wasfound onlyin microaerobic incubations of water from 13.5 m depth.

Mastigamoeba sp.2 (Fig. 17,a) was found infrequently in incubations with waterfrom 13.5, 15 and 18 m depth. It is drop-shaped and tends not to form pseudopo­dia. The triangular nueleus is situated in the po in ted anterior end ofthe cell eloseto the insertion of the 7-8 p.m long flagellum. In contrast to Mastigamoeba sp. 1, itdoes have visible inelusions in the cytoplasm in the form of ingested bacteria.

Ciliates

With the exception of the tintinnids, which were identified principally from thefeatures of their loricae, the definitive ciliate identifications were made from thesilver-stained infraciliature (Fernändez-Galiano 1976, Esteban et al. 1995). In

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MICROBIAL LIFE IN AN ANOXIC FJORD 79

Cyclidium xenium(cells ml")2 3 4

Cristigera cirrifera(cells ml")

0,2 0,4 0,6 0,8 0--'-----'--' , , tl---'-~~~~~~-~

Cyclidium glaucoma(cells ml- 1)

2 3

10

~

11 S

20

2S

Fig. 19. Vertical displacement ofpeaks in abundance offour small scuticociliates. Abundance datafrom formalin-fixed material; species identifications from silver-carbonate-impregnated material,

most cases, sufficient information was obtained from the ciliates retrieved fromthe original water samples. In the case ofthe six small scuticociliate species in thegenera Cyclidium, Uronema and Cristigera, conclusive discrimination and identifi­cation of species depended on additional silver-stained preparations of erudelaboratory cultures.

Close scrutiny of silver-stained natural samples is time-consuming. The spe­cies which are relatively abundant are quickly found but it is often difficult to con­firm the absence ofa rare species. Nevertheless, on the basis ofpast experience ofsimilar studies (e.g. Finlay et al. 1993), we feel relatively confident that few if anyciliate species completely avoided discovery. We also feel eonfident therefore incombining our information on species identities with the eruder identificationsand quantitative information obtained using other methods. The displaced peaksin abundance of 'small scuticociliates' recognised in forrnalin-fixed materialcould, with the benefit of information from silver-stained preparations, beascribed to individual ciliate speeies having little or no overlap in their verticaldistributions (Fig. 19).

In total, 37 species were recognised in the water column. Eight ofthese couldnot be identified further than genus level and only one speeies (Cyclidium xenium)was definitely 'new'. Thus, the ciliates stand in marked contrast to the anaerobicand microaerobic flagellates (see above), ofwhich the majority offorms recordedin Mariager have not previously been described. Fig. 20 illustrates the verticaldistribution of all ciliate species recorded. The new species, together with thosewhich are represented in the literature only as poor or incomplete descriptions,are dealt with in more detail below.

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80 ruM FENCHEL ET AL.

~~ '" '"~ ~~I ~ ~~. ~~ ~

~ s e;, ~ '5 5 e;:s ~ '-:: E: ~ ti ~ ~ ~ Si ~ ~ E ~ .saat~~~'~I~~ ~~~.~~~·al ~.~.; .~~.g8.~ ~~ ~

~ g. s~ F"s:: ] ~ .s 5 8 C:i § ~ ~ § ~ ~ 8 8 ci.~ ~ ~ ~ 6 ~ ~ § ~ f g.:§ ~ ~~~§~·ö~~~§~~i~~~~E~~~E~~~~~~~~~EE.~~E:~~E: ';:: f,J ~ l::l Ei: 0 ~ 1: E ":l C""J ·S ~ ~ E: '_ t:::s ...... :;: .~ ~ .r:s :::s ..... "qJ ::s ~ E .:: _:: ~ E.:: ~i~i~~·~ä~~~~~~~.~.§·!]i]~~i~~]~~·~.!·5.52i~.~!~~o~~]~E=~b~~~b~]~~~~g~~b~~~E]115~o5~c:s '\J - sc ~ (,J , <:) ~ (.) ........... <:::l '0 Cl ~ ~ Cl 9.. (;:) (;:) ...... 9..~ ..... c.J:: '::: ~ u "'l ~ ..... ..:::: C") ~ 0

~~~~8G6~~G~8~SS~G~~~~~~~~~~~~G~~~~~QS

ilj: t :I• '

t'l I:lt'• : I1 I I~ I I I I

• I I l. ~ I c" , ~ I I' I I.. -.. ' I I I I I: • •• , e •• J

±I : I I I ~ I ,

~I"~,I~"I 00 •• 10•• • I e •••• I ••• r• 0 411 •• 1

1. 0 iO • • •I

I ' I~ ~ ), 'I ,

.. -

+,.. ~

il1 /.. .. ..

~ J ~

I I.'•• I. • CI

I I : .. I : II I I I I I II I ,I I ~I I , I I ....I I I I I 1 ~J I • I I e •

• • e 0 •••• •••• •••.. .. "

..i++++++ 1rI f!....

2

4

01 • ... dti .. • !a

8

6

'E' 10 ; : e :

4) • • • •~ : : : :S 12 - - ....'-' "1---'" - .. -'+J • • •fr 14 : : ::A ,I " " - .... "

r • • • •••16 ' : - - .... "- :.'::T"

181- :' ;. 1"11" 120f-. '! t I'

, I

••221-' i. j i

24f-. t lli t26' --.1

Fig. 20. Vertical distribution in the water column of all ciliate species.

Similar to the flagellate community, the ciliate community in the water columncan be divided into three fairly distinct communities, each corresponding to oneof the three principal water strata (Fig. 21): the upper, mixed aerobic layer; themicroaerobic layer (which also supports significant salinity and temperature gra­dients, i.e. density gradients); and the underlying an aerobic zone. The uppermixed water layer was dominated by aerobic forms, especially three species of tin­tinnids, oligotrichs (Strombidium spp.), haptorids (especially Mesodinium spp.), andthe scuticociliates Uronema jilificum Kahl and Cyclidium glaucoma OFM. Themicroaerobic community was the most species rich. Several species (e.g. Colepstesselatus Kahl, Uronychia transfuga OFM) were found only in a tight zone around9.5m and the remainder (e.g. Pleuronema coronatum Kent, Euplotes zenkeuntchi Bur­kovsky) were restricted to the 9-15m layer. Note however that some of these speciesshow abi-modal vertical distribution. This is very obvious with Plagiopogon lorica-

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MICROBIAL LIFE IN AN ANOXIC FJORD

cells rnl'

81

O· 07010.91O~OOl01 Oll OlOOHO:lO~O)J O~1 ~ 6 I 10 110 0,\ I I I I 1.1 J ~ 0 \ OJ 0.) OJ 0' •,: " " ~ ;,. '~--'r--- I i ISr 1 I I

"-~ ~ ~- ~l -.. L.> ------- ~-~-~_. ~ I( ~. -==:;:: I

]' "1" I' ~ ~..<,- ="'-~E ~. :; .~ _~_- " , I ~ .

1 "I I I J_._____~~1"I j _-

Eutintinnus lusus-undae Helicostomella subulata Peritromus faurel Plagiopogon loricatus Plagiopyla frontala

Fig. 21. Quantitative vertical distribution of selected ciliate species, being restricted to the upper,aerobic water (Eutintinnus lusus-undae; Helicostomella subulata), the microaerobic region (Peritromus

faurei; Plagiopogon loricatus) and the deep, an aerobic water (Plagiopylafrontata.)

tus and Euploteszenkewitchi, with peaks around 9.5 m, and again around 14m. Weinterpret these distributions in terms of (a) the ehemosensory response ofmieroaerobie eiliates whieh enables them to aggregate in water with a low oxygentension, and (b) the faet that two superimposed density gradients, due to ternper­ature and to salinity, both oeeurred within the mieroaerobie region. The densitygradients will aet as a barrier to the vertieal mixing ofwater. Thus, the mieroaero­bie eiliates were first of all eoneentrated in the mieroaerobie region beeause oftheir innate tendeney to seek out a low oxygen tension. This aggregation was thenloeally amplified in two depth ranges (around 9.5m and around 14m) by the twodensity gradients.

The eommunity ofeiliates eonsisting offorms found solely in anoxie water, wasnot rieh in speeies. Six eiliates were identified: Spathidium sp., Lacrymaria sp., Meto­pus contortus Quennerstedt, Plagiopylafrontata Kahl, Cristigera cimfera Kahl and Cy­clidium xenium n.sp. We did explore the possibility that Cardiostomatella vermijormewas anaerobie but we eould not entiee eells to reproduee in anaerobie eulturesand we found no evidenee of autofluoreseing endosymbiotie methanogens.

Although the lack of other similar published work makes direet eomparisonsimpossible, an exploratory eomparison with the results of a similar study in astratified freshwater pond (Priest Pot; Finlay et al. 1988) does raise some interest­ing points. The Mariager Fjord water column yielded 37 eiliate species (Fig. 20):Priest Pot yielded 29. The latter however is probably an underestimate: the studydid not benefit from the ability to diseriminate between small seutieociliate spe­eies, and the anaerobie speeies were, at the time of sampling, still in the sediment(they move up into the overlying water later in the summer; Guhl et al. 1994). Itis possible therefore that the potential number of identifiable eiliate speeies in thewater eolumn of Priest Pot is similar to that in Mariager Fjord.

There are also obvious similarities in the eiliate faunas of the two water bodies.

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82 TOM FENCHEL ET AL.

The surface waters of both are the province of tintinnids, oligotrichs and sm allhaptorids (e.g. Askenasia, Mesodinium). The only difference is that in Priest Pot (asin other freshwater bodies) sm all filter-feeding rotifers occupy the niche thatmight otherwise be occupied by a diversity of tintinnid species, as in marinehabitats. The comparison between the two microaerobic communities is particu­larly illuminating. In removing sampIes from progressivdy greater depths inboth water bodies, one is struck by the sudden appearance ofa different commu­nity of ciliates as soon as the O 2 tension falls to a low value (at 9.5 m in the caseofMariager Fjord). With the possible exception ofsome scuticociliates, it is likelythat no ciliate species were present in both Priest Pot and Mariager Fjord but atthe genus level, there were many cases where they were identical e.g. the generaColeps, Prorodon and Euplotes(Table 5). Even where this did not happen, it was ap­parent that a ciliate of a similar functional biology was occupying the equivalentniche: Frontoma in Priest Pot and Cardiostomatella from the fjord are ciliates ofabout the same size, they both probably feed on small algae, using mouths whichare similar in size and function. A similar correspondence can probably also bedrawn between the marine Peritromus and the freshwater Hypotrichidium.

However, one major difference between the two habitats concerns the role ofthe proximity and nature of the sediment. In Priest Pot, the underlying sedimentis organic and flocculent and its surface aerobic for a large part of the year, whenit provides a suitable habitat for interstitial ciliates. One consequence is that itsupports a rich ciliate fauna induding the karyorelictid Loxodes which, beingmicroaerophilic, becomes planktonic during the summer months. Many of themicroaerobic ciliates in Priest Pot spend the winter months (following the col­lapse ofwater column stratification) in the sediment; in Mariager Fjord benthicanoxia is almost permanent.

The following species, all found in Mariager Fjord, have either not previouslybeen described, or the published descriptions are incomplete.

Cyclidium plouneounDragesco, 1963. The cell is 30 - 35 p,m long, and character­ized by the large paroral membrane, which curves upwards and to the left (Figs22,a,b; 23). This characteristic permits rapid identification of silver-stained cellsofthis Cyclidium species. The paroral membrane has 40-45 kinetosomes in zigzagformation. There are 10 - 11 sornatic kineties with double kinetosomes in the an­terior half of the cell. In the posterior half, the kineties consist of three widely­spaced individual kinetosomes (four in kinety 1) with very long kinetodesmalfibres. There is a single caudal cilium. All somatic kineties, except the last one,encircle the posterior end of the cell. This feature is easy to observe in silver car­bonate-stained specimens where the very long kirretodesmal fibres and associatedstructures of the kinetosomes almost connect with each other, producing a con­tinuous stained ring at the posterior pole. The last kinety is absent in the posteriorhalf and terminates at the cell equator. Oral polykinetid 1 has three rows ofkinetosomes lying obliquely to the long oral axis. A short row of two kinetosomes

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MICROBIAL LIFE IN AN ANOXIC FJORD 83

Table 5. Miereaerobic ciliate genera in the water column and their probable functional equivalentsin Priest Pot, a productive freshwater pond (see Finlay et al. 1988).

Mariager Fjord Priest Pot

EuplotesColepsProradonUrotrichaPlagiopogonCardiostomatellaStrombidium/StrobilidiumPeritromus/UronychiaCyclidium spp.Tintinnids

EuplotesColepsProradonUrotricha

small Prorodon/EnchelysFrontonia/Disematostoma

Strombidium/Strobilidium/HalteriaHypotrichidiumCyclidium spp.(Rotifers)

on the left of the polykinetid is also sometimes observed. Oral polykinetid 2 isformed by four rows of kinetosomes, one of them being longer, to join with oralpolykinetid 1. The third row of oral polykinetid 2 is in separate parts, with onepart (having two or three kinetosomes) lying elose to polykinetid 3. Polykinetid3 has two parallel rows ofkinetosomes. The scuticovestige lies in two parts, at theend of and beneath the bend in the paroral mernbane. Cells have one to threemacronuclei and one micronucleus, all in the anterior part. C. plouneouri wasfound at depths of 9 to 16 meters.

The oral polykinetids and somatic infraciliature in C. plouneoun from MariagerFjord resemble those described from the USA by Borror (1965). The somatic in­fraciliature also resembles that described from lakes in Ontario (Canada) by Wil­bert (1986). However, ihe paroral membrane in the Danish organisms is muchlarger, and resembles the brackish water (Roseoff, France) C. plouneouridescribedby Dragesco (1963).

Uronemafilificum Kahl, 1931. The ciliate is 40 - 55 p.m long (Fig. 22,c). It fastensto detritus particles by means of a secreted mucoid thread, which also enables itto rotate around the longitudinal cell axis. Oral polykinetid 1 is formed by threeto six pairs of kinetosomes aligned in parallel and difficult to observe in thestained organism. Oral polykinetid 2 has four or five short rows of kinetosomesoriented approximately perpendicular to the long axis of the oral area. The in­fraciliature of the paroral membrane has an anterior part as a linear file startingat the level of oral polykinetid 2 and formed by seven kinetosomes; and a zig­zagged posterior part with 11 dikinetids starting at the level of oral polykinetid 3.The scuticovestige is usually C-shaped. There are 17 to 19 somatic kineties. Thesomatic kineties do not reach either pole of the cell, All kineties apart from the lastone (kinety n) begin at the same level below the anterior pole; the last kinety be­gins further down, it is Ionger than the rest, and it ends elose to the caudal kineto­some. The caudal cilium is displaced from the centre of the posterior pole. Kinety1 is sometimes longer than the other kineties at the posterior end of the cell, but

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MICROBIAL LIFE IN AN ANOXIC FJORD 85

»>

,,.oo

~",

o.o.,

\,",

Z\

:

,.

,.... ", ",,•·:

:,•

,~,,

f

•I

"·,·

.r-«"....

.o

;,; ,/: I.... ,

/i :,,: :f

\ \ \ l { !\ \ l ( ! I

e

\ \ \ l I /\ ~ I

': .,.. .! I

/!,

i ,

! :

"

o·o.

··•·

o··o

·'. "\\ "\

o ", .o,

•••

','-, e.~ '.

,...~','

••

t

.."

\.

..:

(.

Fig. 23. Infraciliature ofthe ventral (Ieft) and dorsal (right) sides of Cyclidium plouneouri. Note thecharacteristic shape of the paroral membrane (arrow) and the length of the kinetodesmal fibres,

shorter than kinety n. The number ofdikinetids and kinetosomes varies betweensomatic kineties. The anterior halfof the cell contains only dikinetids with pairedkinetosomes. On the ventral surface, the four kineties on each side of the oral ap­paratus (kineties 1-4, and kineties 16-19 in a cell with 19 kineties) have doublekinetosomes in both the anterior and the posterior halfof the cell. The total num-

.-Fig. 22. Silver carbonate impregnation ofsome ofthe scuticociliates found in the water column. Allscale bars = 10 tun. a) ventral surface, and b) dorsal surface of Cyclidium plouneouri. Arrow to theparoral membrane. c) Uronema filificum. Arrows to the three oral polykinetids and caudal ciliumcomplex. d) Cyclidiumglaucoma. Arrows to the three oral polykinetids and to the scuticovestige. e),f) ventral and dorsal surface, respectively, of Cyclidium xenium n. sp, Arrows to the oral polykinetidsand the scuticovestige (small arrows) (e), and to variation in the kinetosome arrangement within thesomatic kineties (f). g) Cristigera cirnfera. White arrows to the somatic kineties in the posterior halfofthe cell body. Note also the ventral depression. Black arrows to the coat ofectobiotic bacteria. h),i) Cardiostomatella oermiforme. White arrow-head to the pre-oral suture (h); i) structure ofthe oral ap-

paratus, with arrows to the oral polykinetids.

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86 ruM FENCHEL ET AL.

d

e/rFig. 21. Various ciliates found in the water column. a) - c) formalin-fixed; d) - g) silver carbonateimpregnation. All scale bars = 20 !Lm. a) Tintinnopsis sp. b) Helicostomella subulata. c) Eutintinnus lu­sus-undae. d) Proradon discolor. arrow to the 'brasse'. e) Euplotes zenkeunichi, black arrows to the cirri;white arrow to the paroral membrane (the rounded shape is a fixation artefact). f) Askenasia stellans,big arrow to the extrusomes; medium single arrow to the equatorial belt of kineties, and small ar­rows to the circumoral wreath of argentophilic granules. g) Plagiopogon loricatus.

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MICROBIAL LIFE IN AN ANOXIC FJORD 87

..... .• . ... .. '

~ . .•• •. t

~ '\~

,I• .

••

,•

•,.,, .. ...,

,,,,

.' .. '.' .,/ "

/'/

'.~

//

// :

/I •(;'

i

\• •

:

:

•.•...

....

'I ... ., ..• •

/" .. .. -

I oD. e.. 0- •• 11 •

I: o' ••••. ..' ..

" .I

. •.. .I

' •. .{' : ~

• ~. Gi

, .' 0. .e' ••i •••• •I : •• 'I =.. · 'I '\ .. ., .: ..' .\ .. .\ : .\ I .'. ..\ . :, .

'\ • CI.

\ '. ·0 .1

\

• " Oe ••., M.' .e.··~.

Fig. 25. Infraciliature of the ventral (Ieft) and dorsal (right) sides of Cyclidium xenium n. sp.

ber of dikinetids in these kineties is always more than 20 per kinety, being closerto each other in the posterior half of the cell, This is easily observable on the leftside. The remaining kineties are formed by single kinetosomes in the posteriorhalf of the cell and are constituted by 13-15 kinetids per kinety. The cytoproct isventral and posterior to the oral region. The contractile vacuole pore lies at theend ofkinety 2. A single macronucleus, with associated single micronucleus, liedose to the centre of the cell. This ciliate is virtually identical to the Uronemafiliji­cum described by Wilbert and Kahan from Solar Lake (1981) and to astrain isolat­ed from Beggiatoa-mats in Nivä Bay (Fenchel, unpublished) although the latterform seems to require a lower P02 (around 4 % atm. sat.) than that indicated bythe vertical distribution in Mariager Fjord (Fig. 20).

Cyclidiumxenium n. sp. [Gk. ~EjJLOjJ, trans.: a present given by ahost to his guest].This scuticociliate is 30-35 p.m in length, with 7-10 somatic kineties (Figs 22,e,f;25). Kinetosomes are double and single in the anterior and posterior halvesrespectively. Kineties 1 and 10 have 14 kinetids, the last four or five lying closerto each other. The remaining kineties have nine kinetids, of which only two arein the posterior half of the cell. The oral region occupies most of the ventral side.There is a single micronudeus, associated with one or two macronudei, both in

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88 TOM FENCHEL ET AL.

.. , ...

." .".'

,_.- .. \ .. (' .:')..::// .::' »:. -:--, .:,1\ j i

.... .- f ,.... __ . ~ ;/

/"~..•-~/

-6", ,v.

Fig. 26. Ventral (Ieft) and dorsal (right) sides of Cristigera cirrifera (silver carbonate impregnation).Cilia drawn on only some ofthe kinetosomes ofthe ventral surface. Ectobiotic bacteria (B) cover

most of the cell surface.

the anterior part ofthe cell. The paroral membrane has approximately 30 kineto­somes in zigzag formation. The scuticovestige is in two parts, one with three orfour kinetosomes at the end of the paroral membrane, the other with two kineto­somes beneath the bend in the paroral mernbrane. The ciliate was discovered inthe anaerobic zone (14.5 to 25m depth) ofMariager Fjord, Denmark.

Cyclidium porcatum is the scuticociliate bearing the dosest resemblance to C.xenium (see Esteban et al. 1993). There are, however, several important differ­ences: (1) C. porcatum typically swims slowly and continuously, in C. xenium, typi­cal swimming consists of repeated short jumps, (2) C. xenium usually has 10 so­matic kineties; C. porcatum always has 7 or 8 kineties, (3) there are significant

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MICROBIAL LIFE IN AN ANOXIC FJORD 89

. . , ...... .,.

:)

. . '.",;:,. ::'

'. ".'. "

. :,', :-:','::.:., .',',.,',", '. " '.'.

........ ', .' "

,,' ... .: :::':: " . . . . , .....: ::.

Fig. 27. Cardiostomatella oermiforme. Left) silver carbonate impregnated. Right) close-up of oral poly­kinetids and pre-oral suture (arrows).

differences in the spatial arrangement of kinetosomes in the kineties and in theoral polykinetids, and (4) C. porcatum has endosymbiotic methanogenic bacteria;these were not observed in C. xenium.

Cristigera cirrifera Kahl, 1928. This ciliate is probably the organism described byKahl (1930-35) and also found in other anaerobic marine habitats by Fenchel &Finlay (1991). However, we have observed that the oral infraciliature is unlike thetypical Cristigera type. and its somatic infraciliature is also reduced (Figs 22,g;26).

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90 IDM FENCHEL ET AL.

Furthermore, the oral polykinetids are not arranged as one kinetosomal file aswould be the case in Cristigera, but as three files. A variable number of kineto­somes (usually four) are located between oral polykinetids 1 and 2 (Fig. 26). Theparoral membrane starts at the level of the middle of oral polykinetid 1, and has30-35 zigzagged kinetosomes with an indentation in its course at the level of oralpolykinetid 3. The scuticovestige is in two parts: at the end of the paroral mem­brane and below the bend ofthe latter. The somatic kineties are interrupted, leav­ing a large kinetosome-free gap between the anterior and posterior halfofthe cell.The kineties in the anterior half each consist of 9 - 10 paired kinetosomes,although a few small organisms (- 18JLm) have fewer. In the posterior half, somekineties are missing. Esteban et al. (1993) and Esteban and Finlay (1994) founda Cyclidium-like scuticociliate in a sulphate-rich solution lake. This ciliate, whichwas bigger than Cristigera, and with a different arrangement ofthe oral and somat­ic infraciliature, was named Isocyclidium, but in some respects it resemblesCristigera cirrijera. Thus a similar morphology was described for Isocyclidium globo­sum (Esteban & Finlay 1994), with paired kinetosomes along the whole kinetylength, 8 - 9 kineties in the anterior half, and 7 - 8 in the posterior half. In Cristigeracimfera from Mariager Fjord, the number of paired kinetosomes per posteriorkinety varies, from one to three (Fig. 26). Again, a similar pattern was observedin I. globosum. Cristigera curifera was found exclusively in anoxie water, in the depthrange 16-25m in Mariager Fjord, Denmark. A specific characteristic ofthis cili­ate is the anterior-posterior depression bordered by the last two kineties on the leftside (Fig. 26). Similar indentations, aIthough less evident, have been described inother Cristigera species. There is either one or two macronuclei, and one associat­ed micronucleus. The ciliate has a single caudal cilium, and cilia in the posteriorhalf of the cell are stiff when the ciliate stops swimming. The entire cell surfaceapart from the oral area is covered with ectobiotic rod-shaped bacteria. Thesebacteria vary in length from 5.7 to 7.6 JLm.

Cardiostomatella vermijorme (Kahl, 1928) Corliss, 1960. This is a large ciliate, 300- 500 JLm in length. The shape and mode oflocomotion of the ciliate superficiallyresembles that ofParamecium, although the cell is variable in shape, and has a dis­tinctive dark anterior region. Apreoral suture arises from the disposition of theanterior somatic kineties. It extends from the mouth up to the dorsal surface atthe anterior end. (Figs 22,h and 27). The right ventral somatic kineties insert onthe preoral suture (Fig. 27). There are four to six or seven postoral kineties; ofthese, the first one on the right is half as long as the rest and probably ends at thecontractile vacuole pore. There are three oral polykinetids, which are difficuIt toobserve because of their position inside the oral cavity. Polykinetid 1 is situatedat the same level as the beginning of the paroral membrane and is formed by threeparallel rows of kinetosomes. Each row usually has seven kinetosomes. Poly­kinetid 2 lies parallel to polykinetid 1 (Figs 22,i and 27) and consists of threeparallel rows of seven or eight kinetosomes. Polykinetid 3 is difficuIt to observe.

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MICROBIAL LIFE IN AN ANOXIC FJORD 91

It is placed at the end of the oral cavity, behind oral polykinetids 1 and 2. It isoriented obliquely to the other oral polykinetids and formed by two parallel rowsoffour to six kinetosomes each. The paroral membrane has 20 - 30 paired kineto­somes in zigzag formation. There are over 100 (usually 110) somatic kineties.Trichocysts are present. The macronucleus is variable in shape and number,ranging from almost ovoid and located in the centre of the cell, to elongated withsevcral constrictions. Individuals with two macronuclei have been observed.When a single macronucleus is present, there is usually one micronueleus next toit. Otherwise, up to eight or more micronuclei are arranged around the rnacro­nucleus. It was found feeding on tiny diatoms at depths of9 to 23 metres in Mari­ager Fjord.

Regenerating forms were frequently encountered. These were extremely vari­able in shape and size. As a consequence, the cells from Mariager Fjord resern­bled the other described species (C. mononucleata, Dragesco, 1963; C. chesapeakensis

Small & Lynn, 1985). Although with a greater number ofkineties, C. vermiforme

from Mariager Fjord is otherwise identical to the cells described by Czapik andJordan (1977) from the Baltic Sea. It may be realistic to combine all Cardiostomatel­

las together in the one species C. vermiforme.

Euplotes zenkewitchi Burkovsky, 1970. This elongated ciliate is approximately 80p.m long. The ventral somatic infraciliature is formed by 9 frontoventral, 5 trans­verse, and 3 caudal cirri, the last of these being smaller than the rest. Two caudalcirri lie closer to each other, at the cell posterior. The remaining cirrus lies on theleft side of the cell, below the adoral zone of membranelles (AZM) (Fig. 24,e).There are 10dorsal somatic kineties, the AZM has 50 - 55 membranelles, and theargyrome is double patella-type. The C-shaped macronucleus has a posteriorelongated region and one micronueleus lies next to it, as described by Curds(1975). The ciliate was found living at depths of 9 to 15.5 metres in MariagerFjord.

Askenasia stellaris (Leegaard, 1920) Kahl, 1930. The cell has a flattened (anteri­or-posterior) shape, with tapering anterior and pointed posterior ends. All cellsexamined were approximately 40 p.m in maximum dimension. The kinetosomesin each equatorial belt of kineties lie very elose to each other (Figs 24,f and 28).There are 70 kineties in this belt; the anterior part - or pre-equatorial kinety belt,corresponding to the pectinelles, is formed by approximately 10 individualkinetosomes. The posterior part of the kineties, corresponding to the cirri, isformed by 12 - 15 zigzagged kinetosomes. Kineties in the sub-equatorial kinetybelt, which supports the bristles, each have three kinetosomes per kinety, thesebeing oriented obliquely to thc kinety axis. Close to the anterior end, each pre­equatorial kinety terminates in basal body-like argyrophilic granules which en­circle the cell (Fig. 28). These probably correspond to the circumoral wreath ofgranules. There are long extrusomes, about half the length of the cell, grouped intwo whorls of six bundles, each with 12-20 tightly packed extrusomes. There are

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92 IDM FENCHEL ET AL.

, .

~E

. , .. '

.:~CWG

.j ..::.~:.:<;;, .' ~< :.;;\::

l l \ \, \ \. . ';" ': ' ..

\;.\\\\;~::S.'·::·;;• 0" ....

( ::. ' ..

" :' -: e ,

"

" " ~"

.' " " .: -,.' " ;' '. :'

".: :.;, i;, ".". -,

Fig. 28. Infraciliature of Askenasia stellaris. E, bundles of extrusomes; CWG, circurnoral wreath ofgranules; PKB pre-equatorial kinety belt; EKB, equatorial kinety belt; SKB, sub-equatorial kinety

belt.

two sausage-shaped maeronuclei, and one mieronucleus elose to the center ofeaeh maeronucleus. This eiliate was found at 8 - 16 metres depth in MariagerFjord. Askenasia stellanshas previously been reported from the North Sea, BaltieSea, Florida, and the White Sea (Kahl 1931, Borror 1965, Krainer and Foissner1990 and referenees therein).

Vertical distribution oJmicrobial biomass and microbialJood uiebs

It is not surprising that photoautotrophs dominated the protist biomass in themixed zone (Fig. 29) whereas they oeeurred in low numbers below - 8m (in thisand following figures biomass is expressed in terms of volume fraetion as ppm).The biomass of phytoplankton in the oxie layer was high and dominated by asmall unieellular diatom, Stephanodiscus hantzschii, whieh aeeounted for about 97% of the autotrophie biomass. Other phototrophie protists included crypto-

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MICROBIAL LIFE IN AN ANOXIC FJORD 93

Scuticoclliates(lO·2ppm)

80 Z 4 6 8

Bacterivorous f1agellates(lO·l ppm)

o Z 4 6 __I ::?e I .... Lu.. ,o

20

25

10§t...~ 15

Fig. 29. The bio-volume distribution in the water column ofthe major groups of microbial planktonin Mariager Fjord on August 9, 1994.

phytes, chlorophytes and euglenoids, but only few genera and speeies were identi­fied: Apedinella spin ifera, Cymbomonas sp., Ochromonas sp., and Pyramimonas sp.Although the majority of the taxa were exclusively observed in the oxie layer, S.hantzschii, A. spinifera and some eryptomonads and eyanobaeteria were founddown to the anaerobie zone.

Seeehi disk reeordings showed that the light eompensation point (1 % surfaeelight intensity) was reaehed at about 4m depth, indicating that the phytoplanktonpopulation in the mixed zone was light limited (about halfof the phototrophie bi­omass being below the eompensation point at any time. The phytoplankton eellswhieh were found below the pyenocline and into the anoxie zone must representsinking eells.

The total biovolumes of baeteria, (heterotrophie) flagellates and eiliates arealso shown in Fig. 29. We were unable to quantify rhizopods, although they wereobserved in living samples. The eoneentration of heterotrophie baeteria wasabout 106 eells ml-1 throughout the water eolumn. However, as previously shownfor anoxie basins in the Baltie (Gast & Goeke 1988) and for Mariager Fjord (Fen­ehel et al. 1990) the mean size of baeteria inereased with the deereasing p02; theaverage cell volume was 0.15 p,m 3 at the surfaee and 0.70 p,m 3 in the anoxie zone.Thus, the total baeterial biomass inereased with depth.

In the oxie zone, the biomass of phagotrophie protists was high (ea 3 ppm) and

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94 TOM FENCHEL ET AL.

almost equally shared between ciliates and flagellates, aIthough the ciliates domi­nated at the top and the flagellates at the bottom ofthe zone (Fig. 29). Tintinnidseontributed most to ciliate biomass. Heterotrophie dinoflagellates aecounted forabout halfof the flagellate biomass in the upper 5m of the oxic zone and their im­portanee inereased with depth between 5 and 8m (Fig. 30).

In the mieroaerobic zone ( - 9- - 16 m), more or less distinet maxima ofphago­troph biomass were due mainly to flagellates (Fig. 29). The most conspicuouspeak, found between 9 and 10m, was due mainly to the dinoflagellate Oxyrrhis ma­

rina, although several ciliate speeies also eontributed. The total biomass of'phago­trophie protists in this narrow peak was similar to that found in the upper oxiezone (Fig. 29). Two additional ciliate and flagellate biomass peaks could also beobserved in the mieroaerobic zone (between 11 and 16m). In the anoxie zone, thebiomass of protists was low and dominated by flagellates and a few speeies ofeili­ates (Fig. 29).

The vertieal distribution profiles ofdifferent, funetionally defined groups (Fig.30), yield some information on the food web strueture in the three main strata ofthe water eolumn. The nanoflagellates found in the water eolumn were generallysmall « 6 p.m) and they ean be eonsidered to be predominantly baeterivorous(Fenehel1982, 1986, Andersen & Serensen 1986, Eecleston-Parry & Leadbeater1994). Nevertheless, in the oxie zone several nanoflagellate speeies (mostly > 6p.m) were found, whieh feed on other protists: Leucocryptos remigera, Telonema subtile,Quadricilia rotundata and Ebria tripartita (Thomsen 1992, Vars 1992).

Seuticoeiliates, hypotriehs, heterotriehs and triehostomatids were eonsideredto be mainly baeterivorous. Due to their re1atively low biomass, the three lattergroups were lumped together in Fig. 30. The seuticoeiliate Cardiostomatella ver­miformewas observed to feed on small diatoms and the Euplotesspp. probablyalsofeed on nanoflagellates.

Nanoflagellates were the dominant eonsumers of bacteria throughout thewater eolumn, but espeeially so in the oxie layers where baeterivorous ciliates(with the exeeption of Cyclidiumglaucoma) were absent. In eontrast, baeterivorouseiliates played a substantial role in the mieroaerobie zone. In the anaerobie zone,the biovolume ofbaeterivorous eiliates and flagellates was about an order ofmag­nitude lower than that in the oxie and mieroaerobie layers.

The heterotrophie dinoflagellates and representatives of some eiliate groupswere the rnain eonsumers ofphototrophie and heterotrophie protists. The hetero­trophie dinoflagellates are known to feed on large prey: diatoms, nanoflagellates,other dinoflagellates and/or ciliates (Gaines & Elbrächter 1987, Lessard 1991,Hansen 1991,1992). Among the eiliates, the (naked) suspension-feedingoligotriehs and tintinnids feed mainly on algae (Verity 1986, ]onsson 1986, Ber­nard & Rassoulzadegan 1990, 1993), and the prostomatids and the haptorids aremainly raptorial feeders (Fenchel 1968, 1969, Corliss 1979).

Oligotriehs dominated the oxic zone, but played a relatively sm aller role in the

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MICROBIAL LIFE IN AN ANOXIC FJORD 95

10g,:;g.

Q IS

20

2S

Tintinnids

(ppm)3 0

Othcr oligotrichs

(IO·'ppm)4 6 8 10

Haptorids and prostomatids(lO·'ppm)

i s 0 2 4 60I 1

Dinoflagellatex

Ippm)1

Bactcr-la (ppm)20 0 2 4

Ftageüates (ppm), 2 3 0

Ctttatcs Ippm}1 2

_10

E-"c.Q

IS

20

"ZS ~ <

Fig. 30. The bio-volume distribution in the water column of some important functionally or taxo­nomically defined groups in Mariager Fjord on August 9, 1994.

mieroaerobie zone. The high population densities of tintinnids and heterotrophiedinoflagellates in the oxie zone refleeted the large supply of phytoplankton food.The tintinnids seemed to depend on the dominating diatom Stephanodiscus hantz­schii. The trophie role of the heterotrophie dinoflagellates in the oxie zone was lessclear. One ofthe dominant speeies, Polykrikos schwartzii, is known to eo-oeeur withlarge (> 25 /l-m) autotrophie dinoflagellates (Carreto et al. 1986) and it has so faronly been grown sueeessfully on this diet in cultures (P.]. Hansen, unpubl.); theonly organisms of an appropriate size left as food for P schwartzii, were other het­erotrophie dinoflagellates and perhaps some eiliates.

Another dinoflagellate, Oxyrrhis marina, was found in the oxic zone in low num-

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bers. This dinoflagellate is known to feed on a variety oforganisms within the sizerange of2-1O /Lm (Goldman et al. 1989). In the oxic zone, it was observed to feedon Stephanodiscus hantzschii. However, 0. marina had its maximum abundance in anarrow layer at the top ofthe microaerobic zone where there were very few dia­tom cells. Oxyrrhis marine is not known as a microaerophilic species. Thus the rea­son for its success in this zone might be explained by the absence of predators(Polykrikos schwartzii and Tintinnopsis tubulosa); that is, the upper part of themicroaerobic zone may serve as arefuge for 0. manna.

The general picture of the food web in the three major zones of the watercolumn can be summarised as folIows. The mixed, oxic (and in part photic zone)is dominated by the production of phytoplankton cells, but the food web is rela­tively complex due to variation in prey size and to bacterial production. Themicroaerobic zone is dominated by bacterivory and chemolithotrophic bacterialproduction probably serves to nourish a significant degree of phagotrophic ac­tivity. Predators and species depending on sedimenting algal cells are also impor­tant. In the anoxic zone the relatively inefficient anaerobic energy metabolismmeans a low growth efficiency. Food chains are therefore short and largely con­fined to one step (bacteria -+ bacterivores); see Fenchel & Finlay (1990) and Fen­chel et al. (1990). Even so, a few predators apparently did occur, including the cili­ate Lacrymaria sp. and the dinoflagellate Hemidinium sp. The latter was found tofeed on nanoflagellates (it was maintained in cultures with the anaerobic flagel­late Hexamita inflata as food).

Compared to the earlier investigation ofMariager Fjord (Fenchel et al. 1990),the peak of protozoan biomass at the chemocline was much less pronounced in1994. While a maximum ofbacterivorous protozoa did occur, it was quantitative­ly overshadowed by the unusually dense populations ofprotozoa in the oxic layer.However, while the chemolithotrophic production detected at the chemoclineprobably reflects the annual average of the biological production in the system,production in the surface layers varies from week to week and our investigationtook pIace at the end of a bloom event which supported very high heterotrophicactivity.

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