Bacterial biomass and respiratory electron transport system activity in the oyster ground area (North Sea) in 1981

Netherlands Journal of Sea Research 18 (1/2)." 71-81 (1984)

71

BACTERIAL BIOMASS AND RESPIRATORY ELECTRON TRANSPORT SYSTEM ACTIVITY IN THE OYSTER GROUND AREA (NORTH S E A ) I N 1981 by B. VAN DER W E R F and G. N I E U W L A N D Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg 7~xel, The Netherlands

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Water sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. ETS activity measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. ATP analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Bacterial numbers and biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 72 72 72 72 73 74 78 80 80

1. I N T R O D U C T I O N D u r i n g 1981 a chemical-biological research p r o g r a m m e was carried out in the N o r t h Sea on the O y s t e r G r o u n d in the n e i g h b o u r h o o d of 4 ° 3 0 ' E , 5 4 ° 3 0 ' N . This area was chosen because it was t h o u g h t to be suitable for a study of the structure a n d functioning of an ecosystem from the f o r m a t i o n of a thermocline to its breakdown. Therefore, the p r o g r a m m e consisted of three cruises: the first one in May, d u r i n g which period an algal spring b l o o m is to be expected in this area (CoLEBROOK, 1979), a second one in J u l y when the water c o l u m n usually is stratified, a n d a third one in S e p t e m b e r when mixing of the water mass is due to start. In this p a p e r some aspects c o n c e r n i n g microbiological p a r a m e t e r s of the water c o l u m n are described a n d discussed. Since most of the bacteria in coastal sea water are a s s u m e d not to be attached to particles ( a c c o r d i n g to ZIMMERMANN, 1977 up to 96%) only the fraction smaller than 50 # m has been considered. T h e respiratory activity of microorganisms in this fraction was d e t e r m i n e d by m e a n s of m e a s u r i n g the m a x i m u m rate of electron transport t h r o u g h the electron transport system (ETS) on the basis of which estimates of in situ d e g r a d a t i o n of

72

B. V A N

DER

WERF

& G.

NIEUWLAND

organic matter have been made. For estimation of bacterial biomass, counting of bacteria by means of epifluorescence microscopy was combined with volume estimates on the basis of scanning electron microscopy photographs. For total living biomass in the fraction smaller than 50/xm the total amount of ATP was determined. Since hydrographical and chemical data were collected in the same area, together with biological data of phytoplankton, zooplankton and benthos, it was possible to estimate the contibution of bacteria to the flow of organic matter. Acknowledgements.--We thank Messrs W.L. Jongbloed and R Havinga of the Medical Department of Electron Microscopy of the University of Groningen for giving the opportunity to work with SEM equipment and for assistance in making the photographs. Discussions with W.W.C. Gieskes and J.H. Vosjan are gratefully acknowledged. 2. MATERIALS AND METHODS 2.1. W A T E R

SAMPLING

The water samples were collected as close as possible near a marker buoy (TIJSSEN & WETSTEYN, 1984a). Subsamples were taken from 30 litre PVC bottles. These bottles were fixed on a rosette sampler together with a Conductivity-Temperature-Depth sensor which recorded continuously these three parameters during downcast. According to these recordings a decision could be made regarding depths of sampling. The water was directly filtered over a 50/xm sieve before handling in the ship's laboratory. 2.2. E T S A C T I V I T Y

MEASUREMENTS

Electron Transport System activities were determined according to the method of PACKARD(1971) as modified by OLAI~CZUK-NEYMAN~: VOSJAN (1977). Incubation time was about 30 minutes. The incubation temperature was 20°C. The activation energy used in the Arrhenius equation was the one determined by VOSJAN(unpublished results) in North Sea water (43 900 J-mol-1). 2.3. A T P A N A L Y S I S

ATP analyses were done immediately after sampling on board of the ship. We used a modified version of the method of HOLM-HANSEN & BOOTH (1966). From the prefiltered water (50/xm sieve) a volume vary-

BACTERIAL

BIOMASS

AND

ETS

ACTIVITY

73

ing between 10 and 150 ml, d e p e n d i n g on the expected ATP content, was filtered over a 0.2 # m m e m b r a n e filters (Sartorius, d i a m e t e r 11.0 mm). A T P was set free from the cells by m e a n s o f a nucleotide releasing reagent ( N R B , L u m a c , Cat. No. 4015), i n c u b a t i o n lasting 3 ½ minutes. T h r e e different concentrations o f A T P ( L u m a c ) were added as an internal standard. L u m i t P M was used as the highly purified luciferineluciferase reagent c o m b i n a t i o n with a H e p e s - L u m i t buffer ( p H 7.75). T h e bioluminescence luciferase assay was carried out in a L u m a c ( M 2000) B i o c o u n t e r at a t e m p e r a t u r e of 20°C. 2.4. B A C T E R I A L

NUMBERS

AND BIOMASS

Bacterial biomass was estimated by m e a n s of a c o m b i n a t i o n of countings u n d e r an epifluorescence microscope and d e t e r m i n a t i o n of average bacterial volumes in the same samples on the basis of scanning electron microscope photographs. Epifluorescence countings were m a d e based on the methods given by HOBIEe/al. (1977) and ZIMMERMANN(1977).O n b o a r d of the ship, after filtration (50 #m), the samples were stored in vials of 50 ml at a t e m p e r a t u r e of 4 to 6°C, fixed with formaldehyde to a final concentration of 1%; 1 to 3 ml water ( d e p e n d i n g on the bacterial n u m b e r ) was filtered over a polycarbonate nuclepore filter ( d i a m e t e r 25 m m , 0.2/zm pore size) which was stained with S u d a n Black (1 : 15000). T h e filter was incubated with acridine orange (1 : 10000). After filtration and rinsing with pyrogen-free water, the filter was air-dried. T h e bacteria were c o u n t e d u n d e r a Zeiss epifluorescence microscope fitted with a H B O W M e r c u r y lamp plus a 500 dichroic b e a m splitting filter 510, L P 520. T h e magnification was 1000 x; a fluorescence-free i m m e r s i o n oil was used; 42 r a n d o m square fields of 40 x 40 # m were counted. A discrimination was only m a d e on the basis of cell shapes: rods and cocci. For d e t e r m i n a t i o n of the cell volumes a scanning electron microscope ( J E O L J S M - 3 5 C ) was used. T h e bacterial cells were fixed with a mixture of formaldehyde and glutaraldehyde, 0.2% and 5% respectively, in a sodium cacodylate buffer (0.2 m o l . d m - 3 , p H 7.2). After rinsing with a decreasing percentage sea water, diluted with distilled water (75%, 50%, 25% and 0%), the filters were air-dried and preserved over silicagel until f u r t h e r t r e a t m e n t in the laboratory. All the dilutions were bacteria-free (filtered over 0.1/zm polycarbonate filters). Subsequently the filters were sputtercoated with a gold layer (thickness a b o u t 1500 nm). T h e preparations were viewed at magnifications of 5 0 0 0 x to 10 000 x. P h o t o g r a p h s of suitable bacteria were m a d e (Ilford Pan F). T h e average bacterial area of the rods and cocci was m e a s u r e d by

74

B. VAN DER W E R F &

NIEUWLAND

G.

means of a Mop Kontron semiautomatic particle analyser. Bacterial volumes were determined using the solid of revolution of these pictures. Corresponding density of the bacteria was assumed to be 1.07 g.ml-2 while a dry weight to net weight ratio of 0.23 and a carbon to dry weight ratio of 0.34 were used (BOWDEN, 1977). 3. R E S U L T S

Respiratory electron transport system activities at in situ temperature of organisms smaller than 50 #m were rather low and distributed homogeneously throughout the water column in May (Fig. 1; as 02, 21.5 #mol.m-3.h-]). In July the activity was slightly higher, with a clear maximum found at a depth between 20 and 30 m. In September, on the other hand, most of the activity took place in the upper 20 m. Notice that in September even at a depth of 40 m the activity was equal to or even higher than the highest values during both previous cruises. An indication of the degree of difference between consecutive casts can be found in Fig. 2, in which we present observations made during diurnal cycles in July and September to look for short-term difference in activity between light and dark periods. Neither in July nor in September a clear day-night rhythm was observed. Again obvious are the maximum at a depth of 20 to 30 m in July, and highest activities between the surface and 20 m in September. As found byPACKARD & WILLIAMS(1981) and VOSJAN(1982a, 1982b), ETS activity can be related to real respiration expressed in mg C . m - 9 . d =1. The results of the exercise of relating ETS-activity to in situ mineralization of organic carbon are for May 139 mg.m =2.d-1, for July 170 mg.m=~-d -1, while in September the mineralization rate of )J rnol m-3.h -1 I0

20

30 40

50

0

20

40

60

I00

200 300

O"

,o.)

,o

,,

20-

40 m

o.i

2o-

"

,°/

\

/.

%

}

20.. I

.

40-

May

40"

July

September

Fig. 1. Mean ETS activities expressed as 0 2 consumed (/xmol-m-3.h-1) in May (11 casts), July (20 casts) and September 1981 (20 casts). Notice differences in scale.

BACTERIAL

BIOMASS

AND

ETS

ACTIVITY

75

organic matter by organisms smaller than 50 /*m proved to be the highest: 430 mg C.m-2.d-1. The distribution of biomass responsible for the mineralization discussed in the previous paragraphs is reflected by vertical profiles of ATP concentrations during July and September (Fig. 3; no data are available for the May cruise). Just as in the ETS profiles, a maximum

,o 0

04

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04 time(hours)

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26-27 July

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o

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~ 4o

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o

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d

5-6 Sept

e

io ii Sept

Fig. 2. Vertical distribution o f E T S activities ( 0 2 values i n / z m o l . m - 3 . h h o u r stations in July and September 1981.

1) d u r i n g 2 5

76

B. V A N 0

200

,o

',.

0

20 ~

WERF

NIEUWLAND 200

0

,o

o/

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20

p

40m

& G.

400¢ug.m -3

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/

DER

/

400

600

800 ./ug.m-3

.//"

./

40. July

September

m

Fig. 3. Mean ATP concentrations (#g.m -3) at different depths (m) in July 1981 (a) and September 1981 (b).

between 20 and 25 m was f o u n d in July, and highest values n e a r the surface in September. Regularity in the daily variation was not observed, neither in J u l y nor in S e p t e m b e r (Fig. 4); on the contrary, irregular variation i n d e p e n d e n t of any day-and-night cycle was the rule. This variation is a reflection of patchiness. Patchiness is also d e m o n s t r a t e d by the difference in A T P c o n c e n t r a t i o n over a short section of 5500 m, sailed in about 15 minutes (Fig. 5). Bacterial biomass showed a clear t e n d e n c y of increace from J u l y towards S e p t e m b e r (Fig. 6) in accordance with ATP results. A T P concentrations give i n f o r m a t i o n on total microbial biomass (PAERL & WILLIAMS, 1976). An u n k n o w n part of this biomass is contributed by bacteria. Actually, the vertical distribution of bacterial biomass (Fig. 7) is not quite similar to the A T P pattern (Fig. 3). T h e vertical distribution of the bacteria c o m p a r e d with E T S (Fig. 1) shows a discrepancy between the location of the m a x i m a , especially in July. T h e r e is a striking difference between the vertical distribution of J u l y and September.

TABLE

1

Comparison of own counts of bacteria and calculated carbon biomass (mg.m 3) with literature data of both these parameters. A tea

Elbe estuary Kiel Fjord N.W. Atlantic Ocean Oyster Ground Oyster Ground

Source

Depth

Cells

Biomass

SALTZMAN(1980) ZIMMV.RMAN(1977) SIEBURTH(unpubl) this paper (.July) this paper (Sept)

Surface Surface Surface Water column Water column

115 28 10.4 l(I 7

200 13 5.3 13.0 22.3

B A C T E R I A L B I O M A S S AND ETS A C T I V I T Y

04

f

08

'---.

12

•

•

20

~ o -

24

""VSc,_ . . . . . . . . . . . . . . . . .

-~-'---~20(~

•

3ol . . . .

16

•

04

77

time(hours)

-'-

•

..........

__'~-_32of ........ 150

(]

0

~o

16 . ~,~..

/

2 6 - 27 July

20

24 .

04

•

08

°

12 lime(hours)

•

,

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50

40 ~oO~ 50

b

m

14

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,..l~o.. 28-29 July

18

. . . . . .

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02

06

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~

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2O 30

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l

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.

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20

5 - 4 Sept

24

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: 300 20:• :__~_:=---~.. ~ : O~ "o' - ~ 30

..............

150 .

. . . . . . . . . . . .

20

time(hours)

" ~ - - - - ~ :-

""--...~

,oo

..........

5O m

d

7e

sept

Fig. 4. Vertical distribution of ATP concentrations (~g.m 3) during 24 hour stations in July and September 1981.

78

B. V A N D E R

WERF

& G. N I E U W L A N D

jug -m-3 900

700.

// Y ! .._... ' 1 ,~

600.

~oo400.

/ "~-

V I

",,

i

~'; i",,I

6

55bo m

Fig. 5. Horizontal variation in ATP concentration (#g.m 3) at a depth of 2 m over a distance of 5500 m (6 September 1981).

4. D I S C U S S I O N T h e bacterial n u m b e r s are in reasonable a g r e e m e n t with the results presented by SALTZMANN(1980), ZIMMERMANNN(1977) a n d SIEBURTH et al. ( q u o t e d by VAN Es & MEYER-RFJL, 1982). T h e bacterial b i o m a s s figures are i n t e r m e d i a t e between the high figures f o u n d in coastal waters a n d the lower ones in o p e n ocean waters (Table 1). T h e contribution of b a c t e r i a to the total a m o u n t of particulate organic c a r b o n ( P O C ) present in the w a t e r (PoSTMA & ROMMETS, 1984) was high in July, a b o u t 20%. In S e p t e m b e r , on the other hand, less t h a n 10% of P O C was f o u n d to be b a c t e r i a - c a r b o n . mg.m-3 25-

2oi

J

/\

10

z'2"a:4~6"2'6 4" ~ ~ J b ih July

September

Fig. 6. Estimate of bacterial biomass (mean of the water column) expressed as for dates in July and September 1981 (in mg-m-3).

BACTERrAL BIOMASS AND ETS ACTIVITY 0

I0 i

,o.

\

0

50, 40. m

20 i

30 i

79

m g , m -3

J'

~o

Fig. 7. Mean bacterial biomass (carbon mg.m -,3) estimated for different depths (m) in July (@) and September 1981 (0).

The clear tendency of increasing heterotrophic activity from May to September (Fig. 1) was followed by a decrease to the level of the May situation in November 1981, when the same area was revisited. The heterotrophic activity measured was of course not only due to bacteria; all organisms smaller than 50 #m contribute to this activity. Yet the mineralization rates found are much lower than values estimated by BAARS & FRANSZ(1984: table 8) on the basis of oxygen consumption measurements; our values were only 42% of total respiration in May, 20% in July, and 43% in September. It should be realized that organisms larger than 50 #m (copepods, algae and others) also contribute to total respiration. For example, in May, and even more so in July, most phytoplankton was larger than 50 #m: Mesodinium and diatoms in May, dinoflagellates in July (GIEsKEs & KRAAY, 1984), SO in our prefihered samples we did not measure the heterotrophic activity of a.o. the larger phytoplankton. Apparently, the contribution of organisms smaller than 50/~m to total respiration was highest in July. The large difference between respiration of the less-than-50/~m fraction and total respiration is not in agreement with the observation of WILLIAMS (1981) who states that the major component of plankton respiration is usually associated with organisms smaller than 5 to 10 /~m. Notice, however, that we may have lost the potential respiration activity of heterotrophs smaller than 0.2/~m (MEL'NmOV, 1975). Also aggregates of particles larger than 50 #m containing attached heterotrophs may have been lost during prefiltration (AzAM et al., 1983). In spite of the intensity of the sampling programme it turned out to be impossible to find significant diurnal variations in the microbiological parameters that we measured. As also pointed out by others (TIJSSEN & WETSTEYN, 1984b; POSTMA & ROMMETS, 1984;

80

B. VAN DER WERF & G. NIEUWLAND

GIESKES • KRAAY, 1984), considerable horizontal variability existed in the water mass close to the marking drogue. The highest and lowest values of ATP concentration during the section of 5500 m that we sampled (Fig. 5) differed by a factor 2.2, and the pattern is similar to those in chlorophyll and oxygen concentration change over the same stretch, which also shows an increase (GIEsKES & KRAAY, 1984; TqSSEN & WETSTEYN, 1984b). This similarity in the small-scale distribution of ATP, chlorophyll and oxygen concentrations is not unexpected, and the profiles in both ATP and ETS (Figs 1 and 3) are similar to the primary production profiles (PosTMA ~: ROMMETS, 1984; GIESKES & KRAAY, 1984). However, the vertical distribution of bacterial biomass differed slightly from that of ATP and ETS in that there were also considerable numbers of bacteria just below the maxima in these profiles. Apparently, the substrate for bacterial growth was not confined to the layer with maximum primary production, but was also found deeper down in the water column. ETS and ATP concentration gradients are, however, not necessarily similar to those in bacterial biomass, as ETS and ATP are associated also with other organisms. 5. SUMMARY Heterotrophic activity and bacterial biomass were measured in May, July and September 1981 in the central North Sea (Oyster Ground area). Bacterial carbon biomass was calculated to be 13 m g . m - 3 in July and 22 m g . m - 3 in September. The ETS activity registered in May (as 02, about 20/zmol.m 3-h-I near the surface)was much lower than in September (up to 200/xmol.m 3,h-1 near the surface) and July (maxima of 60 # m o l . m - 3 . h -1 centering at depths between 20 and 30 m). Mineralization rates of the fraction smaller than 50 #m were (in carbon) 139 m g . m - 2 - d -1 in May, 170 m g . m - 2 . d -1 in July and 430 m g . m - 2 . d -1 in September. These values are less than 45% of the total respiration estimated by colleagues in the same water. Clearly, organisms smaller than 50 #m played a minor role in community respiration. The contribution to total POC by bacteria was as high as 20% in July, less than 10% in September.

6. REFERENCES AZAM,F., T. FENCHEL,J.G. FIELD,J.S. GRAY,L.A. MEVER-REIL& E TtIINGSTAD,1983. The ecological role of water-column microbes in the sea.--Mar. Ecol. Prog. Ser. 10: 257-263. BAARS,M.A. & H.G. FRANSZ,1984.Grazing pressure ofcopepodson the phytoplankton stock on the central North Sea.--Neth. J. Sea Rex. 18 (1/2): 82-96.

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81

BOWDEN, W.B., 1977. Comparison of two direct-count techniques for enumerating aquatic bacteria.--Appl, environm. Microbiol. ,$3: 1229-1232. COLEBROOK, J.M., 1979. Continuous plankton records: seasonal cycles of phytoplankton and copepods in the North Atlantic Ocean and the North Sea.--Mar. Biol. 51: 23-32. Es, EB. VAN & L.A. MEVER-REIL, 1982. Biomass and metabolic activity of heterotrophic marine bacteria.--Adv. Microbiol. Ecol. 6: 111-170. GIESKES, W.W.C. & G.W. KRAAY, 1984. Phytoplankton, its pigments, and primary production at a central North Sea station in May, July and September 1981.--Neth. J. Sea Res. 18 (1/2): 51-70. HOBIE, J.E., R.J. DALEV & S. JASPER, 1977. Use of nuclepore filters for counting bacteria by fluorescence microscopy.--Appl, environm. Microbiol. 33: 1225-1228. HOLM-HANsEN, O. & C.R. BOOTH, 1966. The measurement of adenosine triphosphate in the ocean and its ecological significance.--Limnol. Oceanogr. 11: 510-519. MEL'NIKOV, N.A., 1975. Comparison of the magnitude of the microplanktonic biomass determined from ATP and by direct microscopy.---Oceanology 16: 181-183. OLANCZUK-NEYMAN, K.M. & J.H. VOSJAN, 1977. Measuring respiratory electron transport system activity in marine sedimenc--Neth. J. Sea Res. 11: 1-13. PACKARD,T.T., 1971. The measurement of respiratory electron transport system activity in marine phytoplankton.--J, mar. Res. 29: 235-244. PACKARD, T.T. & P.J. LEB. WILLIAMS, 1981. Rates of respiratory oxygen consumption and electron transport in surface sea water from the North West Atlantic.-Oceanol. Acta 4: 351-358. PAERL, H.W. & N.J. WILLIAMS, 1976. The relation between adenosine triphosphate and microbiol biomass in diverse aquatic ecosystems.--Int. Revue ges. Hydrobiol. 61: 659-664. POSTMA, H. & J.W. ROMMETS, 1984. Variations of particulate organic carbon in the Central North Sea.--Neth. J. Sea Res. 18 (1/2): 31-50. SALTZMAN,H.A., 1980. Untersuchungen fiber die Ver/~nderungen der Mikroflora beim Durchgang von Brackwasser durch die Kfihlanlagen von Kraftwerken. Thesis, University of Kiel, ER.G. TIJSSEN, S.B. & F.J. WETSTEVN, 1984a. Hydrographic observations near a subsurface drifter in the Oyster Ground, North Sea.--Neth. J. Sea Res. 18 (1/2): 1-12. - - - - , 1984b. Diurnal pattern, seasonal change and variability of oxygen in the water column of the Oyster Ground (North Sea) in spring-summer 1981.--Neth. J. Sea Res. 18 (1/2): 13-30. VOSJAN, J.H., 1982a. Sauerstoffaufnahmegeschwindigkeit und ETS-Aktivit/it im Niederliindischen Wattenmeer. In: I. DAUSNER. III Internationales Hydromikrobiologisches Symposium, Smolenice, 3-6 J u n i 1980. Verlag der Slowakischen Akademie der Wissenschaften, Bratislawa: 355-367. , 1982b. Respiratory electron transport system activities in marine environments.--Hydrobiol. Bull. 16: 61-68. WILLIAMS, P.J. LEB., 1981. Microbial contribution to overall marine plankton metabolism: direct measurements of respiration.--Oceanol. Acta 4: 359-364. ZIMMERMAN, R., 1977. Estimation of bacterial number and biomass by epifluorescence microscopy. In: L. RHEINHEIMER. Microbial ecology of a brackish water environment. Springer-Verlag, Berlin: 103-120.