CN101899481A - Method and system for producing heterotrophic alga in high density - Google Patents

Abstract

The invention provides a method and a system for producing heterotrophic alga in high density. In the method, unique biological characteristics of a microbial system are utilized, and the accumulation of lipid is increased by simulating cell division and the uncoupling of cell growth/amount. A process for producing polyunsaturated fatty acid (PUFA) by microalgae fermentation comprises three different stages: (1) a cell proliferation stage for increasing the number of cells; (2) a biomass and lipid accumulation stage for increasing cell biomass, particularly the amount of the lipid; and (3) a lipid concentration increasing stage by refining for producing more PUFA by adjusting the steering of the lipid. Various variables such as the types and concentration of a carbon source and nutritional factors, dissolved oxygen and temperature are changed continuously through a three-stage alga growing process to produce maximum biomass, lipid and docosahexaenoic acid (DHA) by final induction. Therefore, most cell viability leads to the cell division and the rapid formation of a large number of cells, so that the biomass and the lipid yield are integrally enhanced finally.

Description

The method and system that heterotrophic alga in high density is produced

Technical field

The present invention relates to utilize little algae to produce the method for omega-fatty acid.Specifically, the present invention has proposed a kind of multistage research and production method for little algae high-density growth of heterotrophism and production omega-fatty acid.This method has been removed cell fission by its optimum condition of control in reactor separately, growth and the coupling of producing omega-fatty acid.This uncoupling and optimized method make marine alga, and the yield and the throughput of lipid and polyunsaturated fatty acid (PUFA) have obtained whole raising.In addition, by utilize discarded starting material for example raw glycerine (by product that biofuel is produced) wait as main carbon source, with this process integration in biological diesel oil refining technology.Consider that the product in the marine alga process of growth not only comprises the PUFA lipid, and comprise non-many unsaturated lipids, and the latter can be utilized in the production process of biofuel, so adopt original position transesterification reaction and fractionation process, can form the little algae-biofuel system fatty acid methyl ester of production biofuel simultaneously and the polyunsaturated fatty acid methyl esters of an integration.

Background technology

Society and expanding economy at present strengthened high-level efficiency and constantly promoted the method for bio-based products production and utilization.Yet a large amount of energy concentrates on the utilization to existing agricultural-food, and these means have produced the undesired second-order effect of people.Especially, when forwarding useful chemical substance of production and material to, food finally will cause product cost to increase by producing in the arable land.This just causes people to increase utilizing marginal land production and/or utilizes waste or development interest that low value material is produced high-value product.For example, raw glycerine (glycerol or glycerin) is by the by product in the transesterification reaction production biofuel process with vegetables oil.Because the raising of biofuel output recently, world market has produced a large amount of raw glycerines at present.Many people have proposed the various methods of utilizing raw glycerine.The raw glycerine purifying is obtained refining glycerine can be used as animal-feed (being particularly useful for pig feed); Perhaps, raw glycerine can be used as the substrate lactic acid producing in next life of anerobe (for example clostridium Clostridium sp.) fermentation, the product that propionic acid etc. are useful, in addition, also can utilize effective means to change depleted glycerine into frostproofer, the precursor of biological polymer and soap.Yet still old excessive raw glycerine produces, handle by simple burning usually, and also be a kind of pollution technology.Therefore need to seek in addition a kind of method of constructively handling discarded raw glycerine.

In addition, people have recovered the interest of utilizing marine alga to produce, and especially to the little algae of heterotrophism, a large amount of lipids can be produced and store to known its in frond, proved the omega-3 lipid of health-care effect especially.(ω-3PUFAs) is that gang comprises two or more pairs of keys to omega-3 polyunsaturated fatty acids, and the lipid acid of last double bond position on terminal the 3rd carbon atom of methyl.Epidemiology survey has long ago just disclosed the beneficial functional of ω-3PUFAs, and the Eskimos of edible a large amount of abyssal pelagic fishes seldom suffers from a heart complaint.

(DHA 22:6) is made of 22 carbon and 6 two keys docosahexenoic acid, is one of of paramount importance omega-3 polyunsaturated fatty acids, and has been generally believed that brain and eyes to fetus and baby have special beneficial functional.The World Health Organization (WHO) is recommended in strongly and adds DHA (the FAO/WHO Committee of Experts, 1994) in the infant formula.In addition, research shows that also the people beyond the baby needs DHA equally, and the absorption of DHA has reduced suffers from the neurological disorder risk relevant with the age, as Alzheimer's disease and dementia etc.The result shows that DHA is not only the additive in the infant formula, and the diet and the drink that also are used for being grown up comprise cheese, and yogourt is in the cereal food of food companion's sauce and breakfast.It should be noted that there is bigger growth potential in these markets than infant food, so improved whole DHA market potential simultaneously.

The tradition source of ω-3PUFAs mainly is fish oil and seal oil.Cod, salmon, sardines, mackerel, catfish, tapertail anchovy and yaito tuna are normally used for fish oil production.The quality of fish oil depends on kind, the season of fish and catches the area.Because ocean fish fish oil is the complex mixture that the lipid acid by different chain length and degree of unsaturation constitutes, DHA must be from fish oil the refining healthcare products/medicine that comes out just to can be used as.Purification DHA difficulty is big and cost is high from inferior grade fish oil.In addition, the ocean fish reserves are subjected to the influence of season and climatic variation, are difficult to feed rate that provides stable and the demand that satisfies growing DHA.

As everyone knows, fish can not de novo synthesis PUFA as the mankind.Their primary producer of most PUFA from ocean environment: little algae or alga microbial.Occurring in nature has a large amount of little algaes can produce especially DHA of various PUFA, has the potentiality that form suitability for industrialized production but only there is a few little algae to be proved.This mainly is that specific growth rate and cell concn are all very low because little algae can only grow under the photoautotrophy condition mostly.

To this slightly the further investigation of algae throughput researchist and industry member are directed to the process that little algae heterotrophism is produced DHA.Soon before, for commercial applications and two kinds of marine algas that show huge commercial promise are heterotrophism dinoflagellates, Crypthecodinium cohnii Crythecodinium cohnii and traustochytrid marine bacterial classification.It is that (10-50% w/w) also produces the DHA (30-70%) that accounts for the high per-cent of TL because they can accumulate high-load oil in biomass that these two kinds of marine algas are used for the commercialization process.The carbon fed batch cultivation of marine species C.cohnii can obtain the DHA of high bacterium dense (nearly 109g/L) and 20g/L, though incubation time prolongs (400h).Research has confirmed that the throughput of this strain bacterium DHA can reach 1-1.5g/ (L days).

Yet best microbe-derived of DHA is Thraustochytrids section, and especially Thraustochytrium belongs to and the Schizochytrium genus.Thraustochytrium belongs to and the Schizochytrium genus is unicellular algae or algae protobiont, Thraustochytriales order, Thraustochytriaceae section, the member that Thraustochytrium or Schizochytrium belong to.Schizochytrium by the successive bipartition duplicate with sporocyst in discharge zoospore and duplicate, and the Thraustochytrium bacterial classification only duplicates by the formation of sporocyst/zoospore.The outstanding bacterial classification that these two kinds of ocean protobionts are industrial production DHA has been determined in research to thalassiomycetes thraustochytrids.Preliminary study is under the condition of low relatively cell density (5-20g/L), and the Thraustochytrium kind can accumulate the DHA more than 50% TL, produces greater than every liter of 1g DHA substratum, the about 0.2g/ of throughput (dying).Be separated to and had the more Schizochytrium kind of high growth rates.Cultivate at glucose and nitrogenous source batch feeding, add the main sodium source of sodium sulfate conduct and control glucose concn, under the condition of pH and oxygen supply level, selected bacterial strain can reach high-cell density (100g/L) at bob ferment cycle (90-100h), accumulation 40-45g/L DHA, and the throughput of DHA is greater than 10g/ (dying).These outstanding performances make schizochytrium limacinum Schizochytrium become the industrial producing strain of DHA.

Thraustochytriale Thraustochytrium genus and special industrial application and the going through of commercialization culture condition of schizochytrium limacinum Schizochytrium genus production PUFA are seen U.S. Patent No. 5,130,242.' 242 and associated documents in, US Patent No ' s 5,340,742 and 6,977,167, the marine alga of these two genus is cultured in the airtight reactor, control salt, oxygen, temperature and carbon nitrogen source concentration reach the maximum production of PUFA.Yet carbon nitrogen source that is to say that from the agricultural-food of high added value the application of raw glycerine is not included.

Based on these progress, more study attention and turn to the increase biomass and improve PUFA product yield.US Patent No ' s 5,130,242 and 6,977,167 elaborations, by keeping relative high concentrations of phosphoric acid salt, thalline can be kept growth, can partly reach high-density culture and produce.In addition, by the special control of nitrogenous source, or for some time restriction nitrogenous source (control is added or initially supplied with) also can improve the output of PUFA before cell harvesting.Same, at United States Patent (USP) 6,607, in 900, set forth a two-part fed-batch fermentation process, wherein first section is to improve cell density, second section production lipid products.' describing in 900, be similar to US Patent No ' s 5,130,242 and 6,977,167, the substratum in biomass accumulation period contains carbohydrate and amino acid derivative (as nitrogenous source), the substratum in lipid accumulation period mainly contains carbohydrate, contains limited amino acid derivative in addition, and monitoring shows that the oxyty of lipid accumulation phase is lower than the biomass accumulation phase.

Less for the specific function relation concern of trophic factor and cell state at present, but also can inquire some information.At United States Patent (USP) 5,340, different growth patterns has been described in 742, adopt no chlorion to exist, especially under the situation of vitriol and/or subchloride concentration, provide 1) reduce the decline speed of fermentation and cell harvesting, 2) have only less cell concentration increase when reaching high PUFA concentration.Use the process of describing in the United States Patent (USP) 6,607,900, adopt the fed-batch fermentation pattern, stream adds competent carbon nitrogen source can obtain the biomass of 100g/L at least.In this process, fermentation condition comprises the stage of biomass increase and the stage of lipid production.Carbon source and nitrogenous source were all added in the biomass increase stage, and only added carbon source in the lipid production process.Biomass increases the stage oxygen dissolving level at least about 4% in the fermenting process, and the lipid production process then is lower than 3%.The optimal temperature of fermentation is about 30 ℃.Lipid production rate in the process can reach 1.0g/L/hr, and DHA wherein about 40%.According to United States Patent (USP) 6,607,900, the productivity of DHA can reach 0.5g/L/hr basically.

Briefly, United States Patent (USP) 6,607,900 suggestions are cultivated little algae (for example Thraustochtriales and the schizochytrium limacinum Schizochytrium of thraustochytriale Thraustochytrium genus) with two-phase method.At first be the stage that a biomass density increases, wherein dissolved oxygen is better at least about 8%, 4%.On the contrary, second " production " stage, the primary activity of marine alga is not to increase biomass but produce lipid, and it is 1% or 0% better that oxygen supply is reduced to.United States Patent (USP) 6,607, at least 20 ℃ of 900 suggestion culture temperature, 25 ℃ are better, and 30 ℃ are best, do not describe which kind of culture temperature in detail and are fit to which cultivation stage.Yet 6,607,900 point out, because cold water has higher concentration of oxygen gas than hot water, " higher fermention medium temperature has the attendant advantages that reduces dissolved oxygen amount ".Therefore, 6,607,900 suggestions are in the production phase, and when needs lower concentration oxygen or not during aerobic gas, high slightly temperature is more favourable.6,607,900 also pointed out total fermentation time about 90 to 100 hours.In addition, 6,607,900 point out the preferably non-alcohol compound of carbon source of this process, preferably adopt for example carbohydrate of corn steep liquor.These guiding opinions are got rid of the use (glycerine is a kind of alcohol) of raw glycerine.

In addition, United States Patent (USP) 5,130,242 have discussed the technology of one two stage marine alga fermentative production lipid.Fs is an exponential phase of growth, and subordinate phase is a stable lipid production phase.In subordinate phase, the restriction nitrogenous source stimulates the production of lipid.Yet 5,130,242 think that the nitrogen restricted period is short relatively, for example 6-24 hour.According to 5,130,242, first-selected carbon nitrogen source is respectively cereal and certain hydrolysis refuse, as vinasse, and the refuse that a kind of corn fermentation ethanol process produces.Yet, the application of raw glycerine is not discussed." raw glycerine " difference " glycerine " is that as the by product in the production of biodiesel process, raw glycerine comprises the glycerine of 70-80%, is mixed in the pollutent of commercial run, as the free-fat acid ion, and single and two-glyceryl ester, pure and mild salt.These materials or bring, or the reaction of fuel production process incomplete component from starting material.

United States Patent (USP) 6,582,941 have further disclosed Schizochytrium genus SR21 bacterial strain can the high yield unsaturated fatty acids.Glycerine is a kind of carbon source of using of advising.Yet, the application of by product such as raw glycerine is not discussed.

At last, international patent application book WO 2004/083442 has described a kind of method that increases PUFAs output in the nutrient solution of thalassiomycetes thraustochytrids, they is stored at low temperatures after 2-5 days in growth.Raw glycerine is not proposed as carbon source.

Summary of the invention

In view of this, the invention provides a kind of improvement and efficiently the high-cell-density cultivation method produce ω-3PUFAs, DHA especially.This method has utilized microflora unique biological characteristic to produce DHA, increases the accumulation of lipid by the uncoupling of analog cell division and cell growth/amount.Consider and improve the accumulation that cell concentration or concentration can farthest suppress lipid and PUFA, especially DHA subsequently, adopt therefore that the control process parameter realizes uncoupling in specialized apparatus separately.The result has shown to have higher potentiality on yield and throughput.In addition, this method provides the way of handling excessive raw glycerine, and the main carbon source that this method is used is exactly a raw glycerine.The use of raw glycerine not only helps to have consumed this material, and has reduced the fermentation costs of marine alga, because no longer need the raw material of purer costliness.In addition, little algae produce some can be separated as the lipid of the non-ω of being rich in-3 and as the moiety of biofuel (also being known as biofuel here), therefore can produce more raw glycerine again as raw material.At last, being rich in ω-3 can realize by original position transesterification reaction and fractionation process subsequently with the separating of lipid of non-being rich in-ω-3, this process does not need oil to be extracted from water surrounding or separate, produce two kinds of methyl esters products: a kind of nutritional additive of omega-fatty acid methyl esters and methyl esters of a kind of non-ω of being rich in-3 of being rich in can be used as biodiesel fuel.Therefore, only a file of the present invention just fully combines the production of biofuel and the production of ω-3PUFAs.

As described herein, the process of utilizing little algae to produce PUFA comprises three different stepss: (1) cell increment phase is to increase cell quantity; Be that (2) biomasss and lipid accumulation stage are to increase the amount of existing biomass, particularly lipid then; Next be that (3) shorter " making with extra care " steps improve lipid concentration, and adjusting it, to turn to the more PUFA of production mainly be DHA.Various variablees, as the type and the concentration of carbon source and trophic factor, dissolved oxygen amount and temperature produce maximum biomass, lipid and DHA by constantly changing finally to induce in this syllogic marine alga process of growth.

In embodiment, this syllogic process can realize in same container that growth conditions can be at suitable time-controllable therein.Embodiment comprises the process segmentation more specifically, utilize continuous three different growth phases of method natural separation that combine with fed-batch fermentation, that is to say that this process realizes that by two or more placed in-line special purpose reactors each stage in the process can each self-optimizing.In some embodiment, little algae that fermenting process uses belongs to from Schizochytrium, and that a special example that provides here adopts is Schizochytrium limacinum SR 21.Obtaining a large amount of cells is vital for lipid production, because the maximum weight of each cell is limited, and each cell only can be preserved a certain amount of lipid in cell.Many more more biomass and the more substantial lipids of will obtaining at last of cell concentration in the fermented liquid of per unit volume.The invention provides a kind of method and make the quick formation of a large amount of then cell concentrations of cell fission of at first leading of most cell viability, rather than utilize cell viability lead simultaneously cell fission and growth.The result is, in the latter half, adds nutrition to reach maximum cell and lipid amount to the cell of high density, and the integral body that finally obtains biomass and lipid yield improves.

It should be noted that this whole process can be incorporated in the biofuel refining process effectively.This is because the main raw material that little algae utilizes, and the selection purpose of design of this all processes and specified microorganisms is exactly raw glycerine, and it is the by product of biofuel refining process, rather than carbohydrate or other non-alkylol cpds.After pre-treatment on the spot, raw glycerine just can be used as the carbon source marine alga of fermenting.The marine alga that obtains just can produce by the original position transesterification reaction in downstream and the processing of fractionation separates process then: the methyl esters that is rich in ω-3 of (1) food/trophology scale; (2) methyl esters of the non-ω of being rich in-3 that uses in the production process of biofuel.In biofuel production process subsequently, raw glycerine is recovered the carbon source as fermentation and marine alga production in the future again.At last, the discarded biomass of marine alga can be used as nutrition during the fermentation, or directly is used as animal-feed.

The detailed description of embodiment of the present invention

The present invention is based on and utilize little algae production to be rich in improving one's methods of ω-3PUFA.To discovering of marine alga Schizochytrium, in fact the metabolic mechanism of cell proliferation and lipid accumulation is non-coupling connection.The growth of marine alga can be divided into two different stepss: the breeding stage that (1) cell number increases, during the propagation of the cell cell quantity that increases sharply, but cell volume and weight there are not growth; The increase stage of (2) cell volumes, during cell reduce or stop breeding, owing to the accumulation of lipid acid becomes big.It should be noted that, the optimal culture conditions difference in these two stages, linear dependence not, data see Table 1, and culture condition is as follows: Schizochytrium limacinum SR 21 bacterium, and substratum is by 100g/L glycerine, the 5g/L corn syrup solids, all mineral salts that exist in 1g/L ammonium acetate and the seawater are formed, 25 ℃ of cultivations, and shaking speed 175rpm shook 7 days.The results are shown in Table 1.The cell inoculation amount is about 2 * 10 ⁶Individual cell/ml.In initial 48 hours, cell density increases sharply and reaches 42.5 * 10 ⁶Individual cell/ml.Afterwards, cell density slowly increases, and finally increases to 58.5 * 10 ⁶Individual cell/ml.On the contrary, biomass concentration, promptly dry cell weight increased in initial 48 hours slowly, and the 6.9g/L biomass was only arranged in the time of 48 hours, and after this, dry cell weight is kept growth and is finally reached 24.6g/L.Obtain " the cell space weight " of each cell with biomass concentration divided by cell density, the cell space weight in the time of 48 hours is 0.161ng/ cell, and final cell space weight is 0.420ng/ cell, and it is many that this shows that cell volume has increased twice.

Cell quantity in table 1. substratum, dry cell weight, cell space weight and time relation

Incubation time (my god)	1	2	3	5	7
						Cell quantity (10 6Individual cell/ml)	25.2±1.9	42.5±4.2	47.4±11.9	64.0±7.8	8.5±7.75
Dry cell weight (g/L)	3.5±0.1	6.9±0.2	10.1±0.6	16.4±0.8	24.6±1.6
						Cell space weight (ng/ cell)	0.137	0.161	0.214	0.256	0.420

Can see in this shake-flask culture of not optimizing experiment, cell quantity increased at first day and enters plateau subsequently very soon (perhaps even fall into a decline, as the 5th day to the 7th day) and cell space weight only increases significantly (after as the 3rd day, especially between the 5th day to the 7th day) in the fermentation later stage.By the Fermentation Process of Parameter in the close controlling reactor, the above-mentioned nonlinear relationship initial stage that can be adjusted to almost is cell fission, is cell vegetative period almost completely subsequently, at last can whole yield and the throughput that improves biomass and lipid.Find based on this, developed a kind of discrete culture condition and optimized two stages, and significantly increased cell density and lipid accumulation by the strategy of " conversion " between two stages.Also comprise the purification step in the 3rd step, control condition makes the lipid maximum production and generates especially DHA of more PUFA.

The metabolism stream in breeding stage is very big, because nucleic acid, protein, nitrogen and amino acid that other material require in enzyme and the cell is a large amount of.Thereby being consumed fast, oxygen make the carbon source oxidation provide low-molecular-weight carbon and energy to make cell proliferation.In addition, in breeding, in order to keep the high slightly culture temperature of the suitableeest enzyme employing alive.On the contrary, in the lipid accumulation phase, low temperature and hypoxemia consumption are more suitable.The biosynthesizing of enzyme needs nitrogen, and the accumulation of lipid is that carbon source is converted into longer chain fatty acid, needs nitrogen hardly.Therefore the accumulation of cell proliferation and lipid is different.

The invention provides a kind of two stages, integration, the method for the little algae of ω-3 is rich in high cell density fermentation production, mainly concentrate on the uncoupling of cell proliferation and cell growth, and the purification step of follow-up phase III is to obtain better lipodogramme.Method of the present invention can realize in single reactor, by the growth conditions in the controlling reactor.Yet in a specific embodiments, this three stage process is used isolating reactor, brings into play the growth characteristics of marine alga uniqueness by the operational condition of controlling each reactor respectively.A syllogic fermentation condition that is suitable for this process is seen Fig. 1, and wherein 10 representatives are head tank with the raw glycerine.Head tank 10 can comprise that also a groove 12 is used for regulating pH and makes the lipid acid layering, and 14 represent the raw glycerine storage tank.Groove 12 and 14 can be incorporated on one independent jar.Fs realizes that in first reactor 20 concentrating increases cell quantity, controls culture condition with this.Subordinate phase realizes that in second reactor 30 concentrating increases biomass and lipid acid accumulation, and control condition is to satisfy these demands.Control the condition of the 3rd reactor 40, bring out lipid acid and further transfer more a high proportion of DHA and/or omega-fatty acid to.Also the culture condition in second reactor 30 can be adjusted into the phase III growth conditions and needn't transitional cell in other reactor.The fermentation pattern that this technology and the present invention adopt is similar, batch feeding cultivate or cultured continuously in must controlling reactor inside condition, stir thalline, and add or remove the transformation with realization condition such as cell.

Induce the condition of maximum production to comprise type and concentration, dissolved oxygen amount and the temperature of carbon source and nutrition source in two stage controls and conversion.The condition control of first fermentor tank is as follows: high temperature, promptly 28-33 ℃; Low carbon source concentration, 10-40g/L; High nitrogen concentration, 1.0-1.5g/L; High oxyty, 20%-50%.Operating method is that batch feeding is cultivated or cultured continuously.After about 24 hours, the cell density in first fermentor tank reaches about 400 * 10 ⁶Individual cell/ml, about 600 * 10 ⁶Individual cell/ml is better, and about 1000 * 10 ⁶Individual cell/ml is best.

After the fs fermentation ends, cell disposable (fed-batch fermentation) or continuous (continuously fermenting) are transferred to second fermentor tank.In second fermentor tank, cell becomes bigger and begins to accumulate lipid.Second fermentor tank still can fed-batch fermentation or continuously fermented.If the fed-batch fermentation pattern, at first, temperature need be controlled at 30 ℃; Dissolved oxygen (DO) is controlled at 3%-5%; Carbon source concentration 30-50g/L; Nitrogen concentration 0.5-1.0g/L.When the speed of biomass concentration increase slows down (as, when cell passed through " logarithm " or enters stationary phase exponential phase of growth, cell number was constant relatively, and the on-line Control of fermenting process can prove), the about 13-17% of the content of DHA in the biomass.And DHA also can further increase in the stage of the 3rd " making with extra care ".Phase III can independently carry out in the fermentor tank, also can be by reducing fermentation jar temperature to 20-25 ℃, and dissolved oxygen also stops to mend nitrogen fully to 0%-0.5% is induced it in same reactor.When whole end of processing, after about 5 days, nitrogen is exhausted fully, and DHA content is about the 20-22% of dry mycelium.If adopt the pattern of continuously fermenting, the temperature of second fermentor tank need be controlled at 25-30 ℃; DO is 1%-3%; Carbon source concentration is 30-50g/L; Nitrogen then is controlled at 0.1-0.5g/L by feed supplement.The inoculation time of second fermentor tank is about 3-4 days under these conditions.The about 17-20% of the content of DHA in biomass during fermentation ends.

This syllogic fermenting process is typical 5 a days fermentation mechanism, obtains high-cell density (about 150g/L) high productive capacity (about 1.2g/L hr) and the total fatty acids of marine alga, and PUFA, DHA are respectively about 50,25 and 20% yield.Perhaps, the present invention's these numerical value of reason owing to technology controlling and process when production practice a little bias can occur.For example, total fatty acids output generally accounts for the 40%-60% of dry mycelium, mainly is 55%-65%.Equally, the yield of PUFAs accounts for the 12-26% of dry biomass, and it is better to reach 20%-30%.Similarly, the yield of DHA is the 6%-22% of dry biomass, and it is better to reach 20%-25%.The throughput of dry biomass be 1g/L/hr to 2g/L/hr, it is better to 3.0g/L/hr to reach 1.8g/L/hr, then from 0.3g/L/hr to 0.45g/L/hr, it is better to 0.5g/L/hr to reach 0.44g/L/hr for DHA.

About the innovation and application of raw glycerine, the successful application of this process of the present invention will produce great effect to the biofuel industry, because it has solved the handling problem of useless glycerine in the biofuel industrial production.Raw glycerine head tank 10 is integrated into this syllogic system, as shown in Figure 1.If the useless/raw glycerine that produces in the biofuel production process can not appropriately be handled and will threaten environment structure, and because its negative value forms the burden into the biofuel industry.The open market value of raw glycerine is 5 cents/pound at present, can infer the increase decline more (commodity market is quoted as proof) along with output.Changing useless glycerine into added value biological product definitely is the better selection of handling this glycerine.Because the utilization of this biofuel refuse will increase the economic vaibility of biofuel industry, and, so the present invention is not only favourable to biotechnology, also be beneficial to industry, safety and energy industry owing to handled source of waste and also can reduce to environment potential influence.

The most of lipid acid that contains in the marine alga is not ω-3 and similar useful product, as the lipid/oil component in the production of biodiesel process.Therefore, another object of the present invention is, produces lipid via 1 by marine alga) use the by product 2 in the production of biodiesel process) moiety of production biofuel, the same good with PUFA and/or DHA in the biofuel refining of integrating.It is methyl esters that proper method (as solvent extraction) that lipid acid extracts and transesterification (dry and pure, alkaline purification as standard) can be used to transform lipid acid in biomass.This mixtures of methyl esters can be separated with existing still-process on the refining industry subsequently, produces the methyl esters that is rich in ω-3 and sells as food/dietetic product but purpose becomes, and the methyl esters of the non-ω of being rich in-3 is sold as biofuel.It should be noted that the PUFA product that the PUFA methyl esters here is different from other marine alga is that its chemical species is methyl esters rather than lipid acid.By downstream separation process separation of fatty acids and/or fatty acid ester from the seaweed bio amount completely, and subsequently with different lipid acid or fatty acid ester separately, the lipid acid that is suitable for biofuel industry is inserted the biofuel system again and omega-fatty acid is retained following and then market-oriented.So far, a real biofuel-marine alga system has just formed, and not only the by product raw glycerine has obtained utilization, but also is used to delivery capacity outer biofuel (and raw glycerine) and new omega-fatty acid.Utilizing heterotrophic alga to produce DHA as can be seen from the present invention will benefit, because raw glycerine can be significantly reduced the expense (seeing embodiment 3 cost analyses) of marine alga culturing process as carbon source.Though the ω-3 of heterotrophic alga production at present product plays an increasingly important role, fish oil still is the realistic threat (Martek annual report, 2004) of this technology of development because of its lower production cost.In addition, several companies have also produced the fish oil product of Microcapsule, claim in the purge process of avoiding costliness also to have solved the smell that exists in the fish oil, stability and flavour problems.Therefore, the production cost that reduces the marine alga production process still is a requisite job for heterotrophic alga production DHA, and the present invention just provides a kind of real method of doing so at present, by utilizing the carbon source of raw glycerine as economy.

Fig. 3 sees in the little algae-biofuel system that integrates among the present invention.Can see biofuel refinery 100 production biofuel 110 and by product raw glycerine 120.Raw glycerine 120 obtains glycerine raw material 130 through pre-treatment.Pre-treatment comprises acid interpolation control pH and lipid acid.A kind of sour example hydrochloric acid is by stirring or other method and raw glycerine mixing.When pH reduces to 7.0, especially be lower than at 4.0 o'clock, stop to stir, the fatty acid radical ion just changes free fatty acids into and forms one deck at glycerine on mutually.Just can remove free fatty acids by separating these two kinds of different liquid phases.Other impurity such as various salt then remain in the glycerine.Residual salt pair marine alga is harmless, perhaps can be used as the nutritional support growth.In addition, along with this production process of production of biodiesel person's refining, the raw glycerine by product may contain impurity still less, thereby has simplified preprocessing process.Under an initial stage culture condition, cell quantity 140 increases when glycerine raw material 130 and other nutritive ingredient add in the marine alga.Second section further cultivation has increased cell volume 150 and lipid accumulation.Stage 3 is of short duration purification step, makes the condition of jar two change the more lipid that is suitable for producing the increasing of PUFA ratio into.Biomass 160 that finally obtains and then the processed ω of obtaining-3 methyl esters 170 are used to produce nutritious prod and other product (190).Change its course from other methyl esters 180 of seaweed bio amount and to get back to the production of biofuel 110.Nutritious prod of producing from biofuel 110 and extra glycerine can be changed its course participates in the marine alga fermenting process, or sells as nutritive ingredient, as animal-feed etc.

In embodiment of the present invention, any suitable little algae can be used to produce the lipid that is rich in ω-3.In general, can use Thraustochytrids section, especially utilize little algae that Thraustochytrium belongs to and Schizochytrium belongs to.In a concrete embodiment, little algae that fermenting process adopts is the bacterial classification of 21, one known high-cell densities of Schizochytrium limacinum SR, and effective metabolism glycerine is as the unique property of carbon source.By selecting a known bacterial classification that utilizes glycerine, this system produces about marine alga and the utilization of raw glycerine becomes very effective.

The process that described herein three stage cultivations are used to produce is because utilize raw glycerine to be more widely used as raw material.This method can be used to Unicell Oils and Fats (SCO) process, including, but not limited to marine alga production biofuel, has the maximum prospect that SCO uses.

The little algae production process of ω-3 that is rich in that present method is described serves many purposes.Under some condition, little algae self of being rich in lipid can be utilized, as, animal-feed, the human food prods, medicine or nutritious prod, makeup or different sanitising agents, or as foodstuff additive.Perhaps, lipid, especially DHA can separate from little algae and purifying is used for similar purposes.At present, fish oil is the main component of the feed made with ocean fish and shrimp, mainly comprises omega-fatty acid because it provides a series of lipid acid, and it has satisfied fish to energy, the needs of growth and breeding.Yet the level of PUFA depends on the kind of fish in the fish oil, the condition of extracting method and preservation.In fact, the fish oil of standard specifications does not provide enough DHA ratios to satisfy fully the nutritional needs of breeding and seedling.Therefore, the fish oil DHA content that ocean fish produces is high to be because it is from special fish tissue or by special method of purification, and therefore suggestion use in parent population raising and juvenile fish breeding, but the use of these high DHA content fish oil is limited, produce costliness, and it is not too friendly to environment to feed fish with fish.So culture fishery is considered with terrestrial plant and unicellular organism such as little algae, yeast, mould, bacterium and fungi just at the alternative nutrition source of active development.

The main advantage that the unicellular organism that the present invention describes is used is under the condition of may command and environmental safety, they can be easy to be used for industrial production, studies have shown that the diet of adding heterotrophism algae and mould can efficiently provide indispensable fatty acid, by food or the direct diet that enriches larva by adult fish.Unlike fish oil, comprise different ω-3 and other polyunsaturated fatty acid (PUFA), depend on the kind of fish, extracting method and storage conditions, exsiccant heterotrophic alga and yeast have stable PUFA productive rate usually.In addition, DHA can be produced separately by marine alga not of the same race, and also the lipid acid that can mix production wider scope by marine alga not of the same race is with the paraspecific dietary requirements of more effective satisfied kind, and this is that fish oil is irrealizable.

Another potential use of heterotrophism algae preparation is that it helps finwhale in the sea, and shrimp and different mollusks (shellfish, freshwater mussel, mouthful shellfish etc.) are when hatching, and part replaces or the marine alga diet alives of additional larva, if utilize light and marine alga then suitable costliness.

The present invention also will promote " organic fish " industrial expansion, and it will provide cheap ω-3 series in a kind of non-fish meal source.In fact, nutritious prod and food industries are widely used in pharmacy though ω-3 is serial, and its maximum market share is the feed of fish.At present, the output of fish oil is annual to amount to about 1,000,000 tons, and wherein 70-80% is used for the feed of fish.The general fish of culturing only obtains omega-fatty acid from fish meal.Along with the increase of fish meal demand and the minimizing of ocean fish resource, utilize fish meal to support that the growth of aquatic products fish is unpractical.In addition, the omega-fatty acid source in the non-fish meal of the development need of this organic fish industry source.Therefore, add the unique selection that recipe almost become water industry of the marine alga of omega-fatty acid as organic fish is rich in utilization.

The biomass and/or the extract oil that are rich in DHA also can be used as human food prods or additive or nutritious prod.PUFAs can extract from microorganism and be used as nutritious prod or medicament.For example, be rich in the DHA microbial oil and be rich in the ARA microbial oil, all can be used for nutritional additive and foodstuff production, as the infant or baby food prescription by what filamentous fungus Mortierellaalpina produced by what dinoflagellate Crypthecodinium cohnii produced.Similarly, the microbial oil that is rich in DHA of Schizochytrium production also can be used for nutrition interpolation and food ingredient.What deserves to be mentioned is, from biomass, extract PUFAs also nonessential extraction before foodstuff production of purifying.Can be according to this oil (as being used as dry powder or solution) of further purifying again of the concrete purposes in the foodstuff production.

Be rich in the little algae of PUFA and/or the PUFAs of this little algae production and also can be used for other purposes, include but not limited to makeup, sanitising agent etc.

Description of drawings

Fig. 1. the equipment diagram of multistage fermentation.

The control of Fig. 2 A-C. fs reaction parameter.

Fig. 3. the marine alga of integration-biofuel system diagram.

Embodiment

The regulation and control of 1. fs of embodiment reaction parameter

The fermentation condition that effective control of the fs reaction parameter that the present invention describes is set up is that the cell growth increases the cell number index with limited cell resource conversion.See Fig. 2 and table 2.

Fig. 2 shows that dissolved oxygen (DO) brings into play main effect (change mutually coordinate with temperature and carbon/nitrogenous source) in process control.Can see that dissolved oxygen raises, cellular energy then turns to synthetic cell to divide necessary chemical substance, therefore significantly increases cell quantity and the almost no influence of pair cell size.Otherwise, reduce dissolved oxygen, will make cellular energy concentrate on cell growth and lipid accumulation.

This process control obtains 181 * 10 after 24 hours when 50%DO ⁶Cell/ml, 48 as a child but reduced to 22.5 * 10 ⁶Cell/ml.The cell density that the 10%DO value obtains was 1/2 of 50%DO value approximately, at preceding 24 hours about 98.4 * 10 ⁶Cell/ml.What is interesting is that whole process keeps the biomass that high DO (50%) can not produce high density.Cell density and biomass concentration began to reduce after 24 hours.Final cell space weight is the 0.057ng/ cell only, and at the 48th hour up to the 0.066ng/ cell.This breeding that high DO control pair cell obviously is described is favourable, but is unfavorable for lipid accumulation.The later stage pH value of cultivating in high DO value is near 3.0, and this is likely that organic acid accumulates and causes.DO is controlled at that the growth and the shaking flask inoculation of 10% cell is near, and the final cell dry weight is 26.5g/L, and final cell space weight is the 0.290ng/ cell.Though the cell density that low DO value cultivation obtains only 22.5 * 10 ⁶Cell/ml, even be lower than bottle level of shaking, but its biomass sustainable growth obtained the biomass of 5.2g/L in 6 days, and final cell space weight is the 0.315ng/ cell.Continue to cultivate and perhaps can further improve output.The DO value that this description of test is lower causes lower cell density, but favourable to lipid accumulation.

Above conclusion continues shake-flask culture by the cell of the high DO of control after (50%) 72 hour and is proved.High DO cultivated after 72 hours in the fermentor tank, and the nutrient solution of 50ml takes out to place from fermentor tank and shakes bottle, supposes low DO concentration, continued to cultivate the low 20.6g/L of final biomass concentration.

It should be noted that low many of cell density that 10%DO obtains than 50%DO.United States Patent (USP) 6,607,900 think that dissolved oxygen level is 4% of saturation value at least in the fermention medium.Obviously, this is far from being enough for high cell density fermentation because even 10%DO all be insufficient.Keep DO and can only obtain seldom cell, cause all can only obtaining seldom biomass in cell volume build phase and lipid accumulation stage in this level.

Generalized theory, we verified (1) high DO is suitable for the cell growth and hangs down the increase that DO is suitable for lipid accumulation or biomass; (2) if postvaccinal preceding 24 hours control 10%DO (similar) to the technology controlling and process of 4-8%DO before, rather than 50%DO preferably, the increase of cell quantity is not optimum, and this will finally cause low biomass/lipid yield, because the maximum value of cell volume is certain.When obtaining more cell quantity, more glycerine (depleted carbon) is utilized, and produces more lipid, but this just may realize when having only the marine alga process of growth initial stage (as initial 24 hours) to obtain the cell of maximum,

The regulation and control of table 2. fs reaction parameter

Table 2 has illustrated the result of this theoretical procedure.In the experiment of these group data, control condition obtains the cell quantity comparison according to (58.5 * 10 ⁶The final after a little while thalline quantity (24.6 * 10 of cell/ml) ⁶Cell/ml, low by 10%) also less, identical control condition is used in contrast, except the thalline that two are shaken in the bottle is combined into one bottle and cell quantity is doubled, trophic factor, substratum and oxygen-supplying amount remain unchanged.Higher cell quantity (114.7 * 10 when just having caused fermentation ends after cell quantity doubles in the experiment ⁶Cell/ml) and dry cell weight (30.8g/L).The cell space weight of noticing contrast is 0.42ng/ cell and the cell space weight of experimental bacteria is the 0.268ng/ cell.Break away from theoretical constraint, this result is likely owing to all parameters except cell quantity all are consistent (for example, the carbon nitrogen source level are suitable), so there is not enough nutrition to make cell volume grow to maximum value.Perhaps add carbon/nitrogenous source, the cell space weight of experimental bacteria can reach maximum value during fermentation ends, and just as contrast, and overall yield also can significantly increase above 10.4% of observation.Should be noted that these results do not rely on the variation of time.Therefore, if add optimum carbon nitrogen source level, cell volume that may be all can increase simultaneously so, does not just need extra time maximization cell volume.

Embodiment 2.

Initial medium comprises 40g/L glycerine, and the 5g/L yeast extract is as other unessential factor in the ammonium acetate of the 5g/L of nitrogenous source and the seawater.Cell in the less culturing bottle is inoculated in the fermentor tank with the inoculum size of 10% (v/v).Temperature is controlled at 30 ℃, and dissolved oxygen is controlled at 20%-50%.Fs, also make the cell quantity increase stage continue 24 hours, the inner cell quantity growth is very fast during this period of time, and final cell density is 400-800 * 10 ⁶Cell/ml, and high more good more.When cell quantity stopped to increase, subordinate phase began, and began to accumulate lipid acid.This stage D O is controlled at 3-5%, and glycerine and nitrogen concentration are controlled at 30-50g/L and 0.5-1.0g/L respectively by feed supplement.Temperature still is 30 ℃, and this stage lasts till 60-80 hour of whole process, stops to increase up to dry cell weight.Stop to add nitrogenous source then, 30g/L is still added and be controlled to glycerine.The final stage time-temperature will be reduced to 20-25 ℃, and control DO increases the accumulation of lipid acid and higher PUFA ratio is provided with this about 0.5%.Final stage begins the about 24-40 hour cell dry weight in back and begins to reduce, and at this moment whole end of processing is collected thalline.

Fs, high temperature and DO are more suitable because cell 30 ℃ than 25 ℃ look fast (not listing data), and the oxygen consumption is very high during cell proliferation.Therefore, fs maintenance high temperature and high DO are the keys that obtains high-cell density.When cell quantity stopped to increase, cell volume will increase owing to the accumulation of lipid acid.This stage, low DO suited, because high DO is harmful to the synthetic of lipid acid, and the conversion carbon source is other product.The nitrogen concentration in this stage should maintain below the 1.0g/L, and experimental data shows that the nitrogen concentration cell growth that is higher than this value is harmful.In fact, the nitrogen restriction is extremely important for the accumulation naturally of lipid acid.Yet certain density nitrogenous source is necessary in this stage, is used for keeping the cell eubolism, cyto-architectural growth and enzyme synthetic.This stage, the about 13-17% of the content of DHA in biomass was if reduction temperature and this value of DO can further improve when finishing.During whole end of processing, nitrogen is with depleted, and low temperature and low DO will further increase the content of DHA to 20% of dry mycelium.The difference of three different steps controlled variable sees Table 3.Whole fermentation process was controlled at about 5 days.

The variation of table 3. syllogic process parameter

Time (h)	0～24	24～48	80～120
				DO (dissolved oxygen)	50～20％	5～3％	0.5％
Temperature (℃)	30	30	20
				Glycerol concentration (g/L)	30～50	30～50	30
Nitrogen concentration (g/L)	1.5～1.0	1.0～0.5	0.5～0

Table 4 has been described the throughput of typical 5 days syllogic fermenting processs at the 5-L fermentor tank, utilizes above-described processing parameter, pretreated raw glycerine.Final biomass concentration is 137g/L, and average whole transformation efficiency is 46.2%, and average biomass production ability is 1.14g/L/hr.Yet the investigator believes that the result at additional optimal conditions next stage I can further improve.Reason is under certain condition of determining of 5L jar, can obtain very high cell quantity (800 * 10 ⁶Cell/ml) and final average cell space weight (500pg/ cell), but owing to the problem of constant feed supplement and ventilation is difficult to keep.

Can believe to concentrate at present to separate and optimize the syllogic fermentation process in the stage of cell number and cell concentration and can reach or approaching existing commercial-scale yield and throughput, if feed supplement in follow-up research, mix and ventilation can further be controlled and optimize, then present method has with existing and produced

Time (hour)	12	16	24	37	47	57	70	81	95	106	120
												Cell quantity (10 6Cell/ml)	212	288	683			582					455
Dry cell weight (g/L)	7.4	13.0	26.8	55.1	70.4	81.6	102	126	145	143	137
												Cell space weight/pg		45	40			140					301

Dry cell weight throughput (g/L/hr)		1.40	2.15	1.94	1.53	0.76	1.57	2.18	1.37
												Glycerine wear rate (g/L/hr)			3.53	4.46	4.42	4.61	2.29	3.69	1.77	1.39	1.82
Transformation efficiency (%)			60.9	43.5	34.6	16.5	68.6	59.1	77.6

The potentiality of Cheng Jingzheng.

Table 4. syllogic 7 typical yield of fermentation and energy for growth

The dry biomass that obtains in the analysis fermentor tank obtains important parameters and successfully produces lipid acid so that better understand this fermentation, moiety such as the protein and the amino acid whose process of DHA and other important water industry feed.Table 5-7 has summarized the analytical results that utilizes the fermentation of seaweed industry scale.The marine alga of test is tested from 30-L.Representational, the fatty acid content in the seaweed bio amount in little 1L and 5L reactor more than 50% (data are not provided).Lipid acid is similar to the Schizochytrium biomass with amino acid, and the latter has been used for commercial production, and for example, the website is Www.aquafauna.com

The seaweed bio component analysis that table 5. is produced by raw glycerine

Parameter	Massfraction
		Lipid acid	34.41
Protein	37.93
		Robust fibre	1.80
Ash content	8.51
		Carbohydrate	19.19
Calcium	0.45
		Phosphorus	1.92
Potassium	1.38
		Carotenes	835IU?A/lb

The fatty acid profile of the seaweed bio amount that table 6. is produced with raw glycerine

Parameter

The % mass percent

% accounts for lipid acid per-cent

Humidity (88.9%)
			Saturated fatty acid	15.23	44.241
Monounsaturated fatty acids	0.36	1.047
			Shift lipid acid	0.36	1.047
Polyunsaturated fatty acid	16.94	49.215
			Total fatty acids	34.41	100.000
C 14:0 tetradecanoic acid	0.73	2.110
			C 15:0 pentadecanoic acid	0.56	1.640
C 16:0 palmitinic acid	13.77	40.000
			C 17:0 heptadecenoic acid	0.24	0.710
C 18:0 stearic acid	0.55	1.610
			C 18:1n9C oleic acid	0.27	0.780
C 18:2n6 linolenic acid	0.10	0.280
			C 20:0 eicosanoic acid	0.07	0.190
C 18:3n3 linolenic acid	0.04	0.130
			The C18:4n3 therapic acid	0.05	0.140
C20:3n6 homotype-gamma linolenic acid	0.07	0.190
			The C22:1n9 erucic acid	0.07	0.190
C 20:4n6 arachidonic acid	0.07	0.200
			C 20:4n3 eicosatetraenoic acid	0.17	0.480
C 20:5n3 timnodonic acid	0.31	0.900

The C24:1n9 Selacholeic acid	0.06	0.160
			The C22:5n6 clupanodonic acid	3.89	11.300
The C22:5n3 clupanodonic acid	0.21	0.600
			The C22:6n3 docosahexenoic acid	12.60	36.600
Other	0.44	1.270

The aminogram of the seaweed bio amount that table 7. is produced with raw glycerine

Parameter	The % mass percent	% accounts for the per-cent of total amino acid
			Protein	37.93
Aspartic acid	2.52	8.946
			Threonine	1.35	4.792
Serine	1.44	5.112
			L-glutamic acid	4.50	15.974
Proline(Pro)	1.26	4.473
			Glycine	1.35	4.792
L-Ala	2.25	7.987
			Xie Ansuan	2.07	7.348
Methionine(Met)	0.63	2.236
			Isoleucine	1.17	4.153
Leucine	2.16	7.668
			Tyrosine	1.08	3.834

Phenylalanine	1.17	4.153
			Histidine	0.54	1.917
Methionin	1.71	6.070
			Arginine	1.98	7.029
Halfcystine	0.54	1.917
			Tryptophane	0.45	1.597

Embodiment 3. energy consumptions relatively

Matlab-Simulink software is the energy consumption of this system relatively.5 tons of fermentor tanks of simulation marine alga fermentation, basic with the data of 5L and 30L experiment as assessment.

The basic parameter of this process setting is: the fermentor tank of 5 cubic metres useful volume; The fed-batch fermentation scheme; 30 ℃ of temperature of reaction; 25 ℃ of average room temperature; Ventilation Rate is 0.5vvm and 1.0vvm, electricity charge electricity charge $0.046/kwh.The basic setup parameter of heating system is: thermosteresis is equivalent to, and q is a thermosteresis, and h is the thermal conductivity of steel, and A is an interior region, the real internal temperature of Ti, and T is an outside temperature; The heat that needs is equivalent to, and Q is the heat that needs, and M is the total mass of solution, and Cp is a thermal capacity, and T is the target of internal temperature, and Ti is real internal temperature.Other parameter of heating system is: M=5,000kg; Cp=3.95kJ/kgK; Thickness=the 0.005m of steel; Thickness=the 0.02m of isolator; Thermal conductivity=the 45W/mK of steel; Thermal conductivity=the 0.4W/mK of isolator; Surface area=55m ²The basic parameter of stirring system comprises that a disk diameter is six blade turbomachines of 3.48m, and the diameter of turbine is 1.16m; Soltion viscosity is 100cp; Stir speed (S.S.) is 300-1,000rpm.The basic parameter of stirring system is that for the system of 1vvm, piston type air compressor is 170cfm; Theoretical energy input 50kw; Efficient 0.7; Maximum gauge pressure is 450 pounds/square inch.0.5vvm system then be respectively only 100cfm, 27kw, efficient 0.7,450 pounds/square inch of maximum gauge pressures.

Simulator based on above-mentioned parameter is summarised as table 8.Can see for this blow and vent system and need 52 cents/kg marine alga.With the adding and see Table 9 of expense of raw materials, producing 1kg seaweed bio amount needs about 1 dollar of total ventilation and feed supplement cost.Though this only is rough estimation, and perhaps other factors can increase total cost, if seaweed bio amount wholesale price can Da Dao $10/kg (retail price reduces＞50%) still had positive effect for output.

The simulation output of table 8. fermenting process energy expenditure

Ventilation Rate (VVM)	1	0.5
			Fermentor tank volume (m 3)	5	5
Stir speed (S.S.) (rpm)	800	800
			Temperature of reaction (℃)	35	35

Reaction times (hr)	96	96
			Seaweed bio amount (kg, dry weight)	860	860
Total energy cost ($)	450	350
			Energy expenditure (dollar/kg marine alga)	0.52	0.41

The estimation of table 9. material cost

Transformation efficiency	46％
		Glycerine (kg)	2.2
Price ($/kg)	0.12
		Glycerine cost ($)	0.26
Nitrogen and other salt ($) (supposing to be equivalent to the carbon source cost)	0.26
		Total raw material cost (dollar/kg marine alga)	0.52

The invention describes preferential embodiment, skilled with operating procedure, the present invention can be corrected within the scope of the claims.Therefore, the present invention should not only limit to above-mentioned embodiment, and should comprise that this paper describes all corrections in the purport scope.

Claims (27)

1. a method of producing lipid acid may further comprise the steps

I) adopting the condition that improves cell density to carry out little algae cultivates;

Ii) promoting to cultivate above-mentioned little algae under the condition that cell volume increases and lipid acid accumulates;

Iii) promoting to cultivate above-mentioned little algae, wherein said step I under the condition that lipid acid increases), ii) and culture condition iii) have nothing in common with each other.

2. the method for claim 1, lipid acid wherein is meant the lipid acid that is rich in ω-unsaturated double-bond more than 3.

3. method as claimed in claim 2, wherein omega-3 unsaturated fatty acid comprises docosahexenoic acid (DHA).

4. the method for claim 1, wherein this method main source of carbon is a raw glycerine.

5. the method for claim 1, wherein high-cell density is about 200,000, and 000 to 1,000, every milliliter of substratum of 000,000 cell.

6. method as claimed in claim 5, wherein high-cell density is every milliliter of substratum of 800,000,000 cell at least.

7. the method for claim 1, wherein marine alga is selected from the Thraustochytrium and the Schizochytrium of same genus.

8. method as claimed in claim 7, wherein little algae is Schizochytrium limacinumSR21.

9. the method for claim 1, wherein promote the condition of high-cell density to comprise following one or more: keeping culture temperature is 25-35 ℃; Keep dissolved oxygen level 10-50% at least; Keep nitrogen level 1.0-5.0g/L.

10. culture temperature is 30 ℃ in the method as claimed in claim 9.

11. dissolved oxygen level is 20% in the method as claimed in claim 9.

12. promote in the method for claim 1 that the method that cell volume increases and lipid acid accumulates comprises following one or more condition: keeping culture temperature is 25-35 ℃; Keeping dissolved oxygen level is 2-10%; Keep nitrogen level 0.5-1.0g/L.

13. method as claimed in claim 12, wherein culture temperature is 30 ℃.

14. method as claimed in claim 12, wherein dissolved oxygen level is 3-5%.

15. the method for claim 1, wherein promote the condition of lipid acid accumulation to comprise following one or more: keeping culture temperature is 20-25 ℃; Keeping dissolved oxygen level is 0-0.5%; Stop to mend nitrogen.

16. the method for claim 1 promotes that wherein little algae was cultivated 12-36 hour under the condition of high-cell density.

17. method as claimed in claim 16 promotes that wherein little algae was cultivated 24 hours under the condition of cell density.

18. the method for claim 1, wherein promote cell volume to increase and the condition of lipid acid accumulation under little algae cultivation 60-80 hour.

19. the method for claim 1, wherein step I), ii) and iii) all in a fermentor tank, carry out.

20. the method for claim 1, wherein step I) in first fermentor tank, carry out step I i) and iii) in second fermentor tank, carry out.

21. the method for claim 1, wherein step I), ii) and iii) in fermentor tank separately, carry out respectively.

22. produce the method for lipid, may further comprise the steps

I) under the condition that promotes every milliliter of substratum of cell density 800,000,000 cell, cultivate little algae at least;

Ii) under the condition that promotes cell volume increase and lipid accumulation, cultivate little algae;

Iii) under the condition that promotes lipid accumulation, cultivate little algae.

23. method as claimed in claim 22, lipid acid wherein is rich in omega-3 polyunsaturated fatty acids.

24. method as claimed in claim 23, omega-3 polyunsaturated fatty acids wherein comprise docosahexenoic acid (DHA).

25. a method of producing by microorganism integration biofuel and lipid acid may further comprise the steps the production biofuel, wherein production process produces by-product and gives birth to raw glycerine;

The pre-treatment raw glycerine is to form the glycerine raw material;

Cultivate the microorganism that produces lipid with glycerine as carbon source and produce the seaweed bio amount that is rich in lipid acid;

The seaweed bio amount that lipid acid is rich in processing obtains omega-fatty acid and non-omega-fatty acid;

From non-omega-fatty acid, separate omega-fatty acid;

In above-mentioned biofuel production process, use above-mentioned non-omega-fatty acid, use above-mentioned non-omega-fatty acid can produce extra raw glycerine in the wherein above-mentioned biofuel production process, integrate the production process of biofuel and lipid acid thus.

26. a method of integrating biofuel and omega-fatty acid production may further comprise the steps

A) obtain raw glycerine from the biofuel refining process;

B) utilize raw glycerine as carbon source production be rich in the seaweed bio amount of lipid acid, comprise omega-fatty acid and non-omega-fatty acid;

C) collect omega-fatty acid;

D) utilize non-omega-fatty acid to produce biofuel and raw glycerine;

E) repeating step b-d.

27. the method that integration biofuel as claimed in claim 26 and omega-fatty acid are produced, wherein step b) may further comprise the steps

I) under the condition that promotes high-cell density, cultivate little algae;

Ii) promoting to cultivate above-mentioned little algae under the condition that cell volume increases and lipid acid accumulates;

Iii) cultivating above-mentioned under the condition that promotes the lipid acid accumulation is little algae, and wherein each the step condition in three steps is all inequality.

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