Biodegradation of 2,4-dichlorophenol by activated sludge microorganisms

Wat. Res. Vol. 23, No. 11, pp. 1439-1442, 1989 Printed in Great Britain. All rights reserved

0043-1354/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press plc

BIODEGRADATION OF 2,4-DICHLOROPHENOL BY ACTIVATED SLUDGE MICROORGANISMS J. CHUDOBA I t s , J. ALBOKOV,~ l, B. LENTGE 2 a n d R. Kf3MMEL2 ~Department o f Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Suchb~tarova 5, Prague 16628, Czechoslovakia and 2Department of Chemistry, CarlSchorlemmer Technical University, Otto-Nuschke-Strasse, Merseburg, 4200, G.D.R.

(First received October 1988; accepted in revised form May 1989) AMtract--Experiments with the degradation of 2,4-dichlorophenol (2,4-DCP) were carried out in completely-mixed systems at constant volumetric loading (By) and variable solids retention time (SRT). A mixed substrate consisted o f 2,4-DCP (20% of total COD) and methanol (MA, 80% o f total COD). The maximum specific rate of 2,4-DCP removal, qm~, related to the total biomass in the cultivation system, increased with the decreasing SRT from 0.0118 h -~ at the SRT of 10.8 d up to 0.0783 h -t at the SRT of 0.6 d. The following values of kinetic constants o f microorganisms responsible for the degradation of 2,4-DCP were found (20°C and p H = 7 . 5 ) : YCOD=0.37, Ks.coD=0.6mgl -j, q ~ x = 0 . 2 3 3 h -l, b = 0.036 h -I, /tm~x = 0.086 h -1 and Ox.~ = 0.83 d.

Key words--biodegradation, kinetic constants, activated sludge, 2,4-dichlorophenol

NOMENCLATURE

EXPERIMENTAL

b = biomass decay rate constant (T - I ) B v = volumetric loading (M L -3 T -I ) 2,4-DCP = 2,4-dichlorophenol K s = half-velocity constant (M L - 3) M A = methanol qmax= maximum specific substrate removal rate (T- 1) qv = volumetric substrate removal rate (M L -a T -~ ) qv.pot=potential volumetric substrate removal rate (M L -3 T - l ) SRT = solids retention time (T) So = initial substrate concentration (M L -3) S r = substrate removed (ML -3) X = biomass concentration (M L -3) Xocp = concentration of 2,4-DCP-degrading organisms (M L -3) XMA = concentration of methanol-degrading organisms (M L -3) Y = yield constant (--) ~t = constant (--) fl = constant (T) 0, = solids retention time (T) 0~.cr = critical value of SRT (T) 0x. mi, = minimum value of SRT (T) ~max = maximum specific growth rate (T-I).

Experiments were carried out with a mixture of 2,4-diclorophenol (2,4-DCP) and methanol (MA). Methanol was used for the simple reason that it does not provoke filamentous bulking in completely-mixed reactors (Chudoba et al., 1985). The composition o f the two-component substrate is given in Table 1. The substrate solution was dosed by means of a peristaltic pump. Two completely-mixed systems were operated at the technological parameters given in Table 2. Both systems were inoculated with activated sludge from the Prague Central Sewage Treatment Plant. An adaptation period lasted 4 weeks and consisted in a gradually increasing influent concentration of 2,4-DCP up to 88.6mgl -~ (100mgl -t as COD). Values o f the potential volumetric removal rate, qv,pot, the yield coefficient, Y, and the half-velocity constant, K s, were determined by a respirometric method ((~ech et aL, 1984). The solids retention time (SRT) was calculated as described previously (Chudoba et al., 1989). Table 1. Composition of the substrate used in the experiments. All components were dissolved in tap water Component 2,4-Dichlorophenol Methanol KH2PO 4 NaHCO3 NH4HCO 3

INTRODUCTION A m e t h o d o f e n a b l i n g t h e d e t e r m i n a t i o n o f kinetic c o n s t a n t s o f a c t i v a t e d sludge m i c r o o r g a n i s m s r e s p o n sible for t h e d e g r a d a t i o n o f x e n o b i o t i c s was develo p e d a n d p u b l i s h e d in t h e a c c o m p a n y i n g p a p e r ( C h u d o b a et al., 1989). T h e a i m o f t h e p r e s e n t p a p e r is to s h o w t h a t the m e t h o d c a n also be a p p l i e d to p r i o r i t y p o l l u t a n t s . 2 , 4 - D i c h l o r o p h e n o l , w h i c h is k n o w n to be easily d e g r a d a b l e (Pitter, 1975; B e l t r a m e et al., 1982; W a t k i n a n d E c k e n f e l d e r , 1989), was c h o s e n f o r t h e e x p e r i m e n t s as a r e p r e s e n t a t i v e o f the priority pollutants.

PROCEDURES

Concentration (mg I ~) As compound As COD 88.6 266.7 25 84 83

100 400

Table 2. Technological parameters of the activated sludge systems. 0x varied during the experiments as shown in Table 3 Parameter Units Values Aeration volume Volume of settlers Hydraulic retention time Volumetric loading with total COD Volumetric loading with 2,4-DCP COD Volumetric loading with MA COD Recirculation ratio 1439

I 1 h kgm-3d t mg 1- ~h i mgl-~h t --

4 1.5 12.0 1.0 8.3 33.3 1.0

J. CHUBODA et al.

1440

Table 3. Results obtained from the experiments. All values are expressed in COD units 0~ (d)

Compound 2,4-DCP

q~,pot (mgl -t h -I)

10.8 6.9 5.9 3.8 1.7 0.6 • 10.8 6.9 1.7 0.6 •

MA

qm, (h -j x 103)

19.8 + 4.2 19.3+4.7 14.2_+4.1 14.7 _+2.2 12.0_+4.4 6.5? 119_+ 17 113_+11 80+35 27~"

K, (mgl -t)

11,8 + 1.8 15.3_+4,3 12.0_+4.4 19.8 _+2.6 30.8_+9.9 78.3 82_+20 112_+5 205-+74 268

YCOD (--)

0.64 _-4-0.26 0.55_+0.24 0.55_+0.48 0.47 _+0.28 0,57_+0.25 -2.3_+0.43 2.2_+0.20 3.1 -+ 1.24 0.6 _+0.37

0.36 + 0.03 0.41_+0.03 0.35_+0.02 0.35 _+0.02 0.39_+0.04 -0.37_+0.11 0.24_+0.02 0.36_+0.05 0.40 _+0.04

*0 = 14.4 h (chemostat). tCalculated according to the formulae: q~.pot= (So - $2TOD)/0 (TOD = theoretical oxygen demand). Table 4. Kinetic constants of the microorganisms responsible for the degradation of 2,4-DCP and MA Compound

Ycoo (--)

2,4-DCP MA

0.37 0.32

K~coo (mgl -I)

qmax (h -l)

b (h i)

/~,~ (h-t)

0~,¢, (d)

0.6 2.5

0.233 0.479

0,036 0.086 0,039 0.153

0.83 0.37

Analytical methods 2,4-Dichlorophenol was determined by means of u.v. speetrophotometry after being previously distilled out from an acidified solution. The COD was determined by a dichromate semimicromethod (Jirka and Carter, 1975). The concentrations of SS in the reactors and secondary effluents were determined by means o f millipore filters with a mean porosity o f 0.6 pm.

i n c r e a s i n g f r o m 0.020 to 0 . 0 3 2 h -] with the S R T d e c r e a s i n g f r o m 10.7 to 2.1 d a n d W a t k i n a n d E c k e n felder (1989) gave a value o f 0 . 0 2 7 h - l ( 0 . 6 4 8 d -1) w i t h o u t specifying t h e S R T o f the m i x e d c u l t u r e used. All kinetic c o n s t a n t s f o u n d for t h e m i c r o o r g a n i s m s d e g r a d i n g 2 , 4 - D C P a n d M A are s u m m a r i z e d in Table 4. T h e c o n s t a n t s were c a l c u l a t e d by m e a n s o f the following e q u a t i o n s ( C h u d o b a et al., 1989):

RESULTS AND DISCUSSION

qv

1

--=~+ T h e results are s u m m a r i z e d in T a b l e 3 a n d g r a p h ically p r e s e n t e d in Figs 1 a n d 2. It c a n be seen in Fig. 1 t h a t t h e kinetic b e h a v i o u r o f 2 , 4 - D C P is similar to t h a t f o u n d w i t h t h e o t h e r x e n o b i o t i c s s t u d i e d ( C h u d o b a et al., 1989). T h e m a x i m u m specific rates o f 2 , 4 - D C P r e m o v a l , qm~, i n c r e a s e d f r o m 0.0118 h -~ at t h e S R T o f 10.8 d u p to 0.078 h - j at t h e S R T o f 0.6 d. T h e s e values a r e in g o o d a g r e e m e n t with t h o s e p u b l i s h e d in t h e literature. Pitter (1975) f o u n d 0.0105 h -~ w i t h t h e m i x e d c u l t u r e c u l t i v a t e d at the S R T o f 5 d, B e l t r a m e et al. (1982) f o u n d values

(1)

w h e r e ~ = b/Yqmax a n d fl = 1/Yqmax.

qmax=l/r'fl

(2)

b --- ~/#

(3)

0 .... ~--~/(1 - ~ )

(4)

flmax"~- Yqmax"

(5)

30O

120t 20

I00 |

•

16

80

"r.C: ";'--

L: so~

200

~0 12

r

250

I00t

-

60 "~

"

8

40

G"

4

20

' ~'~'

"-:

~ ~ ~',~ ®x ,d

Fig. 1. Dependence of qv.pot (I) and q ~ (2) on 0x obtained for 2,4-dichlorophenol. (3) B~ = 8.3 mg 1-I h -t.

150

:'°I/ ,°I o

I

~

"7 "~ N

| I00 0"

SO P

;

• ]

~'

~','*

®x , d Fig. 2. Dependence of qv pot (1) and qmax (2) on 0x obtained for methanol, i3) Bv = 33.3 mgl -I h -L.

Biodegradation of 2,4-dichlorophenol

1441

Table 5. Steady-stateconcentrationsof total biomassmeasuredand responsiblemicroorganisms calculatedaccordingto equation (6) X°ce 100

X,o,

XM~A100

XMA

X ~ r + XMA 100

0,

X,o,

xocp

X,o,

(d)

(gl -] )

(g 1-')

(%)

(gl -I )

(%)

X,o, (%)

10.9 6.9 5.9 3.8 1.7

1.65 1.27 1.08 0.64 0.34

0.077 0.073 0.027 0.066 0.051

4.7 5.7 6.7 10.3 15.0

0.249 0.237 0.228 0.213 0.168

15.1 18.7 21.1 33.3 49.4

19.8 24.4 27.8 43.6 64.4

Xoce= concentration of DCP-utilizingorganisms. XuA = concentration of methanol-utilizingorganisms. A critical value of the SRT was found to be 0.83 d for 2,4-DCP. The values of qmaxand #m~xindicate that the microorganisms responsible for the degradation of 2,4-DCP grow faster than those responsible for the degradation of morpholine, sulphanilic acid and nitrilotriacetic acid (Chudoba et al., 1989). Examples of the graphical presentation of equation (1) are given in Fig. 3. The increase of the qmx values with the decreasing SRT can be explained by a gradual washing out of other slow-growers from the system, especially predators, which led to the enrichment of the mixed culture with responsible microorganisms. This is supported by the following facts. The steady-state concentrations of microorganisms responsible for the degradation of 2,4-DCP and MA can be calculated from the following equation (Lawrence and McCarty, 1970) X =

YO~S~

(6)

o(1 + box)

where S r is the value of the tested substrate removed, Y is the yield coefficient and b is the decay coefficient of the responsible microorganisms. Using the values of both constants given in Table 4, one can easily estimate the steady-state concentrations of the responsible microorganisms. The results are given in Table 5. It can be seen in Table 5 that with the decreasing SRT the proportions of both groups of responsible microorganisms in the total biomass increased.

I

2

A course of the gradual washing out of rotifers from the cultivation system with the decreasng SRT is presented in Fig. 4. The decrease of rotifers resulted in the increase of Holotrich. The data in Table 5 further show that even at the SRT of 1.7 d, the sum of XDCP and XMA made only 64% of the total biomass. The rest of the biomass consisted of predators and, maybe, other bacterial species. During the experiments, which lasted 6 months, there arose a problem of mite predation. In both aeration tanks, a mass growth of mites (Acarina) appeared. The mites reduced a number of 2,4-DCPdegrading microorganisms to such an extent that the effluent concentrations of 2,4-DCP increased from 0.5 up to 8 mg 1- t. The problem was solved by a batch dose of 2,4-DCP so that its actual concentration in both aeration tanks was 125 m g l -~. All mites were killed and both systems restored after 1 week. The values of the decay coefficient, b, obtained for the microorganisms responsible for the degradation of 2,4-DCP and M A are too high, namely 0.036 h (0.864 d - I ) and 0.039 h - l (0.936 d - ~), respectively, which is believed to be due to the predation. Lawrence and McCarty (1970) quote a value of 0.05 d - i. The measured effluent concentrations of 2,4-DCP are present in Table 6. It can be seen that most

N

0.8

i

0.6

O"

o"

d

0.4 0.2

02 0

1 / 0 x , d "1

Fig. 3. Plots of the qv/qv.po, ratio vs l/0x. (I) 2,4-DCP and (2) MA.

2

4

6

8

10

®~, d

Fig. 4. Dependence of the counts of Rotatoria (I) and Holotricha (2) in 1 ml of mixed liquor on the SRT.

J. CttUaODA et al.

1442

Table 6. Concentrations of 2,4-DCP and values of soluble COD measured in effluents 0x

2,4-DCP

2,4-DCP as COD

Soluble COD

(d)

(rag 1- i )

(rag I- i )

(rag 1- i )

10.8 6.9 5.9 3.8 1.7 0.6

0.48 + 0.23 0.56 _ 0.24 0.34 + 0.26 0.07_+0.03 0.37 _ 0.28 5.70 _+2.30

0.54 0.63 0.38 0.08 0.42 6.44

34 _ 14 38 _ 21 40 __. 14 44_+14 45 _+ 12 102 _+52

Table 7. Effluent concentrations of 2,4-DCP as measured and predicted according to equations (7) and (8) Measured Calculated values (mg I- ~) values (mgl -I) Equation (7)* Equation (7)f Equation (8) 10.8 6.9 5.9 3.8 1.7 0.6

0.48 0.56 0.34 0.07 0.37 5.70

0.44 0.49 0.51 0.61 1.21 -2.80

-2.0

0.41

- 1.75 - 1.04 - 1.21 -0.93 -0.89

0.40 0.74 0.68 1.16 9.9

*Constant values of q,,.~ and b as given in Table 4. fVariable qmaxas given in Table 3 and b = 0.05 d i.

organics present in the effluents are microbial products and not remaining 2,4-DCP. The kinetic constants obtained make it possible to predict the effluent concentrations of 2,4-DCP. The prediction was carried out according to the equation derived from a mass balance of biomass (Lawrence and McCarty, 1970) ScoD -

Ks(l +

box)

Ox(Yqmax-b)-

I

(7)

maximum specific rate of 2,4-DCP removal, qm~, related to the total biomass in the cultivation system, increased with the decreasing SRT from 0.0118 h -1 at the SRT of 10.8d up to 0.0783h -1 at the SRT of 0.6 d. The following values of kinetic constants of microorganisms responsible for the degradation of 2,4-DCP were found (20°C, pH = 7.5): Ycoo= 0.37, Ks.COD 0.6 mg 1-1, qmax= 0.233 h - 1, b = 0.036 h-l, ~max 0.086 h -1 and 0.... = 0.83 d. A microorganism capable of degrading 2,4-DCP was isolated and at present identification procedures are being carried out. ~-

a n d a c c o r d i n g to the e q u a t i o n derived f r o m a m a s s b a l a n c e o f s u b s t r a t e ( C h u d o b a et al., 1989) SCOD -~-

~--

So -- qv, pot0 -- K s _ x / ( K s + qv, pot0 - S 0)2 + 4 K s So

2

REFERENCES

(8) The predicted effluent concentrations are summarized in Table 7. It can be seen that equation (7) can be used for the prediction only with the values qm~x and b obtained for the responsible microorganisms and only in the region above 0x,cr. Equation (8) predicts the effluent concentrations at all values of the SRT. SUMMARY

Experiments with the degradation of 2,4-dichlorophenol (2,4-DCP) were carried out in completelymixed systems at constant volumetric loading (By) and variable solids retention time (SRT). A mixed substrate consisted of 2,4-DCP (20% of total COD) and methanol (MA, 80% of total COD). It has been found that the kinetic behaviour of 2,4-DCP is similar to that observed with the other xenobiotics during the previous experiments. The

Beltrame P., Beltrame P. L., Carniti P. and Demetrio P. (1982) Kinetics of biodegradation of mixtures containing 2,4-dichlorophenol in a continuous stirred reactor. Wat. Res. 16, 429-433. (~ech J. S., Chudoba J. and Grau P. 0984) Determination of kinetic constants of activated sludge microorganisms. Wat. Sci. TechnoL 17, 259-272. Chudoba J., (~ech J. S. and Chudoba P. (1985) The effect of aeration tank configuration on nitrification kinetics. J. Wat. Pollut. Control Fed. 57, 1078-1083. Chudoba J., Albokov~t J. and ~ech J. S. (1989) Determination of kinetic constants of activated sludge microorganisms responsible for degradation of xenobiotics. Wat. Res. 23, 1431-1438. Jirka A. M. and Carter J. J. (1975) Micro semi-automated analysis of surface and wastewaters for chemical oxygen demand. Analyt. Chem. 47, 1397-1402. Lawrence A. and McCarty P. L. (1970) Unified basis for biological treatment design and operation. J. sanit. Engng Div., Am. Soc. cir. Engrs 96, 757-778. Pitter P. (1975) Determination of biological degradability of organic substances. Wat. Res. I0, 231-235. Watkin A. T. and Eckenfelder W. W. Jr (1989) A technique to determine unsteady-state inhibition kinetics in the activated sludge process. Wat. Sci. Technol. 21,593~02.