School of Chemical and Life Sciences, University of Greenwich, Wellington Street, London SE18 6PF, UK1
Department of Biology, Imperial College of Science Technology & Medicine, Prince Consort Road, London SW7 2BB, UK2
Author for correspondence: Patricia J. Harvey. Tel: +44 181 331 9972. Fax: +44 181 331 8305. e-mail: p.j.harvey{at}greenwich.ac.uk
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ABSTRACT |
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Keywords: cellulose, lignin peroxidase, Phanerochaete chrysosporium, oxidant stress, ultrastructure
Abbreviations: LiP, lignin peroxidase; NBT, nitro blue tetrazolium; TEM, transmission electron microscopy
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INTRODUCTION |
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In the natural environment, the fungus differentiates a thin film of mycelium to degrade lignin and metabolize cellulose. By contrast, in liquid cultures (suitable for industrial fermentation) the fungal spore inoculum develops into mycelial pellets, which are agglomerates of hyphae trapped together during germination of the spores (Gerin et al., 1993 ). The pellet habit represents a significant obstacle to oxygen diffusion into the hyphae (Leisola et al., 1983
; Michel et al., 1992
), which is essential for ligninolysis and LiP production (Kirk et al., 1978
; Bar-Lev & Kirk, 1981
; Faison & Kirk, 1985
). The role of oxygen has been ascribed to either its direct participation in ligninolysis (Kirk et al., 1978
) or its capacity to promote the synthesis of one or more enzymes involved in lignin degradation (Bar-Lev & Kirk, 1981
). Alternatively, the necessity to expose fungal pellets or mats in liquid culture to a pure oxygen atmosphere has been ascribed to the need to ameliorate conditions of oxygen starvation (Leisola et al., 1983
;Michel et al., 1992
). To overcome the oxygen limitation to LiP production, a practice has evolved that involves exposing hyphal pellets to an atmosphere of pure oxygen when the culture medium has been depleted of carbon (supplied as glucose), nitrogen or sulphur (Kirk et al., 1978
; Bar-Lev & Kirk, 1981
; Leisola & Fiechter, 1985
; Faison & Kirk, 1985
; Jager et al., 1985
; Asther et al., 1987
; Michel et al., 1990
; Dosoretz et al., 1990
; Dass & Reddy, 1990
; Bonnarme et al., 1993
).
Oxygen has, however, the potential to give rise to toxic oxygen-free radicals that are capable of oxidizing, fragmenting and cross-linking proteins, carbohydrates, lipids and nucleic acids (Wolff et al., 1986 ). In this context, surprisingly few reports on the fungitoxic effects of exposing fungal cultures to elevated concentrations of oxygen have been made. Reid & Seifert (1980)
suggested that increasing oxygen concentration as distinct from increasing atmospheric pressure might be fungitoxic. Later, Leisola et al. (1984)
found that high oxygen tensions were fungitoxic at low (0·1%, w/v) glucose levels, but not at high glucose levels. They proposed that the supply of higher concentrations of glucose allowed the formation of thicker mycelia, which protected against the toxic effects of oxygen. The majority of workers in the field continue to use fungal culture conditions with excess carbon (glucose) and limiting nutrient nitrogen to trigger ligninolysis. Under this regime the fungus secretes copious amounts of extracellular polysaccharide (Buchala & Leisola, 1987
). However, frequently exposing the cultures to oxygen ultimately achieves its metabolic degradation (Dosoretz et al., 1990
) so that sufficient oxygen may diffuse into the hyphae.
In this study, we looked at the intracellular architecture of fungal hyphae from agitated liquid cultures, to learn more about the interrelationship between carbon supply and oxygen in triggering ligninolysis under these conditions. We found that cultures maintained on limiting glucose and exposed to an atmosphere of pure oxygen as well as cultures maintained on cellulose showed a similar massive loss of internal organization of structure. Both cultures also produced LiP, suggesting that LiP synthesis may arise in response to oxidant stress.
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METHODS |
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Conidiospores (2x107) of P. chrysosporium were used to inoculate 600 ml culture medium in 2 l Erlenmeyer flasks. Semi-static cultures (carbon-limited) were obtained by incubating flasks in an air atmosphere at 37 °C on a rotary shaker at 150 r.p.m., 2·5 cm diameter cycle, for the primary phase of growth. Culture flasks were fitted with rubber stoppers through which two glass tubes fitted with gas-sterilizing filters were inserted. When glucose was depleted, fungal pellets were concentrated threefold for the enzyme production phase by decanting off 400 ml medium as in Leisola et al. (1985) . Flasks were then purged with 100% oxygen (10 min at 1 l min-1), sealed under a positive pressure of oxygen and incubation was continued at 60 r.p.m. to encourage the formation of mats of mycelial growth. In cellulose-maintained cultures, flasks were closed with foam stoppers, and continuously agitated at 130 r.p.m. (2·5 cm diameter cycle) (37 °C) in an air atmosphere throughout growth and enzyme production phases.
Enzyme assays.
LiP was measured in the extracellular medium with veratryl alcohol as substrate, according to Tien & Kirk (1984) . One unit (U) of activity is defined as the amount of enzyme catalysing the oxidation of 1 µmol veratryl alcohol min-1. Extracellular glucose and H2O2 were determined by enzyme-coupled assay based on the Trinder colorimetric method, with 4-aminophenazone as substrate, horseradish peroxidase and, for glucose determinations, glucose oxidase, which oxidized glucose with the stoichiometric production of H2O2. The concentration of H2O2 solutions in double-distilled water was determined using
=0·036 mM-1 cm-1. Residual cellulose was filtered from culture broth and from pellets extensively washed with distilled water and was estimated with K2Cr2
reagent according to Wood & Bhat (1988)
. For intracellular catalase, superoxide dismutase and protein analyses, 56 g mycelia was harvested, rinsed and dried with absorbent paper and mechanically ruptured in 1 ml extract buffer (50 mM potassium phosphate buffer, pH 7·5; 0·1 mM EDTA; 0·5 mM PMSF) over liquid nitrogen. Samples were clarified at 10000 g for 1 min, and assayed immediately. Catalase was measured with H2O2 (100 mM) as substrate in 50 mM potassium phosphate buffer (pH 7·8), 0·1 mM EDTA, by measuring the initial (100200 s) linear rate of decrease in A240 (Chance et al., 1979
). One unit of catalase was defined as that amount of enzyme able to decompose 1 µmol H2O2 min-1, at 25 °C, using
=0·036 mM-1 cm-1 for H2O2. Superoxide dismutase was assayed according to McCord & Fridovitch (1969)
by measuring the inhibition of reduction of nitro blue tetrazolium (NBT) by superoxide anions, generated by the xanthine/xanthine oxidase system. The assay mixture contained 0·1 mM EDTA, 0·15 mM xanthine, 0·15 mM NBT and 10 µl xanthine oxidase in 50 mM potassium phosphate buffer (pH 7·5). The rate of reduction of NBT was monitored at 560 nm. One unit of superoxide dismutase was defined as that amount of enzyme able to cause half-maximal inhibition of NBT reduction. The carbonyl content of proteins was measured according to Reznick & Packer (1993)
using 2,4-dinitrophenylhydrazine. Protein-bound hydrazones were detected spectrophotometrically with a Perkin Elmer 555 spectrophotometer and the carbonyl content was calculated from the A370 using an absorption coefficient for 2,4-dinitrophenylhydrazine of 22000 M-1 cm-1. Protein was measured by the Bradford method (1976)
with ovalbumin as standard.
Transmission electron microscopy (TEM).
For TEM, samples were fixed for 4 h at room temperature in 2% (v/v) paraformaldehyde containing 2·5% (v/v) glutaraldehyde in 0·1 M phosphate buffer (pH 4·5). The material was washed twice with 0·1 M phosphate buffer and post-fixed in 1% (v/v) osmium tetroxide in phosphate buffer (pH 4·5) for 1 h at 4 °C, then rinsed with buffer, then distilled water, and stained in 2% (w/v) uranyl acetate in 70% (v/v) ethanol for 15 min at room temperature. Thereafter, samples were dehydrated in an ethanol series (70100%, 10% steps for 10 min) and embedded in Agar 100 resin (Agar 100 Resin kit from Agar Scientific). The material was sectioned with a Reichert Ultracut. Hyphal sections were obtained from the outer cortex of the pellets. Initially, 2-µm-thick sections were cut and observed under a light microscope. The staining with 0·5% (w/v) methylene blue, 0·5% (w/v) azure II, 0·5% borax revealed when sections contained fungal material, at which point thin sections for TEM studies (5070 nm thick) were collected on nickel grids. At least 25 sections of each type of fungal material were observed under the electron microscope. Before TEM observations, grids were stained in 2% (w/v) uranyl acetate and lead citrate (Reynolds, 1963 ). TEM studies were carried out with a Philips 400T transmission electron microscope, using an accelerating voltage of 80 kV.
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RESULTS AND DISCUSSION |
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In light of these results, it was of interest to investigate the intracellular architecture of cultures that synthesized LiP without exposing them to a pure oxygen atmosphere. For this purpose, we selected liquid cultures agitated in air with cellulose as the carbon source. Sections were taken from the outer region of pellets sampled on day 4 when LiP was detected in the extracellular medium (see Fig. 3c). The representative micrographs in Fig. 3(a
, b
) illustrate that these hyphae showed a remarkable similarity to those exposed to pure oxygen. Irregularly shaped electron-translucent areas or voids (cv) in the cytoplasm that are not surrounded by a membrane are evident, as well as electron-dense particles, possibly cytoplasmic protein aggregates. Cytologically normal hyphae as depicted in Fig. 2(a
, b
) were never observed. Mitochondria (m) maintained an orthodox organization, as before, with the cristae visible as lamellae parallel to the long axis of the organelle, in a configuration typical of filamentous fungi (Markham, 1995
). In this micrograph, the presence of a nucleus (n) indicates that the cell is part of the younger mycelium, and not of an ageing hyphal network.
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ACKNOWLEDGEMENTS |
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Received 16 August 1999;
revised 18 November 1999;
accepted 2 December 1999.