UNIVERSITI PUTRA MALAYSIA

ENVIRONMENTAL FRIENDLY ALTERNATIVE METHODS FOR THE RECOVERY OF INTRACELLULAR POLYHYDROXYALKANOATES

(PHA)

VOO PHOOI TEE.

FBSB 2005 28

ENVIRONMENTAL FRIENDLY ALTERNATIVE METHODS FOR THE RECOVERY OF INTRACELLULAR

POLYHYDROXYALKANOATES (PHA)

VOON PHOOI TEE

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

May 2005

SPECIALLY DEDICATED TO MY SON

LIM KEN JI

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

ENVIRONMENTAL FRIENDLY ALTERNATIVE METHODS FOR THE RECOVERY OF INTRACELLULAR POLYHYDROXYALKANOATES

VOON PHOOI TEE

May 2005

Chairman: Professor Mohd. Ali Hassan, PhD

Faculty: Biotechnology and Biomolecular Sciences

Polyhydroxyalkanoates (PHA) are intracellular polymers that can be produced by

bacteria as energy reserve material. This biodegradable material has properties

similar to synthetic thermoplastics. However, the process recovery of PHA using

organic solvents such as chloroform is expensive and not environmental-friendly.

Although most of the organic solvent is recovered for reuse, it still causes serious

damage to health and environment. Thus, alternative methods that are

environmental-friendly are needed for the recovery of PHA. Fermentation was

carried out using a 50 L bioreactor at pH 7, 30°C, with agitation speed of 200 rpm

and 1 vvm aeration rate to maintain aerobic condition. Ralstonia eutropha ATCC

17699 was chosen as the PHA production bacteria. Twenty g/L mixture of acetic and

propionic acids were fed into the broth as carbon sources and PHA was produced in

nitrogen limited condition with C/N = 50. Cells were harvested by centrifugation and

the pellets were then dried in oven at 60°C, grinded and stored at 4OC for recovery of

PHA.

Biomass containing PHA with the concentration of 0.32 glg biomass was treated

with various chemicals such as alkaline solutions (NaOH, KOH), surfactants

(sodium dodecyl sulfate or SDS, sodium salt of a-sulfonate methyl esters derived

from palm stearin or a-SMEPS, Tween 20, Tween 80 and betaine anhydrous) and

enzyme (lysozyme) to digest non-PHA cellular material (NPCM) at dried cells

concentration of 5 g/L. Mechanical methods such as ultrasonic sonication and

homogenization were also used for further cell disruption. Combined treatment of

alkali and homogenization was also investigated. After treatment, PHA granules

were separated from cell debris by centrifugation at 3500 rpm for 10 min. PHA

granules recovered were rinsed twice with deionized water to avoid floatation,

centrifuged and air-dried. Pellet was analyzed by using HPLC and supernatant was

analyzed by the presence of protein.

Combined treatment of NaOH and homogenization were found to give the highest

PHA purity and yield of 97% and 94%, respectively, compared to other methods.

The purity of the final PHA increased with the released of cellular protein. PHA

could be recovered from biomass by combined NaOH pretreatment, (0.2 M, 60 min)

and homogenization (18 min) to achieve cell disruption. This method is simple,

economical, environmental friendly, non-toxic and suitable for larger scale

production. The product obtained was white in colour and ready to be accepted by

end user for commercialization. Thus, combined NaOH treatment and

homogenization can replace chloroform for the recovery of PHA.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

KAEDAH-KAEDAH ALTERNATIF YANG MESRA ALAM UNTUK PEMULIHAN INTRASELULAR POLIHIDROKSIALKANOAT

Oleh

VOON PHOOI TEE

Mei 2005

Pengerusi:

Fakulti:

Profesor Mohd. Ali Hassan, PhD

Bioteknologi dan Sains Biomolekul

Polihidroksialkanoat (PHA) merupakan polimer intraselular yang boleh dihasilkan

oleh bakteria sebagai bahan tenaga simpanan. Bahan yang boleh dibiodegradasi ini

mempunyai sifat yang sama dengan termoplastik sintetik. Walau bagaimanapun,

proses pemulihan PHA dengan menggunakan pelarut organik seperti kloroform

adalah mahal dan tidak mesra alam. Walaupun kebanyakan pelarut organik dipulih

kembali dan digunakan semula, ia masih merbahayakan kesihatan dan alam sekitar.

Oleh demikian, kaedah-kaedah alternatif yang mesra alam amat diperlukan untuk

pemulihan PHA. Fermentasi telah dijalankan dengan menggunakan 50 L bioreaktor

pada pH 7, 30°C dengan laju pengacauan sebanyak 200 rpm dan 1 vvm kadar

pengudaraan untuk mengekalkan keadaan yang aerobik. Ralstonia eutropha ATCC

17699 telah dipilih sebagai bakteria penghasilan PHA. Dua puluh gl L asid asetik

dan asid propionik ditambahkan ke dalam medium sebagai sumber karbon dan PHA

dihasilkan dalam keadaan sumber nitrogen terhad dengan nisbah karbonlnitrogen

sebanyak 50.

Sel-sel bacteria selepas ferrnentasi dikumpulkan dengan teknik pengemparan

kemudian pelet dikeringkan dalam oven pada 60°C, dikisar halus dan disimpan pada

4OC untuk proses pemulihan PHA.

Sel yang mengandungi PHA dengan kepekatan 0.32 g/g sel dirawat dengan pelbagai

bahan kimia seperti larutan beralkali (NaOH, KOH), surfaktan-surfaktan (Natrium

dodesil sulfat atau SDS, garam natrium a-sulfonat metil ester yang diperolehi

daripada stearin sawit atau a--SMPES, Tween 20, Tween 80 dan betain anhidrat)

dan enzim (lisozim) untuk menguraikan bahan selular bukan-PHA (NPCM) pada

kepekatan sel kering 5 g/L. Kaedah-kaedah mekanikal seperti sonikasi ultrasonik dan

penghomogenan juga digunakan untuk pemusnahan sel selanjutnya. Rawatan

kombinasi alkali dan penghomogenan juga diselidik. Selepas rawatan, granul-granul

PHA dipisahkan dari baki sel dengan pengemparan pada 3500 rpm selama 10 min.

Granul-granul PHA yang didapati dibilas dua kali dengan air-nyahion untuk

mengelakkan pengapungan, diempar dan dikeringkan dalam udara. Pelet dianalisis

dengan menggunakan HPLC dan supernatan yang mengandungi protein dianalisis

dengan kehadiran protein.

Kaedah kombinasi rawatan NaOH dan penghomogenan didapati memberi ketulenan

PHA yang tertinggi (97%) dengan hasil sebanyak 94% berbanding dengan kaedah-

kaedah lain yang digunakan. Ketulenan PHA akhir bertambah dengan pembebasan

selular protein. PHA boleh dipulihkan dari sel dengan kaedah pra-rawatan dengan

NaOH (0.2 M, 60 min) dan penghomogenan (18 min) untuk mencapai pemusnahan

sel. Kaedah ini adalah ringkas, ekonomi, mesra-alam, tidak

bertoksik dan sesuai untuk penghasilan secara besar-besaran. Produk yang didapati

adalah benvarna putih dan sedia diterima oleh pengguna-akhir untuk

dikomersialkan. Oleh demikian, kombinasi rawatan NaOH dan penghomogenan

boleh menggantikan kloroform bagi pemulihan PHA.

vii

ACKNOWLEGEMENTS

I wish to express my deepest appreciation and sincere gratitude to my supervisor,

Prof. Dr. Mohd. Ali Hassan and members of the supervisory committee Prof. Dr.

Mohamed Ismail Abdul Karim and Associate Prof. Badlishah Sham Baharin, for

their invaluable guidance, comments and suggestions throughout my study. A

special thanks to Prof. Dr. Yoshihito Shirai (Kyushu Institute of Technology, Iizuka,

Fukuoka, Japan) for his advice, guidance, help and technical support from time to

time. To Dr. Raha, thank you for helping to purchase ATCC 17699.

To my senior laboratory members: Dr. Phang Lai Yee, Dr. Nor'aini, Mr.

Shahrakbah, Sim Kean Hong, Cheong Weng Chung, Wong Kok Mun, Zaizuhana

and friends, Ooi Kim Yng, Rafein, Cyril and Munir, who always share their views

and comments on my project. Special thanks also dedicated to laboratory staff, Mr.

Rosli Aslim, Mrs. Renuga alp Panjamurti and Mrs. Aluyah Marzuki, thank you for

your help and cooperation.

To my grandparent, your support is always in my mind. Also to my parent and

sisters for your help and understanding which encouraged me to continue my study.

Acknowledgement is also dedicated to those who involved directly or indirectly in

the completion of this study. Last but not least, to my dear, Lim W.H., thank you for

sharing your love and happiness throughout my study.

. . . V l l l

I certify that an Examination Committee met on 1 9 ~ May 2005 to conduct the final examination of Voon Phooi Tee on her Master of Science thesis entitled "Environmental Friendly Alternative Methods for the Recovery of Intracellular Polyhydroxyalkanoates" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

ARBAKARIYA ARIFF, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

SURAINI ABD. AZIZ, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

RUSSLY ABDUL RAHMAN, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner)

VIKINESWARY SABARATNAM, PhD Professor Faculty of Science Universiti Malaya (External Examiner)

Profess GuLy r/D&ufi s Dean School bf Graduate Studies Universiti Putra Malaysia

Date: 2 1 JUL 2005

This Thesis submitted to the Senate of Universiti Putra Malaysia and was accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows:

MOHD. ALI HASSAN, PhD Professor Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Chairman)

MOHAMED ISMAIL ABDUL KARIM, PhD Professor Faculty of Engineering Universiti Islam Antarabangsa Malaysia (Member)

BADLISHAH SHAM BAHARIN Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor 1 Dean School of Graduate Studies Universiti Putra Malaysia

Date: 1 1 AUG 2005

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS

CHAPTER

I INTRODUCTION

LITERATURE REVIEW 2.1 Polyhydroxyalkanoates (PHA) and Market Potential 2.2 Physiology and Biochemistry of PHA Synthesis

2.2.1 Pathway of Polyhyroxybutyrate (PHB) Synthesis 2.2.2 Polyhydroxy(butyrate-co-valerate) (PHB-co-HV)

Copolymer Synthesis Production of PHA PHA Properties 2.4.1 Biodegradation of PHA Application and Prospects of PHA Conventional Methods of Recovery and Encountered Problems Cells Disruption Techniques 2.7.1 Microbial Cell Wall 2.7.2 Methods of Cells Disruption

2.7.2.1 Ultrasonication 2.7.2.2 Homogenization 2.7.2.3 Chemical Permeabilization 2.7.2.4 Enzymatic Disruption 2.7.2.5 The Combination of Mechanical

and Chemical Processes

i i . . . 111

v ... V l l l

ix xi xvi xvii xix xxi

xii

GENERAL MATERIALS AND METHODS 3.1 Chemical Reagents 3.2 Experimental Design 3.3 Microorganisms and Preparation 3.4 Preparation of Production Medium 3.5 Preparation of Inoculum for PHA Fermentation 3.6 Production of PHA by Fed-Batch Fermentation 3.7 Harvest and Storage of Biomass Containing PHA 3.8 Recovery of PHA

3.8.1 Chemical Methods PHA Recovery by Chloroform Extraction and Hexane Precipitation PHA Recovery by Simple Digestion Using Various Chemical

3.8.1 Biological Methods 3.8.2 Mechanical Methods

Single Sonication Single Homogenization

3.8.3 Combined NaOH Treatment and Homogenization Method

3.9 Analytical Methods 3.9.1 Organic Acids Determination 3.9.2 PHA Determination 3.9.3 Cell Dried Weight (DCW) 3.9.4 Ammoniacal Nitrogen (AN) 3.9.5 Samples Preparation for

Scanning Electron Microscopic (SEM) 3.9.6 Determination of Protein Released

Lowry Method (195 1) 3.9.7 Examination for Contamination from

Fermentation Broth Negative-Gram Stainning

3.10 Statistical Analysis

PRODUCTION OF POLYHYDROXYALKANOATES (PHA) FROM ORGANIC ACIDS BY FED-BATCH FERMENTATION 4.1 Introduction 4.2 Materials and Methods 4.3 Results and Discussion

4.3.1 Examination for Contamination from Fermentation Broth

4.3.2 PHA Production from Synthetic Organic Acids 4.3.3 Effect of Cells Drying on Recovery

4.4 Conclusion

. . . Xll l

ALTERNATIVE METHODS FOR THE RECOVERY OF PHA 75 5.1 Introduction 75 5.2 Materials and Methods 77 5.3 Results 77

Effect of Various Chemical Treatment for the Recovery of PHA

PHA Recovery by Chloroform Extraction and Hexane Precipitation

Digestion of NPCM with Various Surfactants

Effect of SDS Concentration for the Recovery of Intracellular PHA

Effect of 8 mM SDS Treatment Time for the Recovery of Intracellular PHA

Effect of Alkali Treatment for the Recovery of Intracellular PHA

Effect of Biological Enzyme for the Recovery of PHA

Effect of Mechanical Methods for the Recovery of PHA

Effect of Single Ultrasonic Sonication for the Recovery of Intracellular PHA

Effect of Single Homogenization for the Recovery of Intracellular PHA

Combined NaOH Treatment and Mechanical Disruption

Effect of Combined 0.2 M NaOH and Homogenization Method for the Recovery of Intracellular PHA

Effect of Various Recovery Methods to the Protein Released from Biomass Containing PHA

Effect of Various Recovery Methods to the Purity of PHA Obtained 95

Effect of Various Recovery Methods to the PHA Yield

xiv

5.4 Discussion 5.5 Morphology Changes and Product Outlook 5.6 Conclusion

CONCLUSIONS AND SUGGESTION FOR FUTURE WORK 112 6.1 Conclusions 112 6.2 Suggestion for Future Works 114

REFERENCES APPENDICES BIODATA OF THE AUTHOR

LIST OF TABLES

Table Page

Occurrence of poly-l3-hydroxybutyrate (PHB)

in microorganism species

Polymer property comparison: PHB and PHBIHV

compared with conventional plastics ( Bryom, 1994)

Application for PHB and other PHA

Methods of chemical permeabilization

Compositions of growth medium (GM)

Medium compositions for production of PHA

using R. eutropha ATCC 17699

Trace elements compositions

The Effect of various recovery methods for the recovery of

intracellular PHA with maximum purity (%), yield (%) and

protein released (glg biomass) obtained after certain

treatment time

xvi

LIST OF FIGURES

Figure

General structure of monomers and PHA polymers

Cyclic metabolic pathway of the biosynthesis and

degradation of P(3HB)

Copolymer synthesis from glucose and propionate

Physical state of PHA

Methods of microbial cell disruption

Diagram of general experimental design

Cell dried weight (CDW), PHA concentration and PHA content

profiles of fed-batch fermentation using synthetic organic acids 68

Acetic, propionic acids and ammoniacal nitrogen (AN) profiles

of fed-batch fermentation using synthetic organic acids 70

Effect of various surfactants for the recovery of intracellular PHA 80

Effect of SDS concentration for the recovery of intracellular PHA 82

Effect of 8 mM SDS treatment time for the recovery of

intracellular PHA

Effect of NaOH and KOH concentration for the recovery of

intracellular PHA

Effect of 0.5 M NaOH treatment time for the recovery of

intracellular PHA

Effect of lysozyme treatment time for the recovery of

intracellular PHA

Page

5

xvii

5.7 Effect of single sonication for the recovery of intracellular PHA 89

Effect of single homogenization for the recovery of

intracellular PHA

5.9 Effect of combined treatment of 0.2 M NaOH treatment and

homogenization method for the recovery of intracellular PHA 92

5.10 Effect of various recovery methods to the protein released

from biomass containing PHA 94

5.1 1 Effect of various recovery methods to the purity of PHA obtained 95

5.12 Effect of various recovery methods to the PHA yield 9 6

xviii

LIST OF PLATES

Plate

2.1 Electron microscope view of the accumulation of polymer

granules, PHA in cells of the species R. eutropha

3.1 BIOSTAT U (B-Braun) for the production of PHA

in fed-batch culture

3.2 Ultrasonicator with super sonabox

3.3 Probe for ultrasonicator

3.4 Rotor-stator homogenizer

3.5 Digested sample containing PHA with concentrated H2SO4

3.6 Diluted sample from digested sample before subjected to

HPLC analysis (Dilution: 50 X)

4.1 R. eutropha ATCC 17699 from fermentation broth

4.2 Fine cells after grinded containing PHA

5.1 Dried biomass containing PHA

5.2 PHA obtained from chloroform extraction and

hexane precipitation

5.3 PHA film obtained from PHA granules

5.4 PHA granules obtained from SDS treatment

5.5 PHA obtained from combined 0.2 M NaOH treatment and

homogenization method after air-dried in centrifuge tube

5.6 Ground PHA granules obtained from combined 0.2 M NaOH

reatment and homogenization method with the purity of 97%

5.7 PHA standard

Page

xix

5.8 Dry biomass containing PHA

5.9 PHA obtained by chloroform extraction and hexane precipitation 106

5.10 PHA obtained by combined treatment of 0.2 M NaOH

and homogenization

5.11 PHA obtained by combined treatment of 0.2 M NaOH

and homogenization with 97% purity

5.12 Plastic film obtained from PHA granules with 97% purity

LIST OF ABBREVIATIONS

AN

C

CDW

CMC

Da

g

g/L

GC

GCMS

GM

h

HB

HPLC

HV

L

M

min

mL

nm

NPCM

"C

OD

Yo

P

P(3HB-CO-3HV)

PHA

PHB

PHV

POME

- Ammoniacal nitrogen

- Carbon

- Cell dried weight

- Critical micelle concentration

- Dalton

- Gram

- Gram per litre

- Gas chromatography

- Gas chromatography mass spectrometry

- Growth medium

- Hour

- Hydroxybutyrate

- High performance liquid chromatography

- Hydroxyvalerate

- Litre

- Molar

- Minute

- Millilitre

- Number of replication

- Nanometer

- Non-PHA-cellular materials

- Degree Celsius

- Optical density

- Percent

- Phosphorous

- Poly(3 hydroxybutyrate-co-3 hydroxyvalerate)

- Polyhydroxyalkanoate

- Polyhydroxybutyrate

- Polyhydroxyvalerate

- Palm oil mill effluent

rpm

S

S

sd

Ulmg

v/v

VFA

vvm

wlv

- Rotation per minute

- Sulphur

- Second

- Standard deviation

- Unit per miligram

- Volume per volume

- Volatile fatty acids

Volume of air per volume of liquid per minute

Weight per volume

xxii

CHAPTER 1

INTRODUCTION

Plastics are commonly in used ranging from manufacturing industry to household

products. It is a synthetic polymer with molecular weight ranges 50 -1000 KDa

which can be chemically manipulated to have a wide range of strengths and shapes.

The synthetic thermoplastic such as polypropylene, polyethylene, polyvinyl chloride

and polystyrene can be easily molded into any desired shapes including fibers and

thin films which is highly chemical resistance and popular in many durable.

disposable goods and packaging materials. Although non-biodegradable synthetic

plastics are useful, however owing to its recalcitrant property resulted in high land

requirement for disposal in the landfill. While incineration may pollute the

environment by releasing harmful chemicals such as hydrogen cyanide and hydrogen

chloride are released during incineration (Reddy et al., 2003). Moreover, the use of

non-renewable resources as a basic for the synthesis of petrochemical-based-plastics

awaken the public concern that an alternative material which is more environmental

friendly is required to replace the conventional plastic due to oil reserve depletion.

Polyhydroxyalkanoates (PHA) is a biodegradable, biocompatible, microbial

thermoplastic which has potential to replace petroleum-derived thermoplastics (Sei

et al., 1994; de Koning et al., 1997). The molecular weight of PHA is in the range of

50-1000 KDa that have polymer characteristics that are similar to conventional

plastics such as polypropylene (Reddy et al., 2003). Moreover, PHA are produced

from a large variety of renewable resources (sucrose, starch, cellulose,

triacylglycerols), fossil resources (methane, mineral oil, lignite and hard coal),

byproducts (molasses, whey, glycerol), chemicals (acetic acid, propionic acid,

butyric acid) and carbon dioxide.

However, the used of biologically produced polymers is currently limited because of

high production costs. P(3HB-co-3HV), a copolymer of 3-hydroxybutyrate (3-HB)

with 3-hydroxyvalerate (3-HV) is produced as B I O P O L ~ ~ at U S $ ~ K ~ - ' (Lee, 1996a)

which is more expensive than polypropylene ( U S $ ~ K ~ - ' ) and not popular among

consumer even though it is biodegradable. Significant contributors to the cost of

production are the productivity of PHAs by the chosen bacteria strain, carbon source

and downstream processing. The commercial biopol recovery process involved

thermal treatment, enzyme and surfactant to rupture and solubilize all cell

components apart from Ralstonia eutropha in order to recover PHB (Sei et al.,

1994). These methods are efficient but required additional digestion or solvent

extraction steps to increase the product purity rendering the recovery cost higher.

The difficulty of PHB recovery from microorganisms has been the primary obstacle

to its commercial exploitation. The majority of separation processes that had been

carried out involved the extraction of PHB from the cells with solvents or

chlorinated based chemicals which is expensive, not environmental friendly and

toxicated. For example, PHB can be extracted from bacterial cells using methylene

chloride, propylene carbonate, dichloroethane or chloroform. The polymer solution