DEFINITION OF FOOD FERMENATION:
Microbial(1) activities(2), usually anaerobic(3), on suitable substrate under controlled(4) or uncontrolled (5) conditions resulting(6) in the production of desirable (7) foods or beverages which are characteristically more stable(8), palatable(9) and nutritious(10) than the raw substrate.
What do fermentation scientist do:
1-Fermentation industry beer, wine, dairy fermentation, pickling, oriental fermented foods
2- Industrial fermentations organic acids, antibiotics, medicine
3-New product development genetic engineering
4-Teaching, Research, Extension University, Government, Industry
History of Fermentation
1-Pre-scientific ERA– before 17th century
n a-traditional microbiological process( beer, wine, bread, vinegar, cheese, pickling
n b-traditional food preservation sun drying, salting, freezing
n c-food borne diseases and spoilage accepted as fate or act of God
n a- Elucidation of biological processes through scientific developments in microbiology ,
n -improvement of traditional processes
n -development of new industries based on m.o. not previously exploited
n -antibiotics, enzyme biotechnology
n b-new preservation procedures freeze drying, microwave, radiation
n c-control of food spoilage and food borne diseases by understanding mechanism better
n a- continue to improve traditional methods
n c-new technologies
Some cornerstones of fermentation
n Discovery of microorganism by Antonie von Leewenhoek
n 1810 Canning by Appert
n 1854 Pasteur showed that microbes are responsible for fermentation.
n Fermentation is the conversion of sugar to alcohol to make beer and wine.
n Microbial growth is also responsible for spoilage of food. Bacteria that use alcohol and produce acetic acid spoil wine by turning it to vinegar (acetic acid).
General classification of microorganisms
n Microorganisms that carry out their metabolism using oxygen are referred to as aerobic microorganisms.
n Some microorganisms can substitute nitrate, others sulfate or ferric ion, for oxygen and thus grow in the absence of oxygen. These microorganisms are referred to as anaerobic.
n Obligate (or strict) aerobes: the presence of oxygen is required
n Obligate (or strict) anaerobes: the absence of oxygen is required, oxygen is toxic to the cells
n Facultative anaerobes: can survive with or without oxygen
n Microaerophiles: require low concentrations of oxygen and don’t do well either at atmospheric oxygen concentrations or without oxygen
n Biotechnology: Biotechnology is the scientific activity concerning the integrated application of biochemistry, microbiology and process technology on biological systems on the behalf of industrial processes and environmental management..
n Biochemical engineering The application of engineering principles to biologically based processes (in fermentation processes: manipulating living cells so as to promote their growth and production in a desired way); Maximize productivity Minimize costs
n Fermentation , the act or process of fermenting; a slow decomposition process of organic substances induced by microorganisms, or by complex nitrogenous organic substances (enzymes) of vegetable or animal origin, usually accompanied by evolution of heat and gas, e.g. alcoholic fermentation of sugar and starch, and lactic fermentation.
n All microorganisms need for their microbial activity the presence of several nutrients.
n Water: Major component of all fermentation process is consistent, clean water. PH, dissolved salts and microbial contamination are important factors. Long before plants are set up near by good water sources. Now water treatment is applied. There might be different characteristic water requirement for different type of food production.
n Hard water for English Burton beer, high carbonate water for darker beers.
n In SCP production large amount of water is used so recycling should be applied.
n -Carbohydrates Carbohydrates are capable of being used by all microorganisms,.
n Glucose is the most readily metabolized sugar.
n Most fungi can use disaccharide’s.
n Most common carbohydrate is starch( from maise, cereals and potatoes)
Sugar cane, sugar beet.
n Corn steep liquor which is by product of starch production from corn.
n -Hydrocarbons : petroleum products
n either from light(photosynthesis)or oxidation of medium components
n Most industrial organisms are chemoorganotrophs( common
energy source organic materials.
n Purines and pyrimidines :
n It is generally only in bacteria that cases of purine and pyrimidine metabolism have been reported.
Vitamins and growth factors
n There is considerable species variation in the requirements of vitamins and related factors by other microorganisms.
n Many of the natural carbon and nitrogen sources contain all or some of the required vitamins. Generally, vitamins A, C, D, and K are not necessary for growth. In glutamic acid production biotin is required.
n Inorganic nitrogen sources: ammonia gas and ammonium nitrates( cause alkaline drift), ammonium salts ( cause acidic drift)
n Organic nitrogen amino acids, proteins, urea
Chemical elements and inorganic ions
n Mineral nutrients required by microorganisms are species dependent but consists generally of Fe, K, Mg, Mn.
n Sometimes S, N, Ca, Co, Cu, P, Zn are required.
Microbial cells consist of a wide variety of chemical substances which have to be synthesized or taken up from outside the cell.
n These processes require a lot of energy.
n Each cell has to provide the necessary energy and different possibilities for its supply are developed: a number of organisms can use light energy; most microorganisms, of course, obtain it from the oxidation of chemical compounds.
n Chemicals used as sources for energy are metabolized and energy is conserved either by substrate-level phosphorylation or by building up an electrochemical gradient across the cytoplasmic membrane.
The major carbohydrate-metabolizing pathways are:
n Embden–Meyerhof–Parnas (EMP) pathway, also called glycolysis
n Entner–Doudoroff (ED) pathway
n pentose phosphate (PP) pathway.
n The three pathways differ in many ways, but two generalizations can be made:
n 1. All three pathways convert glucose to glyceraldehyde 3-phosphate (GAP) by different routes.
n 2. The GAP is converted to pyruvate via reactions that are the same in all three pathways.
Transport into the Cell
n 1. Passive transport: facilitated diffusion (e.g. Zymomonas mobilis and erythrocytes). As facilitated diffusion is only found in a few organisms or cell types, it will not be discussed further here.
n 2. Active transport: symport with a proton, protons or sodium ions. In this process of active transport the substrate can accumulate to a high concentration in the cytoplasm in a chemically unaltered form. Active transport requires energy and is linked to energy available from ion gradients or ATP hydrolysis.
n 3. Group translocation: phosphotransferase system (PTS). Group translocation is the process whereby a substance is transported while simultaneously being chemically modified, generally by phosphorylation.
n The process of sugar breakdown is called glycolysis.
n The enzymatic reactions of a glycolytic pathway will form pyruvate coupled to ATP synthesis by substrate-level phosphorylation.
n The overall reaction is:
n Glucose+2 ADP3−+2 Pi2−+2 NAD+→2 Pyruvate−+2 ATP4−+2 NADH+2 H++2 H2O
n There is a second important pathway for the breakdown of carbohydrates which is only found in prokaryotes.
n It was first discovered in 1952 by Entner and Doudoroff in Pseudomonas saccharophila.
n The ED pathway viewed as an alternative to the EMP pathway
n Prokaryotes, which carry out the ED pathway, lack the key enzyme phosphofructokinase of the EMP pathway.
n Because of the net production of only 1 mole ATP per mole glucose fermented, this pathway is usually found in aerobic bacteria
n The overall reaction is:
n Glucose+NADP++NAD++ADP3−+Pi2−→2 Pyruvate−+NADPH+NADH+3H++ATP4−
Pentose Phosphate Pathway
In bacteria, about 80% of the glucose is degraded aerobically via the EMP and ED pathway,about 20% enters the PP pathway for generation of ATP, regeneration of NADPH and the synthesis of precursors for nucleotide
and aromatic amino acid biosynthesis. So many variations of the PP pathway are possible, depending on the need of the growing cell.
The overall reaction is:
Glucose+ATP4−+2 NADP++H2O→Pentose 5-P2−+CO2+ADP3−+2 NADPH+3 H+
Distribution of Embden–Meyerhof–Parnas (EMP),
Entner–Doudoroff (ED), and pentose phosphate (PP) pathway in bacteria and some eukarya
n Oxidation of Pyruvate to Acetyl Coenzyme A
n In all the major carbohydrate catabolic pathways, pyruvate is a common product,
n oxidized during aerobic growth by the pyruvate–dehydrogenase complex:
n Pyruvate−+NAD++CoASH→AcetylCoA +CO2+NADH
The Tricarboxylic Citric Acid Cycle
To finish the respiratory metabolism of glucose, acetyl coenzyme A enters the tricarboxylic acid (TCA) cycle to produce carbon dioxide, water, reduced coenzymes and ATP
The overall reaction is:
Acetyl CoA+ADP3−+Pi2−+Q+2 H2O+NADP++2 NAD+ → 2 CO2+ATP4−+QH2+NADPH+2 NADH++H++3 H++CoASH
n Other Substrates as Sources for Metabolic Activity
n Living organisms can use a variety of substrates for growth:
n almost every naturally occurring organic compound can serve as a source for cell carbon or energy.
n These can be low-molecular-mass compounds or polymers, such as glycogen, starch, cellulose, polysaccharides, lipids, fatty acids and proteins.
n Polymers cannot enter the cell; they must be cleaved outside into monomers and dimers which are small enough to be transported into the cell and then enter the metabolic pathways.
Carbohydrates Glucose is not the only carbohydrate that can be converted to pyruvate by glycolysis
Anaerobic Breakdown of Carbohydrates
n The terms glycolysis and fermentation have been applied to the anerobic decomposition of carbohydrate to the level of lactic acid.
n The final product in some organisms is lactic acid; in others, the lactic acid is further metabolized anaerobically to butyric acid, butyl alcohol, acetone and propionic acid.
n The two most common forms of fermentation are lactic and alcoholic.
n In glycolysis, the same reactions occur whether oxygen is present or not.
n The products are primarily pyruvic acid, NADH and ATP.
n The essential difference between aerobic and anaerobic processes occurs with pyruvic acid and NADH.
n In the case of fermentation reactions, pyruvic acid is converted to a variety of organic compounds.
n These reactions involve the transfer of electrons and hydrogen from NADH to organic compounds.
n Fermentation is a major source of energy for those organisms that can only survive in the absence of air (obligate anaerobes).
n Other fermentative organisms that can grow in the presence or absence of air (facultative anerobes) use fermentation as a source of energy only when oxygen is absent.
n In fermentation, energy gain is very low and occurs as a result of substrate-level phosphorylation.
n The synthesis of ATP in fermentation is restricted to the amount formed during glycolysis.
n During glycolysis, glucose is oxidized to pyruvic acid, which is the physiologically important first intermediate product in the aerobic or anaerobic dissimilation of glucose.
n Pyruvate may also be reached via the metabolism of sugars other than glucose or the metabolism of fatty acids and amino acids.
n Pyruvate is a sort of Grand Central Station, in that it is the point of arrival and departure of a wide variety of metabolic substrates and products.
n Pyruvate is reduced to lactic acid.
n It may also be decarboxylated and reduced to ethyl alcohol.
n Conversely, it may serve as the source of amino acids, fatty acids and aldehydes.
n The anaerobic system of biological oxidations that does not use oxygen as the final acceptor of electrons is called anaerobic respiration.
n In anaerobic respiration, compounds such as carbonates, nitrates and sulphates are ultimately reduced.
n Many facultative anaerobic bacteria can reduce nitrate to nitrite under anaerobic conditions.
n This type of reaction permits continued growth when free oxygen is absent, but the accumulation of nitrite which is produced by the reduction of nitrate is eventually toxic to the organisms.
n Certain species of Bacillus and Pseudomonas are able to reduce nitrite to gaseous nitrogen. This process occurs when aerobic organisms are grown under anaerobic conditions.
n The organisms which reduce sulphate and carbonate are strictly anaerobic
n Desulfovibrio desulfuricans reduces sulphate to hydrogen sulphide as it oxidizes carbohydrate to acetic acid.
n Methanobacterium bryanti is able to couple the reduction of CO2 to methane with the oxidation of carbohydrate to acetic acid.
n Some organisms (strict aerobes) are enzymically equipped to use only free oxygen as the final hydrogen (e−) acceptor, but others (facultative aerobes) are equipped to use as the final hydrogen (e−) acceptor either free oxygen or some reducible inorganic substrate, commonly a nitrate.
n In fermentations, usually only NAD or NADP functions as the hydrogen (e−) carrier. Flavine adenine dinucleotide (FAD) and cytochrome systems are not required since the final hydrogen (e−) acceptor is not oxygen but an organic substance – commonly pyruvic acid.
n Fermentation may be caused by facultative organisms under anaerobic conditions, e.g. Saccharomyces cerevisiae, by strictly anaerobic organisms, e.g. Clostridium or by organisms that do not utilize free oxygen, e.g. species of Lactobacillus.
n Depending on the conditions of growth, the substrate and the organisms involved, the end products of fermentation vary greatly.
The products of glucose fermentation by all species of Streptococcus, many species of Lactobacillus and several other species of bacteria are mainly lactic acid with minor amounts of acetic acid, formic acid
n Several species of Streptococcus produce more than 90% of lactic acid based on the sugar used, and hence this type of fermentation is referred to as homolactic fermentation.
n The homolactic fermentation which forms only lactate is the characteristic of many of the lactic acid bacteria, e.g. Lactobacillus casei, Streptococcus cremoris and pathogenic streptococci, a heterolactic fermentation converts only half of each glucose molecule to lactate.
n Both these fermentations are responsible for the souring of milk and pickles.
n The heterofermentative metabolic sequence found in Leuconostoc and some species of Lactobacillus ferments glucose according to the equation: Glucose→Lactate+Ethanol+CO2
The heterofermentative metabolic sequence in Leuconostoc and some species of
n The major substrates yielding ethanol are the sugars which in yeasts are degraded to pyruvate by the EMP or glycolytic pathway.
n There is a net yield of one ATP for each pyruvate formed from glucose.
n The identical metabolic route for ethanol formation is found in the bacterial species Sarcina ventriculi, Erwinia amylovora and Zymomonas mobilis, which also possess the enzymes pyruvate decarboxylase and alcohol dehydrogenase.
n The butyric fermentation is initiated by a conversion of sugars to pyruvate through the EMP pathway.. The ATP yield per mole of glucose fermented is 2.5 moll
n Fermentation of glucose to butyrate by Clostridium butyricum, C. kluyveri and C. pasteurianum. FdF, Ferredoxin F
Mixed Acid (Formic) Fermentation
n This is a characteristic of most Enterobacteriaceae.
n These organisms dispose of their substrate in part by lactic fermentation
but mostly through pyruvate breaking down into formate and acetyl-CoA, which in turn generates an ATP.
n The formic fermentation yields three ATP per mole of glucose fermented (compared with two in lactic fermentation.
n This pathway extracts additional energy from the substrate.
n Pyruvate is carboxylated to yield oxaloacetate, which is reduced to yield succinate and is then decarboxylated to yield propionate.
n The lactate is first oxidized to pyruvate; part is then reduced to propionate and the rest is oxidized to acetate and CO2:
n This process of extracting energy from lactate yields only one ATP per nine carbon atoms fermented.
n Hence, propionic acid bacteria grow slowly
General pathways for the formation of fermentation products from glucose by various organism
Overview of fermentation products formed from pyruvic acid by different bacteria.
Butanol and acetone fermentation in Clostridium acetobutylicum.(strict anaerobe)
Metabolic regulators added
to media Precursors
: Some intermediary chemicals, when added to fermentation medium directly incorporated to product Example : phenylethylamine, phenylacetic acid in penicillin fermentation
: Compounds that increase production of final products during fermentation.( Common in enzyme fermentations)Example: To induce alphaamylase production via fermentation add starch( an inducer and substrate for the enzyme )
Inhibitors: When certan inhibitors are added to fermentations more of a specific product may be produced or a metabolic intermediate which is normally metabolised is acumulated. Example : Ethanol fermentation may be modified to produce glycerol by adding sodiumbisulphite (acetaldehyde is electron acceptor in forming NAD+ from NADH2 , if sodiumbisulphite is added it forms complex with acetaldhyde and it is no longer electron acceptor, instead dihydroxyacetonephosphate act as electron acceptor then when it is reduced glycerol is formed)
Typical Inoculum Preparation
CLASSIFICATION OF FERMENTATION PROCESSES
BASED ON DEPENDENCE OF PRODUCT FORMATION ON ENERGY METABOLISM
Type I: The product is derived directly from primary energy metabolism. ( growth, carbohydrate metabolism and product formation runs parallel.
Examples: Single cell production, ethanol, gluconic acid production
________________ specific growth rate
————————- specific carbohydrate consumption rate
…………………………… specific product formation rate
Type II: The product is also derived from the substrate used for the primary energy metabolism, but production takes place in a secondery pathway which imary meatabolism
Examples : citric, itaconic and some amino acids productions
Primarymetabolism functions first,accomponied by substrate consumption and growth. Afterwards the product is formed by the rections of intermediary metabolites.
Example: antibiotics and some vitamin productions
n Industrial fermentations may be carried out either batchwise, as fed-batch operations, or as continuous cultures.
n Batch and fed-batch operations are quite common, continuous fermentations being relatively rare.
n For example, continuous brewing is used commercially, but most beer breweries use batch processes.
(A) Batch fermentation.
(B) Fed-batch culture.
(C) Continuous-flow well-mixed fermentation.
(Feed: F, Xf, Sf ,Harvest : F, X, S, P, Volume: V time: t)
(D) Continuous plug flow fermentation, with and without recycling of inoculum.
Typical growth profile of microorganisms in a batch submerged culture
n A batch fermentation can be considered to be a closed system.
n At time t=0 the sterilized nutrient solution in the fermentor is inoculated with microorganisms and incubation is allowed to proceed.
n In the course of the entire fermentation, nothing is added, except oxygen (in case of aerobic microorganisms), an antifoam agent, and acid or base to control the pH.
n The composition of the culture medium, the biomass concentration, and the metabolite concentration generally change constantly as a result of the metabolism of the cells.
Physicochemical equilibration between microorganism and the environment following inoculation with very little growth.
By the end of the lag phase cells have adapted to the new conditions of growth. Growth of the cell mass can now be described quantitatively as a doubling of cell number per unit time for bacteria and yeast’s, or a doubling of biomass per unit time for filamentous organisms as fungi.
By plotting the number of cells or biomass against time on a semilogarithmic graph, a straight line results, hence the term log phase.
Although the cells alter the medium through uptake of substrates and excretion of metabolic products, the growth rate remains constant during the log phase. Growth rate is independent of substrate
concentration as long as excess substrate is present.
As soon as the substrate is metabolized or toxic substances have been formed, growth slows down or is completely stopped.
The biomass increases only gradually or remains constant during this stationary phase, although the composition of the cells may change.
Due to lysis, new substrates are released which then may serve as energy sources for the slow growth of survivors.
The various metabolites formed in the stationary phase are often of great biotechnological interest.
In this phase the energy reserves of the cells are exhausted. A straight line may be obtained when a semilogarithmic plot is made of survivors versus time, indicating that the cells are dying at an exponential rate.
The length of time between the stationary phase and the death phase is dependent on the microorganism and the process used.
The fermentation is usually interrupted at the end of the log phase or before the death phase begins
Cell growth modeling in a batch reactor
The simplest way to model cell growth will be to consider an unstructured, unsegregated model for cell growth. For this kind of model,
rx = dX/dt = µX
rx = rate of cell generation (g/l-hr)
X = cell concentration (g/l)
µ = specific growth rate (hr-1)
µ = µmaxS/(Ks +
µ= specific growth rate (hr-1)
µmax = maximum specific growth
S = substrate concentration (g/l)
KS = saturation constant for
substrate (g/l) ( substrate concentration at ½ of µmax)
Figure depicts the dependence of µ on S according to Monod’s equation.
One should note that Monod’s equation is empirical and does not have any mechanistic basis. The equation is only valid for an exponentially growing culture under condition of balanced growth.
The equation does not work well in transient conditions. Despite its simplicity and no fundamental basis, it works
Diauxic growth :
When two fermentable substrates are available readily fermentable one will be used first and when it is exhausted second one will be used.
(when glucose and galactose are exist in lactic fermentation glucose will be used first and then galactose)
An enhancement of the closed batch process is the fed batch fermentation. In the fed-batch method the critical elements of the nutrient solution are added in small concentrations at the beginning of the
fermentation and these substances continue to be added in small doses during the production phase.
n d(VX)/dt= VµX
n X* dV/dt+ V*dX/dt= VµX
n Since F= dV/dt
n µ= F/V+ 1/X*(dX/dt)
n D=F/V ( dilution rate)
nIn continuous fermentation, an open system is set up.
Sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously taken out of the system.
n In the chemostat in the steady state, cell growth is
n controlled by adjusting the concentration of one substrate.
n In the turbidostat, cell growth is kept constant by using turbidity to monitor the biomass concentration and the rate of feed of nutrient solution is appropriately adjusted.
Continuous bioreactor dynamics
n For a continuously fed bioreactor, the cells are continuously supplied substrate at growth limiting level, and hence they remain in the exponential phase.
nSince the cells remain in the exponential phase, Monod’s equation can be applied.
A cell balance on the reactor can be written
FX – FXf +
V(dX/dt) = rx (3)
F = volumetric flow rate (l/hr)
X = cell concentration inside the reactor and in the outlet stream
Xf = cell concentration in the feed (g/l)
V = reactor volume (l)
rx = rate of cell generation (g/l-hr)
nFor a sterile feed (Xf = 0), and noting that the reaction rate can be written in terms of the specific growth rate (rx = µX), equation (3) can be reduced to
dX/dt = (µ-
D = dilution rate = F/V (hr-1)
nA balance on the substrate yields the following equation
FS – FSf + V(dS/dt) =
F = volumetric flow rate (l/hr)
S = cell concentration inside the bioreactor and in the outlet stream (g/l)
Sf = substrate concentration in the feed (g/l)
V = reactor volume (l)
rs = rate of substrate consumption (g/l-hr)
parameter (Yx/s) is defined that relates the amount of cell mass produced per
amount of substrate consumed, and is mathematically represented as
Yx/s = mass
of cells produced/mass of substrate consumed = rx/-rs
equations (1), (5), and (6) yields
dS/dt = D(Sf – S) –
(continuous stirred tank bioreactor) is now completely described by equations
(4) and (7)
At SS (with fixed Sf and D), µ
= D (8)
S = DKS/(µmax –
X = Yx/s (Sf – S)
D must be less than µmax for a realistic value of S to be achieved.
Relationship between dilution rate and specific growth rate for a steady
n X = 0 (11) and S = Sf (12)
n Equation (11) and (12) define a situation called washout.
nThis situation is encountered whenever the value of dilution rate equals or exceeds µmax.
nA rigorous discussion of washout would point to the fact that whenever µ (Sf), i.e., µ evaluated at Sf , is less than µmax, then the critical dilution rate for washout will occur at D = µ (Sf), and not at D = µmax.
nStart up is an important consideration as well. The general procedure in the start up avoiding washout would be to initiate cell growth in a batch mode until the exponential phase is reached.
nAt this point, the sterile feed would be started with a dilution rate such that D < µ (Sf).
n A non washout steady state would be reached after a transient phase.