CN110951819A

CN110951819A - Method for improving HAU-M1 photosynthetic bacteria flora to produce hydrogen by utilizing corn straw fermentation

Info

Publication number: CN110951819A
Application number: CN202010064821.7A
Authority: CN
Inventors: 路朝阳; 张全国; 胡建军; 王毅; 荆艳艳; 贺超; 金鹏; 刘泽先; 张学婷; 岳田
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2020-04-03
Anticipated expiration: 2040-01-20
Also published as: CN110951819B

Abstract

The invention belongs to the technical field of microbial fermentation and biological hydrogen production, and particularly relates to a method for improving HAU-M1 photosynthetic bacteria flora and producing hydrogen by utilizing corn straw fermentation. According to the method, the corn straws subjected to cellulose degradation are used as hydrogen production substrates, the catalyst is added to promote the HAU-M1 photosynthetic bacteria to produce hydrogen efficiently, and the fermentation way in the hydrogen production process of the corn straw photosynthetic organisms is changed by adding the active carbon, the diatomite and the zeolite with different concentrations, so that the hydrogen production amount of photosynthesis is obviously improved. The diatomite obviously improves the photosynthetic hydrogen production effect of the corn straws, and the hydrogen production amount is improved by 15.93 percent compared with that of a control group; the zeolite obviously reduces the concentration of propionic acid in the reaction solution, changes the way of corn straw light fermentation, and improves the hydrogen yield by 33.33 percent compared with a control group. The method has the advantages of reasonable design, clear steps, simplicity and feasibility, and can improve the hydrogen production efficiency of the photosynthetic bacteria group HAU-M1 by using the corn straws.

Description

Method for improving HAU-M1 photosynthetic bacteria flora to produce hydrogen by utilizing corn straw fermentation

Technical Field

The invention belongs to the technical field of microbial fermentation and biological hydrogen production, and particularly relates to a method for improving HAU-M1 photosynthetic bacteria flora and producing hydrogen by utilizing corn straw fermentation.

Background

Hydrogen is a clean energy source with high calorific value, and has important functions in many fields. Compared with the traditional industrial hydrogen production, the biological hydrogen production has better prospect, does not need to consume a large amount of non-renewable resources, does not generate a large amount of greenhouse gas, and can degrade pollutants such as agricultural biomass wastes, food processing plant sewage, livestock and poultry manure and the like. The biological hydrogen production mainly comprises two modes of dark fermentation and light fermentation. Compared with the hydrogen production by the dark fermentation organisms, the hydrogen production by the light fermentation has the advantages of stable hydrogen production rate, long hydrogen production period, high substrate conversion efficiency and the like. A great deal of research on hydrogen production by photosynthetic organisms is carried out by the predecessors, such as temperature, pH value, illumination intensity, cellulose concentration, inoculum size, colony culture and other single factors, and interaction of the single factors and multi-factor optimization.

However, in the experiment of hydrogen production by photosynthetic organisms, a plurality of problems still exist, the most important is that the metabolic pathway of photosynthetic bacteria is unstable, so that the problems of low energy conversion rate, serious acidification of reaction liquid and the like are caused. Photosynthetic organism hydrogen production reactorSoluble volatile fatty acids, including mainly acetic, propionic, and butyric acids, are present in large amounts in the solution. In different metabolic pathways of photosynthetic bacteria, 1mol of glucose can generate 4 mol of H in the metabolic pathways of acetic acid and butyric acid respectively₂And 2 mol of H₂Whereas in the butyrate metabolic pathway, 2 mol H would be consumed by 1mol glucose₂. The failure to control the metabolic pathway of photosynthetic hydrogen production results in low hydrogen production rate and waste of resources.

If the directional metabolism of photosynthetic bacteria in the hydrogen production process of photosynthetic organisms can be realized, the photosynthetic hydrogen production performance can be greatly improved. Experiments were conducted to investigate the effect of catalysts on the performance of photosynthetic hydrogen production by adding catalysts, including activated carbon, diatomaceous earth, zeolites, etc., to the reaction solution. The activated carbon is specially treated, both the activated carbon and the diatomite have the characteristics of developed pores, large specific surface area, many surface chemical groups and strong adsorption performance, the activated carbon can adsorb harmful substances such as furfural, vanillin and the like in the reaction liquid of the dark fermentation biological hydrogen production so as to improve the hydrogen production amount, and the activated carbon can also be used as a fiber carrier for bacteria immobilization so as to improve the hydrogen production performance. The zeolite has certain adsorbability to metal ions, pollutants and the like, and can remove ammonium ions in the dark fermentation wastewater in the process of hydrogen production by combining dark/light and organisms, thereby improving the hydrogen production. Besides improving the hydrogen production, the catalyst for photosynthetic hydrogen production has other uses, such as adsorbing suspended matters in the reaction liquid to improve the light transmittance of the reaction liquid, adsorbing volatile fatty acids in the reaction liquid to buffer the change rate of the pH value of the reaction liquid, adsorbing harmful ions in the reaction liquid to reduce the harm of the harmful ions to photosynthetic bacteria, and maintaining the stability of hydrogen production.

The method takes HAU-M1 photosynthetic bacteria as hydrogen-producing bacteria, takes corn straws as hydrogen-producing substrates, takes parameters such as gas production rate, hydrogen concentration, pH value, reducing sugar concentration, volatile fatty acid and the like as assessment indexes, and researches the influence of the catalyst on the change of the hydrogen production metabolic pathway of photosynthetic organisms.

Disclosure of Invention

The invention provides a method for promoting efficient hydrogen production of HAU-M1 photosynthetic floras by adding a catalyst by taking corn straws subjected to cellulose degradation as a hydrogen production substrate aiming at specific HAU-M1 photosynthetic floras.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving HAU-M1 photosynthetic bacteria flora to produce hydrogen by utilizing corn straw fermentation comprises the following steps:

(1) pretreatment of corn straws: drying corn stalks, and crushing to 80-100 mu m for later use;

(2) pretreatment of the catalyst: crushing the catalyst to 80-100 mu m, then carrying out acid treatment on the crushed catalyst for 12-24h, and washing for later use;

(3) photosynthetic hydrogen production of HAU-M1 photosynthetic flora: placing the crushed corn straws and cellulase in a citric acid-sodium citrate buffer solution, adding a hydrogen production culture medium to obtain a mixed solution, adjusting the pH to be neutral, adding a catalyst, adding HAU-M1 photosynthetic bacteria strain liquid, shaking up, sealing, performing illumination culture to produce hydrogen, collecting gas and measuring the hydrogen production amount;

the HAU-M1 photosynthetic flora comprises Rhodospirillum rubrum, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, rhodobacter sphaeroides, and rhodobacter capsulata;

the catalyst is active carbon, diatomite or zeolite.

Specifically, the acid treatment in the step (2) is to place the crushed catalyst in 1-1.5mol/L H₂SO₄And (4) treating.

Specifically, the volume of the citric acid-sodium citrate buffer solution in the step (3) is 150mL, and the pH value is 4.8.

Specifically, the concentration of each component in the hydrogen production culture medium in the step (3) is as follows: NaCl 2 g/L, NH₄Cl 0.4 g/L, yeast extract 0.1 g/L, MgCI₂0.2 g/L，K₂HPO₄0.5 g/L and 3.56 g/L of sodium glutamate.

Specifically, in the step (3), the cellulase is the cellulase Ctec2 of danish novacin (Novozymes Biotechnology co., Ltd), the cellulase is a liquid enzyme preparation, and the addition volume is 1-7 mL.

Specifically, the solid-liquid mass ratio of the corn straws to the cellulase in the step (3) is 5: 1-7.

Specifically, the adding amount of the catalyst in the step (3) is 0.5g-2.5 g.

Specifically, the liquid-solid mass ratio of the HAU-M1 photosynthetic flora bacterial liquid to the corn stalks in the step (3) is 6-4: 1.

specifically, in the HAU-M1 photosynthetic bacteria bacterial liquid in the step (3), the volume ratio of rhodospirillum rubrum bacterial liquid to rhodopseudomonas capsulatum bacterial liquid to rhodopseudomonas palustris bacterial liquid to rhodobacter sphaeroides bacterial liquid to rhodobacter capsulatum bacterial liquid is respectively 27: 25: 28: 9: 11; the viable count of the Rhodospirillum rubrum bacterial liquid is 12.0 × 10⁸Per mL, the content of rhodopseudomonas capsulata is 11.0 multiplied by 10⁸The number per mL of rhodopseudomonas palustris is 12.5 multiplied by 10⁸Per mL, rhodobacter sphaeroides is 4.0X 10⁸Per mL, the content of rhodobacter capsulatus is 5.0X 10⁸one/mL.

Compared with the prior art, the method has the beneficial effects that:

the method provided by the invention designs a method for promoting the high-efficiency hydrogen production of the HAU-M1 photosynthetic bacteria by adding a catalyst by using corn straws subjected to cellulose degradation as hydrogen production substrates, and the fermentation approach in the hydrogen production process of the corn straw photosynthetic organisms is changed by adding activated carbon, diatomite and zeolite with different concentrations in the experiment of the invention, so that the photosynthetic hydrogen production amount is obviously improved. The diatomite obviously improves the photosynthetic hydrogen production effect of the corn straws, and the hydrogen production amount is improved by 15.93 percent compared with that of a control group; the zeolite obviously reduces the concentration of propionic acid in the reaction solution, changes the way of corn straw light fermentation, and improves the hydrogen yield by 33.33 percent compared with a control group.

The method has the advantages of reasonable design, clear steps, simplicity and feasibility, and can improve the efficiency of producing hydrogen by utilizing the corn straws for the photosynthetic bacteria group HAU-M1.

Drawings

FIG. 1 is a graph showing the effect of cellulase addition on the production of hydrogen from corn stalks by fermentation with HAU-M1 photosynthetic flora;

FIG. 2 is the effect of cellulase addition on the production of hydrogen soluble metabolites by the photo-fermentation of HAU-M1 photosynthetic bacteria;

FIG. 3 is the effect of activated carbon on the production of hydrogen by the photo-fermentation of HAU-M1 photosynthetic bacteria;

FIG. 4 is the effect of activated carbon on the production of hydrogen soluble metabolites by photo-fermentation of HAU-M1 photosynthetic bacteria;

FIG. 5 is the effect of diatomaceous earth on the production of hydrogen by the photo-fermentation of HAU-M1 photosynthetic bacteria;

FIG. 6 is the effect of diatomaceous earth on the production of soluble metabolites of hydrogen by photo-fermentation of a group of HAU-M1 photosynthetic bacteria;

FIG. 7 is the effect of zeolite on the production of hydrogen by the photo-fermentation of a group of HAU-M1 photosynthetic bacteria;

FIG. 8 is the effect of zeolite on the production of hydrogen soluble metabolites by photo-fermentation of HAU-M1 photosynthetic bacteria.

Detailed Description

The following examples are carried out on the premise of the technical scheme of the invention, and detailed embodiments and specific operation processes are given, but the scope of the invention is not limited by the following examples.

Test materials

The HAU-M1 photosynthetic bacteria colony is obtained by a method in the literature (korean. separation and identification of photosynthetic hydrogen producing bacteria colony and analysis of hydrogen producing characteristics thereof [ D ]. yunnan agriculture university, 2011), can decompose organic substances under illumination to produce hydrogen, and mainly consists of rhodospirillum rubrum (r.hospiranum), rhodopseudomonas capsulata (r.capsulata), rhodopseudomonas palustris (r.pulastis), Rhodobacter sphaeroides (r.hosibacter haloides), and Rhodobacter capsulata (Rhodobacter capsulatus).

The adopted catalyst is active carbon, diatomite and zeolite, and the diatomite is purchased from Tianjin Guangfu Fine Chemical Research Institute in Tianjin; activated carbon was purchased from cigarette-desk double chemical co (shuangshuang chemical co., Ltd.); zeolite was purchased from Tianjin Dingshengxin Chemical Co., Ltd.

The cellulase is Ctec2 of Novozymes (Novozymes Biotechnology Co., Ltd.) of Denmark, which is a liquid enzyme preparation with enzyme activity of 51 FPU/mL and specification of 20 mL.

Photosynthetic hydrogen production test conditions: the photosynthetic organism hydrogen production illuminance is 3000Lux, the temperature is 30 ℃, and the initial pH = 7.0.

The concentration of each component in the hydrogen production culture medium is as follows: NaCl 2 g/L, NH₄Cl 0.4 g/L, yeast extract 0.1 g/L, MgCI₂0.2 g/L，K₂HPO₄0.5 g/L and 3.56 g/L of sodium glutamate. During the preparation process, the components are dissolved in water to prepare the hydrogen production culture medium.

The preparation method of the citric acid-sodium citrate buffer solution comprises the following steps: solution A: accurately weighing C₆H₈O₇.H₂Dissolving O21.014 g in a 500mL beaker by using a small amount of deionized water, and then diluting to 1000mL to obtain 0.1mol/L citric acid solution; and B, liquid B: accurately weighing Na₃C₆H₅O₇.2H₂Dissolving O29.412 g in a 500mL beaker by using a small amount of deionized water, and then diluting to 1000mL to obtain 0.1mol/L sodium citrate solution; and (3) taking 230mL of the solution A and 270mL of the solution B, fully mixing, transferring into a 1000mL volumetric flask, metering to 1000mL by using deionized water, fully mixing, and refrigerating and storing in a refrigerator at 4 ℃.

Examples

1.1 test methods

A method for improving HAU-M1 photosynthetic bacteria flora to produce hydrogen by utilizing corn straw fermentation comprises the following specific steps:

(2) pretreatment of the catalyst: pulverizing the catalyst to 80-100 μm, and placing the pulverized catalyst in 1-1.5mol/L H₂SO₄Performing intermediate treatment for 24 hours, and cleaning for later use;

(3) photosynthetic hydrogen production of HAU-M1 photosynthetic flora: in the test, a 150mL conical flask is selected as a reactor, 5g of corn straw, 1-7 mL of cellulase (liquid enzyme preparation), 120 mL of citric acid-sodium citrate buffer solution (pH 4.8) and a hydrogen production culture medium are added into the conical flask, 5mol/L of NaOH is used for adjusting the pH value to 7, active carbon, diatomite or zeolite with different qualities are added as catalysts, the addition amounts are 0.5g, 1g, 1.5g, 2g and 2.5g respectively, finally 20mL of HAU-M1 photosynthetic bacteria colony liquid is added, the opening is sealed, nitrogen is used for purging for 5 min, the culture box is placed at 30 ℃ for photosynthetic fermentation to produce hydrogen for 84h, and the hydrogen production amount is sampled every 12 h.

In the HAU-M1 photosynthetic bacteria bacterial liquid in the step (3), the volume ratio of rhodospirillum rubrum bacterial liquid, rhodopseudomonas capsulata bacterial liquid, rhodopseudomonas palustris bacterial liquid, rhodobacter sphaeroides bacterial liquid to rhodobacter capsulata bacterial liquid is respectively 27: 25: 28: 9: 11; the viable count of the Rhodospirillum rubrum bacterial liquid is 12.0 × 10⁸Per mL, the content of rhodopseudomonas capsulata is 11.0 multiplied by 10⁸The number per mL of rhodopseudomonas palustris is 12.5 multiplied by 10⁸Per mL, rhodobacter sphaeroides is 4.0X 10⁸Per mL, the content of rhodobacter capsulatus is 5.0X 10⁸one/mL.

1.2 kinetic analysis of Hydrogen production

The modified Gompertz equation is used in the paper to describe photosynthetic hydrogen production, and is as follows:

。

from the cumulative hydrogen production, H (mL), as a function of time t given above, we can find 3 variables. Wherein H is the accumulated hydrogen production (mL), P is the maximum potential hydrogen production (mL), Rm is the maximum hydrogen production rate (mL/H), lambda is the lag phase (H), t is the time (H), and e is the natural constant of 2.718. In the experiment, P, Rm and lambda are calculated by 1stOpt 15PRO software.

The average hydrogen production rate (Roverall) is an important index for investigating the maximum cumulative hydrogen production and time, as follows:

。

wherein V is the working volume of the reactor, P is the maximum potential hydrogen production (mL), Rm is the maximum hydrogen production rate (mL/h), and lambda is the lag phase (h).

2.1 test methods

Using 721 Spectrophotometer (Shanghai)Cyanine scientific and technological instrument limited) for measuring OD of photosynthetic bacteria bacterial liquid_660nmPutting the photosynthetic bacteria colony into a 50mL centrifuge tube, centrifuging at 6000 r/min, pouring out supernatant, drying sediment at 65 ℃ to constant weight, weighing, and drawing dry weight of the photosynthetic bacteria colony versus OD_660nmStandard curve of values. By testing OD in the growth process of the photosynthetic flora_660nmAnd calculating the change of the dry weight of the photosynthetic bacteria group.

The gas is collected by a gas collecting bag, and the concentration of the hydrogen is measured by a 7890B gas chromatograph. Experimental data measurements were made every 12 hours.

3, measurement result:

3.1 Effect of cellulase addition on the Hydrogen production by fermentation of HAU-M1 photosynthetic flora

FIG. 1 is a graph showing the effect of cellulase addition on hydrogen production from corn stover fermented by HAU-M1 photosynthetic flora, wherein (a) hydrogen concentration; (b) the hydrogen production rate; (c) accumulating the hydrogen production; (d) the concentration of reducing sugar; (e) the pH value.

Figure 1 lists the effect of cellulase load on maize straw hydrogen production. As can be seen from FIG. 1a, only 6mL and 7mL of experimental groups had hydrogen concentration of 40% at 12h from the beginning of hydrogen production, and when the cellulase was added in an amount of 1-5 mL, the hydrogen concentration was 17-20%. This is probably because the corn stalks can be rapidly degraded into sugars when the amount of cellulase is large and then used for photosynthetic hydrogen production, and the corn stalks cannot produce enough sugars when the amount of cellulase is small. Then at 24h, the hydrogen concentration of all experimental groups reached a large value, which remained between 45-50%, which is a more desirable condition. This state continued until 48 h, after which the hydrogen concentration gradually decreased and the experimental group with low cellulase addition ended hydrogen production relatively quickly. At 48 h, the maximum hydrogen concentration of 50.54% was reached at a cellulase addition level of 5 mL.

FIG. 1b shows the influence of the cellulase addition amount on the hydrogen production rate, and it is seen from FIG. 1b that the peak hydrogen production period of the experimental group is centered at 36-60 h, at this time, the experimental group has a higher hydrogen concentration, and shows a large slope in the cumulative hydrogen production amount, and when the cellulase addition amount is 5-7 mL, a higher hydrogen production rate value is obtained, wherein the maximum value is 28.27 mL/h (6 mL).

As can be seen from FIG. 1c, when the cellulase addition amount is 1 mL, a lower cumulative hydrogen production amount of 213.59 mL is obtained, and as the cellulase addition amount increases, the cumulative hydrogen production amount shows a continuous rising trend, and when the cellulase addition amount is 5mL, a maximum cumulative hydrogen production amount of 601.56 mL is obtained, corresponding to a specific hydrogen production amount of 120.31 mL/g straw. The cumulative hydrogen production amount decreased to 590.65 mL with the increase in cellulase addition amount of 7 mL. The proper enzyme adding amount is shown to be beneficial to improving the photosynthetic hydrogen production, but the excessive cellulase can cause the inhibition of the enzyme. In the previous research results, the maximum value of the hydrogen production of the corn straws obtained by using the solid cellulase is 40.62 mL/g, the research improves 196.18%, and the liquid cellulase has a better enzymolysis hydrogen production effect on the corn straws. In the following experimental studies, we used 5mL of liquid cellulase as the additive amount.

As can be seen from FIG. 1d, the maximum reducing sugar concentration was obtained at 12h, followed by a drop and essentially a plateau. The maximum reducing sugar concentration of 4.95 g/L is obtained by 6mL of the experimental group at 12h, the reducing sugar concentration of 3.13 g/L is obtained by 1 mL, and in the subsequent hydrogen production process, the reducing sugar concentration and the cellulase basically show positive correlation. As can be seen from fig. 1c and 1d, this indicates that cellulose concentration has a promoting effect on enzymatic hydrolysis, but excess cellulase enzyme inhibits hydrogen production.

FIG. 1e shows the change of pH during the hydrogen production process, wherein the initial pH is 7, the pH value rapidly decreases with the beginning of hydrogen production, then slowly decreases, and slowly rises again with the end of hydrogen production. As the addition amount of cellulase increases, the pH value of the reaction solution tends to decrease. This is because, at the initial stage of the reaction, the cellulase rapidly degrades the cellulose expressed by the straw into a saccharide, and then the photosynthetic bacteria degrade the saccharide into a soluble metabolite such as a volatile fatty acid, thereby lowering the pH value in the reaction solution. Then, the photosynthetic flora utilizes the speed of the volatile fatty acid, the speed of cellulose degradation by cellulase to form the sugar substances and the speed of the sugar substances to be decomposed into the volatile fatty acid, and the three realize dynamic balance, so that the pH value in the reaction liquid is basically stable. With the completion of hydrogen production, the fatty acid in the reaction solution is gradually used up by the photosynthetic bacteria, and the pH value gradually rises.

FIG. 2 shows the effect of cellulase addition amounts on the production of hydrogen soluble metabolites by the photo-fermentation of HAU-M1 photosynthetic bacteria, wherein the addition amounts are 1 mL, 2 mL, 3 mL, 4 mL, 5mL, 6mL and 7mL, respectively.

Fig. 2 shows the influence rule of the cellulase addition amount on the soluble metabolites in the process of producing hydrogen by photosynthesis, and it can be seen that the main components in the metabolites are acetic acid and n-butyric acid, and then propionic acid, ethanol and a small amount of isobutyric acid, which indicates that the metabolic pathway of the HAU-M1 photosynthetic bacteria is acetobutyric acid type fermentation. Comparing fig. 1c, it can be seen that volatile fatty acids in the reaction solution rapidly increased as hydrogen production rapidly increased for 36 to 48 hours, because a large amount of the saccharides were degraded into hydrogen gas and substances such as acetic acid and butyric acid as hydrogen production proceeded (equations (1) and (3)). Subsequently, volatile fatty acids in the reaction solution showed a tendency of stabilization and decrease, and the pH of the reaction solution also slowly rose. Comparing different addition amounts in fig. 2, it can be seen that the total volatile fatty acid concentration in the reaction solution shows a growing trend along with the increase of the addition amount of the cellulase, and in combination with fig. 1d, it can be found that the reducing sugar concentration in the reaction solution can be effectively increased by the increase of the cellulase, and the reducing sugar in the reaction solution is further decomposed into substances such as volatile fatty acid. However, as can be seen from FIG. 2 (7 mL), the concentration of volatile fatty acids decreased, and the amount of hydrogen produced began to decrease with the addition of 7mL of cellulase in FIG. 1c, indicating that excess cellulase had some inhibitory effect on hydrogen production. As can be seen from the observation of FIG. 2 (1 mL), the concentrations of acetic acid and butyric acid show a trend of increasing and then decreasing, which indicates that both the dark fermentation glucose metabolic pathway (equations (1) - (2)) and the acetic acid/butyric acid light fermentation hydrogen production metabolic pathway exist in the HAU-M1 photosynthetic bacteria bacterial flora light fermentation hydrogen production process, which indicates that the HAU-M1 photosynthetic bacteria flora is a complex mixed flora and can sufficiently degrade the hydrogen production substrate. Among the volatile fats in the reaction solution we detected a certain content of propionic acid, the formation of which reduces the hydrogen yield. In the process of photosynthetic hydrogen production, the hydrogen production should be increased by suppressing the production of propionic acid as much as possible.

Equations (1) - (3) are the fermentation hydrogen production with the metabolites acetic acid, propionic acid and butyric acid, respectively, equation (4) is the fermentation metabolic pathway with the metabolite ethanol, and equations (5) and (6) are the acetic acid and butyric acid light fermentation metabolic pathways.

C₆H₁₂O₆+ 2 H₂O → 2 C₂H₄O₂+ 2 CO₂+ 4 H₂(1)

C₆H₁₂O₆+ 2 H₂→ 2C₃H₆O₂+ 2 H₂O (2)

C₆H₁₂O₆→ C₄H₈O₂+ 2 CO₂+ 2 H₂(3)

C₆H₁₂O₆→ C₂H₆O + 2 CO₂(4)

C₂H₄O₂+2H₂O → 4H₂+2CO₂(5)

C₄H₈O₂+6H₂O → 10H₂+4CO₂(6)

3.2 influence of activated carbon on the light fermentation of HAU-M1 photosynthetic bacteria to produce hydrogen.

FIG. 3 is a graph showing the effect of activated carbon on the production of hydrogen by the photo-fermentation of a HAU-M1 photosynthetic bacteria population, wherein (a) the hydrogen concentration; (b) the hydrogen production rate; (c) accumulating the hydrogen production; (d) the concentration of reducing sugar; (e) the pH value.

FIG. 3 depicts the effect of the added amount of activated carbon on the properties of the photosynthetic hydrogen production gas and the properties of the liquid, and at 12h, the reaction liquid has a higher concentration of reducing sugars (FIG. 3 d), the hydrogen production activity is just started, the gas production rate is lower (4.5 mL/h), but the hydrogen concentration is already higher (about 40%), and the pH value is rapidly reduced from initial 7 to about 5.6-6. Cellulose is degraded into saccharides by cellulase within 0-12 h, then the saccharides are converted into fatty acid under the action of photosynthetic flora, and the photosynthetic flora gradually adapts to the environment of reaction liquid to deposit energy for self growth and hydrogen production.

In 12-36 h, the photosynthetic bacteria group begins to produce hydrogen stably and rapidly in the reaction liquid, the rate of degrading reducing sugar by the photosynthetic bacteria group is greater than the rate of degrading cellulose into reducing sugar by cellulase, and the concentration of the reducing sugar in the reaction liquid shows the trend of descending first and then slowly rising. The pH also shows a tendency to rise back slightly and then continue to fall, as can be evidenced from the volatile fatty acid concentration of fig. 4. The relatively stable fermentation liquid environment ensures that the hydrogen production concentration and the hydrogen production rate are in a stable state.

In 36-60 h, the concentration of reducing sugar in the reaction solution shows a slow rising trend and is maintained between 2-3 g/L; at this time, the total amount of volatile fatty acids in the reaction solution tended to increase more and more (fig. 4), which resulted in a continuous decrease in pH, and the pH was maintained at about 5.3 for the experimental group with 2.5g of activated carbon, except for 4.76. The hydrogen concentration is greatly improved at the moment, and the hydrogen concentration is improved from about 44 percent to the maximum value of 55.92 percent. The hydrogen production rate also reaches the maximum value of 22.04 mL/h.

And in the time of 60-72 h, the hydrogen production reaction is finished, and the hydrogen concentration and the hydrogen production rate are rapidly reduced to 0. This results in an increase in the concentration of reducing sugars, as no photosynthetic bacteria population continues to consume sugars. The pH showed a slow downward trend. In this time period, the photosynthetic bacteria group dies rapidly due to the change of the reaction liquid environment, and the metabolic activity of hydrogen production stops. The cellulase continuously degrades the corn straws into reducing sugar, and the concentration of the reducing sugar in the reaction liquid slowly rises.

From the whole time period of hydrogen production, the hydrogen concentration is higher in the middle peak period (12-60 h) of hydrogen production, the hydrogen production rate is more stable in the early stage, the hydrogen production rate is greatly increased in the later stage, the accumulated hydrogen production quantity graph has good evidence, the concentration of reducing sugar is in a rapid descending trend at the beginning and then in a slow rising trend, and the pH value is in a continuous descending trend due to the accumulation of volatile fatty acid in the reaction liquid.

When the amount of the activated carbon added was 1.5g, 602.12 mL, which is the maximum cumulative hydrogen production, was obtained, and there was almost no increase in the amount of the activated carbon added as compared with 601.56 mL, which is the maximum cumulative hydrogen production in FIG. 1 (c). The activated carbon can provide more fixed carriers for the photosynthetic bacteria, the pH value of the solution caused by volatile fatty acid in the buffer reaction solution is reduced, and the proper hydrogen production condition of the photosynthetic bacteria is maintained, but the light transmittance of the reaction solution is greatly reduced by adding the excessive activated carbon, so that the growth and the hydrogen production of the photosynthetic bacteria are not facilitated. The active carbon can promote the hydrogen production amount of the dark fermentation bio-hydrogen production, but can not improve the hydrogen production amount of the light fermentation. And the production of propionic acid cannot be well inhibited by activated carbon.

FIG. 4 shows the effect of activated carbon on the production of hydrogen soluble metabolites by the photo-fermentation of HAU-M1 photosynthetic bacteria, wherein the amounts of activated carbon added are 0.5g, 1g, 1.5g, 2g and 2.5g, respectively.

As can be seen from fig. 4, the overall volatile fatty acid content showed a slight decrease as the concentration of activated carbon increased, which is probably due to the adsorption of volatile fatty acids by activated carbon. The main volatile fatty acids in the reaction solution were still acetic acid and butyric acid. In addition, the production of high-concentration propionic acid and ethanol has a poor effect on hydrogen production. At 12h, the concentration of volatile fatty acids in the reaction solution showed a tendency to decrease with the increase in the concentration of activated carbon, because a part of the fatty acids was adsorbed by activated carbon.

The addition of activated carbon at 1.5g gave a higher propionic acid yield (1.84 g/L) than the maximum propionic acid yield (1.37 g/L) of the control, and the formation of propionic acid resulted in a reduction in hydrogen gas (equation (2)), which explains to some extent that the addition of activated carbon did not significantly increase the hydrogen yield. In addition, the maximum concentration of the ethanol is 1.01g/L which is much higher than the maximum concentration of the control group by 0.66 g/L, and the generation of the ethanol can compete with the hydrogen for H⁺This results in a reduction in hydrogen production. Excess production of propionic acid and ethanol results in a decrease in hydrogen production.

3.3 Effect of diatomaceous Earth on the production of Hydrogen by photo-fermentation of HAU-M1 photosynthetic bacteria

FIG. 5 is a graph showing the effect of diatomaceous earth on the production of hydrogen by the photo-fermentation of a group of HAU-M1 photosynthetic bacteria, wherein (a) the hydrogen concentration; (b) the hydrogen production rate; (c) accumulating the hydrogen production; (d) the concentration of reducing sugar; (e) the pH value.

FIG. 5 shows the influence of diatomaceous earth on the hydrogen production by the light fermentation of HAU-M1 photosynthetic bacteria, the photosynthetic bacteria gradually adapt to the environment of the reaction solution within 0-12 h, and hydrogen production begins, at this time, the hydrogen concentration gradually increases to about 37%, the hydrogen production rate is about 1.8 mL/h, the reducing sugar concentration reaches the maximum value of about 4.1 g/L, and the pH value rapidly decreases to about 5.7. At the moment, the cellulose on the surface layer of the straw is quickly degraded into saccharides by the cellulase in the reaction liquid, and the concentration of the reducing sugar in the reaction liquid reaches a larger value. And the photosynthetic bacteria group quickly degrades a part of carbohydrate into volatile fatty acid, so that the pH value in the reaction liquid is quickly reduced. And then, hydrogen production is carried out, the hydrogen production stabilization period of the photosynthetic bacteria group is 12-60 h, the hydrogen concentration is kept to be a large value, the maximum value reaches 58.39% (1 g, 36 h), and the value is higher than that of other experimental groups. The hydrogen production rate was also kept high, with a maximum of 38.17 mL/h (0.5 g, 36 h), with the maximum increase seen at 24-36 h also being found by the cumulative hydrogen production in FIG. 5 (c). The concentration of reducing sugar is in a descending trend at 12-36 h and then begins to increase, because the hydrogen production rate is too fast at 36h, and the rate of cellulose degradation and saccharide production by the cellulase is less than the rate of the carbohydrate utilization by the photosynthetic bacteria. In this process, the pH value is in a substantially slow decreasing tendency because of the accumulation of volatile fatty acids in the reaction solution.

As can be seen from fig. 5 (c), when the additive amount of the diatomite is 1g, the cumulative hydrogen production of the photosynthetic bacteria group reaches the maximum value of 697.37mL (139.4 mL/g), which is increased by 15.93% compared with the control group 601.56 mL, and the significant amplification is achieved, indicating that the diatomite is favorable for the hydrogen production of the photosynthetic bacteria group. The diatomite has a good pore structure, can provide more landing growth space for the photosynthetic bacteria, can adsorb a part of volatile fatty acid, and stabilizes the change of the pH value in the reaction solution.

FIG. 6 shows the effect of diatomaceous earth on the hydrogen production soluble metabolites by the photo-fermentation of HAU-M1 photosynthetic bacteria, wherein the addition amount of diatomaceous earth is 0.5g, 1g, 1.5g, 2g, and 2.5g, respectively.

From fig. 6, it can be seen that the total content of volatile fatty acids in the hydrogen production process shows a trend of increasing first and then decreasing, in the photosynthetic hydrogen production process, the concentration of volatile fatty acids will change continuously with the progress of hydrogen production, and at 12 hours after the beginning of hydrogen production, the concentration of volatile fatty acids is low, at this time, the concentration of reducing sugars in the reaction solution is high, and most of the carbohydrate substances are not converted into fatty acids by the photosynthetic bacteria group. Then, the concentration of reducing sugar in the reaction solution begins to decrease rapidly, and a large amount of reducing sugar is converted into volatile fatty acid by the photosynthetic flora, so that the concentration of the volatile fatty acid in the reaction solution is increased. Finally, as the rate of volatile fatty acid utilization by the photosynthetic flora is greater than the rate of fatty acid production, the concentration of fatty acids in the reaction solution begins to decrease.

Comparing the several legends in FIG. 6, the concentration of volatile fatty acids in the reaction solution decreased with increasing diatomaceous earth concentration at 12 h. The diatomite has a certain adsorption effect on fatty acid, can buffer the rapid reduction of the pH value of the reaction solution caused by volatile fatty acid, and keeps the reaction solution environment suitable for photosynthetic hydrogen production. The volatile fatty acids generally show a tendency of increasing first and then decreasing, which is consistent with the result that the pH of the reaction solution decreases first and then slowly rises. When the adding amount of the diatomite is 1g, the accumulated hydrogen production of the photosynthetic bacteria group reaches the maximum value of 697.37mL, and the maximum propionic acid concentration obtained in the reaction liquid is 3.12 g/L at the moment, which shows that more carbohydrate in the reaction liquid is diverted to the propionic acid metabolism direction, so that the hydrogen yield is reduced; in addition, the maximum concentration of 0.79 g/L of ethanol is higher than that of the control group by 0.66 g/L, and the metabolic direction of ethanol is not beneficial to the generation of hydrogen. If the production of ethacrynic acid could be reduced so that the carbohydrate would be metabolized more toward acetic and butyric acids, more hydrogen would be available.

3.4 Effect of Zeolite on the photo-fermentation of HAU-M1 photosynthetic bacteria population to produce Hydrogen

FIG. 7 shows the effect of zeolite on the photo-fermentation of HAU-M1 photosynthetic bacteria population to produce hydrogen, wherein, (a) hydrogen concentration; (b) the hydrogen production rate; (c) accumulating the hydrogen production; (d) the concentration of reducing sugar; (e) the pH value.

FIG. 7 shows the effect of zeolite on gas and liquid properties in the hydrogen production process by photo-fermentation of HAU-M1 photosynthetic flora. The hydrogen concentration is rapidly increased along with the hydrogen production, and then the larger value is kept about 50% in 24-60 h, which is a relatively ideal hydrogen production state, and the maximum value reaches 56.66%. The burst period of hydrogen production is concentrated at about 36h, and the period reaches a maximum value of 45.52 mL/h, which is far higher than 26.95 mL/h of a control group (FIG. 1 (b)). The maximum hydrogen production rate at the moment of 36h in fig. 7b can also be seen in fig. 7c, the slope of the cumulative hydrogen production amount about 36h is the largest, then the slope is gentle, hydrogen production is completely stopped at 84h, when the addition amount of zeolite is 1g, the maximum cumulative hydrogen production amount is obtained of 802.06 mL, the specific hydrogen production amount corresponding to 160.41 mL/g is increased by 33.33% compared with 120.31 mL/g of the control group, and the fact that zeolite has an obvious promoting effect on the photosynthetic hydrogen production of corn straws is shown. Compared with the maximum value of 120.42 mL/g of the activated carbon experimental group and 139.47 mL/g of the diatomite experimental group, the zeolite has better effect. The value is even higher than the photosynthetic hydrogen production effect (111.85 mL/g TS) of the apples rich in the carbohydrate. As seen from FIG. 7c, the concentration of reducing sugar rapidly increases to about 2.8 g/L with the start of photosynthetic hydrogen production, and the concentration of reducing sugar in the reaction solution rapidly increases with the hydrogen production rate, and shows a downward trend within 12-36 h, reaches the lowest peak within 36h, and has the minimum value of 2.04 g/L, at this time, the photosynthetic hydrogen production rate reaches the maximum, and a large amount of sugar substances are rapidly absorbed by the photosynthetic bacteria for hydrogen production. As seen from FIG. 7c, the pH value rapidly decreased as hydrogen production started, because the photosynthetic bacteria transformed a large amount of the saccharides into volatile fatty acids, lowering the pH value in the reaction solution. And then the hydrogen production rate of the photosynthetic bacteria is increased within 12-24h, the utilization rate of the volatile fatty acid is greater than the generation rate, and the dynamic balance causes the pH value in the reaction solution to rise. Then, as the photosynthetic flora decays and volatile fatty acid in the reaction solution is accumulated, the pH value in the reaction solution shows a slow and stable trend, which is consistent with the previous situation.

FIG. 8 shows the effect of zeolite on the production of hydrogen soluble metabolites by the photo-fermentation of HAU-M1 photosynthetic bacteria, wherein the amount of zeolite added is 0g, 0.5g, 1g, 1.5g, 2g, 2.5g, respectively.

FIG. 8 shows the effect of zeolite on soluble metabolites in the light fermentation hydrogen production process of HAU-M1 photosynthetic flora, acetic acid and butyric acid being the major metabolically volatile fatty acids in the light fermentation hydrogen production process, followed by butyric acid and a small amount of ethanol, with almost no propionic acid detected. This shows that HAU-M1 photosynthetic flora light fermentation hydrogen production belongs to acetic acid and butyric acid type fermentation, and the two hydrogen production efficiencies are higher. In the previous research, the existence of propionic acid is found in the hydrogen production process of light fermentation of different substrates (corn straws, wheat straws, rice straws, corncobs and sorghum straws). Higher hydrogen production was obtained in the laboratory than in the control, activated carbon and diatomaceous earth, possibly due to the absence of propionic acid as an intermediate product, large hydrogen molecules consumed by propionic acid type metabolism (equation (2)), and the progress of propionic acid metabolic pathways was inhibited by zeolite. As can be seen from fig. 8, as the hydrogen production proceeds, the concentration of volatile fatty acid increases rapidly, resulting in a decrease in pH, and then a substantially steady dynamic equilibrium state is maintained, so that the dynamic equilibrium of the HAU-M1 photosynthetic bacteria population degrading carbohydrates into carbohydrates and carbohydrates into fatty acid is achieved.

Table 1 hydrogen production kinetics parameters under different catalyst conditions.

Table 1 describes the variation of the hydrogen production kinetics parameters under different conditions. It can be seen that at low cellulose concentrations there is a significant effect on the cumulative amount of hydrogen produced by photosynthesis in the corn stover, but excessive cellulose concentrations in turn inhibit the amount of hydrogen produced by photosynthesis. The addition of activated carbon, diatomaceous earth and zeolite all had a significant effect on the cumulative hydrogen production. The excessive activated carbon greatly reduces the light projection of the reaction solution, thereby sharply reducing the cumulative yield. The photosynthetic bacteria group has the strongest tolerance to the activated carbon, reaches the maximum potential value of 723.56 mL when the photosynthetic bacteria group is added in 2g, has smaller tolerance to the diatomite and the zeolite, and respectively reaches the maximum accumulated hydrogen production of 703.8 mL and 808.3 mL when the photosynthetic bacteria group is added in 1 g. When the adding amount of the zeolite is 1.5g, the maximum hydrogen production rate of 70.46mL/h is obtained, which shows that the zeolite has great promotion effect on improving the maximum hydrogen production rate of photosynthetic flora. In the continuous photosynthetic organism hydrogen production process, zeolite can be used as an additive to provide the overall hydrogen production rate of the reactor. The hydrogen production delay period (lambda) after the three substances are added is obviously shortened, the minimum hydrogen production delay period is obtained when the adding amount of the activated carbon is 2.5g, and the activated carbon has an obvious promotion effect on shortening the hydrogen production delay period. The determination coefficient (R2) of each experimental group is very close to 1, which shows that the hydrogen production kinetic parameters have good description effect on the experiment. The average hydrogen production rate (Roverall) is determined by the accumulated hydrogen production and the hydrogen production time, and compared with a control group, the three additives have great promotion effect on providing the average hydrogen production rate. The specific hydrogen production amount reflects the hydrogen production amount per unit mass, and the maximum specific hydrogen production amount of 160.41 mL/g was obtained at a zeolite addition amount of 1 g.

4 conclusion

The invention changes the fermentation way in the hydrogen production process of the photosynthetic organisms of the corn straws by adding the active carbon, the diatomite and the zeolite with different concentrations, and obviously improves the photosynthetic hydrogen production. The test result shows that although the activated carbon has good adsorbability, the activated carbon has no obvious influence on the photo-synthesis hydrogen production because the light transmission in the reaction liquid is reduced; the diatomite obviously improves the photosynthetic hydrogen production effect of the corn straws, and the hydrogen production amount is improved by 15.93 percent compared with that of a control group; the zeolite obviously reduces the concentration of propionic acid in the reaction solution, changes the way of corn straw light fermentation, and improves the hydrogen yield by 33.33 percent compared with a control group.

The foregoing examples are illustrative of embodiments of the present invention, and although the present invention has been illustrated and described with reference to specific examples, it should be appreciated that embodiments of the present invention are not limited by the examples, and that various changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A method for improving HAU-M1 photosynthetic bacteria flora to produce hydrogen by utilizing corn straw fermentation is characterized by comprising the following steps:

the catalyst is active carbon, diatomite or zeolite.

2. The method of claim 1, wherein the acid treatment in step (2) is carried out by exposing the crushed catalyst to 1 to 1.5mol/L of H₂SO₄And (4) treating.

3. The method as claimed in claim 1, wherein the volume of the citric acid-sodium citrate buffer solution in the step (3) is 150 mL.

4. The method of claim 1, wherein the concentrations of the components in the hydrogen-producing medium in step (3) are: NaCl 2 g/L, NH₄Cl 0.4 g/L, yeast extract 0.1 g/L, MgCI₂0.2 g/L，K₂HPO₄0.5 g/L and 3.56 g/L of sodium glutamate.

5. The method of claim 1, wherein the cellulase in step (3) is a liquid enzyme preparation and is added in a volume of 1-7 mL.

6. The method of claim 1, wherein the solid-liquid mass ratio of corn stover to cellulase in step (3) is 5:1 to 7.

7. The method of claim 1, wherein the catalyst is added in an amount of 0.5g to 2.5g in step (3).

8. The method of claim 1, wherein in the HAU-M1 bacterial liquid of photosynthetic bacteria in step (3), the volume ratio of rhodospirillum rubrum bacterial liquid, rhodopseudomonas capsulatum bacterial liquid, rhodopseudomonas palustris bacterial liquid, rhodobacter sphaeroides bacterial liquid to rhodobacter capsulatum bacterial liquid is 27: 25: 28: 9: 11; the viable count of the Rhodospirillum rubrum bacterial liquid is 12.0 × 10⁸Per mL, the content of rhodopseudomonas capsulata is 11.0 multiplied by 10⁸The number per mL of rhodopseudomonas palustris is 12.5 multiplied by 10⁸Per mL, rhodobacter sphaeroides is 4.0X 10⁸Per mL, the content of rhodobacter capsulatus is 5.0X 10⁸one/mL.

9. The method of claim 1, wherein the liquid-solid mass ratio of the HAU-M1 photosynthetic bacteria flora bacterial liquid to the corn stalks in the step (3) is 6-4: 1.