2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P35 AERObIC MTbE bIODEGRADATION by
PAeCilOMyCes vAriOTii
BLAZO LALEVIC a , VERA RAICEVIC a , LJUBInKO
JOVAnOVIC b , DRAGAn KIKOVIC c and MIOMIR
nIKSIC a
a Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-
Zemun,
b Institute for Multidisciplinary Research, Kneza Viseslava,
11000 Belgrade,
c Faculty of science, Lole Ribara 29, 38220 Kosovska Mit-
rovica
lalevicb@yahoo.com
Introduction
Methyl tert-butyl ether (MTBE) has been used since the
1970s as a gasoline additive and octane booster to replace
lead and other toxic additives and to improve combustion
efficiency of gasoline. Because of useful properties, MTBE
has become one of the organic compounds with the highest
production in the world 1 . In 1999, about 3.3 million tons had
produced in the European Union 2 . In the United States, in
1998, MTBE was the fourth-most-produced chemical 3 . However,
its extremely water solubility, mobility and volatilization
have resulted in its contamination of surface soils, groundwater
and sediments mainly from leaky tanks and spills 4 . A
report by the U.S. Geological Survey identified MTBE as the
second most common contaminant of urban aquifers in the
USA 5 . The U.S. Environmental Protection Agency 6 has listed
MTBE as a possible human carcinogen and recommended
concentration in drinking water below 40 μg dm –3 .They also
proposed the methods for removal of MTBE from environments.
Bioremediation is methods, which use natural biological
activity of microorganisms and plants to transform or
destroy different toxic contaminants 7 . Previous bioremediation
studies reported little or no biodegradation of MTBE
under aerobic 8,9 and anaerobic 10,11 condition. However,
authors 4,9,12,13 reported that pure and mixed bacterial cultures
have been capable for MTBE biodegradation. There are only
a few reports of fungal strains capable of biodegradation of
MTBE 14,15,16 so far.
The aim of this paper was to investigate the capacity of
fungal strain Paecilomyces variotii, isolate 129b, for MTBE
biodegradation at different MTBE concentration in laboratory
conditions.
Material and Methods
I s o l a t i o n a n d I d e n t i f i c a t i o n o f
F u n g u s P a e c i l o m y c e s V a r i o t i i
The fungal strain of Paecilomyces variotii, isolate
129b, was isolated from the wastewater of API separator in
Oil Refinery Pancevo, Serbia. The cultures maintained onto
rose bengal streptomycin agar (RBSA) plates 17 at 26 °C. The
identification based on fungal morphological characteristics
s406
growing in malt agar and Czapek yeast agar (CYA), using the
key for identification 18 .
D e g r a d a t i o n E x p e r i m e n t
Liquid suspension cultures grown axenically in 250ml
glass bottles. The growth medium (80 ml) was mineral salts
medium 14 . The medium sterilized at 121 °C for 15 min and
inoculated with 10 % (v/v) of suspension of Paecilomyces
variotii conidia (13.8 × 10 5 ). After addition of different MTBE
concentrations, the liquid suspension cultures incubated for
19 days at 26 °C and shaken at 120 rpm. All the experiments
were conducted in triplicate.
T h e Y i e l d o f P a e c i l o m y c e s
V a r i o t i i M y c e l i a
The yields of mycelia measured after 19 days by means
of dry weight. Liquid suspension cultures were vacuum filtered
onto filter papers. Mycelia and filters were dried at 65 °C
for 24 h and reweighed. The control variant was without
MTBE as an sоle energy and carbon source. The experiments
were conducted in triplicate.
A n a l y t i c a l M e t h o d s
The MTBE used in these experiments originates from
industrial facilities of Oil Refinery Pancevo. Consumption of
MTBE was monitored by a Agilent Technologies 6890n gas
chromatograph (GC) fitted with a flame ionization detector. A
30 m × 0.53 mm ID, 3.0 μm DB-624 column was used (J&W
Scientific, Folsom, CA, USA). The temperature program was
50 °C for 2 minutes, ramp 8 °C min –1 to 100 °C, hold 3 minutes.
The injector temperature was 170 °C and detector (FID)
temperature 300 °C. The flow hydrogen was 40 ml min –1 ,
flow air 450 ml min –1 and make up gas n 2 25 ml min –1 .
The headspace (Agilent 7694E Head Sampler) was:
vial equilibration time 30 min; bath temp. 80 °C; valve/temp
loop 85 °C; transfer line temp. 120 °C; loop size 1 ml; pressure
time 0.00 min; loop fille time 0.050 min; loop eq. time
0.05 min; inject time 1.0 min. Internal standard was acetonitrile
(retention time 2.951 min). The MTBE retention time
was 3.310 min. In experiment, headspace samples (100 μl)
were taken with gas-tight syringes.
The consumption of MTBE was measured in the beginning
of the experiment and after 5; 12; 15 and 19 days.
Results
The soils of Oil Refinery Pancevo are heavy polluted
with different organic substances and it can be expected to
find out big diversity of fungus and bacteria whose use pollutants
there as sole source of carbon and energy. During different
experiments, about 40 bacteria and 14 different fungi
strains were identified and isolated. One of the fungi founded
there, after testing, showed high ability to consume MTBE.
That fungus is isolated and identified as Paecilomyces variotii,
isolate 129b, and used in experiments. Ours results
showed that degradation rate was depending on initial MTBE
concentration (10.85; 34.34 and 83.15 ppm used) and time