CN103642729B - 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 field of comprehensive utilization of waste water resource, relate in particular a kind ofly utilize the high saliferous amino acid wastewater fermentation production of fodder method of subtilis.
Background technology
According to World Health Organization's investigation, there is the people of 70% to drink tap water less than safety and sanitation in the world, have 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 the people of 82% to drink shallow well and river, and what wherein bacterial contamination exceeded hygienic standard accounts for 75%, is subject to the drinking-water population about 1.6 hundred million of Organic pollutants.Wherein the sewage of industrial discharge is the main source of water pollutions.Through investigation statistics, the annual wastewater discharge 659.2 hundred million tons in the whole nation.Wherein, discharged volume of industrial waste water 230.9 hundred million tons, accounts 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 wastewater discharge amount is difficult to statistics.
China is amino acids production big country, the production method that the enterprise that major part produces halfcystine takes is all hair Acid hydrolysis method, the high saliferous amino acid wastewater not only enormous amount of annual generation, 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 caused thus has become the key of restriction amino acids production industry value chain for intractability and burden.But, high-concentration waste water in amino acids production is containing there being again a lot of valuable resource, as the solid matter such as amino acid, protein suspended substance, plurality of inorganic salt, organic acid and reducing sugar etc., full nitrogen (N) content is up to 75-150g/L, and aminoacids content is up to 150-300g/L.Do not contain the difficult poisonous and harmful substances decomposed in this waste water simultaneously.This large amount of discharges containing amino acid wastewater not only pollute environment and also waste amino acid much with practical value.Under current international economy amplification turns slow overall background, attempting " energy-saving and emission-reduction " cleaner production theory and bring new opportunity by for amino acids production enterprise, is also the great scientific research task that fermentation industry faces.
At present, for the process of high saliferous amino acid wastewater, there is no both at home and abroad ripe can the treatment process of popularity.The amino acid wastewater treatment process reported 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, safeguard that the shortcoming such as inconvenient limits the popularization of the method; And biochemical process because of its investment and running cost low, technology maturation, does not produce secondary pollution, operational management facilitate etc. advantage in treatment of Organic Wastewater, obtain global concern.Current this method Problems existing is that centering sewage of low concentration is comparatively suitable, but is not suitable for the amino acid wastewater processing high density.The amino acid wastewater of high density is because of higher concentration NH
3the salt of-N and high density, can produce strong restraining effect to anaerobism and aerobic microorganism, make biochemical reaction effect poor, the not easily stably reaching standard discharge of the water after process.If by sewage dilution for many times, considerably increase treatment capacity again, the quality amino acids resource simultaneously in waste water has all wasted.At present also someone adopts and first carries out pre-treatment (its aminoacid component of concentration and recovery) to high-purity amino acid waste water, and then with the composite waste of biotechnology process lower concentration.This method is a kind of effective ways of process amino acid wastewater, has both reclaimed amino acid, and has again reduced the burden of biological treatment.But it is high whole treatment process to exist concentration process cost, time and effort consuming, and resource reclaim disconnects mutually with wastewater treatment, and the shortcoming such as after having high temperature evaporation concentrated organism is apt to deteriorate.
Summary of the invention
The object of the invention is to be that providing a kind of utilizes the high salt amino acid wastewater fermentation production of fodder method of subtilis, its technique is simple, with low cost, the process of high salt amino acid wastewater and fermentative production are directly combined, eliminate all middle pre-treatment step, realize the direct biological utilisation of high-concentration sewage, while realizing at low cost turning waste into wealth, also reduce discharge, economic benefit and social benefit remarkable.
In order to realize above-mentioned object, the present invention adopts following technical measures:
Utilize a high salt amino acid wastewater fermentation production of fodder method for subtilis, the steps include:
(1) continuous domestication of feed bacterial strain is cultivated, specific as follows: first preparation is containing the liquid nutrient medium of 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, the solid medium being inoculated into above-mentioned different concns gradient successively carries out continuous passage cultivation (culture temperature is 15-35 DEG C), progressively improves the salt resistance ability of bacterial classification.Through bimestrial cultured continuously, final bacterial strain can account in the above-mentioned substratum of substratum about total mass 28-38% at high saliferous amino acid wastewater and grow.
(2) ferment: the bacillus subtilis strain of step (1) being tamed is seeded to in the high saliferous amino acid wastewater fermention medium that is nitrogenous source by inoculum size 0.5-5%, at 15-35 DEG C, rotating speed is in fermentor tank, carry out liquid fermenting under 100-300rpm/min condition, ventilation coefficient is that 0.2-0.8(per minute passes into fermentating liquid volume 0.2-0.8 filtrated air doubly), be placed in 70 DEG C of thermostatic drying chambers after the centrifugal 10min of 4000r/min collects thalline after fermentation 2d, weigh after being dried to constant weight.As calculated, according to said method often raise saliferous amino acid wastewater and can obtain 35g subtilis thalline through bio-transformation is the highest.
Described high saliferous amino acid wastewater produces the waste water produced in amino acid whose enterprise industrial production from hair Acid hydrolysis, 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 Microbiological Culture Collection 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 one or more following 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 standard enzymatic hydrolysis be prepared from (or see Liu Yawei. production of corn starch and transformation technology [M]. Beijing: Chemical Industry Press, 2003:296.)
Described high saliferous amino acid wastewater is the fermention medium of nitrogenous source, and 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 one or more following 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 DEG C, 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 DEG C, 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, can grow fast using high density height saliferous amino acid wastewater as nitrogenous source, so just successfully the purifying treatment of high saliferous amino acid wastewater and fermentative production are directly combined, eliminate middle pre-treatment step, achieve the low cost recycling of amino acid wastewater, both can reclaim a large amount of useful matter, reduce the cost of fermentative production, also reduce the discharge of sewage simultaneously, there is very strong competitive edge.For Wuhan halfcystine manufacturing enterprise, it is with various animal hair for each seed amino acid of raw material production, and year produces waste liquid about 10,000 tons; As waste liquid utilizes completely, can year fermentative production 350 tons of feed subtilises, reach the annual value of production of 1,750 ten thousand, can also reduce the discharge of high cod values waste water, alleviate waste disposal plant load, Benefits in economic and social terms is remarkable simultaneously.
The salt-enduring strain obtained in the present invention carries out domestication with traditional feeding bacillus subtilis strain for starting strain traditional method to cultivate and create physiological compatibility and obtain, do not change its legacy feature, there is extraordinary biological safety, can production be directly used in.
The salt-enduring 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 often liter of waste water stoste output 35g thalline, and single just have very high application potentiality economically.
The present invention is utilized to tame the bacterial strain of cultivation, the process of high saliferous amino acid wastewater and fermentative production directly can be combined, eliminate all middle pre-treatment step, realize the direct biological utilisation of high-concentration sewage, while realizing at low cost turning waste into wealth, also reduce discharge, economic benefit and social benefit remarkable.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated; But the present invention is not limited to these embodiments.
The microorganism used in following examples is all the conventional bacterial classification that the public can obtain.Obviously, those skilled in the art also can use other candida strain to verify technical scheme of the present invention.Described technical scheme, if not otherwise specified, is conventional techniques, and agents useful for same is this area common agents.
Embodiment 1: the seed selection of feed Bacillus subtilis strain.
1. fetch from Wuhan Amino Acid Factory, hair Acid hydrolysis produces the waste liquid after halfcystine, as the use of high saliferous amino acid wastewater required below.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 |
Learn from upper table, 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 high saliferous amino acid wastewater concentration is respectively 125g/L, 250g/L, 330g/L(high saliferous amino acid wastewater of the present invention is in mass) liquid nutrient medium of different gradient, wherein amino acid wastewater is only nitrogen source, described liquid nutrient medium also adds other chemical matrix needed for 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 change into solid medium after partially liq substratum being added interpolation 20g/L agar (calculating by 1000ml water).
3. after by feed, with subtilis, access YEPD substratum activates respectively according to a conventional method, the concentration be inoculated into successively containing amino acid wastewater is respectively 125g/L, 250g/L, the amino acid wastewater solid medium of 330g/L carries out continuous passage cultivation (culture temperature is 28 DEG C), reach the object that domestication is cultivated.Cultivated by domestication, bacterial strain can be the cultured on solid medium of 330g/L in high saliferous amino acid wastewater concentration.
Described subtilis is bought in Chinese industrial Microbiological Culture Collection administrative center: bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum) CICC20037.
Embodiment 2:
Subtilis take glucose as carbon source, fermentation in the amino acid wastewater liquid nutrient medium that is nitrogenous source.
1. preparing high saliferous amino acid wastewater concentration is the liquid fermentation medium of 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 chemical matrix needed for 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. be seeded in the liquid fermentation medium that step 1 prepares by 2% inoculum size by the bacillus subtilis strain of having tamed obtained from embodiment 1, at 31 DEG C in fermentor tank, rotating speed is 220rpm/min, and ventilation coefficient is liquid fermentation and culture 2d under the condition of 0.5.Cultivation terminates the centrifugal 10min of rear 6000r/min and collects thalline, is placed in 70 DEG C of thermostatic drying chambers, dry until weigh after constant weight.Found that, after converting, adopt the fermentation that above-mentioned substratum carries out, often liter of waste water stoste output 35g yeast thalline.
Embodiment 3:
Subtilis be carbon source with W-Gum enzymolysis solution, fermentation in the amino acid wastewater liquid nutrient medium that is nitrogenous source.
1. preparing high saliferous amino acid wastewater concentration is the liquid fermentation medium of 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 chemical matrix needed for 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. be seeded in the liquid fermentation medium that step 1 prepares by 5% inoculum size by the bacillus subtilis strain of having tamed obtained from embodiment 1, at 31 DEG C in fermentor tank, rotating speed is 220rpm/min, and ventilation coefficient is liquid fermentation and culture 2d under the condition of 0.5.Cultivation terminates the centrifugal 10min of rear 6000r/min and collects thalline respectively, is placed in 70 DEG C of thermostatic drying chambers, dry until weigh after constant weight.Found that, after converting, adopt the fermentation that above-mentioned substratum carries out, often liter of waste water stoste output 32g yeast thalline.
Described W-Gum enzymolysis solution, the method for enzymatic hydrolysis is see Liu Yawei. production of corn starch and transformation technology [M]. and Beijing: Chemical Industry Press, 2003:296.
Although made detailed description to the present invention and quoted some specific embodiments as proof, to those skilled in the art, only otherwise it is obvious for leaving that the spirit and scope of the present invention can make various changes or revise.
Claims (4)
1. utilize a high salt amino acid wastewater fermentation production of fodder method for subtilis, the steps include:
(1) continuous domestication of feed bacterial strain is cultivated, specific as follows: first preparation is containing the liquid nutrient medium of the high saliferous amino acid wastewater of different concns gradient, adds 1.5%-2.5% mass volume ratio agar and makes solid medium; After being activated according to a conventional method with subtilis by feed, the solid medium being inoculated into above-mentioned different concns gradient successively carries out continuous passage cultivation, and culture temperature is 15-35 DEG C, progressively improves the salt resistance ability of bacterial classification; Through bimestrial cultured continuously, final bacterial strain can account in the above-mentioned substratum of substratum total mass 28-38% at high saliferous amino acid wastewater and grow;
(2) ferment: the bacillus subtilis strain of step (1) being tamed is seeded to in the high saliferous amino acid wastewater fermention medium that is nitrogenous source by inoculum size 0.5-5%, at 15-35 DEG C, rotating speed is in fermentor tank, carry out liquid fermenting under 100-300rpm condition, ventilation coefficient is 0.2-0.8, be placed in 70 DEG C of thermostatic drying chambers after the centrifugal 10min of 4000r/min collects thalline after fermentation 2d, weigh after being dried to constant weight;
Described high saliferous amino acid wastewater produces the waste water produced in amino acid whose enterprise industrial production from hair Acid hydrolysis, total nitrogen 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 Microbiological Culture Collection 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 125g/L, 250g/L, 330g/L, other chemical matrix components are: carbon source: 30-90g/L, potassium primary phosphate: 3-15g/L, yeast powder: 0-10g/L, magnesium sulfate: 0.3-9g/L, calcium carbonate: 0-2g/L, ferrous sulfate: 0-4g/L; Medium pH is 7-8; Wherein, carbon source is the mixture of one or more following carbohydrates: glucose; Sucrose; Molasses; W-Gum enzymolysis solution; Sweet potato starch enzymolysis solution or tapioca (flour) enzymolysis solution;
Described high saliferous amino acid wastewater is the fermention medium of nitrogenous source, its formula is: high saliferous amino acid sewage 125g/L-330g/L, carbon source: 30-90g/L, potassium primary phosphate: 3-15g/L, yeast powder: 0-10g/L, magnesium sulfate: 0.3-9g/L, calcium carbonate: 0-2g/L, ferrous sulfate: 0-4g/L, medium pH is 6-8; Wherein, carbon source is the mixture of one or more following 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 DEG C, 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 DEG C, and incubation time is 2d.
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| CN107227288B (en) * | 2017-06-30 | 2020-11-24 | 齐齐哈尔龙江阜丰生物科技有限公司 | Recycling method of amino acid production wastewater |
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| CN108034616B (en) * | 2018-01-19 | 2021-03-02 | 中国农业大学 | Fermentation method of bacillus |
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| CN109234322A (en) * | 2018-09-25 | 2019-01-18 | 广西中粮生物质能源有限公司 | A kind of method that high-salt wastewater is used for Rice Powder alcohol prepared by fermenting |
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