Cellulase Production in Continuous Culture by Trichoderma reesei on XyloseBased Media D.W. Schafner and R.T. Toledo* Department of Food Science and Technology, University of Georgia, Athens, GA 30602 Received December 29, 1990/Accepted October 29, 1991 Trichoderrna reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m3 occurred at ph 40 and a dilution rate of 391 h' where residual substrate was 0.43 kg/m3, but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at ph 3.5 and a dilution rate of 110 h', where cell mass production was 2.56 kg/m3 and residual substrate was kg/m3. Monod kinetic constants, corrected for endogenous metabolism, were 91 h', 0.469 kg/m3, 0923 h', and 0.470 kg cells/kg xylose at ph 3.5, for the maximum specific growth rate, MichaelisMenten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing ph. Key words: cellulase xylose Trichoderrna reesei continuous culture a growth modeling INTRODUCTION The hemicellulosic portion of lignocellulosic biomass is an underutilized resource in many conversion operations. Sorghum bagasse, a waste product from syrup production, contains a significant portion of hemicellulose, which can be a source of fermentable sugars for fermentation if separated from the lignin and cellulose fractions. Trichoderrna reesei grows and produces cellulase when batchcultured on xylo~e,"~'~ the predominant hemicellulosic sugar in sorghum cane.7 This investigation was conducted to examine the feasibility of producing cellulase from xylose by continuous culture techniques. Productivity, uniformity of operation, and ease of automation are some of the advantages possible with continuous culture.6 Specific objectives of this study were to: determine the maximum specific growth rate, endogenous metabolism coefficient, MichaelisMenten coefficient, and xylose yield coefficient for this fermentation system; ascertain the possibility of i? reesei producing cellulase in continuous culture on xylosebased media; and to compare the enzyme productivity of continuous culture to that of batch culture. * To whom all correspondence should be addressed. MATERIALS AND METHODS Culture Maintenance and lnoculum Preparation i? reesei (strain QM 9414) was obtained from the American Type Culture Collection, maintained on potato dextrose agar (PDA) slants, and transferred monthly. The fermentor inoculum was prepared in the following manner: a PDA plate was inoculated and incubated (25 C) for 7 days, at which time a good spore crop was evident. Ten milliliters of sterile distilled water was added to the plate, swirled about, and then withdrawn. This suspension contained 1 x lo7 conidia/ml. Two milliliters of this suspension was used to inoculate 100 ml of mineral salts medium (see below), with xylose as sole carbon source contained in a 250mL Erlenmeyer flask. The culture was incubated in a shaking water bath (25 C) for 3 days at 150 rpm. This preinoculum was then added to 1 L of xylosemineral salts medium contained in a 3L flask and incubated an additional 2 days at 25 C and 100 rpm. The culture in the 3L flask was used to inoculate the fermentor. Media and Culture Conditions The mineral salts medium is a modification of that used by Tangnu et a1.,16 and has been described e1~ewhere.l~ A microferm model CMF128s (New Brunswick Scientific, Edison, NJ) fermentor was used. The operating volume was 10 L and agitation rate was 250 rpm. The ph was controlled by addition of 2N NaOH and temperature was maintained at 28 C. Prior experiments with a galvenic steam sterilizable dissolved oxygen probe showed that an aeration rate of 2 VVM was sufficient to maintain dissolved oxygen level at a minimum of 20% saturation value for the medium. Mohagheghi et al." reported no adverse effects on growth rate when dissolved oxygen was 20% of saturation value, and Brown and Sainudeen4 reported no oxygen limitation under similar conditions. Fermentation media were prepared in 5gal glass carboys and sterilized for 1.5 h at 121 C. Used media were collected in sterile 5gal carboys, and samples for analysis were obtained from a steamsterilized fer Biotechnology and Bioengineering, Vol. 39, Pp. 865869 (1992) 0 1992 John Wiley & Sons, Inc. CCC 00063592/92/08036505$04.00
mentor port. Antifoam (polypropylene glycol, M W = 2000) was added as required. Table I. Experimental results. Analyses A 100mL sample was collected in a sterile dilution bottle using an aseptic technique. The bottle was immediately placed on ice and used for all analyses. Dilutions and were plated on PDA agar to check for contamination prior to subsequent analyses. Cell Mass Two 40mL aliquots of the sample were withdrawn and centrifuged at 20,000 rpm for 20 min. The supernatant was discarded, the pellet washed twice with distilled water, and then placed in a tared pan to dry (24 h) at 100 C. Substrate Substrate concentration was determined by measuring the reducing sugars (DNSA methodg) in the filtered fermentation medium. Culture filtrate was diluted prior to the addition of DNSA reagent in sodium citrate buffer (5 mol/l, ph 5.0) to yield an absorbance in the range to 0.5 at 550 nm. Substrate concentrations were ascertained using standard curves. Enzyme Cellulase activity was measured by using the Filter Paper (FP) assay. Onemilliliter culture filtrate was diluted to an appropriate concentration' in sodium citrate buffer (5 mol/l,ph 5.0), and then incubated with a 1 x 6cm strip (50 mg) Whatman No. 1 filter paper for 1 h at 50 C. After incubation, reducing sugars liberated were measured by the DNSA method: RESULTS AND DISCUSSION The fermentor was run in continuous culture for several months to obtain the data in this study. A summary of the steadystate data is presented in Table I. Microbial Growth Kinetics In 1949, Monod proposed a model for cell growth that was based on the MichaelisMenten expression for enzyme kinetics. The basic form of Monod's model is: prn * S r, = ~ KS+S*' This expression is often written in a simpler form: where (1) r,=p*x (2) (3) 3.0 110 195 320 391 598 3.5 110 195 320 391 598 704 811 4.0 110 195 320 391 598 704 811 0.9060 2.27 3.14 3.08 3.38 2.95 2.56 3.18 3.48 4.00 3.51 3.02 9 2.73 3.70 4.16 4.54 4.50 3.92 3.49 0.21 0.14 0.18 0.43 0.64 2.41 0.20 0.31 0.80 1.49 2.90 9.82 0.19 0.41 0.43 1.1 1.5 3.7 9.2 0.26 0.23 0.69 0.29 0.13 0.35 0.10 The Monod equation is intended for use only with organisms which reproduce by binary fission. While hyphal growth in fungi does not occur in this fashion, a number of reports have, nevertheless, shown the relationship to hold tr~e.',~,'~ It has been suggested that this is the case, because turbulent shear in the fermentor breaks the hyphae into very short strands.13 Microscopic examination of the fermentor contents have confirmed this hypothesis previously3 and in these experiments also. This basic form of the Monod equation [eq. (3)] can be modified to account for various special situations including: double substrate limitation, substrate inhibition, and growth inhibition. Another concept which can be included in the Monod model is that of maintenance energy or endogeneous respiration that quantity of energy that must be expended by the cell in order to maintain itself without growing. The modification of the Monod equation can take several forms, but the one we will use is: r, = (p K )* x (4) Another important quantity which we need to specify is the rate of substrate utilization, defined as: r,= P*X (5) In continuous culture, it is also important to define the dilution rate: F D= V (6) With these definitions and a few assumptions we can define expressions for both the steadystate cell mass and steadystate substrate concentration in continuous 866 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992
culture. First, we can modify our expression for rate of cell growth to take into account the effect of continuous dilution: r, = [(p K) * x] [D * x] Under steadystate conditions, no growth occurs, so setting the above equation equal to zero and solving for the specific growth rate (p): (7) p = K + D (8) So the specific growth rate is equal to the dilution rate plus the endogenous metabolism coefficient. Now, modifying the equation for rate of substrate utilization to take into consideration continuous culture: r,= * + D *(Si S) Assuming that steadystate has been attained, we set this equation equal to zero and solve for cell mass (x): P*X D * (S, S) x = D * (Sj S) * Xyl El. (9) (10) Rearranging and using the result for specific growth rate in continuous culture: amount of substrate in the feed), eq. (12) can be rearranged to give: A plot of l/x versus 1/D allows us to determine K and KYl if we know that S, is inlet concentration of the substrate (in this case, 10 kg m ). A similar transformation of eq. (18) yields: And, likewise, a plot of I/(D + K ) versus 1/S will allow us to determine K, and p,,,. Once the necessary constants are determined, these values can be plugged into the equations for steadystate cell mass and substrate concentration, and a plot of these variables versus dilution rate can be generated. Figures 1 and 2 show such plots, with the experimental data superimposed. The solid lines represent values calculated using the model and are extrapolations in some cases. The constants determined by linear regression (least squares method) are presented in Table 11. 5, A r) 4. Now, we need to repeat the process solving for the substrate concentration. First, using the expanded version of the Monod equation in the expression for rate of cell growth, and including the effects of dilution rate: 0,000 20 40 60 80 0.100 And, once again, assuming steadystate condition, no net growth, and setting the equation equal to zero: o= Prn * S K, + S (K + D) and then, solving for the substrate concentration (S): (Ks (K, + S) * (K + D) = pm * S (15) * K ) + (K,* D) + (S * K ) + (S * D) = p,,, * S (16) Ks * (D + K ) = S * [pm (D + K)] (17) K, * (D + K ) S= Ltm (D + K ) And thus we have the final 2 expressions for cell mass and substrate concentration at steadystate in continuous culture, i.e., eqs. (12) and (18). If we assume that S = 0 when Si >> S (i.e., when there is very little substrate in the reactor relative to the Dilution Rate, D (l/hr) Figure 1. Cell mass vs. dilution rate at ph 3.0 (A), 3.5 (El), and 4.0 (=I. c 2 v C. 0 c e * c (u c a 0 (u 0 x X 12 Dilution Rate, D (l/hr) Figure 2. Xylose concentration vs. dilution rate at ph 3.0 (A), 3.5 (O), and 4.0 (W). DO SCHAFNER AND TOLEDO: CELLULASE PRODUCTION BY TRICHODERMA 867
Table 11. Monod kinetics constants. 2 O3 0.8 I J a LL v 3.0 0861 0.414 0.397 80 3.5 0923 0.470 0.469 91 4.0 1315 0.603 0.484 0.107 The Effect of ph on Specific Growth Rate The specific growth rate, p,,,, varies with ph. Brown and Halstead3 proposed that the relationship between specific growth rate and ph is a linear one. Linear regression (least squares method) of data in Table I1 shows the relationship between the specific growth rate of T. reesei (on a xylosebased medium at 28 C) and ph was: pm = 0.104 25.3[H+] (21) It is interesting to note that this equation predicts that growth will cease (pm = 0) at a ph of 2.39, in agreement with the prediction of Brown and Zainudeen4 for T. reesei on 2.5% glucose at 28 C. Product Formation Product formation in batch and continuous culture can be modeled by the following equation': p = Kp * x * e(d"") (22) The constants for this model (at 2 of the 3 ph values, where sufficient data was available) have been determined (Table 111), and the plot of cellulase activity versus dilution rate is shown in Figure 3. The data points are superimposed on curves generated using the constants in Table 111, the data from Table I, and maturation time equation for product concentration shown above. It has been suggested that maturation time is lowered at higher ph val~es,~ and the data in Table I11 support that conclusion. Productivity of Continuous and Batch Culture Productivity can be expressed as either product produced per unit weight of cells per unit time, or as product produced per unit volume of fermentation medium per unit time. Cellulase activity is typically expressed as units of activity per unit volume of medium Table 111. 3.0 3.5 4.0 Maturation time model parameters. [I KP 0.66 0.25 92.4 76.9 0 1 2 3 c04 5 0 )6 Dilution Rate, D (1 /hr) Figure 3. Cellulase production vs. dilution rate at ph 3.0 (A), 3.5 (El),and 4.0 (m). per unit time. This reflects the extracellular nature of the enzyme and the costs associated with recovering it from the fermentation broth. The productivity of batch production of cellulase by T. reesei on xylosebased medium can be calculated from other data14 to be 13 FPU L' h', while this data shows that, unfortunately, the productivity in continuous culture is only 7.6 FPU L' h'. Possibilities for improving cellulase productivity in continuous culture include: the use of a twostage reactor," utilizing xylose in the first stage and xylose plus an inducer (such as sorbose) in the second stage, where enzyme production occurs; the production of mutants, which hyperproduce cellulase'* when grown on xylose; or adaptation of other fermentation variations such as fedbatch or cellrecycle.' CONCLUSIONS The parameters for a Monod model of microbial growth kinetics including endogenous metabolism were obtained for T. reesei (QM 9414) at 3 different ph values in continuous culture. Estimates of maximum specific growth rate, endogenous metabolism coefficient, MichaelisMenten coefficient, and yield coefficient for growth on xylose were calculated. Growth rate was found to change linearly with [H'], and the ph at which growth was predicted to cease agreed with results of others. A maturation time model, fitted to the data, indicated that maturation time decreased with rising ph. Enzyme productivity was lower in continuous culture than in batch culture. NOMENCLATURE dilution rate (h') flow rate through the system (L h') endogenous metabolism coefficient (h') product formation rate constant (FPU kg cells') saturation constant (kg substrate m3) rate of cell growth (kg cells m3 h') rate of substrate utilization (kg xylose h') limiting substrate concentration (kg substrate 6') inlet substrate concentration (kg m3) 868 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992
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