Sub-micron particles in northwest Atlantic shelf water

0198-{)149192 S5.00 + 0.00 Pergamon Press pic

Deep-Sea Research, Vol. 39, No. I, pp, 1-7, 1992. Printed in Great Britain.

RAPID RESPONSE PAPER Sub-micron particles in northwest Atlantic shelf water A. R. LONGHURST,* I. KOIKE,t w. K. W. LI,* J. RODRIGUEZ,:!: P. DICKIE,* P. KEPAY,* F. PARTENSKY,§ B. BAUTISTA,:!: J. RUIZ,:!: M. WELLSII and D. F. BIRD~ (Received 31 December 1990; in revised form 5 July 1991; accepted 31 July 1991)

Abstract-The existence of numerous (1.0 x 107 ml- I ) sub-micron particles has been confirmed in northwest Atlantic shelf water. These particles were counted independently by two different resistive-pulse instrumenls, and their existence confirmed by our ability to reduce their numbers by ultracentrifugation, serial dilution and surface coagulation in a bubbling column. There are important implications for the dynamics of DOM in seawater if, as seems probable, these particles represent a fraction of "dissolved" organic material in seawater.

INTRODUCTION 7

HIGH numbers (1.0 x 10 ml ") of particles in the range 0.4-1.0 Jim equivalent spherical diameter (ESD) have been reported in seawater in the western Pacific Ocean. These counts were obtained with a resistive-pulse particle counter, and their existence was confirmed by ultra-filtration (KOIKE et ai., 1990). Because a significant fraction of the dissolved organic material in seawater probably overlaps the size of the electronically sensed sub-micron particles, this result blurs the distinction between the particulate and dissolved organic matter (DOM) in the ocean (TOGGWEILER, 1990). Because it is believed that the dynamics of the DOM pool holds the key to understanding carbon flux in the ocean (JACKSON, 1988; SUGII\IURA and SUZUKI, 1988; TOGGWEILER, 1988), it was a matter of urgency to confirm and extend this finding, both in other situations and with other instruments. We therefore investigated the population of sub micron particles in the northwest Atlantic shelf water off Nova Scotia in relation to numbers of living cells in the same size range.

'Biological Oceanography Division, Bedford Institute of Oceanography, Department of Fisheries and Oceans, Dartmouth, Nova Scotia, Canada B2Y 4A2. tBiochemical Research Laboratory, Ocean Research Institute, University of Tokyo. :j:Departamento de Ecologia, Universidad de Malaga, Malaga, Spain. §Station Biologique, Roscoff, France. [Marine Research Division, 0220, Scripps Institution of Oceanography, University of California, La Jolla, California, U.S.A. f1Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, Canada.

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MATERIALS AND METHODS

During a lO-day NATO-supported workshop, we brought together two resistive-pulse particle counters: the instrument used in the original investigations off Japan (Elzone 80XY, 12fim orifice, sensing range 0.36-1.0 fim) and a Coulter Multisizer II equipped with a 14 11m orifice, sensing range 0.42-1.0 fim. We used a standard suspension of 0.91 11m fluorescent latex beads to calibrate the counts from the two particle counters. Counts made with an EPICS flow-cytometer, by epifluorescence microscopy and with the two resistive-pulse particle counters agreed within 10%. Although resistive-pulse instruments are not often used for particles in the sub-micron range, they already have been used to count 0.7 11m marine bacteria (MOREL and AHN, 1990) and particles to a lower limit of about 0.3 fim (RASSOULZADEGAN and SHELDON, 1986). Moreover, the technique has a significant advantage over photon correlation spectroscopy for analysis of very small particles (PAUL and JEFFREY, 1984), because estimates of absolute particle abundance can be made. Water was obtained daily (11 = 6) at an offshore (50 m) station off Halifax using a nontoxic Niskin bottle from 10 m (routinely) and 30 m (occasionally) from 25 September to 5 October 1990; the surface water mass was of Gulf of St Lawrence origin, while shelf water lay below a sharp pycnocline at 20 m. On 3 days of foul weather, water was obtained at 10 m closer to the coast, where the 10 m water resembled the sub-pycnocline water farther offshore (Table 1). Water was returned to the laboratory within 1 h by high-speed launch, and withdrawn when required from the sampler ashore, using all the precautions against contamination and agitation described by KOIKE et al., 1990 and KOGURE and KOIKE, 1987. Before counting, natural seawater was prescreened through 3-4 fim Nuclepore filters, using gravity filtration, an essential procedure when working with extremely small orifices, though there is a risk of losing sub-micron particles thereby. Adsorption may occur when small particles pass through some filters with up to Slim pores (SHELDON, 1972; JOHNSON and WANGERSKY, 1985), though for Nuclepore filters larger than 2.0fim this should not be a significant loss. The opposite case, the risk of artificially creating particles by fracture of larger floes, is unimportant since flow through Nuclepore orifices of the dimension used is at extremely low Reynolds numbers: while very large (lOO,lm-l mm) floes would probably disintegrate on contacting the filter, no mechanism exists to produce sufficiently nonlaminar flow through the orifices to fragment the material to sub-micron size. These assumptions have been tested subsequently with both Elzone and Multisizer equipment at sea: with neither instrument was there a significant change in the number of sub-micron particles in unfiltered or in 3-4 11m filtered seawater. We made counts each day (Table 1) of bacteria, cyanobacteria and photosynthetic nanoplankton with epifluorescent microscopy and flow cytometry (Coulter EPICS Vand Bectron-Dickinson FACS analyser). Modifications have been made to our Coulter EPICS to enable detection of picoplanktonic prochlorophytes (CHISHOLM et al., 1988), but none of these cells were evident in the shelf water. Since marine viral particles are in the size range 0.03--0.1 fim, they cannot be sensed using particle counters. RESULTS AND DISCUSSION

Counts of sub-micron (0.36-1.0 fim) particles obtained with the Elzone 80XY were similar to those obtained previously in the western Pacific Ocean (KOIKE et al., 1990), and

TaMe 1. Characteristics of tire water masses and biota attire offshore (WBSW = Whistle Buoy Sea Water) and inshore (CHSW = Chebucto Head Sea Water)stations, respectively, ami for two depths, Particulate C and N counts are 0.7-D.8pm equivalent GFFfiltered. Tire Elzonc counts are given as tirefull rangecounted (0.32-1.00 pm) and also as a count truncatedto covertire same rangeas tire Multisizcr (0.42-1.00 pm). EPICS and B-D FACS indicatetire two flow cytometers used Station data Latitude (N) Longitude (W) Z(m) ("C) (ppt)

Chlorophyll a Phaco-pigmcnts NO) Si0 2 P0 4

(mg m- 3) (mg m- 3) (11m I-I) (urn I-I) (11m I-I)

Synechococcus (Microscope) Syncchococcus (EPICS V) Synechococcus (B-D FACS)

S.D. WBSW 10 m

WBSW30 m

Mean CHSW 10m

S.D. CHSW 10m

CHSW30m

44~2.5'

44°22.5' 63°2'

44°22.5' 63°2'

44°31.5' 63°3'

44°31.5' 63°3'

44~8.5' 63~'

10 16.33 30.83

0.41 (U8

30 12.80 31.986

10 14.47 31.097

2.83 0.43

30 12.40 31.247

1.10 0.44 0.00 0.50 0.27

0.11 (l.05 (l.00 0.05 0.01

1.297 0.807 0.363 0.859 0.400

1.556 0.558 0.504 1.273 0.409

0.08 0.15 0.87 0.80 0.19

1.784 1.191 1.376 2.575 0.616

63°2'

Depth Temperature Salinity

Procaryotes Bacteria

Mean WBSW 10m

CIl

l::

er

3

n'

a :::l

."

'-'

::'.

a:<>

V>

(No. (No. (No. (No.

ml- 1) ml- 1) ml") ml- t )

1.05 1.21 1.11 1.19

x x x x

10(, 105 lOs lOs

2.72 1.34 1.31 2.14

X X X X

105 104 104 104

9.90 4.37

X X

-

4.00

X

105 104 104

1.12 X 106 7.17 x 104 5.64 X 104 6.45 X 104

7.03 2.92 2.28 2.94

X X X X

104 104 104 104

6.42 6.03 3.95 6.50

X X X X

105 104 104 104

S'

~ ;;; g o' V>

::r

Eucaryotcs Small cucaryotes (EPICS V) Small eucaryotes (B-D FACS) Large eucaryotes (EPICS V) Large euearyotes (B·D FACS) P-Nanotlagellates (Microsc) H-Dinotlagellates (Microse) H-Nanotlagellates (Microsc)

(No. (No. (No. (No. (No. (No. (No.

GFF Particulates Particulate carbon Particulate nitrogen

(ug ml- I ) (ug ml- I )

om

0.18

0.04 0.01

Resistive-pulse counter sub-micron 0.42-1.0/Im Multisizer 0.42-1.01 pm Elzone 0.36-1.01 pm Elzone

particles (No. rnl"") (No. ml- I ) (No. ml- I )

1.43 x 107 1.59 x 107 2.35 x 107

5.34 x 10(, 3.30 X 10(, 5.24 X 10(,

mJ- 1) ml- t ) ml- 1) ml- I ) ml- 1) ml ") ml- 1)

9.99 1.25 1.81 2.89 6.10 6.60 6.60

x x x x x x x

103 104 103 103 102 102 10'

2.33 x 103 5.37 X 103 6.73 X 102 5.11 X 102

5.60 X 9.01

X

103 102

X X

X X X X

103 103 102 103

0.17 0.02

0.12 0.01

7.80 1.06

4.64 4.48 9.80 3.11

106 107

6.26 1.10 1.51

X X X

1.98 2.85 2.53 1.20

X X X X

103 103 102 103

0.04 0.01 106 107 107

9.25 4.76 6.47

X X X

6.49 1.26 1.06 1.25

X X X X

103 104 103 103

~ ~

'-'

...r>

0.19 0.02 105 106 106

5.60 7.94

X X

106 106

II,,;.)

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4

1.510

MULTISIZER II '3. ~

.,; II:

4

1.0 10

W

Q.

en w

--'

~ 5.0103 II: < Q. 0.010° 0.3

1.275 0.625 0.95 EQUIVAlENTSPHERICAlPARTICLE DIAMETER (J.I.m)

1.6

4

1.5 10

ELZONE BOXY '3. q 0 ~

1.0104

II:

w

Q.

en

w --'

~ 5.010

3

II:

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Q.

0.0 10° 0.3

0.625 0.95 1.275 EQUIVAlENTSPHERICAL PARTICLE DIAMETER (J.I.m)

1.6

Fig. 1. Representative particle size distributions from the Elzone and Multisizer equipment, from the offshore station at 10 m depth. Open symbols, 27 September 1990; closed symbols, 2 October 1990. Anomalously high counts in smallest channels of Elzone represent electronic noise in channels
were higher than the counts of bacterial and cyanobacterial cells by a factor of 1-2 orders of magnitude. The counts of sub-micron particles obtained with the Coulter Multisizer were more variable because the instrument was being worked up, but were within the same range as those from Elzone 80XY. The overall mean counts of sub-micron particles from these two instruments for the 10 m offshore samples, when corrected for the different size ranges covered by the two instruments, were remarkably similar. Size distributions of the sub-micron particles at 10 m depth obtained with both counters was similar, with a peak abundance at about 0.5 Jim and a sharp decrease in numbers with increasing particle size (Fig. 1). Progressive dilution with 0.22 Jim Sterivex-filtered NaCl (isotonic with seawater) confirmed that physical entities were indeed present in the natural seawater in the numbers indicated by the Multisizer II (Fig. 2). Had the the apparent particle count been caused by an instrumental artifact (such as electronic interference, which must be carefully avoided in setting up resistive particle counters) this dilution effect could not have been observed. The dilution curves, when corrected for the dilution factor, show some quenching by coincident passage of particles in full seawater. This was confirmed by dilution experiments with 0.91 Jim latex beads, and we suggest that seawater samples should be diluted

Sub-micron particles in Atlantic shelf water

w

a:

~

7 1.5 1 0

~

7 1.0 1 0

- - - MS/280990-1

MS/031090 --0- MS/270990-2 - 0 - MS/270990-1 -e-- MS/280990-2

:J

...J

:§

a:

w aw ...J

5

m U

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<:

a-

6

5.0 10

LL

0

a: w

lD

:?;

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0.0 10° 10

DILUTION FACTOR FORSEAWATER IN NaCI

Fig. 2. Progressive dilution experiments done with the Multisizcr equipment in which seawater was progressively diluted with particle-free isotonic NaCi. Replicate data from 27 and 28 October 1990, and data from single experiment on 3 October 1990 arc shown.

prior to Multisizer analysis of sub-micron particles in the same manner as described for Elzone equipment (KOGURE and KOIKE, 1987). We also were able to remove the particles progressively by ultra-centrifugation (Beckman L7, 150,000g), so that after 5.2 h more than 90% of the 0.36-1.0 pm particles had been spun down from the supernatant fluid in the centrifuge tubes. The progressive disappearance of particles during this process is seen in Fig. 3. After ultracentrifugation there was no accumulation of discrete 0.4-1.0 11m particles onto charged forrnvar-coated TEM specimen grids at the bottom of the centrifuge tubes. Instead, there was a progressive accumulation of a film, or matte, composed of a diffuse, electron-transparent material, we think because we did not dilute the samples sufficiently. Identifiable particles within this film were mainly bacteria, viruses, and some cell fragments. Few mineral particles were observed. We think this film was formed by the progressive accumulation of discrete, very diffuse organic particles recorded by the resistive-pulse counter as 0.4-1.0 11m ESD. This suggestion is consistent with observations (KOIKE et al., 1990) that the electronicallycounted particles >O.4/lm ESD are not removed by gentle 0.2/1m filtration, indicating that they have a highly flexible, amorphous nature. If this is the case, the particles (or aggregates) may be considerably more labile than organic colloids such as fulvic acids (SCHNITZER and KODAMA, 1975) with respect to microbial remineralization, and thus they may be a significant pool of re-cyclable carbon and nitrogen. Finally, we were also able to reduce the numbers of particles by surface coagulation in a bubbling column (KEPKAY and JOHNSON, 1988), though we were not able to replicate our results with this technique during the time available to us. We have not attempted to extrapolate from our findings to calculate (for example) the possible carbon contribution of these particles to total particulate carbon. The reason for this should be obvious. We would require an independent means of confirming that the dimension indicated by the resistive-pulse particle counters is correct. There still remains the significant possibility that shear at the orifice of the encounters may distort the dimensions of these "flexible" particles (or perhaps aggregates) in a manner not seen for

6

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LO:>GIIURST

12000

TO Tl ---<>- T2.5 ----(r- T6.2

--0--

~

-a. 0 z

et al.

-0--

9000

Qi

.0

E

::J

6000

c: 0

"0

iii

3000

a,

a 1.00 Particle diameter (11m)

~

600

TO T1 ---<>- T2.5 ----(r- T6.2 --0--

'-

::!.

0

'": ME ::!.

-0--

500 400

Q)

E ::J

300

(5

> 0

200

"0

t

III

100

ll.

a 0.10

3

1.00

Par ticle vo lume (J.1m )

Fig. 3. Pro gressive removal of sub-micron particles fro m offshore 10 m seawater by ultracentr ifugation at 150,000g for a total of 6.2 h . Part icle distributions during the time-co urse o btai ned with the Elzone equ ipment . Electronic noise can again be seen in the smallest chann els.

living cells, whose dim ensions (indicated by a Coulter counter) can be checked microscopically. We await th e publication of electron micrographs of intact sub-micron particles (and adirect determination of th eir elemental composition) before we can calculat e their qu antitative significance in th e particulate carbon and nitrogen budget of the ocean. Thus, both these results and th e ori ginal observations off J ap an suggest th at within a micro bial ecosystem , otherwis e stru ctur ed as we would expect it to be with no rm al po pul ations of viru s particles , bacteria , cyanobacteria and sm all euca ryo tes, th ere exists a hitherto unsusp ected and extre me ly numerous population of organ ic particles of dimension eq uivale nt to th e sma llest procaryotes. If these part icles are labile , and if th ey do indeed represent th e lar gest fraction of "disso lved" o rgan ic material defined by the new analytical techniques , th en part of the newly enlarged DOM pool (J ACKSON, 1988) may be ava ilable for recycling mo re ra pidly th an we have assum ed . Th e new sub-micro n part icles appear to differ significantly from particles previously thought to be the smallest entities in

Sub-micron particles in Atlantic shelf water

7

marine detritus: if the inference from previous experiments, that the new particles result from feeding of microheterotrophs on bacteria (KOIKE et al., 1990), is supported by further experiments, then their protistan origin may explain their unusual nature. Previously, one might have thought that the only significant organic contribution of protists to marine organic detrital particles would be whole cells. Acknowledgements-Our workshop was supported by NATO Science Committee grant 0732/88, ONR contract NOOOI4-90-J·1166, CICYT grant no. PA86-401, and PAl grant no. 9/88, and also by other support from our respective institutions. We thank Trevor Platt and Glen Harrison for helpful comments. The work was carried out in laboratories operated at Bedford Institute of Oceanography as part of the climate research programme of the Canadian Department of Fisheries and Oceans.

REFERENCES CHISHOLM S. W., R. OLSm" E. R. ZETTLER, R. GOERICKE, J. B. WATERBURY and N. A. WELSCmlEYER (1988) A novel free-living prochlorophyte abundant in the oceanic photic zone. Nature, 334, 340-343. JACKSON G. A. (1988) Implications of high dissolved organic matter concentrations for oceanic properties and processes. Oceanography, 1,28-33. JOHNSON D. D. and P. J. WANGERSKY (1985) Seawater filtration: particle flow and impaction considerations. Limnology and Oceanography, 30, 966-971. KEPKAY P. E. and B. D. JOHNSON (1988) Coagulation on bubbles allows microbial respiration of oceanic dissolved organic carbon. Nature, 338, 63-65. KOGURE K. and I. KOIKE (1987) Particle counter determination of bacterial biomass in seawater. Applied and Environmental Microbiology, 53, 274-277. KOIKE I., S. HARA, T. TERAUCIII and K. KOGURE (1990) Role of sub-micrometer particles in the ocean. Nature, 345,242-244. MOREL A. and Y.-H. AHN (1990) Optical efficiency factors of free-living marine bacteria: influence of bacterioplankton upon the optical properties and particulate organic carbon in oceanic waters. Journal of Marine Research, 48,145-175. PAUL J. H. and W. H. JEffREY (1984) Measurements of diameters of estuarine bacteria and particulates in natural water samples by use of a sub-micron particle analyzer. Current Microbiology, 10,7-12. RASSOULZADEGAN F. and R. W. SHELDON (1986) Predator-prey interactions of nanozooplankton and bacteria in an oligotrophic marine environment. Limnology and Oceanography, 31,1010-1021. SCHNITZER M. and H. KODAMA (1975) An electron microscopic examination of fulvic acid. Geoderma, 13, 279-287. SHELDON R. W. (1972) Size separation of marine seston by membrane and glass filters. Limnology and Oceanography, 17, 49~98. SUGmURA Y. and Y. SUZUKI (1981) A catalytic oxidation method for the determination of total nitrogen dissolved in seawater by direct injection of liquid samples. Marine Chemistry, 24,105-131. TOGGWEILER J. R. (1988) Is the downward dissolved organic matter flux important in carbon transport? In: The productivity ofthe ocean: present and past, W. H. BERGER, V. S. S~IETACEK and G. WEFER, editors, Wiley. Chichester. TOGGWEILER J. R. (1990) Diving into the organic soup. Nature, 345, 203-204.