Adaptability to submerged culture and amtno acid contents of certain fleshy fungi common in Finland Mar j a L i is a H a t t u l a and H . G. G y ll e n b e r g Department of Microbiology, University of Helsinki, Finland The literature on the possibilities of sub­ must be considered when the suitability of merged culture of macrofungi is already fungi for submerged production is evaluated. voluminous. However, the interest has merely The present work concerns a collection of been restricted to the traditionally most va­ fungi common in Finland which have been lued fungi, particularly members of the gene­ investigated according to the criteria descri­ ra Agaricus and M orchella (HuMFELD, 1948, bed above. 1952, HuMFELD & SuGIHARA, 1949, 1952, Su­ GIHARA & HuMFELD, 1954, SzuEcs 1956, LITCHFIELD, 1964, 1967 a, b, LITCHFIELD & al., MATERIAL 1963) . The information obtainable from stu­ dies on these organisms is not especially en­ The material consisted of 33 species of fun­ couraging because the rate of biomass pro­ gi, 29 of which were obtained from the duction seems to be too low to compete fa­ Department of Silviculture, University of ,·ourably with other alternatives of single cell Helsinki, through the courtesy of Professor P. protein production (LITCHFIELD, 1967 a, b). Mikola and Mr. 0 . Laiho. Four species were On the other hand, the traditional use of fun­ isolated by the present authors by taking a gi as human food all over the world is a sterile piece of the fruiting body at the point significant reason for continued research on where the cap and the stem are joined and the submerged production o.f fungal mycel­ by transferring it on a slant of suitable agar. ium. If the fungal mycelium is comparable The species considered a!'e listed below fol­ with other single-cell protein preparations as lowing the nomenclature of M. MosER to its protein content and amino acid com­ (1967). (For the species indicated by an position, it seems to gain preference over asterisk fruiting bodies were collected for other alternatives due man's traditional know­ comparison whereas the species with the ledge and experience concerning the toxicity + label were isolated in pure culture by characteristics and other conditions influen­ the pl'esen t authors. ) cing the edibility of fungi. A particular aspect requires attention in this connection. Clavaria ligula The traditional «mushroom« used a.s food Cantharellus · cibarius * concerns the fruiting bodies whereas the sub­ Suillus grevillei ( elegans) merged cultivation produces preparations of S. luteus * mycelium. Therefore it seems that besides the S. bovinus * adaptability to submerged growth the simi­ S. variegatus * larity in composition of the mycelial pre­ Boletus edulis * + paration to the corresponding fruiting bodies L eccinum testaceo-scabrum * + 39 L . sp. («Boletus versipellis«) how rapidly the fungi used the sugar in the L. scabrum * medium. The first screening experiment to Paxillus incolutus * omit the most slowly growing species was P. atrotomentosus made by growing the mycelia in the three Hygrophoropsis aurantiaca («Cantharellus following media: a. «) a ) 5 % malt extract, Tricholomopsis rutilans (Tricholoma r.) b) Modess.' medium (MoDEss 1941) , Tricholoma flavobrunneum c) Reusser's medium (REusSER & al., 1958) T. imbricatum the composition of which is as follows: Lyophyllum loricatum Armillariella mellea (Armillaria m.) * Glucose 40 g, Ammonium tartrate 8 g, KH2P04 Collybia dryophila * 0.5 g, MgSo ·7 H 0 0.,5 g, CaC1 50 mg, FeS04 ·7 C. butyracea 4 2 2 H 2 0 2 mg, MnS03 • H 20 2 mg, Pyridoxine 1 mg, A1icromphale perforans ( M arasmius p.) p-Aminobenzoic acid 1 mg, Thiamine 0.5 mg, Marasmius androsaceus Distilled water 1000 ml. The glucose was sterilized Macrolepiota procera (Lepiota p.) separately and the vitamins were added in separate Agaricus arvensis * + solution which was sterilized by filtration. All three Cystoderma amianthinum media were bottled in 250 ml ·Erlenmeyer-flasks, Coprinus comatus * + 80. m1 in each, and they were sterilized at ll6°C Hypholoma capnoides for 20 minutes. Kuehneromyces mutabilis (Pholiota m.) The inoculation was made by transferring C ortinarius hemitric h us two agar slants of every species into 80 ml Lactarius rcpraesentaneus ·* 5 % malt extract. The flasks were incubated L. necator* 10 days at room temperature as stationary L. deliciosus aggr. * cultures. The grown mycelium was washed L. rufus* with sterile distilled water and was then broken by use of a sterile Ultra-Turrax­ The pure cultures were maintained on Modess' machine. The homogenized mycelium was agar the composition of which is the following suspended into 80 ml of sterile water and 10 (MonEss, 1941): KH2 P04 0.5 g, MgS04 ·7 H 2 0 ml of this suspension was used to inoculate 0,5 g, NH4 Cl 0,5 g, Malt extract 5 g, Glucose 5 g, each flask. Because of the different amount of , FeCl3 1 % sol. 0.5 ml, Agar. 15 g, Distilled water growth of the test organisms the suspensions 1000 ml. The medium was sterilized at 116 o C for employed as inocula were made visually as 20 minutes. similar in density as possible. Every organism The mycelia were inoculated on new agar slants and medium was tested in duplicate. The once a month and the tubes were kept four weeks flasks were incubated for two weeks in a at room temperature (22-24°C) after which they rotary shaker (200 rpm) at 28 °C. After this were transferred to refrigerator temperature. For period the growth was scored visually. Table sixteen of the test organisms (indicated by in the 1 shows the results of the experiment. above list) fruiting bodies were collected. These On the basis of the results of the experi­ were cleaned of dust and sand by scraping and no ment indicated in Table 1, the following water was used for cleaning. The fruiting bodies species were omitted from further study: were cut into small pieces and dried at 40°C until Paxillus involutus, P. atrotomentosus, Tricho­ they were easy to grind to fine powder. The powder loma flavobrunneum, Tricholomopsis ru­ was kept in a closed wessel in tfue refrigerator. tilans, Lactarius repraesentaneus, L. deli­ ciosus, Mar.asmius perforans, M . androsaceus, Cystoderma amianthinum, Boletus edulis and RESULTS Boletus «versipellis«. An experiment concerning the sugar con­ Adaptability to submerge d culture sumption ·was made with the remaining species in order to evaluate the utilization of The adaptability to submerged culture was carbon and the ylield of mycelium per quan­ investigated by some experiments where the tity of sugar used. Reusser's medium, which mycelia were grown on different media. The contains glucose, and Modess' medium, which primary criterion in these experiments was contains maltose in addition to glucose, were 40 Table I. Submerged growth of fungi in different Table 2. The efficiency of mycelium production, media. + + + growth excellent, + + good gram of mycelium/ I 00 grams of mgar. growth, + weak grofth, - no growth (the amount mycelium equal to the inocolum). Mushroom Reusser's Modess' medium medium Cantharellus cibarius 54.6 46.7 Suillus grevillei 42.7 42.2 "'8 ..;: 8 ~ ::l 1;l ::l Suillus luteus 12.9 24.3 Species V>·~ "8~ ;:l"d Suillus bovinus 53.3 22.6 ~8 ..::"' 8"' Suillus variegatus 42.4 16.9 Clavaria ligula ++ +++ +++ Leccinum testaceo-scabrum 36.4 36.2 Cantharellus cibarius +++ +++ +++ Leccinum scabrum 1.5 3.7 Suillus grevillei ++ ++ ++ Hygrophoropsis aurantiaca 26.8 33.5 Suillus luteus ++ + ++ Tricholoma imbricatum 1.0 5.4 Suillus bovinus +++ ++ +++ Lyophyllum loricatum 48.7 36.7 Suillus variegatus + + + A rmillariella melle a 43.8 32.4 Boletus edulis + + Collybia dryophila 8.8 24.5 Leccinum testaceo-scabrumt- + + +++ +++ Collybia butyracea 1.3 8.2 «Boletus versipellis« ++ Macrolepiota procera 12.1 Leccinum scabrum ++ ++ Agaricus arvensis 1.8 6.9 Paxillus involutus + + + Coprinus comatus 0.8 2.6 Paxillus atrotomentosus Hypholoma capnoides 3.9 4.8 Hygrophoropsis Kuehneromyces mutabilis 6.0 28.0 aurantiaca + + + + Cortinarius hemitrichus 57.4 50.2 Tricholomopsis rutilans -+ ++ Lactarius necator 4.5 13.4 Tricholoma Lactarius rufus 0.7 2.5 flavobrunneum + Clavaria ligula 1.0 1.9 Tricholoma imbricatum ++ Lyophyllum loricatum + + + the suspension was inoculated into 80 ml of Armillariella mellea +++ +++ +++ the medium in a 250 ml Erlenmeyer flask. Collybia drJ•ophila + + + +++ The flasks were incubated in a rotary shaker Collybia butyracea + + ++ ++ (200 rpm) at 28°C. Incubation was con­ Micromphale perforans + + + tinued until a considerable amount of rapidly Marasmius androsaceus + + + growing mycelium had developed. During the M acrolepiota pro cera + + ++ ++ incubation the volume of the medium was Agaricus arvensis + + + controlled by adding distilled water to com­ Cystoderma amianthinum + + pensate for evaporation losses. The mycelium Coprinus comatus + + + was separated by Buchner-filtration, and was Hypholoma capnoides + + ++ + then dried to constant weight. Table 2 shows Kuehneromyces mutabilis + + + the efficiency of submerged growth expressed Cortinarius hemitrichus - ++ +++ as grams of mycelium per 100 grams reducing Lactariusrepraesentaneus - sugar. Lactarius necator ++ ++ ++ The results clearly indicated that several of Lactarius deliciosus the species included in this experiment grow aggr. too slowly to be useful for submerged my­ Lactarius rufus + ++ celium production. The growth rate seemed to depend on the medium as well as on the chosen for this experiment. Sugar deter­ species. However, both the media emploved minations were made (a) before inoculation are commonly used in the submerged culti­ and (b) at the time the incubation was in­ vation of fungi. In either or both of the terrupted according to the method of Ber­ media the following ten species yielded trand ( GR:oSSFELD, 1935). A two weeks old mycelium to an amount higher than 25 % stationary culture of mycelium was used as of the weight of the utilized sugar and might inoculum. The mycelium was broken by thus be worth further attention: C antharellus Ultra-Turrax into sterile water and 10 ml of cibarius, Hyprophoropsis aurantiaca, Suillus 41 bovinus, S.