Laurent Rollet

Philippe Nabonnand

Adam [ Appell

[ Appell

Mathematics and Mathematicians in Nancy during World War I

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Introduction

In 1870, Nancy had 43.000 inhabitants. Forty years later, in 1914, it had 120.000 inhabitants. Between 1870 and 1914, Nancy was one of the few French cities to undergoa strong demographic growth (140%). Two elements can explain this situation. One of the consequences of the Industrial Revolution after 1850 was the development of mining, iron and steel industries in Lorraine and the increasing needs of workforce. But this appeared to be a minor factor in the case of Nancy because of its geographic situation. The second explanation finds its source in France's defeat in the war of 1870. After the war the governments of the new Third Republic gave a strategic role to Nancy through the installation of many troops and regiments. The town quickly became a ville-frontière and a garrison town. 2 Moreover a large number of Alsatians and Lorrains (more than 13.000 in 1877) refused German nationality and decided to settle in proximity to their homeland. This demographic shift had an intellectual and a scientific impact. The loss of the University of Strasbourg largely benefited Nancy: a great number of professors left Strasbourg to reinforce Nancy's intellectual institutions. 3 Furthermore, the University of Nancy had to deal with a large number of students coming from Alsace and Moselle. 4 The benefit for the University of Nancy was evident, especially in the scientific disciplines. For instance, the École pratique de médecine was transformed into a Faculty of Medicine and this had important intellectual and cultural repercussions outside the university. Paradoxically, the defeat of 1870 led to the emergence of an intellectual and artistic golden age symbolized by the avant-garde École de Nancy.

The First World War put an end to this brilliant period. In 1918, Nancy returned to its status of one among other ordinary provincial towns with its university losing some of its specific characteristics acquired in the aftermath of the 1870 war.

The purpose of this article is not so much to present a broad picture of the history of the University of Nancy between 1870 and 1918 but to analyse the situation of mathematics and the role played by mathematicians in Nancy during World War I. Despite the restricted scope, this history will cover various topics: the increasing pre-war importance of applied research and engineer training in Nancy, the tight collaborations between mathematicians and physicists to create an aerodynamic institute, the relationships between the Faculty of Science and the army, the involvement of mathematicians in the life of the City, etc. The study of these issues boils down to analysing the workings of a very small community (in 1914 there were only 4 chairs in mathematics in Nancy) and the capacity of its actors to elaborate ambitious projects and obtain massive support from the University, government ministries and the general public.

The first part will be devoted to a survey of the 'golden age' of the Faculty of Science of Nancy before World War I, especially the pioneering development of technical institutes and the role played by mathematicians in this process. The second part will deal more precisely with the involvement of the mathematical community in the war, both on the military and the home fronts, and the damaging consequences of the war. The third part will finally examine a peculiar consequence of the conflict, i.e. the demise of the aeronautics curriculum in Nancy and the leadership role of Gaston Floquet in its pre-World I development.

II. The Faculty of Science before World War I

At the turn of the nineteenth century the evolution of the University of Nancy was characterized by a scientific project centred on the development of applied sciences in a patriotic perspective. The historian Edgar Quinet, in 1873, gave the following vivid description of this patriotic agenda: Convinced that the loss of the war was partly due to lack of research in the applied sciences and to the incapacity of science to help industrial development, many universities decided to create technical institutes devoted to the training of technicians and engineers for the local industries. The creation of these institutes was financed by the Ministry of Public Instruction, by local political institutions (conseil général, municipal council of Nancy, etc.) and by industrial firms. The University of Nancy was part of this movement towards engineer education; the period from 1887 and 1914 saw the founding of the institut chimique (1887), a school of brewery (1893), the institut électrotechnique (1900), the institut botanique agricole et colonial and the very ephemeral institut d'aérodynamique. 5 The most influential actors of this history were the Alsatian chemist Albin Haller (1849-1925), who was the first director of the school of chemistry, and the physicist Ernest Bichat (1845-1905). As president of the conseil général de Meurthe-et-Moselle, Bichat had political and economical networks in Lorraine; he was moreover a strong promoter of close relationships between science and industry and his action led to the creation of the institut chimique and the institut électrotechnique. From 1888 until his death he was both the dean of the Faculty of Science and member of the conseil supérieur de l'instruction publique. His successor to the deanship, the mathematician Gaston Floquet (1847-1920), continued his work by promoting the development of aeronautics and meteorology. The Belgian manufacturer Ernest Solvay (1838-1922) also played a leading role in this process by giving generous subsidies to the technical institutes between 1890 and 1914.

As an auxiliary discipline, mathematics was a decisive component of the curricula in the technical institutes. This situation raised the problem of mathematical training in engineer studies, a central pedagogical concern within the mathematical community was to find the correct balance between pure and applied mathematics in engineer education knowing that engineering students did not necessarily have a good initial mathematical training. 6 One of the consequences of the 1896 university reforms (which gave official status to the University of Nancy) was the creation of the certificats de licence. Thanks to new subsidies from the municipal council and the conseil général, the Faculty of Science was able to develop new courses in applied physics and applied mechanics. This contributed to the strengthening of relationship with local industries. Industrial physics courses were created in 1899 by the physicist F. Perreau, who was then maître de conférences, and the mathematician Henri Vogt, who was elected to the chair of applied mathematics in 1899 and was in charge of applied mechanics courses. Vogt was actively involved in pedagogical reflections on the connections between mathematics and the applied sciences. 7 Before World War I, mathematical curricula were divided in two main parts. In order to obtain a licence de mathématiques students had to pass several certificates (differential and integral calculus, rational mechanics, higher geometry, analysis and astronomy), some of which were quite difficult to obtain. Engineering students could prepare this diploma. But the new curriculum, certificat de mathématiques générales, created in 1908 became the core of their mathematical training. During their studies, engineers also had to obtain certificates in other scientific disciplines (such as general and applied physics, rational mechanics, etc.).

Before 1914, universities paid special attention to adapting mathematical training to the needs of a new student population: indeed, one of the consequences of the reforms of the 1890's was the opening up of the Faculties of Science to students who did not come from classes de mathématiques spéciales. The University of Nancy in particular had an important foreign student population much of which could not follow the certificat de calcul differential et integral for lack of the prerequisite training. In many universities (be it in Paris or in province) there were debates and discussions concerning the best way to receive these students. The solution adopted was the creation of the certificate de mathématiques générales, a new certificate the curricula of which were analogous to those of the classes de mathématiques spéciales. Nevertheless, this was not enough: in Nancy, the large influx of foreign students without solid initial training in mathematics and the sciences led to the creation of new courses in 1908 organized into a one-year prerequisite preparation to the technical institutes. The academic community of the Faculty of Science was keenly aware that foreign students constituted one of the keys of the success of these institutes and accordingly devoted the time and money needed to cater for and retain this new student population.

Was the beginning of the 20 th century a kind of golden age for the University of Nancy? Before 1914, the University of Nancy was one of the most active provincial universities, especially in the scientific and medical fields. Prior to the N-Rays affair, René Blondlot was enjoying international reputation for his electromagnetic experiments of the 1890's. From 1907, Edmond Rothé was closely involved in the research on wireless telegraphy (this technique allowed the Observatoire de Paris to communicate the exact time to the Faculty of Science in 1910). He also shared with Floquet great interest in aerodynamics and meteorology. 8 In 1912, Victor Grignard was awarded the Nobel Prize in chemistry. New buildings and new laboratories were erected: in 1897, Haller obtained a very important subsidy from the Société industrielle de l'Est for the creation of a laboratory devoted to electrochemistry and physical chemistry. Solvay offered 100 000 Francs for this project to be followed, three years later, by an equivalent subsidy for the creation of a laboratory devoted to electricity and electrical engineering. In 1906, following Bichat's project, the institut de mathématiques et de physique to be dedicated to pure science was constructed close to the technical institutes. In 1913, Solvay offered another important subsidy (500 000 Francs) which allowed the extension of the electrical engineering laboratory, the creation of a chair of electrical engineering (attributed to Alexandre Mauduit) and the transformation of the previous maîtrise de conferences in electrical engineering into a maîtrise de conferences in physics on condition that the professor also taught mathematics. This chair was held by the physicist Grumbach.9 At the same time, Louis Maurice Roy was recruited as chef de travaux pratiques de mathématiques avec charge de conférence.

Consequently, the material conditions were quite good for research and educational activities. Nevertheless this situation was in a way artificial. As in other provincial faculties, the number of students was rather low: good students from classes préparatoires usually opted for the Parisian grandes écoles. As a result, the technical institutes were obliged to widen their recruitment requirements to accept best students from the école primaire supérieure or from the école professionnelle de l'Est. To solve this demographic and financial problem, the Faculty of Science and its institutes opened up to the admission of foreign students from 1902 following the model of the German technische Hochschulen. The University of Nancy was one of the first to develop an active policy to attract foreign students with the help of the Société des amis de l'université which was engaged in active lobbying outside France. The first consequence of this drive was the demographic weight of foreign students in Nancy. In 1914 for instance, of the 1089 students enrolled at the Faculty of Science, more than 600 were foreign students most of which were enrolled in the schools of chemistry and electricity. 10 The second consequence was the important rate of failure in the exams for the scientific certificates (see tables 1 and2 below).

Despite these problems, official discourses before 1914 was very optimistic and its rhetoric praised the harmonious relationship between academic research, industrial development and patriotism. Nancy was thus said to be in the forefront of modernity and dynamism. In 1912, the chemist Paul Petithe was then the assessor of the dean of the Faculty (Floquet)expressed this satisfaction very clearly: L'outillage, si complet déjà, de notre Institut, s'est augmenté d'appareils pour les essais de résistance des matériaux […]. Comme on le voit, l'Institut de mécanique conserve sa réputation justifiée d'Institut modèle et en même temps il tient à rendre à l'industrie régionale tous les services de son ressort. En Allemagne, les entrepreneurs font constamment usage des essais de résistance pour les matériaux qu'ils utilisent et notre Institut sera maintenant en mesure de répondre aux demandes qui lui avaient été déjà adressées. 11

III. Mathematicians during the war

Before World War I, there were 4 chairs of mathematics in Nancy: analysis (Floquet), applied mathematics (Vogt), rational mechanics (Husson) and differential and integral calculus (Husson). 12 In addition to the three professors, four part-time lecturers were in charge of courses, especially in the technical institutes. Bertrand was a professor at the École professionnelle de l'Est and had the responsibility for practicals at the institute of electrical engineering (from as early as 1906 to 1920). Chanzy, who was professor at the lycée of Nancy 13 , was in charge of practicals, mathematical conferences and graphical works, from 1903 to 1908 at the institut chimique, and then at the institut electrotechnique (as late as 1920). Pol Simon, a science graduate from the Faculty of Science, carried out practical works in mathematics at the chemistry school. He continued these courses at the newly founded institut métallurgique et minier after the war. Finally, the engineer Louis Roy was recruited in 10 Most of which came from Russia. See: [Birck 2006], pp. 37-41; [Gouzévitch & Gouzévitch 2006].

11 Comptes rendus de l'université de Nancy, 1912, p. 29. 12 Actually, Husson had been elected in 1914 on the chair of rational mechanics. Nevertheless, since he was mobilized during the war he did not hold this chair between 1914 and 1918 and kept the responsibility of the chair of integral and differential calculus. The chair of rational mechanics remained thus unoccupied during the war.

6 1913 as chief of practical works in mathematics. Roy graduated from Supelec, where he obtained a diploma in engineering and a doctorate in science. After the war, he specialized in rational and applied mechanics and made a brilliant career at the University of Toulouse.

The two categories of professors constituted the mathematical workforce in Nancy at the beginning of the war. Of course, some physicists, such as Grumbach, could be involved in mathematical training (see supra), but the seven persons mentioned above were at the centre of mathematical training at the faculty of science. Two points are worth noting. On the one hand, in the light of the historical materials at our disposal, there wasn't any maître de conférences in mathematics at that time. On the other, because of the military mobilization the workforce was substantially reduced during the war. Nevertheless, as we shall see in the next section, the episodes of the construction of the institut d'aérodynamique show quite clearly that the mathematical perimeter was much larger involving close collaborations with physicists (Camille Gutton, Edmond Rothé, Maurice Guéritot, Alexandre Mauduit, Ernest Hahn or Faillot) as well as with local military officers.

In light of the official chart of the faculty (Comptes rendus de l'université de Nancy) and of the administrative decrees of nominations, Bernard was no longer in the faculty after November 1914. 14 Roy probably had to leave the faculty at the end of 1915 or at the beginning of 1916. Despite his poor health, Pol Simon enlisted in the army at the beginning of the conflict as lieutenant instructor in the infantry. After the war he was mentioned in dispatches for his courage during a combat. As for Chanzy, his presence seems to be attested as late as 1916. 15 At the beginning of the war, Edouard Husson (integral and differential calculus) was put on active service as a sergeant in the territorial regiment of the infantry in Toul near Nancy. This situation allowed him perhaps to maintain some of his activities in Nancy and it is likely that he kept a part-time activity at the university until November 1916. 16 He then became a technical attaché in an aviation centre and finally rejoined the school of aviation in Pau as technical instructor. As for the physicists, Alexandre Mauduit (electrical engineering) was mobilized as an engineering captain in Toul while Edmond Rothé joined the radiographic service of the military hospital in Nancy (and continued to teach at the onset of the war). Apart from military mobilization, some professors of the Faculty of Science were also involved in inventing various technical devices for the army. Camille Gutton constructed a grenade detector, Rothé built a field radiographic car and an anemometer, Guéritot, who was in charge of practicals in electricity, developed with Rothé a mechanism for the use of wireless telegraphy in aeroplanes. René Blondlot, who was professor emeritus, worked on periscopes.

In concrete terms, Vogt and Floquet, who were of the older generation, were in charge of all the courses during the war. In 1914-1915, Vogt gave more than 8 courses per week and Floquet was in charge of differential and integral calculus.17 In 1915-1916, Floquet was in charge of rational mechanics and advanced analysis while Vogt taught general mathematics. During the next academic year, Floquet taught differential and integral calculus as well as astronomy and Vogt kept the charge of general mathematics. Vogt also gave courses in applied mathematics and ensured (with Chanzy?) practical works in mathematics. The first consequence of the war was a reduction in the number of professors. In 1915, the faculty of science lost 12 of its 22 professors. 18 The second consequence was a sharp drop in the number of students at the Faculty of Science. In 1913-1914, there were 1089 students with a large proportion of foreign students. A year later, there were only 114 students with not a single foreign student left (see Table 1 below). 19 And this was not without repercussions on the number of diplomas and certificates delivered (see Tables 2 and3 1910-1911 1911-1912 1912-1913 1913-1914 1914-1915 1915-1916 1916-1917 1917-1918 1918-1919 1919-1920 1920-1921 Number of students Foreign Students

Table 3 -Number of various certificates delivered in mathematics and physics at the Faculty of Science of Nancy (1900-1920) -A (differential and integral calculus) -B (general mathematics) -C (higher analysis) -D (higher algebra) -E (higher geometry) -F (astronomy) -G (rational mechanics) -H (applied mechanics) -I (general physics) -J (applied physics) 22

During the war, the academic discourse praised the professors who served in the army and those who, because of their age, had to stay in Nancy, on the home front. Perhaps in order to evade ill fortune, this situation was even seen as a chance for the university as shown by this quotation of Arthur Meyer in November 1915 at the start of academic year: The situation of the university was presented in very similar optimistic terms. Fortified by its past achievements, trustful in its future, the university maintained faith in its high mission and contributed to the creation of an entirely purified moral atmosphere. 24 Until the end of 1915, the material conditions at the Faculty of Science were apparently satisfactory.

Nevertheless, the installation of a high range 380 mm. howitzer in Hampont (Moselle) in January 1916 changed the situation. This canon, a Krupp type SK-L/45 Max (which was called le gros Max by the inhabitants of Nancy) made Nancy and Lunéville its targets. Moreover, during the war, Nancy suffered severe air shelling by planes and dirigibles. These bombings were responsible for much of the destruction and deaths (the 101 bombardments of Nancy between 1914 and 1918 brought about 181 deaths and 310 injured). 25 In November 1916, the tone of the academic discourse did not change much. In his report, the biologist Lucien Cuénot explained that Nancy had effectively received several hundred bombs but he was very pleased about the limited consequences for the Faculty of Science: some buildings 22 Source: Comptes rendus de l'université de Nancy. See also Ibidem. 23 Comptes rendus de l'université de Nancy, 1916. 24 This was the whish expressed in November 1914 by the professor of law François Geny. Comptes rendus de l'université de Nancy, 1915. 25 [Anonyme 1918].

suffered important degradations (broken glasses at the Chemical Institute, the Institute of Physics and the School of Brewery) and the bombardments imposed the holding of the baccalauréat exams in the cellars of the Faculty of Science; fortunately there were no losses among the students and the professors.

Lucien Cuénot was in fact over-optimistic: much of the destruction of the university occurred in 1917 and 1918. A ministerial decree of February 11 th , 1917 ordered the closing of the university because of the bombing. In 1917, the laboratory of electrical engineering, which had benefited from an expensive development thanks to Solvay, was hit by a torpedo and was almost totally destroyed. On October 31 st , 1918, two incendiary bombs partly burnt down the University Library destroying 56.000 out of its 113.000 books.

The University library after the fire 26 Actually, the last months of the conflict and its consequences were to be catastrophic for the University of Nancy and its faculties. First of all the heavy losses led to a demographic bleeding of the faculty of science and its technical institutes (see Table 4 below). After the war, because of the destructions suffered during the war, the financial needs of the faculties and of the engineering school were enormous. Two problems had to be faced: the level of financing (which had not evolved for thirty years) was identical to that of 1914 and was therefore insufficient in face of prevailing inflation ; moreover, there wasn't enough students and their fees (especially those paid by foreign students) could not cover the reconstruction expenditures. At the end of the war, the municipality urged the University to reopen and to follow the example of Lille where the consequences of the war were far more important. In a decision of December 14 th , 1918, the municipal council allowed 15 days for the official procedures of reopening. Furthermore, it enjoined the Faculty of Arts to restart French courses for foreign students and asked the government for a quick demobilization of the professors. 27 This demobilization was very disappointing for Nancy because of its slowness 28 and also because of the return of Alsace-Moselle to the French fold. The reopening of the University of Strasbourg offered very interesting opportunities for many professors from Nancy. As a consequence, Paul Müller (physical chemistry), Louis Hackspill (industrial chemistry) and Edmond Rothé chose to pursue their career in Strasbourg. As we shall see, the departure of the latter coupled with the death of Floquet in 1920, probably signed the death warrant of the pursuit of the aerodynamic course in Nancy.

IV. Some consequences of the war: the short-lived Institut d'Aérodynamique et de Météorologie

The central role of the technical institutes in Nancy favoured a strong cooperation between 'applied' and 'pure' disciplines. Before 1914, the project of an Aerodynamic Centre launched a strong collaboration between mathematicians, physicists and engineers. It also mobilized advanced partnerships with the army and civilian associations.

On July 12 th , 1914, on the military aerodrome of Villers-les-Nancy, the Société des Amis de l'Aviation officially presented to the army two double aviation hangars and a workshop 27 Procès verbaux des délibérations du conseil municipal de Nancy, 1918, pp. 250-253. 28 After the armistice (November 1918), the Faculty of Science organized several courses for 35 students of the American army: wireless telegraphy (Camille Gutton), applied geology (Jules Thiébaut) and aeronautics (Vogt).

hangar for the maintenance of aeroplanes. 29 This donation, which represented 105 000 francs, was made possible thanks to several grants and subscriptions obtained by the association since 1912: the cities of Nancy and Saint-Dié (Vosges) gave important subsidies, so did the Société Générale des Étudiants de Nancy and journals such as l'Est républicain and l'Étoile de l'Est. These solemn festivities were conceived as part of the celebrations of the French National Day of July 14 th (involving official and public torchlight processions, republican banquets, etc.). The hangar ceremony was followed by another donation from the Société des Amis de l'Aviation to the University of Nancy: a five-hectare ground for the construction of a future Institut d'Aérodynamique et de Météorologie, next to the military buildings. The inauguration speeches were pronounced by the Minister of Public Instruction, Jean-Victor Augagneur, and by the originators of the project, Edmond Rothé and Edouard Floquet.

Actually this ceremony was the result of a long commitment of the two professors. On March 20 th , 1912, the Republican Union of Nancy invited Paul Painlevé for a public lecture on aviation. At that time, Painlevé was both a member of the Academy of Science, a deputy for Paris, the president of the Commission Nationale d'Aéronautique and the vice-president of the Ligue Nationale Aérienne. 30 He was accompanied to Nancy by captain Albert Étévéwho was then chief of the school of military aviation in Versaillesand by the lieutenant Jean Étienne Cheutin. 31 During his lecture, Painlevé talked about several aspects of the necessary development of aviation in the province; indeed, the army needed more aeroplanes but also aerodromes and hangars. After this lecture, a hangar committee was created by Floquet and Rothé with the double purpose of raising funds and buying a large field near Nancy. A few days later (April 7 th and 8 th ), the Ligue Aérienne de l'Est, which was chaired by Floquet, organized on the aerodrome of Jarville (a village close to Nancy) a meeting in favour of military aviation. This event furnished the occasion for the merger of the league and the hangar committees since the two boards partially consisted of the same members and shared the same objectives. The result of this merger was a new association, the Société des Amis de l'Aviation, whose aims were to promote aviation in Nancyespecially military aviationthrough the establishment and the installation of aerodromes, the construction of hangars, the creation of an aerodynamic institute and the organization of aviation contests and festivals.

The passion of Rothé and Floquet for aerodynamics and the promotion of aviation was quite old. Since his nomination in 1905, Rothé's researches were focused on this domain and the third part of his physics course was devoted to aerology and aerodynamics [Rothé 1917].

Moreover, Floquet, as founder and director of the Ligue Aérienne de l'Est (later continued as the Société des Amis de l'Aviation), spared no effort in setting up aeronautics in Nancy. In 1909, the organization of the very ambitious Exposition Industrielle de l'Est in Nancy provided him the occasion to propose to the Commission Permanente Internationale d'Aéronautique the candidature of the town as the venue for the IV th international congress of aeronautics. This event lasted 5 days (from September 18 th until September 23 rd ) and was accompanied by several displays of air navigation (especially that of the dirigible "La ville de Nancy", which appeared to have been a severe fiasco). 32 In 1909-1910, Floquet actively campaigned for the choice of Nancy as a staging point of the Circuit de l'Est, an important aviation race "Paris / Toyes / Nancy / Charlevilles-Mézières / Douai / Amiens / Paris" organized by the journal Le matin and rewarded by a 100 000 Francs prize. This event took place in Nancy during a sports week (from August 7 th until 14 th ) and was linked with an aviation festival ending with a cavalcade and the crowning of the aviation queen. 33 The board of the Société des Amis de l'Aviation chaired by Floquet (Rothé being in charge of a technical secretariat) brought together military officers (colonel Lecomte) and local economic, social or political leaders, such as the glass-artist Antonin Daum, Villain (the president of the very influential Société Industrielle de l'Est) or Brun (who was adviser on foreign commerce). The idea of creating an aerodynamic school in Nancy was initiated by Rothé with the aim of creating a framework favourable for a rapprochement between the Army and civilian organizations. Rothé clearly expressed this aim in an article of the Est républicain:

Ce qui donne en effet à la création du Centre de Nancy un caractère tout particulier, c'est que ses fondateurs ont eu l'intention de faire d'abord une oeuvre militaire en dotant Nancy d'escadrilles et de hangars de premier ordre, et aussi une oeuvre civile d'enseignement général en aidant la Faculté des sciences à créer un nouvel Institut doté de cours, de laboratoires de recherches et de postes d'observations. A cet effet, au voisinage de l'aérodrome militaire, la Société a remis à l'Université de Nancy un terrain sur lequel seront édifiés les bâtiments du nouvel institut. Les officiers de l'escadrille y auront librement accès pour y faire les observations et expériences qu'ils jugeront utiles. 34 The conclusion of the article was quite clear concerning the patriotic dimension of the event: "In the presence of the progress achieved by Germany, of the threats of German aviation, our Society will be keen to arouse new contests, new subscriptions". 35 In 1912, the priority for Hirschauer was the strategic separation between what he called school centres (with the finest equipment), depot centres and annexes. At the time Nancy did not appear as a possible school centre and Hirschauer proposed to develop the Châlons and Avor camps, Pau and Versailles Saint-Cyr). A year later, in another report, he nevertheless estimated the military part of the cost of constructing Nancy's aviation centre at almost 86 000 Francs. This amount was nothing in comparison with the estimated costs of the aviation centres for Dijon, Versailles or Lyon (more than 500 000 Francs for each). 39 The project for an institute of aerodynamics and meteorology was first elaborated by Rothé and became a reality in November 1912. As director of this school, he also obtained in June 1913 the creation of a diplôme d'études supérieures aérodynamiques which could be delivered after a one-year degree course. One of the aims of this new diploma was to train plane designers and aviators.

L'enseignement de l'Institut convient à tous les étudiants qui s'intéressent à l'aéronautique dans le but d'entrer dans les maisons de construction ; il est utile aux officiers qui désirent être versés dans les troupes aéronautiques et aux jeunes gens qui veulent y accomplir leur service militaire ; il répond en effet au programme théorique du brevet de pilote militaire. 40 In 1912, amongst the 37 students, several possessed an engineering diploma from the institute of electrical engineering and applied mechanics and 8 were non-commissioned officers or officers. 41 In the absence of a specific building on the aerodrome of Villers-les-Nancy, the courses took place in the mathematics and physics institute. Floquet was in charge of general lectures about aeronautics. Rothé taught aerodynamics, meteorology and wireless telegraphy. Edouard Husson dealt with aeroplane equilibrium and the different types of flight. The curricula also consisted of lectures on engines and screw-propellers (Hahn), on strength of materials (Dumas), on electrical engines (Mauduit) or on meteorology (Marsal). Simon and Guéritot proposed some physics and mathematics classes. The training was completed by several demonstrations on the aerodrome of Villers-les-Nancy (planes, engines or kites) and a talk by Captain Loubigniac 42 , the chief of Nancy's air squadron.43

39 Service Historique de l'Armée de l'Air, AA 13163, doc. AA14

40 Discourse of the chief education officer Charles Adam, Comptes rendus de l'université de Nancy, 1913, pp. 143-144. 41 In 1913-1914, the War Ministry would send 3 lieutenants to the institute (Frelut, Guillemin and Rigaut).

42 Marie-Joseph Loubigniac (1877-1937) Since the beginning of the aeronautics institute project, Rothé defended the idea that the teaching of aerodynamics had to be coupled with the teaching of meteorology. According to him, these two sciences shared historical and conceptual relationships; moreover, inasmuch as the aim of the institute was to train aviators and plane designers and constructors, this association was all the more crucial. In the introduction to the 3 rd volume of his Cours de physique devoted to aerology and aerodynamics Rothé recalled his teaching in Nancy:

Je disais vers la même époque à mes étudiants de Nancy, futurs aviateurs : « C'est aujourd'hui un devoir pour les physiciens de se tourner vers la physique de l'atmosphère et d'apporter une contribution à l'aviation par des recherches nouvelles, recherches qui ne pourront être fructueuses que par une collaboration étroite entre l'aviateur et le physicien ». 44 He pleaded for a methodological and experimental shift to meteorology in order to propose solutions to the problems raised by the development of aviation (« Il faut chercher à résoudre les problèmes que les aviateurs nous soumettront ; il faut en particulier tracer la carte de l'air, comme on a tracé la carte marine… » 45 ). His point of view was far from the romantic visions of the beginnings of aviation and he emphasized the fact that most of the heavier-than-air pioneers were engineers and that the first aeronautic feats relied on previous studies concerning birds, gliders or kites. Rothé insisted on the experimental nature of his teachings in aerodynamics and the only notions of mechanics introduced in his book were those that were necessary for the understanding of experimental methods. He was clearly conversant with the most important works on the resistance of air (Eiffel, Dubuat, Joukowski, Levi-Civita and Villat) and propounded a theory of the flow of air along surfaces. He also commented the fundamental formula of the theory of resistance of air,

R = KSV 2 ,
where R is the resistance, S the surface and V the speed. The constant K depends of temperature, pressure and hygrometry. Rothé argued that with this constant, we can see the fundamental link between aerodynamic and aviation because the characteristics of a plane must be measured under standard atmospheric conditions:

Le fonctionnement du groupe propulseur, moteur et hélice dépend de ces facteurs [pressure, temperature and hygrometry] à un point tel que les essais d'un avion ne doivent pas être effectués dans des conditions quelconques ; ils doivent se ramener à des conditions type, à une atmosphère dite standard. 46 A few weeks before the beginning of the war, Floquet and Rothé were very confident about the future of the aerodynamic institute. The project was supported by the University, the municipality, the economic leaders, the War Ministry and the Public Instruction Ministry. The architect E. André, who was in charge of university buildings 47 , had proposed a 18 500 Francs detailed estimate for the construction of the buildings near the hangars of Villers-les-Nancy.

44 [Rothé 1917], p. VII.

45 Ibidem, p. VII.

46 Ibidem, page X.

47 [Choffel-Mailfert 2007].

Rothé obtained a 20 000 Francs subsidy from the municipal council. André's estimate was approved on June 1 st both by the chief education officer Charles Adam and by… Floquet (the dean of the Faculty of Science).

The war put an end to this project and the building never saw the day. After the war, Rothé went to Strasbourg and the institute existed only virtually through several courses. The airforce left the aerodrome of Villers and created its own meteorological stations. In 1920, the Société des Amis de l'Aviation was moribund: it still counted 214 members but on July 9 th , 109 members voted in favour of its dissolution. Floquet was appointed as a liquidator and he decided to give the remaining money (10 000 Francs) to the Faculty of Science. 48 He died on October 7 th 1920.

V. Conclusion

1914-1918 constituted a turning-point for the University of Nancy. The Belle Époque which was characterized by a strong intellectual effervescence and claims of modernity was followed by a very difficult period. After 1918, the Faculty of Science had to face serious obstacles: many professors left for more attractive posts at the University of Strasbourg. These departures weakened Nancy's scientific institutions which, during interwar years, had to grapple with demographic drain and financial crisis.

An important consequence of the war was the emergence of new strategies centred on the development of engineer training geared to local or regional needs. Before 1914, Bichat, Haller or Floquet fervently stressed the patriotic dimensions of the shift towards applied sciences and aeronautics; these developments were part of an avant-garde national and international strategy. After the war, ambitions became much more modest. Following the recommendations of the ministry enjoining universities to reinforce their links with local industry, a new Institut Métallurgique et Minier was created at the Faculty of Science in 1919.

It was conceived as an adaptation to the needs of regional industry and was partly financed by the Nancy Chamber of Commerce and Industry. 49 The short historical sketch presented in this article highlights the crucial role played by several individuals. Floquet's death and Rothé's departure to Strasbourg definitely closed the aeronautics parentheses. 50 Nevertheless, independently of their importance, it appears that in 1918 only the older and the more solid academic institutions were able to survive; this was the 48 Archives départementales de Meurthe-et-Moselle, 4 M 86. 49 Paul Petit, who was the dean of the Faculty of Science in 1919 thus declared: « Jusqu'ici l'État n'est intervenu en rien, ni pour la première installation, ni pour le fonctionnement de l'Institut métallurgique et minier ; nous espérons que cette création semblant répondre tout à fait aux vue exprimées par M. le Ministre de l'Instruction publique, celui-ci voudra bien accorder largement son appui financier au nouvel Institut, comme aussi aux autres Instituts, constitués dans la même pensée d'adaptation aux besoins de la région. » Comptes rendus de l'université de Nancy, 1919. 50 As for him, Vogt chose to concentrate on the development of electrical engineering as a director of the school of electricity and mechanics and Husson apparently did not try to resume the aeronautics projects.

Faites

  de Nancy un autre Metz, un autre Strasbourg au point de vue moral et intellectuel. Vous le pouvez en attirant les générations nouvelles d'Alsace dans un grand centre d'enseignement scientifique que nous fonderons sur notre nouvelle frontière.

  In 1910, the letterhead of the Société Nationale Aérienne contained the following injunction: "Join the league, enrol your parents, enrol your children […], the German Naval League has a million members". Allegorical plate of Victor Prouvé for the inauguration of the Centre d'aérodynamique: "Beyond the Clouds, for Peace" 36 What was the position of the Army concerning the development of aeronautics in Nancy? The military authorities largely took advantage of the popular fascination for aeronautics. A national fund was launched to support the development of military aeronautics in France. In 1912, colonel Edouard Hirschauer 37 , who was then the permanent inspector of military aeronautics, concluded his official report concerning the strategic organization of French air squadrons by stressing the mobilization of several cities such as Nancy: Beaucoup de Municipalités ou de Départements ont apporté leur concours à la souscription nationale pour l'Aviation Militaire sous forme d'offres de terrains d'atterrissage et de hangars. D'autre part, le Comité de souscription paraît tout à fait désireux de consacrer une partie des fonds de la souscription au développement de cette organisation. C'est ainsi que déjà la ville d'Arras a élevé un hangar sur le terrain de manoeuvres, que la ville de Bar le Duc a acheté un terrain et y a fait édifier des hangars, qu'à Nancy le Comité local se propose également d'avoir son terrain, et ses hangars, etc… Il suffit d'encourager ces mouvements, de les canaliser afin d'avoir ces terrains et ces hangars jalonnant les principaux parcours à travers la France pour posséder un outil aérien tout à fait remarquable. 38

Table 1 -French and foreign students at the Faculty of Science of Nancy (1910- 1921) 20

 1

					Candidates		Certificates delivered
	50
	0
	1910	1911	1912	1913	1914	1915	1916	1917	1918	1919	1920

Table 2 -Evolution of the certificates de licence de sciences at the University of Nancy (1900-1920) for all disciplines 21

 2

	Year	A	B	C	D	E	F	G	H	I	J
		3		0	0	3	4			9	10
		16		0	1	0	1			2	1
		16		2	0	1	3			8	3
		16		0	0	0	6			10	5
		22		1	2	0	4			7	11
		14		4	2	0	2			16	8
		20		0	1	3	0			9	8
		29		4	0	1	9			20	8
		0	29	4	0	0	4		6	15	14
		8	10	6	0	0	1		7	12	12
		3	18	4	0	0	0		5	11	19
		3	21	4	0	0	2	4	8	13	7
		5	22	4	0	0	5	7	7	20	10
		11	14	7	0	2	7	9	4	11	13
		4	14	5	0	3	3	9	7	11	0
		5	10	3	0	0	3	1	0	2	0
		2	7	2	0	0	0	6	2	2	1
		3	9	0	1	0	3	0	5	2	0
		1	0	0	0	0	0	0	0	1	0
		4	19	0	0	0	0	8	7	0	0
		6	96	0	0	3	0	10	19	10	6

  Loin de nous la pensée de nous plaindre de cette diminution momentanée : ceux qui sont partis remplissent aujourd'hui le plus noble des devoirs ; quant à ceux qui ont pu demeurer, MM. Les Doyens sont unanimes à se féliciter de leur travail et de leur assiduité. Les résultats ont été excellents. Souvent la leçon habituelle devient un entretien pour ainsi dire particulier, pour le plus grand profit de l'élève ; telle séance de travaux pratiques ressemble à l'expérimentation de recherche que le maître fait avec ses quelques assistants de laboratoire ; ailleurs, l'enseignement clinique se confond avec le travail clinique auquel se livrent, sous l'oeil du chef, les élèves devenus des collaborateurs pour les soins aux blessés. Parfois, une leçon, un examen sont momentanément interrompus par le tocsin et la canonnade signalant une incursion d'avions ; l'incident passé, la séance continue ; et l'on ne saurait trop insister sur la fermeté tranquille avec laquelle nos élèves se sont adaptés aux conditions peu ordinaires de la vie universitaire à Nancy.Cette rapide esquisse de l'activité de nos collègues non mobilisés serait incomplète, si le rapport annuel n'insistait pas sur la part qu'ils prennent, en dehors de leurs occupations, aux oeuvres plus directement encore en rapport avec la défense nationale, dans les Commissions, dans les OEuvres d'assistance, dans les hôpitaux de la Commission Administrative ou de la Croix-Rouge, dans les services techniques des Instituts pour les questions de chimie, de radiographie, d'aérostation, de météorologie[…].23

  joined the army in 1895 as a private soldier (2 nd class dragoon). He made a quick career. In 1912 he was obtained the rank of captain and was assigned to the service of aviation. He organized the aviation centre of Épinal and his results were highly praised by general Hirschauer. In 1913, he then moved to Nancy to achieve the same task. During the war he worked in the staff headquarters of the War Ministry. The end of his military career was almost entirely devoted to the development and administration of aeronautics.

	Engagé volontaire en 1895 avec un grade peu élevé. Progresse rapidement dans l'armée. En 1912, il organise le
	centre d'aviation d'Épinal avec un résultat qui lui vaut les félicitations du général Hrischauer. En 1913 il
	s'occupe de Nancy.

[Grelon & Birck 1998],[Birck & Grelon 2006],[Rollet & Choffel-Mailfert 2007].

Baccalauréat was not compulsory to study in those technical institutes and students did not always come from the classes préparatoires aux Grandes Ecoles.

Concerning the aims of mathematical training in engineer education, he thus declared in 1911: « Il ne s'agit donc pas d'affaiblir l'enseignement des mathématiques, mais de le diriger vers le but à atteindre ; s'il est réduit en étendue, il doit être approfondi et approprié aux applications; il doit être poussé jusqu'à la détermination des valeurs numériques et de l'approximation qu'elles peuvent comporter ».[Nabonnand 2006].

As we shall see, he would be the first (and only) director of the institut aérodynamique, officially founded in 1913.

[Birck 2006]. See also:[Choffel-Mailfert 2007].

Were they sent to the army? This hypothesis is indeed plausible, but one could also suppose that their parttime lectures were suppressed because of the decrease of the number of students. Despite the lack of precise information concerning the two teachers it seems also reasonable to assume that they were more than 40 years old.

 15 But the official charts of the Faculty of Science contain many errors. For instance, Simon is mentioned until 1917 although he was in the army since

1914.16 His name is present in the organization chart of the faculty but the Comptes rendus de l'université de Nancy don't mention any of his courses, which seemed to be made by Floquet.

At the beginning of 1914 academic year, all the courses in which some students had been registered had been opened. For lack of students the courses of rational mechanics, astronomy, aerodynamics and brewery had been suppressed.

The situation was quite comparable in the other faculties: 7 out of 17 at the Faculty of Law, 5 out 18 at the Faculty of Medicine, 7 out of 14 at the Faculty of Arts and 9 out of 10 at the École supérieure de pharmacie. Comptes rendus de l'universitéde Nancy,

19[START_REF] Grelon | [END_REF]In -1915, the Faculty of Law had 66 students, the Faculty of Medicine 27, the Faculty of Arts 18 and the École supérieure de pharmacie 25. Comptes rendus de l'universitéde Nancy, 1916. 20 Source: Comptes rendus de l'université de Nancy.

Source: Comptes rendus de l'université de Nancy. See also[Nabonnand 2006].

Photo Dufey, reproduite dans Nancy sous les obus, images de guerre,Nancy, Albert Barbier imprimeur, 1918.

This aerodrome had been created in 1913. At the beginning, its squadron was made of 6 Farman planes.

The league was presided by René Quinton and gathered several members of the Academy of Science, such as Paul Appell, Jean Bouquet de la Grye, Pierre Léauté, etc.

Albert Étévé (1880Albert Étévé ( -1976) ) and Jean EtienneCheutin (1880Cheutin ( -1938) ) were two pioneers of military aviation. Cheutin had been of the first to obtain his pilot's licence (licence n° 233). He left active service in 1932 with the rank of general. Étévé was at the time of his conference in Nancy the director of Versailles' military aviation school. As an engineer in aeronautics he developed various important devices such as an anemometer (anemometer Étévé) or a machine gun turret (système Étévé). He was to become a general inspector of aeronautics during the Interwar period.

Menu of the banquet of July 1918, 12 th . Archives municipales de Nancy, 1 I-302.

Auguste Édouard Hirschauer (1857-1943) was one of the fathers of modern military aviation. During the war, he gained major successes during the battle of Craonne in 1917. After 1919, he retired the army and began a political career as a senator.

Service Historique de l'Armée de l'Air, AA 13163, doc. AA5.

Comptes rendus de l'universitéde Nancy, 1913de Nancy, , 1914de Nancy, and 1915. .


Anjing Yang

Xiaoli Zhang
email: xlzhang@yic.ac.cn

Hélène Agogué

Christine Dupuy

Jun Gong
email: jgong@yic.ac.cn

Contrasting spatiotemporal patterns

Keywords:

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

Introduction

Microbe-driven nitrification, denitrification, and the anaerobic ammonium oxidation (anammox) play a pivotal role in nitrogen cycling in estuarine and coastal systems [START_REF] Spencer | Distribution and partitioning of heavy metals in estuarine sediment cores and implications for the use of sediment quality standards[END_REF][START_REF] Howarth | Nitrogen as the limiting nutrient for eutrophication in coastal marine ecosystems: evolving views over three decades[END_REF][START_REF] Lam | Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea[END_REF]). These processes can relieve nitrogen load delivered to coastal waters, reducing the risk of eutrophication [START_REF] Howarth | Coastal nitrogen pollution: a review of sources and trends globally and regionally[END_REF].

The spatial and temporal patterns of the diversity and distribution of N-cycling functional groups underlie the changes of N-cycling processes. Based on the amoA gene (encoding ammonia monooxygenase), Ammonia oxidation was previously thought to be restricted to ammonia-oxidizing bacteria (AOB), whichare mostly presented by the β-proteobacterial Nitrosomonas and Nitrosospira, and a few in the γ-proteobacterial Nitrosococcus [START_REF] Purkhold | Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys[END_REF][START_REF] Horz | Ammonia-oxidizing bacteria respond to multifactorial global change[END_REF]). Dang et al. (2010b) reported that nearby wastewater treatment plants and polluted rivers could have significant impact on AOB composition and distribution of Jiaozhou Bay estuary. Afterwards, ammonia-oxidizing archaea (AOA) belonging to the Crenarchaeota Group 1.1a and Group 1.1b (now known as a separate clade, Thaumarchaeota, [START_REF] Brochier-Armanet | Mesophilic crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota[END_REF]) were revealed to be critical for the global nitrogen cycle, which also had functional amoA gene [START_REF] Francis | New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation[END_REF][START_REF] Zhang | Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils[END_REF]. [START_REF] Francis | Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[END_REF] observed that AOA were pervasive in marine water columns and sediments, and diverse and distinct AOA communities were associated with each of these habitats. 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] Scala | Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments[END_REF] investigated the diversity of nosZ gene in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves and found that denitrifier communities might be restricted geographically. Like denitrification, anammox contributed significantly to the production of N 2 . The anammox bacterial specific 16S rRNA (AMB 16S rRNA) gene in Mai Po estuary sediment performed strong seasonal dynamics due to the anthropogenic and terrestrial inputs [START_REF] Li | Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes[END_REF].

Salinity has been reported as the most important driver for ammonia oxidizer communities. The shift from low-salinity Nitrosomonas communities to high-salinity Nitrosospira communities has been observed in many estuarine systems [START_REF] Francis | Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments[END_REF][START_REF] Bernhard | Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system[END_REF][START_REF] Jin | Diversity and quantity of ammonia-oxidizing archaea and bacteria in sediment of the Pearl River Estuary, China[END_REF]. AOA amoA sequences often formed distinct groups according to salinity (Moister and Francis 2008;[START_REF] Abell | Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary[END_REF]. Additionally, AOA were proposed to be important actors in low-nutrient, low-pH, and sulfide-containing environments [START_REF] Erguder | Environmental factors shaping the ecological niches of ammonia-oxidizing archaea[END_REF]). There were spatial and temporal variations of denitrifying communities at the Fitzroy River and San Francisco Bay estuaries, and salinity, organic carbon, nitrogen, chlorophyll-α and some metals were found to be factors influencing the community structure [START_REF] Abell | Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary[END_REF]Moister and Francis 2010). The anammox bacterial distributions presented strong spatial and seasonal variations along the Cape Fear River estuarine gradient, which were also highly correlated with salinity variation [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF]). Nevertheless, our knowledge about spatiotemporal patterns and controls of N-cycling microbial communities in tidal flats with high anthropogenic disturbance is limited. The dynamics of these nitrogen-removal groups are likely to be complex and tightly coupled when they compete for a common ecological niche. However, studies examining and comparing the seasonal and spatial patterns of ammonia oxidizers, denitrifiers, and anammox bacteria in a single survey are still rare.

Estuarine sediments are also sinks of metals from different origins. Trace amounts of some metals, which are necessary cofactors in enzymes or co-enzymes and electron transport chains, can be stimulatory to microbial activity [START_REF] Granger | Accumulation of nitrogen oxides in copper-limited cultures of denitrifying bacteria[END_REF]Ward 2003, Yang et al. 2013). There are evidences that metals may be an important factor in regulating nitrogen transformations in sediment habitats. For example, in the Douro River estuary (north-west Portugal), the transcription diversity of nosZ gene showed a drastic decrease with the increase of Cu concentration [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]). The metal toxicity was modulated by sediment properties (metal concentrations, grain size, organic carbon to nitrogen ratio, etc.), and denitrification revealed high sensitivity to heavy metals Cu, Cr, Pb, Zn and Cd in sandy sites but not in muddy sites [START_REF] Magalhães | Impact of trace metals on denitrification in estuarine sediments of the Douro River estuary, Portugal[END_REF]. However, the effect of heavy metals on nitrogen cycling populations in sediments has not been studied sufficiently, and little is known about the linkage between metals and the nitrogen microbial community structure in estuarine systems.

Estuarine tidal flats of the Laizhou Bay (LZB), a semi-enclosed bay of the Bohai Sea, northern China, have been hypernutrified due to high level of dissolved inorganic nitrogen (DIN), organic pollutants and heavy metals discharged from coastal industries (e.g. aquaculture, subsurface brine industries and dyeing industries) and agriculture [START_REF] Hu | Polycyclic aromatic hydrocarbons in surface sediments of Laizhou Bay, Bohai Sea, China[END_REF][START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF]. These tidal flats thus present ideal environments for studying the multiple environmental stresses on nitrogen-removal microorganisms. We have carried out an ecological study on benthic microbial nitrogen cycling in this area, and demonstrated that abundances of N-cycling functional groups respond differently to variations of environmental conditions, and metals Cu and Cd affect AOA/AOB dominance [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF]. As another contribution, this study focused on the following objectives: (1) to investigate the relative importance of spatial (locations, layers) and seasonal controls on the distribution of ammonia oxidizers, denitrifiers, and anammox bacteria, and (2) to test the hypothesis that metals impact the diversity and community composition of nitrogen microbial community in the estuarine system.

Materials and Methods

Sampling and environmental parameters analysis

The physical conditions of three hypernutrified estuarine tidal flats in mouths of Jiaolai River (JL), Bailang River (BL) and Di River (Di) of the Laizhou Bay has been described previously in detail [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF]. These three rivers have different pollution history and discharge sources (e.g. dying industries, mariculture and brine industries). The sampling and analysis of the environmental parameters including dissolved oxygen, pH, salinity, and temperature of overlying water, and nitrate, nitrite, ammonium, total organic carbon and nitrogen contents, sediment grain size, and trace metal As, Co, Cd, Cr, Cu, Ni, Pb, Zn levels of sediments, were also performed as described in the previous publication [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF].

In brief, surface sediment samples were collected in 2010 winter (November) and 2011 summer (August), and three sediment replicates (JL1-3, BL1-3 and Di1-3) were randomly sampled in each estuarine tidal flat. The sediment cores were sectioned into the upper layer (0-2 cm) and the lower layer (2-5 cm). The sediment samples were referred to as the location, layer and season collected (e.g. JL-U-W and JL-L-W).

DNA extraction and PCR amplification

The environmental genomic DNA was extracted from 0.5 g of sediment using the Ultra Clean Soil DNA Isolation kit (Mo-Bio, USA). DNA concentration was quantified using a NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). The DNA was diluted 10 times before the PCR amplification.

PCR primers used in this study were shown in Table 1. The PCR amplification procedure was performed with a Tprofessional Thermocycler (Biometra, Germany) using the PCR kit DreamTaq TM Green PCR Master Mix (Fermentas, USA). The reactions were set up in volumes of 25 μl containing 1 μl template DNA, 400 nM of each primer, and 12 μl of PCR Master Mix. PCR programs were as follows: 95°C for 3 min, 30-35 cycles of 95°C for 30 s, 57°C (amoA1F/amoA-rnew) or 56°C (Arch-amoAF/Arch-amoAR) or 53°C (nosZF/nosZ1622R) or 58°C (Pla46f-GC/Amx368r) for 40 s, followed by 72°C for 40 s, and finally 72°C for 10 min.

Denaturing gradient gel electrophoresis, cloning and sequencing PCR products of AMB 16S rRNA gene were analyzed by denaturing gradient gel electrophoresis (DGGE) with a DCode mutation detection system (Bio-Rad, USA). Forty μl PCR products (100-200 ng/μl) were loaded onto a 6% polyacrylamide gel with a denaturant gradient between 20% and 80% (100% denaturant containing 7 M urea and 40% formamide). Electrophoreses were run at a constant voltage of 200 V and 60°C for 5h. Subsequently, the gels were stained for 30 min in 1× GeneFinder (Bio-V, China), and then visualized in an imaging system (Syngene, USA). The main bands were excised and incubated overnight at 4°C in 30μl sterilized deionized water as templates for reamplification. The PCR products were checked for single bands on DGGE, purified using a Purification Kit (Tiangen Biotech, China), and sequenced by a commercial company (Sangon, Shanghai, China). DGGE images were analyzed using Quantity One 2.1 (Bio-Rad, USA) to generate a densitometric profile. The peak areas of the fingerprint patterns were used to indicate the intensities. Bands with a relative intensity of less than 0.5% of the sum of all band intensities were discarded.

As for AOB amoA, AOA amoA and nosZ genes, triplicate of PCR products were pooled, ligated into the pTZ57R/T vector (Fermentas), and transferred into competent Escherichia coli TOP10 cells (Tiangen). Therefore, 12 clone libraries of each gene were constructed for 3 sampling locations, 2 layers and 2 seasons. Positive recombinants were selected using X-Gal-IPTG LB indicator plates amended with ampicillin (100 mg/ml). The insertion was determined by PCR amplification with the universal primer set M13F and M13R. Amplicons of correct size were digested separately with endonucleases (Fermentas) HhaI, RsaI for amoA gene [START_REF] Jin | Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR[END_REF], and MspI, RsaI for nosZ gene [START_REF] Rich | Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils[END_REF]. Restriction fragments were resolved by electrophoresis on 2.5% agarose gels. Each restriction fragment length polymorphism (RFLP) pattern was defined as an operational taxonomic unit (OTU), and representative clones were randomly selected for sequencing (Sangon).

Alpha diversities (local diversities of a given community), such as Shannon (H) and Simpson (D) indices, were calculated based on number and intensities of DGGE bands, or number and relative abundance of OTUs in libraries.

These indices were calculated with an online diversity calculator (http://www.changbioscience.com). The coverage (C) of clone libraries was calculated as C=[1-(n1/N)]×100, where n1 is the number of unique (frequency=1) RFLP pattern detected in a library and N is the total number of clones in the same library [START_REF] Mullins | Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities[END_REF].

Phylogenetic analysis and sequence deposition

Possible chimerical DNA sequences were checked with programs CHIMERA_CHECK [START_REF] Gontcharova | Black box chimera check (B2C2): a windows-based software for batch depletion of chimeras from bacterial 16S rRNA gene datasets[END_REF] and Bellerophon [START_REF] Huber | Bellerophon: a program to detect chimeric sequences in multiple sequence alignments[END_REF]). Nucleotide sequences were aligned with GenBank sequences using ClustalW [START_REF] Thompson | Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequencing weighting, position sequence gap penalties and weight matrix choice[END_REF]. Phylogenetic trees were constructed with MEGA 5.0 [START_REF] Tamura | MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony ethods[END_REF]) using the neighbor-joining and method, and bootstrap resampling analysis for 1,000 replicates was performed to estimate the confidence of the tree topologies.

The nucleotide sequences obtained in this study have been deposited in the GenBank database under accession numbers JX465173 to JX465197 (AMB 16S rRNA), JX465198 to JX465201 (AOA amoA), JX465202 to JX465230 (AOB amoA), and JX465231 to JX465276 (nosZ).

Statistical analyses

Mean values of alpha diversities were compared with the pairwise t-test or one-way ANOVA analysis following by a least significance difference (LSD) test at the 0.05 confidence level. Spearman's correlation coefficient (ρ) was calculated to explore the relationship between alpha diversities and environmental variables. These analyses were performed using the statistic software SPSS 13.0 for windows (SPSS, Chicago, USA).

Community clustering of nitrogen functional groups was analyzed with the principal coordinate analysis (PCoA) using the UniFrac program [START_REF] Lozupone | UniFrac: a new phylogenetic method for comparing microbial communities[END_REF], according to the instructions at the UniFrac website (http://bmf2.colorado.edu/unifrac/index.psp). Differences in community composition clustered by sampling location, layer and season, were pairwise or globally tested based on weighted UniFrac metric. Relationships between microbiota and environmental factors were analyzed using the software CANOCO (version 4.5, Microcomputer Power, Ithaca, USA) [START_REF] Ter-Braak | CANOCO reference manual and CanoDraw for windows user's guide: software for[END_REF]. A detrended correspondence analysis (DCA) was conducted in order to decide whether a canonical correspondence analysis (CCA) or redundancy analysis (RDA) should be used in ordination [START_REF] Ysebaert | Spatial and temporal variation in benthic macrofauna and relationships with environmental variables in an estuarine, intertidal soft-sediment environment[END_REF]. The statistical significance of the variable added was tested using a Monte Carlo permutation test (999 permutations).

Results

DGGE and clone library analyses

A total of 321 bands were detected in DGGE gels of AMB 16S rRNA (Fig. S1). The bands from triplicate samples were combined for subsequent analyses, and then the number of bands per sample varied between 7 and 16 (Table S1). Of 12 amoA genes of AOB and AOA, and nosZ gene clone libraries, 375, 394 and 380 insert-positive clones were identified, resulting in 29, 4 and 46 unique OTUs, respectively. The numbers of OTUs ranged from 2 to 9 (for AOB), from 2 to 4 (for AOA), and from 4 to 10 (for nosZ) (Table S1). The coverage (C) values of bacterial and archaeal amoA and nosZ gene libraries were more than 80%, indicating that most ammonia oxidizer and denitrifier had been detected. Archaeal amoA gene exhibited relatively lower diversities (H 0.10 ~ 0.60; D 0.08 ~ 0.28), while AMB 16S rRNA genes appeared highly diverse (H 1.86 ~ 2.68; D 0.83 ~ 0.93) (Table S1).

Spatial and seasonal patterns of alpha diversities ANOVA and t-test analyses were performed to compare the alpha diversity indices of ammonia oxidizers, denitrifiers and anammox bacteria from different sampling locations (JL, BL and Di), layers (upper and lower) and seasons (winter and summer) (Table 2). The results showed that the alpha diversities of AMB varied greatly among sampling locations, layers, or seasons, with significantly lower indices in Di tidal flat (vs. JL and BL) (P=0.004 for H; P=0.001 for D), in upper layer (vs. lower) (P=0.003 for H; P=0.018 for D) and in winter (vs. summer) (P=0.015 for H; P=0.022 for D).

Alpha diversities of AOB amoA gene were significantly higher in winter than in summer (P=0.025 for H; P=0.048 for D). Nevertheless, AOA amoA gene was more diverse in the lower than in the upper layer in terms of D index (P=0.043), but not in H (P=0.068). No differences were found for alpha diversities of nosZ gene.

Correlations between alpha diversities and environmental factors

In order to explore the relationship between gene diversities and environmental variables, Spearman's correlations were performed (Table 3). Among the eight metals determined, six (Co, Cr, Cu, Ni, Pb and Zn) were collinear (ρ> 0.

Phylogenetic analyses

The phylogenetic tree based on partial AOB amoA gene sequences revealed that no OTUs were common across all samples (Fig. S2). BLASTing against GenBank revealed that A large proportion (246 out of 375) of the clones closely matched with these found from hypernutrified sedimentary environments including San Francisco Bay [START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF]), Pearl River [START_REF] Jin | Diversity and quantity of ammonia-oxidizing archaea and bacteria in sediment of the Pearl River Estuary, China[END_REF], Jiulong River, and Jiaozhou Bay (Dang et al. 2010b), indicating anammox bacterial assemblages in these coastal environments are phylogenetically similar regardless of geographic locations. All

OTUs of AOB amoA were affiliated to β-Proteobacteria and distributed into 8 clusters. Two clusters containing 120 clones appeared to be Nitrosospira-related, with the remaining clusters Nitrosomonas-related. In the Nitrosospira-related cluster, some OTUs were first detected in this study, probably representing novel Nitrosospira lineages. A Nitrosomonas-related OTU (AOB8) was closely related to sequences from Jiaozhou Bay A5 station, which was characterized by high efflux of heavy metals from wastewater treatment plant (Dang et al. 2010b).

Phylogenetic analysis of AOA amoA gene showed that a majority (364 out of 394) of sequences were placed in the "marine sediment/water" Crenarchaeota clade along with Nitrosopumilus maritimus (HM345611), a representative of Crenarchaeota group 1.1a (Fig. S3). Phylogenetically, these sequences were closely related to those from sediments of Jiaozhou Bay estuary [START_REF] Dang | Diversity and spatial distribution of sediment ammonia-oxidizing crenarchaeota in response to estuarine and environmental gradients in the Changjiang Estuary and East China Sea[END_REF]), Bahia del Tobari estuary [START_REF] Beman | Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahia del Tobari, Mexico[END_REF] as well as wastewater treatment plants (WWTP). Only 30 clones (7.6%) fell exclusively into the "hot spring/soil" Crenarchaeota clade, which includes the Crenarchaeota group 1.1b clone Candidatus Nitrososphaera gargensis GA15P03. Sequences between two groups exhibited only 79~80% similarities at the nucleotide level. The most dominant OTU AOA1 (88.8%) occurred in all clone libraries. Interestingly, OTU AOA4 was never recovered from the Di tidal flat.

All nosZ OTUs were grouped into 3 major clusters in the phylogenetic tree (Fig. S4). Cluster 1 was mainly comprised of clones closely related to α-and β-Proteobacteria, cluster 2 mostly to γ-Proteobacteria, and cluster 3 to α-Proteobacteria only. There were no common genotypes across all samples and no OTUs appeared particularly dominant. More than half of nosZ sequences were affiliated with the α-proteobacterial genera, such as Ruegeria, Nisaea, Polymorphum, Rhodobacter, Azospirillum, Mesorhizobium and Bradyrhizobium. These genera were previously found in marine sediment habitats, polluted soils, and activated sludge [START_REF] Huang | Effect of addition of Rhodobacter sp. to activiated-sludge reactors treating piggery wastewater[END_REF][START_REF] Urios | Nisaea denitrificans gen. nov., sp. nov. and Nisaea nitritireducens sp. nov., two novel members of the class Alphaproteobacteria from the mediterranean sea[END_REF][START_REF] Huo | Ruegeria marina sp. nov., isolated from marine sediment[END_REF][START_REF] Nie | The genome sequence of Polymorphum gilvum SL003B-26A1 T reveals its genetic basis for crude oil degradation and adaptation to the saline soil[END_REF]. The second most abundant sequences were related to γ-Proteobacteria, including genera Halomonas, Marinobacter, Pseudomonas and Alcaligenes, which were used in discoloration [START_REF] Mabinya | Halomonas sp. OKOH-a marine bacterium isolated from the bottom sediment of Algoa Bay-produces a polysaccharide bioflocculant: partial characterization and biochemical analysis of its properties[END_REF], heavy metals biosorption [START_REF] Hussein | Biosorption of heavy metals from waste water using Pseudomonas sp[END_REF] or reduction of chemical oxygen demand (COD) [START_REF] Rajeshkumar | Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7[END_REF]) from high salt wastewater.

A total of 25 DGGE gel bands of AMB in different positions were excised and sequenced. All sequences grouped into 4 clusters in the phylogenetic tree based on the 16S rRNA genes (Fig. S5). A large proportion (15 out of 25) of our sequences was grouped into the cluster 1, a distinct clade with <87% sequence identity against GenBank, suggesting novel genetic diversity of AMB species in the Laizhou Bay estuaries. In cluster 2, four sequences affiliated to Candidatus Scalindua were only recovered from winter samples. None of our sequences was placed in Cluster 3, which included other well-known AMB (e.g., Kuenenia, Brocadia, Jettenia asiatica, and Anammoxoxoglobus propionicus).

Cluster 4, a putative anammox or Planctomycetes cluster, contained the sequences only occurring in summer.

Spatial and seasonal patterns of community structure

According to weighted UniFrac significance tests, the community structure of AOB (P=0.03) and AMB (P<0.01) was seasonally different. However, the community differences in AOA or nosZ-type denitrifiers were insignificant across locations, layers or seasons (P > 0.10) (Table 4). These results were consistent with the PCoA plots (Fig. 1), in which AOB and AMB were evidently separated into winter and summer groups, with the first principal coordinates (P1) explaining 73.02% and 77.93% of the total community variability respectively, whereas no distinct patterns could be recognized for either AOA or denitrifiers.

Environmental factors influencing community turnover

To further understand how environmental factors impacted target gene distributions, the weighted CCA analyses were made. DCA results showed that CCA model better approximated AOB amoA and nosZ species relationship to the explanatory variables (maximum gradient length greater than 4), but RDA model should be selected for AMB 16S rRNA and AOA amoA (maximum gradient length less than 3). The optimal models were produced with manual deselection of collinear variables and manual forward selection via Monte Carlo permutation significance tests. Of all the environmental factors analyzed, salinity was significantly correlated with community changes of AOB (P=0.009; Fig. 2A). Sediment grain size was the most important factor influencing the community structure of AOA (P=0.048) (Fig. 2B). The metal As had the strongest correlation with nosZ-type denitrifier community changes, though no variables were significant (P=0.16) (Fig. 2C). For AMB, several environmental factors, such as temperature (P=0.001), dissolved oxygen (P=0.007), pH (P=0.016) and metals As (P=0.029) and Cd (P=0.03), presented significant correlations (Fig. 2D); summertime samples of the Di tidal flat with high pH values grouped separately from these of JL and BL (Fig. 2D).

Discussion

Sediments provide a range of niches for N-cycling microbes which could co-occur or spatiotemporally isolate. Our study investigated the alpha diversities and community structure of four functional groups, which are responsible for interlinked N-cycling processes in sediment samples. We explored their spatial and seasonal patterns and associations with environmental conditions in estuarine tidal flats. It should be noted that the methodologies (i.e., DGGE and clone library analysis) we employed in this study may have some limitations. These include the non-intensive sampling of communities, which leads to the incapability to detect uncommon or rare phylotypes in the communities and thus underestimation of richness [START_REF] Bent | The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity[END_REF]. Nevertheless, Shannon and Simpson indices can be estimated more accurately because rare phylotypes generally have a smaller relative numerical impact, and the use of fingerprinting methods for many of samples, followed by cluster analysis or clone library analysis, are still useful in revealing distributional patterns and composition of microbial communities [START_REF] Bent | The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity[END_REF]. In this study, we found that ammonia oxidizers, denitrifiers, and anammox bacteria had contrasting distributional patterns across locations, layers or seasons, which provides an integrated view of the community assembly and underlying mechanisms of N-cycling functional groups in the these typical coastal habitats.

Ammonia oxidizers

Differences in ammonia oxidizers distribution in estuaries are undoubtedly determined by the complex interplay of biological and environmental variables. In this study, both ANOVA and UniFrac significance tests indicated that AOB diversity and community turnover in sediment exhibited distinct seasonal patterns (Fig. 1, Table 2). Furthermore, our correlation analysis indicated that the lower diversity of AOB in summer could be due to higher concentrations of ammonium and As in the sediment and the higher temperature (Table 3). The higher temperature will increase the activity of heterotrophic microbes in sediment, leading to the increase of remineralization which supplies more ammonium for ammonia oxidizers, of which increase of abundance should be expected. In contrast, we observed a negative correlation between AOB abundance and temperature [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF]. Despite that AOB diversity was demonstrated to be higher in low-salinity sites than in high-salinity sites [START_REF] Sahan | Diversity and spatio-temporal distribution of ammonia oxidizing archaea and bacteria in sediments of the Westerschelde estuary[END_REF], no significant correlations between salinity and alpha diversities were found in our study. These indicate that temperature should represent a highly limited factor for AOB diversity and community size, for which differentiated ecology of AOB clades at different temperatures could be accounted. For instance, [START_REF] Sahan | Diversity and spatio-temporal distribution of ammonia oxidizing archaea and bacteria in sediments of the Westerschelde estuary[END_REF] showed that Nitrosomonas spp.

often was dominant in high-temperature (May, July and September) seasons, while Nitrosospira was abundant in low-temperature seasons in estuarine sediments. [START_REF] Tourna | Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms[END_REF] demonstrated that in slurry incubation, Nitrosospira cluster 1 completely disappeared at high temperature (30°C). Our CCA showed that salinity was the most influential physicochemical factor structuring AOB community composition (Fig. 2), which is consistent with previous studies [START_REF] Francis | Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments[END_REF][START_REF] Bernhard | Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system[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] Jin | Diversity and quantity of ammonia-oxidizing archaea and bacteria in sediment of the Pearl River Estuary, China[END_REF]). AOB at the high-salinity sites were most similar to Nitrosospira clade, but at the mid-and low-salinity sites were distributed among Nitrosospira and Nitrosomonas sequences [START_REF] Bernhard | Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system[END_REF]. Taken together, these data suggest that alpha diversity and community composition of AOB in surface sediment are primarily shaped by temperature and salinity, respectively, which, in turn, account for the seasonal patterns of AOB distribution in these environments.

Our data suggest that AOA have rather stable diversity and composition in estuarine sediments, regardless of reason and location (Table 2,4). The only significant factors we observed for Simpson index and community structure lie in the vertical distribution (upper and lower layers) and grain size of sediment (Table 2, Fig. 2). Nevertheless, although this study demonstrates clearly the distributional patterns of AOA in both diversity and composition in estuarine sediments, the underlying environmental mechanisms remain inconclusive, as the different layers and grain sizes of sediment are closely related to other environmental parameters (Andrieux-Loyer and Aminot 2001), such as dissolved oxygen and pH, which were not measured for sediment in this study. In fact, sediment pH was found to be important for AOA distribution in sediments and soils [START_REF] Erguder | Environmental factors shaping the ecological niches of ammonia-oxidizing archaea[END_REF][START_REF] Li | Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes[END_REF].

The negative correlation between AOB diversity and metal As found in this study is coincident with our recent report that the AOB abundance was negatively correlated with As in the studied samples [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF], suggesting that AOB are sensitive to As, as have been demonstrated in a laboratory study that As (III and V) decreased soil potential nitrification activity and was toxic to AOB [START_REF] Gong | Assessment of pollution-induced microbial community tolerance to heavy metals in soil using ammonia-oxidizing bacteria and biolog assay[END_REF]. The metal As is toxic to bacteria by inhibiting basic cellular functions, which are often linked with energy metabolism, microbial biomass C and respiration [START_REF] Ghosh | Effect of arsenic contamination on microbial biomass and its activities in arsenic contaminated soils of Gangetic West Bengal, India[END_REF]). Nevertheless, no correlation between AOA and metals was found in this study, suggesting that AOA could be more tolerant to As than AOB. In contrast, metal As contributed the most to AOA distinction in polluted mangrove sediments [START_REF] Cao | Diversity and abundance of ammonia-oxidizing archaea and bacteria in polluted mangrove sediment[END_REF], possibly because As presented with much higher concentrations in the mangrove sediment.

Denitrifiers

Similar to AOA, nosZ-type denitrifiers exhibited neither spatial nor seasonal patterns of diversity and composition in this study. This is contrast to the notion that salinity is a major driver of denitrifying communities in aquatic environments on the global scale [START_REF] Jones | Ecological and evolutionary factors underlying global and local assembly of denitrifier communities[END_REF]. The insignificant effect of salinity on community turnover of nosZ-type denitrifiers could be due to the fact that most of our samples were much more saline (36.3-50.3 psu) than generally expected for estuarine samples. The positive correlation between salinity and nosZ gene diversity in this study remain unexplainable (Table 3), as many bacteria cannot survive in high-salinity environments, thus the high salinity should limit the bacterial diversity.

Pb was negatively correlated with alpha diversities of nosZ gene (Table 3), suggesting a potentially toxic effect of Pb on nosZ-type denitrifiers. The links between heavy metals and denitrifiers have been demonstrated in previous studies. For example, increase of Pb concentration in soils led to greater sensitivity of N 2 O reductase [START_REF] Bollag | Effect of heavy metals on the denitrification process in soil[END_REF]. Pb also affected the diversity of soil nirK-type denitrifiers even at the low concentration (1 ppm) [START_REF] Sobolev | Effects of heavy metal contamination upon soil microbes: lead-induced changes in general and denitrifying microbial communities as evidenced by molecular markers[END_REF]. In San Francisco Bay estuary, Pb was also closely correlated with nirS-type denitrifier abundance (Moister and Francis 2010). Nevertheless, these potential effects on denitrifiers should be only attributed to

Pb, the associations with other metals (Cu, Co, Cr, Ni and Zn), which are collinear with Pb, are also potential contributors [START_REF] Cao | Microbial community composition and denitrifying enzyme activities in salt marsh sediments[END_REF].

Anammox bacteria

Comparing with other estuarine environments, such as Cape Fear River estuary [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF], Mai Po Nature

Reserve estuary [START_REF] Li | Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes[END_REF] and Yodo River estuary [START_REF] Amano | Detection of anammox activity and diversity of anammox bacteria-related 16S rRNA genes in coastal marine sediment in Japan[END_REF], the diversity of AMB in Laizhou Bay estuaries was relatively lower, as only the "Scalindua"-like phylotypes were recovered. This result is consistent with well-known adaptation of "Scalindua" to saline environments, and "Kuenenia" and "Brocadia" are low-salinity taxa [START_REF] Dale | Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary[END_REF][START_REF] Junier | Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment[END_REF]. Similar to a previous study of Jiaozhou Bay estuaries (Dang et al. 2010a), this study of LZB estuaries also revealed some phylotypes which presented as putatively new clusters of AMB within the phylum Planctomycetes (Fig. S5). Despite of the lower alpha diversities, the abundance of AMB was high in the investigated sites [START_REF] Zhang | Relative abundance of ammonia oxidizers, denitrifiers, and anammox bacteria in sediments of hypernutrified estuarine tidal flats and in relation to environmental conditions[END_REF], suggesting an important role of AMB in N 2 production in the sediments of LZB tidal flats.

In the high-salinity estuaries of LZB, we identified temperature and pH were significantly correlated with community changes of AMB (Fig. 2). High temperature environments provide special niches for some AMB, such as the presence of "Brocadia" and "Kuenenia" phylotypes in hot spring and deep-sea hydrothermal vents [START_REF] Byrne | Presence and a ctivity of anaerobic ammonium-oxidizing bacteria at deep-sea hydrothermal vents[END_REF][START_REF] Jaeschke | 16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs[END_REF], and a novel "Scalindua sinooilfield" clade in high-temperature oil reservoirs [START_REF] Li | Molecular detection of anaerobic ammonium-oxidizing (anammox) bacteria in high-temperature petroleum reservoirs[END_REF].

pH can affect the equilibrium of NH 3 /NH 4 + in environments. Higher pH is conducive to the formation of free NH 3 , of which higher concentrations could inhibit anammox [START_REF] Aktan | Inhibitory effects of free ammonia on Anammox bacteria[END_REF]. This explains the negative correlations of pH with diversity (Table 3), and composition of AMB (Fig. 2), and a separate AMB assemblage in the Di estuary where a large amount of alkaline water was discharged. In the meantime, our data showed that higher C/N ratio could promote the diversity of AMB. However, [START_REF] Hu | Distribution and diversity of anaerobic ammonium-oxidizing bacteria in the sediments of the Qiantang River[END_REF] observed higher diversity of AMB in samples with lower organic contents. The strong competition or cooperation for nitrite may occur between AMB and denitrifiers in low organic environments, leading to higher diversity of AMB [START_REF] Dalsgaard | Production of N 2 through anaerobic ammonium oxidation coupled to nitratereduction in marine sediments[END_REF][START_REF] Rysgaard | Denitrification and anammox activity in Arctic marine sediments[END_REF]). In addition, heavy metals (e.g. Pb, As and Cd) also strongly influenced AMB diversity and composition in LZB sediment samples (Fig. 2, Table 3). Similarly, a significant correlation between Pb and hzo gene (a functional gene of AMB) diversity was revealed [START_REF] Li | Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes[END_REF].

Conclusions

In summary, we investigated the spatial and seasonal patterns of alpha diversities and community turnover of co-occurring AOA, AOB, nosZ-type denitrifiers and AMB in sediment samples of LZB estuarine tidal flats. Generally speaking, these four N-cycling functional groups exhibited contrasting distribution patterns: seasonality was evident for diversities and community composition of AOB and AMB, but not for AOA and nosZ-type denitrifiers; diversities of these N-cycling groups seldom varied with location of sampling sites or sediment layer, except for AMB which exhibited significant difference among locations and between layers. Further analyses identified different sets of physicochemical parameters that could explain the distributional patterns of these functional groups. In particular, we found that the distributions of bacterial N-cycling players (e.g., AOB, nosZ-type denitrifier and AMB) were potentially related to several heavy metals in sediments, which, however, was never found for AOA in this study. Overall, our results highlight the differentiated distributional patterns of co-occurring N-cycling functional microbes in coastal sediments.

Tables

Figure Captions Figure 1 Figure 2

 Captions12 Figure Captions

64 ,

 64 P<0.05), and thus only Pb, As and Cd was retained for subsequent correlation analyses. H and D of bacterial amoA were

	negatively correlated with the concentration of NH 4	+ -N (ρ=-0.76 and -0.72, P<0.01, respectively), temperature (ρ=-0.69,
	P<0.05; ρ=-0.72, P<0.01, respectively) and metal As (ρ=-0.61, P<0.05), whereas H and D of archaeal amoA exhibited
	no correlation with all environmental variables examined. NosZ gene diversities had positive correlations with
	concentration of NO 2
	with Pb (ρ=0.74 and 0.76, P<0.01), ratio of organic carbon to nitrogen (C/N, ρ=0.73 and 0.72, P<0.01, respectively) and
	NH 4	+ -N (ρ=0.70 and 0.71, P<0.05, respectively), but negatively with pH of overlying water (ρ=-0.72 and -0.75, P<0.01,
	respectively) and DO (ρ=-0.59, P<0.05).

--N (ρ=0.60, P<0.05 for H) and salinity (ρ=0.69, P<0.05 for H), and negative correlations with Pb (ρ=-0.64 and -0.68, P<0.05, for H and D, respectively). H and D of AMB 16S rRNA genes were positively correlated

Table 1

 1 Characteristics of the PCR primers used in the study

	Application	Target gene	Primer	Sequence (5′-3′)	Length of	Reference
					amplicon (bp)
	Cloning	AOB amoA	amoA1F	GGGGTTTCTACTGGTGGT	490	Agogué et al.
			amoArnew	CCCCTCBGSAAAVCCTTCTTC		2008
		AOA amoA	Arch-amoAF	STAATGGTCTGGCTTAGACG	635	Agogué et al.
			Arch-amoAR	GCGGCCATCCATCTGTATGT		2008
		nosZ	nosZ-F	CGYTGTTCMTCGACAGCCAG	267	Zhou et al.
			nosZ1622R	CGSACCTTSTTGCCSTYGCG		2011
	DGGE	AMB16S rRNA	Pla46f	GGATTAGGCATGCAAGTC	453	Sanchez-Melsio
			AMX368r-GC *	CCTTTCGGGCATTGCGAA		et al. 2009
	GC * =CCGCCGCGCGGCGGGCGGGGCGGGGCACGGGGGG

Table 2

 2 Comparison of Shannon (H) and Simpson (D) diversities of target genes across sampling locations or layers or seasons using the one-way ANOVA or t-test analyses*

		AOB amoA	AOA amoA	nosZ		AMB 16S rRNA
		H	D	H	D	H	D	H	D
	Location
	JL	1.14±0.17	0.63±0.06	0.38±0.06	0.18±0.04	1.31±0.26	0.63±0.10	2.43±0.13 a	0.9±0.01 a
	BL	1.10±0.31	0.56±0.10	0.31±0.10	0.14±0.05	1.35±0.09	0.71±0.02	2.48±0.06 a	0.9±0.01 a
	Di	1.21±0.23	0.61±0.09	0.20±0.05	0.09±0.02	1.65±0.18	0.76±0.05	1.96±0.04 b	0.84±0.00 b
	P-value	0.951	0.844	0.270	0.264	0.417	0.379	0.004	0.001
	Layer
	Upper	0.99±0.16	0.55±0.06	0.21±0.05	0.09±0.02	1.43±0.11	0.71±0.04	2.23±0.12	0.88±0.01
	Lower	1.31±0.19	0.64±0.06	0.38±0.06	0.18±0.03	1.45±0.20	0.69±0.07	2.35±0.12	0.89±0.01
	P-value	0.194	0.283	0.068	0.043	0.901	0.782	0.003	0.018
	Season
	Winter	1.43±0.13	0.70±0.03	0.29±0.05	0.13±0.03	1.50±0.14	0.74±0.03	2.17±0.10	0.87±0.01
	Summer	0.87±0.16	0.50±0.06	0.31±0.08	0.14±0.04	1.38±0.17	0.65±0.07	2.41±0.13	0.90±0.01
	P-value	0.025	0.048	0.848	0.845	0.661	0.344	0.015	0.022

*Values are provided as mean and standard errors. Different superscript letters indicate significant pairwise difference (P<0.05) in multiple comparison following ANOVA. All significant differences (P<0.05) are highlighted in bold.

Table 3

 3 Spearman's correlation coefficients (ρ) between environmental factors and Shannon (H) and Simpson indices (D) 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.

				AOB amoA		AOA amoA		nosZ	AMB 16S rRNA
				H	D	H	D	H	D	H	D
	As	-0.61	-0.61	ns	ns	ns	ns	ns	ns
	Cd	ns	ns	ns	ns	ns	ns	ns	ns
	Pb	ns	ns	ns	ns	-0.64	-0.68	0.74	0.76
	C/N	ns	ns	ns	ns	ns	ns	0.73	0.72
	DO	ns	ns	ns	ns	ns	ns	-0.59	-0.59
	Grain size	ns	ns	ns	ns	ns	ns	ns	ns
	NH 4	+ -N	-0.76	-0.72	ns	ns	ns	ns	0.70	0.71
	NO 2	--N	ns	ns	ns	ns	0.60	ns	ns	ns
	NO 3	--N	ns	ns	ns	ns	ns	ns	ns	ns
	pH	ns	ns	ns	ns	ns	ns	-0.72	-0.75
	Salinity	ns	ns	ns	ns	0.69	ns	ns	ns
	Temperature	-0.69	-0.71	ns	ns	ns	ns	ns	ns

*

*C/N=ratio of organic carbon to nitrogen; DO = dissolved oxygen; ns= not significant.

Table 4

 4 Comparisons of community composition of target genes among different sampling stations, layers or seasons using weighted UniFrac significance test*

	Group pairs		P-value
		AOB amoA	AOA amoA	nosZ	AMB 16S rRNA
	Location
	JL vs. BL	0.96	1.00	0.99	1.00
	BL vs. Di	1.00	1.00	1.00	1.00
	JL vs. Di	1.00	1.00	1.00	0.45
	Layer
	Upper vs. Lower	0.73	0.78	0.42	0.99
	Season
	Winter vs. Summer	0.03	0.96	0.68	≤ 0.01
	*The significant correlations (P<0.05) are highlighted in bold.

Table S1

 S1 Shannon (H) and Simpson (D) diversity and coverage (C%) estimates for AMB16S rRNA, AOB amoA, AOA amoA, and nosZ genes 608

	Figure S2
			DGGE								Clone libraries
	Sample	AMB 16S rRNA				AOB amoA				AOA amoA					nosZ
		Bands	H	D	No. of	OTUs	C%	H	D	No. of	OTUs	C%	H	D	No. of	OTUs	C%	H	D
					clones					clones					clones
	JL-U-W	9	2.11	0.87	43	6	97.7	1.57	0.76	37	4	97.3	0.43	0.19		5	96.2	1.42	0.72
	JL-L-W	11	2.32	0.90	30	5	96.7	1.25	0.67	37	3	97.3	0.42	0.22		8	96.3	1.99	0.86
	BL-U-W	11	2.31	0.89	27	4	91.3	1.12	0.62	36	3	94.4	0.20	0.08		7	97.7	1.44	0.74
	BL-L-W	13	2.46	0.91	32	9	87.5	1.98	0.82	32	3	96.9	0.27	0.11		4	96.0	1.07	0.65
	Di-U-W	7	1.86	0.83	35	5	94.3	1.21	0.65	31	2	96.8	0.10	0.04		7	100.0	1.83	0.83
	Di-L-W	8	1.96	0.85	33	9	85.8	1.48	0.65	30	3	96.7	0.32	0.15		6	97.7	1.27	0.68
	JL-U-S	15	2.60	0.92	25	4	96.0	0.90	0.53	29	3	93.1	0.20	0.08		5	97.4	1.00	0.53
	JL-L-S	16	2.68	0.93	50	3	100.0	0.83	0.54	33	3	100.0	0.48	0.24		5	97.4	0.85	0.40
	BL-U-S	14	2.53	0.91	34	3	100.0	0.58	0.39	26	2	100.0	0.17	0.08		5	97.3	1.42	0.73
	BL-L-S	15	2.60	0.92	44	3	100.0	0.73	0.41	40	4	97.5	0.60	0.28		7	95.5	1.48	0.70
	Di-U-S	8	1.97	0.85	33	2	100.0	0.55	0.37	30	3	93.3	0.20	0.08		6	88.9	1.45	0.69
	Di-L-S	9	2.06	0.85	36	6	97.2	1.60	0.77	33	3	93.9	0.20	0.08		10	86.7	2.05	0.85

Acknowledgements

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