The Development of Inocula for Bacterial Processes

The Development of Inocula for Bacterial Processes

Introduction

• The main objective of inoculum development for traditional bacterial fermentations is to decrease lag phase.

• A long lag phase is not only is wastage of time but also medium is consumed in maintaining a viable culture prior to growth.

• The length of the lag phase is affected by the size of the inoculum and its physiological condition.  

• Bacterial inocula should be transferred, when the cells are still metabolically active.

• The age of the inoculum is particularly important in the growth of sporulating bacteria, for sporulation is induced at the end of the logarithmic phase and the use of an inoculum containing a high percentage of spores would result in a long lag phase in a successive fermentation.

• 5% inoculum of thermophilic Bacillus in logarithm phase is used for the commercial production of proteases.

• A two-stage inoculum development programme is used for the production of proteases by Bacillus subtilis.  Inoculum for a seed fermenter was grown for 1 to 2 days on a solid or liquid medium and then transferred to a seed vessel where the organism was allowed to grow for a further ten generations before transfer to the production stage.

• The lag phase in plant fermenters could be almost completely eliminated by using inoculum medium of the same composition as used in the production fermenter and employing large inocula of actively growing seed cultures in the production of bacterial enzymes.

• The inoculum development programme for a pilot-plant scale process for the production of vitamin B12 from Pseudomonas denitrificans is shown below (Spalla et al., 1989).

STOCK CULTURE

Lyophilised with skim milk

↓

MAINTENANCE CULTURE

Agar slope incubated 4 days at 28°

↓

SEED CULTURE - FIRST STAGE

2 dm³ flask containing 0.6 dm³ medium inoculated with culture from one slope; incubated with shaking for 48h at 28°

↓

SEED CULTURE - SECOND STAGE

40 - 80 dm³ fermenter containing 25 - 50 dm³ medium inoculated with 1 - 1.2% first stage seed culture. Incubated 25 - 30h at 32°

↓

PRODUCTION CULTURE

• 500 dm³ fermenter with 300 dm³ medium inoculated with 5% second stage seed culture. Incubated at 32° for 140 - 160 h

• The acetic-acid bacteria used in the vinegar process are extremely sensitive to oxygen starvation therefore it is essential to use an inoculum in an active physiological state.

• The cells at the end of fermentation are used as inoculum for the next batch by removing approximately 60% of the culture and restoring the original level with fresh medium.

• In this process there are enough chances of strain degeneration and contaminant accumulation.

• However, strain stability is a major concern in inoculum development for fermentations employing recombinant bacteria.

• Plasmid stability and productivity in E. coli biotin fermentation was improved if stationary, rather than exponential phase, cells were used as inoculum due to loss of plasmid in fermentation.

• In the lactic-acid fermentation the producing organism may be inhibited by lactic acid. Thus, production of lactic acid in the seed fermentation may result in generation of poor quality inoculum.

• High quality inoculum of Lactococcus lactis 10^-1 on a laboratory scale is obtained using electrodialysis which reduced the lactate in the inoculum and reduced the length of the lag phase in the production fermentation.

Development of Inocula for Anaerobic Bacterial Processes

• Clostridial Acetone-Butanol fermentation is anaerobic process.

• Though the process was outcompeted by the petrochemical industry but there is still considerable interest in reestablishing the fermentation.

• The inoculum development programme described by McNeil and Kristiansen (1986) is given as below

Heat-shocked spore suspension inoculated

into 150 cm³ of potato glucose medium

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Stage 1 culture used as inoculum for 500 cm³molasses medium

↓

Stage 2 culture used as inoculum for 9 dm³ molasses medium

↓

Stage 3 culture used as inoculum for 90,000 dm³ molasses medium

 

• The stock culture is heat shocked to stimulate spore germination and to eliminate the weaker spores.

• The production stage is inoculated with a very low volume.

• The use of such small inocula necessitates the achievement of as near perfect conditions as possible to prevent contamination and to avoid an abnormally long lag phase.

References

• Principles of Fermentation Technology

             (2^nd  edition,  by Peter F. Stanbury, Allan Whitaker and Stephen J. Hall, Butterworth-Heinemann, An  imprint of Elsevier Science.)

• Industrial Microbiology

          (By Casida L. E.New Age international (P) ltd publications)

• A Text Book of Industrial Microbiology

               (2^nd edition By Wulf Crueger & Anneliese Crueger)

• Biotechnology: Food Fermentation Microbiology, Biochemistry & Technology Vol. 1 & 2

           (By V.K. Joshi & Ashok Pandey)

• Manual of Industrial Microbiology and Biotechnology

(2^nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H.   Sherman, Richard C. Willson and J. H. David Wu)

• Industrial Microbiology-An introduction

(By Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary Higton)

• Comprehensive Biotechnology-The Principles, Applications and Rugulations of Biotechnology in Industry, Agriculture and Medicine

(By Mrray Moo Young)

• Fermentation Technology : Up Stream Fermentation Technology- Vol-I

(By H. A. Modi-Pointer Publications)

• Fermentation Technology : Down Stream Fermentation Technology- Vol-II

(By H. A. Modi-Pointer Publications)

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(4^th edition, edited by Gerald Reed, CBR publications)

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  (By M.L. Srivastava, NAROSA publications)

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(By A.H. Patel)

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(4^th edition. By James G. Chappuccino & Natalie Sherman)

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(By F.J. Baker)

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(By Gunasekaran)

• Mannual of Industrial Microbiology and Biotechnology

(2^nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu)

Web references

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