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A sensitive method for naphthalene oxygenase assay in whole cells
ELSEVIER
Journal of Microbiological
Methods
Journal ofMicrobiological Methods
26 (1996) 27-33
A sensitive method for naphthalene oxygenase assay in whole cells V.Riis”,
D. Miethe, W. Babel
Centre for Environmental Research Leipzig-Halle Ltd., Permoserstr. 15, D-04318 Received
8 October
1995; revised 2 December
1995; accepted
, Leipzig, Germany
18 December
1995
Abstract The naphthalene oxygenase activities of strains degrading this compound are frequently low. Spectrometric methods allow determination only down to a minimum of 10 nmol/min/mg protein. Difficulties also result from the fact that the measurements mostly require whole cells, which involves a high background signal. We developed a new sensitive method which enables high cell concentrations to be used because the naphthalene is determined after extraction from the reaction mixture with cyclohexane. This approach has other advantages which are discussed. The method was applied to different strains which degrad,e naphthalene at varying rates and to a community. The values determined coincided with those calculated from the rate of oxygen consumption. Keywords:
Activity;
Cyclohexane
extraction;
Naphthalene
dioxygenase;
1. Introduction The activity levels of enzymes catalyzing the oxidation of hydrocarbons are generally lower than those of common cellular metabolism [ 11. Naphtbalene dioxygenase (NO) is known to be an NAD(P)H-dependent system consisting of 3 components: a reductase containing flavine and iron, ferredoxin and an iron-sulphur-protein [2], which catalyzes the following reaction: naphthalene + 02+NADH+H+ + cis- 1,2-dihydro- 1,Zdihydroxynaphthalene + NAD + . The primary product is immediately oxidized to 1,2_dihydroxynaph,thalene by a subsequent NADlinked enzyme (unless this step is blocked); thus the NADH/NAD couple cannot be used for the spectrometric monitoring of NO activity. NO assays are *Corresponding 2352247.
author.
Tel.:
+49
341 2352347;
fax: t49
341
0167-7012/96/$15.00 0 1996 Elsevier Science BY. All rights reserved PIZ SO167-7012(96)00833-O
Pseudomonads
based on the UV spectrometric determination of the concentration of the substrate or reaction products [3,4], on measuring the oxygen consumption [5] and on the radiometric determination of the non-volatile metabolites of [ 14C]naphthalene after their separation by thin-layer chromatography [2]. Cidaria et al. [4] measured the formation of cis- 1,Zdihydro- 1,2-dihydroxynaphthalene using a blocked mutant of P. putida; they estimated the detection limit of their method to be 10 nmol/min/mg dry cell weight. Because of the higher absorptivity of this compound, this method is more sensitive than determining the decrease of naphthalene concentration. Shamsuzzaman and Bamsley [3] could not measure the naphthalene dioxygenase activity of some Pseudomonads which grew very slowly on naphtbalene. They used whole cells; with crude cell-free extracts they found only 5% of the activity of whole cells. Other authors [4,6] also used whole cells for NO measurement.
28
V. Riis et al. I Journal of Microbiological
We have developed a method which allows the determination of specific activities
2. Materials and methods
2.1. Organisms and cultivation Pseudomonas putida 4476, P. jborescens 6506, P. stutzeri 6083, R oleovorans 1045 and the Sphingomonas sp. 6900 were obtained from the German Collection of Microorganisms and Cell Cultures (DSM, Braunschweig) and P. putida 9816 from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK). Rhodococcus rhodochrous B376 originated from our own collection. The microorganisms were grown at 30°C in shaking flasks on medium I or II (the medium used in each case is specified in Table 2) with the addition of naphthalene and/or salicylate (20 mg/flask containing 150 ml medium; in the case of naphthalene, the quantity exceeds solubility). P. putida 4476, harboring the NAH7 plasmid, was grown on a basal medium advised by DSM for degradative Pseudomonads with the addition of 0.05% w/v salicylate. The strains Sphingomonas sp. and P. jkorescens grew slowly on medium I supplemented with naphthalene; therefore succinate was added as a carbon source (20 mg/150 ml medium), as well as a small amount of yeast extract (15 mg/150 ml). A community from a soil contaminated with mineral oil for a long period was also examined in terms of its NO activity. This community was grown on medium II (without yeast extract) +O.l% w/v diesel fuel as the carbon source. The media contained the following components per 1 1 deionized water: Medium I (mineral medium No. 442 in the DSM catalogue, 1993): 9 g Na,HPO,*2H,O; 1.5 g KH,PO,; 2 g NH&l; 0.2 g MgSO;7H,O; 3 g MnSO,.H,O; 0.2 g ZnSO,.7H,O; 10 pug CoSO,; 5 g NH,Fe(III)-citrate; 10 mg titriplex I; 50 mg tryptophan. Medium II: 762 mg NH,Cl; 870 mg K,HPO,; 680 mg KH,PO,; 5.5 mg CaCl,*6H,O; 71.5 mg MgSO,. 7H,O; 0.8 mg CuSO;SH,O; 0.4 mg ZnSO,*7H,O; 0.8 mg MnS0;4H,O; 0.25 mg Na,MoO,*2H,O;
Methods 26 (1996) 27-33
5.0 mg FeS0,.7H,O; 1 g yeast extract (unless otherwise specified). Basal medium for degradative Pseudomonads (DSM recipe): 1600 mg K,HPO,; 400 mg KH,PO,; 5000 mg MgSO;7H,O; 10 mg CaCl,.2H,O; 1000 mg (NH,),SO,; 50 mg NH,Fe(III)-citrate; 50 mg tryptophan. Following the increase of the optical density E (6,,0 nm, , cmj to >l.O, the suspension was filtered through a wide-pore paper filter (Schleicher and Schuell, round filter no. 1573) in order to remove the residual crystals of naphthalene. After that the cells were harvested by centrifugation (10 min, 6000 X g). The pellet was washed twice with 50 mM potassium phosphate buffer pH 7.0 and resuspended in a few ml (depending on the mass) of the same buffer. Filtration was not always possible; in these cases the final suspension was Potter-homogenized to better distribute any residual naphthalene. The determination of NO activity was always carried out immediately. For two cultures the oxygen consumption was followed with the aid of an automatically recording respirometer (Sapromat D12, Voith GmbH Heidenheim, Germany). The quantity of naphthalene degraded was measured by gas chromatography after extraction with cyclohexane. We used the Shimadzu GC-14A gas chromatograph equipped with a J&W Scientific DBS fused-silica capillary column (30 m X 0.25 i.d., film thickness 0.25 pm) and FID. Other conditions were: carrier gas N,, 1.0 ml/mm, split 1:70, injector and detector temperature 270°C heating program of the column oven from 90” to 140°C at 10”/min. The culture medium (250 ml) was extracted with 25 ml cyclohexane containing the internal standard adamantane. The protein contents were determined using the method of Lowry et al. [7] with bovine serum albumin as the calibration standard. 2.2. Naphthalene
dioxygenase
assay
Reagents 1. 50 mM potassium phosphate buffer pH 7.0 2. Solution of naphthalene (of reagent grade) ethanol, 25 mM (3.2 mg/ml) 3. Cyclohexane
in
29
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
Two hundred ~1 of the naphthalene solution are added to 30 ml bufl’er, the mixture is shaken and 4 ml of the mixture are transferred to each of six lo-ml volumetric flasks with tight stoppers (standard ground) or screw carps. One hundred ~1 each of the cell suspension are added to 3 of the flasks. The cell suspension should contain about 20 mg of protein/ ml or, diluted 1:200, display an optical density of E 600 nm. 1 cm >I.& The flasks are immediately closed, shaken and kept at 25°C in the dark. Ground stoppers are sealed with a little silicone grease. The reaction in the flasks containing cell material is stopped after 3 h by adding 0.4 ml 1 N hiydrochloric acid. After that, 4 ml of cyclohexane are added to each of the flasks, which are shaken intensively on a shaker for 30 min. After separation of the phases, 3 ml of the cyclohexane phase are pipetted directly into a stoppered l-cm cuvette and the absorbance is recorded between 230 and 380 nm against the solvent. It is recommended for evaluation that the absorbance in the maximum (at 275 nm) be referred to the base line at 380 nm. Because the microorganisms can contain residual naphthalene (if grown in the presence thereof), a blank must be determined. For this purpose a mixture of 4 ml of buffer +lOO ~1 of cell suspension +0.4 ml 1 N HCl is extracted with 4 ml cyclohexane and the absorbance A, at 275 nm is measured. The determination of the blank and the start of incubation must be effected simultaneously.
cprot, protein content of the cell suspension in mg/ml. t, incubation time in min. 5.53, absorptivity (absorption coefficient) of naphthalene in mmol-’ ml cm-r.
3. Results and discussion 3.1. Development
of the method
The high volatility of naphthalene impedes the determination of low NO activities because of the prolonged incubation time which is necessary to ensure a measurable turnover. Fig. 1 shows the time-dependent decrease of naphthalene concentration in a cuvette loosely covered with a lid. Significant losses also result from the repeated decanting of a naphthalene solution (stored in a well-sealed flask) into a cuvette (e.g. for hourly measurements) and refilling. The use of, for instance, polyethylene hollow stoppers to close the reaction vessels caused additional losses, as their replacement by glass stoppers revealed. The naphthalene vapour evidently diffuses through the polyethylene. In addition to the mostly low activities of enzymes
0.5 vi 9 0.4
2.3. Evaluation T
The initial concentration of naphthalene is given by the sum of the mean absorbances of the reference samples (buffer + naphthalene) A, and the blank A,. The mean of the absorbances of the samples containing cells A,, must be subtracted. The specific activity is calculated using the following equation: Activity =
nMo1
___[ min X lmgprot
(A,+A,-A,)
5.53 X V-c, X c,,,,
0.2
0.1
1
X V,,
0.3
X 1000 X t
VEXM, volume of the extraction medium in ml. Vcs, volume of the cell suspension in ml.
0 230
250
270 -
290 wsve len@h
310 [nm]
Fig. 1. Evaporation of naphtbalene in a cuvette loosely covered with a lid. Spectra at 0, 1, 2, 3 and 4 h (from above to below); 15 ~1 of a 25 mM solution of naphthalene in 4 ml buffer.
V: Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
30
oxidizing hydrocarbons, we have to consider that the enzyme concentration of whole cells related to the protein content is 2-3 orders of magnitude lower than with crude cell-free extracts. The fact that nevertheless higher activities are found with whole cells may result from other causes (e.g. no enrichment of the enzymes in the crude extract, instability at room temperature). Thus, experiments with crude extracts of the strains P. putida 4476 and P. &orescem 6506 (cultivated on media which only allow slow growth) did not provide measurable values, whereas NO activities of whole cells could be determined. The activity of the crude cell-free extract of the community amounted to l/5 of that of the intact cells. Measurement at higher cell density would be desirable because of the low activity. However, limits are imposed by the high natural absorbance of the cells. Fifteen ~1 of a cell suspension with 10 mg protein/ml diluted with 4 ml buffer has an absorbance at 275 nm of 0.94 (ebiomass = 25 mg-’ ml cm-i). This value is increased by the contribution of the naphthalene (~0.8). Measurement at absorbances > 1.5 is not recommended because of the large error incurred. Table 1 shows the decrease in the absorbance at 275 nm after 10 min for different activities using 0.15 mg cell protein/4 ml reaction mixture (with an absorbance of 0.94 the maximum possible concentration). The calculation is based on an absorptivity of 2000 mmol - ’ ml cm ’ (see Section 3.2). It may be seen that activities 510 nmol/min/mg protein are barely determinable. Using more cell mass to increase the sensitivity of the method is only practicable if the cells are removed before measuring the absorbance or if the
Table 1 Decrease of absorbance (AA) at the maximum possible cell concentration in 10 min for different naphthalene dioxygenase levels Activity (nmol/min/mg 1 5 10 20 50 100
protein)
AA 0.00075 0.00375 0.0075 0.0150 0.0375 0.075
non-degraded naphthalene is extracted and determined in the organic phase. During centrifugation, naphthalene adsorbed on the cells is displaced, simulating a higher turnover rate than occurs in reality. Possible losses due to adsorption would have to be determined by equivalent inhibited samples. Extraction with a solvent such as cyclohexane rules out such an effect and is also more favourable because the evaluation is based on the exact absorption coefficient of the naphthalene (5530 mmoll’ ml cm-i). The extraction eliminates any uncertainty regarding the coefficient to be used. The naphthalenediols formed remain in the aqueous phase during extraction. When directly measuring the reaction mixture the difference of the absorption coefficients of naphthalene and the reaction products (1,2- and 2,3_naphthalenediol) must be taken into account. This amounts to about 2000 mmol-l ml cm-’ if the reaction products accumulate. A premise for the measurement of the dioxygenase activity using the reaction products is their accumulation, as is the case for the method of Cidaria et al. [4]. Errors will be introduced if these products are oxidized to further products. The fate of the reaction products determines the difference of the absorption coefficients at 375 nm which can vary between 2000 (the diols are stable and remain in stoichiometric quantity) and 5530 mmol-’ ml cm-’ (further degradation of the diols to non-aromatic compounds). However, the difference in absorptivity is decisive when calculating the activity. This effect is frequently neglected. The proposed method eliminates this problem. The error of the method was proved by several series of six parallel determinations. The maximum variation coefficient (= relative standard deviation) amounted to 15% for the sample and 8.0% for the reference run. Because of the error, we recommend triplicate determinations. The method developed reaches its lower limit of detection at about 0.2 nmol/min/mg protein. For activities >20 nmol/ min/mg protein, directly measuring the naphthalene decrease in the reaction medium (in the presence of the cells) is advisable. To check the reliability of the method, the cells were pretreated with HgC12. Under such conditions the samples in fact showed no differences to the reference values containing only buffer + naph-
31
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
thalene. Consequently by the cell material.
there are no adsorption
effects
3.2. Application of the method Naphthalene dioxygenase activities for different strains and a consortium utilizing mineral oil are given in Table 2. Measurements were carried out immediately after harvesting the cells. Cells frozen for several days showed a lower activity, decreasing progressively with the storage time. The activities measured are scattered over a wide range: 0.2-35 nmol/min/mg protein. We found high values for the strains P. juoresccns 6506 and P. putida 9816, which grow on naphthalene as the sole source of carbon and energy. The NO activities of P. juorescens 6506 and Sphingomonas sp. 6900 were monitored over time after introduction onto medium II with the addition of naphthalene as the principal carbon source (+ traces of yeast extract). The highest values were always recorded in this phase of cultivation. The time taken to reach this phase
Table 2 Naphthalene
dioxygenase
activity
of various
strains and a,consortium
differed for the strains P. Juorescens 6506 and Sphingomonas sp. 6900 (94 h and 18 h, respectively). It was apparent that the medium used and the physiological state of the cultures played a crucial role. Activity quickly decreased after the exhaustion of the substrate. The microbial consortium from mineral oil-contaminated soil showed low but still distinct activity (1.6 nmol/min/mg protein). It must therefore contain either a larger number of species degrading naphthalene or a relatively few strains with very high degradative activities. We failed to find NO activity >35 nmol/min/mg protein as reported in the literature [3,4]. However, such high values ought only to occur rarely, as the following rough calculation demonstrates. If we postulate merely 20% conversion of the naphthalene carbon into biomass and mineralization of 80%, the following equation results: 2 C,,H,(naphthalene)
+ 19.75 0,
+
NH, -+ C,H70,,5N,
+ 16 CO, + 6H,O
(whole cells)
Strain
Grown on
Duration of cultivation (days)
Activity (nmol/min/mg)
Rhodococcus rhodochrous B376
Medium II + succinate + naphthalene Medium II + succinate + naphthalene + salicylate Medium II + succinate + naphthalene Medium I + naphthalene Medium I + naphthalene (very slow growth) Medium II + succinate + naphthalene after this precultivation, separation of the biomass and change on medium II + naphthalene + 0.008% yeast extract
3 5 3 10 14 5 t2 +4 +8 f14” 8 6 +0.5 +3b +8 + 10 3 4 1 0.5 2
co.20 0.25 0.31 0.80 co.20 0.57 3.5 34.2 25.1 0.45 CO.20 0.23 3.3 5.1 2.1 10.20 1.6 3.1 24.0 1.6 1.1
P. oleovorans 1045 P. stufzeri 6083 P. jluorescens 6506
Sphingomonas sp. 6900
Medium I + naphthalene (very slow growth) Medium II + succinate + naphthalene after this precultivation, separation of the biomass and change on medium II + naphthalene + 0.008% yeast extract
P. putida 4416 (with NAH7 plasmid) P. putida 9816 Consortium from contaninated
Basal medium Basal medium Medium II + Medium II +
soil
“3 days after the exhaustion of the carbon source. b.lust the time of the exhaustion of the naphthalene.
+ salicylate (0.05%) + salicylate + naphthalene succinate + naphthalene diesel fuel + naphthalene
32
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
The formula C,H70,,5N, stands for the biomass [S]. Accordingly 0.364 g biomass is formed from 1 g naphthalene and 2.46 g 0,/g naphthalene are consumed. In Table 3 we have calculated the growth of biomass (starting with 1 mg protein, corresponding to about 1.7 mg cell dry mass) and the consumption of naphthalene for an activity of 50 nmol/min/mg protein. This corresponds to a turnover of 0.385 mg naphthalene per h and mg cell protein. For this activity and with a ratio of biomass formation to mineralization of 20:80, the cells ought to multiply 5-fold in 12 h and convert 12 mg naphthalene/mg cell protein at the beginning. Such high degradation efficiency is scarcely attainable in practice. Limitations caused by the low solubility of the growth substrate naphthalene are conceivable, but only with a high starting biomass (> 100 mg/l) and beyond 10 h. The correctness of the values determined by our method was checked by means of the turnover of naphthalene and oxygen of the strain Sphingomonas sp. and the consortium, which both grew on naphthalene alone. Investigations were carried out with the respirometer ‘Sapromat’. In the case of Sphingomonas sp. we inoculated 250 ml medium II with cell material equivalent to 48 mg protein and added 20 mg yeast extract and 30 mg
naphthalene. The microorganisms utilized 17.2 mg naphthalene in 12 h. Considering the low growth of cells of about 4 mg (as protein), an activity of 3.7 nmol/min/mg protein results. This value conforms with that determined at the same time by our method: 3.3 nmol/min/mg protein. The slow growth is confirmed by the constant rate of oxygen consumption and by the specific consumption of 2.24 mg O,/mg naphthalene (= 9 mol O,/mol naphthalene) which roughly corresponds to the 4: 1 mineralization/biomass formation ratio assumed above. However, the naphthalene dihydrodiol initially formed is further metabolized to carbon dioxide, water, biomass or intermediates. Analogous experiments with the consortium of microorganisms yielded an activity of 2.2 nmol/min/ mg protein - a value which corresponds to that of 1.6 nmol/min/mg protein determined by the method. Oxygen consumption during this time amounted to 6.2 mol/mol naphthalene, which again points to the progressive metabolization of the naphthalene dihydrodiol. Because of the established high sensitivity, the reported method ought to allow the determination of the naphthalene degradation potential of the microbial communities of contaminated as well as pristine soils. This is the subject of further investigations.
References Table 3 Cell growth and naphthalene consumption for an activity of 50 nmol/min/mg cell protein and a 20% turnover of the available carbon in biomass Time (h)
Biomass as protein (mg)
0
8 9 10 11 12
1.000 1.140 1.299 1.481 1.688 1.924 2.209 2.518 2.870 3.271 3.728 4.249 4.879
Degraded
naphthalene
HI Geary, P.J., Mason, J.R. and Joannou, C.L. (1990) Benzene
(mg)
per h
total
0.385 0.439 0.500 0.570 0.650 0.741 0.850 0.969 1.105 1.259 1.435 1.636 1.878
0.385 0.824 1.324 1.894 2.544 3.285 4.135 5.104 6.209 7.468 8.903 10.540 12.420
PI
r31
[41
[51
dioxygenase from Pseudomonas putida ML2 (NCIB 12190). In: Methods in Enzymology Vol. 188, Hydrocarbons and methylotrophy (Ed. M.E. Lidstrom). Academic Press, NY, pp. 52-55. Ensley, B.D. and Haigler, B.E. (1990) Naphthalene dioxygenase from Pseudomonas NCIB 9816. In: Methods in Enzymology Vol. 188, Hydrocarbons and methylotrophy (Ed. M.E. Lidstrom). Academic Press, NY, pp. 46-52. Shamsuzzaman, K.M. and Barnsley, E.A. (1974) The regulation of naphthalene oxygenase in Pseudomonads. J. Gen. Microbial. 83, 165-170. Cidaria, D., Deidda, F. and Bosetti, A. (1994) A rapid method for naphthalene dioxygenase assay in whole cells of naphthalene cis-dihydrodiol dehydrogenase blocked Pseudomonas jkorescens: screening of potential inducers of dioxygenase activity. Appl. Microbial. Biotechnol. 41, 689-693. Catterall, F.A. and Williams, PA. (1971) Some properties of the naphthalene oxygenase from Pseudomonas sp. NCIB 9816. J. Gen. Microbial. 67, 117-124.
K Riis et al. I Journal of Microbiological [6] Yen, K.-M. and Gunlralus, I.C. (1982) Plasmid gene organization: naphthalenei salicylate oxidation. Proc. Natl. Acad. Sci. USA 79, 874-878. [7] Lowry, O.H., Rosebrough, NJ., Farr, A.L. and Randall, R.J. (195 1) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
Methods
26 (1996) 27-33
33
[8] Harris, R.F. and Adams, S.S. (1979) Determination of the carbon-bound electron composition of microbial cells and metabolites by dichromate oxidation. Appl. Environ. Microbiol. 37, 237-243.
Journal of Microbiological
Methods
Journal ofMicrobiological Methods
26 (1996) 27-33
A sensitive method for naphthalene oxygenase assay in whole cells V.Riis”,
D. Miethe, W. Babel
Centre for Environmental Research Leipzig-Halle Ltd., Permoserstr. 15, D-04318 Received
8 October
1995; revised 2 December
1995; accepted
, Leipzig, Germany
18 December
1995
Abstract The naphthalene oxygenase activities of strains degrading this compound are frequently low. Spectrometric methods allow determination only down to a minimum of 10 nmol/min/mg protein. Difficulties also result from the fact that the measurements mostly require whole cells, which involves a high background signal. We developed a new sensitive method which enables high cell concentrations to be used because the naphthalene is determined after extraction from the reaction mixture with cyclohexane. This approach has other advantages which are discussed. The method was applied to different strains which degrad,e naphthalene at varying rates and to a community. The values determined coincided with those calculated from the rate of oxygen consumption. Keywords:
Activity;
Cyclohexane
extraction;
Naphthalene
dioxygenase;
1. Introduction The activity levels of enzymes catalyzing the oxidation of hydrocarbons are generally lower than those of common cellular metabolism [ 11. Naphtbalene dioxygenase (NO) is known to be an NAD(P)H-dependent system consisting of 3 components: a reductase containing flavine and iron, ferredoxin and an iron-sulphur-protein [2], which catalyzes the following reaction: naphthalene + 02+NADH+H+ + cis- 1,2-dihydro- 1,Zdihydroxynaphthalene + NAD + . The primary product is immediately oxidized to 1,2_dihydroxynaph,thalene by a subsequent NADlinked enzyme (unless this step is blocked); thus the NADH/NAD couple cannot be used for the spectrometric monitoring of NO activity. NO assays are *Corresponding 2352247.
author.
Tel.:
+49
341 2352347;
fax: t49
341
0167-7012/96/$15.00 0 1996 Elsevier Science BY. All rights reserved PIZ SO167-7012(96)00833-O
Pseudomonads
based on the UV spectrometric determination of the concentration of the substrate or reaction products [3,4], on measuring the oxygen consumption [5] and on the radiometric determination of the non-volatile metabolites of [ 14C]naphthalene after their separation by thin-layer chromatography [2]. Cidaria et al. [4] measured the formation of cis- 1,Zdihydro- 1,2-dihydroxynaphthalene using a blocked mutant of P. putida; they estimated the detection limit of their method to be 10 nmol/min/mg dry cell weight. Because of the higher absorptivity of this compound, this method is more sensitive than determining the decrease of naphthalene concentration. Shamsuzzaman and Bamsley [3] could not measure the naphthalene dioxygenase activity of some Pseudomonads which grew very slowly on naphtbalene. They used whole cells; with crude cell-free extracts they found only 5% of the activity of whole cells. Other authors [4,6] also used whole cells for NO measurement.
28
V. Riis et al. I Journal of Microbiological
We have developed a method which allows the determination of specific activities
2. Materials and methods
2.1. Organisms and cultivation Pseudomonas putida 4476, P. jborescens 6506, P. stutzeri 6083, R oleovorans 1045 and the Sphingomonas sp. 6900 were obtained from the German Collection of Microorganisms and Cell Cultures (DSM, Braunschweig) and P. putida 9816 from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK). Rhodococcus rhodochrous B376 originated from our own collection. The microorganisms were grown at 30°C in shaking flasks on medium I or II (the medium used in each case is specified in Table 2) with the addition of naphthalene and/or salicylate (20 mg/flask containing 150 ml medium; in the case of naphthalene, the quantity exceeds solubility). P. putida 4476, harboring the NAH7 plasmid, was grown on a basal medium advised by DSM for degradative Pseudomonads with the addition of 0.05% w/v salicylate. The strains Sphingomonas sp. and P. jkorescens grew slowly on medium I supplemented with naphthalene; therefore succinate was added as a carbon source (20 mg/150 ml medium), as well as a small amount of yeast extract (15 mg/150 ml). A community from a soil contaminated with mineral oil for a long period was also examined in terms of its NO activity. This community was grown on medium II (without yeast extract) +O.l% w/v diesel fuel as the carbon source. The media contained the following components per 1 1 deionized water: Medium I (mineral medium No. 442 in the DSM catalogue, 1993): 9 g Na,HPO,*2H,O; 1.5 g KH,PO,; 2 g NH&l; 0.2 g MgSO;7H,O; 3 g MnSO,.H,O; 0.2 g ZnSO,.7H,O; 10 pug CoSO,; 5 g NH,Fe(III)-citrate; 10 mg titriplex I; 50 mg tryptophan. Medium II: 762 mg NH,Cl; 870 mg K,HPO,; 680 mg KH,PO,; 5.5 mg CaCl,*6H,O; 71.5 mg MgSO,. 7H,O; 0.8 mg CuSO;SH,O; 0.4 mg ZnSO,*7H,O; 0.8 mg MnS0;4H,O; 0.25 mg Na,MoO,*2H,O;
Methods 26 (1996) 27-33
5.0 mg FeS0,.7H,O; 1 g yeast extract (unless otherwise specified). Basal medium for degradative Pseudomonads (DSM recipe): 1600 mg K,HPO,; 400 mg KH,PO,; 5000 mg MgSO;7H,O; 10 mg CaCl,.2H,O; 1000 mg (NH,),SO,; 50 mg NH,Fe(III)-citrate; 50 mg tryptophan. Following the increase of the optical density E (6,,0 nm, , cmj to >l.O, the suspension was filtered through a wide-pore paper filter (Schleicher and Schuell, round filter no. 1573) in order to remove the residual crystals of naphthalene. After that the cells were harvested by centrifugation (10 min, 6000 X g). The pellet was washed twice with 50 mM potassium phosphate buffer pH 7.0 and resuspended in a few ml (depending on the mass) of the same buffer. Filtration was not always possible; in these cases the final suspension was Potter-homogenized to better distribute any residual naphthalene. The determination of NO activity was always carried out immediately. For two cultures the oxygen consumption was followed with the aid of an automatically recording respirometer (Sapromat D12, Voith GmbH Heidenheim, Germany). The quantity of naphthalene degraded was measured by gas chromatography after extraction with cyclohexane. We used the Shimadzu GC-14A gas chromatograph equipped with a J&W Scientific DBS fused-silica capillary column (30 m X 0.25 i.d., film thickness 0.25 pm) and FID. Other conditions were: carrier gas N,, 1.0 ml/mm, split 1:70, injector and detector temperature 270°C heating program of the column oven from 90” to 140°C at 10”/min. The culture medium (250 ml) was extracted with 25 ml cyclohexane containing the internal standard adamantane. The protein contents were determined using the method of Lowry et al. [7] with bovine serum albumin as the calibration standard. 2.2. Naphthalene
dioxygenase
assay
Reagents 1. 50 mM potassium phosphate buffer pH 7.0 2. Solution of naphthalene (of reagent grade) ethanol, 25 mM (3.2 mg/ml) 3. Cyclohexane
in
29
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
Two hundred ~1 of the naphthalene solution are added to 30 ml bufl’er, the mixture is shaken and 4 ml of the mixture are transferred to each of six lo-ml volumetric flasks with tight stoppers (standard ground) or screw carps. One hundred ~1 each of the cell suspension are added to 3 of the flasks. The cell suspension should contain about 20 mg of protein/ ml or, diluted 1:200, display an optical density of E 600 nm. 1 cm >I.& The flasks are immediately closed, shaken and kept at 25°C in the dark. Ground stoppers are sealed with a little silicone grease. The reaction in the flasks containing cell material is stopped after 3 h by adding 0.4 ml 1 N hiydrochloric acid. After that, 4 ml of cyclohexane are added to each of the flasks, which are shaken intensively on a shaker for 30 min. After separation of the phases, 3 ml of the cyclohexane phase are pipetted directly into a stoppered l-cm cuvette and the absorbance is recorded between 230 and 380 nm against the solvent. It is recommended for evaluation that the absorbance in the maximum (at 275 nm) be referred to the base line at 380 nm. Because the microorganisms can contain residual naphthalene (if grown in the presence thereof), a blank must be determined. For this purpose a mixture of 4 ml of buffer +lOO ~1 of cell suspension +0.4 ml 1 N HCl is extracted with 4 ml cyclohexane and the absorbance A, at 275 nm is measured. The determination of the blank and the start of incubation must be effected simultaneously.
cprot, protein content of the cell suspension in mg/ml. t, incubation time in min. 5.53, absorptivity (absorption coefficient) of naphthalene in mmol-’ ml cm-r.
3. Results and discussion 3.1. Development
of the method
The high volatility of naphthalene impedes the determination of low NO activities because of the prolonged incubation time which is necessary to ensure a measurable turnover. Fig. 1 shows the time-dependent decrease of naphthalene concentration in a cuvette loosely covered with a lid. Significant losses also result from the repeated decanting of a naphthalene solution (stored in a well-sealed flask) into a cuvette (e.g. for hourly measurements) and refilling. The use of, for instance, polyethylene hollow stoppers to close the reaction vessels caused additional losses, as their replacement by glass stoppers revealed. The naphthalene vapour evidently diffuses through the polyethylene. In addition to the mostly low activities of enzymes
0.5 vi 9 0.4
2.3. Evaluation T
The initial concentration of naphthalene is given by the sum of the mean absorbances of the reference samples (buffer + naphthalene) A, and the blank A,. The mean of the absorbances of the samples containing cells A,, must be subtracted. The specific activity is calculated using the following equation: Activity =
nMo1
___[ min X lmgprot
(A,+A,-A,)
5.53 X V-c, X c,,,,
0.2
0.1
1
X V,,
0.3
X 1000 X t
VEXM, volume of the extraction medium in ml. Vcs, volume of the cell suspension in ml.
0 230
250
270 -
290 wsve len@h
310 [nm]
Fig. 1. Evaporation of naphtbalene in a cuvette loosely covered with a lid. Spectra at 0, 1, 2, 3 and 4 h (from above to below); 15 ~1 of a 25 mM solution of naphthalene in 4 ml buffer.
V: Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
30
oxidizing hydrocarbons, we have to consider that the enzyme concentration of whole cells related to the protein content is 2-3 orders of magnitude lower than with crude cell-free extracts. The fact that nevertheless higher activities are found with whole cells may result from other causes (e.g. no enrichment of the enzymes in the crude extract, instability at room temperature). Thus, experiments with crude extracts of the strains P. putida 4476 and P. &orescem 6506 (cultivated on media which only allow slow growth) did not provide measurable values, whereas NO activities of whole cells could be determined. The activity of the crude cell-free extract of the community amounted to l/5 of that of the intact cells. Measurement at higher cell density would be desirable because of the low activity. However, limits are imposed by the high natural absorbance of the cells. Fifteen ~1 of a cell suspension with 10 mg protein/ml diluted with 4 ml buffer has an absorbance at 275 nm of 0.94 (ebiomass = 25 mg-’ ml cm-i). This value is increased by the contribution of the naphthalene (~0.8). Measurement at absorbances > 1.5 is not recommended because of the large error incurred. Table 1 shows the decrease in the absorbance at 275 nm after 10 min for different activities using 0.15 mg cell protein/4 ml reaction mixture (with an absorbance of 0.94 the maximum possible concentration). The calculation is based on an absorptivity of 2000 mmol - ’ ml cm ’ (see Section 3.2). It may be seen that activities 510 nmol/min/mg protein are barely determinable. Using more cell mass to increase the sensitivity of the method is only practicable if the cells are removed before measuring the absorbance or if the
Table 1 Decrease of absorbance (AA) at the maximum possible cell concentration in 10 min for different naphthalene dioxygenase levels Activity (nmol/min/mg 1 5 10 20 50 100
protein)
AA 0.00075 0.00375 0.0075 0.0150 0.0375 0.075
non-degraded naphthalene is extracted and determined in the organic phase. During centrifugation, naphthalene adsorbed on the cells is displaced, simulating a higher turnover rate than occurs in reality. Possible losses due to adsorption would have to be determined by equivalent inhibited samples. Extraction with a solvent such as cyclohexane rules out such an effect and is also more favourable because the evaluation is based on the exact absorption coefficient of the naphthalene (5530 mmoll’ ml cm-i). The extraction eliminates any uncertainty regarding the coefficient to be used. The naphthalenediols formed remain in the aqueous phase during extraction. When directly measuring the reaction mixture the difference of the absorption coefficients of naphthalene and the reaction products (1,2- and 2,3_naphthalenediol) must be taken into account. This amounts to about 2000 mmol-l ml cm-’ if the reaction products accumulate. A premise for the measurement of the dioxygenase activity using the reaction products is their accumulation, as is the case for the method of Cidaria et al. [4]. Errors will be introduced if these products are oxidized to further products. The fate of the reaction products determines the difference of the absorption coefficients at 375 nm which can vary between 2000 (the diols are stable and remain in stoichiometric quantity) and 5530 mmol-’ ml cm-’ (further degradation of the diols to non-aromatic compounds). However, the difference in absorptivity is decisive when calculating the activity. This effect is frequently neglected. The proposed method eliminates this problem. The error of the method was proved by several series of six parallel determinations. The maximum variation coefficient (= relative standard deviation) amounted to 15% for the sample and 8.0% for the reference run. Because of the error, we recommend triplicate determinations. The method developed reaches its lower limit of detection at about 0.2 nmol/min/mg protein. For activities >20 nmol/ min/mg protein, directly measuring the naphthalene decrease in the reaction medium (in the presence of the cells) is advisable. To check the reliability of the method, the cells were pretreated with HgC12. Under such conditions the samples in fact showed no differences to the reference values containing only buffer + naph-
31
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
thalene. Consequently by the cell material.
there are no adsorption
effects
3.2. Application of the method Naphthalene dioxygenase activities for different strains and a consortium utilizing mineral oil are given in Table 2. Measurements were carried out immediately after harvesting the cells. Cells frozen for several days showed a lower activity, decreasing progressively with the storage time. The activities measured are scattered over a wide range: 0.2-35 nmol/min/mg protein. We found high values for the strains P. juoresccns 6506 and P. putida 9816, which grow on naphthalene as the sole source of carbon and energy. The NO activities of P. juorescens 6506 and Sphingomonas sp. 6900 were monitored over time after introduction onto medium II with the addition of naphthalene as the principal carbon source (+ traces of yeast extract). The highest values were always recorded in this phase of cultivation. The time taken to reach this phase
Table 2 Naphthalene
dioxygenase
activity
of various
strains and a,consortium
differed for the strains P. Juorescens 6506 and Sphingomonas sp. 6900 (94 h and 18 h, respectively). It was apparent that the medium used and the physiological state of the cultures played a crucial role. Activity quickly decreased after the exhaustion of the substrate. The microbial consortium from mineral oil-contaminated soil showed low but still distinct activity (1.6 nmol/min/mg protein). It must therefore contain either a larger number of species degrading naphthalene or a relatively few strains with very high degradative activities. We failed to find NO activity >35 nmol/min/mg protein as reported in the literature [3,4]. However, such high values ought only to occur rarely, as the following rough calculation demonstrates. If we postulate merely 20% conversion of the naphthalene carbon into biomass and mineralization of 80%, the following equation results: 2 C,,H,(naphthalene)
+ 19.75 0,
+
NH, -+ C,H70,,5N,
+ 16 CO, + 6H,O
(whole cells)
Strain
Grown on
Duration of cultivation (days)
Activity (nmol/min/mg)
Rhodococcus rhodochrous B376
Medium II + succinate + naphthalene Medium II + succinate + naphthalene + salicylate Medium II + succinate + naphthalene Medium I + naphthalene Medium I + naphthalene (very slow growth) Medium II + succinate + naphthalene after this precultivation, separation of the biomass and change on medium II + naphthalene + 0.008% yeast extract
3 5 3 10 14 5 t2 +4 +8 f14” 8 6 +0.5 +3b +8 + 10 3 4 1 0.5 2
co.20 0.25 0.31 0.80 co.20 0.57 3.5 34.2 25.1 0.45 CO.20 0.23 3.3 5.1 2.1 10.20 1.6 3.1 24.0 1.6 1.1
P. oleovorans 1045 P. stufzeri 6083 P. jluorescens 6506
Sphingomonas sp. 6900
Medium I + naphthalene (very slow growth) Medium II + succinate + naphthalene after this precultivation, separation of the biomass and change on medium II + naphthalene + 0.008% yeast extract
P. putida 4416 (with NAH7 plasmid) P. putida 9816 Consortium from contaninated
Basal medium Basal medium Medium II + Medium II +
soil
“3 days after the exhaustion of the carbon source. b.lust the time of the exhaustion of the naphthalene.
+ salicylate (0.05%) + salicylate + naphthalene succinate + naphthalene diesel fuel + naphthalene
32
V. Riis et al. I Journal of Microbiological Methods 26 (1996) 27-33
The formula C,H70,,5N, stands for the biomass [S]. Accordingly 0.364 g biomass is formed from 1 g naphthalene and 2.46 g 0,/g naphthalene are consumed. In Table 3 we have calculated the growth of biomass (starting with 1 mg protein, corresponding to about 1.7 mg cell dry mass) and the consumption of naphthalene for an activity of 50 nmol/min/mg protein. This corresponds to a turnover of 0.385 mg naphthalene per h and mg cell protein. For this activity and with a ratio of biomass formation to mineralization of 20:80, the cells ought to multiply 5-fold in 12 h and convert 12 mg naphthalene/mg cell protein at the beginning. Such high degradation efficiency is scarcely attainable in practice. Limitations caused by the low solubility of the growth substrate naphthalene are conceivable, but only with a high starting biomass (> 100 mg/l) and beyond 10 h. The correctness of the values determined by our method was checked by means of the turnover of naphthalene and oxygen of the strain Sphingomonas sp. and the consortium, which both grew on naphthalene alone. Investigations were carried out with the respirometer ‘Sapromat’. In the case of Sphingomonas sp. we inoculated 250 ml medium II with cell material equivalent to 48 mg protein and added 20 mg yeast extract and 30 mg
naphthalene. The microorganisms utilized 17.2 mg naphthalene in 12 h. Considering the low growth of cells of about 4 mg (as protein), an activity of 3.7 nmol/min/mg protein results. This value conforms with that determined at the same time by our method: 3.3 nmol/min/mg protein. The slow growth is confirmed by the constant rate of oxygen consumption and by the specific consumption of 2.24 mg O,/mg naphthalene (= 9 mol O,/mol naphthalene) which roughly corresponds to the 4: 1 mineralization/biomass formation ratio assumed above. However, the naphthalene dihydrodiol initially formed is further metabolized to carbon dioxide, water, biomass or intermediates. Analogous experiments with the consortium of microorganisms yielded an activity of 2.2 nmol/min/ mg protein - a value which corresponds to that of 1.6 nmol/min/mg protein determined by the method. Oxygen consumption during this time amounted to 6.2 mol/mol naphthalene, which again points to the progressive metabolization of the naphthalene dihydrodiol. Because of the established high sensitivity, the reported method ought to allow the determination of the naphthalene degradation potential of the microbial communities of contaminated as well as pristine soils. This is the subject of further investigations.
References Table 3 Cell growth and naphthalene consumption for an activity of 50 nmol/min/mg cell protein and a 20% turnover of the available carbon in biomass Time (h)
Biomass as protein (mg)
0
8 9 10 11 12
1.000 1.140 1.299 1.481 1.688 1.924 2.209 2.518 2.870 3.271 3.728 4.249 4.879
Degraded
naphthalene
HI Geary, P.J., Mason, J.R. and Joannou, C.L. (1990) Benzene
(mg)
per h
total
0.385 0.439 0.500 0.570 0.650 0.741 0.850 0.969 1.105 1.259 1.435 1.636 1.878
0.385 0.824 1.324 1.894 2.544 3.285 4.135 5.104 6.209 7.468 8.903 10.540 12.420
PI
r31
[41
[51
dioxygenase from Pseudomonas putida ML2 (NCIB 12190). In: Methods in Enzymology Vol. 188, Hydrocarbons and methylotrophy (Ed. M.E. Lidstrom). Academic Press, NY, pp. 52-55. Ensley, B.D. and Haigler, B.E. (1990) Naphthalene dioxygenase from Pseudomonas NCIB 9816. In: Methods in Enzymology Vol. 188, Hydrocarbons and methylotrophy (Ed. M.E. Lidstrom). Academic Press, NY, pp. 46-52. Shamsuzzaman, K.M. and Barnsley, E.A. (1974) The regulation of naphthalene oxygenase in Pseudomonads. J. Gen. Microbial. 83, 165-170. Cidaria, D., Deidda, F. and Bosetti, A. (1994) A rapid method for naphthalene dioxygenase assay in whole cells of naphthalene cis-dihydrodiol dehydrogenase blocked Pseudomonas jkorescens: screening of potential inducers of dioxygenase activity. Appl. Microbial. Biotechnol. 41, 689-693. Catterall, F.A. and Williams, PA. (1971) Some properties of the naphthalene oxygenase from Pseudomonas sp. NCIB 9816. J. Gen. Microbial. 67, 117-124.
K Riis et al. I Journal of Microbiological [6] Yen, K.-M. and Gunlralus, I.C. (1982) Plasmid gene organization: naphthalenei salicylate oxidation. Proc. Natl. Acad. Sci. USA 79, 874-878. [7] Lowry, O.H., Rosebrough, NJ., Farr, A.L. and Randall, R.J. (195 1) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
Methods
26 (1996) 27-33
33
[8] Harris, R.F. and Adams, S.S. (1979) Determination of the carbon-bound electron composition of microbial cells and metabolites by dichromate oxidation. Appl. Environ. Microbiol. 37, 237-243.