Thursday, 23 February 2017

The Developement of Inocula for Mycelial Processes

Introduction

The majority of the industrial fermentation processes carried out using mycelial (filamentous) organisms like fungi and Streptomycetes

Vegetative fungi and spores of fungi used as inoculum.

The majority of industrially important fungi and Streptomycetes are capable of asexual sporulation so it is common practice to use a spore suspension as inoculum during an inoculum development programme.

A major advantage of a spore inoculum is that it contains far more 'propagules' than a vegetative culture.

A major advantage of a spore inoculum is that it contains far more 'propagules' than a vegetative culture.
  • Spores development [Sporulation] on solidified media
  • Spores development [Sporulation] on solid media
  • Spores development [Sporulation ]  on submerged culture media
First, we will see production of spores and then use of spore as inoculum.

Sporulation on Solidified Media

Most fungi and Streptomycetes will sporulate on suitable agar media but a large surface area must be employ to produce sufficient spores.
 
Roll bottle technique given by Parker (1950) for the production of spores of Penicillium chrysogenum on solid media.
 
In this technique three hundred cubic centimeters medium containing three percent agar sterilized in one cubic decimeter cylindrical bottles, which then, cooled to forty-five degree and rotated on a roller mill so that the agar set as a cylindrical shell inside the bottle.
 
These bottles inoculated with a spore suspension from a sub-master slope and incubated at twenty-four degree for six to seven days.

Sporulation on Solid Medium

Many filamentous organisms will sporulate freely on the surface of cereal grains from which the spores harvested.
 
Substrates such as barley, hard wheat bran, ground maize, and rice are all suitable for the sporulation of a wide range of fungi.
 
The sporulation of a given fungus affected by the amount of water added to the cereal before sterilization and the relative humidity of the atmosphere, which should be as high as possible during sporulation.
 
Fungi can produce relatively large number of spores on wheat bran or barley bran compared to solidified media like Nutrient agar and Sabouraud agar at particular temperature and humidity.
 
Humidity is very important for the growth of fungi and production of spores about ninety to ninety-eight percent of humidity is required.
 
Sporulation in Submerged Culture
 
Many fungi will sporulate in submerged culture provided a suitable medium is employed and suitable condition provided.
 
This technique is more convenient than the use of solid or solidified media because it is easier to operate aseptically and it may apply on a large scale.
 
The technique first adopted by Foster et al. (1945). He induced submerged sporulation in Penicillium notatum by including two point five percent calcium chloride in a defined nitrate-sucrose medium.
 
Medium components and other conditions favor the sporulation of fungi in submerged culture. According to Rhodes et al. (1957) the conditions necessary for the submerged sporulation of the griseofulvin-producing fungus Penicillium patulu, and the nitrogen level had to be limited to between point zero five and point one percent weight by volume and that good aeration had to maintain.
 
Most Actinomycetes do not sporulate in submerged culture due to this limitation they are more suitably cultivated using solid or solidified media for the production of spore inocula.
 
The Use of the Spore Inoculum
 
For the production of product at large scale, spore itself or vegetative cells developed from the spores used depending on the organism’s fermentation quantity and processes.
 
Some fermentation process can proceed with both either spore or vegetative cell produced from the spore.
 
In the clavulanic acid process the spore inoculum used to, inoculate the final seed stage. In the chlortetracycline process, a vegetative stage of fungi is use for the fermentation process.
 
Direct spore inoculation would avoid the cost of installation and operation of the seed tanks whereas the use of germinated spores would reduce the fermentation time of the final stage thus allowing a greater number of fermentations to carry out per year.
 
However, labor costs for the production of the vegetative inoculum could be almost as high as for the final fermentation although some of these costs may recover.
 
Inoculum Development for Vegetative Fungi
 
Some fungi will not produce asexual spores and therefore such process must use an inoculum of vegetative mycelium.
 
Gibberella fujikuroi is a fungus used for the commercial production of gibberellin. Cultures grow on long potato dextrose agar slants for one week at twenty-four degree.
 
Growth from three slants scraped off and transferred to a nine cubic decimeter. This medium aerated for seventy-five hours, at twenty-eight degree before transfer to a hundred cubic decimeter seed fermenter containing the same medium.
 
The major problem in using vegetative mycelium as initial seed is the difficulty of obtaining a uniform standard inoculum.
 
The procedure may improve by fragmenting the mycelium in a homogenizer such as a Waring blender prior to use as inoculum.

The Effect of the Inoculum on the Morphology of Filamentous Organisms in Submerged Culture
 
When filamentous fungi grown in submerged culture they can grow as 'pellet' form consisting of compact discrete masses of hyphae or as the filamentous form in which the hyphae form a homogeneous suspension dispersed through the medium.
 
When they form pellet they will not be able to grow properly due to nutrients and oxygen-limiting conditions inside the pellet while if they form filamentous forms than their distribution may not be proper in production medium to their filamentous growth.
 
The information available on the morphology of Actinomycetes in submerged culture is very limited compared with that on fungi.

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