CN114410694B - Preparation method and application of microalgae biological hydrogen production system - Google Patents

Abstract

The invention provides a preparation method of a long-acting microalgae biological hydrogen production system, which comprises the following steps: s1: adding organic carbon compound and flocculant into microalgae liquid culture with pH of 6-8, wherein the organic carbon compound is used in an amount to make its concentration 0.5-500 mM, and the flocculant is used in an amount to make its concentration 0.1-100 g/L; s2: and then, sealing the culture system, and standing in an environment with the illumination intensity of 1000-5000Lux and the temperature of 20-30 ℃ to enable microalgae to form aggregates and enter an overall anaerobic condition for continuous growth, so that hydrogen is efficiently produced for a long time. The microalgae biological hydrogen production system adopts a compatible culture mode to culture microalgae aggregates, can realize long-term and efficient hydrogen production at low cost, solves the problem that the microalgae hydrogen production cost and efficiency are difficult to be compatible in the prior art, and has large-scale practical potential.

Description

Preparation method and application of microalgae biological hydrogen production system

Technical Field

The invention relates to the field of microalgae biological hydrogen production, in particular to a preparation method of a microalgae biological hydrogen production system.

Background

Hydrogen is considered an ideal fossil fuel substitute due to its advantages of zero carbon emissions and high heating value. However, hydrogen currently used commercially in the market is still produced from traditional fossil energy sources such as natural gas, which requires a large energy input and is accompanied by a large carbon dioxide production, thus making it difficult to achieve net zero carbon emissions. In order to assist in achieving the 'double carbon' target, it is important to develop a hydrogen preparation method with low energy consumption and zero emission.

The biological hydrogen production can catalyze the hydrogen production under the condition of normal temperature and normal pressure by using renewable organisms as a catalyst, and is a green hydrogen preparation method with low energy consumption. Especially microalgae biological hydrogen production, because microalgae is a single-cell photosynthetic organism which is easy to culture in a large scale, the method not only can produce hydrogen with low energy consumption under the conditions of normal temperature and normal pressure, but also has the potential of converting light energy and consuming carbon dioxide, thereby being a hydrogen preparation method with application prospect and clean zero carbon emission in the whole process. However, microalgae require an anaerobic environment to be active in producing hydrogen, but microalgae photosynthesis inevitably produces oxygen. Therefore, how to construct an anaerobic environment with low cost under the illumination condition is a bottleneck problem of microalgae hydrogen production development and application.

Recently, a biological hydrogen production scheme based on microalgae aggregate can realize hydrogen production by utilizing light energy without adding an oxygen scavenger or depriving sulfur element, which provides a new idea for low-cost biological hydrogen production of microalgae. The biological hydrogen production system based on microalgae aggregate is based on the principle that microalgae cells in the aggregate lack illumination, so photosynthesis is inhibited to stop releasing oxygen, but respiration is normally carried out without being influenced by the illumination, so that the cells in the aggregate consume the original oxygen through self respiration to form an anaerobic environment for producing hydrogen.

However, this type of anaerobic environment formed based on self respiration has a limited ability, and only microalgae at the core of the aggregate can produce hydrogen efficiently, so that the hydrogen production efficiency of the microalgae aggregate as a whole is low (CN 10496258 5B). Although the addition of dimethyl sulfoxide can promote the respiration of microalgae, and can form a larger anaerobic environment inside the microalgae aggregate, so that the hydrogen production yield and rate of the microalgae aggregate can be improved, dimethyl sulfoxide is a toxic substance and the dosage needs to be strictly controlled. In addition, although dimethyl sulfoxide can promote the respiration of microalgae cells, the microalgae culture mode is not changed, namely the microalgae are still in the photoautotrophic culture mode, the light energy is the only energy source, and the microalgae in the aggregate are severely limited due to lack of illumination, so that the cell activity is difficult to maintain for a long time, and the long-acting performance of hydrogen production is limited (CN 1072673 95B).

CN113736829a provides a simple photobiological hydrogen production system, and a preparation method and application thereof, microalgae are gathered together by a flocculant to form a local anaerobic environment inside the aggregate for hydrogen production. Although the difficulty of preparing microalgae aggregate to prepare hydrogen is reduced, the large environment of the obtained culture system is still in an aerobic environment, so that only cells in a local anaerobic environment inside the aggregate have hydrogen preparation capability, and the hydrogen preparation efficiency of the patent system is lower. In addition, the microalgae aggregate disclosed in the patent mainly depends on light energy, however, cells in the aggregate lack of illumination, so that the growth energy is insufficient, and the cell activity is difficult to maintain for a long time, so that the hydrogen production time of the patent is relatively short.

Forming an overall anaerobic environment can improve hydrogen production efficiency, but the cost of forming an overall anaerobic environment is expensive because of the addition of oxygen scavengers or desulfurization of the culture system; and the contradiction is that microalgae hydrogen production is unfavorable for microalgae growth, because microalgae growth requires oxygen, which is also why heterotrophic culture systems require artificial oxygen supplementation. It is therefore desirable to find a simple, low cost solution that can create an overall anaerobic environment and also allow for sustained growth of microalgae.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a preparation method of a microalgae biological hydrogen production system. The microalgae biological hydrogen production system adopts a compatible culture mode to culture microalgae aggregates, can realize long-term and efficient hydrogen production at low cost, solves the problem that the microalgae hydrogen production cost and efficiency are difficult to be compatible in the prior art, and has large-scale practical potential.

The technical scheme of the invention is as follows:

a preparation method of a microalgae biological hydrogen production system comprises the following steps:

s1: adding organic carbon compound and flocculant into microalgae liquid culture with pH of 6-8, wherein the organic carbon compound is used in an amount to make its concentration 0.5-500 mM, and the flocculant is used in an amount to make its concentration 0.1-100 g/L;

s2: and then, sealing the culture system, and standing in an environment with the illumination intensity of 1000-5000Lux and the temperature of 20-30 ℃ to enable microalgae to form aggregates and enter an overall anaerobic condition for continuous growth, so that hydrogen is efficiently produced for a long time.

Preferably, the size of the microalgae aggregate obtained in the step S2 is 50-5000 μm.

More preferably, the size of the microalgae aggregate obtained in the step S2 is 3500-5000 μm.

The invention provides a microalgae biological hydrogen production system prepared by the method.

The invention also provides application of the microalgae biological hydrogen production system in new energy development.

Further, the organic carbon compound is any one or a combination of at least two of glucose, fructose and acetic acid. It should be understood that other organic carbon compounds that can bring microalgae into a mixotrophic culture can be used in the present invention as well.

Further, the flocculant is any one or a combination of at least two of cationic etherified starch, cationic chitosan and cationic lignin. It should be understood that other flocculants capable of flocculating microalgae at near neutral conditions may be used in the present invention as well.

Further, the microalgae are green algae, including any one or a combination of at least two of chlamydomonas reinhardtii, chlorella vulgaris or chlorella pyrenoidosa. It should be understood that other microorganisms that can generate hydrogen in anaerobic environments using light energy and that can flocculate are equally suitable for use in the present invention.

Further, the microalgae in the microalgae liquid culture in the step S1 are cultured in a liquid culture medium, and the liquid culture medium is any one of TAP culture medium, SE culture medium or BG11 culture medium. Typically, the pH of the medium is adjusted to a pH in the range of 6-8 and maintained at a pH of 6-8 throughout to ensure that the microalgae have catalytic activity for hydrogen production. Because a suitable pH is a necessary condition for another microalgae to produce hydrogen in addition to anaerobic conditions.

Further, the microalgae aggregate in the step S2 is directly formed in the original microalgae liquid culture without any separation and enrichment and other additional treatment steps. For example, microalgae aggregates with hydrogen production capability can be formed without collecting the microalgae aggregates by centrifugation. Because the flocculant provided by the step S1 can directly and efficiently flocculate microalgae cells in the microalgae liquid culture to generate microalgae aggregates with larger volume and better stability, anaerobic environment can be effectively formed inside the aggregates, and the microalgae aggregates are not easy to be permeated by external oxygen. The method has remarkable superiority for a large-scale hydrogen production system, reduces operation steps, reduces production energy consumption and reduces production cost.

Furthermore, the microalgae aggregate in the step S2 is in a mixotrophic culture mode, and the whole culture system can be in an anaerobic environment without adding an additional respiration promoter or an oxygen scavenger, and can maintain the cell activity inside the aggregate for a long time. For example, the whole culture system can be anaerobically realized to realize high-efficiency hydrogen production without adding dimethyl sulfoxide or glucose oxidase and catalase. Because the organic carbon compound provided in the step S1 can enable microalgae aggregates to enter a concurrent culture mode, one side promotes the growth rate of microalgae, enhances the overall respiration, greatly improves the oxygen consumption capacity of microalgae aggregation, and can form an overall anaerobic environment to produce hydrogen with high efficiency; on the other hand, the energy supply of the microalgae cells in the aggregate can be maintained, so that the good activity of the microalgae cells in the aggregate can be ensured, and the hydrogen production time can be effectively prolonged. The method has remarkable superiority for practical application of microalgae hydrogen production, reduces production requirements and improves production efficiency.

Step S2, microalgae form obvious cell aggregates and enter a concurrent culture mode, the respiration of the microalgae is enhanced, the microalgae in the aggregates can obtain energy supply, and the whole culture system enters an anaerobic environment and maintains higher cell activity, so that long-term and efficient biological hydrogen production is realized.

In practice, the amount of organic carbon compound and the amount of flocculant may be adjusted according to the specific microalgae cell concentration, so long as the system can ensure that obvious microalgae cell aggregates can be produced, and an effective mixotrophic culture mode can be entered.

It should be understood that the microalgae cell aggregate refers to a three-dimensional multicellular structure formed by the mutual adhesion and agglomeration of microalgae cells, and when the microalgae cells form the aggregate, the microalgae cells in the aggregate consume oxygen through self respiration so as to form an anaerobic environment required for hydrogen production; the culture mode of the culture system is that microalgae in the culture system can be autotrophized by utilizing light energy, and can also be heterotrophized by utilizing organic carbon compounds, so that on one hand, the growth of the microalgae can be accelerated, the consumption of oxygen is promoted, and the whole culture system can be in an anaerobic environment, so that the hydrogen production efficiency of the microalgae is improved; on the other hand, the microalgae cells in the aggregate can be ensured to obtain a growth energy source, and the long-term hydrogen production activity can be maintained.

The beneficial technical effects of the invention are as follows:

1. the invention uses organic carbon compound and flocculating agent to directly generate microalgae cell aggregate in microalgae liquid culture and make the microalgae cell aggregate enter a mixotrophic culture mode, thereby realizing the construction of a low-cost long-acting microalgae hydrogen production system. On one hand, the culture microalgae aggregate can accelerate the growth of microalgae and promote the consumption of oxygen, so that the whole culture system can be in an anaerobic environment to improve the hydrogen production efficiency of the microalgae; on the other hand, the microalgae cells in the aggregate can be ensured to obtain enough growth energy source, so that the microalgae cells can keep growing under the condition of lack of illumination, and the hydrogen production activity can be maintained for a long time.

2. The present invention achieves an overall anaerobic environment without the addition of any oxygen scavengers (similar to oxygen scavenging species such as glucose oxidase). The invention can effectively supply enough growth energy for microalgae only by simple illumination without additional oxygen input, thereby greatly reducing the demands of microalgae for dissolved oxygen and illumination and realizing high-efficiency and rapid growth.

3. The experimental result proves that the continuous hydrogen production time of the microalgae biological hydrogen production system is longer than 31 days, the yield is 22.46 micromoles per milliliter, the problem that the hydrogen production cost and the hydrogen production efficiency are difficult to be compatible in the common microalgae biological hydrogen production system is well solved, and the low-cost large-scale practical application development of the microalgae biological hydrogen production is hopefully promoted.

Drawings

FIG. 1 is a schematic diagram of the biological hydrogen production of a culture-compatible microalgae cell aggregate according to the invention;

FIG. 2 is a graph showing the results of analysis of the daily hydrogen production of the culture system in example 1 of the present invention;

FIG. 3 is a graph showing the results of analysis of oxygen content in the culture system of example 1 of the present invention;

FIG. 4 is a graph showing the cumulative hydrogen production statistics of the Chlorella pyrenoidosa aggregates cultured concurrently with 50mM glucose for 31 days in example 1 of the present invention;

FIG. 5 is a graph showing the result of light absorption at 750nm of the upper layer solution of the hydrogen production system obtained by the production method according to example 1 of the present invention in example 2 of the present invention;

FIG. 6 is a graph showing the observation result of an optical microscope of a chlorella pyrenoidosa aggregate of a hydrogen production system obtained by the preparation method of example 1 according to the present invention in example 3;

FIG. 7 is a graph showing the statistical result of the size of the chlorella pyrenoidosa aggregates of the hydrogen production system obtained by the preparation method of example 1 according to the present invention in example 3;

FIG. 8 is a graph showing the pH test results of the hydrogen production system according to the preparation method of example 1 of the present invention in example 4 of the present invention;

FIG. 9 is a graph showing the results of analysis of the cell activity of Chlorella pyrenoidosa, a hydrogen production system, obtained by the preparation method of example 1 according to the present invention in example 5;

FIG. 10 is a graph showing the chlorophyll content analysis result of Chlorella pyrenoidosa, a hydrogen production system, obtained by the preparation method of example 1 according to the present invention, in example 6 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the drawings and examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1:

this example provides a method for preparing a microalgae biological hydrogen production system, it being understood that the microalgae, liquid medium, flocculant, and organic carbon compound selected in this example are by way of example only and not limitation, and are Chlorella pyrenoidosa, TAP medium, cationic etherified starch, and glucose, respectively.

First, chlorella pyrenoidosa is cultured by using a TAP culture medium, when the light absorption of the culture reaches 0.3 at 750nm (OD 750 = 0.3), glucose and cationic etherified starch are added into the chlorella pyrenoidosa liquid culture, so that the chlorella pyrenoidosa aggregates are flocculated under the near neutral pH condition and enter a concurrent culture mode, and then the content of oxygen and hydrogen in the culture system is monitored by using a gas chromatograph. And monitoring the change condition of the oxygen and hydrogen content in the photobiological hydrogen production system by using a gas chromatograph. It should be understood that the microalgae biological hydrogen production system provided in accordance with the present invention is also suitable for larger scales well beyond 7mL, provided the feedstock is sufficient, the scale is not limited.

Wherein the gas chromatograph is Agilent 8860 type gas chromatograph (Agilent technology in America), and is equipped with TCD detector for simultaneously analyzing oxygen and hydrogen content, and carrier gas is high-purity nitrogen with flow rate of 3-30mL/min.

Wherein glucose (CAS number 14431-43-7) is purchased from Guogong chemical reagent Co., ltd; cationic etherified starch (abbreviated as CS; degree of substitution: 0.025-0.03, viscosity: 460-1200, spot < 0.5) was purchased from Anhui Kuer Bioengineering Co., ltd; cell activity assay kit (HR 0489) and chlorophyll content assay kit (GL 3175) were purchased from beijing berlaibo technologies limited; chlorella pyrenoidosa (GY-D12) and TAP medium were purchased from Shanghai optical Biotech Co.

Glucose and CS are added into the chlorella pyrenoidosa liquid culture under the illumination condition, so that obvious chlorella pyrenoidosa aggregates can be directly produced in an original culture system, and a mixotrophic culture mode capable of utilizing light energy for autotrophy and utilizing glucose for heterotrophy is entered. The growth rate of the chlorella pyrenoidosa under the mixotrophic culture is accelerated, the respiration is greatly improved, and the volume of the formed aggregate is also increased, so that the oxygen in the culture system can be effectively consumed, and the whole aggregate can be in an anaerobic environment to produce hydrogen with high efficiency. In addition, the mixotrophic culture can also effectively overcome the problem of energy supply shortage of cells in the aggregate due to lack of illumination, and can maintain the activity of the chlorella pyrenoidosa in the aggregate, so that long-term efficient hydrogen production of the chlorella pyrenoidosa aggregate can be realized, as shown in fig. 1. After a biological hydrogen production system based on the culture microalgae aggregate is established, a gas chromatograph is used for monitoring the content of oxygen and hydrogen in the hydrogen production system.

And (5) testing the optimal glucose dosage for hydrogen production by cultivating microalgae aggregate in a concurrent manner. An airtight transparent glass tube with a volume of 7mL was used as a reaction vessel, and 3mL of the Chlorella pyrenoidosa culture with OD750=0.3 was added to each glass tube. Next, 0mg (0 mM) was added to each of the different glass test tubes; 6.75mg (12.5 mM); 13.5mg (25 mM); 27mg (50 mM); 54mg (100 mM) glucose was used as a conditional control at various glucose concentrations. The experimental group was based on the above control group with different conditions, and 30mg of CS was added to each glass tube. Culturing under illumination condition of 25deg.C and 1500Lux after sealing the test tube, extracting 100 μl of test tube headspace gas at fixed time point by using airtight needle, and injecting into gas chromatograph sample inlet for analyzing hydrogen and oxygen content in hydrogen production system, and detecting conditions: TCD detector 200 ℃, column temperature box 100 ℃, carrier gas flow rate 3mL/min.

As a result, as shown in FIG. 2, 25mM, 50mM and 100mM of glucose, which are the best results, were obtained by the CS flocculation of Chlorella pyrenoidosa forming aggregates, and the long-acting hydrogen production was achieved, as shown in Table 1. The chlorella pyrenoidosa which is not flocculated by CS can generate hydrogen under the action of glucose, but the hydrogen yield is lower. Under the condition that glucose is not added, the yield of hydrogen of the chlorella pyrenoidosa forming the aggregate through CS flocculation is low, the hydrogen production time is short, and the natural free chlorella pyrenoidosa without CS flocculation does not generate hydrogen. However, at a glucose concentration of 12.5mM, the Chlorella pyrenoidosa flocculated to form aggregates by CS did not exhibit the ability to produce hydrogen for a long period of time, probably because 12.5mM glucose accelerates the proliferation of Chlorella pyrenoidosa, but at a relatively low glucose concentration, the respiration of Chlorella pyrenoidosa as a whole is not enhanced by photosynthesis, but rather the increase in cell number results in failure of CS to flocculate Chlorella pyrenoidosa effectively to form aggregates, and thus failure to form an effective anaerobic environment in the culture system, thereby failing to produce hydrogen for a long period of time.

TABLE 1

Additive components	Hydrogen production time (Tian)	Hydrogen accumulation yield (micromolar)
			0mM glucose+0 mg/mL CS	0	0
0mM glucose+10 mg/mL CS	15	0.98
			12.5mM glucose+0 mg/mL CS	15	4.43
12.5mM glucose+10 mg/mL CS	15	2.05
			25mM glucose+0 mg/mL CS	15	4.68
25mM glucose+10 mg/mL CS	21	19.12
			50mM glucose+0 mg/mL CS	15	3.22
50mM glucose+10 mg/mL CS	31	67.37
			100mM glucose+0 mg/mL CS	9	0.20
100mM glucose+10 mg/mL CS	21	31.40

This inference can be confirmed from the results of the analysis of the oxygen content in the reaction system (FIG. 3), and 12.5mM glucose can significantly increase the oxygen content in the Chlorella pyrenoidosa culture system, and the overall oxygen concentration is maintained above 20% despite the addition of CS to reduce the oxygen content in the system, so that the system fails to produce hydrogen. Glucose in 25mM, 50mM and 100mM can form anaerobic environment in the culture of Chlorella pyrenoidosa flocculated and aggregated by CS, and can maintain the anaerobic environment for a long period of time, so that long-term hydrogen production can be realized. In addition, 25mM and 100mM glucose, the hydrogen production capacity of the culture protein Chlorella pyrenoidosa aggregate is lower than that of 50mM glucose, and the reasons are probably caused by factors such as pH and cell activity of the system. In conclusion, the experimental results show that the system can effectively enter the whole anaerobic environment by concurrently culturing the chlorella pyrenoidosa aggregate under the proper concentration of the organic carbon compound, thereby realizing high-efficiency hydrogen production.

After determining that 50mM glucose is the optimal concentration for hydrogen production by the culture protein and chlorella pyrenoidosa aggregate, testing the accumulated hydrogen production effect of the system, and using 50mM glucose culture protein and chlorella pyrenoidosa aggregate, as shown in figure 4, realizes microalgae biological hydrogen production for up to 31 days, and the accumulated hydrogen yield reaches 67.37 micromoles and the yield reaches 22.46 micromoles per milliliter;

example 2:

and (5) analyzing the effect influence of different glucose concentrations on CS flocculation protein chlorella pyrenoidosa. The absorbance of the supernatant of the hydrogen production system prepared in example 1 at 750nm was measured by an enzyme-labeled instrument to analyze the flocculation of Chlorella pyrenoidosa in the system.

As shown in FIG. 5, 12.5mM glucose was able to significantly enhance the absorbance at 750nm of the supernatant of the culture system of C.pyrenoidosa without CS flocculation, and the absorbance at 750nm of the supernatant was also higher than that of the other culture system with CS flocculation, indicating that 12.5mM glucose was able to promote growth of C.pyrenoidosa, thereby reducing the flocculation effect of limited amounts of CS on C.pyrenoidosa. Although 25mM glucose was also significantly enhanced, the supernatant of the culture system of Chlorella pyrenoidosa which had not been flocculated by CS was absorbed photometrically at 750nm, but the flocculation effect of CS was not significantly affected. Glucose at 50mM and 100mM does not have negative effects on CS flocculation of Chlorella pyrenoidosa, and has the ability to induce flocculation of Chlorella pyrenoidosa itself. In a word, it can be shown from the above experimental results that the flocculation effect of CS on Chlorella pyrenoidosa is not affected by the proper organic carbon compound concentration, and the system can flocculate with high efficiency;

example 3:

and (5) observing and analyzing the chlorella pyrenoidosa aggregate under the mixotrophic culture. The hydrogen production system prepared in example 1 was analyzed by observation with an optical microscope, and the generation of chlorella pyrenoidosa aggregates and the size of the aggregates were measured.

Microscopic observations are shown in FIG. 6 (scale bar 300 μm), and significant Chlorella pyrenoidosa aggregates were produced in the primary culture system after CS flocculation of Chlorella pyrenoidosa in the culture medium. Wherein, the formation of the chlorella pyrenoidosa aggregate in the culture system is most obvious at the concentration of 50mM glucose. The concentration of 100mM, 25mM, 12.5mM glucose was followed sequentially. In addition, at a concentration of 12.5mM glucose, CS flocculates Chlorella pyrenoidosa to form aggregates, unlike Chlorella pyrenoidosa aggregates formed by CS flocculation under photoautotrophic culture. No obvious Chlorella pyrenoidosa aggregates were produced in either of the culture systems without CS.

As a result of statistical analysis of the size of the above-mentioned Chlorella pyrenoidosa aggregates, the size of the Chlorella pyrenoidosa aggregates produced by CS flocculation was concentrated and distributed around 300. Mu.m, and the maximum size diameter exceeded 3500. Mu.m, at a concentration of 50mM glucose, as shown in FIG. 7. In the culture system with the rest glucose concentration, the average size of the chlorella pyrenoidosa aggregates generated by CS flocculation is below 300 mu m. This demonstrates that in a 50mM glucose culture system, chlorella pyrenoidosa forms larger sized aggregates most readily under CS, thereby facilitating the formation of anaerobic conditions for hydrogen production. Although the size of the formed chlorella pyrenoidosa aggregate is relatively large in a 100mM glucose culture system, the hydrogen production effect is less than that of a 50mM glucose culture system, and the pH or cell activity in the system is probably unfavorable for hydrogen production;

example 4:

pH analysis of the chlorella pyrenoidosa system under the culture of the mixotrophic strain. The culture of the hydrogen production system prepared in example 1 was subjected to pH analysis, and after shaking the culture sufficiently, the pH of the culture was measured using a pH meter.

As shown in FIG. 8, 50mM and 100mM glucose can significantly lower the pH of the culture of Chlorella pyrenoidosa in a natural free state, and CS flocculation to form Chlorella pyrenoidosa aggregates can slow down the pH of the culture system, but the pH of the culture system is still reduced to about 5 under the action of 100mM glucose, and the pH of the culture system of Chlorella pyrenoidosa cultured by 50mM glucose is maintained at about 6. Because the optimal pH range of the hydrogen production by the microalgae is 6-8, 100mM glucose is used for culturing the chlorella pyrenoidosa aggregate, and the hydrogen production effect is less than that of 50mM glucose;

example 5:

and (5) analyzing the activity of the chlorella pyrenoidosa cells in the mixotrophic culture. Cell activity analysis was performed on the chlorella pyrenoidosa culture in the hydrogen production system prepared in example 1, after shaking the culture sufficiently, 200 μl of the culture was taken out and mixed with 20 μl of the cell activity analysis liquid, and incubated for 4 hours at room temperature with shaking at a dark place, followed by centrifugation at 1 ten thousand revolutions for 15 minutes using a centrifuge, and the supernatant was measured for light absorption value at 490nm (OD 490) using a microplate reader, and then the following calculation formula was adopted:

cell activity (%) = (OD 490e-OD490 b)/(OD 490c-OD490 b) ×100;

OD490e is the experimental group, namely, the CS flocculated chlorella pyrenoidosa is cultivated in a mixotrophic way, and the sample treated by the cell activity analysis reagent solution absorbs light at 490 nm;

OD490c is the control group, namely, photoautotrophically culturing the naturally free dispersed Chlorella pyrenoidosa, and the light absorption of the sample treated by the cell activity analysis reagent solution at 490 nm;

OD490b is blank TAP culture solution, and the sample treated by the cell activity analysis reagent solution absorbs light at 490 nm;

obtaining the cell activity of the chlorella pyrenoidosa in the culture system;

as shown in FIG. 9, 12.5mM and 25mM glucose were used to culture the naturally free Chlorella pyrenoidosa in a concurrent manner, and the cell activity was increased, while 50mM glucose had little effect on the cell activity of the naturally free Chlorella pyrenoidosa, and 100mM glucose was used to decrease the cell activity. After CS flocculation to form aggregates, the cell activity of the system is correspondingly improved, wherein the improvement is most remarkable for a 25mM glucose culture system, the improvement is next to a 50mM glucose culture system, and the improvement is least for a 100mM glucose culture system.

From the above results, it can be demonstrated that the cell activity of the 50mM glucose culture protein Chlorella pyrenoidosa aggregates is significantly higher than that of the 100mM glucose culture system, and therefore, the hydrogen production effect of the 50mM glucose culture system is superior to that of the 100mM glucose culture system. Although the cell activity of the 25mM glucose culture protein chlorella pyrenoidosa aggregate is highest, the system also forms an anaerobic environment, but the hydrogen yield is lower than that of the 50mM glucose culture system, probably because 25mM glucose enables the protein chlorella pyrenoidosa to enter a normal respiratory sugar metabolism path, and because the aggregate volume in the system is smaller, the illumination of algae cell contact is more sufficient, and the algae cell contact is easy to exchange substances with a solution, so that more protein chlorella pyrenoidosa in the system is probably in a state of dynamic balance of oxygen release and oxygen consumption, and more glucose is used for tricarboxylic acid circulation, so that more ATP is generated, high cell activity is shown, and a smaller amount of hydrogen is generated;

example 6:

and (5) analyzing chlorophyll content of the chlorella pyrenoidosa under the mixotrophic culture. Chlorophyll content analysis was performed on the chlorella pyrenoidosa culture in the hydrogen production system prepared in example 1, after shaking the culture sufficiently, 200. Mu.L of the culture was taken out and mixed with 200. Mu.L of chlorophyll content analysis solution, incubated for 4 hours at room temperature with shaking at a dark place, followed by centrifugation at 1 ten thousand revolutions for 15 minutes using a centrifuge, and the supernatant, i.e., chlorophyll crude, was taken and the light absorption values of the samples at both 665nm (A665) and 649nm (A649) were measured with a microplate reader. According to the following formula:

total chlorophyll content (mg) =ct×n×v;

CT＝6.63×A665+18.08×A649；

n=dilution factor;

v = sample system (L);

obtaining the total chlorophyll content result of the chlorella pyrenoidosa;

as shown in FIG. 10, the results show that 25mM, 50mM and 100mM glucose-concurrently cultured Chlorella pyrenoidosa all reduce the chlorophyll content, and the higher the concentration, the more remarkable the reduction, while the formation of an aggregate through CS can slow down the reduction of the chlorophyll content, and the more remarkable the system for the culture with the concentration of 50mM and 100mM glucose. Since microalgae cultivated in a mixotrophic culture are reduced in dependence on light energy and the higher the glucose concentration, the less dependent the microalgae on photosynthesis productivity, the chlorophyll content decreases with increasing glucose concentration. In addition, high concentrations of glucose can promote metabolism of acidic products by microalgae cells, resulting in a decrease in system pH, which is an acidic pH environment that is detrimental to chlorophyll biosynthesis. The formation of microalgae aggregates by CS can reduce to some extent the direct contact of microalgae cells with high concentration glucose, thus avoiding the production of a large amount of acid metabolites, slowing down the inhibition of chlorophyll synthesis and therefore exhibiting a higher chlorophyll content than microalgae in the free state.

Although the embodiments of the present invention have been disclosed in the foregoing description and drawings, it is not limited to the details of the embodiments and examples, but is to be applied to all the fields of application of the present invention, it will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.

Claims (3)

1. The preparation method of the microalgae biological hydrogen production system is characterized by comprising the following steps of:

s1: adding an organic carbon compound and a flocculating agent to the microalgae liquid culture at pH 6-8, wherein the organic carbon compound is used in an amount such that the concentration of the organic carbon compound is 25mM-50mM, and the flocculating agent is used in an amount such that the concentration of the flocculating agent is 10g/L;

the organic carbon compound is glucose; the flocculant is cationic etherified starch;

s2: then, sealing the culture system, standing in an environment with the illumination intensity of 1000-5000Lux and the temperature of 20-30 ℃ to enable microalgae to form aggregates and enter an overall anaerobic environment for continuous growth, so as to generate hydrogen;

the size of the microalgae aggregate obtained in the step S2 is 300-3500 mu m;

the microalgae is Chlorella pyrenoidosa.

2. The method according to claim 1, wherein in the microalgae liquid culture in step S1, the microalgae is cultured in a liquid medium, and the liquid medium is any one of TAP medium, SE medium or BG11 medium.

3. Use of the method for preparing a microalgae biological hydrogen production system as claimed in claim 1 or 2 in hydrogen energy development.