JOURNAL OF BIOSCIENCE AND BIOENGDJEERING

Vol. 92, No. 5,453-458.2001

Efficient Dechlorination of Tetrachloroethylene in Soil Slurry by Combined Use of an Anaerobic Desdfitobacterium sp.

Strain Y-5 1 and Zero-Valent Iron TAEHO LEE,‘* TAKESHI TOKUNAGA: AKIKO SUYAMA,’

AND KENSUKE FURUKAWA’

Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan’ and Fukuoka Institute of Health and Environmental Sciences, Mukaizano, Daza& Fukuoka 8184135, Japan2

Received 17 August 200UAccepted 11 September 2001

A laboratory test was conducted to examine the combined effect of bioaugmentation of an anaerobic bacterial Desuljitobacterium sp. strain Y-51 and addition of zero-valent iron (Fe@) on the reductive dechlorination of tetrachloroethylene (PCE) in a non-sterile soil slurry. Introduction of a strain Y-51 culture in soil (3 mg vss (volatile suspended solids)/kg soil) containing PCE (at 60 pmol/kg soil) led to complete conversion of PCE to cis-1,2-dichloroethylene (cis-DCE) within 40 d. Treatments of the same soil slurry with Fe0 (O.l-1.0%) resulted in extended PCE dechlori- nation to ethylene (ETH) and ethane (ETA). The combined use of a strain Y-51 culture and Fe’ showed effective dechlorination of PCE than did the individual use. The cis-DCE produced from biological PCE dechlorination by strain Y-51 was totally converted to non-chlorinated end prod- ucts by the following chemical reduction by FeO. Furthermore, anaerobic corrosion of Fe0 was found to stimulate the biological reductive dechlorination of PCE by keeping proper levels of pH and oxidation-reduction potential (ORP) and by producing cathodic hydrogen, which might be used as an electron donor for respiratory PCE dechlorination. These findings suggest that the combined use of bacterial strain Y-51 and Fe0 is effective for practical treatment of PCE and other chlorinated ethylenes in contaminated sites.

[Key words: dechlorination, tetrachloroethylene (PCE), Desulfitobacterium sp., zero-valent iron]

Chlorinated ethylenes such as tetrachloroethylene (PCE) and trichloroethylene (TCE) are ubiquitous contaminants in soil and groundwater, due to their widespread application in dry-cleaning, degreasing, and chemical production. In addi- tion to their persistence to both biotic and abiotic degrada- tion under subsurface environments (1,2), their toxicity and potential carcinogenesis have led to the considerable neces- sity to remove these chlorinated solvents from contaminated sites. Because highly chlorinated compounds (such as PCE) are extremely resistant to oxidative degradation and many contaminated sites are anaerobic, reductive dechlorination is the most promising strategy for remediating groundwa- ters and soils contaminated with chlorinated ethylenes.

A biological reductive dechlorination approach has re- cently received considerable attention as a low cost reme- diation technique (2, 3). PCE and TCE can be reductively dechlorinated to less chlorinated ethylenes by cometabolic processes carried out by sulfate-reducing, methanogenic, and homoacetogenic microorganisms (4). However, the de-

* Corresponding author. e-mail: lee-th@aist.go.jp phone: +81-(0)298-61-6591 fax: +81-(0)298-61-6587 Present address: Microbial and Genetic Resources Research Group, Research Institute of Biological Resources, National Institute of Ad- vanced Industrial Science and Technology, l-l-l Higashi, Tsukuba, Ibaraki 305-8566, Japan.

chlorination rates of the cometabolic processes are generally low, and their contribution to the reductive dechlorination of chlorinated solvents in natural environments is supposed to be very limited. PCE and TCE can also be reduced in re- spiratory processes mediated by some isolated bacteria, such as Dehalospirillum multivorans, Dehalobactor restictus strains PER-K23 and TEA, Dehalococcoides ethenogenes, Enterobacter sp. strain MSI, Desulfitobacterium sp. strain PCE-S (4) and Desulfiobacterium sp. strain Y-51 (5). In contrast to cometabolic processes, respiratory processes that utilize chlorinated compounds as electron acceptors in energy metabolism are fast and generally accepted as ma- jor contributive processes to biological reductive dechlori- nation in anaerobic environments (2, 6). Therefore, the in- troduction of halorespiring bacteria is expected to be a cost- effective approach to the remediation of PCE-contaminated sites. A number of studies have reported the usefulness of introducing dechlorination activities into environments con- taminated with chlorinated compounds (7-9).

The other reductive dechlorination approach that has also recently gained substantial interest is the chemical reduction of chlorinated solvents by zero-valent metals such as alumi- num, zinc, copper and iron (10, 11). Iron is relatively inex- pensive and nontoxic; it was proposed that it could be useful for remediation of contaminated sites (12, 13). In the pres- ence of a proton donor such as water, chlorinated solvents,

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454 LEE ET AL.

R-Cl, are reductively dechlorinated on the surface of metal- lic iron, FeO, according to the following equation:

FeO+R-Cl+H’ -+ Fe2’+R-H+Cl- (1)

However, water alone can serve as the oxidant, and thus, corrosion occurs under anaerobic condition according to the following reaction:

Fe0+2H,0 + Fe*‘+20H-+H, (2)

The corrosion of iron in anaerobic water is one way that hydrogen can be released into aqueous solution (14). The cathodic hydrogen can be used as an energy source for the autotrophic growth of pure cultures of methanogenic (15), homoacetogenic ( 16), and sulfate-reducing bacteria ( 16) and denitrifying bacteria (17). Weathers et al. reported that a mixed methanogenic culture used cathodic hydrogen as an electron donor for reductive dechlorination of chloroform (18). Hydrogen is also generally considered to be the ulti- mate electron donor to stimulate the reductive dechlorina- tion of chloroethylenes (19). However, the contribution of cathodic hydrogen to the reductive dechlorination of PCE by respiratory PCE-reducing bacteria has not been reported yet.

In this study, we examined (i) the usefulness of the in- troduction of reductive PCE dechlorination activity into non-sterile soil slurry by inoculation with a pure anaerobic Desulftobacterium sp. strain Y-51 as a halorespiring bac- terium and (ii) the effectiveness of combined uses of strain Y-5 1 and Fe0 addition on the total dechlorination of PCE.

MATERIALS AND METHODS

Organism and culture condition A halorespiring bacterium Desuljtobacterium sp. strain Y-5 1, capable of dechlorinating PCE stoichiometrically to cis-1,2-dechloroethylene (cis-DCE) at con- centrations as high as 960 pM and as low as 0.6 pM within 2 d, was originally isolated from a PCE-contaminated soil at a dry- cleaning factory in Fukuoka, Japan. The characteristics of strain Y- 5 1 were reported previously (5). The bacterium was maintained by periodic transfer into a 125-ml volume of a sealed vial containing 100 ml of a defined anaerobic medium which contained (per liter of deionized water): K$IPO,, 7.0 g; KHIPO,, 2.0 g; MgSO,, 7H,O, 0.1 g; (NH,)$O,, 1 .O g; trisodium citrate-2 hydrate, 1 .O g; sodium pyruvate, 0.5 g; sodium fumarate 0.7 g; yeast extract, 1.5 g; 10% sodium resazurin 100 ~1; pH 7.2 with 150 mg/Z of PCE. The sub- culture had undergone at least 2 serial transfers at 3O’C before it was used. Five ml of the culture, which had almost completely re- duced PCE added, was used as the inoculum for the bioaugmenta- tion experiments.

Soil Throughout this study, Sahara soil originating from Saga

J. BIOSCI. BIOENG.,

prefecture and not previously exposed to PCE was used. The na- ture of the soil was 85% sand, 12% silt, and 3% clay with a pH of 6.0 and a density of 2.7 g/cm’.

Soil slurry treatments Duplicate loo-gram samples of non- sterilized Sahara soil were placed in 125-ml volume vials and sus- pended in 25 or 30 ml of the medium described above. A descrip- tion of the soil treatments is shown in Table 1. Three sets (set 1 to 3), which received 30 ml of medium, were flushed with pure N, gas (>99.9%) and, of these, sets 2 and 3 were treated with fine- grained iron powder (FeO, >95%, 325 mesh; Wako Chemicals Co., Osaka) at 0.1 and 1 .O% (w/w) of the soil, respectively, and well mixed to remove air bubbles in the soil slung. The other three sets (sets 4 to 6), containing 25 ml of medium, were placed into an anaerobic chamber (atmosphere: N, 9O%+CO, 5%+H, 5%) and inoculated with 5 ml of the culture of strain Y-5 1 to a final concen- tration of ca. 3 mg vss (volatile suspended solids)/kg soil. Sets 5 and 6 were treated with the fine-grained iron powder at 0.1 and 1.0% (w/w) of the soil, respectively, and well mixed. All of the sets, then, were sealed with Teflon-lined butyl rubber septums and fixed with crimped aluminum caps. PCE dechlorination was started after these vials were supplemented with PCE at a final concentration of 60 pmol/kg soil. PCE was added by syringe from stock solutions diluted with NJ-dimethylformamide. The tops of the vial septums were sealed with solidified paraffin, and the vials were then incubated at 3O”C, placed upside down in a dark box. The concentration of PCE and its metabolites were periodically de- termined.

Analytical methads The concentrations of chlorinated ethyl- enes in the vials were measured by injecting 100 pl of headspace gas into a gas chromatograph (GC%A, Shimadzu, Tokyo) equip- ped with a flame ionization detector (FID). A glass column (3-m length; 3-mm inner diameter; GL Science Inc., Tokyo) packed with Silicone DC-550 was used with N, (99.999%) as the carrier gas (50ml/min). The column temperature was raised from 80°C (1 min) to 120°C (3 min) at the rate of lO”C/min, and the injector and detector temperatures were maintained at 250°C. The final products from PCE, ethylene (ETH) and ethane (ETA), were deter- mined by gas chromatography (HITACHI 165, Hitachi, Tokyo). A glass column (3 m length; 3 mm inner diameter; GL Science Inc., Tokyo) packed with Chromosorb 10 1 was used with N, (99.999%) as the carrier gas (20 ml/min). The temperatures of the column, injector and detector were 4O”C, 15O”C, 1 50°C, respectively. The concentration of hydrogen gas in the headspace of the vials was analyzed with a gas chromatograph (GC-14A, Shimadzu) equipped with a thermal conductivity detector (TCD). Argon (99.999%) used as the carrier gas flowed throughout a SUS column (3 m length; 3 mm inner diameter; packed with SHINCARBON T; GL Science Inc.) at the flow rate of 40 ml/min. The change in ORP in the soil slurry was measured using the combination of an Ag/AgCl reference electrode with a platinum button and a Shibata Model POT-200 ORP meter. The millivolt reading was converted to Eh, using the electrode reading plus 203 mV, the standard potential of the Ag/AgCl electrode at 30°C. In an anaerobic chamber, the elec-

TABLE 1. Description of soil slurry treatments

Amendments Inoculation” Treatment Medium PCE Fe’ Desul$tobacterium sp.

(ml) (pmol/kg soil) (% (w/w)) strain Y-5 1 (ml)

Set 1 (Control) 30 60 Set 2 (Fe’ 0.1%) 30 60 0.1 Set 3 (Fe0 1 .O%) 30 60 1.0 Set4(Y51) 25 60 5 Set 5 (Y51+Fe” 0.1%) 25 60 0.1 5 Set 6 (Y5 1 +Fe” 1 .O%) 25 60 1.0 5

a Final concentrations of inoculants in the soil slurries were about 3.0 mg vsskg soil.

VOL. 92,200l TETRACHLOROETHENE DECHLORINATION BY DESULFZTOBACTERIUM SP. Y5 1 AND Fe0 455

trade was inserted through a perforated Teflon septum into the soil slurry in the vial, and the vial was then sealed with a silicon bond and Teflon tape. The pH measurement was also conducted in the same manner. The concentration of the strain Y-S 1 cells was deter- mined as vss according to the Standard Method 15th edition for the examination of waste and wastewater (20).

RESULTS

Biological dechlorination of PCE in soil slurry by inoculation with strain Y-51 Two sets of vials contain- ing a non-sterile soil slurry, supplemented with PCE (60 umol/kg soil), were incubated for 40 d. Strain Y-51 cells were introduced into one set (set 4) but not in the other set (set I), in order to investigate the usefulness of the augmen- tation of Y-5 1 in reductive PCE dechlorination activity. The control vials containing soil slurry only (not treated with the Y-5 1 cells) showed ca. 30% decrease in the initial PCE con- centration over the duration of the test (Fig. la). Although abiotic loss was thought to be a main possible cause of the decrease, there might have been an indigenous population that is capable of degrading PCE to some extent, because small amounts of chlorinated metabolites were detected during the incubation. In contrast, the treatments contain- ing the Y-5 1 cells obviously dechlorinated PCE to c&DCE within 40 d via TCE without accumulation of any other me- tabolites (Fig. Id).

Chemical dechlorination of PCE in soil slurry by addition of Fe0 Two sets of vials were treated with two different concentrations of iron powder (FeO). PCE was re- moved in the treatments using either 0.1% (w/w) or 1.0% (w/w) of FeO. However, there was a significant difference in the PCE reduction rate between the two sets; although the treatment with 0.1% Fe0 (set 2) showed ca. 60% degradation of total PCE added during 40 d of incubation (Fig. lb), the treatment using 1 .O% Fe0 (set 3) removed most of the added PCE within 20 d (Fig. lc). Some PCE dechlorination inter- mediates were detected in the treatment with 0.1% FeO, but not in the treatment with 1.0% FeO. Both sets of treatments with Fe0 showed apparent ETH accumulation as a final product of PCE reduction. Further detailed analysis of the end products of PCE dechlorination by Fe0 showed ETA ac- cumulation as an additional product, and other chlorinated ethylenes, such as tram- 1,2 dichloroethylene, were also de- tected but the amounts were negligible (data not shown).

PCE dechlorination in soil slurry by the combined use of Y-51 cells and Fe0 The combined use of Y-5 1 cells and Fe0 significantly improved the rate of PCE removal and af- fected the fate of PCE in the vials. The treatments with Y-5 1 cells and 0.1% Fe0 (set 5) converted PCE within 30 d (Fig. le), where about 75% of the PCE added was biologically transformed into cis-DCE, which was not detected as in- termediate of chemical PCE reduction, and about 10% was chemically transformed into ETH that was not produced in

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FIG 1. Reductive dechlorination of PCE by various treatments; control without Y-5 1 cells and Fe” (set 1: a); treatment with 0.1% (w/w) Fe0 (set 2: b); treatment with 1.0% (w/w) Fe” (set 3: c); treatment with Y-51 cells (set 4: d); treatment with Y-51 cells and 0.1% (w/w) Fe” (set 5: e); treatment with Y-5 1 cells and 1 .O% (w/w) Fe0 (set 6: f). Symbols: closed circle, PCE; closed square, TCE; closed triangle, cis-DCE; closed dia- mond, VC; open circle, ETH.

J. BIOSCI. BIOENG., 456 LEE ET AL.

TABLE 2. Changes of pH in soil slung treatments

Treatment

Set 1 (Control) Set 2 (Fe 0.1%) Set 3 (Fe 1 .O%) Set4(YSl) Set 5 (YSl+Fe 0.1%) Set 6 (YSl+Fe 1.0%)

PH Initial* Finalb

6.3 kO.2 6.6+0.2 6.3 kO.2 6.8+0.2 6.3+0.2 8.4+0.3 6.2f0.2 6.7kO.2 6.3f0.2 7.1 kO.3 6.2f0.2 8.3 kO.3

a Although the initial pH of the medium was 6.8, pH was decreased to 6.3k0.2, when it was added to soil.

b pH after 40 d of incubation.

the treatment using Y-51 alone (see Fig. Id). The other set using the strain Y-5 1 and 1 .O% Fe0 (set 6) exhibited signifi- cant reduction of PCE, where PCE was rapidly dechlori- nated into cis-DCE within 6 d of incubation, and the cis- DCE produced was subsequently converted to ETH chemi- cally within 12 d by Fe’. The amount of ETH as the final product was one seventh of the initial PCE amount. ETA was also detected as an additional end product (data not shown). Although a small amount of VC was detected after 6 d of incubation, it was slowly decreased within the experi- mental period (Fig. If).

0 2 4 6 8 10 12 I4

Time (d)

FIG. 2. Changes of oxidation reduction potential (ORP) in various treatments; control without Y-51 cells and Fe” (closed circle); treat- ment with Y-51 cells (closed triangle); and treatment with Y-51 cells and 0.5% (w/w) Fe’ (closed square). The value of ORP was measured using an Ag/AgCl electrode at 3O’C.

incubation and became stable after 5 d (Fig. 2). The ORP values thus reached were as follows: -130mV in the non- amended control, -250 mV with Y-5 1 cells, and -330 mV with Y-5 1 cells and 0.5% Fe’.

Effects of anaerobic Fe0 corrosion on biological dechlorination In order to evaluate the effect of Fe0 addi- tion on PCE dechlorination by strain Y-5 1, changes in pH, ORP (Eh), and hydrogen production were measured. Intro- duction of Fe0 significantly affected pH changes in either the treatment with Y-51 cells or without cells. pH changes after 40 d of incubation were affected by the amount of Fe0 added. The control treatment, without FeO, and the treatment with only Y-5 1 cells showed a slight increase in pH 6.3 kO.2 to 6.6kO.2. Treatments with 0.1% Fe0 showed a slight in- crease in pH from 6.3kO.2 to 6.8kO.2 and treatments with 1.0% Fe0 showed pH changes to 8.3f0.3 (Table 2). When the pH change was monitored in the treatment with 1.0% Fe’, a gradual increase in pH was observed, from 6.3 f0.2 to 8.3 f0.3 during 40 d of incubation (data not shown).

The concentration of cathodic H, produced by the Fe0 corrosion was measured. The addition of 1 .O% Fe0 resulted in a gradual increase in H, concentration (Fig. 3a). When both Y-5 1 and 1.0% Fe0 were added simultaneously, the concentration of H, was increased to 420 pmol/vial at day 2 but decreased to zero at day 4. The H, concentration rapidly increased again after day 6 (Fig. 3a). The PCE dechlorina- tion followed by formation and degradation of TCE and cis- DCE in the treatment with Y-5 1 cells and 1 .O% Fe0 are pre- sented in Fig. 3b. While strain Y-51 dechlorinated PCE to cis-DCE during day 2 to day 6, hydrogen concentration, as shown in Fig. 3a, was also decreased.

DISCUSSION

The effect of Fe0 on the ORP was also observed. The Reductive dechlorination has been studied and applied ORP decreased rapidly within the first one or two days of as a major process for remediation sites contaminated with

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FIG. 3. Production (a) and utilization (b) of hydrogen during the treatment of PCE with Y-5 1 cells and Fe“: (a) control without Y-51 cells and Fe0 (closed square), treatment with 1.0% (w/w) Fe0 (closed triangle), and treatment with Y-51 cells and 1.0% (w/w) Fe0 (closed circle); (b) PCE dechlorination in the treatment with Y-5 1 cells plus 1 .O% (w/w) Fe’. Symbols: closed circle, PCE; closed square, TCE; closed triangle, cis-DCE.

VOL. 92.2001 TETRACHLOROETHENE DECHLORINATION BY DESULFITOBACTERZUM SP. Y5 1 AND Fe0 457

chlorinated ethylenes (2, 21-27). Although biological re- ductive dechlorination for PCE and chemical reduction by zero-valent metal such as Fe0 have received great attention, the effect of combined biological and chemical processes on PCE removal in soil slurry has remained to be elucidated. In this study, the individual introduction of biological dechlori- nation activity or zero-valent iron and the combined intro- duction into a non-sterile soil slurry were compared and evaluated.

The introduction of respiratory PCE dechlorinating activ- ity into a non-sterile soil slurry using the strain Desuljh- bacterium sp. Y-5 1 showed apparent reductive PCE dechlo- rination to cis-DCE (Fig. Id). The result suggests that bio- augmentation with halorespiring bacteria is a promising re- mediation technique, although it accumulates intermediates from PCE. The accumulated cis-DCE can be readily de- graded by a subsequent process using various aerobic bac- teria (29) or abiotic reduction by Fe0 (10,27).

The treatment with Fe0 alone also successfully dechlori- nated PCE to ETH (Fig. lb, c) and ETA (data not shown) and no other chlorinated ethylenes as intermediates were apparently accumulated. The very low concentrations of in- termediate products suggest that PCE reduction by Fe0 oc- curred through direct contact between PCE and the iron sur- face and the intermediate molecules might remain attached to the iron surface for a sufficient time for the sufficient electrons to be transferred. However, the amount of ethyl- ene as the final product was approximately one seventh. This may be due to the formation of other end products such as acetylene, propene, propane, 1-butene, butane, and etc. (27) or utilization of ethylene and other end products by indigenous microorganisms in soil. Although there is rela- tively little knowledge on the mechanism of how Fe0 re- duces PCE because of the heterogeneous nature of the re- action, it is generally believed that reduction of chlorinated solvents by Fe0 corrosion depends on the surface area con- centration of Fe0 (10, 12, 13). Actually, the rate coefficient of PCE dechlorination was proportional to the concentration of Fe0 added (increase in surface area concentration of FeO): 0.01 d-’ (0.1% Fe’) to O.O8d-’ (1.0% Fe’) (Table 3). PCE dechlorination rate coefficients mean pseudo-first-order rate coefficients (k) that are slopes of plotting log(C/C,) versus time, t (d-l), i.e., ln(C/C,)=-k.t where C is the concentra- tion of PCE at time t and Co is the initial concentration of PCE. Although the data of the treatment with 0.1% FeO, the treatment with 1.0% FeO, and the treatment with Y-5 1 cell

and 1.0% Fe0 produced apparent linear plots, data plots of the treatment with Y-51 cell and the treatment with Y-51 and 0.1% Fe0 were not linear. For convenient comparison, non-linear plots of data in lag phase and steady state did not include and the remained linear plots were used to calculate rate coefficients.

The combined use of Y-51 and Fe0 showed the most significant effect on the PCE dechlorination rate coefficient and on the end products in the soil slurry. The cis-DCE pro- duced from biological PCE dechlorination by strain Y-51 was further converted into non-chlorinated products by a subsequent chemical reduction by Fe0 (Fig. le, f). The PCE dechlorination rate coefficient (0.23 d-‘) by the combined method was about three times those of the individual treat- ments using only strain Y-51 (0.08 d-l) or using only 1.0% Fe0 (0.08 d-l) (Table 3).

Furthermore, PCE dechlorination by Y-5 1 and Fe0 seemed to be synergistic (17, 18). The combined method may offer great advantages for the application of halorespiring bac- teria to contaminated sites where PCE is present with other chlorinated and/or inhibitory compounds, because the ad- dition of Fe0 can convert a broad range of chlorinated compounds such as chlorinated-methanes, -ethenes, and -ethanes (12), and also remove competitive electron ac- ceptors such as nitrate (17), where strictly anaerobic condi- tions (Eh=below -150mV) would be favorable for PCE dechlorination (Fig. 2).

Iron corrosion could also enhance biological dechlorina- tion by increasing the pH gradually in the highly buffered soil slurry (Table 2). This result is important in the field ap- plication of the combined system, because the increase in pH beyond the tolerance range of the applied bacteria would be lead to a potential limitation of the combined Fe’-micro- bial system (17).

Production of cathodic hydrogen from anaerobic Fe0 cor- rosion offers great advantages to halorespiring bacteria, be- cause H, is one of the most favorable electron donors for respiratory dechlorination (19). The treatments combined with Y-51 cells and Fe0 resulted in the decrease in concen- tration of cathodic hydrogen produced from Fe0 corrosion (Fig. 3a) while PCE was dechlorinated (Fig. 3b). The possi- ble explanation of this could be as follows. While PCE was converted to cis-DCE by strain Y-51, the strain seemed to utilize the HZ, produced by Fe0 corrosion, for the dechlorina- tion of PCE into cis-DCE as electron donor. After that, the H, concentration was increased again at day 6 (Fig. 3a). It

TABLE 3. Transformation of PCE in soil shu-ry treatments

Treatment PCE concentrations (lmolkg soil)

Initial Final” PCE removal efficiency PCE dechlorination rate

(%) coeffkientb (d-l)

Set 1 (Control) 57.9 39.1 30.2 0.004 (0.90) Set 2 (Fe0 0.1%) 57.5 23.6 58.9 0.01 (0.96) Set 3 (Fe0 1.0%) 63.5 0.6 99.1 0.08 (0.96) Set4(Y51) 66.1 10.1 299.8 0.08 (0.99) Set 5 (Y51+Fe” 0.1%) 65.0 10.1 299.8 0.13 (0.99) Set 6 (Y51 +Fe” 1.0%) 69.5 so.1 299.8 0.23 (0.97)

’ PCE concentration after 40 d of incubation. b PCE dechlorination rate coeffkients mean pseudo-first-order rate coeffkients which are slopes of plotting In (C/C,) versus time, t (d-l), where

C is the concentration of PCE at time 1 and C,, is the initial concentration of PCE. The plots did not include non-linear data in lag phase and steady state. The numbers in parentheses are regression coeffkients.

458 LEE ET AL. J. BIOSCI. BIOENG.,

should be noted that the amount of H, consumed was much higher than that of H, to be required for the dechlorination of PCE to cis-DCE. This may be due to H, consumption by other soil indigenous microorganisms such as methanogenic bacteria and by strain Y-5 1 for additional metabolic process such as fumarate reduction. In addition, the combined treat- ments led to a higher production rate of hydrogen than that of the treatments with Fe0 alone (Fig. 3a), which is in agree- ment with a report that the removal of the cathodic hydro- gen layer from the surface of Fe0 by bacteria enhances the corrosion of Fe0 and, thus, the flow of electrons (15).

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In summary, the results obtained in this study were: (a) bioaugmentation with halorespiring bacteria, strain Y-5 1, apparently dechlorinated PCE to c&DCE in a soil slurry; (b) the c&DCE produced was completely dechlorinated to non-toxic end products such as ETH and ETA by a sub- sequent chemical reductive process with Fe0 corrosion; and, (c) Fe0 corrosion could accelerate the biological reductive dechlorination of PCE by maintaining the proper levels of pH and ORP and by producing cathodic hydrogen which can be used as an electron donor for biological dechlorina- tion. These findings suggest that the combined use of strain Y-5 1 and Fe0 could be practically applied to the treatment of PCE and other chlorinated ethylene-contaminated sites.

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ACKNOWLEDGMENTS

We are grateful to Dr. Y. Kamagata for helpful suggestions dur- ing the preparation of the manuscript. This work was financially supported in part by the Japanese Society for the Promotion of Science and CREST (Core Research for Evolution Science and Technology) of the Japan Science and Technology Corporation.

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