Bioprocessing of Agrofood Industrial Wastes for the

4 Bioprocessing of Agrofood Industrial Wastes for the Production of Bacterial Exopolysaccharide J. Kanimozhi, V. Sivasubramanian, Anant Achary, M. Vasanthi, Steffy P. Vinson, and R. Sivashankar CONTENTS 4.1 Introduction .................................................................................................. 68 4.2 Microbial Polysaccharides .......................................................................... 68 4.2.1 Bacterial Exopolysaccharides .......................................................71 4.2.2 Bacterial Alginate ...........................................................................72 4.2.3 Bacterial Cellulose..........................................................................72 4.2.4 Curdlan............................................................................................72 4.2.5 Glucans ............................................................................................76 4.2.6 Gellan and Related Polymers (Sphingans).................................76 4.2.7 Hyaluronan .....................................................................................76 4.2.8 Levan ...............................................................................................77 4.2.9 Succinoglycan .................................................................................77 4.2.10 Xanthan Gum .................................................................................77 4.3 Bacterial Exopolysaccharides Biosynthesis Pathway.............................. 78 4.4 Agroindustrial Wastes ................................................................................ 79 4.5 Bioprocessing of Agroindustrial Wastes ..................................................83 4.5.1 Solid-State Fermentation...............................................................84 4.6 Low-Cost Agrowastes for Exopolysaccharide Production .................... 85 4.6.1 Sugarcane Molasses .......................................................................87 4.6.2 Whey-Dairy Industry ....................................................................88 4.6.3 Pomace.............................................................................................89 4.6.4 Cereals and Cereal Bran ................................................................ 90 4.6.5 Glycerol ...........................................................................................90 4.7 Constraints and Improvements ................................................................. 91 References............................................................................................................... 92

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Bioprocess Engineering for a Green Environment

4.1 Introduction The demand for natural polysaccharides has progressively increased over the past few decades due to their far-reaching commercial applications in various industrial sectors such as food, chemicals, pharmaceuticals, agriculture, and medicine. Due to their diverse structure, biological functions, and material properties, industrial microbial polysaccharides have overtaken traditional polysaccharides in the current polymer market. A useful biopolymer cannot find its proper place in the polymer market unless it is produced economically. In microbial polysaccharide production, the shift in feedstock utilization requires intensive research activities for the application of innovative concepts on a large scale. These concepts could involve novel resources and pretreatments as well as fermentation and downstream processing techniques. Bioprocessing may play an important role by providing adequate pretreatment, coagulation, dewatering, and modification of alternatives. Agrowastes are a renewable, natural, and inexpensive resource that can be employed for the cost-effective production of value-added products, and there has been an increasing trend toward more efficient utilization of industrial agrowastes. In recent years, in order to decrease the costs of bacterial exopolysaccharide (EPS) production, there has been a growing interest in developing culture media based on other sources of sugars, for example, industrial agrofood wastes such as peels and pomace of fruits, vegetables, and sugarcane molasses. These items are rich in sugars such as glucose, fructose, and sucrose, and they also contain nitrogen and vitamins that are useful for EPS biosynthesis. The agrofood industry is developing new technologies to use waste as raw materials for biochemical production to promote economic advantages and diminish environmental pollution. This chapter attempts to extend the use of industrial agrowastewater for bacterial EPS production, with a focus on the bioprocessing techniques.

4.2 Microbial Polysaccharides Polysaccharides are high-molecular-weight carbohydrate molecules composed of long chains of monosaccharide units bound together by glycosidic linkages. Polysaccharides are an important class of biological polymers that are natural, nontoxic, and biodegradable, and that cover the surface of most cells and serve as a storage and structural entity of all cells (Kumar et al. 2007). Polysaccharides can be classified based on the available sources, monomer composition, glycosidic linkage, or biological function. Chemically, polysaccharides may be classified as homopolysaccharides or

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Bioprocessing of Agrofood Industrial Wastes

heteropolysaccharides; on hydrolysis, they produce a single monosaccharide and a mixture of monosaccharides, respectively. Some polysaccharide molecules are linear, while others are branched; as the degree of ramification is increased, there are corresponding changes in physical properties such as water solubility, viscosity, and gelling behavior. The classification of bacterial polymeric substance is shown in Figure 4.1. Microbial polysaccharides are water-soluble polymers and may be ionic or nonionic. The repeating units of microbial polysaccharides are regular, branched or unbranched, and are connected by glycosidic linkages. Some microbial polysaccharides are commercially accepted for the human use by the U.S. Food and Drug Administration (FDA). Eventually, polysaccharides are either extracted from biomass resources such as algae and higher-order plants or produced through microbial fermentation or enzymatic conversion. Currently, a small number of microbial biopolymers are produced industrially on a large scale for commercial purposes. There is

Polysaccharide Source

Higher plant cell wall and seeds Starch, cellulose, pectins, potato starch, exudates gum

Derived and modified

Microbial fermentation

Seaweed extracts Agar, alginate, carrageenas

Microorganisms

Fungal

Bacterial Environment

Intracellular polymeric substance

Galactosaminogalactan, N-acetylglucosamine, pullulan

Extracellular polymeric substance IPS

Inorganic polyanhydrides Polyphosphates

Capsular polysaccharides (bound)

EPS

Polysaccharides

Polyesters

Alginate, cellulose, curdlan, levan, xanthan

Polyhydroxybutyrate

Exopolysaccharides (unbound)

Klebsiella sp., Erwinia sp., Neisseria sp.

Monomer composition

Homopolysaccharides

CMC, sodium alginate, propylene glycol (PG)

Lipopolysaccharides O-antigen Salmonella

Heteropolysaccharide

Dextran, levan

FIGURE 4.1 Classification of bacterial polysaccharides.

Xanthans, gellans

Polyamides Poly-ε-lysine (ε-PL)

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competition among microbial and plant polysaccharides for industrial applications. Nevertheless, the designer polymers of extracellular polymers produced by microorganisms exhibit different eminent material properties and have revolutionized applications in many sectors in addition to plant polysaccharides. Apart from structural diversity, microbial polysaccharides are preferred over plant and algal polysaccharides because the former 1. Enable fast production and high yields 2. Have a process condition that can be fully controlled 3. Have a process that is not affected by geographical or seasonal variations 4. Are of short duration 5. Are energy efficient, in the case of microalgae (production uses solar energy) 6. Have the possibility of utilizing industrial agrowastes Bacterial biopolymers are rapidly emerging as industrially important and are becoming economically competitive with natural gums produced from marine algae and other plants (Bajaj et al. 2007). Different classes of microbial taxa tend to secrete different structural, functional, and beneficial extracellular polymeric substances (Geesey and White 1990) into the environment and are responsible for the formation of biofilms within the extracellular matrix. Polysaccharide biosynthesis and accumulation generally take place after the microorganism’s growth phase. The polysaccharides produced by microorganisms can be classified into three main groups according to their location in the cell (Donot et al. 2012): 1. Cytosolic polysaccharides, which provide a carbon and energy source for the cell 2. Cell wall polysaccharides, which make up the cell wall (e.g., peptidoglycans, teichoic acids, and lipopolysaccharides) 3. EPS that are exuded into the extracellular environment in the form of capsules or biofilm EPS are often favored for commercial use because they are naturally exuded by microorganisms into the extracellular environment, and easier product recovery is facilitated. The demand for bacterial EPSs has great commercialization potential predominantly due to their structural diversity and peculiar characteristics. Today, EPS could be used in many industrial applications, especially in the food and pharmaceutical industries. Industrially, most bacterial EPS are produced via microbial fermentation. However, owing to the development of enzyme immobilization, industry is producing dextran and

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Fermentation • Microbial • Enzymatic conversion

Extraction • Cell removal • Centrifugation • Filtration • Polymer precipitation • Methanol • Ethanol • Isopropanol

Purification • Reprecipitation • Ethanol • Methanol • Isopropanol • Deproteinization • Salting out • Protease • Ultrafiltration

Drying • Freeze drying • Lyophilization • Drum drying

Characterization • Chemical structure • Monomer analysis • GC-MS, FTIR, NMR • Size exclusion chromatography

FIGURE 4.2 General strategies for the production of bacterial exopolysaccharides.

levan through enzymatic conversion. The strategies involved in producing bacterial EPS via microbial fermentation are presented in Figure 4.2. Traditional strategies to improve the microbial fermentative production of bacterial EPS include the following: 1. Improved strain selection 2. Optimization of cultivation conditions 3. Metabolic engineering—manipulation of the gene that encodes the enzyme involved in the metabolic pathway of polysaccharide synthesis 4. Alteration of the regulatory pathways that affect gene expression and enzyme activity 5. Control over biosynthetic process 4.2.1 Bacterial Exopolysaccharides Bacteria have a tendency to produce varied polysaccharides with diverse chemical properties via utilization of carbon sources. Bacterial extracellular polysaccharides are as structurally and functionally diverse as the bacteria that synthesize them. Nevertheless, the greatest potential of bacterial EPSs is related to their use in high-value market niches, such as cosmetics, pharmaceuticals, and biomedicine, in which traditional polymers fail to meet the required degree of purity or lack some specific functional properties. Such markets provide opportunities for the development of up-and-coming bacterial EPSs, providing that they have the specific desirable physicochemical properties. In such high-value applications, product quality wholly surpasses cost production and product yield issues. In particular, downstream processing requirements are highly demanding in these applications. Separation processes become especially challenging when polymers are considered for medical applications because a high-purity product is required. Bacterial EPSs are presented in many forms, including cell-bound capsular polysaccharides, unbound “slime,” and the O-antigen component of lipopolysaccharides, with an equally wide range of biological functions. The biological functions of polysaccharides include a variety of diverse functions

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such as adhesion, cell-to-cell interactions, biofilm formation, and cell protection against environmental extremes. Bacterial EPSs are loosely associated with the cell surface and are secreted into the extracellular environment in the form of slime. They are one of the most important classes of potential probioactive molecules (Bazaka et al. 2011). Some commercially important bacterial EPSs and their industrial applications are presented in Table 4.1. 4.2.2 Bacterial Alginate Alginate is a linear hetropolysaccharide composed mainly of two uronate sugars such as mannuronic and guluronic acids. They form block structures of poly-mannuronic acid sequences, poly-guluronic acid sequences, and mixed sequences. Alginates are produced mainly by liquid bacterial cultures of the genera Azotobacter and Pseudomonas up on the utilization of glucose/ fructose and xylose, respectively. For large-scale industrial production of alginates, Azotobacter species are mainly used due to their competent nature (Morris and Harding 2009). Currently, alginates, salts of alginates, and algenic acid are widely used in the food industry as thickeners and stabilizers due to their gelling property, as per generally recognized as safe (GRAS). In addition, they have anti-inflammatory and detoxifying properties. 4.2.3 Bacterial Cellulose Bacterial cellulose (BC) exemplifies a promising alternative to plant-derived cellulose for specific applications in biomedicine, cosmetics, high-end acoustic diaphragms, paper-making, the food industry, and other applications. Cellulose from plants is normally mixed with lignin and hemicelluloses; however, BC contains sets of parallel chains composed of d-glucopyranose units interlinked by intermolecular hydrogen bonds and is identical in chemical composition to plant cellulose. BC displays many unusual physicochemical and mechanical properties, including higher purity, higher crystallinity, higher degree of polymerization, and higher water absorbing and holding capacity (Mohammadkazemi et al. 2015). BC is found in gram-negative bacteria such as Gluconacetobacter xylinus, Agrobacterium, Achromobacter, Aerobacter, Azotobacter, Pseudomonas, and Rhizobium as well as gram-positive bacteria such as Sarcina. G. Xylinus is one of the most commonly used and studied bacterial species in the production of BC. 4.2.4 Curdlan Curdlan is a neutral, water-insoluble, linear biopolysaccharide that is composed primarily of β (1-3)-linked glucose. It is synthesized by pure culture fermentation using Rhizobium radiobacter and other related bacteria under nitrogen-limiting conditions. Curdlan was given its name because of its ability to “curdle” when heated, a property that makes it a good gelling material

Guluronic acid Mannuronic acid Acetate

Glucose

Glucose

Glucose

Fucose Galactose Glucose Acetate Succinate Pyruvate

Cellulose

Curdlan

Dextran

FucoPol

Components

Alginate

EPS

Anionic

Neutral

Neutral

Neutral

Anionic

Charge

(2.0−10.0) × 106

106−109

5 × 104−2 × 106

∼106

(0.3–1.3) × 10 6

Molecular Weight

Viscous shear thinning solutions in aqueous media Film-forming Emulsifying capacity Flocculating capacity Biological activity due to fucose content

Gel-forming ability Water insolubility Edible and nontoxic Biological activity Nonionic Good stability Newtonian fluid behavior

High crystallinity Insolubility High tensile strength Moldability

Hydrocolloid Gelling capacity Film-forming

Main Properties

Commercially Used Bacterial Exopolysaccharides with Their Potent Applications

TABLE 4.1

(Continued)

Food hydrocolloid Medicine Surgical dressings Wound management Controlled drug release Foods (indigestible fiber) Biomedical Wound healing Tissue-engineered blood vessels Audio speaker diaphragms Foods Pharmaceutical industry Heavy metal removal Concrete additive Foods Pharmaceutical industry: blood volume expander Chromatographic media Hydrocolloid for use in: Food and feed Cosmetics Pharmaceuticals and medicine Oil recovery Source of fucose and fuco-oligosaccharides

Main Applications

Bioprocessing of Agrofood Industrial Wastes 73

Levan

Hyaluronan

Gellan

GalactoPol

EPS

Fructose

Galactose Mannose Glucose Rhamnose Acetate Succinate Pyruvate Glucose Rhamnose Glucuronic acid Acetate Glycerate Glucuronic acid Acetylglucosamine

Components

Neutral

Anionic

Anionic

Anionic

Charge

3.0 × 106

2.0 × 106

5.0 × 105

(1.0–5.0) × 10 6

Molecular Weight

(Continued)

Medicine Solid culture media

Biological activity Highly hydrophilic Biocompatible Low viscosity High water solubility Biological activity: Antitumor activity Anti-inflammatory Adhesive strength Film-forming capacity

Food (prebiotic) Feed Medicines Cosmetics Industry

Foods Pet food Pharmaceutical research: agar substitute and gel electrophoresis

Hydrocolloid for use in: Food and feed Cosmetics Pharmaceuticals and medicine Oil recovery Coatings and packages

Main Applications

Hydrocolloid Stability over wide pH range Gelling capacity Thermoreversible gels

Viscous shear thinning solutions in aqueous media Film-forming Emulsifying capacity Flocculating capacity

Main Properties


TABLE 4.1 (Continued)

74 Bioprocess Engineering for a Green Environment

Glucose Galactose Acetate Pyruvate Succinate 3-hydroxybutyrate Glucose Mannose Glucuronic acid Acetate Pyruvate

Components

Anionic

Anionc

Charge

(2.0–50) × 106

3

LMW < 5 × 10 HMW > 1 × 106

Molecular Weight

Source: Freitas, F. et al., Trends Biotechnol., 29, 388–398, 2011.

Xanthan

Succinoglycan

EPS

Hydrocolloid High viscosity yield at low shear rates even at low concentrations Stability over wide temperature, pH, and salt concentrations ranges

Viscous shear thinning aqueous solutions Acid stability

Main Properties


TABLE 4.1 (Continued)

Foods Petroleum industry Pharmaceuticals Cosmetics and personal care products Agriculture

Food Oil recovery

Main Applications

Bioprocessing of Agrofood Industrial Wastes 75

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to improve the textural quality, water-holding capacity, and thermal stability of various commercial products. In fact, curdlan has often been reported to be a useful additive for a variety of food products such as noodles, sauces, frozen foods, and packaged meats (Salah et al. 2011b). 4.2.5 Glucans Glucans are homopolysaccharides comprises of d-glucose monomers linked by glycosidic linkages. They also demonstrate variability in structural and functional properties depending on the type of glycosidic linkage, degree and type of branching, length of chain, molecular mass, and polymer conformation. Glucan has a six-sided arrangement, where d-glucose rings are joined linearly and contain carbons at varying positions. Glucans are classified as β-glucans and α-glucans. The α-glucans include the EPSs such as dextran, mutan, alternan, and reuteran, and are produced primarily because the microorganisms belong to the family of lactobacillus. α-glucans are produced by the utilization of a sucrose-rich source by the extracellular enzyme produced by the bacteria. 4.2.6 Gellan and Related Polymers (Sphingans) Gellan is a linear anionic microbial heteropolysaccharide secreted by the nonpathogenic genus Sphingomonas. The members of genus Sphingomonas produce a group of structurally related EPSs such as gellan, welan, rhamsan, and diutan, and their backbone is comprised primarily of tetrasacchariderepeating units of two molecules of d-glucose, one of l-rhamnose, and one of d-glucuronic acid. Gellan, welan, rhamsan, and diutan demonstrate structural and functional variations in composition and linkage of their side chains (e.g., gellan contains two acetyl substituents such as O-acetate and l-glycerate, whereas welan side group branches contain a rhamnose or mannose) (Coleman et al. 2008). Among these, gellan gum is one EPS broadly used among sphingans due to the variable functions produced by the strains. Gellan acquired a major place in the current polymer market and is mainly produced by C. P. Kelco in Japan. It is approved as a food additive by the FDA and is marketed under four different names: Kelcogel, Gelrite, Phytagel, and Gel-Gro. Kelcogel is used as thickener, while Gelrite, Phytagel, and Gel-Gro are used as solidifying agents for culture media as a substitute for agar (Bajaj et al. 2007). Gellan, produced by the specific strain S. paucimobilis, has gained much attention because of its unambiguous property of forming thermoreversible gels and it has great commercial prospects in the food and pharmaceutical industries (Bajaj et al. 2007). 4.2.7 Hyaluronan Hyaluronan (HA) is high-molecular-mass extracellular linear polysaccharide with disaccharide repeating units composed of glucuronic acid and


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N-acetylglucosamine (Ruffing and Chen 2006). HA exhibits a range of functional properties. It can interact with proteins that help in the organization of the cellular matrix. The genera Pseudomonas and Streptococcus are the main producers of HA. Due to its various biological functions, scientists have developed various functional biomaterials and tissue constructs that have gained major applications in medicine. In contrast, HA is widely used in regenerative medicine and the cosmetic industry due to its high immunocompatibility, water binding capacity, and retention capacity. 4.2.8 Levan Levan is a highly branched and complex homopolysaccharide of fructose. It is generally composed of d-fructofuranosyl residues attached together by β (2–6) and β (2–1) linkages. Levans are biosynthesized by the action of the enzyme levansucrase. Levan is synthesized from sucrose via the catalytic action of levansucrase, the enzyme responsible for both sucrose hydrolysis and the transfer of d-fructosyl residues from fructose to the levan chain by transfructosylation. Levans are primarily produced by the genera Bacillus, Rahnella, Aerobacter, Erwinia, Streptococcus, Pseudomonas, and Zymomonas (Bahl et al. 2010). Owing to the ease of production, levans have more advantages, as they are economically and industrially feasible with numerous applications. Apart from its biodegradability and biocompatibility properties, it has excellent biomedical properties; it is an anticarcinogenic, a hyperglycemic inhibitor, an anti-AIDS agent, an antioxidant, and an anti-inflammatory (Dahech et al. 2011). Due to its tremendous medicinal and polymeric properties, microbial levan is considered to be a valuable biopolymer with high potential. 4.2.9 Succinoglycan Succinoglycan is a highly branched EPS with glucose and galactose in the main chain and side chain containing tetrasaccharide that are composed of modified sugar residues. Succinate, pyruvate, and acetate are commonly found as monosaccharide substituents. It is produced by several soil bacteria, for example, Rhizobium, Alcaligenes, Pseudomonas, and Agrobacterium (Glenn et al. 2007). Depending on the source organism, succinoglycan contains substituents acetyl and succinyl to varying degrees. 4.2.10 Xanthan Gum Xanthan gum is a complex heteropolysaccharide and is considered to be the first commercialized and widely accepted biopolysaccharide produced via the fermentation process. It is synthesized primarily by bacteria of the genus Xanthomonas as a part of their metabolism. The main chain consists of glucose residues with trisaccharide side chains containing glucuronic acid,

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mannose, pyruvil, and acetyl residues. It has various commercial applications in the food and pharmaceutical industries due to its high viscosity at very small concentrations. Due to its stabilizing and thickening properties, it can be used as a significant material in liquid foodstuffs. With FDA approval as a food additive, the thinning property of xanthan gum provides high consistency in low-calorie drinks in which sugars are replaced by artificial sweeteners.

4.3 Bacterial Exopolysaccharides Biosynthesis Pathway Bacteria produce an extensive variety of carbohydrate polymers (EPSs) that are synthesized via four different biosynthesis pathways: (1) the so called Wzx/Wzy-dependent pathway, (2) the ATP-binding cassette (ABC) transporter-dependent pathway, (3) the synthase-dependent pathway, and (4) extracellular synthesis by use of a single sucrase protein (Schmid et al. 2015). Gellan and xanthan EPSs are synthesized via the Wzx/Wzydependent pathway, where Wzx and Wzy are flippase and polymerase, respectively. Bacterial capsular polysaccharide (CPS) is produced by the ABC transporter-dependent pathway, which is not a characteristic pathway of EPS. The synthase-dependent pathway secretes complete polymer strands across the membranes and the cell wall, and is independent of a flippase for translocating repeat units. The polymerization as well as the translocation process is performed by a single synthase protein, which in some cases (alginate, cellulose) is a subunit of an envelope-spanning multiprotein complex (Rehm 2010). Most bacterial EPSs are synthesized intracellularly and exported to the extracellular environment as macromolecules. There are a few exceptions (e.g., levans and dextrans) for which synthesis and polymerization occur outside the cells by the action of secreted enzymes that convert the substrate into the polymer in the extracellular environment (Rehm 2010). Bacterial EPS can be synthesized by either biological or chemical synthesis. EPS biosynthesis can be divided into three main steps (Donot et al. 2012): 1. Assimilation of a carbon substrate–substrate uptake through either a passive or an active transport system 2. Intracellular synthesis of the polysaccharides in which the substrate is either catabolized by intracellular phosphorylation or transported and oxidized through a direct oxidative periplasmic pathway and polysaccharide synthesis 3. Exopolysaccharide exudation out of the cell


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4.4 Agroindustrial Wastes In spite of the advantages of EPS, fermentation must be cost-competitive with chemical synthesis, and many of the potential applications that have been considered for EPSs depend on whether they can be carried out economically. The fermentation medium can represent almost 50% of the cost for a microbial fermentation (Küçükaşik et al. 2011). Employing complex media for growth is not economically attractive because of the high amount of necessary expensive nutrients such as yeast extract, peptone, and salts. Thus, selecting agrowastes as a raw material for EPS production may reduce production costs. Agroindustries are real contributors to overall worldwide industrial pollution. However, the vast quantities of agricultural and agroindustrial residues that are generated as a result of diverse agricultural and industrial practices represent one of our most important energy-rich resources. Wastes from numerous agrofood industries are hazardous to the environment and require suitable and extensive management approaches. Every year, a large amount of waste is generated from the food and agricultural industries throughout the world. The food, agricultural, and forestry industries produce large volumes of waste every year. The disposal of these wastes is highly problematic in countries where the economy largely depends on agriculture. Worldwide, environmental regulatory authorities are setting strict criteria for wastewater discharge from industries. As regulations become stricter, there is a need to treat and utilize these wastes quickly and efficiently. Significant recent research has been dedicated to managing wastes from food-processing agroindustries. Agroindustries, particularly food-processing industries such as cereal, breweries, dairy, sugarcane, and fruits and vegetables generate large amounts of liquid, solid, and gaseous wastes that emerge not only from processing operations but also from their treatment and disposal. The types of waste generated from different food-processing industries are shown in Figure 4.3. In most countries, wastewater from food and agroproduct industries such as distilleries, sugar factories, dairies, fruit canning, meat processing, and pulp and paper mills is discharged into bodies of water. The agroindustrial wastes depend very much on the processing materials, operations, and operational procedure. Wastewater from agroindustry, predominantly raw-material processing wastes, contains carbohydrates, nutrients, oil and grease, chlorides, sulfates, and heavy metals with high values of Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). The wastewater generated by the food and agricultural industries contributes excessive volumes of agroindustrial wastes around the world. These wastes can contribute to a high pollution load if they are discharged without treatment, thus posing pollution problems for both aquatic and terrestrial ecosystems (Rodríguez-Couto 2008). When making valuable biochemicals from microorganisms, a major part of the production cost is

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Cereal and brewery industry Corn cob Corn-steep liquor Bran Starch Spent grain

Sugar industry Wastewater Molasses Sugarcane bagasse Press mud

Agro industry (food processing industry)

Dairy industry Cheese whey Wastewater

Fruit and vegetable industry Peel Seeds Pomace Wastewater

FIGURE 4.3 Waste generation from different agrofood processing industries suitable for bacterial EPS production.

the fermentation media and associated processes. Therefore, to minimize production costs and address industrial demands and challenges, a variety of microorganisms and cheap agroindustrial substrates have been tested. Agrowastes are rich in sugars that can be readily assimilated by microorganisms, resulting in transforming organic matter into biological products. The process of using agroindustrial waste for biochemical production by either submerged or solid-state fermentation is presented in Table 4.2. These processes make such wastes an appropriate choice as raw materials in the production of bacterial EPSs. Currently, these agrowastes are allowed to decay naturally in the fields, or they are burned. However, they could be used as substrates for microbial conversion via Solid State Fermentation (SSF) into value-added products. In addition, the use of such wastes is an environmentally friendly method of managing waste because their disposal presents an added cost to processors, and direct disposal into soil or landfills causes serious environmental problems. Therefore, the investigation and development of potential value-added processes for biological wastes is highly attractive. Recent investigations were carried out to produce EPSs for biotechnological applications at a lower cost. Using agroindustrial wastes as substrates with the goal of more cost-effective production greatly reduces dependence on nonrenewable fuels and other resources, reduces the pollution potential of industrial processes

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TABLE 4.2 Agroindustrial Waste Biomass Used for High-Value Biochemical Production Agroindustrial Wastes Sugarcane Industry Bagasse

Value-Added Products Protease

Microorganism

References

Fruity aroma Biosurfactant

Bacillus spp tk1 and tk2 (SSF) Aspergillus oryzae (SSF) Clostridium butyricum tistr1032 Pleurotuseryngii T. harzianum L04 Penicillium chrysogenum (SSF) Aureobasidium pullulans Ceratocystis Bacillus subtilis (SSF)

Press mud

Vermicompost

Eisenia fetida

Waste water

Hydrogen

Rhodobacter sphaeroides (SSF)

Dairy Industry Whey

Ethanol

Lactococcus lactis (SmF) Rhodopseudomonas Dietzia natronolimnaea Leuconostoc mesenteroides S. paucimobilis ATCC 31461 Aspergillus nigerATCC9642 Aspergillus oryzae

Liu et al. (2016)

Trichoderma atroviride 676 (SmF)

Marques et al. (2014)

A. flavus A. oryzae (SSF)

Thangaratham and Manimegalai (2014)

Aspergillus foetidus (SSF)

Tran et al. (1998)

Glucoamylase Biohydrogen Animal feed Cellulose Penicillin Molasses

Pullulan

Hydrogen Canthaxanthin Dextran Gellan Citric acid

Waste water

Galactooligosacchrides Lipase

Fruit and Vegetable Industries Peel Pineapple Pectinase Citric acid

Kuberan et al. (2010) Parbat and Singhal (2011) Plangklang et al. (2012) Okano et al. (2007) Benoliel et al. (2013) Gonzalez et al. (1993) Israilides et al. (1999) Rossi et al. (2009) Makkar and Cameotra (1997) Pandit and Maheshwari (2012) Yetis et al. (2000)

Singh et al. (1994) Khodaiyan et al. (2008) Santos et al. (2005) Fialho et al. (1999) El-Holi and Al-Delaimy (2003) Sheu et al. (1998)

(Continued)

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TABLE 4.2 (Continued) Agroindustrial Waste Biomass Used for High-Value Biochemical Production Agroindustrial Wastes

Value-Added Products

Orange

Pectinase Cellulase Xylanase Invertase

Grape

Pullulan

Pea

Cellulose

Seeds Jatropha curcas seed cake Grape seeds Pomace Apple pomace

References

Fusarium oxysporum A. niger Neurospora crassa Penicillium (SSF) Aureobasidium pullulans Trichoderma reesei (SSF)

Mamma et al. (2007)

Protease Lipase Laccase

Pseudomonas aeruginosa (SSF) T. hirsute (SSF)

Mahanta et al. (2008)

Single cell protein

Kloechera apiculata and Rahmat et al. (1995) Candida utilis Rhizopus (four Christen et al. (2000) different strains) (SSF)

Volatile carbons as flavors, acetaldehyde, ethanol, propanol, esters Pectinase Cereal and Brewing Industry Spent grain Citric acid

Cellulase Corncob

Microorganism

Israilides et al. (1999) Verma et al. (2011)

Rodríguez Couto et al. (2006)

Bacillus sp.

Kashyap et al. (2003)

Aspergillus niger & Saccharomyces cerevisiae (SmF) Aspergillus niger FGSCA733 (SSF) Streptomyces rimosus (SSF)

Femi-Ola and Atere (2013)

Oxytetracycline Tetracycline Fructo-oligosacchride Aspergillus japonicus

Corn steep liquor

Riboflavin β-carotene

Wheat bran

Plant growth hormone Gibberellic acid Fruity aroma and Ceratocystis fimbriata banana aroma

Ashbya gossypii Blakeslea trispora (fungus) Gibberella fujikuroi

Ncube et al. (2015) Yang (1996) Mussatto and Teixeira (2010) Lim et al. (2001) Papaioannou and Liakopoulou (2010) Bandelier et al. (1997)

Christen et al. (1997) (Continued)

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TABLE 4.2 (Continued) Agroindustrial Waste Biomass Used for High-Value Biochemical Production Agroindustrial Wastes Wheat and rice brans

Value-Added Products Xylanase Neomycin

Hydrolyzed potato starch

Enzyme pectinase Cephalosporin C Lactic acid Pullulan

Starchy wastewater

Poly(βhydroxybutyric acid) (PHB)

Microorganism Aspergillus terreus A. niger (SSF) Streptomyces marinensis Bacillus sp. Cephalosporium sp. Lactobacillus delbrueckii (SSF) Aureobasidium pullulan Alcaligenes latus

References Gawande and Kamat (1999) Ellaiah et al. (2004) Kashyap et al. (2003) Ellaiah et al. (2002) Anuradha et al. (1999) Barnett et al. (1999) Yu (2001)

and products, enables environmental remediation via safe destruction of accumulated pollutants, improves economies of production, and promotes sustainable production of existing and novel products.

4.5 Bioprocessing of Agroindustrial Wastes Bioprocessing involves the complete use of microorganisms for the manufacture of valuable products and the bioconversion of valuable waste resources to build a sustainable future. Bioprocessing agrowaste using microorganisms is an alternative way to address this problem. Through the development of new innovations, different bioprocesses are employed in the utilization of agrowaste residues in various products. Using harsh chemical and physical processes to synthesize value-added products from waste resources becomes an expensive, hazardous, and nonrenewable proposition. Term related to using wastes through bioprocessing includes the following: 1. Bioconversion, also known as biotransformation, which facilitates the conversion of organic matter such as plant or animal waste into appropriate commodities or bioenergies by biological processes or agents such as microorganisms 2. Biorefinery, which is a concept related to transforming waste biomass into value-added chemicals, power, and fuels 3. Biotransformation, which involves microorganisms modifying chemical compounds

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Using microorganisms to synthesize value-added biochemicals from biomass is a promising alternative to harsh chemical synthesis processes that employ expensive, hazardous, and nonrenewable raw materials. It is crucial to lower production costs. Ways to reduce production costs could involve using cheaper substrates, improving product yield by optimizing fermentation conditions or developing higher yielding strains (e.g., by mutagenesis or genetic manipulation), and optimizing downstream processing. Several industrially important biochemical products have been produced via bioprocessing techniques that use different biological wastes as support substrates. The goal of these technologies is to use the waste to develop value-added products, thereby reducing environmental pollution and solving issues associated with waste disposal. Bioprocessing agroindustrial wastes can be carried out both by submerged fermentation (SmF) and solid-state fermentation (SSF), the latter being the preferred method of production in the industrial sector due to its high productivity, simplicity, and concentrated products. Sugar industry wastes can be processed either way. However, the trend has begun to shift toward SSF because different agroindustrial wastes are being used as a source of lowcost carbon and nitrogen, thereby reducing production costs. Additionally, SSF has many advantages such as low effluent generation, simpler fermentation equipment, and direct applicability of the fermented product for feeding (Yang et al. 2001). 4.5.1 Solid-State Fermentation SSF is the growth of microorganisms on moistened solid substrate in which enough moisture is present to maintain microbial growth and metabolism, but there is no free-moving water (Rahardjo et al. 2006). The materials used in SSF can be divided into two categories: inert (synthetic materials) and noninert (organic materials). The former acts only in attachment places, whereas the latter functions as a source of nutrients (hence the term support substrates). Using support substrates presents several advantages, for example, reduced production costs because these substrates supply some nutritive substances to the microorganisms. Biological wastes are a good example of this kind of material. The main advantages of SSF over the commonly used submerged fermentation (SmF) are: 1. Lower energy requirements 2. Lower risk of contamination and absence of complex machinery and complex control systems 3. Due to lack of free water, smaller fermenters needed to make downstream processing easier


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Obstacles to commercial applications of SSF include limited knowledge related to the design and operation of large-scale bioreactors along with difficulties in controlling important culture parameters such as mass transfer and heat removal. Various bioreactor types have been used in SSF processes, including packed beds, rotating drums, gas–solid fluidized beds, and other stirred bioreactors.

4.6 Low-Cost Agrowastes for Exopolysaccharide Production Recently, a great deal of attention has been focused on the potential of converting agricultural and industrial wastes into single-cell proteins and polysaccharides. In this section, agroindustrial wastes suitable to serve as the fermentation substrate for microbial polysaccharide production are discussed (see Table 4.3). The progressive development of recent research aims to determine how to reuse and valorize agrowastes into useful end products and find alternative solutions for agrowaste disposal. The improper disposal of agrowaste residues is not ecologically sound for either industry or the environment. Agroresidues represent a large amount of organic matter that is rich in biomolecules is easily bioconvertible. Today’s scientists and researchers have learned new ways to maneuver waste into a usable resource by converting it into valuable products with the aim of creating a sustainable future. Microorganisms can naturally produce a wide range of industrially important products such as chemicals, vitamins, organic acids, antibiotics, pharmaceuticals, and biofuels. New bioprocess technologies will demand agroresidues as substrates for the biological conversion of products of high marketable interest. Substrate costs account for more than 40% of total production costs for value-added products (Kumar and Mody 2009); consequently, waste residues are an alternative source for substrates that can reduce overall production costs. Regarding agroindustrial wastes, more attention has been paid to wastes from sugarcane, dairy products, breweries, and fruit and vegetable production. These wastes include mainly lignocellulosic materials, cheese whey, molasses and glycerol-rich products, pomace, and bran. These wastes are rich in sugars, which due to their organic nature are easily assimilated by the microorganisms. This makes such wastes very appropriate to be exploited as raw materials in the production of industrially relevant compounds under SSF conditions. In addition, the reutilization of biological wastes is of great interest for legislative and environmental reasons; industry is increasingly being forced to find alternative uses for residual matter. Moreover, the use of these wastes considerably reduces production costs. Therefore, SSF is being increasingly applied in the production of value-added products from wastes. Sugars are the most generally used carbon sources for the production of

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TABLE 4.3 Low-Cost Agroindustrial Wastes or By-Products Used for Bacterial EPS Production EPS EPS

Organisms Bacillus subtilis Zunongwangia profunda SM-A87 Streptococcus Thermophilus BN1 Rhizobium leguminosarum

Alginate

Bacterial cellulose

Curdlan

Dextran

Pseudomonas oleovorans NRRL B-14682 Azotobacter chroococcum Azotobacter vinelandii Azotobacter chroococcum Gluconacetobacter xylinus Gluconacetobacter swingsii sp. Gluconacetobacter xylinus Komagataeibacter sp. Gluconacetobacter hansenii UAC09 Gluconacetobacter xylinus Rhizobium radiobacter ATCC 6466 Cellulomonas flavigena UNP3 Agrobacterium Weissella sp. Leuconostoc mesenteroides Leuconostoc mesenteroides BD1710 Leuconostoc mesenteroides B512

Agroindustrial Waste Resources Cane molasses Rice bran Whey Soybean meal Skimmed milk Whole milk Cheese whey Wastewater from oil company and fish processing industries Glycerol

Whey broth Wheat bran (7.5%) Corn steep liquor (2%) Whey Molasses Date syrup Food-grade sucrose Pineapple peel juice and sugarcane juice Rotten fruits and milk whey Soya bean whey Coffee Cherry husk Corn steep liquor Date syrup

References Razack et al. (2013) Sun et al. (2014) Rabha et al. (2012)

Sellami et al. (2015)

Freitas et al. (2010)

Khanafari and Sepahei (2007) Saeed et al. (2016) Pandurangan et al. (2012) Mohammadkazemi et al. (2015) Cristina Castroa et al. (2011) Jozala et al. (2015) Suwanposri et al. (2014) Usha Rani and Anu Appaiah (2013) Moosavi-Nasab and Yousefi (2011) Salah et al. (2011b)

Date palm juice by-products Groundnut oil

Arli et al. (2011)

Prairie cord grass Sugar from sugarcane Molasses

West and Peterson (2014) Tayuan et al. (2011) Vedyashkina et al. (2005)

Tomato juice

Han et al. (2014)

Carbo pod extract Cheese whey

Santos et al. (2005) (Continued)

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TABLE 4.3 (Continued) Low-Cost Agroindustrial Wastes or By-Products Used for Bacterial EPS Production EPS Gellan

Levan Xanthan

Organisms Sphingomonas paucimobilis ATCC 31461 Halomonas sp. Xanthomonas campestris Xanthomonas pelargonii Xanthomonas campestris Xanthomonas campestris Xanthomonas campestris Xanthomonas campestris Xanthomonas campestris Xanthomonas campestris pv. campestris Xanthomonas campestris (genetically modified) Xanthomonas campestris

Agroindustrial Waste Resources

References

Cheese whey

Fialho et al. (1999)

Molasses Cheese whey (lactose)

Küçükaşik et al. (2011) Niknezhad et al. (2015)

Date syrup

Moosavi-Nasab et al. (2009); Salah et al. (2010, 2011a) Moosavi and Karbassi (2010) De Sousa Costa et al. (2014) Savvides et al. (2012)

Sugar beet molasses Shrimp shell Whey permeate medium hydrolyzed (WPH) Cheese whey

Gilani et al. (2011)

Whey

Fu and Tseng (1990)

Sweet powder whey

Ghazal et al. (2011)

Apple pomace Grape pomace

Stredansky and Conti (1999)

bacterial EPS. However, cheaper substrates, such as agrofood or industrial wastes and byproducts, have been shown to contain adequate quantities of sugars for the production of several bacterial EPS (e.g., molasses, cheese whey, glycerol by-product). These low-cost substrates are a suitable carbon source for the production of both polymers. 4.6.1 Sugarcane Molasses Molasses is the ultimate effluent, or residual syrup, obtained from the sugarcane industry after the recovery of sugar crystals by repeated crystallization of sugarcane or sugar beet juice. It is a dark, heavy, viscous liquid obtained after extraction processes. Different grades of molasses are produced via repeated processing and boiling. This waste residue is still loaded with

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sucrose content, so it can be used as the paramount low-cost raw material for the production of many valuable biological compounds. Due to a highly concentrated sugar content, sugarcane molasses acts as a most excellent carbon source for the microbial fermentation of valuable compounds. India is one of the largest producers of molasses, where it was used as a popular sweetener in the past. In India, nearly 10–12 million tons of molasses are produced annually. The fermentable sugar content of molasses contributes nearly 48.3% of the total sugars. Molasses is rich in sucrose, glucose, fructose, water, phosphates, calcium, and minerals. It is currently used as compost, an animal feed ingredient, a binder, and a source of energy. In 1970, Brazil started to produce second-generation fuels, that is, bioethanol, from sugarcane molasses, largely out of concern over increasing fossil fuel prices and the environmental impact of greenhouse gas emissions. Biofuels from sugarcane molasses can be used to address these concerns. Using different microorganisms, molasses acts as a substrate for both enzymes and oligosaccharide production (Ghazi et al. 2006). Due to the presence of phenolic compounds, molasses has antimicrobial properties. It also exhibits strong antioxidative and tyrosinase-inhibitory activities (Takara et al. 2002, 2003, 2007). Molasses was successfully used for fermentative production of commercial polysaccharides such as curdlan (Lee et al. 2003), xanthan (Kalogiannis et al. 2003), dextran (Vedyashkina et al. 2005), and gellan (Banik et al. 2007). In addition, molasses can be used in the preparation of edible syrups, potassium salts, and activated carbon. Commercial products made by molasses fermentation include ethyl alcohol, citric acid, baker’s yeast, monosodium glutamate, itaconic acid, acetone, butyl alcohol and so on. 4.6.2 Whey-Dairy Industry Whey is the greenish-yellow liquid obtained from milk after the removal of fat and casein. Cheese whey is one of the major by-products obtained in large amounts from the dairy industry after the processing of milk products. On average, the dairy industry generates 500 m3 of waste per day (Demirel et al. 2005). Dumping of these wastes is a critical environmental concern for the dairy industry due to high biological oxygen demand, transportation problems, and spoilage due to the action of bacteria and fungus. On the other hand, whey contains a high amount of recyclable nutrients that serve as a prominent culture medium for the growth of many microorganisms. Two main types of whey are produced from raw milk: whey permeate and whey retentate. Whey permeate is rich in lactose and has various applications in the pharmaceutical industry. Whey retentate is rich in proteins and residual lactose that can be used for various biotechnological applications (Nath et al. 2008). The nutrient composition of whey may vary depending on the composition of the milk, how it is processed, and the final product. Nutrient-rich


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whey contains 4%–5% lactose, 0.2% lactic acid, 0.8%–1% proteins, fats, minerals, vitamins, growth factors, some small organic molecules, and water. Water is the most abundant constituent present in whey, so it provides a cheap and renewable source of carbon and nitrogen to produce various exopolysaccharides such as dextran (Santos et al. 2005), xanthan gum (Silva et al. 2009), and gellan (Fialho et al. 1999). Fialho et al. (1999) evaluated the production of gellan gum by the S. paucimobilis ATCC 31461 strain in media containing lactose, glucose, and sweet cheese whey as substrates. The strain was known to produce highly viscous gellan directly from lactose (Pollock 1993). Cheese whey has also been investigated as a potential substrate for dextran production by L. mesenteroides NRRL B512 cultures (Santos et al. 2005). Alternatively, the cheese whey acts as a basic medium for the fermentation of useful products of industrial importance. Fermentation for the large-scale utilization of whey was first investigated in 1930s and 1940s. Various researches focused on the bioconversion of whey into useful products such as ethanol, baker’s yeast, methane, single-cell proteins, lactate, propionate, vitamins acetate, citric acid, and Polyhydroxy Butyrate (PHB). 4.6.3 Pomace Pomace is the residue produced after the extraction of juice, flavors, and concentrates from fruits or vegetables. Pomace consists of peel, core, and pulp, which are usually used as animal feed or fertilizer. Another food industryderived process is the direct conversion of pomace into snacks, cereals, and pet foods via extrusion process. (Paraman et al. 2015). However, due to the presence of carbohydrates and other biomolecules, the waste pomace can no longer be considered to be waste. The dry or pulpy substance is rich in dietary fibers, polyphenols, bioactive compounds, and natural antioxidants that make it an attractive source for human diet supplements. Due to the presence of dietary fibers, it contains a lot of health-promoting ingredients as well as value-added products such as organic acids, enzymes, alcohols, biofuels, bioadsorbents, flavors, and pigments. Among all the types of pomace, apple pomace has been the most widely studied and has been utilized using SSF to produce ethanol and crude protein for animal feed (Joshi and Sandhu 1996). The presence of pectin in apple pomace substrate induces the production of pectin esterase (Joshi and Attri 2006). Grape pomace is the residue left from grapes after the wine-making process. It is widely used for the production of various hydrolytic enzymes, but the productivity may change depending on the weather and the type of grape used. To overcome this problem and to reach optimum productivity, grape pomace is used along with orange peels (Ndubuisi Ezejiofor et al. 2014). Grape pomace is primarily used to produce xylanase by Aspergillus awamori in SSF (Botella et al. 2007). Pomace has a large potential for bioconversion into several value-added products in an economically feasible way.

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4.6.4 Cereals and Cereal Bran India is one of the largest cultivators of cereals, which are considered as a staple food for many populations. Main cereals such as rice, wheat, corn, barley, oats, sorghum, and millet are grown in India. Bran and germ are the main by-products that come out as waste after the milling process. These waste components are rich in proteins, nutritional fibers, minerals, natural antioxidants, and micronutrients. Using these by-products is an opportunity to reduce waste and produce value-added chemicals of industrial importance. Bran provides a nutrient medium for the growth of many microorganisms that help in the fermentation process. Reducing the particle size of bran and making some other modifications enables it to be a suitable substrate for the synthesis of valuable compounds. The germ contains 25% protein, 18% sugar, and 16% lipids. The sugars are mainly sucrose and raffinose, and it is also rich in B vitamins and many enzymes (Hoseney 1986). Rice and wheat brans have found various applications in the food and pharmaceutical industries. The presence of micronutrients in rice bran such as oryzanol, tocotrienol, and phytosterol have high potential application in nutraceuticals, pharmaceuticals, and cosmetics. Wheat bran is known as brown gold due to the major role it plays in the medical sector, especially in reducing cholesterol levels and cardiovascular diseases. It is also used in the production of valuable compounds by replacing expensive substrates in the fermentation process. Christen et al. (1997) evaluated wheat bran as possible substrate for aroma/flavor production by Ceratocystis fimbriata. Sandhya et al. (2005) performed a comparative study on the production of neutral protease by A. oryzae using several agroindustrial residues such as wheat bran, rice husk, rice bran, spent brewing grain, coconut oil cake, palm kernel cake, sesame oil cake, jackfruit seed powder, and olive oil cake as substrates in SSF and SMF. They found that wheat bran was the best substrate in both systems. 4.6.5 Glycerol Glycerol, also known as glycerine or propane-1,2,3-triol, is a by-product of many industrial processes, mainly from biodiesel plants and soap manufacturing. Biodiesel is considered to be a green fuel and an alternative to fossil fuels. But large amounts of glycerol come from biodiesel plants and are disposed of without any conversion, which creates environmental pollution. Turning crude glycerol into an economically valuable product resolves waste management problems and also diminishes the cost of biodiesel. 1,3-propanediol is a simple organic chemical and has a variety of applications in the production of polymers, cosmetics, foods, lubricants, and medicines. Dipankar et al. (2012) have suggested the production of hydrogen from crude glycerol using a strain of Rhodopseudomonas palustris via photofermentation. The n-butanol


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acts as an ideal solvent for antibiotics, vitamins, and hormones in pharmaceuticals and as a feedstock for production of various polymers. Clostridium pasteurianum is immobilized on Amberlite to convert crude glycerol into n-butanol by anaerobic fermentation and yielded maximum n-butanol at 25 g/L of initial glycerol concentration (Swati et al. 2013). Glycerol acts as a carbon source in the fermentation process for the production of extracellular polysaccharide (EPS). As far as it concern the idea of conversion of glycerol into useful products is demanded while considering the market values. Although bioprocesses have been used to harness the power of several types of agroindustrial waste in the production of various valuable biochemical productions, the full potential is yet to be investigated. This approach can lower the cost of EPS production and simultaneously reduce environmental problems associated with industrial wastes. In any case, stagnant research is required to use industrial and agricultural wastes for valuable chemical production and to reduce disposal efforts and pollution hazards.

4.7 Constraints and Improvements Because they are cheaper, the bacteria may undergo diverse metabolic pathways due to different nutrient composition and tend to produce undesirable by-products and structural changes in polymers. Nonreacted components might accumulate in the broth and eventually become inhibitors, which lowers product yield. For specific high-value applications in which high-purity and high-quality products are needed, usually good-quality substrates must be used to reduce the risk of impurity carryover to the final product. Therefore, in such cases, the use of wastes or by-products might not be an option or, if they are used, higher investment must be put in downstream procedures. Although the composition and amount of EPS produced by bacteria are genetically determined traits, they are highly influenced by media components and cultivation conditions. For most EPS, the basic carbohydrate structure does not change significantly with growth conditions, but its content in substituent groups can vary extensively, thus changing polymer properties. Exceptions to this behavior have been reported for some EPS-producing strains, such as Rhizobium and Pseudomonas, and it allows for the tailoring of polymer composition. However, many low-cost wastes and by-products are apparently promising for production of many bacterial EPS. Moreover, with advances in research and development (R&D), new technologies have been developed to minimize the cost of EPS production.

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