The influence of different soil types on the fate of phenol and its biodegradation products

Chemosphere, VoI.17, No. II, pp 2199-2205, Printed in Great Britain

1988

OO45-6535/88 $3.00 + .OO Perqamon Press plc

THE INFLUENCEOF DIFFERENT SOIL TYPES ON THE FATE OF PHENOLAND ITS BIODEGRADATIONPRODUCTS

John P. Knezovich*, Joy M. Hlrabayashi, Dorothy J. Bishop, and Florence L. Harrison

Environmental Sciences Division

L-453

Lawrence Livermore National Laboratory Llvermore, California

94550

ABSTRACT

The degradation of [14C]-pheno1 by Pseudomonas putida was determined tn the presence of three d i f f e r e n t sorbents. Phenol degradation resulted in an increased retention of the restdual radioactive compounds by each of the sorbents. Catechol, which is more sorptlve than phenol, was the only aromatic degradation product that was i d e n t i f i e d in the aqueous phase of each experiment. The greater sorptlon of catechol indicates that compounds that are degraded via catechol intermediates may be less mobile and more persistent in the subsurface environment. INT~OUCTI~

Phenollc compounds may enter the environment as a result of pesticide degradation, from releases associated with manufacturing processes, or from the production of energy (1-3). These compounds are r e l a t i v e l y water soluble and are l i k e l y to be mobile in the subsurface where they may contaminate drtnklng-water supplies. soil bacteria has been demonstrated (4, 5), l i t t l e

Although the degradation of phenol by information is available on the influence

of microbial degradation on the retention of i t s degradation products

in s o i l .

Such

information is required to accurately define the fate of phenolic contaminants in the subsurface so that r e a l i s t i c assessments of environmental hazard can be made.

2199

2200

The objectives of this study were to obtain a better understanding of ( l ) how the presence of different sorbents affects the rate of phenol degradation and (2) how the degradation process affects the fate of phenol and i t s metabolites in saturated soil systems. Information derived from these studies w i l l be used ultimately to understand how biodegradation processes influence the transport of phenolic compounds in the subsurface environment. MATERIALS AND METHODS

Chemicals Phenol was obtained from Mallinckrodt, Inc. (Paris, KY). Resorcinol, hydroqulnone, and catechol were obtained from Eastman Kodak Co. (Rochester, NY). Uniformly rlng-labeled [14C]-phenol (specific a c t i v i t y - ll.61 mCi/mM) was obtained from Pathfinder Laboratories, Inc. (St. Louis, MO). All of these compounds were found to be greater than 98% pure by HPLC analysis.

O t h e r chemlcals and solvents were either Analytlcal Reagent Grade from

Mallinckrodt, Inc. (St. Louis, MO) or HPLC-grade from Burdlck and Jackson Laboratories, Inc. (Muskegon, MI). Bacteria A pure strain of a soll bacteria (Pseudomonas putlda) that had been shown to degrade phenol (6) was used in a11 experiments.

P. putlda (ATCC strain 31303) was accllmated for the

aerobic degradation of phenol by continuous culture In a solutlon of nutrient broth (7) that contained phenol (50 mg/L) as the sole carbon source. Bacterlal cultures were maintained in shake flasks at 27°C and were transferred to fresh nutrlent-phenol solutlons at weekly Intervals. Sorbents We used three sorbents that spanned a range of chemical and physlcal properties in the study of phenol blodegradatlon (Table l ) .

A high iron oxide containing soll (Georgia) was a

B-horlzon soll from Early County, GA, and the high organic content soil (Sherman Island) was from the San Joaquln Delta near Rio Vista, CA.

Blue i111te clay was obtained from mine

spoils in E1izabethtown, IL.

Table 1.

Properties of sorbents used in studies of phenol btodegradatlon.

pH

Organic Carbon (%)

CECa

Sand

% Sllt

Clay

Georgia

5.3

0.09

6

19

38

43

Blue i l l i t e

8.3

l.l

14

0

0

I00

Sherman Island

4.4

14.5

38

51

47

2

Soll

acatton exchange capacity (meq/lO0 g).

2201

Experimental Procedures A11 experiments were conducted in 500-mL screw-top culture bottles (Gibco Laboratories, Grand Island, NY). Each bottle cap was f i t t e d with a Teflon seal and two Teflon ports, one of which was connected by silicone tubing to a O.20-~m s t e r i l e f i l t e r

(Gelman Acrodlsc;

Gelman Sciences, Inc., Ann Arbor, MI) and then to a supply of C02-free a i r .

Each outlet port

was connected to a shut-off clamp and then by glass tubing to a CO2 trap that consisted of a 20-mL s c i n t l l l a t i o n vial containing 15 mL of Carbosorb I I (Packard Instrument Co., Downers Grove, IL).

RadlolabeledCO2 produced as a result of phenol mineralization was collected by

passing a i r through each bottle and into the trapping solution for ten mln. Carbon dioxide production was subsequently quantified by adding a 1-mL aliquot of the trapping solution to 15 mL of s c i n t i l l a t i o n cocktail (Permafluor V, Packard Instrument Co.) and counting in a Packard Tri-Carb liquld s c i n t l l l a t i o n counter (Model 4530). Samples were corrected for background radioactivity and quenching by an external standard. The blodegradatlon of phenol In the presence and absence of the three experlmental sorbents was determined in two replicate experiments.

Forty-mL solutions of mineral media

that contained 50 ~g phenol/mL and approxlmately 100,000 dpm/mL of radlolabeled phenol were added to each of twelve, autoclaved culture bottles.

All sorbents were autoclaved before use

and three, 5-g aliquots of each were added to separate bottles. did not receive any sorbents and served as aqueous controls.

The three remaining bottles Twenty-five mL of the

phenol-adapted P. putida culture was then centrifuged at 1,300 X g for 10 mln and II mL of the supernatant was subsequently decanted. The remaining suspension of bacteria was then mixed and added to the test chambers in l-mL allquots.

One culture bottle from each sorbent

set did not receive a bacterlal innoculum and served as a control for phenol distribution in the absence of blodegradatlon. All bottles were placed on a shaker table in an incubator for nine days at 27 ± l°C. contents of the CO2 trap on each bottle were analyzed and replaced on a daily basis.

The

At the

end of the incubation period, the contents of each bottle were a11owed to settle and three l-mL a11quots of each solution were passed through 0.22-~m f l l t e r s (Swlnnex-GS; Millipore Corp., Bedford, MA) and analyzed for total radioactivity by liquid s c i n t i l l a t l o n counting. An addltlonal three aliquots were f i l t e r e d and analyzed for aromatic breakdown products by high performance liquid chromatography (HPLC). A Waters Assoc., Inc. (Milford, MA) Model 6000 HPLC equipped with a Spherisorb 5-~m C18 column (Custom LC, Houston, TX), a IO0-~L injection loop, and a UV (274 nm) absorbance detector was used. The buffered (0.5 M KH2PO4) mobile phase (methanol/water/acetic acid; 40:60:I, v/v/v) was run at a flow rate of I mL/mln and eluent fractions were collected every minute for the f i r s t 30 min in s c i n t i l l a t i o n vials that contained 15 mL of s c i n t i l l a t l o n cocktal1. Areas of the chromatograms that had radioactivity greater than 1.~"k background were identified by co-chromatography with authentic standards. The amount of radioactivity that was not measured in solution or as evolved CO2 was considered to be sorbed to the test sorbent.

The e x t r a c t a b i l i t y of the sorbed radioactivity

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was determined for each experiment.

The water in each culture bottle was decanted, and the

soil was extracted overnight with 25 mL of methanol in a soxhlet extractor. radioactivity was quantified by Iiquld s c i n t i l l a t i o n .

The extracted

The relative distributions of phenol

and i t s degradation products were then calculated by dividing the concentration

of

radioactivity in the sorbed and,aqueous phases by the control values. RESULTS AND DISCUSSION Phenol was mineralized extensively by P. putida in the presence or absence of sorbents (Fig. I ) .

Mineralization proceeded most rapidly in the absence of sorbents and accounted for

47% of phenol degradation by day nine.

Mineralization in the presence of Georgia soil and

blue i111te clay occurred more slowly and accounted for 24% and 27% of the degraded phenol, respectively.

Phenol degradation in the presence of Sherman Island soil occurred following a

four-day lag period after which 30"/. was mineralized during the next five days. The slower rates of mlnerallzatlon that occurred in the presence of the test sorbents were probably due to the reduced b l o a v a i i a b i l l t y of the sorbed phenol.

In addition, the lag in phenol

degradation that occurred in the presence of Sherman Island soil may have been due to the metabolism of more labile carbon sources that were present in this organic-rlch s o i l .

Phenol

degradation by microorganisms other than P. putida was insignificant; less than I% of the phenol was mlnerallzed in each of the autoclaved control sorbents. The biodegradatlon of phenol altered the distribution of radioactivity between the sorbent and water phases of each system (Fig. radioactivity were substantially lower for a l l incubation period.

2).

The aqueous concentrations

of

experiments at the end of the nine-day

In the experiments with Georgia s o i l , the aqueous radioactivity was 3% of

the control value and the radioactivity that was associated with the soil was 156% greater than the control value.

Similar results were obtained for experiments with blue i l l i t e

clay

in which the aqueous radioactivity was 23% of the control value and in the soll fraction was 250% of the control.

For experiments with Sherman Island s o i l , the aqueous radioactivity was

reduced by 87% whlle the amount associated with the soil remained essentially unchanged.

In

each of the blodegradation experiments, the soil/water distribution of phenol-derived carbon was much greater than the distribution coefficients that have been reported for similar ( s t e r i l e ) sorbents (8).

For example, Scott et a1. (5, 9) reported an increase in the

"apparent adsorption" of phenol on soils that occurred as a result of biodegradation.

In

their studies, the decrease of radioactivity in the solution phase was assumed to be a result of sorption or degradation processes. Because the blodegradatlon of phenol may result in an extensive liberation of CO2, however, this fraction of carbon must be accounted for before the distribution of residual compounds can be determined accurately. At the termination of the biodegradation experiments, sorbents were extracted with methanol to assess the influence of biodegradation on the potential for mobilization of phenol and phenol-related carbon residues in the subsurface.

Soxhlet extractions yielded low

recoveries of radioactive compounds that were sorbed to the test sorbents at the end of each experiment. In experiments conducted with autoclaved sorbents, the recoveries of

2203

60 No Sorbent 5O

40 o n

30

0 20

i,o ~

0

0

2

4

6

8

DAY

Figure 1.

The mineralizatlon of phenol by P. putida in the presence and absence of three test sorbents. 50 /

40= I

30 A

=E L

2O

Z O n,~

lO

0

[]

Control

[]

p. Dutlda

A Q U E O U S PHASE

/

~ m

o u,,i

400

SORBED PHASE u,.i c3

"

0

300

Z

a. 200

0

GEORGIA

BLUE ILLITE

SHERMAN ISLAND

Figure 2. The concentrations of phenol and i t s breakdown products in the sorbed and aqueous phases of experiments conducted with and without the addition of phenol-degrading _P. putida.

2204

r a d i o a c t i v i t y accounted for 74, 69, and 61~ of the r a d i o a c t i v i t y present in the Georgia, blue lllite,

and Sherman Island sorbents, respectively.

For experiments in which P. putida was

added to the sol)s, the recovery of r a d i o a c t i v i t y was s i g n i f i c a n t l y lower and accounted for only 3, 10, and 16~ of the r a d i o a c t i v i t y present in Georgia, blue l l l i t e , and Sherman Island sorbents.

It

is apparent that microbial a c t i v i t y was responsible for

the decreased

exactabtltty and conversely, the increased retention of phenol-related carbon residues by each of the experimental sorbents.

These decreases in e x t r a c t a b i l i t y may be due to the

incorporation of phenol-derived carbon by microbes attached to the sorbent surfaces and/or to enzymatic polymerization reactions (10). The i d e n t i t i e s of the aromatic compounds that remained in the aqueous phase of each experiment were determined by HPLC. Phenol was the only aromatic compound present in the aqueous phase of the control experiments that were conducted with autoclaved sorbents, and i t accounted for ~96~ of a l l the r a d i o a c t i v i t y present in these fractions. conducted with P. putlda but no sorbent, catechol

In experiments

was i d e n t i f i e d as the only aromatic

constituent of the aqueous phase and was present at a final concentration of 0.12 gg/mL. The btodegradatton

of phenol in the presence of the experimental sorbents also yielded

catechol as the only aromatic degradation product (Georgia and Sherman Island = 0.04 gg/mL, Blue I l l i t e = 0.29 gg/mL). Other aromatic degradation products (e.g., hydroquinone) were not detected in any experiments.

resorcinol and

Catechol is an i n i t i a l product of phenol degradation and i t s formation'is requtred for the subsequent cleavage of the aromatic ring (4, 6).

Although catechol is more water soluble

than phenol, i t s sorptton to soils has been shown to be greater than that predicted by i t s partitioning properties (2).

This greater sorption is most l i k e l y due to the formation of

hydrogen bonds that are optimized by the ortho-orientatlon of the hydroxyl groups (1).

Due

to i t s stronger sorption, catechol may be less btoavatlable than phenol in the presence of soil.

In addition to the posslble formation of phenolfc po(ymers, this factor may also

account for the decreases in extractable residues that were observed in our experiments. This process is important because many aromatic compounds that are degraded via catechol intermediates (11, 12) may become less mobile and more persistent as a result of t h e i r blotransformation in the subsurface. ACKNONLEDGMENT Hork performed under the auspices of the Ecological Research Division of the U.S. Department of Energy by the Lawrence Llvermore Natlonal Laboratory under Contract H-7405-Eng-48.

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1.

Southworth, G.R., Herbes, S.E., Franco, P.J., and Glddlngs, 3.M., Pollut., 24, 283 (1985).

Hater, Air, and Soil

2.

Boyd, S.A., Soil Science, 134, 337 (1982).

3.

Harrison, F.L., Bishop, D.J., and Mallon, B.J., Environ. Sci. Technol., 19, 186 (1985).

4.

Varga, J.M., and Neujahr, H.Y., Plant and Soil, 33, 565 (1970).

5.

Scott, H.D., Holf, D.C., and Lavy, T.L., J. Environ. Qual., 12, 91 (1983).

6.

Bayly, R.C., and Higmore, G.3., O. Bacterloloqy, If3, 1112 (1973).

7.

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8.

Bishop, D.J., Knezovich, 3.P., and Harrison, F.L., Environ. Scl. Technol., (submitted).

9.

Scott, H.D., Holf, D.C., and Lavy, T.L., 3. Environ. Qual., I I , 107 (1982).

10.

Berry, D.F., and Boyd, S.A., Soil Scl. Soc. Am. O., 48, 565 (1984).

If.

Gibson, D.T., Sclence, 16l, 1093 (1968).

12.

Masunaga, S., Urushlgawa, Y., and Yenezawa, Y., Chemosphere, 12, 1075 (1983). (Received in Germany 6 August 1988; accepted 20 September 1988)