Wat. Re5. V01. 30, N0. 7, pp.

1736-1744, 1996
C0pyr19ht ~c~)1996 E15ev1er5c1ence Ltd
~ Per9am0n Pr1nted 1n 6reat 8r1ta1n. A11r19ht5re5erved
P1h 50043-1354(96)00067-X 0043-1354/96 $15.00 + 0.00

DEC0L0R12A710N AND 810DE6RADA710N 0F ME7HYL

R E D 8 Y K L E 8 5 1 E L L A P N E U M 0 N 1 A E R5-13

P. K. W 0 N 6 ~ a n d P. Y. Y U E N 6
Department 0f 810109y, 7he Ch1ne5e Un1ver51ty 0f H0n9 K0n9, 5hat1n, N.7., H0n9 K0n9

(F1r5t rece1red 0ct06er 1995; accepted 1n rev15edf0rm Fe6ruary 1996)

A65tract--8acter1a1 5tra1n5 1501ated fr0m dye-c0ntam1nated 51ud9e dec010r12ed a t0x1c a20 dye, name1y,
methy1 red (MR). K1e651e11apneum0n1ae R5-13 wa5 5e1ected 6ecau5e 0f 1t5 6etter a6111t1e5t0 c0mp1ete1y
dec010r12e and de9rade MR under aer061c c0nd1t10n5. Effect5 0f phy51c0-chem1ca1 parameter5, 5uch a5:
temperature and aerat10n, c0ncentrat10n5 0f 91uc05e, ethan01, amm0n1um 5u1fate and pH 0f the cu1ture
med1um 0n the MR de9radat10n 6y the 6acter1um and a prev10u51y 1501ated aer061c MR-de9rad1n9
6acter1um, Acet06acter 114uefac1en5 5-1, were determ1ned. K. pneum0n1ae R5-13 had h19her MR
de9radat10n a6111ty than A. 114uefac1en55-1. Under 0pt1ma1 c0nd1t10n5, K. pneum0n1ae R5-13 c0mp1ete1y
dec010r12ed and de9raded 100 m9 1 • 0f MR 1n cu1tura1 med1um. 7he h19h MR de9radat10n a6111ty and
10w nutr1ent and env1r0nmenta1 re4u1rement5 0f K. pneum0n1ae R5-13 ena61e th15 6acter1um t0 6e u5ed
1n the treatment 0f 1ndu5tr1a1 eff1uent c0nta1n1n9 a20 dye5. C0pyr19ht • 1996 E15ev1er 5c1ence Ltd

Key w0rd5--dec010r12at10n, 610de9radat10n, methy1 red, K1e651e11apneum0n1ae

1N7R0DUC710N rep0rted that a20 dye5 c0u1d 6e de9raded 6y 6acter1a

5ynthet1C dye5 have 6een U5ed 1nCrea51n91y 1n text11e
a n d dye1n9 1ndU5tr1e5 6eCaU5e 0f the1r ea5e a n d C05t
effect1Vene55 1n 5ynthe515, f1rmne55, a n d Var1ety 1n
C010r C0mpared W1th natura1 dye5 (6r1ff1th5, 1984).
A m 0 n 9 the m a n y C1a55e5 0f 5ynthet1C dye5 U5ed 1n
the5e 1ndU5tr1e5, a20 dye5 are the 1ar9e5t and the m05t
Ver5at11e, a n d they p1ay a pr0m1nent r01e 1n a1m05t
every type 0f app11Cat10n ( 6 0 r d 0 n a n d 6re90rY,
1983). A20 dye5 are a 9r0Up 0f C0mp0Und5
CharaCter12ed 6y the pre5ence 0f 0ne 0r m0re a20
60nd5 ( - - N - - N - - ) 1n a550C1at10n W1th 0ne 0r m0re
ar0mat1C 5y5tem5 (50, 1989). A20 dye5 are 5ynthet1C
a n d re1at1Ve1y re515tant t0 6101091Ca1 a n d Chem1Ca1
de9radat10n5. H0WeVer, m a n y 5tUd1e5 1nd1Cate that
m05t 0 f the a20 dye5 are t0X1C (H01me, 1984) a n d / 0 r
CarC1n09en1C (Ame5 et a1., 1975; An11ker, 1979;
ChUn9 et a1., 1981); thU5, 1ndU5tr1a1 eff1Uent
C0nta1n1n9 a20 dye5 mU5t 6e treated 6ef0re 1t 15
d15Char9ed 1nt0 the enV1r0nment.
M05t 6101091Ca1 de9radat10n 0f a20 dye5 are Carr1ed
0Ut 6y anaer061C 6aCter1a (R0X0n et a1., 1967; Lar50n
et a1., 1976; ChUn9 et a1., 1978; 2 1 m m e r m a n n et a1.,
1984; Raf11 et a1., 1990; Raf11 a n d Cern1911a, 1993).
A20redUCta5e5, a C1a55 0f en2yme5 1nV01Ved 1n the
redUCt1Ve C1eaVa9e 0f the a20 6 0 n d ( - - N : N - - ) , are
U5ed 1n anaer061C de9radat10n5 6y Var10U5 0r9an15m5
(61n9e11 and Wa1ker, 1971; Wa1ker a n d Ryan, 1971;
ChUn9 et a1., 1978; Meyer, 1981). 0n1y a few 5tUd1e5
"Auth0r t0 wh0m c0rre5p0ndence 5h0u1d 6e addre55ed.
6pre5ent addre55: Department 0f M1cr0610109y, Un1ver51ty
0f 7enne55ee, Kn0xv111e, 7N 37916, U.5.A.
u n d e r aer061c c0nd1t10n5 (1daka et a1., 1982; 50 et a1.,
1990; Y a t 0 m e et a1., 1990; Pa52c2yn5k1 et a1., 1991;
60v1nda5wam1 et a1., 1993). 1n a prev10u5 5tudy,
Acet06acter 114ueJac1en5 5-1, wh1ch wa5 1501ated fr0m
dye-c0ntam1nated 51ud9e, dec010r12ed a n d de9raded
methy1 red ( M R , F19. 1), a t0x1c a20 dye, u n d e r
aer061c c0nd1t10n5 (50, 1989; 50 et a1., 1990). 7 h e
6acter1um de9raded M R 1nt0 tw0 c010r1e55 c0m-
p0und5, name1y, 2-am1n06en201c ac1d ( A 8 A ) and
N,N•-d1methy1-p-pheny1ened1am1ne ( D M P D ) (F19.
1). 1n the pre5ent 5tudy, a 6acter1a1 5tra1n, K1e651e11a
pneum0n1ae R5-13 dec010r12ed and de9raded M R
aer061ca11y fa5ter t h a n A. 114ueJac1en5 5-1, wa5
1501ated fr0m dye-c0ntam1nated 51ud9e. 7 h e effect5 0f
var10u5 phy51c0-chem1ca1 fact0r5 0n the de9radat10n
0f M R 6y K. pneum0n1ae R5-13 and A. 114uefac1en5
5-1 were determ1ned a n d c0mpared. 7 h e app11cat10n
0 f K. pneum0n1ae R5-13 1n the t r e a t m e n t 0f 1ndu5tr1a1
eff1uent c0nta1n1n9 a20 dye5 w111 6e d15cu55ed.
MA7ER1AL5AND ME7H0D5
1501at10n 0f methy1 red-dec010r1=1n9 6acter1a
Dye-c0ntam1nated 51ud9e wa5 c011ected fr0m the wa5te-
water 0ut1et 0f an 1ndu5tr1a1 area 1n H0n9 K0n9 1n wh1ch
eff1uent fr0m the dye1n9 1ndu5try wa5 the maj0r 50urce 0f
wa5tewater. Appr0x1mate1y 1 9 0f 51ud9e wa5 5u5pended 1n
10 m1 5ter11e 0.85% 50d1um ch10r1de (R1ede1-deHaen) and
m1xed th0r0u9h1y. 7he m1xture wa5 5er1a11y d11uted w1th
5ter11e 0.85% 50d1um ch10r1de. A114u0t5 0f 0.1 m1 0f 10
10 : and 10 3d11ut10n5were 5pread 0nt0 M1n1mum Med1um
(MM) p1ate5 c0nta1n1n9 0.1 9 1 ' 0f 91uc05e (Un1var), 0.1 9
1 ~0f amm0n1um 5u1fate ((NH43•,504, Un1var), 0.05 M 0f
p0ta551um ph05phate d16a51c (Un1var, the pH 0f the cu1ture
med1um wa5 adju5ted t0 7 w1th 1M HC13 and 15 9 1 • 0fa9ar

1736
 810de9radat10n 0f methy1 red
(D1fc0) w1th 100 9 1 t 0f methy1 red (MR, 8DH). A11 p1ate5
were 1ncu6ated at 30°C f0r 3 day5. C010n1e5 5urr0unded 6y
ha10 (dec010r12ed) 20ne5 were p1cked and 5treaked 0nt0 M M
p1ate5 c0nta1n1n9 100 9 1-~ 0f M R . 7 h e p1ate5 were a9a1n
1ncu6ated at 30°C f0r 3 day5 t0 c0nf1rm the1r a6111t1e5 t0
dec010r12e MR.
Dec010r1:at10n 0f methy1 red 6y 5e1ected 6acter1a 1n 114u1d
med1um
F1ve 6acter1a1 1501ate5 w1th h19her M R dec010r12at10n
a6111t1e5 (1.e. 1ar9er dec010r12ed ha10 5urr0und1n9 the c010ny
0n M R p1ate5) were 5e1ected. 5t0ck cu1ture5 0f the5e 6acter1a
were prepared 6y 9r0w1n9 a 51n91e c010ny 1n 125 m1 c0n1ca1
f1a5k5 c0nta1n1n9 10 m1 0f M M at 30°C 1n a r0tary 5haker
at 200 rpm f0r 3 day5. An a114u0t 0f 1 m1 0f each 1501ate wa5
1n0cu1ated 1nt0 a 500 m1 c0n1ca1 f1a5k c0nta1n1n9 100 m1 0f
M M w1th 100 m9 1-~ 0f M R . 7he5e cu1ture5 were 1ncu6ated
under the 5ame c0nd1t10n5 f0r 7 day5.
0 n the 7th day, 10 m1 a114u0t5 0f the5e cu1ture5 were
5amp1ed and ce115 were pe11eted 6y centr1fu9at10n w1th a
5534 r0t0r at 20,000 x 9 at 4°C f0r 10 m1n 1n a 50rva11
RC5C refr19erated centr1fu9e. 7 h e a650r6ance5 at 430 n m
(wh1ch 15 the a650rpt10n max1mum f0r the 0ran9e c010r 0f
methy1 red) 0f the cu1ture 5upernatant5 were determ1ned 6y
a M11t0n R0y 5pectr0n1c 3000 5pectr0ph0t0meter.
1dent1f1cat10n 0f 5e1ected methy1 red-dec010r121n9 6acter1a
8acter1a1 1501ate5 w1th h19her M R dec010r12at10n a6111t1e5
1n M M w1th 100 m9 1-~ 0f M R were 5u6jected t0 the 6 r a m
5ta1n, and further 1dent1f1cat10n5 were perf0rmed 6y the
810109 M1cr05tat10n 5y5tem (810109 1nc., Hayward). A11
rea9ent5 and acce550r1e5 were purcha5ed fr0m the 810109
1nc. and u5ed acc0rd1n9 t0 the 1n5truct10n5 0f the
manufacturer.
C0rre1at10n5 0f ce11ma55 and ce11den51ty 0f 5e1ected 6acter1a
7 h e 6acter1a1 1501ate w1th the h19he5t M R dec010r12at10n
a6111ty (1.e. cu1ture 5upernatant w1th the 10we5t a650r6ance
at 430 nm) wa5 5e1ected, and Acet06acter 114uefac1en5 5-1, an
aer061c MR-de9rad1n9 6acter1um 1501ated prev10u51y (50
et a1., 1990), wa5 1nc1uded a5 a reference f0r further 5tud1e5.
L1near re1at10n5h1p5 6etween the ce11 ma55 and ce11 den51ty
0f the5e 6acter1a1 cu1ture5 9r0wn 1n M M w1th 100 m9 1-~ 0f
M R were e5ta6115hed 6y determ1n1n9 the dry we19ht5 0f
100m1 a114u0t5 0f 6acter1a1 cu1ture5 w1th d1fferent ce11
den51ty determ1ned 5pectr0ph0t0metr1ca11y at A520 nm.
1dent1f1cat10n 0f de9radat10n pr0duct5 0f methy1 red 6y HPLC
ana1y515
7 h e 5e1ected 6acter1um and A. 114uefac1en5 5-1 were
9r0wn 1n 100 m1 0 f M M w1th 100 m9 1 ~0 f M R at 30°C w1th
5hak1n9 f0r 7 day5. After 1ncu6at10n, 20m1 0f 2M
hydr0ch10r1c ac1d (Ajax) wa5 added 6ef0re the rem0va1 0f
6acter1a1 ce115 6y centr1fu9at10n. 0 n e hundred m1 ac1d1f1ed
cu1ture 5upernatant wa5 c0ncentrated t0 a60ut 20 m1 1n a
r0tary evap0rat0r at 70°C. A c0ncentrated 5amp1e wa5 then
extracted w1th e4ua1 v01ume 0f d1ch10r0methane (DCM,
Ma111nckr0dt) 3 t1me5. 7 h e D C M extract5 were p001ed and
<~N =N-~N(CH3)2 D- (~-NH 2
C00H
METHY1. RED 2..AM1N08EN201C
F19. 1. Reduct1ve c1eava9e 0f methy1 red.
1737
evap0rated t0 5 m1 at 40°C 1n a r0tary evap0rat0r and then
tran5ferred t0 a te5t tu6e. 7 h e rema1n1n9 D C M wa5 rem0ved
6y evap0rat10n at r00m temperature and p1aced 1n a h00d
0vern19ht. 7 h e extracted re51due wa5 d15501ved 1n 10 m1
acet0n1tr11e (Ma111nckr0dt) and f11tered thr0u9h a 0.2/~m
ny10n f11ter (A11tech). 7wenty #1 0f each 5amp1e wa5
ana1y2ed 6y a 8 e c k m a n H P L C 5y5tem e4u1pped w1th a
m0de1 163 var1a61e wave1en9th detect0r (detect10n wave-
1en9th wa5 250 nm) and 8 e c k m a n U1tra5phere 0 D 5 c01umn
(4.6 m m x 150 mm). 7 h e me5h 512e 0f the pack1n9 0f the
c01umn wa5 5 # m and the m0611e pha5e c0mp05ed 0f 25 m M
ph05phate 6uffer (pH = 3.0) and acet0n1tr11e w1th var10u5
rat10 chan91n9 fr0m 77:23 t0 0:100 (ph05phate
6uffer:acet0n1tr11e). 7 h e 501vent 9rad1ent wa5 26%/m1n.
Effect5 0f phy51c0-chem1ca1fact0r5 0n methy1 red de9radat10n
and ce11 ma55 0f cu1ture5 0f 5e1ected 6acter1a
Effect5 0f temperature and aerat10n. 5t0ck cu1ture5 0f the
5e1ected 6acter1um and A. 114uefac1en5 5-1 were prepared 6y
9r0w1n9 a 51n91e c010ny 1n 100 m1 0f M M 1n 500 m1 c0n1ca1
f1a5k5 f0r 3 day5 at 30°C w1th 5hak1n9. A n a114u0t 0f 1 m1
0f each 6acter1um wa51n0cu1ated 1nt0 a 500 m1 c0n1ca1 f1a5k
w1th 100m1 0f M M p1u5 100m9 1-~ 0f MR. 7 h e cu1ture5
were 1ncu6ated at var10u5 temperature5 (23, 30, 37 and
45°C) 1n r0tary 5haker5 runn1n9 at 2 0 0 r p m (5hak1n9
c0nd1t10n) 0r w1th0ut 5hak1n9 (5tat1c c0nd1t10n). At 5evera1
t1me 1nterva15, 10 m1 a114u0t5 0f each cu1ture were 5amp1ed
and the1r ce11 den51t1e5 were mea5ured 5pectr0ph0t0metr1-
ca11y at A520 n m 1n 0rder t0 determ1ne the ce11 ma55. Ce115
were then pe11eted 6y centr1fu9at10n, and the 5upernatant
wa5 10-f01d d11uted 6ef0re the c0ncentrat10n 0f M R wa5
determ1ned 5pectr0ph0t0metr1ca11y at 430 nm.
Effect5 0f c0ncentrat10n5 0f 91uc05e and ethan01. Pr0-
cedure5 t0 determ1ne the effect5 0f the c0ncentrat10n5 0f
91uc05e and ethan01 0n the M R de9radat10n and ce11 ma55
0f the cu1ture5 0f the 5e1ected 6acter1um and A. 114uefac1en5
5-1 were 51m11ar t0 th05e 0f temperature and aerat10n,
except the med1um u5ed wa5 MM1 (1.e. M M w1th0ut
91uc05e) w1th 100 m9 1 ~ 0f MR. Var10u5 c0ncentrat10n5 0f
91uc05e (0.1, 0.5, 1, 2.5 and 5 9 1 ~) 0r ethan01
(Ma111nckr0dt; 0.1, 0.5, 1.0, 2.5 and 5.09 1-~) were added
1nt0 the med1um. 7 h e cu1ture5 were 1ncu6ated at 30°C under
5hak1n9 c0nd1t10n f0r 2 day5 6ef0re the determ1nat10n5 0f
ce11 den51ty (1.e. ce11 ma55) and M R c0ncentrat10n.
Effect 0f c0ncentrat10n 0f amm0n1um 5u1fate. Pr0cedure5
t0 determ1ne the effect5 0f c0ncentrat10n 0f amm0n1um
5u1fate 0n the M R de9radat10n and ce11 ma55 0f the cu1ture5
0f 60th 6acter1a were 51m11ar t0 th05e 0f 91uc05e and
ethan01, except the med1um u5ed wa5 M M 2 ( M M w1th n0
amm0n1um 5u1fate) w1th 100 m9 1-~ 0f M R , and var10u5
c0ncentrat10n5 (0.1, 0.5, 1, 2.5 and 5 9 1 ~) 0f amm0n1um
5u1fate were added 1nt0 the med1um.
Effect 0f pH 0f cu1ture med1um. Pr0cedure5 t0 determ1ne
the effect5 0f pH 0n the M R de9radat10n and ce11 ma55 0f
the cu1ture5 0f 60th 6acter1a were 51m11art0 th05e 0f 91uc05e
and ethan01, except the med1um u5ed wa5 M M w1th 100 m9
1-~ 0 f M R . pH 0 f t h e med1um wa5 adju5ted t0 6, 6.5, 7, 7.5
0r 8 w1th 1M HC1.
+ H2N-~-N(CH3) 2
C00H
AC1D N,N•-D1ME7HYL-P-
PHENYtJ~1ED1AM1NE
1738
7a61e 1. 1dent1f1cat10n 0f methy1 red-dec010r121n9 6acter1a 6y the
810109 6N m1cr0p1ate5
8acter1a1 1501ate 5pec1e5 1dent1f1ed
3-5 5errat1a marce5cen5
R5-10 K1e651e11ap1ant1c01a
R5-13 K1e651e11apneum0n1ae
85-3 5a1m0ne11a5pp.
85-5 K1e651e11aterr09ena
~51m11ar1ty15the pr0duct 0f pr06a6111t1e5 f0r each 0f the matche5 0f
5amp1e we1150f the m1cr0p1ate c0mpared t0 the data 6a5e t0
expre55 the re11a6111tyand c0nf1dence 0f the 1dent1f1cat10n. 7he
1dent1f1cat10n15accepta61e when the 51m11ar1ty151ar9er than 0.5.
hUncerta1n 1dent1f1cat10n.
RE5UL75
1501at10n 0f methy1 red-dec010r121n9 6aCter1a
F0rty-f0Ur 6aCter1a1 1501ate5 Wh1Ch deC010r12ed
M R 0n M M p1ate5 Were 1501ated fr0m dye-c0ntam1-
nated 51ud9e. A m 0 n 9 the5e 6acter1a, 5 6acter1a1
1501ate5 w1th h19her M R dec010r12at10n a6111ty 1n
M M p1ate (data n0t 5h0wn), name1y, 3-5, R5-10,
R5-13, 85-3 and 85-5, were 5e1ected f0r further
5tudy.
1dent1f1cat10n 0 f the 5e1ected methy1 red-dec010r121n9
6acter1a
F1ve 5e1ected MR-dec010r121n9 6acter1a1 1501ate5
were 5u6jected t0 the 6 r a m 5ta1n and a11 were
6ram-ne9at1ve. 7hey were then further 1dent1f1ed
6y the 810109 6 N m1cr0p1ate5. 7he re5u1t5 0f
1dent1f1cat10n 0f the5e MR-de9rad1n9 6acter1a are
115ted 1n 7a61e 1. A m 0 n 9 the5e 5 1501ate5, 3 were
K1e651e11a 5pp., 1 wa5 5errat1a 5pp. and 1 wa5
5a1m0ne11a 5pp. K1e651e11apneum0n1ae R5-13, wh1ch
had the h19he5t M R dec010r12at10n a6111ty 1n 114u1d
med1um (data n0t 5h0wn), wa5 5e1ected f0r further
5tudy.
C0rre1at10n5 6etween ce11 ma55 and ce11 den51ty 0f
5e1ected 6acter1a
7he c0rre1at10n5 0f ce11 ma55 (dry we19ht 0f ce11
1n m9 m1-~) and ce11 den51ty (a650r6ance at 520 nm)
0f K. pneum0n1ae R5-13 and A. 114uefac1en5 5-1
were 11near. 7he r 2 0f the c0rre1at10n5 0f ce11 ma55
and ce11 den51ty f0r 60th K. pneum0n1ae R5-13
and A. 114uefac1en5 5-1 were 0.99 (data n0t 5h0wn).
1n the 5u65e4uent exper1ment5, the ce11 ma55
0f the5e 6acter1a1 cu1ture5 were e5t1mated 6y
mea5ur1n9 the1r ce11 den51t1e5 5pectr0ph0t0metr1ca11y
at 520 nm.
1dent1f1cat10n 0 f de9radat10n pr0duct5 0 f methy1 red 6y
H P L C ana1y515
H P L C ana1y515 0f the cu1ture 5upernatant5 0f
K. pneum0n1ae R5-13 and the p051t1ve c0ntr01 5tra1n,
A. 114uefac1en5 5-1, 1nd1cated that 60th 6acter1a
de9raded M R and pr0duced 2-am1n06en201c ac1d
P. K. W0n9 and P. Y. Yuen
(A8A) and N,N•-d1methy1-p-pheny1ened1am1ne
( D M P D ) a5 de9radat10n pr0duct5 (F195 2(a) and
51m11ar1ty"
2(6)).
0.770
0.427~
0.884
0.877 Effect5 0f phy51c0-chem1ca1 fact0r5 0n methy1 red
0.891 de9radat10n and ce11 ma55 0f cu1ture5 0f 5e1ected
6acter1a
K. pneum0n1ae R5-13 c0mp1ete1y de9raded M R
at 23, 30 and 37°C under 5tat1c and 5hak1n9
c0nd1t10n5 w1th1n 2 day5 0f 1ncu6at10n a1th0u9h the
M R de9radat10n wa5 c0mparat1ve1y 510wer under
5tat1c c0nd1t10n5 (F195 3(a)-(f)). A. 114uefac1en5 5-1
c0mp1ete1y de9raded M R 0n1y at 30°C after 10n9er
1ncu6at10n (1.e. 7 day5) under 5hak1n9 c0nd1t10n5
(F19. 3(d)). Ne1ther 6acter1a de9raded M R at
45°C (F195 3(9) and 3(h)) 6ecau5e 0f the1r p00r
9r0wth at th15 h19h temperature (F195 4(9) and 4(h)).
7he ce11 ma55 0f 60th 6acter1a1 cu1ture5 wa5 9reater
under 5hak1n9 c0nd1t10n (F195 4(a)-(f)), and the ce11
ma55 0f the cu1ture 0f K. pneum0n1ae R5-13 wa5
9reater than that 0f A. 114uefac1en5 5-1 under 5hak1n9
c0nd1t10n5 (F195 4(6), 4(d) and 4(f)). Under 5tat1c
c0nd1t10n5, the ce11 ma55 0f 60th 6acter1a1 cu1ture5
5h0wed n0 519n1f1cant d1fference (F195 4(a), 4(c) and
4(e)).
K. pneum0n1ae R5-13 c0mp1ete1y de9raded M R
1n the pre5ence 0f 0.5 t0 5 9 1 ~ 0f 91uc05e, wh11e
A. 114uefac1en5 5-1 0n1y c0mp1ete1y de9raded M R
1n the pre5ence 0f 5 9 1-~ 0f 91uc05e (F19. 5(a)). 7he
ce11 ma55 0f the cu1ture5 0f K. pneum0n1ae R5-13
1ncrea5ed a5 the c0ncentrat10n 0f 91uc05e 1ncrea5ed
fr0m 0.1 t0 2.5 9 1-~, wh11e the ce11 ma55 0f the
cu1ture5 0f A. 114uefac1en5 5-1 wa5 n0t 519n1f1cant1y
affected 6y the c0ncentrat10n 0f 91uc05e (F19. 6(a)).
A. 114uefac1en5 5-1 c0mp1ete1y de9raded M R 1n the
pre5ence 0f 0.1 t0 5 9 1-~ ethan01, wh11e K.
pneum0n1ae R5-13 d1d n0t de9rade M R at a11 te5ted
c0ncentrat10n5 0f ethan01 (F19. 5(6)). 7he ce11
ma55 0f the cu1ture5 0f A. 114uefac1en5 5-1 1ncrea5ed
w1th the c0ncentrat10n 0f ethan01 fr0m 0.1 t0
1.0 9 1-~ and the 1ncrea5e 0f the c0ncentrat10n 0f
ethan01 d1d n0t 1ncrea5e the ce11 ma55 0f the
cu1ture5 0f K. pneum0n1ae R5-13 (F19. 6(6)).
K. pneum0n1ae R5-13 ach1eved c0mp1ete de9ra-
dat10n 0f M R 1n the pre5ence 0f any te5ted
c0ncentrat10n 0f amm0n1um 5u1fate, wh11e A.
114uefac1en5 5-1 d1d n0t c0mp1ete1y de9rade M R 1n
the pre5ence 0f any te5ted c0ncentrat10n 0f am-
m0n1um 5u1fate (F19. 5(c). 1n fact 60th M R
de9radat10n and ce11 ma55 0f the5e tw0 6acter1a
cu1ture5 d1d n0t 5h0w any 519n1f1cant dependence
0n the c0ncentrat10n 0f amm0n1um 5u1fate (F195
5(c) and 6(c)).
K. pneum0n1ae R5-13 c0mp1ete1y de9raded M R at
p H 6 . 0 t0 8.0, wh11e A. 114uefac1en5 5-1 0n1y
c0mp1ete1y de9raded M R at pH 6.5 (F19. 5(d)). 7he
ce11 ma55 0f 60th 6acter1a1 cu1ture5 d1d n0t 5h0w any
519n1f1cant dependence 0n the pH 0f the cu1ture
med1um (F19. 6(d)).
 810de9radat10n 0f methy1 red 1739

D15CU5510N n0t very 501u61e 1n water. 50 et a1. (1990) d15501ved

1501at10n, 5e1ect10n and character12at10n 0f methy1
red-dec010r121n9 6acter1a
Dye-c0ntam1nated 51ud9e wa5 u5ed a5 a 50urce f0r
1501at1n9 MR-de9rad1n9 6acter1a. 51nce m05t a20
dye-de9rad1n9 m1cr00r9an15m5 reduct1ve1y c1eave the
a20 60nd(5) 0f re5pect1ve a20 dye and pr0duce
dec010r12ed pr0duct5, pre11m1nary 5e1ect10n 0f MR-
de9rad1n9 6acter1a wa5 6a5ed 0n the dec010r12at10n 0f
M R 0n MM p1ate. MM, a cu1ture med1um w1th 10w
1eve15 0f car60n and n1tr09en 50urce5 (0.1 9 1-~ 0f
91uc05e and 0.1 9 1- ~0f amm0n1um 5u1fate), wa5 u5ed
1n the pre5ent 5tudy 6ecau5e prev10u5 5tud1e5 (09awa
et a1., 1981, 1986; 09awa and Yat0me, 1990)
1nd1cated that 6acter1a had 6etter a20 dye de9rad1n9
a6111t1e5 under p00r nutr1t10na1 c0nd1t10n5. M R wa5
M R 1n a6501ute ethan01 t0 prepare the 5creen1n9
med1um (5M) f0r the 1501at10n 0f MR-de9rad1n9
6acter1a. 7he MR-5M p1ate c0nta1ned a60ut 1% 0f
ethan01, wh1ch fav0red the 9r0wth 0f ethan01-ut1112-
1n9 6acter1a 5uch a5 Acet06acter 114uefac1en5 5-1
(Kr1e9 and H01t, 1984; 50, 1989; 50 et a1., 1991). 1n
the pre5ent 5tudy, 91uc05e wa5 u5ed a5 car60n and
ener9y 50urce5 1n the M R - M M p1ate. M0re
MR-de9rad1n9 6acter1a were 1501ated 51nce m0re
d1ver51f1ed 9r0up5 0f 6acter1a u5ed 91uc05e a5 car60n
and ener9y 50urce5.
1dent1f1cat10n 0f the 5e1ected methy1 red-dec010r121n9
6acter1a
0 f the 5 1501ated MR-dec010r121n9 6acter1a, 3 were
K1e651e11a 5pp. H19her 0ccurrence 0f K1e651e11a

(a)

~
2. 2 ~
-~" u n 2 . 7 8

~ -~-8. e5

•,... 1 a ~:~;
~11. 48
~..~9712.71

(61

~ -- 7.99

1e
1 2 . 77
( 1 4 . 09ER e

F19. 2. HPLC ana1y515 0f pr0duct5 0f the de9radat10n 0f methy1 red 6y (a) K1e651e11apneum0n1ae R5-13,
and (6) Acet06acter 114uefac1en5 5-1. 7he c01umn wa5 a 8eckman U1tra5phere 0D5 c01umn
(4.6 mm x 150 mm, the pack1n9 w1th me5h 512e 0f 5/~m) and the m0611e pha5e c0mp05ed 0f 25 mM
ph05phate 6uffer (pH = 3.0) and acet0n1tr11e w1th var1a61e rat10 chan91n9 fr0m 77:23 t0 0:100 (ph05phate
6uffer:acet0n1tr11e). 7he 501vent 9rad1ent wa5 26%/m1n. 7he retent10n t1me5 f0r N,N•-d1methy1-p-
pheny1ened1am1ne (DMPD), 2-am1n06en201c ac1d (A8A) and methy1 red (MR) were 2.28-2.29 m1n,
7.99-8.05 m1n and 11.4 m1n, re5pect1ve1y.
 1740 P. K. W0n9 and P. Y. Yuen
5 7 A 7 1 C c0nd1t10n 5 H A K 1 N 6 c0nd1t10n
a) 23°C (6) 230C
11H1 11H1
75 75
511 511
25 25
11 0
1 1

11 511 11H1 1511 21111 11 511 11N1 1511 21N1

(c) 30°C (d) 300C
100 11111
75 75
••0 /
511 511
25 25

¢1.
0
0 1 , , 0 1 1 1 1

11 511 11m 150 21H) 11 511 1011 1511 21111

° ~0
(e) 37°C f) 37"C
1. 1110 11H1
e~ 75 75
511 511
25 25
0 1 1
11 1 1

11 511 11111 1511 21111 11 511 11111 1511 21H•

(9) 45°C h) 45"c

100 100
75 75
511 511
25 25
11 0
0 511 11111 1511 21111 0 511 100 1511 21111

71mc (hr)
F19. 3. Effect5 0f temperature and aerat10n 0n the de9radat10n 0f methy1 red 6y K1e651e11apneum0n1ae
R5-13 ([[]) and Acet06acter 114uefac1en55-1 (C)) 1ncu6ated 1n MM at (a) 23°C, (c) 30°C, (e) 37°C and
(9) 45°C under 5tat1c c0nd1t10n5; and at (6) 23°C, (d) 30°C, (f) 37°C and (h) 45°C under 5hak1n9 c0nd1t10n5.
Each p01nt repre5ent5 mean •+ 5tandard err0r 0f tr1p11cate5.

5pp. am0n9 the 5e1ected MR-de9rad1n9 6acter1a may
6e 6ecau5e 0f the1r m1n1mum re4u1rement5 f0r 9r0wth
(Kr1e9 and H01t, 1984). 1n add1t10n, a11 5 6acter1a1
1501ate5 6e10n9 t0 the fam11y 0f Enter06acter1acea. 1t
wa5 6ecau5e m05t mem6er5 0f th15 fam11y are
facu1tat1ve anaer06e5 wh1ch are a6undant 1n the
5amp1e5 0f dye-c0ntam1nated 51ud9e u5ed 1n the
pre5ent 5tudy.
K1e651e11a pneum0n1ae R5-13 wa5 5e1ected f0r
further 5tudy 6ecau5e 1t had the h19he5t M R
dec010r121n9 a6111ty 1n 114u1d med1um. A. 114uefac1en5
5-1, a prev10u51y 1501ated aer061c MR-de9rad1n9
6acter1um (50 et a1., 1990), wa5 1nc1uded a5 a
reference.
1dent1f1cat10n 0f de9radat10n pr0duct5 0f methy1 red 6y
H P L C ana1y515
Re5u1t5 0f H P L C ana1y515 1nd1cated that K.
pneum0n1ae R5-13, a5 we11 a5 A. 114uefac1en5 5-1,
dec010r12ed M R 6y c1eav1n9 1t 1nt0 2-am1n06en201c
ac1d ( A 8 A ) and N,N•-d1methy1-p-pheny1ened1am1ne
( D M P D ) (50 et a1., 1990). De9radat10n 0f M R 1nt0
the c010r1e55 pr0duct5, A 8 A and D M P D , 6y
m1cr00r9an15m5 c0u1d 6e 4uant1f1ed 6y the de9ree 0f
dec010r12at10n 0f M R (50 et a1., 1990). 7he u5e 0f
H P L C ana1y515 t0 5tudy the effect5 0f var10u5
phy51c0-chem1ca1 fact0r5 0n the M R de9radat10n 6y
the5e tw0 6acter1a wa5 very t1me c0n5um1n9, 50
 810de9radat10n 0f methy1 red 1741

5pectr0ph0t0metr1c ana1y515 6y determ1n1n9 the
chan9e5 0f 0ran9e c010r (1.e. dec010r12at10n) 0f MR
1n cu1ture 5upernatant wa5 u5ed 1n the pre5ent 5tudy.
Effect5 0f phy51c0-chem1ca1fact0r5 0n the methy1 red
de9radat10n and ce11 ma55 0f cu1ture5 0f 5e1ected
6acter1a
80th K. pneum0n1a R5-13 and A. 114uefac1en5 5-1
had h19h MR de9radat10n at 23°C, 30°C and 37°C
under 5tat1c c0nd1t10n5 0r under 5hak1n9 c0nd1t10n5.
H0wever, the MR de9radat10n 0f 60th 6acter1a wa5
510wer under 5tat1c c0nd1t10n5. 1t wa5 6ecau5e 60th
6acter1a 9rew fa5ter under 5hak1n9 c0nd1t10n5 (F195
4(a)-(9)). A 1ar9er p0pu1at10n 0f ce115 c0u1d certa1n1y
de9rade m0re m01ecu1e5 0f MR. Near1y n0 MR
de9radat10n wa5 065erved 1n cu1ture5 0f the5e
6acter1a 9r0wn at 45°C under 5tat1c 0r 5hak1n9
c0nd1t10n5 51nce 60th 6acter1a 9rew very p00r1y at
th15 temperature.
7he MR de9radat10n a6111ty 0f K. pneum0n1ae
R5-13 1n the pre5ence 0f 91uc05e wa5 h19her than that
0f A. 114uefac1en5 5-1 6ecau5e 0f the 6etter 9r0wth 0f
the f0rmer 1n the MM w1th 100 m9 1-~ 0f MR. 7he
pre5ence 0f 0.1 9 1-~ 0f 91uc05e pr0v1ded 5uff1c1ent

57A71C c0nd1t10n 5HAK1N6 c0nd1t10n

,4 23°C (6) 230C
0.4 0.4
0.3 0.3
0.2 0.2
0.1 (L1
0.0 0.0
1 1 1 1 1 1 1 1

0 50 1(H1 1511 2011 (1 511 11M1 150 2(H)

(c) 3(HC (d) 30•C
0.4 0.4
0.3 0.3
/0
0.2 0.2
0.1 0.1
0.0 0.0
1 1 1 1

0 50 1(H) 150 2(1(1 0 50 1(H1 150 200

e) 37°C 13 370c
.~ 0.4 (1.4
"~ 0.3 0.3
~ 0.2 1).2
-0--- -0•- - - ~1~0
0.1 1L1
0.0 0.0
1 1 1 1 1 1 • 1

0 50 100 150 200 1) 511 1(H1 1511 2(1(1

(9) 450c (h) 450c

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 1•--1
--

1
--0--
[]
1
-- -0

1
0 0.0
1 1 1 1
11 511 11H1 1511 211(1 11 50 1(H1 150 2(H1

71me (hr)
F19. 4. Effect5 0f temperature and aerat10n 0n the ce11ma55 0f cu1ture5 0f K1e651e11apneum0n1ae R5-13
(1-4) and Acet06acter 114uefac1en55-1 (•) 1ncu6ated 1n MM at (a) 23°C, (c) 30°C, (e) 37°C and (9) 45°C
under 5tat1c c0nd1t10n5;and at (h) 23°C, (d) 30°C, (f) 37°C and (h) 45°C under 5hak1n9c0nd1t10n5. Each
p01nt repre5ent5 mean • 5tandard err0r 0f tr1p11cate5.
1742 P.K. W0n9 and P. Y. Yuen
(a) (6)
100 [] 1-42 100 0-0 -0- -- -0 0
1
Y
./
75 75

50 50

25 25

0 1 1 1 1 1 1 0 P°-1P-----1-~ , t
0 1 2 3 4 5 0 1 2 3 4 5

61uc05e (91-1) Ethan01 (9 1-1)

0

0
~4

1• (c) (d)

100 [] [] [] [] []
100
---%,
75 75
ha
/ •
50 50
9 °
25 25

0 1 1 1 1 1 1 0 1 1 1 1 1

0 1 2 3 4 5 6 5.5 6.0 6.5 7.0 7.5 8.0 8.5

(NH4)2804 (9 1-1) pH
F19. 5. Effect5 0f (a) c0ncentrat10n 0f 91uc05e, (6) c0ncentrat10n 0f ethan01, (c) c0ncentrat10n 0f
amm0n1um 5u1fate and (d) pH 0f the cu1ture med1um 0n the de9radat10n 0f methy1 red 6y K1e651e11a
pneum0n1ae R5-13 ([5]) and A cet06acter 114uefac1en55-1 (0). 7he cu1ture5were 1ncu6ated1nm0d1f1edMM
at 30~C under 5hak1n9c0nd1t10n5f0r 2 day5. Each p01nt repre5ent5 mean + 5tandard err0r 0f tr1p11cate5.

car60n and ener9y 50urce5 f0r K. pneum0n1ae R5-13
t0 de9rade MR. 7he 1e55 ce11 ma55 w1th 5u65tant1a11y
h19h MR de9radat10n 6y A. 114uefac1en5 5-1 1n the
pre5ence 0f h19h c0ncentrat10n5 (1.e. 3% and 5%) 0f
91uc05e 1nd1cated that th15 6acter1um ut1112ed m05t 0f
the ener9y 9enerated fr0m the 91uc05e de9radat10n
f0r the reduct1ve c1eava9e 0f MR. K. pneum0n1ae
R5-13 c0u1d n0t u5e ethan01 f0r 9r0wth, 50 n0
519n1f1cant MR de9radat10n wa5 065erved. 7he
9r0wth and MR de9radat10n 6y A. 114uefac1en5 5-1
were 6etter 1n the pre5ence 0f h19her c0ncentrat10n 0f
ethan01 51nce th15 6acter1um c0u1d u5e ethan01 a5
car60n and ener9y 50urce5 (Kr1e9 and H01t, 1984).
7he c0ncentrat10n 0f amm0n1um 5u1fate had n0
519n1f1cant effect 0n MR de9radat10n, and the 9r0wth
0f 60th 6acter1a 1nd1cated that 0.1 91- ~0f amm0n1um
5u1fate wa5 5uff1c1ent f0r the5e funct10n5.
A. 114ueJac1en5 5-1 5h0wed h19her a6111ty 1n MR
c1eava9e at pH 6.5-7.0 and 0n1y c0mp1ete1y c1eaved
MR at pH6.5 (F19. 4(d)). K. pneum0n1ae R5-13
c0mp1ete1y de9raded MR at a 6r0ad pH ran9e (F19.
4(d)), a1th0u9h effect5 0f m0re ac1d1c (1.e. < 6.0) 0r
a1ka11ne ( > 8.0) pH 0n the MR de9radat10n c0u1d n0t
6e te5ted w1th the ph05phate 6uffer u5ed 1n the
pre5ent 5tudy. 7he5e re5u1t51nd1cated that MR de9ra-
dat10n 6y K. pneum0n1ae R5-13 wa5 1e55 5en51t1ve t0
the chan9e 0f the pH 0f the cu1ture med1um.
C0NCLU510N
(1) K1e651e11a pneum0n1ae R5-13, wh1ch had a
c0mparat1ve1y h19her aer061c methy1 red (MR)
de9radat10n a6111ty c0mpared w1th 0ther a20
dye-dec010r121n9 6acter1a (09awa et a1., 1981, 1986;
50 et a1., 1990; Yat0me et a1., 1990), wa5 1501ated
fr0m dye-c0ntam1nated 51ud9e c011ected fr0m an
1ndu5tr1a1 area 1n H0n9 K0n9. 7h15 6acter1um
c0mp1ete1y de9raded 100 m9 1 ~0f MR 1n the cu1ture
med1um under 5tat1c and 5hak1n9 c0nd1t10n5.
(2) HPLC ana1y515 0f the MR de9radat10n
pr0duct5 0f the cu1ture5 0f K. pneum0n1ae R5-13 and
A. 114uefac1en5 5-1 (50 et a1., 1990) 1nd1cated that
 810de9radat10n 0f methy1 red 1743

(a) (6)
1.2 1.2

0.8 0.8

0.4 0.4
0/
0.0 0.0 D 0
4" 1 1 1 1 1 1 1 1 1 1 1 1

0 1 2 3 4 5 0 1 2 3 4 5

61uc05e (91 "1) Ethan01 (91 "t)

(c) (d)
1.2 1.2

0.8 0.8

0.4 0.4
~ 0.~ ---0---- - 0
0..•.--43-
0.0 0.0
1 1 1 1 1 1 1 1 1 1 1

0 1 2 3 4 5 6 5.5 6.0 6.5 7.0 7.5 8.0 8.5

(NH4)2804 (91-1) pH
F19. 6. Effect5 0f (a) c0ncentrat10n 0f 91uc05e, (6) c0ncentrat10n 0f ethan01, (c) c0ncentrat10n 0f
amm0n1um 5u1fate and (d) pH 0f the cu1ture med1um 0n the ce11 ma55 0f cu1ture5 0f K1e651e11apneum0n1ae
R5-13 (EJ) and Acet06acter 114uefac1en55-1 ( 0 ) . 7he cu1ture5 were 1ncu6ated 1n m0d1f1ed MM at 30°C
under 5hak1n9 c0nd1t10n5 f0r 2 day5. Each p01nt repre5ent5 mean + 5tandard err0r 0f tr1p11cate5.

6 0 t h 6acter1a reduct1ve1y c1eaved M R 1nt0 tw0 REFERENCE5

c010r1e55 pr0duct5, name1y, 2-am1n06en201c ac1d
(A8A) and N,N•-d1methy1-p-pheny1ened1am1ne
(DMPD).
(3) K. pneum0n1ae R5-13 de9raded M R m u c h
fa5ter a n d m 0 r e eff1c1ent1y t h a n A. 114uefac1en5 5 - 1 . 1 n
add1t10n, the M R de9radat10n 6y K. pneum0n1ae
R5-13 re4u1red 1e55 nutr1ent5 a n d wa5 1e55 5en51t1ve t0
the env1r0nmenta1 c h a n 9 e t h a n t h a t 0f A. 114uefac1en5
5 - 1 . 7 h e 5 e advanta9e5 m a k e the u5e 0 f K. pneum0n1ae
R5-13 m0re 5uper10r t h a n A. 114uefac1en5 5-1 1n the
t r e a t m e n t 0 f a20 dye5 1n 1ndu5tr1a1 eff1uent w1th
ever-chan91n9 phy51ca1 a n d chem1ca1 pr0pert1e5.
(4) 1n 0rder t0 app1y the 5ma11 512e 6acter1a1 ce115
1n a c0nt1nu0u5 pr0ce55 f0r the de9radat10n 0f a20
dye5 1n 1ndu5t1ra1 eff1uent, 1mm061112at10n 0 f ce115 0 f
K. pneum0n1ae R5-13 wa5 1n1t1ated.
Ackn0w1ed9ement--7h15 5tudy wa5 5upp0rted 6y a 9rant
fr0m the U P 6 C Earmarked 6 r a n t f0r Re5earch 0f the
H0n9 K0n9 60vernment.
Ame5 8. N., McCann J. and Yama5ak1 E. (1975) Meth0d5
f0r detect1n9 carc1n09en5 and muta9en5 w1th the
5a1m0ne11a/mamma11an-m1cr050me muta9en1c1ty te5t.
Mut. Re5. 31, 347-364.
An11ker A. (1979) Ec0t0x1c0109y 0f dye5tuff5--A j01nt eff0rt
6y 1ndu5try. Ec0t0x1e01. Env1r0n. 5afety 3, 59-74.
Chun9 K. 7., Fu1k 6. E. and E9an M. (1978) Reduct10n 0f
a20 dye5 6y 1nte5t1na1 anaer06e5. App1. Env1r0n.
M1cr06101. 35, 558-562.
Chun9 K. 7., Fu1k 6. E. and Andrew5 A. W. (1981)
Muta9en1c1ty te5t1n9 0f 50me c0mm0n1y u5ed dye5. App1.
Env1r0n. M1cr06101. 42, 641-648.
61n9e11 R. and Wa1ker R. (1971) Mechan15m5 0f a20
reduct10n 6y 5trept0c0ccu5faeca115.11.7he r01e 0f 501u61e
f1av1n5. Xen0610t1ca 1, 231-239.
6r1ff1th5 J. (1984) Deve10pment5 1n the f19ht a650rpt10n
pr0pert1e5 0f dye5--C010r and ph0t0chem1ca1 react10n5.
1n Deve10pment5 1n the Chem15try and 7echn0109y 0f
0r9an1c Dye5 (Ed1ted 6y 6r1ff1th5 J.), pp. 1-30. 50c1ety 0f
Chem15try 1ndu5try, 0xf0rd.
6 0 r d 0 n P. F. and 6re90ry P. (1983) 0r9an1c Chem15tr)• 1n
C010ur. 5pr1n9er-Ver1a9, New Y0rk, 322 pp.
60v1nda5wam1 M., 5chm1dt 7. M., Wh1te D. C. and L0per
J. C. (1993) Phy109enet1c ana1y515 0f a 6acter1a1 aer061c
de9rader 0f a20 dye5. J. 8acter101. 175, 6062-6066.
1744 P. K. W0n9 and P. Y. Yuen
H01me 1. (1984) Ec01091ca1 a5pect5 0f c010r chem15try. 1n
Deve10pment5 1n the Chem15try and 7echn0109y 0f 0r9an1c
Dye5 (Ed1ted 6y 6r1ff1th5 J.), pp. 111-128. 50c1ety 0f
Chem15try 1ndu5try, 0xf0rd.
1daka E., 09awa 7. and H0r1t5u H. (1982) De9radat1ve
pathway 0f p-am1n0a206en2ene 6y 8ac111u55u6t1115. Eur.
J. App1. M1er06101. 15, 141-143.
Kr1e9 N. R. and H01t J. 6. (1984) 8er9ey•5 Manua1 0f
5y5temat1c 8acter10109y, V01. 1, W1111am and W11k1n5,
8a1t1m0re, 964 pp.
Lar5en J. C., Meyer 7. and 5che11ne R. R. (1976) Reduct10n
0f 5u1ph0nated water-501u61e a20 dye5 6y caeca1
m1cr00r9an15m5 fr0m the rat. Acta Pharmac01. 70x1c01.
38, 353-357.
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