WO2023125546A1 - 一种利用韧皮纤维生产pha材料的方法 - Google Patents

一种利用韧皮纤维生产pha材料的方法 Download PDF

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WO2023125546A1
WO2023125546A1 PCT/CN2022/142384 CN2022142384W WO2023125546A1 WO 2023125546 A1 WO2023125546 A1 WO 2023125546A1 CN 2022142384 W CN2022142384 W CN 2022142384W WO 2023125546 A1 WO2023125546 A1 WO 2023125546A1
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fermentation
pha
enzymolysis
product
bast fibers
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PCT/CN2022/142384
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French (fr)
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张玟籍
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常州柯纳生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biological materials and their preparation, and in particular relates to a method for producing PHA materials using bast fibers.
  • PHA Polyhydroxyalkanoate
  • the purpose of this invention is to provide a kind of method that utilizes bast fiber to produce PHA material.
  • the first aspect of the present invention provides a kind of method utilizing bast fiber to produce polyhydroxyalkanoate (PHA) material, comprises enzymolysis step and fermentation step, and described enzymolysis step comprises steps (a)-(a3 ):
  • Described fermentation step comprises steps (b)-(c):
  • the third enzymatic sugar solution is diluted with water.
  • the raw material of the PHA material comes from the bast part of bast fiber crops.
  • the bast fiber crop is hemp.
  • the hemp is selected from the group consisting of kenaf, jute, ramie, or combinations thereof.
  • step (a3) the sugar content of the third enzymatic sugar solution is 18-22%.
  • step (c) fed-batch feeding is adopted in the fermentation process, and the total sugar content in the fermentation broth is kept not lower than 15% within 24 hours.
  • step (b) the sugar content of the fermentation bottom liquid is 15-20%.
  • step (c) the fermentation temperature is 35-42°C.
  • the fermented strain is Escherichia coli engineering bacteria.
  • the Escherichia coli engineered bacteria produce PHA.
  • step (c) the fermentation time is 40-60h.
  • the bast fibers are shredded and presoaked, wherein the shredding includes the following steps:
  • the dry bast fiber refers to the bast fiber with a moisture content of 8-20%.
  • step (Z1) after sieving, the size of the crushed bast fibers is 1-10 cm, preferably 1.5-6 cm.
  • the broken bast fibers are separated by a cyclone separation device.
  • the size of the ultrafine bast fiber is 0.02-2 mm, preferably 0.05-0.5 mm.
  • step (Z3) steam explosion is performed with a steam explosion device.
  • the steam explosion temperature is 150-280°C, preferably 180-250°C.
  • the holding time is 2 min-15 min, preferably 3-10 min.
  • step (Z3) the steam explosion pressure is 1.2-2.5 MPa, preferably 1.6-2.2 MPa.
  • the presoaking solution is selected from the group consisting of dilute hydrochloric acid with a pH of 5.0-6.0, sodium hydroxide solution or clear water with a pH of 8.0-9.0.
  • the presoaking time is 2-12 hours.
  • the temperature of the prepreg is 40-85°C, preferably 60-75°C.
  • the rotational speed of the high-shear homogeneous stirring is 8,000-20,000 rpm.
  • the total time for enzymatic hydrolysis is 48-72 hours.
  • the fermentation product is centrifuged and dehydrated, then extracted with ethanol under pressure, and then dried and granulated to obtain a PHA product.
  • the PHA product is in the form of powder or granular solid.
  • the dry matter concentration of PHA in the fermentation product is 150-300 g/L, wherein the PHA content in the total solid product of the fermentation is not less than 50%.
  • the second aspect of the present invention provides a kind of polyhydroxyalkanoate (PHA) material that utilizes bast fiber to produce, and described polyhydroxyalkanoate (PHA) material is prepared with the method described in the first aspect of the present invention .
  • PHA polyhydroxyalkanoate
  • the present inventor After extensive and in-depth research, the present inventor has firstly developed a novel method for producing PHA material using bast fibers through a large number of screenings.
  • the preparation process of the present invention is simple, and high-yield hemp fibers are used as raw materials, which avoids the embarrassment of competing for food when using starch crops such as corn as raw materials, and significantly reduces the cost.
  • the present invention due to the excellent components of raw materials, the Final product quality and overall production efficiency. On this basis, the present invention has been accomplished.
  • the present invention also provides a method for producing PHA material by using bast fiber.
  • the method uses bast fiber from hemp crops as a raw material, avoiding the defects of conventional grain crop preparation.
  • the enzymolysis method of the present invention can obtain an enzymolysis liquid with a higher sugar content, so as to carry out continuous fermentation.
  • the bast fibers used are derived from hemp crops, such as the outer bark of bast fiber crops such as kenaf and jute.
  • the hemp crop is kenaf.
  • the steps of the present invention specifically include an enzymolysis step and a fermentation step, and the enzymolysis solution is provided through the enzymolysis step as the bottom solution in the fermentation step.
  • the enzymolysis step specifically includes steps (a)-(a3):
  • the third enzymolysis sugar solution is fermented, and the fermentation step includes steps (b)-(c):
  • described operating method comprises the following steps:
  • the hemp fiber After the hemp fiber is dried to a moisture content of 8-20%, it is sent to a shredder for cutting. After cutting, the length is 1-10 cm, preferably 1.5-6 cm. After sieving, it is sent to a cyclone separation device, and then sent to a super Carry out superfine pulverization in the fine pulverizer, pulverization size is 20-800 mesh, more preferably is 50-300 mesh.
  • the pulverized material is sent to the steam explosion device for steam explosion.
  • Pre-soaking treatment is carried out before the steam explosion.
  • the pre-soaking temperature is 30-60°C
  • the pre-soaking time is 2-12 hours
  • the steam explosion temperature is 150-280°C More preferably 180-250°C, steam explosion pressure 1.2-2.5Mpa, more preferably 1.6-2.2Mpa.
  • the material after high-speed steam explosion is quickly and directly sent to the enzymolysis tank for enzymolysis treatment.
  • emulsification and homogenization are used, and the method of exoglucose-endoglucose-glucosidase is added step by step.
  • Carry out enzymatic hydrolysis, and the enzymatic hydrolysis time is 48-72 hours. During this period, the pH and temperature need to be adjusted dynamically to meet the needs of different enzyme preparations.
  • the initial substrate concentration is 15-20%
  • the strain is PHA-producing Escherichia coli engineered bacteria
  • the fermentation temperature is 35-42°C
  • fed-batch feeding is adopted during the process
  • the total sugar concentration is kept not less than 15% within 24 hours.
  • the total fermentation time is 40-60 hours
  • the dry matter concentration after fermentation is 150-300g/L, of which the PHA content is not less than 50%.
  • the material is sent to the centrifugal equipment for dehydration and then extracted with ethanol under pressure to obtain the finished product of PHA.
  • the main advantages of the present invention include:
  • the high-concentration sugar enzymatic hydrolyzate feeding process ensures the high efficiency of the whole process of PHA production and realizes the final high-content PHA acquisition.
  • Kenaf bast fiber naturally dried after harvest, with a moisture content of 14%, without any other treatment.
  • Use a shredder to cut the kenaf fiber and sieve it to obtain kenaf fiber fragments with a length of no more than 10 cm, with an average length of about 8 cm.
  • After sieving use a cyclone separation device to remove impurities and dust.
  • the obtained powder is subjected to pre-dipping treatment, the pre-dipping treatment solution is a dilute hydrochloric acid solution with a pH of 5.5, the pre-dipping temperature is 65° C., and the pre-dipping time is 6 hours.
  • the pre-dipping treatment solution is a dilute hydrochloric acid solution with a pH of 5.5
  • the pre-dipping temperature is 65° C.
  • the pre-dipping time is 6 hours.
  • the steam explosion temperature is 200°C
  • the pressure is 2.1Mpa
  • the pressure holding time is 6 minutes.
  • the steam-exploded raw materials are directly sent into the enzymolysis tank through the pipeline, the substrate concentration is 40%, the exoglucose is added after adjusting the pH to 5.8, the high-shear homogenizer is turned on, the speed is 16,000rpm, and the enzymolysis temperature is 40°C, after 12 hours of enzymatic hydrolysis, adjust the pH to 5.8 and then add endoglucose, the speed of the high shear homogenizer is 20,000rpm, the enzymatic hydrolysis temperature is 45°C, after 12 hours of enzymatic hydrolysis, adjust the pH to 5.0 and then add glucoside
  • the speed of the high-shear homogenizer is 16,000 rpm, and the enzymatic hydrolysis temperature is 45°C. After 12 hours, the speed of the high-shear homogenizer is 12,000 rpm. After 24 hours, the enzymatic hydrolysis ends, and the total enzymatic hydrolysis time is 60 hours.
  • Proportion the enzymatic hydrolysis solution into a fermentation bottom solution with a sugar concentration of 20% add PHA engineered strains, and then carry out fermentation.
  • the fermentation temperature is 35°C.
  • the sugar content of the fermentation process is dynamically detected and the feed is added to ensure that the sugar content of the substrate is within 48 hours. The content is not less than 15%, and the fermentation stops after 54 hours.
  • the fermentation product in the lower layer was centrifuged and dehydrated to obtain a fermentation product with a fermentation yield of 26.1%.
  • the PHA product was obtained by ethanol pressure extraction, and the PHA accounted for 63% of the dry weight.
  • Kenaf bast fiber naturally dried after harvest, with a moisture content of 14%, without any other treatment.
  • Use a shredder to cut the kenaf fiber and sieve it to obtain kenaf fiber fragments with a length of no more than 10 cm, with an average length of about 8 cm.
  • After sieving use a cyclone separation device to remove impurities and dust.
  • the powder obtained is subjected to a pre-preg treatment.
  • the pre-soaking solution is clear water, the pre-soaking temperature is 70°C, and the pre-soaking time is 6 hours.
  • After prepreg it is sent to an intermittent supersonic steam explosion device, the steam explosion temperature is 220°C, the pressure is 2.2Mpa, and the pressure holding time is 8min.
  • the steam-exploded raw materials are directly sent into the enzymolysis tank through the pipeline, the substrate concentration is 40%, the exoglucose is added after adjusting the pH to 5.8, the high-shear homogenizer is turned on, the speed is 16,000rpm, and the enzymolysis temperature is 40°C, after 6 hours of enzymatic hydrolysis, adjust the pH to 5.8, then add endoglucose, the speed of the high shear homogenizer is 20,000rpm, the enzymatic hydrolysis temperature is 45°C, after 6 hours of enzymatic hydrolysis, adjust the pH to 5.0, then add glucoside
  • the speed of the high-shear homogenizer is 12,000 rpm, and the enzymatic hydrolysis temperature is 45°C. After 12 hours, the speed of the high-shear homogenizer is 8,000 rpm. After 24 hours, the enzymatic hydrolysis ends, and the total enzymatic hydrolysis time is 48 hours.
  • Proportion the enzymatic hydrolysis liquid into a fermentation base liquid with a sugar concentration of 20% add PHA engineering strains, and then carry out fermentation.
  • the fermentation temperature is 35°C.
  • the sugar content of the fermentation process is dynamically detected and the feed is added to ensure that the sugar content of the substrate is within 40 hours. The content is not less than 15%, and the fermentation stops after 48 hours.
  • the fermentation product in the lower layer was centrifuged and dehydrated to obtain a fermentation product with a fermentation yield of 23.4%.
  • the PHA product was obtained by ethanol pressure extraction, and the PHA accounted for 58% of the dry weight.
  • Corn stalks are naturally dried after harvesting, with a moisture content of 18%, without any other treatment.
  • the obtained corn stalks are fed into an air flow cutting superfine pulverizer, and the obtained products are sieved to obtain corn stalk powders with a size below 0.1 mm.
  • the obtained powder is subjected to pre-soaking treatment, the pre-soaking treatment solution is clear water, the pre-soaking temperature is 65° C., and the pre-soaking time is 6 hours. After prepreg, it is sent to an intermittent supersonic steam explosion device.
  • the steam explosion temperature is 200°C
  • the pressure is 2.1Mpa
  • the pressure holding time is 8 minutes.
  • the steam-exploded raw materials are directly sent into the enzymolysis tank through the pipeline, the substrate concentration is 40%, the exoglucose is added after adjusting the pH to 5.8, the high-shear homogenizer is turned on, the speed is 12,000rpm, and the enzymolysis temperature is 40°C, after 12 hours of enzymatic hydrolysis, adjust the pH to 5.8, then add endoglucose, the speed of the high shear homogenizer is 16,000rpm, the enzymatic hydrolysis temperature is 45°C, after 12 hours of enzymatic hydrolysis, adjust the pH to 5.0, then add glucoside
  • the speed of the high-shear homogenizer is 16,000 rpm, and the enzymatic hydrolysis temperature is 45°C. After 12 hours, the speed of the high-shear homogenizer is 12,000 rpm. After 24 hours, the enzymatic hydrolysis ends, and the total enzymatic hydrolysis time is 60 hours.
  • the enzymatic solution in step (2) has too low sugar content, it can only be mixed with a fermentation bottom solution with a sugar concentration of 15%. Fermentation is carried out after adding PHA engineering strains. The fermentation temperature is 35°C. Stop after continuing for 60 hours, because of the fusel alcohol contained in the enzymolysis solution, the fermentation is not sufficient. After a little natural sedimentation, the fermentation product in the lower layer was centrifuged and dehydrated to obtain a fermentation product with a fermentation yield of 6.1%. The PHA product was obtained by ethanol pressure extraction, and the PHA accounted for 43% of the dry weight.

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Abstract

一种利用韧皮纤维生产聚羟基脂肪酸酯(PHA)材料的方法。对韧皮纤维进行粉碎、酶解和发酵，制备聚羟基脂肪酸酯产品。该方法降低成本，原料利用率高，产品质量优。

Description

一种利用韧皮纤维生产PHA材料的方法 技术领域
本发明属于生物材料及其制备领域，具体涉及一种利用韧皮纤维生产PHA材料的方法。
背景技术
聚羟基脂肪酸酯(PHA)是一类具有良好力学性能的生物聚合材料，完全由微生物发酵而产生，其可以在土壤、堆肥和海洋等各种环境中快速完全降解，是目前公认的替代塑料的最佳选择之一。然而，由于原料、成本等因素，PHA始终没有得到大规模的生产。
发明内容
本发明的目的是提供了一种利用韧皮纤维生产PHA材料的方法。
本发明的第一方面，提供了一种利用韧皮纤维生产聚羟基脂肪酸酯(PHA)材料的方法，包括酶解步骤和发酵步骤，所述的酶解步骤包括步骤(a)-(a3)：
(a)提供含有韧皮纤维的酶解底物；
(a1)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用外切葡萄糖酶对所述的酶解底物进行酶解，得到第一酶解糖液；
(a2)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用内切葡萄糖酶对所述的第一酶解糖液进行酶解，得到第二酶解糖液；
(a3)在35-50℃下，调节体系pH为4.5-5.5，在高剪切均质搅拌条件下，用葡萄糖苷酶对所述的第二酶解糖液进行酶解，得到第三酶解糖液；
所述的发酵步骤包括步骤(b)-(c)：
(b)对所述的第三酶解糖液进行稀释，得到发酵底液；
(c)对所述的发酵底液进行发酵，得到含有PHA产品的发酵产物。
在另一优选例中，用清水对第三酶解糖液进行稀释。
在另一优选例中，所述的PHA材料的原料来自于韧皮纤维作物的韧皮部分。
在另一优选例中，所述的韧皮纤维作物为麻类。
在另一优选例中，所述的麻类选自下组：红麻、黄麻、苎麻，或其组合。
在另一优选例中，在步骤(a3)中，所述的第三酶解糖液的糖度为18-22％。
在另一优选例中，在步骤(c)中，发酵过程采取流加补料，在24h内保持发酵液中总糖度不低于15％。
在另一优选例中，在步骤(b)中，所述的发酵底液的糖度为15-20％。
在另一优选例中，在步骤(c)中，发酵温度为35-42℃。
在另一优选例中，在步骤(c)中，发酵的菌种为大肠杆菌工程菌。
在另一优选例中，所述的大肠杆菌工程菌产PHA。
在另一优选例中，在步骤(c)中，发酵时间为40-60h。
在另一优选例中，在酶解前，对韧皮纤维进行切碎处理和预浸处理，其中，所述的切碎包括如下步骤：
(Z1)将干燥的韧皮纤维切碎，然后过筛，得到碎韧皮纤维；
(Z2)分离所述的碎韧皮纤维，然后进行超细粉碎，得到超细韧皮纤维；
(Z3)对所述的超细韧皮纤维进行预浸处理，然后进行汽爆，得到待酶解的韧皮纤维物料。
在另一优选例中，在步骤(Z1)中，所述的干燥的韧皮纤维是指含水量为8-20％的韧皮纤维。
在另一优选例中，在步骤(Z1)中，过筛后，所述的碎韧皮纤维的尺寸为1-10cm，较佳为1.5-6cm。
在另一优选例中，在步骤(Z2)中，用旋风分离装置进行碎韧皮纤维分离。
在另一优选例中，在步骤(Z2)中，所述的超细韧皮纤维的尺寸为0.02-2mm，较佳为0.05-0.5mm。
在另一优选例中，在步骤(Z3)中，用蒸汽爆破装置进行汽爆。
在另一优选例中，汽爆温度为150-280℃，较佳为180-250℃。
在另一优选例中，汽爆时，若压力不足可通入压缩空气。
在另一优选例中，保压时间为2min-15min，较佳为3-10min。
在另一优选例中，在步骤(Z3)中，汽爆压力为1.2-2.5MPa，较佳为1.6-2.2MPa。
在另一优选例中，预浸液选自下组：pH为5.0-6.0的稀盐酸、pH为8.0-9.0的氢氧化钠溶液或清水。
在另一优选例中，预浸时间为2-12h。
在另一优选例中，预浸温度为40-85℃，较佳为60-75℃。
在另一优选例中，所述的高剪切均质搅拌的转速为8,000-20,000rpm。
在另一优选例中，酶解总时间为48-72h。
在另一优选例中，对所述的发酵产物离心脱水，然后用乙醇加压提取后进行干燥造粒，得到PHA产品。
在另一优选例中，常温下，所述的PHA产品呈粉末状或颗粒状固体。
在另一优选例中，所述的发酵产物中PHA干物质浓度为150-300g/L，其中发酵总固形产物中PHA含量不低于50％。
本发明的第二方面，提供了一种利用韧皮纤维生产的聚羟基脂肪酸酯(PHA)材料，所述的聚羟基脂肪酸酯(PHA)材料用本发明第一方面所述的方法制备。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例) 中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过广泛而深入的研究，经过大量筛选首次开发了一种新颖的利用韧皮纤维生产PHA材料的方法。本发明的制备流程简单，原料采用了高产量的麻类纤维，避免了使用玉米等淀粉作物为原料时与人争粮的窘境，并明显降低了成本，同时由于原料的优异组分，提高了最后产品的质量与总体生产效率。在此基础上，完成了本发明。
利用韧皮纤维生产PHA材料的方法
本发明还提供了一种利用韧皮纤维生产PHA材料的方法，所述的方法采用来自于麻类作物的韧皮纤维作为原料，避免了常规采用粮食作物制备的缺陷。且通过本发明的酶解方法可以获得具有较高糖度的酶解液，从而进行连续发酵。
本发明中，使用的韧皮纤维来源于麻类作物，例如红麻、黄麻等韧皮纤维作物的外皮。一个优选的实施方式中，所述的麻类作物为红麻。
本发明的步骤具体包括酶解步骤和发酵步骤，通过酶解步骤提供酶解液，从而作为发酵步骤中的底液。所述的酶解步骤具体包括步骤(a)-(a3)：
(a)提供含有韧皮纤维的酶解底物；
(a1)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用外切葡萄糖酶对所述的酶解底物进行酶解，得到第一酶解糖液；
(a2)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用内切葡萄糖酶对所述的第一酶解糖液进行酶解，得到第二酶解糖液；
(a3)在35-50℃下，调节体系pH为4.5-5.5，在高剪切均质搅拌条件下，用葡萄糖苷酶对所述的第二酶解糖液进行酶解，得到第三酶解糖液。
在酶解后，对所述的第三酶解糖液进行发酵处理，所述的发酵步骤包括步骤(b)-(c)：
(b)对所述的第三酶解糖液进行稀释，得到发酵底液；
(c)对所述的发酵底液进行发酵，得到含有PHA产品的发酵产物。
本发明的一些具体实施方式下，所述的操作方法包括如下步骤：
将麻类纤维干燥至水分8-20％后，送入切碎机进行切断，切断后长度为1-10cm，更佳的为1.5-6cm，过筛后送入旋风分离装置，随后送入超细粉碎机中进行超细粉碎，粉碎尺寸为20-800目，更佳的为50-300目。
粉碎后的物料送入蒸汽爆破装置中进行汽爆，汽爆前进行预浸处理，预浸温度为30-60℃，预浸时间为2-12个小时，汽爆温度为150-280℃，更佳的为180-250℃，汽爆压力为1.2-2.5Mpa，更佳的为1.6-2.2Mpa。
通过高速汽爆后的物料，迅速直接送入酶解罐中进行酶解处理，酶解过程中采用乳化均质处理，采用外切葡萄糖酶-内切葡萄糖酶-葡萄糖苷酶分步投放的方式进行酶解，酶解时间为48-72小时，期间需动态调整PH与温度以适应不同酶制剂的需求，酶解后的糖液经过封闭式过滤沉淀后，送入发酵罐中进行发酵，发酵初始底物浓度为15-20％，菌种为产PHA的大肠杆菌工程菌，发酵温度为35-42℃，过程中采取流加补料，在24h内保持总糖浓度不低于15％，总发酵时间为40-60h，发酵后干物质浓度为150-300g/L，其中PHA含量不低于50％，将物料送入离心装备脱水后进行乙醇加压提取，获得PHA成品。
与现有技术相比，本发明的主要优点包括：
(1)采用韧皮纤维为原料，避免了使用粮食原料；
(2)对高聚合度和高纤维含量的韧皮作物的外皮进行粉碎和预浸，使得酶解底物浓度与效率大大提高；
(3)采用了特殊的分步酶解工艺，从而对高纤维底物实现了高效率酶解，得到高糖度的酶解液，适用于连续发酵；
(4)高浓度糖酶解液流加工艺，保证产PHA的全程高效并实现最后的高含量PHA获取。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
(1)预处理步骤
红麻韧皮纤维，收获后自然干燥，含水量为14％，未经过其他任何处理。采用切碎机将红麻纤维切断后过筛，获得长度不超过10cm的红麻纤维小段，平均长度约为8cm，在过筛后用旋风分离装置进行去杂去尘。将获得的麻皮纤维送入带前置粗碎的气流式切割超细粉碎机中，所获得的产品过筛，获得尺寸在0.1mm以下的麻皮粉末。将获得的粉末进行预浸处理，预浸处理液为PH为5.5的稀盐酸溶液，预浸温度为65℃，预浸时间为6h。预浸后送入间歇式超音速蒸汽爆破装置，汽爆温度为200℃，压力为2.1Mpa，保压时间为6min。
(2)酶解步骤
汽爆后的原料通过管道直接送入酶解罐中，底物浓度为40％，调节PH至5.8后加入外切葡萄糖酶，打开高剪切均质机，转速为16,000rpm，酶解温度为40℃，酶解12h后，调节PH至5.8后加入内切葡萄糖酶，高剪切均质机转速为20,000rpm， 酶解温度为45℃，酶解12h后，调节PH至5.0后加入葡萄糖苷酶，高剪切均质机转速为16,000rpm，酶解温度为45℃，12h后，高剪切均质机转速为12,000rpm，24h后酶解结束，酶解时间共计60h。
(3)发酵步骤
将酶解液配比成20％糖浓度的发酵底液，加入PHA工程菌种后进行发酵，发酵温度为35℃，动态检测发酵过程的糖度并采用流加补料，保证底物糖度在48h内不低于15％，发酵持续54小时后停止。稍经自然沉降后将下层发酵物离心脱水后得到发酵产物，发酵得率为26.1％，采用乙醇加压提取的方法得到PHA产品，PHA占干重的63％。
实施例2
(1)预处理步骤
红麻韧皮纤维，收获后自然干燥，含水量为14％，未经过其他任何处理。采用切碎机将红麻纤维切断后过筛，获得长度不超过10cm的红麻纤维小段，平均长度约为8cm，在过筛后用旋风分离装置进行去杂去尘。将获得的麻皮纤维送入带前置粗碎的气流式切割超细粉碎机中，所获得的产品过筛，获得尺寸在0.2mm以下的麻皮粉末。将获得的粉末进行预浸处理。预浸处理液为清水，预浸温度为70℃，预浸时间为6h。预浸后送入间歇式超音速蒸汽爆破装置，汽爆温度为220℃，压力为2.2Mpa，保压时间为8min。
(2)酶解步骤
汽爆后的原料通过管道直接送入酶解罐中，底物浓度为40％，调节PH至5.8后加入外切葡萄糖酶，打开高剪切均质机，转速为16,000rpm，酶解温度为40℃，酶解6h后，调节PH至5.8后加入内切葡萄糖酶，高剪切均质机转速为20,000rpm，酶解温度为45℃，酶解6h后，调节PH至5.0后加入葡萄糖苷酶，高剪切均质机转速为12,000rpm，酶解温度为45℃，12h后，高剪切均质机转速为8,000rpm，24h后酶解结束，酶解时间共计48h。
(3)发酵步骤
将酶解液配比成20％糖浓度的发酵底液，加入PHA工程菌种后进行发酵，发酵温度为35℃，动态检测发酵过程的糖度并采用流加补料，保证底物糖度在40h内不低于15％，发酵持续48小时后停止。稍经自然沉降后将下层发酵物离心脱水后得到发酵产物，发酵得率为23.4％，采用乙醇加压提取的方法得到PHA产品，PHA占干重的58％。
对比例1：
(1)预处理步骤
玉米秸秆，收获后自然干燥，含水量为18％，未经过其他任何处理。采用秸 秆粉碎机将玉米秸秆粉碎至0.5-1cm的碎片，在过筛后用旋风分离装置进行去杂去尘。将获得的玉米秸秆送入气流式切割超细粉碎机中，所获得的产品过筛，获得尺寸在0.1mm以下的玉米秸秆粉末。将获得的粉末进行预浸处理，预浸处理液为清水，预浸温度为65℃，预浸时间为6h。预浸后送入间歇式超音速蒸汽爆破装置，汽爆温度为200℃，压力为2.1Mpa，保压时间为8min。
(2)酶解步骤
汽爆后的原料通过管道直接送入酶解罐中，底物浓度为40％，调节PH至5.8后加入外切葡萄糖酶，打开高剪切均质机，转速为12,000rpm，酶解温度为40℃，酶解12h后，调节PH至5.8后加入内切葡萄糖酶，高剪切均质机转速为16,000rpm，酶解温度为45℃，酶解12h后，调节PH至5.0后加入葡萄糖苷酶，高剪切均质机转速为16,000rpm，酶解温度为45℃，12h后，高剪切均质机转速为12,000rpm，24h后酶解结束，酶解时间共计60h。
(3)发酵步骤
由于步骤(2)中的酶解液糖度过低，故只能配比成15％糖浓度的发酵底液，加入PHA工程菌种后进行发酵，发酵温度为35℃，采用一次性加料，发酵持续60小时后停止，因酶解液中含有杂醇，发酵不充分。稍经自然沉降后将下层发酵物离心脱水后得到发酵产物，发酵得率为6.1％，采用乙醇加压提取的方法得到PHA产品，PHA占干重的43％。
结果显示，采用现有技术中的玉米秸秆发酵液的情况下，酶解液糖度过低，因此无法进行连续发酵，然而，在采用本发明的红麻韧皮纤维作为原料的情况下，酶解得到的酶解液的糖度较高，因此可以进行连续发酵，从而提升生产工艺中的总体产量，提升发酵得率。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种利用韧皮纤维生产聚羟基脂肪酸酯PHA材料的方法，其特征在于，包括酶解步骤和发酵步骤，所述的酶解步骤包括步骤(a)-(a3)：
    (a)提供含有韧皮纤维的酶解底物；
    (a1)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用外切葡萄糖酶对所述的酶解底物进行酶解，得到第一酶解糖液；
    (a2)在35-50℃下，调节体系pH为5-6.5，在高剪切均质搅拌条件下，用内切葡萄糖酶对所述的第一酶解糖液进行酶解，得到第二酶解糖液；
    (a3)在35-50℃下，调节体系pH为4.5-5.5，在高剪切均质搅拌条件下，用葡萄糖苷酶对所述的第二酶解糖液进行酶解，得到第三酶解糖液；
    所述的发酵步骤包括步骤(b)-(c)：
    (b)对所述的第三酶解糖液进行稀释，得到发酵底液；
    (c)对所述的发酵底液进行发酵，得到含有PHA产品的发酵产物。
  2. 如权利要求1所述的方法，其特征在于，在步骤(a3)中，所述的第三酶解糖液的糖度为18-22％。
  3. 如权利要求1所述的方法，其特征在于，在步骤(c)中，发酵过程采取流加补料，在24h内保持发酵液中总糖度不低于15％。
  4. 如权利要求1所述的方法，其特征在于，在步骤(b)中，所述的发酵底液的糖度为15-20％。
  5. 如权利要求1所述的方法，其特征在于，在酶解步骤前，对韧皮纤维进行切碎处理和预浸处理，其中，所述的切碎包括如下步骤：
    (Z1)将干燥的韧皮纤维切碎，然后过筛，得到碎韧皮纤维；
    (Z2)分离所述的碎韧皮纤维，然后进行超细粉碎，得到超细韧皮纤维；
    (Z3)对所述的超细韧皮纤维进行预浸处理，然后进行汽爆，得到待酶解的韧皮纤维物料。
  6. 如权利要求5所述的方法，其特征在于，预浸液选自下组：pH为5.0-6.0的稀盐酸、pH为8.0-9.0的氢氧化钠溶液或清水。
  7. 如权利要求1所述的方法，其特征在于，对所述的发酵产物离心脱水，然后用乙醇加压提取后进行干燥造粒，得到PHA产品。
  8. 如权利要求7所述的方法，其特征在于，常温下，所述的PHA产品呈粉末状或颗粒状固体。
  9. 如权利要求1所述的方法，其特征在于，所述的发酵产物中PHA干物质浓度为150-300g/L，其中发酵总固形产物中PHA含量不低于50％。
  10. 一种利用韧皮纤维生产的聚羟基脂肪酸酯(PHA)材料，其特征在于，所述的聚羟基脂肪酸酯(PHA)材料用权利要求1所述的方法制备。
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