Effects of bioremediation on residues, activity and toxicity in soil contaminated by fuel spills

Sod Bwl. Blochem. Vol. 22. No. 4. pp. 501-505. 1990

oo?&0717 90 53.00 + 0.00 Copyright C 1994 Perg;imon Press plc

Pnn~cd in Great Bntain. All nghls reserved

EFFECTS OF BIOREMEDIATION ACTIVITY AND TOXICITY IN SOIL FUEL SPILLS XIAOPIKG Department

of Biochemistry

WASG

and RICHARD

and Microbiology. Cook NJ 08903-0231, (Acceprcd

ON RESIDUES. CONTAMINATED

BARTHA’

College. U.S.A.

I5 Ocroher

BY

Rutgers

University.

New

Brunswick.

1989)

Summary-Triplicate outdoor lysimeters were contaminated by 1.3 ml cm-r spills of jet fuel. heating oil and diesel oil, respectively. One of each set of triplicates was left untreated. one was tilled only. and one received complete bioremediation treatment consisting of liming, fertilization and weekly tilling. For 20 weeks during summer. hydrocarbon residues were monitored by quantitative gas chromatography. Microbial activity was measured by fluorescein diacetate hydrolysis. Toxicity was assessed by Microtox measurements, seed germination and plant growth bioassays. Persistence and toxicity of the fuels increased in the order ofjet fuel < heating oil c diesel oil. In each case. bioremediation treatment strongly decreased fuel persistence and toxicity and increased microbial activity as compared to contaminated but untreated soil. Tilling alone had a favorable but more limited effect. Good correlations were found between residue decline. microbial activity and toxicity reduction. Persistence and toxicity also correlated with the hydrocarbon composition of these three fuels. Our findings indicate that biorcmediation trcatmcnt can restore fuel spill contaminated soils in 4 -6 weeks to a degree that they can support plant cover. Recovery of the soil is complete in 20 weeks.

MATISIAIS

INTROI>CCTION ruptures, tank failures and various other production. storage and transportation accidents crcatc hydrocarbon contarninatcd soils (Bosscrt and Bartha, 1984). occasionally on a very large scale. As an cxamplc, the 1975 fJilurc of the Continental Pipeline near Crosswicks. N.J. rcsultcd in the spill of cu I.9 million I. kerosene that inundated I.5 ha agricultural land (Dibble and Bartha. 1979). Hydrocarbon contaminated soil tails to support plant growth and is a source of groundwater contamination. Cleanup by excavation and incineration or transport and burial of the soil in secure chemical landfills is prohibitively expensive when large amounts soil are involved. Bioremediation of such contaminated soils by a process similar to the land treatment disposal of oily wastes may ofTcr a less expensive yet ecologically-acceptable alternative (Bartha. 1986). Bioremediation optimizes conditions for microbial hydrocarbon degradation through mixing, aeration, pH control and mineral nutrient (fertilizer) addition. Biorcmcdiation of polluted soil has been performed on emergency basis (Dibble and Bartha, 1979). but to date no controlled studies have been performed to rigorously document the effectiveness of the trcatmcnt for various fuel contaminants. In this study, simulated spills of three hydrocarbon fuels (jet fuel. heating oil and dicscl oil) were applied to soil in outdoor lysimctcr units. Changes in hydrocarbon concentration and toxicity and the accompanying changes in microbial activity with time were measured with or without bioremediation treatment. Pipclinc

of

‘To

whom all correspondence

should be addressed. 501

AN0 METIIOIIS

Biorcmcdiation expcrimcnts wcrc‘ made in outdoor lysimctcrs (Fig. I). The 90 x 90 cm surface area of the lysimcters allowed a dcsircd scale up of laboratory cxpcrimcnts under ambient outdoor climate conditions. Containment of the polluted soil in lysimctcrs avoided any danger of groundwatcr pollution and any hydrocarbon removed by gravity flow or leaching rather than by degradation could be collected and measured. Although open on the sides, a glass roof prevented precipitation from reaching the lysimctcrs and, instead, they were watered weekly by hand at 2.6 ml cm-‘. a rate projected from the average local precipitation (I I2 cm yr-‘). The 20 week experiments were conducted during the summer season from late April to late September of 1988. The average monthly soil temperatures at IOcm depth were 16. 22, 24. 25 and l9C for May, June, July, August and Scptember, respectively, (Department of Meteorology, Rutgers University). Ten lysimcters were available for these experiments and filling them as depicted in Fig. I required I.7 tonnes of sand and 4.5 tonnes of soil. Pilot experiments conducted in the previous (1987) season revealed that soil delivered in bulk from nearby construction sites was not comparable to the hand-collected topsoil we had used in our laboratory experiments (Song et al.. 1990) and needed improvement to approach the water holding capacity and organic matter content of topsoil. The upgrading was achieved by adding to the soil at the approximate rate of 5% by volume a peat-sand-perlite mixture (equal parts). This “upgraded” soil had a texture of 68% sand. 16% silt and 16% clay, 2.2% organic matter and a pH of 6.7. With mechanical compacting and watering. the density of undisturbed field soil was

502

XIAOPISG WASC and RICHARD BARTHA

I

9Ocm

-I

Fig. I. Cross section of a lysimeter unit used m fuel spill bioremediation experiments. Triplicate units were contaminated by JF. HO or DO. The tenth unit served as uncontaminated control.

approximated. To sets of three lysimeters, filled with semi-moist soil in the described manner simulated spills of jet fuel (JF). heating oil (HO) and diesel oil (DO) were applied at the rate of 2.3 ml cm -:. No breakthrough of the fuels to the drainage spout was observed after application or during subsequent waterings. while extensive breakthrough had been observed in the previous season’s pilot experiment that used inferior subsoil (data not shown). No fuel was applied to the last (No. IO) lysimctcr that served as source of uncontaminated control soil. From the sets or three identically contaminated lysimctcrs, one was Icli untrcatcd cxccpt for watering. One was. in addition, tilled wcskly by a hand shovsl to a depth of I5 cm. The third rcccivcd full biorcmcdiation treatment consisting of pH control by liming (55 mg powdered agricultural limestone cm .‘) and fcrtilixcr. Limcs1onc application W;IS based on a soil liming curve and raised the pH from 6.7 10 7.4. Fertilizer addition (10.0 mg urea and 4.3 mg superphosphate cm “) was designed to establish in the fuel contaminated soil approximate C: N and C: P ratios of 200:1 and lOOO:l, rcspcctively (Bar1ha. 1986). These lysimeters were also tilled weekly. No treatment except for watering was applied to the uncontaminated soil control. Periodically, the lysimeter soils were sampled for analysis by removing 5 scoops of soil from different parts of the lysimeter to a depth of I5 cm, then mixing and sieving these subsamples to prepare one composite. A 78 g amount (60g by dry wt) of the composite soil sample was mixed with sufficient anhydrous Na,SO, to absorb moisture and was Soxhlctextracted with I50 ml CH,CI, for 6 h. The extract was brought to volume. As internal standards, octadecanc was added to extracts containing JF and tctracosanc to extracts containing HO and DO. Analysis was performed using a IO m x 0.53 mm fused silica macrobore capillary column with an immobilized polydimcthyl siloxane phase (Alltcch Associates. Deerfield, Ill.) in a Hewlett-Packard Model 5890 gas chromatograph (Avondale. Pa) with Model 3392A integrator. Nitrogen carrier flow was 30 ml min-‘, hydrogen and air for the FID detector were 40 and 200 ml min-‘. respectively. Injection port was I50 C. FID 250 C. The initial oven temperature of 50-C was programmed at 4 C min-’ to 200 C for JF and 205 C for HO and DO.

Parallel with hydrocarbon residue analysis. microbial activity in the collected soil samples was also assessed. Microbial activity was measured by the fluorescein diacetate (FDA) hydrolysis assay (Schnfirer and Rosswall, 1982). FDA was dissolved in acetone (2 mg ml -‘) and stored at -20 C. Fresh soil (I g wet wt) was placed in a I25 ml Erlenmeyer flask containing 50 ml of 60 mM phosphate buffer (pH 7.6). To this soil suspension was added 0.5 ml of FDA stock solution (final cont. 20!1I ml-‘). After I h at 27 C on a rotary shaker (New Brunswick Scientific Co., Edison. N.J.) at 200 rev min-‘. 50 ml of acetone was added to stop further FDA hydrolysis. Soil was removed from the suspension by centrifugation for 5 min at 6000 rev min.’ followed by filtration through Whatman No. 3 tilter paper. This produced a clear solution with a low background absorbance. The amount of FDA hydrolyzed was measured as absorbance at 490nm with a spcctrophotometer (Bausch & Lomb. Spectronic 2000. Rochester. N.Y.). As a measure of acute toxicity of the fuclcontaminated soil we used the Microtox assay (King, 1984) as applied to hydrocarbon-contaminated soils by Matthews and Hastings (1987). A Microlox Model 2055 instrument was used according to the Microtox Manual operating proccdurcs (Microblcs Corporation. Carlsbad, Calif.). EC,,, values (5 min) wcrc calculated by a programmed Sharp EC-5 I50 calculator. All numhors in Microtox dctcrminations rcfcr 10 the weight of dry soil used in the preparation of the extract. Since rc-vegetation of soils contaminatcd by fuel spiils is often a dcsirahlc goill, seed germination and plant growth bioassays using the monocotylcdonous rycgrass (Scccrl~~wrtwk) and the dicotylcdouous soybean (G!,+rc~ rn~.~) wcrc also made. f:or thcsc assays. surface soil from the lysimctcrs was removed to a depth ol’ IS cm, sieved, mixed and placed into nursery flats. Each nursery flat was sccdcd with 100 seeds of either ryeyrass or soybean. To exclude cffccts of lime and fertilizer on plant and “tilled only” growth, to “control ” “untreated” soils the same proportional amount of lime and fertilizer was applied just prior to planting as the “treated” soil had received. The tlats were kept in a grcenhousc under favorable conditions for plant growth and the perccntagc of seed germination was counted after IO days. Subsequently. the plants were allowed to develop. At 30 days. they were harvested, dried a1 70 C for 3 days and the plant biomass dry weight was recorded. Plan1 development and morphology was also rccordcd photographically.

Figures 2A. 3A and 4A show the time course of hydrocarbon disappcarancc from the variously trcatcd soils. Certain similarities and differences are cvidcnt. In each case, tilling alone increased disappearance as compared to untreated soil, but not to the same degree as complete bioremediation treatmcnts consisting of liming. fertilization and tilling did. Pcrsistcncc of the fuels increased in the order of JF < HO < DO, and the proportional effect of biorcmcdiation treatment appeared to increase in the same order. However, 20 weeks of biorcmediation trcatmcnt lowered hydrocarbon concentration to

Effects

of fuel spills

503

0 lJntm . TIlled

.

TIW A TmW rbltlml

A Trecitd Acantrd

(A)

(A)

-.-.

II

I

I

I

. I

I

I

I

I

(B)

II 2

4

III 6

6 Time

10

II 12

14

II

16

1e

I

20

(w66ks)

Fig. 2. Changes in hydrocarbon (IX) residues (A). microbial FDA hydrolysis activily (B). and Microtox EC.,, values (C) with time in soil contaminated by a 2.3 ml cm -! jet fuel spill. The “unlrc;rtcd” soil was contaminated but rcccivcd

no further treatment. The “tilled” soil was Mud weekly to I5 cm depth. The “trcarcd” sample rcccivcd lime, fcrtilizcr and weekly tilling. The “control” soil was not contaminated and rcceivcd no treatment. Tht: symbols apply to all three porrions

(A, B. C) of the figure.

below Smg g-l soil for even the most persistent DO, while at the same time in untreated soil DO concentration was still at 21.8 mg g-l. In laboratory-scale experiments, ic is common lo differentiate biodegradation from evaporation and other physico-chemical losses by the use of poisoned controls (Pramer and Bartha. 1972). The amount of poison required and disposal problems with the poisoned soil precluded this approach in these experiments. Tilling, in addition to increasing biodegradation by increased availability of oxygen, may also have contributed to evaporative losses. Therefore, it was of special interest lo measure the activity of the soil microbial community in response lo the fuel spills. Flat or depressed microbial activity would signify little or no microbial involvement while strong positive responses would indicate a large biodegradative contribution. Figures 29. 39 and 49 revealed strong positive changes in FDA hydrolysis activity in response lo fuel spills. The strongest increases in activity were evident with complete bioremediation treatment, the smallest ones with contaminated but untreated soil. Microbial activity in uncontaminated control soil

Time (weeks) Fig. 3. Changes in tIC rcsiducs (A), microbial FDA hydrolysis activity (B) and Microtox EC? values (C) with time in soil contaminated by a 2.3 ml cm- heating oil spill. Treatments and symbols as in Fig. 2.

showed only very slight fluctuation. As one would expect, microbial activity was inversely correlated with fuel persistence. JF stimulated microbial activity the most; HO and DO to lesser degrees. The parallel hydrocarbon residue and microbial activity measurements lent strong support 10 the argument that biodegradation was the dominant component of the rcmediation process. Curious, however, was the lag in microbial activity during the first 6 weeks of the measurements when actually most of the hydrocarbon disappeared. This apparent paradox was resolved when separate experiments revealed that the hydrocarbon biodegradation products strongly inhibited, most likely by a competitive mechanism, the FDA hydrolysis reaction. The observed increases occurred in spite of this inhibition because an up lo three orders of magnitude increase in microbial numbers (Song and Bartha. 1990) more than compensated for this inhibition. However, a clear expression of the stimulation became apparent only after most of the inhibitory hydrocarbon biodegradation intermediates had disappeared. Therefore, in this expcrimcnt FDA activity peaked c I2 weeks. Microtox measurements. performed only on untreated. treated and uncontaminated soil samples, are shown in Figs 2C. 3C and 4C. In the undegraded state at time 0, all three fuels exhibited toxicity (EC, = 80-90 mg of contaminated

moderate soil). The

and RICHARD BARTHA

XIAOPISGWAS

I

(C)

loo: 50

0

respectively. Toxicity was higher but lasted for a shorter time with bioremediation-accelerated degradation. causing the crossover pattern of the curves. By week 20, both the treated and untreated soils had returned to background toxicity values. DO (Fig. 4C) exhibited a similar toxicity pattern to HO. but in an even more pronounced manner. Also. in this case toxicity declined to soil background amounts by 20 weeks only in bioremediation-treated samples. Significant residual toxicity was still evident in the untreated soil, correlating with the detection of polycyclic aromatic residues in this but not in the treated soil sample (Wang ef al.. 1990). Seed germination and plant growth data (Tables 1 and 2) were consistent with the hydrocarbon residue and Microtox measurements (Figs 2A. C-4A. C). The severity of seed germination and plant growth inhibition (JF < HO c DO) increased in the same order as persistence and Microtox toxicity values. At 4 weeks after the spill, JF inhibited seed germination in untreated but not in treated soil. Inhibition was stronger in case of HO and DO. but bioremediation at least partially restored the ability of the soil to support seed germination and plant growth. Morphological ctfects such as reverse geotropism were noted on some partially cmcrgcd seedlings. Similar cfi’ccts wcrc noted in oil sludge treated soil (Bosscrt and Bartha, 1985) and arc ascribed to the growth hormone-like action of somo polycyclic aromatic hydrocarbon components of the HO and DO. Al I4 weeks. no seed germination and plant growth inhibition was cvidcnt in the fuel contaminated soils cvcn without biorcmediation trcatmcnt cxccpt in the cast of the most persistent and toxic DO. Compared with the rcsiduc concentrations (Table I), it appears that phytotoxicity becomes insignificant after hydrocarbon rcsiducs from JF. HO and DO arc reduced from the initial SO-70 mg g-” soil lo < IS mg g-’ soil. At summer soil tcmpcraturcs and with biorcmcdiation treatment, these conditions can be attained in 4-6 weeks and at that time the rc-vegetation of a spill site can be attempted. The parallel measurements on hydrocarbon residues, microbial activity and residual toxicity in fuel spill contaminated soil mutually support each

I

2

I

4

I

I

6

e

Time

I

10

I

I

12

Iweeks

14

I

I

16

1e

I

20

1

Fig. 4. Chungcs in IIC rcsiducs (A). microbi;ll FDA hydrolysis activity (n) and Microran EC, wlucs (C) with time in soil contaminated by a 2.3 mlcm ’ diesel oil spill. Treatments and symbols 3s in Fig. 2.

subsequent behaviors of the three fuels diffcrcd in terms of toxicity. JF (Fig. 2C) was rapidly detoxified, Ecu, values returning to the control soil value of I20 mg in 2 weeks in case of treated, and in 6 weeks in case of untrcatcd soil. respectively. For HO (Fig. 3C). toxicity first increased during the initial phase of biodegradation. but started to decrease after 6 and I2 weeks for the treated and untreated soil samples, Table

I, Seed germination

after

IO days

in fuel-cont;rminatcd

I4 weeks after the will Germmation

FUCI Treatments

oroduclr JF

HO

Ryeyrars

Rycrtrass

Soybean 7X

95

Unlrcalcd

66

?O

TlllCd

7H

67

67

76

Treated

95

73

72

n3

Untreated

I?

?

65

xx

Tilled

73 77

26

73

X9

JS

7s

x0

6

0

6X

55

Umreucd Tdled

5X

JI

92

no x9

TrGlted

67

6X

93

None

Control

X0

X0

70

‘“L’nlrcalcd”

l~r~mcfers

rccewd

adjuswd

wtth

“Ttllcd”

solI was tilled

and “tilled” Ihc influence spell and

il(

14 weeks 1%)

-

Sovbean

TfCAlCd Do

4 and

soil.

Germinalwn

irl

weeks (%)

4

I)simercr

hmc lo pH

7.5. rcccivcd

ucckly

no trcatmenl.

N and P fcrCltzcr

but rcwved

sods. lime and fcrMizcr of these fxrors

n7

a fuel spdl only. The rod of”tre~ted”

on

no lime or

war applied

lysimclers

and was tdled

wi(s

weekly.

fcrrhrer. To “untreated”

at Ihe ttme of planriny

plmr yield. The “control”

IO cwlude

soil received

no fuel

Effects of

fuel spills

505

Table 2. Plant dry wright yields after 30 days of growth in fuelcontammawd lywmcter soil. 4 and I4 weeks after the spill FWI producls

Dry WI (g) at 4 weeks

Dry wt (g) ar I4 weeks soybean

Rycgrass

JF

L’nueated Tilled Treated

8.17 8.41 13.43

0.07 0.32 0.65

IO.32 IO.22 IO.28

0.30 0.25 2.20

HO

Cntreated Tilled Treated

0.00 3.80 9.70

0.00 0.03 0.20

15.96 IO.16 10.07

0.30 0.15 0.20

Do

Umrwed Tilled Treated

0.00 4.13 14.15

0.00 0.09 0.15

9.67 10.10 9.95

0.10 0.30 0.25

None

Conlrol

I4 53

2.70

9.82

2.00

Treatments’

SoybCNl

Ryegrass

‘See footnote lo Table I

other in describing the fate and effects of a particular fuel spill. They also allow comparisons between the three tested fuel products that are consistent with their known composition (Gary and Handwerk, 1984; Song YI al., 1990). Of the three fuels. JF has the lowest boiling range and highest proportion of aliphatics. DO has the highest boiling range and is richest in aromatics, including many polycyclic aromatic components. HO is intermediate but closer in its charateristics to DO than to JF. Aliphatics in the medium carbon number range (C,2-C2,) arc the most readily mctabolizcd hydrocarbons (Rartha, 1986) and thus our findings make good scnsc also in terms of structure-biodegradability correlation. Our cxpcrimcnts show that under favorable tcmpcraturc conditions hiodcgradation can restore soil contaminated by JF. HO and DO spills to a nontoxic, low-rcsiduc condition and a simple biorcmcdiation treatment consisting of pH control. fertilization and tilling can accomplish this in less than one growing season. Although the scale of these experiments does not allow a realistic cost analysis, for most situations the described bioremediation treatment appears ecologically and financially more attractive than incineration or storage in “secure” chemical landfills. Aclitro,~,/n/~c,,~~~nr-This iment Station Publication by SI~I~ funds.

New Jersey Agricultural ExperNo. D-01 502-02-X9 was supported

REFERESCES Bartha R. (1986) biodegradation.

Biotechnology of petroleum pollutant .\licrohiu/ Eco/o,q~ 12. 155-172.

Bossert I. and Bartha R. (1984) The fate of petroleum in soil ecosystems. In /‘err&urn Microbiology (R. M. Atlas. Ed.). pp. 453-473. Macmillan, New York. Bossert 1. and Bartha R. (1985) Plant growth in soils with a history of oily sludge disposal. Soil Science 140, 75-77. Dibble J. T. and Bartha R. (1979) Rehabilitation of oilinundated agricultural land: a case history. Soil Science 128. 56 -600. Gary J. H. and Handwerk G. E. (1984) Refinery products. In Petroltwm Rt’Jinin~: Techndogy nnd Economic.~. 2nd edn (J. H. Gary, Ed.). pp. S-15. Marcel Dekker, New York. King E. F. (1984) A comparative study of methods assessing the toxicity of bacteria of single chemicals and mixtures, In Ttrxicify Scrcming Procrdurt~r Uxin.q Buck~riul Sy.~tw~s (D. L. Liu and B. J. Dutka. Eds), pp. 175-194. Marcel Dckkcr. New York. Matthews E. and Hastings L. (19X7) Evaluation of toxicity test proccdurc for screening trcatahility potential of waste in soil. Tuxiciry As.w.~smenr 2. 265 -2X I. Prdmcr D. and Bartha R. (1972) Preparation and processing of soil samples for biodegradation studies. Enrironnwnrd Lrllers 2, 217-224. Schniirer J. and Rosswall T. (1982) Fluorescein diacetatc as a measure of total microbial activity in soil and litter. Applied und En~ironmmtol Microbiokq~ 43, I2561261. Song H.-G. and Bartha R. (1990) Effects ofjet fuel spills on the microbial community ofsoil. Applirduncf Enrironnwnrul Microbiolugy (in press). Song H.-G.. Wang X. and Bartha R. (1990) Bioremediation potential of terrestrial fuel spills. Appliedund Enrironmenrul Microbiology (in press). Wang X., Yu X. and Bartha R. (1990) Effect of bioremcdiation on polycyclic aromatic hydrocarbon residues in soil. Enrironmenrul Science und Technology (in press).