Introduction

Nitriles compounds are cyanide-substituted carboxylic acids. Naturally occurring nitriles are found in plants, bone oils, insects, and microorganisms (Digeronimo and Antoine 1976). Synthetic nitriles, on the other hand, have been extensively used in the manufacture of feedstock, solvents, extractants, pharmaceuticals, drug intermediates (chiral synthons), pesticides (e.g., dichlobenil, bromoxynil, ioxynil, buctril), etc. (Banerjee et al. 2002). They are also important intermediates in the organic synthesis of amines, amides, amidines, carboxylic acids, esters, aldehydes, ketones, and heterocyclic compounds (Banerjee et al. 2002). The direct discharge of wastewater containing some of these nitrile compounds could possibly cause severe health hazards because most of them are highly toxic and some are mutagenic and carcinogenic (Nawaz et al. 1991; Pollak et al. 1991). Therefore, the wider use of these toxic compounds could lead to an environmental problem in the future.

Despite that chemical methods for treating these compounds are known, the cost of applying this technology is prohibitive (Nawaz et al. 1991). Bioremediation, an inexpensive technology, can eliminate such compounds by degrading them to harmless intermediates or, ultimately, to carbon dioxide and water (Nawaz et al. 1991). Although microbial degradation of individual nitrile compounds and their derivatives has been reported by various groups of researchers using different strains, such as Pseudomonas (Dhillon and Shivaraman 1999), Nocardia rhodochrous (DiGeronimo and Antoine 1976), Arthrobacter (Yamada et al. 1979), Brevilbacterium (Bui et al. 1984), Pseudomonas putida (Nawaz et al. 1989), Pseudomonas sp. strain K9 (Yamada et al. 1980), Arthrobacter sp. IPCB-3 (Ramkrishna and Desai 1993), Pseudomonas marginalis (Babu et al. 1995), P. putida NRRL-18668 (Fallon et al. 1997), Pseudomonas aeruginosa (Nawaz et al. 1991), Rhodococcus erythropolis BL1 (Langdanhl et al. 1996), Rhodococcus rhodochrous LL100-21 (Dadd et al. 2001), R. rhodochrous J1 (Nagasawa et al. 1988), R. rhodochrous NCIMB 11216 (Tauber et al. 2000), Candida guilliermondii CCT 7202 (Dias et al. 2001), C. guilliermondii UFMG-Y65 (Dias et al. 2000), Comamonas testosteroni and Acidovorax sp. (Wang et al. 2004), Paracoccus thiophilus (Lee and Wang 2004), Pseudomonas stutzeri (Wang and Lee 2001), and strain AAS6 (Wang et al. 2001), there are no reports in the literature about the biodegradation of nitriles by K. oxymora isolated from the cyanide-containing wastewater in Taiwan. Thus, the present study was undertaken to determine the ability of this bacterium to utilize some organic nitriles as a sole source of nitrogen for growth. To have an insight into the pathway of transformation of the cyanide compounds by Klebsiella oxytoca, the biotransformation of these cyanide compounds to their corresponding intermediates and end products by the growth cells of this culture was also studied.

Materials and methods

Organism and culture conditions

The isolation of K. oxytoca SYSU-011 from industrial wastewater was described in our previous study (Kao et al. 2003). Cells were grown on a nitrogen-free glucose (NFG) medium containing Na2HPO4 2H2O (50 mM), KH2PO4 (100 mM), MgSO4 (1 mM), CaCl2 (0.1 mM), and glucose (0.8%). The pH value of this medium was adjusted to 7.0. Filter-sterilized nitriles including acetonitrile, benzonitrile, butyronitrile, methacrylonitrile, glutaronitrile, phenylacetonitrile, propionitrile, valeronitrile, and succinonitrile at indicated doses were added as nitrogen sources. K. oxytoca grown in NFG medium containing butyronitrile (25 mM) was incubated in a Gyrotory shaker at 30°C until the log phase growth appeared. This bacterium was subsequently harvested by centrifugation and washed twice in Na–K phosphate buffer (pH=7.0). The cell pellets were resuspended in the same buffer at OD600nm=1.0 (dry weight=0.105 mg/ml) and used as the inoculated cells in nitrile biotransformation experiments.

Determination of MIC

The minimal inhibitory concentration (MIC) of nitrile compound was determined from the inoculation of K. oxytoca in the NFG medium with each tested nitrile at different concentrations. This performance was conducted by monitoring the growth of K. oxytoca estimated by optical density (OD600 nm) after 120 h of incubation at 30°C. The MIC was defined as the lowest concentration of the inhibitor at which no growth was observed (Silva-Avalos et al. 1990).

Oxidation of substrates (oxygen uptake rate determination)

The oxygen uptake rate was determined following the procedures described in Babu et al. (1995). The oxidation of different nitriles by the cells of K. oxytoca was studied by measuring the oxygen uptake with an oxygraph. One milliliter of cell suspension (A 600=1.0) was injected into the cell chamber followed by the injection of substrate (1 ml) with the concentration of 25 mM. The suspension was then stirred and equilibrated at 30°C for 20 min before reading was recorded. The oxygen uptake for different substrates was recorded for a reaction period of 15 min. All data were corrected for endogenous respiration (Babu et al. 1995).

Enzyme assays (specific activities of nitrile biotransformation enzyme)

The specific activity of nitrile-hydrolyzing enzyme was determined in terms of ammonia production. This enzyme activity was assayed after the incubation of bacterial suspension with different nitriles (25 mM for each) at 37°C for 30 min. The reaction of the enzyme was terminated by heating at 65°C for 1 min. Moreover, the produced ammonia was also determined. Ammonia was quantified by a colorimetric method (APHA 1995). Protein concentration was measured by a modified Lowry method using bovine serum albumin as the standard (Lowry et al. 1951).

Determination of the dry weight of biomass

The culture broth (10 ml) was centrifuged at 6,500×g for 20 min. The pellet was washed twice with double-distilled water and filtered through a millipore membrane filter (0.45 μm pore size). The filter paper was dried to a constant weight at 103°C, and the dry weight of the biomass was determined (Dhillon and Shivaraman 1999). The obtained dry weight biomass was the average of triplicate samples (Table 1).

Table 1 Measurement of MIC, specific activity, and oxygen uptake by K. oxytoca on various nitriles
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Determination of products from nitriles degradation

Nitriles and their degradative products (e.g., amide and carboxylic acid) were determined by capillary gas chromatography (GC) using a supelco wax column with flame-ionized detector. The operational conditions of GC were as follows: oven temperature (50–230°C), injector temperature (220°C), and detector temperature (250°C). The carrier gas was N2 at 4 ml/min. Two microliters of filtered supernatant of reaction mixtures was analyzed using the method described above.

Results

MIC values of nitriles

The MIC values of nitriles for K. oxytoca were presented in Table 1. Among the tested compounds, the values of MIC for butyronitrile, methacrylonitrile, propionitrile, and succinonitrile were over 1,000 mM. This indicated that these compounds exhibit no toxicity effects on the growth of K. oxytoca. Results also show that the MIC value of acetonitrile was found to be 750 mM followed by valeronitrile (570 mM), glutaronitrile (260 mM), phenylacetonitrile (4 mM), and benzonitrile (2 mM). This reveals that these nitrile compounds cause significant inhibition effects on the growth of K. oxytoca.

Specific activities of nitrile biotransformation enzyme

The cell-free extracts of K. oxytoca were used for the analysis of specific activities of nitrile biotransformation enzyme from different nitrile compounds. Table 1 shows the specific activities of nitrile hydrolyzing enzymes in each tested nitrile. The most suitable substrates to activate nitrile biotransformation enzyme were found to be butyronitrile, glutaronitrile, methacrylonitrile, propionitrile, succinonitrile, and valeronitrile. However, the specific activities of nitrile biotransformation enzyme were not significantly activated for acetonitrile, benzonitrile, and phenylacetonitrile because no ammonia production was observed.

Oxygen uptake rates

Results of the respirometric studies (Table 1) indicate that the bacterial cells have the ability to degrade a majority of nitrile compounds. The levels of oxygen uptake rates (nanomoles per minute per milligram dry weight) for these tested compounds were ranked as follows: valeronitrile (4.78)>glutaronitrile (4.20)>propionitrile (4.17)>succinonitrile (4.10)>butyronitrile (3.96)>methacrylonitrile (3.70)>acetonitrile (1.75)>benzonitrile (0.66)>phenylacetonitrile (0.35).

Effects of nitrile concentrations on the growth of K. oxytoca

Figure 1 shows the effects of butyronitrile concentration on the growth of K. oxytoca in the NFG growth medium. This indicates that butyronitrile can be used as the alternative nitrogen source for bacterial growth, although 100 mM butyronitrile induced an apparent lag phase in bacterial growth. Figure 2 shows the utilization of 1 mM of benzonitrile and 1 mM of phenylacetonitrile by K. oxytoca. This indicated that both benzonitrile and phenylacetonitrile could be used as the nitrogen sources by K. oxytoca.

Fig. 1
figure 1

The growth curves of K. oxytoca in NFG media. (♦) 25 mM of butyronitriles, (□) 50 mM of butyronitriles, (▴) 100 mM of butyronitriles

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Fig. 2
figure 2

The growth curves of K. oxytoca in NFG media. (♦) 1 mM of benzonitrile, (□) 1 mM of phenylacetonitrile

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Accumulation of metabolites

Figure 3 shows that the degradation of acetonitrile and sequential formation of acetamide, acetic acid, and ammonia were observed during the growth of K. oxytoca. Results show that acetonitrile dropped from 23 to 13 mM after 125 h of incubation, then continued to drop to 8 mM at 150 h. After 150 h of incubation, 5 mM of acetamide was detected. The concentration of acetamide increased to 9 mM (maximum concentration) after 175 h. The maximum amounts of acetic acid were detected at 198 h. At 200 h, a trace amount of ammonia was produced. Ammonia concentration increased to 0.5 mM after 235 h of incubation. Growth of K. oxytoca showed a lag period of 150 h. The start of logarithmic growth was concomitant with the appearance of acetic acid, acetamide, and ammonia in the culture medium. Figure 4 shows the metabolism of propionitrile by K. oxytoca. Results demonstrated that the metabolism rate of propionitrile was more efficient than the rate observed for acetanitrile. Moreover, the growth phase of K. oxytoca was concomitant with the production of propionamide, propionic acid, and ammonia in the culture medium. Maximum accumulation of ammonia was observed after 34 h of incubation, whereas propionamide disappeared within 50 h of incubation. The production of propionic acid increased with the increase of incubation time. No ammonia, propionamide, and propionic acid were observed in the uninoculated medium (data not shown). This confirms that the formation of these products resulted from the biotransformation of propionitrile.

Fig. 3
figure 3

Time course of substrate disappearance and product formation of acetonitrile degradation during assay with growth cells of K. oxytoca. (▪) acetonitrile, (⋄) acetamide, (▴) acetic acid, (○) ammonia, (*) growth curve

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Fig. 4
figure 4

Time course of substrate disappearance and product formation of propionitrile degradation during assay with growth cells of K. oxytoca. (♦) propionitrile, (▪) propionamide, (▵) propionic acid, (○) ammonia, (*) growth curve

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Discussion

Results from our study show that K. oxytoca is able to utilize nitriles as the sources of nitrogen. This demonstrates that K. oxytoca possesses the necessary enzymatic mechanisms for the metabolism of these nitriles. The cells utilizing nitriles showed a significant oxygen uptake, which is an indirect and approximate measure of enzymatic activity of K. oxytoca. No enzymatic activity was observed in cells treated with 25 mM of acetonitrile, benzonitrile, or phenylacetonitrile (Table 1). A high toxicity of benzonitrile and phenylacetonitrile (as judged by quite low MIC values) would lead to the inactivation of enzyme, whereas the low-enzyme activity of acetonitrile would be due to the fact that acetonitrile was not readily oxidized. However, it was still observable that 1 mM of benzonitrile and 1 mM of phenylacetonitrile were oxidizable (Fig. 2). Furthermore, when the incubation time was extended, 25 mM of acetonitrile can also be degraded (Fig. 3). Butyronitrile at indicated doses was readily utilized, although the rate of degradation of this compound at 100 mM decreased (Fig. 1). Results from this study indicated that the toxicity of nitriles had significant effects on the degradation of these tested nitriles by K. oxytoca.

Results also show that the breakdown of acetonitrile (Fig. 3) and propionitrile (Fig. 4) by K. oxytoca involves a two-step enzymatic mechanism. It is possible that nitrile hydratase (EC 4.2.1.84), the primary enzyme, transforms the nitrile compounds to their respective amides, and the amide is then degraded by amidase (EC 3.5.1.4). A similar sequential formation of the corresponding amide, carboxylic acid, and ammonia by microbial transformation of nitrile compounds was reported earlier (Nawaz et al. 1991; Rezende et al. 1999).

Acetonitrile was metabolized by N. rhodochrous into acetamide, acetic acid, and ammonia (DiGeronimo and Antonoine 1976). This bacterium could also biotransform propionitrile to propionic acid and ammonia, but the production of propionamide was not detected in the culture medium. Similarly, acetonitrile was degraded to acetic acid and ammonia via nitrile hydratase and amidase by P. marginalis (Babu et al. 1995) or R. erythropolis BL1 (Langdanhl et al. 1996), and no acetamide was detected in the culture medium. Dias et al. (2000) pointed out that they failed to detect the respective amide because it was rapidly utilized and the detection limit for acetamide was 100 μM by the GC method used. Dias et al. (2000) showed that C. guilliermondii UFMG-Y65 growing on acetonitrile can rapidly metabolize benzonitrile and acrylnitrile to benzamide/benzoic acid and acrylamide/acrylic acid, respectively, and suggested that the detection of these compounds would be intermediates or end products of their respective nitriles metabolism. Similarly, in this study, the production of acetamide or propionamide was suggested to be the intermediates of acetonitrile or propionitrile metabolism.

Microbial metabolism of nitrile compounds has become a subject of considerable interest from the point of amide production (Babu et al. 1995). The application of microorganisms as biocatalysts for the production of amides and organic acid has attracted commercial interest (Babu et al. 1995; Dias et al. 2000). Biocatalysts are highly specific and efficient in the transformation of nitrile compounds to their corresponding amides or organic acid. Because K. oxytoca is able to produce amide and carboxylic acid via the process of nitriles biotransformation, this bacterium can be applied in the industry of amides/organic acids production. Thus, biotreatment systems inoculated with K. oxytoca have the potential to be developed into an environmentally and economically acceptable waste-treatment scheme for the treatment of nitrile-containing waste with the production of economic byproducts. Results from this study will be useful in designing a scale-up system for practical application.