Isolation, Characterization, and Physiology of Bacteria Able to Degrade Nitrilotriacetate

THOMAS EGLI* and HANS-ULRICH WEILENMANN, Swiss Fed - era1 Institute for Water Resources and Water Pollution Control, Swiss Federal Institutes of Technology, CH-8600 Dubendorf, Switzerbnd

Abstract

Until recently, only three obhgately aerobic bacteria (affiliated with the genus Pseudo- mnasJ that can grow with nitrilotriaoetate ~ N T A J as their only murce of carbon, nitrogen, and enerm have k e n isolated and studied in pure culture. By employing a different isolation strategy than w m used previously. several nonpseudomonads were isolated in pure culture from bolh soil and wastewater that ate able to utilize NTA under aerobic growth conditions. Additionally, a denittifying bacterium was isolated from river sediment that is able to utilize NTA in the absence oroxygen. These isolates have been charackrized with respect to their cell morpholom and physiolow. The data colIected MI far do not allow classification of both the gram-negakive and the P a m - p i - tive strains idated, and the taxonomic position of the isolates remains obscure. However, properties like C, utilization, production of acetoin, and nonmotility clearly indicate that the gram-negative strains do not belong to the genus Pseudomonos. Information i s presented on the regulation of NTA-metabolizing enzymes in isolate TE 1 suggesting that these enzymes are inducible in this bacterium.

INTRODUCTION

In most aquatic ecosystems phosphate is the growth-limiting nutrient for primary biomass production. Consequently, the extensive use of phosphates in detergents and agricultural fertilizers has resulted in widespread eutrophication of lakes and rivers (Stumm and Morgan, 1981). In order to reduce the load of phosphate discharge into surface waters, the use of tripolyphosphate (TPP) in detergents has been either restricted or banned in several countries such as Canada, Sweden, West Germany, and more recently, in Switzerland. In household detergents TPP is replaced mainly by nitrilotriacetic acid INTA).

Under aerobic conditions NTA is readily biodegradable and sev- eral reports on the isolation of pure cultures of NTA-utilizing microor- ganisms can be found in the literature (Focht and Joseph, 1971; Cripps

* To whom correspondenoe should be addressed.

Toxicity h a s m e n t : An International Journal Vol. 4,23-34 (1989) C 1989 John Wiley & Sons, Inc. CCC 0884-81 8 1/8910 1OO23- 12$04.00

24/EGLI AND WEILENMANN

and Noble, 1973; Tiedje et al., 1973; Pickaver 1976; Madson and Alexander, 1985; Kakii et al., 1986). Also, bacteria able to grow with NTA in the absence of oxygen have been isolated from natural waters (Enfors and Molin, 1973). The majority of these isolates have been assigned to the genus of Pseudomonas, although identification of Bacillus, Vibrio, Listeria, and a yeast species has also been reported. For most of these isolates, little more than their gram-stain reaction and cell shape have been published, and the three strains that have been characterized to a greater extent, i.e., Pseudomonas sp. ATCC 27109 (isolated by Focht and Joseph, 19711, Pseudomonas sp. ATCC 29600 (Tiedje et al., 1973), and Pseudomonas sp. T23 (Cripps and Noble, 1973), seem to be virtually identical (Firestone and Tiedje, 1978). To date it is generally assumed that the capacity to degrade NTA is restricted to only a small number of specialist bacteria (Mottola, 1974; Bernhardt et al., 1984; Anderson et al., 1985).

Although Firestone and Tiedje (1978) reported that their isolate did not fit any of the Pseudomonas spp. recognized in Bergey's Manual of Determinative Bacteriology, no further attempt was made to investi- gate the exact taxonomic position of these bacteria. In the present paper we report on the isolation of several pure bacterial cultures able to grow with NTA. These isolates were characterized extensively and compared to two NTA-degrading reference strains-Pseudornonas spp. ATCC 27109 and ATCC 29600.

MATERIALS AND METHODS

The synthetic medium (SM) used for both batch and chemostat enrichment cultures contained the following, per liter: MgS04 * 7H20, 1.0 g; CaC12 * 2H20, 0.20 g; Na2HP04 . 2H20, 0.41 g; KH2P04, 0.26 g; 1 mL of trace element stock solution as described by Pfennig et al. (1981) with NTA (5.2 g L-') as the chelating agent; and 1 mL of vitamin stock solution (which contained the following, per liter: pyridoxin . HC1, 100 mg; 50 mg each of thiamine . HC1, riboflavin, nicotinic acid, D-Ca-pan- tothenic acid, p-amino benzoic acid, lipoic acid, nicotinamide, vitamin B12; biotin, 20 mg; and folic acid, 20 mg). This medium was supple- mented with either NTA (Fluka, purissimum) or other carbon and/or nitrogen sources as described in the individual experiments. For agar plates the medium was supplemented with 1.5% of agar (Agar Noble, Difco).

For isolation and growth, Plate Count Agar (PCA) from Difco and SM/NTA (0.5 g L-') agar plates were used.

The utilization of different carbon sources was tested in liquid

NTA-UTILIZING BACTERIA/25

culture using SM supplemented with NH&l (0.54 g L-'1 and the carbon source (200 mg L-'). Growth was recorded as positive only if the cells were still able to grow after three successive transfers into fresh medium.

The gram reaction was tested with the classical staining proce- dure and by testing for the presence of L-alanine aminopeptidase (Merck, bactident strips).

Test strips (20B) and OF-test ampoules from API were used for the characterization of the isolates (API System S.A., Montalieu Vercieu, France). The following tests were performed as described in the literature: presence of catalase and growth with cellulose (Skerman, 1967), pigment production on King's medium A or B (Stolp and Gadkary, 19811, and growth on tellurite agar plates (Barksdale, 1981). The ability to grow a t 41°C was tested on PCA and SM/NTA agar plates.

Acetate was measured by gas chromatography (Shimadzu GC R1A fitted with a flame ionization detector; column, 0.5% carbowax 0.1% H3P04 on 80-100 carbopack C, 90 cm long; injection temperature 200°C; oven temperature 120°C; detector temperature 175°C; N2 flow 40 mL min-'1. The detection limit was 1 mg L-'. NTA (deviation < 5%) and NH; (deviation < 3%) was measured as described previously (Egli and Weilenmann, 1986).

Growth was determined spectrophotometrically by following the optical density of the culture at 578 nm (Uvikon 860). Parallel samples showed less than 2% deviation.

Electron micrographs were taken with a Philips EM 301 electron microscope. All strains were grown in shake flasks on complex medium (yeast extract, peptone, glucose 1 g L-', each; sodium acetate, 2 g L-'; potassium phosphate buffer, 20 m mol L-', pH 7.0). The cells were grown at 30°C and harvested in the midexponential growth phase. The procedure described by Valentine et al. (1968) was used for negative staining. The standard procedure as described by Muller et al. (1980) and Moor (1969) was used for jet freezing and deep etching, respec- tively. For thin sectioning, cells were fixed in glutaraldehyde (2% final concentration), embedded in low-gelling agarose, postfixed for two hours at 4°C using 1% Os04 in 0.05 mol L-' cacodylate buffer (pH 7.4). After washing with distilled water the gel blocks were kept overnight in 0.5% unbuffered uranyl acetate solution. After dehydration, gel blocks were embedded in Epon 812. Thin sections were stained with uranyl acetate (2% for 5 min) and lead citrate (5 min) as described by Reynolds (1963). Rotary shadowing of air-dried cells with platinum carbon was done at an angle of 25".

26/EGLI AND WEILENMANN

RESULTS AND DISCUSSION

Isolation Strategy

The strategies employed so far to isolate NTA-utilizing microor- ganisms were batch-enrichment cultures with NTA as either the sole carbon or sole carbon and nitrogen source. However, in order to support microbial growth, at least 50% of the carbon present in NTA has to be dissimilated to C 0 2 for energy production. Hence, NTA becomes for the cell a substrate overrich in nitrogen and deficient in carbon. Therefore, one can speculate that NTA is used primarily as a source of nitrogen if additional suitable carbon sources are present in the growth medium. The observation that NH: was produced during growth of bacteria with NTA as the sole source of carbon further supports such speculation (Focht and Joseph, 1971). Consequently, batch enrichments for NTA-degrading microbes were carried out with media containing NTA plus a mixture of easily degradable carbon sources (glucose, acetate, and methanol, 500 mg L-' each). These enrichments had to be carried out in closed bottles filled with a gas mixture of 02/He (20/80%, v/v) in order to prevent the growth of N2-fixing bacteria on the carbon sources. Enrichment cultures were set up in 250-mL bottles containing 50 mL of medium at a range of different pH values (5.0,6.0, and 7.0) and NTA concentrations (0.1,0.5, and 1.0 g L-'). They were inoculated with one drop of soil extract (supernatant of 20 g of garden soil suspended in 50 mL of enrichment medium sonicated mildly for 10 s) and effluent from a model waste- water treatment plant. Within a period of 7-10 days development of turbidity was observed at all concentrations used at pH 6.0 and 7.0. When the initial pH was 5.0, the growth obtained was negligible. All samples showing positive growth were diluted appropriately and plated out on SM/NTA agar plates.

Based on their morphology, colonies of different appearance were picked and further purified on agar plates using several media of different compositions. Throughout this purification procedure, the capacity to degrade NTA was always confirmed in liquid culture. Using this technique, nine pure cultures of NTA-degrading bacteria were isolated (strains TE 1-3 and 5-10).

Strain TE 4 was isolated from a chemostat enrichment culture. This organism became dominant after two months of continuous operation at D = 0.03 h-' (pH was kept constant at 6.5 and the NTA inlet concentration was 1.0 g L-'). Purification of this isolate was performed as described for strains from batch-enrichment cultures.

Additionally, a continuous culture was run at a dilution rate of D = 0.02 h-', supplying nitrate and sulfate as potential electron ac-

NTA-UTILIZING BACTERIA/27

ceptors and NTA as the only source of carbon and energy. With this culture we were able to enrich a denitrifying microbial population from river sediment (Egli and Weilenmann, 1986). From this enrich- ment culture a pure culture of a denitrifying, NTA-degrading bacte- rium was isolated (strain A 1) using a similar procedure as described above for the aerobic isolates.

Characterization of Isolates

All isolates and the two reference strains Pseudomonas spp. 27109 and 29600 from the American Type Culture Collection (ATCC) were characterized using both API 20B test strips (for the identification of aerobic heter krophic bacteria) and conventional methods. The results are summarized in Tables I and 11. Based on these results and on cell morphology (Fig. 11, three distinctly different groups of NTA-degrad- ing bacteria were recognized.

The first group consisted of two gram-negative strains that dif- fered in colony morphology when grown on PCA plates (isolate TE 1 formed big mucous colonies in comparison to isolate TE 2). However, with respect to cell shape and physiology both isolates were identical. Cells were nonmotile, short, sometimes almost coccoid rods, which occurred mostly in pairs during exponential growth (Fig. 1A). Some of their most striking features were their inability to grow on all of the sugars tested, their vitamin requirement, and their optimum growth temperature (35-37'0.

The second group consisted of all the remaining gram-negative isolates (strains TE 4-10), including the two Pseudomonas reference strains and the denitrifying isolate (strain A 1). All these isolates were rod shaped and motile (Fig. 1 0 . In cultures of isolates TE 4-8, L- or Y-shaped cells were frequently observed (Fig. 1D). The pleomorphism of these strains is unlikely to be an artefact resulting from unsuitable chemical fixation because i t was found in thin-sectioned, freeze-frac- tured, and negatively stained preparations. The similarity of two typical species isolated in our laboratory, i.e., strains TE 4 (from chemostat enrichment) and TE 5 (from batch enrichment), to the two Pseudomonas reference strains and the denitrifying isolate is demon- strated in the nutritional and biochemical properties listed in Tables I and 11.

For both groups of gram-negative, NTA-degrading isolates, the API 20B test results did not allow either identification or tentative allocation of the strains to an existing genus. In addition, several properties exhibited by these organisms, such as cell shape and nonmotility (isolate TE l), formation of acetoin (isolates TE 4, 5, A 1,

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3O/EGLI AND WEILENMANN

Fig. 1. Morphology of NTA-degrading isolates. (A) Negatively stained cell of isolate TE 1. (B) Freeze fracture of a cell cluster of isolate TE 3. (C) Rotary shadowed cell of strain TE 5. (D) Thin section of cells of isolate TE 5.

or Pseudomonas sp. ATCC 296001, growth with the C1 compound methylamine as the sole carbon source most probably excludes them from the genus Pseudomonas (Stolp and Gadkary, 1981). In order to establish the taxonomic position of these isolates, further investiga- tions, such as determination of the molar G + / C - ratio of DNA, identification of quinones present in the cells, or analysis of cell wall components, have been initiated.

The gram-positive isolate TE 3 was nonmotile. The barrel-like rods frequently did not separate completely after cell division and V formation was observed (Fig. 1B). The isolate is able to utilize cellulose as a carbon source. Because the biochemical and physiological proper- ties of this coryneform bacterium do not fit the description of any of the Cellulomonas species presently characterized (Stackebrandt and Kandler, 1979) its taxonomic affiliation still has to be clarified.

NTA-UTILIZING BACTERIA/3 1

Regulation of NTA Utilization in Cells of Isolate TE 1

In order to understand and predict the behavior of NTA-degrading microorganisms during wastewater treatment and in nature, the influence of environmental parameters on the regulation of enzymes involved in the uptake and metabolism of NTA in these organisms must first be understood. In this respect, not only physical and chemical parameters, such as pH or temperature, but also the availability of alternative nutrients, e.g., carbon and/or nitrogen sources, must be taken into consideration.

Some aspects of the regulation of NTA degradation were investi- gated in cells of isolate TE 1. During batch growth (3OoC, pH was controlled within a range of 6.8-7.0) with NTA as the only source of carbon, nitrogen, and energy, the maximum specific growth rate constant of this organism was 0.07 h-' with approximately 60% of the nitrogen present in NTA excreted into the culture medium in the form of NH,' . As a consequence of NH; excretion during growth with NTA only, the pH in batch cultures increased gradually with time. In cultures containing initially 1 g L-' NTA, the final pH increased to between 9.0 and 9.2, where growth ceased. When acetate was supplied as an additional carbon source, the bacterium utilized both acetate and NTA simultaneously, and the growth rate constant exhibited was 0.12 h-l. In this case, no NH: production was observed and the pH of the culture was constant, which suggests that uptake of the two substrates is carefully balanced. In order to investigate whether cells growing exponentially with acetate and NH,' (initial concentrations 200 and 180 mg L-', respectively) are able to metabolize NTA, cells from such a culture were harvested, washed with phosphate buffer, and resus- pended in a growth medium containing 50 mg L-' NTA as the only source of carbon and nitrogen. The results in Fig. 2 show that the cells exhibited an extended lag phase and slowly started to utilize NTA only after 20-30 h (indicated by the production of NH;). After 60 h of cultivation, all NTA was utilized but no significant increase in OD578

was observed. However, if acetate was supplied in a similar experi- ment in addition to NTA, the cells were able to grow, albeit slowly, and started to metabolize NTA after a period of 4-5 h (Fig. 3). A similar behavior was observed when a batch culture of isolate TE 1 growing exponentially with acetate/NH: was pulsed with NTA (Hamer et al., 1985). These experiments clearly demonstrate that enzymes involved in the metabolism of NTA had to be induced in the cells. Whether this regulation takes place at the level of NTA transport or enzymes involved in the subsequent metabolism or both needs to be elucidated.

32/EGLI AND WEILENMANN

0.124 /D

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150- -

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NTA-UTILIZING BACTERIA/33

The results presented in Figs. 2 and 3 demonstrate the advantageous effect of the presence of a suitable carbon and energy source on the cell's ability to synthesize the enzymes necessary for the utilization of NTA. Acetate was probably used primarily as an energy source to support the cell-internal rearrangement of the enzymic protein pattern in synthesizing enzymes necessary for NTA metabolism from proteins that had become obsolete.

CONCLUDING REMARKS The data reported clearly demonstrate the importance of isolation strategy and careful characterization of strains with special biodegra- dative properties. In addition, the physiological experiments presented also demonstrate the importance of the physiological status of mi- crobes on their ability to express specific metabolic capacities.

It must be assumed that regulatory phenomena as described here for an NTA-degrading bacterium are the rule rather than the excep- tion, particularly under transient (nonstable, changing) environmen- tal conditions. Especially in biodegradation and toxicological studies using microbial systems-no matter if based on either oxygen uptake or other parameters-such physiological effects must be considered since most likely they are responsible for a considerable part of the variability in results reported. Up to now such effects have been largely ignored and an urgent need exists for more physiological experimentation.

The authors are indebted to E. Wehrli for the preparation of the electron micrographs, and to G. Hamer for his help and support throughout this study. The work was financed by EAWAG (project No. 20-842).

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