Short note - MUSE - Museo delle Scienze

Studi Trent. Sci. Nat., 86 (2009): 63-65
ISSN 2035-7699
63
© Museo Tridentino di Scienze Naturali, Trento 2009
Atti XVIII Convegno Gadio 2008: Un mondo che cambia: successioni ecologiche, invasioni biologiche ed alterazioni antropiche
Sessione 4 - Poster
Short note - Nota breve
Characterization of the microflora in polluted soils by using culture-dependent and
indipendent methods
Elisa GAMALERO1, Arturo Fabiani2, Chiara Musso1, Maurizio Castaldini2, Silvia Landi2, Gian
Paolo Cossa3 & Graziella Berta1*
Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale “Amedeo Avogadro”, Viale Teresa
Michel 11, 15121 Alessandria, Italy
2
Istituto Sperimentale di Studio e Difesa del Suolo, Piazza D’Azeglio 30, 50121 Firenze, Italy
3
ARPA Piemonte, Polo Bonifiche, Spalto Marengo 38, 15100 Alessandria, Italy
*
Corresponding author e-mail: graziella.berta@mfn.unipmn.it
1
rIASSUNTO - Caratterizzazione della microflora in suoli contaminati con l’impiego di metodi coltura dipendenti ed indipendenti - Lo
scopo del lavoro è stato caratterizzare la comunità microbica di suoli inquinati del sito ACNA. I campioni provenivano dalla base (1a),
dalla sommità (1b) e dall’interno di una collinetta di rifiuti (2a, substrato macinato da alcuni anni; 2b, substrato macinato fresco). Sono
stati valutati l’attività dei suoli, la struttura della comunità dei batteri coltivabili, e degli Eubatteri totali ed attivi. Le differenze tra i siti a
livello di composizione ed attività della microflora erano ascrivibili alla presenza di inquinanti diversi. In accordo con le analisi chimiche,
i dati ottenuti indicavano i siti 1a e 2a come i più degradati.
Key words: polluted soil, metabolic profile, enzymatic activities, microbial community structure, DGGE, Liguria (Italy)
Parole chiave: suolo contaminato, profilo metabolico, attività enzimatiche, struttura della comunità microbica, DGGE, Liguria (Italia)
1.
INTRODUCTION
Fifteen national environmentally critical sites have
been identified in Italy. Among them, ACNA (Azienda
Colorifici Nazionali Affini) was an industrial plant active
in the production of different chemicals, mainly intermediates for the synthesis of explosives and dyes, for more
than 100 years, until 1998. This kind of activity led to
a heterogeneous soil pollution characterized by the simultaneous presence of both aromatic hydrocarbons and
heavy metals (D’Annibale et al. 2006).
A first attempt in characterizing the soil microflora
inside the ACNA site, was performed through the use of culture-dependent methods (Avidano et al. 2005). However, it
has been estimated that only 1% of the total soil microflora
is culturable (Torsvick et al. 1990). In this work a microbiological characterization of four soil sites has been performed
by coupling culture dependent and independent methods.
2.
METHODS
Sampling was performed both at the bottom (site
1a; pH 8.23, Total Organic Carbon 1.98%, Soil Organic
Matter 6.52%, sand 84.88%, silt 10.89%, clay 4.23%) and
the top (site 1b; pH 8.45, Total Organic Carbon 1.58%,
Soil Organic Matter 4.09%, sand 86.22%, silt 13.26%,
clay 0.51%) of a waste hill. Inside the zone of waste preliminary treatment (site 2), a substrate previously removed
from the core of a waste hill and left to stand for several
years (site 2a; pH 9.96, Total Organic Carbon 0.83%,
Soil Organic Matter 2.57%, sand 80.43%, silt 9.63%,
clay 9.95%) and a freshly milled substrate (site 2b; pH
8.87, Total Organic Carbon 1.32%, Soil Organic Matter
3.43%, sand 83.62%, silt 12.77%, clay 3.62%) were sampled. Determination of metal concentrations have been
performed following the EPA (Environmental Protection
Agency) method n° 6020a. Measurement of semivolatile
organic compounds and PAHs (Polycyclic Aromatic Hydrocarbons) was performed following the EPA methods n°
8270d and 8280b.
Biological culture-dependent analyses (bacterial
density, composition and structure evaluation, carbon utilization pattern of the soil microflora) were performed as
described by Avidano et al. (2005). The evaluation of soil
microbial activity was assayed through fluorescein diacetate hydrolysis (Adam & Duncan 2001).
The DNA was extracted from the whole community of
culturable bacteria by the CTAB method (Ausubel et al. 1987)
and directly from soil using the UltraClean™ Soil DNA Kit
64
Gamalero et al.
Characterization of the microflora in polluted soils
Tab. 1 - Frequence of culturable bacterial species identified in the four sites.
Tab. 1 - Frequenza delle specie batteriche coltivabili identificate nei quattro campioni di suolo.
Identification
Actinomyces sp.
site 1a
site 1b
8.33
6.06
Arthrobacter histidinolovorans
site 2b
9.68
3.70
6.06
Bacillus amyloliquefaciens
4.17
Bacillus cereus / thuringensis
4.17
Bacillus fastidiosus
3.03
Bacillus halodurans
Bacillus laevolacticus
site 2a
3.22
3.22
4.17
3.22
Bacillus licheniformis
9.68
Bacillus maroccanus
6.06
32.26
Bacillus megaterium
6.45
Bacillus mycoides
6.45
Bacillus sphaericus
8.33
Bacillus subtilis B
4.17
Brevibacillus brevis
12.50
3.22
21.21
Brevibacterium otitidis
7.41
3.22
Burkholderia cepacia
7.41
Burkholderia glumae
4.17
Buttiauxella gaviniae
12.50
Curtobacterium pusillum
3.70
4.17
3.70
Kocuria rosea
Kurthia sibirica
14.81
3.70
3.03
Bacillus thermoglucosidasius
Flavimonas oryzihabitans
14.81
3.70
4.17
Paenibacillus larvae
3.70
Paenibacillus pabuli
3.22
Paenibacillus polymyxa
3.22
Pantoea stewartii
4.17
Providencia alcalifaciens
4.17
3.70
3.70
Pseudomonas alcaligenes
3.70
Pseudomonas aurantiaca
3.70
Pseudomonas fluorescens
12.50
39.39
7.41
Pseudomonas syringae
4.17
6.06
3.70
Ralstonia solanacearum
3.03
Rhodococcus equi
3.70
Roseomonas fauriae
3.70
Sphingomonas capsulata
Staphylococcus gallinarum
6.06
4.17
Staphylococcus pasteuri
6.45
Staphylococcus xylosus
6.45
Total
100
100
100
100
Studi Trent. Sci. Nat., 86 (2009): 63-65
A)
C)
65
green and industrial area. A medium functional diversity
was measured in samples 1a, 1b and 2b (51.6%, 44.2%,
63.2% of positive wells on Biolog microplate), while no
metabolic profile has been observed for 2a site. In addition a low microbial activity has been recorded only in
sites 1b and 2b. The bacterial densities (measured as log
colony forming units/g of soil dry weight) differed among
the sites, with the highest value in site1b (6.40) and the
lowest in site 2a (3.42). In addition, in site 2a, only Gram
positive bacteria have been detected. All the selected
bacterial strains were identified by Biolog system (Tab.
1). Although no bacterial species was shared among all
the four soil sites, the samples 1a and 2b were the most
similar. On the contrary, site 2a, sharing a low number of
species with the other sites, clustered alone. The DGGE
fingerprintings of Eubacteria were quite different among
microbial communities of the four sites. In particular, soils
1a and 2a showed a low number of bands and a reduced
biodiversity.
4.
B)
D)
Fig. 1 - DGGE banding pattern and cluster analysis of culturable
Eubacterial 16SrDNA (A and B) and total Eubacterial 16S rDNA
and rRNA (Cand D). The letter M on the gel images indicates the
marker.
Fig. 1 - Profili DGGE ed analisi cluster dell’rDNA 16S degli Eubatteri coltivabili (A e B) e dell’rDNA e RNA 16S degli Eubatteri
(C e D). La lettera M sull’immagine dei gel indica il marker.
Mega Prep (MO BIO Laboratories, Inc.). RNA extraction
from each soil was performed with RNA PowerSoil™ Total RNA Isolation Kit (MO BIO Laboratories, Inc.) following the manufacturer’s instructions. For DGGE analysis of the Eubacterial community, V6-V8 regions of 16S
rDNA were amplified with primers GC986f and Uni1401r
as described by Felske et al. (1998). The 16S rRNA was
reverse-transcribed with reverse transcriptase enzyme
ImProm II (Promega) with Uni1401r primer. DGGE gels
were analyzed by GelCompare II software, using UPGMA (Unweighted Pair-Group Arithmetic Mathematical
Average) and Dice’s coefficient.
3.
RESULTS
Eighteen pollutants (12 organic and 6 inorganic)
have been detected in the four samples. Soils 1a and 2a
showed a heterogeneous pollution involving both organic
and inorganic compounds, exceeding guideline values for
DISCUSSION
The four sites showed a heterogeneous pollution.
The microbial culturable fraction in site 2a was characterized by the lowest density and by the absolute dominance
of Gram positive strains. The low similarity of the culturable fraction among the four sites suggests that each sample was characterized by a specific microflora. According
to these results, DGGE analysis showed huge differences
in the composition of Eubacterial communities present in
each site, probably related to the different pollutants found,
as well as to the different managements of these soils.
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