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  3. Biopolymer production of polyhydroxyalkanoates and checking the possibility of using them in polymer nanocomposites

Biopolymer production of polyhydroxyalkanoates and checking the possibility of using them in polymer nanocomposites

Number of pages: 129 File Format: word File Code: 31799
Year: Not Specified University Degree: Master's degree Category: Chemical - Petrochemical Engineering
Tags/Keywords: Biopolymer production - Kinetic model - Non-continuous cultivation - Polyhydroxyalkanoate - Polymer nanocomposites - Semi-continuous cultivation
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  • Summary of Biopolymer production of polyhydroxyalkanoates and checking the possibility of using them in polymer nanocomposites

    PhD thesis in the field of Chemical Engineering-Biotechnology

    Abstract

    The aim of this study is to produce biopolymer polyhydroxyalkanoates using carbon sources of glucose, fructose, Molasses and whey by microorganisms Azohydromonas lata DSMZ 1123, Azotobacterbeijerinckii DSMZ 1041, Cupriavidus necator DSMZ 545, Hydrogenophaga pseudoflava DSMZ 1034. In the first stage, in order to screen microorganisms and select the target microorganism for biopolymer production, suitable temperature conditions, age of inoculation and intensity of stirring were determined for each microorganism. In optimal conditions, each carbon source was used alone to determine the type and amount of biopolymer produced by each.  According to the obtained results, C. necator was selected as the suitable microorganism for the continuation of the research due to having favorable conditions (proper and effective growth on the desired environments, stability of biological activity compared to other micro-organisms investigated and significant production of biopolymer). In the discontinuous process of biopolymer production in a flask using C. necator on glucose, fructose and molasses sources, the production rate of polyhydroxybutyrate biopolymer was 3.3, 5.9, and 1.3 g/liter, respectively, and the rate of better efficiency was 0.07, 0.08, and 0.03 g/liter per hour. In addition, acetate (sodium acetate with an optimal concentration of 10 g/liter) was used as a carbon source supplement along with molasses to produce biopolymer, which resulted in the production of 7.2 grams per liter of polyhydroxybutyrate/hydroxyvalerate copolymer. The amount of polyhydroxybutyrate and polyhydroxyvalerate produced in the copolymer was 6.9 and 0.32 g/liter, respectively. In the second stage, by examining the growth kinetics in the discontinuous process and predicting the growth and production process, the discontinuous and semi-continuous process in the bioreactor for the production of biopolymer was investigated. First, in the non-continuous process on glucose, the amount of polyhydroxybutyrate was 4.2 g/L per consumption of 16 g/L carbon source, and the oxygen transfer coefficient was 0.16/s and the specific growth intensity of the microorganism was 0.17/h. Then, the semi-continuous process was investigated using two methods of constant and variable step feeding of carbon and nitrogen sources at concentrations of 300 and 10 g/liter. The amount of polyhydroxybutyrate production in constant feeding was 8.2 g/l and in variable feeding was 11.8 g/l, which increased by about 40%. The productivity of non-continuous and semi-continuous processes with constant and variable feeding was 0.04, 0.085, and 0.137 g/liter/hour, respectively. In the third stage, the possibility of producing polymer nanocomposites using the produced biopolymer was investigated. Using the stabilization method in the solvent, the solution of polyhydroxybutyrate/hydroxyvalerate copolymer was placed in chloroform along with hydroxyapatite nanoparticles. The results indicated that the production of nanocomposite using ultrasonication gave better results and the nanoparticles were uniformly stabilized on the biopolymer surface.

    Key words: Polyhydroxyalkanoate, Cupriavidus necator DSMZ 545, non-continuous culture, semi-continuous culture, kinetic model, biopolymer nanocomposite

    Introduction

    The use of polymers and plastics in most human devices, from the smallest to the largest, is undeniable. The reason for this abundant use of polymers and plastics in human life is their many properties. Per capita consumption of plastic in Europe is 60 kg and in America 80 kg per year [1]. Despite the many benefits of polymers and plastics, their use has caused many environmental problems, and this has caused mankind to think about the production of biodegradable polymers and the biodegradation of polymers and plastics.

    The internal mechanisms and self-regulation ability of nature cannot decompose these pollutants because they are unfamiliar with these materials. This has caused many countries to start developing biodegradable plastics. According to one estimate, more than 100 million tons of plastic are produced every year.40% of this amount is transferred to landfills and several hundred thousand tons are dumped into marine environments and accumulate in ocean areas every year. Burning plastics is one of the options in disposing of plastics; But in addition to being expensive, it is also dangerous [1-2].

    Plastics that are completely degradable are relatively new and promising because of the use of bacteria to form biopolymers, which mainly include polyhydroxyalkanoates[1], polylactic acids[2], aliphatic polyesters[3], polysaccharides[4], or a combination of these materials[1].

    1- Types of biodegradable polymers

    Many biodegradable polymers have been identified and one of the most important of them is polyhydroxyalkanoates. The use of this group of biodegradable polymers has received much attention in agriculture and pharmaceutical industries, etc., due to its compatibility with the environment and vital systems[2].

    Polyhydroxyalkanoates are biodegradable polymers and are formed as intracellular particles in various microorganisms[3]. The molecular weight of these polymers is in the range of 2*105 to 3*106 daltons. The molecular weight changes according to the type of microorganism and growth conditions [3].

    One of the most important polyhydroxyalkanoates is polyhydroxybutyrate. Polyhydroxybutyrate is a linear polymer of 3-hydroxybutyrate and is present in various particle sizes within the cell. Polyhydroxybutyrate is a source of energy and carbon storage for microorganisms, and under conditions such as nitrogen, phosphorus, oxygen, ions, etc. limitations, it accumulates inside the cell, and polyhydroxybutyrate decomposes when these limitations are removed.

  • Contents & References of Biopolymer production of polyhydroxyalkanoates and checking the possibility of using them in polymer nanocomposites

    List:

    First- An overview of previous studies

    1-1- Microorganisms producing polyhydroxyalkanoates. 7

    1-2- Hydroxyalkanoate copolymers. 11

    1-3- How to synthesize hydroxyalkanoate biopolymers. 14

    1-4- Inexpensive carbon sources in the production of PHA polymers. 15

    1-5- Synthesis of polyhydroxyalkanoates in plants. 16

    6-1- Quantitative measurement Biopolymers. 18

    1-7- Physical properties and uses of biopolymers. 19

    1-8- Degradability of polyhydroxyalkanoates. 21

    1-9- Production process of polyhydroxyalkanoates. 23

    1-9-1- Discontinuous process. 23

    1-9-2- Semi-continuous and continuous process. 24

    1-10- Growth kinetic model Microorganism. 1-10-1- Investigation of growth kinetics in discontinuous process. 31- 1-11- Determination of oxygen transfer coefficient in bioreactor. 33- 1-11-1- Measurement methods. 33- Title of page 1-12- Use. Polyhydroxyalkanoates in industries. 36

    1-13- Application of biopolymers in polymer nanocomposites. 39

    1-13-1- Types of polymer nanocomposites. 39

    1-13-2- Manufacturing methods of polymer nanocomposites. 41

    Chapter II- Materials and methods

    2-1- Microorganism.45

    2-2- Transfer of microorganism from dry ice state to primary culture medium.47

    2-3- Storage medium.47

    2-4- Inoculation medium.48

    2-5- Fermentation culture medium.48

    2-6- Preparation of inoculum culture.49

    2-7- Fermentation and sampling conditions. 49

    2-8- Preparation of the calibration curve of cellular dry weight-absorption. 50

    2-9- Preparation of calibration curves to determine the amount of carbon sources. 51

    2-9-1- How to prepare DNS reagent solution. 51

    2-9-2- Drawing the calibration curve of convertible sugars. 51

    Title

    2-10-Gas chromatographic conditions for measuring polyhydroxyalkanoates. 52

    2-10-1- Preparation of internal standard. 53

    2-10-2- Preparation of calibration curves of methylhydroxybutyrate, methylhydroxyvalerate

    Methylhydroxyhexanoate. 53

    2-10-3- Biopolymer extraction and sample preparation for injection into the GC device. 54

    2-10-4- Biopolymer identification and confirmation method by 13C NMR, 1H NMR. FT-IR.55

    2-10-4-1- Infrared Spectroscopy (FT-IR) 56

    2-11-2- Semi-continuous cultivation process. 56

    2-11-2-1- Semi-continuous cultivation process with constant feeding of carbon and nitrogen source. 57

    2-11-2-2- Semi-continuous cultivation process with variable feeding of carbon and nitrogen source. 57

    2-11-3- Determining the oxygen transfer coefficient in the bioreactor. 57

    2-12- Production of polyhydroxybutyrate hydroxyvalerate

    /hydroxyapatite nanocomposite. 59

    Title

    Page

    Chapter 3-Results and discussion

    3-1- Microorganism Hydrogenophaga pseudoflava DSMZ 1034.62

    1-3-1- Examining the biological process conditions.62

    3-1-2- Using glucose as the only carbon source.63

    3-1-3- Using fructose as the only carbon source. 65

    3-1-3- Using whey as the only carbon source 66

    3-2- Microorganism Cupriavidus necator DSM 545.68

    3-2-1- Examining the biological process conditions. 68

    3-2-1-2- Examining the effect of nitrogen to carbon ratio. 69

    3-2-2- Using glucose as the only carbon source. 73

    3-2-3- Using fructose as the only carbon source.74

    3-2-4- Using molasses as the only carbon source.75

    3-2-5- The effect of acetate on the growth of microorganism and biopolymer production.77

    3-2-5-1 - The combination of molasses and acetate as carbon sources.77

    3-3- Microorganism 77

    3-3- Microorganism Azotobacter beijerinckii DSMZ 1041.80

    3-3-1- Examining the biological process conditions. 80

    3-3-2- Using glucose as the only carbon source. 82

    3-3-3- Using fructose as the only carbon source. 83

    3-3-4- Using whey as the only carbon source. 84

    3-4- The microorganism Azohydromonas lata DSMZ 1123.85

    Title

    3-4-1- Examining the biological process conditions. 85

    3-4-2- Using glucose as the only carbon source. 87

    3-4-3- Using fructose as the only carbon source. 88

    3-4-4- Using whey as the only carbon source. 89

    3-5- General results of comparison of four microorganisms in biopolymer production. 92

    3-6- Investigating the growth kinetics of microorganism in biopolymer production. 92

    3-7- Discontinuous culture process in bioreactor. 95

    3-7-1- Determining the oxygen transfer coefficient in the bioreactor. 97

    3-8- Semi-continuous cultivation process with constant feeding in the bioreactor. 98

    3-9- Semi-continuous cultivation process with variable (step) feeding in the bioreactor. 99

    3-10- Biomass efficiency. 100

    3-11- Efficiency 102

    3-12- Production efficiency. 103

    3-13- Diagnostic tests to confirm the produced biopolymer. 105

    3-13-1- Infrared Spectroscopy (FT-IR) 105

    3-13-2- Nuclear Magnetic Resonance Spectroscopy (NMR) 106

    3-14- Investigating the possibility of using the biopolymer produced in nanocomposites. 108

    Title

    Chapter 4-Conclusions and suggestions

    4-1- Conclusion. 113

    4-2- Suggestions.116

    References.117

    English abstract.127

    Appendices.

    Source:

     

    [1] C.S.K. Reddy, R. Ghai, Rashmi, V.C. Kalia, Polyhydroxyalkanoates: an overview, Bioresource Technology, 87 (2003) 137-146.

    [2] M. Shimao, Biodegradation of plastics, Current Opinion in Biotechnology, 12 (2001) 242-247.

    [3] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Progress in Polymer Science, 25 (2000) 1503-1555.

    [4] S.Y. Lee, Bacterial polyhydroxyalkanoates, Biotechnology and Bioengineering, 49 (1996) 1-14.

    [5] S. Khanna, A.K. Srivastava, Optimization of nutrient feed concentration and addition time for production of poly([beta]-hydroxybutyrate), Enzyme and Microbial Technology, 39 (2006) 1145-1151.

    [6] B. Panda, P. Jain, L. Sharma, N. Mallick, Optimization of cultural and nutritional conditions for accumulation of poly-[beta]-hydroxybutyrate in Synechocystis sp. PCC 6803, Bioresource Technology, 97 (2006) 1296-1301. [7] L. Sharma, A. Kumar Singh, B. Panda, N. Mallick, Process optimization for poly-[beta]-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology, Bioresource Technology, 98 (2007) 987-993.

    [8] S. Ciesielski, A. Cydzik-Kwiatkowska, T. Pokoj, E. Klimiuk, Molecular detection and diversity of medium-chain-length polyhydroxyalkanoates-producing bacteria enriched from activated sludge, Journal of Applied Microbiology, 101 (2006) 190-199.

    [9] J.M. Luengo, B. Garc?a, A. Sandoval, G. Naharro, E.R. Olivera, Bioplastics from microorganisms, Current Opinion in Microbiology, 6 (2003) 251-260.

    [10] M. Bassas, E. Rodr?guez, J. Llorens, A. Manresa, Poly(3-hydroxyalkanoate) produced from Pseudomonas aeruginosa 42A2 (NCBIM 40045): Effect of fatty acid nature as nutrient, Journal of Non-Crystalline Solids, 352 (2006) 2259-2263.

    [11] G. Braunegg, G. Lefebvre, K.F. Genser, Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects, Journal of Biotechnology, 65 (1998) 127-161.

    [12] Y. Dai, L. Lambert, Z. Yuan, J.

Biopolymer production of polyhydroxyalkanoates and checking the possibility of using them in polymer nanocomposites
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