Photosynthetic activity and community shifts ofmicrophytobenthos covered by green macroalgaeemi4_335 316..325

Emilio García-Robledo,1* Alfonso Corzo,1

Sokratis Papaspyrou1,2 and Edward P. Morris1,3

1Dpto. Biología. Facultad de Ciencias del Mar yAmbientales, Pol. Río San Pedro s/n. 11510 PuertoReal, Cádiz, Spain.2Unidad asociada de Oceanografía Interdisciplinar. UCA– Instituto de Ciencias Marinas de Andalucia(ICMAN-CSIC), Pol. Rio San Pedro s/n, 11510 PuertoReal, Cádiz, Spain.3Department of Ecology and Coastal Management,Instituto de Ciencias Marinas de Andalucía(ICMAN-CSIC), Pol. Rio San Pedro s/n, Puerto Real,Cádiz, Spain.

Summary

Macroalgae blooms, a frequent consequence ofeutrophication in coastal areas, affect the photo-synthetic activity of sediments dominated by micro-phytobenthos (MPB). Light spectra, steady-state(after 1 h) microprofiles of O2, gross photosynthesis(Pg), community respiration in light (RL) and net com-munity photosynthesis (Pn) were measured in diatom-and cyanobacteria-dominated communities belowincreasing layers of Ulva. Photosynthetic photon flux(PPF) decreased exponentially with increasing layersof algae and the light spectrum was increasinglyenriched in the green and deprived in blue and redregions. Sediment Pg, Pn and RL decreased as thenumber of Ulva layers increased; however, 1.6 timeshigher macroalgal density was necessary to fullyinhibit cyanobacteria Pg compared with diatoms, indi-cating that cyanobacteria were better adapted to thislight environment. Long-term (3 weeks) incubationsof diatom-dominated sediments below increasinglayers of Ulva resulted in a shift in the taxonomiccomposition of the MPB towards cyanobacteria.Hence, changes in the light climate below macroalgalaccumulations can negatively affect the photosyn-thetic activity of sediments. However, spectral nichedifferentiation of MPB taxonomic groups and concur-rent changes in the MPB community may provide

sediments with increased resilience to the detrimen-tal effects of eutrophication.

Introduction

The increase in magnitude and frequency of ephemeralmacroalgal blooms is a widespread symptom of chroniceutrophication in coastal areas (Morand and Briand,1996; Schramm, 1999). Nevertheless in shallow systems,even at relatively high nutrient loads enough light reachesthe benthos to support primary producers, which continueto play a role in benthic–pelagic nutrient cycling (McGlath-ery et al., 2007). Still, as opportunistic macroalgal bloomsaccumulate, they increasingly become strong structuringcomponents, often negatively affecting several other eco-system compartments and functions (Sundbäck et al.,1990; Raffaelli et al., 1998).

Microphytobenthos (MPB), which include benthicdiatoms, green algae, dinoflagellates, euglenoids, cyano-bacteria and photosynthetic bacteria (Admiraal, 1984),are the most ubiquitous benthic primary producers,extending from the high intertidal to depths of up to 200 m(Cahoon, 1999; McGee et al., 2008). Microphytobenthoscan represent a substantial proportion of coastal primaryproduction (MacIntyre et al., 1996), are important forcoastal foodwebs (Evrard et al., 2010) and stronglycontrol benthic–pelagic nutrient transport (Sundbäck andMiles, 2000; Dalsgaard, 2003), meaning they play animportant role in the biogeochemistry of shallow coastalareas throughout the globe (Duarte and Cebrian, 1996).Hence, understanding how macroalgal blooms affectbenthic microbial communities is important for defining theresponse of shallow coastal ecosystems to eutrophication(Sundback and McGlathery, 2005).

Generally, increasing macroalgal densities result inlower sediment light levels due to shading and a decreaseor complete inhibition of MPB photosynthetic activity(Sundbäck et al., 1990; Corzo et al., 2009; Garcia-Robledo and Corzo, 2011). Sediments become increas-ingly anoxic, resulting in altered benthic–pelagic nutrientfluxes (Krause-Jensen et al., 1999) and negative effectson the faunal community (Raffaelli et al., 1998). On theother hand, enhancements in MPB production related tophotoacclimation and an increase in heterotrophicmetabolism have also been reported (Trimmer et al.,2000; Garcia-Robledo and Corzo, 2011), suggesting that

Received 18 October, 2011; accepted 20 February, 2012. *For cor-respondence. E-mail emilio.garcia@uca.es; Tel. (+34) 956 016177;Fax (+34) 956 016019.

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Environmental Microbiology Reports (2012) 4(3), 316–325 doi:10.1111/j.1758-2229.2012.00335.x

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd

the mechanisms driving these changes are complex andnot fully resolved. Previous studies have not distinguishedbetween the effects of shading and other biogeochemicalinteractions between macroalgae and the sediment com-munity, such as direct competition for, and exchange of,organic and inorganic nutrients (McGlathery et al., 2001;Garcia-Robledo and Corzo, 2011). Furthermore, MPB andmacroalgae can also interact indirectly, via the accumula-tion and remineralization of macroalgal organic matter inthe sediment and the resulting release of nutrients(García-Robledo et al., 2008; Corzo et al., 2009).

In the present study, we aimed to exclude these inter-actions and focus only on the effect of changes in themagnitude and spectra of downwelling irradiance causedby different densities of green macroalgae. The changesin light climate are likely to favour MPB functional groupswith a suitable light-niche, such as cyanobacteria (Jor-gensen et al., 1987; Stomp et al., 2007). This will poten-tially produce a shift in community composition that willaffect the photosynthetic activity of the sediment (Bar-ranguet, 1997). Acting as a type of community photo-acclimation, this mechanism may help to keep sedimentsnet autotrophic during shading by macroalgae and, thusprovide increased resilience against the detrimentaleffects of eutrophication. We conducted two experimen-tal manipulations of sediment cores (short-term, 1 h; andlong-term, 3 weeks) so as to examine two specifichypotheses: (i) the light spectrum below increasing den-sities of green macroalgae favours the photosyntheticperformance of cyanobacteria compared with diatomdominated MPB communities; (ii) this change in lightclimate causes a shift from a diatom to a cyanobacteria-dominated community that results in relatively higherrates of benthic photosynthesis at comparable macro-algal densities.

Results

Light attenuation by Ulva

Photosynthetic photon flux (PPF) decreased exponen-tially with increasing layers of Ulva (Fig. 1A). Fitting thelight attenuation curves to the Beer–Lambert equationresulted in an attenuation coefficient of 0.47 � 0.02layer-1 or 0.019 � 0.001 m2 g DW-1. Spectral bands cor-responding to chlorophylls (Chl) a, b and carotenoidswere preferentially absorbed, with the blue (400–490 nm)and red regions of the spectra (620–690 nm) almost com-pletely extinguished below 3–4 layers of Ulva (Fig. 1B).Light attenuation coefficients between 400–490 and 620–690 nm were > 3 times higher than at 750 nm (Fig. 1C).Between 490 and 620 nm attenuation was reduced, with alocal minimum of 0.47 � 0.02 layer-1 observed at 560 nm(Fig. 1C).

Short-term response of microphytobenthos to shadingby Ulva

The diatom- and cyanobacteria-dominated communitieswere both autotrophic in the absence of Ulva (Fig. 2).However, maximum photosynthetic rates were 3 timeshigher in the cyanobacteria compared with thediatom-dominated community (t = 2.71, d.f. = 7, P < 0.05),resulting in higher oxygen concentrations below thesediment surface (Fig. 2). The photic depth (zph) of thecyanobacteria-dominated community (850 mm) wasalso significantly deeper than the diatom-dominated

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Fig. 1. Photosynthetic photon flux (A) measured at the sedimentsurface under an increasing number of Ulva layers in theshort-term (black dots) and long-term experiment (white dots). Lightspectra reaching sediment surface under increasing Ulva layers (B)measured in the short-term experiment. Numbers in (B) representUlva layers. Data are means � SE (n = 2–3). Variation of lightextinction coefficients (k) for the Ulva sp. canopy along light spectra(C), expressed as number of layers-1 � SE (n = 5).

317 E. García-Robledo, A. Corzo, S. Papaspyrou and E. P. Morris

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 316–325

community (500 mm) (t = 3.52, d.f. = 7, P < 0.05) (Fig. 2,Table 1). Although sediment [Chl a] were not significantlydifferent, areal gross production rate (Pg

A) was about 5times higher in the cyanobacteria compared with thediatom-dominated community (t = 3.49, d.f. = 7, P < 0.05,t-test) (Table 1). However, because of the high ratesof community respiration in the light (RL) of thecyanobacteria-dominated community (Table 1), the differ-ence in net community production rates (Pn) was smaller(about 3 times, t = 3.10, d.f. = 7, P < 0.05, t-test).

Under short-term shading by increasing densities ofUlva, the typical subsurface O2 maximum progressivelydisappeared and oxygen depletion became strongerwith depth (Fig. 2A and B). For both communities, the

successive addition of Ulva layers significantly reducedPg (cyanobacteria-dominated: F7,207 = 7.63, diatom-dominated: F4,324 = 41.45, both P < 0.01, two-way ANOVA,factor Ulva layers) and zph (cyanobacteria-dominated:F7,15 = 4.32, diatom-dominated: F4,34 = 10.77, both P <0.05, one-way ANOVA) (Fig. 2C and D). However, thenumber of Ulva layers needed to completely inhibit Pg inthe two communities was different; 5 layers (120 �

5 g DW m-2, Figs 2C and 3A) and 8 layers (195 �

9 g DW m-2, Figs 2D and 3B), for the diatom-dominatedand cyanobacteria-dominated communities respectively.

Decreases in RL of both communities were correlatedwith increasing densities of Ulva and thus decreases inPg

A (Fig. 3E and F). While initial values of RL were clearly

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Fig. 2. Oxygen concentration and gross production rates (Pg) profiles under increasing number of Ulva layers in diatom-dominated (A and C)and cyanobacteria-dominated sediment (B and D). Profiles are means � SE (n = 2–3).

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higher for the cyanobacteria-dominated community(Table 1), rates of sediment community respiration in thedark (RD) were not significantly different (t = 0.51,P > 0.05, t-test) (Table 1).

Net community production rates (Pn) significantlydecreased with increasing number of Ulva layers (Fig. 3Cand D, cyanobacteria-dominated: F8,17 = 6.80, diatom-dominated: F5,25 = 11.14, both P < 0.01, one-way ANOVA).For the diatom-dominated community, Pn became nega-tive (-0.44 � 0.08 mmol O2 m-2 h-1) with 3 Ulva layers(72 � 3 g DW m-2), whereas, for the cyanobacteria-dominated community 6 layers of Ulva (145 �

6 g DW m-2) were required to cause negative Pn (-0.50 �

0.12 mmol O2 m-2 h-1).

Long-term response of microphytobenthos to shadingby Ulva

The initial MPB community used in this experiment wasan autotrophic, intertidal diatom-dominated community(both in terms of biomass and biovolume), similar to thatdescribed in the previous section. After 3 weeks, the sedi-ment remained net autotrophic in both the control and 2Ulva layers treatments, with clearly visible subsurface O2

and Pg peaks at 200–300 mm depth (Fig. 4). However, O2

profiles in cores with 4 and 6 Ulva layers showed noapparent oxygen production peak, although Pg was stillmeasurable (Fig. 4B). Significant differences in Pg

A, RL

and Pn (Fig. 5A) were observed between the lower (0 and2 layers) and higher (4 and 6 layers) shading treatments[P < 0.05, Tukey’s honest significant difference (HSD)].

Shading by Ulva canopies significantly changed sedi-ment pigment concentrations (Fig. 5B). Both Chl a andChl c were higher in cores shaded by 2 and 4 Ulva layers

compared with the control treatment (P < 0.05, Tukey’sHSD test), whereas no difference was found between thecontrol and cores shaded with 6 Ulva layers (Fig. 5B).

The abundance of diatoms did not change betweentreatments (F3,7 = 0.11, P > 0.05, one-way ANOVA)(Fig. 5C). The most common genera were Gyrosigma,Navicula, Pleurosigma and Pseudonitzschia. On the con-trary, the abundance of cyanobacteria, mainly Leptolyng-bya, was significantly higher in the 4 and 6 layercompared with the 0 and 2 layer treatments (P < 0.05,Tukey’s HSD test). The ratio of diatoms to cyanobacteriaabundance (D:C ratio, Fig. 5C) was close to 1 (1.1 � 0.1)for cores incubated with 0 layers of Ulva, whereas fortreatments with 4 and 6 Ulva layers the ratio decreased to0.3 � 0.1.

Discussion

Our experimental design allowed us to clearly demon-strate how the taxonomic composition and photosyntheticactivity of intertidal microphytobenthos communitieschanged in response to reductions in the quantity andquality of light below macroalgal blooms. Any mass trans-fer or competition for nutrients between the macroalgaeand the benthic community, by direct contact or throughthe water phase, was deliberately avoided.

Photosynthetic photon flux at the sediment surfacedecreased exponentially with increasing numbers of Ulvalayers (Fig. 1A), and extinction coefficients were similar toprevious values found for planar Ulva species (Vergaraet al., 1997). Nevertheless, attenuation coefficients ofmacroalgae are strongly controlled by photo-physiologicalprocesses such as chloroplast movement, changingpigment contents and thalli structure (Britz and Seliger,1973; Britz and Briggs, 1987; Vergara et al., 1997).Hence, these attenuation coefficients probably vary daily,seasonally and between locations. Light reaching thesediment surface was progressively impoverished inthe blue (430 nm) and red regions (650 and 675 nm) ofthe spectrum (Fig. 1C), corresponding to the in vivoabsorption peaks of Chl a, b and carotenoids typicalof Ulva, and relatively enriched in green and near infraredas shading by macroalgal biomass was augmented.Based on the measured attenuation coefficients andassuming a maximum intertidal sediment surface PPF(2000 mmol photons-1 s-1), a Ulva biomass of 450 g m-2

would be necessary to completely attenuate all lightreaching the sediment surface, which is in the mid-rangeof the densities reported for natural algal blooms(Hernández et al., 1997; Pihl et al., 1999; Corzo et al.,2009).

The reductions in the magnitude and quality of lightbelow increasing numbers of Ulva layers caused adecrease in the rates of gross photosynthesis (Pg) of

Table 1. Comparison of diatom-dominated and cyanobacteria-dominated communities illuminated at 500 mmol photons m-2 s-1

without Ulva cover.

Diatom-dominatedCyanobacteria-dominated

Chl a 25 � 5 33 � 4Pg

A 3.26 � 0.62 15.65 � 6.41*Pn 1.69 � 0.34 4.86 � 1.60*RD 0.52 � 0.04 0.54 � 0.11RL 1.90 � 0.42 10.79 � 8.01Pg

A/RL 1.83 � 0.23 2.25 � 1.08Pn/RD 3.01 � 0.90 8.76 � 1.23*zph 0.49 � 0.05 0.85 � 0.05*

Chlorophyll a concentration (Chl a) expressed as mg m-2; areal grossphotosynthesis rate (Pg

A), areal net photosynthesis rate (Pn), arealdark respiration rate (RD) and areal light respiration rate (RL)expressed as mmol O2 m-2 h-1 and photic depth (zph) expressed inmillimetres. Data are mean � standard deviation of 4 and 2 cores forthe diatom-dominated and the cyanobacteria-dominated communityrespectively. Significant differences are marked by asterisks (t-test,P < 0.05).

319 E. García-Robledo, A. Corzo, S. Papaspyrou and E. P. Morris

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 316–325

both MPB communities (García-Robledo et al., 2008;Corzo et al., 2009). However, 8 Ulva layers (about200 g DW m-2) were needed to completely inhibit photo-synthetic activity in the cyanobacteria-dominated sedi-ment compared with only 5 layers (corresponding to about120 g DW m-2) in the diatom-dominated sediment (Figs 2and 3). These densities were roughly equivalent to asediment surface PPF of 10 and 50 mmol photons m-2 s-1

respectively. While these light levels are not particularlylow considering the wide range of photo-acclimationobserved in benthic diatoms (Sundback and Granéli,1988; Mercado et al., 2004), the available light was mostlygreen, a region of the spectrum where diatoms haverelatively low absorption cross-sections (Mercado et al.,2004; Morris et al., 2008).

Benthic cyanobacteria, due to their photosynthetic pig-ments (phycocyanin and phycoerythrin), are betteradapted to the light environment below green macroalgalmats (Jorgensen et al., 1987). Comparison of photosyn-thetic action spectra between a diatom and Microcoleuscyanobacterial community showed that maximum photo-synthesis rates were measured at 430 and 670 nm andat 580 and 650 nm respectively (Jorgensen et al., 1987).Hence, cyanobacteria have a higher photosyntheticefficiency compared with diatoms below Ulva canopies,which results in higher rates of Pg

A at equivalentmacroalgal densities and, presumably, a competitiveadvantage.

On the other hand, rates of community respiration in thelight were much higher for the cyanobacteria-dominated

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Fig. 3. Areal gross production (PgA), net production in light (Pn) and light respiration (RL) under an increasing number of Ulva layers

(expressed as biomass of Ulva in g DW m-2) for a diatom-dominated community (A, C and E) and a cyanobacteria-dominated community (B,D and F). Data are means � SE (n = 2–3).

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sediment. RL represents the fraction of PgA that is con-

sumed in the light (Epping and Jørgensen, 1996), plusthe basal respiration rate in dark (RD). Hence, RL waslinearly dependent on Pg

A in both communities (diatom-dominated: RL = 0.51 + 0.36 ¥ Pg

A, r2 = 0.81, P < 0.001;cyanobacteria-dominated: RL = 0.54 + 0.69 ¥ Pg

A, r2 =0.89, P < 0.001). The slope of these regressions repre-sents the proportion of Pg

A that was consumed withineach community: 36% and 69% for the diatom-and cyanobacteria-dominated communities respectively.Because oxygenic photosynthesis and aerobic respirationare not carried out in separate organelles, cyanobacteriatend to have higher rates of RL compared with other algae(Schmetterer, 2004). Still, considering the very tight cou-pling between heterotrophic bacteria and primary produc-ers in sediments, differences in the stimulation of bacterialrespiration (and thus RL) via rapid transfer of C-rich exu-

dates may also help explain this effect (Epping and Kühl,2000).

The ratio of PgA to RL (Pg

A/RL) for the cyanobacterial-dominated sediment did not decrease as steeply as thediatom community with increasing Ulva shading, remain-ing autotrophic (> 1) up to 5 Ulva layers (Fig. 6). Thecritical PPF values for the switch from net autotrophic toheterotrophic in the diatom- and cyanobacteria dominatedsediments were roughly 140 and 35 mmol photons m-2 s-1

respectively. Therefore, assuming an incident PPF

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respiration (RL) (A); chlorophylls a and c contents in the sediment(B); and microphytobenthic cell abundance (C) in the sedimentunder an increasing number of Ulva layers after 3 weeks. Data aremeans � SE (n = 3).

321 E. García-Robledo, A. Corzo, S. Papaspyrou and E. P. Morris

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of 2000 mmol photons m-2 s-1, cyanobacteria-dominatedsediments would be able to remain net autotrophic undera larger macroalgal biomass (216 g DW m-2) than diatom-dominated sediments (135 g DW m-2). Although as men-tioned above, differences in algal attenuation coefficientsneed to be considered, this biomass is in the low range ofvalues (200–420 g DW m-2) reported typically in manycoastal areas affected by seasonal or permanent greenmacroalgal blooms (Hernández et al., 1997; Pihl et al.,1999; Corzo et al., 2009). Thus, even at low densitiesshading by macroalgal canopies can negatively affect theprimary production of MPB and in consequence the redoxstatus of surface sediments.

The temporal scales over which macroalgal bloomsmay affect the microphytobenthic community may varyconsiderably. At time scales of hours to days, MPBspecies may photo-acclimatize to the light conditionsprevailing below the macroalgal canopy increasing thecell content of photosynthetic pigments and their effec-tive absorption cross section (Blanchard and Cariou-LeGall, 1994; Barranguet et al., 1998). At longer timescales, changes in the taxonomic composition of theMPB community are expected (Barranguet, 1997).Indeed, our results suggest that incubation of sedimentsbelow different densities of macroalgae for a period of3 weeks was long enough to induce both of theseacclimation mechanisms. Cyanobacteria increased inabundance (30–160%) when shaded by Ulva. This con-tributed to an increase in sediment Chl a in the interme-diate shading treatments, but not at the highest density,suggesting adjustments in cell pigment contents. Fordiatoms this was confirmed by a doubling of Chl c in the2 and 4 layers shading treatments with no changes incell abundance. These photo-acclimation responsesincreased the light-capture capacity of the MPB commu-

nity, resulting in no change in sediment PgA, Pn or RL

when shaded by 2 Ulva layers (sediment surface PPFreduced to about 100 mmol photons m-2 s-1) for 3weeks.

At higher Ulva densities (4 and 6 layers, correspondingto PPF of about 60 and 20 mmol photons m-2 s-1)increases in diatom photosynthetic pigment contentswere unable to compensate for the reduction in light avail-ability; essentially because diatoms have no specializedphotosynthetic pigments in the green region of the spec-trum and pigment packaging eventually limits cell absorp-tion capacity (Mercado et al., 2004). Hence, long-termshading resulted in decreases in sediment Pg

A, causing Pn

to become negative. Nevertheless, PgA/RL ratios were sig-

nificantly higher than in the diatom-dominated communityand similar to the cyanobacteria-dominated communityshaded by 6 Ulva layers in the short-term experiment(Fig. 6). This compensatory effect could be mainly attrib-uted to the change in taxonomic composition of the MPBcommunity, particularly in the highest density shadingtreatment. Hence, the photosynthetic activity of cyano-bacteria appeared to lessen the detrimental effects ofshading by Ulva canopies on sediments.

In natural systems this general response will also beaffected by feedbacks between macroalgae and MPBsuch as competition for inorganic nutrients, transfer ofdissolved organic matter and stimulation of sediment het-erotrophic metabolism by labile particulate organic matter.Indeed, MPB are readily able to metabolize labile DOMand thus, may well benefit from organic nutrients exudedby macroalgae. Furthermore, interactions with sedimentfauna are also likely to be important; diatoms seem to bebetter adapted to intense grazing than cyanobacteria,then again, sub-oxic sediments negatively affect grazers.

In summary, our results demonstrate that changes inthe quantity and quality of light caused by shading ofsediments by green macroalgae may induce a change inthe taxonomic composition of natural MPB communities,favouring MPB functional groups with more suitable spec-tral niches, such as cyanobacteria (Stomp et al., 2007).This community photo-acclimation may provide sedi-ments with increased resilience to the detrimental effectsof eutrophication, supporting the hypothesis that thediversity of benthic primary producers is critical for deter-mining the response of shallow bays and intertidal areasto environmental perturbations.

Experimental procedures

Sample collection and preparation

Sediment cores (i.d. 5.4 cm, length 20 cm) and planar Ulvasp. were collected in July 2009 from inner Cadiz Bay (SWSpain). Sediment was a silty-mud with no macroscopic mac-roalgal or seagrass material. Cores and macroalgae were

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cyanobacteria-dominated (Cyanobacteria) communities and thenumber of Ulva layers in the short-term experiment and after threeweeks in the long-term experiment (Long term). Data aremeans � SE (n = 2–3).

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© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 316–325

maintained separately in flow through aquariums with in situsea water. Cores were incubated at a PPF of 300 and500 mmol photons m-2 h-1 (long-term and short-term experi-ment respectively) with a 14 light : 10 dark photoperiod atabout 20°C until treated. Planar Ulva sp. was maintained withthe same photoperiod and temperature at a PPF of200 mmol photons m-2 h-1.

Short-term experiment

Six cores were incubated as previously described until analy-sis. In situ seawater was amended with inorganic nutrients(20 mmol NO3

- l-1 and 5 mmol PO43- l-1). Three cores were

processed within 3 days after collection whereas the otherthree were incubated for 2 weeks to allow the development ofa cyanobacteria-dominated community. Addition of nitrateand phosphate, but not silicate, and the absence of macro-faunal grazers favour the development of cyanobacteria-dominated communities (Fenchel, 1998; Garcia de Lomaset al., 2005). After 2 weeks, dense cyanobacterial patchescomposed mainly by Microcoleus sp. developed in two of thecores.

Cores were processed at a rate of one per day. The upper2 cm of sediment was carefully transferred to a Plexiglas tube(i.d. 5.4 cm and 5 cm length) and the bottom of the tube wassealed using parafilm and aluminium foil. The tube was filledwith sea water to a height of 2 cm above the sedimentsurface (Fig. 7) and continuously renewed using an openwater flow system (approximately 10 min hydraulic retentiontime). A Petri dish with thick white paper on the bottom wasplaced on top of the core and filled with sea water (1 cmheight) to help diffuse the downwelling irradiance. Thesystem was illuminated from above with a halogen lamp(Novaflex, World Precision Instruments) at a PPF of470 � 20 mmol photons m-2 s-1, measured at the sedimentsurface (spherical sensor, LiCor). The spectrum of lightreaching the sediment surface was measured using aUSB2000 spectrometer (Ocean Optics) connected to an opticfibre (300 mm diameter and a light acceptance angle of 180°,i.e. cosine, BFL22-200, Thorlabs) positioned close to thesediment surface through the bottom of the core (Fig. 7).After setting up the core, the system was left for I h to reachsteady state before measurements of O2 and Pg profiles wereinitiated. Once these were completed under the initial condi-tions (0 layers), the oxygen microsensor was positionedbelow the oxic-anoxic interface. A single layer of a large,planar Ulva sp. frond was placed so as to fit perfectly withinthe Petri dish diffuser above the core. After one hour, thedownwelling irradiance spectrum was recorded, followed byO2 and Pg profiles. This procedure was repeated, addingsuccessive disks of Ulva, until no Pg could be measured. Thecore was then immediately subsampled in the same positionwhere the profiles were measured using a truncated syringe(i.d. 1.6 cm, depth 2 cm) in order to measure Chl a and c.

Long-term experiment

Twelve sediment cores wrapped in black plastic on the sideswere placed inside a temperature controlled aquarium at20°C and maintained under a 14 h light : 10 h dark photope-

riod for 1 day before the start of the experiment. Light wasprovided by a halogen lamp (Philips 7748S, 250W), at a PPFof 320 � 15 mmol photons m-2 s-1 measured at the sedimentsurface (LiCor spherical light sensor). The lower PPF used inthis experiment was chosen to avoid strong desiccation andbleaching of the macroalgal cover. A continuous open flowsystem (approximately 6 h hydraulic retention time) wasestablished in each core using in situ seawater, which hadlow nutrient conditions (c. 2, 0.5 and 6 mmol l-1 for NH4

+, PO43-

and SiO32- respectively) typical of Cadiz Bay in summer.

As for the short-term experiment a glass Petri dish with1 cm of seawater and white paper was placed on top of eachcore. Three cores were used as Controls (0 layers), whereasin the remaining 9 cores, 2, 4 and 6 Ulva layers were addedto the Petri dishes to have 3 cores per macroalgal density.Ulva layers were replaced with fresh material every week tominimize the effects of Ulva deterioration and the consequentmodification of light conditions below the canopy (Vergaraet al., 1997). Based on the short-term experiment, the den-sities of Ulva were chosen to roughly inhibit MPB photosyn-thetic rate by 0%, 50%, 100% and 150%.

After 3 weeks, two vertical profiles of O2 and Pg weremeasured in each core without disturbing the light climate ofthe treatment. The Petri dish was displaced laterally lessthan 1 cm just enough to introduce the oxygen microsensorwith an angle of 30°. After completion of the profiles the

A

B

C

D

E

Fig. 7. Set-up for short-term experiment. A core with 2 cm ofsediment (A) was covered with 2 cm of seawater (B) in an openwater flow system. A Petri disch (C) under an increasing number ofUlva layers was positioned on top of the core. The oxygenmicrosensor (D) was moved from the bottom to the top andsteady-state O2 and Pg profiles were measured. An optic fibre (E)connected to a spectrometer was positioned close to the sedimentsurface.

323 E. García-Robledo, A. Corzo, S. Papaspyrou and E. P. Morris

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 316–325

upper 5 mm of the sediment was removed, homogenizedand subsampled to measure chlorophyll and microalgal cellabundance.

MPB chlorophyll and cell densities

Chlorophylls a and c were extracted as in Thompson andcolleagues (1999) and estimated using the equations ofRitchie (2008). Sediment subsamples were fixed with glut-araldehyde (2.5% in 0.22 mm filtered sea water) and lateranalysed by optical microscopy. Individual diatoms andcyanobacterial colonies were counted by epifluorescencemicroscopy of a diluted subsample. Empty diatom frustuleswere not counted in order to avoid overestimation of thediatom community.

Oxygen microprofiles and calculation ofphotosynthetic rates

In order to minimize the disturbance by making repeatedmeasurements in the same position during measurement ofoxygen profiles, specially built sensors with a tip diameter of10 mm, long and very slender shaft (4 mm) and fast responsetime (< 0.3 s) were used (Unisense, Denmark). In addition,the microsensors were filled with an electrolyte immobilizedin agarose in order to avoid bubbles in the sensor tip whichwould modify the sensor response when used upside down.Oxygen microsensors were connected to a picoammeter(PA2000, Unisense) and the signal was recorded using anA/D converter. Profiles were automated using a motorizedmicromanipulator connected to a computer controlled usingthe software Sensor TracePro (Unisense).

Gross photosynthesis (Pg) profiles, net community produc-tion (Pn) and community respiration in light (RL) were calcu-lated according to Revsbech and Jorgensen (1983) and Gludand colleagues (1992). Community respiration in darkness(RD) was calculated from the O2 gradient at the diffusiveboundary layer in darkness, i.e. under a sufficient number ofmacroalgal layers where Pg became zero. The photic depth(zph) was estimated as the maximum depth with measurablePg.

Statistical analysis

Significant effects of treatments were examined using one-way and two-way analysis of variance (ANOVA) followed by aTukey’s HSD test for multiple pairwise comparison. Non-linear curve fitting was used to estimate light attenuationcoefficients of algal material. Analyses were considered sig-nificant at P < 0.05. Statistics were performed using the soft-ware Statgraphics Centurion XVI.

Acknowledgements

Authors thank Dr Ignacio Moreno for his help with identifica-tion of benthic diatoms. The research was funded by grantsCTM 2009-10736 (Ministry of Education and Science, Spain),P06-RNM-01787 and P06-RNM-01637 (Andalusian RegionalGovernment). E. García-Robledo was funded by a FPU grant

(AP2005-4897) from Ministry of Education and Science andby the project ECOLAGUNES (SOE1/P2/F153, InterregSUDOE, European Union). E.P.M. was funded by a project(FUNDIV, P07-RNM-2516) granted by the AndalusianRegional Government. S. Papaspyrou was funded by a JAE-Doc fellowship (Programa JAE, JAE-Doc109, SpanishNational Research Council) and a Marie Curie ERG action(NITRICOS, 235005, European Union).

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