MICROBIAL CULTURES Group : Cysteine Tiah Chai Ching Norilyana Ramli Mohd Akhmam Yusof Nurul Maisyarah binti Samsudin

Types of Microbes

Microbial Growth Requirement for microbial growth Physical Requirement Chemical Requirement

Physical requirement Temperature –Microbes grow in a small range of temperature. –Their maximum temperature is 50 o C –Their minimum temperature is 20 o C –Growth best at 20 o C-30 o C pH –pH most bacteria grow best near neutral 6.5  7.5. –For yeast is at

Chemical Requirement Sources of carbon Source of nitrogen Source of oxygen Source of hydrogen Source of sulphur Source of phosphorous Source of trace elements

Nitrogen, Sulfur and Phosphorus –DNA synthesis needs nitrogen and phosphorus. –Protein synthesis need nitrogen and some sulphur –ATP need phosphorus –Trace element (example: iron. Copper, zinc)

Remember !! Microbes that introduced into a culture Innoculum Microbes that grow in/on a culture Culture

Simple Fungi Most yeast are aerobes Grow best on 20 o C to 30 o C Maximum operating temperature- 50 o C Need water in high concentration for growth and metabolism Operating pH 4.5 – 6.5 Media acidified with organic acid(acetic) are more inhibitory to yeast grow than media acidified with mineral acid(hydrochloric)

Characteristic of yeast  Yeast refers to the unicellular phase of the life cycles of many different fungi, but it is used more commonly as a generic term for fungi that have only a unicellular phase.  Yeast has simple nutritional needs.  Unable to carry out photosynthesis, they require a reduced carbon source which can be as simple compound as acetate and nitrogen source such as ammonium sulfate.

Yeasts can use a variety of organic nitrogen compounds (including urea and various amino acids) The only other complex compound that they require is the vitamin, biotin. they also require a variety of salts and trace elements. One of the more well known characteristics is the ability to ferment sugars for the production of ethanol. The most well-known and commercially significant yeasts are the related species and strains of Saccharomyces cerevisiae.Saccharomyces cerevisiae These organisms have long been utilized to ferment the sugars of rice, wheat, barley, and corn to produce alcoholic beverages and in the baking industry to expand, or raise dough.

The yeast's function in baking is to ferment sugars present in the flour or added to the dough. This fermentation gives off carbon dioxide and ethanol. The carbon dioxide is trapped within tiny bubbles and results in the dough expanding, or rising. Saccharomyces cerevisiae is commonly used as baker's yeast and for some types of fermentation. One yeast cell can ferment approximately its own weight of glucose per hour. Under optimal conditions S. cerevisiae can produce up to 18 percent, by volume, ethanol with 15 to 16 percent being the norm.

Requirement of Microbial Bioreactor

Bioreactor

Bioreactor used in the industry

Bioreactor used for yeast is stirred tank bioreactor. Conventional mixing vessel has low capital and low operating costs. Vessel with volume up to 20 litres is made from glass Larger volumes, used stainless steel. Height: Diameter ratio of the vessel is between 2:1 and 6:1, depending on the amount of heat to be removed. Stirrer may be on top or bottom

Baffles Tank is fitted with baffles to prevent central vortex and improve mixing. D<3m, 4 baffles of 6-8 inches are used. Larger vessel needed 6-8 baffles. The width of the baffle is usually between T/10 and T/12. ( T = tank diameter)

Baffled tank with six-blade turbine agitator with disk showing flow patterns: (a) side view, (b) bottom view, (c) dimensions of turbine and tank.

Volumes 75% of the designed volume is used as working volume. Eg.: In fermentation vessel about 75% of the total CSTR volume is filled with liquid, the remaining 25% is used for gas space. If foaming takes place, no chance for immediate contamination.

Agitators Controls the operation of the bioreactor, so careful consideration has to be given when designing the agitator Vessel with H=D, one agitator is sufficient. If H=2D or more, additional sets of agitators should be mounted on the shaft, separated by a distance, φ. Multi-sets of impellers improves mixing and enhances mass transfer

types of agitators: (a) high-efficiency, three- blade impeller (b) double-helical-ribbon, (c) helical-screw.

types of agitators: (a) four-blade paddle, (b) gate or anchor paddle, (c) six-blade open turbine, (d) pitched- blade (45 ) turbine.

Common type used four-blade disk turbine In shear-sensitive system where solids present, advantages in using inclined bladed turbine No. of agitator mounted on the shaft depend on the height of liquid in vessel

Impellers Installed to improve mixing and mass transfer

Spargers Located near the bottom of the vessel with a distance D i /2 below the agitator. (D i = diameter of the impellers) For efficient mass transfer, multiple-orifice ring sparger is used with gas outflow diameter of 3 D i /4 for more better, used single open-pipe sparging; located centrally beneath the disk.

Power Input Power input per unit volume of fermentation vessel for a normal fermenter should be greater than 100 W/m 3. Impeller tip speed (πND i ) > 1.5 m/s or a Froude number (N 2 D i /g) > 0.1

Troubleshooting High agitation and aeration cause foaming Foaming may lead to contamination Antifoaming can’t always be used, why? Because it may have inhibitory effect to microorganism growth So, the simplest device have rakes mounted on stirrer shaft, located on the surface of fluid

More sophisticated device is the ‘Funda-foam system’; foam is destroyed by centrifugal forces If heat removal is a problem, in bioreactor > 100m 3, 12 baffles can be used through which coolant passes

High turbulence is required for efficient mixing; created by vortex field Centrifugal force will draw the gas into the system, which ensures that sufficient turbulence To maintain the sterility, bioreactor must have minimum number of openings For Small opening made with O- ring(leak-proof),for larger opening fitted with gaskets

Most difficult areas to seal effectively is where the agitator shaft passes into the vessel; used double mechanical shaft seal Avoid any direct connection between the non-sterile and sterile area; that is sampling devices and injection port must be accommodated in steam-sterilisation closures.