阅读说明：本技术 一种耦合废水净化产藻红蛋白和紫球藻多糖的方法 (Method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification ) 是由 黄建科 于 2021-08-13 设计创作，主要内容包括：本发明涉及一种海洋微藻培养与农业废水净化相耦合产藻红蛋白和紫球藻多糖的方法,其要点包括如下步骤：步骤一、采用微藻兼养培养模式培养紫球藻藻种；步骤二、向畜禽农业废水中接入紫球藻藻种液进行微藻光自养培养；步骤三、待培养结束后,采收获得紫球藻,并将培养后的清液回用或排放；步骤四、通过分离提取方法获得紫球藻藻细胞中的藻红蛋白以及紫球藻多糖。本发明通过紫球藻快速利用畜禽养殖等农业废水中的有机物、氮及磷等营养物质进行生长与繁殖,达到去除农业废水中的COD、BOD并实现脱氮除磷的作用,实现废水高效净化处理,同时通过紫球藻提取高附加值微藻生物质藻红蛋白和紫球藻多糖,具有较高的应用价值。(The invention relates to a method for producing phycoerythrin and porphyridium polysaccharide by coupling marine microalgae culture and agricultural wastewater purification, which is characterized by comprising the following steps of: step one, cultivating porphyridium algae seeds by adopting a microalgae mixotrophic cultivation mode; step two, inoculating porphyridium algae liquid into the livestock and poultry agricultural wastewater to perform microalgae photoautotrophic culture; step three, after the culture is finished, collecting and obtaining porphyridium, and recycling or discharging the cultured clear liquid; and step four, obtaining phycoerythrin and porphyridium polysaccharide in the porphyridium cell by a separation and extraction method. According to the invention, porphyridium is used for rapidly utilizing organic matters, nitrogen, phosphorus and other nutrient substances in agricultural wastewater of livestock and poultry breeding and the like to grow and reproduce, so that COD and BOD in the agricultural wastewater are removed, nitrogen and phosphorus removal are realized, efficient purification treatment of the wastewater is realized, and meanwhile, high-added-value microalgae biomass phycoerythrin and porphyridium polysaccharide are extracted through the porphyridium, so that the high-added-value microalgae biomass phycoerythrin and porphyridium polysaccharide have high application value.)

1. A method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification is characterized by comprising the following steps:

step one, cultivating porphyridium algae seeds by adopting a microalgae mixotrophic cultivation mode;

step two, inoculating porphyridium algae liquid into the livestock and poultry breeding wastewater to perform microalgae photoautotrophic culture;

step three, after the culture is finished, collecting and obtaining porphyridium, and recycling or discharging the cultured clear liquid;

and step four, obtaining phycoerythrin and porphyridium polysaccharide in the porphyridium cell by a separation and extraction method.

2. The method for producing phycoerythrin and porphyridium polysaccharide through coupled wastewater purification according to claim 1, wherein in the first step, the mixotrophic culture mode employs glycerol as a carbon source and nitrate as a nitrogen source, the concentration of the carbon source is 5-10 g/L, and the concentration of the nitrate is 1-3 g/L.

3. The method for producing phycoerythrin and porphyridium polysaccharide through coupling wastewater purification according to claim 1, wherein in the second step, the livestock and poultry breeding wastewater is diluted to a COD concentration of 100-500 mg/L, a total nitrogen concentration of 10-60 mg/L and a total phosphorus concentration of 2-20 mg/L, and the dilution method is to use seawater with a salinity of 15-30 ‰.

4. The method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification according to claim 1, wherein the temperature for microalgae cultivation is 20-25 ℃ and the cultivation period is 15-25 days.

5. The method for producing phycoerythrin and porphyridium polysaccharide through coupling wastewater purification according to claim 1, wherein the pH of the livestock and poultry breeding wastewater is adjusted to 6-8.

6. The method for producing phycoerythrin and porphyridium polysaccharide by coupled wastewater purification according to claim 1, wherein the inoculation amount of the porphyridium strain liquid is 20% of the volume of the diluted livestock and poultry breeding wastewater.

7. The method for producing phycoerythrin and porphyridium polysaccharide through coupled wastewater purification according to claim 1, wherein before the second step, the livestock and poultry breeding wastewater is physically settled and membrane-filtered to remove particle suspended matters and impurities, and the livestock and poultry breeding wastewater is subjected to disinfection and sterilization treatment, wherein the disinfection and sterilization is performed by any one or a combination of two or more of high-temperature steam sterilization, sodium hypochlorite method, ultraviolet disinfection method and microfiltration membrane filtration method.

8. The method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification according to claim 1, wherein the porphyridium is cultured under illumination conditions, and when the porphyridium is cultured under the illumination conditions, the illumination intensity is 3000-5000 lux; the device adopted by the culture is a shake flask, a fermentation tank or a sterilizable photobioreactor; when the culture device is a shake flask, the rotating speed of the shaking table is controlled between 100rpm and 200 rpm; when the culture device is a fermentation tank or a sterilizable photobioreactor, stirring and ventilation are started, and the dissolved oxygen is controlled to be not less than 10%.

9. The method for producing phycoerythrin and porphyridium polysaccharide by coupled wastewater purification according to claim 1, wherein porphyridium is obtained by any one of centrifugation, air flotation and flocculation in the third step.

10. The method for producing phycoerythrin and porphyridium polysaccharide by coupled wastewater purification according to claim 1, wherein in the fourth step, phycoerythrin and porphyridium polysaccharide are extracted from porphyridium by ultrasonic crushing extraction and ethanol extraction, respectively.

Technical Field

The invention relates to the field of agricultural wastewater treatment and algae culture, and discloses a method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification.

Background

At present, agricultural wastewater is rich in nitrogen, phosphorus, organic matters and the like, is discharged to a peripheral water area without being effectively treated, and is easy to destroy the ecological balance of the water environment of the peripheral water area, so that the water eutrophication is caused. Accumulation of nutrient salts such as nitrogen, phosphorus and the like constitute inorganic pollutants in agricultural wastewater, which lower the oxygen concentration of aquatic organisms and trigger the enrichment of algae. For soluble nutritive salt, physical or chemical treatment methods such as precipitation, filtration and the like are mostly adopted at present, but the effect is not ideal, the cost is high, secondary pollution is easy to cause, the biological treatment method is relatively economic and effective, and the microalgae is widely used for bioremediation of wastewater. Microalgae have a high rate of photosynthetic activity and, if combined with bioproduct production, may become an economic route to sustainable energy production in the future, and from an economic and environmental point of view, the cultivation of microalgae in wastewater is considered to be an ecologically friendly, more suitable method than bacterial-based treatments.

The unicellular or simple multicellular photoautotrophic microorganism of the microalgae taking nitrogen and phosphorus as main nutrient sources has the characteristics of high light energy utilization efficiency, high propagation speed, strong environmental adaptability, short growth period and the like, is rich in various nutrient components such as protein, carotenoid, unsaturated fatty acid, vitamin, mineral elements and the like, and is widely applied to various fields such as biological medicine, nutrient functional food, food and food additives, feed and animal health care, aquaculture and the like. Meanwhile, the microalgae can rapidly utilize organic matters, nitrogen, phosphorus and other nutrient substances to grow and propagate, so that the microalgae can be used for removing COD and BOD in wastewater and realizing nitrogen and phosphorus removal. A microalgae-based wastewater treatment technology is a hotspot of current domestic and foreign researches, and the technology can realize the purification of wastewater and synchronously obtain high-added-value microalgae biomass which is directly used as feed, bait and fertilizer; or separating and extracting natural active substances in microalgae, such as porphyridium polysaccharide, phycoerythrin, unsaturated fatty acid, etc., to obtain high value-added product.

Meanwhile, phycoerythrin is a food pigment with excellent water solubility, has the advantages of high fluorescence intensity, oxidation resistance, free radical removal, high chroma and the like, has wide application in the industries of food, cosmetics, medicines and the like, has excellent physical properties, is a high-fluorescence substance, has high light stability, has small influence of pH value on the fluorescence quantum yield, has larger Stokes shift, and can reduce the interference of Rayleigh scattering and Raman scattering to the maximum extent, thereby having excellent performance on molecular detection. The fluorescence intensity of the fluorescent dye is dozens of times stronger than that of the traditional chemistry, and the fluorescent dye can be used as a fluorescent marker to be applied to the fields of molecular biology, clinical medicine and the like; the porphyridium polysaccharide is usually sulfate polysaccharide, mainly consists of monosaccharides such as xylose, glucose and galactose, generates amylopectin type and amylopectin type a-polyglucose, has the function of preventing virus adsorption, and has special efficacy on treating tumors. In addition, the porphyridium polysaccharide also has the functions of preventing and treating cardiovascular diseases, reducing blood fat, treating diseases of liver, gallbladder, pancreas and the like, and is widely applied to the health product industry, so the porphyridium polysaccharide is a metabolite with wide application range and obvious functional effect.

Accordingly, the prior art does not disclose a method or a technology for applying a wastewater treatment technology based on marine microalgae to the treatment of agricultural wastewater, improving the agricultural wastewater treatment efficiency, reducing the treatment difficulty and realizing high resource utilization of the agricultural wastewater, aiming at the problems of difficult agricultural wastewater treatment, low resource utilization and the like.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide a method for producing phycoerythrin and porphyridium polysaccharide by coupling wastewater purification in the field, so that the technical problems of complex treatment process, high cost, low resource recovery and utilization rate and low additional output value of the existing agricultural wastewater are solved. The purpose is realized by the following technical scheme.

A method for producing phycoerythrin and porphyridium polysaccharide by coupling marine microalgae culture and agricultural wastewater purification is characterized by comprising the following steps: step one, cultivating porphyridium algae seeds by adopting a microalgae mixotrophic cultivation mode; step two, inoculating porphyridium algae liquid into the livestock and poultry agricultural wastewater to perform microalgae photoautotrophic culture; step three, after the culture is finished, collecting and obtaining porphyridium, and recycling or discharging the cultured clear liquid; and step four, obtaining phycoerythrin and porphyridium polysaccharide in the porphyridium cell by a separation and extraction method.

In the first step, the mixotrophic culture mode adopts glycerol as a carbon source and nitrate as a nitrogen source, the concentration of the carbon source is 5-10 g/L, and the concentration of the nitrate is 1-3 g/L.

In the second step, the livestock and poultry breeding wastewater is diluted to a COD concentration of 100-500 mg/L, a total nitrogen concentration of 10-60 mg/L and a total phosphorus concentration of 2-20 mg/L, and the dilution method adopts seawater with salinity of 15-30 per mill.

The temperature for culturing the microalgae is 20-25 ℃, and the culture period is 15-25 days.

And adjusting the pH value of the livestock and poultry breeding wastewater to 6-8.

The inoculation amount of the porphyridium liquid is 20 percent of the volume of the diluted livestock and poultry breeding wastewater.

Before the second step, removing particle suspended matters and impurities from the agricultural wastewater after physical sedimentation and membrane filtration, and performing disinfection and sterilization treatment on the livestock and poultry breeding wastewater, wherein the disinfection and sterilization is performed by any one or a combination of more than two of high-temperature steam sterilization, a sodium hypochlorite method, an ultraviolet disinfection method and a microfiltration membrane filtration method.

The porphyridium is cultured under the illumination condition, and when the porphyridium is cultured under the illumination condition, the illumination intensity is 3000-5000 lux; the device adopted by the culture is a shake flask, a fermentation tank or a sterilizable photobioreactor, and when the culture device is the shake flask, the rotating speed of a shaking table is controlled to be 100-200 rpm; when the culture device is a fermentation tank or a sterilizable photobioreactor, stirring and ventilation are started, and the dissolved oxygen is controlled to be not less than 10%.

In the third step, porphyridium is obtained by any one of a centrifugal method, an air floatation method and a flocculation method.

In the fourth step, phycoerythrin and porphyridium polysaccharide are respectively extracted by an ultrasonic crushing extraction method and an ethanol extraction method.

According to the invention, porphyridium is used for rapidly utilizing organic matters, nitrogen, phosphorus and other nutrient substances in agricultural wastewater of livestock and poultry breeding and the like to grow and reproduce, so that COD and BOD in the agricultural wastewater are removed, nitrogen and phosphorus removal effects are realized, high-efficiency purification treatment of the wastewater is realized, and meanwhile, high-added-value microalgae biomass phycoerythrin and porphyridium polysaccharide are extracted through the porphyridium, and the porphyridium polysaccharide has a plurality of remarkable effects, such as oxidation resistance, cancer resistance, cardiovascular disease treatment and the like, so that the porphyridium polysaccharide is quite wide in application and has higher application value; phycoerythrin has the advantages of high fluorescence intensity, oxidation resistance, free radical removal, high chroma and the like. In addition, the porphyridium culture solution in the wastewater treatment realizes low-cost and high-agricultural wastewater treatment efficiency through repeated recycling, and greatly improves the resource utilization rate. It is suitable for being used as the prior agricultural wastewater treatment method or the improvement of the similar wastewater treatment method.

Drawings

FIG. 1 is a graph showing the change of the dry cell weight, COD, total nitrogen and total phosphorus concentrations of the algal cells used in the culture of Porphyridium according to example 1 of the present invention.

FIG. 2 is a graph showing the variation of phycoerythrin and porphyridium polysaccharide concentrations in the culture of porphyridium according to example 1 of the present invention.

FIG. 3 is a graph showing the change of the dry weight, COD, total nitrogen and total phosphorus concentrations of algal cells used in the culture of Porphyridium according to example 2 of the present invention.

FIG. 4 is a graph showing the variation of phycoerythrin and porphyridium polysaccharide concentrations in the culture of porphyridium in example 2 of the present invention.

Detailed Description

The invention will now be further described with reference to the accompanying drawings.

Example 1: firstly, a microalgae mixotrophic culture mode is adopted to culture porphyridium algae seeds, and the mixotrophic culture mode adopts glycerol as a carbon source and nitrate as a nitrogen source, wherein the concentration of the carbon source is 10g/L, and the concentration of the nitrate is 2 g/L. Standing and settling the livestock and poultry breeding wastewater for 3 hours, then filtering the livestock and poultry breeding wastewater by using a microfiltration membrane, performing ultraviolet disinfection, diluting the livestock and poultry breeding wastewater by 1 time with seawater with the salinity of 30 per mill to ensure that the COD concentration in the livestock and poultry breeding wastewater is 500mg/L, the total nitrogen is 55mg/L, the total phosphorus is 12mg/L, and the pH is adjusted to 6 ℃8. 1000mL of this liquid was charged into a 1.2L-sized photobioreactor, and then sterilized at 115 ℃ for 25min at high temperature. Cooling, inoculating 200mL of porphyridium seed liquid with initial inoculation density of 0.67g/L, introducing 2% CO₂The culture temperature was controlled at 25 ℃.

In the above process, the change process of the algal cell concentration, COD, total nitrogen and total phosphorus under the dry weight condition is measured, as can be seen from figure 1, the algal cells grow rapidly after inoculation, and the concentration of the porphyridium reaches 9g/L after culture to the 24 th day; COD of the wastewater is reduced from 500mg/L to 30.23mg/L, and total nitrogen and total phosphorus are reduced from 55mg/L and 12mg/L to 4.23mg/L and 0.47mg/L respectively. Meanwhile, as can be seen from FIG. 2, the phycoerythrin content and the porphyridium polysaccharide content of the porphyridium culture solution rapidly increased from 4mg/L and 0.17/L to 55.25mg/L and 1.91g/L, respectively. Therefore, the porphyridium can quickly remove COD, total nitrogen and total phosphorus in the wastewater, realize the purification of the wastewater and simultaneously can produce natural products phycoerythrin and porphyridium polysaccharide with high added values.

Example 2: firstly, a microalgae mixotrophic culture mode is adopted to culture porphyridium algae seeds, and the mixotrophic culture mode adopts glycerol as a carbon source and nitrate as a nitrogen source, wherein the concentration of the carbon source is 10g/L, and the concentration of the nitrate is 2.5 g/L. The livestock and poultry breeding wastewater is statically settled for 5 hours, then is filtered by a microfiltration membrane and is subjected to ultraviolet disinfection, and is diluted by 4 times with seawater with salinity of 25 per mill, so that the COD concentration in the agricultural wastewater is 170mg/L, the total nitrogen is 18mg/L, and the total phosphorus is 4 mg/L. 200mL of porphyridium algae seeds are inoculated into a 1.2L photobioreactor (liquid loading volume is 1000 mL), the initial inoculation density is 0.67g/L, and 2% of CO is introduced₂The culture temperature was controlled at 25 ℃.

In the above process, the change process of the algal cell concentration, COD, total nitrogen and total phosphorus under the dry weight condition is measured, as can be seen from FIG. 3, the algal cells grow rapidly after inoculation, and the concentration of the porphyridium reaches 7.63g/L after culture to the 24 th day; COD of the wastewater was reduced from 170mg/L to 7.9mg/L, and total nitrogen and total phosphorus were reduced from 18mg/L and 4mg/L to 1.91mg/L and 0.32mg/L, respectively. Meanwhile, as can be seen from FIG. 4, the phycoerythrin and porphyridium polysaccharide concentrations of the porphyridium culture solution rapidly increased from 4mg/L and 0.17g/L to 45.45mg/L and 1.71g/L, respectively. Therefore, the porphyridium can quickly remove COD, total nitrogen and total phosphorus in the wastewater, realize the purification of the wastewater and simultaneously can produce natural products phycoerythrin and porphyridium polysaccharide with high added values.

In conclusion, the invention aims at the problems of difficult agricultural wastewater treatment, low resource utilization and the like, applies the wastewater treatment technology based on the marine microalgae to the treatment of the agricultural wastewater, greatly improves the agricultural wastewater treatment efficiency, reduces the treatment difficulty and realizes the high resource utilization of the agricultural wastewater. Meanwhile, the livestock and poultry wastewater is used for culturing microalgae porphyridium to realize wastewater purification and obtain natural active substances with high added value, namely phycoerythrin and porphyridium polysaccharide; and the existing agricultural wastewater treatment method is improved, and porphyridium is adopted to purify the agricultural wastewater and produce phycoerythrin and porphyridium polysaccharide.

The above description is intended to illustrate the technical means of the present invention, and not to limit the technical scope of the present invention. Obvious modifications or alterations to the present invention in combination with the prior art knowledge will be apparent to those skilled in the art and fall within the scope of the appended claims.