CN105754889B - A kind of Enterococcus faecium and its application in hydrogen production by anaerobic fermentation - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, in particular to an Enterococcus faecium and its application in hydrogen production by anaerobic fermentation. The strain of Enterococcus faecium is INET2, and the classification name is Enterococcus faecium. The Enterococcus faecium is isolated from the anaerobic digestion sludge treated by 5kGy γ-ray irradiation, and is a new Enterococcus faecium with hydrogen-producing ability. The Enterococcus faecium has the characteristics of rapid growth, high hydrogen production efficiency, drug resistance, heat resistance, acid and alkali resistance, etc.; it can reduce the environmental requirements of the fermentation hydrogen production system, and improve the economic benefits of the entire system; at the same time, it can use organic wastes The production of clean energy hydrogen for the substrate can achieve the dual benefits of waste disposal and energy production.

Description

A kind of Enterococcus faecium and its application in hydrogen production by anaerobic fermentation

technical field

The invention belongs to the technical field of bioengineering, in particular to an Enterococcus faecium and its application in hydrogen production by anaerobic fermentation.

Background technique

With the progress of urbanization and industrialization, energy crisis and environmental pollution have become two major problems restricting the further development of human beings. Fossil fuels, as the main energy supplier today, have their reserves declining continuously; and the environmental problems caused by pollutants (such as carbon dioxide, sulfur dioxide, PM2.5, etc.) accompanying the combustion of fossil fuels are also becoming more and more serious. Hydrogen has the characteristics of high calorific value and non-polluting combustion, making it a reasonable choice for clean energy.

Traditional hydrogen production methods include steam reforming of methane and other hydrocarbons (SRM/SRH), non-catalytic partial oxidation (POX) of fossil fuels, thermal reforming combining SRM and POX, and electrolysis of water. However, the above methods are accompanied by a large amount of energy consumption and a certain degree of environmental pollution. Compared with the above-mentioned traditional methods, biological hydrogen production has more advantages: mild reaction conditions, low energy consumption and high environmental protection. Biological hydrogen production includes photosynthetic biological hydrogen production, photo-fermentative biological hydrogen production and dark fermentation biological hydrogen production. Compared with other biological methods, the dark fermentation method has simpler equipment structure, easier control of reaction conditions and simpler operation, so it has been favored by researchers. their more attention. In addition, the use of organic waste as a substrate for dark fermentation can also achieve the dual purpose of waste treatment and energy recovery and regeneration.

The main body of biological hydrogen production by dark fermentation is the hydrogen-producing microorganisms in the system. Commonly used microbial flora include Clostridium, such as: Clostridium butyricum, Clostridium tyrobutyricum, Clostridium thermocellum, etc.; Enterobacter, such as Enterobacter cloacae, Enterobacter aerogenes, etc.; Escherichia coli, etc.; and some other species, such as Ethanoligenens harbinese, Ruminococcus albus, Caldicellulosiruptor saccharolyticus, etc.

Until now, research on E. faecium has focused on its drug resistance and its probiotic properties. In view of the complexity of actual waste, such as municipal waste, municipal sludge, agricultural waste, etc., common microorganisms are often inhibited by the poisons in them, and the drug resistance and strong viability of Enterococcus faecium have reduced the resistance to bacteria. Substrate selectivity. Therefore, the use of Enterococcus faecium for hydrogen production by fermentation has significant advantages. So far, there is no relevant research report on the application of Enterococcus faecium to hydrogen production by fermentation.

SUMMARY OF THE INVENTION

The invention provides a kind of Enterococcus faecium and its application in anaerobic fermentation hydrogen production, and the specific technical scheme is as follows:

An Enterococcus faecium, the strain of Enterococcus faecium is INET2, the classification name is Enterococcus faecium, and the deposit number in the General Microbiology Center of China Microorganism Culture Collection Management Committee is CGMCC1.15321.

The 16S rDNA gene sequence of the Enterococcus faecium is shown in SEQ ID No. 1, and the atpA gene sequence is shown in SEQ ID No. 2.

The Enterococcus faecium was isolated from the anaerobic digestion sludge treated with 5kGy γ-ray irradiation.

The biological characteristics of the Enterococcus faecium are: under the microscope, the cells are oval or ball-shaped, without spores and without flagella; the strain is Gram-positive, facultatively anaerobic; the strain is anaerobic at 25-40° C. After culturing for 20-48h, milky white colonies with smooth surface, bulges and neat edges can be formed, and the colony diameter is 1-1.5mm.

Application of Enterococcus faecium as described above in hydrogen production by anaerobic fermentation.

The steps of the described Enterococcus faecium fermenting hydrogen production under anaerobic conditions are:

Inoculate Enterococcus faecium into the enriched medium, and culture it anaerobic at 25-40°C and 80-120r/min to the logarithmic phase of bacterial growth; inoculation ratio of 5-30% to initial pH=5 -10 fermentation medium, anaerobic fermentation for 20-48h at a temperature of 25-40°C and 80-120r/min;

The components of the enrichment medium are: organic waste COD=50g/L, peptone 10g/L, yeast powder 0.5g/L, nutrient solution 10mL/100mL;

The components of the fermentation medium are: organic waste COD=5-20g/L, nutrient solution 10mL/100mL.

Both enrichment medium and fermentation medium were sterilized by high pressure water bath at 115℃ for 30min.

Hydrogen production by fermentation under the anaerobic conditions does not require a light source.

In the organic waste, the carbohydrate concentration is COD=5-20g/L; the components of the nutrient solution are: NaHCO ₃ 40g/L, NH ₄ Cl 5g/L, NaH ₂ PO ₄ 2H ₂ O 5g/L L, K ₂ HPO ₄ ·3H ₂ O 5g/L, FeSO ₄ ·7H ₂ O 0.25g/L, MgCl ₂ ·6H ₂ O 0.085g/L, NiCl ₂ ·6H ₂ O 0.004g/L.

In the range of the initial pH=5-10 of the fermentation medium, when the initial pH=7, the maximum amount of hydrogen produced by fermentation.

In the range of inoculation ratio of 5-30%, when the inoculation ratio was 10%, the hydrogen production by fermentation was the largest.

In the temperature range of 25-40 °C, when the temperature of anaerobic fermentation is 35 °C, the hydrogen production of fermentation is the largest.

The beneficial effects of the present invention are:

A new strain of Enterococcus faecium was screened out. The Enterococcus faecium has the characteristics of rapid growth, high hydrogen production efficiency, drug resistance, heat resistance, acid and alkali resistance, etc. It can reduce the environmental requirements of the fermentation hydrogen production system and improve the overall The economic benefits of the system; at the same time, clean energy hydrogen can be obtained from organic waste as a substrate, which can achieve the dual benefits of waste disposal and energy production.

Instructions for the Preservation of Biological Materials

Taxonomy Name: Enterococcus faecium; Strain Number: INET2

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: June 3, 2015

The registration number of the collection center: CGMCC1.15321.

Instruction drawings

Figure 1 is a biological evolutionary tree diagram of Enterococcus faecium INET2.

Figure 2 is a growth curve diagram of Enterococcus faecium INET2.

Figure 3 is a diagram of a hydrogen production device for fermentation of Enterococcus faecium INET2.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1: Isolation and strain identification of Enterococcus faecium INET2:

Sampling from the primary sludge digester of a municipal sewage treatment plant in Beijing, the obtained anaerobic digested sludge was irradiated with gamma rays emitted by the radionuclide ⁶⁰ Co at room temperature at a dose of 5kGy. The irradiated sludge is anaerobic enrichment culture in the enrichment medium for 20-48h, fermented in the medium with glucose as the sole carbon source to produce hydrogen for 20-48h, and then the bacterial liquid is diluted and spread on a solid plate for culture base. After 20-48h anaerobic culture at 25-40℃, the isolated single colonies were picked, inoculated into the fermentation medium, fermented and cultured at 25-40℃ for 20-48h, using gas chromatography (112A, Shanghai, thermal conductivity detector, packed column TDX-01 3000*3mm, column temperature 160°C, detector 110°C, injector 180°C) to detect the gas components generated in the fermentation flask, after a large number of strains screening process, separation A strain INET2 with strong hydrogen production ability and low oxygen sensitivity was obtained.

For the isolated and screened strains, the General Microbiology Center of China Microorganism Culture Collection and Management Committee (CGMCC) was entrusted to carry out strain identification, and comprehensive analysis was carried out according to the experimental data such as cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and atpA gene sequence. , The identified strain belongs to Firmicutes (Firmicutes), Enterococcus faecium (Enterococcus faecium) of Enterococcus (Enterococcus), named INET2.

The biological characteristics of the strain INET2 are: under the microscope, the cells are oval or bulbous, without spores and without flagella; the strain is Gram-positive, facultatively anaerobic; the strain is cultured under anaerobic conditions at 25-40 °C In 20-48h, milky white colonies with smooth surface, raised and neat edges can be formed, and the colony diameter is 1-1.5mm.

The identification results of the physiological and biochemical characteristics of the strain INET2 are shown in Table 1, the length of the 16S rDNA sequence is 1437kp, the full-length sequence is shown in SEQ ID NO.1, the length of the atpA sequence is 861kp, and the full-length sequence is shown in SEQ ID NO.2 Show.

According to the obtained 16S rRNA sequence, a matching analysis was performed in the GenBank of the ezbiocloud website, and its biological evolutionary tree was drawn (as shown in Figure 1), and the position and relationship of the strain in the biological evolution process were obtained. It can be seen from Figure 1 that E. faecium INET2 (CGMCC 1.15321) is closely related to E. albicans, and E. albicans is a recognized species with high hydrogen production capacity.

Table 1 Physiological and biochemical characteristics identification results

index result index result a positive Acid production from carbohydrates (continued) cell shape spherical L-arabinose + spore formation - L-Sorbitose - contact enzyme - L-Rhamnose - oxidase - lactose + growing in air + sucrose + 50℃ growth - maltose + 45℃ growth + trehalose + 15℃ growth + honeybiose + 6.5% NaCl growth + cellobiose + Carbohydrate acid production Songsanose - D-glucose + Raffinose + D-Fructose + Sorbitol - D-Mannose + Mannitol + D-ribose + Sodium Gluconate - D-xylose - Aesculus + L-Xylose - salicin + D-galactose + Amygdalin + D-arabinose - starch -

Example 2: Drawing of the growth curve of Enterococcus faecium INET2

Enterococcus faecium INET2 was inoculated into the enriched medium, incubated under constant temperature shaking at 36°C, 100 r/min, sampling was performed every 2 hours, the absorbance at 600 nm was measured, and the growth curve of the strain was drawn. ,as shown in picture 2.

Example 3: Fermentation hydrogen production of Enterococcus faecium INET2 at different temperatures

The strains in the logarithmic growth phase were taken and inoculated into the fermentation medium at an inoculation ratio of 10%, wherein the glucose concentration was 10 g/L, and the initial pH of the fermentation medium was 7.0. Under different temperature conditions (25-40° C.) and constant temperature oscillation reaction at 100 r/min, the reaction apparatus is shown in FIG. 3 . The gas generated in the reaction process is first washed with sodium hydroxide to absorb carbon dioxide in the gas, and then the remaining gas is collected by the drainage gas collection method, and the content of hydrogen in the collected gas is determined by gas chromatography. After 48h fermentation reaction, the fermentation hydrogen production under different temperature conditions is shown in Table 2.

Table 2 Fermentation hydrogen production of Enterococcus faecium INET2 at different temperatures

Example 4: Fermentative hydrogen production of Enterococcus faecium INET2 at different initial pH

The strains in the logarithmic growth phase were taken and inoculated into the fermentation medium at an inoculation ratio of 10%, wherein the glucose concentration was 10g/L, and the cultured with constant temperature shaking at 35°C and 100r/min. The initial pH value (5.0-10.0) was adjusted with 1 mol/L hydrochloric acid and sodium hydroxide solution, and the fermentation hydrogen production of the strains under different initial pH conditions was compared. The results obtained are shown in Table 3.

Table 3 Fermentation hydrogen production of Enterococcus faecium INET2 at different initial pH

Example 5: Fermentative hydrogen production of Enterococcus faecium INET2 under different glucose concentrations

The strains in the logarithmic growth phase were taken and inoculated into the fermentation medium at an inoculation ratio of 10%, and the initial pH of the fermentation medium was 7.0. Incubate with constant shaking at 35°C and 100 r/min. Comparing the fermentation hydrogen production of strains under different initial glucose concentration (5-20g/L) conditions, the obtained results are shown in Table 4.

Table 4 Fermentative hydrogen production of Enterococcus faecium INET2 under different glucose concentrations

Example 6: Fermentative hydrogen production of Enterococcus faecium INET2 under different inoculation ratios

The strains in the logarithmic growth phase were taken and inoculated into the fermentation medium at different inoculation ratios (5-30%), wherein the glucose concentration was 10 g/L, and the initial pH of the fermentation medium was 7.0. Incubate with constant shaking at 35°C and 100 r/min. The fermentation hydrogen production of the strains under the conditions of different initial strain inoculation ratios was compared, and the results obtained are shown in Table 5.

Table 5 Fermentation hydrogen production of Enterococcus faecium INET2 under different inoculation ratios

Claims (7)

1. a kind of manure enterococcin strain, enterococcus faecium (Enterococcus faecium) bacterial strain is INET2, in China The deposit number of General Microbiological Culture preservation administrative center is CGMCC No.1.15321.

2. application of the manure enterococcin strain described in claim 1 in hydrogen production through anaerobic fermentation.

3. application according to claim 2, which is characterized in that manure enterococcin strain fermentation and hydrogen production under anaerobic The step of are as follows:

Manure enterococcin strain is seeded in enriched medium, Anaerobic culturel is to bacterium under the conditions of 25-40 DEG C, 80-120r/min The logarithmic phase of strain growth；With the inoculation of 5-30% than accessing in the fermentation medium of initial pH=5-10, at 25-40 DEG C of temperature, Anaerobic fermentation 20-48h under the conditions of 80-120r/min；

The component of the enriched medium are as follows: debirs COD=50g/L, peptone 10g/L, yeast powder 0.5g/L, nutrient solution 10mL/100mL；

The component of the fermentation medium are as follows: debirs COD=5-20g/L, nutrient solution 10mL/100mL；

The component of the nutrient solution are as follows: NaHCO₃40g/L, NH₄Cl 5g/L, NaH₂PO₄·2H₂O 5g/L, K₂HPO₄·3H₂O 5g/L, FeSO₄·7H₂O 0.25g/L, MgCl₂·6H₂O 0.085g/L, NiCl₂·6H₂O 0.004g/L。

4. application according to claim 3, which is characterized in that the fermentation and hydrogen production under the anaerobic condition does not need light source.

5. application according to claim 3, which is characterized in that the initial pH=7 of the fermentation medium.

6. application according to claim 3, which is characterized in that the inoculation is than being 10%.

7. application according to claim 3, which is characterized in that the temperature of the anaerobic fermentation is 35 DEG C.