CN104561139A - Method for increasing final cell density of microorganisms and shortening culture time - Google Patents

Abstract

The invention relates to a method for increasing the final cell density of microorganisms and shortening culture time. The method comprises strain aerobic culture which comprises primary and secondary seed culture, wherein the primary seed culture adopts an index carbohydrate supplementation method, the secondary seed culture adopts a constant carbohydrate supplementation method, and finally, low-level culture of a seed liquid is realized. The invention further relates to a method for shortening the time of a microorganism anaerobic fermentation production process.

Description

A kind ofly improve the whole cell density of microorganism and shorten the method for incubation time

Technical field

The invention belongs to technical field of bioengineering.

Background technology

Succinic acid (succinic acid) is widely used in the industries such as medicine, agricultural chemicals, dyestuff, spices, paint, food and plastics, simultaneously as outstanding carbon four platform chemicals, can be used for synthesizing BDO, tetrahydrofuran (THF), γthe organic chemicals such as-butyrolactone and poly butylene succinate (PBS) class Biodegradable material, have huge market potential.

As carbon four platform chemicals most important in biorefinery product engineering, it is mainly the petrification of raw material with butane that current succinic acid is produced, employing be through MALEIC ANHYDRIDE MIN 99.5 electrolysis production by butane.And biological process utilizes fermentation using bacteria by carbon dioxide fixation, and sugar is converted into the biotechnology of succinic acid, compared with traditional petrification, bio-based succinic acid has following advantage: (1) adopts the metabolic pathway of biological fermentation production succinic acid clear and definite, meta-bolites is simple, therefore the purity of crystallized product succinic acid is high, not containing undersaturated diprotic acid and single acid, therefore high as polymerization single polymerization monomer quality, and petrification succinic acid is under a series of catalyst action, through oxidation, hydrogenation, the steps such as reduction obtain, therefore can containing micro-insatiable hunger diprotic acid and single acid in product succinic acid, these impurity to be polymerized with dibasic alcohol for succinic acid that to produce polyester be disadvantageous.(2) biological process succinic acid often produces 1t succinic acid in producing, and will have the CO of 0.37t ₂utilized by thalline, be conducive to the discharge reducing greenhouse gases.(3) biosynthesizing succinic acid can utilize renewable biomass resource, if various straw wastes etc. are as raw materials for production, can utilize abundant agricultural-forestry biomass resource, guarantees bio-based succinic acid by the impact of oil price fluctuation.(4) Biological preparation succinic acid can reduce the consumption of the Nonrenewable resources such as oil, coal, reaches the effect of energy-saving and emission-reduction, makes significant contribution for the development of China's recycling economy and Green GDP increase.

The production bacterial strain of succinic acid mainly comprise Actinobacillus succinogenes ( actinobacillus succinogenes), recombination bacillus coli ( escherichia coli) and Anaerobiospirillum succinoproducens ( anaerobiospirilum succiniciproducens).Intestinal bacteria, because genetic background is clear, easy to operate, easy-regulating, substratum require simple and grow the advantages such as rapid, are widely used in research in recent years to obtain the outstanding bacterial strain of succinic acid-producing.

Recombination bacillus coli is under aerobic conditions, and thalli growth is rapid, and glycosyl raw material is consumed mainly through tricarboxylic acid cycle, and generate energy material is used for somatic cells amount reproduction; Under anaerobic fermentation conditions, thalli growth is slow, mainly through reduction tricarboxylic acid pathway and glyoxylate pathway accumulation organic acid.Therefore, utilizing recombination bacillus coli to grow in succinic acid process at present adopts first aerobic to cultivate the method for thalline post anaerobic fermentation succinic acid.The people such as Jiang are in utilization e. coliin AFP111 two benches fermentation production of succinic acid process, anaerobic stages succinic acid product yield >90%, concentration >100 g/L, throughput rate reaches 1.8 ~ 2.5 g/Lh(Applied and Environmental Microbiology. 2010,76 (4): 1298-1300).In aerobic culturing process, the growth curve of bacterial strain, whole cell density is to seed liquor progression, and namely the magnification of equipment has important directive significance.

Summary of the invention

The present invention relates to a kind of by carrying out the preparation of specific seed liquid to microorganism to reduce the method that seed liquor cultivates progression, the method effectively can reduce facility investment, reduces material consumption and the energy consumption of product.

The present invention relates to and a kind ofly improve the whole cell density of microorganism and shorten the method for incubation time, described method comprises aerobic and cultivates thalline, described aerobic is cultivated thalline and is comprised firsts and seconds seed culture, the benefit sugar mode that wherein first order seed is cultivated is index feed supplement, and the benefit sugar mode that secondary seed is cultivated is constant feed supplement.

The present invention relates to and a kind ofly improve the whole cell density of microorganism and shorten the method for incubation time, described method comprises aerobic and cultivates thalline, described aerobic is cultivated thalline and is comprised firsts and seconds seed culture, the benefit sugar mode that wherein first order seed is cultivated is index feed supplement, and the benefit sugar mode that secondary seed is cultivated is constant feed supplement.

In one embodiment, product of the present invention is selected from succinic acid.

In one embodiment, the microorganism that the present invention uses is selected from intestinal bacteria.

In one embodiment, the microorganism that the present invention uses is selected from recombination bacillus coli.

In one embodiment, the carbohydrate supplemented in the method for the invention is selected from glucose.

In one embodiment, in first order seed is cultivated, dissolved oxygen maintains more than 20%.

In one embodiment, the present invention relates to a kind of succinic acid bacterial strain logarithmic phase that extends and reduce the method that seed liquor cultivates progression, comprise the following steps:

(1) seed liquor activation: get single bacterium colony from LB flat board, be inoculated in LB liquid nutrient medium, at 37 DEG C, shake-flask culture 6 ~ 8 h under 200 r/min conditions, to cell density (OD600) value between 3 ~ 5.

(2) primary seed solution preparation: by inoculum size 5 ~ 10% switching JSM primary-seed medium, adding glucose to starting point concentration is 5 ~ 10 gL ^-1, treat bacterial strain spend lag phase and glucose consumption to lower than 0.5 ~ 1 gL ^-1after, the feed supplement of glucose start index, and culturing process makes dissolved oxygen maintain more than 20% by raising revolution and air flow.

(3) secondary seed solution preparation: by 1 ~ 2% switching JSM secondary seed medium, adding glucose to starting point concentration is 25 ~ 30 gL ^-1, treat bacterial strain spend lag phase and glucose consumption to lower than 0.5 ~ 1 gL ^-1after, glucose starts by 2 ~ 3g L ^-1h ^-1constant speed feed supplement, maintains the increase by 2 ~ 3 per hour of cell OD value, when the decline of OD value raising speed causes <1, turns anaerobically fermenting.

(4) fermentation succinic acid-producing process: by whole for secondary seed solution culture transferring in anaerobic fermentation tank, with anaerobic environment in the ventilation flow rate of 0.1 ~ 0.2 vvm continuously logical carbon dioxide maintenance fermentor tank, and using sodium carbonate as acid neutralizing agent control ph more than 6.4, control temperature at 35 ~ 40 DEG C, transforming glucose synthesizing succinic acid.

Unless otherwise defined herein, the present invention is correlated with, and to have the implication that those of ordinary skill in the art understand usually usual for the Science and Technology term that uses, the name that use relevant to field of fermentation engineering as herein described and technology, be this area as everyone knows and generally use those.Except as otherwise noted, term below should be understood to have following implication:

As described herein, term " index feed supplement " refer to by regulate glucose add rate-controlling specific cell growth rate at 0.1 ~ 0.2h ^-1.

As described herein, term " constant speed feed supplement " refers to and makes raw materials of glucose become restricted growth factor with constant glucose fed speed, and makes specific cell growth rate at 0.03 ~ 0.07h ^-1.

The present invention, by effective technology and control, by extending the logarithmic phase of bacterial strain in seed liquor culturing process, and improving whole cell density, effectively reducing the cultivation progression of seed liquor, reducing the material consumption energy consumption of facility investment and product.

Embodiment

The bacterial strain used in embodiment and substratum as follows:

Bacterial strain: escherichia coliaFP111(Applied Environmental Microbiology. 2002,68,1715-1727)

Substratum:

(1) LB shake-flask seed liquid culture medium: yeast powder 5 gL ^-1; Peptone 10 gL ^-1; NaCl 5 gL ^-1.

(2) JSM substratum: citric acid 3 gL ^-1; Na ₂hPO ₄7H ₂o 3 gL ^-1; KH ₂pO ₄8 gL ^-1; (NH ₄) ₂hPO ₄8 gL ^-1; NH ₄cl 0.2 gL ^-1; (NH ₄) ₂sO ₄0.75 gL ^-1; MgSO ₄7H ₂o 1.00 gL ^-1; CaCl ₂2H ₂o 10.0 mgL ^-1; ZnSO ₄7H ₂o 0.5 mgL ^-1; CuCl ₂2H ₂o 0.25 mgL ^-1; MnSO ₄h ₂o 2.5 mgL ^-1; CoCl ₂6H ₂o 1.75 mgL ^-1; H ₃bO ₃0.12 mg; Al ₂(SO ₄) ₃. xh ₂o 1.77 mgL ^-1; Na ₂moO ₄2H ₂o 0.5 mgL ^-1; Ironic citrate 16.1 mgL ^-1; VB ₁20mg L ^-1; Vitamin H 2 mgL ^-1.

(3) volume of seeding tank and fermentor tank when two-stage is cultivated: first class seed pot 350L, secondary seed tank 35m ³, anaerobic fermentation tank 95m ³.

(4) volume of seeding tank and fermentor tank during third stage culture: first class seed pot 350L, secondary seed tank 3.5m ³, three grades of seeding tank 35m ³, anaerobic fermentation tank 95m ³.

The following examples elaborate to the present invention, but do not limit the present invention.

Embodiment 1

When adopting third stage culture, firsts and seconds seed culture medium is JSM substratum and adds the glucose of 10 ~ 12g/L, and three grades of seed culture are JSM substratum and add the glucose of 70g/L.With anaerobic environment in the ventilation flow rate of 0.1 ~ 0.2 vvm continuously logical carbon dioxide maintenance fermentor tank after three grades of seed culture terminate, and using sodium carbonate as acid neutralizing agent control ph more than 6.4, control temperature at 35 ~ 40 DEG C, transforming glucose synthesizing succinic acid.It the results are shown in Table 1.

Cell density, production concentration at the end of respectively cultivating during table 1 third stage culture

	Whole OD 600	Incubation time (h)	Succinic acid (g/L)
				First order seed	4.3	26	0
Secondary seed	4.8	18	0
				Three grades of seeds	65	40	0
Fermentation	32	45	65

Embodiment 2

When considering bacterial strain third stage culture, operation easier is large, and adds the probability of microbiological contamination, therefore changes two-stage seed culture into.But need the final cell density improving seed when one-level is cultivated in process, therefore have adjusted and mend sugared mode, initial interpolation glucose to concentration is 5 ~ 10 gL ^-1, treat bacterial strain spend lag phase and glucose consumption to lower than 0.5 ~ 1 gL ^-1after, the feed supplement of glucose start index, and culturing process makes dissolved oxygen maintain more than 20% by raising revolution and air flow, the results are shown in Table 2.Result shows, changes to mend after sugared mode most high-cell density and be increased to OD ₆₀₀be increased to about 40, and total incubation time of seed shortens on the contrary, can realize producing sour result with like third stage culture phase simultaneously.

Cell density, production concentration at the end of respectively cultivating when table 2 secondary is cultivated

	Whole OD 600	Incubation time (h)	Succinic acid (g/L)
				First order seed	40	30	0
Secondary seed	65	40	0
				Fermentation	32	45	64

Embodiment 3

Secondary culturing process dissolved oxygen remains most important to strain fermentation, under having investigated the different dissolved oxygen condition of maintenance, bacterial strain is produced to the impact of acid, the results are shown in Table 3.Result shows, it is ensure that bacterial strain has the prerequisite of better producing sour efficiency that dissolved oxygen maintains more than 20%.

Dissolved oxygen	The whole OD of fermentation previous stage seed 600	Incubation time (h)	Succinic acid (g/L)
				5%	63	50	38
10%	63	43	53
				20%	65	40	64
30%	65	40	67

Although the embodiment of reference example describes the present invention in detail in this article, should be understood that, the invention is not restricted to described embodiment.There is this area common skill and can obtain the personnel instructed herein will appreciate that within the scope of the present invention other change, amendment and embodiment.Therefore, the present invention as one man broadly should explain with claim described below.

Claims (10)

1. one kind is improved the whole cell density of microorganism and shortens the method for incubation time, described method comprises aerobic and cultivates thalline, described aerobic is cultivated thalline and is comprised firsts and seconds seed culture, the benefit sugar mode that wherein first order seed is cultivated is index feed supplement, and the benefit sugar mode that secondary seed is cultivated is constant feed supplement.

2. one kind is shortened the method for the time of microbiological anaerobic fermentation production process, described method is carried out aerobic and is cultivated thalline before being included in anaerobically fermenting, described aerobic is cultivated thalline and is comprised firsts and seconds seed culture, the benefit sugar mode that wherein first order seed is cultivated is index feed supplement, and the benefit sugar mode that secondary seed is cultivated is preferably constant feed supplement.

3. according to the method for claim 1 or 2, wherein said index feed supplement refer to by regulate glucose add rate-controlling specific cell growth rate at 0.1 ~ 0.2h ^-1.

4., according to the method for claim 1 or 2, wherein said constant speed feed supplement refers to and makes raw materials of glucose become restricted growth factor with constant glucose fed speed, and makes specific cell growth rate at 0.03 ~ 0.07h ^-1.

5. method as claimed in one of claims 1-4, wherein said product is selected from succinic acid.

6. method according to claim 5, wherein said microorganism is selected from intestinal bacteria.

7. method according to claim 6, wherein said microorganism is recombination bacillus coli.

8. method as claimed in one of claims 1-7, the carbohydrate wherein supplemented is selected from glucose.

9. method any one of claim 1-8, wherein in first order seed is cultivated, dissolved oxygen maintains more than 20%.

10. extend succinic acid bacterial strain logarithmic phase and reduce the method that seed liquor cultivates progression, comprise the following steps:

(1) seed liquor activation: get single bacterium colony from LB flat board, be inoculated in LB liquid nutrient medium, at 37 DEG C, shake-flask culture 6 ~ 8 h under 200 r/min conditions, to cell density (OD600) value between 3 ~ 5;

(2) primary seed solution preparation: by inoculum size 5 ~ 10% switching JSM primary-seed medium, adding glucose to starting point concentration is 5 ~ 10 gL ^-1, treat bacterial strain spend lag phase and glucose consumption to lower than 0.5 ~ 1 gL ^-1after, the feed supplement of glucose start index, and culturing process makes dissolved oxygen maintain more than 20% by raising revolution and air flow;

(3) secondary seed solution preparation: by 1 ~ 2% switching JSM secondary seed medium, adding glucose to starting point concentration is 25 ~ 30 gL ^-1, treat bacterial strain spend lag phase and glucose consumption to lower than 0.5 ~ 1 gL ^-1after, glucose starts by 2 ~ 3g L ^-1h ^-1constant speed feed supplement, maintains the increase by 2 ~ 3 per hour of cell OD value, when the decline of OD value raising speed causes <1, turns anaerobically fermenting;

(4) fermentation succinic acid-producing process: by whole for secondary seed solution culture transferring in anaerobic fermentation tank, with anaerobic environment in the ventilation flow rate of 0.1 ~ 0.2 vvm continuously logical carbon dioxide maintenance fermentor tank, and using sodium carbonate as acid neutralizing agent control ph more than 6.4, control temperature at 35 ~ 40 DEG C, transforming glucose synthesizing succinic acid.