Mucilage microcosms

www.elsevier.com/locate/scitotenv

Science of the Total Environm

Mucilage microcosms

Paola Del Negro a,*, Erica Crevatin a, Chiara Larato a, Carla Ferrari b, Cecilia Totti c,

Marinella Pompei d, Michele Giani e, Daniela Berto e, Serena Fonda Umani a

a Laboratorio di Biologia Marina (LBM), Via A.Piccard, 54, 34010 Trieste, Italyb ARPA Emilia Romagna, Struttura Oceanografica Daphne, Via A.Vespucci, 2, 47042 Cesenatico (FO), Italy

c Dipartimento di Scienze del Mare, Universita Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italyd Centro Ricerche Marine, Via A.Vespucci, 2, 47042 Cesenatico (FO), Italy

e Istituto Centrale per la Ricerca Scientifica e Tecnologica Applicata al Mare (ICRAM), Brondolo, 30015 Chioggia (VE), Italy

Available online 19 October 2005

Abstract

In the summers of 2000, 2001 and 2002, large amounts of sticky mucilaginous material aggregated to form masses of

impressive dimensions over large areas of the Adriatic Sea, particularly in its northern part. Aggregates differing in size were

sampled by SCUBA divers and submitted to chemical (nutrient and organic matter concentrations) and biological analysis (virus,

bacteria and phytoplankton abundances and bacterial metabolism).

Suspended and sinking mucilaginous aggregates were biota-rich environments where the abundance of planktonic organisms

and the concentration of nutrients were orders of magnitude higher than in the surrounding seawater. The embedded phytoplankton

was mostly composed of diatoms, but the dinoflagellate Gonyaulax fragilis, previously reported in association with the aggregates,

was also present. A variety of processes occurred within the mucilaginous aggregates which resulted in the transformation of the

organic matter composition and lability and contributed to a partial degradation of mucilage. For an efficient degradation of

mucilage, several conditions are necessary: high bacterial abundance and activity and an efficient recycling of nutrients within the

aggregates. Most of these conditions, appear to change depending on the type and age of the aggregate.

During the first phase of aggregation (cobwebs and ribbons), bacterial activities addressed the degradation of organic matter,

particularly that of the nitrogen fraction. The degradation products were rapidly taken up by bacteria, supporting an increase in their

abundance and production. In aged mucilage (clouds), the degradation processes decreased and the bacterial metabolism suggested

the presence of new organic labile compounds probably due to phytoplankton production. On the basis of our results, stringers,

generally considered the first step of the aggregation process, seemed to be the result of a mechanical disruption of other types of

aggregates.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Mucilage; Northern Adriatic Sea; Organic matter; Enzymatic activities; Bacterial production; Respiration

1. Introduction

Episodes of massive mucilage formation in the

Northern Adriatic Sea have been recorded since 1729

0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.scitotenv.2005.09.018

* Corresponding author. Tel.: +39 040224989; fax: +39

0402249870.

E-mail address: [email protected] (P. Del Negro).

and occurred at irregular intervals in time from at least

the 19th century until the first-half of the 20th century

(Fonda Umani et al., 1989). In the summer of 1988,

apparently after more than 30 years, mucilage re-

appeared throughout the Northern Adriatic Sea. The

phenomenon was observed again in 1989, 1990,

1991, 1997, 2000, 2001 and 2002 with different tem-

poral and spatial distribution.

ent 353 (2005) 258–269

P. Del Negro et al. / Science of the Total Environment 353 (2005) 258–269 259

Mucilage is constituted by organic gelatinous mate-

rial which forms aggregates characterised by varying

sizes and shapes and by their vertical distribution along

the water column. The temporal sequence of the phe-

nomenon is characterised by an initial formation of

flocks in the water column, followed by stringers, and

then by the development of subsurface layers (bfalsebottomQ) and dense clouds. At all three stages aggre-

gates can sink to the bottom or rise to form surface

layers (Stachowitsch et al., 1990; Precali et al., 2005—

this issue).

Researches of this phenomenon have been dominat-

ed by the idea that polysaccharides are copiously ex-

uded or released by the lysis of diatoms (Myklestad,

1995) during scarce riverine inputs (Degobbis et al.,

1995), unbalanced nutrient availability (Kaltenbock and

Herndl, 1992; Obernosterer and Herndl, 1995; Maes-

trini et al., 1997), low grazing pressure (Malej and

Harris, 1993; Najdek, 1997; Bochdansky and Herndl,

1999), and/or massively released by lysis during virus

infection (Baldi et al., 1997). Azam et al. (1999) argued

that bacteria also play multifaced, varied and dominant

roles in the production of long-lived dissolved and

colloidal polysaccharides.

Recently, Engel et al. (2004) showed, by means of a

combined experimental and modelling study, that the

formation of polysaccharide particles can be described

in terms of aggregation kinetics. Cross-linking of poly-

saccharides can produce aggregates and mucilage. They

entrain detritus and both pelagic and benthic organisms

but it is not an inert accumulation of largely senescent

material but a highly active microbial community in

which the activities of heterotrophs and phototrophs are

mutually supportive (Fogg, 1995; Najdek et al., 2002).

Nevertheless, the aggregates’ biological composition

reflects the compositional spectrum of the water col-

umn in which they appear, undergo pronounced

changes and species successions, and interact in various

ways with the surrounding organisms, particles and

dissolved matter. This community is self-sustained

and capable of resisting outside solicitation (Decho

and Herndl, 1995; Najdek et al., 2002). Bacteria

achieve higher abundances inside the aggregates than

in the surrounding water and the bacterial community

appears characterised by a high diversity (Rath et al.,

1998). These organisms contribute only to a minor

fraction of the bulk organic carbon in the aggregates;

they play the most important role in the decomposition

process. Particulate organic matter solubilization, sub-

strate hydrolysis and uptake, biomass production, res-

piration and substrate release into the surrounding water

are the major microbial processes which transform the

aggregated organic matter. The transformation of or-

ganic matter from the particulate form, due to copious

extracellular enzymes produced by bacteria-colonizing

aggregates, results in the production of more soluble

DOM (dissolved organic matter). DOM is released

much faster than the bacteria can use it and this

leads to the production of a rich plume of substrate

(Azam and Long, 2001). Kiørbe and Jackson (2001)

predicted that free-living bacteria can take advantage

of this plume and use it to fuel a population explosion.

Release of DOM contributes to a partial degradation

of mucilage. Changes in the mucilage composition,

however, were complicated by the adsorption of other

types of DOM on the mucilage, and by the develop-

ment and succession of microbial communities within

the mucilage.

To our knowledge, relationships between bacterial

activities and biomass, and organic matter concentra-

tions have not yet been directly determined on sus-

pended macroscopic organic aggregates of different

types.

In this study we investigate the composition and

microbial dynamics in aggregates collected in the

Northern Adriatic Sea during mucilage events in the

spring–summer in 2000, 2001 and 2002 in order to

assess the changes in microbial community metabolism

of the mucilaginous aggregates in relation to their age.

2. Materials and methods

2.1. Sampling

Aggregates were collected during June–July 2000,

June 2001 and June–July 2002 along three transects in

the Adriatic Sea from the Italian to the Croatian coast

(Fig. 1, Table 1).

Individual aggregates were collected at depths of

relatively high aggregate concentrations (usually at sur-

face, between 10 and 20 m, and at the bottom) deter-

mined from underwater video camera profiles by

SCUBA divers, using large 50 ml polyester syringes

with the forward ends cut off or 1500 ml polymetha-

crylate syringes. Water samples were collected at the

same depths using 12 l Niskin bottles of the CTD

rosette sampler (Sea-Bird Electronics 9/11 Plus).

Mucilage samples were classified according to Pre-

cali et al. (2005—this issue).

2.2. Analytical methods

On board, each size class of aggregates was

separated from the seawater by decantation and

Fig. 1. Sampling locations in the Northern Adriatic Sea.

P. Del Negro et al. / Science of the Total Environment 353 (2005) 258–269260

pooled in a centrifuge tube. After the mucilage pore-

water was extracted by centrifugation (3000 rpm, 15

min), the pellets and supernatant were added to

artificial sterilized sea water (ASSW). The pellets

were gently disrupted in a glass tissue grinder; sub-

samples of the resulting particle suspension were

taken to carry out chemical and microbiological

analyses.

Table 1

Sampling dates, stations, depths and different types of aggregates

collected

Sampling dates Stations Depths (m) Type of mucilage

20-Jun-00 B03 12 Clouds


20-Jun-00 BC01 5 Clouds

06-Jul-00 B15 45 Clouds


04-Jul-01 A04 20 Stringers


05-Jul-01 A07 8 Clouds

05-Jul-01 A07 14 Ribbons

04-Jun-02 B06 15 Stringers

04-Jun-02 B06 19 Stringers

05-Jun-02 C04 22 Cobwebs

24-Jun-02 C02 10 Clouds

24-Jun-02 C04 2 Ribbons

26-Jun-02 C12 10 Cobwebs

27-Jun-02 B15 20 Cobwebs








2.2.1. Nutrients, total dissolved (N-TOT) and phospho-

rus (P-TOT)

In seawater and aggregate porewater total nitrogen

and total phosphorus were determined as nitrate and

orthophosphate, respectively. Samples were analysed

after the addition of potassium persulfate and the oxi-

dation by autoclaving at 120 8C for 40 min according

to Koroleff (1983). Orthophosphate and nitrate (re-

duced to nitrite by a copper–cadmium reduction col-

umn) concentrations were determined following the

colorimetric methods of Hansen et al. (1983) using a

multichannel continuous flux analyser (Braan Luebbe

Autoanalyzer III).

The mucilage pellet was split into at least five por-

tions and then each subsample was oxidized in artificial

seawater and analysed as described for porewater. In

order to verify the complete sample oxidation, different

volumes (from 1 to 25 ml) of each subsample were

oxidized with the same number of replicates.

2.2.2. Dissolved organic carbon (DOC)

Seawater and porewater samples were filtered on

board through precombusted (4 h at 480 8C) glass

fibre filters (GF/F Whatman) and transferred directly

into precombusted glass vials. The dissolved organic

carbon (DOC) concentration was measured using a

Shimadzu TOC 5000 Analyzer with a 1.2% Pt on silica

as catalyst (Cauwet, 1994) at 680 8C. Samples were

acidified (pH 2) with 2N HCl and sparged with pure air

for 10 min immediately prior to analysis. DOC concen-

tration was calculated by subtracting the system blank

and dividing by the slope of the calibration curve

(Thomas et al., 1995). Potassium hydrogen phthalate

was used as standard. One hundred microliter of the

sample were injected for each analysis and the concen-

tration was calculated as the average of three to five

replicates of the same sample. The average instrumental

blank was 8.7F0.9 Amol l�1. The reproducibility of

the analytical system was lower than 2–3%.

2.2.3. Particulate organic carbon (POC)

For measurements of the CHN elemental composi-

tion, aggregates were pooled and filtered onto precom-

busted glass fibre filters (GF/F Whatman). Particulate

organic carbon (POC) and total particulate nitrogen

(TPN) were determined with a CHN Elemental Ana-

lyzer (Carlo Erba Mod. EA1110), after acidification

with HCl (1 N) to remove the inorganic carbonate

fraction. Acetanilide was used as standard. The repro-

ducibility of the analytical system was lower than 2%.

2.2.4. Bacterial abundance

Bacteria on aggregates, in the porewater and in the

surrounding water were counted by epifluorescence mi-

croscopy (Olympus BX 60) after DAPI staining (Porter

and Feig, 1980). Two milliliter subsamples of seawater

and porewater, fixed with 2% (final concentration) of

borate-buffered formalin prefiltered onto a 0.2 AmAcro-

disc filter, were filtered directly onto 0.2 Am black-

stained polycarbonate filters (Nuclepore). Aggregate-as-

sociated bacteria were detached prior to filtration by

ultrasonic treatment of 2 ml aliquots of mucilage sus-

pension containing Na-pyrophosphate (Velji and Alb-

right, 1986). The size of the organisms was measured

using a calibrated ocular micrometer. Bacterial density

was converted into carbon equivalents using the conver-

sion factor of 20 fg C cell�1 (Lee and Fuhrman, 1987).

2.2.5. Virus abundance

The abundance of virus-like particles (VLP) was

estimated by using SYBR Green I (Molecular Probes)

according to the procedure described by Noble and

Fuhrman (1998). Five ml samples (porewater, sur-

rounding water or aggregate suspension) were pre-

served with 0.02 Am filtered formalin (1% final

volume). Subsamples were filtered through a 0.02

Am Anodisc filter. The filters were then placed on

100 Al of a solution of SYBR Green I (1 :2500 final

concentration) and incubated for 15 min in the dark.

Following incubation, the backs of the filters were


dried gently with a kimwipe tissue and mounted onto

glass slides with antifade solution (Noble and Fuhr-

man, 1998).

The enrichment factor (EF) was calculated for each

category of microorganisms according to the following

equation where (n=number of organisms): EF=(n ml�1)

aggregates / (n ml�1) surrounding water.

2.2.6. Hydrolytic enzyme activity

Hydrolytic enzyme activities were measured with

fluorogenic analogs of natural substrates (Hoppe,

1993): l-leucine-4-methylcoumarinile-7-amide (MCA,

aminopeptidase), methyl umbelliferyl-h-d-glucoside(MUF, h-d-glucosidase), methyl umbelliferyl phos-

phate (MUF, phosphatase).

Hydrolysis by free-living bacteria was measured by

incubating 2.5 ml subsamples with 200 AM MUF-h-glucoside and leucine-MCA substrates and 50 AMMUF-phosphate for 1 h at in situ temperature in the

dark. Hydrolysis by aggregate-associated bacteria was

measured on 2 ml mucilage suspension diluted with

sterilized ASSW (1 :100) with 200 and 50 AM sub-

strates as described above. A control, heat-killed at 80

8C for 20 min, was used for both free-living and

aggregate-associated bacteria. Fluorescence was mea-

sured at 380/365 nm excitation and 440/455 nm emis-

sion for MCA/MUF substrates by using a fluorometer

(Shimadzu RF 1501).

2.2.7. Bacterial carbon production

Bacterial carbon production (BCP) was measured

by the incorporation of 3H-leucine (Leu) (Kirchman et

al., 1985). Triplicate 1.7 ml aliquots and two controls

killed with 100% trichloracetic acid (TCA) were

amended with a 20 nM radiotracer and incubated at

in situ (F2 8C) temperature in the dark. Incubations

were stopped with 100% TCA after 1 h. The extrac-

tion with 5% TCA and 80% ethanol was carried out

using the microcentrifugation method (Smith and

Azam, 1992). Activity in the samples was determined

by a h-counter (Packard Tri-Carb 300) after the addi-

tion of 1 ml scintillation cocktail (Ultima Gold MV;

Packard). Incorporation of 3H-leucine was converted

into carbon produced via bacterial protein production

according to Simon and Azam (1989), assuming a 2-

fold isotope dilution for Leu.

The bacterial growth efficiency (BGE) was calculat-

ed from bacterial carbon production (BCP) and bacte-

rial respiration (BR) (Søndergaard and Theil-Nielsen,

1997) according to the following equation: BGE=

BCP/ (BCP+BR) where BCP+BR represents the total

bacterial carbon demand (BCD).

2.2.8. Respiration

Respiration of aggregate-associated bacteria and of

free-living bacteria was measured by following the

changes in dissolved oxygen concentration in sealed

bottles during 22–24 h incubations. Dissolved oxygen

concentrations were estimated with a Mettler DL 21

titrator for automated Winkler titrations based on po-

tentiometric end-point detection (Graneli and Graneli,

1991).

Whole aggregates or part of aggregates were sus-

pended in seawater filtered onto 0.02 Am membranes in

acid-cleaned (10% HCl) and distilled water-rinsed 60

ml BOD bottles in the dark at in situ temperature (F2

8C). Oxygen concentrations were determined in tripli-

cate bottles. Controls consisted of the parallel incuba-

tions of seawater samples filtered through 0.02 Amfilters.

For seawater and porewater measurements, three

replicate samples were collected in 60 ml BOD bottles.

Replicate measurements differ by b10%. Respired C

was estimated from dissolved oxygen utilization, as-

suming a respiratory quotient of 1 (Chin-Leo and Ben-

ner, 1992).

2.2.9. Phytoplankton

The aggregates sampled for phytoplankton counts

were preserved with 1.6% hexamethylene tetramine-

buffered formalin (Throndsen, 1978) and stored at 4 8C.Qualitative and quantitative analyses of phytoplank-

ton were performed following the Utermohl (1958)

method as described by Hasle (1978) using an inverted

microscope (Zeiss Axiovert 135) equipped with phase

contrast and epifluorescence apparatus. In order to

count and identify thecate dinoflagellates, samples

were stained with Calcofluor (Innamorati et al.,

1990), a specific stain for cellulose and h-glucans(Fritz and Triemer, 1985). Only qualitative analyses

were carried out on the samples collected during 2000

because it was difficult to count the extremely dense

samples.

2.2.10. Statistical analysis

Statistical comparisons were performed using an

ANOVA test.

3. Results and discussion

3.1. Biological composition

Bacteria were found on nearly all types of aggre-

gates but their abundance varied with aggregate types,

ranging from 2 d 109 cell l�1, in cobwebs, to 2 d 1011

Table 3

Means and standard deviation of viral and bacterial abundance, and

virus :bacteria ratio (VBR) in surrounding water, porewater and mu-

cilage (n—number of determinations)

n Virus

(1010 VLP l�1)

Bacteria

(109 cells l�1)

VBR

Surrounding water 7 0.3F0.1 0.7F0.1 4.7F0.1

Porewater 16 25.7F4.2 88.9F9.8 2.9F0.1

Mucilage 17 92.2F69.0 87.9F1.0 72.0F0.2


cell l�1, in clouds (Table 2). Ploug and Grossart (2000)

showed that the number of bacteria per unit surface area

was inversely correlated with the aggregate size prob-

ably due to the aggregate fractal nature. Bacterial abun-

dance in aggregates and in porewater was higher

(EF=126–127) than that of free-living bacteria in the

surrounding water (Table 3). Bacterial abundance in the

aggregates varied within the range reported by Herndl

(1988) for the Adriatic mucilage. No data were avail-

able about porewater bacteria. Due to the more favour-

able nutritive conditions, bacteria in aggregates, in

porewater and particularly in clouds were often larger

in size (up to 1.0 Am3) than free-living bacteria in the

surrounding water as previously recorded by Herndl

(1988) and Simon et al. (1990).

Virus-like particles (VLP) abundance was 300-fold

higher in aggregates than in the surrounding water,

reaching an average value of 9 d 1011 VLP l�1 (Table

3). A high viral abundance was also found in marine

snow (Peduzzi and Weinbauer, 1993) but very little

information was available on the significance of viral

infection on aggregate-associated bacteria (Proctor and

Fuhrman, 1991). Our results showed a very high

virus :bacteria ratio (VBR) on aggregates, reaching

an average value of 72 (Table 3). The water values

(VBR=4.7) were in the same range as values reported

from studies on the dynamics of virus and free-living

bacteria (Wommack and Colwell, 2000). VLP varied

according to aggregate size, ranging from 7 d 109 VLP

l�1 in stringers to 3.7 d 1012 VLP l�1 in clouds (Table

2). In stringers and cobwebs the VBR was in the same

range as water samples and only in clouds was it

higher than 100. VBR is higher in more productive

systems (Weinbauer, 2004): the higher nutrient con-

centrations and thus the more efficient metabolism of

bacteria inside the aggregates allowed to produce more

viral progeny per cell, as already observed in culture

studies (Weinbauer, 2004). However, burst size also

increases with cellular size indicating that larger cells

can contain more phages.

Table 2

Means and standard deviation of viral and bacterial abundance, and

virus :bacteria ratio (VBR) in different types of aggregates (n—num-

ber of determinations)

n Virus

(1010 VLP l�1)

Bacteria

(109 cells l�1)

VBR

Stringers 4 0.7F0.2 6.9F1.4 1.8F1.3

Cobwebs 2 0.9F0.8 2.3F0.7 3.8F2.2

Ribbons 4 2.6F0.4 3.3F0.9 5.9F0.8

Clouds 7 370.0F270.0 210.0F20.0 169.3F4.9

Aggregates were often highly enriched in microal-

gae compared to the surrounding water. The highly

diverse autotrophic component consisted of pelagic

and benthic species, and the species composition was

similar to that of the surrounding water. During the

2000 mucilage event, diatom abundances were higher

in the mucilage than in the surrounding water and

Chaetoceros spp., Cylindrotheca closterium, Proboscia

alata and Pseudo-nitzschia delicatissima were always

present. Most of the diatoms were found intact, while

empty dinoflagellate thecae were abundant, particularly

in June. Living dinoflagellates, mostly belonging to

Gonyaulax fragilis, were particularly abundant in the

earliest stage of the mucilage phenomenon. When the

gelatinous masses aged, G. fragilis decomposed and

become unrecognisable (Pompei et al., 2003). The

presence of G. fragilis was reported for several muci-

lage events affecting the Adriatic Sea (Cabrini et al.,

1992; Honsell et al., 1992; Viviani et al., 1995; Pompei

et al., 2003), although this organism was never consid-

ered to be a producer of polysaccharide exudates. Re-

cently, Mackenzie et al. (2002) attributed the mucilage

accumulation observed along the coast of New Zealand

to polysaccharides produced by G. hyalina, a very

similar species to G. fragilis. This confirms the obser-

vations of Alldredge et al. (1988) and Passow (2002),

which referred to the occurrence of cohesive marine

snow and TEP (transparent exopolymeric particles)

largely dominated by dinoflagellates and agreed with

the new results of Pistocchi et al. (2005—this issue).

During 2002 phytoplankton abundance inside the

mucilage was 1–4 orders of magnitude higher than in

the surrounding water. Small flagellates and diatoms

were abundant as well as Pseudo-nitzschia delicatis-

sima, Chaetoceros spp. and C. closterium. Dinoflagel-

lates were mostly represented by athecate species and

by Prorocentrum balticum, but Gymnodinium spp. and

G. fragilis were also found.

These observations suggest that the aging process of

aggregates may have caused a selection among the

species inside the mucilage and the preferential growth

of highly silicified pennate diatoms, like Cylindrotheca,

Table 4

Dissolved organic carbon (DOC) concentrations in the porewater

n DOC POC TOC TPN POC:TPN

AM AM AM AM mol/mol

Stringers 4 206F38 787F578 992F612 81F72 12F4

Cobwebs 3 166F58 260F253 405F317 24F19 10F3

Ribbons 4 143F39 627F347 770F382 62F45 11F3

Clouds 4 501F168 2445F1135 2946F1692 162F106 16F5

Particulate organic carbon (POC), total organic carbon (TOC) and total particulate nitrogen (TPN) concentrations in the aggregates and their molar

ratio (mol/mol). Means and standard deviation are reported (n—number of determinations).


may be related to a more persistent nutrient input,

resulting from a continuous renewing of the aggregate

organic matter by internal recycling (Kaltenbock and

Herndl, 1992; Decho and Herndl, 1995) or to algal

ability to grow at low light intensity (Sdrigotti et al.,

1999).

3.2. Carbon, nitrogen and phosphorus

Inside the aggregates the organic matter concentra-

tion reached values 100-fold higher than in the sur-

rounding water (Giani et al., 2005—this issue). Total

organic carbon concentration ranged between 405 and

2946 Amol l�1 and increased with the aggregate size

(Table 4). Dissolved organic carbon in the porewater

comprised from 17% to 41% of the total organic

carbon and was a substantial fraction of the organic

carbon. The DOC:POC ratio was generally b1 since

organic carbon was mostly present in a particulate or

colloidal form. The POC:TPN increased with the

aggregate size, varying from 10 :1 in cobwebs to

16 :1 in clouds (Table 4). These data are in agreement

with the findings of Alldredge (1998) on marine snow

and the ratio could increase with time as observed in

the Adriatic aggregates (Muller-Niklas et al., 1994),

suggesting that clouds are older than cobwebs, ribbons

and stringers.

Dissolved total phosphorus and total nitrogen were

significantly higher (by one order of magnitude) in the

aggregate porewaters than in the surrounding water

(Table 5) and increased with the size of the aggregates

(Table 6). The average value of the N:P ratio inside the

Table 5

Means and standard deviation of total dissolved N and P concentra-

tions and their molar ratio in the porewater of the aggregates and in

the surrounding water (n—number of determinations)

n P-TOT N-TOT N:P

AM mM mol/mol

Surrounding water 7 50F31 0.9F0.6 18F6

Porewater 16 802F307 10.6F5.8 13F4

aggregates, lower than the value outside ( p b0.01), was

a further indication of the high nutrient recycling. The

N:P ratio in cobwebs and ribbons porewater was

around 14. Only in stringers was the N:P ratio higher

than the Redfield ratio (Redfield et al., 1963) and

primary production could be occasionally limited by

phosphorus.

3.3. Microbial activities

Bacteria associated with aggregates highlighted a

very high potential enzymatic hydrolysis rate per cell

(Table 7), implying that labile biopolymers in aggre-

gates were hydrolysed faster than in the surrounding

water, in agreement with observations of Simon et al.

(2002). In the porewater the availability of low molec-

ular weight compounds resulting from the cleavage

inside the aggregates did not induce any relevant enzy-

matic activity.

Among extracellular enzymatic activities assayed on

mucilage samples, aminopeptidase (AMA) showed the

highest potential activity level, implying that proteins

were decomposed more rapidly than polysaccharides,

as also suggested by Simon et al. (2000). A preferential

decomposition of organic nitrogen rather than organic

carbon was observed also by Grossart and Ploug (2001)

and resulted in a progressive increase in carbon inside

the aggregates. High per cell hydrolysis rates of alkaline

phosphatase (APA) could be important phosphorus

able 6

eans and standard deviation of total dissolved N and P concentra-

ons and their molar ratio in the porewater of different types of

ggregates (n—number of determinations)

n P-TOT N-TOT N:P

AM AM mol/mol

tringers 2 117F79 2367F370 20F19

obwebs 1 91 1296 14

ibbons 4 133F30 1820F237 14F3

louds 3 672F464 13,085F8391 19F7

T

M

ti

a

S

C

R

C

Table 7

Means and standard deviation of enzymatic activities in surrounding water, porewater and mucilage, expressed as attomoles cell�1 h�1 (n—number

of determinations)

n h-Glucosidase (h-Glu) Aminopeptidase (AMA) Phosphatase (APA)

(amol cell�1 h�1) (amol cell�1 h�1) (amol cell�1 h�1)

Surrounding water 7 11.8F0.3 133.0F12.8 135.5F12.7

Porewater 16 1.7F0.1 39.4F4.1 50.7F4.9

Mucilage 18 37.2F0.4 1741.1F328.3 1057.8F721.0


supply both to heterotrophic and autotrophic microor-

ganisms. Phytoplankton and bacteria can release signif-

icant amounts of phosphatases and it is not possible to

separate algal from bacterial sources of this enzyme in

the field (Nausch, 1998). The h-glucosidase activity,

significantly lower than APA and AMA, could be

inhibited by readily available monomeric compounds

(Middelboe and Søndergaard, 1993). We can therefore

hypothesize that the phytoplankton cells embedded in

the aggregates might release exopolysaccharides thus

satisfying the bacterial sugar demand.

The highest level of enzymatic activities was ob-

served in ribbons (Table 8). The APA was the sum of

bacteria and phytoplankton APA activities which hy-

drolyzed organic matter to overcome the apparent

phosphorus limitation. Only in ribbons was the

alkaline phosphatase activity : aminopeptidase activity

(APA:AMA) ratio higher than 1 and reached the max-

imum average value of 2.2 (Table 9) suggesting a P-

limitation inside this kind of aggregate. The

APA:AMA activity ratio has been recently proposed

as a proxy of P deficiency in microbial assemblages

(Sala et al., 2001). However in the ribbons’ porewater

the N:P ratio was b16 suggesting P availability.

According to Azam and Long (2001), the colonizing

bacteria produce phosphate much faster than they can

use it, so the aggregates leave a rich-plume in the

porewater. The very low AMA:h-glucosidase activity

ratio was the result of strong decomposition of organic

nitrogen rather than organic carbon.

Aged aggregates (clouds) showed reduced hydroly-

tic activities (Table 9). Among enzyme activities, AMA

Table 8

Means and standard deviation of extracellular enzymatic activities in differen

of determinations)

n h-Glucosidase (h-Glu)

(amol cell�1 h�1)

Stringers 4 15.5F0.8

Cobwebs 3 0.7F0.6

Ribbons 3 776.2F0.7

Clouds 8 25.1F0.8

had the highest potential activity level suggesting the

beginning of an N limitation. The APA:AMA ratioN1

(Table 10) suggested that P concentrations were able to

sustain primary production. In the porewater, however,

the N:P ratio was N16 (Redfield et al., 1963), confirm-

ing a complete uptake of P by organisms embedded in

mucilage. The phytoplankton could release labile or-

ganic matter and consequently bacteria benefit from

released molecules, decreasing their hydrolytic activi-

ties. The new carbon production seemed to satisfy the

bacteria demand resulting in a high AMA:h-Glu ratio

(Table 9). Bacteria did not work on old glucidic sub-

strate and clouds were enriched in organic carbon

(Table 4).

Probably as a result of the high hydrolytic activities

of aggregate-associated bacteria, concentrations of

DOC and total nitrogen and phosphorus were signifi-

cantly higher in the porewater than in the surrounding

water (Tables 4 and 5). The high nutrient concentration

in the porewater and the spatially tight coexistence of

organisms within the aggregate matrix, enhanced the

uptake of the hydrolysis products by the aggregate-

associated bacteria and enhanced biomass production

rates. Significantly high rates of bacterial biomass

production estimated by the leucine method were

found for aggregate-associated bacteria (Table 11a).

As reported by Muller-Niklas et al. (1994), in the

Adriatic Sea, aggregate-associated bacterial production

exceeded that in the surrounding water (Table 11b). Per

cell bacterial carbon production evaluated in the dif-

ferent aggregate types was significantly correlated

( p =0.05) with enzymatic activity, suggesting the im-

t types of aggregates, expressed as attomoles cell�1 h�1 (n—number

Aminopeptidase (AMA) Phosphatase (APA)

(amol cell�1 h�1) (amol cell�1 h�1)

1068.0F0.5 458.5F0.7

432.7F1.0 60.6F1.0

6894.6F0.4 15,989.4F0.6

957.7F0.9 896.2F0.8

Table 9

Phosphatase activity (APA) :aminopeptidase activity (AMA) ratio and

aminopeptidase activity (AMA):h-Glucosidase activity (h-Glu) ratioin different types of aggregates

APA:AMA AMA:hGlu

Stringers 0.4 68.7

Cobwebs 0.1 600.3

Ribbons 2.2 9.0

Clouds 0.9 38.1

Table 11

(a) Means and standard deviation of total bacterial carbon

production (BCP) and per cell BCP in different types of

aggregates (n—number of determinations)

n BCP BCP

Ag C 1�1 h�1 fg C cell�1 h�1

Stringers 6 8.7F5.3 1.3F0.1

Cobwebs 2 4.2F0.8 1.8F0.1

Ribbons 4 39.8F19.5 12.1F0.5

Clouds 8 275.5F119.4 1.3F0.1

(b) Means and standard deviation of total bacterial carbon

production (BCP) in surrounding water, porewater and mucilage

n BCP

Ag C 1�1h�1

Surrounding water 7 0.5F0.1

Porewater 16 23.7F1.3

Mucilage 20 82.0F11.8

(c) Bacterial growth efficiency (BGE) in different types of aggregates

n BGE %

Stringers 3 4

Ribbons 3 6

Clouds 3 30


portant role played by low molecular weight organic

carbon produced by the hydrolytic process. The highest

carbon production values were due to ribbon-associated

bacteria that showed the highest levels of enzymatic

activity.

The respiration of aggregate-associated microbes

was determined through oxygen changes measured

by Winkler titration (Table 12). Respiration rates in-

creased with increasing aggregate size but if respiration

on aggregates was predominantly due to bacteria and

when the results were normalized per bacterial cell,

respiration proved to be inversely correlated to aggre-

gate size, as found by Ploug et al. (1999) and Ploug

(2001). The high respiration rate per bacterial cell

indicated that aggregate-associated bacteria were cata-

bolically highly active. In general, respiration rates

were higher than bacterial biomass production rates,

indicating that respiration was an important pathway

for aggregate decomposition. Combined measurements

of bacterial biomass production and respiration in the

same aggregates allowed an estimate of the bacterial

growth efficiency (BGE) of the aggregate-associated

bacteria. The few measurements carried out so far have

shown that the growth efficiency was variable, with its

highest values in clouds. In contrast, Grossart and

Ploug (2000) showed values decreasing in aged aggre-

gates. In their study, aggregates were mostly composed

of detritus and increasing fractions of hard-to-degrade

substrates such as lignin and humic substances of

terrestrial origin, unlike the Adriatic aggregates in

which usually no terrestrial organic matter was found

(Berto et al., 2005—this issue). This could explain

Table 10

Phosphatase activity (APA) :aminopeptidase activity (AMA) ratio and

aminopeptidase activity (AMA):h-Glucosidase activity (h-Glu) ratioin surrounding water, porewater and mucilage

APA:AMA AMA:hGlu

Surrounding water 1.0 11.3

Porewater 1.3 23.6

Mucilage 1.1 92.2

why BGE on aggregates decreased over time. During

this research BGE varied between 4% and 30% (Table

11c). This variability indicates that aggregate-associat-

ed bacteria utilized a multitude of substrates. Broader

ranges of BGE were evaluated by Amon and Benner

(1996) who found BGE varying between 8% and 39%

for bacteria degrading high molecular weight DOC and

16–66% for bacteria degrading low molecular weight

DOC. BGE at lower than 8%, in combination with the

highest ectoenzymatic activities, as found in ribbons,

indicates lower substrate quality (Smith et al., 1995;

Ploug et al., 1999) probably due to the presence of

high molecular weight polymers. High BGE in com-

bination with high bacterial production in aged aggre-

gates such as clouds, demonstrated that at least the less

refractory fraction of the POC was rapidly and effi-

ciently used by attached bacteria. Inside clouds bacte-

Table 12

Means and standard deviation of respiration rate expressed as total

and per cell value (n—number of determinations)

n Respiration

Ag C l�1h�1 fg C cell�1 h�1

Stringers 3 210.6F90.5 30.5F2.8

Ribbons 3 588.5F62.3 178.3F16.3

Clouds 3 649.3F64.1 3.1F0.5


ria might rapidly uptake labile molecules produced by

embedded phytoplankton.

Acknowledgements

We wish to thank Bela Jagic for the SCUBA-diving

assistance and our colleagues of LBM microbiology

group involved in the field activity: Paola Ramani,

Massimiliano Lupieri, Mahela Rorato, Pietro Rossin,

Anna Degani, Ana Karuza and Alessandro Bensi.

The research was supported by the Ministry for

Environment in the frame of the MAT (Mucilage in

Adriatic and Tyrrhenian Sea) Project.

The authors thank the crews of the R/V Vila Velebita

(Center for Marine Research, Rudjer Boskovie Insti-

tute, Rovinj, Croatia), R/V Dalla Porta (Istituto di

Scienze Marine-CNR, Ancona, Italy) and R/V Thetis

(Sopromar, Italy) for their collaboration.

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