Optimisation and scale-up of L(+)-lactic acid production using … · 2010-06-07 · Optimisation...

Optimisation and Scale-up of a Biotechnological Process

for Production of L(+)-Lactic Acid from Waste Potato

Starch by Rhizopus arrhizus

Zhanying Zhang

The University of Adelaide

May 2008

Optimisation and Scale-up of a Biotechnological Process for

Production of L(+)-Lactic Acid from Waste Potato Starch by

Rhizopus arrhizus

Zhanying Zhang

B.Eng. (Biochemical Engineering)

M.Eng. (Biochemical Engineering)

Thesis Submitted for the Degree of

Doctor of Philosophy

School of Chemical Engineering

The University of Adelaide

May 2008

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page I

TABLE OF CONTENTS

Table of Contents ...................................................................................................................... I

Abstract................................................................................................................................... VI

Declaration............................................................................................................................VIII

Acknowledgements ..................................................................................................................X

Preface..................................................................................................................................... XI

Chapter 1. Introduction............................................................................................................1

1. L(+)-Lactic Acid and Its Application .................................................................................2

2. Waste Potato Starch and Environmental Concerns ............................................................2

3. Biological Production of L(+)-Lactic Acid.........................................................................3

4. Technical and Economic Chanllenges Associated with L-Lactic Acid Production

by Rhizopus fungi ...............................................................................................................4

5. Aim of This Project.............................................................................................................6

6. References...........................................................................................................................7

Chapter 2. Literature Review: Production of Lactic Acid from Renewable Materials

by Rhizopus Fungi ..................................................................................................9

Statement of Authorship .......................................................................................................10

Abstract .................................................................................................................................11

Contents ................................................................................................................................11

1. Introduction.......................................................................................................................12

2. Substrates for Lactic Acid Production .............................................................................12

2.1 Starch ..........................................................................................................................13

2.2 Lignocellulose.............................................................................................................13

3. Simultaneous Saccharification and Fermentation (SSF) ..................................................13

4. Bioprocess Parameters in Lactic Acid Production............................................................14

4.1. Nutrients.....................................................................................................................14

4.1.1. Nitrogen ..............................................................................................................14

4.1.2. Inorganic Salts ....................................................................................................14

4.2. Morphology................................................................................................................15

4.3. Immobilization...........................................................................................................15

4.4. pH...............................................................................................................................16

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page II

4.4.1. Effect of pH.........................................................................................................16

4.4.2. Neutralizing Agents ............................................................................................16

4.5. Oxygen Supply...........................................................................................................16

4.6. Temperature ...............................................................................................................17

5. Bioreactor System and Scale-up .......................................................................................17

6. Process Modelling.............................................................................................................18

7. Economic Evaluation of Fermentation Processes.............................................................18

8. Mechanisms of Lactic Acid Production by Rhizopus species ..........................................19

8.1 Lactate Dehydrogenase Enzymes ...............................................................................19

8.2 Molecualr Genetic Strategies to Increase Lactic Acid Production .............................19

8.3 Metabolic Flux Analysis .............................................................................................20

9. Conclusions.......................................................................................................................20

References.............................................................................................................................20

Chapter 3. Experimental Materials and Methods ...............................................................24

1. Microorganism and Media................................................................................................25

1.1 Microorganism............................................................................................................25

1.2 Preculture Medium......................................................................................................25

1.3 Production Medium ....................................................................................................25

2. Bioreactor Systems ...........................................................................................................26

3. Cultivation Conditions ......................................................................................................28

3.1 Precultures...................................................................................................................28

3.2 Experimental Set Up and Operation ...........................................................................28

4. Measurement and Analysis ...............................................................................................28

4.1 Measurement of Initial kLa..........................................................................................28

4.2 Analysis of Morphology and Biomass........................................................................29

4.3 Analysis of Samples by HPLC ..................................................................................29

4.4 Calculation of L(+)-Lactic Acid Yield and Productivity............................................30

4.5 Data Analysis ..............................................................................................................31

5. References.........................................................................................................................31

Chapter 4. Production of Lactic Acid and Byproducts from Waste Potato Starch

by Rhizopus arrhizus: Role of Nitrogen Sources ............................................32

Statement of Authorship .......................................................................................................33

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page III

Summary ...............................................................................................................................34

Introduction...........................................................................................................................34

Materials and Methods..........................................................................................................35

Microorganism...................................................................................................................35

Culture Media and Cultivation Methods ............................................................................35

Sample Preparation and Analytical Methods .....................................................................36

Results and Discussion ..........................................................................................................36

Effect of Nitrogen Sources.................................................................................................36

Kinetics of the Formation of Lactic Acid, Fumaric Acid and Ethanol ...............................39

Conclusion.............................................................................................................................41

References .............................................................................................................................41

Chapter 5. Production of Lactic Acid Using Acid-Adapted Precultures of

Rhizopus arrhizus in a Stired Tank Reactor ......................................................42

Statement of Authorship ........................................................................................................43

Abstract .................................................................................................................................44

Introduction...........................................................................................................................44

Materials and Methods..........................................................................................................45

Microorganism...................................................................................................................45

Preculture Medium ............................................................................................................46

Production Medium ...........................................................................................................46

Preparation of Acid-Adapted Precultures...........................................................................46

Lactic Acid Production in the STR ....................................................................................46

Analytical Methods............................................................................................................46

Results ...................................................................................................................................47

Effect of pH on Precultures................................................................................................47

Effect of Acid-Adapted Precultures on the Morphology of R. arrhizus in the STR ...........48

Kinetics of Lactic Acid Production in the STR..................................................................50

Summary of Production of Lactic Acid and By-Products in the STR ................................50

Discussion .............................................................................................................................53

References .............................................................................................................................55

Chapter 6. Enhancement of Lactic Acid Production Using Acid-Adapted Precultures

of Rhizopus arrhizus in a Bubble Column Reactor ..........................................56

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page IV


Abstract .................................................................................................................................58

1. Introduction.......................................................................................................................59

2. Materials and Methods......................................................................................................60

2.1 Microorganism and Media ...........................................................................................60

2.2 Acid-Adapted Precultures ............................................................................................62

2.3 Lactic Acid Production in the BCR..............................................................................62

2.4 Simulation of Scale-up Processes in the BCR..............................................................63

2.5 Analytical Methods......................................................................................................63

3. Results ...............................................................................................................................64

3.1 Effect of Precultures on the Morphology of R. arrhizus...............................................64

3.2 Effects of Precultues on Dissolved Oxygen (DO) Level, Lactic Acid Production

and Starch Consumption ..............................................................................................66

3.3 Summary of Production of Lactic Acid, Fumaric Acid and Ethanol ............................69

3.4 Simulation of Scale-up Processes for Lactic Acid Production .....................................73

4. Discussion..........................................................................................................................75

References .............................................................................................................................77

Chapter 7. Effect of Cultivation Parameters on the Morphology of Rhizopus arrhizus

and the Lactic Acid Production in a Bubble Column Reactor..........................81


(Abstract) ..............................................................................................................................83

1 Introduction........................................................................................................................83

2 Materials and Methods.......................................................................................................84

2.1 Microorganism and Media ...........................................................................................84

2.2 Cultivation Conditions .................................................................................................84

2.3 Analytical Methods......................................................................................................84

3 Results and Discussion .......................................................................................................84

3.1 Glucose and Waste Potato Starch.................................................................................84

3.2 pH Value......................................................................................................................86

3.3 Starch Concentration....................................................................................................87

3.4 Spargers and Aeration Rate..........................................................................................87

4 Conclusions ........................................................................................................................88

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page V

References .............................................................................................................................88

Chapter 8. Conclusions and Future Direction. ......................................................................90

1. Conclusions .......................................................................................................................91

1.1 A Brief Introduction........................................................................................................91

1.2 Major Achievements.......................................................................................................91

1.3 Summary .........................................................................................................................93

2. Future Direction.................................................................................................................93

2.1 Enhancement of Lactic Acid Yield and Productivity .................................................93

2.2 Scale-up of Bench-Scale Process to Pilot Plant..........................................................95

3. References .........................................................................................................................95

Appendix. Production of Fungal Biomass Protein Using Microfungi from Winery

Wastewater Treatment................................................................................................97

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VI

ABSTRACT

L(+)-Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use

in food and food-related industries, and its potential for the production of biodegradable and

biocompatible polylactate polymers.

The aim of this study was to optimize and scale-up a biotechnological process of L(+)-lactic

acid production by suspended cells of R. arrhizus DAR 36017 with waste potato starch as the

substrate. Commonly used inorganic and organic nitrogen sources, including ammonium

sulphate, ammonium nitrate, urea, yeast extract and peptone, were assessed in conjunction

with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio

enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio led to

production of more fumaric acid as a by-product. The use of organic nitrogen sources (yeast

extract, peptone and urea) resulted in a significant reduction of lactic acid yields by 15 % - 34

% with a decrease of C:N from 168 to 28. The use of inorganic nitrogen sources (ammonium

nitrate and ammonium sulphate) led to a high lactic acid yield of 84 % - 91 % at a C:N below

168. Therefore, ammonium nitrate and ammonium sulphate were considered to be better

nitrogen sources for lactic acid production.

Small pellets are the favoured morphological form for many fermentation processes by

filamentous fungi. However, to control filamentous Rhizopus sp in the pellet form in a

submerged fermentation system is difficult due to its filamentous characteristics. An acid-

adapted preculture technique was developed to induce the formation of the pellet form in

bioreactors. Using the acid-adapted precultures, the fungal biomass can be controlled in small

dispersed pellets as a dominant morphological form. With these small pellets, a lactic acid

yield of 86-89%, corresponding to a concentration of 86-89g/L, was obtained in a laboratory

scale process using a stirred tank reactor (STR) and a bubble column reactor (BCR). A batch

bioprocess for lactic acid production was successfully scaled-up from shake flasks to

laboratory scale bioreactors. Results from a simulated scale-up process revealed that the

concentration and productivity of lactic acid decreased with the increase of the scale-up steps

because of increased pellet size. This suggested that a one-step scale-up process using the

acid-adapted preculture may be feasible in an industrial-scale bioreactor system.

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VII

A comprehensive investigation of the impact of cultivation parameters on the morphology of

R. arrhizus and lactic acid production was carried out in the BCR. The results showed that the

fungal morphology was significantly influenced by carbon sources, pH, starch concentrations,

sparger designs and aeration rates. The favoured morphology for lactic acid production was

freely dispersed small pellets, which could be retained as a dominant morphology under

operation conditions at pH 5.0 – 6.0, starch concentrations of 60 – 120 g/L and aeration rates

of 0.2 – 0.8 vvm, using a sintered stainless steel disc sparger. The optimal cultivation

conditions at pH 6.0 and aeration rate of 0.4 vvm resulted in the formation of the freely

dispersed small pellets and production of 103.8 g/L lactic acid, with a yield of 87 %, from 120

g/L liquefied potato starch in 48 h.

This study shows a technically feasible and economically promising process for the production

of lactic acid from waste potato starch. The use of waste potato starch instead of pure glucose

or starch as substrate can significantly reduce the production cost, making this technology

environmentally and economically attractive.

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page VIII

DECLARATION

This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution and, to the best of my knowledge and

belief, contains no material previously published or written by another person, except where

due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis (as

listed below*) resides with the copyright holder(s) of those works.

*List of publications contained in this thesis and copyright holder(s):

1. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Production of lactic acid from renewable materials

by Rhizopus fungi (review). Biochemical Engineering Journal 35:251-263. Copyright for this

paper belongs to Elsevier B.V..

2. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Production of lactic acid and byproducts from waste

potato starch by Rhizopus arrhizus: role of nitrogen sources. World Journal of Microbiology

and Biotechnology 23:229-236. Copyright for this paper belongs to Springer Science &

Business Media.

3. Z.Y. Zhang, B. Jin, J.M. Kelly. 2008. Production of L(+)-lactic acid using acid-adapted

precultures of Rhizopus arrhizus in a stirred tank reactor. Applied Biochemistry and

Biotechnology. D.O.I., 10.1007/s12010-007-8126-7. Copyright for this paper belongs to

Humana Press.

4. Z.Y. Zhang, B. Jin, J.M. Kelly. 2007. Effects of cultivation parameters on the morphology

of Rhizopus arrhizus and the lactic acid production in a bubble column reactor. Engineering in

Life Sciences 7:490-496. Copyright for this paper belongs to WILEY-VCH Verlag GmbH &

Co. KGaA, Weinheim.

An additional publication which employs similar research methodologies, not included in this

thesis directly but presented as an appendix of this thesis,

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page IX

Z.Y. Zhang, B. Jin, Z.H. Bai, X.Y. Wang. 2008. Production of fungal biomass protein using

microfungi from winery wastewater treatment. Bioresource Technology 99: 3871-3876.

Copyright for this paper belongs to Elsevier Ltd.

Zhanying Zhang

Signed………………………………………

Date…………………………………………

ACKNOWLEDGEMENTS

I would like to express my gratitude to all those who accompanied and supported me to

complete this thesis. Without their kind help, it would have been impossible for me to finish

the research.

Firstly, my greatest appreciations are given to my principal supervisor, Associate Professor Bo

Jin, who gave me the opportunity to study in Australia. During my PhD study, Bo has

impressed me deeply by his enthusiasm for research and insight into science. His

encouragement and personal guidance have provided outstanding support for the present thesis.

Besides being an excellent supervisor, Bo is also a close friend to me and provides me with a

lot of help beyond work.

I would like to warmly thank my co-supervisor Dr. Joan M. Kelly as well for her important

support throughout the project, especially for her detailed and constructive advice on this

project. I am also grateful to my labmates in the Water Environment Biotechnology

Laboratory: Dr. Richard Haas, Dr. Zhihui Bai, and PhD candidates Adrian Hunter, Xiaoyi

Wang, Meng Nan Gabriel Chong, Vipasiri Vimonses and Florian Zepf for their help on this

project.

I also wish to thank the University of South Australia and the University of Adelaide, in which

I have done my PhD project and from which I have received scholarships. I would like to

acknowledge the Australia Research Council, which contributed to this project substantially

by funding the project.

Finally, I would like to give my special thanks to my wife Shuang Zhou for her love and

understanding and to my parents and parents-in-law for their encouragement and support from

China during my PhD study.

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page X

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page XI

PREFACE

This thesis contains eight chapters, of which five chapters (Chapter 2, 4, 5, 6 and 7) comprise

the main body. In Chapter 1, a general introduction to this project and thesis is outlined.

Chapter 2 contains a comprehensive literature review, which has been published. Chapter 3

contains a general introduction to the experimental materials and methods used in this study.

Specific details of the methods are given in the relevant chapters. This project aimed to

optimize and scale-up the lactic acid production process using waste potato starch by Rhizopus

arrhizus. The research outcomes and findings are presented thoroughly in Chapters 4 to 7. In

Chapter 4, the focus is on identification of the role of nitrogen sources, and optimization of the

ratio of C:N for lactic acid production in shake flasks. In Chapter 5, a newly developed

inoculation strategy using acid-adapted precultures as the inoculum is described. With this

strategy, the lactic acid production process was successfully scaled up from shake flasks to a

stirred tank reactor (STR). Furthermore, the newly developed inoculation strategy was applied

to a self-designed bubble column reactor (BCR) resulting in successful process scale-up,

described in Chapter 6. In addition, the scale-up between reactors was simulated in this BCR

to determine the ideal scale-up steps for lactic acid production. The results from Chapter 5

and Chapter 6 also proved that the BCR is more suitable for lactic acid production in terms of

yield and productivity. Therefore, in Chapter 7, the results of further comprehensive

optimization of cultivation conditions carried out in the BCR are presented. Chapter 8 draws

the conclusions from each individual published paper and discusses the possible prospects of

this project.

Chapters of 2, 4, 5 and 7 have been published or accepted for publication in refereed academic

journals. Chapter 6 will be submitted to a refereed academic journal. All the papers are closely

related to the research field of this work.

CHAPTER 1

INTRODUCTION

Production of L(+)-lactic acid by Rhizopus arrhizus Z.Y. Zhang Page 1

1. L(+)-lactic acid and its application

Lactic acid (2-hydroxypropionic acid, CH3CHOHCOOH) is the most widely occurring

hydroxycarboxylic acid. It exists naturally in two optical isomers: D(-)-lactic acid and L(+)-

lactic acid. As humans have only L-lactate dehydrogenase that metabolizes L(+) lactic acid,

L(+)-lactic acid is the preferred isomer in the food and pharmaceutical industries and elevated

levels of D(-)-lactic acid are harmful to humans (Expert Committee on Food Additives, 1967).

Lactic acid is the most important multifunctional organic acid due to its versatile applications

in food, pharmaceutical, textile, leather, and chemical industries (Vickroy, 1985). A review

published in 1995 stated that 85% of lactic acid in the USA was used in food and food-related

applications (Datta et al., 1995). An emerging application of lactic acid is its use for

production of biodegradable and biocompatible polylactate polymers, which provide an envi-

ronmentally friendly alternative to biodegradable plastics derived from petrochemical materials

The growth of lactic acid demand is expected to come from the development of new, large-

volume uses, particularly as a feedstock for biodegradable polymers, ‘green’ solvents and

oxygenated chemicals (Datta & Henry, 2006). With the development and commercialization of

biodegradable polymers, their use has increased considerably, and 20–30% of the 120,000

tonnes global production of lactic acid was estimated to be used in these new applications in

2005 (Datta and Henry, 2006). Lactic acid global demand is expected to shoot up to 200,000

tonnes world wide by the end of year 2011 (Ramesh, 2001).

2. Waste potato starch and environmental concerns

Potatoes are the world's fourth largest crop after wheat, rice and maize. The world’s

production of potato is 314.37 million tonnes with the world's largest producer, China,

producing 70.34 million tonnes in 2006 (Food and Agricultural Organisation, 2008).

Internationally, Australia ranks 35th of the list of potato growing countries, with potato

production totalling over 1,200,000 tonnes since 2001 (Australian Bureau of Statistics, 2008).

The data from the Australian Bureau of Statistics show that the gross value of potatoes was

$513.7 million in 2006-2007, accounting for 3.2 % of the total principal agricultural

commodities. The potato processing industry consumes millions of tonnes of potato while


generating large amounts of potato wastewater which contains high starch content. This

wastewater can significantly pollute the environment if it is discharged directly into an

ecosystem without treatment. This practice results in large losses of valuable nutrient

resources. With increased environmental awareness, people are increasingly concerned about

the disposal of wastes.

Waste potato starch collected from wastewater in a potato processing industry can be used as

animal feed or supplied as a substrate for alcohol production by fermentation conventionally,

as the main component is a biodegradable material — starch (Natu et al., 1991). In addition,

the amounts of protein, vitamins, minerals and other nutrients in the potato starch may be

sufficient to meet the growth requirements for many microorganisms, especially for fungi.

Therefore, the biotechnological process converting waste potato starch to other valuable end-

products, eg. microbial biomass protein (MBP) and organic acids, by fungi only needs the

addition of a small amount of other nutrients such as nitrogen and inorganic salts (Collen &

Kenneth, 1987; Jin et al., 1999; Tsao et al., 1999). Therefore, reuse of the waste potato starch

not only can reduce and eliminate potential damage to the environment, but also may lower

the production cost.

3. Biotechnological production of L(+)-lactic acid

Lactic acid can be produced by either chemical synthesis or a biotechnological process, such

as fermentation. Chemical synthetic production relies on using expensive chemicals from non-

renewable raw materials, such as petroleum, via a high energy-cost process, and results in a

racemic mixture of the two isomers. Biotechnological production has an ability to yield an

optically pure form of lactic acid alone or a racemate, depending on the microorganisms and

culture conditions used (Ǻkerberg et al., 1998; Huang et al., 2003; Yin et al., 1997). In

addition, fermentative production of lactic acid can use cheap, renewable resources, such as

whey, molasses, corn and potato, as well as starch wastes from starch processing plants

(Aristidous & Penttila, 2000; Huang et al., 2003; Jin et al., 2003; Richter & Berthold, 1998;

Tsao et al., 1999).


Lactic acid can be produced using bacteria and fungi. Lactic acid producing bacteria (LAB)

have received wide interest because of their high growth rate and product yield. However,

LAB have complex nutrient requirements because of their limited ability to synthesize B-

vitamins and amino acids (Chopin, 1993), making supplementation of sufficient nutrients such

as yeast extracts to production media necessary. In addition, the difficulty in separating

fermentation broth containing lactic acid from bacterial biomass increases the overall cost of

production process.

Fungal Rhizopus species have attracted a great attention in recent decades, and have been

recognized as suitable candidates for lactic acid production. Unlike the LAB, lactic acid

producing Rhizopus strains generate L-lactic acid as a sole isomer of lactic acid (Yin et al., 1997;

Soccol et al., 1994; Yu & Hang, 1989). Rhizopus strains grow better under nitrogen-limited

environments than the LAB (Rosenberg & Krištofícová, 2003). When a starch-based material

is used as the substrate, only small amounts of inorganic salts and inorganic nitrogen are

needed for the lactic acid production using Rhizopus fungi. Separation of the fungal biomass

from the fermentation broth is easy because of their filamentous or pellet forms, leading to a

simple and cheap downstream process. Furthermore, as a by-product from the lactic acid

production, the fungal biomass from Rhizopus strains can be used in bioadsorption processes for

purification of contaminated effluents (Fourest, 1994), for fungal chitosan production

(Pochanavanich et al., 2002; Yoshihara et al., 2003) and as an additive in animal feeds to

improve the feed quality (Kusumaningtyas et al., 2006).

4. Technical and economic challenges associated with lactic acid production by Rhizopus

fungi

Although Rhizopus fungi have been widely investigated for lactic acid production, there are

some significant technical challenges associated with the biotechnological process for the

production of lactic acid:

(1) The cost of lactic acid production is high. It has been estimated that substrate cost is one of

the major operational costs, representing 30–40% of total production costs (Saito et al.,

2003). Commercial glucose and starch from agricultural products (wheat, barley, corn, etc.)


were the main carbon sources used for lactic acid production by either lactic acid bacteria

or Rhizopus sp. in most previous investigations and industrial processes. Therefore, there is

no doubt that the use of low cost substrate, such as starch wastes, can lower the total

production cost significantly. However, there are only a few studies focusing on the studies

of lactic acid production using starch wastes (Huang et al., 2003; Jin et al., 2003).

(2) Lactic acid yield by Rhizopus sp. is low. A lactic acid yield less than 80 % with suspended

cells of Rhizopus species was reported commonly in the literature. The low yield of lactic

acid is due to the production of by-products, such as ethanol and other organic acids, which

competitively consume the substrate. Among these by-products, ethanol is an anaerobic

metabolite and can be accumulated in large amounts if the production process is limited by

oxygen supply. Other by-products are mainly organic acids, such as fumaric acid, malic

acid and succinic acid, which are aerobic metabolites present in small amounts compared to

lactic acid, even under good oxygen supply conditions. Therefore, it is critical to improve

aeration efficiency in the submerged fermentation process. However, the improvement of

oxygen supply and mixing is frustrated because of the difficulty in controlling the

morphology of Rhizopus in the desirable form.

(3) Morphology control is a crucial and difficult task for a submerged fermentation system

using filamentous fungi. The filamentous fungi can easily grow in “filamentous” mycelial

forms. The growth of filamentous fungi in submerged cultures as filamentous mycelia

results in increasing viscosity of the fermentation broth. The highly viscous fermentation

broth makes the bioreactor into a heterogeneous system, and therefore, can significantly

reduce the gas–liquid mass transfer efficiency in the reactor. The filamentous mycelial

biomass can possibly block aeration nozzles, cling to impellers and baffles, and form large

cakes, leading to poor mixing and mass transfer performance in the bioreactor system.

Immobilization has been recognised as an alternative technique to improve the oxygen and

mass transfer efficiency in the submerged fermentation system. However, the use of an

immobilization system results in an expensive and technically complex process. The study

on scale-up of an immobilization fermentation process is missing, which may limit its


application in industry. Therefore, it is worth studying how to enhance the lactic acid yield

using suspended cultures of Rhizopus cells.

It has been proven that growth of filamentous fungi in pellet forms can overcome the

problems caused by filamentous mycelial growth. Enormous efforts have been given to

control the morphological form of fungal cells to pellets, especially to small uniform pellets.

This is because the large pellets may limit internal mass transfer, resulting in a decrease in

the production rate. Therefore, control of the formation of small uniform pellets is a

prerequisite for industrial applications to ensure adequate mass and heat transfer, and

metabolite production. Methods to control the formation of pellets for the lactic acid

production by Rhizopus sp. mainly focus on the control of inoculum size, culture medium,

addition of calcium, cultivation time or a combination of these operational conditions. The

control strategies reported in the literature are either too complex or inefficient for lactic

acid production. The lactic acid yields with these methods are low, less than 80 %.

5. Aim of this project

The aim of this research was to develop a cost-effective biotechnological process for the

production of L(+)-lactic acid using a cheap substrate, waste potato starch, by filamentous

fungus R. arrhizus strain DAR 36017. Research has focused on the technical challenges to

control fungal morphology, to minimize the accumulation of by-products, such as ethanol and

fumaric acid, and consequently to maximize the yield of lactic acid. The specific objectives of

this study was to

(1) investigate the role of nitrogen source in the fermentation of waste potato starch by R.

arrhizus for lactic acid production,

(2) develop a preculture technique and control strategy to obtain desirable morphological

forms of R. arrhizus for lactic acid production in reactors,

(3) scale-up the lactic acid production process from shake flask culture to a bioreactor process,

and select a suitable bioreactor for further optimization, and

(4) optimize lactic acid production processes in the selected bioreactor system


6. References

Ǻkerberg, C., Hofvendahl, K., Zacchi, G., Hahn-Hägerdal, B., 1998. Modelling the influence

of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid

production by Lactococcus lactis ssp Lactis ATCC 19435 in whole-wheat flour. Appl.

Microbiol. Biotechnol. 49, 682–690.

Aristidous, A., Penttila, M., 2000. Metabolic engineering applications to renewable resource

utilization. Curr. Opin. Biotechnol. 11, 187-198.

Australian Bureau of Statistics, 2008. Year Book Australia. pp 497-500.

Chopin, A., 1993. Organization and regulation of genes for amino acid biosynthesis in lactic

acid bacteria. FEMS Microbiol. Rev. 12, 21–38.

Collen, S.A., Kenneth, F.G., 1987. Production of microbial biomass protein from potato

process waste by Cephalosporim eichhoriae. Appl. Environ. Microbiol. 53, 824-829.

Datta, R., Tsai, S.P., Bonsignor, P., Moon, S., Frank, J., 1995. Technological and economic

potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol. Rev. 16, 221-

231.

Datta, R., Henry, M., 2006. Lactic acid: recent advances in products, processes and

technologies- a review. J. Chem. Technol. Biotechnol. 81, 1119–1129.

Expert Committee on Food Additives., 1967. Lactic acid. WHO Food Addit. Ser. 29, 144–148.

Food and Agricultural Organisation., 2008. International Year of the Potato,

http://www.potato2008.org/en/world/index.html.

Fourest, E., Canal, C., Roux, J., 1994. Improvement of heavy metal biosorption by mycelial

dead biomasses (Rhizopus arrhizus, Mucor miehei and Penicillium chrysogenum): pH

control and cationic activation. FEMS Microbiol. Rev. 14, 325–332.

Huang, L.P., Jin, B., Lant, P., Zhou, J., 2003. Biotechnological production of lactic acid

integrated with potato wastewater treatment by Rhizopus arrhizus. J. Chem. Technol.

Biotechnol. 78, 899-906.

Jin, B., Huang, L.P., Lant, P., 2003. Rhizopus arrhizus-a producer for simultaneous

saccharification and fermentation of starch waste materials to L(+)-lactic acid. Biotechnol.

Lett. 25, 1983-1987.


http://www.potato2008.org/en/world/

Jin, B., van Leeuwen, H.J., Patel, B., Doelle, H.W., Yu, Q., 1999. Production of fungal protein

and glucoamylase by Rhizopus oligosporus from starch processing wastewater. Process

Biochem. 34, 59-65.

Kusumaningtyas, E., Widiastuti, R., Maryam, R., 2006. Reduction of aflatoxin B 1 in chicken

feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination.

Myopathology 162, 307–311.

Natu, R.B., Mazza, G., Jadhav, S.J., 1991. Potato: production, processing, and products, in

Waste Utilizaiton, Edited by Salunkhe, D.K., Kadam, S.S., Jadhav, S.J., CRC Press, Boca

Raton, FL, pp175-201.

Pochanavanich, P., Suntornsuk, W., 2002. Fungal chitosan production and its characterization.

Lett. Appl. Microbiol. 35, 17–21.

Ramesh, M.V., 2001. A wonder chemical that will help make biodegradable plastic, why India

needs to milk the full potential of lactic acid. India markets empowering business. April 2.

Richter, K., Berthold, C., 1998. Biotechnological conversion of sugar and starchy crops into

lactic acid. J. Agri. Eng. Res. 71, 181-191.

Rosenberg, M., Krištofícová, L., 1995. Physiological restriction of the l-lactic acid production

by Rhizopus arrhizus. Acta Biotechnol. 15, 367–374.

Saito, K., Kawamura, Y., Oda, Y., 2003. Role of the pectinolytic enzyme in the lactic acid

fermentation of potato pulp by Rhizopus oryzae. J. Ind. Microbiol. Biotechnol. 30, 440–444.

Soccol C.R., Marin, B., Raimbault, M., Lebeault, J.M., 1994. Potential of solid state

fermentation for production of L(+)-lactic acid by Rhizopus oryzae. Appl. Microbiol.

Biotechnol. 41, 286–290.

Tsao, G.T., Cao, N.J., Du, J., Gong, C.S., 1999. Production of multifunctional organic acids

from renewable resources. Adv. Biochem. Eng./Biotechnol. 65, 243-279.

Vickroy, T.B., 1985. Comprehensive biotechnology. Dic Pergamon, Toronto.

Yin, P.M., Nishina, N., Kosakai, Y., Yahiro, K., Park, Y., Okabe, M., 1997. Enhanced

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Yoshihara, K., Shinohara, Y., Hirotsu, T., Izumori, K., 2003. Chitosan productivity

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Yu, R.C., Hang, Y.D., 1989. Kinetics of direct fermentation of agricultural commodities to L(+)

lactic acid by Rhizopus oryzae. Biotechnol. Lett. 11, 597–600.


CHAPTER 2

LITERATURE REVIEW

PRODUCTION OF LACTIC ACID FROM RENEWABLE MATERIALS BY

RHIZOPUS FUNGI

Z.Y. Zhang a, B. Jin a,b, J. M. Kelly c

a SA Water Centre for Water Sciences and Systems, The University of South Australia,

Mawson Lakes, SA 5095, Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia

c School of Molecular and Biomedical Science, The University of Adelaide,

SA 5005, Australia

Biochemical Engineering Journal 2007, 35: 251-263.


STATEMENT OF AUTHORSHIP

PRODUCTION OF LACTIC ACID FROM RENEWABLE MATERIALS BY

RHIZOPUS FUNGI

Biochemical Engineering Journal 2007, 35: 251-263.

Zhang, Z.Y. (Candidate) Performed literature review; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date……………………….. Jin, B.

Supervision of manuscript preparation; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..

Kelly, J. M.

Supervision of manuscript preparation; manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..


Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Production of lactic acid from renewable materials by Rhizopus fungi Biochemical Engineering Journal, v.35 (3), pp. 251-263, August 2007

NOTE: This publication is included on pages 9 – 31 in the print

copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at: http://dx.doi.org/10.1016/j.bej.2007.01.028

http://dx.doi.org/10.1016/j.bej.2007.01.028�

CHAPTER 3

EXPERIEMNTAL MATERIALS AND METHODS


1. Microorganism and media

1.1 Microorganism

R. arrhizus DAR 36017, purchased from Orange Agricultural Institute, Sydney, Australia, was

used for lactic acid production in this project. R. arrhizus WEBL 0401 in Chapter 4 was the

same strain as R. arrhizus DAR 36017, but was named after the abbreviation of our laboratory

name (WEBL, Water Environment Biotechnology Laboratory). Previous research revealed

that this strain could produced L(+)-lactic acid as the sole isomer (Jin et al., 2003). The strain

was maintained and grown for spore production on potato dextrose agar slants at 30 oC for 7

days and stored at 4 oC. Spores were harvested using an inoculation loop, suspended in

sterilized water, and counted under a microscope (BA 400, Mitoc, USA).

1.2 Preculture medium

The preculture medium was prepared based on the medium described by Huang et al. (2003),

and contained (g/L): soluble starch, 10; peptone, 5.0; yeast extract, 5.0; KH2PO4, 0.2;

MgSO4·7H2O, 0.2. The preculture medium had a pH around 6.8. For acid-adapted precultures,

the pH of the preculture medium was adjusted according to the experimental requirements

(Chapter 5 and Chapter 6).

1.3 Production medium

Production medium consisted of waste potato starch or glucose, nitrogen source, KH2PO4

(0.25 g/L), MgSO4·7H2O (0.15 g/L), ZnSO4·7H2O (0.04 g/L). Waste potato starch was

collected from Smith’s Chips Ltd. In the shake flask experiments in Chapter 4, 40 g/L waste

potato starch was used with the addition of 20 g/L CaCO3 as a neutralizing agent. The

production medium for shake flask cultures was gelatinized and sterilized at 100 oC for 40

minutes in an autoclave (HV-110, HIRAYAMA, Japan). For reactor use, 600 – 700 g/L waste

potato starch was liquefied at 95 – 100 oC for 4 h with the addition of 0.05 % α-amylase

(Termamyl® Classic, Novoenzymes, Denmark). The liquefied waste potato starch was stored

in a freezer for later use. When the liquefied starch was used in the reactor, frozen starch was

thawed first and diluted to required concentrations with the addition of other medium

components, followed by sterilization at 121 oC for 20 min in the autoclave.


2. Bioreactor systems

A 3.3 L standard stirred tank reactor (STR) with a working volume of 2.5 L (Bioflo III, New

Brunswick, USA) and a 11.5 L bubble column reactor (BCR) with a working volume of 7.5 L

were used in this study. The STR is shown in Figure 1. The BCR was designed by ourselves

and made by Stainless Steel Welding Services Pty. Ltd (Australia). The BCR system and

design scheme are shown in Figure 2.

Figure 1 Stirred tank reactor (Bioflo III, New Brunswick, USA).


A B

Sterile air Sampling port

580 mm

405 mm

Ø=155 mm

Ø=195 mm

Probes

Air sparger

Figure 2 Bubble column reactor. (A) photograph of the BCR system; (B) design scheme.

The STR was equipped with two conventional 6-flat-bladed turbine impellers and an annular

stainless steel sparger (Ø = 55 mm) with five evenly distributed pores (Ø = 0.5 mm). The

aeration for the BCR was provided by either an annular stainless steel sparger (Ø = 70 mm)

with eight evenly distributed pores (Ø = 0.5 mm) or a sintered stainless steel disc sparger (Ø =

75 mm; pore size, 50 – 70 μm). The BCR was equipped with a biocontrol system (LabVIEW

7.0, National Instruments, USA) to control and monitor the experiment parameters (pH,

dissolved oxygen level and temperature). The electrodes of pH, dissolved oxygen (DO) and

temperature used in the both STR and BCR were purchased from METTLER TOLEDO

(Switzerland).


3. Cultivation conditions

3.1 Precultures

For preparation of the inocula (except acid-adapted precultures), a 5 mL spore suspension was

transferred into a 250 mL shake flask with 100 mL preculture medium under aseptic

conditions. The seed cutlures were incubated for 18 h before inoculation for production use.

The detailed procedures for preparation of the acid-adapted precultures are described in

Chapter 5. Precultures were conducted in an incubator (OM15, Orbital incubator, Ratek,

Australia) at 150 rpm and 30 °C.

3.2 Experimental set up and operation

For shake flask experiments, 5 mL preculture was transferred into a 250 mL shake flask with

100 mL of production medium. The cultivation conditions for shake flask experiments were

set up in the same way as those for the precultures described at 3.3.1.

In the reactor experiments, the inoculum size was 5 % (v/v) for both STR and BCR (except the

scale-up simulation experiments in the BCR). For example, 0.125 L preculture was inoculated

to the STR with 2.375 L production medium (total glucose of 250 g). All the experiments in

the reactors were carried out at 30 °C. The agitation speed and aeration rate for operating the

STR were set at 300 rpm and 1.0 vvm, respectively, through the experiments. The aeration

rate of the BCR was set up at 0.1 – 1.0 vvm, depending on the experimental design. 10 M

NaOH was pumped to neutralize the organic acids produced in reactors during cultivations.

Operation pH was controlled at pH 6.0 in the STR, while the growth pH was controlled

according to the requirements in the BCR.

4. Measurement and analysis

4.1 Measurement of initial kLa

The initial volumetric oxygen transfer coefficient (kLa) in the BCR was determined by the

dynamic gassing-out method (Bandyopadhyay et al., 1996). The measurement of initial kLa

was carried out in water phase at 30 oC under a series of aeration rates. During the


measurement, a DO probe was used to record the change of DO level. The reactor was firstly

aerated with nitrogen. When the DO level was below 5 %, the addition of nitrogen was

stopped, and aeration started at a required flow rate. The DO change was recorded at an

interval of 5 – 15 sec. The initial kLa was calculated using the following equation:

ln C* − CL

C*= −kLa (1)

4.2 Analysis of morphology and biomass

The morphology of R. arrhizus DAR 36017 was observed and recorded using a camera

(PowerShot A95, Canon, Japan). A series of stainless steel sieves (Mining grade, Labtech Essa,

Australia) with an aperture of 1.0 mm, 1.4 mm, 1.7 mm, 2.0 mm and 2.8 mm were used to

collect the pellets with different diameters. Biomass weights were determined after drying at

60 oC for 72 h.

4.3 Analysis of samples by HPLC

High performance liquid chromatography (HPLC) was used to analyse the concentrations of

organic acids, ethanol and residual sugar in the fermentation broth. The HPLC system (Model

350, Varian, Australia; Fig. 3) was equipped with a Rezex ROA-Organic Acid analysis

column (300 × 7.8 mm, Phenomenex, Australia), a refractive index detector (Model 350,

Varian, Australia) and an autosampler (Model 400, Varian, USA). Mobile phase was 4 mM

H SO2 4 solution. Water used for mobile phase was from a water purifying machine

(NANOpure DiamondTM, Barnstead, USA). Fermentation broth samples were centrifuged at

10,000 rpm by a centrifuge (5415R, Eppendorf, USA). 0.5 mL supernatant from centrifuged

sample was diluted 10 times and mixed with 37 % HCl at a ratio of 10:1 (Tay & Yang, 2002).

The mixture was autoclaved at 121 oC for 1 h, and filtered for glucose analysis. Another 0.5

mL supernatant was diluted 25 times and filtered for analysis by HPLC.


Figure 3 High performance liquid chromatography system.

4.4 Calculation of L(+)-lactic acid yield and productivity

The yield of lactic acid was calculated according to the following equation.

Lactic acid concentration

Initial starch concentration (as glucose)Lactic acid yield = (2)

Lactic acid productivity =

Lactic acid concentration

Cultivation time (3)


4.5 Data analysis

The results presented in this thesis were the means of triplicate experiments in shake flasks

and at least duplicate experiments in the STR and BCR. To evaluate the experiment errors, the

standard deviation (SD) was calculated. The calculation of SD was conducted automatically

using the function of STDEV included in Microsoft Office software, Excel.

5. References

Bandyopadhyay, B., Humphrey, A.E., Taguchi, H., 1996. Dynamic measurement of the

volumetric oxygen transfer coefficient in fermentation systems. Biotechnol. Bioeng. 51,

511-519.

Huang, L.P., Jin, B., Lant, P., Zhou, J., 2003. Biotechnological production of lactic acid

integrated with potato wastewater treatment by Rhizopus arrhizus. J. Chem. Technol.

Biotechnol. 78, 899-906.

Jin, B., Huang, L.P., Lant, P., 2003. Rhizopus arrhizus-a producer for simultaneous

saccharification and fermentation of starch waste materials to L(+)-lactic acid. Biotechnol.

Lett. 25, 1983-1987.

Tay, A., Yang, S.T., 2002. Production of L(+)-lactic acid from glucose and starch by

immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor, Biotechnol.

Bioeng. 80, 1–12.


CHAPTER 4

PRODUCTION OF LACTIC ACID AND BYPRODUCTS FROM WASTE POTATO

STARCH BY RHIZOPUS ARRHIZUS: ROLE OF NITROGEN SOURCES

Z.Y. Zhang a, B. Jin a,b, J. M. Kelly c

a SA Water Centre for Water Sciences and Systems, The University of South Australia,

Mawson Lakes, SA 5095, Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia

c School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,

Australia

World Journal of Microbiology and Biotechnology 2007, 23: 229-236.



PRODUCTION OF LACTIC ACID AND BYPRODUCTS FROM WASTE POTATO

STARCH BY RHIZOPUS ARRHIZUS: ROLE OF NITROGEN SOURCES

World Journal of Microbiology and Biotechnology 2007, 23: 229-236. Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date………………………..

Jin, B.

Interpreted data; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..

Kelly, J. M.

Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..


Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Production of lactic acid from and byproducts from waste potato starch by Rhizopus arrhizus: role of nitrogen sources. World Journal of Microbiology and Biotechnology, v.23 (2), pp. 229-236, February 2007

NOTE: This publication is included on pages 34 - 41 in the print


It is also available online to authorised users at:

http://dx.doi.org/10.1007/s11274-006-9218-1

http://dx.doi.org/10.1007/s11274-006-9218-1�

CHAPTER 5

PRODUCTION OF L(+)-LACTIC ACID USING ACID-ADAPTED PRECULTURES

OF RHIZOPUS ARRHIZUS IN A STIRRED TANK REACTOR

Z.Y. Zhang a, b, B. Jin a, b, c, J. M. Kelly d

a School of Earth and Environmental Sciences, The University of Adelaide, Australia

b School of Chemical Engineering, The University of Adelaide, Australia c Australian Water Quality Centre, Bolivar, SA 5095, Australia

d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,

Australia

Applied Biochemistry and Biotechnology 2008, D.O.I.: 10.1007/s12010-007-8126-7.



PRODUCTION OF L(+)-LACTIC ACID USING ACID-ADAPTED PRECULTURES

OF RHIZOPUS ARRHIZUS IN A STIRRED TANK REACTOR

Applied Biochemistry and Biotechnology 2008, D.O.I.: 10.1007/s12010-007-8126-7. Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date…………………….…

Jin, B.

Interpreted data; manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date…………………….…

Kelly, J. M.

Manuscript evaluation I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……


Zhang, Z.Y., Jin, B. and Kelly, J.M. (2008) Production of L(+)-lactic acid using acid-adapted precultures of Rhizopus arrhizus in a stirred tank receptor. Applied Biochemistry and Biotechnology, v. 149 (3), pp. 265-276, June 2008




http://dx.doi.org/10.1007/s12010-007-8126-7

http://dx.doi.org/10.1007/s12010-007-8126-7�

CHAPTER 6

ENHANCEMENT OF L(+)-LACTIC ACID PRODUCTION USING ACID-ADAPTED

PRECULTURES OF RHIZOPUS ARRHIZUS IN A BUBBLE COLUMN REACTOR





Australia

Manuscript to be submitted.



ENHANCEMENT OF L(+)-LACTIC ACID PRODUCTION USING ACID-ADAPTED

PRECULTURES OF RHIZOPUS ARRHIZUS IN A BUBBLE COLUMN REACTOR

Manuscript to be submitted.

Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date………………….……

Jin, B.

Manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………….……

Kelly, J. M.



Enhancement of L(+)-lactic acid production using acid-adapted precultures

of Rhizopus arrhizus in a bubble column reactor

Zhan Ying Zhang a,b, Bo Jin* a, b, c , Joan M. Kelly d

a School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA

5005, Australia b School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia

c Australian Water Quality Centre, Bolivar, SA 5095, Australia d School of Molecular and Biomedical Science, The University of Adelaide, SA 5005,

Australia * To whom correspondence should be addressed.

Tel.: +61 8 8303 5996. Fax: +61 8 8303 4308. Email: [email protected].

Abstract

Controlling the morphology of filamentous fungi in certain forms in submerged cultures is a

prerequisite for the successful production of metabolites. In this study, an acid-adapted

preculture method was successfully developed to manipulate the morphology of Rhizopus

arrhizus in a bubble column reactor (BCR). The morphology of R. arrhizus in the BCR varied

from fluffy mycelia to freely dispersed small loose pellets and big compact pellets, depending

on the acid-adapted precultures inoculated. With the formation of freely dispersed small loose

pellets, a high concentration of 87.9 – 88.7 g/L lactic acid from 100 g/L potato starch in the

BCR was achieved in 42 h fermentation. Results from a simulated scale-up process revealed

that the concentration and productivity of lactic acid decreased with an increased number of

scale-up steps because pellet size increased, indicating that a one-step scale-up process using

the acid-adapted preculture appears to be feasible in an industrial-scale bioreactor system.

Keywords

Lactic acid; Rhizopus arrhizus; morphology; acid-adapted precultures; bubble column reactor;

scale-up


mailto:[email protected]

1. Introduction

Filamentous fungi are morphologically complex microorganisms. The cultivations of

filamentous fungi in submerged cultures for the production of biomass protein and metabolites

such as organic acids, antibiotics and enzymes have been investigated and applied widely over

the past several decades. The successful production of a metabolite often requires a certain

morphological form of the fungus used. However, it is not easy to control the morphology of

filamentous fungi, which can vary from a filamentous form to a pellet form, depending on the

species and cultivation conditions [1].

Strains of filamentous fungus, Rhizopus arrhizus (syn. Rhizopus oryzae), have attracted great

interest because of their capacity to produce pure L(+)-lactic acid, which is a widely used

multifunctional organic acid in the food, pharmaceutical and chemical industries [2], as well

as their amylolytic ability and low nutrient requirements [2-6]. However, due to low lactic acid

yield and productivity, the application of R. arrhizus in industrial production is still in

question. One of the main reasons causing low yield and productivity is the difficulty in

controlling the morphology of R. arrhizus in bioreactors [7-9]. Although immobilization is

considered to be an effective approach to produce lactic acid [9-11], extra operational and

material costs limit its application in an industrial process. Therefore, exploration of

economical and stable operational techniques to control the morphology of R. arrhizus in a

submerged fermentation system is a challenge in creating an industrial process for efficient

production of lactic acid.

It has been demonstrated that pellets are a promising morphological form in industrial

fermentation processes. The pellet biomass results in a low viscosity of the fermentation broth,

and consequently a simple downstream process required to separate the fungal biomass from

the fermentation broth [1, 12, 13]. However, big pellets may limit nutrient and oxygen transfer

to the pellet interior, leading to a low production yield [12, 14]. Therefore, the development of

an engineering strategy to control the fungal morphology in a small pellet form to ensure

adequate mass and heat transfer, and efficient metabolite production, is a prerequisite for

industrial applications [12]. To achieve a pellet form of R. arrhizus, most research has focused

on controlling inoculum size [15-18]. Yang and co-workers obtained small pellets in a stirred


tank reactor (STR) using precultures growing on xylose [19]. However, in most previous

studies, either lactic acid yield or productivity was low.

In a previous study, we have developed an acid-adapted preculture method to control the

morphology of R. arrhizus in pellet forms successfully in a STR [20]. The yield of lactic acid

in the STR reached over 86 % with a productivity of 1.6 g/L/h (Table 1). In this study, the

application of an acid-adapted preculture method to control the morphology of R. arrhizus

DAR 36017, and consequently to enhance the lactic acid yield and productivity was further

investigated in a self-designed BCR. The BCR can generate considerably less shear stress than

conventional STRs since mechanical agitation is not introduced, and therefore it is more

suitable for cultivation of filamentous fungi sensitive to shear force. A simulation experiment

of scale-up process was also carried out to investigate the effects of scale-up steps on the

morphology of R. arrhizus and lactic acid production.

2. Materials and methods

2.1 Microorganism and media

R. arrhizus DAR 36017, obtained from the Orange Agricultural Institute, Sydney, Australia,

was used in this research. This strain was maintained and grown for spore production on

potato dextrose agar slants at 30 oC for 7 days and stored at 4 oC.

The preculture medium was prepared based on the medium described by Huang et al. [21],

and contained (g/L): soluble starch, 10; peptone, 5.0; yeast extract, 5.0; KH2PO4, 0.2;

MgSO4·7H2O, 0.2. The pH of preculture medium was adjusted to an initial value ranged from

2.5 to 5.5, as required, before sterilization. pH in the control preculture medium was 6.8

(unadjusted).The production medium used in the BCR consisted of 100 g/L waste potato

starch, 3.0 g/L (NH4)2SO4, 0.25 g/L KH2PO4, 0.15 g/L MgSO4·7H2O and 0.04 g/L

ZnSO4·7H2O, which was same as that used previously in a STR [20]. Waste potato starch used

in this study was from Smiths Chips Ltd (Australia), where potato starch was separated from

wastewater. Approximately 600 – 700 g/L waste potato starch was liquefied at 95 – 100 oC for


Table 1 Summary of lactic acid production with Rhizopus species in batch fermentations.

Productivity ConcentrationReactor Morphology Substrate

Yield

(%) (g/L/h) (g/L) Reference

89 2.1 88.7 This study BCR

Small pellets (acid-adapted precultures)

Waste potato starch

86

1.6

85.7

20

STR 86 1.7 103.6 7

ALR

Cotton-like flocs via

immobilization

Glucose

87

1.8

104.6

8

STR

Small pellets (precultures growing on

xylose)

Glucose 41 0.6 33 19

STR

Small pellets (precultures by

control of inoculum size and addition

time of CaCO3)

Glucose 76 — 76.1 15

ALR 85 1.4 102.3 18

ALR


control of inoculum size)

Corn starch

77 1.9 92 17

ALR


control of inoculum size)

Glucose 80 2.2 95 16

Shake flask

Immobilization on polymer

support

Glucose

65

–

–

11

Glucose 94 5.0 112.7 Shake flask

Immobilization on poly(vinyl

alcohol)-cryogel

Starch 52 1.8 56.7 10


4 h by the addition of 0.05 % α-amylase (Termamyl® Classic, Novoenzymes, Denmark).

When production medium was prepared, the concentration of the liquefied starch solution was

adjusted to 100 g/L. The potato starch concentration was calculated and expressed as glucose

in this paper. The preculture and production media were autoclaved at 121 oC for 20 min.

2.2 Acid-Adapted precultures

The precultures were prepared in a 250 mL shake flask containing 100 mL precutlure medium.

The procedures for preparation of the acid-adapted precultures were described previously [20].

Spores were harvested from slants using a platinum loop and suspended in sterilized water.

The precultures were acidified at an initial pH from 2.5 to 5.5 with an inoculum size of 105

spores/mL. The 1st precultures with spores were incubated for 18 h at a designated pH of 2.5 –

5.5. The 2nd precultures at a designated pH were prepared using 5 mL of the 1st preculture as

the inoculum and grown for 12 h. In this paper, pH of the precultures refers to the initial pH

unless otherwise stated. The control preculture was inoculated with the same amount of spores

as acid-adapted precultures. All the precultures were cultivated at 30 oC in an orbital shaker at

150 rpm.

2.3 Lactic acid production in the BCR

Production of lactic acid was carried out in a self-designed 11.5 L stainless steel bubble

column reactor with a 7.5 L working volume. The BCR was equipped with a sintered stainless

steel sparger (pore size, 50 – 70 µm) at the bottom. The inoculation was conducted by

transferring 375 mL of acid-adapted preculture into reactors with 7.125 L production medium

(total glucose of 750 g). The temperature and aeration rate in the BCR were maintained at 30

oC and 0.4 vvm throughout the experiments. The cultivation pH was controlled at pH 6.0 with

the addition of 10 M NaOH solution. 0.1 % antifoam (v/v) (Dow Corning® 1510, BDH, UK)

was added to the reactor before sterilization. A few drops of antifoam were added if necessary

during the cultivations. All the cultivations were stopped at 60 h.


2.4 Simulation of scale-up processes in the BCR

The BCR was used for simulation of one-step and multi-step batch processes for lactic acid

production. One-step scale-up cultivation is a process which was scaled-up from shake flask to

reactor. To simulate the two-step scale-up, about 750 mL 18 h culture in the BCR remained

under aeration (so that pellets did not sediment) following the addition of 6.75 L of new

production medium. The cultivation for the two-step scale-up was further carried out for 60 h.

This process was serially repeated for the three-step scale-up. Aeration rate, cultivation

temperature and pH were controlled described in section above.

2.5 Analytical methods

30 to 50 mL samples were taken from the BCR at 6 h to 12 h intervals. The morphology of R.

arrhizus DAR 36017 was recorded in a 9-cm petri-dish using a digital camera (PowerShot

A95, Canon, Japan). For pellet distribution analysis, a series of sieves with an aperture of 1.0

mm, 1.4 mm, 1.7 mm, 2.0 mm and 2.8 mm were used to collect the pellets with a diameter less

than 1.0 mm, between 1.0 mm and 1.4 mm, between 1.4 mm and 2.0 mm, and above 2.8 mm.

Biomass was harvested after filtration and washed three times using tap water. Biomass

weight was determined after drying at 60 oC for 72 h. A Rezex ROA-Organic Acid analysis

column (300×7.8 mm, Phenomenex, Australia) and a refractive index detector (Model 350,

Varian, Australia) were used to analyze all organic compounds, including glucose, lactic acid,

fumaric acid and ethanol. The mobile phase for HPLC was 4 mM H2SO4 solution with a flow

rate of 0.6 mL/min. The column temperature was maintained at 70 oC. 0.5 mL of supernatant

from the centrifuged sample was diluted 10 times and mixed with 37 % HCl at a ratio of 10:1

[9]. The mixture was autoclaved at 121 oC for 1 h, and filtered for glucose analysis by HPLC.

Another 0.5 mL of supernatant was diluted 25 times and filtered for analysis of lactic acid,

fumaric acid and ethanol by HPLC. All the experiments were carried out at least twice and the

results presented were the means of at least duplicate experiments.


3. Results

3.1 Effect of precultures on the morphology of R. arrhizus

Fig. 1 shows the representative morphological forms of R. arrhizus in the BCR using different

precultures. It was observed that inoculation of different precultures significantly affected the

morphology of the fungal biomass. When the control preculture was used, big and irregular

pellets were formed, leading to a range of pellet sizes between 5 and 15 mm (Fig. 1d). In

contrast, the morphology of R. arrhizus changed from coalesced fluffy mycelia to freely

dispersed small pellets in the BCR inoculated with the 1st precultures. The fluffy mycelia

(Figs. 1b1. and 1c1) were formed in the reactor which was inoculated with the 1st precultures

adapted at pH 3.5 – 5.5 (pH referred to the initial pH of precultures in shake flasks unless

otherwise stated, and the cultivation pH in the BCR was 6.0 in the BCR in all cases), whereas

the freely dispersed small pellets appeared in the reactor using the 1st precultures at pH 2.5 and

3.0 (Figs. 1a1). The use of the 2nd precultures at pH 4.0 – 5.5 resulted in forming freely

dispersed small pellets as the dominant morphological form in the BCR (Fig. 1c2). Freely

dispersed compact and big pellets (Fig. 1a2) were formed with the 2nd precultures at pH 2.5

and 3.0 while fluffy mycelia were observed in the reactor using the 2nd preculture at pH 3.5

(Fig. 1b2). It was found that the freely dispersed small pellets were loose and “light” while the

big pellets were compact and “heavy”.

Analysis of pellet distribution of dispersed pellets at 48 h shows that when the 1st preculture at

pH 2.5 was used, the total pellets smaller than 2.0 mm made up 93 % of the total biomass

(w/w) while pellets over 2.0 mm were only 7.0 % (Fig. 2). The use of the 2nd preculture at pH

2.5 and pH 3.0 resulted in a significant reduction in the percentage of the small pellets and an

increase in the numbers of the big pellets. For instance, only 4 % small pellets (< 2.0 mm) but

96 % big pellets ( > 2.0 mm) were formed in the reactor using the 2nd preculture at pH 2.5

(Fig. 2). About 90 % small pellets (< 2.0 mm) were also formed in the BCR using the 2nd

precutlures at pH 4.5 to 5.5. It was found that the fluffy mycelia dominated in the BCR with

the 1st preculture at pH 5.0 (Fig. 2). Pellets smaller than 1.0 mm were only 17 % with the 2nd

preculture at pH 5.0, much less than 42 % with the 1st preculture at pH 2.5. Pellets between 1.4

mm and 2.0 mm were 40 % with the 2nd preculture at pH 5.0, higher than 16 % with the 1st


stpreculture at pH 2.5 (Fig. 2). In general, the use of either the 1 precultures pH 2.5 and 3.0 or

the 2nd precultures at pH 4.5 to 5.5 led to the formation of small pellets whereas fluffy mycelia

were found in the BCR which was inoculated with other 1st nd and 2 precultures.

Figure 1 Effect of precultures on the morphology of R. arrhizus in the BCR. The precultures

used were the 1st precultures adapted at pH 2.5 (a1), pH 3.5 (b1), pH 5.0 (c1), the

2nd precultures adapted at pH 2.5 (a2), pH 3.5 (b2) and pH 5.0 (c2), and the control

preculture (d). Photos were taken at 48 h from a 9.0 cm petri-dish.

d

10 mm

a1

a2 c2

b1 c1

b2

d


0

15

30

45

60

<1.0 1.0-1.4 1.4-2.0 2.0-2.8 >2.8Pellet size (mm)

Pelle

t dist

ribut

ion

(%

Figure 2 Effect of precultures on pellet distribution (48 h). The precultures used were the 1st

preculture adapted at pH 2.5 (grey bar), and the 2nd precultures at pH 2.5 (dark bar)

and pH 5.0 (blank bar).

3.2 Effects of precultures on dissolved oxygen (DO) level, lactic acid production and

starch consumption

Fig. 3 and Fig. 4 display the representative kinetic profiles of DO level, lactic acid production

and starch consumption in the BCR using the acid-adapted precultures. Starch consumption

was expressed as reduction in residual sugar (Fig. 4). Obviously, the change of morphology of

R. arrhizus significantly affected the DO level, lactic acid production and starch consumption

under the operation conditions. The lowest DO level (<30%) was measured in the broth which

had coalesced fluffy mycelia, while the broth with freely dispersed small pellets corresponded

to a DO level above 45 % (Fig. 3). Approximately 85 % DO level was obtained in the broth

dominated by big pellets (Fig. 3b).


0

20

40

60

80

100

0 12 24 36 48 60 72Time (h)

DO

(%)

0

20

40

60

80

100

0 12 24 36 48 60 72Time (h)

DO

(%)

a

b

Figure 3 Effect of precultures on process DO. The precultures inoculated were the 1st (a) and

the 2nd (b) precultures adapted at pH 2.5 ( ), pH 3.5 ( ) and pH 5.0

( ).


0

20

40

60

80

100

0 12 24 36 48 60 72Time (h)

Lact

ic a

cid

and

resid

ual s

ugar

(g/L

0

20

40

60

80

100

0 12 24 36 48 60 72Time (h)

Lact

ic a

cid

and

resid

ual s

ugar

(g/L

a

b

Figure 4 Kinetics of lactic acid production and starch consumption using different precultures.

The precultures inoculated were the 1st (a) and 2nd (b) precultures at pH 2.5 (square),

pH 3.5 (triangle) and pH 5.0 (circle); open symbols, lactic acid production; dark

symbols refer to starch consumption.


The kinetics of lactic acid production was affected by the morphology and DO level in the

BCR (Fig. 4). Using the 1st preculture at pH 2.5 and 2nd preculture at pH 5.0 as inoculum,

lactic acid concentration reached 88.7 g/L and 88.1 g/L at 42 h associated with the formation

of freely dispersed small pellets. The formation of big pellets with the 2nd preculture at pH 2.5

produced 82.3 g/L lactic acid at 48 h. The lactic acid concentration dropped significantly with

the 1st and 2nd precultures at pH 3.5, and 1st preculture at pH 5.0, which resulted in the

formation of coalesced fluffy mycelia (Fig. 1 and Fig. 4). A low starch consumption rate

corresponded to high residual sugar left in the reactor (Fig. 4). This reveals that low DO

caused by fluffy mycelia inhibited the consumption of sugar by R. arrhizus and, consequently,

slowed down the lactic acid production.

3.3 Summary of production of lactic acid, fumaric acid and ethanol

Fig. 5 shows a summary of the maximum lactic acid concentration and productivity in the

BCR using the 1st and 2nd acid-adapted precultures at pH 2.5 – 5.5. Only 45.9 g/L lactic acid

was produced in the BCR inoculated with the 1st preculture at pH 5.5, corresponding to a

productivity of 0.8 g/L/h. The lactic acid concentration in the BCR was enhanced significantly

from 45.9 g/L to 88.7 g/L with the reduction of the adaptation pH of the 1st precultures from

5.5 to 2.5. The use of 2nd precultures at pH 4.0 – 5.5 produced 87.9 g/L to 88.5 g/L lactic

acid. The lactic acid productivity reached 2.1 g/L/h with the 1st precultures at pH 2.5 and pH

3.0, and 2nd precultures at pH 4.5 – 5.5, higher than others. The high lactic acid concentration

and productivity were associated with the formation of freely dispersed small pellets, whereas

low lactic acid concentration and productivity were accompanied with the growth of compact

big pellets or fluffy mycelia. When the BCR was inoculated with the control preculture, less

lactic acid (74.1 g/L) but more ethanol (10.7 g/L) was accumulated due to the formation of big

pellets (Fig. 1d).


0

20

40

60

80

100

2.5 3.0 3.5 4.0 4.5 5.0 5.5Adapted pH

Lact

ic a

cid

conc

entra

tion

(g/L

0.0

0.5

1.0

1.5

2.0

2.5

Lact

ic a

cid

prod

uctiv

ity (g

/L/h

Figure 5 Concentration and productivity of lactic acid. The precultures inoculated were the 1st

(grey) and the 2nd (dark) precultures; Symbols: lactic acid concentration (bar), lactic

acid productivity (square).

The fumaric acid concentration varied between 1.5 g/L and 3.2 g/L when freely dispersed

small pellets were formed but it was below 1.0 g/L when other forms of biomass appeared

(Fig. 1 and Fig. 6a). More ethanol was produced in the fermentation with fluffy mycelia than

with freely dispersed small pellets (Fig. 6b). When the 2nd precultures adapted at pH 2.5 and

pH 3.0 were used as the inoculum, 2.8 – 3.5 g/L ethanol was produced, which was comparably

higher than that measured in the BCR inoculated with the 1st precultures at pH 2.5 and pH 3.0

and the 2nd precultures at pH 4.5 – 5.5 (Fig. 5b). However, the DO level in the bulk broth of

the BCR with the 2nd precutlure at pH 2.5 and pH 3.0 was much higher than that with the 1st

preculture at pH 2.5 and pH 3.0, and the 2nd precultures at pH 4.5 – 5.5 (Fig. 3), indicating that

the accumulation of more ethanol was attributed to the increase in pellet size because of the

poor oxygen transfer inside the big pellets.


0

1

2

3

4

2.5 3.0 3.5 4.0 4.5 5.0 5.5Adapted pH

Fum

aric

aci

d (g

/L)

0

1

2

3

4

5

2.5 3.0 3.5 4.0 4.5 5.0 5.5

Adapted pH

Etha

nol (

g/L)

a

b

Figure 6 Maximal concentration of fumaric acid and ethanol. a, fumaric concentration; b,

ethanol concentration; The precultures inoculated were the 1st (grey bar) and 2nd

(dark bar) precultures.


3.4 Simulation of scale-up processes for lactic acid production

The 7.5 L bench-scale reactor was used to simulate scale-up processes to determine the effect

of the dilution of seed culture on lactic acid production, and the appropriate scale-up

approaches. The 2nd preculture adapted at pH 5.0 was used as the inoculum in one-step scale-

up process from shake flasks to the 7.5 L reactor.

Fig. 7 shows the biomass production during the simulation processes and pellet distribution at

48 h. After the cultivation was scaled up from shake flask to reactor (one-step), the biomass

increased from 0.1 g/L to 2.3 g/L at 18 h. 10 % of culture in the BCR, which was still in

exponential phase, was used for the next scale-up step. The seed cultures were diluted in the

two-step scale-up process. The biomass growth rate slowed down from 0.14 g/L/h to 0.09

g/L/h in 24 h fermentation (Fig. 7a). Three-step scale-up resulted in the further decrease of

biomass growth rate to 0.06 g/L/h in 24 h fermentation (Fig. 7a). In addition, the final biomass

concentration also reduced from 4.6 g/L (one-step) to 3.0 g/L (two-step) and 2.6 g/L (three-

step).

Analysis of pellet distribution at 48 h showed that the increase in the scale-up steps promoted

the growth of pellet size (Fig. 7b). 90 % of the pellets were smaller than 2.0 mm after one-step

scale-up while two-step and three-step scale-up processes resulted in only approximately 39 %

and 9 % small pellets (<2.0 mm), respectively. These results showed that the dilution of seeds

associated with the scale-up processes corresponded to a decrease in biomass production, but

an increase in the pellet size. Furthermore, pellets became loose and light after one-step scale-

up, but compact and heavy after two- and three-step scale-up. As expected, the decrease of

biomass concentration and increase of pellet size resulted in a reduction of the sugar

consumption rate and the lactic acid production rate (Fig. 8a). The production rate of lactic

acid after one-step scale-up was 3.3 g/L/h in 12 – 36 h. However, it was only 2.6 g/L/h and 1.9

g/L/h after two- and three-step scale-up, respectively. The lactic acid concentration after one-

step scale-up was 88.1 g/L, which was higher than 85.1 g/L after two-step scale-up. Further

scale-up (three-step) lead to a prolonged fermentation time over 60 h to achieve a maximum

lactic acid concentration of 81.4 g/L. The increase of scale-up steps promoted ethanol

production (Fig. 8b). The highest ethanol concentration was only 1.6 g/L (42 h) after one-step


scale-up, but it reached 2.2 g/L (42 h) and 4.1 g/L (60 h) after two- and three-step scale-up,

respectively. 1.9 g/L furmaic acid was produced in the one-step scale-up process. The two-

and three-step scale-up processes resulted in producing less than 1.0 g/L fumaric acid.

0

1

2

3

4

5

0 12 24 36 48 60 72

Time (h)

Bio

mas

s (g/

L)

0

15

30

45

60

<1.0 1.0-1.4 1.4-2.0 2.0-2.8 >2.8Pellet size (mm)

Pelle

t dist

ribut

ion

(%

a

b

Figure 7 Effects of scale-up processes on biomass production and pellet distribution (48 h). a,

biomass production in one-step (square), two-step (triangle) and three-step (circle)

scale-up; b, pellet distribution; Symbols: one-step (grey bar), two-step (dark bar) and

three-step (blank bar).


0

20

40

60

80

100

0 12 24 36 48 60 72

Time (h)

Lact

ic a

cid

and

resid

ual s

ugar

(g/L

0

1

2

3

4

5

0 12 24 36 48 60 72Time (h)

Etha

nol (

g/L)

a

b

Figure 8 Kinetics of lactic acid production, starch consumption and ethanol production in

different scale-up processes. a, lactic acid production and starch consumption; b,

ethanol production; Symbols: one-step (square), two-step (triangle) and three-step

(circle).


The DO profiles of three scale-up processes are presented in Fig. 9. After one-step scale-up,

the lowest DO level was 46 %. The DO levels were approximately 64 % and 79 % in the two-

and three-step scale-up processes. It is well known that production of lactic acid and fumaric

acid by Rhizopus is an aerobic process and accumulation of ethanol by Rhizopus takes place in

an anaerobic process. The high DO level was accompanied by low yields of lactic acid and

fumaric acid but high yield of ethanol, indicating that the internal oxygen transfer of pellets

was hampered due to the increased pellet size.

0

20

40

60

80

100

0 12 24 36 48 60 72

Time (h)

DO

(%)

Figure 9 Effect of scale-up processes on DO. Symbols: one-step ( ), two-step ( )

and three-step ( ) scale-up.

4. Discussion

It has been demonstrated that fungal morphology affects nutrient and oxygen uptake rate in

submerged cultures due to the change of viscosity of the fermentation broth [22, 23]. Fluffy

mycelia lead to an increase in the viscosity of the fermentation broth, causing a reduction in


efficiency of mixing and oxygen supply [23]. This can explain why the DO level in the BCR

with fluffy mycelia and coalesced fluffy pellets was low. Growth of filamentous fungi in

freely dispersed pellets decreases the viscosity and improves mixing and mass transfer

capacity [23], which was also demonstrated by our observation.

Fumaric acid and ethanol are the main by-products produced during lactic acid production by

Rhizopus fungi [3, 9, 20]. Synthesis of L-lactic acid, fumaric acid and ethanol occurs in the

cytosol, with pyruvate at the crossroad [24]. Both fumaric acid and lactic acid are produced in

aerobic processes by Rhizopus fungi [24]. In our study, the highest lactic acid yield of 89 %

was achieved with a highest fumaric acid yield of 3.2 % when the freely dispersed small

pellets were formed. A fumaric acid yield of 3.2 % equated to consumption of less than 3.5 %

of the glucose, considering that a maximum of 0.93 g fumaric acid is produced from 1.0 g

glucose [25]. In contrast, high ethanol yields of 2.8 – 4.1 % produced with big pellets and

fluffy mycelia of R. arrhizus could consume 5.5 – 8.0 % of the glucose (theoretical ethanol

yield is 0.51 g/g glucose consumed). Therefore, controlling of the morphology of R. arrhizus

in small loose pellets instead of big compact pellets or fluffy mycelia is favourable for

converting more glucose to lactic acid. 87.9 – 88.7 g/L lactic acid was produced from 100 g/L

starch within a shorter fermentation time (42 h), with less than 1.8 g/L ethanol accumulated in

the presence of freely dispersed small loose pellets. In contrast, less lactic acid and more

ethanol was produced with big compact pellets when the 2nd precultures at pH 2.5 and pH 3.0,

and two- or three-step scale-up were applied.

Although the lactic acid yield of 89 % in this study is slightly higher than that obtained in the

STR using the same inoculation method, the productivity was enhanced by 30 % (Table 1).

The significant increase in the productivity was attributed to the faster biomass growth rate in

the BCR (data not shown). The enhanced biomass growth may be due to the lower shear stress

generated in the BCR. Using waste potato starch as substrate, the lactic acid yield and

productivity achieved in this study were also higher than those in most previous studies using

commercial starch materials and glucose as substrates with the control of inoculum size and

with immobilized Rhizopus cells in batch fermentations (Table 1).


Freely dispersed loose small pellets formed with the inoculation of acid-adapted precultures

made the oxygen transfer easier and required a low aeration rate with sintered stainless steel

sparger. It was found that the aeration rate of 0.4 vvm was enough to maintain the DO level

above 45 % (above 3.4 ppm, 30 oC) in the BCR. The ethanol yield was no more than 1.8 g/L

based on 100 g/L potato starch. In contrast, high oxygen supply conditions were always

required by fermentations with immobilized cells of Rhizopus. Tay and Yang aerated over 1.0

vvm of the mixture of air and pure oxygen (5:1) to maintain a DO level at 90 % [9]. However,

an ethanol yield was as high as 5.9 % because of the difficulty of oxygen diffusion crossing

the mycelial layer. A high ethanol concentration of 5 g/L was also detected with 120 g/L

glucose in the immobilized fermentation with an aeration rate from 0.5 vvm to 2.0 vvm (to

maintain 3 – 5 ppm DO level) by Park et al., despite a lactic acid concentration of 104.6 g/L

lactic acid [8].

The results achieved in this study reveal that the application of acid-adapted precultures is a

promising and economical engineering approach to control R. arrhizus in pellet forms for

lactic acid production. The use of BCR instead of STR results in a higher lactic acid yield and

productivity, indicating that the BCR is more suitable for cultivation of filamentous R.

arrhizus. The results from a simulation experiment of scale-up process reveal that a one-step

scale-up process may be preferred in terms of lactic acid yield and productivity.

Acknowledgement

We greatly acknowledge the research fund from Australian Research Council Discovery grant

(DP0452516).

5. References

[1] Whitaker A, Long P A (1973) Fungal pelleting. Process Biochem 8:27 – 31

[2] Zhang, Z Y, Jin, B, Kelly, J M (2007). Production of lactic acid from renewable

materials by Rhizopus fungi (review). Biochem Eng J 35:251-263


[3] Zhang, Z Y, Jin, B, Kelly, J M (2007) Production of lactic acid and byproducts from

waste potato starch by Rhizopus arrhizus: role of nitrogen sources. World J Microbiol

Biotechnol 23:229-236

[4] Rosenberg M, Krištofíková L (1995) Physiological restriction of the L-lactic acid

production by Rhizopus arrhizus. Acta Biotechnol 15:367 – 374

[5] Soccol C R, Stonoga V L, Raimbault M (1994) Production of L-lactic acid by Rhizopus

species. World J Microbiol Biotechnol 10:433 – 435

[6] Yu R C, Hang Y D (1989) Kinetics of direct fermentation of agricultural commodities to

L(+) lactic acid by Rhizopus oryzae. Biotechnol Lett 11:597 – 600

[7] Kosakai Y, Park Y S, Okabe M (1997) Enhancement of L(+)-lactic acid production

using mycelial flocs of Rhizopus oryzae. Biotechnol Bioeng 55:461 – 470

[8] Park E Y, Kosakai Y, Okabe M (1998) Efficient production of L-(+)-lactic acid using

mycelial cotton-like flocs of Rhizopus oryzae in an air-lift bioreactor. Biotechnol Prog

14:699 – 704

[9] Tay A, Yang S T (2002) Production of L(+)-lactic acid from glucose and starch by

immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor. Biotechnol

Bioeng 80:1 – 12

[10] Efremenko E N, Spiricheva O V, Veremeenko D V, Baibak A V, Lozinsky V I (2006)

L(+)-Lactic acid production using poly(vinyl alcohol)-cryogel-entrapped Rhizopus oryzae

fungal cells. J Chem Technol Biotechnol 81:519 – 522

[11] Tamada M, Begum A A, Sadi S (1992) Production of L(+)-lactic acid by immobilized

cells of Rhizopus oryzae with polymer supports prepared by gamma-ray induced

polymerization. J Ferment Bioeng 74:379 – 383

[12] Zhou Y, Du J, Tsao G T (2000) Mycelial pellet formation by Rhizopus oryzae ATCC

20344. Appl Biochem Biotechnol 84 – 86:779 – 789

[13] Žnidaršič P (2006) The influence of some engineering variables upon the morphology of

Rhizopus nigricans in a stirred tank bioreactor. Chem Biochem Eng 20:275 – 280


[14] Pirt S J (1967) A kinetic study of the mode of growth of surface colonies of bacteria and

fungi. J Gen Microbiol 47:181 – 197

[15] Bai D M, Jia M Z, Zhao X M, Ban R, Shen F, Li X G, Xu S M (2003) L(+)-lactic acid

production by pellet-form Rhizopus oryzae R1021 in a stirred tank fermentor. Chem Eng

Sci 58:785 – 791

[16] Liu T, Miura S, Yaguchi M, Arimura T, Park E Y, Okabe M (2006) Scale-up of L-lactic

acid production by mutant strain Rhizopus sp. MK-96-1196 from 0.003 m3 to 5 m3 in airlift

bioreactors. J Biosci Bioeng 101:9 – 12

[17] Miura S, Arimura T, Hoshino M, Kojima M, Dwiarti L, Okabe M (2003) Optimization

and scale-up of L-lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in

airlift bioreactors. J Biosci Bioeng 96:65 – 69

[18] Yin P M, Nishina N, Kosakai Y, Yahiro K, Park Y, Okabe M (1997) Enhanced L(+)-

lactic acid production from corn starch in a culture of Rhizopus oryzae using an air-lift

bioreactor. J Ferment Bioeng 84:249 - 253

[19] Yang W C, Zhong J L, Tsao G T (1995) Lactic acid production by pellet-form Rhizopus

oryzae in a submerged system. Appl Biochem Biotechnol 51-52:57 – 71

[20] Zhang Z Y, Jin B, Kelly J M (2008) Production of L(+)-lactic acid using acid-adapted

precultures of Rhizopus arrhizus in a stirred tank reactor. Appl Biochem Biotechnol.

D.O.I.: 10.1007/s12010-007-8126-7

[21] Huang L P, Jin B, Lant P, Zhou J (2003) Biotechnological production of lactic acid

integrated with potato wastewater treatment by Rhizopus arrhizus. J Chem Technol

Biotechnol 78:899 – 906

[22] Gibbs P A, Seviour R J, Schimid F (2000) Growth of filamentous fungi in submerged

culture: problems and possible solutions. Crit Rev Biotechnol 20:17 – 48

[23] Papagianni M (2004) Fungal morphology and metabolite production in submerged

mycelia processes. Biotechnol Adv 22:189 – 259


[24] Wright, B E, Longacre, A, Reimers, J, (1996) Models of metabolism in Rhizopus oryzae J

Theor Biol 182:453–457

[25] Tsao, G. T., Cao, N. J., Du, J. and Gong, C. S., (1999) Production of multifunctional

organic acids from renewable resources. Adv Biochem Eng Biotechnol 65:243–279


CHAPTER 7

EFFECT OF CULTIVATION PARAMETERS ON THE MORPHOLOGY OF

RHIZOPUS ARRHIZUS AND THE LACTIC ACID PRODUCTION IN A BUBBLE

COLUMN REACTOR





Australia

Engineering in Life Sciences 2007, 7: 1-8.



EFFECT OF CULTIVATION PARAMETERS ON THE MORPHOLOGY OF

RHIZOPUS ARRHIZUS AND THE LACTIC ACID PRODUCTION IN A BUBBLE

COLUMN REACTOR

Engineering in Life Sciences, 7: 1-8.

Zhang, Z.Y. (Candidate) Performed experiment design, analysis of samples; interpreted data; manuscript evaluation; wrote manuscript. Signed………………………………………………………………Date…………………….…

Jin, B.

Manuscript evaluation; acted as corresponding author. I give consent for Z.Y. Zhang to present this paper for examination towards the Doctor of Philosophy Signed………………………………………………………………Date………………………..

Kelly, J. M.



Zhang, Z.Y., Jin, B. and Kelly, J.M. (2007) Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor. Engineering in Life Sciences, v. 7 (5), pp. 490-496, October 2007




http://dx.doi.org/10.1002/elsc.200700002

http://dx.doi.org/10.1002/elsc.200700002�

CHAPTER 8

CONCLUSIONS AND FUTURE DIRECTION


1. Conclusions

1.1 A brief introduction

High production cost and low lactic acid yield are the key issues which limit lactic acid

production by Rhizopus sp in an industrial process. One of the major production costs is the

substrates used, which can be approximately a quarter of the total production cost if wheat-

flour is used (Åkerberg & Zacchi, 2000). In this study, waste potato starch from a local food

company was used to produce L(+)-lactic acid, which can significantly reduce the costs for

lactic acid production. The low lactic acid yield produced by the filamentous Rhizopus species

is mainly attributed to the formation of ethanol, a major by-product during lactic acid

production. The accumulation of ethanol is caused by poor mass transfer performance,

especially poor oxygen supply, due to the formation of viscous filamentous mycelia, or big

biomass aggregates and pellets during the fermentation processes. These forms of fungal

biomass can significantly reduce the biochemical reaction rate, resulting in low lactic acid

productivity. Therefore, the aim of this thesis study was to develop an efficient and cost-

effective biotechnological process for the production of lactic acid by R. arrhizus using waste

potato starch. The research focused on the optimization and scale-up of the lactic acid

production process.

1.2 Major achievements

Determination of suitable nitrogen sources

To enhance the lactic acid production by R. arrhizus DAR 36017, five commonly used organic

and inorganic nitrogen sources, ammonium sulphate, ammonium nitrate, urea, yeast extract

and peptone, were tested in terms of C:N ratio in order to select a technically and

economically suitable nitrogen source for lactic acid production. It has been found that the

lactic acid concentrations produced from waste potato starch using inorganic nitrogen sources

(ammonium nitrate and ammonium sulphate) were high and stable compared to organic

nitrogen sources (urea, yeast extract and peptone). As an important process operation

parameter, nitrogen sources were studied in a number of investigations. This investigation was

one of few studies which employed C:N ratio, rather than total nitrogen amount in lactic acid


production media. The results reveal that the C:N is a precise nitrogen-related parameter,

showing precisely the role of nitrogen in a fermentation process.

Development of a new inoculation method

In suspended cultures of R. arrhizus small pellets are the preferred morphological form for

lactic acid production in submerged reactor systems. However, the formation of pellets of

filamentous R. arrhizus depends upon the inoculation strategies and cultivation conditions,

which make the control of morphology complicated and difficult. The major achievement of

this research was the development of a new inoculation strategy, namely, using acid-adapted

precultures of R. arrhizus DAR 36017 as inoculum for lactic acid production. This inoculation

method is effective and an easy technique to control the morphology of R. arrhizus in a

desirable form, such as small pellets, in the reactors.

Scale-up of the lactic acid production process to reactor systems

The use of the acid-adapted precultures resulted in the success of scale-up processes from

shake flask cultures to bioreactor systems. This was one of few studies comparing the physical

morphologies and metabolic activities in lactic acid fermentation systems employing two

commonly used bioreactors, namely, a stirred tank reactor and a bubble column reactor. In the

both reactor processes, over 90% fungal biomass was formed as small pellets. The success in

controlling the fungal morphology led to the lactic acid yields of 86 - 89 % based on 100 g/L

waste potato starch. The accumulation of ethanol, a major by-product during lactic acid

production by Rhizopus strains, was minimized due to the formation of small pellets. The

results also showed that the BCR was a more suitable bioreactor for the cultivation of

filamentous Rhizopus for lactic acid production than the STR in terms of the lactic acid yield

and productivity. The better performance of the BCR was possibly attributed to the simpler

reactor structure and lower shear stress environment generated in the BCR, which favoured the

cultivation of Rhizopus. Further study on a simulated scale-up process revealed that the

increase of the scale-up steps resulted in the growth of big and compact pellets, and

consequently, resulted in low lactic acid yield. Therefore, a one-step scale-up process may be

feasible for an industrial-scale bioreactor system.


Comprehensive optimization of cultivation conditions

The optimization of the cultivation conditions using the newly developed inoculation method

was carried out in the laboratory scale BCR process. The cultivation parameters, including

carbon sources, waste potato starch concentration, growth pH, sparger design and oxygen

supply were optimized comprehensively in the BCR. The results further proved the

relationship between the morphological form, and the lactic acid yield and productivity. A

high lactic acid concentration of 103.8 g/L, with a yield of 87 %, was achieved in 48 h

cultivation with the morphological form growing as uniform small pellets.

1.3 Summary

This project successfully developed an inoculation strategy, optimized the cultivation

conditions, and scaled-up the lactic acid production process from shake flask cultures to two

different bioreactor systems. The lactic acid yield achieved in this study is comparable to those

reported processes, which either used complicated and expensive immobilization systems or

other low efficient morphology control strategies by Rhizopus fermentations. It is worth noting

that waste potato starch was used as the substrate for lactic acid production in this study,

which will significantly reduce the production cost and make the process more competitive in

an industrial process.

2. Future direction

2.1 Enhancement of lactic acid yield and productivity

Firstly, the high lactic acid yield of 94 %, with a productivity of 5.0 g/L·h, achieved using a

immobilized Rhizopus strain (Efremenko et al., 2006) indicates that it is possible to further

enhance the lactic acid yield and productivity via bioprocess optimization, including

optimization of cultivation parameters such as inoculum size, agitation speed, impeller design

in the STR and improvement of pellet characteristics in the STR and BCR.

The research on optimization of cultivation parameters is recommended to combine the

quantitative study on the characterisation of pellet formation of R. arrhizus and of the


rheology of the fermentation broth. Uniform small pellets are preferred for lactic acid

production by Rhizopus strains. The pellet’s characteristics such as size and porosity have

been proved to be important for the performance of fermentation broth (Cui et al., 1998). In

the literature, intensive studies have been performed on quantitative analysis of the

morphological variation of filamentous Aspergillus fungi, which are widely used for

production of citric acid, amylase and other enzymes (Papagianni, 2004). Studies on the

formation of pellets of the filamentous fungus, R. arrhizus, may improve the characteristics of

the fermentation broth rheology, which influences significantly the mixing, mass and heat

transfer capacity in a reactor. The research results can provide more precise information on

how to efficiently control the morphology of R. arrhizus in desirable forms and maintain the

rheology of the fermentation broth in a good condition benefiting lactic acid production.

Secondly, the lactic acid yield may be improved by using genetically modified Rhizopus

strains. Yields of lactic acid are compromised primarily because Rhizopus also produces

ethanol with fermentative growth. The production of lactic acid from pyruvate is catalysed by

lactate dehydrogenase (LDH) under aerobic conditions. On the other hand, pyruvate

decarboxylase (PDC) catalyses the conversion of pyruvate to acetaldehyde, which is

subsequently reduced to ethanol by alcohol dehydrogenase (ADH) under anaerobically

stressed growth conditions that may result from inadequate aeration or mycelial clumping.

Therefore, the production of lactic acid competes for available pyruvate with ethanol

fermentation. Consequently, it is expected that lactic acid fermentation could become more

effective if the LDH activity is increased, and activities of ADH and PDC are decreased at the

same time.

Skory and his coworkers (1998, 2004) have developed several mutants with modified ADH

activity and transformants with modified LDH in order to enhance lactic acid yield. Some of

these modified strains did produce more lactic acid than the parent strains. However, the lactic

acid yields achieved using genetically modified Rhizopus were lower than those obtained by

controlling the morphology of Rhizopus. Therefore, research on using genetically modified

strains of Rhizopus should be combined with research on morphology control and

characterisation in order to maximise the yield and productivity of lactic acid.


2.2 Scale-up of bench-scale process to pilot plant

The scale up of the lactic acid production process into a pilot plant is an essential step towards

a commercial process. So far, research on scale up of lactic acid production between reactors

was only reported by Miura and his coworkers (2003, 2006). The results from this project

have shown that a one-step scale-up process is optimal because high lactic acid yield can be

achieved. However, it may not be industrially feasible to scale-up lactic acid production from

shake flask cultures to reactors directly. A promising approach may be to prepare the

precultures in bench-scale or pilot-scale reactors, or avoid pellet formation in precultures until

precultures are inoculated into industrial reactors. Therefore, further study on scale-up of the

lactic acid production process by R. arrhizus from bench-scale to pilot-scale is necessary,

which needs a better understanding of the mechanism of pellet formation as well.

3. References

Åkerberg, C., Zacchi, G., 2000. An economic evaluation of the fermentative production of

lactic acid from wheat flour. Biores. Technol. 75: 119-126.

Cui, Y.Q., van der Lans, R.G.J.M., Luyben, K.Ch.A.M., 1998. Effects of dissolved oxygen

tension and mechanical forces on fungal morphology in submerged fermentation.

Biotechnol. Bioeng. 57: 409-419.

Efremenko, E.N., Spiricheva, O.V., Veremeenko, D.V., Baibak, A.V., Lozinsky, V.I., 2006.

L(+)-Lactic acid production using poly(vinyl alcohol)-cryogelentrapped Rhizopus oryzae

fungal cells. J. Chem. Technol. Biotechnol. 81, 519–522.

Liu, T., Miura, S., Yaguchi, M., Arimura, T., Park, E. Y., Okabe, M., 2006. Scale-up of L-

lactic acid production by mutant strain Rhizopus sp. MK-96-1196 from 0.003 m3 to 5 m3

in airlift bioreactors. J. Biosci. Bioeng. 101, 9–12.

Miura, S., Arimura, T., Hoshino, M., Kojima, M., Dwiarti, L., Okabe, M., 2003. Optimization

and scale-up of L-lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in

airlift bioreactors. J. Biosci. Bioeng. 96, 65–69.

Papagianni, M., 2004. Fungal morphology and metabolite production in submerged mycelia

processes. Biotechnol Adv. 22, 189–259.


Skory, C.D., 2004. Lactic acid production by Rhizopus oryzae transformants with modified

lactate dehydrogenase activity. Appl. Microbiol. Biotechnol. 64, 237–242.

Skory, C.D., Freer, S.N., Bothast, R.J., 1998. Production of l-lactic acid by Rhizopus oryzae

under oxygen limiting conditions. Biotechnol. Lett. 20, 191–194.


APPENDIX

PRODUCTION OF FUNGAL BIOMASS PROTEIN USING MICROFUNGI FROM

WINERY WASTEWATER TREATMENT

Zhan Ying Zhang a, Bo Jin a,b, Zhi Hui Bai a,c, Xiao Yi Wang a

a School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA 5005,

Australia b Australian Water Quality Centre, Bolivar, SA 5095, Australia

c Research Centre for Eco-Environmental Sciences, Beijing 100085, China

Bioresource Technology 2008, 99: 3871-3876.


Zhang, Z.Y., Jin, B. and Kelly, J.M. (2008) Production of fungal biomass protein using microfungi from winery wastewater treatment. Bioresource Technology, v. 99 (9), pp. 3871-3976, June 2008

NOTE: This publication is included on pages 97 – 103 in the print



http://dx.doi.org/10.1016/j.biortech.2006.10.047

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