CN106399387A - Nitrogen defect control method for production of ethanol through fermentation of synthesis gas with mixed bacteria - Google Patents

Abstract

The invention belongs to the field of bioenergy, and in particular relates to a method for controlling nitrogen deficiency by fermenting synthetic gas with mixed bacteria to produce ethanol, comprising the following steps: the first stage: cultivating mixed bacteria to grow to a steady state in a fermentation medium, that is, the output of fermentation products Stable; the second stage: the mixed bacteria grown to a steady state in the first stage are cultivated in the second stage, and the nitrogen concentration of the fermentation medium controlled at this stage is lower than that in the first stage, and the mixed The bacteria are homoacetogenic bacteria, and its sources include cow dung, urban surplus sludge and its enrichment, because ethanol is a non-growth coupled product of acetogenic bacteria, and the lack of nitrogen nutrients is likely to lead to abnormal cell metabolism, resulting in The cells enter the non-growth state or the dormant state, and create conditions unfavorable for the growth of acetogenic bacteria through nitrogen nutrition deficiency, thereby changing the metabolic pathway from the acid-producing phase to the solvent-producing phase, and achieving the purpose of regulating ethanol production.

Description

A Nitrogen Deficiency Regulation Method for Ethanol Production by Fermenting Syngas with Mixed Bacteria

technical field

The invention belongs to the field of biological energy, and in particular relates to a nitrogen deficiency control method for producing ethanol by fermenting synthetic gas with mixed bacteria.

Background technique

Syngas fermentation to produce ethanol is a new technology to produce ethanol from biomass, that is, biomass is completely gasified to obtain synthesis gas with CO, H ₂ , and CO ₂ as the main components, and then microorganisms are used to convert the synthesis gas into ethanol. This technology does not require expensive enzymes and reagents such as acids and alkalis, which reduces production costs, and in theory microorganisms can completely utilize gases, which is an environmentally friendly technology.

Mixed culture biotechnology is a very promising technology in recent years. Its concept is to combine traditional elements of environmental biotechnology from the perspective of bioengineering, to maximize products like industrial biotechnology while treating waste. Mixed culture biotechnology uses the viewpoint of ecology to regulate the ecology of microorganisms through selection or regulation pressure. Compared with pure culture-based industrial biotechnology, mixed culture technology has the following advantages: (1) No sterilization is required; (2) Adaptability brought by microbial diversity; (3) The ability of microorganisms to utilize mixed substrates; (4) ) possibility of a sustainable process.

The research on synthesis gas fermentation ethanol production technology has just started in China. Due to the low solubility of CO, CO ₂ and H ₂ in liquid, the absorption of gas by microorganisms is small, and there are problems such as low ethanol yield. At present, researchers at home and abroad have adopted different control methods to increase the production of ethanol, mainly focusing on the control of fermentation conditions, including optimization of the medium, control of pH, control of gas mass transfer efficiency, and gas components and components. Gas partial pressure control, etc. Some scholars have found that the concentration of yeast extract plays an important role in increasing the ethanol yield of Clostridium autoethanogenum; it has been reported that low yeast extract concentration is beneficial to the sequential batch fermentation of Clostridium ljungdahlii to increase the molar ratio of ethanol to acetic acid. Studies have shown that different sources of nitrogen nutrition and nitrogen nutrition The concentration of substances will affect the concentration and ratio of acetic acid and ethanol, the metabolites of acetogenic bacteria to a certain extent. However, these reports are all based on the effects of yeast extracts on the ethanol production from synthetic gas transformed by pure bacteria, and do not involve the regulation of nitrogen deficiency in the production of ethanol from synthetic gas fermented by mixed bacteria.

The operation method of the synthesis gas fermentation method (patent number CN104822836A) and the method of reducing _CO2 emission and improving alcohol production rate in the synthesis gas fermentation (patent number CN104812904A) invented by Ineos Biological Co., Ltd. do not involve the regulation method based on nitrogen deficiency A method for improving the production of ethanol from synthetic gas fermented by mixed bacteria. A composite flora proposed by the University of Science and Technology of China and its application in syngas fermentation and alcohol production (CN105087441A) also does not involve nitrogen deficiency regulation methods.

In view of the above-mentioned defects, the designer is actively researching and innovating in order to create a nitrogen-deficiency control method for the production of ethanol by fermentation of synthetic gas with mixed bacteria, so that it has more industrial value.

Contents of the invention

In order to solve the above-mentioned technical problems, the object of the present invention is to provide a nitrogen deficiency regulation method for producing ethanol by fermentation of synthetic gas by mixed bacteria. Ethanol is a non-growth coupled product of acetogenic bacteria, and insufficient nitrogen nutrients may lead to abnormal cell metabolism. As a result, the cells enter a non-growth or dormant state, creating conditions unfavorable for the growth of acetogenic bacteria through nitrogen nutrition deficiency, thereby changing the metabolic pathway from the acid-producing phase to the solvent-producing phase, and achieving the purpose of regulating ethanol production.

The present invention proposes a nitrogen deficiency control method for ethanol production by fermenting synthetic gas with mixed bacteria, comprising the following steps:

The first stage: the culture of mixed bacteria grows to a steady state in the fermentation medium, that is, the output of the fermentation product is stable;

The second stage: the mixed bacteria grown to a steady state in the first stage are cultured in the second stage, and the nitrogen concentration of the fermentation medium in this stage is controlled to be lower than that in the first stage;

The mixed bacteria are homoacetogenic bacteria, and its source includes cow dung, urban surplus sludge and its enrichment.

Further, in the first stage and the second stage, the fermentation medium includes a nitrogen source and basic components, the nitrogen source includes yeast extract or NH ₄ Cl, and the concentration of the yeast extract in the first stage is 1.0～10.0g/L, the concentration in the first stage>the concentration in the second stage≥0.05g/L; the concentration of the NH ₄ Cl in the first stage is 0.5～1.0g/L, in the first stage The concentration of > the concentration in the second stage ≥ 0g/L.

Further, the basic components include K ₂ HPO ₄ 0.25g/L, KH ₂ PO ₄ 0.25g/L, MgCl ₂ ·6H ₂ O 0.3g/L, BES 10.5g/L.

Further, during the cultivation process of the first stage and the second stage, syngas is filled to standard atmospheric pressure, and the syngas includes CO, CO ₂ , and H ₂ at a volume ratio of 4:3:3.

Further, the culture temperature of the first stage and the second stage is 30-55° C., and the pH is 7.0-8.0.

By virtue of the above scheme, the present invention has at least the following advantages: the present invention can achieve the purpose of increasing the ethanol production of mixed bacteria syngas fermentation through the regulation of nitrogen nutrition deficiency, because ethanol is a non-growth coupled product of acetogenic bacteria, and nitrogen nutrients Insufficiency is likely to lead to abnormal cell metabolism, resulting in cells entering a non-growth state or a dormant state, creating conditions that are not conducive to the growth of acetogenic bacteria through nitrogen nutritional deficiencies, thereby changing the metabolic pathway from the acid-producing phase to the solvent-producing phase to achieve regulation The purpose of ethanol production; this method can be applied to the synthesis gas fermentation of sources such as lignin and cellulose, and the production of new energy ethanol can make waste resources, and reduce the emission of greenhouse gases such as _CO2 , which is in line with the principle of sustainable development; The large-scale industrial application of ethanol production by gas fermentation provides important theoretical value and practical guiding significance.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Fig. 1 is the ethanol output of synthetic gas fermentation of yeast extract deficiency regulation mixed bacteria in embodiment 1;

Fig. 2 is the ethanol output of the syngas fermentation of mixed bacteria regulated by NH ₄ Cl deficiency in Example 2;

Fig. 3 is the ethanol output of the syngas fermentation of the yeast extract defect regulation mixed bacteria in embodiment 3;

Fig. 4 shows the ethanol production of syngas fermentation of mixed bacteria regulated by NH ₄ Cl deficiency in Example 4.

detailed description

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

Add 50g of homoacetogenic bacteria enrichment to 500mL liquid medium containing 1.0g/L yeast extract, fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure, adjust pH=7.0 , at 37°C for the first stage of cultivation, and after 6 days of cultivation, the homoacetogenic bacteria grow to a steady state and enter the second stage. Take 10g of the enrichment after the first stage of cultivation and inoculate it into 100mL of basal medium containing 0.05g/L yeast extract, and place it on a constant temperature shaker at 37°C at 100rpm. The components and concentrations of the basic elements in the medium are the same as The first stage is the same: K ₂ HPO ₄ 0.25g/L, KH ₂ PO ₄ 0.25g/L, MgCl ₂ ·6H ₂ O 0.3g/L, BES (bromoethanesulfonate) 10.5g/L. Fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure to maintain pH 7.0. Gas consumption and organic product formation were measured by gas chromatography. The results showed that the ethanol accumulation in the second stage was 12.6mmol/L, which was 58.3% higher than the ethanol concentration in the first stage (Fig. 1). The above shows that the two-stage culture method in which the concentration of yeast extract in the second stage is controlled to be lower than that in the first stage can greatly increase the ethanol production.

The screening method for homoacetic acid bacteria is as follows:

Inoculate 200g of cow dung into a 1000mL anaerobic bottle, add 500mL of ingredients (L, pH 7): CH ₃ COONa (3.5g), NH ₄ Cl (0.5g), KH ₂ PO ₄ (0.25g), K ₂ HPO ₄ 3H ₂ O (0.25g), MgCl ₂ 6H ₂ O (0.3g), FeCl ₃ 6H ₂ O (25mg), NiSO ₄ 6H ₂ O (16mg), CaCl ₂ (25mg), ZnCl ₂ (11.5 mg), CoCl 6H ₂ O (10.5 mg), CuCl ₂ 2H ₂ O (5 mg), MnCl ₂ 4H ₂ O (15 mg) and BES (10.5 g) anaerobically treated enrichment medium . The headspace was filled with N ₂ (99%) for 20 minutes to a standard atmospheric pressure, placed on a shaker at 37° C. and 100 rpm for culture, and the headspace gas was replaced every 24 hours. When the fhs gene copy number increased and stabilized, the enrichment ended, and the enrichment was used as the inoculum for subsequent syngas bioconversion experiments.

Example 2

Add 50g of homoacetogenic bacteria enrichment to 500mL liquid medium containing 0.5g/L NH ₄ Cl, fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure, adjust pH=7.0 , at 37°C for the first stage of cultivation, and after 6 days of cultivation, the homoacetogenic bacteria grow to a steady state and enter the second stage. Take 10 g of the enrichment after the first-stage culture and inoculate it into 100 mL of basal medium without NH ₄ Cl (other than the nitrogen source, other basic components and concentrations are the same as in Example 1), and place it on a constant temperature shaker at 37°C and 100 rpm to cultivate. Fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure to maintain pH 7.0. Gas consumption and organic product formation were measured by gas chromatography. The results showed that the maximum ethanol accumulation in the second stage was 7.8mmol/L, which was 40.2% higher than the maximum ethanol accumulation in the first stage (Fig. 2). The above indicated that the two-stage culture method of regulating inorganic nitrogen auxotrophy was beneficial to the accumulation of ethanol.

The screening method for homoacetic acid bacteria is the same as in Example 1.

Example 3

Add 50g of homoacetogenic bacteria enrichment to 500mL liquid medium containing 10g/L yeast extract, fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure, adjust pH=7.0, The first stage of cultivation was carried out at 37°C, and after 6 days of cultivation, the homoacetogenic bacteria grew to a steady state and entered the second stage. Take 10 g of the enrichment after the first-stage culture and inoculate it into 100 mL of basal medium containing 0.1 g/L yeast extract (basic element components and concentrations are the same as in Example 1), and place it on a constant temperature shaker at 37°C and 100 rpm for cultivation . Fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure to maintain pH 7.0. Gas consumption and organic product formation were measured by gas chromatography. The results showed that the ethanol accumulation in the second stage was 10.9mmol/L, which was 45.9% higher than the ethanol concentration in the first stage (Fig. 3). The above shows that the two-stage culture method in which the concentration of yeast extract in the second stage is controlled to be lower than that in the first stage can greatly increase the ethanol production.

The screening method for homoacetic acid bacteria is the same as in Example 1.

Example 4

Add 50g of homoacetogenic bacteria enrichment to 500mL liquid medium containing 1.0g/L NH ₄ Cl, fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure, adjust pH=7.0 , at 37°C for the first stage of cultivation, and after 6 days of cultivation, the homoacetogenic bacteria grow to a steady state and enter the second stage. Take 10 g of the enrichment after the first-stage culture and inoculate it into 100 mL of basal medium with a NH ₄ Cl concentration of 0.25 g/L (except for the nitrogen source, other components and concentrations are the same as in Example 1), and place at 37°C, 100 rpm Cultured on a constant temperature shaker. Fill with CO/CO ₂ /H ₂ (4:3:3) to a standard atmospheric pressure to maintain pH 7.0. Gas consumption and organic product formation were measured by gas chromatography. The results showed that the maximum ethanol accumulation in the second stage was 6.2 mmol/L, which was 25.8% higher than the maximum ethanol accumulation in the first stage (Fig. 4). The above indicated that the two-stage culture method of regulating inorganic nitrogen auxotrophy was beneficial to the accumulation of ethanol.

The screening method for homoacetic acid bacteria is the same as in Example 1.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (5)

1. a kind of mixed fermentation synthesis gas producing and ethanol nitrogen defect regulation and control method it is characterised in that：Comprise the following steps：

First stage：Culture Mixed Microbes grow to stable state in the fermentation medium, and that is, the stable yield of tunning is constant；

Second stage：The Mixed Microbes growing to stable state in the described first stage are carried out the culture of second stage, controlling should The nitrogen concentration of the fermentation medium in stage is less than the described first stage；

Described Mixed Microbes are homoacetogenic bacteria, and its source includes cow dung, city excess sludge and its enriched substance.

2. mixed fermentation synthesis gas producing and ethanol according to claim 1 nitrogen defect regulation and control method it is characterised in that：Institute State in first stage and second stage, fermentation medium includes nitrogen source and basis, described nitrogen source include yeast extract or NH₄Cl, described yeast extract in the concentration in the first stage be 1.0～10.0g/L, the concentration in the first stage>Second-order Concentration >=0.05g/L in section；Described NH₄Cl in the concentration in the first stage be 0.5～1.0g/L, the concentration in the first stage >Concentration >=0g/L in second stage.

3. the mixed fermentation synthesis gas producing and ethanol stated according to claim 2 nitrogen defect regulation and control method it is characterised in that：Described Basis includes K₂HPO₄0.25g/L、KH₂PO₄0.25g/L、MgCl₂·6H₂O 0.3g/L、BES 10.5g/L.

4. mixed fermentation synthesis gas producing and ethanol according to claim 1 nitrogen defect regulation and control method it is characterised in that：Institute State in first stage and the incubation of second stage, be filled with synthesis gas to standard atmospheric pressure, described synthesis gas includes volume ratio For 4:3:3 CO, CO₂、H₂.

5. mixed fermentation synthesis gas producing and ethanol according to claim 1 nitrogen defect regulation and control method it is characterised in that：Institute State the first stage and the cultivation temperature of second stage is 30-55 DEG C, pH is 7.0-8.0.