- Email: [email protected]
Simazine degradation by immobilized and suspended soil bacterium
Intwrurt;onnl
PII:
ELSEVIER
SO964-8305(97)00049-S
Bi,~~l~,tcuiorutif,n & Bir,~l~,~ro~luti~~r1. Vol. 40, No. 24
( I YY71 Y3-YY
( 1997 Elnevier Science Limited All rights reserved. Printed m Great Britain OYh4-8305197 $I 7.00 + 0.00
Simazine Degradation by Immobilized and Suspended Soil Bacterium Diego Martin-Montalvo a, Gerard0 Mengs a, Estrella Ferrer (I, J. Luis Allende’, Ram6n Alonso’ & Margarita Martin a* “Dpt. Bioyuimica y Biologiu MoleculnrIV. Madrid, Spain hiJ/nidad de Fisica Aplicada, F. Veterinaria. Universidad Cotnplutense, 28040 Madrid, Spain “Utlidad de Estadktica, ET% Agrdnomos. UPM Madrid, Spain
Simazine is one of the most heavily used herbicides for weed control in the production of a variety of agricultural crops. Few microorganisms have been isolated that metabolize S-triazines such as simazine at rates that are suitable for environmental remediation. DSZI strain cells were immobilized by adsorption onto ceramic supports. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended cells. The effect of substrate concentration and inoculum size/support ratio on kinetic parameters was investigated. Physiological status of immobilized cells was assessed by measuring their capacity for degrading the herbicide and this capacity was compared to that of free-living cells under different experimental conditions. La simazina es uno de 10s herbicidas mis utilizados en una amplia variedad de cultivos agricolas. Hasta la fecha, son pocos 10s microorganismos aislados que tienen capacidad para metabolizar triazinas coma la simazina, y que por tanto puedan ser utilizados en sistemas de descontaminacion medioambiental. La estirpe DSZI ha sido inmovilizada sobre un soporte ceramico. Se han estimado 10s parimetros cineticos de cultivos de celulas en suspension e inmovilizadas. utilizando metodos no lineales de estimation. Se ha estudiado el efecto de la concentration de sustrato y de1 tamaiio de inoculo sobre 10s parametros cineticos. La capacidad de las celulas de esta estirpe para degradar el herbicida en diferentes condiciones ha confirmado el mantenimiento de su viabilidad una vez inmovilizadas. c 1998 Elsevier Science Ltd. All rights reserved
The detection of simazine in groundwater and soils has prompted environmental concern about pollution. Few microorganisms have been isolated that metabolize alkylated S-triazines such as simazine (Mulbry, 1994) at rates that are suitable for environmental remediation. Cook and Hutter (1984) isolated and characterized a strain of Rhodococcus corallinus that dechlorinates and some alkylated S-triazines. The deaminates isolated strain shows a hydrolase activity that is involved in degradation, and Mulbry (1994) reported the induction effect on triazine hydrolase activity of different nitrogen sources; cyanuric acid is one of them, which is the dechlorinated and deaminated product of simazine or atrazine. The hydrolase activity is induced by cyanuric acid but cells cannot be grown on this compound as a carbon source. Our laboratory is involved in the of bacterial activities for characterization remediating contaminated soils and water. We
INTRODUCTION The S-triazine herbicides have been used in a variety of weed control programs with major crops, but herbicides containing an S-triazine ring are relatively persistent in the environment. This group of herbicides, such as simazine and are included in the EU Priority atrazine, Pollutants List, which coincidentally contains the of compounds as the US same number Environmental Protection Agency’s list (132). The use of microorganisms (Martin el al., 1995: Laine and Jorgensen, 1996) for the bioremediation of sites contaminated with toxic pollutants has become the focus of considerable attention because of the potential for restoring soils at a with saving compared considerable cost technologies such as incineration. *To whom correspondence should be addressed 1-3943823. E-mail: ralonso@ ccupm.upm.es
at: Tel.: + 34-
93
94
D. Martin-Montalvo
have recently isolated a bacterial strain DSZl capable of growing on simazine as a sole carbon and energy source. This strain was isolated from a contaminated soil by enrichment culture. The main aim of this study was to perform a bioremediation system, using the isolated strain DSZl, taking into account its kinetic parameters for growth rate and substrate uptake, when these cells are used in suspension or immobilized. The problems showed by continuous fermentation processes could be solved by the use of immobilized cells as biocatalysts (Ferrer et al., 1996). A methodology is described for cell immobilization and the physiological status of immobilized cells was assessed by measuring cell growth and substrate utilization compared to that of free-living cells.
MATERIALS AND METHODS Isolation of bacteria
et al.
then inoculated with DSZl strain (lo6 cells); plate counts were taken from dilutions of the inoculum and the initial substrate concentrations were measured immediately after inoculation. Cells were grown at 30°C to log phase. The bacterial population was examined by plating and light microscopy to ascertain culture purity throughout the experimental conditions. Data analysis The parameters of the logistic curve fitted to the substrate utilization data were estimated using NLIN, the nonlinear parameter estimation procedure of the statistical software SAS@. Kinetic parameters of the DSZl strain, suspended or immobilized, growing on SZ were estimated according to Robinson and Tiedje (1983): 4 = dS/dt X-‘( specific substrate uptake rate) /L= dX/dt X-’ (specific growth rate) q(day-‘)
The bacterial strain capable of growing on Simazine (SZ) as sole source of carbon and energy, DSZl, used in this work, was isolated from a contaminated soil by enrichment culture. This soil is located in an agricultural site, and has a history of exposure to a wide variety of xenobiotics, including SZ. Different soil samples (5 g), were incubated in a mineral medium PJC (Hareland et al., 1975) at 30°C and SZ was added at a concentration of 10ppm. After 25 days, aliquots of the enrichment cultures were plated on agar plates containing 5ppm simazine as carbon source; the resulting isolates were plated on agar-LB plates (Miller, 1972) and tested for purity. By this technique, a pure culture designed strain DSZl was obtained, which showed the capability of growing on the herbicide. Media and culture conditions The isolated bacterium was grown aerobically in PJC medium, under the conditions mentioned above. Carbon sources were sterilized separately and added to give: 2-20ppm simazine, 1 mM glucose, 2-20 ppm cyanuric acid. Biodegradation experiments were performed using PJC as mineral minimal media, and Simazine (SZ) was added as the sole carbon substrate at 2-20ppm final concentration. It was
and
p(day-‘)
are both given by
Monod’s equation. Analytical methods Simazine concentration was analysed by highpressure liquid chromatography (HPLC). Aqueous samples from the cultures were filtered and 10-25~1 aliquots were injected onto a Novapack C-18 (3.9x150mm) column, using a mobile phase consisting of 40% acetonitrile in water at a flow rate of 0.5 ml min-’ and measuring at 220nm. Analyses were performed by using a Waters model 616PDA996, equipped with a data analysis Millennium 20/ 10. Cell immobilization A ceramic material, sepiolite, was chosen as support. This ceramic material was cut into 1 cm sided cubes (monoliths), placed into PJC medium and autoclaved. After 24 h, the mineral medium was replaced by a new sterile medium. Cells were harvested at the exponential phase of growth, using SZ as carbon source. These cells were used as the inoculum. Immobilized culture cells were examined by electron microscopy. Cells were fixed with 1% glutaraldehyde solution and washed three times with 50mM cacodylate buffer, pH 6.8. The
Simazine degradation by immobilized and suspended soil bacterium
samples were coated with colloidal gold examined by scanning electron microscopy.
and
Chemicals
Simazine was obtained from Ciba-Geigy (Barcelona, Spain). Cyanuric acid was purchased from Sigma Chemical Co. (St Louis, Missouri). All the chemical compounds were of the highest purity commercially available.
95
substrate concentration and the absence of growth at 20ppm SZ. To determine the effect of other carbon sources on the metabolism of SZ, glucose (2OOpg ml-‘) was also added to the medium. The addition of glucose shortened the lag phase (data not shown), however, the biomass formation was not modified. Cyanuric acid utilization as a carbon source by the DSZl strain was tested by using PJC medium and different concentrations of this substrate (220 ppm). Cells grew with 5 ppm cyanuric acid, but the formation of biomass was lower.
RESULTS Suspension culture experiments Isolation of DSZl
strain and growth on simazine
A variety of soil samples suspended in mineral medium were screened for possible SZ degradation. Two millilitre of the suspensions were dispensed into 50ml culture flasks, adding SZ (IOppm), and incubated at 30°C. Transfer of the enrichment culture consistently showed that there was a 10% loss of SZ within 20 days. Control samples containing autoclaved soil displayed no significant SZ degradation. Simazine utilization as carbon source by DSZl strain was tested by adding the substrate to media at different concentrations: 2-20ppm. Figure 1 shows the bacterial growth, using different SZ The growth of DSZl in the concentrations. presence of 2, 5 and 10 ppm SZ started after a lag period and resulted in the disappearance of the substrate and the formation of biomass. Figure 1 shows a short lag phase (1 day) at the lower
Figure 2 shows the depletion of SZ in batch using Sppm cultures, SZ. When the SZ concentration in the medium was 20ppm, no formation of biomass could be detected, and the amount of substrate in the culture remained invariable during the incubation time. Table 1 shows the herbicide degradation results obtained at different SZ concentrations and inoculum sizes. The rate of degradation was not dependent on cell density. For this reason, we decided to use IO6cells ml-’ as inocula for further experiments. The optimum rate of SZ degradation as well as biomass formation was achieved when SZ was added at 5ppm concentration (Fig. 2). At these growth conditions, strain DSZl degrades 70% of the initial SZ in 19 days; biomass formation was consistent with this result, and the maximum ODeoO was obtained. The growth curve obtained when DSZl uses 5 ppm SZ as the carbon
Bacterial growth, using SIMAZINE -2
ppm +5 ppm *lO
as sole carbon source: ppm *20
ppm
time (days) Fig. 1.
Cell growth
of DSZl strain in batch cultures, using different simazine concentrations as the sole carbon expressed as percentages relative to corresponding average values in sterile controls.
source. Data are
D. Martin-Montalvo
96 DSZl
et
al.
Strain
80
0
5
10
15
20
0
t Pays1
5
20
15
10 concentration (ppm)
Fig. 2. Cell growth of DSZl strain using 5ppm simazine in batch cultures and resultant depletion of simazine. Experimental and fitted curves.
Fig. 3. Effect of substrate concentration parameters. l/X dS/dt was calculated as substrate utilization rate.
source (Fig. 2) has been fitted and kinetic parameters (Table 2) have been determined according to the Monod model (Robinson and Tiedje, 1983). Figure 3 shows the effect of substrate growth concentration on the specific substrate utilization rate. The optimum growth conditions of DSZ strain are achieved at 5ppm SZ concentration, associated with a degradation rate of 70%. Although this culture was grown in the constant presence of SZ prior to this experiment, a lag phase of six days was observed after 5ppm SZ exposure. However, the culture rapidly reached the log phase and its maximum biomass production. An important result is that
DSZl can grow at low SZ concentrations (2ppm), because often herbicides are present in soils and waters at very low concentrations.
Table 1. Herbicide Inoculum
Initial
Immobilized cells experiments The ceramic material used in these experiments has the structure of a honeycomb support and its characteristics have been reported previously (Martin er al., 1995). When autoclaved monoliths were added under aseptic conditions to an exponentially growing culture of DSZl strain on 5 ppm SZ, cells were quickly adsorbed onto the surface of the monoliths without losing their
Degradation
Herbicide
size (cells ml-‘)
concentration
by the Strain DSZl” % Removed
(ppm) Final
6x lo5
2 5 10 20
0.99 1.60 8.89 18.2
51.5 68 11 9
IO6
2 5 10 20
0.88 1.42 8.65 18.45
66 71.6 13.5 1.1
10’
2 5 10 20
0.95 1.56 8.69 18.56
52.5 68.8 13.1 7.2
“Results
are given as mean of triplicate
measurements,
on kinetic the specific
after 20 days of incubation.
Simazine degradation by immobilized and suspended soil bacterium Table 2. Kinetic
K (ppm cell h-‘)
td (d) ~Lmax(d-l) & (ppm) Yield (cell/ppm)
Parameters of DSZI Simazine
Strain
Growing
Suspension culture
Immobilized culture
0.14x lo8 5 0.6 3.5 0.4x IOR
0.18x lo8 3.6 1.2 4.3 1.6x lo9
on
K: maximum specific substrate utilization rate; td: cell doubling time; p,,,: maximum specific growth rate; Kd:substrate utilization associated half velocity.
viability. The natural tendency of these microorganisms to adhere to solid surfaces can be observed in the progressive colonization of the longitudinal square channels of the monoliths. The size of the inoculum (5 x lo6 cells g- ’ support) initially used could achieve complete loading of the monolith; Table 3 shows that a cell loading to 300mg biomass g-’ of support was measured, at these inocula size conditions. The kinetic rate constants for immobiilized cell growth and Sppm SZ utilization are summarized in Table 2. Some differences were observed in growth and substrate utilization parameters between immobilized and suspended cells, and a 40-fold increase in the yield (Table 2) is observed at the same experimental conditions. The capacity of the immobilized cells for degrading SZ, keeping their viability, has been calculated by transferring the monoliths loaded with the formed biomass to aqueous solutions containing SZ at two different concentrations; 2 and Sppm. In both experiments, for over 50 days the immobilized cells kept their viability, with a degradation rate near to 70%. In this case, the final amount of substrate degraded was not dependent on the initial substrate concentration used in the experiment. Maintenance of the activity of the immobilized cells over several days may suggest that cell attachment does not negatively affect simazine biodegradation; nevertheless, changes in physiology or morphology of different immobilized cells have been reported (Ghommidh et al., 1982; Sheve and Vogel, 1993). In this way, scanning electron micrographs were taken of immobilized (Fig. 4) DSZl cells, using SZ as substrate. The cell size characteristics were determined from this and additional electron micrographs (Table 3). Examination of the electron micrographs showed no apparent morphological changes upon immobilization.
97
DISCUSSION
Due to the excessive utilization of pesticides, especially in areas of intensive agriculture, many sources of water as well as soils contain high levels of these synthetic compounds. The ability of indigenous strains of soil bacteria to degrade a wide variety of xenobiotic compounds is well documented. When these compounds are used as growth substrates, their presence defines a unique niche which can be occupied by microorganisms possessing the requisite metabolic, physiological and kinetic characteristics (Greer et al., 1992). This report describes the isolation of a soil bacterium which has the ability to degrade simazine. Microorganisms in other environments (Cook and Hutter, 1986; Mulbry, 1994) or in isolated cultures (Mandelbaum et al., 1993) have been reported to metabolize or cometabolize simazine, but ring cleavage is rarely reported, and other carbon sources such as citrate or sucrose were added. As reported above, DSZl is solely responsible for metabolism of the herbicide and can use it as a sole source of carbon and energy. Cyanuric acid can be used by strain DSZl as substrate, only at Sppm concentration, with a low formation of biomass. This result suggests that this compound is an intermediate in the Simazine degradative pathway, but alternative experiments have been carried out to design the catabolic pathway. Mulbry (1994) and Cook and Hutter (1984) reported that the Rhodococcus sp. hydrolase activity which dechlorinated and deaminated S-triazines could be induced by cyanuric acid, but this intermediate as well as others assayed in the induction experiments could not be used by the bacterium as the carbon source. The capacity of DSZl strain has several implications for the fate of S-triazine herbicides in the environment. A general problem with these molecules is that the ring portion persists in the environment. In our system, simazine is metabolized by the DSZl strain, nevertheless we are currently attempting to address this problem mineralization occurs by using [14C] simazine. When the DSZl strain is immobilized onto sepiolite monoliths, cells maintain their viability and capacity to degrade simazine. After the initial adsorption of DSZI cells onto the surface of the monolith they colonize the support very efficiently, showing similar substrate utilization to that showed for suspended cells. It is appropriate to underline the greater capacity of the
D. Martin-Montaho
98
Fig. 4.
Electron
micrographs
of DSZI cells immobilized
immobilized cells for degrading SZ (more than 90 days) compared to the capacity of the suspended cells (15 days). Morphological and physiological Table 3. Characteristics
of Immobilized
Biomass loading” Biomass retainment Physiological status
Operational
DSZl
Cells 300 High
width of cell length of cell
No change 0.57pm I .275 pm
dead cells growing cells
< 6% > 60%
stability
‘Based on dry w/g-’
of support.
et al.
onto sepiolite
monoliths.
using simazine
as substrate.
changes caused by the immobilization process (Klein and Ziehr, 1990; Sheve and Vogel, 1993; Willaert et al., 1996) mean a problem when this system is applied to biotechnological processes. The results of this work show that biodegradation performance (Table 2) obtained when the DSZl strain is immobilized in the selected conditions is slightly higher than that obtained for suspended cells. From this analysis, the maximum SZ utilization did not change upon rate immobilization and the maximum specific growth rate is two-fold higher for the immobilized cells. These results are not surprising if it is taken into account that the DSZ strain has been isolated from
a soil intensively treated with simazine. The intensive use of this herbicide and its relative persistence led to instances in which microorganisms able to use it as carbon source are selected. With a continuous supply of substrate, there is obviously selective pressure to become immobilized in the soil structure. The ceramic material used as a support improves the process implementation. Therefore, the optimization of culture before design of conditions, the the bioremediation process, must be a prior objective; once the biodegradation performance is defined, the conditions of the working treatment can be tried out.
Propachlor suspended
and alachlor
degradation
by immobilized
and
Pseudomonas cells. In Immohiked cells: Basics attti Applicutiotts. Elsevier Science, B.V., p. 762.
Ghommidh. C.. Navarro. for the immobilization Bioettgitteer-ing
J. M. & Durnad. G. (1982) Methods of microbial cells. Biotechnology and
24, 605-6
17.
Greer. L. E., Robinson. J. A. & Shelton. D. R. (1992) Kinetic comparison of seven strain of 2.4-dichlorophenoxyacetic acid degrading bacteria. Applied Ettvironmetztal MicrohiolO,qA’58, 1027-1030. Hareland. W. A.. Crawford. R. L., Chapman. P. J. & Dagley, S. (1975) Metabolic function and properties of 4-hydroxyphenylacetic acid I-hydroxylase from P.~&~nzonas Jourtd
u&hot-ms.
of Bacteriolqy
121, 379-185.
Klein, J. & Ziehr, H. (1990) Immobilization of microbial cells by adsorption. Jourttul of’ Biorechnnl~~g~~ 16, l-1 6. Laine. M. N. & Jorgensen, K. S. (1996) Straw compost and bioremediated soil as inocula for the bioremediation of chlorophenol-contaminated soil. Applied Ettvirwttttet~tuI Microhiolo~~~~ 62, I507-
I 513.
Mandelbaum. R. T.. Wackett, L. P. & Allan, D. T. (1993) Mineralization of the s-triazine ring of atrazine by stable bacteria1 mixed cultures. ,4pplied Ett~~irottmrtttal Microhiol-
ACKNOWLEDGEMENTS
og>’ 59, I695-
The CICYT Grant AMB94-0483, and the Comunidad Autbnoma de Madrid Grant COR0008/94, provided funding for this work. The authors would like to express their appreciation to Ciba-Geigy for providing samples of Simazine.
170I
Martin. M.. Ferrer, E.. Alonso, R. & Fernlindez. J. ( 1995) Bioremediation of soil contaminated by propachlor using native bacteria. internatiottal Biodeterioration and Biociegrofiufiott
35, 2 13-335.
Miller, J. M. ( 1972) E-1.peritnettts in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Mulbry. W. W. (1994) Purification and characterization of an inducible s-triazine hydrolase from Ritorlococcus corallinus NRRL B- 15444R. .4pplied Ettvirottmetttctl Microbiology 60, 613-618.
Robinson. J. A. & Tiedje. J. M. (1983) Nonlinear estimation of monod growth kinetic parameters from a single substrate depletion curve. Applied Ett~~irottttletttal Microhiolog~~ 45(5),
REFERENCES Cook, A. & Hutter, R. (1984) Deethylsimazine: bacterial dechlorination, deamination and complete degradation. Journal
of Agricdture
and Food Clzemistry
32, 58 l--585.
Cook, A. & Hutter, R. (1986) Ring dechlorination of deethylsimazine by hydrolases from Rhodococcus corallinus. FEMS
Ferrer.
Microhiolog~~
E., Blanco.
Lerters
J.. Alonso,
34, 335-338.
R. and
Martin.
M. (1996)
1453-1458.
Sheve. G. S. & Vogel, T. M. (1993) Comparison of substrate utilization and growth kinetics between immobilized and suspended Psc~u~lotttonLts cells. Biotechnolog~~ utd Bioengitiwritig 41, 370-379. Willaert. R. G., Baron. G. V. and De Backer, L. (1996) htmwhili.wtl Living Cell Swtems. Wiley and Sons. UK.
PII:
ELSEVIER
SO964-8305(97)00049-S
Bi,~~l~,tcuiorutif,n & Bir,~l~,~ro~luti~~r1. Vol. 40, No. 24
( I YY71 Y3-YY
( 1997 Elnevier Science Limited All rights reserved. Printed m Great Britain OYh4-8305197 $I 7.00 + 0.00
Simazine Degradation by Immobilized and Suspended Soil Bacterium Diego Martin-Montalvo a, Gerard0 Mengs a, Estrella Ferrer (I, J. Luis Allende’, Ram6n Alonso’ & Margarita Martin a* “Dpt. Bioyuimica y Biologiu MoleculnrIV. Madrid, Spain hiJ/nidad de Fisica Aplicada, F. Veterinaria. Universidad Cotnplutense, 28040 Madrid, Spain “Utlidad de Estadktica, ET% Agrdnomos. UPM Madrid, Spain
Simazine is one of the most heavily used herbicides for weed control in the production of a variety of agricultural crops. Few microorganisms have been isolated that metabolize S-triazines such as simazine at rates that are suitable for environmental remediation. DSZI strain cells were immobilized by adsorption onto ceramic supports. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended cells. The effect of substrate concentration and inoculum size/support ratio on kinetic parameters was investigated. Physiological status of immobilized cells was assessed by measuring their capacity for degrading the herbicide and this capacity was compared to that of free-living cells under different experimental conditions. La simazina es uno de 10s herbicidas mis utilizados en una amplia variedad de cultivos agricolas. Hasta la fecha, son pocos 10s microorganismos aislados que tienen capacidad para metabolizar triazinas coma la simazina, y que por tanto puedan ser utilizados en sistemas de descontaminacion medioambiental. La estirpe DSZI ha sido inmovilizada sobre un soporte ceramico. Se han estimado 10s parimetros cineticos de cultivos de celulas en suspension e inmovilizadas. utilizando metodos no lineales de estimation. Se ha estudiado el efecto de la concentration de sustrato y de1 tamaiio de inoculo sobre 10s parametros cineticos. La capacidad de las celulas de esta estirpe para degradar el herbicida en diferentes condiciones ha confirmado el mantenimiento de su viabilidad una vez inmovilizadas. c 1998 Elsevier Science Ltd. All rights reserved
The detection of simazine in groundwater and soils has prompted environmental concern about pollution. Few microorganisms have been isolated that metabolize alkylated S-triazines such as simazine (Mulbry, 1994) at rates that are suitable for environmental remediation. Cook and Hutter (1984) isolated and characterized a strain of Rhodococcus corallinus that dechlorinates and some alkylated S-triazines. The deaminates isolated strain shows a hydrolase activity that is involved in degradation, and Mulbry (1994) reported the induction effect on triazine hydrolase activity of different nitrogen sources; cyanuric acid is one of them, which is the dechlorinated and deaminated product of simazine or atrazine. The hydrolase activity is induced by cyanuric acid but cells cannot be grown on this compound as a carbon source. Our laboratory is involved in the of bacterial activities for characterization remediating contaminated soils and water. We
INTRODUCTION The S-triazine herbicides have been used in a variety of weed control programs with major crops, but herbicides containing an S-triazine ring are relatively persistent in the environment. This group of herbicides, such as simazine and are included in the EU Priority atrazine, Pollutants List, which coincidentally contains the of compounds as the US same number Environmental Protection Agency’s list (132). The use of microorganisms (Martin el al., 1995: Laine and Jorgensen, 1996) for the bioremediation of sites contaminated with toxic pollutants has become the focus of considerable attention because of the potential for restoring soils at a with saving compared considerable cost technologies such as incineration. *To whom correspondence should be addressed 1-3943823. E-mail: ralonso@ ccupm.upm.es
at: Tel.: + 34-
93
94
D. Martin-Montalvo
have recently isolated a bacterial strain DSZl capable of growing on simazine as a sole carbon and energy source. This strain was isolated from a contaminated soil by enrichment culture. The main aim of this study was to perform a bioremediation system, using the isolated strain DSZl, taking into account its kinetic parameters for growth rate and substrate uptake, when these cells are used in suspension or immobilized. The problems showed by continuous fermentation processes could be solved by the use of immobilized cells as biocatalysts (Ferrer et al., 1996). A methodology is described for cell immobilization and the physiological status of immobilized cells was assessed by measuring cell growth and substrate utilization compared to that of free-living cells.
MATERIALS AND METHODS Isolation of bacteria
et al.
then inoculated with DSZl strain (lo6 cells); plate counts were taken from dilutions of the inoculum and the initial substrate concentrations were measured immediately after inoculation. Cells were grown at 30°C to log phase. The bacterial population was examined by plating and light microscopy to ascertain culture purity throughout the experimental conditions. Data analysis The parameters of the logistic curve fitted to the substrate utilization data were estimated using NLIN, the nonlinear parameter estimation procedure of the statistical software SAS@. Kinetic parameters of the DSZl strain, suspended or immobilized, growing on SZ were estimated according to Robinson and Tiedje (1983): 4 = dS/dt X-‘( specific substrate uptake rate) /L= dX/dt X-’ (specific growth rate) q(day-‘)
The bacterial strain capable of growing on Simazine (SZ) as sole source of carbon and energy, DSZl, used in this work, was isolated from a contaminated soil by enrichment culture. This soil is located in an agricultural site, and has a history of exposure to a wide variety of xenobiotics, including SZ. Different soil samples (5 g), were incubated in a mineral medium PJC (Hareland et al., 1975) at 30°C and SZ was added at a concentration of 10ppm. After 25 days, aliquots of the enrichment cultures were plated on agar plates containing 5ppm simazine as carbon source; the resulting isolates were plated on agar-LB plates (Miller, 1972) and tested for purity. By this technique, a pure culture designed strain DSZl was obtained, which showed the capability of growing on the herbicide. Media and culture conditions The isolated bacterium was grown aerobically in PJC medium, under the conditions mentioned above. Carbon sources were sterilized separately and added to give: 2-20ppm simazine, 1 mM glucose, 2-20 ppm cyanuric acid. Biodegradation experiments were performed using PJC as mineral minimal media, and Simazine (SZ) was added as the sole carbon substrate at 2-20ppm final concentration. It was
and
p(day-‘)
are both given by
Monod’s equation. Analytical methods Simazine concentration was analysed by highpressure liquid chromatography (HPLC). Aqueous samples from the cultures were filtered and 10-25~1 aliquots were injected onto a Novapack C-18 (3.9x150mm) column, using a mobile phase consisting of 40% acetonitrile in water at a flow rate of 0.5 ml min-’ and measuring at 220nm. Analyses were performed by using a Waters model 616PDA996, equipped with a data analysis Millennium 20/ 10. Cell immobilization A ceramic material, sepiolite, was chosen as support. This ceramic material was cut into 1 cm sided cubes (monoliths), placed into PJC medium and autoclaved. After 24 h, the mineral medium was replaced by a new sterile medium. Cells were harvested at the exponential phase of growth, using SZ as carbon source. These cells were used as the inoculum. Immobilized culture cells were examined by electron microscopy. Cells were fixed with 1% glutaraldehyde solution and washed three times with 50mM cacodylate buffer, pH 6.8. The
Simazine degradation by immobilized and suspended soil bacterium
samples were coated with colloidal gold examined by scanning electron microscopy.
and
Chemicals
Simazine was obtained from Ciba-Geigy (Barcelona, Spain). Cyanuric acid was purchased from Sigma Chemical Co. (St Louis, Missouri). All the chemical compounds were of the highest purity commercially available.
95
substrate concentration and the absence of growth at 20ppm SZ. To determine the effect of other carbon sources on the metabolism of SZ, glucose (2OOpg ml-‘) was also added to the medium. The addition of glucose shortened the lag phase (data not shown), however, the biomass formation was not modified. Cyanuric acid utilization as a carbon source by the DSZl strain was tested by using PJC medium and different concentrations of this substrate (220 ppm). Cells grew with 5 ppm cyanuric acid, but the formation of biomass was lower.
RESULTS Suspension culture experiments Isolation of DSZl
strain and growth on simazine
A variety of soil samples suspended in mineral medium were screened for possible SZ degradation. Two millilitre of the suspensions were dispensed into 50ml culture flasks, adding SZ (IOppm), and incubated at 30°C. Transfer of the enrichment culture consistently showed that there was a 10% loss of SZ within 20 days. Control samples containing autoclaved soil displayed no significant SZ degradation. Simazine utilization as carbon source by DSZl strain was tested by adding the substrate to media at different concentrations: 2-20ppm. Figure 1 shows the bacterial growth, using different SZ The growth of DSZl in the concentrations. presence of 2, 5 and 10 ppm SZ started after a lag period and resulted in the disappearance of the substrate and the formation of biomass. Figure 1 shows a short lag phase (1 day) at the lower
Figure 2 shows the depletion of SZ in batch using Sppm cultures, SZ. When the SZ concentration in the medium was 20ppm, no formation of biomass could be detected, and the amount of substrate in the culture remained invariable during the incubation time. Table 1 shows the herbicide degradation results obtained at different SZ concentrations and inoculum sizes. The rate of degradation was not dependent on cell density. For this reason, we decided to use IO6cells ml-’ as inocula for further experiments. The optimum rate of SZ degradation as well as biomass formation was achieved when SZ was added at 5ppm concentration (Fig. 2). At these growth conditions, strain DSZl degrades 70% of the initial SZ in 19 days; biomass formation was consistent with this result, and the maximum ODeoO was obtained. The growth curve obtained when DSZl uses 5 ppm SZ as the carbon
Bacterial growth, using SIMAZINE -2
ppm +5 ppm *lO
as sole carbon source: ppm *20
ppm
time (days) Fig. 1.
Cell growth
of DSZl strain in batch cultures, using different simazine concentrations as the sole carbon expressed as percentages relative to corresponding average values in sterile controls.
source. Data are
D. Martin-Montalvo
96 DSZl
et
al.
Strain
80
0
5
10
15
20
0
t Pays1
5
20
15
10 concentration (ppm)
Fig. 2. Cell growth of DSZl strain using 5ppm simazine in batch cultures and resultant depletion of simazine. Experimental and fitted curves.
Fig. 3. Effect of substrate concentration parameters. l/X dS/dt was calculated as substrate utilization rate.
source (Fig. 2) has been fitted and kinetic parameters (Table 2) have been determined according to the Monod model (Robinson and Tiedje, 1983). Figure 3 shows the effect of substrate growth concentration on the specific substrate utilization rate. The optimum growth conditions of DSZ strain are achieved at 5ppm SZ concentration, associated with a degradation rate of 70%. Although this culture was grown in the constant presence of SZ prior to this experiment, a lag phase of six days was observed after 5ppm SZ exposure. However, the culture rapidly reached the log phase and its maximum biomass production. An important result is that
DSZl can grow at low SZ concentrations (2ppm), because often herbicides are present in soils and waters at very low concentrations.
Table 1. Herbicide Inoculum
Initial
Immobilized cells experiments The ceramic material used in these experiments has the structure of a honeycomb support and its characteristics have been reported previously (Martin er al., 1995). When autoclaved monoliths were added under aseptic conditions to an exponentially growing culture of DSZl strain on 5 ppm SZ, cells were quickly adsorbed onto the surface of the monoliths without losing their
Degradation
Herbicide
size (cells ml-‘)
concentration
by the Strain DSZl” % Removed
(ppm) Final
6x lo5
2 5 10 20
0.99 1.60 8.89 18.2
51.5 68 11 9
IO6
2 5 10 20
0.88 1.42 8.65 18.45
66 71.6 13.5 1.1
10’
2 5 10 20
0.95 1.56 8.69 18.56
52.5 68.8 13.1 7.2
“Results
are given as mean of triplicate
measurements,
on kinetic the specific
after 20 days of incubation.
Simazine degradation by immobilized and suspended soil bacterium Table 2. Kinetic
K (ppm cell h-‘)
td (d) ~Lmax(d-l) & (ppm) Yield (cell/ppm)
Parameters of DSZI Simazine
Strain
Growing
Suspension culture
Immobilized culture
0.14x lo8 5 0.6 3.5 0.4x IOR
0.18x lo8 3.6 1.2 4.3 1.6x lo9
on
K: maximum specific substrate utilization rate; td: cell doubling time; p,,,: maximum specific growth rate; Kd:substrate utilization associated half velocity.
viability. The natural tendency of these microorganisms to adhere to solid surfaces can be observed in the progressive colonization of the longitudinal square channels of the monoliths. The size of the inoculum (5 x lo6 cells g- ’ support) initially used could achieve complete loading of the monolith; Table 3 shows that a cell loading to 300mg biomass g-’ of support was measured, at these inocula size conditions. The kinetic rate constants for immobiilized cell growth and Sppm SZ utilization are summarized in Table 2. Some differences were observed in growth and substrate utilization parameters between immobilized and suspended cells, and a 40-fold increase in the yield (Table 2) is observed at the same experimental conditions. The capacity of the immobilized cells for degrading SZ, keeping their viability, has been calculated by transferring the monoliths loaded with the formed biomass to aqueous solutions containing SZ at two different concentrations; 2 and Sppm. In both experiments, for over 50 days the immobilized cells kept their viability, with a degradation rate near to 70%. In this case, the final amount of substrate degraded was not dependent on the initial substrate concentration used in the experiment. Maintenance of the activity of the immobilized cells over several days may suggest that cell attachment does not negatively affect simazine biodegradation; nevertheless, changes in physiology or morphology of different immobilized cells have been reported (Ghommidh et al., 1982; Sheve and Vogel, 1993). In this way, scanning electron micrographs were taken of immobilized (Fig. 4) DSZl cells, using SZ as substrate. The cell size characteristics were determined from this and additional electron micrographs (Table 3). Examination of the electron micrographs showed no apparent morphological changes upon immobilization.
97
DISCUSSION
Due to the excessive utilization of pesticides, especially in areas of intensive agriculture, many sources of water as well as soils contain high levels of these synthetic compounds. The ability of indigenous strains of soil bacteria to degrade a wide variety of xenobiotic compounds is well documented. When these compounds are used as growth substrates, their presence defines a unique niche which can be occupied by microorganisms possessing the requisite metabolic, physiological and kinetic characteristics (Greer et al., 1992). This report describes the isolation of a soil bacterium which has the ability to degrade simazine. Microorganisms in other environments (Cook and Hutter, 1986; Mulbry, 1994) or in isolated cultures (Mandelbaum et al., 1993) have been reported to metabolize or cometabolize simazine, but ring cleavage is rarely reported, and other carbon sources such as citrate or sucrose were added. As reported above, DSZl is solely responsible for metabolism of the herbicide and can use it as a sole source of carbon and energy. Cyanuric acid can be used by strain DSZl as substrate, only at Sppm concentration, with a low formation of biomass. This result suggests that this compound is an intermediate in the Simazine degradative pathway, but alternative experiments have been carried out to design the catabolic pathway. Mulbry (1994) and Cook and Hutter (1984) reported that the Rhodococcus sp. hydrolase activity which dechlorinated and deaminated S-triazines could be induced by cyanuric acid, but this intermediate as well as others assayed in the induction experiments could not be used by the bacterium as the carbon source. The capacity of DSZl strain has several implications for the fate of S-triazine herbicides in the environment. A general problem with these molecules is that the ring portion persists in the environment. In our system, simazine is metabolized by the DSZl strain, nevertheless we are currently attempting to address this problem mineralization occurs by using [14C] simazine. When the DSZl strain is immobilized onto sepiolite monoliths, cells maintain their viability and capacity to degrade simazine. After the initial adsorption of DSZI cells onto the surface of the monolith they colonize the support very efficiently, showing similar substrate utilization to that showed for suspended cells. It is appropriate to underline the greater capacity of the
D. Martin-Montaho
98
Fig. 4.
Electron
micrographs
of DSZI cells immobilized
immobilized cells for degrading SZ (more than 90 days) compared to the capacity of the suspended cells (15 days). Morphological and physiological Table 3. Characteristics
of Immobilized
Biomass loading” Biomass retainment Physiological status
Operational
DSZl
Cells 300 High
width of cell length of cell
No change 0.57pm I .275 pm
dead cells growing cells
< 6% > 60%
stability
‘Based on dry w/g-’
of support.
et al.
onto sepiolite
monoliths.
using simazine
as substrate.
changes caused by the immobilization process (Klein and Ziehr, 1990; Sheve and Vogel, 1993; Willaert et al., 1996) mean a problem when this system is applied to biotechnological processes. The results of this work show that biodegradation performance (Table 2) obtained when the DSZl strain is immobilized in the selected conditions is slightly higher than that obtained for suspended cells. From this analysis, the maximum SZ utilization did not change upon rate immobilization and the maximum specific growth rate is two-fold higher for the immobilized cells. These results are not surprising if it is taken into account that the DSZ strain has been isolated from
a soil intensively treated with simazine. The intensive use of this herbicide and its relative persistence led to instances in which microorganisms able to use it as carbon source are selected. With a continuous supply of substrate, there is obviously selective pressure to become immobilized in the soil structure. The ceramic material used as a support improves the process implementation. Therefore, the optimization of culture before design of conditions, the the bioremediation process, must be a prior objective; once the biodegradation performance is defined, the conditions of the working treatment can be tried out.
Propachlor suspended
and alachlor
degradation
by immobilized
and
Pseudomonas cells. In Immohiked cells: Basics attti Applicutiotts. Elsevier Science, B.V., p. 762.
Ghommidh. C.. Navarro. for the immobilization Bioettgitteer-ing
J. M. & Durnad. G. (1982) Methods of microbial cells. Biotechnology and
24, 605-6
17.
Greer. L. E., Robinson. J. A. & Shelton. D. R. (1992) Kinetic comparison of seven strain of 2.4-dichlorophenoxyacetic acid degrading bacteria. Applied Ettvironmetztal MicrohiolO,qA’58, 1027-1030. Hareland. W. A.. Crawford. R. L., Chapman. P. J. & Dagley, S. (1975) Metabolic function and properties of 4-hydroxyphenylacetic acid I-hydroxylase from P.~&~nzonas Jourtd
u&hot-ms.
of Bacteriolqy
121, 379-185.
Klein, J. & Ziehr, H. (1990) Immobilization of microbial cells by adsorption. Jourttul of’ Biorechnnl~~g~~ 16, l-1 6. Laine. M. N. & Jorgensen, K. S. (1996) Straw compost and bioremediated soil as inocula for the bioremediation of chlorophenol-contaminated soil. Applied Ettvirwttttet~tuI Microhiolo~~~~ 62, I507-
I 513.
Mandelbaum. R. T.. Wackett, L. P. & Allan, D. T. (1993) Mineralization of the s-triazine ring of atrazine by stable bacteria1 mixed cultures. ,4pplied Ett~~irottmrtttal Microhiol-
ACKNOWLEDGEMENTS
og>’ 59, I695-
The CICYT Grant AMB94-0483, and the Comunidad Autbnoma de Madrid Grant COR0008/94, provided funding for this work. The authors would like to express their appreciation to Ciba-Geigy for providing samples of Simazine.
170I
Martin. M.. Ferrer, E.. Alonso, R. & Fernlindez. J. ( 1995) Bioremediation of soil contaminated by propachlor using native bacteria. internatiottal Biodeterioration and Biociegrofiufiott
35, 2 13-335.
Miller, J. M. ( 1972) E-1.peritnettts in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Mulbry. W. W. (1994) Purification and characterization of an inducible s-triazine hydrolase from Ritorlococcus corallinus NRRL B- 15444R. .4pplied Ettvirottmetttctl Microbiology 60, 613-618.
Robinson. J. A. & Tiedje. J. M. (1983) Nonlinear estimation of monod growth kinetic parameters from a single substrate depletion curve. Applied Ett~~irottttletttal Microhiolog~~ 45(5),
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