FERMENTATION MEDIA

• Bacteria have to be grown (cultured) for them to be identified.

• By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study.

History

• The original media used by Louis Pasteur – urine or meat broth

• Liquid medium – diffuse growth

• Solid medium – discrete colonies.

Colony – macroscopically visible collection of millions of bacteria originating from a single bacterial cell.

• Cooked cut potato by Robert Koch – earliest solid medium

• Gelatin – not satisfactory

- liquefy at 24oC

Agar• Frau Hesse

• Used for preparing solid medium

• Obtained from seaweeds.

• No nutritive value

• Not affected by the growth of the bacteria.

• Melts at 98oC & sets at 42oC

• 2% agar is employed in solid medium

Types of culture media

I. Based on their consistency

a) solid medium

b) liquid medium

c) semi solid medium

II. Based on the constituents/ ingredients

a) simple medium

b) complex medium

c) synthetic or defined medium

d) Special media

Special media• Enriched media

• Enrichment media

• Selective media

• Indicator media

• Differential media patogensitas

• Sugar media

• Transport media

• Media for biochemical reactions

III.Based on Oxygen requirement

- Aerobic media

- Anaerobic media

Solid media – contains 2% agar

• Colony morphology, pigmentation, hemolysis can be appreciated.

• Eg: Nutrient agar, Blood agar

Liquid media – no agar.

• For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture.

• Eg: Nutrient broth

Semi solid medium – 0.5% agar.

• Eg: Motility medium

Simple media / basal media

- Eg: NB, NA- NB consists of peptone (5g), meat extract (3g), NaCl, - NB + 2% agar (15g)= Nutrient agar akuades 1 L

Complex media

• Media other than basal media.

• They have added ingredients.

• Provide special nutrients

Synthetic or defined media

• Media prepared from pure chemical substances and its exact composition is known

• Eg: peptone water – 1% peptone + 0.5% NaCl in water

Basal media

• Nutrient Agar, di larutkan dengan aquadest dalam Erlenmeyer (Erlenmeyer di beri label).

• Erlenmeyerdi beri media di tutup rapat kemudian panaskan kurang lebih 10 menit pada suhu 50 derajat celcius.

• Media di dinginkan kemudian media tersebut di bagi 2. Mulut erlenmeyer di sumbat dengan kapas, di tutup dengan kertas dan ikat dengan tali.

• Media di sterilkan dalam Autoclave selama 15 menit dengan suhu 121 derajat celcius.• Erlenmeyer pertama di perkirakan suhunya 80 derajat celcius, di tambahkan darah

vena sebanyak 3 cc dengan cara di semprotkan melalui dinding Erlenmeyer.• Media di kocok (jangan sampai terbentuk busa) dan di tuang ke dalam cawan petri

sebanyak detengah dari volume cawan petri.

Enriched media

• Substances like blood, serum, egg are added to the basal medium.

• Used to grow bacteria that are exacting in their nutritional needs. lysis of red blood

• Eg: Blood agar (Streptococcus pneumonia), Chocolate agar (Hemorhylus influenza)

Blood agar Chocolate agar

BLOOD AGAR• Utk membedakan kelompok mikro organisme yang melisis atau tidak

melisiskan butir darah merah.• Agar darah terdiri dari bahan dasar yang mengandung 5% darah domba. • Lisis butir darah merah terlihat sebagai wiayah jernih di sekitar koloni. Bila

proses lisis sempurna akan terlihat di wilayah yang benar-benar jernih dan jenis hemalisisnya di sebut beta – hemolisis.

• Bila proses lisis tidak sempurna dan media berwarna kehijauan maka jenis hemolisisnya di sebut Alpha Hemolisis.

• Bakteri yang tidak mampu melisiskan butir darah dan tidak menyebabkan perubahan nyata pada media tersebut di sebut gamma hemolisi.

• Kelompok mikro organisme yang sering di bedakan berdasarkan kemampuan melisiskan butir darah merah adalah sreptococcus dan staphylococcus, proses hemolisis di sebabkan oleh enzim yang di lepas mikro organisme.

• Komposisi : heart extract (10 g), tryphtose (10 g), NaCl (5 g), agar (15 g), akuades 1L

Enrichment media

• Liquid media used to isolate pathogens from a mixed culture.

• Media is incorporated with inhibitory substances to suppress the unwanted organism.

• Eg: • Selenite F Broth – for the isolation of

Salmonella, Shigella

• Alkaline Peptone Water – for Vibrio cholerae

Selective media

• The inhibitory substance is added to a solid media.

Eg:

• Mac Conkey’s medium (MCA) for gram negative bacteria (ex :Enterobacteriateae)

• Blue Suchrose Salt Agar (TCBS) – for V.cholerae

• LJ medium – M.tuberculosis

• Wilson and Blair medium – S.typhi

• Potassium tellurite medium – Diphtheria bacilli

• Salmonella Shigella Agar (SSA)

Komposisi TCBS :

- Yeast extract = 5 gram- Bacteriological Peptone = 10 gram- Sodium thiosulphate = 10 gram- Sodium Citrate = 10 gram- Ox bile = 8 gram- Sucrose = 20 gram- Sodium Chloride = 10 gram- Broom Thymol Blue = 0,04 gram-Thymol Blue = 0,04 gram- Agar = 14 gram- Aquadest = 1000mL- PH = 8,6

MCA media

• Persenyawaan utama dalam media ini adalah laktosa, garam empedu dan merah netral.

• Media ini menghambat pertumbuhan bakteri garam positif yang di sebabkan oleh garam empedu dan kristal violet, bakteri gram negatif yang tumbuh dibedakan dalam kemampuannya memfermentasekan laktosa.

• Koloni dari bakteri yang memfermentasikan laktosa berwarna merah bata dan di kelilingi oleh endapan garam empedu. Endapan ini di sebabkan oleh penguraian laktosa menjadi asam yamg bereaksi dengan garam empedu.

• Bakteri yang tidak memfermentasekan laktosa biasanya bersifat pathogen.

• Komposisi : pepton yeast extract (17 g), protease pepton (3 g) lactose (10 g), bile salt no 3(1.5 g), NaCl (5g), agar (13.5g), netral red(0.03 g), crystal violet (0.001g), akuades 1L

Potassium Tellurite media LJ media

Indicator media

• These media contain an indicator which changes its colour when a bacterium grows in them.

• Eg: • Blood agar

• Mac Conkey’s medium

• Christensen’s urease medium

• Lactose fermenters – Pink colonies

• Non lactose fermenters – colourless colonies

Urease medium

Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease.

Transport media

• Media used for transporting the samples.

• Delicate organisms may not survive the time taken for transporting the specimen without a transport media.

• Eg: • Stuart’s medium – non nutrient soft agar

gel containing a reducing agent

• Buffered glycerol saline – enteric bacilli

Anaerobic media

• These media are used to grow anaerobic organisms.

• Eg: Robertson’s cooked meat medium, Thioglycolate medium.

Type of energy sources and characteristic for a large

scale media

INTRODUCTION TO THE MEDIA OF FERMENTATION

•All micro-organisms require water, sources of energy, carbon,nitrogen, mineral element and vitamin plus oxygen in their growthmedium.

•On a small scale, it is simple to device a medium containing purecompounds, but the resulting medium although satisfy the growth,may be unsuitable for use in a large scale process.

On a large scale one must use sources of cheap nutrient to create a medium which will meet as many as possible of the following criteria:

1. It will produce a maximum yield of product at biomass per gram of substrate used.

2. It will produce a maximum concentration of product or biomass.

3. It will permit the maximum rate of product formation.4. It will be the minimum yield of undesired product.5. It will be cheap and of a consistent quality and is

readily available throughout the year.6. It will cause minimal problem in other aspects of

production and agitation, extraction, purification and waste treatment.

Use of molasses, cereal grain, glucose, sucrose and lactose as carbon sources

and ammonium salts, urea, nitrates, soya bean meal, slaughter-house waste and fermentation residues as nitrogen source

have tended to meet the above criteria for production media.

• The medium selected will affect the design of the fermenter to be used.

• A laboratory medium may not ideal in a large fermenter with a low gas-transfer pattern.

• Media with a high viscosity will also need a higher power input for effective stirring.

• Besides the requirement for growth and product formation, medium may also influence pH variation, foam formation, oxidation-reduction potential and the morphological form of the organisms.

• It may also be necessary to provide precursors or metabolic inhibitors.

Typical Media

MEDIUM FORMULATION• Medium formulation is an essential stage in the design of

~successful laboratory experiments

~pilot-scale development

~manufacturing processes

• The constituents of a medium must satisfy

~the elemental requirements for cell biomass and metabolite production

~must be an adequate supply of energy for biosynthesis and cell

maintenance

• Equation based on the stoichiometry for growth and product formation.Carbon and energy source + nitrogen source + other requirements cell biomass + products + CO2

+ H2O + heat

• This equation should be expressed in quantitative terms, which is economical design of media if component wastage is to be minimal.

• It should be possible to calculate~the minimal quantities of nutrients which will be needed to produce a specific amount of biomass

~the substrate concentration in order to produce required product yield

• A knowledge of the elemental composition of a named micro-organisms, which should include the content of C, H, O, N, S, P, Mg and K, is required in the solution of the elemental balance equation.

• Elemental composition of micro-organism in medium.

• From Table 4.2:

~the absolute minimum quantities that needed to include in an initial medium recipe are N, S, P, Mg and K.

~trace elements may also be needed in smaller quantities such as Fe, Zn, Cu, Mn, Co, Mo, B.

• An analysis of relative concentrations of individual elements in bacterial cells shows

~ some nutrients are frequently added in substantial excess of that required e.g. P, K

~others are often near limiting values, e.g. Zn, Cu.

• Some micro-organisms cannot synthesize specific nutrients such as amino acid, vitamins or nucleotides.

• Once a specific growth factor has been identified it can be incorporated into a medium in adequate amounts as a pure compound or as a component of a complex mixture.

• The role of carbon substrate involved in

~biosynthesis

~energy generation

Water• Major component of all fermentation media

• Factors need to be consideredpH

Dissolve salt

Effluent contamination

• Mineral water content of water is very important in brewing, and most critical in the mashing process, and historically influenced the siting of breweries and the type of beer produced.

• Nowadays, the water may be treated by deionization or other techniques and salts added, or pH adjusted, to favour different beers so that breweries are not so depend on the local water sources.

• In large continuous-culture SCP plants it is common practice to recycle the water streams.

• It may even be possible to reuse all the water in the fermenter fed with appropriate adjustment of nutrient levels.

Energy Sources• Energy for growth comes from either the oxidation of

medium components or from light.

• Most industrial micro-organisms are chemoorganotrophs, therefore the commonest sources of energy will be the carbon source such as carbohydrates, lipids and proteins.

• Some micro-organisms can use methane or methanol as carbon and energy sources.

Carbon Source• Carbon requirement for the medium is normally

provided by:

Nutrient Raw material

Sucrose Sugarcane, sugar beet molasses

Glucose Corn sugar, starch, cellulose

Lactose Milk whey

Fats Vegetable oil

Starch Maize grains, cereals, potatoes and casava

Hydrocarbons Petroleum fractions

Cont..

Barley grains

It may be partially germinated and heat treated to give material known as malt.

Malt:

i. Contains a variety of sugar besides starch

ii. Is the main substrate for brewing in many countries.

iii. Malt extract may also be prepared from malted grain.

Sucrose

• Obtained from sugar cane and sugar beet

• Commonly used in fermentation media in very impure form as beet or cane molasses

Lactose and crude lactose(milk whey)

• Now is extremely limited in media formulation since the introduction of continuous-feeding process.

• Whey is used as a substrate for biomass production

Vegetable oil

• Example: olive, maize, cotton seed, linseed, soya bean, etc.

• Source of carbon : oleic, linoleic and linolenic acid.

• As an antifoam in association with a surface active agent.

Other carbon sources:

• Example:

i. Alcohols

ii. Simple organic acids with alkanes

-more expensive than equivalent quantities of crude carbohydrate.

• These compound can be obtained in a pure form

-by simplify subsequent recovery and purification process

• For methane, methanol and n-alkalines:

- Utilized as a substrates for biomass production.

Vitamins• Biotin

also known as vitamin H or B7

chemical formula :

SONHC 321610

Jelaskan langkah pembuatan media :

• Blood agar

• Chocholate agar

• Mac Conkey’s medium

• Blue Suchrose Salt Agar (TCBS)

• LJ medium

• Wilson and Blair medium

• Potassium tellurite medium

• Salmonella Shigella Agar (SSA)

Jelaskan langkah pembuatan produk:

• Antibiotic (penicillin)

• Vitamin and amino acid

• Enzyme

• Biofuel (bioethanol /biogas)

• Industrial product (ex : wine, MSG)