CN102089434A - Integrated system for productioin of biofuel feedstock - Google Patents

Integrated system for productioin of biofuel feedstock Download PDF

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CN102089434A
CN102089434A CN2009801281461A CN200980128146A CN102089434A CN 102089434 A CN102089434 A CN 102089434A CN 2009801281461 A CN2009801281461 A CN 2009801281461A CN 200980128146 A CN200980128146 A CN 200980128146A CN 102089434 A CN102089434 A CN 102089434A
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algae
heterotrophism
autotrophy
class
seed
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迟占有
郑玉彬
本·路克
陈树林
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University of Washington
Washington State University Research Foundation
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    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/30—Wastewater or sewage treatment systems using renewable energies
    • Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy

Abstract

Disclosed is a culture system for the production of algae biomass to obtain lipid, protein and carbohydrate. By integrating heterotrophic processes with a phototrophic process in parallel, this system provides year around production in colder climates. By integrating heterotrophic processes with a phototrophic process in series, this system creates a two-stage, separated mixed- trophic algal process that uses organic carbon and nutrients for the production of seed in the heterotrophic process, followed by release of cultured seed in large-scale phototrophic culture for cell biomass accumulation. Organic carbon source including waste materials can be used to feed the heterotrophic process. The production capacity ratio between the heterotrophic and the phototrophic processes can be adjusted according to season and according to the availability of related resources. The systems are used for producing and harvesting an algal biofuel feedstock as well as other potential high-value products. The sequence and approach enhances utilization of carbon and nutrient waste-streams, provides an effective method for controlling contamination, adds flexibility in regard to production and type of available products, and supplies greater economic viability due to maximized use of available growth surface areas.

Description

The integrated system of biofuel raw material production
The application serial that the present patent application claim has benefited from July 30th, 2008 application is 60/084,708 the interim patent of the U.S., its relevant complete content document that sees reference.
Technical field
The present invention relates to a culture systems of producing little algal biomass in order to obtain grease, protein and sugar.By the training mode of heterotrophism and autotrophy parallel connection, this culture systems can be in the weather whole year production of cold.By heterotrophism and the placed in-line training method of autotrophy, native system has been set up one two stage, the little algae culturing process of mixotrophism independently, promptly utilize organic carbon source and nutritive substance heterotrophism to cultivate production nutrition algae seed, then with this slightly the algae seed carry out large-scale autotrophy and cultivate to obtain cellular biomass.Wherein organic carbon source comprises the waste that can be used for the heterotrophism cultivation.The turnout ratio of heterotrophism process and autotrophy process can be regulated according to the operability of season and related raw material.Native system is used for producing and obtaining algae bio fuel feedstocks and other high value added products.This cultural method can be strengthened carbon and organic useless nutraceutical utilization, and polluting for controling environment provides an effective means, increases the handiness of production and enabled production type, thereby and because maximum using can provide stronger Economic Force with cultivated area.
Background technology
Adopt microbial method (as utilizing algae and yeast) to produce grease, unit surface is than output of oil-bearing crop height (Pienkos and Darzins, 2009).Studies show that algae might substitute the fossil oil of a large portion and do not change a large amount of land uses or cause great effect.According to Chisti (Chisti, 2007), the U.S. only need about 1.1% and 2.5% existing cultivated area (suppose respectively grease account for the algae dry biomass 70% and 30%) can satisfy the communications and transportation demand for fuel of the U.S. 50%.If utilize means as CO2, every pound of algae bio amount is the CO2 of 1.6-1.8 lb (Chisti, 2007) fixedly.In addition, as a kind of form of aquaculture, algae culture provides new opportunity for agricultural, for the producer, processor and dealer bring economic interests.Obviously, with as the relevant new technology of the algae production of biofuel raw materials for production provide opportunity very greatly for satisfying social demand, response environment variation and economic sustainable development to energy security.
According to the difference as the carbon source kind of energy carrier and alga cells skeleton, algae culture can be divided into three types.First type is that autotrophy is cultivated, and CO2 is as carbon source, and sunlight is as energy derive.Second type is that heterotrophism is cultivated, and organic carbon such as glucose or organic acid are used as the carbon source and the energy.The third type is that mixotrophism is cultivated, and CO2 and organic carbon all are used as carbon source.At present, owing to lack the technology that enables and the technology barrier that is present in each link, there is not a kind of training mode to be used to commercially produce fuel.
Comparatively speaking, consider cultivation scale and cost of investment, the autotrophy of algae is cultivated productivity lower (Singh and Ward, 1997) (Wen and Chen, 2003) production cost height.In addition, because penetrating with cell concn of light is inversely proportional to, the restriction of light can not overcome fully.The accumulation of bioreactor nutrient solution middle and high concentration oxygen also is another open question.The autotrophy cultivation cost of investment of algae is low in the open pond, but productivity is also lower, and is subject to pollution (Rusch andChristensen, 1998 of other microorganisms and insect; Rusch and Malone, 1998).Up to now, the autotrophy algae that can be used for commercially producing is only used for producing high value added products such as pigment, carotenoid and healthcare products, large-scale cultivation autotrophy algae is used for its cost of investment of production biofuel and compares the output value also high a lot (Pienkos and Darzins, 2009).
Different with the autotrophy algae is that growth does not need illumination in oil-containing microorganism such as oily algae or the zymic heterotrophism culturing process, and can carry out high-density culture in larger container.The advantage that heterotrophism is cultivated also has growth velocity fast, output height, easily results.Utilize microorganism to replace plant lipid and albumen as far back as the eighties in 20th century exploitation by yeast fermentation production Unicell Oils and Fats (SCO) attempt as human foods and animal-feed, thereby commercially produce SCO and single cell protein (SCP) (Ward and Singh, 2005).The early stage SCO concentration of production is in high value greases such as cocoa butter, and some zymic zymotechniques have also been realized industrialization subsequently.Since the nineties in 20th century, the little algae of a collection of heterotrophism is used to the poly-unsaturated fatty acids (PUFA) of production high value, is used for replacing unsaturated fatty acids in traditional fish oil as ω-3 family lipid acid.Wherein, some algae kinds such as fragmentation vibrios and Crypthecodinium cohnii (Schizochytrium sp.andCryptocodinium cohnii) are used for suitability for industrialized production DHA (Bailey et al., 2003) and baby formula milk powder by success.In recent years, some researchs that the little algae of yeast and heterotrophism is cultivated are in the news and have shown this processing method have broad application prospects (Easterling et al., 2009; Xiong et al., 2008; Xue et al., 2008).Yet, utilize the obstacle of heterotrophic microorganism production biofuel to be that its culturing process cost of investment is too high and the carbon source raw material cost is too high.Sugar is about 16.7～25% to the transformation efficiency of fat, depend on content of oil and grease in yeast or the microalgae cell, this is because a part of carbon source is used to provide energy, and the carbon of other form is used for forming cytoskeleton and biomass (Kyle and Ratledge, 1992).Therefore, produce 1 ton of grease and need 4～6 tons of glucose approximately.Utilizing fibre source sugar to carry out grease production is to reduce a kind of selection of raw materials cost, but delays suitability for industrialized production as seen from cellulosic ethanol, because some technology barrier causes cost still very high.A kind of selectable solution that reduces cost is exactly to be used for heterotrophism by the organic waste that sugar and/or starch are rich in utilization as carbon source to cultivate (Kapdan and Kargi, 2006).Limited although originate, it is competent, ideal that these organic wastes are used for heterotrophism seed tank culture (the open pond autotrophy that does not comprise the back is cultivated) as carbon source, and the two all is cores of following novel training mode.
Summary of the invention
Advantage of the present invention is to adopt cell high Density Heterotrophic and low-cost extensive autotrophy to cultivate the pattern that combines, and recycle carbon waste, nutrition and organic waste.This training method handiness is strong, can overcome the deficiency that seasonal climate changes, and reduces and pollutes, and reduces the requirement of pond size and cost of investment.At first, according to different needs, but native system heterotrophism and autotrophy are cultivated parallel running, and correspondingly utilize organic and inorganic carbon source to expand production capacity and the stability of system, especially when seasonal severe weather and/or other biological pollution, algae autotrophy culturing process will be interrupted, but heterotrophism process such as saccharomyces olei and heterotrophism algae can utilize organic waste to grow for raw material, greasy yeast or algae bio matter are rich in generation, are processed to biofuel then.The most important thing is, heterotrophism and the integrated system of autotrophy culturing process series connection can carry out the algae mixotrophism and cultivate, two kinds of training methods are independently carried out respectively in the mixed culture, and wherein the heterotrophism process is used to cultivate little algae seed, and the autotrophy process is used for accumulates biomass and grease then.This series connection culture systems can realize by the high yield grease autotrophy photosynthetic algae of heterotrophism of holding concurrently, these algae comprise many bead algae (Chlorella sp) (Hermsmeier et al., 1991), Antarctic Ice algae (Chlamydomonas sp.) (Boyle and Morgan, 2009), grid algae (Scenedesmus sp.) (Abeliovich and Weisman, 1978), and many diatomss (Lewin, 1953).But but production algae bio matter is cultivated in the algae industrialization that these double nutritions are cultivated, and utilizes the integrated culture systems of these double nutrition algae to have following many advantages:
1) training mode that combines with autotrophy of heterotrophism can guarantee that algae is in the throughput of cold season and strengthen production stability in selected place.In order to reach identical purpose, the heterotrophism culture systems is applicable to yeast too.
2) the seed heterotrophism is cultivated the seed autotrophy be better than adopting usually and is cultivated, but this is because the heterotrophism culturing process more effectively produces a large amount of seed thalline within a short period of time.
3) heterotrophism is cultivated the organic materials that can utilize in organic carbon and the organic waste, or even the discarded glycerine that reclaims, and these organic wastes all are facile usually, is the rubbish that produces in the province's product and the course of processing sometimes.The production that utilizes organic carbon to finish part algae bio amount makes this process have more handiness and diversity, can be according to different environment and the content of industrial CO2, or in parallel again or utilize specific heterotrophism reactor separately according to the generation of season and pollution.The waste solid material preferably can and be hydrolyzed into the organic carbon that easily utilizes in the algae culture process through pre-treatment.Culture temperature is 10～40 ℃, and 20～35 ℃ better, and 30～35 ℃ best.Oxygen is passed in the culture vessel in this culturing process, and dissolved oxygen is controlled at 0～90% of fermented liquid saturated oxygen concentration, and 5～50% is better.Fermentor tank that adopts in this process or bio-reactor are one or more in the following culture vessel: stirred tank fermentor, bubble tower bio-reactor, airlift bioreactor, perhaps other any bio-reactors that can be used for the cultivation of microorganism heterotrophism.Here, being used for the reactor that heterotrophism cultivates can be fermentor tank, reactor or the bio-reactor of dark sealing.
4) because the ability of heterotrophism cultivation seeding can be significantly improved under the situation of needs, this provides a kind of solution route for the biological pollution in the autotrophy culturing process of control downstream.Studies show that cultivating the seed category, quantity and the inoculum size that produce through heterotrophism directly has influence on the ability that open autotrophy cultivation overcomes pollution.
In whole growth process, the utilization of carbon and nutritive substance can be optimized control by remaining nutritive substance in the CO2 that produces and the heterotrophism nutrient solution, nutritive substance remaining in these CO2 and the heterotrophism nutrient solution can be used to the autotrophy culturing process, makes that finally the input of pharmaceutical chemicals and solid matter and sewage disposal are more efficient.
Carry out seed culture by a special heterotrophism process, increased autotrophy cultivation pool surface-area productivity, thereby made this process have more economy.
Because temperature and/or sunlight density and the climatic region that can not carry out algae culture can be used to cultivate algae bio matter now, i.e. utilization by organic carbon in this zone remedied the deficiency of inorganic carbon in utilizing before the utilization of this process and several kinds of carbon source made.Its result is significant, and this process is from being feasible with environment economically, can more effective recovery and utilize carbon and nutritive substance, thus improved the sustainability of system.
This heterotrophism autotrophy culturing process provides a kind of method of novelty, in the past the patent that is used for the biofuel raw material about algae bio matter all around independent autotrophy cultivate, independent heterotrophism cultivates (Chen and Chen, 2006) and/or autotrophy-and various technological processs that heterotrophism is cultivated launch, and heterotrophism-autotrophy training mode does not then appear in the newspapers.Especially for the latter, United States Patent (USP) 20080160593-A1 (Oyler, 2008) (document sees reference) has described an autotrophy-heterotrophism technological process that algae produces biofuel.The core of heterotrophism among the present invention-autotrophy culturing process is the utilization of the facultative heterotrophism ability of autotrophy algae.Chen and Johns (1991) are when the facultative heterotrophism ability of some algae of research, studied C/N than and the influence of ventilating heterotrophism Chlorella sorokinina lipid acid being formed, do not reach a conclusion but these algae or its nearly edge algae are used for heterotrophism of the present invention-autotrophy culturing process.A significant characteristic of the order culture method among the present invention is exactly that native system can reduce pollution in the culturing process of open pond, downstream.Theegala etc. (1999) have reported a kind of method of cleaning pollutent in the permanent alga reactor of continuous hydraulic pressure tubular type, utilize in the reactor flow and the residence time to control pollutent, also can not serve the same role but mention inoculum size.
The invention provides a kind of method of producing the biofuel raw material.This method may further comprise the steps: utilize heterotrophism cultivation algae or class alga microbial with seeding, these algae and class alga microbial have the physiological mechanism of autotrophy and heterotrophic growth; The seed of these heterotrophism cultivations carries out the autotrophy cultivation with accumulation biomass and lipid then.In an example, but the heterotrophism cultivation stage comprise and utilize waste water and waste solid material to cultivate algae or class alga microbial as organic carbon source with autotrophy and heterotrophic growth physiological mechanism in opaque fermentor tank or bio-reactor.These waste water and waste solid material include but are not limited to: organic composition, the residual nuclear of algae bio body of agricultural wastes, industrial paper pulp, municipal wastewater, perhaps any organic waste that contains the organic carbon source that can be used for microorganism culturing.This method comprises that specifically the CO2 that will produce in the heterotrophism culturing process carries out autotrophy as carbon source and cultivates.In some instances, algae or class algae microorganism such as Chlorella sp., Chlamydomonas sp., and/or the seed liquor cell concn that Scenedesmus sp. cultivates through heterotrophism is 107～109 cell/ml or more.This method comprises that specifically heterotrophism cultivation algae or class algae microorganism are with accumulation biomass and grease.
The present invention also provides a kind of method of producing biofuel, and this method may further comprise the steps: heterotrophism is cultivated algae with autotrophy and heterotrophic growth physiological mechanism or class algae microorganism with seeding; The seed of these heterotrophism cultivations carries out the autotrophy cultivation with accumulation biomass and grease then; From these algae or class algae microorganism cells, extract grease as biofuel.In an example, but the heterotrophism cultivation stage comprise and utilize waste water and waste solid material to cultivate algae or class alga microbial as organic carbon source with autotrophy and heterotrophic growth physiological mechanism in dark fermentor tank or bio-reactor.These waste water and waste solid material include but are not limited to: organic composition, the residual nuclear of algae bio body of agricultural wastes, industrial paper pulp, municipal wastewater.In some instances, this method can comprise that also utilizing the CO2 that produces in the heterotrophism culturing process to carry out autotrophy as carbon source cultivates.These algae or class algae microorganism include but are not limited to: Chlorellasp., Chlamydomonas sp., and/or the seed liquor cell concn that Scenedesmus sp. cultivates through heterotrophism is about 107～109 cell/ml or more.
The present invention also provides the system that produces the biofuel raw material.This system comprises: have at least an equipment to be used for heterotrophism and cultivate algae with autotrophy and heterotrophic growth physiological mechanism or class algae microorganism with seeding; Having at least an open pond or reactor to be used for autotrophy cultivates by heterotrophism and cultivates the algae that obtains or class algae microorganism seed with accumulation biomass and grease.Dispose an open pond or reactor and cultivate algae or the class algae microorganism seed that obtains from the heterotrophism of at least one fermentor tank with storage.This system can comprise one or more test sets with detect with open pond or reactor are provided in output under the different growth conditionss, can comprise further that also one or more operating devices cut out or open operation in open pond or the reactor automatically in order to the output that provides according to detector.The parameter (growth conditions) of monitoring include but are not limited to sunlight amount, temperature and pollutent existence whether.
Description of drawings
Fig. 1 is heterotrophism-autotrophy algae culture integrated system general introduction;
Fig. 2 is the mass balance of heterotrophism-autotrophy algae culture integrated system;
Fig. 3 is the yeast culture system that utilizes food hydrolysis waste to carry out;
Fig. 4 carries out adding when saccharomyces olei is cultivated the glucose culture systems for utilizing municipal wastewater;
Fig. 5 utilizes castoff to carry out the growth kinetics of C.sorokiniana seed culture;
Fig. 6 is that the multiplication rate of different seed growth processes compares;
Fig. 7 is the growth curve of ChlorellaSorokiniana and EG-1-2;
Fig. 8 carries out the comparison of open pond cultivation multiplication rate for heterotrophism and autotrophy seed liquor;
(Fig. 9 a) and construction investment relatively (Fig. 9 b) for heterotrophism and autotrophy seed carry out the biomass growth fraction of autotrophy when cultivating (identical inoculum size) for Fig. 9;
Figure 10 is that the economy of mixed culture system and pure autotrophy culture systems compares.
Embodiment
The invention describes one two stage alga cells high-density culture technology, wherein the heterotrophism process is used to cultivate the algae seed, carries out autotrophy then and cultivates.Producing used bacterial classification is the algae with autotrophy and heterotrophic growth physiological mechanism.Bead algae (Chlorella sp.) particularly, Antarctic Ice algae (Chlamvdomonassp.), grid algae (Scenedesmus sp.) but and the diatoms cultivated of many double nutritions.The ability that double nutrition is cultivated in this cultivates to produce in the algae bio matter in industrialization has greater advantage.
The meaning of " biomass production " or " biomass accumulation " is the increasing and/or generate in culturing process or the increase of a certain Special Thing quality of synthetic of organism quantity in the nutrient solution.In other words, " biomass " also can refer to the composition of organism itself, or the material of organism generation.These materials can accumulate in vivo, or the part of organism, or the appurtenant of organism, or organism is secreted into the material in the nutrient solution.Such material can include but are not limited to as lipid, protein, carbohydrate, sugar, amino acid, carotenoid etc.These materials can be generated in the biology growing process, and its growing amount in nutrient solution increases with the increase of organism quantity.In addition, the production of these materials is subjected to the influence of culture condition or other environmental factorss (lacking as nitrogen).Particularly the lipid composition in the organism can be used as biofuel.
In an example, discarded carbon source at first is used as nutrition source in the heterotrophism culturing process.The product of first growth phase is as the inoculum of second growth phase.First growth phase still retains enough nutrition and is used for the subordinate phase autophyting growth, in addition the CO of Chan Shenging after finishing in the nutrient solution 2Can be recovered utilization (Fig. 1).
In this invention, one or more organic carbon sources are used to the heterotrophism culturing process.In an example, organic carbon source is a waste material.These wastes can be waste water and/or the solid waste that contains the organic carbon source that can be utilized by heterotroph.The advantage of this example is that raw materials cost is lower, and can effectively utilize waste.If necessary, organic waste (as organic moiety, the residual nuclear of algae bio body, various waste water etc. in agricultural wastes, industrial paper pulp, the municipal waste) at first is collected (P-01 through suitable pre-treatment and hydrolysis in particular device, Fig. 1), be used for the production (E1) of glycogen material then.In some instances, a kind of scheme of hydrolysis organic waste is to carry out enzymic hydrolysis then with dilute acid hydrolysis earlier.The dilute sulphuric acid pre-treatment is considered to the method for most economical effective hydrolysis timber and agricultural wastes, and it can destroy the structure of hemicellulose, reduces cellulosic degree of crystallinity, for bigger porosity is created in the enzymic hydrolysis of back.Under acidic conditions, some albumen can be hydrolyzed to amino acid simultaneously, are utilized by algae and yeast then.After sour pre-treatment, utilize the enzymic hydrolysis process of cellulase and hemicellulase to can be used to produce sugar.The raw glycerine and the remaining biomass in the follow-up esterification process (E-7) that reclaim also can be used as carbon source and nutritive substance (P-20 and P-19).Sugar and/or glycerine are sent in (P-03 and P-04) two heterotrophic fermentation jars (E2 and E3) then, one of them is used for heterotrophism and cultivates production high value added product (P-17), and another is used to the open pond autotrophy process (E-5) of subordinate phase to cultivate seed (P-07).In addition, non-discarded carbon source and/or non-waste water also can be used for the supplementary carbon source of this process.
Except biofuel, the heterotrophism algae also can produce high value added product such as carotenoid, enzyme, and DHA and other ω-3 family lipid acid.The process of describing in this heterotrophism culturing process and the United States Patent (USP) 12/132,131 (document sees reference) is similar.The high value added product that the heterotrophism algae produces can be used as a kind of algae raw material and is utilized and sells, perhaps further separated extraction.In the example of a separation and Extraction, algae bio body (P-18) can be converted into mixed methyl aliphatic ester (FAME) (P-21) and other by products (P-19) by esterification, and do not need grease to extract or dry (E-7), as (document sees reference) described in the PCT/US08/50799.In some special examples, high value added product is made up of ω-3 family lipid acid, and mixing FAME (P-21) then can be separated by the method for distillation (E-8), and the healthcare products ω-3FAME that obtains and non-ω-3FAME can be used as biofuel (P-22) and sold.
Most of autotrophy algae also are facultative heterotrophism algaes, and they have this specific character and have the ability to utilize and transform organic carbon.In this technological process, autotrophy is cultivated by facultative heterotrophism algae in two stages.Fs is that the heterotrophism process of closure is used to cultivate the seed of high-cell density (as 10 7～10 9Individual cell/ml or more), this process and United States Patent (USP) 12/132,131 method similar (document sees reference).The nutrition of fs and high-density cells are used to the open pond or the photoreactor autotrophy culturing process of subordinate phase.Two stage culture process make the pollution in the open pond have very big controllability by the high-density cells inoculation that utilizes the fs to cultivate in this.Fs cultured cells density is about 10 7～10 9Individual cell/ml or bigger, this can keep the viability stronger than other biological in the open autotrophy culturing process of subordinate phase as seed.Under many situations, the algae seed culture is carried out in photoreactor and/or open pond.Yet this needs certain lighting area, cost height not only, and also because the contradictory relation between high-density culture and the illuminating area makes that closed photoreactor can not be used to commercially produce.Algae grows needs high surface-area and volume ratio in the heterotrophic fermentation jar unlike the autotrophy culturing process, and therefore, there is not the conflicting problem of high-density culture and illuminating area in easier expansion scale production yet.Another advantage of heterotrophism seed culture is to have the ability by nitrogenous source control seed cell chlorophyll content.Control by the cell chlorophyll content can not reach the purpose that improves culture light compositing ability by genetic manipulation.
The subordinate phase autotrophy is cultivated the seed that utilizes closed heterotrophism process to cultivate and is carried out (P-07).In a specific examples, at the open cultivation pool (E-5) that is used to this culturing process or similarly have in the alloy vessel of high surface area, algae can contact with external environment wholly or in part.The fermented liquid of (P-06 and P-10) can be used to the nutrition source that cultivate in the open pond of autotrophy in the heterotrophism seed culture process.The exhausted fermented liquid utilizes after can concentrating by the centrifugal mode again in the heterotrophism culturing process, also can be delivered directly in the open cultivation pool with seed cell without separation.In addition, although the volume ratio heterotrophic fermentation jar of autotrophy reactor is much bigger, the concentrated nutrition thing in the heterotrophic fermentation liquid remains a kind of important input thing in the open pond of autotrophy.The alga cells of (P-14) (E-06) is collected in the autotrophy cultivation pool.In the example that extracts about downstream separation of a first-selection, algae bio amount (P-18) is converted into mixed methyl aliphatic ester (FAME) (P-21) and other by products (P-19) by esterification reaction process, and this process does not need greasy extraction and drying (E-7) (PCT/US08/50799).Yet other fuel conversion and fuel method for processing such as pyrolysis, liquefaction, vaporization etc. can be used to process the algae bio body (Li et al., 2008) of recovery.
, carried out the cultivation of algae and will play very important synergy by near the waste water manufactory such as animal rearing field, food processing plant or municipal wastewater treatment plant underestimated at some.
● adopt the nutrient recovery system that is similar in the United States Patent (USP) 12/132,016 (document sees reference) to set up a relevant anaerobic digestion (AD) unit, also can be used to provide nutritive ingredient.Industry, agricultural, compost or municipal wastewater can be handled by AD and produce the liquid that can be converted into the biogas with heat and energy and have prototroph composition (except some organic carbons).In this example, AD can produce heat and energy as the processing unit of a discarded organic carbon, and these heats and energy partly can be used for the cultivation of algae, and eutrophic wastewater also can be used for the cultivation of algae.In addition, open autotrophy pond also needs inorganic carbon source CO 2(P-13), these CO 2Part is cultivated the waste gas that produces in the reactor (P-05) from AD with waste gas (P-09 and P-11) and heterotrophism that relevant (E-09) equipment produces.Temperature in the open pond can be carried out part control by utilizing the refuse producer among the AD (P-23).Required power consumption can be used as the attached load of AD (P-24) producer electric power output.The concentrating cells of results extracts biofuel (P-22) through aforementioned in-situ extraction device (E-07) then from settling tank (P-16).
● the training mode that the heterotrophism process combines with the autotrophy process can utilize waste gas organic carbon and other resources such as water, CO 2, sunlight etc. produces algae bio matter, therefore improved throughput.And atrocious weather or when utilizing organic carbon source to be subjected to polluting to cause autotrophy algae productivity low, heterotrophism and autotrophy process can be carried out alone respectively or parallel running simultaneously.
At last, the integrated system among the present invention can provide one or more detectors (transmitter) and/or controller to monitor situation in one or more reactors, and can be to operator's prompting to the favourable or disadvantageous condition of growing.For example, the autotrophy reactor can be equipped with one and can measure or the transmitter of perception sunlight or other light sources amount (as lumen), this transmitter can be measured or any moment of perception is incident upon the sunlight on the autotrophy nutrient solution or the amount of other light sources, and (as one minute, one hour and one day etc.) project the accumulation volume of the sunlight on the autotrophy nutrient solution in the perhaps a certain special time period.Whether Design of Sensor should help the operator and can determine the autotrophy culturing process to continue or interrupt, stop, close fully and change the heterotrophism cultivation stage over to according to output data.In addition, Design of Sensor can comprise or information is passed to two-position controller, but this two-position controller automatic switchover system by the autotrophy training mode to the heterotrophism training mode, vice versa, as switch back autotrophy and heterotrophism pattern, such as opening the autotrophy pattern.In some instances, include only the autotrophy reactor.Yet, in other examples, because the condition difference comprises autotrophy reactor and/or heterotrophism reactor.Even the heterotrophism reactor does not utilize sunlight, but still can being equipped with the transmitter of monitoring correlated condition, its design is used for recorded information and output data is provided, cultivate or change back the autotrophy training mode to convert heterotrophism automatically to.In addition, this detector can be connected by electronics or manual mode with the reactor of two training modes.
Level or perceived light except monitoring light, this detector also can be used for monitoring (tracking, perception, measurement etc.) other parameters, comprise that the existence that is not limited in temperature, pollutent, season are (as under the situation of the amount of not considering illumination and temperature, automatically be transformed into the heterotrophism training mode during autumn), the condition in one or two reactors of also can reflecting change (as when the heterotrophism reactor owing to keep in repair and failure and other reasons can not operate the time, the autotrophy reactor also will be closed).This ability of this detector can be monitored many useful parameters, and these parameters include in the present invention.In addition, can realize the automatic conversion between the autotrophy heterotrophism training mode, can utilize computer system record input parameter, provide corresponding output data then, for example parameter information or the instruction between the execution training mode that the operator sends, changed.In addition, these detectors and controller can be used for the fine-tune reactor, for example monitor and inform the situation (as the seed production status) in user's heterotrophism reactor, so that regulate the level of growth in the autotrophy reactor, vice versa.In other words, the adjusting of activity level is not disposable in two reactors that carry out according to transmitter, but by manually or automatically regulating gradually.In fact, when adopting multiple reactor, what detectors controller can detect in preset time be exercisable, and they are with which kind of mode operation (autotrophy or heterotrophism).For example, in summer, the partial function of heterotrophism process (30%) is used for seed culture, and most of function (70%) is used for the algae bio amount and produces; When taking place to pollute, the algae bio amount is produced will be converted to seed production.In the winter time, autotrophy production can stop fully, and seed production part will be converted to the algae bio amount and produce.This operation strategy will make equipment be used more effectively, and guarantee the supply whole year of algae bio amount.Describe among this aspect content of the present invention such as Fig. 1, wherein detector 10 is dynamically connected certainly with controller 20.
In addition, in some instances, heterotrophism is cultivated and also be can be used for biomass and lipid accumulation.For example, in order to utilize detector to greatest extent, heterotrophism reactor or heterotrophism are cultivated and are not used further to seed production (or from beginning just to be not used in seed production), but are used for the accumulation of biomass and lipid, for example, heterotrophism described herein/autotrophy process parallel running pattern.In addition, if produced the enough algae seeds level or the quantity of the needs of the system that can satisfy particular season (as reach), the heterotrophism reactor can then be used for the accumulation of biomass and lipid, simultaneously, the autotrophy process is also in the accumulation of carrying out biomass and lipid.
Example
Example 1. is given the schema of fixed system
Fig. 1 has gathered the culturing process that heterotrophism and autotrophy combine, and comprising by the production unit of algae to biofuel, table 1 has been listed key equipment and pipeline.
Equipment among table 1 Fig. 1 and pipeline tabulation
Figure BPA00001299999000121
Figure BPA00001299999000131
Fig. 2 has described the mass balance figure of each technological process of the laboratory of this integrating process that produces 1,000 ten thousand gallons of biofuel and known output and pilot plant level.By mass balance figure as can be known, when producing the 10MMg/yr biofuel by this technological process, the input of the organic carbon that all required nutrition and inorganic carbon can be satisfied the demand substantially.The further organic waste in hydrolysis reactor, handled of analysis revealed or directly utilize raw glycerine can satisfy 1/5 organic carbon input.Extra input part with hydrolysis reactor and anaeroic digestor form can comprise tipping fees by additional income, and electric power and value-added product remedy.
Example 2. heterotrophism are cultivated
Various organic wastes can be used as the raw material of heterotrophism culturing process.Through pre-treatment in various degree, can obtain to contain the product of oleosacchara, short chain fatty acid and/or glycerine etc.Heterotrophic fermentation can utilize these carbon sources to carry out in the large scale fermentation jar, and pH, dissolved oxygen and Controllable Temperature in this fermentor tank are so that provide the growth of cell and obtain the optimal conditions of maximum cell density, and these are that the technological operation personnel are known.Most of carbon and a part of nitrogen, phosphorus are consumed in this process, yet a certain amount of COD, nitrogen and phosphorus also will remain in the effluent liquid.Fortunately, effluent liquid will can be used as the nutrition source of autotrophy algae culture, thereby can reduce cost, the recycling that the increase system drops into.By that analogy, the effluent liquid of final autotrophy culturing process will have the level of lower COD, nitrogen and phosphorus, and this is because the autotrophy algae can utilize and absorb these materials, therefore, has improved the water quality in downstream.Detailed relevant information is provided in this example: (1) algae and class algae biology have the ability of utilizing organic waste in growth and heterotrophism seed cell culturing process; (2) organic waste of various carbon containings, nitrogen and phosphorus can get in the whole integrated system.
Utilize the cultivation of the saccharomyces olei that municipal solid food waste material carries out.As shown in Figure 3, five kinds of saccharomyces oleis are used to the production (E2 among Fig. 1) that heterotrophism is cultivated Unicell Oils and Fats.This process utilizes organic waste as raw material, and wherein food waste is exactly an exemplary of this organic waste.The food waste hydrolyzed solution is used as basic medium (culture medium A), and substratum B adds 5g/L peptone and 5g/L yeast extract paste on this basis, and culture medium C adds 10g/L glucose again on the basis of B.The substratum of water preparation is formed 5g/L 5g/L peptone, 5g/L yeast extract paste and 20g/L glucose with comparing.Every kind of saccharomyces olei is cultivated with these four kinds of substratum, observes their growth potentials in food waste hydrolyzed solution substratum then.Only use the food waste hydrolyzed solution as substratum, saccharomyces olei Rhodotorulaglutinis biomass is 10g/L, the biomass of Cryptococcus curvatus and Yarrowia lipolytica is all compared according to high (Fig. 3), this shows selected saccharomyces olei, and algae and class algae biology have the ability that the discarded organic resource of very strong utilization is carried out the cell high-density growth.
Utilize the cultivation of the saccharomyces olei that municipal wastewater carries out.Data show, if enough carbon and nitrogenous sources are provided, selected these three kinds of saccharomyces oleis (also may comprise other algae and class algae biology) all can utilize the good growth of municipal wastewater, and (＞15g/L) (Fig. 4), this shows that municipal wastewater external carbon and nitrogenous source can be used as the substratum of the potentialization of yeast, algae and class algae biological growth.
The two step saccharomyces olei culture process that utilize food waste and municipal wastewater to carry out.Table 2 has provided the main component of used food waste hydrolyzed solution thing in this cultivation.
Table 2 food waste hydrolyzate moiety
Figure BPA00001299999000141
The food waste hydrolyzate mixes the substratum as saccharomyces olei with municipal wastewater.The cultivation of fs continues 6 days, produces most of nutritive substance and saccharomyces olei organism in this process.After this stage, the 90% yeast bio body that produces is collected, and 10% is left in the nutrition source that is used for further growth and follow-up cultivation in the fermented liquid.More biomass obtains in subordinate phase is cultivated, and subordinate phase is lower than fs efficient, but the concentration of COD, nitrogen and phosphorus is reduced to very low level.The fermented liquid of this process is used to the autotrophy culturing process of C.sorokiniana then because in the fermented liquid still higher nitrogen and the phosphorus of oil-containing can be used for algae grows.This process production autotrophy algae bio amount, the concentration of COD, nitrogen and phosphorus are reduced to and can be discharged in the environment and not cause the level (table 3) of pollution.
The two stage yeast culture processes that table 3 utilizes food waste and municipal wastewater to carry out
Figure BPA00001299999000151
The C.sorokiniana heterotrophism seed cell that utilizes food waste and municipal wastewater to carry out is cultivated.Food waste and waste water are used as the substratum of the seed cell of C.sorokiniana.Cultivate through 6 days heterotrophism, the cell concn of C.sorokiniana can reach 353 and 366 * 10 6Individual cell/ml, nutritive substance is consumed (table 4) in this process.
The C.sorokiniana heterotrophism seed cell that table 4 utilizes food waste and waste water to carry out is cultivated
Figure BPA00001299999000152
Then, the seed cell cultivated of heterotrophism and substratum are inoculated in the autotrophy substratum with 1% and 0.1% inoculum size.Cultivate through 7 days autotrophys, obtain the algae bio amount (table 5) of 0.16g/L and 0.15g/L respectively.
The C.sorokiniana autotrophy seed culture that table 5 utilizes food waste to carry out
Figure BPA00001299999000161
The growth kinetics of C.sorokiniana as shown in Figure 5.Data show and have only C.sorokiniana that (but double nutrition is cultivated one of biological representative) can effectively utilize food waste and carry out the production of heterotrophism seed, and these seed cells also can proceed nutrition and transform, and effectively growth in the autotrophy process.
Example 3. can be applicable to other algae bacterial classifications of this process
Except the Chlorella sorokiniana in example 2,4 and 5, much little phycomycete kind of other kinds also can be cultivated under heterotrophism and autotrophy condition, and can be used in example 1 and 2 processes of describing.Although this phycomycete kind testing data of cultivating by this process slightly is not provided here, this slightly algae can be used as the production bacterial classification because they have the ability of autotrophy heterotrophic growth.
The algae that table 6 can be grown under heterotrophism and/or autotrophy condition
Figure BPA00001299999000162
Figure BPA00001299999000171
The growth velocity of example 4. heterotrophism and autotrophy seed culture relatively
A key of the present invention is exactly to enter the ability that the seed in the open autotrophy cultivation pool cultivates by the heterotrophism process obviously to be better than the autotrophy culturing process.C.sorokiniana (UTEX 1602) as one of canonical biometric that is used for this process, is inoculated in 250ml shaking in the bottle of Kuhl substratum is housed with identical inoculum size, carry out autotrophy and heterotrophism then respectively and cultivate (table 7).
Table 7Kuhl substratum is formed
Figure BPA00001299999000172
The heterotrophism seed culture is under 27 ℃ in dark carries out, and the autotrophy seed culture is at continuously logical CO 2Situation under under 27 ℃ of illumination, carry out.Cultivate after 6 days, growth velocity more as shown in Figure 6.The doubling time of autotrophy seed culture is to increase by 1 times every day, and the heterotrophism seed culture is to increase by 2 times every day.This result shows that the efficient of heterotrophism seed culture process is 2 times of autotrophy process.The advantage that this heterotrophism is cultivated only is enhanced (strengthening owing to ventilating and stirring its ability) when autotrophy is amplified scale (illumination restriction) and used the large scale fermentation jar.
Example 5. pollutes control by heterotrophism seed culture (laboratory culture)
Cultivate for extensive little algae, it is low that cost is cultivated in open pond, but be difficult to accomplish single algae culture of planting usually.Some assorted bacterium such as other algae, bacterium etc. may enter open cultivation pool with low concentration growth but can consume nutritive substance and other resources with little algae competition, thereby cause pollution, and are final to destroy single algae culture systems than the growth velocity of tool advantage.A kind ofly control open culture systems and pollute, keep the method for specific algae kind growth vigor can quicken the growth of algae kind, and just can collect algae kind cell during to certain density at other varied bacteria growings.In some instances, early begin to enter exponential phase of growth, when other assorted bacterium reach harmful cell density, enter stationary phase sooner then thereby the inoculum size that increases the algae kind can shorten lag phase.In these examples, the heterotrophism culture system of foregoing description can provide the seed of enough high-cell density to reach the purpose that control is polluted.Degree and the density thereof of test design in order to measure the heterotrophism seed culture, the big I of suitable cell effectively prevents pollution.
The algae kind that C.sorokiniana (CS) (UTEX 1602) cultivates as desire, northwest Pacific primary algae kind EG-1-2 and E.coli are used as pollution microbes.CS is inoculated into identical shaking with EG-1-2 and carries out the autotrophy cultivation in the bottle.The inoculum size of EG-1-2 is 1 * 10 6Individual cell/ml, and the inoculum size of change CS.The culture experiment design is identical therewith altogether with E.coli for CS.The result shown in Fig. 7-8, by among the figure as can be known, when the inoculum size of CS only is 0.1 * 10 6During individual cell/ml, the growth of EG-1-2 takes advantage, yet when the CS inoculum size increased, EG-1-2 lost growth vigor gradually.These results show the pollution that can control other assorted bacterium by the inoculum size that increases desire cultivation algae kind, particularly utilize heterotrophism seed increasing inoculum size more to help the control of polluting.It is because the algae seed liquor that heterotrophism is cultivated can be brought certain organic carbon into when being inoculated into open pond that bacterium E.coli is introduced into as assorted bacterium.As shown in Figure 8, when the CS inoculum size only be 0.1 * 10 6During individual cell/ml (organic carbon of bringing into is minimum), a little less than the E.coli growth.Along with the increase of CS inoculum size, the cell density of E.coli also increases thereupon, yet E.coli can only keep growth at first day, and after this, its cell density is reduced to a very low level.In addition, no matter inoculum size is much, E.coli does not have tangible negative interaction to the growth of CS.These results stand good for the heterotrophism seed culture strategy of big inoculum size and foregoing description.
The growth velocity and the productivity of example 6. integrated systems
The seed cell that is used for extensive little algae cultivation is most important for the successful cultivation of this system.A major objective of integrated system is to reduce the production cost of relevant individual system in the method for another system by the product application that utilizes a refuse and a system.In the present invention, utilize the organic waste of other system to carry out the cultivation of seed heterotrophism, not only can strengthen inoculum size and also can strengthen contamination resistance, but importantly keeping to reduce running cost under the finished product output situation suitable with productivity.Fig. 9 has described the seed of cultivating by heterotrophism or autophyting growth and has been inoculated in the situation of cultivating in the open pond of 50L and the comparison of growth velocity.
Example 7. little algae mixotrophisms are cultivated and the cost of independent autotrophy culture systems compares
Computer simulation can help industrial analysis to theoretical yield and actual output, training measures, collection effciency, extraction efficiency, nutritive substance recovery, segmentation measure, cost etc. to a great extent.This example has carried out economic evaluation to two kinds of culture systems (mixed culture and independent autotrophy are cultivated) on the data in literature basis of existing relevant devices cost, process cost, productivity.Its calculating is to have 24 open ponds at one (area in each open pond is 80,000m 2, the degree of depth is 0.15m) practice in factory carry out.Figure 10 has provided final analysis, and as seen from the figure, Comparatively speaking, although the equipment input of these two systems and other indirectly attributable expenses are variant slightly, actual overall cost of ownership is suitable.
Though the example that the description among the present invention has the document support to provide, those skilled in the art can make amendment on the basis of claims.
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Claims (22)

1. method of producing the biofuel raw material may further comprise the steps:
Utilize heterotrophism cultivation algae or class phycomycete kind to be used for seeding, but described algae or class phycomycete kind have the physiological mechanism of autotrophy and heterotrophic growth; Then
Utilization is cultivated the seed that obtains through above-mentioned heterotrophism and is carried out the autotrophy cultivation to accumulate the biomass of described algae or class phycomycete kind.
2. the method for claim 1, wherein said biomass comprises lipid.
3. the method for claim 1, wherein said heterotrophism is cultivated and is comprised and utilize discarded starting material as organic carbon source, but autotrophy and heterotrophism are cultivated described algae or class phycomycete kind with autotrophy and heterotrophic growth physiological mechanism in opaque fermentor tank or bio-reactor.
4. method as claimed in claim 3, wherein said discarded starting material are selected from waste water and waste solid material.
5. method as claimed in claim 4, wherein said waste water and waste solid derive from agricultural wastes, industry paper pulp, the organic composition of municipal garbage and the residual nuclear of little algae organism and any other contain the organic waste of the available organic carbon of microorganism.
6. the method for claim 1, concrete steps comprise that also the carbonic acid gas that produces in the described heterotrophism culturing process of input is to described autotrophy culturing step.
7. the method for claim 1, described algae or class phycomycete kind derive from Chlorella sp., Chlamydomonas sp. and Scenedesmus sp.
8. the method for claim 1 is 10 from the seed cell concentration of described heterotrophism culturing process 7～10 9Individual cell/ml or bigger.
9. the method for claim 1, concrete steps comprise that adopting described algae or class phycomycete kind to carry out heterotrophism cultivates with the accumulation biomass.
10. method of producing biofuel specifically may further comprise the steps:
Utilize heterotrophism cultivation algae or class phycomycete kind with seeding, but described algae or class phycomycete kind have the physiological mechanism of autotrophy and heterotrophic growth; Then
Utilization is cultivated the seed that obtains through above-mentioned heterotrophism and is carried out the autotrophy cultivation to accumulate the biomass of described algae or class phycomycete kind; Then
From algae or class phycomycete kind organism, reclaim grease as biofuel.
11. method as claimed in claim 10, wherein, but the heterotrophism culturing step comprises and utilizes waste material to cultivate described algae and class phycomycete kind with autotrophy and heterotrophic growth physiological mechanism as organic carbon source in dark fermentor tank or bio-reactor.
12. method as claimed in claim 11, wherein said scrap feed material come from waste water and waste solid material.
13. method as claimed in claim 12, wherein said waste water and waste solid material come from the organic composition and the residual nuclear of little algae organism of agricultural wastes, industrial paper pulp, municipal garbage.
14. method as claimed in claim 10, concrete steps comprise that the carbonic acid gas that produces in the described heterotrophism culturing process of input is to described autotrophy culturing step.
15. method as claimed in claim 10, described algae or class phycomycete kind derive from Chlorella sp., Chlamydomonas sp. and Scenedesmus sp.
16. method as claimed in claim 10 wherein, is 10 from the seed cell concentration of described heterotrophism culturing process 7～10 9Individual cell/ml or bigger.
17. a system that produces the biofuel raw material comprises:
At least one equipment that is used for heterotrophism cultivation algae or class phycomycete kind is used for seed production, but wherein said algae or class phycomycete kind have the physiological mechanism of autotrophy and heterotrophic growth; Then
At least one open pond or reactor are used for autotrophy and cultivate the heterotrophism seed accumulating the biomass of described algae or class phycomycete kind, and wherein said at least one open pond or reactor are used for receiving and come from algae or the class phycomycete kind seed that described heterotrophism culture device is cultivated.
18. system as claimed in claim 17 comprises that specifically at least one is used for heterotrophism and cultivates the biomass accumulation that the equipment of algae or class phycomycete kind is used for these bacterial classifications.
19. system as claimed in claim 17, wherein said biomass comprises grease.
20. system as claimed in claim 17 comprises that specifically one or more detectors are to provide relevant growth conditions data output in described at least one open pond or the reactor.
21. system as claimed in claim 20 comprises that specifically one or more controllers are in order to cut out automatically according to the described output that is provided by detector or to start operation in described at least one open pond or the reactor.
22. system as claimed in claim 20, wherein said growth conditions comprises the existence of the quantity of illumination, temperature and pollutent.
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