The Submerged Aerobic Culture of Fusarium Sp. IMI397470 Isolated in Sarawak, for the Production of Prolyl Oligopeptidase Inhibitor

Home , Polyozellus

The submerged aerobic culture of Fusarium sp. IMI397470 isolated in Sarawak, for the production of Prolyl Oligopeptidase Inhibitor

Ng Lee Tze

A thesis

presented in fulfillment of the requirements for the degree of

Doctor of Philosophy

at Swinburne University of Technology

2013 Abstract Fusarium sp. was isolated in 2007 in the Malaysian state of Sarawak and later deposited at the CABI Culture Collection (UK) as IMI397470. Crude extracts from the liquid culture of the fungus exhibited high inhibitory activity against prolyl oligopeptidase (POP). Evidence from many studies show the potential of the inhibition of POP as a strategy to ameliorate the symptoms of neurological disorders such as Huntington’s, Parkinson’s and Alzheimer’s Disease. In order to further understand the potential of Fusarium sp. IMI397470, a project to develop fermentation methods for the production of the inhibitor by the fungus resulted in the work now reported. Prior to this project, the known medium for producing POPI inhibitor (POPI) was Potato Dextrose Broth. Through modifications of the physicochemical conditions of culture, a 17‐fold improvement in POPI yield was obtained in shake flask culture by using a semi‐defined mineral salts medium with glucose (10 g L‐1) as the carbon source (Parker Broth). Increasing the incubation temperature from 25o to 30oC resulted in a large improvement in POPI titre obtained.

By examining the relationship between max (maximum specific growth rate) and Qpmax (maximum rate of POPI production), it was determined that the production of POPI by Fusarium sp. IMI397470 is growth‐dissociated i.e. it is only produced significantly after growth rate tends to zero. Thus, POPI has the production pattern of a secondary metabolite. Thus, a production process using immobilized mycelia in repeat batch cultures could be rationally examined. POPI from Fusarium sp. IMI397470 can be produced in a process where mycelia of the fungus is first grown within hydrogel beads (growth phase) which are then subsequently used in repeat batches from which POPI may be recovered (production phase). In this process, the carbon substrate added to Parker Broth for growth is glucose and that for POPI production is soluble starch. Other conditions (for both the growth and production phases) for the production of POPI in a bubble column bioreactor were an incubation temperature of 25oC; culture pH held at 5.5.; a C:N ratio of 190; air supplied at 3.33 vvm; a bead:medium volume ratio of 1; and a bead diameter of 2.8 mm. POPI production using the system developed could be sustained for a minimum of 5 repeat batches. ACKNOWLEDGEMENTS

I thank the following who have contributed in ways large and small to the completion of my research project:

 My supervisor Professor Clem Kuek who gave me an invaluable opportunity to work with his research group at Swinburne University of Technology Sarawak and who provided guidance to me in the design of the project, its implementation and in editing this thesis;  The Senior Research Officer, Sarawak Biodiversity Centre, Dr. Charlie T.C. Yeo for his willingness to become my co‐supervisor, and who provided guidance and support for my application for study leave, project implementation and thesis editing;  My employer the Sarawak Biodiversity Centre and its Chief Executive Officer, Dr. Rita Manurung who supported my application for in‐service training;  The Jabatan Ketua Menteri Sarawak for its approval of my study leave for in‐service training and the provision of funds for supporting the operational costs of my research project;  Other members of Prof. Kuek’s research group, Jennifer Ho, Tay Yea Lu, and Caroline Tang, and Swinburne Sarawak Laboratory Officer Chua Jia Ni for their companionship and help;  My best friend, Ms Lai Lee San for her help and support;  My family for their encouragement and support throughout my candidature.

Swinburne University of Technology Sarawak is acknowledged for its provision of a fee waiver for the duration of my studies under its Memorandum of Understanding with the Sarawak Biodiversity Centre.

Declaration

I hereby declare that this thesis contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of this thesis. To the best of the candidate’s knowledge contains no material previously published or written by another person except where due reference is made in the text of this thesis. Where the work is based on joint research or publications, I have disclosed the relative contributions of the respective workers or authors.

Ng Lee Tze

2 March 2013 TABLE OF CONTENTS

Abstract Acknowledgments Declaration Table of contents Conference presentations List of figures List of tables

CHAPTER 1 INTRODUCTION 1.1 Prolyl Oligopeptidase 1 1.1.1 Prolyl oligopeptidase and Alzheimer’s Disease 1.1.2 Inhibition of prolyl oligopeptidase as potential drug therapy 2 in combating Alzheimer’s Disease 1.2 A producer of prolyl oligopeptidase inhibitor from Borneo’s microbial biodiversity 1.3 Objectives 4 1.4 References

CHAPTER 2 LITERATURE REVIEW 2.1 The genus Fusarium 9 2.1.1 Fusarium species as a producer of secondary metabolites 2.2 The relationship between metabolite synthesis and growth 11 2.3 Submerged aerobic culture 13 2.3.1 Batch culture 2.3.2 Phase separated, repeat batch culture 2.4 Immobilized cell culture as a production approach 14 2.5 Physicochemical influences on culture 16 2.5.1 Medium composition 2.5.1.1 Carbon source 2.5.1.2 The carbon to nitrogen ratio 18 2.5.2 pH 20 2.5.3 Agitation and aeration 21 2.5.4 Temperature 22 2.5.5 Influences on immobilized cell fermentation 23 2.5.5.1 Bead size 24 2.5.5.2 Bead to medium volume ratio 25 2.6 References 27

PART A: PRELIMINARIES

CHAPTER 3 Identity, short‐term maintenance and long‐term preservation of the culture 3.1 Introduction 47 3.2 Materials and methods 48 3.3 Results and discussion 51 3.4 References 53

CHAPTER 4 Initial observations on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 in submerged aerobic culture 4.1 Introduction 55 4.2 Materials and methods 56 4.3 Results 58 4.4 Discussion 62 4.5 References 63

CHAPTER 5 An assay for prolyl Oligopeptidase inhibitor based on units of inhibitory potential 5.1 Introduction 66 5.2 Materials and methods 67 5.3 Results 68 5.4 Discussion 72 5.5 References 73

CHAPTER 6 The activity of prolyl oligopeptidase inhibitor from Fusarium sp. IMI397470 towards recombinant human prolyl oligopeptidase 6.1 Introduction 74 6.2 Materials and methods 75 6.3 Results 77 6.4 Discussion 78 6.5 References 79 CHAPTER 7 The relationship between growth of Fusarium sp. IMI397470 and its production of prolyl oligopeptidase 7.1 Introduction 82 7.2 Materials and methods 83 7.3 Results 7.4 Discussion 86 7.5 References 87

PART B: STUDIES ON FREE MYCELIA IN SHAKE FLASK CULTURE

CHAPTER 8 The effect of the quantity of supplied glucose on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 8.1 Introduction 89 8.2 Materials and methods 90 8.3 Results 8.4 Discussion 95 8.5 References 96

CHAPTER 9 The effect of temperature on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 9.1 Introduction 97 9.2 Materials and methods 9.3 Results 98 9.4 Discussion 100 9.5 References 101

CHAPTER 10 Production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 in a 10 L batch in a stirred tank bioreactor 10.1 Introduction 103 10.2 Materials and methods 10.3 Results 105 10.4 Discussion 108 10.5 References 109

PART C: STUDIES ON IMMOBILIZED MYCELIA IN SHAKE FLASK CULTURE

CHAPTER 11 Initial observations on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in shake flask culture 11.1 Introduction 110 11.2 Materials and methods 111 11.3 Results 113 11.4 Discussion 121 11.5 References 123

CHAPTER 12 Effect of supplying the same amount of glucose in the growth and production phases in the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in shake flask culture 12.1 Introduction 125 12.2 Materials and methods 12.3 Results 126 12.4 Discussion 129

CHAPTER 13 The growth of immobilized mycelia of Fusarium sp. IMI397470 in shake flask culture

13.1 Introduction 130 13.2 Materials and methods 131 13.3 Results 132 13.4 Discussion 134 13.5 References

CHAPTER 14 Effect of temperature on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 14.1 Introduction 135 14.2 Materials and methods 136 14.3 Results 137 14.4 Discussion 139 14.5 References 140

CHAPTER 15 Effect of shaking speed on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 15.1 Introduction 142 15.2 Materials and methods 143 15.3 Results 15.4 Discussion 145 15.5 References 146

CHAPTER 16 Effect of the type of carbon source on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 16.1 Introduction 149 16.2 Materials and methods 150 16.3 Results 151 16.4 Discussion 154 16.5 References 155

CHAPTER 17 Effect of soluble starch on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470: Quantity and when supplied 17.1 Introduction 156 17.2 Materials and methods 17.3 Results 157 17.4 Discussion 160 17.5 References

CHAPTER 18 Effect of bead size on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 18.1 Introduction 162 18.2 Materials and methods 18.3 Results 164 18.4 Discussion 18.5 References 165

CHAPTER 19 Effect of the addition of Tween 80 on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 19.1 Introduction 167 19.2 Materials and methods 168 19.3 Results 169 19.4 Discussion 170 19.5 References

CHAPTER 20 The storage life of immobilized Fusarium sp. IMI397470 20.1 Introduction 172 20.2 Materials and methods 20.3 Results 173 20.4 Discussion 175 20.5 References 176

PART D: STUDIES ON IMMOBILIZED MYCELIA IN BUBBLE COLUMN CULTURE

CHAPTER 21 Effect of controlled pH on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 21.1 Introduction 177 21.2 Materials and methods 178 21.3 Results 182 21.4 Discussion 183 21.5 References 184

CHAPTER 22 Effect of bead to medium volume ratio on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 22.1 Introduction 187 22.2 Materials and methods 188 22.3 Results 189 22.4 Discussion 190 22.5 References

CHAPTER 23 Effect of carbon to nitrogen ratio in the medium on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 23.1 Introduction 192 23.2 Materials and methods 193 23.3 Results 23.4 Discussion 195 23.5 References 197

CHAPTER 24 Effect of air flow on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 24.1 Introduction 198 24.2 Materials and methods 199 24.3 Results 200 24.4 Discussion 201 24.5 References 202

CHAPTER 25 Sustained production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor 25.1 Introduction 204 25.2 Materials and methods 205 25.3 Results 25.4 Discussion 206 25.5 References 207

CHAPTER 26 General discussion 209

Conference presentations

Ng, L.T.; Kuek, C. and Yeo, Y.T.C. (2012) Production of prolyl oligopeptidase inhibitor via repeat batch culture of immobilized Fusarium sp. CMI397470. Sixty second Annual Meeting of the Society of Industrial Microbiology and Biotechnology; 12 ‐ 16 Aug 2012; Washington DC, USA. URL: http://sim.confex.com/sim/2012/webprogram/Session2419.html [28 Feb 2013]

Ng, L.T.; Kuek, C. and Yeo, Y.T.C. (2011) Shake flask production of prolyl oligopepetidase inhibitor by Fusarium sp. CMI397470. In: "Proceedings of the International Congress of the Malaysian Society for Microbiology 2011"; 8 ‐ 11 December 2011, Penang, Malaysia; R.A. Rahim; W.Z. Saad; S.C. Chin et al. (eds.), Malaysian Society for Microbiology, ISBN 978‐ 983‐99873‐1‐7; pp. 492 ‐ 495. LIST OF FIGURES

Figure 2.1 Schematic representations of the relationship between cell growth synthesis of primary and secondary metabolites 12 2.2 The relationship between specific growth rate (µ) and rates of metabolite production in growth‐associated and growth‐dissociated patterns of synthesis 12 2.3 Schematic representation of a phase separated, repeat batch fermentation process 14 3.1 7‐day old cultures of Fusarium sp. IMI397470 on Parker Agar (+ 10 g L‐1 glucose) 52 3.2 A flame‐sealed glass ampoule containing L‐dried progagules of Fusarium sp. IMI397470 in vacuo 52 4.1 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Potato Dextrose Broth 59 4.2 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Parker Broth (+ 10 g L‐1 potato starch) 60 4.3 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Parker Broth (+ 10 g L‐1 glucose) 61 4.4 The maximum biomass (a) and POPI titre (b) in the shake flask culture of Fusarium sp. IMI397470 using 3 different media 62 5.1 Optical density at 414 nm as an indicator of the concentration of 4‐ nitroaniline in 0.1 M Phosphate Buffer 69 5.2 POPI production profiles in a shake flask culture of Fusarium sp. IMI397470 revealed using two different units for expressing inhibition of POP 71 6.1 The inhibitory activity exhibited by crude extract of POPI against human recombinant POP 77 6.2 The inhibitory activity exhibited by crude extract of POPI against Flavobacterium sp. POP 78

7.1 POPI production by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) in shake flask culture showing the relationship between the biomass and POPI profiles 85 7.2 The relationship between the Specific Growth Rate (µ) of Fusarium sp.

IMI397470 and its Specific Rate of Production of POPI (Qp) 86 8.1 The effect of the quantity of supplied glucose on biomass accumulation by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 91 8.2 The effect of supplied glucose on POPI yield of Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 92 8.3 The effect of supplied glucose supply on the maximum POPI titres and biomass obtained from Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 93 8.4 The effect of supplied glucose on the efficiency of the conversion of

glucose to biomass (Yx/s) and POPI (Yp/s) by Fusarium sp. IMI397470 94 9.1 The effect of incubation temperature on biomass accumulation by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 98 9.2 The effect of incubation temperature on the POPI yield of Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 99 9.3 The effect of incubation temperature on the maximum POPI titres and biomass obtained from Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) 99 9.4 The effect of incubation temperature on the efficiency of the ratios

between glucose consumed to biomass (Yx/s) and POPI (Yp/s)

produced, and between POPI and biomass produced (Yp/x) in the fermentation for POPI by Fusarium sp. IMI397470 100 10.1 Day 5 of the 10 L fermentation for POPI by Fusarium sp. IMI397470 using a New Brunswick Scientific BioFlo 310 104 10.2 10 L bioreactor scale production of POPI by Fusarium sp. IMI397470 106 10.3 Shake flask production of POPI by Fusarium sp. IMI397470. This fermentation was the same 30oC run reported in Chapter 9 107 11.1 A summary of the procedure for preparation of immobilized mycelia of Fusarium sp. IMI397470 and their use in subsequent repeated batches for the production of POPI 112 11.2 A flask of Fusarium sp. IMI397470 immobilized in calcium alginate beads 4 days after shaken incubation in Parker Broth (+ 20 g L‐1 glucose) at 25oC and 200 rpm 114 11.3 Beads of immobilized Fusarium sp. IMI397470 at 20X magnification 114 11.4 A thin slice of a bead of immobilized Fusarium sp. IMI397470 (40X magnification) showing mycelium entrapped within the calcium alginate matrix 115 11.5 POPI production by immobilized Fusarium sp. IMI397470 (20 g L‐1 glucose) 116 11.6 POPI production by immobilized Fusarium sp. IMI397470 (40 g L‐1 glucose) 117 11.7 POPI production by immobilized Fusarium sp. IMI397470 (80 g L‐1 glucose) 118 11.8 POPI production by immobilized Fusarium sp. IMI397470 (120 g L‐1 glucose) 119 11.9 The maximum POPI titres obtained in production batches where various amounts of glucose were supplied in Parker Broth. (a) Production batch 1; (b) Production Batch 2 120 11.10 The efficiencies of the conversion of carbon substrate to POPI by Fusarium sp. at various glucose concentrations in Parker Broth when added to beads. Calculated at the point of maximum POPI titre in Production Batch 2 120 12.1 The maximum POPI titres obtained in immobilized shake flask culture of Fusarium sp. IMI397470 across three sequential production batches. At each level of glucose treatment, both the growth and production batches were provided the same glucose quantity 127

12.2 A comparison of the maximum POPI titres obtained in immobilized shake flask culture of Fusarium sp. IMI397470 as influenced by the amount of glucose provided in the growth and production batches 128 12.3 Residual glucose in production batches when supplied with 80 g L‐1 or 120 g L‐1 glucose in Parker Broth in both the growth and production phases 128 13.1 Growth of immobilized Fusarium sp. IMI397470 on Parker Broth (+ 20 ‐1 ‐1 g L glucose, and 4.32 g L CaCl2.2H2O) as estimated by nitrogen content 132 13.2 Growth of immobilized Fusarium sp. IMI397470 on Parker Broth (+ 80 ‐1 ‐1 g L glucose, and 4.32 g L CaCl2.2H2O) as estimated by nitrogen content 133 14.1 The maximum POPI titres produced by immobilized Fusarium sp. IMI397470 grown at 25oC and then at the indicated temperatures during the production batches 138 14.2 The maximum POPI titres produced by immobilized Fusarium sp. IMI397470 grown at 30oC and then at the indicated temperatures during the production batches 138 15.1 The effect of flask shaking speed on the production of POPI by immobilized Fusarium sp. IMI397470 in production phase. (a) 100 rpm vs. 200 rpm; (b) 300 rpm vs. 200 rpm 144 15.2 The POPI titres obtained in production batch at either 100 or 300 rpm relative to those at 200 rpm 145 16.1 The maximum POPI yield obtained with Parker Broth and different carbon sources in production batch 152 16.2 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 lactose) 152 16.3 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 glucose) 152 16.4 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 soluble starch) 153 16.5 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 soluble starch) 153 17.1 POPI production resulting from the use of soluble starch (80 g L‐1) in both the Growth and Production Batches 158 17.2 POPI production resulting from use of glucose (80 g L‐1) in the Growth Batch and soluble starch (80 g L‐1) in the Production Batch 158 17.3 The response in POPI titre in the production batch of fermentation with immobilized Fusarium sp. IMI397470 to a range of soluble starch supplied as the main carbon source 159 17.4 The POPI productivity (titre per day) obtained in production batch when different quantities of soluble starch were supplied to fermentation with immobilized Fusarium sp. IMI397470. Productivity was calculated at the time of POPI peak in each fermentation 159 18.1 The diameters of calcium alginate beads immobilizing Fusarium sp. IMI397470 made by needles of various gauge sizes Beads made with 21G needles were the standard in all studies reported in previous chapters 163 18.2 Production phase POPI profiles of Fusarium sp. IMI397470 immobilized in alginate beads of diameters smaller and larger than 2.8 mm 164 19.1 The relative effectiveness of various Tween 80 addition strategies in the growth and production phases of by immobilized Fusarium sp. IMI397470 on the maximum POPI titre obtained 169 20.1 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) before storage; (b) after 30‐day storage; (c) after further 30‐day storage 174

20.2 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) Before storage; (b) after 60‐day storage 174 20.3 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) Before storage; (b) after 106‐day storage 175 21.1a The bubble column bioreactor rig used in this work 179 21.1b A close‐up of the bubble column with a production batch of immobilized Fusarium sp. IMI397470 underway 179 21.1c Details of the configuration at the bottom of the bubble column bioreactor 180 21.1 The production of POPI by immobilized Fusarium sp. IMI397470 at various controlled culture pH values 183 22.1 POPI yield from the production batch of immobilized Fusarium sp. IMI397470 in a bubble column at various bead to medium volume 189 ratios 23.1 POPI production by immobilized Fusarium sp. IMI397470 using Parker broth (+ 40 g L‐1 soluble starch) variations with different C:N ratios 194 23.2 The maximum POPI titres resulting from the use of Parker Broths (+ 40 g L‐1 soluble starch) variations with different C:N ratios in the fermentation for POPI by immobilized Fusarium sp. IMI397470 195 24.1 POPI production by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor under different rates of sparging with air 200 24.2 The maximum POPI titres in bubble column fermentations with immobilized Fusarium sp. IMI397470 under different rates of sparging with air 201 25.1 POPI production by immobilized Fusarium sp. IMI397470 over 5 sequential production batches 206 26.1 The operating conditions identified in this work for the production of POPI using immobilized mycelia of Fusarium sp. IMI397470 in phase separated repeat batch culture in a bubble column bioreactor 217

LIST OF TABLES

Table 1.0 Examples of potential POP‐inhibitors isolated from different origins 3 2.1 Examples of metabolites that have been isolated from Fusarium solani 10 2.2 Examples of secondary metabolites that have been isolated from Fusarium spp. 10 2.3 Examples of bioreactions mediated by microorganisms immobilized in calcium alginate hydrogel 15 2.4 Examples of the use of various carbon sources in the production of metabolites by different Fusarium species 19 2.5 Incubation temperatures which have been used in the culture of Fusarium species 23 2.6 Examples of bead diameters used fermentations with immobilized cells 26 2.7 Examples of studies where the ratio between the volume of immobilized aggregations and medium volume were investigated 27 3.1 The composition of the maintenance medium (Parker Medium) used for Fusarium sp. IMI397470 49 3.2 The revivability of L‐dried propagules of Fusarium sp. IMI397470 53 5.1 The preparation of 4‐nitroaniline standards 67 6.1 The range of concentrations of POPI from Fusarium sp. IMI397470

used to determine its IC50 against both recombinant human and Flavobacterium sp. POP 75 12.1 Glucose regimes for the preparation of immobilized mycelia and their subsequent use in POPI production 126 14.1 The combinations of incubation temperatures for examining the production of POPI by Fusarium sp. IMI397470 136 14.2 The relative effectiveness of various temperature regimes in the growth and production phases of immobilized Fusarium sp. IMI397470 on the maximum POPI titre obtained 139

16.1 The sole carbon sources used in Parker Broth for the production batches in POPI production by immobilized Fusarium sp. IMI397470 150 19.1 The concentrations of Tween 80 used in Parker Broth (+ 40 g L‐1 soluble starch) to test its effect on POPI yield in the production phase 168 21.1 The sequence of controlled culture pH under which POPI production was studied 181 22.1 The relative volumes of beads to medium in production batches to study effect of the ratio on the bubble column production of POPI by immobilized Fusarium sp. IMI397470 188 22.2 Summary of the fermentation conditions used in Chapter 22 188 23.1 Summary of the fermentation conditions used in Chapter 23 193 24.1 The range of air sparging rates used in fermentations with hydrogel immobilized cells in pneumatically stirred bioreactors 198 24.2 The fermentation volume to air flow rate variations used to set three different aeration levels in the bubble column bioreactor 199 24.3 Summary of the fermentation conditions used in Chapter 24 199 25.1 Examples of sustained repeat batch production of metabolites by hydrogel immobilized microorganisms 204 25.2 Summary of the fermentation conditions used in Chapter 25 205 26.1 Manipulation of physicochemical conditions which improved POPI 213 yield from fermentation with free mycelia of Fusarium sp. IMI397470 26.2 Manipulation of physicochemical conditions which improved POPI yield from fermentation with immobilized mycelia of Fusarium sp. IMI397470 216 26.3 Yield and productivity of POPI in production from immobilized Fusarium sp. IMI397470 via repeat batch culture in a bubble column bioreactor (Chapter 25) 218

CHAPTER 1

Introduction

1.1 Prolyl Oligopeptidase

Prolyl oligopeptidase (EC 3.4.21.26) (POP), also known as prolyl endopeptidase, belongs to a family of serine peptidases classified as S9 and belonging to the SC clan. The distribution of POP has been reported widely in mammals such as the human, rat, and rabbit (Dresdner et al., 1991; Kalwant and Porter, 1991; Dauch et al., 1995). It is expressed in various organs and tissues such as brain, liver, heart, kidney spleen, lung and muscle (Matsubara et al., 1998; Myohanen et al., 2008; Myohanen et al., 2009). The highest enzyme activity has been detected in the brain of mammals (Garcia‐Horsman et al., 2006).

1.1.1 Prolyl oligopeptidase and Alzheimer’s Disease

Research over the last twenty years has linked abnormal mammalian POP activity to neurological disorders (Lawandi et al., 2010). Elevated levels of serum POP activity have been found in patients with bipolar affective disorder (BD) or with schizophrenia (Maes et al., 1995). Corollary evidence of Breen et al. (2004 ) showed that lithium‐treated BD patients showed statistically significant reduction in POP activity. Serum POP activity is also implicated in Huntington’s, Parkinson’s and Alzheimer’s Disease (AD)(Mannisto et al., 2007). For example, elevated levels of POP are found in the patients with AD (Aoyagi et al., 1990). The presence of POP in the brain tissue of AD patients is believed to be associated with either amyloid‐precursor protein (Toide et al., 1997; Rossner et al., 2005), or neuronal degeneration (Laitinen et al., 2001). Major advancement in the research on the association between POP and AD were the findings

1 that the expression of POP correlates with age and that POP inhibitors can reverse drug‐ or lesion‐induced amnesia in animal models (Mannisto et al., 2007).

1.1.2 Inhibition of prolyl oligopeptidase as potential drug therapy in combating Alzheimer’s Disease

Inhibition of POP activity as a strategy to ameliorate neurological disorders has received a lot of research attention (Lawandi et al., 2010; Mannisto et al., 2007). There is evidence from animal and human trials that POP inhibitors can improve cognitive function and is therefore of potential application in treating AD. Toide et al. (1997) found that the POP inhibitor JTP‐4819 could improve memory and learning‐related tests with rats. Working with the same inhibitor and animal model, Miyazaki et al. (1998) found improvement in spatial learning deficits and Shinoda et al. (1999) reported that the inhibitor ameliorated memory impairment. Morain et al. (2002) showed that the POP inhibitor S 17092 improved memory in aged mice, and rodents and monkeys with induced amnesia or spontaneous memory deficits. When clinically tested in humans, S 17092 improved a delayed verbal memory task. The association between AD and POP underlies the interest of pharmaceutical companies in the development of drugs for combating the disease via inhibition of POP activity.

1.2 A producer of prolyl oligopeptidase inhibitor from Borneo’s microbial biodiversity

Hundreds of synthetic or natural compounds from natural resources have been tested for their POP‐inhibitory activity (De Nanteuil et al., 1998). Potential POP‐inhibitors have been reported from different origins including microorganisms and plants (Table 1). Borneo with its tropical rainforest climate, is very rich in biodiversity and has a vast amount of natural resources. The Sarawak Biodiversity Centre (SBC) is a government agency with the responsibility for research on biological resources of Sarawak. One of the

Table 1 Examples of potential POP‐inhibitors isolated from different origins

Origin Author Potential POP inhibitor

Epicatechin, aesculitannin B, linderene, linderene Kobayashi et al. (2002) Plants acetate, linderalactone and isolinderalactone

Chung et al. (2003) Ellagitannin, corilagin

Atack et al. (1991) SUAM‐1221

Toide et al. (1997) JTP‐4819 Microorganisms Katsube et al. (1996) ONO‐1603

Barelli et al. (1999) S 17092

Centre’s R & D programmes focuses on making discoveries in biological resources that would lead to the development of pharmaceutical drugs for combating diseases such as cancer and those caused by infectious agents. The SBC screens plants and soil for microbial compounds that may have industrial potential. One of its screening programs was on endophytic fungi because of reported successes by others such as an antimicrobial compound from a fungus isolated from Taxus wallichiana (Gogoi et al., 2008), paclitaxel‐ producing fungi from a yew tree (Xu et al., 2006) and a taxol‐producing fungus from Taxus mairei (Wang et al., 2000). In 2007, an endophytic fungus was isolated from a yam plant collected at a village called Kampung Semadang (1°17'18"N 110°16'21"E) in the Penrissen area about 25 km from Kuching, Sarawak. Molecular profiling of the fungus undertaken by CABI (Nosworthy Way, Wallingford, Oxfordshire, OX10 8DE, the United Kingdom) identified the fungus as a Fusarium sp. (CABI collection number IMI397470). The molecular profile of IMI397470 places it in the wider Fusarium solani aggregate but not completely matching any Fusarium solani. It is likely that IMI397470 is a new Fusarium species (Cannon, pers. comm.). Butanol extracts from culture filtrates of this fungus were found to have high inhibitory activity against POP of Flavobacterium sp. origin. The

3 discovery of POP inhibitor from the Fusarium genus is new and has never been previously reported.

1.3 Objectives

With its discovered ability to express POP inhibitory activity in its culture filtrates, Fusarium sp. IMI397470 became the subject of efforts to understand its culture requirements in order that sufficiently large quantities of its POP inhibitor(s) (POPI) can be obtained for further study. One effort was the PhD work described in this thesis. The overall aim of the work described was to develop fermentation methods for the production of POPI by Fusarium sp. IMI397470. This comprised the following component objectives: a. To characterize the production of POPI by Fusarium sp. IMI397470 in terms of biomass growth and metabolite production. b. To determine the physicochemical cultural conditions for Fusarium sp. IMI397470 and their effects on the production of POPI in submerged aerobic culture. c. To examine cell immobilization as a means for the repeat batch production of POPI if the production pattern of the latter follows that of a secondary metabolite.

1.4 References

Aoyagi, T.; Wada, T.; Nagai, M.; Kojima, F.; Harada, S.; Takeuchi, T.; Takahashi, H.; Hirokawa, K. and Tsumita, T. (1990) Increased gamma‐aminobutyrate aminotransferase activity in brain of patients with Alzheimer’s disease. Chemical and Pharmaceutical Bulletin (Tokyo) 38: 1748 ‐ 1749.

Atack, J.R.; Suman‐Chauhan, N.; Dawson, G. and Kulagowski, J.J. (1991) In vitro and in vivo inhibition of prolyl endopeptidase. European Journal of Pharmacology 205: 157 ‐ 163.

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Dauch, P.; Vincent, J.P. and Checler, F. (1995) Molecular cloning and expression of rat brain endopeptidase 3.4.24.16. Journal of Biological Chemistry 270: 27266 ‐ 27271.

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CHAPTER 2

Literature review

The following areas of literature are considered (a) the producer microorganism and its production of metabolites; (b) the relationship between the patterns for growth and product synthesis and how these fall into the categories “growth‐ associated” or “growth‐dissociated” is then considered; (c) immobilized cells and their use in repeated batches is presented as a strategy to exploit growth‐dissociated metabolite production; (d) the factors which may affect the submerged aerobic culture of microorganisms are considered as the experimental work will involve choices in cultural parameters to investigate.

2.1 The genus Fusarium

The genus Fusarium was first introduced by Link in 1809. Major species in this family are plant‐pathogenic fungi that produce secondary metabolites with wide range of use. Some of these secondary metabolites are associated with plant diseases, but some were found as drugs or plant and animal growth promoters (Soon‐Ok et al., 2003; Hiramatsu et al., 2006; Chakravarthi et al., 2008). Despite of its characteristic as a plant pathogen, the species is able to produce a wide range of useful enzymes reported in many papers (Table 2.1)

2.1.1 Fusarium species as producers of secondary metabolites

Numerous secondary metabolites such as mycotoxins, plant growth regulators, antioxidants and antibiotics have been isolated from Fusarium spp. (Table 2.2). To date, a few POP inhibitors of fungal origin have been found e.g. Moeszia

Table 2.1 Examples of metabolites that have been isolated from Fusarium solani

Metabolite Reference

Lipase Eddine et al. (2001) Cyanide degradation Dumestre et al. (1997) Thermostable α‐dialkylamino acid aminotransferase Esaki et al. (1994) Fusarubin Gerber and Ammar (1979) Solaniol Ishill et al. (1971)

Antimicrobial metabolites Tayung et al. (2011)

Table 2.2 Examples of secondary metabolites that have been isolated from Fusarium spp. Metabolite Type Species References Bekele et al. Trichothecene Mycotoxins F. sporotricbioides (1991) F. moniliforne Musser et al. Fumonisins Mycotoxins F. proliferaturn (1995) Plant growth Soon‐Ok et al. Gibberellins F. moniliforme regulators (2003) Neofusapyrone, fusapyrone and Hiramatsu et al. Antimicrobial Fusarium sp. deoxyfusapyrone (2006)

Chakravarthi et al. Paclitaxel Anticancer Fusarium sp. (2008) Shiono et al. Fusaristatins A and B Cytotoxic activity Fusarium sp. (2007) Inhibitor of mammalian Hamano et al. Aquastatin A Fusarium sp. Adenosine (1993) Triphosphatases

lindtneri (Heinze et al., 1997) and Polyozellus multiplex (Hwang et al., 1997) but none have been from Fusarium spp. Therefore, the discovery of a POP inhibitor from Fusarium spp. is novel.

2.2 The relationship between metabolite synthesis and growth

Whether the synthesis of a metabolite is associated with growth (growth‐ associated) or not associated with growth (growth‐dissociated) determines how the production process of that metabolite may be required to be. Growth‐associated synthesis of a metabolite means that its appearance in a culture accompanies growth of the microorganism, or that it is produced during the growth phase (trophophase) (Fig. 2.1). Growth‐dissociated synthesis of a metabolite means that its appearance is delayed until growth slows or ceases. In terms of the specific growth rate, growth‐ dissociated metabolites are elaborated when the specific growth rate is tending towards zero (Fig. 2.2) while the converse is true for growth‐associated metabolites. Growth‐dissociated metabolites are typically secondary metabolites (also known as idiolites as they are associated with the idiophase). Secondary metabolites are one of the most economically important compounds produced by microorganisms (Demain, 2000). These metabolites are usually products with unknown functions, and can be deleterious to us such as mycotoxins, or beneficial such as antibiotics (Demain and Fang, 2000). The relationship between growth of Fusarium sp. IMI 397470 and its POP inhibitor will be elucidated early in this work. If POP inhibitor from Fusarium sp. IMI397470 proves to have the production pattern of a secondary metabolite, then it would be an appropriate strategy to try and exploit this characteristic by employing immobilized cell culture. This is because immobilization allows the retention a batch of pre‐grown cells for repeated use. These cells with their specific growth rate at zero or close to it are by definition ideal for the production of growth‐dissociated (secondary) metabolites.

cell number [metabolite]

a. Primary metabolites are growth‐ associated as reported in studies such as those of Paul et al. (1999) and dos Santos et al. (2013)

b. Secondary metabolites are growth‐ dissociated as reported in studies such as those of Toda et al. (1992) and Hamano et al. (1993)

Fig. 2.1 Schematic representations of the relationship between cell growth synthesis of primary and secondary metabolites

Growth-associated Growth-dissociated

• •

Metabolite production

• • max

 Fig. 2.2 The relationship between specific growth rate (µ) and rates of metabolite production in growth‐associated and growth‐dissociated patterns of synthesis 12

2.3 Submerged aerobic culture

A microorganism can be cultivated via different methods such as surface culture, semi‐solid or solid culture method, and submerged aerobic culture. The latter is culture within a liquid that is agitated to enhance oxygen transfer into the liquid. It is a well‐established technique and is used for industrial production of most of the important metabolites such as antibiotics, amino acids, ethanol, organic acids and others (Prabhakar et al., 2005). It has been used for the cultivation of many fungi such as Penicillium marinum (Wigley et al., 2008), Laccaria laccata (Kuek, 1996), Aspergillus spp. (Kuek, 1991; Shah et al., 2009). With Fusarium spp., compounds such as mycotoxins (Jackson and Bennet, 1990; Branham, 1993; Geraldo et al., 2006), enzymes (Kuek, 1991; Rapp, 1995), those antifungal in nature (Altomare et al., 2000), and hormones (Srivastava et al., 2003) have been produced via submerged aerobic culture. Therefore, this form of culture is relevant in the development of a production process for POPI from Fusarium sp. IMI397470.

2.3.1 Batch culture

Batch culture refers to a fermentation which is carried out in a closed system. During the process, nothing is added or removed from the fermenter. Batch culture mode is used in most fermentation studies e.g. in the production of citrinin by Penicillium chrysogenum (Devi et al., 2009), protease by Laccocephalum mylittae (Zhou et al., 2009) and exo‐polysaccharides by Cordyceps militaris C738 (Kim et al., 2003).

2.3.2 Phase separated, repeated batch culture

Secondary metabolites have the ideal synthesis pattern to be exploited by a dividing of the production process into two separated sequential phases (Kuek, 1986) viz. (i) growing of cells; (ii) subsequent retaining of the grown cells for re‐use of the in the synthesis of the secondary metabolite in repeat batches (Fig. 2.3). Immobilization 13

of the cells is a means to enable its recovery and repeated use. Such a production philosophy will be used to develop the fermentation process for POPI from Fusarium sp. IMI397470 should its production pattern prove to be growth‐dissociated.

Growth Phase Production Phase

Growth Production medium medium Cultural conditions optimized for growth

Bioreaction to Cells Bioreaction for produce cells product synthesis Repeated as required

Spent liquor Spent liquor Cultural conditions Optimized for product synthesis Waste Product recovery

Fig. 2.3 Schematic representation of a phase separated, repeat batch fermentation process

2.4 Immobilized cell culture as a production approach

A good definition of cell immobilization is “the physical confinement or location of intact cells to a certain region of space with retention of desired metabolic activity, and which can be repeatedly used” (Kuek, 1986). The main advantage of culturing cells in immobilized form is that it confers a higher density to a microbial aggregation thus making it easier to separate culture liquor from biomass for the purpose of re‐use of that biomass. It has also been often observed that immobilized cells have higher productivities compared to free cells (e.g. Yang and Yueh, 2001; Cruz et al., 2004; Kim et al., 2006; Abdel‐Naby et al., 2011). Examples of where the immobilized cell technique has been successfully applied include the production of cephalosporin (Cruz et al., 2004), Cyclosporin A (Sallam et al., 2005), gibberellic acid (Duran‐Paramo et al., 2004), β‐Mannanase (El‐Naggar et al., 2006); and glucoamylase (Kuek, 1991). 14

In immobilization, entrapment of cells in polymeric hydrogels including alginate, ‐carrageenan and agarose have been used (Wang and Hettwer, 1982; Kuek, 1991; Patil et al., 2009). Immobilization via calcium alginate has been widely used (Table 2.3). Its simplicity and successful outcomes is probably the reason for its popularity. Thus, it was the hydrogel of choice for the experimental part of this work.

Table 2.3 Examples of bioreactions mediated by microorganisms immobilized in calcium alginate hydrogel

Microorganism Product Reference

Aspergillus phoenicus Glucoamylase Kuek (1991)

Cephalosporium acremonium Cephalosporin C Araujo et al. (1999)

Unidentified basidiomycete Decolorization of Zhang et al. (1999) Orange II

Pseudomonas putida Phenol degradation Hannaford and Kuek (1999)

Monascus purpureus Pigment Fenice et al. (2000)

Microcystis Removal of Cu2+ Pradhan and Rai (2000)

Streptomyces rimosus Oxytetracycline Yang and Yueh (2001)

Piromyces sp. KSX1 and β‐glucosidase McCabe et al. (2003) Orpinomyces sp. 478P1

Thermomucor indicae‐ glucoamylase Kumar and Satyanarayana seudaticae (2007)

Aureobasidium pullulans Pullulan production Ürküt et al. (2007)

Fusarium solani Xylanase Gupta et al. (2009)

Bacillus cereus Cyclodextrin Abdel‐Naby et al. (2011) glucosyltransferase

2.5 Physicohemical influences on fermentation

A fermentation process is defined as the utilization of microorganisms to convert a substrate (solid or solute) into a desired product(s). Microorganisms have their unique environmental envelops which may differ slightly or greatly from each other. Accordingly, a fermentation process will be usually defined by a particular set of cultural parameters such as the chemical composition of the medium, pH, incubation temperature, degree of agitation, and level of dissolved oxygen/carbon dioxide.

2.5.1 Medium composition

Medium formulation is crucial in developing a successful fermentation process for biomass growth and subsequent product biosynthesis (Kumar and Jana, 2009). Since Fusarium sp. IMI397470 was a new find as a result of isolation from the field, a typical medium for fungi was used initially viz. commercially prepared Potato Dextrose Agar and its broth version. This medium supported both good growth of Fusarium sp. IMI397470, and culture filtrates from cultivation of the fungi in Potato Dextrose Broth were found to be inhibitory against POP. However, commercial Potato Dextrose Broth is not a viable industrial medium and so in this work a more suitable medium was used as soon as possible. A semi‐defined salts medium with a carbon source used for studies on fungi notably Aspergillus phoenicus (Kuek, 1991) was chosen as a base medium for this work. A more defined medium allows medium components to be exactly changed. Further, manufacturer and batch variations associated with complex media such as Potato Dextrose Broth can be avoided with defined media.

2.5.1.1 Carbon source

Carbon sources used in a fermentation process can be ranging from monosaccharides, disaccharides to polysaccharides. Glucose is an excellent carbon source for growth of fungi although it regulates the biosynthesis of some secondary

metabolites (Demain, 1986) for instance antibiotics (Gallo and Katz, 1972; Haavik, 1974) while El‐banna (2006) reported that glucose strongly enhanced the antimicrobial activity of Corynebacterium kutscheri and Corynebacterium xerosis, and Mojsov (2010) found it produced a good biomass response but poor production of amylolytic enzyme with Aspergillus niger. Lactose, maltose and sucrose are disaccharide sugars. Lactose was found to produce the maximum yields of hexaene H‐85 and elaiophylin by Streptomyces hygroscopicus CH‐7 (Ilić et al., 2010). Maltose has been used as carbon source in the production alpha‐amylase by Penicillum fellutanum (Kathiresan and Manivannan, 2006). Sucrose was found as the most suitable carbon source in maximizing the biomass of Cordyceps militaris C738, Tuber sinense and Cordyceps ophioglossoides L2 (Kim et al., 2003; Tang et al., 2008; Xu et al., 2009). Starch requires breakdown by amylolytic enzymes before it can be utilized by microorganism in a fermentation process. The use of starch as carbon source in fermentation medium for the production of metabolites such as glucoamylase (Kuek, 1991) and pigment (Gunasekaran and Poorniammal, 2008) has been reported. Hexoses such as glucose, mannose, galactose, and fructose were found to be rapidly fermented by Fusarium oxysporum in shake flask culture (Suihko, 1983). When the nutritional requirements of Fusarium moniliforme were studied, glucose was found to be the best carbon source when it, fructose, lactose, starch, galactose and xylose were studied (Suruliranjan and Sarbhoy, 2000). A wide range of choices of carbon source is available amongst the many which have been used in the fermentation of Fusarium spp. (Table 2.4). Glucose ought to be a suitable carbon source for Fusarium sp. IMI397470 on the basis of evidence from the literature, because Potato Dextrose Broth has been used with the fungus in screening by the Sarawak Biodiversity Centre where it was discovered (Yeo, pers. Comm). However, the literature also alerts that use of glucose has to be used with knowledge of its potential for affecting the yield of some secondary metabolites. Therefore, the testing of a range of carbon sources for Fusarium sp. IMI397470 in this work is indicated.

2.5.1.2 The carbon to nitrogen ratio

The carbon to nitrogen ratio has been shown to influence the production of secondary metabolites. On the one hand, the favorable effect of a higher supply of C compared to N has been reported in the production lovastatin by Alternaria alternate (Casas Lopéz et al., 2003). This observation was ascribed to the reasonable idea that in the idiophase (when secondary metabolites are synthesized) the requirement for N is limited whereas excess C can be shunted into secondary metabolism. Similarly, Brzonkalik et al. (2011) found that an excess of C (in relation to N) had a positive effect on the production of alternariol. Added evidence for the superiority of a high C:N ratio comes from the production of polyketide toxins of Fusarium oxysporum which was seen to increase when C:N ratios were increased by decreasing the amount of N added to the medium (Bell et al., 2003). However, there is also conflicting evidence. When the effect of the C:N ratio in media for the production of laccase by Fusarium proliferatum was studied, a low C:N ratio (= 0.69) in the medium was significantly better than one that is high (= 6.9) (Kwon and Anderson, 2001). These findings were confirmed by another study where laccase production was better and occurred earlier when the medium was low in carbon compared to nitrogen (1.3 > 13) (Anderson et al., 2005). Thus, a converse situation has also been found where high C supply compared to N is not favorable. Apart from an effect on growth, the C:N ratio of a medium can also affect fungal sporulation. Zhang et al. (2001) found that sporulation of Plectosporium tabacinum in submerged liquid culture can be influenced by the C:N ration of the medium. The importance of this is that the Fusarium species are sporulating fungi and that the onset of fruiting in fungi may be accompanied by metabolic changes especially for secondary metabolites. The studies reported here point to a necessity to investigate the C:N ratio appropriate of the production of POPI by Fusarium sp. IMI397470. This is especially so when studies reported were all done in batch mode where production was

Table 2.4 Examples of the use of various carbon sources in the production of metabolites by different Fusarium species Carbon source Fusarium Metabolite Reference Fusarium Audhya and Russell Glucose enniatin sambucinum (1975) Fusarium (R)‐1‐ Glucose Uzura et al. (2001) moniliforme Phenylpropanol 8‐O‐Methy‐ Glucose Fusarium solani Kimura et al. (1981) javanicin Fusarium Glucose Farooq et al. (2005) oxysporum Glucose Fusarium sp. Naphthoquinones Kimura et al. (1988) Glucose Fusarium sp. Enniatins Tomoda et al. (1992) Fusarium Panagiotou et al. Glucose Ethanol oxysporum (2005b) Glucose Fusarium roseum Cyclosporine A Ismaiel et al. (2010) Fusarium fujikuroi Glucose Gibberreric acid Uthandi et al. (2010) SG2 Glucose and Antifungal Fusarium solani Sawai et al. (1981) starch substance Glucose and Kobayashi et al. Fusarium sp. Fusarielin A starch (1995) Glucose and Antifungal Fusarium sp. Nihei et al. (1998) starch cyclodepsipeptides Glucose and Fusarium Boonyapranai et al. Naphthoquinones potato verticillioides (2008) Glucose and Fusarium Fusacandins Jackson et al. (1995) mannitol sambucinum A and B Glucose, corn starch, soybean Fusarium sp. Fusaric acid Hidaka et al. (1969) meal Glucose or fructose Fusarium Lipase Rifaat et al. (2010) (biomass); olive oxysporium oil (lipase)

Table 2.4 Examples of the use of various carbon sources in the production of metabolites by different Fusarium species (continued) Carbon source Fusarium Metabolite Reference Glycerol and Fusarium Aquastatin A Hamano et al. (1993) potatoes aquaeductum Monoolein Fusarium sp. Lipase Mase et al. (1995) Dextrin and Fusarium sp. Saricandin Chen et al. (1996) molasses Trehalose Fusarium sp. trehalases Dekker et al. (1997) Fusarium Extracellular Gelatine oxysporum var. trypsin‐like Barata et al. (2002) lini ATCC 10960 protease Fusarium Panagiotou et al. Cellulose Cellulose oxysporum F3 (2005a) Fructose Fusarium solani glucoamylase Bhatti et al. (2007) Fusarium Ramanathan et al. Lactose Cellulose oxysporum (2010) Fusarium Saccharose Cytotoxic alkaloids Ding et al. (2012) incarnatum Fusarium solani Cholesterol Fusarubin Ammar et al. (1979) (Mart.) Sacc. cytotoxic cyclic Rice Fusarium solani pentadepsipeptide, Lee and Lee (2012) neosansalvamide

determined in a growing culture. With the intent in this work to use separated, repeat batch culture where the use of pre‐grown mycelia is a feature, the C:N is probably going to turn out to be different from that required in cultures with growing mycelia.

2.5.2 pH

Culture pH is one of the factors limiting the growth of a fungus and production of high yields (Demain, 1999). Boonyapranai et al. (2008) state that the pH of the medium

may affect cell membrane function, cell morphology and structure, the solubility of salts, the ion state of substrates and the uptake of various nutrients and product biosynthesis. It was established early (1940s and 50s) in the development of fungal fermentation processes for penicillin that pH is an important process parameter. It was found that growth of the producer fungus should be operated at pH 4.5 – 5 while it was known that penicillin production was maximal around pH 7 – 7.5 (Gaden, 2000). With the genus Fusarium, much like other fungi, acidic conditions of culture were preferred. Kuhad et al. (1998) found that pH 3 – 3.5 to be optimal for the production of xylanase by Fusarium oxysporum. Srivastava et al. (2011) found culture pH dropped quickly to 3.2 during growth of Fusarium oxysporum. Culture pH of 5 – 5.5 was found to be optimal for the production of endoglucanase (Chellapandi and Jani, 2009). In a situation similar to the work reported in this thesis, a novel endophytic Fusarium sp. isolated from a tree (Taxus wallichiana) was reported to growth best at pH 6 (Gogoi et al., 2008). In pigment production by Fusarium verticillioides the highest biomass yield was obtained at pH 5, while production of napthoquinone was best obtained at pH 8 (Boonyapranai et al., 2008). Thus, study of the effect of pH on the production of POPI by Fusarium sp. will be required within this work with indications already that an acidic condition may be favorable and that the pH for growth may be different from that for POPI production.

2.5.3 Agitation and aeration

Agitation plays an important role in submerged aerobic culture. It determines the adequacy of mixing and effect of diffusional limitations in gas, heat and mass transfer. The growth rate of fungal mycelium and metabolite production can be affected by the amount of oxygen in a liquid medium in an aerobic fermentation (Barberel and Walker, 2000; Žnidaršič and Pavko, 2001; Abd‐Aziz et al., 2008; Garcia‐ Ochoa and Gomez, 2009). Although the higher transfer rates can be obtained via

higher agitation, in the case of impeller stirred systems higher shear forces deleterious to microorganisms may result. The degree of agitation can also affect the morphology of filamentous microorganisms causing them to grow in dispersed or pellet forms of various sizes depending on agitation as reported by Abd‐Aziz et al. (2008) in work with Trichoderma virens. The many examples of studies reporting that agitation affects either growth or production rates of metabolites include: levan production by immobilized Bacillus subtilis (Shih et al., 2010); biodegradation of benzo(a)pyrene by a Fusarium sp. (Chulalaksananukul et al., 2006; isoflavone conversion by immobilized Rhizopus spp. (Cheng et al., 2010); pullulan production by immobilized Aureobasidium pullulans (Ürküt et al., 2007); and production of inulinase by immobilized Aspegillus niger (Skowronek and Fiedurek, 2006). Further, Chen and Lin (2007) found that decolorization of azo dye by Pseudomonas luteola was less sensitive to changes in agitation rates (dissolved oxygen) when in immobilized than in free cell form. Zhen and Yu (1998) found that oxygen starvation of immobilized Phanerochaete chrysosporium led to increased release of protease which resulted in denaturation of the desired end‐ product ligninase. It is therefore clear that in any typical submerged aerobic culture, agitation and the resultant oxygenation derived is a key culture parameter which affects the bioreaction through direct (biomass quantity; biomass morphology; synthesis rates) and indirect effects (presence of other by‐products affecting yield of the desired product). Accordingly, it will be a culture parameter to be investigated in the development of a process for POPI production from Fusarium sp. IMI397470

2.5.4 Temperature

Temperature has a fundamental effect on the kinetic energy available for biochemical reactions of the metabolism of a microorganism. Laboratory incubation of the Fusarium group usually carried out at 25oC (Leslie and Summerall, 2006). Growth

and production of metabolites from various Fusarium species have been produced at a range of incubation temperatures (Table 2.5). The literature and experience with Fusarium sp. IMI397470 suggests investigation of incubation temperatures in this work for the growth of the fungus and its production of POPI in a range bounded by 25 – 30oC.

Table 2.5 Incubation temperatures which have been used in the culture of Fusarium species Incubation Fusarium temperature Product Reference species (oC) 28 solani pigments Ammar et al. (1979) 26 aquaeductuum aquastatin Hamano et al. (1993) 28 oxysporum Lipase Rifaat et al. (2010) 30 Panagiotou and oxysporum aldose reductases Christakopoulos (2004) Roseum Cyclosporin Ismaiel et al. (2010) oxysporum Mycelial growth Farooq et al. (2005) verticillioides Naphthoquinones Boonyapranai et al. (2008) 25 ‐ 30 globulosum Lipase Gulati et al. (2005) 25 ‐ 30 moniliforme Biotransformation Uzura et al. (2001)

2.5.5 Influences on immobilized cell fermentation

Additional considerations impact a fermentation using immobilized cells. Hydrogels are typically shaped into beads when used to immobilize cells. Thus, when used as such the fermentation would comprise cells within beads which are suspended in a liquid medium. With a gel component distinct from the medium, at least two factors needs to be considered: (a) the size of each bead as this affects the diffusion gradient within the bead which in turn affects mass and gas transfer to and from the cells immobilized in the gel matrix; (b) the ratio of the volume of beads to volume of medium as this affects the productivity per unit bioreaction volume.

2.5.5.1 Bead size

In fermentations with cells immobilized in hydrogel, mass and gas transfer have to occur in a second zone following the liquid medium. This second zone is the gel matrix itself. Two major factors can be deduced to be influential on fermentation rate. The first is the distance over which mass and gas diffusion has to occur where the smaller the distance the easier will it be for cells to receive nutrients and export metabolic products. The second is the surface area to volume ratio of each bead where the larger the surface area compared to volume of the bead, the better will be transfer of mass and gas between bulk liquid and the gel matrix. Both these factors are controlled by bead size. Further, in response to better mass and gas transfer at the periphery rather than the interior of beads, when higher concentrations of cells grow nearer to the exterior of immobilized aggregations (Eikmeier et al., 1984; Pashova et al., 1999), zones of depletion can occur thus causing poor bioactivity within aggregations. Given the preceding deduction, it is not surprising that various different studies with different microorganisms show the superiority of smaller‐sized beads. Ahmed (2008) found that invertase production by Bacillus macerans was better when alginate bead size was smallest in the range 2 to 6 mm in diameter. Similarly, Charumathi and Das (2010) found that dye removal by Candida tropicalis was inversely proportional to bead size. Idris and Suzana (2006) in their work with immobilized Lactobacillus delbrueckii reported of three bead sizes tested, the smallest at a diameter of 1 mm gave the best rates and yield of lactic acid. Similarly, studies by Srinivasulu et al. (2003) and Mordocco et al. (1999) both showed that the production of neomycin from Steptomyces marinensis and degradation of phenol by Pseudomonas putida respectively were inversely related to bead size. However, the evidence is not clear cut. Zain et al. (2011) reported that the larger of two bead sizes tested gave a higher bioethanol yield from a yeast. Wang et al. (2001) found that middle‐sized beads in the range 2, 3 and 5 mm in diameter gave the best degradation of quinoline by Burkholderia sp. Kumaravel and Gopal (2010) also found optimal performance with

beads of a medium size in protein production by Bacillus amyloliquifaciens, as did Hameed (2007) in the removal of ammonia, nitrate and phosphate by Chlorella vulgaris. Various bead diameters have been used in studies of metabolites production, as summarized in Table 2.6. It appears that bead diameters are typically around 3 mm. This suggests that there is no advantage of diameters less than, and the disadvantage of it being greater than this size. This remains to be tested for the production of POPI by Fusarium sp. IMI397470.

2.5.5.2 Bead to medium volume ratio

The ratio of bead number or volume (and therefore quantity of biomass) to volume of medium is an important parameter because the optimal ratio will result in the highest productivity per unit bioreactor volume given that this is related to biomass number and its access to solute and gas inputs. Too low a ratio will mean low rate of production as the biomass quantity per unit bioreaction volume will be low. Too high a ratio may mean that the aggregate demand rate for solute and gas inputs by the biomass will be higher than can be supplied. Studies which recognized the relevance of the bead to medium volume ratio and investigated this factor in fermentation include those shown in Table 2.7. How the bead volume to medium volume ratio affects the production of POPI by immobilized Fusarium sp. IMI397470 is seen as importance and will be investigated in this work.

Table 2.6 Examples of bead diameters used in fermentations with immobilized cells

Diameter Hydrogel Product Microorganism Reference (mm)

Lactobacillus Mirdamadi et al. Alginate 2 Lactic acid casei (2008)

Candida Alginate 2 Ethanol Abbi et al. (1996) shehatae

Lactobacillus Alginate 2.5 to 3.0 Lactic acid Shen and Xia (2006) delbrueckii

‐ Duran‐Paramo et al. 3 α‐amylase Bacillus subtilis carrageenan (2000)

Bacillus Mohapatra et al. Alginate 3 Tannase licheniformis (2006)

Thermomucor Kumar and Alginate 3 Glucoamylase indicae‐ Satyanarayana (2007) seudaticae

Streptomyces Adinarayana et al. Alginate 3.24 Neomycin marinensis (2004)

Alginate ca. 4 Lipase Aspergillus niger Ellaiah et al. (2004)

Aspergillus Alginate 3 to 5 Cyclosporin A Sallam et al. (2005) terreus

Table 2.7 Examples of studies where the ratio between the volume of immobilized aggregations and medium volume were investigated Immobilized Microorganism Bioreaction aggregation: Reference Medium ratio Yeast species Ethanol production 1:1.67 Zain et al. (2011) Candida Branco et al., Xylitol production 1:2.5 optimal guilliermondii (2007) Pseudomonas Hannaford and Phenol degradation 1:3 optimal putida Kuek (1999) 1:3 to 1:11 at Pseudomonas Mordocco et al. Phenol degradation different D in putida (1999) continuous culture Saccharomyces Navaratil et al. Mead production 1:3 optimal cerevesiae (2001) 1:11.8 optimal Bacillus (300 beads to 50 Mohapatra et al. Tannase production licheniformis mL medium; 3 mm (2007) diameter beads) 1:71 optimal Tolypocladium (100 beads per 100 Survase et al. Cyclosporin A inflatum mL medium; 3 mm (2010) diameter beads)

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CHAPTER 3

Identity, short‐term maintenance and long‐term preservation of the culture

3.1 Introduction

The maintenance and preservation of microbial cultures are essential elements in fermentation studies and processes. Effective preservation methods are required to ensure that good viability, and phenotypic and genotypic integrity of cultures are maintained over time. The preservation of cultures can be divided into two terms, short and long term. Short term preservation usually refers to a method involving continual sub‐ culture. Typically, cultures are kept on agar slants at their regular incubation temperature for a certain short period, or stored at 4oC after some period of growth. Sub‐culturing is performed at regular intervals to ensure continued viability of the cultures. This method allows quick recover of the culture for fermentation and it is inexpensive. However, it requires sub‐culturing and storage under conditions where physiological processes are not completely suspended. Both situations may lead to the possibility of genetic variation (Kidby, 1977), and also risk of contamination. Lyophilization is a method used for long term preservation of the strain. Freeze drying, and L‐drying (Lapage et al., 1970) are two of the commonly used methods. Lyophilization is an often used method for the preservation of Fusarium for extended period of time (20+ years) (Jong and Davis, 1979; Leslie and Summerell, 2006). L‐drying as originally developed by Annear (1956, 1958) is a low temperature method for the removal of water to bring cultures to a dried state in vacuo without freezing. As L‐drying was assessed to be the method with the least potential for damaging cell physiology, it was chosen to be the method for long‐term preservation of the culture used in this study to ensure reproducibility and assurance of culture

identity and genetic fidelity. An accompanying strict regime for short‐term culture maintenance and inoculum production was adhered during this study.

3.2 Materials and methods

3.2.1 Identification of the culture The fungus used in this study was isolated in the Malaysian state of Sarawak on the island of Borneo, from the flower of a Yam plant. The fungus was sent for identification by CABI UK (Bakeham Lane, Egham, Surrey TW20 9TY, United Kingdom; previously known as the Commonwealth Mycological Institute). Molecular profiling via Internal Transcribed Spacer (ITS) suggests that the closest identity is Fusarium solani but only 94% similarity was achieved. Further identification was carried out by targeting the translation elongation factor 1‐alpha (TEF) gene. Again there was 94% similarity with Fusarium solani. The molecular profiles of the fungus places it in the wider Fusarium solani aggregate but the profile does not completely match that of any Fusarium solani. CABI advised that that the fungus is most likely a new Fusarium species (Cannon, pers. comm.). The CABI collection number IMI397470 was assigned to the fungus as an unspeciated culture.

3.2.2 Short‐term culture maintenance Master agar cultures. Fusarium sp. IMI397470 was maintained on Parker Agar (+10 g L‐1 glucose) (Table 3.1). A master culture was sub‐cultured to produce a new set of 10 master culture slants when it reached 3 months of normal storage at 4oC. A block of mycelium was cut from the master culture by a knife loop, and aseptically transferred to a new agar slant. All culture transfers were effected within a laminar flow cabinet (Exco). The agar slants were incubated in a static incubator (Binder KB53) at 25oC. After a 7‐day incubation, the slants were stored at 4oC until required. No master culture was allowed to be sub‐cultured through more than one cycle. When no more agar master cultures were available for sub‐culture, a fresh line of master cultures was initiated

using L‐dried ampoules of the fungus (see below for the procedure of reviving L‐dried cultures).

Table 3.1 The basal composition of Parker Medium*+ used for Fusarium sp. IMI397470^ Ingredient Quantity (g L‐1) Monosodium glutamate 0.55 Yeast extract (Difco) 0.5 Agar 15.0 (when required for solidification) Ingredient Quantity (mg L‐1)

MgSO4∙7H2O 200

Na2SO4∙10H2O 200

KH2PO4 130

K2HPO4 70

FeSO4∙7H2O 20

MnSO4∙H2O 10

ZnSO4∙7H2O 2

CuSO4∙7H2O 2 Autoclaving conditions: 121oC; 15 minutes. * In this study this medium is referred to as Parker Broth when in liquid form and as Parker Agar when solidified. Suffixes after the name indicate the type and quantity of main carbon source added. + This medium is referred to as a semi‐defined medium because one of the ingredients (yeast extract) is not exactly defined. ^The version of this medium used for maintenance of master and working cultures contained 10 g L‐1 of glucose.

Working agar cultures. A master culture as prepared above was used was sub‐cultured into 10 agar slants which after incubation for 7 days were then designated as working cultures. Working cultures were kept in incubation at 25oC until needed. Working cultures were routinely used to commence inoculum production, or as a source for more working cultures. If used as a source for more working cultures, the operating policy was that only those cultures which were less than 3 months old can be used. A working culture was discarded whenever it reached 3 months in age.

3.3.3 Long term preservation In the course of studies with immobilized Fusarium sp. IMI397470 reported in later chapters, it was observed microscopically that liquors from cultures with the immobilized fungus contained numerous spores and mycelial fragments. It was decided that such liquors could be the source of propagules for L‐drying. The term “propagule” is used here because besides the spores, some of the mycelial fragments would have been potentially viable. Preparation of culture liquor containing propagules of Fusarium sp. IMI397470 via shake flask culture of its immobilized mycelia Inoculum preparation. This is described in Section 4.2 of Chapter 4. Preparation and culture of immobilized mycelia. This is described in Section 11.2 of Chapter 11. L‐drying of culture liquor. After seven days of incubation of the inoculated hydrogel beads, a drop of culture liquor was placed onto a slide and observed under a microscope at 400x magnification. This observation was repeated daily until the presence of abundant spores was confirmed at which event the culture liquor was considered ready for L‐drying. L‐drying method. All the transfers mentioned were aseptic and effected within a laminar flow cabinet (Exco). Small glass ampoules (Thermo Fisher; 60 mm length x 5 mm inner diameter) were plugged with cotton and autoclaved. A drop of culture liquor was aseptically deposited into an ampoule via a sterile Pasteur pipette. This was followed with a drop of cryoprotectant (10% sucrose + 10% monosodium glutamate solution) and then the cotton bung of the ampoule was replaced. The cotton‐bunged end of the ampoule was then plugged onto an ampoule manifold of a vacuum drier (Labconco FreeZone Plus 12 litre). The ampoules were allowed to evacuate to 0.009 millibar overnight. The dried ampoule was removed from the vacuum drier and flame torched to form a capillary constriction. The ampoule was then plugged back to the vacuum drier and evacuated to 0.009 millibar at which stage the ampoule was ready to be flame torch cut from the drier by burning through the glass constriction previously

formed. In this manner, ampoules of L‐dried propagules of Fusarium sp. IMI397470 in vacuo were produced. Ampoules thus produced were stored at room temperature. Revival of L‐dried propagules and testing of their viability. From each batch of 24 prepared ampoules, three ampoules were sampled. These were surface sterilized with 70% alcohol before being cut open to recover the dried propagules within. A drop of Parker Broth (+ 10 g L‐1 of glucose) was added to the dried propagules within each of the opened ampoule via a sterile Pasteur pipette. After five minutes, the propagule suspension from each ampoule was drop inoculated onto three Parker Agar (+ 10 g L‐1 of glucose), plates and incubated at 25oC for 7 days or until growth was visible. To give an indication of how well the ampouled cultures will store, an “accelerated storage test” (Tommerup and Kidby, 1979) was conducted. A separate set of 3 prepared ampoules were placed in a 60oC oven for 60 minutes before the procedure for revival and viability testing as previously described was performed.

3.3 Results and discussion

Mycelial cultures of Fusarium sp. IMI397470 appear whitish in color in the early stages of incubation. They settle into a biege or light brown color upon maturity, normally after 7 days incubation in the dark. Colonies have dry, powdery look (Fig. 3.1). The protocol for master and working cultures were described more for the record to give confidence that effort was made to control the risk of genetic variation in the fungus through the course of the study. The choice of 3 months as the age for discarding cultures in short‐term preservation of Fusarium sp. IMI397470 was effective as no loss of viability of the master or working agar slant cultures was observed through the entire study period. L‐dried propagules of Fusarium sp. IMI397470 were capable of producing colonial growth after revival. Growth from revival usually appeared after 3 days of incubation. No ampoule (Fig. 3.2) has failed to be revived successfully and this has been confirmed over a period so far of 3 years (Table 3.2). The viability of dried cultures stored in vacuo is correlated to their ability to be revived after heat treatment

Fig. 3.1 7‐day old cultures of Fusarium sp. IMI397470 on Parker Agar (+ 10 g L‐1 glucose)

Fig. 3.2 A flame‐sealed glass ampoule containing L‐dried progagules of Fusarium sp. IMI397470 in vacuo

(Tommerup and Kidby, 1979). This is explained in part by how much residual water is left in the preserved cultures. The less dry a preserved culture is, the higher is the potential for heat treatment to damage its viability. Propagules of Fusarium sp. IMI397470 L‐dried in the manner described in this work were all revivable after heat treatment. This indicates that the preserved cultures have the potential to be stored for long period without loss of viability.

Table 3.2 The revivability of L‐dried propagules of Fusarium sp. IMI397470 Heat Treatment Number of ampoules tested % revival Batch 1 ‐ 3 100 + 3 100 ‐ 3 100 Batch 2 + 3 100 ‐ 3 100 Batch 3 + 3 100 Batch 3 ‐ 3 100 (after 3 years + 3 100 storage) Total no. of ampoules produced in each batch = 24

3.4 References

Annear, D.I. (1956) The preservation of bacteria by drying in peptone plugs. Journal of Hygiene 54: 487 ‐ 508.

Annear, D.I. (1958) Observations on drying bacteria from the frozen and from the liquid state. Australian Journal of Experimental Biology 36: 211 ‐ 222.

Jong, S.C. and Davis, E.E. (1979) Conservation of reference strain of Fusarium in pure culture. Mycopathologia 66: 153 ‐ 159.

Kidby, D.K. (1977) Culture maintenance and productivity. In: “Extra‐cellular Microbial Polysaccharides”; P.A. Sandford and A. Laskin (eds.); American Chemical Society, Washington, D.C.; pp. 1 ‐ 13.

Lapage, S.P.; Shelton, J.E.; Mitchell, T.G. and Mackenzie, A.R. (1970) Culture collections and the preservation of bacteria. In: “Methods in Microbiology Volume 3A”; J.R. Norris and D.W. Ribbons (eds.); Academic Press, London; pp. 135 ‐ 228.

Leslie, J.G. and Summerell, B.A. (2006) The Fusarium laboratory manual. Blackwell publisher Ltd.

Tommerup, I.C. and Kidby, D.K. (1979) Preservation of spores of vesicular‐arbuscular endophytes by L‐drying. Applied and Environmental Microbiology 37: 831 ‐ 835.

CHAPTER 4

Initial observations on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 in submerged aerobic culture

4.1 Introduction

In 2007, Fusarium sp. IMI397470 was isolated from a yam plant near Kuching, Sarawak in a program conducted by the Sarawak Biodiversity Centre to screen for potentially useful microbial compounds. Butanol extracts from culture filtrates of this fungus was found to have high inhibitory activity against prolyl Oligopeptidase (POP). Given the potential of inhibitors of prolyl oligopeptidase (POPI) as beneficial drug therapy for ameliorating symptoms of brain neurological disorders as reviewed in Chapter 2, and therefore conjecturing that such a compound(s) and their microbial producers would be of interest to pharmaceutical companies, studies on the fermentation of this fungus for POPI was commenced. According to Demain (2006), microorganisms isolated from the natural environment usually produce metabolites at “extremely low levels”. Natural selection favoring the genetic variants which have a robust feedback control for the conservation of cellular resources is probably one reason why microorganisms isolated from nature produce metabolites at low levels. An important step in the development of industrial processes for microbial metabolites is to understand the physiology of the microorganism and the effect of the environment presented to it. This contributes to the determination of optimal fermentation conditions and will aid in cost‐ and time saving in industrial production. The aim of this first study was to determine the baseline productivity of POPI in the shake flask culture of Fusarium sp. IMI397470. In particular, the prime fermentation 55

profiles for biomass, POPI titre, and culture pH will be revealed for the first time. Two types of media were used: a complex and a semi‐defined medium. The complex medium Potato Dextrose Broth (PDB) widely used medium for the cultivation of fungi (also in its solid form, Potato Dextrose Agar). The semi‐defined medium Parker Broth has been successfully used for metabolite production by Aspergillus phoenicus (Kuek, 1991) which like the Fusarium species is in the same fungal phylum, the ascomycota.

4.2 Materials and Methods

Inoculum preparation. Each inoculum flask (250 mL Erlenmeyer capped with a 38 mm silicon foam closure [Sigma Aldrich C1046]) contained 50 mL of Parker Broth (+ 10 g L‐1 glucose) (Table 3.1) and a glass marble (1.5 mm diameter). The flasks were inoculated with 6 agar plugs (5 mm diameter) cut from a 7‐day old plate cultures of Fusarium sp. IMI397470. The flasks were incubated at 25oC for 5 days with shaking at 200 rpm in a gyratory incubator (NBS Innova 44R; 2 inch orbit diameter). Inoculum was prepared by homogenizing the contents of 5 inoculum flasks in a sterile semi‐micro blender container (Eberbach E8581) and blender drive (Waring 8011S, Model HGB2WTS3) for (5 seconds at low speed followed by 5 seconds at high speed. After homogenization 5 mL aliquots of the blend served as inoculum for fermentation in shake flask culture. Fermentation. Sufficient flasks were prepared to provide 3 replicates for each sampling point. Each flask (250 mL Erlenmeyer capped with a 38 mm silicon foam closure [Sigma Aldrich C1046]) comprised 45 mL of liquid medium inoculated with 5 mL of inoculum. Inoculated flasks were incubated 25oC and 200 rpm (New Brunswick Scientific Innova 44R; 2 inch orbit diameter). Media. One complex medium, Potato Dextrose Broth (PDB) (Difco) and one semi‐defined medium, Parker Broth ( + 10 g L‐1 glucose or potato starch) (Table 3.1) were used as media for the production of POPI.

Sampling. The contents of three flasks sampled at each 24h interval for the analyses described in the following sections. The standard errors associated with analyses from the three replicates were determined and displayed with each datum point in the figures. Determination of biomass (dry cell weight). Flasks contents were filtered through a piece of pre‐weighed Whatman No. 1 filter paper placed on a sintered glass Buchner funnel. The filtrate was used for the determination of pH, glucose and POPI titre. The filter paper containing mycelial biomass was dried overnight in a 60oC oven before weighing. Drying and weighing were repeated until constant weight was found. Glucose assay. The quantity of residual glucose was determined enzymatically using the glucose oxidase and peroxidase method (Bergmeyer and Bernt 1974). Culture pH. The pH of the culture was measured using a combined electrode with a pH meter (Mettler Toledo, S20). Butanol extraction of POPI from the culture filtrate. Where extraction needed to be performed in order to raise the concentration of POPI, 5 mL of culture liquor was added to an equal volume of butanol in a 15 mL Falcon® tube (Axygen Scientific). The mixture was shaken vertically in a gyratory shaker (NBS Innova 44R; 2 inch orbit diameter) for 1 hour, before being spun at 4000 rpm in a centrifuge (Eppendorf 5702). The butanol layer (2 mL) was decanted to a 5‐mL beaker, and allowed to dry in fumehood overnight. Assay of POP inhibition. The assay method used is that which reports inhibition in terms of percentage inhibition compared to a control without the inhibitor added (Aoyagi, 1991; Kwak et al., 1999; Chung et al., 2003). The dried butanol extract was re‐suspended in 500 µL of 10% DMSO (four‐fold concentration in strength). The reaction mixture comprised 390 µL of phosphate buffer (0.1 mM, pH 7), 50 µL of Prolyl Oligopeptidase from Flavobacterium sp. (PSP‐101; Toyobo Specialties Trading Co., Osaka, Japan) solution (0.3 U mL‐1 of 0.1M phosphate buffer at pH7), and 60 µL of butanol extract as test sample. Three replicates of reaction mixture were prepared for each sample. The reaction mixture was pre‐incubated at 30oC in a waterbath for 10 minutes. Assay was initiated with the addition of 100 µL of substrate solution, 5.0 mM carbobenzoxy‐gly‐pro‐ρ‐nitroanilide (GL Biochem [Shanghai] Ltd., Shanghai, China) in 40% dioxane after which assay mixtures were

incubated for 15 minutes before optical densities were determined at a wavelength of 414 nm using a spectrophotometer (Varian Cary UV/VIS 50). The inhibitory activity was then calculated by the following formula: Percentage of inhibition

  ODsamplePOPI   1  100       ODsample without POPI  

4.3 Results

Fusarium sp. IMI397470 grew in filamentous form under the study conditions with all the media used. It grew better in PDB than Parker Broth with either 10% glucose or starch (Figs. 4.1 and 4.2; Fig. 4.4a). Parker Broth with either 10% starch or glucose resulted in about the same amount of biomass yield over time (Figs. 4.2 and 4.3; Fig. 4.4a). However, fermentation with PDB yielded less than half the maximum POPI titre than when Parker Broth was used (Fig. 4.4b). The other significant difference found with the two types of media is that culture pH remained low (between 4.5 ‐ 5.0) with PDB but with Parker Broth, culture pH stayed high (ca. 8.0 ‐ 8.5). It appears that POPI titres peak after the end of growth phase (Figs. 4.1 to 4.3). A delay in POPI production is also discernible at the early of the biomass curve when growth is most active. The glucose profile for the semi‐defined medium containing this sole carbon source (Parker Broth + 10 g L‐1 glucose), showed a trace reciprocal to the appearance of biomass and almost all of this carbon source was consumed (exhaustion coincided with the biomass plateau) (Fig. 4.2). The biomass accumulation curve was a reciprocal of the glucose curve (Fig. 4.2). The POPI titre obtained in Parker broth, despite type of carbon source used was higher compared to PDB (Fig. 4.4). In contrast, the biomass assimilated in PDB was obviously higher than that of with Parker broth (Fig. 4.4).

100 9 90 8 8 80 7 7 70 6 6 60 5 DW)

-1 5 50 4 4 pH 40 3

Biomass ( g L g Biomass ( 3 30

POPI titre (% POP Inhibition) POP (% titre POPI 2 2 20

1 10 1

0 0 0 012345678

Days Fig 4.1 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Potato Dextrose Broth

100 9 90 8 8 80 7 7 70 6 6 60 5

DW) 5 -1 50 4 4 pH 40 3 Biomass (g L Biomass (g 3 30

POPI titre (% POP Inhibition) POP (% titre POPI 2 2 20

1 10 1

0 0 0 0123456789 Day Fig 4.2 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Parker Broth (+ 10 g L‐1 potato starch)

100 9 9 90 8 8 8 80 7 7 7 70 6

6 6 ) 60 -1 5

DW) 5 5 -1 50 4 4 4 pH 40 3 Biomass (g L 3 30 3 Residual glucose (g L 2 2 20 POPI titre (% POP inhibition) 2

1 10 1 1

0 0 0 0 0123456789 Days Fig 4.3 Shake flask culture of Fusarium sp. IMI397470 for prolyl oligopeptidase with Parker Broth (+ 10 g L‐1 glucose)

5 80

4 DW)

-1 60 3 40 2

Biomass (g L 20 1 POPI titre (% POP Inhibition)

0 0 PDB Starch Glucose PDB Starch Glucose Media Media

a b Fig. 4.4 The maximum biomass (a) and POPI titre (b) in the shake flask culture of Fusarium sp. IMI397470 using 3 different media

4.4 Discussion

Potato Dextrose Broth proved to be a good medium for producing biomass of Fusarium sp. IMI397470 as it grew the best compared to the results with the two Parker Broths. However, this good yield in biomass was not correlated with good POPI yield. The Parker Broths which are chemically semi‐defined basal nutrients with either glucose or starch as the carbon source yielded twice the maximum titre of POPI of the complex medium (PDB). Rapp (1995), Dekker et al. (1997), Tamerler and Keshavarz (2000), Hajjaj et al. (2001), Kim and Oh (2003), and Panagiotou et al. (2005) similarly found that basal or mineral nutrients, with the addition of carbon sources such as simple sugars, polysaccharides, or oils were more suitable than complex media such as PDB. Two important leads arose from this study. Firstly, the relationship between biomass and POPI production and suggest that active growth or high biomass accumulation is not necessarily associated with high POPI yield. Secondly, poorer yields of 62

POPI appear to be obtained when culture pH is on the acidic side (ca. 4.5 ‐ 5.0). Both these observations and inferences were investigated further and reported later in this work.

4.5 References

Aoyagi, T.; Nagai, M.; Ogawa, K.; Kojima, F.; Okada, M.; Ikeda, T. and Takeuchi, T. (1991) Postatin, a new inhibitor of prolyl endopeptidase, produced by Streptomyces viridochromogenes MH534‐30F3. The Journal of Antibiotics 44: 949 ‐ 955.

Bergmeyer, H.U. and Bernt, E. (1974) Determination with glucose oxidase and peroxidase. In: “Methods of Enzymatic Analysis”, Volume 3 (2nd ed); H.U. Bergmeyer (ed); Academic Press Inc.; New York; pp. 1205 ‐ 1212.

Chung, S.K.; Nam, J.A.; Jeon, S.Y.; Kim, S.I.; Lee, H.J.; Chung, T.H. and Song, K.S. (2003) A prolyl endopeptidase‐inhibiting antioxidant form Phyllanthus ussurensis. Archives of Pharmacal Research 12: 1024 ‐ 1028.

Dekker, R.F.H.; Tirl, M.V.; Narayanasamy, R.D. and de Melon Barbosa, A. (1997) Screening for microbial tehalases: extracellular trehalases produced by Fusarium species. Water Journal of Microbiology and Biotechnology 13: 73 ‐ 79.

Demain, A.L. (2006) From natural products discovery to commercialization: a success story. Journal of Industrial Microbiology and Biotechnology 33: 486 ‐ 495.

Hajjaj, H.; Niederberger, P. and Duboc, P. (2001) Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium. Applied and Environmental Microbiology 67: 2596 ‐ 2602.

Kim, T.B. and Oh, D.K. (2003) Xylitol production by Candida tropicalis in a chemically defined medium. Biotechnology Letters 25: 2085 ‐ 2088.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Kwak, J.Y.; Rhee, I.K.; Lee, K.B.; Hwang, J.S.; Yoo, I.D. and Song, K.S. (1999) Thelephoric acid and kynapcin‐9 in mushroom Polyzellus multiflex inhibit prolyl endopeptidase in vitro. Journal of Microbiology and Biotechnology 9: 798 ‐ 803.

Panagiotou, G.; Christakopoulos, P. and Olsson, L. (2005) Simultaneous saccharification and fermentation of cellulose by Fusarium oxysporum F3 ‐ growth characteristics and metabolite profiling. Enzyme and Microbial Technology 36: 693 ‐ 699.

Prabhakar, A.; Krishnaiah, K.; Janaun, J. and Bono, A. (2005) An overview of engineering aspects of solid state fermentation. Malaysian Journal of Microbiology 1: 10 ‐ 16.

Rapp, P. (1995) Production, regulation, and some properties of lipase activity from Fusarium oxysporum f. sp. vasinfectum. Enzyme and Microbial Technology 17: 832 ‐ 838.

Shah, P.; Bhavsar, K.; Soni, S.K. and Khire J.M. (2009) Strain improvement and up scaling of phytase production by Aspergillus niger NCIM 563 under submerged fermentation conditions. Journal of Industrial Microbiology and Biotechnology 36: 373 ‐ 380.

Tamerler, C. and Keshavarz, T. (2000) Lipolytic enzyme production in batch and fed‐batch cultures of Ophiostoma piceae and Fusarium oxysporum. Journal of Chemical Technology and Biotechnology 75: 785 ‐ 790.

Wigley, L.J.; Perry, D.A. and Mantle, P.G. (2008) An Experimental strategy towards optimising directed biosynthesis of communesin analogues by Penicillium marinum in submerged fermentation. Mycological Research 112: 131 ‐ 137.

CHAPTER 5

An assay for prolyl oligopeptidase inhibitor based on units of inhibitory potential

5.1 Introduction

The common assay for inhibitors of prolyl oligopeptidase is based on comparing activity of the enzyme with and without an inhibitor added to the reaction (see Aoyagi, 1991; Kwak et al., 1999; Chung et al., 2003). The reaction is assayed colorimetrically using a chromogenic substrate thus:

prolyl oligopeptidase carbobenzoxy-gly-pro-p-nitroanilide p-nitroaniline released Colorless Yellow

When an inhibitor of POP is added to the reaction, the amount of color generated is attenuated and the difference between the inhibited and the non-inhibited reactions is determined to give a measure of inhibitory power expressed as percentage inhibition thus:  Optical density at end -point of reaction with inhibitor  1 −  ×100  Optical density at end -point of reaction without inhibitor  A significant limitation with this assay method is that it cannot detect inhibition greater than 100% i.e. the reaction is saturated when too much inhibitor is present for the amount of substrate and POP fixed in the system. A way around would be to dilute the inhibitor sample and then running the assay again. However, this raises the semantic problem of values of inhibition greater than 100% (as they would be after correcting for

66 dilution). Thus, an assay that avoids the limitation of 100% as the maximum value and one which can accommodate the dilution of samples of inhibitor was developed. An assay which reports inhibition in terms of the attenuation of the amount of the chromogen released (p-nitroaniline) would be suited for the studies in this project. Within limits, POP should cleave the substrate and release a set quantity of p-nitroaniline in a set time. When inhibitors are added to the reaction, the amount of p-nitroaniline released would be reduced in proportion to the quantity of inhibitor present. Thus, the reduction in the quantity of p-nitroaniline released is a measure of the inhibition exerted and can be expressed as prolyl oligopeptidase inhibitory units or PIU. This study reports on the development of such an assay method.

5.2 Materials and Methods

5.2.1 The 4-nitroaniline standard curve Spectrometry of 4-nitroaniline. Triplicate solutions of 4-nitroaniline of various concentrations were prepared using a stock of 1.0 mM 4-nitroaniline (Sigma Aldrich) in 2% (w/v) ethanol (Table 5.1). The optical densities of the various solutions were determined at 414 nm using a spectrophotometer (Varian Cary 50).

Table 5.1 The preparation of 4-nitroaniline standards 4-nitroaniline 0.1 mM 4-nitroaniline 0.1 M Phosphate Total standard Buffer, pH 7.0 volume mM mL mL mL 0 0 5.0 5.0 0.05 0.25 4.75 5.0 0.10 0.50 4.5 5.0 0.20 1.0 4.0 5.0 0.30 1.5 3.5 5.0 0.40 2.0 3.0 5.0 0.50 2.5 2.5 5.0 67

5.2.2 POPI assay reporting in PIU The assay protocol for POPI is as described in Section 4.2, Chapter 4. However, the optical densities observed were translated to Prolyl Oligopeptidase Inhibitory Units (PIU) where: 1 PIU is that amount of substance that reduces the release of 1 mmol of 4-nitroaniline per minute at 30°C, pH 7.

5.2.3 Production of POPI Shake flask cultures of Fusarium sp. IMI397470 to produce POPI were conducted using the protocol described in Section 4.2, Chapter 4 except that only Parker Broth (+ 10 g L-1 glucose) was used. Culture liquors were sampled daily and treated as previously described except that the samples were not butanol extracted but assayed directly i.e. without dilution or concentration.

5.3 Results

5.3.1 The optical density of 4-nitroaniline solutions of various concentrations The relationship between optical density and concentration of 4-nitroaniline in 0.1 M Phosphate Buffer was found to be linear between 0.05 and 0.50 mM, the range tested (Fig. 5.1). The relationship is described by: y = 1.95298x + 0.00674 with a correlation coefficient of 0.998.

5.3.2 How 4-nitroaniline quantity assayed is used to indicate strength of inhibition (the PIU) In an assay reaction between the chromogenic substrate and POP, the quantity of 4-nitroaniline released is proportional to POP activity. Where POP activity is inhibited, the quantity of 4-nitroaniline released will be reduced. The relationship

1.0

0.8 ) 414

0.6

Optical Density (OD Density Optical 0.4

r = 0.998

0.2

0.0 0.0 0.1 0.2 0.3 0.4 0.5 4-nitroaniline (mM) Fig. 5.1 Optical density at 414 nm as an indicator of the concentration of 4-nitroaniline in 0.1 M Phosphate Buffer

shown in Fig. 5.1 enables the quantity of 4-nitroaniline in the assay to be estimated. Thus, the difference in quantity of 4-nitroaniline released with and without the addition of inhibitor can be determined. This allows an expression of inhibitory power thus: 1 Prolyl Oligopeptidase Inhibitory Unit (PIU) is that amount of substance that reduces the release of 1 mmole mL-1 of 4-nitroaniline in a 15 minute incubation at 30°C and pH 7. Calculation of PIU using the relationship between quantity of 4-nitroaniline and optical density. In an assay for POPI, the quantity of 4-nitroaniline prevented from being released by the presence of the inhibitor is indicated by the difference in optical density:

ODno POPI − ODwith POPI This is the differential OD The amount of 4-nitroaniline indicated by the differential OD can be resolved using the relationship between OD and quantity of 4-nitroaniline: y = 1.95298x + 0.00674 where y = the differential OD; x = quantity of 4-nitroaniline prevented from release Solving for x:

ODdiff = 1.95298x + 0.00674

ODdiff – 0.00674 = 1.95298x

 ODdiff − 0.00674  x =    1.95298  = mmoles mL-1 of nitroaniline prevented from being released in a 15 minute incubation at 30oC and pH 7. = Prolyl Oligopeptidase Inhibitory Unit (PIU) mL-1

This calculation was used to determine the PIU values in the following sections and the rest of this work.

5.3.3 Inhibitory power of POPI from the same culture liquor samples indicated by the two methods

Quantitation of POPI in units reported as percentage inhibition results in a trend curve which shows saturation at Day 3 (Fig. 5.2). In contrast, quantifying POPI in terms of units of inhibition displays no saturation and identifies a peak at Day 6.

80 800

60 600 ) -1

40 400

30 POPI titre (PIU L (PIU POPI titre POPI titre (% inhibition)

20 200

0 0 0 1 2 3 4 5 6 7 Days

Fig. 5.2 POPI production profiles in a shake flask culture of Fusarium sp. IMI397470 revealed using two different units for expressing inhibition of POP

5.4 Discussion

The optical density of 4-nitroaniline in solution is suitable as an indicator of its concentration as the relationship between the two is linear and has a high correlation coefficient. This supports the concept of translating the difference between the optical densities of a reaction with POPI and one without, into a quantity of 4-nitroaniline which is prevented from being formed through inhibition of the cleaving action of POP. This quantity of 4-nitroaniline is used to derive what is called in this study, the Prolyl Oligopeptidase Inhibitor unit. When the active sites on POP are saturated by the quantity of POPI added in the reaction mixture of the assay, the indicated inhibition value tends towards 100%. At that point, the assay will not be able to detect more POPI beyond the saturation amount. The sample could be diluted and re-assay but after allowing for the correction factor, the result would have to show a value higher than 100% (maximal value). This is difficult semantically. On the other hand, with a method which has inhibition units based on quantity of 4-nitroaniline in solution, dilution of samples will not create a problem as there is no “maximum” value to exceed. The absolute value of the indicated inhibition is infinitely scalable. An assay of enzyme inhibition similarly based on the same principle of quantifying the arrest of the development of color can be found for alpha-amylase (Deshpande et al., 1982; Giancarlo et al., 2006; Makkar et al. 2007). The assay reporting in PIU is capable of producing more useful data regarding the production of POPI than when POPI titre is estimated in terms of inhibition percentage. This is evident when the latter method was not capable of identifying the day in the fermentation that POPI titre had peaked. The percentage inhibition reporting assay estimated about same POPI titre after Day 3 whereas the PIU reporting method was still defining differences up to Day 7 where POPI titre peaked and showed that it declined after that point. Therefore, the PIU method is better and was chosen as the assay method for the rest of this work as a result of the data presented here.

5.5 References

Chung, S.K.; Nam, J.A.; Jeon, S.Y.; Kim, S.I.; Lee, H.J.; Chung, T.H. and Song, K.S. (2003) A prolyl endopeptidase-inhibiting antioxidant form Phyllanthus ussurensis. Archives of Pharmacal Research 12: 1024 - 1028.

Deshpande, S.S.; Sathe, S.K.; Salunke, D.K. and Cornforth, D.P. (982) Effects of dehulling on phytic acid, polyphenols, and enzyme inhibitors of dry beans (Phaseolus vulgaris L.). Journal of Food Science 47: 1846 - 1850.

Giancarlo, S.; Rosa, L.M.; Nadjafi, F. and Francesco, M. (2006) Hypoglycaemic activity of two spices extracts: Rhus coriaria L. and Bunium persicum Boiss. Natural Product Research 20: 882 - 886.

Kwak, J.Y.; Rhee, I.K.; Lee, K.B.; Hwang, J.S.; Yoo, I.D. and Song, K.S. (1999) Thelephoric acid and kynapcin-9 in mushroom Polyzellus multiflex inhibit prolyl endopeptidase in vitro. Journal of Microbiology and Biotechnology 9: 798 - 803.

Makkar, H.P.; Siddhuraju, P. and Becker, K. (2007) α-Amylase inhibitor. In: “Methods in Molecular Biology, Volume 393: Plant Secondary Metabolites; H.P.S. Makkar, P. Siddhuraju and K. Becker (eds.); Humana Press Inc., Totowa, New Jersey; pp. 11 - 14.

CHAPTER 6

The activity of prolyl oligopeptidase inhibitor from Fusarium sp. IMI397470 towards recombinant human prolyl oligopeptidase

6.1 Introduction

Since the finding of Yoshimoto et al. (1987) that of inhibitors prolyl oligopetidase have an anti‐amnesic effect through regulation of activity of that enzyme in the brain, the search for such inhibitors have resulted in many potential candidates. These inhibitors are either synthetic or natural in origin, the latter being isolated from both microorganisms and plants (García‐Horsman et al., 2007). Screening for these compounds has mainly been on the basis of activity against POP of bacterial origin (Flavobacterium sp.) because it was a readily available source until the recent advent of recombinant POP. To date, only a few POP inhibitors sourced from microorganisms have been confirmed to have activity against human POP. These include eurystatin A and B (Toda et al., 1992), poststatin (Aoyagi et al., 1991) and lipohexin (Heinze et al., 1997). No prolyl oligopeptidase inhibitors are currently on the market although some have passed early phases of clinical trials (Lambeir, 2011). Like the majority of other studies on POPI, the POPI from Fusarium sp. IMI397470 in this work was assayed using POP from Flavobacterium sp. The basis for the acceptability of the use of bacterial POP for assaying an inhibitor being developed for use against an enzyme of human origin is that both human and bacterial POP are highly conserved in their active site (PROSITE: PDOC00587). Despite this, it needed to be confirmed whether POPI produced by Fusarium sp. IMI397470 is effective against human POP. The aim of this study is to discover such confirmation if any. This will validate the use of bacterial POP as a substitute and also unequivocally establish the potential value of POPI from Fusarium sp. IMI397470. 74

6.2 Materials and methods

Culture liquor was obtained from shake flask cultures of immobilized mycelia. The protocol for inoculum preparation, preparation and fermentation of immobilized mycelia is described in Section 9.2 of Chapter 9. Extraction. To 20 mL of culture liquor was added equal volume of butanol in a 50 mL Falcon® tube. The tube and contents were shaken vertically at 200 rpm (New Scientific Brunswick Innova 44R; 2 inch orbit) at 25oC for an hour. The mixture was the centrifuged at 4000 rpm (Eppendorf 5702), for 20 minutes. The butanol layer was then transferred into a pre‐weighed 10 mL beaker, and air dried overnight in a fumehood. The weight of the dried extract was obtained by deducted the weight of the extract and beaker from beaker. Preparation of a range of POPI concentrations. Dried crude extract was re‐dissolved in 10% DMSO. Various concentrations of the crude extract were prepared in order to obtain

IC50 value for both human recombinant and Flavobacterium sp. POP (Table 6.1)

Table 6.1 The range of concentrations of POPI from Fusarium sp. IMI397470 used to determine its IC50 against both recombinant human and Flavobacterium sp. POP Used for inhibition assay against Used in inhibition assay against human recombinant POP (g mL‐1) Flavobacterium sp. POP (g mL‐1) 0.675 5.438 6.75 10.875 27.0 21.75 216.25 43.5 865.0 87.0 1730 174.0

Prolyl oligopeptidase inhibition assay. The assay protocol was based on that of Toda et al. (1992). The two assays, one using recombinant human POP and the other Flavobacterium

POP were identical except that the total reaction volume for the former assay was smaller because of the price of recombinant human POP. The percentage inhibition was calculated by the following equation:  Differential OD of test sample  Inhibition (%) = 1    100%  Differential OD of control 

Protocol with bacterial POP (Flavobacterium sp.). A total of 60 µL crude extract (1.74 mg mL‐1, containing 10% of DMSO) is transferred to a test tube, added with 390 µL of phosphate buffer (0.1 mM, pH 7), and 50 µL of POP enzyme (0.3 U/mL). The mixture is pre‐incubated in 30oC waterbath for 10 minutes, after that the reaction is initiated with addition of 100 µL of substrate (5 mM Z‐gly‐pro‐ρ‐ nitroanilide) for 15 minutes. After incubation, the reaction was stopped by adding of 600 µL of stopper (Acetate buffer, pH 4) before read at OD wavelength 414 nm via spectrophotometer (Varian Cary UV/VIS 50). Protocol with human recombinant POP. A total of 18 µL crude extract (17.4 mg mL‐1, containing 10% of DMSO) is transferred to a test tube, added with 132.85 µL of phosphate buffer (0.1 mM, pH 7), and 0.15 µL of POP enzyme (1.4 mg mL‐1). The mixture is pre‐incubated in 30oC waterbath for 10 minutes, after that the reaction is initiated with addition of 30 µL of substrate (5 mM Z‐gly‐pro‐ρ‐nitroanilide) for 15 minutes. After incubation, the reaction was stopped by adding of 181 µL of Acetate buffer before read at OD wavelength 414 nm via spectrophotometer (Varian Cary UV/VIS 50).

6.3 Results

Crude POPI from Fusarium sp. IMI397470 exhibited inhibition activity against both human recombinant and Flavobacterium sp. POP (Figs. 6.1 and 6.2). IC50 values were ‐1 derived from the inhibition curves and the indicated IC50 was 60 g mL for Flavobacterium sp. POP and 128 g mL‐1 for recombinant human POP.

100

40 Inhibitory activity (%) 30

0 0.00.20.40.60.81.01.21.41.61.8 Concentration of crude POPI (mg mL-1)

Fig 6.1 The inhibitory activity exhibited by crude extract of POPI against human recombinant POP

100

40 Inhibitory activity (%) activity Inhibitory 30

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Concentration of crude POPI (mg mL-1)

Fig 6.2 The inhibitory activity exhibited by crude extract of POPI against Flavobacterium sp. POP

6.4 Discussion

Even in its crude form from butanol extracts, POPI from Fusarium sp. IMI397470 is confirmed to be effective against recombinant human POP. However, it appears to be twice more effective against Flavobacterium POP than recombinant human POP on the basis of the relative quantities required to cause IC50. Lipohexin is similarly more potent against bacterial POP than human POP (Christner et al., 1997). On a weight basis, POPI from Fusarium sp. IMI397470 is more potent than at least one other reported POPI, kynapcin‐13. Only 60 g mL‐1 of POPI from Fusarium sp. IMI397470 was required to inhibit 50% of Flavobacterium sp. POP whereas 640 mg mL‐1 of kynapcin‐13 could only inhibit

it by 15% (Kim et al., 2002). The confirmation of effectiveness against recombinant human POP means that there is justification for continued study of POPI from Fusarium sp. IMI397470 because of its potential to be developed into a pharmaceutical drug. The comparatively higher sensitivity of Flavobacterium sp. POP towards POPI confirms its suitability as a choice in the assay used in this and other studies because it means that it should be able to detect relatively lower levels of POPI titre. This differential in sensitivity is probably predictable because the primary screening of isolates from the field was based on POP from Flavobacterium sp. Thus, isolates identified as superior as was Fusarium sp. IMI397470 would have a higher activity against the bacterial rather than human POP. Crude extracts usually contain a large amount of other components. Strategies such as partition and fractionation have been commonly used in order to remove the unnecessary materials and also in aid to concentrate the desired compound (Dai et al., 2010). Pernitsky et al. (2011) and Zeeshan et al. (2012) reported the increment of the potency of bioactive compounds when the crude extracts were purified via partial fractionation and preparative thin layer chromatography, respectively. Thus, the relatively lower activity of POPI from Fusarium sp. IMI397470 against recombinant human POP compared to Flavobacterium sp. POP may yet increase upon purification.

6.5 References

Aoyagi, T.; Nagai, M.; Ogawa, K.; Kojima, F.; Okada, M.; Ikeda, T. and Hamada, M. (1991) Poststatin, a new inhibitor of prolyl endopeptidase, produced by Streptomyces viridochromogenes MH534‐30F3 I. Taxonomy, production, isolation, physico‐chemical. The Journal of Antibiotics 44: 949 ‐ 955.

Christner, C.; Zerlin, M.; Gräfe, U.; Heinze, S.; Küllertz, G. and Fischer, G. (1997) Lipohexin, a new inhibitor of prolyl endopeptidase from Moeszia lindtneri (HKI‐0054) and

Paecilomyces sp. (HKI‐0055; HKI‐0096). 2. Inhibitory activities and specificity. The Journal of Antibiotics 50: 384 ‐ 389.

Dai, J. and Mumper, R.J. (2010) Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 15: 7313 ‐ 7352.

García‐Horsman, J.A.; Männisto, P.T. and Venäläinen, J.I. (2007) On the role of prolyl oligopeptidase in health and disease. Neuropeptides 41: 1 ‐ 24.

Heinze, S.; Ritzau, M.; Ihn, W.; Hulsmann, H.; Schlegel, B.; Dornberger, K.; Fleck, W.F.; Zerlin, M.; Christner, C.; Gräfe, U.; Küllertz, G. and Fischer, G. (1997) Lipohexin, a new inhibitor of prolyl endopeptidase from Moeszia lindtneri (HKI‐0054) and Paecilomyces sp. (HKI‐0055; HKI‐0096) .1. Screening, isolation and structure Elucidation. The Journal of Antibiotics 50: 379 ‐ 383.

Kim, S.I.; Park, I.H. and Song, K.S. (2002) Kynapcin‐13 and ‐28, new benzofuran prolyl endopeptidase inhibitors from Polyozellus multiplex. The Journal of Antibiotics 55: 623 ‐ 628.

Lambeir, A.M. (2011) Translational research on prolyl oligopeptidase inhibitors: the long road ahead. Expert Opinion Therapeutic Patents 21: 977 ‐ 981.

Pernitsky, K.; Mason, Q.D.; Cinel, B. and Friedman, M.R. (2011) Discovery and partial purification from lodgepole pine dwarf mistletoe (Arceuthobium americanum) active against Gram positive organisms including methicillin‐resistant Staphylococcs aureus (MRSA). Journal of Medicinal Plants Research 5: 1711 ‐ 1727.

Toda, S.; Obi, Y.; Numata, K.; Hamagishi, Y.; Tomita, K.; Komiyama, N.; Kotake, C.; Furumai, T. and Oki, T. (1992) Eurystatins A and B, new prolyl endopeptidase inhibitors I. Taxonomy, production, isolation and biological activities. The Journal of Antibiotics 45: 1573 ‐ 1579.

Yoshimoto, T.; Kado, K.; Matsubara, F.; Koriyama, N.; Kaneto, H. and Tsuru, D. (1987) Specific inhibitors for prolyl endopeptidase and their anti‐amnesic effect. Journal of Pharmacobio‐Dyn 10: 730 ‐ 735.

Zeeshan, M.; Rizvi, S.M.D.; Khan, M.S. and Kumar, A. (2012) Isolation, partial puridication and evaluation of bioactive compounds from leaves of Ageratum houstonianum. EXCLI Journal 11: 78 ‐ 88.

CHAPTER 7

The relationship between growth of Fusarium sp. IMI397470 and its production of prolyl oligopeptidase

7.1 Introduction

The relationship between the growth and POPI production curves found in the initial observations on the production of POPI by Fusarium sp. IMI397470 in shake flask culture (Chapter 4) suggest that POPI production may have a growth‐dissociated pattern (Enatsu and Shinmyo, 1978). This type of pattern is where production of a microbial compound does not occur during the active growth phase. Production of a growth‐ dissociated compound (as may be revealed by the Specific Rate of Product Formation, Qp) only maximizes as the growth rate of the producing microorganism (as may be revealed by its Specific Growth Rate, ) tends to zero. Secondary metabolites are compounds which are commonly defined as those which are not essential for the biochemistry of growth. Accordingly, in terms of the microbial growth curve, microbiologists generally agree that since secondary metabolites are not essential for growth, those compounds which are largely produced in the idiophase (when growth is complete), are secondary metabolites. Indeed, in microbiology, secondary metabolites are also defined in terms of their relationship to phases in the growth curve (Bunch and Harris, 1986; Barrios‐Gonzalez et al., 2005) or growth rate (Demain, 1986). Putting the two parts of the preceding information together, if the production of a microbial substance has a growth‐dissociated pattern of production, then by inference, it is likely that the substance is a secondary metabolite. The aim in this study is to run a

82 fermentation for POPI using Fusarium sp. IMI397470 and collect enough data points to calculate the indicated Specific Growth Rates () of the fungus and its Specific Rate of

Production of POPI (Qp) over the course of the batch in order to determine whether the production of POPI is growth‐associated or growth‐dissociated via an examination of the relationship between  and Qp.

7.2 Materials and methods

Inoculum preparation, fermentation, sampling, determination of biomass, glucose assay, and culture pH from the culture filtrate followed the protocols described in Section 4.2, Chapter 4. Assay of POP inhibition. This followed the protocol described in Section 4.2, Chapter 4 except that butanol extraction was not conducted. PIU was calculated as explained in Section 5.3.2, Chapter 5.

7.3 Results

7.3.1 POPI fermentation using Fusarium sp. IMI397470 In the first 1 to 1.5 days of culture, fungus displayed normal transition from lag to exponential growth phase (Fig. 7.1). However, during this period, POPI titres did not rise from their baseline value. POPI titres only showed a rapid increase between Days 1.25 and 2.25 at which time fungal growth was slowing down towards stationary phase. POPI production appeared to peak in the stationary phase of the culture. To examine this observation in greater detail, the Specific Growth Rate (µ) of the fungus was calculated for

83 each sample time by firstly determining its Growth Rate (Rx ) thus:

 xt1  xt1  (Sinclair and Cantero, 1990) Rx    t (t 1)  (t -1)   Where x = dry cell weight; t = sampling time

and then deriving the Specific Growth Rate () indicated thus: R xt (Sinclair and Cantero, 1990) t = xt

The Specific Rate of POPI Production per unit biomass (Qp) for each sample point was calculated using the following formula.

 titre at (t 1)  titre at (t -1)    (t 1)  (t -1)  Qp at t =   dry cell weight at t

When the relationship between µ and Qp is examined, it is notable that the maximum

Specific Rate of Production of POPI (Qpmax) and the maximum Specific Growth Rate (µmax) of Fusarium sp. IMI397470 show distinct peaks separated by about a day (Fig. 7.2).

8 10 7

500 7

6 8 6 400 5 )

5 -1

) 6 DW) DW) -1

300 -1 4 4 pH

3 3 4 200 BiomassL (g POPItitre (PIU L Residual glucose (g L 2 2

2 100

1 1

0 0 0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Days

Fig. 7.1 POPI production by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose) in shake flask culture showing the relationship between the biomass and POPI profiles

max

200 3 Qpmax 

150

100 Specific Growth Rate Rate Specific Growth

50 Specific Rate ofProduction of POP-inhibitor Q

0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Days Fig 7.2 The relationship between the Specific Growth Rate (µ) of Fusarium sp. IMI397470

and its Specific Rate of Production of POPI (Qp). Data from Fig. 7.1 was converted

to µ and Qp

7.4 Discussion

The study of the production pattern of a compound with respect to the growth of the cells which produce it is important because it enables the characterization of the pattern as that of either a primary or secondary metabolite. This has implications for process development because production patterns may suggest particular routes of

86 exploitation. For example, continuous culture has less application for secondary metabolites because growth at some rate is required to replace biomass lost through washout. A process where growth is limited will be more suitable for secondary metabolites.

Since the production of POPI (as indicated by Qpmax) is maximal after growth rate has begun to decrease i.e. after the maximum Specific Growth Rate (µmax) has occurred, its pattern of production may be classified as growth‐dissociated (Enatsu and Shinmyo, 1978). This pattern suggests that the POPI inhibitor produced by Fusarium sp. IMI397470 is a secondary metabolite (going by the definition that such metabolites are formed in the idiophase or when growth rates are low at the end of growth). Reports of other time course studies of POPI production by microorganisms are limited. However, time course data from one paper (Toda et al., 1992) showed that POPI inhibitor produced by Streptomyces eurythermus has a similar growth‐dissociated pattern as that which is being reported now. Knowledge that a production pattern is growth‐dissociated enables the fermentation to utilize immobilized cells in phase‐separated repeat batch culture (Kuek, 1986) such as that reported for glucoamylase production (Kuek, 1991). This is a process where the culture has an initial phase for cell growth followed by repeated batches where fermentation of the growth‐dissociated product occurs under conditions optimized for synthesis. Exploitation of Fusarium sp. IMI397470 in the manner described will be examined in studies which follow (Chapter 9 onwards).

7.5 References

Barrios‐Gonzalez, J.; Fernadez, F.J.; Tomasisni, A. and Mejia, A. (2005) Secondary metabolite production by solid‐state fermentation. Malaysian Journal of Microbiology 1: 1 ‐ 6.

Bunch, A.W. and Harris, R.E. (1986) The manipulation of micro‐organisms for the production of secondary metabolites. Biotechnology and Genetic Engineering Reviews 4: 117 ‐ 144.

Demain, A.L. (1986) Regulation of secondary metabolism in fungi. Pure and Applied Chemistry 58: 219 ‐ 226.

Enatsu, T. and Shinmyo, A. (1978) In vivo synthesis of enzymes.Physiological aspects of microbial enzyme production. Advances in Biochemistry Engineering and Biotechnology 9: 111 ‐ 144.

Kuek, C. (1986) Immobilized living mycelia for the growth‐dissociated synthesis of chemicals. International Industrial Biotechnology 6: 123 ‐ 125.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Sinclair, C.G. and Cantero, D. (1990) Fermentation modelling. In: “Fermentation: A practical approach”, B. McNeil and L.M. Harvey (eds.), IRL Press; pp. 65 ‐ 112.

Toda, S.; Obi, Y.; Numata, K.; Hamagishi, Y.; Tomita, K.; Komiyama, N.; Kotake, C.; Furumai, T. and Oki, T. (1992) Eurystatins A and B, new prolyl endopeptidase inhibitors. I. Taxonomy, production, isolation and biological Activities. The Journal of Antibiotics 45: 1573 ‐ 1579.

CHAPTER 8

The effect of the quantity of supplied glucose on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470

8.1 Introduction

A good fermentation can be characterized by the consumption of all the major substrates supplied, an efficient conversion of substrates to product, and high productivity (Kuek, 1996). The carbon source is one of the major components in a fermentation medium. It is a key component because it provides the source of energy for the production of microbial biomass and for the synthesis of compounds. Carbohydrates are commonly used as carbon sources in the formulation of fermentation media. As reviewed in Section 2.5.1.1, Chapter 2, glucose is a good initial choice as carbon substrate for Fusarium sp. IMI397470 and initial studies in this work has already demonstrated growth and POPI production on that substrate (Chapters 4 and 7). Alongside the choice of a carbon substrate is the question of the amount of that substrate to supply in a fermentation. Supply at too low a quantity would make availability of this substrate a rate‐limiting step. An over‐supply would result in cost inefficiency as there would tend to be residual substrate at the end of fermentation. The objective of this study is to determine an optimal starting concentration of glucose in Parker Broth for the production of POPI by Fusarium sp. IMI397470 in shake flask culture.

8.2 Materials and methods

Inoculum preparation, fermentation, sampling, determination of biomass, glucose assay, and culture pH from the culture filtrate followed the protocols described in Section 4.2, Chapter 4. The medium used was Parker Broth (+ glucose ranging from 5 to 20 g L‐1). Assay of POP inhibition. This followed the protocol described in Section 4.2, Chapter 4 except that butanol extraction was not conducted. PIU was calculated as explained in Section 5.3.2, Chapter 5.

8.3 Results

The fungus exhibited normal growth curves at all the concentrations of tested (Fig. 8.1) and although no particular trend was discernible, the lowest quantity of glucose (5 g L‐1) appeared to favour higher biomass accumulation (Figs. 8.1 and 8.3). At this level of supply, while biomass quantity was maximal, POPI titre was minimal. The two higher glucose concentrations (10 and 20 g L‐1) resulted in higher and similar yields (Fig. 8.2 and 8.3). Analysis of yield coefficients at a glucose supply of 5 g L‐1 indicate that conversion of glucose to biomass was most favoured whilst its translation into POPI yield was least favoured (Fig. 8.4). As the amount of supplied glucose was increased from 5 to 20 g L‐1, the efficiency of its conversion to biomass (Yx/s) was reduced (Fig. 8.4). The production of POPI from each unit of glucose substrate (Yp/s) and the production of POPI per unit of biomass ‐1 (Yp/x) was the most efficient with glucose supplied at 10 g L .

4 DW) -1

Biomass (g L (g Biomass 2 5 g L-1 10 g L-1 1 20 g L-1

0 01234567 Days

Fig. 8.1 The effect of the quantity of supplied glucose on biomass accumulation by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose)

1000

800 ) -1 600

400 POPI titre (PIU L (PIU titre POPI 5 g L-1 -1 200 10 g L 20 g L-1

0 012345678

Days

Fig. 8.2 The effect of supplied glucose on POPI yield of Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose)

1400

5 1200 ) 4 1000 -1

800 DW)

-1 3

600

2 400 Biomass (g L Biomass (g

1 200 (PIU L POPI titre Maximum

0 0 0 5 10 15 20 25 Glucose supplied (g L-1)

Fig. 8.3 The effect of supplied glucose supply on the maximum POPI titres and biomass obtained from Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose). This is a re‐presentation of data from Fig. 8.1 and 8.2

100

0.8 200 80

180 0.6 60 160

0.4 140

40 (PIU/biomass) p/x

(biomass/glucose consumed) (biomass/glucose 120 (PIU/ glucose consumed) x/s 0.2 p/s 20 POPI Y 100 POPI Y Biomass Y 0.0 0 80 0 5 10 15 20 Glucose supplied (g L-1)

Fig. 8.4 The effect of supplied glucose on the efficiency of the conversion of glucose to

biomass (Yx/s) and POPI (Yp/s) by Fusarium sp. IMI397470. The yield coefficients were calculated using the maximum values found for biomass and POPI at each glucose level

8.4 Discussion

Glucose was confirmed as suitable for the production of POPI by Fusarium sp. IMI397470 when supplied as the sole carbon source. A POPI was similarly successfully produced by Moeszia lindtneri and Paecilomyces sp. using a medium containing glucose albeit together with glycerol (Heinze et al., 1997). Boonyapranai et al. (2008) reported that the quantity of Fusarium mycelia increased proportionally with increase in glucose supplied in the range of 10 to 50 g L‐1. This was not found in the current study. On the contrary, the lowest level of glucose (5 g L‐1) appeared to be best for the accumulation of cell mass and the most efficient conversion of the substrate to biomass (it gave the ‐1 highest Yx/s). Between 5 and 10 g L of glucose supplied, as the Yx/s became poorer, there was a corresponding increase in the amount of POPI resulting from each unit of glucose consumed (Yp/s). An interpretation of this observation is that as glucose supply was increased, relatively more of it was shunted to metabolic use which resulted in more POPI output, than for the metabolic requirements resulting in growth. In terms of a fermentation for POPI, this is what would be desired: efficient use of the carbon substrate for synthesis of the end‐product rather than any other. From the relationship between glucose input and resulting POPI titre alone, the optimal glucose supply appears to be at middling concentrations indicated by the POPI titre at 10 g L‐1. The advantage of characterizing fermentations by yield coefficient data is revealed here because the choice of 10 g L‐1 is supported by the finding that conversion of the substrate to POPI at 10 g L‐1 was more efficient than when 20 g L‐1 of glucose was supplied. While POPI titres resulting from the supply of glucose at 10 and 20 g L‐1 were similar, both Yx/s and Yp/x revealed that at the higher level of the two, POPI yields per unit glucose consumed and per unit biomass were lower. This can be interpreted to mean that there is repression of POPI production when high levels of glucose are used. Thus, 10 g L‐1 was chosen as the quantity to use in Parker Broth for the fermentation for POPI by Fusarium sp. IMI397470.

8.5 References

Boonyapranai, K.; Tungpradit, R.; Lhieochaiphant, S. and Phutrakul, S. (2008) Optimization of submerged culture for the production of naphthoquinones pigment by Fusarium verticillioides. Chiang Mai Journal of Science 35: 457 ‐ 466.

Demain, A.L. (2006) From natural products discovery to commercialization: a success story. Journal of Industrial Microbiology and Biotechnology 33: 486 ‐ 495.

Heinze, S.; Ritzau, M.; Ihn, W.; Hulsmann, H.; Schlegel, B.; Dornberger, K.; Fleck, W.F.; Zerlin, M.; Christner, C.; Gräfe, U.; Küllertz, G. and Fischer, G. (1997) Lipohexin, a new inhibitor of prolyl endopeptidase from Moeszia lindtneri (HKI‐0054) and Paecilomyces sp. (HKI‐0055; HKI‐0096) .1. Screening, isolation and structure elucidation. The Journal of Antibiotics 50: 379 ‐ 383.

Kuek, C. (1996) Shake flask culture of Laccaria laccata, an ectomycorrhizal basidiomycete. Applied Microbiology and Biotechnology 45: 319 ‐ 326.

Suihko, M.L. (1983) The fermentation of different carbon sources by Fusarium oxysporum. Biotechnology Letters 5: 721 ‐ 724.

Suruliranjan, M. and Sarbhoy, A.K. (2000) Effect of carbon and nitrogen sources on growth and sporulation of Fusarium moniliforme. Journal of Mycopathological Research 38: 25 ‐ 28.

CHAPTER 9

The effect of temperature on the production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470

9.1 Introduction

The isolation of Fusarium sp. IMI397470 from its host was via plate culture at 25oC and this temperature was subsequently used for routine plate cultures and the initial shake flask studies. This choice is consistent with the literature on an appropriate incubation temperature for growth and metabolite production for Fusarium species. Metabolites from various Fusarium spp. have been produced at incubation temperatures which ranged from 25 ‐ 30oC (see Section 2.5.1.4, Chapter 2). The aim of this experiment was to examine the relationship between incubation temperature, growth of Fusarium sp. IMI397470 and its production of POPI in shake flask culture.

9.2 Materials and methods

Inoculum preparation, fermentation, sampling, determination of biomass, glucose assay, and culture pH from the culture filtrate followed the protocols described in Section 4.2, Chapter 4. The medium used was Parker Broth (+ 10 g L‐1) and the incubation temperatures used ranged from 25o to 35oC. Assay of POP inhibition. This followed the protocol described in Section 4.2, Chapter 4 except that butanol extraction was not conducted. PIU was calculated as explained in Section 5.3.2, Chapter 5.

9.3 Results

Fusarium sp. IMI397470 grew equally well at 25o and 30oC but growth was inhibited at 35oC (Fig. 9.1). However, despite growth being similar at the 2 lower temperatures examined, POPI production at 30oC was twice as high as that at 25oC (Fig. 9.2 and 9.3). POPI production was insignificant at 35oC. Conversion of glucose o o substrate to biomass (Yx/s) was similar at 25 and 30 C but its translation to POPI (Yp/s) was approaching three times as efficient at 30oC (Fig. 9.4). The each unit of biomass produced about 2.5 times more POPI at 30oC than at 25oC. An even higher efficiency per unit biomass resulted at 35oC but this can be discounted in comparative analysis because this high value is a function of the much smaller quantity of biomass (insignificant growth) which resulted at this temperature.

3 DW) -1

o 2 25 C 30oC 35oC Biomass (g L

0 0123456789 Days

Fig. 9.1 The effect of incubation temperature on biomass accumulation by Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose)

25oC 2000 30oC 35oC )

-1 1500

1000 POPI titre (PIU L POPI titre

500

0 0123456789 Days

Fig. 9.2 The effect of incubation temperature on the POPI yield of Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose)

7 2500

6 2000

5 )

1500 -1 4 DW) -1

3 1000

2 Biomass L (g POPI yield (PIU L 500 1

0 0 25 30 35 Temperature (oC)

Fig. 9.3 The effect of incubation temperature on the maximum POPI titres and biomass obtained from Fusarium sp. IMI397470 on Parker Broth (+ 10 g L‐1 glucose). This is a re‐presentation of data from Fig. 9.1 and 9.2

0.8 140 700

120

0.6 600 100

80 500 0.4

60 (PIU/biomass)

400 p/x (biomass/glucose consumed)

( PIU / g glucose consumed)

40 x/s 0.2 p/s Y POPI Y 300 20 POPI Y

Biomass 0.0 0 200 25 30 35 Temperature (oC)

Fig. 9.4 The effect of incubation temperature on the efficiency of the ratios between

glucose consumed to biomass (Yx/s) and POPI (Yp/s) produced, and between

POPI and biomass produced (Yp/x) in the fermentation for POPI by Fusarium sp. IMI397470. The yield coefficients were calculated using the maximum values found for biomass and POPI at each temperature

9.4 Discussion

For Fusarium sp. IMI397470, 35oC proved to be too high for either growth or POPI production. The fungus can grow well at either 25o or 30oC and at these temperatures conversion of glucose substrate to biomass were equally as good. A fermentation temperature of 30oC was twice as effective as 25oC for POPI production in terms of POPI titre. This result is somewhat in concordance with at least two other optimization studies on the production of metabolites by Fusarium spp. (Ismaiel et al., 2010; Rifaat et al., 2010) in that they found optima in the range 28 ‐ 30oC. However, unlike the findings now reported, these studies did not find differences between the optimal temperatures for biomass and for POPI production.

100

Apart from just POPI titre evidence, the optimal temperature of 30oC for POPI production by Fusarium sp. IMI397470 is supported by yield coefficient analysis. Both the translation of glucose substrate to POPI (Yp/s) and the POPI titre produced per unit o o o biomass (Yp/x) at 30 C was better than twice that found at 25 C. Thus, 30 C was identified as the best incubation temperature in the range tested.

9.5 References

Ammar, M.S.; Gerber, N.N. and McDaniel, L.E. (1979) New antibiotic pigments related to Fusarubin from Fusarium solani (Mart.) Sacc. I. Fermentation, isolation, and antimicrobial activities. The Journal of Antibiotics XXXII: 679 ‐ 684.

Hamano, K.; Kinoshita‐Okami, M.; Minagawa, K.; Haruyama, H.; Kinoshita, T.; Hosoyac, T.; Furuya, K.; Kinoshita, K.; Tabata, K.; Hemmi, A. and Tanzawa, K.(1993) Aquastatin A, An inhibitor of mammalian adenosine triphosphatases from Fusarium aquaeductuum. Taxonomy, fermentation, isolation, structure determination and biological properties. The Journal of Antibiotics 46: 1648 ‐ 1657.

Ismaiel, A.A.; El‐Sayed, E.A. and Mahmoud, A.A. (2010) Some optimal culture conditions for production of Cyclosporin A by Fusarium roseum. Brazilian Journal of Microbiology 41: 1112 ‐ 1123.

Kobayashi, H.; Sunaga, R.; Furihata, K.; Morisaki, N. and Iwasaki, S. (1995) Isolation and structures of an antifungal antibiotic, Fusarielin A, and related compounds produced by a Fusarium sp. The Journal of Antibiotics 48: 42 ‐ 52.

Panagiotou, G. and Christakopoulos, P. (2004) NADPH‐dependent D‐Aldose reductases and xylose fermentation in Fusarium oxysporum. Journal of Bioscience and Bioengineering 91: 299 ‐ 304.

101

Rifaat, H.M.; El‐Mahalawy, A.A.; El‐Menofy, H.A. and Donia, S.A. (2010) Production, optimization and partial purification of lipase from Fusarium oxysporum. Journal of Applied Sciences in Environmental Sanitary 5: 39 ‐ 53.

Uzura, A.; Katsuragi, T. and Tani, Y. (2001) Optimal conditions for production of (R)‐ 1‐ Phenylpropanol by Fusarium moniliforme strains MS31. Journal of Bioscience and Bioengineering 92: 288 ‐ 293.

102

CHAPTER 10

Production of prolyl oligopeptidase inhibitor by Fusarium sp. IMI397470 in a 10 L batch in a stirred tank bioreactor

10.1 Introduction

Up to this stage of the work, shake flask cultures were successfully used to demonstrate the production of POPI by Fusarium sp. IMI397470 (Chapters 4, 7, 8 and 9). The aim of this study was to confirm on a larger scale, the fermentation profiles associated with the production of POPI on a larger scale. This study is not considered as systematic scaling‐up because the change in scale of bioreaction is not based on holding a known parameter constant e.g. in oxygen transfer rate; mixing time. Further, there is a big step change in the shift from the cultural environment from the shake flask to that of a stirred tank bioreactor.

10.2 Materials and methods

All media and apparatus were sterilized at 121oC for 15 minutes and all transfers were conducted aseptically in a laminar flow cabinet. Inoculum preparation. Parker Agar (+ 10 g L‐1 glucose) (Table 3.1) plates were inoculated with a 5 mm x 5 mm slice excised from a Fusarium sp. IMI397470 working culture and incubated for 7 days at 25oC. From these plates 6 agar plugs (5 mm diameter) were cut and placed in each of 22 Erlenmeyer flasks (250 mL capped with a 38 mm silicon foam closure [Sigma Aldrich C1046]) containing 50 mL of Parker Broth (+ 10 g L‐1 glucose) (Table 3.1) and a glass marble (1.5 mm diameter). The flasks were incubated in a gyratory incubator (NBS Innova 44R; 2 inch orbit diameter) at 25oC and 200 rpm for 5 days. Contents of enough flasks to prepare 1 L of inoculum blend were 103 homogenized in batches using a semi‐micro blender container (Eberbach E8581) and blender drive (Waring 8011S, Model HGB2WTS3) for (5 seconds at low speed followed by 5 seconds at high speed). Inoculum blend was transferred into a sterile inoculum transfer bottle and was thus ready for bioreactor inoculation. Bioreactor culture. The bioreactor used was a New Brunswick Scientific BioFlo 310 with a 14 L vessel with Rushton turbine impellers (Fig. 10.1). The bioreaction was started by inoculating 9 L of Parker Broth (+ 10 g L‐1 glucose) with 1 L of inoculum blend. The bioreactor/bioreaction set up was: incubation temperature = 30oC; air flow was 1.0 vol. vol.‐1 min‐1 at 101 kPa; agitation = 700 rpm; impellers = 2 at 5 cm intervals from vessel bottom; pH was uncontrolled.

Fig. 10.1 Day 5 of the 10 L fermentation for POPI by Fusarium sp. IMI397470 using a New Brunswick Scientific BioFlo 310

104

Sampling. Five mL of culture liquor was sampled daily from the bioreactor vessel via the sampling port. Analysis. The determination of biomass, glucose assay, and culture pH of the daily samples followed the protocols described in Section 4.2, Chapter 4. Assay of POP inhibition. This followed the protocol described in Section 4.2, Chapter 4 except that butanol extraction was not conducted. PIU was calculated as explained in Section 5.3.2, Chapter 5.

10.3 Results

Fermentation profiles of the bioreactor culture (Fig. 10.2) were compared with those of the shake flask culture (Fig. 10.3) at the same incubation temperature of 30oC from the experiment reported in Chapter 9. The bioreactor culture resulted in more biomass formation (ca. 3.5 g L‐1 in shake flasks; ca. 5.5 g L‐1 in the bioreactor) and over a longer growth period. Glucose was completely exhausted in both cultures except that consumption was slower in bioreactor culture. Culture pH in bioreactor culture appeared to be marginally lower than in shake flask culture. The delay between change from high growth to stationary phase and when POPI production is high is greater in bioreactor culture than in shake flask culture. POPI titre took longer to peak in bioreactor culture. Shake flask culture gave a higher POPI titre (ca. 1.5 times) than bioreactor culture.

105

2400 14 9 2200 7

8 2000 12 6 1800 7 10 1600 5

6 ) ) -1 1400 -1

DW) 8

-1 5 4 1200 pH 4 1000 6 3

800 L (PIU titre POPI Biomass (g L 3 4

Residual glucoseL (g 2 600 2 400 2 1 1 200

0 0 0 0 01234567 Days

Fig. 10.2 10 L bioreactor scale production of POPI by Fusarium sp. IMI397470. Conditions: Parker Broth (+ 10 g L‐1 of glucose); inoculum size = 10% (v/v); agitation = 700 rpm; incubation temperature = 30oC; air flow = 1.0 vol. vol.‐1 min‐1 at 101 kPa; pH uncontrolled

106

2400 14 9 2200 7

8 2000 12 6

7 1800 10 1600

) 5

6 -1 )

1400 -1

DW) 8

-1 5 4 1200 pH 4 1000 6 3

800 L POPI titre (PIU Biomass (g L 3 4 Residualglucose (g L 2 600 2 400 2 1 1 200

0 0 0 0 01234567 Days

Fig. 10.3 Shake flask production of POPI by Fusarium sp. IMI397470. Conditions: Parker Broth (+ 10 g L‐1 of glucose); inoculum size = 10%; shaking speed = 200 rpm; incubation temperature = 30oC; pH uncontrolled. This fermentation was the same 30oC run reported in Chapter 9

107

10.4 Discussion

POPI production on a much larger scale and in a different culture mode was confirmed and the associated fermentation profiles were time‐course delayed versions of those in shake flask culture. Shake flask culture gave a higher POPI titre than culture via bioreactor. This reinforces the accepted view in fermentation that scale‐up is a challenge. Hsu and Wu (2002) observed that many large scale fermentation processes give a lower yield that would be expected from laboratory results and gave the studies of George (1998) and Bylund et al. (2000) as examples. Biomass accumulated more slowly and went to a greater quantity in bioreactor culture whereas in shake flask culture, entry into the stationary phase was obvious after 2 days. The shake flask culture probably reached the oxygen transfer limit of the system after 2 days which caused the biomass to peak and stay stationary. In the bioreactor, biomass concentration could go higher than that reached in shake flasks probably because the aeration system could supply a higher rate of oxygen transfer and/or higher dissolved oxygen value. What appeared to be marginally more acidic culture pH through the course of bioreactor culture tends to support the idea that oxygenation was not as limiting as in the shake flask culture. However, better cell growth is not what is required in POPI production. Shake flask culture produced less biomass but more POPI. Having inferred in Chapter 7 that POPI production is growth‐dissociated and therefore better produced when  tends to zero, it may be possible to explain the higher POPI yield in shake flask culture by reason that cells in shake flask culture were in growth phase for a shorter time than those in the bioreactor culture. Of course it may be possible that oxygenation per se influences the production of POPI but there is insufficient data from this experiment to infer anything meaningful.

108

10.5 References

Bylund, F.; Castan, A.; Mikkola, R.; Veide, A. and Larsson, G. (2000) Influence of scale‐ up on the quality of recombinant human growth hormone. Biotechnology and Bioengineering 69: 119 ‐ 128.

George, S.; Larsson, G.; Olsson, K. and Enfors, S.‐O. (1998) Comparison of the Baker’s yeast process performance in laboratory and production scale. Bioprocess Engineering 18: 135 ‐ 142.

Hsu, Y.L. and Wu, W.T. (2002) A novel approach for scaling‐up a fermentation system. Biochemical Engineering Journal 11: 123 ‐ 130.

109

CHAPTER 11

Initial observations on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in shake flask culture

11.1 Introduction

Kuek (1986) proposed that the production of growth‐dissociated metabolites may be advantaged by separating the growth and metabolite synthesis phases thus allowing the respective events to occur optimally. The logical extension of this idea is that in the metabolite synthesis phase, pre‐grown cells are retained and used repeatedly, thus obviating the need to grow biomass for each fermentation as is the practice in conventional batch fermentation. Retention and re‐use of living cells is enabled by the technique of immobilization of which entrapment in hydrogel is common practice (see Section 2.4, Chapter 2). The sol sodium alginate and its gel calcium alginate selected for this study have been successfully used for the immobilization of filamentous microorganisms (Kuek, 1991; Yang and Yueh, 2001; Srinivasulu et al., 2003). Evidence that the production of POPI by Fusarium sp. IMI397470 is growth‐ dissociated pattern is presented in Chapter 7. Given this evidence, it was hypothesized that it should be possible to use immobilized living mycelia of Fusarium sp. IMI397470 to produce POPI in repeated batch cultures. To date, there have been no reports of POPI production via immobilized cells of any kind. The aim of this study was to produce immobilized Fusarium sp. IMI397470 and have it produce POPI by re‐using the pre‐grown mycelia in subsequent repeated batch cultures.

110

11.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 min. The procedure for the production of immobilized mycelia and fermentations with them are summarized in Fig. 11.1. Inoculum preparation. This is described in Section 4.2 in Chapter 4. Production of mycelia immobilized in hydrogel beads. Triplicate batches were prepared to allow for 3 replicates at each type of medium investigated. Two flasks of inoculum culture were combined and homogenized in a sterile Waring Semi‐Micro blender vessel blender for 5 seconds low speed and another 5 seconds high speed. After homogenization, 5 mL aliquots of the blend were inoculated into triplicate lots of 45 mL 2% sodium alginate (Manugel GMB, Kelco AIL Sydney, Australia) solution into which a sterile magnetic stirrer bar had been added. The alginate solution was then placed on magnetic stirrer plate and kept under stirring. The alginate solution was pumped through sterile silicon rubber tubing via a peristaltic pump (ATTO Perista AC‐ 2110) and a sterile 21 gauge hypodermic needle to form beads of alginate with entrapped inoculum which were allowed to drop into sterile 0.1M of CalCl2∙2H2O. After beading, the beads remained another 10 minutes in the calcium chloride solution before they were washed three times with sterile 0.01M CaCl2∙2H2O. After the last wash, the solution was drained and the beads were transferred to the sterile flasks containing 50 mL of double‐strength Parker Broth (+ 20 g L‐1 glucose)(see “Note on glucose concentration” below) supplemented with CaCl2∙2H2O to 0.01M in the medium (4.32 g L‐1). The flasks were incubated in a gyratory incubator (New Brunswick Scientific Innova 44R; 2 inch orbit) at 25oC and 200 rpm for 4 days. After incubation, the beads were aseptically washed with 3 aliquots of sterile Wash Medium and then drained clear. The beads were then ready for use in repeated use in production of POPI. Fermentation for POPI. To the beads prepared as described above (50 mL) was added 50 mL of double‐strength Parker Broth (+ 20, 40, 80 or 120 g L‐1 glucose) supplemented ‐1 o with CaCl2∙2H2O to 0.01M in the medium (4.32 g L ). The flasks were incubated at 25 C and 200 rpm in a gyratory incubator (New Brunswick Scientific Innova 44R; 2 inch orbit). At the end of each batch run, spent medium was drained and the beads were

111

Fig. 11.1 A summary of the procedure for preparation of immobilized mycelia of Fusarium sp. IMI397470 and their use in subsequent repeated batches for the production of POPI

112 washed with 3 lots of 50 mL Wash Medium. Fresh medium was then added to the beads and the flask returned to incubation to start another batch run. A note on glucose concentration and double‐strength Parker Broth: In previous experiments, Fusarium sp. IMI397470 was cultured in Parker Broth containing 10 g L‐1 and in Chapter 8, this was shown to be best for POPI production. The use of 20 g L‐1 in the current experiment as the base concentration for both growth and for POPI production results from the need to allow for the fact that the hydrogel beads are around 95% water (after allowing for the alginate and biomass components), and that they comprised 50% of the total flask contents. Therefore, the double strength of glucose used in this experiment would be reduced to single strength after the medium was added to the beads. This was also the reason why double‐strength Parker Broth was used. Sampling. Before samples were removed from the culture flasks, the weight of the flasks was adjusted back to a previously recorded weight by the aseptic addition of sterile deionised water. This was to replace water lost during shaking incubation. Culture liquor (1.5 mL) was aseptically withdrawn from each of 3 replicate culture flasks each day for analysis. The standard errors associated with analyses from the three replicates were determined and displayed with each datum point in the figures. Analyses. Culture pH was determined using a pH meter with a combination electrode (Mettler Toledo, S20). Residual glucose was assayed enzymatically using the glucose oxidase and peroxidase method (Bergmeyer and Bernt, 1974). Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

11.3 Results

Immobilized living mycelia of Fusarium sp. IMI397470 were successfully produced (Fig.11.2 and 11.3). The interior of the alginate beads appeared to be fully colonized after 4 days of incubation (Fig. 11.4) as the beads changed in appearance

113

Fig. 11.2 A flask of Fusarium sp. IMI397470 immobilized in calcium alginate beads 4 days after shaken incubation in Parker Broth (+ 20 g L‐1 glucose) at 25oC and 200 rpm

Fig. 11.3 Beads of immobilized Fusarium sp. IMI397470 at 20X magnification under a dissecting microscope

114

Fig. 11.4 A thin slice of a bead of immobilized Fusarium sp. IMI397470 (40X magnification) showing mycelium entrapped within the calcium alginate matrix from being translucent, then to a cream color and finally to a beige/pink color similar to that seen with mature colonies on agar plate. Mycelial growth was mainly confined within the beads (Fig. 11.3). When Fusarium sp. IMI397470 was grown on Parker Broth (+ 20 g L‐1 glucose) to produce mycelia within hydrogel beads, the fermentation profiles (Figs. 11.5 to 11.8) were similar to those obtained with free mycelium (Fig. 7.1, Chapter 7). POPI production was evident during the growth batch. When the immobilized mycelia were washed and then re‐used in the first and second production batches, POPI production by pre‐grown mycelia was confirmed. POPI titre appeared to be lower in the second production batch when glucose was supplied at 40 g L‐1 or less. In the production phase, when the amount of glucose supplied in the medium was raised, POPI titres were influenced higher (Figs. 11.5 to 11.8) although this is mainly evident in the second batch (Fig. 11.9). The efficiency with which the supplied glucose was translated into

POPI titre (Yp/s) was highest at the lowest glucose input but slowly rose again after the

115

Growth Production Production Batch Batch 1 Batch 2 8 800 14

12 6

600 ) -1 )

-1 10

8 4 400

6 pH

200 4 2 POPI titre (PIU L (PIU POPI titre

2 Residual glucose (g L

0 0 0 012345678910111213 Days

Fig. 11.5 POPI production by immobilized Fusarium sp. IMI397470 Media: Growth batch = Parker Broth (+ 20 g L‐1 glucose); Production batches = Parker Broth (+ 20 g L‐1 glucose)

116

Growth Production Production Batch Batch 1 Batch 2 8 800 30

600 ) -1 ) -1 20 4 400 pH

10 200 2 POPI titre (PIU L (PIU titre POPI Residual glucose (g L

0 0 0 012345678910111213 Days

Fig. 11.6 POPI production by immobilized Fusarium sp. IMI397470 Media: Growth batch = Parker Broth (+ 20 g L‐1 glucose); Production batches = Parker Broth (+ 40 g L‐1 glucose)

117

Growth Production Production Batch Batch 1 Batch 2 8 800 80

600 ) 60 -1 ) -1

4 400 40 pH

2 200 20 POPI titre (PIU L (PIU POPI titre Residual glucose (g L

0 0 0 012345678910111213141516 Days

Fig. 11.7 POPI production by immobilized Fusarium sp. IMI397470 Media: Growth batch = Parker Broth (+ 20 g L‐1 glucose); Production batches = Parker Broth (+ 80 g L‐1 glucose)

118

Growth Production Production Batch Batch 1 Batch 2 8 800

80 6

600 ) -1 )

-1 60

4 400

40 pH

200 2

POPI titre (PIU L (PIU titre POPI 20 Residual glucose (g L

0 0 0 012345678910111213141516 Days

Fig. 11.8 POPI production by immobilized Fusarium sp. IMI397470 Media: Growth batch = Parker Broth (+ 20 g L‐1 glucose); Production batches = Parker Broth (+ 120 g L‐1 glucose)

119

800 a 800 b ) ) -1 -1 600 600

400 400

200 200 Maximum POPI titre (PIU L titre (PIU POPI Maximum Maximum POPI titre (PIU L POPI titre (PIU Maximum 0 0 0 20406080100120 0 20 40 60 80 100 120 -1 Glucose (g L-1) Glucose (g L ) a b Fig. 11.9 The maximum POPI titres obtained in production batches where various amounts of glucose were supplied in Parker Broth (a) Production batch 1; (b) Production Batch 2

800 )

20 -1 600

400 10 glucose consumed) glucose

200 (PIU / (PIU

p/s 0 0 0 20 40 60 80 100 120 L (PIU titre POPI Maximum -1 POPI Y Glucose ( g L )

Fig 11.10 The efficiencies of the conversion of carbon substrate to POPI by Fusarium sp. at various glucose concentrations in Parker Broth when added to beads. Calculated at the point of maximum POPI titre in Production Batch 2

120 minimum at 40 g L‐1 although never matching the efficiency at low glucose input (Fig. 11.10). The residual glucose concentrations found at the end of fermentations with the three highest glucose inputs were 0, 19 and 43 g L‐1.

11.4 Discussion

This study showed that Fusarium sp. IMI397470 can be cultured within beads of calcium alginate. Macroscopically, the bead surfaces were clear of mycelial growth similar to that achieved with Aspergillius phoenicus (Kuek and Armitage, 1985; Kuek, 1991) and unlike surface mycelial growth on beads reported by Federici et al. (1991) with Aureobasidium pullulans, Sammiah and Chambers (1997) with Trametes versicolor, and Yalcinkaya et al. (2002) with Pleurotus sapidus. Having “clean” bead surfaces means that apparent broth viscosity will be lower (leading to better mass and gas transfer) and less possibility of the breaking of mycelia from bead surfaces to form free mycelial growth. It was demonstrated that the pre‐grown immobilized mycelia can be re‐used to produce POPI. Reports of hydrogel immobilization of Fusarium spp. are few but at least those by Kumar et al. (2011) with Fusarium solani for the production of alpha‐amylase, by Saucedo et al. (1989) with Gibberella fujikuroi (Fusarium moniliforme) for the production of gibberellic acid, and by Wang et al. (2008) with Fusarium sp. for the degradation of pyrene and benzo(a)‐pyrene can be found. Immobilized mycelia of Fusarium sp. IMI397470 appear to require some acclimation before POPI production is maximal. This may be a situation where cellular processes need to switch over from those most required for growth to those which support the production of POPI. Apart from acclimation, a certain amount of glucose is required to be supplied in the medium for high POPI production. The second production batches gave higher POPI titres especially when the amount of glucose provided was higher. Below 40 g L‐1 of glucose in the medium POPI yields were not only lower but production appeared not to be sustained at the previous level into the second batch. This suggests that a

121 certain amount of glucose is required just to maintain the physiology of the living mycelia. The indicated threshold amount for high production of POPI was 80 g L‐1 in the range tested. Going to 120 g L‐1 did not significantly improve POPI titre. Bearing in mind that the glucose concentrations reported here are about double what the final concentrations were after addition to the beads, the amounts of glucose associated with good production of POPI are similar to the findings of glucose optimality at 30 g L‐1 (Kimura et al. 1981, and Ismaiel et al., 2010) and 50 g L‐1 (Kimura et al., 1988) for the production of various metabolites. It may have been possible that the amount of mycelia in the beads increased in response to increasing the glucose supply from 40 to 80 g L‐1 and this then resulted in a higher POPI titre. However, this is likely to be a minor contribution if any because as was later determined, the increase in glucose from 20 to 80 g L‐1 resulted in a 20% increase in biomass (Chapter 13; Figs. 13.1 and 13.2 ) whereas the increase in POPI titre effected was 147% (Fig. 11.10) The best glucose concentration to use in Parker Broth for POPI production was identified as 80 g L‐1 when added to washed beads. This was because there was only a marginal increase in POPI titre when the glucose concentration was increased to 120 g L‐1. This is despite the apparent better yield coefficient for the translation of glucose into POPI because the amount of residual glucose at the end of fermentation with 120 g L‐1 was more than double that with 80 g L‐1. The relatively large amounts of residual (unused) glucose associated with the higher levels of glucose studied is likely to have input cost and waste treatment implications for a future production process. A question which arose as a result of this study was what the POPI production would be if the glucose supply were kept the same in both growth and production phases. One possible advantage may be that the living mycelia would not be subject to a large change in glucose environment when they were switched between the growth and production phases. POPI production may be better when there is no need for acclimation to glucose concentration between the two phases. This question was studied as reported in Chapter 12.

122

11.5 References

Bergmeyer, H.U. and Bernt, E. (1974) Determination with glucose oxidase and peroxidase. In: “Methods of Enzymatic Analysis”, Volume 3 (2nd edition); H.U. Bergmeyer (ed); Academic Press Inc.; New York; pp. 1205 ‐ 1212.

Federici, F.; Petruccioli, M.; Federici, R.G. and Miller, M.W. (1991) Scanning electron‐ microscopy of Ca‐alginate immobilized Aureobasidium pullulans grown under various culture conditions. Mycologia 83: 595 ‐ 600.

Ismaiel, A.A.; El‐Sayed, E.A. and Mahmoud, A.A. (2010) Some optimal culture conditions for production of Cycosporin A by Fusarium roseum. Brazilian Journal of Microbiology 41: 1112 ‐ 1123.

Kimura, Y.; Shimada, A.; Nakajima, H. and Hamasaki, T. (1988) Structure of naphthoquinones produced by the fungus, Fusarium sp., and their biological activity toward pollen germination. Agricultural and Biological Chemistry 52: 1253 ‐ 1259.

Kuek, C. (1986) Immobilized living fungal mycelia for the growth‐dissociated synthesis of chemicals. International Industrial Biotechnology 56: 123 ‐ 125.

Kuek, C. and Armitage, T.A. (1985) Scanning electron microscopic examination of calcium alginate beads immobilizing growing mycelia of Aspergillus phoenicus. Enzyme and Microbial Technology 7: 121 ‐ 125.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

123

Kumar, D.; Muthukumar, M. and Garg, N. (2011) Kinetics of fungal extracellular alpha‐ amylase from Fusarium solani immobilized in calcium alginate beads. Journal of Environmental Biology 33: 1021 ‐ 1025.

Sammiah, P. and Chambers, R.P. (1997) Characterization of a Ca‐alginate‐immobilized Trametes versicolor bioreactor for decolorization and AOX reduction of paper mill effluents. Bioresource Technology 60: 1 ‐ 8.

Saucedo, J.E.N.; Barbotin, J.N. and Thomas, D. (1989) Physiological and morphological modifications in immobilized Gibberella fujikuroi mycelia. Applied and Environmental Microbiology 55: 2377 ‐ 2384.

Srinivasulu, B.; Adinarayana, K. and Ellaih, P. (2003) Investigations on neomycin production with immobilized cells of Strepmyces marinensis NUV‐5 in calcium alginate matrix. AAPS PharmaSciTech 4: 1 ‐ 6.

Wang, X.; Gong, Z.; Li, P.; Zhang, L. and Hu, X. (2008) Degradation of pyrene and benzo(a)pyrene in contaminated soil by immobilized fungi. Environmental Engineering Science 25: 677 ‐ 684.

Yalcinkaya, Y.; Arica, M.Y.; Soysal, L.; Denizli, A.; Genc, O. and Bektas, S. (2002) Cadmium and mercury uptake by immobilized Pleurotus sapidus. Turkish Journal of Chemistry 26: 441 ‐ 452.

Yang, S.S. and Yueh, C.Y. (2001) Oxytetracycline production by immobilized Streptomyces rimosus. Journal of Microbiology, Immunology and Infection 34: 235 ‐ 242.

124

CHAPTER 12

Effect of supplying the same amount of glucose in the growth and production phases in the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in shake flask culture

12.1 Introduction

It was previously demonstrated that Fusarium sp. IMI397470 can be grown immobilized within hydrogel beads and then the immobilized mycelia be used in following separate batches for the production of POPI (Chapter 11). In the previous study, immobilized mycelia were produced using Parker Broth (+ 20 g L‐1 glucose). The immobilized mycelia were used with a range of glucose supply in subsequent batches to produce POPI. It was found that 80 g L‐1 of glucose in Parker Broth yielded high titres. The aim of this study was to determine if POPI yield would be improved if both the growth (of the fungus) and production (of POPI) phases were supplied the same amount of glucose.

12.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 minutes. Inoculum preparation. This is described in Section 4.2 in Chapter 4. Production of mycelia immobilized in hydrogel beads. This was as described in Section 11.2, Chapter 11 except note below, the glucose variations of Parker Broth used. Fermentation for POPI. This was as described in Section 11.2, Chapter 11 except note below, the glucose variations of Parker Broth used.

125

Glucose regimes in the growth and production batches. The amount of glucose supplied was kept the same in both the growth and production phases (Table 12.1).

Table 12.1 Glucose regimes for the preparation of immobilized mycelia and their subsequent use in POPI production

Growth Batch Production Batch (Double‐strength Parker Broth (Double‐strength Parker Broth Treatment with CaCl2.H2O at 0.01 M) with CaCl2.2H2O at 0.01 M) + Glucose (g L‐1) + Glucose (g L‐1) 1 40 40 2 80 80 3 120 120

A note on glucose concentration and double‐strength Parker Broth: The glucose concentrations mentioned are the strengths of media at the point of addition to beads. These concentrations are double‐strength to allow for the fact that the hydrogel beads are around 95% water (after allowing for the alginate and biomass components), and that they comprised 50% of the total flask contents. Therefore, the double strength of glucose used in this experiment would be reduced to single strength after the medium was added to the beads. This was also the reason why double‐strength Parker Broth was used. Sampling. This was as described in Section 11.2, Chapter 11. Analysis. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11. Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

12.3 Results

Despite glucose being supplied at the same level in the growth and production phases, as has been previously observed (Figs. 11.7 and 11.8, Chapter 11) there appeared to be acclimation required for the immobilized mycelia to transit from the 126 growth culture to being POPI productive because Production Batch 1 was lower in POPI titre than the subsequent batches (Figs. 12.1 and 12.2). Where the glucose supply was the same between the growth and production phases, higher glucose supply at 80 and 120 g L‐1 gave better POPI titres although the standard error associated with the POPI values indicated overall that there was probably no differences between the two treatments. In comparison with the process where glucose supply was different between the growth and production phases (Chapter 11), supplying the same amount of glucose in the growth and production phases gave about 1.5 times better POPI yield at the 2 higher glucose levels (Fig. 12.2). As was found previously (Chapter 11), at a glucose feed of 120 g L‐1, this carbon substrate was not completely exhausted at the end of fermentation (Fig. 12.3). At both 80 and 120 g L‐1 of glucose more of the substrate was used by the immobilized mycelia with each successive production batch.

1250 1250 1250 ) ) ) -1 -1 -1 1000 1000 1000

750 750 750

500 500 500 POPI titre (PIU L (PIU titre POPI POPI titre (PIU L (PIU titre POPI POPI titre (PIU L (PIU titre POPI 250 250 250

0 0 0 40 80 120 40 80 120 40 80 120 -1 Glucose (g L-1) Glucose (g L-1) Glucose (g L )

Production Batch 1 Production Batch 2 Production Batch 3

Fig 12.1 The maximum POPI titres obtained in immobilized shake flask culture of Fusarium sp. IMI397470 across three sequential production batches. At each level of glucose treatment, both the growth and production batches were provided the same glucose quantity

127

1400 1400

1200 1200 ) ) -1 -1 1000 1000

800 800

600 600

400 400 L (PIU titre POPI POPI titre (PIU L (PIU titre POPI

200 200

0 0 0 20406080100120 0 20406080100120 -1 Glucose (g L-1) Glucose (g L )

Production Batch 1 Production Batch 2

Fig. 12.2 A comparison of the maximum POPI titres obtained in immobilized shake flask culture of Fusarium sp. IMI397470 as influenced by the amount of glucose provided in the growth and production batches. In one treatment ( — ), the glucose provided in the growth batch was 20 g L‐1 followed by 40, 80 or 120 g L‐1 in the production batch (data from Chapter 11). In the

other treatment (—), 40, 80 or 120 g L‐1 was used in both the growth and production batches

Growth Production Production Production Batch Batch Batch Batch 110

100

90 ) -1 80

30 Residual glucoseL (g 20

0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Days

Fig 12.3 Residual glucose in production batches when supplied with 80 g L‐1 (—) or 120 g L‐1 (—) glucose in Parker Broth in both the growth and production phases

128

12.4 Discussion

This study has shown that at all the three glucose levels tested, POPI yield is better when the same amount of glucose is supplied in the growth phase. The improvement was in the order of 1.5 times. While POPI yield was improved by altering the glucose strategy, the gain did not appear to be the result of obviating the need for glucose acclimation where the amount supplied was different between the growth and production phases. This is because there was clearly still acclimation occurring between the growth and production batches and also between production batches. As has already been observed previously (Chapter 11), POPI production was relatively lower in the first production batch after the growth phase. Acclimation is supported by the residual glucose profiles. Glucose was more completely consumed as the production batches progressed. This could simply be related to the amount of POPI produced i.e. the higher consumption of glucose through successive production batches was related to more POPI being produced as the batches progressed. The maximum POPI titres associated with glucose supply at 80 and 120 g L‐1 were not significantly different. It is argued that 80 g L‐1 represents the optimal in the range tested not only on the latter basis but also because there is more efficient use of glucose at (lower residual value at the end of fermentation) when supplied at the lower of the two levels. This study has provided data to cause alteration of the production protocol for POPI to one where the glucose supply was provided at 80 g L‐1 in both the growth and production batches. This glucose supply regime was followed through the rest of the work to be reported. Since acclimation appears to be a requirement when switching from growth to production batches, for reporting purposes after this chapter, the first production batch after the growth batch will not be described. Only data from the second production batch onwards will be reported and the batch will be numbered as 1.

129

CHAPTER 13

The growth of immobilized mycelia of Fusarium sp. IMI397470 in shake flask culture

13.1 Introduction

POPI is produced by Fusarium sp. IMI397470 in both free mycelial (Chapter 4 and 7 – 10) and immobilized mycelial culture (Chapters 11 and 12). In free mycelial culture it is relatively easy to characterize POPI production in terms of both biomass and POPI titre profiles. However, in immobilized cell cultures it is not possible to directly estimate the quantity of biomass as it is confined within the immobilizing matrix. Visual observations during the cultures of hydrogel beads inoculated with Fusarium sp. IMI397470 showed colonization of the bead matrix through 4 to 5 days. The aim of this experiment is to quantify the biomass in the hydrogel beads used for the production of POPI using immobilized mycelia. With free mycelial cultures, biomass can be directly estimated by determining the dry weight of washed samples (see Section 4.2, Chapter 4). With immobilized mycelia, an indirect method is required. Kuek (1991) estimated the quantity of immobilized mycelia of Aspergillus phoenicus by determining the nitrogen content of hydrogel beads through a period of incubation. The rationale used is that nitrogen content is a proximate estimate of protein content which in turn was an indicator of the amount of fungal biomass present. This approach was used in this study.

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13.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 minutes. Inoculum preparation. The method used was previously described in Section 4.2 in Chapter 4. Preparation of mycelia immobilized in hydrogel beads. The method used was previously described in Section 11.2, Chapter 11 except that the media used were Parker Broth (+ ‐1 ‐1 either 20 or 80 g L glucose; and 4.32 g L CaCl2∙2H2O) and incubation was for 6 days. The 20 g L‐1 broth was to allow comparison with free mycelial culture at that concentration (Chapter 9) while the 80 g L‐1 broth was to find biomass information to accompany the POPI production profile found optimal at that concentration (Chapter 11). Estimation of fungal biomass in hydrogel beads. At daily intervals, three flasks were removed and both the bead contents and culture liquors were collected. The standard errors associated with the following estimation and analyses from each set of three replicates were determined and displayed with each datum point in the figures. The culture liquor was reserved for analysis (see below) and the beads were estimated for biomass content via nitrogen analysis according to the protocol of Kuek (1991) except that the combustion method rather than the Kjeldahl method was used. The beads were rinsed clean of culture liquor with Parker Broth deionized water equivalent to twice the volume of the culture liquor (100 mL). The beads were then dehydrated in the three changes of absolute methanol (200 mL each change) over 6 hours. Subsequently, the beads were allowed to dry in a fumehood before being transferred to 60oC oven to dry. The dried beads were assayed for nitrogen content via the combustion method (AOAC Official Method 990.03). Analyses. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11. Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

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13.3 Results

Biomass of Fusarium sp. IMI397470 as indicated by nitrogen contents appeared to accumulate faster at the same incubation temperature with the same medium when the fungus is immobilized (Figs. 13.1 and 13.2 cf. Fig. 9.1). Nitrogen content in immobilized cultures peaked by Day 1 with either 20 or 80 g L‐1 glucose supplied in Parker Broth whereas with culture of free mycelia it peaked a day later. Biomass quantity at stationary phase was about 13% more (based on the average value at stationary phase) when the higher glucose quantity was supplied but this amount of glucose was not completely exhausted unlike when the lesser amount was provided.

0.8 7 10 120

100 8 0.6

) 5 -1

80 ) -1 6 4 0.4 60

3 pH 4 40 2

0.2 POPI titre (PIU L Residual glucose (g L 2 20 1 Biomass as indicated by % Nitrogen Biomass as indicated by %

0.0 0 0 0 0123456 Days Fig 13.1 Growth of immobilized Fusarium sp. IMI397470 on Parker Broth (+ 20 g L‐1 ‐1 glucose, and 4.32 g L CaCl2∙2H2O) as estimated by nitrogen content 132

0.8 7 120 50 6 100 0.6 40

) 5 -1 )

80 -1 4 30 0.4 60 3 pH 20 40

POPI titre (PIU L 2 0.2 Residual glucose (g L 10 20 1 Biomass as indicated by % Nitrogen

0.0 0 0 0 0123456 Days Fig 13.2 Growth of immobilized Fusarium sp. IMI397470 on Parker Broth (+ 80 g L‐1 ‐1 glucose, and 4.32 g L CaCl2∙2H2O) as estimated by nitrogen content

133

13.4 Discussion

This study confirmed quantitatively that Fusarium sp. IMI397470 is able to colonize hydrogel beads formed from sol mixed with propagules. Growth within beads peaked faster than in free mycelia culture. The standard incubation period of 4 days for the preparation of immobilized mycelia is appropriate as the hydrogel beads would be well colonized within that time. The POPI profile associated with the biomass curve confirms that it was POPI‐ producing biomass which was estimated. The POPI production curve showed the same asynchrony between the biomass and POPI peaks: POPI production peaked after growth had entered the stationary phase. The growth‐dissociated nature of POPI production is thus also confirmed for immobilized mycelia. This study also showed that while the supply of 80 g L‐1 of glucose in production batches to immobilized mycelia results in good POPI titre (Chapter 12), the same amount of glucose supplied to mycelia growing within hydrogel beads in growth batches produces comparatively little POPI. Thus, it can be concluded that the positive effect of high glucose supply on POPI production only applies to pre‐grown mycelia i.e. mycelia grown for re‐ use. This is another confirmation of the value of a separated batch process for POPI production.

13.5 References

Association of Official Analytical Chemists (2005) Protein (Crude) in Animal Feed: Combustion Method (990.03). In: “Official Methods of Analysis of AOAC” (18th ed.); Arlington, VA.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

134

CHAPTER 14

Effect of incubation temperature on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470

14.1 Introduction

The study on the effect of temperature on the POPI production by Fusarium sp. IMI397470 is reported in Chapter 9. However, that study was based on free mycelia and now that an immobilized mycelia process is being examined, it is necessary to check the previous results. Higher metabolic activity has been claimed for immobilized cells (in reviews such as those by Cassidy et al., 1996 and Couto, 2009) over free cells. Gupta et al. (2010) working with Aspergillus niger found that the production of ‐amylase was optimal at 30oC with free cells but 35oC with immobilized cells. A re‐examination of the temperature effect on POPI production this time by immobilized mycelia was therefore warranted. The aim of this study was to determine the optimum incubation temperature for POPI production by immobilized Fusarium sp. IMI397470 and how this compares with that previously identified for free mycelia of the fungus. Fusarium sp. IMI397470 grows equally well at either 25o or 30oC in free cell culture (Chapter 9). However, POPI production was found to be better at 30oC than at 25oC. In this study, the fungus will be cultured within hydrogel beads at both those temperatures after which they will be used in production batches at 25o, 30o and 35oC. This should reveal whether the temperature used for growth has consequence for in the production phase.

135

14.2 Materials and methods

Table 14.1 The combinations of incubation temperatures for examining the production of POPI by Fusarium sp. IMI397470 Set Treatment Growth phase (oC) Production phase (oC)

A Control 25 25 1 25 30 2 25 35 B Control 25 25 1 30 30 2 30 35

Sampling. This was as described in Section 11.2, Chapter 11. Analysis. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11. Culture liquor was assayed directly for POPI using the protocol

136

described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

14.3 Results

For immobilized mycelia grown at 25oC, subjecting them to 30o and 35oC in the production phase did not improve POPI titres over the practice of continuing the 25oC incubation temperature of the growth phase into the production phase (Fig. 14.1). A production batch temperature of 35oC for mycelia grown at 25oC depressed POPI yield significantly. The response of immobilized mycelia cultured at 30oC and then incubated at either 30o or 35oC in POPI production was similar to that of mycelia grown at 25oC (Fig. 14.2). For mycelia cultured at 25oC, the standard errors associated with the POPI titres suggest that there was no difference between subsequent production held at either 25o or 30oC (Figs. 14.1). Similarly, no difference was found between those two production temperatures for mycelia grown at 30oC (Fig. 14.2). The relative effectiveness of each temperature treatment confirm the observations on POPI titres yielded (Table 14.2).

137

500

400 ) Fig. 14.1 The maximum POPI titres -1 produced by immobilized 300 Fusarium sp. IMI397470 grown 200 at 25oC and then at the POPI titre (PIU L indicated temperatures during 100 the production batches

0 25 30 35 Incubation temperature (oC)

Fig. 14.2 The maximum POPI titres produced by immobilized Fusarium sp. IMI397470 grown 200

o ) at 30 C and then at the -1 indicated temperatures during the production batches 100 (—). The maximum POPI titre of the control (immobilized POPI titre (PIU L mycelia incubated at 25oC for both the growth and production 0 25 30 35 batches) is shown () but not o Incubation temperature ( C) included in the response curve

138

Table 14.2 The relative effectiveness of various temperature regimes in the growth and production phases of immobilized Fusarium sp. IMI397470 on the maximum POPI titre obtained

Growth phase Production phase Treatment Relative Effectiveness (oC) (oC)

Control 25 25 1

1 25 30 0.98

2 25 35 0.49

Control 25 25 1

3 30 30 1.04

4 30 35 0.16

Max. POPI titre of treatment Relative Effectiveness  Max.POPI titreof control

14.4 Discussion

Contrary to the findings in Chapter 9 where 30oC was found to be best for POPI production with free mycelia, when this temperature was applied to pre‐grown immobilized mycelia POPI production was not improved. This situation was true irrespective of whether the immobilized mycelia were first cultured at 25o or 30oC. With immobilized mycelia, POPI production appeared to be optimal at either 25o or 30oC. The results with free mycelia (Chapter 9) indicate that the biochemistry in a culture where the growth and POPI production are not separated requires a higher incubation temperature for the latter to be optimal. Immobilization appears to obviate the need for increasing process temperature for POPI production to 30oC from 25oC. Differences between free mycelial and immobilized cells culture will also no doubt contribute to what has been observed in this study. Broth viscosity is lower with immobilized fungi than when mycelia grow in filamentous form in liquid resulting in better mass and

139

oxygen transfer (Gbewonyo and Wang, 1983; Thongchul and Yang, 2003). Immobilized cells are also claimed to be more stable against environmental shifts such as in pH (Adlercreutz et al., 1985; Buzas et al., 1989; Shin et al., 2002). The higher cell densities achievable (per unit volume of bioreaction) by immobilization is a reason given for superior performance in metabolite production compared to that via free cells (Kim et al., 2006). Lastly, it would be expected that within the matrix of a hydrogel which immobilizes a microorganism, mass and oxygen transfer rate would be different to that available to free cells in liquid. Since no difference was found between the temperature strategies of culturing at 25o or 30oC and then production at one of those two temperatures, the lower temperature was chosen as the operating temperature for later studies in this work.

14.5 References

Adlercreutz, P.; Holst, O. and Mattiasson, B. (1985) Characterisation of Gluconobacter oxydans immobilized in calcium alginate. Applied Microbiology and Biotechnology 22: 1 ‐ 7.

Buzas, Z.; Dallman, K. and Szafani, B. (1989) Influence of pH on the growth and ethanol production of free and immobilized Saccharomyces cerevisiae cells. Biotechnology and Bioengineering 34: 882 ‐ 884.

Cassidy, M.B.; Lee, H. and Trevors, J.T. (1996) Environmental applications of immobilized cells: a review. Journal of Industrial Microbiology 16: 79 ‐ 101.

Couto, S.R. (2009) Dye removal by immobilized fungi. Biotechnology Advances 27: 227 ‐ 235.

140

Gbewonyo, K. and Wang, D.I.C. (1983) Enhancing gas‐liquid transfer rates in non‐ newtonian fermentations by confining mycelial growth to microbeads in a bubble column. Biotechnology and Bioengineering 25: 2873 ‐ 2887.

Gupta, A.; Gautam, N. and Modi, D.R. (2010) Optimization of α‐amylase production from free and immobilized cells of Aspergillus niger. E3 Journal of Biotechnology and Pharmaceutical Research 1: 001 ‐ 008.

Kim, C.J.; Lee, S.J.; Chang, Y.K; Chun, G.T.; Jeong, Y.H. and Kim, S.B. (2006) Repeated batch culture of immobilized Gibberella fujikuroi B9 for gibberellic acid production: An optimization study. Biotechnology and Bioprocess Engineering 11: 544 ‐ 549.

Shin, M.; Nguyen, T. and Ramsay, J. (2002) Evaluation of support materials for the surface immobilization and decoloration of amaranth by Trametes versicolor. Applied Microbiology and Biotechnology 60: 218 ‐ 23.

Thongchul, N. and Yang, S.T. (2010) Controlling filamentous fungal morphology by immobilization on a rotating fibrous matrix to enhance oxygen transfer and L(+)‐lactic acid production by Rhizopus oryzae. In: “Fermentation Biotechnology. ACS Symposium Series Volume 862; B.C. Saha (ed.); American Chemical Society; pp: 36 ‐ 51.

141

CHAPTER 15

Effect of shaking speed on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470

15.1 Introduction

Agitation is one of the important factors in submerged aerobic culture. It enables oxygen supply to the cultivated microorganism in order to reach the desired yield in production. In general, agitation in batch fermentation often has a strong impact on the development of fungal morphology (Žnidaršič and Pavko, 2001; Bakri et al., 2011). However, the effect of agitation on fungal morphology is not as significant where the mycelia are confined within immobilization matrices. Since the mycelia is protected within a matrix, it is less affected by shear forces exerted by agitation, does not contribute to broth viscosity, and the fermentation need not be concerned about whether filamentous or pelletized growth forms are preferred. This is particularly true for the immobilization achieved with Fusarium sp. IMI397470 in this work because the mycelium is largely confined within the hydrogel beads and the bead surfaces are visibly clear of mycelial growth (Fig. 11.3, Chapter 11). In this situation, a study on the effect of agitation is more about its influence on mass and liquid transfer through the bulk liquid of the medium and the immobilization matrix. Although static culture is also used for metabolite production by Fusarium spp. (e.g. Fusarielin A studied by Chakravarthi et al., 2008 and paclitaxel by Kobayashi et al., 1995), Ismaiel et al. (2010) and Rifaat et al. (2010) showed that agitated is better than static culture in the production of the metabolites. Studies on metabolite production by Fusarium spp. in free mycelial form have been carried out at shaking speeds ranging from 120 to 220 rpm (Breinholt et al., 1997; Chaiet et al., 1989; Srivastava et al., 2003; and 142

Gupta et al., 2009). The aim of this study was examine the effect of shaking speed on the production of POPI by immobilized Fusarium sp. IMI397470.

15.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 minutes. Inoculum preparation. This was as previously described in Section 4.2 in Chapter 4. Preparation of mycelia immobilized in hydrogel beads. This was as previously described in Section 11.2 Chapter 11, except that the Parker Broth used for both the Growth and ‐1 ‐1 Production Phases was + 80 g L glucose and 4.35 g L CaCl2∙2H2O. After the growth batch, the next immediate batch (in the production phase) was run for acclimation of the immobilized mycelia and this batch was not described in the results. Fermentation for POPI. This was as described in Section 11.2, Chapter 11 except that the Parker Broth used for both the Growth and Production Phases was + 80 g L‐1 glucose and ‐1 4.35 g L CaCl2∙2H2O. The experiment was conducted in two runs each with 200 rpm as the control (standard used in all studies previous to this one) viz. one run was 100 compared with 200 rpm, and the other run was 300 compared with 200 rpm. Sampling. This was as described in Section 11.2, Chapter 11. Analysis. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11. Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

15.3 Results

The POPI production appeared to be influenced by flask shaking speed. Both 100 and 300 rpm resulted in poorer POPI production than that at 200 rpm (Fig. 15.1). When

143 examined as a proportion of the POPI titre obtainable at 200 rpm (the standard used in all fermentations to date), yields with 100 and 300 rpm were lower throughout the fermentation except at the end when POPI values had already peaked in any case (Fig. 15.2). Although production of POPI was slower at 100 rpm, it resulted in a 9% higher peak titre than that at 200 rpm which occurred 3 days earlier.

300 400

300 ) ) -1 -1 200

200 POPI titre (PIU L POPI (PIU titre POPI titre (PIU L (PIU titre POPI 100

100

200 rpm 200 rpm 100 rpm 300 rpm

0 0 0123456789 012345678 Days Days

a b

Fig. 15.1 The effect of flask shaking speed on the production of POPI by immobilized Fusarium sp. IMI397470 in production phase. (a) 100 rpm vs. 200 rpm; (b) 300 rpm vs. 200 rpm

144

100

Relative POPI titre (%) titre POPI Relative 20 100 rpm 300 rpm

0 0123456 Days Fig. 15.2 The POPI titres obtained in production batch at either 100 or 300 rpm relative to those at 200 rpm POPI titre at n rpm at Day Relative POPI titre (%)  t 100 POPI titre at 200 rpm at Dayt

15.4 Discussion

The degree of agitation in shake flasks affects POPI production. This is not surprising because a variety of shaking speeds have been reported for the production of various metabolites by microorganisms. For example Ismaiel et al. (2010) reported that 120 rpm was optimal for the production of Cyclosporin A by Fusarium roseum and noted that Moussaif et al. (2003) concurred while optima were found at 180 rpm (Kobel and Traber, 1982 cited by Ismaiel et al., 2010) and 200 rpm (Evers et al., 1999 cited by Ismaiel et al., 2010 ) by others. However, shaking speed data should be interpreted carefully as they are not directly comparable unless the flask types, flask volume to medium volume, and orbit diameter of the gyratory incubator were equivalent (the last parameter is most often not specified).

145

In the case of POPI production by Fusarium sp. IMI397470, the optimum shaking speed was found to be what had already been used as the standard in the work to this point viz. 200 rpm. Shaking at either a lower of higher speed did not improve productivity. The lower speed of 100 rpm did ultimately give a marginally higher POPI titre but at the expense of fermentation time (the peak was 3 days later). Thus, 200 rpm was chosen as the best shaking speed for the production of POPI under the conditions used and it was applied for the rest of the shake flask studies in this work. In terms of mass and gas transfer, it is possible to perceive that at a shaking speed of 100 rpm, supply of nutrients and output of POPI and other metabolites is lower than what is required and matched at 200 rpm. However, this explanation would imply that such transfer is at excess at 300 rpm such that it affects POPI production negatively. A more plausible explanation may be that at the high shaking speed of 300 rpm bead to bead abrasion disrupts the fungal mycelia in the proximity of bead surfaces (see Fig. 11.4, Chapter 11) and thus their productive capacity. Even though macroscopically the beads appear to be devoid of mycelial growth, fungal filaments can be seen at the interface of the bead surface and medium. Kuek and Armitage (1985) revealed via scanning electron microscopy of hydrogel bead surfaces that culture in shake flasks does result in surface abrasion. The effect of gas transfer/mixing on POPI production will be investigated in another way later in this work when bubble column studies are reported (see Chapter 25).

15.5 References

Bakri, Y.; Mekaeel, A. and Koreih, A. (2011) Influence of agitation speeds and aeration rates on the xylanase activity of Aspergillus niger SS7. Brazilian Archives of Biology and Technology 54: 659 ‐ 664.

146

Breinholt, J.; Ludvigsen, S.; Rassing, B.R. and Rosendahl, C.N. (1997) Oxysporidinone: A aovel, antifungal N‐Methyl‐4‐hydroxy‐2‐pyridone from Fusarium oxysporum. Journal of Natural Products 60: 33 ‐ 35.

Chaiet, L.; Fromtling, R.A.; Garrity, G.M.; Monaghan, R.L.; Hensens, O.D.; Valiant, M.E. and Zink, D.L. (1989) L‐681,572‐A new antifungal agent isolation, characterization, and biological activity. The Journal of Antibiotics 42: 1718 ‐ 1721.

Gupta, V.K.; Gaur, R.; Yadava, S.K. and Darmwal, N.S. (2009) Optimization of xylanase production from free and immobilized cells of Fusarium solani F7. BioResources 4: 932 ‐ 945.

Ismaiel, A.A.; El‐Sayed, E.A. and Mahmoud, A.A. (2010) Some optimal culture conditions For production of Cycosporin A by Fusarium roseum. Brazilian Journal of Microbiology 41: 1112 ‐ 1123.

Kuek, C. and Armitage, T.A. (1985) Scanning electron microscopic examination of calcium alginate beads immobilizing growing mycelia of Aspergillu phoenicus. Enzyme and Microbial Technology 7: 121 ‐ 125.

147

Moussaïf, M.; Jacques, P.; Schaarwächter, P.; Budzikiewicz, H.; Thonart, P. (1997) Cyclosporine C is the main antifungal compound produced by Acremonium luzulae. Applied and Environmental Microbiology 63: 1739 ‐ 1743.

Rifaat, H.M.; El‐Mahalawy, A.; El‐Menofy, H.A. and Donia, S.A. (2010) Production, optimization and partial purification of lipase from Fusarium oxysporium. Journal of Applied Sciences in Environmental Sanitation 5: 39 ‐ 53.

Srivastava, A.C.; Ahamad, S.; Agarwal, D.K. and Sarbhoy, A.K. (2003) Screening of potential gibberellin producing Fusarium strains for the hybrid rice production. Food, Agriculture and Environment 1: 250 ‐ 253.

Žnidaršič, P. and Pavko, A. (2001) The Morphology of filamentous fungi in submerged cultivations as a bioprocess parameter. Food Technology and Biotechnology 39: 237 ‐ 252.

148

CHAPTER 16

Effect of the type of carbon source on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470

16.1 Introduction

Previous chapters in this work report studies which have found that Fusarium sp. IMI397470 produced good POPI yield in Parker Broth with glucose as the carbon source. However, as reviewed by Demain (1986), carbon source regulation of secondary metabolism occurs in fermentations. A readily metabolized carbon source such as glucose appears to be a cause of metabolite repression. Examples given by Demain (1986) include the repression of the production of penicillin, cephalosporin, alkaloids when undepleted glucose remains in production media. When more slowly metabolized carbon substrates such as lactose is used, production of penicillin (Revilla et al., 1984) and of enniatin (Audhya and Russell, 1975) is favoured. One of the advantages of the separated batch culture process used in this work is that the growth and production batches are in different parts of the process and can be optimized differently. A glucose substrate which is highly suitable for growth can be supplied in the growth phase while more slowly metabolized (and therefore less repressive) substrates can be fed in the production phase. The aim of this study was to examine the effect of different carbon substrates on POPI production by pre‐grown immobilized mycelia of Fusarium sp. IMI397470.

149

16.2 Materials and methods

Table 16.1 The sole carbon sources used in Parker Broth for the production batches in POPI production by immobilized Fusarium sp. IMI397470

Polysaccharides Disaccharides Monosacharride Soluble starch Sucrose Glucose (Fisher Scientific) Lactose Maltose All carbon sources were added at a concentration of 80 g L‐1

Sampling. This was as described in Section 11.2, Chapter 11. Analysis. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11, except that residual glucose for media with maltose and lactose were assayed using the dinitrosalicylic acid method (Miller, 1959). Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

150

16.3 Results

All the carbon sources supplied in the production phase to pre‐grown immobilized mycelia resulted in POPI production. Lactose and maltose were equally poor as substrates in delivering POPI yield (Fig. 16.1). Sucrose and glucose gave “middling” maximum POPI titres while soluble starch gave the best and significantly different POPI titre (1.5‐fold better than the titre with glucose). In contrast with the fermentations fed the disaccharides and monosaccharide, residual glucose with soluble starch was never significant (Figs. 16.2 to 16.5).

1400

1200 )

-1 1000

800

600 POPI titre (PIU L (PIU titre POPI 400

200

0 12345 Carbon source

Fig. 16.1 The maximum POPI yield obtained with Parker Broth and different carbon sources in production batch 1= lactose; 2 = maltose; 3 = sucrose; 4 = glucose; 5 = soluble starch

151

1400 8 100

1200

80 6 1000 ) ) -1 -1 800 60 4

600 pH 40

400 POPI titre L (PIU 2 20 200 Residualglucose (g L

0 0 0 01234567891011 Days Fig 16.2 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 lactose)

1400 8 100

1200

80 6 1000 ) ) -1 -1 800 60 4

600 pH 40

400

POPI titre (PIU L (PIU POPI titre 2 20

200 Residual glucose (g L

0 0 0 01234567891011 Days Fig 16.3 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 maltose)

152

1400 8 100

1200

80 6

1000 ) -1 ) -1 800 60 4

600 pH 40

400 POPI titre (PIU L (PIU titre POPI 2

20 Residual glucose (g L 200

0 0 0 01234567891011 Days Fig 16.4 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 glucose)

1400 8 100

1200

80 6

1000 ) -1 ) -1 800 60 4

600 pH 40

400 POPI titre (PIU L 2

20 Residual glucose (g L 200

0 0 0 01234567891011 Days Fig 16.5 Residual glucose in the production batch during POPI fermentation with immobilized Fusarium sp. IMI397470 and Parker Broth (+ 80 g L‐1 soluble starch)

153

16.4 Discussion

The polymeric glucose in the form of soluble starch appears to be best for the production of POPI by pre‐grown immobilized Fusarium sp. IMI397470. Soluble starch was superior to the best of the other carbon sources, glucose by about 1.5 fold. When fed soluble starch, the fungus only ever encountered low levels of glucose which did not exceed 2 g L‐1 unlike when fermentations were fed glucose, maltose and lactose. The implication is that the presence of glucose in a certain quantity was repressive towards POPI production. That a more complex and therefore more slowly metabolized carbon substrate than simple sugars like glucose is better for metabolite production is consistent with many previous studies (e.g. Revilla et al., 1984; and Audhya and Russell; 1975 cited in the introduction) going as far back as Reese et al. (1969). The identification of soluble starch as a more suitable carbon substrate than glucose for the production phase means that a two‐substrate process is now indicated: glucose used in the growth batches followed by soluble starch in the production batches. This process strategy conforms with other examples. In the separated repeated batch production of glucoamylase, Kuek (1991) showed that the use of the carbon substrate soluble starch in the growth phase followed by Dextran T‐10 in the production phase optimizes production of the enzyme by Aspergillus phoenicus. The commercial production process for shikonin from Lithospermum erythrorhizon was made successful in the early 1980s by the use of a two‐stage culture where the first stage was biomass proliferation followed by metabolite production (Fujita, 1988). The two stages had different medium compositions designed to optimize each stage separately. These results raise two questions: (a) the effective quantity of soluble starch to use in the production phase; (b) whether having soluble starch in both the growth and production phases would be advantageous. These questions were examined in the study reported in Chapter 17.

154

16.5 References

Audhya, T.K. and Russell, D.W. (1975) Enniatin production by Fusarium sambucinum: Primary, secondary, and unitary metabolism. Journal of General Microbiology 86: 327 ‐ 331.

Demain, A.L. (1986) Regulation of secondary metabolism in fungi. Pure and Applied Chemistry 58: 219 ‐ 226.

Fujita, Y. (1988) Industrial Production of Shikonin and Berberine. In: “Ciba Foundation Symposium 137 ‐ Applications of Plant Cell and Tissue Culture”; G. Bock and J. Marsh (eds.); John Wiley & Sons, Ltd., Chichester; pp. 228 ‐ 235.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31: 426 ‐ 428.

Reese, E.T.; Lola, J.E.; and Parrish, F.W. (1969) Modified substrates and modified products as inducers of carbohydrases. Journal of Bacteriology 100: 1151 ‐ 1154.

Revilla, G.; Lopez‐Nieto, M.J.; Luengo, J.M. and Martin, J.F. (1984) Carbon catabolite repression of penicillin biosynthesis by Penicillium chrysogenum. Journal of Antibiitocs (Tokyo) 37: 781 ‐ 789.

155

CHAPTER 17

Effect of soluble starch on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470: Quantity and when supplied

17.1 Introduction

It was established in Chapter 16 that substituting soluble starch for glucose in the production phase improves POPI yield by Fusarium sp. IMI387470. Since soluble starch is better than glucose as a carbon source in the production phase, this raises the question of whether POPI production would be better if immobilized mycelia were grown on soluble starch in place of glucose. For one, there would be less acclimation required because there is no change in the carbon substrate fed. In the study reported in Chapter 16, glucose was substituted with soluble starch at the same concentration. Examination of the relationship between the quantity of soluble starch supplied and effect on POPI production would be a logical extension of the previous study. The aim of this study was to (a) determine whether feeding soluble starch in both the growth and production phases is better than a glucose/soluble starch strategy in the fermentation for POPI with immobilized Fusarium sp. IMI397470; (b) examine a range of concentrations of soluble starch for effect on POPI titre in the production phase.

17.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 minutes. Inoculum preparation. This was as previously described in Section 4.2 in Chapter 4.

156

Preparation of mycelia immobilized in hydrogel beads. This was as previously described in Section 11.2, Chapter 11 except that two types immobilized mycelia were produced: (a) those produced in the manner standard until this study i.e. with Parker ‐1 Broth (+ 80 g L glucose and CaCl2.2H2O); (b) those produced using Parker Broth (+ 80 g ‐1 ‐1 L soluble starch and 4.35 g L CaCl2.2H2O). After the growth batch, the next immediate batch (in the production phase) was run for acclimation of the immobilized mycelia and this batch was not described in the results. Fermentation for POPI. This was as described in Section 11.2, Chapter 11. In the production phase, Parker Broth with various concentrations of soluble starch as ‐1 ‐1 required (20, 40 or 80 g L ) plus 4.35 g L CaCl2.2H2O was used. Sampling. This was as described in Section 11.2, Chapter 11. Analysis. Culture pH and residual glucose were determined as previously described in Section 11.2, Chapter 11. Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

17.3 Results

Effect of carbon substrate feed strategy. Using soluble starch as the main carbon source in both the growth and production phases of the fermentation process did not improve POPI yield (compare Fig. 17.1 with Fig. 17.2). Not only was the maximum POPI titre higher with glucose‐grown immobilized mycelia fed soluble starch in the production phase, but time to peak was also faster.

Effect of the quantity of soluble starch supplied in production batch to immobilized mycelia grown on glucose. A clear improvement of yield was observed when the supplied soluble starch was increased from 20 to 80 g L‐1 (Fig. 17.3). Although the best titre obtained was with 80 g L‐1, this maximum titre was slower to peak slower than with 40 g L‐1. POPI productivity (titre per day) calculated at time of POPI peak was highest when the supply of soluble starch was 40 g L‐1 (Fig. 17.4).

157

1400 0.8

1200

1000 0.6 ) -1 ) -1 800

0.4 600 Glucose (g L Glucose (g POPI titre (PIU L (PIU POPI titre

400 0.2

200

0 0.0 0123456789 Days

Fig. 17.1 POPI production resulting from the use of soluble starch (80 g L‐1) in both the Growth and Production Batches

1400 0.8

1200

1000 0.6 ) -1 ) -1 800

0.4 600 GlucoseL (g POPI titre (PIU L (PIU POPI titre

400 0.2

200

0 0.0 0123456789 Days

Fig. 17.2 POPI production resulting from use of glucose (80 g L‐1) in the Growth Batch and soluble starch (80 g L‐1) in the Production Batch

158

1400

1200 ) -1 1000

800

600

400 Maximum POPI titre (PIU L (PIU titre POPI Maximum

200

0 0123456 Days

Fig. 17.3 The response in POPI titre in the production batch of fermentation with immobilized Fusarium sp. IMI397470 to a range of soluble starch supplied as the main carbon source. ( ) 20 g L‐1; ( ) 40 g L‐1; ( ) 80 g L‐1

250 ) -1 day

-1 200

150

100

POPI productivity (PIU L 50

0 20 40 60 80 Soluble starch supplied (g L-1) Fig. 17.4 The POPI productivity (titre per day) obtained in production batch when different quantities of soluble starch were supplied to fermentation with immobilized Fusarium sp. IMI397470. Productivity was calculated at the time of POPI peak in each fermentation

159

17.4 Discussion

Providing soluble starch to both the growth and production phases in the production of POPI by immobilized Fusarium sp. IMI397470 did not improve yield. This is despite a reasonable expectation that such a strategy might be advantageous by obviating the need for acclimation of the mycelia. It seems that other mechanisms at play are more influential. This may include the lower catabolite repression property of starch compared to glucose (Reese et al., 1969; Demain, 1986). The acclimation previously reported to be observed in the first batch between growth and production might be explained as the cells switching their metabolism from one geared for growth to one for producing idiolites. Thus, as a result of this study, the POPI fermentation process was modified to one where the readily metabolized carbon substrate glucose is supplied in the growth phase, while the more slowly metabolized soluble starch is supplied in the POPI synthesis phase. This two‐medium process joins the type previously reported by Kuek (1991) and Fujita (1988). With regard to the quantity of soluble starch selected to be supplied, 40 rather than 80 g L‐1 was chosen despite the latter being associated with a higher maximum POPI titre. This is because (i) 80 g L‐1 of soluble starch results in a highly viscous medium which is hard to work with; (ii) POPI productivity with 40 g L‐1 is 15% better than with 80 g L‐1. Depending on the process economics of a fully developed process, productivity rather than maximum titre could be the more critical selection criterion. In studies from this point on, the process for POPI fermentation using immobilized Fusarium sp. IMI397470 consists of a growth phase fed glucose at 80 g L‐1 and a production phase fed 40 g L‐1 soluble starch.

17.5 References

Demain, A.L. (1986) Regulation of secondary metabolism in fungi. Pure and Applied Chemistry 58: 219 ‐ 226.

160

Fujita, Y. (1988) Industrial production of shikonin and berberine. In: “Ciba Foundation Symposium 137 ‐ Applications of Plant Cell and Tissue Culture”; G. Bock and J. Marsh (eds.); John Wiley & Sons, Ltd., Chichester; pp. 228 ‐ 235.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Reese, E.T.; Lola, J.E.; and Parrish, F.W. (1969) Modified substrates and modified products as inducers of carbohydrases. Journal of Bacteriology 100: 1151 ‐ 1154.

161

CHAPTER 18

Effect of bead size on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470

18.1 Introduction

In an immobilized cell system mass and gas transfer have a bigger effect than in a free cell system. Solutes and dissolved gases have to move through the immobilizing matrix apart from the bulk liquid of the medium. The effect of bead size on fermentations was reviewed in Section 2.5.5.1, Chapter 2. A deduction based on the relationship between diffusional distances within beads and their surface area to volume ratio would predict that better bioactivity can be expected of beads of smaller sizes even though there is some contrary experimental evidence. Thus, discovering what the situation might be with the beads used in POPI fermentation would be pertinent. The aim of this study was examine to the effect of the size of beads immobilizing Fusarium sp. IMI397470 on the production of POPI.

18.2 Materials and methods

All apparatus and media were autoclaved at 121oC for 15 minutes. Inoculum preparation. The method is previously described in Section 4.2 in Chapter 4. Production of mycelia immobilized in hydrogel beads. Triplicate sets of beads of ranging from 2.0 to 4.5 mm in diameter were prepared by using hypodermic needles of various gauge sizes (Fig. 18.1). Each set of beads of different sizes were made from a

162

total volume of 45 mL sodium alginate (Manugel GMB, Kelco AIL Sydney, Australia) plus 5 mL inoculum.

30G (ca. 2 mm) 21G (ca. 2.8 mm) None (ca. 4.5 mm)

Fig 18.1 The diameters of calcium alginate beads immobilizing Fusarium sp. IMI397470 made by needles of various gauge sizes. Beads made with 21G needles were the standard in all studies reported in previous chapters

The rest of the procedure for the production of immobilized mycelia was as described in Section 11.2, Chapter 11 except that the Parker Broth used was (+ 80 g L‐1 glucose). After the growth batch, the next immediate batch (in the production phase) was run for acclimation of the immobilized mycelia and this batch was not described in the results. Fermentation for POPI. This was as described in Section 11.2, Chapter 11 except that the Parker Broth used was + 40 g L‐1 soluble starch). The effect of bead size was run as paired tests where fermentation with a test bead size was run at the same time as those made with a 21G needle (2.8 mm diameter) as the control. Sampling. This is described in Section 9.2 of Chapter 9. pH. This is described in Section 4.2 of Chapter 4. Glucose assay. This is described in Section 4.2 of Chapter 4. POP‐inhibition assay. POPI was assayed directly from culture liquors. The assay protocol is described in Section 4.2 of Chapter 4. The calculation of inhibitory activity is described in Section 5.3.2 of Chapter 5.

163

18.3 Results

Beads smaller than the standard 2.8 mm diameter did not significantly change POPI production profile by the fungus (Fig. 18.2). The maximum POPI titre obtained was marginally lower. On the other hand, while increasing the bead diameter from 2.8 to 4.5 mm did not significantly change the maximum POPI titre obtained, POPI productivity (POPI/time) was lower with bigger beads.

600 600 ) ) -1 -1

400 400 POPI titre (PIU L (PIU titre POPI POPI titre (PIU L 200 200

0 0 01234567 01234567 Days Days Fig 18.2 Production phase POPI profiles of Fusarium sp. IMI397470 immobilized in alginate beads of diameters smaller and larger than 2.8 mm. ( ) 2.8 mm control; () 2.0 mm; ( ) 4.5 mm

18.4 Discussion

The use of beads of 2.8 mm in diameter as the standard in this work proved to be the best size in the range tested. The use of beads 30% smaller in diameter did not improve POPI productivity as may be predicted on the basis of improved mass and gas transfer. The use of beads 60% larger did not change the maximum POPI titre attainable but increased fermentation time to reach the peak. Thus, no improvements were found through the bead size changes. 164

Mordocco et al. (1999) found that while the surface area to volume ratio of beads improves linearly with decreases in bead size, their work on phenol degradation by Pseudomonas putida showed that improvements in bioactivity do not further improve beyond a threshold bead size. This phenomenon is probably related to the fact that below a certain bead diameter, mass and gas transfer across the gel matrix ceases to be an influence (below this diameter reaction rates anywhere within the bead would be similar). Such an explanation might explain why beads of immobilized Fusarium sp. IMI397470 2.0 mm in diameter did not perform better than those of 2.8 mm in diameter. This explanation is supported by the data with the beads of 4.5 mm diameters which show lower POPI productivity than beads 2.8 mm in diameter. The larger beads may have been limited in their production rates by the poorer mass and gas transfer that is predictable with a greater expanse of gel matrix to transverse. The best bead diameter of 2.8 mm found in this study lies in the middle of the range of optimum diameters reported in other studies: 1 ‐ 2 mm (Mordocco et al., 1999); 2 mm (Ahmed, 2008); 2 mm (Charumathi et al., 2010); 3 mm (Wang et al., 2001); 4 mm (Hameed, 2007); 5 mm (Zain et al., 2011). In the remaining studies in this work, bead diameter remained unchanged at 2.8 mm.

18.5 References

Ahmed, S.A. (2008) Invertase production by Bacillus macerans immobilized on calcium alginate beads. Journal of Applied Science Research 4: 1777 ‐ 1781.

165

Eikmeier, H.; Westmeier, F. and Rehm, H.J. (1984) Morphological development of Aspergillus niger immobilized in Ca‐alginate and K‐carrageenan. Applied Microbiology and Biotechnology 19: 53 ‐ 57.

Hameed, M.S.A. (2007) Effect of algal density in bead, bead size and bead concentrations on wastewater nutrient removal. African Journal of Biotechnology 6: 1185 ‐ 1191.

Mordocco, A.; Kuek, C. and Jenkins, R. (1999) Continuous degradation of phenol at low concentration using immobilized Pseudomonas putida. Enzyme and Microbial Technology 25: 530 ‐ 536.

Pashova, S.; Slokoska, L.; Sheremetska, P.; Krumova, E.; Vasileva,L. and Angelova, M. (1999) Physiological aspects of immobilised Aspergillus niger cells producing polymethylgalacturonase. Process Biochemistry 35: 15 ‐ 19.

Wang, J; Han, L.; Shi, H. and Qian, Y. (2001) Biodegradation of quinolone by gel immobilized Burkholderia sp. Chemosphere 44: 1041 ‐ 1046.

Zain, M.M., Kofli, N.T. and Yahya, S.R.S. (2011) Bioethanol production by calcium alginate‐immobilised ST1 yeast system: effects of size of beads, ratio and concentration. IIUM Engineering Journal 12: 11 ‐ 19.

166

CHAPTER 19

Effect of the addition of Tween 80 on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470

19.1 Introduction

Although its mode of action is not well‐understood, Tween 80 has long been used by industrial microbiologists to improve fermentation yield e.g. as reported in an early paper by Reese and Maguire (1968). Surfactants such as Tween 80 have proven to be effective in the production of useful metabolites in bacteria, fungi and medicinal mushrooms albeit sometimes with inconsistent results (Zhang and Cheung, 2011). Their beneficial use in fermentation have included the production of cellulases, amylases and ligninases (Galindo and Salcedo, 1996), a lipase from Fusarium solani (Murphy et al., 1998), exopolysaccharide from Cordyceps sinensis (Liu and Wu, 2012) and α‐amylase from Thermomyces lanuginosus (Arnesen et al., 1998). The mode of action of Tween 80 is thought to be changes in microbial physiology, membrane permeability and membrane‐associated enzymatic functions (Galindo and Salcedo, 1996; Zhang and Cheung, 2011). Mizrahi and Miller (1969) concluded from their data that Tween 40, 60 and 80 facilitated the transport of fermentation substrates into mycelia of Claviseps paspali in the production of ergot alkaloids. This conclusion must logically also extend to the possibility that the transport of metabolites out of cells into the culture liquor. The aim of this study was to examine whether the production of POPI by immobilized Fusarium sp. IMI397470 can be improved by the use of Tween 80.

167

19.2 Materials and methods

Table 19.1 The concentrations of Tween 80 used in Parker Broth (+ 40 g L‐1 soluble starch) to test its effect on POPI yield in the production phase

Concentration (% v/v) Phase 0.01 Production only 0.01 Growth and production 0.10 Production only

Sampling. This is described in Section 9.2 of Chapter 9. pH. This is described in Section 4.2 of Chapter 4. Glucose assay. This is described in Section 4.2 of Chapter 4. POP‐inhibition assay. POPI was assayed directly from culture liquors. The assay protocol is described in Section 4.2 of Chapter 4. The calculation of inhibitory activity is described in Section 5.3.2 of Chapter 5.

168

19.3 Results

The addition of Tween 80 at 0.01% (v/v) to production medium did not increase the maximum POPI titre attained in the production phase. The yield obtained was poorer than the non‐Tween 80 treated control (Fig. 19.1; addition strategy 1). Increasing the Tween 80 concentration by ten‐fold only served to lower the POPI yield even more, to about 20% of the maximum yield of the untreated control (Fig. 19.1; addition strategy 3). The addition of Tween 80 to both the growth and production phases (at 0.01% v/v) also did not improve POPI yield (Fig. 19.1; addition strategy 2).

1.0

0.8

0.6

0.4

Relative Effectiveness Relative 0.2

0.0 123 Addition strategy

Fig 19.1 The relative effectiveness of various Tween 80 addition strategies in the growth and production phases of by immobilized Fusarium sp. IMI397470 on the maximum POPI titre obtained. (1) Tween 80 at 0.01% (v/v) added in production phase only; (2) Tween 80 at 0.01% (v/v) added in both growth and production phases; (3) Tween 80 at 0.1% (v/v) added at production phase only Max.POPI titreof  Tween80 fermentation Relative Effectiveness  Max.POPI titreof ‐ Tween80 fermentation

169

19.4 Discussion

The addition of the surfactant Tween 80 was not found to improve the yield of POPI in the production phase. Adding it earlier in the growth phase as well as in the production phase also failed to improve POPI yield. Since the use of a relatively low concentration (0.01% v/v) of Tween 80 compared to concentrations reported by others to be beneficial (up to 1.3% w/v [Arnesan et al., 1998]) lowered POPI yield, higher concentrations apart from 0.1% (v/v) were not tested in this study. The use on Tween 80 at 0.1% (v/v) in the production medium was deleterious to POPI production as it reduced the maximum POPI titre obtained to only 20% of the untreated control. POPI production appears to be negatively affected by Tween 80 at concentration much lower than those which proved beneficial in other fermentations e.g. at 0.1% (w/v) reported by Murphy et al. (1998); 0.3 (w/v) by Zhang and Cheung (2011); 0.5% (w/v) by Galindo and Salcedo (1996); and 1.3% (w/v) by Arnesan et al. (1998). The negative results with Tween 80 suggest that the production of POPI by Fusarium sp. IMI397470, is not sensitive to changes in membrane permeability and membrane‐associated enzymatic functions which are thought to be two of the mechanisms involved in yield improvement by the surfactant (Galindo and Salcedo, 1996; Zhang and Cheung, 2011).

19.6 References

Arnesen, S.; Eriksen, S. H.; Olsen, J. and Jensen, B. (1998) Increased production of a‐ amylase from Thermomyces lanuginosus by the addition of Tween 80. Enzyme and Microbial Technology 23: 249 ‐ 252.

Chen, H. B.; Huang, H.C.; Chen, C. I.; Yet‐Pole, I. and Liu, Y. C. (2010) The use of additives as the stimulator on mycelial biomass and exopolysaccharide productions in

170

submerged culture of Grifola umbellate. Bioprocess Biosystems Engineering 33: 401 ‐ 406.

Galindo, E. and Salcedo, G. (1996) Detergents improve xanthan yield and polymer quality in cultures of Xanthomonas campestris. Enzyme Microbial and Technology 19: 145 ‐ 149.

Liu, Y.S. and Wu, J.Y. (2012) Effects of Tween 80 and pH on mycelial pellets and exopolysaccharide production in liquid culture of a medicinal fungus. Journal of Industrial Microbiology and Biotechnology 39: 623 ‐ 628.

Mizrahi, A. and Miller, G. (1969) Role of glycols and Tweens in the production of ergot alkaloids by Claviseps paspali. Journal of Bacteriology 97: 1155 ‐ 1159.

Murphy, C. A.; Cameron, J. A.; Huang, S. J. and Vinopal, R. T. (1998) A second polycaprolactone depolymerase from Fusarium, a lipase distinct from cutinase. Applied Microbiology and Biotechnology 50: 692 ‐ 696.

Reese, E.T. and Maguire, A. (1969) Surfactants as stimulants of enzyme production by microorganisms. Applied Microbiology 17: 242 ‐ 245.

Zhang, B. B. and Cheung, P. C. K. (2011) Use of stimulatory agents to enhance the production of bioactive exopolysaccharide from Pleurotus tuber‐regium by submerged fermentation. Journal of Agricultural Food Chemistry 59: 1210 ‐ 1216

171

CHAPTER 20

The storage life of immobilized Fusarium sp. IMI397470

20.1 Introduction

The advantage of immobilized cells in fermentation is that they can be re‐used from batch to batch. An added utility may be that of being able to store the immobilized cells between production batches until needed. Kuek et al. (1992) working with immobilized fungal mycelia found that Hebeloma westraliense retained its viability after storage in water at 4oC for 8 months, while Elaphomyces sp. stored poorly. However, the re‐usability of immobilized cells for metabolite production after a period of storage does not appear to have been reported before. An appreciation of the storage capacity of Fusarium sp. IMI397470 will extend the utility of the fungus. Therefore, the aim of this study was to examine the extent to which immobilized Fusarium sp. IMI397470 can be effectively stored at 4oC.

20.2 Materials and methods

172

beads were drained and washed thrice with 50 mL aliquots of Parker Broth (‐ carbon source) and then a further 50 mL of the same medium was added. The flasks were then stored at 4oC for various periods after which the capacity of the beads for POPI production was tested. One set was stored for 30 days. Another set was stored for 60 days. The third set was stored for 106 days. At the end of the storage period, the beads were drained and then used in POPI production. Fermentation for POPI. This was as described in Section 11.2, Chapter 11 except that the Parker Broth used was + 40 g L‐1 soluble starch). Sampling. This is described in Section 9.2 of Chapter 9. pH. This is described in Section 4.2 of Chapter 4. Glucose assay. This is described in Section 4.2 of Chapter 4. POP‐inhibition assay. POPI was assayed directly from culture liquors. The assay protocol is described in Section 4.2 of Chapter 4. The calculation of inhibitory activity is described in Section 5.3.2 of Chapter 5.

20.3 Results

Immobilized Fusarium sp. IMI397470 retained its ability to produce POPI after storage for 30 days at 4oC (Fig. 20.1). Further storage of the fungus for another 30 days resulted in a maximum POPI titre of only 30% of the original capability. The 60‐day storage test confirmed this finding where maximum POPI titre after storage was never more than 25% of that prior to storage (Fig. 20.2). The longest term storage examined of 106 days showed that most of the capability to produce POPI had been lost after storage (Fig. 20.3). Consistently across the three sets of storage tests, post‐storage production of POPI appears to occur under more acidic conditions than before the immobilized mycelia were stored (Figs. 20.1 to 20.3). The 106‐day stored mycelia which showed no post‐storage ability to produce POPI had the lowest culture pH in production batches (ca. pH 4.5).

173

1000 1000 1000

6 6 6 800 800 800 ) ) ) 1 1 - - -1

600 4 600 4 600 4 pH pH pH 400 400 400

2 2 2 POPI titre (PIU L POPI titre POPI titre (PIU L (PIU titre POPI 200 200 L (PIU POPI titre 200

0 0 0 0 0 0 01234 012345678 012345 Days Days Days a b c Fig 20.1 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) before storage; (b) after 30‐day storage; (c) after further 30‐day storage

1000 7 1000 7

6 6 800 800 ) 5 ) 5 -1 -1

600 4 600 4

3 3 pH 400 400 pH 2 2 200 200

POPI titre (PIU L (PIU titre POPI 1

0 0 0 0 012345 0246810121416182022 Days Days a b Fig 20.2 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) Before storage; (b) after 60‐day storage

174

800 800 6 6 ) ) -1 -1 600 600 4 4 400 400 pH pH

2 2 200 200 POPI titre (PIU L (PIU POPI titre POPI titre (PIU L POPI titre

0 0 0 0 01234567 012345678910 Days Days a b Fig 20.3 POPI production by immobilized Fusarium sp. IMI397470 before and after storage at 4oC. (a) Before storage; (b) after 106‐day storage

20.4 Discussion

Immobilized Fusarium sp. IMI397470 is re‐usable for POPI production if stored at 4oC for no longer than 30 days. Stored between 30 and 60 days, its capacity for POPI production remains but reduced to between 25 ‐ 30% of that prior to storage. POPI production capacity is essentially lost by 106 days of storage. That physiological change had occurred to the mycelia during storage is indicated by the lower pH found in post‐ storage fermentation. The process for the production of POPI using immobilized Fusarium sp. IMI397470 as developed in this work now not only includes re‐use of pre‐grown mycelia in repeat batches but batching can be temporarily suspended and the mycelia stored at 4oC about 30 days for later resumption of production.

175

20.5 Reference

Kuek, C.; Tommerup, I.C. and Malajczuk, N. (1992) Hydrogel bead inocula for the production of ectomycorrhizal eucalypts for plantations. Mycological Research 96: 272 ‐ 277.

176

CHAPTER 21

Effect of controlled culture pH on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor

21.1 Introduction

Elucidation of POPI production by Fusarium sp. IMI397470 was first studied using free mycelia in shake flask culture (Chapters 4, 7, 8 and 9). Recognition of the implication of the growth‐dissociated nature of POPI production led to studies with immobilized mycelia, also in shake flask culture (Chapters 11 to 19). While pH profiles were often determined in these studies, in had not been hitherto possible to properly study the effect of controlling culture pH using shake flask cultures. One approach in shake flask cultures might have been to use the “Good” buffers. These are synthetic buffers which have limited permeability of biological membranes (Good et al., 1966) and are thus ideal for culture studies as they do not affect the biochemistry of the cells being studied. However, even with buffers, a certain amount of pH drift occurs in cultural studies. The traditional approach in fermentation is to conduct pH investigations with a bioreactor equipped with automatic pH control. This was the approach taken as it was in any case, within the natural progression of process development to move from shake flask cultures to a bioreactor. Indeed, this study marks the change from shake flask investigations to those using a bubble column. Thus, one of the aims of this study was to establish a baseline for POPI production by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor. A pneumatically agitated bioreactor was chosen for this work because it was assessed that it has the least potential for eroding or damaging the hydrogel beads containing immobilized mycelia. This is because pneumatically agitated bioreactors provide a low‐shear environment in comparison with mechanically agitated 177 bioreactors (Wang and Zhong, 2007). The bubble column bioreactor which was the configuration chosen for this work has been successfully used for the production of metabolites by immobilized fungi e.g. Kuek (1991); Manolov (1992); Dierkes et al. (1993); Abdul Karim and Mohamad Annuar (2009); Castillo‐Carvajal et al. (2012). In a study with Fusarium verticillioides, Boonyapranai et al. (2008) set the initial pH of media over a range of values and found that a high initial pH was not favourable for the biomass accumulation. However, as the initial pH of the medium was increased, metabolite (naphthoquinone) production increased until a setting of pH 8. An inverse relationship was thus found between the effect of pH on biomass and metabolite production. Gaden (2000) reported that growth in a penicillin fermentation is optimal around pH 4.5 – 5 whereas production of the antibiotic is optimal at the higher pH of 7 – 7.5. In contrast, with Fusarium roseum biomass accumulation and metabolite (Cyclosporin A) production were both optimal at around pH 6 (Ismaiel et al., 2010). Although the studies reported were both not where the growth and metabolite phases were separated, they provide some evidence that pH for metabolite production by pre‐grown biomass may be different from optimized differently from the requirement for growth. The other aim of this study was to study the effect of controlled pH on the production of POPI by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor.

21.2 Materials and methods

All apparatus and media were sterilized at 121oC for 15 minutes and all transfers were aseptically made. The bubble column bioreactor used. A custom‐blown glass bubble column reactor (3.0 cm internal diameter; 200 ml working volume) and its ancillary parts were used (Figs. 22.1a and 22.1b). The sparger in the bioreactor was downward pointing (0.5 cm from the bottom of the reactor) with a single orifice 0.5 mm in diameter (Fig. 21.1c). Air was double‐filtered firstly with an oil filter (Festo LOE‐D‐Mini X943; 40 m) and then down to 0.2 m via an AcroPak 300 Capsule with PTFE membrane filter (Pall Corp.).

178

Fig. 21.1a The bubble column bioreactor rig used in this work

Fig. 21.1b A close‐up of the bubble column with a production batch of immobilized Fusarium sp. IMI397470 underway

179

Fig. 21.1c Details of the configuration at the bottom of the bubble column bioreactor

Filtered air was humidified prior to entry in the bubble column by passage through a jacketed vessel containing water. Culture pH was maintained by the automatic addition of 0.05 M H2SO4 or 0.1 M NaOH regulated by a pH controller. The electrode was sterilized by immersion in 70% (v/v) ethanol for 15 min before use. Electrode drift was corrected daily by comparisons of pH controller readings against an external pH meter. Culture temperature was maintained by water flow from a recirculating bath through the water jacket of the reactor. Fresh medium was added to the reactor when required by aseptic transfer from reservoirs. Inoculum preparation. This was as described in Section 4.2, Chapter 4. Preparation of immobilized mycelia. For each bubble column run, 150 mL of beads were produced using the protocol described in Section 11.2, Chapter 11 except that incubation was in the bubble column itself with 150 mL of double‐strength Parker

180

Broth (+ 80 g L‐1 glucose, becoming 40 g L‐1 when added to an equal volume of beads) as the Growth Medium, at 25oC and an air flow of 1.0 L min‐1. Incubation was for 3 days, chosen because POPI titre always peaked before Day 3 which by the growth‐ dissociated nature of POPI production is an indication that stationary phase of growth had been reached. pH regimes tested. Production of POPI by immobilized cells was carried out in four sequential production batches after the growth batch. In each production batch, culture pH was automatically controlled to a set value in the sequence outlined in Table 21.1.

Table 21.1 The sequence of controlled culture pH under which POPI production was studied Sequence Phase Batch pH controlled at (±0.05) 1 Growth ‐ Nil 2 Production ‐ Nil 3 1 6.5 4 2 5.5 5 3 4.5 6 4 5.5

Bubble column production of POPI. Each batch culture comprised 150 ml of double‐ strength Parker Broth Medium (+ 40 g L‐1 soluble starch, becoming 20 g L‐1 when added to an equal volume of beads) as the POPI Production Medium, and 150 mL of beads. Air was supplied at 1.0 L min‐1. Temperature was maintained at 25oC. Samples (1.5 ml) were withdrawn each day with a sterile hypodermic needle through a sampling port which was sealed with a septum. At the end of each production batch, liquor was drained, and the beads were washed within the reactor with Wash Medium (Parker Broth without carbon source) equivalent to the volume of liquor. The next production batch was begun with the addition of 150 ml of fresh Parker Broth Medium (+ 40 g L‐1 soluble starch). Immediately after the growth batch (production of immobilized mycelia), one batch with Production Medium was run for 5 days as an acclimation batch. The experimental production batches reported in this study were run after that.

181

Analysis. Residual glucose was assayed enzymatically using the glucose oxidase and peroxidase method (Bergmeyer and Bernt, 1974). Culture liquor was assayed directly for POPI using the protocol described in Section 4.2, Chapter 4. The calculation of inhibitory activity is described in Section 5.2 of Chapter 5.

21.3 Results

Immobilized Fusarium sp. IMI397470 grew well in the bubble column, with POPI titre peaking as early as at Day 1 of the growth batch (Fig. 22.1). POPI production was observed in all the production batches following the growth batch. POPI production was highest when culture pH was controlled to pH5.5 (Production Batch 2). In case the lower production observed at pH4.5 (Production Batch 3) was caused by loss of viability of the immobilized mycelia, fermentation was carried on to another batch at pH 5.5. POPI titre recovered in this batch although not to the same level as the previous run at the same culture pH.

182

Production Batch 1 Production Batch 2 Production Batch 3 Production Batch 4 7 pH6.5 pH5.5 pH4.5 pH5.5 500

400 5 ) -1 300 4

3 pH 200

POPI titres (PIU L 2

100 1

0 0 01234567891011121314151617181920 Days

Fig. 21.1 The production of POPI by immobilized Fusarium sp. IMI397470 at various controlled culture pH values

21.4 Discussion

POPI production by immobilized Fusarium sp. IMI397470 responded to the pH at which the fermentation was controlled. POPI production was highest when pH was controlled to 5.5. At pH 4.5, the lowest value tested, the lower level of POPI production was demonstrated to be not due to loss of cell viability by the return to a higher maximum POPI titre immobilized mycelia when the culture was returned to pH 5.5. Although POPI titre in this batch (Production Batch 4) exceeded the maximum titre at pH 4.5, the maximum titre was lower than when previously fermented at pH 5.5. This observation may be an indication that fermentation at pH 4.5 caused an irreversible deterioration of POPI production capability.

183

There are various examples of fungi favouring acidic conditions in fermentation, for example in the production of glucoamylase (Kuek, 1991), lysozymes (Parra et al., 2005), manganese peroxidase (Urek and Pazarlioglu, 2007) and α‐amylase (Gupta et al., 2010). With regard to Fusarium spp., Gupta et al. (2009) found the optimal pH for xylanase production to be around 5 for immobilized Fusarium solani. Audhya and Russell (1975) found that pH 6 was optimal for the production of enniatin by Fusarium sambucinum. Ismaiel and workers (2010) reported the optimal pH for cyclosporin A production by free mycelia of Fusarium roseum was between 5.5 and 6.0. These findings support the finding of pH 5.5 as the optimum POPI production by immobilized Fusarium sp. IMI397470, and it became the operating pH in subsequent studies reported in this work. The maximum POPI titre found in this first bubble column run was only between 30 to 40% of the maximum found in previous shake flask runs (ca. 1000 – 1300 PIU L‐1). Between shake flask and bubble column studies, at least two changes occurred which would have contributed to the difference in POPI yield: (a) the volume of the fermentation increased from 100 mL to 300 mL; (b) the cultural conditions within the shake flask and the bubble column would substantially different. Nevertheless, moving from shake flask to bioreactor studies is necessary step in developing fermentation processes and in the transition, larger scale fermentations initially give a lower yield (Hsu and Wu, 2002) than expected.

21.5 References

Abdul Karim, M.A. and Mohamad Annuar, M.S. (2009) Novel application of coconut husk as growth support matrix and natural inducer of fungal laccase production in a bubble column reactor. Asia‐Pacific Journal of Molecular Biology and Biotechnology 17: 47 ‐ 52.

184

Audhya, T.K. and Russell, W. (1975) Enniatin production by Fusarium sambucinum: primary, secondary, and unitary metabolism. Journal of General Microbiology 86: 327 ‐ 331.

Castillo‐Carvajal, L.; Ortega‐González, K.; Barragán‐Huerta, B.E. and Pedroza‐Rodríguez, M. (2012) Evaluation of three immobilization supports and two nutritional conditions for reactive black 5 removal with Trameter versicolor in air bubble reactor. African Journal of Biotechnology 11: 3310 ‐ 3320.

Dierkes, W.; Lohmeyer, M. and Rehm, H.J. (1993) Long‐term production of ergot peptides by immobilized Claviseps purpurea in semicontinuous and continuous culture. Applied and Environmental Microbiology 59: 2029 ‐ 2033.

Gaden, E.L. (2000) Fermentation process kinetics. Biotechnology and Bioengineering 67: 413 ‐ 635.

Good, N.E.; Winget, G.D.; Winter, W.; Connolly, T.N.; Izawa, S. and Singh, R.M.M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5: 467 ‐ 477.

Gupta, V.K.; Gaur, R.; Yadava, S.K. and Darmwal, N.S. (2009) Optimization of xylanase production from free and immobilized cells of Fusarium solani F7. BioResources 4: 932 ‐ 945.

185

Hsu, Y.L. and Wu, W.T. (2002) A novel approach for scaling‐up a fermentation system. Biochemical Engineering Journal 11: 123 ‐ 130.

Ismaiel, A.A.; El‐Sayed, E.A. and Mahmoud, A.A. (2010) Some optimal culture conditions For production of Cycosporin A by Fusarium roseum. Brazilian Journal of Microbiology 41: 1112 ‐ 1123.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Manolov, R.J. (1992) Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble column bioreactor. Applied Microbiology and Biotechnology 37: 32 ‐ 36.

Parra, R.; Aldred, D. Magan, N. (2005) A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain

Applied Microbiology and Biotechnology 67: 336 ‐ 344.

Urek, R.O. and Pazarlioglu, N.K. (2007) Enhanced production of manganese peroxidise by Phanerochaete chrysosporium. Brazillian Archives of Biology and Technology 50: 913 ‐ 920.

Wang, S.J. and Zhong, J.J. (2007) Bioreactor engineering. In: “Bioprocessing for value‐ added products from renewable resources”; S.T. Yang (ed.); Elsevier B.V.; pp. 131 ‐ 162.

186

CHAPTER 22

Effect of bead to medium volume ratio on the production of prolyl oligopeptidase by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor

22.1 Introduction

The influence of the bead to medium volume ratio on fermentation with immobilized cells was introduced in Section 2.5.5.2 Chapter 2. Table 2.7 (Chapter 2) shows six examples of studies where a range of bead:medium ratios were examined. All the studies referred to found optimal ratios where the beads were more “fluidized” (there was more medium in proportion to beads) than the standard ratio of 1:1 used in this work to date. Several other studies used a ratio which was more fluid viz. 1:5 e.g. El‐Naggar et al. (2006) in the production of β‐Mannanase from Aspergillus niger, Shen and Xia (2006) in the production of lactic acid production by Lactobacillus delbrueckii, and Matošiƈ et al. (1998) in the production of ergot‐alkaloid by Claviceps paspali. The choice of a starting operating ratio to use at the beginning of the development of a process is arbitrary as this can be optimized during process development as is now the focus of this study. The objective of this study was to examine the amount of POPI production by Fusarium sp. IMI397470 when the bead:medium volume ratio was varied from the standard of the 1:1 used to date.

187

22.2 Materials and methods

All protocols used were as described in Section 21.2, Chapter 21 except that the quantities of beads and medium used were varied as required (Table 22.1) and the fermentation conditions as summarized below (Table 22.2).

Table 22.1 The relative volumes of beads to medium in production batches to study effect of the ratio on the bubble column production of POPI by immobilized Fusarium sp. IMI397470 Bead volume Medium volume Total Bead: medium ratio (mL) (mL) Fermentation Volume (mL) 200 100 300 2:1 150 150 300 1:1 100 200 300 1:2 75 225 300 1:3 The 1:1 bead to medium ratio was the standard used in all studies reported to this point.

Table 22.2 Summary of the fermentation conditions used in Chapter 22

Growth Medium Parker Broth (+ 80 g L‐1 glucose)

Production Medium Parker Broth (+ 40 g L‐1 soluble starch)

Growth batch temperature 25oC

Production batch temperature 25oC

pH controlled to pH5.5 (±0.05)

Air flow 1.0 volume volume‐1

188

22.3 Results

POPI production by immobilized Fusarium sp. IMI397470 was evident at a bead to medium ratio of 2:1 (Fig. 22.1) but this was significantly at a lower rate than the best production found with the ratio at 1:1. More “fluidized” ratios greater than 1:2 or 1:3 were examined as these already indicated low yields of POPI. A higher bead volume than 2:1 was not examined because at 2:1, the beads were not evenly distributed by the set air sparging (a large portion of the beads resided at the bottom of the bubble column at any one time).

1000 ) -1 800

600

400 POPI titre (PIU L POPI titre (PIU

200

0 012345678 Days

Fig 22.1 POPI yield from the production batch of immobilized Fusarium sp. IMI397470 in a bubble column at various bead to medium volume ratios: ( ) 2:1; () 1:1; () 1:2; and ( ) 1:3.

189

22.4 Discussion

In the range studies, production of POPI by immobilized Fusarium sp. IMI397470 was clearly optimal a bead to medium volume ratio of 1:1. At the ratio below it where there was twice as much beads as medium, the poorer performance is could be due to the relatively larger amount of biomass representing a sink for nutrients and oxygen that could not be sufficiently supplied. This was probably contributed to by the poorer fluidization of the beads in the bubble column at the 2:1 ratio. Many studies (Table 2.7, Chapter 2; El‐Naggar et al., 2006; Shen and Xia, 2006; Matošiƈ et al., 1998) reported better results with the use of a bead:medium volume ratio greater than the 1:1 found to be optimal in this study. This suggests that under the conditions used for the fermentation for POPI in the bubble column, a higher biomass loading can be sustained i.e. the mass and gas transfer demand by pre‐grown Fusarium sp. IMI397470 was relatively lower such that more biomass can be present per unit volume of bioreactor volume. This is a reasonable deduction because the comparative studies referred to were all batch cultures where growth of the biomass and metabolite production were expected to occur together. As such, the demand on mass and gas transfer would be higher thus setting a lower limit for the amount of biomass which can be effectively supported per unit bioreactor working volume. Thus, another advantage of using immobilized pre‐grown mycelia as with Fusarium sp. IMI397470 is that more biomass per unit volume can be packed into a bioreactor with resultant higher potential for volumetric productivity. In the remaining bubble column studies, a bead:volume ratio of 1:1 was used.

22.5 References

190

Matošiƈ , S.; Mehak, M.; Ercegoviƈ, L.; Brajkoviƈ, N. and Šuškoviƈ, J. (1998) Effect of surfactants on the production of ergot‐alkaloids by immobilized mycelia of Claviceps paspali. World J. Microbiol. Biotechnol. 14: 447 ‐ 450.

Shen, X. and Xia, L. (2006) Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii. World Journal of Microbiology and Biotechnology 22: 1109 ‐ 1114.

191

CHAPTER 23

Effect of carbon to nitrogen ratio in the medium on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor

23.1 Introduction

Parker Broth, the medium used in this work (Table 3.1, Chapter 3) is a semi‐ defined basal salts medium to which a selected carbon source is added. It has been successfully used in phase separated, repeat batch fermentations with Aspergillus phoenicus (Kuek, 1991), a fungus in the same phylum as Fusarium sp. IMI397470. The variant of this medium as developed thus far to this chapter, has a C:N ratio of 190:1. This C:N ratio is high compared to that used in some other studies e.g. beauvericin production by Fusarium oxysporum where it was found to be optimal at 4 (Lee et al., 2008) or ‐amylase production by Bacillus licheniformis where it was found to be optimal at 1 (Dharani Aiyer, 2004). Despite this, it has been shown that Parker Broth is capable of supporting good production of POPI by Fusarium sp. IMI397470 (Chapters in Part B; Chapters 21 and 26). Given that a review of the literature (Section 2.5.1.2; Chapter 2) revealed examples of good results in metabolite production with both high and low C:N ratios and the fact that in a phase separated, repeat batch culture, the ratio requirement is likely to be different because pre‐grown cells are used, it would be necessary to check the appropriateness of the C:N ration in the Parker Broth variant developed thus far. The aim of this study was to examine C:N ratios arbitrarily lower (95:1) and higher (381:1) than that in the variant of Parker Broth developed to this point in the work (190:1), for their effect on the production of POPI by immobilized Fusarium sp. IMI397470.

192

23.2 Materials and methods

All protocols used were as described in Section 21.2, Chapter 21 except that two variations of double‐strength Parker Broth (+ 80 g L‐1 soluble starch) were used. One variant had a C:N of 95:1 while in the other the ratio was 381:1. The variation was achieved by only altering the N components of the medium while keeping the C content constant. For comparison against a standard, POPI productivity data from a comparable run with a C:N ratio of 190:1 (reported in Fig. 22.2, Chapter 22 ) was used. The fermentation conditions were as summarized below (Table 23.1).

Table 23.1 Summary of the fermentation conditions used in Chapter 23

Growth Medium Parker Broth (+ 80 g L‐1 glucose) Production Medium Parker Broth (+ 40 g L‐1 soluble starch) with N component altered to achieve the required C:N ratio Growth batch temperature 25oC Production batch temperature 25oC pH controlled to pH 5.5 (±0.05) Air flow 1.0 L min‐1

23.3 Results

The time course for POPI production (Fig. 23.1) and comparison of the maximum POPI titres obtained (Fig. 23.2) clearly show that the C:N ratios lower or higher than 190 gave significantly poorer results.

193

1000

800 ) -1

600

POPI titre (PIU L (PIU titre POPI 400

200

0 0123456

Days Fig. 23.1 POPI production by immobilized Fusarium sp. IMI397470 using Parker broth (+ 40 g L‐1 soluble starch) variations with different C:N ratios: ( ) C:N = 95:1; ( ) C:N = 190:1 (data from Fig. 22.2, Chapter 22);

() C:N = 381:1

194

1000 )

-1 800

600

400 POPI titre (PIU L (PIU POPI titre

200

0 95:1 190:1 380:1

Carbon: Nitrogen ratio Fig. 23.2 The maximum POPI titres resulting from the use of Parker Broths (+ 40 g L‐1 soluble starch) variations with different C:N ratios in the fermentation for POPI by immobilized Fusarium sp. IMI397470. Data for the 190:1 ratio was from Fig. 22.2, Chapter 22

23.4 Discussion

In alterations of the C:N ratio of Parker Broth arbitrarily lower or higher than 190:1, the variations were significantly poorer. This means that the standard ratio of 190:1 as developed thus far is best. Studies which report good metabolite yields with low C:N ratios such as 1 (‐ amylase; Dharani Aiyer, 2004) or 40 (lovastatin; Casas López et al., 2003) indicate situations where the relative requirement for N must have been higher. It could be that the higher requirement for N is related to both ‐amylase and lovastatin having a

195 correspondingly high N component in their molecular structures, or for the support of growth since growing cells were used in the studies referred to. The finding that a proportionally high C to N content in Parker Broth is suitable for POPI production is supported by the study of Brzonkalik et al. (2011) who showed that having an excess of C in relation to N had a positive effect of the production of alternariol. Similarly, production of polyketide toxins was improved when N was decreased in the medium (Bell et al., 2003). Even the study of Casas López et al. (2003) where the optimal C:N ratio (at 40) was much lower than 190:1, revealed that ratios with higher N content (<40) were poorer in producing lovastatin. In a study with pre‐ grown mycelia of Gibberella fujikuroi re‐suspended in fresh medium, production of Fumonisin B1 was repressed when additional N was added to the medium 120 h into the culture (Shim and Woloshuk, 1999). This finding adds weight to the reasoning that says metabolites such as lovastatin are produced under nitrogen limited conditions (Casas López et al., 2003). Thus, these reports all indicate the benefit of a medium composition which is apparently one suited for cells which by virtue of a lesser requirement for N, are not in active or rapid growth. Such cells are those which are poised for the production of secondary metabolites, which POPI is indicated to be in Chapter 7. The lesser performance of the Parker Broth formulation with the highest C content compared to N (380:1) may possibly be explained by the carbon source being in excess of the requirement to energize POPI production and at which level, sufficient residual glucose accumulates in the culture liquor to be repressive of POPI production. The latter is a speculation because it is not known whether POPI is indeed subject to carbohydrate catabolite repression. The finding from this study meant that a Parker Broth with a C:N ratio of 190:1 was confirmed for use in the rest of the studies in the work.

196

23.5 References

Brzonkalik, K.; Herrling, T.; Syldatk, C. and Neumann, A. (2011) Process development for the elucidation of mycotoxin formation in Alternaria alternata. AMB Express 1: 27 ‐ 35.

Casas López, J.L.; Sánchez Pérez, J.A.; Fernández Sevilla, J.M.; Acién Fernández, F.G.; Molina Grima, E. and Chisti, Y. (2003) Production of lovastatin by Aspergillus terreus: effects of the C:N ratio and the principal nutrients on growth and metabolite production. Enzyme Microbial and Technology 33: 270 ‐ 277.

Dharani Aiyer, P.V. (2004) Effect of C:N ratio on alpha amylase production by Bacillus licheniformis SPT27. African Journal of Biotechnology 3: 519 ‐ 522.

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Lee, H.S.; Song, H.H.; Ahn, J.H.; Shin, C.G.; Lee, G.P. and Lee, C. (2008) Statistical optimization of growth medium for the production of the entomopathogenic and phytotoxic cyclic depsipeptide beauvericin from Fusarium oxysporum KFCC11363P. Journal of Microbiology and Biotechnology 18: 138 ‐ 144.

Shim, W.B. and Woloshuk, C.P. (1999) Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi. FEMS Microbiology letters 177: 109 ‐ 116.

197

CHAPTER 24

Effect of air flow on the production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor

24.1 Introduction

In bubble columns sparging with air not only serves top effect gas transfer but also stirring. Both are inter‐related as stirring controls diffusion gradients. To meet higher oxygen demands, sparging rate has to increase and this causes increased stirring with an attendant increase in shear forces in the fermentation. Both high and low air sparging rates have been used with hydrogel immobilized cells in fermentation with pneumatically stirred bioreactors (Table 24.1).

Table 24.1 The range of air sparging rates used in fermentations with hydrogel immobilized cells in pneumatically stirred bioreactors vvm* Microorganism Product Reference 0.17 ‐ 1.0 Aspergillus clavatus Ribonuclease Manolov (1992) 0.4 Rhizopogon nigrescens Mycelium de Oliveira et al. (2006) 0.5 Rhizopus oryzae Lipolytic enzyme Lopez et al. (2008) 0.5 ‐ 3.0 Kidney cell line Urokinase Devi et al.(2007) 1.33 Candida guilliermondii Xylitol Branco et al. (2007) 3.125 Aspergillus phoenicus Glucoamaylase Kuek (1991) 3.33 Fusarium sp. IMI397470 POPI Current work cyclodextrin Kunamneni et al. 4.0 Bacillus sp. glucanotransferase (2007) *volume of air (volume of fermentation)‐1 min‐1

The range in reported sparging rates reflects the different oxygen requirement of the mediating microorganisms (growth and metabolite synthesis) coupled with the sensitivity of the hydrogel material to abrasive damage which can occur even in

198 pneumatically stirred bioreactors as observed by Kuek and Armitage (1991) via scanning electron microscopy, and by others e.g. Lin and Lee (2008). Thus, it was necessary to examine the effect of air sparging rate on the production process for POPI being developed especially a feature of the process is the repeated use of pre‐grown mycelium. The aim of this study was to compare the relative POPI yields resulting from sparging rates lower and higher than the standard of 3.33 vvm used up to this point in the work.

24.2 Materials and methods

All protocols used were as described in Section 21.2, Chapter 21 except that three different sets of beads were prepared and used in three separate fermentations supplied three different levels of aeration/mixing (Table 24.1). The fermentation conditions were as summarized in Table 24.2.

Table 24.2 The fermentation volume to air flow rate variations used to set three different aeration levels in the bubble column bioreactor Run Working volume (L) Air flow (L min‐1) Aeration rate (vvm) 1 0.3 0.5 1.67 2 0.3 1.0 3.33 3 0.3 1.5 5.00

Table 24.3 Summary of the fermentation conditions used in Chapter 24

Growth Medium Parker Broth (+ 80 g L‐1 glucose) Production Medium Parker Broth (+ 40 g L‐1 soluble starch) Growth batch temperature 25oC Production batch temperature 25oC pH controlled to pH 5.5 (±0.05) Air flow Varied as required

199

24.3 Results

POPI production was clearly superior at an air sparging rate of 3.33 vvm (Fig. 24.1 and 24.2). Neither increasing the quantity of air supplied nor decreasing it by 0.5 L min‐1 made any significant difference to POPI production. At 1.67 vvm the beads were poorly stirred by the air flow while 5 vvm was the maximum rate that could be effectively used because the high turbulence caused by air exiting the culture deposited beads on the upper walls of the bioreactor, and the large throughput of air resulted in high loss of water from the fermentation.

1000

800 ) -1

600

POPI titre (PIU L (PIU POPI titre 400

200

0 012345678

Days

Fig. 24.1 POPI production by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor under different rates of sparging with air ( ) 1.67 vvm [0.5 L min‐1]; () 3.33 vvm [1.0 L min‐1]; () 5.0 vvm [1.5 L min‐1]

200

1000 )

-1 800

600

400 POPI titre (PIU L (PIU POPI titre

200

0 1.67 3.33 5 Air sparging rate (vvm)

Fig. 24.2 The maximum POPI titres in bubble column fermentations with immobilized Fusarium sp. IMI397470 under different rates of sparging with air

24.4 Discussion

The optimal air sparging rate for POPI production by Fusarium sp. IMI397470 is at the higher end of rates previously reported in studies by others. The higher optimum sparging rate for POPI production reflects a higher oxygen/stirring demand than the fermentations in those other reports. This is despite the fact that POPI production in this process is via pre‐grown mycelia. Some studies on the effect of sparging rate reported a proportional response to its increase. For instance, Manolov (1992) showed that when sparged at 0.17, 0.5 to 1 vvm, ribonuclease from immobilized Aspergillus clavatus was best at the highest rate of air supply. This was not the finding in this POPI study. When sparging was increased beyond 3.33 to 5.0 vvm POPI yields became significantly poorer. This situation is 201 similarly to that reported by Devi et al. (2007) in the production of urokinase by immobilized a kidney cell line. In the range 0.5 – 3.0 vvm in an airlift bioreactor, optimum production was found at 2.0 vvm. In free cells a similar response to sparging rate was reported for the growth of Porphyra haitanensis (Zhang et al., 2006) where specific growth increased with sparging until 1.2 vvm after which it decreased. In the case of alginate immobilized Fusarium sp. IMI397470 a probable explanation for the poorer yield at the highest sparging rate used could be one previously advanced in the discussion of results in Chapter 15. This explanation says that in an environment of high mixing as would be occurring at 5 vvm, bead to bead abrasion (Kuek and Armitage, 1985) disrupts the fungal mycelium established in the proximity of bead surfaces (see Fig. 11.4, Chapter 11) and thus their productive capacity. The finding in this study suggested no change to the standard sparging rate used to date in this work and 3.33 vvm was confirmed as the operating sparging rate for the POPI process.

24.5 References

Branco, R.F.; Santos, J.C.; Murakami, L.Y.; Mussatto, S.I.; Dragone, G. and Silva, S.S. (2007) Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration. Process biochemistry 43: 258 ‐ 262. de Oliveira, L.P.; Rossi, M.J.; Furigo, A.; Silva G.N. and de Oliveira V.L (2006) Viability and infectivity of an ectomycorrhizal inoculum produced in an airliftbioreactor and immobilized in calcium alginate. Brazillian Journal of Microbiology 37: 251 ‐ 255.

Devi, R.B.; Kunamneni, A.; Prabhakar, T.; Plou, J.F. and Ellaiah, P. (2007) Continuous production of urokinase by alginate‐immobilized cells of a mutated MPGN kidney cell line in an airlift bioreactor. Communicating Current Research and Educational Topics and Trends in Applied Microbiology, A. Méndez‐Vilas (Ed.) 263 ‐ 270.

202

Kuek, C. (1991) Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads. Applied Microbiology and Biotechnology 35: 466 ‐ 470.

Kuek, C. and Armitage, T.M. (1985) Scanning electron microscopic examination of calcium alginate beads immobilizing growing mycelia of Aspergillus phoenicus. Enzyme Microbial and Technology 7: 121 ‐ 125.

Kunamneni, A.; Prabhakar, T.; Jyothi, B. and Ellaiah, P. (2007) Investigation of continuous cyclodextrin glucanotransferase production by the alginate‐immobilized cells of alkalophilic Bacillus sp in an airlift reactor. Enzyme and Microbial Technology 40: 1538 ‐ 1542.

Lin, T.J. and Lee, Y.C. (2008) High‐content fructooligosaccharides production using two immobilized microorganisms in an internal‐loop airlift bioreactor. Journal of the Chinese Institute of Chemical Engineers 39: 211 ‐ 217.

Lopez, E.; Delve, F.J.; Longo, M.A. and Sanroman, M.A. (2008) Lipolytic enzyme production by immobilized Rhizopus oryzae. Chemical Engineering Technology 31: 1555 ‐ 1560.

Manolov, R. J. (1992) Batch and continuous ribonuclease production by immobilized Asperyillus clavatus cells in a bubble‐column bioreactor. Applied Microbiology and Biotechnology 37: 32 ‐ 36.

Zhang, W.; Gao, J.T.; Zhang, Y.C. and Song Qin, S. (2006) Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble‐column bioreactor. World Journal of Microbiology and Biotechnology 22: 655 ‐ 660.

203

CHAPTER 25

Sustained production of prolyl oligopeptidase inhibitor by immobilized Fusarium sp. IMI397470 in a bubble column bioreactor

25.1 Introduction

The aim of this study was to combine all the conditions found optimal in previous studies and test the potential of the process developed for sequential repeat batch cultures in the manner reported in similar studies with other microorganisms immobilized in hydrogel (Table 25.1).

Table 25.1 Examples of sustained repeat batch production of metabolites by hydrogel immobilized microorganisms Microorganism No. of Days in Reference repeat production batches Rhizopus oryzae 4 20 López et al. (2008) Aspergillus clavatus 4 35 Manolov (1992) Aspergillus phoenicus 5 19 Kuek (1991) Bacillus subtilis 5 15 Shih et al. (2010) Bacillus halodurans 9 9 Shrinivas and Kumar (2012) Streptomyces marinensis 9 36 Srinivasulu et al. (2003) Monascus purpureus 9 55 Fenice et al. (2000) Bacillus licheniformis 13 19 Mashhadi‐Karim et al. (2011)

204

25.2 Materials and methods

All protocols used were as described in Section 21.2, Chapter 21. The fermentation conditions were as summarized in Table 25.2.

Table 25.2 Summary of the fermentation conditions used in Chapter 25

25.3 Results

The production of POPI by immobilized Fusarium sp. IMI397470 was sustained over 21 days over 5 sequential batch runs (excluding the growth batch and the acclimation batch which followed immediately after). The maximum POPI titre increased over the production batches (Fig. 25.1). It was about twice the titre of the initial batch by the Batch 5. The time to POPI peak shortened as batch number increased (ca. 4 days becoming 2 days).

205

Production Production Production Production Production 2000 Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 7

1800

6 1600

1400 5 )

-1 1200 4

1000 pH 3 800 POPI titre (PIU L (PIU titre POPI 600 2

400

1 200

0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Days Fig. 25.1 POPI production by immobilized Fusarium sp. IMI397470 over 5 sequential production batches

25.4 Discussion

Fusarium sp. IMI397470 immobilized in calcium alginate can sustain POPI output in a bubble column for as long as 5 batches if not more, over 21 production days (not counting growth and acclimation time). The study was ended after having established sufficient repeatability and as the maximum POPI titre was still rising at Batch 5, further batches were likely to have been successfully run. The sustainability of POPI production compares well with the lower range reported in other similar studies in which batch repeats numbered between 4 and 13 over production periods ranging from 9 to 55 days (Table 25.1).

206

Mycelia of Fusarium sp. IMI397470 immobilized in calcium alginate beads became more productive for POPI as batch number increased. While this may indicate further acclimation to production conditions, it may also be due to the indirect effect of other changes. For example, bead size may have been reduced through bead to bead abrasion through time. Unfortunately, bead size at the start of the batch run and after the last batch was not measured although it was observed that the beads appeared smaller after 5 batches. As bead size reduces, it would be expected that mass and gas transfer to mycelia entrapped within will be improved through an improving surface area to volume ratio for each bead. This improvement may be the basis for POPI yield improving through the batches. López et al. (2008) also found that the yield of lipolytic enzyme from calcium alginate immobilized Rhizopus oryzae also increased with batch number. Similar increases with batch number were also reported by Mashhadi‐Karim et al. (2011) and Shrinivas and Kumar (2012) although in both cases yield dropped in the last batch. On the other hand in other similar studies yield was reported to decrease over batch number (Fenice et al., 2000; Srinivasulu et al., 2003). In the latter study, bead disintegration as reported was probably a cause for decreasing yield. Occurring with an increase in POPI yield over batch number was a shortening of the time to POPI peak. The speculations previously made on why there was an improvement in POPI yield are also extended to this observation. Kim et al. (2006) working with the production of gibberellic acid by celite immobilized Gibberella fujikoroi also found that fermentation time decreased over the number of batches repeated (over 11 batches in a 70 day period) as did yield of the acid.

25.5 References

Fenice, M.; Federici, F.; Selbmann, L. and Petruccioli, M. (2000) Repeated‐batch production of pigments by immobilised Monascus purpureus. Journal of Biotechnology 80: 271 ‐ 276.

207

López, E.; Deive, F.L.; Longo, M.A. and Sanromán M.Á. (2008) Lipolytic Enzyme production by immobilized Rhizopus oryzae. Chemistry Engineering and Technology. 31: 1555 ‐ 1560.

Kim, C.J.; Lee, S.J.; Chang, Y.K.; Chun, G.T.; Jeong, Y.H. and Kim, S.B. (2006) Repeated‐batch culture of immobilized Gibberella fujikoroi B9 for gibberellic acid production: An optimization study. Biotechnology and Bioprocess Engineering 11: 544 ‐ 549.

Manolov, R.J. (1992) Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble‐column bioreactor. Applied Microbiology and Biotechnology 37: 32 ‐ 36.

Mashhadi‐Karim, M.; Azin, M.; Gargari, S.L.M. (2011) Production of alkaline protease by entrapped Bacillus licheniformis cells in repeated batch process. Journal of Microbiology and Biotechnology 21: 1250 ‐ 1256.

Shih, I.L.; Chen, L.D. and Wu, J.Y. (2010) Levan production using Bacillus subtilis natto cells immobilized on alginate. Carbohydrtae Polymers 82: 111 ‐ 117.

Shrinivas, D. and Kumar, R. (2012) Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. Journal of Industrial Microbiology and Biotechnology 39: 93 ‐ 98.

Srinivasulu, B.; Kunamneni, A. and Ellaiah, P. (2003) Investigations on neomycin production with immobilized cells of Streptomyces marinensis Nuv‐5 in calcium alginate matrix. AAPS Pharmacology Science and Technology 4: 449 ‐ 454.

208

CHAPTER 26

General discussion

This general discussion is to bring together the discussions of results in the preceding individual chapters each with their specific aims. The overall objective of the work described in this thesis was to develop fermentation methods for the production of POPI by Fusarium sp. IMI397470. This general discussion is themed around the three component objectives of the overall aim.

26.1 Characterization of the production of POPI by Fusarium sp. IMI 397470 in terms of biomass growth and metabolite production

The elucidation of the production pattern for a metabolite is important in the development of fermentation processes because the pattern may suggest a particular route of exploitation. For metabolites which are associated with the active growth of cells (primary metabolites), an optimal fermentation process would feature rapid growth rates in short batches or continuous culture at high dilution rates, both of which allow high volumetric productivity. On the other hand continuous culture is less suitable for secondary metabolites because growth has to be limited whereas in a continuous culture some rate is required to replace biomass lost through washout. A process where growth is limited will be more suitable for secondary metabolites. Thus, knowledge of the production pattern of a metabolite is of key importance in fermentation development. Even at the beginning of this work when the relationship between growth of Fusarium sp. IMI397470 and POPI production was examined by simple juxtaposition of curves for the two, the classical appearance of a growth‐dissociated pattern for the production of POPI was apparent (Fig. 4.2, Chapter 4) ‐ there was a lag in POPI production 209 with respect to biomass accumulation. When later the matter was determined quantitatively in terms of Specific Growth Rate for Fusarium sp. IMI397470 () and the

Specific Rate of POPI Production (Qp), the data distinctly showed that max occurred earlier than Qpmax (Fig. 7.2, Chapter 7), indicating that maximum production of POPI occurs after growth rate of the fungus slows down (or conversely that it is produced at low rates when growth rates are high). The production of POPI by Fusarium sp. IMI397470 is not associated with growth. The production of POPI is maximal in the idiophase of the fungus in liquid culture. By classical definition in terms of max and Qpmax, it is likely that there will be general agreement that POPI is a secondary metabolite. The industrial exploitation of this knowledge lies in a fermentation process that maintains the physiology of pre‐grown mycelia of Fusarium sp. IMI397470 while the latter is re‐used in repeat batches from which POPI is recovered from each batch. The concept of this approach was confirmed in studies of the production of POPI using immobilized Fusarium sp. IMI397470 (Section 26.3).

26.2 Determination of the physicochemical cultural conditions for Fusarium sp. IMI 397470 and its production of POPI in submerged aerobic culture (shake flask and bioreactor).

The first submerged aerobic cultures of Fusarium sp. IMI397470 employed Potato Dextrose Broth (PDB) because the fungus was isolated on Potato Dextrose Agar. Indeed, PDB proved to be a good medium for the culture of the fungus with good biomass yield obtained. However, it was clearly demonstrated that PDB is a poor medium for POPI production and that the semi‐defined Parker Broth which comprised mineral salts and either glucose or starch (10% w/v) was a better medium for POPI production by at least 2‐ fold (Fig. 4.4, Chapter 4). This gave a clue that PDB may lack certain mineral salts and that manipulation of the carbon source may influence POPI outcomes. Incubation temperature was found to be an important factor in the production of POPI by free mycelia of Fusarium

210 sp. IMI397470. Incubation at 30oC resulted in a maximum POPI titre which was 2.5 times better than at the baseline temperature of 25oC (Fig. 9.2, Chapter 9). This simple change in a physical process parameter was much more effective than a chemical change (glucose concentration) in improving POPI titre (Table 26.1). The effectiveness of temperature on

POPI production is reinforced by the fact that the Yp/x (POPI yield/cell weight) associated with the maximum POPI titre effected by optimum temperature to be more than twice that of that associated with the maximum POPI titre effected by glucose optimum (Table 26.1) i.e. the cells at the temperature optimum were better at producing POPI than those at the glucose optimum. With a fermentation process where immobilized mycelia are used, the study of physicochemical conditions of culture more directly on POPI production without the confounding effect of growth is possible. In such a process, the immobilized mycelia are grown in a separate phase and the pre‐grown mycelia are then re‐used in a subsequent phase where the conditions can be adjusted to suit POPI production. Thus, when a range of carbon substrates ranging from mono‐, di‐ and polysaccharides in Parker Broth were compared for their effectiveness in the production of POPI by pre‐grown immobilized Fusarium sp. IMI397470, soluble starch was found to be clearly better than glucose by 1.5‐ fold and better still than sucrose, maltose and lactose (Fig. 16.1). The polymeric form of glucose appears to be best for POPI production and this is speculated to be possibly due to less catabolite repression than may be exerted by monomeric glucose. However, glucose still has an important role in that it was found to be a better substrate in the biomass phase of the POPI production process i.e. during the production of immobilized mycelia, the fermentation should contain glucose rather than soluble starch but in subsequent repeat batches in the POPI production phase, soluble starch should be used as this strategy yielded higher titres of POPI (Fig. 17.1 and 17.2, Chapter 17). It would appear that having a substrate which is relatively easier to metabolize in the growth phase of the process is more advantageous than lessening the requirement of acclimation to a different substrate when switching between glucose and soluble starch when the process is moved into the POPI production phase.

211

Table 26.1 Manipulation of physicochemical conditions which improved POPI yield from fermentation with free mycelia of Fusarium sp. IMI397470 Physicochemical Max. POPI Productivity Chapter Best condition Biomass yield Yield coefficient, Yp/x culture variable titre (PIU L‐1 day‐1) indicated (g L‐1 DW) (POPI yield/dry cell weight) examined (PIU L‐1) Baseline initial Potato Dextrose 119.5 6.80 17.5 19.9 * observation Broth Glucose concentration 10 g L‐1 821.8 4.00 205.4 137.0 8 (5, 10 and 20 g L‐1) Temperature 30oC 2060.3 3.64 566.0 515.1 9 (25, 30 and 35oC) *This culture run was conducted as an additional study because the original PDB run at the beginning of this work (Chapter 4) was performed using an assay which reported in % inhibition. The PIU assay developed in this work was used for this run.

213

The distinct effect of incubation temperature on POPI production seen with free mycelia was not seen when mycelia were immobilized in hydrogel because incubation at 25o and 30oC resulted in similar POPI titres (Fig. 14.1; Chapter 14). It was a disappointment not to have an equivalent improvement in POPI yield through an increase in incubation temperature but it would appear that the differences in the environments of free and immobilized mycelia restrict a positive response in POPI output to temperature by immobilized cells. POPI production by immobilized mycelia appears to require relatively much lower N than C because it was found to occur best with a C:N of 190 (Fig. 23.2). This finding supports the contention made in this work that POPI production is growth‐dissociated because if it were growth‐associated, the lower C:N ratio of 95:1 would be preferred since more N would be available for assimilation into materials required for growth. The influence of gas transfer on POPI production by immobilized cells when studied via shake flask cultures is induced from the effect of shaking speed. As would be predictable for aerobic culture certain rate of gas transfer is required for good POPI productivity as shaking below 200 rpm does not give enough agitation (Fig. 15.2, Chapter 15). However, it appears that shaking can be in excess as well because POPI production at 300 rpm was also found to be poorer than at 200 rpm. A possible reason for this is that agitation about a certain threshold causes too much disruption of the fungal mycelia at the proximity of bead surfaces. Phase separated repeat batch culture enabled the examination of the effect of controlled culture pH on POPI production by pre‐grown immobilized mycelia. In a range of pH values tested, 5.5 was found to be optimal (Fig. 21.1, Chapter 21). This value was indicated in earlier studies with batch cultures where pH was uncontrolled because POPI peaks were seen to occur at times when culture pH was within range of this value especially when abundant carbon substrate was supplied (Production Batch 2, Fig. 11.7; Production Batch 2, Fig. 11.8, Chapter 11). A medium manipulation that did not improve POPI yield by immobilized cells was supplementation with Tween 80. This surfactant was effective in increasing titres of

214 various metabolites from different microorganisms but proved ineffective with Fusarium sp. IMI397470. This leads to the inference that POPI production is not rate‐limited by membrane permeability or membrane‐associated enzyme functions. In terms of improving POPI fermentation, through physicochemical manipulations in this work, POPI production using free mycelia of Fusarium sp. IMI397470 was improved 17‐fold in terms of the maximum POPI titre obtained and 25‐fold in terms of POPI productivity (Table 26.1). With immobilized mycelia, improvement in terms of maximum POPI titre obtained was 9‐fold and 3.7‐fold in terms of POPI productivity (Table 26.2). Judicious choice of some of the values set for the culture variables at the outset e.g. for C:N ratio (190), shaking speed (200 rpm), bead:medium volume ratio (1:1); bead size (2.8 mm), and air sparging rate(3.33 vvm) meant that the process was already near to optimal even as cultural manipulations were attempted.

26.3 Examination of cell immobilization as a means of the repeat batch production of POPI

With POPI established as having a growth‐dissociated pattern of production, it was appropriate to develop a repeat batch culture process where the first part of the process was for the production of immobilized mycelia and the second part the repeated use of that biomass for the sustained production of POPI. Mycelia of Fusarium sp. IMI397470 grew well within hydrogel beads which were used to immobilized them (Fig. 13.2, Chapter 13) especially when supplied the amount of glucose used as standard from Chapter 12 onwards (80 g L‐1 in the medium, or 40 g L‐1 after allowing for dilution after addition to an equal volume of beads). The immobilized mycelia could be used in repeated production batches to produce POPI. The best bead diameter in the range tested was 2.8 mm as going smaller gave no advantage as may be predicted by a superior bead surface to volume ratio and going larger resulted in lower yield (Fig. 18.2, Chapter 18).

215

Table 26.2 Manipulation of physicochemical conditions which improved POPI yield from fermentation with immobilized mycelia of Fusarium sp. IMI397470

POPI yield Productivity Physicochemical culture variable examined Best condition indicated Chapter (PIU L‐1) (PIU L‐1 day‐1)

Baseline initial observation 20 g L‐1 in growth phase; 129.5 64.8 11 20 g L‐1 in production phase

Glucose concentration in production phase 120 g L‐1 618.5 103.1 11 (20, 40, 80, and 120 g L‐1)

Same glucose concentration in growth and production 80 g L‐1 in growth and 957.9 119.7 12 phases production phase (40/40, 80/80, and 120/120 g L‐1)

Type of carbon substrate in production phase (at 80 g L‐1) Soluble starch 1190.0 238.0 16 (lactose, maltose, sucrose, glucose, and soluble starch) Quantity of soluble starch in production phase 80 g L‐1 soluble starch 1190.0 238.0 17 (20, 40, and 80 g L‐1) (40 g L‐1 soluble starch) (821.68) (273.89)

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In the bubble column, a bead to medium volume ratio of 1:1 was better than having either less or more medium compared to bead volume (Fig. 22.1, Chapter 22). Suspension of beads in the bioreactor was difficult the ratio was less than 1 because there was insufficient fluidization. This was also an issue with the examination of the optimal rate of air flow to employ. At a rate lower than the optimal of 3.33 vvm, beads were poorly fluidized in the bioreactor. If a high rate of 5 vvm is used, POPI yield will be poor and significant amounts of water will be lost from the ferment through entrapment in the air stream or evaporation. The various studies in developing the process for the production of POPI using immobilized mycelia of Fusarium sp. IMI397470 elucidated the bubble column operating conditions shown in Fig. 26.1.

Growth Phase Production Phase

Parker Broth Parker Broth (+80 g L-1 glucose) (+40 g L-1 soluble starch

Conditions to produce immobilized mycelia Conditions to o 25 C; produce POPI Air at 3.33 vvm; Bioreaction in 25oC; pH uncontrolled; bubble column Air at 3.33 vvm; Repeated Bead size = 2.8 mm pH 5.5; as required Bead:medium ratio = 1 Bead = 2.8 mm Spent liquor Spent liquor Bead:medium ratio = 1; C:N ratio = 190

Waste Product recovery

Fig. 26.1 The operating conditions identified in this work for the production of POPI using immobilized mycelia of Fusarium sp. IMI397470 in phase separated repeat batch culture in a bubble column bioreactor

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POPI production in the system developed could be sustained for at least 5 repeat batches (Fig. 25.1, Chapter 25). More production batches could probably have been supported as the mycelia were producing more POPI as the batch number increased. This showed that they were more than sufficiently viable in terms of POPI synthesis not only in terms of the maximum POPI titre obtained but also in POPI productivity. The latter was increased with batch number indicating that culture times to POPI peaks were shortening (Table 26.3). Thus, cell immobilization is demonstrated to be a good means of exploiting the growth‐dissociated pattern of POPI production in Fusarium sp. IMI397470 via a process where the growth and metabolite production phases are separated and where the latter is operated repeatedly to enable multiple batches from which the metabolite may be recovered.

Table 26.3 Yield and productivity of POPI in production from immobilized Fusarium sp. IMI397470 via repeat batch culture in a bubble column bioreactor (Chapter 25) Batch No. Maximum POPI yield Productivity (PIU L‐1) (PIU L‐1 day‐1) 1 841 168 2 1021 340 3 1088 362 4 1376 458 5 1814 907 Fermentation conditions: 25oC for both growth and production phases; Parker Broth (+80 g L‐1 glucose) in growth phase; Parker Broth (+40 g L‐1 soluble starch) in production phase; pH 5.5 in production phase; bead:medium ratio = 1; air at 3.33 vvm.

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26.4 Conclusions and suggestions for future work

In submerged aerobic culture, the production of POPI by Fusarium sp. IMI397470 is growth‐dissociated and produced in highest quantities in the idiophase of growth. A two‐ phase production process where immobilized mycelia of the fungus was grown and then re‐used in a repeated batch optimized for POPI production proved appropriate to exploit the production pattern determined for POPI. Culture of the fungus and its production of POPI were both responsive to physicochemical manipulations of culture. The advantage of the repeat batch two‐phase production process is the ability to re‐use the same biomass repeatedly and the higher productivity of POPI (shorter fermentation times to peak tire). Future work to extend this study could include further optimization of the bubble column physicochemical conditions for POPI production e.g. should the incubation temperatures for the growth and POPI production phases be the same? Besides fermentation studies, fractionation to purify POPI and elucidation of its structure and properties would need to be undertaken to further understand its potential as a compound for attenuating oligopeptidase activity in mammalian systems as part of the early steps in discovering a drug for ameliorating neurological disorders.

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