LACTIC ACID PRODUCTION BY IMMOBILIZED RHIZOPUS ORYZAE IN A ROTATING FIBROUS ...

265 Pages·2004·7.15 MB·English

LACTIC ACID PRODUCTION BY IMMOBILIZED RHIZOPUS ORYZAE
IN A ROTATING FIBROUS BED BIOREACTOR

DISSERTATION

Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University

By

Nuttha Thongchul, M.S.

*****

The Ohio State University

2005

Dissertation Committee:
Approved by
Professor Shang-Tian Yang, Advisor

Professor Jeffrey J. Chalmers
__________________________________
Professor James F. Rathman Advisor
Graduate Program in Chemical Engineering

ABSTRACT

Lactic acid has long been widely used in food and pharmaceutical industries. Due
to the evolution of new applications in solvent replacement, biodegradable polymer, and
oxygenated chemicals, the estimated worldwide market of lactic acid, especially the
L(+)-isomer, tends to increase rapidly. Lactic acid bacteria have been extensively used in
industrial production of lactic acid because of their high growth rate and product yield.
However, the major limitations including costly substrates and complicated product
recovery make bacterial fermentation economically unattractive. In contrast, filamentous
fungi, Rhizopus oryzae, can produce the optically pure L(+)-lactic acid from complex
carbohydrates present in agricultural residues and plant biomass without prior treatment;
therefore, can overcome the problems in bacterial fermentation.
However, cultivation of filamentous fungi in a stirred tank bioreactor is usually
troublesome because of the diversity and change in morphology during fermentation
which in turn affects lactic acid production. Therefore, in this research, fungal
morphology was controlled by immobilization. Contrary to free cell culture in the stirred
tank bioreactor, the fermentation carried out in a Rotating Fibrous Bed bioreactor
(RFBB) resulted in good control of morphology, and improved oxygen transfer and lactic
acid production from glucose. The improved oxygen transfer obtained in the RFBB not
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only increased lactic acid production rate, but also limited undesirable ethanol production
and allowed the bioreactor to be operated for long-term production.
To minimize the production cost, the feasibility of using low-value substrates
derived from agricultural residues and plant biomass was studied. It was found that R.
oryzae was capable of utilizing both starchy materials present in agricultural residues and
pentose sugars which were abundant in plant biomass. Lactic acid yields obtained from
these substrates were comparable to the yield from glucose. However, the production rate
obtained from fermentation of pentose and insoluble starch was lower than that obtained
from fermentations of glucose and soluble starch because of the complicated pentose
metabolism and poor oxygen transfer in the cultivation with insoluble starch.
Process engineering techniques were also used to improve lactic acid production
in the RFBB. Previous study reported the critical demand of oxygen for lactic acid
production. In this research, it was found that increasing oxygen transfer rate led to the
increase in lactic acid productivity. Although high oxygen transfer rate was maintained,
ethanol production and the estimation of the critical biofilm thickness indicated an anoxic
condition in the overgrown immobilized fungal cells on the rotating fibrous matrix. In
this study, growth of immobilized cells was controlled by shaving-off the mycelia with
rotational shear rate and by limiting the concentration of the nitrogen source in the
medium.
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In order to achieve controlled growth and immobilization of productive cells for
stable long-term operation, spore germination and cell immobilization at the initial phase
was studied. The effect of rotational speed on spore immobilization on different fibrous
matrices was investigated. The mechanisms of spore immobilization on different fibrous
matrices were elucidated.
The knowledge gained in these process engineering techniques is important to the
development of the RFBB and its scale-up for lactic acid production from sugars.

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Dedicated to my mother

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ACKNOWLEDGMENTS

My great appreciation goes to my advisor, Dr. Shang-Tian Yang, for intellectual
support, encouragement, and enthusiasm throughout my study, and for his patience in
correcting both my stylistic and scientific errors. I have gained a lot from his insights and
I have enjoyed my staying at Ohio State as his student.
I would like to acknowledge with sincere gratitude to the members of my
dissertation committee, Dr. Jeffrey J. Chalmers and Dr. James F. Rathman. I am grateful
for their helpful advices on various topics.
I wish to thank Dr. Nalin Nilubol, Dr. Suraphong Nawankasattusat, and Dr.
Amorn Petchsom for providing me help and opportunity to pursue my Ph.D. study.
The scholarship from Chulalongkorn University is gratefully acknowledged.
Financial supports from the U.S. department of agriculture (SBIR), Consortium for Plant
Biotechnology Research, Inc., and Environmental Energy, Inc. to various phases of this
work are also acknowledged.
I also appreciate Mr. Carl Scott, Mr. Paul Green, and Mr. Leigh Evrard for their
technical assistances.
My colleagues in my research group, especially Ms. Supaporn Suwannakham,
Ms. Suwattana Pruksasri, Dr. Liping Wang, and Mr. Anli Ouyang, offered many kinds of
support and help over the last 4 years. I benefited a lot from discussions with them.
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I feel indebted to my best friend, Mr. Kovit Visessompak, for his helps in many
ways during the last 4 years. I also wish to thank Mr. Taradon Luangtongkum, who is
always by my side, for his warm support and understanding.
Special thank goes to my family for their love and support. Without their love, I
would not be able to deal with ups and downs during my study. Finally, my heartfelt
gratitude goes to my mother, Ms. Sudathip Thongchul, for her love and inspiration. My
graduation could have not been possible without her warm support and understanding.

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VITA

October 5, 1974(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).. (cid:133)(cid:133)(cid:133)(cid:133)..Born (cid:150) Bangkok, Thailand

July, 1995(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).....B.S. Chemical Engineering
Chulalongkorn University
Bangkok, Thailand

August, 1998(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)M.S. Bioprocess Technology
Asian Institute of Technology
Pathumthani, Thailand

September, 1998 (cid:150) present(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)...Researcher
Chulalongkorn University
Bangkok, Thailand

PUBLICATION

Thongchul, N. and Yang, S.-T., Controlling filamentous fungal morphology by
immobilization on a rotating fibrous matrix to enhance oxygen transfer and L(+)-lactic
acid production by Rhizopus oryzae. In Fermentation Biotechnology, ed. B.C. Saha.
Oxford University Press, MA, 2003, pp. 36-51.

FIELD OF STUDY

Major Field: Chemical Engineering

Specialty: Biochemical Engineering

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TABLE OF CONTENTS

Page
Abstract(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).................(cid:133)(cid:133)ii
Dedication(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).........(cid:133)(cid:133)v
Acknowledgments(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..(cid:133).vi
Vita(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)....(cid:133)viii
List of Tables(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)....................(cid:133).xiii
List of Figures(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)...(cid:133)..xv

Chapters:

1. Introduction(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133).. (cid:133)..1

2. Literature Review(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..(cid:133).13

2.1 Significance of Filamentous Fungal Fermentation(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)...(cid:133)..13
2.2 Fungal Morphology in Submerged Fermentation(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..15
2.2.1 Substrate(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)...(cid:133)..20
2.2.2 pH(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..24
2.2.3 Shear rate(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..26
2.2.4 Dissolved oxygen(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133).31
2.3 Reaction Systems Used in Submerged Filamentous Fungal Fermentation(cid:133)..(cid:133)..37
2.4 L(+)-Lactic Acid Production(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..41
2.4.1 Metabolic pathway of R. oryzae(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..(cid:133)..42
2.4.2 L(+)-lactic acid production by R. oryzae(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..43
2.5 Conclusion(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..44
2.6 References(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..46

3. Controlling Filamentous Fungal Morphology by Immobilization on a Rotating
Fibrous Matrix to Enhance Oxygen Transfer and L(+)-Lactic Acid Production
by Rhizopus oryzae(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..69

Summary(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..69
3.1 Introduction(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)...(cid:133)..71
3.2 Materials and Methods(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..(cid:133)..74
3.2.1 Culture, inoculum preparation, and medium compositions(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..74
3.2.2 Rotating Fibrous Bed Bioreactor(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).(cid:133)..74
3.2.3 Determination of volumetric oxygen transfer coefficient(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)..75
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