Xiaoli Zhang

Hélène Agogué

Christine Dupuy

Dr Jun Gong
email: jgong@yic.ac.cn

Relative Abundance of Ammonia Oxidizers, Denitrifiers, and Anammox Bacteria in Sediments of Hypernutrified Estuarine Tidal Flats and in Relation to Environmental Conditions

Keywords: AMB, anammox bacteria, amoA, ammonia monooxygenase gene, AOA, ammonia-oxidizing archaea, AOB, ammonia-oxidizing bacteria, C/N, ratio of carbon to nitrogen, DIN, dissolved inorganic nitrogen, DO, dissolved oxygen, LZB, Laizhou Bay , cobalt, Cd, cadmium, Cr, chromium, Cu, copper, Ni, nickel, Pb, lead, Zn, zinc. Fe, iron Environment factors, heavy metals, nitrogen cycle, community size, abundance, gene copy number ratio

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Introduction

A tidal flat is a multifunctional ecosystem characterized by high primary production, intense nutrient remineralization and pollutant transportation and transformation in sediments. In estuarine tidal flats, microbial processes play an important role in self-purification by transformation, degradation, and alleviation of these pollutant and nutrient overloads [START_REF] Alvarez-Salgado | Nitrogen cycling in an estuarine upwelling system, the Ria de Arousa (NW Spain). I. Short-time-scale patterns of hydrodynamic and biogeochemical circulation[END_REF][START_REF] Spencer | Distribution and partitioning of heavy metals in estuarine sediment cores and implications for the use of sediment quality standards[END_REF]. Microorganism-mediated nitrogen cycling processes are of particular concern with respect to the ecosystem functions and services provided by estuaries [START_REF] Howarth | Nitrogen as the limiting nutrient for eutrophication in coastal marine ecosystems: Evolving views over three decades[END_REF] given that nitrogen limits primary oceanic production, and nitrogen overloading may be implicated in coastal eutrophication.

Nitrification, denitrification, and anaerobic ammonium oxidation (anammox) are tightly coupled nitrogen cycling processes in marine sediments, in which nearly 50% of marine nitrogen removal occurs [START_REF] Codispoti | The oceanic fixed nitrogen and nitrous oxide budgets: moving targets as we enter the Anthropocene?[END_REF].

Ammonia oxidation is the first and rate-limiting step of nitrification in which ammonia (NH 3 ) is oxidized to nitrite (NO 2 -) by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). The ammonia monooxygenase gene (amoA) has been used extensively as a molecular marker for studying both types of ammonia oxidizers in environmental samples [5, 15, 18-21, 33, 34, 37, 39, 40, 42-45]. Denitrification is an anaerobic reduction of nitrate, through nitrite and to nitrous oxide (N 2 O) and eventually dinitrogen (N 2 ). Two functionally similar but structurally different genes, nirK and nirS, encoding the copper (NirK) and cytochrome cd1-containing nitrite reductase (NirS), have not been found in the same organism and show different distributions in environments [START_REF] Zumft | Cell biology and molecular basis of denitrification[END_REF]. Both nirK and nirS genes are responsible for the rate-limiting step (NO 2 -to NO), and the nosZ gene codes for nitrous oxide reductase which catalyzes the reduction of N 2 O to N 2 , the final step of denitrification [START_REF] Francis | New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation[END_REF][START_REF] Zhou | Response of denitrification genes nirS, nirK, and nosZ to irrigation water quality in a Chinese agricultural soil[END_REF]. Like denitrification, anammox represents a net loss of nitrogen from the system by the production of N 2 via the reduction of NO 2 -coupled to the oxidation of ammonium (NH 4 + ). Specific primers targeting 16S rRNA genes have been used for detecting anammox bacteria in environments [START_REF] Francis | New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation[END_REF].

It has been shown that community size or abundance of a functional community involved in nitrogen cycling is frequently correlated with the process rate, which reflects the ecosystem functions provided by the community [START_REF] Hallin | Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment[END_REF][START_REF] García-Lledó | Genetic poential for N 2 O emission from the sediment of a free water surface constructed wetland[END_REF][START_REF] Petersen | Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska[END_REF]. For example, [START_REF] Hallin | Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment[END_REF] found that abundance, rather than composition, correlated with denitrification rates and nitrification rates by ammonia-oxidizing archaea in agricultural soils [START_REF] Hallin | Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment[END_REF]. Bacterial amoA gene abundance and ammonium content best explain potential nitrification rates in Alaskan soils [START_REF] Petersen | Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska[END_REF], whereas nosZ, nirS and nirK gene abundance and nitrate best explain potential denitrification rates in estuarine sediments [START_REF] Dong | Changes in benthic denitrification, nitrate ammonification, and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne Estuary, United Kingdom)[END_REF]. Denitrifying gene abundances can be used as proxies for predicting N 2 O emissions in soil and wetland environments [START_REF] García-Lledó | Genetic poential for N 2 O emission from the sediment of a free water surface constructed wetland[END_REF][START_REF] Morales | Bacterial gene abundances as indicators of greenhouse gas emission in soils[END_REF]. A positive correlation between anammox cell number and activities was reported in both water columns and sediments, the abundance of anammox bacteria therefore can be considered as a proxy for anammox activities in environments [START_REF] Kuypers | Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation[END_REF][START_REF] Lam | Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea[END_REF][START_REF] Schmid | Anaerobic ammonium-oxidizing bacteria in marine environments: widespread occurrence but low diversity[END_REF][START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF].

Earlier studies of marine sediments from a variety of estuarine locations have shown different patterns of AOA/AOB dominance [START_REF] Beman | Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahía del Tóbari, Mexico[END_REF][START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF][START_REF] Santoro | Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary[END_REF][START_REF] Abell | Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary[END_REF]. For denitrifying genes, nirS was more abundant than nirK in the San Francisco Bay estuary and subtropical Fitzroy estuary [START_REF] Abell | Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary[END_REF][START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF]. The contribution of anammox to N 2 production was estimated to be relatively low in coastal environments, and dissolved oxygen, nutrient load and salinity have been suggested as controls of the contribution of anammox to nitrogen removal [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF][START_REF] Teixeira | Potential rates and environmental controls of anaerobic ammonium oxidation in estuarine sediments[END_REF].

However, only a few studies have examined the abundances of ammonia oxidizers, different types of denitrifiers, and anammox bacteria in a single survey; and the genetic potential for N 2 O emissions and contribution of anammox to N 2 production and their spatiotemporal pattern have seldom been explored for estuarine tidal flats.

Recent studies have showed that heavy metal accumulation may be an important factor in regulation of nitrogen transformations, because some critical enzymes involved in the microbial nitrogen cycle are metalloenzymes [START_REF] Morel | The biogeochemical cycles of trace metals in the oceans[END_REF]. For example, the AMO coded by amoA gene is a Cu/Fe containing enzyme [START_REF] Yu | Elemental profiling of single bacterial cells as a function of copper exposure and growth phase[END_REF], and the NIR coded by nirK gene has been shown to contain Cu [START_REF] Nojiri | Structure and function of a hexameric copper-containing nitrite reductase[END_REF]. The effect of heavy metals on nitrogen cycling populations in sediments have not been studied sufficiently [START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF]. Moreover, most of the studies that have been performed have targeted only the top layer of sediments, and few have examined variability in N-cycling microbes' distributions in deeper layers. Surprisingly, AOB 16S rRNA genes had been detected at depths of up to 40 cm in sediments, and rates of nitrification down to 8 cm depth were also detectable, whereas the depth distribution of AOA in estuarine sediments is largely unknown [START_REF] Freitag | Community structure of ammonia-oxidizing bacteria within anoxic marine sediments[END_REF][START_REF] Mortimer | Anoxic nitrification in marine sediments[END_REF]. Recently, it was also demonstrated that the greatest potential for anammox was localized to the upper 2 cm and could be detected down to a depth of 4.5 cm in the sediments [START_REF] Rooks | The depth-specific significance and relative abundance of anaerobic ammonium-oxidizing bacteria in estuarine sediments (Medway Estuary, UK)[END_REF].

In the past decades, the Laizhou Bay (LZB), Bohai Sea, northern China has been hypernutrified due to huge interrestrial input (most commonly, dissolved inorganic nitrogen, DIN) from about 20 rivers of the coastal zone [START_REF] Zhang | Eutrophication status, mechanism and its coupling effect with algae blooming in Bohai[END_REF][START_REF] Luo | Distribution and pollution assessment of heavy metals in surface sediment in Laizhou Bay[END_REF]. In the meantime, in order to supply more tideland for economic developments (e.g. aquaculture, industry, harbor construction) in the coastal-line region of southern bank of LZB, tidal flat reclamation projects have been extensively planned and will be implemented in the near future. Thus, the environmental and ecological impacts of these reclamation projects are of great concern. In this study, we aimed to provide a baseline examination of ecosystem functions provided by nitrogen cycling microbes in sediments of LZB estuarine tidal flats, by using the gene abundance as indicators. For this purpose, abundances of N-cycling gene markers (amoA, nirS, nirK, nosZ and anammox 16S rRNA) for ammonia oxidization, denitrification, and anammox processes were determined by quantitative PCR (qPCR). Copy number ratios of related genes and their relationship with environmental factors were calculated in order to explore the environmental impact on dominance of functional phylotypes, genetic potentials for N 2 O emission, and contribution of anammox to N 2 production in coastal sediments. In addition, our study provides additional data for the links between microbial nitrogen cycle and trace metal levels.

Materials and Methods

Study Sites and Sampling

Estuarine tidal flats of Bailang River (BL), Di River (Di) and Jiaolai River (JL), which flow into the Laizhou Bay (LZB), Bohai Sea, were investigated (Fig. 1). These three threes have different pollution history. The Bailang River is 127 km long and drains a catchment of about 1,237 km 2 ; it used to receive a lot of sewage and industrial wastewater as it flowed through Weifang City. However, the execution of a remediation project has significantly improved water quality of this river since 2006. The estuarine area covering three sampling sites (BL1-3) has been enclosed for mariculture since May 2011 and this led to less tidal exchange and higher salinity due to evaporation especially in summer. The Jiaolai River is about 130 km long, with a catchment area of 5478 km 2 , receiving extensive agricultural and industrial discharges. The Di river is small sewage river (23 km long, catchment area 119 km 2 ), loaded with a large amount of wastewater from dyeing and subsurface brine industries, resulting in relatively high alkalinity and salinity.

In each tidal flat (location), three sites (JL1-3, BL1-3 and Di1-3) located in lower tidal area were sampled (Fig. 1). Sediment samples were collected in November 2010 (winter) and August 2011 (summer).

Sediment cores (7.5 cm internal diameter) were taken with a custom-made corer. The top/upper layer (0-2 cm) and the lower layer (2-5 cm) of sediments were sliced and put into sterile plastic bags. Samples were stored in an ice box (4°C) and immediately frozen at -80°C after transferred to the laboratory.

Analysis of Environmental Factors

Dissolved oxygen (DO), pH, salinity, and the temperature of overlying water were measured on site with a Hydrolab MS5 Water Quality probe (HACH, USA). Lyophilized sediments were leaching liquor with KCl

DNA Extraction and Quantitative PCR Assay

Total genomic DNA of the sediment samples was extracted using the UltraClean Soil DNA Isolation kit (MO-BIO, USA) according to the manufacturer's instructions. To examine the spatiotemporal variation of community size of N-cycling groups in estuarine sediments, the abundances of several genes (anammox bacterial 16S rRNA, AOB-amoA, AOA-amoA, nirS, nirK and nosZ) were quantified using quantitative PCR (qPCR). Primers used were as listed in Table 1. The PCR cycle started with 2 min at 50°C and 10 min at 95°C, followed by a total of 40 cycles of 30 s at 95°C, 40s at 57°C for nirS genes (55°C for AMB 16S rRNA gene, 58°C for AOB-amoA and AOA-amoA genes, and 51°C for nirK gene), and 40 s at 72°C.

Melt curves were obtained to check the specificity of amplification. The PCR amplification efficiencies were 85-113%, and correlation coefficients (R 2 ) for all assays were more than 0.99.

Statistical analysis

Student's t-test (two-tailed) was used to compare mean values of environmental variables as well as gene abundance between tidal flats, seasons, or layers. Spearman's correlation coefficient (ρ) between microbial abundances and environmental variables were calculated. All these analyses were performed using the program SPSS 13.0 for windows (SPSS, Chicago, USA).

Results

Environmental Characteristics

Thirty-six sediment samples were taken from surface sediments of LZB also showed similar levels of metal concentrations, which were lower than other coastal area in China (e.g. Jiaozhou Bay, Hongkong coast) and oversea (e.g. Thermaikos Gulf, Greece) [START_REF] Luo | Distribution and pollution assessment of heavy metals in surface sediment in Laizhou Bay[END_REF].

Nevertheless, among the three tidal flats, JL generally had the most high levels of heavy metals. Significant differences in concentrations of Cd, Cu, Ni, Pb and Zn were only found between BL and Di (P<0.05, Fig. S1). No significant differences were detected for Cr or Co among all these three tidal flats (P>0.05). The average concentration of As was 2.84 mg/kg at BL, representing the highest among the three areas. Metal concentrations seemed higher in the summer than in the winter, but only As concentrations were significantly different between the two seasons (P<0.05, Fig. S1). In addition, there was no significant effect of sediment layer on heavy metal concentrations (Fig. S1).

Among the eight heavy metals determined, six metals (Co, Cr, Cu, Ni, Pb and Zn) were collinear (ρ>0.64, P<0.05). In contrast, As and Cd did not follow the pattern of other metals, as significant correlations were only observed between As and temperature (ρ=0.74, P<0.01), NH 4 -N (ρ=0.61, P<0.05)

and C/N (ρ=0.66, P<0.05), and between Cd and nitrite (ρ=-0.73, P<0.01) and pH (ρ=-0.76, P<0.01).

Abundances and Ratios of N-cycling Gene Markers

Comparison of gene abundances is shown in Fig. 2 (for raw data of copy numbers are Table S1). The copy numbers of anammox bacterial 16S rRNA and N-cycling functional genes were determined for the 36 sediment samples. AOA-amoA genes varied between 1.0 × 10 3 and 3.3×10 5 copies g -1 sediment (Fig. 2A-C; Table S1), which was between 0.5 to 26.6 times the level of AOB-amoA found in the samples (Table 2).

Compared with AOB-amoA, AOA-amoA appeared to be more abundant across all the samples (P<0.05, n = 36) (Fig. 2A-C). Among the denitrifying genes, nirK appeared to be the most abundant, with copy numbers ranging from 2.7 × 10 3 to 1.6 × 10 7 g -1 sediment (Fig. 2D-F). The mean abundance of nirS was about half of nirK but 1.0 to 217.7 fold as much as that of nosZ genes (Table 2). The ratio between nitrite and nitrous oxide reductases (nirK+nirS)/nosZ varied greatly from 1.3 to 473.0. The abundance of AMB 16S rRNA gene ranged from 2.2 × 10 3 to 1.3 × 10 3 copies g -1 sediment (Fig. 2A-C), with a mean value approaching to that of nosZ genes, whereas their ratios varied greatly from 0.1 to 11.6 (Table 2).

Correlation Analysis

In order to explore the relationship between gene abundances and environmental variables, Spearman's correlations were performed (Table 3). Because the six heavy metals (Co, Cr, Cu, Ni, Pb, and Zn) were collinear, only Cu was considered as a representative here. For all gene abundances examined, most were negatively correlated with temperature (ρ=-0.75 to -0.91, P<0.01), except for AOA amoA and AMB 16S rRNA genes which showed no significant correlations (P>0.05). AOA also showed no correlation with other environmental variables, whereas AOB amoA was negatively correlated with the metal As (ρ=-0.62, P<0.05). Among the denitrifying genes and their combination examined, only nosZ was significantly correlated with Cd (ρ=0.63, P<0.05) and NO 2 -N (ρ=-0.74, P<0.01). Notably, significant correlations between abundances of AMB 16S rRNA genes and Cu (ρ=0.82, P<0.01), nitrite, pH and salinity (ρ=-0.64 to -0.79, P<0.05) were observed (Table 3).

The correlations between ratios of gene copy numbers and environmental variables may give a clue to understanding the regulating mechanisms for genes of identical functions. The AOA/AOB amoA copy number ratios were significantly correlated with pH (ρ=-0. 3).

Correlations between different genes in samples cross locations and seasons were analyzed (Table 4).

When all 36 sediment samples from both the upper and lower layers were considered, there was no correlation between AOA and AOB amoA genes; whereas nirS and nirK abundances were significantly correlated (ρ=0.93, P<0.01). Interestingly, all the three denitrifying genes were positively correlated with AOB amoA gene (ρ=0.42-0.63, P<0.05), whereas only nirS and AMB 16S rRNA genes were positively correlated with AOA amoA (ρ=0.38, P<0.05). No correlation was observed between AMB and AOB. A similar pattern of these correlations was identified when only the upper layer samples were analyzed.

Nevertheless, significant correlations between nirK and AOA (ρ=0.59, P<0.05) were only obtained from the dataset for the upper layers; correlations between AOA and nosZ or AMB were observed from the dataset for both layer samples, but not the upper layer samples (Table 4).

Discussion

Ammonia Oxidizers

In this study, both ammonia oxidizers had significantly higher abundance in winter than in summer (Fig. 2).

This seasonal change may be explained by strongly negative correlation between AOB and temperature (Table 3). However, because no significant correlations were found between AOA and the environmental factors measured in this study, factors contributing to seasonal changes of AOA in LZB estuaries remain unclear. This contrasts with previous studies showing that both AOA and AOB abundance were negatively correlated with the salinity gradient ranging from 0.6 to 31 psu in the San Francisco Bay estuary [START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF], and that the highest AOA and AOB abundance were recorded at 20 psu when a similar salinity range was examined in the Plum Island Sound estuary [START_REF] Bernhard | Abundance of ammonia-oxidizing archaea and bacteria along an estuarine salinity gradient in relation to potential nitrification rates[END_REF]. This discrepancy may be due to the high salinity (36.3-50.3 psu) for most of our samples. Nevertheless, the high salinity explains that both AOB and AOA amoA gene copy numbers determined in this study were about two to three order of magnitude lower than these reported for other estuaries [START_REF] Jin | Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China[END_REF][START_REF] Dang | Diversity, abundance, and spatial distribution of sediment ammonia-oxidizing Betaproteobacteria in response to environmental gradients and coastal eutrophication in Jiaozhou Bay, China[END_REF][START_REF] Bernhard | Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system[END_REF][START_REF] Woebken | Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system[END_REF].

AOA outnumbered AOB in a majority of our samples, which is consistent with previous studies which showed that AOA usually dominated in more saline environments [START_REF] Jin | Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China[END_REF][START_REF] Caffrey | Ammonia oxidation and ammonia-oxidizing bacteria and archaea from estuaries with differing histories of hypoxia[END_REF][START_REF] Park | Comparative analysis of archaeal 16S rRNA and amoA genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments[END_REF][START_REF] Beman | Diversity of ammonia-oxidizing archaea and bacteria in the ediments of a hypernutrified subtropical estuary: Bahia del Tobari, Mexico[END_REF]. However, AOB dominated in samples from Di and JL with salinity around 50 psu, suggesting that the salinity is not the only determining variable in the LZB estuaries, other variables such as nitrite, pH, and C/N ratio may also impact the abundances of ammonia oxidizers [START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF][START_REF] Bernhard | Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system[END_REF][START_REF] Sahan | Diversity and spatio-temporal distribution of ammonia-oxidizing archaea and bacteria in sediments of the Westerschelde estuary[END_REF][START_REF] Nicol | Crenarchaeal community assembly and microdiversity in developing soils at two sites associated with deglaciation[END_REF]. Our further analysis indicated a negative correlation between AOA/AOB ratio and pH (Table 3), which is consistent with previous notion that AOA outnumbers AOB in soils with lower pH [START_REF] Prosser | Relative contributions of archaea and bacteria to aerobic ammonia oxidation in the environment[END_REF]. Furthermore, we found a negative correlation between As (0.84-3.91 mg/kg) and AOB abundance, and that the AOA/AOB ratio was also significantly positively correlated to both Cd (0.03-0.08 mg/kg) and Cu (1.32-11.06 mg/kg) in the LZB estuarine sediments (Table 3), contrasting to the study in the San Francisco Bay estuary, where Ni and Pd showed strong correlations with abundance of AOA [START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF]. Nevertheless, our results suggest different responses of AOA and AOB to heavy metal contaminated sediments, with AOB seemingly being more sensitive to As contamination. A similar pattern has been observed in highly contaminated soils (As, 10000 mg/kg; Pb, 200 mg/kg; Cu, 1600-2400 mg/kg) [START_REF] Li | Do ammonia-oxidizing archaea respond to soil Cu contamination similarly as ammonia-oxidizing bacteria?[END_REF][START_REF] Ollivier | Abundance and diversity of ammonia-oxidizing prokaryotes in the root-rhizosphere complex of Miscanthus x giganteus grown in heavy metal-contaminated soils[END_REF], where AOB was negatively affected whereas AOA seemed more tolerant.

Denitrifiers

Of the three types of denitrifiers examined, the nirK-type was more abundant than nirS-type and nosZ, especially in BL and JL, suggesting a dominant role of nirK-type denitrifiers in denitrification in the LZB estuaries. The lowest nirK-type abundances were recorded in summertime samples from Di where both the water salinity (50.3 psu) and pH (8.61) were the highest. The dominance of nirK-type and lowest abundance of the nosZ-type denitrifiers in LZB estuarine tidal flats are consistent with a study on sediment of a constructed wetland [START_REF] García-Lledó | Genetic poential for N 2 O emission from the sediment of a free water surface constructed wetland[END_REF], but contrast with several reports that nirS-type dominated in estuarine sediments [START_REF] Abell | Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary[END_REF][START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF][START_REF] Magalhães | Impact of trace metals on denitrification in estuarine sediments of the Douro River estuary, Portugal[END_REF]. This is consistent with the idea that nirS-and nirK-type denitrifiers have different habitat preferences, as suggested by several studies [START_REF] Hallin | Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment[END_REF][START_REF] Junier | Habitat partitioning of denitrifying bacterial communities carrying nirS or nirK genes in the stratified water column of Lake Kinneret, Israel[END_REF]. In sediments of the hypernutrified Colne estuary, the decrease of nirS gene abundance in the more marine sites might be related to nitrate and ammonia gradient [START_REF] Smith | Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA) and their transcripts in estuarine sediments[END_REF], whereas nirS was highest in the more marine sites and nirK abundance was highest in riverine sites [START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF].

In this study, however, the higher ratio of nirK/nirS was shown to be related to lower pH and NO x -N. The effects of these environmental variables on dominance of the two denitrifier types are thus confounding at present.

Trace metal availability may affect denitrification through the metalloenzymes and toxicity to benthic denitrifiers [START_REF] Morel | The biogeochemical cycles of trace metals in the oceans[END_REF], and more generally, negative relationships between metals and the activity or the abundance of denitrifiers are found. For example, nirK was negatively correlated with Ag and methyl-mercury in the San Francisco Bay estuary [START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF]; both the abundance of nosZ gene and the denitrification rates in sediment decreased by the amendment of Cu (up to 79 mg/kg of wet sediment) in the Douro River estuary [START_REF] Magalhães | Impact of trace metals on denitrification in estuarine sediments of the Douro River estuary, Portugal[END_REF][START_REF] Magalhães | Impact of copper on the diversity, abundance and transcription of nitrite and nitrous oxide reductase genes in an urban European estuary[END_REF];

lowest Cu concentrations (0 -0.277 mg/kg of dry sediment) could yield a drastic decrease in the abundance of nirK, nirS and nosZ genes in salt marsh sediments [START_REF] Cao | Microbial community composition and denitrifying enzyme activities in salt marsh sediments[END_REF]. Nevertheless, a positive correlation between nirS and Pb was also observed [START_REF] Mosier | Denitrifier abundance and activity across the San Francisco Bay estuary[END_REF]. In this study, we found a significantly positive correlation between Cd (0.03 -0.08 mg/kg) and nosZ and the ratio of nirK/nirS in these three tidal flats (Table 3). Such previous data along with ours, thus suggest that heavy metals, even presenting at relatively low levels, may be important factors influencing specific denitrifying populations or their relative abundance in estuarine sediments.

The high abundance of nirS and nirK genes compared with nosZ gene is an indicator of the genetic capacity of the system to potentially accumulate the N 2 O intermediary [START_REF] García-Lledó | Genetic poential for N 2 O emission from the sediment of a free water surface constructed wetland[END_REF]. Therefore, the value of nirS+nirK-nosZ or the ratio of (nirS+nirK)/nosZ could be used as predictors of the potential for N 2 O emission. The seasonal effect on nirS+nirK-nosZ was significant (t-test, P<0.01; Fig. 2D), predicting a

greater N 2 O emission potential in winter than in summer in the LZB estuarine tidal flats. This seasonal pattern of N 2 O emission has long been observed [START_REF] Kieskamp | Seasonal variation in denitrification rates and nitrous oxide fluxes in intertidal sediments of the western Wadden Sea[END_REF], although the N 2 O flux rate was not determined in this study. Nevertheless, no significant correlations were found between metals and nirS+nirK-nosZ or (nirS+nirK)/nosZ in our study, contrasting to a previous investigation showing that N 2 O production was stimulated with the progressive increase of metals (Cu, Zn, Cr and Cd) to estuarine sediments [START_REF] Magalhães | Impact of trace metals on denitrification in estuarine sediments of the Douro River estuary, Portugal[END_REF].

Relatively lower concentrations of heavy metals in the LZB estuarine tidal flats might explain this discrepancy.

Anammox bacteria

We found that AMB abundances were negatively correlated with salinity, pH and nitrite, and positively with Cu in the LZB estuarine sediments. This result is basically consistent with several previous studies which have showed that salinity [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF], and nitrite concentration are significantly correlated with abundance of anammox bacteria [START_REF] Dang | Environmental factors shape sediment anammox bacterial communities in hypernutrified Jiaozhou Bay, China[END_REF]. Both high salinity and nitrite toxicity can inhibit anammox bacteria, which is understandable as the higher salinity challenges microorganisms with higher osmotic pressure, and nitrite suppresses the anammox process at certain threshold concentrations [START_REF] Jin | The inhibition of the Anammox process: a review[END_REF]. The inhibition on physiology of AMB may eventually lead to population decreases due to grazing pressure in the benthic microbial food webs. As for the positive correlation between Cu and AMB abundance found in this study, we are unable to give a proper explanation, since few studies have been carried out on effect of heavy metals on AMB [START_REF] Jin | The inhibition of the Anammox process: a review[END_REF].

Nevertheless, it is noteworthy that a previous study on closed marine systems has demonstrated that the addition of trace metals (Fe, Mn, Cu, Zn and Mo) enhances the denitrification processes [START_REF] Labbé | Addition of trace metals increases denitrification rate in closed marine systems[END_REF], which could supply more NO 2 -as substrate for anammox.

To our knowledge, this study for the first time investigated the abundance ratio between a marker gene of AMB (i.e. specific 16S rRNA gene) and a nitrous oxide reductase gene (i.e. nosZ) in sediment samples, as a potential indicator of the relative contribution of anammox to N 2 production (%R amx ). The rationale is that both the AMB and the nosZ-type denitrifiers are responsible for N 2 production, the abundance of these marker genes may be proxy of the capability of their functions. Although the relative contributions of these two processes were not investigated using 15 N stable isotope labeling in this study, the variations of AMB/nosZ in relation to environmental factors might give a clue to further studies. Our correlation analysis showed that the ratio of AMB/nosZ was significantly and positively correlated with C/N ratio, temperature, concentrations of As and NH 4 -N in sediments, and negatively with dissolved oxygen in the overlying water (Table 3). In agreement, the %R amx was positively correlated with both the organic carbon content of the sediment and the concentration of NO 3 -in the overlying water from intertidal flats in southeast England [START_REF] Nicholls | Wide spread occurrence of the anammox reaction in estuarine sediments[END_REF].

In the Chesapeake Bay, the %R amx ranged from 0 to 22%, with the highest rate in the freshwater portion of the main stem of upper Chesapeake Bay, where water column NO 3 -concentrations are consistently high [START_REF] Rich | Anaerobic ammonium oxidation (anammox) in Chesapeake Bay sediments[END_REF].

Nevertheless, it has also been shown that higher bottom water oxygen concentrations inhibited dissimilatory nitrate reduction to ammonium (DNRA) and denitrification but stimulated both anammox activity and the %R amx [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF]. Therefore, the usefulness of the abundance ratio of AMB 16S rRNA/nosZ as an indicator of AMB's contribution to N 2 production is partly supported by these previous observations. Further studies are needed to reveal the relationships between this or alternative indicators (e.g. the ratio of anammox functional gene to nosZ copy numbers) and %R amx , which could benefit to large-scale investigations and our understanding on the spatiotemporal variations on the contribution of anammox to N 2 production in coastal ecosystems.

In summary, we have examined the spatiotemporal pattern of the abundance of N-cycling microbes in sediments of three hypernutrified LZB tidal flats where the heavy metal contamination were at relatively low levels. Overall, function-similar groups have showed niche specialization in the LZB estuarine sediments.

AOA are more abundant than AOB in most cases, and their relative abundance is correlated significantly with the concentration of heavy metals (e.g. Cd and Cu) and pH. For denitrifiers, nirK-type usually outnumbers nirS-type, and nosZ-type is the lowest. The variations in abundances of nirS and nirK show a similar pattern by correlating with temperature, whereas their copy number ratio is correlated with pH, nitrite, nitrate, and cadmium. The variation of the ratio of nirK and nirS abundance depends on nitrogen load and cadmium. Abundance of nosZ-type is correlated with not only temperature, but also nitrite and Cd in sediments. Anammox bacterial abundances are strongly correlated with salinity, pH, nitrite and Cu.

Furthermore, we have explored for the first time the associations between environmental factors and combinations or ratios of copy numbers of nitrogen cycling genes. For example, the abundance ratio of anammox bacterial 16S rRNA gene to denitrifying nosZ gene, which could be a potential indicator for the contribution of anammox to N 2 production, is correlated to temperature, dissolved oxygen in overlying water, and ratio carbon to nitrogen and concentrations of ammonium and As in sediments. For the two indicators of genetic potentials for N 2 O emission, (nirS+nirK-nosZ) and (nirS+nirK)/nosZ, the former is only correlated to the temperature, whereas the latter shows no correlations with any variables determined. Taken together, our study stresses that abundances of N-cycling functional groups respond differently to variations of environmental conditions, and multiple factors including heavy metals with relatively low concentrations may play a role in shaping nitrogen cycling processes in these estuarine tidal flats.

(

  2M), then nitrate (NO 3 --N), nitrite (NO 2 --N), and ammonium (NH 4 + -N) were measured with a nutrient AutoAnalyser (Seal, Germany). Total organic carbon and nitrogen contents of sediments were measured with a Vario Micro Cube Elemental Analyser (Elementar, Germany). A Marlvern Mastersizer 2000F granulometer (Malvern, England) was used for sediment grain size analysis. Sediments were treated with 1M HCl [32], and concentrations of eight heavy metals (As, Co, Cd, Cr, Cu, Ni, Pb and Zn) were determined with an ELAN DRC II plasma mass spectrometry (ICP-MS) (PerkinElmer, Hong Kong).

  Plasmids containing cloned gene PCR amplicons were extracted with the TIANpure Midi Plasmid Kit (Tiangen, Beijing, China) for use in standard curves: archaeal amoA clone AOALZB-1, bacterial amoA clone AOBLZB-8, anammox bacterial 16S rRNA clone AMX-LZB-11, nirS clone NIRSLZB-7, nirK clone NIRKLZB-3, and nosZ clone NOSZLZB-6. Concentrations of sediment and standard DNA were measured with a NanoDrop 2000C spectrophotometer (Therno, Wilmington, DE, USA). A serial tenfold dilutions (10 -2 to 10 -7 ) were used to obtain standard curves. All sample and standard reactions were performed in triplicate with an ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA), and an average value was calculated. The quantification was based on the fluorescent dye SYBR Green I. Each reaction was performed in a 25 μl volume containing 1 μl of DNA template, 0.5 μl of each primer (10 μM) and 12.5 μl of Maxima SYBR Green/ROX qPCR Master Mix 2x (Fermentas, USA).

Figure 1 .

 1 Figure 1. Location of the sampling sites in estuarine tidal flats of Laizhou Bay.

Figure 2 .

 2 Figure 2. Copy numbers of anammox bacterial 16S rRNA and AOB-amoA, AOA-amoA (A-C), nirS, nirK and nosZ genes (D-F) determined for sediment samples (g -1 wet wt) from Laizhou Bay estuaries. Standard errors of the mean are indicated. Different letters above the bars indicate significant differences (P<0.05) between sampling time or sampling layer or sampling site. Samples from all sites were subjected to seasonal or layer comparisons.

  [START_REF] Ollivier | Abundance and diversity of ammonia-oxidizing prokaryotes in the root-rhizosphere complex of Miscanthus x giganteus grown in heavy metal-contaminated soils[END_REF], P<0.05) and the metals Cd (ρ=0.62, P<0.05) and variables examined were not supported. AMB/nosZ, a potential indicator for the relative contribution of anammox and denitrification processes to N 2 production, was correlated with C/N (ρ=0.78, P<0.01), temperature (ρ=0.70, P<0.05), dissolved oxygen (ρ=-0.68, P<0.05) and NH 4 -N (ρ =0.59, P<0.05). In addition, a positive correlation was identified between AMB/nosZ and As (ρ=0.61,

and Cu (ρ=0.58, P<0.05). The nirK/nirS ratios showed positive correlation with Cd (ρ=0.87, P<0.01), and negative with NO 2 -N (ρ=-0.85, P<0.01), NO 3 -N (ρ=-0.58, P<0.05) as well as pH (ρ=-0.71, P<0.01).

However, correlations between other denitrifying gene ratios (i.e. nirS/nosZ, nirK/nosZ and (nirS+nirK)/nosZ) P<0.05) (Table

Table 1 .

 1 Primers for target genes used in this study.

	Target gene	Primer	Sequence (5′-3′)	Length of	Reference
				amplicon (bp)
	AOA-amoA	Arch-amoA-for CTGAYTGGGCYTGGACATC	256	[33]
		Arch-amoA-rev TTCTTCTTTGTTGCCCAGTA
	AOB-amoA	amoA1F	GGGGTTTCTACTGGTGGT	490	[34, 35]
		amoAr new	CCCCTCBGSAAAVCCTTCTTC
	nirS	Cd3aF	GTSAACGTSAAGGARACSGG	425	[36]
		R3cd	GASTTCGGRTGSGTCTTGA
	nirK	F1aCu	ATCATGGTSCTGCCGCG	472	[37]
		R3Cu	GCCTCGATCAGRTTGTGGTT
	nosZ	nosZ2F	CGCRACGGCAASAAGGTSMSSGT 267	[38]
		nosZ2R	CAKRTGCAKSGCRTGGCAGAA
	Anammox bacterial	AMX808F	ARCYGTAAACGATGGGCACTAA	232	[17]
	16S rRNA	AMX1040R	CAGCCATGCAACACCTGTRATA

Table 2 .

 2 Ratios or relative abundance between target genes involved in N-cycling of Laizhou Bay estuarine tidal flats*. AOB = bacterial amoA gene; AMB = anammox 16S rRNA gene; AOA = archaeal amoA gene; L = lower layer; U = upper layer.

				JL				BL			Di
		Winter	Summer	Winter	Summer	Winter	Summer
		U	L	U	L	U	L	U	L	U	L	U	L
	AOA/AOB	1.0	3.3	0.9	6.6	3.7	1.0	26.6	6.4	0.5	1.7	0.8 0.7
	nirK/nirS	2.6	2.2	1.7	4.4	1.8	2.0	2.3	0.2	1.2	0.7	0.2 0.2
	nirK/nosZ	13.4	12.3	21.2	16.2 121.7 16.1	47.2	16.7 255.3 66.5	0.2 0.3
	nirS/nosZ	5.2	5.6	12.5	3.7	67.6	8.2	20.9	68.0 217.7 90.6	1.0 1.4
	(nirK+nirS)/nosZ 18.6	17.9	33.7	19.9 189.3 24.3	68.1	84.8 473.0 157.1 1.3 1.6
	AMB/nosZ	0.1	0.1	4.3	4.1	1.7	0.4	7.3	11.6	2.9	1.2	0.1 2.2

*

Table 3 .

 3 Spearman's correlation coefficients (ρ) between environmental factors and gene abundance and ratio of target genes across seasons, locations and layers*. Only the significant correlations (P<0.05) are shown, and the highly significant correlations (P<0.01) are highlighted in bold.

		As	Cd	Cu	C/N	DO	Grain	NH4-N	NO2-N	NO3-N	pH	Salinity	Temp
	Abundance
	AOA	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	AOB	-0.62	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	-0.75
	nirS	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	-0.75
	nirK	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	-0.91
	nosZ	ns	0.63	ns	ns	ns	ns	ns	-0.74	ns	ns	ns	-0.76
	nirS+nirK-nosZ	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	-0.85
	AMB	ns	ns	0.82	ns	ns	ns	ns	-0.79	ns	-0.64	-0.78	ns
	Ratio
	AOA/AOB	ns	0.62	0.58	ns	ns	ns	ns	ns	ns	-0.52	ns	ns
	nirK/nirS	ns	0.87	ns	ns	ns	ns	ns	-0.85	-0.58	-0.71	ns	ns
	nirK/nosZ	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	nirS/nosZ	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	(nirS+nirK)/nosZ	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	AMB/nosZ	0.61	ns	ns	0.78	-0.68	ns	0.59	ns	ns	ns	ns	0.70

**AOA = archaeal amoA gene; AOB = bacterial amoA gene; AMB = anammox 16S rRNA gene; DO = dissolved oxygen; Grain = sediment grain size; ns = not significant; Temp = temperature.

Table 4 .

 4 Spearman's correlation between N-cycling gene abundances across season, site and layer (lower triangle) or upper layer only (upper triangle)*. *Only the significant correlations (P<0.05) are shown, and the highly significant correlations (P<0.01) are highlighted in bold. AOA=archaeal amoA gene; AOB=bacterial amoA gene; AMB=anammox bacterial 16S rRNA gene; ns = not significant.

		AOB	AOA	nirS	nirK	nosZ	AMB
	AOB	-	ns	0.80	0.63	ns	ns
	AOA	ns	-	0.51	0.59	ns	ns
	nirS	0.63	0.38	-	0.92	0.50	ns
	nirK	0.54	ns	0.93	-	0.67	ns
	nosZ	0.42	ns	0.67	0.78	-	0.49
	AMB	ns	0.34	ns	ns	0.35	-

*

Acknowledgments

This work was supported by the CAS Knowledge Innovation Project (No. KZCX2-YW-JC203), the Natural Science Foundation of China (No. 41206155), the CAS Scientific Innovation Program -Interdisciplinary Field, the One Hundred Talents Program of CAS, and International Partnership Program for Creative Research Teams. Thanks are due to Ms. Xiaohong Guo, Beibei Xing and Drs. Kexin Zhao and Xiaobo Liu for helps in sampling and laboratory assistance.

Conflict of Interest

The authors have declared no conflict of interest.

Supporting Information

Table S1. Copy numbers (mean ± standard error) of anammox bacterial 16S rRNA and AOB-amoA, AOA-amoA, nirS, nirK and nosZ genes determine for sediment samples (g -1 dry wt) from the estuarine tidal flats of Laizhou Bay*.

. Environmental parameters of the overlying water (A-B) and sediment samples; mean values of dissolved oxygen (DO, μM), pH, salinity (Sal, psu), temperature (T, °C), heavy metals (Cr, Co, Ni, Cu, Zn, Cd, Pb, As, mg/kg), nitrate (NO 3 -N, mg/kg), nitrite (NO 2 -N, mg/kg), ammonium (NH 4 -N, mg/kg), ratio of organic carbon to organic nitrogen (C/N), and sediment grain size (d0.5, μm). Significant differences were tested at P<0.05. Letters indicate significant difference. Error bars show standard deviation. Samples from all sites were subjected to seasonal or layer comparisons.


domain: phys chim

text:
Mohand Chalal

Françoise Ehrburger-Dolle

Isabelle Morfin

Françoise Bley

Maria-Rosa Aguilar De Armas

María-Luisa López Donaire

Julio San

Nimet Bölgen

Erhan Pişkin

Omar Ziane

Françoise Ehrburger-Dolle A

Maria- Rosa Aguilar De Armas

Roger Casalegno

SAXS investigation of the effect of temperature on the multiscale structure of a macroporous poly(N-isopropylacrylamide) gel

Keywords: thermosensitive cryogel, pNIPA, SAXS, volume phase transition, isoscattering point

Introduction

Poly(N-isopropylacrylamide) (pNIPAm) gel is a typical example of a temperature sensitive gel as it exhibits a volume phase transition at a critical temperature (Tc) of about 34 °C in aqueous media [1]. Below Tc, pNIPA hydrogels are swollen, hydrated, and hydrophilic. Above Tc, the gels shrink due to the distortion of the hydrophilic/hydrophobic balance in the network structure. The rate of  Corresponding author; e-mail: fehrburg@spectro.ujf-grenoble.fr response of pNIPA hydrogels is low due to the formation of a dense "skin layer" of the shrunken gel, which prevents the mass transport of water out of the shrinking gel [2]. One possibility to circumvent this problem inherent to macroscopic gels (or macrogels) is by decreasing the size of the gels to mesoscopic lengths. Thus a large amount of work dealing with synthesis and characterization of colloidal microgels has been published [3,[START_REF] Kausar | Microgels from Smart Polymers[END_REF]. An alternative way to accelerate the response of pNIPA gels consists in the synthesis of a macroporous gel [START_REF] Okay | Macroporous Hydrogels from Smart Polymers[END_REF]. The macropores serve as channels that facilitate convective transport of liquid released during the shrinkage of the gel constituting the thin macropore walls. An efficient way to obtain macroporous gels is the cryotropic gelation technique [START_REF] Lozinsky | [END_REF], implying polymerization at a sub-zero temperature, yielding cryogels. These highly porous polymeric materials exhibit a broad variety of porosities and morphologies [7][8][9][10][11] allowing the preparation of macroporous gels with properties tailored for a given application, particularly in the field of biochemistry or bioengineering (e.g., tissue engineering scaffolds [12]).

As information about the internal structure of the gels and its variation as a function of temperature is essential for using gels as functional materials, this field has been widely investigated since the last 30 years [START_REF] Arndt | Hydrogel Sensor and Actuators: Engineering and Technology[END_REF]. It appears that Small-Angle Scattering measurements [14] constitute experimental methods that are well adapted to investigate the multiscale structure of polymer gels.

The usual q-domain for Small Angle X-ray Scattering (SAXS) or Small Angle Neutron Scattering SANS (10 2 to 1 nm 1 ) allowing structural information between roughly 100 and 1 nm in the real space can be extended up to a few µm by Small Angle Light Scattering (SALS) for transparent gels.

The first extensive study of pNIPA gels (macrogels) near the volume phase transition by SANS was reported by Shibayama et al. in 1992 [15]. Since that time, several papers reporting SAXS or SANS studies of thermally induced structural modifications in pNIPA gels [16][17][18][19][20] have been published.

Because of their fast kinetic response, microgels [21][22] or core-shell colloidal particles [23][24] were also extensively investigated. The first paper devoted to the comparison of SAXS curves obtained for macrogels and cryogels appeared recently [25]. It was shown that combining SAXS and Wide-Angle X-ray Scattering (WAXS) by extending the q-domain generally investigated (0.05 -1 nm 1 ) up to larger q-values (12 nm 1 ) brings new information about the changes in the molecular arrangement below and above the transition temperature.

The aim of the present paper is to show the structural modifications occurring during the stepwise increase of temperature not only at the mesoscale but also, for the first time, at the molecular scale.

The choice of a macroporous gel for this study offers the possibility to investigate a thermosensitive gel having a relatively small thickness (a few micrometers) yet larger than microgels (a few hundred nanometers) but without the need for stabilization of colloidal particles. The macroscopic structure (macropore size distribution and wall thickness) of this cryogel and its change with temperature have been investigated previously by Two-Photon Fluorescence Microscopy [26]. The wall thickness (i.e., the thickness of the gel investigated here by SAXS) was shown to decrease from 122 µm at 23°C to 102 µm at 28°C and to 4 µm at 34°C. The present paper is devoted to the study of the internal structure of these walls, at the meso and nanoscale at different temperatures between 18 and 37°C. The observed features will be related to that occurring at the macroscopic scale.

Materials and experimental method

2.1. Cryogel sample preparation. The pNIPA cryogel was prepared in tubular-shaped glass molds (0.5 cm diameter) by free radical polymerization. The total monomer (NIPA) concentration within the medium (distilled water) was 6% wt/v. The amount of cross-linker, N,N′methylenebisacrylamide (MBAA), was 6.6 wt% of the total amount NIPA, corresponding to 1 M for 15 M of NIPA yielding a degree of cross-linking larger than for the pNIPA cryogel we have investigated previously (1/40 in ref. [25]) and for the gel investigated by Shibayama et al. [16].

Nitrogen was passed-through the solution for 15 min prior to polymerization. For reaction activation, N,N,N',N'-Tetramethylethylenediamine (TEMED) (1 wt%) was added and the solution was cooled in an ice bath for 5 min. Then the radical initiator, ammonium persulfate (APS) (1 wt%), was added and the reaction mixture was stirred during about 1 minute. 1 ml of the reaction mixture was injected into each glass mold. Extensive details of the cryogelation (or cryopolymerization) are given elsewhere [25]. The solution in the mold was frozen at -20ºC in about 1 h in order to attain the polymerization reaction conditions (-12°C) as fast as possible for avoiding polymerization to begin before of all components were frozen. Chemical polymerization starts at -20°C and completes during storage of the sample at -12°C for 16 hours. The sample was subsequently thawed at room temperature. The yield of the polymerization reactions generally exceeds 85%. The cryogel matrix in the glass mold was washed by passing distilled water to remove any possible unreacted monomers and other ingredients and dried in air until to reach a constant weight. Re-swelling of the dry cryogel in water occurs within a few minutes [11].

SAXS measurements. SAXS experiments were performed at the European Synchrotron

Radiation Facility (ESRF) a Grenoble, France on the French CRG beamline D2AM. The incident energy was set to 16 keV (  0.77 Å). The detector, an indirect illumination CCD camera (Princeton Instruments), with pixel size equal to 50 µm was located at distances of 2 and 0.3 m a http://www.esrf.eu/ from the sample. These configurations provided data for wave vectors q (  

           2 sin 4    q
) ranging between 3.510 2 and 12 nm -1 . The beam-stop was a small pillar (2 mm diameter lead wire). The swollen sample was placed in a stainless steel holder closed by two mica windows 1 mm apart. The sample holder was inserted in a small furnace allowing temperature to vary between 10 and 40°C with an accuracy better than 0.2 °C. Heating was provided by a Thermocoax wire coiled around the furnace concentrically to a cold water circuit. Temperature control was achieved by means of an Eurotherm controller and sample temperature was measured by a Pt resistance located close to the cryogel sample. SAXS measurements were performed at 9 temperatures (18, 21, 24, 26, 28, 30, 32, 34 and 37°C). The duration of each temperature plateau was 16 minutes. After about 8 minutes, the shape of the curves did not change anymore as equilibrium was reached. Temperature control and data acquisition were automated. The images were processed by means of the software bm2img available on the beamline b . All intensity curves were corrected by taking into account the flat field and the dark current. Scattering of the same thickness of pure water was subtracted from the total intensity.

Results and Discussion

3.1. Evolution of the SAXS curves with temperature. Figure 1 shows the double logarithmic plots of the SAXS intensity I(q) obtained for the cryogel at the different temperatures. The curve (arbitrarily shifted along the intensity axis) obtained for the dry cryogel is also plotted in Figure 1.

The comparison between the latter and the curves obtained for the swollen sample below (T=18°C)

and above (T=37°C) the transition temperature, reveals the same trends as previously reported for other series of cryogels [25]: a strong increase of the high-q correlation peak which appears as a bump at low temperature as in gels [19,25] and a significant decrease of the scattered intensity in the intermediate q domain above 30°C. For the dry sample, the intensity scattered in the low-q region is much larger than for the collapsed hydrogel. This feature reveals the macroscopic collapse after drying yielding a strong increase of the wall surface per unit volume. These questions will be discussed in details in the next paragraphs. The interesting feature shown in this figure is the existence of an isoscattering point more clearly seen in the insert. This point, located at q iso 3.633 nm -1 and vanishing above 30°C was never mentioned before as this q-domain was never investigated. Isoscattering points have been reported in several phase change processes driven by temperature as the smectic C to smectic A transition in liquid crystals [27], the isothermal crystallization of cocoa butter between 19 and 23°C [28] and more generally in microphase b http://www.esrf.fr/exp_facilities/BM2/BM2.html separation processes [29][30][31]. The origin of the isoscattering point observed here will be discussed in a next paragraph.

Scattering functions and fitting of the experimental curves.

In the q domain investigated by a large majority of researchers (q1 nm 1 ), small-angle scattering of neutrons (SANS) or X-rays (SAXS) by neutral gels is assumed to result from the sum of two contributions [15]:

-the scattering from density fluctuations which exist also in semi-dilute polymer solutions (liquidlike or solutionlike fluctuations) and which are described by the Ornstein-Zernike (OZ)

equation 2 2 1 ) 0 ( ) ( OZ OZ  q I q I   ( 1
)
where  is the correlation length;  represents the range of the spatial correlation of concentration fluctuations in the gel (polymer rich domains) that will be named T-blobs in the following as they depend on temperature; I OZ (0) is the asymptotic value of the O-Z intensity when q0

-the scattering from solidlike density fluctuation due to the gel cross-linking often described by a Guinier equation

          3 exp ) 0 ( ) ( 2 2 G G G q R I q I (2)
where R G is the Guinier radius of gyration related to the diameter a by

3 / 5 2 G R a 
for spherical objects and I G (0) is the asymptotic value of the Guinier intensity when q0.

In the present case, experiments were extended in the WAXS domain in order to investigate the chain-chain correlation peak recently revealed in cryogels and gels [25]. Without the support of a relevant model, such peaks can be fitted with a pseudo-Voigt equation [27] writing as

                             2 exp 1 1 ) ( 2 1 2 1 2 1 2 1 V   q q c q q c a q I ( 3
)
where a corresponds to the maximum of the peak located at q 1 (assumed to correspond to

1 / 2 q d  
in the real space), c is the fraction of the lorentzian contribution and  1 is related to the size of the organized domains.

Finally, because the gel objects investigated are the walls (12 µm thick at room temperature) of the macropores, a low-q intensity turn-up resulting from the scattering of the gel surface is expected and actually observed. Thus, the low-q part of the intensity curve I(q) will be described by the following power law

p q I q I   0 P P ) ( (4)
where the exponent p is related to the surface fractal dimension D s by p D s   6

and I P 0 is the prefactor.

It follows that the equation I fit (q) used for the fit of the experimental curve I(q) consists in the sum of four equations

) ( ) ( ) ( ) ( ) ( V OZ G P q I q I q I q I q I fit     (5)
involving 10 parameters in a non-linear regression. The regression procedure was achieved by means of the Marquardt-Levenberg algorithm provided by SigmaPlot 10.0. Because the data in I(q) extend over more than 4 decades, a weight w=1 [I(q)] 2 was used. The upper q limit for the regression was fixed at 6.2 nm 1 in order to avoid the contribution of expected scattering features occurring at higher q-values. Figure 2 shows, as an example, the result of the fit for the intensity curve measured at 28°C. The variation of the residuals as a function of q is also plotted (insert). As shown in the second insert, the residual distribution is gaussian and the peak is located very near zero (9.1110 5 ) and its half-width equals 0.01. A linear regression of the variation of the predicted versus measured intensity (not shown) yields a slope equal to 1.006 and extrapolates to 0.012 (r 2 0.9998). The same analysis of the fit results was performed for all experimental curves and yielded similar conclusions. The value of the parameters, the statistical errors and the dependencies obtained for the above fit are indicated in Table 1.

Figure 2. Fit of the experimental data obtained at 28°C with equation ( 5). The parameters extracted from the fit are given in Table 1. The upper insert shows the variation of the residuals with q. Their distribution is shown in the lower insert. The red line is the fit of the residuals with a Gaussian distribution (r 2 0.963). The position of the maximum is equal to 9.12e-5.

As expected in a regression with four equations and ten parameters, it is essential to avoid incorrect solutions resulting from a local minimum in the sum of squares. To this end, firstly, a very small tolerance value must be used: in the present case tolerance was set to 10 20 . Secondly, the initial parameters were modified until a solution yielding not only a good regression coefficient (r 2 close to 0.999) but also dependencies that are all strictly smaller than 1. It was shown, by changing the value of the initial parameters, that the solution with dependencies strictly smaller than 1 is unique.

Thus the fitting equation obtained by the non-linear regression can be considered as describing satisfactorily the experimental data and the analysis of the extracted parameters presented in the next paragraph will make sense.

  2 ) ( / 1 q I w 
was used and tolerance was set to 10 20 . The regression coefficient r 2 is equal to 0.9997.

For the pseudo-Voigt equation c equals 1. It means that the distribution is lorentzian. In fact, in most cases, c remains equal or close to unity. Thus this parameter will not be discussed in the following. Figure 3 shows logarithmic plots of the curves calculated with the parameters extracted from the fits at different temperatures. It appears that the contribution of the Ornstein-Zernike scattering extends over a very broad range of q values. This feature affects the contribution of all other regimes yielding a dependency larger for the OZ parameters than for the other ones and, therefore, a larger statistical error (see Table 1). This observation also explains the failure of attempts aiming to reduce the number of parameters by performing fits over a restricted range of q values (up to 1nm 1 , corresponding to the experimental conditions of ref. [15]). The evolution with temperature of the OZ and pseudo-Voigt calculated curves is shown on Figures 4a and4b. Below 32°C (Figure 4a), the intensity of the chain-chain correlation peak increases.

Above 32°C (Figure 4b), the OZ contribution drops significantly while the peak remains nearly unchanged and similar to that obtained for the dry cryogel as already observed previously in other cryogels [25]. Figure 4c shows the sum of the OZ and pseudo-Voigt curves calculated from the fit.

These curves display (see insert) the same isoscattering point as the experimental curves plotted in Figure 1. This result strengthens the validity of the fitting procedure and proves that the isoscattering point is related to the structure of the T-blobs. Actually, such a result is also expected from the curves plotted in Figure 3 which show that the power law and the Guinier scattering do not contribute anymore to the intensity in the isoscattering point region. 6) and (7). The dashed lines drawn through the points obtained at 18 and 21°C extrapolate to 13.3°C (arrow).

By means of swelling and calorimetry experiments, Li and Tanaka [32] have shown that pNIPA gels belong to the Ising universality class. In these conditions, one expects 0.631 and 1.238. These exponents are slightly larger than the ones involved in the mean field model (0.5 and 1 for  and  respectively). In any case, Equations ( 6) and (7) imply that  scales as [I OZ (0)]  with  close to 0.5. This remark allows the determination of the temperature domain in which the fit to Equations ( 6) and ( 7) is relevant. As shown in Figure 6, this scaling law is verified only for three temperatures (26, 28 and 30°C) yielding an exponent equal to 0.586 (may be four if the point at 24°C is taken into account) which is quite small. Nevertheless, a non-linear regression with Equations ( 6) and (7) performed over the three point yields (i), the same T c value (31.35 and 31.36 in Equations ( 6) and (7), respectively) and (ii), critical exponents 0.712 and 1.22. Including the point at 24°C for the non-linear regression yields a significant difference between the values of T c obtained from the fit by equation ( 6) (32.30°C) and equation ( 7 Further, the critical temperature (31.4°C) deduced from Differential Scanning Calorimetry (DSC) measurements (Figure 7) and resulting from the "melting" of the water cages around the isopropyl groups [33] agrees perfectly with that determined by the non-linear regression performed between 26 and 30°C. Thus, despite the fact that the number of data points is very low, the result agrees with an Ising model for describing the transition in the present pNIPA cryogel as it was for the gels investigated by Shibayama [15], yet the value of  is slightly larger than the theoretical one (0.631).

It could be observed in Figure 5 that the lines drawn through the points corresponding to 18 and 21°C for  and I OZ (0) extrapolate to the same temperature (13.3°C). Also, the slope of this line in the logarithmic plot of  versus I OZ (0) (Figure 6) is very close to 1. These features do not prove a linear increase of  and I OZ (0) in the low temperature domain but incites one to further investigate this question in the future.

Equation (1) indicates that the intensity I OZ (q) scale as q 2 for q1, in the asymptotic regime. In fact, this asymptotic regime is not visible because of the high-q extra scattering resulting from an inter-chain correlation revealed by the broad peak fitted with the pseudo-Voigt equation (Equation 3). The variation of the peak position q 1 and the peak height a as a function of temperature are plotted on Figure 8. Figure 8. Evolution of the position q 1 (left axis) and the intensity a (right axis) of the high-q peak resulting from the fit with the pseudo-Voigt equation (Equation 3). The solid lines result from a linear regression of the data (closed symbols).

The two other parameters obtained from the fit of the peak with the pseudo-Voigt equation, i.e., c, the fraction of the Lorentzian versus Gaussian distribution and  1 , related to the extent of the organized domains, are plotted as a function of temperature in Figure 9. It appears that the overall fit is not very sensitive to c, as also suggested by large statistical errors. Thus, the variation of c between 0.7 and 1 (pure Lorentzian distribution) with temperature may not be relevant. The same comment could apply to the variation of  1 with temperature yet the increase of  1 by a factor two may not be artifactual but this point will not be discussed further. In contrast with these two parameters, the peak position q 1 which can be easily checked without any fit and the peak height a deserve further comments. 3) with the blob size  (Equation 1).

Figure 8 shows that the peak height a increases linearly with temperature up to 24°C and remains nearly constant above 32°C. Between 24 and 32°C, the rate of increase becomes larger. It appears (Figure 10) that a scales as the blob size  with an exponent equal to 0.26. The physical meaning of the value of this exponent is not obvious. Meanwhile, the scaling law suggests that the increase of a corresponds to the increase of the number of scatterers resulting from the increase of the size of the T-blobs. Conversely, the peak position q 1 displays a weak monotonous linear increase (between 5.18 and 5.64 nm 1 ) with temperature up to 34°C. The values of q 1 are very similar to that obtained for the different cryogels investigated earlier [25] below and above the critical temperature but slightly larger than the value (5.7 nm 1 ) reported for a weakly cross-linked pNIPA gel at 40°C [19].

The increase of q 1 (Figure 8) corresponds, in the real space, to a decrease of the distance d between the dehydrated (i.e., without "water cages" around the isopropyl terminal groups in the side-chain) polymer chains. d can be estimated by means of the Bragg equation

1 / 2 q d  
from 1.21 to 1.12 nm. As discussed earlier [25], such lengths are less than or nearly twice that of the side chain. It follows that the peak may actually originate from a local packing by means of hydrophobic association of isopropyl groups of two neighbor chains and/or hydrogen bonds between amide groups [33]. Thus, it may be assumed that the scattering object from which this peak originates are the collapsed hydrophobic clusters (or nanopockets) revealed by Ahmed et al. [34] by means of UV Resonance Raman experiments, which are present in the T-blobs.

The weak variation of q 1 (or d) with temperature could be attributed to tiny changes in the conformation of the pNIPA chains resulting from the change of hydrogen bonding of water at the C=O or N-H sites. The strong increase of a (concordant with the increase of ) between 26 and 30°C could be related to the abrupt spectral change in the amide (AmI) bands observed between 30 and 40°C in the pNIPA nanoparticles investigated in ref. [34], for which the transition temperature is 33°C. Figures 5,6 and 8 show that collapsed clusters are present at lower temperatures, between 18 and 24°C. This feature is consistent with the ones reported in ref. [34]. It is also consistent with the conclusions drawn about the peak intensity in a gel as compared to the cryogel at room temperature [25]. Meanwhile, it is difficult to explain why the values of  obtained at 18°C (0.543 nm) and 21°C (0.886 nm) are smaller than the corresponding value of d (1.21 and 1.19, respectively) deduced from the peak position q 1 . For the dry sample, q 1 (=5.56 nm 1 ) is slightly smaller than in the fully collapsed cryogel at 37°C (q 1 =5.63 nm 1 ). Accordingly, the inter-chain distance would be slightly larger in the dry sample (1.13 or 1.03 nm) than in the collapsed aqueous cryogel (1.11 or 1.02 nm). The analysis of the UV Raman resonance spectra of the dry pNIPA led Ahmed et al. [34] to suggest the existence of interchain hydrogen bonding between pendent amides.

Conversely, in the collapsed hydrogel, amides seem to be hydrogen bonded to water and not directly to each others. At this point, and because of the very small differences in the values of d, it is difficult to go further into this discussion.

Finally, (i), the analysis of the OZ and pseudo-Voigt equation, (ii), of the corresponding parameters (I OZ (0),  and a), and (iii), their correlation render the origin of the isoscattering point, i.e., the existence of a point (q iso, I iso ) independent of T, more straightforward. In the q-range investigated (q2 nm 1 ), the variation of the scattered intensity I(q,T) is equal to I OZ (q,T)+I V (q,T). Both functions contain temperature dependent parameters for which the temperature dependence is described by equations that remain the same over a given range of temperature. It follows that I(q,T) is expressed by a linear combination of two functions, in which case, an isoscattering point q iso ,I iso is expected [35]. The isoscattering point does no longer exist above T c because the temperature dependence of the parameters in I OZ (q,T) changes. In the present case, q iso equals 3.633 nm 1 . The physical meaning of this value is still unclear. It must be noted that this isoscattering point does not indicate that the system transforms from one state to another one as generally reported and aforementioned. Here, the isoscattering point results from the fact that the number of nanopockets giving rise to the high-q peak is directly related to the size of the T-blobs (diverging at T c 31.4°C). In the domain of temperature ranging between 32 and 37°C, i.e., above the temperature of the transition occurring at the molecular level and characterized by the divergence of the O-Z parameters, it is still necessary to include an O-Z equation to fit the experimental curves (Figures 3 and4). Meanwhile, the intensity in quite small and the values of  vary between 1 and 2.5 nm (Figure 5). This feature makes sense because the system is still a gel.

However, the gel network is different from what it was below T c and the isoscattering point vanishes accordingly. Figure 12. Variation of the exponent p in the power law (Equation 4) resulting from the scattering of the gel surface. The surface fractal dimension D s is equal to 6-p.

Figures 11 and12 show that, within the domain of temperature (18-30°C) investigated above, the parameters related to the equations describing the scattering at lower q-values (power law and Guinier equations) do not vary much. Because the Guinier equation describes the solidlike inhomogeneities arising from the cross-links, R G is supposed to be independent of the temperature.

However, as in ref. [15], R G increases slightly between 18 and 32°C, i.e., above T c . It can be noticed that these values, ranging between 2.5 and 5.4 nm are much smaller than that reported for gels [15,16]. This feature could be related to the particular method of polymerization (cryopolymerisation) used for cryogels. Assuming a spherical shape for these static inhomogeneities arising from the cross-link regions, their diameter d st can be evaluated by 3

/ 5 2 G st R d 
. It follows that d st varies between 6.45 and 13.93 nm when T increases from 18 to 32°C. Between 32 and 34°C, R G jumps and seems to reach a plateau at 34°C (R G 16.5 nm or d st 42.6 nm). Also at 32°C, the exponent p in the power law (Equation 4) drops from 3.80 to 3.34 revealing a sharp increase of the fractal dimension D s of the gel surface from 2.20 to 2.66 (Figure 12). This feature could be related to the concept of the crumpled globule state describing the collapse of polymer gels [36] or the protein folding [START_REF] Dewey | Fractals in molecular biophysics[END_REF] associated to microphase separation. It follows that the radius of gyration R G measured at 34°C could be related to the size of the polymer rich domains (microphases) near cross-linking points. . Figure 13 shows the variation of sr for the pNIPA cryogel studied. The amplitude of the jump is mainly related to the swelling ratio at room temperature (the lower the degree of cross-linking, the larger the variation of sr) in gels [START_REF] Shibayama | [END_REF], in microgels [21] and in cryogels [25]. Further, the degree of cross-linking seems also to influence the shape of the transition [32]. As mentioned in paragraph 2.1., the degree of cross-linking of the present cryogel is quite large since [MBAA]/[NIPA] equals 1/15. Thus, the swelling and the swelling jump is much smaller than for the pNIPA cryogel investigated earlier [25] for which [MBAA]/[NIPA] was equal to 1/40.

Relation between features observed at nano

Between 18 and 24°C, the swelling ratio decrease is nearly linear and small. At 26°C, i.e., at the beginning of the phase transition domain, the swelling ratio begins to decrease more intensively and attains nearly half of its value at T c . The insert in Figure 13 indicates that the effect of the temperature elevation on deswelling is the strongest near the critical temperature (T c =31.4°C) deduced from the physico-chemical events shown by DSC and SAXS (divergence of the blob size ). Because the cryogel is a macroporous gel, it is strongly heterogeneous at the scale of a few or tens micrometers. SAXS data result from the averaging over several macropore (filled with water) and pore walls (the gel investigated) since the size of the beam is about 200 µm. The swelling measurement yields the total amount of water rejected from the sample, i.e., from the macropore and from the gel walls with no discrimination. In order to get a more precise information about the effect of temperature on the pore wall thickness and on the macropore size distribution, Two-Photon Fluorescence Microscopy [26] measurements were performed, as mentioned in the Introduction. The value of the mean pore size and of the wall thickness measured at 24, 28 and 34°C are indicated on Figure 13. It must be noted that the mean pore size obtained at 28°C is larger than that measured at 24°C as a result of a significant decrease of the contribution of the smaller pores yielding a non-symmetrical distribution. The diminution of small pores initiating the deswelling of the macroporous gels could result from local collapses of walls for reasons that are not clear yet. On the opposite, the deswelling occuring above T c , could result from the decrease of the wall size (the gel). The physico-chemical processes, revealed at the mesoscale by the divergence of  and the endothermic effect would induce a stress field in the gel. The sharp decrease of the fractal dimension D s of the gel surface around 32°C could result from the existence of a distribution of local collapsed domains (the crumpled globules mentioned above). The relaxation of the increasing stress could be achieved by the formation of large collapsed domains yielding the second part of the macroscopic volume decrease (Figure 13) going on up to 37°C.

Summary and conclusion

The modifications of the structure of a pNIPA gel constituting the walls in a cryogel with temperature were investigated over a broad range of length by SAXS. The experimental curves were fitted by means of a sum of four equations in order to take into account the scattering of each component of the system:

-the scattering of the surface of the gel is fitted with a power law yielding a surface fractal dimension different from 2

-the scattering of the solidlike inhomogeneities arising from the cross-links and characterized by a

Figure 1 .

 1 Figure 1. Evolution of the SAXS curves with temperature and comparison with the curve obtained for the dry cryogel for which the intensity was arbitrarily shifted. The insert shows the curves plotted in linear coordinates displaying an isoscattering point (I0.02450.0002) located at 3.6330.012 nm 1 .

Figure 3 .

 3 Figure 3. Logarithmic plots of the four curves contributing to the fit (black continuous line) of the experimental data (dark grey circles) and calculated by using the fitting parameters.

Figure 4 .

 4 Figure 4. Evolution of the Ornstein-Zernike and the pseudo-Voigt curves calculated by using the parameters obtained from the fit (a) between 18 and 32°C; (b) at 32, 34 and 37°C and for the dry cryogel; (c) sum of the O-Z and Voigt curves displaying the same isoscattering point (insert) as the experimental curves.

3. 3 .Figure 5 .

 35 Figure 5. Variation of the correlation length  (left axis) and the asymptotic intensity I OZ (0) (right axis) with temperature. The solid lines result from the fit with equations (6) and(7). The dashed

  ) (31.90°C), and unrealistic exponents (1.015 and 1.523).

Figure 6 .

 6 Figure 6. Logarithmic plot of the variation of the parameter  with the I OZ (0). The solid line results from the linear regression (r 2 0.9999991) of the data measured at 26, 28 and 30°C. The slope is equal to 0.586. The slope of the dashed line joining the points at 18 and 21°C is equal to 0.99.

Figure 7 .

 7 Figure 7. Differential Scanning Calorimetry (DSC) curves measured at a heating rate equal to 0.5 °C/min (SETARAM Micro DSC III).

Figure 9 .

 9 Figure 9. Evolution of the parameters  1 (left axis) and c (right axis) resulting from the fit with the pseudo-Voigt equation (Equation3).

Figure 10 .

 10 Figure 10. Variation of the pseudo-Voigt intensity a (Equation3) with the blob size  (Equation1).

Figure 11 .

 11 Figure 11. Variation of the Guinier radius of gyration R G (Equation 2) with temperature.

  -and mesoscopic scale by SAXS and macroscopic observations. The macroscopic evidence of the physico-chemical phase transition is revealed by significant decrease of the sample volume (the volume phase transition) related to the decrease of the degree of swelling or swelling ratio. The swelling ratio sr is generally determined by the mass of water per unit mass of dry gel:

Figure 13 .

 13 Figure 13. Variation of the swelling ratio with temperature. The insert indicates that the temperature determined by DSC (T=31.4°C) corresponds to the inflexion point of the curve sr(T). The mean macropore diameter and the wall thickness obtained by Two-Photon Fluorescence Microscopy [26] are indicated on the top of the figure.

  of the liquidlike or thermal inhomogeneities described by an Ornstein-Zernike equation diverging at a critical temperature as a result of the growth of instabilities induced by "melting" of the bound water molecules surrounding the side chains -the scattering of hydrophobic nanopockets formed in the T-blobs, resulting from the formation of local inter-chain bonds and yielding the high-q correlation peak fitted by a pseudo-Voigt equation.The analysis of the parameters yielding structural information at the meso and nano-scale are compared with the macroscopic volume change and the thermodynamic effects over the same range of temperature. It is shown that the critical temperature T c 31.4°C determined from the divergence of the Ornstein Zernike equation is the same as that of the endothermic effect measured by DSC

Table 1 .

 1 Value of the parameters obtained by fitting the experimental curve at 28°C with equation (5). The regression procedure was achieved by means of the Marquardt-Levenberg algorithm provided by SigmaPlot 10.0 (www.systat.com ). A weight

	Parameter					Value	Standard Error	CV (%) a	Dependency b
	I p0					2.6710 3	0.0410 3	1.42	0.994
	p					3.755	0.006	0.15	0.998
	I G (0)					0.142	0.009	6.29	0.992
	R G (nm)					3.89	0.04		0.96	0.922
	I OZ (0)					0.32	0.02		6.37	0.999
	 (nm)						2.7	0.1		3.72	0.999
	a				3.65710 2	0.00410 2	0.11	0.693
	c					1.00	0.03		3.18	0.974
	q 1 (nm 1 )					5.477	0.005	0.09	0.855
	 1 (nm)					0.452	0.004	0.77	0.986
	dependency	 1		variance variance	of of	the the	other other parameter, parameter,	parameters parameters	changing constant

a CV(%) is the parameter coefficients of variation, expressed as a percentage. This is the normalized version of the standard errors: CV  Standard Error100 /parameter value.

b

The dependence of a parameter is defined to be:

Acknowledgements

Preparation of the cryogels investigated was performed in the context of the FP6-Network of Excellence: EXPERTISSUES, "Novel Therapeutic Strategies of Tissue Engineering of Bone and Cartilage Using Second Generation Biomimetic Scaffolds". Partial financial support by CICYT-MAT2007-63355 is acknowledged. The authors are grateful to the ESRF, Grenoble, for access to the French CRG beamline D2AM (proposal No.0201702) and they acknowledge the help of its technical staff, Jean-François Berar, Nathalie Boudet, Bernard Caillot, Stephan Arnaud. The authors wish to thank Isabelle Grillo (ILL) for her help in the DSC measurements and Jean-Louis Chemin (SIMAP) for the design of the furnace used for the SAXS measurements.

resulting from the "melting" of the water cages surrounding the side chains (dissociation of the hydrophobic solvatation). The evolution of the OZ parameters  and I OZ (0) with temperature confirms the validity of an Ising model for describing the transition in this pNIPA cryogel. The scaling law between  and the intensity a of the chain-chain correlation peak shown here for the first time gives new insights in the origin of the isoscattering point observed up to T c . The parameters characteristic of the structure at a larger length scale, i.e., the radius of gyration of the solid-like heterogeneities, R G and the exponent p of the power law (related to the fractal dimension of the gel surface) begin to change significantly with temperature only above T c . This feature suggests that, above T c , the volume decrease results from the contraction of the system allowing the relaxation of the stress induced by the conformational changes (physico-chemical effect) occurring below T c . Further investigations are in progress in order to study the influence of the degree of cross-linking on these mechanisms.


Zhibin Zhou
email: zhibin.zhou@ecole-navale.fr

Franck Scuiller

Jean Frédéric Charpentier

Mohamed Benbouzid
email: mohamed.benbouzid@univ-brest.fr

Tianhao Tang
email: thtang@shmtu.edu.cn

An Up-to-Date Review of Large Marine Tidal Current Turbine Technologies

Keywords: Tidal current turbine, large machine, review

Owning to the predictability of tidal current resources, marine tidal current energy is considered to be a reliable and promising renewable power source for coastal areas or some remote islands. During the last 10 years, various original horizontal axis and vertical axis marine current turbines (MCT) have been developed around the world. Although various projects have been reported in the state-ofthe-art research papers in recent years, many of these projects were only at the design stage when the papers were published. In fact, some projects do not have any further developments during the several years after the first reporting. In this paper, up-todate information about large tidal turbine projects over 500 kW is focused. The newest achievements of these large tidal current turbine technologies are presented. These technologies represent the industrial solutions for several pre-commercial MCT farm projects in the coming years. This paper provides a useful background for researchers in the marine turbine energy domain.

I. INTRODUCTION

One of the main advantages of marine current energy is related to the predictability of the resource. The astronomic nature of tides is driven by the gravitational interaction of the Earth-Moon-Sun system and makes marine tidal currents highly predictable with 98% accuracy for decades [START_REF] Benelghali | A simulation model for the evaluation of the electrical power potential harnessed by a marine current turbine[END_REF]. There are basically two ways of generating electricity from marine tidal energies: either by building a tidal barrage across an estuary or a bay, or by extracting energy from free flowing tidal currents. The main drawback of the solution is that large barrage system would change the hydrology and may have negative impacts on the local ecosystem [START_REF] Pelc | Renewable energy from the ocean[END_REF]. Therefore during the last few decades, developers have shifted towards technologies that capture the kinetic energy from tidal-driven marine currents. The exploitable marine current energy with present technologies is estimated about 75 GW in the world and 11 GW in Europe [START_REF] Boye | Rapport de la mission d'étude sur les énergies marines renouvelables[END_REF].

In fact, various original horizontal axis and vertical axis marine current turbines (MCT) have been developed around the world in recent years [START_REF] Rourke | Marine current energy devices: Current status and possible future applications in Ireland[END_REF][START_REF] Bahaj | Generating electricity from the oceans[END_REF][START_REF] Zhang | State of the art in tidal current energy extracting[END_REF]. The majority of MCT devices are horizontal axis turbines with rotation axis parallel to the This work is supported by Brest Métropole Océane (BMO) and the Shanghai Maritime University. current flow direction. The main disadvantages associated with vertical axis turbines are relative low self-starting capability, high torque fluctuations and generally lower efficiency than horizontal axis turbine design. Currently, only horizontal axis MCTs appear to be the most technologically and economically solution for large-scale marine current turbines with power capacity over 500 kW.

Although various turbine projects have been reported in some state-of-the-art research papers, many of these projects were only at the design stage when the papers were published [START_REF] Rourke | Marine current energy devices: Current status and possible future applications in Ireland[END_REF], [START_REF] Benelghali | Marine tidal current electric power generation technology: State of the art and current status[END_REF]. However, some projects are abandoned or never have been built, for instance, the Lunar Energy Tidal turbine. And some projects do not have further developments during the several years after the first announcement, such as Atlantis Resource Nereus and Solon turbines. Therefore, an up-to-date review of industrialized turbine technologies is necessary.

In this paper, up-to-date information and the newest achievements of industrialized large tidal current turbine technologies (over 500 kW) will be focused. In Section II, the pilot pre-commercial MCT farms information and the industrialized large turbine technologies are presented. The conclusion and perspective are then given In Section III.

II. LARGE MARINE CURRENT TURBINE TECHNOLOGIES

Several horizontal axis turbine technologies are developed more than one or two generations and have been chosen by the industrial communities to realize pilot demonstrative MCT farms before the final commercial stage. These projects illustrate the up-to-date developments of large MCT technologies which will provide electricity to coastal or island areas in the coming years. Table I summarizes the main information about some of these pilot MCT farm projects and their planned/estimated operational dates. From this table, it can be seen that most of these turbine technologies have attended megawatt-level power capacity.

A. OpenHydro Turbine Technology

OpenHydro is an open-center turbine technology; a 250 kW prototype was installed and tested at European Marine Energy Center (EMEC) off Orkney islands in Scotland and was connected to the UK national grid in 2008. This turbine

B. HS1000 and AR1000 Turbine Technologies

The 1MW pre-commercial turbine HS1000 (Fig. 2) was tested by Andritz Hydro Hammerfest (original Hammerfest Strøm) at EMEC tidal test site at the end of 2011. It started delivering energy to the grid in 2012. The HS1000 turbine is based on the technology of a smaller prototype HS300 (300 kW, reported as E-Tide turbine project in [START_REF] Zhang | State of the art in tidal current energy extracting[END_REF][START_REF] Benelghali | Marine tidal current electric power generation technology: State of the art and current status[END_REF]) which was installed in Norway and connected to the public grid in 2004. This megawatt-level HS1000 turbine technology is planed to be used in a 10 MW commercial array in the Sound of Islay on the west coast of Scotland; the tidal resource and seabed surveys are completed [13]. This technology is also reported to be chosen in the first phase of the MeyGen tidal current project in Inner Sound of the Pentland Firth [9].

Another candidate for the MeyGen project is the AR1000 turbine (Fig. 3) technology developed by Atlantis Resources Corporation [14]. The Atlantis AR1000 turbine features fixed pitch configuration and is rated at 1 MW at 2.65 m/s current velocity. The AR1000 can be rotated in the slack period between tides using a yaw drive for optimally facing the tides. The first AR1000 was successfully deployed and tested at the EMEC facility during the summer of 2011. The AR1000 system is also scheduled to be installed on Daishan demonstration site in China during 2014. A larger turbine called AR1500 (1.5 MW at 3.0 m/s) is under development for future installation in the Pentland Firth in Scotland and the Bay of Fundy in Canada. The SeaGen S turbine (showed in Fig. 4) developed by Marine Current Turbine Ltd. (owned by Siemens since 2012) is a well-known system that can generate electricity from marine current energy. This technology has a twin axial-flow turbine supported on a structure with the ability to raise the moving components out of the water for maintenance [16]. It is the world first grid-connected megawatt-level MCT system. The 1.2 MW SeaGen S system (2×600 kW) was installed in Strangford Lough in Northern Ireland in 2008 and has generated 8 GWh electricity since the installation. During a strong spring tide, the SeaGen S system can deliver more than 20 MWh in a single day. It is reported that up-scaled SeaGen S systems with 20 meter rotor and 2 MW power rating will be installed in two commercial arrays in UK waters (as listed in Table 1) from 2015. The Voith Hydro turbine adopts robust design: it uses fixed-pitch blades and permanent magnet synchronous generator to achieve compact structure. Specially designed symmetric blade profile can be operated for bidirectional tidal currents avoiding pitch and yaw requirements. The nacelle system uses seawater lubrication technology to avoid grease and seals for the bearings. The electrical machine is protected from corrosion and marine fouling by proven coatings and sacrificial anodes. The low-maintenance characteristics makes the estimated system lifetime up to 20 years [18]. The 1 MW turbine has a rotor diameter of 16 m and reaches the rated power at a current velocity of 2.9 m/s.

D. Voith Hydro Turbine Technology

E. Sabella Turbine Technology

One of the potential MCT applications is to provide electricity for remote islands not connected to the continental electric grid. As example, in the near future, several MCTs are planed to be installed in the Fromveur passage (near Brest) in France to supply part of the load demand of Ouessant Island. For the Fromveur project, the Sabella tidal turbine technology will be used [19]. The Sabella D10 turbines have a rotor diameter of 10 m and a power capacity of 0.5~1.1 MW for 3.0~4.0 m/s current velocities. Figure 6

F. Alstom Tidal Turbine Technologies

In 2010, Alstom announced a 1 MW turbine project named Beluga 9 for deployment in Canada's Bay of Fundy in 2012 [21]. Beluga 9 was a joint project with Canadian company Clean Current. It was intended for very powerful currents (up to 4.5 m/s) and for sites with depths of 30 meters or more. However, with the termination of the licensing agreements with Clean Current at the end of 2012, there is no further news about this megawatt turbine project. Clean Current has decided to focus on river turbines and shallower water tidal turbines (< 20 meters). While Alstom's intention is to pursue the larger utility scale opportunities [22].

In 2013, Alstom successfully installed a 1 MW tidal current turbine at EMEC tidal test site after the acquisition of Tidal Generation Limited (TGL) [23][24]. Figure 7 shows the Alstom's 1 MW tidal turbine system. This tidal turbine weighs 150 tonnes and has three pitchable blades, a rotor diameter of 18 m and a nacelle length of 22 m. A standard drive train and power electronics device is inside the nacelle. This turbine system can be installed in a water depth between 35 and 80 meters. It can generate electricity with a tidal current between 1 m/s and 3.4 m/s, and reaches the rated power for a tidal current speed of 2.7 m/s. This 1 MW Alstom tidal turbine is under extensive testing and analysis in different operational conditions off Orkney islands throughout 2013 over an 18 month period. Although the Alstom's 1 MW tidal current turbine seems similar to a standard wind turbine system (with three pitchable blades and gearbox-integrated drive train), this tidal turbine have some special characteristics. Its buoyancy enables the turbine to be easily installed and retrieved by using small vessels, thus reducing the installation and maintenance costs. It has an intelligent nacelle which can be rotated by the water thrusts to orientate the turbine system to face the tide direction; that enables the system to harness energy efficiently from both ebb and flood tides

III. CONCLUSIONS AND PERSPECTIVES

The turbines presented above represent the newest achievements in the megawatt-level marine current turbine technologies. The comment point is that they are all horizontal axis turbine. And it should be noted that more than half of these turbine technologies adopt the fixed pitch solution for the blades.

In addition to these industrialized large turbine technologies, there are other megawatt turbine projects under development. For example, the TidalStream Triton technology mounts several turbines on a semi-submersible buoying platform to achieve high power harness capacity. It should be noted that in FORCE, there are also several megawatt-level current turbine plans which are strongly related to the turbine technologies presented in this paper. High tidal current speed (up to 5.5m/s) and Nova Scotia's feed-in-tariff make FORCE one of the most attractive and economic tidal sites in the world [26][27]. Siemens and Bluewater are jointly developing a 2 MW floating tidal current turbine, called SeaGen F, to be installed in Bay of Fundy and it will supply electricity for 1,800 Nova Scotia households. OpenHydro, the DCNS tidal subsidiary, will proceed with plans for a grid-connected 4 MW tidal array to be installed at FORCE in later 2015. This array will consist of two 2 MW turbines with 16 m rotor diameters.

  technology has been chosen by the French companies EDF and DCNS to build a demonstrative MCT farm off the coast of Paimpol-Bréhat in Brittany, France. The first 500 kW OpenHydro turbine (Fig.1) was tested in September 2011 near Brest. This 850 tonnes turbine has a diameter of 16 m and is supposed to be installed at a depth of 35 meters. This technology uses multi-blades fixed between the open-center rim and the outside shell in a rim-driven configuration. The permanent synchronous generator is integrated into the outside rim shell. The planed MCT farm with 4 turbines is reported to be in operation in 2014[10]. However, some delays in the final farm operation could still be envisaged.

Fig. 1 .

 1 Fig. 1. DCNS-OpenHydro turbine © [11].

Fig. 2 .

 2 Fig. 2. Andritz Hydro Hammerfest HS1000 turbine © [12].

French

  energy company GDF Suez has plans to install pilot tidal energy farms at Raz Blanchard off the coast of Lower Normandy and in the Fromveur passage off the coast of Finistère in Brittany. These two sites represent 80% of the marine current energy potential in France [17]. For the Raz Blanchard project, GDF Suez has recently confirmed to use Voith Hydro HyTide turbine and Alstom tidal turbine technology. The Voith Hydro turbine system (shown in Fig. 5) is developed by German hydropower equipment maker Voith Company. The first test turbine of 110 kW has been in operation near the South Korean island of Jindo since 2011. The up-scaled version of 1 MW turbine is now installed and tested at EMEC tidal test site.

  illustrates the perspective Sabella turbine farm. The Sabella D10 is based on the first French marine current turbine Sabella D03 (3 m rotor diameter) which was tested at Bénodet estuary near Brest in 2008. The prototype D10 turbine is now completing the construction and is scheduled to be installed in the Fromveur passage at the end of 2014. Larger turbines D12 and D15 with power capacities of 1~2 MW are under design for future turbine farm applications [19-20].

Fig. 3 .

 3 Fig. 3. Atlantis AR1000 turbine © [14].

Fig. 4 .

 4 Fig. 4. SeaGen S turbine © [15].

Fig. 5 .

 5 Fig. 5. Voith Hydro turbine © [18].

Fig. 6 .

 6 Fig. 6. Sabella turbine farm illustration © [20].

Fig. 7 .

 7 Fig. 7. Alstom tidal turbine © [23].

  The 1/23rd scale and 1/10th scale turbines (Triton T6 and Triton T3) were successfully tested in 2009 and 2011 respectively [25]. The first full-scale version Triton T36 system (with 2.5 MW power rating) is planned to be built at the Fundy Ocean Research Center for Energy (FORCE) tidal test site in Nova Scotia, Canada.

TABLE I PILOT

 I MCT FARMS IN THE COMING YEARS [3], [8-10].

	Companies	Location	Turbine Name	Pitchable Blades	Turbine Number	Total Capacity (MW)	Operational Year
	DCNS, EDF	Paimpol-Bréhat	OpenHydro	No	4	2	2014/2015
	MeyGen	Pentland Firth (Scotland)	HS1000 or AR1000	Yes No	6	6	2015/2016
	MCT, Siemens	Kyle Rhea (Scotland)	SeaGen S	Yes	4	8	2015
		Anglesey (Wales)	SeaGen S	Yes	5	10	2015
	Andritz Hydro Hammerfest	Sound of Islay (Scotland)	HS1000	Yes	10	10	N/A
	GDF Suez, Eole	Raz Blanchard	Voith Hytide	No	3~6	3~12	2016
	Generation	Fromveur	Sabella	No	N/A	N/A	2016