CN113512567B - Method for improving oil yield of microalgae in heterotrophic system - Google Patents
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Abstract
The invention discloses a method for improving oil yield of microalgae in a heterotrophic system, which comprises the following steps: expanding culture of microalgae in autotrophic culture medium, centrifuging, and suspending in heterotrophic culture medium to obtain microalgae solution; expanding culture of rhodobacter capsulatus in photosynthetic bacteria culture medium, centrifuging and suspending in photoheterotrophic culture medium to obtain rhodobacter capsulatus bacterial liquid; inoculating the microalgae solution and the rhodobacter capsulatus bacterial solution into a photoheterotrophic culture medium for photoheterotrophic culture; the invention improves the oil yield of the photoheterotrophic culture microalgae by carrying out symbiotic culture on the microalgae and the rhodobacter capsulatus in the photoheterotrophic culture medium, the biomass yield of the culture system can be improved by more than 65 percent compared with that of a pure microalgae system, and the fatty acid yield can be improved by 30 percent compared with that of the pure microalgae system.
Description
Technical Field
The invention belongs to the technical field of microalgae biology, and relates to a method for improving microalgae oil yield under a photoheterotrophic condition.
Background
Biodiesel, namely fatty acid methyl ester, is a biomass energy source and has the characteristic of being renewable. It mainly comes from oil-producing crops such as soybean, sunflower seed, etc., but the disadvantages of low yield, high cost, land competition with other crops, etc. limit the further development. Some microalgae are rich in grease, and have short growth cycle and do not occupy cultivated land, so that the microalgae become a new source of biodiesel. In the commercialization process of microalgae oil production, high production cost is a primary bottleneck problem faced by microalgae for producing biodiesel. How to reduce the commercial cost of algae oil production becomes a very research-value topic, wherein, improving the oil yield of microalgae is an effective solution.
At present, most of researches on microalgae oil production adopt a photoautotrophic culture mode. However, photoheterotrophic culture can overcome this disadvantage due to the low cell density of the photoautotrophic culture. The photoheterotrophic culture can also absorb organic carbon in the wastewater to grow and accumulate grease, so that win-win effect of wastewater treatment and oil production is achieved.
However, microalgae are cultured alone under heterotrophic conditions, and the oil yield of microalgae is low.
Disclosure of Invention
The invention aims to provide a method for improving the oil yield of microalgae under the photoheterotrophic condition, which improves the oil yield of the microalgae by carrying out symbiotic culture on the microalgae and rhodobacter capsulatus in a photoheterotrophic culture medium.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for increasing oil production rate of microalgae in a heterotrophic system, the method comprising the steps of:
(1) Expanding culture of microalgae in autotrophic culture medium, centrifuging, and suspending in heterotrophic culture medium to obtain microalgae solution;
(2) Expanding culture of rhodobacter capsulatus in photosynthetic bacteria culture medium, centrifuging and suspending in photoheterotrophic culture medium to obtain rhodobacter capsulatus bacterial liquid;
(3) Inoculating the microalgae solution and the rhodobacter capsulatus bacterial solution into a photoheterotrophic culture medium for photoheterotrophic culture.
In the step (1), the biomass in the microalgae solution is 10-15g/L; in the step (2), the biomass in the rhodobacter capsulatus bacterial liquid is 1.0-1.5g/L.
In the step (3), the weight ratio of the microalgae liquid to the rhodobacter capsulatus bacterial liquid is 10:1-10:3; after inoculation, the initial biomass in the heterotrophic medium is 400-500 mg/L.
In the step (3), the conditions of the heterotrophic culture are as follows: culturing at 22-26 deg.c and illumination intensity of 6000-10000 lux for 6 days.
The illumination intensity is preferably 7000 to 9000lux, more preferably 7500 to 8500lux, most preferably 8000lux.
The microalgae is Chlorella of Chlorophyta.
The microalgae is Chlorella vulgaris or Chlorella pyrenoidosa.
The autotrophic culture medium is a modified C culture medium.
The photosynthetic bacteria culture medium is 112VanNiel's yeat agar culture medium.
The heterotrophic culture medium is an improved C culture medium added with sodium acetate.
The heterotrophic culture medium comprises the following raw materials: sodium acetate, ca (NO) 3 ) 2 ·4H 2 O、KNO 3 、C 3 H 7 Na 2 O 6 P·5H 2 O、MgSO 4 ·7H 2 O, vitamin B 12 Biotin, thiamine hydrochloride, tris (hydroxymethyl) aminomethane, na 2 EDTA·2H 2 O、FeCl 3 ·6H 2 O、MnCl 2 ·4H 2 O、ZnCl 2 、CoCl 2 ·6H 2 O and Na 2 MoO 4 ·2H 2 O。
The contents of the raw materials in the heterotrophic culture medium are as follows: sodium acetate 2-10 g/L, ca (NO) 3 ) 2 ·4H 2 O100～200mg/L、KNO 3 500～1500mg/L、C 3 H 7 Na 2 O 6 P·5H 2 O 25～75mg/L、MgSO 4 ·7H 2 O20-60 mg/L and vitamin B 12 0.05-0.15 mug/L, biotin 0.05-0.15 mug/L, thiamine hydrochloride 5-15 mug/L, tris (hydroxymethyl) aminomethane 250-750 mg/L, na 2 EDTA·2H 2 O 2～5mg/L、FeCl 3 ·6H 2 O 500～700μg/L、MnCl 2 ·4H 2 O 100～120μg/L、ZnCl 2 25～35μg/L、CoCl 2 ·6H 2 O10-15 mug/L and Na 2 MoO 4 ·2H 2 O 5～10μg/L，pH 6.0～8.0。
The sodium acetate content is preferably 2g/L to 8g/L, more preferably 4g/L to 6g/L, most preferably 5g/L.
The Ca (NO) 3 ) 2 ·4H 2 The content of O is preferably 125mg/L to 175mg/L, more preferably 140mg/L to 160mg/L, and most preferably 150mg/L.
The KNO 3 Preferably 750mg/L to 1250mg/L, more preferably 900mg/L to 1100mg/L, most preferably 1000mg/L.
The C is 3 H 7 Na 2 O 6 P·5H 2 The content of O is preferably 40mg/L to 60mg/L, more preferably 45mg/L to 55mg/L, and most preferably 50mg/L.
The MgSO 4 ·7H 2 The content of O is preferably 30mg/L to 50mg/L, more preferably 35mg/L to 45mg/L, and most preferably 40mg/L.
The vitamin B 12 Preferably 0.07. Mu.g/L to 0.13. Mu.g/L, more preferably 0.09. Mu.g/L to 0.11. Mu.g/L, most preferably 0.1. Mu.g/L.
The biotin content is preferably 0.07. Mu.g/L to 0.13. Mu.g/L, more preferably 0.09. Mu.g/L to 0.11. Mu.g/L, most preferably 0.1. Mu.g/L.
The thiamine hydrochloride content is preferably 7 μg/L to 13 μg/L, more preferably 9 μg/L to 11 μg/L, and most preferably 10 μg/L.
The content of tris (hydroxymethyl) aminomethane is preferably 400mg/L to 600mg/L, more preferably 450mg/L to 550mg/L, and most preferably 500mg/L.
The Na is 2 EDTA·2H 2 The content of O is preferably 2mg/L to 4mg/L, more preferably 2.5mg/L to 3.5mg/L, most preferably 3mg/L.
The FeCl 3 ·6H 2 The O content is preferably 550. Mu.g/L to 650. Mu.g/L, more preferably 550. Mu.g/L to 600. Mu.g/L, most preferably 588. Mu.g/L.
The MnCl 2 ·4H 2 The content of O is preferably 100. Mu.g/L to 115. Mu.g/L, more preferably 105. Mu.g/L to 110. Mu.g/L, most preferably 108. Mu.g/L.
The ZnCl 2 Preferably 28 μg/L to 32 μg/L, more preferably 30 μg/L to 32 μg/L, most preferably 31.2 μg/L.
The CoCl 2 ·6H 2 The content of O is preferably 11. Mu.g/L to 14. Mu.g/L, more preferably 11. Mu.g/L to 13. Mu.g/L, and most preferably 12. Mu.g/L.
The Na is 2 MoO 4 ·2H 2 The content of O is preferably 6 mug/L to 9. Mu.g/L, more preferably 7. Mu.g/L to 8. Mu.g/L, most preferably 7.5. Mu.g/L.
The pH of the heterotrophic medium is preferably 6.5 to 7.5, more preferably 6.8 to 7.2, most preferably 7.0.
Compared with the scheme in the prior art, the invention has the following advantages:
(1) According to the invention, the oil yield of the photoheterotrophic culture microalgae is improved by carrying out symbiotic culture on the microalgae and the rhodobacter capsulatus in the photoheterotrophic culture medium, the biomass yield of the culture system can be improved by more than 65% compared with that of a pure microalgae system, and the fatty acid yield can be increased by 30% compared with that of the pure microalgae system;
(2) When the weight ratio of the microalgae liquid to the rhodobacter capsulatus bacterial liquid is 10:1-10:3, the addition of rhodobacter capsulatus can obviously improve the oil yield of the microalgae.
Drawings
FIG. 1 shows the variation of biomass according to example 1;
FIG. 2 shows fatty acid content and yield for example 1;
FIG. 3 shows the variation of biomass according to example 2;
FIG. 4 shows the fatty acid content and yield of example 2.
Detailed Description
The present invention will be described in detail with reference to examples.
The microalgae in the embodiments of the invention are all Chlorella genus of Chlorella phylum, purchased from NiES japonica algae seed stock;
rhodobacter capsulatus in the examples of the present invention was rhodobacter, purchased from the collection of microorganism strains in Guangdong province.
The modified C medium, 112VanNiel's yeat agar medium, and heterotrophic medium used in the examples of the present invention were each composed of:
improvement C culture medium: ca (NO) 3 ) 2 ·4H 2 O 150mg/L、KNO 3 1000 mg/L、C 3 H 7 Na 2 O 6 P·5H 2 O 50mg/L、MgSO 4 ·7H 2 O40 mg/L, vitamin B 12 0.1. Mu.g/L, biotin 0.1. Mu.g/L, thiamine hydrochloride 10. Mu.g500mg/L, na of/L tris (hydroxymethyl) aminomethane 2 EDTA·2H 2 O 3mg/L、FeCl 3 ·6H 2 O 588μg/L、MnCl 2 ·4H 2 O 108μg/L、ZnCl 2 31.2μg/L、CoCl 2 ·6H 2 O12. Mu.g/L and Na 2 MoO 4 ·2H 2 O7.5 mug/L, pH 7.0, and distilled water as solvent;
112Van Niel's yeat agar medium: k (K) 2 HPO 4 1 g/L、MgSO 4 0.5 g/L and Yeast extract10g/L, pH 7.0, and distilled water as solvent.
Photoheterotrophic medium: sodium acetate 5g/L, ca (NO 3 ) 2 ·4H 2 O 150mg/L、KNO 3 1000 mg/L、C 3 H 7 Na 2 O 6 P·5H 2 O 50mg/L、MgSO 4 ·7H 2 O40 mg/L, vitamin B 12 0.1. Mu.g/L, biotin 0.1. Mu.g/L, thiamine hydrochloride 10. Mu.g/L, tris (hydroxymethyl) aminomethane 500mg/L, na 2 EDTA·2H 2 O 3mg/L、FeCl 3 ·6H 2 O 588μg/L、MnCl 2 ·4H 2 O 108μg/L、ZnCl 2 31.2μg/L、CoCl 2 ·6H 2 O12. Mu.g/L and Na 2 MoO 4 ·2H 2 O7.5 mug/L, pH 7.0, and distilled water.
Example 1
A method for improving oil yield of microalgae in a heterotrophic system comprises the following steps:
(1) Adding the modified C culture medium into 1L photosynthetic reactor, sub-packaging with 0.6L culture medium, sterilizing with 121 deg.C high pressure steam for 20 min, inoculating Chlorella vulgaris to ultra-clean bench to start autotrophic enlarged culture, and introducing CO 2 The mixed gas with air enters the reactor after being sterilized by a filter of 0.22 mu m at the top end of the reactor, and the aeration rate is 0.5v/v/min, wherein CO 2 The concentration is 4%, the temperature is controlled at 24+/-2 ℃, and the illumination intensity is 8000lux; and (3) culturing for 7d, centrifuging for 10 minutes at 6000 rpm after culturing is finished, collecting microalgae, and suspending the collected microalgae in a heterotrophic culture medium to obtain microalgae liquid, wherein the biomass in the microalgae liquid is 10-15g/L.
(2) Adding 112Van Niel's yeat agar culture medium into 250mL screw conical flasks, subpackaging 0.25L each flask, sterilizing with high-pressure steam at 121deg.C for 20 min, inoculating rhodobacter capsulatus bacterial liquid into an ultra-clean workbench when the culture medium is cooled to room temperature, and culturing for 7d at 24+ -2deg.C and 8000lux illumination intensity; and centrifuging for 10 minutes at 8000 rpm after the culture is finished to collect the rhodobacter capsulatus, and suspending the collected rhodobacter capsulatus in a photoheterotrophic culture medium to obtain rhodobacter capsulatus bacterial liquid, wherein the biomass in the rhodobacter capsulatus bacterial liquid is 1.0-1.5g/L.
(3) Adding a heterotrophic culture medium into 1L conical flasks, subpackaging 0.7L each flask, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, inoculating microalgae liquid and rhodobacter capsulatus bacterial liquid according to a weight ratio of 10:1 when the culture medium is cooled to room temperature, enabling the initial biomass of the system to be 330mg/L, starting the heterotrophic culture under the illumination intensity of 8000lux at 24 ℃ for 6 days, and enabling the stirring rate in the culture process to be 200 revolutions per minute.
After six-day heterotrophic culture, the biomass yield of the algae-bacteria mixed system is 201mg/L/d, which is higher than the sum of the biomass yields of the pure bacteria system (42 mg/L/d) and the pure microalgae system (121 mg/L/d). The final fatty acid yield of the algae bacteria mixed system is 33mg/L/d, which is 1.32 times of the fatty acid yield (25 mg/L/d) of the pure microalgae system.
The culture process of the pure bacterial system and the pure microalgae system is respectively as follows:
the cultivation process of the pure bacterial system comprises the following steps: adding a heterotrophic culture medium into 1L conical flasks, sub-packaging each flask with 0.7L, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, inoculating the rhodobacter capsulatus bacterial liquid in the step (2) when the culture medium is cooled to room temperature, so that the initial biomass of the system is 30mg/L, starting the heterotrophic culture under the illumination intensity of 8000lux at 24 ℃, wherein the culture time is 6d, and the stirring rate in the culture process is 200 revolutions per minute.
The culture process of the pure microalgae system comprises the following steps: adding a heterotrophic culture medium into 1L conical flasks, sub-packaging each flask with 0.7L, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, inoculating the microalgae solution in the step (1) when the culture medium is cooled to room temperature, enabling the initial biomass of the system to be 300mg/L, starting the heterotrophic culture at 24 ℃ and under 8000lux of illumination intensity, wherein the culture time is 6d, and the stirring rate in the culture process is 200 revolutions per minute.
Example 2
A method for improving oil yield of microalgae in a heterotrophic system comprises the following steps:
(1) Adding the modified C culture medium into 1L photosynthetic reactor, sub-packaging with 0.6L culture medium, sterilizing with 121 deg.C high pressure steam for 20 min, inoculating Chlorella pyrenoidosa to ultra-clean bench, and culturing under autotrophic growth, CO 2 The mixed gas with air enters the reactor after being sterilized by a filter of 0.22 mu m at the top end of the reactor, and the aeration rate is 0.5v/v/min, wherein CO 2 The concentration is 4%, the temperature is controlled at 24+/-2 ℃, and the illumination intensity is 8000lux; and (3) culturing for 7d, centrifuging for 10 minutes at 6000 rpm after culturing is finished, collecting the chlorella pyrenoidosa, and suspending the collected chlorella pyrenoidosa in a heterotrophic culture medium to obtain a microalgae solution, wherein the biomass in the microalgae solution is 10-15g/L.
(2) Adding 112Van Niel's yeat agar culture medium into 250mL screw conical flasks, subpackaging 0.25L each flask, sterilizing with high-pressure steam at 121deg.C for 20 min, inoculating rhodobacter capsulatus bacterial liquid into an ultra-clean workbench when the culture medium is cooled to room temperature, and culturing for 7d at 24+ -2deg.C and 8000lux illumination intensity; and centrifuging for 10 minutes at 8000 rpm after the culture is finished to collect the rhodobacter capsulatus, and suspending the collected rhodobacter capsulatus in a photoheterotrophic culture medium to obtain rhodobacter capsulatus bacterial liquid, wherein the biomass in the rhodobacter capsulatus bacterial liquid is 1.0-1.5g/L.
(3) Adding a heterotrophic culture medium into 1L conical flasks, sub-packaging each flask with 0.7L, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, inoculating microalgae liquid and rhodobacter capsulatus liquid according to a weight ratio of 10:1 when the culture medium is cooled to room temperature, starting the heterotrophic culture with initial biomass of 330mg/L at 24 ℃ and illumination intensity of 8000lux for 6d, and stirring at a speed of 200 revolutions per minute in the culture process.
After six-day heterotrophic culture, the biomass yield of the algae-bacteria mixed system is 176mg/L/d, which is higher than the sum of the biomass yields of the pure bacteria system (42 mg/L/d) and the pure microalgae system (105 mg/L/d). The final fatty acid yield of the algae bacteria mixed system is 35mg/L/d, which is 1.30 times of the fatty acid yield (27 mg/L/d) of the pure microalgae system.
The culture process of the pure bacterial system and the pure microalgae system is respectively as follows:
the cultivation process of the pure bacterial system comprises the following steps: adding a heterotrophic culture medium into 1L conical flasks, sub-packaging each flask with 0.7L, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, inoculating the rhodobacter capsulatus bacterial liquid in the step (1) when the culture medium is cooled to room temperature, so that the initial biomass of the system is 30mg/L, starting the heterotrophic culture under the illumination intensity of 8000lux at 24 ℃, wherein the culture time is 6d, and the stirring rate in the culture process is 200 revolutions per minute.
The culture process of the pure microalgae system comprises the following steps: adding a heterotrophic culture medium into 1L conical flasks, sub-packaging each flask with 0.7L, sterilizing with high-pressure steam at 121deg.C for 20 min, inoculating microalgae solution when the culture medium is cooled to room temperature to make the initial biomass of the system 300mg/L, starting heterotrophic culture at 24deg.C under 8000lux illumination intensity for 6d, and stirring at 200 rpm.
Comparative example 1
Comparative example 1 with respect to example 1, microalgae solution and rhodobacter capsulatus solution were inoculated in a weight ratio of 10:5, and after six days of heterotrophic culture, the biomass yield of the mixed algal-bacterial system was 152mg/L/d, which was lower than the sum of the biomass yields of the pure bacterial system 42mg/L/d and the pure microalgae system 121 mg/L/d. The final fatty acid yield of the algae bacteria mixed system is 23mg/L/d, which is 0.92 times of the fatty acid yield (25 mg/L/d) of the pure microalgae system.
Comparative example 2
Comparative example 2 with respect to example 2, microalgae and rhodobacter capsulatus bacteria were inoculated at a weight ratio of 10:5, and after six days of heterotrophic culture, the biomass yield of the mixed algal bacteria system was 137mg/L/d, which was lower than the sum of the biomass yields of the pure bacterial system 42mg/L/d and the pure microalgae system 105 mg/L/d. The final fatty acid yield of the algae bacteria mixed system is 26mg/L/d, which is 0.96 times of the fatty acid yield (27 mg/L/d) of the pure microalgae system.
The measurement and calculation methods of the cell dry weight, fatty acid content and biomass yield referred to in each of the above examples and comparative examples are as follows:
cell dry weight measurement: drying 0.45 μm acetate fiber film to constant weight, recording mass as m 0 (g) Extracting culture solution VmL every 24 hr during cell culture, filtering, oven drying to constant weight, and recording mass as m 1 (g) A. The invention relates to a method for producing a fibre-reinforced plastic composite The dry weight C can be calculated according to the following formula: c (g/L) = (m) 1 -m 0 )*1000/V。
Biomass yield = dry cell weight/days of culture at sampling.
Oil content and composition determination: after the culture is finished, the algae bacteria are harvested, freeze-dried for 48 hours, 25mg of freeze-dried algae bacteria powder is weighed and put into a digestion tube, 2mL of esterification reagent consisting of acetyl chloride and methanol according to the volume ratio of 1:9 is added, and the reaction is carried out in a water bath at 80 ℃ for 2.5 hours. After cooling to room temperature, 1mL of 0.58% NaCl solution was added to stop the esterification reaction. Then, 2mL of n-hexane (dissolved with methyl benzoate as a standard substance, the concentration of which is 0.36 mg/mL) was added, the mixture was thoroughly mixed and centrifuged at a low rotation speed, and after the solution was separated, the supernatant was taken for gas phase analysis to test the content of fatty acid (oil content).
Oil yield: calculated from biomass and oil content.
Wherein, the unit of biomass is mg/L, the unit of oil content is mg/L, and the unit of oil yield is mg/L/d.
The above detailed description of a method for increasing oil production rate of microalgae under heterotrophic conditions with reference to examples is illustrative and not limiting, and several examples can be listed according to the scope defined thereby, and therefore, variations and modifications without departing from the general inventive concept shall fall within the scope of protection of the present invention.
Claims (4)
1. A method for increasing the oil yield of microalgae in a heterotrophic system, comprising the steps of:
(1) Expanding culture of microalgae in autotrophic culture medium, centrifuging, and suspending in heterotrophic culture medium to obtain microalgae solution;
(2) Expanding culture of rhodobacter capsulatus in photosynthetic bacteria culture medium, centrifuging and suspending in photoheterotrophic culture medium to obtain rhodobacter capsulatus bacterial liquid;
(3) Inoculating the microalgae solution and the rhodobacter capsulatus bacterial solution into a photoheterotrophic culture medium for photoheterotrophic culture;
the microalgae is chlorella vulgaris or chlorella pyrenoidosa;
in the step (3), the weight ratio of the microalgae liquid to the rhodobacter capsulatus bacterial liquid is 10:1-10:3; after inoculation, the initial biomass in the heterotrophic culture medium is 400-500 mg/L;
the contents of the raw materials in the heterotrophic culture medium are as follows: sodium acetate 2-10 g/L, ca (NO) 3 ) 2 •4H 2 O 100 ~200 mg/L、KNO 3 500 ~ 1500 mg/L、C 3 H 7 Na 2 O 6 P•5H 2 O 25~75 mg/L、MgSO 4 •7H 2 O20-60 mg/L and vitamin B 12 0.05-0.15 [ mu ] g/L, biotin 0.05-0.15 [ mu ] g/L, thiamine hydrochloride 5-15 [ mu ] g/L, tris (hydroxymethyl) aminomethane 250-750 mg/L, na 2 EDTA•2H 2 O 2 ~ 5 mg/L、FeCl 3 •6H 2 O 500~700 μg/L、MnCl 2 •4H 2 O 100~120 μg/L、ZnCl 2 25~35 μg/L、CoCl 2 •6H 2 O10-15 mug/L and Na 2 MoO 4 •2H 2 O 5~10 μg/L，pH 6.0~8.0；
The conditions of the heterotrophic culture are as follows: and culturing for 6 days at 22-26 ℃ under the illumination intensity of 6000-10000 lux under stirring.
2. The method according to claim 1, wherein in step (1), the biomass in the microalgae solution is 10-15g/L; in the step (2), the biomass in the rhodobacter capsulatus bacterial liquid is 1.0-1.5g/L.
3. The method of claim 1 or 2, wherein the autotrophic medium is a modified C medium.
4. The method of claim 1 or 2, wherein the photosynthetic bacteria medium is 112Van Niel's yeat agar medium.
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| CN104480151A (en) * | 2014-11-20 | 2015-04-01 | 中国科学技术大学 | Method for improving yield of heterotrophic microalgal fatty acid with sodium acetate |
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| CN101363005A (en) * | 2008-09-16 | 2009-02-11 | 广东绿百多生物科技有限公司 | Method for coculturing fine algae and photosynthetic bacteria |
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