MEDIUM DESIGN OF FERMENTATION

Industrial Fermentation Medium The basic ingredients of media include carbon source nitrogen source inorganic salt growth factor and distilled water etc.

Medium Development

To maintain economic competitiveness, low-cost crude materials are frequently used

Levels of minerals and growth factors may be critical

Medium design should be a relatively simple process, but is often complicated by factors such as cost: availability of substrates, reliability of substrate supply, handling, storage, ease of preparation and storage, transportation of components, health and safety considerations, \

 It is often easy to forget that the primary consideration is really quite simple: to supply the microorganism or cell line with a source of necessary nutrients in a readily utilisable form. In an ideal world all medium components should readily dissolve in water in order to be available to the cell.

 Many media, especially at industrial scale, depart from this ideal, and contain suspended solids and oil phases that add to the complexity of sterilisation and fermenter operation.

 Many scientists have applied the technique of response surface methodology in order to optimise medium. Although undoubtedly a useful technique, especially in the very earliest stages of process definition, the same improvements in yields can be obtained simply by applying sound logic to the answers to the above questions.

 Select the correct carbon and nitrogen sources, add the required macro and micronutrients and growth factors, and the yield of cells should be very good.

 A simple calculation of how muchbiomass is wanted and how much product is required, and applying simple microbial kinetics can determine the optimum levels of the substrates added. The key point here is that ideally, though, understanding of cell physiology, and quantitative approaches to the latter, are probably all that is needed to formulate a suitable medium for

 Applying PlackettBurman design to identify significant factors (i.e., identify key nutrients which can be carried out by finding out the elemental composition of the cell line chosen), followed by central composite design (CCD) to determine the optimal level of medium constituents and their interaction

Media Type
Media can be solid, semi-solid or liquid. Most liquid media can, of course, readily be converted to solid media by addition of a suitable gelling agent, such as agar or gellan. Just make sure the gelling agent is fully dissolved and dispersed before attempting to sterilise the medium.

Microbiological Culture Media

Media used in bioprocessing can be broadly divided into three categories based on their composition: 1.synthetic, 2.semi-synthetic and 3.complex

Synthetic Media
Synthetic media are fully chemically defined. All the components are known, as is the specific concentration of each of the components. Such media are usually quite simple, containing a carbon/energy source, nitrogen source, and a range of salt (usually inor-ganic);

 Synthetic media are useful in research and laboratory situations where experimental accuracy is paramount and data interpretation needs to be clear. In general, such media are much more costly than other media types, especially if specific components such as vitamins and growth factors are required, as these ingredients tend to be very expensive when supplied in the pure form.

 Yields of cells tend to be much lower than those obtained when the same cell line is grown in either semisynthetic or complex media, as the synthetic media often do not match an organisms or cell lines exact requirements. It is, of course, easier to investigate the impact of single nutrients upon cell growth and physiology using these media than the more complex ones

Semi-synthetic Media
Semi-synthetic media are largely chemically defined, as above, but include one or more poorly specified component(s) of variable but controlled composition, for example, yeast extract, which is particularly useful if a cell line requires a range of B vitamins.

 Such media are useful in research and laboratory situations where a particular organism has a requirement for a substrate that would be too expensive to supply in the pure form on a routine basis.

 Such components are also valuable where the specific nutritional needs are not well known, and a rich source of many nutrients such as yeast extract is used in a shotgun approach to provision of minor nutrients.

 Plant, animal, fish and microbial extracts have routinely been used in the past to supply vitamins and essential growth factors for specific organisms. The current trend in medium design is to avoid extracts of animal origin, especially bovine sources, as there are potential health risks associated with these products.

Complex Media
Complex media are largely composed of substances that are usually of plant or animal origin. These materials vary from batch to batch, and composition is influenced by time of year, location of origin, and small changes in production methods.

 They are usually relatively cheap, can be supplied in bulk and the source should be reli-able. Substrates such as beet and cane molasses, corn steep liquor, soya bean meals and extracts, whey powders, hydrolysed starches and a range of many different oils all fall into this category.

Medium component
Carbon Nitrogen Other substrates: elements, trace elements, growth factors

Nutrient sources for industrial fermentation

Carbon & energy source + nitrogen source + O2 + other requirements Biomass + Product + byproducts + CO2 + H2O + heat

Fermentation media
Nutrient Carbon Nitrogen Raw material molasses, starch corn steep liquor, soybean meal, pure ammonia or ammonium salts, urea, nitrate salts, phosphate salts Vitamins biotin, yeast extract, beef and growth extract, corn steep liquor, factors wheat germ meal

Carbon source
A carbon source is necessary to provide the cell with energy as well as the material with which to grow and synthesise arange of primary and secondary metabolites. There is obviously a wide range of carbon sources and the one chosen should be appropriate to the organism but also to the economics of the process

GLucose
Glucose is universally acceptable for growth of most cell lines, be they animal, plant or microbial. Supplied as a powder in the pure form, the substrate is readily available, reliable, easily stored, easily handled and has no signifi cant implications for health and safety. These qualities make glucose a popular choice of carbon source.

Drawbacks og glucose utilization

(i) the possibility of the organism suffering from the Crabtree effect if glucose is over supplied in the initial stages of growth; (ii) the loss of available substrate to the Maillard reaction, which can occur if glucose is sterilised with a nitrogen source presen

Sucrose
Often the sugar of choice in research laboratories, sucrose, is utilised by many but not all cell lines. Commercially available sucrose comes in many forms and grades, from pure granulated forms to complex molasses solutions

 Sucrose does not tend to suffer from the same drawbacks as glucose and, although it can be subject to caramelisation when over sterilised, generally it can be autoclaved/sterilised with nitrogen compounds without the same problems as glucose.

LACTOSE
Lactose can only be utilised by a few cell types, e.g. Escherichia coli, and is usually only metabolised very slowly. The sugar is only really useful for some commercial processes that use the complex substrate whey, a byproduct of the dairy industry, which contains both lactose (approximately 50 g lactose per litre of whey and 4%

Other Sugars
Other sugars can be used as substrates but tend to be very specifi c to the cell line chosen, e.g. melobiose is sometimes used for the cultivation of yeast cells. Sugars falling into this category tend to be too expensive to use on a routine basis. If choosing an unusual sugar the questions to be posed are Why is this sugar required? and What

Oils
A number of oils are now routinely used as carbon sources in the bioprocess industries. Rapeseed oil (canola oil in the USA/Canada) and methyl oleate are two popular choices. Representing both a carbon and an energy source, oils are economical and readily avail-able.

1. Carbon sources (1) Starch (corn, wheat, potato )

Widely used in fermentation industry Starch dextrin glucose Advantages: cheaper than glucose

(2) Molasses
Byproduct of cane or beet sugar production A dark viscous syrup containing 50% -75% fermentable sugars (mainly sucrose) with 2% nitrogen, vitamins and minerals
Cheaper

NITROGEN SOURCE
The amount of nitrogen in any medium really determines the amount of biomass that will be achieved for the particular cell line, given that plenty of carbon is available and all required nutrients are present in the initial medium. Nitrogen is required for growth and synthesis of, for instance, proteins and nucleic acids.

2. Nitrogen source (1) Inorganic nitrogen source

Ammonia, ammonium and nitrate
Microbes can utilize it faster. After it is utilized, the pH of medium will be changed. (NH4)2SO4 2NH3 + H2SO4 NaNO3 + 4H2 NH3 + 2H2O +NaOH

(2) Organic nitrogen sources

Urea Yeast extract Peptones Corn steep liquor Soybean cake powder Peanut powder Bran hydrolysis liquid
corn steep liquor powder

Trace elements
Trace elements are regarded as micronutrients, being required only in tiny amounts, usually g quantities per litre. The functions of trace elements within the cell are many and varied but are usually associated with enzyme activity, where the trace element forms part of the enzyme or functions as a catalyst for the

 where complex or semi-synthetic media are utilised, it may not be necessary to supply these elements separately. Local water supplies may even contain adequate amounts of these elements. If, however, a very pure synthetic medium utilised in the laboratory, or if a cell has an absolute requirement for a particular element or ele-ments, they must be added to the medium.

Growth factors
Small amount of organic compounds necessary for microbial growth. e.g. amino acids, vitamins, biotin Sources: normally organic nitrogen source e.g. corn steep liquor

Corn steep Liquor, CSL

Corn steep liquor is the water extract byproduct resulting from the steeping of corn during the commercial production of corn starch and other corn products.

Different medium for different food products

Chinese distilled spirit solid medium Fruit spirit fruit juice or fruit sauce Beer wort Alcohol : starch mash Amino acid: starch mash Citric acid starch mash Lactic acid starch mash

Unit 2 Preparation of Starch Sugar

Starch is a large molecule that is made of branching chains of glucose molecules. It can be broken down into glucose by hydrolysis.

Starch found in grains, potatoes, beans, peas, cassava Typically 20-30% amylose, remainder amylopectin

Characteristics of starch
Insoluble in hot water Blue complex compound by reaction with iodine. Insoluble in 30% of ethanol solution or above. Hydrolyzed by acid and enzyme, the ultimate product is glucose.

starch granule

IV.A.ii.-52

Starch: Amylose

Amylose is a linear glucan with [1,4] glycosidic linkages

CH 2 OH O OH OH CH 2 OH O OH OH CH2 OH O OH OH OH Linear

CH2 OH O OH OH O OH Dglucose

O .....

[2 ]linkage ,2

reducingend

IV.A.ii.-53

Starch: Amylopectin

Amylopectin is a branched glucan with [1,4] and [1,6] glycosidic linkages

CH2 OH O OH OH CH2 OH O OH OH CH 2 OH O OH OH O Branched

.....

O .....

[2 ]linkage ,2 reducingend CH 2 OH O OH OH OH

Dglucose

[1 ]linkage ,1

.....

CH 2 O OH OH

O ...

Starch: Amylopectin
[1,6] branch points result in tree-like structure less extended (more compact) than amylose larger MW

[1,6] linkages

reducinge nd

Why should starch be hydrolyzed to glucose?

1. Most microbes could not utilize starch directly. 2. To obtain sugar from the starch of many different plants, rather than just sugar beets or sugar cane.

Conversion of starch to fermentable sugars 1. Acid hydrolysis method

Starch slurry is acidified to a pH value and then heated in a converter under pressure. (C6H10O5)n (C6H10O5)x C12H22O11 C6H12O6
amylum dextrin oligosaccharidesglucose

Due to heat and acid, there are

Glucose combination reaction

and decomposition
reaction

starch
acid

glucose Combination reaction oligosaccharide ( difficult to utilize )

Total lost of glucose 7

Decomposition reaction colour content (difficult to decolor) <1

How to avoid combination and decomposition reactions occur?

Concentration of starch Concentration of hydrolysis acid Reaction temperature
not too high

Advantage
Simple Time saving

Disadvantage More by-product, DE value only 90 Requiring fine starch Need corrosion resistant materials Need more energy for heating

Extent of hydrolysis measured using dextrose equivalents (DE ) value reducing sugars DE value 100 total solids

The higher the DE, the more sugars and less dextrins are present. The DE value of starch is zero and that of dextrose is 100.

DE

A B C

time

remove after

add sodium bicarbonate

Starch hydrochloric acid

and

minutes

test Benedicts cool

with

test sample iodine

with

2. Enzyme hydrolysis method Which enzyme is used to break down starch into sugar syrup?
Amylase Glucoamylase Also called: double enzyme hydrolysis

Endoenzymes

amylase

attacks [1,4] bonds to produce various oligosaccharides

Lower MW!

pullulanase attacks [1,6] bonds to produce various oligosaccharides (amylose fragments)

Exoenzymes(non-reducing end)

-amylase

attacks [1,4] bonds at non-reducing end to Sweet! produce maltose

glucoamylase attacks [1,4] bonds at non-reducing end to produce glucose

High MW!

Enzymes in processing starch

Enzyme
-amylase

Source
Bacillus subtilis

Action
Only -1,4-oligosaccharide links are cleaved to give dextrins with maltose and up to 50% (w/w) glucose Only -1,4-links are cleaved, from non-reducing ends, to give limit dextrins and maltose -1,4 and -1,6-links are cleaved, from the non-reducing ends to give glucose

-Amylase

Malted barley

Glucoamylase

Aspergillus niger

Pullulanase

B. acidopullulyticus Only -1,6-links are cleaved to give straight-chain maltodextrins

There are two basic steps in enzymatic starch conversion: liquefaction and saccharification

Production of Glucose Syrups from Starch

Corn Starch Slurry
Liquefaction Thermostable -amylase Gelatinization (105C, 5 min) Dextrinization (95C, 2h)

Liquefied Starch DE 10-15

Saccharification Glucoamylase (60C, pH 4.0-4.5, 24-72 h)

Glucose Syrups DE 95-96

1) Liquefaction
Higher temperature hydrolysis of starch involve a starch gelatinization process, dissolution of the nanogram-sized starch granules (swells and bursts)to form a viscous suspension.

During liquefaction, long-chained starch

molecules are partial hydrolysis into smaller branched and linear chains of glucose units (dextrins) , with concomitant loss in viscosity. Enzyme: -amylase End-point : DE vaule 20 30(why?) iodide brown

Why should be two steps, liquefaction and saccharification? a. Low DE value b. Retrogradation (ageing) c. Substance size

Q:Whats the difference between gelatinization and retrogradation?

2) Saccharification
Involving the production of glucose and a little maltose by further hydrolysis. Enzyme: glucoamylase End-point : DE is the highest

Advantage
Higher quality Save energy Protect environment

Disadvantage
Complex operation Time consuming

How to make high-fructose corn syrup?

Need 3 steps and 3 enzymes
alfa-amylase->shortening the starch
(Liquefaction)

glucoamylase->produces glucose
(saccharification)

glucose isomerase-> convert to fructose

(isomerization)

3. Acid-enzyme combination method

(Two-stage hydrolysis) Slurry is only partially converted by acid and then treated with an appropriate enzyme or enzymes until the conversion is complete.

1) Acid-enzyme hydrolysis Acid--- liquefaction Glucoamylase----- saccharification Suitable for: starch granule is hard

2) Enzyme- acid hydrolysis

-Amylase (thermostable) --- liquefaction Acid----- saccharification Suitable for: starch granule size is irregular

Production of Glucose from Starch

Items DE value Color Processing condition Energy consuming By-product Cycle time acid 91 10 HTP high acid/enzym enzyme e 95 0.3 HTP high 98 0.2 NPT low little long

more moderate short moderate