Application of Continuous Culture Technology for the Development of Explosives-degrading...

Application of Continuous Culture Technology for the Development of

Explosives-degrading Microorganisms” D. GUNNISON: H. L. FREDRICKSON: D. L. KAPLAN,’

A. L. ALLEN,’ C. M. MELLO,’ J. E. WALKER,’ GLENN MYRICK,” W. E. EVANS,’’ AND M. OCHMAN“

hU.S. Army Engineer Waterways Experiment Station 3909 Halls Ferry Road

Vicksburg, Mississippi 39180-6199 ‘ U.S. Army Natick Research, Development,

and Engineering Center Kansas Street

Natick, Massachusetts 01 760-5020 “American ScientiJic International Corporation

McLean, Virginia 22101

Microorganisms exposed to anthropogenic substances have sometimes responded by acquiring new genes for degradation of these compounds, either for detoxification or to enable the microbe to use the contaminant as a source of energy to meet metabolic needs.’ However, even in these microorganisms, the pathways necessary for degradation may lack sufficient genetic control to obtain maximum biological destruction. As Chakrabarty’ has indicated, community catabolism of the contaminant may decrease effectiveness in catabolism by individual microorganisms as a result of self-imposed constraints on rapid degradation of common metabolites by natural communities participating jointly in the degradation of the compound.

Previously, application of continuous culture technology as a process termed “plasmid-assisted molecular breeding” was used to facilitate development of bacte- rial strains capable of complete degradation of anthropogenic compounds, such as the xcnobiotic pesticidc 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).* More rccently, it has been suggested that application of continuous culture technology, coupled with more intensive selective pressures may accelerate development of new metabolic capabilities in a microorganism or enhance acquisition of such capabilities from other bacteria having the requisite biodegradative genes.’

We report here the application of continuous culture tcchnology utilizing chemical gradients coupled with other environmental selective pressures, based on the anticipated physiological capabilities required for a single specics of microorganism

a This work was supported by work units in the Environmental Quality and Technology Program of the U S . Army and the Strategic Environmental Rcsearch and Development Program of the US . Department of Defense. Permission to present and publish this work was obtained from the Chief, US . Army Corps of Engineers.

230

GUNNISON et al.: EXPLOSIVES-DEGRADING MICROORGANISMS 231

to mineralize highly refractory explosive compounds. Our goal is to apply our understanding of potential metabolic pathways to force microorganisms to evolve enhanced degradative capabilities for these compounds.

This approach is essentially a variation of continuous culture, except that by use of selective addition and removal of chemicals in the culture feed and a variety of incubation conditions, additional biochemical and physiological pressures are placed on the cultures over that normally used in continuous culture. This technology is applied to the development of microbial isolates capable of mineralizing 2,4,6- trinitrotoluene (TNT) and related compounds under aerobic, anaerobic, and microaerophilic conditions. Through application of a gradually changing scenario of chemical growth substrates over time, the cultures are forced to use in sequence 1) compounds related to the nitroaromatic target compounds as sources of nitrogen and possibly also carbon; 2) the target nitroaromatic compounds as nitrogen sources and structurally related cometabolite(s) as a source of carbon; and 3) the target nitroaromatic compounds as both carbon and nitrogen sources. We trace the development of increasingly complex degradational capabilities as first the mixed cultures, then individual microbial consortia and/or microorganisms develop the ability to mineralize explosives.

RESULTS AND DISCUSSION

Changes in Microbial Populations over Time in Three Different Continuous Cultures

All continuous cultures were conducted in M-8 medium, modified from that used by Abriel et aL3 M-8 medium contained the following ingredients, per liter of glass distillcd water: NaH2P0, . 7H20, 60 g; K2HP04, 60 g; N H Q , 2.50 g; NaCI, 5.0 g; MgS04, 0.520 g; and A9 solution, 2.5 mL. A9 solution contained (mg/L of glass distilled water): HBO?, 300; ZnCI,, SO; MnC12. 4H20, 30; CoCI2, 200; CuC1,. 2Hz0, 10; NiClz . 6H,O, 20; NaMoO, . 2H20, 30. For preparation of the inoculum, the equivalent of 100 g dry weight of each of the following materials was suspended in 2000 mL final volume of M-8 medium: a) sediments from lakes and freshwater and saltwater ponds in eastern MA, and b) explosive-contaminated soils from the vicinity of Volunteer Army Ammunition Plant, Chattanooga, TN; U.S. Navy Hastings East Industrial Park, Hastings, NE; U.S. Navy Submarine Base Bangor, Bangor, WA; and U.S. Navy Crane Naval Weapon Station, Crane, IN. This suspension was shaken vigorously for 1 min, allowed to settle for 15 sec, shaken again, and allowed to settle 10 sec. Then 150 mL of the partially clarified supernatant was used per liter of aerobic, anaerobic, and microaerophilic culture medium.

Aerobic and anaerobic continuous cultures were cultivated using continuous Row in 1- and 5-liter BioFlo I1 fermenters, respectively (New Brunswick Scientific). Aerobic cultures were initially supplemented with five mg of each of thc following explosives: 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and TNT per liter of M-8 medium. To encourage the microbial communities to utilize nitrogen from the explosives, very little inorganic nitrogen was supplied; instead 20 mg each of 2,4-dinitroanilinc and dinitro-o-cresol (collectively termed “inducer compounds”) were added to each liter of M-8 medium. To encourage microorganisms to utilize aromatic compounds as sources of carbon and energy, 1.44 g of sodium benzoate was added to each liter of M-8 medium to serve as the cometabolite. Nutrient exchange in the aerobic culture was accomplished by using the nutrient pump built into the fcrmenter to add medium and allowing excess medium to leave the culture

232 ANNALS NEW YORK ACADEMY OF SCIENCES

via an overflow drain in the side of the fermenter vessel. The anaerobic culture was cultivated in the same medium as the aerobic continuous culture, except that 5 g of soluble starch was also added to each liter of medium to support development and maintenance of anaerobic fermentation. Continuous nutrient exchange for the anaerobic culture was maintained using an outboard double-headed peristaltic pump to add and remove equal volumes of fresh and spent medium simultaneously. In both systems, the flow rate of medium was approximately 1% of the culture volume per day. Aerobic cultures were maintained at 30°C and aerated with 0.5 liters per minute (LPM) of filter-sterilized air at an agitation speed of 350 rpm. Anaerobic cultures were initially stripped of oxygen by sparging with nitrogen and then held at 30°C at an agitation speed of 350 rpm. The soluble starch present in the anaerobic medium created an oxygen demand of sufficient intensity to remove any oxygen leaking into the fermenter. All anaerobic sample collections were performed under a nitrogen atmosphere. Inflowing culture medium was continuously sparged with nitrogen gas at 0.20 LPM in the feed vessel (500 mL).

Microaerophilic continuous cultures were cultivated in batch mode in a static 2800 mL Fernbach flask. One-thousand five-hundred mL of M-8 medium as formu- lated for the anaerobic culture was placed into the flask and inoculated with starter inoculum, shaken vigorously for 2 min, and then incubated aerobically for 1 week under static conditions at 30°C. At this time, the culture was again shaken vigorously and 75 mL of the culture was removed by decantation. Seventy-five mL of fresh M-8 medium was added, the culture shaken again, and replaced into the 30°C incubator. Replacement of 75 mL of the medium was repeated at weekly intervals over the entire course of the work. Measurement of dissolved oxygen levels in the microaerophilic cultures revealed no detectable dissolved oxygen below 1 cm depth, both immediately before and within 1 hour following weekly nutrient exchanges.

The pattern for cometabolite addition, the levels and duration of inducer compound addition, and the regime for TNT addition were changed incrementally over

1 o4

1 o3

1 o2

1 0'

1 oo

I I I I I I I I I

Concentmtlon d Sub.tnt.s In Inflow Medlum

1 I -_-

I

I _ _ _ 7 I

I I

I I

TNT I / -l / I -_

_-- ,2f L-- 7 Inducer Compounds

I I I I I I \ I I, I

0 50 100 150 200 250 300 350 400

incubation, Days

FIGURE 1. Changes in nutritional status imposed on continuous cultures over the course of incubation.

GUNNlSON et al.: EXPLOSIVES-DEGRADING MICROORGANISMS 233

I I I I I I I 10 Aemblc Culture

0 yc a s 8 s g incubation Period

Total Heterotrophic Microorganisms B TNT-Resistant Microorganisms EBB Benzoate-Degrading Microorganisms

0 TNT-Degrading Microorganisms TNT-Benzoate Degrading Microorganisms

FIGURE 2. Levels of microbial populations present in the aerobic, microaerophilic, aid anaerobic continuous cultures over selected time intervals.

the course of incubation for each of the continuous cultures using the general pattern illustrated in FIGURE 1. The soluble starch added to the anaerobic culture was sufficient to develop and maintain a highly reduced state, as indicated by consistent oxidation-reduction potential measurements in the range of -240 to -260 mV (corrected with reference to the hydrogen electrode). Development of microbial populations in the presence of gradually increasing levels of TNT with time under aerobic, anaerobic, and microaerophilic conditions was monitored by periodic dilution and spread plating of these cultures onto a variety of media (FIG. 2). Counts obtained on nutrient agar were used to estimate total heterotrophs. Counts using nutrient agar containing 50 mg of TNTlL were used to determine the total number of heterotrophs able to grow in the presence of TNT. The following three


media were based on an M-8 mineral salts solution: solidified with 15 g of Noble agar/L. M-8 salts agar containing 100 mM sodium benzoate and 2.5 g of ammonium chloride per liter was used to enumerate benzoate-degrading microorganisms. M-8 salts agar containing 100 mM of sodium benzoate and 50 mg of TNT/L was used to indicate microorganisms possibly utilizing TNT as a nitrogen source and sodium benzoate and/or TNT as carbon sources. M-8 salts agar containing SO mg of TNT/ L was used to support microorganisms able to utilize TNT as a sole source of carbon and nitrogen.

Under all three growth conditions, the trends followed by the total heterotrophic and TNT-tolerant microorganisms were similar, although the anaerobic populations reached the highest initial population densities, then fell to the lowest average levels of the three cultures (FIG 2). By contrast, these two groups of microorganisms behaved nearly identically in the aerobic and microaerophilic cultures. Patterns for the populations of total benzoate degraders and benzoate degraders utilizing the nitroaromatics as a nitrogen source were also quite similar in the three cultures. Microorganisms isolated on M-8 medium containing TNT as the sole carbon and nitrogen source first began to appear in the microaerophilic culture (160 d). Then these microorganisms appeared in the aerobic and anaerobic cultures at the same time (although the sampling frequency during this time was very low) and coin- cidental with TNT levels in the feed media to these cultures reaching 45 mg/L. These microorganisms reached the highest levels in the aerobic and microaerophilic cultures, but reached slightly lower levels in the anaerobic culture. These observations suggest that 1) microaerophilic conditions supported TNT-degrading microbial populations somewhat better than either the aerobic or anaerobic conditions, 2) populations of microorganisms in the aerobic and microaerophilic cultures were quite likely dominated by TNT degraders towards the end of the first year of incubation, and 3) benzoate supported high population levels during the inter- mediate term of the incubation process (30 days to 160 days), but were still very high at the end of one year of incubation.

Changes in TNT Mineralization Capability over Time in the Three Continuous Cultures

The ability of each of the cultures to mineralize TNT to carbon dioxide was evaluated periodically by incubating 10 mL continuous culture samples with 10 mL of fresh culture medium in the presence of 0.199 $3 of U-ring-[I4C]-TNT. The fresh culture medium contained explosives at the level present in the inflowing medium at the time the sample was taken. Each culture was examined for 14C02 production over a period of 20 days during incubation in biometer flasks with a 1 N KOH trap. The aerobic and microaerophilic cultures showed development of the ability to mineralize TNT over the course of several months (FIG. 3), with the microaerophilic culture apparently achieving the highest overall average of the three continuous cultures. However, owing to the extensive standard errors observed for the microaerophilic cultures, the effective mineralization by this culture at nine months cannot be said to greatly exceed that of the aerobic culture. Some of the extensive variation observed in the microaerophilic mineralization studies may have resulted from the tendency of these cultures to produce clumps of microbial growth. Despite repeated attempts to break up the clumps by forcing thcin through the narrow passage of a pipet tip, even dispersions of cultures in the biometer flasks was of limited success.


80

loo I - -- --- 1

MICROAEROP€ULIC CULTURE ~

AEROBIC CULTURE 80 c

Continuous Culture Afier FourMoQths - Seven Months

A NmeMouths 40

2oLe 0 0 5 I L- I ~ ~~

10 15 20

Incubation, Days

FIGURE 3. Mineralization activity iu the aerobic and microaerophilic continuous cultures as determined in assay with U-'4C-labcled TNT. No substantive levels of radiolabeled carbon dioxide were formed in the anaerobic continuous culture.

The radioassays for the anaerobic culture were run with a nitrogen atmosphere in the headspaces of the biometer flasks. The anaerobic cultures did not demonstrate substantial levels of TNT mineralization based on I4CO2 formation. However, the ability of the anaerobic culture to evolve radiolabeled methane and/or organic acids and alcohols was not examined in these studies. The period of greatest increase in T N T mineralization activity in the aerobic and microaerophilic cultures occurred following the time when the level of TNT in the infusion medium had reached 45 mg/L. Since the anaerobic culture did not demonstrate substantial levels of T N T mineralization under these conditions, the relationship of TNT level in the infusion medium to mineralization activity in the anaerobic culture cannot be determined.

After h mo of incubation, thc level of carbon in the aerobic culture inflow likely limited the microbial population in the cultures, as the sodium benzoate in the inflow was quite low at this point. This was not the case for the anaerobic and microaerophilic cultures, where starch was present in the inflow at all times. The population measurements indicated that the microbial numbers in each of the cultures were substantial. However, mixing of the culture samples with the fresh culture medium containing TNT at the level of inflowing medium may have produced a toxic situation for these microorganisms, because the TNT levels in the continuous cultures were not as high as in the inflows. While the microorganisms in the aerobic and microaerophilic continuous cultures overcame this as the incubation progressed, the anaerobic microbial population may not have been able to d o this within the time frame considered here.


Nitroaromatie Transformation Products Observed in the Continuous Cultures

Chemical samples of each of the cultures were taken periodically, freed of soil and cells by centrifugation, extracted with ether, and evaporated under nitrogen. Initially, duplicate samples containing soil and cells were also extracted with ether and evaporated under nitrogen. However, this procedure never produced explosives or TNT transformation products any different from those obtained from the culture supernatant alone. Therefore all remaining extractions were performed on the culture supernatant only. The ether residue was taken up in one one-hundredth the original volume of absolute ethanol. Samples were analyzed by reverse phase high performance liquid chromatography (HPLC) using a 3.9 X 300 mm C-18 column on a Waters HPLC. The separations were developed using a linear gradient from 85% water-15% methanol to 100% methanol over 70 minutes, and the separation was monitored at 254 and 233 nm using a Waters photodiode array detector.

TABLE 1. Major Organic Compounds Identified in Aerobic, Microaerophilic, and Anaerobic Cultures

Peaks Detected, as Percent of Total Peak Height

Time Aerobic Microaerophilic Anaerobic (months) Compound" Culture Culture Culture

One

Two

Five to Six

Sodium benzoate or Starch-benzoate*

D A N T MPEP TNT DNT ADNT HOADNT Azoxytoluenes

Sodium benzoate or Starch-benzoate

DANT MPEP TNT DNT ADNT HOADNT Azoxytoluenes



81.3

ND ND ND ND ND ND 7.28

72.3

2.61 ND 1.91 5.00 ND ND 5.85

73.3

4.81 ND 3.30 1.81 ND 3.73

19.0

33.6

7.38 3.28

12.3 6.56

31.2 ND 5.74

57.3

5.83

7.77 5.83 ND ND 7.77

15.5

22.0

8.06 44.7 4.40

12.1 ND ND 8.79

93.8

ND ND ND ND

3.53 ND ND

48.8

ND ND ND ND 4.65 ND ND

27.6

0.100 ND 6.58 ND ND

31.6 23.7


TABLE 1. Continued

Time

Nine to Ten

(months) Compound“





Twelve

Peaks Detected, as Percent of Total Peak Height

Aerobic Culture

21.1

18.2 ND ND ND ND

60.7 Trace

23.2

17.4 ND ND ND ND

59.4 Trace

Microaerophilic Anaerobic Culture Culture

63.8 47.22

8.51 ND 6.81 ND 14.5 2.38 ND ND ND ND ND 47.6 6.38 1.20

NS

NS NS NS NS NS NS NS

44.8

11.9 ND ND ND 4.88

ND 40.6

~

Abbreviations: DANT = diaminonitrotoluene; MPEP = 2,4-diphenyl-4-methyI-2-pentene; TNT = 2,4,6-trinitrotoluene; DNT = 2.4- + 2,6-dinitrotoluene; ADNT = aminodinitrotoluene; HOADNT = hydroxylaminodinitrotoluene; azoxytoluenes = 2,2‘,6,6‘-tetranitro-4,4‘-azoxytoluene + 4,4’,6,6’-tetranitro-2,2’-azoxytoluene.

* Sodium benzoate was used alone in aerobic cultures. Starch plus sodium benzoate were used in microaerophilic and anaerobic cultures and came out as a complex peak.

Only those compounds repeatedly occurring in the same or two or more different cultures are shown. Several compounds, including bis-(t-butyl-4-dimethylbenzyl phenol) (DMBP) occurred only once, although at this time the compound comprised a substantial portion of the total peak height. Aminonitrotoluene (ANT) occurred only in the aerobic culture. These compounds were added to the totals used to compute peak height percents.

Calibration standards used included 2,4,6-TNT; 2,4-diamino-6-nitrotoluene (2,4- DA-6NT); 2,4- and 2,6-dinitrotoluene (2,4- and 2,6-DNT); 4-amino-2,6-dinitrotoluene (4A-2,6-DNT); 2-amino-4,6-dinitrotoluene (2A-4,6DNT); 2,2’,6,6‘-tetranitro-4,4’-azoxytoluene and 4,4’,6,6’-tetranitro-2,2’-azoxytoluene. In some in- stances, high levels of unknown compounds appeared in the HPLC chromatograms of the culture fluid. To identify these compounds, the samples were extracted with ether, dried under nitrogen, taken up in water, and derivitized with trimethyl silane prior t o separation and analysis on a Hewlett-Packard gas chromatograph-mass spectrometer.

The pattern and relative abundance of the nitroaromatic compounds detected in the three continuous cultures over the course of 12 months are listed in TABLE 1. These values were obtained by summing the peak heights for each HPLC run and then dividing individual peak heights by the value for the total. Major observations for these compounds are as follows.


Aerobic Culture

Major products other than the sodium benzoate, TNT, and the DNTs (the compounds added to the inflow), were diaminonitrotoluene, hydroxylaminonitrotoluene, and the azoxynitrotoluenes. These are the typical intermediates expected during aerobic incubation of DNT and TNT with cultures from a variety of soil and aqueous media." Sodium benzoate consistently comprised a significant portion of the total peak height until this compound was tapered off in the inflow mixture and finally discontinued at 11 months. Principal products observed beginning at the first month were low levels of the azoxynitrotoluenes. These peaked by 6 mo and declined to trace levels by 10 months. Surprisingly, detectable levels of the mononitrotoluenes were not observed. Instead, the diaminonitrotoluenes appeared at the second month and steadily increased until they comprised nearly twenty percent of the peak heights observed at twelve months. The hydroxylaminonitro- toluenes formed substantial portions of the products beginning in the sixth month. These compounds reached approximately 60% of the total peak heights by the end of one year of incubation. TNT and DNT levels rose and then declined over the first 6 mo of incubation, but were not found after this. This may reflect a gradual increase in TNT mineralization capability, as indicated by the increased production of I4CO2 with time in the TNT radioassay.

Microaerophilic Culture

The starch-benzoate levels found in the microaerophilic culture remained sub- stantially lower than the sodium benzoate levels in the aerobic culture throughout the incubation, except for a sharp rise in peak height at the ninth month. As for the aerobic and anaerobic cultures, sodium benzoate levels in the inflow were tapered off beginning at 230 days and stopped completely by the eleventh month. However, starch, which showed up in the same region on the HPLC as sodium benzoate, was added to the culture continuously over the incubation. All of the remaining compounds, except for hydroxylaminodinitrotoluene, appeared in this culturc at some time during the course of the incubation. Of particular interest was the predominant role of the compound identified by GC-mass spectrometry as 2,4- diphenyl-4-methyl-2-pentene (MPEP). The exact role of this compound in explosives degradation pathways, if any, is unknown. DANT levels rose slowly, but steadily over the course of the incubation, while the azoxynitrotoluenes rose and then fell, never reaching nine percent ol the total peak heights. The data thus suggest a period of highly mixed production for the first 6 mo, after which product formation activity fell off considerably. This does not appear to agree with the effectiveness of the microaerophilic culture in mineralization, as mentioned previously. Also in contradiction to the gradually increased mineralization capability is the apparent increase in TNT lcvels in the culture over the 9 mo for which the culture analysis data are available. One possible explanation is that the culture gradually developed an enhanced cometabolic capability, utilizing starch as the primary carbon source, while the explosives were mineralized very gradually.

Anaerobic Culture

The starch-benzoate addition pattern for this culture was the same as for the microaerophilic culture, but the behavior of these compounds in the anaerobic

GUNNISON ef al.: EXPLOSIVES-DEGRADING MICROORGANJSMS 239

culture was quite difkrent from that observed for the microaerophilic culture. In contrast to the other cultures, the diaminonitrotoluenes never comprised more than 0.1% of the products. The monoaminodinitrotoluenes never exceeded 6.7% of the peak height of the total products present. The azoxy compounds never exceeded 23.7% of the peak height at 6 mo, and then disappeared completely. TNT was occasionally detected after the first month, but only at low levels, and disappeared completely after 10 months of incubation. The hydroxylaminonitrotoluene levels rose steadily for the first 10 mo of incubation, then fell back slightly; these were the highest levels achieved by this compound in any of the cultures. The data suggest that a large portion of the TNT present is converted to hydroxylaminonitrotoluene, but no indication as the inability of the anaerobic culture to mineralize radiolabeled TNT is given.

SUMMARY

All three continuous cultures eventually developed microbial populations able to grow on a selective medium TNT as a sole carbon, energy, and nitrogen source. However, by the end of the first twelve months of incubation, the colonies developing on this medium were quite small. Initial attempts to isolate pure cultures of TNT- mineralizing microorganisms from colonies obtained on plates of M-8 salts agar with TNT have not yet proven successful. Upon startup of the continuous cultures, none of the microorganisms present was able to grow on M-8 salts medium with T N T or benzoate as carbon and energy sources. This ability was clearly acquired over the course of the first year of continuous culture. Whether this ability developed as the result of a series of beneficial mutations or through the lateral transfer of suitable genetic material, or both, cannot be determined from the work presented here. However, samples of each of the cultures were frozen at various intervals, providing material for later examination with gene probes to determine the points of origin of the necessary genetic potential. All three continuous cultures are still running at this point. Indications are that one or more individual TNT-mineralizing strains are emerging in the aerobic and microaerophilic cultures. The observation that strains able to utilize a given target compound emerge slowly from an evolving mixed culture having the ability to use the compound as a group is common in continuous culture technology (A. M. Chakrabarty, University of Illinois Medical Center at Chicago, IL, personal communication, August 1995). We have been periodically attempting to isolate strains from the continuous cultures using the enrichment scheme depicted in FIGURE 4. By applying this technique, we have been successful to date in obtaining two different rapidly growing microorganisms able to use 2.6-DNT as a sole carbon, energy, and nitrogen source. The cultural character- istics and ecophysiology of these microorganisms are presently being explored.

The gradual development of enhanced TNT mineralization capability in concert with the appearance of the diaminomonitro-compounds is clearly evident in the aerobic culture. The same process is evident in the microaerophilic culture, although the formation and disappearance of both the monoaminodinitro-compounds appeared to fall off after the second month. The failure to demonstrate extensive mineralization in the radioassay, coupled with the disappearance of the parent explosives in the absence of all but the hydroxylaminonitrotoluene transformation product, suggests a different fate for the nitroaromatic explosives in the anaerobic culture. Assay of the liquid and cellular material remaining in the biometer flasks after the anerobic radioassay indicated that more than 95% of the radioactivity was


ENRlCHMENT MICROORGANlSMS ABLE TO DEGRADE SOIL FRACTIONS CONTAINING

TNT CONJUGATES

SAMPLE SOILS FROM WHICH FRACTIONS WERE DERIVED AND USE COLD FRACTIONS AS

A CARBON SOURCE

T-sFER T o LrQum I D , , WITH s o i L FRACTION AS THE SOLE CARBON SOURCE

INCUBATE 1 1 WEE ,THEN RETRANSFER 1 r RETRANSFER TO LIQ f ID MEDIUM CONTALNING

J

REPEAT T CE MORE

ISOLATE ON AGAR

ONLY THE SOIL FRACTION AS CARBON SOURCE

~ ASSAY FOR C-14 CO,

_,- '._ +,--

.;ROWSON SOU. F&CrlON ANU RELEASES RADIOLABELED CARBON DIOXIUE: BOL AD

FORM OF TST IS AVAILABLE

GROWS Oh SOU. B R A C ~ O N , BUT PRODUCES YO RAJMOLABELED CARIWN DIOXIDE:

COJIPLEXED FORM OFTNT NOT AVAILABLF

FIGURE 4. Enrichment procedure used to isolate individual explosives-mineralizing rnicro- bial strains from the continuous cultures.

present at the end of the incubation period, with about half of this activity present in the cell mass. Whether or not the fate of the radiolabeled TNT involved fermentation of the compound to organic acids and alcohols, as observed in Crawford's laboratory: awaits further experimentation.

The ability of continuous culture technology to develop microorganisms capable of utilizing compounds previously considered refractory is evident from the work presented here. Further development of the technology to obtain strains suitable for a variety of biotreatment applications is underway.

ACKNOWLEDGMENTS

We thank Professor A. K. Chakrabarty, University of Illinois Medical Center, Chicago, IL, for helpful discussions at the onset of this work, near the end of the first year, and again after a year and a half of experimentation. We also thank Drs. Judith C. Pennington and James M. Brannon of the USAE Waterways Experiment Station for reviewing the manuscript.

REFERENCES

1.

2.

CHAKRABARTY, A. M. 1995. Microbial degradation of toxic chemicals: Evolutionary in- sights and practical considerations. ASM News 62: 130-739.

KELLOGG, S. T., D. K. CHATTERJEE & A. M. CHAKRARARTY. 1981. Plasmid-assisted molecular breeding: New technique for enhanced biodegradation of persistent toxic chemicals. Science 214: 1133-1135.

GUNNISON ef al.: EXPLOSIVES-DEGRADING MICROORGANISMS 241

3. ABRIEL, M.-A,, C. MICHAN, K. N. TIMMIS & J. L. RAMOS. 1989. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J. Bacteriol. 171:

GEIGER, P.-G. & H.-J. KNACKMUSS. 1994. Basic knowledge and perspectives on biodegradation of 2,4,6-trinitrotoluene and related nitroaromatic compounds in contaminated soil. In Biodegradation of Nitroaromatic Compounds. J. C. Spain, Ed.: 1-18. Plenum Press. New York.

CRAWFORD, R. L. 1994. Biodegradation of nitrated munition compounds and herbicides by obligately anaerobic bacteria. In Biodegradation of Nitroaromatic Compounds. J. C. Spain, Ed.: 87-98. Plenum Press. New York.

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