Microorganisms in Fermentation

Abstract

Microorganisms [bacteria, fungi (yeasts and mold)] have been adopted successfully in a wide range of industries, from food and beverage processing industries to pharmaceutical operations. Additionally, microorganisms offer tremendous unexploited potential for value- added products such as amino acids, nucleotides and nucleosides, vitamins, organic acids, alcohols, exopolysaccharides, antibiotics, antitumor agents, etc., through various fermentation processes and parameters. This chapter reviews the involvement of various groups of microorganisms in fermentation. The measurement of microbial biomass, growth and kinetics, and factors affecting fermentation processes are also explained. The roles of microorganisms (bacteria and yeasts) involved in fermentation processes [solid-state fermentation (SSF) and submerged fermentation (SmF)] mostly related in processing industries are discussed.

FormalPara What You Will Learn in This Chapter

Microorganisms [bacteria, fungi (yeasts and mold)] have been adopted successfully in a wide range of industries, from food and beverage processing industries to pharmaceutical operations. Additionally, microorganisms offer tremendous unexploited potential for value- added products such as amino acids, nucleotides and nucleosides, vitamins, organic acids, alcohols, exopolysaccharides, antibiotics, antitumor agents, etc., through various fermentation processes and parameters. This chapter reviews the involvement of various groups of microorganisms in fermentation. The measurement of microbial biomass, growth and kinetics, and factors affecting fermentation processes are also explained. The roles of microorganisms (bacteria and yeasts) involved in fermentation processes [solid-state fermentation (SSF) and submerged fermentation (SmF)] mostly related in processing industries are discussed.

1.1 Introduction

The term “fermentation” is borrowed from the Latin word fevere which means “to boil.” According to Louis Pasteur, fermentation was defined as “La vie sans l’air”, i.e., life without air, and the science of fermentation is also known as zymology or zymurgy. However, fermentation is one of the oldest food storage/preservation methods throughout the world dating back to the Neolithic period (10,000 years BC) [39, 41]. In biochemical sense, it is the metabolic measure in which complex organic compounds (particularly carbohydrates) are broken down into simpler compounds without the involvement of oxygen (exogenous oxidizing agent) along with the generation of energy (ATP molecules).

The end products of fermentation differ depending on the microorganism. The scientific motivation behind fermentation started with the naming and identification of microorganisms (in 1665) by Leeuwenhoek and Hooke [15]. Thereafter, Pasteur dismantled the “spontaneous generation theory” in 1859 by ideally designed experimentation. However, the performance of an exclusive bacterium Lactococcus lactis in fermented milk was searched out by Sir Joseph Lister in the year 1877 [44]. Generally, microbes in the form of distinct cell or group of cells, usually bacteria, sometimes fungi, algae, or cells of animal or plant origin, are involved in the process of fermentation. Several parameters are associated with the fermentation process, such as concentration of microbial cells and their constituents (enzymes), and conditions like pH, temperature, and fermented medium (aerobic/anaerobic). In general, the microbial fermentation is divided into four categories, viz., (i) generation of biomass (viable cellular material); (ii) production of metabolites; (iii) synthesis of enzymes, vitamins, and proteins; and (iv) transformation/conversion of substrate into value-added products [56]. The current chapter provides an overview of the microorganisms involved in fermentation, growth and kinetics, factors affecting fermentation, bio-products (primary and secondary metabolites) derived from fermentation processes, and uses of probiotics in food fermentation.

1.2 Microbial Diversity: an Overview

There is no universal accord on how to categorize/classify microorganism and such classification is rather arbitrary. However, there are two basic/primary cell types: prokaryotic and eukaryotic. The fundamental difference/characteristic between them is the presence or absence of membrane around the cell’s genetic information.

Prokaryotes are unicellular organism and have simple structure with a single chromosome. Prokaryotic cell lacks membrane-bound organelles (i.e., mitochondria and endoplasmic reticulum) and also lacks nuclear membrane.

Eukaryotes have a more complex internal structure, with more than one chromosome (DNA molecule) in the nucleus. Eukaryotic cells have a true nuclear membrane and hold membrane-bound organelles (i.e., golgi apparatus, mitochondria, endoplasmic reticulum) and a variety of functional/specialized organelles. A detailed contrast/comparison of prokaryotes and eukaryotes is presented in ◘ Table 1.1. Evidence/data recommends that a universal/common ancestor (family history) gave rise to three distinctive/inherent branches of life: Eukaryotes, Eubacteria (or “true bacteria”), and Archaebacteria. ◘ Table 1.2 complies some of the inherent aspects/features of these groups.

Table 1.1 Comparison between Prokaryotes and Eukaryotes [48]

Table 1.2 Primary subdivision of cellular organisms

1.2.1 Prokaryotes

The sizes of maximum or most prokaryotes differ from 0.5 to 3 micrometers (μm) in length (or equivalent radius). Different species have different shapes such as spiral or spirillum (e.g., Rhodospirillum), spherical or coccus (e.g., staphylococci), or cylindrical or bacillus (e.g., Escherichia coli). Prokaryotic cells grow rapidly, with usual doubling times of one-half hour to several hours. Moreover, prokaryotes can use/take advantage of a variety of nutrients as carbon source, including hydrocarbons, carbohydrates, proteins, and carbon dioxide [48].

1.2.1.1 Eubacteria

Eubacteria (genus of gram-positive bacteria in the family Eubacteriaceae) are microscopic single-celled organisms, characterized by rigid cell wall, and grow in diverse environments. The Eubacteria can be divided into several groups. One differentiation is established on the gram stain—the name comes from Danish bacteriologist Hans Christian Gram who developed the technique in 1884. The staining process first requires fixing/mending the cells (by heating), and then crystal violet (basic dye) is added; all bacteria will stain purple. Next, iodine is added (binds crystal violet), followed by the rapid decolorization/addition of ethanol (or acetone). Gram-positive cells stay purple, while gram-negative cells become colorless. Finally, counterstaining with safranin or fuchsine leaves gram-positive cells purple, while gram-negative cells are red. This ability to react with the gram stain differentiates bacteria by the chemical and physical properties of their cell wall (or structure of the cell envelope) [48].

A typical/ideal gram-negative cell is E. coli (◘ Fig. 1.1). It has an outer membrane supported by a thin peptidoglycan (also known as murein) layer. Peptidoglycan is a polymer of sugars (polysaccharide) and amino acids that forms a structure similar to a mesh/chain-link fence/layer. Peptidoglycan provides structural strength as well as resists (opposing action) the osmotic pressure of the cytoplasm. A second membrane (the inner or cytoplasmic membrane) prevails and is detached from the outer membrane by the periplasmic space. The cytoplasmic membrane (bacterial plasma membrane) is composed of phospholipid bilayer (50% protein, 30% lipids, and 20% carbohydrates). The cell envelope (capsule of polysaccharide) serves to protect, reserve/retain important cellular compounds, and in turn exclude/prevent undesirable compounds in to the cell environment. It provides structural/membrane integrity and loss of membrane integrity that leads to cell lysis and cell death. The cell envelope is essential to the transport of preferred material in and out of the cell [30].

Fig. 1.1

A typical/ideal gram-negative cell (E. coli)

A typical gram-positive cell is Bacillus subtilis. Gram-positive cells do not have an outer membrane. Rather they have a very thick, rigid cell wall with multiple layers of peptidoglycan and are made up of glycan strands that are cross-linked by peptide side chains. Gram-positive cells also contain teichoic acids covalently bonded to the peptidoglycan (wall teichoic acids) or are attached to the lipid membrane (lipo teichoic acids). Because gram-positive bacteria have a single cytoplasmic membrane, they are often much better suited/appropriate to extraction of proteins. Such extraction can be technologically advantageous when the protein is a desired product. Spore-forming gram-positive bacteria (B. subtilis) produce morphologically specific daughter cells by asymmetric cell division. The cell wall of spores varies from that of mother cells and has specific sets of proteins [36]. Unlike eukaryotic DNA, which is neatly packed into a cellular compartment called the nucleus (within the nucleus), bacterial DNA floats freely in the cytoplasm, in a twisted thread-like mass called the nucleoid [48].

Actinomycetes are bacteria (generally gram-positive, anaerobic), but, morphologically, they resemble molds with their long filaments and highly branched hyphae. However, the lack of a nuclear membrane and the composition of the cell wall require/appropriate classification as bacteria. Actinomyces, Thermomonospora, Streptomyces, Microbacterium, Bifidobacterium, Brevibacterium, Corynebacterium, Micrococcus, and Brachybacterium are examples of the genera convenient to this group [62]. Actinomycetes are essential sources of antibiotics. Over 500 species of Streptomyces bacteria have been recognized as the builders of abundant bioactive metabolites, such as antibacterials, antivirals, antifungals, and enzyme inhibitors. Certain actinomycetes possess amylolytic [23, 26], pectinolytic [25], and cellulolytic [6] enzymes and are useful in enzymatic hydrolysis of starch, pectin, and cellulose, respectively.

Other/alternative characteristics within the Eubacteria can be made based on cellular nutrition and energy metabolism. One important example is photosynthesis. The cyanobacteria (cyanophyta or blue green algae) obtain their energy through photosynthesis and have chlorophyll, fix CO₂ into sugars, and are able to produce oxygen. Some of the cyanobacteria are nitrogen-fixing (fix atmospheric nitrogen in anaerobic condition) by means of specialized cells called heterocysts.

Prokaryotes may have other visible structures/organizations when observed/viewed under the microscope, such as ribosomes, storage granules, spores, and volutins. Ribosomes are the site of biological protein synthesis (translation). Prokaryotes have 70S ribosomes, are made of 50S and 30S subunits, and are highly complex cellular structures [48].

Storage granules (which are not present in every bacterium) act as reservoir—nutrients can be stored in the cytoplasm. Storage granules are source of basic/key metabolites in the form of polysaccharides (glycogen), polyphosphates, lipids, nitrogen, and sulfur granules. Volutins (polyphosphate bodies) are another granular intracytoplasmic/intracellular energy-rich compound, composed of inorganic polymetaphosphates, present in some species (e.g., Agrobacterium tumefaciens and Rhodospirillum rubrum) [47]. It acts as a reserve store of energy and of phosphate, and, in some cases, nucleic acid, protein, and lipids are associated with these granules. Some photosynthetic bacteria (e.g., Rhodospirillum rubrum) have chromatophores which are large inclusion bodies (pigment-containing and light-reflecting cells) utilized in photosynthesis for the absorption of light [30].

Some bacteria make intracellular spores or endospores. Bacterial endospores (seed like) are dormant, tough, and nonreproductive structures and are produced as a resistance to adverse conditions (i.e., high temperature, radiation, and toxic chemicals). Spores can germinate under favorable growth conditions to yield actively growing bacteria. Certain bacteria (e.g., Streptococcus, Bacillus antracis) have a coating or outside cell wall called capsule, which is usually a polysaccharide (lipopolysaccharides) or polypeptide (lipoproteins). Many bacterial cells secrete some extracellular material in the form of slime layer which protects the bacterial cells from environmental changes (e.g., antibiotics and desiccation). Unlike bacterial capsule, which is attached tightly to the bacterium and has definite boundaries, the slime layer is loosely associated and can be easily washed off [48]. ◘ Table 1.3 summarizes the architecture of most bacteria.

Table 1.3 Characterization of various components in bacteria [48]

1.2.1.2 Archaebacteria

Archaebacteria are similar to eukaryotes, but these cells differ greatly at the molecular level. Archaebacteria and Eubacteria or “true” bacteria both are considered to be part of same kingdom of “single-celled” organisms. However, sophisticated genetic and molecular studies allowed the major biochemical differences between Archaebacteria and Eubacteria. The differences between Archaebacteria and Eubacteria are as follows:

• 1. Archaebacteria are called ancient bacteria, whereas the Eubacteria are called “true” bacteria.

• 2. Eubacteria are usually found in soil and water and living in and on of large organism, whereas Archaebacteria are found in extreme environments and possess unusual metabolism (e.g., salt brines, ocean depths, and hot springs).

• 3. Eubacteria are divided into two groups known as gram-positive and gram-negative; whereas three types of Archaebacteria are found: methanogens (methane-producing bacteria), halophiles (grow at very strong salt solutions), and thermophiles (grow at high temperatures and low pH values).

• 4. Archaebacteria have no peptidoglycan.

• 5. The nucleotide sequences in the rRNA are similar within the Archaebacteria and distinctly different from Eubacteria.

• 6. The lipid composition of the cytoplasmic membrane is very different for the two groups [48].

1.2.1.3 Proteobacteria

Proteobacteria is a major phylum of gram-negative bacteria. Under this phylum, Acetobacter sp. and Gluconacetobacter sp. are the main bacterial groups consisted with nine and eight species, respectively. They are chiefly employed in the making of vinegar but also of significance in the fermentation of coffee and cocoa [46].

1.2.2 Eukaryotes

Fungi (yeasts and molds), algae, protozoa, and animal and plant cells comprise the eukaryotes. Eukaryotes are five to ten times larger than prokaryotes in diameter (e.g., yeast about 5 μm, animal cell about 10 μm, and plant cell about 20 μm). Eukaryotes have a true nucleus (enclosed within membrane) and a number of cellular organelles (e.g., mitochondria, golgi apparatus etc.) inside the cytoplasm [48]. ◘ Figure 1.2 is a schematic representation of two typical eukaryotic cells.

Fig. 1.2

Schematic representation of two typical eukaryotic cells

In cell wall and cell membrane structure, eukaryotes are similar to prokaryotes. The plasma membrane is made of proteins and phospholipids that form a bilayer structure (lipid bilayer with embedded protein). Major proteins for the membrane are hydrophobic and are embedded in the phospholipids (50% of all lipids) matrix. Sterols are essential in all eukaryotic cell/cytoplasmic membranes. One major difference is in the presence of sterols (third major class of membrane lipid after phospholipids and glycolipids) in the plasma/cytoplasmic (plasmalemma) membrane of the eukaryotes. For instance, cholesterol is the major sterol in animal plasma membrane. Sterols increase rigidity and strengthen the membrane structure (reduce membrane fluidity and permeability) and make the membrane less flexible [48].

The cell wall of eukaryotic cells (e.g., algae, plant, fungi) shows considerable variations. Some eukaryotes (e.g., algae) have polysaccharides and cellulose in their cell wall, while others have chitin and other polysaccharides (e.g., fungi). The true fungi do not have cellulose in their cell wall. The plant cell wall is composed of cellulose fibers and is linked via hemicellulose adhere/edge to form the network which is embedded in pectin aggregates. The cellulose-hemicellulose-pectin network imparts strength to the cell wall. Animal cells do not have a cell wall but only a cell/plasma/cytoplasmic membrane. For this logic, animal cells are very shear-sensitive and fragile (delicate) [48]. This aspect significantly complicates/upsets the design of large-scale bioreactor for animal cells [30].

The nucleus of the eukaryotic cells (except mammalian red blood cells) contains chromosomes as nuclear material (DNA-associated proteins), surrounded by a membrane. The nuclear membrane/envelope, a double membrane, consists of concentric and porous (nuclear pores) membranes. The nuclear envelope completely encloses the nucleus and separates the cell’s genetic material from the surrounding cytoplasm. The nuclear pores are required to regulate nuclear transport of the molecules across the envelope. The nucleolus is an area in the nucleus that stains differently and is the site of ribosome and rRNA synthesis. It is not surrounded by a membrane. Nevertheless, many chromosomes have small amounts of RNA and basic proteins called histones adhered to the DNA. Each chromosome contains a single linear DNA molecule on which the histones are attached [48].

The mitochondria are double membrane-bound organelle and are the powerhouses (generate most of the cell’s supply of adenosine triphosphate) of a eukaryotic cell, where respiration and oxidative phosphorylation occur. The mitochondria have a nearly cylindrical (vary considerably in size and structure) shape 1 μm in diameter and 2–3 μm in length. The regular structure of mitochondria is shown in ◘ Fig. 1.3. The external membrane is made of a phospholipid bilayer with proteins embedded in the lipid matrix. It has a protein-to-phospholipids ratio very much alike to that of eukaryotic plasma membrane (approx. 1:1). The mitochondria contain complex system of inner membranes (compartmentalized) called cristae. A gel-like matrix containing large amounts of protein (mixtures of enzymes) fills the space inside the cristae. Some enzymes of oxidative respiration (oxidation of pyruvate, fatty acids, and citric acid cycle) are bound to the cristae. Mitochondria have its own DNA which is organized as several copies of single, usually circular chromosome and have protein-synthesizing system and reproduces independently [48].

Fig. 1.3

Regular structure of mitochondria

The endoplasmic reticulum (ER) is a complex, convoluted/tangled membrane system of interconnected network of flattened, sac- or tubelike structure known as cisternae. There are two types of ER, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER). The RER (granular) contains ribosomes on the inner surfaces (giving a rough appearance) and is the site of protein synthesis and modifications/corrections of protein structure after synthesis. The SER (agranular) is more engaged with lipid, phospholipids, and steroids synthesis.

Lysosomes are very small membrane-bound organelle that contain and release digestive/hydrolytic enzymes. Lysosomes add to the digestion of nutrients and invading substances/biomolecules (e.g., peptides, nucleic acids, carbohydrates, and lipids).

Peroxisomes (also known as microbody) are similar to lysosomes in their structure, but not in function. Peroxisomes contain oxidative enzymes (carry out oxidative reactions) that produce hydrogen peroxide.

Glyoxysomes (specialized peroxisomes found in plants) are also very small membrane-bound particles that contain the enzymes of glyoxylate cycle.

Golgi bodies (golgi apparatus or golgi complex) are endomembrane system in the cytoplasm and are composed of membrane aggregates/combinations engaged for the secretion of certain proteins. Golgi bodies are sites/centers where proteins (glycosylation enzymes) are modified by the addition of various sugars monomers in a process called glycosylation. Such modification/conversion is essential to protein function (responsibility) in the body.

Vacuoles are membrane-bound organelles (filled with cell sap) of low density (have no basic shape or size) and are enclosed by a membrane called tonoplast. Vacuoles are responsible for food digestion, osmotic regulation (hydrostatic pressure or turgor within the cell), and waste product storage. Vacuoles may occupy a large fraction of cell volume (up to 90% in plant cells) and maintain an acidic internal pH.

Chloroplasts (known as plastid) are comparatively large, chlorophyll-containing organelles that are responsible for photosynthesis in photosynthetic eukaryotes (algae and plant cells). Every chloroplast is characterized by its two membranes, an outer membrane and a large number of inner membranes called thylakoids. Chlorophyll molecules are combined with thylakoids, which contain a regular membrane structure with lipid bilayers. Chloroplasts are autonomous units consisting of their own DNA (ctDNA) and protein-synthesizing machinery.

Certain prokaryotes and eukaryotic organisms contain flagella—long, filamentous structures that are connected to one end of the cell and are responsible for the motion of the cell. Eukaryotic flagella contain two central fibers surrounded by 18 (nine fused pairs of microtubule) peripheral fibers, which exist in doublets (so-called “9 + 2” arrangement). Fibers are in a tube structure called microtubule and are composed of proteins called tubulin. The whole fiber assembly is installed/embedded in an organic matrix and is surrounded by a membrane. Cilia are flagella-like structures but are numerous and shorter. Only one group of protozoa, called ciliates (e.g., Paramecium), contains cilia. Eukaryotic cilia are structurally identical to eukaryotic flagella, although differentiations are made according to length and/or functions. Ciliated organisms move much faster than flagellated ones. The cytoskeleton (in eukaryotic cells) refers to filaments that provide an internal framework to organize the cell’s internal activities and control its shape. These filaments are essential in cell movement and separation of chromosomes into the two daughter cells during cell division and transduction of mechanical forces into biological response. The types of fibers present are actin filaments, intermediate filaments, and microtubules.

1.2.2.1 Microscopic Eukaryotes

Fungi are heterotrophs (obtain their food by absorbing dissolved molecules) that are widespread in distribution and grow in a wide range of habitats. Fungal cells (fungal mycelia) are larger than bacterial cells, and their typical internal structures, such as nucleus and vacuoles. The fungal mycelia can become visible to naked eye and can also be seen easily with a light microscope. Two major/dominant groups of fungi are yeasts and molds.

Yeasts are eukaryotic and single-celled microorganism of 5–10 μm size. Yeast cells are usually cylindrical, spherical, or oval. Yeasts can reproduce by asexual or sexual modes. Asexual reproduction (vegetative growth) is by either budding or fission. In budding, a small bud (known as “bleb” or daughter cell) forms on the parent/mother cell; this progressively enlarges/swells and detaches from the mother cell. Asexual reproduction by fission is identical to that of bacteria. Only a few species of yeast (e.g., Saccharomyces pombe) can reproduce by fission. In fission, the cell grows to a definite size and divides into two equal cells. Sexual reproduction of yeasts concerns with the formation of a zygote (a diploid cell) from fusion of two haploid cells, each having a single set of chromosomes. The nucleus of the diploid cells divides several times to form ascospores. Each ascopspore finally becomes a new haploid cell and may reproduce by budding or fission. The life cycle/life process of typical yeast cell is pictured in ◘ Fig. 1.4. The classification (analysis) of yeast is based on reproductive means (e.g., budding or fission) and the nutritional requirement/demand of cells. The most widely used yeast Saccharomyces cerevisiae is one of the main microorganisms of industrial interest such as baking, distilling, and wine making. S. cerevisiae is used in alcohol fermentation/formation under anaerobic or low-oxygen conditions (e.g. in beer, wine, and whisky making) and also for baker’s yeast (leavening agent) production under aerobic conditions, where it converts fermentable sugar (present in dough) into carbon dioxide [48].

Fig. 1.4

Life cycle/life process of typical yeast cell

Molds are filamentous fungi and have a mycelial structure (i.e., network of tubular branching), the highly branched system (mycelium) of tubes that contains cytoplasm with many nuclei. Long and thin multicellular filaments on the mycelium are called hyphae. Certain branches of mycelium may grow in the air, and the asexual spores (or differentiation at the ends of hyphae) formed on these aerial branches are called conidia. Conidia are nearly spherical in structure and are often colored/pigmented. Some molds reproduce by sexual modes and form sexual spores. These spores provide resistance against heat, drying, freezing, and some chemical agents. Both sexual and asexual spores of molds can germinate and form hyphae. ◘ Figure 1.5 describes the structure and asexual reproduction of molds. Molds usually form long, highly branched cells, and easily grow on moist, solid nutrient surface. The growth of molds in the form of pellets can be an alternative in industrial fermentation processes. The growth of mold in submerged culture often forms cell aggregates and pellets. The pellets reach a diameter of 1.0–2.0 mm at the end of fermentation. Pellet formation can cause some nutrient transfer (mainly oxygen) problems inside the pellet. However, pellet formation reduces broth viscosity, which can improve bulk oxygen transfer. Molds are used for the production of citric acid (Aspergillus niger), and the pellet growth is being widely used in the production of citric acid. Several groups of antibiotics such as penicillin are produced by mold (Penicillium notatum-chrysogenum group) [48].

Fig. 1.5

Structure and asexual reproduction of molds

Algae are usually unicellular (eukaryotic) organisms (e.g., chlorella, diatoms). The size of a typical unicellular alga is 10–30 μm. However, multicellular algae (e.g., giant kelp, spirogyra, and stonewort) sometimes form a branched or unbranched filamentous structure. Some algae with multicellular structure are present in marine water (e.g., seaweeds). All algae are photosynthetic and contain chloroplasts (similar in structure to cyanobacteria), which commonly impart/transmit a green color to the organisms. The chloroplasts are the sites/centers of chlorophyll pigments and are important for photosynthesis. Some algae contain calcium carbonate or silica in their cell wall. Diatoms containing silica in their cell wall are used as filter aids/supports in industry. Some algae, such as Chlorella, Spirulina, Scenedesmus, and Dunaliella, are used for wastewater treatment with simultaneous single-cell protein production. Certain gelling agents (e.g., alginic acid or alginate) are obtained from marine/brown algae and seaweeds. Some algae are brown or red due to the occupancy of other pigments [30, 48].

Protozoa are unicellular, motile, either free-living or parasitic, and comparably large (1–50 mm) eukaryotic cells that lack cell walls. Protozoa are normally heterotrophic and obtain food by ingesting other small organisms or organic particles. Protozoa are usually uninucleate and reproduce by sexual or asexual modes. They are classified on the basis of their motion. The amoeba move by amoeboid motion, whereby the cytoplasm of the cell flows forward to form a pseudopodium (false foot), and the rest of the cell flows toward this lobe. The flagellates move using their flagella. Trypanosomes move by flagella and cause a number of diseases in humans. The ciliates move by motion of a large number of small appendages on the cell surface called cilia. These protozoa do not engulf/imbibe food particles, but absorb dissolved food contents through their membranes. The sporozoans are nonmotile and contain members that are human and animal parasites. Protozoa cause some diseases (human pathogens), such as malaria, giardiasis, and amoebic dysentery. Some protozoa are helpful in removing/eliminating bacteria from wastewater in biological wastewater treatment methods and obtaining clean effluent [48].

1.3 Methods of Fermentation

Fermentation has been classified into liquid fermentation (LF) or submerged fermentation (SmF) and solid-state fermentation (SSF) mainly based on the level of water used during the fermentation (◘ Table 1.4).

Table 1.4 Comparison between liquid and solid substrate fermentations

1.3.1 Submerged Fermentation (SmF) or Liquid Fermentation (LF)

SmF exploits/utilizes free-flowing liquid substrates, broths, and molasses. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly and hence need to be constantly replaced/supplemented with nutrients. This fermentation method is suitable for microorganisms such as bacteria that need high moisture content (◘ Table 1.5). An additional choice of this technique/method is that purification/refining of products is easier. SmF is mainly used in the extraction of secondary metabolites that necessitate to be used in liquid form.

Table 1.5 Examples of main groups of microorganisms involved in SSF and SmF processes

1.3.2 Solid-State Fermentation

SSF utilizes solid substrates, like bran, bagasse, and paper pulp. The main interest/advantage of using these substrates is that nutrient-rich waste materials can be easily/efficiently recycled as substrates. In this fermentation method/technique, the same substrate can be used for long fermentation periods and can be utilized very slowly and steadily. Henceforth, this technique supports controlled release of nutrients. SSF is best suited/adapted for fermentation techniques including fungi and microorganisms that depend on limited moisture content [6]. Nevertheless, it cannot be used in fermentation processes involving organisms that require high a_w (water activity), such as bacteria [4].

Bacteria and yeasts are equally involved in SmF and SSF, whereas fungi are mostly concerned with SSF processes. The roles of bacteria and yeasts in SmF are mostly related to food and beverage processing industries and will be discussed in detail in the subsequent chapters. Filamentous fungi are best suited for SSF owing to their physiological, biochemical, and enzymological properties and dominate in oriental foods, ensiling and composting processes [24].

The hyphal means/modes of fungal growth and their good tolerance to low a_w and high osmotic pressure conditions make fungi more efficient for bioconversion of solid substrates. Koji (in Japan) and Tempeh (in Indonesia) are the two most successful applications of SSF using filamentous fungi. In Koji production, the fungus Aspergillus oryzae is grown on soybean and wheat bran, which is the first step of soy sauce and citric acid production. Koji is an unpurified form of several hydrolytic enzymes required in further steps of the fermentation process. Similarly, Tempeh is a fermented food produced by the growth of Rhizopus oligosporus on soybeans. In Tempeh fermentation, the hydrolytic enzymes released by R. oligosporus degrade the anti-nutrients such as trypsin inhibitors and hemagglutinins (lectins) in crude soybean and improve taste, flavor, and nutritive quality. Further, the hyphal mode of growth has an added advantage to filamentous fungi over unicellular microorganisms in colonization of solid substrate and better utilization of nutrients [5]. The basic mode of fungal growth is a combination of apical extension of hyphal tips and the mycelium ensures a firm and solid structure. The hydrolytic enzymes are excreted at the hyphal tip, without large dilution like in the case of SmF.

1.4 Growth Measurement During Fermentation Process

In order to grow successfully, microorganisms must have a supply of water as well as numerous other substances including mineral elements, growth factors, and gas, such as oxygen. Virtually all chemical substances in microorganisms contain carbon in some form, whether they are proteins, fats, carbohydrates, or lipids.

1.4.1 Growth in SmF

Microbial growth is defined as the increase in all chemical components in the presence of suitable medium and the culture environment. Growth of the cell mass or cell number can be described quantitatively as a doubling of cell number per unit time for bacteria and yeast, or a doubling of biomass per unit time for filamentous organisms such as fungi. After the inoculation of a sterile nutrient solution with microorganisms and cultivation under physiological conditions, four typical phases of growth are observed as indicated in the ◘ Fig. 1.6. Growth is the result of consumption of nutrients.

Fig. 1.6

Four typical phases of microbial growth

The cells consume a part of the substrate/reactant and essential nutrients from the medium of fermentation. The cells initially multiply and grow. Depending on the type of cells, whether they are unicellular or molds, the growth pattern varies. For example, the unicellular organisms, which divide when they grow, will increase the number of cells or increase the biomass. As they increase in number, they consume more and more of substrate. The other types of cell (e.g., mold) will not increase in number, but they increase in size which also results in increase in viscosity of the broth.

Numerous procedures in biology require cells to be counted. By counting the cells in a known volume of a culture, the concentration can be assessed. For example, the concentration of microorganisms/cell number (e.g., bacteria, virus, and other pathogens) in blood or body fluids can reveal about the progress of an infectious disease and/or about a person’s immune system. Knowing the cell concentration is important in molecular biology experiments in order to adjust the amount of reagents and chemicals applied to the experiment.

The various methods of measuring microbial growth are discussed below.

1.4.1.1 Plate Count

A viable cell count (number of colony-forming bacteria) allows one to identify the number of actively dividing/growing cells in a liquid sample. In this method, fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of sterile Petri dish (containing nutrient medium) using a sterile pipette (◘ Fig. 1.7). The bacteria grow as a colony (i.e., cluster of cells or clones which arise from single bacterium by asexual reproduction) on a nutrient medium. When sample is plated, each colony that grows represents a single cell or spore in the original sample. The colony becomes visible to the naked eye and the number of colonies (colony count multiplied by the dilution factor) on a plate can be counted.

Fig. 1.7

Plate count of viable colony-forming bacteria

1.4.1.2 Serial Dilution

A serial dilution is the series of sequential dilution of a substance in solution. Each dilution will reduce the concentration of sample by a specific amount to a more suitable (usable) concentration. The first step in making a serial dilution is to take a known volume (usually 1 ml) of stock and place it into a known volume of distilled water (usually 9 ml) to make a ten-fold (0.1 M) serial dilution (◘ Fig. 1.8). The progressive tenfold serial dilution could be 0.01 M, 0.001 M, 0.0001 M, etc. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.

Fig. 1.8

Serial dilution/series of sequential dilution of stock solution

A tenfold dilution reduces the concentration of a solution or a suspension by a factor of 10, that is, one-tenth the original concentration. A series of tenfold dilutions is described as tenfold serial dilutions. They are carried out in small sterile test tubes and are usually made of glass, and it is preferable if they have fitted lids to minimize the risk of contamination during the dilution.

The dilution factor is the number which multiplies the final concentration to get actual concentration (cell/ml) and thus decreases the final concentration of stock solution (◘ Fig. 1.9).

Fig. 1.9

Graph representing optical density (O.D.) with dilution factor

1.4.1.3 Most Probable Number Method

The MPN method is a method used to estimate the concentration of viable microorganism in a sample by means of replicating liquid broth growth in tenfold or twofold (10× or 2×) dilutions. MPN analysis is a statistical method based on random dispersion of microorganisms per volume in the given sample (◘ Fig. 1.10). In this method, measured volumes of water are added to a series of tube containing a liquid indicator growth medium. The media receiving one or more indicator bacteria show growth and characteristic color change. The color change is absent in those receiving an inoculums of water without indicator bacteria. It is commonly used in estimating microbial populations in soils, waters, agricultural products and is particularly useful with samples that contain particulate material that interferes with plate count enumeration methods.

Fig. 1.10

Most probable number (MPN) method of microorganisms per volume of sample

1.4.1.4 Optical Density

The optical density/spectrophotometer is usually taken to measure the concentration of growing bacteria. This method is completely based on Lambert–Beer’s law. Optical density is directly proportional to the biomass in the cell suspension in a given range that is specific to the cell type. Cell suspensions/cell concentrations are turbid and absorb and scatter the intensity of light. The higher the cell concentration, the higher is the turbidity. The cell culture is placed in a transparent cuvette and the absorbance is measured relative to medium alone.

The Lambert–Beer Law

When a ray of monochromatic light of initial intensity (I_o) passes through a solution in a transparent vessel, some of the light is absorbed so that intensity of transmitted light “I” is less than “I_o”. There is some loss of light intensity from scattering by particles in the solution and refection at the interfaces.

The absorbance of an electronic transition depends on two external factors:

• 1. The absorbance is directly proportional to the concentration (C) of the solution of the sample used in the experiment.

$$ \mathrm{A}\propto C $$

• 2. The absorbance is directly proportional to the length of light path (l), which is equal to the width of the cuvette.

$$ \mathrm{A}\propto l $$

Combing the two relationships,

$$ \mathrm{A}=\varepsilon C\ l $$

This proportionality can be converted into an equation by including a constant.

This formula is known as the Beer–Lambert law (◘ Fig. 1.11), and the constant ε is called molar absorptivity or molar extinction coefficient and is a measure of the probability of the electronic transition. The larger the molar absorptivity, the more probable is the electronic transition. In UV spectroscopy, the concentration of the sample solution is measured in mol/L and the light path in cm. Thus, given that absorbance is unit-less, the units of molar absorptivity are L/mol/cm.

Fig. 1.11

The Beer-Lambert law represents A = εC l; A, absorbance; constant ε is called molar absorptivity or molar extinction coefficient; C, concentration of the solution; l, light path (l), which is equal to the width of the cuvette

Turbidimetry is an established method used to study bacterial growth (◘ Fig. 1.12) since optical density measurements make it possible to follow bacterial population growth in real time. The electronic counting chambers or Coulter counter numbers are used to measure size distribution of cells. Turbidity measurement is done by estimations of large number of bacteria in clear liquid media and broth.

Fig. 1.12

Turbidimetry method (optical density with duration of incubation) of bacterial growth (Aeromonas sp., Klebsiella sp., Pseudomonas sp.,)

1.4.1.5 Kinetic Models for Cell Growth

It is generally believed that the growth rate of the cells at any time in the growth phase is proportional to the number of cells present at the time, which is popularly described by Malthus’ law [63].

$$ \frac{dx}{dt}=\mu x,\kern1.625em x={x}_o\kern0.5em at\kern0.5em t=o $$

(1.1)

where x is the mass of the cells per unit volume, μ is the proportionality constant known as specific growth rate in (hour)⁻¹, and t is the time in hours.

On integration, we get

$$ In\frac{x}{x_o}=\mu t\kern0.5em \mathrm{or}\kern0.5em x={x}_o{e}^{\mu t} $$

(1.2)

$$ In\ x= In\ {x}_o+\mu t $$

(1.3)

The time required to double the microbial mass is given by Eq. (1.2). The exponential growth is characterized by a straight line on a semi-logarithm plot of In x versus time.

$$ {\tau}_{\mathrm{d}}= In\frac{2}{\mu }=\frac{0.693}{\mu } $$

(1.4)

Where τ_d is the doubling time of cell mass [40].

1.4.1.5.1 Monod Model

The growth phase passes through various phases, viz., high growth phase, low growth phase, and finally cessation. In other words, the specific growth rate varies with the residual concentration of the limiting substrate. The relation is well explained by an empirical equation proposed by Monod (1949) [34].

$$ \mu =\frac{\mu_{\mathrm{m}}S}{k_{\mathrm{s}}+S} $$

(1.5)

where μ_m is the maximum growth rate achievable and k_s is the limiting substrate concentration when specific growth rate is equal to half the maximum specific growth rate, i.e., μ = μ_m/2.

This is evident by substituting μ_m/2 for μ in Eq. (1.5), i.e.,

$$ \frac{\mu_{\mathrm{m}}}{2}=\frac{\mu_{\mathrm{m}}S}{k_{\mathrm{s}}+S} $$

(1.6)

By cancelling μ_m on both sides and on arrangement, k_s = S.

In Eq. (1.5), if S >  > k_s, the denominator may be equated to simply S.

$$ \mu =\frac{\mu_{\mathrm{m}}S}{k_{\mathrm{s}}+S}=\frac{\mu_{\mathrm{m}}S}{S}={\mu}_{\mathrm{m}} $$

(1.7)

In other words, the above equation predicts the percentage in μ_m as compared to μ, when S is very large [40].

Example 1.1

A particular organism follows kinetics growth equation (Monod 1949). The kinetic parameter μ_m = 0.5 h⁻¹, k_s = 2 g/L and S = 3.5 g/L. Determine the value of μ.

Ans: $\mu =\frac{0.5\times 3.5}{2+3.5}=\frac{1.75}{5.5}=0.318\kern0.28em {\mathrm{h}}^{-1}$

1.4.2 Growth in Solid-State Fermentation

Direct determination of biomass in SSF is very difficult due to the problem of separating the microbial biomass from the substrate. This is especially true for SSF processes involving fungi, because the fungal hyphae penetrate into and bind tightly to the substrate. On the other hand, for the calculation of growth rates and yields, it is the absolute amount of microbial biomass which is important. However, in the case of bacteria and yeasts, the microbial population (colony-forming units) can be calculated by serially diluting the fermented mash and pour plating on specific culture medium. Desgranges et al. [13] and Terebiznik and Pilosof [55] have outlined the following methods for biomass estimation of fungi (also applicable to bacteria) in SSF.

1.4.2.1 Metabolic Measurement of Biomass

1.4.2.1.1 Respiratory Metabolism

Oxygen (O₂) consumption and carbon dioxide (CO₂) evolution resulting from respiration are the metabolic processes by which aerobic microorganisms derive most of their energy for growth. Carbon compounds within the substrate are metabolized, which are converted into microbial biomass and carbon dioxide. Production of CO₂ causes the weight of fermenting substrate to decrease during growth, and the amount of weight lost can be correlated to the amount of growth that has occurred. The measurement of either CO₂ evolution or O₂ consumption is more powerful when coupled with the use of a correlation model. If both the monitoring and computational equipment are available then these correlation models provide a powerful means of biomass estimation since continuous on-line measurements can be made. Other advantages of monitoring effluent gas concentrations with paramagnetic and infrared analyzers include the ability to monitor the respiratory quotient to ensure optimal substrate oxidation, the ability to incorporate automated feedback control over the aeration rate, and the nondestructive nature of the measurement procedure.

1.4.2.1.2 Production of Primary Metabolites

Usually, a growing cell will use its primary metabolites for growth and not export them to the medium. However, in fermentation processes designed for production of primary metabolites, i.e., amino acids, enzymes, organic acids, or nucleotides, their production is often growth-related. It has been frequently observed that there is a good correlation between mycelial growth and organic acid production, which can be measured by the pH analysis or a posteriori correlated by HPLC analysis on extracts. In the case of Rhizopus, Sauer et al. [45] demonstrated a close correlation between fungal protein (biomass) and organic acids (citric, fumaric, lactic, or acetic). However, the ratio of product formed per unit cell mass is not necessarily constant and as a consequence correlation with cell mass is poor.

1.4.2.1.3 Biomass Components

1.4.2.1.3.1 Protein Content

The most readily measured biomass component is protein. The ^*biuret method is easy, with good reproducibility since it measures peptide bonds, but it is not very sensitive. ^$Kjeldahl nitrogen when multiplied by 6.25 to obtain crude protein is subject to error from nonprotein nitrogen. The Folin–Ciocalteu method (^**Lowry’s method) is more sensitive, but since it responds strongly to the aromatic amino acids, it can give an erroneous value unless samples and reference protein are similar in composition [20].

[^$Kjeldahl method or Kjeldahl digestion in analytical chemistry is a method for the quantitative determination of nitrogen contained in organic substances plus the nitrogen contained in the inorganic compounds ammonia and ammonium (NH₃/NH₄⁺)].

[*Biuret method: The chemical text used to assess the concentration of protein (i.e., the presence of peptide bonds). The intensity of color or absorbance measured at 540 nm is directly proportional to the protein concentration (according to the Lambert–Beer law).]

[^**Lowry’s method: The biochemical assay for determining the total level of protein in a solution. The intensity of color or absorbance measured at 600 nm is directly proportional to the protein concentration.]

1.4.2.1.3.2 Nucleic Acid

Nucleic acid methods based on DNA or RNA determination are reliable only if there is little nucleic acid in the substrate and if no interfering chemicals are present.

1.4.2.1.3.3 Glucosamine

A useful method for the estimation of fungal biomass in SSF is the glucosamine method. This method takes advantage of the presence of chitin in the cell walls of many fungi. Chitin is a poly-N-acetylglucosamine. Interference with this method may occur with growth on complex agricultural substrates containing glucosamine in glycoproteins.

1.4.2.1.3.4 Ergosterol

Ergosterol is the predominant sterol in fungi. Glucosamine estimation was therefore compared with the estimation of ergosterol for determination of the growth of Agaricus bisporus.

1.5 Factors Affecting Microbial Growth in Fermentation

The growth of microbes is influenced by various internal and external factors, viz., pH, temperature, composition of the media, etc.

1.5.1 Temperature

Microorganisms need optimum temperature for growth. If grown at a temperature below the optimum, growth occurs slowly resulting in a reduced rate of cellular production (◘ Fig. 1.13). The bacteria can be divided into following groups based on their optimum growth temperature.

Fig. 1.13

Bacterial growth (growth rate vs. temperature in °C) based on temperature (thermophiles, hyperthermophiles, mesophiles, and psychrophile)

Thermophiles are heat-loving bacteria (i.e., Thermus aquaticus, Thermococcus litoralis, and Bacillus stearothermophilus). The optimum growth temperature is between 45 and 70 °C and is commonly found in hot springs and in compost heaps. Hyperthermophiles are bacteria that grow at very high temperatures (i.e., genus Sulfolobus). Their optimum temperature is between 70 and110 °C. They are usually members of the Archaea and are found growing near hydrothermal vents of great depths in the ocean (◘ Fig. 1.14b, d).

Fig. 1.14

Members of the Archae found near hydrothermal vents of great depths in the ocean (b, d) and Arctic and Antarctic regions (a, c) in the streams fed by glaciers

Mesophiles are bacteria that grow best at moderate temperatures (i.e., Pesudomonas maltophilia, Thiobacillus novellus, Streptococcus pyrogenes, Streptococcus pneumoniae, Clostridium kluyveri etc). Their optimum growth temperature is between 25 and 45 °C. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body.

Phychrophiles are cold-loving bacteria (i.e., Arthrobacter sp., Psychrobacter sp.). Their optimum growth temperature is between −5 to 15 °C. They are usually found in the Arctic (◘ Fig. 1.14a, c) and Antarctic regions and in the streams fed by glaciers. These bacteria are of little importance in fermentation.

1.5.2 pH

Most microorganisms grow optimally between pH 5 and 7 (fungi and yeast grow in acidic conditions) (◘ Fig. 1.15). Microorganisms can be placed in one of the following groups based on their optimum pH requirements. Neutrophiles grow best at a pH range of 5 and 8. Acidophiles grow best at a pH below 5.5. Alkaliphiles grow best at a pH above 8.5.

Fig. 1.15

Microorganisms growing in different pH range

1.5.3 Oxygen

Oxygen (aeration) is an important factor for aerobic organisms and is very strongly related to growth rate. Optimum growth of many microorganisms usually requires large amounts of dissolved oxygen. As oxygen is sparingly soluble in water (8.4 mg/L at 25 °C), it needs to be supplied continuously (generally in the form of sterilized air) to a growing culture. The air produces bubbles and the stirrer is used to break up the bubbles and mix content in fermentation. If airflow is inadequate or the air bubbles are too large, the rate of transfer of oxygen to the cells is low and is insufficient to meet oxygen demand.

1.5.4 Constituents of Growth Medium

1.5.4.1 Macro- and Microelements

All microorganisms require certain elements for growth and metabolism. Macro-elements are needed in concentrations larger than 10⁻⁴ M. Carbon, nitrogen, oxygen, hydrogen, sulfur, phosphorus, Mg²⁺, and K⁺ are major elements. Carbon compounds in growth medium are major sources of cellular carbon and energy. However, micro-elements are needed in concentration less than 10⁻⁴ M. Trace elements such as Mo²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Ca²⁺, Na⁺ and vitamins, growth hormones, and metabolic precursors are micro-elements.

1.5.4.2 C:N Ratio of Growth Medium

Carbon-to-nitrogen ratio (C/N ratio or C:N ratio) is a ratio of the mass of carbon to the mass of nitrogen in a substrate. The microorganisms decomposing organic matter with a higher C:N ratio are confronted with a surplus of C in relation to N and microorganisms confronted with a lower C:N ratio are facing a lack of C in relation to N [16]. The most common carbon sources in industrial fermentations are molasses (sucrose), starch (glucose, dextrin), corn syrup, and waste sulfite liquor (glucose). In laboratory fermentation, glucose, sucrose, and fructose are the most common carbon sources. Methanol, ethanol, and methane also constitute cheap carbon sources for the fermentation. Most industrially used microorganisms can utilize inorganic or organic sources of nitrogen. Inorganic nitrogen may be supplied as ammonia gas, ammonium salts, or nitrates. Ammonia has been used for pH control and as the major nitrogen source in a defined medium [52]. Ammonium salts such as ammonium sulfate usually produce acid conditions in the growth medium. However, ammonium nitrates normally cause an alkaline drift (provide basic environment) as they are metabolized.

1.5.4.3 Growth Factors

Some microorganisms cannot synthesize a full complement of cell components and therefore require preformed compounds called growth factors. Growth factors stimulate the growth and synthesis of some metabolites. Vitamins, hormones, and amino acids are the major growth factors. Some commonly used vitamins are thiamine (B₁), riboflavin (B₂), pyridoxine (B₆), biotin, cyanocobalamine (B₁₂), folic acid, lipoic acid, p-amino benzoic acid, and vitamin K. The growth factors most commonly required are vitamins, but there may also be a need for specific amino acids, fatty acids, or sterols. Depending on the organism, some or all of the amino acid may need to be supplied externally. Some fatty acids, such as oleic acids and sterols, are also needed in small quantities by some organisms. Many natural media formulations contain all or some of the required growth factors. For example, in processes used for the production of glutamic acid, limited concentrations of biotin must be present in the medium. Some production strains may also require thiamine (B₁).

1.6 Starter Culture(s)

1.6.1 Concept of Starter Cultures

A starter culture may be defined as a preparation containing large numbers of desired microorganisms, used for accelerating the fermentation process. The preparations may contain some unavoidable residues from the culture substrates and additives (such as antifreeze or antioxidant compounds), which support the vitality and technological functionality of the microorganisms. A typical starter after being adapted to the substrate facilitates improved control of a fermentation process and predictability of its products [18, 41]. Basically there are three categories of starter cultures: (1) single-strain cultures, contain only one strain of a species; (2) multi-strain cultures, contain more than one strain of a single species; and (3) multi-strain mixed cultures, contain different strains from different species [58].

1.6.2 History and Subsequent Development of Starter Culture

Microorganisms are naturally omnipresent and hence observed in raw substrates. This was the basis of the idea of spontaneous fermentation. Backslopping was the important technological phenomenon used in spontaneous fermentation by inoculating the raw material with a small quantity of a previously performed successful fermentation. Hence, the dominance of the best adapted strains resulted in backslopping. This technology is still used for production of foods and beverages where the ecology and concrete knowledge about microbial population and role are not clearly known. This is also an economical and reliable method of production of fermented foods.

1.6.2.1 Recombinant Starter Culture

Recombinant technology and genetic manipulation offer extensive possibilities to incorporate new traits into organisms and have positive applications in food and pharmaceutical industries. The recombinant Lactococcus is starter strains to produced peptidases from Lactobacillus helveticus 53/7 for potential use as an additional enzyme supply for accelerated proteolysis during cheese ripening [22]. It is possible to increase the production of expolysaccharides (EPS) by Streptococcus thermophilus strains through genetic engineering of galactose metabolism. In situ production of EPS by starter sutures (S. thermophilus) is an alternative to the addition of biothickeners of fat-free yogurts [42]. Bohmer et al. [8] reported both LABs (Lactobacillus plantarum NC8 and Lactobacillus casei) as potential expression hosts for recombinant enzyme [hyperthermophilic β-glycosidase (CelB)] production.

1.7 Facets of Fermentation

Fermentation process is used by human beings for various processes from industrial applications to food and agriculture, bioremediation of organic wastes to biovalorization of food and agricultural wastes, etc.

1.7.1 Industrial Fermentation

Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make a product useful to humans.

1.7.1.1 Production of Biomass

Microbial cells or biomass is sometimes the intended product of fermentation, for example, single-cell protein (SCP), baker’s yeast, lactobacillus, etc. SCP refers to edible unicellular microorganism. The biomass (protein extract) from pure or mixed cultures of algae, yeasts, fungi, or bacteria may be used as an ingredient or a substitute for protein-rich foods and is suitable for human consumption or as animal feeds. Baker’s yeast (Saccharomyces cerevisiae) is commonly used as a leavening agent in baking bread and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol.

1.7.1.2 Production of Extracellular Metabolites

Metabolites can be divided into two groups: primary metabolites (ethanol, citric acid, glutamic acid, lysine, vitamins, and polysaccharides) and secondary metabolites (penicillin, cyclosporin-A, gibberellins, and lovastatin) (described earlier).

1.7.1.3 Production of Intracellular Components

The intracellular components are several microbial enzymes, such as catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicelluase, lipase, lactase, streptokinase, etc. Moreover, the industrially synthesized recombinant proteins, such as insulin, hepatitis B vaccine, interferon, and granulocyte colony-stimulating factor, play an important role in health care.

1.7.2 Food Fermentation

Fermentation of food is the ancient process of making bread, curds, idli, dosa, cheese, etc., from thousand years ago. However, similar process is employed specifically to the conversion of sugars into ethanol producing alcoholic drinks such as wine, beer, and cider and in the preservation of sour foods with the production of lactic acid such as in sauerkraut and yogurt. More localized foods prepared by fermentation may also be based on beans, grain, vegetables, fruit, honey, dairy products, fish, meat, or tea [7].

1.7.3 Biofuels

Fermentation is the main source of ethanol in the production of ethanol fuel (i.e., ethyl alcohol) and butanol (butyl alcohol). Common crops such as sugarcane, potato, cassava, and corn are fermented by yeast to produce ethanol and are further processed to produce motor fuel or biofuel additive to gasoline.

1.7.4 Sewage Treatment

Sewage treatment (also called “wastewater treatment”) is the process of removing contaminants from municipal wastewater (containing mainly household sewage plus some industrial wastewater). In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria.

1.7.5 Animal Feeds

A wide variety of agricultural waste products can be fermented to use as food for animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility.

1.8 Representative Metabolites Produced by Microorganisms

Microbial metabolites represent an incredibly diverse array of chemistry. Microbes can make molecules that synthetic chemists cannot access. While over 25,000 microbial metabolites have been reported in the scientific literature, fewer than 2% of these have ever been readily available to the wider research community. Most metabolites have only ever existed in small quantities in the research laboratory in which they were discovered, and their biological activity has never been fully investigated. Representative metabolites produced by various microbial species are shown in ◘ Table 1.6.

Table 1.6 Representative metabolites produced by different groups of microorganisms

1.8.1 Primary Metabolites

Primary metabolites are vital molecules (e.g., amino acids, vitamins, nucleotides, solvents, or organic acids) of all living cells and are the end product of metabolic pathways or their intermediate products or are domicile of essential macromolecules or are converted into coenzymes.

1.8.1.1 Amino Acids

The amino acid market is over $6 billion and has been growing at 5–10% for each year [9]. For instance, monosodium glutamate, an effective flavor enhancer, is a leading amino acid in terms of tonnage. Various species of the genera Corynebacterium and Brevibacterium (e.g., Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum) are claimed to be producers of glutamate.

1.8.1.2 Nucleotides and Nucleosides

The nucleotide/nucleoside fermentations are due to the activity of two purine ribonucleoside 5′-monophosphates, [i.e, guanylic acid (5′-GMP) and inosinic acid (5′-IMP)]. Three primary processes are involved: (i) hydrolysis and enzymatic deamination of yeast RNA to IMP, (ii) synthesis and phosphorylation of the nucleoside (inosine and guanosine) by Bacillus subtilis mutants, and (iii) direct fermentation of sugar to IMP (by Corynebacterium glutamicum mutants) and conversion of guanine to GMP (using intact cells of Brevibacterium ammoniagenes). The intracellular AMP and GMP are limited for effective accumulation of purine nucleotides and are mostly explained by control feeding of purine auxotrophs [35], for instance, adenine-needing mutants deficient of nucleotide degrading enzymes (e.g., adenylosuccinate synthetase), which caused accumulation of inosine or hypoxanthine, which resulted from breakdown of intracellular accumulated IMP.

1.8.1.3 Vitamins

Several microbial strains are responsible for the production of vitamins or vitamin-like compounds (e.g., β-carotene, vitamin B12, riboflavin, vitamin C, linolenic acid, vitamin F, and ergosterol) [51]. Riboflavin (vitamin B2) has been produced commercially for many years by both fermentation and chemical synthesis; however, fermentation is the major route today. For instance, two yeastlike molds, Eremothecium ashbyii and Ashbya gossypii, were found to synthesize riboflavin in concentrations more than 20 g/L. Vitamin B12 (Cyanocobalamin) is formed industrially with Propionibacterium shermanii and Pseudomonas denitrificans [49]. The overproduction of vitamin B12 is absolutely dependent upon addition of betaine (the mechanism of control is unknown). The P. freudenreicheii can produce 206 mg/L of vitamin B12 and is not still a dominant industrial producing organism.

1.8.1.4 Organic Acids

Microbes have been widely used for the commercial production of organic acids (e.g., citric acid, acetic acid, lactic acid, gluconic acid, pyruvic acid, malic acid, tartaric acid, succinic acid, and itaconic acid). Among different types of organic acids, citric acid (CA) is easily palatable, assimilated, and has low toxicity. Therefore, it is extensively used (about15%) in the pharmaceutical and food industry (as an antioxidant, acidifying and flavor-enhancing agent). The CA is produced via the Embden–Meyerhof pathway (EMP) and the first step of the tricarboxylic acid (TCA) cycle. Harmsen et al. [17] reported that a high level of CA production is also linked with an increased level of fructose 2,6-biphosphate (an activator of glycolysis).

Vinegar production is best carried out with species of Gluconacetobacter sp. and Acetobacter sp. [61]. However, vinegar is consisting of about 5–20% acetic acid (AA). Fermentation has virtually waived chemical synthesis of lactic acid (LA). The LA is produced anaerobically with a 95% (w/w) yield based on carbohydrate, a titer of over 100 g/L, and a productivity of over 2 g/L. This is comparable to processes employing LA bacteria. Lactobacilli produce mixed isomers, whereas Rhizopus forms L -(+)- LA exclusively. Rhizopus oryzae is favored for formation since it makes only the stereochemically pure L -(+)-LA. Itaconic acid is used as a comonomer in resins and synthetic fibers and is produced from the selective fungal (i.e., Candida) sp. [61]. Although microbial processes exist for the other acids, they have not been exploited commercially on a large scale. Succinic acid can be made from the rumen organism Actinobacillus succinogenes at 110 g/L [64]. Pyruvic acid is formed at 69 g/L at 56 h with a yield of 0.62 g/g glucose using Torulopsis glabrata [29].

1.8.1.5 Alcohols

Ethyl alcohol (C₂H₅OH) is a primary metabolite that can be formed from fermentation of a carbohydrate/sugar or a polysaccharide that can be depolymerized to a fermentable sugar. Yeasts are preferred for these fermentations, but the species used depends on the substrate employed. Saccharomyces cerevisiae is used for the fermentation of hexose, whereas Candida sp. or Kluyveromyces fragilis may be employed if pentose or lactose, respectively, is the substrate. Ethanol is produced in Brazil from cane sugar at 12.5 billion liters/year and is used as a 25% fuel blend or as a pure fuel. With regard to beverage ethanol, some 60 million tons of beer and 30 million tons of wine are produced each year. Production of glycerol is usually done by chemical synthesis from petroleum feedstocks, but better fermentations processes are available [60]. Osmotolerant yeast strains (Candida glycerinogenes) can produce up to 130 g/L of glycerol. Six hundred thousand tons of glycerol are produced annually by recovery as a by-product of the fat and oil industries, by synthesis from propylene, and, to a small extent, by glucose fermentation using Saccharomyces cerevisiae [54].

1.8.2 Secondary Metabolites

Microbial secondary metabolites are compounds produced by strains of certain microbial species (mainly by actinomycetes and fungi), usually late in the growth cycle (idiophase). The production of secondary metabolites starts when growth is limited with exhaustion of one key nutrient source (i.e., carbon, nitrogen, or phosphate). For example, penicillin biosynthesis by Penicillium chryosgenum starts when glucose is exhausted from the culture medium and the fungus starts consuming lactose, a less readily utilized glucose [57]. A characteristic of secondary metabolites is that they are usually not produced during the phase of rapid growth (trophophase) but are synthesized during a subsequent production stage (idiophase). The difference/comparison between primary and secondary metabolites is given in ◘ Fig. 1.16 and ◘ Table 1.7. The secondary metabolites have an enormous range of biological activities and are extremely important to our health and nutrition [10]. A group that includes antibiotics, toxins, pesticides, animal and plant growth factors, and other medicines has tremendous economic importance [11].

Fig. 1.16

The difference/comparison between primary and secondary metabolites

Table 1.7 Differences between primary and secondary metabolites

1.8.2.1 Antibiotics

The best known/recognized secondary metabolites are antibiotics (antimicrobial agent). The antibiotic revolutionized medicine in twentieth century and its market involves about 160 antibiotics and derivatives, (e.g., β-lactam peptide antibiotics, the macrolide polyketides and polyketides, tetracyclines, aminoglycosides, and others). The anti-infective market is framed of 62% antibacterials, 13% sera immunoglobulins and vaccines, 12% anti-HIV antivirals, 7% antifungals, and 6% non-HIV antivirals. Titers of penicillin with Penicillium chrysogenum have reached 70 g/L, whereas those of Cephalosporin-C by Acremonium chrysogenum are over 30 g/L. Published data on clavulanic acid production by Streptomyces clavuligerus indicate the titer to be above 3 g/L [21, 38].

1.8.2.2 Antitumor Agents

Ever since the discovery/origination of the Actinomycins by Waksman and Woodruff [59] and the use of Actinomycin-D against the Wilms tumor in children, microbes have served as a prime source of anticancer agents. The essential microbial molecules are mitomycin-C, bleomycin, daunorubicin, doxorubicin, etoposide, and calicheamicin, all are produced by Actinomycetes. Taxol (paclitaxel) is a very productive compound against ovarian and breast cancer, produced by the endophytic fungi [1]. Another plant product is camptothecin (CPT), produced by certain angiosperms, which is a modified monoterpene indole alkaloid and is active against type I DNA topoisomerase. Its water soluble derivatives irinotecan and topotecan are used against cancer. It also can be made by endophytic fungi.

1.9 Concluding Remarks

A wide variety of organic chemicals like enzymes, amino acids, and antibiotics can be formed by fermentation. Fermentation is a metabolic process that converts organic substrates (mainly carbohydrates) into useful products (organic acids, gases, or alcohol) using diverse groups of microbes [bacteria, fungi (yeasts and mold)]. Most microorganisms used in commercial fermentation need disaccharides or six-carbon sugars as substrates, even though the microbial world contains organism that can break down essentially any organic compound. There has been a significant improvement in industrial products by strain selection/manipulation when related with the processes that were used in the first half of the twentieth century. However, further studies should aim to gather knowledge concerning several beneficial/microbial strains and processes involved in the formation of high-value fermented products.

Take-Home Messages

• Presence (in the case of eukaryotes) or absence (in the case of prokaryotes) of membrane around the cell’s genetic materials is the fundamental difference between eukaryotes and prokaryotes.

• Gram staining differentiates bacteria into two groups, Gram +ve or Gram –ve, based on chemical and physical properties of their cell walls.

• Water level or water activity is the main difference between solid and submerged fermentation.

• Microorganisms have a supply of water as well as numerous other substances including mineral elements, growth factors, and gas such as oxygen to grow.

• Various methods of measuring microbial growth are plate count, serial dilution, most probable number (MPN), and optical density/spectrophotometer method.

• Monod equation can be used to measure microbial growth of microorganisms.

• Metabolic measurement of biomass includes respiratory metabolism, production of primary metabolites, biomass components of protein content, DNA or RNA components, and glucosamine method for the estimation of fungal biomass in solid- state fermentation.

• The growth of microbes is influenced by various internal and external factors, viz., pH, temperature, composition of the media, etc.

• In spontaneous fermentation, there is no control over the fermentation process and is mediated by natural microflora present on the surface of the substrate(s), whereas in controlled fermentation, the fermentation process is optimized for pH, temperature, incubation time and is mediated by specific microorganism or a group of microorganisms (starter culture).