methods of enhancement in cellulase production

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Review

Fungal solid-state fermentation and various

methods of enhancement in cellulase production

Li Wan Yoon, Teck Nam Ang, Gek Cheng Ngoh

^*

, Adeline Seak May Chua

Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o

Article history:

Received 30 August 2013 Received in revised form 11 May 2014

Accepted 16 May 2014 Available online 10 June 2014

Keywords:

Cellulase

Solid state fermentation White-rot fungi Brown-rot fungi

Enzyme production enhancement

a b s t r a c t

Cellulase serves vast applications in the industries of biofuel, pulp and paper, detergent and textile. With the presence of its three components i.e. endoglucanase, exoglucanase andb^- glucosidase, the enzyme can effectively depolymerize the cellulose chains in lignocellulosic substrate to produce smaller sugar units that consist of cellobiose and glucose. Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. Solid state fermentation (SSF) by fungi is a preferable production route for cellulase as it imposes lower cost and enables the production of cellulase with higher titre. This article gives an overview on the major aspects of cellulase production via SSF by applying white-rot fungi (WRF) and brown-rot fungi (BRF), which include the type of lignocellulosic substrates for cellulase production, inoculum preparation and process conditions applied in SSF. The parameters that affect SSF production of cellulase such as fermentation medium, duration, pH, temperature and moisture content are highlighted. In addition, potential methods that can improve cellulase production, namely genetic modification, co-culture of different fungal strains, and development of bioreactors are also discussed.

1. Introduction

Cellulase is the enzyme that plays a key role in hydrolyzingb^- 1,4-glycosidic linkage in cellulose, a dominant component in plant cell wall. Cellulase which contributed to a large pro- portion in the global market of industrial enzymes signifies its status as an important enzyme class in the market. In fact, cellulase is the third largest industrial enzymes by dollar volume[1]and accounts for approximately 20% of the total enzyme market in the world[2]. The strong demand of cellulase is attributed to its major applications in the pulp and paper, textile, food and beverages, detergent and animal feed

industries [1,3]. It has been forecasted that the demand of cellulase will be strongly driven by the commercial production of biofuel in near future[1]. This will further boost the production of cellulase due to the skyrocketing demand from the biofuel industry.

Products obtained from hydrolysis of lignocellulosic substrate by cellulase consist of mainly glucose, cellobiose and cello-oligosaccharides[1]. Among them, glucose is the most desirable product as this basic subunit of cellulose could serve as valuable feedstock for a large variety of specialty chemicals such as ethanol, organic acid and single cell protein[4,5]. To facilitate a complete hydrolysis of cellulose into glucose, a cellulase system consists of endoglucanase, exoglucanase and

*Corresponding author. Tel.:þ60 3 79675301; fax:þ60 3 79675371.

E-mail address:ngoh@um.edu.my(G.C. Ngoh).

Available online atwww.sciencedirect.com

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b-glucosidase are required to be present in an appreciable amount[6].

At present, cellulase can be produced via biological route by means of bacterial or fungal fermentation. There are a wide range of microorganisms capable of producing cellulase such as aerobic and anaerobic bacteria, anaerobic fungi, soft rot fungi, white rot fungi (WRF) and brown rot fungi (BRF)[2,7,8]. Most of the fungi are able to produce a complete cellulase system as compared to bacteria [9]. The commercial cellulase is most commonly produced from two strains of soft rot fungi (SRF), namelyTrichoderma reeseiandAspergillus niger[1], via submerged fermentation[10]. Although fungi are able to produce a complete cellulase system, cultivation of eitherT. reeseiorA. nigerresulted in deficiency on a particular cellulase components. For example, T. reeseiis not capable of producing substantial amount ofb^- glucosidase, meanwhile endoglucanase and exoglucanase are found to be lacking in the cellulase system ofA. niger[2,11]. Be- sides that, submerged fermentation suffers from a major drawback that is associated with the low concentration of end products [12], and thus, further purification is needed. The additional downstream processes required for the submerged fermentation contribute to higher cost of cellulase production.

Due to the shortcomings mentioned, researchers are focusing on how to improve the titre of cellulase as well as to reduce the production cost of cellulase. One of the solutions is by applying solid state fermentation (SSF) as an alternative production route for various industrial enzymes[12]because it closely resembles the conditions of the natural habitat of filamentous fungi. Besides, the titre of enzymes produced from SSF is more superior compared to the titre produced via submerged fermentation [13,14]. Several successful cases have been reported for cellulase production byA. nigerandT.

reesei via SSF [15e20]. As an example, when A. niger was cultivated on wheat bran, corn bran and kinnow peel in the ratio of 2:1:2, a higher cellulase activity of 10.81 U$g¹was recorded from SSF compared to 5.54 U$g¹from submerged fermentation[18]. Similar result was also obtained through the cultivation ofT. reeseivia SSF whereby cellulase activity in the range of 250e430 IU$g¹ was obtained compared to 160e250 IU$g¹obtained from liquid state fermentation[15].

Apart from the commonly applied soft rot fungi (SRF), white rot fungi (WRF) and brown rot fungi (BRF) have also been applied in cellulase production by means of solid state fermentation. In view of the lack of compiled literatures related to fungal solid-state fermentation in cellulase production, this review highlights the potential of WRF and BRF in cellulase production, the selection of suitable lignocellulosic substrate and the fungal inoculum preparation for SSF.

Furthermore, the major process parameters affecting SSF are presented. The enhancement of cellulase production via genetic modification, co-culture of fungi and improvement of bioreactor designs are also discussed in detail.

2. Wood rotting fungi and their ability in degrading lignocellulose

Wood rotting fungi can be classified into three categories, namely white-rot fungi (WRF), brown-rot fungi (BRF), and soft- rot fungi (SRF). These fungi have the ability to depolymerize

lignocellulosic materials. Among them, the SRF especiallyT.

reeseiandA. nigerare the prominent cellulase producers that possess a complete cellulase system. Besides SRF, some of the WRF and BRF such asPhanerochaete chrysosporiumandGloeo- phyllum trabeum are also competent cellulase producers [21,22]. Most of the WRF are categorized under the phylum of Basidiomycota while only a few are from the phylum of Ascomycota[23]. On the other hand, all the BRF belongs to the phylum of Basidiomycota [23]. The WRF and the BRF that belong to the class of Basidiomycetes are reported to be able to degrade cellulose by releasing cellulase enzymes[24]. How- ever, the fungi act differently in the degradation of lignocellulosic substrates in terms of the decay pattern and the structural changes of the degraded substrates. The WRF tend to degrade all the components namely lignin, cellulose and hemicellulose in lignocellulosic substrate once they colonize the lignocellulosic substrate [8] whereas BRF preferentially degrade the cellulose and hemicellulose contents with the lignin content being modified but not degraded[23,25].

Based on the degradation pattern, the WRF can be classified into two categories, according to their ability to either undergo selective delignification or simultaneous degradation of lignin and cellulose[8,23]. Under selective delignification, lignin and hemicellulose in the substrate are broken down before cellulose [25]. In contrast to the selective delignification, all the components in the substrate including cellulose are degraded by the WRF in accordance with the simultaneous degradation mechanism[25,26].P. chrysosporiumandCeripor- iopsis subvermisporaare the examples of WRF that experience simultaneous degradation and selective delignification mechanisms respectively[27].

The relationship between the degradation pattern of WRF and BRF with the yield and activity of cellulase remains unknown. The degradation pattern as deduced from lignocellulosic components loss was found to have no apparent correlation with cellulase production[28,29]. Degradation of lignocellulosic substrate might not be taking place readily though a significant amount of cellulase enzymes has been produced[29]. Despite this, the production of enzymes might be dependent on the initial content of cellulose, hemicellulose and lignin in the lignocellulosic substrate. Liu et al.[30]has reported that cellulase was preferably secreted from lignocellulosic substrate with a higher initial cellulose content and lower lignin content. Similar observation was also stated by Philippoussis et al.[31] whereby endoglucanase production was affected by the initial hemicellulose content of the lignocellulosic substrate.

3. Cellulose hydrolysis

Cellulose is the major structural polysaccharides found in plant cell wall of lignocellulosic substrate[32]. It is commonly present together with hemicellulose and lignin in lignocellulosic substrate such as sugarcane bagasse, rice husk and wheat straw [33]. Cellulose chains are single-type polymers made up of glucose monomers and these monomers are linked together by b-1,4-glycosidic bond [2]. The chains contain highly ordered crystalline regions and some amor- phous regions at an irregular interval[33]. Due to the presence b i o m a s s a n d b i o e n e r g y 6 7 ( 2 0 1 4 ) 3 1 9e3 3 8

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of the crystalline structure, plant cell wall is resistant to microbial and enzymatic attack.

Theb-1,4-glycosidic bonds in cellulose can be broken apart effectively by cellulase enzymes produced by the WRF and the BRF. To efficiently break down cellulose into its monomers, the cellulase system applied should consist of three components, i.e. endoglucanase, exoglucanase and b-glucosidase that act synergistically[6,34]. As explained by Zhang et al.[35], the hydrolysis of lignocellulosic substrate is divided into primary hydrolysis and secondary hydrolysis. Primary hydrolysis generally happens on the surface of substrate, aiming to fractionate the chains of cellulose into soluble sugars with degree of depolymerization less than 6. The sugar released into liquid phase as a consequence of the catalytic action provided by endo- and exoglucanase during primary hydrolysis. Endoglucanase hydrolyzes cellulose chains to form new chain ends and these chain ends are further broken down into cellobiose and cello-oligosaccharides by exoglucanase[6,36].

The cellobiose is then fractionated into glucose with the aid of b-glucosidase in secondary hydrolysis. The overall mechanism of the cellulose hydrolysis is depicted inFig. 1.

The BRF have an enzyme system that operates differently from the WRF and with this, a more superior performance in cellulase production might be demonstrated by BRF. As pro- posed by Tewalt and Schilling[37], BRF degrade lignocellulosic substrate in two steps. In the initial stage, the cell wall of lignocellulosic substrate is modified in the absence of enzymes.

Thereafter, cellulase enzyme is secreted by BRF to break down the cellulose into glucose. Low molecular weight degradation agents such as hydrogen peroxide (H2O2) play an important role in the first stage of lignocelluloses degradation by BRF[38]. The production of hydrogen peroxide by BRF was induced by cellulose in lignocellulosic substrate, preferably the crystalline cellulose[39]. These low molecular weight agents penetrate through the cell wall of the lignocellulosic substrate and react with endogenous iron or other transition metals to produce hydroxyl radical via Fenton reaction [39,40]. The hydroxyl radical produced degrades the lignocelluloses in the substrate and this degradation mechanism is also known as BRF

oxidative degradation in many contexts. In the subsequent step, cellulase is released by BRF to further break down the remaining cellulose in the cell wall. The cellulase system of BRF has sufficient amount of endoglucanase andb-glucosidase but deficient in exoglucanase[41]. Hence, oxidative degradation is a crucial step to complement the incomplete cellulase system of BRF to enhance the overall conversion of cellulose into glucose.

4. Fungal SSF in cellulase production

Although cellulase is produced via submerged fermentation commercially, these enzymes can also be produced via solid state fermentation (SSF). The latter has the potential to be up- scaled for greater volume production. SSF is carried out without the apparent presence of free water[10]but with sufficient moisture provided to support the growth of fungi on lignocellulosic substrate [14]. Due to this reason, the cost of dewatering in downstream processing can be greatly reduced.

The production cost of SSF can be further reduced down to ten- fold compared to submerged fermentation[42]due to the lower energy consumption[13]and the employment of suitable low cost lignocellulosic substrate[1]. SSF also offers advantages in terms of higher concentration of enzymes, higher fermentation productivity and lower demand on the sterility of the equip- ments[13,43]. The crude enzyme solution obtained from SSF can be applied directly to hydrolyze the lignocellulosic substrate.

SSF is particularly suitable for filamentous fungi as they can be cultivated under the absence of free water[43]. It was confirmed that filamentous fungi is a better cellulase producer under SSF than submerged fermentation[44]. Apart from the operational advantages, cellulase produced by BRF,G. trabeum in SSF has better performance in terms of hydrolysis efficiency compared with those produced from the liquid culture[37].

Table 1 summarizes the potential white-rot and brown-rot fungal strains for cellulase production.

From the fungal strains shown inTable 1,P. chrysosporium which is reported as one of the best cellulase producers[63]is among the more commonly used WRF in cellulase production.

Fig. 1eMechanism of cellulose hydrolysis by cellulase.

b i o m a s s a n d b i o e n e r g y 6 7 ( 2 0 1 4 ) 3 1 9e3 3 8

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However, only one species under Phanerochaete genus was examined for producing cellulase. Unlike WRF in the genus of Phanerochaete, more than five species from the generaTrametes and Pleurotusare excellent cellulase producers. Conversely, despite the proven ability of BRF in cellulase production, very limited numbers of BRF strains had been tested for cellulase production to date.

It should be noted that not all the WRF and BRF are effective in producing cellulase. Some of them might be more superior in producing other oxidative and hydrolytic enzymes.

For instance,Lentinula edodeswhich is also known as shiitake mushroom produced higher amount of ligninolytic enzymes whereasFunalia trogiiandPleurotus dryinusare inclined to the accumulation of a higher amount of cellulase under SSF[54].

Besides that, the production of cellulase is hypothesized to be greatly affected by the fungal morphology as reviewed by Singhania et al. [1]. Fungi with filamentous mycelia are reported to have enhanced cellulase production[64]. However, the actual relationship between fungal morphology and the productivity of enzymes is still remained unknown[65].

5. Types of lignocellulosic substrate for cellulase production

One significant aspect in SSF is the type and composition of lignocellulosic substrate applied. The selection of a suitable

lignocellulosic substrate which is able to support the fungal growth and simultaneously stimulate the production of cellulase is desirable. Lignocellulosic substrate that contains sufficient nutrients to supplement the fungal growth is preferable. Besides the amount of nutrients, substrate that enables the anchorage of fungi during its growth is another important criterion to be considered when choosing the substrate for SSF[46]. The potential substrates to be applied in cellulase production by WRF and BRF are shown inTable 2. To facilitate the reproducibility of cellulase production via uniform substrates preparation, as many details to the best of our knowledge on the lignocellulosic substrates were compiled in Table 2with reference to a checklist suggested by Barton[66].

The lignocellulosic substrates applied in cellulase production can be classified according to their woody and herbaceous nature as presented inTable 2. Larger varieties of herbaceous substrates have been tested for cellulase production compared to the woody substrates. This is attributed to most of the herbaceous substrate are produced in substantial amount in many countries and these agricultural residues are low in cost. In terms of processing method, woody substrate was mostly de-sized by cutting the substrate into smaller pieces whereas grinding and sieving are preferable in reducing the size of herbaceous substrate before SSF. As a result, woody substrate with larger size is more commonly applied compared to herbaceous substrate. Besides, the age of woody substrate might affect the reproducibility of cellulase production as the compositions of the substrate might change as it ages although this aspect is often of lesser importance when herbaceous substrate is used. Regarding to the storage of substrate, the processed substrate is preferably stored in dry condition to reduce the risk of contamination. By keeping the substrates under dried condition, the storage time could be prolonged.

In the aspect of cellulase production, Eucalyptus wood chip is a more commonly applied woody substrate whereas wheat straw is the most investigated among the herbaceous substrate (Table 2). The chemical compositions of wheat straws and wood chips are suitable to support the fungal growth as well as to facilitate the production of cellulase. In general, woody substrate has a higher percentage of lignin content compared to herbaceous plant. Some substrates such as mandarin peels and wheat bran with a low composition of lignin can also boost cellulase production[48]. This might due to the presence of other compounds such as free sugars and organic acids[48]. Furthermore, material with high content of starch such as sago waste is also suitable for cellulase production[55].

Also, it is important to use the substrate with desired structural properties to support fungal growth. Most of the lignocellulosic substrates have recalcitrant structure due to the presence of the complex interaction between the carbo- hydrate and lignin in the substrates. The recalcitrant structure might impede the accessibility of microorganisms to the cellulose and hemicellulose portion of the substrate[33]. Thus, pretreatment is often viewed as a means to alter the originally complex and recalcitrant chemical structure of lignocellulosic substrates [68]. However, pretreatment of substrate prior to cellulase production might not be necessary as it probably does not aid the production of enzymes as reported by Table 1ePotential white-rot and brown-rot fungal

strains in cellulase production via solid state fermentation.

Genus Species Reference

White-rot fungi

Phanerochaete chrysosporium [21,44e47]

Trametes versicolor [29,48,49]

trogii [50]

pubescens [51]

hirsuta [48]

ochracea [48]

Pleurotus ostreatus [48,49,52,53]

dryinus [53,54]

tuberregium [53,54]

sajor-caju [55]

pulmonarius [52]

Lentinus edodes [31,47,53,54,56,57]

tigrinus [51,58]

Cerrena maxima [51]

Funalia trogii [51,54]

Coriolopsis polyzona [51]

Pycnoporus coccineus [29,51]

sanguineus [59]

Bjerkandera adusta [59]

Fomes fomentarius [48]

Psedotremella gibbosa [48]

Trichaptum biforme [48]

Irpex lacteus [60]

Ceriporiopsis subvermispora [61]

Brown-rot fungi

Laetiporeus sulfurous [29]

Fomitopsis e [62]

Wolfiporia cocos [29]

Piptoporus betulinus [49]

Gloeophyllum trabeum [22,44]

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Table 2eThe potential woody and herbaceous substrates applied in cellulase production by WRF and BRF via SSF.

Substrate Substrate definition Fungal strain Cellulase production^a Reference

Location Age of sample (years)

Method of processing Method of storage Composition of substrate

Woody substrates Eucalyptus wood

chips

Pulp mill from Lorena, Brazil

8 Wood chips were cut to 2.5 cm1.5 cm0.2 cm and immersed in water for 16 h. The water was drained and the chips were autoclaved prior to SSF.

e e Ceriporiopsis

subvermispora

CMCase: 31.2 U$kg¹ b-glucosidase: 43.6 U$kg¹

[61]

Fiberboard mill from Lorena, Brazil

8 Cut to

2.5 cm1.5 cm0.2 cm.

Chips immersed in water for 12 h. After drainage of water the chips were autoclaved.

Air dried to final humidity of 8e10%.

Stored in dry condition.

42.8±0.8% glucan, 13.0±0.2% xylan, 26.3±0.7% lignin

Laetiporeus sulfurous FPase:

15.4 U$culture¹ b-glucosidase:

7.6 U$culture¹

[29]^,b

Trametes versicolor FPase:

4 U$culture¹ b-glucosidase:

3.8 U$culture¹

[29]^,b

Pine wood chips Lorena, Brazil 28 Cut to

3.5 cm2.0 cm0.3 cm and immersed in water for 16 h. Chips were autoclaved

e e Ceriporiopsis

subvermispora

CMCase: 15.3 U$kg¹ b-glucosidase: 53 U$kg¹

[61]

Beech leaves (Fagus sylvatica)

Brussels, Belgium e Desized to 0.1e0.5 cm. e e Pleurotus dryinus CMCase:

284 U$g¹ FPase: 32 U$g¹

[54]

Herbaceous substrates Wheat straw

(Triticum aestivum)

e e e e e Lentinus tigrinus CMCase:

~1200 U$g¹ b-glucosidase:

~1950 U$g¹

[58]

Brussels, Belgium e e e e Pleurotus dryinus CMCase:

401 U$g¹ FPase: 41 U$g¹

[54]

e e Ground until homogenous

powder and obtained size in the range of 0.5e3 mm.

e e Bjerkandera adusta CMCase:

~2.4 U$mg¹protein Exoglucanase:

~0.3 U$mg¹protein b-glucosidase:

<0.5 U$mg¹protein

[67]

e e Soaked in water for 12e24 h.

Supplied 20% of

supplements to substrate.

e 80.03% cellulose, 6.32% hemicellulose, 8.51% lignin

Lentinus edodes CMCase:

0.78 U$g¹substrate dry weight

[31]

(continued on next page)

biomassandbioenergy67(2014)319e338

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Table 2e(continued)

Substrate Substrate definition Fungal strain Cellulase production^a Reference

Location Age of sample (years)

Method of processing Method of storage Composition of substrate

Wheat straw (Triticum aestivum)

e e Shredded into 4e6 cm in

length. The chips were soaked in water for 24 h.

10% (w/w) of gypsum was added to straw before autoclaved.

e e Lentinus edodes CMCase:

~55 mU$g¹cultivation substrate

b-glucosidase:

~120 mU$g¹cultivation substrate

[57]

Wheat bran New Delhi, India e Dried, ground and separated by 20 mesh size.

e e Fomitopsissp. CMCase:

71.699 U$g¹substrate FPase:

3.492 U$g¹substrate b-glucosidase: 53.679 U$g¹ substrate

[62]

Corn fiber Wet corn milling plants, Illinois, USA

e Hot water steeping and sulfur dioxide

pretreatment. Sample was dried at 80C for 4 days prior to be used.

Stored under desiccated condition

18% cellulose, 35%

hemicellulose, 17%

starch, 18% lignin, 7% moisture

Phanerochaete chrysosporium

CMCase: 3.42 (m^g

glucose)$(mg protein.min)¹ Exoglucanase: 0.23 (m^g glucose)$(mg protein.min)¹

[21]

Corn stover Enshi city, China e Air-dried and ground to the size in the range of 0.425 e0.85 mm.

e e Irpex lacteus Cellobiohydrolase:69.3

U$g¹culture

CMCase: ~2.5 U$g¹culture b-glucosidase: 11.1 U$g¹ culture

FPase: ~2.5 U$g¹culture

[60]

Reed grass (mixture of Typha angustifolia, Carex pseudocyperus and Phragmites australis)

Supplied 20% of

Lentinus edodes CMCase: 0.97 U$g¹dry substrate

[31]

Bean stalk (Phaseolus coccineus)

Supplied 20% of

Lentinus edodes CMCase: 1.39 U$g¹dry substrate

[31]

Sago waste Johor, Malaysia e Air-dried and sieved through 2 mm sieve.

Stored at room temperature.

e Pleurotus sajor-caju CMCase:

2 85 U$g¹ Low Fpase andb^- glucosidase

[55]

a Cellulase production reported is at the maximum activities and the units of cellulase produced were reported in the same format as in the original articles. 1 U of cellulase activity is equivalent to 1m^{mol min}¹of hydrolysis product formed under the assay condition. CMCase represents carboxymethyl cellulose activity, FPase represents filter paper activity,b-glucosidase represents b-glucosidase activity, Endoglucanase represents endoglucanase activity and Cellobiohydrolase represents cellobiohydrolase activity.

b The cellulase activity in the unit of U$culture¹was based on 50 g ofEucalyptus grandiswood chips per culture.

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Brijwani and Vadlani[69]. Cen and Xia[9]have also stated that pretreatment is not a prerequisite for SSF as compared to submerged fermentation. These could be the reasons that only a few groups of researchers pretreat their substrates prior to SSF as indicated inTable 2. In spite of that, most of the researchers autoclaved their substrates before inoculation.

Besides sterilizing the substrates, autoclaving can also serve as a pretreatment step since the structure of the substrates can be disrupted during pressurized steaming process. Simi- larly, inoculating the substrate with WRF in SSF can also be considered as a pretreatment step because WRF are prominent in degrading the lignin content and thus, the complex structure of lignocellulosic substrate is greatly modified during the course of fermentation[6,70].

It is apparent that some lignocellulosic substrates are un- able to support cellulase production under SSF by WRF and BRF. Hong et al.[46]has proven thatP. chrysosporiumcould not grow well on substrates such as paddy husk, coconut fiber, wood dust, coconut meal, palm kernel cake, sugarcane bagasse and oil palm trunk. Cellulase activities were also failed to be detected when Pleurotus ostreatus and Trametes versicolorwere cultivated on a mixture of tomato pomace and sorghum stalk respectively[71]. Hong et al.[46]deduced that substrate contains secondary metabolites that act as antimi- crobial agents can inhibit the growth of fungus and cellulase production [46]. In addition, lignocellulosic substrates with low porosity might not be able to support fungal growth and cellulase production due to the decrease in the availability of oxygen between the moist solid particles[69].

Furthermore, WRF and BRF might also be substrate specific as the same fungal strain that failed to produce cellulase from a particular substrate could have produced cellulase when it is cultivated on another substrate. For instance, P. ostreatus produced a significant amount of cellulase when it was cultivated on the residues of ethanol production from wheat grains (REP) but not on maple leaves[48]. Similarly, different fungal strains cultivated on the same substrate might result in a significant difference in cellulase titre produced. As shown inTable 2, a relatively higher cellulase activity can be detected whenLentinus tigrinusinstead ofP. dryinuswas cultivated on wheat straw. Hence, it is important to search for the suitable combination of fungal strain and lignocellulosic substrate for optimal cellulase production.

It is rather difficult to compare the cellulase activities produced from different substrates presented inTable 2. Besides different combination of fungal strain and lignocellulosic substrate applied in the SSF system, cellulase enzymes produced was reported in different enzyme activity units and hence, direct comparison might not be applicable. However, the commonly applied enzyme loading for efficient hydrolysis of lignocellulosic substrate is in the range of 10e50 U$g¹FPase on cellulose and 20e40 U$g¹b-glucosidase on cellulose when the solid loading of 2e5% is applied in enzymatic hydrolysis[72].

6. Inoculum preparation for solid state fermentation

Inoculum preparation is another important aspect in solid state fermentation. There are several ways of preparing fungal

inoculum for SSF. To identify the most suitable type of inoculum to be employed in SSF, both the nature of fungi involved and the purpose of studies have to be taken into consider- ation. The commonly applied inoculum preparation methods for SSF include spore suspension, mycelia disc, mycelia suspension and pre-inoculated substrates.

Spores suspension can be prepared by washing the surface of fungi cultured on Petri dish with sterilized water or salt solution[46,73]. In the preparation of spore suspension, the fungal biomass is macerated and spores suspension is then obtained after filtration of the washing liquid. The number of spore count in the suspension can be adjusted with the aid of hemocytometer and the inoculum density can then be adjusted by adding water or salt solution into the suspension to achieve the desired spore concentration [63,70]. Spores suspension with concentration of approximately 10⁶cm³is often used as the inoculum for SSF[46,63,70,73,74]. Besides the ease of inoculum preparation, spore suspension can also be stored for a longer duration and thus, mishandling during transfer of inoculums can be prevented. The drawback of this method is that the spore counting process is time consuming and it might not be suitable for some of the fungi that are sporeless or lack of spore such as the mutants of Pleurotus sajor-caju [75], Pleurotus eryngii [76]and Lentinus edodes [77].

Fungal growth might also be slower if the spore suspension was used for inoculation due to its longer lag phase.

Inoculum in the form of mycelia disc is prepared by cutting the agar plug from the periphery of the actively grown fungi [31]. The mycelia disc can be directly used to inoculate the substrate. Mycelia disc inoculation is a more convenient method compared to spore suspension, but it might not be advisable to be used in comparison study involving different types of fungi. This is because the growth rate of different fungi might be different and hence, the mycelia density of the fungi grown on the agar plug varies with the types of fungi.

This contributes to the difficulties in determining the density of fungi and a fair comparison for cellulase production for different fungi cannot be established.

For WRF or BRF that is sporeless or lack of spores, mycelia suspension appears as a preferable choice of inoculum in SSF.

Inoculation of substrate by mycelia suspension can greatly eliminate the lag phase experienced by cultivating the fungi from its spore suspension. As a result, mycelia suspension inoculation was employed by many researchers and it has become the most popular choice of inoculum preparation method in cellulase production via SSF[21,29,51,54,56,61,62].

However, this method involves many preparation steps that are tedious and time consuming. First, the mycelia mat or mycelia disc from an agar plate with actively grown fungi needs to be transferred into a liquid medium before incu- bating it for 5e7 days [29,48]. After the incubation period, washing and homogenization of the fungal pellets is per- formed[21,48,61,62].

Some researchers used pre-inoculated substrate as the inoculum for SSF [52,55,58,60,78]. In general, this type of inoculum was prepared by transferring the mycelia disc onto the cooked or autoclaved wheat grains[52,55,78]. It was then incubated at room temperature for a period of time ranges from 6 to 21 days, depending on the amount of wheat grain and mycelia disc used[45,52,55,78]. Calcium carbonate was

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Table 3eProcess conditions applied in SSF for cellulase production by WRF and BRF.

Microorganisms Lignocellulosic substrate

Process conditions Reference

Fermentation medium Duration pH Temperature Moisture content

White rot fungi

Phanerochaete chrysosporium Soybean hull 2% (by weight) urea 6 days for CMCase and FPase

4 25C e [63]

Phanerochaete chrysosporium Corn fiber 0.25 g dm³KH2PO4

0.063 g$dm³ MgSO4$7H2O

0.013 g$dm³CaCl2$2H2O

1.25 cm³$dm³of trace element solutions

2 days for endocellulase and exocellulase production

e 37C 25 g of substrate with 75 cm³ of medium

[21]

Phanerochaete chrysosporium Corn stover 0.5% (NH4)2SO4

0.25 g$dm³KH2PO4 0.063 g$dm³ MgSO4$7H2O 0.013 g$dm³ CaCl2$2H2O

1.25 cm3$dm³of trace element solutions

4 days of FPase 4.5e4.8 37C 2 g of substrate with 7 cm3 of medium

[82]

Phanerochaete chrysosporium 28% rice straw, 2%

wheat flour

Mineral solution 4 days for FPase and

CMCase

e 32C 70% [73]

Pleurotus sajor-caju Sago waste 0.2% KH2PO4

0.05% MgSO4$7H2O 0.38% urea

7 to 9 days for CMCase and FPase

e 25C 83% [55]

Pleurotus ostreatus Tree leaves 1 g$dm³NH4NO3

0.8 g$dm³KH2PO4

0.2 g dm³Na2HPO4

0.5 g dm³MgSO4$7H2O 4 g$dm³yeast extract

Peptone with concentration equivalent to 20 mmol$dm³nitrogen

10 to 14 days for CMCase 6 27C 4 g of substrate with 12 cm³ of medium

[51]

Pleurotus dryinus Beech leaves 1 g$dm³NH4NO3

0.8 g$dm³KH2PO4

0.2 g$dm³Na2HPO4

0.5 g$dm³MgSO4$7H2O 4 g$dm³yeast extract

Peptone with concentration equivalent to 20 mmol$dm³nitrogen

10 to 14 days for CMCase 6 27C 4 g of substrate with 12 cm3 of medium

[54]

Pleurotus dryinus Wheat straw 1 g$dm³NH4NO3

0.8 g$dm³KH2PO4

0.2 g$dm³Na2HPO4

0.5 g$dm³MgSO4$7H2O

4 g$dm³yeast extract (NH4)2SO4with concentration equivalent to

20 mmol dm³nitrogen

10 to 14 days for CMCase 6 27C 4 g of substrate with 12 cm³ of medium

[54]

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Pseudotremella gibbosa Residue of ethanol production from wheat grains

2 g$dm³ammonium tartrate 0.8 g$dm³KH2PO4

0.2 g$dm³K2HPO4

0.5 g$dm³MgSO4$7H2O 4 g$dm³yeast extract

0.1 mmol$dm³CuSO4$5H2O and MnSO4$H2O

9 to 14 days for CMCase 5.5 27C 4 g of substrate with 12 cm3 of medium

[44]

Lentinula edodes 79% of eucalyptus sawdust, 20% of rice bran

e 11 days for FPase17 days for

CMCase

e 25C 60% [52]

Lentinula edodes Reed grass e 35 days for endoglucanase

production

5.34 26C 62e65% [27]

Bjerkandera adusta 2% wheat straw on agar plate

7.8 mg$dm³CuSO4$5H2O 18 mg$dm³FeSO4$7H2O 500 mg$dm³MgSO4$7H2O 10 mg$dm³ZnSO4

50 mg$dm³KCl 1 g$dm³K2HPO4

2 g$dm³NH4NO3

6 days for CMCase 5 28C e [63]

Irpex lacteus Corn stover e 5 days for FPase e 28C 75% [56]

Pycnoporus sanguineus 2% wheat straw on agar plate

7.8 mg$dm³CuSO4$5H2O 18 mg$dm³FeSO4$7H2O 500 mg$dm³MgSO4$7H2O 10 mg$dm³ZnSO4

50 mg$dm³KCl 1 g$dm³K2HPO4

2 g$dm³NH4NO3

8 days for CMCase 5 28C e [63]

Trametes versicolor Eucalyptus grandis chips

2% malt extract 15 days for total cellulase production60 days forb^- glucosidase production

e 28C e [25]

Brown rot fungi

Fomitopsissp.RCK2010 Wheat bran 0.5 g$dm³(NH4)2SO4

0.5 g$dm³KH2PO4

0.5 g$dm³MgSO4

0.05% N2 equivalent of urea 2 mmol$dm³zinc and nickel ions 0.2% (by weight) Triton X-100

11 days for CMCase15 days forb-glucosidase16 days for FPase

5.5 30C Substrate-to-moisture ratio of 1:3.5

[58]

Gloeophyllum trabeum Corn fiber 0.250 g$dm³KH2PO4

0.063 g$dm³ MgSO4$7H2O

0.013 g$dm³CaCl2$2H2O

1.25 cm3$dm³trace element solution

2 days for endocellulase and exocellulase production

e 30C 25 g of substrate with 80 cm3 of medium

[18]

Piptoporus betulinus Wheat straw Distilled water 42 days for endoglucanase,

b-glucosidase and cellobiohydrolase

e 28C Approximately 75% [45]

Laetiporeus sulfureus Eucalyptus grandis chips

2% malt extract 15 days for total cellulase production120 days forb^- glucosidase production

e 28C e [25]

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added to the cooked wheat grain before inoculation to adjust the pH of the wheat grain into a range which is suitable for the particular fungus to grow[55]. By using this inoculum preparation method, the inoculum size is relatively difficult to be quantified and this might hinder the work which involves direct comparison of the performance of different fungi in cellulase production. On the other hand, one of the advantages associated with this method is that the substrate with grown fungus after SSF can be blended with a portion of fresh inoculum and use to re-inoculate a new batch of substrate.

This can be seen as a step to minimize the use of fresh inoculum and also the waste generated from the SSF process.

Other than the abovementioned inoculum preparation methods, a user-friendly and cost-effective inoculum preparation method developed specifically for solid-state fermentation called Cellophane Film Culture (CFC) technique has been reported [79]. This technique employed agar plated overlaid with cellophane film to ease the separation of viable fungal biomass, which is used subsequently as inoculum in fermentation. Similar to inoculum preparation via mycelia suspension, inoculum prepared from CFC technique can be quantified. It is reported that the technique has added advantages such as it requires less stringent handling condition and has lower risk of contamination during the inoculum preparation process compared to spore and mycelia suspension methods[79]. In addition, inoculum prepared from CFC technique exhibits homologous morphology which permits the quick colonization following inoculation on the solid substrate.

Besides the types of inoculum as mentioned, cellulase production is also affected by the inoculum size applied[80].

Colonization of fungi on lignocellulosic substrate might take a relatively longer time if a low dosage of inoculum is used. This

might correspondingly raise the risk of contamination where other fast growing fungi might colonize the substrate in a faster rate compared to that of the intended microbial species.

Higher inoculum size might accelerate the fungal growth rate but at the same time increase the rate of nutrient depletion.

Upon nutrients depletion, the growth of the fungi is affected and this might not be helpful in improving the yield of cellulase[55].

7. Process conditions in SSF

Apart from the preparation of inoculum, the operating conditions of SSF, such as the composition of fermentation medium, fermentation duration, pH, temperature and moisture content of the substrate can affect the cellulase production.

There is a direct relationship between the operating condition and the titre of cellulase obtained. For instance, the presence of unfavorable humidity and temperature gradient affect cellulase production negatively[81]. Hence, a suitable range of operating conditions must be selected carefully during solid state fermentation. The process conditions applied for cellulase production by WRF and BRF via SSF was summarized in Table 3.

7.1. Fermentation medium

Fermentation medium used in SSF influences the types and titres of enzymes produced using WRF or BRF. Despite cellulase production was attempted with simple medium such as 2% malt extract[29], fermentation medium used by most of the researchers for cellulase production consists of carbon source, nitrogen source, phosphorus source, trace element

Table 4eNitrogen sources for the production of different cellulase enzyme components by WRF and BRF via SSF.

Cellulase components Nitrogen source Nitrogen source concentration Remarks Reference Total cellulase system Casein Equivalent to 0.05% N2 Maximum FPase obtained is

4.682 U$g¹

[62]

Urea 2% (by weight) dry soy hull Increase the cellulase activities by nearly two fold

[63]

Endoglucanase Urea Equivalent to 0.05% N2 Maximum CMCase obtained is

81.832 U$g¹

[62]

Peptone Final concentration in

fermentation medium equals to 20 mmol$dm³of nitrogen

CMCase increased from 20 U$cm³ to 28e35 U$cm³whenP. ostreatus was cultivated on tree leaves after addition of peptone.

[51]

Peptone concentration higher than 18 g$dm³

CMCase of approximately 450.6 U$cm³was obtained.

[50]

Final concentration in

Induced CMCase to 284 U$g¹when P. dryinuswas cultivated on beech leaves

[54]

Ammonium sulfate [(NH4)2SO4]

Final concentration in

Induced CMCase to 401 U$g¹when P. dryinuswas cultivated on wheat straw.

[54]

Exoglucanase Peptone Peptone concentration higher than

18 g$dm³

Cellulase activity of approximately 36 U$cm³was obtained

[50]

b-glucosidase Soybean meal Equivalent to 0.05% N2 Maximumb-glucosidase is 69.083 U$g¹

[62]

Peptone Induced by peptone concentration

approximately 10 g$dm³ b-glucosidase activity of approximately 3.58 U$cm³was obtained

[50]

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solution and other minerals (Table 3)[83]. In general, there was no apparent variation between the fermentation medium required by WRF and BRF (Table 3).

Cellulose in lignocellulosic substrate acts as the essential carbon source with a role as inducer for cellulase production [49]. Besides, cellulase and fungal biomass production can also be stimulated by the nitrogen source present in fermentation medium[54].Table 4shows a general trend whereby peptone is the potential nitrogen source that stimulates the production of all the components in a complete cellulase system.

Different cellulase components were probably induced by different types of nitrogen source. This can be deduced from the enhancement of endoglucanase production from 20 U$cm³ to a range of 28 U$cm³e35 U$cm³ when the fermentation medium was supplemented with peptone, which was in contrast with the depletion of endoglucanase activity to a range of 13 U$cm³e17 U$cm³when peptone was substituted with ammonium sulfate [(NH4)2SO4] in the cultivation ofP. ostreatuson tree leaves[51].

Although peptone could generally enhance the production of all the cellulase enzyme components, the effect of supple- menting this nitrogen source in different lignocellulosic substrates has varied outcome. As shown in an investigation by Kachlishvili et al.[54], peptone was the most suitable nitrogen source for CMCase production whenP. dryinuswas cultivated on beech leaves whereas (NH4)2SO4 was a better nitrogen source when wheat straw was applied. This suggests that a particular nitrogen source might have interactive effects with certain lignocellulosic substrates in stimulating cellulase production. Therefore, it is essential to have a proper combination of nitrogen source, lignocellulosic substrate and fungal strain for maximizing the production of cellulase via SSF.

Besides nitrogen source, the fermentation medium for cellulase production was also supplemented with phosphorus source, trace elements and other minerals. Phosphorus source is essential in supporting the growth of fungus as it aids the formation of phospholipid bilayer in the fungal cell membrane [6]. Potassium dihydrogen phosphate (KH2PO4) is a commonly applied phosphorus source (Table 3). Furthermore, cellulase production could be enhanced by adding surfactant such as Tween 80 and triton X-100 into the fermentation medium.

These surfactants improve the permeability of fungal cell membrane by allowing the secretion of cellulase in a more rapid manner[84,85].

Trace elements such as zinc, nickel, manganese and copper might serve as cofactors for cellulase enzymes, and they are proven to be capable in enhancing the production of cellulase [62,70]. Fermentation medium with copper (II) ion (Cu^2þ) concentration in the range of 6.5 mmol dm³e11 mmol dm³enhanced endoglucanase and b-glucosidase production whereas higher amount of exoglucanase was obtained when medium with Cu^2þconcentration in the range of 2 mmol dm³e4 mmol dm³was applied[50].

Although some trace elements are essential for fungal growth and enzymes production, they could be toxic to the fungi when present in excess[86]. Heavy metals such as mercury and lead (Pb) imposed adverse effect on the production of cellulase [74,87e89]. Mercury in the range of 0.05e0.25 mmol dm³ hampers fungal growth[90]whereas the fungal biomass growth was inhibited when Pb with

concentration higher than 50 mg dm³is present[86]. It was also discovered that the carboxymethyl cellulase activity (CMCase) was inhibited by Pb^2þunder the investigated range of 30, 200 and 400 mg Pb^2þ$kg¹straw, as reported by Huang et al.[74]. These heavy metals affect the growth rate of fungi by prolonging the lag phase and causes morphological changes to the fungi[86]. Cellulase production on the tran- scriptional and translational level is also affected when excessive heavy metals were penetrated into the cell[86].

7.2. Fermentation duration

When WRF and BRF are cultivated on lignocellulosic substrate under suitable condition, fungi starts to grow by extending their hyphal tips[83]. The fungal mycelium is then spread throughout the substrate forming a network of mycelia. Pri- mordial formation which involves the development of dense and progressive mycelium mat takes place next until the surface of the substrate is almost colonized by the mycelium mat[91]. The first sign of fungal growth was reported on day 2 of SSF in most of the studies involving cellulase production [48,52,55]. After 7e11 days, the fungus cultivated might have completely colonized the substrate, depending on the amount of substrate used[48,52,55]. At the final stage of fungal growth, the basidiome and fruiting body of the fungi might have developed[91].

During colonization phase of fungal growth, extracellular enzymes are produced to degrade the lignocellulosic substrate into smaller soluble molecules that could be utilized as nutrients for growth[46,91]. Due to the hydrolytic action of the enzymes produced, depolymerization of lignocellulosic components occurs. Cellulase was first detected on day 2 of SSF [21,48,54] and its production peaked within 6e16 days as frequently reported (Table 3)[48,52,57,62]. Different fermentation duration might be required for the specific cellulase components to reach their maximum activity. Machuca and Ferraz[29]reported that the maximum total cellulase activity was shown on the 15th day whereas 120 days were required forb-glucosidase to reach its maximum activity whenLaeti- poreus sulfureuswas cultivated onEucalyptus grandischips.

Cellulase activity is peaked during the colonization phase of fungal growth and it rapidly decreases at the later stage during the formation of fruiting body[58,91]. The maximum cellulase activities could have occurred when the metabolic activity of fungi and the concentration of water-soluble sugar were both at their highest value[57]. On the other hand, BRF colonizes lignocellulosic substrate at a faster rate with ten- dency to produce a higher titre of cellulase in comparison with WRF[29]. However, there was no significant difference in the fermentation duration required by WRF and BRF for optimum cellulase production even though BRF required a lesser time to colonize the lignocellulosic substrate (Table 3)[29].

7.3. pH

Due to the difficulty in monitoring the pH throughout SSF, the pH is normally not a controlled parameter during SSF and it is adjusted at the beginning of SSF. If the initial pH value of the fermentation medium is adjusted, the variations of pH during the SSF were normally not taken into account [92]. The pH

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might vary slightly during the course of SSF and this variation is related to the metabolic activities of the fungi[73]. It has been reported that during SSF, the initial pH dropped after 4 days of fermentation and increased again at the end of SSF on the 8th day[73]. The decrease of pH might be due to the formation of organic acids and the consumption of ammonium salt in the fermentation medium[73,83].

In general, an initial pH of approximately 5 is preferable for cellulase production by most of the WRF and BRF (Table 3) [9,50,55,58,62,67]. It is worth noting that most of the lignocellulosic substrates have buffering property in which they can minimize the variation of pH during SSF[9]. The nitrogen- containing inorganic salt in the fermentation medium such as urea can also offset the variation in pH throughout the SSF process[92]. Due to these collective factors, it is not uncom- mon for cellulase production to be conducted without pH adjustment in the initial stage (Table 3)[21,29,49,55,56,60,73].

7.4. Temperature

Temperature is a fungal-dependent parameter that influences cellulase production. For instance, white-rot fungusP. chrys- osporiumis normally cultivated at 37C[21,45,74]whereasL.

edodesis cultivated at a much lower temperature (25e27C) in order to produce cellulase optimally via SSF[31,51,54,56,57].

The suitable temperature for cellulase production by other commonly reported WRF and BRF was stated inTable 3.

The optimum temperature for cellulase production and fungal growth might be different.Pcynoporus sanguineusach- ieved optimum growth at 37C but 28C is more suitable for cellulase production by this WRF[67]. The optimum temperature for cellulase production normally falls within 25e30C (Table 3). SSF carried out at high temperature imposes an adverse effect on cellulase production because the enzymes produced could be denatured[62]. This suggests that a desirable fermentation temperature should be a compromise between the optimum temperature for cellulase production as well as fungal growth.

7.5. Moisture content

The moisture content of the substrate is another parameter worth to be examined in SSF. When the moisture content is lower than the required level, the solubility of nutrients is limited and it hinders effective nutrients uptake by the fungi [18,62]. On the contrary, when the moisture content is too high, the particles of substrate will be surrounded by a thick layer of water. Consequently, the particles tend to stick together and this limits air diffusion between the particles and the surrounding[62,93]. In addition, the risk of contamination is greater if higher moisture content is applied in the SSF as the condition encourages the growth of unfavorable microorganisms.

The amount of moisture required is directly related to the structure of lignocellulosic substrate. The porosity and specific surface area of the solid particles govern the efficiency of air diffusion and water holding capacity of the substrate[92].

When determining the moisture content for optimal cellulase production, it is necessary to take into account the nature of the substrate applied in SSF [94]. Some lignocellulosic

substrates have the ability to absorb more water and these substrates with higher water absorbability are preferable because the moisture content can be adjusted easily upon the addition of fermentation medium[94]. As shown inTable 3, moisture content lower than 60% and higher than 80% is less favorable for both fungal growth and cellulase production in SSF, regardless of whether WRF or BRF was applied in the SSF system.

8. Enhancement of cellulase production

Besides the suitable substrate and operating conditions, one of the alternatives that can enhance the cellulase production by fungal solid-state fermentation is to genetically modify the fungal strain. Fungal strain can be modified to become more robust in producing higher titre of better quality cellulase.

Moreover, co-culture of different fungal strains is also deemed possible to enhance cellulase production. Apart from that, the application of suitable bioreactor with advanced control features can further facilitate the production of cellulase in a larger scale.

8.1. Genetic modification of fungal strain

In the industry, some genetically modified fungal strains have been applied in cellulase production via submerged fermentation[13]. Genetic modification of fungal strains is aimed to achieve dual purposes. Firstly, it is to improve the yield of cellulase and secondly, to enhance the quality of cellulase in terms of thermostability, tolerance to inhibitors and resistance to environmental changes[2,36]. The enhancement of cellulase quality has a significant impact on the hydrolysis rate that converts cellulose to glucose[95,96]. Despite its potential benefits, genetic engineering is relatively complex and the biosafety issue of the genetic modified fungal strain should be properly prioritized.

In genetic modification of fungi, several techniques such as mutagenesis of fungi and heterologous expression of cellulase enzyme can be used to develop robust fungal strains or fungal systems[2,36]. Mutagenesis process creates mutants that are able to withstand harsher environmental conditions or possess higher resistance to environmental changes throughout their cultivation period. There are two commonly applied mutagenesis approaches namely ultraviolet (UV) mutagenesis and chemical mutagenesis [97]. As the name suggest, UV mutagenesis randomly modifies the genes by exposing the fungi to UV ray [98]; chemical mutagenesis modifies the fungal genes by subjecting the fungi to DNA alkylating chemicals such as N-methyl-N⁰-nitro-N-nitro- soguanidine (MNNG) and ethyl methane sulfonate (EMS) [97,98]. Despite being a more convenient genetic modification technique, mutagenesis is not commonly practiced in enhancing cellulase production by WRF and BRF and thus, report related to the technique is scarce till date.

Heterologous expression of cellulase enzyme is another potential genetic modification technique. In this technique, the gene of the fungal strains that governs the production of high activity cellulase was cloned and expressed in other microorganisms via this technique [36]. Before cellulase b i o m a s s a n d b i o e n e r g y 6 7 ( 2 0 1 4 ) 3 1 9e3 3 8

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production, heterologous expression begins with protein sequencing and isolation of the genes[96,99,100]. Quiroz et al.

[59]suggested that protein with small molecular weight which contributes to the cellulolytic activity is of particular interest for heterologous expression. As an example, the protein that shows clear cellulolytic activity on the 25 kDa zymogram band is suitable for heterologous expression due to its small size [59]. Several heterologous expression of cellulase which involved WRF has been attempted. One of the researches outlined the cloning of endoglucanase gene fromT. versicolor and expressing the gene as a functional recombinant enzyme in a yeast strain,Pichia pastoris[95]. The yeast produced fungal endoglucanase as an active enzyme but in low yield. The successful attempt in terms of heterologous expression leads to further optimization in improving the recombinant endoglucanase yield[95].

In a similar study, Bey et al.[24]heterologously expressed the gene for cellobiose dehydrogenase (CDH) production from a WRF Pycnoporus cinnabarinus into P. pastoris. The purified recombinant enzymes byP. pastoriscomplemented with the commercialT. reeseicocktail were tested on the saccharification of wheat straw. The result from the work referred showed that heterologous expression of cellulase inP. pastoriswas an effective technique to produce high CDH activities with excellent performance in the saccharification process. More- over, the recombinant CDH ofP. cinnabarinuswas found to be thermostable as it was able to withstand a much higher temperature at 70C during saccharification[24].

Furthermore, metabolic engineering can also serve as an alternative to mutagenesis and heterologous expression of cellulase technique to enhance the metabolic pathway that stimulate cellulase production and suppress those that act in an opposite manner[36,101]. Besides focusing on the specific biochemical and transporting steps in cellulase production, the overall metabolic network should be taken into account as the variations in these pathways would affect cellulase production[92]. However, cellulase regulation in WRF and BRF was not researched extensively as the precise mechanism related to cellulase induction and repression is not available to date[102].

8.2. Co-culture of fungi

Improvement in cellulase production can also be achieved via co-culture of different fungi. Co-culture is beneficial in cellulase production via SSF as the fungi are normally co-existed symbiotically on solid substrates in nature[13]. Besides that, co-culture also offers advantages such as higher productivity, adaptability and substrate utilization compared to pure and monoculture [36]. The enzymes system produced by co- culturing different fungi could complement each other and form a complete cellulase system that is favorable for lignocellulosic substrate hydrolysis. As shown by Kalyani et al.

[103], the deficiency ofb-glucosidase in the cellulase system of Sistotema brinkmanniicaused the accumulation of cellobiose.

The accumulated cellobiose served as a strong inducer forb^- glucosidase production by the co-cultured strain, Agaricus arvensis. With cellulase system that complements each other, the cellulase activity of 1.6 FPU$cm³was recorded by the co- culturingS. brinkmanniiandA. arvensis. This cellulase activity

was 2.3e3 fold higher than the cellulase activity obtained from either A. arvensis (0.5 FPU$cm³) or S. brinkmannii (0.3 FPU$cm³).

The performance of co-culturing fungi for cellulase production was also reported by Hu et al.[104]wherebyb^-glucosidase and cellobiohydrolase was enhanced when the WRFP.

chrysosporiumwas co-cultured with another strain of soft rot fungi, such asA. nigerorAspergillus oryzaeon wheat bran. It was also evident from a report that the initially low level of endoglucanase activity with 0.45 U$g¹produced byPleurotus djamor was improved to 1.4 U$g¹when it was co-cultured with another fungal strain,Trichoderma viride[105]. The co- culture ofT. viride and Ganoderma leucidumhas also shown positive results in exoglucanase andb-glucosidase production [106]. Lignocellulosic components were depolymerized to a greater extent when the fungi were co-cultured on lignocellulosic substrate during SSF[107].

When the fungi were co-cultured for cellulase production, the undesirable competition between the fungi should be avoided. The co-cultured strains should not be imposing any significant negative effect on the growth of each other, as described by Hu et al.[104]. Besides, the time point of inoculating the fungal strains also has an impact on cellulase production. It was shown by the co-cultivation ofT. reeseiRUT-30 andP. chrysosporiumBurdsall produced the maximum cellulase activity when inoculation time was delayed for 1.5 day [108]. Similarly, the maximum cellulase activity of 3.2 IU$g¹ was obtained whenA. oryzaewas co-cultured on soybean fiber withT. reeseiandP. chrysosporiumon the 36th hour[109]. Co- culturing fungi is viewed as an economical and feasible approach to improve the yield of cellulase production.

8.3. Improvement on bioreactor design

To date, cellulase production by WRF and BRF has mainly been confined to lab scale. Though various types of WRF and BRF had been screened for their ability in producing cellulase coupled with the parameters that affect the process in lab scale, little has been reported on larger scale bioreactors with appropriate measurement to monitor the process variables.

As for the operating mode, batch process is the most preva- lently examined mode compared to fed batch and continuous processes in cellulase production via SSF[43].

There are limited studies detailed on the bioreactor system for the production of cellulase by WRF and BRF, and the production of cellulase by fungi is mostly carried out in Erlen- meyer flask scale. Since lab scale production of cellulase by WRF and BRF is limited, bioreactor designs used in the production of cellulase by soft rot fungi are applicable to the WRF and BRF system due to their similar nature. Tray bioreactor, packed bed bioreactor, rotary drum bioreactor and fluidized bed bioreactor are the most commonly used lab scale bioreactors for SSF[110,111]. The schematic diagrams of these bioreactors are shown inFig. 2.

Table 5summarizes the comparison based on the beneficial features and limitations exhibited by the bioreactors shown in Fig. 2. Some potential methods to overcome the limitations of the bioreactors were also listed. Among the bioreactors examined for cellulase production, tray bioreactor is the most widely employed in lab, pilot and even industrial

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scale[111]. The application of tray bioreactor in the industrial kojiprocess adopting SSF symbolizes its stability and feasi- bility for large scale production [110]. Furthermore, rotary drum[112e114]and packed bed bioreactors[115,116]are also prominent for the purpose of cellulase production via SSF. On the other hand, fluidized bed bioreactor has not received extensive research in the aspect of cellulase production although the production of other enzymes by using fluidized bed reactor was deemed feasible[9].

Bioreactor with appropriate designs is able improve the cellulase production by overcoming the heat and mass transfer problem which becomes more prevalent in large scale. When the fungus is cultivated during SSF, effective diffusion of nutrients and oxygen is the key factor that en- sures optimal fungal growth. As fermentation progress, depletion of nutrients occurs and heat is generated due to the metabolic activity of fungus[110]. The metabolic heat cannot be easily dissipated because of the poor thermal conductivity of lignocellulosic substrate[43,119]. As a result, temperature gradient builds up across the substrate bed[110]and the nutrients supply to the fungi is not uniform. The temperature of the interior part of the substrate bed can be elevated to a critical value whereby an increase of 8 cm in the bed height causes an increase the temperature by approximately 20C [120]. This can hamper cell growth and denature the cellulase enzymes produced [110]. When the air passes through the substrate bed with temperature gradient, it tends to be warmed up and causes the substrate bed to dry up[110]. The agglomeration of substrate particles during fungal mycelium

growth and the shrinkage of substrate bed would further complicate the mass and heat transfer problem in SSF as these restrict air flow and nutrients diffusion[110,111].

Due to the aforementioned mass and heat transfer prob- lems, local environment in the bioreactor such as oxygen concentration, nutrients concentration, pH and temperature deviates from their respective optimal values during SSF[110].

It is easier to maintain the local environment in a small scale bioreactor such as lab scale bioreactor compared to large scale reactor. This is because the surface area of substrate bed to volume ratio in small scale bioreactor is larger compare to the large scale bioreactor[110]. Heat can be dissipated more easily from the substrate bed in small scale bioreactor to maintain the temperature within the system[110]. Besides the scale of bioreactor, the mass and heat transfer problem are also dependent on the types of bioreactor applied in SSF. When packed bed bioreactor is employed, there will be an increase in pressure drop with increased bed height and this leads to channeling effect that would obstruct effective heat and mass transfer[110]. Conversely, pressure drop and channeling are usually less common in tray bioreactor.

Mixing the substrate bed intermittently might reduce the mass transfer problem [121] or it might cause the moist lignocellulosic substrate particles to aggregate and form larger particles[44]. This prevents effective fungal penetration into the substrate during its growth. Fungal morphology might also experience some changes as mixing can damage the fungal mycelium[110]. Apart from that, mass transfer problem can be resolved by placing some inert materials in the Fig. 2eSchematic diagram of commonly employed bioreactors for SSF: (a) tray bioreactor, (b) rotary drum bioreactor, (c) packed bed bioreactor and (d) fluidized bed bioreactor (adapted from Mitchell et al.[111]).