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Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans
Chemosphere 62 (2006) 3–8 www.elsevier.com/locate/chemosphere
Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans Przemysław Bernat, Jerzy Długon´ski
*
Department of Industrial Microbiology and Biotechnology, University of Ło´dz´, Banacha 12/16, PL 90-237 Ło´dz´, Poland Received 4 October 2004; received in revised form 4 April 2005; accepted 15 April 2005 Available online 15 June 2005
Abstract In this study, we have examined the effects of synthetic medium ingredients and culture incubation conditions on growth and tributyltin chloride (TBT) degradation activity of the fungus Cunninghamella elegans. The best efficiency of TBT conversion to less toxic derivatives: dibutyltin and monobutyltin was noticed on media which contained glucose, NH4Cl, K2HPO4 and MgSO4. Next, the constructed M3 medium (with the above components) ensured vigorous growth of C. elegans and allowed the reduction of 80% of the initial TBT content (10 mg l 1), after 3 d of biodegradation. The further acceleration of the biocide utilization by C. elegans was achieved by additional oxygen supply (pO2 P 20%) to the growing fungus (89% after 2 d of incubation in the BioFlo II bioreactor). The efficient xenobiotic biodegradation was related to the intensity of fungal growth. The obtained results suggest a cometabolic nature of TBT utilization by C. elegans. 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Cometabolism; Cunninghamella elegans; Tributyltin
1. Introduction Butyltin compounds have been widely used as stabilizers for chlorinated polymers, catalysts for a variety of chemical reactions, and biocides for boat paints (White et al., 1999; Hoch, 2001). Of particular importance to the environment is the high toxicity of tributyltin chloride (TBT). Present knowledge indicates that TBT is among the most toxic compounds known for aquatic ecosystems (Hoch, 2001). Several in vivo studies have
* Corresponding author. Tel.: +48 42 6354460; fax: +48 42 6655818. E-mail address: [email protected] (J. Długon´ski).
shown that low concentrations of TBT (0.1–0.5 lg l 1) are immunotoxic, neurotoxic and hepatotoxic (Zaucke et al., 1998). The European Union has decided to specifically include TBT compounds in its list of priority compounds in water in order to control its fate in natural systems (Behra et al., 2003). Additionally, International Maritime Organization bans the application of tin biocides as anti-fouling agents on ships after January 1, 2003 and prohibits the presence of tin biocides as antifouling agents after January 1, 2008. It has been estimated that between 70% and 80% of the 28 038 ships in global commerce use TBT (Champ, 2003). Cleaning ships with a TBT-painted bottom generates large volumes of TBT-contaminated wastewater. Nowadays there is a great danger that more TBT would be released
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.04.045
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
4 Table 1 TBT degradation by microorganisms Microorganisms
Initial TBT content
Coniophora puteana Pseudomonas diminuta Chlorella vulgaris Cunninghamella elegans
2.5 mg l 8 lg l 1 100 lg l 10 mg l
1
1 1
to ports and harbours in the five-year compliance period than has been released from ships in the past 40 years to the same waters. This would be devastating to the marine environment. However, restrictions of TBT uses did not largely diminish its consumption, as a change in applications from anti-fouling paints to wood and variety of other materials (e.g. PVC) preservations seems to occur at present. Nowadays, some recent studies have also shown the presence of TBT in sewage sludges as well as in soils and ground waters (Behra et al., 2003). Much of evidence suggests that biotic degradation is the major pathway for the removal of TBT from the environment (Gadd, 2000). Microorganisms such as microalgae, bacteria, cyanobacteria are responsible for biocide removal. Nevertheless, only a limited number of these investigations have involved fungi (Table 1). Previously we revealed that microscopic fungus Cunninghamella elegans IM 1785/21Gp was able to degrade 70% of TBT (added at 10 mg l 1), to less toxic derivatives dibutyltin (DBT) and monobutyltin (MBT) during 7 d of growth in Sabouraud medium. It was also indicated that C. elegans used a cytochrome P-450 system for TBT debutylation (Bernat and Długon´ski, 2002). In the attempt to speed up and increase the bioconversion of TBT, in this paper we optimise TBT degradation by examining how selected culture parameters: synthetic medium ingredients and incubation conditions, influence the xenobiotic removal by C. elegans.
Degradation (%)
Literature
40 90 78 70
White et al. (1999) Kawai et al. (1998) Tsang et al. (1999) Bernat and Długon´ski (2002)
2. Materials and methods 2.1. Chemicals TBT, dibutyltin dichloride (DBT), monobutyltin trichloride (MBT) were purchased from Aldrich. Tetrabutyltin (TTBT) was obtained from Supelco. Other chemicals were from J.T. Baker, Fluka and POCh (Gliwice, Poland). All the chemicals were high purity grade reagents. Stock solutions of TBT were prepared at 10 mg ml 1 ethanol. 2.2. Microorganism C. elegans (Lender) IM 1785/21Gp from the Department of Industrial Microbiology and Biotechnology fungal strain collection was the subject of our work. The features of this strain had been described in our earlier paper (Bernat and Długon´ski, 2002). 2.3. Growth conditions and TBT utilization Spores originating from 14-d old cultures on ZT slants were used to inoculate 20 ml Sabouraud medium (Difco) (in 100 ml Erlenmeyer flasks). The cultivation was carried out at 28 C on rotary shaker (200 rpm) for 24 h. The precultures were transferred to fresh medium (in the ratio 1:9) and incubated for the next 24 h. Next, mycelium was filtered and moved to the same amount of selected synthetic medium (Table 2). Three
Table 2 Composition of applied synthetic media Ingredients
Content (g l 1)
Medium (number) 1
2
3
4
5
6
7
K2HPO4 KH2PO4 NH4Cl MgSO4 Æ 7H2O MnSO4 FeSO4 Æ 7H2O CaCl2 Æ 2H2O KCl (NH4)2HPO4 Glucose Distil water
4.36 1.7 2.1 0.2 0.05 0.01 0.03 5.55 5.55 40 up to 1000 ml
+ + + + + + +
+ +
+ + + + +
+
+ +
+ + + + + +
+ + +
+ + + +
+ + + +
+
+ +
+ +
+ +
+ +
+ +
8
+ + +
+ + + + +
+ + + + + +
+ +
+ +
+ +
9
+ + + + + + + +
10
11
+ + + +
+ + + + + + +
+ + +
+
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
milliliter of the homogenous preculture was introduced into 17 ml of medium with TBT (10 mg l 1) or without the xenobiotic in the appreciate control cultures. The initial pH was 7. The cultures were incubated for 7 d. Fungal mycelia were separated from culture media by filtrating through Sartourius filter (0.25 lm) and then dried at 105 C to reach a constant weight. 2.4. The batch experiment The fungal preculture (150 ml) (obtained as described above) was introduced into 850 ml of M3 medium with the following ingredients: glucose (40 g), NH4Cl (2.1 g), K2HPO4 (5.55 g), MgSO4 Æ 7H2O (0.2 g) and distilled water (up to 1 l) and with TBT (10 mg l 1). The biodegradation was performed in stirred tank bioreactor (Bioflo II, New Brunswick, USA) for 3 d, with controlled aeration, agitation, temperature and level of dissolved oxygen (pO2). The following experimental conditions were maintained in bioreactor: constant flow
5
1 l air min 1, 400 rpm, 28 C, pO2 P 20%, the initial pH of the medium was 7. 2.5. The organotin analysis The organotin contents in fungal cultures were determined according to the procedure of Binato et al. (1998) with modifications. The sample was homogenised (MISONIX) with acetone (20 ml) and concentrated hydrochloric acid (pH 2–3), and then centrifuged at 500g for 10 min. Internal standard TTBT (100 lg) was added to the supernatant and the sample was extracted twice with tropolone/hexane (3:10 000, w/v). The extracts were dried over anhydrous sodium sulphate and evaporated. Two ml of hexane were added and the organotins in the extracts were methylated with 500 ll of methyl magnesium bromide. The reaction was quenched with ammonium chloride (2:10, w/v). Methylated organotins were determined by gas chromatography (a Hewlett-Packard Model 6890) equipped with a 5973 Mass
Fig. 1. (A) TBT degradation by C. elegans in the synthetic media with TBT (10 mg l 1). TBT (j), DBT (h), MBT ( ). (B) Dry weight of mycelium of C. elegans on synthetic medium after 7 d incubation with TBT (10 mg l 1) (j) control (h). (1) A synthetic basic growth medium, (2–11) a synthetic growth medium without: (2) NH4Cl, (3) MnSO4, (4) FeSO4, (5) CaCl2, (6) MgSO4 Æ 7H2O, (7) KH2PO4, (8) K2HPO4, (9) phosphorus, (10) potassium, (11) glucose. Results are mean values of three experiments with standard deviations (SD).
6
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
Detector. The separation was carried out in a capillary column HP 5 MS methyl polysiloxane (30 m · 0.25 mm id · 0.25 lm ft). The column temperature was maintained at 60 C for 4.5 min, then increased to 280 C at 20 C min 1. Finally, the column temperature was maintained at 280 C for 3 min. Helium was used as the carrier gas at flow rate of 1.2 ml min 1. Injection port temperature was 250 C. Split injection (2 ll) was employed. The individual butyltin species could be distinctly separated and they were identified on the basis of their retention times and characteristic ions. Quantitative analyses were performed on single ions at m/z 165 for MBT, 151 for DBT, 193 for TBT and 179 for TTBT (int. std.).
Fig. 2. Degradation of TBT (A) and growth curve (B) on synthetic M3 medium by C. elegans (initial TBT concentration 10 mg l 1). (A) TBT (), DBT (j), MBT (m); (B) dry weight (·) and pH (s). Results are mean values of three experiments with standard deviations (SD).
3. Results The TBT degradation activity of C. elegans on a basic synthetic growth medium (Lobos et al., 1992) (Table 2, medium 1) and next, on media without particular ingredients was examined (Table 2, medium 11). The best yields of TBT depletion were observed in the culture media (Erlenmayer flask experiments) which included glucose, NH4Cl, K2HPO4 and MgSO4 (Fig. 1A, media 1, 3, 4, 5). Taking into account the obtained results, the synthetic growth M3 medium was constructed. The culture inspection showed that C. elegans grew vigorously in M3 synthetic medium and reduced 80% of initial TBT content (10 mg l 1) within 3 d of shake flasks incubation (Fig. 2). At the end of the experiment about 95% of TBT was eliminated in the contaminated medium. The major amount of TBT was converted to xenobiotic derivatives between 1 and 3 d of C. elegans incubation. Additionally, the most vigorous growth of the fungus combined with the medium acidification, was observed in the same period of culture. The growth of aerobic microorganisms depends on dissolved oxygen (pO2) in the culture medium. Oxygen could positively influence the product kinetics with increasing values by acting as a direct parameter of product formation (Kapat et al., 2001). In our earlier studies (data not published) it was observed that the minimal pO2 level which limited C. elegans growth is 20%. Therefore, in the next step of our investigation the xenobiotic biodegradation was performed in the stirred tank bioreactor (Bioflo II, New Brunswick, USA) with controlled aeration, agitation, temperature and level of dissolved oxygen (pO2 P 20%). The results of the experiment presented in Fig. 3 showed that the
Fig. 3. Degradation of TBT on M3 medium by C. elegans in a bioreactor (initial TBT concentration 10 mg l 1) TBT (), DBT (j), MBT (m), dry mass (s), pO2 (- - -). The sample was taken three times.
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
growth of C. elegans and degradation of TBT were significantly enhanced. Additionally, after 2 d of incubation, 89% of the initial amount of the xenobiotic was converted to TBT derivatives and the highest increase in the fungus biomass was noticed on the second day of the culturing.
4. Discussion As shown in our previous investigation (Bernat and Długon´ski, 2002), C. elegans degraded TBT when grown in Sabouraud liquid medium. However, the Sabouraud nutrient is rather inconvenient for the study of the optimisation of the xenobiotic biodegradation. Therefore, in the present paper a synthetic growth medium, which allowed checking an effect of a particular nutrients ingredients on TBT removal by the fungus, was applied. It was found that the presence of the source of carbon, nitrogen, sulphur, phosphorus and magnesium in the growth medium is necessary for the enhancement of TBT degradation activity of C. elegans. Vroumsia et al. (1999), who investigated the role of glucose and nitrogen on 2,4-dichlorophenoxyacetic acid (2,4-D) degradation (100 mg l 1) by C. elegans, found also a strong influence of these two elements on the xenobiotic biodegradation. Singh and Joshi (1991) revealed that phosphate ions were needed for the bioaccumulation of TBT into Escherichia coli cells. The important role played by magnesium in the growth and metabolism of microorganisms was described by Walker (1994). We also found that potassium was one of the crucial nutrients, which played a positive role in TBT biodegradation. Although the growth of C. elegans in this medium without the xenobiotic was observed (Fig. 1B), a significant reduction of TBT degradation efficiency in culture medium without that ion was noticed. It is very interesting, because TBT disturbs cell membranes integrity. The release of K+ from cells, arising from increased cytoplasmic membrane permeability of yeasts Debaromyces hansenii and Candida maltosa, was used to monitor organotin toxicity, implicating the cytoplasmic membrane as the site of action (Cooney et al., 1989; Laurence et al., 1989; Tobin and Cooney, 1999). It was also observed, that media with lower (6–10) and higher (1, 3–5) buffering capacity presented higher biomass level after 7 days of culturing without TBT (Fig. 1B). Among low buffering capacity media supplemented with the xenobiotic, only medium 7 had biomass >3 g l 1, while media 1, 3–5 had biomass >3 g l 1. The importance of the above elements was confirmed by TBT biodegradation in M3 medium (Fig. 2). The yield of the xenobiotic elimination was significantly higher than the one observed earlier (Bernat and Długon´ski, 2002) on Sabouraud medium (95% and 70% respectively after 7 days incubation). Additionally,
7
gradual culture acidification and followed by very low pH value of the M3 medium at the end of the experiment was observed (Fig. 2). Probably ammonia was taken up in the form of NH3 by specific transport system and the process must have been completed with excretion of cations (Nielsen, 1992). Our results indicate that the lack of nutrients results in the cell growth limitation and the TBT debutylation by the fungus. It seems to suggest that biocide degradation by C. elegans is a cometabolic phenomenon. The nongrowth (secondary) substrate—TBT, can only be transformed in the presence of a growth (primary) substrate—glucose. Our observations are in agreement with the studies of other authors. Kawai et al. (1998) found that debutylation of TBT by Pseudomonas diminuta was enhanced by the supplement of organic nutrient broth into experiment medium. Also, degradation of TBT by algae (Tsang et al., 1999; Tam et al., 2002) was present in a rich growth medium. The acceleration of C. elegans growth by additional oxygen supply (pO2 P 20%) (Fig. 3) stimulated TBT elimination from the M3 medium (89% after 2 d of incubation in the bioreactor). The efficient xenobiotic biodegradation was related to the intensive fungal growth (Fig. 3) which confirms our suggestion that TBT elimination by C. elegans has a cometabolic nature.
References Behra, P., Lecarme-Theobald, E., Bueno, M., Ehrhardt, J.J., 2003. Sorption of tributyltin onto a natural quartz sand. J. Colloid Interf. Sci. 263, 4–12. Bernat, P., Długon´ski, J., 2002. Degradation of tributyltin by the filamentous fungus Cunninghamella elegans, with involvement of cytochrome P-450. Biotech. Lett. 24, 1971– 1974. Binato, G., Biancoott, G., Piro, R., Angeletti, R., 1998. Atomic absorption spectrometric screening and gas chromatographic-mass spectrometric determination of organotin compounds in marine mussels: an application in samples from Venetian Lagoon. Fresen. J. Anal. Chem. 361, 333– 337. Champ, M.A., 2003. Economic and environmental impacts on ports and harbors from the convention to ban harmful marine anti-fouling systems. Mar. Pollut. Bull. 46, 935–940. Cooney, J.J., de Rome, L., Laurence, O., Gadd, G.M., 1989. Effects of organotin and organolead compounds on yeast. J. Ind. Microbiol. 4, 279–288. Gadd, G.M., 2000. Microbial interactions with tributyltin compounds: detoxification, accumulation, and environmental fate. Sci. Total Environ. 258, 119–127. Hoch, M., 2001. Organotin compounds in the environment—an overview. Appl. Geochem. 16, 719–743. Kapat, A., Jung, J.K., Park, Y.H., 2001. Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition. J. Appl. Microbiol. 90, 216–222.
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Kawai, S., Kurokawa, Y., Harino, H., Fukushima, M., 1998. Degradation of tributyltin by a bacterial strain isolated from polluted river water. Environ. Pollut. 102, 259– 263. Laurence, O.S., Cooney, J.J., Gadd, G.M., 1989. Toxicity of organotins towards the marine yeast Debareyomyces hansenii. Microb. Ecol. 17, 275–285. Lobos, J.H., Leib, T.K., Su, T.M., 1992. Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium. Appl. Environ. Microbiol. 58, 1823– 1831. Nielsen, J., 1992. Modelling the growth of filamentous fungi. Adv. Biochem. Eng. Biotechnol. 46, 187–223. Singh, K., Joshi, L., 1991. Analysis of organotin uptake in Escherichia coli K-12. Ecotoxicol. Environ. Safety 21, 235– 239. Tam, N.F., Chong, A.M., Wong, Y.S., 2002. Removal of tributyltin (TBT) by live and dead microalgal cells. Mar. Pollut. Bull. 45, 362–371.
Tobin, J.M., Cooney, J.J., 1999. Action of inorganic tin and organotins on a hydrocarbon-using yeast, Candida maltosa. Arch. Environ. Contam. Toxicol. 36, 7–12. Tsang, C.K., Lau, P.S., Tam, N.F.Y., Wong, Y.S., 1999. Biodegradation capacity of tributyltin by two Chlorella species. Environ. Pollut. 105, 289–297. Vroumsia, T., Steiman, R., Seigle-Murandi, F., Benoit-Guyod, J.L., 1999. Effects of culture parameters on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) by selected fungi. Chemosphere 39, 1397– 1405. Walker, G.M., 1994. The roles of magnesium in biotechnology. Crit. Rev. Biotechnol. 14, 311–354. White, J.S., Tobin, J.M., Cooney, J.J., 1999. Organotin compounds and their interactions with microorganisms. Can. J. Microbiol. 45, 541–554. Zaucke, F., Zo¨ltzer, H., Krug, H.F., 1998. Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds. Fresen. J. Anal. Chem. 361, 386–392.
Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans Przemysław Bernat, Jerzy Długon´ski
*
Department of Industrial Microbiology and Biotechnology, University of Ło´dz´, Banacha 12/16, PL 90-237 Ło´dz´, Poland Received 4 October 2004; received in revised form 4 April 2005; accepted 15 April 2005 Available online 15 June 2005
Abstract In this study, we have examined the effects of synthetic medium ingredients and culture incubation conditions on growth and tributyltin chloride (TBT) degradation activity of the fungus Cunninghamella elegans. The best efficiency of TBT conversion to less toxic derivatives: dibutyltin and monobutyltin was noticed on media which contained glucose, NH4Cl, K2HPO4 and MgSO4. Next, the constructed M3 medium (with the above components) ensured vigorous growth of C. elegans and allowed the reduction of 80% of the initial TBT content (10 mg l 1), after 3 d of biodegradation. The further acceleration of the biocide utilization by C. elegans was achieved by additional oxygen supply (pO2 P 20%) to the growing fungus (89% after 2 d of incubation in the BioFlo II bioreactor). The efficient xenobiotic biodegradation was related to the intensity of fungal growth. The obtained results suggest a cometabolic nature of TBT utilization by C. elegans. 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Cometabolism; Cunninghamella elegans; Tributyltin
1. Introduction Butyltin compounds have been widely used as stabilizers for chlorinated polymers, catalysts for a variety of chemical reactions, and biocides for boat paints (White et al., 1999; Hoch, 2001). Of particular importance to the environment is the high toxicity of tributyltin chloride (TBT). Present knowledge indicates that TBT is among the most toxic compounds known for aquatic ecosystems (Hoch, 2001). Several in vivo studies have
* Corresponding author. Tel.: +48 42 6354460; fax: +48 42 6655818. E-mail address: [email protected] (J. Długon´ski).
shown that low concentrations of TBT (0.1–0.5 lg l 1) are immunotoxic, neurotoxic and hepatotoxic (Zaucke et al., 1998). The European Union has decided to specifically include TBT compounds in its list of priority compounds in water in order to control its fate in natural systems (Behra et al., 2003). Additionally, International Maritime Organization bans the application of tin biocides as anti-fouling agents on ships after January 1, 2003 and prohibits the presence of tin biocides as antifouling agents after January 1, 2008. It has been estimated that between 70% and 80% of the 28 038 ships in global commerce use TBT (Champ, 2003). Cleaning ships with a TBT-painted bottom generates large volumes of TBT-contaminated wastewater. Nowadays there is a great danger that more TBT would be released
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.04.045
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
4 Table 1 TBT degradation by microorganisms Microorganisms
Initial TBT content
Coniophora puteana Pseudomonas diminuta Chlorella vulgaris Cunninghamella elegans
2.5 mg l 8 lg l 1 100 lg l 10 mg l
1
1 1
to ports and harbours in the five-year compliance period than has been released from ships in the past 40 years to the same waters. This would be devastating to the marine environment. However, restrictions of TBT uses did not largely diminish its consumption, as a change in applications from anti-fouling paints to wood and variety of other materials (e.g. PVC) preservations seems to occur at present. Nowadays, some recent studies have also shown the presence of TBT in sewage sludges as well as in soils and ground waters (Behra et al., 2003). Much of evidence suggests that biotic degradation is the major pathway for the removal of TBT from the environment (Gadd, 2000). Microorganisms such as microalgae, bacteria, cyanobacteria are responsible for biocide removal. Nevertheless, only a limited number of these investigations have involved fungi (Table 1). Previously we revealed that microscopic fungus Cunninghamella elegans IM 1785/21Gp was able to degrade 70% of TBT (added at 10 mg l 1), to less toxic derivatives dibutyltin (DBT) and monobutyltin (MBT) during 7 d of growth in Sabouraud medium. It was also indicated that C. elegans used a cytochrome P-450 system for TBT debutylation (Bernat and Długon´ski, 2002). In the attempt to speed up and increase the bioconversion of TBT, in this paper we optimise TBT degradation by examining how selected culture parameters: synthetic medium ingredients and incubation conditions, influence the xenobiotic removal by C. elegans.
Degradation (%)
Literature
40 90 78 70
White et al. (1999) Kawai et al. (1998) Tsang et al. (1999) Bernat and Długon´ski (2002)
2. Materials and methods 2.1. Chemicals TBT, dibutyltin dichloride (DBT), monobutyltin trichloride (MBT) were purchased from Aldrich. Tetrabutyltin (TTBT) was obtained from Supelco. Other chemicals were from J.T. Baker, Fluka and POCh (Gliwice, Poland). All the chemicals were high purity grade reagents. Stock solutions of TBT were prepared at 10 mg ml 1 ethanol. 2.2. Microorganism C. elegans (Lender) IM 1785/21Gp from the Department of Industrial Microbiology and Biotechnology fungal strain collection was the subject of our work. The features of this strain had been described in our earlier paper (Bernat and Długon´ski, 2002). 2.3. Growth conditions and TBT utilization Spores originating from 14-d old cultures on ZT slants were used to inoculate 20 ml Sabouraud medium (Difco) (in 100 ml Erlenmeyer flasks). The cultivation was carried out at 28 C on rotary shaker (200 rpm) for 24 h. The precultures were transferred to fresh medium (in the ratio 1:9) and incubated for the next 24 h. Next, mycelium was filtered and moved to the same amount of selected synthetic medium (Table 2). Three
Table 2 Composition of applied synthetic media Ingredients
Content (g l 1)
Medium (number) 1
2
3
4
5
6
7
K2HPO4 KH2PO4 NH4Cl MgSO4 Æ 7H2O MnSO4 FeSO4 Æ 7H2O CaCl2 Æ 2H2O KCl (NH4)2HPO4 Glucose Distil water
4.36 1.7 2.1 0.2 0.05 0.01 0.03 5.55 5.55 40 up to 1000 ml
+ + + + + + +
+ +
+ + + + +
+
+ +
+ + + + + +
+ + +
+ + + +
+ + + +
+
+ +
+ +
+ +
+ +
+ +
8
+ + +
+ + + + +
+ + + + + +
+ +
+ +
+ +
9
+ + + + + + + +
10
11
+ + + +
+ + + + + + +
+ + +
+
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
milliliter of the homogenous preculture was introduced into 17 ml of medium with TBT (10 mg l 1) or without the xenobiotic in the appreciate control cultures. The initial pH was 7. The cultures were incubated for 7 d. Fungal mycelia were separated from culture media by filtrating through Sartourius filter (0.25 lm) and then dried at 105 C to reach a constant weight. 2.4. The batch experiment The fungal preculture (150 ml) (obtained as described above) was introduced into 850 ml of M3 medium with the following ingredients: glucose (40 g), NH4Cl (2.1 g), K2HPO4 (5.55 g), MgSO4 Æ 7H2O (0.2 g) and distilled water (up to 1 l) and with TBT (10 mg l 1). The biodegradation was performed in stirred tank bioreactor (Bioflo II, New Brunswick, USA) for 3 d, with controlled aeration, agitation, temperature and level of dissolved oxygen (pO2). The following experimental conditions were maintained in bioreactor: constant flow
5
1 l air min 1, 400 rpm, 28 C, pO2 P 20%, the initial pH of the medium was 7. 2.5. The organotin analysis The organotin contents in fungal cultures were determined according to the procedure of Binato et al. (1998) with modifications. The sample was homogenised (MISONIX) with acetone (20 ml) and concentrated hydrochloric acid (pH 2–3), and then centrifuged at 500g for 10 min. Internal standard TTBT (100 lg) was added to the supernatant and the sample was extracted twice with tropolone/hexane (3:10 000, w/v). The extracts were dried over anhydrous sodium sulphate and evaporated. Two ml of hexane were added and the organotins in the extracts were methylated with 500 ll of methyl magnesium bromide. The reaction was quenched with ammonium chloride (2:10, w/v). Methylated organotins were determined by gas chromatography (a Hewlett-Packard Model 6890) equipped with a 5973 Mass
Fig. 1. (A) TBT degradation by C. elegans in the synthetic media with TBT (10 mg l 1). TBT (j), DBT (h), MBT ( ). (B) Dry weight of mycelium of C. elegans on synthetic medium after 7 d incubation with TBT (10 mg l 1) (j) control (h). (1) A synthetic basic growth medium, (2–11) a synthetic growth medium without: (2) NH4Cl, (3) MnSO4, (4) FeSO4, (5) CaCl2, (6) MgSO4 Æ 7H2O, (7) KH2PO4, (8) K2HPO4, (9) phosphorus, (10) potassium, (11) glucose. Results are mean values of three experiments with standard deviations (SD).
6
P. Bernat, J. Długon´ski / Chemosphere 62 (2006) 3–8
Detector. The separation was carried out in a capillary column HP 5 MS methyl polysiloxane (30 m · 0.25 mm id · 0.25 lm ft). The column temperature was maintained at 60 C for 4.5 min, then increased to 280 C at 20 C min 1. Finally, the column temperature was maintained at 280 C for 3 min. Helium was used as the carrier gas at flow rate of 1.2 ml min 1. Injection port temperature was 250 C. Split injection (2 ll) was employed. The individual butyltin species could be distinctly separated and they were identified on the basis of their retention times and characteristic ions. Quantitative analyses were performed on single ions at m/z 165 for MBT, 151 for DBT, 193 for TBT and 179 for TTBT (int. std.).
Fig. 2. Degradation of TBT (A) and growth curve (B) on synthetic M3 medium by C. elegans (initial TBT concentration 10 mg l 1). (A) TBT (), DBT (j), MBT (m); (B) dry weight (·) and pH (s). Results are mean values of three experiments with standard deviations (SD).
3. Results The TBT degradation activity of C. elegans on a basic synthetic growth medium (Lobos et al., 1992) (Table 2, medium 1) and next, on media without particular ingredients was examined (Table 2, medium 11). The best yields of TBT depletion were observed in the culture media (Erlenmayer flask experiments) which included glucose, NH4Cl, K2HPO4 and MgSO4 (Fig. 1A, media 1, 3, 4, 5). Taking into account the obtained results, the synthetic growth M3 medium was constructed. The culture inspection showed that C. elegans grew vigorously in M3 synthetic medium and reduced 80% of initial TBT content (10 mg l 1) within 3 d of shake flasks incubation (Fig. 2). At the end of the experiment about 95% of TBT was eliminated in the contaminated medium. The major amount of TBT was converted to xenobiotic derivatives between 1 and 3 d of C. elegans incubation. Additionally, the most vigorous growth of the fungus combined with the medium acidification, was observed in the same period of culture. The growth of aerobic microorganisms depends on dissolved oxygen (pO2) in the culture medium. Oxygen could positively influence the product kinetics with increasing values by acting as a direct parameter of product formation (Kapat et al., 2001). In our earlier studies (data not published) it was observed that the minimal pO2 level which limited C. elegans growth is 20%. Therefore, in the next step of our investigation the xenobiotic biodegradation was performed in the stirred tank bioreactor (Bioflo II, New Brunswick, USA) with controlled aeration, agitation, temperature and level of dissolved oxygen (pO2 P 20%). The results of the experiment presented in Fig. 3 showed that the
Fig. 3. Degradation of TBT on M3 medium by C. elegans in a bioreactor (initial TBT concentration 10 mg l 1) TBT (), DBT (j), MBT (m), dry mass (s), pO2 (- - -). The sample was taken three times.
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growth of C. elegans and degradation of TBT were significantly enhanced. Additionally, after 2 d of incubation, 89% of the initial amount of the xenobiotic was converted to TBT derivatives and the highest increase in the fungus biomass was noticed on the second day of the culturing.
4. Discussion As shown in our previous investigation (Bernat and Długon´ski, 2002), C. elegans degraded TBT when grown in Sabouraud liquid medium. However, the Sabouraud nutrient is rather inconvenient for the study of the optimisation of the xenobiotic biodegradation. Therefore, in the present paper a synthetic growth medium, which allowed checking an effect of a particular nutrients ingredients on TBT removal by the fungus, was applied. It was found that the presence of the source of carbon, nitrogen, sulphur, phosphorus and magnesium in the growth medium is necessary for the enhancement of TBT degradation activity of C. elegans. Vroumsia et al. (1999), who investigated the role of glucose and nitrogen on 2,4-dichlorophenoxyacetic acid (2,4-D) degradation (100 mg l 1) by C. elegans, found also a strong influence of these two elements on the xenobiotic biodegradation. Singh and Joshi (1991) revealed that phosphate ions were needed for the bioaccumulation of TBT into Escherichia coli cells. The important role played by magnesium in the growth and metabolism of microorganisms was described by Walker (1994). We also found that potassium was one of the crucial nutrients, which played a positive role in TBT biodegradation. Although the growth of C. elegans in this medium without the xenobiotic was observed (Fig. 1B), a significant reduction of TBT degradation efficiency in culture medium without that ion was noticed. It is very interesting, because TBT disturbs cell membranes integrity. The release of K+ from cells, arising from increased cytoplasmic membrane permeability of yeasts Debaromyces hansenii and Candida maltosa, was used to monitor organotin toxicity, implicating the cytoplasmic membrane as the site of action (Cooney et al., 1989; Laurence et al., 1989; Tobin and Cooney, 1999). It was also observed, that media with lower (6–10) and higher (1, 3–5) buffering capacity presented higher biomass level after 7 days of culturing without TBT (Fig. 1B). Among low buffering capacity media supplemented with the xenobiotic, only medium 7 had biomass >3 g l 1, while media 1, 3–5 had biomass >3 g l 1. The importance of the above elements was confirmed by TBT biodegradation in M3 medium (Fig. 2). The yield of the xenobiotic elimination was significantly higher than the one observed earlier (Bernat and Długon´ski, 2002) on Sabouraud medium (95% and 70% respectively after 7 days incubation). Additionally,
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gradual culture acidification and followed by very low pH value of the M3 medium at the end of the experiment was observed (Fig. 2). Probably ammonia was taken up in the form of NH3 by specific transport system and the process must have been completed with excretion of cations (Nielsen, 1992). Our results indicate that the lack of nutrients results in the cell growth limitation and the TBT debutylation by the fungus. It seems to suggest that biocide degradation by C. elegans is a cometabolic phenomenon. The nongrowth (secondary) substrate—TBT, can only be transformed in the presence of a growth (primary) substrate—glucose. Our observations are in agreement with the studies of other authors. Kawai et al. (1998) found that debutylation of TBT by Pseudomonas diminuta was enhanced by the supplement of organic nutrient broth into experiment medium. Also, degradation of TBT by algae (Tsang et al., 1999; Tam et al., 2002) was present in a rich growth medium. The acceleration of C. elegans growth by additional oxygen supply (pO2 P 20%) (Fig. 3) stimulated TBT elimination from the M3 medium (89% after 2 d of incubation in the bioreactor). The efficient xenobiotic biodegradation was related to the intensive fungal growth (Fig. 3) which confirms our suggestion that TBT elimination by C. elegans has a cometabolic nature.
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