Fermentation Technology PDF

ChE 412 – Biochemical Engineering Fermentation Technology I. Introduction The term “fermentation” originates from the Latin verb “fervere,” meaning “to boil.” While biochemists view fermentation as a catabolic process that generates ener...

ChE 412 – Biochemical Engineering Fermentation Technology I. Introduction The term “fermentation” originates from the Latin verb “fervere,” meaning “to boil.” While biochemists view fermentation as a catabolic process that generates energy, industrial microbiologists define it as the large- scale cultivation of microorganisms to convert substrates into valuable products through aerobic or anaerobic processes. As depicted in Figure 1, fermentation involves a series of steps, from inoculation to product recovery. Fermentation technology is widely employed to produce a variety of products, including organic solvents (acetone, alcohols), fermented beverages (wine, beer, whiskey), enzymes, amino acids, vitamins, and pharmaceuticals. The success of fermentation processes hinges on optimal microbial growth, which is influenced by numerous biochemical and physical factors. This chapter delves into the growth characteristics of microorganisms, explores different types of fermentation processes, and discusses their applications. Figure 1 Schematic diagram of fermentation technology (Kaur, 2024) II. Microbial Growth A key factor in successful fermentation is achieving optimal microbial growth. Microorganisms require specific conditions, including pH, temperature, oxygen levels, minerals, energy sources, and raw materials, to complete their life cycle, which is typically divided into six phases as illustrated in Figure 2: a. Lag Phase: This initial phase is characterized by a period of adaptation where microorganisms acclimate to their new environment. During this time, there is no significant increase in cell number. b. Acceleration Phase: In this phase, microorganisms begin to actively divide and their population starts to increase. c. Log Phase: This is the period of exponential growth, where microorganisms rapidly divide and consume nutrients at a high rate. d. Stationary Phase: As nutrient levels decline and waste products accumulate, microbial growth slows and eventually ceases. The population remains relatively stable during this phase. e. Death Phase: In this final phase, microorganisms begin to die off due to unfavorable conditions, such as nutrient depletion and toxic waste accumulation. The overall growth curve is typically sigmoidal. The optimal harvest time depends on the desired product. For instance, if the goal is to obtain cell biomass, the microorganisms are harvested during the exponential phase. Conversely, if the desired product is a secondary metabolite, harvesting occurs during the stationary phase when these compounds are often produced.  Figure 2. Growth curve of unicellular organisms: (A) lag phase; (B) accelerated growth phase; (C) exponential growth phase; (D) decelerated growth phase; (E) stationary phase; (F) death phase. (Dutta, 2008) Figure 2 illustrates the typical growth curve of microorganisms in a batch fermentation system. In this closed system, a single inoculation of microorganisms and nutrients is introduced into the fermenter. The system remains sealed throughout the entire process, from the initial lag phase to the final death phase. Once the desired product is formed, it is harvested, and the fermentation cycle is complete. Batch Fermentation: A Closed-System Approach In contrast to batch fermentation, fed-batch fermentation involves the periodic addition of nutrients to the culture medium. This controlled nutrient feeding strategy allows for optimization of product yield and productivity by preventing nutrient inhibition and maintaining optimal growth conditions. For instance, substrates like ethanol, methanol, or acetic acid, which can be inhibitory at high concentrations, can be added gradually during later growth phases. Fed-Batch Fermentation: A Controlled Nutrient Supply Continuous fermentation is the most efficient method for large-scale production. In this process, fresh medium is continuously fed into the fermenter, while an equal volume of culture containing products and cells is simultaneously withdrawn. This maintains a steady-state environment, promoting continuous exponential growth and maximizing product yield. Continuous fermentation is particularly suitable for the production of primary metabolites such as organic acids, amino acids, and single-cell protein. Continuous Fermentation: A Steady-State Process Continuous fermentation is the most efficient method for large-scale production. In this process, fresh medium is continuously fed into the fermenter, while an equal volume of culture containing products and cells is simultaneously withdrawn. This maintains a steady-state environment, promoting continuous exponential growth and maximizing product yield. Continuous fermentation is particularly suitable for the production of primary metabolites such as organic acids, amino acids, and single-cell protein. III. Fermentation Process Fundamentals of Fermentation  Fermentation, once solely associated with anaerobic processes carried out by yeast-like organisms, now encompasses both anaerobic and aerobic techniques. While anaerobic fermentation relies on the natural gas exchange within the fermentation vessel, aerobic processes necessitate the controlled supply of oxygen. To maintain optimal conditions, fermenters are designed to provide aseptic environments, regulate temperature and pH, and ensure efficient mixing and aeration. Principle of Fermentation During fermentation, microorganisms extract energy from carbohydrates without oxygen. Initially, glucose is partially broken down into pyruvate through glycolysis. Subsequently, pyruvate is transformed into either alcohol or acid, while simultaneously regenerating nicotinamide adenine dinucleotide (NAD+). This regenerated NAD+ is crucial for glycolysis to continue and produce more adenosine triphosphate (ATP). However, fermentation is significantly less efficient than aerobic respiration, yielding only about 5% of the energy. Figure 3. Common metabolic pathways used by various organisms to produce different fermentation end products from glucose. (Magar, 2021) Anaerobic Process. Fermentation is a biochemical process that occurs without oxygen. Energy Production. It extracts energy from the partial oxidation of glucose or other carbon sources. Metabolic Pathways. The oxidation of the substrate involves either the Embden-Meyerhoff-Parnas (EMP) or Entner-Doudoroff (ED) pathways. Pyruvate Production. These pathways lead to the production of pyruvate, ATP, and NAD(P)H. NAD+ Regeneration. In the absence of oxygen, pyruvate is reduced to regenerate NAD+(P). Product Formation. This reduction step is essential for the fermentation process to continue and results in the formation of various products, including ethanol and organic acids. ATP Generation. ATP is the primary energy currency produced during fermentation through substrate-level phosphorylation. NADH Re-oxidation. NADH is re-oxidized back to NAD+ in the second phase of fermentation. Product Diversity. The specific product formed depends on the organism and its metabolic capabilities. o Lactic Acid Fermentation. In bacteria like Streptococcus lactis, pyruvate is converted to lactic acid, regenerating NAD+ and producing ATP. o Alcoholic Fermentation. In yeasts like Saccharomyces cerevisiae, pyruvate is converted to ethanol, also regenerating NAD+ and producing ATP. Types of Fermentation 1. Lactic acid homofermentation Homolactic Fermentation - A type of fermentation where glucose is primarily converted into lactic acid. Key Organisms - Bacteria from genera Lactococcus, Enterococcus, Streptococcus, Pediococcus, and some Lactobacillus species. Process - These bacteria ferment glucose to produce lactic acid as the main end product. 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝑙𝑎𝑐𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 Industrial Application - Lactococcus species are widely used in dairy starter cultures for various fermented dairy products. 2. Lactic acid heterofermentation Heterolactic Fermentation - A type of fermentation where glucose is converted into a mix of products. Key Organisms - Bacteria from genera Leuconostoc, Oenococcus, Weissella, and heterofermentative Lactobacillus species. Process - These bacteria ferment glucose to produce lactic acid, ethanol/acetic acid, carbon dioxide, and water as end products. 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝑙𝑎𝑐𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑒𝑡ℎ𝑦𝑙 𝑎𝑙𝑐𝑜ℎ𝑜𝑙 + 2𝐶𝑂2 + 𝐻2 𝑂 3. Propionic acid fermentation Propionic Acid Fermentation - A type of fermentation where glucose is converted into a mix of products. Key Organisms - Bacteria from the genus Propionibacterium and the species Clostridium propionicum. Substrate Utilization - Both sugars and lactate can be used as starting materials for this fermentation. Metabolic Pathway - When sugars are available, the Embden-Meyerhof-Parnas (EMP) pathway is used to produce pyruvate. Pyruvate is then converted to propionate through a series of reactions involving oxaloacetate, malate, fumarate, and succinate. End Products - In addition to propionic acid, acetic acid and carbon dioxide are also produced as byproducts. 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝑙𝑎𝑐𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑝𝑟𝑜𝑝𝑖𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐶𝑂2 + 𝐻2 𝑂 4. Diacetyl and 2,3-Butanediol Fermentation This fermentation pathway involves the production of two key compounds: diacetyl and 2,3- butanediol. Diacetyl Production o Citric acid is metabolized into pyruvic acid and acetylmethylcarbinol. o Diacetyl is a byproduct of this reaction. 2,3-Butanediol Production o Diacetyl can be further reduced to 2,3-butanediol. o This reduction step is carried out by microorganisms from genera such as Enterobacter, Erwinia, Hafnia, Klebsiella, and Serratia. o The process involves a double decarboxylation step. 𝐷𝑖𝑎𝑐𝑒𝑡𝑦𝑙 ↑ 𝐶𝑖𝑡𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 → 𝑃𝑦𝑟𝑢𝑣𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐴𝑐𝑒𝑡𝑦𝑙𝑚𝑒𝑡ℎ𝑦𝑙𝑐𝑎𝑟𝑏𝑜𝑛 ↓ 2,3 − 𝐵𝑢𝑡𝑦𝑙𝑒𝑛𝑒 𝑔𝑙𝑦𝑐𝑜𝑙 It's important to note that diacetyl, while a precursor to 2,3-butanediol, can contribute off-flavors in certain fermented beverages like beer if not controlled. 5. Alcoholic Fermentation Process - Glucose is converted into ethyl alcohol. 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝐸𝑡ℎ𝑦𝑙 𝑎𝑙𝑐𝑜ℎ𝑜𝑙 Key Organisms - Yeasts and certain fungi and bacteria. Initial Step - Pyruvate is formed, either through the EMP pathway (yeasts) or the ED pathway (bacteria like Zymomonas).  Redox Balance - NAD+ is regenerated during the reduction of acetaldehyde to ethanol, maintaining redox balance in the fermentation process. Figure 4. Alcohol Fermentation. (Magar, 2021) 6. Butyric Acid Fermentation Process - Glucose is converted into acetic acid and butyric acid. 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝐴𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑏𝑢𝑡𝑦𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 Key Organisms - Obligate anaerobic bacteria, primarily from the genus Clostridium. Metabolic Pathway - Pyruvate is oxidized to acetyl-CoA, producing CO2 and H2. Product Formation - A portion of the acetyl-CoA is converted into acetic acid, generating ATP. Acetone-Butanol Fermentation - Certain bacteria, like Clostridium acetobutylicum, can produce fewer acids and more neutral products, leading to acetone-butanol fermentation. The Fermenter A fermenter is a specialized vessel engineered to facilitate microbial growth and product formation. It must fulfill several critical functions: Preventing the escape of microorganisms and ensuring aseptic conditions. Continuously tracking key parameters like pH, temperature, and pressure. Providing a stable environment with minimal energy and labor requirements. Enabling the transition from laboratory-scale to industrial-scale production. Essential Components of a Fermenter A typical fermenter comprises the following components: 1. Agitator a. Ensures uniform mixing of the culture medium. b. Promotes efficient mass transfer of oxygen and nutrients. c. Minimizes shear stress on cells. d. Common types of agitators include disc turbines, vaned discs, open turbines, and propellers. 2. Aeration System a. Delivers oxygen to the microbial culture. b. Spargers, such as orifice or nozzle spargers, are used to introduce air into the medium. c. The choice of aeration system depends on the specific requirements of the fermentation process. 3. Baffles a. Prevent vortex formation and improve mixing efficiency. b. Typically, four to eight baffles are installed along the vessel wall. By carefully designing and operating fermenters, it is possible to optimize microbial growth and product yield, making fermentation a vital tool in biotechnology and industrial production.  Figure 5. Typical design of a fermenter (Banerjee, 2023) IV. Types of Fermenters The choice of fermenter depends on various factors, including the type of microorganism, the nature of the product, and the desired scale of production. Here are some common types of fermenters: 1. Stirred Tank Fermenter a. A cylindrical vessel equipped with impellers for mixing and spargers for aeration. Figure 4 Figure 6. Stirred-tank fermenter (Butler, 2003) b. Widely used for batch fermentation of various products, including antibiotics, enzymes, and biofuels. c. Advantages: i. Good mixing and mass transfer. ii. Easy to control process parameters. iii. Suitable for a wide range of microorganisms. d. Disadvantages: i. High energy consumption due to agitation and aeration. ii. Potential for shear damage to cells, especially sensitive ones. 2. Bubble Column Bioreactor  Figure 7. Bubble Column Bioreactor (Abufalgha, Pott, & Clarke, 2021) a. A tall, cylindrical vessel with air sparged from the bottom. b. Used for aerobic fermentations of microorganisms that are not sensitive to shear stress. c. Advantages: i. Simple design and low operating costs. ii. High oxygen transfer rates. iii. Suitable for large-scale production. d. Disadvantages: i. Limited mixing efficiency, especially for viscous broths. ii. Potential for cell damage due to bubble coalescence 3. Airlift Fermenter Figure 8. Principle of Airlift Bioreactor (Barragán, Figueroa, Durán, & González, 2016) a. A cylindrical vessel divided into a draft tube and a downcomer. Air is sparged into the draft tube, creating a density difference that drives the circulation of the liquid. b. Used for aerobic fermentations where high oxygen transfer rates are required. c. Advantages: i. High oxygen transfer efficiency. ii. Low shear stress on cells. iii. Simple design and low operating costs. d. Disadvantages: i. Less precise control over mixing and mass transfer compared to stirred tank bioreactors. 4. Cylindro-Conical Fermenter Figure 9. Cylindrical-conical fermentation tanks, while more complex and expensive to build than simple cylindrical tanks, offer superior yeast separation. This reduces the need for multiple beer purification cycles, minimizing beer loss. ( Beer Tanks.eu , 2024) a. A cylindrical vessel with a conical bottom, commonly used in the brewing industry. b. Used for batch fermentation of beer and other alcoholic beverages. c. Advantages: i. Efficient yeast sedimentation and recovery. ii. Suitable for long fermentation periods. d. Disadvantages: i. Limited mixing and aeration capabilities. ii. Not suitable for all types of fermentation processes. 5. Fluidized Bed Bioreactors Figure 10. Fluidized bed bioreactor (Tiwari, Sonwani, & Singh, 2023) a. Specialized type of bioreactor where solid particles, often immobilized microorganisms or catalysts, are suspended in a fluid stream. b. Advantages: i. The fluidized bed configuration ensures efficient mixing and contact between the solid particles and the liquid phase, leading to improved mass and heat transfer rates.  ii. The fluidized bed nature helps maintain a uniform temperature distribution throughout the reactor, preventing localized hot or cold spots. iii. Fluidized bed bioreactors are well-suited for continuous operation, allowing for steady- state production. c. Disadvantages: i. Maintaining fluidization requires significant energy input, particularly for large-scale operations. ii. The design and operation of fluidized bed bioreactors can be more complex than other types of bioreactors. 6. Photobioreactors Figure 11 (a) Schematic diagram of a photobioreactor (Jochum, Moncayo, & Jo, 2018), (b) commercially available bioreactor (Everflow, 2024) a. Specifically designed for cultivating photosynthetic microorganisms, such as algae and cyanobacteria. b. These reactors provide a controlled environment with optimal light conditions, temperature, and nutrient supply. c. Common types of photobioreactors include: i. Tubular Photobioreactors: These consist of long, transparent tubes arranged in various configurations. ii. Bubble Column Photobioreactors: These use airlift to circulate the culture and provide oxygen. iii. Airlift Photobioreactors: These combine features of airlift and tubular photobioreactors. d. Key considerations for photobioreactor design include: i. Light Intensity and Quality: Proper illumination is crucial for photosynthetic organisms. ii. Temperature Control: Maintaining optimal temperature is essential for efficient growth and product formation. iii. Gas Exchange: Adequate oxygen supply and carbon dioxide removal are necessary. iv. Nutrient Supply: A balanced nutrient solution is required to support microbial growth. V. Applications of Fermentation Technology Fermentation technology has a wide range of applications, including: 1. Food and Beverage Production a. Beer, wine, and other alcoholic beverages b. Bread, yogurt, and other dairy products c. Vinegar, soy sauce, and other fermented foods 2. Pharmaceutical Production 3. Antibiotics (penicillin, streptomycin) 4. Vaccines 5. Enzymes 6. Vitamins  7. Biofuel Production a. Ethanol b. Biodiesel 8. Chemical Production a. Organic acids (citric acid, lactic acid) b. Amino acids 9. Industrial enzymes VI. Microbial Production of Specific Products Beer Production The brewing process involves several key steps: 1. Malting - Barley grains are germinated and dried to activate enzymes. 2. Mashing - The malted grain is mixed with hot water to extract sugars. 3. Lautering - The wort is separated from the spent grain. 4. Boiling - The wort is boiled with hops to add bitterness and flavor. 5. Fermentation - Yeast ferments the sugars in the wort, producing alcohol and carbon dioxide. a. Fermentation processes are categorized based on temperature requirements: i. Warm Fermentation - Conducted using Saccharomyces cerevisiae yeast. - Optimal temperature range: 15-20°C. - Commonly used for producing ales and certain lagers. ii. Cool Fermentation - Conducted using Saccharomyces pastorianus yeast. - Optimal temperature range: around 10°C. - Typically used for producing lagers, which undergo a longer maturation period. iii. Spontaneous Fermentation: - Relies on wild yeast and bacteria present in the environment. - No specific yeast strain is added. - Often used in traditional brewing methods, such as Belgian lambic beers. 6. Lagering Process - Some beers, particularly lagers, undergo a maturation period known as lagering. During this process, the beer is stored at low temperatures (near freezing) to allow for further clarification, flavor development, and the formation of a smooth, clean taste profile. 7. Maturation - The beer is aged to develop flavor and clarity. Figure 12. Installation of cylindroconical fermentation tanks in a brewery ( Beer Tanks.eu , 2024)  Xanthan Gum Production Xanthan gum is a complex carbohydrate composed of repeating sugar units. Its unique structure, with negatively charged side chains, allows it to form highly viscous solutions when mixed with water. This property makes it useful in various industries, including food, pharmaceuticals, and cosmetics. Xanthan gum is soluble in both hot and cold water but is insoluble in most organic solvents. It is also stable under freezing and thawing conditions. Figure 10. Structure of Xanthan gum Microbial Strain: Xanthomonas campestris Fermentation Process: Submerged, aerobic fermentation Product Recovery: Precipitation and drying Figure 13. Xanthan gum manufacturing steps (Brova, 2023) VII. Advantages and Disadvantages of Fermentation Technology Advantages: 1. Fermentation can improve the taste, texture, and nutritional value of food. 2. Fermentation can reduce the risk of foodborne illness by inhibiting the growth of harmful bacteria. 3. Fermentation processes often have low energy requirements and can utilize low-cost substrates. 4. Fermentation can contribute to sustainable practices by reducing waste and producing renewable products. Disadvantages: 1. Fermentation processes are susceptible to contamination by unwanted microorganisms. 2. Fermentation can be influenced by various factors, leading to variability in product quality. 3. Some fermentation processes, such as traditional winemaking, can take several months. References Beer Tanks.eu. (2024). Cylindrically-conical fermentation tanks for the fermentation and maturation of beer. Retrieved from Beer tanks: http://www.beertanks.eu/offer/tanks/cylindroconical-fermentation-tanks-pressure/ Abufalgha, A., Pott, R., & Clarke, K. (2021). Quantification of oxygen transfer coefficients in simulated hydrocarbon-based bioprocesses in a bubble column bioreactor. Bioprocess and Biosystems Engineering, 44, 1-9. doi:10.1007/s00449-021-02571-1 Banerjee, S. (2023, August 3). Design of a Fermenter. Retrieved from Microbe Notes: https://microbenotes.com/design- of-a-fermenter/ Barragán, L. P., Figueroa, J., Durán, L. R., & González, C. A. (2016). Chapter 7 - Fermentative Production Methods. In P. Poltronieri, & O. F. D'Urso, Biotransformation of Agricultural Waste and By-Products (pp. 189-217). Elsevier. doi:https://doi.org/10.1016/B978-0-12-803622-8.00007-0. Brova. (2023, 27 September). Xanthan Gum Manufacturing Process. Retrieved from Brova: https://brova.co/blog/xanthan-gum-manufacturing-process/ Butler, M. (2003). Hybridomas, Genetic Engineering of. In R. A. Meyers, Encyclopedia of Physical Science and Technology (Third Edition) (pp. 427-443). Academic Press. doi:https://doi.org/10.1016/B0-12-227410-5/00319-7 Dutta, R. (2008). Fundamentals of Biochemical Engineering. New Delhi: Ane Books India. Everflow. (2024). Photobioreactors (PBRs). Retrieved from Everflow: https://everflowglobal.com/photobioreactor/ Jochum, M., Moncayo, L., & Jo, Y.-k. (2018). Microalgal cultivation for biofertilization in rice plants using a vertical semi- closed airlift photobioreactor. PLOS ONE, 1-13. doi:10.1371/journal.pone.0203456 Kaur, H. (2024). 19 Fermentation Technology. Retrieved from Environmental Microbiology & Biotechnology: https://ebooks.inflibnet.ac.in/esp15/chapter/fermentation-technology/ Magar, S. T. (2021, 22 August). Fermentation- Principle, Types, Applications, Limitations. Retrieved from Microbe Notes: https://microbenotes.com/fermentation/ Tiwari, H., Sonwani, R., & Singh, R. H. (2023). Bioremediation of dyes: a brief review of bioreactor performance. Environmental Technology Reviews, 12(1), 83-128. doi:10.1080/21622515.2023.2184276