CN111518698A

CN111518698A - Method for rapidly denitrifying by using microalgae and application thereof

Info

Publication number: CN111518698A
Application number: CN202010278141.5A
Authority: CN
Inventors: 魏东; 骆小英; 杨润青
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-08-11
Anticipated expiration: 2040-04-10
Also published as: CN111518698B

Abstract

The invention discloses a method for quickly denitrating by using microalgae and application thereof. The method comprises the following steps: s1, culturing the microalgae cells to a logarithmic phase to obtain microalgae seed liquid; s2, inoculating the microalgae seed liquid into a fermentation tank filled with a fermentation medium containing high nitrate nitrogen for fermentation, and when the concentration of glucose in the fermentation medium is lower than 5g/L, supplementing glucose to make the concentration of glucose in the fermentation system higher than 10g/L, and controlling the pH value to be 6.0-7.5; wherein NO is present in the fermentation medium containing high nitrate nitrogen₃ ^‑Is higher than 3000 mg/L. The invention can also recover, wash and freeze-dry the microalgae after fermentation culture to obtain the microalgae biomass containing high protein. The method can synchronously realize rapid denitration, microalgae high-density culture and high-protein biomass production, and reduce the production ofThe discharge of the high nitrate nitrogen wastewater is realized, and the resource recycling of the nitrate nitrogen is realized.

Description

Method for rapidly denitrifying by using microalgae and application thereof

Technical Field

The invention belongs to the technical field of industrial biology, and particularly relates to a method for quickly denitrating by using microalgae and application thereof.

Background

The accelerated development of the human industrialization process brings serious water pollution while improving the life quality of peopleDyeing, in which the waste water from the food, leather, paper and fertilizer industries results in the emission of large amounts of nitrogen-containing compounds, in particular nitrate. A large amount of nitrogen oxides (NOx) also exist in flue gas discharged in the industrial production process, and are subsequently converted into high-concentration nitrogen-containing waste liquid such as nitric acid, nitrate and the like; flue gas denitration towers also produce large amounts of waste nitric acid, which, together with high nitrate wastewater, is referred to as high nitrate nitrogen industrial wastewater. High-nitrate-nitrogen industrial wastewater usually contains more than 200 mg/L of NO₃ ^-N, and industrial waste waters from the production of explosives, fertilizers, pectins, cellophane and metalworking contain more than 1000mg/l of NO₃ ^--N. NO in the industrial wastewater according to the discharge standard of copper and nickel cobalt industrial pollutants released by the state₃ ^-The direct emission standard of (1) is not more than 15mg/L, and the indirect emission standard of (2) is not more than 40 mg/L. If the high-nitrate-nitrogen industrial wastewater is directly discharged into a water body without being treated, not only can eutrophication of a water environment be caused, but also drinking water or other ways can enter human bodies, diseases such as methemoglobinemia, carcinogenesis and gene mutation can be induced, and the health of human bodies can be threatened. Therefore, how to treat the high-nitrate nitrogen industrial wastewater with high efficiency is an urgent problem in the current industry.

The common domestic and foreign modes for treating high-nitrate-nitrogen industrial wastewater mainly include physicochemical treatment and biochemical treatment. The physical and chemical treatment includes chemical denitrification, catalytic denitrification, reverse osmosis denitrification, ion exchange denitrification and the like, and the biochemical treatment mainly adopts a biological denitrification method. The methods have different advantages and disadvantages, but have obvious limitations in the aspects of wastewater treatment efficiency, treatment capacity, nitrogen recycling and the like. In the biological treatment method, particularly the biological treatment method based on microalgae culture, the nitrate in the wastewater can be quickly absorbed and converted into protein in the biomass, and the method has the advantages and characteristics of low cost and capability of turning waste into wealth, and has wide application prospects in the aspects of treatment of high-nitrate-nitrogen industrial wastewater and resource utilization thereof.

The most outstanding characteristics of microalgae are that the growth speed is fast, the environmental adaptability is strong, the culture mode is various, and various nitrogen elements in the water body can be utilized to be converted into high-value microalgae nitrogen-containing compounds (such as protein, chlorophyll and the like). Therefore, it is feasible to reduce the nitrogen content in wastewater by efficiently denitrifying microalgae. The Chlorella pyrenoidosa (Chlorella pyrenoidosa) serving as a primary producer in the water body can convert rich nutrients such as nitrogen and phosphorus in the water body into high-quality microalgae biomass, and the content of intracellular protein can reach 30-68% of dry weight. At present, the chlorella pyrenoidosa is widely applied to water adjusting agents in the culture process of fisheries and baits of aquatic animals such as fishes and shrimps. Chlorella pyrenoidosa has been approved as a new resource food in 2012 in China and can be used as a functional substitute of vegetable protein sources such as soybean and the like. In recent years, a great deal of research has been carried out to combine chlorella pyrenoidosa with wastewater treatment, so as to realize wastewater emission reduction and obtain microalgae biomass resources rich in high-valued products.

In the chinese patent application 201811416938.6, when chlorella/scenedesmus obliquus is used to treat reeling waste water, protein-containing microalgae biomass is obtained (the culture period is 16-24 d by using a light shaking table), but the total nitrogen content, especially nitrate nitrogen content, in the reeling waste water is low, and meanwhile, the protein yield of the chlorella in the application is only 7.70-14.41 mg/L. Therefore, although the application realizes wastewater treatment and microalgae biomass co-production, the denitration capacity and the protein yield are low, and the requirements of practical application cannot be met.

Chinese patent application 201810266297.4 discloses a method for treating monascus fermentation wastewater and co-producing microalgae protein feed by using chlorella pyrenoidosa (the culture period is 7-10 days), although the content of crude protein in biomass obtained after the chlorella pyrenoidosa in the patent application treats wastewater can reach 63.77%, the total nitrogen content of the original wastewater in the patent application after pretreatment is only 910mg/L (ammonia nitrogen accounts for 55%, and the content of nitrate nitrogen is not given), meanwhile, the culture systems of the two patents are both in a shaking flask system, are not subjected to scale tests, have significant difference with the culture of a fermentation tank system, and cannot be used in industrial wastewater treatment.

Chinese patent application 201811502958.5 discloses a method for treating cleaning wastewater in microalgae production from nitric acid waste liquid, which adopts an autotrophic mode and has a culture period of 1-36 days; chinese patent application 201811502952.8 discloses a method for treating high nitrate wastewater by using microalgae, which comprises fermenting (culture period is 1-36 d), reacting, concentrating and drying to make the microalgae absorb nitrate ions in the high nitrate nitrogen wastewater and obtain microalgae biomass. The two patents apply that the culture period of the microalgae is long, the denitrification capability of the microalgae and the quality such as protein content of the obtained microalgae biomass are not evaluated, and the nutritive value of the microalgae cannot be judged.

Therefore, the prior art does not organically combine the rapid and efficient treatment of high-nitrate-nitrogen wastewater and the co-production of high-protein biomass by using microalgae, and is not favorable for realizing the resource efficient utilization of the wastewater.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for quickly denitrating by using microalgae.

The invention also aims to provide application of the method for rapidly denitrifying by using the microalgae.

The purpose of the invention is realized by the following technical scheme:

a method for rapid denitration by using microalgae comprises the following steps:

s1, culturing the microalgae cells to a logarithmic phase to obtain microalgae seed liquid;

s2, inoculating the microalgae seed liquid into a fermentation tank filled with a fermentation medium containing high nitrate nitrogen to perform intermittent feeding and nourishing fermentation, and when the concentration of glucose in the fermentation medium is lower than 5g/L (preferably lower than 10g/L), supplementing glucose to make the concentration of glucose in the fermentation system higher than 10g/L, and controlling the pH value to be 6.0-7.5; wherein NO is present in the fermentation medium containing high nitrate nitrogen₃ ^-Is higher than 3000 mg/L.

The microalgae in the step S1 is at least one of chlorella, scenedesmus, gloeococcus and chlorochlorella; preferably Chlorella pyrenoidosa (Chlorella pyrenoidosa).

The microalgae seed solution described in step S1 is preferably obtained by the following method:

(1) inoculating the microalgae cells to the inclined plane of a basic culture medium for culturing to obtain microalgae lawn;

(2) and (2) transferring the microalgae lawn obtained in the step (1) to a liquid basal culture medium for culture to obtain microalgae seed liquid.

The conditions for the culture described in step (1) are preferably: the culture temperature is 30-35 ℃ (preferably 30 ℃), and the illumination is 10 mu mol/m²/s。

The liquid basic culture medium in the step (2) comprises the following components: 10g/L of glucose; EDTA 500 mg/L; FeCl₃·7H₂O 49.8mg/L；Co(NO₃)₂·6H₂O 15.7mg/L；H₃BO₃114.2mg/L；NaNO₃1250mg/L；MgSO₄·7H₂O 1000mg/L；CaCl₂·2H₂O 111mg/L；ZnSO₄·7H₂O 88.2mg/L；KH₂PO₄1250mg/L；CuSO₄·5H₂O 15.7mg/L；MnCl₂·H₂O 14.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；pH 6.1。

The conditions for the culture described in step (2) are preferably: the culture temperature is 30-35 ℃ (preferably 30 ℃), and the illumination is 10 mu mol/m²And/s, the rotating speed is 150rpm, and the culture time is 3-5 days.

The inoculation amount of the microalgae seed liquid in the step S2 is 1 × 10 according to the final concentration of the microalgae seed liquid in the fermentation system⁶～1×10¹⁰The final concentration of the strain in the fermentation system is 5 × 10 percent (calculated by CFU/mL addition (namely the inoculation amount is 5-10 percent (v/v)))⁶～1×10⁸The final concentration of CFU/mL is 1 × 10, more preferably⁸CFU/mL (i.e., inoculum size of 8% (v/v)) was added for calculation.

NO in the high nitrate nitrogen containing fermentation Medium as described in step S2₃ ^-The concentration of (A) is higher than 3600 mg/L; preferably 3600-25600 mg/L; more preferably 3600-11000 mg/L; most preferably 3647-10941 mg/L.

The fermentation medium containing high nitrate nitrogen in the step S2 preferably comprises the following components: 0-50 g/L of glucose; NaNO₃5～35g/L；KH₂PO₄1.25g/L；EDTA500mg/L；MgSO₄·7H₂O 1000mg/L；CuSO₄·5H₂O15.7mg/L；FeCl₃·7H₂O 49.8mg/L；CaCl₂·2H₂O 111mg/L；MnCl₂·H₂O 14.2mg/L；Co(NO₃)₂·6H₂O 15.7mg/L；ZnSO₄·7H₂O 88.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；H₃BO₃114.2mg/L。

The composition of the fermentation medium containing high nitrate nitrogen in the step S2 is more preferably as follows: 5-20 g/L of glucose; NaNO₃5～15g/L；KH₂PO₄1.25g/L；EDTA500mg/L；MgSO₄·7H₂O 1000mg/L；CuSO₄·5H₂O15.7mg/L；FeCl₃·7H₂O 49.8mg/L；CaCl₂·2H₂O 111mg/L；MnCl₂·H₂O 14.2mg/L；Co(NO₃)₂·6H₂O 15.7mg/L；ZnSO₄·7H₂O 88.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；H₃BO₃114.2mg/L。

The composition of the fermentation medium containing high nitrate nitrogen described in step S2 is most preferably: 5g/L of glucose; NaNO₃10g/L；KH₂PO₄1.25g/L；EDTA500mg/L；MgSO₄·7H₂O 1000mg/L；CuSO₄·5H₂O 15.7mg/L；FeCl₃·7H₂O 49.8mg/L；CaCl₂·2H₂O 111mg/L；MnCl₂·H₂O 14.2mg/L；Co(NO₃)₂·6H₂O15.7mg/L；ZnSO₄·7H₂O 88.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；H₃BO₃114.2mg/L。

The fermentation medium containing high nitrate nitrogen in the step S2 is a fermentation medium subjected to high-temperature and high-pressure sterilization treatment.

The high-temperature and high-pressure sterilization treatment is preferably realized by the following steps: continuously spraying steam at 110-121 ℃ for sterilization or sterilizing in a high-temperature high-compaction tank; preferably autoclaving at 121 deg.C for 15 min.

The fermentation tank in the step S2 is a light fermentation tank (the filling coefficient is 70-80%); preferably 1-50000L light fermentation tank; more preferably 5 to 50L.

The illumination intensity of the fermentation culture in the step S2 is 20-2000 mu mol/m²(ii)/s (light intensity in the center of the fermenter); preferably 120 to 560. mu. mol/m²S; more preferably 120 to 210. mu. mol/m²/s。

The light source adopted by the fermentation culture in the step S2 is LED white light, warm white light or natural white light; preferably, it is a white positive light.

The conditions of the fermentation culture described in step S2 are: the temperature is 30-35 ℃ (preferably 30 ℃), the stirring speed is 100-250 rpm, the fermentation period is 60-84 h, the ventilation volume is 0.5-2.0 vvm (preferably 1.5vvm), and the culture is stopped when the content of nitrogen or phosphorus in the fermentation system is 0.

The glucose supplementation in the step S2 is preferably performed by feeding a glucose solution; the concentration of the glucose solution is 450-700 g/L (preferably 500 g/L).

The glucose supplement in the step S2 is preferably glucose supplement so that the concentration of glucose in the fermentation system is 10-50 g/L; preferably 10-20 g/L; more preferably 10 to 15 g/L.

The pH value described in step S2 is preferably 6.5.

The pH value regulator in the step S2 is preferably a nitric acid solution; preferably 0.5-1 mol/L nitric acid solution.

Defoaming agent may be added to the fermentation culture described in step S2 for defoaming (appropriately fed-batch depending on the foaming condition).

The defoamer is preferably a PPE polyether defoamer.

The concentration of the PPE polyether defoaming agent is 3 per mill of mass-volume ratio.

The method for rapidly denitrating by using microalgae further comprises the following steps after the step S2:

s3, separating and washing the microalgae after the fermentation is finished, and freeze-drying to obtain the microalgae biomass containing high protein.

The freeze-drying described in step S3 is preferably vacuum freeze-drying at-40 ℃.

The method for rapidly denitrifying by using microalgae is applied to wastewater treatment or production of microalgae biomass rich in protein.

The wastewater is high-nitrate nitrogen industrial wastewater; preferably NO₃ ^-Industrial wastewater with concentration higher than 3000 mg/L; further preferably NO₃ ^-Industrial wastewater with the concentration higher than 3600 mg/L; still more preferably NO₃ ^-Industrial wastewater with the concentration of 3600-25600 mg/L; more preferably NO₃ ^-Industrial wastewater with the concentration of 3600-11000 mg/L; most preferably NO₃ ^-The concentration of the industrial wastewater is 3647-10941 mg/L.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a new method for treating high-nitrate-nitrogen industrial wastewater and recycling the high-nitrate-nitrogen industrial wastewater. Obtaining a facultative fermentation culture medium containing high nitrate nitrogen by improving a basic culture medium, inoculating chlorella pyrenoidosa after sterilization for high-density optical fermentation, intermittently supplementing carbon and nitrogen sources (glucose and nitric acid), wherein the biomass concentration can reach 23.40-30.10 g/L after fermentation is finished, and the protein content in the harvested microalgae biomass can reach 50.06-52.88% of the dry weight; the highest average consumption rate of nitrate in the light fermentation process can reach 5.15g/L/d, the rapid denitration, the high-density culture of microalgae and the output of high-protein biomass are synchronously realized, a novel, efficient and economic method is provided for realizing the combination of the resource utilization of high-nitrate nitrogen industrial wastewater and the production of algae-based high-protein feed raw materials, the discharge of the high-nitrate nitrogen wastewater is reduced, and the resource recycling of nitrate nitrogen is realized.

(2) According to the invention, high-concentration nitrate radicals in the culture medium are removed through microalgae high-density culture, and nitrogen resource conversion and utilization are realized. The method utilizes the chlorella pyrenoidosa to be cultured in an improved basic culture medium containing high nitrate nitrogen, optimizes the culture conditions of the chlorella pyrenoidosa by mixotrophic high-density culture, aims to realize the high rate of removing nitrate in the culture medium of the chlorella pyrenoidosa, and finally obtains the chlorella pyrenoidosa biomass with high protein content. On one hand, the method can realize the rapid treatment of the high-nitrate nitrogen industrial wastewater by the microalgae, reduce the sewage discharge and save water resources; on the other hand, the method can also obtain protein-rich microalgae biomass, realizes economic production of high-value microalgae biomass, is a new technical approach of green recycling economy, and has good environmental benefit and social benefit.

(3) The invention carries out microalgae culture in a fermentation tank system, the change of the culture system causes the regulation and control of the illumination intensity and the ventilation quantity to be different from the shaking bottle system, and simultaneously, the fed-batch strategy of the fermentation tank system can not be rapidly and aseptically realized in the shaking bottle system, therefore, the success of the shaking bottle system can not be copied in the fermentation tank system, but in the fermentation tank system of the invention, the biomass yield and the denitration capacity are superior to those of the shaking bottle system, and the fermentation system can be smoothly enlarged to the industrial scale,

drawings

FIG. 1 is a graph of the growth of heterotrophic cells and nitrate depletion of Chlorella pyrenoidosa at different inoculum densities in shake flasks.

FIG. 2 is a graph of the growth of heterotrophic cells and nitrate depletion of Chlorella pyrenoidosa in shake flasks at various sodium nitrate concentrations.

FIG. 3 is a graph of the growth of heterotrophic cells and nitrate depletion of Chlorella pyrenoidosa in shake flasks at different light intensities.

FIG. 4 is a graph of the increase in biomass of C.mixotrophicus at different initial glucose concentrations in a 5L light fermentor.

FIG. 5 is a graph showing the change in protein content in Chlorella mixotrophica cells at different initial glucose concentrations in a 5L light fermentor.

FIG. 6 is a graph of biomass yield, nitrate assimilation rate, and protein yield of C.mixotrophicus at different initial glucose concentrations in a 5L light fermentor.

FIG. 7 is a graph of the increase in biomass of Chlorella mixotrophica and the consumption of phosphate at different initial sodium nitrate concentrations in a 5L light fermentor.

FIG. 8 is a graph showing the change in protein content in Chlorella mixotrophica at different initial sodium nitrate concentrations in a 5L light fermentor.

FIG. 9 is a graph of biomass yield, nitrate assimilation rate, and protein yield of C.mixotrophicus in 5L light fermentors at different initial sodium nitrate concentrations.

FIG. 10 is a graph of the increase in biomass of C.mixotrophicus at different pH's in a 5L light fermentor.

FIG. 11 is a graph showing the change in protein content in Chlorella mixotrophica at different pH in a 5L light fermentor.

FIG. 12 is a plot of the mixotrophic Chlorella pyrenoidosa biomass yield, nitrate assimilation rate, and protein yield at different pH in a 5L light fermentor.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The detection method adopted in the embodiment of the present invention can be performed with reference to the following descriptions:

determination of the Biomass concentration in Chlorella pyrenoidosa

The biomass concentration determination method during the culture of Chlorella pyrenoidosa (Chlorella pyrenoidosa) adopts a dry weight method for determination. Sucking 2mL of algae liquid, placing the algae liquid into a 2mL centrifugal tube accurately weighed, repeatedly cleaning and centrifuging the obtained algae mud twice by using ultrapure water after centrifugation, and then placing the centrifugal tube containing the algae mud into a constant-temperature oven at 60 ℃ to dry to constant weight. And weighing by using an analytical balance and calculating to obtain the weight of the dried algae powder. The biomass and biomass yield (units: g/L/d) were calculated according to the formula:

biomass (g L)^-1)＝(m₂-m₁)/0.002；

In the formula: m is₁Empty tube mass (g); m is₂Mass (g) of centrifuge tube containing dried algal mud.

Biomass yield ═ DW₂-DW₁)/₂-t₁)；

In the formula DW₁，DW₂Respectively represents t₁And t₂Biomass at time point.

NO in Chlorella pyrenoidosa (II)₃ ^-、PO₄ ^3-And determination of glucose concentration

1)NO₃ ^-And PO₄ ^3-Determination of concentration

The measurements were performed using a multi-parameter water quality analyzer, HANNA HI83200, italy. Selecting a proper range (0-30 mg/L), diluting a sample to be detected to a determination range (0-30 mg/L), adding corresponding HI93728 nitrate radical and HI93717 phosphate radical reagents into a cuvette according to instrument instructions, determining and reading. After the reading is finished, the reading is multiplied by the dilution times to obtain the NO in the culture medium₃ ^-And PO₄ ^3-The content of (a). NO₃ ^-The average assimilation rate (unit: g/L/d) was calculated according to the formula:

average rate of assimilation ═ N_t2-N_t1)/(t₂-t₁)；

In the formula N_t1、N_t2Respectively represents t₁And t₂Nitrate concentration at time point.

2) Determination of glucose concentration

The glucose concentration is measured by adopting an SBA-40D biosensing analyzer, and the correction range is 0.5-1.0 g/L. Before measurement, the supernatant of a sample to be measured is taken, filtered by a 0.45 mu m filter membrane and diluted to be within the glucose concentration correction range. And (3) performing calibration by using 1.0g/L glucose standard solution, after the standard solution passes through the calibration, sucking 25 mu L of treated supernatant by using a micro-syringe for determination, repeatedly determining each sample for three times, then taking an average value, and multiplying the determination reading by the dilution factor to obtain the glucose concentration of the sample to be determined.

(III) determination of protein content in Chlorella pyrenoidosa

The protein content was determined using a semi-automatic Kjeldahl apparatus from FOSS.

Protein yield was calculated using the following formula:

protein yield (g/L/d) ═ DW₂×PC₂-DW₁×PC₁)/(t₂-t₁)；

In the formula, DW₁、DW₂Respectively represents t₁And t₂Biomass concentration at time point, PC₁、PC₂Respectively represents t₁And t₂Protein content at time point.

Example 1 Effect of different initial cell densities on Chlorella pyrenoidosa growth and nitrate assimilation rates in Shake flasks

1.1 Strain activation and seed liquid preparation

Inoculating Chlorella pyrenoidosa strain to slant containing basic culture medium, culturing at 30 deg.C under illumination of 10 μmol/m²And/s, culturing for 3-5 days and observing the growth condition of the chlorella pyrenoidosa. Inoculating Chlorella pyrenoidosa with inoculating loop into triangular flask (liquid content of 100mL) containing basic liquid culture medium, culturing at 30 deg.C and 10 μmol/m²Culturing in a constant-temperature shaking table (the rotating speed is 150rpm) for 3-5 days to serve as seed liquid.

Wherein the basic culture medium (pH 6.1) has the following components (unit is mg/L) as shown in the following table 1:

TABLE 1 liquid Medium formulation

Components	Content (mg/L)	Components	Content (mg/L)	Components	Content (mg/L)
						Glucose	10000	NaNO₃	1250	KH₂PO₄	1250
EDTA	500	MgSO₄·7H₂O	1000	CuSO₄·5H₂O	15.7
						FeCl₃·7H₂O	49.8	CaCl₂·2H₂O	111	MnCl₂·H₂O	14.2
Co(NO₃)₂·6H₂O	15.7	ZnSO₄·7H₂O	88.2	Na₂MoO₄·2H₂O	11.92
						H₃BO₃	114.2	/		/	/

Note: EDTA is ethylenediamine tetraacetic acid

1.2 culture of Chlorella pyrenoidosa at different initial cell densities

Transferring the cultured Chlorella pyrenoidosa seed liquid to an improved basal culture medium, and controlling the initial cell density to be 1 × 10 respectively⁶、5×10⁶、1×10⁷、5×10⁷And 1 × 10⁸CFU/mL, placing the shake flask in a constant-temperature shaking table, and observing the growth of the chlorella pyrenoidosa and the influence of nitrate assimilation. The formulation of the modified basal medium used therein is different from the formulation in table 1 in that: initial glucose and NaNO₃The concentration is respectively 50g/L and 3.75 g/L; meanwhile, in a fed-batch mode, when the glucose concentration in the culture medium is less than 10g/L, glucose (the mother liquor concentration is 500g/L) is fed in a fed-batch mode, so that the glucose concentration in the culture medium is 50 g/L. The culture temperature is 30 ℃, the rotating speed is 150rpm, and the illumination intensity is 120 mu mol/m²And/s, culturing for 5 days.

Collecting algae liquid every day during the culture process, centrifuging, washing algae mud for multiple times, drying at constant temperature to constant weight, and measuring biomass; centrifuging, taking supernatant, measuring the glucose concentration, and determining the volume of the fed-in glucose mother liquor; determination of NO₃ ^-To calculate NO₃ ^-The rate of assimilation.

1.3 results of the experiment

The variation of the biomass and nitrate concentration of Chlorella pyrenoidosa at different starting cell densities is shown in FIG. 1 the results show that when the starting cell density is higher than 5 × 10⁶The biomass concentration of the chlorella pyrenoidosa is not significantly different at CFU/mL, and is basically kept above 21.75g/L, nitrate assimilation rate is gradually increased along with the increase of initial cell density, and the cell density is 1 × 10 when the culture is cultured to the 2 nd day⁸The average assimilation rate of nitrate radical reaches the maximum value at CFU/mL, and is 2.51g/L/d, which is obviously higher than that of other groups (p)<0.01)。

Example 2 Effect of different sodium nitrate concentrations on Chlorella pyrenoidosa cell growth and nitrate assimilation rates in Shake flasks

2.1 algal seed activation and seed liquid preparation

Culturing Chlorella pyrenoidosa for 3-5 days by the culture method of 1.1 to serve as seed liquid.

2.2 culture of Chlorella pyrenoidosa with different sodium nitrate concentrations

The cultured Chlorella pyrenoidosa seed solution is processed as described in 1.2, with initial cell density of 1 × 10⁸CFU/mL, dispensed into medium with different sodium nitrate concentrations (final concentrations of 5, 15, 25 and 35g/L) for culture. The illumination was 210. mu. mol/m²And/s, the rest conditions were the same as 1.2, and the culture was carried out for 4 days.

The collection and treatment of samples during the culture and the determination of relevant indices are referred to the method described in 1.2.

2.3 results of the experiment

The biomass and nitrate concentration of Chlorella pyrenoidosa at different sodium nitrate concentrations were varied as shown in FIG. 2. The results show that when the concentration of the sodium nitrate exceeds 25g/L, the biomass of the chlorella pyrenoidosa is slowly increased; when the concentration of the sodium nitrate is 5-15 g/L, the biomass concentration of the chlorella is 14.88-21.08 g/L, and a high average assimilation rate of nitrate (1.81-2.15 g/L/d) is obtained.

Example 3 Effect of different light intensities on the growth of Chlorella pyrenoidosa and nitrate assimilation Rate in Shake flasks

3.1 algal seed activation and seed liquid preparation

3.2 culture of Chlorella pyrenoidosa under different light intensities

The cultured Chlorella pyrenoidosa seed liquid is processed according to the method of 1.2, and the initial cell density is controlled to be 1 × 10⁸CFU/mL, the illumination intensity is 120, 150, 180, 210 mu mol/m respectively²And s. At the same time, the initial glucose and NaNO in the culture system₃The concentrations were 20g/L and 15g/L, respectively, and when the glucose concentration in the medium was < 10g/L, glucose was fed to the feed stream (mother liquor concentration was 500g/L) so that the glucose concentration in the medium was 20 g/L. The culture was carried out for 6 days under the same conditions as 1.2.

3.3 results of the experiment

The biomass and nitrate concentration of Chlorella pyrenoidosa under different light intensities are shown in FIG. 3. The result shows that when the illumination intensity is 150-210 mu mol/m²At the time of/s, no significant difference exists between the biomass, and the biomass is 33.75-35.22 g/L. Meanwhile, the assimilation rate of nitrate radical is 120-210 mu mol/m²The obvious difference does not exist between the concentrations of the components per second and is 1.14-1.38 g/L/d, so that the illumination intensity of the chlorella pyrenoidosa cultured under the shake flask mixotrophic condition is 210 mu mol/m²And the nitrate ions are more favorable for cell proliferation in the second time, so that the microalgae is promoted to assimilate nitrate ions efficiently.

Example 4 Effect of different initial glucose concentrations in a 5L light fermentor on Chlorella pyrenoidosa growth, nitrate assimilation Rate and protein production

4.1 Strain activation and seed liquid preparation

4.2 Co-culture of Chlorella pyrenoidosa in 5L light fermenter at different initial glucose concentrations

Inoculating the cultured Chlorella pyrenoidosa seed liquid according to an inoculation volume (fermentation volume) of 8%3.5L) were inoculated into sterilized modified basal medium with initial cell density of 1.0 × 10⁸CFU/mL, setting initial glucose concentrations at 5 and 20g/L, respectively. The medium and the feeding bottle were autoclaved at 121 ℃ for 15 min. In the fermentation process, a glucose mother liquor (450-700 g/L) is fed in a flowing mode to maintain the glucose concentration to be 10-20 g/L in the fermentation process, and a specific glucose feeding strategy is as follows: when the initial glucose concentration is 5g/L, supplementing glucose to maintain the concentration at 10g/L in 12 hours, and supplementing glucose to maintain the concentration at 15-20 g/L every 12 hours after 36 hours; when the initial glucose concentration is 20g/L, the initial glucose concentration is<And when the concentration is 5g/L, supplementing glucose to maintain the concentration at 25-30 g/L. The aeration rate in the light fermenter was maintained at about 1.5 vvm. 3 per mill (m/v, antifoaming agent/culture medium) of an antifoaming agent (PPE polyether antifoaming agent, Jinan Xinglong Fei chemical Co., Ltd.) is prepared, and when foam is higher than a foam position in the fermentation process, a proper amount of antifoaming agent is added to reduce the interference of the foam. In the fermentation process, a 0.5mol/L nitric acid solution is adopted for adjustment, and the pH is controlled to be 6.5. The modified basal medium used in this example has a formula different from the formula in table 1 in that: NaNO starting material₃The concentration was 15 g/L. The culture temperature in this example is 30 deg.C, the light source is placed side by side in series by LED white light hard lamp strips, when the initial glucose concentration is 5g/L, the illumination intensity is 120 μmol/m²S; when the initial glucose concentration was 20g/L, the light intensity was from 120. mu. mol/m depending on the cell growth with the culture time²The/s is gradually increased to 560. mu. mol/m²And s. The culture time is 72 h.

Collecting algae liquid every 12h and every 8h under the conditions of 5g/L glucose and 20g/L glucose in the culture process, centrifuging to obtain algae mud, washing for multiple times, drying a part of algae mud at constant temperature to constant weight, and measuring biomass; vacuum freeze-drying the other part of the algae mud at-40 ℃ to obtain freeze-dried algae powder, and measuring the protein content of the algae powder; centrifuging, taking supernatant, measuring the glucose concentration, and determining the volume of the fed-in glucose mother liquor; determination of NO₃ ^-To calculate NO₃ ^-Average rate of assimilation.

4.3 results of the experiment

The biomass, protein content, biomass yield, nitrate assimilation rate and protein yield of Chlorella pyrenoidosa at different initial glucose concentrations are shown in FIGS. 4-6. As shown in FIG. 4, the Chlorella pyrenoidosa exhibited a lag phase of 24h in both groups, while the maximum biomass concentration (37.2g/L) was obtained at the end of the culture at an initial glucose concentration of 20 g/L. As shown in FIG. 5, the protein content of Chlorella pyrenoidosa was always higher than 20g/L in the culture process at a glucose concentration of 5g/L, and was 33% higher than 20g/L at 48 h. At the end of the culture, the protein content of Chlorella pyrenoidosa under 5g/L glucose conditions was 48.47% of the dry weight. As shown in FIG. 6, when the glucose concentration was 5g/L, the maximum nitrate average assimilation rate of Chlorella pyrenoidosa was 4.47g/L/d, which is higher than 27% under the condition of 20 g/L. Although the biomass concentration was not increased significantly at a glucose concentration of 5g/L, the protein content and the nitrate average assimilation rate were both higher than those at a glucose concentration of 20 g/L. Therefore, the initial glucose concentration of 5g/L can be selected to realize rapid denitration and co-production of high-protein biomass in a high-nitrate nitrogen culture medium, and meanwhile, the production cost in industrialization can be reduced.

Example 5 different initial NaNO in 5L light fermentor₃Effect of concentration on Chlorella pyrenoidosa growth, nitrate assimilation Rate and protein production

5.1 Strain activation and seed liquid preparation

5.2 different initial NaNO in 5L light fermenter₃Co-culture high-density culture of chlorella pyrenoidosa under concentration

Preparation and Sterilization of the relevant solution at the beginning of the fermentation and inoculation of the cells at the beginning of the fermentation are referred to the procedure described in 4.2. Setting initial NaNO in fermentation System₃The concentrations were 5, 10 and 15g/L, respectively, and the initial glucose concentration was 5 g/L. When the concentration of glucose is<10g/L, supplementing a glucose mother liquor (450-700 g/L) in the first 24h to maintain the sugar concentration at 10-15 g/L, and maintaining the sugar concentration at 15-20 g/L after 24h and after the culture is finished. Wherein at 36h, NaNO is initially present₃The sugar supplement concentration of 15g/L is 10-15 g/L; the illumination intensity at the beginning of the culture is 100-120 mu mol/m for different initial concentrations of sodium nitrate²S, increasing the illumination intensity to 140-160 mu mol/m in the first 12h after finishing the culture²And s. The pH regulator used in the fermentation process is 1mol/L nitric acid solution, and the pH is controlled to be 6.5; the rest conditions are the same as 4.2, and the culture lasts for 60-84 h (when the PO is initially generated)₄ ^3-The culture was terminated at a concentration of 0).

Collection and determination of samples during culture was as described in reference to 4.2.

5.3 results of the experiment

At different initial NaNO₃The biomass, protein content, biomass yield, nitrate assimilation rate and protein yield of Chlorella pyrenoidosa at the concentrations are shown in FIGS. 7-9. Wherein, when NaNO is initiated₃The time points for terminating the culture at concentrations of 5, 10 and 15g/L were 60, 60 and 84 hours, respectively. As can be seen from FIG. 7, the initial NaNO at 10g/L₃Under the experimental conditions, although PO₄ ^3-When the concentration is reduced to 0 in 60h, the biomass of the chlorella pyrenoidosa can still continue to grow, and the highest biomass concentration (33.07g/L) is obtained in 84 h; as shown in FIG. 8, when NaNO is used₃When the concentration is 5-10 g/L, the protein content can reach 51.48% -52.36% when the culture is stopped. At the same time, in the initial NaNO₃Under the condition of 10g/L, the biomass yield, the average assimilation rate of nitrate and the protein yield of the cells at the time of terminating the culture were respectively 7.88g/L, 5.15g/L/d and 4.51g/L/d, which were significantly higher than those of the other groups. This indicates that at an initial glucose concentration of 5g/L, the initial NaNO₃Under the condition of the concentration of 10g/L, the chlorella pyrenoidosa can obtain higher protein yield while obtaining higher average assimilation rate of nitrate (figure 9).

Example 6 Effect of different pH on Chlorella pyrenoidosa growth, nitrate assimilation Rate and protein production in a 5L light fermentor

6.1 Strain activation and seed liquid preparation

6.2 Co-culture of Chlorella pyrenoidosa in 5L light fermenter under different pH in fermenter

Preparation and Sterilization of the relevant solution at the beginning of the fermentation and inoculation of the cells at the beginning of the fermentation are referred to the method described in 5.2. Setting initial glucose concentration and initial NaNO in fermentation System₃The concentrations were 5g/L and 10g/L, respectively, and the pH values were set to 6.0, 6.5 and 7.5, respectively, and the other conditions were the same as 5.2.

Culture conditions and measurement methods can be specifically referred to the method described in 5.2.

6.3 results of the experiment

The cell biomass, protein content, biomass yield, nitrate assimilation rate and protein yield of Chlorella pyrenoidosa at different pH are shown in FIGS. 10-12. Wherein, when the initial pH was 6.0, 6.5 and 7.5, respectively, the time points for terminating the culture were 72, 60 and 72 hours, respectively. FIG. 10 shows that there was no significant difference between the biomass of Chlorella pyrenoidosa at different pH's. As can be seen from FIG. 11, under the condition of pH 6.5, the protein content in the Chlorella pyrenoidosa cells at 60h was 51.48% of the dry weight of the cells, which is higher than that in the other groups; but at 72h, higher protein content (52.88%) was obtained with chlorella pyrenoidosa at pH 7.5. FIG. 12 shows that the average assimilation rate of nitrate was 5.15g/L/d at pH 6.5, which is 1.34 and 6.44 times higher than that at pH6.0 and pH 7.5.

The comprehensive examples 1-6 show that under the condition of mixotrophic fermentation, the initial glucose and NaNO are₃The concentrations are respectively 5g/L and 10g/L, when the fermentation tank is maintained at a pH value of 6.5, the chlorella pyrenoidosa can obtain a high average assimilation rate of nitrate nitrogen of 5.15g/L/d, and the highest protein content of 51.48% of the dry weight is obtained.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for rapidly denitrating by using microalgae is characterized by comprising the following steps:

s2, inoculating the microalgae seed liquid into a fermentation tank filled with a fermentation medium containing high nitrate nitrogen to perform intermittent feeding and nourishing fermentation, and when the concentration of glucose in the fermentation medium is lower than 5g/L, supplementing glucose to make the concentration of glucose in a fermentation system higher than 10g/L, and controlling the pH value to be 6.0-7.5; wherein NO is present in the fermentation medium containing high nitrate nitrogen₃ ^-Is higher than 3000 mg/L.

2. The method for rapid denitrification using microalgae according to claim 1, wherein:

the microalgae in the step S1 is at least one of chlorella, scenedesmus, gloeococcus and chlorochlorella;

NO in the high nitrate nitrogen containing fermentation Medium as described in step S2₃ ^-The concentration of (A) is higher than 3600 mg/L;

and the glucose supplement in the step S2 is glucose supplement, so that the glucose concentration in the fermentation system is 10-50 g/L.

3. The method for rapid denitrification using microalgae according to claim 2, wherein:

the microalgae described in step S1 is Chlorella pyrenoidosa (Chlorella pyrenoidosa);

NO in the high nitrate nitrogen containing fermentation Medium as described in step S2₃ ^-The concentration of (A) is 3600-25600 mg/L;

and the glucose supplement in the step S2 is glucose supplement, so that the glucose concentration in the fermentation system is 10-20 g/L.

4. The method for rapid denitrification using microalgae according to claim 1, wherein:

the fermentation medium containing high nitrate nitrogen in the step S2 comprises the following components: 0-50 g/L of glucose; NaNO₃5～35g/L；KH₂PO₄1.25g/L；EDTA 500mg/L；MgSO₄·7H₂O 1000mg/L；CuSO₄·5H₂O 15.7mg/L；FeCl₃·7H₂O 49.8mg/L；CaCl₂·2H₂O 111mg/L；MnCl₂·H₂O 14.2mg/L；Co(NO₃)₂·6H₂O15.7mg/L；ZnSO₄·7H₂O 88.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；H₃BO₃114.2mg/L。

5. The method for rapid denitrification using microalgae according to claim 4, wherein:

the fermentation medium containing high nitrate nitrogen in the step S2 comprises the following components: 5-20 g/L of glucose; NaNO₃5～15g/L；KH₂PO₄1.25g/L；EDTA 500mg/L；MgSO₄·7H₂O 1000mg/L；CuSO₄·5H₂O 15.7mg/L；FeCl₃·7H₂O 49.8mg/L；CaCl₂·2H₂O 111mg/L；MnCl₂·H₂O 14.2mg/L；Co(NO₃)₂·6H₂O15.7mg/L；ZnSO₄·7H₂O 88.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；H₃BO₃114.2mg/L。

6. The method for rapid denitrification using microalgae according to claim 1, wherein:

the inoculation amount of the microalgae seed liquid in the step S2 is 1 × 10 according to the final concentration of the microalgae seed liquid in the fermentation system⁶～1×10¹⁰Calculating the addition of CFU/mL;

the illumination intensity of the fermentation culture in the step S2 is 20-2000 mu mol/m²/s；

The conditions of the fermentation culture described in step S2 are: the temperature is 30-35 ℃, the stirring speed is 100-250 rpm, the fermentation period is 60-84 h, the ventilation volume is 0.5-2.0 vvm, and the culture is stopped when the content of nitrogen or phosphorus in the fermentation system is 0;

the pH value in step S2 is 6.5;

the fermentation medium containing high nitrate nitrogen in the step S2 is subjected to high-temperature and high-pressure sterilization treatment;

the high-temperature and high-pressure sterilization treatment is realized by the following modes: continuously spraying steam at 110-121 ℃ for sterilization or sterilizing in a high-temperature high-compaction tank;

the fermentation tank in the step S2 is a light fermentation tank;

the glucose supplement in the step S2 is carried out in a mode of feeding glucose solution;

the concentration of the glucose solution is 450-700 g/L;

the pH value regulator in the step S2 is a nitric acid solution;

defoaming by adding a defoaming agent in the fermentation culture in the step S2;

the defoaming agent is PPE polyether defoaming agent.

7. The method for rapid denitrification using microalgae according to claim 1, wherein:

the microalgae seed liquid in the step S1 is obtained by the following method:

(2) transferring the microalgae lawn obtained in the step (1) to a liquid basic culture medium for culture to obtain microalgae seed liquid;

the culture conditions in the step (1) are as follows: the culture temperature is 30-35 ℃, and the illumination is 10 mu mol/m²/s；

The liquid basic culture medium in the step (2) comprises the following components: 10g/L of glucose; EDTA 500 mg/L; FeCl₃·7H₂O 49.8mg/L；Co(NO₃)₂·6H₂O 15.7mg/L；H₃BO₃114.2mg/L；NaNO₃1250mg/L；MgSO₄·7H₂O 1000mg/L；CaCl₂·2H₂O 111mg/L；ZnSO₄·7H₂O 88.2mg/L；KH₂PO₄1250mg/L；CuSO₄·5H₂O 15.7mg/L；MnCl₂·H₂O 14.2mg/L；Na₂MoO₄·2H₂O 11.92mg/L；pH 6.1；

The culture conditions in the step (2) are as follows: the culture temperature is 30-35 ℃, and the illumination is 10 mu mol/m²And/s, the rotating speed is 150rpm, and the culture time is 3-5 days.

8. The method for rapid denitrification using microalgae according to claim 1, further comprising the following steps after step S2:

9. The use of the method for rapid denitrification using microalgae according to any of claims 1 to 8 in wastewater treatment or production of protein-rich microalgae biomass, wherein the method comprises the following steps:

the wastewater is high-nitrate nitrogen industrial wastewater.

10. Use according to claim 9, characterized in that:

the wastewater is NO₃ ^-Industrial waste water with a concentration higher than 3000 mg/l.