Marine Chemistry 42 (1993) 237-251 237

Elsevier Science Publishers B.V., Amsterdam

Monosaccharide composition of particulate hydrolysable sugar

fraction in surface microlayers from brackish and marine waters

Anne-Marie Compiano a, Jean-Claude Romano a, Frederic Garabetian a, Pierre Laborde b and

Isabelle de la Giraudirre a
aCentre d'Oc~anologie de Marseille, Station Marine d'Endoume, Rue Batterie des Lions, 13007 Marseille, France
bLaboratorie de Biologie Marine, Av. des Facult~s, 33405 Talence, Cedex, France
(Received 26 November 1991; revision accepted 24 August 1992)

ABSTRACT

Compiano, A.-M., Romano, J.-C., Garabetian, F., Laborde, P. and de la Giraudirre, I., 1993. Monosaccharide composition of
particulate hydrolysable sugar fraction in surface microlayers from brackish and marine waters. Mar. Chem., 42: 237-251.

Twenty-nine sea-surface microlayer samples, associated with their related bulk water, were collected in coastal waters of the
northwestern Mediterranean Sea, and examined for the concentration and composition of their particulate sugar fraction. The
monosaccharide content was assayed by high-performance liquid chromatography (HPLC) and fluorescence detection after
dansylhydrazine precolumn derivatization. Samples were collected in both marine areas influenced by fresh water. As compared
with bulk waters, accumulation of particulate organic matter in the surface microlayer was higher in marine than in brackish
waters (about three-fold higher for particulate organic carbon (POC) and ten-fold higher for sugars). However, in brackish bulk
waters, the concentrations of organic compounds (POC, particulate organic nitrogen (PON), chlorophyll a and sugars) were
higher than in marine bulk waters. In all the samples the sugar content appeared to be directly linked to POC. All the surface
microlayers were enriched in bacteria but phytoplanktonic pigments were less represented in microlayers than in other
particulate organic fractions. In brackish waters, chlorophyll a concentrations were correlated with the percentage of mono-
saccharide carbon in POC, but the microbiomass could explain only a small part (5-10%) of the sugar concentrations recorded
in surface microlayers. In all cases, glucose was the major monosaccharide form. In brackish waters, no change was noted in
monosaccharide composition of the sugar fraction in surface microlayers as compared with their related bulk waters. In marine
samples, a slight but significant depletion of glucose was recorded in the microlayers, and three forms (mannose, arabinose and
rhamnose) exhibited higher percentages ( + 4%, + 4% and + 15%, respectively) in marine sea-surface microlayers. During mild
acid hydrolysis of the particulate organic matter, the release of the 'Dubois-reactive compounds' was faster in marine microlayer
samples than in related bulk waters, and the relative concentration of glucose increased with time of hydrolysis in the two types
of water. When hydrolysis was not complete, a compound which eluted at the beginning of the chromatogram and remained
at a constant level throughout the hydrolysis, was detected.

INTRODUCTION surface of the sea during fine weather periods

Surface microlayers are characterized not
only by enhancement of organic matter loadings,
as compared with bulk waters, but also by
changes in composition. Accumulation processes
are particularly enhanced inside slicks, the
smooth blue-grey stripes which appear on the
Correspondence to: A.-M. Compiano, Centre d'Ocranologie de
Marseille, Station Marine d'Endoume, Rue Batterie des Lions,
13007 Marseille, France.
(Ewing, 1950; Lafond and Lafond, 1972) and are
sites of modification of some physical properties
of surface water by organic matter accumulation
(Garrett et al., 1974).
Several studies have previously been carried
out on a microlayer lipid fraction which was
suspected of having a major surfactant influence
(Daumas et al., 1976; Marty et al., 1979; Barbier
et al., 1981). Other studies (Williams et al., 1986)
have shown that other compounds such as

0304-4203/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved

238
proteins or carbohydrates (Henrichs and
Williams, 1985) may have a strong influence on
the surface-active properties of sea-surface
microlayer samples (Baier, 1970; Baier et al.,
1974) and may account for enrichment of
organic matter in surface films (for reviews, see
Garrett (1972) and Sieburth (1983)). These films
are also the site of photochemical reactions,
(Wangersky, 1976), and of accumulation of
bacteria and the consequent biodegradation
based on particulate organic matter (Harvey and
Young, 1980; Mimura et al., 1988).
The monosaccharide composition of marine
suspended particulate organic matter has been
studied (Mopper et al., 1980; Sakugawa and
Handa, 1983, 1985), but it is rather difficult to
compare data because of the unreliability of the
available analytical techniques when applied to
seawater samples. In seawater, the amounts of
suspended particulate matter are generally low;
consequently, isolation and purification steps are
usually necessary before the assaying pro-
cedures, with consequent losses and changes in
material (Dawson and Mopper, 1978). Recent
availability of sugar-specific fluorochroms
(Avigad, 1977) and developments in high-perfor-
mance liquid chromatography (HPLC) (Mopper
and Johnson, 1983), have facilitated monosacch-
aride separation and detection at low concentra-
tion levels. Using this technique, we have
examined the monosaccharide composition of
the hydrolysable polysaccharide fraction of the
particulate organic matter accumulated in
surface films formed in marine or brackish water
areas and compared them with the related bulk
water samples. In parallel, we have studied, in
some samples, the degradability of the whole
sugar fraction by means of acid hydrolysis
kinetics.
MATERIALS AND METHODS
Sample collection
All the microlayer samples were collected
using Harvey's skimmer (Harvey, 1966), and the
A.-M. COMPIANO ET AL.
related bulk waters were sampled at 0.5 m depth
by pumping with acid-cleaned silicone tubing.
Sample collection was carried out in various
coastal areas (Fig. 1), mainly on the French
Mediterranean coast, either at marine sites or in
coastal areas under the influence of continental
water inflows (marked by arrows in the maps):
five samples (H) were collected on the French
Atlantic coast, in a small temperate lagoon with
high primary production rates (Collos et al.,
1988) and natural surface films. The dates and
times of sampling, wind speeds and bulk water
temperatures are given below in Table 2, for each
sample. Samples are here treated together, to
compare the composition of marine and
brackish water microlayers.
Total sugar fraction analysis
Measure~ments were performed on particles
collected by filtration on precombusted (for 5 h
at 550°C) Whatman (Hillsboro, OR, USA) GF/
F filters and treated by the phenol-sulphuric acid
method of Dubois et al. (1956), according to the
procedure of Gerchakov and Hatcher (1972),
and measured with a spectrophotometer at
485 nm. For calibration a glucose solution was
used as the standard. On the basis of 11 of filtered
seawater, the limit of detection was less than
2/~gl -l and precision was about 15% of the
measured values.
Kinetics of acid hydrolysis
We have studied the release of the Dubois-
reactive compounds and monosaccharide com-
position during mild acid .hydrolysis on five
microlayer and related bulk water samples col-
lected in a marine coastal area. For each sample,
six subsamples of particulate matter were col-
lected as above. One was treated as a whole by
the Dubois colorimetric method. Five were
hydrolysed at 100°C by 5 ml of 0.5 M HESO 4. At
10, 20, 40, 60 and 150rain, the hydrolysis was
stopped by immersion in ice, followed by cen-
trifugation to remove the filter. Part of the super-
PARTICULATE HYDROLYSABLESUGAR FRACTION IN SURFACEMICROLAYERS 239

N 5q E 47°N.
Hl to H51

A B- Atlantic
Ocean
7

~Bordeaux
44°I~

1~: continental water]

inflows ]

eR3
eR1

eR4
Mediterranean Sea

~ ; "*'-
, .

5,20"E
°"
~

1
E

Fig. 1. Map of sampling locations.

natant was measured as above and part was
assayed by HPLC.
H P L C measurements
The particulate matter, collected on filters as
above, was treated with 5 ml of 1.86 N H 2SO4 at
100°C for 4h (Mopper, 1977). As reported by
Mopper et al. (1980), stability was good under
these conditions. After centrifugation, the super-
natant (neutralized to pH 4.0) was subjected to
HPLC, using the method of Mopper and
Johnson (1983), by precolumn fluorochrom
derivatization at 65°C for 20min; the fluoro-
chrom reagent was dansylhydrazine (DNS
(Sigma Chemical Co., St. Louis, MO, USA, ref.
D,7634); Avigad, 1977) and was used at 5% w/v.
Depending on the carbohydrate concentrations
of the samples, hydrolysed volumes in the
reactions varied from 20 to 200 pl, but the water/
DNS ratio was kept constant by the addition of
a sufficient volume of bidistilled water to obtain
a final reaction volume of 640#1. Both the hyd-
rolysis extracts from acid kinetics experiments
and the completely hydrolysed water samples
were treated in the same way. Separation was
carried out on a Beckman (Berkeley, CA, USA)
Ultrasphere CIs-ODS column (5 pm,
250mm x 4.4mm). Eluant A was 0.08 M acetic
acid (pH 4.0), eluant B was acetonitrile and the
flow rate was 1.2mlmin -~ . The gradient used
was: 22-30% B for 14min, 30-70% B for 2min,
240 A.-M. COMPIANOET AL.

isocratic at 70% B for 3 min, and 70-30% B for TABLE 1

3min. The following monosaccharides were Release of sugar compounds during hydrolysis kinetics, at
detected: galactose (Gal), glucose (Glc), various times (T: 10, 20, 40, 60 and 150 min) in microlayer
(MIC) and bulk water (BW) samples
mannose (Man), xylose (Xyl), arabinose (Ara),
ribose (Rib), fucose (Fuc) and rhamnose (Rhm). /'10 T20 T4o r~0 L_~o
Slight changes in elution gradient or in
Dubo& assays
the pH of eluant A were sometimes necessary to A MIC 11 11 14 15 17
achieve optimum xylose/arabinose separation or BW 11 11 15 16 18
when monosaccharide concentrations were low H P L C assays
B MIC 5 38 73 76 100
relative to that of DNS (see Compiano and
BW 16 25 64 73 100
Romano, 1988). Fructose, which reacts poorly C MIC nd 27 32 33 37
with DNS (Avigad, 1977; Mopper and Johnson, BW nd 21 19 32 43
1983), often coelutes with arabinose; its response D MIC 41 42 22 23 33
BW 20 17 20 25 24
factor was normalized to 1/27 with arabinose. Its
concentrations were always low and it is not A: Percentages of sugar released at each time (T) of hydrolysis,
considered here. On a basis of 11 of filtered in reference to the total sugar weight directly assayed by the
Dubois method on a subsample.
seawater, the detection limit varied from B: Percentages of the HPLC-assayed monosaccharide sum re-
5.5nmoll -~ for rhamnose to 30nmoll -~ for covered at each time of hydrolysis related to the assayed sum at
glucose, and reproductibility varied from 1% for the end of hydrolysis (150min).
C: Glucose recovery at each time of hydrolysis, related to the
glucose to 6% for arabinose.
HPLC-assayed monosaccharide sum; nd: not detected.
The following HPLC apparatus was used: two D: Relative size (in arbitrary fluorescence units; afn) of the
Altex (Berkeley, CA, USA) pumps (model 110A) 'polysaccharide' peak to total compounds resolved by HPLC
monitored by an Altex gradient programmer
(model 420), a Rheodyne (Berkeley, CA, USA) France) analyser. Salinity was measured with a
injection valve fitted with a 100/~1 loop, and a Beckman salinometer. Bacterial counts were
Schoeffel (Westwood, USA) fluorescence carried out by epifluorescence microscopy,
detector (model 97). The fluorescent signal of the according to Hobbie et al. (1977), with DAPI
derivatized monosaccharides was read with an (4',6-diamidino-2-phenylindole), a DNA-
excitation wavelength of 370 nm and a 540 nm binding fluorochrom, as reagent (Porter and
cut-off secondary filter for emission. Feig, 1980). To compare percentage values or
HPLC measurements of chlorophyll a con- poorly normalized data distributions, the non-
centrations were carried out with the same equip- parametric Mann-Whitney test (Siegel, 1956)
ment, on G F / F filters, by extraction with 5 ml of was used.
90% acetone in water, according to the method RESULTS
of Mantoura and Llewellyn (1983), as modified
by de la Giraudi6re et al. (1989), using fluores- Kinetics of hydrolysis
cence detection with a wavelength of 430 nm for
excitation and a 580nm cut-off filter for First we compared the behaviour of the sugar
emission. fraction during mild hydrolysis, in five marine
samples of surface microlayers and their corres-
Other seawater analyses ponding bulk waters, followed by phenol-sul-
phuric acid colorimetric reaction (Dubois et al.,
Particulate organic carbon (POC) and par- 1956) and HPLC assays. The results are pre-
ticulate organic nitrogen (PON) analyses were sented in Table 1.
conducted on precombusted Whatman GF/C The percentage of the Dubois-reactive
filters using a Perkin-Elmer (Bois-Colombes, compounds released during mild hydrolysis,
PARTICULATE HYDROLYSABLE SUGAR FRACTION IN SURFACE MICROLAYERS
related to the total particulate sugar fraction,
directly assayed by the Dubois method, varies
from 11 (Tin) to 18% (T150). There is no signifi-
cant difference on hydrolysis between microlayer
and bulk water samples.
If the summed amounts of HPLC-assayed
monosaccharides are considered, the percentages
of released monosaccharides increase regularly
during the hydrolysis procedure. They increase
more quickly in microlayer samples than in bulk
waters. For the first bulk water hydrolysis period
the accuracy of the HPLC assays is close to the
detection threshold as a consequence of the low
released monosaccharide concentration levels.
When sugar concentrations are low (in bulk
water samples and at the beginning of the hyd-
rolysis process), glucose is generally the best-
resolved monosaccharide form in the chromato-
grams, and it is possible to follow its progression
as a fraction of the total detected amount of
monosaccharides. During the first 60-150 min of
hydrolysis, glucose is not the major form
released: it ranges from 20 to 27% at T20min,but
its peak is sometimes difficult to distinguish
during the first 20 min of hydrolysis.
During the chromatographic assays of the
hydrolysis kinetic samples, at the beginning of
the chromatograms a large and well-resolved
peak appeared (retention time 7 min), which was
never present when the particulate sugar fraction
was completely hydrolysed by 1.86 N H2SO 4. A
mild hydrolysis was conducted on cellulose using
0.5N H2SO 4. After 15min of hydrolysis, the
hydrolysate was treated and assayed by HPLC.
On the chromatogram we have detected only the
presence of this peak and glucose. On the basis of
this 'rough' calibration, and considering its
relative retention time in a C,8-ODS column
under our chromatographic conditions, we have
estimated that this peak involved small poly-
saccharides, which were not yet hydrolysed. Its
trend during the mild hydrolysis is presented in
Table 1, with concentrations expressed in 'arbi-
trary fluorescent units' (afu); it appears to be
markedly different from that of the other
fractions. Its relative concentration remains
241
roughly stable in bulk water samples, and in
microlayers there is a slight decrease between T40
and T60. The rate of hydrolysis remained
constant; we have supposed that large polymers
were first broken into smaller units (the peak),
then, during the course of hydrolysis, the latter
were split into their monosaccharide com-
ponents as if they were continuously released and
consumed during the hydrolysis.
Natural samples
Table 2 shows sampling conditions and hyd-
rological data for the collected surface micro-
layer and bulk water samples. Most of the
samples exhibit organic matter enrichment in the
surface microlayers as compared with the bulk
waters. On the other hand, some of them (HI,
H2, B1 and B3), collected in brackish waters,
show a slight depletion in the concentrations
recorded in the microlayer. If the whole set of
samples is considered, average enrichments of
microlayers POC and PON are recorded (enrich-
ment factors (EF values) are respectively 3.6 and
3.0), with parallel accumulation of bacteria. The
mean enrichment factor for the monosaccharide
sum is weaker (1.5). The two C/N ratio values
are similar at the two sampling depths, but a
wider range of variation is recorded in the bulk
water samples (10.6 ___ 14.6) as compared with
the microlayers. Statistically, no enrichment of
pigments occurred in microlayers, as indicated
by the mean chlorophyll a concentrations, which
are similar in the microlayer (6.30 #g 1-1 ) and the
bulk water samples (6.20 /~g1- 1). As will be
shown below, this is the consequence of marked
differences in the pigment content of marine and
brackish waters.
Assuming a carbon/monosaccharide weight
ratio of 0.4, the percentage of glucidic carbon in
POC appeared to be slightly lower in the
microlayers (9.8% +__ 7.2; n = 21) than in the
related bulk waters (12.7% ___ 9.1; n = 21);
however, this difference is not significant at the
P0.05 level.
The distribution of the total monosaccharide
242 A.-M. C O M P I A N O ET A L

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0~
c'q "~

,2 t~
P A R T I C U L A T E H Y D R O L Y S A B L E S U G A R F R A C T I O N IN S U R F A C E M I C R O L A Y E R S 243

i.
..d
~5
V
O
.g

"3

0)

,.o

I
.o -~
244
~i l -- ii1~ &3I i illlllJl
~4"~ 0.5 t,O/
~72t- o //
"~ ! ~ Imi~,-otoye~s:,I
lb
'-" V - - ~
~SJPv,,
-, °
Ib~t~a{ers:
i ............
o I
f of salinity data is weak for the marine samples
0i ~ - ," , ~ L
0 5 10 15 P.O,O.(mg/I)
Fig. 2. Relationship between POC and the weight of mono-
saccharide in surface microlayer and bulk water samples.
The relationship is linear, and is highly significant at the
P0.00~ level. - - - - - , MS --- f ( P O C ) ; - - - , POC = f ( M S ) .
weight fraction is well correlated with POC
values, with the exception of two samples col-
lected in the brackish waters of a pond. These
exhibit parallel increases of all per-liter mono-
saccharide concentrations (as compared with
other samples) in the two sampling layers;
however, this increase was coupled with a
depletion in the microlayer as compared with the
bulk waters (EF < 1) and the presence of a
phytoplanktonic bloom, as attested by the high
chlorophyll a concentrations recorded. The best
fit of the relationship is a power form, and is
significant at the P0.00~ level (monosaccharide
sum (MS) --- 0.85 × POC°82; r = 0.818;
n = 42) when all the samples are considered.
However, power and linear forms are equivalent
and significant at the P0.00~ levels when the two
above-mentioned samples are discarded (Fig. 2):
MS = (0.26 × POC) - 3359; r = 0.818; n =
36. From this relationship, the sugar weight in
POC may vary from 20 to 30% of the POC.
The recorded range of salinity variations is
wide (Table 2). Standard deviation values are
12.3 and 12.2, respectively, for all surface
microlayer and bulk water samples. This is the
consequence of the various environmental situa-
tions examined here. Half the samples were col-
lected in coastal areas under the influence of
continental water inflows. Consequently, we
have studied the samples after collecting them
into two groups, above and below a 36%o salinity
A.-M. COMPIANO ET AL.
value, which may be considered as the threshold
between brackish and marine waters in Mediter-
ranean.
Particulate organic matter and total sugar
fraction in brackish and marine samples
After grouping of the samples, the dispersion
set, but remains high for the brackish water set.
This is the consequence of the varying distance of
the sampling locations into the dilution plume of
continental waters.
In microlayers collected in brackish water
areas (salinity less than 36%0o), particulate
organic matter accumulation is less than in the
marine samples group (salinity greater than
36%o). This is shown by the respective EF values
(Table 3): 3.1 and 10.2 for POC values in the
brackish water and marine samples, respectively,
and 0.96 (no enrichment) and 11.1 for the mono-
saccharide sum.
On the other hand, the brackish waters (both
surface microlayers and bulk waters) are richer
in POC than the marine waters. The percentage
of the monosaccharide fraction (Table 3) in POC
has its highest value (33%) in brackish bulk
waters and its lowest value (18.6%) in marine
bulk waters. If surface microlayers are con-
sidered alone, this difference remains between
brackish and marine samples: the monosacch-
aride percentages were 25.8 % for the former and
20.9% for the latter. If all the brackish water
samples are considered, the percentage of mono-
saccharide in POC is well correlated in a linear
fashion with the chlorophyll a concentrations
(r = 0.832; n = 41; P < 0.001). Conversely, in
marine waters (S > 36%o), this relationship was
not significant at the Po.o5 level.
When microlayer samples alone are compared
with regard to their salinity classification (Table
3), no differences were recorded, in terms of the
general characterization of their particulate
organic matter content. Differences in con-
centrations appear not to be significant, at the
P0.05 level, in microlayers collected in brackish
PARTICULATE HYDROLYSABLE SUGAR FRACTION IN SURFACE MICROLAYERS 245

TABLE 3
C o m p a r i s o n s o f brackish (S < 369/00) a n d m a r i n e water (S > 36960) samples f r o m surface m i c r o l a y e r ( M I C ) a n d bulk water (BW)
samples

Monosaccharide Chl.a Bacterial S(%o) POC PON C/N MS/POC

s u m (#g1-1) (#gl - I ) count (pgl - I ) (pgl i)
(logl0cell I l)

S < 369/00
MIC Mean 1709 8.5 9.68 20.65 9903 866 10.3 25.8
SD 2519 15.0 0.35 11.25 11700 835 5.4 18.5
n 19 13 9 19 19 18 18 19
BW Mean 1772 11.7 9.54 19.19 3162 315 12.2 33.4
SD 3524 17.0 0.32 10.32 3802 304 16.5 25.7
n 16 10 7 16 16 16 16 16
EF Mean 0.96 0.73 1.01 3.1 2.7
P(Ho) ns ns ns ns ns ns ns ns

S > 36%o
MIC Mean 1225 1.8 9.85 37.78 5100 439 9.8 20.9
SD 1186 2.1 0.40 0.28 3635 176 0.3 7.8
n 9 6 6 9 9 3 3 9
BW Mean 110 1.0 9.01 37.88 502 131 5.7 18.6
SD 105 1.5 0.21 0.19 287 37 1.4 7.7
n 12 10 8 12 12 5 5 12
EF 11.1 1.9 1.I 10.2 3.4
P(Ho) 0.001 ns 0.001 ns 0.01 ns ns ns

C o m p a r i s o n s o f s a m e s a m p l i n g levels b e t w e e n salinity g r o u p s
MIC 'U' 85 16 19 0 81 21 17 77
P(Ho) ns ns ns 0.001 ns ns ns ns
BW 'U' 29 8 6 0 22 35 15 17
P(Ho) < 0.001 < 0.001 < 0.001 < 0.001 <0.01 ns ns ns

ns, no significant difference.

water as compared with those from marine areas. recommended by Cowie and Hedges (1984b), the
On the other hand, bulk water samples differ relative concentrations of glucose are referred to
greatly (Table 3) in the two types of environ- the total monosaccharide sum, and those of the
ment. The brackish bulk waters are richer than other forms are referred to the total sum minus
the corresponding marine waters in sugars, the glucose concentration. In samples with
chlorophyll a arid bacteria, but not in POC and salinity values below 36%0, the composition of
PON, perhaps as a consequence of the wide the monosaccharide fraction shows no signifi-
dispersion recorded for these parameters in the cant difference between the two sampling layers.
brackish bulk water group. However, in marine waters, half the monosacch-
aride percentages are significantly different when
Monosaccharide composition surface microlayers and underlying waters are
compared: glucose and rhamnose are higher in
Within the two salinity groups, glucose is bulk waters, and mannose and arabinose are
always the main monosaccharide form in micro- lower, as compared with their percentages in the
layers and bulk waters. However, its percentage corresponding microlayers.
(Table 4) is generally higher in bulk waters than For samples with salinity value below 36%0,
in the microlayer samples. The high glucose the order of relative concentrations of the
levels mask the evolution of minor forms. As measured monosaccharides, is Glu > Gal >
246 A,-M. COMPIANO ET AL.

TABLE 4

Comparisons of brackish (S < 36%0) and marine (S > 36%0) water samples

Glc Gal Man Ara Xyl Rib Fuc Rhm

S < 36%0
MIC Mean 46.6 27.9 18.0 5.9 14.3 8.0 11.8 16.5
SD 12.0 5.9 4.9 4.4 3.7 4.9 5.5 4.9
n 19 19 19 19 19 19 19 19
BW Mean 51.9 28.1 15.5 4.6 15.9 6.6 14.4 17.8
SD 17.1 7.2 3.9 3.6 3.8 2.7 3.9 6.2
n 16 16 16 16 16 16 15 16
EF Mean 0.7 1.5 2.2 1.7 2.0 1.8 1.5 2.0
P(Ho) as ns ns as ns ns ns ns

S > 36%o
MIC Mean 48.0 22.2 25.3 10.2 14.8 6.7 5.7 15.7
SD 7.0 5.2 3.3 3.4 5.7 3.4 2.8 4.0
n 9 9 9 9 9 9 8 9
BW Mean 55.3 18.2 21.5 6.6 14.3 7.5 4.8 31.0
SD 10.3 9.4 4.6 2.3 6.7 6.7 4.4 18.5
n 12 12 12 12 12 12 9 11
EF Mean 8.5 25.8 18.4 19.5 16.0 16.0 41.4 5.2
P(Ho) 0.05 ns 0.05 0.01 ns ns ns 0.05

Comparisons of same sampling levels between salinity groups

MIC ~U' 66 42 20 43 82 76 17.5 73
P(Ho) ns < 0.05 < 0.001 ns ns ns < 0.05 ns
BW 'U' 71 42 26 68 95 88 4 53
P(Ho) ns < 0.05 < 0.01 ns ns ns < 0.001 ns

Percentages of monosaccharides are related to the total monosaccharide sum for glucose or to the total minus glucose for the other
forms.
P(Ho), probability of risk of rejection of null hypothesis (identity of the medians), given by the 'U'-test; the difference is significant for
P(Ho) < 0.05.

Man > Rhm > Xyl > Fuc > Rib > Ara for
the surface microlayer and Glu > Gal > Rhm >
Man > Xyl > Fuc > Rib > Ara for the bulk
waters, and for the two marine samples the order
is Glu > Man > Gal > Rhm > Xyl > Ara >
Rib > Fuc for the microlayers and Glu >
Rhm > Man > Gal > Xyl > Rib > Ara >
Fuc for the underlying waters.
For the brackish water sample group, the
order of mean relative concentrations does not
exhibit great changes between microlayer and
bulk water samples: the best represented mono-
saccharides are glucose and galactose and the
least represented are ribose and arabinose. Some
permutations (at the 2-3 % level) are at the limit
of significance. Changes in order are more
marked for the marine sample set; although the
least represented are still ribose, fucose and ara-
binose, galactose becomes less abundant, in
favour of mannose and rhamnose, in marine
bulk waters.
To test the influence of bulk water particulate
matter composition upon the dynamics of
accumulation processes, we have compared
(Table 4) the compositions of sugar fractions,
sampling layer by sampling layer, between
brackish and marine waters. The mean percen-
tage values of galactose, mannose and fucose
are found to be significantly different, by
the Mann-Whitney U-test, both in microlayers
and in bulk water samples (at the 0.05, 0.001 and
0.001 P levels in microlayer samples and at
the 0.01, 0.001 and 0.001 P levels in bulk
water samples, respectively), when values on
both sides of the 36%0 salinity threshold are
compared.
PARTICULATE HYDROLYSABLE SUGAR FRACTION IN SURFACE MICROLAYERS
DISCUSSION
The surface microlayers sampled differ from
their underlying bulk waters, not only as a site of
organic matter accumulation, but also in the
changes that occur in the composition of organic
fraction. This is demonstrated here for the sugar
fraction of particulate matter.
In brackish waters (S < 36%o), surface films
exist, as shown by the EF values. However,
accumulation of organic compounds in micro-
layers is lower than in marine environments, and
changes in the composition of particulate
organic matter of the microlayer as compared
with that of bulk waters are low or even statistic-
ally insignificant. More significant changes were
recorded in surface microlayers collected in
marine waters (S > 36%0). The fact that surface
films are, partly, formed by accumulation of
organic matter from bulk waters (Dietz and
Lafond, 1950; Sieburth, 1983) provides a
possible explanation for this difference between
marine and brackish microlayer water samples.
The higher particulate matter loading previously
prevailing in brackish bulk waters could mask
the relative enrichment of organic matter in the
corresponding surface films. This result was
previously reported (Williams et al., 1986); the
higher the initial concentration in bulk waters,
the lower the recorded EF in surface microlayers.
The high EF values for organic compounds
recorded in marine waters could be also the
consequence of an enhancement, in marine
areas of the intensity of hydrodynamic processes
(convection cells or Langmuir circulation),
which are considered to be of primary impor-
tance in surface film formation (Langmuir,
1938; Dietz and Lafond, 1950; Sutcliffe et al.,
1963).
Our bulk water samples collected in areas
under the influence of continental water inflows
have higher organic matter contents than our
marine samples. They are also enriched in
chlorophyll a and particulate carbohydrates.
Continental water inflows into the sea contain
terrestrial detrital organic matter (Cowie and
247
Hedges, 1984b). Cellulosic compounds from
terrestrial higher plants (support tissues) have
been proposed as a possible source of relative
glucose enrichment (the main monomeric form)
in marine sediments (Degens and Mopper, 1975;
Delmas, 1983; Cowie and Hedges, 1984a). For
this reason, glucose concentrations have been
compared with concentrations of other minor
forms (Ittekkot et al., 1982), or, more particu-
larly, those of galactose (Cowie and Hedges,
1984a,b), ribose (Degens and Mopper, 1975) or
fucose (Delmas, 1983). In our water samples, the
percentages of glucose in the monosaccharide
content of particulate matter are slightly lower in
continentally influenced samples than in marine
samples. We note only a trend of decreasing
relative glucose concentration between the
surface microlayer and the bulk water in the two
types of environment. However, if data disper-
sion is taken into account, there appears to be no
significant statistical difference, although we
must consider that changes in composition may
also result from exchange processes between the
particulate and the dissolved pools of organic
material; the latter is an important fraction
which was not examined here.
Nevertheless, other differences appear in mon-
osaccharide composition of the particulate hyd-
rolysable sugar fraction when brackish and
marine samples are compared. Enhancement of
galactose and fucose percentages relative to total
monosaccharides, and a decrease in relative
mannose concentrations, are recorded in
brackish water samples as compared with marine
samples, in both microlayer and bulk water
samples. The occurrence of surface film forma-
tion by accumulation processes does not signifi-
cantly modify the differences previously
recorded in monosaccharide composition
between brackish and marine bulk waters.
Changes in the composition of the particulate
sugar fraction related to the (continental or
marine) origin of the organic matter appear to be
predominant compared with the modifications in
monosaccharide composition that result from a
possible chemical selection in the accumulation
248
processes of the organic matter in sea surface
microlayers.
The relative galactose and fucose enhance-
ments recorded in low-salinity samples may
perhaps be explained by the origin of the sus-
pended particulate matter. Galactose is present
in terrestrial higher-plant pectins (Aspinall,
1970). Fucose is reported to be present at higher
concentrations in freshwater diatoms than in
brackish water species (Hecky et al., 1973), but it
is generally found in sediments at lower levels
(less than 2%) (Cowie and Hedges, 1984b) than
occurred in our samples.
On the other hand, galactose is also one of the
major components of diatom cell walls (Percival,
1968; Hecky et al. 1973; Haug and Myklestad,
1976), which are abundant in all types of water
samples.
As shown by chlorophyll a concentrations,
and some phytoplankton cell counts (de la
Giraudi6re et al., 1989), fresh water and brackish
water samples contain not only higher-plant
detritus but also phytoplankton cells. In these
waters, monosaccharide concentrations in POC
seem to be the direct consequence of the high
phytoplankton biomass, as indicated by the close
relationship between MS/POC ratios and
chlorophyll a values. The weight of carbohy-
drates to phytoplanktonic carbon may vary, de-
pending on species and environmental con-
ditions, from 10 to 30% (Percival, 1968). This
range of MS/POC ratio was confirmed by sugar
HPLC analysis of 22 phytoplankton cultures
(Compiano, 1990). An estimation of the phyto-
planktonic carbon from concentration of
chlorophyll a is rather difficult if the ecological
conditions prevailing in microlayers are con-
sidered (de Souza-Lima and Romano, 1983).
However, assuming an average phytoplankton
carbon equivalent (Cp = [Chl.a] x 60), the
phytoplankton carbon contribution to POC
could represent only a few per cent in microlayer
samples (5% and 2% in brackish and marine
waters, respectively) and 22% and 11% for the
two groups o f bulk water samples, respectively.
A different choice of equivalence value, applied
A.-M. C O M P I A N O ET AL.
to microlayer samples, does not significantly
change the contribution of phytoplanktonic
carbon to POC. Thus we may suppose that, in
microlayers, a large part of the measured par-
ticulate sugars is not associated with phyto-
planktonic biomass.
On the other hand, in marine samples (either
in microlayers or bulk waters), where
chlorophyll a contents are lower than in low-
salinity samples, no significant relationship exists
between chlorophyll a and the sum of mon-
osaccharide concentrations.
In marine samples, high EF values for organic
matter in microlayers as compared with bulk
waters are associated with significant changes in
monosaccharide composition. Decrease of the
mean glucose relative concentrations in micro-
layers, in relation to a greater speed of hydro-
lysis, could be evidence for the occurrence of
degradation and consumption of organic matter
by bacteria. Bacterial populations are currently
reported to be enhanced in surface films
compared with levels in bulk waters (for reviews,
see Sieburth (1983) and Romano (1989)). In
marine microlayers, bacterial activity is also gen-
erally enhanced (Williams et al., 1986), with high
oxygen consumption (Mimura et al., 1988) and
CO2 production (Garabetian, 1991). Moreover,
after breakage of polymers, glucose should be
preferentially consumed as a substrate, with a
higher assimilation rate than xylose or arabinose
(Garabetian et al., 1993), and/or bound, as a
monomer, to humic matter, for instance.
As mannose plays a role either as a reserve or
as a component of cellular walls of various
species of bacteria, the mannose enrichment in
microlayers could be related to the recorded cor-
responding enhancement in bacterial numbers.
However, mannose is also a constituent of gym-
nosperm hemicellulose (Cowie and Hedges,
1984a), and the levels of bacterial carbon, based
on counts, cannot alone explain mannose enrich-
ment between bulk water (ll/~g1-1) and
microlayer samples (161/~g l- ~). Mannose
microlayer enrichment may also be the con-
sequence of phytoplankton cell degradation.
PARTICULATE HYDROLYSABLE SUGAR FRACTION IN SURFACE MICROLAYERS
Handa and Yanagi (1969) have reported that in
three diatom species, mannose is the main con-
stituent of the residual sugar fraction remaining
after water extraction. The presence of dead
phytoplankton cells in surface microlayers is
shown by microscopy observations (de Souza-
Lima, 1985) or enrichment in chlorophyll
degradation products (de la Giraudi6re et al.,
1989).
One of the most interesting points is the
recorded enrichment of arabinose in marine
surface microlayers. Arabinose was considered
by Handa and Yanagi (1969) to be a constituent
of the marine residual sugar fraction, and its
concentrations have been reported to increase in
waters after a phytoplankton bloom (Ittekkot et
al., 1982). Moreover, arabinose is considered in
its monomeric form as a complexing reagent for
metals (Cowie and Hedges, 1984a), which are
well known to accumulate in surface microlayers
(Hoffman et al., 1974). We may consider that
arabinose enrichment may result from phyto-
plankton cell degradation, as shown by the
reported increase of degrading pigments in
microlayers (de la Giraudi6re et al., 1989). For a
batch of 20 samples, the percentages of arabi-
nose are directly linked to the pheophorbid/
chlorophyll a ratios at the P0.05level (Spearman's
test, r5 = 0.408). Arabinose could then be chem-
ically stabilized in particles by metal complexa-
tion processes. Moreover, during a hydrolysis
kinetic experiment carried out on one marine
microlayer sample, more than 75% of the arabi-
nose content of the sugar fraction was released
during the first 20min; arabinose would thus
appear to be engaged in more labile molecular
structures than most of the other monosacch-
arides.
In this study, it must be borne in mind that the
assayed particulate hydrolysable carbohydrate
fraction represents only a part of the whole
microlayer sugar fraction. As was previously
reported (Jullien et al., 1982; Williams et al.,
1986; Garabetian et al., 1993), in surface
microlayers, POC values are much higher than
the sum of lipid, protein and sugar carbon of
249
biological origin. The presence of this 'complex'
organic fraction, and the difficulty, at present, of
accurately measuring the monosaccharide com-
position of the dissolved sugar fraction, may
have a substantial influence upon the car-
bohydrate budget of sea surface microlayers.
CONCLUSION
Carbohydrates are currently reported as one
of the types of organic compounds that accu-
mulate in surface microlayers. They can play a
role in some physical properties of surface films
(Garrett, 1972; Baier et al., 1974; Wangersky,
1976), and also as a trophic resource for bacterial
growth (Sieburth, 1983). However, at present
determinations of the sugar fraction composition
of particulate matter of the sea surface micro-
layer are rare in the literature. Recent improve-
ments in HPLC determination of monosacch-
arides by fluorochrom derivatization will help in
a more extensive screening of surface microlayer
composition. Surface microlayers are usually
considered as a whole. The results presented here
provide evidence for differences in sugar enrich-
ment of surface films between brackish and
marine waters. However, we cannot determine
whether these differences result from the com-
position of the initial bulk waters or from dif-
ferences in accumulation processes for the two
environments considered. As was previously
reported for the water column (Handa and
Yanagi, 1969; Delmas, 1983; Sakugawa and
Handa, 1983), carbohydrates may provide geo-
chemical information on microlayer properties,
not only from the study of monosaccharide com-
position but also from the knowledge of their
polymerization state. Unfortunately for their
examination, sugars are highly reactive
compounds and microlayer samples exhibit
rapid changes after collection (Laborde et al.,
1986). The coupling of HPLC analytical pro-
cedures with kinetics of hydrolysis may reduce
the number of steps and the time between sample
collection and treatment, and help to minimize
the risk of possible modifications. At present,
250
direct HPLC determination of dissolved mono-
saccharides in natural waters is not possible with
sufficient accuracy. With DNS, fluorochrom
problems appeared at low concentrations
(Compiano and Romano, 1988), and potentio-
metric detection does not have the sensitivity
necessary for natural water samples. In future,
expected progress in fluorochrom chemistry will
allow the examination of the interactions
between dissolved and particulate sugar
fractions which may occur during accumulation
processes of organic matter in surface microlayers.
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