Gut Microbiota from Lower Groups of Animals: An Upcoming Source for Cellulolytic Enzymes with Industrial Potentials

Cellulosic plant materials are a reliable source of renewable energy. Cellulose-based plant materials are now being used for bioenergy production as alternatives to fossil fuels. The traditional way of converting lignocellulosic materials to ethanol and other bioenergy is an expensive and environmentally unsafe process. Several research works have been conducted to find outsource of lowcost cellulolytic enzymes. Initially, fungal species were considered as sources of cellulolytic enzymes. Later on, several studies showed that bacterial species are a more potent source of cellulose-degrading enzymes. Phytophagous lower invertebrates are a good source of cellulolytic gut bacteria. They utilize a wide variety of plant materials as their food source. In this review, thorough literature studies have been made to explore the invertebrate groups that are novel sources of cellulolytic gut bacteria with high efficacy for enzyme production. This study also encompasses a brief description of cellulose, the activity, and cellulase enzyme application in industrial aspects.


Introduction
Lignocellulosic biomass is the most abundant biological macromolecules in nature. It can be a promising source of renewable raw materials for the production of biofuels and various chemicals [1][2][3][4][5][6][7][8][9][10][11][12]. The plant cell wall comprises 35-50% cellulose and 20-35% hemicellulose with 5-30% lignin that together provides 90% of the dry weight of plant materials [2]. These huge amounts of biomasses are ultimately disposed of as waste materials in nature. But proper processing of these lignocellulosic wastes can mitigate the environmental and energetic demand for sustainable and renewable bioenergy [3,[13][14][15][16]. Recent trends have adopted cellulosic raw materials over fossil fuels [10,13,17,18] because of their several drawbacks. Brazil is the pioneered country in utilizing renewable energy and produces ethanol from sugarcane since the 1970s [13]. But the processing of these energy resources through instrument intensive, the thermochemical treatment process is very expensive [19] and needs an alternative one. The traditional way of converting lignocellulosic materials to ethanol requires acid-reliant hydrolysis and fermentation steps, which ended up with the formation of forces together make robust and stable cellulose crystals. At ambient temperature, these hydrogen bonds of cellulose molecules can only be hydrolyzed by the cellulase enzyme system's synergistic action. Cellulase is a multienzyme system, which consists of three major components: 1, 4-β-endoglucanase (EC 3.2.1.4), 1,4-β-exoglucanase (EC 3.2.1.91) and β glucosidase (EC.3.2.1.21) (β-D-glucoside glucohydrolase or cellobiase) [42]. Endoglucanase causes random cleavage of β-1,4-glycosidic bonds along a cellulose chain, liberating a new end. Exoglucanase imparts an exo-attack at the reducing or non-reducing end of microcrystalline cellulose and produces glucose or cellobiose as the end product. β glucosidase is responsible for cellobiose hydrolysis, producing glucose as the end product [43] (Figure.1).The synergistic and sequential action of all these three enzymes facilitates the complete hydrolysis of cellulose to glucose.
Symbiotic microorganisms within the insect gut have a significant contributions to the nutritional ecology of insects [44]. The persistent association of microorganisms in the insect digestive tract provides nutritional advantage through several physiological activities, including digestion and detoxification of specific foodstuff, synthesis of essential amino acids, vitamins, sterol, and nitrogen fixation, and production of pheromone [2,44,45]. Woodborer and plant-eating insects cannot digest their foodstuffs easily as cellulosic plant materials are very stable polymer and require enzymatic attack for degradation [44]. Partial degradation during insect chewing makes some cellulose of foodstuffs available for cellulase enzyme. Endoglucanases or CMCases from different microbial sources consist of catalytic modules of glycosyl hydrolase families (GH) 5-9, 12, 44, 45, 48, 51, and 74. Bacterial endoglucanases possess multiple catalytic modules, carbohydrate-binding modules (CBMs), and other modules, while fungal endoglucanases possess a catalytic module with or without a CBM [43]. Most of the exoglucanases are cellobiohydrolases (CBHs), which are produced in different forms by bacteria and fungi. The catalytic modules of CBHs belong to the glycosyl hydrolase family of 5,6,7,9,48, and 74 [43]. The glycoside hydrolase family's exoglucanases 48 mainly act on crystalline cellulose and induce its hydrolysis, which is mediated by bacterial cellulase systems. β -glucosidase (BGs) does not possess CBM in catalytic modules and hydrolyze soluble cellodextrins and cellobiose to glucose. Cellobiose is an inhibitor of endoglucanase and CBH. Different microorganisms produce various BGs with catalytic modules belonging to families 1, 3, and 9.Generally, aerobic fungi produce BGs extracellularly, but BGs of anaerobic bacteria remain within their cytoplasm [43]. Microbial cellulase within the anaerobic insect gut is associated with the large enzyme integrating protein scaffoldin, which contains multiple copies of cohesin modules to integrate the different enzymes and other components. These entire components together form a multienzyme cellulosome complex [46]. Cellulase and other enzymes contain a complementary cohesin-docking domain that specifically binds to the cohesin modules of scaffoldin. Scaffoldin modules also have carbohydrate-binding domains that facilitate the cellulosome complex ( Figure. 2) to bind with cellulosic substrates for degradation [46]. Cellulosome complex in association with several cellulases promotes the degradation of most recalcitrant cellulose molecules into monomeric glucose molecules utilized by insects and herbivorous animals as an energy source.
Besides these environmental sources, many phytophagous lower invertebrates' gut microbiota has been empirically studied to obtain microorganisms with cellulolytic potential. Cellulase activity within the invertebrate digestive tract has been determined in the long past [63]. Literature reflects that the following invertebrate groups have been studied previously for cellulolytic gut bacterial source:

Arthropods-insects.
Diversified habitat and plant fiber-based diet make Arthropods a potent reservoir of several gut microbial communities. Literature survey depicts that various Arthropoda species have been explored thoroughly in search of gut microbiota with cellulolytic potential [2,64]. Among Arthropods, insects are the most studied group regarding obtaining novel gut bacterial strain, which can synthesize cellulase enzyme with industrial potential [20,44,65]. Due to the wide range of diversity and multitrophic relationships between insect groups and plant hosts, insect species harbor symbiotic bacterial communities within their digestive tract [44,66,67]. Diverse ecological niches and the phytophagous nature of insects have raised interest in studying the digestion mechanism of insect species involving microbial and endogenous cellulase [20,68]. Insect group, termites have evolved with symbiotic systems [69] that efficiently degrade lignocellulosic foodstuffs [70,71,72] and thus make the termite group a promising source of cellulolytic enzymes. Termite consists of 2000 described species that are subdivided into two groups, namely 'higher' and 'lower' group [70,73,74]. Both groups are involved in symbiotic relationships with prokaryotes, but lower groups are also the protists' host [70,73,74]. As most of the termites are wood and soil dwellers, symbiotic relationships with protozoan and prokaryotic fauna within their gut help them turn over the complex biopolymer of wood and other cellulosic and lignocellulosic foodstuffs [73,75].Termites are more potent in cellulose degradation and assimilation than other cellulose utilizing invertebrates [69]. Termites are also found to utilize fungus derived cellulolytic enzyme by making an intriguing symbiotic relationship with fungal species [76]. Cellulolytic gut bacteria have been screened in many species of termites, including Zootermopsis angusticollis [75], Nasutitermes lujae [77], Macrotermes gilvus [78], Coptotermes gestroi [79], Cryptotermes sp. [80], Coptotermes formosanus [81], Coptotermes heimi [82], Cryptotermes brevis [83], Psammotermes hypostoma Desneux [84], Amitermes evuncifer [85], Macrotermes gilvus [86], Coptotermes curvignathus [87].
Most of the termite species utilize microbial cellulase for degradation of the cellulosic foodstuffs, but the existence of endogenous cellulase has been reported within the gut of subterranean termite Reticulitermes speratus [110]. Apart from termite, endogenous cellulase activity has been found in other insect order also [19]. Most of the study has prioritized isolation and quantification of cellulolytic bacteria from different insect gut regions; some work has been focused on metagenomic and pyrosequencing approaches to identify cellulase-encoding genes. Termites are the insects in which cellulase genes have been first discovered [111], followed by other insect species belonging to the order Coleoptera [112,113], Hymenoptera [114], Orthoptera [115], and Hemiptera. βglucosidase and endo -β-1,4 glucanase activities have been estimated in the gut of Nasutitermes takasagoensis [116]. Moreover, through the metagenomic approach, 45 different glycoside hydrolases (GH family) genes have been reported in higher termite Nasutitermes takasagoensis [117]. Researchers have identified endogenous cellulolytic systems within the beetle larvae also. With the aid of transcriptomic technology, one cellulase of glycoside hydrolase family 45 (GH45) and seven GH5 cellulases have been identified from the beetle larvae of Mesosa myops [118]. Two β-glycosidases (βGly1 and βGly2) have been purified from the midgut lumen of beetle Tenebrio molitor larvae [119]. Endogenous cellulase activities have also been detected in the gut homogenate of several cockroach species [64,120]. Cellulose digesting activity has also been determined in the digestive fluids of some other insects, including grasshopper Dissosteira carolina [121] and Schistocerca gregaria [64], longhorn beetle Hylotrupes bajules, Crickets Acheata domesticus, Stick insects Eurycanta calcarata [64], and locusts species [122].

Molluscs.
Apart from insects, some empirical studies have also been conducted to determine cellulolytic bacteria in snail species. Land snails (Gastropoda: Pulmonata) include several distinct lineages of terrestrial gastropods, which utilize various resources of the terrestrial ecosystem that make them efficient in exploiting the available niches, which is why the realized diversity is quite high. They are generally herbivorous, feed upon a wide range of plant materials, and many of them are the pests of agricultural and horticultural plants [137]. As most land snail species consume cellulosic and lignocellulosic materials, they can be a viable and potential source of cellulolytic gut microbe fauna. Pioneered study on bacterial cellulase in the animal gut has been conducted on land snail Helix pomatia [138,139], which has been followed by Florkin and Lozet 1949 [140]and Jeuniaux 1950Jeuniaux , 1955, who worked on the contribution of microbial cellulase and chitinase respectively in the degradation of plant material in the gut of H. pomatia. The African giant snail Achatina fulica (Mollusca-Gastropoda)is the most studied snail species in this respect. The existence of endogenous cellulase within the gut of A. fulica is evident from the work of Soedigdo et al. 1970 [143] and Dar et al. 2020 [144]. Microbial communities with cellulolytic potential have been isolated from Achatina fulica [7,[145][146][147] and Arachatina marginata [148,149]. Few works have been aimed to investigate the physiochemical environment of the gut of helicid snails [150], the occurrence of fermentative bacteria in edible snail Helix pomatia and Cornu aspersum (Gastropoda: Pulmonata) [151], and homolactic intestinal bacteria of Helix aspersa [152], but the detailed works emphasizing microbial contribution in the digestion of cellulose biopolymer in several other gastropod snail guts are yet to be deciphered. Other molluscan species such as marine turban shell Batillus cornutus has been found to possess polysaccharide digesting gut bacteria [153] and wood-boring bivalvia Bankia setacea also depends on nitrogen-fixing cellulolytic endosymbionts for wood degradation in the marine environment [154][155][156][157].The cellulolytic activity within the different areas of the gut of the land slug Arionater had been detected through the CMC zymography and esculin hydrate activity gel assays, which revealed the existence of endoglucanase and βglucosidase enzymes [158] within their gut. Further study was carried out to isolate and identify cellulolytic bacterial colony within the Ariongut, which was the main source of enzyme activity within the gut [158]. Four endo-β -1,4glucanases (21 K, 45 K, 65 K, and 95 K cellulase) and 2 β -glucosidases (110 K and 210 K) were purified from the digestive fluid of sea hare Aplysia kurodai [159]. These enzymes were able to hydrolyze CMC, filter papers, and lichenan, and these all cellulase were able to digest seaweeds, mainly sea lettuce Undaria pinnatifida [159].
Literature survey reflects that insect species are prioritized for the investigation of the gut bacterial community. Other phytophagous species such as terrestrial snails or algae or seaweed consuming aquatic snails and geophagous earthworms can also be efficient model species. Exploration of more invertebrate species may be helpful for the discovery of novel microorganisms with cellulolytic potentials. Lists of lower invertebrates and their gut bacterial strains with cellulolytic potentials (Table 1) and the specific activity of gut bacterial cellulolytic enzymes (Table 2) are presented in this review.

Biotenchnology and Industrial Application of Cellulase
Biotechnological approaches have been adopted in the long past since the 1980s to apply cellulase in the food industry, followed by several other commercial and industrial parts

Mollusca-Gastropoda-Stylommatophora-Achatinidae
Many genera had been isolated which were belonged to three phyla, namely Proteobacteria, Actinoibacteria, and Firmicutes   [153] To obtain efficient hydrolytic potential, cellulase enzymes should possess some desired attributes, including high specific activity, high catalytic activity against crystalline cellulose, high thermostability, resistance to end-product inhibition, and stability against shear force [164]. Various genetic tools are being used for microbial strain improvement to achieve these attributes and enhance enzyme production. Several industrially used fungal strains such as A. niger, T. reesei, Saccharomyces cerevisae, Pichia pastoris, and bacterial strains like Escherichia coli, Bacillus subtilis [164] have subjected to genetic engineering for the production of a recombinant enzyme with high potential for industrial application. Homologous and heterologous expression techniques have been adopted in the recent era to overexpress microbial cellulase and other hydrolytic enzymes [164]. Owing to the genetic engineering of the cellulolytic microbial strain, cellulose-degrading enzymes' efficient production has enhanced its biotechnological potential in various industrial fields. A brief account of the application of cellulase and allied enzymes have been discussed here.

Food processing industry.
The application of enzymes in the extraction of fruit juices and pulps mitigates the problem of low yield, stability, and clarity of product, which are the main difficulties faced by the food industries in the early 1930s. Later on, progressive research on enzyme technology leads to the production of cellulase, hemicellulase, and pectinase from the food-grade microorganisms A. niger and T. reesei. A combination of these enzymes (pectinase, cellulase, hemicellulase), also called macerating enzymes, plays an important role in the extraction and clarification of vegetable and fruit juices [1] also improves the stability and textures of the purees and pulp. A mixture of pectinase and a low level of hemicellulase and cellulase, commercially know as Olivex is used to extract olive oil from olive seeds. The use of Olivex improves the quality of olive oil extract by enriching extra virgin olive oil with vitamin E and antioxidants, reducing the induction of rancidity and lowering oil content in the wastewater [165]. Infusion of pectinase enzyme helps in peeling of citrus food by reducing its bitterness. Application of β-glucosidase and pectinase ameliorate the texture, aroma, flavor, and volatiles compounds of specific fruits and vegetables [166]. Microbial enzymes are long being used in the quality improvement of bakery products also. Amylases and proteases are mainly used in the bakery industry [167], but recently the use of hemicellulase and endo-xylanase helps in equal distribution of water in dough and bread by hydrolyzing arabinoxylan present in dough [168]. This redistribution of water facilitates the enhancement of flavor, volume, softness, texture, and bakery products' stability.

Brewery and winery industry.
The application of exogenous enzymes in wine and beer biotechnology playsa key role in quality control and production rate. α and β-amylase, carboxypeptidase, and β-glucanase are endogenously synthesized during the germination of barley before malting and synergistically act hydrolyze seed reserves during the malting process. But their improper activities often result in un-malted and poor quality barley. Application of microbial β-glucanase facilitates hydrolysis of β-glucan and reduces the wort viscosity during the maceration and fermentation process of barley. In the winery, exogenous enzymes hemicellulase, pectinase, β-glucanase are used for better maceration, improved color extraction, filtration and clarification, and wine stability and quality [165]. Furthermore, the β-glucosidase enzyme application modifies glycosylated precursors that enhance the aroma of wine [169].

Paper and pulp industry.
Application of biomechanical pulping process using enzymes instead of the only mechanical process reduces the energy expenditure during grinding and refining of the woody material in pulps. Mixtures of endoglucanase I and II and hemicellulase have been used to better drainage and beat ability in the paper mills before or after beating pulp, which in turn increases the overall production rate [1]. Cellulase and xylanase enhance the bleaching and deinking of several types of paper wastes [170]. Overall addition of several hydrolytic enzymes ameliorates fiber brightness, strength properties, pulp freeness, and cleanliness.

Textile and laundry industry.
The application of cellulase in the bio-stoning process of denim and jeans products has achieved great success. Usage of cellulase in bio polishing of cotton fabric also has an advantage as an enzyme can readily remove surface fibers and fuzz, resulting in the glossy, smooth, and brighter appearance of cotton garments [1,165]. Cotton garments usually become fluffy and dull after repeated wash. The addition of cellulase enzyme in household detergents helps remove fluffy fibrils from cotton, boosting the appearance and brightness of the garments [1].

Animal feed.
In the animal feed industry, cellulase plays a key role in removing Anti-nutritional Factors (ANF) from the cereals, grains, and vegetables used for animal feed in poultry, cattle, and fish farming. Pretreatment with cellulase and hemicellulase induces partial digestion of lignocellulosic materials and β-glucans, dehulling cereal grains, which improves the cereal quality and ensures a high yield of milk and meat production [165].

Research development and agriculture.
A combination of hydrolytic enzymes, including cellulase, hemicellulase, ligninase, have an immense effect on plant growth and plant disease control [1]. Cellulases and βglucanases can degrade the cell wall and inhibit the germination of spores of some phytopathogens. Mixtures of different hydrolytic enzymes facilitate the digestion of desired plant or fungal cell walls to produce protoplast, which can be used to make hybrid strains of desired properties for research purposes [23].

Waste management.
As cellulose is the most abundant biomolecules in the plant, a large number of wastes of leaf litter and other lignocellulosic materials are generated from forests, agricultural fields, and agro-industries. These wastes containing a large amount of raw cellulose may cause environmental pollution. But nowadays, with the help of enzyme technology, these unutilized or underutilized cellulosic sources are being converted to produce several biofuels and bio commodities, sugars, and alcohol [1, 171,172]. Application of garden snail (Cornu aspersum) cellulase in paper waste saccharification is empirical evidence of cellulase activity in waste management [173].

Conclusion and Future Prospect
Cellulase and allied enzymes are getting attraction worldwide due to their wide range of applications in vast areas of industries. Although in the past, fungal-based enzymatic systems have been used for cellulolytic enzyme production, later many research works have been carried out in search of more efficient microbial enzymatic systems as a source of cellulolytic enzymes. Bacterial enzymatic systems are more promising due to enzyme complexity, extreme habitat variability, and low production cost. Researchers are focusing on bacterial strain improvement to obtain tailor-made cellulolytic enzymes with high specific activity and catalytic efficiency with the aid of biotechnology and enzymology. Moreover, identifying newer sources of cellulose-degrading microorganisms is essential for the isolation of novel cellulolytic genes. Previous studies assert that the gut of phytophagous and herbivorous invertebrates is the host of the cellulolytic bacterial niche. In the future, further exploration of such invertebrates is necessary for the isolation of novel bacteria, which will bring great prospects in the industrial application of cellulolytic enzymes.

Funding
This research received no external funding.