阅读说明：本技术 一种硫酸新霉素清洁生产发酵方法 (Clean production and fermentation method of neomycin sulfate ) 是由 刘铭 江兵 曾勇 谭小芳 胡珂 方钰霏 于 2020-12-08 设计创作，主要内容包括：本发明涉及一种硫酸新霉素清洁生产发酵方法,硫酸新霉素清洁生产培养基包括发酵基础培养基和补料氮源培养基；其中,发酵基础培养基包括氯化铵0.2%-0.73%(w/v),补料氮源培养基包括28-35％(w/v)的氯化铵。本发明在不降低硫酸新霉素产量及增加杂质的情况下,降低生产废水中的硫酸根离子的量,减轻其在后续废水处理过程中对活性污泥的毒害影响。该发明通过使用氯化铵替换发酵基础培养基以及补料中的硫酸氨,并控制发酵过程中的pH来进行发酵。发酵废水进行稀释后泵入装有污泥的厌氧发生器。最终发酵效价和发酵组分与原硫酸铵配方相当,厌氧活性污泥处理污水能力略有优势。该发明对硫酸新霉素发酵清洁生产具有重要意义。(The invention relates to a neomycin sulfate clean production fermentation method, wherein a neomycin sulfate clean production culture medium comprises a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the fermentation basal culture medium comprises 0.2-0.73% (w/v) of ammonium chloride, and the supplemented nitrogen source culture medium comprises 28-35% (w/v) of ammonium chloride. The method reduces the amount of sulfate ions in the production wastewater and lightens the toxic effect of the sulfate ions on activated sludge in the subsequent wastewater treatment process under the conditions of not reducing the yield of neomycin sulfate and increasing impurities. The invention carries out fermentation by replacing ammonium sulfate in a fermentation basal medium and a supplementary material by ammonium chloride and controlling the pH value in the fermentation process. The fermented waste water is diluted and pumped into an anaerobic generator filled with sludge. The final fermentation titer and the fermentation components are equivalent to the original ammonium sulfate formula, and the sewage treatment capability of the anaerobic activated sludge is slightly superior. The invention has important significance for the fermentation and clean production of the neomycin sulfate.)

1. A neomycin sulfate clean production culture medium is characterized by comprising a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the fermentation basal culture medium comprises 0.2-0.73% (w/v) of ammonium chloride, and the supplemented nitrogen source culture medium comprises 28-35% (w/v) of ammonium chloride.

2. The neomycin sulfate clean production medium of claim 1, wherein said fermentation basal medium further comprises: 1-7.0% (w/v) of soybean cake powder, 5.0-9.0% (w/v) of rice flour, 0.1-0.6% (w/v) of corn steep liquor, 0.50-3.0% (w/v) of oral sugar, 0.3-0.6% (w/v) of NaCl0.3-0.6% (w/v) of light calcium carbonate, 0.3-0.6% (w/v) of KH₂PO₄0.01~0.05%（w/v），Na₂HPO₄0.02-0.10% (w/v), 0.02-0.05% (w/v) of alpha-amylase, and 0.0001-0.3% (w/v) of silicone oil.

3. A method for fermentation of neomycin sulfate clean production using the neomycin sulfate clean production medium of any one of claims 1-2, comprising the steps of:

(1) inoculating sand soil spores to a slant to prepare a slant spore suspension;

(2) inoculating the slant spore suspension into a seed tank filled with a seed culture medium, and performing fermentation culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank filled with a fermentation basal culture medium, and fermenting by supplementing a supplemented culture medium and ammonium chloride in the fermentation process to obtain a fermentation liquid;

(4) putting the fermentation liquor into an adsorption tank, adding resin to adsorb neomycin, filtering, and collecting wastewater;

(5) collecting the filtered wastewater after adsorption, and treating the filtered wastewater by using activated sludge;

and finishing the clean production of the neomycin sulfate.

4. The method according to claim 3, wherein the slant spore suspension is obtained by culturing in step (1) at 26-29 ℃ for 4-8 days; the fermentation culture time in the step (2) is 35-40 h.

5. The method according to claim 3, wherein the pH is adjusted to 6.2-6.4 by using sodium hydroxide solution during the fermentation in the step (3); the fermentation time is 150-180 h.

6. The method according to claim 3, wherein in the step (3), the ammonia nitrogen concentration of the fermentation broth is controlled to be 1.4-25g/100ml by feeding ammonium chloride.

7. The method of claim 3, wherein in the step (4), the saturated resin is washed with a detergent, low-concentration ammonia water and high-concentration ammonia water in sequence, and decolorized to prepare the product.

8. The method of claim 7, wherein the detergent comprises 0.01-0.25 mol/L hydrochloric acid and 0.01-0.1mol/L ammonium chloride; the saturated resin is washed sequentially with low-concentration ammonia water with the concentration of 0.04-0.4mol/L and high-concentration ammonia water with the concentration of 2.0-3.5 mol/L.

9. The method of claim 3, wherein: and 5) diluting the COD concentration of the wastewater to 4000mg/L by using tap water, wherein the sludge loading is 0.8-1.2L, the flow rate is 8.5-8.9L/d, and treating by using activated sludge.

Technical Field

The invention relates to the technical field of neomycin sulfate, in particular to a neomycin sulfate clean production fermentation method.

Background

Neomycin sulfate fermentationSulfate ions with higher concentration exist in the wastewater, and the sulfate is reduced by microorganisms in the anaerobic activated sludge to produce S^2-It has the functions of inhibiting and poisoning microbe. The main sources of sulfate ions are the medium base material in the fermentation process and ammonium sulfate in the feed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a fermentation method for clean production of neomycin sulfate, ammonium chloride is used for replacing nitrogen source ammonium sulfate, the emission of sulfate ions is reduced from a fermentation source, and S is relieved^2-Toxic effects on activated sludge. Meanwhile, the fermentation yield and the impurity ratio of the neomycin sulfate are not increased.

The technical scheme adopted by the invention is that,

a neomycin sulfate clean production culture medium comprises a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the fermentation basal culture medium comprises 0.2-0.73% (w/v) of ammonium chloride, and the supplemented nitrogen source culture medium comprises 28-35% (w/v) of ammonium chloride.

Preferably, the fermentation basal medium further comprises: 1-7.0% (w/v) of soybean cake powder, 5.0-9.0% (w/v) of rice flour, 0.1-0.6% (w/v) of corn steep liquor, 0.50-3.0% (w/v) of oral sugar, 0.3-0.6% (w/v) of NaCl0.3-0.6% (w/v) of light calcium carbonate, 0.3-0.6% (w/v) of KH₂PO₄0.01～0.05％(w/v)，Na₂HPO₄0.02-0.10% (w/v), 0.02-0.05% (w/v) of alpha-amylase, and 0.0001-0.3% (w/v) of silicone oil.

The method for performing neomycin sulfate clean production fermentation by using the neomycin sulfate clean production culture medium comprises the following steps:

(1) inoculating sand soil spores to a slant to prepare a slant spore suspension;

(2) inoculating the slant spore suspension into a seed tank filled with a seed culture medium, and performing fermentation culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank filled with a fermentation basal culture medium, and fermenting by supplementing a supplemented culture medium and ammonium chloride in the fermentation process to obtain a fermentation liquid;

(4) putting the fermentation liquor into an adsorption tank, adding resin to adsorb neomycin, filtering, and collecting wastewater;

(5) collecting the filtered wastewater after adsorption, and treating the filtered wastewater by using activated sludge;

and finishing the clean production of the neomycin sulfate.

Preferably, the bevel spore suspension is obtained by culturing at 26-29 ℃ for 4-8 days in the step (1); the fermentation culture time in the step (2) is 35-40 h.

Preferably, sodium hydroxide solution is used for adjusting the pH value to 6.2-6.4 during the fermentation process in the step (3); the fermentation time is 150-180 h.

Preferably, in the step (3), the ammonia nitrogen concentration of the fermentation liquor is controlled to be 1.4-25g/100ml by feeding ammonium chloride.

Preferably, in the step (4), the saturated resin is washed by using a detergent, low-concentration ammonia water and high-concentration ammonia water in sequence, and the saturated resin is decolored to prepare the product.

More preferably, the detergent contains 0.01-0.25 mol/L hydrochloric acid and 0.01-0.1mol/L ammonium chloride; the saturated resin is washed sequentially with low-concentration ammonia water with the concentration of 0.04-0.4mol/L and high-concentration ammonia water with the concentration of 2.0-3.5 mol/L.

Further preferably, in the step 5), tap water is used for diluting the COD concentration of the wastewater to 4000mg/L, the sludge loading is 0.8-1.2L, the flow rate is 8.5-8.9L/d, the wastewater is treated by activated sludge, the wastewater is replaced once every 2-4 days, the diluted COD is measured before water enters, the treated effluent wastewater is collected every day from the fourth day after sample injection, the COD is measured, and the removal rate of the COD is calculated. Volatile acidic fatty acids (VFAs) of the effluent wastewater were measured.

The invention has the following effects:

1. ammonium chloride is used for replacing nitrogen source ammonium sulfate, so that the emission of sulfate ions is reduced from a fermentation source, and S is relieved^2-Toxic effects on activated sludge. Meanwhile, the fermentation yield and the impurity ratio of the neomycin sulfate are not increased.

2. Because the toxic action borne by the activated sludge is reduced, the replacement frequency of the sludge can be further reduced, and the fermentation cost is saved to a certain extent.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples. These examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1

A neomycin sulfate clean production culture medium comprises a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the nitrogen source feeding medium comprises 28% (w/v) ammonium chloride.

In the fermentation basal medium: soybean cake powder 4.0% (w/v), rice flour 6.0% (w/v), corn steep liquor 0.5% (w/v), oral sugar 1.8% (w/v), ammonium chloride 0.53% (w/v), NaCl0.5% (w/v), light calcium carbonate 0.4% (w/v), KH₂PO₄0.04％(w/v)，Na₂HPO₄0.08% (w/v), 0.04% (w/v) of alpha-amylase and 0.015% (w/v) of silicone oil.

Preferably, the method comprises the steps of:

(1) inoculating sand soil spores to a slant to prepare a slant spore suspension;

(2) inoculating the slant spore suspension into a seed tank filled with a seed culture medium, and performing fermentation culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank filled with a fermentation basal culture medium, and fermenting by supplementing a supplemented culture medium and ammonium chloride in the fermentation process to obtain a fermentation liquid;

(4) putting the fermentation liquor into an adsorption tank, adding resin to adsorb neomycin, filtering, and collecting wastewater;

(5) collecting the filtered wastewater after adsorption, and treating the filtered wastewater by using activated sludge;

and finishing the clean production of the neomycin sulfate.

Further preferably, the slant spore suspension is obtained by culturing at 26 ℃ for 4 days in the step (1); the time of fermentation culture in the step (2) is 35 h.

Further preferably, the pH is adjusted to 6.2 by using sodium hydroxide solution during the fermentation in the step (3); the fermentation time was 150 h. Resulting in excessive pH reduction of the fermentation liquor and abnormal metabolism of the streptomyces fradiae.

Further preferably, in the step (3), the ammonia nitrogen concentration of the fermentation liquor is controlled to be 1.4g/100ml by feeding ammonium chloride.

Further preferably, in the step (4), the saturated resin is washed with a detergent, low-concentration ammonia water and high-concentration ammonia water in sequence, and the product is decolorized to prepare the product.

Still more preferably, the detergent comprises 0.01mol/L hydrochloric acid and 0.01mol/L ammonium chloride; the saturated resin was washed with a low-concentration ammonia water concentration of 0.04mol/L and a high-concentration ammonia water concentration of 2.0mol/L in this order.

Further preferably, in the step 5), tap water is used for diluting the COD concentration of the wastewater to 4000mg/L, the sludge loading is 0.8L, the flow rate is 8.5L/d, activated sludge is used for treatment, the wastewater is replaced once every 2 days, diluted COD is measured before water enters, treated effluent wastewater is collected every day from the fourth day after sample injection, COD is measured, and the removal rate of the COD is calculated. Volatile acidic fatty acids (VFAs) of the effluent wastewater were measured.

Example 2

A neomycin sulfate clean production culture medium comprises a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the nitrogen source feeding medium comprises 35% (w/v) ammonium chloride.

In the fermentation basal medium: soybean cake powder 7.0% (w/v), rice flour 9.0% (w/v), corn steep liquor 0.6% (w/v), oral sugar 3.0% (w/v), ammonium chloride 0.73% (w/v), NaCl0.6% (w/v), light calcium carbonate 0.6% (w/v), KH₂PO₄0.05％(w/v)，Na₂HPO₄0.10% (w/v), alpha-amylase 0.05% (w/v), silicone oil 0.3% (w/v).

Preferably, the method comprises the steps of:

(1) inoculating sand soil spores to a slant to prepare a slant spore suspension;

(2) inoculating the slant spore suspension into a seed tank filled with a seed culture medium, and performing fermentation culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank filled with a fermentation basal culture medium, and fermenting by supplementing a supplemented culture medium and ammonium chloride in the fermentation process to obtain a fermentation liquid;

(4) putting the fermentation liquor into an adsorption tank, adding resin to adsorb neomycin, filtering, and collecting wastewater;

(5) collecting the filtered wastewater after adsorption, and treating the filtered wastewater by using activated sludge;

and finishing the clean production of the neomycin sulfate.

Further preferably, the bevel spore suspension is obtained by culturing at 26-29 ℃ for 4-8 days in the step (1); the fermentation culture time in the step (2) is 35-40 h.

Further preferably, the pH is adjusted to 6.4 by using sodium hydroxide solution during the fermentation in the step (3); the fermentation time is 180 h. Resulting in excessive pH reduction of the fermentation liquor and abnormal metabolism of the streptomyces fradiae.

Further preferably, in the step (3), the ammonia nitrogen concentration of the fermentation liquor is controlled to be 25g/100ml by feeding ammonium chloride.

Further preferably, in the step (4), the saturated resin is washed with a detergent, low-concentration ammonia water and high-concentration ammonia water in sequence, and the product is decolorized to prepare the product.

Still more preferably, the detergent comprises 0.25mol/L hydrochloric acid and 0.1mol/L ammonium chloride; the saturated resin was washed successively with a low-concentration ammonia water concentration of 0.4mol/L and a high-concentration ammonia water concentration of 3.5 mol/L.

Further preferably, in the step 5), tap water is used for diluting the COD concentration of the wastewater to 4000mg/L, the sludge loading is 1.2L, the flow rate is 8.9L/d, activated sludge is used for treatment, the wastewater is replaced once every 4 days, diluted COD is measured before water enters, treated effluent wastewater is collected every day from the fourth day after sample injection, COD is measured, and the removal rate of the COD is calculated. Volatile acidic fatty acids (VFAs) of the effluent wastewater were measured.

Example 3

A neomycin sulfate clean production culture medium comprises a fermentation basal culture medium and a supplemented nitrogen source culture medium; wherein the nitrogen source feeding medium comprises 32.5% (w/v) ammonium chloride.

In the fermentation basal medium: 7.0% (w/v) of soybean cake powder, 3.6% (w/v) of rice flour, 0.5% (w/v) of corn steep liquor, 2.8% (w/v) of oral sugar, 0.73% (w/v) of ammonium chloride, 0.5% (w/v) of NaCl0.4% (w/v) of light calcium carbonate, KH₂PO₄0.04％(w/v)，Na₂HPO₄0.08% (w/v), 0.04% (w/v) of alpha-amylase and 0.1% (w/v) of silicone oil.

Preferably, the method comprises the steps of:

(1) inoculating sand soil spores to a slant to prepare a slant spore suspension;

(2) inoculating the slant spore suspension into a seed tank filled with a seed culture medium, and performing fermentation culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank filled with a fermentation basal culture medium, and fermenting by supplementing a supplemented culture medium and ammonium chloride in the fermentation process to obtain a fermentation liquid;

(4) putting the fermentation liquor into an adsorption tank, adding resin to adsorb neomycin, filtering, and collecting wastewater;

(5) collecting the filtered wastewater after adsorption, and treating the filtered wastewater by using activated sludge;

and finishing the clean production of the neomycin sulfate.

Further preferably, the slant spore suspension is obtained by culturing at 28 ℃ for 5 days in the step (1); the time of fermentation culture in the step (2) is 38 h.

Further preferably, the pH is adjusted to 6.3 by using sodium hydroxide solution during the fermentation in the step (3); the fermentation time was 160 h. Resulting in excessive pH reduction of the fermentation liquor and abnormal metabolism of the streptomyces fradiae.

Further preferably, in the step (3), the ammonia nitrogen concentration of the fermentation liquor is controlled to be 15g/100ml by feeding ammonium chloride.

Further preferably, in the step (4), the saturated resin is washed with a detergent, low-concentration ammonia water and high-concentration ammonia water in sequence, and the product is decolorized to prepare the product.

Still more preferably, the detergent comprises 0.15mol/L hydrochloric acid and 0.08mol/L ammonium chloride; the saturated resin was washed successively with a low-concentration ammonia water concentration of 0.3mol/L and a high-concentration ammonia water concentration of 2.5 mol/L.

Further preferably, in the step 5), tap water is used for diluting the COD concentration of the wastewater to 4000mg/L, the sludge loading is 1L, the flow rate is 8.6L/d, activated sludge is used for treatment, the wastewater is replaced once every 3 days, diluted COD is measured before water enters, the treated effluent wastewater is collected every day from the fourth day after sample injection, COD is measured, and the removal rate of the COD is calculated. Volatile acidic fatty acids (VFAs) of the effluent wastewater were measured.

Example 3 was compared with comparative examples, which were cultured in the media shown in tables 1 and 2.

TABLE 1 fermentation basal Medium

TABLE 2 Nitrogen source supplemented Medium

1. Compared with the formula of the comparative example, the ratio of fermentation units to main impurities is basically equivalent, and the quality of the neomycin sulfate is basically consistent before and after change, as shown in the following table 3.

TABLE 3 comparison of fermentation quality of inventive and comparative formulations

2. The results of the activated sludge on wastewater treatment are shown in tables 4 and 5, and the COD removal rate of the formula of the invention is reduced slowly compared with that of the formula of the comparative example, and the sludge treatment capacity is slightly stable.

TABLE 4 fermentation waste water of comparative example formulation

Number of days	Sample introduction COD mg/L	Effluent CODmg/L	COD removal rate	VFA mM
					1	12952	-	-	-
4	-	5750	55.6％	9.95
					5	-	3250	74.9％	7.62
6	11150	4050	63.6％	10.58
					7	-	5650	49.3％	21.58
8	-	4825	56.7％	19.26
					9	10550	4797	54.5％	22.85
10	-	5360	49.2％	28.14
					11		4020	61.9％	12.48
12	9940	4000	59.8％	19.68
					13		5620	43.5％	34.70
14		5506	44.6％	37.66
					15	11130	5392	51.6％	38.93
16		5652	49.2％	45.07
					17		6550	41.15％	48.45

TABLE 5 example 3 formulation of fermentation wastewater

The above-described embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention is defined by the claims, and equivalents including technical features described in the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.