Aerobic microbial degradation of aromatic sulfur-containing compounds and effect of chemical structures

Chemosphere. Vol. 36, No. 15, pp. 3033-3041, 1998

Pergamon

0 1998 Elsevier Scmce Ltd All rights reserved. Printed in Great Britain CO454535/98 $19.00+0.00

PII: SO0456535(98)00004-6

AEROBIC MICROBIAL DEGRADATION OF AROMATIC SULFURCONTAINING COMPOUNDS AND EFFECT OF CHEMICAL STRUCTURES

A. Q. Zhang,

S. K. Han.

Department of Environmental

J. Ma,

X. C. Tao’,

L. S. Wang*,

Science and Engineering, Nanjing University,

People’s Republic of China

Nanjing 210093,

’Department of Chemistry, Anhui Normal University,

Wuhu 241000,

People’s Republic of China

(Received in Germany 8 August 1997; accepted 25 November 1997)

ABSTRACT Batch data of aerobic phenylsulfonyl

acetates

microbial

degradation

have been determined,

rate constants and the qualitative

chemical structures were analyzed. The phenylthio followed by the phenylsulfonyl those with a isopropoxy biodegradability, the ortho-position.

group. The nitro-group

of phenylthio, relationships

phenylsullinyl

between

and

their Kb and

acetates were most subject to microbial transformation,

acetates. The compounds

while the nitro-group

&

with a methoxy group were easier degraded than

and chloro-group

at the para-position

on benzene

were shown to lower the

had stronger side effect on degradation

than at

01998 Elsevier Science Ltd. All rights reserved

INTRODUCTION Microbial chemicals,

degradation

is not only one of the most important

but also a key process for the chemical recycling in the environment.

the study on the biodegradability discharged

transformation

of priority pollutants

mechanisms

for organic

Much attention is paid to

as well as organic chemicals

frequently

used or

through the industry production”’ 21. On the contrary, little is known about the distribution

removal

of the newly synthesized

difftcult.

The investigation

organic

compounds,

on the biodegradability

which will make environment

of new types of chemicals

and

risk assessment

is in need to provide

a

scientific basis for the application of their analogues. Twenty-live

aromatic sulfur-containing

acetates, 6 phenylsulfonyl structures

acetates investigated

can be expressed

* To whom correspondence

compounds

by Figure

including 9 phenylthio

here are products synthesized

1. It is of interest

should be addressed 3033

acetates,

10 phenylsulfinyl

in the laboratory. The general

to note that these

recently

synthesized

3034 compounds

have much in common except the oxidation states of the sulfur atom, the substituting groups on

phenyl ring and the ester alkyl groups. In addition. these compounds slow hydrolysis

rate at neutral

pH at room temperature”)

have quite strong hydrophilicity,

and comparatively

Photobacterium phosphoreum14’. However. no literature about the biodegradability

very

weak acute toxicity

to

of their analogues can be

found.

One of the purpose of this study was to determine sulfur-containing process.

compounds

the biodegradation

and evaluate whether the biodegradation

The other aim was to analyze the potential relationships

kinetic data of these aromatic

play an important

between

role in removal

the biodegradation

rates and

chemical structures to prepare for the follow-up studies of degradation pathway.

MATERIALS

AND METHODS

R2

R, - a substituting

group on phenyl ring at the ortho-position (R,=H,

R2 -

a substituting group on phenyl ring at the para-position (Rx=H, Cl. NO?)

RI

a ester alkyl group (R&HI.

-

CH(CI-IT):)

Figure 1. The general structures

Twenty-five

synthesized

aromatic sulfur-containing

by silica gel column chromatography

ofthe tested

compounds

compounds

were used as test materials. Purification

resulted in greater than 98% p~rity’~‘. During the experiment,

purities were monitored by high performance

liquid chromatography

grade solvent methanol

was redistilled

their

(HPLC) to assure that no interference

peak had occurred. The structures and code number of these compounds

inorganie chemicals

Cl, NO>)

are listed in Table 1. Analytical

before use. Water was doubly distilled

were all in A. R. grade. Yeast extract was a biochemical

in quartz. Glucose

and

reagent. A neutral buffer

solution was prepared using NazHP04. NaHzPO4 and pure water.

The mineral salts culture medium consisted of the sterile stock solution of 1M/L MgS04 (1mL per liter)

3035 mixed with the axenic mineral salts solution containing (per liter of redistilled water) 2.688 of NaIHPOa, 1.56g of NaHzPOa, 2.728 of NH4Cl and 2.03g of NaCl. As a carbon source for the bacterium, stock solutions of yeast extract and glucose, after being autoclaved at 115 C, were added to the above salts medium to make the nutrient medium, while the final concentrations of yeast extract and glucose were lg/L and 5g/L, respectively. Solid medium contained 1.5 to 2.0 percent (wt/v) agar. The pH of all kinds of culture medium used in this study was adjusted to 7.0 + 0.2@].

The bacterial strain used here was originally isolated by the faculty of the Teaching and Research Section of Environmental Biology in Nanjing University, who tentatively identified it as Pseudomonas sp. Pure cultures were maintained on nutrient agar slants. In order to adapt Pseudomonas sp. to the utilization of tested compounds as sole carbon source, the strain was grown in a 150-mL Erlenmeyer flask containing 50 mL of nutrient medium on a rotary shaker at 30 ‘c. After the postexponential

phase was reached

(approximately 48h according to the growth curve), In& of the culture was transferred into fresh medium in which 10% of the yeast extract and glucose was replaced by the tested aromatic sulfur-containing compounds. This shit? was repeated when turbidity can be observed until the tested chemical was the sole organic carbon source.

A fresh liquid mineral salts medium with the tested chemicals as sole carbon source was inoculated with the preacclimated organisms and incubated at 30 ‘c at 60rpm. The bacterial growth was monitored by measuring the optical density at 55Omn, in addition, the number of living cells was determined by plate count. When the cell density reached the level of 108cells/mL, aerobically grown cells were harvested by centrifugation, washing twice with the buffer, and resuspended in a fresh mineral salts medium at 30 C . The suspension was then incubated at 30 C for 2hr in an effort to remove residual substrates and to deplete intracellular reserves before being used as the final inocula.

The tested chemicals were added to the corresponding 150-mL Erlemneyer flasks containing sterile mineral salts medium separately. The flasks were put in an ultrasonator for 15 min to dissolve the compounds. The final concentrations of organic chemicals were approximately 3OmglL. These aqueous solutions were then incubated at 30 “c overnight to make sure that they were germ-free. Subsequently, qualified chemical solutions were inoculated in the proportion of one part of inocula to nine parts of solution. The initial cell density was around 106cells/mL. AAer thoroughly homogenized by shaking the flasks, the inoculated solutions were transferred into the test tubes, each tube contained 2 to 3-mL liquor. The test tubes were incubated at 30 C on a rotary shaker as mentioned above. At the same time, the qualified test solutions with autoclaved inocula and the sterile mineral salts medium without any test material inoculated with the same inocula served as control sets I and II, respectively. At least triplicates were run for each test

3036 material and the number of controls were twice the number of replicates used per test compound.

At appropriate intervals to provide a minimum of five time points, test tubes were taken from the shaker. The content in tubes, after filtration

through

0.22-p

Industry Research Institute). was stored in a refrigerator Liquid Chromatography

equipped

(Beijing Analytical Instruments The chromatographic

filters immediately

(Shanghai

Medical

until analyzed by a SY-5000 High Performance

with an UV detector (254nm) and a 15cm x 0.4cm id RP-18 column

Factory) to determine the amounts of tested compounds

condition was as follows: column temperature,

methanol : water (80:2Ovlv); flow rate, lmlimin.

Logarithms

pore-size

of the remaining concentrations

30 C; composition

in aqueous phase. of mobile phase,

Triplicates were also run for HPLC analysis.

of the organic compounds

were plotted against time, and

with linear regression analysis first-order rate constants were obtained from the slope of the regression lines that were through the points of the active transformation

phase. Degradation

rate constants determined

for

rate constants. Different models on biodegradation

are

each treatment were the averages from all replicates of that treatment.

RESULTS AND DISCUSSION Aerobic Microbial deeradation normally

used for various

Pseudo-first-order

reasons.

In most environmental

models,

pragmatic

first-order

kinetics

are

assumed, ds dt

where s is the concentration

I

s=k

of the substance at the beginning (so) or at time t (s,),

k is the first-order rate

constant and 7’l,z is biological half-life time. In this study, results of initial range-finding pseudo-first-order

kinetics could be applied over the concentration

tests show that 60 percent of the compounds Reactions could be approximated

range around 30mg/L. The degradation

could be removed from the aqueous phase after 6 or 7 days.

to the pseudo-first-order

reaction. The first-order rate constants of test set

(&Q~~) are calculated as described above. The general average coefficient The worst squared correlation coefficient for linear regressions demonstrating

of variation for replicates is 4%.

(R’) is 0.842. Besides, the probabilities

are all less than 0.01, at which the significance

that the Kd,rawvalues are significantly

of control set

corresponding

level of Student’s

different from zero and Pseudomonas

the compounds as its sole carbon source after acclimation. each of the first-order rate constants

test indicate that

On the contrary, the difference

to t-values

t test is set,

sp. could utilize between zero and

I (K,,,,) obtained using the same method is not significant

3037 Table 1 The chemical structures, aerobic biodegradation rate constants and degradation half-lives of the tested compounds

No.

RI

Rl

R3

S-01

NO2

NO2

CH3

s-02

NO2

NO2

CWCH3)2

NO2

CH3

s-03 s-04 S-06 s-07 S-08 s-09 S-IO so-01 so-02 so-03 so-04 so-05 SO-06 so-07 SO-08 so-09 so-10 SG2-03 sol-04 sol-05 SG2-07 SO*-08 sol-09

Cl Cl H

1

1.94

NO2

WCH3h

NO2

CWH3)2

NO2

Cl Cl H H

CHS CH(CH& CH, CWCH&

NO2

NO2

CH3

NO2

NO2

CWH3)2

NO2 NO2 NO2

Cl Cl H H NO2

NO2 NO2 NO2

Cl Cl H NO2 7-2 NO2

7’402

CH3

NO2

CHW-W2

NO2

CH3

NO2

Cl Cl H H

CWH3)2 CH3

CH(CH3h CH3

CH(CH&

NO2

CH3

NO2

CWH3)2

NO2

CH3

Cl Cl H

Td4

CH3

CWCH,h CH3

0.264 0.353 0.298 0.511 0.404 0.401 0.546 0.535

2.63 1.96 2.33 1.36 1.72 1.73 1.27 1.30

0.391 0.327 0.275 0.241 0.408 0.378 0.402 0.393 0.412 0.403

1.77 2.12 2.52 2.88 1.70 1.83 I .72 1.76 1.68 1.72

0.433 0.362 0.467 0.436 0.403 0.479

1.60 1.91 1.48 1.59 I .72 1.45

* S - phenylthio acetates; SO - phenylsultinyl acetates; SO2 - phenylsulfonyl acetates.

at the 0.01 level, which means the chemical hydrolysis and other abiotic influences under the test condition

are very weak. The corrected rate constants I&,,% calculated by subtracting KautOfrom &SW for eliminating abiotic contribution are listed in Table 1. The data of control set II shows that the starved-culture is under the endogenous condition after centrifugation, washing and incubation procedure. In other words, inocula will not change the concentrations of the tested compounds or the remaining concentrations in control tubes are under the HPLC limit of detection. Obviously, it is biodegradation that changes the compounds ir est tubes. The degradation half-lives are also illustrated in Table 1.

Effects of Ester Alkvl Grottos on biodeeradation.

phenylthio acetates, phenylsultinyl

It is noted that there are 3 types of compounds,

acetates and phenylsulfonyl acetates, while each type has different

number of chemicals. The number of each structure type is so small that neither the assumptions of normality nor the central limit theorem seems to be applicable”‘. Under this circumstance, parametric statistical methods. together with nonparametric procedures, are employed to tackle the problem and analyze

3038 the effect of chemical

structures

on biodegradation.

others on account of the complex employed

Since the biological

factors involved.

the significance

in all cases was 0.05 instead of 0.01. Concerning

biodegradation.

tested compounds

test has greater deviation

level for one-sided

(one-sided alternative).

to their structure similarities,

By reason that each pair of compounds

the bio-hydrolysis

The difference

bond cleavage of the compounds.

signed-ranks

using paired t test

with a distinguished

retard in reactivity.

Since an isopropyl has a large bulk than a esters to approach

due to the great steric hindrance

esterst’l. Wilcoxon matched-pairs

in

path. It is assumed that the hydrolyase in cells

methyl. it is more difficult for reaction center in isopropyl catalyzing

respectively.

has same structure features except the ester

that the isopropyl is associated

That is supposed to be related to their possible degradation would help the acyl-oxygen

SO*-03 and SO*-04,

in each pair is significant at o =0.05 level of significance

alkyl group, it can be concluded

test

S-01 and S-02. S-03 and S-04, S-07 and S-08, S-09 and S-10, SO-01 and

were paired according

Kd,c0rrbetween two components

statistical

the effect of the ester alkyl groups on

SO-02, SO-03 and SO-04. SO-05 and SO-06. SO-07 and SO-08, SO-09 and SO-lo, SOz-07 and Sol-08

than

the active site in enzyme

about the acyl group than that in methyl

test. the nonparametric

analogue to the paired t test. results

in the same conclusion.

Effects of Different Substituents paired-sample

study design is adopted preferentially

rid of the influence discussed,

of the other confounding

compounds

individually

on Phenvl Ring. As for the type and the position of substituents, rather than the independent

factors. For example,

the

samples I test in order to get

when the effect of nitro group is

S-01 and S-09, S-02 and S-10, SO-02 and SO-lo, SO-01 and SO-09 were matched

in that both chemicals

in each pair containing

almost identical

oxidation state of sulfur, the ester alkyl group. the type and position of substituents existence of a nitro group in a specific position. Likewise. compounds

structure

features like the

on phenyl ring except the

S-04 and S-06. S-07 and S-09, S-08

and S-10, SO-03 and SO-05 SO-04 and SO-06. SO-07 and SO-04. SO-08 and SO-lo. SO>-03 and S02-05, Sol-07

and SC&09 are related separately

on the basis of the same matching

importance of existence of chlorine in degradation. pairs of compounds

principle

to represent

the

In accordance with the above matching characteristic.

4

(S- 10 and S-06, SO-09 and SO-05, SO- 10 and SO-06. Sol-O9 and S02-05) are used to

investigate the effect of substituting position of a nitro group. Neither the one-tailed p-values of these paired t tests nor the corresponding confirms

ones of Wilcoxon

the tendency that the nitro-group

biodegradability

sign rank test are above the significance

and the chloro-group

while the nitro-group at the para-position

level, which

on phenyl ring are shown to lower the

has stronger side effect on degradation than that

at the ortho-group.

To gain an insight into the effects of the substituents

on degradation

of the tested compounds,

it is

necessary to investigate the structure of benzene. There are two circular lobes in the n-orbitals of benzene, one above and one below the plane of the ring. The 6 rt electrons in benzene are completely delocalized over

3039 the entire ring[gl, so the electron-withdrawing

substituents will decrease the density of electron cloud on

phenyl ring because of resulting further delocalizat,on of the positive charge over the x-orbitals of phenyl ring. Then the electron transfer from ring to molecular oxygen leading to the ring oxidation is relatively hindered as well as the oxidation of the sulfur-containing

carbon chain affected by the resonance and

inductive effects of the substituents, which accounts for the decrease in reactivity of bidegradation of aromatic compounds [“I . It is known that the nitro-group is of strong electronegativity, the Hammett constants of nitro are 0,=0.71, q~O.78. When a nitro-group occupies at the para-position, the nitrogenoxygen double bond overlaps the x-orbitals of phenyl ring, which allows for the further delocalization of the x electrons and decrease the density of electron cloud. As a result, the oxidation process where there is electron transfer from the compounds to molecular oxygen is retarded, and the degradation of the tested nitroaromatics occurred at a relative low rate. Similarly, chlorine is also more electronegative than carbon. The electron-withdrawing

power of the chlorine imparts a partial positive charge at the phenyl ring

rendering it less electron-releasing, which results in the stabilization of the tested halogenated compounds. On the other hand, the addition of a nitro-group at the ortho-position less significantly retards the biodegradation than that at the para-position, suggesting that the steric bulk of the nitro group is of great importance. The large volume nitro group flanked by the adjacent chain can no longer be coplanar with the ring, and the conjugative effect is reducedt”‘. Accordingly, the stabilization of the ring by resonance interaction with nitro group is weakened, and the biodegradation of ortho-substituting nitroaromatics occurs more actively than that of para-substituting ones. Although the occurrence of peaks with short retention times on HPLC was observed in our studies, these peaks have not yet been totally identified. Analyses to determine possible metabolites on the basis of the present study are in progress.

Effects of the Oxidation State of

Sulfur Atom on biodeuradation. Similar to the changes in the ester

alkyl groups and substituting groups on phenyl ring, changes in the oxidation states of sulfur play an important role in degradation. Using the one-sided paired-sample study design as described above, we find that the phenylthio and the phenylsulfonyl acetates both are more susceptible to microbial transformation than the corresponding phenylsulfinyl acetates, but there is no significant difference in KQ,,,, between the phenylthio and phenylsulfonyl acetates. However, the study of Han Shuokui et aLD1 indicates that the hydrolysis rate constants at neutral pH decrease in a different order : -S- > -SO*- > -SO-, which implies that there are some other competitive degradation pathways involved covering up the effect of neighboring group participation of -S- group on the alkyl ester hydrolysis. Due to the high reactivity of the methylene group, the carbon-sulfur

bond cleavage might become a potential pathway. Guided by this concept, a

benzenesulfonic acid has been detected. Specific mechanism study is underway.

3040 CONCLUSION From the experiments compounds.

it can be seen that Pseudomonas sp. strain cells can be used to degrade the tested

The average degradation half-life is about 2 days. This means the original forms of the aromatic

sulfur-containing

compounds

can not persist in the environment

ways of removal for these compounds, containing compounds

and biodegradation

The microbial degradation

have shown the reaction to be first-order

effect and the steric effect of the substituents

is one of the major

kinetics of the tested aromatic sulfur-

and primarily depended on the electronic

on phenyl ring and the groups about the carbonyl group,

obviously inconsistent

with a simple hydrolysis

mechanism.

Since the mechanism

work in the preceding

stage, there is little evidence provided

study is not our aim of

for the specific process for transformation.

Although the effect of groups discussed above explains most experimental

observations,

it is no more than a

working hypothesis at this point and should be refined as more information about the degradation pathway is obtained. A further study involved in bacterial metabolism laboratory

in an effort

to establish

the quantitative

on these compounds relationships

is being carried out in our

between

structural

features

and

biodegradability.

Acknowledgments

This project was funded by the National Natural Science Foundation

of P. R. China

and the State Key Laboratory of Pollution Control and Resource Reuse of Nanjing University.

REFERENCES

1.

A. M. Martin, Biological

Degradation

of Wasters.

Elsevier

Applied

Science,

London and New

York (1991) 2.

G. Rheinheimer,

Aquatic

3.

S. K. Han, L. Q. Jiang, and phenylsulfonyl

4.

Microbiology

(3rd Edn), p. 145. John Wiley & Sons, New York(1985)

L. S. Wang and Z. Zhang.

acetates,

and neighboring

Hydrolysis

group effect.

of phenylthio,

Chemosphere,

phenylsulfinyl

25, 643-649 (1992)

L. S. Wang, Q. G. Huang, S. K. Han. Z. Zhang and H. Gao, The toxicity

of sulfur derivatives

of

Environ Chem,

chloronitrobenzene

and

quantitative

structure-activity

relationships.

13,

123-128 (1994) 5.

X. Q. Xu. W.Q.Zhou acetates

6.

and Z. Zhang,

with sodium perborate.

D. Jenkins and B. H. Olson,

Selective

oxidation

of phenylthio

Chemicul’ Journal of Chinese Water and wastewater

acetates

Universities.

microbiology.

Water

to phenylsultinyl

13, 490-492 (1992) Sci.

Technol..

20,

53 (1988) 7.

B.

Roser,

Massachusetts 8.

Fundamentals

of

Biostatistics

(2nd Edn),

~~278-293.

Duxbury

Press,

Boston,

(1986)

M. S. Matta and A. C. Wilbraham,General, -573. The Benjamin/Cummings

Publishing

Organic, Company,

and Biological California

Chemistry(2nd

(1986)

Edn), pp555

3041 9.

S. N. Eie,

Organic Chemistry, p.66.

D. C. Health and Company,

Lexington, Massachusetts/

Toronto (1984) 10. Y. H. Yang, X. M. Hua, S. L. Chen and S.J. Hu, Catabolic pathways for aromatic hydrocarbon biodegradation and their molecular genetic analysis. Advances in Environ. Sci., 3, 31-43 (1995) 11. N. S. Isaacs, Physical Organic Chemistry, p.290. Longman Group UK Limited, England (1987)