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Phenol and Naphthalene Degradation by Mixed Culture of Microorganisms
P H E N O L AND NAPHTHALENE DEGRADATION B Y M I X E D CULTURE
OF M I C R O O R G A N I S M S N.S.
MANUKOVSKI,
M.I.
TEREMOVA,
I n s t i t u t e o f Biophysics,
660036, K r a s n o y a r s k , U S S R
GUREVICH and I . M .
PAN’KOVA
U S S R Academy o f S c i e n c e s ,
Wastewater c o n t a i n s , ces;
Yu.L.
as a r u l e ,
a mixture o f organic substan-
i t ’ s b i o d e g r a d a t i o n i s r e a l i z e d by m i c r o b i a l c o m m u n i t i e s .
The
composition o f t h e communities i s e s t a b l i s h e d spontaneously under non-sterile
c o n d i t i o n s o f no e x t e r n a l c o n t r o l
impact.
T h i s communication d w e l l s upon t h e problem o f t h e p u r p o s e f u l f o r m a t i o n o f t h e m i c r o o r g a n i s m community t h a t i s c a p a b l e t o p u r i f y w a t e r more e f f i c i e n t l y .
The o b j e c t u n d e r i n v e s t i g a t i o n was t h e wa-
t e r o f a cokechemical i n d u s t r y .
B i o d e g r a d a t i o n o f p h e n o l a n d na-phtha-
l e n e was m o s t i n t e r e s t i n g t o u s . We h a v e s t u d i e d t w o ways t o i m p r o v e t h e c o m p o s i t i o n o f m i c r o b i a l community, which p u r i f i e d wastewater
-
from aromatic hydrocar-
bons.
1. One c a n i n t r o d u c e a new s t r a i n i n t o t h e m i c r o b i a l c o m m u n i t y . The s t r a i n c o u l d be o b t a i n e d by a r e c o m b i n a n t DNA,
or s p e c i a l l y se-
l e c t e d , or found i n a d i f f e r e n t environment.
2.
The s p e c i e s c o m p o s i t i o n o f a c o m m u n i t y i s n o t a f f e c t e d
(i.e. n o new s p e c i e s o r s t r a i n i s i n t r o d u c e d ) ,
but the r a t i o o f the
densities o f the species present i s appropriately varied. Community c o m p o s i t i o n s h o u l d b e c h a n g e d w i t h r e s p e c t t o t h e feature o f both microorganisms i n t e r a c t i o n ,
and t h e o x i d a t i o n k i n e -
t i c s o f t h e mixed s u b s t r a t e . S p e c i e s and q r o w t h c o n d i t i o n s . monas s p .
and N o c a r d i a sp.
The b a c t e r i a l c u l t u r e o f P s e u d o -
have been i s o l a t e d from t h e wastewater
on t h e s e l e c t i v e media c o n t a i n i n g p h e n o l , g u i a c o l , The g l a s s f e r m e n t o r
o f t h e 0.45-0.6
and
naphthalene
1 e f f e c t i v e v o l u m e was e q u i p p e d
w i t h an a u t o m a t i c d e v i c e t o m a i n t a i n t h e pH a n d t e m p e r a t u r e . was m a i n t a i n e d w i t h i n t h e 6.7-7.5
range,
The pH
a n d t h e c u l t i v a t i o n tempe-
r a t u r e was e q u a l t o 20
156
a n d 30
OC
OC
f o r N o c a r d i a sp.,
b u t 32-34
OC
f o r Pseudomonas s p . Analysis.
The c o n c e n t r a t i o n o f b o t h p h e n o l a n d g u i a c o l was d e -
termined c o l o r i m e t r i c a l l y over 4-aminosntipyrine 2,3-oxygenase
as t h e i n d i c a t o r ;
a c t i v i t y i n c e l l s was d e t e r m i n e d c o l o r i m e t r i c a l l y o v e r
c a t e c h o l ; n a p h t h a l e n e c o n c e n t r a t i o n was d e t e r m i n e d b y t h e c h r o m a t o graph.
I n addition,
s u p e r n a t a n t a b s o r p t i o n maximum was d e t e r m i n e d
-
w i t h i n t h e r e g i o n o f 370
420 n m a t pH 1 1 . 0
111.
1. D e g r a d a t i o n o f a s i n g l e s u b s t r a t e The c u l t u r e s s t u d i e d h a v e shown d i f f e r e n t a b i l i t i e s t o o x i d i z e the substrate
( s e e T a b l e 1). B o t h t h e d e p e n d e n c e o f t h e c e l l s den-
s i t y and t h e s u b s t r a t e c o n c e n t r a t i o n on t h e d i l u t i o n r a t e ,
and t h e
dependence o f t h e s p e c i f i c r a t e o f s u b s t r a t e c o n s u m p t i o n on t h e g r o w t h r a t e f i t i n Monod's and P i r t ' s models ( s e e F i g .
1
-
3).
TABLE 1 Degradation o f aromatic hydrocarbons as s o l e sources o f carbon i n c h e m o s t a t c u l t u r e s o f Pseudomonas a n d N o c s r d i a Culture, substrate
Substrate input
output
Gs/ll
Dilution rate
[h-']
1000800
< 1
N o c a r d i a spp. guiacol
250
1-5
0.065-
Pseudomonas sp. + N o c a r d i a sp. p h e n o l
BOO
c1
0.140.20
Pseudomonas sp. phenol
Pseudomonas spp.
20004000
Growth yield
0.070.27
c 0.5
0.6
0.12
+ +
0.0750.12
N o t d e t e r m i n e d f o r w a l l g r o w t h and i n s o l u b i l i t y o f n a p h t h a l e n e .
We h a v e m e a s u r e d t h e d e p e n d e n c e o f t h e s p e c i f i c r a t e o f s u b s t r a t e
consumption on t h e d i l u t i o n r a t e . g r o w t h and c e l l s a g g r e g a t i o n ,
The o c c u r r e n c e o f t h e a t t a c h e d
has been measured as w e l l .
o x i d a t i o n d a t a a r e p r e s e n t e d i n T a b l e 2;
I t s h o u l d be n o t e d ,
we'll
d i s c u s s them b e l o w .
t h a t t h e observed v a l u e o f t h e economic
c o e f f i c i e n t i s l e s s tl- n t h e t h e o r e t i c a l one, spent f o r maintenance i s c l o s e t o zero.
a l t h o u g h t h e energy
One c a n e a s i l y see t h a t
d u r i n g t h e p h e n o l d e g r a d a t i o n b y Pseudomonas sp. was a l w a y s c o l o r e d g r e e n ,
The p h e n o l
or b r o w n .
t h e c u l t u r a l medium
-
157
-
0.1
0.2
0.3 DIWTDN RATE [ h-'1
Fig. 1. C h e m o s t a t c u l t u r e o f P s e u d o m o n a s s e . with phenol a s a lim i t i n g substrate. 1 - b i o m a s s , 2 - output c o n c e n t r a t i o n o f phenol
--
- 0.200,
J
0
0.15
-
0.10
.
0.05
-
0
0.05
0.1
0.15
DILUTION RATE
1 0 .h-']
Fig. 2. C h e m o s t a t c u l t u r e o f N o c a r d i a s p . w i t h g u i a c o l a s a limiting s u b s t r a t e ( S o = 0.25 9/11. 1 biomass, 2 autput concentration o f guiacol; a r r o w 8 i n d i c a t e c h a n g e s o f d i l u t i o n r a t e
-
-
-
-
158
'p
1.0
c
-
2
P
n
F i g . 3. S p e c i f i c u p t a k e r a t e o f p h e n o l ( 1 ) a n d g u i a c o l ( 2 ) i n r e l a t i o n t o t h e d i l u t i o n r a t e i n c h e m o s t a t c u l t u r e o f Pseudomonas s p . and N o c a r d i a spp.
N o t e some d i s c r e p a n c i e s b e t w e e n t h e e v i d e n c e a n d t h e m o d e r n theories:
1. N o n - s i n g l e - v a l u e d
r e s u l t o f t h e s u b s t r a t e d e g r a d a t i o n under
c o n s t a n t c u l t i v a t i o n c o n d i t i o n s was o b s e r v e d i n a s e r i e s o f e x p e r i m e n t s (see dash l i n e i n F i g . as w e l l ,
2.
as exp.
7va expa.
Pseudomonaa ap.
2 f o r t h e case o f q u i a c o l o x i d a t i o n ,
1-6 and 13-16 i n Table 2).
c u l t u r e s a l w a y s show m e t e - c l e a v a g e
pathway
f o r catechol oxidation a t the i n i t i a l stage o f c u l t i v a t i o n . (rather long)
2.
t i m e i t disappeared b o t h f o r mixed,
I n some
and m o n o - c u l t u r e s .
Substrate mixture degradation I n s p i t e o f some i n e x p l i c a b l e f e a t u r e s i n s i n g l e s p e c i e s c u l -
t u r e behavior,
t h a t oxidase phenol,
guiacol,
t r i e d t h e m i x e d c u l t u r e o f Pseudomonas aD. them.
+
and naphthalene, N o c a r d i a sp.
we
t o treat
We assumed t h e enzyme k i n e t i c s o f d i f f e r e n t m i c r o o r g a n i s m s t o
d i f f e r f o r v a r i o u s a r o m a t i c h y d r o c a r b o n s . So, complement each o t h e r .
t h e c u l t u r e s should
-
159
-
TABLE 2 Parameters o f phenol d e g r a d a t i o n process i n chemostat ~
NN
Dilution r a p ]
-
Substrate input
hdil
Biomass
output
[mg/l]
1
0.07
1000
0.2
594
2
0.07
1000
0.2
600
Cleavage pathway, absorbtion p e a k [nm]
Economical coef f i c i e n
b/gJ 0.59 0.60
375 meta-
3
0.07
1000
0.3
630
4
0.07
1000
0.2
490
5
0.07
1000
0.2
550
6
0.07
80 0
0.2
510
7
0.07
1000
0.45
440
8
0.08
800
0.2
478
9
0.09
800
0.6
474
10
0.12
1000
0.8
58 2
11
0.14
BOO
0.5
400
12
0.15
1000
0.3
590
13
0.17
880
19.0
454
14
0.17
800
0.5
467
15
0.19
800
21.0
384
0.49
16
0.20
1000
0.5
700
0.70
17
0.23
1000
0.4
510
18
0.26
1000
1.5
672
19
0.27
800
0.9
512
20
0.27
800
1.0
21
0.07
800
0.36
0.63 375
0.55 375 380
0 . 1 h-'.
0.59 0.58 meta-
0.50 0.59 0.50
375
0.58
375
0.51 0.67 0.64
450
410
0.56
610
s m a l l meta
0.76
9/11 and n a p h t h a l e n e because o f t h e e x t r e -
The d i l u t i o n r a t e was e q u a l t o
T h i s i s t h e c r i t i c a l f l o w f o r N o c a r d i a sp.,
i s u t i l i z e d moat e f f i c i e n t l y ;
0.44 0.60
375
t h i s i s the calculated concentration,
mely poor s o l u b i l i t y o f naphthalene).
0.63 ortho-
The m i x e d a u b s t r a t e c o n t a i n e d p h e n o l ( 0 . 5
( 2 g/l);
0.49
that culture didn't
a t which phenol
o x i d i z e t h e naph-
thalene. F o r t h e e x p e r i m e n t we u s e d Pseudomonas ap.
naphthalene.
adapted t o u t i l i z e
The a d d i t i o n o f p h e n o l t o t h e n a p h t h a l e n e u t i l i z i n g
c u l t u r e r e s u l t e d b o t h i n t h e i n t e n s i v e brown c o l o r appearance i n t h e culture,
a n d an i n c r e a s e d r e s i d u a l p h e n o l c o n c e n t r a t i o n .
-
160
-
The m i x e d c u l t u r e was a b s o l u t e l y c o l o r l e s s ,
but the residual
c o n c e n t r a t i o n s o f b o t h p h e n o l and n a p h t h a l e n e were l e s s t h a n 0.2 mg/l.
I t s h o u l d be s t r e s s e d ,
t h a t N o c a r d i a sp.
successfully u t i l i z e d
phenol a t r e l a t i v e l y h i g h i n p u t concentrations o f the former,
with rather great d i l u t i o n rates, The No.
and
mixed c u l t u r e .
2 m e t h o d was i l l u s t r a t e d w i t h t h e f o l l o w i n g e x p e r i m e n t .
The n a p h t h a l e n e o x i d i z i n g c u l t u r e ,
i s o l a t e d from wastewater d i s c h a r -
ged from p h e n o l i c w a t e r b i o l o g i c a l t r e a t m e n t p l a n t , i n t o a n a e r o t a n k i n a s p e c i f i c way.
As s r e s u l t ,
d u a l p h e n o l c o n c e n t r a t i o n d e c r e a s e d a s much a s 30
was i n o c u l a t e d
t h e average r e s i -
X,
b u t the process
s t a b i l i t y o f a sewage b i o l o g i c a l t r e a t m e n t e n h a n c e d ( t h e v a r i a t i o n c o e f f i c i e n t d e c r e a s e d 2.8
4
times).
8
These d a t a s r e shown i n F i g .
12
4.
20
16
MONTMS
F i g . 4. D y n a m i c s o f p h e n o l a t t h e o u t p u t o f b i o l o g i c a l p u r i f i c a t i o n o f wastewater i n coke and b y - p r o d u c t p l a n t . Arrow i n d i c a t e sowing o f nsphth a l e n e - o x i d i z i n g b a c t e r i a Thus,
t h e e x p e r i m e n t s c a r r i e d o u t show t h a t t h e c h a n g e s i n t h e
q u a l i t y a n d number c o m p o s i t i o n o f a m i c r o b i a l c o m m u n i t y , n a l l y s t a b l e c o n t i n u o u s c u l t u r e have a p o s i t i v e e f f e c t , community r e a r r a n g e .
with functioi.e.
make t h e
-
161
-
Now, w e ’ l l d i s c u s s t h e s e i n e x p l i c a b l e f e a t u r e s m e n t i o n e d a b o v e i n t h e s i n g l e species c u l t u r e behaviour. DlSCUSSION
Both b i o c h e m i s t r y and g e n e t i c s of t h e m i c r o b i a l o x i d a t i o n o f
it is
a r o m a t i c h y d r o c a r b o n s are r a t h e r well s t u d i e d . N e v e r t h e l e s s ,
s t r a n g e , t h a t t h e e v i d e n c e on t h e c a t a b o l i s m m u l t i r o u t i n e s s are n o t i n c l u d e d i n t o w a s t e w a t e r t r e a t m e n t , when c o n s i d e r e d a t t h e p o p u l a t i o n and community l e v e l s .
T h i s e v i d e n c e m a k e s us d i s c u s s t h e p r o -
blem of a m u l t i r o u t i n e s s . Jones e t a l . I 2 1 found t h a t t h e r e g i o n o f m i c r o b i a l growth lim i t a t i o n a s w e l l a s i t s i n h i b i t i o n b y p h e n o l h a d t h e i r s p e c i f i c economic c o e f f i c i e n t s . These r e g i o n s a r e n ’ t o f any i n t e r e s t and have n o g e n e r a l b o u n d a r y , a s i t i s shown i n F i g . 5 [2, 3 1 . Assume t h e g r o w t h r a t e d e p e n d e n c e on s u b s t r a t e c o n c e n t r a t i o n b e a s s h o w n i n F i g . 6 . Q u i t e a few r e s e a r c h e r s s u p p o r t t h i s a s s u m p t i o n , T h e maximum o f regions.
p ( S ) belong t o both t h e l i m i t a t i o n , and i n h i b i t i o n
There is no corresponding p o i n t for i t i n Fig.
5, or a
t r a n s i t i o n a r e a from l i n e 1 t o l i n e 2 . Hence, t h e b a s i c assumption presented a t Figs.
5, 6 should be changed.
The p o s s i b l e c h a n g e f o r
i t i s shown i n F i g s . 5 a n d 6 i n d a s h l i n e s .
0
1
2
5
3
6
7 1l p
f h-’I
F i g . 5. R e c i p r o c a l p l o t o f g r o w t h y i e l d a n d g r o w t h r a t e w i t h p h e n o l limitation, 2 inhibition, oxidizing b a c t e r i a (from /3.2/). 1 3 h y p o t h e t i c a l l i n e o f p a s s a g e from l i m i t a t i o n t o i n h i b i t i o n
-
-
-
- 162
-
Taking i n t o account k i n e t i c features o f t h e aromatic hydrocarone c a n e a s i l y show t h e e x i s t e n c e o f t h e a r e a o f t h e
bons o x i d a t i o n , bi-stability i n Fig.
i n behaviour.
I t ’ s evident,
i n part,
f r o m o u r d a t a shown
3 a n d T a b l e 2.
P o s i t i o n 7 i n T a b l e 2 shows t h e r e s u l t s t o b e s t a t i s t i c a l l y d i f f e r e n t from t h o s e p r e s e n t e d i n p o s i t i o n s 1-6. meta-cleavage
o f t h e c a t e c h o l wasn’t
s i t i o n 7 v s p o s i t i o n s 1-6
The c o m p a r i s o n o f
shows t h a t m e t a - c l e a v a g e
l e s s output phenol concentration; p o s i t i o n 21,
observed.
I n t h i s case t h e po-
pathway p r o v i d e s
t h e aame shows t h e e v i d e n c e s i n
obtained from another s e r i e s o f long-time
experiments.
W
t-
a
n
SUBSTRATE
F i g . 6. S p e c i f i c growth r a t e vs. s u b s t r a t e c o n c e n t r a t i o n (hypothemeta-cleavage and 2 - o r t h o - c l e a v a g e t i c a l curves; f o r example, 1 pathways o f phenol, dash l i n e - o t h e r h y p o t h e s i s )
-
O n t h e o t h e r hand, p o s i t i o n s 1 3 - 1 6 d o n ’ t 2,3-oxygenase way)
(which i s the f i r s t -
activity
o f r e g a r d l e s s s i g n i f i c a n t v a r i a t i o n s i n t h e o u t p u t p h e n o l con-
centration.
Probably,
enzymes ( n a m e l y ,
2,3-
we j u s t o b s e r v e d a v a r i a t i o n o f t h e d i f f e r e n t and 1,Z-oxygenase)
complete e l i m i n a t i o n of ta, Fig.
show t h e d e c a y o f
s t e p enzyme o f m e t a - p a t h -
activities,
t h e c a t a b o l i c pathway.
contrary t o
Our e x p e r i m e n t a l d a -
unfortunately, f a i l s t o prove i t . One c a n e a s i l y d e s c r i b e t h e a r e a s o f m u l t i - s t a b l e
2 ( e a r d i a spe.)
and F i g .
3 (Pseudomonas s p . ) .
behaviour i n
Genetic features
o f m i c r o o r g a n i s m s used t o degrade t h e a r o m a t i c h y d r o c a r b o n s have resulted i n the following. t u r e changed meta-pathway
The p h e n o l o x i d i z i n g Pseudornonas sp. f o r ortho-pathway
shown i n p o s i t i o n 2 1 o f T a b l e 2.
i n some t i m e ;
cul-
that’s
When o x i d i z i n g n a p h t h a l e n e ,
we’ve
-
163
-
i n o c u l a t r e d t h e clones showing a g r e a t a c t i v i t y o f i n t o a culture,
b u t ortho-pathway
2,3-oxygenase
h a s b e g u n t o d o m i n a t e soon.
The d e l a y o f t r a n s i t i o n f r o m m e t e - c l e a v a g e
i n t o the orthoclea-
vage p a t h w a y i s e v i d e n c e o f t h e i n t e r a c t i o n b e t w e e n t h e c e l l s m i c r o b i a l p o p u l a t i o n and s u b s t r a t e b i o d e g r a d a t i o n p l a s m i d s , t h e enzyme a c t i v i t i e s t u r n o u t . e x p e r i m e n t s 1-7,
rather than
T h i s l a t t e r c o u l d be observed i n
T a b l e 2 , t h a t were c a r r i e d o u t as a p i l o t e x p e r i -
ment. Hence,
i t i s evident,
t h a t some f e a t u r e s o f a s u b s t r a t e c a t a -
b o l i s m ( a t i n t r e c e l l u l a r l e v e l ) h a v e a s t r o n g i m p a c t on t h e w a s t e w a t e r p r o c e s s i n g on t h e w h o l e . REFERENCES
1 2 3
M a n u a l o f M e t h o d s f o r G e n e r a l B a c t e r i o l o g y . P. G e r h a r d t e.a. ed., W a s h i n g t o n 1 9 8 1 . G.L. J o n e s , F. J a n s e n and A . J . McKay, S u b s t r a t e I n h i b i t i o n o f t h e g r o w t h o f b a c t e r i u m NCIB 8250 b y p h e n o l . L . Gen. M i c r o b i o l . , 7 4 ( 1 9 7 3 ) N I , p. 139-148. Yu.L. G u r e v i c h , S t a b i l i t y a n d R e g u l a t i o n o f G r o w t h i n M i c r o b i a l P o p u l a t i o n s . N o v o s i b i r s k , Nauka, 1 9 8 4 , 165 p .
OF M I C R O O R G A N I S M S N.S.
MANUKOVSKI,
M.I.
TEREMOVA,
I n s t i t u t e o f Biophysics,
660036, K r a s n o y a r s k , U S S R
GUREVICH and I . M .
PAN’KOVA
U S S R Academy o f S c i e n c e s ,
Wastewater c o n t a i n s , ces;
Yu.L.
as a r u l e ,
a mixture o f organic substan-
i t ’ s b i o d e g r a d a t i o n i s r e a l i z e d by m i c r o b i a l c o m m u n i t i e s .
The
composition o f t h e communities i s e s t a b l i s h e d spontaneously under non-sterile
c o n d i t i o n s o f no e x t e r n a l c o n t r o l
impact.
T h i s communication d w e l l s upon t h e problem o f t h e p u r p o s e f u l f o r m a t i o n o f t h e m i c r o o r g a n i s m community t h a t i s c a p a b l e t o p u r i f y w a t e r more e f f i c i e n t l y .
The o b j e c t u n d e r i n v e s t i g a t i o n was t h e wa-
t e r o f a cokechemical i n d u s t r y .
B i o d e g r a d a t i o n o f p h e n o l a n d na-phtha-
l e n e was m o s t i n t e r e s t i n g t o u s . We h a v e s t u d i e d t w o ways t o i m p r o v e t h e c o m p o s i t i o n o f m i c r o b i a l community, which p u r i f i e d wastewater
-
from aromatic hydrocar-
bons.
1. One c a n i n t r o d u c e a new s t r a i n i n t o t h e m i c r o b i a l c o m m u n i t y . The s t r a i n c o u l d be o b t a i n e d by a r e c o m b i n a n t DNA,
or s p e c i a l l y se-
l e c t e d , or found i n a d i f f e r e n t environment.
2.
The s p e c i e s c o m p o s i t i o n o f a c o m m u n i t y i s n o t a f f e c t e d
(i.e. n o new s p e c i e s o r s t r a i n i s i n t r o d u c e d ) ,
but the r a t i o o f the
densities o f the species present i s appropriately varied. Community c o m p o s i t i o n s h o u l d b e c h a n g e d w i t h r e s p e c t t o t h e feature o f both microorganisms i n t e r a c t i o n ,
and t h e o x i d a t i o n k i n e -
t i c s o f t h e mixed s u b s t r a t e . S p e c i e s and q r o w t h c o n d i t i o n s . monas s p .
and N o c a r d i a sp.
The b a c t e r i a l c u l t u r e o f P s e u d o -
have been i s o l a t e d from t h e wastewater
on t h e s e l e c t i v e media c o n t a i n i n g p h e n o l , g u i a c o l , The g l a s s f e r m e n t o r
o f t h e 0.45-0.6
and
naphthalene
1 e f f e c t i v e v o l u m e was e q u i p p e d
w i t h an a u t o m a t i c d e v i c e t o m a i n t a i n t h e pH a n d t e m p e r a t u r e . was m a i n t a i n e d w i t h i n t h e 6.7-7.5
range,
The pH
a n d t h e c u l t i v a t i o n tempe-
r a t u r e was e q u a l t o 20
156
a n d 30
OC
OC
f o r N o c a r d i a sp.,
b u t 32-34
OC
f o r Pseudomonas s p . Analysis.
The c o n c e n t r a t i o n o f b o t h p h e n o l a n d g u i a c o l was d e -
termined c o l o r i m e t r i c a l l y over 4-aminosntipyrine 2,3-oxygenase
as t h e i n d i c a t o r ;
a c t i v i t y i n c e l l s was d e t e r m i n e d c o l o r i m e t r i c a l l y o v e r
c a t e c h o l ; n a p h t h a l e n e c o n c e n t r a t i o n was d e t e r m i n e d b y t h e c h r o m a t o graph.
I n addition,
s u p e r n a t a n t a b s o r p t i o n maximum was d e t e r m i n e d
-
w i t h i n t h e r e g i o n o f 370
420 n m a t pH 1 1 . 0
111.
1. D e g r a d a t i o n o f a s i n g l e s u b s t r a t e The c u l t u r e s s t u d i e d h a v e shown d i f f e r e n t a b i l i t i e s t o o x i d i z e the substrate
( s e e T a b l e 1). B o t h t h e d e p e n d e n c e o f t h e c e l l s den-
s i t y and t h e s u b s t r a t e c o n c e n t r a t i o n on t h e d i l u t i o n r a t e ,
and t h e
dependence o f t h e s p e c i f i c r a t e o f s u b s t r a t e c o n s u m p t i o n on t h e g r o w t h r a t e f i t i n Monod's and P i r t ' s models ( s e e F i g .
1
-
3).
TABLE 1 Degradation o f aromatic hydrocarbons as s o l e sources o f carbon i n c h e m o s t a t c u l t u r e s o f Pseudomonas a n d N o c s r d i a Culture, substrate
Substrate input
output
Gs/ll
Dilution rate
[h-']
1000800
< 1
N o c a r d i a spp. guiacol
250
1-5
0.065-
Pseudomonas sp. + N o c a r d i a sp. p h e n o l
BOO
c1
0.140.20
Pseudomonas sp. phenol
Pseudomonas spp.
20004000
Growth yield
0.070.27
c 0.5
0.6
0.12
+ +
0.0750.12
N o t d e t e r m i n e d f o r w a l l g r o w t h and i n s o l u b i l i t y o f n a p h t h a l e n e .
We h a v e m e a s u r e d t h e d e p e n d e n c e o f t h e s p e c i f i c r a t e o f s u b s t r a t e
consumption on t h e d i l u t i o n r a t e . g r o w t h and c e l l s a g g r e g a t i o n ,
The o c c u r r e n c e o f t h e a t t a c h e d
has been measured as w e l l .
o x i d a t i o n d a t a a r e p r e s e n t e d i n T a b l e 2;
I t s h o u l d be n o t e d ,
we'll
d i s c u s s them b e l o w .
t h a t t h e observed v a l u e o f t h e economic
c o e f f i c i e n t i s l e s s tl- n t h e t h e o r e t i c a l one, spent f o r maintenance i s c l o s e t o zero.
a l t h o u g h t h e energy
One c a n e a s i l y see t h a t
d u r i n g t h e p h e n o l d e g r a d a t i o n b y Pseudomonas sp. was a l w a y s c o l o r e d g r e e n ,
The p h e n o l
or b r o w n .
t h e c u l t u r a l medium
-
157
-
0.1
0.2
0.3 DIWTDN RATE [ h-'1
Fig. 1. C h e m o s t a t c u l t u r e o f P s e u d o m o n a s s e . with phenol a s a lim i t i n g substrate. 1 - b i o m a s s , 2 - output c o n c e n t r a t i o n o f phenol
--
- 0.200,
J
0
0.15
-
0.10
.
0.05
-
0
0.05
0.1
0.15
DILUTION RATE
1 0 .h-']
Fig. 2. C h e m o s t a t c u l t u r e o f N o c a r d i a s p . w i t h g u i a c o l a s a limiting s u b s t r a t e ( S o = 0.25 9/11. 1 biomass, 2 autput concentration o f guiacol; a r r o w 8 i n d i c a t e c h a n g e s o f d i l u t i o n r a t e
-
-
-
-
158
'p
1.0
c
-
2
P
n
F i g . 3. S p e c i f i c u p t a k e r a t e o f p h e n o l ( 1 ) a n d g u i a c o l ( 2 ) i n r e l a t i o n t o t h e d i l u t i o n r a t e i n c h e m o s t a t c u l t u r e o f Pseudomonas s p . and N o c a r d i a spp.
N o t e some d i s c r e p a n c i e s b e t w e e n t h e e v i d e n c e a n d t h e m o d e r n theories:
1. N o n - s i n g l e - v a l u e d
r e s u l t o f t h e s u b s t r a t e d e g r a d a t i o n under
c o n s t a n t c u l t i v a t i o n c o n d i t i o n s was o b s e r v e d i n a s e r i e s o f e x p e r i m e n t s (see dash l i n e i n F i g . as w e l l ,
2.
as exp.
7va expa.
Pseudomonaa ap.
2 f o r t h e case o f q u i a c o l o x i d a t i o n ,
1-6 and 13-16 i n Table 2).
c u l t u r e s a l w a y s show m e t e - c l e a v a g e
pathway
f o r catechol oxidation a t the i n i t i a l stage o f c u l t i v a t i o n . (rather long)
2.
t i m e i t disappeared b o t h f o r mixed,
I n some
and m o n o - c u l t u r e s .
Substrate mixture degradation I n s p i t e o f some i n e x p l i c a b l e f e a t u r e s i n s i n g l e s p e c i e s c u l -
t u r e behavior,
t h a t oxidase phenol,
guiacol,
t r i e d t h e m i x e d c u l t u r e o f Pseudomonas aD. them.
+
and naphthalene, N o c a r d i a sp.
we
t o treat
We assumed t h e enzyme k i n e t i c s o f d i f f e r e n t m i c r o o r g a n i s m s t o
d i f f e r f o r v a r i o u s a r o m a t i c h y d r o c a r b o n s . So, complement each o t h e r .
t h e c u l t u r e s should
-
159
-
TABLE 2 Parameters o f phenol d e g r a d a t i o n process i n chemostat ~
NN
Dilution r a p ]
-
Substrate input
hdil
Biomass
output
[mg/l]
1
0.07
1000
0.2
594
2
0.07
1000
0.2
600
Cleavage pathway, absorbtion p e a k [nm]
Economical coef f i c i e n
b/gJ 0.59 0.60
375 meta-
3
0.07
1000
0.3
630
4
0.07
1000
0.2
490
5
0.07
1000
0.2
550
6
0.07
80 0
0.2
510
7
0.07
1000
0.45
440
8
0.08
800
0.2
478
9
0.09
800
0.6
474
10
0.12
1000
0.8
58 2
11
0.14
BOO
0.5
400
12
0.15
1000
0.3
590
13
0.17
880
19.0
454
14
0.17
800
0.5
467
15
0.19
800
21.0
384
0.49
16
0.20
1000
0.5
700
0.70
17
0.23
1000
0.4
510
18
0.26
1000
1.5
672
19
0.27
800
0.9
512
20
0.27
800
1.0
21
0.07
800
0.36
0.63 375
0.55 375 380
0 . 1 h-'.
0.59 0.58 meta-
0.50 0.59 0.50
375
0.58
375
0.51 0.67 0.64
450
410
0.56
610
s m a l l meta
0.76
9/11 and n a p h t h a l e n e because o f t h e e x t r e -
The d i l u t i o n r a t e was e q u a l t o
T h i s i s t h e c r i t i c a l f l o w f o r N o c a r d i a sp.,
i s u t i l i z e d moat e f f i c i e n t l y ;
0.44 0.60
375
t h i s i s the calculated concentration,
mely poor s o l u b i l i t y o f naphthalene).
0.63 ortho-
The m i x e d a u b s t r a t e c o n t a i n e d p h e n o l ( 0 . 5
( 2 g/l);
0.49
that culture didn't
a t which phenol
o x i d i z e t h e naph-
thalene. F o r t h e e x p e r i m e n t we u s e d Pseudomonas ap.
naphthalene.
adapted t o u t i l i z e
The a d d i t i o n o f p h e n o l t o t h e n a p h t h a l e n e u t i l i z i n g
c u l t u r e r e s u l t e d b o t h i n t h e i n t e n s i v e brown c o l o r appearance i n t h e culture,
a n d an i n c r e a s e d r e s i d u a l p h e n o l c o n c e n t r a t i o n .
-
160
-
The m i x e d c u l t u r e was a b s o l u t e l y c o l o r l e s s ,
but the residual
c o n c e n t r a t i o n s o f b o t h p h e n o l and n a p h t h a l e n e were l e s s t h a n 0.2 mg/l.
I t s h o u l d be s t r e s s e d ,
t h a t N o c a r d i a sp.
successfully u t i l i z e d
phenol a t r e l a t i v e l y h i g h i n p u t concentrations o f the former,
with rather great d i l u t i o n rates, The No.
and
mixed c u l t u r e .
2 m e t h o d was i l l u s t r a t e d w i t h t h e f o l l o w i n g e x p e r i m e n t .
The n a p h t h a l e n e o x i d i z i n g c u l t u r e ,
i s o l a t e d from wastewater d i s c h a r -
ged from p h e n o l i c w a t e r b i o l o g i c a l t r e a t m e n t p l a n t , i n t o a n a e r o t a n k i n a s p e c i f i c way.
As s r e s u l t ,
d u a l p h e n o l c o n c e n t r a t i o n d e c r e a s e d a s much a s 30
was i n o c u l a t e d
t h e average r e s i -
X,
b u t the process
s t a b i l i t y o f a sewage b i o l o g i c a l t r e a t m e n t e n h a n c e d ( t h e v a r i a t i o n c o e f f i c i e n t d e c r e a s e d 2.8
4
times).
8
These d a t a s r e shown i n F i g .
12
4.
20
16
MONTMS
F i g . 4. D y n a m i c s o f p h e n o l a t t h e o u t p u t o f b i o l o g i c a l p u r i f i c a t i o n o f wastewater i n coke and b y - p r o d u c t p l a n t . Arrow i n d i c a t e sowing o f nsphth a l e n e - o x i d i z i n g b a c t e r i a Thus,
t h e e x p e r i m e n t s c a r r i e d o u t show t h a t t h e c h a n g e s i n t h e
q u a l i t y a n d number c o m p o s i t i o n o f a m i c r o b i a l c o m m u n i t y , n a l l y s t a b l e c o n t i n u o u s c u l t u r e have a p o s i t i v e e f f e c t , community r e a r r a n g e .
with functioi.e.
make t h e
-
161
-
Now, w e ’ l l d i s c u s s t h e s e i n e x p l i c a b l e f e a t u r e s m e n t i o n e d a b o v e i n t h e s i n g l e species c u l t u r e behaviour. DlSCUSSION
Both b i o c h e m i s t r y and g e n e t i c s of t h e m i c r o b i a l o x i d a t i o n o f
it is
a r o m a t i c h y d r o c a r b o n s are r a t h e r well s t u d i e d . N e v e r t h e l e s s ,
s t r a n g e , t h a t t h e e v i d e n c e on t h e c a t a b o l i s m m u l t i r o u t i n e s s are n o t i n c l u d e d i n t o w a s t e w a t e r t r e a t m e n t , when c o n s i d e r e d a t t h e p o p u l a t i o n and community l e v e l s .
T h i s e v i d e n c e m a k e s us d i s c u s s t h e p r o -
blem of a m u l t i r o u t i n e s s . Jones e t a l . I 2 1 found t h a t t h e r e g i o n o f m i c r o b i a l growth lim i t a t i o n a s w e l l a s i t s i n h i b i t i o n b y p h e n o l h a d t h e i r s p e c i f i c economic c o e f f i c i e n t s . These r e g i o n s a r e n ’ t o f any i n t e r e s t and have n o g e n e r a l b o u n d a r y , a s i t i s shown i n F i g . 5 [2, 3 1 . Assume t h e g r o w t h r a t e d e p e n d e n c e on s u b s t r a t e c o n c e n t r a t i o n b e a s s h o w n i n F i g . 6 . Q u i t e a few r e s e a r c h e r s s u p p o r t t h i s a s s u m p t i o n , T h e maximum o f regions.
p ( S ) belong t o both t h e l i m i t a t i o n , and i n h i b i t i o n
There is no corresponding p o i n t for i t i n Fig.
5, or a
t r a n s i t i o n a r e a from l i n e 1 t o l i n e 2 . Hence, t h e b a s i c assumption presented a t Figs.
5, 6 should be changed.
The p o s s i b l e c h a n g e f o r
i t i s shown i n F i g s . 5 a n d 6 i n d a s h l i n e s .
0
1
2
5
3
6
7 1l p
f h-’I
F i g . 5. R e c i p r o c a l p l o t o f g r o w t h y i e l d a n d g r o w t h r a t e w i t h p h e n o l limitation, 2 inhibition, oxidizing b a c t e r i a (from /3.2/). 1 3 h y p o t h e t i c a l l i n e o f p a s s a g e from l i m i t a t i o n t o i n h i b i t i o n
-
-
-
- 162
-
Taking i n t o account k i n e t i c features o f t h e aromatic hydrocarone c a n e a s i l y show t h e e x i s t e n c e o f t h e a r e a o f t h e
bons o x i d a t i o n , bi-stability i n Fig.
i n behaviour.
I t ’ s evident,
i n part,
f r o m o u r d a t a shown
3 a n d T a b l e 2.
P o s i t i o n 7 i n T a b l e 2 shows t h e r e s u l t s t o b e s t a t i s t i c a l l y d i f f e r e n t from t h o s e p r e s e n t e d i n p o s i t i o n s 1-6. meta-cleavage
o f t h e c a t e c h o l wasn’t
s i t i o n 7 v s p o s i t i o n s 1-6
The c o m p a r i s o n o f
shows t h a t m e t a - c l e a v a g e
l e s s output phenol concentration; p o s i t i o n 21,
observed.
I n t h i s case t h e po-
pathway p r o v i d e s
t h e aame shows t h e e v i d e n c e s i n
obtained from another s e r i e s o f long-time
experiments.
W
t-
a
n
SUBSTRATE
F i g . 6. S p e c i f i c growth r a t e vs. s u b s t r a t e c o n c e n t r a t i o n (hypothemeta-cleavage and 2 - o r t h o - c l e a v a g e t i c a l curves; f o r example, 1 pathways o f phenol, dash l i n e - o t h e r h y p o t h e s i s )
-
O n t h e o t h e r hand, p o s i t i o n s 1 3 - 1 6 d o n ’ t 2,3-oxygenase way)
(which i s the f i r s t -
activity
o f r e g a r d l e s s s i g n i f i c a n t v a r i a t i o n s i n t h e o u t p u t p h e n o l con-
centration.
Probably,
enzymes ( n a m e l y ,
2,3-
we j u s t o b s e r v e d a v a r i a t i o n o f t h e d i f f e r e n t and 1,Z-oxygenase)
complete e l i m i n a t i o n of ta, Fig.
show t h e d e c a y o f
s t e p enzyme o f m e t a - p a t h -
activities,
t h e c a t a b o l i c pathway.
contrary t o
Our e x p e r i m e n t a l d a -
unfortunately, f a i l s t o prove i t . One c a n e a s i l y d e s c r i b e t h e a r e a s o f m u l t i - s t a b l e
2 ( e a r d i a spe.)
and F i g .
3 (Pseudomonas s p . ) .
behaviour i n
Genetic features
o f m i c r o o r g a n i s m s used t o degrade t h e a r o m a t i c h y d r o c a r b o n s have resulted i n the following. t u r e changed meta-pathway
The p h e n o l o x i d i z i n g Pseudornonas sp. f o r ortho-pathway
shown i n p o s i t i o n 2 1 o f T a b l e 2.
i n some t i m e ;
cul-
that’s
When o x i d i z i n g n a p h t h a l e n e ,
we’ve
-
163
-
i n o c u l a t r e d t h e clones showing a g r e a t a c t i v i t y o f i n t o a culture,
b u t ortho-pathway
2,3-oxygenase
h a s b e g u n t o d o m i n a t e soon.
The d e l a y o f t r a n s i t i o n f r o m m e t e - c l e a v a g e
i n t o the orthoclea-
vage p a t h w a y i s e v i d e n c e o f t h e i n t e r a c t i o n b e t w e e n t h e c e l l s m i c r o b i a l p o p u l a t i o n and s u b s t r a t e b i o d e g r a d a t i o n p l a s m i d s , t h e enzyme a c t i v i t i e s t u r n o u t . e x p e r i m e n t s 1-7,
rather than
T h i s l a t t e r c o u l d be observed i n
T a b l e 2 , t h a t were c a r r i e d o u t as a p i l o t e x p e r i -
ment. Hence,
i t i s evident,
t h a t some f e a t u r e s o f a s u b s t r a t e c a t a -
b o l i s m ( a t i n t r e c e l l u l a r l e v e l ) h a v e a s t r o n g i m p a c t on t h e w a s t e w a t e r p r o c e s s i n g on t h e w h o l e . REFERENCES
1 2 3
M a n u a l o f M e t h o d s f o r G e n e r a l B a c t e r i o l o g y . P. G e r h a r d t e.a. ed., W a s h i n g t o n 1 9 8 1 . G.L. J o n e s , F. J a n s e n and A . J . McKay, S u b s t r a t e I n h i b i t i o n o f t h e g r o w t h o f b a c t e r i u m NCIB 8250 b y p h e n o l . L . Gen. M i c r o b i o l . , 7 4 ( 1 9 7 3 ) N I , p. 139-148. Yu.L. G u r e v i c h , S t a b i l i t y a n d R e g u l a t i o n o f G r o w t h i n M i c r o b i a l P o p u l a t i o n s . N o v o s i b i r s k , Nauka, 1 9 8 4 , 165 p .