Termite's Biomass Degradation Strategy And

TERMITE’S BIOMASS DEGRADATION STRATEGY AND

BIOMIMIC TREATMENT

SHUAI ZHANG

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Biological Systems Engineering

JULY 2017

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of SHUAI

ZHANG find it satisfactory and recommend that it be approved.

______Shulin Chen, Ph.D., Chair

______Yang Bin, Ph.D.

______Hanwu Lei, Ph.D.

______Su Ha, Ph.D.

ii ACKNOWLEDGEMENT

Life is colorful and exciting with the polish of difficulties in livelihood and decoration of dream in our heart, just as the old poem saying “Let life be beautiful like summer flowers”. My student experience has come to the end, at the moment of accomplishing my PhD thesis. However, this doesn’t mean that this is the end of study. Keeping touch with new knowledge is the basic requirement for a researcher, no matter I am going to work as a post-doctor in a university, an engineer in industry or a researcher in an institution. I luckily have obtained a lot of help towards my graduate program at Washington State University from my best Professors and friends. I would like to take this opportunity to show my great appreciation.

Firstly, I want to give great thanks to my dear advisor Shulin Chen. He offered me the opportunity to study at WSU and get advanced education in USA. In his group, hardworking and creative thinking are treated as the soul of research. He did his best to push me to reach the goal and help me to design my Ph.D work. During the study, I deeply felt the pressure from courses and research work, but I really enjoyed his encouragement. What I know was that he tried to help and protect each his students, even when his finance is tight. I would also thank all my committee members, Dr. Yang Bin, Dr. Su Ha and Dr. Hanwu Lei. They were patient and kindly to provide suggestions and revise my dissertation. I was impressed with their professionalism when I asked for their help in solving experiment problems, such as electrochemistry and delignification.

I would like to show my appreciation to all my friends in the Bioprocessing Bioproduct

Engineering Laboratory (BBEL). We all make great efforts to build atmosphere for open communication, research sharing and assistantship. I would like specially thank to Dr. Xiaochao

iii Xiong, Dr, Jing Ke and Dr. Dongyuan Zhang. They separately trained me when I stayed in BBEL of WSU and TIB of Tianjin. They taught me a lot in how to work in professional way in their fields. I would like to thank Jonathan Lomber. He spent a lot of time to teach me how to use equipment and help me fix all the problems in using it. Also, I want to give great thanks to Ms.

Joanna Dreger. She always provided me timely suggestions in my course and give me kindly guidance.

Finally, I want to show my great thanks to my family, especially my parents Jianjun Zhang and Shuping Lou. I could not finish my Ph.D study without your encouragement and support in my daily-life.

iv TERMITE’S BIOMASS DEGRADATION STRATEGY AND

BIOMIMIC TREATMENT

Abstract

by Shuai Zhang, Ph.D. Washington State University July 2017

Chair: Shulin Chen

Termite, C. formosanus, is well known for its efficient wood digestion ability. The purpose of this dissertation reesasrch is to firstly investigate the related mechanism used by termite in wood digestion and then develop the bio-mimic process for treating the different components of lignocellulosic biomass. In the first part of the study, we proved the hydroquinone-production route in termite by expression of β-glucosidases (Glu1B) in E.coli Rosetta and further hydrolysis arbutin for hydroquinone generation. Arbutin was found as a compound existing in southern pine extractives with the concentration of 0.08 mg/g and the enzyme activity of Glu1B to arbutin was shown as 0.034 IU/ml at 28 oC. This suggested a pathway for HO· production in termite: arbutin in wood particles is initially hydrolyzed by β-glucosidases to release hydroquinone, which is further catalyzed by iron and lignin oxidase for HO· generation. With this knowledge, we developed an electro-Fenton process to mimic termite’s strategy in the second part of the study. In the electro-Fenton process, we used electricity for electron supply to oxygen through cathode reduction. This simulated H2O2 conversion process, in which oxygen extracted electrons from hydroquinone. Then, the generated H2O2 was further decomposed by iron for HO· production. In

v the next step, we used electro-Fenton process in lignin treatment. It was discovered that electro-

Fenton process was able to transform lignin to long-chain fatty acids. The possible mechanism was that lignin was initially oxidized to volatile fatty acids (VFAs) and then, the VFAs were synthesized to long-chain fatty acids through Kolbe reaction. After process optimization, the yield

of long-chain fatty acids reached to 8.68 mg/L palmic acid and 8.56 mg/L octadecanoic acid. In the last part of the study, we applied electro-Fenton process to cellulose degradation. It was found that it could effectively destroy microalgae cell and degrade cellulose, the major component in microalgae cell wall. Under optimal conditions, electro-Fenton process enhanced lipid extraction from 40 to 87.53% (wt/wt, total lipid). Also, the waste water generated from electrolysis could be used for microalgae cultivation without inhibitory effect.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT ...... iii ABSTRACT ...... v LIST OF FIGURES ...... xii LIST OF TABLES ...... xv

1 INTRODUCTION ...... 1

1.1 Introduction to sustainable biofuels ...... 2

1.2 Current pretreatment technologies for saccharification ...... 5

1.3 Termite-a good natural example for lignocellulose degradation ...... 7

1.4 Hydroquinone-a functional compound in termite ...... 9

1.5 Arbutin-Possible source of hydroquinone in termite ...... 10

1.6 H2O2-a key oxidant for lignocellulose degradation in termite ...... 11

1.7 Electro-Fenton reaction–a termite mimicking process...... 13

1.8 Effects of different factors on electro-Fenton performance ...... 15

1.9 Electro-Fenton process application in lignin treatment...... 17

1.10 HO· for cellulose degradation ...... 18

1.11 Dissertation Overview ...... 20

vii 1.12 Publications ...... 23

1.13 References ...... 24

2 MECHANISMS OF HYDROQUINONE PRODUCTION IN COPTOTERMES

FORMOSANUS ...... 30

2.1 Abstract ...... 31

2.2 Introduction ...... 32

2.3 Materials and Methods ...... 35

2.3.1 Preparation of Southern yellow pine extract ...... 36

2.3.2 Thin-layer chromatography separation...... 36

2.3.3 HPLC analysis ...... 37

2.3.4 Recombinant β-glucosidase expression and purification ...... 37

2.3.5 Enzymatic digestion of the recombinant β-glucosidase...... 38

2.4 Results ...... 39

2.4.1 HPLC results for p-arbutin detection ...... 39

2.4.2 Purification of the recombinant Glu1B ...... 41

2.4.3 Hydrolysis of arbutin and PNPG with purified recombinant β-glucosidase ...... 41

2.5 Discussion ...... 43

2.6 Acknowledgement ...... 45

2.7 References ...... 46

viii 3 CONVERSION LIGNIN INTO LONG-CHAIN FATTY ACIDS WITH THE

ELECTRO-FENTON REACTION ...... 51

3.1 Abstract ...... 52

3.2 Introduction ...... 53

3.3 Materials and methods ...... 56

3.3.1 Materials and apparatus ...... 56

3.3.2 Electrochemical oxidation of lignin and lignin derived aromatics ...... 57

3.3.3 Fatty acid analysis and quantification ...... 58

3.3.4 Guaiacol electrolytes analysis ...... 59

3.3.5 Free chlorine analysis ...... 59

3.3.6 Hydroxyl radical analysis ...... 60

3.3.7 Adipic acid electrolysis ...... 61

3.3.8 Lignin electro-conversion ...... 61

3.4 Results and Discussion ...... 63

3.4.1 Lignin degradation and conversion through electro-Fenton reaction ...... 63

3.4.2 Production of long chain fatty acids from lignin derived model aromatics ...... 65

3.4.3 The proposed mechanism in the conversion of lignin-derived aromatics ...... 66

3.4.3.1 The key intermediators analysis in guaiacol degradation ...... 67

3.4.3.2 Free chlorine detection in electro-Fenton reaction ...... 71

ix 3.4.3.3 Hydroxyl radical detection in the electro-Fenton reaction ...... 71

3.4.3.4 Fatty acids synthesis from adipic acid electro-Fenton reaction ...... 72

3.4.4 The effect of different factors on electro-conversion ...... 74

3.4.5 Lignin electrochemical conversion to long chain fatty acids ...... 78

3.5 Feature of this technique process ...... 80

3.6 Conclusion ...... 82

3.7 Acknowledgement ...... 82

3.8 References ...... 83

4 ELECTRO-FENTON FOR SIMULTANEOUS MICROALGAE HARVEST AND LIPID

EXTRACTION ENHANCEMENT ...... 88

4.1 Abstract ...... 89

4.2 Introduction ...... 90

4.3 Materials and methods ...... 93

4.3.1 Strain and culture condition ...... 93

4.3.2 Neutral Lipid extraction and Fatty Acid Analysis ...... 94

4.3.3 Cell disruption ...... 95

4.3.4 Optimization of electro-Fenton cell disruption conditions ...... 95

4.3.5 Hydroxyl radical detection ...... 96

4.3.6 Iron detection ...... 96

x 4.3.7 Electron microscopy and Fluorescence microscope analysis ...... 97

4.3.8 Reuse of electrolysis waste water for subsequent microalgae cultivation ...... 98

4.4 Results and discussion ...... 98

4.4.1 Growth property ...... 98

4.4.2 Optimization of different factors on electro-Fenton cell disruption ...... 99

4.4.3 Confirmation of predicted optimum condition ...... 100

4.4.4 Fatty acid (FA) composition analysis during electro-Fenton reaction ...... 102

4.4.5 Microalgae cell disruption mechanism in electro-Fenton reaction ...... 103

4.4.5.1 Hydroxyl radical generation in electro-Fenton reaction ...... 103

4.4.5.2 TEM of algae cell wall and cytomembrane in electro-Fenton process ...... 104

4.4.5.3 Fluorescence microscope (FM) of algae during electro-Fenton reaction ...... 106

4.4.6 Iron distribution ...... 108

4.4.7 Microalgae cultivation in the electro-Fenton waste water ...... 111

4.4.8 Feature of this technology ...... 111

4.5 Conclusions ...... 113

4.6 Acknowledgement ...... 114

4.7 References ...... 115

5 CONCLUSION ...... 120

5.1 Concluding Remarks ...... 121

xi 5.2 Perspectives and Future Directions ...... 122

xii LIST OF FIGURES Page Chapter 1

Figure 1. 1. Roadmap vision of bioenergy electricity generation by region...... 2

Figure 1. 2. The general microbial-based process for bio-fuel production...... 4

Figure 1. 3. The mechanism of hydroquinones oxidation and H2O2 generation...... 9

Figure 1. 4. Schematic pathway of HO· generation through EF reaction in divided cell. RH stands for an unsaturated compound, while Ar represent aromatic compounds...... 14

Figure 1. 5. A previous proposed cellulose cleavage pathway initiated by HO·...... 19

Figure 1. 6. The suggested mechanism of cellulose random cleavage induced by HO·...... 19

Chapter 2

Figure 2. 1. The hydroquinone based pathway of H2O2 generation in the fungi system...... 34

Figure 2. 2. HPLC results of TLC purified sample (a) and arbutin (98%) standard solution (b) with the mobile phase: Methanol: 0.1% acetic acid = 10:90...... 40

Figure 2. 3. SDS-PAGE results of whole E.coli cell protein extractives with Glu1B and purified

Glu1B...... 41

Figure 2. 4. The results of enzyme activity of the purified recombinant β-glucosidase (Glu1B) against PNPG and arbutin...... 42

Figure 2. 5. Thin layer chromatography of arbutin hydrolytic products, arbutin and hydroquinone. Lane 1, arbutin; Lane 2, hydroquinone; A and B indicate arbutin and hydroquinone, respectively. Each was loaded at approximately 10 µg. Lane 3, arbutin hydrolytic products hydrolyzed by BGL1B (at 28 oC, 30 min)...... 43

xiii Chapter 3

Figure 3. 1. Experimental apparatus: 1. DC Power Supply, 2. Digital Ampermeter, 3. Digital

Voltmeter, 4. Anode (graphite), 5. Cathode (graphite), 6. Magnetic bar, 7. Magnetic stirring controller, 8. Compressed purified oxygen, 9. Activated carbon-based catalyst...... 58

Figure 3. 2. Fatty acids production from lignin-derived aromatics with electro-Fenton reaction. 66

Figure 3. 3. Proposed pathway for fatty acids formation from guaiacol through electro-Fenton process...... 71

Figure 3. 4. GC results of adipic acid electro-Fenton reaction (A) Adipic acid, (B) its resultant products...... 74

Figure 3. 5. Effect of different parameters on the conversion of guaiacol to palmic acid and octadecanoic acid...... 77

Figure 3. 6. The column plot for palmic acid and octadecanoic acid production from lignin electrolysis versus electrolysis time...... 79

Figure 3. 7. Conversion scheme of lignin to long chain fatty acids...... 80

Chapter 4

Figure 4. 1. The growth curve of N. oceanica IMET1 in fresh seawater BG-11...... 99

Figure 4. 2. Respond surface curve standing for the interactive effects of FeSO4 concentration, current density and time on the lipid extraction yield: (A) effect of FeSO4 concentration and current density; (B) effect of FeSO4 and time; (C) effect of current density and time...... 102

Figure 4. 3. Effects of electrolysis time on the composition of extracted fatty acid from N. oceanica IMET1...... 103

Figure 4. 4. 2,3-dHBA and 2,5-dHBA production versus time in electro-Fenton reaction ...... 104

xiv Figure 4. 5. TEM for microalgae cells...... 106

Figure 4. 6. FM of microalgae cells...... 108

Figure 4. 7. (a) Iron distribution in the supernatant and precipitation; (b) Averaged relative abundance of iron in each phase. IOE stands for iron abundance on the surface of electrode. IIP stands for iron abundance in the precipitation mixture. IIS stands for iron abundance in the supernatant...... 110

Figure 4. 8. The growth curve of N. oceanica IMET1 in electro-Fenton resulting waste water...... 111

xv LIST OF TABLES Page Chapter 1

Table 1. 1. Classification of biofuels based on production techniques ...... 3

Table 1. 2. Summary of various pretreatment processes, their advantages/disadvantages, pretreatment conditions and percent yield ...... 5

Table 1. 3. Standard Reduction Potentials in Aqueous Medium of the Most Commonly Reported

Oxidizing Agents ...... 12

Chapter 2

Table 3. 1. Experiments for the hydroxyl radical analysis ...... 60

Table 3. 2. Identification of lignin degraded compounds through electro-Fenton reaction ...... 63

Table 3. 3. The related identified compounds in the GC-QTOF ...... 67

Table 3. 4. Spectrum absorption in HO· analysis ...... 72

Chapter 4

Table 4. 1. Experimental design matrix for the optimization of electro-Fenton conditions...... 101

xvi

DEDICATION

This dissertation is dedicated to my parents, Jianjun Zhang and Shuping Lou.

xvii 1 INTRODUCTION CHAPTER ONE

INTRODUCTION

1 1.1 Introduction to sustainable biofuels

Fossil fuels play important roles in the world’s energy supply. It is estimated that fossil fuel will still provide about 84% of energy demand around the world according to the claim from World

Energy Outlook (WEO) 1 and continue to be the major energy source for the next few decades.

However, the expected depletion time for fossil fuel is computed to 2112 1. To solve this problem, a worldwide research is carried out aiming to discover other reliable energy resources. Among all options, bioenergy is treated as one of the potential reliable energy resources 2. Bioenergy is defined as a kind of energy produced from biological sources, which includes a variety of byproducts from agriculture process. As estimated, by 2050, it could supplies 3000 TWh of electricity, about 75% of world’s electricity generation (Figure 1. 1)3.

Figure 1. 1. Roadmap vision of bioenergy electricity generation by region.

Biofuel if a category of bioenergy that is known as solid, liquid and gaseous fuels, which are mainly generated from sustainable bio-based feedstocks. Bioethanol and bio-diesel are two major renewable transportation fuels aiming to replacing gasoline and diesel, because of energy demands

2 and environmental concerns. According to production techniques, biofuels are classified to three generation biofuels, which are shown in the table 1.1 4. Particularly, the SGBs and TGBs are also known as advanced biofuels, in which SGBs are produced from lignocellulosic biomass, such as wheat straw, bagasse and wood; TGBs are mainly made from algae. These biofuels could avoid many problems faced by FGBs, such as competing with food supplement, high cost, etc.

Table 1. 1. Classification of biofuels based on production techniques

Generation Feedstock Fuel forms

Bioalcohols, vegetable oil, First generation biofuels Sugar, starch, vegetable biodiesel, biosyngas, (FGBs) oils, or animal fats biogas

Bioalcohols, bio-oil, bio- Non-food crops, wheat Second generation biofuels DMF, biohydrogen, bio- straw, corn, wood, solid (SGBs) Fischer–Tropsch diesel, waste, energy crop wood diesel

Third generation biofuels Algae Vegetable oil, biodiesel (TGBs)

Bio-ethanol and bio-diesel have attracted world wide attention, because they have great potential applications. A good example for bio-fuel application nowadays is to mix 10% of bio-ethanol into gasoline to decrease fossil fuel consumption 5. There are several advantages of bio-fuels over other kinds of bioenergy sources. First, they are relatively pure carbon based chemicals with more industrial applications, such as solvent 6 and synthesis 7. Secondly, they have less ash content and are much easier to burn completely without char generation in the burning process. Thirdly,

3 through pretreatment and fermentation process, the separated lignin derivatives, lipids and other kinds of byproducts could be used for chemical production.

The general process for microbial-based bio-fuel production is shown in the Figure 1. 2 8. Basically, microbes are employed to use sugars, which come from lignocellulose, for bio-fuel fermentation.

Then, the generated bio-fuel needs to refined and/or upgraded before becoming a qualified fuel.

However, the natural lignocellulose is not easy to be digested by microbial and enzymes, due to its lignin-carbohydrate complexity.

Figure 1. 2. The general microbial-based process for bio-fuel production8.

Lignin, the secondary natural abundant polymer in the biomass, forms covalent linkages to hemicellulose and cross linkages with plant polysaccharides. Such structural association strengthens plant cell walls mechanically and protects plants from damage caused by external factors 9. Lignin is heterogeneously composed of phenylpropanoid monomers 10 and the resulting poly-dispersed polymer in plant cell walls creates resistance to chemical and biological degradation 11. This phenylpropanoid polymer also develops the lignin-hemicellulose matrix

4 through covalent linkages surrounding cellulose in the lignocellulosic microfibrils 12. Thus, natural lignin has the capability to protect the cellulose from enzymatic saccharification. This recalcitrance too poses severe limitation on bioconversion of lignocellulose to biofuels and other bio-chemicals

13.

1.2 Current pretreatment technologies for saccharification

Pretreatment processes are often required to efficiently unlock the lignin associated matrix in plant cell wall and expose cellulose to enzymes for saccharification and biofuel production 14. Based on the mechanism, the pretreatment technologies were divided into four kinds: physical pretreatment, chemical pretreatment, physico-chemical pretreatment and biological pretreatment 15. The criteria for effective pretreatment should be characterized as: preserving hemi-cellulose fraction, limiting generation of inhibition for the downstream process, minimize energy input and being cost- effective 16. However, none of those pretreatment technologies can be treated as the best, due to their intrinsic advantages and disadvantages, shown in the table 1. 2 16, 17.

Table 1. 2. Summary of various pretreatment processes on corn stover, their advantages/disadvantages, pretreatment conditions and percent yield.

Pretreatme Advantage Disadvantage Treatment Yield (%) Citation nt process condition

5 Diluted Increase in Synthesis of 158 °C and 1.8% 90% of 18 acid porosity/incre furfural/need for sulfuric acid for 5 glucose treatment ase recycle min with 30% wt yield enzymatic solid loading hydrolysis Alkaline Removal of Formation of 0.5 g Ca(OH)2/g, 91.3 % of 19 treatment lignin/hemicellu salts of calcium 55 °C for 4 weeks glucose lose hydrolysis and magnesium with aeration yield Ammonia Removal of Removal of first stage 190 °C, 78 % of 20 treatment lignin/decrystalli ammonia/costly 5.0 mL/min, 30 glucose zing cellulose min for hot water yield treatment and 170 °C, 5.0 mL/min, 60 min for aqueous ammonia treatment AFEX Lignin Costly/not 120 °C, 60 % 91.2 % of 21 removal/hydroly employed for moisture content, glucose sis of high lignin 1:1 kg ammonia/kg yield hemicellulose/de content of dry matter and crystallization of 120 psi cellulose Oxidative Low energy Formation of delignific intensity, less acids which act pH=11.5, 0.25g 83% of 22 ation equipment and as enzyme maintenance inhibitors/costly/ H2O2/g biomass, glucose cost hemicellulose 10% solid yield degradation loading,48h treatment

Wet Treatment of Costly 195 oC, 15 min, 12 83.4 of 23 -1 oxidation wastes/Fast bar O2, 2 g/L glucose Na2CO3 and 6% yield solid loading Biological Cheap/ecofriend A part of white rot fungus 66.4 % 24 ly fermentable Irpex lacteus CD2, glucose sugars are 60 days yield utilized as

6 carbon source/slow process

Most of the chemical and physical pretreatment technologies treat biomass fast with high sugar yield. However, inhibition generation and high energy input (e.g. high temperature and high pressure) are the limitations for these kinds of pretreatment methods. On the other hand, although bio-pretreatment treats the biomass with less inhibition and energy input, it needs much longer time to treat the biomass, which makes it cost-ineffective. Based on these reasons, the possible better ways to treat biomass are to combine the advantages of them. There are two ways: (1) Bio- pretreatment combines with the other chemical or physical pretreatments and make two or more stage-pretreatment technologies; (2) exploring the mechanism happened in the bio-pretreatment and mimicking the reactions chemically or physically with lower amount of energy input and producing less inhibition . In other words, the understanding of bio-pretreatment in the nature will allow us to modify the existing chemical or physical pretreatments to be more desirable for industry perspectives.

1.3 Termite-a good natural example for lignocellulose degradation

Termite is a good model for this natural inspired research, due to its high biological lignocellulosic degradation efficiency 25. It could digest wood and utilize 95% of cellulose and 80% of hemicellulose in 24 h. Termites seem to develop effective strategies in the digestion system to treat lignocellulosic biomass and utilize the nutrients from it. To explore these strategies, many studies

7 have been carried out. The digestion system of termite consists of grinding process and three parts of guts, foregut, midgut and hindgut. The function of grinding process is to reduce the particle size of their food to less than 50 µm through chewing process 26. Besides, enzymes (e.g. cellulase, xylase and phenoloxidase) and some small molecule chemicals (e.g. hydroquinone) are secreted from saliva gland and mixed with chewed particles 26, 27. One of the most important functions of foregut and midgut is to increase contact area between cellulase and cellulose to enhance hydrolysis. One of the ways is to further reduce the particle size to 19.6 µm and 9.6 µm in the foregut and midgut, respectively 28. Another way is to modify and degrade lignin and expose more cellulose out of lignin-carbohydrate matrix. It is reported that natural lignin experiences initial lignin-polysaccharide dissociation, Cα oxidation/carboxylation and phenolic dehydroxylation in the foregut and further phenolic carboxylation, demethoxylation and carbonylation in the midgut

29. The function of hindgut is mostly related to cellulose digestion and lignin further condensation

28, 30. The previous literature shows that lignin oxidation, such as Cα oxidation/carboxylation, phenolic carboxylation and carbonylation, is an important way to dissociate lignin matrix 29 in the termite. In the lignin oxidation process, oxygen is usually required as the non-toxic oxidant. To confirm this, Ke et al. explored the oxygen distribution in the guts of termite and eventually discovered that oxygen mainly exists in foregut and midgut 31. This finding points out that aerobic degradation of the food particles takes place in the gut system, which gives an explanation of lignin oxidation reaction. In the meantime, another oxidative species, hydrogen peroxide, was found with relatively high concentration (60 µM) in the mid gut 32. Based on hydrogen peroxide generation pathways in the nature, it is possible to produce H2O2 by oxygen reduction. The mechanism is summarized in the Figure 1. 3 33.

Figure 1. 3. The mechanism of hydroquinones oxidation and H2O2 generation.

Hydroquinones are firstly oxidized to be semiquinones through extracellular oxidation by phenoloxidase. Then, semiquinones are furtherly auto-oxidized to benzoquinones. In the meantime,

·- oxygen obtains electrons and forms superoxide anion radical (O2 ), which spontaneously turns to

H2O2. At last, quinone reductase makes a redox cycling to reduce benzoquinones back to hydroquinones 34. However, this mechanism has not been confirmed in the termite yet. But based on some evidences, it was hypothesized that this mechanism possibly takes place in the gut of termite.

1.4 Hydroquinone-a functional compound in termite

Hydroquinone (1,4-dihydroxybenzene) is discovered broadly existing in the saliva gland of both workers and soldiers in C. formosanus 27. It is commonly regarded as the pheromone and weapon produced by termites. With the help of hydroquinone, termites mark and share the food resources with each other. For example, in the range of 5–10 ng/µl saliva, hydroquinone can be regarded as phagostimulant for M. darwiniensis and in addition to antioxidant BHT(10% ratio to hydroquinone), the phagostimulating effect even greatly increases 35. On the other hand, termites

9 repel their food covered on high concentration of hydroquinone 35. For example, C. formosanus, using filter paper as the food, 2ng/cm2 of filter paper hydroquinonedose increases in the feeding

(phagostimulant), but 20 ng/cm2 of filter paper dose decreases in the feed (repellent) in one of the colonies 36. In another aspect, hydroquinone could be used as poison to protect themselves, because the soldiers produce more than workers do in the same colony and they perform more aggressively than the workers 27. In addition, phenoloxidase, one of the key enzymes for hydroquinone oxidation, is also discovered in the C. formosanus 37 and R. flavipes 38. The R. flavipes’s pheoloxidase specific enzyme activity to hydroquinone is 43.43 nmol/min/mg. Although there is no report about C. formosanus’ phenoloxidase specific enzyme activity to hydroquinone in the literature, the enzyme activity data from R. flavipes could be used as reference, due to the close relationship between the two kinds of termites. In other words, the factors for H2O2 generation mechanism exist in C. formosanus: hydroquinone, phenoloxidase and oxygen. Thus, it is likely that H2O2 is produced with this mechanism.

1.5 Arbutin-Possible source of hydroquinone in termite

If hydroquinone is an important intermediator for H2O2 generation, how can termite obtain it in nature? A logical hypothesis is from their food. Hydroquinone can be synthesized in plants and work as antioxidant in fruit with a relatively high concentration 39. However, due to two hydroxyl groups on the benzene ring, it is easily to be oxidized to benzoquinone under neutral and alkaline conditions to generate benzoquinone as the product. Benzoquinone is toxic to cells and could evenly kill partial of insects and bacterial 40. For this reason, hydroquinone usually loses one

10 equivalent water and combines with glucose to generate p-arbutin, which is more stable and common in the plants. When it is necessary to release out hydroquinone, arbutin could be hydrolyzed by β-glucosidase and release equal equivalent glucose and hydroquinone 41. Southern pine is one of the favorite food for C. formosanus 42. Thus hydroquinone existing in C. formosanus is hypothesized to be released from arbutin in the southern pine via enzymatic action of β- glucosidase produced from termite. In order to test this hypothesis, arbutin should be detected in the southern pine. HPLC is proper equipment for arbutin measurement. Due to its aromatic ring structure, arbutin has good absorption in the 280 nm 43. For this reason, HPLC method was developed for arbutin detection in pear and bearberry leaf extracts 39, 44. Secondly, it should be confirmed that arbutin could be hydrolyzed by β-glucosidase produced by termite In the C. formosanus, β-glucosidase is discovered and divided into 5 different species (Glu1A-1E), belonging to family 1 45. Among the five kinds of β-glucosidase, Glu1B was thought to be the most effective enzyme to hydrolyze cellubiose in the termite’s gut system 46. Further, to test its enzyme activity in vitro, Glu1B gene was redesigned for E.coli and the recombinant β-glucosidase was produced 47. Through specific activity analysis, it shows broad activity to both glycosyl and aryl

(or akyl) substrates. This makes arbutin hydrolysis probable.

1.6 H2O2-a key oxidant for lignocellulose degradation in termite

Except for hydroquinone-derived hydrogen peroxide generation mechanism in the termite, how to employ hydrogen peroxide to degrade lignin efficiently in the woody biomass also draws our

11 attention. Hydrogen peroxide is a powerful oxidant, whose oxidative potential is shown in the

Table 1. 3 48.

Table 1. 3. Standard Reduction Potentials in Aqueous Medium of the Most Commonly Reported

Oxidizing Agents

Oxidant Reduction reaction Eo/V vs SHE

+ - fluorine F2(g)+2H +2e HF 3.05

+ - hydroxyl radical •OH+H +e H2O 2.80

+ - ozone O3(g)+2H +2e 2.075 O2(g)+H2O

+ - hydrogen peroxide H2O2+2H +2e H2O 1.763

- - chlorine Cl2(g)+2e Cl 1.358

+ - oxygen O2(g)+4H +4e H2O 1.229

Compared with other oxidative regents in the table 1.3, H2O2 is less toxic than fluorine and ozone, more stable than hydroxyl radical and has higher oxidative ability than chlorine and oxygen. From this standpoint, it is suitable to be employed by ‘termite for lignin oxidation.

H2O2 utilization in termite could be one of biomass degradation strategies. Based on its function in the brown rot fungi, it can be speculated that Fenton or Fenton like reaction could take place in

49 gut system . The definition of Fenton reaction is that the decomposition rate of H2O2 is enhanced by ferrous/ferric catalyst and simultaneously generate HO·, which has high oxidative potential

(2.8V). The mechanism is shown as follows 50:

12 2+ 3+ − -1 -1 Fe + H2O2 → Fe + HO• + OH k=76 M s (1)

3+ 2+ + -1 -1 Fe + H2O2 → Fe + HOO• + H k=0.01 M s (2) It is reported that hydroxyl radical is the major oxidative and the most powerful species generated in the Fenton reaction. HO· prefers to react with aromatic ring and unsaturated bond through electrophilic reaction and cause side chain oxidation, demethoxylation, aromatic ring hydroxylation and ring opening 51. In order to investigate the function of hydroxyl radicals in lignin degradation, scientists applied Fenton reaction on the biomass pretreatment. They confirmed that the function of hydroxyl radical is to increase the pores and access hydrolysate area of woody fibers, decrease the crystalline of cellulose and make lignin more hydrophilic through demethoxylation, hydroxylation and Cα side chain cleavage 52-54. In addition, due to high oxidative potential, short half-life time (10-9 s) of hydroxyl radical and high decomposition rate of hydrogen peroxide in the Fenton reaction, lignin oxidation reaction could happen without additional heat or pressure and almost no toxic oxidant remaining in the liquor phase. Thus, Fenton reaction based process might be a promising pretreatment method.

In related research on termite, Fenton reaction has already drawn attention. Due to its gut condition,

Fenton reaction is supposed to take place in the wood digestion process 55. In the C. formosanus, the measured content of ferric/ferrous ions was up to 2.03 µg/µl of gut fluid in the foregut and midgut 32. In addition, the pH range of foregut and midgut was between 6.5 and 7.5 31. In this pH range, HO· could be effectively produced by Fenton reaction 56.

1.7 Electro-Fenton reaction–a termite mimicking process

13 Through understanding the hydroquinone-drived H2O2 generation mechanism in the C.

- formosanus, in which hydroquinone transfers an electron to oxygen and converts it to ·O2 . For lignin degradation, Electro-Fenton (EF) reaction is proposed as a promising way to mimic this process. EF process is an advanced oxidation technology studied mainly in wastewater treatment

- applications. In the reaction, oxygen similarly obtains electrons from cathode to generate ·O2 and

57, 58 then produces H2O2 on site, which is shown in the Figure 1. 4 .

Figure 1. 4. Schematic pathway of HO· generation through EF reaction in divided cell. RH stands for an unsaturated compound, while Ar represent aromatic compounds.

There are two obvious advantages of H2O2 generation on site over addition of H2O2 for Fenton reaction: it eliminates acquisition, shipment and storage of H2O2 and diluted concentration of H2O2 improves its safety during handling 59. In addition, due to the slower reduction rate of ferric ions to ferrous ions, ferrous ions generation reactions are the limiting steps in the Fenton reaction 60.

From this standpoint, EF reaction improve Fenton reaction through efficiently and directly ferric

14 ions reduction on the cathode. In the meantime, Hydroxyl radical could generated on the anode through water oxidation 48.

1.8 Effects of different factors on electro-Fenton performance

Generally, EF reaction includes two parts: (1) hydrogen peroxide production, and (2) hydroxyl radical production. In hydrogen peroxide production electro-chemistry, the materials of cathode, catalyst and oxygen concentration in the reactor are the major factors. The cathode material is the most important factor for oxygen reduction. It decides the reaction surface and current efficiency.

In previous research, active carbon fiber (ACF), graphite, reticulated vitreous carbon (RVC) and carbon-PTFE O2 diffusion are reported to be excellent cathode material for hydrogen peroxide production 48. In some cases, to furtherly increase the reaction surface of cathode, three- dimensional electrodes, such as activated carbon 61 and foam nickel 62, are chosen to work as the third electrode in the electrolysis reactor through electro-polarity. In addition, oxygen concentration directly affects hydrogen peroxide generation rate. In the electrolysis process, oxygen diffuses on the surface of cathode and obtains electrons to generate hydrogen peroxide.

The mechanism is shown as follows:

+ - O2 + 2H +2e H2O2 For this reason, enough oxygen in the reactor makes it occupy the cathode surface efficiently. The oxygen in the reactor could be divided into three parts: dissolved oxygen, oxygen generated from anode and the fed oxygen. At the 25 oC, the solubility of oxygen in water is 8.3 mg/L, which is not adequate for hydrogen peroxide production. Although, there are some other studies about

15 hydrogen peroxide production from water electrolysis 63, oxygen is produced from anode and needs to diffuse from anode to cathode. The diffusion limitation decreases the efficiency of electrolysis. For this reason, additional air or pure oxygen is required to feed into the electrolysis reactor 61.

For hydroxyl radical production, it depends on anode material, electrolyte and its environment and the concentration of ferric/ferrous ions. On the anode surface, the mechanism of hydroxyl radical production is shown as follows:

+ - 2H2O 2H + 2OH· +2e (1) OH- HO· + e- (2) There are two kinds of electrodes can be applied to EF reaction. One is non-active electrode, which has physico-absorption of hydroxyl radicals (3), such as Pt, graphite electrode; another one is

52, 64 active electrode, which has chemisorption of hydroxyl radicals (4), such as Ti/IrO2, Ti/SnO2 .

The mechanism is show as follows:

M + HO· M (HO·) (3) M + HO· MO + H+ + e- (4) In oxidative anode, MO is a more stable oxidant, instead of the unstable HO·:

MO + R RO + M (5) In the oxidation process, due to no substrate radical generation, coupling reaction or other side- reactions can be decreased. Another factor affecting hydroxyl radical production is electrolyte.

- 2- - Some electrolytes can work as hydroxyl radical scavengers, such as HCO3 , CO3 , H2PO4 and

2- 65 HPO4 . Previous studies show that 0.05 M NaClO4 , NaCl and Na2SO4 could be used as the electrolyte for EF reaction and hydroxyl radical production performance in those electrolytes are

16 - - 2- 66 ClO4 > Cl >> SO4 . Also, the environment of electrolyte and concentration of ferrous/ferric ions are important factors, it is reported that temperatures up to 35-40 oC, pH condition near 3.0

2+ 3+ 2+ and optimized Fe /Fe concentration (0.1-1mM, depending on Fe regeneration rate on the cathode) 48.

1.9 Electro-Fenton process application in lignin treatment

Hydroxyl radical is reported as an effective radical employed for degradation of lignin and its derivatives and biomass pretreatment 51, 67. Oturan et al. and Zeng et al. explained the possible oxidation mechanism: first, lignin was degraded to the oligomers aromatics and ring-opening products, such as short chain dicarboxylic acids (C1-C6), and then the resulting products were

58, 68 furtherly mineralized to CO2 and H2O . EF reaction has been illustrated as one of effective way for HO· generation and aromatics mineralization 48. Lglesias et al. employed heterogeneous electro-Fenton reaction for winery wastewater treatment, including polyphenols and lignin 69. Iron loaded activated carbon showed the highest degradation performance with 82% chemical oxygen demand (COD) at 15V. On the other hand, for the purpose of increasing the reaction surface,

Ugurlu et al. successfully applied NaCl as electrolyte and activated carbon as the third electrodes to remove 90% of lignin and 96% of phenol from pulp waste water, which was induced by HClO and HO· 70. However, short chain dicarboxylic acids and lignin mineralization doesn’t meet the requirement of biofuel quality, which prefers employing long-chain carboxylic acids (C12-C22) such as biodiesel 71. Weimer et al. successfully used short chain carboxylic acids from polysaccharide fermentation to synthesize longer chain alkane (C4-C8) with electro-catalysis 72.

17 In principle, Naber et al. proved that carboxylic acids could be polymerized through decarboxylation and coupling reaction during electro-synthesis 73, which is described as electro-

Kolbe reaction as follows:

R1-COOH + R2-COOH R1-R2 + 2CO2 (6)

All these results encourage considering possibility of controlling the operating parameters in lignin or aromatics electro-degradation to elong the carbon bone length of short chain carboxylic acids by electro-oxidation of aromatics through electro-Kolbe reaction, instead of complete mineralization. This could benefit lignin valorization with biodiesel production instead of complete mineralization into CO2.

1.10 HO· for cellulose degradation

HO· shows poor selectivity for delignification and cellulose degradation 74. Except for lignin oxidation, HO· is proposed to degrade cellulose through random chain cleavage. With high redox potential, HO· is proposed to attack C-2 position on the glucose units and abstract their hydrogen atoms to induce the random cleavage of cellulose Figure 1. 5 75.

Figure 1. 5. A previous proposed cellulose cleavage pathway initiated by HO·. On the other hand, Guay et al. suggested a new explanation on HO· induced cellulose random cleavage by means of photochemical way 76. In their theory, HO· targeted at C-1 position on the glucose units to break down the 1,4-glucosidic bond (Figure 1. 6).

Figure 1. 6. The suggested mechanism of cellulose random cleavage induced by HO·.

19 Cellulose is one of the major components in the microalgae cell wall, which takes up about 33% in microalgae dry cell weight 77. Since cellulose has crystal area and resistance to chemicals, it makes microalgae cell wall a rigid structure to inhibit neutral lipid existing in the lipid body extracted out by organic solvents 78. Due to the function of HO· in cellulose degradation and its widely application in nature for cellulose crystal structure disruption and its further degradation 79,

Kim et al. and Concas et al. successfully employed Fenton reaction for HO· generation and microalgae cell wall disruption to increase lipid extraction yield to 79%-94.5% (%, total lipid) by organic solvents 80, 81. During Fenton oxidation, microalgae was not only attacked by HO· to form holes on its cell wall, but also coagulated by positive charged Fe2+ for further harvest 82. However, there are still several problems remaining: (1) Lack of advanced techniques by controlling Fenton reaction. Notably, the resulting HO· not only caused microalgae cell wall degradation, but also led to lipid oxidation and furtherly decreased its extraction yield. For the purpose of realizing reaction termination, inhibitors and ethanol dilution are required. (2) Continuous H2O2 requirement in the microalgae treatment increases the chemical cost and safety concerns during its handling, shipment and storage 59. Taking these in account, the electro-Fenton reaction is preferred for avoiding these problems through H2O2 production on site and electricity-driven reaction. From this point of view, electricity is not only used to drive electro-Fenton process, but also sustainably produce H2O2 on site to disrupt the cell wall.

1.11 Dissertation Overview

20 The work in this dissertation research aims at bio-mimicking lignocellulosic biomass degradation strategy in termite and further employing it for lignin and cellulose treatment, respectively. We seek to discover one of termite-based strategies and develop the process according to this strategy.

In addition, the reaction process was employed in the lignin and cellulose degradation, respectively.

In order to accomplish the first task, we proposed (Chapter 2) the hypothesis that hydroquinone was one of the key intermediators introducing HO· production for southern pine digestion in termite. Through wood chewing for particle size reduction, termite can simultaneously hydrolyze arbutin, existing as southern pine extractives, by β-glucosidase and releasing hydroquinone for further oxidation. To confirm this, we use HPLC to analyze the acetone extraction of southern pine, aiming to Arbutin discovery and quantification. Next, we expressed Glu1B, recognized as the key

β-glucosidase related to cellubiose hydrolysis in Coptotermes formosanus. Finally, we employed thin layer chromatography (TLC) and Ionic chromatography (IC) to analyze the products generating from arbutin hydrolysis by the expressed β-glucosidase. Glucose and hydroquinone were detected in the hydrolysate, which proved that termite could obtain hydroquinone from arbutin hydrolysis. On the other hand, iron and laccase were discovered in the termite, based on the previous literature. They could catalyze hydroquinone for oxygen reduction and induce

HO· production.

In the Chapter 3, we successfully employed electro-Fenton process for bio-mimicking. In principle, oxygen obtains electrons from cathode, in which oxygen catches electrons from hydroquinone for

2+ H2O2 production. Then, the generated H2O2 is catalyzed by Fe for HO· production. HO· has high oxidation potential, which has the ability to completely mineralize lignin and its derivatives to CO2 and H2O. In this chapter, we first employed electro-Fenton process for lignin treatment and

21 discovered that long-chain fatty acids, such as palmic acid and octadecanoic acid, were produced as intermediators during lignin degradation. In order to explain this phenomenon and simplify the mechanism, four lignin model compounds (e.g. guaiacol, salicylic acid, ferulaic acid and phthalic acid) were employed. We discovered that long-chain fatty acids could be produced during all the four compounds degradation, while its yield from guaiacol degradation was much higher than other ones. Next, we chose guaiacol as the lignin model compound for the mechanism research and process optimization. In the mechanism explanation, we inferred that short-chain organic acids

(e.g. adipic acid) generating from lignin aromatic ring opening reactions experienced Kolbe synthesis reaction to enlong carbon bond and form long-chain fatty acids. Finally, we employed the optimal conditions to convert lignin to long-chain fatty acids.

In the chapter 4, we used the bio-mimicking electro-Fenton process for microalgae cell wall disruption. As we shown above, electro-Fenton process is an effective way for HO· production.

Besides lignin degradation, HO· can also cause degradation of cellulose, which is the main composition in microalgae cell wall. In this chapter, we hypothesized that electro-Fenton process could effectively disrupt microalgae cell wall for lipid extraction enhancement and simultaneously assist cell’s harvest. In order to confirm this hypothesis, we first optimized electro-Fenton process with respond surface methodology (RSM) and obtained the optimal conditions of FeSO4 concentration, current density and electrolysis time. Then, we not only used salicylic acid to capture HO· and HPLC to quantify their amounts, but also employed transmission electron microscopic (TEM) and Fluorescence microscope (FM) to observe cell wall disruption and lipid distribution in the electro-Fenton process. Furthermore, iron distribution detection and waste water re-cultivation lead us to know how to recycle iron and waste water for cost reduction.

22 1.12 Publications

The following manuscripts have been prepared according to the work in this dissertation:

1. Shuai Zhang, Xiaochao Xiong, Yonghong Meng, Yuxiao Xie, Na Sa, Shulin Chen,

“Mechanisms of hydroquinone production in Coptotermes formosanus”.

2. Shuai Zhang, Le Gao, Bowen Liu, Jiaren Zhang, Yiqing Yao, Dongyuan Zhang, Wenya

Wang, Shulin Chen, “Conversion lignin into long-chain fatty acids with the electro-Fenton

reaction”.

3. Shuai Zhang, Yuyong Hou, Kaiyang Chen, Sanyuan Shi, Mengdi Zhang, Dongyuan

Zhang, Wenya Wang, Shulin Chen, “Electro-Fenton for simultaneously microalgae harvest

and lipid extraction enhancement”.

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2 MECHANISMS OFCHAPTER HYDROQUINONE TWO PRODUCTION IN

COPTOTERMES FORMOSANUS

MECHANISMS OF HYDROQUINONE PRODUCTION IN

COPTOTERMES FORMOSANUS

30 2.1 Abstract

Hydroquinone may be a potentially important mediator for lignin modification in termites because of its potential roles in radical generation. The hydroquinone-production route in termites has not been revealed. This study aimed at filling the information gap. To elucidate this mechanism, one of the β-glucosidases (Glu1B) from Coptotermes formosanus was expressed in E.coli Rosetta.

Hydroquinone and glucose were detected in the hydrolysate of arbutin containing the enzyme mixture by using Thin Layer Chromatography and Ionic Chromatography. This result indicated that hydroquinone was produced from hydrolyzing arbutin by the recombinant Glu1B. To further verify arbutin hydrolysis in C. formosanus, arbutin in the Southern pine, a favorable food of termites, was quantified at a concentration of 0.08 mg/g. The findings suggest that termites can release hydroquinone from the arbutin by means of Glu1B in lignocellulosic biomass.

2.2 Introduction

Lignin, the secondary natural polymer abundant in the biomass, forms covalent linkages to hemicellulose and cross linkages with plant polysaccharides. Such structural association strengthens plant cell walls mechanically and protects plants from damage caused by external factors 1. Lignin is heterogeneously composed of phenylpropanoid monomers 2 and the resulting polydispersed polymer in plant cell walls creates resistance to chemical and biological degradation

3. This phenylpropanoid polymer also develops the lignin-hemicellulose matrix through covalent linkages surrounding cellulose in the lignocellulosic microfibrils 4. Thus, natural lignin has the capability to protect the cellulose from enzymatic saccharification. This recalcitrance too poses severe limitation on bioconversion of lignocellulose to biofuels and other biochemicals 5.

Pretreatment processes are often required to efficiently unlock the lignin associated matrix in plant cell wall and expose cellulose to saccharification by enzymes to release sugars for use in production of lignocellulose-based biofuel 6. Currently, industrial pretreatment technologies including chemical and physical methods have various limitations, including high pretreatment costs and negative environmental impact. In the efforts seeking for better pretreatment options, wood-feeding termites (WFTs) have been proposed as a promising model for systems created by nature for degrading lignocellulose 7. Termites have a highly efficient wood-digestion rate (65%-

99% polysaccharides consumption within 24h) in natural conditions 8-10. This wood digestion process inspires innovative designs for efficient pretreatment technologies.

Researchers have long sought solutions to explain WFT’s efficient wood digestion 11. For this purpose, all parts of termites related to wood feeding process were explored from mouse and saliva gland to hindgut 12-14. Studies show that both lignin and polysaccharides (cellulose and hemicellulose) degradation are important to efficient wood utilization in the digestion process 15,

16. In order to efficiently digest wood, termites mechanically chew wood into small particles

(approximately 50 µm) and simultaneously mix them with host digestion enzymes. These enzymes include polysaccharides hydrolytic enzymes (e.g. cellulase, β-glucosidase and hemicellulolytic enzymes) 12, 17 and partial lignin degradation enzymes (e.g. laccase) 18 secreted from saliva glands at the beginning of the wood digestion process 12. Next, the chewed mixture is transported to the foregut and midgut of termites, where most of the lignin degradation occurs 19.

Lignin is degraded in termites with the help of hosts and microbes 20, 21. Through this process, natural lignin is modified and degraded by initial lignin-polysaccharide dissociation, Cα oxidation/carboxylation and phenolic dehydroxylation in the foregut and further phenolic carboxylation, demethoxylation and carbonylation in the midgut 22. Cellulase and β-glucosidase secreted from saliva glands, are thought to hydrolyze amorphous parts of cellulose and released glucose from pine lignocelluloses when they pass through the foregut and midgut 21, 23. The final portion of wood digestion occurs in the hindgut by symbiotic protistan communities secreting more complex cellulosic enzymes 24, 25. Thus, both polysaccharide hydrolysis enzymes and lignin degradation play important roles in effective lignocellulosic biomass utilization. Understanding the mechanisms for lignin-degradation is critical to the design of termite-based pretreatments.

Natural lignin degradation requires high oxygen tension 23, 26, including free oxygen and stored

27 oxygen in H2O2 participates . Some lignin degradation enzymes in termites such as Hemocyanin

28 require free oxygen as an oxidant , while others use H2O2,, such as manganase peroxidase (MnP),

29 30, 31 . In addition, both oxygen and H2O2 have been found in termite guts . Besides, Coy notes that H2O2 has the other function in the wood degestion, except for working as oxidant, which increases the enzyme activity of laccase in the termite species Reticulitermes flavipes 18. This

32 differs from the well-known H2O2-independent laccase . In all likelihood, H2O2 is an important chemical in termites. However, the mechanisms for H2O2 generation in the lower wood feeding termite is poorly understood. In white-rot fungi, hydroquinones play an important role in driving

33 H2O2 production . Figure 2.1 displays the entire proposed mechanisms in the fungi system and it shows as follows: hydroquinone is firstly liberated by β-glucosidase from the arbutin structure 34.

Then the product is activated by laccase or peroxidase to generate semi-quinone, which is an intermediate that automatically converts to benzoquinone with O2 oxidation. At the same time, the

•- 33, 35, 36 superoxide anion radical (O2 ) produced from oxygen spontaneously converts to H2O2 .

Figure 2. 1. The hydroquinone based pathway of H2O2 generation in the fungi system.

In C. formosanus, one of the most efficient lignocellulose degradation lower termites 31, hydroquinone (benzene-1,4-diol) is mainly secreted from the salivary gland for food-marking

37 pheromones and self-defense . Similarly, termites may share this H2O2 generation mechanism in the wood digestion process, based on hydroquinone oxidation by the recombinant laccase in vitro

18. However, the mechanism for hydroquinone production has not yet been revealed for the termite species C. formosanus. We hypothesized that β-glucosidase in the termite C. formosanus breaks down the sugar-aromatic conjugate of arbutin in their food, releasing hydroquinone. β-glucosidase of Glu1B is the most effective cellobiase in this kind of termite 38, 39. Belonging to the GH family

1 (GH1) 40, 41, both glycosyl and aryl (or akyl) β-glucosidase activity are shown on this types of enzyme 42. Thus, arbutin may be a potential substrate for Glu1B. On the other hand, it may be present in termite food, working as as common anti-oxidant in the plants 43-45.

To vertify this hypothesis, we reconstituted this reaction in vitro. First, arbutin was detected in the

Southern pine, a favorite food of the termite C. formosanus 46, with Thin Layer Chromatography separation (TLC) and High-Performance Liquid Chromatography (HPLC). Then Glu1B was expressed in E.coli and purified by Fast Protein Liquid Chromatography (FPLC). The purified enzyme was characterized in terms of arbutin hydrolosis. Finally, both hydroquinone and glucose were detected in the hydrolysate to determine whether Glu1B could hydrolyze the arbutin structure.

This study aimed at clarifying the additional functions of this type of enzyme in wood degradation along with the hydrolysis function of oligosaccharide metabolites. The findings provide indirect evidence of H2O2 production in termite. This work advances our knowledge of termite-based pretreatment and may provide new insights for developing new process for biofuel production from lignocellulose.

2.3 Materials and Methods

2.3.1 Preparation of Southern yellow pine extract

Southern yellow pine was obtained from American Wood Fiber (Columbia, Ohio) and welly milled in the Wood Materials and Engineering Laboratory at Washington State University.

Particles then were sieved through 35 mesh (0.40 mm) and 42 mesh (0.35mm) Tyler Standard screens. The resulting particles between 35 and 42 mesh were used for ethanol extraction.

Approximately 15 g of screened material was extracted twice with 75% ethanol (150 ml) under reflux for 1h and then centrifuged. The supernatant was Rota-evaporated to about 50 ml and transferred to a separation funnel. The solution was extracted twice with 30 ml petroleum ether, and ether extracts were discarded. The remaining solution was evaporated to dryness and the residue was dissolved into 10 ml methanol.

2.3.2 Thin-layer chromatography separation.

The TLC plates (silica gel, 0.2 mm on aluminum, Sigma) were prewashed and activated as described in other report 47. Activated plates were manually spotted with 10 µl of samples and 10

µl of arbutin (98% purity, Sigma) methanol (50 ppm) solution. The mobile phase with ethyl acetate-formic acid-water (88:6:6) was selected, according to the literature 48. About 100 ml of the mobile phase was used for development. A TLC chamber with a distance of 10 cm was developed in the mobile phase for 15 min at 20±1 oC. After development, all plates were dried in a current air by an air drier. Then the dried TLC plates were colored with iodine vapor (99.8% purity, Sigma).

To compare with the arbutin standard, the Rf valve was calculated. Next, residues (Rf =0.384) were collected for HPLC analysis. All collected residues were dissolved in methanol overnight to ensure that all samples were transferred into methanol. Finally, they were passed through a 0.45 µm membrane filter into sample vials for HPLC analysis.

2.3.3 HPLC analysis

The chromatography for arbutin detection was performed on a Varian Pro Star 230 HPLC equipped with a vacuum degasser, a quaternary pump, a thermostatic column compartment, and a diode array detector. HPLC analysis was carried out on a Varian Microsorb-MV 100-5 C18 column (4.6 x 250 mm, 5.0 µm) at 25 oC. Acetic acid at the concentration of 0.1% (v/v) was employed as mobile A. Methanol was used as a mobile phase B. The methanol acetic acid solution

(10:90) was pumped through the column. The flow rate was 1.0 ml/min, detection λ=280 nm and the scan range was from 200 to 400 nm. The arbutin standard solution was prepared in a series of concentrations of 50, 100, 200, 500 and 1000 ppm to create a linear curve for arbutin concentration calculation in the sample. The injection volume was 20 µl each time.

2.3.4 Recombinant β-glucosidase expression and purification

Gene encoding the recombinant β-glucosidase, Glu1B, from C. formosanus with the optimized codon of E. coli was synthesized by GenScript (NJ, USA), based on a sequence reported by Zhang

37 et al. 39. Recombinant Glu1B was expressed in the E.coli Rosetta (DE3) and purified with the

AKTA FPLC system, as described previously 39, 49. The purification column was exchanged to a nickel-charged 1-ml HiTrap IMAC column. The column was equilibrated by running the gradient from a 100% binding buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole, pH 7.4,

0.5 mM Tris[hydroxypropyl] phosphine, 10% glycerol) to a 100% elution buffer (20 mM, Tris-

HCl, pH 8.0, 300 mM NaCl, 500 mM imidazole, pH 7.4, 0.5 mM Tris[hydroxypropyl]phosphine,

10% glycerol). The extracted proteins were loaded in the binding buffer and then eluted by the elution buffer with a linear gradient from 0 to 100%. The eluted target protein was desalted by the desalting buffer (100mM Sodium acetate, pH 5.6, 0.5 mM Tris[hydroxypropyl]phosphine, 10% glycerol) on a PD-10 column, aliquoted and flash-frozen. The purified β-glucosidase was stored at -80 oC until use. The concentration of the purified protein was determined by Bradford Regent

(Sigma), using bovine serum albumin (BSA) as reference. The purity of the purified protein was detected by means of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andcompared with the protein profile of the crude extracts.

2.3.5 Enzymatic digestion of the recombinant β-glucosidase

Arbutin (98% purity, Sigma) and 4-Nitrophenol-β-D-glucopyranoside (PNPG) (98% purity,

Sigma) were dissolved in a 50 mmol/L citrate buffer (pH 5.0) at 5.0 mg/ml. Aliquots of 5 µl of the purified recombinant β-glucosidase (0.8 µg) was added to 100 µl of PNPG and arbutin solution at a concentration of 0.1% sodium azide, respectively. The mixtures were incubated at 28oC for 30 min and the enzymes were denatured at 95oC for 10 min. The hydrolysate of PNPG solution was

38 used for glucose detection, and the hydrolysate of arbutin was used for hydroquinone and glucose detection.

Hydroquinone detection was performed on the TLC plate. The TLC plate was prewashed and activated according to the procedure above. Aliquots of 2 µl of arbutin hydrolysates, arbutin solution (5 mg/ml) and hydroquinone (99% purity, Sigma) solution (5 mg/ml) were manually spotted on the activated plate, respectively. The mobile phase was chosen as chloroform-methanol

(84:16). Approximately 100 ml of mobile phase was employed for the developing process. A TLC chamber with a distance of 10 cm was used and developed in the mobile phase for 15 min at 20±1 oC. After development, the TLC plate was dried with an air drier and colored by iodine vapor.

The sugar concentration was analyzed with a Dionex ICS-3000 Ion Chromatograph. Hydrolysates were filtered through a 0.45mm nylon filter before sugar analysis. The separation of sugars was carried out with a CarboPac PA20 Guard column (4 × 50 mm) and an IonPac AS11-HC analytical column (4 × 250 mm) at room temperature (25°C), and the following detection was achieved by an ED40 electrochemical detector. An AS40 sampler was employed for continuous running, and

Dionex PeakNet 5.1 chromatography software was used for data analysis.

2.4 Results

2.4.1 HPLC results for p-arbutin detection

Arbutin was found in Southern yellow pine with a relative concentration of 0.08 mg/g with HPLC detection. This is a common way to examine this type of chemical 50. Figure 2. 2 shows HPLC results for the TLC purified ethanol extractives and arbutin standard. Arbutin in the TLC purified ethanol extractives eluted at the retention time (RT) of 4.573 min (Figure 2. 2 (a)) and RT of the standard arbutin (1000 pm) appeared at 4.627 min (Figure 2. 2 (b)). There was an acceptable 0.05 min delay in RT between the two peaks 51. Although three kinds of impurities remained in the TLC purified extractives, the arbutin peak could be separated with the impurities. This confirms that arbutin exists in Southern pine. Based on the relative arbutin standard curve (the relative arbutin standard solution wasn’t calibrated by the quality controlled solution) and the TLC recovery factor of arbutin (α=0.861), the content of arbutin in the Southern pine was estimated at 0.08 mg/g.

Figure 2. 2. HPLC results of TLC purified sample (a) and arbutin (98%) standard solution (b) with the mobile phase: Methanol: 0.1% acetic acid = 10:90.

2.4.2 Purification of the recombinant Glu1B

The recombinant Glu1B was successfully expressed and purified from E.coli. The target protein,

Glu1B, showed a single band in the purified protein channel, compared with several different bands in the E.coli cell protein extractives channel (Figure 2. 3). In addition, the molecule weight

(MW) of the target protein was 50 kda, which is consistent with the theoretical result calculated from the amino acid sequence of Glu1B.

Figure 2. 3. SDS-PAGE results of whole E.coli cell protein extractives with Glu1B and purified

Glu1B.

2.4.3 Hydrolysis of arbutin and PNPG with purified recombinant β-glucosidase

The enzymatic activity of β-glucosidase was reflected by 1µmol of released glucose per minute per ml of purified enzyme. Figure 2. 4 shows the enzyme activity of the purified recombinant β- glucosidase (Glu1B) against pNPG and arbutin. The released glucose was detected in both hydrolysate of pNPG and arbutin (Figure 2. 4). The recombinant Glu1B showed enzyme activity against pNPG is 2 IU/ml and 0.034 IU/ml at 28 oC, meaning that its enzyme activity against pNPG was about 60 fold of that of arbutin. In addition, Figure 2. 5 shows the TLC results of hydrolysate of arbutin. Compared with the positons of arbutin and hydroquinone standard solution on the TLC plate, both arbutin and hydroquinone were detected in the hydrolysate of arbutin.

Figure 2. 4. The results of enzyme activity of the purified recombinant β-glucosidase (Glu1B) against PNPG and arbutin.

Figure 2. 5. Thin layer chromatography of arbutin hydrolytic products, arbutin and hydroquinone.

Lane 1, arbutin; Lane 2, hydroquinone; A and B indicate arbutin and hydroquinone, respectively.

Each was loaded at approximately 10 µg. Lane 3, arbutin hydrolytic products hydrolyzed by

BGL1B (at 28 oC, 30 min).

2.5 Discussion

This study identified a new function of β-glucosidase (Glu1B) in the termite, C. formosanus, on arbutin hydrolysis in Southern pine. This was accomplished by analyzing recombinant Glu1B’s enzyme activity against the arbutin test in vitro.

This study treated P- arbutin (glycosylated hydroquinone) as a potential source for releasing hydroquinone in termites. The results reveal arbutin in Southern pine, along with other kinds of wood 52. The arbutin content of 0.08 mg/g Southern pine is lower than in pears and bearberry 44,

53. Therefore, Southern pine is not poisonous to termites and provides them as food 46, 53, 54. In addition, P-arbutin is a type of phenol derivative with the highest UV absorption at 280 nm and a weak acidity. Therefore, we used 0.1% acetic acid to adjust the pH for a peak result that is sharper in the HPLC 50. The separation of p-arbutin and other impurities was conducted according to the different polarities in the developed solvent. Due to its polarity, a C-18 reverse column packed with based-deactivated silica was used to prevent peak-tailing caused by the interaction between polar and silanol groups 51. Aqueous methanol showed good performance to yield and separate p- arbutin as the mobile phase and 10% of methanol-water solution was shown to be effective for separating the p-arbutin peak from other remaining impurity peaks in the TLC-purified samples.

However, both higher and lower concentrations of methanol compromised the resolution among the four peaks.

This study identified a new function of the recombinant β-glucosidase (Glu1B) on arbutin hydrolysis. We found that Glu1B originated from C. formosanus showed enzyme activity against arbutin in vitro, since both glucose and hydroquinone were detected in the hydrolysate. This was contrary to previous findings 55. This result demonstrates the ability of Glu1B to break down the aryl-glycosyl bonds in the arbutin structure. This finding was consistent with the classification of this type of enzyme belonging to the glycosyl hydrolase family 1 (GH1) 39. Both glycosyl β- glucosidase and aryl (or akyl) β-glucosidase activities were shown on this type of enzyme 42. In addition, the enzyme activity of Glu1B against PNPG was 60 times higher than that against arbutin.

This is similar with other studies reported that β-glucosidase from other species has higher enzymatic activity against PNPG than that against arbutin 56. This may serve as a protective mechanism, making hydroquinone release gradually from arbutin and protecting termites from

44 high concentrations of liberated hydroquinone in their guts 54. Due to the low enzyme activity against arbutin, the recombinant Glu1B usually took longer to accumulate hydrolytic products from arbutin for enzyme detection. Therefore, we extended hydrolysis to 30 minutes to achieve this reaction.

These findings reveal the mechanism of hydroquinone generation in the termite C. formosanus.

This provides a better understanding of H2O2 generation in termites. Through arbutin detection in the Southern pine and the arbutin-hydrolysis function confirmation of Glu1B, this study demonstrated that C. formosanus can liberate hydroquinone from arbutin provided by their food.

This study represents the first step to provide hydroquinone for the H2O2 generation next in the process, which has been detected in relatively high concentrations in the mid-gut of termites 31.

This could be one of the pathways in the termite. Further understanding the H2O2 production mechanism for termites will elucidate how the termite C. formosanus carries out efficient, wood- based food digestion. This can lead to development of a new, efficient lignocellulosic pretreatment method based on the cooperation of chemicals and enzymes. This study confirms a new function of this enzyme; however, more research is needed on whether this enzyme works on arbutin in termites. Further research will investigate the hydrolysis reactions of arbutin in vivo.

2.6 Acknowledgement

This research is based on work supported by grants from the National Science Foundation, USA

(Grant No. 1231085) and Washington State University.

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3 CONVERSION LIGNINCHAPTER INTO LONG THREE-CHAIN FATTY ACIDS WITH THE ELECTRO-FENTON REACTION

CONVERSION LIGNIN INTO LONG-CHAIN FATTY ACIDS WITH THE

ELECTRO-FENTON REACTION

3.1 Abstract

The lack of effective lignin valorization technology remains a major barrier to utilizing lignocellulose for producing biofuels and biochemicals. In this work, it was first discovered that lignin could be converted into fatty acids in an electro-Fenton reactor, the precursor of biodiesel or advanced biofuels. The possible conversion pathway was further revealed experimentally:

Guaiacol was used as the lignin model compound to be subjected to electrolysis treatment. The key intermediators produced during the electrolysis process were analyzed using GC-QTOF. The results suggested that Cl2 and HO· initially induced aromatic ring opening to generate muconic acid, adipic acid and succinic acid. The short chain dicarboxylic acids were further coupled and synthesized to long chain fatty acids through Kolbe reaction. The proposed pathway was verified by detecting Cl2, HO· and by carrying out adipic acid coupling reaction. Finally, six related factors

(time, current, oxygen flow rate, catalyst loading, NaCl and Fe2+ concentration) in electro-Fenton process were studied and the highest yield of palmic acid and octadecanoic acid (mainly components in long chain fatty acids) reached to 62 mg L-1 and 43 mg L-1, respectively. This study proved that lignin and its derived aromatics could be converted into palmic and octadecanoic acid through Electro-Fenton reaction. This technology provided a new route to use lignin for the production of bio-chemical and biofuels. This is the first report on converting lignin into fatty acids through electro-Fenton electrolysis.

3.2 Introduction

Lignocellulose is an abundant source of feedstock for biofuel production that can not only contribute to meet the energy demands, but also address climate change concerns.1, 2 Sugars derived from cellulose and hemi-cellulose in the lignocellulosic biomass can be converted through fermentation to vary of liquid fuels such as ethanol or fuel precursor such lipids.3-5 However, the high production cost of cellulosic biofuels is currently limiting its wide application at industry scale. A commonly recognized approach for overcoming this barrier is to produce high value co- products in addition to fuel. A major opportunity in this approach is alternative use of lignin.

Accounting for 10-25% dry weight of lignocellulose,6 lignin is usually burned as a low grade energy source after separation form the cellulose and hemi-cellulose.7 Numerous efforts have been devoted to valorize lignin by converting it to biofuels and high-valuable bioproducts.

Lignin and its derived aromatics can be converted to biofuels through either physicochemical or biochemical techniques. The former route includes hydrothermal hydrogenation, oxidation, pyrolysis and catalytic pyrolysis.8, 9 In such processes, lignin is firstly degraded to low-molecular aromatics, such as phenolics. Next, hydrogenation and deoxygenation are carried out to upgrade the lignin derived aromatics mainly into cyclic hydrocarbon.10 For example, Runnebaum et al.11 discovered that lignin derived aromatics can be converted into hydrocarbon by Pt/γ-Al2O3 in the

12 presence of H2. Wang et al. furtherly confirmed that alkali extracted corn stover lignin were able

13 to be employed to produce cyclic hydrocarbons with Ru/Al2O3 and acidic zeolite. Kleinert et al. successfully accomplished the single step to produce biofuel by combination of lignin depolymerization and removal of oxygen process. However, the harsh operating conditions, such

53 as high temperature and pressure, has been a major technical challenge for physicochemical technique, along with further purification cost, catalyst thermal instability and deactivation and safety considerations.8, 14 Biological conversion of lignin using microbes and enzymes can be accomplished under much milder conditions. In a typical biological conversation process, lignin derived aromatics are firstly metabolized into 3-carboxymuconate through the “funneling pathway”;15 and then the 3-carboxymuconate entered the TCA-cycle and fatty acid synthesis pathway to produce lipid accumulating in cells.16 It was reported that lignin derived aromatics were utilized by oleaginous Rhodococci to reach lipid content of 4.08%.17 A major drawback of biological conversion, however, is that it usually takes fermentation time, which makes it less effective in industrial application.

Recently, several new technologies have been tested to convert lignin and its derived aromatics to biofuels, including microwave pyrolysis,18 electrochemical conversion,19 photo-catalyzed conversion,20, 21 etc. In these new developments, electrochemical conversion is recognized as one of promising environmental-friendly ways.22, 23 The principle of the process is based on electricity- driven oxidation in which electrons are extracted from the aromatics thus activating the aromatics to related radicals to cause further oxidation.24-26 At the same time, cathodic reduction occurs that donate electrons to aromatics and unsaturated bond to produce hydrocarbons.27-29 Electrochemical oxidation is one of the electrochemical conversion approaches and has been broadly investigated for the degradation of lignin and its derived aromatics.22, 30-34 It has been widely investigated to decrease chemical oxygen demand (COD) in industrial effluent. In such an application, the aromatics in waste water are directly mineralized to CO2 through electro-oxidation reaction, including anode oxidation,35, 36 three phase-three dimensional oxidation37, 38 and advanced

54 oxidation process (AOP), such as electro-Fenton reaction.39, 40 An example is that Oturan et al.41 employed carbon felt electrode to generate HO· in situ by electro-Fenton reaction to mineralize

2,4-dichlorophenolacetic acid into CO2 and H2O. Interestingly, some ring opening products were identified as the key intermediators before its final mineralization. One the basis of these results, a two-phage reaction was proposed as the electro-degradation mechanism of aromatic mineralization: firstly, the aromatics were oxidized to the ring-opened products and then the

42 resulting products were furtherly completely mineralized to CO2 and H2O. Later, Shao et al. and

Permentel et al.25 furtherly confirmed that aromatics ring opening products were composed of volatile fatty acids (VFAs), which included short chain carboxylic acids (C1~C6), such as muconic acids, maleic acid, oxalic acid, etc.25, 42 However, for the purpose of lignin valorization to biodiesel, no matter lignin mineralization or the short chain carboxylic acids (C1~C6) are not meet the its

43 quality requirements, which commonly employ long-chain carboxylic acids (C12~C22). Luckily,

Naber et al.44 theoretically demonstrated that the length of carbon bone in the carboxylic acids could be enlonged through decarboxylation and coupling reaction, named Kolbe reaction. Weimer

45 et al. used short chain carboxylic acids to synthesize longer chain alkane (C4~C8) with electro- catalysis, which furtherly proved the way of transforming short-chain carboxylic acids into biodiesel. Taken together, these prior works inspired us to speculate that perhaps the production of long chain carboxylic acids and alkanes from lignin and its derived aromatics could be realized through the initial electro-oxidation for VFAs production and their carbon bone length further elongation by the operating parameters regulation, instead of complete mineralization of the aromatics. In this study, the three-phase three-dimensional electro-Fenton reaction was used to oxidize lignin and its derived aromatics, during which the operating parameters was controlled to prompt the synthesis of long-chain fatty acids from VFA intermediates instead of degrading the

VFAs to CO2. A series of experiment including adipic acid coupling reaction and measurements including HO· detection and Gas chromatography-quantitative time of flight mass spectrometry

(GC-QTOF) analysis were conducted to illustrate the reaction mechanism. The results suggested that lignin-based VFAs produced by the electro-oxidation were enlonged to generate long chain acids through Kolbe reaction. This study provided new insight in developing a green pathway to convert lignin and its derivatives from bio-refinery process and pulp industry to fatty acid-based biofuels and bioproducts.

3.3 Materials and methods

3.3.1 Materials and apparatus

Lignin (lignin, alkali, Sigma-Aldrich, USA) was used in this research without further purification.

All regents were analytic grade. The derivatization regents were 14% BF3-methanol solution and

N-methyl-N-(trimethysilyl)trifluoroacetamide (MSTFA) (Sigma-Aldrich, USA). The activated carbon based catalyst was produced in our own laboratory. All the electrolytes were prepared with deionized water.

Gas chromatography (GC) experiments were carried out with a Shimadzu GC Solution system

(Shimadzu, Kyoto, Japan) equipped with a 100 m0.25 mm i.d., film thickness 0.20 µm, sp-2560 capillary column (Supelco, USA) and flame ionization detector (FID). Nitrogen was used as carrier gas with a flow rate at 1 ml min-1.

Gas chromatography-quantitative time of flight mass spectrometry (GC-QTOF) analysis was performed on 7890A GC/7200 Q-TOF MS equipped with a 30 m×250 μm i.d., film thickness 0.25

μm, Agilent 19091S-433 HP-5MS capillary column (Agilent Technologies, Inc.). Helium was used as carrier gas with a flow rate at 1.2 ml min-1.

3.3.2 Electrochemical oxidation of lignin and lignin derived aromatics

The electrochemical oxidation was carried out in the undivided three-phase three-dimensional electrode reactor (Figure 3. 1), based on Uğurlu’s method with modification.37 Briefly, graphite electrodes were employed as both cathode and anode. The whole electrolysis volume was 40 ml and the distance between electrodes (3 cm2 effective area) was situated at 2 cm. The reaction system was stirred at 350-400 rpm to enhance mass transfer. Compressed oxygen was supplied to the reactor from bottom of the cathode through a PVC tube. The galvanostatic condition was performed by a DC power supply (APS3005S-3D, Nanjing Guorui Atten Technologies Industrial

Corporation, China). Guaiacol, salicylic acid, phthalic acid and ferulaic acid were chosen as lignin derived aromatics. The initial electrolysis was performed in 40 ml NaCl solution (0.1M) containing

0.1% (wt/v) lignin or its derived aromatics, 3% activated carbon based catalyst (wt/v) and pH remaining at 3 with 0.5 A current and 350-400 rpm magnetic stirring. Lignin was electrolyzed for

20 min; while lignin derived aromatics were electrolyzed for 8 min. After electrolysis, the resulting electrolyte were extracted three times with 50 mL ethyl acetate, which was furtherly used for fatty acid analysis.

During the optimization process, electrolysis time, current, air flow, NaCl concentration, catalyst amount and Fe2+ concentration were selected as the parameters for optimization. The effect of each parameters was determined based on the yield of fatty acid. All the experiments in this research were carried out in triplicates. During the optimizing process, gas generated from the reactor was collected and used for determination of the composition.

Figure 3. 1. Experimental apparatus: 1. DC Power Supply, 2. Digital Ampermeter, 3. Digital

Voltmeter, 4. Anode (graphite), 5. Cathode (graphite), 6. Magnetic bar, 7. Magnetic stirring controller, 8. Compressed purified oxygen, 9. Activated carbon-based catalyst.

3.3.3 Fatty acid analysis and quantification

The composition and amounts of fatty acids were analyzed by GC, based on Zhang’s method with modification.46 Briefly, 5 mL extractives were derivatized with methyl esterification and determined by GC (GC-20, Shimadzu, Japan). The injector temperature was 250 oC. The column temperature was initially held at 165 oC for 5 min, and then it was programmed from 165 oC to

180 oC at 5 oC min-1, finally held at 180 oC for 5 min. After that, the temperature of column was

o o o -1 o furtherly increased from 180 C to 240 C at 5 C min and held at 240 C for 5 min. The temperature of the FID detector was set at 260 oC. The flow rate of hydrogen and air were set at

40 and 400 mL min-1, respectively.

3.3.4 Guaiacol electrolytes analysis

GC-QTOF was used to separate and identify resultant intermediators in the guaiacol electrolysis.

The NIST 8.0 database was used to interrupt the MS results. All the samples were silylated based on Zhou’s method.47 1µL sample was injected in GC-QTOF in each run. The GC-QTOF was operating in EI mode at 70 eV. The temperature of inlet, source and transmission were 250 oC, 230 oC and 280 oC, respectively. The temperature programing was: 120 oC, 2 oC min-1 up to 200 oC and holding it for 5 min.

3.3.5 Free chlorine analysis

Free chlorine was detected with KI-starch solution, according to Mallmann’s method.48 Briefly,

KI-starch solution was prepared by adding 5 g KI into 100 mL cold starch solution (10%, wt/v) and continuously diluting to 2000 mL. 50 mL KI-starch solution was mixed with the gas generating from electro-Fenton reaction and keep at room temperature for 4 min, the color of KI-starch solutions were measured to detect the amount of free chlorine. Gas from the reactor without electrolysis was used as the control.

3.3.6 Hydroxyl radical analysis

Salicylic acid is employed as the probe to determine hydroxyl radical in the electrolysis, according to Yan’s method with modification.49 Briefly, 9 mM salicylic acid-ethanol solution was used to capture HO· in the electro-Fenton reaction. The experimental design was shown in Table 3. 1.

After reaction, 2mL electrolyte was taken and centrifuged to obtain the supernatant. Then, the OD of all the supernatants were measured at 510 nm with a spectrophotometer. Hydroxyl radical removal rate (HRA) was calculated with the formula as follows:

Absorpton −Absorption HRA= HD HRA × 100% AbsorptionHD−AbsorptionBG

Table 3. 1. Experiments for the hydroxyl radical analysis

NaCl electrolysis electro-Fenton reaction Valuable Ia IIb IIIc IVa Vb VIc

-1 O2 (L min ) 2 2 2 2 2 2

Stirring rate (rpm) 350 350 350 350 350 350

Catalyst* (%, wt/v) 3 3 3 3 3 3

NaCl (M) 0.1 0.1 0.1 0.1 0.1 0.1

Time (min) 4 4 4 4 4 4

Current (A) - 0.5 0.5 - 0.5 0.5

Guaiacol (%, wt/v) - - 0.1 - - 0.1

Fe2+ (mM) - - - 1 1 1

* Catalyst is activated carbon based catalyst. Ia and IVa are treated as background (BG); IIb and

Vb are used for HO· detection (HD); IIIc and VIc are used for HO· removal rate detection (HRA).

3.3.7 Adipic acid electrolysis

40 mg adipic acid was mixed with 40 mL reaction buffer (0.1M NaCl, 3% catalyst, 1 mM FeSO4), kept at 0.5 A and 2 L min-1 for 4 min. After electrolysis, 50 mL ethyl acetate was used to extract fatty acids for three times and then the extractives used for fatty acids analysis.

3.3.8 Lignin electro-conversion

The lignin electro-conversion was carried out in the same way as adipic acid electrolysis. 40 mg lignin was mixed with 40 mL reaction buffer and samples were taken at 0, 4, 7, 10, 13, 16, 19, 22,

25, 28 and 31 min. After electrolysis, 50 mL ethyl acetate was used to extract fatty acids for the further analysis.

In summary, the research sequence of electro-Fenton reaction converting lignin to fatty acids was that:

1. The three-dimensional three-phase electro-Fenton was employed in lignin valorization. Aiming to obtain oxidative coupling products, the electrolysis parameters including time and working current were regulated and GC-QTOF was used to analyze the resulting compounds in the treatment.

2. Some fatty acids were identified and furtherly proved by four kinds of lignin derivatives, including guaiacol, salicylic acid, phthalic acid and ferulaic acid. GC was used to quantify the amount of generated fatty acids.

3. The lignin derivative above with the highest fatty acids conversion yield was selected as the model compound and its intermediators resulted from electro-Fenton reaction identified by GC-

QTOF analysis was used to describe the possible scheme for fatty acids production, which was furtherly confirmed by HO· (salicylic acid detection), Cl2 (KI-starch detection) and electro-fatty acids synthesis from adipic acid (GC detection).

4. Due to the influence of electrolysis parameters (e.g. electrolysis time, current, air flow, NaCl concentration, catalyst amount and Fe2+ concentration) largely on fatty acids production, optimization was furtherly carried out.

5. Under optimal condition, lignin was valorized by electro-Fenton reaction and the resulting fatty acids were quantified by GC.

3.4 Results and Discussion

3.4.1 Lignin degradation and conversion through electro-Fenton reaction

Long-chain fatty acids were discovered as interesting intermediators during lignin electro- oxidation. Table 3. 2 displays the compounds identified during lignin degradation in electro-

Fenton reaction. The results show that partial of lignin was degraded to be single aromatic ring derivatives, such as 4-hydroxybenzoic acid, acetovanillone, 3-vanilpropanol, etc, through its ether bond cleavage and side chain oxidation, which furtherly formed poly aromatic rings, such as 4H-

1-Benzopyran-4-one, 3-(3,4-dimethoxyphenyl)-6,7-dimethoxy, isopimaric acid, abietic acid, etc, through oxidative coupling reaction. Notably, long-chain fatty acids, such as oleic acid, myristic acid, etc, were discovered in the resulting product, including palmic acid and octadecanoic acid as the main components. These fatty acids were considered as important products in lignin valorization, due to its possible utilization for biofuel production. For the sake of further explaining its production mechanism and increasing its yield, lignin model compounds were chosen to illustrate the possible pathway and study the related factors in the electro-Fenton conversion process.

Table 3. 2. Identification of lignin degraded compounds through electro-Fenton reaction

NO. RT (min) Compound Molecular

Formula

1 5.780 Butanedioic acid, 2TMS derivative C10H22O4Si2

2 6.973 Hydroquinone, 2TMS derivative C12H22O2Si2

3 8.683 4-Hydroxybenzoic acid, 2TMS derivative C11H16O3Si

4 11.800 Acetovanillone, TMS derivative C12H18O3Si

5 12.219 1-(4-((tert-Butyldimethylsilyl)oxy)-3,5- C16H26O4Si

dimethoxyphenyl)ethanone

6 12.925 2,6-dimethoxybenzene-1,4- C14H26O4Si2

bis(trimethylsilyloxy)-

7 14.915 Vanillic Acid, 2TMS derivative C14H24O4Si2

8 16.091 3-Vanilpropanol, bis(trimethylsilyl)- C16H30O3Si2

9 16.841 Myristic acid, TMS derivative C17H36O2Si

10 18.693 4-Coumaric acid, 2TMS derivative C15H24O3Si2

11 20.863 Palmitic Acid, TMS derivative C19H40O2Si

12 23.828 Linoelaidic acid, trimethylsilyl ester C21H40O2Si

13 23.949 Oleic Acid, (Z)-, TMS derivative C21H42O2Si

14 24.452 Stearic acid, TMS derivative C21H44O2Si

15 25.220 Benzene, 1,1'-(1,2-ethynediyl)bis[2,4- C18H18O4

dimethoxy-

16 25.693 11,14-Eicosadienoic acid, (Z)-, TMS C23H44O2Si

derivative

17 25.898 Isopimaric acid, TMS C23H38O2Si

18 26.190 Abietic acid, TMS derivative C23H38O2Si

19 26.721 Callitrisic acid, trimethylsilyl ester C23H36O2Si

20 27.256 4H-1-Benzopyran-4-one, 3-(3,4- C19H18O6

dimethoxyphenyl)-6,7-dimethoxy-

21 27.703 Vanillylmandelic acid, 3TMS derivative C18H34O5Si3

22 28.476 benzene, 1,1'-methylenebis[3-(1,1- C29H48O2Si2

dimethylethyl)-5-methyl-4-

[(trimethylsilyl)oxy]-

23 30.376 2-Hydroxyestrone, 2TMS derivative C24H38O3Si2

3.4.2 Production of long chain fatty acids from lignin derived model aromatics

In the initial study, lignin was employed to generate long chain fatty acids in electro-Fenton reactor.

After analyzing the products by GC-QTOF, palmic acid and octadecanoic acid were discovered as two major valorized products. In order to confirmed that palmic acid and octadecanoic acid could also be produced from lignin model aromatics, four other lignin models (guaiacol, salicylic acid, ferulaic acid and phthalic acid) were chosen for electrolysis treatment. Figure 3. 2 shows fatty acids production from four different lignin derived aromatics through electro-Fenton process.

After 4 min electrolysis, palmic acid yield from salicylic acid, ferulaic acid, phthalic acid and guaiacol were 2.55 mg L-1, 2.88 mg L-1, 6.21 mg L-1 and 44.40 mg L-1, respectively; octadecanoic

65 acid yield from the four aromatics were 1.54 mg L-1, 1.88 mg L-1, 4.49 mg L-1 and 31.53 mg L-1, respectively. These results indicated that the lignin derived aromatics were able to generate fatty acids through electro-Fenton process, but the yield of fatty acids varied significantly. Guaiacol was much easier to realize this electrochemical conversion, which was about 11 folds and 16 folds of salicylic acid and ferulaic acid and 7 folds and 6 folds of phthalic acid in palmic acid and octadecanoic acid production, respectively. One of the possible reasons was that guaiacol had higher solubility in the electrolyte, which led to higher mass transfer during the electro-Fenton reaction. Because guaiacol, the common product in lignin degradation,50, 51 showed higher fatty acids yield, in the next stage of this work, the factors related to guaiacol conversion in electro-

Fenton process were evaluated, which could provide data for natural lignin electro-conversion and the illustration of electro-Fenton mechanism in model aromatic degradation.

Figure 3. 2. Fatty acids production from lignin-derived aromatics with electro-Fenton reaction.

3.4.3 The proposed mechanism in the conversion of lignin-derived aromatics

3.4.3.1 The key intermediators analysis in guaiacol degradation

Guaiacol was discovered to experience ring opening oxidation in its initial degradation and the resulting dicarboxylic acids took place Kolbe reaction for long-chain fatty acids synthesis in electro-Fenton treatment. Table 3. 3 lists the major chemicals detected by GC-QTOF, classified into 5 reactions.

Table 3. 3. The related identified compounds in the GC-QTOF

Reaction NO. RT Compound Molecular (min) Formula Chlorination 4 7.213 4-Chloro-2-methoxyphenol, C10H15ClO2Si trimethylsilyl ether

Hydroxylation 1 5.457 1,4-Benzenediol, 2-methoxy- C7H8O3

Hydroxylation 2 6.084 Silane, [1,2- C12H22O2Si2 and phenylenebis(oxy)]bis[trimethyl-] demethoxylation 5 7.711 1,3-Bis(trimethylsiloxy)benzene C12H22O2Si2

Ring opening 3 6.884 2,4-Hexadienedioic acid (E,E)-, C12H22O4Si2 bis(trimethylsilyl) ester 10 6.481 2-Butenedioic acid (E)-, C10H20O4Si2 bis(trimethylsilyl) ester 11 9.295 Hexanedioic acid, C12H26O4Si2 bis(trimethylsilyl) ester Coupling 6 8.264 Decanoic acid, tert- C13H28O2Si reaction butyldimethylsilyl ester

7 17.203 Tridecanoic acid, trimethylsilyl C16H34O2Si ester 8 21.191 Hexadecanoic acid, trimethylsilyl C19H40O2Si ester 9 24.811 Octadecanoic acid, trimethylsilyl C21H44O2Si ester

Based on these compounds, the possible route of guaiacol electrochemical degradation and fatty acids synthesis in the electro-Fenton process were presented in the Figure 3. 3. Initially, guaiacol

(compound 1) was oxidized by Cl2 and HO· to undergo demethoxylation and hydroxylation, forming catechol (compound 2) in which Cl2 and HO· were generated from NaCl electrolysis and

2+ electro-Fenton process. Then, the catechol was oxidized by O2 with Fe as catalyst in the acidic condition to generate o-quinone (compound 3). Since quinone was easy to be degraded by HO·, o-quinone was converted into muconic acid (compound 4), which was further reduced to adipic acid (compound 5). Meanwhile, muconic acid was also degraded to 2-butenedioic acid (compound

6), which was furtherly reduced to succinic acid (compound 7). In addition, adipic acid could also generate succinic acid through electro-oxidation. Compound 4, 5, 6 and 7 were named as volatile fatty acids (VFAs). There were two kinds of oxidation reaction for VFAs: First, VFAs were completely mineralized to produce H2O and CO2 by HO·; Second, HO· drove VFAs to polymerize each other and synthesize fatty acids through oxidation coupling reaction (Kolbe reaction).44

Consequently, there were two pathways of fatty acids synthesis. The first one was octadecanoic acid synthesis: two equivalents of adipic acids were coupled with each other, generating one equivalent of decanoic acid (compound 8). Further, one equivalent of decanoic acid condensed with equal equivalent of adipic acid to produce one equivalent of n-pentadecanoic acid (compound

9). Then, one equivalent of n-pentadecanoic acid was coupled with one equivalent of succinic acid to form one equivalent of octadecanoic acid (compound 10). The other one was palmic acid synthesis: two equivalents of adipic acids polymerized each other, generating one equivalent of decanoic acid (compound 8). Furthermore, one equivalent of decanoic acid was coupled with equal equivalent of succinic acid to produce one equivalent of n-tridecanoic acid (compound 11). Then,

68 one equivalent of n-tridecanoic acid was coupled with one equivalent of succinic acid to generate one equivalent of palmic acid (compound 12).

Figure 3. 3. Proposed pathway for fatty acids formation from guaiacol through electro-Fenton process.

3.4.3.2 Free chlorine detection in electro-Fenton reaction

Cl2 was discovered as the coproduct releasing from electrolysis. The resulting gas caused the color of KI solution changed from white to dark blue in the test group, while the color of control remained white. Cl2 could oxidize KI in the neutral condition to generate I2, which showed blue color by chelating the starch. However, O2 could only oxidize KI to produce I2 in acidic condition, so that it didn’t turn the color of KI to blue in the neutral condition. These phenomena indicated that there was a large amount of chlorine gas generated in the electro-Fenton process of guaiacol degradation. It was because that NaCl was employed as electrolyte and Cl2 produced on the surface

52 of anode. The resulting Cl2 could oxidized guaiacol through chlorination to form 4-chloro-2- methoxyphenol and perform demethoxylation reaction.53

3.4.3.3 Hydroxyl radical detection in the electro-Fenton reaction

HO· was confirmed to form in the electro-Fenton process and furtherly attack guaiacol. Table 3. 4 showed that the absorption HD and absorption BG in the electro-Fenton process were 0.614 and

0.035, respectively, whereas they were 0.071 and 0.025 in the NaCl electrolysis process. These results indicated that electro-Fenton generated much more HO· than that from NaCl electrolysis.

Meanwhile, the value of HRA was 20% after guaiacol addition in the electro-Fenton process, which demonstrated that guaiacol was attacked by HO· and removed about 20% of total HO· from the electro-Fenton reaction. These explained that HO· could oxidized guaiacol and caused hydroxylation reaction to generate catechol and hydroquinone, which were further oxidized into ring opening products.54

Table 3. 4. Spectrum absorption in HO· analysis

Electrolysis Group Absorption (510 nm)

NaCl electrolysis Ia 0.025±0.009

IIb 0.071±0.014

IIIc 0.024±0.005

Electro-Fenton IVa 0.035±0.005

Vb 0.614±0.004

VIc 0.501±0.013

Note: Ia and IVa are treated as background (BG); IIb and Vb are used for HO· detection (HD); IIIc and VIc are used for HO· removal rate detection (HRA).

3.4.3.4 Fatty acids synthesis from adipic acid electro-Fenton reaction

Based on Figure 3. 3, guaiacol could be electronically degraded into adipic acid as the important intermediators, which was proposed to furtherly form palmic and octadecanoic acid. In order to confirm this critical synthesis step, adipic acid was employed in the electro-Fenton reaction to analyze the final product and the GC results were shown in the Figure 3. 4 (A) and (B). Compared with Figure 3. 4 (A), there was an obvious peak at 15.146 min and 18.201 min, representing palmic acid and octadecanoic acid, and an obvious decreasing peak at 14.224 min, representing adipic acid (Figure 3. 4 (B)). The peak at 19.667 min representing nonadecylic acid, which was used as an interior label. These results demonstrated that adipic acid underwent oxidative coupling and formed palmic acid and octadecanoic acid through electro-Fenton process. In addition, palmic acid was much easier to be synthesized than octadecanoic acid in this reaction.

Figure 3. 4. GC results of adipic acid electro-Fenton reaction (A) Adipic acid, (B) its resultant products.

3.4.4 The effect of different factors on electro-conversion

The effect of these factors on guaiacol conversion were presented in Figure 3. 5. The optimal conditions for guaiacol electro-conversion were in 0.1 M NaCl with 1 mM Fe2+, 3% (wt/v) activated carbon-based catalyst loading with 0.7 A current and 2 L min-1 oxygen flow rate during

4 min electrolysis, which led to the highest concentration of palmic acid and octadecanoic acid were 62 mg L-1 and 43 mg L-1, respectively. Any value of these factors that were either higher or lower than the optimal conditions would decrease the yield of fatty acids production (Figure 3. 5

(a)-(f)).

Oturan et al.39 suggested the mechanism of electro-Fenton as: the oxygen was transferred to cathode to produce H2O2 in presence of electricity, and then H2O2 was converted into

HO· catalyzing by Fe2+, which had been described in reaction (1), (2) and (3). Organic compounds were initially activated by HO·, and then attacked by oxidative species (O2 and HO·) to generate

55, 56 CO2 and H2O as final products (reaction (4) and (5)):

+ - O2 + 2H + 2e → H2O2 (1)

2+ 3+ - Fe + H2O2 → Fe + OH + HO· (2)

Fe3+ + e- → Fe2+ (3)

R-H + HO· → R· + H2O (4)

R· + O2 → ROO· → degradation production→ CO2 + H2O (5)

Based on the above mechanism, it was suggested that oxidative species, such as hydroxyl radicals and O2, played important roles in guaiacol degradation and long chain fatty acids synthesis. In the guaiacol degradation reaction, oxygen was employed as the reactant and captured electrons on the

23 -1 cathode to produce H2O2. Low oxygen flow rate (< 2 L min ) was not suitable for H2O2 production, but higher one caused mass transfer problems in the electro-Fenton system, which inhibited oxygen to obtain electrons from cathode. On the other hand, graphite electrode could not supply sufficient reaction surface. To overcome this problem, activated carbon catalyst was polarized and became the third electrodes in high current density and voltage environment. It had been proven that the voltage should be higher than 10 V to make activated carbon effectively polarize and 3% catalyst loading was used to guarantee sufficient electro-chemical reaction areas.37

In our experiment, average voltage in 0.5 A and 0.1 M NaCl environment was higher than 15 V, which made third electrodes (activated carbon- based catalyst) work well. Furtherly, the resultant

2+ H2O2 was sustainably catalyzed by Fe to generate HO·, which drove guaiacol oxidation and long chain fatty acids synthesis. Guaiacol was firstly degraded into VFAs,55, 56 and then, these VFAs were polymerized each other through Kolbe reaction to elongate their carbon bone for palmic acid and octadecanoic acid production. Higher current density benefited this process, which was already proven by Schäfer et al.57 and Naber et al.44 On the other hand, HO· could also attack those long chain fatty acid molecules and cause them to be completely mineralized, consequently the manipulation of electrolysis time was quite important for the synthesis of long chain fatty acids from guaiacol.

Figure 3. 5. Effect of different parameters on the conversion of guaiacol to palmic acid and octadecanoic acid.

3.4.5 Lignin electrochemical conversion to long-chain fatty acids

The optimal time of conversion lignin to long-chain fatty acids were longer than that of guaiacol.

The increase in electrolysis time from 0 to 16 min led to increase in two fatty acids concentrations, whereas further extending time decreased the concentration (Figure 3. 6). The highest concentration of palmic acid and octadecanoic acid reached to 8.68 mg L-1 and 8.56 mg L-1, respectively. Based on the result obtained earlier in the study, lignin was successfully converted to long-chain fatty acids and the optimized electrolysis time was 16 min, instead of 4 min. On the other hand, the yields of these fatty acids were lower than that of guaiacol. The possible explanation for this was that lignin was firstly required to decrease its molecular to simple oligomers through oxidation reaction, such as monomer and dimers; and then they were further converted to quinones, which experienced ring cleavage and resulted in adipic acid and succinic acid, according to Shao’s conclusion.42 Finally, the resultant acids would follow the possible long chain fatty acids pathway to synthesize palmic and octadecanoic acid (Figure 3. 7). Longer degradation pathway led to longer reaction time.

Figure 3. 6. The column plot for palmic acid and octadecanoic acid production from lignin electrolysis versus electrolysis time.

Figure 3. 7. Conversion scheme of lignin to long-chain fatty acids.

3.5 Feature of this technique process

In this research, three-dimensional electro-Fenton reaction, a kind of AOP treatment, was novelly employed to valorize lignin to long-chain fatty acids. The proposed scheme for lignin conversion was described as: lignin was initially degraded to small fragments, such as phenolic compounds; then the resulting fragments experienced ring opening reactions to form dicarboxylic acids (e.g. adipic acid), which were furtherly converted to long-chain fatty acids through Kolbe synthesis. In the traditional electro-Fenton approach, dicarboxylic acids releasing from lignin and its derivatives

41, 42, 58 through electro-oxidation was completely mineralized to CO2, while our process

80 successfully balanced lignin electro-oxidation and the resulting dicarboxylic acids coupling reaction through the regulation of six different factors in the electrolysis. Similar strategy is also reported in the lignin natural degradation process. Vardon et al.16 discovered that lignin subunits, e.g. p-coumarate and ferulate, could not be directly used as the carbon source by P. putida. They typically occurred aromatic ring oxidation and opening for β-ketoadipate generation before consumed in TCA-recycle and fatty acid biosynthesis. This β-ketoadipate pathway from aromatics to fatty acid were also reported in Pseudomonas, Acinetobacter and Rhodococcus.59 This makes our lignin electro-conversion technique a bio-mimic process. It showed us a new approach to effectively convert lignin to its high-value added products. In addition, it belonged to an environmental-friendly route for lignin treatment, due to its widely employment in effluent disposal in room temperature and atmosphere pressure.60 This might furtherly decrease the cost in equipment, its maintenance and waste water treatment.

However, the conversion rate of lignin cannot still satisfy our requirement. Clearly, question on how to efficiently oxidize lignin to low molecular aromatics remains the bottleneck for this process, due to its low solubility into water phase and limitation of mass transfer to the reaction surface.

Taking this into account, we will trying to combine other technologies to enhance the water solubility of lignin before its electro-conversion in our future work. Supercritical water (SCW), which has been already scaled up for industrial demonstration,61 is a promising process to degrade lignin to phenolics. It was reported that 20-25 mol% components were water-soluble containing catechol and other phenolic compounds.62 These water-soluble components are much easier to diffuse around cathode and have better mass transfer in electro-Fenton conversion. For these

81 reason, combining SCW lignin treatment with electro-Fenton process offers a strategy for increasing conversion yield of lignin to long chain fatty acids.

3.6 Conclusion

The three-dimensional electro-Fenton reaction was used to convert lignin and lignin-derived aromatics into palmic acid and octadecanoic acid for their valorization. Through parameters regulation, including Fe2+ and NaCl concentration, electrolysis time, catalyst loading, applied current and oxygen flow rate, the yields of palmic acid and octadecanoic acid under optimal conditions reached to 62 mg L-1 and 43 mg L-1 from 0.1% guaiacol solution, respectively. The mechanism was suggested as that lignin and its derived compound were firstly degraded to VFAs, such as muconic acid and butadiene acid, with Cl2 and HO· supplement and then, the resulting

VFAs were coupled to generate palmic acid and octadecanoic acid through Kolbe reaction. This study provided a new pathway for direct conversion from lignin to palmic and octadecanoic acid as a strategy to valorize this undervalued component in lignocellulosic biomass.

3.7 Acknowledgement

We thank the grants from National Science Foundation, USA to support this work (Grant No.

1231085).

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CHAPTER FOUR

4 ELETRO-FENTON FOR SIMULTANEOUS MICROALGAE HARVEST AND

ELETRO-FENTONLIPID FOR EXTRACTION SIMULTANEOUS ENHANCEMENT MICROALGAE HARVEST AND LIPID EXTRACTION ENHANCEMENT

4.1 Abstract

Currently, lipid extraction remains a major bottleneck in microalgae technology for biofuel production. In this study, we developed an effective cell wall disruption technique based on the electro-Fenton reaction in order to enhance lipid extraction from the wet biomass of N. oceanica

IMET1. Taking economic feasibility and lipid extraction yield into account, we obtained the

-2 optimal conditions with 9.1 mM FeSO4 in a 16.4 mA·cm current density for 37.0 min. In the optimal condition, over 87% of the neutral lipid was extracted. During the 37 min electrolysis,

0.75 mM HO· was artificially produced, resulting in cell wall disruption and lipid dispersion in the entire cell. This was measured by transmission electron microscope (TEM) and Fluorescence microscopy (FM) observation. We also found that the electrolysis wastewater, including 88 mg·L-

1 of total iron, did not inhibit further cultivation of microalgae.

4.2 Introduction

Microalgae is a sustainable source for third-generation biofuel production that can meet energy demands as well as addressing environmental concerns 1. Driven by solar energy, microalgae can

2 biofix CO2 (4-14%, v/v) and other greenhouse gas (GHG) into biomass that can be used for lipid production and GHG abatement 3. However, several economic and technical constraintslimit the industrial applications of microalgae development. These include high costs and low lipid extraction yield 4, 5. Triacylglycerols are the major components of microalgae lipid, which take up

20%-50%, 6, 7 and disperse in the cytoplasm, bounded by a rigid cell wall. These are the most useful sources for biodiesel production 8-11. However, the rigid cell wall largely inhibit lipid extraction process. Hence, effective extraction techniques are required to extract lipid from microalgae cells.

Currently, several laboratory-scale methods have been investigated for lipid extraction, such as

12 13 14 15 organic solvents , supercritical CO2 , subcritical water extraction and milking . Among these techniques, organic solvent extraction is a practical method of lipid extraction in industrial applications 16. Halim et al. proposed that organic solvents extract lipid from the microalgae cell by diffusing into cytoplasm and dissolved most lipids. Lipids undergo counter-diffusion through microalgae cell to the bulk solvent for the downstream process 17, 18. Organic solvent extraction techniques include the dry and wet routes, depending on the water content of the microalgae. The dry route requires over 85% of the water removal in the microalgae. Lardon et al. report that nearly

85% of the total energy is consumed in this process 19. Furthermore, the potential fossil energy ratio (FER) of the wet and dry routes reaches 1.82 and 2.38, respectively. This indicates that the

90 wet route is more feasible for piloting 20. However, cell wall disruption becomes a new bottleneck for lipid liberation and extraction in the wet route 21. The cell wall is constructed of complex carbohydrates and glycoproteins, and has high mechanical strength and chemical resistance.

Pretreatments is usually required in the cell wall disruption, and is conceptually divided into mechanical and non-mechanical techniques. The former includes high-pressure homogenization

(HPH) 22, 23, high-speed homogenization (HSH) 24, hydrodynamic cavitation 25, microwaving 26-

28, ultrasonication (USN) 29-31, and other techniques. These processes destroy the rigid cell wall with strong external forces, which address high energy concerns 32, 33. Grimi et al. report energy consumption ranging from 12 kJ·kg-1 dry weight (DW) for USN treatment to 1.5 MJ·kg-1 DW for

HPH process in Nannochloropsis sp. cell disruption 32. On the other hand, non-mechanical methods are known as lower energy consumption, with direct physical energy transfer and additional chemical reactions for cell disruption 22. Non-mechanical methods include acid hydrolysis 34, ionic liquid extraction 35-37, steam explosion 38, etc. However, further challenges include continuous chemical supplement, lipid degradation and waste liquor treatment, limiting their development in industrial experimentation 22.

To address these issues, recent research has focused on making the process more cost-effective and controlled, reducing energy consumption below algae’s potential energy storage of 21 kJ·g-1, as well as improving the mildness, adaptability and recoverability of products 22. Fenton cell disruption techniques represent a new trial that effectively disrupt the microalgae cell wall. This method uses HO· (3~5 min) in mild temperatures and atmospheric pressures and employ a low level of toxic reactants 39, 40. Moreover, iron catalysts contributes to efficient microalgae harvest

-1 35 with 0.56 g·L FeCl3 coagulation . Also, the Fenton treatment is a promising technique in

91 effluent disposal. In fact, it can remove over 90% of the chemical oxygen demand (COD) in combined industrial and domestic wastewater for water recycling 41. These reduce the costs of equipment, maintenance and waste water treatment, as well as energy consumption. However, continuous H2O2 supplement remains a bottleneck of the Fenton treatment due to safety concerns in the handling and shipping process and higher operation costs 42, 43. Meanwhile, the hard controllability of Fenton reaction fails to meet the requirement of industrial applications. The excessive reaction usually leads to lipid degradation and markedly decreases the lipid extraction yield. The traditional way to solve this problem is to add organic solvents and inhibitors in order to terminate the reaction 39, 40, further raising costs 40, 44.

To address these issues, this study aimed to employ the electro-Fenton reaction as an alternative technique for microalgae cell wall disruption. Through cathodic oxygen reduction, H2O2 is continuously generated on the surface of the cathode, which addresses the continuous

45 supplementation of H2O2 as well as safety concerns . Furthermore, the electro-Fenton process can be terminated through current and electrolysis time regulation with convenient manual regulation.

In this study, we examined the effects of different parameters (e.g. FeSO4 concentration, current density and time) on microalgae cell wall disruption with response surface methodology (RSM).

We used their models in the optimization process to identify the optimal conditions for lipid extraction. We also analyzed lipid composition using a gas chromatograph for a quality evaluation during the electro-Fenton process. We also measured the amount of hydroxyl radical, the distribution of iron, and monitored changes in the cell wall and lipid droplets using High

Performance Liquid Chromatography (HPLC), a spectrophotometer, florescence microscope (FM)

92 and a transmission electron microscope (TEM), respectively. Finally, we used the wastewater directly from electrolysis for microalgae re-cultivation and the amount of microalgal cells reached

3 g·L-1. Findings indicated that this new method is an efficient and low cost method of extracting lipid from microalgae.

4.3 Materials and methods

4.3.1 Strain and culture condition

N. oceanica IMET1 was kindly provided by Dr. Yubin Ma from the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. Seed cultures were maintained in 250- mL Erlenmeyer flasks with 150 mL of seawater BG-11 medium under continuous artificial illumination. In the experiments, 50 mL logarithmic phase seed cells and 350 mL fresh medium were transferred to glass columns (4.1 cm in diameter, 37 cm in height) with 2% (v/v) CO2.

Aeration flow for each column was 10 mL·min-1. Light intensity and temperature were maintained at 50 μM·m–2·s –1 and 25 oC with an initial pH of 7.2, respectively. For the dry cell weight (DCW) measurement, the microalgal culture samples (10 mL) were filtered by pre-weighed 0.8 µm microporous filter papers, and then dried overnight at 105 oC. DCW was calculated using the difference between the final and beginning weights of the filter papers 46.

4.3.2 Neutral Lipid extraction and Fatty Acid Analysis

The microalgal total neutral lipid and lipid extraction yield from electrolysis were based on Fajardo et al.’s and Steriti et al.’s method 40, 47 , respectively. The yield of extracted neutral lipids was calculated using the ratio between the weight of extracted lipids and the weight of total lipids in the microalgae.

For the fatty acid methyl ester (FAME) reaction, 1 mL of the KOH-methanol solution (0.5 M) with nonadecanoic acid (0.1 mg·mL-1) was added to the lipid extract at 65 oC for 15 min, and shaken every 5 min. Next, 2 mL of 14% BF3-methanol solution was added at 65 oC for 2 minutes with continuous shaking. Subsequently, 2 mL of hexane and 1 mL of saturated NaCl solution was employed, and the FAMEs were dissolved in the hexane layer. For reproducibility, triplicates were carried out in each experimental condition.

FAME analysis was carried out using GC (GC2010, Shimadzu) equipped with a FID detector and a SP-2560 column (100m×0.25mm×0.2µm, Supelco, USA), based on Zhang et al. 48. 1 µL methylated sample was injected at 250 oC. The column temperature was initially set at 165 oC and held for 5 min. Subsequently, it was programmed to 180 oC at the rate of 5 min-1 and held for 5 min. Next, the temperature was increased from 180 oC to 240 oC at the rate of 5 min-1 and held for another 5 min. The temperature of FID detector remained at 260 oC. The flow rate of hydrogen and air were supplied at 40 and 400 mL·min-1, respectively.

4.3.3 Cell disruption

Microalgae cells with 26 days cultivation were chosen as the material for electro-Fenton reaction.

50 mL cell samples were added into 100-mL glass beakers along with different amounts of FeSO4.

Next, two graphite electrodes were dipped into the medium (3 cm2 effective area) and the distance between them was situated at 2 cm. A magnetic stirrer (350-400 rpm) was employed to enhance mass transfer in the electro-Fenton reaction. Compressed oxygen (2 L·min-1) was fed to the cathode for H2O2 production. Finally, electro-Fenton reaction was terminated by cutting off the electricity. After electrolysis, microalgae precipitate was collected for lipid extraction according to Method 4.3.2.

4.3.4 Optimization of electro-Fenton cell disruption conditions

Several parameters affecting microalgae cell wall disruption were evaluated through single-factor experiments by calculating the lipid extraction yield. These included electrolysis time, FeSO4 concentration and current density. Three trials were run for each combination: FeSO4 (5-15 mM), current density (5-30 mA·cm-2) and electrolysis time (0-60 min). Design expert (version 8.0.6) was used to design experiments and analyze data. Experimental designs of the electro-Fenton conditions are shown in Table 4. 1.

4.3.5 Hydroxyl radical detection

The amount of HO• formation in the electro-Fenton reaction was monitored using the modified salicylic acid method 49. The experimental apparatus was the same as that in section 4.3.3. Based

-2 on optimal results, the electro-Fenton reaction was carried out at 9.1 mM FeSO4, 16.4 mA·cm current density, 2 L·min-1 oxygen feeding at 350-400 rpm in room temperature. In addition, 5 mL salicylic acid-ethanol solution (90 mM) was added into the electro-Fenton reactor, which also contains a 40 mL BG11 cultural medium, and the pH of the solution was adjusted to 6.0, which is the same as the final pH value of microalgae medium in electrolysis. For the detection, samples were collected at 0, 5, 15, 37 and 60 min, respectively. They were filled through a 0.22 µm luer syringe filter before analysis with HPLC (Shimadzu, LC-20AD).

HPLC analysis was performed on an ODS-BD C-18 column (Sinochrom, 4.6260 mm, 5.0 µm) at 35 oC. In the mobile phase, an acetic acid-water-methanol solution (1:79:20) was passed through the column with a flow rate of 1 ml·min-1. The UV detector monitored salicylic acid and its derivatives at 296 nm. The injection volume was 10 µL each time 49.

4.3.6 Iron detection

The iron concentration in the supernatant and microalgae precipitation was detected with the modified 1,10-phenanthroline method 50. 1 mL samples collected at 26 days were taken with electrolysis at the optimized conditions of 0, 5, 15, 37 and 60 min, respectively. Samples without

96 the addition of FeSO4 were treated as a control. All experiments were performed in triplicate. The experimental cells underwent flocculation for 30 minutes after electrolysis. Next, the supernatant and precipitate were collected. Before iron detection, each precipitate was added in 50 µL concentrated HCl (37%, v/v), and 1 mL of water mixed by ultrasonic treatment for 5 min to dissolve the precipitated iron. In the detection, 2.5 mL ammonium acetate buffer (mixing 40 g ammonium acetate, 50 ml glacial acetic acid and water to 100 mL) was added into each supernatant and mixture. Then, 1.25 mL hydroxylamine hydrochloride solution (10%) and 2.5 mL 1,10- phenanthroline (0.5%, m/v) were used to chelate the iron. Sequentially, the volume of solution was supplied to 25 mL with e-pure water. After 10 min, the color development reaction was complete.

Finally, samples were measured with a spectrophotometer at 510 nm. The iron detection standard curve was required in each batch.

4.3.7 Electron microscopy and Fluorescence microscope analysis

To observe the cell wall disruption process, electro-Fenton analyses ranging from 0 to 60 min were performed, respectively. Transmission electron microscopic (TEM) studies were carried out with

TEM (JEM-1400; JEOL,Tokyo, Japan) according to Hou et al. 46. To elucidate the lipid extraction process under electro-Fenton system, the microalgae cells stained by Nile red 51, 52 were examined with a fluorescence spectrometer (Leica DM5000B microscope) with BP 516-560 nm excitation filter, 580 nm dichoic mirror and LP 590 nm emission filter (Leica filter cube N2.1) at 25 oC 53.

4.3.8 Reuse of electrolysis waste water for subsequent microalgae cultivation

For comprehensive utilization of wastewater from the electro-Fenton reaction, culture medium recycling was carried out. The resulting supernatant was centrifuged for further cultivation with replenished fresh BG-11 nutrients, except for FeSO4. Cultivation conditions were consistent with methods described in Section 4.3.1. DCW was used to evaluate the effects of wastewater on cell growth.

4.4 Results and discussion

4.4.1 Growth property

Since N. oceanica IMET1 is one of microalgal strains with high lipid accumulation 6, it was selected as the model strain for later cell disruption and lipid extraction research. The cell growth curve during N. oceanica IMET1 cultivation was measured by dry weight analysis shown in the

Figure 4. 1. It was observed that this strain reached the stationary stage with the biomass concentration about 3.10 g·L-1 after 26 days with neutral lipid content about 30.47% (wt/wt, DCW).

Before arriving at stationary stage, microalgae began to turn yellow on 22th day (data was not shown), which meant gradual lipid accumulation in cells. These phenomenon were similar to Ma’s results 6. The lack of nutrients and accumulation of toxic catabolites inhibited the growth of microalgae and induced cell apoptosis in the further cultivation. In order to keep microalgae

98 healthy cell state to evaluate the effect of electro-Fenton treatment on its cell disruption, microalgae fluid was taken at 26th day.

Figure 4. 1. The growth curve of N. oceanica IMET1 in fresh seawater BG-11.

4.4.2 Optimization of different factors on electro-Fenton cell disruption

The electro-Fenton pretreatment enhancing the yield of microalgae lipid extraction was shown in

Table 4. 1. Based on the previous research, FeSO4 concentration, current density and electrolysis time largely influenced lipid extraction yield (data is not shown). The optimum experimental

-2 conditions were 10 mM FeSO4, 17.5 mA·cm current density and 30 min (Std. order 15 in Table

4. 1). The lipid extraction yield reached to the maximum 88.6% (wt%, total neutral lipid) without cells freeze-drying. In other Electro-Fenton pretreatment with either less or more treatment conditions lower lipid extraction yield. In this process, the three factors played important roles on

99 lipid extraction by varying HO· amounts and its contract time with microalgae. Pimentel et al.

(2008) discovered that Fe2+ concentration and current density were directly related to

HO· production rate 54. In addition, contact time between HO· and microalgae largely affected the cell wall structure disruption and lipids extraction yield 40, 55. Less HO· didn’t meet the requirement of cell wall disruption, however, excessive HO· caused lipids consumption. The way for

HO· production was thus needed to control strictly. In this research, electro-Fenton is precisely controlled by electricity supply regulation for higher lipids extraction yield. The lipids extraction yield of untreated N. oceanica IMET1 was about 40% (%, total neutral lipid) under the same hexane/hydroalcoholic extraction. Therefore, electro-Fenton pretreatment played an important role in enhancing lipids extraction.

4.4.3 Confirmation of predicted optimum condition

The predictive quadratic model for lipid extraction yield were based on actual parameters as follows:

Y = -37.83889 + 12.64796  X1 + 3.79849  X2 + 1.91768  X3-0.040689  X1  X2 - 0.027345 

X1  X3 – 0.00590643  X2  X3 - 0.60049  X1 ^ 2 - 0.097653  X2 ^ 2 - 0.021420  X3 ^ 2;

Where Y represents lipid extraction yield (wt%, total neutral lipid), X1 represents FeSO4

-2 concentration (mM), X2 represents current density (mA·cm ) and X3 represents time (min). The predicted maximum lipid extraction yield calculated by the predictive model was 86.33% with the

-2 optimum conditions, including 9.1 mM FeSO4, 16.4 mA·cm current density and 37.0 min

100 electrolysis (Figure 4. 2). This model described the experimental data well. The lipid extraction yield verified under the predicted optimum conditions was 87.53%, which was close to the predicted lipid extraction yield. In the optimal conditions, electro-Fenton enhanced the yield from

40 % to 87.53%, equal to from 12.2% (wt%, DCW) to 26.7% (wt%, DCW). This result is comparable to the 23% (wt%, DCW) lipid extraction yield which resulted from Ti4O7-based membrane anodic oxidation 56.

Table 4. 1. Experimental design matrix for the optimization of electro-Fenton conditions.

Std. order Run order Fe2+ conc. (mM) Current density Time Extraction

(mA·cm-2) (min) yield (wt%)

17 1 10.00 17.50 30.00 84.00 11 2 10.00 5.00 60.00 64.97 6 3 15.00 17.50 0.00 43.31 3 4 5.00 30.00 30.00 58.41 10 5 10.00 30.00 0.00 40.36 12 6 10.00 30.00 60.00 54.80 13 7 10.00 17.50 30.00 81.71 16 8 10.00 17.50 30.00 84.99 4 9 15.00 30.00 30.00 46.60 15 10 10.00 17.50 30.00 88.60 14 11 10.00 17.50 30.00 85.64 5 12 5.00 17.50 0.00 46.27 7 13 5.00 17.50 60.00 66.28 1 14 5.00 5.00 30.00 57.75 2 15 15.00 5.00 30.00 56.11 9 16 10.00 5.00 0.00 41.67 8 17 15.00 17.50 60.00 46.92

101

4.4.4 Fatty acid (FA) composition analysis during electro-Fenton reaction

The 15 individual FAMEs identified were shown in Figure 4. 3. In the control one, the major FAs of IMET1 were fatty acids containing 16 carbon atoms, including palmitic acid (C16:0) and hexadecenoic acid (C16:1), comprising 32.5% and 26.2% of the total FAs, respectively. The third most abundant was oleic acid (C18:1), accounting for 22.9%. Notably, compared with FAs composition in the control, there were no obvious changes during a 1h electro-Fenton reaction.

Carocho et al. (2013) found that monounsaturated (MUFA) and saturated fatty acids (SFA) were more resistant to HO· than polyunsaturated fatty acids (PUFA), due to their fewer double bonds

57. The SFA and MUFA, the major contents in the IMET1, reached 40.5% and 49.4%, respectively.

This made it difficult for HO· to change the FAs composition through peroxidation and degradation 58, 59. These suggested that the FAs produced from IMET1 were suitable to employ this technology for algae cell wall disruption.

102

Figure 4. 3. Effects of electrolysis time on the composition of extracted fatty acid from N. oceanica IMET1.

4.4.5 Microalgae cell disruption mechanism in electro-Fenton reaction

4.4.5.1 Hydroxyl radical generation in electro-Fenton reaction

HO• was continuously produced during electro-Fenton process. Figure 4. 4 presented the line plots for salicylic acid-captured HO• products vs. electrolysis time. The production rates of 2,5-dHBA and 2,3-dHBA were initially kept at 15.76 µM·min-1 and 7.65 µM·min-1, respectively. After 15min, they started to decrease. On the other hand, results showed no obvious accumulation of these products after electricity was powered off. This indicated that the production of HO· could be effectively terminated through electricity regulation. Meanwhile, Jen et al. (1998) showed that one

103 equivalent of HO· drove one equivalent of hydroxyl group addition on the salicylic acid 49.

According to this theory, HO· generation during electrolysis was calculated. At the optimal time

(37 min), 0.75 mM HO· was formed in the electro-Fenton process.

Figure 4. 4. 2,3-dHBA and 2,5-dHBA production versus time in electro-Fenton reaction

4.4.5.2 TEM of algae cell wall and cytomembrane in electro-Fenton process

Microalgae cell structure was gradually degraded in the electrolysis. Figure 4. 5 displayed the microalgae cell status vs. time during electrolysis. Compared with untreated microalgae cells

(Figure 4. 5a and b), iron ions were obvious absorbed on the surface of the cell wall, and further diffused to the cytomembrane and even the lipid body in FeSO4 group (Figure 4. 5c). However, these microalgae cell structures were kept intact without degradation by the absorbed iron in the

1h treatment (Figure 4. 5d). In addition, we used electrolysis to treat microalgae cells for 15 mins.

It was discovered that most of microalgae were dead, and some organelles, such as chloroplast,

104 were destroyed (Figure 4. 5e). At the same time, a pore formed by HO· degradation on the cytomembrane (Figure 4. 5f). We presumed that the thickness of cell wall was thicker than that of the cytomembrane, which firstly caused cytomembrane degradation. However, cell wall disruption was the true bottleneck in microalgae lipid wet extraction 16. We found that a 15 min electro-

Fenton reaction did not destroy the cell wall (Figure 4. 5d), which was consistent with previous results showing that 15 min was not the optimized electrolysis time for lipid extraction. In addition, when electrolysis time reached 37 min, most of organelles, including the lipid body, were destroyed (Figure 4. 5g). Fig. 5h depicted a pore that appeared on the cell wall. This indicated that cell wall disruption occurred in 37 min. Results showed that cell wall degradation changed the shape of microalgae cell and resulted in a higher lipid extraction yield. Finally, most of microalgae cell structure was completely destroyed after 1h of electro-Fenton treatment (Figure 4. 5i). The cell wall and cytomembrane were completely disrupted, and the cellar contents leaked out (Figure

4. 5j). In the meantime, lipid was directly exposed to electro-Fenton agents as it diffused into the electrolyte, which resulted in lipid degradation by HO·. These results were consistent with Steriti et al.’s research 40. It meant that the disruption degree of cell wall could be regulated by electrolysis time, which aided sequential extraction of various products from the microalgal cell.

105

Figure 4. 5. TEM for microalgae cells.

4.4.5.3 Fluorescence microscope (FM) of algae during electro-Fenton reaction

Microalgae cells showed obvious coagulation with iron addition and its lipid was degraded by excessive HO• treatment. Figure 4. 6a showed that the untreated microalgae cells dispersed in the culture medium and were separated from each other. The lipid body with red fluorescence displayed the lipid position in the intact microalgae cells. The addition of FeSO4 caused cell aggregation, and the cells was still completed (Figure 4. 6b). There was no obvious changes in red fluorescence during 1h of FeSO4 treatment. This indicated that cell and lipid degradation did not occur during treatment. In the next experiment, we used the electro-Fenton reaction to disrupt microalgae cells. Figure 4. 6c indicated that red fluorescence appeared on the whole microalgae cells. This meant that the lipid body was disrupted during the 15 min electro-Fenton reaction. It was likely that the lipid body membranes absorbed the iron ions (Figure 4. 5e), which catalyzed

106 the resulting H2O2 from electrolysis to produce HO· on site and destroyed the membranes. When the reaction time was lengthened to 37 min, red fluorescence began to appear outside of the cells, as shown in the FM image (Figure 4. 6d). This was due to cell wall disruption (Figure 4. 5h).

However, a one-hour electro-Fenton reaction disrupted most cells, and their fragments aggregated together (Figure 4. 6e). A large amount of red fluorescence appeared among the resulting fragments with low intensity. This confirmed that HO· was able to completely destroy the microalgae cell structure. The leaked lipids were constantly exposed to HO· and consumed through oxidation reaction.

107

Figure 4. 6. FM of microalgae cells.

4.4.6 Iron distribution

In the electrolysis process, iron worked as the catalyst that induced HO· production 54, and also as the precipitant enhancing microalgae harvest through microalgae surficial charge neutralization 60.

Owning to these functions, our investigation of iron distribution further explained the trend of

HO· production and provide the data for iron recovery in downstream process. The evaluation of

108 iron distribution in the supernatant and precipitate vs. time was shown in the Figure 4. 7a and

Figure 4. 7b. As shown in Figure 4. 7a, iron concentration in the supernatant decreased from 322 mg·L-1 to 22 mg·L-1, while its content in the precipitate increased from 179 mg·L-1 to 320 mg·L-1 during 60 min electrolysis. In addition, part of the iron was absorbed on the surface of cathode through iron reduction. Figure 4. 7b indicated that the iron abundance of supernatant during 1h electrolysis decreased from 64% to 4%. However, the iron abundance of precipitate and electrodes increased from 33% and 2% to 63% and 33%, respectively. These results indicated that there were three main stages of iron distribution. In the first stage, about 33% of the iron was rapidly adhered on the microalgae through physical absorption, and 64% of that remained as free ions in the electrolyte. Metal ions could neutralize the negative charges distributed on the surface of microalgae cell wall 60. This was also confirmed by the fact that iron dispersed on the cell wall, cytomembrane and lipid droplets of microalgae (Figure 4. 5c). In the second stage, 18% of additional iron attached to the microalgae during 5 min of electrolysis. This might be due to chemicals, such as Fe(OH)2 and Fe(OH)3, forming during electrolysis, as well as to enhanced cell coagulation. In the third stage, iron began to accumulate on the surface of cathode by iron reduction and continuously absorbed on the microalgae. This might decrease electrolysis efficiency and inhibit cell disruption by decreasing the contact area between cathode and oxygen. After 37 min of electrolysis, about 95% of the microalgae cells were harvested by co-flocculation through 30 min of natural precipitation (data not shown). This indicated that the electro-Fenton process could achieve simultaneous cell disruption and harvest. Furthermore, more than 60% of iron was removed from the supernatant, suggesting us to recover iron from precipitate residues after lipid extraction.

109

110

4.4.7 Microalgae cultivation in the electro-Fenton waste water

The electro-Fenton process is well-known as a kind of advanced oxidation process (AOP) in wastewater treatment. We discovered that microalgae could grow healthily in the resulting wastewater. Figure 4. 8 displayed the growth curve of IMET1 in the wastewater. Results showed that microalgae could grow in the wastewater at up to 3.0 g·L-1 in 24 days cultivation. After that, microalgae arrived at a stationary stage without obvious biomass accumulation. The growth curve of microalgae cultured with wastewater was similar to that of fresh sea water. This indicated that the quality of wastewater met the requirements for IMET1 growth, and did not negatively affect the growth of microalgae.

Figure 4. 8. The growth curve of N. oceanica IMET1 in electro-Fenton resulting waste water.

4.4.8 Feature of this technology

111

In this study, we employed the electro-Fenton process for the simultaneous cell disruption and harvest of microalgae in wet route. Theoretically, oxygen was directly reduced on the surface of

2+ cathodes driven by electricity for H2O2 production. Next, the resulting H2O2 was catalyzed by Fe to produce HO· for microalgae cell wall degradation through the Fenton reaction. Furthermore,

Fe3+ kept obtaining electrons on the cathode, which inhibited the side reaction of oxygen generation. The related mechanism were shown as follows 42:

Electro-Fenton reaction mechanism:

- + O2 + e + 2H → H2O2 (1)

2+ 3+ - Fe + H2O2 → Fe + OH + HO· (2)

Fe3+ + e- → Fe2+ (3)

Side reaction in the Fenton process:

3+ 2+ + Fe + H2O2 → Fe + 2H + O2 (4)

Taken these into account, the electro-Fenton reaction conquered several problems remaining in the

Fenton reactions: 1. H2O2 was sustainably produced on site by oxygen feeding, eliminating the

43 need for acquisition, shipment and storage of H2O2 , and might further decrease treatment costs

2. The reaction termination and HO· production could be artificially regulated by using electricity without additional inhibitors consumption 39. This could effectively balance microalgae cell wall degradation and lipid oxidation in the Fenton-based wet cell disruption route. 3. Electro-Fenton reaction decreased co-reactions causing by Ferric ions and increased the efficiency of HO• production. On the other hand, FeSO4 also worked as a precipitant for microalgae co-flocculation

112 during electrolysis. In this work, we proposed a new electro-Fenton cell disruption technique to be used with the lipid extraction process. Firstly, the fresh microalgae fluid is treated with our electro-

Fenton process in optimal conditions. Next, natural sedimentation is employed to harvest microalgae from the resulting fluid, and the wastewater is reused for microalgae cultivation. Then, neutral lipids are extracted with a hexane/hydroalcoholic solution and used for downstream lipid upgradation. Finally, the microalgae residue containing over 60% total iron is burnt for iron recovery. However, further research is needed to improve this process. This includes investigation of microalgae cultivated in wastewater for electro-Fenton cell disruption. Due to the high iron content of the culture medium, the optimal FeSO4 supplement may be changed. In addition, comprehensive iron recovery techniques are required to recycle iron for later electro-Fenton usage.

4.5 Conclusions

This study investigated a novel microalgae cell disruption technique for N. oceanica IMET1 lipid extraction based on electro-Fenton reaction. Findings showed that the yield of lipid extraction depends on current density, Fe2+ concentration and electrolysis time. With the optimal conditions of 16.4 mA·cm-2 current density, 9.1 mM Fe2+ and 37.0 min electrolysis time, 87.53% of the lipid was extracted (%, total neutral lipids) , and there were no obvious changes in components. About

0.75 mM HO· was produced during a 37 min electro-Fenton reaction, which caused cell wall, cytomembrane and lipid body disruption and enhanced organic solvents (ethanol/hexane) lipid extraction. After electrolysis, over 60% of the total iron distributed in the microalgae precipitation and on the surface of the electrode, which could be used for iron recovery. Furthermore, the

113 resulting wastewater containing 88 mg·L-1 iron ions met the quality requirements for microalgae cultivation, and no inhibitors obviously affected microalgae growth. This study developed an electro-Fenton reaction to disrupt the microalgae cell wall for lipid extraction and overcome obstacles in the traditional Fenton reaction, such as H2O2 chemical costs, and problems with reaction termination. This electrochemistry technology holds promise as a new approach for microalgae biofuel extraction.

4.6 Acknowledgement

This work was supported partly by the National Science Foundation of USA (Grant No. 1231085), the Key Program for International Cooperation Projects of Sino-Canada (Grant No.

155112KYSB20160030), the National Natural Science Foundation of China (Grant No.

31570047).

114

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CHAPTER FIVE 5 CONCLUSION

CONCLUSIONS

120

5.1 Concluding Remarks

This dissertation study included investigation on a possible pathway for hydrogen peroxide production in termite, and employing Electro-Fenton reaction to mimic the speculated radical- based lignin degradation process in termite for lignin conversion and microalgae cell wall disruption. The main conclusions derived from this study are summarized as follows:

We confirmed a pathway of hydroquinone production related to HO· generation for degrading lignocellulose. Arbutin, the precursor of hydroquinone, was discovered in the southern pine extractives. It was hydrolyzed by the expressed Glu1B, the most efficient β-glucosidase for lignocellulose degradation in C. formosanus. These results conjugated with the evidences reported in the previous literature suggested a mechanism for lignocellulosic biomass degradation: termite secretes β-glucosidase in chewing process to hydrolyze arbutin and forms hydroquinone, which is catalyzed by iron ions and lignin oxidase (e.g. laccase and peroxidase) to supply electrons to oxygen for H2O2 generation. The produced H2O2 is furtherly converted to HO· through iron catalysis. This pathway can be well mimicked by electro-Fenton process, which provide electrons to oxygen by cathode for H2O2 production and the resulting H2O2 is decomposed to HO· by iron.

We then applied this process for lignin and cellulose degradation. In the lignin degradation process, we discovered that electro-Fenton process could convert lignin to long-chain fatty acid. Through regulating parameters, including Fe2+ and NaCl concentration, electrolysis time, catalyst loading, applied current and oxygen flow rate, the yields of palmic acid and octadecanoic acid were optimized. The mechanism was suggested as that lignin was first degraded to VFAs, such as

121 muconic acid and butadiene acid, with Cl2 and HO· supplement and then, the resulting VFAs were coupled to generate palmic acid and octadecanoic acid through Kolbe reaction. This study provided a new pathway for direct conversion from lignin to palmic and octadecanoic acid as a strategy to valorize this undervalued component in lignocellulosic biomass.

In addition, we applied electro-Fenton process to degrade cellulose, the main composition of microalgae cell wall, for enhancement of microalgae lipid extraction. Through parameters regulation (e.g. current density, Fe2+ and electrolysis time), the yield of lipid extraction was optimized. HO· was efficiently produced in the electrolysis and caused the disruption of cell wall, cytomembrane and lipid body membrane. After electrolysis, over 70% of total iron distributed in the microalgae precipitation and, on the surface of electrode, which could be used for iron recovery.

Furthermore, the resulted wastewater containing 88 mg·L-1 iron ions met the quality requirements for microalgae cultivation and had no obviously adverse effect on microalgae growth. The use of electro-Fenton reaction to disrupt microalgae cell wall for lipid extraction solved the problems with using the traditional Fenton reaction, such as H2O2 chemical cost, handling problems and reaction termination problems. It is anticipated that this electrochemical technology can work as a new approach for microalgae oil extraction.

5.2 Perspectives and Future Directions

In this dissertation, one of the mechanisms xisting in termite for HO· production in lignocellulose degradation was investigated. Actually, termite may have develop several different strategies for radical formation. Glucose oxidase is also a well-known strategy related to HO· generation in some

122 species of fungi and bacterial. In the next step of research on termite-based HO· production, the secreted enzymes from saliva gland, foregut and midgut to check whether there is glucose oxidase secreted out during wood digestion or not should be analyze. The results will prove the hypothesized mechanism employed by termite.

In addition, a possible mechanism was discovered to valorize lignin to high-value product through electro-Fenton process. However, the conversion rate of lignin was still too low to satisfy cost reduction requirement. Clearly, question on how to efficiently oxidize lignin to low molecular aromatics remains the bottleneck for this process, due to its low solubility into water phase and limitation of mass transfer to the reaction surface. Taking this into account, a combination of other technologies to enhance the water solubility of lignin before its electro-conversion is recommended for future work. Supercritical water (SCW), which has been already scaled up for industrial demonstration, is a promising process to degrade lignin to water soluble phenolics. These water-soluble components are much easier to diffuse around cathode and have better mass transfer in electro-Fenton conversion. For these reason, combining SCW lignin treatment with electro-

Fenton process offers a strategy for increasing conversion yield of lignin to long-chain fatty acids.

Electro-Fenton reaction was successfully demonstrated to degrade microalgae cell wall and solving several problems remaining in using Fenton reactions for microalgae cell disruption, such as sustainable H2O2 consumption and oxidation rate regulation. However, several topics are still required to be studied to improve the whole process: (1) Investigation of wastewater cultivated microalgae for electro-Fenton cell disruption. Due to high iron content in their culture medium, the optimal FeSO4 supplement could be changed. (2) Comprehensive iron recovery techniques are

123 required to recycle iron for later electro-Fenton usage. (3) Optimal integration of the whole process and estimation of economics are required before this technique is scaled-up.

124