Lecture 1: Fermentation Fermentation Production of ... AWS

Lecture 1: Fermentation. Fermentation. • This is the metabolic processes of deriving energy by the degradation and oxidation of organic compounds in the absence of oxygen. • In the process, by-products are generated (e.g., ethanol, methane, organic acids). These by-products and the biomass are the materials of interest ...

Lecture 1: Fermentation Fermentation   

This is the metabolic processes of deriving energy by the degradation and oxidation of organic compounds in the absence of oxygen. In the process, by-products are generated (e.g., ethanol, methane, organic acids). These by-products and the biomass are the materials of interest in fermentation processes. Various chemicals have been generated by fermentation processes including: o Alcohols and solvents (acetone, butanol, ethanol, fusel oil and glycerol). o Acids (citric, fumaric, gluconic, itaconic, lactic, oxalic, and tartaric) o Amino acids (MSG, l-lysise HCl, DL-methionine, L-trytophane). o Antibiotics, vitamins o Others (yeast, polymers) o Enzymes Why produce ethanol? o Ethanol is an important material as beverage, fuel and starting material for the manufacture of chemicals including acetic acid, acetaldehyde, butanol and ethylene (key intermediate in the petrochemical industry). o The dramatic increase in the price of crude oil and the evidence of declining reserves has prompted the need for alternative fuel. Ethanol, a renewable resource, was seen as particularly promising. The ethanol fuel burns more cleanly and with higher efficiency. o Combustion of ethanol generates less NOx and CO and no SOx compared to gasoline. Brazil has generated 50 billion litres of ethanol in the first decade of its National alcohol program. Since then the production of ethanol globally has escalated. In Australia we use gasoline and ethanol blends (up to 5% ethanol).

Production of ethanol:

Organisms 

The principal organisms that are used in production of ethanol are yeast and bacteria. Alcohol fermentation is achieved commercially using yeast (Saccharomyces cervisiae) and some kinds of bacteria. The “waste” products of this process are ethanol and carbon dioxide (CO2).

Other organisms of primary interest to industrial operations in fermentation of ethanol include S. uvarum, Schizosaccharomyces pombe, and Kluyueromyces sp. and Clostridium sphenoides) o Yeast  Yeasts are single-celled fungi.  As fungi, they are related to the other fungi that people are more familiar with- edible mushrooms, baker’s yeast, and molds  Many consider edible yeast and fungi to be as natural as fruits and vegetables.  One species of yeast, Saccharomyces cerevisiae, has been "domesticated" over the centuries.  The scientific name of the genus of baker’s yeast, Saccharomyces, refers to “saccharo” meaning sugar and “myces” meaning fungus. The species name, cerevisiae, is derived from the name Ceres, the Roman goddess of agriculture. o Bacteria  A great number of bacteria are capable of ethanol formation. Many of these microorganisms, however, generate multiple end products in addition to ethyl alcohol.  These include other alcohols (butanol, isopropylalcohol, 2,3-butanediol), organic acid (acetic acid, formic acid, and lactic acids), polyols (arabitol, glycerol and xylitol), ketones (acetone) or various gases (methane, carbon dioxide, hydrogen).  Those microbes that are capable of yielding ethanol as the major product are mesophilic bacteria including Clostridium sporogenes, Zymomonas mobilis

Requirements for Growth 

pH o The products of microbial growth can cause shifts in pH which can affect the cell growth and metabolism. For ethanol fermentation, the pH must be controlled at pH 4-5.

O2 concentration: o A small concentration of oxygen must be provided to the fermenting yeast as it is a necessary component in the biosynthesis of polyunsaturated fats and lipids. Typical amounts of O2 maintained in the broth are 0.05 – 0.10 mm Hg oxygen tension.

Trace metals o Minerals (i.e. Mn, Co, Cu, Zn) and organic factors (amino acids, nucleic acids, and vitamins) are required in trace amounts.

Minerals o Phosphorus, sulfur, potassium, and magnesium must also be provided but in minor components

Nitrogen o For cell growth and metabolism yeast require include inorganic (e.g., NH4+) or organic sources of nitrogen (e.g., urea, amino acids, and peptides).

Vitamins and growth factors o The most common growth factors for yeasts are biotin, pantothenic acid, inositol, thiamine, nicotinic acid, and pyridoxine.

Water o Water is essential for microbial growth and metabolism. The extent to which water is available for biological metabolism is expressed as water activity (Aw) or, occasionally, as water potential (ratio of equilibrium water vapour pressure of the food to pure water at 20°C). Aw requirements of organism is from 0.70 to greater than 0.99.

Alcohol (Product) Inhibition o Yeasts are highly susceptible to ethanol inhibition. Concentration of 1-2% (w/v) are sufficient to retard microbial growth and at 8-18% (w/v) alcohol, the growth rate of the organism is nearly halted.

Temperature o The conversion of glucose to ethanol and CO2 is an exothermic reaction. The complete fermentation of a 180g/L glucose solution could raise the growth medium temperature by 20°C. o Every 5°C increase in temperature increases the evaporative loss of ethanol by 1.5. Yeast metabolism will also increase at 35°C. o Subsequent rise in temperature between 35-43 °C will reduce their metabolism. Cooling must be implemented if temperature rises above 35°C.

Carbon Feedstock o The feedstock consists of carbohydrates that are prepared to make them readily available for organism assimilation. o Complex polysaccharides (e.g., biomass) are made up of large organic polymers. In order for the organism to access the energy potential of the material, these chains must first be broken down into their smaller constituent parts. o These constituent parts or monomers such as sugars are broken down by hydrolysis. Usually this involves breaking them down to simple sugars (glucose, fructose, galactose, mannose, maltose and maltotirose).  Hydrolysis: can be achieved by:  chemical cleavage achieved with acid and temperature  Enzymes (hydrolases)  Invertase is used industrially to hydrolyze sucrose to so-called invert sugar (glucose + fructose).  Lactase is essential for digestive hydrolysis of lactose in milk.  β-Amylase catalyzes the conversion of starch to maltose (beer)  Cellulase is used in the hydrolysis of cellulose into glucose

o biomass substrate: for the production of alcohols is sugars, starches and cellulose:  Sugars  Sucrose is the primary form of sugar that is used in fermentation of ethanol. It is derived by mechanically crushing and squeezing the juice from sugar cane and stripping and pulping sugar beets. Sucrose is referred to as disaccharides consisting of glucose and fructose.  Yeast is able to break down sucrose into its monosaccharide sugars. It generates an enzyme called invertase to hydrolyse sucrose into glucose and fructose, which are then fermented by the yeast cell. 

Starches  Cornstarch consists of a water soluble fraction (20% amylase) and water insoluble fraction (80%) amylopectin. Cornstarch is derived from milled corn.  The preparation of cornstarch involves boiling the starch in water, which dissolves and makes it susceptible to hydrolysis by the α – amylase.  In the final phase of the preparation glucoamylase is added to catalyse saccharification, hydrolysis of starch to glucose.


 Cellulose is derived from plant or biomass materials (e.g., sugar cane bagasse) by processes including steam explosion and milling.  Both processes disrupt the lignin, hemicellulose and cellulose components of the biomass.  Hemicellulose is dissolved in the steam explosion method and the lignin is extracted either with methanol or dilute NaOH. The remaining cellulose is hydrolysed with an enzyme referred to as cellulose to glucose, which can be subsequently fermented by yeast and or bacteria.

Carbohydrate catabolism 

Microorganisms require substrates to: o Synthesize new cells o Synthesize extracellular products (e.g., ethanol) o Provide energy necessary to:  Drive the synthesis reactions  Maintain concentrations of materials within the cells which differ from those in the environment  Drive recycling (turnover) reactions within the cell.

 

These general reactions are represented by a flowsheet in Figure 2 (picture at the end of this lecture). Carbohydrate metabolism is the breakdown of carbohydrates into smaller units and the release of energy. The steps are: o Step 1: Hydrolysis:  This involves the use of specific enzymes in breaking down complex carbohydrates into simple monomeric sugars like glucose. o Step 2: Conversion of monomers to energy  When the monomer is glucose this process is referred to as Glycolysis. This step breaks down glucose into energy in the form of molecules of ATP (Adenosine-5'-triphosphate).  ATP is a multifunctional nucleotide that is most important as a molecular currency of intracellular energy transfer.

In this role, ATP transports chemical energy within cells for metabolism (cellular respiration, biosynthetic reactions:

o Step 3: Pyruvate fermentation:  The nature of the product formed in pyruvate fermentation will depend on the presence of oxygen and organism.  Anaerobic respiration  is the metabolic pathway in the absence of oxygen.  In the absence of oxygen fermentation of the pyruvate molecule, derived from the glycolysis process will occur.

 Aerobic respiration:  is the pathway where glucose is broken down in the presence of oxygen. When oxygen is present, acetyl-CoA is produced from the pyruvate molecules are generated by glycolysis.  Acetyl-CoA  is an important molecule in metabolism, used in many biochemical reactions.  Its main use is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidised for energy production  The mitochondria will undergo aerobic respiration which leads to the Krebs Cycle or citric acid cycle to generate various organic acids including citric, malic, lactic, oxalic, ketoglutaric acids.

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