Sunflower - Bioenergy Crop Plants - page 724

the addition of metal ions such as cobalt and surfactants such as Tween-80 to obtain ready release
of the enzyme into the broth (Srinivasan and Seetalaxman 1988; Gashel 1992; Kocher et al. 2008).
Cellulosic substrate at a concentration of 1% has been reported to be the best for cellulase production
(Wayman and Chen 1992; Steiner et al. 1994; Kanotra and Mathur 1995; Singh et al. 1995; Reczey
et al. 1996). Szengyel et al. (1997) reported that a 2% carbon source (Willow) gave the highest
cellulase activity, and we found 1% soluble cellulose to be optimal for cellulose production by T.
reesei Rut C-30 NRRL 11460 (Sharma 2000). In literature, various cellulosic substrates have been
evaluated, e.g., crystalline cellulose (Aiello et al. 1996; Geimba et al . 1999), amorphous cellulose
(Busto et al. 1996), cotton, wheat straw, barley husk, wheat bran (Gashel 1992; Maheswari et al.
1993; Steiner et al 1994; Awafo et al. 2000), sugarcane bagasse (De-Paula et al. 1999), soyhull
(Jha et al. 1995), sunflower (Sharma et al. 1996), corn straw (Yong et al. 1998) , corn cobs (Xia and
Cen 1999), etc. Yong et al. (1998) observed that batch cultivation of T. reesei on 3.75% maize straw
gave a cellulase production of 1.76 U/mL (Filter paper (FP) activity) and high cellobiase activity
(0.38 U/mL) in 3 days. A pH of 5-6 is optimal for growth of T. reesei , but as the pH goes down, the
cellulase production increases (Chahal and Wang 1978). However, control of pH is critical because,
if left uncontrolled, enzymes will be inactivated by severely acidic conditions (Kosaric et al. 1980;
Duff et al. 1987) . Abdel Fattah et al. (1995) reported that controlling the pH at 5 in the culture
medium during fermentation increased the levels of cellulases, and we observed the same pH for T.
harzianum (Kocher et al. 2008). Juhasz et al. (2004) reported that nonbuffered media produce high
filter paper but lower β-glucosidase activities than buffered media. In addition to pH, temperature
also plays an important role in cellulase production. Maximal cellulase production by T. reesei
has been observed between 25 and 30°C (Maryam et al. 2007) because higher temperatures
caused reduced excretion of cellulases. Duff et al. (1987) reported that the optimal temperature for
cellulase production by the mixed culture of T. reesei Rut-C 30 and Aspergillus phoenicis in a 10-L
fermenter was 27°C. Janas and Targonski (1995) studied the effect of temperature in the range
of 26-38°C on the production of cellulases by four mutant strains of T. reesei. We observed that
28°C was the optimum for cellulose production by T. reesei in a 15-L fermenter under submerged
conditions (Table 30.1; Sharma 2000).
The supply of nutrients in the form of nitrogen as ammonium ions seems to be the most critical
factor in regulating the pH of the medium for optimal cellulase yield (Martin et al. 1986). Nitrogen
in the form of potassium nitrate (KNO 3 ) has been reported to be better than ammonium chloride
(NH 4 Cl) or urea in facilitating the production of cellulase (Gashel 1992). Ammonium sulfate and
urea have also been recommended as nitrogen sources for cellulase production (Gutierrez Correa

taBle 30.1
Batch Production of cellulase in a 15-l
Fermenter by t. reesei rut c-30

cellulase activities (Iu/g)

time
(days) Filter Paper cmcase b-Glucosidase

1 16 49 6

2 29 167 18

3 53 250 30

4 92 364 40

5 112 424 49

6 145 440 54

Fermentation conditions: temperature 28°C, pH 4.6, cellulose 1%,
inoculum 3% (v/v), aeration 0.5 L/s, and agitation 250 rpm.