Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies.

Accepted Manuscript Title: Solid State Fermentation for Production of Microbial Cellulases: Recent Advances and Improvement Strategies Author: Sudhanshu S. Behera Ramesh C. Ray PII: DOI: Reference:

S0141-8130(15)30091-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.090 BIOMAC 5504

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International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

6-6-2015 28-10-2015 29-10-2015

Please cite this article as: http://dx.doi.org/10.1016/j.ijbiomac.2015.10.090 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Solid State Fermentation for Production of Microbial Cellulases: Recent Advances and Improvement Strategies

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SUDHANSHU S. BEHERA1* and RAMESH C. RAY2

*Corresponding author

E-mail: [emailprotected]

Tel./Fax: +91- 674- 2470528

ICAR- Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 751 019,

Department of Biotechnology and Medical engineering, National Institute of Technology, Rourkela 769008, India.

India

ABSTRACT

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Lignocellulose is the most plentiful non-food biomass and one of the most inexhaustible renewable resources on the planet, which is an alternative sustainable energy source for the production of second generation biofuels. Lignocelluloses are composed of cellulose, hemicellulose and lignin, in which the sugar polymers account for a large portion of the biomass. Cellulases belong to the glycoside hydrolase family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. They are multi-enzymatic complex proteins and require the synergistic action of three key enzymes: endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) for the depolymerization of cellulose to glucose. Solid state fermentation, which holds growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged 1 Page 1 of 56

fermentation. The review summarizes the critical analysis of recent literature covering production of cellulase in solid state fermentation using advance technologies such as consolidated

bioprocessing,

metabolic

engineering

and

strain

improvement,

and

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circ*mscribes the strategies to improve the enzyme yield. Keywords: Bioreactor design,consolidated bioprocessing, gene expressing system, solid state

fermentation, cellulase, strain improvemrnt

1. Introduction

Lignocelluloses are the most generous renewable carbon resource in the world with production rate of 200 billion tons biomass per year [1, 2]. Lignocelluloses are mainly

composed of cellulose (35–50%), hemicelluloses (25–30%) and lignin (25–30%). However, cellulose, the most important cell wall polysaccharide in plants is restored constantly in

nature by photosynthesis [3, 4]. Cellulases are employed in industries for the preparation of medicines, perfumes, resins, starch production, baking, waste treatment [5, 6] and most

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importantly for bioethanol production from lignocellulosic biomass [7]. Moreover, the demand for renewable fuels, particularly bioethanol, is projected to increase 3.4-fold by 2035, worldwide [8, 9]. The U.S. Department of Energy, predicts that 30% of petroleum-based transportation fuels would be refilled with biomass-based fuels by 2025 [10,11]. Considering the industrial potentials of cellulases, an important aspect of cellulose research is to obtain highly active cellulases at low cost.

Solid-state fermentation (SSF) technology is expanding with increasing importance for the production of high value- added products, for instance enzymes, from agro-industrial by-products [2,12]. SSF involves the growth of microorganisms on moist solid substrates in

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the absence of free flowing water. It has gained considerable attention of late due to several advantages over submerged fermentation (SmF) [13, 14].

Successful applications of cellulosic materials as renewable carbon sources are

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dependent on the development of economically feasible process technologies for cellulase production. Reserachers have shown that cellulase production, was the most expensive step

during ethanol production from lignocellulosic biomass, which accounts for approximately

40% of the total cost [15]. Significant cost reduction is required in order to enhance the

commercial viability of cellulase yield [16].

The successful strategy to produce cellulolytic enzymes includes both microorganism

selection, understanding the basic physiology of cellulolytic microorganisms coupled with engineering principles applied to SSF and improved fermentation process conditions [17].

Recent advances in recombinant DNA technology allow the fast identification of novel

cellulase genes, large scale production of cellulases and their genetic modifications to make tailor-made enzymes for various applications [18]. In addition, bioreactor design also gained

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advanced bioprocess technologies for improving cellulase production [14, 19, 20].

In this review we discuss on the production of cellulase in SSF using various potential

and advance methods, i.e. genetic modification, co-culture of different fungal strains, and development (design) and modelling of bioreactors and strategies to improve enzyme yield.

2. Microbial Cellulase

Cellulolytic enzymes are widespread in nature and are found in plants, insects, and microorganisms. Aerobic and anaerobic bacteria are known to produce cellulolytic enzymes as single enzyme or in the form of cellulosomes, multi-enzyme complexes comprising several cellulolytic enzymes bound to a scaffold protein [21,22]. However, the most common

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commercially available cellulases so far are non-complexed native enzyme mixtures derived from fungi, especially Trichoderma or Aspergillus species. For use as enzyme producers, cellulolytic fungi have the great advantage of both utilizing secretory pathways and the

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production of high protein yields [23]. Additionally, other fungi, such as Penicillium, Acremonium and Chrysosporium are viewed as potential and promising alternatives to

Trichoderma [24].

For the efficient hydrolysis of cellulose, basically three types of synergistically acting enzymes are necessary. Cellobiohydrolases, also named as exoglucanases (E.C. 3.2.1.176)

(E.C. 3.2.1.91), attack the crystalline ends of cellulose producing cellobiose [23]. Endoglucanases (EGs) (E.C. 3.2.1.4), split glycosidic bonds within the amorphous part of the

substrate [3]. Finally, the released cellobiose is cleaved by β-glycosidases (BGls) (E.C.

3. SSF

3.2.1.21) into glucose monomers [3, 24,25].

SSF has been known since ancient time in Asian and Western countries [26,27]. In

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SSF, various types of fungi, [3] bactria [5] and actinomycetes [28] have been employed for the production of various bio-products. Classical examples of it are the fermentation of rice by Aspergillus oryzae to initiate the koji process and Penicillium roquefortii for cheese production [5,13,29]. Nevertheless, the importance of SSF was nearly ignored in Western countries, perhaps after the discovery of penicillin using SmF technology in 1940s. However, in last two decades, SSF has attracted regained attention due to several biotechnological advantages such as higher fermentation capacity, higher end-product stability, lower catabolic repression and cost-effective technology [2,30,31]. SSF can be defined as the fermentation in the absence or near absence of free water and intimately provides the natural habitat for the

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growth of microorganism on surface of solid material (or substrate). However, SmF involves fermentation in the presence of surplus water.

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3.1 Cellulase-producing microorganisms in SSF

The majority of microorganisms employed in cellulase production in SSF are fungi,

bacteria and to a smaller extent actinomycetes, which acts upon under specific (aerobic and

anaerobic) conditions.

Several fungi such as Aspergillus

3.1.1 Fungi

niger, Aspergillus nidulans, Fusarium solani,

Humicola insolens, Penicillium brasilianum, Phanerochaete chrysosporium, Trichoderma reesei, Trichoderma viride, Trametes versicolor, Penicillium sp., etc. have been cultivated in

SSF for cellulase production, where basal mineral salts medium is added for moistening the

substrate. A few illustrative examples are cited here. Quiroz-Castaneda et al. [32] analyzed the growth and production of cellulolytic enzymes by two basidiomycete fungi Bjerkandera

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adusta and Pycnoporus sanguineus grown on lignocellulosic materials (wheat straw, rice husk, corn stubble, jatropha ground seed husk, and cedar and oak sawdust) and composition and growth rates and levels of cellulolytic and xylanolytic activities were compared. Similarly, a process of SSF on tomato pomace was developed with the white-rot (basidiomycetous) fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support and

conditions for laccase, xylanase, and protease activity production were

identified [33]. Deswal et al. [34] reported the optimization of various physiological and nutritional parameters for cellulase production from newly isolated brown rot fungus Fomitopsis sp. (RCK2010) using low cost substrates such as wheat straw and rice straw under SSF conditions. Ang et al. [35] isolated a lignocellulosic degrading fungus, Aspergillus

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fumigatus SK1 from cow dung and claimed that the strain SK1 was the strongest xylanases producer with high level of cellulase secretion as compared to 16 other screened fungi, using untreated oil palm trunk as substrate in SSF. Delabona et al. [36] isolated a fungal strain

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(A. fumigates) from the Amazon forest, as an efficient producer of cellulase and xylanase. Flodman et al. [37] investigated the effects of intermittent mechanical mixing in SSF of wet

corn distillers grain with T. reesei (NRRL 11460) for production of cellulase. More recently, Yang et al. [38] reported the improve production of β-1,3-1,4-glaucoma by Rhizomucor

miehei under SSF for industrial applications such as malting process, especially in brewing

industries.

Until now, most of cellulase-producing microbes have been isolated from terrestrial

sources. However, Trivedi et al. [39] made an effort to isolate marine fungus (Cladosporium sphaerospermum) capable of hydrolyzing cellulose through SSF as an economical technique

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3.1.2 Bacteria

and saccharification potential was investigated using green seaweed (Ulva fasciata) biomass.

Several cellulase-producing bacteria such as Acidothermus cellulolyticus, Bacillus

subtilis, Bacillus coagulans, Bacillus pumilus,Clostridium acetobutylicum,Clostridium thermocellum,Cellulomonas fimi,Cellulomonas bioazotea, Cellulomonas uda, etc., have been isolated from different sources and selected due to their xylan-degrading properties [2,4,23,40]. Few examples are cited here. In order to explore the possibility of using banana waste as solid substrate for the production of cellulases, Krishna [41] designed and tested a bioprocess to ascertain the suitability of banana fruit stalk as a solid substrate for SSF employing Bacillus subtilis (CBTK 106). Heck et al. [40] designed a statistical optimization of xylanase activity by an Amazon environment isolated strain of B. coagulans grown in SSF, using an industrial fibrous soybean residue as substrate. Moreover,

Acidothermus

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cellulolyticus is a highly thermotolerant hot spring bacterium that has been investigated as a source of enzyme for biomass hydrolysis. Rezaei et al. [42] demonstrated production of xylanase and cellulase from SSF of switchgrass colonized by A. cellulolyticus. Considering

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the potent bacterial strains for the production of cellulose enzyme, Singh and Kaur [43], isolated an efficient strain, Bacillus sp. JS14 from a total 30 bacterial isolates and claimed a

highest enzyme activity for the production of cellulase.

3.1.3 Ascomycetes

The fungi-like organisms of the phylum ascomycete have the large potential for the production of cellulolytic enzymes [44]. The ascomycete strains have been predominately

isolated from ecological niches in agricultural waste such as wheat, rice, maize and sugar cane etc. [28] .The isolated form of cellulase degrading enzymes and their enzyme activities

under various SSF conditions were investigated by several groups of researchers. One of the

most efficient strains selected from that study was genus Ulocladium [44], which consists of

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mostly saprotrophic species and produce cellulolytic and hemicellulolytic enzymes.

3.2 Advantages of SSF

SSF rapidly builds up interest in the recent years, due to the high titers of enzyme

production employing fungal cultures. It is reported that SSF usually allows more production of crude enzymes as compared to SmF [13,14]. Brijwani and Vadlani [45] claimed SSF is an efficient technique for producing a scheme of enzymes with balanced activities that can competently saccharify lignocellulosic biomass like wheat straw acting as both carbon and energy source. Kumar et al. [46] compared and optimized both pectinase and cellulase production in SmF and SSF systems using response surface modelling and considered both techniques are equally cost-effective using agro industrial wastes as the substrate. In contrast,

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Cunha et. al. [47] reported that cellulase catalyzed liquefaction of sugarcane bagasse enables SmF of A. niger and production of endoglucanase was 12-fold higher than SSF. Inspite of the above facts, microbial cellulases undergo induction and repression

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mechanisms. The repression conditions such as substances formed during the pretreatment of the lignocellulosic feedstock inhibit enzymatic hydrolysis as well as microbial fermentation

steps [48]. Therefore, the process design, media formulation, in situ detoxification by using reducing agents (such as dithionite, dithiothreitol, sulfite) and enzymatic treatments (such as

laccase, peroxidase) are prerequired developmental areas to improve the production of

microbial cellulase [48,49]. One important biological factor in favour of SSF reported by Cadirci et al. [50] is the low catabolite repression, which appeares to be limiting enzyme

production by A. niger in SmF. Moreover, an SSF process facilitates the use of a single reactor for both hydrolysis and fermentation, and also minimizes the contamination risk

because of the presence of ethanol in the fermentation broth. However, a drawback of this

process is that the operating conditions cannot be optimized for both steps simultaneously [5].

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4. Strategies to Improve Production of Microbial Cellulase in SSF

In recent culture studies, considerable efforts have been directed toward the

development of cellulases production by improving the cellulase activities or imparting of desired features to enzymes. Several strategies, such as metabolic engineering and strain improvements, recombinant or heterologous cellulase expression, mixed or co-culture technique and fermentor (bioreactor) design and also modelling of SSF (heat and mass transfer in bioreactors) are gaining importance for the production of microbial cellulase in SSF. The spectrum of enhanced microbial cultures employed for production of cellulases in SSF systems has been presented in the established literature (Table 1).

4.1 Metabolic engineering and strain improvements 8 Page 8 of 56

To maximize cellulase production, suitable strain improvement and optimization of culture conditions should be accomplished [52]. Several researchers claimed that mutagenic agents can achieve strain improvement for the production of microbial cellulases [53,54].

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Different mutagenic agents, such as ethyl-methane sulphonate (EMS), N-methyl-N-nitro-Nnitrosoguanidine (NTG), ultraviolet (UV) irradiation, and gamma irradiation (Co60 γ-rays)

have been applied to several fungal strains to improve cellulase production [55-57]. Dillon et al. [58] developed a mutant strain of A. oryzae NRRL 3484 with higher extracellular cellulase

production by using a multistep mutation strategy. In this context, Vu et al. [52] claimed that

under the optimized medium and SSF conditions, the cellulase yield from the modified fungal strain (Aspergillus sp. SU-M15) was found 8.5-fold more than that of the wild type strain

grown on the basal wheat bran medium. The fungus Chaetomium cellulolyticum NRRL 18756, subjected to various dozes of gamma irradiation (0.5 KGy) showed the highest

extracellular CMCase production which was 1.6-fold higher than that of the wild type [55].

Indigenous strain of T. viride FCBP-142 was selected to develop over-producer of cellulases

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and subjected to mutagenesis with UV and EMS [59].

El-Ghonemy et al. [54] studied the different filamentous fungi (i.e., Aspergillus,

Trichoderma and scopulariopsis) for extracellular cellulases production and optimized SSF medium and culture conditions to increase cellulases production. It was claimed that Aspergillus oryzae NRRL 3484 exhibited relatively higher cellulases production. In another report, El-Ghonemy et al. [57] examined the process of mutagenesis using UV irradiation followed by chemical treatments to improve the biosynthesis of cellulase using A. oryzae NRRL 3484. Recently, Mostafa [56] isolated A. niger from wheat straw that was subjected to various dozes of gamma irradiation (1KGy and 2KGy) to enhance the production of enzymes such as carboxymethyl cellulase (CMCase) and filter paper cellulase (FPA). Catabolite repression is a major concern for production of microbial cellulase in SSF. 9 Page 9 of 56

Raghuwanshi et al. [60] developed a mutant strain of Trichoderma asperellum RCK2011 through UV irradiation for enhanced production of cellulase with reduced sensitivity to catabolite repression. A heterokaryon 28, was developded and derived through inter-specific

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protoplast fusion between Aspergillus nidulans and A. tubingensis (Dal8). Further, the fusion product was taken up through cyclic mutagenesis followed by selection on increasing levels

of 2-deoxy glucose as selection marker. The mutated protoplast fusion products showed many folds enzymatic cellulase activities (endoglucanase, β-glucosidase, cellobiohydrolase,

FPase) and xylanase under the shake flask and SSF systems [61].

Allthough, mutagenic agents are involved in strain improvement for the potential development and production of cellulase as compared to their wild counterparts, their may be

every possibility of undesirable mutation of desirable (beneficial) genes during the process of random mutatation by mutagenic agents. In this context, Lee et al. [62] reported that resulted the simultaneous modification of multiple genes in

mutagenic agents may

Saccharomyces cerevisiae and Klebsiella oxytoca towards improving ethanol production,

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acquiring high stress tolerance, and facilitating a decrease in byproduct formation.

Moreover, to improve enzymatic hydrolysis of cellulose, pretreatment processes have

been optimized to minimize inhibitor formation [63]. Transporters, regulators and enzymes of oxidoreductase categories have been identified that can improve the tolerance of biocatalysts to inhibitors such as furans (from sugar dehydration), acetate, soluble products from liginin formed during pretreatment process and also provide conditions that increase the effectiveness of cellulases [48,63].

Moreover, optimization of SSF medium and culture conditions is essential to enhance cellulases production. Statistical technique of optimazation is one of the relevant methods of SSF conditions followed by several researchers for production of cellulase. Response surface 10 Page 10 of 56

methodology (RSM) is a statistical technique for the modeling and optimization of multiple variables found in SSF medium and culture conditions. By combining experimental designs (multiple variables) with interpolation of first- or second-polynomial equations (in a

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sequential testing procedure) RSM can be applied to determine the optimum process conditions in SSF processes [64]. Generally, RSM is followed by two steps: first step is

known as “Plackett–Burman Design (PBD)” for screening significant factors, affecting enzymatic hydrolysis of substrate and the second step known as “Central Composite Design

(CCD)” for forming the response surface, which is further meant for fitting the model and

predicting the optimum value [65].

Zhang and Sang [66] applied statistical methods such as PBD and CCD to SSF

conditions for enhancing cellulases production by a newly isolated Penicillium chrysogenum QML-2. Compared to unoptimized conditions of cellulases production, the optimization

results found 3.34, 5.12 and 3.75 folds improvement of endoglucanase, FPase, and β-

glucosidase, respectively. Similarly, Dagnino et al. [67] followed a CCD to optimize the pre-

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treatment of rice hulls with diluted acid to obtain fermentable sugars for bioethanol production. The regression equations were formulated considering the function of the variables (acid concentration and heating time). The optimum conditions were achieved by pretreatment of rice hulls with 0.3% (w/v) H2SO4 for 33 min. The substantial purification of glucans was found to increase by 18.7% with the extraction of xylans (14.7%). Moreover, the efficacy of mild enzymatic conversion of glucans to glucose (dissolved sugars) and subsequent conversion to bioethanol production was found to be 50% and 84%, respectively.

4.2 Recombinant Strategy ( Heterologous Cellulase Expression)

An approach with outstanding potential for production of cellulase has been the focus of most research efforts to date, is the “consolidated bioprocessing (CBP)”. In brief, CBP for 11 Page 11 of 56

cellulose production may be defined as “production of cellulase combing hydrolysis of cellulose and fermentation in one step without the need for externally supplied enzymes” [63]. Recent studies of the fundamental principles of CBP reveal the usage of microbial

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cellulase, which are supported by the engineering (recombinant) non-cellulolytic organisms that display high product yields and titers to express a heterologous cellulase system [68,69].

Moreover, microbial evolution resulting from recombinant DNA technology allows the fast identification of novel cellulase genes, large scale production of cellulases and their genetic

modifications to make tailor-made enzymes for various applications [70]. The basic of

recombinat DNA strategy of heterologous protein or enzyme expression has been presented in Fig. 1.

4.2.1 Yeast expression system

The advantages that yeast expression system provides include simple handling in

inexpensive media formulations and rapidly reaching high cell densities, whilst simultaneously being devoid of pyrogens, pathogens or viral inclusions [70,71]. Several yeast

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expression systems including, S. cerevisiae, Pichia pastoris and Kluyveromyces marxianus have been developed for use in the biofuel industry, and are especially of interest as a CBP organisms. Of these, S. cerevisiae is the most well characterized and commonly used yeast for heterologous gene expression, which produces cellulases in sufficient quantities to hydrolyze cellulose.

The limited cellulase secretion capacity of yaest is one of the most significant barriers

for the production of second-generation bioethanol [20,72]. Meanwhile, the current market for FDA-approved therapeutic proteins, obtained from optimization of yeast secretion system has a wider significance [73]. It is therefore clear that an increase in the heterologous protein

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secretion capacity of yeast would benefit not only the renewable energy sector, but the biopharmaceutical protein industry as well.

Njokweni et al. [74] had constructed and compared a recombinant S. cerevisiae strains

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expressing β-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) to S. cerevisiae Y294[SFI] strain, and claimed thet

the S. cerevisiae Y294[Pccbgl1] strain yielded 2.6 times more β-glucosidase activity (under

both aerobic and anaerobic conditions) than the other two strains. Two recombinant strains of S. cerevisiae expressing the BGL1 (β-glucosidase) gene originating from Saccharomycopsis

fibuligera, an industrial strain of S. cerevisiae, was constructed in order to enhance heterologous cellulase protein production at growth enability on non-native substrates by

virtue of heterologous enzyme expression [75]. Similarly, Kitagawa et al. [76] used a plasmid

potential of cellulase production.

harboring the endoglucanase gene from Clostridium thermocellum (Ctcel8A) for increase the

Engineering the yeast S. cerevisiae to utilize cellobiose by co-expressing cellobiose

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transporter and β-glucosidase is a novel strategy only reported recently. Guo et al. [77] developed and expressed BGL1 gene (an intracellular, secreted or cellwall associated), encoding β-glucosidase in S.s fibuligera. The said strain used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11/ h) on cellobiose. Kitagawa et al. [76] introduced a cellulase gene from Clostridium thermocellum (Ctcel8A)

into a hom*ozygous diploid yeast deletion strain collection by

transformation, and identified genes that enhanced β-glucosidase activity when Ctcel8A was heterologously expressed. Moreover, the deletion in genes encoding components of the class

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C core vacuole/endosome tethering (CORVET) complex, responsible for the enhancement of heterologous cellulase protein production, was reported.

Baek et al. [78] adopted a new strategy to produce cellulolytic yeast strains of

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combining three types of yeast cells each displaying different cellulase. Two fungal endoglucanases, Thermoascus aurantiacus EGI and T. reesei EGII, and two bacterial

endoglucanases, C. thermocellum CelA and CelD, were expressed on the yeast surface.

Tailoring the combination ratio of each cell type the production of ethanol was also optimized and showed a 1.3 fold more yield (2.1 g/l) than cells composed of an equal amount of each

cell type, suggesting the suitability of this system for cellulosic ethanol production. More recently, Van Zyl et al. [70] investigated the over-expression of components of the exocytic

SNARE complex, which is a soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor proteins play crucial roles in facilitating protein trafficking system. Further, an

increase was observed in the secretion of the Talaromyces emersonii Cel7A (a

cellobiohydrolase) and the S. fibuligera Cel3A (a β-glucosidase), through the separate and

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simultaneous over-expression of different components of the exocytic SNARE complex in S. cerevisiae.

However, its major limitations as a commercial protein production host are its

relatively low protein yields and tendency to hyperglycosylate certain heterologous proteins, which contribute to a reduced secretion rate [70].

4.2.2 Bacterial Expression System

A wide range of bacterial expression systems have been employed for the significant developments in the production of recombinant cellulase enzyme. These bacterial expression systems, including B. subtilis, Escherichia coli, Corynebacterium glutamicum, Zymomonas

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mobilis etc., among which, E. coli remains the most commonly used system for recombinant cellulase protein production, which are found an extreme habitats or in animal digestive systems [79]. Moreover, the gram-positive B.subtilis or Clostridium species are preferentially

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used due to their stronger secretion abilities and surface display properties [20].

The major limitation, for most of the thermophilic bacteria is that, even under

optimized conditions, obtain a low amount of enzymes of interest as compared to mesophiles.

For example, Geobacillus spp. produced 0.058 U/ml endoglucanase activities at 60oC [80]. To potentiate an enzyme’s industrial utility, one can use recombinant DNA technology for

large scale expression of the enzyme in a heterologous host system, e.g. E. coli. Moreover, to exploit this thermostable microbial cellulase for industrial applications, one needs to

overexpress the enzymes in a suitable host. Graham et al. [81] expressed a recombinant enzyme of multidomain cellulase (90 kDa) and claimed the cellulase under category of

glycosyl hydrolase super family, found optimal activity at 109°C, with a half-life of 5h at

100°C, and also withstands denaturation in strong detergents, high-salt concentrations, and

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ionic liquids. In addition, cellulases found active above 100°C may facilitate in biofuel production from lignocellulosic feedstocks.

Moreover, commercial applications of cellulase require high specific activity, high

thermostability, broad active pH range and wide substrate specificity. Therefore, it is necessary to consider a robust cellulase which can hydrolyze the biomass under extreme industrial processing conditions [82]. Bhalla et al. [83] cloned a gene encoding a GH10 endoxylanase from Geobacillus sp. WSUCF1 and expressed heterologously at a high expression level in E. coli. The results found that recombinant enzyme exhibited high specific activity (461.0 U/mg), high thermostability and high hydrolytic activity (92%) and suggest its potential utility in industrial applications including pulp bleaching and producing biofuels.

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Xylan is a major component of hemicelluloses, which can be enzymatically degraded to useful products like xylose xylitol, and ethanol. Xylan is generally insoluble in nature; however, a number of microorganisms with the help of their endogenous enzymes can readily

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solubilize xylan. D-xylanase is one of the key enzymes required for the degradation of xylan. In this context, Goswami et al. [84] reported the increase in xylanase production by

heterologous expression of Bacillus brevis xylanase gene in E. coli BL21and secreting it in the medium, so that it require minimum downstream processing for its applications in paper Mitra et al. [85] isolated an endo-β-1,4-xylanase gene xynA of a

and pulp industry.

thermophilic Geobacillus sp. WBI from “hot” compost. The gene encoding 407 residues was overexpressed in E. coli, which contributed specifically alkali-thermostability when

incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0.

Das et al. [86] reported a hydrolytic performance of a recombinant cellulolytic

cellulase (Clostridium themocellum) expressed in Escherichia coli cells. The expressed

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results were compared with the performance of naturally isolated cellulases applying various modes of fermentation trials using steam explosion pretreated thatch grass and Z. mobilis. The results revealed that recombinant cellulase in shake flask SSF found seven fold increase of ethanol titre (8.8 g/L) as compared to naturally isolated cellulases (B. subtilis and T. reesei).

However, several on-going problems were associated with recombinant protein

expression in bacterial species. One of them is the persistence of enzyme truncation that may take place due to lack of protective glycosylation of cellulases produced in bacteria which are natively fungal in origin [80]. Investigations into methods to increase enzyme stability are continuing to combat this problem. Codon mismatching is another potential barrier to

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heterologous cellulase expression, and options for codon optimisation are being explored [87]. In contrast to these obstacles, recombinant production of cellulases in bacteria often leads to an increase in enzyme yield, as compared to the original host. Bacterial (E. coli)

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cellulase enzyme is easy to be expressed and purified from bacterial system. Using gene cloning strategies, it is possible to clone genes and get their products for various applications.

Utilizing them for digesting cellulosic materials for ethanol fermentation is one of the several

uses of cellulase.

4.2.3 Plant Expression System

Plants as alternative hosts for the production of recombinant proteins are being

actively traced, taking advantage of their unique characteristics [88,89]. The most obvious benefits of cellulase production in plants are low production costs and the ease of achievable

scale-up. Both are crucial factors for the economically feasible production of lignocellulosic

biofuels due to the high amounts of enzymes needed during the process [90].

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There are several strategies applied in plant expression system for the production of cellulase. One of these is the choice of the (sub)-cellular compartment for heterologous cellulases expression or (sub)-cellular locations for cellulase accumulation in plant systems. Depending on the (sub)-cellular compartment targeted, (such as apoplast,vacuole, endoplasmic reticulum or mitochondria), significant effects on enzyme stability and expression level have been observed [91]. For example, the enzyme endoglucanase E1 from Acidothermus cellulolyticus, showed low expression levels in the cytosol of several plant species, however higher yields of this enzyme (up to 16% of total soluble protein) wasachieved when targeted to different subcellular compartments [92]. Similar results have been shown for bacterial cellulase Cel5A from Thermotoga maritime which was expressed in subcellular compartments of chloroplast, vacuole or apoplast in tobacco plant [93]. 17 Page 17 of 56

The second important factor in the expression of cellulase enzymes through plant systems is in the potential for truncation of the enzyme or enzymes.

One alternative to

dealing with the possible truncation of enzymes is to produce single domain enzymes, such as

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the endocellulase from the hyperthermophilic archeon Sulfolobus solfataricus, which has been successfully expressed in its active form in the endoplasmic reticulum of tobacco plants

[94]. Considering the strength of transgenic rice seeds as a bioreactor for production of enzymes for conversion cellulosic biomass, Zhang et al. [94] investigated a codon-optimized

synthetic gene “endo-1,4-β-glucanase (E1)” (from Acidothermus cellulolyticus), and

transformed into rice (Oryza sativa L. ssp. japonica) under the control of a Gt1promoter (a rice seed storage protein) found encoding enzyme perfectly.

Economical production of fermentable sugars is again not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, there is a

need of plant molecular farming system, where plants are used as bioreactors. The palnt

based bioreactor could be used for the mass production of cell wall degrading enzymes that

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will help to scale down the costs [95]. In this context, Klose et al. [96] reported a novel promising strategies to engineer plant cell walls for improved biomass processing. They compare the heterologous expression of a mesophilic cellulase (endoglucanase TrCel5A) from T. reesei targeted to the endoplasmic reticulum and apoplast, and demonstrated a correct localization combined with high level expression of the active enzyme in both subcellular compartments.

However, the downgrade effect of plant expression system is the considerable increase in protein augmentation that will occur when various optimized parameters are more fully integrated with each other. It has been documented that overproduction of recombinant cellulases might negatively influence plant growth and development, potentially limiting their

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industrial applications. Furthermore, knowing the complex nature of plants, it will take more time and effort to achieve the result than has been the case for the simpler unicellular system such as of yeast. Nonetheless, the potential for plants to become one of the major avenues for

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protein production appears very promising [89].

4.3 Mixed-culture (co-culture) system

Several researchers have used a single strain of pure-culture for bioconversion of biomass. However, utilization of the substrate by a single strain in pure-culture-based

processes have been limited to a narrow range of biomass such starch [97]. Nevertheless, the mixed cultures are structured by highly diverse microbes and constructed from natural

inocula, which facilitate biological conversion of heterogeneous biomass under non-sterile conditions without modifying strain stability.

Degradation of lignocellulosic agro-industrial residues by aid of complex microbial

communities is a favorable approach to provide an efficient biomass decomposition for

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subsequent conversion to value-added products. Wongwilaiwalin et al. [98] established an active thermophilic lignocelluloses degrading microbial consortium, using co-existence of eight major microbes. The diverse group of microbes comprises anaerobic bacterial genera (e.g. Clostridium and Thermoanaero bacterium) along with aerobic/facultative anaerobic (including Rhodocyclaceae bacterium), and also non-culture bacteria for cellulosic biomass degradation, from high-temperature sugarcane bagasse compost. The results revealed that the lignocellulolytic enzyme system is suitable for biomass degradation and conversion in biotechnological industry. Using various agricultural wastes and SSF technology, Dhillon et al. [99] performed SSF to investigate the potential of agricultural residues for the production of cellulase and hemicellulase adopting individual and mixed cultures of A. niger and T. reseei. The results showed that maximum FPase cellulase activity of 13.57 IU/g dry substrate 19 Page 19 of 56

(gds), 22.89 IU/gds and 24.17 IU/gds and β-glucosidase activities of 21.69 IU/gds, 13.58IU/gds and 24.54 IU/gds were obtained with wheat bran medium with Aspergillus niger, Trichoderma reseei and co-cultures of A. niger and T. reseei, respectively.

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Moreover, the co-culture system found within bacteria communities expected increase the growth and activity of cellulolytic bacteria under unfavorable environmental conditions.

Lu et al. [100] determined the effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.)

on the cellulose-degrading activity of a cellulolytic CTl-6 (C. thermocellum) using paper and straw (cellulose materials) as substrate in a peptone cellulose solution medium. The results

reported that in co-culture W2-10 with CTl-6 the CMCase activity was found to increase

from 0.23 U/ml to 0.47 U/ml.

4.4 Fermenter (Bioreactor) Design for Cellulase Production in SSF

Over the years, different types of fermenters (bioreactors) have been employed for

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various purposes, including cellulase production in SSF systems. Laboratory studies are generally carried out in Erlenmeyer flasks, Roux bottles, beakers, jars and glass tubes (as column bioreactor) [13]. Large scale fermentations have been carried out in tray-, drum- or deep tank type fermenters. SSF processes for production of cellulase could be operated in batch, fed-batch or continuous modes, although batch processes are the most common. Various bioreactors have been used to evaluate SSF of lignocellulolytic wastes with different microorganisms [13]. However, Tray Bioreactor (TB), Packed Bed Bioreactor (PBB), Rotary Drum Bioreactor (RDB), Fluidized Bed Bioreactor (FBB) and Instrumented Labscale Bioreactor (ILB) are the most commonly used bioreactors for SSF systems. 4.4.1 Tray bioreactor (TB)

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Among these bioreactors, the static TB, also known as a koji bioreactor, is a simple and common SSF bioreactor [10]. This type of reactor is generally, composed of flat trays, where bioparticle system is arranged to form a layer of about 1.5 or 2 cm of thickness. The

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bioreactor is stored in a chamber at constant temperature with passive aeration [102]. Given conditions of static TB, Brijwani et al. [103] performed cellulase production in SSF using a

co-culture of T. reesei and A. oryzae. The optimum parameters of temperature of 30 oC, pH of 5, and moisture content of 70% were found suitable for cellulase production in this study.

Furthermore,the maximum FPase activity of 10.7 FPU/gds and β-glucosidase of 10.7 IU/gds

were obtained after 96 h of incubation in static TB in agreement with optimized activities at shake flask level. The utilization of a crude inducer lactoserum (source of lactose) as a

strategy to enhance cellulase production by A. niger strains (NRRL 567 and NRRL 2001) using apple pomace as solid substrate in Erlenmeyer flasks and plastic trays was reported

[58].

However, Vu et al. [52] reported SSF in TB that resulted a heat transfer resistance in steep temperature gradients within the solid substrate bed, which in turn adversely affected

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the biochemical reaction and enzyme activity. Moreover, Brijwani et al. [103] have described that “TBs have simple designs but require large areas, are cumbersome to handle, and are highly labor-intensive to operate”.

Another alternative, benefits to cellulase production in SSF is the deep bed bioreactor.

This is a new design and best choice of bioreactor for SSF conditions due to improved internal aeration interface with outside environment along with temperature control [104]. As a result, the choice of bioreactor for SSF is confined to deep-bed systems. Although heat and mass transfer limitations affect deep-bed configurations as well,, their use is encouraged due to better process management and control as compared to TBs.

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4.4.2 Packed Bed Bioreactor (PBB) In SSF, the interaction of transport phenomena with biochemical reactions has a considerable effect on the productivity of the bioreactor [105]. In batch fermentation,

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substrate is fed into the reactor in batch wise; whereas in continuous fermentation, substrate is continuously fed into the reactor and the fermented product is discharged simultaneously.

Besides maintaining a constant reactor volume in continuous fermentation, several researchers suggested the unproductive time (e.g. due to filling, emptying and cleaning) is

reduced, leading to increased volumetric productivity [106].

Given higher volumetric productivity of continuous fermentation, Crespo et al. [107] evaluated the production of ethanol under continuous operational mode carried out in a PBB

using a thermoanaerobic bacterium Caloramator boliviensis (optimum growth temperature of 60 oC) that could efficiently ferment pentose-rich sugarcane bagasse hydrolysates. This

reactor conditions claimed conversion of 98% of substrates with ethanol yields of 0.40–0.46

g/g of sugar were obtained. Abdeshahian et al. [108] carried out to evaluate the use of palm kernel cake in the production of cellulase by the cultivation of A. niger FTCC 5003 in a

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laboratory PBB for seven days. The operating parameters such as incubation temperature, initial moisture content of the substrate, and aeration rate were studied under controlled conditions and their effects were evaluated on the production of cellulase using RSM. The study had reported the production of cellulase with a yield as high as 244.53 U/gds, using 100g of palm kernel cake.

The efficiency of continuous fermentation can be further enhanced through the use of

immobilized cells that enable higher cell densities per unit reactor volume [106,109]. Mathew et al. [106] evaluated the production of bioethanol from oilseed rape straw hydrolysate using S. cerevisiae cells immobilized in Lentikat discs in a continuous-flow packed bed columns, which enabled higher volumetric productivity (12.88 g/ L/h) and a stable quality of product.

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Generally, SSF is implemented as a cylindrical PBB with the wetted solid matrix of inoculated filamentous fungi. Due to the consumption of the substrate, an increase in the concentration of the fungal biomass and of the valuable product was achieved. However, the

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fungal growth was accompanied by heat release due to respiratory activities. The problem of heat accumulation during the course of fermentation has been alleviated to a considerable

extent using a packed-column bioreactor that can inhibit the microorganism growth and affect the production of metabolites. The main reasons of heat generation in PBB is the deficient

the substrate and to the low air flow rates employed.

removal of the metabolically generated heat, due to the low effective thermal conductivity of

Several groups of researchers have attempted to minimize the temperature gradients

caused by heat transfer resistances and limited oxygen transfer during production of cellulase in SSF using PBB. One of the targeted strategies for PBB is the change in the substrate

morphology caused by fungal growth and oxygen transfer within SSF substrates. Wang et al.

[110] reported that with the help of combing the kinetic model of permeability and fractaloxygen exponential model analysis, oxygen transfer was closely related with surface structure

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change in SSF of steam exposed rice straw using Penicillum decumbens JUA10.T.The growth of fungal biomass and oxygen diffusion rate in SSF showed an opposite trends. Thus modelling of PBB towards fungal growth and changed substrate morphology is essetial for the controlled oxygen transfer in SSF. FBBs with forced aeration present a promising alternative in terms of instrumented

bioreactors for SSF processes. Castro et al. [111] investigated a fixed-bed SSF bioreactor with forced aeration for the production of a co*cktail of hydrolases (amylases, cellulases, xylanases and proteases) by A. awamori IOC-3914 using babassu cake as the raw material. More recently, Buck et al. [12] developed a practically relevant feedback control scheme that allows influencing and equalizing the moisture and temperature distributions along the PBB

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during SSF. This prevents locally critical process conditions which may necessitate the shutdown of the operation of the bioreactor. In consensus, PBBs are cost, space and labor effective and are often employed in

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SSFs with the aim of increasing productivity.

4.4.3 Rotary drum bioreactor (RDB)

Among these several types of SSF reactors, RDBs provide relatively gentle and uniform mixing by improving baffle design, since there is no agitator within the substrate

bed. Rotating drums have been used as bioreactors for SSF since the 1930s and are already applied to make many products. The engineering principles of RDB have recently received

materials [112].

renewed interest, making it a viable fermenter for biofuel production using cellulosic

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Using the RDB, Alam et al. [113] evaluated that the production of cellulase in terms of FPase activity by T. harzianum using empty fruit bunches as the major substrate in a RDB. Kim and Kim [79] evaluated production of cellulases by Penicillium verruculosum co*kE4E, a novel fungal strain isolated from bituminous coal. SSF was performed in 2 L tissue culture roller bottles using alkali-treated empty palm fruit bunch fiber, a lignocellulosic biomass, as the sole carbon source. Noratiqah et al. [113] used enzymatic degradation of oil palm empty fruit bunch by using self production cellulase enzyme in RDB. In the first step, oil palm empty fruit bunch was pretreated with 2% (v/v) HNO3 and degraded by A. niger EFB1 crude cellulase. In the second step, a standard RSM was followed to optimize the enzymatic degradation condition of oil palm empty fruit bunch in RDB. The optimal degradation

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condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. However, RDB with a tumbling motion of the solid substrate, aided by baffles on the

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inner wall of the rotating drum, creates particle agglomeration over time [17]. Another problem with this type of bioreactor is in controlling heat and mass transfer inside the bed

with high rotation speed of reactor that leads to potential difficulties in scale-up process

[113]. 4.4.4 Fluidized bed bioreactor (FBB)

A FBB in which the particles move independently like a fluid could easily transfer the generated metabolic heat in substate, and also control the diffusion of O2 and moisture in

substrate [114]. In this context, Foong et al. [115] studied heat and mass transfer of palm kernel cake in FBB and claimed rapid heat transfer from the waste biomass to air was

observed within the first 150s with a temperature drop of 30 °C. Trovati et al. [116] studied

the production of ethanol from cassava starch in an SSF process using co-immobilized

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glucoamylase -S. cerevisiae in pectin gel in an FBR. A significant improvement in volumetric ethanol productivity (11.7 g/L/h) was obtained as compared with traditional batch processes (5.8 g/L/h).

Immobilization in gels is a method of biocatalyst retention in continuous systems

[117]. Ethanol volumetric productivities of more than 60 g/L/h with complete conversion of 15% dextrose feed have been achieved using immobilized Z. mobilis in a FBR [118]. Simultaneous saccharification and fermentation of starch to ethanol has been investigated by several researchers. Applications of the FBR using co-immobilized

glucoamylase-

S. cerevisiae and glucoamylase-Z. mobilis have shown that typical volumetric productivities in the range of 15–40 g/L/h could be achieved [119].

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With aim for improvement of the simultaneous hydrolysis and fermentation steps and in order to overcome the inhibition of S. cerevisiae to high sugar concentrations, Moshi et al. [120] developed a process for the production of high bioethanol titre through fed-batch and

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simultaneous saccharification and fermentation of wild, non-edible cassava, Manihot glaziovii. Fed-batch and simultaneous saccharification and fermentation allowed fermentation

of up to 390 g/L of starch-derived glucose achieving high bioethanol concentration of up to

190 g/L (24% v/v) with yields of around 94% of the theoretical value.

4.4.6 Instrumented Labscale Bioreactor (ILB)

Instrumented lab-scale column type bioreactors have been used for the SSF

operations.Farinas et al. [121] evaluated the effects of operational conditions on endoglucanase production by a selected strain of A. niger cultivated under SSF using an ILB

equipped with an on-line automated monitoring and control system. Using an optimized

conditions of bioreactor system that include substrate initial moisture (72%), flow rate (20 mL/min), and inlet air humidity (70%), the maximum production of endoglucanase of

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56.1 U/gds was achieved.

Similarly, Pirota et al. [123] evaluated the effects of operational conditions on

xylanase production by a new strain of A.oryzae (P6B2) isolated from the Amazon Rain Forest. The new starin was cultivated under SSF using an ILB equipped with an on-line automated monitoring and control system. The effects of initial substrate moisture content and temperature on xylanase production were evaluated and compared to static conditions. Further, the selected consitions for the characterization resulted in the highest production of xylanase (2830.7 IU/g), which was achieved using initial substrate moisture content (80%, 28 °C) and an air flow rate (20 mL/min) with an inlet air humidity (80%).

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5. Process Scale up and Modelling of bioreactor (Heat and Mass transfer in bioreactor) In contempt of several utilities of SSF in industrial applications, the process scale up is limited due to difficulties in monitoring and controlling the different process parameters.

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Inspite of of above facts, the understanding of heat and mass transfer effects in director are among the most critical aspects of SSF. These act challenges and need attention for the

design and operation of bioreactors and scale-up for the commercialization of SSF processes

are essential for modeling and optimizing SSF [101].

[17]. In addition, the effects of temperature and humidity on microbial growth in bioreactors

Given importance to kinetic model of transport phenomena, heat and mass transfer, and operating variables in the whole-tray chamber, Jou and Lo [101] investigated heat and

mass transfer in static tray fermentation using SSF to produce fungal enzymes and compared the effects of temperature and humidity on the growth of Trichoderma by using the traditional

method of static tray fermentation. The results showed that the optimum growth conditions

for static tray fermentation of Trichoderma fungi are a 1 cm bed height, 28 oC, and 95% relative humidity. Moreover, this study has envisioned the applications in large scale static

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tray fermentation of fungi.

Using Le Duy kinetic model, Biswas et al. [51] correlated between microorganism

(thermophilic fungus Thermoascus aurantiacus) growth in wheat straw and production of cellulase and hemicellulases in a ILB, designed and operated in SSF system. The optimum conditions such as moisture content of the medium, growth temperature and air flow rate produced higher enzyme yields compared to those reported in the literature for other SSF bioreactors. Xue et al. [124] implemented a comprehensive computational fluid dynamics model for fast pyrolysis of cellulose in a FBB. This model is general and can be used for any biomass with known compositions of cellulose, hemicellulose, and lignin. However, Sheikhi 27 Page 27 of 56

ety al. [125] introduced a state-of-the-art sequential modular approach towards the modeling of a complex three-phase FBB. Using this modeling pathway, the prediction of non-ideal FBBs at different operating conditions such as a wide range of gas velocity, liquid flow rate,

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biocatalyst particle size, and the concentration of glucose in the feed stream, etc., becomes

possible, even in industrial process simulators.

5.1. Challenges on Process Scale up

Over the last decade, there has been a momentous improvement in operating how to

design, and scale-up SSF bioreactors. However, several problems are associated with SSF operations, such as high solid loading, biomass handling and transfer, washing of pretreated

solids and formation of inhibitors, mismatch in optimum temperatures for enzymatic

saccharification and fermentation, which are addressed during development stages at small

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scale in a laboratory environment [30]. Considering the mismatch in optimum temperatures for enzymatic saccharification and fermentation, the hydrolyzing enzymes requires an optimum temperature around 50 oC, whereas most of the fermenting yeasts have optimum temperatures ranging from 30 to 37 oC [126]. It was reported that higher amount of substrate loading (or solids concentration) in the

fermentation, will increase the ethanol yields, and hence reduce the amount of cellulase in downstream processes. However, high substrate loading causes inefficient heat and mass transfer due to the high viscosity [127]. Considering the above problems, Chen and Li. [126] investigated a novel lignocelluloses bioconversion process of non-isothermal simultaneous solid state saccharification, fermentation and separation (NSSSFS). In this process cellulase

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was produced by T. reesei YG3, the average Fpase activity was found 50–60 FPU/mL. Furthermore, one new 300L pilot scale NSSSFS coupled CO2 gas stripping loop system and one 110 m3 industrial level plant was also established. In the NSSSFS, enzymatic

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saccharification and fermentation operated around 50oC and 37oC, respectively. To minimize the end product feedback inhibition due to ethanol fermentation, formed ethanol was

separated by CO2 gas stripping with adsorption on activated carbon. In addition, the solids

substrate loading was reached to 25%, which increased ethanol yield from 18.96% to 30.29%.

The major problems to overcome in large-scale SSF are heat accumulation and heterogeneous distribution in a complex gas–liquid–solid multiphase bioreactor (or

fermenter) system [17]. Wang et al. [110] developed a mathematical model for RDB, in anaerobic SSF considering the radial temperature distribution in the substrate bed. Validation

experiments were conducted in a 5 m3 pilot plant fermenter for production of fuel ethanol

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from milled sweet sorghum stalks.

5.2. Recent Advances on Process Scale up Scale-up of a bioreactor or setting of operating condition is essential for optimization

of production. Lee et al. [128] designed a novel solid state bioreactor, and evaluated cellulase production, using local isolate A. niger USM AI 1 grown on sugar bagasse and palm kernel cake (1:1 (w/w) ratio). Under optimized SSF conditions of 0.5 kg substrate, 70% (w/w) moisture content, temperature at 30oC, aeration at 4L/h/ g fermented substrate for 5 min and mixing at 0.5 rpm for 5 min, about 3.4U/g of FPase activity was obtained. The results claimed that the performanace of newly designed SSF bioreactor is acceptable and potentially used as prototype for large-scale bioreactor design. 29 Page 29 of 56

Lin et al. [112] demonstrated the scale-up of a RDB to 100 L capacity RDB successfully. In addition, it is reported that the ethanol production from pretreated sugarcane bagasse by SSF using Kluyveromyces marxianus var. marxianus (a thermotolerant yeast

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strain) under 37, 42, and 45 °C. The SSF at 42 °C for 72 h along with 10% of waterinsoluble-solids at pH 5.0 supplemented with 0.2 ml cellulase/g- water-insoluble-solids and

1g/L of thermotolerent yeast, resulted in a final ethanol concentration and theoretical ethanol yield of 24.6g/L and 79%, respectively. These observations indicated that the performance

and the chosen SSF operating conditions for scale-up-drum reactor was as effective as those

attained from flask runs.

Shao et al. [129] developed a scale-up approach of simultaneous saccharification and

fermentation for the conversion of waste paper sludge to ethanol in CSTR configuration. Lab scale experiments were performed to determine the desired degree of mixing (or scale-up

criteria). Computational fluid dynamics simulations were performed to calculate the mixing

power requirement at large scale. Other scale-up criteria such as solid-liquid mass transfer at

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large scale are analyzed using a combination of industrial mixing analysis and computational fluid dynamics simulations.

In order to decrease process costs and increase the efficiency, it is desired to use

thermostable enzymes in industrial processes [2,75]. However, most cellulase are not stable at high temperature and a number of efforts have been made in order to obtain thermostable cellulases [75]. Saqib et al. [130], studied a themostability of a crude endoglucanase produced from

Aspergillus fumigatus using a modified SSF approach which featured a constant

pressure of isolated liquid water inside the fermentation chamber without direct contact with the substrate. Various thermostability parameters such as half lives (T1/2), enthalpies (∆H), entropy (∆S) and free energy (∆G) of activation denaturation were calculated for the crude endogluconase. The optimum results revealed that the half lives (T1/2) of the enzyme 6930, 30 Page 30 of 56

866, and 36min at 60 oC, 70 oC, and 80 oC, respectively. The enthalpies (∆H) of activation denaturation were 254.04, 253.96, and 253.88 K J mole−1, at 60 oC, 70 oC, and 80 oC, respectively, whereas entropy (∆S) of activation denaturation and free energy changes (∆G)

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of activation of denaturation were 406.45, 401.01, and 406.07 Jmole−1 K−1 and 118.69, 116.41, and 110.53 K J/ mole at 60 oC, 70 oC, and 80 oC, respectively. This study opens up

new opportunities to use principles of thermodynamics in fermentor designs for cellulase

production. 5.3. Research gaps

The SSF is an attractive process for the production of enzymes. The production of enzymes including, production of microbial cellulase in SSF is advantageous over SmF, have

been reported in numerous works [2,30,122]. In spite of the several advantages of SSF, their application in industrial processes is limited due to difficulties in monitoring and controlling

the different process parameters and several operating variables which affect microbial

growth and metabolite production. The critical research gap is the commercialization of

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microbial cellulase i.e. novel design and scale-up of bioreactors with lowered expenses with regards to product recovery and energy expenditure [28]. Moreover, industrial (or modern) SSF of cellulase remains a traditional industry and

there are very few designs available for bioreactors operating in SSF on a large-scale. The primary problem for modern SSF is engineering factors and scaling up this technique. The reason mainly associated is difficult and deficit of mass and heat transfer resulting from the continuous gas phase in SSF. In addition, the challenge of producing cellulase is being met with an expansion of research in the biofuel arena. Major improvements have been made as researchers learn more about the optimization of pretreatment processes to minimize inhibitor formation and to improve enzymatic hydrolysis of cellulose. 31 Page 31 of 56

6. Conclusion and Future Prospectives The bio-engineering aspects of SSF of lignocellulosic biomass become the essence of

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future research using cellulases and cellulolytic microorganisms. Although numerous efforts and several years have been invested in cellulase research, high production cost of cellulase

remains to restrict the wide utilization of cellulosic materials. Moreover, cost-effective production of valuable products from lignocellulosic biomass still faces many technical

limitations. To encounter the demand of cellulase, its activities or its desired features could be

improved by protein an engineering approach which is probably an advance area of enzyme technology.

Although, facets open to consideration for process optimization for higher and qualitative cellulase yield, including cheaper technologies for strain improvements and co-

culture systems, DNA recombinant technology for heterologous enzyme expression such as

engineering of genes/enzymes, and designing of bioreactors, which are undoubtedly, serve to

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improve cellulase qualities and quantities towards downstream fermentative operations. However, to understand the basic physiology of cellulolytic microorganisms it is essential for bio-engineering approaches in SSF coupled with engineering principles need for efficient cellulase yield. It is anticipated that SSF wound continues to be studied more with continued focus on engineering parameters with heat and mass transfer aspects for scale-up of bioreactors. Also, the profiteering of diverse agro-industrial residues and cultivating microbes should be oppressed more for developing design and operation of bioreactors and scale-up for the commercialization of production of cellulase in SSF processes.

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Enz.Res. (2012)

http://dx.doi.org/10.1155/2012/196853.

[131] C.R. Silva, T.C. Zangirolami, J.P. Rodrigues, K. Matugi, R.C. Giordano, R.L.C. Giordano, An innovative biocatalyst for production of ethanol from xylose in a continuous bioreactor, Enz. Microb. Technol. 50 (2012) 35-42.

[132] F. Mingardon, A. Chanal, C. Tardif, H.P. Fierobe, The issue of secretion in heterologous expression of Clostridium cellulolyticum cellulase-encoding genes in Clostridium acetobutylicum ATCC 824, Appl. Environ. Microbiol. 77 (2011) 2831-2838.

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[133] R. Kondo, R. De Leon, T.K. Anh, K. Shimizu, I. Kamei, Bioethanol production from alkaline-pretreated sugarcane bagasse by consolidated bioprocessing using Phlebia sp.

Ac ce p

ip t

MG-60, Int. Biodeterior. Biodegrad. 88 (2014) 62-68.

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ip t cr us an M d te Ac ce p Fig. 1. Recombinant strategy of heterologous protein or enzyme expression

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Table 1. Spectrum of enhanced microbial cultures employed for production of cellulases in solid state fermentation (SSF) systems.

Types of Wastes/Substatre

Inoculant

Factor affecting cellulase production

Aspergillus sp. SU14

Cellulase in a yield 8.5-fold [52] Exposure to different exceeding that of the wild doses (0.5~2.5 KGy) type starin grown on the of Co60 γ-rays. basal wheat bran medium

Cellulase productiona

Wheat bran

ip t

Metabolic engineering and strain improvements

Reference s

expression

Cosolidated bioprocessing

Ac ce p

Yeast system

Phlebia sp.

Bioethanol production using Phlebia cellulase

[133)

Alkali-pretreated sugarcane bagasse

Mutagenesis with Trichoderma Develop over-producer of [59] ultra violet (UV) and viride FCBPcellulases [cellulase activity chemical ethyl 142 (122.66 IU/ml)] methane sulfonate (EMS)

Expressed in Saccharomyces cerevisiae BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera

Surface-engineered S. [77] cerevisiae utilized 5.2 g cellobiose/ L [(Yeast Extarct Pentose Dextrose (YEPD) medium] and produced 2.3 g ethanol/ L in 48 h, with growth rate (0.11/ h) on cellobiose than the wild counterpart

Bacterial expression system

Gene encoding 407 residues were overexpressed in E. coli

[85] Endo-β-1,4-xylanase gene xynA of a Specifically alkalithermophilic Geobacil thermostability; incubated at lus sp. WBI 52

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in E. coli 65°C for 1 h under alkaline [132] condition (pH 10.0) and retained 75% activity at pH 11.0.

ip t

Heterologous expression of Clostridium cellulolyticum cellulose encoding genes

2- fold over production of cellulase

Clostridium acetobutylicum

Plant Expression System

CMC as substrate

Expressed in rice (Oryza sativa L. ssp. japonica)

Expressed in Endoglucanase Tobacco plants TrCel5A from the mesophilic fungus,Trichoderma reesei

Ac ce p

Endo-1,4-β-glucanase (E1) from Acidothermus cellulolyticus

Impact on the composition of [95] plant cell wall polysaccharides, although the precise changes were dependent on the subcellular localization of the enzyme [94] Cellulase activity in the transgenic line C19 seeds was estimated at about 830 U/g

Mixed-culture (coculture) system

Aspergillus Rice straw with niger and wheat bran in the Trichoderma ratio of 3:2 reesei

Mixed or Co-culture

For mixed culture ; FPasemax: 24.17 IU/gds Maximum β-glucosidase activities: 24.54 IU/gds

[99]

Bioreactor design

Thermoascus aurantiacus Dry wheat straw

Enzyme yields of 1709 U [51] endoglucanase, 4 U Intermittent agitation cellobiohydrolase, 79 U βrotating drum type glucosidase, 5.5 U FPA, bioreactor 4490 U xylanase and 45 U βxylosidase / gds

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Aspergillus niger NRRL Erlenmeyer flasks and Enhance 567 and A. niger plastic trays production NRRL 2001

[79]

Trichoderma Empty fruit bunches harzianum (EFB)

culture Production of cellulase

ip t

2-L tissue roller bottles Rotary bioreactor

drum The productivity of cellulase activity was 5.1 FPA/gds/day

Empty palm bunch fiber

Penicillum fruit verruculosum co*kE4E

Soybean hulls, wheat bran

Trichoderma reesei and Aspergillus oryzae

Oilseed rape

Saccharomyces cerevisiae cells Continuous-flow immobilised in packed bed columns Lentikat discs

Aspergillus awamori IOC3914

Packed bed bioreactor

Ac ce p

Sugarcane bagasse

Kluyveromyces marxianus var. marxianus (a thermotolerant yeast strain)

Aspergillus niger EFB1 Oil palm empty fruit bunch

FPasemax of 10.7 FPU/gds and β-glucosidase (max) of 10.7 IU/gds were obtained after 96 h incubation

Static tray bioreactor

Caloramator Sugarcane bagasse boliviensis hydrolysates

Babassu cake

cellulases

Fixed-bed SSF

Rotary drum bioreactor (RDB) to 100-L capacity

Rotary drum bioreactor and optimize the enzymatic degradation

[113]

[103]

Apple pomace

[58]

[106]

Volumetric productivity of ethanol (12.88 g/ L/ h) was obtained

ethanol yields of 0.40–0.46 g/g of sugar were obtained

[107]

Maximum activities of [111] exoamylases, endoamylases, proteases, xylanases and cellulases (CMCase) were, respectively, 73.4, 55.7, 31.8, 23.8 and 6.2 U/ gds Reactor was scaled up to [112] (100L, with 10 kg sugarcane bagasse, at 42 °C for 72 h); cellulase and yeast resulted in a final ethanol concentration and the theoretical ethanol yield of 24.6 g/L and 79%, respectively Improvement in reducing [113] sugar and polyoses production 1.07 fold compared to that before optimization in shake flasks culture

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Fed-batch simultaneous saccharification fermentation (

Instrumented scale bioreactor

Ac ce p

Non-edible cassava (Manihot glaziovii) tuber

Aspergillus niger

succeeded

in −1

producing

12 g × L of

ip t

ethanol

[116]

Ethanol productivity (11.7 g/L/h)

Fermentation of up to 390 [120] g/L of starch-derived glucose and achieving high bioethanol concentration of up to 190 and g/L (24% v/v) with yields of around 94% of the theoretical value

S. cerevisiae

Biocatalyst

Cassava starch

Co-immobilized gluco amylaseS. cerevisiae in Fluidized bed reactor pectin gel

[131]

Xylose

Glucose (xylose) isomerase (from Streptomyces rubiginosus) co-immobilized Continuous reactors with S. cerevisiae in calcium alginate gel

Maximum production of [121] endoglucanase (56.1 U/g) was achieved and forced lab- aeration conditions (50.2 IU/g) compared to static conditions (29.8 IU/g) after 72 h of cultivation

[aCellulase 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 1 mmol min-1 of hydrolysis product formed under the assay condition. The cellulase activities included, CMCase: carboxymethyl cellulose activity; FPase: filter paper activity; β-glucosidase: β-glucosidase activity; Endoglucanase: endoglucanase activity and Cellobiohydrolase: cellobiohydrolase activity].

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Filename: Directory:

Cellulase 1.doc

Ac ce p

ip t

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