CN112457994A

CN112457994A - Method for promoting growth of chlorella pyrenoidosa by using volatile fatty acid

Info

Publication number: CN112457994A
Application number: CN202011357848.1A
Authority: CN
Inventors: 宋明明; 苏琨洋; 刘娜; 李雪; 于泽
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-03-09
Anticipated expiration: 2040-11-27
Also published as: CN112457994B

Abstract

The invention belongs to the technical field of microalgae biology, and particularly relates to a method for promoting growth of chlorella pyrenoidosa by using volatile fatty acid. The invention takes BG-11 culture medium as basic culture medium of chlorella pyrenoidosa, the microalgae liquid is centrifugally concentrated when the microalgae grows to logarithmic phase, and acetic acid: propionic acid: butyric acid with concentration of 4-10 g/L mixed Volatile Fatty Acids (VFAs), and the inoculated seed liquid has optical density value of 0.2-1.0 at 680nm, and is cultured at 24-26 deg.C in dark for 2-5 days. The method can obviously shorten the growth period of the heterotrophic pyrenoid chlorella, enables the microalgae to obtain the highest biomass concentration within two days, realizes the rapid accumulation of biomass, improves the conversion efficiency, reduces the problem of high culture cost caused by overlong culture period in the actual production, and provides a new idea for the large-scale production of oil preparation by the microalgae.

Description

Method for promoting growth of chlorella pyrenoidosa by using volatile fatty acid

Technical Field

The invention belongs to the technical field of microalgae biology, and particularly relates to a method for promoting growth of chlorella pyrenoidosa by using volatile fatty acid, in particular to a method for realizing short-term biomass growth of microalgae by adding an external carbon source into a culture medium and mixing the volatile fatty acid for heterotrophic culture in the culture process of the chlorella pyrenoidosa.

Background

In the modern society, with the rapid development of global economy, the demand for fuel is increasing dramatically. The greenhouse effect caused by the release of a large amount of carbon dioxide in the combustion process of the traditional fossil fuel makes the problems of global warming, ecological environment pollution, human health and the like increasingly prominent, and the best means for solving the problems is to seek an economic and environment-friendly renewable energy source for replacing the fossil fuel. Biodiesel has great development potential as a clean renewable energy source, and the biodiesel in China faces the dilemma of low production quantity and high gap quantity. The large-scale production of the biodiesel is realized, and the selection of the algae strains is particularly critical. Microalgae is a main body of third-generation biodiesel with the advantages of high oil content, high photosynthetic efficiency, easy culture, large yield per unit area, no land competition with agriculture and the like, and is increasingly concerned by people, and the large-scale industrial production of microalgae for oil production is limited by high culture cost and low energy conversion rate at present. Therefore, how to reduce the microalgae culture cost and improve the conversion efficiency is a key point to be urgently solved in the current development of microalgae biodiesel.

There are three culture modes for microalgae to synthesize biomass by using carbon source, namely autotrophy, mixotrophy and heterotrophy (Moona M, et al. Mixotropic growth with acetate or zeolite fat acid microorganisms maxima growth and lipid production in Chlamydomonas reinhardtii. Algal Research 2 (2013) 352-. Microalgae is a photoautotrophic microorganism, energy is accumulated in the air by using carbon dioxide as a carbon source, the most commonly used culture mode is provided, but factors such as light limitation and the like in the culture process have low biomass density, and large-scale industrial production is difficult to realize. The defects of the autotrophic culture of the microalgae can be relieved to a great extent by the mixed culture and the heterotrophic culture, the growth speed of the microalgae is improved to a great extent, but the high-cost external carbon source in the culture is a great challenge for limiting the high-density culture of the microalgae, and in order to solve the limitation, the preparation of biomass Energy by using waste is a beneficial attempt in the fields of environmental protection and Energy development (Liang Y., Producing liquid transportation fuels from hydrothermal microorganism, applied. Energy 2013, 104, 860-doped 868), and the development and the utilization of cheap substrates are crucial to the reduction of the production cost of the biological Energy.

The traditional culture medium for oleaginous microorganisms takes sugar (such as monosaccharide, lactose, sucrose and the like) as a basic raw material, can be absorbed and utilized by most oleaginous microorganisms, but has certain limitation and higher cost when being used as a carbon source. The anaerobic fermentation technology is a simple and easy process, and is currently applied to a plurality of organic waste treatment fields, such as municipal waste, municipal sludge, livestock and poultry manure, crop straws and the like. Among them, VFAs are important intermediates in anaerobic digestion process, are short-chain organic acids, which occur after hydrolysis stage and acidogenesis stage of anaerobic digestion and are widely present in anaerobic fermentation process of various organic wastes such as industrial food wastes, agricultural wastes, sewage sludge, etc. If excessive VFAs accumulated in the fermentation waste liquid are not reasonably utilized, the energy production process is inhibited, the energy conversion rate is low, and the volatile acid substances with pungent odor have potential harm to the ecological environment. Therefore, the research on how to reduce the microalgae culture cost and improve the transformation efficiency and the reasonable utilization of VFAs has wide economic and social benefits.

Disclosure of Invention

Aiming at the problems and the defects in the prior art, the invention provides a method for promoting the growth of chlorella pyrenoidosa by using mixed VFAs as an external carbon source, the algae liquid is pretreated, mixed VFAs with different concentrations are added and diluted in proportion to a set optical density for heterotrophic culture, and algae cells grow at a high speed by using the VFAs under heterotrophic conditions; the method can maximize the biomass of the chlorella pyrenoidosa in a short period, realize the high-efficiency and rapid accumulation of the algae cells, and simultaneously reduce the culture cost by using heterotrophic conditions.

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

a method for rapidly promoting the growth of chlorella pyrenoidosa by utilizing volatile fatty acid comprises the following steps:

(1) using BG-11 culture medium as basic culture medium, periodically measuring optical density value of the algae solution to be inoculated every day, drawing growth curve, carrying out centrifugal concentration on the algae solution when the microalgae grows to logarithmic phase, suspending the algae solution by using BG-11 culture medium as seed solution;

(2) adding the mixed VFAs solution into the seed solution prepared in the step (1), wherein the optical density after inoculation is 0.2-1.0, and culturing for 2-5 days at 24-26 ℃ in the dark.

Preferably, the microalgae in step (1) is Chlorella pyrenoidosa strainChlorella pyrenoidosa(FACHB-1216) purchased from the freshwater algae breeder Bank of Chinese academy of sciences.

Preferably, the centrifugal concentration condition in step (1) is that the microalgae reach logarithmic growth phase.

Preferably, the concentration of the mixed VFAs solution of step (2) is 4 to 10 g/L, the mixed VFAs solution consisting of acetic acid, propionic acid and butyric acid; wherein the concentration ratio of the acetic acid to the propionic acid to the butyric acid is 4-6: 1-5: 1-5, respectively.

More preferably, the mixed VFAs solution obtained in the step (2) is added to the seed solution prepared in the step (1), and the concentration ratio of acetic acid, propionic acid and butyric acid in the mixed VFAs solution is 4: 5: 1.

Compared to other glycosyl substrates as carbon sources, VFAs have shorter metabolic pathways, shorter and higher theoretical lipid conversion efficiencies. In addition, the conventional culture period of the microalgae is usually about one week, the invention discovers that the growth speed of the microalgae can be improved while the VFAs are used as the carbon source, the high conversion rate of the microalgae in a short period is realized, a new idea is provided for the large-scale production of oil preparation by the microalgae, the waste recycling is realized, the problem of energy shortage can be effectively relieved, and the invention has important economic and environmental protection significance.

Advantageous effects

(1) The preparation method of the invention obviously shortens the logarithmic growth phase of the chlorella pyrenoidosa, and the microalgae can obtain the highest biomass concentration within two days by using VFAs as an external carbon source under the heterotrophic condition, thereby realizing biomass accumulation.

(2) The VFAs of the invention are used as intermediate products after anaerobic digestion treatment, have wide sources and can be derived from the anaerobic fermentation process of organic wastes such as agricultural wastes, food wastes, sewage sludge and the like. The energy microalgae is cultured by using VFAs generated in the anaerobic digestion process, so that the culture cost of the microalgae can be reduced, substances such as volatile acid in fermentation waste liquid can be reasonably utilized, the deep utilization of a substrate is realized, and the dual effects of renewable energy production and environmental protection are achieved.

Drawings

FIG. 1 growth of Chlorella pyrenoidosa under culture conditions of examples 1-6 at different initial optical densities and different mixed VFAs; wherein plot (a) is the growth of Chlorella heterotropha utilizing different ratios of mixed VFAs at low initial optical densities of examples 1-4; FIG. (b) is a graph showing the growth of the heterotrophic Chlorella pyrenoidosa of examples 5-6 after increasing the initial optical density at an optimum ratio of volatile acid.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The following examples were conducted in accordance with conventional methods and conditions, and experimental methods without specifying specific conditions were used.

The microalgae described in the following examples are Chlorella pyrenoidosa strainsChlorella pyrenoidosa(FACHB-1216) purchased from the freshwater algae breeder Bank of Chinese academy of sciences.

The formulation of BG-11 medium referred to in the following examples is shown in tables 1 and 2.

TABLE 1 BG-11 Medium formulation

Name of medicine	Dosage of medicine/L
		NaNO₃	1.50g
K₂HPO₄	0.040g
		MgSO₄·7H₂O	0.075g
CaCl₂·2H₂O	0.036g
		Citric acid	0.006g
Ferric ammonium citrate	0.006g
		EDTANa₂	0.001g
Na₂CO₃	0.020g
		A₅Solutions of	1 mL

TABLE 2A 5 solution formulation

Name of medicine	Dosage of medicine/L
		MnCl₂·4H₂O	1.86 g
ZnSO₄·7H₂O	0.22 g
		Na₂MoO₄·2H₂O	0.39 g
H₃BO₃	2.86 g
		CuSO₄·5H₂O	0.08 g
Co(NO₃)₂·6H₂O	0.05 g

Example 1

(1) Chlorella pyrenoidosa is cultured by using BG-11 culture medium as basic culture medium (formula shown in Table 1), and optical density is measured every day to observe growth condition of microalgae and draw growth curve.

(2) And when the microalgae grow to the logarithmic phase, centrifuging the algae liquid at 4000 r/min for 10 min, discarding the supernatant, and re-suspending the algae cells by using a BG-11 culture medium to serve as seed liquid.

(3) Inducing the rapid growth of algae cells, and preparing a mixed VFAs solution with the concentration ratio of acetic acid to propionic acid to butyric acid being 6: 3: 1, wherein the concentration ratio of the acetic acid to the propionic acid to the butyric acid is 4 g/L. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at the temperature of 25 ℃ in the dark. After the chlorella pyrenoidosa is inoculated, the chlorella pyrenoidosa directly enters the logarithmic phase for 0.84 d^-1The highest biomass concentration of 0.13 g/L was obtained on day 2 (see FIG. 1, Table 3).

Example 2

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid to propionic acid to butyric acid is 6: 1: 3. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at the temperature of 25 ℃ in the dark. After the inoculation of the chlorella pyrenoidosa, directly entering the logarithmic phase for 0.80 d^-1The highest biomass concentration of 0.13 g/L was obtained on day 2 (see FIG. 1, Table 3).

Example 3

(2) And (3) allowing the microalgae to grow to a logarithmic phase, centrifuging the algae liquid at 4000 r/min for 10 min, removing supernatant, and re-suspending algae cells by using a BG-11 culture medium to serve as seed liquid.

(3) Induction of algal cellsAnd (3) rapidly growing, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of the acetic acid to the propionic acid to the butyric acid is 4: 5: 1. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at the temperature of 25 ℃ in the dark. After the inoculation of the chlorella pyrenoidosa, directly entering the logarithmic phase for 0.91 d^-1The highest biomass concentration of 0.14 g/L was obtained on day 2 (see FIG. 1, Table 3).

Example 4

(3) Inducing the rapid growth of algae cells, and preparing 10 g/L mixed VFAs solution, wherein the concentration ratio of acetic acid to propionic acid to butyric acid is 4: 1: 5. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at the temperature of 25 ℃ in the dark. After the chlorella pyrenoidosa is inoculated, the chlorella pyrenoidosa directly enters the logarithmic phase at 0.87 d^-1The highest biomass concentration of 0.12 g/L was obtained on day 2 (see FIG. 1, Table 3).

Example 5

(1) Culturing Chlorella pyrenoidosa with BG-11 culture medium as basic culture medium (formula shown in Table 1), measuring optical density every day, observing growth condition of microalgae, and drawing growth curve

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid, propionic acid and butyric acid is the optimal mixed VFAs ratio of 4: 5: 1. Adding the mixed VFAs solution into the seeds prepared in the step (2)In the seed solution, the optical density value of the inoculated seed solution at 680nm is 0.8, and the seed solution is cultured for 2.5 days at 25 ℃ under the dark condition. After the inoculation of the chlorella pyrenoidosa, the chlorella pyrenoidosa directly enters the logarithmic phase for 0.61 d^-1The highest biomass concentration of 0.40 g/L was obtained on day 2 (see FIG. 1, Table 3).

Example 6

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid, propionic acid and butyric acid is the optimal mixed VFAs ratio of 4: 5: 1. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.4 at 680nm for 2.5 days at the temperature of 25 ℃ in the dark. The inoculated chlorella pyrenoidosa directly enters the logarithmic phase for 0.70 d^-1The highest biomass concentration of 0.24 g/L was obtained on day 2 (see FIG. 1, Table 3).

Comparative example 1

(3) And (3) inducing the rapid growth of algae cells, adding BG-11 culture solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at 25 ℃ in the dark. Growth was inhibited after Chlorella pyrenoidosa inoculation and the biomass concentration at day 2 was only 0.01 g/L (see Table 3).

Comparative example 2

(3) And (3) inducing the rapid growth of algae cells, adding BG-11 culture solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the temperature of 25 ℃ for 9 days under the illumination condition, wherein the optical density value of the inoculated seed solution at 680nm is 0.2. Chlorella pyrenoidosa grew slowly after inoculation and the biomass concentration at day 2 was 0.06 g/L (see Table 3).

Comparative example 3

(3) Inducing the rapid growth of algae cells, and preparing a mixed VFAs solution with the concentration ratio of acetic acid to propionic acid to butyric acid being 6: 3: 1, wherein the concentration ratio of the acetic acid to the propionic acid to the butyric acid is 4 g/L. Adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 9 days at 25 ℃ under the illumination condition. The growth of the chlorella pyrenoidosa after inoculation is slow, and the specific growth rate is 0.50 d^-1The biomass concentration on day 2 was 0.09 g/L (see Table 3), lower than in example 1.

Comparative example 4

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid to propionic acid to butyric acid is 6: 1: 3. Adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 9 days at 25 ℃ under the illumination condition. The growth of the chlorella pyrenoidosa after inoculation is slow, and the specific growth rate is 0.50 d^-1The biomass concentration on day 2 was 0.10 g/L (see Table 3), lower than that of example 2.

Comparative example 5

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid, propionic acid and butyric acid is the optimal mixed VFAs ratio of 4: 5: 1. Adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 9 days at 25 ℃ under the illumination condition. The growth of the chlorella pyrenoidosa after inoculation is slow, and the specific growth rate is 0.54 d^-1The biomass concentration on day 2 was 0.08 g/L (see Table 3), lower than that of example 3.

Comparative example 6

(3) Inducing rapid growth of algae cells, preparing mixed VFAs solution of 10 g/L, whereinThe concentration ratio of acetic acid, propionic acid and butyric acid is 4: 1: 5. Adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 9 days at 25 ℃ under the illumination condition. The growth of the chlorella pyrenoidosa after inoculation is slow, and the specific growth rate is 0.41 d^-1The biomass concentration on day 2 was 0.08 g/L (see Table 3), lower than that of example 4.

Comparative example 7

(1) Scenedesmus tetracaudatus is cultured by taking BG-11 culture medium as a basic culture medium (the formula is shown in table 1), the optical density of the Scenedesmus tetracaudatus is measured every day, the growth condition of microalgae is observed, and a growth curve is drawn.

(1) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid, propionic acid and butyric acid is the optimal mixed VFAs ratio of 4: 5: 1. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 12 days at the temperature of 25 ℃ in the dark. After the Scenedesmus tetrandra is inoculated, the growth is slow, and the specific growth rate is 0.11 d^-1The biomass concentration on day 2 was 0.09 g/L (see Table 3).

Comparative example 8

(3) Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid, propionic acid and butyric acid is 5: 0.5: 4.5. Adding mixed VFAs solution into the seed liquid prepared in step (2), culturing the inoculated seed liquid at 25 deg.C in the dark at optical density of 0.2 at 680nmAnd 5 days of cultivation. The chlorella pyrenoidosa directly enters the logarithmic growth phase after inoculation, and the specific growth rate is 0.08 d^-1The biomass concentration on day 2 was 0.05g/L (see Table 3).

Comparative example 9

Inducing the rapid growth of algae cells, and preparing 8g/L mixed VFAs solution, wherein the concentration ratio of acetic acid to propionic acid to butyric acid is 7: 1.5: 1.5. And (3) adding the mixed VFAs solution into the seed solution prepared in the step (2), culturing the inoculated seed solution at the optical density value of 0.2 at 680nm for 5 days at the temperature of 25 ℃ in the dark. The chlorella pyrenoidosa directly enters the logarithmic growth phase after inoculation, and the specific growth rate is 0.06 d^-1The biomass concentration on day 2 was 0.07 g/L (see Table 3).

Claims

1. A method for promoting the growth of chlorella pyrenoidosa by utilizing volatile fatty acid is characterized by comprising the following steps:

selecting a BG-11 culture medium as a basic culture medium, regularly measuring the optical density value of the algae liquid to be inoculated every day, drawing a growth curve, carrying out centrifugal concentration on the algae liquid when the microalgae grows to a logarithmic growth phase, and suspending the algae liquid by using the BG-11 culture medium as a seed liquid;

and (2) adding the mixed VFAs solution into the seed solution prepared in the step (1), wherein the optical density of the inoculated seed solution is 0.2-1.0, and culturing for 2-5 days at 24-26 ℃ in the dark.

2. The method of claim 1, wherein the step of removing the metal oxide layer comprises removing the metal oxide layer from the metal oxide layerThe microalgae in the step 1 (1) is Chlorella pyrenoidosa strainChlorella pyrenoidosa（FACHB-1216）。

3. The method of claim 1 wherein the concentration of said mixed VFAs solution of step 1. (2) is 4-10 g/L, said mixed VFAs solution consisting of acetic acid, propionic acid and butyric acid; wherein the concentration ratio of the acetic acid to the propionic acid to the butyric acid is 4-6: 1-5: 1-5, respectively.