CN103642729A - Method for producing Bacillus subtilis for feeds by fermenting high-salt-content amino acid wastewater - Google Patents
Method for producing Bacillus subtilis for feeds by fermenting high-salt-content amino acid wastewater Download PDFInfo
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Abstract
The invention discloses a method for producing Bacillus subtilis for feeds by fermenting high-salt-content amino acid wastewater, which comprises the following steps: 1. habituated culture of feed strain: carrying out continuous habituated culture to enhance the tolerance of the strain to high-salt-content amino acid wastewater; and 2. fermentation of strain: under appropriate fermenting conditions, carrying out liquid ventilation culture in a fermentation tank for some time to obtain the Bacillus subtilis for feeds. The microzyme is centrifugated, collected, dried in a 70 DEG C drying oven to constant weight, and weighed. The calculation indicates that every liter of high-salt-content amino acid wastewater can obtain 35g of Bacillus subtilis at the maximum. The method can effectively utilize amino acids, microelements and growth factors contained in the wastewater, greatly lowers the fermenting cost of the Bacillus subtilis for feeds, greatly reduces the sewage treatment load, achieves the comprehensive utilization effect, and has obvious economic and social benefits.
Description
Technical field
The present invention relates to the comprehensive utilization field of waste water resource, relate in particular a kind of method of subtilis for high saliferous amino acid wastewater fermentative production feed of utilizing.
Background technology
According to World Health Organization's investigation, there is in the world 70% people to drink the tap water less than safety and sanitation, there are every day 2.5 ten thousand people to die from the disease relevant to sewage or severe sanitary condition; To the year two thousand fifty, estimate that the whole world has about 7,000,000,000 people and faces and use water shortage.China has 82% people to drink shallow well and river, and what wherein bacterial contamination surpassed hygienic standard accounts for 75%, is subject to the drinking-water population approximately 1.6 hundred million of Organic pollutants.Wherein the sewage of industrial discharge is the main source that water pollutes.Through investigation statistics, 659.2 hundred million tons of the annual wastewater discharges in the whole nation.Wherein, 230.9 hundred million tons of discharged volume of industrial waste water, account for 35.0% of wastewater emission amount.In fact, water amount of blowdown is considerably beyond this number, because many township and village enterprises industrial sewage quantity discharged is difficult to statistics.
China is amino acids production big country, the production method that most of enterprise that produces halfcystine takes is all hair acid system hydrolysis method, the annual high saliferous amino acid wastewater producing is enormous amount not only, and Pollutant levels are high, its COD concentration is 50～80g/L, ammonia nitrogen concentration is 5～8g/L, total cl concn is 120～150g/L, belong to that typical high density is organic, ammonia nitrogen, high salinity, strongly acid wastewater, greatly, the problem of environmental pollution causing has thus become the key of restriction amino acids production industry Sustainable development for intractability and burden.Yet, high-concentration waste water in amino acids production is containing there being again the resource of a lot of preciousnesses, as solid matter suspended substance, plurality of inorganic salt, organic acid and reducing sugars etc. such as amino acid, protein, full nitrogen (N) content is up to 75-150g/L, and aminoacids content is up to 150-300g/L.The poisonous and harmful substances simultaneously not decomposing containing difficulty in this waste water.Environment has not only been polluted in this large amount of discharges containing amino acid wastewater has also been wasted a lot of amino acid with practical value.Under current international economy amplification turns slow overall background, attempt " energy-saving and emission-reduction " cleaner production theory Jiang Wei amino acids production enterprise and bring new opportunity, be also the great scientific research task that fermentation industry faces.
At present, for the processing of high saliferous amino acid wastewater, there is no the ripe treatment process that can generally promote both at home and abroad.The amino acid wastewater treatment process of reporting at present mainly contains electroosmose process, ultrafiltration process, biochemical process etc., but electrodialysis and ultrafiltration process are because of investment with running cost is high, the popularization of the method for having safeguarded the drawbacks limit such as inconvenient; And biochemical process because of its investment and running cost low, technology maturation, does not produce secondary pollution, operational management such as facilitates in treatment of Organic Wastewater, to obtain global concern at the advantage.The problem that current this method exists is that centering sewage of low concentration is comparatively suitable, but is not suitable for processing the amino acid wastewater of high density.The amino acid wastewater of high density is because of higher concentration NH
3the salt of-N and high density, can make biochemical reaction effect poor to anaerobism and the aerobic strong restraining effect of microorganisms, and the water after processing is difficult for stably reaching standard discharge.If by sewage dilution for many times, greatly increased again treatment capacity, the quality amino acids resource in waste water has all wasted simultaneously.Also someone adopts first high-purity amino acid waste water is carried out to pre-treatment (its aminoacid component of concentration and recovery) at present, and then by biotechnology, processes the composite waste of lower concentration.This method is to process a kind of effective ways of amino acid wastewater, has both reclaimed amino acid, has reduced again the burden of biological treatment.But in whole treatment process, exist concentration process cost high, time and effort consuming, resource reclaims and to disconnect mutually with wastewater treatment, and has the shortcomings such as the concentrated organism afterwards of high temperature evaporation is apt to deteriorate.
Summary of the invention
The object of the invention is to be to provide a kind of method of subtilis for high salt amino acid wastewater fermentative production feed of utilizing, its technique is simple, with low cost, high salt amino acid wastewater is processed and the direct combination of fermentative production, all middle pre-treatment step have been removed from, realize the direct biological utilisation of high-concentration sewage, in realization at low cost, when turning waste into wealth, also reduced discharge, economic benefit and social benefit are remarkable.
In order to realize above-mentioned object, the present invention adopts following technical measures:
Utilize a method for subtilis for high salt amino acid wastewater fermentative production feed, the steps include:
(1) continuous domestication of feed bacterial strain is cultivated, specific as follows: first preparation, containing the liquid nutrient medium of the high saliferous amino acid sewage of different concns gradient, adds 1.5%-2.5%(mass volume ratio) agar makes solid medium.After feed is activated according to a conventional method with subtilis, be inoculated into successively on the solid medium of above-mentioned different concns gradient and carry out continuous passage cultivation (culture temperature is 15-35 ℃), progressively improve the salt resistance ability of bacterial classification.Through bimestrial cultured continuously, final bacterial strain can account in the above-mentioned substratum about substratum total mass 28-38% and grow at high saliferous amino acid wastewater.
(2) fermentation: the bacillus subtilis strain of step (1) domestication is seeded to and is take in the fermention medium that high saliferous amino acid wastewater is nitrogenous source by inoculum size 0.5-5%, at 15-35 ℃, rotating speed is under 100-300rpm/min condition, in fermentor tank, to carry out liquid fermenting, ventilation coefficient is that 0.2-0.8(per minute passes into fermentating liquid volume 0.2-0.8 filtrated air doubly), after the centrifugal 10min of 4000r/min collects thalline after fermentation 2d, be placed in 70 ℃ of thermostatic drying chambers, weigh after being dried to constant weight.As calculated, according to said method every rising saliferous amino acid wastewater can be through the highest 35g subtilis thalline that obtains of bio-transformation.
The waste water producing in enterprise's industrial production of described high saliferous amino acid wastewater from hair acid system hydrolytic producing amino-acid, full nitrogen (N) content is 75-150g/L, and aminoacids content is 150-300g/L, and chloride ion content is 80-180g/L.
Described feed derives from Chinese industrial microbial strains preservation administrative center with subtilis: bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum) CICC20037.
The liquid nutrient medium of the described high saliferous amino acid wastewater containing different concns gradient, the concentration of its high saliferous amino acid wastewater is respectively 125g/L, 250g/L, 330g/L, other chemical matrix components are: carbon source (by glucose meter): 30-90g/L, potassium primary phosphate (KH
2pO
4): 3-15g/L, yeast powder: 0-10g/L, magnesium sulfate (MgSO
4): 0.3-9g/L, calcium carbonate (CaCO
3): 0-2g/L, ferrous sulfate (FeSO
4): 0-4g/L.Medium pH is 7-8; Wherein, carbon source is the mixture of following one or more carbohydrates: glucose; Sucrose; Molasses; W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution.
Described W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution be with conventional enzymatic hydrolysis be prepared from (or referring to Liu Yawei. production of corn starch and transformation technology [M]. Beijing: Chemical Industry Press, 2003:296.)
The fermention medium that described high saliferous amino acid wastewater is nitrogenous source, its formula is: high saliferous amino acid sewage 125g/L-330g/L, carbon source: 30-90g/L, potassium primary phosphate (KH
2pO
4): 3-15g/L, yeast powder: 0-10g/L, magnesium sulfate (MgSO
4): 0.3-9g/L, calcium carbonate (CaCO
3): 0-2g/L, ferrous sulfate (FeSO
4): 0-4g/L, medium pH is 6-8; Wherein, carbon source is the mixture of following one or more carbohydrates: glucose; Sucrose; Molasses; W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution.
As preferably, the culture temperature that in step (1), domestication is cultivated is 20-35 ℃, and the medium pH that domestication is cultivated is 7.2.
As preferably, the fermention medium described in step (2), medium pH is 7.2.
As preferably, the culture temperature in step (2) is 31 ℃, and incubation time is 2d.
Compared with prior art, the present invention has the following advantages:
Bacterial strain in the present invention is after continuous domestication is cultivated, the high saliferous amino acid wastewater of the high density of usining is as nitrogenous source and Fast Growth, so just successfully by the purifying treatment of high saliferous amino acid wastewater and the direct combination of fermentative production, removed middle pre-treatment step from, the low-cost recycling of having realized amino acid wastewater, both can reclaim a large amount of useful matteies, reduced the cost of fermentative production, also reduce the discharge of sewage simultaneously, there is very strong competitive edge.Take Wuhan halfcystine manufacturing enterprise is example, and it take various animal hairs as each seed amino acid of raw material production, year produces approximately 10,000 tons of waste liquids; As waste liquid utilizes completely, can 350 tons of feed subtilises of year fermentative production, reach 1,750 ten thousand annual value of production, can also reduce the discharge of high COD value waste water simultaneously, alleviate waste disposal plant and load, ecology and economy remarkable benefit.
In the present invention, resulting salt tolerant bacterial strain is to take traditional feeding bacillus subtilis strain as starting strain, by traditional method, to tame to cultivate and produced physiological compatibility and obtain, do not change its legacy feature, there is extraordinary biological safety, can be directly used in production.
The salt tolerant bacterial strain vitality of gained of the present invention is strong, and nitrogenous source utilising efficiency is high, under fermentor tank bench scale, can reach the production level of every liter of waste water stoste output 35g thalline, and list just has very high application potentiality economically.
Utilize the present invention to tame the bacterial strain of cultivation, high saliferous amino acid wastewater can be processed and the direct combination of fermentative production, all middle pre-treatment step have been removed from, realize the direct biological utilisation of high-concentration sewage, in realization at low cost, when turning waste into wealth, also reduced discharge, economic benefit and social benefit are remarkable.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated; But the present invention is not limited to these embodiment.
The microorganism of using in following examples is all the conventional bacterial classification that the public can obtain.Obviously, those skilled in the art also can verify technical scheme of the present invention with other candida strain.Described technical scheme, if not otherwise specified, is routine techniques scheme, and agents useful for same is this area common agents.
Embodiment 1: the seed selection of Bacillus subtilis strain for feed.
1. fetch from Wuhan Amino Acid Factory, the waste liquid after halfcystine is produced in the hydrolysis of hair acid system, as the use of required below high saliferous amino acid wastewater.Its component list is as follows:
Table 1 amino acid wastewater coherent element content
| Project name | Unit | Detected result | Method foundation |
| Full nitrogen (N) | ％ | 8.79 | GB/T8572-2010 |
| Sodium (Na) | ％ | 0.52 | ICP method |
| Chlorine (Cl) | g/L | 131.26 | Ag Cl turbidimetry |
| Plumbous (Pb) | mg/kg | 0.91 | ICP method |
| Arsenic (As) | mg/kg | 0.076 | Atomic fluorescence method |
| Mercury (Hg) | mg/kg | 0.014 | Atomic fluorescence method |
| Chromium (Cr) | mg/kg | 1.51 | ICP method |
| Cadmium (Cd) | mg/kg | 0.06 | ICP method |
Table 2 amino acid wastewater aminoacid component and content
| Project name | Detected result % |
| Taurine | 0.02 |
| Aspartic acid | 2.16 |
| Threonine | 1.58 |
| Serine | 3.92 |
| L-glutamic acid | 3.33 |
| Glycine | 3.18 |
| L-Ala | 1.58 |
| Gelucystine | 0.19 |
| α-amino-isovaleric acid | 1.50 |
| Methionine(Met) | 0.10 |
| Isoleucine | 0.56 |
| Leucine | 0.73 |
| Tyrosine | 0.16 |
| Phenylalanine | 1.20 |
| Γ-aminobutyric acid | Do not detect |
| Ornithine | 0.03 |
| Methionin | 0.42 |
| Proline(Pro) | 2.99 |
| Histidine | 0.18 |
| Tryptophane | Do not detect |
| Arginine | 2.07 |
| Oxyproline | Do not detect |
| Summation | 25.9 |
From upper table, learn, its amino acid total mass mark reaches 259g/L, and total chlorine, up to 131.26g/L, is typical high saliferous amino acid wastewater.
2. preparation is containing the liquid nutrient medium of high saliferous amino acid wastewater.Preparing respectively high saliferous amino acid wastewater concentration is 125g/L, 250g/L, 330g/L(high saliferous amino acid wastewater of the present invention is in mass) liquid nutrient medium of different gradients, wherein amino acid wastewater is only nitrogen source, described liquid nutrient medium also adds other required chemical matrix of microorganism, its component is: glucose: 90g/L, KH
2pO
4: 15g/L, yeast powder: 10g/L, MgSO
4: 9g/L, CaCO
3: 2g/L, FeSO
4: 4g/L, medium pH is 7.2.And after being added to interpolation 20g/L agar (calculating by 1000ml water), partially liq substratum changes into solid medium.
3. after feed being accessed respectively according to a conventional method to YEPD substratum and activates with subtilis, the concentration being inoculated into successively containing amino acid wastewater is respectively 125g/L, 250g/L, on the amino acid wastewater solid medium of 330g/L, carry out continuous passage cultivation (culture temperature is 28 ℃), reach the object that domestication is cultivated.By domestication, cultivate, bacterial strain can be grown on the solid medium that be 330g/L in high saliferous amino acid wastewater concentration.
Described subtilis is bought in Chinese industrial microbial strains preservation administrative center: bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum) CICC20037.
Embodiment 2:
Subtilis take the fermentation of glucose in the liquid nutrient medium that carbon source, amino acid wastewater are nitrogenous source.
1. the liquid fermentation medium that the high saliferous amino acid wastewater concentration of preparation is 200g/L, wherein high saliferous amino acid wastewater is only nitrogen source (its source, composition consistent with embodiment 1), described liquid fermentation medium also adds other required chemical matrix of microorganism, its component is respectively: glucose: 50g/L, KH
2pO
4: 9g/L, yeast powder: 6g/L, MgSO
4: 5g/L, CaCO
3: 1.2g/L, FeSO
4: 2.5g/L, medium pH is 7.2.
2. the bacillus subtilis strain of having tamed obtaining from embodiment 1 is seeded to the liquid fermentation medium of step 1 preparation by 2% inoculum size, in fermentor tank, at 31 ℃, rotating speed is 220rpm/min, liquid fermentation and culture 2d under the condition that ventilation coefficient is 0.5.Cultivation finishes the centrifugal 10min of rear 6000r/min and collects thalline, is placed in 70 ℃ of thermostatic drying chambers, dry until weigh after constant weight.Found that, after converting, the fermentation that adopts above-mentioned substratum to carry out, every liter of waste water stoste output 35g yeast thalline.
Embodiment 3:
Subtilis take the fermentation of W-Gum enzymolysis solution in the liquid nutrient medium that carbon source, amino acid wastewater are nitrogenous source.
1. the liquid fermentation medium that the high saliferous amino acid wastewater concentration of preparation is 200g/L, wherein high saliferous amino acid wastewater is only nitrogen source (its source, composition consistent with embodiment 1), described liquid fermentation medium also adds other required chemical matrix of microorganism, its component is respectively: W-Gum enzymolysis solution (according to W-Gum dry weight note): 50g/L, KH
2pO
4: 9g/L, yeast powder: 6g/L, MgSO
4: 5g/L, CaCO
3: 1.2g/L, FeSO
4: 2.5g/L, medium pH is 7.2.
2. the bacillus subtilis strain of having tamed obtaining from embodiment 1 is seeded to the liquid fermentation medium of step 1 preparation by 5% inoculum size, in fermentor tank, at 31 ℃, rotating speed is 220rpm/min, liquid fermentation and culture 2d under the condition that ventilation coefficient is 0.5.Cultivation finishes the centrifugal 10min of rear 6000r/min and collects respectively thalline, is placed in 70 ℃ of thermostatic drying chambers, dry until weigh after constant weight.Found that, after converting, the fermentation that adopts above-mentioned substratum to carry out, every liter of waste water stoste output 32g yeast thalline.
Described W-Gum enzymolysis solution, the method for enzymatic hydrolysis is referring to Liu Yawei. production of corn starch and transformation technology [M]. and Beijing: Chemical Industry Press, 2003:296.
Although the present invention has been made detailed explanation and has quoted some specific embodiments as proof, to those skilled in the art, only otherwise it is obvious leaving that the spirit and scope of the present invention can make various changes or revise.
Claims (4)
1. utilize a method for subtilis for high salt amino acid wastewater fermentative production feed, the steps include:
(1) continuous domestication of feed bacterial strain is cultivated, specific as follows: first preparation, containing the liquid nutrient medium of the high saliferous amino acid sewage of different concns gradient, adds 1.5%-2.5% mass volume ratio agar and makes solid medium; After feed is activated according to a conventional method with subtilis, be inoculated into successively on the solid medium of above-mentioned different concns gradient and carry out continuous passage cultivation, culture temperature is 15-35 ℃, progressively improves the salt resistance ability of bacterial classification; Through bimestrial cultured continuously, final bacterial strain can be grown in high saliferous amino acid wastewater accounts for the above-mentioned substratum of substratum total mass 28-38%;
(2) fermentation: the bacillus subtilis strain of step (1) domestication is seeded to and is take in the fermention medium that high saliferous amino acid wastewater is nitrogenous source by inoculum size 0.5-5%, at 15-35 ℃, rotating speed is under 100-300 rpm/min condition, in fermentor tank, to carry out liquid fermenting, ventilation coefficient is 0.2-0.8, ferment after 2 d after centrifugal 10 min of 4000 r/min collect thalline and be placed in 70 ℃ of thermostatic drying chambers, weigh after being dried to constant weight;
The waste water producing in enterprise's industrial production of described high saliferous amino acid wastewater from hair acid system hydrolytic producing amino-acid, total nitrogen content is 75-150 g/L, and aminoacids content is 150-300 g/L, and chloride ion content is 80-180 g/L;
Described feed derives from Chinese industrial microbial strains preservation administrative center with subtilis: bacillus amyloliquefaciens plant subspecies (
bacillus amyloliquefaciens subsp. plantarum) CICC 20037;
The liquid nutrient medium of the described high saliferous amino acid wastewater containing different concns gradient, the concentration of its high saliferous amino acid wastewater is respectively 125 g/L, 250 g/L, 330 g/L, other chemical matrix components are: carbon source: 30-90 g/L, potassium primary phosphate: 3-15 g/L, yeast powder: 0-10 g/L, magnesium sulfate: 0.3-9 g/L, calcium carbonate: 0-2 g/L, ferrous sulfate: 0-4 g/L; Medium pH is 7-8; Wherein, carbon source is the mixture of following one or more carbohydrates: glucose; Sucrose; Molasses; W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution;
The fermention medium that described high saliferous amino acid wastewater is nitrogenous source, its formula is: high saliferous amino acid sewage 125 g/L-330 g/L, carbon source: 30-90 g/L, potassium primary phosphate: 3-15 g/L, yeast powder: 0-10 g/L, magnesium sulfate: 0.3-9 g/L, calcium carbonate: 0-2 g/L, ferrous sulfate: 0-4 g/L, medium pH is 6-8; Wherein, carbon source is the mixture of following one or more carbohydrates: glucose; Sucrose; Molasses; W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution.
2. method according to claim 1, is characterized in that: the culture temperature of described step (1) is 20-35 ℃, and medium pH is 7.2.
3. method according to claim 1, is characterized in that: the fermention medium pH described in described step (2) is 7.2.
4. method according to claim 1, is characterized in that: the culture temperature in described step (2) is 31 ℃, and incubation time is 2 d.
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