[0001] This application claims priority from U.S. Provisional Patent Application No. 61/440,686, filed February 8, 201 1 , U.S. Provisional Patent Application No. 61/442,261 , filed February 13, 2011 , and U.S. Provisional Patent Application No. 61/532,745, filed September 9, 2011.

TECHNICAL FIELD

[0002] The disclosed embodiments of the subject application are in the field of algal biomass, fermentable sugars, and biofuel production.

BACKGROUND

[0003] Biofuels will play an increasing role in the United States energy market as energy prices increase, political will to establish national energy independence will intensify and apprehension about climate change will continue to grow. The price of petroleum has fluctuated dramatically, reaching record highs of more than US$140 per barrel in 2008. In part, those price increases reflected economic, political, and supply chain uncertainties. Political concerns about the availability of petroleum supplies have led to the realization that the United States' energy independence is of critical strategic importance, both economically and militarily. The release of C0 ₂ from fossil fuel combustion may also substantially contribute to global warming and climate change and efforts are intensifying to develop biofuels to reduce this release. The United States has responded by issuing a renewable fuel standard update (RPS2) that encourages a shift to more advanced biofuels in the market. Additionally, many states have responded by enacting their own renewable portfolio standards mandating electricity providers to obtain a certain percentage of their power from renewable energy sources. As a result of these concerns and RPS requirements, domestically produced biofuels have become an increasingly attractive alternative to foreign fossil fuels. [0004] Microalgae are some of the most productive and therefore desirable sources of biofuels. The Department of Energy (DOE) has determined that biofuel yield per acre from microalgal culture exceeds that of many organisms including land crops. Between the late 1970s and 1990s, the DOE's National Renewable Energy Laboratory (NREL) evaluated the economic feasibility of producing biofuels from a variety of aquatic and terrestrial photosynthetic organisms. Biofuel production from microalgae was determined to have the greatest yield per acre potential of any of the organisms screened. Microalgal biofuel production was estimated to be 8 to 24 fold greater than the best terrestrial biofuel production systems. Current estimates of the potential productivity for algal biofuel production range from 2,000 to 10,000 gallons/acre. According to the DOE, microalgae yield "30 times more energy per acre than land crops such as soybeans." Although existing technologies are promising, there is still a need for systems and methods that create even greater efficiencies in biofuel production from microalgae to meet economic targets needed for successful commercialization.

[0005] The use of microalgae in heterotrophic conditions for the production of oil has been commercialized for use as a nutraceutical. Many algae have the capability for heterotrophic growth and can be utilized in multiple trophic modes for production of useful products. Algae are known to be able to grow using photoautotrophic, mixotrophic, and heterotrophic modes. Another mode, referred to as photoheterotrophic has also been elucidated but has strong similarities to mixotrophic growth. Purely heterotrophic growth has been documented as useful for production of lipids for biofuel production. Mixotrophic growth has been used for production of Chlorella for the nutraceutical market in large open ponds and photobioreactors supplemented with carbon source to enhance growth in the presence of light.

DEFINITIONS

[0006] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary embodiments, suitable methods and materials are described below. In case of conflict, the subject specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0007] "Microalgae" have been variously defined through the ages and it is prudent to describe the microalgae to which this invention could apply. For the purposes of this patent, microalgae include the traditional groups of algae described in Van Den Hoek et al. (Please add full cite to article). This invention can be used in the photosynthetic, heterotrophic, and mixotrophic culturing of microalgae.

[0008] "Heterotrophic boost" and "Hetero boost™" refer to the process wherein a large inoculum of existing biomass is placed into a bioreactor and fed a fixed carbon source to produce rapidly a specific bioproduct or set of bioproducts. The term is used broadly enough to refer to the inoculum originating from biomass produced by any phototrophic organism grown either phototrophically or mixotrophically. Additionally, the term refers to the production of any bioproduct produced in this method.

[0009] "Bioreactor" or "fermentor" are used to refer to the same device herein. The use of fermentor is a term of art that generally does not limit the ensuing bioreaction contained therein to a fermentative (i.e., anaerobic) process; this application also makes this distinction as the bioreactors and fermentors herein are used primarily for aerobic processes.

"Heterotrophic" means growing on fixed organic carbon without need for light.

[0010] "Photoheterotrophic" means growing on fixed organic carbon but requiring, or being advantageous, to have some light present as a trigger or photochemical leaver for improved heterotrophic growth.

[0011] "Phototrophic" and "photoautotrophic" are used interchangeably and refer to growth on simple medium without the use of fixed organic carbon, wherein all carbon is supplied by inorganic carbon (e.g., carbon dioxide, bicarbonate, or carbonate).

[0012] "Mixotrophic" growth is growth in the presence of a fixed carbon source in the light, with inorganic carbon also present, wherein improved productivity is achieved due to the presence of the fixed organic carbon.

[0013] "Lean biomass" is used to describe algal biomass produced with a minimum level of lipid.

[0014] "Lipid extracted algae" or "LEA" is used to describe the residual biomass left after the lipid is extracted from the algae. This is the same as delipidated biomass.

[0015] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the application as a whole. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably. Furthermore, as used in the description of the invention and the appended claims, the singular forms "a", "an", and "the" are inclusive of their plural forms, unless contraindicated by the context surrounding such. The singular "alga" is likewise intended to be inclusive of the plural "algae."

SUMMARY

[0016] Exemplary embodiments of the compositions, systems, and methods disclosed herein improve the process of producing biofuels from microalgae.

[0017] In one embodiment of the present application involves method for production of a bioproduct wherein the organism is first grown in mass culture at low density, the organism is harvested non-destructively, the inoculum is introduced to the bioreactor at high density and the bioreactor produces the bioproduct at high productivity using a fixed carbon source.

[0018] In another embodiment, the organism in the above embodiment is grown in mass culture in the light. This light can be provided either by natural or artificial lighting. Such mass culturing is suitably done fully phototrophically or mixotrophically. The mixotrophy can be done in the full lighted pond acreage or ponds can be combined in a deeper pond and treated mixotrophically before being harvested.

[0019] In another embodiment of the subject application, the organism is suitably a microalga. This microalga can be a green alga. Additionally, it can be selected from strains of Chlorella and, more specifically, can be C lorella kessleri, C. sorokiniana, C. vulgaris, and C. zofingiensis.

[0020] In another embodiment, the photosynthetic organisms are suitably chosen from any algae, cyanobacteria, or photosynthetic bacteria capable of combined trophic states selected from phototrophic, mixotrophic, photoheterotrophic and, required, heterotrophic boost.

[0021] In another embodiment of the subject application, the organism producing the bioproduct suitably uses simple or complex sugars. These sugars can be selected from monosaccharides (such as glucose), polysaccharides (such as polyxylose, starch) and complex mixtures such as crude sugars and biomass hydrolysates.

[0022] In another embodiment of the subject application, the heterotrophic boost will generate bioproducts such as, but not limited to, lipids, polymeric compounds, pigments, nutraceuticals, secondary metabolites, and other high value compounds.

[0023] In another embodiment of the subject application, the production organism is engineered to have improved utilization of sugars common to biomass hydrolysates. Such sugars are galactose, arabinose, xylose, mannose, and polymers of the same. Such recombinant organisms can have introduction of genes selected from D-xylose isomerase, D-xylose kinase, transaldolase, transketolase, and mannose isomerase either expressed constitutively or inducible in the heterotrophic boost bioreactor.

[0024] In another embodiment, genes are introduced into the production organism under tight inducible control such that enzymes are induced in the heterotrophic boost bioreactor that breakdown the cell wall releasing additional sugars to enhance lipid production and improve lipid harvesting.

[0025] In another embodiment, the heterotrophic boost reaction is exposed to metabolic inhibitors subject in the biomass hydrolysate that are either added as the biomass hydrolysates are added or are added as partially purified or purified chemicals to shift the metabolism to favor bioproduct production rather than growth.

[0026] Another embodiment of the subject application uses a recombinant organism that is induced in the heterotrophic boost to express a thioesterase or series thereof, that induces production of hydrocarbons specific to that esterase such as terpenes and alkanes.

[0027] Another embodiment utilizes the heterotrophic boost under the previous embodiments but as a secondary production organism for a co-fermentation, wherein the secondary organism produces more or an additional useful product, but removes sugars that are not used by the primary production strain. This reduces the viscosity of the growth medium while improving the overall yields of the process.

[0028] Yet another embodiment is a heterotrophic boost process wherein the bioreactor is provided small amounts of light to act in a stimulatory method, defined as photoheterotrophic, where additional metabolism is possible using light controlled genes that are not available without this small amount of light.

[0029] Another embodiment uses cultures produced either phototrophical!y or mixotrophically which are harvested non-destructively and cryopreserved for future use as inoculum for the heterotrophic boost bioreaction. Such a system would disconnect inoculum production from the heterotrophic boost to allow all season production of the bioproduct at latitudes where winter production is not possible.

[0030] Another embodiment is a method for production of algal biomass in which an algal culture is fed a fixed carbon source in a single addition step and all of the added fixed carbon is consumed rapidly and predominantly by the algal strain of interest to maximize algal production. The algal culture can be between 0.01 and 1 g dry weight per liter of medium with the fixed carbon added at between 0.05 and 3 grams per liter of medium.

[0031] In a further embodiment, the fixed carbon source is suitably added at the beginning of the dark cycle and thereby offsets the removal of photosynthesis to either equal or exceed the use of energy by the algae in the dark cycle (thereby either preventing biomass decrease at night or adding additional biomass).

[0032] In a further embodiment, the algal culture is suitably fed a fixed carbon source under mixotrophic, photoheterotrophic, or heterotrophic growth conditions.

[0033] Another embodiment is a method for production of algal biomass wherein an algal culture is fed a fixed carbon source in a continuous or semi-continuous fashion, such that all of the fixed carbon is consumed rapidly and predominantly by the algal strain of interest to maximize algal production. [0034] Another embodiment is a method for production of algal biomass wherein an algal culture is fed a complex carbohydrate mixture in the presence of enzymes which release sugars that can be directly utilized by the alga for growth or product synthesis. The complex carbohydrate is suitably a liquefied starch or plant hydrolysate. Suitable plants include, but are not limited to, cassava, corn, sweetpotatoes, soybean, sunflower, rapeseed, or algae. Suitable enzymes suitably include, but are not limited to, alpha-amylase, glucoamylase, and granular starch hydrolyzing enzyme.

[0035] In a further embodiment, the algal culture is grown in an enclosed bioreactor with or without light provided. It can further be carried out in open systems, such as open ponds or raceways.

[0036] In a further embodiment, the algae biomass or plant biomass can be first extracted prior to treatment with the enzymes, for example delipidated algal biomass or soybean meal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] A better understanding of the exemplary embodiments of the subject application will be had when reference is made to the accompanying drawings, and wherein:

[0038] Figure 1 is a diagram illustrating a system for the production of biofuel using mixotrophic growth according to one embodiment of the subject application.

[0039] Figure 2 is a diagram illustrating a system for the production of biofuel using phototrophic growth according to one embodiment of the subject application.

[0040] Figure 3 is a diagram illustrating a bioreactor used in the production of bioproduct according to one embodiment of the subject application.

[0041] Figure 4 is a graph illustrating the three phases of growth in an algal fed batch fermentation.

[0042] Figure 5 is a diagram illustrating the catabolic pathways for xylose and mannose.

[0043] Figure 6 is a diagram illustrating a mixotrophic to heterotrophic growth plan according to one embodiment of the subject application.

[0044] Figure 7 is a graph illustrating a preliminary growth profile for heterotrophic boost batch run through the ponds (photosynthetically grown) and Heteroboost™ (heterotrophically grown) according to one embodiment of the subject application.

[0045] Figure 8 is a diagram illustrating a batch night supplement feed operation for a mixotrophic pond according to one embodiment of the subject application.

[0046] Figure 9 is a graph illustrating glucose supplemented Chlorella protothecoides batch cultures in 5.2 L aquaria with a daily dilution according to one embodiment of the subject application.

[0047] Figure 10 is a graph illustrating operation data from a 400L pond growing Chlorella protothecoides supplemented with glucose during dark cycles according to one embodiment of the subject application.

[0048] Figure 1 1 is a graph illustrating glucose uptake of dark cycle supplemented organic carbon supplemented Chlorella protothecoides culture according to one embodiment of the subject application.

[0049] Figure 12 is a diagram illustrating a continuous night supplement feed operation for a mixotrophic pond according to one embodiment of the subject application.

[0050] Figure 13 is a graph illustrating the effect of dark cycle organic carbon supplement on chlorophyll content of Chlorella protothecoides according to one embodiment of the subject application.

[0051] Figure 14 is a graph illustrating simultaneous saccharification and growth of algae according to one embodiment of the subject application.

DETAILED DESCRIPTION

[0052] With reference now to the drawings wherein the showings are for purposes of illustrating the example embodiments only and not for purposes of limiting same, using the variety of methods of the exemplary embodiments of the subject application, the example embodiments described herein are directed to methods for improved mixed trophic state algal cultures. The example embodiments are particularly applicable to improved methods for producing biofuels from microalgae.

[0053] The subject application is directed to methods for production of a bioproduct. The method comprises producing a biomass phototrophically or mixotrophically in a mass culture system, concentrated to an appropriate density without harming the cells. The biomass or inoculum is then harvested and placed in a bioreactor. The bioreactor is continuously fed a fixed carbon source to provide energy to the cells as they make the bioproduct in large excess. The C02 is removed while oxygen is pumped into the reaction, either from the atmosphere or in concentrated form.

[0054] The bioreactor is commonly referred to in the art as fermentation in a fermentor but is supplied with oxygen to maintain a respiratory rather than a fermentative (anaerobic) state. Herein a fermentor and bioreactor are referred to interchangeably and, unless otherwise stated, respiratory metabolic state. The addition of carbon source after phototrophic growth or mixotrophic growth, concentration, and placing in a fermentor is referred to as a heterotrophic boost or Heteroboost, herein used interchangeably.

[0055] Referring now to Figure 1 , there is shown a diagram illustrating a process for biofuel production using mixotrophic growth according to one embodiment of the subject application. As shown in Figure 1 , a mixotrophic pond 100 is used to produce algae. The mixotrophic pond suitably is at ambient temperature, has a pH ranging from approximately 5 to approximately 9, and has a depth ranging from 0.I m to 0.5 m.

[0056] Suitable nutrients, such as nitrogen, potassium, phosphorus, and C02 are fed into the pond at 102 via a suitable feed inlet. A suitable fixed carbon source feed, such as glucose, glycerol, or acetate, is also fed into the pond at 104 via a suitable feed inlet. A carbon source sensor 106 is employed for determining the appropriate supplementation of fixed carbon to be fed into the mixotrophic pond based on a desired set point. The sensor suitably monitors parameters such as culture density, temperature, dissolved oxygen and ambient light levels (surface and bottom) in order to maintain optimal rates of lean biomass generation. The fixed carbon concentration set point is suitably between 0 to 12 g of carbon per liter. Clarified water is also feed into the mixotrophic pond at 108 via a suitable feed inlet. The algae is produced in the mixotrophic pond to a set concentration, preferably between 0.1 to 3 g/L. The algae is harvested continuously from the mixotrophic pond as shown at 1 10 via a suitable outlet and subjected to a dewatering process 1 12 to concentrate the algae to an appropriate density without harming the algae.

[0057] The concentrated algae is fed into heteroboost bioreactor 1 16 as shown at 1 14 via a suitable feed inlet. A suitable fixed carbon source is fed into the heteroboost bioreactor as shown at 118 via a suitable feed inlet as well as desired amounts of nutrients, antifoam materials, and pH control solution as shown at 120 via a suitable feed inlet. Living delipidated algae may also suitably be fed into the heteroboost bioreactor as shown at 122 via a suitable feed inlet.

[0058] The harvested algae fed into the heteroboost bioreactor produces a bioproduct, high lipid algae, under suitable conditions in the bioreactor. Preferably, the bioreactor maintains a temperature ranging from about 22° C to about 32° C, a pH ranging from about 5 to about 9, and a biomass concentration ranging from about 20 to about 200 g/L.

[0059] The high lipid algae is output from the heteroboost bioreactor as shown by 124 via a suitable outlet and subjected to a suitable non-destructive extraction process 126. The non-destructive extraction process produces live delipdated algae, which may be fed back into the heteroboost reactor as shown at 122, and algae oil as shown at 128. The high lipid algae may suitably also be subjected to a suitable aqueous extraction as shown at 130. The aqueous extraction process is in the presence of suitable extraction chemicals fed into the extraction process as shown at 132. The aqueous extraction process produces algae oil as shown at 128.

[0060] Referring now to Figure 2, there is shown a diagram illustrating a process for biofuel production using phototrophic growth according to one embodiment of the subject application. As shown in Figure 2, a phototrophic shallow pond 200 is used to produce algae. The phototrophic pond suitably is at ambient temperature, has a pH ranging from approximately 5 to approximately 9, and has a depth ranging from 0.I m to 0.3 m.

[0061] Suitable nutrients, such as nitrogen, potassium, and phosphorus, and C02 are fed into the pond at 202 via a suitable feed inlet. Clarified water is also feed into the phototrophic pond at 204 via a suitable feed inlet. The algae is produced in the phototrophic pond to a set concentration, preferably between 0.1 to 3 g/L. The algae is harvested continuously from the phototrophic pond as shown at 206 via a suitable outlet and subjected to a dewatering process 208 to concentrate the algae to an appropriate density without harming the algae.

[0062] The concentrated algae is fed into the heteroboost bioreactor 212 as shown at 210 via a suitable feed inlet. A suitable fixed carbon source is fed into the heteroboost bioreactor as shown at 214 via a suitable feed inlet as well as desired amounts of nutrients, antifoam materials, and pH control solution as shown at 216 via a suitable feed inlet. Living delipidated algae may also suitably be fed into the heteroboost bioreactor as shown at 218 via a suitable feed inlet.

[0063] The harvested algae fed into the heteroboost bioreactor produces a bioproduct, high lipid algae, under suitable conditions in the bioreactor. Preferably, the bioreactor maintains a temperature ranging from about 22° C to about 32° C, a pH ranging from about 5 to about 9, and a biomass concentration ranging from about 20 to about 200 g/L.

[0064] The high lipid algae is output from the heteroboost bioreactor as shown by 220 via a suitable outlet and subjected to a suitable non-destructive extraction process 222. The non-destructive extraction process produces live delipdated algae, which may be fed back into the heteroboost reactor as shown at 218, and algae oil as shown at 224. The high lipid algae may suitably also be subjected to a suitable aqueous extraction as shown at 226. The aqueous extraction process is in the presence of suitable extraction chemicals fed into the extraction process as shown at 228. The aqueous extraction process produces algae oil as shown at 224.

[0065] Figure 3 is a diagram illustrating an example heteroboost bioreactor 300 according to one embodiment of the subject application. The bioreactor is of a typical bubble column reactor design. The bioreactor 300 is generally in the shape of an elongated column having a top 302 and a bottom 304, and having a height of approximately 30 feet and a width of approximately 5 feet. The bioreactor is supported by a skirt design support mechanism at the bottom of the bioreactor.

[0066] The bioreactor suitably includes a cooling jacket 306 comprised of components having suitable cooling properties and surrounding a least a portion of the bioreactor.

[0067] The bioreactor includes a mount 308 for mounting a mixing apparatus for mixing the constituents contained within the reactor. The mount is located at the top of the bioreactor and is preferably 8 inches in diameter.

[0068] The bioreactor also includes an algae feed 310 for receiving the algae from the pond. The algae feed 310 is located near the top of the bioreactor and is preferably 2 inches in diameter.

[0069] The bioreactor further includes an off gas port 312 for releasing gas, such as C02, produced in the bioreactor. The off gas port 312 is located near the top of the bioreactor and is preferably 4 inches in diameter.

[0070] An algae outlet port 314 is located at the bottom of the bioreactor for the output of the algae produced in the bioreactor. Preferably, the algae outlet port has a diameter of 4 inches.

[0071] An air feed inlet 316 is also located at the bottom of the bioreactor for introducing air into the bioreactor. The air feed inlet 316 is preferably 2 inches in diameter.

[0072] The cooling jacket 306 includes a cooling inlet feed 318 for introducing a cooling material into the cooling jacket and a cooling outlet feed 320 for removing the cooling material from the cooling jacket. Preferably, the cooling inlet feed 318 has a diameter of 2 inches and the cooling outlet feed 320 has a diameter of 2 inches.

[0073] A manway 322 is located on one side the bioreactor near the top of the bioreactor for allowing access into the bioreactor. Preferably, the manway 322 has a diameter of 24 inches.

[0074] A glucose feed inlet 324 is located on the side of the bioreactor near the top of the bioreactor for feeding glucose into the bioreactor. The glucose feed inlet 324 preferably has a diameter of 2 inches.

[0075] The bioreactor also includes several glass viewing ports 326 down the side of the bioreactor for viewing the reaction inside the bioreactor. Preferably, the glass viewing ports having a diameter of 6 inches. [0076] An antifoam feed inlet 328 is located near the top of the bioreactor for introducing antifoam material into the bioreactor. Preferably, the antifoam feed inlet is 2 inches in diameter.

[0077] A pH control feed inlet 330 is located near the top of the bioreactor for introducing a pH control material or base into the bioreactor. Preferably, the pH control feed inlet has a diameter of 2 inches.

[0078] High cell density inoculum combined with proper feeding control resulted in significantly improved volumetric productivity and yield of product from glucose when compared to conventional heterotrophic fermentation. Other carbon sources than glucose have been tried at bench scale and appear to provide similar results. The use of crude hydrolysates with a predominance of monosaccharides will provide similar heterotrophic boost results while providing additional economic value in the overall process through lower cost feedstock.

[0079] High cell density inoculum creates an environment that retards contaminating microbial growth preventing costly run shortages due to contamination. High concentrations of phototrophically grown algal biomass, as inoculum obtained from open ponds, performs well in this heterotrophic boost system. Moreover, this phenomenon is more evident with Chlorella protothecoides than with other algae strains. Mixotrophically grown high cell density inoculum will also create an environment that will retard microbial growth to act similarly to that described above for purely phototrophically grown biomass.

[0080] High Quality triacylglyceride containing oil is extracted from high density heteroboosted biomass generated from phototrophically grown inoculum. Control of nutrient feed and other reactor environmental parameters in the heterotrophic boost reactor enables the reduction of bacterial contamination (on a per weight basis) during the production of oil free of impurities that could impact quality.

[0081] Starting with high cell density inocula from phototrophic or mixotrophic algal cultures and using a growth rate-controlling nutrient feed in the heterotrophic boost bioreactor results in lower energy requirements for production of the resulting oil or other bioproduct.

[0082] Using a draw and fill process extended runs resulted in improved

volumetric productivity and yield of product from glucose. This same draw and fill process will provide similar results for other carbon sources.

[0083] The heterotrophic boost process changes the economics for oil production, significantly improving it when compared to conventional heterotrophic fermentation. Heterotrophic boost uses pond grown biomass as the starting inoculum providing a 57% improvement in the oil yield from glucose and a 35% increase in volumetric productivity over conventional algal fermentation process using the same strain.

[0084] Key parameters of impact affecting this heterotrophic boost process are summarized in Error! Reference source not found.. In general, this subject application is focused on improving process conditions to result in high volumetric productivity and maximum yield of lipid from carbon in the hydrolysates, while keeping energy consumption low. Tables 1 and 2 outline cost impact factors and general strategies planned to achieve cost targets.

[0085] The most significant technical barrier is adjusting to the components of the hydrolysates versus purified sugars. The non-fermentable solids will have a negative impact on mass transfer and could impact the downstream process; however, there are a number of process solutions that include controlled glucose feed rate, high overall dilution rate, draw and fill process, and shorter fermentation runs to overcome insufficient sugar quality. [0086] A key economic advantage of the heterotrophic boost process is illustrated in Figure 4. In a typical algal fed batch fermentation, maximum specific growth (logarithmic growth = (μ) = ln(X2/X2) ^» (t2-t1)-1 +D) occurs for the first 75-80 hours and the volumetric oil production rate is very low. When culture growth slows, shifting from logarithmic to linear growth (linear growthl = (X2-X2) ^« (t2-t1 )-1 +D)) an increase in volumetric productivity (Qp rate = g oil ^« (L ^« hr)-1) occurs. This causes a metabolic overflow where intracellular pools of key intermediates are at a maximum resulting in an increase in the fatty acid production rate. Fatty acids are the most energy dense hydrocarbon cellular storage products which is illustrated in Figure 4. Importantly, the heterotrophic boost process is carried out at high cell density using pond grown algal

Table 2 - Substrates tested for growth and lipid

production in shake flasks experiments with

Chlorella protothecoides KRT1007

biomass that does not reach a logarithmic growth rate, resulting in higher cumulative volumetric oil productivities. Oil content surpasses 35% dry cell weight in the heterotrophic boost process approximately 100 hours before a standard fed batch algal fermentation. In Figure 5, the change in dry cell weight is represented by the square shapes and the change in intracellular oil is represented by the diamond shapes.

[0087] In addition to this growth related economic improvement, there are other specific process advantages demonstrated when using the heterotrophic boost process. The percentage of oil in biomass increases from approximately 5% in the inoculum to an average level of 68% dry cell weight at the end of a heterotrophic boost run. This high oil is predominantly non-polar lipid, free of contaminating chlorophyll and polar fatty acids, making oil recovery more efficient. Data supports that simplified extraction is needed, which precludes the need and costs associated with additional oil refining.

[0088] Because of the starting high cell densities in the heterotrophic boost process, the culture

[0089] undergoes a metabolic shift to linear growth conditions that favors improved oil yield and productivity. Figure 4 illustrates the impact of growth rate on productivity when the fermentation culture shifts from log phase growth to linear growth. The heterotrophic boost process starts at high cell densities (targeting 50 g/L) eliminating the exponential growth phase and yielding much higher cumulative volumetric productivity.

[0090] Because of the high starting cell concentration in heterotrophic boost, the algal biomass to microbial contamination ratio is low creating an environment that suppresses contaminating microbial growth for certain strains.

Xylose and Mannose Utilitization

[0091] Xylose and Mannose utilization will be improved based on incorporating genes known to be involved in these pathways from other organisms that are absent in C. protothecoides genome. Xylose and mannose were targeted, being the most abundant hemicellulosic sugars in softwood hydrolysates. When tested in purified form, both xylose and mannose induced some lipid production in shake flasks, although the increase in biomass was negligible and lipid accumulation was less than 10% of that found with glucose. A list of purified sugars used to test substrates for growth and oil production in C. protothecoides is shown in Table 2. Substrate utilization can be improved using identified genes and pathways involved in substrate utilization. Two pathways for each sugar substrate are described in Figure 5. In the case of xylose and mannose utilization, a gene or genes necessary to complete or complement lesions in existing pathways are being introduced. A single gene or multiple genes will be able to complement these lesions. Based on genomic analyses of the existing Chlorella genomes, the fermentation of D-xylose requires coordinate expression of up to four enzymes: D-xylose Isomerase (XI), D- xylulokinase (XK), transaldolase (TAL), and transketolase (TKT). One or more of these genes will be expressed using a native Chlorella promoter active under the heterotrophic boost process.

[0092] For D-mannose, simple expression of the mannose isomerase (Ml) gene to convert D-mannose into D-fructose, which has already been demonstrated as an efficient substrate for producing lipid in C. protothecoides will be sufficient to improve this trait. This will also be sufficient in other microalgal strains such as C. kessleri, C. sorokiniana, C. zofingiensis, Nannochloropsis, C. vulgaris, Haemotococcus, and other microalgal production strains.

[0093] Substrate transport for both xylose and mannose in Chlorella is well characterized and paralogus transporters will be sufficient for xylose and mannose utilization.

[0094] Expression of mannose isomerase, xylose isomerase and xylose kinase will be sufficient to enable their utilization and improve the use of mannose and xylose, respectively in engineered strains.

[0095] DNA sequences are optimized to match the codon preferences of C. protothecoides or other target strain. Regulatory elements (5' and 3' UTRs) are tested to optimize expression and best activity is used for the production strain. Both constitutively expressed and heterotrophic growth specific gene promoters are suitable. Increases in productivity (growth rate and lipid yield) of 5-20 % are provided with this approach. With optimization up to 50% improvement will be provided by the subject method. Delivery of strains that suitably efficiently utilize xylose and mannose, the most abundant hemicellulosic sugars from softwood biomass, will enable utilization of a wider range of sugar sources and introduce new opportunities for locating the algal lipid production operations.

[0096] Engineering the production of a chitinase or other cell wall degrading enzymes (cellulases and hemicellulases, endoglucanases, lysozyme, and etc) at the final stages of heterotrophic growth will release additional monosaccharides for additional lipid production. Additional benefit from degrading the cell wall late in heterotrophic boost lipid production will be that the cells will be easier to break for lipid extraction.

Growth Inhibitors

[0097] A hurdle for the fermentation of crude biomass hydrolysates is the presence of growth inhibitors in the feedstocks. Acetate, furan, furfural, and 5 hydroxy-methyl furfural are all toxic compounds found in biomass hydrolysates. These compounds inhibit fermentation by traditional organisms; however their impact on green algae and other microalgal bioproduct production strains is unknown except for acetate. Acetate is a viable substrate for heterotrophic growth and lipid production in Chlorella and other microalgae (e.g., Chlamydomonas). On a per carbon basis, acetate performs as well as glucose both in yield and response time. Inhibitors to ethanolic fermentations impact heterotrophic boost production of lipid but will have a positive impact on the algal bioproduct being produced in the heterotrophic boost and also inhibit contaminating organisms. Incorporation of these crude hydrolysates and/or inhibitors extracted from crude hydrolysates provides a process improvement to the heterotrophic boost process. Inhibitors reduce or inhibit biomass production, but not oil or other bioproduct accumulation (other bioproducts could include pigments, secondary metabolites, nutraceuticals and pharmaceuticals). In an ideal production scenario, lean biomass growth would be limited to 10% with the remainder of the biomass increase coming from lipid production. Previous studies with E. coli indicate that the strong electrophiles (furfural and 5-MHF) inhibit sulfur assimilation and pyruvate dehydrogenase. This was originally identified through whole genome transcript analysis, wherein an increase in transcript abundance for genes related to cysteine and methionine biosynthesis was identified. Alterations in metabolic or proteomic profiles help identify alternative sites of inhibition. Strategies to reduce negative impacts and maximize benefits of these potential growth inhibitors will be deployed and range from cultures adapted to continuous feed with inhibitor containing hydrolysates, treatment with enzymes (laccase, cellulase, lysozyme, and the like), inclusion of microorganisms that specifically detoxify the inhibitor, to more refined engineered strategies including the repression of NADPH dependent furfural oxidoreductases, or by introducing the NADPH/NADH transhydrogenase (pntAB) to reduce the abundance of the cofactor. [0098] Maximal lipid accumulation (or other bioproduct accumulation) and minimal non-lipid biomass accumulation during the heterotrophic boost phase is achieved. This provides a significant advantage over traditional fermentations where cultures must be maintained in logarithmic growth for high productivity. As such, the impact of growth inhibitors may be reduced or even provide a benefit to the heterotrophic boost process.

Alkane production in heterotrophic algal bioreactor

[0099] Genes from a number of plant thioesterases have been identified that are responsible for the production of terpenes, hydrocarbons, and alkanes in plants These genes have been isolated and provide an opportunity for production of hydrocarbons directly in the algal cells from a heterotrophic boost run. Algae is suitably modified by introduction of these genes in the chloroplast or nucleus of the algal cell using microbiolistic or glass bead transformation methods. Control of the expression of these genes is required using inducible promoters that can be switch on in the fermentative (bioreactor) step. Such metabolic control of the algal production strain would turn on production of the alkanes in the heterotrophic boost bioreactor and divert production from lipid to alkanes. The hydrocarbons would be excreted in the growth medium of the bioreactor and harvested in a non-invasive method, such as by skimming of the top of the culture or by capture using resins or hydrophobic membranes. Use of inducible promoters to turn on lethal, or inappropriate for the pond, genes in the heterotrophic boost bioreactor allows expression of genes that improve processing of the biomass or crude sugar (amylases, cellulases, pectinases, chitinases, and etc) in situ.

Mixotrophic to heterotrophic

[00100] A method is used to produce healthier cultures by using a small amount of fixed organic carbon in open pond culture to stimulate better growth. The open pond cultures can preferably remain very shallow to maximize light penetration and the addition of fixed organic carbon will act in either an additive or synergistic mode to improve the growth in these cultures. Since light only penetrates a few centimeters in a normal culture due to the phenomenon called self-shading (algal cells above preventing light from getting to cells below) the cells are often in a non-productive state due to lack of light. Having small amounts of fixed carbon allows the cells to remain active while out of maximal light. Additionally, even at very low levels (submillimolar) these fixed carbon compounds have a beneficial effect on the cells, adding additional growth and mobilizing metals and minerals required for growth. A diagram of mixotrophic to heterotrophic growth plan is shown in Figure 6. Here cells are added to shallow ponds to stimulate enhanced growth of the culture as discussed above with respect to Figure 1 . The culture is grown to a fairly low concentration (< 1-3 g/L) then transferred with concentration as shown at 602 to the bioreactor 600 where purely heterotrophic growth is initiated. A continuous substrate feed is fed into the bioreactor as shown at 604 as is an oxygen supply as shown at 606. Carbon dioxide produced in the bioreactor is output back to the pond as shown at 608. A high oil algae is produced which is output from the bioreactor as shown at 610. The use of mixotrophic or photoheterotrophic growth in the earlier stage poises this culture for better heterotrophic boost and production of lipid.

Transgenic improvements to production methods

[00101] There are numerous transgenic improvements that may be incorporated into the production methods set forth in the subject application. Examples of transgenic improvements are discussed below, wherein it is understood that such examples are not an exhaustive list, and other transgenic improvements may be incorporated into the production methods set forth in the subject application.

[00102] The use of siRNA in the medium (addition to the culture when concentrated and in the bioreactor) will turn on or reduce expression of targeted genes. Extracellular siRNAs (or functionally analogous pieces of nucleic acids e.g. , hairpins, short RNA tandems, PNA/RNA duplexes and etc) are allowed to permeabilize the cell membranes (such as, microwave power or rapid mixing with impellers to make the cell membranes more porous to the siRNA). The heterotrophic lipid production step is suitably performed under acidic conditions as this improves substrate transport (through hexose/H+ symporters) and inhibit growth of contaminating species. Increase NADPH pool volume and flux to improve availability for lipid production as described in a recent metabolomics report of heterotrophic chlorella growth), and the pentose phosphate pathway is enhanced due to the limitation of available NADPH (Xiong, 2010). When engineering metabolic capability, select strategies that improve NADPH availability.

[00103] Production strains are suitably engineered with genes conferring efficient xylose (xylose isomerase and xylulose kinase), mannose (mannose isomerase) utilization. Use multiple strains in Heteroboost to enable co-utilization of multiple substrates. For example, one strain will use glucose, one will use mannose (glucose deficient), and one will use xylose (glucose deficient). A glucose deficient strain that can use mannose or xylose is suitably engineered by conditionally repressing expression of glucose 6 phosphate isomerase or mutagenize to reduce activity. Alternatively, a strain is suitably engineered to suppress/alter/utilize the glucose suppression mechanism for alternate substrates. (In yeast engineered xylose utilization is still subject to glucose suppression.)

[00104] Utilize Heterotrophic growth specific promoter and terminator sequences for expression of xylose and mannose related genes.

[00105] Utilize growth inhibitors found in lignocellulosic hydrolysates to prevent growth and fermentation of contaminating organisms.

[00106] Utilize growth inhibitors found in lignocellulosic hydrolysates to prevent assimilation of carbon into non-lipid biomass.

[00107] Limit non-lipid biomass accumulation to <20% (ensures efficient Carbon conversion to lipid)

[00108] Prevent biosynthesis of amino acids or protein synthesis (e.g. addition of cycloheximide) to improve carbon assimilation into lipid.

[00109] Use a strain of algae with an engineered pyruvate dehydrogenase (PDH is a target of furfural and 5-hydroxymethyl furfural inhibition) that has enhanced resistance to growth inhibitors from lignocellulosic hydrolysates. The PDH from Galdieria sulphuraria or from other extremophiles may be more resistant to these compounds as they are likely to be present in its natural environment of high temp and low pH.

[00110] Induce radical scavenging proteins (SOD, peroxidase) to alleviate potential oxidative stress.

[00111] Engineer proteins involved in xylose or mannose utilization such they will bind to a scaffolding protein. This will improve flux of metabolites through the pathway.

[00112] Repression of hydrogenase during Heteroboost will prevent the waste of energy thus improving energy storage in the form of lipid (Hydrogenase activity may be enhanced when utilizing lignocellulosic hydrolysates (sulfur assimilation inhibited condition)

[00113] Use genetic switches (tightly controlled gene expression) to regulate expression of genes necessary only under the heterotrophic growth condition to enable more efficient growth under photoheterotrophic conditions. (The reverse is also of merit, i.e. tight regulation of genes required during photoautotrophic growth that are not needed in heterotrophic boost) As mentioned, use of hormone-like molecules that may activate regulatory mechanisms (induction or repression).

[00114] Use magnetic separation and algal strains genetically engineered to respond to magnetic fields through over-expression of iron transporters and iron storage protein complexes (Fea1 and Fer1 from Chlamydomonas reinhardtii respectively) to enable an inexpensive and selective approach to dewatering biomass from photoautotrophic growth for use as seed culture for the heterotrophic process.

[00115] Use magnetic hysteresis and algal strains genetically engineered to respond to magnetic fields through over-expression of iron transporters and iron storage protein complexes (i.e. Fea1 and Fer1 from Chlamydomonas reinhardtii respectively) to enable efficient magnetically induced cell lysis after heterotrophic lipid production.

[00116] Increase oil production in the Heteroboost step by engineering hydrolytic enzymes to digest and degrade algal cell wall polysaccharide components into sugars that can be metabolized into lipid. Transformation constructs capable of expressing a transgene expressing an active hydrolytic enzyme is made, such as lysozyme, chitinase, glucanase, cellulase, hemicellulase, pectinase, or glucuronidase that is able to digest and degrade the polysaccharide components of the algal cell wall into simple sugars. The promoter and other regulatory elements used to drive expression of the transgene will enable controlled expression in transgenic algae via a small molecule inducing agent. This promoter and other regulatory elements will prevent expression unless in the presence of the small molecule inducing agent. The inducing agent is suitably introduced at a late stage of the Heteroboost phase, a point at which the great majority of the lipid has already been produced by the algal cells. Upon inducing expression of the transgene, the hydrolytic enzyme will be synthesized and secreted into the periplasmic space where it can begin to hydrolyze cell wall components. Progressive digestion of cell walls will liberate sugars that will be available for uptake by cells that are still intact, providing more carbon substrate for the production of additional lipid. Addition of the inducing agent is suitably done at a rate that allows optimal production of additional lipid from the newly available sugar substrates generated from the cell walls. This method is suitably used in Chlorella protothecoides, other Chlorella species, Nannochloropsis species, Ostreococcus species, Ankistrodesmus species, Dunaliella species, Haematococcus species, Neochloris species, Phaeodactylum species, Scenedesmus species, Spirulina species, Tetraselmis species and other microalgae.

[00117] Increase oil production in the HeteroBoost step by engineering inhibition of starch synthesis genes via physical addition of inhibitory RNA (RNAi) species. A method that introduces synthetic, specific inhibitory RNA species into the concentrated algal cell biomass during the HeteroBoost is suitably employed. The target of these RNAi species will be endogenous messenger RNA species required for expression of enzymes required for starch synthesis (i.e. starch synthetase, starch branching enzymes, amylopectin synthase, glucanotransferase, isoamylase, 3-phosphoglycerate, ADP-Glucose Pyrophosphorylase, etc.). The effect of inhibiting starch synthesis will be to generate additional oil from carbon that would have gone into starch biosynthesis. RNAi will be synthesized in vitro and introduced to cells during the HeteroBoost® phase. A partial cell disruption technique is suitably employed to facilitate entry of RNAi species into cells where they can have the inhibitory effect on gene expression. The following cell disruption techniques are suitably used independently and/or in combinations: ultrasonication, microwave radiation, rapid mixing with impellers and electroporation. This method will be used in Chlorella protothecoides, other Chlorella species, Nannochloropsis species, Ostreococcus species, Ankistrodesmus species, Dunaliella species, Haematococcus species, Neochloris species, Phaeodactylum species, Scenedesmus species, Spirulina species, Tetraselmis species and other microalgae.

Concentration methods for phototrophic algae as inoculum for heterotrophic fermentation reactors

[00118] Cells grown under phototrophic conditions slow or stop growth at relatively low cell concentrations due to cell-cell signaling and light limitation. Heterotrophic cells are able to maintain growth under significantly higher cell concentrations due to the volumetric nature of the growth reaction on fixed carbon. By concentrating phototrophic or photo-heterotrophic or mixotrophic cells as an inoculum for heterotrophy, therefore, one can limit the number and size of heterotrophic reactor vessels required, the time that the algae must spend in the reactors, as well as the amount of nutrients/sugar necessary for the cells to grow from low to high concentration. Various methods of concentrating algae exist and are in use today, primarily in the field of wastewater treatment. Using any of these methods to prepare algae for heterotrophic growth requires that the method meet the following criteria: insignificant damage to cells, preserved cell viability and optimal or near optimal performance under heterotrophic conditions; and ability to re-use clarified water for algae growth (either as a single unit process or series of processes). This is a critical requirement for a successful and economic production facility due to the high cost of the nutrients and the large water demand that algae projects naturally have. Options that meet these criteria include centrifugation, dissolved air floatation (DAF), autoflocculation, membrane filtration, rotary vacuum drum filtration (RVDF), and magnetic separation.

[00119] Bench scale data confirms the use of centrifugation as a concentration method for algae cells as an inoculum for a heterotrophic growth reactor vessel (0.2 g/L to 80 g/L). However, the high energy costs associated with running centrifuges and the likelihood that further processing will be required to prepare the clarified water for continued growth make centrifugation unlikely to succeed in a full scale oil production facility. [00120] Technologies like dissolved air floatation, autoflocculation, and magnetic separation may have similar issues with recycling water for phototrophic growth, and require significant additional research to prove the capable of cells concentrated by these methods to perform as expected under heterotrophic conditions. Each of these methods implements the strategy of forming clusters of cells to make them easier to separate from the surrounding water. Theory suggests that cells in clusters (formed either by charged inorganic particles, organic polymers, or [para]magnetic interactions) will have difficulty with mass transfer in a heterotrophic reactor vessel. This could significantly limit performance. However, each of these technologies has potential to be effective, as long as the coagulation/flocculation is reversible. Mechanical, chemical, and magnetic methods could be employed as ways to break the cell clusters, but further research must be conducted as to the efficacy of this concept and the productivity of cells concentrated by these methods compared to non-cluster-forming methods.

[00121] Rotary vacuum filtration experiments have shown that this method can be used to concentrate algae, but require the use of a filter aid. This could be prohibitive if the filter aid cannot be removed or used downstream of the phototrophic growth vessels. It may be possible to identify a filter aid that could be consumed by the algae in order to make this process a plausible method to concentrate cells for heterotrophic oil production.

[00122] Hollow fiber membranes, while high in CapEx and OpEx, are suitable for this application. This technology employs the concept of tangential flow filtration, where particles move tangentially across the membrane, which provides a physical barrier for the algae cells. Water is able to permeate across the membrane without requiring a large pressure force. Flux maintenance techniques can be employed to prevent fouling and maintain consistent flow across the membranes over time. It is currently used in the wastewater treatment, biotechnology, and pharmaceutical industries as a way to concentrate cells. Hollow fiber membrane filtration has the added benefit of removing the vast majority of the bacteria and other organisms that could be an issue in the clarified water stream. Empirical data has shown that this type of filtration is capable of concentrating phototrophic algae from 0.2 g/L to >50 g/L in a repeatable and predictable fashion. Economically, this process is less expensive compared to centrifugation due to lower energy costs and the secondary water treatment step required of a centrifugation dewatering to make the water reusable. Additionally, hollow fiber filtration could also be used to condition a medium prior to heterotrophic growth by means of diafiltration. Temperature and shear forces can also be carefully controlled using this type of filtration to ensure cell viability.

[00123] Microfiltration or ultrafiltration allow control of key parameters such as temperature, medium buffer, and optimal transfer cell density for heterotrophic performance.

[00124] The culture can be conditioned from a photosynthetic to heterotrophic metabolism while being concentrated resulting in improved productivity and yields of product from glucose. Microfiltration allows the control of both C02 and dissolved oxygen preventing lags in performance at transfer.

[00125] Nutrients from the filtered pond broth can be recycled back to the ponds free of microbial contaminants. Pond broth can be concentrated in stages slowing growth and conditioning the medium for increased fatty acid productivity before being transferred to Heteroboost.

Secondary fermentation for improvement of the utilization of crude sugars from biomass hvdrolysates

[00126] The heterotrophic boost as described in the subject application provides for the production of a bioproduct in a bioreactor using a large inoculum from either a mixotrophic or phototrophic production of biomass. The use of these crude sugars presents difficulties in that not all production organisms can utilize polymeric sugars, and monomeic sugar use is also dependent on the organism. For instance, the current C. protothecoides strain KRT1007 is not capable of use of polymeric sugars and some of the sugars from cellulose and hemicellulose degradation. In this embodiment of the heterotrophic boost process, the fermentor is allowed to progress until the viscosity of the liquid increases to a level that inhibits mass flow to an economically detrimental level. This level will vary with the bioproduct being produced. That is, if the bioproduct is a commodity one can calculate using a cost model, the level of air that can economically be pushed through the bioreactor and when this level is exceeded this secondary step would be applied. The secondary treatment to the heterotrophic boost is to add native or recombinant oleaginous yeast or fungal strains that use the sugars and produce oil that can be either coextracted with the algal or separated and extracted separately. Such an approach allows one to more efficiently use the sugars provided by the biomass hydrolysates or crude sugar preparations lowering the cost of the overall process. Additionally, this could tailor the lipid profile generated by the process to more closely fit that needed for further refining.

[00127] In one embodiment, the algal heterotrophic boost is run to high viscosity, and a culture of recombinant yeast is added that uses xylose and polymeric xylose that is not used by C. protothecoides KRT1007. This yeast rapidly multiplies over a 24 hour period and consumes the xylose and lowers the viscosity of the culture medium. The algae continues to grow and use glucose. The yeast also uses glucose as well as xylose. Both produce additional lipid and are harvested using the same procedures and processed in batch.

[00128] In another embodiment, the algae are harvested when the viscosity of the medium reaches too high of a level. The remaining culture medium is retained and transferred to a new bioreactor and the yeast added to this medium. The yeast converts the remaining sugars into biomass and additional lipid for extraction in a separate process than the algal lipids. This step can also be used to make alternative bioproducts using yeast that are engineered to produce the specified product, but use these alternative sugars for growth.

[00129] In another embodiment, a filamentous fungus is used for the processes described above (either cofermentation or separate fermentation). The fungus can produce any of a number of bioproducts including oil, pigments, nutraceuticals, pharmaceuticals and the like. The filamentous fungi used in a cofermentation will also serve to simplify the dewatering of the culture medium by providing a mat and collection surface, as has been seen with other algal systems using filamentous cyanobacterial.

Photoheterotrophic growth within reactors [00130] The use of a dim light source (<20 umol/m ² /s; incandescent, fluorescent, or LED) in fermentation reactors provides significant advantages to the bioreaction beyond photosynthetic biomass generation. Light photons allows (triggers) use of certain lignocellulosic carbon sources, including xylose, arabinose and other C5s, by some algae species. Advantage is that C5 sugar feedstocks are available at lower comparative cost as compared to C6s, do not compete with food supply, and reduces overall cost to produce useful biomass and lipids for bioenergy products. Similarly C5 sugars could also be used as a supplement for daytime mixotrophic growth, generating biomass and lipids in open ponds.

Extension of operations to lower light environments

[00131] Operation for generating lipids from algae in higher latitudes locations, or where only seasonal growth is availability, or where only periods of growth available or where availability of fermentors is periodic. Operation grows oleaginous algal strains phototrophically in open ponds and bioreactors during ambient or available periods (spring, summer, fall), with or without carbon supplementation. Biomass is harvested, concentrating biomass to a desired target (10-750 g/L) with centrifuge or microfiltration or some other device known to the art, then freezing biomass. Stockpiled biomass concentrate could involve use of cryoprotectants, such as glycerol or glucose (ex 0.1 to 200 g/L), or none at all. The use of sugars and sugar alcohols would have the benefit of future use as a fixed carbon source for organisms like C. protothecoides that can use glucose or glycerol to produce lipid or other bioproducts. Freezing is done via flash freezing (e.g., plunge liquid nitrogen baths), or gradual freezing in a chilling device. Necessary target temperatures for stable long-term storage (weeks to months) are comparatively high (4 to -20°C) thus reducing electrical costs associated with long-term storage and could use existing large-scale freezer technologies. Algae frozen without cryoprotectants are viable in high numbers by rapid or gentle thawing. Frozen material would be stored in vessels (barrels, tubs) or stacked as frozen bricks sealed in thin plastic sheeting. Frozen cell mass can be inoculated as a thawed concentrate into heterotrophic reactor with supplemented carbon and aeration of a fermentor to generate lipids. Cryoprotectants present in frozen biomass, such as glycerol or glucose, are used as carbon source in future processing. The advantage is that phototrophic growth and heterotrophic growth are disconnected in time, and can be run in separate campaigns rather than continuously. This allows plant construction (downstream processing) to be reduced in size and full year operation of the said plant at higher latitudes where year round algal biomass growth is not possible economically. Similarly, seed materials for fermentation can be stocked stable for long-term storage until available resources or desired inoculum volumes are present to run a fermentation cycle.

Raceway multi-trophic state operation

[00132] Such operation utilizes existing paddlewheel driven raceways (10 to 100 cm deep) and temporarily converts them into fermentation reactors to generate algal biomass and lipid in situ, or alternatively switch reactor gradually over to full heterotrophy. Once ponds achieve biomass >0.8 g/L, parasitic drawdown of supplemented fixed carbon by other microorganisms (e.g., fungi, bacteria) is controllable. This involves as an alternative mode, periodic use of various industrial- scale antibiotics. For rapid switch-over, temporary modification to raceways would involve switching gas feed line from C02 to air to augment dissolved oxygen levels in the medium. Aeration could also be augmented with alternative mixing technologies like those employed by aeration beds in wastewater treatment plants. Operations benefit by beginning at the end of a light period when oxygen levels would be maximal. Raceways would be shaded with light blocking cloth, and surface covered with hollow dark colored floating plastic (e.g., polypropylene, polyethylene) spheres (1-10 cm in diameter) for fully heterotrophic growth. Clear colored spheres and/or sheeting is used when mixotrophic growth is more preferred, depending on the production strain and bioproduct to be produced. Spheres darken growth medium, as well as limit atmospheric release of oxygen, maintaining higher aeration levels in medium driven by submerged injected gas feeds and paddlewheel forward motion. Spheres do not interfere with paddlewheel operation, since they are carried over wheel without entrapment. Similarly, if needed, surface ponds are mixed more vigorously by temporarily placing submerged gas injector pumps, and extra gas injection locations. Supplementation with fixed carbon source and augmented oxygenation is monitored in situ to maintain optimal heterotrophic fermentation conditions, while sampling of lipid levels would determine harvest cycle. A gradual switch from phototrophic to heterotrophic strategy in PBRs involves a gradual increase in supplementation feed of fixed carbon (e.g., 0.1 to 1 , 1 to 10, 10 to 20 g/L glucose) as illustrated in Figure 7. The incremental increases in supplementation are determined by pigmentation changes associated with carbon supplementation. Incremental increases in carbon supplementation also support mixotrophic condition that would generate biomass much quicker than pure phototrophy, as well as a phototrophy to heterotrophy. Gradual loss of chlorophyll would also necessitate the increase in shading, or alternatively, increasing volume/depth of the raceway to prevent unwanted bleaching/stress of cells by natural sunlight. At a certain point, the raceway would be converted to a pure heterotrophic reactor, as described above. The principal advantage of both these strategies is the re-use of capitally expensive open phototrophic reactors as a lower cost fermentor. Further, less dewatering and pumping of medium is needed, and hold-up time would be effectively removed. Clean-out of photo/hetero raceways would be less frequent, further reducing productivity unit down-times.

Batch addition of fixed carbon, harvest and dilution

[00133] Open algae cultures operated with carbon supplementation need to compete with pond contaminants such as bacteria and fungi. One strategy favoring algae utilization is to batch supplement ponds with fixed carbon combined with a daily harvest and dilution as illustrated in Figures 8-10. Figure 8 is a diagram illustrating a batch night feed supplement operation. As shown in Figure 8, a mixotrophic pond 100 is used to produce algae. The mixotrophic pond suitably is at ambient temperature, has a pH ranging from approximately 5 to approximately 9, and has a depth ranging from 0.I m to 0.5 m.

[00134] Suitable nutrients, such as nitrogen, potassium, and phosphorus, and recycled medium are fed into the pond at 802. A suitable fixed carbon source feed, such as glucose, glycerol, or acetate, is also fed into the pond at 804 in one or more batches. C02 and/or air is fed into the pond at 806. The algae is produced in the mixotrophic pond to a set concentration, preferably between 0.1 to 3 g/L. The algae is harvested continuously during phototrophic growth as shown at 810 and in batches after carbon growth as shown at 812.

[00135] Figure 9 illustrates glucose supplemented Chorella protothecoides batch cultures in 5.2 liters aquaria with a daily dilution. Glucose was supplemented at the beginning of the dark cycle. Cultures were then sampled and diluted to 0.3 gdw/L at the end of the dark cycle. The glucose was provided at either 0.375g/L as represented by the triangles or at 0.5 g/L as represented by the X symbol. Figure 10 illustrates operation data from a 400 L pond growing Chorella protothecoides supplemented with glucose during dark cycles. The pond was cut at the start of each light cycle with a biomass concentration set point of 0.3 gdw/L. Glucose was added in batch doses of 0.5 g/L concentrations and C02 was sparged into the pond only during light cycles.

[00136] By supplementing at a single time point at typical algae culture concentrations (0.01 to 1 g/L), carbon supplements (0.05 to 3 g/L) are rapidly removed from the medium (<12 hrs) by the predominant algae minimizing contaminant utilization and growth as illustrated by Figure 1 1. By understanding the supplement to biomass yield values as well as the supplement uptake rate of the algae, a strategy can be devised to add only enough fixed carbon to generate the desired biomass in the desired amount of time. Multiple batch doses could also be envisioned, to control the concentration of supplement of carbon below a set point. Altering the type of carbon supplements fed to ponds could also reduce competitive microorganisms, many of which are inflexible to feed changes. Further benefits of a batch supplementation includes the ability to complete a batch harvest, or to semi- continuously harvest a portion of culture once carbon supplement levels in the pond medium are low or absent thereby minimizing waste.

[00137] In this embodiment, fixed carbon supplemented open ponds are operated as a continuous turbidostat with a maximal biomass density set point designed to maintain an appreciable penetration of light into pond. This is achieved by daily batch or continuous dilution with fresh medium and supplemented with fixed carbon as prior additions are completely utilized by the algae. Harvested biomass and spent medium would be replaced and re-grown for the next harvest interval. The advantages of such a system include a superior lean biomass accumulation rate than that which can be accomplished in photoautotrophic growing conditions (Figure 9). Diurnal batch supplementation may be applied prior to nightfall to maintain or to grow algal biomass overnight, thereby gaining higher yields and making better use of capital pond infrastructure. This strategy or combinations of these could also be applied to periods when weather or light conditions are non-optimal for photoautotrophic growth, as occurs daily (e.g., early morning, late afternoon), inclement weather (e.g., cloudy days), or seasonally (e.g., late fall, winter, early spring) in mid to high latitudes. Ponds operated in this fashion would be partially dewatered with a concentrator and sent to a fermentor where purely heterotrophic growth is initiated.

Continuous supplementation of fixed carbon to open ponds operated as a turbidostat

[00138] Another configuration is to supplement open ponds with variable levels of fixed carbon, as illustrated in Figure 12. As shown in Figure 12, a mixotrophic pond 1200 is used to produce algae. The mixotrophic pond suitably is at ambient temperature, has a pH ranging from approximately 5 to approximately 9, and has a depth ranging from 0.I m to 0.5 m.

[00139] Suitable nutrients, such as nitrogen, potassium, and phosphorus, and recycled medium are fed into the pond at 1202. A suitable fixed carbon source feed, such as glucose, glycerol, or acetate, is also fed into the pond at 1204. A carbon source sensor 1206 is employed for determining the appropriate supplementation of fixed carbon to be fed into the mixotrophic pond based on a desired set point. The fixed carbon concentration set point is suitably between 0 to 12 g of carbon per liter. C02 and/or air is fed into the pond at 1208.

[00140] The algae is produced in the mixotrophic pond to a set concentration, preferably between 0.1 to 3 g/L. The algae is harvested continuously from the mixotrophic pond as shown at 1210 while algae grows in light or on the carbon source.

[00141] Supplementation would be controlled by real-time sensors monitoring parameters such as culture density, temperature, dissolved oxygen and ambient light levels (surface and bottom) in order to maintain optimal rates of lean biomass generation. The type of fixed carbon fed to ponds could also be alternated in time to further reduce competitive microorganisms. With use of rapid dewatering technologies, such as microfiitration or centrifugation, permeate, or centrate, with unused fixed carbon from the medium would be continuously recycled back to the pond to support further growth. During return flows, the medium could be periodically or continuously treated by UV sterilization, subjected to mechanical filtration of debris and contaminants, and/or passed through activated carbon to remove growth inhibitors. Envisioned is a system where biomass is only partially, but rapidly (to reduce parasitic utilization), dewatered by a decentralized concentrating unit dedicated to each production pond. Partially dewatered biomass would be transferred to a centralized concentrator for more complete dewatering, medium recapture, and sent to a fermentor where purely heterotrophic growth is initiated. With the use of carbon and nitrogen supplementation, algae cultures can be manipulated to preferentially generate lean biomass densities (approximately 3 g/L) much higher than what can be achieved through purely photoautotrophic culture. Due to partial depigmentation of algae grown with carbon supplements, penetration of light through ponds is greater allowing use of deeper ponds (15-100 cm), further increasing area productivity of ponds as illustrated in Figure 13.

[00142] Advantages of this system include superior areal and volumetric productivity, growth rate and densities that would be able to maintain a more consistent source of biomass. Such a system would be relatively independent of diurnal, seasonal, and latitudinal light conditions. Other benefits include the ability to run dewatering systems continuously on high concentrations of algae culture, both reducing the capital costs needed for higher through-put dewatering systems, as well as reducing the overall pond areas needed to generate an equivalent biomass with pure photoautotrophy. Cost impacts from evaporation would also be mitigated by a higher volume to surface area ratio. The C02 generated by the heterotrophic and mixotrophic growth on the carbon supplement could also offset some of the need for C02 input during photoautotrophic growth. This reduces the total amount of C02 feed and any C02 purification costs. Similarly, photosynthetic phases of algae would supply some of the oxygen needs of heterotrophy and mixotrophy. However, levels of oxygen present at night would need to be maintained with air sparging, which could use the existing pond piping networks and injection points used to supply carbon dioxide during daylight. Simultaneous saccharification and fermentation (SSF) using starch and amylases for heterotrophic or mixotrophic generation of algae biomass and lipids

[00143] While some algae are capable of utilizing a variety of fixed carbon sources including glycerol, organic acids, and various sugars (C6 and C5), some are also capable of utilizing disaccharides and small oligosaccharides. Use of simple monomers, such as glucose, in industrially grown algae processes allows for the rapid generation of biomass and bio-compounds. However, competing organisms present in mixotrophic ponds or present as contaminates in fermentation vessels are often more effective at monomer utilization. This fact can reduce overall process yields or foul resulting bioproducts if not controlled with additions of antibiotics or other control technologies. One solution to this problem is through the use of a simultaneous saccharification and conversion (SSC) process. SSC has been widely adopted by the ethanol industry to increase overall yields of target compound, minimize parasitic loss of sugar feed, and reduce capital investment by combining process vessels. In this embodiment, algae would be grown in open ponds or fermentors in the presence of raw or liquefied polysaccharide (e.g., starch) as discussed above and an appropriate industrial grade hydrolytic enzyme (e.g. , for starch alpha-amylase, glucoamylase or granular starch hydrolyzing enzyme). Input rates of substrate and enzyme would be kept sufficient to catalyze the generation of smaller polysaccharides and glucose from added starch or other sugar polymer to support growth rates of algae biomass and associated bio-compounds. Benefits of this system would include a lower input feed cost (e.g., unrefined starch) as compared to more refined concentrate sources (e.g., glucose syrups; dextrose powders), superior long term storage stability, and lower associated costs to transport (i.e., syrups approximately 33% water).

[00144] The conversion of starch from cassava tuber or sweet potato uses raw starch hydrolyzing enzymes simultaneously inoculated with algae for a simultaneous saccharification and conversion (SSC) to lipid process. Use of an SSC process may present less stress to the algae, which may not be able to handle the osmotic stress of very concentrated sugar solutions. In addition, the overall level of sugar will be kept low, with monomeric sugar (e.g. , glucose) released by enzyme action being taken up by algae, reducing the chance for growth by contaminant bacteria. Feed solutions of potential sugars provided to the SSC process may need to undergo pre- hydrolysis to produce a less viscous solution for better mass transfer and higher overall titer of lipid from sugar.

Example conditions for hydrolysis comprise:

[00145] Conversion conditions recommended by Genencor for Stargen 002:

pH: 3.3 to 4.5

Dry Solids: 20 to 34% w/w

Pretreatment Hyd ration prior to SSC: recommended at least 48°C

Conversion time: 24 to 76 hours at 20 to 40°C

Particle size: 1.5 to 2.5mm screen hole diameter

[00146] Conversion conditions recommended for heterotrophic boost process: pH: 6.2 to 6.7

2 to 4% glucose concentration

Soluble sugars only

Conversion time: 24 to 76 hours at 28 ± 1 °C

[00147] Conversion conditions recommended for hybrid Simultaneous Starch Conversion (SSC)-heterotrophic boost process:

pH: 3.3 to 6.7, preferred pH 5.5

Dry Solids: 20 to34% w/w, preferred conditions <5%

Pretreatment/Hydration prior to SSC: recommended at least 48°C, higher for complete liquefaction of polysaccharides

2 to 4% glucose concentration, lower with combined process

Conversion time: 24 to 76 hours at 28°C

Particle size: 1.5 to 2.5 mm screen hole diameter

[00148] Experiments have been performed to date showing simultaneous saccharification and conversion of pure cassava starch with algae. Some resulting data are presented in Figure 14. The dark left hand most bar represents the use of glucose liberated from cassava starch. The light color second most left bar represents pure glucose control. The third bar from the left represents simultaneous saccharification of liquefied cassava starch and growth of algae. The bar on the right represents saccharification of liquefied cassava starch with no algae present.

H drolvzed lipid extracted algae (LEA) meal as fixed carbon feedstock for mixotrophic or heterotrophic biofeed

[00149] Costs associated with fixed carbon (e.g., glucose) used to generate algae bioproducts are a major factor affecting scaling of algae based biofuels. Algae are capable of utilizing a variety of fixed carbon sources including glycerol, organic acids, and various sugars (C6 and C5). Industrial sources of these compounds are largely fermentatively or agriculturally derived, traded actively on commodity exchange markets and, thus, often subject to abrupt and uncontrollable price changes due to market speculations. Although issues associated with diverting fixed carbon to biofuels, may affect food security, price instability is the predominate issue that makes scaled biofuel production particularly challenging. One significant by-product generated by algae based biofuel producers is the residual lipid extracted algae (LEA). After removing lipids, LEA is predominately composed of carbohydrates and, to a lesser extent, proteins and minerals. With minimal processing, LEA can be used as an internally generated feed source of fixed carbon used to supply or supplement existing fixed carbon feed sources to generate further algal biomass generated in open or closed bioreactors, either heterotrophically or mixotrophically. Methods available to transform LEA meal to a more bio-available sugar source include enzymatic digestion with amylases, or acid hydrolysis using liquids or solid acid (beads) catalysts. In this process, LEA meal, deproteinated LEA meal, or algae rich in starch, would be allowed to contact a catalyst (e.g., enzymes, liquid or solid acid), generating an algal hydrolysate. Upon neutralization, algae hydrolysate could be substituted directly as a fixed carbon feed used for generating further algae biomass, or to support yeast growth (e.g., ethanol processes), or bacteria (e.g., butanol, lipids) or other bio-based products. [00150] The foregoing description of a preferred embodiment of the subject application has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject application to the precise form disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiment was chosen and described to provide the best illustration of the principles of the subject application and its practical application, to thereby enable one of ordinary skill in the art to use the subject application in various embodiments and with various modifications, as are suited to the particular use contemplated. All such modifications and variations are within the scope of the subject application as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.