CN107653208B

CN107653208B - Hydrogen producing bacteria

Info

Publication number: CN107653208B
Application number: CN201711126954.7A
Authority: CN
Inventors: 刘洪艳; 王红玉; 李凯强
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2017-11-15
Filing date: 2017-11-15
Publication date: 2020-04-21
Anticipated expiration: 2037-11-15
Also published as: CN107653208A

Abstract

The invention provides a strain (seaweed beijerinckii) WANY51 capable of utilizing kelp hydrolysate to grow and efficiently produce hydrogen, and the strain can stably grow and efficiently produce hydrogen under the condition that the kelp hydrolysate is used as a sole culture substrate. The hydrogen production efficiency of the hydrogen-producing bacteria WANY51 under the optimum culture condition is as high as 990.50 ml/L. The hydrogen-producing bacteria WANY51 provided by the invention has higher butanol production efficiency, and the butanol yield under the most suitable condition for producing hydrogen is 130.40 ml/L. The hydrogen production efficiency of the hydrogen producing bacteria WANY51 is obviously higher than that of the existing hydrogen producing strains or mixed flora, and the hydrogen producing bacteria WANY51 has the advantage of wide substrate utilization range, can fully utilize the normal growth of multiple components of kelp hydrolysate and produce hydrogen with high efficiency, and has more obvious economic benefit when being used as hydrogen producing engineering strains.

Description

Hydrogen producing bacteria

Technical Field

The invention relates to the field of microorganisms, in particular to a bacterium capable of utilizing kelp hydrolysate to grow and produce hydrogen.

Background

The biological energy mode of obtaining fuels such as alcohol, diesel oil, methane, hydrogen and the like by taking biomass as a raw material can meet the actual and future energy requirements with the renewable characteristic and becomes a research hotspot, wherein the biological hydrogen production is one of the current alternative ways of fossil energy fuels. The reported hydrogen-producing bacteria mainly comprise Enterobacter, Bacillus and Clostridium, but the hydrogen-producing bacteria separated at present are difficult to enter the industrial production due to the hydrogen-producing capability and the culture cost. In order to reduce the production cost of biological hydrogen production, researchers have produced hydrogen gas from crops or crop wastes as biomass, but the first and second generation biomasses contain a large amount of lignin and are difficult to decompose and utilize by microorganisms. The development of microbial hydrogen production technology based on such biomass has been greatly limited.

The kelp as the third generation biomass has the characteristics of high polysaccharide content, land contention resistance, low lignin content and the like compared with terrestrial plants. The large-scale seaweed yield of China is in the leading position of the world, the world has the largest kelp culture and processing scale, the annual yield can reach 120 ten thousand tons, and the annual yield of the kelp in nearly 10 years accounts for 83-87% of the global total yield (FAO data statistics). The kelp has polysaccharide content of more than 50 percent, and algal polysaccharide can provide rich organic matters for the growth of microorganisms. kim et al (Antimicrobial activity of ethanol extracts of Laminaria japonica microorganisms [ J ]. Anaerobe,2013,21(6):34-38) hydrolyzed Laminaria japonica (Laminaria japonica) by both acid and enzyme methods to study the fermentation of ethanol by microorganisms. Liu et al (nutritional hydrolysis from macro-algae marine japonica, using anaerobic mixed bacteria [ J ]. International Journal of Hydrogen Energy,2014,39(17): 9012) hydrolyzed kelp (marine japonica) by four methods of heat, acid, alkali and ultrasonic wave, and carried out microbial fermentation Hydrogen production research. Relevant researches show that the marine macroalgae polysaccharide has high degradation efficiency and is an effective substrate for the growth and metabolism of microorganisms.

Although kelp and other large-scale algae have natural advantages as third-generation biomass raw materials, the research on energy utilization of the large-scale algae, particularly the biological hydrogen production by taking the kelp as the biomass raw material, is few at present, and the performance of the obtained hydrogen-producing bacteria reported at present is mostly not ideal, so that the hydrogen-producing bacteria are difficult to put into application due to the problems of hydrogen production efficiency and scale cost.

Disclosure of Invention

The invention aims to provide a strain capable of utilizing kelp hydrolysate to grow and efficiently produce hydrogen, and application of the strain in producing hydrogen and butanol.

The strain provided by the invention is hydrogenogen (Clostridium beijerinckii) WANY51, is preserved in China general microbiological culture Collection center (CGMCC), and has the address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, accession number: CGMCC No.14045, preservation date: year 2017, month 4 and day 18.

The hydrogenogen (Clostridium beijerinckii) WANY51 provided by the invention is a gram-positive anaerobic bacterium, the colony form in the solid culture medium is round, milky white, neat in edge, smooth in surface and slightly convex, and the colony size is 1.0-2.0 cm. Under an electron microscope, the strain WANY51 is in a spindle-shaped form, the propagation mode is binary division, and the cell size is 8.19 microns multiplied by 1.45 microns.

The culture characteristics of the hydrogen-producing bacteria WANY51 are as follows: the separation culture medium adopts kelp hydrolysate, and the preparation process comprises the following steps: dissolving the kelp dry powder in distilled water according to the proportion of 5g/L, sterilizing for 20min at the temperature of 121 ℃ under high pressure to obtain heat-treated kelp hydrolysate, wherein the pH value is 7.2-7.4, and adding 2.0% of agar into a solid culture medium (the agar is added into the kelp hydrolysate according to the proportion of 20 g/L).

Taking kelp hydrolysate as a liquid culture medium for hydrogen production, culturing for 24h under the conditions that the initial concentration of the kelp hydrolysate and distilled water (namely the initial volume percentage of the kelp hydrolysate in the liquid culture medium, hereinafter all expressed as the initial concentration of the kelp hydrolysate) is 80%, the initial pH value is 7.2-7.4, and the temperature is 35 ℃, starting to enter a logarithmic phase, and culturing for 72h under the condition that the cell density OD (OD) is the cell density₆₀₀To the highest. The hydrogen production process of the strain WANY51 occurs within 24h to 60h of logarithmic phase, and the gas accumulation amount has no obvious change after 60 h. After the strain WANY51 grows by utilizing kelp hydrolysate and produces hydrogen, the components of fermentation liquor of the strain WANY51 mainly comprise butyric acid, butanol and acetic acid, wherein the content of butyric acid is the highest and is 937.02ml/L, and the strain belongs to typical butyric acid type fermentation.

The hydrogen-producing bacteria WANY51 can utilize kelp hydrolysate as the only culture medium, and the bacterial strain growth and hydrogen-producing characteristics are kept consistent through continuous several generations of culture, and the biological properties are basically stable. The Genebank accession number of 16SrDNA of the hydrogen-producing bacteria WANY51 is KY 197470.

Under the culture condition that the kelp hydrolysate is used as the only culture medium, the culture temperature, the initial pH value and the initial concentration of the kelp hydrolysate required by the bacterial strain WANY51 for hydrogen production are as follows:

the culture temperature is as follows: 20 ℃ to 40 ℃, wherein 35 ℃ is preferred;

the initial pH value conditions are as follows: pH5.0-9.0, preferably pH7.0;

the initial concentration of the kelp hydrolysate is 20-100%, wherein the initial concentration of the kelp hydrolysate is preferably 80%.

The preferable culture conditions for the hydrogen-producing bacteria WANY51 to produce butanol are as follows: the kelp hydrolysate is used as the only culture medium, the culture temperature is 35 ℃, the initial pH value is pH7.0, and the initial concentration of the kelp hydrolysate is 80%.

The invention has the beneficial effects that:

(1) provides a strain WANY51 capable of producing hydrogen with high efficiency, and simultaneously, the strain also has higher butanol production efficiency. The Hydrogen-producing efficiency of the Hydrogen-producing bacteria WANY51 under the optimal culture condition is as high as 990.50ml/L, and the Hydrogen Production is 198.1ml/g calculated by the highest substrate concentration (5g/L) in kelp hydrolysate, which is obviously higher than the Hydrogen Production (83.28ml/g) of mixed flora reported by Park et al (Production of Hydrogen from marine macro-organic using and biotechnological & Bioprocess Engineering,2009,14(3): 307) and the Hydrogen Production (83.28ml/g) of Liu et al (ecological Hydrogen Production of marine-organic laboratory, using microbial mixture of bacterial [ J. Journal of biological 9, 201417 ml) of mixed flora.

(2) The hydrogen-producing bacteria WANY51 provided by the invention has higher butanol production efficiency, and butanol can be used as a substitute fuel of petroleum, especially as a good vehicle substitute fuel. The butanol yield of the strain WANY51 under the optimum hydrogen production condition is up to 130.40ml/L, and the strain can be used as an engineering strain to simultaneously obtain butanol with high efficiency under the optimum hydrogen production condition, thereby being beneficial to fermentation control and production cost reduction.

(3) The strain WANY51 provided by the invention can stably grow and efficiently produce hydrogen under the condition that kelp hydrolysate is used as the only culture substrate, and the strain has the advantage of wide substrate utilization range and can fully utilize multiple components of kelp hydrolysate. The main components of the conventional hydrogen-producing bacteria culture medium are glucose, beef extract, peptone and yeast juice, the price of 1L of the conventional culture medium is about 1.35 yuan, the price of 1L of kelp hydrolysate required by the normal growth and hydrogen production of the strain WANY51 is only 0.52 yuan at most and is 38% of the price of the conventional culture medium, and therefore, the hydrogen-producing bacteria WANY51 has more obvious economic benefits when being used as hydrogen-producing engineering strains.

Description of the drawings:

FIG. 1 shows a single colony of hydrogen-producing bacteria WANY51, which was swelled with paraffin on the solid medium.

FIG. 2 shows the form of hydrogen-producing bacteria WANY51 under an electron microscope.

FIG. 3 shows the hydrogen production efficiency of WANY51 strain at different culture temperatures.

FIG. 4 shows the hydrogen production efficiency of the strain WANY51 under different initial pH values.

FIG. 5 shows the hydrogen production efficiency of the strain WANY51 under the conditions of different initial concentrations of kelp hydrolysate.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples.

1. Isolation of the Strain (Clostridium beijerinckii) WANY 51:

preparing a kelp hydrolysate culture medium, dissolving 5g/L of kelp dry powder in distilled water, stirring for 30min by using a magnetic stirrer, adjusting the pH value to 7.2-7.4, and then carrying out autoclaving at 121 ℃ for 20min to obtain the kelp hydrolysate culture medium.

Sealing the sludge from the intertidal zone, charging nitrogen, and storing at 4 deg.C for 1 month under anaerobic condition. Before inoculation, sludge is heated at 80 ℃ for 10min for heat shock treatment, then 10% of the sludge is inoculated into a closed container containing kelp hydrolysate, nitrogen is filled, and the kelp hydrolysate is cultured in a constant temperature shaking table at 30 ℃ and 120rpm for 24 h. Inoculating the culture solution into the kelp hydrolysate culture medium according to the ratio of 1:100 (V/V), and repeatedly culturing for 3 times according to the same culture conditions to enrich the anaerobic hydrogen-producing mixed flora. After the obtained mixed flora is subjected to gradient dilution, a three-layer flat plate method is adopted for scribing and separating, and the method comprises the following specific steps: adding agar into the kelp hydrolysate according to the proportion of 20g/L to prepare a solid culture medium, sterilizing, spreading the solid culture medium to serve as a first layer of flat plate, cooling and solidifying the first layer of flat plate, uniformly coating the bacterial liquid, and pouring a layer of kelp hydrolysate solid culture medium to serve as a second layer of flat plate after the bacterial liquid is completely absorbed; and cooling and solidifying the second flat plate, pouring a layer of sterilized solid paraffin as a third flat plate, culturing the third flat plate at 30 ℃ for 24 hours after the solid paraffin is fully solidified, and after the flat plate grows out of bacteria, selecting a single bacterial colony (shown in figure 1) with obvious gas production behavior to cause paraffin to bulge, inoculating the single bacterial colony into a screw test tube filled with a kelp hydrolysate culture medium, and culturing at 30 ℃ for 24 hours. The screw test tube is internally sleeved with a fermentation tube, the condition of gas production can be judged by observing bubbles in the fermentation tube through naked eyes, and the bigger the bubbles are, the bigger the gas quantity collected in the fermentation tube is, the gas is fully filled in the fermentation tube, and the strain with high gas yield can be judged. And repeatedly carrying out separation and purification 5-6 times by the three-layer plate method and gradient dilution culture until a pure culture of a single colony is obtained.

2. Identification of hydrogen-producing strains:

the strain was streaked on the above three-layer solid plate, and cultured at 30 ℃ for 24 hours. Through visual observation, the colony morphology of the strain WANY51 is circular, milky white, neat in edge, smooth in surface and slightly convex. Gram staining of strain WANY51 was positive. Under an electron microscope, the strain WANY51 appeared in a shuttle-like shape (FIG. 2), and propagated in a binary division manner, with a cell size of 8.19. mu. m.times.1.45. mu.m.

16SrDNA sequencing and identification, ① bacterial genome DNA is extracted by a kit, ② 16SrRNA gene PCR amplification is carried out, general primers are 27F:5'-AGAGTTTGATCCATGGCTCAG-3' and 1541R:5'-AAGGAGGTGATCCAGCC-3', the reaction program comprises 94 ℃ for 4min, 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 2min, 30 cycles, 72 ℃ for extension for 10min, ③ the gene sequence of the amplification product is determined and provided by Beijing Olympic engineering service company Limited, ④ bacterial strain 16SrRNA sequence is subjected to Blast homologous comparison in NCBI database to determine the species classification of the bacterial strain, an adjacent approach (Neighbour-Joining) is adopted to establish a phylogenetic tree, and the bacterial strain WANYN 51 belongs to Clostridium butyricum (Genebank accession number: KY 197470).

3. And (3) identifying the hydrogen production performance:

the kelp hydrolysate is used as a liquid culture medium, different temperatures, initial pH values and initial concentrations of the kelp hydrolysate are set, and the hydrogen production performance of the strain WANY51 under different culture conditions is measured. And collecting gas generated by the bacterial strains by adopting a drainage method, and quantitatively analyzing the hydrogen production content by utilizing gas chromatography.

(1) And (3) measuring the hydrogen production capacity at different temperatures: culturing WANY51 at different temperatures (20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C) for 48h, wherein the initial pH values are pH7.0, and the initial concentration of herba Zosterae Marinae hydrolysate is 80%. The test result shows that the strain WANY51 can grow and produce hydrogen at different temperatures, and the hydrogen production efficiency is the highest at 35 ℃ and reaches 990.50ml/L (figure 3). Meanwhile, the components and the content of the liquid-phase fermentation product of the strain are also measured under the temperature condition, the liquid-phase fermentation product of the strain mainly comprises butyric acid, butanol and acetic acid, wherein the butanol content is 130.40 ml/L.

(2) Hydrogen production capacity under different initial pH values is determined: the initial concentration of the kelp hydrolysate is set to be 80%, the culture temperature is 35 ℃, and the hydrogen production efficiency is measured after culturing for 48h under the conditions that the initial pH values are 5.0, 6.0, 7.0, 8.0 and 9.0 respectively. The results show that: the strain WANY51 was able to grow at a set pH, and at an initial pH of 7.0, the strain grew best, with a cell density OD₆₀₀And the highest value reaches 1.02. The bacterial strains have obvious hydrogen production behavior within the pH range of 6.0-9.0. When the initial pH is 7.0, the hydrogen production efficiency of the strain by utilizing kelp hydrolysate is highest and reaches 987.07ml/L (figure 4).

(3) And (3) measuring the hydrogen production capacity under different initial concentrations of kelp hydrolysate: the initial concentrations of the kelp hydrolysate were set to 20%, 40%, 60%, 80% and 100%, the temperature was 35 ℃, the initial ph was ph7.0, and the hydrogen production efficiency was determined after 48h of culture. The results show that the strain WANY51 can grow under the different concentrations of the kelp hydrolysate, and the hydrogen production efficiency is the highest and reaches 990ml/L (figure 5) when the initial concentration of the kelp hydrolysate is 80%.

Claims

1. The hydrogenogen bacterium Clostridium beijerinckii WANY51 is preserved in China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 14045.

2. The application of the hydrogenogen CGMCC No.14045 in hydrogen production as claimed in claim 1 is characterized in that the culture medium for hydrogen production is a kelp hydrolysate culture medium, the kelp hydrolysate culture medium is prepared, kelp dry powder is dissolved in distilled water according to the proportion of 5g/L, the pH value is adjusted to 7.2-7.4 after stirring for 30min by a magnetic stirrer, and then the kelp hydrolysate culture medium is obtained after autoclaving at 121 ℃ for 20 min.

3. The application of the hydrogenogen CGMCC No.14045 in butanol production according to claim 1 is characterized in that a culture medium used for butanol production is a kelp hydrolysate culture medium, the kelp hydrolysate culture medium is prepared, kelp dry powder is dissolved in distilled water according to the proportion of 5g/L, the pH value is adjusted to 7.2-7.4 after stirring for 30min by a magnetic stirrer, and then the kelp hydrolysate culture medium is obtained after autoclaving at 121 ℃ for 20 min.

4. The application of the hydrogenogen CGMCC No.14045 in the hydrogen production of claim 2 is characterized in that: under the condition that kelp hydrolysate is used as the only culture medium: the culture temperature is 20-40 ℃; the initial pH value is pH5.0-9.0; the initial concentration of the kelp hydrolysate is 20-100%.

5. The use of the hydrogen-producing bacteria CGMCC No.14045 of claim 1 in butanol production or both hydrogen production and butanol production, wherein: under the condition that kelp hydrolysate is used as the only culture medium: the culture temperature is 35 ℃, the initial pH value is pH7.0, and the initial concentration of the kelp hydrolysate is 80%.