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    • 专利摘要:
    Process for the production of polyhydroxyalkanoates and ectoin by simultaneous saccharification and fermentation from cereal grain hydrolysates. The invention relates to a process for using liquefied cereal grain that makes it possible to obtain polyhydroxyalkanoates (pha) and ectoin by means of a simultaneous fermentation and saccharification process (ssf) using a halophilic bacterium. The procedure allows to flexibilize the operation of an ethanol biorefinery by deriving a fraction of the raw material to produce pha and/or ectoin, depending on the market price of the fuels. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2562377A1
    申请号:ES201531498
    申请日:2015-10-19
    公开日:2016-03-03
    发明作者:María GARCÍA TORREIRO；María LÓPEZ ABELAIRAS；Thelmo Alejandro LÚ CHAU；Juan Manuel Lema Rodicio
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    Procedure for the production of polyhydroxyalkanoates and ectoin by simultaneous saccharification and fermentation from cereal grain hydrolysates
    TECHNICAL SECTOR OF THE INVENTION
    The present invention relates to a process for producing polyhydroxyalkanoates (PHA) and ectoin from cereal grain hydrolysates. The process, object of the present invention can be integrated into a bioethanol production facility in which part of the cereal would be derived, once pretreated, into the production of bioplastics and ectoin. This invention is based on the adaptation of the simultaneous fermentation and saccharification (SSF) process, used in the production of ethanol, for the production of PHA and ectoin
    STATE OF THE TECHNIQUE
    The world production of plastics in 2012 reached 288 million tons. It is estimated that only 6% of plastic waste is recycled. A recent study estimates that some 270,000 tons of plastic waste are drifting in the oceans (Eriksen M. et al., 2014; Plos ONE 9: e111913. Doi: 10.1371 / joumal.pone.0111913). Despite being a material manufactured to resist and last over time, it is often also designed for single-use applications. Biodegradable or biodegradable plastics could replace fossil plastics in many of their applications. The ability to be degraded easily would be an important environmental advantage over fossil plastics
    There are different types of microorganisms capable of producing and accumulating different types of PHA, although those that are most frequently obtained are polyhydroxybutyrates (PHB) and polyhydroxivaleriates (PHV). In general, they are classified as thermoplastic polyesters. These materials are accumulated by various microorganisms inside as reserve material. Depending on the accumulation mechanism or kinetics used, microorganisms can be classified into three categories (Lopar M et al., 2013; Biochem Eng J 79 · 57-70):
    i) Species that show a strict separation between the growth phase and the production phase of PHA under a limitation of nitrogen or phosphorus (eg Methylomonas extorquens).
    ii) Species that accumulate PHA under balanced nutrient conditions, but reach maximum production under nutrient limitation such as nitrogen or phosphorus (eg Cupriavidus necator)
    iii) Species that reach high accumulation rates even without any limitation of essential nutrients (eg Azahydromonas can).
    Halomonas boliviensis belongs to the first type of species, so that in order to change the metabolism towards the accumulation of PHA it is necessary to apply a limitation on some essential nutrient such as nitrogen or phosphorus. In previous studies, it has been indicated that this halobacterium is capable of accumulating PHA in a proportion of up to 80% of its weight, using glucose as a substrate in a fed-batch operation (Quillaguamán J. et al., 2008; Appl Microbiol Biotechnol 78: 227-232). In addition, due to its halotolerant character, ectoin is obtained as an extracellular by-product, a solute compatible with interesting properties for cosmetic products, such as protein stabilizer and other cellular structures against UV radiation and dryness (Pastor JM et al., 2010; Biotechnol Adv 28: 782-801)
    The production of bioplastics still cannot compete in terms of production costs with petroleum plastics. The current production costs of PHA (4-6 € Ikg) are significantly higher than those of synthetic plastics obtained through chemical processes (1-2 € / kg). The factors that most influence this cost are the price of the substrate and the recovery stage of the bioplastic, being able to represent the cost of the substrate up to 50% of the total production cost. This percentage was reduced to 30% when integrated production of PHA and ethanol was carried out using sugar cane as raw material (Nonato R.v. et al., 2001; Appl Microbiol 57: 1-5). When pure cultures are used in the production of bioplastics, maintaining sterile conditions in the bioreactor represents an additional cost to take into account
    Simultaneous saccharification and fermentation is a process frequently used in the production of elanol from cereal. Some of its advantages over the use of a two-stage saccharification and fermentation process (SHF) are: i) reduction of the risk of microbial contamination; ii) lower osmotic stress due to reduced glucose concentration and Hi) greater energy efficiency (Bothast RJ. and Schlicher MA, 2005; Appl Microbiol Biotech 67: 19-25). Some previous patents have used a modified form of this process to produce ethanol using a thermotolerant yeast (Otto E. and Escovar-Kousen J., 2005. US

    2005f0026261 A1), in the production of butanol by the ABE process (Davidov EH. Et al., 2011 WO 2010087737 A3) And in the production of monosodium glutamate, lactic and succinic acid (Bergsma MH et al., 2012_WO 2012019159 A 1) _ Previously, the production of PHA from wheat hydrolysates has been studied using Cupriavidus necator bacteria following a two-stage hydrolysis and fermentation scheme
    5 (Koutinas AA et al., 2007; Enz Microb Technol 40: 1035-1044), but there are no previous reports of its production applying an SSF process
    DESCRIPTION OF THE INVENTION
    The present invention relates to a process for the production of PHA and ectoin by adapting the process of obtaining bioethanol from cereal grain, in an integrated biorefinery scheme. In this way, from a single input current, various products (ethanol, PHA, ectoin) can be obtained
    The invention allows to reduce the costs of PHA production by integrating into an ethanol biorefinery, using a low-cost substrate, the use of a halophilic bacterium, which does not require a sterilization process prior to fermentation, and obtaining a ca-product of high commercial value such as ectoin. Integration in a biorefinery of ethanol and PHA production helps reduce the costs associated with
    15 raw material (cereal) and reagents (enzymes). It also allows the operation of the plant to be flexible for the production of variable proportions of ethanol, PHA and ectoin, depending on the market price of fuels.
    The invention relates to a process for using liquefied cereal grain, characterized in that it comprises the following steps:
    20 a) centrifuge the liquefied cereal grain;
    b) subject the obtained supernatant, cereal must, to a simultaneous fermentation and saccharification (SSF) process in a reactor;
    c) centrifuge the fermented must in the previous stage and separate the bacterial biomass, which contains the PHA, from the supernatant containing the ectoin;
    D) subject the bacterial biomass to a procedure of extraction and purification of the PHA; Y
    e) concentrate the supernatant obtained after centrifugation of the fermented must using evaporation under reduced pressure and purify the ecloin.
    Taking into account the characteristics of the process, centrifugation of the feed stream is essential to eliminate, at least, the larger solids_ Since the polymers accumulate
    30 intracellularly, prior separation of solids from the feed will facilitate the product extraction process. It was found that despite the solid-liquid separation after liquefaction, the sugar yield of saccharification is maintained
    In a preferred embodiment, the initial volume of the fermentation medium, formed by the compounds indicated in Table 2, is 35% with respect to the final working volume. In addition, in a particular embodiment, the fermentation medium is subjected to a sterilization process at a preferred temperature of 115Q C for about 20 minutes.
    The process of saccharification and simultaneous fermentation of cereal must includes:
    a) progressive filling of the reactor; Y
    b) reactor operation in batch mode, also called batch mode.
    The progressive filling of the reactor in the process of simultaneous saccharification and fermentation (SSF) comprises the following steps ·
    a) addition of glucoamylase enzyme, from the beginning of the filling of the reactor until it is completely filled so that its final concentration is in the range 0.15 to 1.5 gfL;
    b) addition of inoculum; Y
    C) addition of the fermentation medium, which comprises cereal wort, salts and sodium glutamalo
    The propagation medium used for the preparation of the inoculum is composed of 50% (vfv) of cereal mass and 50% (vfv) of water with salts, NaCI, MgS04H20, K2HP04, NH4CI, FeS047H20, Tris glutamalo
     19-1 0-2015
    sodium and glucoamylase enzyme. The inoculum is added to the reactor in the form of a pulse, in a proportion of 10% with respect to the final volume of the reactor. Said inoculum is characterized by having an optical density in the range 2.8 to 3. The addition of! Inoculation occurs between two and a half hours and three and a half hours of the rector's start of operation, preferably at three hours of reactor operation.
    5 During the progressive filling of the reactor, air is supplied to the reactor, which in a preferred embodiment is carried out through a sterile filter. The flow rate of the air supplied to the reactor is in the range of 0.5 to 2.5 L air / (L average · min). The pH is maintained throughout the SSF process at a preferred value of 7.5 by the addition of 5M NaOH, while the temperature is preferably maintained at 30Q C.
    The fermentation medium is added between four and a half hours and five and a half hours after the operation of the reactor, preferably in the fifth hour of operation of the reactor. The salts introduced in the filling process comprise NaCI, MgS04 · H20, K2HP04, NH4 CI and FeS047H20
    The process of adding the enzyme and the fermentation medium ends between thirteen and a half hours and fourteen and a half hours of reactor operation, preferably at fourteen hours of reactor operation. The reactor operates in batch mode until approximately 72 hours
    In one embodiment, the PHA extraction and purification procedure is the acid procedure described in López López-Abelairas et al., 2015; Biochemical Engineering Journal 93: 250-259. The stage of extraction and purification of the PHA includes ·
    a) centrifugal separation of bacterial biomass;
    b) lyophilize the biomass resulting from centrifugation;
    C) homogenize the solid resulting from the previous stage by mechanical stirring;
    d) subject the homogeneous suspension of biomass with a determined solids load to an acid digestion process using a solution of H2S04;
    e) adjust the pH of! digestion product at 10, with a 0.5 N NaOH solution;
    f) wash the solid with water; Y
    25 g) apply a bleaching stage with sodium hypochlorite, to remove residual protein
    In one aspect of the invention, the H2S04 solution used in the homogeneous suspension step has a concentration of! 3.5% The acid digestion step is carried out at a temperature in the range 70Q C to 90Q C, preferably at 80 "C; with a duration of between 5 to 7 hours, preferably 6 hours; and with a preferred solid charge of 5% (w / v) In another aspect of the invention the sodium hypochlorite used in the
    The bleaching stage has a concentration of 3% (plv).
    The method for purifying ectoin comprises the following steps (Onraedt et al, 2005; Biotechnology Progress21 · 1206-1212):
    a) concentrate the supernatant obtained after centrifugation of the fermented must using evaporation under reduced pressure and 60Q C;
    B) remove salts and glutamate by washing with ethanol;
    c) crystallize the concentrated liquid resulting from the previous stage;
    d) separating the ectoin crystals by filtration;
    e) wash the formed ectoin crystals with ethanol; Y
    f) crystallize the resulting liquid to obtain purified ectolna.
    In another aspect the invention relates to a biorefinery incorporating the process object of the present invention for the production of polyhydroxyacanoates and ectoin.
    P201S31498 10-19-201
    BRIEF DESCRIPTION OF THE FIGURES
    The modalities described in the figures are illustrated by way of example and not by way of limitation
    Figure 1 shows a scheme of the classic first generation bioethanol production process.
    Figure 2 shows a scheme of the integrated biorefinery process, with the production of bioethanol, PHA and ectoin. The conventional ethanol production process is shown within the marked area.
    Figure 3 shows the scheme of the SSF reactor filling process for the production of PHA and yectoin
    Figure 4 shows the evolution of the growth and accumulation of H. boliviensis during an SSF process with cereal must. Being DCW (Ory Cell Weight), the total weight of the biomass and RCM (Residual Cell Mass), the weight of the biomass, regardless of PHA
    EXAMPLES OF REALIZATION OF THE INVENTION
    The procedure described above was applied to the production of PHA and ecloin using as a substrate corn must obtained after a liquefaction stage. The first stage of filling the reactor lasted 14 hours, during which the progressive filling of the same was carried out while part of the saccharification and fermentation was carried out. After 14 hours of filling, the fermentation continued until the depletion of the substrate. The filling process consists of the following stages, shown in Figure 3:
    to Sterilization (11S · C for 20 min) of the reactor with the corresponding pH, p02 and temperature probes with a volume of fermentation medium, see Table 1, 3S0! or with respect to the final working volume
    b) Time or: The process began with the addition of the enzyme glucoamylase, during the first 14 hours of the process. Enzyme diluted in distilled water was added so that the final enzyme concentration in the reactor was 1, S gIL.
    cl Hour 3: addition of the inoculum with a ratio of 10% (v / v). Prepared in the propagation medium described in Table 2. The optical density of the inoculum used was in the range 2.8 to 3.
    d) Hour S · the addition of the rest of the fermentation medium began until hour 14, together with the remaining enzyme
    e) Hour 14 ended the entry of nutrients and enzyme. Due to the continuous saccharification of glucose, this substrate was always kept in excess. The limiting nutrient was nitrogen, so that, according to the growth characteristics of H. boliviensis, Baclerian growth was observed until the nitrogen failure caused a change in metabolism towards the accumulation of PHA The total duration of the process was 72 hours
    Table 1 Fermentation medium
    Compound Concentration
    Liquefied and centrifuged must 100% vIII
    I was born 4S gil
    MgS04H20 2.8 gIL
    K2HP04 2.2 gIL
    NH4CI 4 gIL
    FeS047H20 O, OOS gIL
    Sodium glutamate 20 gIL
     19-1 0-2015
    Table 2 Propagation medium
    Compound Concentration
    Liquefied and centrifuged must 50% vlv
    Water 50% vlv
    I was born 45 gIL
    MgS04H20 1.4 gIL
    K2HP0 4 0.55 gIL
    NH4CI 2.3 gIL
    FeS047H20 0.005 gIL
    Sodium glutamate 3 gIL
    Tris 15 gIL
    Glucoamylase 146 mglL
    The means used both in the case of propagation and in the SSF reactor are an adaptation for cereal must of the medium used by QuiUaguamán J. et al., 2008; Appl Microbiol Biotechnol78: 227-232.
    5 The SSF process was carried out in a 2-volume laboratory-scale bioreactor that had on-line monitoring and automatic control of temperature, agitation, pH and air flow, and monitoring of% of dissolved oxygen and% C02 in the gas output stream
    The pH was maintained throughout the process at 7.5 by the addition of 5M NaOH, while the temperature was maintained at 30Q C.
    10 Previous studies studied the behavior of the enzyme in these optimal conditions for the growth of the bacteria, in terms of salt concentration, temperature and pH, and it was found that even under these conditions, the decrease in glucoamylase activity was not relevant for the continuity of the process
    Unlike ethanol production, this process is aerobic, so air was supplied to the reactor through a sterile filter. The flow of air supplied varied from 1 UL · min to 5 UL · min, which was progressively increased to maintain an adequate concentration of dissolved oxygen (p02) in the reactor
    The evolution of the SSF process over time is shown in Figure 4. An excess glucose concentration (20-60 gIL) was maintained during the process, as well as the concentration of P_P04-3 (0.1-0.2 giL). Until hour 32 a great bacterial growth (RCM) was observed, reaching a concentration of 22 gIL. From hour 32 it is observed how bacterial growth stops, but the PHA content
    20 begins to increase exponentially, coinciding with the disappearance of ammoniacal nitrogen in the medium. Through the quantification of PHA by gas chromatography it was found that the type of accumulated polymer is formed by PHA units. Although the fermentation was continued until hour 77, at hour 72 the maximum accumulation was reached, 50% (by weight) of PHA with respect to dry biomass (Dry cell weight, DCW) in addition to 4 gIL of ectoin in the liquid medium
     19-1 0-2015 19-1 0-2015
    权利要求:
    Claims (17)
    [1]

    Proceed to take advantage of liquefied cereal grain to obtain ectoin and
    polyhydroxyalkanoates (PHA), characterized in that it comprises the following steps:
    to. centrifuge the liquefied cereal grain;
    5 b. subject the obtained supernatant, cereal must, to a fermentation process and
    simultaneous saccharification in a reactor;
    c centrifuge the fermented must in the previous stage and separate the bacterial bio mass, which
    contains the PHA, of the supernatant containing the ectoin;
    d subject the bacterial biomass to a procedure of extraction and purification of PHA; Y
    10 and concentrate the supernatant obtained after centrifugation of the fermented must using
    evaporation under reduced pressure and purify the ectoin.
    2 The method according to claim 1, characterized in that the saccharification process and
    Simultaneous fermentation of cereal must includes:
    to. progressive filling of the reactor; Y
    fifteen b. reactor operation in batch mode, also called batch mode.
    [3]
    3. The method according to claim 2, characterized in that the progressive filling of the reactor
    understand the stages of
    to. Addition of glucoamylase enzyme, from the beginning of the reactor filling until reaching its
    complete filling;
    twenty b. inoculum addition; Y
    C. addition of the continuous fermentation medium, which comprises cereal must, salts and
    sodium glutamate
    [4]
    Four. The process according to claim 3, characterized in that the salts of the fermentation medium
    they comprise NaCI, MgSO ~ H20, K2HP04, NH4CI and FeS04 · 7H20.
    25 5The process according to claim 1, characterized in that the process fermentation stage
    simultaneous saccharification and fermentation is performed using a halophilic bacterium
    6 The method according to claim 2, characterized in that the cereal must is subjected to a
    sterilization process
    7 The method according to claim 6, characterized in that the sterilization of the must is carried
    30 conducted inside the reactor at a preferred temperature of 115Q C.
    8 The method according to claim 6, characterized in that the sterilization of the must has a
    Preferred duration of 20 minutes.
    [9]
    9. The method according to claim 2, characterized in that the saccharification process and
    Simultaneous fermentation is carried out at a preferred pH of 7.5.
    35 10The process according to claim 2, characterized in that air is supplied to the reactor
    eleven The method according to claim 10, characterized in that the supply of air is produced at
    through a sterile filter
    [12 ]
    12. The method according to claims 10 and 11, characterized in that the air flow
    supplied is in the range of 0.5 to 2.5 L airJ (medium L · min).
    40 13.The process according to claim 3, characterized in that the enzyme glucoamylase is added in
    continuous in the reactor maintaining its final concentration in the range 0.15 1.5 gIL
    The method according to claim 3, characterized in that the inoculum is prepared in a propagation medium composed of 50% (v / v) cereal must and 50% (vfv) of water with the salts, NaCI, MgSO4 · H20 , K2HP04, NH4CI, FeS04 · 7H20, Tris, sodium glutamate and glucoamylase enzyme
    The method according to claim 3, characterized in that the inoculum is added in a proportion 5 of 10% (v / v) with respect to the final volume of the reactor.
    The method according to claim 3, characterized in that the inoculum has an optical density in the range 2.8 to 3.
    [17]
    17. The method according to claim 3 characterized in that the addition of the inoculum is carried out in two and a half hours and three and a half hours of reactor operation
    The method according to claim 17, characterized in that the addition of the inoculum is preferably performed in the third hour of operation of the reactor.
    [19]
    19. The method according to claim 3 characterized in that the addition of the rest of the fennentation medium is carried out within four and a half hours and the five and a half hours of operation of the reaelor
    [20]
    20. The method according to claim 19, characterized in that the addition of the rest of the fermentation medium is preferably carried out in the fifth hour of operation of the reactor.
    [21 ]
    twenty-one . The process according to claim 3, characterized in that the addition of the enzyme, the inocula and the fennentation medium ends between 13:30 and 14:30 hours of reactor operation.
    [22]
    22. The process according to claim 21, characterized in that the addition of the enzyme, the inoculum and 20 of the rest of the fermentation medium preferably ends after 14 hours of operation of the reactor.
    [23]
    2. 3. The method according to claim 3 characterized in that the reactor operates in discontinuous mode until the 72 hour of operation of the reactor.
    [24]
    24. The method according to claim 1, characterized in that the step of extraction and purification of the PHA comprises ·
    25 to separate by centrifugation the Baelerian biomass; b lyophilize the biomass resulting from centrifugation;
    C. homogenize the solid resulting from the previous stage by mechanical stirring;
    d. subject the homogeneous suspension of biomass with a solid charge to an acid digestion process using a solution of H2S04;
    30 e. adjust the pH of the solution obtained in the previous step to 10 using a 0.5N NaOH solution;
    F. wash the solid with water; Y
    g. apply a bleach with sodium hypochlorite to remove residual protein and obtain PHA.
    [25]
    25. The method according to claim 24, characterized in that the H2S04 solution used in the homogeneous suspension stage has a concentration of 3.5%.
    The method according to claim 24, characterized in that the acid digestion step is carried out at a temperature in the range 70 "C to 90" C, preferably at 80 "C
    [27]
    27. The method according to claim 24, characterized in that the acid digestion process has a duration in the range 5 to 7 hours, with a preferred duration of 6 hours.
    The method according to claim 24, characterized in that the acid digestion process is carried out with a 5% solid charge (pfv)
    [29]
    29. The method according to claim 24, characterized in that the sodium hypochlorite used in the bleaching stage has a concentration of 3% pfv

    The method according to claim 1, characterized in that the ectoin recovery and purification step comprises ·
    to. concentrate the supernatant obtained after centrifugation of the fermented must using evaporation under reduced pressure; 5 b. remove salts and glutamate by washing with ethanol;
    c crystallize the concentrated liquid resulting from the previous stage; d separating the ecloin crystals by filtration;
    and. wash the formed ectoin crystals with ethanol; Y
    crystallize the resulting liquid to obtain purified ectoin 10 31 Polyhydroxyalkanoates, PHA, obtained by the process according to claims 1 to 29
    [32]
    32 Ectoin obtained by the process according to claims 1 to 30.
    [33]
    33. Biorefinery that incorporates the procedure according to reinvidications at 1 to 30.
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    同族专利:
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    ES2562377B2|2016-06-08|
    WO2017068217A1|2017-04-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2004046333A2|2002-11-15|2004-06-03|Novozymes North America, Inc.|Ethanol production by simultaneous saccharification and fermentation |
    DE102008045237A1|2008-08-28|2010-03-04|Helmholtz-Zentrum Für Umweltforschung Gmbh - Ufz|Simultaneously producing poly/D-3-hydroxyalkanoate and compatible solute comprises cultivating halophilic bacteria in culture medium e.g. with a carbon source, and extracting the components form the mixture|CN107777780A|2016-08-30|2018-03-09|张继红|Water process preparation, preparation method and applications|
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