WO2023010787A1 - 农业废弃物发酵生产聚羟基脂肪酸酯的方法 - Google Patents

农业废弃物发酵生产聚羟基脂肪酸酯的方法 Download PDF

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WO2023010787A1
WO2023010787A1 PCT/CN2021/142357 CN2021142357W WO2023010787A1 WO 2023010787 A1 WO2023010787 A1 WO 2023010787A1 CN 2021142357 W CN2021142357 W CN 2021142357W WO 2023010787 A1 WO2023010787 A1 WO 2023010787A1
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fermentation
pha
aeration
waste
stage
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French (fr)
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董红敏
王顺利
张万钦
尹福斌
曹起涛
连天境
周谈龙
朱志平
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Institute of Environment and Sustainable Development in Agriculturem of CAAS
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Institute of Environment and Sustainable Development in Agriculturem of CAAS
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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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • 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
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously

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  • the invention belongs to the field of utilization of agricultural waste, and in particular relates to a method for producing polyhydroxyalkanoate by fermentation of agricultural waste.
  • PHA Polyhydroxyalkanoates
  • the current PHA fermentation production process mainly uses glucose, vegetable oil, etc. as carbon sources, and is synthesized under high-carbon and low-nitrogen imbalance conditions.
  • the high cost of raw materials restricts the large-scale production and application of PHA.
  • scholars have studied a series of methods for producing PHA by using waste biomass.
  • CN 104357496B discloses a method for synthesizing PHA by using corn steep liquor hydrolyzate, using hydrolyzed corn steep liquor to provide nitrogen source and reducing the cost of adding nitrogen source in PHA synthesis
  • CN 104357496B 111892699A discloses a device and method for synthesizing PHA from food waste, which first acidifies the food waste and then further synthesizes PHA.
  • the invention provides a method for producing PHA by two-stage fermentation of agricultural waste such as livestock and poultry manure.
  • the object is crop straw and/or fruit and vegetable waste; the method includes:
  • the present invention proposes a plan to produce PHA by using livestock and poultry manure and other agricultural wastes from planting sources as raw materials through a two-stage fermentation process.
  • the main components of PHA are PHB and PHV.
  • the present invention through the selection of different functional strains in each stage, combined with the control of specific conditions in each fermentation stage, enables directional production of lactic acid and a variety of volatile fatty acids with specific ratios in the primary fermentation stage, and then in the subsequent The high-efficiency production of PHA is realized in the secondary fermentation stage.
  • the fermentation temperature in the primary fermentation stage is 35-45° C., and the pH value is 4.5-5.5.
  • This condition can improve the substrate competition ability of dominant lactic acid bacteria, and at the same time, the lactic acid produced by fermentation will reduce the pH of the system, further inhibit the metabolic activity of other microorganisms, and then promote the enrichment of dominant lactic acid bacteria, which is conducive to the realization of organic waste in agricultural waste such as manure. Directed conversion of carbon sources to specific ratios of lactic acid and various volatile fatty acids.
  • the volatile fatty acid of the present invention is acetic acid, propionic acid, butyric acid etc. (there may also be a small amount of isobutyric acid, valeric acid, isovaleric acid, hexanoic acid etc.).
  • the fermentation temperature in the secondary fermentation stage is 22-28° C.
  • the pH value is 7.5-8.5, so as to improve the utilization efficiency of organic carbon sources such as lactic acid by PHA-synthesizing bacteria.
  • the fermentation substrate is a mixture of livestock and poultry manure and fruit and vegetable waste, and the carbon-nitrogen ratio of the mixture is (30-50):1, preferably (35-40):1;
  • the fermentation substrate is a mixture of livestock and poultry manure and crop stalks, and the carbon-to-nitrogen ratio of the mixture is (30-40):1.
  • the present invention finds that when the ratio of livestock and poultry manure and plantation waste in the fermentation substrate is further controlled so that it is at the carbon-nitrogen ratio defined in the present invention, it can help the organic carbon source in the waste to transform into a specific proportion of lactic acid and a variety of volatile fatty acids, combined with the content of N, P and other trace elements in the fermentation broth, to ensure the production of PHA in the secondary fermentation stage.
  • the fruit and vegetable waste in the present invention refers to rotten fruits, seedlings, vines, leaves and tailings in the process of fruit or vegetable planting; crop straw is one or more of corn straw, wheat straw, and rice straw Kind; the total solid content of the fruit and vegetable waste is 2-20%, the volatile solid content is 90-98% TS (based on the total solid content), the total sugar content is 10-20% (based on fresh weight), and the carbon-nitrogen ratio is (40-100): 1; the total solid content of crop straw is 30-70%, the volatile solid content is 85-95% TS (based on total solid content), the crude fiber content is 35-55%, and the carbon-nitrogen ratio is (45-60):1.
  • the aeration rate of the primary fermentation stage is 0-2000mL/L/d;
  • the secondary fermentation stage is continuous air aeration aerobic fermentation, and the air aeration intensity is 0.8-1.2L/L/min.
  • the lactic acid bacteria include one or more of Bifidobacterium, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus bulgaricus.
  • the preparation method of the sludge of described domestication is:
  • the intensity of continuous air aeration is 0.8-1.2L/L/min.
  • the main microbial species in the acclimated sludge are the bacteria that can synthesize PHA.
  • the general acclimatization time is about 4 months, which is subject to the purpose of enriching the bacterial flora that can efficiently produce PHA.
  • the main flora in the domesticated sludge are Paracoccus and Gemmobacter, and the PHA content accounts for 27-42% of the dry weight of bacterial cells.
  • the fermentation concentration of primary fermentation stage is 2%VS-15%VS (calculated as volatile solid); 10 6 -10 9 CFU/mL;
  • the fermentation concentration of secondary fermentation stage is 60-120mmol C/L, is preferably 100-120mmol C/L; (HRT) is 5-36h.
  • the above conditions cooperate with each other to help ensure the final fermentation effect.
  • the invention provides a method for producing PHA by two-stage mixed fermentation of livestock and poultry manure and other agricultural wastes.
  • the yield of PHA is high, and the fermentation substrate is livestock and poultry manure, crop straw, fruit and vegetable waste, etc., without additional addition
  • Exogenous carbon and nitrogen sources wide sources of raw materials, broaden the production sources of PHA, and significantly reduce the cost of raw materials in the PHA production process; at the same time, it also realizes the harmless treatment and high-efficiency of agricultural organic waste such as livestock and poultry manure. Value-based utilization has good application prospects.
  • the content of lactic acid and volatile fatty acid is measured by liquid chromatography and gas chromatography, respectively.
  • liquid chromatography and gas chromatography respectively.
  • PHA The content of PHA was determined by gas chromatography.
  • instrument configuration and test methods please refer to: Pinto-Ibieta F, et al. Strategy for biological co-production of levulinic acid and polyhydroxyalkanoates by using mixed microbial cultures fed with synthetic hemicellulose hydrolysate[J]. Technology, 2020, 309:123323.
  • Embodiment 1 pig manure-fruit and vegetable mixed fermentation produces PHA
  • lactic acid bacteria culture medium MFS broth (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.), inoculate Baishengyou lactic acid bacteria powder (4g/L, purchased from Shanenkang Biotechnology (Suzhou) Co., Ltd. ), control the culture temperature and pH to 35-45° C. and 6.0-7.0, respectively, and cultivate for 36 hours.
  • the fermentation concentration is 4% VS (calculated as volatile solids), the pH is controlled at 4.5-5.5, the fermentation temperature is controlled at 35-40°C, and intermittent aeration keeps the oxidation-reduction potential (ORP) in the environment (fermentation system) at -250mV ⁇ -450mV, the specific aeration intensity is 200mL/L/min, and the aeration time is 5min/d.
  • ORP oxidation-reduction potential
  • the total carboxylic acid output, lactic acid output, and volatile fatty acid output of the material were 932.6, 880.3, and 52.3 mg/gVS respectively, and the output of acetic acid, propionic acid, and butyric acid in the volatile fatty acid was 16.6 mg/gVS respectively. , 1.6 and 25.9 mg/g VS.
  • Sludge domestication use acetic acid as the carbon source, ammonium chloride as the nitrogen source, and the aerobic aeration tank bottom sludge of the sewage treatment plant as the initial PHA strain source, at room temperature (20-30°C), continuous aeration ( 1L/L/min) and pH conditions (6.8-9.3) for 4 months; after domestication in the present invention, the dominant PHA-synthesizing bacteria in the sludge are Paracoccus (30%) and Gemmobacter (36%), PHA The content accounts for 27-42% of the dry weight of bacterial cells.
  • step 4 Use the acidification solution obtained by fermentation in step 2 as raw material, inoculate the acclimated sludge in step 3, the initial inoculation amount is 70% (V/V), and the subsequent continuous operation process is no longer supplemented; the fermentation concentration is 108 mmol C/L , the pH is controlled at 7.8-8.3, the fermentation temperature is controlled at 22-28°C, the continuous aeration is 0.8L/L/min, and the HRT is controlled at 24h.
  • the PHA concentration in the system reached 328mg/L, and the output was 348mg PHA/g VS, wherein the specific components were PHB (97%) and PHV (3%).
  • the production steps of this example are the same as those of Example 1, except that the fermentation raw materials in step 2 are pig manure and corn stalk waste, and the carbon-nitrogen ratio of the mixed material is 35:1.
  • the total carboxylic acid production, lactic acid production and volatile fatty acid production of fermentation raw material unit VS were 640.8, 609.4 and 31.4 mg/gVS, of which the production of acetic acid and butyric acid in volatile fatty acid were respectively 14.4 and 17.0 mg/g VS.
  • This acidified liquid is through two-stage aerobic fermentation (process parameters are the same as step 3 and 4 in embodiment 1), through 3 weeks of continuous fermentation, PHA concentration reaches 314mg/L in the system, and output is 326mg PHA/g VS, wherein concrete composition is PHB (96.9%) and PHV (3.1%).
  • step 2 the fermentation pH is controlled at 3.5, and the fermentation temperature is controlled at 55°C.
  • step 4 fermentation concentration is controlled at 60mmol C/L. After 3 weeks of continuous fermentation, the PHA concentration in the system reached 286mg/L, and the output was 217mg PHA/g VS, wherein the specific components were PHB (94%) and PHV (6%).
  • the production steps of this example are the same as those of Example 1, except that the fermentation temperature in step 4 is controlled at 40°C. After 3 weeks of continuous fermentation, the PHA concentration in the system reached 252mg/L, and the output was 267mg PHA/g VS, wherein the specific components were PHB (95.5%) and PHV (4.5%).
  • the artificially configured acidified solution containing only lactic acid is used as a carbon source to synthesize PHA.
  • the synthesis conditions of PHA are the same as those in Steps 3 and 4 of Example 1.
  • the concentration of PHA in the system reaches 157mg/L, the output is 173mg PHA/g VS, the main component of which is PHB (99.4%).
  • the unit yield of PHA was reduced by 50.3% and 46.9%, respectively, indicating that the acidified solution of agricultural waste obtained in the present invention has a significant advantage in synthesizing PHA.
  • step 4 the HRT is controlled at 4h.
  • Step 2 pig manure and apple waste are used as substrates, and the carbon-to-nitrogen ratio of the mixed material is 55:1.
  • step 2 intermittent aeration keeps the ORP in the system at -220mV, the specific aeration intensity is 500mL/L/min, and the aeration time is 10min/d.
  • the invention belongs to the field of utilization of agricultural waste, in particular to a method for producing polyhydroxyalkanoate by fermentation of agricultural waste.
  • the fermentation substrate is agricultural waste, including a mixture of livestock and poultry manure and planting waste.
  • the method includes: a primary fermentation stage: the fermentation bacteria are lactic acid bacteria, the fermentation temperature is 35-55 ° C, and the pH value is 3.0-6.0, intermittent aeration to maintain the oxidation-reduction potential in the system at -250 ⁇ -450mV; Secondary fermentation stage: use domesticated sludge for aerobic fermentation, the fermentation temperature is 20-40°C, and the pH value is 7.0-9.0 .
  • the present invention efficiently converts organic carbon sources in livestock and poultry manure and planting waste into PHA without additional addition of exogenous carbon sources and nitrogen sources, which can not only broaden the production sources of PHA, reduce the production cost of PHA, and Reducing the potential pollution of livestock and poultry manure and planting waste provides a new way for their high-value utilization.

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Abstract

本发明提供了一种农业废弃物发酵生产聚羟基脂肪酸酯的方法。本发明中发酵底物为农业废弃物，包括畜禽粪污和种植业废弃物的混合物，该方法包括：一级发酵阶段：发酵菌为乳酸菌，发酵温度为35-55℃，pH值为3.0-6.0，间歇曝气使系统中氧化还原电位维持在-250～-450mV；二级发酵阶段：采用驯化的污泥进行好氧发酵，发酵温度为20-40℃，pH值为7.0-9.0。本发明在不额外添加外源碳源和氮源的条件下，将畜禽粪污和种植业废弃物中有机碳源高效转化为PHA，不仅可拓宽PHA的生产来源，降低PHA生产成本，而且减少畜禽粪污和种植业废弃物的潜在污染。

Description

农业废弃物发酵生产聚羟基脂肪酸酯的方法
交叉引用
本申请要求2021年8月2日提交的专利名称为“农业废弃物发酵生产聚羟基脂肪酸酯的方法”的第202110878525.5号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于农业废弃物利用领域，具体涉及一种农业废弃物发酵生产聚羟基脂肪酸酯的方法。
背景技术
聚羟基脂肪酸酯(Polyhydroxyalkanoates，PHA)是一种可生物降解的新型可塑原料，它具备传统塑料制品相似的材料学特性。不同于传统塑料，PHA可在自然环境(如土壤、海水)或生物处理(如厌氧消化、好氧堆肥)条件下被完全降解为CO 2/CH 4和水，是环境友好的可降解材料。PHA的推广应用是解决白色污染和改善生态环境的有效途径，为此PHA的高效生产和应用也得到了广泛关注。
当前PHA发酵生产工艺主要通过葡萄糖、植物油等为碳源，在高碳低氮的失衡条件下进行合成。原料成本高制约了PHA的规模化生产和应用，为降低生产成本，学者们研究了系列利用废弃生物质生产PHA的方法，如中国专利CN 111394398 A公开了一种以高盐糖蜜为原料进行发酵制备PHA的方法，以糖蜜废水替代葡萄糖降低碳源成本；CN 104357496B公开了一种利用玉米浆水解液合成PHA的方法，采用水解玉米浆提供氮源降低了PHA合成中外加氮源的成本；CN 111892699A公开了一种利用餐厨垃圾合成PHA的设备和方法，其将餐厨垃圾首先酸化再进一步合成PHA。畜禽粪便相对餐厨垃圾而言，资源量大且物料特性稳定，但N/P含量更高，易使PHA培养过程达到营养平衡状态，造成PHA被转化利用，不利于PHA的累积，这就导致了采用畜禽粪便生产PHA相对于其他物料 的难度更大，目前尚没有理想的方法。
鉴于此，有必要摸索出一种新的更利于推广的，适于以含有高氮、磷、有机物和矿物质等复杂成分的废弃物为原料合成PHA的方法。
发明内容
本发明为解决PHA生产过程中原料成本高，提供了一种利用畜禽粪污等农业废弃物两阶段发酵生产PHA的方法。
具体地，本发明的技术方案如下：
一种农业废弃物发酵生产聚羟基脂肪酸酯(PHA)的方法，其发酵底物为农业废弃物，所述农业废弃物包括畜禽粪污和种植业废弃物的混合物，所述种植业废弃物为农作物秸秆和/或果蔬废弃物；所述方法包括：
A、一级发酵阶段：发酵菌为乳酸菌，发酵温度为35-55℃，pH值为3.0-6.0，间歇曝气使发酵系统中氧化还原电位(ORP)维持在-250mV～-450mV；
B、二级发酵阶段：采用驯化的污泥进行好氧发酵，发酵温度为20-40℃，pH值为7.0-9.0。
本发明在现有PHA合成技术研究基础上，提出了利用畜禽粪污与其他种植源农业废弃物为原料通过两阶段发酵工艺生产PHA的方案。其中PHA的主要成分为PHB和PHV。
具体地，本发明通过各阶段不同功能菌种的选择，配合各发酵阶段的特定条件控制，使得在一级发酵阶段可定向生成具有特定比例关系的乳酸和多种挥发性脂肪酸，进而在后续的二级发酵阶段实现PHA的高效生产。
优选，一级发酵阶段的发酵温度为35-45℃，pH值为4.5-5.5。这一条件可提高优势产乳酸菌的底物竞争能力，同时发酵产生的乳酸会降低系统pH，进一步抑制其他微生物的代谢活性，进而促进优势乳酸菌的富集，利于实现粪污等农业废弃物中有机碳源向特定比例关系的乳酸和多种挥发性脂肪酸的定向转化。
本发明所述挥发性脂肪酸为乙酸、丙酸、丁酸等(还可能有少量的异 丁酸、戊酸、异戊酸、己酸等)。
优选，二级发酵阶段的发酵温度为22-28℃，pH值为7.5-8.5，以提升PHA合成菌对乳酸等有机碳源的利用效率。
本发明中，所述发酵底物为畜禽粪污和果蔬废弃物的混合物，所述混合物的碳氮比为(30-50):1，优选为(35-40)：1；
或，所述发酵底物为畜禽粪污和农作物秸秆的混合物，所述混合物的碳氮比为(30-40):1。
本发明发现，当进一步控制发酵底物中畜禽粪污和种植业废弃物的比例，使其处于本发明限定的碳氮比时，可有助于废弃物中有机碳源向特定比例的乳酸和多种挥发性脂肪酸的定向转化，并结合发酵液中的N、P及其他微量元素含量，进而保证二级发酵阶段PHA的生产。
本发明所述果蔬废弃物为水果或蔬菜种植过程中产生的腐烂的果实、秧、藤、叶以及加工过程中的尾料；农作物秸秆为玉米秸秆、小麦秸秆、水稻秸秆中的一种或多种；所述果蔬废弃物的总固体含量2-20％，挥发性固体含量90-98％TS(基于总固体含量)，总糖含量为10-20％(基于鲜重)，碳氮比为(40-100)：1；农作物秸秆的总固体含量为30-70％，挥发性固体含量为85-95％TS(基于总固体含量)，粗纤维含量为35-55％，碳氮比为(45-60)：1。
本发明中，作为一个具体实施方式，一级发酵阶段的曝气量为0-2000mL/L/d；
优选，一级发酵阶段的具体曝气强度为0-400mL/L/min，曝气时间为0-10min/d。
本发明中，二级发酵阶段为连续空气曝气好氧发酵，空气曝气强度为0.8-1.2L/L/min。
本发明中，所述乳酸菌包括双歧杆菌、嗜热链球菌、植物乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、保加利亚乳杆菌中的一种或多种。
本发明中，所述驯化的污泥的制备方法为：
以乙酸为碳源，氯化铵为氮源，将污水处理厂好氧曝气池底泥在室温(20-30℃)、空气连续曝气及pH值为6.5-9.5的发酵体系下驯化；
和/或，空气连续曝气的强度为0.8-1.2L/L/min。
经驯化后的污泥中主要微生物种类为可合成PHA的菌。一般驯化时间为4个月左右，以实现富集可高效产PHA的菌群的目的为准。
优选，经驯化后，所述驯化的污泥中主要菌群为副球菌属(Paracoccus)和芽殖杆菌属(Gemmobacter)，PHA含量占细菌细胞干重的27-42％。
本发明中，一级发酵阶段的发酵浓度为2％VS-15％VS(以挥发性固体计)；菌液接种量为2％-10％(V/V)，菌液中的活菌数为10 6-10 9CFU/mL；
和/或，二级发酵阶段的发酵浓度为60-120mmol C/L，优选为100-120mmol C/L；驯化的污泥的接种量为40％-80％(V/V)，水利停留时间(HRT)为5-36h。上述条件相互配合，有助于保证最终的发酵效果。
本发明的有益效果：
本发明提供了一种利用畜禽粪污与其他农业废弃物两阶段混合发酵产PHA的方法，PHA产量高，且发酵底物为畜禽粪污、农作物秸秆、果蔬废弃物等，不额外添加外源碳源和氮源，原料来源广泛，拓宽了PHA的生产来源，并显著降低PHA生产过程的原料成本；同时还实现了畜禽粪污等农业源有机废弃物的无害化处理和高值化利用，具有良好的应用前景。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
本发明具体实施方式部分，乳酸和挥发性脂肪酸含量分别采用液相色谱仪和气相色谱仪测定，具体的仪器配置及测定方法参见：Lian T,Zhang W,Cao Q,et al.Enhanced lactic acid production from the anaerobic co-digestion  of swine manure with apple or potato waste via ratio adjustment[J].Bioresource Technology,2020,318:124237。
PHA含量采用气相色谱仪测定，具体仪器配置和测试方法参见：Pinto-Ibieta F,et al.Strategy for biological co-production of levulinic acid and polyhydroxyalkanoates by using mixed microbial cultures fed with synthetic hemicellulose hydrolysate[J].Bioresource Technology,2020,309:123323。
实施例1猪粪-果蔬混合发酵生产PHA
本实施例包括如下步骤：
1、乳酸菌培养：配置乳酸菌培养基—MRS肉汤(青岛高科技工业园海博生物技术有限公司)，接种佰生优乳酸菌粉(4g/L，购自善恩康生物科技(苏州)有限公司)，控制培养温度和pH分别为35-45℃和6.0-7.0，培养36h。
2、将培养后的乳酸菌的菌液(活菌数约为10 7CFU/mL)接种到以猪粪、苹果废弃物为底物的厌氧发酵罐中，混合物料的碳氮比为(35-40):1，接种量为10％(V/V)。发酵浓度为4％VS(以挥发性固体计)，pH控制在4.5-5.5，发酵温度控制为35-40℃，间歇曝气使环境(发酵系统)中氧化还原电位(ORP)维持在-250mV～-450mV，具体曝气强度为200mL/L/min,曝气时间5min/d。经过2周连续厌氧发酵，物料的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为932.6、880.3和52.3mg/gVS，其中挥发性脂肪酸中乙酸、丙酸、丁酸的产量分别为16.6、1.6和25.9mg/gVS。
3.污泥驯化：以乙酸为碳源，氯化铵为氮源，以污水处理厂好氧曝气池底泥为初始PHA菌种来源，在室温(20-30℃)、连续曝气(1L/L/min)及pH条件下(6.8-9.3)驯化4个月；本发明中经驯化后，污泥中的优势PHA合成菌属为Paracoccus(30％)和Gemmobacter(36％)，PHA含量占细菌细胞干重的27-42％。
4.以步骤2发酵获得的酸化液为原料，接种步骤3驯化后的污泥，初始接种量为70％(V/V)，后续连续运行过程不再补充；发酵浓度为108 mmol C/L，pH控制在7.8-8.3，发酵温度控制为22-28℃，连续曝气0.8L/L/min，HRT控制在24h。经过3周连续发酵，系统中PHA浓度达328mg/L，产量为348mg PHA/g VS，其中具体成分为PHB(97％)和PHV(3％)。
实施例2猪粪-秸秆混合发酵生产PHA
本实施例的生产步骤与实施例1相同，区别仅在于：步骤2中发酵原料为猪粪与玉米秸秆废弃物，混合物料的碳氮比为35：1。经过2周连续厌氧酸化发酵后，发酵原料单位VS的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为640.8、609.4和31.4mg/gVS，其中挥发性脂肪酸中乙酸和丁酸的产量分别14.4和17.0mg/gVS。
该酸化液经过二级好氧发酵(工艺参数同实施例1中步骤3和4)，经过3周连续发酵，系统中PHA浓度达314mg/L，产量为326mg PHA/g VS，其中具体成分为PHB(96.9％)和PHV(3.1％)。
实施例3
本实施例的生产步骤与实施例1相同，区别仅在于：步骤2中发酵pH控制在3.5，发酵温度控制为55℃。
一级发酵结果：经过2周连续厌氧发酵，物料的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为373.4、342.5和30.9mg/gVS，其中挥发性脂肪酸中乙酸、丙酸和丁酸的产量分别为5.0、2.7和23.2mg/gVS。
二级发酵结果：经过3周连续发酵，系统中PHA浓度达277mg/L，产量为275mg PHA/g VS，其中具体成分为PHB(96.5％)和PHV(3.5％)。
实施例4
本实施例的生产步骤与实施例1相同，区别仅在于：步骤4中发酵浓度控制在60mmol C/L。经过3周连续发酵，系统中PHA浓度达286mg/L，产量为217mg PHA/g VS，其中具体成分为PHB(94％)和PHV(6％)。
实施例5
本实施例的生产步骤与实施例1相同，区别仅在于：步骤4中发酵温度控制在40℃。经过3周连续发酵，系统中PHA浓度达252mg/L，产量为267mg PHA/g VS，其中具体成分为PHB(95.5％)和PHV(4.5％)。
对比例1
参照实施例1中酸化液中乳酸浓度，以人工配置的仅含乳酸的酸化液为碳源合成PHA，PHA合成条件同实施例1步骤3和4，经过3周连续发酵，系统中PHA浓度达157mg/L，产量为173mg PHA/g VS，其中主要成分为PHB(99.4％)。比实施例1和实施例2中PHA单位产量分别降低了50.3％和46.9％，表明本发明获得的农业废弃物酸化液在合成PHA方面有显著优势。
对比例2
本实施例的生产步骤与实施例1相同，区别仅在于：步骤4中HRT控制在4h。
二级发酵结果：经过3周连续发酵，系统中PHA浓度达174mg/L，产量为106mg PHA/g VS，其中具体成分为PHB(95.1％)和PHV(4.9％)。
对比例3
本实施例的生产步骤与实施例1相同，区别仅在于：步骤2中发酵pH控制在6.5，发酵温度控制为60℃。
一级发酵结果：经过2周连续厌氧发酵，物料的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为192.7、174.2和18.5mg/gVS，其中挥发性脂肪酸组分中只检测到丁酸，产量为18.5mg/gVS。
二级发酵结果：经过3周连续发酵，系统中PHA浓度达158mg/L，产量为106mg PHA/g VS，其中具体成分为PHB(95.4％)和PHV(4.6％)。
对比例4
本实施例的生产步骤与实施例1相同，区别仅在于：步骤2中猪粪、苹果废弃物为底物，混合物料的碳氮比为55：1。
一级发酵结果：经过2周连续厌氧发酵，物料的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为127.4、119.5和7.9mg/gVS，其中挥发性脂肪 酸组分中只检测到丁酸，产量为7.9mg/gVS。
二级发酵结果：经过3周连续发酵，系统中PHA浓度达168mg/L，产量为120mg PHA/g VS，其中具体成分为PHB(95.5％)和PHV(4.5％)。
对比例5
本实施例的生产步骤与实施例1相同，区别仅在于：步骤2中间歇曝气使系统中的ORP保持在-220mV，具体曝气强度为500mL/L/min,曝气时间10min/d。
一级发酵结果：经过2周连续厌氧发酵，物料的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为320.6、150.8和169.8mg/gVS，其中挥发性脂肪酸中乙酸、丙酸和丁酸的产量分别为85.2、33.6和51.0mg/gVS。
二级发酵结果：经过3周连续发酵，系统中PHA浓度达162mg/L，产量为151mg PHA/g VS，其中具体成分为PHB(94.7％)和PHV(5.3％)。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明属于农业废弃物利用领域，具体涉及农业废弃物发酵生产聚羟基脂肪酸酯的方法。本发明中发酵底物为农业废弃物，包括畜禽粪污和种植业废弃物的混合物，所述方法包括：一级发酵阶段：发酵菌为乳酸菌，发酵温度为35-55℃，pH值为3.0-6.0，间歇曝气使系统中氧化还原电位维持在-250～-450mV；二级发酵阶段：采用驯化的污泥进行好氧发酵，发酵温度为20-40℃，pH值为7.0-9.0。本发明在不额外添加外源碳源和氮源的条件下，将畜禽粪污和种植业废弃物中有机碳源高效转化为PHA，不仅可拓宽PHA的生产来源，降低PHA生产成本，而且减少畜禽粪污和种植业废弃物的潜在污染，为它们的高值化利用提供了新途径。

Claims (4)

  1. 一种农业废弃物发酵生产聚羟基脂肪酸酯的方法，其特征在于，所述聚羟基脂肪酸酯包括PHB和PHV；发酵底物为农业废弃物，所述农业废弃物包括畜禽粪污和种植业废弃物的混合物，所述种植业废弃物为农作物秸秆和/或果蔬废弃物；所述方法包括：
    一级发酵阶段：发酵菌为乳酸菌，发酵温度为35-40℃，pH值为4.5-5.5，间歇曝气使发酵系统中氧化还原电位维持在-250mV～-450mV；
    二级发酵阶段：采用驯化的污泥进行好氧发酵，发酵温度为22-28℃，pH值为7.8-8.3，二级发酵阶段为连续空气曝气好氧发酵，空气曝气强度为0.8-1.2L/L/min；
    所述驯化的污泥的制备方法为：
    以乙酸为碳源，氯化铵为氮源，将污水处理厂好氧曝气池底泥在室温、空气连续曝气及pH值为6.5-9.5的发酵体系下驯化；空气连续曝气的强度为0.8-1.2L/L/min；
    经驯化后，所述驯化的污泥中主要菌群为副球菌属(Paracoccus)和芽殖杆菌属(Gemmobacter)，聚羟基脂肪酸酯含量占细菌细胞干重的27-42％；
    所述发酵底物为畜禽粪污和果蔬废弃物的混合物，所述混合物的碳氮比为(30-50)：1；
    或，所述发酵底物为畜禽粪污和农作物秸秆的混合物，所述混合物的碳氮比为(30-40):1。
  2. 根据权利要求1所述的方法，其特征在于，一级发酵阶段的曝气量为0-2000mL/L/d；一级发酵阶段的曝气强度为0-400mL/L/min，曝气时间为0-10min/d。
  3. 根据权利要求1所述的方法，其特征在于，所述乳酸菌包括双歧杆菌、嗜热链球菌、植物乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、保加利亚乳杆菌中的一种或多种。
  4. 根据权利要求3所述的方法，其特征在于，一级发酵阶段的发酵浓度为2％VS-15％VS；菌液接种量为2％-10％V/V，菌液中的活菌数为10 6-10 9CFU/mL；
    和/或，二级发酵阶段的发酵浓度为60-120mmol C/L；驯化的污泥的接种量为40％-80％V/V，水利停留时间为5-36h。
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CN116144712A (zh) * 2023-04-20 2023-05-23 中国农业科学院农业环境与可持续发展研究所 一种通过生物酸化减少畜禽粪污温室气体排放的方法
CN116144712B (zh) * 2023-04-20 2023-07-21 中国农业科学院农业环境与可持续发展研究所 一种通过生物酸化减少畜禽粪污温室气体排放的方法
CN117228831A (zh) * 2023-11-14 2023-12-15 青岛锦龙弘业环保有限公司 一种反硝化池中除氮的复合碳源的制备方法
CN118006514A (zh) * 2024-04-08 2024-05-10 中国农业科学院农业环境与可持续发展研究所 利用富含乳酸的农业废弃物发酵液富集聚羟基脂肪酸酯合成菌群及生产聚羟基脂肪酸酯的方法

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