CN112126597B

CN112126597B - Method for culturing spirulina based on biogas slurry

Info

Publication number: CN112126597B
Application number: CN202010890868.9A
Authority: CN
Inventors: 赵卫红; 江凤娟; 凌佳怡; 李文麟; 许冰倩; 岑经; 高志刚; 李元广
Original assignee: Dongtai City Spirulina Bio Engineering Co ltd; Yancheng Institute of Technology
Current assignee: Dongtai City Spirulina Bio Engineering Co ltd; Yancheng Institute of Technology
Priority date: 2020-08-29
Filing date: 2020-08-29
Publication date: 2022-08-12
Anticipated expiration: 2040-08-29
Also published as: CN112126597A

Abstract

The invention discloses a method for culturing spirulina based on biogas slurry, which takes waste biomass livestock and poultry manure, namely biogas slurry as a raw material, dilutes the waste biomass livestock and poultry manure by 10 times of the biogas slurry, adds 8% of Zarrouk culture solution, and is used for culturing spirulina, the growth condition of the spirulina is good, the spirulina has good absorption effect on nitrogen, phosphorus and other nutritive salts in water, and the removal rate of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and active phosphorus can reach 58%, 83%, 77.4% and 53.12% respectively. The spirulina is cultured by utilizing the biogas slurry, so that the cost can be saved, the environment can be purified, and the technology has great potential in the aspect of ecological environment protection.

Description

Method for culturing spirulina based on biogas slurry

Technical Field

The invention belongs to the field of waste recycling, and particularly relates to a method for culturing spirulina based on biogas slurry.

Background

The biogas slurry is high-concentration organic wastewater obtained by anaerobic fermentation of organic matters such as livestock and poultry manure and is residual liquid obtained by anaerobic fermentation of biogas, contains a large amount of nitrogen, phosphorus and organic matters, has strong nutritional capability and high nutrient availability, is a multi-element sanitary quick-acting compound fertilizer, and has high application value.

As a big agricultural country, China develops and applies biogas to occupy a large part of China. In the utilization of fermentation residue of a biogas digester, many studies and demonstrations have been conducted in various regions, such as the use of biogas slurry as a fertilizer, feed supplement, seed soaking, biopesticides, plant growth stimulants, and the like. Therefore, the comprehensive problem of widely and reasonably utilizing the residual liquid of the methane tank is beneficial to agricultural production and has obvious positive effect on improving the ecological environment.

Biogas fermentation refers to a series of biochemical reactions of organic matters under anaerobic conditions. The residue of methane fermentation mainly contains three main substances such as mineral substances, active ingredients, nutrient substances and the like. The biogas slurry serving as an organic fertilizer contains various active substances, has the highest content of nutrient components and mainly contains the following substances: firstly, trace elements are in the form of high-activity ions which can penetrate into daughter cells after being fermented by residual liquid biogas and can stimulate the germination of seeds or the growth of plants to provide micronutrients and bioactive substances. And secondly, nucleic acid, gibberellin, cellulase, monosaccharide, unsaturated fatty acid, growth hormone, antibiotic and the like secreted by the microorganism have the functions of promoting plant germination, plant growth, flowering and other functions of adjusting fermentation raw materials in the process of plant growth. And thirdly, nutrients, macromolecular substances in the raw materials are decomposed by microorganisms, and nutrient elements such as nitrogen, phosphorus and potassium are supplied to the plants and can be directly absorbed and utilized by the plants.

The nutritional types of spirulina can be divided into autotrophic, heterotrophic and mixotrophic types. The autotrophic type is a process of utilizing light energy and an inorganic carbon source to carry out photosynthesis so as to enable algae cells to synthesize organic matters by themselves, and aiming at the autotrophic type spirulina, the spirulina grows in the dark by utilizing one or more organic matters as energy sources and carbon sources under the condition of no light, namely heterotrophic propagation is carried out, the growth rate is higher than that of chlorella under the autotrophic condition, and high biomass can be generally obtained, similar to the fermentation of bacteria. In addition, the spirulina can perform mixotrophic growth under the conditions of illumination and utilization of organic carbon sources, and the growth speed and the alga density are high.

The growth condition of spirulina is influenced by various environmental factors including nutrition, environment and ecological factors, and the main influencing factors are as follows: pH, temperature, light cycle and light intensity, etc. At present, the Zarrouk culture medium is used for culturing spirulina finished products, the culture cost is high, and the growth condition of algae is common.

CN105779352A discloses a method for cultivating spirulina by utilizing biogas slurry, belonging to the technical field of spirulina cultivation. According to the method, a film is built around the reservoir, after the reservoir is covered, a stainless steel screen is arranged above the reservoir, attapulgite powder and a soil mixture are laid on the stainless steel screen, the film is insulated by sunlight irradiation, and culture solution is continuously transpired to provide moisture and nutrients for spirulina, so that the method for cultivating spirulina by utilizing biogas slurry is obtained. The example proves that the method is unique and novel, the cultivation is simple and easy, no special equipment is needed, the whole cultivation process is pollution-free, the problems that the heavy metal of the spirulina exceeds the standard and chemical substances are remained are fundamentally solved, the yield of the spirulina reaches over 95 percent, and the method can be popularized and applied on a large scale. Although the invention uses the biogas slurry of the livestock farm as a nutrient source, the pretreatment is complex, and the attapulgite belongs to a rare resource, so the invention is low in environmental friendliness and is not suitable for large-scale industrialized popularization.

CN103275900A discloses a recycling method of biogas slurry after anaerobic treatment of municipal domestic waste, belonging to the technical field of environmental protection. Sorting domestic waste, crushing the selected biomass, pulping, performing anaerobic fermentation, discharging biogas slurry after fermentation, making the biogas slurry into culture solution, and injecting the culture solution into culture ponds in each spirulina culture greenhouse; meanwhile, separating and purifying the collected high-quality algae seeds in the local soda lake, expanding the pure-variety algae seeds, then culturing and expanding propagation in one culture pond, then inoculating the expanded and propagated algae seeds into other culture ponds for production and culture, then collecting the cultured high-quality spirulina, and finally preparing the prepared and separated algae mud into finished product algae powder after spray drying. The spirulina cultivated by using the biogas slurry to replace chemical culture solution is organic algae, and the green production of spirulina products is realized. Solves the problems of high construction cost and high cost of biogas slurry treatment, and reduces the culture cost of the spirulina by 50 percent.

CN107460128A discloses a method for cultivating microalgae by using biogas slurry, wherein microalgae is inoculated in an artificial culture medium, and is collected when the specific growth rate of the microalgae begins to decrease, then the biogas slurry is added in batches, and the microalgae is collected when the growth rate of the microalgae decreases to 60-70% of the growth rate before the biogas slurry is added for the first time; adding the biogas slurry in batches again, and harvesting when the growth rate of the microalgae is reduced to 50-60% of the growth rate before the biogas slurry is added for the first time; and adding the biogas slurry in batches for the third time, and finishing the culture after harvesting when the growth rate of the microalgae is reduced to be less than 50% of the growth rate before adding the biogas slurry for the first time. The invention provides a detailed operation method for reasonably utilizing biogas slurry to realize microalgae cultivation, which improves the yield of microalgae, reduces the microalgae cultivation cost and improves the gain effect of microalgae cultivation and other processes to the maximum extent.

It is known from the combination of the two patents CN103275900A and CN107460128A that the harmful components of the biogas slurry after fermentation in the two prior arts are not determined, and decolorization and deodorization are not performed, so that the cultured spirulina is not only bad in shape, but also not food safety grade, and is not suitable for industrialization. How to utilize biogas slurry to culture spirulina has low cost and high yield, and is a topic worthy of research.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for culturing spirulina based on biogas slurry, which takes waste biomass livestock and poultry manure, namely biogas slurry as a raw material, is used for culturing spirulina after optimized treatment reaches the standard, the growth condition of the spirulina is good, harmful ingredients in the biogas slurry are reduced, and a good purification effect is achieved.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a method for culturing spirulina based on biogas slurry comprises the following steps:

step 1, selecting biogas slurry, and pretreating to obtain supernatant for later use, wherein OD430 of the obtained supernatant is lower than 0.4, and the heavy metal content reaches GB5084-2005 and meets the standards of farmland irrigation water quality;

step 2, selecting spirulina, and culturing until OD560 is 0.9-10 for later use;

step 3, diluting the supernatant by 8-20 times, adding a Zarrouk culture medium into the diluted supernatant, and uniformly stirring to obtain an optimized culture medium for later use;

and 4, adding spirulina liquid into the optimized culture medium, and performing illumination culture.

As an improvement, the biogas slurry pretreatment steps are as follows: firstly, standing and precipitating 5 times of the concentrated chicken manure, namely biogas slurry, and taking supernatant; secondly, placing the activated carbon in pure water overnight by using activated carbon with different specifications purchased from the belonged to Plumbum preparatium waste activated carbon regeneration company, and then drying the activated carbon every other day for use; thirdly, deodorizing and decoloring the supernatant by using activated carbon with different specifications (430, 830 and 1240 specifications) in sequence, and filtering for 2 times at the flow rate of 1.2 ml/min; and finally, sterilizing by using an ultraviolet sterilizer to obtain the proper biogas slurry, and taking the spirulina liquid of the spirulina into the supernatant according to the volume.

As a modification, the volume fraction of Zarrouk medium in step 3 was 8%.

The improvement is that in the step 4, the illumination culture condition is that the temperature is 26 +/-1 ℃, the illumination intensity is 8000lux, the light period L: D (light: dark) is 12:12, and the flask is periodically shaken every morning at 8:00, noon at 12:30 and evening at 18: 30.

In the improvement, the volume ratio of the spirulina liquid to the optimized culture medium in the step 4 is 1: 3.

Has the advantages that:

compared with the prior art, the method for culturing the spirulina based on the biogas slurry has the advantages that the common biogas slurry in life is used as a main nutrient source to culture the spirulina, the nutrient substance plasma in the biogas slurry is detected, the culture medium is optimized, and the culture medium added with the biogas slurry is used for optimizing and amplifying the spirulina culture process, so that the water quality is purified, and the economic efficiency of spirulina production is improved.

(1) The process of culturing the spirulina by using the biogas slurry is actually the process of purifying the biogas slurry by using the spirulina, the spirulina cannot be directly cultured in the biogas slurry, the biogas slurry needs to be diluted and pretreated, and the concentration is most suitable to be 10%;

(2) after the Zarrouk culture solution with different proportions is added into the biogas slurry diluted by 10 times, the nutrient factors required by the growth of the spirulina are supplemented, and the spirulina shows better growth trend. After 8% of Zarrouk culture solution is added, the spirulina has good absorption effect on elements such as nitrogen, phosphorus and the like in water and NH ₄ ⁺ -N、NO ₃ -N、NO ₂ The clearance rates of N and P reach 58.58%, 83.03%, 77.4% and 53.12% respectively;

(3) the biogas slurry can replace zarrouk to be used as nutrient salt for culturing spirulina.

Drawings

FIG. 1 shows the growth of spirulina in biogas slurry with different dilution times;

FIG. 2 shows the growth of Spirulina in biogas slurries containing varying proportions of Zarrouk.

Detailed Description

The Spirulina platensis is provided by Totai century Biotechnology Co., Ltd.

The culture medium (Zarrouk) used in the present invention is a Spirulina platensis formula of Toltitussi Biotechnology Ltd. Sterilizing at 121 deg.C for 20min, cooling to room temperature, and using, the specific formula is shown in the following table 1:

TABLE 1zarrouk Medium

Composition (I)	Finished product formula (g/L)
		NaHCO ₃	10.00
KH ₂ PO ₄	0.40
		K ₂ HPO ₄	0.00
NaNO ₃	1.20
		NaCl	1.00
MgSO ₄ ·7H ₂ O	0.20
		Na ₂ EDTA·2H ₂ O	0.02
FeSO4·7H2O	0.01

Example 1 biogas slurry pretreatment

Collecting 5 times of concentrated chicken manure (laying hen) biogas slurry in 2019 in 1 month from Haian Puhao bioenergy Co.

Firstly, standing and precipitating 5 times of the concentrated chicken manure, namely biogas slurry, and taking supernatant;

secondly, placing the activated carbon in pure water overnight by using activated carbon with different specifications purchased from the belonged to Plumbum preparatium waste activated carbon regeneration company, and then drying the activated carbon every other day for use;

thirdly, deodorizing and decoloring the supernatant by using activated carbon with different specifications (430, 830 and 1240 specifications) in sequence, and filtering for 2 times at the flow rate of 1.2 ml/min;

and finally, sterilizing by using an ultraviolet sterilizer, and performing component analysis, wherein OD430 is lower than 0.4, and the contents of mercury, chromium, arsenic, lead and other heavy metals reach GB5084-2005 (water quality standard for farm irrigation), thus obtaining the applicable biogas slurry.

Example 2 growth of Spirulina in biogas slurries at different dilution times

Algae generally produce organic substances by absorbing inorganic salts such as N, P from the outside through photosynthesis, and these inorganic salts are essential for the growth of algae. However, the content of the nutrient elements is not more and better, and according to the Sherfford resistance law, organisms have resistance ranges to various factors in the growth process, if a certain amount of one essential element in the culture solution exceeds a certain amount, toxic effects can be generated on the algae, the growth and the propagation of the algae are influenced, and serious people can cause the death of the algae.

The biogas slurry filtered in example 1 was centrifuged, and the supernatant (diluted 8, 10, 20 times) was diluted to 150ml biogas slurry. Equal spirulina platensis solution (50ml) is selected from the spirulina platensis solution, mixed culture is carried out with biogas slurry (1:3), 3 groups are arranged in parallel, and OD is measured twice in each culture medium. Controlling the culture conditions in an illumination incubator, wherein the temperature is controlled to be 26 +/-1 ℃, the light intensity is controlled to be 8000lux, and the light period is controlled to be L: d (light: dark) is 12:12, and the growth condition of the spirulina is observed by shaking regularly at 8:00 in the morning, 12:30 in the noon and 18:30 in the evening. Culturing for 10 days, measuring the OD value at 560nm every other day in each bottle for 2 times (using the OD value to represent the biomass), making a growth curve to study the growth condition of the spirulina platensis in biogas slurry with different dilution times, and determining the optimal dilution time of the biogas slurry for the growth of the spirulina platensis to determine the tolerance range of the spirulina platensis.

The growth conditions of spirulina platensis in 5-time concentrated layer chicken manure biogas slurry with the dilution ratio of 8 times, 10 times and 20 times are shown in figure 1 and table 2. Spirulina can grow normally in the concentrated biogas slurry, and has the best growth effect in the 10 times diluted biogas slurry, and then the diluted 20 times biogas slurry, and has poor growth in the 8 times diluted biogas slurry, and 3 kinds of the concentrated biogas slurryOD in concentration ₅₆₀ All showed significant differences after day 2 (P)<0.05). When the spirulina is just inoculated into 3 biogas slurries with different concentrations, the growth of the spirulina is retarded; from day 3 to day 4, the cell proliferation rate increased and the growth of Spirulina platensis entered logarithmic phase. Wherein OD is obtained in the biogas slurry diluted by 10 times in the 4 th day ₅₆₀ The value reaches a peak. The spirulina grown in the biogas slurry with 3 concentrations showed the highest growth rate on day 4. From day 5, the growth of the algae decreased, the growth trend decreased, and the growth of the spirulina entered the stationary phase. The concentration of the spirulina is diluted by 10 times according to the growth condition of the spirulina in the diluted biogas slurry, economic conditions, future use and other conditions.

TABLE 2 growth of Spirulina platensis in biogas slurry at different dilution times

Note: the difference in the upper case letters indicates significant difference between the same columns (p <0.05)

Capital letters with different superscripts indicate significant differences between the same lines (p <0.05)

This shows that the dilution rate has obvious inhibition effect on the growth of the spirulina, on one hand, the organic matter concentration in the biogas slurry is high, the organic matter concentration is not easy to be absorbed by the spirulina, and the spirulina liquid is gradually changed from green to yellow, so that the spirulina is dead; on the other hand, in the high-concentration biogas slurry, the culture solution is dark in color and weakened in light transmittance, so that the culture solution is far away from a light saturation point in an autotrophic culture mode, and the autotrophic growth is inhibited. When the spirulina is just inoculated into the biogas slurry, the spirulina is in a slow growth stage, an in-vivo enzyme system and cell components are continuously perfected in the spirulina to synthesize enzymes necessary for growth, when the spirulina is cultured to the fourth day, the spirulina enters a logarithmic growth stage, the cell metabolic capacity is strong, the spirulina growth reaches a peak value in the sixth day and then enters a slow growth stage, a growth curve is in a descending trend, the cell division speed is reduced, the spirulina grows slowly, and nutrient substances in the biogas slurry are greatly consumed.

Example 3 growth of Spirulina after addition of Zarrouk Medium

According to the embodiment 1 and the embodiment 2, the selected biogas slurry is pre-treated and then diluted by 1 time for standby. The results of the growth of Spirulina platensis after adding different proportions of Zarrouk culture solution are shown in FIG. 2. After 2%, 4%, 6%, 8% and 10% of Zarrouk culture solution is added into the 10-time diluted biogas slurry, the spirulina platensis all show better growth, and the growth effect is positively correlated with the adding proportion of the Zarrouk culture solution. The spirulina platensis has better growth rate within 6 days. The growth effects were relatively better at 6%, 8% and 10% concentrations, and relatively worse at 2% and 4% concentrations. As can be seen from Table 3, OD in the 2% and 4% concentrations ₅₆₀ The values showed significant differences only on days 2 and 4 (P)<0.05) was not significantly different in other days. OD in 4% and 6% concentration ₅₆₀ The difference in value is significant (P)<0.05), indicating that the effect at 6% concentration is clearly better than 4%. The differences between 6% and 8% were significant on

days

2 and 6, not significant on other days, OD in concentrations of 8% and 10% ₅₆₀ The difference is not significant (P is more than 0.05), and the addition proportion of 8 percent is better by comprehensively considering the cost and the growth condition.

TABLE 3 growth of Spirulina platensis in biogas slurries containing varying proportions of Zarrouk

Note: the lower case letter superscripts differ significantly between columns (P <0.05) and the upper case letter superscripts differ significantly between rows (P < 0.05).

Example 4 NH in biogas slurry after Spirulina platensis was cultured in biogas slurry ₄ ⁺ -N、NO ₃ -N、NO ₂ Determination of the-N, P content

Selecting 50ml of spirulina platensis, respectively inoculating the 50ml of spirulina platensis into 2 250ml conical flasks, wherein one flask is filled with 150ml of biogas slurry diluted by 10 times, the other flask is filled with 150ml of biogas slurry diluted by 10 times after 8% Zarrouk is added, and the two flasks are compared to each other and are respectively provided with 3 parallel groups.

Placing all conical flasks in an intelligent illumination incubator for culture, and culturing algae seedsThe parts are as follows: the temperature is 26 +/-1 ℃, the illumination intensity is 8000lux, the photoperiod L: D (light: dark) is 12:12, and the flask is shaken at the timing of 8:00 in the morning, 12:30 in the noon and 18:30 in the evening. The whole culture period is 10 days, 30ml biogas slurry containing Spirulina platensis is taken every other day, centrifugated for 30min at 18 deg.C under 10000rmp by Eppendorf5804R desk-top high-speed large-capacity centrifuge, supernatant is taken, ammonia Nitrogen (NH) is measured by Nashin reagent method, zinc-cadmium reduction method, diazo-azo photometry and molybdenum blue method respectively ₄ ⁺ -N), nitrate Nitrogen (NO) ₃ -N), nitrous Nitrogen (NO) ₂ -N) and active phosphorus (P). Measuring NH in water at intervals by using ultraviolet wind-solar photometer and matched cuvette ₄ ⁺ -N、NO ₃ -N、NO ₂ The absorbance values of N and P are zeroed using pure water as a reference, and the contents are calculated from the respective index standard curves.

NH treatment with excel 2019 ₄ ⁺ -N、NO ₃ -N、NO ₂ Standard curve data for N and P are plotted. The abscissa is the concentration ρ (mg/L) and the ordinate is the corrected absorbance A' of the series of standard solutions.

A'＝b'{ρ}(mg/L)

Due to occasional errors in the experiment, the resulting regression equation will generally not pass through the origin, and there is an intercept a:

A'＝a+b'{ρ}(mg/L)

calculating the content of each index in water:

each treatment was set to 3 replicates and the results averaged. The raw data were statistically analyzed using excel 2019. Mean ± standard error (Means ± SE) of 3 replicates were used as final data; all data were analyzed using single-factor analysis of variance using SPSS12.0 statistical analysis software, with P <0.05 indicating significant differences. Specific results are shown in tables 4 and 5 below.

TABLE 4 NH in biogas slurry ₄ ⁺ -N、NO ₃ -N、NO ₂ Variation in the-N, P content

Note: the difference between the same columns in the superscript is significant (P <0.05)

As can be seen from Table 4, NO in biogas slurry ₃ -N、NO ₂ The contents of-N and P in the first 4 days are decreasing, NH ₄ ⁺ The content of-N decreases with the number of days of cultivation. NH (NH) ₄ ⁺ The content of-N differed significantly between days 0, 2 and 4 (P)<0.05)，NO ₃ -N、NO ₂ The contents of N and P are both significantly different on days 2 and 4 (P)<0.05)。

TABLE 5 NH after addition of 8% Zarrouk ₄ ⁺ -N、NO ₃ -N、NO ₂ Variation in the-N, P content

As shown in Table 5, 8% of Zarrouk culture medium was added to the biogas slurry to obtain NH in the water ₄ ⁺ -N、NO ₃ -N、NO ₂ The contents of-N and P are reduced, and the spirulina platensis can be cultured for 9 days to obtain NH ₄ ⁺ -N、NO ₃ -N、NO ₂ The removal rates of-N and P reach 58.58%, 83.03%, 77.4% and 53.12%, respectively.

In conclusion, the treated biogas slurry can completely replace most of finished spirulina culture media, the cost is saved in large-scale production, economic algae is provided, and a way is developed.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are included in the present invention.

Claims

1. A method for culturing spirulina based on biogas slurry is characterized by comprising the following steps: step 1, selecting biogas slurry, and pretreating to obtain supernatant for later use, wherein OD430 of the obtained supernatant is lower than 0.4, and the heavy metal content reaches GB5084-2005, which meets the standards of farmland irrigation water quality, wherein the pretreatment of the biogas slurry comprises the following steps: firstly, standing and precipitating 5 times of the concentrated chicken manure, namely biogas slurry, and taking supernatant; secondly, placing the activated carbon in pure water overnight by using activated carbon with different specifications purchased from the belonged to Plumbum preparatium waste activated carbon regeneration company, and then drying the activated carbon every other day for use; thirdly, deodorizing and decoloring the supernatant by using active carbon with different specifications in sequence, and filtering for 2 times at the flow rate of 1.2 ml/min; finally, sterilizing by using an ultraviolet sterilizer to obtain the proper biogas slurry, and taking the spirulina liquid of the spirulina into the supernatant according to the volume; step 2, selecting spirulina, and culturing until OD560 is 0.9-10 for later use; step 3, diluting the supernatant by 8-20 times, adding a Zarrouk culture medium into the diluted supernatant, wherein the volume fraction of the Zarrouk culture medium is 8%, and uniformly stirring to obtain an optimized culture medium for later use; and 4, adding a spirulina liquid into the optimized culture medium, wherein the volume ratio of the spirulina liquid to the optimized culture medium is 1:3, performing illumination culture at the temperature of 26 +/-1 ℃ and the illumination intensity of 8000lux, wherein the photoperiod L: D (light: dark) =12:12, and shaking the bottle at the fixed time of 8:00 in the morning, 12:30 in the noon and 18:30 in the evening.