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Internationnl Biodeterioration & Btodegradation (1995) 397408

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Polycyclic Aromatic Hydrocarbon Biodegradation and Kinetics Using Cunninghamella echinulatu var. elegans

Teresa J. Cutright

Department of Civil Engineering, The University of Akron, Akron, OH 44325-3905, USA

(Received 27 April 1995; revised and accepted 11 May 1995)

ABSTRACT

Bioremediation is a process technology that uses microorganisms to degrade specific organic chemicals. In recent years, bioremediation has proven to be successful for the remediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Even though bioremediation has had a high success rate, the associated kinetics are still not fully understood. The kinetics become even more complicated when fungi are used for the remediation. The primary objective of this research was to determine the specific degradation rates for the bioremediation of PAH contaminated soils. Specifically, the kinetics associated with the fungi Cunninghamella echinulata var. elegans in conjunction with three supplemental nutrient solutions were investigated.

NOTATION

ARC: Alberta Research Council cosub: co-substrate concentration e(): total bacterial enzyme concentration MSl: Supplemental mineral solution 1 MS2 Supplemental mineral solution 2 MS3: Supplemental mineral solution 3 K: equilibrium constant K,: half-saturation constant, g/kg soil PAH: Polycyclic aromatic hydrocarbon PPM: Parts per million S: contaminant-substrate concentration, g/kg/day %l,X : maximum utilization rate, g/kg soil

397

398 T. J. Cutright

INTRODUCTION

The kinetics for modeling the bioremediation of contaminated soils can be extremely complicated (Rifai et al., 1991). This is largely due to the fact that the primary function of microbial metabolism is not for the remedia- tion of the environmental contaminants. Instead the primary metabolic function, whether bacterial or fungal in nature, is to grow and sustain more of the microorganism (Lee & Hoeppel, 1991; Bailey & Ollis, 1986; Levin & Gealt, 1993). Therefore, the formulation of a kinetic model must start with the active biomass and factors, such as supplemental nutrients, oxygen source, that are necessary for subsequent biomass growth.

The kinetic formulation is further complicated when co-substrates and secondary substrates are present (Bailey & Ollis, 1986). Researchers are not in agreement as to how the contaminants and supplemental nutrients should be classified. For example, when is each contaminant or supple- ment nutrient a primary, secondary or co-substrate? Can all of the contaminants be ‘lumped’ together as one substrate, or must they be addressed on an individual basis?

Depending on the classification used, the reproducibility and/or validity of the kinetic model will vary. However, it is possible to use kinetics to explain the degradation of soil contaminants. Recent bioremediation studies have indicated that a first order reaction, such as the well known Michaelis-Menton kinetic model, can adequately represent the degrada- tion kinetics (Baker & Herson, 1994; Dhawan et al., 1993; Erickson et al., 1993; Kostecki & Calabrese, 1992; Warith et al., 1992). This paper will discuss how this simplification can be used to estimate the kinetics for soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Specifi- cally, the kinetics associated with Cunninghamella echinulata var. elegans in conjunction with three different supplemental nutrient solutions will be investigated. For each of the preliminary experiments, all of the PAHs will be grouped as one substrate instead of approached individually. Experi- ments where each PAH compound is treated as a separate, individual substrate are currently being investigated.

MATERIALS AND METHODS

Soil and fungus origin

A manufactured gas plant (MGP) site sample labeled FC2-2 was used for all of the bioremediation experiments. The sample originated from the Alberta Research Council (ARC) primary clean-up facility in Alberta,

PAH biodegradation and kinetics 399

Canada. The initial PAH contamination level was determined to vary between 5 and 20% by weight. To eliminate any error due to this variance, the soil was thoroughly homogenized before beginning the experiments.

All three of the experiments used the fungus Cunninghamella echinulata var. elegans. The specific fungal strain 26269 was purchased from the Amer- ican Type Culture Collection (ATCC) in Rockville, MD. The fungal selection was based on an extensive literature search and preliminary experimental results (Atlas & Atlas, 1991; Atlas & Sayler, 1987; Pflug & Burton, 1988; Tabek et al., 1991). Bioremediation experiments were conducted batchwise over an 8 week period. The exact methodology for acclimating the fungus in the experiments was presented in a previous paper (Cutright & Lee, 1994). It is also identical to the methodology that is used in the control blanks, with the exception of adding 5 ml of acclimated fungal solution.

Supplemental mineral salt solutions

The results of a soil characterization determined that the soil was deficient in certain nutrients. Specifically, the soil has a nitrogen concentration of 0.28 wt%, an organic matter content of 13.2%, and a total phosphorus concentration of 0.6 ,ug P/g of soil. It is important to note that most of the phosphorus present in soil is ‘unavailable’ to the microorganisms, there- fore the concentration of 0.6 pg/g is extremely low (Baker & Herson, 1994). This level, in conjunction with the low level of nitrogen present, dictated the need for the addition of supplemental nutrients.

Three supplemental nutrients solutions were used in the experiments. Although these particular solutions have not been previously used with fungi, they have been extremely successful when used with bacteria (Aggarwal et al., 1991). The first solution, MS1 adds nitrogen and phos- phorous to the soil at a concentration of 0.25 g NHdNOs, 0.1 g K2HP04, in 1 1 distilled water (Travis, 1990). The other two media, MS2 and MS3, were developed by Bailey and Coffey (1986) and Gauger et al. (1990), respec- tively. These two solutions contain complex nutrient sources in addition to nitrogen and phosphorous additives. Specifically, MS2 consists of: O-3 g NHdNOs, 0.2 g KzHPOd, 0.2 g MgS04.7H20, 0.2 g CaS04, 2-O g malt extract, 2-O g glucose, 2.0 g casamino acid, and 1 1 distilled water. MS3 is comprised of: 4.0 g K2HP04, 4.0 g Na2HP04, 0.2 g MgC12, 2-O g NH&l, 0.001 g CaCl*, 1.42 g Na2S04, 0.001 g FeCls, and 1 1 distilled water.

Hydrogen peroxide was used as the oxygen source at a concentration of 0.1 g per liter of solution (Morgan & Watkinson, 1992). The hydrogen peroxide was introduced with the supplemental nutrient solutions. This enabled the flasks to be sealed without creating an anerobic atmosphere which would have been detrimental to fungal growth.

400 T. J. Cutright

Contaminant concentration

Contaminant concentration of the extractable PAHs in the untreated soil was determined at the beginning and at weekly intervals by Soxhlet extraction. Ten grams of dry soil were extracted with 350 ml of reagent grade methylene chloride for 15 h (EPA Method 3540). Extract containing the soluble tar was evaporated using a Buchi rotavapor (Model RE-111). The thimble containing the extracted soil was dried at 48°C for 4 h in a NAPCO vacuum oven. After drying, the weights of the thimble and extraction flask were used to determine the percent of hydrocarbons and, specifically, PAHs present in the soil.

Thirty milliliters of reagent grade trichloroethylene were used to re- dissolve the hydrocarbon-PAH contaminants for analysis by high perfor- mance liquid chromatography (HPLC). The HPLC was used to determine the qualitative and quantitative amounts of PAHs present. It was also used to track the individual PAH compounds present in the soil over the entire eight weeks of treatment.

Control blanks

Control blanks were conducted for eight weeks prior to initiating the experiments with Cunninghamella echinulata var. elegans. This was done to determine whether the remediation resulted from the fungus or was due to the enhancement of indigenous microorganisms.

Forty grams of contaminated soil were placed in a 250 ml Erlen- meyer flask. To each flask, 20 ml of distilled water or the appropriate supplemental solution was added. The flasks were then sealed to elim- inate the possibility of erroneously attributing any PAH loss to bior- emediation, when it may have occurred due to the volatilization of lower ringed structures. The resulting mixture was agitated for 1 min to ensure adequate contact, and placed in a darkened drawer for the duration of the experiment. This procedure was repeated for each supplemental nutrient solution. Three replicate flasks were conducted for each treatment. The control blanks and fungal-treated samples were run simultaneousiy.

Biomass concentration

The control blanks indicated that there was no activity from indigenous microorganisms, thus any loss in PAH concentration would be due solely to the presence of the fungi. Therefore, the biomass concentration, x, was determined from fungal spore counts performed at the initiation of each

PAH biodegradation and kinetics 401

experiment. For the fungi Cunninghamella echinulata var. elegans, the ‘normal’ spore count for the initiation of each experiment was 2 x lo8 spores/ml. Spore counts were then converted to biomass concentration by using an average soil density of 2.6 mg/ml (Stainer et al., 1963) and an average weight of one spore equal to 1.5 x lo-” g (Brodkorb & Legge, 1992). This biomass calculation was used in equation 5 of the kinetic formulation.

KINETIC FORMULATION

Analysis of the bioremediation experiments exhibited an exponential degradation trend for all three of the experimental sets (Cutright & Lee, 1994). Michaelis-Menton kinetics is a universal model used to describe the behavior spanning zero to first order reactions. The model can also be used to describe exponential degradation as exhibited by the bioremedia- tion experiments.

The soil used for the experiments contained a relatively high percentage (>50%) of > 4-ringed PAHs. During the remediation of these high mole- cular weight contaminants, the fungi excrete enzymes which initiate the degradation process (Tabak et al., 1991). In this instance, both the contaminants and the supplemental nutrient solution utilized by the fungi are classified as substrates. This two-substrate system is similar to typical enzyme catalyzed reactions. Since the Michaelis-Menton kinetic model is so well known, the preliminary steps for its formulation will not be presented here. The final form for a two substrate system is (Baker & Herson, 1994; Fogler, 1986; Froment & Bischoff, 1990; Levenspeil, 1972; Manahan, 199 1):

with

V max Sl

v=Kl +s1 (1)

(ke0 ~2) V

max = (s2 + K1,2)

and

(2)

(3)

where K is the equilibrium constant, eo and s the total fungal enzyme and substrate concentrations, and the subscripts 1 and 2 refer to the first and second substrates, respectively. From equations (l)-(3), it is apparent that

402 T. J. Cutright

if s2 is held constant and si is varied, the reaction will follow Michaelis- Menton kinetics. If s,,,b > Ks,cosub, at all times, then equation (1) can be rewritten as:

v s ))=--iT?E-

KS + s (4)

where v,, is the maximum utilization rate (g/kg), s is the contaminant- substrate concentration (g/kg/day), K, is the half-saturation constant (g/ kg), and v is the specific substrate utilization rate (per day).

When the biomass concentration (mg/l) is known, the specific substrate utilization rate can be calculated for steady-state systems by:

vmg As -1

v=nt*” * From equation (5), the instantaneous specific substrate utilization rate

can be determined from experimental data. At any point on the PAH- substrate degradation curve, the biomass concentration will be constant and the slope of the tangent line, at that point, will correspond to a constant As/At. With this approach a good approximation of ‘v’ can be determined at the beginning of the remediation treatment, before the degradation has reached a ‘quasi-steady state’. In this application, quasi- steady state refers to when the fungal degradation is occurring at a constant substrate utilization rate. this method can become an accurate determination of the specific substrate utilization rate when actual steady state conditions are present.

Since the biomass concentration was determined at t = 0, it is important to note that this is just an estimation of the substrate utilization rate. In order to develop a truly rigorous model, the biomass be monitored throughout the entire experiment.

would have had to

RESULTS AND DISCUSSION

Control blanks

At the end of the eighth week, the contaminant concentration had decreased by less than l%, thus indicating the inability of the indigenous microorganisms to remediate effectively the soil. The occurrence of d 1% loss can be attributed to the slight volatilization of contaminants during the initial agitation stage. Table 1 contains the weight of total PAH’s present in the untreated soil and after 8 weeks’ treatment with supplement nutrients.

PAH biodegradation and kinetics 403

TABLE 1 Control Blanks for PAH Contaminated Soil after 8 Weeks of Treatment with

Supplemental Nutrients

Sample Total contaminant weight (g PAH/kg soil) a

Percent loss of extractable PAHs

Untreated soil 25402 & 0.003 MS1 25.18 & 1.24 0.88 MS2 25.21 % 0.63 0.75 MS3 2522 & 0.42 0.71

‘See the first column of Table 2 for list of individual PAHs.

Treated soil samples

Table 2 contains the final average contaminant weight after 8 weeks of remediation for all three supplemental mineral salt solutions. The highest loss of extractable PAHs of 97% was achieved using supple- mental solution 1. After treatment with the fungus in conjunction with

TABLE 2 Total Contaminant Weight after 8 Weeks of Remediation with Cunninghamella echinulata

var. elegans

Compound Weight (g/kg soil)

Untreated MS1 MS2 MS3

Napththalene 11.12 Acenaphthylene 1.01 Acenaphthene 3.99 3.04 Pehananthrene 0.25 Anthracene 0.05 Flnoranthene 0.70 0.11 0.26 0.15 Pyrene 0.49 0.10 0.24 0.12 4-Terphenyl-did 2.03 0.82 0.39 Chrysene-diz 2.07 1.17 0.55 Benz(a)anthracene 0.69 0.14 0.42 0.20 Chrysene 0.52 0.10 0.26 0.13 Benzo(b)fluoranthene 0.87 0.13 0.16 0.23 Benzo(k)fluoranthene 0.16 - 0.06 Benzo(a)pyrene 0.22 0.10 Dibenz(ah)anthracene 0.31 -

Benzo(ghi)perylene 0.89 0.21 0.17 Indeno( 123cd)pyrene 0.04 -

Total 25.40 f 0.003 0.79 + 0.004 6.43 +Z 0.063 2.04 & 0.035 % Removal 0.97 0.75 0.92

404 T. J. Cutright

MSl, 11 of the compounds were no longer detected. By the end of the eighth week only fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(ghi)perylene were present. Figure 1 contains the degradation curve for Cunninghamella echinulata var. elegans in conjunction with supplemental solution 1, 2 and 3. As shown in Fig. 1, there was an overall ‘steady’ decrease in the amount of PAHs present over the 8 week period. In fact, most of the loss occurred by the sixth week. Very little degradation occurred during the final 2 weeks, as indicated by the relatively flat curve.

Treatment with MS2 yielded a 73% reduction in extractable PAHs, with the majority of the loss occurring by the second week. Over the remaining 6 weeks of treatment, only 4 g of additional of PAH contam- ination were removed.

As with MS2, there was a general decrease in PAH concentration with the fungus MS3 combination. However, approximately 70% of the remediation was achieved within the first week. The PAH concentration continued to decrease over the subsequent 7 weeks of treatment. By the end of the eighth week, 92% of the PAHs present had been remediated. Unlike the MSl-fungi combination, treatment with MS3 was only able to remediate eight of the PAHs in their entirety. The difference between the two treatment combinations was the inability of MS3 to degrade benzo(a)pyrene, 4-terphenyl-di4, and chrysene-dlz.

30 _I

0 1 2 3 4 5 6 7 8 WCXk

Fig. 1. PAH degradation with Cunninghamelln echinulata var. elegans.

PAH biodegradation and kinetics 405

Kinetics

Table 3 contains the calculated utilization rates for each fungus-supple- mental solution combination for prequasi-steady-state, as well as the quasi-steady-state conditions. It is important to note that the individually tracked PAHs were ‘lumped’ as one substrate for the kinetic formulation. As indicated in Fig. 1 and Table 3, the specific utilization rates, or degra- dation rates, were affected by the choice of supplemental nutrient solution used. For MSl, the first week of treatment comprised the pre-steady state conditions of 1_54/day. After the first week, the PAHs were degraded at an average rate of 0.52/day until the end of the sixth week, and 0*008/day for the final 2 weeks.

MS2 was the only supplemental solution to yield two pre-steady-state conditions. For the first week, the specific utilization rate of 0+33/day was achieved. This first, pre-steady-state rate only enabled 9% of the PAHs present to be degraded. During the second week, an additional 5 1% of the PAHs were remediated. The second pre-steady-state rate had increased to l.l26/day, over three times the initial value. After 60% of the PAHs had been remediated, the utilization rate decreased to the steady-state value of O.O89/day. The occurrence of two pre-steady-state condition can be attributed to components comprising the supplemental solution. MS2 was the only solution that contained glucose and casamino acid. These parti- cular compounds may have inhibited the degradation. Once the fungus had exhausted the glucose and casamino acid, the actual degradation was initiated, as indicated by a higher pre-steady-state utilization rate. The competition of the PAHs with the glucose and casamino acid would also explain the lower remediation efficiency of 73% for this treatment combination.

As with MSl, there was only one pre-steady-state value for treatment with MS3. The pre-steady-state rate of 2_483/day enabled 67% of the PAHs to be remediated within the first week of treatment. The actual steady-state value for the final 7 weeks of treatment was 0*134/day.

TABLE 3 Specific Pre-steady- and Quasi-steady-State Utilization Rates

Utilization rate (day-‘)

Nutrient solution I Nutrient solution 2 Nutrient solution 3

Pre-steady state 1.540 1.126” 2.483 Quasi-steady state 0.008 0.089 0.134

aSecond stage pre-steady-state value.

406 T. J. Cutright

Although this value was higher than the steady-state rates for the previous two experiments, it only remediated an additional 25% of the PAHs. Thus the final overall remediation achieved with MS3 was 92%.

CONCLUSIONS AND RECOMMENDATIONS

After 8 weeks, less than 1% of the PAHs were removed from the control blanks. This indicated that any indigenous microorganisms present were inefficient for the remediation of the contaminants. However, laboratory scale remediation with the fungus Cunninghamella echinulata var elegans had promising results with all of the supplemental nutrient solutions investigated. MS1 achieved 97% loss of extractable PAHs, with MS2 and MS3 reaching 73 and 92%, respectively. The choice of supplemental solution to be implemented would depend on the extent of contamination as well as on time constraints. For instance, if there were time limitations, remediation with MS3 would be optimal since it enabled 70% remediation within the first week. If final, overall remediation values are the driving force, MS1 should be used since it achieved the highest efficiency.

For a first-order reaction system, the rate of change in contaminant concentration is proportional,to the contaminant concentration in the soil. Therefore, the specific substrate utilization rates at steady-state conditions can be used for the prediction of the time required for bioremediation at a larger scale. It should be noted that this particular time prediction tool is dependent on the microorganism, and contaminant type and concentra- tion. In other words, the relationship generated from the specilic substrate utilization rates presented in this paper would be specifically for the time prediction PAH contaminated soil remediated with Cunninghamella echi- nulata var. elegans.

However, there are two areas that may pose a problem for using the fungi over large contaminated areas. The first is associated with the long- term storage of the starter culture. Fungal strains are more temperature sensitive than conventional bacterial strains. Secondly, for sub-surface applications, mass transfer and pumping limitations may occur. For these reasons, fungal treatment should probably be limited to small contami- nated areas or on-site batch treatments.

There are three specific areas that should be investigated further. First, the biomass should be monitored throughout the entire experiment. Respiration studies, with the capability for continuous analysis, will enable the accurate determination of the biomass. This would enable the development of a more rigorous kinetic model as well as decrease some of the experimental uncertainty. The second area is to investigate the kinetics

PAH biodegradation and kinetics 407

without ‘lumping’ the all of the PAH compounds as one substrate. In other words, treat each PAH compound as an individual substrate. The comparison of the individual versus lumped-substrate results would provide insights as to how valid it is to lump the contaminants together. The third area is to evaluate the interaction, if any, between C. echinuluta and the indigenous population. This could be achieved by comparing the degradation kinetics in ‘untreated’ vs sterilized soil samples.

REFERENCES

Aggarwal, P.K., Means, J.L. & Hinchee, R.E. (1991). Formulation of nutrient solutions for in situ bioremediation. In On-Site, Bioremediation: Process for Xenobiotic and Hydrocarbon Treatment, ed. R.E. Hinchee. Butterworth- Heinemann, Boston, MA.

Atlas, R.M. & Atlas, M.C. (1991). Biodegradation of oil and bioremediation of oil spills. Current Opinion in Biotechnology, 2(3), 44&443.

Atlas, R.M. & Sayler, G.S. (1987). Tracking microorganisms and genes in the environment. Proceedings of Reducing Risks from Environmental Chemicals Through Biotechnology, 19-23 July.

Bailey, A.M. & Coffey, M.D. (1986). Characterization of microorganisms involved in accelerated biodegradation of metalaxyl and metolachlor in soils. Can. J. Microbial., 32, 562-567.

Bailey, J.E. & Ollis, D.F. (1986). Biochemical Engineering Fundamentals. McGraw-Hill, New York.

Bailey, N.J., Jobson, A.M. & Rogers, M.A. (1973). Bacterial degradation of crude oil: comparison of field and experimental data. Chem. Geol., 11, 203- 221.

Baker, K.H. & D.S. Heron (eds) (1994). Bioremediation. McGraw-Hill. New York. Brodkorb, T.S. St Legge, R.L. (1992). Enhanced biodegradation of phenanthrene

in oil tar contaminated soils supplemented with Phanerochaete chrysospor- ium. Appl. Environ. Microbial. 58(9), 3117-3121.

Cutright, T.J. & Lee, S. (1994). Remediation of PAH contaminated soil using Achromobacter sp. Energy Sources, 16, 279-287.

Dhawan, S., Erickson, L.E. & Fan, L.T. (1993). Model development and simu- lation of bioremediation in soil beds with aggregates. Ground Water, 31(2). 271-284.

Erickson, D.C., Loehr, R.C. & Neuhauser, E.F. (1993). PAH loss during bior- emediation of manufactured gas plant site soils. Water Res. 27(5), 911-919.

Folger, H.S. (1986). Elements of Chemical Reaction Engineering. Prentice-Hall, New York.

Froment, G.F. & Bischoff, K.B. (1990). Chemical Reactor Analysis and Design, 2nd edn. Wiley, New York.

Gauger, W.K., Kilbane, J.J., Kelley, R.L. & Srivastava, V.J. (1990). Enhance- ment of microbial degradation of hydrocarbons in soil and water. In Gas Oil and Coal Biotechnology ZZ, eds C. Atkins and J. Smith, Institute of Gas Techology.

408 T. J. Cutright

Kostecki, T.P. & Calabrese, E.J. (1992). Contaminated Soils: Diesel Fuel Contamination. Lewis, Michigan.

Lee, R.F. & Hoeppel, R. (1991). Hydrocarbon degradation potential in reference soils and soils contaminated with jet fuel. In On-Site Bioremediation: Process for Xenobiotic and Hydrocarbon Treatment, ed. R.E. Hincheee, Butterworth-Heinemann, Boston, MA.

Levenspiel, 0. (1972). Chemical Reaction Engineering, 2nd edn. J. Wiley, New York.

Levin, M.A. & Gealt, M.A. (1993). Biotreatment of Industrial and Hazardous Waste. McGraw-Hill, New York.

Manahan, SE. (1991). Environmental Chemistry, 5th edn. Lewis, Michigan. Morgan, R. & Watkinson, R.J. (1992). Factors limiting the supply and efficiency

of nutrient and oxygen supplements for the in-situ biotreatment of contaminated soil and groundwater. Water Res., 26(l), 73-78.

Pflug, A.D. & Burton, M.B. (1988). Remediation of multimedia contamination from the wood-preserving industry. In Environmental Biotechnology: Redu- cing Risks from Environmental Chemicals through Biotechnology. Plenum Press, New York.

Rifai, H.S., Long, G.P. & Bedient, P.B. (1991). Modeling bioremediation: theory and field application. On-Site Bioremediation: Process for Xenobiotic and Hydrocarbon Treatment, ed. R.E. Hinchee. Butterworth-Heinemann, Boston, MA.

Stainer, R.Y., Duodoroff, M. & Adelberg, E.A. (1963). The Microbial World. Prentice-Hall, New York.

Tabak, H.H. et al. (1991). Characterization and optimization of treatment of organic wastes and toxic organic compounds by a ligonolytic white rot fungus in bench-scale bioreactors. On-Site Bioremediation: Process for Xenobiotic and Hydrocarbon Treatment, ed. R.E. Hinchee. Butterworth- Heinemann, Boston, MA.

Travis, M.D. (1990). Bioremediation of petroleum spills in Arctic and sub-Arctic environments: a feasibility study. US Department of Commerce, Report no. AK-RD-90-12.

Warith, M.A., Ferehner, R. & Fernandes, L. (1992). Haz. Waste Haz. Mater., 9(2), 137-147.