Bacterial strains and growth conditions
strains were isolated during biofiltration processes of mixtures and
single components of VOCs contaminated air. Two strains, named 14 and
C18, were selected as positive and negative controls for all molecular
reactions. The strains were isolated from a mixed population of an
enriched culture of samples withdrawn from bed biofilter matrices. This
was generally constituted of composted organic matter (food waste,
plant residues and wood chips). Enrichment cultures were set up using
minimal salt basal medium (MSB) (Stanier et al. 1966) supplemented with each single solvent as carbon source and grown at 28°C. The same medium was utilized to determine microbial
growth (O.D. 660 nm) and respirometric assay (Pritchard et al. 1992), while 15 g Noble agar l−1 was added to prepare plate tests with the carbon source distributed in vapor phase.
Escherichia coli TOP10 and BL21 (Stratagene, La Jolla, CA, USA) were used as a further negative control.
DNA extraction from bacterial culture
Genomic DNA of isolated strains grown in LB broth (Triptone 10 g l−1, yeast extract 5 g l−1, NaCl 10 g l−1) was extracted as described previously (Versalovic et al. 1991).
Nucleic acid (DNA and RNA) extraction and purification from organic matter samples
Extractions were performed essentially as described by Griffiths et al. (2000).
Briefly, 100 mg powdered organic matrix, obtained from each bioreactor
after freezing, lyophilization and further homogenization, were
dissolved in 0.1 ml CTAB extraction buffer and 0.1 ml of
phenol/chloroform/isoamyl alcohol (25:24:1 by vol.) (pH 8.0).
extraction-purification steps, pellets containing nucleic acids were
washed with 70% (v/v) ethanol, air dried and resuspended in 30 μl
Tris/EDTA buffer (pH 7.4 and RNase free).
The extracted DNA and RNA quality was further verified by analysis on agarose gel stained with ethidium bromide (0.5 μg/ml).
Preparation of purified RNA for RT-PCR analysis
nucleic acids, 30 μl, were treated with (3 U) DNase (Sigma and
RNase-free) for 1 h at 37°C. The reaction mixture was then mixed with
guanidine thiocianate (GTC) up to 200 μl and 200 μl sodium acetate, 2 M
pH 4.0, followed by 40 μl chloroform/isoamyl alcohol (24:1) and 200 μl
phenol acid. The suspension was then vigorously stirred and placed on
ice for 15 min. Subsequently, it was centrifuged at 13,00 wg for 10 min
(4°C). To the collected supernatant, 1 vol. ethanol was added and then
frozen for 30 min at −20°C, followed by centrifugation at 13,000 wg for
20 min (4°C). The pellet of RNA was further washed in 70% (v/v) ethanol
and again centrifuged at 13,000 g for 5 min (4°C). At the end, the
pellet was resuspended in 30 μl diethylpyrocarbonate (DEPC) treated
The RNA presence and quality was estimated on agarose gel (0.5–1% w/v) in 15% (v/v) formaldehyde, ethidium bromide, 0.5 μg/ml,
run in MOPS buffer (20 mM, pH 7.0, 10 mM sodium acetate; 1 mM EDTA, pH 8.0).
Primers and PCR amplification of 16S rDNA and catabolic enzymes from genomic DNA
16S rDNAs were selectively amplified (Gene Amp PCR System 2400, Perkin
Elmer thermal cycler) from purified genomic DNA or from boiled cells
routinely cultured on LB broth. Oligonucleotide primers were designed
to anneal to conserved positions in the 3′ and 5′ regions of bacterial
16S rRNA genes (Al-Robaiy et al. 2001). The forward primer prbfo corresponding to position 509–525 of E. coli
16S rRNA whose upper sequence was 5′-ACTACGTGCCAGCAGCC-3′. The reverse
primer was prbre 5′-GGACTACCAGGGTATCTAATCC-3′ corresponding to the
complement of position 784–805 of E. coli 16S rRNA. The amplification product thus resulted as 297 bp.
reaction mixture, in a final volume of 100 μl, was placed in 0.2 ml
thin wall Eppendorf test-tubes. The reaction mixture, contained Taq
Polimerase (Perkin-Elmer) 2.5 U, template DNA solution (20–200 ng
genomic DNA), 0.5 μM of each primer, 4% (v/v) DMSO, MgCl2 1.5 μM and 10 μl buffer 10×
(Perkin-Elmer). Amplification conditions were as follows: a preliminary
denaturation of 10 min at 94°C followed by 40 cycles of 45 s at 94°C
(denaturation), 30 sec at 66°C (annealing), 30 s at 72°C (extension)
and a final cycle of 8 min at 72°C. PCR products were electrophoresed
at 10 V cm−1 in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).
primers to amplify catabolic enzyme genes have been chosen to identify
the key enzymes involved on the xylene and toluene aerobic degradation
pathways. Due to the presence of various isoforms of the key enzymes in
different bacterial species, the choice of primers has been limited to
extremely conserved DNA regions using the BLAST program analysis of the
National Center for Biotechnology Information of the National
Institutes of Health (http://www.ncbi.nlm.nih.gov/blast).
For xylene pathways, the sequence of PCR primer, xylfwd, was 5′-CATTCTTTCTTTGGCTTGGCTTAGTGG-3′, corresponding to nucleotide
4988–5014 of xylene monooxygenase hydroxylase component (xylM) of AF019635 sequence, and the primer, xylrev, was 5′-CCTCAATCTTTATCGCATCTTTGACGG-3′, corresponding to nucleotide 5256–5230
of the same sequence. For the toluene degradation pathway, the alpha subunit-terminal oxygenase component, tbmD gene, of toluene/benzene-2-monooxygenase (Johnson and Olsen 1995)
was chosen to design the following primers: tol2fwd primer sequence
5′-CGCTGGACTGACAAGTGGTTCTGG-3′, corresponding to 2838–2861 of L40033
Gene Bank sequence, and tol2rev reverse primer
5′-TTCTCCGAGAGCCATTGCATCTCTT-3′, corresponding to 3148–3124 position of
the same sequence.
The reaction mixture
components were the same as those described above, with amplification
conditions as follows: a preliminary denaturation of 120 s at 94°C
followed by 35 cycles of 45 s at 94°C, 50 s at 58°C, 60 s at 72°C and
8 min at 72°C (extension). PCR products were electrophoresed at 10 V cm−1 in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).
RT-PCR were performed on the extracted RNA, after a preliminary phase
of denaturation at 70°C for 5 min followed by rapid cooling on ice. The
reverse-transcription reactions were performed in 0.2 ml thin wall
Eppendorf tubes in 50 μl final volume, using a mixture of exaprimers
(Invitrogen, Milan, Italy) at 10 ng/μl, 200 μM of every dNTP, 200 U of
M-MLV-RT (Invitrogen, Milan, Italy), 4 mM DTT, 40 U RNase inhibitor
(Invitrogen, Milan, Italy), and 10 μl 5× buffer (Invitrogen, Milan,
Italy). The reverse-transcription reaction was performed on 5 μl of the
extracted RNA at 37°C for 1 h. The PCR was performed on 5 μl of
produced cDNA as described above. Reaction tests where the sample had
been omitted and replaced with an equal volume of water were used as
negative control, and the presence of the rRNA 16S bacterial gene was
tested as positive control.