Page 23 - Volume 6, Issue 2, July 2013
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© Benaki Phytopathological Institute
Biodegradation of diazinon by epiphytic yeasts
71
source at 50
o
C and drying gas (N
2
) at 19 psi
pressure and 250
o
C temperature. The elec-
trospray ionization mode was in positive
mode using nitrogen as nebulizer gas at 50
psi. The needle voltage was 5000 V while the
spray shield voltage was 600 V.
The European guideline SANCO 2011/
12495 document (Method Validation and
Quality Control Procedures for Pesticide
Residues Analysis in Foods and Feeds) was
followed for the validation of the analytical
methods. Different known concentrations of
diazinon and IMP were fortified in the min-
eral salts medium (0.05 μg/mL, 0.1 μg/mL
and 0.5 μg/mL) and tomato fruits (only par-
ent compound) with 5 replicates.
Yeast cultures
Rhodotorula glutinis
was isolated from to-
mato plants while
Rh. rubra
from strawberry
plants. Cultures were developed in mineral
salts medium containing in g/L: 12 NH
4
NO
3,
8 KH
2
PO
4
, 2 Na
2
SO
4
, 4 KCl, 1MgSO
4
.
7H
2
O, 0.5
CaCl
2
, 0.26 ZnSO
4
.
7H
2
O, in 250mL Erlenmeyer
flasks supplemented with diazinon and cells
(each yeast in a separate flask) and incubated
in an orbital incubator at 25
o
C and 150 rpm.
Effects of various factors on diazinon
degradation in liquid cultures
Incubations of the yeasts in 250 mL flasks
containing 50 mL mineral salts medium (NH
4-
NO
3
, KH
2
PO
4
, Na
2
SO
4
, KCl, MgSO
4
.
7H
2
O, CaCl
2
,
ZnSO
4
.
7H
2
O, FeSO
4
.
7H
2
O and Na
2
MbO
4
.
7H
2
O)
with 10
2
, 10
4
and 10
6
cells/mL, in the pres-
ence of 10 μg/mL diazinon, were conduct-
ed at 25
o
C in order to examine the effect of
yeast inoculum on the degradation. To ex-
amine the effect of concentration on the
biodegradation rates, the experiments were
conducted in 250 mL flasks containing 50 mL
mineral salts medium supplemented with
diazinon at concentrations of 10 and 25 μg/
mL. To confirm the influence of temperature,
degradation trials were conducted in 250 mL
flasks containing 50 mL of the mineral salts
medium supplemented with 10 μg/mL di-
azinon at 15
o
C, 25
o
C and 35
o
C, respectively.
The influence of glucose in the nutrient min-
eral medium, as an extra source of carbon,
was also investigated simultaneously with
all the above parameters. In each trial there
were additional 250 mL flasks containing
50 mL mineral salts medium supplemented
with glucose under the certain experimen-
tal conditions and the influence of this fac-
tor was also estimated. All treatments were
in triplicate. Flasks were incubated in an or-
bital incubator at 25
o
C and 150 rpm. At time
intervals of 2 days (samples were taken asep-
tically and analyzed for diazinon residues.
Extraction of diazinon and IMP from
pure cultures and tomato fruits
Subsamples of 5 mL of the of miner-
al salts nutrient medium (50 mL) were tak-
en aseptically and filtered through a What-
man No 2 filter above a 100 mL separating
funnel. Liquid- liquid extraction was carried
out with 20 mL dichloromethane: acetone
(1:1) for three times. Extracts were passed
through anhydrous sodium sulfate, collect-
ed in a 100 mL flat bottom flask and evapo-
rated to dryness on a rotary evaporator. The
dry residue was diluted to 2 mL methanol:
water (30:70) and filtered through a 0.2 μm
film filter in order to be analyzed in the LC-
MS/MS chromatographic system.
The method used for the sample pro-
cessing of tomato fruits has been described
by Bilthoven (1996). 30mL of acetone were
added in an aliquot of 15 g of the homo-
genated sample in a 250 mL PTFE centri-
fuge bottle (Nalgene, Rochester, NY, USA)
and stirred for 1 min in an ultra-turrax ho-
mogenizer at 15000 rpm. 30 mL of dichlo-
romethane and 30 mL of petroleum ether
(40-60
o
C) were added following by a new
stirring step for 1 min. The sample was cen-
trifuged at 4000 rpm for 2 min. 25 mL of the
supernatant were evaporated to dryness on
a water bath at 65-70
o
C and afterwards 3 mL
of methanol: water (30:70) was added. The
extract was placed in an ultrasonic bath for
30 s, filtered through a 0.2 μm film filter and
transferred in a vial with teflon septa, ready
for chromatographic analysis.
Enzymatic assays
One mL of liquid culture was added in an
