Vol. 58, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 3067-30710099-2240/92/093067-05$02.00/0Copyright © 1992, American Society for Microbiology

Role of Heterotrophic Bacteria in Complete Mineralization ofTrichloroethylene by Methylocystis sp. Strain M

HIROO UCHIYAMA,l* TOSHIAKI NAKAJIMA,2 OSAMI YAGI,' AND TADAATU NAKAHARA2National Institute for Environmental Studies, 16-2 Onogawa,' and Institute ofApplied Biochemistry,

University of Tsukuba, 1-1-1 Tennoudai 2 Tsukuba-shi Ibaraki 305, JapanReceived 2 January 1992/Accepted 29 June 1992

Biodegradation experiments with radioactively labeled trichloroethylene showed that 32% of the radioactivecarbon was converted to glyoxylic acid, dichloroacetic acid, and trichloroacetic acid and that the samepercentage was converted to CO2 and CO after 140 h of incubation by a pure culture of a type IImethane-utilizing bacterium, Methylocystis sp. strain M, isolated from a mixed culture, MU-81, in ourlaboratory. In contrast, these water-soluble [14CJtrichloroethylene degradation products were completely orpartially degraded further and converted to CO2 by the MU-81 mixed culture. This phenomenon wasattributed to the presence of a heterotrophic bacterium (strain DA4), which was identified as Xanthobacterautotrophicus, in the MU-81 culture. The results indicate that the heterotrophic bacteria play an important rolein complete trichloroethylene degradation by methanotrophs.

Trichloroethylene (TCE) is a volatile chlorinated organiccompound that has been widely used as an organic solventand degreasing agent. TCE, which is one of the contami-nants most frequently detected in groundwater, is a sus-pected carcinogen (18). Several methods have been em-ployed for reclaiming groundwater, and biological treatmentof pollutants by utilizing bacteria to degrade chlorinatedaliphatics is a low-cost technique that could be used to purifylarge volumes of contaminated groundwater. Recent studieshave shown that several oxygenase-producing bacteria, in-cluding methane-utilizing bacteria (1, 4, 9, 10, 12, 13, 16, 22,25), ammonia oxidizers (2, 28), and toluene oxidizers (20),oxidize TCE. We previously reported TCE degradation by amethanotrophic mixed culture, MU-81 (26), and a type IImethanotroph, Methylocystis sp. strain M (27), which- wasisolated from MU-81. Among these degraders, the ammoniaand toluene oxidizers require ammonia or aromatic com-pounds (such as phenol), which are also environmentalpollutants, to induce the enzyme activities. Although meth-ane is required to activate methanotroph cells, it is anontoxic substrate, and much higher degradation rates couldbe obtained with methanotrophs than with other TCE de-graders. Thus, the use of methanotrophs would be advanta-geous for aquifer bioremediation. To date, however, there isstill little available information about the products of TCEdegradation by whole cells of methanotrophs. It is importantto have this information before methanotrophs can be usedfor in situ cleanup purposes. Little et al. detected glyoxylicand dichloroacetic acids as water-soluble TCE breakdownproducts in the culture of a type I methanotroph, strain 46-1(16). Chloral, 1,1,1-trichloroethanol, and trichloroacetic acidwere also found in Methylosinus trichosporium OB3b byOldenhuis et al. (22) and Newman and Wackett (21). Wepreviously detected 1,1,1-trichloroethanol, glyoxylic, dichlo-roacetic, and trichloroacetic acids as water-soluble productsin Methylocystis sp. strain M (19). Since chloral and chlori-nated acetic acids are known to harbor mutagenic andhepatocarcinogenic properties, respectively (3, 14), the ac-cumulation of these compounds is undesirable. Since the

* Corresponding author.

above experiments were performed with pure cultures, wedecided to investigate the fate of the TCE degradationproducts in mixed cultures. In this study, we first comparedthe profiles of the TCE degradation products by pure (strainM) and mixed (MU-81) cultures. Then we isolated hetero-trophic bacteria from the mixed culture MU-81 and investi-gated their roles in TCE degradation.(A preliminary report of this study was published previ-

ously [25a].)

MATERIALS AND METHODS

Chemicals. [1,2-'4C]TCE (13 mCi; DuPont, Wilmington,Del.) was dissolved in methanol to give a stock solutionconcentration of 10 mg of ['4C]TCE ml-'. Ready-soluv HP(Beckman Instruments, Inc., Fullerton, Calif.), a scintilla-tion fluid for the determination of radioactivity, was used foraqueous samples.Organisms and culture conditions. The methane-utilizing

mixed culture MU-81 and Methylocystis sp. strain M wereisolated in our laboratory. The cultures were grown in amineral salt medium in a methane-air (1:1, vol/vol) atmo-sphere at 30°C as described previously (26).

Radiolabeled TCE biodegradation. TCE degradation stud-ies with pure or mixed cultures were conducted in 155-mlserum bottles containing 30 ml of mineral salt medium. Eachbottle, with a septum cap lined with Teflon, was inoculatedwith 0.2 ml of the precultured strain M, MU-81, or aheterotrophic bacterium and received 50 ml of filter-steril-ized methane (40% of headspace volume) by injection. Atthe start of cultivation, a dose of 10 ,uCi of [1,2-14C]TCE wasused and the culture was incubated at 30°C with shaking at100 rpm. This procedure led to a concentration of 1.0 ppmTCE in the liquid culture medium after equilibration with theheadspace.Measurement of '4C in various fractions of the culture was

based on the method of Little et al. (16). The cultures werefractionated to yield cells, CO2 plus carbon monoxide (CO),and water-soluble fractions. After 140 h of culture, the pH ofthe sample was made alkaline with 2 N NaOH (indicated bythe blue color of thymolphthalein), and headspace 14C02 and14Co were trapped in the medium. Tests with cold CO

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revealed that CO was also trapped and that the trappingefficiency exceeded 96%. Culture samples were then centri-fuged at 5,000 x g for 10 min to yield cells associated with'4C; the cells were washed and suspended in saline, and theirradioactivity was determined. The supernatant was ex-

tracted with n-hexane to remove TCE and acidified with 6 NHCI (indicated by the color change of tropaeolin 00) in aclosed bottle to release 14CO2 and 14CO to the headspace,and the liquid phase was completely removed with a syringethrough the septum. This liquid phase corresponded to thewater-soluble fraction. Into the empty bottle, 1 N NaOHsolution was injected to trap 14C02 and 14CO, and theradioactivity of the solution was determined with a Beckmanmodel LS9000 scintillation counter.

Before the treatment described above, the fate of theradiolabeled TCE degradation products was investigated byassaying headspace 14C02 and 14CO. An aliquot of gassample was removed with a gas-tight syringe and injectedinto the gas chromatography-gas proportional counting sys-tem. A Shimadzu (Kyoto, Japan) model GC-7A gas chro-matograph equipped with a thermal conductivity detectorand a Unibeads C (80/100 mesh) column (3 mm by 2 m) keptat 130°C with He (40 ml/min) as the carrier gas was con-nected to a Aloka (Tokyo, Japan) model RGC-211 gasproportional counter. When the radiolabeled compounds inthe liquid culture medium were determined, 1 ml of theculture was withdrawn with a syringe and centrifuged for10 min at 5,000 x g. An aliquot of the supernatant wasinjected directly into a Waters model 510 high-performanceliquid chromatograph (HPLC) equipped with a TSK-GELSCX(H+) column (Tosoh Co., Tokyo, Japan) and a Watersmodel 481 UV spectrophotometer; the sample was elutedisocratically with 0.01 N sulfuric acid containing 5% aceto-nitrile at a rate of 1 ml/min. Fractions were collected every 5drops and analyzed with the scintillation counter.

RESULTS

Distribution of 14C-labeled TCE degradation products inpure and mixed cultures. TCE degradation in the pure andmixed cultures was confirmed by mineralization studies. Atthe end of the experiment (140 h of incubation), less than 5%of the initial [14C]TCE remained in the reaction bottles. Thefact that the total counts in the control bottle lacking bacteriaremained almost constant during the experiment (deter-mined by radio-gas chromatography) indicated that therewas no loss of 14C-labeled material from the bottle during theexperiment.The distribution of '4C radioactivity appearing in the

washed cells and the water-soluble and C02-plus-CO frac-tions in the pure and mixed cultures is shown in Fig. 1. Thedistribution patterns of 14C radioactivity in the cell fractionsof the pure and mixed cultures were almost identical. On thecontrary, in the pure culture, 32% of the transformed TCEappeared in the water-soluble fraction and the same percent-age appeared in the CO2-plus-CO fraction. In the mixedculture, only 12% of the transformed TCE appeared in thewater-soluble fraction, whereas in the C02-plus-CO fractionthe value increased to 51%.

Isolation and characterization of heterotrophic bacteria inmixed culture MU-81. The distribution patterns were appar-ently different in the pure and mixed cultures (Fig. 1). Todetermine whether such differences were due to the pres-ence of heterotrophic bacteria in the mixed culture MU-81,we isolated and characterized the bacteria. In the mixedculture MU-81, three morphologically different types of

0

M

MU-81

Radioactivity (%)

so InnI I

Cell Woalteir- C02+COsoluble(36) (32) (32)

l (37) 1(12)1 (51)

FIG. 1. Distribution of radioactivity after degradation of [14C]TCE by a pure culture (M) and a mixed culture (MU-81). Experi-ment was performed in 140-h cultures. Numbers within parenthesesindicate the percentages of the total radioactivity of each fraction.

heterotrophic bacteria and a type II methanotroph, Methy-locystis sp. strain M, had already been observed (27). Byrepeated streaking and isolation, we obtained the threeisolates, including strains DA4 and NG2. They grew onnutrient agar and did not require methane for growth.

Strain DA4 formed yellow colonies and appeared as ashort rod-shaped (1.1 to 2.4 ,um in length and 1.0 to 1.2 ,umin width), nonmotile, gram-negative bacterium that utilizedshort-chain fatty acids, including dichloroacetic acid. Theseand additional characteristics of strain DA4 are summarizedin Table 1. Based on these results, the bacterium wassubsequently identified and designated as Xanthobacter au-totrophicus DA4.

Strain NG2 formed white colonies and was a cytochromec oxidase-positive gram-negative rod bacterium that wasmotile in the liquid medium. These properties are in agree-ment with the description of the genus Pseudomonas asgiven in Bergey's Manual of Determinative Bacteriology(23).Although the third isolate was assigned to the genus

Bacillus, it played no apparent role in the degradation of

TABLE 1. Characteristics of strain DA4q

Parameter Result or Parameter value

Physiological characteristics Utilization of:Gram stain Negative Arabinose -

Oxidase + Fructose -

Catalase + Glucose -

Oxidation-fermentation - Mannose -

test Mannitol -

Indole production - Maltose -

P-Galactosidase: - Sucrose -

NO3 reduction to NO2 + Citrate +NO3 reduction to N2 - Malate +H2S production - MalonateEgg yolk agar opacity - Propionate +Acetoin production - Succinate +Methyl red - HistidineArginine dehydrolase - Phenylalaninemol% G+C of DNA 69.5

Hydrolysis of:Casein, gelatin, starch, -

Tween 80Urea + (weak)a Colonies of DA4 are yellow, opaque, shiny, smooth, round, regular, and

entire.

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3

2

1

0

Ac4

:5.C

310a

Ill

O 4 8 12

E- 2 * Bo B0.

1EJ0I.-

*5 0 4 8 12

2

1 -

0~o 4 8 12

2[ D

0 4 8 12Time (min)

FIG. 2. HPLC profiles of water-soluble fractions derived frompure and mixed cultures incubated with ['4C]TCE: A, strain M; B,mixed culture MU-81; C, mixed culture of strains M and NG2; D,mixed culture of strains M and DA4. A small portion of the 140-hculture was centrifuged, and 80 Ill of the supernatant was injectedinto the HPLC. Peaks: I, trichloroacetic acid; II, dichloroaceticacid; III, glyoxylic acid. See the text for details.

TCE. Therefore, only strains DA4 and NG2 were selectedfor further TCE defradation studies.

Determination of 4C-labeled water-soluble products in pureand mixed cultures. Since a significant difference in theradioactivity distribution was observed in the water-solublefractions of the pure and mixed cultures (Fig. 1), we ana-lyzed the radiolabeled TCE degradation products in fourcultural systems (strain M, MU-81, strains M and NG2, andstrains M and DA4) by HPLC. The retention times ofradioactive peaks I, II, and III corresponded to those ofauthentic trichloroacetic, dichloroacetic, and glyoxylic ac-ids, respectively (Fig. 2). The chromatographic separationrevealed that the elution profiles of the three mixed cultures(Fig. 2B, C, and D) were different from that of the pureculture (Fig. 2A). In the mixed cultures of MU-81 and strainsM and DA4, only trichloroacetic acid was detected; theamount of trichloroacetic acid was about half that of the pureculture. In contrast, in the culture of strains M and NG2,trichloroacetic and dichloroacetic acids were still present

1.0 _

E

10

a a:

1.

0.05

II 0

.5 A

.0

1.5 "

OL_-----0

0.

5 B

.0 _

1.5 Fe

O .C~~~~0N0 40 80 120 160 0 40 80 120 160

Time (hr) Tim. (hr)

FIG. 3. Time course of CO2 and CO derived from [14C]TCE bystrain M (A) and a mixed culture of strains M and DA4 (B).Headspace samples (0.2 ml) were assayed with RI-GC system.Symbols: 0, "4C02; 0, 14CO; V, cell growth.

and their amounts did not decrease. The amount of glyoxylicacid was markedly reduced in all three mixed cultures.These data indicated that the heterotrophic bacteria (espe-cially strain DA4) were capable of mineralizing most of thewater-soluble compounds and that the restoration of thedegradability of MU-81 could be completed by the combinedactivity of strains M and DA4.

Fate of 14C-labeled degradation products during TCE deg-radation in pure and mixed cultures. Along with the TCEdegradation, CO2 and CO were produced in both pure andmixed cultures and CO was subsequently oxidized (Fig. 3).After 140 h of incubation in both cultures, the radioactivityof CO was low enough to be neglected; the radioactivity ofCO2 in the mixed culture was about 1.5 times higher thanthat of the pure culture, which is in good agreement with thedata in Fig. 1. TCE degradation occurred during the activephase of cell growth of strain M (50 to 120 h) (data notshown), indicating that the active metabolism of a metha-notroph (especially through the activity of methane mono-oxygenase) was necessary for TCE biodegradation.

Since trichloroacetic, dichloroacetic, and glyoxylic acidswere the major water-soluble products in strain M (Fig. 2A),we monitored the fates of these compounds in pure andmixed cultures (Fig. 4). In the pure culture, trichloroaceticacid was predominant and glyoxylic acid accumulated at themid-log phase and then became oxidized. Although tri-chloroacetic acid was also predominant in the mixed culture,its radioactivity decreased to about half that of the pureculture after 140 h of incubation. In addition, dichloroaceticand glyoxylic acids were subsequently oxidized, and their

80

3:

2

1.2r B

0.9 t

0

O

L. 3.V J

v"

,

y/3*/

0 40 80 120 160Tm (hr) Thm (hr)

FIG. 4. Time course of water-soluble TCE degradation productsderived from ['4C]TCE. The water-soluble products were assayedas described in the legend to Fig. 2. Symbols: 0, trichloroaceticacid; 0, dichloroacetic acid; A, glyoxylic acid; V, cell growth.

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level decreased to almost zero at 160 h incubation. Thedifferences observed in the activity of the chlorinated aceticacids were apparently due to the ability of strain DA4 tometabolize the products resulting from TCE degradation bythe methanotrophs.Based on the observation (Fig. 4A) that the ratio of

radioactivity of trichloroacetic, dichloroacetic, and glyoxylicacids in the water-soluble fraction was approximately 9:4:1after 140 h of incubation, it was deduced that about 21, 9,and 2.3% of the initial radioactivity of [14C]TCE was con-verted to trichloroacetic, dichloroacetic, and glyoxylic ac-ids, respectively (Fig. 1). In contrast, in the mixed culture ofstrains M and DA4, the ratio was 90:8:2 after 140 h ofincubation (Fig. 4B). The degradability of MU-81 was re-stored by strains M and DA4 (Fig. 2B and D). Therefore,about 10% of initial [14C]TCE appeared to be converted totrichloroacetic acid in the mixed culture MU-81 (Fig. 1).

DISCUSSION

Our results demonstrated that glyoxylic and dichloroace-tic acids were completely converted to CO2 by a heterotro-phic bacterium, DA4, whereas trichloroacetic acid wassomewhat resistant to further degradation (although its con-tent decreased). In a carbohydrate utilization test, strainDA4 was able to grow in minimal medium containing 0.1%glyoxylic or dichloroacetic acid as a sole carbon source butnot in minimal medium with 0.01% trichloroacetic acid (datanot shown), implying that trichloroacetic acid is metabolizedvia cooxidation by strain DA4. Based on the results shown inTable 1, strain DA4 was assigned to X. autotrophicus. Theability to degrade halogenated aliphatic compounds is mainlyobserved in the pseudomonads (5, 8) and has not beenreported for chemolithoautotrophic bacteria, except for X.autotrophicus GJ10 (15). Strain GJ10 was reported to be anitrogen-fixing hydrogen bacterium that degraded 1,2-dichlo-roethane and some other halogenated alkanes, includingdichloroacetic acid. For these degradations, strain GJ10constitutively produces a heat-stable dehalogenase that isspecific for halogenated carboxylic acids and converts themto corresponding alcohols and halide ions (15). Strain DA4 ismorphologically and physiologically similar to strain GJ10,except that strain DA4 contains a cytochrome c oxidase andcannot utilize fructose or sucrose as a sole carbon source.Thus, it is speculated that strain DA4 converts dichloroace-tic acid to hydrogen chloride and dihydroxyacetic acidthrough the activity of a haloalkanoate dehalogenase andthat the latter compound is metabolized to CO2 via centralmetabolic pathways.The time course experiment indicated that trichloroacetic

acid was a predominant breakdown product in the water-soluble fraction in the stationary phase (Fig. 4A). Trichloro-acetic acid is considered to be formed by the oxidation ofchloral, which is converted from TCE via a Cl shift catalyzedby methane monooxygenase (6). We previously reportedthat TCE degradation is catalyzed not only by epoxidationfollowed by the spontaneous breakdown of TCE oxide toproduce dichloroacetic acid, etc., but also by Cl shift fol-lowed by the formation of trichloroacetic acid and 2,2,2-trichloroethanol and that the Cl shift pathway seems to besignificant in strain M (19). The observation that trichloro-acetic acid was a predominant dead-end degradation productby strain M supports this assumption (Fig. 4A).

During the TCE oxidation by strain M, glyoxylic acid wasalso one of the predominant breakdown products in themid-log phase but decreased in the stationary phase. Since

strain M cannot utilize glyoxylic acid as a sole carbon source(27), it is possible that glyoxylic acid was assimilated viacooxidation in strain M.Henry and Grbic-Galic reported that CO, an intermediate

in the aerobic degradation of TCE by Methylomonas sp.strain MM2, was subsequently oxidized to CO2, even in theabsence of an exogenous supply of electron donor (e.g.,formic acid) (11). In a whole-cell study of Methylococcuscapsulatus (Bath) (24), the CO produced was not oxidized inthe absence of an exogenous electron donor. In the case ofstrain M, CO was produced in the log phase and thenoxidized, even in the absence of an exogenous electrondonor (Fig. 3A), as in the case of Methylomonas sp. strainMM2. In the cases of both strain M and Methylomonas sp.strain MM2, storage polymers such as lipid granules wereobserved, whereas their presence has not been reported inM. capsulatus (Bath). Additionally, it was proposed thatlipid storage granules act as an endogenous source of elec-trons and enhance TCE oxidation (11). Therefore, the factthat the CO produced was subsequently oxidized to CO2even in the absence of an exogenous electron donor may bedue to the presence of endogenous electron donors asgranules. Such a phenomenon may be a distinct character-istic of methanotrophs containing storage polymers.

After TCE oxidation by strain M had taken place for 100 h,at least 10 mol% of TCE was transformed to CO, based onthe data in Fig. 3A. However the real amount of COproduced may actually have been much larger because offurther oxidation along with the progression of the produc-tion. Regardless of the amount produced, CO is reported toinhibit TCE oxidation in spite of its lack of toxicity to cells(11). In our preliminary experiment, we also observed that150 ,uM CO inhibited the TCE oxidation rate by about 34%in strain M. However, since no difference was observedbetween the fates of CO in the pure and the mixed cultures(Fig. 3), it is concluded that strain DA4 cannot oxidize CO,suggesting that strain DA4 does not contribute to the allevi-ation of the inhibition by CO. Since bacteria capable ofoxidizing CO are ubiquitous in the environment (17), COproduction during TCE oxidation does not prevent fieldapplication of strain M for the cleanup of the environment.

After TCE degradation by the pure culture (Fig. 4A) hadtaken place for 160 h, the radioactivities of the dichloroaceticand trichloroacetic acids in the water-soluble fraction wereabout 9 and 20% of the initial TCE radioactivity, respec-tively. Dichloroacetic acid was reported to be produced byTCE oxidation by a type I methanotroph, strain 46-1 (16),and by soluble methane monooxygenase from M. ticho-sponum OB3b (6). Trichloroacetic acid was also detected inM. trichosporium OB3b by Newman and Wackett (21).Although the stoichiometric amounts produced were notdetermined accurately except for those of soluble methanemonooxygenase, in which dichloroacetic acid was observedat a yield of 5% of the initial TCE concentration, bothchlorinated acetic acids are reported to be hepatocarcino-gens in mice (14). In the case of strain M, although it remainsto be determined whether both chlorinated acetic acids aretoxic at the concentrations calculated above, the amounts ofthese TCE degradation products should be low. Recently,these chlorinated acetic acids have attracted a great deal ofinterest as by-products of the chlorine disinfection of watercontaining natural organic materials, and the U.S. Environ-mental Protection Agency is developing regulations to con-trol these disinfection by-products (7). It is suggested thatthe field application of methanotrophs for the removal ofTCE from the environment requires further studies on the

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ecophysiology of heterotrophic bacteria (e.g., associatedbacterial flora, population dynamics, potential for degrad-ability, etc.) to decrease the final concentrations of the TCEdegradation products.

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