Filip F Pniewski
email: filip.pniewski@ug.edu.pl

Paulina Biskup

Iwona Bubak

Pierre Richard

Adam Latala

Gérard Blanchard

Adam Latała

Gerard Blanchard

Photo-regulation in microphytobenthos from intertidal mudflats and non-tidal coastal shallows

Keywords: microphytobenthos, photoacclimation, photosynthesis, photosynthetic pigments, Aiguillon Bay, Baltic Sea Poland are elsevier_YECSS_4596

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Microphythobenthos is an ecological formation grouping various photosynthetic organisms, i.e. cyanobacteria and algae (e.g. euglenophytes, diatoms, green algae etc.) growing on a variety of substrata in shallow aquatic environments.

Microphytobenthos inhabiting sediments

is traditionally divided into two groups depending on the nature of the sediment, i.e. free-living epipelon and epipsammon attached to the sediment particles [START_REF] Round | Benthic Marine Diatoms[END_REF]. Epipelon is usually dominated by large, biraphid diatoms, whereas epipsammon consists of small-sized diatoms with a substantial contribution of other algal groups, including cyanobacteria and coccoid chlorophytes [START_REF] Round | The Ecology of Algae[END_REF]. Microphytobenthos is responsible for a significant proportion of estuarine primary production, especially in intertidal mudflats which are devoid of higher plants and macroalgae [START_REF] Blanchard | Photosynthetic characteristic of microphytobenthos in Marennes-Oléron Bay, France: preliminary results[END_REF][START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF]. In addition, benthic microalgae can be a substantial source of food, not only to benthic but also pelagic food webs due to the resuspension in the water column during the high tide [START_REF] Lucas | Benthic-pelagic exchange of microalgae at a tidal flat. 2. Taxonomic analysis[END_REF]. They mediate a number of geochemical processes, including nutrients turnover in the sediment and their fluxes at the water-sediment interface (e.g. [START_REF] Sundbäck | The response of shallow-water sediment communities to environmental changes[END_REF][START_REF] Sundbäck | Nitrogen in nontidal littoral sediments: role of microphytobenthos and denitrification[END_REF]. They can also stabilize sediment surface through mucilage production [START_REF] Sutherland | The effect of carbohydrate production by the diatom Nitzschia curvilineata on the erodibility of sediment[END_REF].

The presence and functioning of microalgae in marine sediments is controlled by various environmental factors (e.g. temperature, nutrients, tides, grazing) [START_REF] Barranguet | Factors controlling primary production and photosynthetic characteristics of intertidal microphytobenthos[END_REF]Consalvy et al., 2004 and references therein) with light availability being the major ecological variable affecting microphytobenthos biomass, species composition and photosynthetic pigments, as well as driving its photosynthetic reactions (e.g. [START_REF] Hartwig | Factors affecting respiration and photosynthesis by the benthic community of a subtidal siliceous sediment[END_REF][START_REF] Macintyre | Microphytobenthos: the ecological role of the "secret garden" of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production[END_REF][START_REF] Light | Photosynthetic characteristics of sub-tidal benthic microalgal populations from a temperate, shallow water marine ecosystem[END_REF][START_REF] Underwood | Patterns in microphytobenthic primary productivity: species-specific variation in migratory rhythms and photosynthetic efficiency in mixed-species biofilms[END_REF]. The irradiance must be strong enough to exceed respiration losses and enable net photosynthesis. However, light is quickly attenuated in the sediment, which depends on its granulometry. Consequently, in muddy sediments light is limited to the top approx. millimetre, whereas in sandy sediments it can penetrate deeper, down to a few millimetres [START_REF] Kühl | Light penetration and light intensity in sand marine sediments measured with irradiance and scalar irradiance fiber-optic microprobes[END_REF][START_REF] Macintyre | Microphytobenthos: the ecological role of the "secret garden" of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production[END_REF][START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF]. In addition, light intensity varies within an extremely wide range and it is the fastest changing factor [START_REF] Kühl | Light penetration and light intensity in sand marine sediments measured with irradiance and scalar irradiance fiber-optic microprobes[END_REF]. Therefore, it requires from microphytobenthos a complex adaptive response.

Photoacclimation, which is a dynamic process occurring on different spatio-temporal scales, allows algae to efficiently use available irradiance if limited, and protect themselves from excessive light [START_REF] Copertino | Photophysiology of a turf algal community: integrated responses to ambient light and standing biomass[END_REF]. Epipelic microalgae from intertidal mudflats developed not only physiological acclimation mechanisms but also, due to their motility, a behavioural photoacclimation strategy allowing them to position themselves in the sediment within an optimal light regime (e.g. [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Serôdio | Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass[END_REF]. Epipsammic microalgae have received relatively less attention in this aspect, having no or strongly limited ability to move they have to rely on their physiology when acclimating to changing light conditions. Moreover, photoacclimation at the community level may also depend on species composition, abundance and behaviour [START_REF] Underwood | Patterns in microphytobenthic primary productivity: species-specific variation in migratory rhythms and photosynthetic efficiency in mixed-species biofilms[END_REF]. Therefore, it has been assumed that epipelic and epipsammic assemblages exhibit different photo-regulatory and adaptive strategies [START_REF] Brotas | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF].

The main objective of this study was to assess and compare photosynthetic activity and photo-regulation mechanisms in two types of microphytobenthos assemblages: epipelon from an intertidal mudflat (Aiguillon Bay, the Atlantic, France) and epipsammon from non-tidal sandy coastal shallows (Puck Bay, the Baltic, Poland) across the seasons.

Materials and methods

Research areas, sampling and algal suspension preparation

Sampling was carried out in two different areas. The first location was Esnandes in Aiguillon Bay located on the west coast of France (the Atlantic, 47°00′ N, 1°05′ W). The sampling site consists of mud with a mean grain size ranging from 7 to 13 µm [START_REF] Lorin | Contribution à l'étude des transits sédimentaires dans la partie orientale duPertuis Breton et la baie de l'Aiguillon[END_REF][START_REF] Guarini | Benthic contribution to pelagic microalgal communities in two semi-enclosed, European-type littoral ecosystems (Marennes-Oléron Bay and Aiguillon Bay, France)[END_REF]. The second location was Puck Bay (the Baltic, 54°43′ N, 18°34′ E), which is a subregion of the western part of the Gulf of Gdańsk (Poland). The sampling site -Władysławowo -is located in the western-internal part of Puck Bay and the bottom sediments of this part of the bay are dominated by fine sands with a narrow zone of medium sands along the coast [START_REF] Jankowska | Zatoka Pucka[END_REF]. At the sampling site 69% particles were between 250 and 500 µm.

Sediment samples were taken during three seasons: spring, summer and autumn 2007. At the intertidal site, samples were collected in March (spring) and during two summer and two autumn months, June/July and November/December, respectively. At the non-tidal site, the sampling was carried out in May (spring), August (summer) and October (autumn). At both sites, sampling took place 3 to 5 times each season. During sampling days temperature and light measurements were carried out using Multiparameter WTW meter (WTW GmbH, Weilheim, Germany), and LI-189 and LI-1400 Datalogger (Li-Cor, Lincoln, Nebraska, USA), respectively. In Aiguillon Bay average sediment temperatures varied within the range of 12.6-16.0 °C in spring, 19.2-21.2 °C in summer and 4.1-11.0 °C in autumn. In Puck Bay, the sediment temperatures changed within similar ranges of 11. 9-13.5 °C in spring, 16.8-20.6 °C in summer and 9.6-11.6 °C in autumn. On the intertidal mudflat the maximum solar radiation input was up to approx. 1030, 2700, 560 µmol m -2 s -1 in spring, summer and autumn, respectively. Maximum light intensity at the sediment surface at the non-tidal site was lower and reached 516 µmol m -2 s -1 in spring, 1220 µmol m -2 s -1 in summer and 260 µmol m -2 s -1 in autumn. The measured temperatures and light values reflected their seasonal variation patterns.

In Aiguillon Bay samples were collected during low tide (scrapping the upper approx. 2 mm of the sediment randomly chosen area) as described in [START_REF] Herlory | Methodology of light response curves: application of chlorophyll fluorescence to microphytobenthic biofilms[END_REF]. To collect microphytobenthos samples the method based on the natural ability of microalgae to move was used [START_REF] Eaton | The estimation of numbers and pigment contents in epipelic algal populations[END_REF]. Sediment samples were placed in trays, covered with 2 nylon nets (100 µm mesh) and left till the beginning of the low tide the next day. When algae appeared on the net surface they were washed with filtered sea water of salinity 35. The method was chosen as it allows selecting motile cells actively forming biofilms at the mud surface [START_REF] Haubois | Spatio-temporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oléron Bay, France[END_REF]. In Puck Bay sediment cores (10 cm in diameter) were taken as presented in [START_REF] Dahl | Use of sand-living microalgal communities (epipsammon) in ecotoxicological testing[END_REF]. The top centimetre of the collected sediment cores was cut off and placed in filtered sea water of salinity 6.7. The sediment was then mixed and sonicated for 5 min to detach and resuspend algal cells adhered to sediment particles. Finally, the algal suspension was separated from the sediment and filtered through planktonic net (100 µm) in order to remove as many small invertebrates as possible. Prepared suspensions (approx. 300 ml) were kept overnight and during the next day in a growth chamber at constant light and temperature conditions; low light intensity of approx. 50 µmol m -2 s -1 , 14:10 L:D photoperiod and temperatures corresponding to those observed in the environment, i.e. approx. 10 °C (autumn), 15 °C (spring) or 20 °C (summer).

Species composition

Species composition was analysed in replicated subsamples of prepared microalgal suspensions fixed with Lugol's solution. The material was studied under a Nikon 80i microscope using 40x objective in order to determine the relative abundance of cyanobacteria and microalgae by counting at least 300 cells. In order to perform a detailed analysis of diatom species composition, one of collected subsamples was treated with 10% hydrochloric acid, then rinsed a few times with distilled water, and then treated with hydrogen peroxide at 30-90 °C for 6-9 h. The diatom sample was mounted in Naphrax [START_REF] Battarbee | Diatom analysis[END_REF] and analysed with the same microscope under 100x oil immersion objective, counting at least 300 frustules. The biovolume of observed species was calculated according to [START_REF] Olenina | Biovolumes and size-classes of phytoplankton in the Baltic Sea[END_REF]. The biomass (wet weight) of each taxonomic group was derived based on an assumption of a plasma density of 1 g cm -3 across all taxa (HELCOM, 2013). elsevier_YECSS_4596

Photosynthetic pigment analyses

Five ml aliquots of microphytobenthos suspension were taken and filtered through GF/C Whatman glass filters (25 mm diameter) under low vacuum then frozen and stored at -20 °C until further processing. Pigments were extracted with 4 ml of cold 90% acetone (HPLC grade) at -20 °C for 4 h [START_REF] Strickland | A Practical Handbook of Seawater Analysis[END_REF]. After this time, extracts were centrifuged (7000 rpm, 10 min) and filtered through PTFE filter. Prepared extracts were analysed with KONTRON liquid chromatograph comprising triple system pump Kontron 422, Diode Array Detector 440 (set at 440 nm) and fluorescence detector SFM 25 KONTRON Analytical (Ex. 430 nm; Em. 670 nm). Pigment separation was performed using reverse phase chromatography (RP-HPLC) (250 mm × 4.6 mm Allsphere

[ALLTECH] column packed with Spherisorb ODS2 5 µm particle size) following optimized analytical gradient protocol provided by [START_REF] Wright | Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton[END_REF]. The HPLC system was calibrated using high purity pigment standards purchased from The International Agency for 14 C Determination DHI Institute for Water and Environment in Denmark. Pigments were identified from their absorbance spectra and retention times, and quantified following the procedure described by [START_REF] Mantoura | Calibration methods for HPLC[END_REF].

Photosynthesis-irradiance (P-E) curves

Photosynthesis and respiration rates were determined using the micro-respirometric technique described by [START_REF] Zurzycki | Volumetric methods[END_REF] suitable to obtain reliable results from small biomass sample. The measurements were based on the volumetric principle, i.e. a change in gas volume under constant pressure.

The main part of the apparatus was a plate with micro-chambers (Fig. 1.), each connected to capillaries, which was placed inside a hermetically sealed compensation chamber. All measurements were carried out under the assumption that the plate with research material was kept at a constant temperature completely isolated from the external atmospheric conditions. In this study a plate with 5 micro-chambers was used. Four of them were employed for the gas exchange measurements and the last one was designed as a thermobarometer (the micro-chamber did not contain research material) which was used to eliminate all the possible fluctuations of temperature and pressure within the measuring system. The lighting system consisted of an OSRAM tungsten halogen lamp 20 W/8 V with dichroic reflector emitting white light, a condenser, collector lenses, iris diaphragm and water filter. The light intensity was measured separately for each micro-chamber using Fiber Quantum Sensor of DIVING-PAM (Walz, Effeltrich, Germany). The maximum irradiance level was 1330 µmol m -2 s -1 . A set of neutral filters and diaphragms was used to achieve eleven light levels. A stable temperature was assured by connecting the micro-respirometer to a circulating water bath [START_REF] Zurzycki | Volumetric methods[END_REF]. Gas exchange measurements were performed at temperatures of 20 °C, 15 °C and 10 °C for summer, spring and autumn benthic communities, respectively.

Three to five ml of microphytobenthos suspension aliquots were filtered onto a GF/C Whatman glass filter. After that, small pieces of filters were cut out and placed in micro-chambers moistened with the filtered sea water. The micro-chambers were then covered with a cover glass with a hanging drop of 0.2 M carbonate buffer and sealed with vaseline. The carbonate buffer served as the source of CO 2 for photosynthesis and being maintained at constant level in the chambers enabled gas volume change by oxygen evolution only. The respiration and photosynthesis rates were estimated by measuring the volume of produced or used oxygen in dark and each light level. The changes in oxygen volume were monitored through the movement of kerosene bars coloured with Sudan III and placed in capillaries. During the measurement, in darkness and at each light level, the samples were left for 10 min to adapt to measurement conditions. After the adaptation period, the readings of kerosene bars movement were taken at equal time intervals for the next 10 min (every 5 min) and 9 min (every 3 min) for respiration and photosynthesis, respectively. The final data were calculated according to the equation of [START_REF] Cunningham | A new form of differential micro-respirometer[END_REF] simplified by [START_REF] Zurzycki | Volumetric methods[END_REF], ΔV = F(ΔA-ΔBI), where: ΔV is the volume of the produced gas, F is the active capillary cross section, ΔA and ΔB are the displacements of the kerosene bars inside the measuring capillary and thermobarometer, respectively, and coefficient I is the correction for temperature and pressure changes. The obtained volume readings were converted to mass units using ρ values provided by Šesták et al. (1971): ρ = 1.331 kg m -3 for summer measurements at 20 °C, ρ = 1.355 kg m -3 for spring measurements at 15 °C and ρ = 1.378 kg m -3 for autumn measurements at 10 °C. Calculated photosynthesis and respiration rates were then normalized per total chlorophyll a (Chl a) concentration.

To quantitatively compare the P-E curves inferred from gas exchange measurements empirical data were mathematically fitted to the model of [START_REF] Platt | Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton[END_REF] and photosynthetic parameters, i.e. the photosynthetic capacity (P m ), the maximum light utilization coefficient (α), the light saturation index (E k ), the light saturation point (E m ) and the photoinhibition parameter (β) were estimated [START_REF] Sakshaug | Parameters of photosynthesis: definitions, theory and interpretation of results[END_REF]. modified from [START_REF] Zurzycki | Volumetric methods[END_REF] summer elsevier_YECSS_4596

Statistical analyses

To compare the mean values of the analysed parameters analysis of variance (ANOVA) was used. If it was necessary, particular data sets were transformed (using natural logarithm). If the requirements of ANOVA were not met, non-parametric test, Kruskal-Wallis ANOVA by Ranks was used. Post-hoc comparisons were made with Tukey HSD test. To describe linear relationship between analysed variables, if present, the Pearson correlation coefficient was applied. All statistical analyses were performed using Statistica 10 (StatSoft Inc., USA).

Results

Microscopic observations

From all the samples collected at the intertidal site only 11 diatom species belonging to 6 genera were identified. Species richness varied within the range of 5-8 species per sample and during each season a different taxon dominated the assemblage (Fig. 2). During two seasons a strong domination of one species (over 85% of total cell count) was observed: Pleurosigma aestuarii (Brébisson ex Kützing) W. Smith and Navicula sp. in spring and summer, respectively. In autumn, 4 out of 9 identified species constituted 90.8% of all cells: Plagiotropis tayrecta T. B. B. Paddock (31.4%), Plagiotropis sp. (30.2%), Pleurosigma aestuarii (Brébisson ex Kützing) W. Smith (15.9%) and Gyrosigma sp. (13.3%). Other species were observed occasionally. Contrastingly, the nontidal epipsammic communities exhibited more diversity, with four taxonomic groups, including: cyanobacteria, diatoms, euglenophytes and green algae, being most frequently observed. During all three seasons diatoms dominated the microphythobenthos (Fig. 3.) and contributed to the total community biomass in 50, 51 and 59% in spring, summer and autumn, respectively. In the samples, numerous small-sized diatoms were observed and the total number of 63 species was identified. In summer 50 species were recorded, whereas in spring and autumn it was 33 and 38, respectively. Throughout the year five species were most frequently observed (>57% of cell count), i.e. Amphora coffeaeformis (C. Agardh) Kützing, Fragilaria sopotensis Witkowski & Lange-Bertalot, Nitzschia aurariae Cholnoky, Opephora mutabilis (Grunow) Sabbe & Wyverman and Planothidium delicatulum (Kützing) Round & L. Bukhtiyarova (Fig. 2). The second most common group in spring was cyanobacteria with 38% input to the microphytobenthos biomass, which later strongly decreased (Fig. 3). In summer, cyanobacteria were replaced by green algae (25% of biomass) and euglenophytes (12% of biomass). Subsequently, the biomass of green algae decreased (down to 10%) in autumn, while further increase of euglenophytes biomass, up to 17%, was observed (Fig. 3).

Pigment analyses

The analyses of pigments specific to the observed dominant taxonomic groups showed that there were statistically significant effects of both variables site and season (ANOVA, P < 0.05). Fucoxanthin (fuco) was the most abundant carotenoid for both epipelon and epipsammon (more than 70% and 50% of total amount of carotenoids identified in the microphytobenthos samples of the intertidal and non-tidal site, respectively). The comparison of the fuco/Chla ratio calculated for both sampling sites showed that it was always significantly higher for microphytobenthos from the intertidal site (ANOVA, P < 0.001). In the non-tidal microphytobenthos this ratio reached values an order of magnitude higher when compared to the ratios of other pigments (Fig. 4). Additionally, it was confirmed that fucoxanthin significantly correlated with the biomass of diatoms (r = 0.905, P < 0.001). For both studied assemblage types, the highest values of fuco/Chla were observed in autumn, although no clear seasonal pattern was found (Fig. 4A). The zeaxanthin/Chla (zea/Chla) ratio showed a steady increase throughout the year, also reaching the highest values in autumn (Fig 4B). The highest lutein/Chla (lut/Chla) and Chlb/Chla ratios were observed during the warmest period (Figs. 4C,D). The increased summer values of lut/Chla remained the same next season (Fig. 4C), whereas the Chlb/Chla ratio significantly declined (Fig. 4D). In addition, in autumn strong correlation between chlorophyll b and diatoxanthin was found (r = 0.947, P < 0.001).

Fig. 3

Seasonal changes in the contribution of algal groups to the total community biomass in microphytobehtos from the coastal shallows of the Puck Bay.

elsevier_YECSS_4596

The diatoxanthin/diadinoxanthin (Dt/Dd) ratio values changed across the seasons at both sampling sites revealing different variation patterns (Fig. 4B). In the intertidal assemblages the Dt/Dd ratio varied across the year within a very narrow range (min÷max = 0.03-0.17) with the highest values observed in summer (Fig. 5A). Mean Dt/Dd values for summer and autumn were significantly different (Tukey HSD test, P < 0.05). In contrast, in the non-tidal communities the ratio values were significantly higher (ANOVA, P < 0.001) and their large, but statistically insignificant, variations were observed (min÷max = 0.35-0.75; Tukey HSD test, P > 0.05; Fig. 5A). Identified carotenoids were separated into two groups, namely (i) photosynthetic (PSC; fucoxanthin, peridinin, prasinoxanthin) and (ii) photoprotective (PPC; alloxanthin, β-carotene, diadinoxanthin, diatoxanthin, lutein, neoxanthin, violaxanthin, zeaxanthin) carotenoids (following [START_REF] Stoń | Qualitative and quantitative analysis of Baltic phytoplankton pigments[END_REF], and subsequently their relationships were studied. In the epipelon, the PPC/PSC ratio decreased in summer, while in the epipsammon conversely, an increasing pattern was observed (Fig. 5B).

Moreover, there were no statistically significant differences between summer and autumn in the mean ratio values for both microphytobenthos types (Tukey HSD test, P > 0.05).

Photosynthetic parameters

The adjustment of the mathematical model [START_REF] Platt | Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton[END_REF] was always good and R 2 always exceeded the value of 0.9 (Fig. 6). The photosynthetic response of microphytobenthos varied significantly between seasons and sites (ANOVA, P < 0.05). In both assemblage types the highest P m values were observed in summer, whereas the lowest in autumn. There were no differences between microphytobenthos types within each particular season (Tukey HSD test, P > 0.05). The highest initial slope (α) was measured in spring and no statistically significant difference between intertidal and non-tidal communities was observed (Tukey HSD test, P > 0.05). In summer and autumn, however, α values measured for the non-tidal communities were statistically lower comparing to the corresponding values from the intertidal assemblages (Tukey HSD test, P < 0.001). There were no significant differences in summer and autumn α values in the epipelon (Tukey HSD test, P > 0.05, Fig. 7A), while in the epipsammon photosynthetic efficiency (α) showed a significant decrease over time (Tukey HSD test, P < 0.05, Fig. 7B). The saturation index (E k ) measured for the epipelon and epipsammon exhibited different seasonal variations (Fig. 7C). E k of the intertidal communities varied similarly to P m with the highest value during the warmest season. In the microphytobenthos from non-tidal site, a significantly lower mean E k was found in spring (Tukey HSD test, P < 0.05), and the parameter subsequently increased reaching its maximum in autumn. During summer and autumn, P m and α were changing concomitantly (y = 0.00459x + 0.00851, r = 0.814, P < 0.001), therefore no difference between summer and autumn mean E k values was observed. One of the most apparent differences between P-E curves obtained for algae from the two sampling sites was the fact that in the non-tidal assemblages there was no downturn in photosynthetic rates when exposed to high light (β = 0). Contrastingly, in the intertidal samples β was always present. The autumn β value was significantly higher compared to spring and summer (Kruskal-Wallis test, P < 0.05, Fig. 7D). For light saturation point (E m ), maximum values were calculated for summer (Kruskal-Wallis test, P < 0.05), whereas they were lower for spring and autumn (Fig. 7E).

Discussion

Previous studies showed that microphytobenthos structure depends on the sediment type [START_REF] Round | The epipsammon a relatively unknown freshwater algal association[END_REF][START_REF] Round | A diatom assemblage living below the surface of intertidal sand flats[END_REF]. The species identified in the intertidal samples were typical of muddy sediments (e.g. [START_REF] Ribeiro | Taxonomic survey of the microphytobenthic communities of two Tagus estuary mudflats[END_REF][START_REF] Haubois | Spatio-temporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oléron Bay, France[END_REF]. The assemblages consisted of small and large taxa of diatoms, and across seasons a clear shift in dominance was observed. Navicula genus was the main component of the assemblage in summer, whereas larger diatoms dominated during cooler seasons. The biological activity of small and large diatoms is different, therefore they function within different ecological constraints [START_REF] Snoeijs | The importance of diatom cell size in community analysis[END_REF]. [START_REF] Haubois | Spatio-temporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oléron Bay, France[END_REF] suggested that the high abundance of small species may be due to the resuspension/deposition cycle and redistribution of microphytobenthos during the high tide, which causes losses of biomass and in turn promotes smaller fast-growing taxa. However, although diatoms' growth rates depend on cell size [START_REF] Williams | Division rates of salt marsh diatoms in relation to salinity and cell size[END_REF], it is true at optimal temperatures, while within a low temperature range diatoms of different sizes grow at a similar rate [START_REF] Admiraal | Influence of light and temperature on the growth rate of estuarine benthic diatoms in culture[END_REF]. This could explain the strong dominance of a single species during warmer months and more species comparably contributing to the assemblage structure in autumn and winter seasons. The proportion between small and large diatoms may strongly affect the overall ecophysiological properties of the community. Thus the observed seasonal variability in species dominance may, at least partially explain variations in the photoacclimation of an assemblage.

Diatoms dominated microphytobenthos in Puck Bay considering both the number of species and their biomass, in congruence with previous results (e.g. [START_REF] Witkowski | Mikrofitobentos[END_REF]. This was also reflected in the pigments as fuco/Chla ratio was the highest throughout the year. Microphytobenthos in non-tidal coastal shallows develops more complex structures lasting over longer periods of time as compared to the day-to-day formed biofilms on intertidal mudflats, leading to the higher diversity and evenness of species. Most of the species observed in the non-tidal microphytobenthos were smaller individuals (≤15 µm), which are typical of sandy sediments, as a limited colonization space and the environment dynamics favour the growth of small taxa [START_REF] De Jonge | The occurrence of "epipsammic" diatom populations: a result of interaction between physical sorting of sediments and certain properties of diatom species[END_REF][START_REF] Sundbäck | Effects of nutrient enrichment on microalgal community composition in a coastal shallow-water sediment system: an experimental study[END_REF]. The dominant species were generally the same as reported in other studies (e.g. [START_REF] Witkowski | Diatoms of the Puck Bay coastal shallows (Poland, Southern Baltic)[END_REF][START_REF] Zgrundo | Studies on the biodiversity of contemporary diatom assemblages in the Gulf of Gdańsk, Oceanol[END_REF]. Previous reports on microphytobenthos from the southern Baltic coast (e.g. [START_REF] Witkowski | Mikrofitobentos[END_REF][START_REF] Pliński | Microphytobenthos of the shallow littoral of the southern Baltic[END_REF] showed that cyanobacteria and other algal groups are most abundant in summer, while in other seasons their number is strongly reduced. The results obtained in this study differ from previous observations showing that the input of non-diatom taxa to the micorphytobethos biomass was constant throughout the year, although with a seasonally changing dominant groups. In comparison to the epipelon from intertidal mudflats, in the epipsammic assemblages E k was higher (77-198 µmol m -2 s -1 ), indicating high light acclimation. A significant change in E k was observed during the transition from spring to summer months. No difference was found between summer and autumn values. The lack of E k changes indicates that both contributing parameters i.e. P m and α (E k = P m /α; [START_REF] Talling | The phytoplankton population as a compound photosynthetic system[END_REF] covary (linear correlation, r = 0.814, P < 0.001). The interpretation of data obtained for the non-tidal assemblages is difficult as no single mechanism exclusively responsible for the 'E k -independent' variability was identified (see [START_REF] Behrenfeld | In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis[END_REF].

Photoinhibition is a time-dependent, dynamic process, related to the light dose received and it was previously described as the negative slope of the light response curve (β) at supersaturating irradiance (e.g. [START_REF] Platt | Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton[END_REF]. [START_REF] Henley | Measurement and interpretation of photosynthetic light-response curves in algae in the context of photoinhibition and diel changes[END_REF] claimed that β indicates photoinhibition only during the incubation period and thus it does not reflect true photoinhibition that microalgae experience in their environment. In addition, a decrease in photosynthetic rates under high light may also result from a reversible down-regulation (e.g. xanthophyll cycle; [START_REF] Serôdio | Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos[END_REF]. The decrease in photosynthetic rates was only observed in the epipelic assemblages. In autumn β increased, and light saturation point (E m = 2.8E k ) decreased significantly (Kruskal-Wallis test, P < 0.05, when E m was expressed in terms of E k ) as compared to values observed in spring and summer (approx. E m = 3.7E k ). Several studies have shown that at low temperature plants may increase the level of photoprotective pigments which makes them more resistant to photoinhibition (e.g. [START_REF] Maxwell | Photosystem II excitation pressure and development of resistance to photoinhibition[END_REF]. The same phenomenon was observed in the diatom Fistulifera saprophila (Biskup, unpublished data). A decreased pool of xanthophylls in the epipelon observed in this study (Figs. 5A,B) could have limited its photoprotective potential under lower temperatures leading to a photoinhibitory decrease in photosynthetic rates rather than photoprotective down-regulation.

Laboratory experiments have shown that epipelic diatoms may experience photoinhibition when illuminated with high light for a longer time (t > 90 min; [START_REF] Blanchard | Characterizing and quantifying photoinhibition in intertidal microphytobenthos[END_REF], although it was rarely or never detected in the field (e.g. [START_REF] Blanchard | Photosynthetic response of natural assemblages of marine benthic microalgae to short-and long-term variations of incident irradiance in Baffin Bay, Texas[END_REF][START_REF] Dodds | Photosynthesis-irradiance patterns in benthic microalgae: variations as a function of assemblage thickness and community structure[END_REF]. This was explained by epipelon photoprotective migratory behaviour and dense biofilm formation leading to self-shading, which limits damaging light exposure [START_REF] Blanchard | Characterizing and quantifying photoinhibition in intertidal microphytobenthos[END_REF]. Recent study of [START_REF] Serôdio | Photosynthetic activity, photoprotection and photoinhibition in intertidal microphytobenthos as studied in situ using variable chlorophyll fluorescence[END_REF], however, revealed that microphytobenthos may experience photoinhibiton in its natural environment, and photoprotective mechanisms, both behavioural and physiological, may not be sufficient to prevent it. Whether it is due to the altered physiological state of cells, or structural changes within the biofilm it is still not known.

Conclusions

This study presents structure and photoacclimation mechanisms in two microphytobenthos communities from two different marine ecosystems, i.e., intertidal mudflats (Aiguillon Bay) and non-tidal sandy coastal shallows (Puck Bay).

Photosynthetic activity of both microphytobenthos types changed seasonally indicating their good adjustment to varying environmental conditions. The composition and proportion of photosynthetic and photoprotective carotenoids confirmed low and high light acclimation in the epipelon and epipsammon, respectively. This was further supported by the variations observed in photosynthetic parameters estimated on P-E curves. The epipsammon turned out to be resistant to high irradiance, suggesting that physiological mechanisms were sufficient enough to prevent photoinhibition. In contrast, a downturn in photosynthetic rates was observed in the epipelon subjected to high light intensities revealing its susceptibility to higher irradiance. This fact reinforced the hypothesis that behavioral photoacclimation, accompanying physiological mechanisms, is an essential component of photosynthesis regulation in microphytobenthos from intertidal mudflats. Overall, the observed photosynthetic properties of studied microphytobenthos communities seem to be a resultant of autecological characteristics of species contributing to the assemblage and are governed by site specific environmental conditions.

Uncited reference [START_REF] Seems Behrenfeld | Compensatory changes in Photosystem II electron turnover rates protect photosynthesis from photoinhibition[END_REF][START_REF] Woelfel | Diatoms inhabiting a wind flat of the Baltic Sea: species diversity and seasonal succession[END_REF]

Fig. 1

 1 Fig.1Plate with micro-chamber system used in volumetric micro-respirometer; typical plateview from above (A), cross-section (B); brass plate (1), capillaries (2), plastic tubes (3), measurement capillaries (4), mounting base (5), combs (6);

Fig. 2

 2 Fig. 2 Dominant diatom species at the intertidal site: (A) Gyrosigma sp., (B) Navicula sp., (C) Plagiotropis tayrecta, (D) Plagiotropis sp., (E) Pleurosigma aestuarii, scale bar = 20 µm; and the non-tidal site: (F) Amphora coffeaeformis, (G) Fragilaria sopotensis, (H) Nitzschia aurariae, (I) Opephora mutabilis and (J) Planothidium delicatulum, scale bar = 10 µm.

  elsevier_YECSS_4596

Fig. 4

 4 Fig. 4 Seasonal changes in photosynthetic pigment ratios (n = 9-15, mean ± standard error). (A) fucoxanthin/chlorophyll a in microphytobenthos from the intertidal and non-tidal site; (B) lutein/chlorophyll a; (C) zeaxanthin/chlorophyll a; (D) chlorophyll b/chlorophyll a in microphytobenthos from the nontidal site.

Fig. 5 Fig. 6

 56 Fig. 5 Seasonal changes in photosynthetic and photoprotective pigments in microphytobenthos from the intertidal and non-tidal site (n = 9-15, mean ± standard error). (A) Diatoxanthin/diadinoxanthin ratio; (B) photoprotective/photosynthetic carotenoids ratio.

Fig. 7

 7 Fig. 7 Seasonal variations of photosynthetic parameters (n = 9-15, mean ± standard error). (A) The photosynthetic capacity (P m ); (B) the maximum light utilization coefficient (α); (C) the light saturation index (E k ); (D) photoinhibition parameter (β); (E) the light saturation point (E m ) in microphytobenthos from the intertidal and non-tidal site.

Acknowledgements

This study was supported by the French Ministry of Education and Research under the Eiffel Doctorat grant No 494 709 F and the Polish State Committee for Scientific Research under the grant KBN 0724/B/P01/2008/34. FFP thanks John Breslin for correcting English.

Diagnostic pigments confirmed the presence of particular taxonomic groups, however their concentration did not always correspond to algal groups' biomass, indicating that pigments had other sources or their concentrations were altered by photoacclimation. Strong variations in Chlb/Chla and increased values of lut/Chla suggested that those pigments came also from phanerogam debris as the sampling site is located nearby underwater meadows formed by Chara baltica

Bruzelius,

Myriophyllum spicatum L., Ruppia maritima L. and Zostera marina L., and is surrounded by Phragmites australis (Cav.) Trin. Moreover, a strong correlation between chlb and diatoxanthin in autumn pointed to euglenophytes as a significant source of the former.

Some authors speculated that microalgae from muddy sediments are usually acclimated to rather low light intensities, while the organisms of sandy sediments show features of high light acclimation [START_REF] Barranguet | Factors controlling primary production and photosynthetic characteristics of intertidal microphytobenthos[END_REF][START_REF] Barranguet | Estimating primary production rates from photosynthetic electron transport in estuarine microphytobenthos[END_REF]. This was later proved when [START_REF] Brotas | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF] analysed the pigment composition of benthic communities inhabiting different sediment types and showed that benthic microalgae from mud revealed higher ratios of fuco/Chlc and fuco/Chla and lower Dt/Dd ratios than those inhabiting sands. The same tendencies were recorded in the present study supporting the notion of the epipelon assemblages' preference for low light, and the non-tidal epipsammon for higher irradiances.

Algae acclimated to low light conditions maximise light absorption through the increase in the number or/and size of photosynthetic units and variations in the proportion of photosynthetic and photoprotective pigment content.

Conversely, under high light conditions algae limit light absorption and maximise the utilization of already absorbed photons by increasing the amount and activity of Calvin cycle enzymes [START_REF] Falkowski | Aquatic Photosynthesis[END_REF][START_REF] Copertino | Photophysiology of a turf algal community: integrated responses to ambient light and standing biomass[END_REF].

Photoacclimation is reflected in P-E curve variations commonly involving increased value of the initial slope (α) and decreased P m under low light leading to low E k values. An inverse response is observed under high light resulting in high E k .

Other responses cannot be excluded however, as the behaviour and therefore interpretation of P-E curves depend on the unit of biomass to which photosynthesis rates are normalised (Richardson et al., 1983 and references therein). In ecological studies, photosynthetic rates are usually expressed on a chlorophyll a basis as it is the most common measure of algal biomass [START_REF] Macintyre | Photoacclimation of photosynthesis-irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria[END_REF].

In the epipelon α was quite high, compared to the epipsammon, which is in agreement with the low light photoacclimation model, and varied within a quite limited range, only slightly declining over the year. The pigment analyses revealed that the PPC/PSC ratio was very low in summer and autumn indicating an increased amount of photosynthetic carotenoids. Seasonal decrease in α coupled with the increase in PSC photosynthetic carotenoids/chlorophyll a (PSC/Chl a ratio; data not shown) is still consistent with the shade acclimation as light absorption is a self-limiting process and cell heavy pigmentation leads to the self-shading of pigment molecules, a decrease in optical absorption cross-section and consequently less efficient light harvesting [START_REF] Dubinsky | Photoacclimation processes in phytoplankton: mechanisms, consequences, and applications[END_REF]. Photoacclimation in intertidal mudflats involves not only physiological adjustment of the photosynthetic apparatus but also behavioural photoacclimation [START_REF] Serôdio | Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass[END_REF][START_REF] Mouget | Migration or photoacclimation to prevent high irradiance and UV-B damage in marine microphytobenthic communities[END_REF] connected to the diatoms ability to move. [START_REF] Serôdio | Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass[END_REF] showed that the peak in biomass at the sediment surface was observed when light intensity was within the rage of 100-250 µmol m -2 s -1 and downward migration started at higher light intensities. Microalgae by moving position themselves within the most optimal light regime in the sediment [START_REF] Blanchard | Photosynthetic characteristic of microphytobenthos in Marennes-Oléron Bay, France: preliminary results[END_REF][START_REF] Underwood | Microalgal (microphytobenthic) biofilms in shallow coastal waters: how important are species?[END_REF]. Migratory behaviour in epipelon from Aiguillon Bay has already been well documented [START_REF] Herlory | Microstructure of microphytobenthic biofilm and its spatio-temporal dynamic in an intertidal mudflat (Aiguillon Bay, France)[END_REF], which together with present observations on limited α seasonal variations pointed to the vertical migration as its main photo-regulation mechanism, in agreement with Jesus et al.

(2009).

In the epipsammon an approx. 6-fold decrease in α was recorded across the seasons. This was accompanied by a substantial increase in the xanthophylls pool in relation to chlorophyll a. Elevated levels of PPC/PSC ratio indicated an increase in photoprotective carotenoids (including a 3.5-fold increase in zea/Chl a, Figs. 3B, and a 2-fold diatoxanthin + diadinoxanthin/chlorophyll a [Dt + Dd/Chl a], data not shown). Such pigments (e.g. zeaxanthin, diatoxanthin, β-caroten) do not transfer energy to the reaction centres, thus light absorption takes place with reduced efficiency [START_REF] Falkowski | Aquatic Photosynthesis[END_REF] which allows a safe use of high irradiance. This suggests that adaptations to seasonally changing light regime in epipsammon are based on the physiological adjustment of photosynthetic systems and, as opposed to epipelon, it lacks endogenous rhythms and does not display migratory behaviour [START_REF] Raven | Adaptation, acclimation and regulation in algal photosynthesis[END_REF][START_REF] Brotas | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF].

In both microphytobenthos types, the seasonal variations in P m followed the same pattern with maximum during the warmest summer months which also corresponded with the period of the highest light intensity. This unimodal seasonality is consistent with previous findings (e.g. [START_REF] Light | Photosynthetic characteristics of sub-tidal benthic microalgal populations from a temperate, shallow water marine ecosystem[END_REF]. Increased P m during summer months is in agreement with the photoacclimation theory indicating that microalgae maximize the use of absorbed light [START_REF] Copertino | Photophysiology of a turf algal community: integrated responses to ambient light and standing biomass[END_REF], which is possible due to the increased content of electron transport chain and activity of enzymes involved in carbon metabolism, mainly RUBISCO [START_REF] Davison | Environmental effects on algal photosynthesis: temperature[END_REF].

The relationship between α and P m is described by the E k parameter which does not depend on biomass [START_REF] Henley | Measurement and interpretation of photosynthetic light-response curves in algae in the context of photoinhibition and diel changes[END_REF], and thus seems to be more useful in describing the photoacclimation status of algae [START_REF] Falkowski | Aquatic Photosynthesis[END_REF][START_REF] Light | Photosynthetic characteristics of sub-tidal benthic microalgal populations from a temperate, shallow water marine ecosystem[END_REF].

In the epipelon, the observed E k values were quite low (37-87 µmol m -2 s -1 ), confirming the hypothesis about its shade acclimation. The previously reported unimodal seasonality of E k (e.g. [START_REF] Light | Photosynthetic characteristics of sub-tidal benthic microalgal populations from a temperate, shallow water marine ecosystem[END_REF] was also observed in this study, with values highest in summer and lowest in autumn. The optimisation of P m in relation to α suggests long-term photoacclimation allowing microalgae to use absorbed light efficiently throughout the year [START_REF] Blanchard | Photosynthetic characteristic of microphytobenthos in Marennes-Oléron Bay, France: preliminary results[END_REF][START_REF] Light | Photosynthetic characteristics of sub-tidal benthic microalgal populations from a temperate, shallow water marine ecosystem[END_REF]. In the epipelon, the photoacclimation process involved mainly changes in P m as quite limited variation in α was observed indicating an "E k -dependent" light-photosynthesis relationship [START_REF] Behrenfeld | In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis[END_REF]. The significant correlation between E k and P m (linear correlation, r = 0.811, P < 0.001), and the lack of the correlation between E k and α (linear correlation, r = -0.285, P > 0.05), implied that light intensity may not play such an important role in regulating productivity [START_REF] Guarini | Microphytobenthic potential productivity estimated in three tidal embayments of the San Francisco Bay: a comparative study[END_REF]. Relatively limited α variations in the intertidal epipelon allow the assumption that the overall primary productivity is controlled mainly by temperature.

Myriphyllyum

, or


Igor A Goncharenko

Marian Marciniak
email: m.marciniak@itl.waw.pl

Enhancing the circular waveguide microresonator temperature measurement sensitivity and accuracy by using liquid crystal gap filling

We consider the design methods and principles of operation of a fast-response integrated optical sensor for temperature measurement. The sensitive element consists of a microring resonator based on the double-slot waveguide made from Si 3 N 4 and coated on a SiO 2 buffer layer, which is disposed on a silicon substrate. The nematic 5CB liquid crystal (LC) fills the slots of the ring waveguide. The sensor uses only the optical radiation, and the electrical current is not supplied to the measured matter. That allows applying such sensor for measuring the temperature of highly inflammable liquids and gases. The use of the microresonators based on the double-slot waveguides considerably (more than one order of magnitude) increases the sensor sensitivity. The operating speed of the device is defined by the time of establishing the steady-state regime in the ring microresonator and equals to tens of nanoseconds. The measuring device can consist of a large number of such sensing elements that allows simultaneously controlling the temperature in different points of the fluid flow or the matter volume. The sensor has a micrometer size and can be produced by using routine integrated technologies.

Introduction

The wide use of automated control and executive systems, application of new technological processes, conversion to flexible manufacturing systems stimulate the development of measuring and sensor devices. Sensors also are important elements of fire alarm and disaster protection systems. In addition to high metrological characteristics sensors have to possess a high reliability, durability, stability, small size, mass and power consumption, compatibility with microelectronic information processing devices [START_REF] Garmash | Possibilities, tasks and trends of optical fiber measuring systems in modern instrument engineering[END_REF]. Fiber and integrated optical devices fulfill these requirements in a maximal degree [START_REF] Passaro | Guided-wave optical biosensors[END_REF].

Waveguide microring resonators are quite challenging candidates for measuiring the temperature variation with high sensitivity [START_REF] Goncharenko | A digital optical sensor for the remote monitoring of temperature[END_REF][START_REF] Lin | Simultaneous measurement of refractive index and temperature using a microring resonator[END_REF][START_REF] Xu | Ultra-sensitive chipbased photonic temperature sensor using ring resonator structures[END_REF][START_REF] Yu | Sensitive birefringent temperature sensor based on a waveguide ring resonator[END_REF][START_REF] Wu | The sensing characteristics of plasmonic waveguide with a ring resonator[END_REF][START_REF] Campanella | Localized strain sensing with fiber Bragg-grating ring cavities[END_REF]. In [START_REF] Goncharenko | A digital optical sensor for the remote monitoring of temperature[END_REF] the method of on-line monitoring the variation of the temperature of remote physical objects on the base of waveguide ring microresonators and its schematic structure is proposed. The temperature range measured by the proposed sensor can be as wide as 500 °С for resonators with radius 8 µm. In order to increase the reliability of data communications the sensor output optical signal is converted into digital form. In [START_REF] Lin | Simultaneous measurement of refractive index and temperature using a microring resonator[END_REF] an approach to the simultaneous measurement of refractive-index and temperature changes using liquid-core silica optical ring resonators is proposed and theoretically demonstrated. The silicon-based optical ring resonator devices that can resolve temperature differences of 1 mK using the traditional wavelength scanning methodology is presented in [START_REF] Xu | Ultra-sensitive chipbased photonic temperature sensor using ring resonator structures[END_REF]. Xuhui Yu et al. [START_REF] Yu | Sensitive birefringent temperature sensor based on a waveguide ring resonator[END_REF] demonstrate a sensitive birefringent thermometer based on a SiO 2 waveguide ring resonator. The precision of the sensor is improved by a factor of more than 3, compared to the two-beam polarization interferometer, benefiting from the enhanced birefringence effect from the resonances of two polarization modes in the waveguide ring resonator. A surface plasmon polaritons temperature sensor with temperature sensitivity of 1.36nm/°C is described in [START_REF] Wu | The sensing characteristics of plasmonic waveguide with a ring resonator[END_REF]. The sensor consists of two metal-insulator-metal waveguides coupled to each other by a ring resonator. The transmission properties are numerically simulated by finite element method. The sensing characteristics of such structure are systematically analyzed by investigating the transmission spectrum. The original device for strain sensing was proposed in [START_REF] Campanella | Localized strain sensing with fiber Bragg-grating ring cavities[END_REF]. The device constitutes an optical resonator formed by inserting a Fiber Bragg Grating in a closed fiber loop. This peculiar feature allows to perform cavity-enhanced, local strain measurements with a reduced sensitivity to environmental perturbations. Because both the strain and temperature variations result in the change of the grating period, this device also could be used for temparature measurements.

An interesting design for sensing purpose is the slot waveguide, which consists of two strips of a high-index material separated by a sub-wavelength low-index (slot) region [START_REF] Almeida | Guiding and confining light in void nanostructure[END_REF]. By using the electric field discontinuity at the interface between high-index-contrast materials, increased optical intensity concentration in the slot low-index region can be obtained [START_REF] Xu | Experimental demonstration of guiding and confining light in nanometer-size low-refractive-index material[END_REF]. The unique feature of a slot waveguide combined with a microring resonator has shown a great potential for sensing applications [START_REF] Barrios | Slot-waveguide biochemical sensor[END_REF][START_REF] Barrios | Label-free optical biosensing with slotwaveguides[END_REF]. The slot in such waveguide can be filled with a substance, which is sensitive to a defined parameter, thus increasing the sensor sensitivity [START_REF] Goncharenko | Optical temperature sensor on the base of slot waveguide with LC filling[END_REF]. In last years waveguides with double-slot have been proposed for sensing applications. It has been shown that microrings on the base of such waveguide can tolerate larger fabrication errors than single-slot ones [START_REF] Dell'olio | Optical sensing by optimized silicon slot waveguides[END_REF][START_REF] Kargar | Design and optimization of waveguide sensitivity in slot microring sensors[END_REF].

Refractive indices of a liquid crystal (LC) are fundamentally interesting and practically useful parameters. In addition to the molecular constituents and wavelength, the temperature is the most important factor affecting the LC refractive indices. As the operating temperature changes, the LC refractive indices change considerably [START_REF] Li | Extended Cauchy equations for the refractive indices of liquid crystals[END_REF][START_REF] Belyaev | Temperature investigation of dielectric characteristics of liquid crystal 5СВ in relaxation region[END_REF].

In the paper we consider the operation principle of an optical sensor for temperature measurement on the base of ring microresonator made of double-slot waveguides with LC filling and optimization of the sensor structure for the higher sensitivity and accuracy. Microresonator is characterized by a set of resonance wavelengths. An input signal on the wavelength coinciding with one of the resonance wavelengths couples into the ring waveguide and passes through it to the output. Any variation of the resonator optical length L⋅n eff , where L is the geometric resonator length, n eff is waveguide mode effective index, leads to shifting its resonance wavelength. As a result the intensity of the output signal on the carrier wavelength coinciding with I. Goncharenko, M. Marciniak : Enhancing the circular waveguide microresonator temperature measurement sensitivity and accuracy by using liquid crystal gap filling 1.3 the resonance wavelength of the unperturbed resonator changes. The operation principle of the sensor is based on changing the waveguide properties of the slot waveguide and consequently the resonance conditions of the microresonator in dependence on index of the LC filling the slot. We consider waveguides made from Si 3 N 4 and coated on a SiO 2 buffer layer, which is disposed on a silicon substrate. The use of Si 3 N 4 for the slot-waveguide strips permits a larger slot region to facilitate LC filling of the slot volume [START_REF] Barrios | Slot-waveguide biochemical sensor[END_REF]. The nematic 5CB liquid crystal fills the slot of the ring waveguide. Microresonator radius is 32 or 16 µm and coupling coefficients between the straight and ring waveguides equal to 0.5. A time of establishing a steady-state condition in the microresonator with such parameters is about 50 ps [START_REF] Goncharenko | Optical broadband analog-digital conversion on the base of microring resonator[END_REF]. This time is one of the aspect that defines the operating speed of the sensor.

Structural diagram and principle of operation of the sensor

The total width of all dielectric strips in the double-slot waveguides is equal 1000 nm. The width of the left G l and right G r slots is varied from 50 to 150 nm. The sensitivity of the doubleslot waveguide sensors depends on a relative size of its parts [START_REF] Almeida | Guiding and confining light in void nanostructure[END_REF], thus we calculated double-slot waveguides with a different ratio between the widths of the side and middle strips. The width of the middle strip W m varies from 200 to 600 nm. Consequently, the width of the side strips W l and W r changes from 400 to 200 nm. The height of all strips is 300 nm. The optical radiation wavelength is 1550 nm.

As the radiation source a semiconductor laser can be optimally used, for instance, Mitsubishi Electric Corporation FU-68PDF-V510M61B 1.55 µm DFB-LD module with a polarization maintaining fiber pigtail. The laser output optical power equals to 15 mW and relative intensity noise is -155 dB/Hz. The optical radiation at the output of the ring resonator is measured by a photodetector, for instance, Hamamatsu Corporation G6742-003 InGaAs pin photodiode on a ceramic substrate with a 0.3mm active area [19]. The photodiode sensitivity on the peak sensitivity wavelength 1550 nm is equal to 0.95 A/W and the dark current is 1.5 nA.

Calculating the mode fields and optimizing the sensor design

We used the algorithm based on the Method of Lines [START_REF] Pregla | The method of lines for the analysis of dielectric waveguide bends[END_REF] modified for the structure under investigation for calculation of the mode field distribution and effective index n eff of the bent double-slot waveguides with LC filling.

The temperature variation of n e and n o LC indices can be expressed as [START_REF] Li | Extended Cauchy equations for the refractive indices of liquid crystals[END_REF]:

β β         - - ≈         - + ≈ c o i o c e i e T T G n T n T T G n T n 1 2 ) ( 1 ) ( (1)
where n i is the index of LC in isotropic state, T c is the clearing point (clearing temperature), β is a material parameter depending on the LC molecular structure, G e,o are proportionality constants for the corresponding waves. For 5CB LC these parameters are: n i = 1.5865, T c = 309.3 K, β = 0.1375, G e = 0.1914, G o = 0.1942 [START_REF] Li | Extended Cauchy equations for the refractive indices of liquid crystals[END_REF]. The imaginary part κ of the refractive index of 5CB is smaller than 0.02 and does not exhibit appreciable anisotropy [START_REF] Pan | Temperature-dependent optical constants and birefringence of nematic liquid crystal 5CB in the terahertz frequency range[END_REF]. The real and imagenery parts of the refractive index of Si 3 N 4 equal to 2.55 and 0.001, respectively. The same parameters for SiO 2 are 1.45 and less than 0.0004 at 20 °C. Thus, the calculated propagation loss in the doubleslot LC-filled ring waveguide with bend radius 32 µm is approximatelly 2.16 dB.

Figure 2 shows the transformation of transverse field distribution of the modes polarized along the substrate plane in bent double-slot waveguides with LC filling under changes of the slot separation W m . Width of the slots is equal to 100 nm. Bend radius is 32 µm. The transverse mode field distribution in bent double-slot waveguides with limiting large (600 nm, curve 3) and small (200 nm, curve 1) slot separations and specific intermediate (320 nm, curve 2) slot separation is shown in figure 3. It follows from the figures that mode field distribution in double-slot waveguide with large slot separation (W m > 400 nm, W l = W r < 300 nm) looks like a field of a conventional strip waveguide, because the mode is mostly concentrated in wide middle strip of the double-slot waveguide. At that the mode amplitude in the slot regions is small. The mode field distribution of the double-slot waveguide with a moderate specific slot separation (W m ≈ 320 nm, W l = W r ≈ 340 nm) looks similar to the supermode of three coupled waveguides, because the all three strips have approximately the same width. At that the mode amplitude in the slot regions is substantially high. For a small slot separation (W m < 300 nm, W l = W r > 350 nm) the mode field distribution corresponds to the field of two coupled waveguides but with substantial field amplitude in the slot regions. In this case the waveguide mode is concentrated in side strips, which are wider than the middle strip. The specific slot separation corresponds to the case when all three strips have approximately equal width. In order to check this assumption we have calculated the dependence of the mode field distribution on the slot separation in a double-slot waveguide with a constant width of the side strips. For W l = W r = 300 nm the specific slot separation equals approximately to 300 nm, whereas for W l = W r = 400 nm the specific slot separation is achieved with W m ≈ 400 nm.

Our calculation shows that the variation of the slot width doesn't influence substantially the mode field distribution in the waveguide regions outside the slots. The slot widening leads only to I. Goncharenko, M. Marciniak : Enhancing the circular waveguide microresonator temperature measurement sensitivity and accuracy by using liquid crystal gap filling 1.5

Journal of Electromagnetic Optics, v.2, no. 1 (2014) increasing the part of the optical power in the slot regions. Therefore, one can expect that waveguide with wider slots will be more sensitive to the temperature variation.

Figure 4 shows the optical signal power on the output of the ring resonator with bend radius 32 µm based on double-slot waveguides with a different slot separation. Dashed line 4 presents the optical power on the output of the ring microresonator with bend radius 16 µm based on doubleslot waveguide with W m = 320 nm. It follows from the figure that the ring resonator on the base of double-slot waveguides with the specific medium slot separation (W l = W r ≈ W m ) is the most sensitive to the temperature. At that, the ring resonator with a larger bend radius shows the higher sensitivity to the temperature variation. Using the data from the figure 4 we can estimate the sensor sensitivity to temperature variation. For the laser output optical power 15 mW, photodiode sensitivity 0.95 A/W, 3 dB input/output loss and propagation loss 2 dB, the sensitivity of the sensor based on the waveguide with bend radius 16 µm is equal to 0.14 mA/K in the temperature region from 250 to 270 K and it is 0.04 mA/K for the temperature ranging from 275 to 308 K. The device based on the resonator with radius of 32 µm has the sensitivity to temperature of the order of 0.34 mA/K in temperature range from 245 to 255 K, and only 0.01 mA/K for the temperature from 270 to 295 K.

The operating speed of the device is defined by the time of establishing the steady-state regime in the ring microresonator and the time of response of the sensing element on the temperature variation. The time of establishing the steady-state regime in the ring microresonators with bend radii 8-128 µm is of the order of tens picosecond [START_REF] Goncharenko | Optical broadband analog-digital conversion on the base of microring resonator[END_REF]. The response time of the sensor element τ = C/G, where С is the microresonator thermal capacity, G = kS r is the heat transfer between resonator and substrate, k is the heat transfer coefficient, S r is the area of contact between resonator and substrate [START_REF] Pilipovich | Array converter of infrared images into visible images, based on waveguide microcavities[END_REF]. Thermal capacity of Si 3 N 4 microresonator with bend radius 32 µm, waveguide width 1 µm and thickness 0.3 µm C ≈ 0.128⋅10 -9 Дж⋅K -1 . Heat transfer coefficient for silicon nitride k = 20 W⋅m -2 ⋅K -1 [START_REF] Bechtold | Efficient extraction of thin-film thermal parameters from numerical models via parametric model order reduction[END_REF]. Thus for microresonator with bend radius 32 µm and waveguide width 1 µm the heat transfer between resonator and substrate G is approximately equal to 4.02⋅10 -9 W K -1 . Therefore, the characteristic time defining the time of response of the device on the temperature variation τ is equal to 0.032 s.

The measurement range and sensitivity of the temperature sensor on the base of the slot and double-slot waveguide ring microresonator can also be changed by modifying the microresonator parameters, i.e. the ring radius, coupling coefficients, etc. [START_REF] Goncharenko | A digital optical sensor for the remote monitoring of temperature[END_REF][START_REF] Goncharenko | Liquid concentration sensor based on slot waveguide microresonators[END_REF].

Figure1Figure 1 .

 1 Figure1 shows the structure of double-slot waveguide ring microresonator. Ring microresonator

Figure 2 .

 2 Figure 2. The dependence of the transverse mode field distribution in the bent double-slot waveguides on the slot separation W m .

Figure 3 .

 3 Figure 3. The transverse mode field distribution in the bent double-slot waveguides with LC filling with different slot separation.

Figure 4 .

 4 Figure 4. The temperature dependence of optical signal power on the output of ring resonator based on the double-slot waveguides with the slot width G l = G r = 100 nm and slot separation W m equal to 200 (1), 320 (2,4) and 600 (3) nm. Waveguide bend radius is 32 (1-3) and 16 (4) µm.

Journal of Electromagnetic Optics, v.2, no.1 (2014)

measurement sensitivity and accuracy by using liquid crystal gap filling 1.6 Journal of Electromagnetic Optics, v.2, no.1 (2014)

Conclusion

We have investigated the structural diagram and the operation principles of an integrated optical temperature sensor. The sensor uses only the optical radiation, and the electrical current is not supplied to the measured matter. That allows to apply such sensor for measuring the temperature of highly inflammable liquids and gases. The use of the microresonators based on the double-slot waveguides considerably (more than one order of magnitude) increases the sensor sensitivity. The operating speed of the device equals to 0.03 s.

The mode field distribution in the double-slot waveguide with an LC filling with a small slot separation is similar to the fields of two coupled waveguides. However the amplitude value in the region of slots is sufficiently large. When the slot separation increases the mode field distribution fluently transforms first to the field of three coupled waveguides, and then converts into the fields of a conventional strip waveguide. It turns out that the largest field amplitude in the slot region corresponds to a moderate slot separation when all three strips of the double-slot waveguide have approximately equal width (the case of three coupled waveguides). As a result, such waveguide is found to be the most sensitive to the temperature changes.