Novel applications of Alginate and PHAs.
1. Introduction
Industrial interest in microbial polymers has been stimulated by their unique properties and the opportunity to develop new materials, which can be used for specific applications in medical and pharmaceutical industries.
The intracellular polyester PHB and other PHAs have been drawing attention because they are biodegradable and biocompatible thermoplastics, which can be processed to create a wide variety of consumer products, including plastics, films, and fibers (Aldor & Keasling, 2003). Recently, and based on their properties of biocompatibility and biodegradability, new attractive applications for PHAs have been proposed in the medical field, where the chemical composition and product purity are critical (Williams & Martin, 2005).
The subjects covered in this chapter include research concerning the production of alginate and PHB by
2. Structure and physical properties of alginate and PHAs
Alginates are polysaccharides constituted by variable amounts of β-D-mannuronic acid and its C5-epimer α-L-guluronic acid linked by 1-4 glycosidic bonds (Figure 1). The monomers are distributed in blocks of continuous mannuronate residues (M), guluronate residues (G) or alternating residues (MG) (Smidsrod & Draget,1996).
The capability of alginate to confer viscosity in solution is dependent on its molecular mass (MM). The MM of algal alginates has been found to range from 48 to 186 kDa (Donnan & Rose, 1950). In contrast, some alginates isolated from
The gelling properties of alginate are based on the affinity of the molecule towards certain ions, especially Ca++, and the ability to bind these ions selectively and cooperatively. Selective ion binding is related to the content of G residues, in particular the length of the G-blocks. Alginates rich in G residues show an increased ionic binding, resulting in enhanced mechanical rigidity (Grant et al., 1973). Alginates with a low M/G ratio form strong and brittle gels, while alginates with a high M/G ratio form weaker and softer, but more elastic gels (Skjak-Braek et al., 1986).
Bacterial alginates which are
On the other hand, polyhydroxyalkanoates (PHAs) are aliphatic polyesters generally composed of β-hydroxy fatty acid monomers in which the carboxyl group of one monomer forms an ester bond with the hydroxyl group of the neighboring monomer (Madison & Huisman, 1999; Figure 1). The MM of PHAs is dependent on the bacterial species used and culture conditions but is generally on the order of 50 to 1,000 kDa (Madison & Huisman, 1999). At present, more than 150 different hydroxyalkanoate constituents have been reported in PHAs, as homopolyesters or as copolyesters (Steinbuchel & Lutke-Eversloh, 2003). These highly diverse polymers can be classified according to the size of the comprising monomers. PHAs containing monomers with C4–C5 atoms are categorized as short-chain-length PHAs (scl-PHAs). In contrast, medium-chain-length PHAs (mcl-PHAs) are composed of C6–C14 β-hydroxy fatty acids (Lee, 1996). Most bacteria synthesize either scl-PHAs or mcl-PHAs (Madison & Huisman, 1999). scl-PHAs have properties close to conventional plastics, while the mcl-PHAs are regarded as elastomers and rubbers (Suriyamongkol et al., 2007).
Polyhydroxybutyrate (PHB) is the more abundant PHA and has been studied extensively. This polymer has some mechanical properties similar to conventional plastics like polypropylene or polyethylene, although it is highly crystalline and stiff, leading to brittleness and low elongation to break (Khanna & Srivastava, 2005). Initial biotechnological developments were aimed at making PHAs easier to process. Because the monomeric composition of a PHA is crucial for its mechanical properties, the incorporation in the PHB polymer of secondary monomer units(s) such as β-hydroxyvalerate (3HV) improves the characteristics of the material obtained. For example, a random copolymer of 3HB and 3HV is more ductile, easier to mold, and tougher than PHB homopolymer (Taguchi & Doi, 2004), and it can be used to prepare films with excellent water and gas barrier properties reminiscent of polypropylene, and can be processed at a lower temperature while retaining most of the other mechanical properties of PHB.
3. Novel applications of alginate and PHAs
3.1. Novel applications of alginates
Novel alginates applications have been focused on pharmaceutical and biomedical fields, because they are non-toxic, biocompatible, non-immunogenic, hydrophilic and biodegradable material (Augst et al., 2006; Hernández et al., 2010). Alginate hydrogels can be used as bulking materials for
Some therapeutic applications of alginate microencapsulation are related with drug delivery. For low-molecular weight drugs, regulating drug-alginate interactions in alginate gels allows the control of drug release. This is especially important for drugs that have severe side effects, like antineoplastic agents. Besides, some proteins with therapeutic activities can be alginate-microencapsulated to improve their efficacy and targeting, because alginate encapsulation facilitate a localized delivery without adverse side effects. Alginate microencapsulation has been proven with basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The release of VEGF is controlled by the dissolution of the ionic binding complex between alginate and VEGF and subsequent diffusion, showing a constant release rate for several weeks (Augst et al., 2006).
Alginate has been successfully used for cell microencapsulation, which is of great importance for Diabetes Mellitus type 1 treatment, where several efforts have been made for regulated insulin supply for treating insulin-dependent patients (Hernández et al., 2010). Moreover, there are several new strategies developed to improve the cell-alginate immobilization process (Hoesli et al., 2011), as well as immune protection and oxygen supply to avoid hypoxia problems during transplants (Ludwig et al., 2010). In the tissue engineering field, alginate has been used for bone regeneration therapy using co-immobilization of human osteoprogenitors and endothelial cells in studies
Alginates have also been studied for development of novel immunotherapy strategies for cancer treatment using dendritic cells which are potent initiators of immune response. Calcium cross-linked alginate gels carrying dendritic cells initiated the immune response and allowed the migration of the immune cells through the alginate gel (Hori et al., 2008, 2009).
3.2. Novel applications for PHAs
PHAs have received much attention as candidates to produce biodegradable plastics compatible with the environment, due to their material properties (similar to those of well-known plastics such as polypropylene), their production from renewable sources, and their inherent biodegradability in various environments (Taguchi & Doi, 2004). These biopolyesters are attractive to replace non-biodegradable plastics, especially for those products that usually have single-use applications, such as food packaging.
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Alginate |
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Gel formation/ Biocompatible/ Bioinert/ Biodegradable/ Drug alginate interaction |
Augst et al., 2006 |
Cell immobilization |
Gel formation/ Biocompatible/ Bioinert/ Diffusion |
Hernández et al., 2010 Hoesli et al., 2011 | |
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Gel formation/ Biocompatible/ Bioinert/Diffusivity |
Hernández et al., 2010 | |
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Immunogenicity | Kashef et al., 2006 | |
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Low Viscosity | Draget & Taylor, 2011 | |
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Gel formation/ Biocompatible/ Bioinert/ Diffusivity |
Hori et al., 2008 | |
PHA |
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Thermoplasticity, physical and mechanical resistance/ Biodegradable | Chen, 2009 |
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Thermoplasticity, physical and mechanical resistance/ Biocompatible | Wu et al., 2009 Grage et al., 2009 |
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Biocompatible/ Biodegradable | Chen, 2009 | |
Cell microencapsulation and nanoencapsulation |
Biocompatible/ Biodegradable | Grage et al., 2009 | |
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Biocompatible | Lee et al., 2005 Wang et al., 2008 |
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Biocompatible | ||
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Biocompatible | Grage et al., 2009 |
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Biodegradability/ Methyl esterification capability | Zhang et al., 2009 |
Some of the monomers present in PHAs are known to be present in human and animals. For example, the monomeric component of PHB (β-hydroxybutyrate) is a ketone body normally found in human blood (Williams & Martin, 2005). The biocompatibility, together with the adjustable mechanical properties, and controllable biodegradability of PHAs have raised interesting applications in the medical field. These polymers have been used in artificial organ construction, drug delivery, tissue repair, and nutritional/therapeutic uses (Chen & Wu, 2005; Freier, 2006; Grage et al., 2009; Valappil et al., 2006; Williams & Martin, 2005; Wu et al., 2009; Zinn et al., 2001). Because several PHAs are available now in sufficient quantity, some of them have been used in biocompatibility studies
Very interesting applications for PHAs are found in the fabrication of drug delivery devices. Their biocompatibility, combined with their biodegradation, make them good candidates for this purpose (Chen, 2009). The possibility to create PHAs of various monomeric compositions and molecular weights makes possible the fine control of their degradation rate (Wu et al., 2009). Several drugs have been entrapped or microencapsulated in PHA homopolymers or copolymers, such as, anticancer agents, antihypertensives, hormones, vaccines, etc. (Williams & Martin, 2005).
More recently, new applications have been reported for PHAs, not just as a material but for the PHA granules themselves as micro-nano beads, resulting in applications for protein purification and specific drug delivery (Grage et al., 2009). Affinity protein purification methods make use of an affinity tag fused to the protein of interest. The interaction of the tagged protein with an immobilizing matrix allows the separation of the protein. PHA granules have been used as an inexpensive affinity support, while the phasins, PHA synthase, or PHA depolymerase, proteins naturally associated with the granules, work as the affinity tag for the purification or immobilization of proteins (Lee et al., 2005; Wang et al., 2008). Combining the fusion of the protein of interest with the phasin protein (PhaP), intein mediated self-cleavage, and PHA synthesis in recombinant
PHA nanoparticles have also been used in drug delivery, target specific therapy and as biomarkers or biosensors (Grage et al., 2009). Using the same principle of affinity binding of PHA synthase to PHA granules, Brockelbank et al., (2006) demonstrated the display of antigen fragments at the surface of PHA beads, showing their potential to be used in immunoglobulin G (IgG) purification from human serum. The functional display of antigen or antibodies fragments at the bead surface can be used for diagnostic or therapeutic applications (Grage et al., 2009). Fusion of PHA synthase with streptavidin has shown that these PHA beads can also be used for ELISA, DNA purification, enzyme immobilization and flow cytometry (Peters & Rhem, 2008). Engineered proteins for inorganics, like gold or silica, or IgG, have also been fused to the PHA synthase, and displayed at the surface of PHA granules, so these biobeads can be used for medical bioimaging procedures as inorganic contrast agents (Grage et al., 2009; Jahns et al., 2008).
With respect to targeted drug delivery, Yao et al., (2008) demonstrated that phasins can be fused to ligands recognized by tissue specific receptors, and that the ligand-PhaP-nanobeads are taken up by the correct tissue
Other applications for PHAs include their use as fine chemicals. A high diversity of carboxylic acids, all in the (R)- configuration, can be obtained by depolymerization or by chemical degradation of PHAs, and these can be bulk chemicals for various applications (Chen, 2009). Some of them have been used as starting material for the synthesis of antibiotics, vitamins, aromatics and pheromones (Ruth et al., 2007).
A new field of application for PHAs has been devised in the energy industry. These polymers can be used as biofuels (Chen, 2009). The conversion of PHB or mcl-PHAs to their methyl ester derivatives by acid catalyzed hydrolysis, allowed their use as fuels in blends with ethanol, gasoline, and diesel, with reasonable combustion heats (Zhang et al., 2009).
4. Azotobacter
vinelandii
The majority of nitrogen fixing bacteria are capable of reducing N2 only in anaerobic or microaerobic conditions. In contrast,
5. Genetics and biosynthesis of alginates and PHAs in A. vinelandii
5.1. Biosynthesis of alginates by A. vinelandii
The pathway for alginate synthesis has been well established and it is conserved among brown algae,
The
5.2. Biosynthesis of PHAs by A. vinelandii
PHB in
In
When
The control of PHB biosynthesis in
The regulation of PHB synthesis in
6. Fermentation parameters affecting the production and the composition of alginate
For several decades the synthesis of alginate and PHB by
6.1 Influence of the dissolved oxygen tension (DOT) and the oxygen transfer rate (OTR) on the quantity and quality of alginate
Many studies have shown that aeration and mixing are critical parameters for optimizing the production of microbial polysaccharides (Galindo et al., 2007). It is known that under low dissolved oxygen tension (DOT), the organism accumulates the intracellular storage polymer, PHB; whereas at high DOT,
More recently, Lozano et al., (2011) reported a study about the evolution of the MMM of the alginate produced by
6.2. Influence of the medium components
It is widely known that the components of the culture medium play an important role in the production of alginate by
Our group reported the influence of (3N-morpholino)-propane-sulfonic acid (MOPS), a component used in the medium to keep a constant pH, on the quality of the alginate in terms of the chemical composition and rheological behaviour of alginate-reconstituted solutions (Peña et al., 2006). This compound had an important effect on the acetyl content and physicochemical properties of this polymer. A two-fold higher acetylation degree of alginate was obtained when 13.6 mM MOPS was supplemented to the medium. The higher acetylation resulted in greater viscosity of the alginate solutions, but it exhibited less pronounced pseudoplastic behaviour. These changes in the functional properties of the polymer can have great value in terms of specific applications of alginate in food and pharmaceutical fields.
6.3. Effect of the specific growth rate
Another important culture parameter for the synthesis of alginate is the specific growth rate (Díaz-Barrera et al., 2009, 2010; Priego-Jiménez et al., 2005). Priego-Jiménez et al., (2005) using exponentially fed-batch cultures, found that the specific growth rate of
7. Parameters that affect PHAs production in A. vinelandii
Commercial production of PHAs requires not only high yields and productivities, but also a well defined chemical composition. Fermentation parameters affect the amount of PHAs produced by
PHAs production in different organisms is induced under nutrient limitation (Verlinden et al., 2007). For
7.1. Oxygen limitation
When
On the other hand, there are few reports related to the effect of oxygen on the composition of the PHB produced by
7.2. Medium composition: carbon and nitrogen sources
The high production cost is the main limiting factor for the use of PHAs for commercial purposes. An alternative to reduce costs is the use of cheaper feedstock (
Page, 1992
). Several attempts have been made to improve the culture media composition which depends on the microorganism (Table 2). Although
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Glucose (1%) and acetate (15mM) | NH4 + | 2.37 | 0.25 | N.D | Flasks | Page & Knosp, 1989 |
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Beet Molasses (5%) and sucrose (2%) | NH4 + | 7 | 0.35 | N.D | Flasks | Page, 1992 |
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Glucose (5%) acetate (15mM) |
NH4+ Fish peptone (1%) |
25 | 0.65 | 1700 | Fed batch culture | Page & Cornish, 1993 |
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Glucose (4%) | ----- | 4 | 0.1 | 1660 | Flasks | Myshkina et al., 2008 |
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Glucose (2%) Acetate (20mM) |
----- | 4 | 0.2 | 1100 | Flasks | Myshkina et al., 2008 |
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Molasses (4%) Sucrose (2%) |
----- | 1 | 0.05 | N.D. | Flasks | Myshkina et al., 2008 |
7.3. Addition of alkanoates
For several
For
8. Scaling up of alginate and PHAs production
Trying to reproduce in agitated tank the results obtained in plates or in shake flasks, is troublesome and the variables involved are poorly understood. This is particularly important, because the MM of the polymer drops dramatically when the alginate process is scaled up from shake flasks to fermentors (Peña et al., 1997, 2000). Both, the power input (P/V) and the oxygen transfer rate (OTR), have been used as scaling up parameters (Peña et al., 2008; Reyes et al., 2003). Recently, our group has studied both the evolution of the specific power consumption and oxygen transfer rate, occurring in shake flasks cultures of
These studies have revealed that the power consumption increased exponentially during the course of the fermentation (up to 1.4 kW m-3) due to an increase in the viscosity of the culture broth. Taking these data as a starting point, a scale-up strategy based on the evolution of the power input observed in shake flasks has been evaluated, trying to reproduce in a stirred fermentor culture the MMM of the alginates obtained in shake flasks (Peña et al., 2008). Simulating the evolution of the power input in 14 L fermentors, allowed us to reproduce the MMM and molecular mass distributions of the alginate obtained in shake flasks (Figure 4), a situation that had not been possible to achieve before using other criteria (i.e., initial power input (Reyes et al., 2003)).
Currently PHAs bacterial production, at industrial scale, is mainly conducted using the strain
Fed batch cultures of
In the case of mcl-PHAs production, Hartmann et al., (2005) evaluated chemostat cultures of
9. Design of novel processes to improve alginate and PHA production
Alginate production by fermentation using the
Our research group has carried out studies in fed-batch and multistage fermentation processes (Mejía et al., 2010; Priego-Jiménez et al., 2005) that are able to achieve high biomass concentration, in order to take advantage of the higher specific-alginate production capacities of mutant such as the AT6 strain. Employing a high oxygen concentration (10%) allowed obtaining a maximum biomass concentration of 7.5 g L-1 in the first stage of the cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9.5 g L-1, after 50 h of cultivation. Alginate concentration obtained from the AT6 strain was two fold higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature (Mejía et al., 2010).
Most of the studies using
Two-stage continuous cultures can help establishing a good growth/PHAs synthesis compromise. Jung et al., (2001) used a two-stage continuous cultivation system with two fermentors connected in series, producing cells at a specific growth rate in the first compartment, and establishing conditions to accumulate PHA at higher rates in a second compartment, with a relatively long residence time. Dilution rates of 0.21 h−1 in the first fermentor and 0.16 h−1 in the second fermentor yielded a volumetric PHAs productivity of 0.06 gPHA L−1 h−1, a high productivity for cultures grown on alkanes.
Processes using cheaper substrates have the potential to lower the production costs of PHAs production, but for the use of some of these substrates additional processing is needed. Cerrone et al., (2010) reported an interesting strategy to simultaneously produce PHB and treat olive mil wastewater using
10. Down stream processes
Alginate and PHAs have been proposed for novel applications in pharmaceutical and biomedical fields. However, for these applications it is necessary to ensure products with a high purity, and in most cases with a defined chemical composition. Chemical composition of these polymers can be controlled by the fermentation stage, but their purity will be determined by the down stream processing. Moreover, costs and efficiency of purification procedures could affect the whole process feasibility.
The extraction process of alginate from
The PHAs extraction processes require the separation of the cells containing the polymer by centrifugation. The recovery of intracellular PHAs could be carried out by solvent extraction using acetone, chloroform, methylene chloride or dichloroethane. Although this method is the most used, it is also expensive and environmentally unfriendly (Verlinden et al., 2007; Yasotha et al., 2006). Besides, several alternative methods have been developed to improve PHAs purification. Enzymatic digestion does not need hazardous solvents and it shows high selectivity. Yasotha et al., (2006) proposed an enzymatic method coupled to an ultrafiltration system and achieved a final PHAs purity of 92.6 % with a recovery of almost 90 %. However, this method could be very expensive. Another interesting alternative for PHAs recovery was proposed by Page & Cornish (1993), using fish peptone like nitrogen source for the growth of
11. Conclusions
Based on a better understanding of the biosynthesis and regulation of alginate and PHAs in
References
- 1.
Akaraonye E. Keshavarz T. Roy I. 2010 Production of polyhydroxyalkanoates: the future Green materials of choice. ,85 732 743 . - 2.
Aldor I. Keasling J. 2003 Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Opinion on Biotechnology,14 475 483 . - 3.
Asami K. Aritomi T. Tan Y. Ohtaguchi K. 2004 Biosynthesis of polysaccharide alginate by in a bubble column. Journal of Chemical Engineering Japan,37 1050 1055 . - 4.
Augst A. Joon-Kong H. Mooney D. 2006 Alginate hydrogels as biomaterials. ,6 623 633 . - 5.
Banki M. Gerngross T. Wood D. 2005 Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. , 14(6), 1387-1395. - 6.
Barnard G. Mc Cool J. Wood D. Gerngross T. 2005 Integrated recombinant protein expression and purification platform based on vironmental Microbiolology,71 5735 5742 . - 7.
Brockelbank J. Peters V. Rehm B. 2006 Recombinant strain produces a ZZ domain displaying biopolyester granules suitable for Immunoglobulin G purification. Applied and Environmental Microbiology, 72(11), 7394-7397. - 8.
Campos M. Martinez-Salazar J. Lloret L. Moreno S. Núñez C. Espín G. Soberon-Chávez G. 1996 Characterization of the gene coding for GDP-mannose dehydrogenase () from Azotobacter vinelandii. Journal of Bacteriology,178 1793 1799 . - 9.
Castañeda M. Guzmán J. Moreno S. Espin G. 2000 The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in . Journal of Bacteriology,182 2624 2628 . - 10.
Castañeda M. Sanchez J. Moreno S. Nunez C. Espin G. 2001 The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in . Journal of Bacteriology,183 6787 6793 . - 11.
Cerrone F. Sánchez-Peinado M. Juárez-Jiménez B. González-López J. Pozo C. 2010 Biological teatment of two-phase olive mil wastewater (TPOMW, alpeorujo): Polyhydroxyalkanoates (PHAs) production by strains. Journal of Microbiology and Biotechnology,20 594 601 . - 12.
Chen G. Page W. 1997 Production of poly-β-hydroxybutyrate by in a two-stage fermentation process. Biotechnology Technology,11 347 350 . - 13.
Chen G. Wu Q. 2005 The application of polyhydroxyalkanoates as tissue engineering materials. ,26 6565 6578 . - 14.
Chen G. 2009 A microbial polyhydroxyalkanoates (PHA) based bio-and materials industry. Chemistry Society Reviews,38 2434 2446 . - 15.
Cheze-Lange H. Beunard D. Dhulster P. Guillochon D. Caze A. Morcellet M. Saude N. Junter G. 2002 Production of microbial alginate in a membrane bioreactor. Enzyme Microbiology Technology,30 656 661 . - 16.
Clementi F. 1997 Alginate production by . Critical Reviews in Biotechnology,17 327 361 . - 17.
Conway T. 1992 The Entner-Doudoroff pathway: History, physiology and molecular biology. FEMS Microbiology Reviews,9 1 27 . - 18.
Díaz-Barrera A. Peña C. Galindo E. 2007 The oxygen transfer rate influences the molecular mass of the alginate produced by . Applied Microbiology Biotechnology,76 903 910 . - 19.
Díaz-Barrera A. Silva P. Ávalos R. Acevedo F. 2009 Alginate molecular mass produced by in response to changes of the O2 transfer rate in chemostat cultures. Biotechnology Letters,31 825 829 . - 20.
Díaz Barrera. A. Silva P. Berrios J. Acevedo F. 2010 Manipulating the molecular weight of the alginate produced by in continuous cultures. Bioresource Technology,101 9405 08 . - 21.
Donnan F. Rose R. 1950 Osmotic pressure, molecular weight, and viscosity of sodium alginate.105 113 . - 22.
Dörig G. Pier G. 2008 Vaccines and immunotherapy against , Vaccine,26 1011 1024 . - 23.
Draget I. Taylor C. 2011 Chemical, physical and biological properties of alginates and their biomedical implications.25 251 256 . - 24.
Durner R. . Witholt B. Egli T. 2000 Accumulation of Poly R-β-Hydroxyalkanoates in during growth with octanoate in continuous culture at different dilution rates, Applied and Environmental Microbiology, 66(8), 3408-3414. - 25.
Ertesvag H. Hoidal H. Schjerven H. Svanem B. Valla S. 1999 Mannuronan C-5 epimerases and their application for and in vivo design of new alginates useful in biotechnology. Metabolic Engineering,1 262 269 . - 26.
Freier T. 2006 Biopolyesters in Tissue Engineering Applications.203 1 61 . - 27.
Fukui T. Shiomi N. Doi Y. 1998 Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by . Journal of Bacteriology,180 667 673 . - 28.
Galindo E. Peña C. . Núñez C. . Segura D. Espin G. 2007 Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by . Microbial Cell Factories,6 1 16 . - 29.
Gimmestad M. Ertesvag H. Heggeset T. Aarstad O. Svanem B. Valla S. 2009 Characterization of three new alginate lyases, one of which is involved in cyst germination. Journal of Bacteriology,191 4845 4853 . - 30.
González-López J. . Pozo C. . Martínez-Toledo M. . Rodelas B. Salmeron V. 1996 Production of polyhydroxyalkanoates by H23 in wasteater from olive oil mills (Alpechin), International Biodeterioration and Biodegradation, 38 (3-4), 271-276. - 31.
Grage K. Jahns A. Parlane N. Palanisamy R. Rasiah I. Atwood J. Rehm B. 2009 Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-Beads in biotechnological and biomedical applications. ,10 660 669 . - 32.
Grant G. Morris E. Rees D. Smith P. Thom D. 1973 Biological interactions between polysaccharide and divalent cations: the egg box model. FEBS Letters,32 195 198 . - 33.
Hartmann R. . Hany R. . Pletscher E. . Ritter A. . Witholt B. Zinn M. 2005 Tailor-made olefinic medium chain lenght poly R-hydroxyalkanoates by Gpo1 : Batch versus chemostat production, Biotechnology and Bioengineering, 93 (4), 737-746. - 34.
Hejazi P. . Vasheghani-Farahani E. Yamini Y. 2003 Supercritical fluid disruption of for Poly-β-Hydroxybutyrate recovery, Biotechnology Progress,19 1519 1523 . - 35.
Hernández R. . Orive G. . Murua A. . Pedraz J. 2010 Microcapsules and microcarriers for in situ cell delivery, , 62 (7-8), 711-730. - 36.
Hoesli C. . Raghuram K. . Kiang R. . Mocinecová D. . Hu X. L. . Johnson J. . Lacík I. . Kieffer T. Piret J. 2011 Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation, 108 (2), 424-434. - 37.
Hori Y. Winans A. Huang C. Horrigan E. Irvine D. 2008 Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy. ,29 3671 3682 . - 38.
Hori Y. Stern P. J. Hynes R. Darrell J. 2009 Engulfing tumors with synthetic extracellular matrices for cancer immunotherapy. , 30 (35), 6757-6767. - 39.
Jahns A. Haverkamp R. Rehm B. 2008 Multifunctional Inorganic-Binding Beads Self-Assembled Inside Engineered Bacteria. 19 (10), 2072-2080. - 40.
Jung K. Hazenberg W. Prieto M. Witholt B. 2001 Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates). ,72 19 24 . - 41.
Kashef N. Behzadian-Nejad Q. Najar-Peerayeh S. Mousavi-Hosseini K. Moazzeni M. Gholamreza E. 2006 Synthesis and characterization of alginate-tetanus toxoid conjugate. Journal of Medical Microbiology,55 1441 1446 . - 42.
Kennedy C. Rudnick P. Mac Donald. T. Melton T. 2005 Genus . In: Bergey´s Manual of Systematic Bacteriology, G. M. Garrita, 384-401,2 part B, Springer-Verlag, New York, NY. - 43.
Khanna S. Srivastava A. 2005 Recent advances in microbial polyhydroxyalkanoates. ,40 607 619 . - 44.
Lee S. 1996 Bacterial Polyhydroxyalkanoates49 1 14 . - 45.
Lee S. Park J. Park T. Lee S. Lee S. Park J. K. 2005 Selective Immobilization of Fusion Proteins on Poly(hydroxyalkanoate) Microbeads. ,77 5755 5759 . - 46.
Lozano E. Galindo E. Peña C. 2011 The quantity and molecular mass of the alginate produced by under oxygen-limited and non oxygen-limited conditions are determined by the maximal oxygen transfer rate (OTRmax). Microbial Cell Factories,10 1 13 . - 47.
Ludwig B. Zimmerman B. Steffen A. Yavriants K. Azarov D. Reichel A. Vardi P. Grman T. Shabtay N. Rote A. Evron Y. Neufeld T. Mimon S. Ludwig S. Brendel M. Bornstein S. Barkai U. 2010 A novel device for islet transplantation providing immune protection and oxygen supply. ,42 918 922 . - 48.
Lynn A. Hassid W. 1966 Pathway of alginic acid synthesis in the marine brown alga, Silva. Journal of Biological Chemistry,241 5284 5297 . - 49.
Madison L. Huisman G. 1999 Metabolic Engineering of Poly(3 -Hydroxyalkanoates): From DNA to Plastic. , 63(1), 21-53. - 50.
Manchak J. Page W. 1994 Control of polyhydroxyalkanoates synthesis strain UWD. Microbiology,140 953 963 . - 51.
Mee C. Banki M. Wood D. 2008 Towards the elimination of chromatography in protein purification: Expressing proteins engineered to purify themselves. , 135 (1-2), 56-62. - 52.
Mejía M. Segura D. Espín G. Galindo E. Peña C. 2010 Two stage fermentation process for alginate production by mutant altered in poly-β- hydroxybutyrate (PHB) synthesis. Journal of Applied Microbiology,108 55 61 . - 53.
Ménard M. Dusseault J. Langlois G. Baille W. Tam S. Yahia L. Zhu X. Hallé-P J. 2010 Role of protein contaminants in the inmmunogenicity of alginates. ,DOI:10.1002/jbm.b.31570. - 54.
Morch Y. Holtan S. Donatti I. Strand B. Skjak-Braek 2008 Mechanichal properties of C-5 epimerized alginates. ,9 2360 2368 . - 55.
Myshkina V. Nikolaeva D. Makhina T. Bonartsev A. Bonartseva G. 2008 Effect of growth conditions on the molecular weight of polyhydroxybutyrate produced by 7B, Applied Biochemistry and Microbiology, 44 (5), 482-486. - 56.
Myshkina V. Ivanov E. Nikolaeva D. Makhina T. Bonartsev A. Filatova E. Ruzhitsky A. Bonartseva G. 2010 Biosynthesis of poly-β-hydroxybutyrate-co-hydroxyvalerate copolymer by strain 7B. Applied biochemistry and microbiology, 46 (3), 289.296. - 57.
Noguez R. Segura D. Moreno S. Hernández A. Juárez K. Espín G. 2008 Enzyme INtr, NPr and IIANtr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in. Journal of Molecular Microbiology and Biotechnology15 244 254 . - 58.
Page W. Knosp O. 1989 Hyperproduction of polyhydroxybutyrate during exponential growth of UWD, Applied and Environmental Microbiology, 55(6), 1334-1339. - 59.
Page W. Manchak J. Rudy B. 1992 Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by UWD. Applied and Environmental Microbiology,58 2866 2873 . - 60.
Page W. 1992 Production of polyhydroxyalkanoates by UWD in beet molasses culture, FEMS Microbiology Reviews,103 149 158 . - 61.
Page W. Cornish A. 1993 Growth of UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-hydroxybutyrate. Applied and Environmental Microbiology,59 4236 4244 . - 62.
Page W. Tindale A. Chandra M. Kwon E. 2001 Alginate formation in UWD during stationary phase and the turnover polyhydroxybutyrate. Microbiology,147 483 490 . - 63.
Parente E. Crudele M. Ricciardi A. Mancini M. Clementi F. 2000 Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by DSM576 in batch fermentation. Journal of Industrial Microbiology and Biotechnology,25 242 248 - 64.
Peña C. Campos N. Galindo E. 1997 Changes in alginate molecular mass distributions, broth viscosity and morphology of cultured in shake flasks. Applied Microbiology and Biotechnology,48 510 515 . - 65.
Peña C. Trujillo-Roldan M. Galindo E. 2000 Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of . Enzyme and Microbiology Technology,27 390 398 . - 66.
Peña C. Hernandez L. Galindo E. 2006 Manipulation of the acetylation degree of alginate by supplementing the culture medium with 3-(N-morpholino)-propane-sulfonic acid. Letters of Applied Microbiology,43 200 204 . - 67.
Peña C. Peter C. Büchs J. Galindo E. 2007 Evolution of the specific power consumption and oxygen transfer rate in alginate-producing cultures of conducted in shake flasks. Biochemical Engineering Journal,36 73 80 . - 68.
Peña C. Millán M. Galindo E. 2008 Production of alginate by in a stirred fermentor simulating the evolution power input observed in shake flasks. Processes of Biochemistry,43 775 778 . - 69.
Peña C. Galindo E. Büchs J. 2011 The viscosifying power, degree acetylation and molecular mass of the alginate produced by in shake flasks are determined by the oxygen transfer rate. Process Biochemistry,46 290 297 . - 70.
Peralta-Gil M. Segura D. Guzmán J. Servin-Gonzalez L. Espin G. 2002 Expression of the poly-beta-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and is dependent on the transcriptional activator PhbR. Journal of Bacteriology,184 5672 5677 . - 71.
Peters V. Rehm B. 2008 Protein engineering of streptavidin for assembly of streptavidin beads. Journal of Biotechnology,134 266 274 . - 72.
Pindar D. Bucke C. 1975 The biosynthesis of alginic acid by . Biochemistry Journal,152 617 622 . - 73.
Poole R. Hill S. 1997 Respiratory protection of nitrogenase activity in , roles of the terminal oxidases. Biosciences Reports,17 303 317 . - 74.
Post E. Kleiner D. Oelze J. 1983 Whole cell respiration and nitrogenase activities in growing in oxygen controlled continuous culture. Archives of Microbiology,134 68 72 . - 75.
Priego-Jiménez R. Peña C. Ramírez O. T. Galindo E. 2005 Specific growth rate determines the molecular weight of the alginate produced by . Biochemistry Engineering Journal,25 187 193 . - 76.
Pyla R. Kim T. Silva J. Jung Y. 2009 Overproduction of poly-beta-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF. , 84 (49), 717-724. - 77.
Quagliano J. Miyazaki S. 1997 Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from 6B, Applied microbiology and biotechnology,48 662 664 . - 78.
Remminghorst U. Rehm B. 2006 Bacterial alginates: from biosynthesis to applications. Biotechnology Letters,28 1701 1712 . - 79.
Reyes C. Peña C. Galindo E. 2003 Reproducing shake flasks performance in stirred fermentors: production of alginates by . Journal of Biotechnology,105 189 198 . - 80.
Ruth K. Grubelnik A. Hartman R. Egli T. Zinn M. Ren Q. 2007 Efficient production of (R)-β-hydroxycarboxylic acids by biotechnological conversion of Polyhydroxyalkanoates and their purification. . 8(1), 279-286. - 81.
Sabra W. Zeng A. Sabry S. Omar S. Deckwer-D W. 1999 Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of under microaerobic conditions. Applied Microbiology and Biotechnology,52 773 780 . - 82.
Sabra W. Zeng A. Lunsdorf H. Deckwer-D W. 2000 Effect of oxygen on formation and structure of alginate and its role in protecting nitrogenase Applied and Environmental Microbiology,66 4037 4044 . - 83.
Sabra W. Zeng A. Deckwer W. D. 2001 Bacterial alginate: physiology, product quality and process aspects. ,56 315 325 . - 84.
Sabra W. Zeng A. 2009 Microbial production of alginates: Physiology and Process Aspects, In: , Bernd H. A. Rehm, 153-173, Microbiology Monographs13 Springer Verlag,DOI: 10.1007/978-3-540-92679-5, Berlín-Heidelberg. - 85.
Saude N. Junter G. 2002 Production and molecular weight characteristics of alginate from free and immobilized-cell cultures of . Process Biochemistry 2 (37), 895-900. - 86.
Segura D. Espin G. 1998 Mutational inactivation of a gene homologous to ptsP affects poly-beta-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii. Journal of Bacteriology,180 4790 4798 . - 87.
Segura D. Vargas E. Espín G. 2000 Beta-ketothiolase genes in . Gene,260 113 120 . - 88.
Segura D. Cruz T. Espin G. 2003a Encystment and alkylresorcinol production by strains impaired in polybeta- hydroxybutyrate synthesis. Archives of Microbiology,179 437 443 . - 89.
Segura D. Guzmán J. Espín G. 2003b mutants that overproduce poly-beta-hydroxybutyrate or alginate. Applied Microbiology and Biotechnology,63 159 163 . - 90.
Senior P. Dawes E. 1971 Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in . Biochemistry Journal,125 55 66 . - 91.
Senior P. Beech G. Ritchie G. Dawes E. 1972 The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of . Biochemistry Journal,128 1193 1201 . - 92.
Senior P. Dawes E. 1973 The regulation of poly-β-hydroxybutyrate metabolism in . Biochemistry Journal,134 225 238 . - 93.
Setubal J. dos Santos. P. Goldman B. Ertesvåg H. Espin G. Rubio L. Valla S. Almeida N. Balasubramanian D. Cromes L. Curatti L. Du Z. Godsy E. Goodner B. Hellner-Burris K. Hernandez J. Houmiel K. Imperial J. Kennedy C. Larson T. Latreille P. Ligon L. S. Lu J. Mærk M. Miller N. Norton S. O’Carroll I. Paulsen I. Raulfs E. Roemer R. Rosser J. Segura D. Slater S. Stricklin S. Studholme D. Sun J. Viana C. Wallin E. Wang B. Wheeler C. Zhu H. Dean D. Dixon R. Wood D. 2009 The genome sequence of , an obligate aerobe specialized to support diverse anaerobic metabolic processes. Journal of Bacteriology. 191(14), 4534-4545. - 94.
Skjak-Braek G. Grasdalen H. Larsen B. 1986 Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydrates Research,154 239 250 . - 95.
Smidsrod O. Draget K. 1996 Chemistry and physical properties of alginates. Carbohydrates European,14 6 12 . - 96.
Socolofsky M. Wyss O. 1962 Resistance of the cyst. Journal of Bacteriology,84 119 124 . - 97.
Steinbüchel A. Lütke-Eversloh T. 2003 Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms.16 81 96 . - 98.
Sun H. Pan, Zhigang Y. Shi M. 2007 The immune response and protective efficacy of vaccination with oral microparticle vaccine in mice. International Immunopharmacology,7 1259 1264 . - 99.
Sun Z. Ramsay J. Guay M. Ramsay B. 2007 Fermentation process development for the production of medium chain length poly-β-hydroxyalkanoates. ,75 475 485 . - 100.
Suriyamongkol P. Weselake R. Narine S. Moloney M. Shah S. 2007 Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants- A review. .25 148 175 . - 101.
Taguchi S. Doi Y. 2004 Evolution of polyhydroxyalkanoate (PHA) production system by “Enzyme Evolution”: successful case studies of directed evolution.4 145 156 . - 102.
Trujillo-Roldán M. Moreno S. Segura D. Galindo E. Espín G. 2003 Alginate production by an mutant unable to produce alginate lyase. Applied Microbiology Biotechnology,60 733 737 . - 103.
Trujillo-Roldán M. Moreno S. Espín G. Galindo E. 2004 The roles of oxygen and alginate-lyase in determining the molecular weight of alginate produced by . Applied Microbiology Biotechnology,63 742 747 . - 104.
Valappil S. Misra K. Boccaccini A. Roy I. 2006 Biomedical applications of polyhydroxyalkanoates, an overview of animal testing and in vivo responses. 853-868. - 105.
Vázquez A. Moreno S. Guzmán J. Alvarado A. Espín G. 1999 Transcriptional organization of the algGXLVIFA genes: characterization of algF mutants. Gene,232 217 222 . - 106.
Verlinden R. Hill D. Kenward M. Wiliams C. Radecka I. 2007 Bacterial synthesis of biodegradable polyhydroxyalkanoates, ,102 1437 1449 . - 107.
Wang Z. Wu H. Chen J. Zhang J. Yao Y. Chen G. Q. 2008 A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles. ,8 1957 1962 . - 108.
Wang-H H. Zhou X. Liu Q. Chen-Q G. 2011 Biosynthesis of polyhydroxyalkanoate homopolymers by89 1497 1507 . - 109.
Williams S. Martin D. 2005 Applications of Polyhydroxyalkanoates (PHA) in Medicine and Pharmacy. .DOI: 10.1002/3527600035.bpol4004. - 110.
Wu G. Moir A. Sawers G. Hill S. Poole R. 2001 Biosynthesis of polybeta-hydroxybutyrate (PHB) is controlled by CydR (Fnr) in the obligate aerobe . FEMS Microbiology Letters,194 215 220 . - 111.
Wu Q. Wang Y. Chen G. 2009 Medical application of microbial biopolyesters Polyhydroxyalkanoates. ,37 1 12 . - 112.
Yang J. S. Xie Y. J. He W. 2011 Research progress on chemical modification of alginate: A review.84 33 39 . - 113.
Yao Y. Zhan X. Zhang J. Zou X. Wang Z. Xiong Y. Chen J. Chen G. 2008 A specific drug targeting system based on polyhydroxyalkanoate granule binding protein PhaP fused with targeted cell ligands. , 29(36), 4823-4830. - 114.
Yasotha K. Aroua M. Ramachandran K. Tan I. 2006 Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration,30 260 268 . - 115.
Zapata-Vélez A. Trujillo-Roldán M. 2010 The lack of a nitrogen source and/or the C/N ratio affects the molecular weight of alginate and its productivity in submerged cultures of . Annals of Microbiology,60 661 668 . - 116.
Zhang X. Luo R. Wang Z. Deng Y. Chen G. 2009 Application of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels. ,10 707 711 . - 117.
Zinn M. Witholt B. Egli T. 2001 Occurrence, synthesis and medical application of bacterial Polyhydroxyalkanoate. ,53 5 21 . - 118.
Zinn M. Weilenmann H. Hany R. Schmid M. Egli T. 2003 Tailored synthesis of poly R-β-hydroxybutyrate-co-hydroxyvalerate (PHB/HV) in DSM 428, Acta Biotechnologica,23 309 316 .