CN1844360A - Microorganism flocculant producing strain by using bean dregs and process for producing same - Google Patents
Microorganism flocculant producing strain by using bean dregs and process for producing same Download PDFInfo
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Abstract
This invention relates to a microbial flocculating agent. The invention isolates a high-yield bioflocculation microorganism-Paenibacilluspolymyxa GA1 CCTCC M 206017, and utilizes bean dregs as the substituted culture medium to synthesize the flocculating agent. The culture process comprises: processing the bean dregs at pH11.0 by boiling at high temperature and pressure, adjusting the pH to pH6.0-8.0, inoculating with 0.5-2% seed solution, applying two-stage fermentation process, wherein: at the early state of 24 hours, the culture temperature is 30deg C and the shaker speed is 150r/min; and at the late stage of 32 hours, the culture temperature is 25deg C and the shaker speed is 100r/min. By applying the two-stage fermentation process, the bacteria can make use of nutrients to synthesize the flocculating agent at a maximu rate, with the yield increaed to 7.86g/L and culture cycle decreased to 56h. By using the bean dregs and the two-stage fermentation process, it can lower the cost for producing the microbial flocculating agent, improve the yield, and realize the massive industrial production of microbial flocculating agent.
Description
Technical field
The present invention relates to water conditioner-flocculation agent of using in the industry such as Industrial Wastewater Treatment, foodstuff production and fermentation, be specifically related to a kind of microbial flocculant.
Background technology
Flocculation agent is widely used in the industry such as Industrial Wastewater Treatment, foodstuff production and fermentation.Generally flocculation agent is divided three classes: (1) inorganic flocculating agent, as Tai-Ace S 150, polymerize aluminum chloride, bodied ferric sulfate etc.; (2) organic synthesis polymeric flocculant is as polyacrylamide and derivative thereof, polymine, poly styrene sulfonate etc.; (3) natural macromolecule flocculating agent is as treated starch, poly-glucosamine, chitosan, sodiun alginate, chitin and microbial flocculant.In these flocculation agents, the organic synthesis polymeric flocculant is widely used among the actual production owing to its good flocculating effect and low cost.Polyacrylamide is comparatively commonly used a kind of, but it has refractory organics, evidence suggests that acrylamide monomer is a kind of strong carcinogens, and has the intensive neurotoxicity, residues in that acrylamide monomer easily causes secondary pollution in the environment.Present many countries have forbidden or have limited and use this type of flocculation agent.The use of inorganic flocculating agent can be brought a large amount of mineral ions in the treatment solution to food and fermentation industry, and excessive mineral ion not only influences the local flavor and the mouthfeel of product, also is unfavorable for people's health, especially Al
3+Absorption, with the initiation of at present increasing senile dementia direct relation is arranged; Fe3+ has severe corrosive, and easily residual, can make processed water have color, influences water quality.Though natural macromolecule flocculating agents such as chitosan, sodiun alginate, chitin are nontoxic, the energy safe disposal, its flocculation activity is weak, cost is higher, has limited its widespread use.
Microbial flocculant is that a class can make aggegation, the sedimentary special high molecular polymers such as solid suspended particle, somatic cells and colloidal particle that is difficult for degraded in the water body by what microorganism produced in process of growth.It is easy to separate, the settling efficiency height, and degradable, its degraded product is nontoxic to environment, can not produce secondary pollution, and wide accommodation has good turbidity removal and decolorization, is a kind of flocculation agent of highly effective and safe.
But the domestic and international at present research to microbial flocculant is in the laboratory study stage mostly, does not also reach practical application and suitability for industrialized production stage.The key that restricts its development be output cross low or production cost too high.Its output of bacterium for producing flocculant of being screened as Ye Jingjing etc. is 1.985g/L; Employing pseudomonas GX-4 such as Zhou Xu utilize the fish meal processing waste water to produce flocculation agent, and its output only is 0.612g/L; Its output of bacterium for producing flocculant 0.5～0.9g/L of screening such as Wang Zhen; The genus bacillus of one strain produce flocculant of screening such as the Ma Xiaona of China Agricultural University, though its output up to 12.48g/L, is to use pure chemistry reagent citric acid and L-glutamic acid etc. as substratum, thereby the cost height.Therefore screen a kind of flocculation agent output height, can utilize the microbial strains of cheap raw material again, and study the major issue that its production technique is need solution during microbial flocculant is produced.
Summary of the invention
The objective of the invention is to utilize plant height efficiency flocculating agent generation bacterium, select cheap raw material substratum as an alternative, the produce flocculant culture process that research is best solves the problems referred to above that exist in the microbial flocculant production simultaneously.
The present invention is achieved through the following technical solutions the foregoing invention purpose:
One strain utilizes the production bacterium of Dregs Manufacture microbial flocculant to be Paenibacillus polymyxa (Paenibacillus polymyxaGA1 CCTCC M 206017), utilizes the production method of Dregs Manufacture microbial flocculant to comprise that seed liquor is cultivated, fermented liquid is cultivated and the extraction of flocculation agent.
(1) seed liquor is cultivated: the Paenibacillus polymyxa through slant culture is seeded in the seed culture fluid of sterilization, under 24～32 ℃, the condition of 100～200r/min, cultivated 18～32 hours, to bacterial concentration be 0.5～1.5 * 10
8CFU/mL;
(2) two sections fermentation culture: concentration is that 100～250g/L, pH are 11.0 bean dregs nutrient solution behind 115～125 ℃, 0.07～0.15MPa high pressure-temperature hydrolysis, 20～40min, regulate pH to 6.0～8.0, inoculate 0.5～2% seed liquor by volume, earlier 30 ℃ of temperature, shaking table speed 150r/min condition bottom fermentation 24 hours,, fermented 32 hours under the shaking table speed 100r/min condition 25 ℃ of temperature in the back;
(3) extraction of flocculation agent: fermented liquid is at the centrifugal 20～40min of 5000～8000r/min, collect supernatant liquor, add 1～3: 1 precooling anhydrous propanone by volume, stir and place and staticly settled under 4 ℃ 24 hours, with 4000～6000r/min centrifugal collecting precipitation, with 4 ℃ of dehydrated alcohol dehydrations for several times, vacuum-drying is precipitated to constant weight.
Below in conjunction with accompanying drawing in detail the present invention is described in detail.
Description of drawings
Fig. 1 is the Photomicrograph (1 000 *) of bacterial strain GA1;
Fig. 2 is the influence of bean dregs high pressure-temperature hydrolysis pH value to the GA1 produce flocculant;
The different training methods of Fig. 3 are utilized the influence of bean dregs synthetic flocculant to GA1;
Wherein: (a) cultivate for segmentation;
(b) be 25 ℃ of culture temperature, shaking table speed 100r/in;
(c) be 30 ℃ of culture temperature, shaking table speed 150r/min;
(d) be that three kinds of training method flocculation agent output and growth cycle compare.
Fig. 4 utilizes the technological process of production synoptic diagram of bean dregs synthetic flocculant for Paenibacillus polymyxa GA1.
1, the separation of bacterium for producing flocculant and feature
Bacterium for producing flocculant GA1 is the strain flocculation of adopting conventional bacterium separation method to screen from the soil of Changsha Yue Lu mountain The agent Producing Strain, the outer polymer of the born of the same parents of its generation has good turbidity removal decoloring ability. This bacterial strain is at the beef extract-peptone solid The bacterium colony milky is flat on the agar medium, is irregular diffusion; Containing bacterium colony on the agar medium of glucose Translucent, protuberance, thickness. This strain cell is shaft-like, big or small 0.6 μ m～0.8 μ m * 2 μ m～5 μ m (Fig. 1). Leather is blue Albert'stain Albert is positive, and gemma is arranged, the tool pod membrane; This bacterial strain is chemoheterotrophy, aerobic or amphimicrobian. Can utilize glucose The fermentation and acid aerogenesis, the catalase positive, hydrolyzed starch, the VP test is positive, edwardsiella hoshinae. Utilize universal primer 27F and 1495R increase to the DNA sample of the GA1 bacterial strain that extracts, with the order-checking of the 16S rDNA that obtains The result is submitted to GenBank, and accession number is DQ166375. The Blastn homology search is the result show, the 16S of this bacterial strain The similitude maximum of rDNA and Paenibacillus polymyxa reaches 99%. Comprehensive above morphology, Physiology and biochemistry Feature and 16S rDNA sequence homology analysis qualification result can think that bacterial strain GA1 is Paenibacillus polymyxa, with it Called after Paenibacillus polymyxa GA1. This bacterial classification is submitted to China as the culture that is used for proprietary program Typical case's culture collection center is numbered CCTCC NO:M 206017.
2, the production method of flocculant
(1) seed culture medium and seed liquor are cultivated
Seed culture medium is peptone 10.0g/L, beef extract 3.0g/L, NaCl 5.0g/L, pH7.0,0.1MPa sterilization 30min. Cultivate a little bacterium colony of picking on the inclined-plane in fresh GA1, be inoculated in the seed culture medium, in 24～32 ℃ of temperature, Cultivate 18～32h under the shaking table speed of 100～200r/min, bacterial concentration is about 0.5～1.5 * 108CFU/mL. With this Seed liquor when nutrient solution is inoculated as the GA1 produce flocculant.
(2) preparation of bean dregs nutrient solution
Bean dregs are byproducts of bean product process, multiplexly make feed and fertilizer, even are taken as discarded object and abandon, Fully effectively do not utilized, caused serious environmental pollution. Utilize bean dregs as the bacterium for producing flocculant produce flocculant Substitutive medium can be accomplished this resource of rational exploitation and utilization bean dregs, has wide market potential and application prospect.
The used bean dregs of Paenibacillus polymyxa GA1 synthetic flocculant are that soybean last in the bean curd manufacturing process is residual Slag is the bulk shape solid of white. Moisture content is about 82%～88%, and its nutritional labeling is comparatively complicated, roughly nutritional labeling For every 100g dry weight contains 50g～60g cellulose, 22g～28g crude protein, 9g～12g lipid material and 2g～4g approximately Mineral matter.
The concentration of bean dregs in the nutrient solution (weight in wet base) is 100～250g/L, specifically decides on the water ratio and the bulk program of bean dregs.The bean dregs nutrient solution is first through 115～125 ℃, 0.07～0.15MPa high pressure-temperature hydrolysis, 20～40min under alkaline condition, the back regulates pH6.0～8.0, under 22～32 ℃ of culture temperature, shaking table speed 100～200r/min condition, inoculation seed liquor 0.5～2% is cultivated.Flocculation agent output will increase (Fig. 2) after the high temperature and high pressure hydrolysis pre-treatment, during the high pressure-temperature hydrolysis during with pH11.0 output the highest.The effect of alkalescence high pressure-temperature hydrolysis has: (1) is hydrolyzed into small organic molecule with larger molecular organics, so that better utilized by Institute of Micro-biology; (2) substratum is sterilized; (3) alkali can in and the part acidic substance that produced of bean dregs high temperature and high pressure hydrolysis.
(3) two sections fermentating culturing process
According to Paenibacillus polymyxa GA1 growth characteristics, the flocculation agent of its generation is a secondary metabolites, and thalli growth and flocculation agent synthetic optimal culture condition do not overlap.Best thalli growth condition is the initial pH7.0 of substratum, 30 ℃ of culture temperature, shaking table speed 150r/min and inoculum size 5%; Flocculation agent synthetic optimal culture condition is the initial pH6.5 of substratum, 25 ℃ of culture temperature, shaking table speed 100r/min and inoculum size 1%.Two sections culture process are proposed in view of the above.The bean dregs nutrient solution is after the process high pressure-temperature hydrolysis of pH11.0, transfer to pH6.5, inoculate 1% seed liquor, in the 24h at the initial stage of cultivating, adopt 30 ℃ of temperature, shaking table speed 150r/min, promote the growth of thalline, shorten the lag phase of thalli growth, make that cell concn reaches a higher level within a short period of time in the nutrient solution, for next step synthetic flocculant lays the first stone; Adopt 25 ℃ of flocculation agent synthetic optimum culturing temperatures and best shaking table speed 100r/min at the later stage 32h that cultivates, maximally utilise the nutritive substance synthetic flocculant.Under the different training methods, GA1 utilizes the output of bean dregs synthetic flocculant, and as shown in Figure 3, the result shows: 30 ℃ of temperature, and under the shaking table speed 150r/min, thalli growth is fast, culture cycle short (56h), (6.58g/L) (Fig. 3 c) but flocculation agent yields poorly; 25 ℃ of temperature, under the shaking table speed 100r/min, the output height (7.76g/L) of flocculation agent, but long (72h) (Fig. 3 b) of culture cycle, its reason is that thalline growth metabolism under low temperature and low dissolved axygen level is slow, the bacterial growth cycle is long; And adopt two sections culture methods can promote thalli growth, and make culture cycle shorten to 56h, can make simultaneously the output of flocculation agent keep that (Fig. 3 is a) about 7.86g/L again.Therefore, use two sections culture methods to can be good at solving the thalli growth of bacterial strain GA1 and the contradiction between the produce flocculant, can guarantee that nutrient solution utilizes raw material to synthesize the secondary metabolites microbial flocculant to greatest extent in suitable mycetocyte concentration range, thereby make output remain on higher level, can shorten culture cycle again simultaneously.
(4) extraction of flocculation agent
Nutrient solution centrifugal 20～40min under 5000～8000r/min with Paenibacillus polymyxa GA1, collect supernatant liquor, the adding volume ratio is 1～3: 1 precooling anhydrous propanone (4 ℃), and stirring to place under 4 ℃ staticly settles 24h, makes the precipitation of generation stable; With 4000～6000r/min centrifugal collecting precipitation, with the dehydration of the dehydrated alcohol of precooling for several times, the powder essence of in the dehydration precipitation being milled promotes its dehydration, vacuumizes drying precipitatedly to constant weight then, obtains the thick product of flocculation agent, is buff powder.Fig. 4 is seen in the technical process of flocculation agent production.This flocculation agent is a polysaccharose substance, has very high flocculation activity.When dosage was 0.1g/L, the Kaolin clay suspension flocculating rate to 0.4% reached more than 94%, and slime water, paper waste, burnt black ink etc. are had good turbidity removal decolorizing effect.
What filter out with the present invention is the high yield bacterium of raw material production flocculation agent with bean dregs, adopt two sections fermentating culturing process synthetic flocculants, not only can rationally utilize this resource of bean dregs, reduce the production cost of microbial flocculant, and can improve output, shorten culture cycle, realize the production of microbial flocculant heavy industrialization.
Embodiment
A little bacterium colony of picking from the preservation inclined-plane of bacterial strain GA1 is inoculated in to the 50mL that the 15mL seed culture medium is housed and shakes in the bottle, cultivates 24h under 30 ℃, the shaking table speed of 150r/min, and bacterial concentration is about 1.0 * 10
8CFU/mL.Seed liquor when this nutrient solution is inoculated as the GA1 produce flocculant.
The concentration of bean dregs (weight in wet base, water ratio 85.5%) substratum is 200g/L, and adjust pH is 11.0, with the bean dregs substratum at high pressure-temperature (0.1MPa, 121 ℃) hydrolysis 30min down.
Under aseptic technique, the initial pH value of bean dregs substratum is transferred to 6.5 through the pretreated bean dregs substratum of high pressure-temperature hydrolysis, inoculate 1% seed liquor, in the 24h at the initial stage of cultivating, adopt 30 ℃ of the optimum tempss and the shaking table speed 150r/min of thalli growth, in the 32h of later stage cultivation, adopt 25 ℃ of the optimum tempss and the shaking table speed 100r/min of thalline synthetic flocculant, maximally utilise the nutritive substance synthetic flocculant.
With nutrient solution centrifugal 30min under 6000r/min of Paenibacillus polymyxa GA1, collect supernatant liquor, the adding volume ratio is 2: 1 a precooling anhydrous propanone (4 ℃), stirs to place under 4 ℃ to staticly settle 24h, makes the precipitation of generation stable.With the centrifugal 30min collecting precipitation of 4000r/min, with 4 ℃ of dehydrated alcohol dehydrations for several times, the powder essence of in the dehydration precipitation being milled, promote its dehydration, vacuumize drying precipitated then at normal temperatures to constant weight, obtain the thick product of flocculation agent of yellow powder powder, output is 7.86g/L, and productive rate is the synthetic thick products of 39.3g flocculation agent of the fresh bean dregs of every kg (weight in wet base).
Claims (2)
1, a strain utilizes the production bacterium of Dregs Manufacture microbial flocculant to be Paenibacillus polymyxa (Paenibacilluspolymyxa GA1 CCTCC M 206017).
2, a kind of production method of utilizing the Dregs Manufacture microbial flocculant as claimed in claim 1 comprises that seed liquor is cultivated, fermented liquid is cultivated and the extraction of flocculation agent, the invention is characterized in:
(1) seed liquor is cultivated: the Paenibacillus polymyxa through slant culture is seeded in the seed culture fluid of sterilization, under 24～32 ℃, the condition of 100～200r/min, cultivated 18～32 hours, to bacterial concentration be 0.5～1.5 * 10
8CFU/mL;
(2) two sections fermentation culture: concentration is that the pH of 100～250g/L is 11.0 bean dregs nutrient solution behind 115～125 ℃, 0.07～0.15MPa high pressure-temperature hydrolysis, 20～40min, regulate pH to 6.0～8.0, inoculate 0.5～2% seed liquor by volume, earlier 30 ℃ of temperature, shaking table speed 150r/min condition bottom fermentation 24 hours,, fermented 32 hours under the shaking table speed 100r/min condition 25 ℃ of temperature in the back;
(3) extraction of flocculation agent: fermented liquid is at the centrifugal 20～40min of 5000～8000r/min, collect supernatant liquor, add 1～3: 1 precooling anhydrous propanone by volume, stir to place and staticly settled under 4 ℃ 24 hours, with 4000～6000r/min centrifugal collecting precipitation, with 4 ℃ of dehydrated alcohol dehydrations for several times, vacuum-drying is precipitated to constant weight.
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| CN101870958A (en) * | 2010-05-10 | 2010-10-27 | 新疆农业科学院微生物应用研究所 | Paenibacillus and application thereof to environmental engineering |
| CN101503709B (en) * | 2009-03-13 | 2011-01-05 | 厦门大学 | Method for preparing bioflocculation by Bacillus licheniformis |
| CN101942407A (en) * | 2010-08-18 | 2011-01-12 | 无锡天易生物科技有限公司 | Producing strain and production method for producing microbial flocculant from wheat starch waste water |
| CN101993137A (en) * | 2010-08-06 | 2011-03-30 | 国家海洋局天津海水淡化与综合利用研究所 | Preparation method for seawater microbial flocculants |
| CN101327975B (en) * | 2008-07-31 | 2011-09-28 | 东北大学 | Method for preparing microorganism flocculant |
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| CN104045138A (en) * | 2014-01-17 | 2014-09-17 | 山西潞安环保能源开发股份有限公司 | Coal biological flocculant and preparation method thereof |
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| CN101327975B (en) * | 2008-07-31 | 2011-09-28 | 东北大学 | Method for preparing microorganism flocculant |
| CN101503709B (en) * | 2009-03-13 | 2011-01-05 | 厦门大学 | Method for preparing bioflocculation by Bacillus licheniformis |
| CN101870958A (en) * | 2010-05-10 | 2010-10-27 | 新疆农业科学院微生物应用研究所 | Paenibacillus and application thereof to environmental engineering |
| CN101993137B (en) * | 2010-08-06 | 2012-11-21 | 国家海洋局天津海水淡化与综合利用研究所 | Preparation method for seawater microbial flocculants |
| CN101993137A (en) * | 2010-08-06 | 2011-03-30 | 国家海洋局天津海水淡化与综合利用研究所 | Preparation method for seawater microbial flocculants |
| CN101942407A (en) * | 2010-08-18 | 2011-01-12 | 无锡天易生物科技有限公司 | Producing strain and production method for producing microbial flocculant from wheat starch waste water |
| CN102260729B (en) * | 2011-06-24 | 2013-03-13 | 哈尔滨工业大学 | Bioflocculant fermentation method with mycelium pellet as vector |
| CN102260729A (en) * | 2011-06-24 | 2011-11-30 | 哈尔滨工业大学 | Bioflocculant fermentation method with mycelium pellet as vector |
| CN102559768A (en) * | 2012-02-09 | 2012-07-11 | 湖南大学 | Two-step fermentation production method of microbial flocculant |
| CN102719482A (en) * | 2012-02-28 | 2012-10-10 | 湖南大学 | Method for producing microbial flocculant by utilizing culture wastewater and residual activated sludge |
| CN103184239A (en) * | 2013-03-11 | 2013-07-03 | 宁波飞日水产实业有限公司 | Method for producing microbial flocculant by using surimi processing waste water |
| CN104045138A (en) * | 2014-01-17 | 2014-09-17 | 山西潞安环保能源开发股份有限公司 | Coal biological flocculant and preparation method thereof |
| CN105621572A (en) * | 2016-02-29 | 2016-06-01 | 济南大学 | Light coagulant synthesized by bean dregs |
| CN106365279A (en) * | 2016-10-16 | 2017-02-01 | 谭淞文 | Microbiological product capable of flocculation and coagulation enhancement and preparation method, use method and application thereof |
| CN107381838A (en) * | 2017-08-31 | 2017-11-24 | 常州豪坦商贸有限公司 | A kind of preparation method of compound microbial water purification catridge material |
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