Marine Microbes as a Potential Source of Cellulolytic Enzymes

CHAPTER TWO

Marine Microbes as a Potential Source of Cellulolytic Enzymes N. Trivedi*,†,1, C.R.K. Reddy*,†,2, A.M. Lali{ *Division of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, India † Academy of Scientific and Innovative Research (AcSIR), New Delhi, India { DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India 2 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Classification of Cellulases 3. Marine Microbes as a Source of Cellulases 4. Marine Bacterial Cellulases 5. Marine Fungal Cellulases 6. Marine Yeast Cellulases 7. Conclusions and Future Prospectives Acknowledgment References

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Abstract Marine environment hosts the wide range of habitats with remarkably high and diverse microbial populations. The ability of marine microorganisms to survive in extreme temperature, salinity, and pressure depends on the function of multivarious enzyme systems that in turn provide vast potential for biotechnological exploration studies. Therefore, the enzymes from marine microorganism represent novel bio catalytic potential with stable and reliable properties. Microbial cellulases constitute a major group of industrial enzymes that find applications in various industries. Majority of cellulases are of terrestrial origin, and very limited research has been carried out to explore marine microbes as a source of cellulases. This chapter presents an overview about the types of marine polysaccharases, classification and potential applications of cellulases, different sources of marine cellulases, and their future perspectives.

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Present address: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India.

Advances in Food and Nutrition Research, Volume 79 ISSN 1043-4526 http://dx.doi.org/10.1016/bs.afnr.2016.07.002

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2016 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Oceans cover about 70% of the earth’s surface and contain 97% of our planet’s water. Marine habitats represent the largest reservoir of biodiversity of the planet (Bull, Ward, & Goodfellow, 2000). The microbial diversity of marine origin has developed cellular machinery to thrive well even in extreme conditions. Marine microbes tolerating salt concentration of 1.7 M, temperature stability of 80–108°C, and high pressure of 60 MPa have been already reported in the literature (Marhuenda-Egea & Bonete, 2002; Singh et al., 2014). These properties have impelled worldwide research to target marine microbes as a potential source of novel enzymes. Various enzymes have been characterized from marine microbes isolated from seawater and different marine sediments. The main marine enzymes reported so far includes lipases, proteases, laccases, and polysaccharases. The polysaccharide-degrading enzymes from marine microbes have gained global attention due to novel industrial applications. Apart from the cellulases (Trivedi et al., 2011a) and amylases (Chakraborty, Khopade, Kokare, Mahadik, & Chopade, 2009; Li et al., 2007; Mohapatra, Banerjee, & Bapuji, 1998), marine microbes have also known to produce seaweeddegrading enzymes such as agarases (Gupta, Trivedi, Gupta, Reddy, & Jha, 2013; Suzuki, Sawai, Suzuki, & Kawai, 2003), carrageenases (Lemoine, Collen, & Helbert, 2009; Sarwar, Matoyoshi, & Oda, 1987), alginate lyases (Huang et al., 2013; Kim et al., 2013), fucoidanase (Silchenko et al., 2013), ulvan lyases, etc. (Collen, Sassi, Rogniaux, Marfaing, & Helbert, 2011). The marine polysaccharases have been summarized in Fig. 1. Among all, cellulases have been studied extensively across the globe due to wide applications in fuel, leather, textile, agriculture, food, medical, paper, and pulp industries (Menendez, Garcia-Fraile, & Rivas, 2015; Trivedi et al., 2011b). Potential industrial uses of cellulases have been summarized in Fig. 2. Increasing energy securities and global warming issues due to emissions of greenhouse gases led to finding other renewable energy sources. Among all, plant biomass has been given high priority to be used as a source of transportation fuels (mainly bioethanol) and chemicals. Cellulosic-richbiomass has been well recognized as a major source of energy feedstock or as a raw material for production of high value chemicals (Cherry & Fidantsef, 2003; Perlack et al., 2005). Cellulose, the most abundant and renewable organic polymer, is a linear homopolymer of D-glucose linked

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Fig. 1 Different type of marine polysaccharases.

Fig. 2 Potential industrial uses of cellulases.

with 1,4-beta acetal bond and constitutes primary structural cell wall material in both lower and higher plants (Saha, Roy, Sen, & Ray, 2006). Cellulose has been mainly used to produce ethanol through fermentation. Among other plant cell wall polysaccharide, cellulose is the most

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recalcitrant polymer to catalytic degradation. Cellulose could be hydrolyzed to its monomer, ie, glucose, either by chemical (acid), or enzymatic route. Currently, chemical hydrolysis is the process most widely followed to produce sugars from cellulose. Due to combination of high temperatures and strong acids, chemical hydrolysis leads to the degradation and accumulation of nonsugar by-products such as 5-hydroxymethylfurfural, formic acid, levulinic acid, and acetic acid (organic acids) which pose problems in downstream process (Mussatto & Roberto, 2004). As an alternate, enzymatic hydrolysis represents the greener approach for the saccharification of cellulosic-rich biomass. Cellulase is an inducible enzyme that catalyzes the hydrolysis of cellulose. It belongs to the class of hydrolases and is mainly produced by fungi, bacteria, protozoa, insects, and termites (Ozioko, Ikeyi, & Ugwu, 2013). Microbes produce cellulolytic enzymes as a single unit or in the form of cellulosomes (Bayer, Lamed, & Himmel, 2007). Cellulase has been commercialized during 1960s but still has some shortfalls such as low enzyme activity and stability in certain ecosystems associated with high salt, strong acid, strong alkali, and low temperature. In the global enzyme market, cellulases are the third most important industrial enzymes (15%) after amylase (25%) and protease (18%) (Sajith, Priji, Sreedevi, & Benjamin, 2016). Recently, the research on cellulases has significantly been increased due to its role in bioethanol production from cellulosic-rich biomass. By 2035, the global ethanol demand has been projected to increase by 3.5-fold (Limayem & Ricke, 2012). The high cost of enzyme production is a major limiting factor in commercialization of bioethanol production through enzymatic hydrolysis. Therefore, exploration for finding new and potential sources of cellulase is always desirable for economical enzyme production at industrial scale. Microorganisms with biochemical diversity and possible genetic modification are targeted as a source of potential enzymes. Microbial cellulases isolated from marine environment show higher degree of stability in different harsh conditions such as pH, temperature, and salinity.

2. CLASSIFICATION OF CELLULASES Cellulose hydrolysis via enzymatic route is accomplished synergistically by actions of group of enzymes known as cellulases. Based on their hydrolysis potential, cellulases have been classified into three types (Bhat & Bhat, 1997; Narra, Dixit, Divecha, Madamwar, & Shah, 2012), namely:

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(1) Endo-1-4-β-glucanase (EG, EC 3.2.1.4): Breaks down glycosidic bond in amorphous region (2) Exocellulase/cellobiohydrolase (CBH, EC 3.2.1.91): attaches to crystalline ends of cellulose chain producing cellobiose (3) β-Glucosidase (BGL, EC 3.2.1.21): Breaks down glucosidic bond of cellobiose and cellodextrins into glucose (Bhat & Bhat, 1997) Till date, a majority of cellulase producing microbes have been isolated from terrestrial sources. Some efforts have been made to explore the marine-based sources for cellulase production which are summarized below.

3. MARINE MICROBES AS A SOURCE OF CELLULASES The major cellulases producing bacterial genera include Bacillus, Clostridium, Cellulomonas, Bacteriodes, Acetivibrio, Rumminococcus, Geobacillus, Alteromonas, Vibrio and fungal genera include Aspergillus, Trichoderma, Chrysosporium, Penicillium (Assareh, Zahiri, Noghabi, Aminzadeh, & Khaniki, 2012; Dotsenko, Sinitsyna, Hinz, Wery, & Sinitsyn, 2012; Nandakumar, Thankur, Raghavarao, & Ghildyal, 1994; Roboson & Chambliss, 1989). The cellulolytic activity and stability vary with the source of origin. The cellulases used in industries require higher activity and stability in order to endure diverse harsh conditions. Globally, several research groups have been involved in isolation and characterization of cellulase producing microorganism with higher catalytic activity and stability. In comparison to terrestrial environment, microbes from marine habitat with hyper variable conditions, such as high pH, temperature, pressure, salinity, oxidative stress, metals, radiations, and chemicals could represent the novel source of extracellular enzymes with higher catalytic potential (Dalmaso, Ferreira, & Vermelho, 2015). So far, cellulases with alkali-halotolerant, organic solvent stable, ionic liquid stable, thermostable characteristics have been reported from marine microbes (Rastogi et al., 2010; Trivedi et al., 2011a, 2011b; Trivedi, Gupta, Reddy, & Jha, 2013; Yu & Li, 2015). Table 1 summarizes various cellulases recently studied from marine microbial sources.

4. MARINE BACTERIAL CELLULASES Increasing demand for industrial enzymes with desired characteristics turned attention toward exploration of marine microbes as a potential source. Microorganism surviving in marine environment develops well-evolved

Table 1 Cellulases Most Recently Reported from Marine Microbes pH and Microorganisms Isolated From Temperature

Characteristics

References

Gracilibacillus sp. SK1

Salt lake

8 and 60°C

Alkali stable

Yu and Li (2015)

Aureobasidium pullulans 98

Sea saltern of yellow sea

5.6 and 40°C

Potential CMCase

Yanjun, Liang, Zhenming, and Xianghong (2015)

Bacillus cereus JD0404

Muddy sediments of mangrove swamps

7 and 50°C

Able to degrade agroresidues

Chantarasiri (2015)

Cladosporium sphaerospermum

Deteriorated seaweed Ulva

4 and 25°C

SSF derived

Trivedi, Reddy, Radulovich, and Jha (2015)

Brachybacterium, Brevibacterium, Seaweed Eucheuma Halomonas, Kokuria, Micrococcus, cottonii Nocardiopsis, Pseudomonas, and Streptomyces (genera)

4.8 and 50°C

Lignocellulosic biomass degradation

Santhi, Bhagat, Saranya, Govindarajan, and Jebakumar (2014)

Isoptericola sp. JS-C42

Marine sediments

7.6 and 30°C

Plant biomass degradation

Santhi, Gupta, Saranya, and Jebakumar (2014)

Bacillus VITRKHB

–

7.8 and 25.8°C Good industrial efficacy

Singh et al. (2014)

Bacillus sp. H1666

Seawater samples

7 and 50°C

Seaweed degradation

Harshvardhan, Mishra, and Jha (2013)

Bacillus halodurans CAS 1

Marine sediments

9 and 60°C

Thermostable and haloalkaline

Annamalai, Rajeswari, Elayaraja, and Balasubramanian (2013)

Pseudoalteromonas sp.

Sargassum polycystum (Brown seaweed)

5 and 45°C

Ionic liquid stable

Trivedi et al. (2013)

Aspergillus ZJUBE-1

Soil of sea

4–5 and 65°C

SSF derived

Liu, Xue, He, & Yao (2012)

Bacillus flexus

Deteriorated seaweed Ulva

10 and 45°C

Alkali-halotolerant

Trivedi et al. (2011a)

Bacillus aquimaris

Deteriorated seaweed Ulva

11 and 45°C

Organic solvent stable

Trivedi et al. (2011b)

Aspergillus terreus

Marine water

7 and 37°C

Cellulase production Padmavathi, Agarwal, and through agro-waste residue Nandy (2012)

Aspergillus niger

East China Sea

6 and 28°C

Cellulase production using Xue, Chen, Lin, Guan, and E. crassipes, raw wheat bran, Yao (2012) raw corn cob, raw rice straw

Chaetomium sp.

Leaves of the mangroves

9.7 and 50°C

Cellulase production using agricultural and industrial waste

Mucor plumbeus

Ravindran, Naveenan, and Varatharajan (2010)

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cellular machinery suitable for thriving in extreme conditions of pH, temperature, pressure, and salinity (Singh et al., 2014). Recently, Annamalai et al. (2013) reported an extracellular thermostable, haloalkaline cellulase from Bacillus halodurans CAS. The enzyme showed thermal stability at 80°C and pH stability at 12 along with compatibility with different detergents and organic solvents. Similarly, alkali stable cellulase from halophilic Gracilibacillus sp. SK1 has been used in saccharification of corn stover and rice straw for bioethanol production. The ethanol yield was found to be 0.186 g/g reducing sugar with conversion efficiency of 58.2% (Yu & Li, 2015). Apart from the cellulases of terrestrial origin, microbial cellulases from marine environment have also shown successful conversion of cellulose-based plant biomass to fermentable sugar. Bacillus cereus JD0404 isolated from the muddy sediments of mangrove swamps showed bioconversion of cellulose-based biomass (Chantarasiri, 2015). In another recent study, cellulases from Enhydrobacter sp. ACCA2 were used to hydrolyze different plant biomasses such as bamboo, cumbu leaf, cumbu stem, sorghum leaf, and sorghum stem (Premalatha, Gopal, Jose, Anandham, & Kwon, 2015). Recently, extensive work has been carried out in the field of algal biofuel using cellulases of marine origin. Trivedi et al. (2011a, 2011b, 2013, 2015) focused on isolation of different marine microbes with cellulolytic potential. Among the different marine microbes isolated, B. flexus produced alkali halotolerant cellulase with a recovery of 25.03% and purity fold of 22.31. The optimum pH and temperature were found to be 10 and 45°C, respectively. Another strain Pseudoalteromonas sp. was found to stable in six different ionic liquids. The activity measured at 5% (v/v) was maximum with 1-ethyl-3-methylimidazolium bromide ([EMIM]Br) followed by 1-ethyl-3-methylimidazolium acetate ([EMIM] Ac), 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3methylimidazolium methanesulfonate ([C2MIM][CH3SO3]), 1-butyl-3methylimidazolium trifluoromethanesulfonate ([BMIM][OTF]), and 1-butyl-1-methylpyrrolidinium trifluromethanesulfonate ([BMPL][OTF]) with 115%, 104.7%, 102.2%, 98.33%, 93.84%, and 92.67%, respectively, and >80% activity at 15% (v/v) in all ionic liquids (ILs). Industrial applicability of the cellulase was also evaluated by using green seaweed Ulva lactuca as a carbon source in medium containing different ionic liquids. The specific activity of cellulase in IL containing reaction medium was found to be twofold higher in comparison to aqueous-based reaction medium. Similarly,

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Harshvardhan et al. (2013) also reported cellulase from marine Bacillus sp. H1666 showing saccharification of marine macrophytic green alga U. lactuca in single step. Apart from seaweeds, microalgae have also been hydrolyzed using cellulase for biogas production. Mun˜oz, Hidalgo, Zapata, et al. (2014) used cellulases from nine different marine bacteria belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella for the hydrolysis of cell walls of Botryococcus braunii and Nannochloropsis gaditana. The cellulases have also been reported from microbes isolated from Antarctic region. Ferres, Amarelle, Noya, and Fabiano (2015) studied cellulases from different bacterial genera such as Pseudomonas, Pyschrobacter, Pseudoalteromonas, Loktanella, Flavobacterium, Arthrobacter, Polaromonas, Rhodococcus, Cryobacterium, Janthinobacterium, etc. Microbes surviving in extreme conditions of desiccation and freezing got adapted by metabolic functions and the synthesis of structurally adapted enzymes. Recently, Deep, Poddar, and Das (2016) have successfully cloned, overexpressed, and characterized halostable, solvent-tolerant novel β-endoglucanase from a marine bacterium Photobacterium panuliri strain LBS5T. The enzyme is monomeric and has molecular weight of 53 kDa with an optimum pH and temperature of 4 and 40°C, respectively. Further, the structural analysis of recombinant β-endoglucanase showed presence of 25% helix, 30% sheets, and 56% irregularities. The enzyme showed stability in different organic solvents with an increase in enzyme activity by 1.5-fold. The recombinant enzyme showed stability in 50% v/v concentrations of solvents which was higher than cellulases from B. vallismortis RG-07 (30% v/v), Thalossobacillus sp. LY18 (20% v/v) and B. aquimaris (20% v/v) (Gaur & Tiwari, 2015; Li, Wang, Li, & Yu, 2012; Trivedi et al., 2011b). Extremophilic microorganisms, mainly thermophiles and alkaliphiles, are potential lignocellulolytic enzyme producers (Trincone, 2011). Marine thermophilic microorganisms, such as Pyrococcus sp. (Matsui et al., 2000) and Thermotoga sp. (Duffaud, McCutchen, Leduc, Parker, & Kelly, 1997), have been reported for glycoside hydrolases.

5. MARINE FUNGAL CELLULASES Cellulases isolated from marine fungus have higher saccharification potential over bacterial cellulases. Recently, Trivedi et al. (2015) reported solid state fermentation (SSF)-derived cellulase from Cladosporium sphaerospermum using green seaweed Ulva fasciata as substrate. The optimum

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enzyme production was attained at 25°C with 60% moisture level, pH 4 and incubation period of 4 days. The optimum CMCase and FPase activity were found to be 9.56  0.53 and 8.83  0.37 U/g DW of seaweed. The enzyme was found to be active and stable in the presence of different ILs, namely, [EMIM]Ac, [BMIM]Cl, [BMIM][OTF], and [BMPL][OTF]. At 10% v/v concentration, enzyme retained 72.17–85.04% activity in all the ILs. After 24 h of preincubation in all ILs (10% v/v), enzyme activity was in the range of 73.77–93.67%. The hydrolysis of algal biomass with SSF-derived cellulase was optimized. The hydrolysis of U. fasciata feedstock with enzyme (10 U/g) for 24 h at 40°C and pH 4 gave maximum reducing sugar yield of 112  10 mg/g DW which on fermentation gave an ethanol yield of 0.47 g/g reducing sugar corresponding to 93.81% conversion efficiency. The ethanol yield estimated under optimized conditions was 4.4 kg/100 kg dry algal biomass. Xue et al. (2012) demonstrated an environment-friendly process to produce cellulase (17.80 U/g DW) by a marine Aspergillus niger under SSF using seawater, raw E. crassipes, raw rice straw, raw corn cob, and wheat bran. Several efforts have also been made to produce cellulases using cheap alternative carbon sources such as agricultural and industrial wastes. Padmavathi et al. (2012) explored marine fungal strain for cellulase production using 10 agro-waste residues. Among all, eucalyptus was found to be good carbon source for the cellulase production (733.3 IU/mL) at optimum pH of 7 and temperature of 37°C. Similarly, Ravindran et al. (2010) optimized the cellulase production from marine fungi using cotton seed, sugarcane bagasse, rice bran, and waste paper as a carbon source. The enzyme showed high activity and stability under neutral to alkaline pH and high temperature.

6. MARINE YEAST CELLULASES Marine yeast has gained considerable attention as a source of novel enzymes. Most of the yeast-derived enzymes, namely, amylase, alkaline protease, acid protease, phytase, lipase, inulinase have been isolated from terrestrial microorganisms. These enzymes have various potential applications in food, pharmaceutical, mariculture, and fermentation industries. Similarly, marine yeasts could also play a major role as a source of novel enzymes due to their ability to tolerate extreme harsh conditions. Kudanga and Mwenje (2005) reported endoglucanase and exoglucanase production from some sp. of Aureobasidium pullulans, black yeast. Similarly,

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Zhang and Chi (2007) isolated and screened 19 strains of marine yeasts for cellulase production and optimized the conditions for higher cellulase production. Recently, Yanjun et al. (2015) isolated marine yeast from surface seawater of sea saltern at Yellow sea (China) for cellulase production. The strain 98 showed higher cellulase production (CMCase 4.51 U/mg and FPAase 4.75 U/mg protein) and was identified as A. pullulans. The molecular mass of the purified CMCase from A. pullulans 98 was 67.0 kDa. This strain has ability to survive in different marine environments and therefore play crucial role in biodegradation of polymers in marine environment (Nagahama, 2006). Apart from cellulases, A. pullulans has been shown to produce different enzymes such as protease (Chi, Ma, Wang, & Li, 2007), lipase (Wang, Chi, Wang, Liu, & Li, 2007), and amylase (Li et al., 2007).

7. CONCLUSIONS AND FUTURE PROSPECTIVES Marine microorganisms are being intensively studied worldwide, although systematic knowledge of the physiology, genetics, metabolism, and enzymology is limited. Therefore, it is necessary to explore the marine microorganism for diverse and unique applications with systematic knowledge. With the developments in biotechnology, industrial cellulases market is expected to grow in near future. Recently, focus has been shifted from terrestrial sources to marine-based sources mainly microorganisms for the production of novel cellulases due to their ability to survive in harsh conditions. Cellulases isolated from these organisms have shown their potential for bioconversion processes which have role in bioenergy-based industries. The study of marine microorganisms for cellulases has been significantly strengthened using the synthetic and system biology approach. The major future challenges for cellulase production includes (1) isolation and screening of potential cellulase producers with high stability and activity under extreme conditions; (2) design of improved biomass pretreatment strategies for better cellulase interaction; (3) manipulation of genetic engineering techniques for over production of enzyme with higher activity, stability, and process tolerance; and (4) enzyme production cost.

ACKNOWLEDGMENT Mr. Nitin Trivedi gratefully acknowledges the Department of Scientific and Industrial Research (DSIR), New Delhi for awarding Research Fellowship.

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