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           Ann Microbiol (2015) 65:1–13                                                                      3

           Table 1 Physico-chemical features and bacterial community diversity of Favignana sandy beaches

           Sample            Mean no. T-RFs a  Mean bacterial 16S rRNA  % Humidity  Organic carbon (%)  Texture (%)
                                           gene copies (copies/g of sand) a
                                                                                          Gravel  Sand  Silt  Clay

           Lido Burrone shore-line  9.0±0  3.9±1.2x10 4          0.26      5.93           n.d.   97.9  2.0  n.d.
           Lido Burrone mid-line  6.7±0.6  1.7±0.4x10 4          0.02      5.79           n.d.   99.5  0.5  n.d.
           Lido Burrone upper-line  6.7±1.5  1.2±1.1x10 4        0.1       5.90           0.3    97.6  2.0  0.1
           Faraglioni shore-line  9.7±0.6  1.3±0.6x10 5          0.20      9.76           1.0    99.0  n.d.  n.d.
           Faraglioni mid-line  7.0±0      7.6±1.5x10 4          0.02      8.96           n.d.   100  n.d.  n.d.
           Faraglioni upper-line  5.7±0.6  6.9±0.8x10 4          0.04      8.52           n.d.   100  n.d.  n.d.
           Praja shore-line  8.7±0.6       4.6±1.1x10 4          0.18      6.56           10.8   87.4  1.8  0.1
           Praja mid-line    7.0±0.6       3.2±0.3x10 4          0.03      7.14           n.d.   98.5  1.4  0.1
           Praja upper-line  4.7±1.0       3.4±0.5x10 4          0.01      6.76           0.3    97.8  1.9  0.1

           N.d. not detectable
           a
            The mean number of T-RFs are shown as a proxy of community diversity (ribotypic diversity). Mean bacterial 16S rRNA gene copies are calculated
           with qPCR (see Material and Methods)
           ±, standard deviation


           pmoA/amoA, primer pairs A189F/A682R and A189F/       Massive sequencing was performed by Illumina-Solexa
           mb661R (Bourne et al. 2001;Horzet al. 2005) were used with  technology (Gloor et al. 2010; Bartram et al. 2011)withthe
           the two-steps amplification protocol reported in Horz et al.  pair-end protocol on an Illumina HiSeq2000 machine by
           2005. In particular, A189F/mb661R primer pair specifically  Beijing Genome Institute sequencing service (www.
           amplify pmoA sequences and not amoA sequences, allowing  genomics.cn/). Sequences are deposited in the Bioproject
           for discrimination between methane monooxygenase and am-  database (http://www.ncbi.nlm.nih.gov/bioproject/)with ID:
           monia monooxygenase genes (Bourne et al. 2001). Genomic  234346.
           DNA of Sinorhizobium meliloti was used as a positive control
           for nifH and nosZ amplification, while for pmoA/amoA geno-  Statistical analyses and processing of T-RFLP data
           mic DNA of Nitrosomonas europaea was included as a positive
           control in PCR amplification.                      Analysis of T-RFLP profiles was performed as previously
                                                              reported (Pini et al. 2012). Statistical analyses were performed
                                                              on a binary matrix obtained by linearly combining data from
           Metagenetic analysis of 16S rRNA gene amplicons    the two restriction enzymes as previously reported (Mengoni
                                                              et al. 2009). Ribotypic diversity, as the number of Terminal
           DNA aliquots extracted from Faraglioni beach samples were  Restriction Fragments (T-RFs) identified in each sample, was
           pooled together, with respect to the position along the sea-to-  used as an estimator of richness as reported previously
           land transect to obtain three samples, each composed by the  (Mengoni et al. 2009). Cluster (UPGMA) analysis and
           triplicate samples of shore-line, mid-line and upper-line sam-  Canonical Correlation Analysis (CCA) were done with Past
           ples, respectively. The variable V3 region of the 16S rRNA  software (Hammer et al. 2001) on the Jaccard similarity
           gene pool of total bacterial community was amplified from  matrix obtained from binary T-RFLP profiles. To test the
           each sample DNA with primer pairs V3-338F (5′-ACTCCT  distribution of the variance of T-RFLP profiles within and
           ACGGGAGGCAGCAG-3′)and V3-533R(5′-TTACCGCG          among beaches and on the Y-axis, AMOVA (Analysis of
           GCTGCTGGCAC-3′) (Huse et al. 2008). PCR conditions  Molecular Variance, Excoffier et al. 1992) was applied using
           were as described in Sogin and coworkers (Sogin et al.  Arlequin 3.11 software (Excoffier et al. 2007). AMOVA was
           2006). Ten independent PCR reactions per sample were done,  used as a statistical methodology alternative to the classical
           then pooled together to produce three representative PCR  analysis of variance (ANOVA). AMOVA is more flexible for
           amplicon libraries for each environmental DNA sampling  biological data than classical ANOVA because it does not
           point (shore-line, mid-line, upper-line). Products were re-  require a prior assumption of normality of the dataset and
           solved by agarose gel electrophoresis and bands were purified  statistical significance is computed through a permutation test
           with a MinElute Gel Extraction Kit (Qiagen, Inc.). Quality  (Excoffier et al. 1992; Mengoni and Bazzicalupo 2002), and it
           and quantity of products was assessed on a Biophotometer  has been widely applied to analyze community profiles in
           (Eppendorf).                                       molecular microbial ecology (for examples see Dalmastri
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