WO2017063548A1 - 一株克雷伯氏菌及用它制备微生物絮凝剂的方法 - Google Patents

一株克雷伯氏菌及用它制备微生物絮凝剂的方法 Download PDF

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WO2017063548A1
WO2017063548A1 PCT/CN2016/101793 CN2016101793W WO2017063548A1 WO 2017063548 A1 WO2017063548 A1 WO 2017063548A1 CN 2016101793 W CN2016101793 W CN 2016101793W WO 2017063548 A1 WO2017063548 A1 WO 2017063548A1
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fermentation
microbial flocculant
strain
liquid
klebsiella
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PCT/CN2016/101793
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栾兴社
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青岛耀东生物工程有限公司
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Priority to EP16836142.6A priority Critical patent/EP3196294B1/en
Publication of WO2017063548A1 publication Critical patent/WO2017063548A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12R2001/22—Klebsiella

Definitions

  • the invention relates to a strain of Klebsiella and a method for preparing the same, and belongs to the field of biotechnology and bioengineering.
  • Microbial flocculant is a natural biological macromolecule produced by microorganisms. It has the characteristics of mild production conditions, non-toxicity, high efficiency, biodegradability in the environment, and is an environmentally friendly product. Microbial flocculant is a new type of biological water treatment agent, which is a modern new fermentation product. The research on microbial flocculants has become a hot spot in academic research in the world. Scientific guidance has made microbial flocculants a strong market demand. In 2013, the State Council's “12th Five-Year Plan for Bio-Industrial Development” industrialized and promoted the application of new fermentation products; and vigorously developed high-performance bio-environmental biological preparations as a key development area.
  • CN1616358A (applicant: Yu Xingshe; invention name: method for preparing bioflocculant from Arthrobacter), the applicant screened the bacillus producing microbial flocculant by soil sampling.
  • the selective medium for screening the strain is highly targeted and beneficial.
  • the medium for fermenting the microbial flocculant by this strain is inexpensive.
  • CN102586331A Applicant: Yu Xingshe; inventor name: a method for preparing bioflocculant by high-concentration fermentation
  • the applicant uses the isolated micrococcus DS16 to ferment by the bacterial fermentation method to reach a higher yield ( ⁇ 2.0%).
  • this method has increased investment in equipment, and the fermentation process and operation steps are also somewhat cumbersome. Therefore, for microbial flocculants, a green environmentally friendly biological agent, further improvement based on the existing technology, maintaining high efficiency, increasing scientific and rational processes, increasing production and yield, and reducing production and use costs are industrialization and popularization applications. The technical problems that must be overcome before.
  • One embodiment of the present invention provides a strain of Klebsiella sp. LDX1-1 which is isolated from moist soil under the dead leaves of apricot forest in the southern suburb of Jinan City, Shandong province. Deposited on September 7, 2015 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences), with the preservation number CGMCC No. .11330.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee
  • Another embodiment of the present invention also provides the use of Klebsiella sp. strain LDX1-1 as described above for the preparation of a microbial flocculant, which can be used to prepare a microbial flocculant.
  • a further embodiment of the present invention provides a method for preparing a microbial flocculant by using the Klebsiella strain LDX1-1 described above. Specifically,
  • the activated cultured slanted species are inoculated into a liquid culture medium, and shake cultured at 24-30 ° C, 150-180 r / min for 10-18 h to obtain a liquid strain, and set aside;
  • composition and dosage of the liquid culture medium is (g/L): sucrose 10-15, NH 4 NO 3 1.5-2.5, K 2 HPO 4 0.8-1.3, MgSO 4 0.3-0.8, NaCl 0.3-0.8, FeSO 4 0.01-0.04.
  • the liquid strain is inoculated into the fermentation medium according to the inoculation amount of 2-4% (volume ratio), and the fermentation fermentation method is used for fermentation culture in the fermenter for 24-32 hours, the fermentation liquid is obtained by fermentation, and the fermentation liquid is filtered to remove impurities.
  • a liquid proteoglycan (PPS) microbial flocculant is obtained.
  • the fermentation broth was determined, the microbial flocculant yield was ⁇ 60%, the yield was ⁇ 2.0%, and the flocculation rate was ⁇ 94%.
  • the fermentation control conditions were: temperature 24-30 ° C, aeration ratio of 0.25-0.8 VVM, and stirring speed of 200-600 r/min.
  • composition and dosage of the fermentation medium are (g/L): sucrose 15-30, NH 4 NO 3 1.5-4.5, K 2 HPO 4 0.8-2.0, MgSO 4 0.2-0.7, NaCl 0.2-0.8, FeSO 4 0.01 -0.04, MnSO 4 0.01-0.05, pine needle extract 0.1-0.4, corn starch 6-12, corn syrup 1-3, ZnSO 4 0.018-0.040.
  • the activator component in the above medium is: MnSO 4 and pine needle extract.
  • the method for using the microbial flocculant is directly mixed into surface water, domestic sewage, industrial sewage, ecological lake water, aquaculture wastewater, fermentation culture liquid, and the like in a liquid state.
  • the method for applying the microbial flocculant of the invention to the treatment of domestic sewage is: adding an appropriate amount of flocculant to the domestic sewage in the flocculation device with a stirring function, rapidly mixing for 40 s at a stirring speed of 120 r/min, and then The stirring speed of 60 r/min was slowly mixed for 120 s. After 30 minutes of static, the contaminants precipitated and separated. Aspirate the supernatant for determination.
  • the amount of microbial flocculant PPS dry weight
  • the amount of microbial flocculant PPS dry weight
  • COD removal rate ⁇ 85% heavy metal Mn 2+ removal rate ⁇ 85%.
  • the components in the enriched culture solution formula are formulated into a liquid medium, and 50 mL of each 250 mL triangular flask is dispensed, autoclaved at 121 ° C for 25 min, and cooled to 25-28 ° C. Add the corresponding amount of lysozyme under the conditions of the bacteria and mix.
  • the collected bacteria sample is connected to the cultured enriched culture solution, shaken, shaken at 20-27 ° C, 120-180r / min for 2-3 days, to obtain enriched culture liquid.
  • the selection medium was prepared, and a selection medium plate was prepared using a ⁇ 9 cm dish.
  • the enriched culture liquid is serially diluted, and the appropriate dilution is applied to the selection medium plate, and cultured at 20-27 ° C for 2-3 days to select colonies that grow fast, have a thick viscosity, and have a light color circle.
  • a typical colony was selected and inoculated on a slant medium, and the culture was matured and stored in a refrigerator at 4 °C.
  • Each medium is as follows:
  • the components and amounts (g/L) of the enriched culture solution beef extract 2, peptone 9, NaCl 5, MgSO 4 0.6, lysozyme 0.03 (500,000 U/g), and distilled water 1000 mL.
  • composition and amount of the slant medium g/L: glycerol 12, NaNO 3 2.5, K 2 HPO 4 0.9, MgSO 4 0.6, NaCl 0.6, FeSO 4 0.015, purified agar powder 14.
  • the individual morphological characteristics of the screening strain were: long rod shape 1.5-1.8 ⁇ m ⁇ 2.5-5.0 ⁇ m, short rod shape 1.5-1.8 ⁇ m ⁇ 1.8-2.5 ⁇ m. Single arrangement, rich in mucus layer, no movement, no spores. Aerobic.
  • the culture characteristics of the screening strains were as follows: on the beef extract peptone medium plate, a central protrusion, an opaque colony with a neat edge, a luster, and a large viscosity was produced. A thick, shiny, viscous, pale yellow lawn is formed on the slope.
  • the contact enzyme test was positive. In the peptone water medium, it can strongly ferment glucose, sucrose, maltose, arabinose to produce acid, ferment fructose, xylose, glycerol, lactose to produce acid, slow fermentation of dextrin and starch to produce acid, no Fermentation of xylitol produces acid to produce gas. Methyl red negative, V-P positive. Resistant to 7% NaCl growth. A large ⁇ 17 cm colony was formed on the starch hydrolyzed plate for 2 days. Fat hydrolysis is negative. Gelatin is negatively hydrolyzed. Litmus milk is reduced. Urease positive.
  • the strain is named Klebsiella sp. strain LDX1-1, which was deposited on September 7, 2015 in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Chaoyang District, Beijing) No. 3, No. 1 Beichen West Road, Institute of Microbiology, Chinese Academy of Sciences), the deposit number is CGMCC No.11330.
  • composition and amount of the liquid culture medium sucrose 12, NH 4 NO 3 2, K 2 HPO 4 0.8, MgSO 4 0.5, NaCl 0.4, FeSO 4 0.013.
  • composition and dosage of fermentation medium (g/L): sucrose 23, NH 4 NO 3 2, K 2 HPO 4 1.2, MgSO 4 0.5, NaCl 0.4, FeSO 4 0.015, MnSO 4 0.02, pine needle extract (manufacturing company) For Xi'an Runxue Biotechnology Co., Ltd., the product specifications are proportional extraction 10:1) 0.2, corn starch 7, corn syrup 1.8, ZnSO 4 0.03.
  • the activated cultured slanted species were inoculated into a liquid culture medium, and cultured at 26 ° C, 170 r / min for 13 h, to obtain a liquid strain, and set aside;
  • the liquid strain was inoculated into the fermentation medium according to the inoculation amount of 2.5% (volume ratio), and the fermentation fermentation was carried out for 30 hours in the fermenter.
  • the fermentation control conditions were: temperature 26 ° C, aeration ratio of 0.45 VVM, stirring speed It is 500r/min.
  • the fermentation liquid is obtained by fermentation, and the fermentation liquid is filtered to remove impurities to obtain a liquid proteoglycan (PPS) microbial flocculant.
  • PPS liquid proteoglycan
  • the fermentation broth was determined to have a microbial flocculant yield of 61%, a yield of 2.1%, and a flocculation rate of 95%.
  • the microbial flocculant yield is determined by adjusting the pH of the microbial flocculant to 7.0, mixing and precipitating with 2.5 times absolute ethanol, centrifuging at 5000 r/min for 5 min to obtain a primary precipitate, and then washing the first time with 6 times absolute ethanol.
  • Example 3 Application of microbial flocculant to sewage treatment
  • the microbial flocculant prepared in Example 2 was applied to treat domestic sewage.
  • the operation method for treating domestic sewage is to add appropriate flocculant to the domestic sewage having the stirring function flocculation device, rapidly mix for 40s at a stirring speed of 120r/min, and then slowly mix for 120s at a stirring speed of 60r/min. After 30 minutes of static, the contaminants precipitated and separated. Aspirate the supernatant for determination.
  • the raw water for domestic sewage of Qingdao Chengtou Daren Water Co., Ltd. (chemical oxygen demand (COD) is 859mg/L, total phosphorus is 22.9mg/L, Mn 2+ is 0.434mg/L, Fe 3+ is 84.6mg/L)
  • COD chemical oxygen demand
  • the amount of PPS microbial flocculant (dry weight) is 3-5g per ton of water, and the excellent results are: the flocculation rate of suspended solids (SS) is 93%, the COD removal rate is 93%, and the total phosphorus removal rate is 86%, the removal rate of heavy metal Mn 2+ was 87%, and the removal rate of Fe 3+ was 97%.
  • a highly active microbial flocculant-producing bacteria-Klebsiella LDX1-1 was screened, and the strain was used to produce a microbial flocculant by activating fermentation method with high yield, and the strain has a fast growth rate and strong metabolism ability.
  • the rate is ⁇ 60%), the fermentation cycle is short ( ⁇ 32h), the yield is high ( ⁇ 2.0%), and the flocculation rate is high ( ⁇ 94%).
  • the microbial flocculant prepared by the invention is applied to domestic sewage treatment, the SS flocculation rate is ⁇ 90%, the COD removal rate is ⁇ 85%, the heavy metal Mn 2+ removal rate is ⁇ 85%, etc., and has broad development prospects and good application value. .

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Abstract

提供一株克雷伯氏菌及其发酵制备微生物絮凝剂的方法，该菌株为克雷伯氏菌(Klebsiella sp.)菌株LDX1-1，已于2015年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.11330。

Description

一株克雷伯氏菌及用它制备微生物絮凝剂的方法 技术领域
本发明涉及一株克雷伯氏菌及用它制备微生物絮凝剂的方法，属于生物技术与生物工程领域。
背景技术
随着工业迅速发展、人口急增等世界性问题的出现，造成了水质污染加剧，水资源紧张。我国近几年来每年有600亿立方米工业污水和200亿立方米生活污水排放，长江、黄河等七大水系遭到严重污染，生态湖水、近海地域水质也遭到不同程度的破坏，人民健康和经济发展受到严重威胁，水资源保护形势严峻。我国是世界上最缺水的国家之一，人均淡水资源仅为世界人均水平的四分之一。由于人口众多和社会发展，产生的大量工业和生活污水使有限的水资源遭到严重污染。不少缺水的现象，让人触目惊心，水资源危机的警钟已经敲响。《中华人民共和国水法》从立法的角度确立了保护水资源、节约用水、高效用水等法律。水已经成为了最紧缺的资源。
几十年来，化学絮凝剂在对保障人类社会、经济和生存环境的水资源的可持续性利用中，在对地表水净化、污水处理方面发挥了巨大作用。但科学研究越来越多地发现：化学絮凝剂具化学毒性和难以降解性质，其在使用后形成的残留和积累、迁移转化对人类健康、生物多样性干扰和生态系统平衡方面产生严重危害。
微生物絮凝剂是由微生物产生的天然生物大分子，具有生产条件温和、本质无毒无害、使用高效、环境中生物可降解等特点，是环境友好性产品。微生物絮凝剂是新型生物水处理剂，属当代新型发酵产品。对微生物絮凝剂的研究已成为世界学术研究的热点，科学导向已使微生物絮凝剂形成了强烈的市场需求。2013年国务院《生物产业发展十二五规划》中把新型发酵产品的产业化与推广应用；把大力发展高性能生物环保生物制剂列为重点发展领域。但纵观近30年来微生物絮凝剂的研究现状，仍存在着菌种性能不好、生产基质不经济、发酵工艺不够合理及产品活性低等实际情况，造成产量低、生产成本高、产品价格高，影响了专业化的可操作性和市场的可接受性。
十几年来，本发明专利申请的发明人栾兴社致力于微生物絮凝剂的研发，先后取得了两项专利：其公开号分别为CN1616358A和CN102586331A。其中，CN1616358A(申请人：栾兴社；发明名称：由节杆菌制备生物絮凝剂的方法)中，申请人由土壤采样筛选了产生微生物絮凝剂的节杆菌。筛选该菌的选择性培养基代谢针对性强，利 用该菌种发酵生产微生物絮凝剂的培养基成本低廉。但利用该筛选菌株发酵产生微生物絮凝剂的产率较低(≤42％)和产量也较低(≤0.9％)。CN102586331A(申请人：栾兴社；发明名称：一种高浓度发酵制备生物絮凝剂的方法)中，申请人用分离的微球菌DS16通过菌量优势发酵方法进行发酵达到了产量较高的水平(≥2.0％)。但是该方法在设备方面增加了投资，发酵工艺及操作步骤也略显繁琐。所以，对微生物絮凝剂这一绿色的环保生物制剂来说，在现有技术基础上进一步改善，保持高效、工艺愈加科学合理、提高产量和产率、降低生产与使用成本是产业化和推广应用前必须攻克的技术难题。
发明内容
本发明的一个实施例提供了一种克雷伯氏菌(Klebsiella sp.)菌株LDX1-1，它是从山东省济南市的南郊杏树林枯叶下的潮湿土壤中分离得到的，该菌株已于2015年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏编号为CGMCC No.11330。
本发明的另一个实施例还提供了一种如上所述的克雷伯氏菌(Klebsiella sp.)菌株LDX1-1在制备微生物絮凝剂方面的应用，可以用于制备微生物絮凝剂。
本发明的又一个实施例提供了一种以上述克雷伯氏菌菌株LDX1-1制备微生物絮凝剂的方法，具体的，
(1)液体菌种培养
将活化培养的斜面菌种接种于液体菌种培养基中，在24-30℃、150-180r/min条件下震荡培养10-18h，得液体菌种，备用；
液体菌种培养基的组分及用量为(g/L)：蔗糖10-15，NH4NO3 1.5-2.5，K2HPO4 0.8-1.3，MgSO4 0.3-0.8，NaCl 0.3-0.8，FeSO4 0.01-0.04。
(2)发酵培养
将液体菌种按2-4％(体积比)的接种量接种于发酵培养基，在发酵罐中利用激活发酵法进行发酵培养24-32h，发酵结束得发酵液，将发酵液过滤去除杂质，得液体蛋白多糖(PPS)微生物絮凝剂。发酵液经测定，微生物絮凝剂产率≥60％，产量≥2.0％，絮凝率≥94％。
发酵控制条件为：温度24-30℃、通气比为0.25-0.8VVM、搅拌转速为200-600r/min。
发酵培养基的组分及用量为(g/L)：蔗糖15-30，NH4NO3 1.5-4.5，K2HPO4 0.8-2.0， MgSO4 0.2-0.7，NaCl 0.2-0.8，FeSO4 0.01-0.04，MnSO4 0.01-0.05，松针提取物0.1-0.4，玉米淀粉6-12，玉米浆1-3，ZnSO4 0.018-0.040。上述培养基中的激活剂成分为：MnSO4和松针提取物。
该微生物絮凝剂的使用方法为：以液体状态直接混入地表水、生活污水、工业污水、生态湖水、养殖废水、发酵培养液等中。
将本发明的微生物絮凝剂应用于对生活污水进行处理的操作方法为：向具有搅拌功能的絮凝装置内的生活污水中加入适量絮凝剂，以120r/min的搅拌转速快速混合40s，然后再以60r/min的搅拌转速缓慢混合120s。静止30min，污染物沉淀分离。吸取上清液测定。对生活污水处理，微生物絮凝剂PPS使用量(干重)为每吨水3-5g，SS絮凝率≥90％，COD去除率≥85％，重金属Mn2+去除率≥85％。
具体实施方式
实施例1：菌株的筛选
(1)取样
取济南南郊杏树林枯叶下的潮湿土壤，用无菌取样铲刮开土壤表层，取地表下3-10cm之间的土层放入无菌袋，并在无菌袋上标注取样日期、时间、地点、植被，将土壤菌样尽快放入4℃冰箱保存，当天至两天内进入菌种富集筛选程序。
(2)筛选
将富集培养液配方中的诸成分(溶菌酶除外)配制成液体培养基，分装，每一250mL三角瓶50mL，于121℃高压蒸汽灭菌25min，冷却至25-28℃时，在无菌条件下加入对应量的溶菌酶，混匀。将采集的菌样接入培养好的富集培养液中，摇匀，于20-27℃、120-180r/min进行摇瓶培养2-3天，得富集培养菌液。
配制选择培养基，用Φ9cm平皿制作选择培养基平板。把富集培养菌液进行系列稀释，选择合适的稀释度涂布于选择培养基平板，20-27℃培养2-3天，挑选生长快、粘稠度大、有浅色圈的菌落，经纯化后，挑选典型菌落接种于斜面培养基，培养成熟后于4℃冰箱保存。
各培养基如下：
富集培养液的组分及用量(g/L)：牛肉浸膏2，蛋白胨9，NaCl 5，MgSO4 0.6，溶菌酶0.03(50万U/g)，蒸馏水1000mL。
选择培养基的组分及用量(g/L)：丙三醇25，NaNO3 2.5，K2HPO4 1.2，MgSO4 0.4，NaCl 0.4，FeSO4 0.015，CuSO4 2.0，丙酸钠2.0，纯化琼脂粉15。
斜面培养基的组分及用量(g/L)：丙三醇12，NaNO3 2.5，K2HPO4 0.9，MgSO4 0.6，NaCl 0.6，FeSO4 0.015，纯化琼脂粉14。
(3)菌株鉴定
筛选菌株的个体形态特征为：长杆状1.5-1.8μm×2.5-5.0μm，短杆状1.5-1.8μm×1.8-2.5μm。单个排列，有丰富的粘液层，不运动，没有芽孢。好氧。
筛选菌株的培养特征为：在牛肉浸膏蛋白胨培养基平板上产生中央突起，边缘整齐、有光泽、粘性大的不透明菌落。在斜面上形成稠厚、有光泽、粘性大、淡黄色的菌苔。
筛选菌株的生理生化特征为：接触酶试验呈阳性。在蛋白胨水培养基中能够强烈发酵葡萄糖、蔗糖、麦芽糖、阿拉伯糖产酸产气，发酵果糖、木糖、丙三醇、乳糖产酸产气，缓慢发酵糊精和淀粉产酸产气，不发酵木糖醇产酸产气。甲基红阴性，V-P阳性。耐7％NaCl生长。淀粉水解平板上2天形成Φ17cm庞大菌落。油脂水解阴性。明胶水解阴性。石蕊牛奶还原。脲酶阳性。
该菌株16SrDNA序列测定结果如下(SEQ-1)：
Figure PCTCN2016101793-appb-000001
该菌株命名为克雷伯氏菌(Klebsiella sp.)菌株LDX1-1，它已于2015年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏编号为CGMCC No.11330。
实施例2
液体菌种培养基的组分及用量(g/L)：蔗糖12，NH4NO3 2，K2HPO4 0.8，MgSO4  0.5，NaCl 0.4，FeSO4 0.013。
发酵培养基的组分及用量(g/L)：蔗糖23，NH4NO3 2，K2HPO4 1.2，MgSO4 0.5，NaCl 0.4，FeSO4 0.015，MnSO4 0.02，松针提取物(生产企业为西安润雪生物科技有限公司，产品规格为比例提取10:1)0.2，玉米淀粉7，玉米浆1.8，ZnSO4 0.03。
(1)液体菌种培养
将活化培养的斜面菌种接种于液体菌种培养基中，在26℃、170r/min条件下震荡培养13h，得液体菌种，备用；
(2)发酵培养
将液体菌种按2.5％(体积比)的接种量接种于发酵培养基，在发酵罐中以激活发酵方式进行发酵培养30h，发酵控制条件为：温度26℃、通气比为0.45VVM、搅拌转速为500r/min。发酵结束得发酵液，将发酵液过滤去除杂质，得液体蛋白多糖(PPS)微生物絮凝剂。发酵液经测定，微生物絮凝剂产率为61％，产量为2.1％，絮凝率为95％。
微生物絮凝剂产率的测定方法为：将微生物絮凝剂调pH值为7.0，用2.5倍无水乙醇进行混合沉淀，以5000r/min离心5min获得初次沉淀物，然后用6倍无水乙醇洗涤初次沉淀物，以5000r/min离心5min，获取二次沉淀物，把二次沉淀物在40℃真空干燥6h，称干重。产率＝(絮凝剂干重Kg/碳源干重Kg)×100％。
絮凝率的测定方法为：配制重量比为0.4％的高岭土悬液和5％的CaCl2溶液，在六联搅拌仪的反应杯中加入体积比98％高岭土悬液、2％CaCl2溶液，混匀，制成絮凝体系备用。测定时每100mL絮凝体系加入20μL发酵液，以120r/min的搅拌转速快速混合40s，然后再以60r/min的搅拌转速缓慢混合120s。静止5min。吸取上清液，测定波长550nm下的OD值B。加蒸馏水替代发酵液重复以上操作测得OD值A。絮凝率＝{(A-B)/A}×100％。
实施例3：微生物絮凝剂对污水处理的应用
将实施例2制备的微生物絮凝剂应用于对生活污水进行处理。对生活污水进行处理的操作方法是：向具有搅拌功能絮凝装置内的生活污水中加入适量絮凝剂，以120r/min的搅拌转速快速混合40s，然后再以60r/min的搅拌转速缓慢混合120s。静止30min，污染物沉淀分离。吸取上清液测定。
对青岛城投大任水务有限公司生活污水原水(化学需氧量(COD)为859mg/L，总磷为22.9mg/L，Mn2+为0.434mg/L，Fe3+为84.6mg/L)进行处理，PPS微生物絮凝剂使用量(干重)为每吨水3-5g，达到的优良结果是：悬浮物(SS)絮凝率为93％，COD 去除率为93％，总磷去除率为86％，重金属Mn2+去除率为87％，Fe3+的去除率为97％。
本发明实施例筛选到了一种高活性微生物絮凝剂产生菌-克雷伯氏菌LDX1-1，采用该菌株利用激活发酵法高产量生产微生物絮凝剂，具有菌株生长速度快、代谢能力强(产率≥60％)、发酵周期短(≤32h)、产量高(≥2.0％)、絮凝率高(≥94％)等优点。将本发明制备的微生物絮凝剂应用于生活污水处理，SS絮凝率≥90％，COD去除率≥85％、重金属Mn2+去除率≥85％等，具有广阔的开发前景和很好的应用价值。

Claims (6)

  1. 克雷伯氏菌(Klebsiella sp.)菌株LDX1-1，所述菌株的保藏编号为CGMCC No.11330。
  2. 权利要求1所述的克雷伯氏菌(Klebsiella sp.)菌株LDX1-1在制备微生物絮凝剂方面的应用。
  3. 一种以权利要求1所述的克雷伯氏菌菌株LDX1-1制备微生物絮凝剂的方法，包括：
    (1)液体菌种培养
    将活化培养的斜面菌种接种于液体菌种培养基中，在24-30℃、150-180r/min条件下震荡培养10-18h，得液体菌种，备用；
    液体菌种培养基的组分及用量为：蔗糖10-15g/L，NH4NO3 1.5-2.5g/L，K2HPO4 0.8-1.3g/L，MgSO4 0.3-0.8g/L，NaCl 0.3-0.8g/L，FeSO4 0.01-0.04g/L；
    (2)发酵培养
    将液体菌种按体积比2-4％的接种量接种于发酵培养基，在发酵罐中进行发酵培养24-32h，发酵结束得发酵液，将发酵液过滤去除杂质，得微生物絮凝剂；
    发酵培养基的组分及用量为：蔗糖15-30g/L，NH4NO3 1.5-4.5g/L，K2HPO4 0.8-2.0g/L，MgSO4 0.2-0.7g/L，NaCl 0.2-0.8g/L，FeSO4 0.01-0.04g/L，MnSO4 0.01-0.05g/L，松针提取物0.1-0.4g/L，玉米淀粉6-12g/L，玉米浆1-3g/L，ZnSO4 0.018-0.040g/L。
  4. 如权利要求3所述的制备微生物絮凝剂的方法，其中，步骤(2)的发酵控制条件为：温度24-30℃、通气比为0.25-0.8VVM、搅拌转速为200-600r/min。
  5. 如权利要求3或4所述的制备微生物絮凝剂的方法，其中，所述液体菌种培养基的组分及用量为：蔗糖12g/L，NH4NO3 2g/L，K2HPO4 0.8g/L，MgSO4 0.5g/L，NaCl 0.4g/L，FeSO4 0.013g/L。
  6. 如权利要求3或4所述的制备微生物絮凝剂的方法，其中，所述发酵培养基的组分及用量为：蔗糖23g/L，NH4NO3 2g/L，K2HPO4 1.2g/L，MgSO4 0.5g/L，NaCl 0.4g/L，FeSO4 0.015g/L，MnSO4 0.02g/L，松针提取物0.2g/L，玉米淀粉7g/L，玉米浆1.8g/L，ZnSO4 0.03g/L。
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