Johanne Aubé

Pavel Senin

Patricia Bonin

Olivier Pringault

Céline Jeziorski

Olivier Bouchez

Christophe Klopp

Rémy Guyoneaud

Marisol Goñi-Urriza
email: marisol.goni@univ-pau.fr

Meta-omics provides insights into the impact of hydrocarbon contamination on microbial mats functioning

Keywords: microbial mats, metabolism, metagenomics, metatranscriptomic, functioning, diversity

Photosynthetic microbial mats are stable, self-supported communities. Due to their coastal localization, these mats are frequently exposed to hydrocarbon contamination and are able to grow on it. To decipher how this contamination disturbs the functioning of microbial mats, we compared two mats: a contaminated mat exposed to chronic petroleum contamination and a reference mat. The taxonomic and metabolic structures of the mats in spring and fall were determined using metagenomic and metatranscriptomic approaches.

Extremely high contamination disturbed the seasonal variations of the mat. ABC transporters, two-component systems and type IV secretion systems related genes were overabundant in the contaminated mats. Xenobiotic degradation metabolism was minor in the metagenomes of both mats and only the expression of genes involved in Polyciclic Aromatic hydrocarbon degradation was higher in the contaminated mat. Interestingly, the expression rates of genes involved in hydrocarbon activation decreased during the 1-year study period, concomitant with the decrease in easily degradable hydrocarbons, suggesting a transient effect of hydrocarbon contamination. Alteromonadales and Oceanospirillales hydrocarbonoclastic bacteria appeared to be key in hydrocarbon remediation in the contaminated mat. Overall, the contaminated microbial mat was able to cope with hydrocarbon contamination and displayed an adaptive functioning that modified seasonal behaviour.

Introduction

Photosynthetic microbial mats growing in coastal areas are among the most diverse and complex marine ecosystems [START_REF] Allen | Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay[END_REF]. The overall metabolism of a microbial mat is driven by solar light as the main energy source. Microbial activities generate physical and chemical gradients in the mat that maintain the structure and activity of the mat's community [START_REF] Van Gemerden | Microbial mats: a joint venture[END_REF], leading to a stable, self-supported mat. Microbial mats are highly dynamic and are subjected to strong fluctuations at the diel or seasonal scales [START_REF] Van Gemerden | Microbial mats: a joint venture[END_REF]. The extensive genetic and metabolic diversity of photosynthetic microbial mat-inhabiting organisms enable development in a wide variety of environments, including hot springs, hypersaline and alkaline environments and polar ponds [START_REF] Allen | Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay[END_REF][START_REF] Vincent | Cyanobacterial Dominance in the Polar Regions[END_REF][START_REF] Dillon | Spatial and temporal variability in a stratified hypersaline microbial mat community[END_REF][START_REF] Harris | Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat[END_REF][6]. Due to their localization in coastal zones, microbial mats are frequently subjected to hydrocarbon contamination [START_REF] Barth | The influence of cyanobacteria on oil polluted intertidal soils at the Saudi Arabian Gulf shores[END_REF][START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF]. The response of microbial mats to petroleum contamination has been studied in microcosm experiments and under natural conditions after acute pollution events (accidental or laboratory experiments). Microbial mats can cope with contamination and develop a robust community adapted to petroleum levels [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Bordenave | Effects of Heavy Fuel Oil on the Bacterial Community Structure of a Pristine Microbial Mat[END_REF]. The few studies that have focused on chronic oil pollution have concluded that the structure of microbial mats is related to the level of contamination [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF] and that contaminated mats can degrade hydrocarbons [START_REF] Paissé | Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution[END_REF][START_REF] Paisse | How a Bacterial Community Originating from a Contaminated Coastal Sediment Responds to an Oil Input[END_REF]. Generally, noticeable changes in composition [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Paissé | Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution[END_REF] of high contaminated mats are reported, and some studies described lower diversity in higher contaminated mats [START_REF] Abed | Diversity, Distribution and Hydrocarbon Biodegradation Capabilities of Microbial Communities in Oil-Contaminated Cyanobacterial Mats from a Constructed Wetland[END_REF], but not overabundance of hydrocarbonoclastic microorganisms have been observed. Nevertheless, when these contaminated mats were submitted to fresh oil pollutions, an immediate induction of RHD genes involved in polyaromatic hydrocarbon degradation was observed, accompanied with an efficient degradation of crude oil [START_REF] Paissé | Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution[END_REF]. This metabolic response (associated with a community structural shift [START_REF] Paisse | How a Bacterial Community Originating from a Contaminated Coastal Sediment Responds to an Oil Input[END_REF]) was however quickly reversed, highlighting a fast, adaptive and efficient response of the metabolically active bacterial population. Oil contamination also impacts typical seasonal behaviour of photosynthetic microbial mats, by strongly inhibiting primary production and respiration in spring, but with no significant impact in fall [START_REF] Aubé | The impact of long-term hydrocarbon exposure on the structure, activity, and biogeochemical functioning of microbial mats[END_REF]. As consequences, the typical overabundance of Cyanobacteria in fall compared to spring could not be observed in the higher contaminated mat [START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF]. These studies were performed using taxonomic and/or metabolic gene markers, such as ribosomal genes or genes involved in hydrocarbon degradation [START_REF] Bordenave | Effects of Heavy Fuel Oil on the Bacterial Community Structure of a Pristine Microbial Mat[END_REF][START_REF] Paissé | Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution[END_REF][START_REF] Abed | Microbial Diversity of a Heavily Polluted Microbial Mat and Its Community Changes following Degradation of Petroleum Compounds[END_REF][START_REF] Paisse | How a Bacterial Community Originating from a Contaminated Coastal Sediment Responds to an Oil Input[END_REF][START_REF] Abed | Diversity, Distribution and Hydrocarbon Biodegradation Capabilities of Microbial Communities in Oil-Contaminated Cyanobacterial Mats from a Constructed Wetland[END_REF][START_REF] Paisse | Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?[END_REF].

High-throughput sequencing of metagenomes and metatranscriptomes has provided an unprecedented overview of the functional capacities and gene expression of microbial communities confronting environmental stresses. As the presence and expression of genes involved in hydrocarbon degradation (including complete metabolic pathways) have been observed in oil spill-contaminated seawaters [START_REF] Lu | Microbial gene functions enriched in the Deepwater Horizon deep-sea oil plume[END_REF][START_REF] Mason | Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill[END_REF][START_REF] Rivers | Transcriptional response of bathypelagic marine bacterioplankton to the Deepwater Horizon oil spill[END_REF] and soils [START_REF] De Menezes | Comparative metatranscriptomics reveals widespread community responses during phenanthrene degradation in soil[END_REF][START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF], applying these sequencing techniques to contaminated microbial mats will provide new insights on the adaptation of microbial mats to hydrocarbon contamination. The aim of this paper was to elucidate the impact of long-term petroleum contamination on the functioning of microbial mats. Two photosynthetic microbial mats located in the Berre lagoon, a brackish lagoon bordering the Mediterranean Sea in the South of France, were investigated in this work. These mats feature similar physical and chemical parameters (salinity, temperature, solar irradiation, etc.) [START_REF] Aubé | The impact of long-term hydrocarbon exposure on the structure, activity, and biogeochemical functioning of microbial mats[END_REF] but have been subjected to differing hydrocarbon contamination levels. Specifically, the contaminated mat received hydrocarbon inputs from a petrochemical industry site for more than 80 years. By applying metagenome and enriched mRNA metatranscriptome sequencing, we described the key metabolic pathways of both microbial mats. Comparing the contaminated mat with the reference mat revealed the role of petroleum pollution in microbial community functioning. A deeper analysis of metabolism related to hydrocarbon-degradation pathways was also performed.

Material and methods

Sampling sites and procedure

Berre lagoon is located on the Mediterranean French coast. Microbial mat samples were collected from two sites in the lagoon: a reference station located in the bird reserve "Les salins du Lion" (SL) (43.452570 N 5.230085 E) and a contaminated station located in a retention basin receiving hydrocarbon-contaminated rainfall water from a petrochemical industry site (EDB1) (43.484946 N 5.188452 E) [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Pringault | Contrasted effects of natural complex mixtures of PAHs and metals on oxygen cycle in a microbial mat[END_REF]. At this latter site, the hydrocarbon content reaches 96 µg/g [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF]. In addition to this chronic contamination, an accidental oil spill occurred in September 2009 due to the overflow of a hydrocarbon-polluted water retention pond [START_REF] Beau-Monvoisin | Déversement accidentel d'hydrocarbures, sur l'étang de Berre suite à un débordement des bassins d'orage de la Compagnie Pétrochimique de Berre[END_REF], which increased the hydrocarbon content. Three sampling exercises were performed: one in spring (April 2012) and two in fall (September 2011 and 2012), which were named Apr12, Sept11 and Sept12, respectively. A piece of mats (around 1.5 m 2 ), including the top two centimetres of sediment were collected at mid-day as described in [START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF].

Subsamples for molecular analyses were stored at -80°C. A description of main physicalchemical characteristics and hydrocarbon composition of both sites can be found at Table S1.

Whole meta-genome and meta-transcriptome sequencing and bioinformatics procedures

DNA and RNA were co-extracted in triplicate using an RNA PowerSoil Total RNA Isolation Kit (MO BIO Laboratories, Inc.) coupled with an AllPrep DNA/RNA Mini Kit (QIAGEN) for DNA and RNA separation. RNA was digested with an RNase-Free DNase Set (QIAGEN) for DNA removal, and RNA quality was checked as described by Aubé and coworkers [START_REF] Aubé | The impact of long-term hydrocarbon exposure on the structure, activity, and biogeochemical functioning of microbial mats[END_REF]. Genomic DNA libraries were constructed using a NEXTflex PCR-free DNA Sequencing Kit from BioScientific PCR-free kit following the manufacturer's protocol. RNAseq libraries were prepared according to Illumina's protocol on a Tecan EVO200 liquid handler. An Illumina TruSeq Stranded mRNA sample prep kit was used to analyse RNA after mRNA enrichment using an Epicentre Ribo-Zero rRNA removal kit. RNA was fragmented to generate double-stranded cDNA. Ten cycles of PCR were used to amplify libraries following manufacturer's instructions (Illumina TruSeq Stranded mRNA library prep kit, Cat. No. RS-122-2101), and the libraries were precisely quantified by qPCR using a KAPA Library Quantification Kit. RNA-seq and gDNA sequencing were performed with an Illumina HiSeq2500 instrument using a paired-end read length of 2100 bp with an Illumina TruSeq SBS sequencing kit v3.

The Trim Galore! utility was used for read quality control http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Possible adapter sequences based on the Illumina TruSeq Adapter index were removed from the reads. Data filtering was performed with cutadapt [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF] and FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) by trimming reads with a Q score of less than 15 at the 3' end, trimming unidentified dNTP (N) at the read ends and discarding reads with Ns>10%. Reads shorter than 50 bp with low-quality bases (Q<20) were also discarded.

For functional and taxonomic annotations, all filtered reads (DNA and mRNA) were aligned to the KEGG gene database [START_REF] Kanehisa | KEGG: kyoto encyclopedia of genes and genomes[END_REF] using LAST aligner v392 [START_REF] Frith | Parameters for accurate genome alignment[END_REF]. Each read was then annotated using the single-directional best hit information method [START_REF] Moriya | KAAS: an automatic genome annotation and pathway reconstruction server[END_REF] with an identity cut-off of 60% and a score of 96. Eukaryotic sequences were removed from the dataset. Statistics of the reads at each bioinformatics analysis step are shown in the supplementary material (Table S2).

Statistical analysis

Before statistical analysis, random sampling of filtered data was performed to obtain the same number of reads per sample (i.e. the minimum number of sequences in metagenomic or metatranscriptomic samples (Table S2)) and the means of the pseudo-triplicates were calculated to perform statistical tests. Taxonomic biomarkers of hydrocarbon contamination were detected using the LEfSe algorithm [START_REF] Segata | Metagenomic microbial community profiling using unique clade-specific marker genes[END_REF]. The first analysis step was a non-parametric Kruskal-Wallis (KW) sum-rank test to detect taxa and functional pathways with significant differential abundances. Biological consistency was subsequently investigated using a pairwise Wilcoxon test. Finally, linear discriminant analysis (LDA) was used to estimate the effect size of each differentially abundant taxon. Alpha values of 0.05 were used for the KW and Wilcoxon tests, and a threshold of 2 was used for the logarithmic LDA scores. Gene and transcript abundances for functional data were compared using a two-group White's nonparametric t-test in STAMP [START_REF] Parks | STAMP: statistical analysis of taxonomic and functional profiles[END_REF] by KEGG subsystem. To account for the variation of transcriptional activity, the gene expression rate was calculated as described by Stewart el al [START_REF] Stewart | Microbial metatranscriptomics in a permanent marine oxygen minimum zone[END_REF]. Hydrocarbon content were analysed by principal component analysis (PCA) using the FactoMineR package [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF]; variables with cos2 below 0.5 on each factorial plane are not shown on the corresponding correlation circles of the PCA.

Accession number

The sequence data are available in the Sequence Read Archive of the National Center for Biotechnology Information under accession number SRP063590.

Results and discussion

Microbial mats are highly stratified systems, in which microorganisms' metabolism supports their physical and chemical structure. In return, physical and chemical conditions of the mats favours microbial specific metabolisms: structure and function are thus slightly dependent. The goal of the present study was to determine if pollution changes metabolic profiles and its consequences on coastal mat's structure and functioning.

Structure of microbial mat communities

The metagenomes and metatranscriptomes of the microbial mats were annotated using the KEGG database. Between 24.70% and 46.86% of the metagenome and metatranscriptome sequences, respectively, aligned to the KEGG database (Table S2). The community composition based on the taxonomic affiliations of protein-encoding genes revealed a structure similar to that described by 16S rRNA gene affiliations [START_REF] Aubé | The impact of long-term hydrocarbon exposure on the structure, activity, and biogeochemical functioning of microbial mats[END_REF] and to those of other mats [START_REF] Bolhuis | Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing[END_REF][START_REF] Severin | Diversity of nitrogen-fixing bacteria in cyanobacterial mats[END_REF]; i.e. Proteobacteria, Cyanobacteria, and Bacteroidetes were dominant (Fig. 1) with metagenomes mean relative abundance of 48.1%, 16.0% and 15.5% respectively. The high similarity between the structure described here and those described in previous studies on the same mats based on 16S rDNA sequences [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Mason | Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill[END_REF], supported the robustness of the annotation approach. Oscillatoriales dominated among Cyanobacteria. The relative abundance of Cyanobacteria was highest in the EDB1 Sept12 metagenomes and metatranscriptomes, with a greater abundance of Rivulariaceae (Table S3). Among the Deltaproteobacteria (10.8% of the metagenomes mean relative abundance) most of the sequences were affiliated with sulphate reducers in the order Desulfobacterales (Table S3).

The sequences related to Gammaproteobacteria (15.0% of the metagenomes mean relative abundance) were affiliated with Alteromonadales and Chromatiales; most of the Chromatiales were purple sulphur bacteria. Among the Alpha and Betaproteobacteria (17.1% and 4.3% of the metagenomes mean relative abundance), Rhodobacterales and Burkholderiales dominated, respectively. Consistent with observations in the Guerrero Negro hypersaline microbial mats and Highborne Cay mat (The Bahamas) [START_REF] Robertson | Diversity and Stratification of Archaea in a Hypersaline Microbial Mat[END_REF][START_REF] Mobberley | Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling[END_REF], Archaea were in the minority in the microbial communities, accounting for 1% to 3% of total reads and mainly represented by the methanogens Methanomicrobia. Thus, both mats are classical mats with dominance of photosynthetic bacteria and bacteria involved in the sulphur cycle (sulphate reducers and sulphur oxidizers [START_REF] Van Gemerden | Microbial mats: a joint venture[END_REF]).

The community structures described using metatranscriptomic data differed from those described by metagenomic data (Fig. 1). Remarkably, transcripts related to Cyanobacteria highly dominated the communities in both mats, accounting for 36.5% to 78% of the reads, highlighting the major role of Cyanobacteria compared to other functional groups in the mats [START_REF] Mobberley | Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling[END_REF][START_REF] Wieland | Biogeochemistry of an iron-rich hypersaline microbial mat (Camargue, France)[END_REF].

As microbial mat functioning is dependent on the seasonal period [START_REF] Pinckney | Impacts of seasonality and nutrients on microbial mat community structure and function[END_REF], sampling was performed in spring and fall to assess the seasonal variation of the structure and activity of the mats. The reference mat showed typical seasonal variations characterized by 1) an increase of Cyanobacteria in fall (around 13% of metagenomic data in spring and 19% in fall, Table S3); and, 2) an increase of the sulphur related microorganisms in spring. This increase was observed either at metagenomic (from near 14% in fall to 19% in spring) or at metatranscriptomic level (a mean of 6% in fall and 21% in spring, Table S3) and mainly concerned Desulfobacterales and Chromatiales. On the contrary, no seasonal variations were observed in the contaminated mat: Cyanobacteria highly dominated the community in Sept12 but not in Sept11 samples, and a continuous decrease of the relative abundance of microorganisms related to sulphur cycle, either at metagenomics or metatranscriptomic level, was observed in the contaminated mat (Table S3).

Metabolism of microbial mat communities

A total of 7,009 different KEGG ortholog groups (KOs) were detected in the metagenome, and 6,470 KOs were detected in the metatranscriptome. Among the annotated reads, around 44% of the microbial mat metagenomes matched genes involved in pathways associated with metabolism (Fig. 2); including 10.2-11.1% related to genes involved in carbohydrate metabolism (547 KOs); and 9.9-10.8% related to amino acid metabolism (420 KOs). Energy metabolism-related sequences represented only 4.1-4.5% of the metagenomes but up to 49.9% of the metatranscriptomes (389 and 381 KOs for genes and transcripts, respectively). Among them, photosynthesis metabolism-related genes (i.e. genes encoding proteins of photosystems I and II (P700 and P680) and phycobiliproteins (phycocyanin, allophycocyanin and phycoerythrin subunits)) exhibited maximum expression rates of 1094.

Genes related to nitrogen and sulphur metabolism exhibited high expression rates as well.

Sequences related to nitrogen metabolism were mainly involved in nitrogen fixation (i.e. nitrogenase enzyme complex); among them, nifH exhibited expression rates as high as 5.85.

The sequences related to the sulphur cycle were involved in sulphate reduction: adenylylsulphate reductase and sulphite reductase (AprAB and DsrAB) exhibited maximum expression rates of 8.83 (data not shown).

While reference mat showed a typical seasonal behaviour, contaminated mat did not show the same behaviour. In this case, the comparison of metabolic potentials of both mats in order to define which of them are related to the contamination is challenging. For further analysis on hydrocarbon contamination impact on microbial mat functioning, all the samples for each mat (reference or contaminated) have been averaged. This approach could probably dissimulate some metabolisms, but eliminates the errors originating from a seasonal divergent functioning.

Metabolic and taxonomic differences between sites with focus on metabolic pathways associated with xenobiotic degradation

Microbial communities from coastal environments impacted by punctual oil input have been intensively studied (e.g. [START_REF] Mason | Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill[END_REF][START_REF] Lamendella | Assessment of the Deepwater Horizon oil spill impact on Gulf coast microbial communities[END_REF][START_REF] Atlas | Oil Biodegradation and Oil-Degrading Microbial Populations in Marsh Sediments Impacted by Oil from the Deepwater Horizon Well Blowout[END_REF]), and a decrease in microbial diversity associated with dominance of hydrocarbon-degrading marine bacteria is generally observed following an oil spill [START_REF] Nogales | Anthropogenic perturbations in marine microbial communities[END_REF]. By contrast, in this study, dominance of hydrocarbonoclastic bacteria in the contaminated mat compared to the reference mat was not observed, consistent with previous studies of chronically contaminated sites [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF][START_REF] Cappello | Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study[END_REF]. At the taxonomic level, 36 and 3 differentially abundant taxa were detected based on metagenomic and metatranscriptomic data, respectively (Fig. 3). At the metagenomic level, the main difference was a decrease in the relative abundance of some phylogenetic groups (Fig. 3a), suggesting a sensitivity of those groups to contamination. At the transcriptomic level (Fig. 3b), few taxa appear characteristic of each mat. Only Parvularculales within Alphaproteobacteria, were characteristic taxa of the contaminated mat. Alphaproteobacteria are key contributors to the later stages of oil degradation [START_REF] Kostka | Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill[END_REF]. However, hydrocarbon degradation is strain specific; phylogenetic description based on taxonomic genes such as 16SrRNA (especially when this description is performed at high phylogenetic levels such as the order) has low explanatory value, since two strains belonging to the same taxon can be or not hydrocarbonoclastic. Using short reads approaches, affiliation of sequences below the genus, or even the family is often unattainable.

The greatest differences in metabolic potential at the metagenomics level were related to signal transduction and membrane transport. These pathways were significantly more abundant in the hydrocarbon-contaminated microbial mat, whereas nucleotide metabolism and replication and repair pathways were significantly more abundant in the reference mat (Fig. 4). The contaminated mat was enriched with genes involved in two component systems (signal transduction, TCS) of the OmpR family (involved in copper ion efflux and manganese transport as a response of Mn starvation) and NtrC family (involved in nitrogen availability) (Table S4). The TCS are known to modify microbial physiology in response to multiple environmental signals and thus playing a role in biogeochemical cycles. Indeed, marine bacteria have varied TCS systems, and it has been proposed that lacks of TCS could be a hallmark of oligotrophy in marine systems [START_REF] Held | Unique Patterns and Biogeochemical Relevance of Two-Component Sensing in Marine Bacteria[END_REF]. Contaminated mat is probably enriched with TCS as an adaptive response to contamination. The overabundance of membrane transport related genes concern the ABC transporters and the type IV (T4SS) secretion systems.

Recently, Xu et al. [START_REF] Xu | Dibutyl phthalate alters the metabolic pathways of microbes in black soils[END_REF] described an enrichment in ABC transporters and TCS after dibutyl phthalate contamination in soils. They also observed an acceleration of nitrogen, carbon and sulfate metabolisms and suggested that ABC transporters and TCS were the culprits of this metabolic activation. The T4SS are found in Gram negative bacteria, and are capable of secrete a wide variety of substances across the bacterial membranes including toxic bacterial effectors that result in cell death of rival bacteria and eukaryote [START_REF] Green | Bacterial Secretion Systems: An Overview[END_REF]. T4SS is ancestrally related to bacterial DNA conjugation systems, and the T4SS-mediated genetic horizontal transfer is considered as a major contributor to bacterial genomic mobility [START_REF] Waksman | From conjugation to T4S systems in Gram-negative bacteria: a mechanistic biology perspective[END_REF]. Altogether, enrichment of ABC transporters and T4SS appears as a mechanism to control the community structure and the metabolic potential in the contaminated microbial mat.

Although less pronounced, xenobiotic biodegradation and metabolism pathways were significantly more abundant in the contaminated mat than in the reference mat. At the metatranscriptomic level, no differences in xenobiotic biodegradation and metabolism nor in the others level1 KEGG metabolic pathways were observed between the two mats (p-value>0.05 or difference in mean proportions <0.05%).

To further characterize the hydrocarbon degradation potential of the mats, genes involved in hydrocarbon molecule activation were retrieved from the whole dataset (Fig. 5a, Table S5). These genes are specifically associated with hydrocarbon degradation, whereas those involved in intermediate reactions can be shared with other metabolic pathways (http://www.genome.jp/kegg/pathway.html). The gene encoding alkane 1-monooxygenase (alkB) (the single representative of genes involved in the fatty acid degradation pathway, Table S5), which is responsible for alkane activation, was more abundant (Wilcoxon test, pvalue = 0.03125) than the genes involved in degradation of aromatic compounds (Fig. 5a), as observed after the Deepwater Horizon oil spill [START_REF] Mason | Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill[END_REF]. The relative abundance and taxonomic identification of alkB were similar between the contaminated and reference mats (Fig. 5a andS1). Although the alkB gene has been widely used to investigate alkane degradation [START_REF] Vomberg | Distribution of alkB genes within n-alkane-degrading bacteria[END_REF][START_REF] Van Beilen | Diversity of Alkane Hydroxylase Systems in the Environment[END_REF][START_REF] Beilen Jb Van | Alkane hydroxylases involved in microbial alkane degradation[END_REF][START_REF] Smith | Alkane hydroxylase gene (alkB) phylotype composition and diversity in northern Gulf of Mexico bacterioplankton[END_REF] and as a marker of petroleum degradation potential [START_REF] Mason | Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill[END_REF], its direct relationship with petroleum hydrocarbon degradation is questionable, especially in chronically contaminated environments [START_REF] Paisse | Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?[END_REF]. In fact, alkB is involved in the degradation of alkanes of both biogenic and petrogenic origin. Biogenic alkanes are the consequence of organic matter degradation and are ubiquitous in nature [START_REF] Widdel | Anaerobic biodegradation of saturated and aromatic hydrocarbons[END_REF]. As for alkB, the relative abundance of all the genes involved in the degradation of aromatic hydrocarbons was similar in both mats (Fig. 5a).

Analysis at the metatranscriptomic level revealed significant higher expression of genes involved in PAH activation in the contaminated mat (Fig. 5b). However, the expression of all the genes involved in HC activation in the contaminated mats decreased over time (Fig. 6a). Interestingly, this decrease was concomitant with changes in hydrocarbon composition during the sampling exercises (Fig. 6b). Short-chain alkanes (<C28), medium-weight PAHs, and heavy PAHs dominated the hydrocarbons in the first, second, and last exercises, respectively. The contaminated mat was therefore enriched in recalcitrant molecules in the last exercise [START_REF] Aubé | The impact of long-term hydrocarbon exposure on the structure, activity, and biogeochemical functioning of microbial mats[END_REF], reaching a composition similar to that previously observed at this site [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF].

Overall, these results (chemical modification and a decrease in the expression rates of hydrocarbon activation-involved genes) suggest a progressive degradation of the more easily degradable hydrocarbons released by the overflow spill in September 2009.

A decrease in relative abundances was evident for fatty acid degradation activation genes, namely alkB (Fig. 7a). The taxonomic affiliations of the alkB transcripts suggest Alteromonadales-related bacteria as the main alkane degraders in the first sampling exercise, consistent with a previous study of the Berre lagoon in which Marinobacter aquaeolei was the dominant alkB phylotype [START_REF] Paisse | Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?[END_REF]. The relative abundance of alkB transcripts decreased over time in parallel with the decrease in lower-molecular-weight alkanes. Transcripts involved in PAH degradation also decreased, albeit to a lesser extent, with Oceanospirillales-related bacteria as the main actors (Fig. 7b).

Conclusion

Both mats displayed a typical structure and functioning, with dominance of Cyanobacteria and sulphur cycle related microorganisms.. Genes involved in environmental information processing were overabundant in the contaminated mat compared to the reference one. However, xenobiotic degradation metabolisms represented a minor part of the metagenome and metatranscriptome, and no overabundance of hydrocarbonoclastic bacteria was observed in the contaminated mat. The expression levels of genes associated with hydrocarbon degradation pathways varied between the mats and among sampling exercises.

In the contaminated mat, the expression of genes responsible for hydrocarbon activation was related to hydrocarbon composition, suggesting degradation of easily degradable molecules.

The studied mat appeared robust enough to cope with hydrocarbon contamination but its seasonal behaviour was affected. S5). The plot compares the hydrocarbon contaminated mat (EDB1) in black to the reference (SL) in white. Data represent the mean of the triplicates.

Acknowledgement

Figure captions : Fig. 1 Fig. 2

 :12 Figure captions:

Fig. 3

 3 Fig. 3 LEfSe analysis of the microorganisms differentially abundant (metagenomes; a) and active

Fig. 4

 4 Fig. 4 Comparison (White's non-parametric t-test in STAMP) of functional gene annotations of

Fig. 5

 5 Fig. 5 Extended error plots comparison of the hydrocarbon degradation-related genes or

Fig. 6

 6 Fig.6Expression level of genes involved in hydrocarbon degradation (TableS5) in mats (a);

Fig. 7

 7 Fig. 7 Relative abundance (%) and affiliation of transcripts involved in the activation of fatty

The authors received support with linguistic proofreading from a professional proofreading service. This work was supported by the French National Research Agency [ANR FUNHYMAT ANR11 BSV7 014 01].


H Khelidj

A Portavoce
email: alain.portavoce@im2np.fr

M Bertoglio

M Descoins

L Patout

K Hoummada

A Hallén

A Charaï

M C Benoudia

D Mangelinck

Ge(Sn) growth on Si(001) by magnetron sputtering

Keywords: GeSn, Magnetron Sputtering, Silicon substrate, Epitaxy

The semi-conductor Ge 1x Sn x exhibits interesting properties for optoelectronic applications. In particular, Ge 1x Sn x alloys with x  0.1 exhibit a direct band-gap, and integrated in complementary-metal-oxide-semiconductor (CMOS) technology, should allow the development of Si photonics. CMOS-compatible magnetron sputtering deposition was shown to produce monocrystalline Ge 1x Sn x films with good electrical properties at low cost. However, these layers were grown at low temperature (< 430 K) and contained less than 6% of Sn. In this work, Ge 1x Sn x thin films were elaborated at higher temperature (> 600 K) on Si(001) by magnetron sputtering in order to produce low-cost and CMOS-compatible relaxed pseudocoherent layers with x ≥ 0.1 exhibiting a better crystallinity. Ge 1x Sn x crystallization and Ge 1x Sn x crystal growth were investigated. Crystallization of an amorphous Ge 1x Sn x layer deposited on Si(001) or Ge(001) grown on Si(001) leads to the growth of polycrystalline films. Furthermore, the competition between Ge/Sn phase separation and Ge 1x Sn x growth prevents the formation of large-grain Sn-rich Ge 1x Sn x layers without the formation of -Sn islands on the layer surface, due to significant atomic redistribution kinetics at the crystallization temperature (T = 733 K for x = 0.17). However, the growth at T = 633 K of a highly-relaxed pseudo-coherent Ge 0.9 Sn 0.1 film with low impurity concentrations (< 2 × 10 19 at cm 3 ) and an electrical resistivity four orders of 2 magnitude smaller than undoped Ge is demonstrated. Consequently, magnetron sputtering appears as an interesting technique for the integration of optoelectronic and photonic devices based on Ge 1x Sn x layers in the CMOS technology.

Introduction

Current microelectronic device production is based on the complementary-metal-oxidesemiconductor (CMOS) technology [START_REF] Jeong | Tunnelling-based ternary metal-oxide-semiconductor technology[END_REF][START_REF] Robinson | CMOS Future for Microelectronic Circuits: Low power consumption of complementary metal-oxide-semiconductor integrated circuits drives next stage of ultraminiaturization[END_REF]. This technology has significantly evolved over the years due to device size reduction requirements and partly relies on thin film deposition, growth, and patterning capabilities at the nanometer scale [START_REF] Arunkumar | Thin Film: Deposition, Growth Aspects, and Characterization[END_REF]. It is currently based on Silicon (Si) and Germanium (Ge) semiconductors and their alloys, as well as on the use of different metals (Cu, Ni, Pt…), oxides (SiO 2 , HfO 2 …) and nitrides (Si 3 N 4 , TiN…) [START_REF] Pillarisetty | Academic and industry research progress in germanium nanodevices[END_REF][START_REF] Chang | Formation of copper silicides from Cu(100)/Si(100) and Cu(111)/Si(111) structures[END_REF][START_REF] Mangelinck | Three-dimensional composition mapping of NiSi phase distribution and Pt diffusion via grain boundaries in Ni2Si[END_REF][START_REF] Radisavljevic | Single-layer MoS 2 transistors[END_REF][START_REF] Yeo | Dual-metal gate CMOS technology with ultrathin silicon nitride gate dielectric[END_REF]. This technology provides a high production rate of cost-effective integrated nanostructured devices with high levels of purity and reliability. Photonics is a well-established technology allowing for high speed communication [START_REF] Nagatsuma | Advances in terahertz communications accelerated by photonics[END_REF][START_REF] Vlasov | Silicon CMOS-integrated nano-photonics for computer and data communications beyond 100G[END_REF]. However, the integration of photonic devices and circuits in microelectronic chips would promote the development of new optoelectronic technologies, boosting the capacity of current electronic devices [START_REF] Graydon | The data centre challenge[END_REF][START_REF] Reed | Silicon optical modulators[END_REF][START_REF] Hochberg | Towards fabless silicon photonics[END_REF][START_REF] Chen | Device engineering for silicon photonics[END_REF]. Standard CMOS technology already allows the integration of photonic devices such as waveguides (Si nitrides), SiGe-based optical modulators, and Ge-or SiGe-based photodetectors [START_REF] Cazzanelli | Second-harmonic generation in silicon waveguides strained by silicon nitride[END_REF][START_REF] Lacava | Si-rich Silicon Nitride for Nonlinear Signal Processing Applications[END_REF][START_REF] Kim | Strain-induced enhancement of plasma dispersion effect and free-carrier absorption in SiGe optical modulators[END_REF][START_REF] Michel | High-performance Ge-on-Si photodetectors[END_REF][START_REF] Stavarache | SiGe nanocrystals in SiO 2 with high photosensitivity from visible to short-wave infrared[END_REF]. However, Si and Ge being indirect band-gap semiconductors, an essential photonic element is missing for full integrated CMOS Siphotonic development: a light source (i.e. laser integration). Ge 1x Sn x is a CMOS-compatible IV-IV semiconductor exhibiting interesting properties for infrared photodetector, light emitting diode, and laser fabrication [START_REF] Cong | Silicon Based GeSn p-i-n Photodetector for SWIR Detection[END_REF][START_REF] Oehme | GeSn Heterojunction LEDs on Si Substrates[END_REF][START_REF] Elbaz | Ultra-low-threshold continuous-wave and pulsed lasing in tensile-strained GeSn alloys[END_REF][START_REF] Ghosh | Metal-Semiconductor-Metal GeSn Photodetectors on Silicon for Short-Wave Infrared Applications[END_REF][START_REF] Choe | Displacement Damage Effects in Germanium Tin LEDs[END_REF][START_REF] Zhou | Optically Pumped GeSn Lasers Operating at 270 K with Broad Waveguide Structures on Si[END_REF][START_REF] Elbaz | Reduced Lasing Thresholds in GeSn Microdisk Cavities with Defect Management of the Optically Active Region[END_REF]. Furthermore, Ge 1x Sn x alloys can be used for the fabrication of integrated optical amplifier, Gas sensors [START_REF] Homewood | The rise of the GeSn laser[END_REF], and high-speed thin-film transistors [START_REF] Moto | Imptoving carrier mobility of polycrystalline Ge by Sn doping[END_REF][START_REF] Xu | Enhanced mobility of Sn-doped Ge thin-films (≤50 nm) on insulator for fully depleted transistors by nucleation-controlled solid-phase crystallization[END_REF], and direct band-gap Ge 1x Sn x alloys are of high interest for solar cells [30]. Ge 1x Sn x single crystals are expected to be best suited for all these applications, since grain boundaries (GB) in polycrystalline films create deep band-gap levels and act as effective recombination centers, and impair device efficiency. For example, the electronic behavior of polycrystalline semiconductors is strongly dependent on grain size. GB scattering decreases charge carrier mobility, which can increase linearly with grain size at constant temperature [START_REF] Moto | Imptoving carrier mobility of polycrystalline Ge by Sn doping[END_REF]. In the case of p-doped Ge, an inversion from p-type to n-type can be observed if the trap energy level in GB lies in the upper half of the band-gap [START_REF] Möller | Measurement and calculation of the carrier concentration in polycrystalline germanium thin films[END_REF].

The Ge 1x Sn x alloy is expected to exhibit a direct band-gap for Sn compositions of 10 at% (x = 0.1) and above, as required for laser fabrication [START_REF] Wirths | Lasing in direct-bandgap GeSn alloy grown on Si[END_REF][START_REF] Song | High rectification efficiency direct bandgap Ge1-xSnx Schottky diode for microwave wireless power transfer[END_REF][START_REF]Ex situ n$\mathplus$ doping of GeSn alloys via non-equilibrium processing[END_REF]. However, the maximum Sn solubility limit in Ge is ~ 1.1 at % at 673 K [START_REF]Binary Alloy Phase Diagrams, 2nd Edition -ASM International[END_REF][START_REF] Olesinski | The Ge-Sn (Germanium-Tin) system[END_REF]. Consequently, only metastable Sn-rich Ge 1x Sn x layers can be produced [START_REF] Groiss | Free-running Sn precipitates: an efficient phase separation mechanism for metastable Ge 1-x Sn x epilayers[END_REF]. The microcrystalline, optical and electrical properties of Ge 1x Sn x films may consequently be highly dependent on fabrication techniques and conditions. Furthermore, the large difference of lattice parameters between Si and Ge 1x Sn x alloys increases growth complexity, the lattice parameter of diamond Sn being 19.5% larger than the Si lattice parameter [START_REF]KittelCharles-IntroductionToSolidStatePhysics8ThEd[END_REF]. Semiconductor growth is usually performed using chemical vapor deposition (CVD) [START_REF] Allen | Laser chemical vapor deposition: A technique for selective area deposition[END_REF] or molecular beam epitaxy (MBE) [START_REF] Panish | Molecular Beam Epitaxy[END_REF], to produce defect-free and contaminationfree layers, compatible with microelectronic technology requirements. The sputtering technique is CMOS-compatible and significantly low-cost compared to the CVD and MBE techniques [START_REF] Lee | Fabrication of high-quality single-crystal Cu thin films using radio-frequency sputtering[END_REF].

However, sputtered layers are expected to be highly contaminated, in particular with O and C, and to exhibit poor electrical properties compared to layers grown by MBE or CVD. Si 1x Ge x layers are currently grown by CVD in the CMOS technology [START_REF] Xu | Structural and photoelectronic properties of a-SiGe:H thin films with varied Ge prepared by PECVD[END_REF][START_REF] Zhang | Microstructure and optic-electric performance of SiGe/Si heterostructures[END_REF], while magnetron sputtering is used for ohmic contact fabrication for example [START_REF] Mysliwiec | TiAl-based ohmic contacts on p-type SiC[END_REF][START_REF] Buchholt | Ohmic contact properties of magnetron sputtered Ti3SiC2 on n-and ptype 4H-silicon carbide[END_REF]. However, high quality Ge 1x Sn x material can be obtained by sputtering: Ge 1x Sn x -based photodetectors using a Ge 0.94 Sn 0.06 active layer grown by sputtering were shown to exhibit a dark-current density and a responsivity comparable with similar devices using MBE-grown Ge 1x Sn x layers [START_REF] Zheng | GeSn p-i-n photodetectors with GeSn layer grown by magnetron sputtering epitaxy[END_REF]. The growth of Ge 1x Sn x thin films by sputtering have been investigated less often than using CVD or MBE. Ge 1x Sn x layers directly sputtered on an Si(001) substrate are usually polycrystalline [START_REF] Zhang | Raman scattering study of amorphous GeSn films and their crystallization on Si substrates[END_REF][START_REF] Mahmodi | Formation of nanocrystalline GeSn thin film on Si substrate by sputtering and rapid thermal annealing[END_REF][START_REF] Mahmodi | Synthesis of Ge 1-x Sn x Alloy Thin Films by Rapid Thermal Annealing of Sputtered Ge/Sn/Ge Layers on Si Substrates[END_REF], but monocrystalline sputtered Ge 1x Sn x layers can be grown on Ge(001) and GaAs(001) substrates, using low growth temperatures between 423 and 443 K [START_REF] Ladr N De Guevara | Ge1-xSnx alloys pseudomorphically grown on Ge(001)[END_REF][START_REF] Qian | GeSn/GaAs Hetero-Structure by Magnetron Sputtering[END_REF]. Zheng et al. [START_REF] Zheng | Growth of Crystalline Ge 1-x Sn x Films on Si (100) by Magnetron Sputtering[END_REF] succeeded to grow Ge 1x Sn x single crystals on Si(001) substrates using the growth of a sputtered Ge buffer on the Si substrate before Ge 1x Sn x deposition. The Ge buffer was grown at T = 673 K, but the Ge 1x Sn x film was grown at T = 423 K with x  0.06. Ge 1x Sn x films grown or annealed at higher temperatures generally exhibit better crystal quality and thus better electrical properties [START_REF] Taoka | Electrical and optical properties improvement of GeSn layers formed at high temperature under well-controlled Sn migration[END_REF]. Low temperature growth is necessary to ensure Sn incorporation. CVD or MBE growths of Ge 1x Sn x are usually performed at temperatures comprised between 473 and 723 K, and the obtained films are sometimes annealed at higher temperatures in order to improve the electrical and optical properties [START_REF] Conley | Temperature dependent spectral response and detectivity of GeSn photoconductors on silicon for short wave infrared detection[END_REF][START_REF] Zhang | Crystal Quality Improvement of GeSn Alloys by Thermal Annealing[END_REF].

The indirect-to-direct band gap transition occurring at lower Sn composition for relaxed Ge 1x Sn x films [START_REF] Ghetmiri | Direct-bandgap GeSn grown on silicon with 2230 nm photoluminescence[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF], the present work aims to investigate the low-cost high-temperature growth of relaxed monocrystalline Ge 1x Sn x films on Si(001) with x  0.1 in a commercial magnetron sputtering setup. Indeed, relaxed Ge 0.9 Sn 0.1 films were grown at T = 633 K in pseudoepitaxy on Si(001) using magnetron sputtering. These films contain impurity concentrations lower than 2 × 10 19 at cm 3 and exhibit an electrical resistivity four orders of magnitude lower than that of undoped Ge.

Experiments

Ge and Sn were co-deposited on Si(001) substrates using a 99.9999% pure Ar gas flow to simultaneously sputter in DC mode a 99.999% pure Ge target and a 99.99% pure Sn target, in a commercial magnetron sputtering system exhibiting a base pressure of 10 -8 Torr. In this setup, the sample is mounted on a horizontal rotating sample holder, and three targets can be placed symmetrically (120° angle between each target) above the sample, with an angle of 45° between the normals of the target surfaces and the sample surface. Deposition was performed at the pressure of 2.25  10 3 Torr. The sample was rotating at a speed of 5 rpm and was set to ground level, while a voltage was applied to each target to maintain a constant power during deposition.

The Ge target was sputtered at the power of 15 W during the deposition of the Ge buffer, corresponding to a deposition rate of 0.09 nm s 1 . Ge 1x Sn x was deposited during 96 s with a power of 150 W for the Ge target and a power of 15 W for the Sn target, corresponding to a deposition rate of 0.84 nm s 1 for Ge and of 0.21 nm s 1 for Sn. The Ge and Sn fluxes were calibrated separately, using X-ray reflectivity to determine the thicknesses of sputtered films deposited at room temperature (RT) in different conditions. In the following, the nominal composition of the films, i.e. calculated from the Ge and Sn fluxes taking into account the atomic volume difference between Ge and Sn (  Sn / Ge ~ 1.2), will be given unless otherwise specified.

The Si substrates were dipped in a 5% HF solution in order to remove the Si native oxide before being loaded in the growth chamber. The structure of the Ge 1x Sn x films was studied by X-ray diffraction (XRD) in the Bragg-Brentano geometry (-) using a Cu K  source ( K = 0.154 nm) in a PANalytical X'Pert PRO setup equipped with an X'Celerator detector designed for high speed data collection. This setup was also used to perform in situ XRD measurements during amorphous Ge 1x Sn x film crystallization. In this case, the temperature was raised from 423 to 853 K following a heating ramp made of 5 K per minute steps separated by 5 min long XRD measurements at constant temperature, corresponding to an average heating ramp of 1 K min 1 .

Structural observations were also performed using transmission electron microscopy (TEM).

Scanning TEM Dark-Field (STEM-DF) and Energy Dispersive X-Ray Spectrometry (EDS) analyses were acquired at 200 keV using a LaB 6 Thermo Fisher Tecnai TEM equipped with a silicon drift detector OXFORD X-max80. High-Resolution TEM images were performed at 200 keV using a field emission gun Thermo Fisher Titan microscope equipped with a spherical aberration (Cs) correction system. We consider the Cs value in an order of magnitude between 0 and 0.05 mm allowing a point-to-point resolution around 1 Å to be reached. The surface topography of the layers was studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM). AFM images were obtained in non-contact mode using a PSIA XE-100 in air microscope. The composition of the layers was determined using Rutherford backscattering (RBS) and atom probe tomography (APT). For the RBS, 2.0 MeV alpha particles were used with a backscatter detector positioned at 170°. SEM images as well as the TEM and APT sample preparations were performed in an FEI dual beam HELIOS 600 nanolab setup. APT measurements were performed at T = 50 K using a laser-pulsed CAMECA LEAP 3000XHR microscope, with a laser pulse frequency of 100 kHz and a laser power of 0.2 nJ. The resistivity of the films was measured at RT by the four probe method. Fig. 1a presents in situ XRD measurements recorded during annealing a 100 nm-thick Ge 0.83 Sn 0.17 film deposited at room temperature on Si(001). No diffraction peaks are observed after deposition, the film being amorphous. Three diffraction peaks at 2 = 26.92°, 44.70°, and 52.87° appear simultaneously during the annealing ramp, when the temperature reaches T = 733 K. They respectively correspond to the atomic planes (111), (220), and (311) of the Ge 1x Sn x diamond structure (fig. 1b). Surprisingly, one can note that their intensity decreases when the temperature increases, and the peaks disappear concurrently, leaving no diffraction signal at T = 853 K. Fig. 2a shows the intensity variation of the (111) peak recorded during the annealing ramp. The peak is detected at T = 733 K and its intensity increases with temperature up to T = 778 K. This stage corresponds to the nucleation followed by the growth of the Ge 1x Sn x crystal from the initial amorphous film. For temperatures higher than T = 778 K, the intensity of the (111) diffracted peak decreases and finally disappears at T = 818 K, without the apparition of other diffraction peaks until the end of the annealing at T = 873 K. In order to understand this behavior, the variations of the full width at half maximum (FWHM) of the (111) peak were analyzed using the Scherrer equation, giving the average grain size in the direction perpendicular to the film surface [START_REF] Holzwarth | The Scherrer equation versus the 'Debye-Scherrer equation[END_REF]. The "thickness" variations versus temperature of the Ge 1x Sn x grains with (111) planes oriented parallel to the film surface are shown in fig. 2b (solid squares, right axis). The data should be interpreted qualitatively since parameters other than grain size could also modify the FWHM of diffraction peaks. However, one can note that the grain size is significantly smaller than the film thickness. The grain size is less than 3 nm when the diffraction peak is first detected, and increases with temperature to reach a maximum size of about 30 nm at T = 783 K, in agreement with the nucleation and growth of the Ge 1x Sn x polycrystalline film up to this temperature. The size of the Ge 1x Sn x grains stays constant after T = 783 K, despite the decrease of the diffraction peak intensity (fig. 2a). The variations of the diffraction angle 2 of the (111) peak were investigated considering the polycrystalline Ge 1x Sn x layer fully relaxed, and its lattice parameter a GeSn following the corrected-Vegard law:

Results and discussion

Ge(Sn) Crystallization

(1) a Ge = 0.565 nm, a Sn = 0.649 nm, and b = 0.0041 nm are respectively the Ge and Sn lattice parameters in the diamond structure, and the bowing coefficient [START_REF] Mahmodi | Formation of nanocrystalline GeSn thin film on Si substrate by sputtering and rapid thermal annealing[END_REF][START_REF] Mahmodi | Synthesis of Ge 1-x Sn x Alloy Thin Films by Rapid Thermal Annealing of Sputtered Ge/Sn/Ge Layers on Si Substrates[END_REF][START_REF] Ladr N De Guevara | Ge1-xSnx alloys pseudomorphically grown on Ge(001)[END_REF][START_REF] Qian | GeSn/GaAs Hetero-Structure by Magnetron Sputtering[END_REF][START_REF] Zheng | Growth of Crystalline Ge 1-x Sn x Films on Si (100) by Magnetron Sputtering[END_REF][START_REF] Taoka | Electrical and optical properties improvement of GeSn layers formed at high temperature under well-controlled Sn migration[END_REF][START_REF] Conley | Temperature dependent spectral response and detectivity of GeSn photoconductors on silicon for short wave infrared detection[END_REF][START_REF] Zhang | Crystal Quality Improvement of GeSn Alloys by Thermal Annealing[END_REF][START_REF] Ghetmiri | Direct-bandgap GeSn grown on silicon with 2230 nm photoluminescence[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Holzwarth | The Scherrer equation versus the 'Debye-Scherrer equation[END_REF][START_REF] Gencarelli | Crystalline Properties and Strain Relaxation Mechanism of CVD Grown GeSn[END_REF]. The average Sn content of the crystalized Ge 1x Sn x layer was deduced from this equation, using the Bragg equation [START_REF] Kim | Polycrystalline GeSn thin films on Si formed by alloy evaporation[END_REF] and the experimental diffraction angle 2 to determine a GeSn . Fig. 2b presents the variation of the Sn concentration in the Ge 1x Sn x grains versus temperature. The Sn concentration is about 11% at the beginning of crystallization, and continuously decreases as temperature increases, reaching 4% before the disappearance of the diffraction peak. Ge 1x Sn x crystallization at 733 K allowed Sn incorporation above 10% to be obtained. However, atom mobility is fast enough to support Ge and Sn phase separation at this temperature and above. As expected, the in situ XRD measurements reveal a strong competition between Sn incorporation and Ge 1x Sn x crystallization.

As soon as the nucleation process is completed, the crystal growth rate as well as the Ge/Sn separation rate increase with temperature. One can note that the grain size and their Sn composition stay almost constant while the diffraction peak intensity decreases. This phenomenon can be explained by a lateral shrinking of the 30 nm-thick Ge 0.96 Sn 0.04 grains. Since Ge 1x Sn x grains lose Sn, an Sn-rich alloy should grow. However, this Sn-rich phase shows no diffraction peak, meaning that either its grains exhibit crystallographic orientations not compatible with the - diffraction geometry, or the phase is actually amorphous or liquid. Diffraction peaks corresponding to β-Sn are detected. Thus, the Ge 1x Sn x phase was probably consumed by the growth of liquid Sn, as the Sn bulk melting temperature is ~ 505 K and can be lower for nanocrystals [START_REF] Liu | Size-Dependent Melting Behavior of Colloidal In, Sn and Bi Nanocrystals[END_REF]. However, no diffraction peak corresponding to crystalline Ge is detected at RT, suggesting that the mechanism of Ge/Sn phase separation at high temperature in the thin film leads to the formation of amorphous Ge. Fig. 3b and3c present SEM and AFM images of the sample surface after annealing. Large islands are observed with a lateral size between 94 and 470 nm, an average height ~ 110 nm, and a surface density ~ 8.4 × 10 8 cm 2 . The root-mean-square (RMS) surface roughness is ~ 26 nm. These islands probably correspond to the Sn-rich phase that crystallized during sample cooling. Fig. 4 presents STEM Dark-Field observations performed on the sample after the in situ XRD annealing. Fig. 4a shows a cross-section view between two islands similar to that observed on the sample surface by SEM (Fig. 3b) and AFM (Fig. 3c). The Pt layer was deposited on the sample surface for TEM sample preparation. The islands are in direct contact with the Si substrate, as confirmed by High-Resolution TEM observations (Fig. 4b). One can note that the island/Si interface is flat and abrupt, which suggest that this interface corresponds to the initial Si substrate surface. This is in agreement with the Si-Sn phase diagram [START_REF]Binary Alloy Phase Diagrams, 2nd Edition -ASM International[END_REF], showing phase separation between Si and Sn. The maximum solubility of Sn in Si is 0.1 at% at 1339 K, while the solubility limit of Si in -Sn is less than 0.5 at%. The stack of two layers (noted A and B in Fig. 4a) is observed between the two islands. Fig. 4c presents a High-Resolution TEM image of the interface between these two layers. The layer A is amorphous and its average thickness is ~ 25 nm. As shown in the inset of Fig. 4c, a short-range order exists in the layer, corresponding to a distance of 0.32 nm (radius of the ring in the FFT pattern in the inset), which is significantly higher than the nearest neighbor distance (~ 0.245 nm) in the Ge crystal. Below this amorphous layer, a bright contrast is observed with an average thickness of ~ 30 nm, corresponding to the layer B. Fig. 4c shows that this layer is crystalline and coherent with the Si crystal. EDS analyses (not shown) confirmed that the islands contain only Sn atoms (in the detection limit of the technique). However, the overlapping of Ge and Pt signals prevented the Ge distribution in the sample to be determined.

The TEM observations show that the mechanism of Ge/Sn phase separation is complex. Sn atoms are located in large islands exhibiting a height similar to the thickness of the amorphous Ge 0.83 Sn 0.17 layer initially deposited on Si (Fig. 4a). However, the Ge atoms seem to have been incorporated in the Si Substrate, part in an amorphous layer and part in the Si lattice, as the bright contrast of the layer B can be due to the presence in this region of atoms heavier than Si. Ge evaporation from the Si substrate and Ge lattice diffusion in Si cannot occur at 853 K. However, Sn migration in Ge is effective at this temperature, Sn atoms living the Ge 1x Sn x film to form liquid Sn droplets. Thus, Ge evaporation from the surface of melted Sn cannot be ruled out.

According to Ge self-diffusion [START_REF] Portavoce | Manganese diffusion in monocrystalline germanium[END_REF][START_REF] Chroneos | Diffusion of n-type dopants in germanium[END_REF], Ge atom mobility is limited at 853 K (few nanometers after one hour), which can be the reason for the Ge layer to stay amorphous once emptied of the Sn atoms. The Ge/Si intermixing located in the layer B is difficult to explain considering equilibrium kinetic parameters such as lattice diffusion. This intermixing could occur only through short-circuit paths, such as interface diffusion/redistribution, or due to unusual high concentrations of point defects. XRD in situ isothermal annealing at T = 788 K was performed on the same as-deposited Ge 0.83 Sn 0.17 film. The thermal annealing was stopped as soon as Ge 1x Sn x diffraction peaks were detected. AFM measurements (fig. 6) give an RMS surface roughness ~ 1.3 nm, and an average lateral grain size ~ 25 nm, which is in agreement with the Ge 1x Sn x grain size determined from the XRD peaks, considering the lateral inaccuracy involved with AFM in air due to tip effects. Obviously, these holes correspond to the locations of the -Sn(Ge) alloy and confirm that the Ge/Sn phase separation process should occur laterally in the layer, with the formation of a liquid -Sn(Ge) alloy, probably developing droplets on the Ge 1x Sn x surface.

These results show that the atomic mobility and the Ge/Sn phase separation kinetic are too high at the Ge 1x Sn x crystallization temperature to easily obtain large-grain Sn-rich polycrystalline Ge 1x Sn x layers without the formation of -Sn from the crystallization process.

The limiting phenomenon is probably nucleation, since crystalline Ge 1x Sn x layers can be grown at lower temperatures.

Ge buffer growth

As reported in the literature, Ge 1x Sn x layers deposited on Si are usually polycrystalline.

However, monocrystalline Ge 1x Sn x layers can be grown on Si substrate using the deposition of a Ge buffer on the Si substrate before the growth of the Ge 1x Sn x layer. Fig. 7 presents the diffractogram measured at RT on a 100 nm-thick Ge layer deposited at RT on the Si(001) substrate and annealed at 873 K for 30 min under vacuum (P < 10 6 mbar). The Ge layer is polycrystalline as the (111), (220), and (311) diffraction peaks of the Ge diamond structure are detected at 2 = 27.29°, 45.54°, and 53.82°, respectively. One can note that the polycrystalline Ge film exhibits the same peaks and the same peak intensity ratios as the polycrystalline Ge 1x Sn x films that are characteristics of a randomly oriented polycrystal with a diamond structure (Si, Ge, Ge 1x Sn x …). Indeed, thin film growth is highly dependent on surface and interface energies, and the surface and interface contributions in the overall film energy increases as the film thickness decreases. Depending on atom mobility during growth (growth conditions), the film tends to exhibit a texture allowing for energy minimization, as much as possible. The {111} and {113} surfaces are known to be low-energy surfaces of the diamond structure, while {110} facets are more easily observed on surfaces exhibiting low-level contamination of O and C. {111} and {113} facets are the equilibrium facets of relaxed Si and Ge crystals [START_REF] Bermond | The equilibrium shape of silicon[END_REF] and are observed on relaxed Si-Ge islands [START_REF] Portavoce | Sb-surfactant mediated growth of Ge nanostructures[END_REF][START_REF] Portavoce | Control of homoepitaxial Si nanostructures by locally modified surface reactivity[END_REF]. Consequently, if the surface contamination level stays low, the texture of polycrystalline Si, Ge and Ge 1x Sn x films generally shows a large fraction of grains exhibiting the {111} and {113} crystallographic orientations in the direction parallel to the film surface. The relaxed Ge lattice parameter a rel is expected to be ~ 0.565 nm at RT [START_REF] Yu | Ge/GeSn heterostructures grown on Si (100) by molecular-beam epitaxy[END_REF]. In the - XRD geometry used in this study, the measured plane spacing corresponds to plane spacing in the direction perpendicular to the film surface and is thus denoted

d  exp .
The Ge film lattice parameter in the direction perpendicular to the surface a  exp was thus determined from the diffraction angles of the three diffraction peaks Ge(111), Ge(220), and Ge(311) using the Bragg equation. In average a  exp = 0.565 nm, corresponding to a fully relaxed Ge polycrystalline layer. The Si lattice parameter was also determined from the Si(400) diffraction peak observed in the same diffractogram (fig. 7) for comparison. For the Si substrate a  exp = 0.543 nm, as expected for relaxed Si [START_REF] Moontragoon | The direct and indirect bandgaps of unstrained SixGe1-x-ySny and their photonic device applications[END_REF].

The crystallization of a Ge layer deposited at RT on the Si(001) substrate cannot be used for growing a monocrystalline Ge 1x Sn x film, since it leads to the formation of a polycrystalline Ge buffer. Instead, the Ge buffer should be grown at a sufficiently high temperature to allow the Ge crystal to form in partial coherence with the Si substrate due to a high enough atomic mobility, but sufficiently low to prevent extensive Si/Ge intermixing and interdiffusion. Note that the epitaxy is only partially coherent because of the large thickness of the Ge layer and the large misfit between Si and Ge that lead to the nucleation of misfit dislocations. Fig. 7 shows XRD measurements (bottom diffractogram) performed at RT on a 100 nm-thick Ge layer deposited by magnetron sputtering on Si(001) at T = 633 K. This diffractogram corresponds to a Ge layer that is pseudo-coherent with the Si substrate: a single Ge diffraction peak is now observed at 2 = 65.98°, corresponding to the same orientation as the substrate Si(400) diffraction peak. RBS measurements (not shown) confirmed that the layer contains only Ge and that its thickness is ~ 100 nm. XRD measurements give a  exp = 0.566 nm, which indicates that the Ge layer is compressively strained on the Si substrate [START_REF] Wirths | Tensely strained GeSn alloys as optical gain media[END_REF]. Considering that a rel = 0.565 nm for Ge, the perpendicular distortion   exp of this pseudo-coherent Ge layer was found to be   exp = 0.0018, using eq. 2 [START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF].

(

) 2
Considering that the Poisson's ratio of Ge  ~ 0.271 [START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF], the parallel distortion  // exp of the Ge lattice was determined using eq. 3 [START_REF] Berbezier | SiGe nanostructures: new insights into growth processes[END_REF].

(

) 3
 // exp = 0.0024 corresponding to a // exp = 0.564 nm (eq. 2), and thus, corresponding to a relative relaxation rate R = 95.5% (eq. 4).

(

) 4
The Ge film is significantly relaxed and should contain a high concentration of dislocations [START_REF] Zheng | Growth of Crystalline Ge 1-x Sn x Films on Si (100) by Magnetron Sputtering[END_REF][START_REF] Taoka | Electrical and optical properties improvement of GeSn layers formed at high temperature under well-controlled Sn migration[END_REF][START_REF] Conley | Temperature dependent spectral response and detectivity of GeSn photoconductors on silicon for short wave infrared detection[END_REF][START_REF] Zhang | Crystal Quality Improvement of GeSn Alloys by Thermal Annealing[END_REF][START_REF] Ghetmiri | Direct-bandgap GeSn grown on silicon with 2230 nm photoluminescence[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Holzwarth | The Scherrer equation versus the 'Debye-Scherrer equation[END_REF][START_REF] Gencarelli | Crystalline Properties and Strain Relaxation Mechanism of CVD Grown GeSn[END_REF][START_REF] Kim | Polycrystalline GeSn thin films on Si formed by alloy evaporation[END_REF][START_REF] Liu | Size-Dependent Melting Behavior of Colloidal In, Sn and Bi Nanocrystals[END_REF][START_REF] Chroneos | Diffusion of n-type dopants in germanium[END_REF][START_REF] Toko | 70 °C synthesis of high-Sn content (25%) GeSn on insulator by Sn-induced crystallization of amorphous Ge[END_REF][START_REF] Bermond | The equilibrium shape of silicon[END_REF][START_REF] Portavoce | Sb-surfactant mediated growth of Ge nanostructures[END_REF][START_REF] Portavoce | Control of homoepitaxial Si nanostructures by locally modified surface reactivity[END_REF][START_REF] Yu | Ge/GeSn heterostructures grown on Si (100) by molecular-beam epitaxy[END_REF][START_REF] Moontragoon | The direct and indirect bandgaps of unstrained SixGe1-x-ySny and their photonic device applications[END_REF][START_REF] Wirths | Tensely strained GeSn alloys as optical gain media[END_REF][START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF][START_REF] Berbezier | SiGe nanostructures: new insights into growth processes[END_REF][START_REF] Takeuchi | Growth of highly strain-relaxed Ge1-xSnx/virtual Ge by a Sn precipitation controlled compositionally step-graded method[END_REF]. The height of these islands varies typically between 1 and 3 nm (fig. 8c), and the RMS of the surface is ~ 0.71 nm, which are significantly smaller than the thickness of the film. Similar to the SEM images, depressions (~ 3 nm) surrounded by three to five islands are also observed (white circle in fig. 8b). The surface structure displayed in the SEM and AFM images has been also observed by Zhang et al. [START_REF] Zhang | Structural properties of GeSn thin films grown by molecular beam epitaxy[END_REF] on the surface of Sn-rich Ge 1x Sn x layers grown by MBE on Ge(001) substrates at T = 473 K. They interpreted this surface structure as the formation of Sn islands on the Ge 1x Sn x layer surface due to Ge/Sn phase separation. However, in our case, the same structure is found on the surface of the pure Ge buffer, and thus cannot be attributed to Sn.

This structure should be related to stress relaxation through elastic and plastic relaxation mechanisms. For example, similar surface states can be observed on Si(Ge) layers grown in epitaxy on Si(001) [START_REF] Berbezier | SiGe nanostructures: new insights into growth processes[END_REF][START_REF] Takeuchi | Growth of highly strain-relaxed Ge1-xSnx/virtual Ge by a Sn precipitation controlled compositionally step-graded method[END_REF][START_REF] Zhang | Structural properties of GeSn thin films grown by molecular beam epitaxy[END_REF][START_REF] Volpi | Nucleation and evolution of Si 1-x Ge x islands on Si(001)[END_REF]. The layers studied by Zhang et al. were shown to be partially relaxed and to contain threading dislocations. Considering the high relaxation level of the Ge layer, this structure should be related to both the weak residual epitaxial stress located in the film, and threading dislocations. In the case of Sn-rich Ge 1x Sn x layers with low residual strain grown by low-pressure CVD on a Ge buffer grown on Si(001), Nicolas et al. [START_REF] Nicolas | Dislocation pipe diffusion and solute segregation during the growth of metastable GeSn[END_REF] showed thanks to APT measurements that quasi-vertical dislocation lines are passing through the Ge 1x Sn x layer up to the surface. The depressions observed on the film surface could correspond to similar dislocations reaching the surface and locally modifying the elastic strain, promoting the formation of several island-like ripples around the end of the dislocation line. In this case, the density of the surface depressions (1.2  10 11 cm 2 ) should correspond to the threading dislocation density in the Ge buffer. The presence of a surface structure on a buffer layer is not desired, as this structure may prevent the epitaxial growth of a following film and can promote the formation of structural defects at the buffer/film interface. However, in the present case, the surface roughness of the Ge buffer is very low (~ 5 Ge atomic steps in the (001) direction), and such a Ge buffer can be used as virtual substrate to grow pseudo-coherent Sn-rich Ge 1x Sn x layers, as reported in the case of CVD and MBE growth [START_REF] Conley | Temperature dependent spectral response and detectivity of GeSn photoconductors on silicon for short wave infrared detection[END_REF][START_REF] Zhang | Crystal Quality Improvement of GeSn Alloys by Thermal Annealing[END_REF][START_REF] Ghetmiri | Direct-bandgap GeSn grown on silicon with 2230 nm photoluminescence[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Tran | Ion beam synthesis and photoluminescence study of supersaturated fullyrelaxed Ge-Sn alloys[END_REF][START_REF] Holzwarth | The Scherrer equation versus the 'Debye-Scherrer equation[END_REF][START_REF] Gencarelli | Crystalline Properties and Strain Relaxation Mechanism of CVD Grown GeSn[END_REF][START_REF] Kim | Polycrystalline GeSn thin films on Si formed by alloy evaporation[END_REF][START_REF] Liu | Size-Dependent Melting Behavior of Colloidal In, Sn and Bi Nanocrystals[END_REF][START_REF] Toko | 70 °C synthesis of high-Sn content (25%) GeSn on insulator by Sn-induced crystallization of amorphous Ge[END_REF][START_REF] Bermond | The equilibrium shape of silicon[END_REF][START_REF] Portavoce | Sb-surfactant mediated growth of Ge nanostructures[END_REF][START_REF] Portavoce | Control of homoepitaxial Si nanostructures by locally modified surface reactivity[END_REF][START_REF] Yu | Ge/GeSn heterostructures grown on Si (100) by molecular-beam epitaxy[END_REF][START_REF] Moontragoon | The direct and indirect bandgaps of unstrained SixGe1-x-ySny and their photonic device applications[END_REF][START_REF] Wirths | Tensely strained GeSn alloys as optical gain media[END_REF][START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF][START_REF] Berbezier | SiGe nanostructures: new insights into growth processes[END_REF][START_REF] Takeuchi | Growth of highly strain-relaxed Ge1-xSnx/virtual Ge by a Sn precipitation controlled compositionally step-graded method[END_REF].

Ge(Sn) single crystal growth

Fig. 9 presents X-ray diffractograms measured at RT on different 100 nm-thick Ge 0.83 Sn 0.17 films grown on the Ge buffer in different conditions. The diffractogram (a) corresponds to a Ge 0.83 Sn 0.17 film deposited at the same temperature as the Ge buffer without growth interruption. The chosen growth temperature T = 633 K is 100 K lower than the Ge 0.83 Sn 0.17 crystallization temperature determined in the XRD in situ ramp annealing experiments (T = 733 K), aiming to increase Sn incorporation, but is 200 K higher than the growth temperature used in former studies (T = 423 K) for growing Ge 0.94 Sn 0.06 layers by sputtering, aiming to improve the layer crystalline properties. This temperature is actually in the range used to grow Sn-rich Ge 1x Sn x layers by CVD or MBE. The (004) diffraction peak is the only one corresponding to the diamond structure detected for the Si substrate, as well as for the Ge and Ge 1x Sn x layers, displaying the pseudo-coherent relation between the substrate and the two stacked layers. However, one can note that the Ge 1x Sn x (400) diffraction peak is less intense than that of the Ge(400). A small diffraction peak corresponding to -Sn(101) is also detected, meaning that Sn droplets are probably present on the sample surface (Fig. 3). The formation of the phase -Sn can be suppressed using a modified growth process consisting of switching off the sample heater before growing the Ge 1x Sn x layer, without growth interruption between the Ge buffer and the Ge 1x Sn x layer. In this case, XRD measurements (diffractogram (b) in Fig. 9) show no sign of the -Sn phase, and the Ge 1x Sn x (400) and Ge(400) diffraction peak intensities are similar. The diffractogram (c) shown in Fig. 9 was acquired on a sample for which the sample heater was switched off after the growth of the Ge buffer and a growth interruption of 10 min was performed before Ge 1x Sn x growth. The -Sn phase is not detected in this case, but the Ge 1x Sn x (400) peak intensity is smaller than the Ge(400) peak intensity, and additional Ge 1x Sn x diffraction peaks are observed, corresponding to the three diamond lattice orientations (111), (220), and (311), usually detected for Ge and Ge 1x Sn x layers crystallized from an amorphous layer deposited on Si(001) (Figs. 1b, 5a, and 7).

Obviously, the Ge 1x Sn x layer grew in pseudo-epitaxy on the Ge buffer at the beginning, but due to the temperature decrease, the end of the Ge 1x Sn x growth promoted the formation of a polycrystalline layer. However, according to the significant difference of diffraction peak intensity, the top polycrystalline layer should be significantly thinner than the monocrystalline layer in contact with the Ge buffer. The last diffractogram (d) in Fig. 9 was acquired after the crystallization of an amorphous Ge 0.83 Sn 0.17 film deposited at RT on the Ge buffer. In this case, the Ge buffer is pseudo-coherent with the Si(001) substrate, but the Ge 1x Sn x film is entirely polycrystalline and the Ge 1x Sn x (400) peak characteristic of epitaxial growth is not detected.

As expected, the growth of a pseudo-coherent Ge 1x Sn x layer on Si(001) without the formation of the -Sn phase is highly dependent on kinetics, and growth conditions generally need to be determined empirically. Fig. 10 shows SEM and AFM measurements performed on the surface of the Ge 1x Sn x layer coherently grown on the Ge buffer (diffractogram (b) in Fig. 9). A surface structure is observed similar to that displayed on the Ge buffer before Ge 1x Sn x deposition, in agreement with the observations of Zhang et al. [START_REF] Zhang | Structural properties of GeSn thin films grown by molecular beam epitaxy[END_REF]. The pattern is less organized than that on the Ge buffer surface. The ripples/islands exhibit a similar average height ~ 3 nm (Fig. 10c), but they are larger with an average width ~ 75 nm (Figs. 10a and10b). The RMS of the surface is ~ 1.24 nm. One can note that 3 nm-high islands on the Ge surface should have been easily covered with the 100 nm-thick Ge 1x Sn x layer. The fact that this topography is also observed on the Ge 1x Sn x surface is thus in agreement with the effect of extended defects, such as dislocations, which can propagate in the Ge 1x Sn x layer from its pseudo-coherent interface with the Ge buffer up to the surface. The surface density of the depressions surrounded with islands is reduced to ~ 3.3  10 10 cm 2 on the Ge 1x Sn x surface. Consequently, the Ge 1x Sn x layer may contain a threading dislocation density almost one order of magnitude smaller than that of the Ge buffer. This might be related to the lower misfit between Ge 1x Sn x and Ge (about 1.5%) than between Ge and Si (about 4%).

However, the precise determination of the misfit requires knowledge of the composition of Sn in Ge 1x Sn x . Fig. 11a presents the Sn and Ge atomic distributions determined by APT in the Ge 1x Sn x layer. The Sn and Ge distributions (solid symbols) follow random distributions (solid lines), and are centered on 10 at% and 90 at% for Sn and Ge, respectively. Thus, the film corresponds to a Ge 0.9 Sn 0.1 solid solution free of Sn clusters. Interestingly, no contaminants are detected in the Ge 0.9 Sn 0.1 layer. Indeed, Fig. 11b shows part of the mass spectrum corresponding to the Ge 0.9 Sn 0.1 layer. The impurities C and O usually incorporated in sputtered layers are not detected, meaning that their concentrations are lower than 2 × 10 19 at cm 3 (~ 0.04 at%) in the film [START_REF] Descoins | PdGe contact fabrication on Se-doped Ge[END_REF]. The Sn composition determined by APT is in agreement with RBS measurements performed on the same sample (Fig. 11c). Furthermore, the RBS measurements are consistent with the stack of a 100 nm-thick Ge 0.9 Sn 0.1 layer and a 100 nm-thick Ge layer on the Si substrate. However, one can note that the Sn concentration of this film is significantly lower than that of the expected Ge 0.83 Sn 0.17 film, corresponding to Ge and Sn fluxes used during growth. Since no Sn cluster was detected by APT in the Ge 0.9 Sn 0.1 layer, this lack of Sn should be mainly due to Sn desorption from the sample surface during growth. Indeed, the growth was performed at a temperature about 100 K higher than the Sn bulk melting temperature, at a pressure P ~ 3 × 10 -3 mbar of Ar. The lattice parameter of the Ge 0.9 Sn 0.1 layer in the direction perpendicular to the sample surface was found to be a  exp = 0.576 nm from the angle of the Ge 1x Sn x (400) diffraction peak in the clearly evidenced the competition between Sn incorporation and crystal growth. The high crystallization temperature of Ge 1x Sn x , compared to the atomic diffusion kinetic and to the Sn melting temperature, drastically limits the fabrication of crystalline Sn-rich Ge 1x Sn x films.

Furthermore, the crystallization of an amorphous Ge 1x corresponding to the structure 100 nm-thick Ge 0.0 Sn 0.1 /100 nm-thick Ge /Si(001).

FIG. 1 .

 1 FIG. 1. a) XRD measurements performed during the in situ annealing of an amorphous Ge 0.83 Sn 0.17 film deposited on Si(001), following an average ramp of ~ 1 K min 1 from 423 to T = 853 K; and b) diffractogram acquired at T = 778 K during the in situ annealing (red line in a).

FIG. 2 .

 2 FIG. 2. XRD measurements performed during the in situ annealing of an amorphous Ge 0.83 Sn 0.17 film deposited on Si(001), following an average ramp of ~ 1 K min 1 from 523 to T = 853 K: a) integrated and normalized intensity of the Ge 1x Sn x (111) diffraction peak versus temperature, and b) average grain size determined from the half-maximum width of the (111) peak using the Scherrer equation (solid squares, right axis), and Sn concentration determined from the diffraction angle 2 of the (111) peak (solid triangles, left axis).

FIG. 3 .

 3 FIG. 3. Analyses performed at RT on the sample that experienced the XRD in situ annealing presented in Fig. 1 and 2: a) X-ray diffractogram, b) SEM measurements, and c) AFM measurements.

Fig

  Fig.3ashows the diffractogram measured on the sample at RT after the ramp annealing.

FIG. 4 .

 4 FIG. 4. Cross-section TEM measurements performed on the sample that experienced the XRD in situ annealing presented in Fig. 1 and 2: a) STEM Dark-Field image, b) and c) High-Resolution TEM images. The inset in c) presents the local FFT obtained on the layer A.

FIG. 5 .

 5 FIG. 5. Diffractograms measured at RT on an amorphous Ge 0.83 Sn 0.17 film deposited on the native silicon oxide formed on Si(001) and annealed at T = 788 K for 6 min, before (top) and after (bottom) chemical etching in a 5% HF solution (a), and SEM images obtained on the same sample before (b) and after (c) chemical etching.

Fig. 5a showsFIG. 6 .

 6 Fig. 5a shows the diffractogram (top) measured at RT on this sample after annealing. The

FIG. 7 .

 7 FIG. 7. X-ray diffractograms measured at RT on a 100 nm-thick Ge film deposited at RT on the Si(001) substrate and annealed at 873 K for 30 min (top) and a 100 nm-thick Ge film deposited on Si(001) at T = 633 K (bottom).

Fig. 8a presentsFIG. 8 .

 8 Fig. 8a presents an SEM image of the Ge layer surface. A structure made of undulations

FIG. 9 .

 9 FIG. 9. X-ray diffractograms measured at RT on different 100 nm-thick Ge 0.83 Sn 0.17 films grown in different conditions on a 100 nm-thick Ge buffer grown at T = 633 K on Si(001): a) deposition at the same temperature as the Ge buffer without growth interruption, b) deposition without growth interruption but stopping sample heating before Ge 0.83 Sn 0.17deposition, c) deposition after stopping sample heating at the end of the Ge buffer deposition and with a growth interruption of 10 min before Ge 0.83 Sn 0.17 deposition, and d) crystallization of an amorphous Ge 0.83 Sn 0.17 film deposited at RT on the Ge buffer.

FIG. 10 .

 10 FIG. 10. Analyses performed on the surface of a 100 nm-thick Ge 0.83 Sn 0.17 film deposited on a 100 nm-thick Ge buffer grown at T = 633 K on Si(001) in the conditions corresponding to Fig. 9b (no growth interruption, sample heater off): a) SEM image, b) AFM image, and c) AFM profile measured along the line presented in b).

FIG. 11 .

 11 FIG. 11. Chemical characterization of the 100 nm-thick Ge 0.83 Sn 0.17 film deposited on a 100 nm-thick Ge buffer grown at T = 633 K on Si(001) in the conditions corresponding to Fig. 9b (no growth interruption, sample heater off): a) Ge and Sn atomic distributions determined by APT in the Ge 1x Sn x layer (solid symbols) compared to random distribution (solid lines) centered on 90 and 10 at%, respectively, b) part of the mass spectrum acquired by APT in the Ge 1x Sn x layer, and d) RBS measurements performed on the same sample (black) and simulations (red solid line) corresponding to the structure 100 nm-thick Ge 0.0 Sn 0.1 /100 nm-thick Ge /Si(001).

FIGURE CAPTIONS FIG. 1 .FIG. 2 .

 CAPTIONS12 FIGURE CAPTIONS

FIG. 3 .FIG. 4 .FIG. 5 .FIG. 6 .FIG. 8 .FIG. 9 . 35 FIG. 10 .FIG. 11 .

 345689351011 FIG. 3. Analyses performed at RT on the sample that experienced the XRD in situ annealing

  Sn x layer deposited directly on a clean Si(001) substrate, or on a Ge buffer grown on the Si(001) substrate, does not allow the growth of a pseudo-coherent Ge 1x Sn x layer, probably due to crystal nucleation starting at the surface of the amorphous Ge 1x Sn x layer instead of at the Ge 1x Sn x /Si(001) or Ge 1x Sn x /Ge(001) interface. The Ge buffer and the Ge 1x Sn x layer must be grown at a temperature allowing for crystal growth and dislocation nucleation without significant atomic transport, in order to grow a monocrystalline layer without Ge/Sn phase separation. This temperature can be significantly lower than the Ge 1x Sn x crystallization temperature, as the nucleation process is unnecessary when the layers are grown on a single crystal. The low-cost and CMOS-compatible magnetron sputtering technique can be used to grow, at similar temperature as for CVD or MBE growth, relaxed Sn-rich Ge 1x Sn

x layers with x ≥ 0.1, displaying low impurity concentrations and reasonable electrical properties.

While Sn incorporation is mainly limited by Ge/Sn phase separation kinetics during Ge 1x Sn x crystallization, Sn surface desorption appears as the main limiting phenomenon during growth at temperatures lower than the crystallization temperature.

Khelidj et al.Figure7

Acknowledgements

This work was supported by the French-Algerian cooperation program PROFAS B+. The Ion Technology Centre ITC, Uppsala University, Sweden, is acknowledged for the RBS facilities.

diffractogram (b) in Fig. 9. Considering that a 0.1 GeSn = 0.574 nm (eq. 1), the perpendicular distortion of the Ge 0.9 Sn 0.1 lattice parameter was found to be   exp = 0.0035 (eq. 2).

(

According to eq. 5 [START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF], the Poisson's ratio of Ge 0.9 Sn 0.1 is  0.1 GeSn = 0.280 ( Ge = 0.271 and  Sn = 0.360 [START_REF] Aubin | Impact of thickness on the structural properties of high tin content GeSn layers[END_REF]), leading to  // exp = 0.0045 (eq. 3) and a // exp = 0.571 nm (eq. 2). Consequently, the relative relaxation rate of the Ge 0.9 Sn 0.1 film compared to that of the Si substrate is R = 90.3%

(eq. 4). The Ge 0.9 Sn 0.1 film is significantly relaxed, but not as relaxed as the Ge buffer, which is in agreement with a lower dislocation density in the Ge 0.9 Sn 0.1 layer. The resistivity of the undoped Ge 0.9 Sn 0.1 film was measured at RT. It was found to be  0.1 GeSn = 8 × 10 4  cm. For comparison, the resistivity of undoped monocrystalline Ge at RT is  Ge ~ 50  cm [START_REF] Chan | Development of procedures for obtaining clean, low-defect-density Ge(100) surfaces[END_REF].

Despite the unusual deposition technique employed for semiconductor growth, and the significant threading dislocation density probably contained in the layers, the resistivity of the Ge 0.9 Sn 0.1 film is found to be four orders of magnitude lower than that of Ge. Magnetron sputtering can be used to grow a pseudo-coherent Ge 0.9 Sn 0.1 film on Si(001) at a relatively high temperature (T = 633 K), displaying a high level of relaxation (R = 90.3%) and relatively low impurity concentrations (< 2 × 10 19 at cm 3 ), exhibiting interesting electrical properties ( 0.1 GeSn = 8 × 10 4  cm).

Conclusion

Magnetron sputtering growth of Ge 1x Sn x layers (x > 0.6) on Si(001) was performed at similar temperatures as usually used for MBE and CVD growth. XRD in situ measurements Khelidj et al.