Effect of different nitrogen supplementations on fermentation of bagasse by Sporotrichum pulverulentum wild type and mutants

Notes and brief articles support of this idea was the observation that mCLAM and DNP stimulated respiration to similar extents. In our study, stimulation of C. geophilum oxygen uptake by DNP (Fig. 1, A) was substantially less than that obta ined with SHAM. SHAM has also been reported to stimulate respiration in Candida albicans (Kot et al., 1976). However, in this case SHAM increases oxygen uptake of cyanide-sensitive oxidases by removing the competitive or regulatory effect of the alternative pathway. Regardless of the mechanism underlying SHAM-stimulated oxygen uptake, these observations make it difficult to estimate the in vivo contribution of the alternative pathway using conventional procedures. The results of this study, using pure cultures of an ectomycorrhizal fungus, confirm earlier studies ofwillow and beech ectomycorrhizas with regard to the presence of cyanide-insensitive respiration. The possible ph ysiological sign ificance of th is alternative pathway in these tissues is at pres ent unclear.

REFERENCES

ANTIBUS, R. K. (1980). Mechanisms of acclimation to lowered growth temperatures in isolates of arctic and temperate ectomycorrhizal fungi . Ph.D. Dissertation, Virginia Polytechnic Institute & State University, Blacksburg, VA.

ANTIBUS, R. K. , TRAPPE, J . M . & LINKINS, A. E . ( 1980). Cyanide resistant respiration in Salix nigra endomycorrhizae. Canadian Journal of Botany 56, 14-20. COLEMAN, J. O. D. & HARLEY, J . L. (1976). Mitochondra of mycorrhizal roots of Fagus syloatica. New Phytologist 76,3 17-33°. HARLEY, J. L. & REES, T. ap. (1959). Cytochrome oxidase in mycorrhizal and uninfeeted roots of Fagus sy luatica. Ne w Phytologist 58, 364-386. HARLEY, J. L., MCCREADY, C. C., BRIERLEY, J. K. & JENNINGS, D. H . (1956). The salt resp iration of excised beech mycorrhizas II. The relationship between oxygen consumption and phosphate absorption . New Phytologist 55, 1-28. HARLEY, J . L. , MCCREADY, C. C. & WEDDING, R. T . (1977). Control of respiration of beech mycorrhizas during ageing. New Phytologist 78, 147-159. KOT, E. J., OLSON, V. L., ROLEWIC, L. J. & MCCLARY, D. O. (1976). An alternate respiratory pathway in Candida albicans. Antonie van Leeuwenhoek 42 , 33-48. LAMBERS, H . (1980). The physiological significance of cyanide-resistant respiration. Plant Cell and Environment 3, 293-302. LAMBERS, H. (1982). Cyanide-resistant resp irati on : A non-phosphorylating electron transport pathway acting as an energy overflow. Physiologia Plantarum 55, 47 8-485 . LATIES, G. G. (1982). The cyanide-resistant, alternative path in higher plant respiration. Annual Review of Plant Physiology 33, 519-555. SOLMOS, T . (1977). Cyan ide resistant respiration in higher plants. Annual Review of Plant Physiology 28, 279-297·

EFFECT OF DIFFERENT NITROGEN SUPPLEMENTATIONS ON FERMENTATION OF BAGASSE BY SPOROTRICHUM PULVERULENTUM WILD TYPE AND MUTANTS BY FAIEZ AL-ANI AND JOHN E. SMITH

Department of Bioscience and Biotechnology, Applied Microbiology Division, University of Strathclyde, Glasgow GIIXW Supplementation of bagasse with peptone and malt extract, followed by fermentation by Sporotrichum pulverulentum wild type, increased the in vitro digestibility of bagasse from 27 to 36 and 39 % respectively. Yeast extract and simple nitrogen sources, e.g. NH 4N03 , NH 4 CI and asparagine, reduced the in vitro digestibility of bagasse below the untreated control value. With all the above nitrogen sources, the mutants 44-2 and 63-2 of S. pulverulentum also reduced the digestibility of bagasse below the control value. In most readily available sources of lignocellulose, e.g. wood, straw, bagasse, etc. the presence oflignin severely restricts the availability of cellulose for ruminant digestion. Biological delignification using lignin-degrading micro-organisms such as the white-rot fungi could make this carbohydrate available if rapid lignin removal could be achieved without extensive carbohydrate loss (Reid, 1983). Tran s. Br . mycol. S oc. 86 (1), (1986)

The role of nitrogen supplementation on microbial delignification of lignocellulosic materials has been extensively examined with culture media (Zeikus, 1981), woody materials (Reid, 1983) and straw (Zadrazil & Brunnert, 1980). The present study examines the role of nitrogen supplementations on lignin degradation of sugarcane bagasse by Sporotrichum puluerulentum Novobranova (AT CC

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166

Notes and brief articles

32 629 ) and its mutants 63-2, a third generation mutant, non-sporulating , cel" , xyl", and 44- 2 a selected eel" revertant of mutant 44, nonsporulating. Cultures were kindly supplied by Professor Karl-Eriksson, Swedish Forest Products Research Laboratory, Box 5604, S-22486, Stockholm, Sweden and have been used successfully in wood delignifying studies (E riksson, Johnsrud & Vallander, 1983). The sugarcane bagasse used in this study was obtained from the Egyptian Sugar Company, Cairo (crop 1979/1980). The bagasse was pulverized in a coffee mill (M oulinex), passed through a sieve (20 mesh) and stored at 4 °C in an air-tight container until used, A modified z.z-dimethyl succinate (DMS) medium (F enn & Kirk, 1979) was used throughout this study. Mycelial inocula were prepared by cultivating each fungus in 100 ml DMS medium in 500 ml Erlenmeyer flasks and incubating in a controlled temperature shaker at 200 rev, min-I for 1 week , Incubation temperatures were 34° for the mutants and 39° for the wild type. The mycelium was harvested and gently macerated in a sterilized Waring blender. The resultant suspension containing intact mycelial fragments was transferred into sterilized screw-eapped bottles and used d irectly or sto red at 4°. Semi-solid substrate fermentations in static flasks were used, Pulverized bagasse (10 g) was placed in 500 ml wide neck Erlenmeyer flasks fitted with non-absorbent cotton-wool plugs . Baga sse was moistened with 55 ml aqueous solutions of yeast extract, peptone, malt extract, NH 4N03 , NH4Cl or asparagine (each containing 1 % nitrogen), adjusted to pH 4'5 . Bagasse enriched with 55 ml DMS medium or water was prepared for comparison. The flasks were autoclaved at 121° for 30 min, cooled and inoculated with 5 ml mycelium suspension, so that the final ratio of solid: liquid was 109: 60 ml. The flasks were incubated for 2 weeks at the specified temperatures, At the completion of the experiment, the flask contents were dried on aluminium foil at room temperature and the fermentation evaluated by determining the in vitro digestibility (Jones & Hayward, 1973, 1975) and acid detergent fibre (AD F ) and acid detergent lignin (AD L) (van Soest, 1963). The effects of the different nitrogen sources on the in vitro digestibility, acid detergent fibre (AD F ) and acid detergent lignin (AD L) of bagasse fermented by the wild type are shown in Table 1, Increases in in vitro digestibility, when compared to the untreated control (27'05 % ), occurred in decreasing order of magnitude with DMS (39'22 % ), malt extract (38'73 % ), water (37'15 % ) and peptone (36' 19 %). Decreases in digestibility below that of the untreated control occurred with Trans. Br . my col. S oc, 86 (1), (1986)

Table 1. Effect of diffe rent nitrogen supplementations on fermentation of bagasse by Sporotrichum pulverulentum Addition Unfermented control Water NH.N03 Asparagine NH.C1 Yeast extract Peptone Malt extract DMS

Digestibility"

ADFt

27-05 ± 0'29

50'°5 ± 0'01

7'57 ± O' l l

37-15±0-29 13-22±0 -07 15-84±0-08 16-70±O '06 18-94±0-67 36'19±0-38 38-73±O'66 39'22±O'21

52'40±O'45 51'10 ± 0-58 53-11 ±0-35 49'41±0-80 51-79 ±O'75 42-92±o'81 49'34±0-32 51'80 ± O'39

8'04 ±0-32 1O-05 ±o-20 ll '75 ±0-01 9'48 ±0-44 1l -ZZ ±O '29 10-Z0±0-32 8'43 ±0-14 7'09 ±0-88

• % solubility (digestibility) of dr y matter.

t % acid

detergent fibre of dry matter, AD lignin of dry matter, Each value is the mean of three determinations,

*%

NH4N03 (13'22 % ), asparagine (15'84 % ), NH 4CI (16'70 %) and yeast extract (18'94 % ). The measured lignin content (AD L ) of baga sse increased after fermentation in all samples except with DMS supplementation where the lignin content showed a small decrease. In general, there were onl y slight differences in ADF values after fermentation in all cases, except for bagasse enriched with peptone where the ADF value dropped to 42 '92 %, The effects of NH 4N0 3 , peptone, DMS and water supplementation on th e fermentation of bagasse by the mutants are shown in Table 2. With both mutants supplementations with DMS or water gave closely similar digestibilities but still below the untreated control values. Mutant 44-2 gave extremely low digestibility results with NH 4N0 3 (10' 48% ) and peptone (12'41 % ), while mutant 63-2 gave low digestibility with NH 4N0 3 (14'68 % ) but much higher results with peptone (25'75% ), although this value was still below the control value (27'05 %), The ADL values or measured lignin content increased with all supplementations for both mutants , The highest values for mutant 63-2 were with DMS (10'03 % ) and water (10'04 % ) whereas with mutant 44-2 the highest values occurred with NH4N03 (11'49%) and peptone (12'63 % ). The ADF values increased with all supplementations for both mutants. The highest values for each mutant occurred with DMS and water supplementation, Thus, the present results of fermenting bagasse with the wild type of S, puluerulentum showed reduced digestibility with simple nitrogen sources but increased digestibility with the more complex

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Notes and brief articles Table 2. Effect of different nitrogen supplementations on fermentation of bagasse by Sporotrichum pulverulentum mutants 63-2 and 44-2 Addition Digestibility ADF Unfermented 27'05±O'29 50'05±O'Ol control Mutant 63-2 14'68±o'08 54"42± 1'04 NH.NO. Peptone 25'75±O'37 50'7 0±o'45 DMS 19'62 ±O'23 57'94± 1'11 Water 20'63 ±o'44 59'4 6±O'98 Mutant 44-2 NH.NO. 10'48±o'53 54'56±o'20 Peptone 12"41± 0'23 55'37 ± 0'20 DMS 19'77±O'27 57'54±O'64 Water 20,66±o'18 57'62±O'52 Results calculated as in Table 1.

ADL 7'57±O'l1

9'15±o'34 9'26±o'14 10'°3 10'04±O'34 11"49±O'05 12'63 ± 0'24 9'89±O'25 10'06±o'14

forms of nitrogen, except yeast extract. These results are in substantial agreement to those of Reid (1983) with Phanerochaete chrysosporium on wood and Ander, Eriksson & Yu (1983) wth S. pulverulentum also growing on wood. It has been suggested that vitamins or growth factors present in peptone and malt extract, could be responsible for the stimulation of bagasse digestibility (Reid, 1983)· Zadrazil & Brunnert (1980) similarly found that supplementation with NH4N0 3 reduced the digestibility of straw fermented by some white-rot fungi. They considered the reduction in digestibility to be due to the cessation of degradation at a point where cellulose was already available for the fungus, but before most of the lignin had been metabolized, The decrease in digestibility of bagasse supplemented with NH4 Cl could also be due to the decrease in pH. Reid (1983) considered that supplementation with NH 4C1 decreased the pH far enough from the optimal to decrease lignin breakdown. In the present study the highest digestibility and lowest lignin content were obtained with bagasse supplemented with DMS medium and this may support the role of pH in lignin degradation since the pH is effectively controlled by the z.z-dimethyl succinate (Reid, 1979; Fenn & Kirk, 1979). It should be further noted that bagasse with only water and fermented by S. puluerulentum gave the third highest digestibility after those supplemented by DMS medium and malt extract (Table 1) and also the second lowest lignin content after that supplemented with DMS. Reid (1983) found that water only supplemented wood consistently gave the highest ratio of lignin degradation to the total weight loss and consequently the most digestible Trans. Br. mycol. Soc. 86 (1), (1986)

product. Zadrazil & Brunnert (1982) similarly found that S. pulverulentum increased the digestibility of straw most in the absence of NH 4N0 3 • Reid (1979) has considered that increasing the nitrogen supply could inhibit lignin degradation in two ways, viz. by repressive concentrations of nitrogen delaying the appearance and limiting the maximum rate of lignin degradation, or by increasing the rate of carbohydrate depletion and thereby shortening the period of lignin metabolism. Mutants 44-2 and 63-2 were derived by Eriksson et al. (1983) and produce higher phenol oxidase activity than the parent strains, i.e, wild type and mutant 44, are positive for xylanase and show increased ligninolytic activity with wood. However, the present studies showed that the activity of these mutants towards enhancing the digestibility and lignin degradation of bagasse were inferior to the wild type. Although many results, including those of the present study, would indicate that nitrogen repression is common for lignin degradation by white-rot fungi, it cannot be taken as the rule, since the ligninolytic activity of two important ligninolytic fungi, namely Lentinus edodes and Pleurotus ostreatus are not inhibited by high nitrogen concentrations (Leatham & Kirk, 1983; Ander et al., 1983). REFERENCES

ANDER, P" ERIKSSON, K.-E. & Yu, H.-S. (1983). Physiologicalrequirementsfordegradationoflignin substances by Sporotrichum pulverulentum. European Journal of Applied Microbiology and Biotechnology 18,374-380.

ERIKSSON, K.-E., JOHNSRUD, S. C. & VALLANDER, L. (1983). Degradation of lignin and lignin model compounds by various mutants of the white-rot fungus Sporotrichum puluerulentum. Archives of Microbiology 135, 161-168. FENN, P. & KIRK, T, K. (1979). Ligninolytic system of Phanerochaete chrysosporium: inhibition by o-phthalate. Archives of Microbiology 123, 307-309. JONES, D. I. H. & HAYWARD, M. V. (1973). A cellulase

digestion technique for predicting the dry matter digestibility of grasses. Journal of Science of Food and Agriculture 24, 1419-1426,

JONES, D, I. H. & HAYWARD, M. V. (1975), The effectof pepsin pretreatment of herbage on the prediction of dry matter digestibility from solubility in fungal cellulase solutions, Journal of Science of Food and Agriculture 26, 7 11-7 18, LEATHAM, G, F. & KIRK, T, K. (1983). Regulation of ligninolytic activity by nutrient nitrogen in white-rot Basidiomycetes, FEMS Microbiology Letters 16, 65-67. REm, I. D. (1979). The influence of nutrient balance on lignin degradation by the white-rot fungus Phanerochaete chrysosporium. Canadian Journal of Botany 57, 2050-2058.

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Notes and brief articles

REID, 1. D . (1983). Effects of nitr ogen supplements on degradation of aspen wood lignin and carbohyd rate comp onents by Phanerochaete chrysosporium. Applied and Environmental M icrobiology 45,830-837. VAN SOEST, P. J. (1963). Use of detergents in the anal ysis of fibrous feeds. II . A rapid method for th e determination of fibre and lignin. J ournal of the A ssociation of Official Analy tical Chemists 46, 829-835. ZADRAZIL, F . & BRUNNERT, H. ( 1980). The influence of amm onium nitrate supplementation on degradation and in vitro digestibility of straw colonised by higher fung i. European J ournal of A pplied Mic robiology and Biotechnology 9, 37- 44·

ZADRAZIL, F . & BRUNNERT, H. (1982). Solid state fermentat ion of lignocellulo se cont aining plant residues with S porotrichum puluerulentum Nov. and Dichomitus squalens (Karst.) Red . European J ournal of Applied Microbiology and Biotechnology 16,45-51. ZEIKUS, J. G. (1981). Lignin metabolism and the carbon cycle: polymer biosynthesis, biodegradation and environm ent al recalcitrance. In A dva nces in Mic robial Ecology , vol. 5 (ed. M . Alexander), pp . 211- 243. New York and Lond on : Plenum Pr ess.

ANTIBIOTIC ACTIVITY OF OO SPOREIN FROM VERTICILLIUM PSALLIOTAE BY M. WAINWRIGHT, R. P . BETTS

Department of Microbiology, University of Sheffield AN D D. M . TEALE

Department of Virology, University of Sh effield, Sh effield SlO 2 TN, U.K. Verticillium psalliotae produces a deep red pigment, the dibenzoquinone oosporein, in Czapek-Dox medium. Studies on the antibiotic act ivity of this compound are reported here. During studies in which some of John T yndall's experiments on microbial antagonism were repeated (Wainwright, 1985) a fungus was isolated which produced a deep red pigment in the culture medium. The crude ether extract of the acidified medium was shown to have weak antibiotic activity . The isolate was identified as Verticillium psalliotae Treschow, and the red pigment as th e dibenzoquinone oosporein. V . psalliotae is a pathogen of the commercial mushroom, while oosporein is of particular int erest because it is a mycotoxin. The fungus was isolated as described previously (Wainwright, 1985 ), transferred to Czapek-Dox (Oxoid) agar and grown for 10 days at 25 DC. A mycelial disc (6 mm ) was then inoculated into Czapek-Dox medium (Oxoid, 1 1 in a 5 I Erlenmeyer flask). The flasks were initially incubated at room temperature (18°), but subsequent studies showed that maximum pigment formation occurred at 25° in the dark. The isolate gre w on Czapek-Dox agar as a white colony, whose underside was or igin ally also white, but after about 10 days a pink anthocyanin-like colouration appeared, darkening to purple red. The pigment eventually disappeared, and distinct, broadly concentric, gre y saltation zones appeared on th e underside of the colony . The isolate initially app eared sterile, but vert icillate spore-bearing phialides eventually appeared in groups of three. Spore pr oduction was more Tran s. B r. my col. Soc . 86 (1), (1986)

pronounced when the isolate was grown on corn meal agar (Oxoid) . It produced two sizes of conidia and was difficult to distinguish from V . lamellicola (F . E . V. Smith) W . Gams, but was classified as V. psalliotae on the basis of its ability to form a deep red pigment , and becau se of th e cur ved appearance of the larg er conid ia. The isolate grew on the surface of liquid Czapek-Dox medium on standi ng, as a white mycelial mass, with a buff-coloured underside. A deep red pigment was excreted into the medium. At room temperature, in the light, the fungus produced various pigmentations, including grey, light pink, yellow and dark red. This variability in pigment formation is difficult to explain, since agar discs taken from the same inoculum grew into mycelium which produced different coloured pigments, even though the medium and incubation conditions were identical. On further incubation however , these pigments all eventually turned to the more typ ical red-purple colour. The formation of a red pigment in the growth medium is typical of V. psalliotae, and was described at some length by Treschow (1941) in his or iginal description of th e species, and later confirmed by Atkins (1947). Wh en acidified to pH 2'0, the filtered culture medium turned a deeper red colour, and thi s pigment could be dissol ved in ether (in which it turned yellow). A mixture of red and yellow residues remained when the ether evapora-

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