Faculty of Resource Science and Technology
Feasibility of Bioethanol Waste Stream for Production of Laccase in Pichia pastoris
Farah Wahida Binti Mamat
Master of Science 2022
Feasibility of Bioethanol Waste Stream for Production of Laccase in Pichia pastoris
Farah Wahida Binti Mamat
A thesis submitted
In fulfillment of the requirements for the degree of Master of Science (Fermentation Technology)
Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK
2022
i
DECLARATION
I declare that the work in this thesis was carried out in accordance with the regulations of Universiti Malaysia Sarawak. Except where due acknowledgements have been made, the work is that of the author alone. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.
………
Signature
Name: Farah Wahida Binti Mamat
Matric No.: 18020178
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
Date :
ACKNOWLEDGEMENT
Foremost, I am gratitude to Almighty Allah SWT, for His grace and blessing, in giving me the strength to complete this thesis. I would like to express my sincere thanks to my supervisor, Dr Nurashikin binti Suhaili, and my co-supervisor, Assoc. Prof. Dr Dayang Salwani binti Awang Adeni, for their guidance, patience and motivation throughout my study. My sincere thanks also goes to Dr Ngieng Ngui Sing for his guidance and advice in completing my research work. I am extremely grateful and indebted to these individuals for imparting their knowledge and expertise in this study.
My deepest gratitude to my beloved parents and siblings, for their endless love, continuous support and always have faith in me. Their encouragement and advice when the times got rough are much appreciated and duly noted. I would also like to take this opportunity to record my sincere thanks to my friends and colleagues at the R&D Biochemistry Lab for their assistance, moral support and memories. Last but not least, my sincere gratitude to the management of the Universiti Malaysia Sarawak for making it possible for me to complete my study here in Sarawak. Thank you all.
iii ABSTRACT
Laccases are multipurpose enzymes that have wide biotechnological applications. The present study seeks to evaluate the feasibility of spent fermentation broth generated upon the bioethanol production from sago hampas, as a feedstock for production of laccases in recombinant Pichia pastoris GS115. Characterisation of the sago bioethanol liquid waste (SBLW) indicated glycerol as the main component, along with glucose and lactic acid.
Evaluation of SBLW as a fermentation feedstock for laccase production in P. pastoris GS115 showed that the cell growth was generally feasible when SBLW was used as a feedstock.
The activity of laccases reached the highest (0.00076 U mL-1 ± 3.5 × 10-5)in fermentations that employed 40% (v/v) SBLW. This represented 73% of that obtained using the standard synthetic medium. Supplementation of 40% (v/v) SBLW with 1.0% (w/v) yeast extract (YE) yielded enhancements of 1.2-fold and 1.5-fold of biomass concentration and laccase activity, respectively. The expression of laccases was further enhanced when the 40% (v/v) SBLW and 1.0% (w/v) YE was supplemented with 2.0% (v/v) glycerol. The highest laccase activity recorded was 0.00206 U mL-1 ± 5.8 × 10-5. Both of the biomass and laccase production were increased by 1.9-fold and 2.1-fold in comparison to that achieved by the standard synthetic medium. The efficacy of laccases produced by the optimised SBLW medium was evaluated in terms of the capability of the enzymes to decolourise Remazol Brilliant Blue R (RBBR) dye. The enzyme showed a decolourisation percentage of 68.6%, which represented 91% of the decolourising capability of laccases produced using standard BMMH medium. This indicates the promising efficacy of laccases produced from SBLW. In summary, this work gives an important insight into exploitation of SBLW for production of value-added products. Moreover, this work contributes to the development of recombinant laccase production using low cost and eco-friendly feedstock.
Keywords: Bioethanol spent broth, bioproducts, laccase, Pichia pastoris, sago
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Kebolehsanaan Sisa Bioetanol untuk Penghasilan Laccase dari Pichia pastoris ABSTRAK
Laccases adalah enzim serbaguna yang mempunyai aplikasi bioteknologi yang luas. Kajian ini bertujuan untuk menilai kebolehlaksanaan menggunakan kaldu fermentasi yang dihasilkan selepas penghasilan bioetanol dari sagu hampas sebagai bahan baku bagi produksi laccases di rekombinan Pichia pastoris GS115. Pencirian sisa cecair bioetanol dari sagu (SBLW) menunjukkan gliserol sebagai komponen utama, bersama dengan glukosa dan asid laktik. Penilaian SBLW sebagai bahan baku fermentasi untuk pengeluaran laccase di P. pastoris GS115 menunjukkan bahawa pertumbuhan sel umumnya dapat dilaksanakan ketika SBLW digunakan sebagai bahan baku. Aktiviti laccases mencapai tahap tertinggi (0.00076 U mL-1 ± 3.5 × 10-5) dalam fermentasi yang menggunakan 40% (v/v) SBLW. Ini mewakili 73% daripada yang diperoleh menggunakan media sintetik standard. Penambahan 40% (v/v) SBLW dengan 1.0% (w/v) ekstrak ragi (YE) menghasilkan peningkatan 1.2 kali ganda dan 1.5 kali ganda kepekatan biojisim dan aktiviti laccase. Ekspresi laccases diperbanyakkan lagi ketika 40% (v/v) SBLW dan YE 1.0% (w/v) ditambah dengan gliserol 2.0% (v/v). Aktiviti tertinggi laccase yang dicatatkan ialah 0.00206 U mL-1 ± 5.8 × 10-5. Kedua-dua pengeluaran biojisim dan laccase meningkat sebanyak 1.9 kali ganda dan 2.1 kali ganda berbanding dengan yang diperoleh oleh medium sintetik standard. Keberkesanan laccases yang dihasilkan oleh medium SBLW yang dioptimumkan dinilai dari segi kemampuan enzim untuk menyahwarnakan pewarna Remazol Brilliant Blue R (RBBR).
Enzim menunjukkan peratusan penyahwarnaan 68.6%, yang mewakili 91% dari kemampuan penyahwarnaan laccases yang dihasilkan menggunakan medium BMMH standard. Ini menunjukkan keberkesanan laccases yang menjanjikan yang dihasilkan dari SBLW. Ringkasnya, kajian ini memberikan gambaran penting mengenai eksploitasi SBLW
untuk pengeluaran produk bernilai tambah. Lebih-lebih lagi, kajian ini menyumbang kepada pengembangan penghasilan laccase rekombinan menggunakan bahan makanan kos rendah dan mesra alam.
Kata kunci: Bioproduk, cecair sisa bioetanol, laccase, Pichia pastoris, sagu
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TABLE OF CONTENTS
Page
DECLARATION i
ACKNOWLEDGEMENT ii
ABSTRACT iii
ABSTRAK v
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xv
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 4
2.1 Bioethanol 4
2.1.1 Applications of Bioethanol 5
2.1.2 Bioethanol Production 7
2.1.3 Utilisation of Bioethanol Waste 11
2.2 Laccases 13
2.2.1 Applications of Laccases in Bioremediation 16
2.2.2 Laccase Production 18
2.2.2.1Laccase Production by Different Substrates 18
2.2.2.2Laccase Production by Different Hosts 20 2.2.2.3Factors affecting optimum production of laccases by fungi 23 2.2.3 Remazol Brilliant Blue R (RBBR) Decolourisation Test 27
CHAPTER 3: MATERIALS AND METHODS 32
3.1 Microorganisms 32
3.2 Media 32
3.2.1 Sago Hampas 32
3.2.2 Media Preparation 33
3.2.2.1Yeast Malt Broth (YMB) 33
3.2.2.2Buffered Methanol Glycerol Histidine (BMGH) 33
3.2.2.3Buffered Minimal Methanol Histidine Medium (BMMH) 33
3.3 Enzymatic Hydrolysis of Sago Hampas 33
3.4 Bioethanol Fermentation 34
3.4.1 Preparation of Inoculum 34
3.4.2 Fermentation 34
3.4.3 Recovery of SBLW 35
3.5 Pichia pastoris GS115 Fermentation 35
3.5.1 Preparation of Inoculum 35
3.5.2 Fermentation 36
3.5.3 Optimisation of Laccase Production 37
ix
3.6 In vitro RBBR Decolourisation 37
3.7 Analytical Methods 38
3.7.1 Dry Cell Weight (DCW) Analysis 38
3.7.2 Measurement of Optical Density 39
3.7.3 Laccase Assay 39
3.7.4 High Performance Liquid Chromatography (HPLC) 40
3.7.5 Statistical Analysis 40
CHAPTER 4: RESULTS AND DISCUSSION 42
4.1 Bioethanol Fermentation Using SHH as a Medium 42
4.2 Characterisation of Sago Bioethanol Liquid Waste (SBLW) 43 4.3 Evaluation of SBLW as a Feedstock for Laccase Production in P. pastoris
GS115 46
4.3.1 Effect of Different Formulation of SBLW on Recombinant P. pastoris
GS115 Growth 46
4.3.2 Effect of Different Formulation of SBLW on Laccase Activity 50
4.4 Optimisation of Laccase Production Using SBLW 52
4.4.1 Effect of Supplementation of 40% SBLW with YE on P. pastoris GS115
Growth 52
4.4.2 Effect of Supplementation of 40% SBLW with YE on Laccase Activity 55 4.4.3 Effect of Supplementation of 40% SBLW with Peptone on P. pastoris
GS115 Growth 56
4.4.4 Effect of Supplementation of 40% SBLW with Peptone on Laccase Activity 58
4.4.5 Effect of Supplementation of 40% SBLW with 1.0% YE and Glycerol on
P. pastoris GS115 Growth 61
4.4.6 Effect of Supplementation of 40% SBLW with 1.0% YE and Glycerol
on Laccase Activity 65
4.5 Determination of the Efficacy of Laccases Produced from the
Optimised SBLW Medium 68
CHAPTER 5: CONCLUSION AND FUTURE RECOMMENDATIONS 71
5.1 Conclusion
5.2 Future Recommendations
REFERENCES APPENDICES
71 72 74 109
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LIST OF TABLES
Page Table 2.1 Applications of bioethanol in various sectors 6 Table 2.2 Summary of previous works that adopted RBBR decolourisation test
for evaluating the efficacy of laccases 28
Table 4.1 Composition of SBLW 44
Table 4.2 Kinetic parameters for P. pastoris GS115 growth when cultured for
BMMH and SBLW at different concentrations 49
Table 4.3 Kinetic parameter for P. pastoris GS115 growth when cultured on BMMH, unsupplemented 40% SBLW and 40% SBLW
supplemented with different concentrations of YE 54 Table 4.4 Kinetic parameters for P. pastoris GS115 growth when cultured on
BMMH, unsupplemented 40% SBLW and 40% SBLW supplemented
with different concentrations of peptone 58
Table 4.5 Kinetic parameters for P. pastoris GS115 growth when cultured on BMMH, 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and glycerol at
different concentrations 63
Table 4.6 Summary of the comparison of maximum biomass concentration and maximum laccase activity obtained from P. pastoris GS115
fermentations using different media 67
LIST OF FIGURES
Page Figure 2.1 Process of bioconversion of biomass to bioethanol 8 Figure 2.2 Ethanol fermentation pathway by S. cerevisiae 10
Figure 2.3 Catalytic mechanism of laccase enzyme 13
Figure 2.4 Potential uses of laccases for industrial and biotechnological purposes 16 Figure 2.5 Mycoremediation mechanism of RBBR in wastewater 29 Figure 3.1 Bioethanol fermentation broth after 48h of fermentation 35
Figure 3.2 P. pastoris GS115 grown on YPD agar 36
Figure 3.3 BMMH standard medium and 100% SBLW medium 37
Figure 3.4 General overview of the research project 41
Figure 4.1 Growth profile of S. Cerevisiae, glucose consumption and bioethanol production during bioethanol fermentation using sago hampas
hydrolysate as a feedstock 43
Figure 4.2 Comparison of batch fermentation kinetics of P. pastoris GS115
cultured using BMMH medium and SBLW at different concentratio 47 Figure 4.3 The P. pastoris GS115 culture when cultivated using BMMH medium
on (a) day 0;(b) day 8. The P. pastoris GS115 culture when cultivated
using 40% (v/v) SBLW medium on (c) day 0; (d) day 8 48 Figure 4.4 The ABTS assay consisting of crude laccases produced on day 8 from
fermentations using (a) BMMH medium and (b) 40% (v/v) SBLW 50 Figure 4.5 Comparison of laccase activity from P. pastoris GS115 fermentation
susing BMMH and SBLW at different concentrations 52
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Figure 4.6 Comparison of batch fermentation kinetics of P. pastoris GS115 when cultured using BMMH and 40% SBLW supplemented with YE at
different concentrations 53
Figure 4.7 Comparison of laccase activity from P. pastoris GS115 fermentations using BMMH, unsupplemented 40% SBLW and 40% SBLW
supplemented with YE at different concentrations 56 Figure 4.8 Comparison of batch fermentation kinetics of P. pastoris GS115 when
cultured using BMMH, unsupplemented 40% SBLW and 40% SBLW
supplemented with peptone at different concentrations 57 Figure 4.9 Comparison of laccase activity from P. pastoris GS115 fermentations
using BMMH, unsupplemented 40% SBLW and 40% SBLW
supplemented with peptone at different concentrations 60 Figure 4.10 Comparison of batch fermentation kinetics of P. pastoris GS115 when
cultured in BMMH, unsupplemented 40% (v/v) SBLW, 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and glycerol at different
concentrations 61
Figure 4.11 Comparison of laccase activity of P. pastoris GS115 when cultured in BMMH, unsupplemented 40% (v/v) SBLW, 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and 40% (v/v) SBLW supplemented with 1.0% (w/v) YE and glycerol at different
concentrations 66
Figure 4.12 The absorbance spectrum of RBBR dye before and after the decolourisation by crude laccases obtained from fermentations using
BMMH and optimised SBLW medium 68
Figure 4.13 The RBBR decolourisation test using crude laccases obtained from fermentations using BMMH and optimised SBLW medium on (a) day
1 and (b) day 15 69
xv
LIST OF ABBREVIATIONS
ABTS BMGH BMMH BOD CO2
cm COD Cu
°C DCW gdcw L-1 g
g L-1 HPLC H HMF IU g-1 IU mL-1
2,2’-azinobis-(3-ethylbenzthiazoline 6-sulfonic acid) Buffered minimal glycerol histidine medium
Buffered minimal methanol histidine medium Biochemical oxygen demand
Carbon dioxide Centimeter
Chemical oxygen demand Copper atom
Degree celcius Dry cell weight
Dry cell weight in gram per liter Gram
Gram per liter
High Performance Liquid Chromatography Hour
Hydroxymethylfurfural International units per gram International units per milliliter
L µL µmol µ (h-1) mg L-1 mL mL min-1 mm mM min M Nm n OD h-1 min-1 M-1 cm-1
% PDA
Liter Microliter Micromole Micro per hour Milligram per liter Milliliter
Milliliter per minute Millimeter
Millimolar Minutes Molar Nanometer
Number of variable Optical density Per hour Per minute
Per molar per centimeter Percentage
Potato dextrose agar
xvii RBBR
rpm SBLW SHH SSF TOC U U g-1 U mL-1 U L-1 v/v w/v YE YMB YNB YPD
Remazol Brilliant Blue R Revolution per minute Sago bioethanol liquid waste Sago hampas hydrolysate Solid state fermentation Total organic compound Unit
Unit per gram Unit per milliliter Unit per liter
Volume per volume Weight per volume Yeast extract Yeast malt broth Yeast nitrogen base
Yeast extract peptone dextrose
CHAPTER 1
INTRODUCTION
1.1 Introduction
Exploitation of agricultural waste as alternative feedstocks for bioethanol production has gained increasing interests among biorefineries. One of the potential agricultural wastes in Malaysia is sago fibre, which composed of approximately 50% to 60% of residual starch along with other lignocellulosic components (Mohammad et al., 2020). The use of hydrolysate of sago fibre as a substrate for producing bioethanol has been reported by Awang-Adeni and co-workers (Mohammad et al., 2020).
Given a far-sighted context, with the increasing demand for bioethanol at the industrial level in the future, it is estimated that the production of the waste stream following bioethanol distillation will also increase. Direct disposal of the waste stream may lead to serious environmental pollutions due to the high organic content, dissolved solids and other toxic compounds (Espana-Gamboa et al., 2011). Therefore, there is an emerging need to investigate the fate and potential of the waste stream generated following the bioethanol production.
Research in Brazil, India and Europe have shown the feasibility of exploiting vinasse, the glycerol-based stillage discharged after the industrial production of bioethanol using molasses derived either from starch crops (wheat and cassava), sugar crops (beet and sugarcane) or cellulosic material (harvesting crop residues and wood) (Christofoletti et al., 2013), as feedstocks for production of several products such as polyhydroxyalkanoates (Bhattacharyya et al., 2012; Pramanik et al., 2012) volatile fatty acids and biohydrogen
2
Similar potential could also be envisaged in the case of the stillage generated from the bioethanol production using sago hampas as a substrate. To date, the exploitation of the waste stream generated following the production of bioethanol from sago fibre are poorly explored. Thus, a fundamental investigation into properties and applications of the waste stream will shed some light on its rational fate and direction.
One of the industrially relevant enzymes laccases, were used as the model case throughout this study. Laccases are polyphenol oxidases that are widely used as bioremediators for treating wastewater generated from paper and pulp, petrochemical and textile industries (Chandra & Chowdhary, 2015), bioremediation of xenobiotics and as biosensors in medical diagnostics (El-Batal et al., 2015). The demand for laccases for industrial purposes has been rapidly increased, hence necessitates the development of recombinant laccase production. Previously, a laccase producing strain, P. pastoris GS115 was established by Sing (2017). The laccase gene from the fungus Marasmius cladophyllus UMAS MS8 was expressed heterologously in P. pastoris GS115 with the aim of getting an increased level of laccase enzyme expression as compared with the native fungus M.
cladophyllus (Sing, 2017). Nonetheless, development of strategies for optimal and inexpensive production of laccases by P. pastoris GS115 is still under-examined since the strain is relatively new.
Previous studies of laccase production were based on a synthetic medium, Buffered Methanol-Complex Medium (BMMH) (Maestre-Reyna et al., 2015), which is fossil-based and often deemed as expensive. Hence, there is a need for developing a cost-effective and sustainable strategy for producing laccases in P. pastoris GS115. Then, the efficacy of laccases produced in P. pastoris GS115 was evaluated in terms of the capability of the enzymes to decolourise Remazol Brilliant Blue R (RBBR). RBBR dye is an example of an
anthraquinone dye, which has a structural resemblance to some polycyclic aromatic hydrocarbons (Bohacz, 2020). The decolourisation of RBBR dye by ligninolytic enzymes of white rot fungi such as laccases have been widely reported (Adak et al., 2016; Syafiuddin &
Fulazzaky, 2021), and the RBBR dye was decolourised the fastest as compared to the other dyes (Sing et al., 2013; Noman et al., 2020).
The main aim of this study is to explore the feasibility of liquid waste generated after the production of bioethanol from sago hampas, as a fermentation feedstock for recombinant laccase production in P. pastoris GS115. The specific objectives of this research are:
i. To characterise the composition of liquid waste generated upon the production of bioethanol from sago hampas with particular emphasis on its fermentable sugars and inhibitory compounds;
ii. To investigate the feasibility of sago bioethanol liquid waste as a fermentation feedstock for producing recombinant laccases in P. pastoris GS115; and iii. To optimise the production of recombinant laccases in P. pastoris GS115
using sago bioethanol liquid waste (SBLW) as a feedstock.
4 CHAPTER 2
LITERATURE REVIEW
2.1 Bioethanol
Bioethanol is an alcohol produced by microbial fermentation. According to Cripwell et al. (2020), bioethanol is commonly produced from sugary and starchy crops such as corn and sugarcane. It is clear and colourless liquid, biodegradable and has low toxicity. Due to the huge demand worldwide, the global production of bioethanol is rapidly increasing. The world bioethanol production was increased about 23% from 90 billion L in 2012, to 117 billion L in 2014 and is expected to reach approximately 158 billion L by 2023 (Toor et al., 2020).
According to Lisin (2020) more than 70% of the ethanol production in the world is produced by Brazil and the United States, which are known as two major ethanol producers.
Both countries have started mass production of bioethanol derived from sugarcane and corn since early 1970s. There is a rising emphasis in the production of bioethanol from lignocellulosic biomass for the past few years. More cellulosic ethanol will be eventually produced from various types of feedstocks, but intensive research are needed to reduce the cost production of ethanol to make them more economical (Chandel et al., 2019). The bioethanol production can be minimised if renewable substrates are utilised. Thus, the use of biomass as substrates for bioethanol production is seen as an interesting option.
Some developing countries are able to reduce the notable amount of oil imported and replace their existing fossil-based fuel by producing bioethanol (Yusoff et al., 2021).
Bioethanol is widely known as an alternative energy source with high potential in automotive applications since the natural energy resources such as fossil fuel, petroleum and coal are
estimated to deplete within the next 40 - 50 years (Abbas et al., 2020). Besides, bioethanol result in improved vehicle performance, reduce the consumption of the fossil fuels, improve air quality and less toxicity (Du et al., 2016).
2.1.1 Applications of bioethanol
Bioethanol is widely used as liquid biofuel in power automobiles in which the application is about 75% of the total bioethanol produced in the world. It is currently used as the major additive for gasoline in automobile industries, and these combination helps to reduce the hydrocarbon and carbon monoxide emissions from vehicles (Jhang et al., 2020).
The combination of ethanol and gasoline can be at any concentration up to 100% ethanol.
This has vastly minimised the consumption of petroleum fuel. Air pollution can also be reduced too as bioethanol is generally CO2 neutral. Moreover, bioethanol is also used in other sectors such as in the chemical, transportation and pharmaceuticals industry. Table 2.1 outlines the summary of applications of bioethanol in different sectors.
