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jgCLASSIFPD SECURITY CLASSIFICATION OF THIS PAGE (Whtt Data Kntvrrd)

KgPORT DOCUMENTATION PAGE 1. REPORT NUMBER 2. COVT ACCESSION NO

4. TITLE (mtä Subllllm)

BIODEGRADATICN OF AIPRA TNT AND ITS PRODUCTION IS0MER5

7. AL'THORf«,

Richard W. ^raxler

f. PERFORMING Ofic "ATION NAME AND AOCRESS

Dept. of Plsu.c Pathology - Entomology University of Phode Island, Kingston, RI 02881

II. CONTROLLING OFFICE NAME ANO ADDRESS

US Army Natick Development Center Food Sciences Laborptory, AMXNM-TPB lfRtiete. Mass. 01760

I«. MONITORING AGENCY NAME % AODRESSf" dllltrmnl from Controlling Olli cm)

READ INSTRUCTIONS BEFORE COMPLETING FORM

S. RECIPIENT'S CATALOG NUMBER

S. TYRÄ OF REPORT A PERIOD COVERED

Interim July 73 -July Jh

6. PERFORMING ORG. REPORT NUMBER

8. CONTRACT OR GRANT NUMB'.Rfa}

DAAGI7-73-C-O276

10. PROGRAM ELEMENT. PROJECT, TASK AREA ft WORK UNI I' NUMBERS

Project No. 6U-00037-T253

12. REPORT OATE

11. NUMBER OF PAGES

IS. SECURITY CLASS, fof Ulla raporf;

Unclassified

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16. DISTRIBUTION STATEMENT (ol It, m Rtpott)

Approved for public 1»lease, distribution unlimited. Destroy this report w'ien no longer needed. Do not return to the originator.

17. DISTRIBUTION STATEMENT (ol thl afcalraci' anlaracf In Block 20. II dllttjrent from Rmporl)

I«. SUPPLEMENTARY NOTES Reproduced by

NATIONAL TECHNICAL INFORMATION SERVICE

US Department ")! Commerce Spiincliald, VA. 22151

19. KEY WORDS (Conllnum on ravmrmm mid* II nmcmmmry mnd Htmntlty by block numoer)

TNT, nitrotoluene, nitrohodies, pink wat«r, red weter, pollution abatement, munitions wastes

20. ABSTRACT (Conllnum an ravarm» tie* II nacaaaary and Idmntlly ty «lock mimbar)

Gram negative bacteria have been isolated from several sources which utilize 2, U,6-trinitrotoluene (a-TNT) as the sole source of cnrbor and nitrogen for growth There is a direct relationship between the a-TNT concentration in the growth medium and growth response of isolate I-2WT as well as.the percentage of TNT degradation by this organism. Studies using ring UL- C-TNT suggested possible

;e (Modegradation) of TNT as 1 C activity wan released as carbon ring cleavage dioxide and also found,in the cells. Heterotrophic carbon dioxide fixation was demonstrated using NaH CO3 added to a culture growing on unlabeled TNT.

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BIODEGRADATION OF ALPHA TNT

ANT)

ITS PRODUCTION ISOMERS

Annual Report-U

by:

Richard W. Traxler

University of Rhode Island

Kingston, RI 02881

Contract No. DAAG17-T3-C-0276

July 197^

U. b. Army Natick Derelopment Center

I'atick, Massachusetts

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TABLE OF CONTENTS

:,-■;

>:■■

II.

III.

Li et of Tables

Ust of Illustrations

Statement of the Problem

Background

Materials and Methods

A. Enrichment culture techniques B. Analytical techniques

TV. Resulte

A. Aerobic enrichments B. Anaerobic enrichments C. Growth studies with Isolate IIBX D. Growth studies with isolate I-2WT E. Other growth and utilization studies F. Metabolic studies

V. Conclusions

VI, Literature Cited

LIST OF TABLES

1. Mineral Salts Basal Media

2. Enrichment isolates, by source and substrate(s)

3. Effect of yeast extract concentration on growth of IIBX

Page

3

1»

5

7

7 8

9 9 9

Ik 16 18

23

23

7

10

14

k. Relationship between substrate concentration and percent utilization lU

5. Growth of sewage isolates on M-9(-N) supplemented with 0.01$ yeast extract 16

lfc. 6. TNT (ring-UL-x C) distribution with IIBX and I-2WT growing on TNT in M-9 medium 20

7. Percentage of labeled TNT metabolized by IIBX and I-2WT in ammonia- free medium 20

8. Heterotropliic carbon dioxide fixation by 1-2-5 22

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LIST OF ILLUSTRATIONS

1. Growth of UK on TNT and Yeast Extract

2. Effect of temperature on growth of XX» on TNT and percent degradation

3. Growth of I-2WT and mutant 1-2-5 on TUT

U. Effect of ammonia and yeast extract on growth of 1-2-5 on TNT

5. Effect of TNT concentration on growth response of 1-2-5 and percent

degradation

6. Oxygen uptake by I1BX

T. «ated ax» as carton dioxide »itn IIBX and 1-2-5 8™in6 0= «

(ring-UL-ll+C)

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I. Statement of the Problem

The vaste products from the manufacture and shell loading of munitions re-

presents a serious disposal problem for the Department of Defense. In the manu-

facture of TNT, the plant wash -water contains quantities (50-100 ppm) of TNT; while

the waste from the Sellite Process contains TNT and the production iscmers of TOT.

It has been shown that these nitrobodies can be absorbed into an XAD2 column

and eluted from the XAD2 column with acetone (now in critical supply) cr other

organic solvents. This treatment may be an effective solution to this waste disposal

problem in that it will generate a nitrobody free effluent water. The XAP2 system

is not destructive, therefore, it has not solved the problem of nitrobody disposal,

but results in a concentrated waste which may be burned unless explosiv? dangers or

serious air contamination problems are associated with the burning process. If

these nitrobodies are found to be biodegradable, they could be rendered ecologically

safe by biological treatment.

II. Background

Recalcitrant molecules have specific structural characteristics which prevent

microbial metabolism of the molecules, but at this time microbiologist.s are not

able to describe all of these structural characteristics. It can be stated that

naturally occurring organic molecules are biodegradable by some microbial form

whereas the truly recalcitrant molecules are found in broad classes of the synthetic

organic compounds. In nature, many compounds which are potentially biodegradable,

are slowly degradable or totally undegradable because of their interaction in the

soil or the influence of environmental factors.

In studies of nitrophenols, Raymond and Alexander (l) isolated a bacterium

able to metabolize p-nitrophenol and showed the appearance of stoichiometric amounts

of nitrite in the medium and the formation of catechol. Tewfik and Evans (2)

demonstrated the biodegradatlon of 3,5-dinitro-o-cresol via reduction of the 3-nitro

grout to an amino group which is then replaced by a hydroxy] s^oup. Ring fission

occurs after the formation of the trihydroxyl species. These observations clearly

indicate that substitution of nitro-groups to the benzene ring does not necessarily

confer recalcitrance to the molecule.

One of the first serious studies of TNT biodegradatlon by microorganism was

reported by Osmon and Klausmeier (3). In their study they tested for degradation

on agar containing 1$ yeast ext- '■ and 1$ glucose plus 100 mg/liter of TNT. Their

measurement of degradation was the loss of TNT from the medium which can only be

considered as presumptive evidence of degradation. In no case were they able to

detect the disappearance of TNT in flasks containing only mineral salts and TNT.

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II. Background (continued)

A recent study by Won, Heckly, Glover and Hoffsommer (h) describes metabolic

disposition of TNT. Again they describe the disappearance of TNT but have also

included in their media readily oxidizab.le substrate such as glucose (0.1$), and

yeast extract (O.^). They have demonstrated oxygen uptake by organism suspensions

in the presence of TNT. Analysis of culture filtrates shows the conversion cf TNT

to 2,2' ,6,6'-tetranitro-1*,'+'-azoxytoluene; 2,2' ,k,U '-tetranitro-6,6'-asc,-/toluene;

U-amino-2,6-dinitrotoluene; 2-amino-l+-hydroxylaminotoluene; and 2-amino-l*,6-dini-

trotoluene. None of these intermediates indicate biodegradation cf the TNT molecule,

but only biotransformation of the ring substituted nitrogen groups.

A review of the literature demonstrates that none of the other laboratories

•working on TNT degradation have yet isolated an organism that will grow in a mineral

salts medium with TNT present as the sole source of organic carbon and energy.

-6-

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III. Materials and Methode

A. Enrichment culture techniques.

Inocula for enrichment cultures have been obtained from Narragansett Bay

sediments, University of Rhode Island boiler plant effluent and raw sewage

obtained at the inlet pipe of the University of Rhode Island treatment plant.

The basal media used for enrichment isolations are described in Tablfi 1.

For aerobic isolations either M-9 or M-9(-N) were used as enrichment media.

In the anaerobic isolations, T (") medium was used as it provided nitrate as an

electron acceptor.

Table 1. Mineral Salts Basal Media

Component M-9* M-9(-N) T1**

NH4CI (g)

Na2HP04 (g)

KH2P04 (g)

NaCl (g)

MgS04 (g)

NH4NO3 (g)

CaCl2 (mg)

FeS04 (mg)

MnS04 (mg)

Co(N03)2

(NE^sMoyO^

Distilled water (ml)

1.0

6.0 6.0 1

3.0 3.0 0.5

b.o 5.0

0.1 0.1 0.5

2.5

20.0

1.0

0.01

0.005

0.1

000 1000 lOOO

*Reference 5

**Reference 7

In all isolations 50 ml of medium containing 100 ug/ml of MJT was used in a

250 ml Erlenmyer flask or a 300 ml Bellco side-arm flask. These were inoculated

with 0.1 g of sediment or 1.0 ml of liquid and incubated at 25, 30 and 35°C on

gyrorotary shakers at 125 rpm. The primary enrichment cultures were incubated until

visible turbidity developed, then 0.1 ml of the primary culture was transferred, to

a fresh flask of the same medium. When turbidity developed in the secondary enrich-

ment flask, the bacterial population was streaked for isolation onto mineral salts-

TI\iT medium solidified with 1. 5$ washed agar and the same medium supplemented with

0.01$ Dlfco yeast extract. When indicated 0.01$ yeast extract was included in the

primary and secondary enrichment flasks.

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5 )4 The pure cultures obtained by this isolation scheme were inoculated at 10 -10

viable cells per ml (80-100 Klett Units) into mineral salts broth containing 100

ug/ml cf TNT with or without 0.01$ yeast extract supplement. The growth response

in this test flask was followed by turbidity measurement in the Klett-Summerson

photo-electric colorimeter using the No. H2 blue filter.

Anaerobic isolations have used only T medium, and all incubations were carried

out in a National Appliance Company, Model 36^0 anaerobic incubator using N^ as

the inert replacement gas.

Enrichments from se'age and the initial sewage degradation studios were done

in a continuous flow fermentor. The fermentor vessel was charged with 250 ml of

double strength M-9 medium containing 100 ug/ml of TNT and 250 ml of raw sewage.

Aeration was set at 500 cc/minute and the incubation temperature was ;>0°C. This

mixture was allowed to incubate for 2 hours, then an M-9 + 100 ug/ml TNT solution

was fed through the fermentor vessel at a rate of 2 ml/minute. At intervals,

0.1 ml of 10"^ 10 and. 10~5 dilutions from the fermentor vessel were . pread

plated onto M-9 TNT washed agar with and without 0.01$ yeast extract supplement.

Representative of each colony type developing on the isolation plates were

selected and their growth response on TNT determined in liquid medium.

B. Analytical techniques.

Studies were performed on a gas Chromatographie method for the quantitation

of TNT but discontinued as we did not feel the values had good reprcducibility.

We have adopted, a method using abscrbance at 224 run in the Carey model 1^ spec-

trophotometer. A standard curve was prepared from the O.D. readings of varying

concentrations of alpha-TNT at 224 nm. The standard curve was prepared with

known concentrations of TNT in petroleum ether and also with known concentrations

of TNT in mineral salts medium, extracted into petroleum ether.

Residual TNT in culture media has been determined by comparison to the stand-

ard curve after different treatments of the culture. The bacterial cells were

removed by centrifugation, then the clarified supernatant was extracted twice

with 25 ml of petroleum ether. The combined extracts were placed in a 100 ml

volumetric flask and diluted to 100 ml with petroleum ether. Further dilutions

of this extract were made as required to obtain readings within the range of the

s t and ar d curve.

A second method of quantification has involved lysis of the cells by treating

25 ml of the culture with 5 ml of concentrated H2S04. After lysis, the same

extraction procedure was used to determine residual TNT. The lysis procedure was

designed to release any cell-bound TNT.

Thin layer chromatography has been used for the detection of TNT and its

related nitro-aromatic compounds. The support used in all these experiments was

Eastman No. 13179, 20X20 cm Silica Gel sheets without fluorescent indicator.

-8-

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Developing Solvents;

a. Chloroform, Methanol, ülac. Acetic Acid, 80:20:1

b. Benzene, Hexane, Acetone, 50:50:10

Indicator Sprays:

a. 0.5$ Fast Black in distilled water followed by 0.1 N Ne

b. Ethylenediamine

IV. Results

A. Aerobic enrichments:

A total of 5? aerobic enrichment cultures (Table 2) have been examined for

their ability to utilize TNT. Of these 57 isolates, only 19 isolates have pa-sed

the first proof step which was increased turbidity upon inoculation into M-9 cr

modified M-9 medium with TNT as the sole source of carbon. In some sases the

cultures passing the first proof step were mixed cultures which, when purified,

did not show a positive growth response. At this time, advanced studies have

used isolate A25-7-B-X-II (simplified code HEX) and isolate A7WASN-1 (simplified

code 1-2).

B. Anaerobic enrichments:

Repeated attempts at the isolation of TNT utilizing organisms under anaerobic

conditions from various inocula have failed to yield any isolates cf interest to

this study.

C. Growth studies with isolate HEX:

Figure 1 shows the typical growth response of isolate HEX in M-9 medium

containing 100 ug/ml of TNT and 0.01$ (100 ug/ml) yeast extract aa a supplement.

If yeast extract was not present in the medium, there was no turbidity increase

beyond the inoculum value. This plot demonstrates that 0.01$ (100 ug/ml) of

yeast extract will not support growth of isolate HEX.

Further evidence for the stimulatory nature of yeast extract is provided

in Table 3- Yeast extract at even 100 ug/ml doe- not allow growth of the text

organism, but in the presence of 100 ug/ml of TNT provides significantly better

growth than lower concentrations of this supplement with TNT.

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150

z

»- I- Ul .J * 50

TNT +

YEAS! EXTRACT,

Figure

12 3 4 5 6 7 TIME IN HOURS

Growth of 11BX on TNT and yeast extract.

125 r

to t 75

ID _J

0 —o

// / ^-nf

^_, ^_- -m

/ // j/\s Temperature °C % TNT Used / // Syr •—• 25 14

//^JV^ ■—■ 30 15 //y^^^ o—o 35 13 *B^"^ a—a 40 16

«—* 45 10

i i 1_ ,. i i -j—

25 -

I 2 3 4 5 6 HOURS

Figure 2. Effect of temperature on growth of II BX on TNT and percent degradation.

13

«■■I I 'lil»i«Mhtil tUttm - mil|- ' ' "■ i ' ilifiU—Mfi li In in-1 i uuuiua

S.IH. .IIJUI.I , Ml ,.M», i IIIHIIKIIII ii ■ nuuiip.j..,iu»iiituiiu i marriM'»» ^,.^ygMW.»^«^lt,..-^^ji...llllliyj,l|ut;^

"able 3« Effect of yeast extract concentration on growth of IIBX.

Yeast Extract TNT Initial Final

(ug/mi) (ug/ml) Klett Units Klett Units

100 - 21 30

50 - 20

10 - 14 11

100 100 40 lU2 1

50 100 33 115

10 100 33 57

The effect of temperature on the growth of the organism and disappearance of TNT

was investigated using isolate IIBX (Figure 2). Flasks of M-9 + yeast extract

(100 ug/ml) + TNT (100 ug/ml) were incubated at 25, 30, 55, kO and k^C. There is an

increased growth rate and final cell density with each 5C increase in the incubation

temperature up to 40C. The exponential growth phase is shorter at 1+5C than at any

other temperature and the final cell density is lower. The percentage of the TNT

used at each temperature is not significantly different except for possibly k^C,

which is the lowest percent utilization.

The relationship between initial TNT concentration in the medium and the percent

TNT utilization is inversely related to TNT concentration (Table h). This inverse

effect suggests either TNT inhibition or inhibition by metabolic products.

Table k. Relationship between substrate concentration and percent utilization.

TNT #TNT

ug /ml Utilized

25 kh

50 32

100 28

D. Growth studies with isolate I-2WT and its mutants:

Isolate I-2VJT (Full -:oäe A7WASN-1) was obtained from the main boiler plant

effluent at the University of Rhode Island. This organism was used for growth

studies as the wild type and after exposure to UV irradiation for 5> 10, 15 and 20

seconds, the UV irradiated cells were plated on M-9 medium without yeast extract for

out growth. Figure 3 sho^s the growth response or the initial isolate (I-2WT) and

the mutant obtained from the 5 second exposure (1-2-5) on M-9 medium supplemented

with yeast extract. The 5 and 20 second mutants were grown in M-9 medium without

-Ik-

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200 r

10 I* 20

TIME IN HOURS Figure 3. Growth of I-2WT and mutant 1-2-5 on TNT.

250 r

Z D K I- Ul

Figure 4. Ef fee' of ammonia and yeast extract on growth of 1-2-5 on TNT.

15

i "-■---■■ ■■■■-. .._ jg^gBUjfrfl .,. ^ , ,.■■^^.^■^■«1,^1^=.^^^■■••'^'-■-rlfii|-rtiJ 8t I - '■*■*• iw'iUtiMi,**!! \t-r,;.M. mi

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■ c--,>J.iH.,-.-p„iu!'>n»»iitiM'm

yeast extract supplement. The growth response of the 10 second mutant was essen-

tially the same as the 5 second mutant and the 15 second mutant was like that of the

20 second mutant. The growth of the 5 second mutant (1-2-5) was faster than the wild

type or 20 setond mutant and reached % greater cell density.

Organism 1-2-5 was used to determine the effect of ammonia and yeast extract on

the utilization of TNT. Figure k shows that in the presence of yeast extract that a

slightly better final cell yield is obtained in the absence of ammonia in the medium.

Yeast extract is not required for the growth of 1-2-5 but has a definite stimulatory

effect.

The effect of varying TNT concentration on the growth response and percent TNT

utilization is shown in Figure 5> There is a direct relationship between substrate

concentration and the amount of growth obtained ->ith 1-2-5- Substrate concentration

is definitely limiting below 100 ug/ml, and at 25 ug/ml supports very little growth

of the organism. In this expeiiment the TNT served as both the sole source of carbon

and nitrogen. With this isolate there is a direct relationship between substrate

concentration And percentage of TNT utilized as contrasted to isolate HEX in which

there was.- an inverse relationship.

E. Other growth and utilization studies:

Nine of the sewage isolates have been screened for growth on TNT (lOO ^ig/ml) in

M-9(-N) with 0.01$ yeast extract as a supplement. The nature of the growth curves

for these isolates is the same as those obtained with isolated I-2WT and HEX, there-

fore, we will report only initial and final turbidity readings on these isolates

(Table 5).

All nine isolates gave a good growth response in this series. There was no

essential difference in the growth rate of any of these isolates with the exception

of S-9-SY-UN-1 and S-9-SY-UN-5 which had higher growth rates and the greatest

increase in cell density.

Table 5- Growth of sewage isolates on M-9(-N) supplemented with 0.01$ yeast extract.

Isolate Number

S-9-SY-UN-2 "~~

S-9-SY-UN-6

S-9-SY-UN-1

S-9-SY-UN-5

S-9-SY-UN-3

3-9-3Y-UN-U

S-9-UY-UN-12

3-9-UY-UN-DFW

S-9-UY-UN-5 _

Kiett Heading 0 Hours

Klett Heading U8 Hours

1*5 315

165 500

135 Uio

125 1+20

135 305

195 350

1U0 500

125 280

185 335

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250 lOOppm

200 50ppm

ISO 25ppm m

100 TNT DEGRADATION

50

lOOppm, 62% 50ppm 48% 25pp(n| 30%

. i I ■i

20 25 5 10 15 TIME IN HOURS

Figure 5. Effect of TNT concentration on growth response of 1-2-5 and percent degradation.

a> JC o a. Zi c a> o» >> K o

TNT Endogenous

_L X

t Tip

10 20 30 40 MINUTES

50 60

Figure 6. Oxygen uptake by IIBX.

17

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F. Metabolic studies:

Results of respirometric studies ''.ndicate the oxidation of TNT by isolate IIBX

(Figure 6). Oxidation proceeded without a lag following the tip of organisms.

During the JO minutes prior to the break, the 02 consumption by isolate IIBX on TNT

was 3^% greater than the endogenous uptake. The oxygen uptake by the endogenous

control for the 30 minute period -was 25 uliters compared to 38 uliters by TNT. Sub-

tracting the endogenous value indicates a net oxygen uptake of 13 uliters er O.38

nmcles of oxygen per 0.88 p.moles (200 ug) of TNT in the Warburg flask. It is un-

likely that all the TNT was oxidized in this experiment, therefore it is possible

that 1 Mmole of oxygen is taken up for each uniole of TNT oxidized. This data is not

conclusive since the amount of gas exchange is so low that a large margin for error

can exist. The amount of carbon dioxide evolved was extremely low and could not be

measured accurately in this experiment.

An endogenous repression study was performed by growing the cells on TJJ'J- C

glucose to label the cellular reserve materials. The labeled cell suspension was

placed in phosphate buffer (O.OIM, pHT-0) with and without TNT. Both flasks were

swept with air during metabolism to carry ajiy produced carbon dioxide into a

methanol-ethanolamine trap. The dpm for the test and control flask were the same

(25^ and 256) for the initial period of the experiment, indicating no repression of

the endogenous metabolism by TNT, At 100 minute of incubation (after the oxygen

break in this experiment), there was an observed 35% greater CO2 evolution in the

absence of TNT than in its presence indicating a 35% repression of the endogenous

metabolism by the presence of TNT. Since the repression occurs following the oxygen

break, it would appear that calculations made prior to the break are valid. It does,

however, indicate an effect by TNT or its metabolic products on secondary metabolic

events in isolate IIBX.

Wien (ring-UL- C) TNT became available, experiments were performed to determine

the fate of TNT. Isolates IIBX and I-2WT were grown in flasks of M-9 supplemented

with TNT (100 ng/ml) and 0.01% yeast extract (Figure 7). TNT (ring-UL-^C) at 6.1+

x 10 dpm was added to each flask. The accumulative dpm collected as carbon dioxide

are shown in this plot. Table 6 shows the activity obtained in the filtrate after

1+20 minutes incubation. The recovery of C was only 3.8% for isolate IIBX and 5-9%

for isolate I-2WT of the initial activity added to the flask. In the experimental

protocol, the cells from the reaction flask were recovered on a Millipore membrane

and the filtrate activity measured. The cells were then washed with a large volume

of water to remove any unmetabolized TNT which might have attached to the cells.

-18-

■ 1 ;u<imi*imäa^ut^miu**n*mMM, • m\, mt^^im^iii I ■■ »"-"■ ----- - .L. I- ■- ■ ■ -:- -■-,:■;:. j ...

ffflirwir».ii»i .,

1

12

9 I o

2 6 a o

CVJ o o

3 -

J—I—I 1—_l I ■ ■ ■ 1 90 180 270

MINUTES 360

Figure 7 Accumulated dpm as carbon d'oxide with IIBX and 1-2-5 growing on TNT (ring-UL-l4C).

19

in ■niMiimiiain ■m niihlliHillllTii aaUk ■ i -ii ii ■ itintiiiMiaiMfctorii.in m*%jü.amauu - Ail-2vr :',-'.-■-.■ J j.^^r-uViJ- „■■.:,.-,■■ '.i^u^jtlMM

^wuiiiiijm.nuwm IP .1 t(, , i wiw ,1.1,111-mm . mini|, jji IIPW»H mi.,<L_ ,i I,I i ijwi,,. i mum. UU^T

Ta"ble 6. TNT (ring-UL- C) distribution with IIBX and I-2WT growing on TNT in M-9 medium.

ßajnpxe TOT X.-C W dpm $ Recovered

Activity

dpn: % Pecovered

Activity

Initial fla-k

Final Filtrate

Cells

Accumulated CO2

Total Activity Recovered

6.k x iob 6.U X 10°

2.1 x 105 87.0 3-5 '■: 10' 93-0

3.0 x 10^ 12.5 2.^ x 1Ö* 6.6

1.2 x 105 0.5 1.2 x 10-' 0.3

2.U x 10' 3.8 3.8 X 10' 5.^

The measured percent utilization of TNT by isolate IIBX in previous growth

systems has varied from 12-28$ of an initial concentration of 100 ug'ml. The 13.0L£

of the recovered label incorporated into the cells and released as carbon dioxide in

this experiment by isolate IIBX is, therefore, at the lower range of TNT utilization

as shown by the spectrophotometric method. In this experiment flask agitation was

not mechanically possible and aeration depended only on the air sweep, thus cutting

'".he aeration efficiency of the system below our normal value. It appears that the

C values for isolate I-2WT are low when compared to utilization measurements by

the spectrophotometer. This ^C data indicates about 7$ utilization versus the

20-27$ utilization by our standard method.

The experiment was repeated with two important modifications. The reaction was

run for 22 hours in a shaker water bath, which slightly increased the aeration

efficiency of the system and NH4CI was not used in the medium. The results are

reported in Table "J.

Table 7. Percentage of labeled TNT metabolized by IIBX and I-2VJT in ammonia-free

medium.

Sample IIBX I-2WT

dpm %

1.3 x 10° -

k 2.1 x 10 9-6

2.0 x 105 89.3

2.7 x 105 1.2

2.2 x 105 17. U

dpm

Initial Activity

Cells

Filtrate

C02

Total Recovered

1.3 x 10

2.6 x 10

3.9 x 105

3-5 x 105

k.2 x 105

6.1

93.1

.8

32.6

The percentage of the initial activity recovered in this experiment was low

-20-

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WJ»llM['UJWiW!iP^^^PMWWWMNWWPW^^ ,iU/,, ^^^^^„q^p^^..-

(if.ki for HEX and 32.6^. for I-2WT). We know fron; previous experiments that neither

isolate can utilize the majority of the TNT in these batch culture systems. Also,

the aeration efficiency of the C apparatus is much lover than our normal growth

system, therefore, we expect a reduction In TNT utilization. The low percentage

recovery of C activity is due to the lose of TNT which has been absorbed to the

cells during incubation and its removal by the vigorous wash of the cells. For this

reason it is logical to calculate percentages based on recovered activity rather than

initial activity. Current and future experiments will include counts of the wash

water to substantiate the fate of the "lest" activity.

The data in Table 7 shows 9-6% uptake of recovered label "by the HEX cells and

6.1% uptake by the cells of I-2WT grown on the labeled TNT. The trapped carbon

dioxide contained 1.2$ and 0.8$ of the recovered activity. Since the TNT was

labeled only in the ring, any labeled carbon dioxide must result from cleavage of

the benzene ring. Thin layer chromatography examination of the labeled TNT' doe:-

not detect any impurities in this preparation. This experiment is not definitive

proof of ring metabolism but is suggestive of ring fission and further experiments

are planned to confirm this activity. Warburg respirometric studies confirmed the

low level of carbon dioxide production by these isolates. Emphasis will be placed

on the study of the cellular products as an indication of TNT utilization rather

than carbon dioxide evolution because of the low amounts of carbon dioxide released

in this system.

These organisms are required to obtain their carbon for growth only from TNT

which is present at low substrate levels (100 ug/ml). It is likely, therefore, that

most of the metabolized carbon is used for the synthesis of cellular material. It

is expected that in the course of metabolism, decarboxylations do occur in the over-

all metabolic process, which should result in a net production of measurable amounts

of carbon dioxide. The low levels of carbon dioxide detected with these isolates

suggested that heterotrophic carbon dioxide fixation might be an important

phenomenon in this system (6).

Ik To test this hypothesis, NaHC O3 was added to a culture of organism 1-2-5

growing en unlabeled TNT in the absence of NH4CI. The inocula cells were grown on

p-hydroxybenzoate and nutrient agar. The flask contained an excess of bicarbonate

(20 mg) carrying 1.59 x 10 dpm of C activity. Carbon dioxide produced from the

metabolism of TNT equilibrates with the C bicarbonate added as an exogenous supply

in these experiments. Dilution experiments based on specific activity would be

required to determine the percentage contribution of each source of carbon dioxide.

The TNT concentration was 100 ug/ml or a total flask content of 5 mg. The cells

were collected at the end of the growth period (22 hours) and counted for ' C

incorporation (Table 8). The cells grown in the presence of the labeled carbonate

incorporated 2.0% (2.2 x 10 dpm) of the activity or O.k mg of carbonate. The flask

contained 5 mg of TNT (22 umoles) and showed the fixation of 9 umoles cf carbon

dioxide or kl% heterotrophic carbon dioxide fixation. Previous experiments have

indicated that organism 1-2-5 only utilized 6o-?0# of the TNT at a substrate

concentration cf 100 ug/ml suggesting that -the actual percentage of heterotrophic

-21-

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qj^^^gjgQI^Qqgggqfg^ ■jni;jiiii-»»i | M

carbon dioxide fixation is much higher. There was no significant difference in the

values obtained in this experiment based upon the substrate used to grow the inoculum

ceils.

Table 6. Heterotrophic carbon dioxide fixation by 1-2-5-

Sample p-OH Ben:

dpm

,oate Grown

$ Recovery

Nutrient Agar Grown

dptn H> Recovery

Initial NaHC03 1.6 x io6 - 1.6 x 106 -

Cells k

5.2 x 10 2.0 1+

5-5 x 10 2.0

Filtrate 8.5 x 105 55.0 1.0 x 10° 65.O

Total Recovery 8.9 x 105 56.0 1.1 x 106 69.O

Cells based on Rec overy - u.o - 5-0

Cells grown on TNT agar demonstrate a definite reddish-brown colony color that is

not apparent in nutrient agar grown cells of any of the isolates. Also, in liquid

medium this same reddish-brown color is apparent in the filtrate. The color is not

identical to the red color which developes in TNT media exposed to light. The ■L C- TI1T experiments all indicate a significant incorporation of labeled carbon from TNT

into the cell. The ^C incorporated from TNT could be as cellular components,

accumulated TNT, partial degradation products of TNT or metabolic transformation

products of TNT. In an effort to determine the nature of the incorporated C and

also the reddish-brown color described above, organism 1-2-5 was grown on carrier TNT

with ring-UL- C-TNT. After growth, the cells were collected and counted for activity.

The dpm from a 25 ml aliquot of cells was 98^0. The cells from the remaining 25 ml

cf the culture were lysed with 5 ml of concentrated H2S04. The lysed cells were

extracted twice with 25 ml of petroleum ether, the extracts combined, evaporated to

dryness and resuspended in 1.0 ml of petroleum ether. The concentrated extract was

spotted (200 lambda) onto prewashed silica gel TLC plates and developed in Benzene:

Hexane:Acetone (50:50:10), dried, and sprayed with ethylenediamine. A purple spot

was obtained at an R.f of 0.80 which corresponded to 2,2' ,6,6' -tetranitro-^,1*' -

azoxytoluene in our known mixture. In the killed cell control, the same spot was

found as well as a second spot identified as alpha-TNT. The spots were removi from

the TIC plate and extracted into toluene, flor added and courted in the scintillation

spactrophotometer. The activities in the two spots of azoxy compound were *+ dpm

and the TNT spot was 10 dpm. These low counts are not conclusive but indicate that

these components probably have no significance as the cellular incorporated ^C

(981*0 dpm) from the labeled substrate. Earlier experiments without labeled TNT have

indicated the same 2,2 ' ,6,6'-tetranitro-l+,^ '-azoxytoluene component in TNT grown cells

and an additional pink spot at an Rf of O.76 that does not correspond to any compound

in our reference mixture.

-22-

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mm. wmmmmmmmm WPNfllHi^ffüW? imijffui■! ijui .,j.g i,pj.umw,n i nisa^iMfiii^wy .*"py"^~

7. Conclusions

1. Bacteria have teen isolated which can utilize 2,1+,6-trinitrotoluene (INT)

as a sole source of carbon and nitrogen for growth. All isolates obtained -je Grain

negative rods. Yeast extract (0.01$) is stimulatory and is required for grov*'h of

all isolates. Mutants of isolate I-2WT do not require yeast extract for growth but

are stimulated by its addition. Colonies of the various isolates have beer, obtained

on mineral salts medium containing 100 ug/ml of TNT, 0.01$ yeast extract and

solidified with 1.5$ washed agar.

2. Using spectrophotometric analysis of TNT, it has been demonstrated that the

various isolates cause a 10-72$ disappearance of TNT from the growth u-edlum. The

percentage of TNT utilized is related to the initial substrate concentratijn in the

growth medium.

3. Studies with labeled TNT (ring-UL- C) have shown the incorporation of

6-12$ of the label into the cells and the release of 0.8-1.2$ of the label as carbon

dioxide. Since the TNT is labeled only in the ring, this suggests TNT ring cleavage

by the bacteria. It is concluded, therefore, that isolated IIBX and I-2WI may be

capabLe of TNT biouegradation.

k. Studies with NaHC^ 03 demonstrate heterctrophic carbon dioxide fixation by

organism 1-2-5 growing on TNT. This isolate growing on 22 umoles of TNT fixed 9

umoles of cpjbon dioxide.

5. Preliminary experiments suggest that the C label from TNT' metabolism

which is incorporated into the cell is not in the form of nitroaromatic compounds.

VI. Literature Cited

1, Raymond, D.G.M. and M. Alexander, 1971. Microbial metabolism and co-

metabolism of nitrophenols. Pest. Biochem. Physiol., 1:123-130.

2. Tewfik, M.S. and W.C. Evans, 1966. The metabolism of 3,5-dinitro--"-cresol

(DNOC) by soil microorganisms. Biochem. J. 99:3i»

3. Osmon, J.L. and B.E. Klausmeier, 1972- The microbial degradat

explosives. Develop. Ind. Microbiol., I^:2i+7-252.

:n cf

U Won, W.D., E.J. Heckly, D.J. Glover and J.C. Hoffsommer, 197^- Metabolic

disposition of 2,lj-,6-trinitrotoluene. Appl. Microbiol., 27: 513-516.

5. Roberts, R.B., D.B. Cowie, P.H. Abelson, E.T. Bolton and R..J. Britten, 19:>

Studies cf biosynthesis in Escherichia coli, Carnegie Inst. of Washington

Publication No. 607.

6. Leadbetter, E.R. and J.W. Foster, i960. Bacterial oxidation of gaseous

alk.anes. Arch. Microbiol., 35:92-10U.

7. Traxler, R.W. and J.M. Bernard, 1969. The utilization of n-alkanes by

Pseudomonas aerugincsa under conditions of anaerobicsis. I. Preliminary observations.

Int. Biodetn. Bull., 5:21-25-

-23-

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