BR102016015367A2 - ESTABLISHMENT OF PARAMETERS FOR THE OBTAINATION OF HIGH MOLECULAR AND LOW GRAY CRYSTALLINITY POLYHYROXIALCANOATE FROM GROSS GLYCERINE - Google Patents

Abstract

The present patent document (pi) relates to the establishment of parameters of a batch method for producing high molecular weight and low crystallinity polyhydroxyalkanoates (phass) from the bacterial fermentation of crude glycerine. The parameterized method results in phas with high molecular mass, reduced 3-hydroxybutyrate content in the chain and therefore with low crystallinity. the crystallinity degree of the phas can range from 47% to 68% and the molecular mass from 630 to 920 kda, depending on different aeration rates and agitation speeds, origin of glycerine from biodiesel (vegetable or animal), microorganism and need for supplementation of the culture medium. It is noted that the formation of low crystallinity phas and high molecular mass is associated with a combination of these process variables. It is noteworthy that the main feature that differentiates this method from those found in the state of the art is the use of aeration and agitation as control variables to favor the synthesis of high molecular weight and low crystallinity phas, thus representing an alternative to make this method easier. material more suitable for processing and consequently expand its possibilities for industrial applications.

Description

INVENTION PATENT DESCRIPTIVE REPORT

SETTING PARAMETERS TO OBTAIN

POIJHTDROXIALC AN HIGH MOLECULAR MASS ATTACK AND LOW GRADE CRYSTALLINITY FROM RAW GLYCERIN

FIELD OF INVENTION

[01] The present invention is a method for obtaining high molecular weight and low crystallinity polyhydroxyalkanoates (PHAs) from the bacterial fermentation of crude glycerin. The material obtained can be used as raw material in tissue engineering, pharmaceutical sector and, preferably, for the manufacture of biodegradable packaging.

BACKGROUND OF THE INVENTION

[02] Polyhydroxyalkanoates (PHAs) are polyesters accumulated by various microorganisms as stockpiles, subject to limitation of an essential nutrient for growth, and excess carbon source (KHARDENAVIS, AA; KUMAR, MS; MUDLIAR, SN; CHAKRABARTI, T. Biotechnological conversion of agro-industrial wastewater into biodegradable plastic, polyhydroxybutyrate, Biosource Technology, v. 98, pp. 3579-3584, 2007; LAYCOCK, B.; HALLEY, P.; Pratt, S.; WERKERC, A LANTA, P. The chemomechanical properties of microbial polyhydroxyalkanoates (Progress in Polymer Science, p.1-48, 2012). The constituents of PHAs may be short chain hydroxy esters, which contain from 3 to 5 carbon atoms in the chain; medium chain, which comprise hydroxy esters containing from 6 to 16 carbon atoms in the chain; and long chain, which comprise hydroxy esters with 17 or more carbon atoms in the main chain (SQUIO, CR; ARAGÃO, GMF. Cultivation strategies for the production of poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate) biodegradable plastics. -co-3-hydroxyvalerate) by bacteria., Chemistry Nova, v. 27, no. 4, pp. 615-622, 2004).

[03] PHA polymers have thermoplastic and physicochemical properties very similar to various conventional polymers derived from nonrenewable fossil resources such as polypropylene and polyethylene (MIZUNO, K .; OHTA, A .; HYAKUTAKE, M .; ICHINOMIYA, Y. TSUGE, T. Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4. Polymer Degradation and Stability, v. 95, n.8, pp. 1335-1339, 2010). In addition, they are completely biodegradable and biocompatible, coming from renewable, recyclable and non-toxic raw materials, which make them highly applicable to petrochemical polymers. PHAs can be employed in tissue engineering, pharmaceuticals and biodegradable packaging manufacturing (LEE, SY; CHOI, J. Effect of fermentation performance on the economics of poly (3-hydroxybutyrate) production by Alcahgenes latus. Polymer Degradation and Stability, v 59, no. 1-3, pp. 387-393,1997).

[04] Despite the wide range of applications in the industry, PHAs are about four times more expensive to produce than petrochemical polymers (FALCONE, D. B. B .; AGNELLI, JAM. area of biodegradable polymers Polymers: Science and Technology, v. 17. η 1. p. 5-9, 2007). Given this, different strategies of economic viability of the production of PHAs have been investigated. The literature cites the use of glucose and fructose as conventional carbon sources for biopolymer production (SUTHERLAND, I. A Sticky Business: Microbial Polysaccharides: Current Products and Future Trends. Microbiology Today, v. 29, pp. 70-71, 2002). On the other hand, the use of low cost alternative substrates in fermentative processes, such as industrial residues, allows the reduction of production costs, and simultaneously collaborates to minimize environmental problems, as it helps in the destination of these residues.

[05] Crude glycerin is an important byproduct of the biodiesel production process. Every 45.3 kg of biodiesel produced 4.53 kg of residual glycerine is generated (CARDONA, C.; POSADA, J .; MONTOYA, M. Use of Glycerol from Biodiesel Production: Conversion to Added Value Products. In: Proceedings of European Congress of Chemical Engineering, v. 1, pp. 16-20, 2007). It is estimated that between 2008 and 2013, the biodiesel industry will have a surplus of 70,000 tons of glycerine per year, thus increasing the possibilities of environmental impacts (ALBARELLI, JQ; SANTOS, DT; HOLLAND, MR Energetic and economic evaluation of waste glycerol cogeneration in Brazil, Brazilian Journal of Chemical Engineering, v. 28, no. 04, pp. 691-698, 2011). Crude glycerin is mainly composed of ethanol or methanol residues, glycerol, residual fatty acids and minerals, which are direct precursors for bacterial synthesis of biopolymers such as PHAs (JOHNSON, DT; TACONI, KA). added conversion of crude glycerol resulting from biodiesel production Environmental Progress, v. 26, no. 4, pp. 338-348, 2007; ANDRE, B.; LANGE, AB; ROBENEK, H.; STEINBUCHEL, A. Conversion of Glycerol to Poly (3-Hydroxypropionate) in Recombinant Escherichia coli (Applied and Environmental Microbiology, v. 76, no. 2, pp. 622-626, 2010.).

[06] The PHA family has a wide range of mechanical properties, from strongly crystalline to elastic, depending on the composition and especially the size of the hydroxy ester units that make up the polymer. These biopolymers can exhibit polymerization degree of up to 30,000, confirmed by the high molar masses. Thus, PHA polymers with high molecular weight and low amount of short chain hydroxy esters increase the thermoplastic capacity of the PHA polymer (FORMOLO, MC Biodegradable polymer biosynthesis: thermal and spectroscopic characterization. Annals of the VII Brazilian Polymer Congress. Belo Horizonte 2003. Brazilian Association of Polymers), and, consequently, its commercial suitability (SIM, SJ, SNELL, KD, HOGAN, SA, et al. PHA synthase activity Controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. , v. 15, pp. 63-67, 1997).

[07] In this context, the degree of crystallinity of the different PHA polymers depends on the polymer composition, being 60-80% for short chain hydroxy esters such as P (3HB) and decreasing to 30-40% for PHA copolymer. medium and long chain, such as hydroxyvalerate (HV) (SERAFIM, LS; LEMOS, PC; ALBUQUERQUE, MG; KINGS, A. A. Strategies for PHA production by mixed cultures and renewable waste materials. Applied and Environmental Microbiology, v.81, pp.615-628, 2008). PHA polymers with units containing more than 6 carbons and medium side chain (PHAMSC) have lower crystallinity level and are more elastic (MADISON, LL; HUISMAN, GW) Metabolic engineering of poly (3-ydroxyalkanoates): from DNA to plastic. Microbiology and Molecular Biology Reviews, v. 63, η 1, pp. 21-53,1999).

[08] Thus, the present innovation deals with a batch process to obtain high molecular weight, low percentage short chain hydroxy esters and low crystallinity polyhydroxyalkanoates (PHAs) obtained from submerged cultivation in medium containing crude glycerin.

[09] A method has been developed to increase the molecular weight of the polymer while minimizing the synthesis of short chain hydroxy esters during the process. The result is a long-chain, low-crystallinity polymer, which is therefore more thermally processable.

[10] The novelty of the invention is in the method of conducting the bacterial culture, in which the aeration rate and agitation rate of the medium is controlled to provide the synthesis of high molecular weight and low crystallinity PHAs. The joint action of these characteristics makes this material more suitable for thermal processing and, thus, expands its industrial applications, especially in the packaging sector, besides contributing to the reduction of polymer production costs and enabling an alternative use of glycerine in gross form.

BRIEF DESCRIPTION OF THE INVENTION

[11] The present patent document relates to the production of high molecular weight and low crystallinity polyhydroxyalkanoates (PHAs) by controlling the aeration and agitation of the submerged culture in medium containing crude glycerin, whose main purpose is to make this material more suitable for thermal processing. Specifically, the invention relates to a method of submerged cultivation of PHA-producing bacteria in medium containing residual glycerine from biodiesel, in which control of the erection rate and agitation rate allows the production of long chain PHAs, which results in increased molecular weight of the biopolymer, as well as minimizing the synthesis of short-chain hydroxy esters, which gives the PHA polymer greater crystallinity. These characteristics together enable the thermal processability of PHAs obtained from crude glycerine fermentation and its various industrial applications, especially in the packaging sector. Thus, what is proposed in this patent document has novelty parameter, since to date, no scientific or technical work understood in the state of the art has a similar mechanism of production, functionality and application, and can preferably be used for packaging manufacture. biodegradable.

BRIEF DESCRIPTION OF THE FIGURES

[121 Figure 1 shows a chromatogram obtained by high performance liquid chromatography with molecular exclusion column, which was used to quantify the molecular mass of polyhydroxyalkanoates from the calibration curve of polystyrene molecular mass standards. Figure 2 shows an X-ray difiatogram used to calculate crystallinity index of polyhydroxyalkanoate sample from the crystalline peak area.

DETAILED DESCRIPTION OF THE INVENTION

[13] In order that the process of the invention may be better understood and evaluated, its detailed description will be given below.

[14] The invention is the development of a method whose establishment of process parameters enables the production of high molecular weight, low short chain hydroxy esters PHAs from submerged cultivation in medium containing crude glycerin in order to of taking this cost-effective thermally processable biopolymer and can compete with conventional plastics and be widely applied in various industry sectors, especially for packaging production.

[15] The present invention discloses all parameters related to the process of obtaining high molecular weight and low crystallinity PHAs from the establishment of the aeration and stirring speed variables of the fermentative cultivation step. Thus, it is proposed in this patent document, a product with more thermoplastic characteristics, as a more viable alternative for thermal processing of this biopolymer, since to date there is no commercially available method that relates process variables such as aeration and velocity. stirring and obtaining said product characteristics.

[16] In order to prove the application of aeration and agitation control to obtain high molecular weight and low crystallinity PHAs produced in medium containing crude glycerin, experiments were carried out with PHA-producing microorganisms and crude glycerin. . PHA polymers are obtained using two precultures and the final culture. In the first preculture, 10 µl of the microorganism colony should be inoculated in nutrient broth and incubated on an orbital shaker at 28 to 32 ° C with agitation of 150 rpm for a period of 24 hours. From this preculture a spinning of about 8 to 12% is transferred to an unrestricted mineral medium, preferably composed of nitriloacetic acid, ferrous ammonium citrate, MgS04.7H20, CaCl2.2H20, (NH4) 2S04, ΚΗ2Ρθ4 and Na2HP04 12H20, which must be incubated on an orbital shaker at a temperature of 30 ° C, shaking at 150 rpm for a period of 24 hours. The second preculture should be transferred to a mechanically shaken bioreactor containing limited media preferably composed of Na2HP04.12H20, ΚΗ2Ρθ4, H3BO3, CoC12.6H20, ZnS04.7H20, Na2Mo04.2H20, NiCl2.6H20, CuS04. 5H20 from 10.0 to 15.0% crude glycerin and initial pH 7.0. The process should be conducted for a period of 72 h, with the system temperature maintained at 35 ° C, aeration 0.5 to 1.5 wm and medium stirring speed 200 to 750 rpm.

[17] At the end of the process, the fermented medium should be centrifuged, the biomass collected, washed with distilled water and oven dried (20 to 40 ° C). Recovery of the biopolymer may be conducted from cell drying and extraction with the aid of chloroform and sodium hypochlorite (2: 1 v / v) under heating at 50 to 90 ° C and subsequent evaporation of the solvent. It can also be with chloroform (50: 1 v / v) for 2 h at 60 ° C, drying the cells by lyophilization followed by extraction with chloroform and by submitting the cells with chloroform in ultrasound for 2 h at 30 ° C, among others. .

[18] The experiments of the described method result in the production of medium chain or low chain hydroxy esters PHA polymers with molecular weight ranging from 600 to 1000 kDa; and polymers with low 3-hydroxybutyrate content in the chain, which may have a degree of crystallinity of 40% to 80%, due to different aeration rates and agitation speed, origin of crude glycerine (animal or vegetable), microorganism and need for supplementation of the culture medium.

[19] The description thus far of obtaining high molecular weight and low crystallization PHAs from the control of the aeration and agitation parameters of the crude glycerin compound medium refers to one of its preferred embodiments. It should, however, once again be made clear that the invention is not limited to the disclosed embodiment, as those skilled in the art will immediately realize that changes and substitutions may be made within this inventive concept described herein. Accordingly, it cannot in any way be construed as limiting the invention, which is limited to the scope of the following claims. EXAMPLE 1: [20] As a non-restrictive demonstrative reference, crude glycerin from oil and residual fats was donated by the Pilot Plant of the Federal University of Bahia, Salvador, Brazil; from castor oil, donated by Comanche Combustíveis da Bahia LTDA, Simões Filho, BA, Brazil and from soybean oil, donated by Petrobras, Camaçari, BA, Brazil. PHA-producing bacterial strains were donated by the Institute for Technological Research (IPT), São Paulo, Brazil. EXAMPLE 2: [21] As a non-restrictive demonstrative reference, the conservation and maintenance of the bacterial strains Cupriavidus necator IPT 026, Cupriavidus necator IPT 027 and Burkholderia cepacia IPT 438, used for PHA accumulation, was obtained by subculturing every 15 days, in tubes containing nutrient broth composed of 5.0 g L '1 meat peptone 3.0 g L1 meat extract and 15 g L1 agar, incubated at 28 to 32 ° C for 48 hours and then packaged under refrigeration (4 ° C). For cell propagation a solution of inoculum with microorganisms in nutrient broth was produced, incubated for 24 hours in an orbital shaker at 30 ± 2 ° C and shaking at 150 rpm. Inoculum samples were collected every 3 hours and analyzed for growth by absorbance reading at 620 nm on a spectrophotometer and colony counting by depth technique on nutrient agar, totaling in 24 hours 1012 CFU mL'1. EXAMPLE 3: [22] As a non-restrictive demonstrative reference, 8-12% of the inoculum obtained according to EXAMPLE 2 was transferred to 250 mL Erlenmeyer flasks containing 80 mL of a 19.1 g nitrogen-free mineral solution composed of nitrogen. L1 of nitrilotriacetic acid, 10 g L "1 ferrous ammonium citrate, 50 g L" 1 MgSO4.7H20, 5 g L1 CaCl2.2H20, 200 g L1 (NH4) 2 SO4 and 223.8 g L1 Na2HP04. 12H20, pH of medium adjusted to 7 and incubated on orbital shaker at 30 ± 2 ° C, 150 rpm. After 24 hours, this medium was aseptically transferred to a bioreactor containing nitrogen-limiting mineral medium (50 g L "1 of (NH4) 2 SO4) and 8 to 20 g L1 crude glycerin. The pH of the medium was adjusted to 7. Cultures were tested with agitation at 200 to 900 rpm and aeration at 0.3 to 2.0 wm After 96 hours of fermentation, the cells were separated from the medium and the biopolymer extracted EXAMPLE 4: [23] With reference demonstrative and non-restrictive, the PHA samples obtained according to EXAMPLE 3 were dissolved in chloroform, PTFE membrane filtered and subjected to molecular mass analysis in a GPC-HPLC system equipped with a refractive index (RI) detector. The molecular weights of the PHA polymers, depending on the cultivation conditions, ranged from 600 to 960 kDa using 15 g L'1 of crude glycerin, 200 rpm and 0.3 wm. , 600 kDa was obtained and using 10 g L1 of crude glycerin, 500 rpm and 1.0 wm, gave 960 kDa. EXAMPLE 5: [24] With demonstrative and non-restrictive reference, PHA polymer samples obtained according to EXAMPLE 3 were dried, subjected to methanolysis and separated to the organic phase for analysis of chemical composition in GC-MS library system. NIST The mass spectrometer was programmed to scan in the range of 50 to 550 m / z and temperature from 80 ° C to 200 ° C (20 ° C min -1). PHA monomers were identified by comparison with standard retention time and mass spectra. The composition of the PHA polymers varied with the process conditions. The use of 15 g L'1 crude glycerin, 200 rpm and 0.3 wm generated 40% 3-hydroxybutyrate and 60% 8-hydroxynanoate, while the use of 10.0 g L'1 crude glycerine, 500 rpm and 1.0 wm generated 25% 3-hydroxybutyrate and 30% 3-hydroxypentanoate. EXAMPLE 6: [25] For demonstrative and non-restrictive reference, PHA samples obtained according to EXAMPLE 3 were subjected to percent crystallinity index analysis, determined by X-ray diffractometer with λ = 1.5433 Â and scanning. between 5.0 ° and 50 ° and the results used to calculate PHA crystallinity. The crystallinity percentages ranged from 45 to 70%, depending on the cultivation conditions. Using 15 g L'1 crude glycerin, 200 rpm and 0.3 wm, obtained 70% crystallinity index PHAs polymer and using 10 g L'1 crude glycerin, 500 rpm and 1.0 wm, obtained 45% crystallinity.

Claims (4)

[01] SETTING PARAMETERS TO OBTAIN POLEHEDROXIALC IN HIGH MOLECULAR MASS AND LOW CRYSTALLINITY ACT FROM THE RAW GLYCERIN CHARACTERIZED BY utilizing the aeration and agitation rate of the minimally supplemented crude glycerine containing medium the synthesis of high molecular weight polyhydroxyalkanoates and low short chain hydroxy esters, and consequently lower degree of crystallinity, which makes this biopolymer more suitable for thermal processing. [02] SETTING PARAMETERS TO OBTAIN POLEHEDROXIALC IN HIGH MOLECULAR MASS AND LOW CRYSTALINITY ACT ACCORDING TO Claim 1, CHARACTERIZED BY USING COMPOSED CULTURE MEDIA PREFERREDLY FROM 1.0 TO 15.0 % of crude glycerin, which may originate from plant or animal sources, as the main carbon source for accumulation of PHAs in cells, as well as sources of nitrogen, phosphorus, calcium, magnesium, among other trace elements. [03] ESTABLISHMENT OF PARAMETERS TO OBTAIN HIGH MOLECULAR MASS POLEHEDROXIALCANOATE AND LOW CRYSTALINITY DEGREE, ACCORDING TO Claim 1 and 2, CHARACTERIZED BY Obtaining polyhydroxyalkanoates from broth with bacterial media in a specially bred bacterial culture medium pH between 6 and 8, in an aeration range of 0.3 to 3.0 wm, agitation speed between 150 and 900 rpm, temperature between 28 and 37 ° C for a period ranging from 24 to 120 h of cultivation. [4] SETTING PARAMETERS FOR POLEHEDROXIALC ACHIEVING HIGH MOLECULAR MASS AND LOW CRYSTALINITY ACT FROM RAW GLYCERIN according to claim 1, 2 and 3, CHARACTERIZED BY separating cells from the medium by centrifugation Flocculation, among others, should subsequently be dried in an oven, lyophilizer, spray-dryer, or other drying method, and extract the biopolymer from the interior of cells under heating with the aid of organic solvents such as chloroform, dichloromethane, propylene carbonate. dichloroethane, combination of these solvents, combination of chloroform and sodium hypochlorite, among other organic solvents and combinations thereof, followed by evaporation; Dry cells may also be subjected to treatments with organic solvents and hypochlorite, followed by ultrasound heating; extraction by acid hydrolysis, action of alkaline hypochlorite, enzyme cocktails, detergents, among other agents.