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ISOLATION AND IDENTIFICATION OF POLYHYDROXYALKANOATE BIOPOLYMERS PRODUCING PSEUDOMONADS FROM DIFFERENT SOURCES AND OPTIMIZING CONDITION FOR MAXIMUM BIOPOLYMER PRODUCTION USING OILY HYDROCARBON COMPOUNDS

تعداد97 صفحه در فایل word

Ph.D. Thesis in

Bacteriology

ISOLATION AND IDENTIFICATION OF POLYHYDROXYALKANOATE BIOPOLYMERS PRODUCING PSEUDOMONADS FROM DIFFERENT SOURCES AND OPTIMIZING CONDITION FOR
MAXIMUM BIOPOLYMER PRODUCTION USING OILY HYDROCARBON COMPOUNDS

Abstract

Bacteria are diverse and abundant in soils, but only a few bacteria have known to growth on hydrocarbon contaminated areas and utilize complex carbon source such as crude oil for the synthesis of Polyhydroxyalkanoate (PHA) that giving them both bioremediation potential and the ability to produce important biopolymers. Bioremediation is the conversion of chemical compounds by living organisms, especially microorganisms, into energy, cell mass and biological waste products. Some of the most important of these bioremediation products are PHAs, which are a family of biopolymers produced by some bacteria and accumulated intracellularly as carbon and energy storage material. The main objective of this study was to isolate and characterize PHAs-producing bacteria in petroleum hydrocarbon-contaminated areas from different regions of Iranian South-Western refineries. Also, the ability of isolates to produce PHAs from crude oil as the sole carbon source was studied and then optimizes the culture condition such as incubation time, temperature, culture media and carbon sources for maximum growth and PHA production. From 45 isolated Pseudomonads, Fifteen PHA-producing strains were identified and their morphological, physiological, genomic, and 16S rRNA gene sequence properties were studied. Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 2% (v/v) Gachsaran crude oil. The repeated monomer composition of the copolymer was determined by GC–MS. Approximately 1/3 of the isolates were able to produce PHA using petroleum as a carbon source and produced biopolymer composites contained monomers of: C8 (3-hydroxyoctanoate), C10 (3- hydroxydecanoate), C12 (2-hydroxydodecanoate), C14 (3-hydroxytetradecanoate) and C16 (3- hydroxydecahexanoate) which are known as biopolymers. Finally, T=30 ^oC, incubation time=7day, PHA production media and 2% (v/v) crude oil were selected as the optimum conditions for highest bacterial growth and PHA accumulation. Under the optimal conditions, the highest bacterial growth (OD 600nm) and PHA content (w/w %) were 0.35±0.05 and 22.25±0.13, respectively. This study indicates that stressed environments like oil-contaminated sites can be potential sources for PHA producers and these isolates could be used in future bioremediation of hydrocarbons.

Key words: Polyhydroxyalkanoate, Pseudomonads, Bioremediation

دسته: زبان خارجه, زبان خارجه برچسب: Bioremediation, Key words: Polyhydroxyalkanoate, Pseudomonads

توضیحات

Table of Content

Content Page

Chapter One: Introduction

1-Bioplastics from microorganisms. 2

1-1- Biomaterials and bioplastics. 2

1-2- Monomers of bacterial plastics synthesized by microorganisms. 3

1-3-Polymerization of the bacterial plastics. 4

1-4-Applications of the bacterial plastics. 4

2- Polyhydroxyalkanoate (PHA) 5

2-1- Occurrence and diversity. 5

2-2- Structure and composition of PHA granules. 8

2-3- PHA biosynthesis and genes involved. 10

2- 4- Regulation of PHA metabolism.. 14

2- 5- Isolation of PHAs. 16

2-6- PHA producer in Soil 19

2-7- Potential applications of PHA.. 20

2-7-1- PHA as alternative plastic (“bulk” applications) 20

2- 7- 2- PHA granules as beads. 22

3- Pseudomonas. 26

3-1- overview.. 26

3-2- PHA production by Pseudomonas. 27

4- Aims and objectives. 28

Chapter Two: Materials and Methods

2-1- Bacterial detection and identification. 31

2-1-1- Sample collection. 31

2-1-2- Reference strains. 33

Content Page

2-1-3- Isolation of bacterial strains. 33

2-1- 4- Molecular identification. 33

2-2- PHA production assay. 35

2-2-1- Screening of PHAs producing microorganisms. 35

2- 2- 2- Quantitative analysis. 36

2-2-3- PA characterization. 37

2-3- Optimization of culture conditions for cell growth and PHA
accumulation. 37

2-3-1- Culture media. 37

2-3-2- Incubation time and temperature. 39

2-3-3- Carbon source (crude oil) concentration. 39

2-4- Statistical analysis. 39

Chapter Three: Results

3-1- Primary screening of bacteria for PHA production. 42

3-2- Quantitative analysis of the PHA produced by Pseudomonas isolates. 45

3-3– PHA characterization. 48

3-4- Optimization of culture conditions for cell growth and PHA
accumulation. 55

3-4-1- Culture media. 55

3-4-2- Incubation time and temperature. 56

3-4-3- Carbon source (crude oil) concentration. 57

Chapter Four: Discussion

4-1- PHA production by pseudomonads isolated from contaminated areas. 60

4-2- Primary screening of bacteria for PHA production. 62

4-3- Quantitative analysis. 63

4-4- Optimization of culture conditions for cell growth and PHA
accumulation. 63

4-4-1- Culture media. 63

4-4-2- Incubation time and temperature. 64

Content Page

4-4-3- Carbon source (crude oil) concentration. 64

4-5- PHA characterization. 65

4-6- conclusion. 66

4-7- Comments. 67

Reference. 68

List of Tables

Table Page

Table 1- 1. Overview of the four classes of PHA synthases. 13

Table 2- 1. Geographic location and type of contamination prevalent in different
sampling sites. 32

Table 2-2. Pseudomonas genus – specific primers PA – GS (16S rDNA-based primer set) 33

Table 2-3. Synthetic modified PHA production medium with their composition. 38

Table 2-4 Gachsaran oil analysis using GC/MS. 40

Table 3-1. Main characteristics of the isolated Pseudomonas spp. 42

Table 3 – 2. Quantitative analysis of 15 PHA positive isolates that were selected by Sudan Black B and Nile Red A staining. 46

Table 3 – 3. PHA Biosynthesis by four Pseudomonas strains using glucose (2% v/v)
as sole carbon source. 47

Table 3-4. GC-MS analysis of methanolyzed samples of PHAs produced by Pseudomonas spp. grown in a crude oil mineral medium under aerobic condition at
30 ^◦C for 7 Days. 49

Table 3-5. Effect of Carbon source (crude oil) concentration on bacterial growth and PHA production 57

List of Figures

Figure Page

Figure 1 – 1. Most common bio-based polymer building blocks for bacterial plastics. 3

Figure 1 – 2. Most common bio-based polymer molecular structures. 4

Figure 1 – 3. Common PHA monomer structures. 6

Figure 1 – 4. Electron-microscopic pictures of PHA-rich Cupriavidus necator DSM.. 8

Figure 1 – 5. Schematic view of a PHA granule 9

Figure 1 – 6. Cyclic metabolic nature of P(3HB) biosynthesis and degradation in bacteria.. 11

Figure 1 – 7. Reaction catalysed by PHA synthase. 11

Figure 1 – 8. Structural organisation of a PHA granule and metabolic interconnections between the different pathways involved in the biosynthesis and catabolism of PHBs and PHAs. . 12

Figure 1 – 9. Surface modification of PHA granules using GAPs as anchors / immobilisation tags and possible applications of these functionalised beads. 24

Figure 1 -10. Potential applications for PHA granules. 25

Figure 2- 1. Standard Crotonic acid curve. 36

Figure Page

Figure 3-1. Three different species of Pseudomonas on Cetrimide agar. 43

Figure 3-2. Molecular confirmations for Pseudomonas spp. isolated from contaminant areas using genus specific primers. 43

Figure 3 – 3. Sudan Black B staining of PHA granules (black section). 44

Figure 3 – 4. Fluorescent Nile Red staining of cells accumulating polyhydroxylalkanoic acids (PHAs) on agar plates. 45

Figure 3 – 5. Mass spectra of Methyl 3-hydroxyoctanoate. 50

Figure 3 – 6. Mass spectra of Methyl 3-hydroxydecanoate. 51

Figure 3 – 7. Mass spectra of Methyl 3-hydroxydodecanoate. 52

Figure 3 – 8. Mass spectra of Methyl 3-hydroxytetradecanoate. 53

Figure 3 – 9. Mass spectra of Methyl 3-hydroxyhexadecanoate. 54

Figure 3-10. Effect of culture media on bacterial growth (a) and PHA production (b). 55

Figure 3-11. Effect of incubation time on bacterial growth. 56

Figure 3-12. Bacterial growth rate (a) and PHA accumulation (b), during cultivations of Pseudomonas spp. in PHA production medium supplemented with 2% (v/v) crude oil in different temperature. 57

Figure 3-13. Effect of carbon source (crude oil) concentration on bacterial growth and PHA production. 58

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