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Polysaccharide Production by Submerged Fermentation

  • Óscar J. SánchezEmail author
  • Sandra Montoya
  • Liliana M. Vargas
Living reference work entry

Abstract

This chapter describes the importance of the polysaccharides obtained from different natural sources as well as their natural occurrence. An overview on the different biological activities of the polysaccharides obtained from fungi, bacteria, and algae is provided. The potential of microbial organisms, especially microfungi and bacteria, as well as macromycete fungi, during production of bioactive polysaccharides at large scale is recognized. The features of the submerged fermentation technology are disclosed considering that this cultivation technique has played and continues playing a crucial role in the industrial production of different polysaccharides with diverse biological activities. Main topics related to the production of these compounds by submerged fermentation making emphasis in the production of fungal and bacterial polysaccharides are briefly described as well. Finally, some words on the significance of the research and development on production of bioactive polysaccharide are presented.

Keywords

Microbial polysaccharides Polysaccharides from macromycetes Bioactive polysaccharides Submerged cultivation Fermentation feedstocks Cultivation media Bioreactors 

1 Introduction

Polysaccharides are compounds widely distributed in the nature. They are present in bacteria, fungi, algae, plants, and animals. These natural biological polymers are nontoxic and have different functions, the best known of which is to be an energy reserve. They are also structural molecules as cellulose in plants, β-glucans in microorganisms (fungi and bacteria), and chitin in arthropods. In addition, these biomolecules are essential for the metabolism of living beings along with proteins and polynucleotides. They also play an important role in cellular communication, adhesion, and molecular recognition for immune system, fertilization, pathogenesis prevention, and blood coating, among others (Yang and Zhang 2009; Zong et al. 2012; Öner 2013).

Currently, people seek foods that are not only nutritive, safe, nice in sight, and with good taste but also offer health benefits like treatment and prevention of diseases. Research advances have allowed the identification of biologically active compounds of different nature in foods like peptides, carbohydrates, polyphenols, carotenoids, phytosterols, fatty acids, etc. (Mazza 2000; Webb 2007; Cheung 2008; Aluko 2012). In particular, a group of polysaccharides have gained a special interest not only because they are an important energy source in human diet but also because they exhibit special features: their chemical structures with glycosidic linkages make them resistant to the digestion; they can be used as prebiotics; they show diverse biological activities such as antitumor, anticarcinogenic, immunomodulatory, protective against mutagens, and hypoglycemic activities, among others; and they can be used in food, cosmetic, agricultural and pharmaceutical industries (Izydorczyk et al. 2005; Mantovani et al. 2008; Brown and Williams 2009; Yang and Zhang 2009; Patel et al. 2010; Lee et al. 2012; Zong et al. 2012; El Enshasy and Hatti-Kaul 2013; Öner 2013; Zhang et al. 2013).

Submerged fermentation is the most employed procedure for the production of polysaccharides. This is explained by the facility to manipulate and optimize the operating parameters during this type of fermentation (Xiao et al. 2010). Submerged fermentation has been used to cultivate different bacteria and fungi in order to obtain intracellular and extracellular polysaccharides exhibiting diverse properties and biological activities (Suárez and Nieto 2013).

The objective of this chapter is to describe the importance of the polysaccharides obtained from different natural sources and provide main topics related to the production of these compounds by submerged fermentation and their applications in food and pharmaceutical industries.

2 Polysaccharides

The polysaccharides, also named glycans, are polymers condensed from a large number of monosaccharides. These monosaccharides or simple derivatives from them can be obtained by complete hydrolysis using acids or specific enzymes. d-glucose is the predominant monosaccharide in polysaccharides although d-fructose, d- and l-galactose, d-xylose, and d-arabinose are also common. Some monosaccharide derivatives can also be found as hydrolysis products of natural polysaccharides as d-glucosamine, d-galactosamine, d-glucuronic acid, N-acetylneuraminic acid, and N-acetylmuramic acid. In these polymers, monosaccharides are linked by glycosidic bonds between the glycosyl residue of the hemiacetal and the hydroxyl residue of another monosaccharide. Polysaccharides differ from each other in their repeated monosaccharidic units, length of their chains, and degree of branching. They can be linear or branched, and with the exception of cyclic polysaccharides known as cycloamyloses or cyclodextrins, their chains have a nonreducing (terminal) end and a reducing end. The general formula of polysaccharides is C x (H2O), where x is usually a number between 200 and 2,500 (Izydorczyk et al. 2005; Aluko 2012; Zong et al. 2012).

Polysaccharides can be divided into two classes according to the number of different monosaccharides in their structure: homopolysaccharides containing a single type of monosaccharide and heteropolysaccharides containing two or more different monosaccharidic units. The homopolysaccharides are linear or branched chains, are commonly named by the repeating monosaccharidic unit, and have the same or different glycosidic bonds as presented in Table 1. The glycosidic linkages may have α- or β-configurations in several positions. On the other hand, heteropolysaccharides can have different bond types within each monosaccharidic unit as well as different types and sequences of glycosidic linkages.
Table 1

Main natural homopolysaccharides

Type

Polysaccharide

Repeating unit

Glycosidic linkage/Monosaccharidic unit

Linear

Amylose

α-(1→4)-Glc

Cellulose

β-(1→4)-Glc

Xylan

β-(1→4)-Xyl

Inulin

β-(2→1)-Fru

Levan

β-(2→6)-Fru

Laminarin

β-(1→3)-Glc

Chitin

β-(1→4)-Glc-N-Ac

β-Glucan

β-(1→4, 1→3)-Glc

Curdlan

β-(1→3)-Glc

Branched

Amylopectin

α-(1→4, 1→6)-Glc

Dextran

α-(1→2, 1→3, 1→4, 1→6)-Glc

Levan

α-(2→1, 2→6)-Fru

Pullulan

α-(1→6)-maltotriose

Scleroglucan

β-(1→3, 1→6)-Glc

Glycogen

α-(1→4, 1→6)-Glc

Lentinan

β-(1→3, 1→6)-Glc

Grifolan

β-(1→3, 1→6)-Glc

Schizophyllan

β-(1→3, 1→6)-Glc

According to another classification, polysaccharides can be divided into neutral, acid, or basic. Neutral polysaccharides like amylose, cellulose, and amylopectin are composed only of glucose. Acid or anionic polysaccharides contain derivatives of acid sugars, and their structures have negative charge as pectin and alginates. To date only one cationic polysaccharide is known, the chitosan obtained by modification of the animal polysaccharide chitin. The chitosan is a polymer made of β-(1→4)-2-amino-2-desoxi-d-glucopiranose, which has positive charges at pH between 6 and 7 (Izydorczyk et al. 2005).

2.1 Overview of Biological Activity of Polysaccharides

Over the last years, different research efforts have been done to study the in vitro and in vivo biological activity of different polysaccharides obtained both from natural sources and at industrial level. Among the pharmacological activities reported for polysaccharides, antibiotic, antioxidant (Dahech et al. 2011), antimutagenic, anticoagulant, immunomodulatory (McIntosh et al. 2005; El Enshasy and Hatti-Kaul 2013), anticarcinogenic (Zong et al. 2012), antitumor (Silbir et al. 2014), hypoglycemic, and hypocholesterolemic (Belghith et al. 2012) activities may be highlighted. Different polysaccharides obtained from seaweeds like alginates, carrageenan, and ulvan have shown antibacterial, antiviral, and antifungal activities, among others. For this reason, their use has been evaluated as a dietetic supplement for a variety of fish (Peso-Echarri et al. 2012).

β-Glucans are biopolymers exhibiting a wide range of biological activities. The macromolecular structure of the β-glucans depends on the source and isolation method. Microbial β-glucans are mainly composed of a central linear chain linking d-glucose through β-(1→3) linkages and branches of several sizes originated by β-(1→6) linkages. These branches are presented at different intervals along the central chain. On the other hand, β-glucans derived from cereals are polysaccharides based on glucose with β-(1→3) and β-(1→4) linkages. Different factors like the primary structure, solubility, degree of branching, molecular weight, polymer charge, and structure in aqueous media are related to the biological activity of β-glucans. Some β-glucans obtained from microorganisms (fungi and bacteria) and cereals like oats and barley have exhibited immunomodulatory activity, which depends on the structure, molecular weight, and branching of these polymers. The (1→3)-β-glucans, especially from microbial cells, have shown their ability to stimulate the innate immunity and activate the proinflammatory response, i.e., they exhibit anti-inflammatory properties in vitro and in vivo (Kogan and Kocher 2007; Mantovani et al. 2008; Brown and Williams 2009). In several reports on β-glucans, their immunomodulatory activity has been related to the induction of the production of tumor necrosis factor alpha (TNF-α), γ-interferon, interleukin 8 (IL-8), and nitric oxide; the activation of macrophages and lymphocytes; and the stimulation of CD8 membrane cells (El Enshasy and Hatti-Kaul 2013).

Some β-glucans having structure with β-(1→3, 1→4) linkages from cereals have presented antimicrobial, antiparasitic, hypocholesterolemic, antithrombotic, and antimutagenic activities. Those β-glucans with β-(1→3, 1→4) obtained from the yeast Saccharomyces cerevisiae present antiparasitic, antibacterial, antiviral, antifungal, antimutagenic, and antitumor activities, among others (Zong et al. 2012). Those ones isolated from different fungi such as Auricularia polytricha, Grifola frondosa, Candida albicans, Poria cocos, Agaricus blazei, Lentinus edodes, Schizophyllum commune, and Coriolus versicolor exhibit antitumor, immunomodulatory, and antimutagenic properties (Mantovani et al. 2008; Lin et al. 2010; Carneiro et al. 2013; Zhang et al. 2013).

The polysaccharides or polysaccharide extracts from bacteria like Penicillium jiangxiense, some extracts from plants like Achyranthes bidentata, and polysaccharides from plants like Angelica sinensis (Oliv.) Diels and Panax ginseng C. A. Meyer (ginseng), as well as the pectic polysaccharide obtained from Angelica gigas Nakai, have shown anticarcinogenic and antitumor properties and have been traditionally used as medicines in China. In recent years, the interest to develop drugs from polysaccharides against cancer has become evident. Some in vitro studies have shown the potential of these compounds although more in vivo tests are required to confirm the in vitro results (Aluko 2012; Zong et al. 2012; Wasser 2013).

2.2 Natural Occurrence of Bioactive Polysaccharides

2.2.1 Bacteria

Bacteria are often employed as producer of extracellular polysaccharides (exopolysaccharides) at industrial level. Bacterial β-(1→3)-d-glucans are mostly linear glucans, while β-(1→3, 1→6)-d-glucans are branched polymers and β-(1→3, 1→2)-d-glucans have cyclic structures. About 30 species of lactobacilli are exopolysaccharide producers (Badel et al. 2011). The most common are L. casei, L. acidophilus, L. brevis, L. curvatus, L. delbrueckii bulgaricus, L. helveticus, L. rhamnosus, L. plantarum, and L. johnsonii although their productivities are much lower than species of Alcaligenes, Xanthomonas, Sphingomonas, and Leuconostoc, which are the microorganisms most used in industry for the production of curdlan, xanthan, gellan, and dextran (McIntosh et al. 2005; Öner 2013).

The xanthan gum is a component of dietary fiber and is also employed for the formulation of liquid or solid products in the pharmaceutical industry, in the latter case as an agent for controlled release (Tungland and Meyer 2002; Pooja et al. 2014). Gellan gum is composed by repeated tetrasaccharide units of d-glucose, d-glucuronic acid, and l-rhamnose, is obtained from bacterium Pseudomonas elodea, and has been proved as a release vehicle of pharmaceuticals, cell carrier, material for guided bone regeneration, and wound dressing (Lee et al. 2012).

The curdlan is a polysaccharide produced by fermentation using the bacterium Alcaligenes faecalis. The effectiveness of the biological activity of curdlan, as in the case of other β-(1→3)-d-glucans (see Table 2), depends on the chemical structure, molecular weight, and conformation. Several reports on this polysaccharide suggest that the structure influences the type of biological activity, especially in the case of anti-inflammatory and antitumor activities. It has been reported that curdlans with a degree of branching less than 50 are not considered as effective antitumor agents. On the other hand, the carboxymethyl ether and sulfate and phosphate esters of curdlan show higher solubility in water and increased biological activity. Curdlan sulfates present anticoagulant and anti-HIV activities as well as inhibitory effects on the development of malaria parasites in vitro. For this reason, curdlan has been proposed for formulation of cosmetic products and as a protective agent for fish farming (McIntosh et al. 2005; Zong et al. 2012).
Table 2

Main bacterial bioactive polysaccharides

Name

Composition

Source

Health benefits

Reference

Curdlan

β-(1→3)-d-glucose

Alcaligenes faecalis

Immunomodulating and pharmacological responses include anti-tumorigenicity; anti-infective activities against bacterial, fungal, viral, and protozoal agents; anti-inflammatory activity; wound repair; protection against radiation; and anticoagulant activity

McIntosh et al. (2005), Zong et al. (2012)

Hyaluronan

Repeating units of β-(1→4)-linked disaccharides of β-d-N-acetyl-glucosamine-β-(1→4)-d-glucuronic acid

Pseudomonas aeruginosa

Because of its very high immunocompatibility and water binding and retention capacity, hyaluronan is widely used in regenerative medicine and cosmetic applications

Öner (2013)

Pasteurella multocida

Fructan: levan type

Repeating fructofuranosyl rings connected by β-(2→6) links

Zymomonas mobilis, Bacillus spp., Streptococcus, Pseudomonas, Xanthomonas, and Aerobacter

Immunomodulator, antitumor and antioxidant agent, hypocholesterolemic, hypolipidemic, hypoglycemic, and a blood plasma substitute

Dahech et al. (2011), Belghith et al. (2012), Silbir et al. (2014)

The levan-type EPS1 exopolysaccharide was isolated from the bacterium Paenibacillus polymyxa EJS-3 and is composed of fructofuranosyl residues with β-(2→6) linkages and branching due to β-(2→1) linkages. This compound showed antiproliferative activity against tumor cells. On the other hand, acetylated, phosphorylated, and benzylated modified EPS1 exhibited and improved antiproliferative activity. Other exopolysaccharide from Rhizobium sp. N613 (REPS) that is a β-glucan composed of a main chain of glucose linked by β-(1→4) bonds with β-(1→6) branching can significantly suppress tumor formation and improve the immune response in mice (Zong et al. 2012).

2.2.2 Fungi

The use of medicinal mushrooms for the prevention and treatment of human diseases is very old. Different bioactive compounds can be obtained from fungi such as polysaccharides, polyphenols, fatty acids, colorants, and thickeners, among others (Cheung 2008; Wasser 2013). The polymeric carbohydrates from fungi can be intracellular (intrapolysaccharides) or extracellular. They are typically glycans composed of glucose (glucans) that have different structures, molecular weights, and compositions depending on the organism from which are isolated or on the medium to which are excreted. Thus, when fungi are grown in a liquid medium, several of these compounds are released into the medium, and then they can be recovered by means of different extraction processes. Moreover, a significant amount of intrapolysaccharides makes part of the fungal biomass either within the basidioma’s structure or in the mycelium (Montoya et al. 2013).

Most bioactive polysaccharides synthesized by fungi are homoglucans although some compounds can form peptidoglycan or glycoprotein complexes. The structure of the fungal cell wall is depicted in Fig. 1. It is composed of homoglucans like the chitin, β-(1→3)-glucan, β-(1→6)-glucan, α-(1→3)-glucan, heteroglucans, and other compounds as the mannoproteins (Becerra-Jiménez et al. 2011). Fungal β-glucans are typically found in the intermediate layer of the cell wall adjacent to the plasmatic membrane and have the function of maintaining the rigidity and shape of the cells. Some of the most fungal β-glucans known for their pharmacological applications are lentinan, schizophyllan, and krestin (usually abbreviated as PSK or polysaccharide K) (Mantovani et al. 2008). These polymers are β-(1→3, 1→6)-glucans as shown in Table 3. Lentinan obtained from Lentinus edodes has a triple-helix structure and a molecular weight between 400 and 800 kDa. Schizophyllan obtained from the fungus Schizophyllan commune has also a triple-helix structure and a molecular weight of about 450 kDa. PSK is a proteoglycan composed of 25–38 % protein residues and β-(1→4)-glucan with β-(1→6)-glucopyranosyl side chains, has a molecular weight of 94 kDa, and is obtained from Coriolus versicolor (El Enshasy and Hatti-Kaul 2013).
Fig. 1

Structure of the fungal cell wall (Modified from Becerra-Jiménez et al. 2011)

Table 3

Main fungal bioactive polysaccharides

Bioactive compounds

Source

Health benefits

Reference

Heteroglucans and homoglucans

Agaricus blazei, Ganoderma lucidum, Lentinus edodes, Grifola frondosa, Coriolus versicolor, Schizophyllum commune

Anticancer and antitumor activities

Mantovani et al. (2008), Ziliotto et al. (2009), Ramberg et al. (2010), Hirahara et al. (2012), Wu et al. (2012), Yamanaka et al. (2012), Yue et al. (2012), El Enshasy and Hatti-Kaul (2013), Zhang et al. (2013), Wang et al. (2014)

β-(1→3)-glucan with β-(1→6) branching, β-(1→3)-branched β-(1→2)-mannan, β-(1→3)-glucan, cordyglucan, lentinan, glucan, mannoglucan, galactomannan, grifolan, schizophyllan, acidic polysaccharides, heterogalactan, krestin, heteroglycan, scleroglucan (β-(1→6)-monoglucosyl-branched β-(1→3)- glucan), heteroglucans

Agaricus blazei, Cordyceps sinensis, Cryptoporus volvatus, Ganoderma lucidum, Grifola frondosa, Hericium erinaceus, Inonotus obliquus, Lentinus edodes, Morchella esculenta, Phellinus linteus, Pleurotus ostreatus, Polystictus versicolor, Schizophyllum commune, Sclerotinia sclerotiorum, Tremella aurantialba

Immunomodulators. The immunostimulating effect of β-glucan is probably associated with the activation of cytotoxic macrophages and T helper and natural killer (NK) cells and with the promotion of T-lymphocyte differentiation and activation for the alternative complement pathway

Moradali et al. (2007), Mantovani et al. (2008), (El Enshasy and Hatti-Kaul 2013)

β-glucan and exopolysaccharides

Agaricus blazei, Cordyceps sinensis, Lentinus edodes, Phellinus baumii, Tremella fuciformis

Antidiabetic effect

Silva et al. (2012)

Prepared derivatives of (1→3)-β-d-glucan

Saccharomyces cerevisiae

Antibacterial, antimutagenic, antioxidant, antitumor, and immunostimulating activities

Kogan and Kocher (2007)

Pullulan: α-(1→6)-linked polymer of maltotriose subunits

Synthesized by fermentation of liquefied starch, coconut by-products, beet molasses, and agro-industrial waste with Aureobasidium pullulans Chemical reactions

Pullulan has been used for liver and tumor target delivery of drug. Pullulan has the application of targeting drug to liver and cancer cells

Prajapati et al. (2013)

EPS2

Polysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

EPS2 exhibited profound free radical-scavenging activities

Laurienzo (2010)

Exopolysaccharides

Marine fungus Penicillium sp. F23-2

Three polysaccharides from Penicillium sp. F23-2 possessed good antioxidant properties, especially scavenging abilities on superoxide radicals and hydroxyl

Laurienzo (2010)

The pharmacological applications of the fungal polysaccharides represent a great interest for both scientific community and industry. For instance, the white-rot fungus Ganoderma lucidum (Reishi), known as “Lingzhi” in China, “Reishi” in Japan, and “Youngzh” in Korea, has been widely employed as a tonic to promote the longevity and health in China and other Asian countries for more than 2,000 years ago. Bioactive compounds from the different strains of G. lucidum can become very diverse depending on the geographic distribution of this fungus, growth conditions, and feedstocks (substrate). According to several reports (Pan et al. 2012, 2013), the polysaccharidic extracts from G. lucidum present antioxidant activity and improve the immunity, so they have been suggested for the treatment of gastric cancer. The β-glucan from the macromycetes Grifola frondosa (known as Maitake beta-glucan or MBG) has been applied as coadjuvant for cancer treatment like chemotherapy (Lin et al. 2010).

In general, the pharmacological activities of fungal polysaccharides are strongly related to their molecular weight and degree of branching. Recently, Wang et al. (2014) researched five polysaccharide fractions obtained from the fruiting body of Lentinus edodes, which showed different structures to those ones reported before. The in vitro and in vivo antitumor tests indicated that the five fractions played a double role. Firstly, they regulate the immune system, and, secondly, they directly kill the cancer cells exhibiting less secondary effect than the chemotherapeutic drugs.

The nutraceutical and chemopreventive properties of the fungus Agaricus blazei (Agaricus brasiliensis) are related to the presence of β-glucans. In particular, the chemical modification of these β-glucans has been proposed in order to improve their scientific and commercial application, for instance, by increasing their solubility. The sulfation has gained special interest because the β-glucans increase their solubility avoiding granuloma formation. In recent years, several authors have reported the activity of these polysaccharides in healing wounds and burns as well as the antimutagenic, anticarcinogenic, and immunomodulatory activities (Angeli et al. 2009; Sui et al. 2010; Yamanaka et al. 2012).

2.2.3 Algae

An important variety of polysaccharides such as alginates, agar, agarose, and carrageenan may be obtained from marine algae (Laurienzo 2010), some of them with pharmacological applications as can be observed in Table 4. For instance, the alginate consumption in humans has allowed the decrease of cholesterol and glucose in the blood. In addition, the alginates have shown its prebiotic activity, ability to mobilize fatty acids, immunostimulating activity, ability to reduce the blood pressure and enzymatic activity in the intestine, and preventive effect against cancer. Some alginates have been reported as coadjuvant for reparation of the intestinal mucosa. The prebiotic activity of the alginates, carrageenan, and ulvan has enabled the improvement of the intestinal microbiota in fishes as well (Peso-Echarri et al. 2012).
Table 4

Main algal bioactive polysaccharides

Name

Composition

Source

Health benefits

Alginate

Guluronic and mannuronic acid

Marine brown algae (Phaeophyceae) and as capsular polysaccharides in soil bacteria

Alginate dressings for wound healing have been successfully applied for many years to cleanse a wide variety of secreting lesions, and they still remain widely used in many circumstances. Alginates have been shown to be useful also as hemostatic agents for cavity wounds

Agar/agarose

Agar (or agar-agar) is a phycocolloid, which is constructed from complex saccharide molecules (mainly β-d-galactopyranose and 3,6-anhydro-α-l-galactopyranose units). Agar and its variant agarose contain also variable amounts of sulfate, pyruvate, and uronate substituents

Agar is extracted from certain species of red algae: Gelidium, Gelidiella, Pterocladia, Gracilaria, Gracilariopsis, and Ahnfeltia

Suspending agent for radiological solutions (barium sulfate), as a bulk laxative as it gives a smooth and nonirritating hydrated bulk in the digestive tract, and as a formative ingredient for tablets and capsules to carry and release drugs

Exopolysaccharides

Soluble polymers in the culture medium (RPS)

Spirulina platensis

Antiviral activity

Source: Laurienzo (2010)

3 Techniques for Cultivation of Microorganisms

Although many polysaccharides can be extracted from other living organisms like plants or even animals, the microorganisms and macromycetes are the most employed source of bioactive polysaccharides. In order to produce these biopolymers at industrial level, fermentation technologies are being implemented. In principle, the organisms that contain or synthesize polysaccharides can be cultivated under controlled conditions in special fermentation systems named bioreactors. However, only a few of these organisms are suitable to be grown under such conditions, which can substantially differ from those of the ecosystems where these organisms naturally grow. The microbial organisms (mostly microfungi, yeasts, and microalgae) have the ability to quickly propagate not only in plates and flasks but also in large bioreactors with volumes of thousands of liters. This can be explained by their small sizes (from 0.5 to 10 μm) implying high surface/volume ratios that allow fast exchange of nutritional compounds and metabolites between the cytoplasm and the surrounding medium. The accumulation of very different substances inside the microbial cells enables their transformation into end or intermediate products through enzyme-catalyzed reactions. In this way, the metabolism of microorganisms tends to be very intense compared to higher macroscopic organisms. In addition, the microorganisms have a wide range of nutritional substances and substrates for their growth. Thus, they can be cultivated using culture media that contain cheap components, e.g., agricultural or agro-industrial residues.

For specific types of polysaccharides, other organisms can be cultivated like the macrofungi (macromycetes), which have a behavior similar to their microscopic counterparts when grown in liquid fermentation media in bioreactors. If the macromycetes are cultivated on the surface of solid materials containing the nutrients required for their development, special biomass structures are formed such as the basidiomata or fruiting bodies. In the particular case of fungi, fungal cells form a filamentous structure named mycelium when liquid media are utilized. If this medium is to be stirred, the diffuse mycelium is converted into spherical cell agglomerations called pellets. When micro- or macrofungi are grown on solid materials, the fungal cells colonize the substrate taking from it the nutrients (in some cases, the solid particles are merely carriers for cells, and the nutrients are taken from the liquid medium that impregnates the solid material). For macromycetes, this growth process is named vegetative stage; after this, the macromycetes form the fruiting bodies that represent the reproductive stage of the growth cycle of the fungi. Evidently, the fructification is not possible when macromycetes are grown in liquid media. The polysaccharidic composition of the fungal biomass obtained from either liquid culture media or solid materials is quite different, and the growth rate of the latter process is usually slower.

As mentioned above, the microorganisms can be cultivated on the surface of solid substrate. This type of aerobic fermentation is called solid-state fermentation (SSF). SSF has been commercially implemented mainly in China, Japan, and other Asian countries for the production of different compounds like fermented foods, hydrolytic enzymes, biopesticides, and other value-added products (Pandey et al. 2000). In fact, worldwide production of the most consumed mushroom, Agaricus bisporus, is a special type of SSF where the fungal cells colonize previously composted solid substrates. On the other hand, some biological agents used against insects attacking economically important crops are cultivated on cereal grains in several tropical countries. For instance, the entomopathogenic fungus Beauveria bassiana is commercially produced in Colombia by SSF of rice grains in order to control the damage caused by the coffee berry borer (Hypothenemus hampei). However, SSF has not traditionally received a big attention in Western Europe and North America except for some specific cases in the food industry.

The Western microbiologic industry has been based on the utilization of liquid culture media for the growth of microorganisms. For aerobic fermentations, the microorganisms can develop on the surface of a liquid nutritive medium disposed in trays that, in turn, are located in shelves; purified air is supplied to the cultivation rooms, and the cells take the oxygen directly from the air. This type of cultivation technology is called surface fermentation and played an important role in the mid-twentieth century especially in Western Europe. For instance, main part of citric acid was produced by using this technology. However, the surface fermentation was abandoned due to the high risk of infection and elevated labor costs.

The submerged fermentation is the most used cultivation technology for the production of cell biomass and value-added products from it (metabolites) in the world. In this case, the microbial cells do not grow on the surface of the liquid medium, but they do in the bulk of the liquid, which is contained in a stainless steel vessel (fermenter). The microorganisms take the nutrients they need from the liquid medium in a solubilized way. If they are aerobic, the air should be supplied to the fermenter by pumping and sparging for the oxygen to transfer from air bubbles to the liquid medium. This technology offers several advantages: low risk of infection, hermeticity and compactness, facility of control and automation, low labor costs, higher reproducibility of the process for different production cycles, and versatility in the usage of a wide range of biological agents (bacteria, molds, yeasts, macromycetes, microalgae, plant cells, animal cells). Nevertheless, the capital costs of the submerged fermentation are higher due to the higher degree of sophistication of the bioreactors (fermenters), and the products tend to be dilute in the liquid medium at the end of fermentation. However, current trends for the development of more effective strains of microorganisms exhibiting elevated titers of the products (e.g., by the development of genetically modified microorganisms) have enabled to neutralize these drawbacks.

3.1 Features of the Submerged Fermentation

Several factors should be defined before the implementation of a submerged fermentation process at industrial scale. Among these factors, selection of the time regime of fermentation, preparation of the culture medium, selection of the process microorganism, aeration, heat transfer, type of bioreactor, process control, cell recovery and downstream operations, and treatment and final disposal of wastes should be highlighted (see Fig. 2). Some of these factors are briefly described below.
Fig. 2

Schematic diagram of the main aspects affecting the submerged fermentation

3.1.1 Time Regime of Fermentation

Submerged fermentations can be organized depending on how the liquid medium and fermented broth are supplied or removed from the fermenter. During batch fermentation, a series of operating procedures are periodically repeated to ensure the growth and development of process microorganisms. These procedures can include the washing and disinfection of the fermenter, fermenter filling-up with the culture medium, sterilization of such medium, inoculation of microbial cells, fermentation, and unloading of the bioreactor content at the end of cultivation process (Cardona et al. 2010). The main drawback of this regime consists in the operating and feedstock costs needed during each fermentation batch to ensure the cell biomass propagation until a concentration high enough to allow appropriate rates of cell growth and product biosynthesis. In addition, the cells are not reutilized that implies not to employ all the potential of cell biomass formed during the process. In general, the production of polysaccharides is accomplished through batch fermentation using bacterial or fungal cells.

Fed-batch fermentation is one of the most employed cultivation regimes when process microorganisms present catabolic repression, i.e., when high substrate concentrations inhibit specific metabolic processes like those related to cell growth rate. For this reason, the microorganisms grow faster at low substrate concentrations. To implement such a process, conventional batch fermentation is performed through employing a less concentrated medium. Once the sugars have been consumed, the bioreactor is fed with portions of fresh medium or by adding a small amount of medium permanently until the end of fermentation. This continuous feeding of the medium can be done in a linear way (with a constant feeding rate) or according to a more complex function defining the rate with which the fresh medium is added to the fermenter, e.g., by an exponential feeding rate (Sánchez and Cardona 2008). Control of flow rate of medium feeding is quite advantageous because the inhibitory effect caused by high concentrations of substrate or product in fermentation broth is neutralized.

Continuous fermentation consists in the cultivation of cells in a bioreactor to which the fresh medium is permanently added and from which an effluent stream of culture broth is permanently removed. The microorganisms are reproduced within the bioreactor at a grow rate that offsets the cells’ withdrawal with the effluent achieving the corresponding steady state. To ensure the system homogeneity and reduce concentration gradients in culture broth, continuous stirred-tank reactors (CSTR) are employed. In this way, a constant production of fermented wort can be obtained without the need of stopping the bioreactor operation in order to perform the periodic procedures typical of batch processes like filling-up and unloading. This allows a remarkably increase of volumetric productivity compared to batch or fed-batch processes. Unfortunately, the microorganisms used to produce bioactive polysaccharides do not exhibit fast growth rates, so the continuous regime is difficult to achieve. In addition, the continuous cultivation during several months can lead to the loss of the ability to produce the target polysaccharides (genetic instability) since the microbial cells tend to form biomass under these conditions and to revert to the native strain in the case of mutants or genetically engineered microorganisms.

3.1.2 Preparation of the Culture Medium

The function of the liquid fermentation medium is to supply the nutritional substances required by the microorganisms to grow and synthesize the desired products. This depends on the metabolism type the process microorganism exhibits under the cultivation process. For instance, if the organism has a fermentative type of metabolism where there is no final electron acceptor (oxidizing agent) added to the medium and the energy source (the compound undergoing oxidation) is organic, there is no need to pump air into the fermenter and therefore the bioreactor design is simpler. The prevalent metabolism type in microorganisms producing polysaccharides is aerobic respiratory metabolism, where an energy source (mainly sugars) is oxidized to CO2, an electron acceptor (oxygen contained in the air) is added to the medium, a nitrogen source should be present to ensure the synthesis of proteins and nucleic acids, and a series of micro- and oligoelements are also added along with the carbon source (again the sugars) to form new cell biomass. Many bacteria and fungi (micro- and macromycetes) exhibit an aerobic respiratory metabolism. In particular, the fungi utilize as energy/carbon source several plant polysaccharides like starch, cellulose, or pectin thanks to their ability to release hydrolytic enzymes (amylases, cellulases, or pectinases). In the case of the microalgae, the energy source is the sunlight, and the carbon source is the atmospheric carbon dioxide.

Metals play an important role during the metabolic processes leading to the biomass growth and biosynthesis of polysaccharides. In general, they act as cofactor of many enzymes and related proteins such as ligninases, amylases, cytochromes (electron carriers), and cellulases, among others. They also can inhibit many enzymes, so their concentration in the culture medium should be thoroughly determined.

Culture media may be synthetic or complex. In the first case, the composition of the medium is clearly established. Simple sugars are often used as energy source in these media. In the case of complex media, the exact composition is not known since materials like molasses or hydrolyzates are employed. These media are preferred because of their low cost.

3.1.3 Air Supply

The aeration levels should be defined in order to improve the fermentation process. The oxygen contained in the air is needed to oxidize the organic compounds for microorganisms with aerobic respiratory metabolism. These microorganisms exhibit high growth rates, have elevated oxygen consumption rates, and release significant amounts of heat. As the oxygen has a low solubility in the culture broth (about 6 mg/L), high air flow rates should be used to supply the oxygen needed (between 0.5 and 2.0 vvm or volumes of air per volume of broth in a minute). The aerobic fermenters are limited by the mass transfer of oxygen to the liquid medium. For this reason, many bioreactors are provided with a stirrer with one or more impeller in order to intensify the dissolution of oxygen in the water. In addition, a sparger is mounted below the lower impeller with the same purposes. This gas–liquid system has a tendency to form foam that implies negative effect on the fermentation performance, so antifoam substances or devices are included in the fermenter.

3.1.4 Heat Transfer

All the living organisms transform the energy released by the oxidation of organic or inorganic substances, or collected from the sunlight, into other forms of useful energy, which is employed for the metabolic functions of the cell and for reproduction. These energy transformations are not 100 % efficient. Although the living matter is much more efficient than the artificial human devices (engines, turbogenerators, photovoltaic cells), some portion of energy cannot be utilized and is dissipated in the form of heat. When microbial cells grow and propagate in the culture medium, metabolic heat is produced. If this heat is not removed, the temperature of the system increases, reaching values not optimal for the growth rate or biosynthesis of the target products. High temperatures can even lead to cell death. To avoid this situation, the bioreactors are provided with some devices for heat exchange such as jackets in small fermenters (up to 1,000 L), cooling coils (up to 50–100 m3), or external heat exchangers with recirculation of liquid medium (above 100 m3). All these devices have pipelines inside where cooling water flows; the water is not in contact with the liquid culture medium but removes the heat through the metal walls of the pipelines by conduction and convection. The heat transfer in cylindrical tanks has been well studied, so the commercial fermenters for polysaccharide production do not present big difficulties on this matter.

3.1.5 Type of Bioreactors

One bioreactor for fermentation (fermenter) is a complex equipment that should ensure all the optimal conditions for the growth of microbial cells being, at the same time, flexible and versatile in order to be used for different purposes. Most of the bioreactors for submerged fermentation are standard apparatuses that have several internal devices and accessories to intensify the mass transfer of oxygen, ensure the homogenization of the culture broth, remove the metabolic heat, avoid the contamination with non-desired organisms, and maintain the required values of pH, temperature, and oxygen concentration, among other operating parameters. There exist in the market several commercial firms offering standard bioreactors to not only cultivate microorganisms but also macromycetes, plant cells, and animal cells.

Agitation and aeration are very important topics to be considered during the design and construction of bioreactors for submerged fermentation as mentioned above. In addition, the control (automated or not) of the bioreactor is crucial to attain the success in the fermentation industry. The different devices for process control (probes, transducers, controllers, actuators) along with the software are quite expensive and can add up to 80 % to the main cost of the bioreactor (without control devices). However, some fermentation processes for the production of polysaccharides require a strict pH and temperature control. For this reason, an evaluation of the process viability should be performed in each case to establish if the sophistication level of a given bioreactor justifies its utilization considering the price and market of the target product.

Finally, the target product remains in the culture broth (exopolysaccharide) or inside the cells (intrapolysaccharide). A series of different unit operations are required to separate and purify these polysaccharides without loss of their biological activities. An example of a sequence of those downstream operations to recover a bioactive exopolysaccharide could be as follows: centrifugation, liquid–liquid extraction, adsorption using activated carbon, liquid chromatography, evaporation, drying, and packing. These operations are complex and difficult to design. A more detailed description of this issue is out of the objectives of this chapter.

4 Submerged Fermentation for Production of Fungal Polysaccharides

Submerged fermentation involves the development of microorganisms in a liquid medium enriched with nutrients and with high oxygen concentrations (aerobic conditions). In the case of fungal cells, the hyphal development (especially in basidiomycetes) in submerged cultures results in the uncontrolled development of the mycelium (set of hyphae). The extension of the fungal biomass has significant effects on the mass transfer, growth rate, and product secretion. The fungal mycelium can form pellets causing their proliferation in the whole culture medium and increasing the viscosity, which limits the mass transfer of oxygen. All these drawbacks limit the operation of the bioreactors (Rodriguez-Couto and Toca-Herrera 2007).

The production of extracellular polysaccharides using macromycetes by submerged fermentation is influenced by the process time, temperature, composition of the culture medium, agitation speed, initial pH, and inoculum size, among other factors. The macromycetes can grow under varied environmental conditions, e.g., at a wide range of temperature (Shu et al. 2007; Suárez and Nieto 2013). However, the submerged fermentations are usually carried out at temperature between 26 °C and 36 °C considering that the temperature increase can accelerate the fungus metabolism and diminish the solubility of oxygen in the medium. On the other hand, the aeration also influences the concentration of dissolved oxygen. This can be controlled by fixing the volume of air supplied to the culture medium during the process (Kim et al. 2008; Suárez and Nieto 2013). To accomplish the follow-up and control of all these variables demands time, efforts, and money in order to obtain the experimental data required for the comprehensive evaluation of the interaction of these variables with the production of polysaccharides.

Regarding the culture medium for polysaccharide production from fungi, different reports published in the last decade evidence the importance of the presence of vegetable oils rich in oleic acid as promoters of fungal biomass formation and, consequently, of their constituent polysaccharides (Yang et al. 2000; Hsieh et al. 2008; Hao et al. 2010). When choosing the type of vegetable oil to be used as inductor of polysaccharide production, it is necessary to take into account the fatty acid composition of the oil since several researchers have reported that important amounts of linoleic acid suppress the production of biomass and polysaccharides, while the presence of oleic promotes their production (Park et al. 2002; Hsieh et al. 2006, 2008). Soy and olive oils have been used as promoters of cell growth and polysaccharide production.

More research is needed about the control of submerged fermentation processes using macromycete fungi. For instance, Wu et al. (2006) found that it was necessary to control such variables as substrate composition, pH of the medium, temperature, and other environmental conditions to increase and maintain constant the production of cell biomass and exopolysaccharides by submerged fermentation using the macromycetes Auricularia auricular. Previous studies have demonstrated that the pH control plays a very important role during the development of mycelial biomass and polysaccharide production (Fang and Zhong 2002). On the other hand, some reports evidence the effect of pH on the chemical structures and molecular weight of the polysaccharides obtained from fungi. This is particularly important since other studies suggest a clear relationship between the biological properties of the polysaccharides and their molecular weights (Shu et al. 2003, 2004; Hamedi et al. 2012; Zhang et al. 2013). For instance, Shu et al. (2003) concluded that the polysaccharides found in the culture broth where Agaricus blazei was grown strongly depended on their molecular weight.

The dispersion of the mycelium in liquid industrial fermentations implies the homogenization and agitation of the branched hyphae. This dispersion produces a broth with a non-Newtonian behavior, and its apparent viscosity increases with the agitation speed. This reduces the transport of nutrients as well as the transfer of oxygen and heat, therefore increasing the operating costs (Prosser and Tough 1991). Thus the agitation plays a very important role regarding the mycelium integrity. If the agitation is very strong, the breakdown of mycelium is produced, and, therefore, pellets formation decreases. At the same time, the biomass formation is affected as well as the target metabolites like the polysaccharides (Cui et al. 1997; Suárez and Nieto 2013).

5 Submerged Fermentation for Production of Bacterial Polysaccharides

The submerged fermentation to obtain polysaccharides from bacteria is used universally. For example, the curdlan exopolysaccharide is obtained by submerged fermentation of Agrobacterium sp. or Alcaligenes faecalis under nitrogen-limiting conditions. For the production process of this biopolymer, critical factors such as the carbon source, nitrogen source, phosphate concentration, pH, and agitation rate, among other factors, should be optimized. Moreover, although the conventional nitrogen source for production of curdlan is NH4Cl, studies with NaNO3, urea, and yeast extract have been reported as well (Jiang 2013).

Badel et al. (2011) studied the main conditions for the production of dextran, levan, inulin, mutan, and reuteran from some lactobacilli species as well as their monosaccharide composition (see Table 5). The cultivation temperature varied between 32 °C and 42 °C, the process time for biopolymer production was between 18 and 72 h, and the pH lied in the range between 5 and 6. The most effective bacteria for their high yields in the production of polysaccharides were L. rhamnosus 9595, L. rhamnosus 9595 M, L. delbrueckii bulgaricus, and L. helveticus.
Table 5

Principal producers and culture conditions for lactobacillus polysaccharide production

Microorganisms

Media

Temperature (°C)

Time (h)

pH

Yield (mg/L)

L. rhamnosus 9595 M

BMMa

32–37

72

6

~1,000

L. delb. bulgaricus RR

Whey

38

24–28

5

95–100

L. rhamnosus R

BMMa

37

72

6

500

L. delb. bulgaricus

Milk

42

24

/

110

L. delb. bulgaricus

MRS

40

18

/

263

L. rhamnosus GG

Milk

37

20

/

80

L. delb. bulgaricus 291

Skimmed milk

37

22

/

80

L. casei CG11

BMMa

25

48

/

130

L. helveticus

Skimmed milk

37

60

5

730

L. delb. bulgaricus

Whey (protein-free)

37

18

6

800

L. rhamnosus 9595

Whey permeate supplemented

37

24

6

2,775

L. paracasei

BMMa

32–37

72

6

~80

Source: Badel et al. (2011)

a BMM basal minimal medium, MRS Man Rogosa Sharpe

6 Conclusion

Bioactive polysaccharides are value-added products with important applications, especially in the pharmaceutical industry due to their key biological activities: antibiotic, antioxidant, antimutagenic, anticoagulant, immunomodulatory, anticarcinogenic, antitumor, hypoglycemic, and hypocholesterolemic activities. The world market of nutraceuticals and natural pharmaceuticals is steadily increasing. To meet the worldwide demand, especially in the developed countries, it is necessary to not only find new sources and applications of these polysaccharides but also to improve the technology for the large-scale production of these biopolymers.

The submerged fermentation is the most developed technology in the world to produce a wide range of bio-based products. Fortunately, the organism naturally producing bioactive polysaccharides can be cultivated by fermentation using liquid culture medium. This chapter attempted to provide an overview on the features of the submerged fermentation applied to the production of bioactive polysaccharides from fungi and bacteria. The possibilities to continue the scientific research and technological development on this matter are bright and wide. Undoubtedly, the engineering approach to improve this type of technology in the case of bioactive polysaccharides has the key to reduce production costs and increase their production levels worldwide.

Notes

Acknowledgments

The authors want to thank all the members of the research group on Food and Agribusiness of Universidad de Caldas (Colombia) for the knowledge generated during the last years on the theme of fermentation technologies and macromycete cultivation. The financial support for the different research projects of the Vice-rectorate for Research and Graduate Students of Universidad de Caldas and of the General Royalties System of Colombia is also acknowledged.

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Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Óscar J. Sánchez
    • 1
    Email author
  • Sandra Montoya
    • 1
  • Liliana M. Vargas
    • 1
  1. 1.Bioprocess and Agro-industry Plant, Institute of Agricultural BiotechnologyUniversidad de CaldasManizalesColombia

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