Food and Industrial Microbiology

Lesson 22. Lesson 22 TYPES OF FERMENTATION- SUBMERGED, SOLID AND SURFACE TYPE

Module 6. Fermentation process strategies

Lesson 22
TYPES OF FERMENTATION- SUBMERGED, SOLID AND SURFACE TYPE

22.1 Introduction

Fermentation is one of the oldest technologies used for food preservation. Over the centuries, it has evolved, been refined and diversified. Today a variety of fermented foods is produced both in industrialised and developing countries using this technology at the s. A wide range of raw materials is used as substrates and panoply of products is concocted. Foods derived from fermentation are major constituents of the human diet all over the world. Although advances in food science and technology have given rise to a wide range of new food technologies, fermentation has remained an important technology throughout the history of mankind. Many benefits are attributed to fermentation. It preserves and enriches food, improves digestibility, and enhances the taste and flavour of foods. It is also an affordable technology and is thus accessible to all populations. Furthermore, fermentation has the potential of enhancing food safety by controlling the growth and multiplication of a number of pathogens in foods. Thus, it makes an important contribution to human nutrition, particularly in developing countries, where economic problems pose a major barrier to ensuring food safety.

Fermentation systems may be liquid, also known as submerged or solid state, also known as surface. Most fermentors used in industry are of the submerged type, because the submerged fermentor saves space and is more amenable to engineering control and design.

Fig. 22.1 Submerged fermentation unit (www.biocon.com)

22.2 Submerged Liquid Fermentations

Submerged liquid fermentations are traditionally used for the production of microbially derived enzymes. Submerged fermentation involves submersion of the microorganism in an aqueous solution containing all the nutrients needed for growth.

Fermentation takes place in large vessels (fermenter) with volumes of up to 1,000 cubic metres. The fermentation media sterilises nutrients based on renewable raw materials like maize, sugars and soya. Most industrial enzymes are secreted by microorganisms into the fermentation medium in order to break down the carbon and nitrogen sources. Batch-fed and continuous fermentation processes are common. In the batch-fed process, sterilised nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilised liquid nutrients are fed into the fermenter at the same flow rate as the fermentation broth leaving the system. Parameters like temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled to optimize the fermentation process.

Next in harvesting enzymes from the fermentation medium one must remove insoluble products, e.g. microbial cells. This is normally done by centrifugation. As most industrial enzymes are extracellular (secreted by cells into the external environment), they remain in the fermented broth after the biomass has been removed. The enzymes in the remaining broth are then concentrated by evaporation, membrane filtration or crystallization depending on their intended application. If pure enzyme preparations are required, they are usually isolated by gel or ion exchange chromatography.

Several types of submerged type of fermentors are known and they may be grouped in several ways: shape or configuration, whether aerated or anaerobic and whether they are batch or continuous. The most commonly used type of fermentor is the Aerated Stirred Tank Batch Fermentor.

22.2.1 Aerated stirred tank batch fermentor

A typical fermentor of this type (as shown in Fig 22.1 and Fig. 22.2) is an upright closed cylindrical tank fitted with one or more baffles attached to the side of the wall, a water jacket or coil for heating and/ or cooling, a device for forcible aeration (known as sparger), a mechanical agitator usually carrying a pair or more impellers, means of introducing organisms and nutrients and of taking samples, and outlets for exhaust gases. Modern fermentors are highly

automated and usually have means of continuously monitoring, controlling or recording pH, oxidation-reduction potential, dissolved oxygen, effluent O2 and CO2, and chemical components. Further diagrams of stirred tank fermentors are shown below

22.2.1.1 Aeration system (Sparger)

Sparger is a device for introducing air into fermenter. Aeration provides sufficient oxygen for organism in the fermenter. Fine bubble aerators must be used. Large bubbles will have less surface area than smaller bubbles which will facilitate oxygen transfer to a greater extent. Agitation is not required when aeration provides enough agitation which is the case Air lift fermenter. But this is possible with only for medium with low viscosity and low total solids.

For aeration to provide agitation the vessel height/diameter ratio (aspect ratin) should be 5:1.

Air supply to sparger should be supplied through filter.

There are three types of sparger viz. porous sparger, orifice sparger and nozzle sparger.

1. Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non agitated vessel. The size of the bubble formed is 10-100 times larger than pore size. There is a pressure drop across the sparger and the holes tend to be blocked by growth which is the limitation of porous sparger.

2. Orifice sparger: used in small stirred fermenter. It is a perforated pipe kept below the impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air holes drilled on the under surfaces of the tubes and the holes should be at least 6mm diameter. This type of sparger is used mostly with agitation. It is also used with out agitation in some cases like yeast manufacture, effluent treatment and production of SCP.

3. Nozzle sparger: Mostly used in large scale. It is single open/partially closed pipe positioned centrally below the impeller. When air is passed through this pipe there is lower pressure loss and does not get blocked.

4. Combined sparger agitator: This is air supply via hallow agitator shaft. The air is emitted through holes in the disc or blades of agitator.

Fig. 22.2 An aerated stirred tank fermentor (Najafpour, 2007)

1. Lid, 2. air-outlet, 3. fermenter, 4. module base permeable for air,
5. cultivation substrate, 6. cooling device, 7. air-inlet, 8. water supply,
9. water dischage, 10. heat resistant seal, 11. support device for the module base, 12. edge of the module base,
13. quick coupling, 14. pipe for the inflow and outflow of the cooling liquid, 15. heat resistat seal, 16. exterior ring, 17. coupling

22.3 Surface Fermentation

In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry. A. niger forms a mycelium layer on the liquid surface of the aluminum or stainless steel trays. These trays are stacked in fermentation rooms supplied with filtered air which serves both to supply oxygen and to control the temperature of fermentation. Surface fermentation is easy to control and to implement. It needs no aeration or agitation of the fermentation broth, so it needs no instrumentation for aeration and agitation. The separation of citric acid from the mycelium is easy because the microorganism is not dispersing into the medium. Only the temperature and humidity of the fermentation chamber need controlling. It can be used easily in small plants as well as in third world countries. With surface fermentation, the fermentation broth is concentrated due to a high evaporation rate during fermentation. Thus, expenses and losses during recovery and purification are low. However, surface fermentation has the following disadvantages: Building investment costs are high. Personnel expenses are high in developed industrial countries with extremely high wages. Fermentation time is long and therefore productivity is low.

Submerged fermentation is favored over surface fermentation for the following reasons

• Lower total investment costs;
• Improved process control;
• Reduced fermentation time;
• Reduced floor space requirements;
• Lower labour costs;
• Simpler operations; and
• Easier maintenance of aseptic conditions on an industrial scale.

However, submerged fermentation has some disadvantages compared to surface fermentation: expenses for equipment are higher; consumption of electrical energy is higher; and the process is very sensitive to short interruptions or breakdowns in aeration and vulnerable to infections, which result not only in losses of yield, but also in a total breakdown of respective batches

22.4 Solid-State Fermentation (SSF)

The origin of Solid-state fermentation can be traced back to bread-making in ancient Egypt. Solid substrate fermentations also include a number of well known microbial processes such as soil growth, composting, silage production, wood rotting and mushroom cultivation. In addition, many familiar western foods such as mold-ripened cheese, bread, sausage and many foods of Asian origin including miso, tempeh and soy sauce are produced using SSF. Beverages derived from SSF processes include ontjom in Indonesia, shao-hsing wine and kaoliang (sorghum) liquor in China and sake in Japan.

SSF is used for the production of bioproducts from microorganisms under conditions of low moisture content for growth. The medium used for SSF is usually a solid substrate (e.g., rice bran, wheat bran, or grain), which requires no processing. In order to optimize water activity requirements, which are of major importance for growth, it is necessary to take into account the water sorption properties of the solid substrate during the fermentation. In view of the low water content, fewer problems due to contamination are observed. The power requirements are lower than submerged fermentation. Inadequate mixing, limitations of nutrient diffusion, metabolic heat accumulation, and ineffective process control renders SSF generally applicable for low value products with less monitoring and control. There exists a potential for conducting SSF on inert substrate supports impregnated with defined media for the production of high value products.

It involves the growth of microorganisms on moist solid particles, in situations in which the spaces between the particles contain a continuous gas phase and a minimum of visible water. Although droplets of water may be present between the particles, and there may be thin films of water at the particle surface, the inter-particle water phase is discontinuous and most of the inter- particle space is filled by the gas phase. The majority of the water in the system is absorbed within the moist solid-particles the more general term “solid-substrate fermentation” is used to denote any type of fermentation process that involves solids, including suspensions of solid particles in a continuous liquid phase and even trickling filters.

Advantages of Solid State Fermentation over Submerged Fermentation

1. Higher volumetric productivity
2. Usually simpler with lower energy requirements
3. Might be easier to meet aeration requirements
4. Resembles the natural habitat of some fungi and bacteria
5. Easier downstream processing
6. The fungal hyphae are bathed in a liquid medium and do not run the risk of desiccation;
7. Temperature control is typically not overly difficult, such that the organism is exposed to a constant temperature throughout its growth cycle;
8. The availability of O₂ to the biomass can be controlled reasonably well at a particular level of saturation of the medium
9. The availability of the nutrients to the organism can be controlled within relatively narrow limits if desired, through the feeding of nutrient solutions.
10. Although shear forces do occur within mechanically stirred bioreactors, the nature and magnitude of these forces are well understood and it is possible to use bioreactors that provide a low-shear environment, if the organism is highly susceptible to shear damage, such as bubble columns or air lift bioreactors;
11. pH control is relatively easy to provide.

In contrast, the environment in SSF can be quite stressful to the organism. For example:

1. Fungal hyphae are exposed to an air phase that can desiccate them;
2. Temperatures can rise to values that are well above the optimum for growth due to the inadequate removal of waste metabolic heat. In other words, the temperature to which the organism is exposed can vary during the growth cycle;
3. O₂ is typically freely available at the surface of the particle, however, there may be severe restrictions in the supply of O₂ to a significant proportion of the biomass that is within a biofilm at the surface or penetrating into the particle;
4. The availability of nutrients to the organism may be poor, even when the average nutrient concentration within the substrate particle, determined after homogenizing a sample of fermenting solid particles, is high. In other words, there tend to be large concentration gradients of nutrients within the particles; movement of the particles of the solid substrate can cause impact and shear damage. In the case of fungal processes the hyphae can suffer severe damage
5. It may be difficult to provide pH control under some circumstances.

However, there are certain instances in which, despite being more problematic, SSF may be appropriate:

1. When the product needs to be in a solid form (e.g., fermented foods);
2. When a particular product is only produced under the conditions of SSF or, if produced in both SLF and SSF, is produced in much higher levels in SSF. For example, certain enzymes are only induced in SSF and some fungi only sporulate when grown in SSF, in which the hyphae are exposed directly to an air phase. For example, Monascus pigment and many fungal spores are produced in much higher yields in SSF
3. If it is desired to use genetically unmodified organisms in a process for the production of such a product, then SSF may be the only option;
4. When socio-economic conditions mean that the fermentation process must be carried out by relatively unskilled workers. Some SSF processes can be relatively resistant to being overtaken by contaminants;
5. When the product is produced in both SSF and SLF, but the product produced in SSF has desirable properties which the product produced in SLF lacks. For example, spore-based fungal biopesticides produced in SSF processes are usually more resistant to adverse conditions than those produced in SLF, and are therefore more effective when spread in the field;
6. When it is imperative to use a solid waste in order to avoid the environmental impacts that would be caused by its direct disposal. This is likely to become an increasingly important consideration as the ever-increasing population puts an increasing strain on the environment.

Some examples of traditional SSF processes are:

1. Tempe, which involves the cultivation of the fungus Rhizopus oligosporus on cooked soybeans.
2. The koji step of soy sauce manufacture, which involves the cultivation of the fungus Aspergillus oryzae on cooked soybeans.
3. ‘ang-kak’, or “red rice”, which involves the cultivation of the fungus Monascus purpureus on cooked rice.

Beyond this, over the last three decades, there has been an upsurge in interest in SSF technology, with research being undertaken into the production of a myriad of different products, including:

1. Enzymes such as amylases, proteases, lipases, pectinases, tannases, cellulases, and rennet;
2. Pigments;
3. Aromas and flavor compounds;
4. “Small organics” such as ethanol, oxalic acid, citric acid, and lactic acid;
5. Gibbrellic acid (a plant growth hormone);
6. Protein-enriched agricultural residues for use as animal feeds;
7. Animal feeds with reduced levels of toxins or with improved digestibility;
8. Antibiotics, such as penicillin and oxytetracycline;
9. Biological control agents, including bioinsecticides and bioherbicides;
10. Spore inocula (such as spore inoculum of Penicillium roqueforti for blue cheese production).
11. Decolorization of dyes;
12. Biobleaching;
13. Biopulping;
14. Bioremediation.

Bacteria, yeast and fungi can all grow on solid substrates and have applications in SSF processes. However, filamentous fungi are the best adapted species for SSF and dominate in the research and practical applications around the world. Bacterial SSF fermentations are rarely used for large scale enzyme production, but are very important in nature and in the fermented food industry. Filamentous fungi are the most important group of microorganisms for enzyme production in SSF. The hyphal mode of growth gives a major advantage to filamentous fungi over unicellular microorganisms in the colonization of solid substrates and the utilization of available nutrients. The filamentous fungi have the power to penetrate solid substrates. Hydrolytic enzymes are excreted at the hyphal tip, without large dilution. This makes the action of hydrolytic enzymes very efficient and allows penetration into most solid substrates. This is critical for the growth of the fungi. Fungi cannot transport macromolecular substrates across the cell wall, so the macromolecule must be hydrolyzed externally into soluble units that can be transported into the cell.

Fundamentally, there are 6 types of solid-state fermenters:

1. Tray bioreactor

2. Packed bed bioreactor

3. Rotary drum bioreactor

4. Swing solid state bioreactor

5. Stirred vessel bioreactor

6. Air solid fluidized bed bioreactor

The simplest SSF reactor is the tray. In a tray bioreactor a relatively thin layer of substrate is spread over a large horizontal area. There is no forced aeration, although the base of the tray may be perforated and air forced around the tray. Mixing, if any, is by simple automatic devices or manual. Internal temperature may vary with ambient temperature; or the tray may be placed in a temperature-controlled room. Tray bioreactors have been used successfully at laboratory, pilot, semi-commercial and commercial scale.

Fig. 22.3 Structure of a tray type solid state fermentation system (Okafor, 2007)

22.4.1 Factors affecting enzyme production in solid state fermentation systems

The major factors that affect microbial synthesis of enzymes in a SSF system include: selection of a suitable substrate and microorganism; pre-treatment of the substrate; particle size (inter-particle space and surface area) of the substrate; water content and a _w of the substrate; relative humidity; type and size of the inoculum; control of temperature of fermenting matter/removal of metabolic heat; period of cultivation; maintenance of uniformity in the environment of SSF system, and the gaseous atmosphere, i.e. oxygen consumption rate and carbon dioxide evolution rate.

Current trends on SSF have focused on application of SSF for the development of bioprocess such as bioremediation and biodegradation of hazardous compounds, biological detoxification of agro-industrial residues, biotransformation of crops and crop-residues for nutritional enrichment, biopulping, and production of value-added products such as biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. SSF systems, which during the previous two decades were termed as a ‘low-technology’ system appear to be a promising ones for the production of value-added ‘low volume–high cost’ products such as biopharmaceuticals. SSF processes offer potential advantages in bioremediation and biological detoxification of hazardous and toxic compounds.