Isolation and Characterization of an Enterobacter cloacae Strain That ...

APPLIED AND ENVIRONMENTAL MICROBIOIOGY. July 1989, p. 1665-1669

Vol. 55. No. 7

0099-2240/89/071665-05$02.00/0

Isolation and Characterization of an Enterobacter cloacae Strain That Redqces Hexavalent Chromium under Anaerobic Conditions PI-CHAO WANG,1 TSUKASA MORI.'t KOHYA KOMORIL'T MASANORI SASATSU,2 KIYOSHI TODA,' AND HISAO OHTAKE14' Inistitiute of Appliedl Mi(-obiology, Univ'e1sit! of Tokyo, Bunikvo-ki, Toky 113, 1 andic Depirtnelnt of MicWrobiology, o

Toklyo College of Pharmacy, Tokyo 192-03,

2

Jlp(l1l

Received 8 February 1989/Accepted 5 April 1989

An Enterobacter cloacae strain (HOI) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chropiate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).

Microbial transformations for many metallic minerals are known (5, 11, 13). These transformations include redox conversions of inorganic forms and conversions from inorganic to organic form and vice versa (21). Bacterial reduction has been found in metallic minerals such as manganese (12, 23), metal iron (3, 16), mercury (15, 27), selenite (7. 13), and tellurite (5, 22, 24). Some microbial transformations enable the bacteria to increase their tolerance toward toxic heavy metals (5, 27). Hexavalent chromium (chromate) is toxic and mutagenic in bacterial test systems (16, 26). Bacterial resistance to chromate has been found jn several Pseiudomiionatis strains (2, 4, 20). Ohtake et al. (14) reported that the chromate resistance in Pseludotinonas fluiores(cens LB300(pLHB1) was related to the decreased uptake of chromate. Chromate was transported mainly via the sulfate active transport system. Kinetics of chromate uptake by P. fluor-es(cens LB300 with and without the plasmid pLHB1 showed that the Vml,x for chromate uptake with the resistant strain was 2.2 times less than that for the sensitive strain, whereas the K,,, was the same for both. However, the molecular basis for the reduced uptake of chromate in the resistant strain was not elucidated. Pseiudomnonias and Aer-omtionatis strains have been reported to reduce anaerobically hexavalent chromium to trivalent form (10, 11, 18). Trivalent chromium is less toxic than hexavalent chromium, and at a neutral pH, it readily forms less-soluble chromium hydroxides. The biochemistry of anaerobic reduction of chromate in these strains was not studied, and it was not determined with certainty whether the organisms used toxic chromate as a terminal electron acceptor anaerobically. In this paper we describe the isolation and characterization of a chromate-resistant strain of Eniteirobacter- cloacuae that reduces chromate under anaerobic conditions. *

MATERIALS AND METHODS Isolation of bacterial strains. Chromate-resistant bacteria were isolated from activated sludge samples taken from a

municipal wastewater treatment plant. Enrichment cultures prepared in KSC medium containing (grams per liter of distilled water) NH4Cl (0.03), K,HPO4 (0.03), KH2PO4 (0.05), NaCI (0.01), sodium acetate (2.0), MgSO4- 7H,O (0.01), CaCO3 (0.005), FeCI3 7H.O (0.005), and Casamino Acids (Difco Laboratories, Detroit, Mich.) (1.0) and tap water (100 ml). For the enrichment culture, 10 ml of the sludge sample was dispersed in 90 ml of KSC medium with various concentrations of potassium chromate and incubated anaerobically at 30°C. The anaerobic conditions were established by passing nitrogen gas through the culture before the start of incubation. The beginning of the reduction of chromate was judged by the color change of the culture; yellow turned white in the presence of hexavalent chromium. About 3 weeks after the start of incubation, the reduction of chromate was observed in one enrichment culture. About 10 ml of the culture was transferred into 90 ml of fresh KSC medium with 0.5 mM potassium chromate and incubated until the chromate was completely reduced. After being plated on nutrient agar (Difco), single colonies were isolated and purified by cycles of liquid culture and single-colony isolation. Purity was determined by microscope and by naked-eye examination of colony morphology. E. cloacae IAM 1624 was obtained from the culture collection of the Institute of Applied Microbiology, University of Tokyo. Susceptibility testing. Overnight cultures were diluted 1:100 in nutrient broth (Difco) with various concentrations of chromate, and incubation proceeded at 30°C for 24 h under aerobic and anaerobic conditions. The growth was assayed by measuring turbidity with a spectrophotometer at 660 nm (model 101: Hitachi, Tokyo, Japan). Batch cultures. Sterile glass bottles (120-mI volume) were filled with 100 ml of fresh KSC medium, inoculated with an overnight grown culture (20%), and incubated at 30°C for 24 h. Fermenters (500 ml; luchi Inc., Tokyo, Japan) were also used for long-term experiments. Analytical methods. Chromate was determined spectrophotometrically by using diphenylcarbazide (25). Total chrowere

Corresponding author.

Present address: Chemical Process Technology Department. Ishikawajima-Harima Heavy Industries Co., Ltd., Yokohama 235. Japan. t Present address: Public Works Research Institute. Ministry of Construction. Tsukuba. Ibaraki 305. Japan.

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TABLE 1. Morphological and biochemical characteristics of strain HO1 Test or characteristic

Result

0.8_

Gram stain reaction ............................Cell type ............................ Rod Mobility ............................ + Nitrate reductase ............................+ (strong) Phenylalanine deamination ............................ Hydrogen sulfide ............................Indole ........................... -

Voges-Proskauer ...........................

B0,.2L

+

Citrate ............................+ o-Nitrophenyl-,-D-galactopyranoside .........................+ Urease .............................-

-

mium was assayed with an automatic absorption spectrophotometer (type AA-880; Nippon Jarrell Ash, Kyoto, Japan). RESULTS Isolation of chromate-reducing bacterium. The bacterium isolated from activated sludge was a short, gram-negative, motile rod. This bacterium grew at pH 6 to 8 (optimum pH, 7.0) and at 20 to 40°C (optimum, 30°C). This bacterial strain was designated HO1. It was determined that the strain HO1 belongs to the family Enterobacteriaceae because it is oxidase negative and nitrate reductase positive. The tests for identification of the species were carried out by using the Minitek Numerical Identification System (Becton Dickinson and Co., Paramus, N.J.). The characteristics of strain HO1 are shown in Table 1. On the basis of the morphological and biochemical properties, this bacterium was identified as E. cloacae at a confidence level of 99%. Resistance to neomycin, kanamycin, and nalidixic acid was observed in strain

HO1. Effect of chromate on growth. The effect of chromate on the growth of E. cloacae HO1 was tested under aerobic and anaerobic conditions (Fig. 1). The growth was measured in nutrient broth (Difco) containing various concentrations of potassium chromate. Strain HO1 could grow above 10 mM chromate under both aerobic and anaerobic conditions, though growth was slow under anaerobic conditions. Resistance to chromate is not a common property of E. cloacae strains. For example, E. cloacae IAM 1624 was much more sensitive to chromate than the HO1 strain was (Fig. 1). Several strains of Escherichia coli, Serratia marcescens, and Enterobacter aerogenes were also tested for chromate resistance, but these enteric bacteria could not grow in the presence of 1 mM chromate (data not shown).

0~~~~~

=

Lysine decarboxylase ............................Arginine dihydrolase ............................+ Ornithine decarboxylase .............................+ Dextrose ........................... + Malonate ........................... + (weak) Adonitol ........................... + (weak) L-Arabinose ........ .......................... + Inositol .............................+ (weak) Raffinose ........................... + D-Sorbitol ........................... + Lactose ...........................+ L-Rhamnose ...........................+ Sucrose ...........................+ Oxidase ............................Maltose ...........................+ Mannite ........................... + Xylose ...........................+ DNase ...........................-

0~-0

0.0

0

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CrO2 (mM) FIG. 1. Chromate resistances in E. cloacae H01 and IAM 1624. Cells were grown for 24 h at 30°C under aerobic and anaerobic conditions in nutrient broth containing various concentrations of potassium chromate. Growth was assayed by measuring turbidity at 660 nm. Symbols: *, aerobic culture of HO1; 0, anaerobic culture of H01; A, aerobic culture of 1AM 1624; A, anaerobic culture of IAM 1624.

Reduction of hexavalent chromium. Though E. cloacae HOI was resistant to chromate under both aerobic and anaerobic conditions, only the anaerobic culture showed chromate reduction (Fig. 2). In this experiment, HO1 cells were grown aerobically and anaerobically in KSC medium containing 0.5 mM potassium chromate. Clearly, the growth of cells was poor in KSC medium, compared with that in nutrient broth (Fig. 1). The yellow of the anaerobic culture turned white with chromate, and the turbidity was observed to increase as the chromate concentration decreased (Fig. 2B). Though cells grew aerobically, no chromate decrease was detected (Fig. 2A). Total chromium concentrations remained unchanged in both aerobic and anaerobic cultures. Since chromate is a strong oxidizing agent, it was thought that some of the chromate reduction could be a result of chemical redox reactions rather than biochemical activity of HO1 cells. However, inhibition of cell growth by adding penicillin, cycloserine, or chloramphenicol resulted in a simultaneous loss of chromate reduction under anaerobic

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grown in KSC medium with an initial concentration of 0.5 mM potassium chromate at 30°C under aerobic (A) and anaerobic (B) conditions.

CHROMATE-REDUCING E. CLOACAE STRAIN

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FIG. 3. Changes of cell viability of E. (loacae HO1 after addition of potassium chromate. E. cloacae HO1 cells were grown in KSC medium aerobically (a) and anaerobically (b). Potassium chromate (0.5 mM) was added to 1-h cultures, as indicated by arrows.

conditions (data not shown). Cell-free KSC medium did not show any reduction of chromate (see Fig. 4). Turbidity increase in the anaerobic culture continued until chromate reduction was completed. Thus, the final turbidity of the anaerobic culture became greater than that of the aerobic culture. The addition of chromate led to a transient decrease in cell viability, regardless of whether the culture was aerobic or anaerobic (Fig. 3). In this experiment, 0.5 mM chromate was added to aerobic and anaerobic cultures 1 h after the start of incubation. The aerobic growth soon resumed, but the anaerobic cell growth started several hours after the addition of chromate. Factors affecting chromate reduction. The rate of chromate reduction was dependent on cell density, i.e., the higher the cell density, the greater the reduction rate (Fig. 4). At a

E

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0.1

0.0

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FIG. 4. Effect of viable-cell density on chromate reduction. KSC medium with 0.5 mM potassium chromate was inoculated with various concentrations of E. cloacaie HO1 cells and incubated at 30°C under anaerobic conditions. Symbols (E. clO(wace cells per 50 * 5 x 10":O0. 1 x 0: milliliter): A, control (no cells), O1 io0. A, 5 x

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FIG. 5. Changes of turbidity (a) and chromate concentration (b) of anaerobic cultures of E. cloacae HO1 before and after addition of chromate. Cells were grown in KSC medium at 30'C. Potassium chromate was added at 30 h. as indicated by arrows. x 108 cells per ml, 0.5 mM chromate was completely reduced within 5 h. However, the specific rate of chromate reduction was maximum at a density of 1 x 107 cells per ml, and the maximum specific rate was about 5 x 10' nmol of CrO42- per h per cell. No significant reduction of chromate was detected with cell-free control culture. The rate of chromate reduction was also dependent on the amount of added chromate (Fig. 5). Various amounts of potassium chromate were added to anaerobic cultures that had been grown without chromate for 30 h. The time required for complete reduction increased in proportion to the amount of added chromate. When the chromate concentration exceeded 3 mM, complete reduction did not occur. Turbidity increase was clearly dependent on the amount of added chromium. The turbidity increased as the reduction proceeded, and the final turbidity levels were proportional to the amount of added chromate. Addition of chromate again led to a transient decrease in cell viability, and the cell growth was delayed depending on the amount of added chromate (data not shown). Chromate reduction in E. clkoai((ie HOI was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C). Cells grown with acetate or glycerol reduced chromate more rapidly than those grown with glucose.

density of 5

DISCUSSION Bacterial resistance to toxic chromate has been found in PseuIdotinoniais amt1bign(a (9), P. fluoresceens (2), P. aerIiugin(os(i (3, 20), and StreptoC'ocitus lac(tis (6). Except in the P. mibigiiti strain, the chromate resistance in these bacteria was plasmid determined. This is the first report of a chromate-resistant enteric bacterium. E. (loac(twe HO1 isolated from activated sludge can grow aerobically in nutrient broth with as much as 10 mM potassium chromate, whereas normal bacteria cannot tolerate chromate of this level (Fig. 1). We do not know whether this chromate resistance is plasmid determined, although this bacterium does have plasmids of four different sizes (data not shown). Attempts to cure strain HO1 by treatment with EtBr and acridine orange were unsuccessful, because this bacterium was highly resistant to both the reagents. E. cloacaie HO1 was resistant to chromate under both aerobic and anaerobic conditions. The anaerobic growth was

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accompanied by the decrease of toxic chromate in culture medium (Fig. 2 and 3). Yellow became white in the presence of chromate, and turbidity increased without delay as chromate decreased. This initial turbidity increase was not accompanied by cell growth. It is known that the stable forms of chromium are the trivalent and hexavalent forms (1). Trivalent chromium is much less toxic than the hexavalent form (17, 21), and it forms less-soluble chromium hydroxide at a normal pH. Though a direct method to assay chromium hydroxide is unavailable, it seems likely that toxic hexavalent chromium was reduced to trivalent chromium and insoluble chromium hydroxide was formed in anaerobic cultures of E. cloacae HO1. The initial turbidity increase which was not accompanied by cell growth was believed to be caused by the formation of insoluble chromium hydroxide. Bacterial reduction of chromate in Pseuidomonas strains (8, 11, 18, 19) and in an Aeromnonas strain (4) has been reported. P. amnbigua Gl (8) and Pseudomonas strain K21 (19) were observed to decrease chromate under aerobic conditions. Horitsu et al. (8) have found chromate-reducing activity in a cell extract of P. ambiguca Gl, and this activity required NADH as a hydrogen donor. Anaerobic chromate reduction was also found in Pseiudomonas strains (11, 18). Lebedeva and Lyalikova (11) isolated chromate-reducing Pseudomonas strains from industrial sewage and observed that the pseudomonads adhered to form aggregates and precipitated as chromate reduction proceeded. Though these bacteria were facultative anaerobes, chromate reduction occurred only under anaerobic conditions. Physiological studies of the anaerobic reduction of chromate have not been done, and its mechanism is still unknown. E. cloacae HO1 reduced chromate only under anaerobic conditions. This bacterium probably utilized toxic chromate as an electron acceptor anaerobically, because (i) the anaerobic growth of HO1 cells accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurs more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. However, strict anaerobic conditions were not required for supporting the chromatereducing activity of HOI. Flushing the culture with purified nitrogen gas for 10 to 20 min allowed the cells to start chromate reduction. When the cell densities were as high as 107 cells per ml, the anaerobic conditions could also be established by cell respiration, and gassing the culture with purified nitrogen was not required before the start of chromate reduction. Ohtake et al. (14) reported that the chromate resistance in P. fliuorescens LB300(pLHB1) was related to the decreased uptake of chromate under aerobic conditions. Decreased chromate uptake is also probably the mechanism of chromate resistance in E. cloacae HO1 under aerobic conditions. There was a clear difference in 5lCrO42- accumulation between E. cloacae HO1 and IAM 1624 under aerobic conditions; the chromate-sensitive IAM 1624 cells accumulated about three times more 51CrO42- than did strain HOI (C. Cervantes, unpublished data). Chromate uptake experiments have not been done with anaerobic cultures of E. cloacae HO1. However, it is possible that decreased chromate uptake protected cells to some extent from the toxicity of chromate even under anaerobic conditions, because the resistant cells did not lose their viability before detoxification of chromate started. Further investigation is being undertaken to determine whether the ability of HO1 to reduce chromate enables the cells to increase their tolerance toward this toxic oxyanion.

ACKNOWLEDGMENTS We thank Aiko Hirata for transmission electron microscopy and Simon Silver for helpful discussions. This work was supported by grants from the Ministry of Education, Science and Culture, Japan, and from the Nippon Life Insurance Foundation, Osaka, Japan. LITERATURE CITED 1. Bell, C. F., and K. A. K. Lott. 1966. Modern approach to

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