Determination of Monod Kinetics of Toxic Compounds by

Chapter 9 D e t e r m i n a t i o n of M o n o d K i n e t i c s o f Toxic C o m p o u n d s by R e s p i r o m e t r y for Structure—Biodegradability R e l a t i o n s h i p s 1

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Downloaded by FUDAN UNIV on February 19, 2017 | http://pubs.acs.org Publication Date: March 8, 1990 | doi: 10.1021/bk-1990-0422.ch009

Sanjay Desai , Rakesh Govind , and Henry Tabak

1Department of Chemical Engineering, University of Cincinnati, Cincinnati, OH 45221 Risk Reduction Engineering Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268 2

The key to the evaluation of the fate of toxic organic chemicals in the environment is dependant on evaluating their susceptibility to biodegradation. Biodegradation is one of the most important mechanisms in controlling the concentration of chemicals in an aquatic system because toxic pollutants can be mineralized and rendered harmless. Experiments using an electrolytic respirometer have been conducted to collect oxygen consumption data of toxic compounds from the list of RCRA and RCRA land banned chemicals (phenols and phthalates). The estimation of Monod kinetic parameters were obtained for all the compounds by a graphical method. The f i r s t order kinetic constants for the substituted phenols were related to the structure of the compounds by the group contribution method. It has been estimated that 50,000 organic chemicals are commercially produced in the United States and a large number of new organic chemicals are added to the production l i s t each year (1). The presence of many of these chemicals in the environment could be attributed to inadequate disposal techniques. Since many of these hazardous chemicals can be detected in wastewater, their fate in wastewater treatment system is of great interest. Of many factors that affect the fate of these compounds, microbial degradation is probably the most important. Biodégradation can eliminate hazardous compounds by biotransforming them into innocuous forms, degrading them 0097-6156/90/0422-0142$06.00/0 © 1990 American Chemical Society Tedder and Pohland; Emerging Technologies in Hazardous Waste Management ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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by m i n e r a l i z a t i o n t o carbon d i o x i d e and water. P r e d i c t i o n of the c o n c e n t r a t i o n of t o x i c chemicals i n n a t u r a l and engineered environments and a s s e s s i n g t h e i r e f f e c t s on human and other s p e c i e s due t o p o s s i b l e exposure r e q u i r e s information on the k i n e t i c s of biodégradation. Howard e t a l . (2) have reviewed the d i f f e r e n t techniques f o r measuring k i n e t i c s of biodégradation. Due t o the l a r g e number of chemicals, i t i s imperative t h a t the method s e l e c t e d should not be l a b o r i n t e n s i v e and time consuming. A l s o , the parameters obtained should be i n t r i n s i c ; t h a t i s , dependant only on the nature of the compound and the degrading m i c r o b i a l community and not on the r e a c t o r system used f o r data c o l l e c t i o n . I f t h i s c o n d i t i o n i s s a t i s f i e d , then the parameters obtained can be used f o r any r e a c t o r c o n f i g u r a t i o n and can be used i n mathematical models t o estimate the f a t e of the t o x i c chemicals. I t i s possible to obtain i n t r i n s i c k i n e t i c parameters from batch experiments f o r a s i n g l e s u b s t r a t e , provided t h a t the i n i t i a l c o n d i t i o n s are p r e c i s e l y known (3,4). Gaudy e t a l . (5) have shown t h a t i t i s p o s s i b l e t o use oxygen consumption t o determine s u b s t r a t e removal because oxygen u t i l i z a t i o n during m i c r o b i a l energy p r o d u c t i o n . So, i t i s p o s s i b l e t o o b t a i n i n t r i n s i c k i n e t i c parameters from oxygen consumption data from s i n g l e batch experiments. Grady e t a l . (6) a l s o demonstrated t h a t the values of the parameters obtained from oxygen uptake are i n general agreement with values determined by more t r a d i t i o n a l methods* The measurement of oxygen consumption i s one of the o l d e s t means of a s s e s s i n g b i o d e g r a d a b i l i t y , and respirometry i s one technique f o r measuring oxygen consumption. In r e s p i r o m e t r i c methods s u b s t r a t e samples, along with biomass, are kept i n contact with a gaseous source of oxygen. Oxygen uptake by microorganisms over a p e r i o d of time i s then measured by change i n volume or pressure of the gas phase. An a l k a l i i s i n c l u d e d i n the apparatus t o absorb carbon d i o x i d e produced d u r i n g biodégradation. Samples are u s u a l l y incubated a t constant temperature and are kept away from l i g h t , but the procedures vary with d i f f e r e n t instruments. E l e c t r o l y t i c respirometry i s the most commonly employed method f o r biodégradation s t u d i e s . G e n e r a l l y i t i s used f o r biochemical oxygen demand (BOD) determination with the exception of s t u d i e s by D o j l i d o (7), Oshima e t a l . (8), Gaudy et a l . (9) and Grady et a l . (6), who used i t f o r determination of biodégradation k i n e t i c s . The data c o l l e c t i o n i s automatic with e l e c t r o l y t i c respirometry, so manual c o l l e c t i o n and a n a l y s i s of samples i s not r e q u i r e d t o f o l l o w the course of s u b s t r a t e degradation. Since the respirometer can be automated, the l e n g t h of the experiment i s l e s s important and thus unacclimated biomass can be used i n the experiments. The o b j e c t i v e of t h i s study was t o f i n d degradation r a t e s of the Resource Conservation and Recovery Act

Tedder and Pohland; Emerging Technologies in Hazardous Waste Management ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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EMERGING TECHNOLOGIES IN HAZARDOUS WASTE MANAGEMENT

(RCRA) and RCRA land banned compounds with the use o f mixed c u l t u r e s obtained from a wastewater treatment p l a n t , so t h a t the r a t e s obtained i n the l a b o r a t o r y could be used t o p r e d i c t degradation r a t e s i n the environment. Chemicals were obtained from A l d r i c h Chemical Company (Milwaukee, WI) and were reported 99+% pure. The phenols and p h t h a l a t e s t e s t e d were phenol, o - c r e s o l , m-cresol, pc r e s o l , 2,4-dimethyl phenol, dimethyl p h t h a l a t e , d i e t h y l p h t h a l a t e , d i p r o p y l phthalate, d i b u t y l p h t h a l a t e and b u t y l benzyl p h t h a l a t e .


Experimentation E l e c t r o l y t i c respirometry s t u d i e s were conducted u s i n g an automated continuous oxygen measuring V o i t h Sapromat B-12 e l e c t r o l y t i c respirometer (Voith-Morden, Milwaukee, WI). T h i s system c o n s i s t s o f a temperature c o n t r o l l e d water bath which contains the measuring u n i t s , a recorder f o r d i g i t a l indication, a p l o t t e r for d i r e c t presentation of the oxygen uptake curves o f s u b s t r a t e s , and a c o o l i n g u n i t f o r c o n d i t i o n i n g and continuous r e c i r c u l a t i o n o f water bath contents. The system used had 12 measuring u n i t s each connected t o the recorder. Each u n i t , as shown i n F i g u r e 1, c o n s i s t e d o f a r e a c t i o n v e s s e l A, with a carbon d i o x i d e absorber (soda lime) mounted i n a stopper, an oxygen generator B, and a pressure i n d i c a t o r C. Interconnected by hoses, the v e s s e l s form a s e a l e d measuring system so t h a t barometric pressure f l u c t u a t i o n s do not adversely a f f e c t the r e s u l t s . The magnetic s t i r r e r i n the sample t o be analyzed provides vigorous a g i t a t i o n , thus ensuring e f f e c t i v e gas exchange. Microorganism a c t i v i t y i n the sample c r e a t e s a pressure r e d u c t i o n t h a t i s recorded by the pressure i n d i c a t o r which c o n t r o l s both the e l e c t r o l y t i c oxygen generation and p l o t t i n g o f the measured v a l u e s . The C O 2 generated i s absorbed by soda lime. The N 2 / O 2 r a t i o i n the gas phase above the sample i s maintained constant throughout the experiment by an on/off feedback c o n t r o l loop. As oxygen i s depleted, pressure r e d u c t i o n i s created i n the sample and, as a r e s u l t , the l e v e l o f the s u l p h u r i c a c i d i n the pressure i n d i c a t o r r i s e s and comes i n contact with a platinum e l e c t r o d e . T h i s completes the c i r c u i t and t r i g g e r s the generation o f oxygen by e l e c t r o l y s i s . The e l e c t r o l y t i c c e l l provides the r e q u i r e d amount o f oxygen t o the r e a c t i o n v e s s e l by e l e c t r o l y t i c d i s s o c i a t i o n o f a C U S O 4 H2SO4 s o l u t i o n thus a l l e v i a t i n g the negative pressure. As the l e v e l o f the e l e c t r o l y t e i n the pressure i n d i c a t o r decreases, the contact with the e l e c t r o d e i s broken. T h i s switches o f f the e l e c t r o l y t i c c e l l . The amount o f oxygen s u p p l i e d t o the sample i s recorded d i r e c t l y i n m i l l i g r a m s per l i t e r by the recorder. The r e c o r d e r i s connected t o a microcomputer which records data from the measuring u n i t s every 15 minutes. The n u t r i e n t s o l u t i o n i s made as per O r g a n i z a t i o n o f



Reaction vessel.

C.

1. 2. 3. 4. 5. 6. 7.

Magnetic stirrer. Sample (250 ml) Carbon dioxide absorber Pressure indicator. Electrolyte. Electrodes. recorder.

F i g u r e 1. Schematic o f a Measuring U n i t

Pressure indicator.

Β. Oxygen generator.

A.

Β


3

S

S!

ι

es

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Economic Cooperation and Development (OECD) g u i d e l i n e s (10). I t c o n t a i n s 10 ml of s o l u t i o n A and 1 ml of each of the s o l u t i o n s Β t o F per l i t r e of s y n t h e t i c medium : s o l u t i o n A - KH P0 8.5 g, K HP0 21.75 g, Na HP0 .2H 0 33.4 g, and NH C1 2.5 g; s o l u t i o n Β - MgS04.7H 0 22.5 g; s o l u t i o n C - C a C l 27.5 g; s o l u t i o n D - FeCl3.6H 0 0.25 g; s o l u t i o n Ε - MnS0 .4H 0 39.9 mg, H3BO3 57.2 mg, ZnS0 .7H 0 42.8 mg, ( Ν Η ) Μ θ 7 θ 34.7 mg and FeCl3.EDTA 100 mg; and s o l u t i o n F - Yeast e x t r a c t 150 mg. The chemicals f o r each of the s o l u t i o n s A, B, C, D, Ε and F, are d i s s o l v e d i n 1000 ml of d e i o n i z e d water. The s o l u t i o n D i s f r e s h l y prepared immediately before the s t a r t of an experiment. The m i c r o b i a l inoculum was an a c t i v a t e d sludge sample from The L i t t l e Miami wastewater treatment p l a n t i n C i n c i n n a t i , Ohio, which r e c e i v e s predominantly domestic sewage. The sludge was allowed t o s e t t l e f o r about an hour, decanted, and then aerated a t room temperature f o r 24 hours. The dry weight of sludge was determined by d r y i n g samples, i n d u p l i c a t e s , of 1 ml, 2 ml and 3 ml a t 105°C overnight. A c o n c e n t r a t i o n of 30 mg/1 of sludge as dry matter was used i n the experiment. The t o t a l volume of the s y n t h e t i c medium i n the 500 ml c a p a c i t y r e a c t o r v e s s e l s was brought up t o a f i n a l volume of 250 ml. The c o n c e n t r a t i o n of the t e s t compounds was 100 mg/1 of medium. A n i l i n e was used as the biodegradable r e f e r e n c e compound at a c o n c e n t r a t i o n of 100 mg/1. The stock s o l u t i o n s were made i n d i s t i l l e d water f o r a n i l i n e and phenol with c o n c e n t r a t i o n of 5 g/1. Other compounds were added d i r e c t l y t o the r e a c t i o n v e s s e l s u s i n g m i c r o l i t e r syringes. The t y p i c a l experimental system c o n s i s t e d of d u p l i c a t e f l a s k s f o r the r e f e r e n c e substance a n i l i n e and the t e s t compounds and a s i n g l e f l a s k f o r t o x i c i t y c o n t r o l ( t e s t compound + a n i l i n e at 100 mg/1) and an inoculum c o n t r o l . The contents of the r e a c t i o n v e s s e l s were s t i r r e d f o r an hour t o ensure a steady s t a t e of the endogenous r e s p i r a t i o n a t the i n i t i a t i o n of oxygen uptake measurements. Then the t e s t compound and a n i l i n e were added. The r e a c t i o n v e s s e l s were incubated a t 25°C i n the temperature c o n t r o l l e d water bath and s t i r r e d c o n t i n u o u s l y throughout the run. The m i c r o b i o t a of the a c t i v a t e d sludge used as inoculum were not p r e a c c l i m a t e d t o the t e s t compounds. The i n c u b a t i o n p e r i o d of the experimental run was between 20 t o 40 days. 2

4

2

4

4


2

2

4

2

Biokinetic

4

2

2

4

2

2

4

6

2 4

Parameters

Although many models have been proposed f o r m i c r o b i a l growth and s u b s t r a t e removal, the Monod r e l a t i o n s h i p i s among the most popular k i n e t i c expressions used today. The Monod r e l a t i o n , i n combination with the l i n e a r law f o r s u b s t r a t e removal, can provide an adequate d e s c r i p t i o n of m i c r o b i a l growth behavior. I t s t a t e s t h a t


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the r a t e o f c e l l growth i s f i r s t order with r e s p e c t t o biomass c o n c e n t r a t i o n (X) and o f mixed order w i t h r e s p e c t to s u b s t r a t e c o n c e n t r a t i o n ( S ) . dX/dt = ^ X ) / ( K m

+ S)

s

(1)

C e l l growth i s r e l a t e d t o s u b s t r a t e removal by the l i n e a r law dX/dt = - Y (dS/dt)

(2)

The k i n e t i c parameters o f i n t e r e s t are the maximum s p e c i f i c growth r a t e μ the h a l f s a t u r a t i o n constant K and the y i e l d c o e f f i c i e n t Y. Here these k i n e t i c parameters were estimated d i r e c t l y from the experimental oxygen uptake curves. The y i e l d c o e f f i c i e n t Y and the h a l f s a t u r a t i o n constant K were determined by the method of Grady e t a l . (6) and the maximum s p e c i f i c r a t e constant, μ , was determined by the method o f Gaudy e t a l . (10). I f the c o n c e n t r a t i o n o f s u b s t r a t e and biomass are expressed i n oxygen e q u i v a l e n t s , then the oxygen uptake O , a t any time i n a batch r e a c t o r may be c a l c u l a t e d from :


ι η /

S /

s

πι

u

0

U

f

= ( S - S) - (X - X ) 0

(3)

0

f

where s u b s c r i p t o denotes i n i t i a l c o n d i t i o n . The y i e l d c o e f f i c i e n t Y was estimated from t h e oxygen consumption a t the beginning o f t h e p l a t e a u o f the oxygen uptake curve by : Y = (1 - 0 / S ) - Y u p

o

(4)

p

where Y i s the product y i e l d and O i s the cumulative oxygen uptake value a t the i n i t i a t i o n o f the p l a t e a u . In t h i s study the product y i e l d was n e g l i g i b l e . The oxygen uptake has reached i t s p l a t e a u when the cumulative oxygen uptake value i s approximately constant over a long p e r i o d o f time. At t h i s p l a t e a u , oxygen uptake i s due t o endogenous r e s p i r a t i o n o f the m i c r o b i o t a r a t h e r than the degradation o f the t e s t s u b s t r a t e . I f the assumption t h a t S » K i s made, then the term /( s ) i - Equation 1 approaches one and s i m p l i f i e s the equation. Combining t h i s s i m p l i f i e d equation with Equation 2 and i n t e g r a t i n g and s u b s t i t u t i n g i n Equation 3 gives p

u p

S

s

K

+

s

n

In

[ X + O / ( l / v - 1)] = l n ( X ) + j i ^ t ...(5) 0

u

0

The p l o t o f l n [ X + 0 /(1/Y - 1)] versus time w i l l g i v e a s t r a i g h t l i n e with slope μ-^. The oxygen uptake v a l u e 0 a s s o c i a t e d with one-half o f the estimate o f μ i s used i n 0

U

f

u

ιη

K

s

= S

0

- 0 " / ( 1 - Y) U

American Chemical Society Library 1155 16th St, N.W. Tedder and Pohland; Emerging Technologies in Hazardous Waste Management ACS Symposium Series; American Chemical Society: Washington, DC, 1990. Washington. OX. 20036

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t o get the estimate of K . The the p l a t e a u i s u t i l i z e d to get t h a t Equation 5 i s v a l i d only degradation when S » K and not period. s

S


Structure-Activity

oxygen uptake curve before t h i s estimate of K . Note i n the i n i t i a l p e r i o d of throughout the degradation s

Relationships

P r e d i c t i v e techniques, based on r e l a t i o n s h i p between s t r u c t u r e and b i o d e g r a d a b i l i t y , are important due t o the i n c r e a s i n g number and type of chemicals e n t e r i n g the environment and the f a c t t h a t a s s e s s i n g b i o d e g r a d a b i l i t y may be time consuming and expensive. In the f i e l d of biodégradation there are s e v e r a l s t u d i e s which have attempted t o c o r r e l a t e some p h y s i c a l , chemical or s t r u c t u r a l property of a chemical with i t s biodégradation. Based on the type and the l o c a t i o n of the s u b s t i t u e n t groups, Geating (11) developed an algorithm to p r e d i c t biodégradation. Q u a l i t a t i v e r e l a t i o n s h i p s f o r d i f f e r e n t compounds have been i n v e s t i g a t e d by others, but q u a n t i f i c a t i o n i s r e q u i r e d f o r r e g u l a t o r y purposes. F i r s t order r a t e constants of biodégradation, or f i v e day BOD values f o r chemicals, have been c o r r e l a t e d using both p h y s i c a l and chemical p r o p e r t i e s . P a r i s , et a l . (12,13) e s t a b l i s h e d a c o r r e l a t i o n between second order biodégradation r a t e constants and the van der Waal s r a d i u s of s u b s t i t u e n t groups, f o r s u b s t i t u t e d a n i l i n e s and f o r a s e r i e s of p a r a - s u b s t i t u t e d phenols. Wolfe, et a l . (14) c o r r e l a t e d second order a l k a l i n e h y d r o l y s i s r a t e constants and biodégradation r a t e constants f o r s e l e c t e d p e s t i c i d e s and phthalate e s t e r s . Several workers have observed a c o r r e l a t i o n between b i o d e g r a d a b i l i t y and l i p o p h i l i c i t y , s p e c i f i c a l l y octanol/water p a r t i t i o n c o e f f i c i e n t s (log P). P a r i s , et a l . (15) found a good c o r r e l a t i o n between biodégradation r a t e constants and l o g P, f o r a s e r i e s of e s t e r s of 2,4dichlorophenoxy a c e t i c a c i d . Banerjee, et a l . (16) obtained a s i m i l a r r e l a t i o n s h i p f o r chlorophenols. Vaishnav, et a l . (17) c o r r e l a t e d biodégradation of 17 a l c o h o l s and 11 ketones with octanol-water c o e f f i c i e n t s using 5-day BOD data. P i t t e r (18) found a dependence of biodégradation r a t e s on e l e c t r o n i c f a c t o r s , l i k e the Hammett s u b s t i t u e n t f a c t o r s , f o r a s e r i e s of a n i l i n e s and phenols. Dearden and Nicholson (19) have c o r r e l a t e d 5-day BOD v a l u e s with modulus d i f f e r e n c e s of atomic charge across a s e l e c t e d bond i n a molecule f o r amines, phenols, aldehydes, c a r b o x y l i c a c i d s , halogenated hydrocarbons and amino a c i d s . A d i r e c t c o r r e l a t i o n between the b i o d e g r a d a b i l i t y r a t e constant and the molecular s t r u c t u r e of the chemical has been used by Govind (20) t o r e l a t e the f i r s t order biodégradation r a t e constant with the f i r s t order molecular c o n n e c t i v i t y index and by B o e t h l i n g (21) t o c o r r e l a t e the biodégradation r a t e constants with the molecular c o n n e c t i v i t i e s f o r e s t e r s , carbamates, ethers, phthalates and a c i d s . 1


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Group C o n t r i b u t i o n Approach T h i s approach i s widely used i n chemical engineering thermodynamics t o assess the v a r i e t y o f pure component p r o p e r t i e s such as l i q u i d d e n s i t i e s , heat c a p a c i t i e s and c r i t i c a l constants f o r organic compounds. T h i s method i s s i m i l a r t o the Free-Wilson model, which i s widely used i n pharmacology and medicinal chemistry. Using a group c o n t r i b u t i o n approach, a l a r g e number o f chemicals o f i n t e r e s t can be c o n s t i t u t e d from perhaps a few hundred f u n c t i o n a l groups. Property p r e d i c t i o n i s based on the s t r u c t u r e of the compound. According t o t h i s method, the molecules o f a compound are s t r u c t u r a l l y decomposed i n t o f u n c t i o n a l groups or t h e i r fragments, each having unique c o n t r i b u t i o n towards the compound p r o p e r t i e s . The b i o d e g r a d a b i l i t y r a t e constant, k, i s expressed as a s e r i e s f u n c t i o n of c o n t r i b u t i o n s , α j , o f each group of the compound. The f i r s t order approximation o f t h i s s e r i e s f u n c t i o n r e p r e s e n t i n g biodégradation r a t e constant can be expressed as L In

(k) = Σ Njotj

(7)

j=l where N-j i s the number of groups o f type j i n the compound, a j i s the c o n t r i b u t i o n of group o f type j and L i s the t o t a l number of groups i n the compound. Using Equation 7 f o r each compound, a l i n e a r equation i s constructed. For a given data s e t t h i s generates a s e r i e s of l i n e a r equations which are s o l v e d for a s using l i n e a r regression analysis. 1

R e s u l t s and D i s c u s s i o n Oxygen uptake curves f o r the t e s t compounds, r e f e r e n c e compound a n i l i n e , the t o x i c i t y c o n t r o l ( a n i l i n e + t e s t compound) and the endogenous c o n t r o l systems were generated over a p e r i o d o f 20 t o 40 days. Within a p e r i o d of 10 days a l l the c o n t r o l s , t e s t compounds and a n i l i n e r e v e a l e d the l a g phase, biodégradation phase and the p l a t e a u r e g i o n . Figures 2 and 3 i l l u s t r a t e the r e p r e s e n t a t i v e oxygen uptake curves generated f o r two o f the t e s t compounds, phenol and d i b u t y l p h t h a l a t e . These data r e v e a l t h a t a l l the t e s t compounds, namely phenol, o - c r e s o l , m-cresol, p - c r e s o l , 2,4-dimethyl phenol, dimethyl phthalate, d i e t h y l p h t h a l a t e , d i p r b p y l p h t h a l a t e , d i b u t y l phthalate and b u t y l benzyl p h t h a l a t e were biodegradable a t the c o n c e n t r a t i o n l e v e l o f 100 mg/1 under the experimental c o n d i t i o n s . According t o OECD (11), the r e s u l t s of the degradation experiment were v a l i d because 60% degradation of c o n t r o l substance a n i l i n e was achieved w i t h i n a p e r i o d o f 28 days. Within a p e r i o d o f 40 days a l l the compounds were degraded between 80% - 95%.




150




9. DESAI ET AL.


151


152


The t o x i c i t y c o n t r o l data ( t e s t compound + a n i l i n e ) r e v e a l e d t h a t , except b u t y l benzyl p h t h a l a t e , none o f the t e s t compounds were i n h i b i t o r y towards the biodégradation of a n i l i n e i n the sludge. B u t y l benzyl p h t h a l a t e causes a s l i g h t i n h i b i t o r y e f f e c t on a n i l i n e degradation by i n c r e a s i n g the a c c l i m a t i o n time. The oxygen uptake curves of a l l the t o x i c i t y c o n t r o l s demonstrated two consecutive degradation phases, one f o r the t e s t compound and another f o r p o s i t i v e c o n t r o l compound a n i l i n e as shown i n F i g u r e 2 and 3, and t h i s behavior i s c h a r a c t e r i s t i c f o r c u l t u r e s i n which the t e s t compound i s not i n h i b i t i n g the biodégradation o f the c o n t r o l s u b s t r a t e . The d i f f e r e n c e between the t o t a l oxygen uptake o f the t o x i c i t y c o n t r o l ( t e s t compound + a n i l i n e ) , and o f the corresponding t e s t compound, was approximately equal t o the t o t a l oxygen uptake o f the c o n t r o l compound a n i l i n e . Estimates o f the Monod k i n e t i c parameters, and the maximum s p e c i f i c s u b s t r a t e uptake r a t e per u n i t biomass, m (^m/Y) phenols, phthalates and a n i l i n e , obtained by the method d e s c r i b e d before, are given i n Table I . k

f

o

r

Table I . Compound

Estimate o f Monod Parameters k^ " (day" )

Ϋ

^ ~ (day" )

(mg/1)

Aniline

16.1

0. 38

6.15

6.10

PHENOLS Phenol o-Cresol m-Cresol p-Cresol 2,4-Dimethyl phenol

16.9 10.0 17.3 18.5 14.4

0. 58 0. 41 0. 46 0. 33 0. 39

9.82 4.10 7. 97 6.11 5. 62

9.43 16 .41 17 .62 27 .78 14 .07

16.4 4.5 12.0 12.0

0. 43 0. 46 0. 48 0. 58

7. 07 3. 00 5. 78 6.95

41 .68 11 .67 15 .81 51 .38

12.8

0. 61

7. 80

36 .25

1

PHTHALATES Dimethyl p h t h a l a t e Diethyl phthalate Dipropyl phthalate Dibutyl phthalate B u t y l benzyl phthalate

1

The measured r a t e constants suggest t h a t , i n comparison t o phenol, the presence o f an o- o r p-methyl group renders a p h e n o l i c compound l e s s biodegradable. S u b s t i t u e n t s i n the para p o s i t i o n have a l e s s pronounced e f f e c t than i n the ortho p o s i t i o n . T h i s a l s o e x p l a i n s the μ value o f 2,4-dimethyl phenol which i s s m a l l e r than the one f o r p - c r e s o l but g r e a t e r than the one f o r o - c r e s o l . The i n t r o d u c t i o n o f a m-methyl group seemed t o have l i t t l e e f f e c t on the b i o d e g r a d a b i l i t y o f the p h e n o l i c 1Ιι


9. DESAI ETAL.


153

compound. Sugatt et a l . (22) have reported t h a t the low molecular weight phthalates degrade s l i g h t l y f a s t e r than the high molecular weight ones, but no such t r e n d was observed i n t h i s study. Comparison of our r e s u l t s with those reported i n the l i t e r a t u r e (22,23,24,25) was not p o s s i b l e because e i t h e r the r a t e s were not reported, or they were second order r a t e s with d i f f e r e n t u n i t s . Our values f o r h a l f s a t u r a t i o n constant K , are h i g h f o r some of the compounds. T h i s r e s u l t might be caused by the l i n e a r i z e d g r a p h i c a l approach used f o r i t s e s t i m a t i o n . I t i s necessary t o have a t l e a s t f i v e data p o i n t s f o r each unknown v a r i a b l e t o get a s t a t i s t i c a l l y v a l i d c o r r e l a t i o n . So i t i s imperative t o ensure t h a t each group f o r which a c o n t r i b u t i o n i s c a l c u l a t e d occurs i n a t l e a s t f i v e compounds. The c o n t r i b u t i o n s of d i f f e r e n t groups were c a l c u l a t e d from l i t e r a t u r e data (26). These data were f i r s t order constants c a l c u l a t e d by the equation :


s

dBOD/dt = k BOD

(8)

The groups and t h e i r c o n t r i b u t i o n parameters are given i n Table I I . The data s e t used t o c a l c u l a t e c o n t r i b u t i o n parameters d i d not i n c l u d e the c r e s o l s , phenol and 2,4dimethyl phenol. The c o n t r i b u t i o n of the p h t h a l a t e group was not c a l c u l a t e d because e i t h e r not enough data were a v a i l a b l e or the only data a v a i l a b l e were f o r those p h t h a l a t e s used i n t h i s study. Table I I . Group C o n t r i b u t i o n Parameters No.

Group

1 2 3 4

Methyl Hydroxy Aromatic Aromatic

a

CH OH ACH AC 3

CH C

J

-3.460 -2.983 -0.8340 1.9730

Using the c o n t r i b u t i o n parameters of Table I I , f i r s t order r a t e constants were p r e d i c t e d f o r phenols. The comparison between the p r e d i c t e d values and the values obtained from experimental data, u s i n g Equation 8 are given i n Table I I I . The p r e d i c t e d values are w i t h i n 10% of the experimental v a l u e s . However, the method d e s c r i b e d i s a f i r s t order approximation which assumes t h a t there are no group i n t e r a c t i o n s . A d d i t i o n a l s t u d i e s are needed to v e r i f y t h i s assumption. Conclusion Based on the oxygen uptake data a l l the t e s t e d phenols and p h t h a l a t e s are biodegradable i n a c t i v a t e d sludge with


154



our experimental conditions. Similar results have been noted in the literature. A l l compounds were degraded in excess of 80%. A methyl group in o- or p- position of phenolic compounds decreases the degradation rate in comparison to phenol, while a m-methyl group does not affect the degradation rate. No such trend was observed with phthalates. With the exception of butyl benzyl phthalate, none of the test compounds inhibit aniline degradation. More data are required to determine whether or not unique group contributions exist with respect to i t s position (o, m, and ρ ) , and to include more groups in the structure-activity relationships. Table I I I . Comparison of Actual and Predicted ln(k) Values Compound Actual Predicted % Error ln(k) ln(k) o-Cresol -6.0890 m-Cresol -5.7706 p-Cresol -5.8659 2,4-Dimethyl Phenol -6.2472 Phenol -5.6891

-5.8330 -5.8330 -5.8330 -6.4860 -5.1800

4.20 -1.08 0.56 -3.82 8.95

Nomenclature k f i r s t order biodégradation constant (hr"" ) k maximum specific substrate uptake rate per unit biomass (day"" ) K half saturation constant (mg/1) L total number of groups Nj number of groups of type j O oxygen uptake (mg/1) °up oxygen uptake at the plateau (mg/1) S substrate concentration (mg/1) X biomass concentration (mg/1) Y biomass yield coefficient Yp product yield coefficient otj contribution of j th group μ maximum specific rate constant (day"" ) 1

m

1

s

u

1

ΙΪΙ

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Determination of Monod Kinetics of Toxic Compounds by

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