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Cas illo López, MÁ.; A agón Re uel a, P.; Cues a Ama , G.; Saba e Ma co, C. (2006).
Biodeg ada ion o he bicide diu on by s ep omyce es isola ed om soil. In e na ional
Biode e io a ion and Biodeg ada ion. 58(3-4):196-202. doi:10.1016/j.ibiod.2006.06.020.
Biodeg ada ion o he he bicide diu on by s ep omyce es
isola ed om soil
M.A. Cas illo
a,
*
, N. Felis
a
, P. A ago´ n
b
, G. Cues a
a
, C. Saba e
a
Abs ac
The diu on deg ading ac i i y o 17 s ep omyce e s ains, ob ained om ag icul u al and non-ag icul u al soils, was de e mined in he
labo a o y. All s ains we e iden ified as S ep omyces sp. by pheno ypic cha ac e is ics and PCR-based assays. The s ains we e cul i a ed in
liquid medium wi h diu on (4 mg L-1) a 25
1
C o 15 days. Biodeg ada ion ac i i y was de e mined by high-pe o mance liquid
ch oma og aphy. The esul s indica ed ha all s ains we e able o deg ade diu on, bu o di e en amoun s. Twel e s ains deg aded he
he bicide by up o 50% and ou o hem by up o 70%. S ain A7-9, belonging o S. albido l clus e , was he mos e ficien o ganism in he
deg ada ion o diu on, achie ing 95% deg ada ion a e fi e days o incuba ion and no he bicide emained a e 10 days. O e all, he s ains
isola ed om ag icul u al soils exhibi ed highe deg ada ion pe cen ages and a es han hose isola ed om non-ag icul u al soils. Gi en he
high deg ada ion ac i i y obse ed he e, he s ep omyce e s ains show a good po en ial o bio emedia ion o soils con amina ed wi h diu on.
Keywo ds: He bicide; Diu on; Ac inomyce es; S ep omyce es; Soil
1. In oduc ion
Mic oo ganisms play a significan ole in he ans o -
ma ion and deg ada ion o pes icides. E en he mos
pe sis en pes icides can be me abolized o some ex en by
mic obial cul u es, ei he by u iliza ion o he compounds
as sou ces o ene gy o nu ien s, o by come abolism wi h
o he subs a es suppo ing mic obial g ow h. This las
p ocess is p obably he mos widesp ead mechanism o
pes icide biodeg ada ion. Comple e mine aliza ion o
pes icides o hei ans o ma ion o non- oxic p oduc s is
desi able, bu i is mo e likely o be ca ied ou by conso ia
o mic oo ganisms han single isola es.
The e is g owing conce n o e he po en ial o
con amina ion o soils, su ace and g oundwa e by he
epea ed use o he bicides. Among he he bicides used o
p e en he g ow h o undesi able plan s, phenylu ea de i-
a i es ha e been widely used since hei disco e y in he
ea ly 1950s. One o hem, diu on (3-(3,4-dichlo ophenyl)-1,
1-dime hylu ea), is employed widely o weed con ol in
non-c op a eas, such as oads, ailways acks, and pa hs,
and o con ol weeds in a ange o ee c ops, such as, pea ,
apple, o es y, o namen al ees and pineapples, suga
cane, co on, al al a and whea (Tomlin, 2003). I kills
plan s by blocking elec on anspo a pho osys em II,
hus inhibi ing pho osyn hesis.
Diu on is applied o soil, whe e i ends o accumula e
because o i s low solubili y in wa e . DT50 in soils a ies
om 90–180 days (Hill e al., 1955; Tomlin, 2003); o he
sou ces indica e ha i can ange om one mon h o one
yea (Field e al., 2003), al hough alues o g ea e han
3000 days ha e been epo ed (Madhum and F eed, 1987).
The a iabili y in calcula ed DT50s is likely o be due o
di e ences in soil composi ion and o he condi ions
(Gailla don, 1996; Rouchaud e al., 2000). Deg ada ion
inc eases mode a ely in soils ea ed wi h diu on o many
yea s. In one expe imen , whe e soils we e ea ed a a a e
o 3 kg ha
-1
yea
-1
o 12 yea s, he hal -li e dec eased by
50% o e his pe iod, sugges ing ha he soil mic obial
communi y was becoming inc easingly able o deg ade i
(Rouchaud e al., 2000).
Much da a ha e been collec ed on he di ec and indi ec
e ec s o diu on on a ge and non- a ge o ganisms, and
diu on is suspec ed o be geno oxic (Canna-Michaelidou
and Nicolau, 1996). The US En i onmen al P o ec ion
Agency (EPA) has classified diu on as a ‘known/likely’
ca cinogen since 1997. The e is conflic ing e idence on
whe he diu on can cause mu a ions. The Na ional
Ins i u e o Occupa ional Sa e y and Heal h in he US
ca ego ized diu on as a mu agen, based on old s udies
(Seile , 1978). I s use in Eu ope is being e iewed.
Se e al epo s conside ed mic obial deg ada ion he
main mechanism o dissipa ion o diu on om soil
(Shee s, 1964; Geissbu
¨ hle e al., 1973), as well as
in aqua ic en i onmen s (Ellis and Campe , 1982;
Howa d, 1991). In a numbe o hese s udies, soil ungi
ha e shown a high deg ada i e ac i i y agains
phenylu eas, wi h occa- sional abili y o deg ade diu on
(Weinbe ge and Bollag, 1972; Tillmanns e al., 1978;
V oumusia e al., 1996; Tixie e al., 2000), bu only a ew
s udies ha e ound deg ada i e ac i i y in bac e ia
(Shel on e al., 1996; Esposi o e al., 1998; Culling on
and Walke , 1999; Tixie e al., 2001, 2002; Widehem
e al., 2002). The main deg ada ion p oduc s and
pa hways o diu on deg ada ion a e e iewed by
Giacomazzi and Coche (2004).
Among bac e ia, ac inomyce es ha e a conside able
po en ial o he bio ans o ma ion and biodeg ada ion
o xenobio ics. De Schi ij e and De Mo (1999) desc ibed
deg ada ion by ac inomyce es o pes icides wi h widely
di e en chemical s uc u es, including o ganochlo ines, s-
iazines, ca bama es, o ganophospha es, sulphonylu eas
and phenylu eas. O he s udies (Al a ez, 2003) showed
ha a ious species o ac inomyce es exhibi ed a high
capaci y o deg ade hyd oca bons.
Few s udies ha e been conduc ed o assess he abili y o
ac inomyce es o deg ade o ans o m diu on. Only li le
da a a e epo ed abou specific s ains. Shel on e al. (1996)
s udied he abili y o S ep omyces (PS1/5) o me abolize a
a ie y o s uc u ally di e en he bicides, including diu on,
which was deg aded by 19% a e 7 days. Esposi o e al.
(1998) ound one unique s ain wi h a good po en ial o
diu on deg ada ion. Mo e ecen ly, o he au ho s desc ibed
A h obac e s ains which ha e been able o deg ade diu on
(Tixie e al., 2001, 2002; Widehem e al., 2002).
The me abolic di e si y o ac inomyce es and hei
pa icula g ow h cha ac e is ics (in asi e mycelial, apid
coloniza ion o selec i e subs a es, e c.) make hem
a ac i e o ganisms o bio emedia ion. The aim o he
p esen s udy was o in es iga e he diu on biodeg ada ion
po en ial o s ep omyce e s ains isola ed om soil.
2.
Ma e ial and me hods
2.1. Mic obiology
2.1.1.
Soils
Ac inomyce e s ains we e isola ed om se en di e en soils: wo
uncul i a ed soils and fi e soils om ci us o cha ds egula ly ea ed wi h
diu on. The uncul i a ed soils we e clay loam, wi h pH 8, high con en s o
calcium ca bona e (41%) and o ganic ma e (5–6%). The cul i a ed soils
we e sil y loam o sandy loam, wi h pH 7.8–8.1, high con en o calcium
ca bona e (38%) and low le el o o ganic ma e (1.5–2%). The samples
we e collec ed om he fi s 10 cm below he su ace, asep ically
ans e ed o s e ile ials and s o ed a 4
1
C un il used.
2.1.2. Mic oo ganisms
A o al o 53 ac inomyce e s ains we e isola ed om soil using
s anda d isola ion me hods. Se en een andomly chosen s ains we e
es ed o diu on deg ading abili y. Twel e ou o hem we e isola ed om
he ag icul u al soils, and fi e s ains om wo uncul i a ed soils. The
isola es o biodeg ada ion assay a e filed a he Mic obiology Labo a o y
o Bio echnology Depa men , Poly echnic Uni e si y o Valencia, as
shown in Table 1. The s ains we e main ained a 4
1
C on ISP-2 aga
medium (yeas ex ac , 4 g; mal ex ac , 10 g; dex ose, 4 g; aga , 20 g;
dis illed wa e , 1000 mL). Biomass o biodeg ada ion s udy was ob ained
a e 7 days o cul i a ion in Pe i dishes a 25
1
C on ISP-2 aga medium,
wi hou any adap a ion o he bicide. Fi e mL o ISP-2 liquid medium was
added o he Pe i dishes o ob ain conidial suspensions. The final conidia
concen a ion was es ima ed using a haemocy ome e (imp o ed Neu-
baue chambe ).
2.1.3. Pheno ypic cha ac e iza ion o ac inomyce e s ains
The ac inomyce e s ains we e main ained on ISP-2 slan s a 4
1
C and
nu ien b o h wi h 20% glyce ol a -20
1
C. In o de o obse e pigmen
p oduc ion and mac oscopic mo phology, he isola es we e cul u ed on
In e na ional S ep omyces P ojec (ISP) media (Shi ling and Go lieb,
1966). Pheno ypic cha ac e is ics we e de e mined by s anda d p oce-
du es, desc ibed p e iously by Williams e al. (1989).
2.1.4.
DNA ex ac ion and pu i ica ion
Cells we e cul u ed on ISP-2 b o h o 3–5 days, washed and collec ed
by cen i uga ion and ozen o 24 h. The hawed pelle was esuspended
in 1 mL o TE bu e (1 mM EDTA, 10 mM T is/HCl) wi h glass beads
(3 mm) and o exed o disagg ega e he clumps o mycelium. Fou
hund ed
m
L o his suspension was incuba ed wi h 100
m
L lysozyme
(50 mg mL
-1
) and 15
m
L o mu anolysine (1330 U mL
-1
) a 37
1
C o
30 min (Lanoo e al., 2005). A e addi ion o 30
m
L SDS (10%), 3
m
L
p o einase K (20 mg mL
-1
) (Sigma) and glass beads (400
m
m), cells we e
o exed and incuba ed a 37
1
C o 1 h. Cell deb is and polysaccha ides
we e selec i ely p ecipi a ed wi h CTAB (ce yl ime hyl ammonium
b omide) a 65
1
C o 10 min. DNA was pu ified wi h equal olumes o
phenol:chlo o o m:isoamylalcohol (25:24:1). The suspension was shaken,
cen i uged and he uppe laye was emo ed o a clean ube. A final
pu ifica ion s ep wi h chlo o o m:isoamylalcohol (24:1) emo ed he
esidual phenol om he suspension. The uppe laye was emo ed o a
clean ube and DNA was p ecipi a ed wi h isop opanol. The solu ion was
cen i uged o pelle he DNA, he supe na an was disca ded and a wash
s ep wi h 70% ice-cold (-20
1
C) e hanol was made and he pelle was d ied
in acuum. TE bu e was added o suspend he d ied DNA.
2.1.5.
PCR ampli ica ion o ac inomyce ales/s ep omyces s ains
PCR p ime s we e designed p e iously and syn hesized by TIB
MOLBIOL (Be lin, Ge many). P ime s 243F and A3R amplified a
Table 1
S ep omyce e isola es o biodeg ada ion assays
O igin
S ain code
a
Non-ag icul u al soil F1-3, F1-5, F1-11, F1-12, F3-8
Ag icul u al soil A1-1, A1-2, A1-5, A2-2, A2-4, A2-6,
A2-11, A3-2, A3-14, A4-2, A5-3, A7-9
a
All isola es a e main ained in he Mic obiology Labo a o y o
Bio echnology Depa men , Poly echnic Uni e si y o Valencia, Spain.
agmen o 1.25 kb in a wide ange o ac inomyce es om soil. P ime s
Sm6F and Sm5R amplified a 600 bp in mos o he ep esen a i e membe s
o he genus S ep omyces (Moncia dini e al., 2002). The amplifica ion
eac ion was ca ied ou in a 50
m
L eac ion olume, con aining 200
m
M
each o he ou dNTPs, 3
m
L o genomic DNA, 1U Taq polyme ase
(Ecogen) wi h i s app op ia e eac ion bu e , 3 mM MgCl
2
o p ime s
243F/A3R and 1.5 mM MgCl
2
o p ime s Sm6F/Sm5R and 1
m
M o each
p ime . PCRs we e ca ied ou in a he mocycle (Techne-P ogene) unde
he ollowing condi ions: o p ime s 243F/A3R he p og am was ini ia ed
by dena u a ion o 95
1
C o 5 min ollowing by 30 cycles o 94
1
C o 45 s,
68
1
C o 2 min, and 72
1
C o 1 min, one cycle o 72
1
C o 10 min, and
holding a 4
1
C. Fo p ime s Sm6F/Sm5R he p og am was ini ia ed by
dena u a ion o 95
1
C o 1 min ollowed by 35 cycles o 94
1
C o 1 min,
65
1
C o 45 s, and 72
1
C o 1 min, one cycle o 72
1
C o 10 min, and
holding a 4
1
C. Con ols wi hou bac e ial DNA we e included o each
eac ion. Ten
m
L o o al 50
m
L PCR samples we e loaded on o 1.5%
aga ose gels and subjec ed o elec opho esis o 1 h a 80–90 V. Amplified
DNA was isualized by e hidium b omide s aining unde UV ligh . A
100 bp DNA ladde (Fe men as) was included as molecula size ma ke
(Moncia dini e al., 2002).
2.2. Biodeg ada ion assay
2.2.1. Chemicals and eagen s
Diu on s anda d was supplied by Riede de-Hae¨ n (Seelze,
Ge many) and was mo e han 99% pu e. All sol en s used we e
HPLC (high- pe o mance liquid ch oma og aphy) g ade. Ul apu e
wa e was p o- duced by Milli Q sys em (Millipo e) and Sep-Pak
Silicagel ca idge was a Wa e s p oduc .
2.2.2. Biodeg ada ion condi ions
T iplica e flasks (500 mL) con aining 200 mL s e ile ISP-2 medium wi h
diu on (4 mg L
-1
) we e inocula ed wi h s ep omyce e conidial suspension
o ob ain a final concen a ion o 1
x
10
6
conidia mL
-1
. Spo e suspension
was no added o con ol flasks o assess abio ic losses. Flasks we e
incuba ed a 25
1
C on a o a y shake o up 15 days. Residual diu on
concen a ions we e measu ed by HPLC a egula in e als (1, 5, 10 and
15 days).
2.2.3. Diu on analyses
He bicide concen a ions we e measu ed by HPLC. Samples (25 mL) o
liquid cul u e we e cen i uged (5 min, 4000 pm). The supe na an was
ex ac ed wi h dichlo ome hane (1:1 / , 3
x
) by shaking in a sepa a ion
unnel. The cell pelle s we e in es iga ed o possible e en ion o diu on.
The combined dichlo ome hane ex ac s we e dehyd a ed wi h anhyd ous
Na
2
SO
4
and e apo a ed o d yness unde a s eam o N
2
, and
subsequen ly dilu ed o a final olume o 5 mL wi h me hanol. The
ex ac was pu ified h ough Sep-Pak Silicagel ca idges p ecoa ed wi h
10 mL o dichlo ome hane and 10 ml o e hyl ace a e, successi ely. Diu on
was elu ed wi h 4 mL e hyl ace a e. The elua e was e apo a ed o d yness
unde s eam o N
2
and dissol ed in 5 mL o me hanol and ese ed o
ch oma og aphic analysis.
HPLC analyses we e pe o med using a Hewle -Packa d HP 1100
sys em p o ided wi h a a iable wa eleng h UV de ec o G1314A. A
e e se-phase column Hype sil ODS (125
x
4 mm, 5
m
m d
p
) p o ec ed wi h
a gua d p ecolumn wi h he same phase was used h oughou . The mobile
phase was me hanol–wa e (70:30 / ) wi h a flow a e o 0.8 mL min
-1
and a wa eleng h o 254 nm. Da a we e analysed wi h a HP-ChemS a ion
so wa e. Re en ion ime o diu on unde hese ch oma og aphic
condi ions was 3.1 min.
Wo king calib a ion s anda ds o diu on we e p epa ed by se ial
dilu ion om indi idual s ock solu ions in me hanol and used o spiking
o liquid cul u e samples o eco e y assays and o ma ion o calib a ion
plo . The calib a ion cu e p o ed o be linea wi hin a ange o
0.5–
2 mg L
-1
. The diu on de ec ion limi in liquid medium (a signal- o-
noise a io o 3:1) was 0.04 mg L
-1
. The eco e y o he ex ac ion and
pu ifica ion me hod was a ound 88%.
3. Resul s and discussion
3.1. Cha ac e iza ion o selec ed s ains
In all isola ed s ains a agmen o 1.25 kb was amplified
wi h p ime s 243F and A3R, which a e a ge ed o a wide
ange o ac inomyce es om soil. This se o p ime s has
been shown o ha e 97% specifici y o he Ac inomyce ales
g oup. A 600 bp agmen was ob ained wi h he p ime s
Sm6F and Sm5R, specific o S ep omyces genus. These
p ime s ha e 98% specifici y o his genus (Moncia dini
e al., 2002).
Pheno ypic and mo phological s udies on selec ed s ains
e ealed a ypical pa e n in membe s o S ep omyces
genus. They de elop ae ial mycelium on spo ula ion media.
The axonomy o S ep omyces emains con used and he
defini ion o species is un esol ed, due o he g ea a ie y
o mo phological, cul u al, physiological and biochemical
cha ac e is ics ha a e obse ed bo h a in e - and in a-
species le el. This emphasizes he impo ance o he
polyphasic axonomy app oach, which can only be
main ained by s anda diza ion (Good ellow, 1997; Ande -
son and Welling on, 2001). This is why we ha e applied he
me hods ecommended in he In e na ional S ep omyces
P ojec (Shi ling and Go lieb, 1966), which is he basis o
he classifica ion o he genus S ep omyces in Be gey’s
Manual o Sys ema ic Bac e iology (Williams e al., 1989).
These pheno ypic s udies allowed an a filia ion o he
selec ed s ains o he majo clus e s desc ibed by Williams
e al. (1989) (Table 2).
3.2. Biodeg ada ion s udies
The esul s o biodeg ada ion, exp essed as pe cen age o
deple ion o diu on, es ima ed by HPLC a egula
in e als, a e shown in Table 3. The esul s indica e ha
all s ains es ed we e able o deg ade diu on, bu wi h
di e en deg ada ion pe cen ages. The e was no e idence
o diu on deg ada ion by abio ic losses in con ol flasks.
Mo eo e , ex ac ion o cell pelle s wi h dichlo ome hane
showed ha no diu on e en ion occu ed in cells.
Among he s ains es ed, A7-9 appea ed o be he mos
e ficien o he deg ada ion o diu on, achie ing 53.7%
deg ada ion a e one day’s incuba ion, 95.6% a e fi e
days and no he bicide emained a e 10 days’ incuba ion.
S ains A1-1, A1-2, A2-4, A2-6 and A2-11 we e also
e ficien , deg ading mo e han 50% diu on by day 5, and
eaching highe pe cen ages a he end o he assays. These
s ains we e isola ed om ag icul u al soils which had
ecei ed some ea men wi h diu on and o he he bicides,
indica ing he abili y o he o ganisms o adap o he
en i onmen al condi ions. On he o he hand, he s ains
isola ed om non-ag icul u al soils had deg aded less han
50% diu on by day 5, al hough s ains F1-11, F1-5 and F3-
8 achie ed up o 50% deg ada ion a he end o he assays
(70.8%, 52.2% and 51%, espec i ely). O e all, he diu on
deg ada ion pe cen age o he s ains isola ed om
Table 2
Pheno ypic cha ac e iza ion o S ep omyces s ains
S ain
Ae ial
mycelium
Subs a e
mycelium
Soluble
pigmen
Spo e chain
mo phology
Melanin
An ibio ic ac i i y agains
C. albicans S. ce e isiae S. mu inus A. nige
B. sub ilis M. lu eus
F1-3
Whi e/yellow
Red/o ange
-
S
a
-
+
+
+
+
+
+
F1-5
G ey
Red/o ange
-
S
-
+
+
-
-
+
+
F1-11
Whi e
Yellow
-
RF
b
-
-
-
+
+
-
+
F1-12
Reddish/g ey
Red/o ange
-
RF
+
+
+
-
-
+
-
F3-8
Da k/g ey
Red/o ange
+
S
-
-
-
-
-
-
-
A1-1
Da k g ey
Yellow/b own
-
S
-
+
+
-
+
-
-
A1-2
Reddish/g ey
Red/o ange
+
S
-
-
-
-
-
-
-
A1-5
Reddish/g ey
Yellow/b own
+
S
-
+
+
-
-
+
-
A2-2
Whi e/yellow
Yellow/b own
-
RF
-
-
+
-
-
+
-
A2-4
Reddish/g ey
Yellow/b own
+
RF
+
+
-
-
-
+
+
A2-6
Da k g ey
Red/o ange
+
S
-
-
-
-
-
-
-
A2-11
G ey
Red/o ange
-
S
-
-
-
-
-
-
-
A3-2
Whi e
Yellow/b own
-
RF
-
-
-
+
+
-
-
A3-14
Da k/g ey
Red/o ange
+
S
-
-
-
-
-
-
-
A4-2
Whi e/yellow
Yellow/b own
-
RF
-
-
+
-
-
+
-
A5-3
G ey
Yellow/b own
-
RF
-
-
-
-
-
-
-
A7-9
Whi e
Yellow/b own
-
RF
-
-
-
+
+
-
-
S ain
Lipolysis
Ni a e
G ow h a
G ow h wi h
Assimila ion o ca bon sou ces Clus e a filia ion
educ ion
45 1C
NaCl (7%)
m-inosi ol
mani ol
L
- hamnose a finose
D
-melibiose
F1-3
+
+
+
+
-
+
+
-
+
S. imosus
F1-5
-
-
-
+
+
+
-
+
+
S. lydicus
F1-11
+
+
-
+
-
+
-
-
-
S. albido la us
F1-12
+
-
-
-
+
+
-
+
+
S. ul issimus
F3-8
+
+
-
+
+
+
+
+
+
S. g iseo ube
A1-1
+
+
-
-
+
+
+
+
+
S. iolaceusnige
A1-2
+
+
-
-
-
+
+
+
+
S. g iseo ube
A1-5
+
-
+
+
+
+
+
-
-
S. g iseo i idis
A2-2
+
+
-
+
-
+
+
-
-
S. anula us
A2-4
-
+
-
-
+
-
-
-
-
S. pu pu eus
A2-6
+
+
-
+
+
+
+
+
+
S. g iseo ube
A2-11
+
-
+
+
+
+
+
+
+
S. ochei
A3-2
+
-
-
+
-
+
-
-
-
S. albido la us
A3-14
+
+
-
+
+
+
+
+
+
S. g iseo ube
A4-2
+
+
-
+
-
+
+
-
-
S. anula us
A5-3
+
-
-
+
-
+
+
-
+
S. hals edii
A7-9
+
-
-
+
-
+
-
-
-
S. albido la us
a
S, spi al.
b
RF, ec iflexible.

Table 3
Disappea ance o diu on (%)
a
in he p esence o S ep omyces s ains in
pu e cul u es, ini ial concen a ion 4 mg
L
-1
a
Mean o h ee alues
7
s anda d de ia ion.
ag icul u al soils was always highe han hose isola ed
om non-ag icul u al soils.
The diu on biodeg ada ion a e o 17 s ains o
s ep omyce es was calcula ed and he esul s a e shown
in Table 4. S ains A7-9 and A2-11 we e he as es
deg ade s, wi h simila hal -li es (0.7 and 1.4 days,
espec i ely). Al hough A2-11 was able o deg ade hal o
he diu on in 1.4 days, i s deg ada ion e ficiency was lowe
han ha obse ed o s ain A7-9, since 32% o diu on
s ill emained in ac a e 15 days. S ains A2-4, A1-2 and
A1-1 exhibi ed a simila deg ada i e beha iou , wi h hal -
li es less han 5 days (2.2, 2.9 and 3.7 days, espec i ely)
and no achie ing o al deg ada ion o diu on by he end o
assay. The hal -li e o s ains A2-6, A5-3, A1-5 and A2-2
was sligh ly highe (5–9 days) and he deg ada ion can be
conside ed mo e g adual. I is in e es ing o no e ha one
non-ag icul u al isola e (F1-11) showed a hal -li e o 6.4
days, compa able o ag icul u al isola es, al hough he
highes diu on deg ada ion a e co esponded mainly o
s ains isola ed om ag icul u al soils. These esul s
sugges ha s ains isola ed om soils egula ly ea ed
wi h he bicide ha e a conside able po en ial o biodeg a-
da ion, since he o ganisms a e adap ed o pes icide
p esence. This is in line wi h Widehem e al. (2002), who
isola ed and cha ac e ized one ac inobac e ia s ain,
A h obac e sp. N2, om a long- e m diu on con ami-
na ed soil. This s ain was able o deg ade diu on
comple ely, sugges ing mic obial adap a ion o he bicide
me abolism.
Du ing ou biodeg ada ion s udies, one uniden ified
HPLC peak, concomi an wi h he disappea ance o
diu on, was obse ed in he ch oma og ams o F1-11 and
A2-4 s ains. Acco ding o he li e a u e, we could expec
ha his p oduc co esponds ei he o he main me aboli e
o diu on biodeg ada ion, 3,4-dichlo oaniline, o o he
Table 4
Biodeg ada ion a e pa ame e s o diu on in pu e cul u es o S ep o-
myces s ains, ini ial concen a ion 4 mg
L
-1
Reg ession equa ion
a,b
R
2
Hal -li e (Days)
y ¼ e
(4.31587-0.0181151x)
y ¼ e
(4.11371-0.0154032x)
y ¼ e
(4.34204-0.0669697x)
y ¼ e
(4.39645-0.0267647x)
y ¼ e
(4.12174-0.01550x)
y ¼ e
(4.12674-0.0574754x)
y ¼ e
(4.00593-0.0327392x)
y ¼ e
(4.15663-0.0329325x)
y ¼ e
(4.31274-0.0429261x)
y ¼ e
(4.01999-0.04947x)
y ¼ e
(4.13322-0.04205x)
y ¼ e
(3.96774-0.0390319x)
y ¼ e
(4.14448-0.01406x)
y ¼ e
(4.67398-0.0422027x)
y ¼ e
(4.11715-0.00834766x)
y ¼ e
(4.18217-0.0470733x)
y ¼ 42.3888-17.9689 ln x
R
2
: Co ela ion coe ficien squa ed (P
o
0.05).
a
Exponen ial model, y ¼ e
a+bx
[y ¼ % deg ada ion, x ¼ ime,
a ¼ in e cep and b ¼ slope].
b
Loga i hmic x model, y ¼ a+b ln x [y ¼ % deg ada ion, x ¼ ime,
a ¼ in e cep and b ¼ slope].
deme hyla ion p oduc s (Ellis and Campe , 1982; Culling-
on and Walke , 1999; Tixie e al., 2000; Widehem e al.,
2002). On he o he hand, he e was no e idence o any
o he HPLC peak di e en om diu on in he ch oma o-
g ams o he o he s ains. The he bicide migh be u he
ans o med in o me aboli es no de ec ed wi h he
analy ical me hod used, o e en mine alized.
Fu he s udies o op imize he diu on deg ada ion
condi ions in he soil mus be ca ied ou . Since p e ious
s udies ha e shown ha S ep omyces s ains can deg ade a
a ie y o pes icides, i is no un easonable o expec ha
hey will also do so in he soil. Culling on and Walke
(1999) obse ed he apid deg ada ion o diu on in soils
when a single bac e ium, able o deg ade i unde
labo a o y condi ions, was added, and sugges ed ha
apid he bicide biodeg ada ion could occu in he soil.
Many o he deg ada ion compounds o diu on exhibi a
highe oxici y han he pa en compound (Tixie e al.,
2000, 2001). Howe e , hei u he biodeg ada ion, ei he
by ungi o bac e ia, has been epo ed in many s udies
(Tixie e al., 2002; T a kin e al., 2003). In soils, a wide
ange o mic oo ganisms a e p esen , and we can expec
ha he simul aneous ac ion o a ious mic oo ganisms
will succeed in deg ading hese compounds (El-Fan oussi,
2000; So ensen e al., 2003; Ba a-Ca acciolo e al., 2005).
I is likely ha diu on could be ans o med sequen ially by
di e en s ains in soil habi a s, including S ep omyces
s ains, o he ac inobac e ia and ungi.
This s udy complemen s he esul s ob ained by o he
au ho s, since a la ge numbe o S ep omyces s ains ha e
shown diu on deg ada i e ac i i y. Ou esul s sugges he
S ain
1 day
5 days
10 days
15 days
S ain
F1-3
25.8
7
2.4
31.7
7
2.0
38.6
7
2.5
42.3
7
1.3
F1-3
F1-5
40.7
7
1.0
42.7
7
1.5
46.3
7
1.2
52.2
7
2.7
F1-5
F1-11
26.1
7
2.5
45.9
7
3.7
62.3
7
1.9
70.8
7
1.6
F1-11
F1-12
20.6
7
1.1
30.6
7
1.1
35.7
7
1.1
46.5
7
2.0
F1-12
F3-8
18.2
7
2.1
20.7
7
1.2
36.5
7
1.1
51.0
7
2.6
F3-8
A1-1
39.6
7
1.0
53.2
7
1.5
68.5
7
1.1
72.1
7
1.6
A1-1
A1-2
42.2
7
1.4
56.9
7
1.2
63.2
7
1.6
63.9
7
1.6
A1-2
A1-5
41.1
7
1.4
43.7
7
1.3
51.2
7
1.4
62.9
7
1.4
A1-5
A2-2
28.2
7
1.9
42.0
7
2.1
47.9
7
2.0
62.0
7
1.4
A2-2
A2-4
48.1
7
2.6
52.1
7
1.3
70.0
7
2.1
71.2
7
0.3
A2-4
A2-6
33.7
7
1.2
56.2
7
2.3
57.2
7
2.3
64.7
7
1.7
A2-6
A2-11
43.5
7
1.6
60.9
7
1.3
66.9
7
1.6
68.1
7
1.5
A2-11
A3-2
38.5
7
1.1
41.2
7
1.3
43.3
7
4.3
47.1
7
5.3
A3-2
A3-14
0.0
7
0.0
9.0
7
1.4
31.2
7
1.3
43.1
7
1.5
A3-14
A4-2
38.7
7
1.1
41.3
7
0.6
44.0
7
1.3
45.5
7
0.5
A4-2
A5-3
40.1
7
1.6
42.9
7
2.1
61.9
7
1.6
67.0
7
1.6
A5-3
A7-9
53.7
7
2.4
95.6
7
2.5
100.0
7
0.0
100.0
7
0.0
A7-9
0.9193
22.3
0.8602
13.1
0.9775
6.4
0.9590
18.1
0.7778
15.2
0.9490
3.7
0.8117
2.9
0.9084
7.4
0.9512
9.3
0.8559
2.2
0.8017
5.3
0.8106
1.4
0.8178
16.5
0.9791
18.1
0.9087
24.6
0.9218
5.7
0.9043
0.7
impo ance o a con inuous sea ch o new s ains wi h
deg ada i e ac i i y gi en he high deg ada ion a es
obse ed. They could be used e ficien ly in bio emedia ion
o pes icide-pollu ed soils, since hey easily su i e and
p oli e a e as a consequence o hei mycelial g ow h and
g ea po en ial me abolic di e si y. The abili y o many
S ep omyces s ains o be gene ically enginee ed is ano he
a ac i e ea u e. Howe e , i is necessa y o s udy hei
adap a ion and unde s and he con ol o exp ession o
ca abolic genes in hei na u al en i onmen (Cases and de
Lo enzo, 2005).
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