Page 4 - Bacci_Pagoto_etali_2015

Author's personal copy

2 Ann Microbiol (2015) 65:1–13

Hernandez et al. 2012), consequently hampering functional conditions present in the beach and then stored at −80 °C
studies and biodiversity estimations in such an “extreme”,but prior to DNA extraction. We cannot exclude that for some
common environment. In fact, the large fluctuations in tem- samples the storage conditions could have affected the relative
perature, humidity, salinity and nutrient of sandy beaches, abundance of some taxa.
especially in a temperate zone, could allow for consideration Physical-chemical characteristics are reported in Table 1.
of these environments as nontypical, when compared to soil or Determination of organic carbon (TOC) present in the test
water environments. Additionally, the supralittoral zone of samples was performed following a standard protocol (Italian
sandy beaches may also contain human pathogens, due to Official Bulletin, G.U. n ° 248 of 10.21.1999) with a Perkin
human impact by recreational use of beaches, or urbanization Elmer elemental analyzer CHNS/O Series II model 2400.
(see for examples Bonadonna et al. 2003; Mudryk 2005; Granulometry was analyzed by particle size analysis using
Ugolini et al. 2008). standard procedures (Bowles 1988;Head 1984).
The aim of this work is to provide a first insight into the
composition of bacterial communities present in supralittoral DNA extraction, T-RFLP profiling, functional genes
sediments of Mediterranean sandy beaches by using three detection and real-time PCR
beaches at Favignana Island (Italy), which are differentially
exposed to wind and water streams and to anthropic impact. DNA was extracted from sediments, after homogenization of
the total sample contained in the 50 ml polypropylene sterile
tube, by using a commercial kit (Fast DNA Spin kit for soil,
Material and methods QBiogene, Cambridge, UK), following manufacturer’s
instructions.
Sampling site description, sampling procedure The 16S rRNA genes were amplified from extracted DNA
and physico-chemical characteristics of all 27 collected samples with primer pairs 27f and 1495r, as
previously reported (Trabelsi et al. 2009). Terminal-
Samplesof subsurfacesand(5cm below the surface) were Restriction Fragment Length Polymorphism (T-RFLP) proce-
taken in summer 2011 in three beaches of Favignana Island dure was then followed on purified amplification products. In
(Italy). The Favignana Islands are part of the Protected Marine particular, amplicons were digested separately with restriction
Area “Isole Egadi”, located in the Southern part of enzymes TaqIand AluI and digestions were resolved by cap-
Mediterranean sea, in close proximity to Sicily Island (Italy) illary electrophoresis on an ABI310 Genetic Analyzer
and is characterized by Lower Pleistocene carbonate (Applied Biosystems, Foster City, CA, USA) using LIZ 500
grainstones. The three main beaches present in the Island were (Applied Biosystems) as a size standard. T-RFLP analysis was
sampled, namely, Praja (37°55′45.62″ N, 12°19′30.66″ E), performed on two technical PCR replicates from each DNA
which is located in close proximity to the main urban centre extract, as previously reported (Mengoni et al. 2005). Only
of the island on the north-east coast; Lido Burrone (37°55′ peaks present in both duplicate runs were considered for
9.67″ N, 12°18′24.67″ E), a touristic beach on the south-west successive analyses.
coast, and Faraglioni (37°56′42.83″ N, 12°16′46.93″ E), a Real-Time PCR for quantification of bacterial cells was
beach with limited touristic use, due to lack of main roads performed estimating the number of 16S rRNA gene copies
for accessing it, on the north-west coast. From every beach, a with bacterial primers Eub341F (5′- CCTACGGGAGGCAG
transect from the damp band to the upper limit of the beach CAG-3′) and Eub515R (5′-TACCGCGGCKGCTGGCA-3′)
(thereafter referred to as the “Y-axis”) was done considering targeting the 16S rRNA gene, as reported by Simmons and
three sampling points: i) the damp band (here named as the coworkers(Simmonsetal. 2007), using genomic DNA of
shore-line), ii) the intermediate zone between the damp band Sinorhizobium meliloti Rm1021 as the concentration standard
and the upper limit of the beach (named as the mid-line) and for copy number calculation (genome size 6.9 Mbp). Data
iii) the upper limit of the beach, 1 m before the dune zone and obtained were compared with one-way ANOVA.
the vegetation (named as the upper-line). For each sampling Functional diversity was evaluated by PCR amplification
point, three samples 20-cm apart from each other, were taken of nifH (encoding the nitrogenase reductase subunit), nosZ
along an ideal line parallel to the shore-line. A total of nine (encoding the nitrous oxide reductase gene) and pmoA/amoA
samples per beach were taken. Sampling consisted of filling (the internal fragment of particulate methane monooxygenase
completely a 50 ml polypropylene sterile tube inserted in and ammonia monooxygenase genes, Holmes et al. 1995). For
sandy sediment from its surface down to the total length of nifH the semi-nested approach described in Widmer et al.
the tube (ca. 10 cm). Due to the impossibility of immediately 1999 was used, following the amplification protocol reported
storing at −80°C or extracting DNA, samples were stored with in Giuntini et al. 2006.For nosZ, primers nosZ-F-1181 and
an open lid at ambient temperature (25°C) for two days in the nosZ-R-1880, were used (Rich et al. 2003) with the amplifi-
dark, trying to simulate as much as possible the natural cation protocol already reported (Pastorelli et al. 2011). For