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Appl Microbiol Biotechnol (1986) 24: 180--185 Applied Microbiology Biotechnology © Springer-Verlag 1986 Screening of white-rot fungi for biological pretreatment of wheat straw for biogas production H. W. Miiller and W. Trfisch Fraunhofer-Institut fiir Grenzfl/ichen- und Bioverfahrenstechnik, Nobelstr. 12, D-7000 Stuttgart 80, Federal Republic of Germany Summary. Twenty two basidiomycetes, mostly white rot fungi, were grown on wheat straw. Lig- nin-, cellulose-, and hemicellulose-degradation was recorded in order to find a species growing on lignin preferably. The "oyster-mushroom" Pleurotus sp. "florida'" showed fastest delignifica- tion of all tested fungi. Straw pretreated by this fungus was fermented anaerobically to biogas. The gas yield produced from "myco-straw" is twice the amount from un- treated straw. Introduction Lignocellulose biomass is continuously produced in great quantities, giving rise, until recently, only to disposal problems. Because of its chemical composition it could be an excellent substrate for biotechnological processes. However, cellulose fi- brils are imbedded into a lignin matrix which pro- tects them from enzymatic attack. Straw, a lignocellulose waste material, can be pretreated chemically or physically to increase its biotechnological substrate value (Chahal et al. 1981; Detroy et al. 1981; Fan et al. 1981). White rot fungi can be used for the biological pretreatment. They are the only microorganisms known for their ability to degrade lignin com- pletely. Therefore, it should be possible to use these microorganisms to degrade the lignin com- ponent in ligno-cellulosic waste material to make the cellulose and hemicellulose components more accessible for further biotechnological use. Some of these fungi produce edible fruiting bodies -- a Offprint requests to: H. W. MOiler fact, that might be important when calculating process economy. The most investigated fungus for lignin de- gradation is Phanerochaete chrysosporium (Keyser et al. 1978; Kirk et al. 1978; Leisola et al. 1983; Tien and Kirk 1983; Ulmer et al. 1983) In the present work other white rot fungi were searched for selective lignin removal. The aim of this inves- tigation is to use the "myco-straw" as a substrate in a further biotechnological process: biogas pro- duction. Previous investigations have mainly been concerned with the use of "myco-straw" as an an- imal fodder (Zadrazil 1980). Materials and methods Organisms. The stock cultures of white rot fungi used in the degradation experiments were mostly isolated from fruiting bodies of wild growing species. Pleurotus sp. "'florida'" was iso- lated from commercial available summer oyster-mushrooms. Lentinus edodes originates from "Deutsche Sammlung for Mi- kroorganismen" (DSM 1899). Some tropical species, found in Brazil and Ecuador, are not yet identified. They were named according to the place of finding. The mutants of Pleurotus sp. "florida", named PFR 343 and PFP 551, were isolated from basidiospores from Pleurotus sp. "florida" according to the method of Eriksson (Eriksson and Goodeil 1974). Saponin was added at the rate of 2 g/1 to induc~ colonial growth of the mycelium. The microorganisms used were: Flammulina velu- tipes (Curt. ex. Fr.) Sing., Fomes marginatus (Fr.) Gill., Ganod- erma applanatum (Pers. ex. Fr.) Pat.,, Hapalopilus rutilans (Pers. ex. Fr.) Karst, Kuehneromyces mutabilis (Schff. ex. Fr.) Kumm, Laetiporus sulfureus (Bull. ex. Fr.) Murill, Lentinus edodes (Berk.) Singer, Nematoloma capnoides (FrO Karst., Phellinus robustus Karst., "Pichincha a'" "'Pichincha b" (tropi- cal species from Ecuador), PIeurotus sp. "florida" PFR 343, PFP 551 (cellulase deficient mutants from P. florida), Poria sp., "Rio rot" (tropical species from Brazil), Stereum hirsutum (Willd.) Pers., Stropharia rugosoannulata Fadow. ex. Murr., Trametes gibbosa (Pers. ex. Fr.) Fr., Tramates versicolor (Lin. ex. Fr.) Pilat., "'Tungurahua'" (tropical species from Ecuador). Fermentation. For solid state fermentations with the basidio- mycetes 300 g moistened wheat straw (water content 75%) inH. W. Mtiller and W. TrSsch: Screening of white-rot
fungi for biological pretreatment 181 1-L beakers was used. After sterilisation (90 min, 121°C) the straw was inoculated with mycelium of white rot fungi grown for 10 days on malt-extract agar slants. After incubation for 30, 60 and 90 days at 25 °C the contents of three beakers, re- spectively, were dried, weighted, milled and analysed. Loss of organic matter was estimated as the difference between the weight of the dried straw at the beginning and at the end of the solid state fermentation. Biogas fermentations were performed in four stirred 1.5-1 fermenters ("Biostat V", Braun Melsungen). An exactly weighed amount of straw was placed in the fermenter. De- pending on experimental conditions a different amount of freeze-dried cow manure was added and the fermenter filled up to 1.41 with tap water. To get comparable results, the same batch of freeze-dried cow manure was used in all fermenta- tions. All solids were added in such an amount, that the total dry matter in each fermenter was always 60 g (e. g., 4% total dry matter). The fermenter content was inoculated by the addi- tion of 100 ml of a bacterial suspension from a good working, laboratory-scaled biogas plant. Temperature was maintained at 37°C, pH was maintained at 7.2 by controlled addition of 5N NaOH. The biogas produced was collected in a graduated cylinder and analysed daily gaschromatographically (Carbo- sieve S-(126/140) column) for the amounts of CO2 and CH4. The enzymatic hydrolysis of straw cellulose was carried out in 50-ml Erlenmeyer flasks containing the various samples of milled straw. The cellulose content of the unfermented straw (control) and the several "mycostraws" had been ana- lysed before. The weight of each sample was adjusted to an equal cellulose content of 100 mg straw cellulose. Citrate buf- fer (8 ml, pH 4.8) and 1 ml cellulase solution (from Oxyporus spec., Merck), standardized to 0.5 filterpaper-units per ml, were added. The flasks were closed and incubated at 30°C in a shaking water bath. Every 24 h samples of the fluid were col- lected and analyzed for glucose by the glucose dehydrogenase test system (Merck). Analyses. Lignin, cellulose and hemicelluloses were deter- mined according to the acid and neutral detergent method of Goering and Van Soest (1970). Total carbon and total nitrogen were determined by combustion of the sample in a CHN-ana- lyzer ("CHN-Rapid', Heraeus). The volatile fatty acids were determined by HPLC (OPS II column with RP 18 Material, 5 Ixm). Results Biological degradation of wheat straw Wheat straw used for degradation studies con- sisted of 28.0% hemicelluoses, 42.9% cellulose, 16.0% lignin and 0.87% nitrogen. 22 basidiomy- cetes, including 2 cellulase deficient mutants of P. florida, were tested for their potential to degrade straw. All of them grew on this substrate, but de- graded it to a very different extent (Fig. 1). Od 30d 60d 90d 30d 60d 90d ~ fdry iO, O ~ i i .... i ~ ~ matter 5.0 Hentcelluloses Cellulose Lignm Solubles untreated straw F velutipes F, margmatus % % % % % % % % % G. applanatum H rutdans K.mutabHts L.sulfureus L. edodes N capno=des % % % % % % P robustus Pichlncha" a ,,Pichincha" b 30d ¢oOd 90d 30d 60d 90d 30d 60d 90d 30d 60d 90d 30d 60d 90d 30d 60d 90d % % % % % % % % % P ostreatus Pleurotussp ..florida" PFR 343 % % % % % % % % % PFP 551 Por=a sp. ..Rio rot" 5,7 S hwautum S.rugosoannulata T g=bbosa % % % % % % T. verslcolor ..Tungurah ua" 30d 60d 90d 30d 600 90d 30d 60d 90d Fig. 1. Degradation of straw (dry matter) by basidiomycetes after incubation periodes of 0, 30, 60 and 90 daysH. W. Miiller and W. TrSsch: Screening of white-rot fungi for biological pretreatment 183 Stropharia rugosoannulata should be the best or- ganisms for biological pretreatment of straw be- cause of their higher affinity for lignin. Digestibility tests With regard to the use of "myco-straw" for me- thane fermentation the enzymatic hydrolysis of cellulose was the most important limiting step. Biodegradability of straw can be measured in an in vitro test system by the amount of glucose lib- erated during incubation with cellulolytic en-
zymes. Figure 2 shows the enzymatic formation of glucose from different pretreated straw samples as a function of time. Untreated straw is only hardly hydrolysable. Straw, pretreated 30, 60 and 90 days with Pleurotus sp. 'florida" shows increas- ing glucose liberation. The amount of glucose formed from straw incubated 90 days with Pleuro- tus sp. 'florida" was as large as the amount of glu- cose liberated from a microcristalline, lignin free cellulose (Avicel). Other straw samples, pretreated by growth of Kuehneromyces mutabilis and Hapalopilus rutilans were markedly harder hydrolysed by cellulase. As a result of these experiments Pleurotus sp. 'flori- da" is the best fungus to pretreat wheat straw for biogas production. The reason, that K. mutabilis- treated straw is hydrolised worse can be ex- plained by less delignification compared to P. flo- rida. Straw pretreated with H. rutilans is hardly hydrolysable, although a good delignification took place. This shows that delignification is not the only parameter by which to estimate the use- fulness of microorganisms for biological pretreat- ment of lignocellulosics. Possibly a new physical barrier is built up by fungal mycelium, which hin- ders the attack of cellulolytic enzymes. Biogas fermentations The first step of biogas production from pre- treated straw is the hydrolysis of carbohydrate components. Anaerobic fermentation of pre- treated straw suspended in tap water together with a N-source (3 g/1 (NH4)2SO4) did not effect high yields of biogas because of a very rapid acid- ification of the slurry in consequence of the low buffer capacity. This problem could not be over- come by neutralisation. Therefore, in the following experiments straw was not fermented as the sole substrate but in combination with cow manure. Manure contains a buffer system and needs, thereforel less addition of NaOH for the maintenance of neutral pH; the nitrogen content of manure was sufficient for un- limited cell growth. Fermentations were carried out with an addition of straw of 50%, 33% and 25% to cow manure. The total dry matter in all fermentations was 4%. A mixture of manure and straw containing 33% straw (in dry matter) has proved to be the most effective. The substrate composition of the fermenters is shown in Table 1 for such experi- ments. When such a mixture of manure and straw was fermented anaerobically for 30 days in batch experiments, considerably more volatile acids were produced in fermenters containing pre- treated straw compared with fermenters contain- ing untreated straw (Fig. 3). The higher acid levels in the fermenters with pretreated straws can be converted quantitatively to methane. Higher yields of methane were ob- tained by conversion of biologically pretreated straw than with untreated straw, as is shown in Fig. 4Table 1. Composition of a mixture of manure and straw (2:1, dry weight) for fermentation to biogas Manure Untreated 30 d with 60 d with 90 d with Straw + P. florida P. florida P. florida Manure treated Straw treated Straw treated Straw + Manure + Manure + Manure Hemiculloses 16.1% 20.0% 16.9% 15.0% 13.8% Cellulose 15.0% 24.3%H.W. MOiler and W. Tr6sch: Screening of white-rot fungi for biological pretreatment mM~6"t J -- -" Untreated straw - -0-g 30 d with P. florida treated straw 1oo -- -- ~ -~ 60 d with P. florida treatmt straw J~ v *-~P-~90 d with P. florida treated straw ../- / \\V. 6o ,e" / / \~": ,0 V .... 0 r-- 2 4 6 # 10
12 14 16 18 20 22 24 26 28 30 doys Fig. 3. Concentration of acetic acid during the fermentation of
a manure/straw mixture (2:1, dry weight). Fermenter volume: 1.5 1, total solids 4% mt/al CH 4 1300 ~200 1100 ~000 900 800 700 600 500 ~00 300 200 100 0 neutr ohzahon f ~ = ~ un=~e s=~* /~ I/\i) L [ i • } J 2 ~, 6 8 10 12 14 16 18 20 22 ~ 26 28 30 days Fig. 4. Methane production rate (ml/d) during a batch fer- mentation of a manure/straw mixture (2:1, dry weight) with 60 g total solids in 1.5 1 fermenter volume The total production of biogas during a batch fermentation of a manure/straw mixture (2:1, dry weight) is shown in Table 2. Due to fungal pretreatment the methane yield as well as the average methane content in the bio- gas increased. Theoretically, 0.5
1 biogas should be produced from 1 g lignocellulosics (Jerger et al. 1982). Considering the fact that in the experi- ments the dry mixture of manure and straw had 84.9 percent of organic dry matter, the gas yield is improved from 0.293 1/g for untreated straw to 0.343 l/g for straw pretreated 90 days with Pleuro- tusflorida (referred to content of organic dry mat- ter). The analysis of the digested fermenter con- tents showed, that the cellulose content in the di- gested mixture of manure and straw was lower and the lignin content was higher compared to the analyses before biogas fermentation (compare Ta- bles
2 and 3). The relative increase of lignin is due to its resistance to microbial attack in an anae- robic environment. The C/N-ratio lowers, be- cause of the loss of carbon as biogas. Discussion In most cases all of the fungi tested grew well on wheat straw, but they increased the digestibility of this substrate to a different extent. All compo- nents of the lignocellulose were degraded by white rot fungi. But only those fungi, which de- grade lignin preferably can be used for biological pretreatment. The digestion experiments with H. rutilans showed, however, that good biological delignification is not in all cases equivalent to good digestibility. Therefore, the digestibility-test Table 2. Yields of methane and biogas during a 30 day batch fermentation of a manure/straw mixture (2:1, dry weight) with 60 g total solids in 1.5 1 fermenter volume, using Pleurotusflorida Untreated 30 d with 60 d with 90 d with Straw + P. florida P. florida P. florida Manure treated Straw treated Straw treated Straw + Manure + Manure + Manure Total gas (1) 14.955 15.255 16.205 17.506 Methane (1) 9.321 10.235 11.017 11.917 Increase by fungal treatment -- 9.8% 18.2% 27.9% Average CH4-content 62.3% 67.1% 68.0% 68.1% Biogas per g org. solid (1) 0.29
3 0.299 0.318 0.343H. W. MOller and W. TrOsch: Screening of white-rot fungi for biological pretreatment Table 3. Composition of the dry matter of the digested fermenter contents after 30 days fermentation 185 Untreated 30 d with 60 d with 90 d with Straw + P. florida P. florida P. florida Manure treated Straw treated Straw treated Straw + Manure + Manure + Manure Cellulose 13.1% 8.0% 7.2% 7.1% Lignin 13.9% 13.4% 13.0% 12.9% % C 35.21 32.65 33.07 32.6
4 % N 2,17 2.28 2.39 2.42 C/N 16.2 14.3 13.8 13.
5 with cellulases is important to estimate the possi- ble usefulness of a fungus for biological pretreat- ment of lignocellulosics. The results show, that "myco-straw" can be better hydrolysed and converted to biogas in com- parison to untreated straw. After biological lignin removal the straw cellulose is better accessible for anaerobic digestion. These results confirm the findings of Bisaria (Bisaria et al. 1983). Besides delignification, the crystallinity of native cellulose seems to be reduced by fungal enzymes, for its di- gestibility increases with the time of fungal pre- treatment. During anaerobic fermentation the im- proved digestibility effects a higher yield in vola- tile acids followed by a higher gas yield. With a mixture of manure and straw (2: 1) an increase of the gas yield of about 30% can be reached. The percentage of "myco-straw" effecting this in- crease was only 33% of the total dry matter. As was demonstrated with Pleurotus sp. "florida", biological pretreatment of wheat straw can dou- ble both digestibility and biogas yield compared with that on untreated straw. This procedure, involving microbial dilignifi- cation and biogas production, offers the possibil- ity of utilizing and removing the waste wheat straw in a completely biological way. The useful products from this process are fungi in the first step and methane in the second step. Because of the good accessibility to cellulose, mycostraw may constitute a technical substrate not only for bio- gas production but also for other biotechnological processes. References Bisaria R, Madam M, Mukhopadhay SN (1983) Production of biogas from residues from mushroom cultivation. Biotech- nol Letters 5:811--812 Chahal DS, Moo-Young M, Vlach D (1981) Effect of physical and physicochemical pretreatments of wood for SCP-pro- duction with Chaetomium
cellulolyticum. Biotechnol Bioeng 23 : 2417--2420 Detroy RW, Lindenfelser LA, Sommer S, Orton WL (1981) Bioconversion of wheat straw to ethanol: chemical modifi- cation, enzymatic hydrolysis and fermentation. Biotechnol Bioeng 23 : 1527-- 1535 Eriksson K-E, Goodell EW (1974) Pleiotropic mutants of the wood rotting fungus Polyporus adustus lacking cellulase, mannanase and xylanase. Can J Microbiol 20:371--378 Fan LT, Gharpuray MM, Lee Y-H (1981) Evaluation of pre- treatments for enzymatic conversion of agricultural resi- dues. Biotechnol Bioeng Symp 11:29--45 Goering HK, Van Soest PJ (1970) Forage fiber analysis. Agri- cultural Handbook Nr. 379, Agriculture Research Service US-Dpt. Agriculture Jerger DE, Dolenc EA, Chynoweth DP (1982) Bioconversion of woody biomass as a renewable source of energy. Bio- technol Bioeng Symp 12:233--285 Keyser P, Kirk TK, Zeikus JG (1978) Ligninolytic enzyme sys- tem of Phanerochaete chrysosporium: synthesized in ab- sence of lignin in response to nitrogen starvation. J Bacte- riol 135:790--797 Kirk TK, Schulz E, Connors WJ, Lorenz LF, Zeikus JG (1978) Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 117:227-- 285 Leisola M, Ulmer D, Fiechter A (1983) Problem of oxygen transfer during degradation of lignin by Phanerochaete chrysosporium. EurJ Appl Microbiol Biotechnol 17:113-- 116 Tien M, Kirk TK (1983) Lignin degrading enzyme from the hymenomycete Phanerochaete chrysosporium. Science 221:661--663 Ulmer D, Leisola M, Puhakka J, Fiechter A (1983) Phanero- chaete chrysosporium: growth pattern and lignin degrada- tion. Eur J Appl Microbiol Biotechnol 18:153--157 Zadra~il F (1980) Conversion of different plant wastes into feed by basidiomycetes. Eur J Appl Microbiol Biotechnol 9:243--248 Received August 26, 1985/Revised January 20, 1986。