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APPLICATIONS OF FOUR BACTERIAL AND FUNGAL SPECIES FOR BIOREMEDIATION OF SELECTED POLYCYCLIC AROMATIC
HYDROCARBONS AND PENTACHLOROPHENOL
Hamid Borazjani, David A. Strobel, Curry Templeton, Jennifer Wagner, Mary Hannigan, Fran Hendrix,
Michele Welch and Lynn Prewitt
Mississippi Forest Products Laboratory P. 0. Drawer FP
Mississippi State, MS 39762
ABSTRACT
In order to test their ability to degrade polycyclic aromatic hydrocarbons (PAHs) and pentachlorophenol (PCP), two fungi ( Cladosporium and Aspersillus m) were isolated from soil contaminated with wood treating wastes and from an automobile fuel filter. Another two test bacteria (JP-Y and JP- W) were isolated from highly contaminated wood-treating waste process water.
In a 120-day bench soil study, Aspersillus was tested for transformation of naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, and benzo(a) pyrene. CladosDorium was tested for degradation of selected PAHs and pentachlorophenol in contaminated soil.
significant reduction in PAHs after 60 and 9 0 days, respectively. Of the two bacterial isolates, only JP-Y showed significant degradation of PAHs and PCP in 5 days. JP-W showed no degradation potential for these compounds.
Cladosporium a and AsDersillus produced a
INTRODUCTION
Microorganisms in natural systems such as soil, sediment, and water are instrumental in the degradation of chemical wastes which enter these systems. Biological degradation of pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and chlorinated phenols, has received widespread attention in recent years. PAHs containing three or fewer aromatic rings have been completely biodegraded in a variety of environments by many different genera of microorganisms that can utilize these compounds as sole sources of carbon and energy (3). Microorganisms from polluted
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environments have been isolated and used to break down chlorinated hydrocarbons (1,2,4,5,7). Mixed bacterial cultures, which originated from soil contaminated by wood preservative chlorinated phenols in sawmills, effectively degraded pentachlorophenol (PCP) ( 8 ) .
This paper reports on laboratory evaluation of four microorganisms on bioremediation of selected PAHs and PCP in contaminated soil and process water.
MATERIALS AND METHODS
BIODEGRADATION STUDY
1) ASPERGILLUS TEST
The fungus Aspersillus was isolated from an automobile fuel filter and was grown in a liquid medium (Potato Dextrose Broth [PDB] from Difco Laboratories, Detroit, Michigan). The sandy loam test soil came from Wiggins, Mississippi. The soil was thoroughly dried under a fume hood, was homogenized by hand mixing, and was screened with a 2 mm sieve.
with 0.5 g naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, and with 0.05 g of chrysene and benzo (a) pyrene (Aldrich Chemical Company Inc., Milwaukee, WI). The chemicals were dissolved thoroughly in methylene chloride. The solvent was allowed to evaporate from the soil under a fume hood. Fifty grams of screened sterilized sawdust was added to the spiked soil, and the mixture was then homogenized for 2 hours in a clean glass jar using a ball mill with 50 revolutions/minute. Two treatments with three replicates: controls with no fungal addition and inoculated with fungal solution added, were used in this study. For the controls, 120 g of soil mixture and 17 mL sterile PDB were added to each of three brown leaded pyrex dishes. For the fungal treatments, 120 g of soil mixture and 17 mL of homogenized fungal solution were added to each dish. Soil in each dish was mixed with a glass rod. A 20 g soil sample for chemical and microbial analysis was taken from each dish for day - 0. Dishes were stored at 22OC. Samples were monitored weekly for moisture adjustment and soil aeration. For a period of 120 days, 20 g soil samples were taken from each dish at 30-day intervals.
One thousand grams of soil was autoclaved and then spiked
EXTRACTION METHOD FOR ASPERGILLUS TEST
Five-gram, air-dried soil samples were weighed into a 50 mL polypropylene screw-cap centrifuge tube. Three mL of 9 , 10- diphenyl anthracene was added as an internal standard (Aldrich Chemical Company Inc., Milwaukee, WI) along with 12 mL methylene chloride to each sample. The samples were mixed using a
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vortexer for five minutes and allowed to settle. Two mL of the methylene chloride extract were filtered through a 0.2 pm 13 mm nylon filtration disc into an autosampler vial. The samples were analyzed for PAHs using a Hewlett Packard 1090A Liquid Chromatograph with a diode-array detector.
2) CLADOSPORIUM TEST
The fungus Cladosporium was isolated from contaminated soil at a wood treating site in California and was cultured in PDB liquid medium. The soil was air-dried and screened with a 2 mm sieve. Six hundred g of screened soil was mixed with 30 g of straw and 30 g of chicken manure. The mixture was then homogenized for 2 hours in a clean glass jar using a ball mill with 50 revolutionslminute. A control treatment and a loaded fungal treatment were used. For controls 100 g of soil mixture and 12.5 mL of sterilized deionized water were added to each of three brown leaded pyrex dishes to provide 1 2 . 5 % moisture content. For the fungal treatment, 100 g of mixture and 12.5 mL of cladosporium fungal solution were added to each dish. Soil in each dish was mixed with a glass rod. A 30 g soil sample for chemical and microbial analysis was taken from each dish for day - 0. Dishes were stored at 22OC, and samples were monitored weekly for moisture adjustment and soil aeration. 30 g soil samples were taken from each dish at 30-day intervals for 60 days. Soil samples were extracted using a soxhlet extraction procedure (EPA methoda3540)(6). The results were analyzed by gas chromatography (EPA method 8040 and 8100) (6).
3) PROCESS WATER TEST
Two bacteria that had been isolated from highly contaminated process water were used in this test. Bacterial isolates (JP-Y and JP-W) were cultured in liquid medium (nutrient broth, Difco Laboratories, Detroit, Michigan). Nine 250 mL flasks with cheese cloth stoppers were sterilized, and a solution of processed water from a wood-treating plant in Missouri was prepared and homogenized in a sterile blender.
Three treatments with three replications each were used as follows: 1.) controls, no bacterial solution 2.) JP-Y bacterial solution added, and 3 . ) JP-W bacterial solution added. For the controls, 100 mL of process water and 15 mL of sterilized nutrient broth were added to each of three flasks. For JP-Y bacterial treatments, 100 mL of process water and 15 mL of bacterial solution were added to the three flasks. The remaining three flasks each contained 100 mL of process water and 1 5 mL of JP-W bacterial solution. The flasks were placed on a wrist- action shaker for a 5-day period at room temperature. At the end of that time, a 5 mL sample was removed from each flask for
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microcounts. The remaining solution in each flask was extracted using a liquid-liquid extraction procedure (EPA Method 3520)(6). The results were analyzed by gas chromatography (EPA 8040, 8100) (6).
MEDIA PREPARATION AND COLONY COUNTS
The media used were nutrient agar (NA), 23 g in one liter of deionized water, NA amended with 5 mg/L of technical-grade PCP (P) (Vulcan Materials Company, Witchita, Kansas), NA amended with 20 mg/L of whole creosote (C), NA amended with a combination of 5 mg/L of PCP and 2 0 mg/L of whole creosote (P + C), potato dextrose agar (PDA), 39 g in one liter of deionized water, PDA amended with antibiotics (PDAA) 120 mg/L of streptomycin sulfate (Nutritional Biochemical, Cleveland, Ohio) and 30 mg/L of chlortetracycline (JCN Biochemicals, Cleveland, Ohio), and PDAA amended with 20 mg/L of whole creosote (PDAAC). The NA and PDA were autoclaved for 20 minutes at 15 psi and 12PC and then cooled to 55OC. Both creosote and PCP were dissolved in methyl alcohol and added to cooled NA and PDA. The antibiotics were added to the cooled liquid medium before pouring into petri dishes. The pH of the media was adjusted to 6.9 to 7.1 before autoclaving. Twenty-five mL of NA, C, PI C + PI PDAA and PDAAC was poured into disposable petri plates and allowed to solidify.
For colony counts, duplicate samples of both loaded and non- loaded soil were used. Colonies were counted after 24 to 96 hours of incubation at 28OC. A Darkfield Quebec Colony Counter (A0 Scientific Instruments, Keene, New Hampshire) was used to count the number of colonies on each plate.
estimate of the total number of bacteria. plates, the count represented the approximate number of bacteria that was acclimated to creosote. On PCP-containing plates, the count represented the approximate number of bacteria that was acclimated to PCP. plates, the count represented the approximate number of bacteria that was acclimated to both creosote and PCP. the count represented the approximate number of total fungi. On PDAAC plates, the count represented the approximate number of fungi that was acclimated to creosote. technique was used to recover microorganisms from process water and contaminated soil.
The number of counts recovered on NA plates provided an On creosote-containing
On combined PCP and creosote-containing
On PDAA plates,
The dilution plate
220
RESULTS AND DISCUSSION
Aspergillus Test. The effects of AsDersillus a treatment on the biological breakdown of selected PAHs during a period of 120 days are summarized in Figures 1-5. These figures clearly show that no degradation occurred during the first two months of this study. PAHs were biodegraded significantly by day 90, and this reduction continued until the end of the test (day 120). This reduction correlates with the significant increase in fungal population on day 90 (Table 1). The fungal population increased significantly in control samples also. Many airborne fungi, as well as aspergillus, cross-contaminated the control samples, which explains the reduction of PAHs in control samples. Bicyclic and pentacyclic PAHs were the most biodegraded compounds (Fig. 1, 4). Half life analysis of the data showed a significant difference between treated soil and control (Table 2).
Cladosporium Test. The effects of CladOSDOriUm on the biological breakdown of selected PAHs and PCP during a 60-day test are summarized in Figures 6-11. In general, the disappearance rate of PAHs and PCP for fungus treatment was good (Fig. 10,ll). Figures 6-9 show a variable pattern of breakdown rates for Ira selected group1@ of PAHs. It is probable that these results were caused by a breakdown of compounds higher in molecular weight than the pentacyclics to lower molecular weight compounds. The microbial population for this study is summarized in Table 3. Microorganisms for both treatments grew steadily throughout the 60 days. The largest increase occurred for the Cladosporium population recovered on PDAA media (Table 3).
Process Water Test. The effects of each experimental treatment on the degradation rate of PAHs and PCP is summarized in Figures 12-13. JP-Y isolate showed a significant degradation of PCP and PAHs. JP-W treatment showed no degradation in the process water study. Bacterial populations remained very high for both JP isolates (Table 4 ) . Possibly, the JP-W could have utilized other organic compounds in the process water instead of the PCP and selected PAHs.
CONCLUSION
Apparently, bacteria or fungi capable of degrading PCP and PAHs can be isolated and manipulated for bioremediation of chemical wastes. However, in most cases where the rapid degradation of PCP and PAHs have been demonstrated, the best source of inoculum was from areas where PAHs and PCP had been used for a long period of time. the JP-W isolate. JP-Y and JP-W were both isolated from the same process water. whereas JP-W has practically no effect on breakdown of PCP or PAHs. This highly contaminated process water contained other compounds besides the compounds analyzed in this study, and the
An exception in this study was
JP-Y has excellent biodegradation ability,
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JP-W may have utilized those chemicals. This explanation can be fully determined only by testing the process water for other organic constituents and monitoring the breakdown rate in the presence of JP-W isolate.
Based on the results of these experiments, contaminated soil and water could be effectively bioremediated by using proper microorganisms and correct methods of treatment. This technique can be applied easily to the field for bioremediation of chemicals in soil and water.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge A & R Foreign Auto in Starkville, Mississippi for providing the AsDeraillus SE+. contaminated fuel filter.
_-
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REFERENCES
1. Bedard., D. L., Unterman, R. , Bopp, L. H. , Brennan, M. J. , Haberl, M. L. and Johnson, C. 1986. Rapid assay for screening and characterizing microorganisms for ability to degrade polychlorinated biphenyls. Appl. Environ. Microbial. 51, 761-768.
2. Edgehill, R. U. and R. K. Finn. 1982. Isolation, Characterization and growth kinetics of bacteria metabolizing pentachlorophenol. Eur. J. Appl. Microbiol. Biotechnol. 16:179-184.
3 . Heitkamp, Michael A., and C. E. Cernigla. 1988. Mineralization of polycyclic aromatic hydrocarbons by a bacterium isolated from sediment below an oil field. Appl. Environ. Microbiol. Vol. 54, NO. 6. 1612-1614.
4 . Larsson, Per, L. Oklan and Lars Tranik. 1988. Microbial degradation of Xenobiotic, aromatic pollutants in humic water. Appl. Environ. Microbiol. Vol. 54, No. 7. 1864-1867.
5. Steiert, J. G. and Crawford R. L. 1985. Microbial degradation of chlorinated phenols. Trends Biotechtol. 3, 300-305.
6. Test Methods for evaluating solid wastes. 1986. U.S. EPA. SW- 846, Third Edition.
7. Valo, R. J. Apajalathis, and M. Salkinoja-Salonen. 1985. Studies on the physiology of microbial degradation of pentachlorophenol. Appl. Microbiol. Biotechnol. 21:313-319.
8. Watanabe, I. 1973. Isolation of pentachlorophenol decomposing bacteria from soil. Soil Sci. Plant Nutr. Tokoyo. 19 (2) : 109-116.
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150
100
50
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Figure 1. Biological breakdown of selected bicyclic PAHs in soil.
CONC. (ppm)
Aspergillus test
DAY 0 DAY 30 DAY 60 DAY 90 DAY 120
DAY
CONTROL - TREATMENT
2 2 8
1000
800
600
400
200
0
Figure 2. Biological breakdown of selected tricyclic PAHs in soil.
CONC. (ppm)
Aspefgi l us test
DAY 0 DAY 30 DAY 60 DAY 90 DAY 120
DAY
CONTROL TREATMENT
229
Figure 3. Biological breakdown of selected tetracyclic PAHs in soil.
CONC. ( p p d 1200
Aspergi l lus test
1000
800
600
400
200
0 DAY 0 DAY 30 DAY 60 DAY 90 DAY 120
DAY
CONTROL TREATMENT
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Figure 4. Biological breakdown of selected pentacyclic PAHs in soil.
40
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30
25
20
15
10
5
0 I
Aspergillus test
DAY 0 DAY 30 DAY 60 DAY 90 DAY 120
DAY
CONTROL TREATMENT
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Figure 5. Biological breakdown of total selected PAHs in soil.
CONC. (ppm) 2500
Aspergillus test
2000
1500
1000
500
0 DAY 0 DAY 30 DAY 60 DAY 90 DAY 120
DAY
a CONTROL TREATMENT
2 3 2
60
50
40
30
20
10
0
Figure 6. Biological breakdown of selected bicyclic PAHs in soil.
CONC. (ppm)
Cladosporium test
nAv n nAY 30 DAY 60 Y , . , " w . . . -- - - - - - -
DAY
CONTROL TREATMENT
2 3 3
500
0
Figure 7. Biological breakdown of selected tricyclic PAHs in soil.
CONC. (ppm) n
I
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DAY 0 DAY 30
DAY DAY 60
CONTROL TREATMENT
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Figure 8. Biological breakdown of selected tetracyclic PAHs in soil.
CONC. (ppm)
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Cladosporium test
DAY 0 DAY 30 DAY 60
DAY
CONTROL TREATMENT
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Figure 9. Biological breakdown of selected pentacyclic PAHs in soil.
CONC. (ppm)
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60
40
20
0
Cladosporium te 8 t
DAY 0 DAY 30
DAY DAY 60
CONTROL TREATMENT
2 3 6
1200
1000
800
600
400
200
0
Figure IO. Biological breakdown of selected total PAHs in soil.
CIadosDorium test
DAY 0 DAY 30
DAY DAY 60
CONTROL TREATMENT
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Figure 11. Biological breakdown of pentachlorophenol in soil.
CONC. (ppm) 1
200
150
100
50
0 DAY 0 DAY 30
DAY
Cladosporium test
DAY 60
CONTROL TREATMENT
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Figure 12. Biological breakdown of PCP in contaminated process water.
CONC. (mg/L) (Thousands)
30
25
* 20
15
10
5
0 CONTROL JP-Y JP-W
TREATMENTS
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25
20
15
10
5
0
Figure 13. Biological breakdown of PAHs in contaminated process water.
CONC. (mg/L) (Thousands) ,/'
CONTROL JP-Y JP-W
TREATMENTS
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