Fermentation: A Potential Strategy for Microbial Metabolite Production

Abstract

Fermentation is derived from a Latin word fermentum, a process of chemical changes in an organic substrate by the action of microbial enzymes. The science of fermentation is called as zymology, and the first zymologist was Louis Pasteur. Study of fermentation is called as fermentation technology. Fermenter or bioreactor is the heart of fermentation technology where the whole study is involved. It is a container with biomechanical and biochemical environment that controls the transfer of oxygen, nutrient to the cells, and metabolic products from the cells. There are different types of fermenters like stirred tank fermenter, airlift fermenter, bubble column reactor, fluidized-bed reactor, packed bed reactor, and membrane bioreactor. There are different processes such as batch, continuous, and fed batch or semi-continuous. Secondary metabolites are produced either through submerged or solid-state fermentation process during the stationary phase of an organism. These metabolites are showing a rising demand in food, cosmetics, drugs, and other industries. The production of these metabolites can be improved by strain improvement through mutagenesis and r-DNA technologies. This chapter focuses on all these areas in detail.

1. Introduction

The fermentation is respiration without air. The word fermentation is a Latin word fermentum. The science of fermentation is called zymology, and Louis Pasteur is considered to be the first zymologist [1]. The process of fermentation is known from ancient years, and also, it is a biological method for food preservation and also gives good organoleptic properties in the final product [2].

Fermentation is used since olden days to preserve and modify foods. For many years fermentation techniques were used without knowing the microbial involvement and mechanisms. Greeks attribute fermentation to one of the gods-Dionysos-a god of fruit fermentations. The first fermentations included the production of beer and fermented milk beverages in 3150 B.C at Babylon, soy sauce in 1000 B.C at China [1]. Fermentation is the natural process in which various microorganisms like yeast, bacteria, and fungi are involved in the exchange of complex substrates into simpler compounds. These compounds are useful to humans on industrial scale [3]. There are different types of fermentation like batch, continuous and fed-batch fermentation, solid-state fermentation, and submerged fermentation. In the presence of oxygen, mixing of substrate takes place in submerged fermentation [4].

Bioreactors are the heart of fermentation technology. Bioreactors are tanks or vessels where the cells or cell-free enzymes convert raw materials into biochemical products [5]. Bioreactors are simple or highly instrumental. It consists of complex pipes, fittings, wires, and sensors [6].

Each bioreactor is different and has some principles. The main aim of designing bioreactor is to consider the required oxygen transfer, low shear stress, and adequate mixing. Nutrients are efficiently provided to the cells, and waste products must be removed. Operating parameters like temperature, pH, dissolved oxygen, and substrate concentration are easy to control and monitor. Always bioreactor should be simple, inexpensive, and should be easily handled without contamination [7].

The sizes of bioreactor differ generally from shake flask (100–1000 ml) to laboratory scale vessel (1- 50 L) to pilot level (0.3–10 m³) to laboratory scale (2–500 m³) for large-scale production and industrial applications. Lab scale fermenter is typically constructed with the glass, and the pilot scale and industrial scale vessel are made of stainless steel [8]. Bioreactor is of different types; they are stirred tank fermenter, airlift fermenter, bubble column reactor, fluidized-bed reactor, packed bed reactor, and membrane bioreactor.

The most important product categories for fermentation fall into the broad areas of primary and secondary metabolites. Primary metabolites are intermediates of pathways directly involved in growth processes. They are small molecules under 1500 Da in molecular weight. Secondary metabolites are also known as idiolites; their molecular weights are more than 1500 Da and have unusual ring structures. They are not involved in growth processes. The secondary metabolites most commonly indicate antibiotics or anti-infective. These compounds are produced by microorganisms [9].

1.1 Types of fermentation based on substrate

1.1.1 Solid-State Fermentation (SSF)

The fermentation takes place in the support of solid substrate with non-specific, natural state, and low moisture content. The substrate used is nutrient-rich waste that can be reused. Substrates like bran, bagasses, and paper pulp are used as solid substrates in solid-state fermentation. It contains less moisture content; hence, this fermentation technology is good for microorganisms like fungi. It is not suitable for bacteria. This type of fermentation is slow and takes long time [10]. Product yield is high in SSF, and the reason for high yield is unknown. The solid material used in SSF is non-soluble and acts both as physical support and source of nutrients [11]. The primary setup of SSF is the “solid substrate bed” that contains the moist solids and an inter particle void space, and the process is aerobic in nature [12]. SSF is used for the development of bioprocess like bioremediation and biodegradation of hazardous compounds, biotransformation of crops, biopulping, production of biologically active secondary metabolites like antibiotics, alkaloids, plant growth factors, enzymes, etc., [13, 14, 15].

1.1.2 Submerged Fermentation (SmF)

In this fermentation, microorganisms need controlled atmosphere for production of high-quality end products. The substrates used in SmF are liquid media, molasses, waste water, vegetables juices, and soluble sugar to extract bioactive compounds [16]. SmF is mostly in use for the production of polysaccharides. It is also used to grow or to cultivate different bacteria and fungi to obtain intracellular and extracellular polysaccharides [17, 18].

1.2 Types of fermentation based on process

1.2.1 Batch fermentation

These processes are broadly used to produce chemicals, biotechnology, pharmaceutical, and agricultural products. Batch culture is a closed system in which the medium, nutrients, and inoculums are added to the fermenter or bioreactor under aseptic conditions. In this cultivation, a known number of microbial cells are inoculated into the bioreactor containing sterilized nutrient medium. A complete batch cultivation consists of many steps like medium formulation, filling the bioreactor, sterilization in place, inoculation, cultivation, product harvesting, and bioreactor cleaning in place. For ingenious performance of batch cultivation, it is essential to reduce all nonproductive steps except cultivation.

The submerged batch cultivation is used for the production of alcoholic beverages like beer, wine, whisky, brandy, rum, etc., and also used in the production of acidifiers or preservatives like vinegar and lactic acids, amino acids, or sweeteners (Figure 1) [20, 21].

Figure 1.

Batch fermentation (Source: [19]).

1.2.2 Fed-batch cultivation

This process represents semi-open system, and in this system one or more nutrients are gradually added by maintaining aseptic condition. The advantages of fed-batch cultivation are the prolonged product synthesis, to achieve higher cell densities and increase in the yield of product which is proportional to the concentration of biomass and capacity to enhance yield or productivity by controlled addition of nutrients. The fed-batch fermentations are used in large scale for the production of baker’s yeast, pure ethanol (Figure 2) [23].

Figure 2.

Fed-batch fermentation (Source: [22]).

1.2.3 Continuous cultivation

This culture represents an open system in which the nutrients are continuously added to the bioreactor by using aseptic conditions; simultaneously the culture broth is removed at the same time. The advantage of this culture is the opportunity to set up an optimum state for long-term production, the ability to attain stable product quality, and reduce any unrecommended time during the bioreactor operation.

There are some problems encountered due to extensive use of continuous fermentation on a large scale; they are larger possibility of contamination due to pumping of the medium in and out of the fermenter, the danger due to genetic mutations in the production strain in a long-term operation, and additional investments may be required for technical facilities (Figure 3) [25].

Figure 3.

Continuous fermentation (Source: [24]).

2. Bioreactors

Bioreactors are tanks or vessels which convert raw materials into biochemical products or lesser waste byproducts. These reactors are cylindrical, ranging in size from liter to cubic meters, and the capacity also differs depending on the design. It is heart of biotechnological systems that are used for agricultural, environmental, industrial, and medical applications [26]. The bioreactor is used for the production of biomass, for metabolic formation, even for the production of an active cell molecule [27]. Fermentors are having many accessory parts with various important roles during the fermentation process (Table 1)

Sl. No.	Part of fermenter	Function
1	Impellor (agitator)	Continuous stirring of media for homogeneous distribution of oxygen throughout the system preventing cells from settling down
2	Sparger (Aerator)	Sparger (Aerator) Introduce sterile air or oxygen to the media during the aerobic fermentation process
3	Baffles (vortex breaker)	Provides better mixing by disrupting the vortex formation
4	Temperature probe	Measures and monitors changing temperature in the medium during the fermentation process
5	Cooling jacket	Maintains temperature throughout the process
6	pH probe	Evaluates and monitors the change in pH of the medium
7	Level probe	Measures the level of medium within the fermenter
8	Foam probe	Detects the presence of the foam
9	Acid	Neutralizes the basic environment and maintains the pH
10	Base	Neutralizes the acidic environment and maintains the pH
11	Sampling point	To acquire samples throughout the process
12	Valves	Regulates and limits the flow of liquids and gases
13	Control panel	Monitors all parameters in the system
14	Inlet air filter	Filtration of air before entering the fermenter
15	Exhaust air filter	Prevention of contaminants from escaping
16	Rotameter	Measures flow rate of liquid or air
17	Dissolve oxygen probe	Measures dissolve oxygen in the fermenter
18	Pressure gauge	Measures internal pressure of the fermenter

Table 1.

Different parts of the fermenter with their significant roles.

(Source: [8]).

2.1 Types of bioreactor or fermenter

2.1.1 Stirred tank fermenter (STF)

This is the more frequently used fermenter, and it is known for its flexibility. The fermenter is cylindrical in shape, the shaft present inside drives the motor, and stir the content inside the tank. It has many benefits like robustness, reliability, and easy operating. The agitator present here is top or bottom driven [28]. Laboratory scale stirrer tank bioreactors are made up with borosilicate glass and stainless steel lid. Its capacity is 1 to 100 L [29]. The stirred tank bioreactors provide high value of mass and heat transfer rates and also good mixing. These system faces problem like high number of variables affect the mass transfer and mixing but most important among these is stirrer speed, number of stirrers, and flow rate of the gas used [30]. The most important role of impellers in STF accomplishes three main tasks like solid suspension, mixing and dissolution of the required atmospheric oxygen into the aqueous phase, and maximizing the interfacial area between gaseous and aqueous phases (Figure 4) [32].

Figure 4.

Stirred tank bioreactor (Source: [31]).

2.1.2 Air lift fermenter (ALF)

ALFs are also known as tower fermenter or reactor. The vessel is cylindrical in shape and in this air is presented at the bottom. ALFs are able to use free or immobilized cells that are suitable for bacteria, yeast, fungi, animal, and plant cells. This fermenter does not contain agitation system unlike other fermenters, but the content inside the vessel is mixed by the air introduced from the bottom. The cell mixture flows all over the column as an implication of the gradient air bubbles. The fluid is separated within two connected regions by means of draft tube or baffles for improving circulation and oxygen transmission and aligning shear forces in the equipment [33]. This fermenter has the benefit of generating liquid mixing and gas transfer without the use of mechanical agitators. The applications of airlift fermenter have increased with the introduction of genetic engineering techniques with the outcome of obtaining new strains of unicellular organisms capable of producing many novel products [34]. To grow yeast rapidly, it is very important to supply oxygen, and also, it is important to remove the heat of the fermentation. One of the experiments succeeded in growing food yeast that is Candidia utilis to produce RNA rapidly. The volume of fermenter was 145 m³, and oxygen transfer rate was 9.9 kg-O₂/m³/h using a superficial gas with a velocity of 30 cm/s. The production rate of yeast in this fermenter was 9.79 kg-dry cell m³/h (Figure 5) [36].

Figure 5.

Airlift bioreactor (Source: [35]).

2.1.3 Bubble column reactor

This fermenter or reactor is one of the simplest types, and it is easy to scale up. This belongs to the general class of multiphase reactors and consists of three main categories such as the trickle bed reactor, fluidized-bed reactor, and the bubble column reactor. Basically, it is a cylindrical vessel having a gas distributor at the bottom. The gas is sparged into a liquid phase or liquid–solid suspension in the form of bubbles. These fermenters are used as multiphase reactors in chemical, petrochemical, biochemical, and metallurgical industries [37]. Microorganisms are used to produce industrially important products such as enzymes, proteins, and antibiotics. Streptomyces cattleya was used for the production of thienamycin by continuously operating bubble column bioreactor [38]. Aureobasidium pullulans was used to produce glucoamylase (Figure 6) [40].

Figure 6.

Bubble column reactor (Source: [39]).

2.1.4 Fluidized-bed reactor (FBB)

FBB has many advantages than other type of reactors, and hence, the use of this reactor has increased. FBB was developed for the biological systems. This reactor constitutes a packed bed containing small-sized particles that can move the fluid. The small particle size accelerates higher rate of oxygen transfer, mass transfer, higher mixing rates, and nutrients availability to the cells [8]. FBB has applied in many industrially important processes. The present applications extend number of physical processes like cooling, heating, drying, sublimation, desublimation, adsorption, desorption, coating and granulation, and many catalytic and noncatalytic reactions. The major advantages of this reactor are: easy to handle and transportation of solids due to temperature distribution due to intensive solids mixing, high transfer of heat between bed immersed heating or cooing surfaces. The disadvantages of this reactor are: solids fluidized with gas require expensive equipment to separate solids and gas, due to high solid velocities, erosion of internals and attri¬tion of solids occurs, possibility of defluidization due to agglomeration of solids making difficult in scaling-up (Figure 7) [42].

Figure 7.

Fluidized-bed bioreactor (Source: [41]).

2.1.5 Packed bed reactor

It is also known as fixed-bed bioreactor and is used in wastewater treatment with biofilms attached. The nutrients are fed from the top or bottom with immobilized biocatalyst. These reactors are commonly treated with the immobilized cells and consist of a bed of packing, made with glass, ceramic, polymer and also exist in various shapes and sizes which allow the flow of fluid from one end to the other [43]. A packed bed reactor has two-phase upward flow used in wide number of multiphase chemical processes [44]. It is a plug flow reactor that belongs to the group where back mixing is not present; but in this reactor small amount of back mixing occurs that affects the attributes of the fermentation (Figure 8) [43].

Figure 8.

Packed bed bioreactor (Source: [45]).

2.1.6 Membrane bioreactor (MBR)

This bioreactor contains hollow fiber system; it is developed for the control of yeast and bacteria. The hollow fibers are made of cellulose acetate or from polysulfone fibers or acrylic copolymer [46]. MBR can transfer the two physical separation processes by filtering the biomass through a membrane [47]. This process is a combination of both biological stage and membrane modules. The specific function of this bioreactor and membrane module is biological degradation of organic pollution and separation of microorganisms from the treated wastewater. The membrane forms a physical barrier for all suspended solids and enables both recycling and also free from bacteria and viruses [48]. The advantages of this bioreactor are biocatalyst regeneration and immediate separation of biomass and the product. The major disadvantages are they are difficult to control and monitor the growth and breakdown of the culture and also extreme growth of microorganisms. The metabolic activity of cells is inhibited by increase of toxic materials or products in the membrane (Figure 9) [50].

Figure 9.

Membrane bioreactor (Source: [49]).

2.2 Secondary metabolites from microorganisms through fermentation

Secondary metabolites produced by microorganisms have a low molecular mass. These are not necessary for growth and serve a variety of ecological roles, working as antibiotics, phytohormones, anticancer agents, enzymes, and producing pigments [51, 52]. Usually, these metabolites are produced during culture’s late growth phase and have unusual structures. Nutrient type and concentration can be changed, including the carbon source, to affect the synthesis of secondary metabolites [53, 54, 55]. Usually, organic substances and secondary metabolites are created when primary metabolites are changed. The period of rapid growth (trophophase) is when secondary metabolites are not formed but are synthesized during the late growth phase of microorganisms (idiophase). The differences between the primary and secondary metabolites are given in Table 2 and Figure 10. The secondary metabolites are vital to human health and nutrition and have many biological properties [56]. A class of extremely significant economic importance includes antibiotics, poisons, pesticides, animal and plant growth hormones, and other medicines. After completion of the microbial growth, they are formed in a fermentation medium, typically during the stationary phase. The generation of secondary metabolites and enzymes has been effectively achieved through the use of solid-state fermentation (SSF), which is associated with simpler downstream processing procedures [57, 58]. Smaller fermenters using SSF have been demonstrated to yield a more stable product while using less energy [54, 59].

Primary metabolite	Secondary metabolite
Produced during the growth phase (Tropophase) of the cell	Produced during no growth phase (Idiophase) of the cell
Accumulated in large quantities	Accumulated in very small quantities

Table 2.

Difference between primary and secondary metabolites.

Figure 10.

Difference between primary and secondary metabolites.

Submerged fermentations (SmF) have been demonstrated to generate more stable products with lower energy requirements in smaller fermenters and still produce secondary metabolites. Recent research has focused on enhancing the production of bioactive secondary metabolites, given their importance in various applications. Once precursors can be obtained, secondary metabolite production can be enhanced in both submerged and solid-state fermentations [8].

2.3 Secondary metabolites: their biosynthesis paths

The growth, development, reproduction, and energy production of organisms do not require secondary metabolites. Because of this, not all microbial species produce these substances. Secondary metabolic pathways are used to create secondary metabolites from primary metabolites like amino acids and acetyl-coenzyme A [60]. Figure 11 illustrates how primary and secondary metabolic pathways are related. Secondary metabolism regulation is a complicated process. Therefore, understanding the metabolic pathways involved is crucial to improving microorganisms’ synthesis of secondary metabolites. The metabolic pathways of malonic acid and shikimic acid are responsible for the synthesis of the precursors of chemical compounds called phenolic. In plants, the shikimic acid pathway is the most significant. In bacteria and fungi, and the malonic acid pathway is an essential source of phenolics. The methylerythritol phosphate pathway or the mevalonic acid pathways are the two metabolic pathways that can be used to synthesize terpenoid precursors. There are mevalonic acid pathways in yeast, fungi, animals, plants, archaea, and some eubacteria. Most bacteria, cyanobacteria, and plant plastids contain the methylerythritol phosphate pathway [60].

Figure 11.

Schematic representation of metabolic pathways involved in the production of secondary metabolites by microorganisms using glucose.

2.4 Solid-state and submerged fermentation for microbial metabolites production

Fermentation is classified into submerged fermentation (SmF) and solid-state fermentation (SSF), which is mostly determined by the quantity of water utilized in the process (Table 3). The process of solid-state fermentation (SSF) is widely used to produce microbial metabolites [63]. With the benefit of a high product concentration and very little energy used, SSF is carried out on a solid substrate with low moisture content [64]. The substrate in a solid matrix absorbs the necessary water content in SSF, which provides additional benefits for the growth of microorganisms for the transfer of oxygen. A variety of agricultural wastes are utilized as SSF substrates, including rice straw, corn cobs, wheat straw, rice hulls, and sugarcane bagasse [59]. Since submerged fermentation (SmF) possesses a simpler downstream procedure than solid-state fermentation (SSF), SmF is the preferred method for producing metabolites in the industrial setting. While there is a knowledge gap in the SSF process, there is a possibility of strong and easy control over many factors in the case of SmF. The microbial morphology is more appropriate for generating secondary metabolites in SSF, although SmF is a simpler procedure to perform. Thus, SSF has several benefits, including simple gaseous transport, low water consumption, utilization of cellulosic waste, pH control, and requirement for smaller fermenters, which reduces the work needed for downstream processing [60, 65].

Factor	Submerged state fermentation	Solid-state fermentation
Substrates	Uses soluble substrates (sugars)	Insoluble substrates such as starch, cellulose, pectins, and lignin are used
Aseptic conditions	Heat sterilization and aseptic control	Vapor treatment, non-sterile conditions
Water	A high volume of water is consumed and effluent is discarded	Limited consumption of water; low Aw, no effluent is formed
Metabolic heating	Easy control of temperature	Low heat transfer capacity
Aeration	Limitation of soluble oxygen. High level of air required	Easy aeration and high surface exchange air/substrate
pH control	Easy pH control	Buffered solid substrates
Mechanical agitation	Good homogenization	Static condition required
Scale up industrial equipment	Industrial equipment is available	Need for engineering and new design equipment
Inoculation	Easy inoculation, continuous process	Spore inoculation, batch process
Contamination	Risk of contamination for single-strain bacteria	Risk of contamination for low-rate growth fungi
Energetic consideration	High energy consuming	Low energy consuming
Volume of equipment	High volumes and cost technology	Low volumes and cost technology
Effluent and pollution	High volumes of polluting effluents	No effluent; less pollution
Concentration of substrate/products	30–80 g/l	100/300 g/l

Table 3.

Comparison between submerged state and solid-state fermentations.

Source: [61, 62].

The standard industrial technique, known as a stirred tank reactor (SmF), involves growing microorganisms in a liquid medium to produce the required output [66]. Large-scale industrial enzyme synthesis is favored by this method since it is easier to manage and promotes superior microbial growth (Figure 12). Since secondary metabolites are generally secreted in the fermentation broth, SmF is appropriate for synthesizing these molecules, which are required to be used in liquid form [3]. While SmF has advantages, recent evidence indicates that SSF has a large impact on productivity, leading to higher yields and improved product characteristics compared to SmF. Precursor availability can enhance the ability of both submerged and solid-state fermentation to produce secondary metabolites [59].

Figure 12.

Solid-state and submerged fermentation to produce value-added products [67].

The same substrate can be utilized extremely slowly and steadily over a prolonged fermentation period in SSF. Thus, this method facilitates the prescribed release of nutrients. Fermentation techniques involving fungi and microorganisms that require restricted moisture content are most suited for the use of SSF [68]. However, it cannot be utilized in fermentation processes involving microorganisms like bacteria that need high a_w (water activity). While fungi are mainly interested in SSF processes, bacteria and yeasts are equally involved in SmF and SSF. The food and beverage processing industries play an essential role in bacteria and yeasts in SmF. Because of their physiological, biochemical, and enzymatic characteristics, filamentous fungi are most suitable for SSF and are widely used in ensiling, composting, and oriental food industries [69]. The group of microorganisms involved in both SSF and SmF techniques are shown in Table 4.

Microorganisms	SSF	SmF
Bacteria
Bacillus sp.	Composting natto, α-amylase	Enzymes (α-amylase, polygalcturonase, phytase, etc.)
Clostridium sp.	Ensiling, food	Pesticide degradation
Lactic acid bacteria	Ensiling, food	Fermented foods (yogurt, lacto-pickle, sausage, etc.)
Pseudomonas sp.	Composting	Xenobiotic degradation
Serratia sp.	Composting
Fungi
Alternaria sp.	Composting
Peniclium notatum, P. roquefortii	Cheese	Penicillin
Lentinus edodes	Shiitake mushroom	—
Pleurotus oestreatus, sajor-caju	Mushroom	—
Aspergillus niger, A. oryzae	Food, enzymes (glucoamylase)	Food enzymes (glucoamylase, amylopullulanase)
Amylomyces rouxii	Cassava tape
Beauveria sp., Metharizium sp.	Bioinsecticide	Bioinsecticide
Phanerochaete chrysosporium	Composting, lignin, degradation	—
Rhizopus sp.	Composting, food, enzymes, organic acid	Food, enzymes, organic acid
Trichoderma sp.	Composting biological control, bioinsecticides	Cellulase
Yeasts
Endomycopsis butronii, Schwanniomyces castelli	Casava tape	—
Saccharomyces cerevisiae	Alcoholic beverages, ethanol	Alcoholic beverages

Table 4.

Group of microorganisms involved in SSF and SmF methods.

Source: [69].

2.5 Microbial metabolites produced in solid-state and submerged fermentations

Microbial metabolites are often produced through submerged and solid-state fermentation (SSF). The efficiency of SSF in a particular process will depend on the kind of microorganisms used. Filamentous fungi like Aspergillus, Fusarium, Penicillium, and Rhizopus, yeasts like Saccharomyces cerevisiae, and bacteria like Bacillus species are used in SSF [70]. These microorganisms produce various metabolites, such as antibiotics, bioactive chemicals, biodiesel, biosurfactants, and bioethanol [70, 71]. SSF is more economical than submerged fermentation and is employed in several sectors, including the food, pharmaceutical, cosmetic, fuel, and textile industries.

2.5.1 Bacteria

Numerous cellulase-producing bacteria, including Cellulomonas fimi, Cellulomonas bioazotea, Cellulomonas uda, Acidothermus cellulolyticus, Bacillus subtilis, Bacillus coagulans, Bacillus pumilus, Clostridium acetobutylicum, Clostridium thermocellum, etc., have been isolated from various sources and can degrade xylan [72, 73, 74]. A statistical optimization of the xylanase activity by an isolated strain of B. coagulans from the Amazon environment, cultivated in SSF with an industrial fibrous soybean residue as substrate, was designed by Heck et al. [75]. Furthermore, Acidothermus cellulolyticus, a highly thermotolerant hot spring bacterium, has been studied as a potential source of hydrolytic enzymes for biomass. Rezaei et al. [76] showed that the SSF of switchgrass colonized by A. cellulolyticus may produce both cellulase and xylanase. Bacillus subtilis is found to be a strong bacterial strains that produce cellulase enzyme through submerged fermentation using grape pomace hydrolysate. Cellulase was produced in the range of 0.175–0.184 IU/ml at a w/v ratio of 15 (w/v), pH 6.0 for 7 days [77]. Protease produced from B. subtilis B22 in submerged fermentation under blue LEDs has a wide range of possible uses, including the food, detergent, leather, and pharmaceutical sectors [78]. Ali et al. [79] reported the optimization for maximum bacitracin production by B. licheniformis PCSIR-410-(5) through submerged fermentation. Numerous human illnesses, including bacterial endocarditis, osteomyelitis, intracranial infections, surgical infections, chronic sinusitis, and disease related to Clostridium difficile, were treated with this antibiotic. The production of Bacillus subtilis WX-17 by using a sole substrate of BGS (brewer’s spent grains) with submerged fermentation was carried out. The beverage had a final cell count of 9.86 CFU/ml. It is shown to be a nutritious beverage, tested to contain an increased amount of amino acids, total phenolic content, and antioxidant activity. The likely use of the liquid as a novel nutritional beverage for human health is justified by its greater nutritional content and the presence of viable B. subtilis in it (Table 5) [80].

Enzymes	Microorganisms	Substrate		Productivity		Reference
		SSF	SmF	SSF	SmF
Esterase	Aspergillus niger I-1472	Sugar beet pulp	Media containing cinnamic acid	20 nkat/mg dry wt.	0.4 nkat/ml	[18]
Cellulase	Trichoderma viride ATCC	Wheat bran	Mandel’s liquid media	60.5 FPU	28 FPU	[18]
Invertase	Aspergillus niger (mutant)	Polyurethane foam	Basal media	Higher	Lower	[18]
Phytase	Aspergillus niger CFR 335	Wheat bran	PDB basal media	Higher	Lower	[18]
Tannase	Aspergillus niger Aa-20	Polyurethane foam	Production media	12,000 IU/l	2500 IU/l	[18]
L-Asparaginase	Streptomyces spp., Serratia marcescens	Soybean meal	Yeast extract medium	49.23 U/ml	24.16 U/ml	[18]
Amylase	Bacillus spp.,	Oil cakes, wheat bran, bagasse	Starch broth	Around 50,000 U/g	400 U/ml	[18]
Xylanase	Thermotolerant Bacillus spp.,	Corn cob and wheat bran	Corn cob and yeast extract	6.18 U/g	16.13 U/ml	[44]
Amylase	Rhodotorula mucilaginosa	Wheat bran	—	250 U/g	—	[50]
Phytase	Rhodotorula minuta	—	Malt yeast extract media	—	91.85 U/ml	[45]

Table 5.

Enzyme production from microorganisms.

2.5.2 Fungi

To produce cellulase, a variety of fungi have been grown in SSF, where the basal mineral salt medium is added to moisten the substrate. These include Aspergillus niger, Aspergillus nidulans, Fusarium solani, Humicola insolens, Penicillium brasilianum, Phanerochaete chrysosporium, Trichoderma reesei, Trichoderma viride, Trametes versicolor, Penicillium sp., etc. [68]. Deswal et al. [79] have reported a study on optimization of several physiological and nutritional parameters for cellulase production from a brown rot fungus Fomitopsis sp. (RCK2010) using cost-effective substrates like wheat and rice straw under SSF conditions. D’Souza et al. [81] reported a pH of 5.0 was shown to be optimal for Aspergillus niger to produce tannase. Both submerged and solid-state fermentation were used to create the enzyme tannase from a variety of submerged media and solid agricultural wastes; however, solid-state fermentation produced higher enzyme yields. Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 were shown by Shivanna and Venkateswaran [82] to be able to accumulate phytase in both submerged and solid-state fermentation media. The results showed that the enzyme activity in SSF was 3–5 times higher than in the SmF medium. Gunashree and Venkateswaran [83] reported on optimization of various parameters for the production of phytase by using Aspergillus niger CFR 335. In both fermentation media, sucrose was shown to be an appropriate carbon source in place of glucose, along with peptone, Tween-20, and NaH2PO4 in SSF and CaCl2 in SmF. Gaykawad et al. [84] have reported a study on the use of animal fat by-products as substrate for the enzymatic production of value-added fatty acids using Mucor circinelloides and Mortierella alpina strains. Both strains demonstrated similar lipid content in shake flask, batch, and fed-batch fermenters. Both the strains produced Gamma-linolenic acid, however, only M. alpina produced arachidonic acid during the exponential growth phase. After 10 days, the agro-industrial waste that had been infected with Calocybe indica produced 5.3 g/l of dry mycelial weight, and 13.73 g/l of the fermented product was reported by Kapri et al. [85] which had a significant improvement in nutraceutical qualities. Furthermore, by applying this optimized fermenting process with edible/medicinal mushrooms, many other types of lingocellulosic agro-industrial wastes can be used for health advantages, as C. indica is known to have anti-cancer properties. Submerged fermentation of Chaetomium globosum DX-THS3 was carried out using silica microparticles to change its shape into small pellets. This approach of using the ideal silica concentration (10 g/L) and size (600 mesh) effectively created a highly active biopellet form that significantly increased the production of 6-d-glucoronidase (GUS). The obtained GUS activity was 680 U/mL, according to the results, which was almost 3.2 times greater than the same GUS activity in a traditional operation without silica [86]. Venkatachalam et al. [87] showed that Box–Behnken experimental design (BBED) coupled with response surface modeling (RSM) was applied with the optimal parameters in the production of maximum pigment and biomass in submerged fermentation of Talaromyces albobiverticillius 30,548. Indiscriminate disposal of agro-wastes into the environment may be reduced considerably by using them for the production of natural bioactive compounds through submerged and solid-state fermentations. The production of EPS from Pleurotus pulmonarius through submerged fermentation with agricultural wastes has been carried out. EPS inhibited the growth of pathogenic microorganisms and sustained the growth of probiotics. Therefore, the bioactivities of EPS make it a superior option for natural products that are widely utilized as preservatives in the food industry [88].

2.5.3 Yeast

Throughout the nineteenth century, natural dyes constituted the primary source of color for textiles. Synthetic dyes have almost entirely superseded natural colors. Synthetic dyes are beautiful because of their wide color range, increased repeatability, improved dying quality, and financial advantages. But it is commonly recognized that some artificial colors are hazardous to the environment and have detrimental effects. Natural pigments or dyes are a valuable substitute for possibly hazardous synthetic ecosystems [89]. Red pigments produced by Monascus yeast are employed as food coloring and textile coloring, according to some research. Furthermore, Monascus species are cultivated on rice grains in some Asian nations and are utilized to color some foods, which raise the need for extremely safe pigments [90]. Velmurugan et al. [91] reported the use of corn cob, an agricultural byproduct for the production of pigments from Monascus purpureus KACC 42430 through SSF. The highest yield of the pigments with corn cob powder shows it as the best substrate for the SSF method. The enzymatic digestibility of Monascus purpureus is enhanced by ground maize cob, as previously mentioned, which leads to increased pigment synthesis. Gowthami and Gunashree [92] report optimization of various physico-chemical parameters for the maximum production of phytase by SmF from Cystobasidium minutum, which is imperative for growth of the organism and metabolite yield. Strain improvement of Rhodotorula gracilis by UV mutation and optimization of different media composition of Czapek Dox broth (CDB), Potato Dextrose broth (PDB), Sabaroud’s Dextrose broth (SDB), Fat Produing Medium (FPM) for the production of hydrolysate phytase enzyme through SSF and SmF method [93]. Saccharomyces cerevisiae BY4742 in glucose fermentation by using wheat straw as solid substrate and the production of ethanol was improved by UV mutagenesis of YUV1–1 and YUV2–2 which preferred high temperature. The yield of the ethanol was higher than the native yeast of S. cerevisiae BY4742. Thermotolerant strains are very useful for raising the temperature of SSF and fermentation [94]. Li et al. [95] have shown in their study that yeast fermentation using feed from food waste can replace crayfish’s conventional diet by 30% which may in turn improve their antioxidant capacity and enhance non-specific immunity. The fermentation conditions were maintained, and initial moisture was maintained at about 60% of food waste bran and inoculated with 2% of the yeast mixture (Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica, 3:2:1). Brewer’s spent grain (BSG) has been studied for its potential as a carbon source substitute for Rhodotorula mucilaginosa (LBP4), which produces biosurfactants. Alkali enzymatic BSG hydrolysate was employed in the study as a carbon source to optimize LBP4’s generation of biosurfactants [96, 97].

3. Conclusion

Fermentation is a potential strategy for microbial metabolite production, and it can be used to produce a variety of primary metabolites such as amino acids, nucleotides, organic acids, and vitamins. Acetic acid (Acetobacter), lactic acid (Lactobacillus), alcohol and carbon dioxide (yeast), and other main metabolites can be used to categorize fermentation. Solid-state fermentation (SSF) is a common technique for the developing of microbial metabolites, and it is carried out on a solid substrate using certain microorganisms (yeasts and fungi) that are grown on a moist, solid, non-soluble organic material that serves as a source of nutrients and support for the microorganisms’ growth when free-flowing water is absent or almost absent. The success of applying SSF to a specific process is affected by the nature of the microorganisms. The majority of microorganisms utilized in SSF are filamentous fungi like Aspergillus, Fusarium, Penicillium, and Rhizopus, as well as yeasts like Saccharomyces cerevisiae and bacteria like Bacillus species. These microorganisms are employed to manufacture an array of metabolites, including antibiotics, bioactive compounds, biodiesel, biosurfactants, and bioethanol. The necessity to find alternative production methods has arisen due to the recent rise in demand for natural medicine. With its many uses and several advantages, fermentation has emerged as an excellent applicant to close this gap. However, there is still more work to be done in terms of comparing these procedures because different fermentation techniques differ from one another. Furthermore, a great deal of research still needs to be done to find sustainable substrates and methods for preserving quality and output. These have the potential to lower the price and increase the production of these chemicals.