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1 Fermentation Technology : Dr.Jehan Abdul Sattar The term fermentation is used to indicate microbial cell propagation and generation of products under either aerobic, microaerobic, or anaerobic conditions. Aerobic indicates condition where air is intentionally mixed with the medium; microaerobic refers to air that is initially present, but is then used up or displaced as microbial growth occurs; while anaerobic indicates a condition where oxygen is removed and intentionally excluded from the fermentation media since it is toxic to the cells. The secretion of metabolites from the inside of microbial cells to the surrounding medium and accumulation of the metabolite in the medium occurs as a consequence of the oxidation of monosaccharides, particularly glucose under both aerobic and anaerobic conditions.

Fermentation Process:

The entire process can be divided in three stages. Stage I : Upstream processing which involves preparation of liquid medium, separation of particulate and inhibitory chemicals from the medium, sterilization, air purification etc., Stage II: Fermentation which involves the conversion of substrates to desired product with the help of biological agents such as microorganisms., Stage III: Downstream processing which involves separation of cells from the fermentation broth, purification and concentration of desired product and waste disposal or recycle. A fermentation process requires a fermenter for successful production because it provide the organism growing within it with optimal pH, temperature, oxygen, and other environmental conditions. *A fermenter is a vessel for the growth of microorganisms which, while not permitting contamination, enables the provision of conditions necessary for the maximal production of the desired products 2

Types of Fermentation Processes

1. Submerged Cultivation

Submerged cultivation of microbial cells in bioreactors guarantees a controlled environment for the efficient production of high-quality end products and to achieve optimum productivity and yield. Industrial bioreactors operated in batch, fed-batch, or continuous mode are utilized to culture different types of microorganisms producing a wide range of products. Different approaches to submerged cultivation of microorganisms in bioreactors are discussed below. -A- Batch Cultivation Batch culture represents a closed system in which the medium, nutrients, and inoculum are added to the bioreactor, mostly under aseptic conditions, at the beginning of cultivation, the volume of the culture broth in the bioreactor is theoretically constant during cultivation (practically, small deviations in culture volume are caused by a low feed rate of acid/base solutions to keep the pH at a desired level and by sampling or introducing air/gas into the culture; on balance, such changes are usually ignored due to their small value relative to the total working volume of the bioreactor). Typically, at the beginning of batch cultivation, a known number of viable cells 3 are inoculated into the bioreactor that is already filled with sterilized medium containing all nutrients. After inoculation, the cell culture follows the classical growth curve described , which is divided into four main phases. ugh the cells are metabolically activethey are adapting their enzymatic apparatus to a new environment; no significant increases in biomass concentration, substrate consumption, or product synthesis are observed), it is desirable to shorten it as much as possible. The length of the lag phase is influenced mainly by the concentration of cells in the inoculum and their physiological state, the composition of the inoculation and cultivation medium (mainly the source of carbon and energy, pH, and temperature), and the size of the inoculum. The exponential (logarithmic growth) phase is characterized by rapid cell proliferation (biomass concentration is an exponential function of time), constant specific growth rate, which is equal to the maximum specific growth rate of the culture under conditions of absence of growth limitation (growth rate is not limited because all nutrients are present in excess, while also not attaining growth- inhibiting concentrations), fast consumption of the source of carbon and energy, and a high rate of primary metabolite production. The depletion of nutrients by the end of the exponential phase (in the case of aerobically grown cultures, these are signaled by a rapid increase in dissolved oxygen concentration) causes a reduction in the specific growth rate and a transition to the stationary phase, characterized by the stagnation of growth and utilization of carbon and energy; this phase is important for the synthesis of secondary metabolites. The whole batch operation consists of several steps, including medium formulation, filling the bioreactor, sterilization in place (SIP systems), inoculation, cultivation, product harvesting, and bioreactor cleaning in place (CIP systems). For efficient performance of batch operation, it is important to minimize all nonproductive steps (all steps listed above except cultivation), achieve a high rate of product synthesis, optimize productivity, and maximize the yield of the end product. Extension of exponential growth is advantageous for the efficient production of biomass es (ethanol, acetic, citric, or lactic acids), whereas in the case of secondary metabolite production, the exponential phase is shortened (by the limitation of one nutrient, usually the source of nitrogen) and the stationary phase is prolonged to achieve the maximum yield of the product. Batch culture systems provide a number of advantages:

1- Reduced risk of contamination or cell mutation as the growth period is short.

2- Lower capital investment when compared to continuous processes for the same

bioreactor volume. 4

3- More flexibility with varying product/biological systems.

4- Higher raw material conversion levels, resulting from a controlled growth

period.

5- Processes using one reactor to make various products.

Disadvantages of Batch Culture

There are several disadvantages of batch culture. The nutrient in the working volume becomes depleted; the other major problem is the limitation and depletion of the substrate. Since there is no flow stream to take effluent out, as the system is closed, toxins form there. A disadvantage related to substrate depletion is that the growth pattern may reach the death phase quickly in an old culture. The long duration of the batch system for slow growth results in exhaustion of essential nutrients and an accumulation of metabolites as by products. The technical problem resulting in changes to media composition may directly affect the microbial exponential growth phase. Inhibition is another factor affecting the bioprocess. As a result, inhibition may slow down bio-catalytic activities. Inhibitory product may block enzyme activities, and the cells became poisoned by the by-product. One common disadvantage of the batch process is that one has to carry out a cycle for production: the product should be sent for downstream processing, then the system has to be cleaned and recharged with fresh feed, so the process is highly labour intensive for downtime and cleaning. 5

B- Fed-Batch Cultivation

Fed-batch culture represents a semi-open system in which one or more nutrients are aseptically and gradually added to the bioreactor while the product is retained inside , the volume of the culture broth in the bioreactor increases within this time. The main advantages of fed-batch over batch cultures are: (a) the possibility to prolong product synthesis, (b) the ability to achieve higher cell densities and thus increase the amount of the product, which is usually proportional to the concentration of the biomass, (c) the capacity to enhance yield or productivity by controlled sequential addition of nutrients, and (d) the feature of prolonged productive n the bioreactor would normally be prepared for a new batch.

Disadvantages include:

1- Lower productivity levels due to time for filling, heating, sterilization, cooling,

emptying and cleaning the reactor.

2- Higher costs in labour and/or dynamic process control for the process.

The typical food fed-batch fermentations are large- yeast, pure ethanol, which is further utilized for alcoholic beverages produced by mixing ingredients such as liquors , and submerged acetification for vinegar production.

Continuous Cultivation

Continuous culture represents an open system in which nutrients are aseptically and continuously added to the bioreactor, and the culture broth (containing cells and metabolites) is removed at the same time , the volume of the culture broth is constant due to a constant feed-in and feed-out rate. Continuous culture is used for a chemostat, represented by a constant specific growth rate of cells, which is equal to the dilution rate and is controlled by the availability of the limiting nutrient, Thus if 100 grams per liter of the limiting nutrients are required for maximum growth but only 90 grams per liter are added, then the rate of growth will be 90% of the maximum, although other types of continuous operation such as turbidistat (a constant concentration of biomass controlled by the dilution rate). If however during the exponential phase of growth, a constant volume is maintained by ensuring an arrangement for a rate of broth outflow which equals the rate of inflow of fresh medium, then the microbial density or turbidity (i.e., cells per unit volume) remains constant. The type nutristat (a constant parameter related to cell growth controlled by the dilution rate) can be employed. In the laboratory and in practice the chemostat is widely used . 6 The main advantages of continuous culture (chemostat) over the batch mode are (a) the possibility to set up optimum conditions for maximum and long-term product synthesis, (b) the ability to achieve stable product quality (the steady state is characterized by a homogeneous cell culture represented by a constant concentration of biomass and metabolites), and (c) a distinct reduction in In spite of these advantages, there are also several problems that hamper the extensive utilization of continuous operation on a large scale. These include (a) increased risk of contamination due to the pumping of the medium in and out of the bioreactor, (b) the danger of genetic mutations in the production strain in a long-term operation, and (c) additional investments may be required for technical facilities. Continuous fermentation systems based on immobilized cell technology have also been studied in beer production.

2. Solid Substrate Fermentation

(SSC) is used for systems where microorganisms are cultured on the surface of a concentrated water-insoluble substrate (usually containing polysaccharides as a carbon and energy source) with a low level of free water. This technique was developed in the Eastern countries, where it has been used for centuries for the production of traditional foods such as soy sauce, miso, or sake, using different substrates and microorganisms. In the Western countries, it has not been widely exploited and its application is limited mainly to the production of industrial enzymes, certain food products, or feed supplements. Solid substrate fermentation is characterized by very low water activity; thus, the main features of this system are substantially different compared with classical submerged cultivation as shown in Table1. There are several advantages of SSF over the conventional submerged technology such as (a) the use of a concentrated medium, resulting in a smaller 7 reactor volume and lower capital investment costs, (b) the lower risk of contamination with yeasts and bacteria due to low moisture levels and substrate complexity, (c) the simplicity of the technology and low production of effluent water from the process, (d) the higher product yield and easier product recovery, and (e) the use of agricultural wastes as substrates for certain applications (e.g., feed supplements and cellulolytic enzymes). Filamentous fungi are preferable for SSF processes, mainly due to their abilities to: (a) grow on substrates with reduced water activity, (b) penetrate their hyphae into the solid substrate, and (c) produce exoenzymes (e.g., amylolytic and cellulolytic enzymes), which decompose the polysaccharides (the main carbon source often present in solid substrates). When using yeast, it is necessary to integrate a material pretreatment step (such as steam explosion, acid or alkali treatment, followed by enzymatic digestion or a combination of these) into the process or to use a mixed culture, where in the complex cellulosic or starchy material is first degraded by other organisms (usually molds) that are able to produce extracellular enzymes and the released glucose is then consumed by yeasts (or less frequently by bacteria) yielding the desired product. Table 1. Main Differences between Solid Substrate Fermentation and Submerged

Cultivation

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