Fumaric Acid Fermentation by Rhizopus oryzae with Integrated Separation Technologies

Dissertation

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

By

Kun Zhang, M.S., B.S.

Graduate Program in Chemical and Biomolecular Engineering

The Ohio State University

2012

Dissertation Committee:

Shang-Tian Yang, Advisor

Jeffrey J. Chalmers

David Wood

Copyright by

Kun Zhang

2012

ABSTRACT

Fumaric acid is a dicarboxylic acid and an indispensible intermediate in the metabolism of many microorganisms. It is used mainly as a food acidulant and as the chemical feedstock for the production of paper resins, unsaturated polyester resins, alkyd resins, plasticizers, and miscellaneous industrial products. Currently, fumaric acid is produced through a petroleum-based chemical method. However, limited deposits of petroleum, increasing production costs and serious environmental repercussions have prompted chemical companies to find an alternative. Fungal fermentation provides a promising solution due to its advantages in environmental friendliness and availability of widespread renewable biomass feedstock.

However, conventional fumaric acid fermentation processes suffer from low product yield, low productivity, and high production cost, whereas the competing chemical production methods are currently more economical. Therefore, the goal of this research was to develop effective and economic fermentation and recovery methods for improved fumaric acid production by Rhizopus oryzae. One of the major technical challenges is to control the fungal morphology and physiology for the overproduction of fumaric acid in a sustainable and scalable way. First, an effective fermentation process with good morphology control was developed using soybean meal hydrolysate as the nitrogen source for cell growth and fumaric acid production by R. oryzae. To improve the

iii productivity, in situ product recovery by coupling the fermentation with an ion exchange column for fumaric acid adsorption was investigated. In addition, an alternative separation process using adsorption with activated carbon followed with desorption and crystallization with acetone was developed to increase the recovery yield and reduce the production cost. The main results and conclusions are summarized below.

First, a simple seed culture medium was developed with soybean meal hydrolysate

(SMH) as the nitrogen source for controlling the morphology of R. oryzae ATCC 20344.

Uniformly dispersed mycelial clumps with a diameter of ~0.1 mm were formed with enhanced subsequent fumaric acid production. The optimal seed culture conditions were initial pH of 3.0, SMH concentration of 30% (v/v), spore concentration of 8×104/mL and glucose concentration of 10 g/L. With an inoculation size of 25%, the fermentation reached a fumaric acid titer of 50.2 g/L with yield of 0.72 g/g glucose. SMH with a high protein content was a good nitrogen source and the formation of protein precipitate acted as the immobilization carriers for cells. The solid-phase protein also provided a novel method for slow/controlled release that allowed the utilization of the nitrogen source by cells for an extended period without losing cell activity.

The fermentation was then studied in a 5-L stirred tank bioreactor (STB) and the results also showed that using SMH as the nitrogen source improved fumaric acid production with increased yield and productivity compared to urea and yeast extract. The effect of CO2 on fumaric acid fermentation was studied. Aeration with air containing 16.7%

CO2 increased the productivity by 76% and final product titer by 13%. A product titer of

35.6 g/L, yield of 40% and productivity of 0.4 g/L∙h were obtained in batch fermentation with an initial seeding density of ~5000 mycelial clumps/ml. With CO2 addition, similar

iv fumaric acid production in the fermentation was obtained with either CaCO3 or NaOH as the neutralizing agent, indicating that CaCO3 can be replaced with NaOH and CO2 to simplify the downstream process.

Separation of fumaric acid from the fermentation broth by adsorption was studied with a strong-basic anion exchange resin IRA900, which was selected because of its high adsorption capacity at the fermentation-favored pH of 5, high selectivity against byproducts (glucose and malic acid) and easy desorption with a high recovery yield. The adsorption isotherm and the mechanism involved in the fumaric acid adsorption onto

IRA900 were investigated. The results showed that fumaric acid adsorption onto IRA900 followed the Langmuir isotherm. Also, the adsorption reaction rate between fumaric acid and functional groups of the resin followed the second-order kinetics, indicating a mechanism of two-site-occupancy adsorption reaction between FA2- and resin active sites.

However, such a mechanism cannot explain the high adsorption capacity at pH 3 with a high initial fumaric acid concentration. Using an intraparticle diffusion model, it was found that the higher adsorption at pH 3 could be attributed to the higher hydrophobic interaction between neutral fumaric acid molecules and the hydrophobic resin backbone.

The separation process using IRA900 ion exchange resin was then scaled up in a fixed bed column with the optimal conditions identified as: the resin in the chloride form, flow rate of 4.10~5.34 ml/min, initial fumaric acid concentration of ~5.0 g/L, and desorption solution of 0.7 mol/L NaCl. It was also found that end product inhibition occurred when the fumaric acid concentration in the medium reached >20 g/L. Therefore, intermittent in situ recovery was used to alleviate the product inhibition and facilitate the subsequent

v product separation. A preliminary study showed that fermentation with in situ recovery enhanced product yield by 25% and productivity by 59%.

Fermentation produced fumaric acid is usually recovered by precipitation and crystallization of the acid at room temperature. In order to recover the low-concentration fumaric acid (<6.3 g/L) present in the filtrate after crystallization, an integrated separation process was developed for fumaric acid recovery and purification by adsorption with activated carbon followed with desorption and crystallization with acetone. Fumaric acid adsorption on activated carbon was found to be governed by non-electrostatic interactions between the undissociated fumaric acid and the activated carbon. The desorption of fumaric acid from activated carbon was studied using acetone to strip all fumaric acid from activated carbon. After removing acetone by evaporation at 70 oC, fumaric acid crystals were obtained with a high purity and recovery yield. Both activated carbon and acetone can be recovered and reused in the adsorption process. The adsorption and desorption process was then evaluated in a fixed bed column to recover fumaric acid from the fermentation broth, achieving a high recovery yield of 93%. Finally, water sweeping was used to further increase the purity of fumaric acid crystals to >98%. A comparative economic analysis showed that this process could significantly reduce the operational costs and enhance the recovery yield, although it would require additional capital investment. Compared to the conventional recovery process using precipitation, the new process is economically and environmentally favorable with good potential for industrial application.

vi

DEDICATION

Dedicated to my wife and parents

vii

ACKNOWLEDGEMENTS

First, I want to give my special thanks to my advisor, Professor Shang-Tian Yang.

Not only his guidance and support throughout my research enabled me to develop a thorough understanding of the subject, but also his patience, understanding and forgiveness taught me how to be a fantastic advisor. It would not have been possible to complete my Ph.D. degree without his invaluable guidance and mentoring.

I would like to thank Dr. Jeffrey Chalmers and Dr. David Wood for their time being on my committee and their valuable advices on my research project.

I want to give my sincere thanks to all the previous and current laboratory members in our group, especially Dr. Baohua Zhang, Dr. Xiang Zou, Dr. Lijie Zhang for their helpful suggestions and support. Also, I want to thank Ms. Yu Chen for her good job in helping some of my experiments.

Financially supports from the United Soybean Board and the Consortium for Plant

Biotechnology Research Inc to this project are also acknowledged.

Finally, I would like to thank my parents and my parents-in-law for all their support.

Special thanks to my wife Qing Zhang as well as our beloved two babies. Without their support, I would have achieved nothing.

viii

VITA November 1982 ………………………………………………Born-Qingdao, PR China June 2005 ………………………………………………………… B.S. Bioengineering Nanjing University of Technology, Nanjing, PR China June 2008 …………………………………………………………… M.S. Microbiology Nanjing University of Technology, Nanjing, PR China

PUBLICATIONS 1. Yang, S.T., Zhang, K., Zhang, B., Huang, H. 2011. Bio-Based Chemicals | Fumaric Acid. In: Murray Moo-Young (ed.), Comprehensive Biotechnology, Second Edition, Volume3, pp.163-177. Elsevier.

2. Zhang, K., Gao, Z., Li, S., Fu, Y., Huang, H., Wei, P., Ji, S. 2008. Multiple target optimizations on the fumaric acid production by Rhizopus oryzae based on Desirability function, China Biotech 28(4): 59-64.

3. Zhang, K., Xu, Q., Li, S., Fu, Y., Gao, Z., Huang, H., 2008. Integrated mutagenesis to screen high fumaric acid producing mutant of Rhizopus oryzae with propylene alcohol resistance. J Anhui Agri Sci 36(23): 9823-9825.

FIELDS OF STUDY

Major Field: Chemical and Biomolecular Engineering

ix

TABLE OF CONTENTS

ABSTRACT ...... iii

DEDICATION ...... vii

ACKNOWLEDGEMENTS ...... viii

VITA ...... ix

PUBLICATIONS ...... ix

FIELDS OF STUDY...... ix

LIST OF TABLES ...... xvi

LIST OF FIGURES ...... xviii

CHAPTER 1 ...... 1

Introduction ...... 1

1.1 Background ...... 1

1.2 Objectives ...... 4

1.3 Scope of study ...... 5

1.4 Reference ...... 7

CHAPTER 2 ...... 10

Literature Review...... 10

2.1 Fumaric acid properties and applications ...... 10

2.2 Fumaric acid production methods ...... 13

2.2.1 Isomerization of maleic acid to fumaric acid ...... 13

2.2.2 Fermentation of sugar to fumaric acid ...... 14

2.3 Fumaric acid fermentation microbiology...... 16

x 2.3.1 Fumaric acid producing microorganisms...... 16

2.3.2 Metabolic pathway for fumaric acid biosynthesis in Rhizopus ...... 18

2.4 Fermentation process development and optimization ...... 19

2.4.1 Cell immobilization and morphology control ...... 20

2.4.2 Medium formulation ...... 24

2.4.3 Effects of pH and neutralizing agent ...... 28

2.4.4 Effects of dissolved oxygen on cell growth and fumaric acid production30

2.4.5 Product inhibition...... 31

2.5 Product separation and purification ...... 32

2.6 Conclusion and future prospects ...... 36

2.7 Reference ...... 37

CHAPTER 3 ...... 57

Effects of Soybean Meal Hydrolysate as the Nitrogen Source on Seed Culture

Morphology and Fumaric Acid Production by Rhizopus oryzae ...... 57

3.1 Introduction ...... 57

3.2 Materials and Methods ...... 59

3.2.1 Cultures and meida ...... 59

3.2.2 Seed culture preparation ...... 60

3.2.3 Fermentation ...... 60

3.2.4 Analytical methods ...... 61

3.3 Results ...... 62

3.3.1 Morphology studies ...... 62

3.3.2 Effects of nitrogen sources in seed culture medium ...... 64

3.3.3 Effects of SMH concentration ...... 65

3.3.4 Effects of seed age on fumaric acid fermentation ...... 67

3.3.5 Effects of inoculation conditions on fumaric acid fermentation ...... 67

xi 3.4 Discussion ...... 68

3.4.1 Comparison of cell morphology to other studies ...... 68

3.4.2 Advantages of soybean meal hydrolysate ...... 70

3.4.3 Comparison with other studies ...... 71

3.5 Conclusion ...... 72

3.6 Reference ...... 73

CHAPTER 4 ...... 86

Optimization on Fumaric Acid Production by Rhizopus oryzae in 5-L Stirred-Tank

Bioreactor ...... 86

4.1 Introduction ...... 86

4.2 Materials and Methods ...... 87

4.2.1 Cultures and meida ...... 87

4.2.2 Seed culture preparation ...... 88

4.2.3 Fermentation ...... 88

4.2.4 Analytical methods ...... 89

4.3 Results ...... 90

4.3.1 Effects of nitrogen source ...... 90

4.3.2 Effects of CO2 addition ...... 91

4.3.3 Effects of cell density...... 93

4.4 Discussion ...... 94

4.4.1 Effects of SMH ...... 94

4.4.2 Effects of CO2 addition ...... 95

4.5 Conclusion ...... 96

4.6 Reference ...... 97

CHAPTER 5 ...... 109

xii Characteristics and Mechanism Study of Fumaric Acid Adsorption onto IRA900 Ion

Exchange Resin ...... 109

5.1 Introduction ...... 109

5.2 Materials and Methods ...... 110

5.2.1 Materials ...... 110

5.2.2 Adsorption experiments ...... 111

5.2.3 Desorption of fumaric acid from IRA900 ...... 112

5.2.4 Analytical methods ...... 112

5.3 Results and Discussion ...... 113

5.3.1 Resin screening ...... 113

5.3.2 Effects of pH and modeling ...... 113

5.3.3 Effect of pH under different initial concentrations ...... 116

5.3.4 Adsorption kinetics study under different pHs and mechanism study .. 117

5.3.5 Selectivity study ...... 123

5.3.6 Desorption process ...... 124

5.4 Conclusion ...... 124

5.5 Reference ...... 126

CHAPTER 6 ...... 140

Intermittent in situ Recovery of Fumaric Acid from Fermentation Broth by Using

IRA-900 Ion Exchange Resins...... 140

6.1 Introduction ...... 140

6.2 Materials and Methods ...... 141

6.2.1 Materials ...... 141

6.2.2 Adsorption and desorption on fixed bed column ...... 141

6.2.3 Fermentation process for end product inhibition study ...... 142

6.2.4 Intermittent in situ recovery ...... 143

xiii 6.2.5 Analytical methods ...... 144

6.3 Results and discussion ...... 145

6.3.1 Adsorption process on fixed bed column...... 145

6.3.2 Desorption process on fixed bed column ...... 148

6.3.3 End product inhibition ...... 149

6.3.4 Intermittent in situ recovery ...... 149

6.4 Conclusion ...... 150

6.5 Reference ...... 151

CHAPTER 7 ...... 163

Fumaric Acid Recovery and Purification from Fermentation Broth by Activated Carbon

Adsorption Followed with Desorption and Crystallization with Acetone ...... 163

7.1 Introduction ...... 163

7.2 Materials and Methods ...... 164

7.2.1 Adsorption of fumaric acid onto activated carbon ...... 164

7.2.2 Stationary desorption of fumaric acid from activated carbon via acetone

...... 165

7.2.3 Separation on fixed bed column ...... 165

7.2.4 Water sweeping ...... 166

7.2.5 Cost analysis ...... 167

7.2.6 Analytical methods ...... 168

7.3 Results and Discussion ...... 168

7.3.1 Fumaric acid adsorption by activated carbon ...... 168

7.3.2 Stationary desorption of fumaric acid by acetone ...... 173

7.3.3 Adsorption and desorption on fixed bed column ...... 174

7.3.4 Water sweeping ...... 175

7.3.5 Cost analysis ...... 176

xiv 7.4 Conclusion ...... 177

7.5 Reference ...... 178

CHAPTER 8 ...... 192

Conclusions and recommendations...... 192

8.1 Conclusions ...... 192

8.1.1 Effects of Soybean Meal Hydrolysate as the Nitrogen Source on Seed

Culture Morphology and Fumaric Acid Production by Rhizopus oryzae ...... 193

8.1.2 Optimization on Fumaric Acid Production by Rhizopus oryzae in 5-L

Stirred-Tank Bioreactor ...... 193

8.1.3 Characteristic and mechanism study of fumaric acid adsorption onto

IRA900 ion exchange resin ...... 194

8.1.4 Intermittent in situ Recovery of Fumaric Acid from Fermentation Broth

by Using IRA-900 Ion Exchange Resins ...... 195

8.1.5 Fumaric acid recovery and purification from fermentation broth by

activated carbon adsorption followed with desorption and crystallization with

acetone ...... 195

8.2 Recommendations ...... 196

8.2.1 Fermentation process development ...... 196

8.2.2 Improvement on in situ product recovery ...... 198

8.2.3 Improvement on fumaric acid recovery by activated carbon adsorption199

8.3 Reference: ...... 200

BIBLIOGRAPHY ...... 202

xv

LIST OF TABLES

Table 2.1 Comparison of fumaric acid production from various substrates and fermentor systems by Rhizopus spp...... 43

Table 2.2 Typical medium compositions used in seed culture preparation and fermentation for fumaric acid production from glucose by Rhizopus oryzae...... 44

Table 2.3 Separation methods for recovering organic acids from fermentation broth. 45

Table 3.1 Fumaric acid production from various morphologies by Rhizopus sp...... 75

Table 3.2 Summary on cell morphology cultured under different conditions ...... 76

Table 3.3 Effects of nitrogen source in seed culture media on cell morphology and subsequent fumaric acid fermentation ...... 77

Table 3.4 Effects of seed age on fumaric acid fermentation (inoculation size of 15%) 78

Table 3.5 Effects of inoculation size and cell density on fumaric acid fermentation ... 79

Table 4.1 Effects of different nitrogen sources on fumaric acid fermentation ...... 98

Table 4.2 Effects of CO2 addition on fumaric acid fermentation under different neutralizing agent (NaOH, CaCO3) ...... 99

Table 4.3 Effects of cell density on fumaric acid fermentation ...... 100

Table 5.1 Adsorption capacity for fumaric acid by different resins under different pHs

...... 128

Table 5.2 Model parameters obtained from fitting the experimental equilibrium data with isotherm model ...... 129

Table 5.3 Comparison of regression results for fumaric acid adsorption kinetics on

IRA900 resin by using different equations ...... 130

Table 5.4 Selectivity study of IRA900 resin adsorbing fumaric acid ...... 131 xvi Table 6.1 Results summary of fumaric acid adsorption on IRA-900 fixed bed column

...... 152

Table 6.2 Results summary of fumaric acid desorption from IRA-900 fixed bed column ...... 153

Table 6.3 Performance of intermittent in situ recovery with two cycles ...... 154

Table 7.1 Model parameters obtained from fitting the experimental equilibrium data with isotherm models...... 179

Table 7.2 Selectivity study of fumaric acid adsorption on activated carbon ...... 180

Table 7.3 Effects of temperature on fumaric acid desorption from 2g activated carbon by using 100 ml acetone ...... 181

Table 7.4 Effects of contacting time on fumaric acid desorption from 2g activated carbon by using 100 ml acetone at (47±1) oC ...... 182

Table 7.5 Comparison between traditional precipitation and newly-developed precipitation and adsorption process for fumaric acid recovery (initial conc. of 30 g/L) from 1 ton fermentation broth ...... 183

xvii

LIST OF FIGURES

Figure 1.1 Research objectives and scope of the study ...... 9

Figure 2.1 Molecular structure of fumaric acid ...... 46

Figure 2.2 Solubility of fumaric acid in water under different temperatures ...... 47

Figure 2.3 Current applications of fumaric acid ...... 48

Figure 2.4 A flow chart for fumaric acid production from maleic acid via isomerilization ...... 49

Figure 2.5 Flowsheet for fumaric acid production via fermentation ...... 50

Figure 2.6 Glucose metabolic pathways in R. oryzae ...... 51

Figure 2.7 Batch fermentation kinetics of Rhizopus oryzae in 250-ml shake-flask ..... 52

Figure 2.8 Different morphologies of R. oryzae in submerged culture ...... 53

Figure 2.9 Schematic diagram of a rotary biofilm contactor with adsorption columns for fumaric acid production and recovery ...... 54

Figure 2.10 Fed-batch fermentation for fumaric acid and ethanol production from glucose by Rhizopus oryzae in a rotating fibrous bed bioreactor (RFBB) ...... 55

Figure 2.11 Extractive fermentation process with immobilized cell fermentation coupled with hollow-fiber membrane contactors for continuous product recovery from the fermentation broth with simultaneous solvent regeneration ...... 56

Figure 3.1 Cell morphology of Rhizopus oryzae developed under different conditions

...... 80

Figure 3.2 Comparison of soybean meal hydrolysate ...... 81

Figure 3.3 Fermentation profiles of the seeds prepared by the three nitrogen sources 82

xviii Figure 3.4 Effects of SMH concentration and spore concentration on cell growth and subsequent fermentation process ...... 83

Figure 3.5 Glucose and dissolved protein profiles of the seed culture ...... 84

Figure 3.6 Kinetics of batch fermentation of glucose with soybean meal hydrolysate by

R. oryzae at pH 5.5 and 32 oC...... 85

Figure 4.1 Metabolic pathways in R. oryzae ...... 101

Figure 4.2 Fermentation kinetics of fumaric acid production by R. oryzae in 5-L stirred tank bioreactor with different nitrogen sources ...... 102

Figure 4.3 Fermentation kinetics with pH control by 200 g/L NaOH (without CO2) 105

Figure 4.4 Fermentation kinetics with pH control by 200 g/L NaOH (with CO2 addition) ...... 106

Figure 4.5 Fermentation kinetics with pH control by CaCO3 and CO2 addition ...... 107

Figure 4.6 Fermentation kinetics with different cell densities ...... 108

Figure 5.1 Isotherms of IRA900 adsorbing fumaric acid at pH 3, 4 and 5 ...... 132

Figure 5.2 Effects of pH on fumaric acid adsorption by IRA900 resin at fumaric acid initial concentration of 5 and 15 g/L ...... 133

Figure 5.3 Dissociation equilibrium of aqueous fumarate solution ...... 134

- 2- Figure 5.4 Mole ratio of three fumarate ion forms (H2FA, HFA , FA ) at different pHs

...... 135

Figure 5.5 Adsorption kinetics of fumaric acid on IRA900 resin at pH 3, 4, 5 ...... 136

Figure 5.6 Plot of the pseudo-second-order equation for the adsorption kinetics of fumaric acid on IRA900 resin at pH 3, 4, 5 ...... 137

Figure 5.7 Plot of the intraparticle diffusion equation for the adsorption kinetics of fumaric acid on IRA900 resin at pH 3, 4, 5 ...... 138

Figure 5.8 Desorption process by NaOH, NaCl and H2SO4 at different concentrations

...... 139

xix Figure 6.1 Apparatus of intermittent in situ recovery from fermentation by ion-exchange ...... 155

Figure 6.2 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption by IRA-900 resin column under two ion forms (OH- and Cl-) ...... 156

Figure 6.3 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption by IRA-900 resin column under different flow rates ...... 157

Figure 6.4 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption by IRA-900 resin column under different initial fumaric acid concentrations

...... 158

Figure 6.5 Stripping curves (A) and outlet pH profiles (B) of fumaric acid desorption from IRA-900 resin column by using NaCl under different concentrations ...... 159

Figure 6.6 Stripping curves (A) and outlet pH profiles (B) of fumaric acid desorption from IRA-900 resin column by using NaOH under different concentrations ...... 160

Figure 6.7 Product inhibition effect on fumaric acid production ...... 161

Figure 6.8 Fermentation kinetics with intermittent in situ fumaric acid recovery by ion exchange ...... 162

Figure 7.1 Fumaric acid solubility at different temperatures ...... 184

Figure 7.2 Adsorption isotherms of fumaric acid on activated carbon at different pHs

...... 185

Figure 7.3 Determination of pHpzc of activated carbon by the pH drift method ...... 186

Figure 7.4 Dissociation equilibrium of aqueous fumarate solution ...... 187

Figure 7.5 Effects of acetone volume on desorption of fumaric acid from 2g activated carbon ...... 188

Figure 7.6 fixed bed column separation of fumaric acid by activated carbon ...... 189

Figure 7.7 fixed bed column separation of fermentation broth by activated carbon .. 190

Figure 7.8 Effects of water sweeping on recovery yield and crystals purity ...... 191

xx CHAPTER 1

Introduction

1.1 Background

Fumaric acid is a dicarboxylic acid and an indispensible intermediate in the metabolism of many microorganisms. Its chemical structure with multiple functional groups allows it to be readily transformed into many other useful chemicals. Thus fumaric acid has been selected by the United States Department of Energy as one of the

“top 12” chemical building blocks that can potentially be produced from abundant renewable biomass (Yang, 2007). Currently, fumaric acid is used mainly as a food acidulant and as chemical feedstock for the production of paper resins, unsaturated polyester resins, alkyd resins, plasticizers, and miscellaneous industrial products (Roa

Engel et al., 2008). It is produced through chemical reactions of n-butene to maleic anhydride and then maleic acid, followed by isomerization (Roa Engel et al., 2008). The total production capacity of fumaric acid in the US is ~50 million pounds (23,000 tons) per year. Although the petroleum-based chemical method can reach a high production yield, increasing oil prices and accompanying pollution problems have prompted the chemical industry to search for environmentally benign, bio-based manufacturing processes using renewable feedstock for fumaric acid production. Compared to the chemical synthesis method, bio-production via fermentation provides the following

1 advantages: the fermentation process with mild conditions is environmentally benign with reduced the greenhouse gas emission and toxic wastes; using renewable biomass and agricultural residues as the feedstock makes the process sustainable and more economical; the bio-based product is green and can be safely used in foods and other consumer products. Several organic acids, including citric acid, gluconic acid and itaconic acid, are currently produced by fungal fermentation in industry (Magnuson and Lasure, 2004).

However, conventional fumaric acid fermentation processes suffer from low product yield, low productivity, and high production cost, whereas the competing chemical production methods are currently more economical.

The major technical challenge in developing a fermentation process for fumaric acid production is how to control the fungal morphology and physiology (metabolism) for the overproduction of fumaric acid in a sustainable and scalable way. Morphology control is important to the submerged fermentation by filamentous fungi because their complex morphologies often cause difficult fermentor operation and poor performance. During submerged fermentation, filamentous fungi can grow into various morphologies, including free mycelia, smooth and hairy pellets, small loose mycelial clumps, and large aggregated mycelia with high density (Paul et al., 1998; Papagianni, 2004). The best morphology for the production of a certain product varies among fungi. Several studies have been carried out on the morphology of Rhizopus oryzae, but few reported the effect of morphology on the subsequent fumaric acid production. Understanding the effects of various process parameters on cell morphology and subsequent fumaric acid production by R. oryzae facilitated the optimization of the fermentation process.

2 Another problem associated with traditional fumaric acid fermentation is low productivity. The reduced production rate happened at the later phase of the fermentation, which was probably resulted from the end product inhibition or declined cell activity

(Yang et al., 2011). In situ product recovery has been proven to effectively reduce end product inhibition and enhance the productivity of the process with wide applications in organic acid production (Ataei et al., 2008; Cao et al., 1996). Nevertheless, there are few reports on applying in situ product recovery to fumaric acid production. Cao et al. (1996) developed an integrated system using rotary biofilm contactor (RBC) coupled with ion exchange column for simultaneous fermentation and separation. High fumaric acid titer

(>75.5 g/L) and productivity (>3.78 g/(L∙h)) were achieved. However, the complexity of this system and the high cost of resin at the industrial scale limit its commercial development.

Minimizing the production costs is essential for the biotechnological process to be competitive with petrochemical production. More than 60% of the total production costs are derived from downstream processing including separation, concentration and purification of the product in the fermentation broth (Bechthold et al., 2008). In a typical fumaric acid batch fermentation process with CaCO3 as the neutralizing agent, due to the low solubility of both calcium fumarate (2.11% at 30 oC, w/v) and fumaric acid (0.7%, w/v), the harvested fermentation broth is first heated to 80 oC and acidified to pH 2.0 by

H2SO4 (Gangl et al., 1990). Fumaric acid is dissolved at this temperature with the formation of calcium sulfate as the precipitate out of solution. Then, fumaric acid crystals are recovered by cooling the filtrate. One problem of the process is that the use of CaCO3 produces large quantities of solid waste calcium compounds causing environmental

3 pollution (Fu et al., 2009). In order to develop an easier and cheaper downstream process, alternative neutralizing agents such as NaOH and Na2CO3 have been attempted due to the high solubility of sodium fumarate (Gangl et al., 1990; Zhou et al., 2002). However, the fumraic acid production yield was significantly reduced in these processes, which was possibly due to the lack of CO2 compared to the use of CaCO3. Another issue is that fumaric acid cannot be totally recovered by cooling and some remains in the filtrate (6.3% at 25 oC, w/v). Re-crystallization is required to completely recover the rest of the fumaric acid. The inorganic impurities in the fermentation broth also reduce the purity of the fumaric acid crystals. An alternative method is necessary to fully recover all the fumaric acid without compromising product purity.

1.2 Objectives

The goal of this research was to develop effective fermentation and recovery methods for fumaric acid production from glucose by R. oryzae. To be more specific, first, a novel and simple seed culture method was developed with better morphology control and enhanced subsequent fumaric acid production in shake-flask fermentation.

The fermentation process was then scaled up and optimized in a 5-L stirred tank bioreactor (STB). Second, an ion-exchange resin IRA900was identified and used for in situ recovery of fumaric acid from the fermentation broth. The adsorption isotherm and reation kinetics were studied to better understand the adsorption process. A novel intermittent in situ product recovery method was developed by coupling the fermentation with the adsorption column. Third, in order to find an alternative method to recover all the fumaric acid with enhanced purity, the separation process by using adsorption with

4 activated carbon followed by desorption and crystallization with acetone was investigated.

Cost analysis was carried out to compare the economics of the process with that of the traditional precipitation method. Figure 1.1 provides an overview of the research objectives, approaches, and scope of the study, which is described below.

1.3 Scope of study

Task 1: Fermentation process development, optimization and scale-up

Fumaric acid production by fermentation involves two phases: seed culture and acid production. Due to little addition of nitrogen source into the fermentation medium, the cell morphology is mainly formed during the seed culture phase. Thus, our first objective is to find an effective method for morphology control with enhanced subsequent fumaric acid production, which is discussed in Chapter 3. The effects on cell morphology and fumaric acid fermentation of several influencing factors, including nitrogen source, pH, and spore concentration, were investigated to optimize the fermentation conditions on the shake-flask level.

However, the process was difficult to scale up in STB: the fumaric acid production was relatively low with a significant decrease in production rate in the later phase of the fermentation. Therefore, adding different concentrations of nitrogen source was studied.

To further optimize the fermentation process, the strategy of using CO2-enriched air for aeration was investigated. The results are given in Chapter 4.

Task 2: Adsorption characteristics study and in situ product recovery

5 Ion-exchange adsorption has been used extensively and successfully for in situ recovery of carboxylic acids. In order to establish an in situ product recovery method for fumaric acid recovery from the fermentation process, first, a suitable resin was determined, which should have a high adsorption capacity at the fermentation-favored pH of 5 and a high selectivity and an easy desorption process with high recovery yield. The adsorption isotherm and reaction kinetics were also studied to elucidate the reaction mechanism involved in the adsorption process (See Chapter 5).

The adsorption and desorption process was then scaled up in a fixed bed column to identify the optimal running conditions for influencing factors, including the rein ion form, flow rate and initial fumaric acid concentration. End product inhibition was identified by adding different amounts of fumaric acid into the initial fermentation medium. The method for in situ product recovery was explored and a novel intermittent in situ recovery was proposed. The details are given in Chapter 6.

Task 3: Alternative purification method by activated carbon adsorption

One problem of the conventional crystallization method is that fumaric acid cannot be totally recovered by precipitation and there will be some fumaric acid remaining in the filtrate (6.3 g/L at 25 oC, w/v). Re-crystallization at a low temperature is necessary; however, the high inorganic impurities in the concentrated fermentation broth may reduce the purity of the fumaric acid crystals. In order to find an alternative method to recover all the fumaric acid with enhanced purity, an integrated separation process by using adsorption with activated carbon followed by desorption and crystallization with acetone was developed. The characteristics of fumaric acid adsorption on activated carbon

6 including pH effects on the adsorption isotherm and selectivity were first studied.

Desorption process with acetone was also studied in order to find the optimal condition for recovery. The separation process was scaled up in a fixed bed column. Water sweeping was applied to further enhance the purity of the final product. The newly developed process was simulated and cost analysis was performed using SuperPro

Designer to compare the economics of the process with that of the traditional precipitation method. The results are discussed in Chapter 7.

1.4 Reference

Ataei S.A., Vasheghani-Farahani E. 2008. In situ separation of lactic acid from fermentation broth using ion exchange resins. J Ind Microbiol Biotech, 35: 1229-1233.

Bechthold I., Bretz K., Kabasci S., Kopitzky R., Springer A. 2008 Succinic acid: a new platform chemical for biobased polymers from renewable resources. Chem Eng Technol, 31(5): 647–654.

Cao N.J., Du J.X., Gong C.S., Tsao G.T. 1996. Simultaneous production and recovery fumaric acid from immobilized Rhizopus oryzae with a rotary biofilm contactor adsorption column. Appl Environ Microbiol 62: 2926-2931.

Fu, Y., Chen, Y., Li, S., Huang, H. 2009. Fixed-bed adsorption study for fumaric acid removal from aqueous solutions by Amberlite IRA-400 resin. Chem Eng Technol 10: 1625-1629.

Gangl, I.C., Weigand, W.A., Keller, F.A. 1990. Economic comparison of calcium fumarate and sodium fumarate production by Rhizopus arrhizus. Appl Biochem Biotechnol. 24-25: 663-677.

Magnuson J.K. and Lasure L.L. 2004. Organic acid production by filamentous fungi. In: Tkacz J and Lange L (eds.) Advances in fungal biotechnology for industry, agriculture and medicine, pp: 307-340. Place: Springer.

Papagianni M. 2004. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22: 189-259.

Paul G.C., Thomas C.R. 1998. Characterisation of mycelial morphology using image analysis. Adv Biochem Eng Biotech 60: 1-59. 7 Roa Engel C.A., Straathof A.J.J., Zijlmans T.W., van Gulik W.M., van der Wielen L.A.M. 2008. Fumaric acid production by fermentation. Appl Microbiol Biotechnol 78: 379-389.

Yang S.T. 2007. Bioprocessing - from biotecnology to biorefinery. In: Yang ST (eds.) Bioprocessing for value-added products from renewable resources - new technologies and applications, pp: 1-24. Place: Elsevier

Yang S.T., Zhang K., Zhang B., Huang H. 2011. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition, 3, 163-177.

Zhou, Y., Du, J., Tsao, G.T. 2002. Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents. Bioproc Biosyst Eng 25: 179-181.

8

Figure 1.1 Research objectives and scope of the study

9 CHAPTER 2

Literature Review

2.1 Fumaric acid properties and applications

Fumaric acid, named after the genus fumaria, can be found in many plants (Roa

Engel et al., 2008). It is also known as (E)-2-butenedioic acid, boletic acid, lichenic acid, allomaleic acid, and trans-1,2-ethylene-dicarboxylic acid. Fumaric acid (C4H4O4) has a chemical structure of two acid carbonyl groups and a trans-double bond (Figure 2.1) with a molecular weight of 116.07. Pure fumaric acid has an appearance of white solid crystal with no odor and a very slight acid taste. It has a density of 1.625 kg/m3 and a melting point of 286oC (Yang et al., 2011).

Fumaric acid has a low solubility of less than 10 g/L in cold water. Figure 2.2 shows its solubilities in water under different temperatures. Different fumarate salts have different solubilities in water. Calcium fumarate is difficult to dissolve in water with a low solubility of 21.1 g/L at 30oC, whereas sodium fumarate is much more soluble with a higher solubility of 220 g/L at 30oC. Based on the large difference in the solubility of different forms of fumarate and its free acid, fumaric acid can be recovered and separated from the fermentation broth after acidification. The solubilities of fumaric acid in organic solvents are also low: 54.4 g/L in 95% ethanol and 16.9 g/L in acetone at 29.7C, 0.03 g/L in benzene and 0.27 g/L in carbon tetrachloride at 25C.

10 Fumaric acid is an important specialty chemical with extensive applications in various fields. Figure 2.3 shows its current industrial applications. The largest use of fumaric acid is to synthesize a variety of resins, including paper resins (35% of annual fumaric acid production), alkyd resins (6%) and unsaturated polyester resins (15%), which have a bright future for continued market growth (Roa Engel et al., 2008).

Although fumaric acid is more expensive than maleic anhydride as the raw material for these resins, it is preferred in the polymer industry because of its nontoxic nature.

Fumaric acid-based resins also have physical advantages, including greater hardness in the polymer structure and higher durability, which makes them superior to other resins within the same price range. Moreover, the increasing trend of synthesizing biodegradable polymeric products benign to the environment favors the use of dicarboxylic acids, such as fumaric acid, as the monomer for polymerization. The additional functionalities of fumaric acid could allow cross-linking and provide a novel type of polymer-based products (Grijpma et al., 2005).

Fumaric acid has been used as an acidulant in foods and beverages since 1946. It is currently used in many food products, including corn and wheat tortillas, refrigerated biscuit doughs, gelatin desserts, gelling aids, fruit juice, nutraceutical drinks, and wine.

Research shows that fumaric acid improves the quality and reduces costs of many food and beverage products. For examples, it can lower the pH of the tortilla dough to increase the effectiveness of a mold inhibitor (e.g., calcium propionate); it can greatly increase the porosity with improved dough machinability for bread baking; it can provide more sourness per unit weight, as compared to other acidulants, in fruit juice drinks and acidify

11 wine without affecting its flavour; it can prolong the shelf life of acid coated candies as it does not absorb moisture during storage and distribution (Yang et al., 2011).

Fumaric acid is used as a feed additive to improve feed efficiency. One example is its use as an additive to piglet feed during the post-weaning period. The addition of fumaric acid and its adjustment of the pH value improve weight gain, food consumption and feed conversion ratio. Its use as a supplement in the cattle feed also leads to a large reduction (up to 70%) in methane emission. This provides an effective way of controlling the methane level in atmosphere as farm animals are responsible for 14% of the methane emission caused by human activity (Mcginn et al., 2004).

As a four-carbon dicarboxylic acid, fumaric acid can serve as the starting material for the synthesis of various chemicals, including maleic acid, malic acid, succinic acid and L-aspartic acid. Also, its ester derivatives have well-established applications in several fields. Monoethyl fumarate and dimethyl fumarate are the most used ones, especially in mold inhibition. However, recently dimethyl fumarate was found as an allergic sensitizer at a low content, causing extensive, serious eczema (inflamed skin conditions) which is difficult to treat (Gimenez-Arnau et al., 2009). Another use of the esters is as the medicine to treat psoriasis, an abnormal skin condition

(Moharregh-Khiabani et al., 2009; Mrowietz et al., 1998). Psoriatic persons are unable to produce fumaric acid normally, so they have to take orally fumaric acid in the form of dimethyl or monoethyl fumarate as supplement. These fumarate esters can also be used to produce transplantation medicines, especially pharmaceuticals for treating, alleviating and suppressing host-versus-graft reactions (Joshi et al., 2000). Last but not the least, many medicines are produced commercially in the form of fumarate salts. Ferrous

12 fumarate is well known as the supplement to prevent and to treat iron deficiencies and iron deficiency anemia with the advantages of high iron content, resistance to oxidation and little side effect (Zlotkin et al., 2001).

2.2 Fumaric acid production methods

2.2.1 Isomerization of maleic acid to fumaric acid

Currently, fumaric acid (trans-butenedioic acid) is mainly produced from its isomer, maleic acid (cis-butenedioic acid) by catalytic isomerization with >90% yield.

Industrially, maleic acid is produced from maleic anhydride [C2H2(CO)2O], which is produced from either benzene or n-butane via catalytic oxidation. These reactions are summarized below (Lorences et al., 2003).

n-Butane oxidation to maleic anhydride: C4H10 + 3.5 O2 → C4H2O3 + 4 H2O

Hydrolysis of maleic anhydride: C4H2O3 + H2O → C4H4O4

It is noted that fumaric acid can be recovered as a byproduct during the production of maleic anhydride. This not only improves the production of maleic anhydride but also turns the waste fumaric acid into valuable product and simplifies the downstream process.

The oxidation of n-butane uses catalysts based on vanadium and phosphorus oxides. For the isomerization of maleic acid to fumaric acid, various types of catalysts have been developed. They can be categorized into three types: mineral acids, peroxycompounds with bromides and bromates, and sulfur-containing compounds such as thiourea and its derivatives (Bachmann and Scott, 1948). The isomerization process has been well established since the 1970s. Figure 2.4 shows a simplified process flowsheet.

Decolorization and filtration are applied to remove the impurities present in the product

13 solution. Fumaric acid with a high purity can be obtained through crystallization, washing and drying (Roa Engel et al., 2008). However, high toxicity of catalysts, harsh production conditions and harmful exhausts generated in this process cause serious pollution and health concerns.

Alternatively, enzymatic conversion of maleic acid to fumaric acid can be carried out with maleate cis-trans isomerase or maleate isomerase under mild conditions.

Microorganisms producing this enzyme include Pseudomonas species, Alcaligenes faecalis and Arthrobacter spcies (Otsuka, 1961; Takamura et al., 1969; Kato et al., 1995).

Instead of using purified enzymes, whole-cell catalysis is preferred for industrial production due to simplified procedures and lower costs for enzymes used in this process.

However, maleate isomerases from these organisms were unstable even at a moderate temperature. Using thermo-stable maleate isomerases derived from Bacillus spp. alleviated this problem and enhanced fumaric acid production (Goto M et al., 1998). A high fumaric acid productivity of 6.98 g/L·h and yield of 95% from maleic acid was achieved using Pseudomonas alcaligenes strain XD-1 cells treated at 70oC for 1 h to inactivate fumarase, which otherwise would convert fumaric acid to malic acid, without affecting the activity of maleate isomerase (Ichikawa et al., 2003).

2.2.2 Fermentation of sugar to fumaric acid

During the 1940’s and before the rising of the petrochemical industry, fumaric acid was produced from sugars by fermentation with Rhizopus arrhizus (Rhodes et al., 1959;

Goldberg et al., 2006). Figure 2.5 shows the flowsheet for fermentation and subsequent downstream processes once used in industry for fumaric acid production from sugar

14 (Gangl et al., 1990). In general, the fermentation process involves two steps: seed culture preparation and acid production. After ~24 h seed culture, cells are harvested and transferred into the production fermentor for fumaric acid production. The downstream process depends on the neutralizing agent used during the fermentation. In the CaCO3 process, the fermentation broth containing calcium fumarate, cells and excessive CaCO3

o is acidified with H2SO4 to pH 1.0 and heated to 160 C. After filtration to remove insoluble particles (cells and CaSO4), the filtrate is cooled to below the room temperature to recover fumaric acid as precipitate via crystallization. When Na2CO3 is used as the neutralizing agent, the fermentation broth is first filtered to remove cells and then acidified to recover fumaric acid crystals. This process is simpler as no heating and cooling steps are involved and no gypsum (CaSO4), a solid waste, is produced.

The conventional industrial fermentation process for fumaric acid production became unfavorable due to the high production cost that can be attributed to the high sugar price (versus petroleum feedstocks) and relatively low product yield (<85% w/w), titer (<70 g/L), and reactor productivity (<1 g/L·h). However, rising oil prices, environmental concern of petroleum-based manufacturing, and demands for green and sustainable products and processes have prompted renewed interests in the production of fumaric acid from bio-based feedstock via fermentation. In the past thirty years, there have been substantial research efforts to improve and optimize the fermentation process on a laboratory scale, including screening for different Rhizopus species and strains, using cheaper biomass feedstock, optimizing fermentation conditions and medium formulation, and developing novel bioreactors, separation methods and integrated fermentation-separation processes. Table 2.1 summarizes and compares the fermentation

15 results from studies using various Rhizopus species and substrates. In general, fumaric acid production in the fermentation varied greatly depending on the species, substrates and fermentation conditions, with the product titer ranging from less than 20 g/L to as high as 130 g/L, product yield from 0.2 to 1.0 g/g glucose consumed, and productivity from less than 0.1 to 4.25 g/L·h. The highest product titer (130 g/L) and yield (1.0 g/g) were achieved by controlling the pH with CaCO3 and oxygen availability at 30%-80% saturation during the fermentation (Ling and Ng, 1989), while the highest productivity

(4.25 g/L·h) was obtained in an immobilized cell fermentation process with simultaneous product separation via adsorption (Cao et al., 1996). Detailed discussions on fumaric acid producing microorganisms, metabolic pathways and enzymes involved in the biosynthesis of fumaric acid, and process parameters affecting the fermentation performance are given in the following sections.

2.3 Fumaric acid fermentation microbiology

2.3.1 Fumaric acid producing microorganisms

Insofar as is known, microbial production of fumaric acid is mainly confined to filamentous fungi. Ehrlich first identified fumaric acid production in Rhizopus nigricans in 1911. Foster and Waksman then screened 41 strains from eight genera of Mucorales and identified Rhizopus, Mucor, Cunninghamella, and Circinella as fumarate producers

(Foster ans Waksman, 1939). Several other fungal cultures outside the order Mucorales, including Penicillium griseo-fulvum, Aspergillus glaucus and Caldariomyces fumago, are also able to produce fumaric acid. Among them, several Rhizopus species (nigricans, arrhizus, oryzae, and formosa) were identified as the best fumarate producers. However,

16 only R. arrhizus and R. oryzae have been extensively studied for their fumaric acid production potential.

Rhizopus spp, which belong to the class of zygomycetes, have had extensive industrial applications for thousands years. They can produce various hydrolyases, including amylases (Amedioha, 1993), endoglucanase (Karmakar et al., 2010), xylanase

(Bakir et al., 2001), and lipases (Fadiloglu et al., 1999), and can degrade starch and starch-based materials to produce various industrial products, including alcohol

(Taherzadeh et al., 2003), lactic acid, and fumaric acid (Ghosh and Ray, 2011). Their cell walls also contain large amounts of chitin and chitosan, which can be valuable byproducts (Liao et al., 2008). R. oryzae and R. arrhizus are the two most studied species for fumaric acid production. However, not all strains within these species can be used to produce fumaric acid. In general, R. arrhizus NRRL 2582 gave the highest product titer and yield, whereas R. oryzae ATCC 20344 gave the highest productivity (see Table 2.1).

In submerged cultures under aerobic conditions, some strains of R. oryzae produce fumaric acid while others produce L-lactic acid as the main metabolic product. Under anaerobic conditions, these strains may produce more ethanol, instead of lactic or fumaric acid. Depending on different organic acid production during aerobic submerged fermentation, R. oryzae can be divided into two groups, lactic acid producers containing two lactate dehydrogenase genes (ldhA and ldhB), and fumaric-malic acid producers containing only one ldhB gene (Abe et al., 2003; Saito et al., 2004). The fumaric acid producing ability of the heterothallic fungi R. nigricans was also found to be related to its sexuality: the male race(+) can produce fumaric acid in high yields, whereas the female race(-) seldom produces any fumaric acid (Gryganskyi et al., 2010).

17 2.3.2 Metabolic pathway for fumaric acid biosynthesis in Rhizopus sp.

Although fumaric acid is an important intermediate in the tricarboxylic acid (TCA) cycle present in most aerobic organisms, its production by Rhizopus is not through this pathway. Initially, a C-2 plus C-2 condensation similar to the reactions in the glyoxylate bypass was hypothesized for fumaric acid biosynthesis (Foster et al., 1949). However, later experiments showed a high fumaric acid molar yield of 140%, exceeding the theoretical molar yield (100%) for the postulated pathway, and thus disapproved this hypothesis (Romano et al., 1967). Also, the high glucose concentration essential for fumaric acid production inhibited the activity of the key enzyme, isocitrate-glyoxylate lyase, of the glyoxylate pathway. Later, a C-3 plus C-1 mechanism with CO2 fixation was proposed as the pathway for fumaric acid biosynthesis, which was supported by the discovery that R. oryzae harbored cytosolic pyruvate carboxylase, malate dehydrogenase and fumarase. The presence of this reductive TCA pathway (rTCA) was further confirmed in 13C nuclear magnetic resonance and enzymatic activity studies (Kenealy et al., 1986). This mechanism was also supported by the finding that the addition of cycloheximide (as inhibitor of the cytosolic fumarase) caused a large decrease in fumaric acid production.

Figure 2.6 shows the metabolic pathways in R. oryzae (Wright et al., 1996). The main pathway for fumaric acid biosynthesis is rTCA, which is located in the cytosol and involves three reactions starting from pyruvate. The first reaction is catalyzed by pyruvate carboxylase, which is ATP-dependent condensation of pyruvate and CO2 to form oxaloacetic acid (OAA). The product OAA is then converted to malate by malate dehydrogenase and then to fumarate by fumarase in cytosol. Fumaric acid is also an

18 intermediate metabolite in the TCA cycle. However, fumarate generated in the TCA cycle is mainly utilized for the biosynthesis of cell constituents and cannot be accumulated in a significant amount during active cell growth. Therefore, the production of fumarate from glucose by Rhizopus is believed to operate entirely through the cytosolic rTCA pathway with a high theoretical molar yield of 200% (Kenealy et al.,

1986). The rTCA pathway can balance the excessive ATP and NADH and adsorb CO2 produced in the oxidative TCA cycle. However, the two pathways compete for the sole pyruvate carbon flux. Under aerobic conditions, C4 (TCA cycle) intermediates can be formed in cytosol for biosynthesis during the growth phase. When nitrogen is limiting and cell growth stops, the continuing metabolism of glucose and CO2 fixation lead to the overproduction of fumaric acid at a theoretical yield of 129% (w/w) from glucose.

Meanwhile, the TCA cycle must remain active in order to provide energy for cellular maintenance and material transportation. Therefore, the actual fumaric acid yield in the fermentation is usually much lower than the theoretical yield. In addition, there are other competing pathways that lead to other products such as lactic acid and ethanol. Ethanol production usually occurs under anaerobic conditions or when oxygen is limited in the culture, and thus can be decreased by providing sufficient oxygen to the culture.

Depending on the strain and culture conditions, additional byproducts including malic acid, citric acid, and succinic acid could also be formed in relatively small amounts.

2.4 Fermentation process development and optimization

Fumaric acid can be produced from renewable resources in aerobic fermentation with Rhizopus. However, the current fermentation process usually suffers from low

19 product yields, titers, and productivities. Batch fermentation produces fumaric acid with ethanol, lactic acid and malic acid as additional byproducts (see Figure 2.7). The submerged fermentation is also difficult to operate in conventional stirred-tank fermentor because of the filamentous morphology of the fungal cells. One major technical challenge in developing a large-scale fermentation process for fumaric acid production is how to control the fungal morphology and physiology (metabolism) for the overproduction of fumaric acid in a sustainable and scaleable way. Important parameters to be optimized in the fermentation process include medium composition and oxygen transfer rate (aeration) or the control of dissolved oxygen in the medium during fermentation. The control of pH to maintain at a proper level with an appropriate neutralizing agent is also important to fumaric acid production. These are discussed below.

2.4.1 Cell immobilization and morphology control

In submerged cultures, depending on the growth conditions, filamentous fungi can exhibit various morphologies: clumps, pellets and filaments (see Figure 2.8). The filamentous morphology increases the viscosity of the broth and negatively affects mass transfer, while the clump morphology suffers from low internal mass and O2 transfer. In general, filamentous fungal fermentations are difficult to operate in large scale bioreactors due to the mycelial morphology and high broth viscosity. The highly branched fungal mycelia cause complex (viscous) broth rheology and difficulty in mixing and aeration in conventional stirred-tank fermentors. Cell immobilization has been used to control cell morphology and improve mass transfer and fumaric acid production. In addition, power consumption in mixing and aeration can also be greatly reduced because

20 the fermentation broth is maintained at a low viscosity under “cell-free” conditions, which also allows the product to be easily recovered from the culture medium. Methods for cell immobilization and morphology control are discussed below.

Pellets formation as self immobilization is desirable in large-scale filamentous fungal fermentation because cells grown in the pellet form, also referred to as self immoblization, has advantages of lower medium viscosity, less possibility of wraping around impellers and better mass transfer. However, the central region of large pellets could undergo autolysis due to nutrient limitation, which would have a negative effect on fumaric acid production (Roa Engel et al., 2011). Hence, small pellets with a diameter of

~1 mm or less are generally desirable in filamentous fungal fermentations (Papagianni.

2004). It is thus important to control the pellet size in preparing the seed culture for the fermentation. Agglomeration is the primary mechanism for pellet formation, which might occur between hyphae, between spores and hyphae, or between solid particles and hyphae.

The pellet formation process can be classified into two types: coagulating and noncoagulating type (Zhou, 1999). The coagulating type represents the process in which the spores aggregate with other small agglomerates and ungerminated spores to form pellets, while the noncoagulating type represents the process in which one spore grows out to one pellet. Depending on the strain and culture conditions, fungal pellets can have very different physical structures (fluffy loose, compact smooth, hollow smooth pellets) that could in term affect their fermentation performance.

Factors affecting cell growth and pellet formation, including the strain, spore inoculum size, and culturing conditions (medium composition, temperature, pH, and agitation speed) have been extensively studied (Papagianni. 2004). Several studies have

21 been done concerning the effects on the pellet formation of Rhizopus. The pH of the culture medium has been reported as an important factor for the pellet formation. Its influence is mostly due to the change of the surface properties of the cells. Different strains may have different sensitivities to the pH. In one study, when the initial pH was in the range of 2.60-3.36, small (diameter <1 mm), uniformly distributed, spherical pellets were formed in shake-flasks (Zhou et al., 2000). Higher initial pH values promoted the formation of large pellets or filamentous forms. In another study, no significant differences were observed on pellet formation at pH 2.5–7 (Liao et al., 2007). The spore inoculum size is recognized as another important factor. Hyphae interaction is deemed as the main mechanism for pellet formation. Generally speaking, a low spore inoculum concentration is preferred. The content of nitrogen source could also influence pellet formation. A certain concentration of the nitrogen source is required to guarantee pellet formation, while low nitrogen content may lead to clump morphology. Different nitrogen sources could also cause different pellet structures (Du et al., 2003). Furthermore, some metal ions (Mg2+, Zn2+, Fe2+), calcium carbonate, agitation, polymer additives and spore storage time can also influence pellet formation (Zhou et al., 2000; Liao et al., 2007; Liu et al., 2008).

Cell immobilization on solid carriers has also been extensively studied for immobilizing fungal mycelia and control cell morphology during fermentation.

Entrapment and adsorption are two most commonly used methods: entrapment is to entrap cells in solid carriers such as calcium alginate beads and polyurethane foam; adsorption is to attach cells to solid carriers via physical or chemical affinity. Compared to entrapment, adsorption has received more attention lately due to its advantages of

22 simple operation and lower costs. However, most of the earlier studies on immobilized

Rhizopus cells for fumaric acid production used entrapment in polyurethane foam cubes, cork pieces, Ca-alginate or polyurethane sponge as the carrier (Buzzini et al., 1995;

Petruccioli et al., 1996). Compared to free-cell fermentation in the corresponding studies, enhanced fumaric acid production was achieved with cell immobilization in the carriers

(Petruccioli et al., 1996). However, the limitation of mass transfer into the inside of the immobilized mycelia resulted in low reactor productivity. In addition, some carriers such as gel beads are mechanically weak and highly susceptible to shear force, and hence they are not suitable for large-scale fermentation.

Biofilm formation on solid carriers via adsorption can overcome the mass transfer limitation occurring to cell entrapment and large cell pellets. Biofilm of filamentous mycelia also has the advantage of better product secretion, which usually occurs at the hyphae tips. A rotary biofilm contactor (RBC) with immobilized R. oryzae as a biofilm on the solid plastic surfaces of rotating discs in the bioreactor was developed for fumaric acid fermentation (Cao et al., 1997). The rotating discs with immobilized cells moved the cells between the gas phase and the liquid phase in the reactor. Enhanced oxygen transfer and increased CO2 fixation were attained by directly exposing the biofilm to the air.

When the biofilm was submerged in the water, the cells took up the substrates and nutrients and excreted the produced fumaric acid. The RBC system coupled with an adsorption column (see Figure 2.9) greatly increased the reactor productivity to 4.25 g/L·h with a high fumaric acid yield of 85% (w/w) from glucose (Cao et al., 1996).

Without the adsorption column, a high fumaric acid product titer of 74 g/L was reached with the productivity of 3.08 g/L·h using CaCO3 as the neutralizing agent to maintain the

23 fermentation pH at around 5.0. The productivity in the RBC reactor was 3 times higher than that of an equivalent stirred vessel fermentor. The RBC could be utilized repetitively for two weeks without losing any biological activity. However, this system based on the available surface area for cell immobilization can be difficult to scale up for industrial use.

Fungal spores and cells have also been immobilized in a rotating fibrous bed bioreactor (RFBB) for fermentation to produce lactic acid from glucose and starch by R. oryzae (Tay and Yang, 2002). In the RFBB, a fibrous matrix is used for spore immobilization and biofilm growth, which is controlled with a proper rotational speed or shear acting on the biofilm. It can achieve high cell density, high mass transfer rate, and high reaction rate. Also, cells immobilized in the RFBB can continuously produce the acid product without any degeneration in a fed-batch fermentation operated for an extended period (Figure 2.10).

2.4.2 Medium formulation

Table 2.2 shows typical medium compositions used in the fermentation for cell growth or seed culture preparation and for acid production. Glucose is usually used in the fermentation medium as the carbon source for fumaric acid production by Rhizopus spp.

Although the best fumaric acid production is achieved using glucose, other carbon sources including starch, sucrose and xylose have also been explored (see Table 2.1). In general, low-cost substrates with a high carbohydrate content can be used in place of the more expensive refined substrate (glucose and sucrose). Rhizopus sp. has the ability to directly ferment starch-based materials, including cassava, corn and potato flour, which

24 can significantly lower the raw material costs (Moresi et al., 1991, 1992; Carta et al.,

1999; Xu et al., 2010; West, 2008). Recently, using abundant lignocellulosic biomass as feedstock in fermentation has become the trend for the emerging biorefinery industry developing green and renewable industrial products to replace petroleum-based products

(Yang, 2007). However, acid pretreatment and hydrolysis of cellulose and hemicellulose in the plant biomass to fermentable sugars are costly and usually generate some toxic compounds that severely inhibit cell growth and fermentation. Also, the utilization of xylose, which is a major component sugar in hemicellulose, is poor and results in much lower product yield, titer and productivity than those with glucose (Kautola et al., 1989).

It is worth mentioning that other high-value products such as chitin can be co-produced with fumaric acid in the fungal fermentation as demonstrated by using the hydrolysate of dairy manure (48% fiber, 16% crude protein) as the main substrate with additional glucose added to 100 g/L (Liao et al., 2008). The fermentation produced 25 g/L fumaric acid and 5.32 g/L biomass with 0.21 g chitin/g biomass.

The accumulation of fumaric acid happens when the fungal cells are cultured under stress with limited cell growth, such as with excess carbon source and limited nitrogen source in the fermentation medium. When the medium contains sufficient nitrogen source, most of the carbon source (e.g, glucose) will be consumed for cell growth instead of fumaric acid biosynthesis (Bulut et al., 2009). Thus, the relative amount of carbon source

(C) to nitrogen source (N) present in the medium is the most important factor in the fermentation. A high C/N ratio of 120 to 250 (w/w) is required for the overproduction of fumaric acid, converting 60~70% of glucose to fumaric acid (w/w) (Magnuson and lasure,

2004). Ding et al. (2011) found that decreasing urea concentration from 2.0 to 0.1 g/L

25 enhanced fumaric acid production from 14.4 to 40.3 g/L. A medium without any nitrogen source have also been successfully applied to obtain high fumaric acid production.

However, cells may lose their productivity over time when cultured under the N-free conditions. Also, substrate inhibition may occur when the content of carbon resource is too high. The sugar consumption rate decreases when the sugar concentration exceeds 10%

(w/v). Fumaric acid production may also be affected by the type of nitrogen source used in the medium. Organic nitrogen source, such as yeast extract, promotes cell growth with little fumaric acid accumulation, whereas inorganic nitrogen source, such as urea and (NH4)2SO4, is superior for fumaric acid production (Ling and Ng, 1989). When imposing a nitrogen limitation during the fermentation is not possible, phosphorus limitation can be used instead (Riscaldati et al., 2000).

As discussed earlier, fumaric acid biosynthesis involves CO2 fixation coupled with the conversion of pyruvate to oxaloacetate, the precursor to malate and fumarate. Adding

CaCO3, which is usually used as the neutralizing agent to maintain the medium pH around 6.0, in the fermentation medium is thus beneficial for the fumaric acid fermentation. The reaction between CaCO3 and fumaric acid liberates CO2, which can be used in the reaction for oxaloacetate formation from pyruvate. With the sufficient supply of CO2, the maximal theoretical yield of fumaric acid from glucose is 2 mol / mol. If no external CO2 is added and the CO2 is soly generated from TCA cycle, the maximal theoretical yield decreased to 1.5 mol / mol. (Roa Engel et al., 2008)

Some trace or heavy metals also play an important role in fumaric acid production.

Many metal ions including Mg2+, Zn2+, Fe2+ are cofactors or activators for some enzymes involved in the central catabolism and biosynthesis of macromolecules (DNA, RNA). A

26 proper level of these metal ions thus should be included in the medium. In addition, trace elements could also affect cell morphology, which has significant effects on the fermentation as discussed before. In one study, the medium containing 500 ppm Mg2+, 4 ppm Zn2+ and 100 ppb Fe2+ was found to be optimal to grow the fungal cell to form small spherical pellets beneficial to fumaric acid production (Zhou et al., 2000).

Other nutrients such as vitamins can also affect the fermentation. Many vitamins serve as coenzymes or enzyme cofactors and play important roles in cell metabolism and biosynthesis. For example, biotin is necessary for cell growth and metabolism of fats and amino acids. Biotin is also the activator of pyruvate carboxylase, which catalyzes the condensation of puruvate with CO2. CO2 fixation is the key step leading to fumaric acid biosynthesis. Riboflavin is the main component of cofactors flavin adenine dinucleotide

(FAD) and flavin mononucleotide (FMN), which play key roles in energy metabolism.

Though there were few studies directly supplementing vitamins into the medium, the addition of vitamin-rich materials such as corn steep liquor (Ling and Ng, 1989; Kang et al., 2010) and yeast extract (Petruccioli et al., 1996) has been applied universally and proven beneficial to fumaric acid production.

Adding methanol was also shown to be beneficial for fumaric acid production

(Petruccioli et al., 1996). It has been shown that in critic acid fermentation by Aspergillus niger, methanol enhanced the permeability of the cell membrane and cells responded to the diminished intracellular citrate level by increasing citrate production via the repression of 2-oxoglutarate dehydrogenase (Maddox et al., 1986). The same mechanism could also contribute to the increased fumaric acid production with methanol addition in some studies. Also, the slight toxicity of methanol would reduce cell growth and thereby

27 improve acid production. A small amount of tartaric acid has also been added in the fermentation medium in some studies; however, it is not clear if and how tartaric acid may affect cell growth and fumaric acid production.

2.4.3 Effects of pH and neutralizing agent

Fumaric acid production can be greatly improved by adding neutralizing agent to maintain the fermentation pH at the desirable range of 5.5~6.0. CaCO3 is the most frequently-used neutralizing agent because it can give a higher fumaric acid yield compared to other neutralizing agents. However, there are several issues limiting its industrial use. First, calcium fumarate has a low solubility of 21 g/L (at 30 oC) and is usually present as precipitate in the fermentation broth. The product aggregates with the cells, forming a highly viscous broth, which negatively affect mixing and mass transfer in fermentation. Second, adding sulfuric acid to the broth after fermentation is required to neutralize the excessive CaCO3 and to recover fumaric acid, which produces CaSO4, a byproduct with little use and causing environmental pollution. In many places, it is prohibited to use a process generating CaSO4 for organic acids production.

Other neutralizing agents, including Ca(OH)2, Na2CO3, NaHCO3 and (NH4)2CO3, have also been used for fumaric acid fermentation pH control (Gangl et al., 1990; Zhou et al., 2002). However, fumaric acid yield and productivity are usually lower as compared to using CaCO3. This is because Ca(OH)2 has a low solubility in water, whereas a high

+ concentration of Na (from Na2CO3) could negatively impact cell metabolism. In addition, sodium fumarate has a relatively high solubility in water and a high fumarate concentration could cause product inhibition and reduce fumaric acid production.

28 Nevertheless, the high solubility of sodium fumarate facilitates the separation of fumarate from cell biomass before its recovery as fumaric acid precipitate after acidification with sulfuric acid. Unlike the process using CaCO3, heating is not required and the downstream process is much simplified. Furthermore, cells present in the pellet form can be easily recycled for the next batch fermentation. These advantages should offset the disadvantages of lower fumaric acid yield and productivity and make the process economically attractive (Gangl et al., 1990; Zhou et al., 2002). (NH4)2CO3 can also be used when cell growth is limited by phosphorous. Though the yield is not comparable to that by using CaCO3, the product ammonium fumarate can be used directly to produce aspartic acid (Riscaldati et al., 2000).

Fermentation process without using a neutralizing agent was proposed to prevent product inhibition and avoid excessive waste salt production during downstream process.

The fermentation pH can be controlled via the removal of fumaric acid during fermentation without using a neutralizing agent. A simultaneous fermentation-separation process that avoids the use of neutralizing agents while preventing product inhibition could greatly enhance the process economics (Cao et al., 1996). This will be discussed in a separate section later. Roa Engel et al. (2011) developed a low pH fermentation strategy for fumaric acid production. He found that swiching off pH control in the later phase of the fermentation did not affect the performance and allowed to reach a low pH of 3.6, at which acid recovery can be simplied with less amounts of inorganic base.

29 2.4.4 Effects of dissolved oxygen on cell growth and fumaric acid production

Fumaric acid is produced mainly under aerobic conditions. It is thus important to maintain a high level of dissolved oxygen (DO) in the fermentation broth. The solubility of oxygen in water is low, only 40 mg/L at 25 oC. Therefore, continuous aeration and agitation are necessary in order to maintain a high DO level and increase the oxygen transfer rate (OTR) to meet the oxygen requirements for cell growth and metabolism. For conventional stirred-tank fermenters, higher agitation speeds could better disperse gas bubbles, break them into smaller ones and extend their residence time, and thereby increase OTR and fumaric acid production. However, high agitation rates also generate high shear rates that could damage fungal cells. Air lift fermenters, which achieve good mixing via liquid recirculation between bubbling and non-bubbling zones, can provide high oxygen transfer rates with minimal shear stress on cells due to the increased frequency of gas-liquid contact. In an airlift reactor with a porous sparger, the generated turbulence of two-phase flow inside the reactor not only provided favorable conditions for mass transfer with a high overall mass transfer coefficient (kLa) and good hydrodynamics, but was also beneficial to the formation of small cell pellets and keeping them well dispersed in suspension (Du et al., 1997). Pressure pulsation can also be applied to reduce the resistance of oxygen transfer from the gas phase to the liquid phase in a stirred-tank fermenter, resulting in higher fumaric acid yield and productivity (Zhou,

1999).

However, too high of the DO concentration could lead to more cell growth at the expense of fumaric acid production (Ling and Ng, 1989). Also, fumaric acid production of 130 g/L was attained by maintaining the DO level at 80% to 100% of saturation during

30 the growth phase and at 30% to 80% during the acid production phase. It is thus suggested that the DO concentration should be controlled at different levels at different phases of the fermentation process. A two-phase DO control strategy that kept DO at 80% in the first 18 h and then switched to 30% afterward improved both fumaric acid yield and productivity as compared to when the DO was kept at a constant level of 30%, 60% or 80% (Fu et al., 2010).

2.4.5 Product inhibition

Product inhibition is a common phenomenon in organic acid fermentations. The accumulation of fumaric acid, both in the salt form and free acid form, inhibits cells and reduces acid production. In particular, the increased concentration of free fumaric acid can decrease the intracellular pH and cause the retrodiffusion of fumaric acid through the membrane and thus inhibit fumaric acid production (Roa Engel et al., 2008). It has been reported that fumarate production by R. arrhizus was repressed when the fumarate concentration was over 34 g/L (Yang et al., 2011). However, another research showed that R. arrhizus could tolerate a high fumarate concentration of 71.3 g/L after adaptation.

It should be noted that although the highest amount of fumarate produced by this organism was reported to be 130 g/L (Ling and Ng, 1989), much of the product calcium fumarate was present as precipitates, instead of in the solution. Strain development through adaptation and metabolic engineering can alleviate product inhibition by increasing cell’s acid tolerance. Another way to resolve this product inhibition problem is to remove the product from the fermentation broth while it is being produced using in situ product removal techniques. Compared to conventional fermentation processes,

31 simultaneous fermentation and separation (SFS) has the advantages of higher productivity and lower separation cost.

2.5 Product separation and purification

Several separation processes have been developed for recovery and purification of non-volatile carboxylic acids, including precipitation, extraction, adsorption and electrodialysis. Table 2.3 summarizes the advantages and disadvantages of methods currently or potentially can be used for carboxylic acid separation (Yang et al., 2007).

The choice of the separation method depends on the type and quality of the carboxylic acid as well as its concentration and purity in the medium. Generally speaking, in order to make the recovery process economical, a product concentration of ~10% (w/v) is necessary.

Precipitation has been extensively used in the production of carboxylic acids, whose calcium salts have low solubilities in water. However, a large amount of H2SO4 is required to acidify the calcium salt, generating much solid waste CaSO4 which is expensive to dispose of by landfill.

Currently, SFS has become the trend for product separation and recovery. In situ product removal from the fermentation broth during carboxylic acid fermentation has many advantages, including ease in pH control without addition of neutralizing agent, prevention of the end product inhibition, and avoidance of the metabolic shift due to overproduction of the end product (Yang et al., 2007). Thus, enhanced productivity, product yield and titer could be achieved through the process. Solvent extraction has thus been applied to separate fermentation-produced carboxylic acids, including citric acid

32 and lactic acid, at an industrial scale (Hong et al., 2001; Wasewar et al., 2004). It has good potential to be an in situ recovery method for simultaneous fermentation and separation (SFS). Meanwhile, a lot of research has been carried out on using ion exchange adsorption (Ataei et al., 2008; Cao et al., 1996; Wang et al., 2000) and electrodialysis (Huang et al., 2007) to separate fermentation produced carboxylic acids, though they are not yet widely used in industry.

Extractive fermentation is to apply an organic solvent to separate carboxylic acids from the fermentation broth. Long-chain aliphatic amines with high distribution coefficients is the most frequently used organic solvent (Wasewar et al., 2004).

Extractive fermentation has attracted much interest because it can produce and recover the desired acid product continuously in one step, reduce the end-product inhibition, and thus result in significant increase of productivity and final product concentration.

Although this technology suffers from solvent toxicity and limitation of suboptimal operating pH (Yang et al., 2007), the newly-developed hollow-fiber membrane-based extractive fermentation process (see Figure 2.11) provides a promising solution (Huang et al., 2004). In the membrane-based extractive fermentation process, an immobilized cell bioreactor was used for fermentation to avoid direct contact of the cell with the toxic solvent; also it can enhance the stability of the fermentation and be suitable for continuous production. In the first extraction column, hydrophobic hollow fiber membrane was applied to imbed the organic solvent in the pores of the microporous support so that the acid product can be extracted while little or no solvent can get into the bioreactor, thus alleviating its toxicity effect on cells. Then, in the second or back-extraction column, the acid was stripped to reach a high concentration in the final

33 product. In this process, only a small amount of solvent is required by circulation between the first and second extraction column and thereby continuously regenerated with a good long-term stability. The fermentor pH can be self-regulated by balancing between the fermentation and extraction and thus does not need to be controlled by base addition. In addition, downstream processing can be simplified without the requiring broth acidification. So far, this technology has been well applied to the production of several organic acids, including lactic, propionic, and butyric acids (Huang et al., 2004;

Jin et al., 1998; Wu et al., 2003). The extractive fermentation process could bring another promising way for economic and effective production of fumaric acid as well.

Electrodialysis fermentation (EDF) is to apply electrodialysis to SFS and to couple the fermentation with an electrodialyzer with ion-exchange membranes for the removal of ionized carboxylates. EDF has been applied to lactic acid production, but with little research on other acids. In general, EDF provides good pH control without the need of base and produces a concentrated and relatively pure product stream, simplifying subsequent downstream processes and improving reactor productivity (Huang et al.,

2007). However, several drawbacks and complexity, including relatively low product yield and concentration, membrane fouling by cells and proteins, and possible removal of nutrients and inorganic ions by electrodialysis, limit further applications of EDF (Yang et al., 2007).

So far, only adsorption has been successfully applied on in situ recovery of fumaric acid from the fermentation broth (Cao et al., 1996; Fu et al., 2009). Various adsorbents were investigated for fumaric acid-fumarate adsorption. Among them, polyvinyl pyridine

(PVP) resin and a strong-base ion-exchange resin IRA-900 exhibited the highest loading

34 capacity of ~0.32 g/g resin. After adsorption, 0.4 N NaOH and 0.4 M NaCl were used as eluents to desorb the adsorbed fumarate from PVP and IRA-900, respectively. After acidification of the eluted solution with HCl, fumaric acid can be generated as crystals with a nearly 100% purity. An SFS process was developed for fumaric acid production in a RBC coupled with adsorption to remove fumaric acid (Cao et al., 1996). During the process, fumaric acid was adsorbed onto the polyvinyl pyridine (PVP) resin, and the anion OH- released from the adsorption column into the circulating fermentation broth maintained the broth pH at ~4.5. Thus, no neutralizing agent was needed in this process.

The process increased both product yield (from 0.75 to 0.85 g/g) and productivity (from

3.78 to 4.25 g/L·h) compared to the same fermentation process without in situ product removal. It should be also noted that, higher pHs around 5.8 to 6.2 favored fumaric acid fermentation while lower pHs of below 5 favored fumaric acid adsorption by PVP. To optimize the SFS process, it is important to maintain the broth pH within a proper range that can provide a good balance between fumaric acid production by cells and its removal from the fermentation broth by adsorption.

The adsorption-desorption process with ion-exchange resins described before is not ideal as it requires large amounts of acid (HCl) and base (NaOH) and produces NaCl as a byproduct. To solve this problem, ammonium hydroxide, instead of NaOH, can be used as the eluent to generate ammonium fumarate, from which fumaric acid can be released after removing ammonia by adsorption with acid Y-zeolite in a packed column (Zhou,

1999). Finally, the Y-zeolite is regenerated to the acid form by heating or steam stripping to release the adsorbed ammonium, which can be concentrated and recycled for use in the process. As a result, the whole recovery system could recover fumaric acid without

35 consuming significant amounts of chemicals, with the only operating cost being energy for heating.

2.6 Conclusion and future prospects

The rising prices of oils and petrochemicals and concerns about the depleting petroleum reserves have prompted the search for bio-based fuels and chemicals.

Filamentous fungal fermentation provides a good alternative way for the production of fumaric acid from renewable feedstocks. However, bioproduction of fumaric acid by filamenous fungi such as Rhizopus oryzae requires further development and optimization to be economically competitive. An earlier economic analysis of fumaric acid fermentation process conducted in late 1980’s concluded that the fermentation route could become competitive with the petrochemical route once the oil prices were above

$34 to $61/barrel (Gangl et al., 1990). In the recent years, the oil prices have risen above $80/bbl and reached as high as $140/bbl. Meanwhile, the prices of corn and glucose have also increased significantly. It would be necessary to conduct a new cost analysis based on current prices. Nevertheless, the fast advances in biotechnology, in particular in the fields of functional genomics and systems biology provide new tools and knowledge that will make major impacts on strain development and the fermentation technology. Coupled with advances in novel bioreactors and separation techniques, the fermentation process for fumaric acid production should be economically attractive in the foreseeable future, if not today.

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42 Table 2.1 Comparison of fumaric acid production from various substrates and fermentor

systems by Rhizopus spp.

Producti Titer Yield Strain Fermenter Substrate -vity Reference (g/L) (g/g) (g/L·h) Gangl. et al. 1990; Shake flask 38~ 0.33~ 0.46~ Ling and Ng, 1989; Glucose stirred tank 130 1.0 2.0 Rhodes et al., 1962; Riscaldati, 2000 Fluidized Molasses 17.5 0.36 0.36 Petruccioli et al., 1996 bed Rhizopus arrhizus Shake flask Xylose 15.3 0.23 0.07 Kautola et al., 1989 Shake flask Potato flour 43.5 0.58 0.42 Moresi et al., 1991

Shake flask Corn starch 71.9 0.60 0.50 Moresi et al., 1992

Shake flask Grape must 24.1 0.23 0.17 Buzzini et al., 1995 Zhang et al., 2008; Zhou et al., 2000; Fu Shake flask 35.8~ 0.33~ 0.48~ Glucose et al., 2010; Ding et stirred tank 66.5 0.67 0.90 al., 2011; Roa Engel et al., 2011 RBC with Glucose 92.0 0.85 4.25 Cao et al., 1996 adsorption Rhizopus RBC Glucose 75.5 0.75 3.78 Cao et al., 1997 oryzae 10L air lift Glucose 37.8 0.75 0.81 Du et al., 1997 Bubble Glucose 37.2 0.53 1.03 Zhou et al., 2002 column Dairy Shake flask 25 - 0.17 Liao, 2005 manure Shake flask Corn straw 27.8 0.35 0.33 Xu et al., 2010 14.7~ 0.50~ Foster et al., 1938; Shake flask Glucose 0.25 Rhizopus 20.0 0.66 Romano et al., 1967 nigricans Shake flask Apple juice 33.1 - 0.23 Podgorska et al., 2004 Rhizopus Cassava Stirred tank 21.3 - - Carta et al., 1999 formosa bagasse

RBC: Rotary biofilm contactor

43 Table 2.2 Typical medium compositions used in seed culture preparation and

fermentation for fumaric acid production from glucose by Rhizopus oryzae.

Seed culture medium for spore Fermentation medium for Components germination and cell growth acid production

Carbon source Glucose 30-50 g/L Glucose 80-130 g/L

(NH4)2SO4 2.2-4.0 g/L (NH4)2SO4 0.25 g/L Nitrogen or Yeast extract ~2.5 g/L or Yeast extract 0.4 g/L source or Urea ~2.0 g/L or None

KH2PO4 0.6-1.6 g/L; KH2PO4 0.3-0.6 g/L; MgSO 0.25-0.4 g/L; MgSO 0.25-0.4 g/L; Metals 4 4 ZnSO4 0.044-0.088 g/L; ZnSO4 0.044-0.088 g/L;

FeCl3 0.0075 g/L FeCl3 0.0075 g/L Neutralizing CaCO 3.0 g/L CaCO 80 g/L agent 3 3 Corn steep liquor 0.5 Other Corn steep liquor 0.5 mL/L mL/L; Supplements Tartaric acid 0.0075 g/L Methanol 15 mL/L Tartaric acid 0.0075 g/L

44 Table 2.3 Separation methods for recovering organic acids from fermentation broth.

Methods Operating Principle Advantages / Disadvantages Relatively inexpensive, can be performed Based on low solubility of with simple equipment; the organic salts, usually in Produce a solid waste when calcium salt is Precipitation the calcium form in water. used; Fumaric acid also has a low solubility in water. Cannot conveniently used as a in situ recovery method Adsorption of undissociated High selectivity, high capacity for charged organic acids to ionic molecules; Can be used for in situ recovery; Ion exchange exchange resins in a packed adsorption Ion exchange resins are expensive and column, followed with regeneration of the resin requires additional desorption. chemicals and/or energy Partition between organic Widely used in industry in the production of and aqueous phases based on lactic acid and citric acid; Easy to operate Solvent different solubilities of the and scale up; Extraction organic acid in the two Solvent can be toxic to cells; Has not been immiscible phases tried with fumaric acid fermentation

Electric current is applied to Organic acid is concentrated in aqueous move negatively charged solution, does not require acid addition to organic acid ions through an adjust the solution pH; Electrodialysis anion-exchange membrane Product purity is low and may require further towards the anode in the purification; high energy input; Membrane electrodialyzer fouling; Difficult to scale up

45

HOOC H C C H COOH

Figure 2.1 Molecular structure of fumaric acid

46 60

50

40

30

20

Solubility (g/L) Solubility 10

0 0 20 40 60 80 100 temperature (OC)

Figure 2.2 Solubility of fumaric acid in water under different temperatures

47 Fumaric acid production 90,000 ton/a

35% paper resins (35%) 17% alkyd resins (6%) unsaturated polyester resins (15%) 22% plasticizers (5%) 6% food and beverage additives (22%) 15% 5% miscellaneous (17%)

Figure 2.3 Current applications of fumaric acid (Roa Engel et al., 2008)

48 Raw material: Decolorization Isomerization Centrifugation Maleic acid (activated charcoal)

Output: Drying Crystallization Filtration Fumaric acid crystal

Figure 2.4 A flow chart for fumaric acid production from maleic acid via isomerilization Fig. 4 Flow chart for fumaric acid production from maleic acid[2]

49 Process with Na2CO3 Seed culture Acidification Filtration (Sulfuric acid to pH 1)

Fermentation

Acidification at 160oC Cooling (Sulfuric acid to pH 1) Filtration to 20 oC

Process with CaCO3

Output: Filtration drying Fumaric acid crystal

Figure 2.5 FlowsheetFig. 5 Flow for chart fumaric for fumaric acid production acid production via fermentation via fermentation (Gangl[7] et al., 1990)

50

Ext- Glucose Glucose Citrate Ext- Acetyl Citrate -CoA G-6-P Oxalo- keto- acetate2 CO2 4 glutarate

Malate2 succinate F-1,6-bP Pyruvate2 3 5 Fumarate2 Ext- Culture Fumarate Mitochondrion Medium Pyruvate1 7 1

CO2 Acetadehyde Cytosol 6 Oxaloacetate1 8 2 CO2 3 EtOH Lactate Malate1 Fumarate1

Ext-Ethanol Ext-lactate Ext-Malate Ext-Fumarate

1 pyruvate carboxylase 2 malate dehydrogenase 3 fumarase 4 pyryvate dehydrogenase complex 5 succnate dehydrogenase 6 lactate dehydrogenase 7 pyruvate decarboxylase 8 ethanol dehydrogenase

Figure 2.6 Glucose metabolic pathways in R. oryzae (Wright et al., 1996)

51 100 40 Shake-flask

80 30

glucose 60 fumarate 20 lactate

40 malate Glucose (g/L) Glucose

ethanol (g/L) Products 10 20

0 0 0 25 50 75 100 125 Time (h)

Figure 2.7 Batch fermentation kinetics of Rhizopus oryzae in 250-ml shake-flask.

Fumaric acid is produced from glucose, with lactate, malate, and ethanol as byproducts.

52

Figure 2.8 Different morphologies of R. oryzae in submerged culture - pellet (left); loose

mycelia (middle), mycelial clump (right)

53 Adsorption Air, feed, column medium

Motor Rotary biofilm contactor Pump

Figure 2.9 Schematic diagram of a rotary biofilm contactor with adsorption columns for Fig. 9 Rotary biofilm contactor coupled with an adsorption column[6] fumaric acid production and recovery (Cao et al., 1996)

54

75 glucose fumarate ethanol 60

45

30 Concentration (g/L) 15

0 0 50 100 150 200 250 300 350 400 Time (h)

Figure 2.10 Fed-batch fermentation for fumaric acid and ethanol production from glucose

by Rhizopus oryzae in a rotating fibrous bed bioreactor (RFBB)

55 Fermentation Extraction Back Extraction Carboxylic acid or Carboxylate

Immobilized pH Hollow- cell probe Fiber Bioreactor Membrane Contactor

Stripping Feed Solution Medium Organic Solvent

Figure 2.11 Extractive fermentation process with immobilized cell fermentation coupled

with hollow-fiber membrane contactors for continuous product recovery from the

fermentation broth with simultaneous solvent regeneration. (Yang et al., 2007)

56 CHAPTER 3

Effects of Soybean Meal Hydrolysate as the Nitrogen Source on Seed Culture

Morphology and Fumaric Acid Production by Rhizopus oryzae

3.1 Introduction

Fumaric acid (HOOCCH=CHCOOH), an organic acid with a trans-double bond and two carboxylic acid groups, is an important specialty chemical with wide industrial applications, including uses as feedstock for the synthesis of polymeric resins and as an acidulant in foods and pharmaceuticals (Roa Engel et al., 2008). Fumaric acid can be readily transformed into other C4 chemicals including malic acid, succinic acid and aspartic acid (Yang et al., 2011), and has been identified by the United States Department of Energy as one of the “top 12” building-block chemicals that can potentially be produced from abundant renewable biomass (Yang 2007). Currently, fumaric acid is mainly produced by petroleum-based chemical synthesis. However, the limited petroleum resource, rising oil prices, and heightened environmental concern of chemical synthesis have prompted renewed interests in the development of bio-based fumaric acid by fermentation using filamentous fungi, such as Rhizopus oryzae and R. arrhizus, which can produce large amounts of fumaric acid from sugars under aerobic conditions (Roa

Engel et al., 2008).

However, the filamentous fungal fermentation process is difficult to scale up for

57 industrial production of fumaric acid due to difficulties in controlling cell morphology and growth in conventional stirred-tank fermentors (Yang et al., 2011). In general, the overproduction of fumaric acid by the fungal cells only take place when they are cultured under stress with sufficient oxygen supply. The production of fumaric acid decreased when there is oxygen limitation and ethanol production significantly increased (Cao et al.,

1996). Extensive research has thus focused on how to control the fungal morphology and physiology (metabolism) for fumaric acid biosynthesis (Fu et al., 2009; Liao et al., 2007;

Liu et al., 2008; Roa Engel et al., 2011; Zhou et al., 2000; Zhou et al., 2011). During submerged fermentation, filamentous fungi can grow into various morphologies, including free mycelia with hyphae dispersed freely and uniformly in the fermentation broth, smooth and hairy pellets with a discernible core of highly entangled networks of hyphae, small loose mycelial clumps, and large aggregated mycelia with high density

(Paul et al., 1998; Znidarisic et al., 2001). Different morphologies in fermentation have significant effects on bioreactor performance and fumaric acid production (see Table 3.1).

In general, small pellets with a diameter of ~1 mm or less are considered as the most desirable morphology because they afford the lower medium viscosity, less possibility of wrapping around impellers, and better mass transfer as compared to freely dispersed mycelia (Papagianni 2004). However, higher fumaric acid production was achieved with dispersed mycelia in free-cell fermentation (Gang et al., 1990; Rhodes et al., 1961) and biofilm immobilized on a rotary biofilm contactor (RBC) (Cao et al., 1996).

The objectives of this study were to explore the relationship between cell morphology of R. oryzae and subsequent fumaric acid fermentation, and develop an effective fermentation process for fumaric acid production. Soybean meal is the residual

58 product after the extraction of oil from whole soybeans with a high protein content. A novel culture approach was developed using soybean meal hydrolysate (SMH) as the nitrogen source. The effects of SMH on seed culture morphology and subsequent fumaric acid production by R. oryzae were studied. Compared to the pellet morphology obtained by using urea or yeast extract as nitrogen source, R. oryzae grew into mycelial clumps with higher fumaric acid production by using SMH.

3.2 Materials and Methods

3.2.1 Cultures and meida

R. oryzae ATCC 20344 used in this study was cultured on YMP (yeast extract, malt extract, and peptone) agar plates containing 3 g/L yeast extract, 3 g/L malt extract, 3 g/L peptone, 20 g/L glycerol and 20 g/L agar. After ~7 day incubation at 32 oC, the spores were harvested by washing with sterile distilled water and collected as a spore suspension, which was stored at 4 oC.

Soybean meal containing 44%-50% crude protein, 20~30% carbohydrate, 0.5% fat,

7% fiber and 12% moisture was obtained from Cargill (Sidney, OH). To prepare the soybean meal hydrolysate (SMH), 20 g of soybean meal were added into 400 mL of 0.25

N HCl, and the mixture was autoclaved at 121 oC for 30 min. The hydrolysate was filtered under sterile conditions, and the filtrate was stored at room temperature for future use. The protein content in the hydrolysate was determined by BioRad protein assay and found to be ~12 g/L.

59 3.2.2 Seed culture preparation

Unless otherwise noted, all seed cultures were prepared in 250 mL shake-flasks each containing 50 mL of the growth medium with 10 g/L glucose and 20% SMH (v/v) as nitrogen source. The pH of the medium was then adjusted to 3.0 using NaOH. After inoculation with the spores in suspension, the cultures were incubated at 35~37 oC for 24 h in a rotary shaker agitated at 220 rpm.

The effects of nitrogen source, initial pH, SMH concentration and spore concentration on cell morphology were studied. The effects of nitrogen source (urea, yeast extract and SMH) on cell morphology were studied. The seed culture medium with

SMH was the same with the above mentioned. The medium with urea contained: 30 g/L glucose, 2 g/L urea, 0.6 g/L KH2PO4, and 0.50 g/L MgSO4. Urea was sterilized by filtration and aseptically added into the medium. The medium with yeast extract contained 30 g/L glucose and 2.5 g/L yeast extract. The cultures were incubated at 35~37 oC for 48 h in a rotary shaker at 220 rpm. Also, the effects of initial pH (2.2, 3.0, 4.0, 5.0,

6.0) and SMH concentrations (4%, 10%, 20%, 30%, 40%, v/v) on cell morphology under two spore concentrations of 4.0×107/L and 8.0×107/L were studied. The experimental conditions and results are summarized in Table 3.2. All the trials were run in three replicates.

3.2.3 Fermentation

Fumaric acid fermentation was carried out in 250 mL shake-flasks. The seeds were first harvested from the seed cultures under the same conditions and then inoculated into the fermentation medium at an inoculation size of 25%. The volume of the medium in the

60 flask was 40 mL. The fermentation medium contained 90 g/L glucose, 0.6 g/L KH2PO4 and 0.5 g/L MgSO4. The pH was maintained above 5.0 by adding 60 g/L CaCO3. The fermentations were performed at 35 oC for 4~5 d in a rotary shaker at 220 rpm.

Using the seed cultured in the mycelial clumps form, the effect of seed age (18, 21,

24 h) on fumaric acid fermentation was studied under the inoculation size of 15%. Each condition had three replicates. The other seed culture conditions were previously identified as favorable for mycelial clumps morphology.

Different inoculation methods (seed age, inoculation size and seeding density) were studied on the selected medium and conditions which were identified previously as the favorable to the formation of mycelial clumps. The seeds were harvested from the seed cultures under the same age and then inoculated into the fermentation medium at a certain inoculation size. The seeds would precipitate at the bottom of the harvested flasks.

Higher seeding density, i.e., the density of mycelial clumps in the medium, can be achieved by removing a certain amount of supernatant medium. Thus, under the same inoculation size, higher seeding density corresponds to more mycelial clumps and less seed culture medium carryover.

3.2.4 Analytical methods

Cell morphology was identified by examining cultures dispersed on Petri dishes. An

Olympus microphotograph (Tokyo, Japan) was used to observe the cell morphology. Cell dry weight was determined after drying the cells at 100 °C overnight.

HPLC was used to analyze the organic compounds, including glucose, lactic acid, malic acid, fumaric acid and ethanol, present in the fermentation broth. Because calcium

61 fumarate was precipitated in the fermentation broth, sample pretreatment was required for

HPLC detection: dilute hydrochloric acid was added to neutralize excessive CaCO3 and acidify fumarate. Due to the low solubility of fumaric acid in water (6.3 g/L at 25 oC), the broth was heated to 80 oC to increase the solubility of fumaric acid. The HPLC system

(Shimazu Scientific Instruments) equipped with a RID-10A refractive index detector and an organic acid analysis column (HPX-87H Bio-Rad, Richmond, CA) was operated under the following conditions: sample volume of 15 μl, mobile phase of 0.005 M H2SO4, flow rate of 0.6 mL/min, and column temperature of 45 oC.

3.3 Results

3.3.1 Morphology studies

The effects of initial pH (2.2, 3.0, 4.0, 5.0, 6.0), different nitrogen sources (SMH, urea, yeast extract) and spore concentrations (4, 8×107 /L) on cell morphology were studied with the morphologies shown in Figure 3.1 and results summarized in Table 3.2.

Nitrogen sources and their concentrations play important roles on cell morphology.

At pH 3, the cell formed non-uniform pellets with varying sizes (d = 0.5~4 mm) (See

Figure 3.1D) by using urea and yeast extract as nitrogen source, whereas the cell formed uniformly dispersed mycelial clumps by using SMH. The micro-morphology (Figure

3.1G, 3.1H) of mycelial clumps showed that there was no obvious compact core in the middle of the clump as in the pellet (Figure 3.1I), though overlapping of hyphae occurred.

In addition, the mycelial clumps were tiny with an equivalent diameter of <0.1 mm. The formation of mycelial clumps might be resulted from the tiny protein precipitates of SMH at pH 3, which may have some immobilization effects on the spore granulation and cell

62 growth. The effects of SMH concentrations (4%, 10%, 20%, 30%, 40%, v/v) under spore concentrations of 4, 8×107 /L were also investigated. Depending on the amount of SMH added in the growth media, cells might form aggregated mycelia (4% SMH, Figure 3.1F), non-uniform morphology with pellets and filaments intertwined (10% SMH, Figure 3.1A) or uniformly dispersed mycelial clumps (30/40% SMH, Figure 3.1C). At the SMH concentration of 20%, high spore concentration favored the uniform morphology while low spore concentration caused the non-uniform morphology.

The initial pH is also of significance to cell morphology. In general, spores were unable to germinate at a low pH of ~2.2, whereas mycelial clumps were obtained at pH

3.2. At pH ~4.2, the morphology became non-uniform with the formation of large aggregated mycelia, pellets and intertwined filaments (Figure 3.1E). Further increasing the pH to 5.3 and 6.3 produced aggregated mycelium (Figure 3.1F).

The spore concentration is another influencing factor to cell morphology. However, there were no significant differences between the cell morphologies under the two levels of spore concentrations (4, 8×107 /L) within the studied SMH concentrations. One reason might be that the studied spore concentration levels were high. However, at SMH concentration of 20%, the higher spore concentration increased the uniformity of the cell morphology as mentioned before. Based on micro-morphology of samples taken from the two spore concentrations (Figure 3.1G and 3.1H), the non-uniform morphology at the lower spore concentration had more filaments interwined among the mycelial clumps.

63 3.3.2 Effects of nitrogen source in seed culture medium

The seeds prepared from the three different nitrogen sources (SMH, urea, yeast extract) were transferred into fermentation medium for fumaric acid production. The seeds from SMH showed morphology of mycelial clumps, whereas the seeds from urea and yeast extract had pellet morphology. It was also observed that the lag phase of seed culture process with SMH was only ~10 h, significantly shorter than the lag phases (> 24 h) with urea and yeast extract. The reason for the mycelial clumps formation and short lag phase was further investigated. When the pH of SMH was adjusted to 3 by adding

NaOH, tiny precipitates were observed in the medium (See Figure 3.2). After centrifugation and washing, these precipitates were obtained and identified by BioRad assay as protein precipitates with a high concentration of 1.28 g/L. The formation of these precipitates is due to the change of medium pH, which is close to the proteins’ isoelectric point where protein has a minimal solubility. These protein precipitates not only acted as the immobilization carriers for the spores but also provided protein in solid phase for the cell growth. The solid-phase protein also provided a novel method for slow/controlled release that allowed the utilization of the nitrogen source by cells for an extended period without losing cell activity.

Figure 3.3 shows the fermentation kinetics for the three cases. For both urea and yeast extract cases, more than 40 g/L glucose was consumed with little fumaric acid production (6~8 g/L) in the first 22 h, whereas for the SMH case, during the same period,

~11 g/L fumaric acid was produced with the yield of 0.5 g/g glucose. Since no nitrogen source was added into the fermentation medium, the protein (~0.1 g/L) in the SMH medium was the carry-over of the SMH from the seed culture, as indicated from the

64 residual protein concentration of 0.2 g/L after 24 h seed culture (data not shown). The decrease of the protein concentration coupled with the increase of cell growth and fumaric acid production at the beginning of the fermentation proved the beneficial effects of the SMH carry-over from the seed culture. Also, high ethanol production (>20 g/L) was found for the urea and yeast extract cases, possibly due to the oxygen limitation inside the large pellets. On the other hand, little ethanol production (~1.7 g/L) in the

SMH case indicated good mass and oxygen transfer of the mycelial clumps morphology.

The results are summarized in Table 3.3. Overall, compared with the seeds prepared from urea and yeast extract, the seeds from SMH had a better morphology with the significantly higher fumaric acid production.

3.3.3 Effects of SMH concentration

The seeds prepared from the four SMH concentrations (10%, 20%, 30%, 40%, v/v) under two spore concentrations (4, 8×107 /L) were transferred into fermentation medium for fumaric acid production. Figure 3.4A describes the effects of SMH and spore concentration on the cell dry weight. In general, the cell dry weight increased with the increase of the SMH concentration from 10% to 30%. However, there was no significant difference in cell dry weight in the medium containing 30% and 40% of SMH.

Additionally, increasing the spore concentration enhanced cell dry weight. But, the two-fold increase of spore concentration did not cause a corresponding two-fold increase of the biomass.

The results of the subsequent fumaric acid fermentation under different cases are compared with the product titer and yield shown in Figure 3.4B and 3.4C, respectively.

65 Overall, increasing the spore concentration significantly improved fumaric acid product titer, especially under lower SMH concentrations of 10% and 20%. The reason can be attributed to the increased seeding density in the fermentation medium. Also, the variations of the fermentation results between the replicates were much smaller at the higher spore concentration. However, the effect of spore concentration on product yield was insignificant. With respect to the effect of SMH concentration, generally, the product titer increased with the increase of SMH concentration from 10% to 30%. A low product titer with high yield was observed under 10% SMH, which can be attributed to the low cell metabolic activities as indicated by the low glucose consumption in fermentations.

The change of the fermentation results under different SMH and spore concentrations can be explained from two aspects: first, the non-uniform morphology of the seed formed under a low SMH and spore concentration increased the possibility of forming large aggregated mycelium during the fermentation resulting in a negative effect on the mass transfer and decrease of the fumaric acid production; second, increasing SMH concentration significantly increased the nitrogen content of the carryover from seed culture to the fermentation medium, resulting in enhanced cell activity and fumaric acid production. Additionally, the product yield decreased from a SMH concentration of 30% to 40% under the two spore concentrations, possibly due to in the fermentation medium the too high nitrogen content introduced from carryover. Overall, the morphology of uniformly dispersed mycelial clumps was favorable to achieve a high and stable fumaric acid production. The optimal values for the SMH and spore concentrations were identified as 30% (v/v) and 8×107/L.

66 3.3.4 Effects of seed age on fumaric acid fermentation

During seed culture process, the soluble protein concentration in the medium decreased gradually, showing the slow utilization of SMH (See Figure 3.5), which is possibly due to the protein precipitates as mentioned before. The three seed ages (18, 21,

24h) corresponded to different residual SMH concentrations (protein concentration of

0.19, 0.16, 0.16 g/L) in the carry-over, hence, different nitrogen availability for the fermentation process. Table 3.4 summarizes the effects of seed age on fermentation. In general, the product titer and yield increased with the increase of seed age. The reason may be that more carbon was switched to the cell growth and aspiration under the high protein concentration. Therefore, the best seed age was 24 h.

3.3.5 Effects of inoculation conditions on fumaric acid fermentation

From the above study, the inoculation process brought a small amount of nutritional substances into the fermentation medium. The experiment in which the cells were washed before inoculation to remove the SMH residues showed a significant decrease of fumaric acid production (<22.0 g/L) and that the production increased with increasing the cell density. It is thus clear that both cell density and the carryover of the seed culture medium played important roles in affecting the fermentation kinetics.

Thus, the effects of cell density under different inoculation sizes were studied.

Increasing inoculation size increased both nitrogen availability and cell density in the fermentation. Generally speaking, the product yield decreased significantly with the increase of inoculation size. The high fumaric acid yield under 15% inoculation size with a less product titer was attributed to the reduced glucose consumption, which was caused

67 by less active cell and decreased cell activities in the later phase of the fermentation.

Increasing inoculation size from 15% to 25% increased both the amount of SMH carryover and active cell. The increased cell density led to the increase of the product titer, whereas the increase of SMH carryover decreased the product yield. But, too much active cell with high amount of nitrogen under a high inoculation size of 35% can cause excessive cell growth and result in a low product yield. Overall, the inoculation size of 25%

(v/v) gave the best results with a high concentration of 48.4 g/L and a yield of 0.63 g/g glucose.

Figure 3.6 shows the fermentation kinetics under the optimal conditions. After a 7 h lag phase with little glucose consumption, the cell began to accumulate fumaric acid.

Although glucose consumption rate decreased gradually in the latter phase of the fermentation, fumaric acid production seemed steady with a productivity of 0.34 g/(L.h).

In ~140 h fermentation, 50.2 g/L fumaric acid was produced with the yield of 0.72 g/g glucose. Malic acid was the major byproduct with the final concentration of 3.2 g/L.

3.4 Discussion

3.4.1 Comparison of cell morphology to other studies

In this study, through the use of SMH as the nitrogen source, R. oryzae was cultured into the morphology of uniformly dispersed tiny mycelial clumps. As discussed by Paul et al. (1998), mycelial clumps are loose aggregates of hyphae. Compared to pellet developed by using urea and yeast extract as the nitrogen source, they are much smaller

(d ~0.1mm), not sufficiently tightly packed and do not have compact core. Such particular morphology as intermediate one between filaments and pellets may be better

68 for fumaric acid production, because it can reduce the broth viscosity with enhanced mass and oxygen transfer compared to filaments, while does not have the problem of low internal mass transfer happened in pellet, as can be seen from Table 3.3. Similarly, Zhou et al. (2011) reported that decreasing pellet diameter increased fumaric acid yield. Roa

Engel et al. (2011) studied the oxygen profiles in the pellet and found the anaerobic core inside the pellet when the diameter was larger than 0.5 mm. Further research on the detailed effects of the morphologies (mycelial clump, pellet, dispersed filaments, etc.) on medium rheology and oxygen transfer will be investigated in future.

The effects of several influencing factors on cell morphology, such as culture pH, inoculation size and nitrogen source, were investigated in this research. Nitrogen source is of great significance to cell morphology. In this study, R. oryzae grow into loose fluffy mycelial clumps when using SMH as nitrogen source, whereas smooth pellets with varying sizes were formed when using urea. Du. et al. (2003) also found that the pellets were fluffy with a loose outer zone when the medium contained corn steep liquor, while the pellets were larger, compact with a smooth surface when ammonium sulfate was used.

In general, inorganic nitrogen source in the medium caused compact smooth pellet growth and rich organic nitrogen source prompted looser structure. Culture pH is another important factor affecting cell morphology. In this study, a high pH caused large aggregated mycelium while a low pH of ~3.0 led to mycelial clumps. Compared to other studies, similar morphology of aggregated mycelium was formed under a high pH of 5.0 as reported by Zhou et al. (2000), who used 2.0 g/L urea as nitrogen source. The morphology was different at a low pH of 3.0, where uniform pellet with diameters around

1mm was formed. Such difference can be attributed to different nitrogen sources used.

69 On the contrary, Liao et al. (2007) found no significant difference in the pellet formation of R. oyzae within a pH range of 3.0-7.0. Besides the strain difference, another reason might be the use of calcium carbonate, because calcium ions can induce mycelia aggregation during fungal growth. The study on effects of spore concentration showed that increased spore concentration prompted the homogeneity of the cell morphology and mycelium suspension, which was in good agreement with Znidarsic et al. (1998).

However, Liao et al. (2007) reported that the spore concentration did not influence the pellet formation of R. oryzae, while Du et al. (2003) found that for strain Rhizopus chinesis, a high spore concentration (1×106 spores /L) led to the production of more hyphae and a low concentration (1×102 spores /L) resulted in a pellet formation. One reason for the different cell morphologies was the level of spore concentration: low spore concentration level (less than 107 spores /L) are favorable for the pellet growth of strains of Rhizopus (Liao et al., 2007), while in this report a higher spore concentration (above

4×107 spores /L) was applied. Also, it is likely that the effect of spore concentration on cell morphology was dependent on other factors with more influences.

3.4.2 Advantages of soybean meal hydrolysate

This study developed a novel simple culture medium for R. oryzae ATCC 20344 with SMH as the nitrogen source. Soybean meal is the most commonly-used protein source for animal feeds with 67% of the market (Dozier III et al., 2011). The cheap price

($0.4/kg), high protein content and metabolizable energy qualify soybean meal to be a good nitrogen source. However, its application as nitrogen source for industrial fermentation process is few. Among previous reports, there were no studies using

70 soybean meal for fumaric acid production. Compared to urea and yeast extract as the nitrogen source, the SMH showed particular advantages: 1) high protein content in SMH

(both precipitates and dissolved) reduced the lag phase of the seed culture process under the low pH, resulting in a short seed culture period; 2) the protein precipitates of SMH not only acted as the immobilization carriers for the spores prompting the mycelial clumps morphology of R. oryzae, but also provided protein in solid phase for the cell growth which provides a novel method for slow utilization of nitrogen source; 3) a certain amount of SMH carry-over from the seed culture medium was beneficial for the fumaric acid fermentation, indicating the favorable nutritional ingredients in SMH; 4)

Better mass and oxygen transfer in the fermentation medium was achieved due to the smaller size of mycelial clumps with less ethanol production. Overall, SMH showed great potential as nitrogen source for industrial fermentation process.

3.4.3 Comparison with other studies

In this study, under the optimal seed culture and inoculation conditions, the fermentation process reached a product titer of 50.2 g/L and yield of 72%. As listed in

Table 3.1, high fumaric acid production was achieved by applying rotary biofilm contactor (RBC) coupled with simultaneous fermentation and separation. Nevertheless, the difficulty in scaling up RBC system limits its commercial development. Among previous reports, the highest fumaric acid production of 103 g/L with the yield of 0.79 g/g glucose was reached by R. arrhizus (Rhodes et al., 1962). However, in recent ten years, the highest reported fumaric acid production by Rhizopus free cell fermentation decreased to 56.2 g/L with a yield of 0.54 g/g glucose (Fu et al., 2010). The cell was in

71 the morphology of pellet, which was formed by using multi-stage preculture strategy (Fu et al., 2009). Also, the fermentation processes applied two-stage dissolved oxygen (DO) control strategy in stirred tank bioreactor. In this case, we successfully achieved a high product yield of 0.72 g/g glucose in shake-flask fermentation without additional control.

Also, the initial glucose concentration in the fermentation medium was less than 90 g/L.

Generally, a high C/N ratio of 120–250 (w/w) is necessary for a high product titer of fumaric acid (Yang et al., 2011). Therefore, it is believed that by adding more glucose and applying sophisticated DO and/or temperature control on bioreactor level together with in situ recovery, the fumaric acid fermentation can be further improved.

3.5 Conclusion

A novel simple culture medium with SMH as the nitrogen source was developed in this study for R. oryzae ATCC 20344. Uniformly dispersed mycelial clumps (d~0.1mm) were formed with enhanced subsequent fumaric acid production. The optimal seed culture conditions were initial pH of 3.0, SMH concentration of 30% (v/v), spore concentration of 8×107 /L and glucose concentration of 10 g/L. With an inoculation size of 25%, the fermentation reached a product titer of 50.2 g/L with a yield of 0.72 g/g glucose. Compared to the results of other reports, this process using mycelial clumps with

SMH as the nitrogen source showed promising potential for improving fumaric acid production.

72 3.6 Reference

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Cao, N.J., Du, J.X., Gong, C.S., Tsao, G.T. 1996. Simultaneous production and recovery fumaric acid from immobilized Rhizopus oryzae with a rotary biofilm contactor adsorption column. Appl Environ Microbiol 62: 2926-2931.

Cao, N.J., Du, J.X., Chen, C.S., Gong, C.S., Tsao, G.T. 1997. Production of fumaric acid by immobilized Rhizopus using rotary biofilm contactor. Appl Biochem Biotechnol 63-65:387-394

Dozier III, W.A., Hess, J.B. 2011. Soybean Meal Quality and Analytical Techniques, In: Hany El-Shemy (eds) Soybean and Nutrition, pp: 111-124. Place: InTech

Du, L.X., Jia, S.J., Lu, F.P. 2003. Morphological changes of Rhizopus chinesis 12 in submerged culture and its relationship with antibiotic production. Process Biochem 38: 1643-1646.

El-Enshasy, H.A. (2007) Filamentous fungal cultures – process characteristics, products, and applications. In: Yang ST (eds.) Bioprocessing for value-added products from renewable resources - new technologies and applications, pp: 225-261. Place: Elsevier.

Fu, Y.Q., Xu, Q., Li, S., Huang, H., Chen, Y. 2009. A novel multi-stage preculture strategy of Rhizopus oryzae ME-F12 for fumaric acid production in a stirred-tank reactor. World J Microbiol Biotechnol, 25: 1871-1876.

Fu, Y.Q., Li, S., Chen, Y., Xu, Q., Huang, H., Sheng, X.Y. 2010. Enhancement of fumaric acid production by Rhizopus oryzae using a two-stage dissolved oxygen control strategy. Appl Biochem Biotechnol, 162: 1031-1038.

Gangl, I.C., Weigand, W.A., Keller, F.A. 1990. Economic comparison of calcium fumarate and sodium fumarate production by Rhizopus arrhizus. Appl Biochem Biotechnol. 24-25: 663-677.

Liao, W., Liu, Y., Chen, S. 2007. Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production. Appl Biochem Biotechnol 136-140: 689-701.

Ling, L.B., Ng, T.K. 1989. Fermentation process for carboxylic acids. US 4,877,731. 31, Oct, 1989.

Liu, Y., Liao, W., Chen, S. 2008. Study of pellet formation of filamentous fungi Rhizopus oryzae using a multiple logistic regression model. Biotechnol Bioeng 99: 117-128.

73 Papagianni, M. 2004. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22: 189-259.

Paul, G.C., Thomas, C.R. 1998. Characterisation of mycelial morphology using image analysis. Adv Biochem Eng Biotech 60: 1-59.

Rhodes, R.A., Moyer, A.J., Smith, M.L., Kelley, S.E. 1959. Production of fumaric acid by Rhizopus arrhizus. Appl Microbiol 7:74-80.

Rhodes, R.A., Lagoda, A.A., Jackson, R.W., Misenhei, T.J., Smith, M.L., Anderson, R.F. 1961. Production of fumaric acid in 20 liter fermentors. Appl Microbiol 10: 9-15.

Roa Engel, C.A., Straathof, A.J.J., Zijlmans, T.W., van Gulik, W.M., van der Wielen, L.A.M. 2008. Fumaric acid production by fermentation. Appl Microbiol Biotechnol 78: 379-389.

Roa Engel, C.A., van Gulik, W.M., Marang, L., van der Wielen, L.A.M., Straathof, A.J.J. 2011. Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae. Enzyme Microb Technol. 48: 39-47.

Yang, S.T. 2007. Bioprocessing - from biotecnology to biorefinery. In: Yang ST (eds.) Bioprocessing for value-added products from renewable resources - new technologies and applications, pp: 1-24. Place: Elsevier.

Yang , S.T., Zhang, K., Zhang, B., Huang, H. 2011. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition, 3, 163-177.

Yu, S., Huang, D., Wen, J., Li, S., Chen, Y., Jia, X. 2012. Metabolic profiling of a Rhizopus oryzae fumaric acid production mutant generated by femtosecond laser irradiation, Bioresource Technol 14: 610-615.

Zhou, Y., Du, J., Tsao, G.T. 2000. Mycelial pellet formation by Rhizopus oryzae ATCC 20344, Appl Biochem Biotechnol 84-86: 777-789.

Zhou, Z., Du, G., Hua, Z., Zhou, J., Chen, J. 2011. Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation. Bioresource Technol 102: 9345-9349.

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74 Table 3.1 Fumaric acid production from various morphologies by Rhizopus sp.

Titer Yield Productivity Strain Morphology Reference (g/L) (g/g) (g/L·h)

R. arrhizus Rhodes et al., Dispersed 103 0.79 1.22 ATCC 1961 mycelia 52918 73.0 0.72 0.50 Gangl et al., 1990 R. delemar ATCC Pellet 39.6 0.40 0.54 Zhou et al., 2011 10260 92.0 0.85 4.25 Cao et al., 1996 Biofilm 75.5 0.75 3.78 Cao et al., 1997 30~38.2 0.35~0.45 - Zhou et al., 2000 31 - 0.32 Liao et al., 2007 R. oryzae ATCC 42.5 0.56 0.51 Fu et al., 2009 Pellet 20344 56.2 0.54 0.7 Fu et al., 2010 Roa Engel et al., 19.8 0.19 0.12 2011 49.4 0.56 - Yu et al., 2012 Mycelial 50.2 0.72 0.33 This study clumps

75 Table 3.2 Summary on cell morphology cultured under different conditions

Spore conc. pH Nitrogen source Morphology Figure 1 (104/mL)

Urea (2 g/L) 8 Yeast extract Pellets (d ≈ 0.5~4 mm) D 8 (2 g/L) SMH (4%, v/v) 4, 8 Large aggregated mycelium F

Pellets interwined with mycelial SMH (10%, v/v) 4, 8 A 3 clumps, dispersed non-uniformly

Pellets interwined with mycelial SMH (20%, v/v) 4 B clumps, dispersed uniformly SMH (20%, v/v) 8 Uniformly dispersed mycelial SMH C 4 clumps (30/40%, v/v)

Large clump and pellet interwined 4 SMH (20%, v/v) 4, 8 with mycelial clumps, dispersed E non-uniformly

5, 6 SMH (20%, v/v) 4, 8 Large aggregated mycelium F

76 Table 3.3 Effects of nitrogen source in seed culture media on cell morphology and

subsequent fumaric acid fermentation

Nitrogen Consumed Fumaric acid FA Yield Malic acid Ethanol Morphology source Glucose (g/L) (g/L) (g/g) (g/L) (g/L) SMH Mycelial clumps 70.9±4.1 38.3±0.2 0.54±0.03 3.7±0.4 1.7±0.4 Urea 90.6±6.3 22.5±0.4 0.25±0.01 2.1±0.0 20.8±1.6 Pellets Yeast (d ≈ 0.5~4 mm) 93.3±6.9 19.3±1.8 0.21±0.02 2.2±0.1 22.9±2.6 exteact

Note: The protein concentrations in seed culture mediums with SMH and yeast extract as nitrogen source were 0.42 g/L and 0.05 g/L, respectively.

77 Table 3.4 Effects of seed age on fumaric acid fermentation (inoculation size of 15%)

Seed Initial protein Consumed Fumaric Yield Malic acid Cell dry age (h) conc. (g/L) Glucose (g/L) acid (g/L) (g/g) (g/L) weight (g) 0.28 0.065 18 0.14 56.9±2.9 15.9±3.4 7.5±4.2 ±0.06 ±0.007 0.46 0.068 21 0.10 57.9±7.7 26.9±10.2 7.2±1.8 ±0.11 ±0.017 0.66 0.067 24 0.07 52.9±1.7 34.8±0.8 4.6±0.0 ±0.03 ±0.003

78 Table 3.5 Effects of inoculation size and cell density on fumaric acid fermentation

Ino Cell density Consumed Fumaric Yield Malic acid Cell dry -size (103/mL) Glucose (g/L) acid (g/L) (g/g) (g/L) weight (g) 2.4±0.5 52.9±1.7 34.8±0.8 0.66±0.03 4.6±0.0 0.067±0.003 15% 6.0±0.9 49.5±0.4 36.9±2.7 0.74±0.06 4.9±0.1 0.156±0.014 4.0±0.8 87.0±2.5 43.1±0.8 0.50±0.02 6.8±0.2 0.089±0.004 25% 10.0±1.0 77.1±1.8 48.4±0.9 0.63±0.02 6.0±0.2 0.146±0.010 35% 14.0±1.2 85.4±3.7 41.1±0.3 0.48±0.02 3.0±0.7 0.162±0.010

79

Figure 3.1 Cell morphology of Rhizopus oryzae developed under different conditions. A, B, C, D, E, F: see Table 3.2 for details; G/H: samples extracted from B/C

show the more/fewer connections among the mycelial clumps; I: pellet (d ~1mm)

80

Figure 3.2 Comparison of soybean meal hydrolysate at natural pH (right) and pH 3

with protein precipitates (left)

81 120 25 Urea

100 20

80 Glucose 15 60 Fumaric acid Malic acid 10

40 Ethanol

Acids (g/L) Acids Glucose (g/L) Glucose 20 5

0 0 A 0 20 40 60 80 100 120 Yeast extract 25

100

20 80

60 Glucose 15 Fumaric acid

40 Malic acid 10 Acids (g/L) Acids

Glucose (g/L) Glucose Ethanol 20 5

0 0 B 0 20 40 60 80 100 100 50 Glucose SMH 80 Fumaric acid 40

Malic acid

60 30

40 20

20 10 (g/L) Acids Glucose (g/L) Glucose

0 0 0 20 40 60 80 100 120 140 160 C Time (h)

Figure 3.3 Fermentation profiles of the seeds prepared by the three nitrogen sources: urea (A), yeast extract (B), SMH (C)

82 0.80 4.00E+07 8.00E+07 0.60

0.40

0.20 Cell dry (g) weightdry Cell 0.00 A 10 20 30 40

50

4.00E+07 40 8.00E+07 30

20

10

Fumaric acid (g/L) acid Fumaric 0 B 10 20 30 40

0.80 4.00E+07 8.00E+07

0.60

0.40

Yield (g/g) Yield 0.20

0.00 C 10 20 30 40 SMH conc. (%, v/v)

Figure 3.4 Effects of SMH concentration and spore concentration on cell growth and subsequent fermentation process A. Effects on cell dry weight in 50 ml cell culture medium; B, C: Effects on subsequent fumaric acid product titer and yield 83 12 0.5

10 0.4

8 0.3 6 0.2

4 Glucose

Proetin (g/L) Proetin Glucose (g/L) Glucose Protein 2 0.1

0 0 0 5 10 15 20 25 Time (h)

Figure 3.5 Glucose and dissolved protein profiles of the seed culture by using soybean

meal hydrolysate (20%, v/v) at pH 3

84 60 Glucose 80 Fumaric acid 45

Malic acid 60

30

40

Acids (g/L) Acids Glucose (g/L) Glucose 20 15

0 0 0 20 40 60 80 100 120 140 160 Time (h)

Figure 3.6 Kinetics of batch fermentation of glucose with soybean meal hydrolysate by R.

oryzae at pH 5.5 and 32 oC

85 CHAPTER 4

Optimization on Fumaric Acid Production by Rhizopus oryzae in 5-L Stirred-Tank

Bioreactor

4.1 Introduction

The accumulation of fumaric acid occurs when fungal cells are cultured under stress with limited cell growth, such as under the condition of excess carbon source and limited nitrogen source in the fermentation medium. However, cells may lose their activities over time when cultured under N-limited conditions (Yang et al., 2011). Thus, the relative amount of carbon source (C) to nitrogen source (N) present in the medium is the most important factor in the fermentation and a high C/N ratio is usually required for overproduction of fumaric acid. Previously we developed a novel seed culture method by using soybean meal hydrolysate (SMH) as nitrogen source for controlling the cell morphology of R. oryzae ATCC 20344 with enhanced fumaric acid production. The fermentation in shake flask reached a product titer of 50.2 g/L with yield of 0.72 g/g glucose. However, the process was difficult to scale up on 5-L stirred tank bioreactor

(STB): the fumaric acid production was much lower with a decrease of cell activity in the later phase of fermentation. Therefore, in the first step, we tried to improve the process by adding nitrogen source to maintain a high cell activity.

Second, CaCO3 is the most frequently used neutralizing agent to maintain medium

86 pH around the fermentation-favored value of 5.0. As shown in the metabolic pathways of

R. oryzae in Figure 4.1 (Wright et al.,1996), fumaric acid biosynthesis involves CO2 fixation by pyruvate carboxylase catalyzing the conversion of pyruvate to oxaloacetate, which is the precursor to malate and fumarate. The reaction between CaCO3 and fumaric acid liberates CO2, which can be used in the reaction for oxaloacetate formation from pyruvate and thus benefits the fumaric acid fermentation. However, the use of CaCO3 produced large quantities of solid waste calcium compounds causing environmental pollution (Fu et al., 2009). Due to the high solubility of sodium fumarate, several attempts have been made to use Na2CO3 or NaOH as neutralizing agent for an easier and cheaper downstream processing (Gangl et al., 1990; Zhou et al., 2002). However, fumraic acid production yield was significantly reduced in these processes, possibly due to the lack of CO2 compared to the use of CaCO3. Roa Engel et al. (2011) found that increasing CO2 content in the inlet gas was helpful to fumaric acid production. Therefore, in this study, we tried to improve the process with NaOH as the neutralizing agent by providing CO2-enriched inlet gas for CO2 fixation.

4.2 Materials and Methods

4.2.1 Cultures and meida

R. oryzae ATCC 20344 used in this study was cultured on YMP (yeast extract, malt extract, and peptone) agar plates containing 3 g/L yeast extract, 3 g/L malt extract, 3 g/L peptone, 20 g/L glycerol and 20 g/L agar. After ~7 day incubation at 32 oC, the spores were harvested by washing with sterile distilled water and collected as a spore suspension, which was stored at 4 oC.

87 Soybean meal containing 44%-50% crude protein, 0.5% fat, 7% fiber and 12% moisture was obtained from Cargill (Sidney, OH). To prepare the soybean meal hydrolysate (SMH), 20 g of soybean meal was added into 400 mL of 0.25 N HCl, and the mixture was autoclaved at 121 oC for 30 min. The hydrolysate was filtered under sterile conditions, and the filtrate was stored at room temperature for future use. The protein content in the hydrolysate was determined by BioRad protein assay and found to be ~12 g/L.

4.2.2 Seed culture preparation

All seed cultures were prepared in 250 mL shake-flasks containing 50 mL of the growth medium with 10 g/L glucose and 20% SMH (v/v) as nitrogen source. The pH of the medium was then adjusted to 3.0 using NaOH. After inoculation with the spores in suspension, the cultures were incubated at 35~37 oC in a rotary shaker agitated at 220 rpm for 24 h.

4.2.3 Fermentation

The fermentation process was carried out in 5-L stirred tank bioreactor (STB).

Unless otherwise noted, the default conditions for fermentation in STB were: the volume of the medium in the tank was 3 L; after 24 h seed culture, all the seeds were harvested, washed by sterile distilled water, and then transferred into bioreactor with an inoculation size of 10%; the fermentation medium contained 80-100 g/L glucose, 40 mL/L SMH, 0.6 g/L KH2PO4 and 0.5 g/L MgSO4; the SMH with pH of ~4.0 adjusted by NaOH was added into the bioreactor separately; the fermentation pH was maintained above 5.0 by

88 adding excessive CaCO3; the rotation speed was 500 rpm and the air flow rate was 2.0 vvm; the fermentation was performed at 37 oC for 4~5 d.

The effects of nitrogen source (seed culture carryover, 0.5 g/L urea, 20, 40, 60 mL/L

SMH) were studied. For the case without additional nitrogen source, the inoculums containing the harvested seeds and seed culture carryover were transferred directly into the bioreactor without washing and the inoculation size was 25%. For the case with 0.5 g/L urea, urea was sterilized separately and added into the bioreactor after sterilization.

The effects of pH neutralizing agents (NaOH and CaCO3), and CO2 addition (with / without CO2) were investigated. For the cases using NaOH, 200 g/L NaOH was applied.

For the cases with CO2 addition, pure CO2 was mixed with air in a ratio of 1:5 as the inlet gas and entered the bioreactor after the membrane filtration.

The effects of cell density (~2500, ~5000, ~8000 mycelial clumps/mL) on fumaric acid fermentation were also studied. With the same inoculation size of 10%, different amounts of seed cultures were prepared to achieve different cell densities in the fermentation process. Due to the removal of seed culture carryover by washing, the only varying factor under the same inoculation size was cell density.

4.2.4 Analytical methods

HPLC was used to analyze the organic compounds including glucose, lactic acid, malic acid, fumaric acid and ethanol present in the fermentation broth. Because calcium fumarate was precipitated in the fermentation broth, sample pretreatment was required for

HPLC detection: dilute hydrochloric acid was added to neutralize excessive CaCO3 and acidify fumarate. Due to the low solubility of fumaric acid in water (6.3 g/L at 25 oC), the

89 broth was heated to 80 oC to increase the solubility of fumaric acid. The HPLC system

(Shimazu Scientific Instruments) equipped with a RID-10A refractive index detector and an organic acid analysis column (HPX-87H Bio-Rad, Richmond, CA) was operated under the following conditions: sample volume of 15 μl, mobile phase of 0.005 M H2SO4, flow rate of 0.6 mL/min, and column temperature of 45 oC.

4.3 Results

4.3.1 Effects of nitrogen source

Figure 4.2A shows typical fermentation kinetics of fumaric acid production in STB without nitrogen source. The nitrogen was mainly introduced from the seed culture carryover. The fumaric acid production almost ceased after ~75 h fermentation.

Decreasing glucose consumption rate and increasing dissolved oxygen (DO) concentration in the latter phase of the fermentation indicated a loss of cell activity. After

140 h fermentation, there was still ~42 g/L glucose remaining. The final product titer was

16 g/L with a low productivity of 0.12 g/(L.h).

In order to improve the cell activity in the latter phase, different strategies of nitrogen source addition were studied. Figure 4.2B shows the kinetics of the fermentation with initial addition of 0.5 g/L urea. The production process was divided into three phases:

0-17 h, cell growth phase with decreased DO level and little fumaric acid production

(<4.5 g/L, yield: 0.19 g/g glucose); 17-49 h, acid production phase with high cell activity and high fumaric acid production (yield: 0.46 g/g glucose, productivity: 0.5 g/(L.h));

49-113 h, latter phase with a high DO level, low cell activity and slow fumaric acid production (yield: 0.22 g/g glucose, productivity: 0.14 g/(L.h)). The final product titer

90 reached 30 g/L with an overall yield of 0.27 g/g glucose and productivity of 0.27 g/(L.h).

Compared to the case without nitrogen source, the product titer and productivity increased significantly. However, due to the low yield in cell growth and latter phases, the overall yield was still low.

Therefore, enhancing the yield during the cell growth phase and prolonging the production phase with high cell activity is the key to increase product titer and yield. For such a purpose, the effects of adding different amounts of SMH (20, 40, 60 ml/L) in the fermentation medium were investigated with the results shown in Figure 4.2 C, D and E.

For both fermentations with 20 and 40 ml/L SMH, higher fumaric acid yields (~0.45 g/g glucose) were observed in the initial cell growth phase. However, too much SMH (60 ml/L) caused excessive cell growth and high cell aspiration rate (high CO2 percentage, data not shown) with a low yield (<0.3 g/g glucose). But, at the lower SMH concentration

(20 ml/L) the decrease in cell activity was observed in the latter phase of the fermentation, while increasing SMH amount to more than 40 ml/L significantly enhanced the cell activity during this period with increased product titer. Additionally, the use of SMH as nitrogen source reduced malic acid production to 3.6 g/L compared to that with urea as nitrogen source (6.9 g/L). The results are summarized in Table 4.1. Overall, with high product titer and yield, 40 ml/L SMH was determined as the best nitrogen source.

4.3.2 Effects of CO2 addition

Figure 4.3 shows the fermentation kinetics with NaOH as the neutralizing agent to control the medium pH above 5. Compared to the fermentation with CaCO3 (shown in

Fig. 4.2D), there was no CO2 generation in this case. The fumaric acid production was

91 low at the beginning of 24 h with titer of 6.3 g/L and yield of 0.2 g/g glucose, whereas in the case of CaCO3 the fumaric acid titer reached 7.2 g/L with yield of 0.38 g/g glucose in

18 h. Then, fumaric acid was produced steadily from 24 h to 115 h with an average yield of 0.48 g/g glucose. The decreased glucose consumption rate indicated a loss of cell activity in the latter phase of fermentation. The fermentation stopped with ~20.8 g/L glucose remaining. The final fumaric acid titer reached 25. 6 g/L with overall yield of

0.39 g/g glucose and productivity of 0.22 g/(L.h).

Figure 4.4 shows the fermentation kinetics with NaOH as neutralizing agent and the addition of pure CO2 into the inlet gas. Aeration with air containing 16.7% CO2 was used.

Compared to the case without CO2, The fumaric acid production at the beginning of 24 h fermentation was enhanced to titer of 8.9 g/L and yield of 0.32 g/g glucose. A higher cell activity was achieved in the latter phase of fermentation, leading to increased product titer of 32.6 g/L and productivity of 0.29 g/(L.h). However, the malic acid production (7.4 g/L) was also increased with CO2 addition.

Figure 4.5 shows the fermentation kinetics with CaCO3 as neutralizing agent and the addition of pure CO2 into the inlet gas. Compared to the fermentation with CaCO3 but without CO2 addition, the major difference lied in the enhanced fumaric acid production rate in the latter phase of fermentation. The productivity after 30 h was similar to the value at the beginning with the average productivity of 0.4 g/(L.h), whereas without CO2 addition the productivity decreased significantly to 0.13 g/(L.h) after 50 h fermentation with the average productivity of 0.23 g/(L.h). Table 4.2 compares the fermentation results of the above four cases. Overall, aeration with air containing CO2 greatly increased the productivity of fumaric acid production, which was more pronounced when CaCO3 was

92 used as neutralizing agent. The reason was possibly the high dissolved CO2 concentration in this case. Additionally, the process using NaOH and CO2 addition had similar fumaric acid production and higher productivity as compared to the process using CaCO3, indicating the possibility of replacing the use of CaCO3 with NaOH and CO2 addition for a simplified downstream process.

4.3.3 Effects of cell density

Previously, we reported that cell density played an important role in fumaric acid production in shake flasks. Thus, the effects of cell density on the fermentation process in

STB were investigated. Figure 4.6 shows the fermentation kinetics under different cell densities (~2500, ~8000 mycelial clumps /mL) with the results summarized in Table 4.3.

The fermentation kinetics of cell density of ~5000 /ml was shown in Figure 4.5. For all the cases, aeration with air containing 16.7% CO2 was applied. For the cases with cell densities of ~2500 and ~5000 /ml, the kinetics of the two fermentation processes were similar with similar product titers and yields, whereas the higher cell density increased productivity by 15%. However, further increasing cell density to ~8000 /mL did not facilitate fumaric acid production. The fumaric acid titer, yield and productivity all dropped by 21%, 17%, and 25%, respectively. The reason can be attributed to high glucose consumption caused by increased cell growth and aspiration. Overall, the best cell density range was identified as 2500~5000 /mL.

93 4.4 Discussion

4.4.1 Effects of SMH

In this study, the effects of nitrogen source on fumaric acid production by R. oryzae were investigated. Previously, it was reported that inorganic nitrogen sources, such as urea and (NH4)2SO4, were superior to fumaric acid production whereas organic nitrogen sources, such as yeast extract, caused excessive cell growth with less fumaric acid production (Yang et al., 2011). On the contrary, this study found that, compared to the inorganic source of urea, the use of SMH can not only increase the fumaric acid yield during cell growth phase but also prolong the cell activity with enhanced product titer.

Soybean meal is the residual product after the extraction of oil from whole soybeans with high protein content. It is the most commonly-used protein source for animal feed with

67% of the market (Dozier III et al., 2011). The rich mineral components of soybean meal eliminated the need for adding extra mineral elements into the medium (Batal et al.,

2010). Also, it was reported that the microbial metabolism can improve the antioxidant activity of soybean meal with enhanced reducing power (Wongputtisin et al., 2007), which is beneficial for fumaric acid production. In addition, it was found that protein precipitates were formed when the pH of SMH was adjusted to >3, as discussed in

Chapter 3. The formation of these precipitates is due to the change of medium pH, which is close to some proteins’ isoelectric point where the protein has a minimal solubility.

These protein precipitates provided protein in solid form, which not only slowed down the protein utilization but also prolonged the availability of nitrogen source during the fermentation process, hence leading to the enhanced cell activity in the latter phase. But, under a high cell density (Figure 4.5B), the protein precipitate was quickly digested by

94 the high amount of the cells with excessive cell growth and glucose consumption at the beginning, resulting in a decrease of fumaric acid production. Overall, with its cheap price ($0.4/kg), high protein content, rich mineral components, high reducing power and slow release of nitrogen source due to protein precipitation at pH >3, soybean meal showed great potential as nitrogen source for industrial fermentation process.

4.4.2 Effects of CO2 addition

As mentioned before, CO2 fixation is essential for high fumaric acid production. The

CO2 produced from the reaction between CaCO3 and acids can be used as a dditional carbon source for oxaloacetate formation from pyruvate. The importance of this reaction was illustrated by Zhou et al. (2002). They compared the effects of different neutralizing agents (Ca(OH)2, CaCO3, NaHCO3) on fumaric acid production. The carbonate salts, both CaCO3 and NaHCO3, significantly enhanced the production compared to Ca(OH)2.

In our studies, the improvement from using NaOH to CaCO3 mostly lied in the enhanced fumaric acid titer with a similar yield and productivity. For the fermentation with NaOH together with CO2 addition, the results were even better than the case with CaCO3 with higher productivity and similar product titer and yield. Roa Engel et al. also studied the effects of CO2 by adding it into inlet gas (2011). They found that the air enriched by CO2 with content less than 10% was beneficial for fumaric acid production, whereas a high concentration of CO2 negatively affected fumarate production probably due to oxygen limitation. Also, as reported by many researchers, the inhibitory effects of CO2 increased with the increase of dissolved CO2 concentration (Mcintyre et al., 1998). Contrary to their findings, no inhibitory effect of CO2 was observed when aeration with air containing 16.7%

95 CO2 was used, instead, the CO2 addition significantly enhanced productivity by 76%. It should be noted that the major difference between this study and the other two studies was that those studies used nitrogen-free medium while this study used 40 mL/L SMH as nitrogen source. Therefore, another important advantage of SMH is to increase the cell

+ tolerance against tough conditions such as high Na / dissolved CO2 concentrations.

4.5 Conclusion

In conclusion, this is the first study using soybean meal as nitrogen source for fumaric acid production. Results showed that using 40 mL/L SMH as nitrogen source successfully improved the production yield during the cell growth phase and prolonged the production phase with high cell activity. The air enriched by CO2 with content of 16.7% significantly enhanced productivity by 76% and product titer by 13%. With a cell density of ~5000 mycelial clumps /ml, a high fumaric acid production was reached with product titer of 35.6 g/L, yield of 0.4 g/g glucose and productivity of 0.4 g/(L.h). Additionally, the process using NaOH and CO2 addition had similar fumaric acid production and higher productivity as compared to the process using CaCO3, indicating the possibility of replacing the use of CaCO3 with NaOH and CO2 addition for a simplified downstream process. It is believed that the fermentation process can be further improved by more sophisticated operations, such as multiple-phase DO control, glucose / nitrogen source feeding strategies, etc. Also, with the advantages of cheap price, high protein content, rich mineral components, high reducing power induced by microbial metabolism, and slow release of nitrogen source due to protein precipitation at pH >3, soybean meal showed great potential as nitrogen source for industrial fermentation process.

96 4.6 Reference

Batal, A.B., Dale, N.M., Saha, U.K. 2010. Mineral composition of corn and soybean meal. J Appl Poultry Res 19: 361-364.

Dozier III, W.A., Hess, J.B. 2011. Soybean Meal Quality and Analytical Techniques, In: Hany El-Shemy (eds) Soybean and Nutrition, pp: 111-124. Place: InTech

Fu, Y., Chen, Y., Li, S., Huang, H. 2009. Fixed-bed adsorption study for fumaric acid removal from aqueous solutions by Amberlite IRA-400 resin. Chem Eng Technol 10: 1625-1629.

Gangl, I.C., Weigand, W.A., Keller, F.A. 1990. Economic comparison of calcium fumarate and sodium fumarate production by Rhizopus arrhizus. Appl Biochem Biotechnol. 24-25: 663-677.

Mcintyre, M. and Mcneil, B. 1998. Morphogenetic and biochemical effects of dissolved carbon dioxide on filamentous fungi in submerged cultivation. Appl Microbiol Biotech 50: 291-298.

Roa Engel, C.A., Straathof, A.J.J., Zijlmans, T.W., van Gulik, W.M., van der Wielen, L.A.M. 2008. Fumaric acid production by fermentation. Appl Microbiol Biotechnol 78: 379-389.

Roa Engel, C.A., van Gulik, W.M., Marang, L., van der Wielen, L.A.M., Straathof, A.J.J. 2011. Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae. Enzyme Microb Tech. 48: 39-47.

Wongputtisin, P., Khanongnuch, C., Pongpiachan, P., Lumyong, S. 2007. Antioxidant activity improvement of soybean meal by microbial fermentation. Res J Microbiol 2(7): 577-583.

Wright, B.E., Longacre, A., Reimers, J. 1996. Models of Metabolism in Rhizopus oryzae. J Theor Biol 182(3): 453-457.

Yang, S.T. 2007. Bioprocessing - from biotecnology to biorefinery. In: Yang ST (eds.) Bioprocessing for value-added products from renewable resources - new technologies and applications, pp: 1-24. Place: Elsevier.

Yang , S.T., Zhang, K., Zhang, B., Huang, H. 2011. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition, 3, 163-177.

Zhou, Y., Du, J., Tsao, G.T. 2002. Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents. Bioproc Biosyst Eng 25: 179-181.

97 Table 4.1 Effects of different nitrogen sources on fumaric acid fermentation

Cons. Glucose Fumaric Productivity Malic acid Nitrogen source Yield (g/L) acid (g/L) (g/L.h) (g/L) Seed culture 40 16 35% 0.117 5.0 carryover 0.5 g/L Urea 110 30 27% 0.272 6.9 20 ml/L SMH 44 16.3 42% 0.209 0.4 40 ml/L SMH 78 31.4 40% 0.227 3.6 60 ml/L SMH 117 33.2 28% 0.292 5.2

98 Table 4.2 Effects of CO2 addition on fumaric acid fermentation under different

neutralizing agent (NaOH, CaCO3)

Cons. Glucose Fumaric acid Productivity Malic acid CO addition Yield 2 (g/L) (g/L) (g/L.h) (g/L)

None (NaOH) 65.9 25.6 39% 0.223 2.1

NaOH + CO2 86.2 32.6 38% 0.286 7.4

CaCO3 78 31.4 40% 0.227 3.6

CaCO3 + CO2 88.6 35.6 40% 0.400 5.6

99 Table 4.3 Effects of cell density on fumaric acid fermentation

Cell density Cons. Glucose Fumaric acid Productivity Malic acid Yield (Mycelial clumps/ml) (g/L) (g/L) (g/L.h) (g/L)

~2500 88.8 33.6 38% 0.348 4.9 ~5000 88.6 35.6 40% 0.400 5.6 ~8000 84 28.1 33% 0.302 3.7

100 Ext- Glucose Glucose Citrate Ext- Acetyl Citrate -CoA G-6-P Oxalo- keto- acetate2 CO2 4 glutarate

Malate2 succinate F-1,6-bP Pyruvate2 3 5 Fumarate2 Ext- Culture Fumarate Mitochondrion Medium Pyruvate1 7 1

CO2 Acetadehyde Cytosol 6 Oxaloacetate1 8 2 CO2 3 EtOH Lactate Malate1 Fumarate1

Ext-Ethanol Ext-lactate Ext-Malate Ext-Fumarate

1 pyruvate carboxylase 2 malate dehydrogenase 3 fumarase 4 pyryvate dehydrogenase complex 5 succnate dehydrogenase 6 lactate dehydrogenase 7 pyruvate decarboxylase 8 ethanol dehydrogenase

Figure 4.1 Metabolic pathways in R. oryzae (Wright et al., 1996)

101 40 100 Glucose DO

Malic acid 30

80

Fumaric acid

60 20

40 DO (%) DO

10 Products (g/L) Products Glucose (g/L) Glucose 20

0 0 0 25 50 75 100 125 150

Time (h)

A Without nitrogen 150 40 Glucose DO 120 Fumaric acid

30

Malic acid

90 20

60

DO (%) DO

Glucose (g/L) Glucose 10 30 (g/L) Products

0 0 0 20 40 60 80 100 120

Time (h)

B With 0.5 g/L Urea

Figure 4.2 Fermentation kinetics of fumaric acid production by R. oryzae in 5-L stirred tank bioreactor with different nitrogen sources (continued)

102 Figure 4.2: continued

100 35 Glucose 30 80 Fumaric acid

25 Malic acid 60 20

40 15

10 Glucose (g/L) Glucose

20 (g/L) Products 5

0 0 0 20 40 60 80 100 120 Time (h) C With 20 ml/L SMH

100 50 Glucose 45

80 Fumaric acid 40

Malic acid 35 60 30 25 40 20

15 Glucose (g/L) Glucose

20 10 (g/L) Products 5 0 0 0 20 40 60 80 100 120 140 Time (h) D With 40ml/L SMH

(continued)

103 Figure 4.2: continued

100 50 Glucose 45 80 Fumaric acid 40

Malic acid

35 60 30 25 40 20 15

Glucose (g/L) Glucose 20 10 Products (g/L) Products 5 0 0 0 20 40 60 80 100 120 Time (h) E With 60 ml/L SMH

104 100 50 Glucose 45 80 Fumaric acid 40 Malic acid

35

60 30 25

40 20 Acid (g/L)Acid

Glucose (g/L) Glucose 15 20 10 5 0 0 0 20 40 60 80 100 120 Time (h)

Figure 4.3 Fermentation kinetics with pH control by 200 g/L NaOH (without CO2)

105 100 50 Glucose 45 80 Fumaric acid 40

Malic acid

35 60 30 25

40 20 Acid (g/L)Acid

Glucose (g/L) Glucose 15 20 10 5 0 0 0 20 40 60 80 100 120 Time (h)

Figure 4.4 Fermentation kinetics with pH control by 200 g/L NaOH (with CO2 addition)

106 100 50 Glucose 45 80 Fumaric acid 40 Malic acid

35

60 30 25

40 20 Acid (g/L)Acid

Glucose (g/L) Glucose 15 20 10 5 0 0 0 20 40 60 80 100 Time (h)

Figure 4.5 Fermentation kinetics with pH control by CaCO3 and CO2 addition

(cell density of ~5000/ml)

107 100 50 Glucose 45 80 Fumaric acid 40

Malic acid 35 60 30 25

40 20 (g/L) Acid 15 Glucose (g/L) Glucose 20 10 5 0 0 0 20 40 60 80 100

Time (h) A low cell density of ~2500/ml 50 100 Glucose 45 Fumaric acid 40 80 Malic acid

35

30 60 25 20 40

15 (g/L)Acid Glucose (g/L) Glucose 20 10 5 0 0 0 20 40 60 80 100

Time (h)

B high cell density of ~8000/ml

Figure 4.6 Fermentation kinetics with different cell densities

108

CHAPTER 5

Characteristics and Mechanism Study of Fumaric Acid Adsorption onto IRA900 Ion

Exchange Resin

5.1 Introduction

A major problem associated with traditional fumaric acid fermentation is low productivity. Reduced production rate was observed at the later phase of the fermentation, which was probably resulted from product inhibition effect and declined cell activity

(Yang et al., 2011). In situ product recovery has been proven to effectively reduce product inhibition effect and enhance the productivity of the process (Cen and Tsao,

1993). Nevertheless, there are few reports on applying the technology of in situ product recovery to fumaric acid production. Cao et al. (1996) developed an integrated system using rotary biofilm contactor (RBC) coupled with ion exchange column for simultaneous fermentation and separation. High fumaric acid titer (>75.5 g/L) and productivity (>3.78 g/(l.h)) were achieved. However, the complexity of this system and the high resin cost during the scale-up limit its commercial development.

Ion-exchange adsorption has been used extensively and successfully on in situ product recovery for carboxylic acid (Ataei et al., 2008; Cao et al., 2002; Wang et al.,

2000). Strong basic anion-exchange resins, Amberlite IRA-900 and IRA400, have been used for fumaric acid adsorption (Cao et al., 1996; Fu et al., 2009). However, the underlying mechanism and dynamics of the adsorption process are still unknown. A

109 comprehensive and insightful study is necessary to understand the interactions between the resins and fumaric acid molecules in order to design an efficient adsorption system.

In this study, our objective is to find a suitable ion exchange resin for in situ fumaric acid recovery from fermentation broth and characterize the adsorption process. Amberlite

IRA-900 was selected due to its high adsorption capacity at the fermentation-favored pH of 5, high selectivity against byproducts (glucose and malic acid) and easy desorption with a high recovery yield. Due to the importance of pH for ion exchange adsorption, the effects of pH on the fumaric acid adsorption process were investigated. The pH isotherm and kinetics were also modeled to study the mechanism of the adsorption process. The fundamental data will be useful to understand and predict the adsorption behavior of fumaric acid on IRA900 and thus give direction for process design.

5.2 Materials and Methods

5.2.1 Materials

In this study, the resin Dowex Optipore L-493 and activated carbon were purchased from Sigma-Aldrich (St. Louis, MO). Resin Amberlite XAD4 and Dowex Optipore SD20 were purchased from SUPELCO (Bellefonte, PA). Resin Amberlite IRA900 was purchased from Acros Organics (Pittsburgh, PA). The fresh resins were first washed and sonicated several times to remove the impurities. Then, all the resins were dried at 37oC for future use.

110 5.2.2 Adsorption experiments

For the resin screening, 0.2 g dry resin was added into 10 ml 5 g/L fumaric acid solution at three different pHs: 2.0, 3.5 and 5.0. The pH was adjusted to the set value using 0.5 M NaOH or HCl solutions before adding resin. Then, all the samples were incubated in a shaking incubator at 200 rpm and 25 oC for 12 h. The residual fumaric acid concentration was detected and the adsorption capacity was calculated as the amount of adsorbed fumaric acid over the dry weight of the resin. All the experiments were repeated in duplicate and the average values were reported.

For the effect of solution pH, 0.2 g dry IRA900 resin was added into 10 ml fumaric acid solution with different pHs (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5) under two initial concentrations (5, 15 g/L). For the pH isotherm, 0.2 g dry IRA900 resin was added into

10 ml fumaric acid solution with different initial concentrations (2.5, 5.0, 7.5, 10, 15, 20,

25, 30 g/L) under pH of 3.0, 4.0 and 5.0. For the effects of byproducts (glucose and malic acid), 0.2 g dry IRA900 resin was added into 10 ml 10 g/L fumaric acid solution with different byproducts concentrations (glucose: 5.0, 10.2, 19.4, 39.6, 76.3 g/L; malic acid:

4.2, 8.7, 17.0 g/L). The following procedures were the same with previously mentioned.

For the kinetics study under pH of 3.0, 4.0 and 5.0, 0.2 g dry IRA900 resin was added into 10 ml fumaric acid solution with initial concentration of 15 g/L. After adding the resin into the solution, samples were taken at incubation times of 7, 12, 17, 22, 27, 32,

45, 61, 92 and 120 min. The amount of adsorbed fumaric acid at different times was calculated.

111 5.2.3 Desorption of fumaric acid from IRA900

Different concentrations of NaOH, NaCl and H2SO4 (0.5, 1.0, 1.5 g/L) were used as the desorption solvent. ~5 g dry IRA900 resin was added to 100 ml 23.6 g/L fumaric acid solution at pH 5. The adsorption process was carried out in a shaking incubator at 200 rpm and 25 oC for 12 h. After adsorption, all the resins were collected and washed with distilled water. Samples were taken to detect the removed fumaric acid amount by washing. ~0.4 g wet resin containing adsorbed fumaric acid was added into different desorption solvents. The desorption process was performed in a shaker at 200 rpm and 25 oC for 12 h. The recovery yield was estimated based on the aqueous fumaric acid amount in the desorption solution over the adsorbed amount of fumaric acid.

5.2.4 Analytical methods

HPLC was used to analyze the organic compounds, including glucose, lactic acid, malic acid, fumaric acid and ethanol, present in the fermentation broth. Because calcium fumarate was precipitated in the fermentation broth, sample pretreatment was required for

HPLC detection: dilute hydrochloric acid was added for neutralization of excessive

CaCO3 and acidification of fumarate. Due to the low solubility of fumaric acid in water

(6.3 g/L at 25 oC), the samples were heated to 80 oC to increase the solubility of fumaric acid. The HPLC system (Shimazu Scientific Instruments) equipped with a RID-10A refractive index detector and an organic acid analysis column (HPX-87H Bio-Rad,

Richmond, CA) was operated under the following conditions: sample volume of 15 μl, mobile phase of 0.005 M H2SO4, flow rate of 0.6 mL/min, and column temperature of 45 oC.

112 5.3 Results and Discussion

5.3.1 Resin screening

In order to find the best ion exchange resin for fumaric acid recovery, the adsorption capacities of various adsorbents were studied at pH 2.0, 3.5 and 5.5 with the results shown in Table 5.1. For most resins, including activated carbon, L-493 and SD20, their adsorption capacities for fumaric acid decreased dramatically when increasing the pH from 2.0 to 5.5. Activated carbon had the highest adsorption capacity of 210.35 mg/g at pH 2. On the other hand, IRA900, a strong-basic anion exchange resin, possessed a high adsorption capacity at both pH 3.5 and 5. Such property is beneficial for in situ product recovery because the optimal pH for fumaric acid fermentation is 5.0 to 6.0. Thus,

IRA900 was selected for further study.

5.3.2 Effects of pH and modeling

Figure 5.1 shows the adsorption isotherms for IRA900 ion exchange resin adsorbing fumaric acid at pH 3.0, 4.0 and 5.0. For all three cases, the adsorption capacities increased with the increase of fumaric acid concentration. At both pH 4 and 5, the adsorption capacities reached the highest value at the equilibrium concentration of >10 g/L; whereas at pH 3, the adsorption capacity increased dramatically with the increase of equilibrium concentration. Consequently, at the low equilibrium concentration of <2.5 g/L, the order for the adsorption capacity at different pHs was: pH 4 > pH 5 > pH 3; at the concentration of 2.5~5.0 g/L, the order was: pH 4 > pH 3 > pH 5; at the high concentration of >5.0 g/L, the order was: pH 3 > pH 4 > pH 5.

113 In order to understand the different adsorption behaviors at different pHs, the experimental equilibrium data were correlated with Langmuir and Freundlich models.

The Langmuir theory is based on the assumption that the solute is adsorbed onto the adsorbent at specific homogeneous sites until a monolayer is formed; the multi-layer formation of adsorbate is impossible due to the electrostatic repulsion between adsorbed molecules and those in solution (Giles and Smith, 1974). The Langmuir equation can be described as follows:

where: Ce (g/L) is the equilibrium concentration of fumaric acid; qe (mg/g) is the surface concentration of fumaric acid at equilibrium; Qmax (mg/g) and KL (L/g) are Langmuir constants related to adsorption efficiency and energy, respectively. The linear form is given by:

Therefore, the two constants Qmax and KL can be obtained by plotting Ce/qe versus Ce, which gives a straight line with slope of 1/ Qmax and intercepts of 1/ KLQmax.

The Freundlich model, typically for heterogeneous surfaces, can be represented as follows:

n where: KF (mg/g (g/L) ) is the adsorption capacity when Ce equals one; n is Freundlich exponent, representing the degree of adsorption dependence on equilibrium concentration.

Similarly, the two constants KF and n can be obtained by plotting lnQe versus lnCe,

114 which gives a straight line with slope of 1/n and intercepts of lnKF. The linear form is represented by:

(4)

Table 5.2 summarizes the results of model fitting. The adsorption behavior under different pHs can be well described from the parameters obtained from fitting the experimental data to the isotherm model. The correlation coefficients (R2) with the

Langmuir isotherm model were high for fumaric acid solution at pH 4 and 5 with values of 0.990 and 0.999, respectively, showing a homogenous adsorption at pH 4 and 5.

2 However, the R (0.956) was lower at pH 3. A higher value of the Langmuir constant KL indicated a more favorable adsorption process. By comparing KL, the adsorption of fumaric acid onto IRA900 was most favorable at pH 5. However, the maximum adsorption capacity at pH 5 was the lowest as indicated from the comparison of Qmax. For the regression by Freundlich model, R2 values for the adsorption at pH 4 and 5 were significantly less than those of Langmuir model; whereas a slightly higher R2 (0.982) was observed at pH 3. As indicated from the values of KF, at a low equilibrium concentration, the extent of adsorption for fumaric acid solution at pH 4 and 5 was significantly higher than that at pH 3. The n values for all the cases were higher than unity, reflecting that under all the three pHs fumaric acid was favorably adsorbed by IRA900 resin, with the highest favorability at pH 5. Overall, the Langmuir isotherm gave the best correlation for fumaric acid adsorption onto IRA900. Based on these isotherm constants, the adsorption process can be mathematically defined to predict the residual concentration after adsorption or amount of adsorbent for desired separation.

115 5.3.3 Effect of pH under different initial concentrations

In order to further explore the effects of solution pH on fumaric acid adsorption, the effects of different pHs (2.5, 3.0, 3.5, 4.0 4.5, 5.0, 5.5) on the adsorption process were investigated under two initial fumaric acid concentrations (5, 15 g/L) with the results shown in Figure 5.2. At a low concentration of 5 g/L, the adsorption capacity increased with the increase of pH from 2.0 to 3.5. Then, the adsorption capacity decreased slowly from pH 4.0 to 5.6. However, the trend was different at a high concentration of 15 g/L.

The adsorption capacity dropped significantly from 2.5 to 5.0. Such phenomena were in good agreement with the pH isotherm: higher adsorption capacity occurred at pH 4 under low concentrations, whereas under high concentrations the adsorption capacity was higher at a lower pH of <3.0.

To better understand such different adsorption behaviors, the fumarate dissociation in aqueous solution at different pHs was studied. In aqueous fumarate solution, unionized fumaric acid (H2FA) exists in equilibrium with fumarate anions (HFA-, FA2-), as shown in Figure 5.3. Fumaric acid, as a diprotic acid, has two pKa values, 3.03 and 4.44. Thus, at a certain pH, the ratio between the three ion forms (H2FA, HFA- and FA2-) can be calculated from the following equations:

(5)

(6)

Figure 5.4 shows the mole ratio of the three fumarate ion forms at different pHs. At pH <3, unionized fumaric acid H2FA was dominant with HFA- as the primary dissociated anion. At 3 < pH < 4, fumarate anions HFA- was dominant; with the increase of pH, the

116 mole percentage of H2FA decreased and that of FA2- increased. At pH > 4, the primary fumaric acid ion form became FA2-. Such results indicated that the varied adsorption behaviors under different pHs were associated with the different ion forms of fumaric acid. At low fumarate concentrations the dominant ion forms of fumarate anions HFA- and FA2- under the pH of >3.5 had higher adsorption capacities by IRA900, while at high fumarate concentration the dominant unionized H2FA under the pH of <3.0 can achieve a significantly higher adsorption capacity.

5.3.4 Adsorption kinetics study under different pHs and mechanism study

In order to elucidate the relationship between the ion forms of fumaric acid and adsorption behaviors, the adsorption kinetics of fumaric acid adsorption on IRA900 under different solution pHs were studied and modeled by different equations. Figure 5.5 depicts the adsorption kinetics at different pHs. The adsorbed fumaric acid amount increased with the increase of the contacting time. For the solution pH of 4 and 5, the equilibria were reached after ~30 min adsorption, whereas the adsorption under the solution pH of 3 reached the equilibrium more slowly after ~50 min. In addition, although pH 3 gave the highest adsorbed fumaric acid amount at the equilibrium, at the beginning of the adsorption (<30 min), the adsorbed amount at pH 3 was less than pH 4.

In order to understand such different adsorption behaviors at different pHs, the adsorption kinetics were modeled by pseudo first-order equation, pseudo second-order equation and intraparticle diffusion models for mechanism study. Generally speaking, the ion-exchange adsorption process often involves four consecutive steps including diffusion processes (Findon et al., 1993; Sag et al., 2000; Özacar et al., 2008): bulk

117 diffusion (solute diffusion from the bulk solution to the surrounding film of the particle), external diffusion (diffusion across the liquid film to the adsorbent particle surface), intraparticle diffusion (diffusion from the surface into the internal active sites of the adsorbent), and adsorption reaction on the active sites via ionic interactions.

Identification of the rate limiting steps in adsorption process is essential to understanding the adsorption mechanism and defining the rate parameters for design purposes (Sag et al., 2000).

The limitation of the adsorption reactions between the functional groups of the resin and the ions was first examined. Pseudo first-order and pseudo second-order models are most commonly used to describe the mechanism controlled by chemical reactions. The pseudo first-order equation, also known as Lagergren’s equation, is the earliest known equation describing the adsorption rate based on adsorption capacity (Kammerer et al.,

2011). It is summarized as below:

where qe and qt are the amounts of fumaric acid adsorbed at equilibrium and time t

(mg/g), respectively; k1 is the rate constant of pseudo first-order equation (1/min). The linear pseudo first-order equation is shown as below:

(6)

By plotting log (qe - qt) versus t, the first-order rate constant k1 and equilibrium adsorption capacity qe can be calculated from the slope (k1/2.303) and the intercepts (log qe).

118 For the pseudo second-order equation, it is a two-site-occupancy adsorption, i.e., the adsorbate molecule reacts with two adsorption sites (Ho, 2006). The equation is summarized as below:

where k2 is the rate constant (g/mg/min). The model is most commonly used in its linearized form by plotting t/qt versus t. The rate constant k2 and qe can be identified from the slope and the intercept.

A comparison of the results of model fitting with the correlation coefficients is shown in Table 5.3. For the first-order kinetic model, the correlation coefficients (R2)

2 obtained at pH 4 and 5 were low. Although the R at pH 3 was high, the calculated qe value (216.4 mg/g) obtained from the model deviated considerably from the experimental value (298.3 mg/g), which was also the case with pH 4 and 5. Such results indicated that the pseudo first-order equation was not suitable to describe the mechanism of fumaric acid adsorption on IRA900. For the pseudo-second-order equation, the plot of t/qt versus t as shown in Figure 5.6 generated good straight lines for all three pHs, with very high R2 values (~0.999) for the model fitting. The calculated qe values were also in good agreement with the experimental data. Such results showed that the adsorption reaction between fumaric acid and functional groups of the resin belongs to the second-order kinetics, indicating a mechanism of two-site-occupancy adsorption. The functional group of the IRA900 ion exchange resin is quaternary ammonium with one positive valence on each adsorption site, whereas fumarate may contain zero (unionized H2FA), one (HFA-)

119 or two (FA2-) negative valences based on the solution pH as we discussed before. From the two-site-occupancy adsorption mechanism, it was the FA2- molecule that reacted with two adsorption sites of the resin. Accordingly, from the comparison of rate constant k2

2- under different pHs, pH 5 with FA as the primary ion form gave the highest k2 value followed by pH 4 and pH 3. Such results agreed well with the high Langmuir constant KL at pH 5. Based on such mechanism, the adsorption capacity should increase with the increase of pH from 3 to 5 due to the increased FA2- concentration. However, as shown from the results of kinetics study and pH-effect study, at a high initial fumarate concentration, a lower pH gave a higher adsorption capacity. This contradiction indicated another controlling mechanism for fumarate adsorption on IRA900.

In a well-stirred batch system, the boundary layer surrounding the resin particle is much reduced with the decrease of external mass transfer coefficient; thus, intrapaticle diffusion probably becomes the rate limiting step (Findon et al., 1993). Intraparticle diffusion model was applied to study its limitation on the overall adsorption process. The model can be represented by the following equation:

-1/2 where kp is the intraparticle diffusion rate constant (mg/g (min) ). If the intraparticle

0.5 diffusion is the sole rate-limiting step, the plot of qt versus t should pass through the origin. If not, the plot may present a multilinearity, indicating that multiple steps, such as boundary layer diffusion or some other processes, take place (Özacar, 2003). For a general multiplinearity plot, the beginning shape part (Stage 1) is possibly due to the bulk diffusion or external diffusion; the second part (Stage 2) describes the gradual adsorption stage by intraparticle diffusion; the third part (Stage 3) shows the final equilibrium stage

120 where intraparticle diffusion begins to slow down, due to the reduced concentration gradient (Özacar, 2003). Figure 5.7 depicts the plots of the linearized form of the intraparticle diffusion model at all pHs studied. The curves were not linear over the whole time range, indicating multiple-process effects on fumaric acid adsorption. Due to the high stirring rate, the external surface adsorption (Stage 1), which is absent in Figure

5.7, was completed before 5 min. The first linear part of the curve belonged to intraparticle diffusion stage (Stage 2). Results showed that pH 3 had a significantly long period of the intraparticle diffusion stage from 5 min to 50 min, whereas the period of

Stage 2 in pH 4 and 5 only lasted from 7 to 27 min. Final equilibrium adsorption (Stage 3) started after intraparticle diffusion stage at 50 min for pH 3 and at 27 min for both pH 4 and 5. The second linear part (Stage 2) was fitted to the intraparticle diffusion model with the results summarized in Table 5.3. The slope of the equation characterized the rate constant of intraparticle diffusion (kp). From the comparison of kp, pH 5 had a

2- significantly lower kp, implying the less intraparticle diffusion rate of ion form FA . As

- indicated by the higher kp of pH 3 and 4, the ion form of H2FA and HFA had higher intraparticle diffusion rates. The high intraparticle diffusion rate and long period during the adsorption process of pH 3 led to a high extent of intraparticle diffusion when the adsorption reached equilibrium. Therefore, the higher adsorption capacity at pH 3 than pH 4 and 5 at high concentrations, as shown previously, was attributed to the higher extent of intraparticle diffusion of neutral fumaric acid ion form than the other two anions

(HFA-, FA2-).

The reason for the high extent of intraparticle diffusion at pH 3 was further elucidated from a molecular level. First, from the perspective of adsorbent, the backbone

121 of the IRA900 resin is the hodrophobic polystyrenic matrix (Li et al., 2004). Li et al.

(2000) described the resin structure as that “each single resin particle includes a huge number of tiny microgels and an interconnected network of pores” and “the functional groups of the resin reside primarily within the microgels” (Li et al., 2000). Therefore, with the high extent of intraparticle diffusion, more active sites, the functional groups of adsorbent, were exposed to fumarate solution for adsorption reaction. This was also the reason why the kinetics still followed the pseudo-second-order model. As can be seen from Equation 7, (qe – qt) is the driving force for pseudo-second-order reaction, which is proportional to the available fraction of active sites (Ho et al., 2006). Therefore, although intraparticle diffusion was the primary controlling mechanism for fumaric acid adsorption, the mechanism still followed the pseudo-second-order model.

From the perspective of the adsorbate, Gregory and Semmens (1971) pointed out that increasing hydrophobic interaction between the resin matrix and the hydrocarbon chains of the adsorbate molecules led to enhanced adsorption capacity. Due to the higher hydrophobicity of ion form H2FA, the increased concentration of H2FA at lower pH increased hydrophobic interactions between the adsorbate and the resin matrix and thus promoted the extent of intraparticle diffusion. Therefore, as compared to pH 4 and pH 5, with a higher percentage of H2FA, pH 3 gave a higher adsorption capacity. For the ion form HFA- and FA2-, although FA2- was more favorable for the adsorption reaction with a high reaction rate, the low hydrophobicity of FA2- resulted in a low extent of intraparticle diffusion, leading to decreased adsorption capacity at a higher pH. Another reason for the high intraparticle diffusion was the high initial concentration. The concentration gradient within the exchanger particle acts as the driving force and controls the overall rate for an

122 intraparticle-diffusion limited adsorption process (Li et al., 2000). As discussed before, the adsorption capacity of fumaric acid at pH 3 under a low concentration of <5.0 g/L was lower compared to pH 4 and 5. Besides the reason of lower FA2- concentration at pH

3, another reason was that the extent of intraparticle diffusion was significantly reduced at a lower concentration with a less concentration gradient.

5.3.5 Selectivity study

Selectivity is critical in the process of in situ fumaric acid recovery from fermentation broth due to the high concentration of glucose at the beginning of the fermentation process and the existence of byproduct malic acid. Based on the fermentation results in our lab, malic acid was the major byproduct with final concentration <5.0 g/L. As shown in Table 5.4, the selectivity of fumaric acid against glucose and malic acid was studied with initial fumaric acid concentration of 10 g/L at pH 5. The selectivity is defined as the adsorption capacity for fumaric acid over that for byproduct. For the effects of glucose concentration, increasing glucose concentration did not influence the fumaric acid adsorption. Due to the increased adsorption capacity for glucose with the increase of glucose concentration, the selectivity for fumaric acid dropped significantly. For the effects of malic acid concentration, the addition of malic acid lowered the adsorption capacity for fumaric acid significantly. Especially at a higher malic acid concentration of 17 g/L, the adsorption capacity for fumaric acid decreased by

28%, whereas the adsorption capacity for malic acid increased to 81.0 mg/g dry resin.

However, the relatively higher adsorption capacity for fumaric acid against glucose and malic acid guaranteed the selective adsorption of fumaric acid on IRA900. The reason for

123 such selective adsorption can be attributed to the fact that compared to the molecules of glucose and malic acid, the higher hydrophobicity of the fumaric acid molecule resulted from carbon double bond significantly increased the hydrophobic interactions with resin hydrophobic matrix, leading to enhanced intraparticle diffusion extent, as discussed before.

5.3.6 Desorption process

For desorption, various eluants (NaOH, NaCl and H2SO4) were evaluated for their ability to strip the adsorbed fumaric acid from the resin IRA900, and the results are shown in Figure 5.8. The results showed that neither strong base (NaOH) nor strong acid

(H2SO4) was effective in desorbing the adsorbed fumaric acid from IRA900. Although the desorption of fumaric acid increased with increasing the NaOH concentration, the efficiency was lower than 50%. The desorption efficiency with H2SO4 was lower than 30% and not affected by its concentration (0.5, 1.0 and 1.5 g/L). In contrast, NaCl at a concentration of 1 g/L was very effective to desorb fumaric acid from IRA900, with a high stripping yield of ~95%. Thus, fumaric acid can be separated from the fermentation broth by adsorption with IRA900 and then desorption with NaCl with a high recovery yield.

5.4 Conclusion

In this study, our objective was to identify a suitable ion exchange resin for in situ product recovery of fumaric acid from fermentation broth and characterize the adsorption process. IRA900 strong-basic anion exchange resin was selected due to its high

124 adsorption capacity at fermentation-favored pH of 5, high selectivity against byproducts

(glucose and malic acid) and easy desorption with high recovery yield of ~95%. In this way, we successfully developed an effective separation method for in situ fumaric acid recovery by using IRA900 for selective adsorption with desorption by using NaCl as stripping agent.

Equilibrium and kinetics studies were studied under different pHs of 3, 4 and 5 for characteristics and mechanism studies. Results found that at low equilibrium concentration of < 5.0 g/L, the adsorption capacity for fumaric acid at pH 3 was lower than that at pH 4 and 5, whereas at the high concentration of >5.0 g/L, pH 3 gave a significantly higher adsorption capacity. It was found that the varied adsorption behaviors under different pHs were associated with the different ion forms of fumaric acid. The

Langmuir isotherm gave the best correlation for fumaric acid adsorption onto IRA900.

Fitting results of kinetics data by pseudo-first-order and pseudo-second-order equations showed that the adsorption reaction between fumaric acid and functional groups of the resin belongs to the second-order kinetics, indicating a mechanism of two-site-occupancy adsorption reaction between ion form FA2- and resin active sites. However, such a mechanism cannot explain the high adsorption capacity at pH 3 with a high initial concentration. By fitting the data with the intraparticle diffusion model, the reason was found to be the high extent of intraparticle diffusion occurred at pH 3. It was further found that such increased extent of intraparticle diffusion at lower pH was attributed to the enhanced hydrophobic interactions between the hodrophobic resin matrix and the unionized H2FA with a high percentage at lower pH, and the driving force of the high concentration gradient.

125 5.5 Reference

Ataei, S.A., Vasheghani-Farahani E., 2008. In situ separation of lactic acid from fermentation broth using ion exchange resins. J Ind Microbiol Biotech 35: 1229-1233.

Cao, N.J., Du, J.X., Gong, C.S., Tsao, G.T. 1996. Simultaneous production and recovery fumaric acid from immobilized Rhizopus oryzae with a rotary biofilm contactor adsorption column. Appl Environ Microbiol 62: 2926-2931.

Cao, X., Yun, H.S., Koo, Y.M. 2002. Recovery of L-(+)-lactic acid by anion exchange resin Amberlite IRA-400, Biochem Eng 11: 189-196.

Cen, P. and Tsao G.T. 1993. Recent advances in the simultaneous bioreaction and product separation processes. Sep Technol 3: 58–75.

Findon, A., Mckay, O., Blair, N.S. 1993. Transport studies for the sorption of copper ions by chitosan. J Environ Sci Health A 28: 173-185.

Fu, Y., Chen, Y., Li, S., Huang, H. 2009. Fixed-bed adsorption study for fumaric acid removal from aqueous solutions by Amberlite IRA-400 resin. Chem Eng Technol 10: 1625-1629.

Giles, C., Smith, D.1974. General treatment and classification of the solute sorption isotherms. J Colloid Interface Sci 47: 755-765.

Gregory, J. and Semmens, M.J. 1972. Sorption of carboxylate ions by strongly basic anion exchangers. J Chemi Soc 68, 1045-1052.

Ho, Y.S. 2006. Review of second-order models for adsorption systems. J Hazard Mater B136, 681-689.

Kammerer, J., Carle, R., Kammerer, D.R. 2011. Adsorption and ion exchange: basic principles and their application in food processing. J Agric Food Chem 59: 22-42.

Li, P., SenGupta, A.K. 2000. Intraparticle diffusion during selective ion exchange with a macroporous exchanger. React Funct Polym 44: 273-287.

Li, P., SenGupta, A.K. 2004. Sorption of hydrophobic ionizable organic compounds (HIOCs) onto polymeric ion exchangers. React Funct Polym 60: 27-39.

Özacar, M. 2003. Equilibrium and kinetic modeling of adsorption of phosphorus on calcined alunite. Adsorpt 9: 125-132.

Özacar, M., Sengil, I.A., Turkmenler, H. 2008. Equilibrium and kinetic data, and adsorption mechanism for adsorption of lead onto valonia tannin resin. Chem Eng J 143: 32-42.

126 Sag, Y., Aktay, Y. 2000. Mass transfer and equilibrium studies for the sorption of chromium ions onto chitin. Proc Biochem 36: 157-173.

Yang , S.T., Zhang, K., Zhang, B., Huang, H. 2011. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition, 3, 163-177.

Wang, J., Wen, X., Zhou, D. 2000. Production of citric acid from molasses integrated with in-situ product separation by ion-exchange resin adsorption, Biores Technol 75: 231-234.

127 Table 5.1 Adsorption capacity for fumaric acid by different resins under different pHs

Fumaric acid in water Adsorption capacity Resins pH (g/L) (mg FA / g dry resin) 2 0.87 ± 0.01 210.4 ± 2.10 Activated Carbon 3.5 3.00 ± 0.02 104.9 ± 1.0 5 4.81 ± 0.01 13.3 ± 0.3 2 3.22 ± 0.01 92.7 ± 0.1 L-493 3.5 4.40 ± 0.00 35.1 ± 0.0 5 5.08 ± 0.00 0.0 ± 0.1 2 4.96 ± 0.01 6.1 ± 0.1 XAD4 3.5 5.02 ± 0.01 4.0 ± 0.4 5 5.08 ± 0.00 0.0 ± 0.1 2 2.42 ± 0.04 133.0 ± 1.9 SD20 3.5 3.42 ± 0.01 84.0 ± 0.6 5 4.50 ± 0.01 28.6 ± 0.6 2 3.08 ± 0.01 75.8 ± 0.3 IRA 900 3.5 1.76 ± 0.01 142.3 ± 0.2 5 2.23 ± 0.40 133.1 ± 8.2

128 Table 5.2 Model parameters obtained from fitting the experimental equilibrium data with

isotherm model

Langmuir model Freundlich model pH 2 2 R Qmax KL R KF n 3 0.956 625.0 0.103 0.982 89.5 1.80 4 0.990 312.5 0.457 0.967 115.6 3.27 5 0.999 212.8 0.644 0.975 119.1 5.86

129 Table 5.3 Comparison of regression results for fumaric acid adsorption kinetics on

IRA900 resin by using different equations

Intraparticle Pseudo-1st-order equation Pseudo-2nd-order equation qe_det diffusion pH (mg/g) qe-cal k qe-cal k kp (mg/g 1 R2 2 R2 R2 (mg/g) (1/min) (mg/g) (g/mg min) (min)-1/2) 3 298.3 216.4 0.004 0.996 297.4 0.0004 0.998 31.1 0.989 4 244.7 89.3 0.041 0.894 250 0.001 0.999 30.3 0.967 5 191.2 79.3 0.034 0.932 199.2 0.0016 0.999 15.2 0.999

130 Table 5.4 Selectivity study of IRA900 resin adsorbing fumaric acid

(fumaric acid initial concentration of 10 g/L, pH 5)

Ini. conc. FA adsorbed Byproduct adsorbed Byproduct Selectivity (g/L) amount (mg/g) amount (mg/g) - - 158.3±5.3 - - Glucose 5.0 148.8±14.3 2.4±2.0 60.9 Glucose 10.2 157.7±7.9 6.6±1.5 23.9 Glucose 19.4 142.2±16.0 17.3±1.7 8.2 Glucose 39.6 162.3±8.1 47.2±5.8 3.4 Gluocse 76.3 160.0±13.6 51.6±5.2 3.1 Malic acid 4.2 149.8±1.0 29.8±0.2 5.0 Malic acid 8.7 117.0±0.2 40.3±0.4 2.9 Malic acid 17.0 114.3±0.2 81.0±0.2 1.4

131 480 pH 3

400 pH 4

pH 5 320

240

160

(mg/g dry resin) dry (mg/g 80 Adsorption capacity capacity Adsorption

0 0 5 10 15 20 25 30 Equilibrium concentration (g/L)

Figure 5.1 Isotherms of IRA900 adsorbing fumaric acid at pH 3, 4 and 5

Points: experimental data; Lines: Langmuir model

132

360

5 g/L

300 15 g/L

240

180

(mg/g dry resin) dry (mg/g 120 Adsorption capacity capacity Adsorption

60 2 2.5 3 3.5 4 4.5 5 5.5 6 pH

Figure 5.2 Effects of pH on fumaric acid adsorption by IRA900 resin at fumaric acid initial

concentration of 5 and 15 g/L

133

Figure 5.3 Dissociation equilibrium of aqueous fumarate solution

134 1

0.8

0.6 FA2- 0.4 HFA-

Mole ratio Mole H2FA 0.2

0 2.5 3 3.5 4 4.5 5 5.5 pH

- 2- Figure 5.4 Mole ratio of three fumarate ion forms (H2FA, HFA , FA ) at different pHs

135 280

240

200

160

(mg/g) 120 pH 3

80 pH 4 pH 5

40 Fumaric acid adsorbed adsorbed acid Fumaric 0 0 20 40 60 80 100 120 140 Time (min)

Figure 5.5 Adsorption kinetics of fumaric acid on IRA900 resin at pH 3, 4, 5

136 0.7

0.6

0.5

0.4

0.3 pH 3

0.2 pH 4 t/qt (g min/mg) (g t/qt 0.1 pH 5

0 0 20 40 60 80 100 120 140 t (min)

Figure 5.6 Plot of the pseudo-second-order equation for the adsorption kinetics of fumaric

acid on IRA900 resin at pH 3, 4, 5

137 280

240

200

160

(mg/g) 120 pH 3

80 pH 4 pH 5

40 Fumaric acid adsorbed adsorbed acid Fumaric 0 2 4 6 8 10 12 1/2 1/2 t (min)

Figure 5.7 Plot of the intraparticle diffusion equation for the adsorption kinetics of fumaric

acid on IRA900 resin at pH 3, 4, 5

138 100

80 NaOH

60 NaCl H2SO4 Yield (%) Yield 40

20

0 0.25 0.5 0.75 1 1.25 1.5 1.75 Concentration (g/L)

Figure 5.8 Desorption process by NaOH, NaCl and H2SO4 at different concentrations

139 CHAPTER 6

Intermittent in situ Recovery of Fumaric Acid from Fermentation Broth by Using

IRA-900 Ion Exchange Resins

6.1 Introduction

A major problem associated with traditional fumaric acid fermentation is low productivity (Roa Engel et al., 2008). One reason was the reduced productivity at the later phase of the fermentation, which was probably resulted from the end product inhibition and declined cell activity (Yang et al., 2011). In situ product recovery has been proven to effectively reduce the end product inhibition effect and enhance the productivity of the process with wide applications in organic acid production (Cen and

Tsao, 1993; Ataei et al., 2008; Wang et al., 2000). Nevertheless, there are few reports on applying the technology of in situ product recovery to fumaric acid production. Cao et al.

(1996) developed an integrated system using rotary biofilm contactor (RBC) coupled with ion exchange column for simultaneous fermentation and separation. High product titer of >75.5 g/L and productivity of >3.78 g/(L.h) were achieved. However, the complexity of this system and high resin cost during process scale-up limited its commercial development.

Previously, we identified a suitable ion exchange resin, Amberlite IRA900, for in situ fumaric acid recovery from fermentation broth due to its high adsorption capacity at

140 the fermentation-favored pH of 5 and high selectivity against byproducts such as glucose and malic acid. With NaCl as stripping solution, almost all the fumaric acid adsorbed on the resin can be recovered. The objective of this study was to apply the resin to develop a novel, economical and scalable in situ recovery method. The separation process

(adsorption and desorption) was first evaluated on a fixed bed column and some influencing factors, such as resin ion forms, flow rate, initial fumaric acid concentrations and different stripping agents, were investigated. The product inhibition effect on fermentation process was studied. Then, a new method of fumaric acid fermentation with intermittent in situ recovery by ion exchange resin was proposed.

6.2 Materials and Methods

6.2.1 Materials

In this study, the resin Amberlite IRA900 was purchased from Acros Organics

(Pittsburgh, PA). The fresh resins were first washed and sonicated several times to remove the impurities, then the wet resins were added into a fixed bed column for fumaric acid adsorption.

6.2.2 Adsorption and desorption on fixed bed column

The adsorption was carried out in a glass column (diameter 1.6 cm; height 19 cm) filled with ~23 g wet IRA900 resin. Fumaric acid aqueous solution was fed to the top of the column at a desired flow rate regulated by a peristaltic pump. Samples were collected from the column outlet every 5 minutes. Breakthrough and outlet pH curves for each condition were obtained. The effects on the adsorption process of some influencing

141 factors, such as resin ion forms (Cl-, OH-), flow rate (2.34, 4.10, 5.34 ml/min), and fumaric acid initial concentrations (2.4, 4.8, 10.0 g/L) were investigated. Several parameters were used to evaluate the performance of the adsorption. T_bre was the breakthrough time when the outlet fumaric acid concentration reached 5% of the initial concentration; T_sat was the saturation time when the outlet fumaric acid concentration reached 95% of the initial concentration; Adsorption capacity was the amount of the adsorbed fumaric acid over the total resin amount; Adsorption percentage was the amount of the adsorbed fumaric acid at T_sat over the total amount of fumaric acid flowing through the column from the beginning to T_sat.

For the desorption process, after the resin was saturated with fumaric acid, different desorption agents (NaCl, NaOH) under different concentrations were fed to the top of the column at the flow rate of (5.4±0.3) ml/min regulated by a peristaltic pump. Samples were collected from the column outlet every 5 minutes. Stripping and outlet pH curves for each condition were obtained. The recovery yield was calculated as the fumaric acid concentration in the total desorption solution over the adsorbed amount.

6.2.3 Fermentation process for end product inhibition study

R. oryzae ATCC 20344 used in this study was cultured on YMP (yeast extract, malt extract, and peptone) agar plates containing 3 g/L yeast extract, 3 g/L malt extract, 3 g/L peptone, 20 g/L glycerol and 20 g/L agar. After ~7 day incubation at 32 oC, the spores were harvested by washing with sterile distilled water and collected as a spore suspension, which was stored at 4 oC for future use. Soybean meal obtained from Cargill (Sidney, OH) was used as nitrogen source. To prepare the soybean meal hydrolysate (SMH), 20 g of

142 soybean meal were added into 400 mL of 0.25 N HCl, and the mixture was autoclaved at

121 oC for 30 min. The hydrolysate was filtered under sterile conditions, and the filtrate was stored at room temperature for future use.

Seed cultures were prepared in 250 mL shake-flasks containing 50 mL growth medium with 10 g/L glucose and 20% SMH (v/v) as nitrogen source. The pH of the medium was then adjusted to 3.0 using NaOH. After inoculation with the spores in suspension, the cultures were incubated at 35~37 oC for 24 h in a rotary shaker agitated at

220 rpm.

Fumaric acid fermentations were carried out in 250 mL shake-flasks. The volume of the medium in the flask was 40 mL. The fermentation medium contained 90 g/L glucose,

0.6 g/L KH2PO4 and 0.5 g/L MgSO4. The seeds were first harvested from the seed cultures under the same conditions and then inoculated into the fermentation medium at an inoculation size of 25%. To study the end product inhibition, different initial concentrations of fumaric acid (0, 0.6, 4.4, 8.3, 20.3, 39.9 g/L) were added to the medium.

The pH was maintained above 5.0 by adding 60 g/L CaCO3. The fermentations were performed at 35 oC for 4~5 day in a rotary shaker at 220 rpm. The produced fumaric acid concentration was the value of the final concentration minus the initial.

6.2.4 Intermittent in situ recovery

Figure 6.1 shows the apparatus set-up for the integrated system. For the fermentation part, the process was carried out in 5-L stirred tank bioreactor (STB). The volume of the medium in the tank was 2 L. The seed culture process was the same as mentioned in 6.2.3.

The seeds were harvested, washed by sterile distilled water to remove the residues, and

143 then transferred into bioreactor with an inoculation size of 10%. The fermentation medium contained 80-100 g/L glucose, 40 mL/L SMH, 0.6 g/L KH2PO4 and 0.5 g/L

MgSO4. The SMH was added into the bioreactor separately with pH of ~4.0 adjusted by

NaOH. The fermentation pH was maintained at 5.0 by automatically adding 1 M Na2CO3.

The rotation speed was 500 rpm and the air flow rate was 2.0 vvm. A cotton filter was installed on the end of the in-situ-recovery sampler to avoid the entrance of the cells into the column. The fermentations were performed at 37 oC for 4~5 d.

For the adsorption part, ~80 g IRA-900 was filled into the column (diameter 1.6 cm; height 52 cm) to recover fumaric acid from 2-L fermentation broth. Before the fermentation, for sterilization purpose, the resin and column were washed with hot autoclaved distilled water for ~ 1 h and then autoclaved 1 M NaCl for ~0.5 h. During the fermentation process, the medium passed through the column at a flow rate regulated by a peristaltic pump. The effluent of the column was recycled back to the fermentor. After

3~5 h when the resins were saturated with fumaric acid, in situ recovery was terminated and 0.7 mol/L NaCl was fed into the column (last 1~2 h) for fumaric acid desorption and resin regeneration. The starting time of each cycle and the total cycling times for the whole process were evaluated based on fermentation kinetics and adsorption breakthrough curves.

6.2.5 Analytical methods

HPLC was used to analyze the organic compounds, including glucose, lactic acid, malic acid, fumaric acid and ethanol, present in the fermentation broth. Because calcium fumarate was precipitated in the fermentation broth, sample pretreatment was required for

144 HPLC detection: dilute hydrochloric acid was added for neutralization of excessive

CaCO3 and acidification of fumarate. Due to the low solubility of fumaric acid in water

(6.3 g/L at 25 oC), the samples were heated to 80 oC to increase the solubility of fumaric acid. The HPLC system (Shimazu Scientific Instruments) equipped with a RID-10A refractive index detector and an organic acid analysis column (HPX-87H Bio-Rad,

Richmond, CA) was operated under the following conditions: sample volume of 15 μl, mobile phase of 0.005 M H2SO4, flow rate of 0.6 mL/min, and column temperature of 45 oC.

6.3 Results and discussion

6.3.1 Adsorption process on fixed bed column

6.3.1.1 Effects of resin ion forms

Figure 6.2 compares the breakthrough curves (A) and outlet pH profiles (B) under the two ion forms (OH- and Cl-) of the IRA900 resin for fumaric acid recovery with the results and conditions summarized in Table 6.1. The resin IRA900 was initially in the form of chloride, and the hydroxide form was obtained by washing the resin first with 1 mol/L NaOH and then distilled water to pH 7. The two ion forms of the resin had similar breakthrough curves for fumaric acid adsorption with similar T_bre and T_sat. Compared to the OH- form, Cl- form gave a higher adsorption capacity with a slightly lower adsorption percentage. The major difference between the two forms was outlet pH. For the Cl- form, outlet pH first increased quickly to 6.3 at 15 min, then decreased sharply to

~4.3 at 30 min, and gradually approached the initial feed pH ~5. For the OH- form, after a sharp increase at the beginning, the outlet pH was maintained at 13.2 for ~30 min, then

145 decreased quickly below 7, and gradually approached the initial pH ~5. The different outlet pH profiles of the two ion forms may significantly affect the coupled fermentation.

Because the favorable fermentation pH for fumaric acid production was in the range of

5~6, Cl- ion form was selected in order to minimize the influence of the column outlet pH.

In addition, the OH- form required extra pretreatment by NaOH solution and additional washing by a large amount of distilled water for neutralization.

Also, we found that the outlet pH can be well applied to anticipate the breakthrough situation of the column. For the Cl- ion form, the T_bre (41 min) corresponded to the low outlet pH 4.7 where the pH began to increase from the bottom, while for the OH- form, the T_bre (38 min) corresponded to the outlet pH ~13.0 where the pH began to drop.

When the resin was close to saturation, the outlet pH approached the initial pH of 5.

Therefore, based on outlet pH, we can conclude some key points immediately, such as 5% breakthrough point and 95% saturation point, without detecting the fumaric acid concentrations afterwards, hence saving a lot of time and labor.

6.3.1.2 Effects of flow rate

Figure 6.3 shows the breakthrough curves (A) and outlet pH profiles (B) under different flow rates (2.34, 4.10 and 5.34 ml/min) with the results and conditions summarized in Table 6.1. With the increase in flow rate, the time for the breakthrough and saturation of the resin was reduced significantly. Too high flow rate (5.34 ml/min) led to a decrease in the adsorption capacity of the resin due to the decrease of the retention time of fumaric acid in the column. Although the lower flow rate (2.34 ml/min) gave a higher adsorption percentage (66.2%), the value was still low, possibly due to the

146 high initial fumaric acid concentration. However, the differences of the adsorption performances between different flow rates were relatively small compared to the change of T_bre and T_sat. Overall, in order to shorten the adsorption period and maintain a good adsorption percentage, the flow rate should be controlled in the range of 4.10~5.34 ml/min. Under such conditions, the resin reached saturation in less than 2 h. In addition, although the global shape of the outlet pH profiles under the three flow rates was similar, there were some differences of key points in the pH curves. For example, as the increase of the flow rate, the pH curve hit the bottom at an earlier time and then approached the initial feed pH more quickly, corresponding to the shortened T_bre and T_sat. Thus, these matching changes once again proved the effectiveness of the outlet pH in predicting the adsorption process.

6.3.1.3 Effects of initial concentration

Figure 6.4 shows the breakthrough curves (A) and outlet pH profiles (B) under different initial fumaric acid concentrations (2.4, 4.8 and 10 g/L) with the results and conditions summarized in Table 6.1. With the decrease of initial fumaric acid concentration, the time for the breakthrough and saturation of the column increased significantly. The lower initial concentration (2.4 g/L) resulted in a decrease of the adsorption capacity of the resin, but the lower concentrations (2.4, 4.8 g/L) significantly enhanced the adsorption percentage by 33%. The reason was that at high concentrations more fumaric acid was washed away without interacting with the resin. Therefore, for the purpose of attaining both high adsorption percentage and short adsorption period, the initial concentration should be controlled at ~5 g/L. It should be noted that the adsorption

147 percentage of 82% was reached at a flow rate of 5.3 ml/min. Reducing flow rate may further increase the adsorption percentage to >90%. In addition, from the outlet pH profiles, the low initial concentration (2.4 g/L) caused a long period (~70 min) of a low outlet pH at 3.2, which may have some negative effects on the coupled fermentation process.

6.3.2 Desorption process on fixed bed column

Figure 6.5 and 6.6 compare the stripping curves and outlet pH profiles of fumaric acid desorption from saturated IRA-900 resin by NaCl and NaOH under different concentrations with the results and conditions summarized in Table 6.2. By using NaOH as stripping solution, the recovery yield was low, ~50%. Increasing NaOH concentration cannot increase the yield. Additionally, the outlet pH was kept at a very basic level of

~13 with a high elute pH, which requires a large amount of acid for acidifying fumarate and distilled water for neutralizing the resin. On the contrary, NaCl gave a much higher recovery yield than NaOH. Especially at a high concentration of 0.7 mol/L, all the fumaric acid adsorbed by the resin can be removed. From the stripping curve, the higher

NaCl concentration, the higher fumaric acid concentration was at the beginning of the stripping process. By using 0.7 mol/L NaCl as the stripping solution, the fumaric acid adsorbed on the resin can be removed almost completely in ~40 min. From the outlet pH profiles, the outlet pH first increased a little bit, and then gradually decreased to the bottom followed by a slow increase. The elute pH, the pH of the collected desorption solution, decreased with the increase of NaCl concentration, thus reducing the amount of required acid for acidification. In addition, the resin after stripping by NaCl was

148 regenerated in Cl- ion form for reuse. Overall, 0.7 mol/L NaCl was identified as the best stripping solution.

6.3.3 End product inhibition

The effect of end product inhibition on fumaric acid production was studied with the results shown in Figure 6.7. Different amounts of fumaric acid (0.6, 4.4, 8.3, 20.3, 39.9 g/L) were added into the medium with zero addition as the control. Without adding fumaric acid, the fermentation process produced ~53 g/L fumaric acid with the yield of

0.68 g/g glucose. Adding <8.3 g/L fumaric acid (0.6, 4.4, 8.3 g/L) decreased the yield to

~0.6 g/g glucose, whereas the influence on product titer was insignificant. Further increase of initial fumaric acid concentration to >20 g/L significantly reduced the product titer and yield by 16% and 30%, respectively. Therefore, the end product inhibition occurred when the fumaric acid concentration in the medium reached > 20 g/L. In situ recovery, hence, is necessary to reduce the fumaric acid concentration from the medium by external adsorption.

6.3.4 Intermittent in situ recovery

In a preliminary study (See the results in Table 6.3 and fermentation kinetics in

Figure 6.8), two cycles of in situ recovery were run. In each cycle, ~7.3 g fumaric acid was adsorbed by the resin equivalent to 3.7 g/L fumaric acid in the fermentor. The adsorption capacity of the resin under such conditions was ~93 mg fumaric acid/g resin.

In total, ~33.7 g/L fumaric acid was produced with yield of 49.1% and productivity of

0.35 g/L.h. The fumaric acid adsorption capacity of the resin was decreased compared to

149 the value in the fixed bed column study, possibly due to impurities in the fermentation broth. The resin performance can be further increased by optimizing flow rate and broth pH. Compared to previous studies (See Figure 4.3), the fermentation with in situ recovery successfully enhanced production yield by 25% and productivity by 59%. The fermentation can be further improved with more recovery cycles and glucose feeding.

Also, compared to traditional in situ recovery, the newly developed intermittent in situ recovery significantly decreased the required resin amount: for example, for a 2-L fermentation process, assuming a total fumaric acid production of 30 g/L and resin adsorption capacity of 90 mg/g, the traditional simultaneous recovery requires a resin amount of 670 g, whereas the intermittent recovery only requires a resin amount of 85 g with 8 cycles. Additionally, the desorbed fumaric acid can be immediately processed with enhanced downstream efficiency. More experiments will be carried out on optimizing this process.

6.4 Conclusion

As a conclusion, we first identified the optimal conditions for fumaric acid adsorption and desorption by IRA-900 resin in a fixed bed column. The favorable ranges for each condition were the resin in chloride form, the flow rate of 4.10~5.34 ml/min, the initial fumaric acid concentration of around 5.0 g/L, and desorption solution of 0.7 mol/L

NaCl. Also, we found that by studying the change of outlet pH, some key parameters during the adsorption process such as T_bre and T_sat can be well predicted. Also, by studying the effect of product inhibition on fermentation, we found that the end product inhibition occurred when the fumaric acid concentration in the medium reached > 20 g/L.

150 Therefore, intermittent in situ recovery was proposed to alleviate the product inhibition and facilitate the separation process. A preliminary study showed that the fermentation with in situ recovery successfully enhanced the production yield by 25% and the productivity by 59%. The fermentation can be further improved by adding more recovery cycles and glucose feeding, with good potential for industrial fumaric acid production.

6.5 Reference

Ataei S.A., Vasheghani-Farahani E. 2008. In situ separation of lactic acid from fermentation broth using ion exchange resins. J Ind Microbiol Biotech, 35: 1229-1233.

Cao N.J., Du J.X., Gong C.S., Tsao G.T. 1996. Simultaneous production and recovery fumaric acid from immobilized Rhizopus oryzae with a rotary biofilm contactor adsorption column. Appl Environ Microbiol 62: 2926-2931.

Cen, P. and Tsao, G.T. 1993. Recent advances in the simultaneous bioreaction and product separation processes. Separ Technol 3:58–75.

Roa Engel C.A., Straathof A.J.J., Zijlmans T.W., van Gulik W.M., van der Wielen L.A.M. 2008. Fumaric acid production by fermentation. Appl Microbiol Biotechnol 78: 379-389.

Wang, J., Wen, X., Zhou, D. 2000. Production of citric acid from molasses integrated with in-situ product separation by ion-exchange resin adsorption, Biores Technol 75: 231-234.

Yang S.T., Zhang K., Zhang B., Huang H. 2011. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition, 3, 163-177.

151 Table 6.1 Results summary of fumaric acid adsorption on IRA-900 fixed bed column

Ion Fumaric Flow rate T_bre Adsorption capacity T_sat Adsorption form acid (g/L) (ml/min) (min) (mg/g) (min) percentage OH- 10 4.1 38 119.6 110 65.2% Cl- 10 4.1 41 130 110 61.3% Cl- 10 2.34 82 129.1 180 66.2% Cl- 10 5.34 26 124.5 82 59.3% Cl- 2.4 5.0 158 112.8 240 81.7% Cl- 4.8 5.3 74 124.4 125 82.0%

Note: T_bre: breakthrough time when the outlet fumaric acid concentration reached 5% of the initial concentration; T_sat: saturation time when the outlet fumaric acid concentration reached 95% of the initial concentration; Adsorption percentage: the amount of the adsorbed fumaric acid at T_sat over the total amount of fumaric acid flowing through the column from the beginning to T_sat.

152 Table 6.2 Results summary of fumaric acid desorption from IRA-900 fixed bed column

Concentration Volume Time Recovery Stripping solution Elute pH (mol/L) (mL) (min) Yield NaCl 0.3 500 98 83.2% 4.95 NaCl 0.5 500 93 83.7% 4.77 NaCl 0.7 500 96 104% 4.40 NaOH 0.5 800 140 51.0% 13.4 NaOH 0.7 500 87 44.0% 13.2

153 Table 6.3 Performance of intermittent in situ recovery with two cycles

Fermentation: Cons. Glucose (g/L) 68.6 Total fumaric acid (g/L) 33.7 Yield (%) 49.1 Productivity (g/L.h) 0.35 Fumaric acid recovery: 1st cycle 2nd cycle Total time (h) 4.5 5.3 Time for adsorption/desorption (min) 3/1.5 4/1.3 Adsorbed fumaric acid (g) 7.3 7.4 Adsorption capacity (mg/g) 92.4 93.1

154 Air in A B Air out

G I J

H

C D E F K

Figure 6.1 Apparatus of intermittent in situ recovery from fermentation by ion-exchange

A, B: Air filter; C: neutralizing agent 100 g/L Na2CO3; D,G, I: Peristaltic pump; E: 5-L stirred tank bioreactor; F: Cotton filter; H: Ion-exchange column filled with IRA-900; J:

Desorption agent 0.7 mol/L NaCl; K: Effluent collector.

155 10

Cl-

8 OH-

6

4

2 Fumaric acid (g/L) acid Fumaric 0 0 20 40 60 80 100 120 A Time (min)

14 Cl- 12 OH-

10 pH 8

6

4 0 20 40 60 80 100 120 B Time (min)

Figure 6.2 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption by

IRA-900 resin column under two ion forms (OH- and Cl-)

156

10

8

6 5.34 4 4.10 2.34

2 Fumaric acid (g/L) acid Fumaric 0 0 40 80 120 160 200 A Time (min)

7 5.34 4.10 2.34

6 pH

5

4 0 40 80 120 160 200 B Time (min)

Figure 6.3 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption

by IRA-900 resin column under different flow rates (2.34, 4.10 and 5.34 ml/min)

157 10

10 g/L 8 4.8 g/L 2.4 g/L 6

4

2 Fumaric acid (g/L) acid Fumaric 0 0 50 100 150 200 250 A Time (min)

7 10 g/L 4.8 g/L

6 2.4 g/L

5 pH

4

3 0 50 100 150 200 250 B Time (min)

Figure 6.4 Breakthrough curve (A) and outlet pH profile (B) of fumaric acid adsorption by IRA-900 resin column under different initial fumaric acid concentrations

(2.4, 4.8 and 10 g/L)

158

50

40

30 0.3 NaCl 0.5 NaCl 20 0.7 NaCl

10 Fumaric acid (g/L) acid Fumaric 0 0 20 40 60 80 A Time (min)

8 0.3 NaCl 0.5 NaCl 6

0.7 NaCl pH

4

2 0 20 40 60 80 B Time (min)

Figure 6.5 Stripping curves (A) and outlet pH profiles (B) of fumaric acid desorption from

IRA-900 resin column by using NaCl under different concentrations

159

30

20 0.5 NaOH 0.7 NaOH

10 Fumaric acid (g/L) acid Fumaric 0 0 30 60 90 120 150 A Time (min)

14

12

0.5 NaOH

10

pH 0.7 NaOH 8

6

4 0 30 60 90 120 150 B Time (min)

Figure 6.6 Stripping curves (A) and outlet pH profiles (B) of fumaric acid desorption from

IRA-900 resin column by using NaOH under different concentrations

160

1

Produced FA 60 0.9 Yield 0.8

50 0.7

40 0.6 Yield 0.5 30 0.4

Pro. fumaric acid (g/L)acid fumaric Pro. 20 0.3 0 10 20 30 40 50 Initial fumaric acid (g/L)

Figure 6.7 Product inhibition effect on fumaric acid production

161

Figure 6.8 Fermentation kinetics with intermittent in situ fumaric acid recovery by ion

exchange

162 CHAPTER 7

Fumaric Acid Recovery and Purification from Fermentation Broth by Activated

Carbon Adsorption Followed with Desorption and Crystallization with Acetone

7.1 Introduction

Low production cost is essential for the biotechnological process in order to be competitive with petrochemical production. More than 60% of the total production costs are derived from downstream processing, including separation, concentration and purification of the product in the fermentation broth (Bechthold et al., 2008; Gluszcz et al., 2004). In a typical fumaric acid fermentation process with CaCO3 as the neutralizing agent, the harvested fermentation broth is first heated to 80 oC and acidified to pH 2.0 by

o H2SO4 due to the low solubility of both calcium fumarate (2.11% at 30 C, w/v) and fumaric acid (0.7%, w/v) (Gangl et al., 1990). Fumaric acid is dissolved at this temperature with the formation of calcium sulfate as the precipitate out of solution. Then, fumaric acid crystals are recovered by cooling the filtrate. However, fumaric acid cannot be totally recovered by cooling and some remains in the filtrate (6.3% at 25oC, w/v).

Re-crystallization is required to completely recover the rest fumaric acid. Nevertheless, the small difference of the solubility at 30oC and 0oC (See Figure 7.1) slowed the re-crystallization process. The inorganic impurities in the fermentation broth reduce the purity of the fumaric acid crystals.

163 In order to find an alternative method to recover the low-concentration fumaric acid

(<6.3 g/L) present in the filtrate after crystallization, adsorption with activated carbon was investigated. Desorption of fumaric acid from the activated carbon was then studied using acetone, which can effectively strip fumaric acid from activated carbon. After removing acetone by evaporation and water sweeping, fumaric acid crystals were obtained with purity of >99%. Both activated carbon and acetone can be recovered and reused in the adsorption process. This process is effective and economical for fumaric acid recovery and purification, and should have great potential in industrial production of fumaric acid.

7.2 Materials and Methods

7.2.1 Adsorption of fumaric acid onto activated carbon

In this study, the activated carbon (purchased from Aldrich) was charcoal-activated and granulated. The granules were 4-10 mm in length and ~4 mm in diameter. The fresh carbon granules were first washed and sonicated several times to remove the black carbon powder. Then, the activated carbon was dried at 37oC for future use.

For the effect of solution pH, 0.4 g dry activated carbon was added into 20 ml fumaric acid solution with different concentrations. The pH was adjusted to the set value

(1.0, 2.0, 3.0) using 0.5 M NaOH or HCl solutions before adding adsorbent. All the samples were incubated in a shaker at 220 rpm for 24 h at 25 oC. The residual fumaric acid concentration was detected and adsorption capacity was calculated as the amount of adsorbed fumaric acid over the dry weight of activated carbon. For the effect of temperature, the initial pH of the solution with varying concentration was adjusted to 2.

164 The adsorption behavior was studied at four temperatures (25, 37, 45, 60 oC). For the effect of byproducts (glucose, malic acid), the initial pH of the solution was 2 and the temperature was 25 oC. All the experiments were repeated in duplicate and the average values are reported.

7.2.2 Stationary desorption of fumaric acid from activated carbon via acetone

Acetone (purchased from Fisher Scientific) was used as the desorption solvent. The effects of acetone volume and incubation temperature on the desorption process were studied. 2 g wet activated carbon with the maximum adsorption of fumaric acid was added into acetone with different volumes. Desorption was performed in a stationary state overnight at various temperatures. Then, the activated carbon was separated by filtration. The desorption solution containing acetone and fumaric acid was treated with a rotary evaporator (purchased from Heidolph Instruments). All the solution was added into the evaporating flask and heated at 60 oC. Acetone was evaporated and recovered in the receiving flask by condensation. Due to the evaporation of acetone, fumaric acid crystallized and adhered to the glass wall. ~100 ml distilled water was added to dissolve the fumaric acid crystals. Samples were taken to measure the concentration of crystallized fumaric acid.

7.2.3 Separation on fixed bed column

A column (1.6×23.5 cm) packed with wet activated carbon (dry weight of 15 g) was used for fixed bed column separation. 1 L fumaric acid solution with a concentration of ~5.0 g/L at pH 2 was applied on the column at a flow rate of 1.87 ml/min by pumping. 165 At the outlet of the column, Effluent samples were taken every 15 minutes to measure the fumaric acid concentration and compose the breakthrough curve. All the effluent was collected to calculate the adsorption capacity of the fixed bed column. For the column desorption by acetone, the column was first heated at 47 oC, then 1.2 L acetone was used at a flow rate of 3.3 ml/min. All the effluent was collected and then treated with rotary evaporator connected with condenser for fumaric acid crystallization and acetone recovery. For the column separation of fermentation broth, 400 ml fermentation broth containing 5.0 g/L fumaric acid, 3.1 g/L glucose and 1.8 g/L malic acid at pH 2.0 was fed into the column at the flow rate of 1.6 ml/min. For desorption, 0.8 L acetone was used at the flow rate of 1.9 ml/min. Other procedures were the same as described before.

7.2.4 Water sweeping

A mimic after-desorption acetone solution (100 ml) containing 4.4 g/L fumaric acid,

0.5 g/L glucose and 0.5 g/L malic acid was prepared. The solution was added into rotary evaporator connected with condenser for solute crystallization and acetone recovery.

After all the acetone was evaporated, the rotary flask was cooled at room temperature.

Then, distilled water at room temperature of different volumes (5, 10 mL) was used for water sweeping. After ~5 min, the liquid which may carry some crystals was poured out and centrifuged. The suspension after centrifugation was collected for HPLC detection.

The residues were dissolved by hot water and added back into the rotary flask. Additional distilled water was added to dissolve all the crystals to measure the ingredient concentration. The purity was calculated as the amount of fumaric acid over total amount of the products.

166 7.2.5 Cost analysis

The cost analysis was performed in order to compare the economics of traditional precipitation process to that of the precipitation-adsorption process developed in this study. SuperPro Designer was used to model the two processes and evaluate the economics. The procedures before crystallization were the same for the two processes: fermentation broth was acidified, heated and filtrated. It was assumed that the filtered fermentation broth of 1 ton contained 30 g/L fumaric acid, 10 g/L glucose and 3 g/L malic acid. For the traditional precipitation, fumaric acid crystallization was carried out at

5 oC by using chilled water (price $0.4/ton) and lasted for ~6 h with a high crystallization yield of 90%. After being separated from the aqueous solution by filtration, the crystal was dried in a rotary dryer with the final product obtained. The filtrate was regarded as liquid waste with the treatment cost of $0.01/kg. For the current method, fumaric acid crystallization was carried out at room temperature by using cooling water (price

$0.05/ton) and lasted for ~2 h with a lower crystallization yield of 80%. The crystal was obtained in the same way as in the precipitation method. Differently, the filtrate flowed through packed bed column filled with activated carbon to absorb the fumaric acid.

Acetone of two-bed volume was used for desorption. The desorption broth was then heated at 70oC for complete acetone evaporation, which was condensed by cooling for future reuse. The fumaric acid crystals were washed to remove the soluble impurities.

The packed bed column and the crystallization tank were considered as the capital investment as well as the activated carbon and acetone due to their reusability. However, there was some weight loss in activated carbon (0.5%) and acetone (1%) during the process. Thus, the loss during each batch was considered as the consumables. For both

167 processes, the fumaric acid selling price of $2.1/kg was assumed with the total revenue calculated. The net difference of the two processes can be roughly estimated by the difference between the two processes of total revenue minus cost.

7.2.6 Analytical methods

HPLC was used to analyze the organic compounds, including glucose, lactic acid, malic acid, fumaric acid and ethanol, present in the fermentation broth. Because calcium fumarate was precipitated in the fermentation broth, sample pretreatment was required for

HPLC detection: dilute hydrochloric acid was added to neutralize excessive CaCO3 and acidify fumarate. Due to the low solubility of fumaric acid in water (6.3 g/L at 25 oC), the broth was heated to 80 oC to increase the solubility of fumaric acid. The HPLC system

(Shimazu Scientific Instruments) equipped with a RID-10A refractive index detector and an organic acid analysis column (HPX-87H Bio-Rad, Richmond, CA) was operated under the following conditions: sample volume of 15 μl, mobile phase of 0.005 M H2SO4, flow rate of 0.6 mL/min, and column temperature of 45 oC.

7.3 Results and Discussion

7.3.1 Fumaric acid adsorption by activated carbon

7.3.1.1 Effects of pH and isotherm modeling

As mentioned before, the target for fumaric acid recovery is the low-concentration fumaric acid (<6.3 g/L) present in the filtrate after crystallization. Thus, the isotherms of fumaric acid adsorption by activated carbon were carried out under low initial concentrations (<6.3 g/L). Figure 7.2 shows the adsorption isotherms at pH 1.0, 2.0 and

168 3.0. For all the three cases, the adsorption capacities increased with the increase of fumaric acid concentration. At the same equilibrium concentration, higher adsorption capacity of activated carbon was observed at a fumaric acid solution of pH 1 than pH 2, whereas the adsorption capacity was significantly lower when the solution pH was increased to 3.

In order to further understand the adsorption mechanism, the experimental equilibrium data was correlated with Langmuir and Freundlich models. The Langmuir theory is based on the assumption that the solute was adsorbed onto the adsorbent at specific homogeneous sites until a monolayer is formed; the multi-layer formation of adsorbate was impossible due to the electrostatic repulsion between adsorbed molecules and those in solution (Giles and Smith, 1974). The Langmuir equation can be described as follows:

where: Ce (g/L) is the equilibrium concentration of fumaric acid; qe (mg/g) is the surface concentration of fumaric acid at equilibrium; Qmax (mg/g) and KL (L/g) are Langmuir constants related to adsorption efficiency and energy, respectively. The linear form is given by:

Therefore, the two constants Qmax and KL can be obtained by plotting Ce/qe versus Ce, which gives a straight line with slope of 1/ Qmax and intercepts of 1/ KLQmax.

The Freundlich model, typically for heterogeneous surfaces, can be represented as follows: 169

n where: KF (mg/g (g/L) ) is the adsorption capacity when Ce equals one; n is Freundlich exponent, representing the degree of adsorption dependence on equilibrium concentration.

Similarly, the two constants KF and n can be obtained by plotting lnQe versus lnCe, which gives a straight line with slope of 1/n and intercepts of lnKF. The linear form is represented by:

(4)

Table 7.1 summarizes the results of modeling. The correlation coefficients (R2) with Langmuir isotherm model were high for fumaric acid solution at pH 1 and 2 with values of 0.994 and 0.994, respectively. The higher value of the Langmuir constant KL indicated a more favorable adsorption process. By comparing KL, the adsorption of fumaric acid onto activated carbon was more favorable at pH 1 than pH 2, also as indicated from the comparison of Qmax. However, the fitting of the isotherm at pH 3 by

Langmuir model had a low R2 (0.802). For the regression by Freundlich model, all three

2 cases had a high R (>0.99). As indicated from the values of KF, the extent of adsorption for fumaric acid solution at pH 1 was higher than that at pH 2, whereas the extent of adsorption at pH 3 was significantly lower. Such results were in agreement with the conclusion made from Langmuir model. The n values for all the cases were higher than unity, reflecting that under all three pHs fumaric acid is favorably adsorbed by activated carbon. Overall, considering the model suitability on fumaric acid solution at pH 3 and the highly heterogeneous surface of activated carbon, the Freunlich model is more suitable for fumaric acid adsorption on activated carbon.

170 In order to elucidate the effects of solution pH on fumaric acid adsorption, the adsorption mechanisms was further investigated. In aqueous fumarate solution, unionized fumaric acid (H2FA) exists in equilibrium with fumarate anions (HFA-, FA2-), as shown in Figure 7.3. Fumaric acid, as a diprotic acid, has two pKa values, 3.03 and 4.44. Thus,

- H2FA and HFA are the two primary forms for aqueous fumarate solution at pH 3, whereas H2FA is dominant at pH 2. Moreover, due to a close-to-pKa solution pH of 3, the concentration of H2FA is equal to the concentration of HFA-. For activated carbon, it has both basic and acidic functional groups (Chun et al., 2004). The combined effects of all the functional groups determine pHpzc at which the net surface charge of activated carbon becomes zero. The carbon surface has a net positive charge at pH < pHpzc and net negative charge at pH > pHpzc (Al-Degs et al., 2007). The pH drift method is used to identify the pHpzc of the activated carbon used in this study. From Figure 7.4, the pHpzc is the pH value (7.63) at the intersection point of the curve pHfinal vs pHinitial and the line pHinitial = pHinitial (Faria et al., 2004). Therefore, at both pH 2 and 3, the activated carbon had a net positive charge. If electrostatic interaction was the sole mechanism for fumaric acid adsorption, the adsorption capacity of pH 3 should be higher than that of pH 2 due to the negative charge of fumarate anion. However, the adsorption capacity at pH 3 was significantly lower, only half the value of adsorption capacity at pH 2 under the same equilibrium concentration. As mentioned before, the initial concentration of neutral

H2FA at pH 3 was half of its initial concentration at pH 2. Combining these findings, a conclusion can be reached that the adsorption of fumaric acid on activated carbon is mostly governed by non-electrostatic interactions such as hydrogen bonding or hydrophobic interactions (Moreno-Castilla, 2004); and the interactions mostly occurred

171 between the undissociated fumaric acid and the activated carbon. The mechanism can be well applied to explain the finding of the increased adsorption capacity at pH 1: the increased H+ concentration increased some interactions such as hydrogen bonding between fumaric acid and activated carbon.

7.3.1.2 Effects of byproducts concentrations

Selectivity is critical in the process of fumaric acid recovery from fermentation broth due to the existence of large amounts of glucose and malic acid. Based on the fermentation results in our lab, malic acid was the major byproduct with a final concentration of <5.0 g/L. As shown in Table 7.2, the selectivity of fumaric acid against glucose and malic acid was studied. In general, the addition of glucose and malic acid lowered the adsorption capacity for fumaric acid to some extents. The selectivity is defined as the adsorption capacity for fumaric acid over that for byproduct. At a lower byproduct concentration (~2.5 g/L), the selectivity values of fumaric acid over glucose and malic acid were 31.0 and 8.3, respectively. With an increase in the glucose and malic acid concentration, the selectivity decreased, especially at a high malic acid concentration.

However, the relatively higher adsorption capacity for fumaric acid over the byproducts guaranteed the selective adsorption on activated carbon. The reason can be attributed to the fact that compared to the molecules of glucose and malic acid, the higher hydrophobicity of the fumaric acid molecule, resulted from a carbon double bond, significantly increases the hydrophobic interactions with activated carbon.

172 7.3.2 Stationary desorption of fumaric acid by acetone

Desorption process for fumaric acid adsorbed on activated carbon was also studied.

In an unreported experiment, the effects of different desorption solutions (water, H2SO4) were studied. Water can wash away ~35% fumaric acid from activated carbon, whereas

~75% fumaric acid can be removed by using H2SO4. Gao et al. used acetone to strip the lactic acid adsorbed on activated carbon (Gao et al., 2011). Thus, the stationary desorption of fumaric acid by using acetone was investigated. Because acetone with a low boiling point (56.1 oC) can be easily evaporated at a high temperature, the acetone solution after 24-h desorption was put into the rotary evaporator (at 70 oC) connected with a condenser for fumaric acid crystallization and acetone regeneration. It was observed that all the acetone was evaporated and fumaric acid crystals were formed. The effects of acetone volume and temperature of the stationary desorption process were studied with results shown in Figure 7.5 and Table 7.3. The overall recovery yield was calculated as the crystallized fumaric acid amount over the adsorbed amount on activated carbon. The recovery yield increased with an increase of acetone volume and temperature.

The results can be explained by the increased solubility of fumaric acid in acetone with the increase of solvent volume and temperature. The best values of acetone volume and temperature were identified as 100 ml (for 2 g activated carbon) and 47 oC, respectively.

Under such conditions, more than 98% fumaric acid can be removed from activated carbon. Comparing to the fumaric acid crystallization in water, its crystallization in acetone by evaporation was much faster, in less than 20 min. Moreover, the evaporated acetone can be recovered by condensation and the activated carbon by washing for future reuse. However, the volume loss of the acetone (12%) was high after a 24-h stationary

173 desorption process at 47 oC. Thus, the effects of contacting time (0.5, 1, 1.5, 2, 2.5, 5, 19 h) were further studied at 47 oC with results shown in Table 7.4. A recovery yield of >90% was achieved after 5 h desorption with volume loss of only 3%. Thus, the decrease of contacting time from 24 h to <10 h successfully prevented too much loss of acetone volume as well as maintained a high recovery yield.

7.3.3 Adsorption and desorption on fixed bed column

The breakthrough curve of fixed bed column separation of fumaric acid by activated carbon is shown in Figure 7.6. Assuming that the column was broken through when the effluent concentration reached 10% of the initial concentration, the breakthrough time was at 370 min with breakthrough volume of 700 ml at a flow rate of 1.87 ml/min. At this point, the adsorption percentage, calculated as the adsorbed amount over the total amount flowing through the column, was 95%. For a 5% breakthrough point, the breakthrough volume decreased to 420 ml with an enhanced adsorption percentage of 99%.

Considering the beneficial effect of flow rate decrease on improving adsorption percentage, 400 ml fermentation broth was applied with a lower flow rate of 1.63 ml/min.

Figure 7.7 shows the breakthrough curve. Although the adsorption capacity was only 132 mg/g due to unsaturated activated carbon adsorption, the fumaric acid concentration in the effluent was controlled under <0.1 g/L with a high adsorption percentage of 99.5%.

For the adsorption of byproducts in the fermentation broth, the adsorption percentages for glucose and malic acid were only 21.3% and 38.1%, while the adsorption capacities were

17.7 and 18.7 mg/g, respectively. Comparing the breakthrough curve of fumaric acid with

174 those of glucose and malic acid, selective adsorption of fumaric acid by column separation was achieved.

The desorption process by acetone on fixed bed column was also studied. For the adsorption case of pure fumaric acid solution, 1.2 L acetone was applied to strip fumaric acid from the activated carbon at a flow rate of 3.3 ml/min and the recovery yield reached

94%. For the adsorption case of 400 ml fermentation broth, 0.8 L acetone was used at a flow rate of 1.9 ml/min and the recover yield reached 93%. The recover yield can be further improved by increasing acetone volume and decreasing flow rate.

7.3.4 Water sweeping

However, the acetone can not only strip fumaric acid out of the activated carbon, but also glucose and malic acid. The crystallization process did not have selectivity for fumaric acid either due to the evaporation of solvent. Consequently, for the fumaric acid recovery from fermentation broth, the crystals after crystallization had a low purity of

<90%. In order to sweep the byproducts, such as glucose and malic acid, out of the crystals, water sweeping was carried out based on the low solubility and slow dissolving rate of fumaric acid. The effect of sweeping water volume was studied with a control without water sweeping. Figure 7.8 shows the recovery yield of fumaric acid, glucose and malic acid as well as the purity of the fumaric acid crystals after water sweeping. By applying water sweeping, the purity of fumaric acid crystals was significantly enhanced whereas the recovery yield for both glucose and malic acid significantly dropped. Also, the purity increased with more water being used. A high purity of 98.2% was reached by using 10% water with trace amounts of glucose and malic acid. Although the recovery

175 yield of fumaric acid was also decreased due to the increased volume of water, the small volume of washing solution can be added into another batch of fermentation broth for recycle. Similarly, the water sweeping process was applied to the case of recovery from fermentation broth. The sweeping water washed out not only these soluble byproducts but also the yellow pigments of the broth. The purity of the final product reached >98%, which might be even higher due to the undetectable trace amount of glucose and malic acid. To reach the requirement of a minimal purity of 99.5% for industrial-grade crystals, such water sweeping process can be further improved by using cold water in which fumaric acid has a lower solubility or by double sweeping.

7.3.5 Cost analysis

Table 7.5 shows the factors with significantly different values between traditional precipitation and the newly-developed precipitation-adsorption process. Compared to the traditional process, although the new process require additional capital investment

(activated carbon, acetone, fixed bed column, crystallization tank), the process had the following advantages: First, by operating the crystallization process at room temperature, the cooling cost was reduced significantly due to the use of cooling water ($0.05/ton) instead of chilled water ($0.4/ton); Second, by targeting the filtrate for fumaric acid recovery, the cost of liquid waste treatment was avoided; Third, although the crystallization yield (80%) was lower, the overall recovery yield (95%) was enhanced by

14.5% with a higher total revenue. The purity of the final crystals was also improved to

99%. The unit net difference of the two processes ($0.255 /kg fumaric acid) was obtained by comparing their values of revenue minus cost over initial fumaric acid amount in the

176 fermentation broth. Thus, for a 500 ton fermentation broth in 100 batches it would be

$383,000, which can offset the cost of capital investment. Therefore, from a long-term consideration, this precipitation and adsorption process was both economically and environmentally favorable with good potential for industrial fumaric acid production.

7.4 Conclusion

The aim of this research was to recover the low-concentration fumaric acid (<6.3 g/L) present in the filtrate after crystallization. An integrated separation process was developed for fumaric acid recovery and purification by adsorption with activated carbon followed with desorption and crystallization with acetone. Fumaric acid adsorption on activated carbon was found to be governed by non-electrostatic interactions between the undissociated fumaric acid and the activated carbon. The desorption of fumaric acid from activated carbon was studied using acetone to strip all fumaric acid from activated carbon.

After removing acetone by evaporation at 70 oC, fumaric acid crystals were obtained with a high purity and recovery yield. Both activated carbon and acetone can be recovered and reused in the adsorption process. The adsorption and desorption process was then evaluated in a fixed bed column to recover fumaric acid from the fermentation broth, achieving a high recovery yield of 93%. Finally, water sweeping was used to further increase the purity of fumaric acid crystals to >98%. A comparative economic analysis showed that this process could significantly reduce the operational costs and enhance the recovery yield, although it would require additional capital investment. Compared to the conventional recovery process using precipitation, the new process is economically and environmentally favorable with good potential for industrial application.

177 7.5 Reference

Al-Degs, Y.S., El-Barghouthi, M.I., El-Sheikh, A.H., Walker, G.M. 2007. Effects of solution pH, ionic strength, and temperature on adsorption behavior of reactive dyes on activated carbon. Dyes Pigments 77: 16-23.

Bechthold, I., Bretz, K., Kabasci, S., Kopitzky, R., Springer, A. 2008. Succinic acid: a new platform chemical for biobased polymers from renewable resources. Chem Eng Tech, 31(5): 647–654.

Chun, Y., Sheng, G., Chiou, C., Xing, B. 2004. Compositions and sorptive properties of crop residue-derived chars. Env Sci Tech 38: 4649-55.

Faria, P., Orfao, J., Pereira, M. 2004. Adsorption of anionic and cationic dyes on activated carbons with different surface chemistries. Water Res 38: 2043-52.

Gangl, I.C., Weigand, W.A., Keller, F.A. 1990. Economic comparison of calcium fumarate and sodium fumarate production by Rhizopus arrhizus. Appl Biochem Biotech 24-25: 663-677.

Gao, M.T., Shimamura, T., Ishiba, N., Takahashi, H. 2011. pH-uncontrolled lactic acid fermentation with activated carbon as an adsorbent. Enzyme Microb Tech 48: 526-530.

Giles, C., Smith, D. 1974. General treatment and classification of the solute sorption isotherms. J Colloid Interface Sci 47: 755-765.

Gluszcz, P., Jamroz, T., Sencio, B., Ledakowicz, S. 2004. Equilibrium and dynamic investigations of organic acids adsorption onto ion-exchange resin. Bioproc Biosyst Eng 26: 185-190.

Mattson, J., Mark, H. Activated carbon: surface chemistry and adsorption from solution. New York: Marcel Dekker, Inc.; 1971.

Moreno-Castilla, C. 2004. Adsorption of organic molecules from aqueous solutions on carbon materials, Carbon 42: 83-94.

178 Table 7.1 Model parameters obtained from fitting the experimental equilibrium data with

isotherm models

Langmuir model Freundlich model pH 2 2 R Qmax KL R KF n 1 0.994 239.6 4.97 0.994 197.0 3.26 2 0.994 223.6 4.12 0.992 170.4 3.62 3 0.802 - - 0.996 54.8 1.09

179 Table 7.2 Selectivity study of fumaric acid adsorption on activated carbon

(fumaric acid initial concentration of 5.5 g/L)

FA adsorbed Ini. conc. Byproduct adsorbed Byproduct Selectivity amount (mg/g) (g/L) amount (mg/g) 221.3±0.5 - - - - 203.2 ±0.9 Glucose 2.6 6.8±1.8 31.0±8.1 197.4±2.6 Glucose 4.2 9.4±0.1 21.1±0.5 203.2±2.6 Glucose 8.6 22.2±4.5 8.1±1.3 190.1±1.6 Glucose 16.5 21.9±4.8 8.7±2.7 194.3±0.4 Malic acid 2.9 23.4±0.8 8.3±0.3 193.4±0.7 Malic acid 4.8 34.1±0.5 5.7±0.1 189.0±2.5 Malic acid 9.6 54.7±0.9 3.4±0.1 179.3±2.4 Malic acid 18.6 70.1±15.8 2.6±0.6

180 Table 7.3 Effects of temperature on fumaric acid desorption from 2g activated carbon by

using 100 ml acetone

Temperature (oC) V_des (mL) FA yield 22 ± 1 96 ± 2 0.77 ± 0.01 33 ± 1 95 ± 2 0.83 ± 0.01 41 ± 1 91 ± 2 0.86 ± 0.03 47 ± 1 88 ± 2 0.98 ± 0.05

Note: V_des: acetone volume after 24 h desorption

181 Table 7.4 Effects of contacting time on fumaric acid desorption from 2g activated carbon

by using 100 ml acetone at (47±1) oC

Time (h) V_des (mL) FA yield 0.5 100 0.63 1 99 0.76 1.5 99 0.80 2 99 0.87 2.5 96 0.89 5 97 0.92 19 91 1.03

182 Table 7.5 Comparison between traditional precipitation and newly-developed precipitation and adsorption process for fumaric acid recovery (initial conc. of 30 g/L)

from 1 ton fermentation broth

Precipitation Adsorption Activated carbon 100 kg; Acetone 2 .0 ton; Capital investment Fixed bed column (height 2.0 m; diameter 0.16 m) Vessel for acetone evaporation Operational cost per batch Cooling for Chilled water Cooling water crystallization 14.5 ton, $5.8 10.6 ton, $0.53 Water 5.0 ton, $0.25 8.0 ton, $0.4 Liquid waste Liquid waste Liquid waste treatment 1.0 ton, $10 12 kg, $0.12 0.5% activated carbon ($0.5); Consumables 1% acetone ($14) Total $16.05 $15.6 Revenue per batch Recovery yield 83% 95% Purity 98% 99% Revenue $52.7 $59.9 (Fumaric acid $2.1/kg) Net difference (capital 0 + $7.65 investment not included) Unit net difference 0 $0.255 /kg fumaric acid

183 60

50

40

30

20

Solubility (g/L) Solubility 10

0 0 20 40 60 80 100 temperature (OC)

Figure 7.1 Fumaric acid solubility at different temperatures

184

280

240

200

160

120 pH 1 80 pH 2 40 pH 3

0 Adsorption capacity (mg/g) capacity Adsorption 0 0.5 1 1.5 2 2.5 3 3.5

Equilibrium concentration (g/L)

Figure 7.2 Adsorption isotherms of fumaric acid on activated carbon at different pHs.

Points: experimental data; Lines: Freundlich model

185 12 11 10 9 pHpzc = 7.63 8

final 7

pH 6 5 4 3 2 2 3 4 5 6 7 8 9 10 11 12 pHinitial

Figure 7.3 Determination of pHpzc of activated carbon by the pH drift method

186

Figure 7.4 Dissociation equilibrium of aqueous fumarate solution

187 1

0.9

0.8

Yield 0.7

0.6

0.5 40 80 120 160 200 Acetone volume (mL)

Figure 7.5 Effects of acetone volume on desorption of fumaric acid from 2g activated

carbon

188

4

3

2

1 Fumaric acid (g/L) acid Fumaric 0 0 100 200 300 400 500 600 Time (min)

Figure 7.6 fixed bed column separation of fumaric acid by activated carbon

189

6

Fumaric acid 5 Glucose 4 Malic acid 3

2

1 Concentration (g/L) Concentration 0 0 50 100 150 200 250 Time (min)

Figure 7.7 fixed bed column separation of fermentation broth by activated carbon

190

1

0.8

Fumaric acid 0.6 Glucose Malic acid 0.4 Purity

0.2 Recovery yield, purity yield, Recovery

0 control 5%H2O 10%H2O

Figure 7.8 Effects of water sweeping on recovery yield and crystals purity

191 CHAPTER 8

Conclusions and recommendations

8.1 Conclusions

In this study, a novel fermentation method for fumaric acid production by Rhizopus oryzae was developed. By using soybean meal hydrolysate (SMH) as nitrogen source for seed culture process, uniformly dispersed mycelial clumps with diameters of 0.1 mm were formed with enhanced subsequent fumaric acid production. The process was also scaled up on 5-L stirred tank bioreactor (STB). With the addition of 40 mL/L SMH and

CO2-enriched inlet gas, the fermentation reached a high fumaric acid production with titer of 35.6 g/L, yield of 40% and productivity of 0.4 g/(L.h). In order to minimize the production cost, alternative separation methods were investigated. IRA900 resin was selected for in situ product recovery due to its high adsorption capacity at fermentation-favored pH of 5.0. Intermittent in situ product recovery was proposed based on the knowledge of separation process on fixed bed column. In order to increase the recovery yield and improve the product purity, a novel integrated separation process involving fumaric acid adsorption by activated carbon followed by desorption and crystallization via acetone was developed. The important results and conclusions obtained in this study are summarized below.

192 8.1.1 Effects of Soybean Meal Hydrolysate as the Nitrogen Source on Seed Culture

Morphology and Fumaric Acid Production by Rhizopus oryzae

Traditional fumaric acid production by fungal fermentation has received much attention, but suffers from low productivity and yield largely because of poor cell morphology limiting mass transfer. In this study, a simple seed culture medium was developed with soybean meal hydrolysate (SMH) as the nitrogen source for controlling the morphology of R. oryzae ATCC 20344. Uniformly dispersed mycelial clumps with a diameter of ~0.1 mm were formed with enhanced subsequent fumaric acid production.

The optimal seed culture conditions were initial pH of 3.0, SMH concentration of 30%

(v/v), spore concentration of 8×104/mL and glucose concentration of 10 g/L. With an inoculation size of 25%, the fermentation reached a fumaric acid titer of 50.2 g/L with yield of 0.72 g/g glucose. SMH with a high protein content was a good nitrogen source and the formation of protein precipitate acted as the immobilization carriers for cells. The solid-phase protein also provided a novel method for slow/controlled release that allowed the utilization of the nitrogen source by cells for an extended period without losing cell activity.

8.1.2 Optimization on Fumaric Acid Production by Rhizopus oryzae in 5-L

Stirred-Tank Bioreactor

The fermentation was then studied in a 5-L stirred tank bioreactor (STB) and the results also showed that using SMH as the nitrogen source improved fumaric acid production with increased yield and productivity compared to urea and yeast extract. The effect of CO2 on fumaric acid fermentation was studied. Aeration with air containing 16.7%

193 CO2 increased the productivity by 76% and final product titer by 13%. A product titer of

35.6 g/L, yield of 40% and productivity of 0.4 g/L∙h were obtained in batch fermentation with an initial seeding density of ~5000 mycelial clumps/ml. With CO2 addition, similar fumaric acid production in the fermentation was obtained with either CaCO3 or NaOH as the neutralizing agent, indicating that CaCO3 can be replaced with NaOH and CO2 to simplify the downstream process.

8.1.3 Characteristic and mechanism study of fumaric acid adsorption onto IRA900 ion exchange resin

The objective was to develop a novel, economical and scalable in situ recovery method for fumaric acid separation. IRA900 strong-basic anion exchange resin was selected due to its high adsorption capacity at the fermentation-favored pH of 5.0, high selectivity against byproducts (glucose and malic acid), and easy desorption process by using NaCl with a high recovery yield of ~95%.The adsorption isotherm and the mechanism involved in the fumaric acid adsorption onto IRA900 were investigated. The results showed that fumaric acid adsorption onto IRA900 followed the Langmuir isotherm. Also, the adsorption reaction rate between fumaric acid and functional groups of the resin followed the second-order kinetics, indicating a mechanism of two-site-occupancy adsorption reaction between FA2- and resin active sites. However, such a mechanism cannot explain the high adsorption capacity at pH 3 with a high initial fumaric acid concentration. Using an intraparticle diffusion model, it was found that the higher adsorption at pH 3 could be attributed to the higher hydrophobic interaction between neutral fumaric acid molecules and the hydrophobic resin backbone.

194 8.1.4 Intermittent in situ Recovery of Fumaric Acid from Fermentation Broth by

Using IRA-900 Ion Exchange Resins

The separation process by using IRA900 ion exchange resin was evaluated on a fixed bed column with the optimal conditions identified. The favorable ranges for each condition were the resin in chloride form, the flow rate of 4.10~5.34 ml/min, the initial fumaric acid concentration of around 5.0 g/L, and desorption solution of 0.7 mol/L NaCl.

Also, we found that by studying the change of outlet pH, some key parameters during the adsorption process such as breakthrough time and saturation time can be well predicted.

It was also found that end product inhibition occurred when the fumaric acid concentration in the medium reached >20 g/L. Therefore, intermittent in situ recovery was used to alleviate the product inhibition and facilitate the subsequent product separation. A preliminary study showed that fermentation with in situ recovery enhanced product yield by 25% and productivity by 59%.

8.1.5 Fumaric acid recovery and purification from fermentation broth by activated carbon adsorption followed with desorption and crystallization with acetone

Fermentation produced fumaric acid is usually recovered by precipitation and crystallization of the acid at room temperature. In order to recover the low-concentration fumaric acid (<6.3 g/L) present in the filtrate after crystallization, an integrated separation process was developed for fumaric acid recovery and purification by adsorption with activated carbon followed with desorption and crystallization with acetone. Fumaric acid adsorption on activated carbon was found to be governed by non-electrostatic interactions between the undissociated fumaric acid and the activated carbon. The desorption of

195 fumaric acid from activated carbon was studied using acetone to strip all fumaric acid from activated carbon. After removing acetone by evaporation at 70 oC, fumaric acid crystals were obtained with a high purity and recovery yield. Both activated carbon and acetone can be recovered and reused in the adsorption process. The adsorption and desorption process was then evaluated in a fixed bed column to recover fumaric acid from the fermentation broth, achieving a high recovery yield of 93%. Finally, water sweeping was used to further increase the purity of fumaric acid crystals to >98%. A comparative economic analysis showed that this process could significantly reduce the operational costs and enhance the recovery yield, although it would require additional capital investment. Compared to the conventional recovery process using precipitation, the new process is economically and environmentally favorable with good potential for industrial application.

8.2 Recommendations

8.2.1 Fermentation process development

In this study, we developed a novel seed culture process by using SMH as nitrogen souce with the formation of well-controlled cell morphology of mycelial clumps. The advantages of mycelial clumps over pellets were discussed mostly based on previous studies and fermentation results. It is recommended to describe the advantages of mycelial clumps in a more quantitative and comprehensive way. For example, confocal laser scanning microscopy together with image analysis provides powerful tools to generate a quantitative description of the cell morphology, which may be used for modeling and prediction of mass transfer in mycelial aggregates (Hile et al., 2005). Also,

196 the cell morphology has significant influence on rheology (Papagianni, 2004). Studying such effects may help to better understand the effects of cell morphology on mass and oxygen transfer.

We found soybean meal to be a good nitrogen source with several advantages.

However, due to the complicated ingredients of soybean meal, we do not know the exact favorable components of soybean meal for fumaric acid production. In addition, we found that protein precipitates were formed when the pH of SMH was adjusted to >3. The research on component analysis of both SMH and protein precipitates as well as the effect of solution pH on protein precipitates and their constitutions may better explain the advantages of SMH and protein precipitates, providing reasonable guidance on using soybean meal. Also, based on the literature, the microbial metabolism on soybean meal can improve the antioxidant activity of soybean meal with enhanced reducing power

(Wongputtisin et al., 2007), leading to increased fumaric acid production. Thus, to better support the point, a quantitative study on the change of the reducing power during the fermentation is necessary.

For the fermentation on STB, we studied the effects of nitrogen source, CO2 addition, and cell density. It is believed that the fermentation process can be further improved from the following ways. First, the percentage of CO2 in the inlet gas can be optimized. Due to the high cost of pure CO2, the possibility of replacing CO2 enriched inlet gas with flue gas can be investigated. Second, the initial glucose level in this study was kept constant in the range of 80~100 g/L. It is reported that a high C/N ratio of 120 to 250 (w/w) is required for the overproduction of fumaric acid (Magnuson and Lasure,

2004). Thus, studying the effects of initial glucose level with different level of nitrogen

197 source is recommended as well as different glucose feeding strategies. Third, two-phase

DO control strategy was demonstrated as a useful way to improve fumaric acid production (Ling and Ng, 1989; Fu et al., 2010). Increasing stirring speed also helps to maintain a high DO level. However, the generated high shear rate may seriously damage the cell and reduce the cell activity. Thus, it is recommended to apply the DO control together with the study on effects of stirring speed. Last but not least, the fermentation results in STB were still not comparable to those in the shake flask. Compared to the fermentation in shake flask, the possibility of stress exposure for the cells was significantly enhanced in STB, especially at a high stirring speed. The increased stress exposure caused detrimental effects and some unpredictable metabolic shifts (Schmidt et al., 2005). To fully understand the fermentation differences between shake flask and STB, we need a comprehensive and quantitative process characterization such as dissolved oxygen and CO2 concentration profiles in culture broth, full metabolite profiles, etc.

From another perspective, different fermentor modes can be applied, such as air lift fermentors. Air lift fermentors achieve good mixing via liquid recirculation between bubbling and non-bubbling zones, providing high oxygen transfer rates with minimal shear stress on cells (Du et al., 1997). Overall, it is believed that the fermentation process can be further improved if the above recommended work can be partly or fully finished.

8.2.2 Improvement on in situ product recovery

In this study, we developed a novel intermittent in situ product recovery method. A preliminary experiment showed an improvement on production yield of fumaric acid by using this method. The process can be further improved by the following ways: First, the

198 adsorption capacity of the IRA900-filled column was lower, possibly due to many impurities in the fermentation broth. Thus, the adsorption performance needs to be optimized regarding flow rate, adsorption and desorption time for each cycle, etc. Second, in order to further improve the efficiency of in situ product recovery, several parameters have to be optimized, such as the starting time of the recovery. If the adsorption starts too early, the high glucose and low fumaric acid concentration may significantly lower the adsorption performance. However, if the adsorption starts too late, the high fumaric acid concentration may cause end product inhibition on fermentation process. In addition, pH is another influencing factor: a lower pH of ~4.0 favored the adsorption process, whereas a higher pH of >5.0 benefited the fermentation process. Third, in situ product recovery strategy can be combined with fed-batch fermentation. Wu et al. (2012) enhanced acetone-butanol-ethanol fermentation by using fed-batch fermentation with in situ recovery by pervaporation. The glucose concentration can be well maintained at a suitable level by using fed-batch fermentation. With the addition of nitrogen source to maintain the cell activity, it is likely to achieve a continuous fermentation.

8.2.3 Improvement on fumaric acid recovery by activated carbon adsorption

In this study, we developed a novel fumaric acid recovery method by activated carbon adsorption followed by desorption and crystallization with acetone. The process can be further improved in the following ways: First, it is recommended to research the effects of temperature on fumaric acid adsorption on activated carbon as well as thermodynamic study. The knowledge may help us better understand the interactions between fumaric acid and activated carbon, thus giving reasonable guidance on process

199 design. Second, in order to further improve the separation performance on fixed bed column, the parameters for the process need to be optimized, such as flow rate, initial fumaric acid concentration, solution pH etc. Third, in order to reach an industrial-grade fumaric acid, the purity of the final products should be further improved. The recommended methods are to apply cold water sweeping or double sweeping. Last but not least, cost analysis showed that this newly-developed process is economically superior to the traditional method from a long-term view. A more comprehensive economic analysis including the fermentation part together with sensitivity test may make the conclusion more reliable and persuasive.

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