UPTEC X 99 045 NOV 1999
Biodegradation of xenobiotic compounds by the white-rot fungus Trametes trogii
Master’ degree project s
Molecular Biotechnology Programme Uppsala University School of Engineering UPTEC X 99 045
Date of issue 1999-11
Biodegradation of xenobiotic compounds by the white-rot fungus Trametes trogii
Title (Swedish) Abstract White-rot fungi play an essential role in the degradation of lignin. Due to their ligninolytic enzyme system they also have the ability to degrade several highly persistent environmental pollutants. In the search for an effective degrader of certain industrial waste the white-rot fungus Trametes trogii was studied. First the activity of ligninolytic enzymes in the fungus was increased by varying conditions of growth. Then its biodegradation of the xenobiotics PCB and anthraquinone-blue was studied. The fungus was able to completely degrade the analysed compounds, even during primary metabolism. The results imply that Trametes trogii is a promising candidate for bioremediation of contaminated soils and sediments. Keywords: white-rot fungus, Trametes trogii, degradation, PCB, anthraquinone-blue, peroxidase, lignin Supervisors
Alberto Viale Department of Biological Chemistry, University of Buenos Aires, Argentina
Matti Nikkola, Uppsala University
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Introduction… … … … … … … … … … … … … … … … … … … ...1 Background… … … … … … … … … … … … … … … … .… … … ... 2
2.1 Basic ecology and fysiology of fungi… … … … … … … … … … .... 2
2.1.1 Characteristics of fungi 2.1.2 Decomposition of organic material 2.1.3 Degradation by fungi compared to bacteria
Lignin and its degradation… … … … … … … … … … … ..… … … . 3
2.2.1 Lignin molecule 2.2.2 Lignin degrading fungi 2.2.3 Mechanism of lignin biodegradation
Lignin-degrading enzymes… … … … … … … … … … … … … … ... 5
2.3.1 Lignin peroxidase 2.3.2 Manganese peroxidase 2.3.3 Laccase
Organic pollutants degraded by ligninolytic fungi… ...… ...… ... 7
2.4.1 PCB 2.4.2 Anthraquinone dyes
Materials and methods… … … … … … … … … … … … … … … .. 9
3.1 3.2 3.3 3.4 3.5 3.6 Organisms and inoculum… … … … … … … … ...… … … … … … .. 9 Culture media – solid… … … … … … … … … … … … … … … … .... 9 Culture media – liquid… … … … … … … … … … … … … … … … ... 9 Chemicals… … … … … … … … … … … … … … … … … … … … … ... 11 Cultivation of fungi… … … … … … … … … … … … … … ...… … … 11 Methods of analyses… … … … … … … … … … … … … … … … … .. 12
3..6.1 Fungi 3.6.2 Activity of ligninolytic enzymes 3.6.3 Degradation of xenobiotics
Results and discussion… … … … … … … … … … … … … … … ..
14 4.1 Varying culture conditions to increase activity of enzymes… ... 14
4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.6 Fixed culture conditions Effect of pH Effect of oxygen availability Results of varying pH and oxygen availability Comparison with another white-rot fungus Effect of sawdust
4.2 4.3 4.4
Degradation of anthraquinone-blue… … … … … … … … … … … 18 Degradation of PCB… … … … … … … … … … … … .… … … … … . 21 Future studies… … … … … … … … … … … … … … … … … … … … 23
Acknowledgements… … … … … … … … … … … .… … … … … ...
References… … … … … … … … … … … … … … … … … ..… … … 25
White-rot fungi play an essential role in the decomposition of dead trees, especially in the degradation of lignin. To be able to degrade the complex lignin molecule, these fungi use various extracellular enzymes with low specificity and strong oxidative activity. Due to the low specificity of the enzymes, white-rot fungi also have an ability to degrade a wide variety of environmental pollutants. Recent studies show that fungi with high ligninolytic activity are capable of degrading pollutants including polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs), dioxines, DDT, industrial dyes etc. White-rot fungi have therefore been expected to be good candidates for bioremediation of contaminated soils and sediments. The white-rot fungus Trametes trogii BAFC 463 produces high levels of the various enzymes involved in the degradation of lignin (Levin 1998). Before this work its degrading ability of xenobiotics (gr. “ xenos” foreign) had not been studied. , Investigations of this ability in other white-rot fungi have for the most part been done on Phanerochaete chrysosporium. Only a few others have been studied and especially rare are studies of white-rot fungi colonizing the southern hemisphere. Trametes trogii is common in South America and due to its high levels of ligninolytic enzymes it was considered a possible degrader of environmental pollutants. The aim of this study was to increase the production of ligninolytic enzymes in Trametes trogii and to investigate its capacity of degrading different xenobiotic compounds.. This was done in two parts. At first the ligninolytic activity of the fungus grown at different conditions was studied. By changing the liquid growth media in different ways the most productive condition was determined. The effect of pH, superfactant and sawdust. in the media was studied. The condition with the highest ligninolytic activity was then used to study the ability of Trametes trogii to degrade PCBs and an industrial polimeric dye, anthraquinone-blue. This study was part in larger project to find new ways to degrade environmentally hazardous compounds in industrial waste.
Basic ecology and fysiology of fungi
2.1.1 Characteristics of fungi Fungi are a very heterogenous group, nevertheless the typical fungi have a range of features that separate them from other organisms. Many of them are filamentous, which means that they consist of hyphae, surrounded by a cell wall. The hyphae grow at their tips, and branch periodically, which creates a network of hyphae called mycelium. All fungi are heterotrophs, and due to their rigid cell wall they must excrete extracellular enzymes to break down complex polymers and then absorb simple nutrients. Fungi are eukaryotic in contrast to bacteria. When growth is restricted in some way, the intermediates of the primary metabolic pathway can be shunted over to other pathways which lead to a production of secondary metabolites. They differ in chemical composition and are often species- or strain-specific. Some secondary metabolites are of commercial and medical interest, like penicillin. Lignin is in most white-rot fungi degraded only during the secondary metabolism. (Boominathan and Reddy, 1992). 2.1.2 Decomposition of organic material The decomposition of dead organic material is always due to a charateristic succession of fungi. In general there is a progressive utilization of the substrates present; simple monomers like sugars and amino acids are used first by weak parasites (“ sugar fungi” that grow on the living leaves. They have a restricted ability ) to degrade polymers like cellulose. When the leaves fall and are incorporated into the litter layer, these fungi are poor competitors with other fungi. Brown-rot fungi are able to degrade polymers like cellulose and hemicellulose and they bring about the major part of the decomposition, leaving the lignin more or less intact as a brown layer. Lignin-degrading fungi seem to colonize much later than the others and their existence is essential for the complete degradation of plant residue and the utilisation of the carbon trapped by lignin. Lignin is not a growth substrate, instead the white-rot fungi want to reach the cellulose and hemicellulose. It is usually slowgrowing members of the basidiomycotina that have the ability to synthesize all the enzymes needed to degrade the complex lignin molecule. 2.1.3 Degradation by fungi compared to bacteria
Most of the work on biodegradation has been carried out on bacteria. Although bacteria are fast growing and can respond to a changing environment by populations
utilizing the energy source present, there are important advantages of using fungi instead of bacteria for biodegradation. Many of the pollutants are toxic to the organisms that are supposed to degrade them. The extracellular enzyme system of fungi enables them to tolerate considerably higher concentrations of a certain xenobiotica than bacteria who have their enzymes inside the cell. Also, many of these chemicals have low water solubility and are therefore not available to the same extent to bacteria. The nonspecific nature of the enzyme system enables the fungi to degrade complex mixtures of pollutants, such as the commercial PCB preparation Arochlor, all the way to CO2. This is very important, because there are always metabolites formed during the degradation that can be as toxic or even more toxic than the original substance. In contrast, many different bacteria may be needed to successsfully and completely degrade the same mixtures. The degrading system of fungi is usually induced by nutrient depletion and not by a particular pollutant. This is important because repression of enzyme synthesis does not occur when the concentration of a chemical is too low for effective enzyme induction. In this way the fungi can degrade very low concentrations of a pollutant and do not have to be preconditioned to it (Barr and Aust, 1994).
Lignin and its degradation
2.2.1 Lignin molecule Lignin is the material that confers the qualities of rigidity and durability that make woody plants “ woody” It makes up about 20-30 % of wood, and is found in cell . walls, in a complex with cellulosic and hemicellulosic polysaccharides. In this natural composite material, the cellulose fibrils provide tensile strength, and the hemicellulose and lignin provide cross-linking, binding the structure together. Lignin is a very large, highly crosslinked and stereochemically complex polymer that is biosynthesized by the polymerization of phenylpropanoid precursors. There are three of these precursors, differing in the number of methoxyl groups on the aromatic ring (fig. 1).
CH2OH CH CH CH2OH CH CH CH2OH CH CH
OCH3 OH OH
Figure 1. Structure of the three lignin precursors: (1) p-coumaryl alcohol, (2) coniferyl alcohol, (3) sinapyl alcohol.
Lignin polymerization takes place in cell walls after the polysaccharides have been deposited, and is initiated by enzymatic oxidation of the precursors to phenoxy radicals. These radicals can couple with eachother and with the growing lignin polymer in numerous ways to form a complex cross-linked network. This random polymerization of lignin gives it a very complex and irregular structure (fig. 2).
Figure 2. Scematic structure of a portion of a lignin molecule (Adler 1977).
2.2.2 Lignin degrading fungi The only organism known to extensively degrade lignin are fungi (Kirk and Farrell, 1987). Because lignin is an insoluble polymer, the initial steps in its biodegradation must be extracellular. Fungi have, in contrast to bacteria, indeed extracellular enzyme systems. Lignin degrading fungi are classified into three major categories based on the type of wood decay caused by these organisms: white-rot fungi, brown-rot fungi and soft-rot fungi. Of these three groups, white-rot fungi are the most effective lignin degraders and have been the most extensively studied group. In this group the most commonly used model organism in lignin biodegradation studies is strains of Phanerochaete chrysosporium (Boominathan and Reddy, 1992). White-rot fungi comprise a heterogeneous collection of several hundreds of species of basidiomycetes (Ainsworth et al., 1973). They are able to completely mineralize both the lignin and carbohydrate components of wood. Despite the fact
that lignin is rich in carbon, it is not a growth substrate. White-rot fungi metabolize lignin only in the presence of an alternate energy source, such as cellulose, hemicellulose or simple carbohydrates. This is called co-metabolism. Most white-rot fungi degrade lignin only during secondary metabolism, which is triggered by a limitation of nutrients. However some fungal species have been reported to degrade lignin also during primary metabolism under conditions of nutrient sufficiency (Leatham and Kirk, 1983). There are many factors involved in the lignin degradation process, but very few of them are completely understood. 2.2.3 Mechanism of lignin biodegradation Because of the random polymerization process that forms it, lignin has a complex and irregular structure. The diversity of the interunit linkages and the irregularity of their arrangement make it difficult for a ligninolytic fungus to produce enzymes that could recognize and cleave all of them. The solution that has evolved in the whiterot fungi is to produce enzymes of low specificity that initiate, but do not direct, oxidative reactions in lignin. Kirk and Farrell (1987) have termed this process enzymatic combustion: the enzyme activates the lignin to overcome an energy barrier and begin a thermodynamically favoured oxidative fragmantation without further control of the reaction pathway by the enzyme. The chemical changes produced by white-rot fungi in lignin include oxidative cleavage of the propanoid side chains and also demethylation and oxidation cleavage of aromatic rings (Chen and Chang, 1985). Lignin biodegradation does not proceed by an orderly removal of the peripheral subunits. Instead it also involves oxidation of the aromatic rings and side chains in the interior of the polymer, increasing the solubility of the polymer core at the same time as fragments of varying size are set free. The disorderly nature of this degradation agrees with the concept of enzymatic combustion (Reid, 1995).
A set of enzymes that will extensively degrade lignin and reproduce the effects of a white-rot fungus has not yet been identified. However, three oxidative enzymes are commonly found extracellularly in ligninolytic cultures of white-rot fungi. It is clear that different combinations of the known enzymes are produced by various lignindegrading fungi, suggesting that there is more than one successful strategy for lignin biodegradation (Hatakka, 1994).
2.3.1 Lignin peroxidase Lignin peroxidase (LiP) was first discovered in Phanerochaete chrysosporium (Glenn et al., 1983; Tien and Kirk, 1983) and it is produced by many, but not all, white-rot fungi. This enzyme is an extracellular hemeprotein, dependent of H2O2, with an unusually high redox potential and low optimum pH (Gold and Alic, 1993). It shows little substrate specificity, reacting with a wide variety of lignin model compounds and even unrelated molecules (Barr and Aust, 1994). It has the distinction of being able to oxidize methoxylated aromatic rings without a free phenolic group (fig. 3), generating cation radicals that can react further by a variety of pathways, including Cα-Cβ cleavage, ring opening, demethylation and phenol dimerisation.
OCH3 Ligninase / H2O2 + OCH3 e
OCH3 (+H2O, -e )
+ 2 CH3OH O
OCH3 Cation radical
Figure 3. The action mechanism of lignin peroxidase was discovered from studies of metoxylated benzenes. They were oxidized by the enzyme to unstable molecules called cation radicals. In the figure the cation radical 1,4-dimetoxibenzene, decomposes in the reaction with H2O, producing methanol and benzenequinona.
2.3.2 Manganese peroxidase Another type of heme peroxidase, manganese peroxidase (MnP), was later found in the culture media of the same fungi (Glenn and Gold, 1985). This enzyme shows a strong preference for Mn(II) as its reducing substrate. The product Mn(III) forms a complex with organic acids and diffuses away from the enzyme to oxidize other materials, such as lignin (fig. 4).
H2 O 2 E Mn(III) Mn(II) lactate substrate
Figure 4. The action mechanism of manganese peroxidase
The redox potential of the Mn peroxidase - Mn system is lower than that of lignin peroxidase and it has only shown capacity for oxidize in vitro phenolic substrates (Vares 1996). Phenolic substrates are oxidized to phenoxy radicals, which can react further by demethylation, alkyl-phenyl cleavage, Cα oxidation or Cα-Cβ cleavage (Tuor et al.,1992). Manganese peroxidase seems to be more widespread among white-rot fungi than lignin peroxidase (Hatakka, 1994) and is readily detected in cultures on real lignocellulosic substrates.
This enzyme is a copper-containing oxidase and it does not require peroxide, H2O2 (Thurston, 1994). Like manganese peroxidase, it normally oxidizes only those lignin model compounds with a free phenolic group, forming phenoxy radicals. However in the precense of the artificial substrate ABTS (2,2´-azinobis(3-ethylbenzthiazoline5-sulphonate) or some other synthetic mediators, laccase can also oxidize certain non-phenolic compounds, veratryl alcohol and Mn (II) (Collins and Dobson, 1997). Laccase is produced by most white-rot fungi (Hatakka, 1994), but normally not in Phanerochaete chrysosporium (Kirk and Farrell, 1987). Laccase has the capability to both depolymerize and polymerize lignin model compounds (Eriksson et al., 1990). Despite the fact that laccase was the first enzyme found to have a function in the degradation of lignin, its role in this process is still not known.
Organic pollutants degraded by ligninolytic fungi
The low specificity and strong oxidative abilities of fungal lignin degradation systems allow them to be applied to the degradation of many organic pollutants. White-rot fungi can metabolize compounds including clorophenols, polycyclic aromatic hydrocarbons, chloroanilines and pesticides such as methoxychlor and DDT (Hammel, 1992). To varying degrees, pollutants in these groups are co-oxidized by the fungi to give CO2 and largely uncharacterized polar metabolites. The xenobiotic oxidations of white-rot fungi are not rapid or efficient, but they are very nonspecific. Moreover, many white-rot fungi are natural inhabitants of soil litter. These considerations make ligninolytic fungi attractive candidates for use in low tech bioremediation programs. Little is known about the mechanisms that white-rot fungi employ for oxidation of organopollutants.
2.4.1 PCB Polyclorinated biphenyls (PCBs) are a large family of compounds produced commercially by the direct chlorination of biphenyl. Chlorines can be placed at any or all of the ten available sites, with 209 different PCB compounds theoretically possible. Of the possible compounds, only about half are actually produced in the synthesis due to steric hindrance. These compounds are manufactured and sold as complex mixtures differing in their average chlorination level. The crude mixtures resulting from the chlorination are fractionally distilled to produce commercial mixtures with the desired properties. The products range from light oily fluids (di-, tri-, and tetra-chlorobiphenyls) to heavy oils (penta-chloro-biphenyls), to greases and waxes (more highly chlorinated). Nowadays these compounds are forbidden in many countries. The excellent physical and chemical properties of PCBs (low vapor pressures, low water solubility, excellent dielectric properties, flame resistance, stability to oxidation) led to their widespread use. In a 50-year period approximately 700.000 metric tons of PCBs were produced. Such extensive application of these chemically stable compounds has resulted in significant global contamination (Abramowicz, 1990). The lipophilic nature of PCBs contributes to their tendency to accumulate in fatty deposits and results in a magnification in the food chain. There have been controversies about the toxicity of PCBs. However it has been proven that PCBs interfere with many biological functions, including the immune system, the nervous system and several endocrine systems. They also cause certain cancers in animals. These observations suggest that the potential human health hazards from PCB exposure have been underestimated (Carpenter, 1998). 2.4.2 Anthraquinone dyes Anthraquinone is the basic unit of this class of dyes. By introducing hydroxyl and amino groups in anthraquinone a wide range of colors are available, among others anthraquinone-blue (fig. 5). It is the quinonoid system that acts as a chromophore. Anthraquinone dyes have excellent fastness properties.
NH O SO3H
Figure 5. Molecular structure of anthraquinone-blue
MATERIALS AND METHODS
Organisms and inoculum
The strains BAFC 463 of Trametes trogii Berk. and BKM-F-1767 of Phanerochaete chrysosporium were used in the experiments. Both of these were obtained from the culture collection of the Faculty of Natural Science, University of Buenos Aires, Argentina. Stock cultures were maintained on malt-agar medium slants at 4° C. In order to obtain the inoculates the fungi were grown on plates with agar medium before the experiments. Trametes trogii were incubated on the plates at 28° C for seven days. Phanerochaete chrysosporium were incubated on the plates at 38°C for three days.
Culture media - solid
Malt-agar medium Malt extract D(+)-glucose Bacto Agar (Difco) Distilled water Agar medium Bacto Agar (Difco) Distilled water 20 g Up to 1000 ml 20 g 20 g 20 g Up to 1000 ml
Culture media - liquid
Malt medium Malt extract Glucose Distilled water 12.7 g 10 g Up to 1000 ml
Materials and Methods
Basal medium Glucose Asparagine MgSO4-7H2O HK2PO4 H2KPO4 CaCl2 CuSO4-5H2O MnCl2-H2O H3BO3 NaMoO4-2H2O FeCl3 ZnCl2 HCl-thiamine Distilled water 10 g 4g 0.5 g 0.6 g 0.5 g 0.1 g 0.4 mg 0.09 mg 0.07 g 0.02 mg 1 mg 2.5 mg 0.1 mg Up to 1000 ml
Kirk medium (Tien and Kirk 1988) Glucose Ammonium tartrate Sodium acetate Thiamine H2KPO4 MgSO4-7H2O CaCl2 CuSO4 MnSO4 H3BO3 NaMoO4-2H2O ZnSO4-7H2O CoCl2 NaCl FeSO4-7H2O AlK(SO4)2-12H2O Distilled water 10 g 0.2 g 3.28 g 2 mg 2g 0.53 g 0.1 g 1 mg 5 mg 0.1 mg 0.1 mg 1 mg 1 mg 10 mg 1 mg 0.1 mg Up to 1000 ml
Materials and Methods
The mixture of PCBs used in the experimnets was manufactured under the name Aroclor 1242, where the figures stand for 12 carbon atoms and 42% chlorine by weight. This corresponds to three chlorines/biphenyl on average. The concentrations used in the media with the fungi were either 50 ppm or 100 ppm. In the experiments concerning the degradation of anthraquinone-blue, the concentration of the dye in the media was 50 ppm. The surfactant Tween 80 (polyoxyethylene sorbitan monooleate) was used at a concentration of 1.7 mM in most of the cultures with basal medium and in all the cultures with Kirk medium. Sawdust from the tree Populus sp. (Poplar) was used at a concentration of 1 g per 25 ml medium in some of the cultures with basal medium as well as Kirk medium.
Cultivation of fungi
All substances used were of analytical grade, with the obvious exceptions of malt extract and sawdust. Glas materials were washed, treated with a mixture of chrome sulphate and then rinsed with distilled water. Culture media were sterilized by autoclavation at 121° 1.2 atmospheres, for at least 20 minutes. C, Cultivation in solid medium were made in Petri dishes, 10 cm of diameter, with 25 ml medium; or in test tubes, 20 cm of length and 2 cm of diameter, with 25 ml medium inclined. Cultivation in liquid medium were made in 250 ml Erlenmeyer flasks containing 25 ml medium. Erlenmeyer flasks and test tubes were plugged with cotton coated with gauze bandage and then covered with aluminium foil. After autoclavation, each flask was inoculated with two pieces (7 mm diameter), that were cut off the edge of mycelial mats of cultures grown on agar medium for 7 days in 25° for Trametes trogii and 3 days in 38° for Phanerochaete chrysosporium. The initial C C mass of the mycelium incorporated into the cultures were considered negligible in the studies of the growth rate. All cultures had an initial pH of 4.6. The incubations of the cultures of Trametes trogii were made in culture chamber New Brunswick Psicrotherm G-27, with a temperature of 28° ± 1 and C constant light. The erlenmeyer-cultures were either incubated at static or agitated conditions. In the latter condition the cultures were agitated constantly by a rotational agitator at 125 r.p.m. The incubations of the cultures of Phanerochaete chrysosporium were always made at static conditions, without light and at a temperature of 38° (Tien and Kirk 1988). C
Materials and Methods
Methods of analyses
3.6.1 Fungi To separate the mycelia from the supernatants the cultures were filtrated through Whatman GP filter papers. The mycelia and the filters were dried together in an oven for 24 hours at 80ºC and then weighed. After the filtration the supernatants from the culture were kept at -20ºC until used for analyses of extracellular enzymes. The filtered mycelia were dried and weighed together with the filter papers. To obtain the dry-weight of the mycelium only, an estimated weight of the dry paper was subtracted. The weight of the dry paper was estimated as the weight before the drying minus five percent. In general the results are expressed as mg of mycelium/25 ml of culture medium. Proteins in the supernatant (extracellular proteins) were measured by the Bradford method (1976), using bovine serumalbumine 1 mg/ml as standard. 3.6.2 Activity of ligninolytic enzymes Lignin peroxidase (LiP) activity was measured using the method described by Tien and Kirk (1984). In this method the increase of absorbance at 310 nm, due to the oxidation of the veratryl alcohol to veratryl aldehyde, is measured. The reaction mixture contained: 2.2 ml of sodium tartrate buffer (50 mM, pH 4,5 at 25ºC), 40 µl of veratryl alcohol (2 mM) and 240 µl of culture supernatant. The reaction was initiated by the addition of 20 µl of H2O2 (0.2 mM). The absorbance was measured immediately (ε310 = 9333 M-1cm-1). One enzymatic activity was defined as the quantity of enzyme that produced 1 µmol of oxidized product. Manganese peroxidase (MnP) activity was measured following the method described by Glenn and Gold (1985). This method is based on the oxidation of Mn(II) to Mn(III), and uses as substrate 2.5 ml of phenol red (0.01%) and MnSo4 (0.1 mM) in sodium succinate buffer (0.1 M). The reaction mixture contained 2.5 ml of substrate and 200 µl culture supernatant. The reaction was initiated by the addition of H2O2 (0.1 mM). After an incubation of 2 min at 30ºC, the reaction was ended by the addition of NaOH (5 M). The absorbance was then measured at 610 nm (ε610 = 22000 M-1cm-1). One enzymatic activity was defined as the quantity of enzyme that produced 1 µmol of oxidized product. Laccase activity was measured using the method described by Bourbounnais et al. (1995), which is based on the oxidation of the substrate ABTS (2,2´-azino-bis(3ethylbenzothiazoline-6-sulphonic acid)) (5 mM). After dissolving the substrate in 2.4 ml sodium acetate buffer (0.1 M, pH 5.0), 100 µl of culture supernatant was added and then the mixture was incubated 2 min at 30ºC. The absorbance was measured at
Materials and Methods
420 nm (ε420 = 36000 M-1cm-1). One enzymatic activity was defined as the quantity of enzyme that produced 1 µmol of oxidized product. 3.6.3 Degradation of xenobiotics The degradation of anthraquinone-blue by the fungi was measured by adding 50 ppm of the dye to the 25 ml erlenmeyer culture, and then at different times take new samples of the supernatant and measure the absorbance at 600 nm. The absorbance was measured after 10 minutes, 30 minutes, 2 hours, 4 hours, 8 hours and 24 hours. This procedure was done with cultures of varying growth time. The minimal inhibitory concentration of PCBs for Trametes trogii was found by investigating the growth at solid malt-agar medium prepared with different concentrations of a PCB-mixture. In the PCB-degradation experiments Trametes trogii were grown for five days and then either 50 ppm or 100 ppm sonificated PCB-mixture was added to the culture. Both liquid malt medium and basal medium were used in the experiments. After a total growth of either 12 or 24 days, the growth was stopped and the cultures were prepared for analysis with gas-liquid chromatography. To stop the growth and the enzymatic activity, the pH of the culture was lowered to around pH=1.0 with 0.5 ml HClO4. As an internal control, 50 ppm of biphenyl dissolved in hexane was added to every culture. In order to separate the remaining PCB from the culture, 10 ml of hexane was added to each one. The cultures were then shaken and stored overnight at 8° The next day as much as possible of the hexane-phase was C. separated from the cultures. To the hexane-phase, 2 g of Na2SO4 was added in order to bind the remaining water. The hexane-phase was then stored at -20° until C the analysis with gas-liquid chromatography. The chromatograph was a Hewlett-Packard, model 5840A, with a HP1 column (cross-linked methyl siloxane) 10 m length, 0.53 mm id. Detector: FID (flame ionizer detector). Injector temperature: 200 ºC. FID temperarure 310 ºC. Initial temperature (of column) 80 ºC, for 2 min. Rate 10 ºC/min. Final temperature 200 ºC, for 15 min.
RESULTS AND DISCUSSION
Varying culture conditions to increase activity of enzymes
The ability of Trametes trogii to degrade lignin has earlier been investigated by Levin (1998). It was then found that the fungi, at certain conditions, produced high levels of ligninolytic enzymes. This implied that Trametes trogii is a possible degrader of environmentally hazardous substances. The aim of my work was to increase its production of ligninolytic enzymes and to decide its degrading ability of these substances, especially PCB. In order to increase the activity of the ligninolytic enzymes, different culture conditions were varied. 4.1.1 Fixed culture conditions The level of nitrogen in the liquid medium is very important and crucial for the synthesis of ligninolytic enzymes in white-rot fungi. These enzymes are in most of these fungi synthesized during the secondary metabolism, which is induced by limitations of nitrogen or carbon (Holzbaur and Kirk, 1981). Consequently the levels of nitrogen in the liquid medium need to be low but still high enough for growth to be possible. Furthermore the level of nitrogen need to favor the production of glucose oxidase, which is essential for the use of glucose as a carbon source. The chosen level of nitrogen is known to favour the synthesis of both glucose oxidase and ligninolytic enzymes in the most studied white-rot fungus, Phanerochaete chrysosporium (Kirk et al., 1978; Kelley and Reddy, 1986). Since the basal media, with asparagine (0,5 g/l) as nitrogen source and glucose (10 g/l) as carbon source, had worked well (Levin, 1998), it was used throughout the experiments. For cultivation of Trametes trogii a temperature around 28ºC is ideal (Levin, 1998). Growth of the fungi, production of proteins and the tested enzymic activities were all at a maximum at this temperature. Therefore this temperature was used in all the experiments with Trametes trogii of this work. 4.1.2 Effect of pH To investigate the effect of pH on the activity of ligninolytic enzymes, successive experiments were done with two different pH of the medium. Generally growth of fungi is ideal at low pH. This is also true for Trametes trogii, which grow at maximal rate at pH=3.1 (Levin, 1998). However the production of extracellular enzymes is normally not very high at such a low pH. As for Trametes trogii some enzymes are produced at a maximum rate between pH=4.0 and pH=5.0, for example exoglucanase and endoxylanase. Other enzymes are favoured at a higher pH,
Results and Discussion
between 5.5 and 6.5, for example β-glucosidase, β-xylosidase and pectine lyase (Levin, 1998). The experiments were therefore done at pH=4.6 and pH=6.0. 4.1.3 Effect of oxygen availability Synthesis of ligninolytic enzymes in Phanerochaete chrysosporium and other white rot fungi is particularly active at high oxygen level (Dosoretz and Grethlein, 1991; Faison and Kirk, 1985). However there are problems of oxygen availability arising from the culture conditions in liquid media. The oxygen level has been reported undetectable in mycelial mats at depths below 2 mm in nonagitated liquid cultures of white rot fungi (Leisola et al. 1983). Several different ways were used in our experiments with Trametes trogii to increase the oxygen availability of the fungus. Shallow cultures, 25 ml media in 250 ml erlenmeyers, were used in order to get more oxygen in the medium due to a high surface/volume relation. Two other ways of increasing the availability of oxygen are to use surfactants in the medium and to cultivate with agitation. The addition of the surfactant Tween 80 to the growth medium results in higher permeability of oxygen through the cell membranes (Lestan et al, 1994). Agitation of the culture increases the level of oxygen in the medium. These two ways were used in these experiments in order to increase the ligninolytic activity in Trametes trogii. The interaction of these ways with eachother and with pH was also studied. 4.1.4 Results of varying pH and oxygen availability To determine at what conditions the ligninolytic activity of Trametes trogii was at a maximum, the cultivations of the fungus were done at varied pH and oxygen availability. The cultivations were done at static condition, with or without superfactant, and at agitated condition, with or without superfactant. These cultivations were all done at the two different pH. Then the weight of the fungi, extracellular proteins and ligninolytic enzymes in the supernatant were measured every three or four days until the maximum levels were determined. All the experiments were done twice. In Table 1 the maximum level is shown for all the experiments. The maximum level of the enzymatic activity for laccase and LiP was reached between day 17 and 28, during the second metabolism. It was clearly shown that the highest ligninolytic activity was at static condition, with added Tween 80 and with a medium at pH 4.6. Both the enzymatic activity of LiP and laccase reached a maximum at these conditions. In the agitated condition Trametes trogii grew very well and also the laccase activity was high, but the protein level and LiP activity were very low. The superfactant Tween 80 had a large overall positive effect. Especially the laccase activity increased by the superfactant. The effect of pH was also clear. Both the growth and ligninolytic activity were higher at the low pH.
Results and Discussion
pH 4.6 pH 6.0 pH 4.6 pH 6.0 pH 4.6 pH 6.0 pH 4.6 pH 6.0 Static condition - without Tween 80 - with Tween 80 Agitated condition - without Tween 80 - with Tween 80 118 123 81 87 5 13 4 7 0.002 0.015 0 0.006 0.61 0.68 0.07 0.22 87 121 93 94 17 31 14 24 0.044 0.063 0.014 0.018 0.25 0.69 0.07 0.15
Table 1. Comparison of weight, extrcellular protein and enzymatic activity in the supernatant for Trametes trogii cultivated at two different pH, 4.6 and 6.0. Experiments were done in static and agitated condition, both with and without superfactant. The shown values are the maximum levels measured during day 17-28 of cultivation, approximately during the secondary metabolism. Growth is expressed in mg dry weight/25 ml media. Proteins are expressed in µg/ml supernatant. LiP and Laccase are expressed in UE/ml supernatant.
Measurable levels of ligninolytic enzymes were only found of laccase and LiP. Manganese peroxidase was not found in any of the conditions, which was somewhat surprising and intriguing since Levin (1998) found at least low levels of this enzyme. At all investigated conditions the growth curve of Trametes trogii had more or less the same form. The fungus grew at an exponential rate up to around day 12 of the culture. Then it grew at a slower rate up to around day 25 and after that went into the phase of autolysis and consequently the weight went down. Second metabolism was initiated in response to limitation of carbonsource. Among whiterot fungi normally the production of LiP and MnP is initiated during the second metabolism, which is due to lack of nutritions (see section 3.2.2). There are exceptions to this and occasionally LiP and MnP are detected during primary metabolism (Eggert et al., 1996). In similar conditions as in this experiment Levin (1998) measured glucose residues in the medium and found that the Trametes trogii had consumed the glucose around day 20 and possibly the second metabolism started as a consequence. However the production of LiP in our experiments was initiated much earlier. Do the second metabolism start earlier or is LiP produced during the primary metabolism? More investigations are needed to answer that. The conclusion is that the maximum ligninolytic activity of Trametes trogii was reached after cultivation at static condition, with Tween 80 and at pH 4.6. These conditions were therfore used throughout the degradation experiments.
Results and Discussion
4.1.5 Comparison with another white-rot fungus In order to put the study of Trametes trogii in perspective, a similar study was done with the most investigated white-rot fungus, Phanerochaete chrysosporium. Some obvious differences were found in the comparison. T. trogii grew much slower and showed a maximum enzymatic activity around day 25 for both LiP and laccase (Graph 1). P. chrysosporium had a maximum LiP activity at day 3, a maximum MnP activity at day 10 and were not producing extracellular laccase at all. It also had more than three times higher activity of the only common ligninolytic enzyme, LiP, than T. trogii (Graph 2). The cultivation of the fungi were done with some differences. T. trogii were grown at the best conditions stated above. P. chrysosporium were grown at the normally used conditions (Tien and Kirk, 1988). This means that differences in cultivation is the temperature and some minerals in the media. Both were grown at static conditions, with Tween 80 and at pH 4.6. The slower growth is a disadvantage for T. trogii in a future use as a degrader of hazardous substances.
0,08 0,07 LiP 0,06 LiP (UE/ml) 0,05 0,04 0,03 0,02 0,01 0 0 5 10 15 20 25 30 Days of growth Laccase
0,6 0,5 0,4 0,3 0,2 0,1 0
Enzymic activity (UE/ml)
0 0 5 Days of growth 10 15
Graph 1. Synthesis of laccase and lignin peroxidase by Trametes trogii grown in basal medium. Enzymic activity is expressed in UE/ml supernatant.
Graph 2. Synthesis of manganese peroxidase and lignin peroxidase by Phanerochaete chrysosporium grown in Kirk medium. Enzymic activity is expressed in UE/ml supernatant.
4.1.6 Effect of sawdust Some of the most efficient inductores of synthesis of ligninolytic enzymes are aromatic compounds and samples of lignin (Collin and Dobson, 1997; Haars and Huttermann, 1993). In this experiment was used sawdust, which obviously contains
Results and Discussion
a lot of lignin, to induce synthesis of the enzymes in Trametes trogii. The sawdust came from the tree poplar, on which Trametes trogii naturally lives. To every erlenmeyer flask, containing 25 ml of media, 1 g of sawdust was added. Other culture conditions were as the most productive stated above. The most important result from this experiment was that the stimulation of the extracellular laccase was notable. The maximum value of laccase was more than three times higher in experiments with sawdust than without. The peak was also reached earlier, day 17 compared to day 24 (graph 3). Unfortunately the method that were used for measuring lignine peroxidase was not suitable for supernatants not completely transparent. Since the sawdust coloured the supernatant, it was not possible to measure the lignin peroxidase in a reliable way. Another problem with the sawdust is that it may not be completely clean and thus makes analyses more difficult.
3 with sawdust without sawdust
0 0 10 20 Days of growth 30
Graph 3. Comparison of synthesis of extracellular laccase (UE/ml supernatant) by Trametes trogii cultivated with and without sawdust.
Degradation of anthraquinone-blue
The degradation of aromatic compounds by Trametes trogii was first studied in experiments concerning the degradation of the dye anthraquinone-blue. The advantage of studying this kind of degradation is that it can easily be displayed by a spectrophotometer. In this experiment anthraquinone-blue was added to the flasks with cultivated Trametes trogii and then the absorbance of the supernatant was measured at different times. This procedure was done to several flasks with cultures
Results and Discussion
of different times of growth. In order to put the study of Trametes trogii in perspective, a similar experiment was also done with Phanerochaete chrysosporium. At first the experiments were done with the common media, basal medium for T. trogii and Kirk medium for P. chrysosporium. The results of the experiments turned out to be a bit surprising. T. trogii degraded the anthraquinone-blue much faster and to a greater extent than P. chrysosporium. In the case of T. trogii the degradation did not seem to have any correlation with the amount of ligninolytic enzymes. It degraded the dye a bit slower when the enzymic levels were close to zero, but the difference was very small (graph 4). On the other hand the degradation by P. chrysosporium seemed correlated with the enzymic activitiy of MnP, since the fastest degredation took place after around 10 days of cultivation (graph 5).
1 0,9 0,8 14 0,7 Abs (600 nm) 0,6 0,5 0,4 0,3 0,2 0,1 0 0 5 10 15 20 25 30
Time of degradation (hours)
3 5 7
19 24 28
0,7 Abs (600 nm) 0,6 0,5 0,4 0,3 0,2 0,1 0 0 5 10 15 20 25
10 14 19
Time of degradation (hours)
Graph 4. Degradation of anthraquinone-blue by Trametes trogii grown in basal medium. The different lines represent the time of cultivation (in days) when the dye was added.
Graph 5. Degradation of anthraquinone-blue by Phanerochaete chrysosporium grown in Kirk medium. The different lines represent the time of cultivation (in days) when the dye was added.
Since the degradation did not seem to correlate with the ligninolytic activity of T. trogii, the experiment was also done with fungi grown in malt medium. This medium is rich in nutrition and therefore the fungus is not supposed to enter the second metabolism and then not produce ligninolytic enzymes. However the degradation of anthraquinone-blue in malt medium was just as fast and extensive as with the basal medium (graph 6). This was a very surprising result. It was then found that laccase was also present in cultures of malt medium, but not lignin peroxidase or manganese peroxidase. Maybe the degradation of anthraquinone-blue is carried out by laccase only, which also could explain the poor degradation by P.
Results and Discussion
chrysosporium. Future studies are needed to decide what enzyme is degrading anthraquinone-blue in T. trogii.
0,8 0,7 0,6 Abs (600 nm) 0,5 0,4 0,3 0,2 0,1 0 0 5 10 15 20 25 Time of growth (hours) 7 10 14 19 24 28
Graph 6. Shows the degradation of anthraquinone-blue by Trametes trogii grown in malt medium. The different lines represent the time of cultivation (in days) when the dye was added.
Attempts were also done to determine whether the enzymes degrading anthraquinone-blue were situated freely in the supernatant or connected closely to the fungus. The supernatant that was used for measuring the absorbance after time “ minutes” , was stored for 24 hours and then the absorbance was measured 10 again. If there wasn´t a difference between the two absorbances the enzymes were situated in the supernatant and still acting during the 24 hour storage. These measurements were done for both T. trogii and P. chrysosporium. A large difference was noticed even here between the two fungi. In the stored supernatant of P. chrysosporium there was hardly any degradation at all. However in the case of T trogii there were not much difference between the degradation in the flask with the fungus and in the supernatant only. This means that the enzymes of T. trogii responsible for the degradation of anthraquinone-blue are for the most part situated freely in the supernatant.
Results and Discussion
Degradation of PCB
In a minimal inhibitory test it was found that Trametes trogii grew almost normal at a concentration of 50 ppm PCB-mixture in the solid media, but very slowly at 100 ppm. The degradation experiments were therefore mostly done at 50 ppm. The degradation of the PCB-mixture Aroclor 1242 was studied by adding the mixture to 5-day old cultures of Trametes trogii and then measure the PCB-content when the culture was either 12 or 24 days old. The two different time-lengths were used to study the effect of differences in the activity of ligninolytic enzymes. In the 12-day cultures the activities of lignin peroxidase and laccase were low (see section 5.1.5). To study the difference in degradation between primary and secondary degradation, the experiments were done both with fungus grown in basal medium and in malt medium. The remaining PCB content was measured with a gas-liquid chromatograph. With this type of chromatograph it was not possible to determine exactly the kind of PCB compounds degraded. However it was clearly showed that the fungus could degrade most PCBs to a very large extent. Especially PCBs with few chlorines were degraded extensively. The remaining PCB content was more or less the same in the cultures of 12-days and 24-days. It shows that the degradation takes place mostly up to day 12. Another important and surprising result was that the degradation was somewhat greater in malt medium than in basal medium. This means that the degradation takes place during the primary metabolism and that lignin peroxidase is of no importance in the primary degradation of PCB. It is important to remember that in these experiments it is only possible to study the degradation of PCB and not of the metabolites that are produced. Lignin peroxidase may be important in the degradation of the sometimes hazardous metabolites. Experiments were also done with 100 ppm PCB-mixture in the medium. Trametes trogii had no problems growing and degrading this higher concentration. Actually the remaining PCB-content was similar to the experiments with lower initial concentration. For the 12-day-old cultures grown in malt medium with 100 ppm PCB the degradation was as high as 90 % (chromatogram 1). In these experiments with PCB it was shown that Trametes trogii is a promising fungus for future use in bioremediation of contaminated soils, but a lot more research is necessary before that may be reality.
Results and Discussion
Chromatogram 1 Degradation of PCB-mixture (100 ppm) by Trametes trogii cultured in malt medium. The upper black line shows the PCB content in the control, where no degradation has taken place. The lower red line shows the PCB content after seven days of degradation, between day 5 and 12 of the culture. The internal standard, biphenyl, is represented by the large peak to the left (3.54). The further to the right in the chromatogram the more chlorinated are the PCBs, normally one more chlorine for every peak.
Results and Discussion
In order to use Trametes trogii for bioremediation it is necessary to know more about - the degradation of xenobiotics when the fungus is grown at larger scale, at fermentor level. - the degradation of other xenobiotics. - the metabolites produced at the degradation of xenobiotics. - what enzymes are responsible for the degradation by T. trogii. - why the degradation is better for the fungus grown in malt medium than in basal medium. Since the results of this work were promising, continous work has been done on these problems at the department.
I would like to express my sincere gratitude to some people who have given me all kind of support during my work. I thank…
Dr. Alberto Viale – my supervisor, for giving me the opportunity to do this work at his lab, for always caring to make my stay as good as possible and for giving me support and guidance whenever I needed it throughout my work! Dr. Flavia Forchiassin – for letting me work in her lab of Experimental Mycology and for introducing me to and teaching me about fungi. Dr. Laura Levin – for all the answers to my questions and for always helping and supporting me when I ran into problems. Dr. Matti Nikkola – for being the examiner of this project. Leandro - for being such a great co-worker! Thanks for stimulating discussions late at night about research and other important things in life. Ramiro, Paula, Gustavo, Laura, Gabriela for all the good times we had together and for showing me the best of Argentina. Lorena – for making my life complete.
_________________________ Christian Haglund
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