Seasonal DOC accumulation in the Black Sea: a regional

explanation for a general mechanism

Gustave Cauwet a,*, Gaelle Deliat a, Anton Krastev b, Galina Shtereva b,Sylvie Becquevort c, Christiane Lancelot c, Andre Momzikoff d,

Alain Saliot e, Adriana Cociasu f, Lucia Popa f

aLaboratoire d’Oceanographie Biologique (UMR CNRS 7621), Observatoire Oceanologique, B.P. 44, 66651 Banyuls sur mer, Cedex, FrancebInstitute of Oceanology, Bulgarian Academy of Sciences, PO Box 152, 9000 Varna, Bulgaria

cEcologie des Systemes Aquatiques, Universite Libre de Bruxelles, Campus de la Plaine, CP 221, Boulevard du Triomphe,

B-1050 Bruxelles, BelgiumdInstitut Oceanographique, URA CNRS 2077, 195 Rue Saint Jacques, F-75005 Paris, France

eLaboratoire de Physique et Chimie Marines, ESA CNRS 7077, Universite P. et M. Curie (Paris VI), 4 Place Jussieu,

75252 Paris cedex 05, FrancefRomanian Marine Research Institute, Brd Mamaia 300, 8700 Constanza, Romania

Abstract

During three cruises in the Black Sea, organised in July 1995 and April–May 1997, biological and chemical parameters that

can influence the carbon budget were measured in the water column on the NW shelf, particularly in the mixing zone with

Danube River waters. We observed in early spring (end of April–May) conditions an important input of freshwater organisms

that enhanced the microbial activity in the low salinity range. High bacterial activity regenerates nitrogen in the form of nitrates,

but is also responsible for an important consumption of ammonium and phosphate, leading to a high N/P ratio and a strong

deficit in phosphorus. The consequence is a limitation of phytoplankton development but also a production of carbohydrates

that accumulate all along the salinity gradient. These mechanisms are responsible for a seasonal accumulation of dissolved

organic carbon (DOC) that increases from 210 AM in winter to about 280 AM in summer. All this excess DOC disappears

during winter, probably degraded by bacterial activity. The degradation of carbon-rich organic matter increases the phosphorus

demand by bacteria bringing limitation to phytoplankton primary production.

D 2002 Published by Elsevier Science B.V.

Keywords: Dissolved organic carbon; Carbon cycling; Accumulation; Productivity; Microbial loop; Nutrients consumption; Carbohydrates;

Black Sea; Danube; Mixing zone

1. Introduction

Dissolved organic matter (DOM) in seawater is now

considered as a major focus to understand marine

biogeochemical processes and must be considered as

an important variable in global carbon cycling

(Ducklow et al., 1995; Guo et al., 1996; Hansell and

0304-4203/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S0304 -4203 (02 )00064 -6

* Corresponding author. Tel.: +33-46888-7356; fax: +33-46888-

5946.

E-mail address: cauwet@arago.obs-banyuls.fr (G. Cauwet).

www.elsevier.com/locate/marchem

Marine Chemistry 79 (2002) 193–205

Carlson, 1998; Jickells et al., 1995; Kirchman et al.,

1991; Toggweiler, 1992). From several recent studies,

we know that an important proportion of the carbon

fixed by phytoplankton through photosynthesis can be

released as DOM during blooms (Biddanda and Ben-

ner, 1997; Biersmith and Benner, 1998; Fajon et al.,

1999; Norrman et al., 1995; Strom et al., 1997). Several

authors have calculated the mean percentage of the

carbon fixed by photosynthesis that is rapidly released

as DOC, and the numbers proposed are ranging

between 10% and 20% (Chen and Wangersky, 1996;

Malinsky-Rushansky and Legrand, 1996; Passow et

al., 1994). In very extreme situation, like the mucilage

events occurring sometimes in northern Adriatic Sea,

the numbers can be more important, up to 70% (Ober-

nosterer andHerndl, 1995).Mucilage DOC is generally

utilised by bacteria (Smith et al., 1995) but there is also

evidence that the DOC released is frequently accumu-

lating in the euphotic zone during summer time (Bor-

sheim and Myklestad, 1997; Borsheim et al., 1999;

Williams, 1995). The net annual accumulation of DOC

in the euphotic zone was calculated by several authors.

In the Sargasso Sea, it was estimated at 0.99–1.21 mol

C m� 2 (Carlson et al., 1994), in the western Medi-

terranean Sea, at 1.23 mol C m � 2 (Copin-Montegut

and Avril, 1993) and up to 1.65–4.18 mol C m � 2 in

north Atlantic waters (Borsheim andMyklestad, 1997).

The accumulated DOM is mainly composed of carbon-

rich molecules like carbohydrates.

Williams (1995) pointed out that the export of C-

rich organic matter provokes a decoupling of the

carbon and nitrogen cycles that could be of some

importance from a geochemical point of view. To

perform the mineralisation of that carbon-rich DOM

in surface and coastal waters, bacteria will need

inorganic nitrogen and phosphorus and then compete

with phytoplankton for these nutrients.

The accumulation of DOM was generally evi-

denced in oligo- to meso-trophic environments (W.

Mediterranean, Sargasso Sea, North Atlantic) but

rarely in eutrophic nutrients-rich coastal environ-

ments. Due to an important inflow of freshwater from

several large rivers (Danube, Don, Dniepr, Dniestr

Rivers) the Black Sea is generally considered a

eutrophicated environment (Aubrey, 1992; Bodeanu,

1992; Gomoiu, 1992; Murray et al., 1991). The low

salinity ( < 20) and the reported high DOC concen-

trations (Coble et al., 1990; Mopper and Kieber, 1991;

Torgunova et al., 1992) make this environment attract-

ing to study the possible DOC accumulation in a rich

environment. In the present paper the DOC accumu-

lation was considered in relation to nutrients utilisa-

tion and recycling, organic carbon distribution and

bacterial activity under different seasonal conditions

(winter, spring and summer) in the NW Black Sea

from 1995 to 1997.

2. Materials and methods

2.1. Sampling

During summer 1995 (17 July–1 August) and in

April–May 1997, three cruises were organised in the

NW Black Sea (Fig. 1), onboard the RV ‘‘Professor

Vodyanitsky’’ (IBSS, Sevastopol, Ukraine). Water

samples were collected at different depths at respec-

tively 26 (July 1995), 50 (April 1997) and 25 (May

1997) stations, with a Go-Flo bottles rosette mounted

on a Mark III–Neil Brown CTD probe. During the

EROS-21 expedition (April–May 1997), three high-

resolution salinity gradients (from 10 to 11 samples)

were obtained between salinity of >0 and 18 in the

Danube River plume. Measurements in the plume

were compared to those within the Black Sea (salinity

>17, marine reference). Salinity sections were

sampled in front of two Danube delta branches. One

in front of the Chilia branch, the northernmost one (15

April) and two in front of the Sulina branch, the

central one (18 April and 2 May). The transect in

front of the Sulina branch was first sampled during

winter while the second one was performed under full

spring conditions. Sampling of each salinity gradient

was conducted ‘‘quasi-synoptically’’, within less than

6 h. Surface samples were collected with clean plastic

buckets or glass bottles and processed later aboard the

RV ‘‘Professor Vodyanitsky’’. We will use the term

‘‘mixing zone’’ for all samples collected at low to

intermediate salinity (0–18), while samples at higher

salinity (18–20), encountered only at depths higher

than 20 m, will be considered as ‘‘marine’’ samples.

2.2. DOC and POC

Water samples were filtered through pre-com-

busted glass fibre Whatman GF/F filters (porosity

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205194

Fig. 1. Map of NW Black Sea with sampling sites. Detailed situation of stations sampled in the salinity gradients.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205 195

0.7 Am), under gentle vacuum (200 mm Hg). Previous

tests have demonstrated that cell breakage and sub-

sequent DOC release are negligible in these condi-

tions (Cauwet, unpublished). The filtrate was stored in

glass tubes, poisoned with mercuric chloride (18.5

AM) and closed with Teflon lined screw caps. This

preservation method has proved to be efficient even

after several months storage. The concentration

chosen (18.5 AM) was determined to be perfectly

efficient on filtered samples without disturbing the

IR measurement. Metallic mercury (Hg0) is very

volatile and can easily form amalgams with many

metals, including gold that covers the inside of the IR

cell of the TOC analyser, but the proportion of that

reduced chemical form produced in the combustion

tube in very oxidative conditions is very low and the

copper wool utilised to trap halogens can also fix the

traces of mercury. Former experiments made in our

laboratory have shown a shifting of the IR baseline,

due to shadowing of the IR cell, when the concen-

tration of mercuric chloride was over 100 AM. No

disturbance was observed at 40 AM.

Further analysis of the dissolved organic carbon

(DOC) was performed using a high-temperature cata-

lytic oxidation (HTCO) technique (Shimadzu TOC

5000). Each filtrate was acidified to pH 2 by 5 N

phosphoric acid and bubbled for 10 min to eliminate

all the inorganic carbon. After CO2 elimination, 100

Al were directly injected into the vertical furnace,

filled with platinum coated silica, and oxidised at

680 jC. The CO2 formed was analysed with a non-

dispersive infrared detector (NDIR). The instrument

blank, estimated from the measurement of milli-

Q + pure water (TOC < 0.4 AM), was in the range

6–11 AM. The concentration was calculated from

the 4-point calibration curve, shifted to the origin

and the blank (average of 3–6 tubes) was subtracted

from this value. The precision is in the rage 2–3%

(Cauwet, 1994). Total dissolved nitrogen (TDN) was

analysed at the same time as DOC by coupling a

Sievers NOA 170B chemiluminescence detector to

the TOC analyser. This method, based on a lab-made

device has proved to be reliable and precise (better

than 2%) by the participation to international inter-

calibration exercises (Sharp and et al., 2000) and is

described elsewhere (Cauwet, 1999). The blanks were

in the range 0.3–0.6 AM and the method for calcu-

lation was the same as for DOC.

The filters were rinsed with milliQ water and dried

at 40–50 jC for 24 h before being analysed for

particulate organic carbon (POC) and nitrogen

(PON). POC and PON were analysed using the same

analytical system and method but with a different

furnace (900 jC+ catalyst). The filters were first

acidified with 5 N phosphoric acid in a silica boat

and dried in an oven at 40 jC for 24 h before being

analysed. The blanks, estimated from clean filters

acidified like samples were below the detection limit,

but filters combusted several weeks before and stored

protected from dust (polycarbonate boxes) but not

from atmosphere exhibited variable blanks from 0 to 7

Ag C. It was also observed that the phosphoric acid

solution utilised to destroy carbonates must be pre-

pared fresh each day, to avoid increasing nitrogen

blanks, which are probably due to the fixation of

ammonium in the atmosphere.

2.3. Nutrients

Inorganic nutrients (NO3, NO2, NH4 and PO4) were

measured onboard immediately after sampling. Nitrate

and nitrite concentrations were determined by spec-

trophotometry according to Grasshoff (1983). Deter-

mination of nitrite or the sum nitrite plus nitrate, after

reduction on a Cd column, is performed after the

formation of a diazonium salt, which couples with a

second aromatic amine to form an azo dye. Ammo-

nium and phosphate concentrations were deter-

mined by spectrophotometry according to Koroleff

(1983a,b). Phosphate was determined by forming a

phosphomolybdate complex, which was then reduced

to a coloured complex with ascorbic acid. Ammonium

reacts with hypochlorite in excess to give a monochlor-

amine which, in the presence of phenol and cataly-

tic amounts of nitroprusside ions, gives indophenol

blue.

2.4. Carbohydrates

The analysis of carbohydrates was performed on

the 0.2-Am filtered fraction, by the TPTZ method of

Myklestad et al. (1997) after a 0.1 N HCl hydrolysis

(100 jC, 20 h in nitrogen atmosphere). In this method,

reduced sugars (monosaccharides) are downgrading

Fe3 + to Fe2 + , which form a coloured complex with

2,4,6-tripyridyl-s-triazin (TPTZ).

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205196

2.5. Bacterial production

Bacterial production was estimated by incorporat-

ing 3H-thymidine into bacterial DNA (Fuhrman and

Azam, 1982). Some 10 ml of seawater samples were

incubated at an in situ temperature in the dark for 60

min with a saturating concentration of 40 nM 3H-

methyl-thymidine (48.2 Ci mmol � 1, Radiochemical

Centre, Amersham, England). After a 1-h incubation

period, cellular macromolecules were precipitated

with ice-cold trichloroacetic acid (TCA) (5%, final

concentration) over 5 min. The cold TCA insoluble

material was collected by filtration onto cellulose

acetate filters (Sartorius, 0.2 Am pore size) and rinsed

five times with 5 ml of ice-cold 5% TCA. The filters

were placed in scintillation vials, and counted by

liquid scintillation (Filtercount PackardR). The thy-

midine incorporation rate was converted into the

bacterial cell production rate, by using a conversion

factor of 0.5� 1018 bacteria produced per mole of

incorporated thymidine (experimentally determined

in this area; cf. Becquevort and Bouvier, unpublished

data). The bacterial biomass production rate was

estimated from the cell production rate by using the

bacterial carbon content estimated from epifluores-

cence microscopy analysis.

2.6. Ectoenzymatic activity

The utilisation of polymeric organic matter

demands a preliminary hydrolysis. To evaluate the

ability of bacteria to hydrolyse biopolymers, potential

ectoenzymatic activity was measured at 20 jC after

adding a saturating concentration of artificial sub-

strates that produce fluorescent products when hydro-

lysed by present ectoenzymes. Two ectoenzymatic

activities were tested: ectoprotease and ecto-h-gluco-sidase.

To determine ectoprotease activity, L-leucyl-2h-naphtylamine hydrochloride (LLhN) was used as

substrate for proteolytic ectoenzymes. It produces

fluorescent naphtylamine after hydrolysis of the pep-

tide-like bond. The experimental procedure was based

on that of Somville and Billen (1982). Water samples

(2 ml) were transferred into a sterile quartz fluorimeter

cell kept at 20 jC; 50 Al of a sterile 40 mM solution

(1000 Am final concentration) of L-leucyl-2h-naphty-lamide was added and the increase of fluorescence at

410- under 340-nm excitation (Kontron SFM 25

fluorimeter) was measured as a function of time over

25–30 min. Enzyme activity was estimated from the

initial slope. Fluorescence of a standard naphtylamine

solution was used for calibration.

To determine ectoglucosidase activity, 4-methyl-

umbelliferyl-h-glucoside (MUF-GLU) was used as a

substrate for h-glucosidase, which produces fluores-

cent 4-methylumbelliferone after hydrolysis of h-linked (1–2, 1–3, 1–4, 1–6) disaccharides of glucose,

celluhexose and carboxymethyl cellulose (Barman,

1969). The procedure was adapted from the protocol

of Hoppe (1983) and Somville (1984). Two-milliliter

water samples were transferred into sterile quartz fluo-

rimeter cells and kept at 20 jC; 250 Al of a sterile 6 mM

solution of 4-methylumbelliferyl-h-glucoside was

added. The increase of fluorescence at 445 nm under

360-nm excitation was again measured as a function of

Table 1

Dissolved inorganic nutrients, DIN/DIP ratio, Chl. a [min�max

(mean)] of the Danube–Black Sea mixing zone and the marine

waters in July 1995 and April–May 1997

Mixing zone,

0.3 < salinity < 17

Marine waters,

salinity>17

July 1995

Number of stations 10 8

NO2–NO3 (AM) nd–28.00 (5.09) nd–0.8 (0.2)

NH4 (AM) 0.24–2.42 (0.88) nd–0.7 (0.3)

PO4 (AM) 0.05–0.47 (0.17) nd–0.1 (nd)

DIN/DIP 3–62 (22) nd–18 (nd)

Chlorophyll-a

(Ag l� 1)

1.32–15.08 (5.15) 0.2–1.8 (0.9)

April 1997

Number of stations 24 7

NO2–NO3 (AM) 13.88–203.05 (94.37) 1.03–25.2 (7.06)

NH4 (AM) 0.14–8.88 (3.99) 0.04–0.80 (0.31)

PO4 (AM) 0.09–2.94 (1.36) 0.01–0.1 (0.06)

DIN/DIP 25–245 (92) 22–1301 (291)

Chlorophyll-a

(Ag l� 1)

0.57–6.64 (4.01) 0.42–3.80 (1.11)

May 1997

Number of stations 20 5

NO2–NO3 (AM) 13.05–72.11 (35.80) 1.00–5.25 (2.52)

NH4 (AM) 0.14–8.43 (2.39) 0.29–0.50 (0.32)

PO4 (AM) 0.01– 2.12 (0.55) nd–0.02 (0.01)

DIN/DIP 55–1808 (321) 131–280 (213)

Chlorophyll-a

(Ag l� 1)

1.15–14.39 (5.75) 0.95–1.92 (1.20)

nd: non-detectable, concentration below the detection limit.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205 197

time over 25–50 min. Enzyme activity was estimated

from the initial slope. Fluorescence of a standard 4-

methylumbelliferone solution was used for calibration.

3. Results

3.1. Environmental conditions

During the April–May 1997 cruise, meteorological

conditions shifted from winter to full spring. Until 25

April, typical winter meteorological conditions pre-

vailed. The river discharge was around 9000 m3 s� 1

(A. Cociasu, personal communication), and the wind

speed varied between 6 and 11 m s� 1 (average 8.5 m

s� 1) with a south–southwestern or north–northwest-

ern direction. No real river plume was formed and the

extension of the mixing zone influenced by Danube

Riverwas less than10milesoffshore.Temperature in the

surface mixed layer ranged between 5.7 and 8.0 jC.

Fig. 2. Distribution of inorganic nutrients (NO3, NH4, PO4) along the salinity gradients at different periods of the year. The line indicates perfect

mixing dilution.

Fig. 3. Dissolved inorganic N/P ratio evolution along the salinity

gradient at different seasons.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205198

After 25 April, stable meteorological conditions

prevailed. Danube River discharge decreased to

8200–8400 m3 s� 1. The predominant south–south-

eastern wind ranged between 0 and 6 m s� 1 (aver-

age 3 m s� 1), enhancing the vertical stratification

and the river plume extended over the shelf up to a

distance of 40 miles offshore. Temperature in the

wind mixed layer ranged between 8.1 and 12.0 jC(average 9.2 jC).

During the July 1995 cruise, river discharge was

around 8000 m3 s� 1. Due to changing wind con-

ditions (predominant south wind speed ranged

between 0 and 12 m s � 1), the river plume was

sometimes destroyed but it rapidly reformed with a

great extension. Temperature in the wind mixed layer

ranged between 22.8 and 25.6 jC (average 24.3 jC).

3.2. Nutrients

In marine waters, the concentration of all nutrients

was never really high but was specially low in July

1995 (Table 1). Concentration in chlorophyll did not

reach 2 Ag l� 1 (with one exception). It was different

in the mixing zone where the river inputs have a great

influence, giving rise to important concentration gra-

dients. Nitrate concentration in the river was very

variable, exceeding 200 AM in April, and only about

Fig. 4. Total suspended matter along the salinity gradient in early

May 1997 (Sulina 2).

Fig. 5. Particulate organic carbon and nitrogen and their ratio in the salinity gradient in April (Sulina 1) and early May 1997 (Sulina 2).

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205 199

70 AM in May and 30 AM in July. In any condition

there was a dilution along the salinity gradient with

low values at high (!) salinity. The same observation

can be made for ammonium and phosphate, where the

highest values are observed in April (8.8 and 2.9 AM,

respectively) and May (8.4 and 2.1 AM, respectively),

much lower in July (2.4 and 0.4 AM, respectively).

But the major difference between seasons for all

nutrients is in the distribution patterns observed along

the salinity gradient (Fig. 2). In winter conditions, the

mixing zone is dominated by the dilution process, and

all nutrients concentrations plotted versus salinity,

fitting very well with the simple conservative mixing.

In contrast with this observation, these parameters did

not follow the dilution curve in May and July. Nitrates

exhibit an excess concentration in the first part of the

gradient and a deficit in the second part. Ammonium

and phosphates show an important consumption in

May (spring conditions). In July, the same behaviour

is observed for phosphate, ammonium being very low

at all times (below 1 AM).

Another interesting observation is the variation of

the inorganic N/P ratio in the river and in the mixing

zone. In the river, it decreased from winter to summer,

passing from 69 in April to 56 in May and to 33 in

summer. In the mixing zone, it remains almost con-

stant in winter conditions (50–80) but increased in

May and July in the first part of the salinity gradient,

reaching values close to 200 at salinity 10 (May 1997,

Sulina branch) and decreased afterwards (Fig. 3).

3.3. Particulate and dissolved organic carbon

Total particulate organic carbon, including detritus

and living organisms, globally decreases with turbid-

ity along the salinity gradient, as can be observed in

most estuaries due to sinking of riverine particles

(Cauwet et al., 1990; Cauwet and Mackenzie, 1993;

Cauwet and Sidorov, 1996; Meybeck, 1989; Milliman

et al., 1984). Variations of POC are visible during the

two conditions, with some increases at higher salinity

(10–15) probably due to marine production (Figs. 4

and 5). While remaining almost constant during

winter (April), the C/N ratio rapidly decreases in the

first part of the gradient (salinity < 3), and then con-

stantly increases up to salinity 15.

DOC behaviour is completely different from what

can be expected along a salinity gradient (Fig. 6).

Whatever is the season, it exhibits almost constant

values all along the mixing zone (salinity, from 0 to

17). While the true marine values, measured at high

(18–20) salinity and in deep-water samples, are about

constant (around 200 AM or slightly less in the central

deep basin), the mean concentration in surface sam-

ples of the mixing zone varies with time. From about

210 AM in April, it increases up to 220–230 AM in

May to reach almost 300 AM in summer.

3.4. Bacterial production and activity

Bacterial production and specific growth rate were

maximal (76.4 mgC m � 3 day � 1 and 3.3 day � 1) in

the Danube–Black Sea mixing zone and decreased in

marine waters. Depending on which area is being

Fig. 6. DOC distribution vs. salinity during the three cruises: April

1997, May 1997, July 1995. Some data from the April cruise belong

to the spring conditions and are slightly higher (240–270 AM).

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205200

considered, bacterial production and growth increased

sharply in July 1995. No clear spatial variations of

bacterial biomass were reported, however, and the

biomass was lower in April–May 1997 than in July

1995 (Becquevort et al., 2002). More interesting is the

ectoenzymatic activity pattern (protease and h-gluco-sidase). In April, in both Chilia and Sulina branches,

the ectoproteolytic activity decreased sharply from 4

AM day � 1 to almost 0, while in May it increased up

to 7 AM day � 1 at salinity 3 to decrease down to 0 at

higher salinity rates (Fig. 7). Ectoglucosidasic activity,

which remained at relatively low values (0.05–0.02

AM day � 1), also decreases along the salinity gradient,

with the exception of a high concentration in early

May at very low salinity.

4. Discussion

The distribution of the studied parameters de-

scribes a series of processes occurring along the sa-

linity gradient and how they evolve with seasons. The

three periods of the year studied during these surveys

correspond approximately to:

– a winter situation, with low temperature and few

biological activity (April 1997),

– a spring situation (May 1997) where biological

processes have started (in the river and the coastal

zone) in relation with an increasing temperature,

– a summer situation (July 1995), representing, at

least partly, the result of the high activity

developed in spring and early summer.

The month of May, being a period of higher

activity when many parameters are changing rapidly,

is mostly used to describe the processes that occurred.

The decrease in suspended matter at low salinity is

characteristic of a high rate of sedimentation, as

observed in many estuaries. While total suspended

matter is decreasing regularly during mixing in April,

we observe a fast decrease between salinity 1.5 and 3

in May (Fig. 4). This rapid sinking generally affects in

priority large, dense, inorganic particles like sand and

silt. These particles are poor in organic matter (less

than 1%) and the sedimentation mechanism has no

great effect on the carbon stock as suspended material

(Cauwet and Mackenzie, 1993).

The decrease observed for POC in spring condi-

tions (Fig. 5b) suggests that, in addition to the organic

matter trapped in the inorganic matrix and lost by

sedimentation, a fraction of particulate organic matter

(40–50 AM C) is rapidly degraded. During the period

of the survey, a high content of freshwater living cells

(mainly diatoms) was observed in the river water.

When higher salinity rates were reached (3–4), these

freshwater organisms seem to be killed by the change

in osmotic pressure and the cells are lysed, as dem-

onstrated by the high concentrations of serine meas-

ured in low salinity samples (Ragueneau et al., 2002).

Besides exudation of organic components, algal

cellular components released through cell lysis may

be an important source of organic matter for bacteria.

Laboratory studies on microbial decomposition of

phytoplankton cells have shown rapid remineralisa-

tion rates. Lysis products are macromolecular biopol-

ymers containing carbohydrates, proteins and lipids

that must be hydrolysed by ectoenzymes (protease and

carbohydrases) before taken up by bacteria (Saliot et

Fig. 7. Proteolytic and Glucosidasic activities in the mixing zone of

Danube River in Late winter (Chilia and Sulina 1) and early spring

(Sulina 2) conditions.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205 201

al., 2002). The relatively high concentration in pro-

tease (Fig. 7) demonstrates that this activity is impor-

tant, suggesting the presence of a rich protein

substrate. The maximum of proteolytic activity in

May appears at salinity 2.8 where the lysis seems to

be at its maximum rate and decreases downstream,

while it was much lower originally and regularly

decreases in April (Fig. 7). At the opposite, the

glucosidase concentration is relatively high at salinity

0 but rapidly decreases down to very low values. The

first consequence is an evolution of the nature of

particulate organic matter, particularly the protein

matter. The increase of the particulate C/N ratio

observed between salinity 2.9 and 15.5 is due, at least

partly, to this selective degradation (Fig. 5b). The

second consequence is the visible change in the

concentration of nutrients. In May, NO3 and PO4

decreased in the river end member by 20–30%

compared to the concentrations measured in April.

In contrast, NH4 increased by 50%. These data

suggest the occurrence of significant biological pro-

cesses of nutrient uptake, elimination and regeneration

in the Danube River and leads to a high planktonic

production corroborating the richness of freshwater

inputs in living cells. Combined with river processes,

hydrolysis and degradation of protein organic matter

at low salinity is regenerating inorganic nitrogen in

the form of nitrates, as demonstrated by the excess

concentration noticed up to salinity 10 (Fig. 2).

Ammonium and phosphate exhibit opposite behav-

iour. The utilisation of these two inorganic nutrients

by algal and bacterial activity is more rapid than the

regeneration rate and the concentration profile shows

an important consumption all along the salinity gra-

dient, to rapidly reach very low concentrations at

higher salinity rates.

These processes, occurring immediately at the river

mouth, have a dramatic importance for the biological

activity in the coastal zone and the carbon budget of

the entire Black Sea. Due to the consumption of

phosphate by microbial loop, phosphorus concentra-

tion is rapidly falling down to extremely low values at

salinity rates higher than 7–10. This low phosphorus

content is not very favourable for primary production

and explains the relatively low chlorophyll concen-

tration measured in this area. It was observed (Rague-

neau et al., 2002) that the planktonic population at

high salinity (10–15) contains a high proportion of

small organisms (picoplankton), which is character-

istic of oligotrophic waters. Moreover, correlation

analyses show a PO4 control on the significance of

the classical food web versus microbial food web in

the NW Black Sea.

Another consequence of these processes is the

increase of the inorganic N/P ratio. It was observed

several times that when the inorganic N/P ratio is

high, while nitrogen and phosphorus are at low

concentration, phytoplankton tends to produce car-

bon-rich organic matter like carbohydrates (Fajon et

al., 1999; Søndergaard et al., 2000). The results

previously obtained during mesocosm experiments

(Fajon et al., 1999) describe this mechanism well

(Fig. 8). If this is occurring here, we should observe

higher carbohydrates concentration in May than in

April, and possibly even more in July. Unfortunately,

carbohydrates were not measured in July but when we

compared the concentration of total dissolved carbo-

hydrates in April and May 1997, we observed a

decrease from 25 to 8 AM C in April (from salinity

2 to salinity 15) and an almost constant value around

27 AM C in May for the same salinity range (Fig. 9).

Obviously, the dissolved carbohydrates present in

April are slowly consumed along the gradient while

they are accumulating during spring (and low phos-

phorus) conditions. The quasi-absence of phosphorus

prevents bacteria from using that carbon-rich organic

matter, as demonstrated in other situations (Fajon et

al., 1999; Søndergaard et al., 2000; Williams, 1995).

The release of carbohydrates by phytoplankton is

generally a second-phase mechanism that occurs after

Fig. 8. Relation between nutrients depletion and the production of

polysaccharides during a mesocosm experiment.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205202

they have accumulated in the cells (and therefore as

particulate carbohydrates). This could justify the

increase of the C/N ratio observed in particulate

matter between salinity 3 and 15 (Fig. 5). When they

are released, they also accumulate in the dissolved

form, thus increasing the DOC content. DOC concen-

tration is globally constant from salinity 0 to 15, but it

must be considered also that the values measured at

salinity 0 are systematically lower than in the mixing

zone. This suggests a source of extra DOC in the

coastal zone. Comparing the DOC concentration in

April and May, we notice an increase of the average

value of about 15 AM C, which is about the same

concentration as that of carbohydrates which accumu-

lated at intermediate salinity. We can suppose that the

accumulation of carbohydrates is going on until

summer and leads to an increase of DOC concen-

tration of about 80 AM, as had been observed. These

opposite mechanisms of production and utilisation of

DOC (proteins degradation and carbohydrates accu-

mulation) could also explain the relatively constant

DOC concentration along the mixing zone. It was

recently suggested (Thingstad et al., 1997) that DOC

accumulation could be due to a malfunctioning of the

microbial loop. We would not say that the microbial

loop is ‘‘malfunctioning’’, but in nutrients deficiency

(particularly phosphorus), bacteria seem to be unable

to utilise a carbon-rich organic substrate like carbohy-

drates. In previous experiments (Fajon et al., 1999),

we have observed that the lack in nutrients (P and N),

the production of carbohydrates, the decrease or

disappearance of glucosidases and the inability of

bacteria to use this DOM occur simultaneously. It is

interesting to note that all factors are converging for

the accumulation of DOC. In the mixing zone of the

Danube–Black Sea system, we never reach complete

absence of nutrients like in mesocosms experiments,

which explains the progressive accumulation of car-

bohydrates and the quite slow evolution of parameters

along the salinity gradient.

Considering that the winter DOC concentration in

the mixing zone is about similar to that in ‘‘marine’’

water at any time, we can suppose that the carbon

accumulating from spring to summer disappears

totally from the water column. What we do not know

precisely is if all of it is recycled in surface waters or

at the sediment–water interface after coagulating and

sinking, or if part of it is escaping to degradation after

reaching the sediment with particles and could con-

stitute a carbon sink. This latter hypothesis is only

probable in two situations: a fast burial of organic

matter with large supply and fast sedimentation of

suspended sediment, which generally occurs in front

of river deltas, or the existence of an anoxic zone in

deep waters, avoiding oxidation of organic matter.

The Black Sea is then a good candidate to be a carbon

sink, with its large shelf receiving the sediments from

several rivers and its large anoxic central basin.

5. Conclusion

These results demonstrate that an initially rich

system can be of limited benefit to primary producers

if bacteria are very active close to the source of

nutrients. High flux of rich organic matter enhances

the biodegradation processes which increase bacte-

rial activity and limit the availability of phosphorus.

This can lead to an accumulation of DOC from

May to September, representing up to 100 AMDOC. In the case of the Black Sea, it is probably

enhanced by the management of the Danube River,

responsible of changes in nutrients supply and

freshwater production (Cociasu et al., 1997). It is

likely that the mechanisms put into evidence during

mesocosms experiments by several workers (Norr-

man et al., 1995, Fajon et al., 1999; Søndergaard et

al., 2000) also occur in natural systems when the

concentration of nutrients decreases and are provok-

ing an important DOC accumulation in the form of

carbohydrates. It gives a very general character to

Fig. 9. Concentration in dissolved carbohydrates along the salinity

gradient in April and May 1997.

G. Cauwet et al. / Marine Chemistry 79 (2002) 193–205 203

the phenomenon and concerns most of oceanic

areas, with probably variable amplitude. There is

evidence that most of the accumulated DOC dis-

appears during winter but we are unable to give a

precise estimation of the fate of this organic supply.

If we can estimate that most of it is recycled

through the microbial loop in surface waters, it

could be of some interest to know how much can

reach the deep ocean, in the dissolved form by

vertical mixing, but also after aggregation as partic-

ulates. In that case, it could be an important vertical

flux of fresh, carbon-rich, DOC, which is rapidly

transferred to the deep ocean and could constitute a

non-negligible carbon sink.

Acknowledgements

We are very grateful to all EROS participants for

their involvement with a special mention to the co-

ordinators N. Panin and J.-M. Martin, to the crew of

the RV «Professor Vodyanitsky» and the Ukrainian

and Romanian pilot boats «Spruit» and «Filioara»,

and to the Chiefs Scientists during the cruises, C.

Lancelot and V. Egorov. We kindly acknowledge the

support of the European Commission under the 4th

Environment Programme (ENV4 CT96-0286). Many

thanks also to the anonymous reviewers who made

very useful comments and suggestions.

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