Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Claims 1-11 and 18-20 are pending. Claims 12-17 are cancelled.
Election/Restrictions
Applicant’s election of Group I, claims 1-11 and 18-20 in the reply filed on 1/23/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 1-11 and 18-20 are examined herein.
Specification
The disclosure is objected to because of the following informalities: in [0078], the top layer is referred to as 32, which is inconsistent with Fig. 1, which shows the top layer as 28.
Appropriate correction is required.
The use of the term sarkosyl in [0063] and [0064], which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology (N-lauroylsarcosine); furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Objections
Claims 1 and 18-19 are objected to because of the following informalities:
In claim 1, the word “phase” is on its own separate line. Phase should appear on the same line as decolorized biomass.
In claim 18, “catenoids” in line 10 is a misspelling of “carotenoids.” And “relatively hydrophobic ionic liquid-based solvent” is repeated in the Markush group for the second solvent.
In claim 19, the term “n- butanol” has an extra space.
Appropriate correction is required.
Claim Rejections – 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-11 and 18-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "the solubilized biomass" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 1 also recites “mixing the alcoholic solvent and supernatant phase.” There is an article missing before supernatant phase, so it is unclear whether “supernatant phase” is any supernatant phase or whether “supernatant phase” specifically refers back to the supernatant phase recited in line 3.
Claim 4 recites “re-exposing the solubilized biomass mixture to a protein-denaturing solution.” The combination of “re-exposing” with “a protein-denaturing solution” leads to ambiguity in the claim scope because it is unclear whether “a protein-denaturing solution” refers specifically to the protein-denaturing solution recited in line 2 of claim 1 or another protein-denaturing solution not recited in claim 1.
Claims 2-11 are rejected for depending from a rejected base claim and not rectifying the source of indefiniteness discussed above.
Claim 7 recites the limitation "the bottom layer" and “the top layer” in lines 2 and 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 7 is further indefinite for “transesterifying the middle phase with fatty esters.” Transesterification is the process of reacting triglycerides with an alcohol to produce fatty acid alkyl esters. However, the claim limitation can reasonably be interpreted as reacting the middle phase with fatty esters (as a reactant) rather than producing fatty esters as the product. Therefore, the claim has multiple reasonable interpretations that conflict with each other. Applicant may consider amending the claim to recite “transesterifying the middle phase to produce fatty esters.”
Claims 8-9 are rejected for depending from a rejected base claim and not rectifying the source of indefiniteness discussed above.
The term “substantially” in claim 8 is a relative term which renders the claim indefinite. The term “substantially” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Therefore the person of ordinary skill in the art would have been unable to determine the required purity of chlorophyll in the powder.
Claims 10 and 19 both recite “a PUTTS buffer.” The term PUTTS buffer is not taught by the prior art, nor is it explicitly defined in the specification, thus the metes and bounds of the claim are not defined. Applicant may obviate this rejection by amending claims 10 and 19 to recite the specific composition of a PUTTS buffer exemplified in the specification in [0063]: 8M urea, 1% v/v Triton-X, 0.2% w/v N-lauroylsarcosine, 100 mM NaH2PO4, and 10 mM Tris-HCI buffer at pH 7.5.
Claim 18 recites the limitation "the second solvent" in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 18 is indefinite for “missing the second solvent with the supernatant.” It is unclear whether “missing” is a typo for “mixing” or whether “missing” implies another action and if so, it is unclear what action is required.
The terms “relatively hydrophobic” and “relatively hydrophilic” in claim 18 are relative terms which render the claim indefinite. The terms “relatively hydrophobic” and “relatively hydrophilic” are not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Therefore the person of ordinary skill in the art would have been unable to determine the hydrophilicity or hydrophobicity of the first and second solvent.
Claim 18 is further indefinite for the modifier “immiscible” used for the first and second solvents (“immiscible relatively hydrophobic aqueous ionic liquid-based solvent,” “immiscible selectively hydrophobic deep eutectic solvent,” and immiscible relatively hydrophobic polar alcohol”). Immiscible refers to liquids not capable of forming a homogeneous mixture when added together. Here, it is unclear whether the requirement is that the first or second solvent is incapable of forming a mixture with the other solvent, with water or with another substance.
Claim 18 is further indefinite for “extracting chlorophyll, carotenoids, lipids and fatty acids, proteins, polysaccharides from the top, middle, and bottom layers.” There is no conjunction separating proteins from polysaccharides, so it is unclear whether these are alternatives, whether they are all required, and whether the list is complete. It is further unclear from which layer each of the components is extracted or whether the components may be extracted from any layer.
Claim 20 recites “wherein deep eutectic solvents are used to de-water and recover polar alcohol solvents.” It is unclear whether the method further comprises a step of de-watering and recovering any polar alcohol solvent, whether this limitation is requiring that the butanol (a polar alcohol) is de-watered, or whether the method further comprises de-watering the butanol with a deep eutectic solvent.
Claims 19-20 are rejected for depending from a rejected base claim and not rectifying the source of indefiniteness above.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-11 and 18-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 recites a chemical extraction method comprising hydrolyzing a decolorized biomass phase into a hydrolyzed product and fermenting the hydrolyzed product into an alcoholic solvent.
Claim 18 recites fermenting the hydrolyzed decolorized biomass but does not specify the resulting product of the fermentation.
The person of ordinary skill in the art would not have recognized that the inventors, at the time the application was filed, had possession of the claimed genus of fermentation conditions.
The specification discloses that the fermentation may be done by organisms such as Escherichia coli, Synechococcus elongatus PCC 7942 and Clostridium acetobutylicum ([0074]). No other information is provided within the specification regarding the fermentation conditions.
Huang et al. (Green Chemistry 22.1 (2020): 153-162) teaches extracting pigments from microalgal biomass using acetone, followed by enzymatic hydrolysis of the pigment-extracted biomass and fermentation to ethanol with recombinant Saccharomyces cerevisiae (Abstract). Huang engineers a cellulase/amylase-displayed S. cerevisiae and then initiates ethanol fermentation by adding the recombinant yeast strains to 20 mL of fermentation media containing microalgal residue in 100 mL closed flasks, equipped with a bubbling CO2 outlet under oxygen-limited conditions (page 156, left column, 2.7 Ethanol fermentation). The fermentation is carried out for 96 h at 37 °C with continuous agitation at 400 rpm (page 156, left column, 2.7 Ethanol fermentation). Huang’s microalgae is Chlamydomonas sp. JSC4 strain (page 154, right column, 2.1 Microalgal strain and growth conditions).
Gao et al. (Bioresource technology 206 (2016): 77-85) prepares ionic liquid-extracted or hexane-extracted algae for acetone, butanol, and ethanol fermentation by Clostridium saccharobutylicum DSM 13864 (Abstract and page 79, right column 2.2.1. Clostridium strain and cultivation conditions). The fermentation medium is 3 g L-1 yeast extract, 5 g L-1 peptone, 5 g L-1 soluble starch, 2 g L-1 ammonium acetate, 2 g L-1 NaCl, 3 g L-1 MgSO4·7H2O, 1 g L-1 of K2HPO4, 1 g L-1 of K2HPO4, and 0.1 g L-1 FeSO4·7H2O, and 80 g L-1 liquid-extracted algae (page 79, right column, 2.2.2 Batch fermentation). The ionic liquid-extracted algae is first boiled to remove dissolved oxygen and transferred into an anaerobic chamber (page 79, right column, 2.2.2 Batch fermentation). Gao’s microalgae is Chlorella vulgaris strain UTEX 2714 (page 78, right column, 2.1.1 Microalgae strain and cultivation conditions).
Given the breadth of the claims, which encompass the fermentation of any decolorized biomass into any alcoholic solvent (claim 1) or the fermentation of the decolorized biomass into any substance (claim 18), and the complete lack of guidance provided within the specification regarding any fermentation conditions, the person of ordinary skill in the art would not have recognized that the inventors had possession of the claimed invention. The state of the art suggests a degree of predictability only with respect to specific microorganisms and solvents. For example, the fermentation conditions to produce acetone, butanol, and ethanol by Clostridium saccharobutylicum from a microalgal biomass are taught by the prior art. However, none of the present claims limit the microorganism to any particular bacterial species and only claim 5 requires that the alcoholic solvent is n-butanol. Note that claim 19 recites that the second solvent is n-butanol, but claim 19 depends from claim 18, which does not recite fermenting the hydrolyzed decolorized biomass into the second solvent.
Claim 9 recites enzymatically processing chlorophyll powder into tocopherols, pheophorbide B, or phytol. However, the specification does not disclose any of the conditions required for processing the chlorophyll powder into tocopherols, pheophorbide B, or phytol. Note that as drafted the claim reads as a solid-state reaction.
Zhao et al. (Renewable Energy 118 (2018): 701-708) teaches that acid hydrolysis of chlorophyll with sulfuric acid between 0.2 and 2.0 M at temperatures between 40 °C and 80 °C produces pheophorbide (page 703, left column 2.5. Acid hydrolysis). However, Zhao does not teach any enzymes that convert chlorophyll into pheophorbide.
Holden (Biochemical journal 78.2 (1961): 359) teaches the enzymatic conversion of chlorophyll in vitro to phytol (page 359, left column, paragraph 1). However, the reaction in vitro requires the presence of a solvent such as acetone or methanol (page 359, left column, paragraph 1).
Therefore, given the absence of enzymatic processing conditions disclosed in the specification and the lack of such conditions for processing a chlorophyll powder taught by the prior art, the person of ordinary skill in the art would not have recognized that the inventors had possession of the claimed genus of enzymatic processing conditions recited in claim 9.
In addition, claim 18 recites a genus of first solvents and second solvents. The person of ordinary skill in the art would have been unable to reasonably predict and visualize the structures of all the species within the claimed genus of first and second solvents.
The first solvent is selected from a relatively hydrophilic aqueous surfactant- and protein denaturant-based solvent, a relatively hydrophilic aqueous ionic liquid-based solvent, a relatively hydrophilic aqueous surfactant, and/or protein denaturant-based deep eutectic solvent, and an immiscible, relatively hydrophobic deep eutectic solvent.
The second solvent is selected from an immiscible, relatively hydrophobic polar alcohol: an immiscible, relatively hydrophobic polymer-based solvent; an immiscible, relatively hydrophobic aqueous ionic liquid-based solvent; an immiscible, relatively hydrophobic, deep eutectic solvent; and an immiscible, relatively hydrophobic aqueous ionic liquid-based solvent.
The specification discloses in Table 2 a list of hydrophilic protein-denaturing solvents that include PUTTS buffer (formulation given in [0063]), choline acetate, choline dihydrogen phosphate, and deep eutectic solvents that include choline chloride-urea, Choline acetate-urea, and choline dihydrogen phosphate-urea ([0063]).
However, in [0065], the specification discloses that hydrophilic protein non-denaturing solutions include choline chloride, choline acetate, and choline dihydrogen phosphate (0065]).
Thus, choline acetate and choline dihydrogen phosphate are listed as both hydrophilic protein-denaturing solvents as well as hydrophilic protein non-denaturing solvents.
In addition, there is a required functional relationship required between the first solvent and the second solvent, which is that the first solvent and the second solvent must partition into top, middle, and bottom layers.
Ruiz teaches a process for extracting proteins, pigments, lipids, and carbohydrates from microalgae in an aqueous two-phase system comprising the polymer polypropylene glycol-400 and a cholinium dihydrogen phosphate ionic liquid (Abstract). Proteins partition into the ionic liquid-rich phase and pigments into the polymer-rich phase, while starch and lipids are recovered at the interface (Abstract). Thus, Ruiz teaches a single species of the claimed solvent system: a first solvent that is a relatively hydrophilic aqueous ionic liquid-based solvent and a second solvent that is an immiscible, relatively hydrophobic polymer-based solvent.
Given the ambiguity in the specification regarding which solvents are protein-denaturing as well as the small number of combinations taught by the prior art and disclosed within the specification, the person of ordinary skill in the art would have been unable to predict and visualize the structures of all the species within the claimed genus of first solvents and second solvents.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-7 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Ruiz et al. (ACS sustainable chemistry & engineering 8.6 (2020): 2441-2452) in view of Gao et al. (Bioresource technology 206 (2016): 77-85), Kongjan et al. (Clean energy and resources recovery. Elsevier, 2021. 421-446), and Xu et al. (Journal of Chemical & Engineering Data 64.8 (2019): 3547-3555) as evidenced by Figueiredo et al. (Phys Chem Chem Phys. 2013 Dec 7;15(45):19632-43).
“Solubilized” is interpreted as making a substance more soluble.
Ruiz teaches a process for extracting proteins, pigments, lipids, and carbohydrates from microalgae in an aqueous two-phase system (ATPS) comprising the polymer polypropylene glycol-400 and a cholinium dihydrogen phosphate ionic liquid (Abstract). Proteins partition into the ionic liquid-rich phase and pigments into the polymer-rich phase, while starch and lipids are recovered at the interface (Abstract).
Ruiz teaches that proteins preferentially migrate to the IL-rich phase, which is the more hydrophilic phase and the pigments prefer to migrate to the opposite phase (page 2445, left column, bottom paragraph).
Ruiz disrupts cells by bead milling to release most of the intracellular material from the microalgal biomass into small fragments including cell wall particles (cell debris) and intracellular material (page 2446, right column, Multistep Process Design). The biomass is suspended in Milli-Q water and disrupted in a bead mill (“solubilized,” page 2442, right column, Microalgae Cultivation, Harvesting, and Cell Disruption).
Although Ruiz teaches producing a suspended biomass mixture (the disrupted biomass mixed with Milli-Q water), Ruiz does not teach exposing the biomass to a protein-denaturing solution, separating the solubilized photosynthetic biomass into a supernatant and decolorized biomass, hydrolyzing the decolorized biomass, or fermenting the hydrolyzed decolorized biomass into a polar alcoholic solvent.
Gao teaches preparing ionic liquid-extracted algae for acetone, butanol, and ethanol fermentation by Clostridium saccharobutylicum DSM 13864 (Abstract and page 79, right column 2.2.1. Clostridium strain and cultivation conditions). The microalgae is Chlorella vulgaris strain UTEX 2714 (page 78, right column, 2.1.1 Microalgae strain and cultivation conditions). Gao mixes the algae with the ionic liquid [C2mim] [EtSO4] (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction), which is the same as “solubilizing photosynthetic biomass.” Residual ionic liquid-extracted algae (ILEA) is recovered by adding water or ethanol to the ionic liquid mixture to precipitate the residual solids (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). The solids are washed with solvent until they are colorless (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). The solution is filtered through and washed with additional solvent to remove the ionic liquid (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). Precipitation necessarily produces a supernatant (the liquid lying above the solid). Gao then hydrolyzes and ferments the biomass into acetone, butanol, and ethanol (Figure 1). Both butanol and ethanol are alcoholic solvents.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Ruiz by first performing mixing the microalgae biomass with the ionic liquid cholinium dihydrogen phosphate, separating the decolorized biomass from the supernatant, and hydrolyzing and fermenting the biomass, as suggested by Gao. The person of ordinary skill in the art would have been motivated to produce the value-added chemicals acetone, butanol, and ethanol. The person of ordinary skill in the art would have had a reasonable expectation of success because [C2mim] [EtSO4] and cholinium dihydrogen phosphate are both ionic liquids. It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to mix the PPG-400 with the supernatant and fractionate the supernatant mixture per the teaching of Ruiz in order to separate pigments, lipids, proteins, and polysaccharides into top, middle and bottom layers. The person of ordinary skill in the art would have had a reasonable expectation of success in the separation.
Ruiz does not teach that cholinium dihydrogen phosphate is a protein-denaturing solution. Ruiz does not teach mixing the alcoholic solvent and the supernatant phase.
Ruiz teaches that aqueous two-phase systems (ATPSs) can be formed by combining a high variety of aqueous solutions, including polymers, salts, alcohols, and ionic liquids (age 2442, left column, paragraph 2).
Gao does not teach that the butanol produced by the acetone, butanol, ethanol fermentation is n-butanol.
Kongjan teaches that 1-butanol (synonym for n-butanol) is produced by ABE fermentation (paragraph 1 on page 425).
Xu teaches that n-butanol forms an aqueous two-phase system(ATPS) with the ionic liquid n-butylpyridine dicyanamide (Abstract).
n-butylpyridine dicyanamide is a protein-denaturing solution due to the presence of the dicyanamide anion as evidenced by Figueiredo (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the ionic liquid of Ruiz with n-butylpyridine dicyanamide and the PPG-400 polymer with n-butanol. The person of ordinary skill in the art would have been motivated by the teaching of Xu, which suggests that n-butanol forms an aqueous two-phase system with the ionic liquid n-butylpyridine dicyanamide, as well as the teachings of Gao and Kongjan that Clostridium saccharobutylicum produces n-butanol. Therefore, the person of ordinary skill in the art would have been motivated to replace the polymer PPG-400 with n-butanol in order to reduce the cost of reagents to perform the method and to recycle the product of the fermentation.
Regarding claims 2-3, Ruiz and Gao does not teach separating the supernatant phase from the decolorized biomass by gravimetric settling or centrifugation.
Gao teaches separating the supernatant phase from the decolorized biomass by precipitation and filtration (page 79, left column, 2.1.4. Hexane/2-propanol and ionic liquid extraction).
Ruiz teaches downstream processes for separation of cell debris include centrifugation, filtration, and chromatography (page 2446, right column, Multistep Process Design, bottom paragraph). Ruiz also teach gravimetric settling to separates phases (page 2442, right column, bottom paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace Gao’s step of precipitation and filtration with either gravimetric settling or centrifugation to separate the supernatant phase from the decolorized biomass. The person of ordinary skill in the art would have had a reasonable expectation of success in either alternative, which are both suggested by Ruiz.
Regarding claim 4, note that the claim does not specify what substance is used to wash the solubilized biomass mixture. Thus, washing the solubilized biomass mixture with a protein-denaturing solution satisfies both claim limitations of “washing the solubilized biomass mixture“ and “re-exposing the solubilized biomass mixture to a protein-denaturing solution.”
Ruiz does not teach washing the solubilized biomass mixture and re-exposing the solubilized biomass mixture to a protein-denaturing solution.
However, Gao teaches extracting biomolecules by mixing microalgae with the ionic liquid (page 79, left column, 2.1.4. Hexane/2-propanol and ionic liquid extraction).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to repeat the step of extracting the biomolecules from the biomass by washing the biomass again with Xu’s n-butylpyridine dicyanamide. The person of ordinary skill in the art would have been motivated to increase the amount of biomolecules extracted from the biomass by using an additional portion of solvent to access any residual biomolecules, such as pigments, proteins, and lipids, in the biomass.
Regarding claim 5, Kongjan teaches that 1-butanol (synonym for n-butanol) is produced by ABE fermentation (paragraph 1 on page 425). Thus, the acetone-butanol-ethanol (ABE) fermentation in the method of Ruiz modified by Gao would have produced 1-butanol.
Regarding claim 6, Ruiz teaches preparing mixtures in the biphasic system and letting the phases equilibrate for at least 12 h to reach complete separation and equilibration of the phases (page 2442, right column, bottom paragraph). Thus, Ruiz teaches gravimetric settling since the phases equilibrate naturally over time.
Regarding claim 7, Ruiz teaches that proteins partition into the ionic liquid-rich phase and pigments into the polymer-rich (hydrophobic) phase, while starch and lipids are recovered at the interface (Abstract).
Ruiz teaches filtering the IL-rich phase into protein and permeate (the flow-through of the filter): see page 2443, left column Recycling of phase forming components and further purification of biomolecules.
Ruiz does not teach transesterifying the middle phase with fatty esters.
Gao teaches that total available lipids in the microalgae are quantified by direct transesterification as fatty acid methyl esters or FAME (page 80, left column , 3.1. Lipid extraction and composition analysis).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to transesterify lipids at the interface in order to quantify the total available lipids. The person of ordinary skill in the art would have had a reasonable expectation of success in transesterifying the lipids at the interface.
Ruiz recovers pigments from the most hydrophobic phase (page 2446, left column, paragraph 1). Ruiz teaches that lutein and chlorophyll b are the most abundant pigmenst in the biomass (Figure S1).
Ruiz does not teach drying the top layer into a powder.
Gao teaches evaporating solvents to determine the mass of extractable lipids (page 79, 2.1.4. Hexane/2-propanol and ionic liquid extraction).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to recover the pigments including lutein and chlorophyll from the n-butanol phase. The person of ordinary skill in the art would have expected the pigments to partition into this layer because n-butanol has a hydrophobic tail, and thus chlorophyll would have partitioned into the more hydrophobic layer. It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to evaporate the n-butanol solvent in order to form a powder of the pigments for mass measurement. The person of ordinary skill in the art would have had a reasonable expectation of success in evaporating n-butanol to form a pigment powder.
Regarding claim 11, Ruiz’s biomass is microalgae (Fig. 1).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Ruiz et al. (ACS sustainable chemistry & engineering 8.6 (2020): 2441-2452) in view of Gao et al. (Bioresource technology 206 (2016): 77-85), Kongjan et al. (Clean energy and resources recovery. Elsevier, 2021. 421-446), and Xu et al. (Journal of Chemical & Engineering Data 64.8 (2019): 3547-3555) as evidenced by Figueiredo (Phys Chem Phys. 2013 Dec 7;15(45):19632-43), as applied to claims 1-7 and 11 above, further in view of Guzman et al. (Journal of agricultural and food chemistry 60.29 (2012): 7238-7244).
See discussion of Ruiz, Gao, Kongjan, and Xu above, which is incorporated into this rejection as well.
“Substantially” is determined as any enriched powder in which the powder is more than 50% chlroophyll.
Regarding claim 8, Ruiz teaches that lutein and chlorophyll b are the most abundant pigments in the biomass (Figure S1). Ruiz does not teach a substantially pure powder of chlorophyll.
Guzman teaches an UPLC method that quickly separates chlorophylls and carotenoids (Abstract). Guzman completely purifies chlorophyll from other pigments (Fig. 2).
Gao teaches evaporating solvents to determine the mass of extractable lipids (page 79, 2.1.4. Hexane/2-propanol and ionic liquid extraction).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to separate the pigments lutein and chlorophyll from each other by applying the method of Guzman to the top layer (n-butanol comprising pigments). The person of ordinary skill in the art would have been motivated to purify the pigments from each other for individual retail. The person of ordinary skill in the art would have had a reasonable expectation of success in purifying the chlorophyll from lutein and the other pigments. It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to evaporate the solvent in order to form a powder of the chlorophyll for mass measurement.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Ruiz et al. (ACS sustainable chemistry & engineering 8.6 (2020): 2441-2452) in view of Gao et al. (Bioresource technology 206 (2016): 77-85).
“Extracting chlorophyll, carotenoids, lipids and fatty acids, proteins, polysaccharides from the top, middle, and bottom layers” is interpreted as extracting chlorophyll, carotenoids, lipids and fatty acids, proteins, or polysaccharides from any of the top, middle, or bottom layers.
“Relatively” is interpreted as not further limiting hydrophilic or hydrophobic in the first or second solvents.
“Solubilized” is interpreted as making a substance more soluble.
Ruiz teaches a process for extracting proteins, pigments, lipids, and carbohydrates from microalgae (“photosynthetic biomass”) in an aqueous two-phase system comprising the polymer polypropylene glycol-400 and a cholinium dihydrogen phosphate ionic liquid (Abstract and Fig. 4). Proteins partition into the ionic liquid-rich phase and pigments into the polymer-rich phase, while starch and lipids are recovered at the interface (Abstract). The pigments include lutein (carotenoid) and chlorophyll (page 2444, left column, Microalgae Cultivation, Harvesting, and Cell Disruption).
Ruiz teaches that proteins preferentially migrate to the IL-rich phase, which is the more hydrophilic phase and the pigments prefer to migrate to the opposite phase (page 2445, left column, bottom paragraph). Thus, the IL solvent (“first solvent”) is a “relatively hydrophilic aqueous ionic liquid-based solvent” and the polymer solvent (“second solvent”) is a “relatively hydrophobic polymer-based solvent.”
Ruiz disrupts cells by bead milling to release most of the intracellular material from the microalgal biomass into small fragments including cell wall particles (cell debris) and intracellular material (page 2446, right column, Multistep Process Design).
Ruiz does not teach solubilizing the photosynthetic biomass in a first solvent, separating the solubilized photosynthetic biomass into a supernatant and decolorized biomass, hydrolyzing the decolorized biomass, or fermenting the hydrolyzed decolorized biomass.
Gao prepares ionic liquid-extracted algae for acetone, butanol, and ethanol fermentation by Clostridium saccharobutylicum DSM 13864 (Abstract and page 79, right column 2.2.1. Clostridium strain and cultivation conditions). The microalgae is Chlorella vulgaris strain UTEX 2714 (page 78, right column, 2.1.1 Microalgae strain and cultivation conditions). Gao mixes the algae with the ionic liquid [C2mim] [EtSO4] (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction), which is the same as “solubilizing photosynthetic biomass.” Residual ionic liquid-extracted algae (ILEA) is recovered by adding water or ethanol to the ionic liquid mixture to precipitate the residual solids (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). The solids are washed with solvent until they are colorless (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). The solution is filtered through and washed with additional solvent to remove the ionic liquid (page 79, left column 2.1.4. Hexane/2-propanol and ionic liquid extraction). Precipitation necessarily produces a supernatant (the liquid lying above the solid). Gao then hydrolyzes and ferments the biomass into acetone, butanol, and ethanol (Figure 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Ruiz by first mixing (“solubilizing”) the microalgae biomass with the ionic liquid cholinium dihydrogen phosphate, separating the supernatant phase from the decolorized biomass, and then hydrolyzing and fermenting the biomass, as suggested by Gao. The person of ordinary skill in the art would have been motivated to produce the value-added chemicals acetone, butanol, and ethanol. The person of ordinary skill in the art would have had a reasonable expectation of success because [C2mim] [EtSO4] and cholinium dihydrogen phosphate are both ionic liquids. It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to mix the PPG-400 with the supernatant and fractionate the supernatant mixture per the teaching of Ruiz in order to separate pigments, lipids, proteins, and polysaccharides into top, middle and bottom layers. The person of ordinary skill in the art would have had a reasonable expectation of success in the separation.
Conclusion
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/CANDICE LEE SWIFT/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657