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Determination-of-the-amount-of-PHB-was-performed-chemicallyusing-

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Determination-of-the-amount-of-PHB-was-performed-chemicallyusing-

Central South University

A grope for optimum incubating conditions of

Acidiphilium DX1-1 to produce poly-β-hydroxybutyrate

Bachelor's degree dissertation of Central South University Catalogue

    Catalogue

    Abstract ................................................................................................................................ i 摘要 .................................................................................................................................... ii Chapter 1 An Overview on Polyhydroxyalkanoates ................................................... 1 1.1 Introduction ..................................................................................................................... 1 1.2 The category of biodegradable plastics ............................................................................ 2 1.3 Polyhydroxyalkanoates(PHAs) ................................................................................... 3 1.4 Properties and practical applications of PHA ............................................................. 4 1.4.1 PHA’s properties ....................................................................................................... 4 1.4.2 PHA’s applications .................................................................................................... 4 1.5 PHA synthesis .............................................................................................................. 5 1.5.1 PHA biosynthesis in natural isolates .......................................................................... 5 1.5.2 PHA production by recombinant bacteria .................................................................. 7 1.5.3 Production of PHA by genetically engineered plants................................................ 11 1.5.4 Production of PHA by anaerobic digestion of biological wastes ............................... 13 1.6 Biodegradability of PHA ........................................................................................... 13 1.6.1 Microorganisms ...................................................................................................... 14 1.6.2 Mechanism ............................................................................................................. 14 1.6.3 Influencing factors of biodegradation of PHA ......................................................... 15 1.7 Economics of PHA production.................................................................................. 15 1.8 Conclusions ................................................................................................................ 16 1.9 The investigation significance, target and content of this research ........................ 17 Chapter 2 Materials and methods ............................................................................... 18 2.1 Materials ..................................................................................................................... 18 2.1.1 Reagents ................................................................................................................. 18 2.1.2 Apparatus................................................................................................................ 18 2.1.3 Microorganism........................................................................................................ 18 2.1.4 Media ..................................................................................................................... 21 2.2 Methods ...................................................................................................................... 21 2.2.1 Analysis of PHB content ......................................................................................... 21 2.2.2 Construction of PHB standard curve........................................................................ 21

Bachelor's degree dissertation of Central South University Catalogue

    2.2.3 Bacteria culture ....................................................................................................... 22 2.2.4 Determination of the optimum carbon and nitrogen source combination .................. 22 2.2.5 Orthogonal experiment............................................................................................ 23 2.2.6 Growth of microorganisms and production of PHB under optimum conditions ........ 24 Chapter 3 Results ........................................................................................................... 26 3.1 PHB standard curve.................................................................................................... 26 3.2 Results of optimum conbination of carbon and nitrogen source experiment ......... 27 3.3 Results of the orthogonal experiment ....................................................................... 29 3.4 Cell growth curve and PHB production curve ......................................................... 31 Chapter 4 Discussion...................................................................................................... 33 Acknowledgment ............................................................................................................... 35 References ......................................................................................................................... 36 Appendix:Translation ....................................................................................................... 40

Bachelor's degree dissertation of Central South University Abstract

    Abstract

Polyhydroxyalkanoates(PHAs) are microbial polyesters synthesized by a variety

    of microorganisms as a temporary storage material of energy and carbon. They are

    gaining more and more importance worldwide due to their close analogy to plastics ,

    biodegradability and biocompatibility. PHBs (poly-β-hydroxybutyrate, a kind of

    PHA) biodegradability makes them extremely desirable substitutes for synthetic

    plastics. The competition between biodegradable plastics and petrochemical plastics

    could be in terms of production cost. Therefore, different sources (natural isolates,

    recombinant bacteria, engineered plants) and other methods are being investigated to

    exert more control over the quality, quantity and economics of PHB production.

    A new bacterial strain, designated as strain DX1-1, was isolated from acid mine

    drainage of DeXing mine, Jiangxi Province, China by our laboratory. The bacterial

    isolate was found to be affiliated to the genus Acidiphilium and was found capable of

    producing a considerable amount of PHB, as confirmed by Fourier transform

    infrared spectroscopy. This experiment had determined the optimum carbon and

    nitrogen source combination(glucose and potassium nitrate(KNO

    ) combination) 3

    which could improve PHB production of strain DX1-1; obtained optimum

    incubating conditions, namely, concentrations of carbon source (glucose: 15g/L),

    nitrogen source (KNO: 6g/L) and initial pH(3.0); determined the PHB productions 3

    changing with time under the optimum incubating conditions and the process of

    PHB production was a growth-associated process. By this experiment the PHB

    production of strain DX1-1 was heightened greatly to 19.75g/L and Acidiphilium

    DX1-1s industrial application becomes more promising.

    Keywords: Acidiphilium DX1-1;bioplastic;polyhydroxybutyrat;incubating conditions

    i

    Bachelor's degree dissertation of Central South University 摘要

    聚羟基烷烃(PHAs)是许多微生物可以合成的生物聚酯,一类用于暂时贮存

    能量和碳的材料。PHAs由于性质类似于塑料,具有生物可降解性和生物相容性

    而越来越受到全世界的关注。PHB(聚-β-羟基丁酸,PHAs的一种)的生物可降

    解性使它成为合成塑料极好的替代品。生物可降解塑料与石油化学合成塑料之

    间的竞争主要是生产成本。因此人们利用不同的来源(自然分离菌种,重组细

    菌,转基因植物)以及其他的方法尽力提高生产PHB的质,量以及经济效益。

    从中国江西省德兴铜矿矿坑水样中分离出一菌株,经鉴定属于Acidiphilium 属,命名为DX1-1。经红外检测已证实该菌株具有生产聚-β-羟基丁酸(PHB)的能力。本试验确定了提高DX1-1菌株PHB产量的最佳碳氮源组合(葡萄糖和硝酸钾(KNO

    )组合),通过正交试验得到了最优培养条件即碳源(葡萄糖:15g/L)3

    氮源浓度(KNO:6g/L)以及pH(3.0),并确定了在最优培养条件下PHB产量随时3

    间的变化以及PHB的生产过程是一个与生长协同性过程。通过本次试验,提高了DX1-1菌株PHB的产量,最高达到19.75g/L,增加了Acidiphilium DX1-1菌株的工业应用前景。

    Acidiphilium DX1-1;生物塑料;聚羟基丁酸;培养条件

    ii

Bachelor's degree dissertation of Central South University Introduction

    Chapter 1 Introduction

    An Overview on Polyhydroxyalkanoates

    1.1 Introduction

    There is no doubt that the utility of plastic materials plays a more and more

    important role in our day-to-day life. Plastics are utilized almost in all manufacturing

    industries ranging from automobiles to medicine. Plastics are very much

    advantageous because as synthetic polymers, their structure can be chemically

    manipulated to have a wide range of strengths and shapes. And their molecular

    weights ranging from 50,000 to 1,000,000Da [1]. The synthetic polyethylene,

    polyvinyl chloride and polystyrene are largely used in the manufacture of plastics.

    Plastics can be easily molded into almost any desired shape including fibers and thin

    films. They have high chemical resistance and are more or less elastic, hence popular

    in many durable, disposal goods and as packaging materials.

    What makes plastics undesirable is the difficulty in their disposal. Petrochemical

    plastics are posing problems in solid-waste disposal and global environment

    management. Plastics being xenobiotic are resistent to microbial degradation [2].

    Excessive molecular size seems to be mainly responsible for the resistance of these

    chemicals to biodegradation and their persistence in soil for a long time. In the recent

    years, there has been increasing public concern over the harmful effects of

    ,petrochemical-derived plastic materials in the environment. Natures built-in

    mechanisms and self-regulation ability cannot tackle novel pollutants since these are

    unfamiliar to it.

    The environmental impact caused by these platics has created much interest in the

    development of biodegradable plastics and this also has prompted many countries to

    start developing biodegradable plastics. According to an estimate, more than 100

    million tons of plastics are produced every year, and which are simultaneously

    accumulating at the rate of 25 million tons per year. This seriously threatens humans

    living space, conditions and qualities.The per capita consumption of plastics in the

    United States of America is 80Kg and 60Kg in the European countries [3]. It has been

    threported that during the 20 century the consumption of plastic products is increasing

    at the rate of 13.8% per year world over, which is four times of the steel industrys

    [4]developing rate. Forty percent of the 75 billion pounds of plastics produced every

    year is discarded into landfills. Several hundred thousand tones of plastics are

    discarded into marine environments every year and accumulate in oceanic regions.

    Incinerating plastics has been one option in dealing with non-degradable plastics,

    but other than being expensive it is also dangerous. Harmful chemicals like hydrogen

    chloride and hydrogen cyanide are released during incineration [5]. Burying is another

    method, but it is only a temporary treatment leaving the plastics still undegraded. And

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Bachelor's degree dissertation of Central South University Introduction

    nether should the underground water pollution and soil contamination be neglected

    which are caused by plastics buried under the ground. Recycling also presents some

    major disadvantages, as it is difficult to sort the wide variety of plastics and there are

    also changes in the plastics material such that its further application parameter is

    [5]limited .

    Replacement of non-biodegradable by degradable plasticis of major is appealing both to decision- makers and the plastic industry. Making eco-friendly products such

    as bioplastics is one such reality that can help us overcome the problem of pollution

    caused by non-degradable plastics. The biodegradable plastics can be completely

    degraded into water and carbon dioxide by microorganisms in various environment

    such as soil, marine and sewage.

    Thus, it becomes inevitable for us to improve upon the method of production, selection of raw materials, recycling, conversion to suitable forms of certain wastes,

    so that we will not discard any material waste into the environment which nature

    cannot stand and keep the earth we are living on clean and beautiful as before.

    1.2 The category of biodegradable plastics

    At present there are three types of biodegradable plastics: photodegradable plastic, semi-biodegradable plastic, and completely biodegradable plastic. Photodegradable

    plastics have light sensitive groups incorporated directly into the backbone of the

    polymer as additives. Extensive ultraviolet radiation (several weeks to months) can

    disintegrate their polymericstructure rendering them open to further bacterial

    degradation [3]. However, majority of landfills lack sunlight and thus they remain

    non-degraded. Even a part of landfills can be exposured to sunlight, they can not be

    degraded completely, forming fragments and powders which cause a secondary

    pollution. Semi-biodegradable plastics are the starch-linked plastics where starch is

    incorporated to hold together short fragments of polyethylene. The idea behind

    starch-linked plastics is that once discarded into landfills, bacteria in the soil will

    attack the starch and release polymer fragments that can be degraded by other

    bacteria. Acturally bacteria indeed attack the starch but starch are turned off by the

    polyethylene fragments, which thereby remain non-degradable [5]. The third type of

    biodegradable plasticis rather new and promising because of its actual utilization by

    bacteria to form a biopolymer. Included are polyhydroxyalkanoates(PHAs),

    polylactides(PLA), aliphatic polyesters, polysaccharides, copolymers and/or blends

    of the above. These biodegradable plastics can be completely degraded into water

    and carbon dioxide by microorganisms in various environment such as soil, marine

    and sewage.

    Since the discovery of the thermoplastic properties of polyhydroxyalkanoates

    (PHAs) and their biodegradability, many attempts have been made to improve

    bioprocesses of the industrial production of PHAs and make them the replacement of

    petrochemical-derived plastics in many countries.

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Bachelor's degree dissertation of Central South University Introduction

    1.3 Polyhydroxyalkanoates(PHAs)

    PHA are polyesters of various hydroxyalkanoates synthesized by many

    gram-positive and gram-negative bacteria from at least 75 different genera. These

    polymers are accumulated intracellularly to levels as high as 90% of the cell dry

    weight in response to conditions of nutrient stress (limition of nitrogen, phosphorus,

    [1]or oxygen) and act as carbon and energy reserve compounds .

    Non-storage PHA that are of low molecular weight, poly(3HB), have been detected

    in the cytoplasmic membrane and cytoplasm of Escherichia coli. It is also a membrane constituent in yeasts, plants and animals. Putative functions include a role

    in voltage-gated calcium channels or DNA transport, protection of the

    macromolecules, to which it is bound, from degradative enzymes [6]. More than 100 different monomer units have been identified as constituents of the storage PHAs

    (Figure 1-1). This creates a possibility for producing different types of biodegradable

    polymers with an extensive range of properties. The molecular mass of PHA is in the

    range of 50,0001,000,000 Da and varies with the PHA producer. The monomer units

    are all in D(-) configuration owing to the stereospecificity of biosynthetic enzymes[6,7].

    Bacterially produced polyhydroxybutyrate and other PHAs have sufficiently high

    molecular mass to have polymer characteristics that are similar to conventional

    [1]plastics such as polypropylene .

     R O

    

    [CH(CH ) CO]2nx

n=1 R= CH P(3B) 3

    R= CHCHP(3HV) 23

     R= (CH)CH P(3HO) 243

    n=2R= HP(4HB)

    R= CHP(4HV) 3

    n=3R= HP(5HV)

    Other R groups CH=CH (CH)CH=CH (CH)CHBr, 2,272,22,4,72

    CHCH(CH) CHCHCH(CH) CHCHCH 233, 23223

    [16]Figure 1-1. Molecular structures of PHAs

Polyhydroxyalkanoates (PHAs) forms one among the major group of biodegradable

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Bachelor's degree dissertation of Central South University Introduction

    plastics. Poly-β-hydroxybutyrate (PHB) is a homopolymer of β-hydroxybutyrate and

    is the most widespread and best characterized member of the PHAs family.

    Copolymers of PHB can be formed by co-feeding of substrates and may result in the

    formation of polymers containing 3-hydroxyvalerate (3HV) or 4-hydroxybutyrate

    (4HB) monomers. The incorporation of 3HV into PHB results in copolymer

    poly(3-hydroxybutyrateco-3-hydroxyvalerate) [P(3HB-3HV)] that is less stiff and

    more brittle than P(3HB)[8].Together, polymers containing such monomers form a

    class of PHA referred to as short-side-chain PHA(ssc-PHA). In contrast, polymers

    composed of C6C16 3-hydroxy fatty acids or aliphatic carbon sources are referred to

    as medium-side-chain PHA (msc-PHA). The composition of the resulting PHA

     [9]depends on the growth substrate used. The msc-PHA are also synthesized from

    [10,11]carbohydrates, but the composition is not related to the carbon source . The

    msc-PHA have a much lower level of crystallinity than PHB or P(3HB-3HV) and are

    more elastic as well. They have a potentially different range of applications compared

    [12,13]to ssc-PHA . The vast majority of microbes synthesize either ssc-PHA containing

    primarily 3HB units or msc-PHA containing 3-hydroxyoctanoate (3HO) and

    [14,15]3-hydroxydecanoate(3HD) as the major monomers .

    PHA are produced from a wide variety of substrates such as renewable resources

    (sucrose, starch,cellulose, triacylglycerols), fossil resources (methane, mineral oil,

    lignite, hard coal), byproducts (molasses, whey, glycerol),chemicals (propionic acid,

    4-hydroxybutyric acid) and carbon dioxide. This can turn many pollutants useful and

    is very good for environmental protection as well as resources recycling.

    1.4 Properties and practical applications of PHA

    1.4.1 PHAs properties

    The PHAs are non-toxic, biocompatible, biodegradable thermoplastics that can be

    produced from renewable resources. They have a high degree of polymerization, are

    highly crystalline, optically active and isotactic (stereochemical regularity in repeating

    units), peizoelectric and insoluble in water. These features make them highly

    [17]competitive with polypropylene, the petrochemical-derived plastics.

    1.4.2 PHAs applications

    PHAs have a wide range of applications owing to their novel features. Initially,

    PHA were used in packaging films mainly in bags, containers and paper coatings.

    Similar applications as conventional commodity plastics include the disposable items,

    such as razors, utensils, diapers, feminine hygiene products, cosmetic

    containers––shampoo bottles and cups. In addition to potential as a plasticmaterial ,

    PHAs are also useful as stereoregular compounds which can serve as chiral precursors

    for the chemical synthesis of optically active compounds[7]. Such compounds are

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Bachelor's degree dissertation of Central South University Introduction

    particularly used as biodegradable carriers for long term dosage of drugs, medicines,

    hormones, insecticides and herbicides. They are also used as osteosynthetic materials

    in the stimulation of bone growth owing to their piezoelectric properties, in bone

    plates, surgical sutures and blood vessel replacements. However, the medical and

    pharmaceutical applications are limited due to the slow biodegradation and high

    hydraulicstability in sterile tissues [18]. The PHAs are considered as a source for the synthesis of chiral compounds (enantiometrically pure chemicals) and are raw

    materials for the production of paints. PHAs can be easily depolymerised to a rich

    source of optically active, pure, bi-functional hydroxy acids. PHB for instance is

    readily hydrolyzed to R-3-hydroxybutyric acid and used in the synthesis of Merck’s

    anti-glaucoma drug ‘Truspot’. In tandem with R-1,3-butanediol, it is also used in the synthesis of β-lactams. Plant derived PHAs can be depolymerized and used, directly

    or following esterification, in the manufacture of bulk chemicals[9]. Besides helping to

    replace the existing solvents, β-hydroxy acid esters and derivatives are likely to find

    growing use as ‘green solvents’ similar to lactic acid esters. The conversion of

    hydroxy acids into crotonic acids such as 1,3-butanediol, lactones, etc. would help

    improve the market value as they have an existing market demand in thousands of

    tonnes.

    1.5 PHAs synthesis

    PHB(poly-β-hydroxybutyrate, a kind of PHAs) can be synthezed in two

    ways–—chemical synthesis and biosynthesis. Chemical synthesis consists of two

    processing routes: one of which is to use β-butyrate as monomers to synthesize PHB; another route is synthesizing PHB with β-hydroxybutyric acid as monomers. The first method is not promising because the conditions of reactions is hard to control. The

    other way bankcrupts due to its high production costs [19]. Both of these ways are not

    suitable for cosmical industrial production. Compare to chemical synthesis ,

    biosynthesis is advantageous by virtue of its simple incubating conditions and security

    during producing.

    1.5.1 PHAs biosynthesis in natural isolates

    1. Microorganism

    PHAs are accumulated as intracellular granules by many prokaryotic organisms

    which are gram-positive or gram-negative when they are under specific unfavourable

    nutritional conditions or during starvation as a reserve carbon and energy source. Not

    all microorganisms accumulate 3-hydroxybutyrate and only a few accumulate it in

    large amounts( Table.1-1). So far over 300 kinds of bacterium of 65 genera have been

    reported to have the abilities of accumulating PHB. When an external carbon source is

    available but the concentration of nutrients such as nitrogen, phosphorus, or oxygen

    5

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