UNIVERSITI PUTRA MALAYSIA ENVIRONMENTAL FRIENDLY ALTERNATIVE METHODS FOR THE RECOVERY OF INTRACELLULAR POLYHYDROXYALKANOATES (PHA) VOO PHOOI TEE. FBSB 2005 28
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
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