STUDIES ON L-GLUTAMIC ACID PRODUCTION USING BREVIBACTER/UM SP.

237 Pages·2014·6.86 MB·English

by  Madhavan Nampoothiri K

STUDIES ON L-GLUTAMIC ACID PRODUCTION
USING BREVIBACTER/UM SP.
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BY
MADHAVAN NAMPOOTHIFII. l(.. M.sc..B.Ed.
UNDER THE SUPERVISION or
Dr. ASHOK PANDEY. M.Sc..D.Phil.
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DECLARATION
I, Madhavan Nampoothiri do hereby declare that the thesis entitled "Studies
on L-glutamic acid Production using Brevibacterium sp." is an authentic work
accomplished by me under the supervision of Dr. Ashok Pandey, Head,
Biotechnology Division, Regional Research Laboratory (Council of Scientific and
Industrial Research), Trivandmm, and also declare that the contents in the thesis
have not previously formed in any form for the award of any degree, diploma,
associateship, fellowship or other similar title or recognition.
In keeping with the general practice of reporting scientific observations, due
acknowledgement has been made wherever the work described is based on the
findings of other investigators. wt
MADHAVAN N AMPOOTHIRI, K.
08.07.1997
Trivandrum
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Council of Scientific & Industrial Research TEFL? (04-,P14)3f9-:1
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REGIONAL REsEARcH LABORATORY 490311.490;
Industrial Estate P.0.. Thiruvananthapuram-695 019. Kerala, India.
Dr. ASHOK PANDEY
Head, Biotechnology Division
July 08, 1997
CERTIFICATE
This is to certify that to the best of my knowledge and belief, the thesis
entitled "Studies on L-glutamic acid Production using Brevibacterium sp.", is a
record of bonafide research carried out by Mr. Madhavan Nampoothiri, under
my supervision and also that the contents in this thesis have not previously formed
in any form for the award of any degree, diploma, associateship or other similar title
or recognition.
A HOK PANDEY
ACKNOWLEDGEMENTS
With great respect, I place on record my sincere gratitude to
Dr. Ashok Pandey, who has kindly consented to supervise my Ph.D. studies.
I extend my submissive thanks to him for the inspiring guidance, timely
help, encouragement and the moral support he has given during the course
of the work. My special thanks are also due to his wife and kids for their
friendly approach and presenting plenty of memorable occasions.
I acknowledge with deep sense of gratitude, Dr. Vijay Nair,
Director and Dr. A.D. Damodaran, former Director, RRL, T rivandrum for
their interest in my studies and also for extending the laboratory facilities.
I appreciate and acknowledge the Council of Scientific and
Industrial Research, New Delhi for granting CSIR Junior and Senior
Research Fellowships.
I take this opportunity to express my sincere gratitude and
thankfulness to Dr.M Chandrasekharan, Dept. of Biotechnology, CUSAT,
Cochin; Dr. C. Balagopalan, CT CRI, Trivandrum; Prof Vzjayammal,
Dept. of Biochemistry, Kerala University; Dr.K. C.M. Raja and Dr. P.
Prema, RRL, T rivandrum, for their help, interest and cooperation.
It is really a pleasure for me to express my deep gratitude to Prof
JM Meyer, University of Louis Pasteur, France, for his outstanding
guidance and thought provoking discussions, dealing with the iron
requirement and the siderophore production studies in Brevibacterium sp.
I express my sincere gratitude to my dear colleagues, 1|/I/s. P.
Selvakumar, Scientist; K. Balakrishnan, Sailas Benjamin and Dr.
Ashakumari, for their cooperation throughout the period. I extend my
thanks to my other colleagues, Section Heads and stafi’ of this laboratory,
for their kind hearted and endless support.
I convey my indebtedness to my beloved parents for their care and
overwhelming encouragement which greatly helped me for the successful
completion of this work. I am also thankful to my sister and relatives. And
above all, I submit myself before the Almighty who has given me the might
to lead a restless life for the pursuit of knowledge.
MADHA VAN NAMPOOTHIRI, K.
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PREFACE
The thesis entitled "Studies on L-glutamic acid Production using
Brevibacterium sp." is carired out by the author at Regional Research Laboratory
(CSIR), Trivandrum during the period from July1992 to June 1997.
Amino acids are now widely used for their flavour enhancing, nutritional,
physiological and chemical properties involving the food, feed, agrochemical and
pharmaceutical industries. Among the various methods for the production of amino
acids, eg. protein hydrolysis, chemical synthesis and microbiological methods, the
latter has the advantage of providing the optically active and biologically required
L-forrn of amino acids from cheap carbon and nitrogen sources and hence the
fermentation technology represents and will remain the key position in the amino
acid industry.
Glutamic acid is the most important commercial amino acid obtained
exclusively by fermentation. It is a typical type of aerobic fermentation and still the
batch/fed batch process is the most popular fermentation method. Owing to the
importance of the particular industrial fermentation much efforts are still going on
in the line and thus, an attempt has been made to develop an indigenous technology
for the production of L-glutamic acid using a strain of Brevibacterium sp.
(DSM 20411).
The non-pathogenic corynefonn bacteria belonging to the genera
Corynebacterium and Brevibacterium are the chief bioconverters used for the large
scale production of amino acids and nucleotides. Apart from that these Coryneforrn
bacteria are widely used in many biotransfonnations such as the transformation of
steroids, terpenoids, degradation of hydrocarbons etc, they are known for the
production of a number of useful enzymes including amidases, nitrilases, proteases
and esterases and all of them can play vital roles in biotransformation experiments.
The thesis comprises a set of experiments mainly focused on the
improvement of L-glutamic acid fennentation. Much attention has been given to
use of locally available raw materials, culturing the organism on inert solid
substrates and also immobilization of the bacterial cells from the view point of long
term utilization of biocatalyst and continuous operation of the stabilized system.
Studies were also carried out for the down stream processing for the extraction and
purification of L-glutamic acid. An attempt was made to study the morphological
features of the microorganism including the cell premeability. In relation with the
accumulation of glutamic acid within the cells an approach was made to study the
behaviour of the Brevibacterium cells when they are exposed to hyper osmotic
environment. Attempts were also made to study the requirement of iron and
production of siderophores by this microbial strain. The search for a suitable
nitrogen source for glutamate fermentation ended with a promising result that they
got a potent urease activity and it can be utilized for many biotransfonnation
studies.
The entire thesis is presented in three sections, viz. introductory section,
experimental section and the concluding section.
The introductory section (A) contains a general intrduction, an extensive
survey of the recent literature available on the subject, followed by the significance
and specific objectives of the present investigation.
The experimental section (B) deals with various experiments conducted and
each one is described under separate sub-sections. In addition to a general materials
and method section (B.1), each sub-section has its own brief introduction, materials
and methods, and results and discussion.
The third and final section (C) contains the summary and prospects of the
present work, bibliography and a synoptic bio-data of the author, including the list
of research publications.
LIST OF TABLES
Page
Table A.1 Microbial strains producing L-glutamic acid
Table A.2 Biotechnological applications of Coryneform
bacteria
Table A.3 General characteristics of the genus
Brevibacterium
Table A.4 L-glutamic acid production from non­ 19
carbohydrate materials
Table A.5 Fermentation conversion of glutamate 20
producing bacteria by various environmental
factors
Table A.6 Sample importers of glutamate - 1995 28
Table A.7 Sample importers of glutamate - April 1996 29
Table B.1 Different media compositions for L-glutamic 45
acid production
Table B.2 Effect of temperature on L-glutamic production 53
by Brevibacterium sp.
Table B.3 Effect of ratio of volume of the medium and 55
flask in glutamic acid production
Table B.4 Effect of agitation on cell growth of 56
Brevibacterium sp.
Table B.5 Perfomiance of glutamic acid fermentation 57
with different agitation speeds
Table B.6 Effect of inoculum size on growth and 59
glutamic acid production by Brevibacterium sp.
Table B.7 Growth of Brevibacterium sp. on various 59
carbon sources
Table B.8 Mixed substrate fermentation for glutamic acid 65
production