CN101921811B

CN101921811B - Method for culturing microalgae

Info

Publication number: CN101921811B
Application number: CN 201010231006
Authority: CN
Inventors: 李岩; 张晓东; 周文广; 华栋梁; 张�杰
Original assignee: Energy Research Institute of Shandong Academy of Sciences
Current assignee: Energy Research Institute of Shandong Academy of Sciences
Priority date: 2010-07-20
Filing date: 2010-07-20
Publication date: 2012-09-05
Anticipated expiration: 2030-07-20
Also published as: CN101921811A

Abstract

The invention relates to a method for culturing microalgae, which is characterized by comprising the following steps of: (1) fermenting to produce acid, enabling the supernatant which is acid liquor obtained after fermentation to flow to a culture device, and collecting the produced gas mixture in a gas storage tank; (2) inoculating the cultured microalgae to the acid liquor contained in the culture device for heterotrophic culture; and (3) circularly introducing the gas mixture in the gas storage tank to a photobioreactor for aerobic autotrophic culture. In the invention, the acid production stage through methane fermentation and the microalgae culturing process are coupled, and heterotrophy and autotrophy are combined; thus, oil-rich microalgae with high biomass is cultured, and meanwhile, a hydrogen-rich gas with high concentration can be obtained. The method is mainly used for culturing the oil-rich microalgae.

Description

Little algae cultured method

Technical field

The present invention relates to little algae culture technique.

Background technology

Along with the continuous aggravation of global energy crisis, and the continuous enhancing of people's environmental consciousness, renewable resourcess such as biofuel receive the many concerns of People more and more.Biofuel is a kind of fatty acid monoester that is obtained through transesterification reaction by plant-animal or microbial oil and low-carbon alcohol; It is renewable, biodegradable; The burning and exhausting performance is good than fossil diesel fuel, is a kind of environmentally friendly energy, becomes the substitute of fossil diesel fuel gradually.Little algae is a kind of one-celled plants, has photosynthetic efficiency height, adaptive capacity to environment is strong, growth cycle is short and living weight is high characteristics.In recent years because the development of biofuel industry; The comprehensive exploitation of the oil-rich microalgae that fat content is higher has caused the many concerns of People more and more with utilizing; Microalgae grease can solve present biodiesel raw material source and lack and problem of unstable; Each country and geographic scientific research institution have carried out the research work of little algae cultivation aspect in succession, make its become gradually many countries increase income energy-conservation, turn bane into boon and protect the important means of environment.

In many characteristics of little algae, its stronger adaptive faculty has a wide range of applications.At present existing research work shows that little algae can grow, like patent CN101525576A in sewage or organic waste water; A kind of method of utilizing sewage and trade effluent to produce little algae is characterized in that, this method may further comprise the steps: the initial-stage culture step; After will passing through one-level processing and filtering sewage adding bio-reactor; In bio-reactor, add the algae kind, stir culture under the condition of illumination, blowing air and carbonic acid gas grows into stationary phase up to microalgae cell; Grease is induced step, and the volume ratio according to 1: 200～1: 1000 in bio-reactor adds the trade waste after sugarcane bagasse is fermented, and induces the oil and fat accumulation in the microalgae cell; Extraction step, oil and fat accumulation reach stable after, extract the microalgae cell in the bio-reactor.Organic sewage still contains nutritive elements such as a large amount of nitrogen, phosphorus after handling through anaerobic-aerobic, wherein feed stack gas after little algae can utilize CO ₂Carry out growth and breeding and accumulate grease, this had both reduced the cultivation cost of little algae, simultaneously sewage was carried out a biological disposal upon, and made it can recycle.

Little algae can also utilize biogas fermentation waste liquid-natural pond liquid to cultivate; Like patent CN 101285075A; The coupling process that a kind of biogas fermentation and autotrophic freshwater microalgae are cultivated is disclosed; Biogas fermentation and the photosynthetic cultivation of autotrophic freshwater microalgae are carried out simultaneously, it is characterized in that: 1) biogas fermentation: fermentation obtains biogas and natural pond liquid; 2) processing of natural pond liquid: the natural pond liquid that obtains behind the biogas fermentation of learning from else's experience, filter and remove remaining solid particulate, be 25%～100% of original content with the water dilution, as the full nutritional medium of autotrophic freshwater microalgae; 3) introducing of the inoculation of autotrophic freshwater microalgae and biogas: the autotrophic freshwater microalgae of packing into of the natural pond liquid after will handling is cultivated the bioreactor of usefulness, inserts the fresh autotrophic freshwater microalgae algae kind that spreads cultivation, and biogas is introduced reactor drum from reactor bottom; 4) autotrophic freshwater microalgae is cultivated and controlled: it is 1000Lx～2000Lx that autotrophic freshwater microalgae is cultivated early stage intensity of illumination, increases light intensity to 4000Lx～8000Lx, intermittence or 24h continuous illumination to cultivating the middle and later periods; 5) results of autotrophic freshwater microalgae cell: after the frustule cessation of growth cessation, stop the illumination ventilation, behind the collection frustule, remove the moisture in the frustule.Little algae is inoculated in the dilution natural pond liquid to a certain degree, and with the CO in the biogas ₂For inorganic carbon source is carried out the autotrophy process; Reach 6g/L through final living weight after 9 days; The coupling of little algae and biogas fermentation had both realized the cultivation of little algae in this process; Improve the purity of biogas, reduced the environmental pollution that the discharging of natural pond liquid brings again, reached the purpose that increases society and economic benefit.But shortcomings such as aforesaid method mainly is the training method that adopts autotrophy, and it is slow to exist the speed of growth, and living weight is relatively low.

Summary of the invention

Technical problem to be solved by this invention is: product acid phase and little algae culturing process of coupling biogas fermentation, heterotrophism combines with autotrophy, in the oil-rich microalgae of cultivating high-biomass, can obtain the higher hydrogen-rich gas of concentration.

For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:

(1) acidogenic fermentation

With animal excrement, agricultural crop straw, city domestic sewage in rural areas by using, industrial and agricultural production organic waste water and waste residue etc. is fermentation substrate; Be placed on the fermentation reaction device; Controlled temperature is at 25 ± 1 ℃ after adding active sludge, the 12-72h that ferments, and the gas mixture of generation is collected in the gas-holder.Said active sludge is meant the microorganism species that is used for the WWT anaerobic ferment process.

(2) little algae is cultivated

The freshwater microalgae that the present invention cultivates comprises the autotrophic freshwater microalgae after wild environments such as coming from fresh water or the domestication, can be in each big algae kind preservation center or commercially available acquisition, like chlorella, grid algae and tenaculat Habenaria etc.Said little algae algae kind is chlorella (chlorella vulgaris); Once be recorded among the U.S. Patent No. ublic specification of application US 20090288223 November 19 in 2009; Also can obtain in Texas ,Usa algae kind preservation center, its deposit number is UTEX 2714.

The cultivation of algae kind: medium component is NH ₄Cl 400mg/L, KH ₂PO ₄56mg/L, K ₂HPO ₄108mg/L, MgSO ₄7H ₂O 100mg/L, Glacial acetic acid min. 99.5 1mg/L, micro-1ml/L; The algae kind is inoculated in the substratum of the bacterium of going out through aseptic technique, places illumination box to leave standstill cultivation then, shake 3-4 every day; Culture temperature is 15-30 ℃, and intensity of illumination is 2000-4000Lux, cultivates 7 days.

Acid solution is cultivated: acidogenic fermentation finishes after the acid solution of screw extrusion press after separating flow to culture apparatus, inoculates to carry out heterotrophism behind little algae and cultivate.Culture condition is temperature 15-30 ℃, cultivates 5 days.

Aerobic culture: after treating that organic carbon source exhausts, carry out autotrophy in gas mixture (staple is hydrogen and carbonic acid gas) the feeding photo-biological reactor with the fermentation generation and cultivate, culture condition is temperature 15-30 ℃, and intensity of illumination is 4000-8000Lux, cultivates 7 days.

Marsh gas fermentation processes roughly can be divided into two stages, and the firstth, produce acid, being about to organic matter degradation is micromolecular VFA such as acetate, propionic acid etc., produces hydrogen simultaneously, CO ₂And minor N H ₃, H ₂Gases such as S; The secondth, produce methane, the i.e. staple of biogas.Because the acidogenic fermentation time is short, several hrs was by several days, and condition is easier to control, and the gas that produces mainly is hydrogen and carbonic acid gas, and it is harsh to produce the methane process condition, and the cycle is longer, generally needs about two months, and misoperation causes the failure of fermentation easily.Little algae not only utilizes organic carbon source such as acetate to grow fast, can also utilize carbonic acid gas in the gas to carry out autotrophy as inorganic carbon source and cultivate.Therefore acid process and little algae two stages of cultivation are combined, compare with biogas fermentation, improved the stability of total system, fermentation time also shortens greatly.To produce acid (hydrogen) fermentation and combine, have following beneficial effect with little algae cultivation:

(1) acidogenic fermentation time weak point only needs several hours to several days, and little algae heterotrophism process growth velocity is high, and the cycle is short, and this system compares with whole biogas fermentation, and cycle and stability all are greatly improved.

(2) the autotrophy process of little algae has improved the concentration of hydrogen, for next step application such as fuel cell etc. provides good condition, and has further increased the living weight of little algae.

(3) this process turns waste into wealth, and has reduced environmental pollution, has realized the recycling of waste.

Description of drawings

Fig. 1 is a process route view of the present invention.

Embodiment

Embodiment 1: step 1: acidogenic fermentation.With 500g cow dung is fermentation substrate; Adding water, to regulate solids content be 6%, fills in the reaction unit, begins fermentation after inoculating 25% active sludge (taking from sewage work); Temperature is controlled at 25 ± 1 ℃; The collection and confinement of gases that produces behind the fermentation 48h, carries out solid-liquid separation with tunning through screw extrusion press in gas-holder.

Step 2: the cultivation of algae kind: the algae kind of employing is chlorella vulgaris (available from Texas ,Usa algae kind preservation center, UTEX 2714).Medium component is NH ₄Cl 400mg/L, KH ₂PO ₄56mg/L, K ₂HPO ₄108mg/L, MgSO ₄7H ₂O 100mg/L, Glacial acetic acid min. 99.5 1mg/L, micro-1ml/L, total fat content can reach more than 40% of dried cell weight in this substratum, is oil-rich microalgae.This algae experiment confirm can survive in acidogenic fermentation gained liquid.Concrete operations are: substratum is joined respectively in the Erlenmeyer flask (the ventilative film that seals of tool), after aseptic technique inserts the algae kind, place illumination box to leave standstill cultivation through 121 ℃ of sterilizations then; Shake every day 3 times; Culture temperature is 28 ℃, and intensity of illumination is 4000Lux (fluorescent lamp), cultivates 7 days.

Acid solution is cultivated: the supernatant of getting after the solid-liquid separation is in acid solution to the fermentation culture device, according to 10% inoculum size (algae kind OD ₆₈₀Be 0.8) cultivate after inserting above-mentioned cultivation gained algae kind.Culture condition is 28 ℃ of temperature, and aeration-agitation cultivates that the sampling and measuring living weight is 15g/L after 5 days, and fat content is 38%.

Embodiment 2: present embodiment and embodiment 1 something in common repeat no more, and difference is can proceed the autotrophy process after the heterotrophism cultivation, on the basis of embodiment 1; Little algae after the heterotrophism cultivation is placed the pot type bioreactor, and the gas mixture that circulation feeds in the gas cylinder carries out the autotrophy cultivation, and culture condition is 28 ℃ of temperature; Intensity of illumination is 6000Lux; Measure micro algae biomass 18g/L after 7 days, fat content is 37%, and hydrogen purity is more than 90%.

Embodiment 3: step 1: acidogenic fermentation.With the 500g corn straw is fermentation substrate; With fill in the reaction unit after 500g cow dung is mixed, adding water, to regulate solids content be 8%, begins fermentation after inoculating 25% active sludge (taking from methane-generating pit); Temperature is controlled at 25 ± 1 ℃; The collection and confinement of gases that produces behind the fermentation 72h, carries out solid-liquid separation with tunning through screw extrusion press in gas-holder.All the other steps are with step 2 among the embodiment, and the sampling and measuring living weight is 18g/L after 5 days, and fat content is 39%.

Embodiment 4: step 1: acidogenic fermentation.With the 2L beer brewery water is that fermentation substrate fills in the reaction unit; Begin fermentation after inoculating 20% active sludge (taking from methane-generating pit), temperature is controlled at 25 ± 1 ℃, and the collection and confinement of gases of generation is in gas-holder; Behind the fermentation 12h, tunning is carried out solid-liquid separation through screw extrusion press.All the other are with the step 2 among the embodiment 1, and the sampling and measuring living weight is 10g/L after 5 days, and fat content is 35%.

Claims

1. little algae cultured method; It is characterized in that it comprises the steps: that (1) is fermentation substrate with cow dung, corn straw and beer brewery water; Add the active sludge acidogenic fermentation, the supernatant after the fermentation ends is that acid solution flow to culture apparatus, and the gas mixture of generation is collected in the gas-holder; (2) will pass through cultured microalgae is inoculated into and carries out heterotrophism in the acid solution in the culture apparatus and cultivate; Said little algae is that Texas ,Usa algae kind preservation center obtains, and deposit number is little algae of UTEX2714; (3) the little algae after the heterotrophism cultivation is placed photo-biological reactor, gas mixture circulation in the gas-holder is passed into the autotrophy of ventilating in the photo-biological reactor cultivates.

2. little algae cultured method according to claim 1; It is characterized in that said acidogenic fermentation is meant that with animal excrement, agricultural crop straw, city domestic sewage in rural areas by using be fermentation substrate; It is mixed placing the fermentation reaction device with an amount of water, add active sludge after controlled temperature at 25 ± 1 ℃, 6-72h ferments; The supernatant of fermentation ends is that acid solution flow to culture apparatus, and the collection and confinement of gases of generation is in gas-holder.

3. little algae cultured method according to claim 2 is characterized in that said active sludge is meant the microorganism species that is used for the WWT anaerobic ferment process.

4. little algae cultured method according to claim 1 is characterized in that being meant that through cultured microalgae medium component is NH in the step (2) ₄Cl 400mg/L, KH ₂PO ₄56mg/L, K ₂HPO ₄108mg/L, MgSO ₄7H ₂O 100mg/L, Glacial acetic acid min. 99.5 1mg/L, micro-1ml/L; The algae kind is inoculated in the substratum of the bacterium of going out through aseptic technique, places illumination box to leave standstill cultivation then, shake 3-4 every day; Culture temperature is 15-30 ℃, and intensity of illumination is 2000-4000Lux, cultivates 7 days.

5. little algae cultured method according to claim 1, it is characterized in that carrying out behind the little algae of said inoculation heterotrophism and cultivate and be meant: culture condition is temperature 15-30 ℃, and aeration-agitation was cultivated 5 days.

6. little algae cultured method according to claim 1; It is characterized in that said ventilation autotrophy cultivation: the gas mixture that feeds the fermentation generation to bioreactor carries out the autotrophy cultivation; Culture condition is temperature 15-30 ℃, and intensity of illumination is 4000-8000Lux, cultivates 7 days.