WO2022100598A1 - 一种农业废弃物通过发酵方式生产中链羧酸的方法与应用 - Google Patents

一种农业废弃物通过发酵方式生产中链羧酸的方法与应用 Download PDF

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WO2022100598A1
WO2022100598A1 PCT/CN2021/129717 CN2021129717W WO2022100598A1 WO 2022100598 A1 WO2022100598 A1 WO 2022100598A1 CN 2021129717 W CN2021129717 W CN 2021129717W WO 2022100598 A1 WO2022100598 A1 WO 2022100598A1
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fermentation
chain carboxylic
acid
carboxylic acid
agricultural waste
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French (fr)
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董红敏
张万钦
尹福斌
曹起涛
连天境
王顺利
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中国农业科学院农业环境与可持续发展研究所
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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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

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  • the invention relates to the field of agricultural waste utilization, in particular to a method and application for producing medium-chain carboxylic acid from agricultural waste by fermentation.
  • Livestock and poultry manure, crop straw, fruit and vegetable waste and other agricultural production and living organic wastes have a large amount of resources and high content of organic components, which are good biomass resources.
  • Anaerobic digestion to produce biogas technology is a commonly used method to deal with this type of waste, but due to the low efficiency of a single biogas product, the market competitiveness is weak.
  • medium-chain carboxylic acids caproic acid, heptanoic acid, octanoic acid, etc.
  • the process mainly includes two steps: 1) One The first-stage fermentation organic waste is directionally converted into short-chain carboxylic acids; 2) The second-stage fermentation uses short-chain carboxylic acids to synthesize medium-chain carboxylic acids through carbon chain extension reactions.
  • Chinese patent CN104357496B discloses a method for synthesizing caproic acid by catalyzing lactic acid by microorganisms, and points out that lactic acid can effectively replace ethanol, drive carbon chain extension reaction, and promote the synthesis of medium chain carboxylic acid.
  • Chinese patent CN105420168B also discloses a Clostridium rumensis that utilizes lactic acid to synthesize caproic acid and an application method thereof, which realizes the synthesis of caproic acid without adding ethanol. However, they did not study and record the scheme of simultaneously preparing and obtaining a variety of medium-chain carboxylic acids without adding ethanol.
  • the first-stage anaerobic acidification process products of organic matter are mainly volatile short-chain carboxylic acids.
  • a method for producing volatile short-chain carboxylic acids such as acetic acid and butyric acid from wastes.
  • There is also Chinese patent CN104911128 B which discloses a method for converting glucose/fructose etc. into lactic acid by using thermophilic anaerobic bacillus.
  • the research on the directional transformation to obtain a variety of medium-chain carboxylic acids has not been reported, and the idea of adding electron donors exogenously in the fermentation process of agricultural wastes such as poultry manure is still used, and the production cost needs to be further reduced.
  • the invention provides a method that can reduce the directional transformation of agricultural source biomass resources. Scheme for the cost of producing medium chain carboxylic acids.
  • a method for producing medium-chain carboxylic acid from agricultural waste by fermentation the fermentation substrate is a mixture of livestock and poultry manure and agricultural waste, and the agricultural waste is crop straw and/or fruit and vegetable waste; the method comprises: :
  • Secondary fermentation stage use domesticated sludge for anaerobic fermentation, the fermentation temperature is 25-40°C, and the pH value is 4.0-7.0.
  • the present invention proposes a method that can realize high-efficiency medium-chain carboxylic acid without adding external electron donors. production plan. Specifically, the present invention selects different starter agents at each stage and controls the fermentation process in different stages, so that lactic acid and a variety of volatile fatty acids with a specific ratio can be directionally generated in the primary fermentation stage. The secondary fermentation stage enables the simultaneous and efficient production of multiple medium-chain carboxylic acids.
  • the volatile fatty acids of the present invention are acetic acid, propionic acid, butyric acid and the like.
  • the medium chain carboxylic acid is n-hexanoic acid, n-heptanoic acid, n-octanoic acid and the like.
  • the fermentation temperature of the primary fermentation stage is 50-55°C, and the pH value is 4.5-5.5.
  • This condition can improve the substrate competition ability of lactic acid bacteria, so as to facilitate the enrichment and fermentation of dominant lactic acid bacteria, and realize Directed conversion of specific ratios of lactic acid to various volatile fatty acids.
  • the fermentation temperature of the secondary fermentation stage is 25-35°C, and the pH value is 5.0-6.5, so as to improve the yield and production efficiency of caproic acid, and further ensure the efficient production of caproic acid and caprylic acid.
  • Reverse beta oxidation yields heptanoic acid.
  • the fermentation substrate is a mixture of livestock and poultry manure and crop straw, and the carbon-nitrogen ratio of the mixture is (30-40): 1;
  • 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 (30-40):1.
  • the ratio of livestock and poultry manure and agricultural waste in the fermentation substrate is further controlled to be in the carbon single ratio defined in the present invention, it can help to obtain a specific ratio of lactic acid and a variety of volatile fatty acids, This in turn ensures the production of the final medium chain carboxylic acid.
  • the crop straw of the present invention is one or more of corn straw, wheat straw and rice straw; the fruit and vegetable wastes are rotten fruits, seedlings, vines, leaves and other wastes produced during the planting of fruits or vegetables.
  • the tailings; the total solid content of the crop straw is 30-70%, the volatile solid content is 85-95% TS (based on the total solid content), the crude fiber content is 35-55%, and the carbon-nitrogen ratio is (45% -60): 1;
  • the lactic acid bacteria include one or more of Bifidobacterium, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus bulgaricus.
  • the fermentation concentration is 2% VS-15% VS (in terms of volatile solids); the inoculation amount of the bacterial liquid is 2%-8% (V/V), and the number of viable bacteria in the bacterial liquid is 10 6 -10 9 CFU/mL.
  • step B the inoculation amount of the domesticated sludge is 5%-20% (V/V), preferably 10%.
  • the preparation method is:
  • the granular sludge in the anaerobic fermenter for treating organic wastewater is acclimated under the fermentation system of 25-40°C and pH value of 5.0-6.5.
  • the main microbial species in the domesticated sludge were carbon chain elongation bacteria.
  • the general domestication time is about 2 months, which is based on the purpose of enriching the carbon chain extension flora.
  • the main bacterial groups in the domesticated sludge of the present invention are Clostridium kluyveri 10-25%, Prevotellaceae 20-30%, Succinivibrionaceae 10-15% %, Bacteroidales 5-10%, Spirochaetaceae 5-10%, Rikenellaceae 2-8%, Fibrobacteraceae 2-5%, Lachnospiraceae 2-5%, Ruminococcaceae 2-5%, other 10-15%.
  • the present invention also provides an application of the above method in the production of medium chain carboxylic acid.
  • the present invention also provides a method for producing short-chain carboxylic acids such as lactic acid by using livestock and poultry manure and other agricultural wastes by high-temperature mixed fermentation, comprising the following steps:
  • the lactic acid fermentation flora in step 1 mainly includes Bifidobacterium, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and the like.
  • short-chain carboxylic acids such as lactic acid in the fermentation stage of step (2), specifically inoculating 2%-8% of the lactic acid bacteria liquid by volume, controlling the fermentation temperature to be 45-60°C, and controlling the pH to be 4.0-6.0.
  • the invention provides a method for producing medium-chain carboxylic acid by using livestock and poultry manure and other agricultural wastes by two-stage gradient temperature-controlled mixed fermentation.
  • the fermentation substrates are livestock and poultry manure, crop straw, fruit and vegetable waste, etc.
  • the conversion process of medium-chain carboxylic acid does not require the addition of external electron donors, the operating cost is low, and the resulting carboxylic acid products have high added value; it can effectively realize the harmless treatment and high-efficiency treatment of agricultural organic wastes such as livestock and poultry manure. Value utilization.
  • the technological operation of the invention is simple, the anaerobic fermentation conditions are easy to control, the economical cost is low, and the invention has a good application prospect.
  • the invention can convert more than 90% of endogenous short-chain carboxylic acid into high-value-added medium-chain carboxylic acid, and provides an economical and feasible new way for high-value utilization of agricultural waste.
  • FIG. 1 is a bar graph of the carboxylic acid yield of material unit VS under different fermentation temperature conditions in Example 1 and Comparative Example 1 of the present invention.
  • Fig. 2 is the transformation trend diagram of lactic acid and medium chain carboxylic acid in Example 2 of the present invention.
  • Fig. 3 is the transformation trend diagram of lactic acid and medium chain carboxylic acid in Example 3 of the present invention.
  • Example 1 High-temperature fermentation produces short-chain carboxylic acids such as lactic acid
  • Lactic acid bacteria culture configure lactic acid bacteria medium - MRS 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. Company), control the culture temperature and pH to be 35-38°C and 6.0-7.0, respectively, and culture for 36h.
  • the total carboxylic acid production, lactic acid production and volatile fatty acid production of the material under high temperature fermentation conditions are 226.8, 214.9 and 23.0 mg/ In gVS, the yields of acetic acid, propionic acid and butyric acid in volatile fatty acids were 6.1, 3.4 and 13.5 mg/gVS, respectively.
  • Example 1 The production steps of this comparative example are the same as those of Example 1, the only difference being that: in step 2, the fermentation temperature is controlled to be 35-38°C. After 2 weeks of continuous anaerobic acidification fermentation, the carboxylic acid production per unit of fermentation raw material VS is shown in Figure 1.
  • the total carboxylic acid production, lactic acid production and volatile fatty acid production of the materials under mesophilic fermentation conditions are 107.9, 81.9 and 25.8 mg/ gVS.
  • the total carboxylic acid yield and lactic acid yield of the material under high temperature conditions Example 1 were 2.1 and 8.3 times higher than those under mesophilic fermentation (Comparative Example 1) conditions, respectively.
  • the production ratio of lactic acid to other volatile fatty acids also changed greatly.
  • Example 2 Two-stage fermentation to produce medium chain carboxylic acid
  • the primary fermentation acidified solution obtained after continuous anaerobic acidification fermentation for 2 weeks in Example 1 was transferred to the secondary fermentor, and the acclimated bacteria group with carbon chain extension (Clostridium kluyveri 23.4%) was inoculated. , Prevotellaceae 22.3%, Succinivibrionaceae 11.6%, Bacteroidales 8.2%, Spirochaetaceae 6.7%, Rikenellaceae 5.5%, Fibrobacteraceae 3.8%, Lachnospiraceae 2.4% , Ruminococcaceae 3.6%, other about 12.5%) sludge, the preparation method of the sludge is: with ethanol and/or lactic acid as electron donors, will treat the granular sludge in the anaerobic fermentation tank of organic wastewater , acclimated for 2 months under the fermentation system of 25-40 °C and pH value of 5.0-6.5.
  • the inoculum amount is 10% (volume ratio)
  • the fermenter is equipped with polyurethane biological filler for microorganism attachment
  • the fermentation temperature is 30 ⁇ 1°C
  • the pH value of the secondary fermentation process is controlled to be 6.0 ⁇ 0.2.
  • medium-chain carboxylic acids such as caproic acid through carbon chain extension (see Figure 2). It can be further obtained by calculation that 866.0 mg of COD medium-chain carboxylic acid can be synthesized correspondingly for every 1 g of lactic acid degraded.
  • Example 2 The production steps of this example are the same as those of Example 2, the only difference is that the pH value in the secondary fermentation process is controlled to be 4.5 ⁇ 0.2. Compared with Example 2, the conversion rate of lactic acid was relatively slow, and only two medium-chain carboxylic acids, hexanoic acid and octanoic acid, were detected in the secondary fermentation product, and the maximum concentrations were 2302.8 and 562.3 mgCOD/L respectively (see Figure 3). It was further concluded that for every 1 g of lactic acid degraded, 409.3 mg of COD medium-chain carboxylic acid could be synthesized correspondingly.
  • the bacterial liquid was Streptococcus thermophilus with a viable count of 10 6 CFU/mL (purchased from Shanenkang Biotechnology (Suzhou) Co., Ltd.), the fermentation concentration in the first fermentation stage was 15% VS, and the bacterial liquid inoculum was 8% (V/V), after 2 weeks of continuous anaerobic acidification fermentation, the total carboxylic acid production, lactic acid production and volatile fatty acid production of the fermentation raw material unit VS were 370.0, 327.1 and 43.9 mg/g VS, respectively.
  • the yields of acetic acid, propionic acid and butyric acid in volatile fatty acids were 18.4, 9.6 and 15.9 mg/g VS, respectively.
  • the above-obtained primary fermentation acidified solution was transferred to a secondary fermentor, and the method of Example 2 was used to produce medium-chain carboxylic acid. , 823.6 and 932.5 mgCOD/L, the medium chain carboxylic acid yield was 791.8 mgCOD medium chain carboxylic acid/g lactic acid.
  • This comparative example adopts a one-step method to directly produce medium chain acid from the substrate, and the specific steps are as follows:
  • the fermentation raw materials and concentrations were the same as those in Example 1, the fermentation temperature was 35 ⁇ 1°C, and the pH value was 5.5 ⁇ 0.5.
  • the medium-chain carboxylic acid was obtained by a one-step method. After 3 weeks of anaerobic fermentation, lactic acid could be detected in the fermentation substrate. , acetic acid, butyric acid and n-hexanoic acid, the concentrations were 550.2, 1360.0, 2470.8 and 620.5 mgCOD/L, respectively.
  • the results show that although medium-chain carboxylic acids can be obtained by one-step fermentation, only n-hexanoic acid can be detected, and the content is significantly low Medium chain carboxylic acid concentration in the two-step fermentation process.
  • the invention belongs to the field of agricultural waste utilization, and in particular relates to a method and application for producing medium-chain carboxylic acid from agricultural waste through fermentation.
  • the fermentation substrate is a mixture of livestock and poultry manure and agricultural wastes, and the agricultural wastes are crop straws and/or fruit and vegetable wastes;
  • the method includes: A. Primary fermentation stage: fermentation bacteria It is lactic acid bacteria, fermentation temperature is 45-60°C, pH value is 4.0-6.0; B, secondary fermentation stage: anaerobic fermentation is carried out using domesticated sludge as inoculum, fermentation temperature is 25-40°C, pH value is 4.0 -7.0.
  • the present invention obtains high value-added medium-chain carboxylic acid from livestock and poultry manure and agricultural waste through a two-stage gradient temperature-controlled fermentation method without adding external electron donors, which is the highest value of agricultural waste. Chemical utilization provides a feasible new way.

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Abstract

提供一种农业废弃物通过发酵方式生产中链羧酸的方法与应用。所述方法发酵底物为畜禽粪污和农业废弃物的混合物，所述农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45-60℃，pH值为4.0-6.0；B、二级发酵阶段：采用驯化的污泥作为接种物进行厌氧发酵，发酵温度为25-40℃，pH值为4.0-7.0。通过两阶段梯度控温发酵的方法在不添加外源电子供体的条件下，从畜禽粪污和农业废弃物中获得高附加值的中链羧酸，为农业废弃物的高值化利用提供一条可行的新途径。

Description

一种农业废弃物通过发酵方式生产中链羧酸的方法与应用
交叉引用
本申请要求2020年11月13日提交的专利名称为“一种农业废弃物通过发酵方式生产中链羧酸的方法与应用”的第202011266252.0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
本发明涉及农业废弃物利用领域，具体涉及一种农业废弃物通过发酵方式生产中链羧酸的方法与应用。
背景技术
畜禽粪污、农作物秸秆、果蔬废弃物等农业生产生活有机废弃物资源量大，有机组分含量高，是良好的生物质资源。厌氧消化产沼气技术是常用的处理该类废弃物的方法，但由于单一沼气产品效能低，市场竞争力较弱。相对于沼气，生物质资源两阶段厌氧发酵工艺转化生产的中链羧酸(己酸、庚酸、辛酸等)具有更广泛的用途和市场价值，该工艺主要包括2个步骤：1)一级发酵有机废弃物定向转化为短链羧酸；2)二级发酵将短链羧酸通过碳链延长反应合成中链羧酸。
近年来，通过有机废弃物转化生产中链羧酸的研究得到了广泛关注，例如，陈哲柯等以剩余污泥为原料，其厌氧发酵液进行链式延长反应初步获得了己酸(陈哲柯.基于厌氧发酵的剩余污泥产中链脂肪酸研究.湖南大学,2018)。也有研究表明，以食品废弃物和城市有机生活垃圾为原料，通过两阶段厌氧发酵工艺可产生高浓度的己酸。
但现有技术中链羧酸转化工艺均需外源添加乙醇作为电子供体，而Chen等通过全生命周期评价分析得出，乙醇是该合成工艺的最主要环境影响因素和成本来源(Chen W S,Strik D P,Buisman C J,et al.Production of Caproic Acid from Mixed Organic Waste:An Environmental Life Cycle Perspective[J].Environmental Science&Technology,2017,51(12): 7159-7168.)。因此，降低乙醇添加量或不添加外源电子供体是提高该工艺经济性的关键。
对此，中国专利CN104357496B公开了一种通过微生物催化乳酸合成己酸的方法，并指出乳酸可有效替代乙醇，驱动碳链延长反应，促进中链羧酸的合成。中国专利CN105420168B也公开了一种利用乳酸合成己酸的瘤胃梭菌及其应用方法，实现了不添加乙醇的己酸合成。但它们对于在不添加乙醇的情况下，同时制备获得多种中链羧酸的方案并没有进行研究记载。
通常地，有机物一级厌氧酸化过程产物以挥发性短链羧酸为主，如中国专利CN105132475 A、CN100526469 C、CN103923951 A、CN103509829 A和CN 105603011 A等公开了利用剩余污泥、餐厨垃圾等废弃物产乙酸、丁酸等挥发性短链羧酸的方法。也有中国专利CN104911128 B公开了一种利用嗜热厌氧杆菌将葡萄糖/果糖等转化为乳酸的方法，但针对直接以畜禽粪污等农业废弃物为原料，在不添加外源电子供体的情况下，定向转化获得多种中链羧酸的研究尚未见报道，现有仍沿用着在禽粪污等农业废弃物发酵过程中外源添加电子供体的思路，生产成本有待进一步降低。
发明内容
本发明为解决畜禽粪污等农业废弃物两级发酵产中链羧酸过程需添加乙醇、乳酸或甲醇等外源电子供体的缺陷，提供了一种可降低农业源生物质资源定向转化生产中链羧酸的成本的方案。
具体地，本发明的技术方案如下：
一种农业废弃物通过发酵方式生产中链羧酸的方法，发酵底物为畜禽粪污和农业废弃物的混合物，所述农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45-60℃，pH值为4.0-6.0；
B、二级发酵阶段：采用驯化的污泥进行厌氧发酵，发酵温度为 25-40℃，pH值为4.0-7.0。
本发明通过对畜禽粪污与其他农业废弃物为原料制备中链羧酸的现有技术进行了深入研究，提出了一种可无需增加外源电子供体，即可实现中链羧酸高效生产的方案。具体地，本发明通过各阶段不同发酵剂的选择，配合对发酵过程的分阶段不同控制，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，从而在后续的二级发酵阶段实现多种中链羧酸的同时高效生产。
本发明所述挥发性脂肪酸为乙酸、丙酸、丁酸等。中链羧酸为正己酸、正庚酸、正辛酸等。
优选本发明中，一级发酵阶段的发酵温度为50-55℃，pH值为4.5-5.5，这一条件可提高产乳酸菌的底物竞争能力，以利于优势乳酸菌的富集和发酵，并实现特定比例乳酸与多种挥发性脂肪酸的定向转化。
二级发酵阶段的发酵温度为25-35℃，pH值为5.0-6.5，以提升己酸的产量和生产效率，并进一步在保证高效生产己酸、辛酸的基础上，实现丙酸和乳酸经反向β氧化作用获得庚酸。
本发明中，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；
或，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(30-40):1。
本发明发现，当进一步控制发酵底物中畜禽粪污和农业废弃物的比例，使其处于本发明限定的碳单比时，可有助于特定比例乳酸与多种挥发性脂肪酸的获得，进而保证最终中链羧酸的生产。
本发明所述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；所述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；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。
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。
本发明步骤A中，发酵浓度为2％VS-15％VS(以挥发性固体计)；菌液接种量为2％-8％(V/V)，菌液中的活菌数为10 6-10 9CFU/mL。
步骤B中，所述驯化的污泥的接种量为5％-20％(V/V)，优选为10％。制备方法为：
以乙醇和/或乳酸为电子供体，将处理有机废水的厌氧发酵罐中的颗粒污泥，在25-40℃，pH值5.0-6.5的发酵体系下驯化。
经驯化后的污泥中主要微生物种类为碳链延长菌。一般驯化时间为2个月左右，以实现富集碳链延长菌群的目的为准。
本发明驯化后的污泥中主要菌群为克鲁维氏梭菌(Clostridium kluyveri)10-25％、普雷沃氏菌(Prevotellaceae)20-30％、琥珀酸弧菌(Succinivibrionaceae)10-15％、拟杆菌(Bacteroidales)5-10％、柔式螺旋体菌(Spirochaetaceae)5-10％、理研菌(Rikenellaceae)2-8％、纤维杆菌(Fibrobacteraceae)2-5％、毛螺旋菌(Lachnospiraceae)2-5％、瘤胃菌(Ruminococcaceae)2-5％、其他10-15％。
本发明还提供一种上述方法在生产中链羧酸中的应用。
另外，考虑到上述畜禽粪污与其他农业废弃物两阶段梯度控温混合发酵产中链羧酸的方法中，在一级发酵阶段获得的短链羧酸在工业中也有广泛的应用价值，本发明还提供一种利用畜禽粪污与其他农业废弃物高温混合发酵生产乳酸等短链羧酸的方法，包括如下步骤：
①接种常用产乳酸的乳酸菌；
②发酵阶段生产乳酸等短链羧酸。
其中，步骤①的乳酸发酵菌群主要包括双歧杆菌、嗜热链球菌、植物乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌、保加利亚乳杆菌等。
其中，步骤②所述发酵阶段生产乳酸等短链羧酸，具体为按体积比2％-8％接种产乳酸菌液，控制发酵温度为45-60℃，控制pH值为4.0-6.0。
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梯度控温混合发酵产中链羧酸的方法，发酵底物为畜禽粪污、农作物秸秆、果蔬废弃物等，原料来源广泛，且中链羧酸转化过程无需添加外源电子供体，运行成本低，产生的羧酸产品附加值高；可有效实现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。本发明的工艺操作简单、厌氧发酵条件易于控制、经济成本低廉，具有良好的应用前景。本发明可使超过90％的内源短链羧酸转化为高附加值的中链羧酸，为农业废弃物的高值化利用提供一条经济可行的新途径。
附图说明
图1为本发明实施例1、对比例1中不同发酵温度条件下物料单位VS的羧酸产量柱状图。
图2为本发明实施例2中乳酸和中链羧酸转化趋势图。
图3为本发明实施例3中乳酸和中链羧酸转化趋势图。
具体实施方式
在以下的实施例中提供了本发明的示例性的实施方案。以下的实施例仅通过示例的方式给出，并用于帮助普通技术人员使用本发明。所述实施例并不能以任何方式来限制本发明的范围。
实施例1高温发酵生产乳酸等短链羧酸
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35-38℃和6.0-7.0，培养36h。
2、将培养后的乳酸菌的菌液(活菌数为10 7CFU/mL)接种到以猪粪、 玉米秸秆为底物的厌氧发酵罐中，混合物料的碳氮比为40:1，接种量为4％。发酵浓度为4％VS，pH控制在4.5-5.5，高温厌氧发酵，发酵温度控制为50-55℃。经过2周连续厌氧酸化发酵后，发酵原料单位VS的羧酸产量见图1，物料在高温发酵条件下的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为226.8、214.9和23.0mg/gVS，其中挥发性脂肪酸中乙酸、丙酸、丁酸的产量分别为6.1、3.4和13.5mg/gVS。
对比例1中温发酵生产乳酸等短链羧酸
本对比例的生产步骤与实施例1相同，区别仅在于：步骤2中发酵温度控制为35-38℃。经过2周连续厌氧酸化发酵后，发酵原料单位VS的羧酸产量见图1，物料在中温发酵条件下的总羧酸产量、乳酸产量和挥发性脂肪酸产量分别为107.9、81.9和25.8mg/gVS。从图1中可知，高温条件下(实施例1)物料的总羧酸产量和乳酸产量分别是中温发酵(对比例1)条件下的2.1和8.3倍。且乳酸与其他挥发性脂肪酸的产量比值也发生了很大变化。实施例2两阶段发酵产中链羧酸
将实施例1中经连续厌氧酸化发酵2周后获得的一级发酵酸化液转移至二级发酵罐中，接种驯化后的具有碳链延长菌群(克鲁维氏梭菌Clostridium kluyveri 23.4％、普雷沃氏菌Prevotellaceae 22.3％、琥珀酸弧菌Succinivibrionaceae 11.6％、拟杆菌Bacteroidales 8.2％、柔式螺旋体菌Spirochaetaceae 6.7％、理研菌Rikenellaceae 5.5％、纤维杆菌Fibrobacteraceae 3.8％、毛螺旋菌Lachnospiraceae 2.4％、瘤胃菌Ruminococcaceae 3.6％、其他约12.5％)的污泥，所述污泥的制备方法为：以乙醇和/或乳酸为电子供体，将处理有机废水的厌氧发酵罐中的颗粒污泥，在25-40℃，pH值5.0-6.5的发酵体系下驯化2个月。接种量为10％(体积比)，在发酵罐中装有聚氨酯生物填料用于微生物附着，发酵温度为30±1℃，二级发酵过程的pH值控制为6.0±0.2。经过1周的连续发酵，95％以上的乳酸经碳链延长转化为己酸等中链羧酸(见图2)，己酸、庚酸、辛酸浓度分别为4039.5、813.0和690.3mgCOD/L，经计算可进一步得出，每降解1g乳酸，可相应的转化合成866.0 mgCOD中链羧酸。
实施例3
本实施例的生产步骤与实施例2相同，区别仅在于：二级发酵过程中pH值控制为4.5±0.2。相对实施例2，乳酸转化速率较慢，二级发酵产物中仅检测到己酸、辛酸2种中链羧酸，且最大浓度分别为2302.8和562.3mgCOD/L(见图3)，经计算可进一步得出，每降解1g乳酸，可相应的转化合成409.3mgCOD中链羧酸。
实施例4
本实施例的生产短链羧酸的步骤与实施例1相同，区别仅在于：底物采用猪粪和果蔬废弃物(TS:6％、VS:92％TS、总含糖量10％鲜重、C/N＝60:1)的混合物，所述混合物的碳氮比为40:1。菌液为活菌数为10 6CFU/mL的嗜热链球菌(购自善恩康生物科技(苏州)有限公司)，一级发酵阶段发酵浓度为15％VS，菌液接种量为8％(V/V)，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370.0、327.1和43.9mg/gVS。其中挥发性脂肪酸中乙酸、丙酸和丁酸的产量分别为18.4、9.6和15.9mg/gVS。
本实施例将上述获得的一级发酵酸化液转移至二级发酵罐中，采用实施例2的方法生产中链羧酸，经过1周的连续发酵，己酸、庚酸、辛酸浓度分别为7732.9、823.6和932.5mgCOD/L，中链羧酸产率为791.8mgCOD中链羧酸/g乳酸。
对比例2
本对比例采用一步法直接由底物生产中链酸，具体步骤如下：
发酵原料及浓度同实施例1，发酵温度为35±1℃，pH值5.5±0.5，通过一步法尝试获取中链羧酸，经过连续3周的厌氧发酵，发酵底物中可检测到乳酸、乙酸、丁酸和正己酸，其浓度分别为550.2、1360.0、2470.8和620.5mgCOD/L，研究结果表明一步法发酵虽可获得中链羧酸，但仅能检测到正己酸，且含量显著低于二步发酵法中中链羧酸浓度。
对比例3
本对比例的生产步骤与实施例2相同，区别仅在于：二级发酵温度为45-48℃。经过1周的连续发酵，仅检测到己酸和辛酸，其浓度分别为443.5和108.7mgCOD/L，中链羧酸产率仅为95.2mgCOD中链羧酸/g乳酸。
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，具体涉及一种农业废弃物通过发酵方式生产中链羧酸的方法与应用。本发明的方法中，发酵底物为畜禽粪污和农业废弃物的混合物，所述农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45-60℃，pH值为4.0-6.0；B、二级发酵阶段：采用驯化的污泥作为接种物进行厌氧发酵，发酵温度为25-40℃，pH值为4.0-7.0。本发明通过两阶段梯度控温发酵的方法在不添加外源电子供体的条件下，从畜禽粪污和农业废弃物中获得高附加值的中链羧酸，为农业废弃物的高值化利用提供一条可行的新途径。

Claims (4)

  1. 一种农业废弃物通过发酵方式生产中链羧酸的方法，其特征在于，发酵底物为畜禽粪污和农业废弃物的混合物，所述农业废弃物为农作物秸秆或果蔬废弃物；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50-55℃，pH值为4.5-5.5；
    B、二级发酵阶段：采用驯化的污泥进行厌氧发酵，发酵温度为25-35℃，pH值为5.0-6.5；
    所述中链羧酸包括正己酸、正庚酸和正辛酸；
    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；
    或，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(30-40):1。
  2. 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。
  3. 根据权利要求2所述的方法，其特征在于，步骤A中，发酵浓度为2％VS-15％VS；菌液接种量为2％-8％(V/V)，菌液中的活菌数为10 6-10 9CFU/mL。
  4. 根据权利要求1所述的方法，其特征在于，步骤B中，所述驯化的污泥的接种量为5％-20％(V/V)，制备方法为：
    以乙醇和/或乳酸为电子供体，将处理有机废水的厌氧发酵罐中的颗粒污泥，在25-40℃，pH值5.0-6.5的发酵体系下驯化。
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