*4.3.3 Fermentation conditions*

Fermentation condition play the main role for the standardization of process parameters such as incubation period, inoculum size, pH, carbon and Nitrogen source, metal ions, etc. Maximum cellulase production may vary from 1 day to weeks. It is usually observed that fungal cultures require longer incubation period for cellulase production than bacterial cultures. The highest cellulase level was achieved 96 hrs of the fermentation while using *T. harzianam* and *P. chrysosporium* [93]. Maximum cellulase production was observed after 96 h by *A. niger* [94].

**69**

*Overview of the Process of Enzymatic Transformation of Biomass*

Optimal cellulase secretion from *Aspergillus niger* was achieved at a time of 72 h in maize straw while 96 and 120 h were the growth period in millet and guinea corn

The age and concentration of inoculum also plays an important role in the production of cellulases. An increase in inoculum size up to an optimum limits results in rapid proliferation and biomass synthesis which leads to produced higher amount of cellulase [96]. On the other hand higher inoculum volume beyond optimum size leads to increases in the water content of medium in case of SSF creating aeration

Bacterial and fungal cellulase production found to be significantly affected by pH. Milala et al. [95] reported maximum cellulase activity at pH 4.0 by *A. niger*. Devi and Kumar [98] optimized condition of cellulase production in fungal strain *A. niger* against the lignocellulosic bio wastes like saw dust, paper cellulose at varying environmental parameters of pH (4.0–7.0) and maximum activity was observed at pH 5. Gao et al. [99] studied the production of extracellular cellulases by a newly isolated thermoacidophilic fungus *Aspergillus terreus* M11 on the lignocellulosic materials in solid-state fermentation (SSF) and the high-level cellulase activity was observed at pH 3.0. However, the results appeared to contradict previous results reported by Solingen et al. [100] of an alkaline novel *Streptomyces* sp. isolated from east African soda lakes that have an optimal pH of 8.0, highlighting the effect of

The fermentation temperature plays a very significant role on the growth and metabolic activity of microbial cells. Optimum temperature for cellulase production under solid-state fermentation by *Trichoderma reesei* RUT C30 was 33°C [101]. Fatma et al. [102] studied ethanol production from rice straw using cellulase produced by *T. reesei* F-418 cultivated in alkali treated rice straw under SSF and reported 162 U/g substrate cellulase activity when fungus was cultivated incubation at 28°C. Maximum enzyme production (3.9 U/ml) was achieved at 45°C temperature by *Aspergillus niger* using paper cellulose [98]. Gao et al. [99] studied production of extracellular cellulases by a newly isolated thermoacidophilic fungus *Aspergillus terreus M11*, on the lignocellulosic materials in solid-state fermentation (SSF) at 45°C. Jang and Chen [103] described a CMCase produced by a *Streptomyces* T3-1 with optimum temperature 50°C. Schrempf and Walter, [104] described a

problems in SSF and it will responsible for reduction in overall yield [97].

alkaline environment on the adaptation of these *Streptomyces*.

CMCase production by *S. reticuli* at an optimum temperature 55°C.

Various carbon sources such as metabolizable sugars, commercial cellulose and agricultural residues/by-products have been used for cellulase production. Some carbon sources resulted good growth with low enzyme production while some supported good growth along with high yield of enzyme secretion. Commercially available carbon sources used for cellulase production were Powdered cellulose by *A. niger* [105], and CM Lactose by *Mucor circinelloides* [81]. Several studies focused on cellulase use in the bioconversion of agro-industrial waste [106]. Chandra et al. [107] studied effect of several carbon sources including groundnut fodder, wheat bran, rice bran and sawdust on cellulase production by *A. niger.* They found that highest titers of cellulolytic enzymes in solid state fermentation on wheat bran. Azzaz, [108] studied effect of several carbon sources including banana wastes, rice straw, wheat straw, corn stalks and pure cellulose powder on cellulase production by *A. niger* and *A. flavus* NRRL 5521. He observed that wheat straw gave the highest cellulase production when fermented with *A. niger* (0.177 U/mL) while rice straw gave the highest (0.046 U/mL) cellulase production when fermented with *A. flavus* NRRL 5521. The lignocellulosic residues offer cheaper substituent of pure cellulose available commercially for the production of cellulase. Mixed substrates like wheat bran and corn cob are used as best carbon source in case of *A. niger* NRRL3 for cellulase production under SSF [109]. Milala et al. [95] used different agricultural

*DOI: http://dx.doi.org/10.5772/intechopen.85036*

straws respectively [95].

*Elements of Bioeconomy*

**4.3 Production of cellulases**

*4.3.1 Submerged fermentation*

method with a commercial kit but when polysaccharide are used a substrate, reducing sugars released is measured by the DNS (dinitrosalicylic acid) method [81].

The technique which are mainly used for the enzyme production are Submerged

When fermentation is performed with some free flowing nutrient media; it is termed as SmF [84]. In industry, enzymes are produced mostly by SmF, primarily due to the much simplified processes associated with scale-up compared to those involved for scale-up in SSF [85]. In fact, some other important factors like indulgence in controlling process parameters, monitoring and downstream processing makes SmF more significant [86]. Only a few designs are available in literature for SSF based bioreactors. This is principally due to several problems encountered in case of SSF for controlling various parameters like pH, temperature, aeration and moisture content. Fungal cellulase production is largely dependent on media composition and culture conditions. Thus development of a suitable fermentation strategy is necessary for full exploitation of potential of microorganism used for fermentation [87]. Several reports are available for cellulase production using SmF. Karthikeyan et al. [88] reported cellulase production from *Penicillium* strain K-P in liquid medium supplemented with different carbon and nitrogen sources at varying pH and temperature, maximum cellulase activity was observed on fifth day (pH 3.0 and 30°C) in the presence of fructose and ammonium nitrate as carbon and nitrogen source respectively. Narasimha et al. [89] reported maximum cellulase production using *A. niger* on medium (pH 5) supplemented with 1% CMC or

When fermentation is performed on nonsoluble materials in the absence of free flowing nutrient media, so that the material used can serve as a platform for support as well as nutrients; it is termed as solid state fermentation. While compared for their potential it was found SSF offers various opportunities over SmF because they are eco-friendly on account of lower energy requirements, produce lesser wastewater and they are based on employment of waste solid biomass [90]. Further advantages of SSF over SmF include prevalence of nonaseptic conditions, a wide variety of substrate are available, low capital cost, inexpensive downstream processing [91], higher product concentration, high reproducibility, lesser space requirements (compact fermenters), easy contamination management [92]. It is observed that production cost was decreased about 10 fold in SSF over SmF.

Fermentation condition play the main role for the standardization of process parameters such as incubation period, inoculum size, pH, carbon and Nitrogen source, metal ions, etc. Maximum cellulase production may vary from 1 day to weeks. It is usually observed that fungal cultures require longer incubation period for cellulase production than bacterial cultures. The highest cellulase level was achieved 96 hrs of the fermentation while using *T. harzianam* and *P. chrysosporium* [93]. Maximum cellulase production was observed after 96 h by *A. niger* [94].

fermentation (SmF) and solid state fermentation (SSF) [83].

**68**

sawdust.

*4.3.2 Solid state fermentation*

*4.3.3 Fermentation conditions*

Optimal cellulase secretion from *Aspergillus niger* was achieved at a time of 72 h in maize straw while 96 and 120 h were the growth period in millet and guinea corn straws respectively [95].

The age and concentration of inoculum also plays an important role in the production of cellulases. An increase in inoculum size up to an optimum limits results in rapid proliferation and biomass synthesis which leads to produced higher amount of cellulase [96]. On the other hand higher inoculum volume beyond optimum size leads to increases in the water content of medium in case of SSF creating aeration problems in SSF and it will responsible for reduction in overall yield [97].

Bacterial and fungal cellulase production found to be significantly affected by pH. Milala et al. [95] reported maximum cellulase activity at pH 4.0 by *A. niger*. Devi and Kumar [98] optimized condition of cellulase production in fungal strain *A. niger* against the lignocellulosic bio wastes like saw dust, paper cellulose at varying environmental parameters of pH (4.0–7.0) and maximum activity was observed at pH 5. Gao et al. [99] studied the production of extracellular cellulases by a newly isolated thermoacidophilic fungus *Aspergillus terreus* M11 on the lignocellulosic materials in solid-state fermentation (SSF) and the high-level cellulase activity was observed at pH 3.0. However, the results appeared to contradict previous results reported by Solingen et al. [100] of an alkaline novel *Streptomyces* sp. isolated from east African soda lakes that have an optimal pH of 8.0, highlighting the effect of alkaline environment on the adaptation of these *Streptomyces*.

The fermentation temperature plays a very significant role on the growth and metabolic activity of microbial cells. Optimum temperature for cellulase production under solid-state fermentation by *Trichoderma reesei* RUT C30 was 33°C [101]. Fatma et al. [102] studied ethanol production from rice straw using cellulase produced by *T. reesei* F-418 cultivated in alkali treated rice straw under SSF and reported 162 U/g substrate cellulase activity when fungus was cultivated incubation at 28°C. Maximum enzyme production (3.9 U/ml) was achieved at 45°C temperature by *Aspergillus niger* using paper cellulose [98]. Gao et al. [99] studied production of extracellular cellulases by a newly isolated thermoacidophilic fungus *Aspergillus terreus M11*, on the lignocellulosic materials in solid-state fermentation (SSF) at 45°C. Jang and Chen [103] described a CMCase produced by a *Streptomyces* T3-1 with optimum temperature 50°C. Schrempf and Walter, [104] described a CMCase production by *S. reticuli* at an optimum temperature 55°C.

Various carbon sources such as metabolizable sugars, commercial cellulose and agricultural residues/by-products have been used for cellulase production. Some carbon sources resulted good growth with low enzyme production while some supported good growth along with high yield of enzyme secretion. Commercially available carbon sources used for cellulase production were Powdered cellulose by *A. niger* [105], and CM Lactose by *Mucor circinelloides* [81]. Several studies focused on cellulase use in the bioconversion of agro-industrial waste [106]. Chandra et al. [107] studied effect of several carbon sources including groundnut fodder, wheat bran, rice bran and sawdust on cellulase production by *A. niger.* They found that highest titers of cellulolytic enzymes in solid state fermentation on wheat bran. Azzaz, [108] studied effect of several carbon sources including banana wastes, rice straw, wheat straw, corn stalks and pure cellulose powder on cellulase production by *A. niger* and *A. flavus* NRRL 5521. He observed that wheat straw gave the highest cellulase production when fermented with *A. niger* (0.177 U/mL) while rice straw gave the highest (0.046 U/mL) cellulase production when fermented with *A. flavus* NRRL 5521. The lignocellulosic residues offer cheaper substituent of pure cellulose available commercially for the production of cellulase. Mixed substrates like wheat bran and corn cob are used as best carbon source in case of *A. niger* NRRL3 for cellulase production under SSF [109]. Milala et al. [95] used different agricultural


#### **Table 3.**

*Effect of supplementation of various carbon sources [106].*

wastes millet, guinea corn straw, rice husks and maize straw as carbon sources for cellulase production by *Aspergillus niger*. According to Mrudula and Murugammal [85] lactose was found to be the best inducer in SmF and SSF (**Table 3**). Prasanna et al. [110] also reported lactose as the most excellent carbon source for cellulase production by *Penicillium* sp. followed by carboxymethyl cellulose and galactose.

Different researchers studied the effect of various nitrogen sources for cellulase production by employing different microbes. Peptone was reported as most effective nitrogen source for Penicillium sp. [110], *Penicillium waksmanii* F10-2 [111], urea for *A. niger* [89] and NH4NO3 for *Trichoderma reesei* NRRL 11460 [112]. Although the addition of beef extract and peptone (as organic nitrogen source) leads to enhanced growth and enzyme production but they were not economically fit because of their higher cost.

Cellulase production by some microorganisms has been found to be influenced by metal ions, chelators, detergents and surfactants. It was reported that usually metal ions such as Ag+ , Cu2+, Hg2+, Fe3+, K+ , Mn2+, Mg2+, and Zn2+ are slightly or completely inhibitory of cellulase, whereas metal ions such as Ca2+, Co2+ and Na+ either stimulate or does not affect the cellulase activity [113]. Addition of Tween20 leads to a significant increase in endoglucanase and xylanase production by *Melanocarpus* sp. MTCC 3922 [114]. Cellulase activity increased with Tween80 and reduced with SDS [115]. Enhancement in enzyme production by Tween80 may be due to increase in permeability of cell membrane allowing rapid secretion and synthesis of the enzymes [116].
