CN103833461A

CN103833461A - Method for extraction of alga liquid fertilizer synergist by genetically engineered bacterium fermentation

Info

Publication number: CN103833461A
Application number: CN201310616623.7A
Authority: CN
Inventors: 王爱云; 陈吉强; 刘云
Original assignee: SHANDONG RUNYIN BIOCHEMICAL CO Ltd
Current assignee: SHANDONG RUNYIN BIOCHEMICAL CO Ltd
Priority date: 2013-11-29
Filing date: 2013-11-29
Publication date: 2014-06-04

Abstract

The invention discloses a method for extraction of an alga liquid fertilizer synergist by genetically engineered bacterium fermentation. The method comprises the following steps of by a molecular biological technology, extracting photosynthetic bacterium, lactobacillus and actinomycete genetic gene molecules which can decompose insoluble macromolecules of alga into soluble micromolecules, connecting the genetic gene molecules to saccharomycete cytoplasm so that a DNA genome is recombined, and carrying out the recombined DNA genome expression in escherichia coli cells to construct a genetically engineered bacterial strain. The method improves bioactivity of microorganisms and improves an alga large-molecule decomposition capability. The method utilizes a high-density fermentation technology, improves fermentation strength and shortens fermentation time. The method utilizes a micro-filtration membrane and ultrafilter membrane separation technology, completely retains biologically-active substances and nutrients of alga, forms a net-type fertilizer-carrying system from the alga extract and urea, delays urea decomposition, provides a plurality of nutrients for crops, promotes crop growth and realizes urea synergism.

Description

A kind of method of genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist

Technical field

The preparation method who the present invention relates to fertilizer synergist is directly involved in a kind of method of genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist.

Technical background

The general rate of fertilizer application of China's agricultural is large at present, and fertilizer efficiency utilization ratio is low, particularly taking urea as main utilization rate of nitrogen fertilizer is below 25%.A large amount of nitrogenous fertilizer decomposes volatilization or outflows with water and ecotope is polluted.Over nearly twenties years, agriculture and fertilizer expert has studied coating controlled-release fertilizer, inhibitor slow-release fertilizer, has added polypeptide fertilizer, has extended fertilizer efficiency period to suppressing urea decomposition, save fertilizer and play active effect, but ubiquity cost is high, the shortcoming such as few has additional nutrients.

Seaweed extracted liquor has greatly retained natural radioactivity component, contain a large amount of non-itrogenous organic substances, 40 several mineral materials and the VITAMIN such as incomparable abundant K, the Ca of land plant, Mg, Zn, I, contain especially Sargassum polysaccharides, alginic acid, highly unsaturated fatty acids and multiple natural phant growth regulator in marine alga, there is very high biological activity, the generation of nonspecific factor in can stimulating plant body, regulates Endogenous hormone balance.In urea, add seaweed extracted liquor, under the effect of hydrogen bond, Seaweed Extract and urea form the fertile body that carries of alpha-helix or macromolecule network structure, can not only delay the decomposition of urea, the more important thing is and provide multiple nutrient for farm crop, promote plant growth, therefore say that seaweed extracted liquor is current best area synergist.Once adopted chemical hydrolysis and flora heap fermentation method both at home and abroad.The activity of chemical hydrolysis havoc marine alga endogenous substance, nutritive ingredient loss is serious; Flora heap fermentation method fermentation time reached about 10 days, was difficult to realize suitability for industrialized production, sought the easily preparation method of the seaweed extracted liquor of row of a kind of high-efficient simple, was technical problem urgently to be resolved hurrily.

Summary of the invention

Object of the present invention is just to provide a kind of marine alga liquid extracting method of efficient, easy, complete reservation marine alga natural activity.

The object of the present invention is achieved like this: genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist production method step is as follows:

The structure of genetic engineering bacterium: selecting the fermentation of seaweed marine alga macromole of degrading is that micromolecular photosynthetic bacteria, milk-acid bacteria are donor, extract the gene group DNA in its cell, select yeast to extract its tenuigenin as carrier, connect recombinant plasmid dna genome, selection intestinal bacteria are host receptor, in recombinant plasmid dna gene insertion Bacillus coli cells, build genetic engineering bacterium.

The cultivation screening of genetic engineering bacterium: the engineering bacteria cell that builds gene is implanted in substratum and cultivated, and good bacterial strain continues to cultivate to filter out growth situation after every cultivation 12h, so cultivates five generations of screening, the excellent species filtering out.

Three grades of seed culture: the strain excellent filtering out is after culture medium culturing, and culture transferring is cultivated 8 hours culture transferrings to shaking table and cultivated 10h to 200L first class seed pot, then culture transferring is cultivated 12h to 1000L secondary seed tank.

Two-stage expands fermentation: to 10t ferment tank 16 hours, 10t tank fermented liquid is transplanted to the 20t fermentor tank employing flow feeding 30h that continuously ferments by 1000L seeding tank culture transferring.

Microfiltration membrane filtering solid impurity: fermented liquid is squeezed into microfiltration membrane separator filtering solid impurity with pump.

Ultra-filtration membrane filtering mycelia: the fermented liquid pump of removing solid impurity is squeezed into ultra-filtration membrane filtering mycelia.

Ion-exchange demineralization: the cleaner liquid of filtering mycelia is squeezed into ion-exchanger desalination with pump.

Negative pressure evaporation is concentrated: the marine alga liquid after desalination carries out the concentrated marine alga liquid that obtains of negative pressure evaporation.

A preferred embodiment of the invention: in three grades of seed culture, the configuration proportion of substratum is: pure water 800g/L, peptone 2.6g/L, yeast powder 2g/L, KH ₂pO ₃2g/L, K ₂hPO ₃1g/L, ZnSO ₄8H ₂o 0.5g/L, MgSO ₄2g/L.

According to another kind of preferred implementation of the present invention: two-stage expands fermentation liquor formulation in fermentation and is: seaweed powder 200 order 25 mass parts, glucose 5 mass parts, inorganic salt 1 mass parts, beans matter albumen 2 mass parts, water 77 mass parts.45 DEG C of leavening temperatures, pH value is 6, dissolved oxygen ratio: 1:4.

Brief description of the drawings:

Accompanying drawing 1 shows the process flow diagram of the method for a kind of genetic engineering bacterium fermented extracted of the present invention marine alga liquid fertilizer synergist.

Embodiment

Accompanying drawing 1 shows the process flow sheet of the method for a kind of genetic engineering bacterium fermented extracted of the present invention marine alga liquid fertilizer synergist.Be described further below in conjunction with accompanying drawing.

The structure 1 of genetic engineering bacterium: selecting fermentation of seaweed can decompose the insoluble macromole of marine alga is the micromolecular photosynthetic bacteria of solubility, milk-acid bacteria, actinomycetes are as donor bacterium, the donor bacterium of selecting is cultivated respectively in substratum, from donor photosynthetic bacteria, milk-acid bacteria, in actinomycetes cell, obtain and there is the genetic molecule that decomposes marine alga characteristic, saccharomycetic tenuigenin is cut as carrier, by the photosynthetic bacteria obtaining, milk-acid bacteria gene combine with carrier, the gene group of restructuring is imported in colibacillary cell, gene are at expression in escherichia coli, so the new microorganism cells building is referred to as to genetic engineering bacterium.

The cultivation screening 2 of genetic engineering bacterium: prepare substratum: pure water 800g/L, peptone 2.6g/L, yeast powder 2g/L, KH ₂pO ₃2g/L, K ₂hPO ₃1g/L, ZnSO ₄8H ₂o 0.5g/L, MgSO ₄2g/L, stirs, and then the genetic engineering bacterium of structure is implanted in substratum and is cultivated sampling after 24 hours, chooses the bacterial strain growing fine and continues to cultivate, and eliminates the weak bacterial strain of growing way, and so cultured continuously screened for five generations, finally selected strain excellent.

Three grades of seed culture 3: the strain excellent of selecting was implanted to shaking table culture medium culturing after 8 hours, culture transferring was to 200L seed tank culture 10 hours, culture transferring was to 1000L seed tank culture 12 hours again, 45 DEG C of three grades of culture temperature, pH value is 6, dissolved oxygen ratio: 1:4, when culture transferring, growth nursery stage is all in logarithmic phase.

Two-stage expands fermentation 4: the preparation of fermented liquid: seaweed powder 200 order 25 mass parts, glucose 5 mass parts, inorganic salt 1 mass parts, beans matter albumen 2 mass parts, water 77 mass parts.Fermented liquid is respectively charged in 10t, 20t fermentor tank, seed liquor culture transferring, to 10t fermentor tank, is fermented 16 hours, growth nursery stage is still in logarithmic phase, grain weight 150g/L, pours one grade fermemtation liquid into second order fermentation tank, takes flow feeding, fermented liquid concentration is remained on to 30%, grain weight keeps 150g/L, 45 DEG C of leavening temperatures, and pH value is 6, dissolved oxygen ratio: 1:4., fermentation time 30 hours.

Microfiltration membrane filtering solid impurity 5: fermented liquid pump is squeezed into microfiltration membrane separator with pump, fermented liquid one microfiltration membrane separator upper cover enters every film pipe clear liquid and after enrichment, discharges from purified liquor outlet in separator cylinder through fenestra, and solid impurity is discharged with the outlet of lower cover impurity.

Ultra-filtration membrane filtering thalline 6: the clear liquid of microfiltration membrane filtering is squeezed into ultra-filtration membrane separator with pump, liquid enters every ultrafiltration membrane pipe from upper cover, seaweed extracted liquor infiltration membranous wall is discharged from cleaner liquid outlet after enrichment in cylindrical shell, and macromole mycelia intercepts in pipe and discharges from the outlet of lower cover mycelia.

Ion-exchange demineralization 7: cleaner liquid is squeezed into the positively charged ions such as cation exchanger Ca, Mg, Na and is absorbed with pump, then is absorbed by negatively charged ion such as anion exchanger lactate, sulfate radicals, reaches the object of desalination.

Negative pressure evaporation concentrated 8: the cleaner liquid after desalination is squeezed into triple effect evaporation unit with pump, at negative pressure-0.99MPa, at 80 DEG C of temperature, the concentration of evaporation concentration to 90%, obtains seaweed extracted liquor.

Principal feature of the present invention is: the genetic engineering bacterium collection photosynthetic bacteria that the means of applied molecular biology build, milk-acid bacteria, actinomycetes, yeast fermentation energy in marine alga decomposes the insoluble macromolecular feature of marine alga in one, at expression in escherichia coli, greatly improve biological activity, strengthen the macromolecular ability of decomposition marine alga, adopt high cell density fermentation, improve ferment strength, shorten fermentation time, adopt microfiltration membrane and ultra-filtration membrane isolation technique, complete reservation biologically active substance and the nutritive substance in marine alga, make seaweed extracted liquor and urea be combined into network and carry fertile system, delay the decomposition of urea, and provide multiple nutrient for farm crop, promote the growth of farm crop, reach the object of urea synergy.

Claims

1. a method for genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist, is characterized in that the step of the method is as follows:

The structure of genetic engineering bacterium: selecting the fermentation of seaweed marine alga macromole of degrading is that micromolecular photosynthetic bacteria, milk-acid bacteria are donor, extract the gene group DNA in its cell, select yeast to extract its tenuigenin as carrier, connect recombinant plasmid dna genome, selection intestinal bacteria are host receptor, in recombinant plasmid dna gene insertion Bacillus coli cells, construct genetic engineering bacterium;

The cultivation screening of genetic engineering bacterium: the genetically engineered mycetocyte of structure is implanted in substratum and cultivated, and good bacterial strain continues to cultivate to filter out growth situation after every cultivation 12h, so cultivates five generations of screening, the excellent species filtering out;

Three grades of seed culture: the strain excellent filtering out is after culture medium culturing, and culture transferring is cultivated 8 hours culture transferrings to shaking table and cultivated 10h to 200L first class seed pot, then culture transferring is cultivated 12h to 1000L secondary seed tank;

Two-stage expands fermentation: to 10t ferment tank 16 hours, 10t tank fermented liquid is transplanted to the 20t fermentor tank employing flow feeding 30h that continuously ferments by 1000L seeding tank culture transferring;

Microfiltration membrane filtering solid impurity: fermented liquid is squeezed into microfiltration membrane separator filtering solid impurity with pump;

Ultra-filtration membrane filtering mycelia: the fermented liquid pump of removing solid impurity is squeezed into ultra-filtration membrane filtering mycelia;

Ion-exchange demineralization: the cleaner liquid of filtering mycelia is squeezed into ion-exchanger desalination with pump;

2. a kind of method of genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist according to claim 1, is characterized in that: the preparation of substratum in three grades of seed culture: pure water 800g/L, peptone 2.6g/L, yeast powder 2g/L, KH ₂pO ₃2g/L, K ₂hPO ₃1g/L, ZnSO ₄8H ₂o 0.5g/L, MgSO ₄2g/L.

3. a kind of method of genetic engineering bacterium fermented extracted marine alga liquid fertilizer synergist according to claim 1, it is characterized in that: two-stage expands fermentation liquor formulation in fermentation and is: seaweed powder 200 order 25 mass parts, glucose 5 mass parts, inorganic salt 1 mass parts, beans matter albumen 2 mass parts, water 77 mass parts, 45 DEG C of leavening temperatures, pH value is 6, dissolved oxygen ratio: 1:4.