Trans. Br. mycol. Soc. 84 (2), 251-258 (1985) Printed in Great Britain EFFECT OF CUL TURAL CONDITIONS ON PRODUCTION OF CELLULASES IN TRICHODERMA LONGIBRACHIATUM By D. K. SANDHU AND M. K. KALRA Department of Biology, Guru Nanak Dev University, Amritsar-is joo e, India The productionof cellulase components which include FP activity, CM-ase and p-glucosidase on carboxymethyl cellulose (CMC) was investigated. The relative distribution of cell free and cell associated enzymes varied with the age of the culture. The optimal pH of the medium for synthesis of enzymes in extracellular, cytosol and cell debris associated states was between 4'5 and 5"0 with optimal temperature being 27°C. Shake cultures gave comparatively low yields of enzymes as compared to stationary cultures. When the medium was supplemented with 1 % lactose, maximum production of cellulolytic activities in the culture fiitrate was achieved, that is, 8'1, 0·6 and 0'13 units per ml of CM-ase, FP activity and ,a-glucosidase respectively. There was a varied response in the induction of cell associated enzymes by different substrates. An increase in substrate concentration (CMC and lactose) had no significant effect on production of extracellular enzymes. In recent years, attention has been focused on decreasing the cost of the enzymatic hydrolysis of cellulosic plant material, either by screening microbial mutants (Montenecourt & Eveleigh, 1977; Farkas et al., 1981) or by manipulation of cultural conditions to improve cellulase enzyme levels. Improvement in cellulase productivity has also been obtained by increasing cellulose concen- tration, addition of glucose to cellulose medium in order to increase cell mass and continuous culture methods (Wilke, Yang & Von Stockar, 1976; Peitersen, 1977). Temperature profiling and pH cycling have also led to increased production of cellulase in Trichoderma species (Mukhopadhyay & Malik, 1980). Trichoderma longibrachiatum Rifai has proved to be a good cellulase producer and a potential source of single cell protein (Sidhu & Sandhu, 1980; Sandhu & Kalra, 1982). The aim of the present study was to characterize the effect of culture conditions on the complete cellulolytic enzyme complex in this species. MATERIALS AND METHODS Organism Trichoderma longibrachiatum Rifai (ATCC 44788) used in the present study was isolated from degrading Mangijera wood. The stock culture was maintained in soil at 4 °C and subcultured in Vogel's glucose agar whenever required. Influence of shake or stationary cultures Vogel's medium (25 ml) (Sandhu & Kalra, 1982) supplemented with 1 % carboxymethyl cellulose (CMC) (Sigma) was dispensed in 100 ml Erlen- meyerflasks. The flasks were autoclaved at 10 p.s.i. for 20 min. Each flask was inoculated with a spore suspension to give a final concentration of 5 x 10 6 ml? of the medium and incubated at 27° as stationary or shake cultures. Six flasks per treatment were analysed daily for 10 days. Incubation temperature and initial pH of medium The medium was inoculated with test organisms and incubated at 15°,22°,27°,32°,37°and 42°. The initial pH was adjusted with 0'1 N-NaOH or 0'1 N-HCl to different values ranging from 2 to 8. After inoculation cultures were incubated at 27°. Flasks were analysed after 5 days of incubation as stationary cultures. Effect of various soluble substrates Carboxymethyl cellulose in Vogel's medium was replaced by 1 % lactose, maltose, sucrose or cellobiose as a sole source of carbon. Sugars were sterilized by seitz filtration before adding to the autoclaved medium. Complex compounds like yeast and malt extract at a concentration of 1 % were used separately and in combination for cellulase production. The effect of substrate concentrations on cellulase production was studied RES ULTS Influence of carbon source Among the carbon sources tested the highest extracellular enzyme activities were recorded with lactose followed by CMC and malt extract (Fig. 4). FP activity and CM-ase in the cytosol fraction were found to be maximum on malt extract whereas p-glucosidase was maximum on maltose and CMC. Cellobiose gave maximum mycelial dry weight and was found to be the best source for cell debris enzymes except for p-glucosidase which had a higher activity in yeast extract. No CM-ase activity was detected on sucrose (Fig. 4b). Of the different concentrations of CMC, t :5% gave the highest Influence of pH and temperature Maximum activity in the culture filtrate was recorded at pH 5"0. The pH values lower than 4'0 and higher than 5'5 had an adverse effect on cellulase production (Fig. 2a-c). The cytosol and cell debris enzymes were maximum between pH 4-5 and negligible at pH 3'0 and 7'0 except for p-glucosidase (Fig. z c-c ). The enzymes in all three fractions were higher at 27° but best . growth was supported at 32° with reasonable amounts of enzymes also (Fig. 3a-c). At minimal and maximal temperatures tested, i.e. 15° and 42°, the enzyme components in the extracellular and cytosol fractions were present but were absent in cell debris excepting p-glucosidase. Effect of shake and stationary culture on e nzy me production Th e production of the three components of cellulase in the culture filtrate was higher in stationary culture being 0'275, 1'64 and 0'10 units ml- 1 and 0'17, 1'0 and 0'08 units rnl'? in shake culture for FP activity, CM-ase and p-glucosidase respectively (Fig. 1 a,d,g ). The enzyme activities in the cytosol and cell debris fractions appeared earlier and were comparable under the two culture conditions (Fig. 1b, c, e,f ,h,1). Enz yme activities have also been expressed for different fractions as activity per flask (T able 1). The relative proportions of the enzyme in the above two fractions were found to be highest for p-glucosidase followed by CM-ase and FP activity. The activities in the cytosol disappeared after the eighth day but persisted in the cell debris beyond the tenth day. The maximum mycelial mass was obtained on 4 and 6 days of incubation in shake and stationary culture respect- ively. The pH of the medium showed a steady increase towards neutrality after an initial decline. Fractionation of samples Each sample was filtered through a sintered glass funnel at 4°. The resulting culture filtrate was centrifuged at 8500 g for 15 min at 4° and stored for analysis of enzyme activity and unused substrate . The cell mass was washed with 0'1 M acetate buffer at pH 5"0 and dried between folds of filter paper . The above mycelium was frozen, crushed with chilled acid washed sand to prepare cytosol extracts and for cell debris fraction, and macerated without sand. Both fractions were suspended in buffer and centrifuged at 22000 g for 30 min at 4°. The supernatant was taken as the cytosol fraction while the pellet of cell debris without sand was resuspended in 5 ml of buffer and stored at - 15° if not immediately analysed. 25 2 Cellulases in Trichoderma longibrachiatum in the case of lactose and CMC. The inoculated flasks were incubated at 27° for 5 days as stat ionary culture s. Dry weight determinations Samples were transferred to dried and preweighed sintered glass funnels and washed thoroughly with cold deionized water. These were dried at 75° to a constant weight. Enzyme unit One unit of enzyme activity is equivalent to 1 pM of product released per min. Cytosol and cell debris enzymes have been expressed in terms of specific activity as units mg ? protein for cytosol and units mg ? dry weight for cell debris. The sol-able protein content was estimated by the method of Lowry et al. (1951). Estimation of enzymes Filter paper activity (FP activity) and carboxy- methyl cellulase (CM-ase) were estimated as described earlier (Sandhu & Kalra, 1982) using filter paper strips and CMC as substrates. For p-glucosidase, to 0'5 ml of the diluted enzyme sample was added 0'5 ml of 5 msr-p-nitrophenyl- p-D-glucopyranoside (PNPG) (Sigma). The mix- ture was incubated at 37° for 30 min. The reaction was terminated by adding 4 ml of 0'2 M-NaOH/ glycine buffer at pH 10·6 and the absorbance read at 420nm. Carboxymethyl cellulose in the culture filtrate was determined with anthrone reagent (U pdegraff, 19 69 ). D. K. Sandhu and M. K. Kalra 253 0·35 2·1 0·28 (a) (d) (g) ~ • r r 1·5 4 1 »- 0·25 l' -, 0·20 ;<;:: E - \- , .~ ::> ~ - ~ a eE )i 4 0.12 »"Q) 0·15 , 0·9 N U I \ <:: '" 1/ .', / • A ~ ~t: >( . ., -V·, \ ~ , j "4-,. It. ' • • l 0·05 0·3 ./, 0·04 /~ ' ./~ / I ~/ , 0 ~. 0 0·21 T" (b) • (e) (h) '2 1\. I I .;; I \ »- _ 0 I \ .~ 0·15 .~ 0 • l ", . \ t) t I \ '" "" :1\ I \ " E ~::> l \ / \ N - 0·09 I • • <:: "0 , . • I \ "-I (!) /! \ I • '. >. !/ \\ ~ l' ~ I ~ I. \ • I 0·03 . ., • If \.\ • ~I '\ / / • 0 (j) 8 10 4 62 (i) ;, . .,{::::., . ! ~ "'. I I I • l / 8 10 6 Days 2 4 e-e_ / '."·"'::1::1-·-·-1 . - /- (c) 8 10 64 2 a 0·02 0·04 0·06 Fig. 1. Production of FP activity (a-c), CM-ase (d-f), p-glucosidase (g-l) in shake ( ) and stationary ( ) cultures on 1 ';;0 CMcellulose. 254 Cellulases in Trichoderma longibrachiatum Table 1 . Distribution of enzymes in three fractions of eellulases Enzyme distribution (units per flask) Enzyme fraction Extracellular Cytosol Cell-debris FP activity 7'0 0'045 0 '11 CM-ase 37'5 0'115 0 '24 ,8-glucosidase 2'3 2 0'12 1'2 i 23 • ~ c::: Co E i c Cl 35 5 IS o (a) <. I "'. • 15 22 27 32 37 42 Temperature (DC ) 0·2 0·3 0·35 0·25 ~ ::E u 0 .)5 .5 r o r bIl E ::: 0·24 ~ ~ 0·20 u 0.16 r E 0·12 2 0.16 . '" :§ 0·08 11 0 ee b >< ~ o 30 I 20 ~ '" ~ OIl 10 E ~ t- O (e) (a) (b) 23456 78 pH 0~ 1~""''''-''''''-'-~~ , ~:~j 1·2 0·8 0·4 0·3 0·2 0·1 ~ B 0·16 » o x i ~ 0·05 r e 0·08 2 . '" :§ 0-<, F 1f r-y ., , ""1 0; o e ';( ~ ~ ~ § 0·35 r bIl E 2 ~ '" ~ 0·25 .5 r o 0.15 Fig. 2. Effect of initial pH of medium on growth and cellulase production in extraceIlular (A.), cytosol (e ), cell-debris (0) fractions; FP activity and growth (a), CM-ase (b), /I-glucosidase (c). CM-ase and FPactivity in cell-debris are plotted as activity x 10- 1 • Fig. 3. Effect of temperature of incubation on growth and cellulase production in extraceIlular (A.), cytosol (e ), cell-debris (0 ) fractions; FP activity and growth (a), CM-a se (b), /I-glucosidase (c). CM-ase and FP activity in cell-debris are plotted as activity x 10 - 1 . D. K. Sandhu and M. K. Kalra 255 < 0-8 ~ D:. (a) '0 <:: 0·6 '" <I) '" '1' 100 ;:;a u 0·4 r- 80 .5 60 S r Ol) 0 ~LJh: 5 0·2 40 ~ x C ~ C ~. Cl '0 a a r Ol) 5 ~ 10 >., ~ :~ °tf 5 .0 (b) o <I) I '" '0 <I) - 5 ;; ~ >. u 0·6 N .• 4 <:: .5 0·5 ~ 2 0 ~ 0-4 Q. . r Ol) 0·3 e . 2 0·2 lL . [jl 0 0·1 • '" .9 • 0 0 >. a o r S 02 1 2 1 i (c) '" ~ "3 0·1 ~ o '" lhl tfB ~ A ~ • 0 ~ • 0 <I) a '-' ~d) '0 o o <:: 1;l '" '" '" '" 1;l <I) 0 <I) <I) 0 >< >< u '" :E '" 5"3 <I) <I) '" <.> .9 .9 .2 0 >.:::: '" ~ o <;; o ~ <I) '" ;; '" ~ '" ;:;a ;:I o <.> <I) ;:;a <I) ~ -l U CI) -e >- <I) "' '" a'l0i u >-;:;a Fig. 4. Effect of various carbon sources on growth and cellulase production in extracellular (~), cytosol (.), cell-debris (0) fractions; FP activity and growth (a), CM-ase (b), p-glucosidase (c). CM-ase and FP activity in cell-debris are plotted as activity x 10-'. yield of all the extracellular enzymes (Fig. 5a-c). A proportional increase in cytosol and cell debris enzymes was recorded with increase in the substrate concentration. All the cellulase compon- ents in the three fractions reached their plateaux at 1-1'5 % of lactose concentration after which the activities were almost constant (Fig. 6a-c). Growth expressed as dry weight increased with increase in substrate concentration both in the case of CMC and lactose, the increase being relatively small above one per cent (Figs sa, 6a). 9 MYC 84 Cellulases in Trichoderma longibrachiatum • 2·5 (e) 1·5 2·01·0 Concn of lactose (%) ____ - e _.l A & /& 0_ 0 0-0 __ 0 0_ 0 0 0 0 (a) .l __ .l __ .l __ .l ./ 120 e I -a e /- 80 .><: '" ea <;:: 00 • E 40 ~ .~ ~ ~ 7- Cl 0 ____ e ___ ._e 0·5 (b) __ .l »>' / j Fig. 6. Production of cellulase and growth at different concentrations oflactose in extracellular (£), cytosol (.), cell-debris (0) fractions; FP activity and growth (a), CM-ase (b), ,8-glucosidase (e). CM-ase and FP activity in cell-debris are plotted as activity x 10-'. 0·7 < c, e ~ I -e 0·5 t:: e '" ~ " '" '" '" <;:: =E 00 u 0·3 E .S ~ i ~ 0 c x 0·1 '; ~ "Cl 12 i "" E 2 10 >. .~"' .L' . £; ~ 8 ~=: " 8 E .• 6 ~.~ t:: ~ 8 0. 0·4 r eo E 2 0·2 "0 '" 0 ~ o r 0·2 E :2 0·12 '" E Q) c '" !:: 0·04 '! 2·50·5 1·0 1·5 2·0 Conen ofCMC (%) o 0·4 40 (a) < 0·3 30 p., ~ ~ "Cl !j <:: 20 '" 0·2 1;l '" =E u .S 0·1 10 r 0 0_0 0 0 o- X '; e -c r bO 2·0 E 2 ~ (b) ",' '': / ~ ~.L' 1·2 " .::: -e "'0= '" " o a " E t:: 0·4;>, N " t:: ~ 8 0·3 / 0. r eo 0·2 ~==_o_o E 2 0·1 0:"' ] 0 ~ o r E 0·24 (e) 2 ' '" "3 0·16 ~ g ;< ~ 0·08 - - " / 0_ 6 _0- t o Fig. 5. Production of cellulase and growth at different concentrations of CMcellulose in extracellular (£), cytosol (.), cell-debris (0) fractions; FP activity and growth (a), CM-ase (b),,8 glucosidase (e). CM-aseand FP activity in cell-debris are plotted as activity x 10-'. DISCUSSION Extracellular enzyme production studies in other fungi (Boretti et ai., 1973; D'Souza & Furtado, 1977) has been shown as linear with growth. The enzymes in the cytosol and cell debris fraction contributed minor proportions of FP activity and eM-ase but reasonable amounts of p-glucosidase. However, relative proportions of the three fractions varied during growth as the cell associated (cytosol and cell debris) reaction was higher in the early phase compared to the late growth phase, thus indicating synthesis and release of these enzymes into the medium which added to the concentration D. K. Sandhu and M. K. Kalra 257 of extracellular enzymes (Berg & Pettersson, 1977; Kubicek, 1981, 1983). Regarding distribution of the enzymes in the three fractions during the late exponential growth phase, most of the FP activity and CM-ase (97'8, 99'1 %, respectively) were located extracellularly. Measurement of fJ-gluco- sidase showed up to 33 % of the total activity to be associated with the mycelium throughout the growth period. Halliwell & Lovelady (1981) have reported that 99'0 % of CM-ase in T. koningii Oudem. is extracellular whereas 90 % of fJ- glucosidase is associated with cells. The active release of CM-ase and FP activity into the medium in early stages of cultivation of T. reesei Simmons was reported by Vaheri, Vaheri & Kaupinen (1979) with fJ-glucosidase being detected mainly in the cell debris which was not released until the cells had autolysed. In different Trichoderma species, which are the most promising fungi for industrial production of cellulases, most of the ,a-glucosidase activity has been shown as localized within the cells (Berg & Pettersson, 1977; Kubicek, 1981) except in the cases of T. harzianumRifai and T. pseudokoningii Rifai where high levels of extracellular enzymes have been reported (Sidhu, 1983). Studies on fungi other than Trichoderma, which include Penicillium janthinellum Biourge and Sporotrichum pulverulen- tum Burds., have also shown the cell free occurrence of fJ-glucanases and fJ-glucosidases with only small amounts associated with the mycelium (Eriksson & Hamp, 1978; Rapp, Grote & Wagner, 19 81). The cultural conditions have a marked effect on the production of enzymes in all three fractions. In shake flasks the enzymes reached their maximum values earlier but the activity was low compared with stationary cultures. This low activity may be due to inactivation of enzymes by shaking, as reported earlier (Reese & Mandels, 1980). Maxi- mum production of cell free and cytosol enzymes was obtained at pH 5'0 except for CM-ase in cytosol fraction which along with all cell debris activities showed a pH optimum at 4'5. Growth and enzyme production was markedly inhibited when the initial pH was below 4'0 and above 6'0. This pH range has also been reported as the most favourable hydrogen ion concentration for cellulolytic enzyme synthesis in several other fungi namely, Sclerotium rolfsii Sacco (Shewale & Sadana, 1978), T. reesei (Andreotti et al., 1980; Mukhopadhyay & Malik, 1980), Pellicularia filamentosa (Pat.) Rogers (Tani- guchiet al., 1980),Eupenicilliumjavanicum (Beyma) Stolk & Scott (Tanaka et al., 1980) and Aspergillus terreus Thom (D'Souza & Volfova, 1982). As reported in studies on T. viride Pers., production of maximum cell mass may not produce maximum cellulase yield (Andreotti et al., 1980; Mukhopad- hyay, 1982). The observations made in the present study also show similar characteristics. Here the growth was maximum at 32° while optimal production of all cellulase components in the three fractions was maximum at 27°. Of the different carbon sources lactose gave the highest yield of cellulase enzymes in the extracellular medium although best growth was supported by cellobiose. In earlier studies on Trichoderma species lactose has been shown to be a good inducer (Andreotti et al., 1980; Kubicek, 1983). In Penicillium species too, it produced higher amounts of fJ-glucosidase but gave low yields of other cellulase components (Lakshmikanthan & jagan- nathan, 1980). Carboxymethyl cellulose proved to be less effective in inducing the enzymes compared with lactose and was inhibitory at higher concen- trations. This may be due to release of high levels of glucose in the culture broth which is a known repressor of cellulase. Increasing the concentration of lactose above 1 % did not bring about a significant increase in enzyme production in the culture filtrate. The three cellulolytic enzymes in the cytosol and cell debris showed differential induction by various substrates. This location of enzymes in association with mycelium or cell free state has previously been shown to depend upon the carbon source (Berg, 1975; Berg & Pettersson, 1977; Chaudhury & Tauro, 1982). 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(2), 251-258 (1985) Printed in Great Britain EFFECT OF CUL TURAL CONDITIONS ON PRODUCTION OF CELLULASES IN TRICHODERMA LONGIBRACHIATUM By D. K. SANDHU AND M. K. KALRA Department of Biology, Guru Nanak Dev. separately and in combination for cellulase production. The effect of substrate concentrations on cellulase production was studied RES ULTS Influence of carbon source Among the carbon sources tested the highest extracellular. An increase in substrate concentration (CMC and lactose) had no significant effect on production of extracellular enzymes. In recent years, attention has been focused on decreasing the cost of