INTRODUCTION

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In order to enhance the biodegradation of chemicals of environmental concern, the inoculation of microorganisms capable of degrading the chemicals has been reported (7, 12). However, the attempts did not always produce desired results. One possible reason for the failure of inoculation is the failure of the responsible microorganisms to grow, since in a natural environment, the concentrations of the chemicals are generally low (5, 9, 10). If the density of the microorganism is insufficient, the degradation of the chemicals is not detectable (16, 24). Therefore, studies of the growth of responsible microorganisms at low chemical concentrations are indispensable to enhance the biodegradation of the chemicals.

Studies of the yield coefficient (YC), i.e., the efficiency of biomass production at the expense of the chemicals of concern which provide energy and carbon sources, are indispensable to the kinetic analysis of degradation of the compounds (19). However, measurements of YC in the laboratory have sometimes been overestimated, especially when the concentration of the chemical of concern was low, because microorganisms utilized not only the chemical of concern but also uncharacterized organic compounds contaminating the inorganic medium from water, glassware, or air (1, 4, 6, 14, 20). Therefore, the YC that was observed reflected the combined influence of the chemicals of concern and uncharacterized organic compounds, rather than the YC of the chemicals alone (1, 14, 20).

In our previous study (20), we proposed a method to eliminate uncharacterized organic compounds by using bacterial communities which did not utilize the chemicals of concern. In this study, by using this method, we estimated the YC of the 2,4-dichlorophenol (DCP)-degrading Pseudomonas sp. strain DP-4 at the expense of DCP alone as a model chemical of environmental concern, and the effect of the concentration of DCP on YC is discussed.

	MATERIALS AND METHODS

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Experimental microorganisms. Pseudomonas sp. strain DP-4 (17) was used as a DCP degrader. Strain DP-4 was isolated from an enrichment culture inoculated with sewage, soil, and activated sludge. This bacterium is gram-negative and rod-shaped and has a polar flagellum (17). DP-4 was cultured in a mineral salts (MS) medium with or without the heterotrophic bacterial community which is described below.

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Cultivation of DP-4. Pure-culture experiments of DP-4 were performed by adding the desired amount of DCP and DP-4 suspension to 1,000 ml of a sterilized MS medium in a 1.2-liter screw-cap glass bottle.

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Analysis of concentration of DCP. The concentration of DCP was analyzed on a high-performance liquid chromatography system (16), consisting of a Yanaco L-4000W pump (Yanagimoto Co., Ltd.), a Shodex M-315 UV-visible variable-wavelength detector (Showa Denko K. K.) set at 254 nm, and a Shodex RSpak DS-613 and RSpak DS-613(p) column (Showa Denko). The eluent consisted, in volume, of 40% ion-exchanged water and 60% methanol. The pH was adjusted to 11.2 by 6.25 mM Na₂HPO₄ and 1.5 mM NaOH. The flow rate was 1.0 ml min¹. If necessary, DCP in a 25-ml subsample from the culture was concentrated with a Sep-pak plus tC18 cartridge (Waters Corp.) to 2.5 ml of sample (20).

The measurement of the density of DP-4 and total heterotrophic bacterial community. The density of DP-4 was measured by a CFU method with DP-4 selective agar medium in which the heterotrophic bacterial community, except for DP-4, could not grow. DP-4 selective agar medium contained 0.7 mg of nutrient agar (Eiken Chemical Co., Ltd.), 8 mg of agar (Kyokuto Co., Ltd.), 200 µg of carbenicillin sodium (Wako), 70 µg of bacitracin (Wako), 15 µg of ampicillin sodium (Wako) and 20 µg of C of DCP (Wako) per ml of modified MS (mMS) medium. Subsamples from the MS medium were diluted with sterilized ion-exchanged water by 10-fold serial dilution. The 1-ml portion thus obtained was poured into DP-4 selective agar medium. The medium was incubated for 7 days.

Composition of MS or mMS medium. The MS medium contained 2.1 µM Na₂HPO₄, 0.9 µM KH₂PO₄, 40 µM NH₄NO₃, 10 µM MgSO₄ · 7H₂O, 10 µM CaCl₂ · 2H₂O, 3 µM Na₂SiO₃, 1 µM MnCl₂ · 4H₂O, 1 µM H₃BO₃, 1 µM Na₂MoO₄ · 2H₂O, 5 nM FeCl₃ · 6H₂O, 5 nM CuSO₄ · 5H₂O, 5 nM ZnSO₄ · 7H₂O, and 5 nM CoSO₄ · 7H₂O in ion-exchanged water. The pH was adjusted to 7.0 to 7.2. The mMS medium contained 2.1 mM Na₂HPO₄, 0.9 mM KH₂PO₄, 2 mM NH₄NO₃, 0.1 mM MgSO₄ · 7H₂O, 0.1 mM CaCl₂ · 2H₂O, 30 µM Na₂SiO₃, 10 µM MnCl₂ · 4H₂O, 10 µM H₃BO₃, 10 µM Na₂MoO₄ · 2H₂O, 50 nM FeCl₃ · 6H₂O, 50 nM CuSO₄ · 5H₂O, 50 nM ZnSO₄ · 7H₂O, and 50 nM CoSO₄ · 7H₂O in ion-exchanged water. The pH was 7.2.

Estimation of YC. In this investigation, the YC of DP-4 was defined as follows: YC = (Bt  B₀)/Sd, where Bt is the density of DP-4 at the end of incubation, B₀ is the initial density of DP-4, and Sd is the amount of DCP degraded per milliliter of medium. Because of the difficulty in measuring the biomass of DP-4 at low density or in mixed culture, YC was expressed as the number of CFU of DP-4 produced at the expense of 1 unit amount of DCP.

	RESULTS

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In pure culture of DP-4 in MS medium (Fig. 1A), all the DCP was degraded when the initial concentration of DCP added was 10, 1, or 0.1 µg of C ml¹. On the other hand, 60% of DCP was degraded during a 30-day incubation period when the initial concentration of DCP added was 0.01 µg of C ml¹. The density of DP-4 increased to more than 10⁵ CFU ml¹, irrespective of the initial concentration of DCP added (Fig. 1B). Even when no DCP was added, the density of DP-4 increased to ca. 10⁶ CFU ml¹. Therefore, the increase of the density of DP-4 was attributable to the expense not only of DCP but also of uncharacterized organic compounds contaminating the MS medium.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Change in concentration of DCP (A) or density of DP-4 (B) in pure culture. The initial concentration of DCP added was 10 (), 1 (), 0.1 (), 0.01 (), or 0 (×) µg of C ml1.

In a mixed culture of DP-4 with the heterotrophic bacterial community (Fig. 2A), all the DCP was degraded when the initial concentration of DCP added was 10, 1, or 0.1 µg of C ml¹. On the other hand, no DCP was degraded during a 30-day incubation period when the initial concentration of DCP was 0.01 µg of C ml¹. The density of DP-4 increased to 1.2 × 10⁷, 1.1 × 10⁶, or 1.9 × 10⁴ CFU ml¹ when the initial concentration of DCP added was 10, 1, or 0.1 µg of C ml¹, respectively (Fig. 2B). When the initial concentration of DCP added was 0.01 µg of C ml¹ or no DCP was added, the density of DP-4 did not increase and was almost constant at ca. 10³ CFU ml¹ during the incubation period. This suggested that uncharacterized organic compounds had almost been eliminated by the heterotrophic bacterial community and that DP-4 utilized DCP as a sole source of carbon and energy. The density of the total heterotrophic bacterial community was ca. 10⁶ to 10⁷ CFU ml¹ during the incubation period, irrespective of the initial concentration of DCP added (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Change in concentration of DCP (A) or density of DP-4 (B) in mixed culture. Symbols are as described in the legend to Fig. 1.

In mixed culture, 80% of DCP was degraded during the 30-day incubation period when the initial concentration of DCP was 0.07 µg of C ml¹ (Fig. 3A). On the other hand, no DCP was degraded when the initial concentration of DCP was 0.05 or 0.01 µg of C ml¹. The density of DP-4 did not increase (Fig. 3B), even when DCP was partially degraded. The density of the total heterotrophic bacterial community was ca. 10⁶ CFU ml¹ during the incubation period, irrespective of the initial concentration of DCP added (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Change in concentration of DCP (A) or density of DP-4 (B) in mixed culture. The initial concentration of DCP added was 0.07 (), 0.05 (), or 0.01 () µg of C ml1. N.D., not detected.

DP-4 in a mixed culture was unable to degrade DCP at a concentration of 0.01 µg of C ml¹ without the addition of glucose (Fig. 4A). However, DP-4 degraded DCP completely by two additions of glucose at a final concentration of 1 µg of C ml¹. The density of DP-4 was almost constant at ca. 10⁵ CFU ml¹, irrespective of the addition of glucose (Fig. 4B). The density of the total heterotrophic bacterial community was ca. 10⁶ CFU ml¹, irrespective of the addition of glucose (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Change in concentration of DCP (A) or density of DP-4 (B) in mixed culture with () or without () the addition of glucose. The arrow (at a and b) indicates the first or second addition of glucose to a final concentration of 1 µg of C ml1, respectively.

In pure culture, the YC of DP-4 apparently increased with the decrease of the initial concentration of DCP added (Fig. 5). In mixed culture, however, the YC of DP-4 decreased discontinuously with the decrease of the initial concentration of DCP added and was estimated by an arithmetic mean to be 1.5, 0.19, or 0 CFU per pg of DCP-C when the initial concentration of DCP added was in the range of 0.7 to 10, 0.1 to 0.5, or 0.07 µg of C ml¹, respectively. The YC could not be estimated when the initial concentration of DCP added was less than 0.05 µg of C ml¹, because neither the degradation of DCP nor the increase in the density of DP-4 was observed.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   Relationship between the initial concentration of DCP added and the YC of DP-4 in pure culture () or mixed culture ().

	DISCUSSION

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The overestimation of YC of DP-4 in pure culture was attributable to the presence of uncharacterized organic compounds contaminating the MS medium, which allowed DP-4 to grow. For example, the amount of uncharacterized organic compounds in synthetic liquid medium was reported to be 2 (6), 0.1 (14), or from 0.1 to 1 (18) µg ml¹ in terms of the amount of carbon, and the density of microorganisms increased to 10⁷, 10⁵, or 10⁵ to 10⁶ cell ml¹, respectively. In our previous study (18), the density of DP-4 increased to 10⁵ cell ml¹, even in double-distilled water. If we assume that the YC at the expense of uncharacterized organic compounds was equal to the YC at the expense of 1 µg of C ml of DCP¹, the amount of uncharacterized organic compounds estimated in this study was ca. 0.7 µg of C ml¹, because the density of DP-4 was 1.1 × 10⁶ CFU ml¹ at the stationary phase in pure culture without DCP (Fig. 1B). Therefore, to estimate the YC at the expense of DCP, the elimination of uncharacterized organic compounds contaminating synthetic medium is indispensable, especially when the concentration of DCP is low.

Mixed culture with a heterotrophic bacterial community, as used in this study, seemed to be a simple and effective way to eliminate uncharacterized organic compounds, because the density of DP-4 was almost constant in a mixed culture without DCP (Fig. 2B). The density of DP-4 in the mixed culture without DCP was ca. 1/1,000-fold lower than that of the pure culture. This suggested that DP-4 could utilize only 0.1% of uncharacterized organic compounds and that the heterotrophic bacterial community then eliminated ca. 99.9% of the uncharacterized organic compounds.

The reason for using a bacterial community from a natural environment to eliminate uncharacterized organic compounds is that in the preliminary study, bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter calcoaceticus, or some isolates which were obtained from groundwater, rivers, or lakes could not effectively eliminate the uncharacterized organic compounds. DP-4 inoculated to a final density of 10³ ml¹ increased to 10⁵ to 10⁶ ml¹ in MS medium cultured with these bacteria (data not shown). The heterotrophic bacterial community used in this study seemed not to affect the growth or activity of DP-4 except for the elimination of uncharacterized organic compounds.

The reason for the discontinuous decrease of YC with the decrease in concentration of DCP is not clear. One possible explanation is that different degradation systems are involved in the DCP degradation, as a number of studies have reported (1, 22). Another hypothesis, such as the effect of maintenance loss (2, 23) of DP-4, is probably negligible. If the maintenance coefficient was almost the same as that of Pseudomonas aeruginosa of 0.26 mg of C per gram of biomass C per hour (15), then 10⁵ DP-4 cells ml¹, whose C biomass per cell is 0.1 pg (21), will consume only 1.9 ng of C ml¹ of DCP for maintenance, even in the 30-day incubation period. There was enough DCP to allow DP-4 to increase to a density of 10⁵ cells ml¹ when 0.1 µg of DCP-C ml¹ was added.

The problem of this method is that we couldn't measure the biomass of DP-4 because of experimental restrictions. Since cell size generally becomes smaller under oligotrophic conditions (11), the YC of DP-4 at the low concentration of DCP is probably even lower than at a high concentration of DCP if the YC of DP-4 was expressed in terms of biomass.

The failure of degradation of 0.01 or 0.05 µg of C ml of DCP¹ in mixed culture (Fig. 3A) was not attributable to the low density of DP-4 of 10³ CFU ml¹, because even a higher density of 10⁵ CFU ml of DP-4¹ did not degrade that level of DCP in mixed culture without the addition of glucose (Fig. 4A). On the other hand, DP-4 degraded 0.01 µg of C ml of DCP¹ in pure culture (Fig. 1A) or in mixed culture with glucose supplementation when the density of DP-4 was 10⁵ CFU ml¹ (Fig. 4). These results suggested that DP-4 required auxiliary organic compounds, rather than high density, to degrade extremely low concentrations of DCP, such as 0.01 µg of C ml¹. Although this phenomenon is similar to cometabolism (3, 8), we couldn't confirm whether DP-4 specifically cometabolized 0.01 µg of C ml of DCP¹. We should have used ¹⁴C-labeled DCP, but it was impossible because of our equipment restrictions. For example, Schmidt and Alexander (13) have reported a similar phenomenon by using ¹⁴C-labeled glucose, in which Salmonella enterica serovar Typhimurium mineralized glucose at a high concentration, but not at a low concentration, as its sole source of carbon and mineralized a low concentration of glucose if a cosubstrate was contained. In their study, however, both significant growth of S. enterica serovar Typhimurium and the acceleration of degradation were observed simultaneously. Interestingly, they observed no differences in the percentage of glucose carbon incorporated into cells between the time when the bacterium grew at a high concentration of glucose alone and when it grew at a low concentration of glucose with a cosubstrate.

The results obtained in this study with DP-4 suggest that the biodegradation of extremely low concentrations of chemicals may be controlled by the presence of higher concentrations of other available substrates, as a number of studies have reported (7, 12, 25). The data also suggest that the inoculation of microorganisms to enhance the biodegradation of chemicals in polluted natural environments will not always give satisfactory results, because the concentrations of available substrates for microorganisms are generally extremely low in natural environments (11). We need more information about ecological or physiological factors which affect the biodegradation of low concentrations of chemicals that are assimilated at higher concentrations.