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 2, 13-16, 18, 20, 23-24, 31 and 33 are canceled. Claim 35 is new.
Claims 1, 3-12, 17, 19, 21-22, 25-30, 32, 34 and 35 are pending and under consideration in this action.
The instant claims are entitled to an effective filing date of 05/12/2021.
Claim Rejections - 35 USC § 112(a)
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, 3-12, 17, 19, 21-22, 25-30, 32, 34 and 35 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. The amendment filed on 08/14/2026 has introduced new matter into the claims.
Claim 1, as filed on 08/14/2026, recites a multi-stage, multi-strain fermentation method, comprising: a first fermentation process, comprising inoculating one or more strains of microorganism(s) selected from Pyrococcus, Metallosphaera, Rhodospirillum, Chloroflexus, Cyanobacteria, Chlorella, Dunaliella, Nannochloropsis, Scenedesmus, and Botryococcus, into a mineral medium, the mineral medium comprising potassium nitrate, ferrous sulfate heptahydrate, and zinc sulfate heptahydrate; incubating the inoculated mineral medium with aeration at 0.01-0.4 vvm; under either light or no light conditions; and with a first primary feed stock wherein carbon dioxide is the primary carbon source; and a which first fermentation process produces a first fermentation broth;
a second fermentation process, comprising inoculatinq one or more strains of microorganism(s) selected from Sordaria, Trametes, Irpex, Lenzite, Phanerochaete, Klebsiella, Ochrobactrum, Dysgonomonas, Sphingobacterium, Bacteroides, Parabacteroides, Flavobacterium, polymorphonuclear Aeromonas, Pleomorphomonas, Arcticibacter, Elizabethkingia, Neisseria, Mycobacterium, Trichoderma, Zymomonas, Stenotrophomonas, Paenibacillus, Nocardia, Nocardiopsis, Bacillus, Rhizobium, Cellulomonas, Vibrio, Cellvibrio, Cytophaga, Alistipes, Aspergillus, Ruminofilibacter, and Clostridium into a second primary feed stock comprising lignocellulosic material, which second fermentation process produces a second fermentation broth, wherein the first primary feed stock and the second primary feed stock are different;
combining the first fermentation broth and the second fermentation broth to form a first mixture;
sterilizing the first mixture to form a sterilized first mixture; and
initiating a third fermentation process, comprising inoculating a culture medium comprising the sterilized first mixture with one or more biosurfactant producing strains of microorganism(s) selected from Pseudomonas, Bacillus, Candida, Acinetobacter, Pantoea, Streptomyces, Rhodococcus, Pseudozyma, Ustilaginales, and Moesziomyces under conditions and for a time sufficient to produce a biosurfactant fermentation broth, which biosurfactant fermentation broth comprises at least one glycolipid or lipopeptide biosurfactant.
Claim 22, as filed on 08/14/2026, recites a multi-stage, multi-strain fermentation method, comprising: a first fermentation process, comprising inoculatinq one or more strains of microorganism(s) selected from Pyrococcus, Pseudomonas, Metallococcus sp., Metallosphaera, Rhodospirillum, Chloroflexus, Aspergillis, Cyanobacteria, Chlorella, Dunaliella, Nannochloropsis, Scenedesmus, and Botryococcus into the first primary feed stock comprising carbon dioxide, which first fermentation process produces a first fermentation broth;
a second fermentation process, comprising inoculating one or more strains of microorganism(s) selected from Sordaria, Trametes, Irpex, Lenzite, Phanerochaete, Klebsiella, Ochrobactrum, Dysgonomonas, Sphingobacterium, Bacteroides, Parabacteroides, Flavobacterium, polymorphonuclear Aeromonas, Pleomorphomonas, Arcticibacter, Elizabethkingia, Neisseria, Mycobacterium, Trichoderma, Zymomonas, Stenotrophomonas, Paenibacillus, Nocardia, Nocardiopsis, Bacillus, Rhizobium, Cellulomonas, Vibrio, Cellvibrio, Cytophaga, Alistipes, Aspergillus, Ruminofilibacter, and Clostridium into a second primary feed stock comprising lignocellulosic material, wherein the one or more strains of microorganism(s) of the second fermentation process are cultured using mineral medium comprising (NH4)2SO4, MgSO4-7H2O, CuSO4-5H2O with an initial mineral medium pH of 7-8.5, and 2-10% lingocellulosic material by volume as the primary carbon source, the second fermentation process maintained at 20-40°C, with stirring at 100-300 rpm, which second fermentation process produces a second fermentation broth, wherein the first primary feed stock and the second primary feed stock are different;
combining the first fermentation broth and the second fermentation broth to form a first mixture;
sterilizing the first mixture to form a sterilized first mixture; and
initiating a third fermentation process, comprising inoculating a culture medium comprising the sterilized first mixture with one or more biosurfactant producing strains of microorganism(s) selected from Pseudomonas, Bacillus, Candida, Acinetobacter, Pantoea, Streptomyces, Rhodococcus, Pseudozyma, Ustilaginales, and Moesziomyces under conditions and for a time sufficient to produce a biosurfactant fermentation broth, which biosurfactant fermentation broth comprises at least one glycolipid or lipopeptide biosurfactant.
Claim 26, as filed on 08/14/2026, recites the method of claim 1, wherein the third fermentation process further comprises an inorganic salt medium, the inorganic salt medium comprising NO3, FeCl2, NaH2PO4, K2HPO4, MgSO4, KCI, choline chloride, yeast extract, and trace elements Zn, Mn, and CI, wherein a carbon source for the third fermentation process is a mixture of the first fermentation broth and the second fermentation broth.
Applicant’s amendment, filed 08/14/2026, directs attention to the specification and the original claims for support and asserts that no new matter has been added. See the remarks p. 9 para. 2. Applicant indicates that support for the medium compositions can be found in at least paragraphs [0007], [0009], [0012], [0052], and [0055]. However, the specification as filed does not provide sufficient written description of the above underlined limitations.
Claim 1 and dependent claims contain new matter because of the limitation in claim 1 requiring the first fermentation process to comprise a mineral medium comprising potassium nitrate, ferrous sulfate hetahydrate, and zinc sulfate heptahydrate, which encompasses mineral media comprising any amount of potassium nitrate, ferrous sulfate hetahydrate, and zinc sulfate heptahydrate. Claim 22 and dependent claims contain new matter because of the limitation in claim 22 requiring second fermentation process to comprise a mineral medium comprising (NH4)2SO4, MgSO4-7H2O, CuSO4-5H2O, which encompasses mineral media comprising any amount of (NH4)2SO4, MgSO4-7H2O, CuSO4-5H2O. Claim 26 contains new matter because of the limitation requiring the third fermentation process to comprise an inorganic salt medium comprising NO3, FeCl2, NaH2PO4, K2HPO4, MgSO4, KCI, choline chloride, yeast extract, and trace elements Zn, Mn, and CI.
The specification, as filed, and the original claims do not provide support commensurate in scope with the mineral medium required in the first fermentation process of amended claim 1. Original claim 20, as filed 05/12/2022, recites “the first culture condition comprise a medium comprising: potassium nitrate of 0.1-0.5%, potassium dihydrogen phosphate of 0.1-0.5%, magnesium sulphate heptahydrate of 0.01-0.2%, calcium chloride dihydrate of 0.01-0.05%, ferrous sulfate heptahydrate of 0.01-0.05%, zinc sulfate heptahydrate of 0.001-0.01%, manganese chloride tetrahydrate of 0.001-0.01%, copper sulfate pentahydrate of 0.001-0.01%, disodium edetate of 0.001-0.01%, and calcium carbonate of 0.1-0.3%”. The specification, in paragraph [0007], teaches fermentation A with a representative medium containing: 0.1-0.5% potassium nitrate, 0.01-0.05% potassium dihydrogen phosphate, 0.01-0.2% magnesium sulphate heptahydrate, 0.01-0.05% calcium chloride dihydrate, 0.01-0.05% ferrous sulfate heptahydrate, 0.001-0.01% zinc sulfate heptahydrate, 0.001-0.01% manganese chloride tetrahydrate, 0.001-0.01% copper sulfate pentahydrate, 0.001-0.01% disodium edetate, and 0.1-0.3% calcium carbonate. The specification, in paragraph [0052], teaches a first fermentation medium embodiment with 0.15% potassium nitrate, 0.1% potassium dihydrogen phosphate, 0.01% magnesium sulphate heptahydrate, 0.01% calcium chloride dihydrate, 0.01% ferrous sulfate heptahydrate, 0.001% seven hydrated zinc sulfate, 0.002% manganese chloride tetrahydrate, 0.001% copper sulfate pentahydrate, 0.001% disodium edetate, and 0.1% calcium carbonate. As such, the specification as filed and original claims do not provide support commensurate in scope with the recited mineral medium in amended claim 1, because the specification as filed and original claims teach specific percentage ranges for each mineral medium component, whereas claim 1 encompasses mineral media comprising any amount of the recited components.
The specification, as filed, and the original claims do not provide support commensurate in scope with the mineral medium required in the second fermentation process of amended claim 22. Original claim 24, as filed 05/12/2022, recites the method of claim 22, wherein the medium [in the second fermentation] comprises: (NH4)2SO4 0.1-0.5%, MgSO4.7H2O 0.01-0.05%, CuSO4.5H2O 0.01-0.03%, MnSO4 0.001-0.005%, and calcium carbonate 0.1-0.5%. The specification, in paragraph [0009], teaches fermentation B that uses a base medium containing: (NH4)2SO4 0.1-0.5%, MgSO4. 7H2O 0.01-0.05%, CuSO4.5H2O 0.01-0.03%, MnSO40.001-0.005%, and 0.1-0.5% calcium carbonate. The specification, in paragraph [0055], teaches a second fermentation medium that contains (NH4)2SO4 0.1%, MgSO4·7H2O 0.01%, CuSO4.5H2O 0.01%, MnSO4 0.001%, and 0.1% calcium carbonate. As such, the specification as filed, and the original claims do not provide support commensurate in scope with the recited mineral medium in amended claim 22.
The specification, as filed, and the original claims do not provide support for the inorganic medium recited in claim 26. Original claim 30, as filed 05/12/2022, recites the culture medium [in the third fermentation] is at pH 6-7 and comprises: NaNO3: 0.1-1.4%, FeCl2: 0.002-0.006%, NaH2PO4: 0.25-1.5%, K2HPO4: 0.25-1.8%, MgSO4.7H2O: 0.005-0.015%, KCl: 0.05%-0.3%; Choline chloride: 0.05%-0.3%; yeast extract: 0.001-0.1%, and trace elements: Zn, Mn, Ca. The specification, in paragraph [0012], teaches fermentation C using a culture medium that includes components: NaNO3: 0.1-1.4%, FeCl2: 0.002-0.006%, NaH2PO4: 0.25-1.5%, K2HPO4 0.25-1.8%, MgSO4·7H2O: 0.005-0.015%, KC1: 0.05%-0.3%; Choline chloride: 0.05%-0.3%; yeast extract: 0.001 to 0.1%; and trace elements: Zn, Mn, Ca. As such, the original claims and the specification as filed does not provide support commensurate in scope with the instantly required inorganic medium.
Such limitations recited in the instant claims 1, 22 and 26 (and required by dependent claims), which did not appear in the specification or original claims, as filed, introduce new concepts and violate the description requirement of the first paragraph of 35 U.S.C 112. Applicant is required to provide sufficient written support for the limitations recited in the instant claims. Applicant can remove the new matter limitations from the claims to obviate this rejection.
Claim Rejections - 35 USC § 112(b)
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 22, 25 and 30 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.
Claims 22 and 30 recite the limitation “the first primary feed stock” in line 5. There is insufficient antecedent basis for this limitation in the claim. Claim 22 is an independent claim and there is no earlier requirement for a first primary feed stock in the claim. Therefore, it is unclear which first primary feed stock is required in claim 22. Claim 30 is an independent claim, and there is no earlier requirement for a first primary feed stock. Therefore, it is unclear which first primary feed stock is required in the first fermentation process of claim 30.
Claim 25 depends from claim 22 and is rejected for the reason set forth above.
Response to Arguments
Applicant's arguments filed 08/14/2026, with respect to the previous rejection of claims 1, 3-12, 17, 19, 21-22, 25-30 and 32-34 under 35 U.S.C. 112(b), have been fully considered but they do not apply to the new grounds of rejection set forth above.
Claim Rejections - 35 USC § 103
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.
Claims 1, 3-6, 17, 19, 21 and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1) in view of Reed (US 2020/0165733).
Regarding claims 1, Koskinen teaches cultivating autotrophic microorganisms in closed bioreactors. Gases containing concentrated CO2 are purged to photobioreactors to provide carbon dioxide to autotrophic microorganisms. See [0123]. Koskinen teaches growing cultures autrophically (i.e. with light). See [0098]. Koskinen teaches autotrophic microorganisms such as cyanobacteria cultivated for oil production. See [0094]. Koskinen teaches an aeration of 0.5-1 vvm, which overlaps with the instantly claimed 0.01-0.4 vvm. See [0218]. Koskinen teaches a second aerobic process using hetero/mixotrophic oil accumulating microorganisms. The second aerobic aerated treatment process is fed with lignocellulosic palm oil production residues (i.e. a second primary feed stock). See [0078]. Koskinen teaches bacteria capable of producing lipids. See [0099]. Bacteria include Bacillus (i.e. a second and third fermentation strain). See [0102]. In example 5, Koskinen teaches cultivating Streptomyces (i.e. a third fermentation strain) G009 in cell waste. Streptomyces cells from previous fermentations are harvested by filtration, and oil-extracted cell residue is supplied to the fermentation broth. See [0217]. Koskinen suggests the medium is sterilized in example 5. See [0218], and see [0109] for Koskinen’s suggestion that sterilization is typically meant for the medium. Furthermore, in example 5, the time for lipids to accumulate is 32-144 h. See [0218]. Koskinen teaches lipids including glycolipids (i.e. a biosurfactant). See [0043].
Koskinen does not teach a first fermentation process using a mineral medium comprising potassium nitrate, ferrous sulfate heptahydrate and zing sulfate heptahydrate.
Koskinen does not teach a first primary feed stock wherein carbon dioxide is the primary carbon source.
Reed teaches culturing microorganisms for the synthesis of nutrients or whole cell products in a medium containing gaseous inorganic carbon as the primary or sole carbon source and without any exposure to light. See [0224]. Reed teaches a consortium of microorganisms that comprise chemoautotrophic microorganisms. See claim 54 of Reed. Chemoautotrophic microorganisms can use CO2 as their sole carbon source to support biological growth. See [0032]. Reed teaches Rhodospirillum rubrum and other Rhodospirillum sp. (i.e. a chemoautotroph) and cyanobacteria including Scenedesmus sp. See [0196], and see [0199] for support that Rhodospirillum is a chemoautotroph. Reed teaches inoculating a bioreactor with a residual volume (i.e. fermentation broth) and turning on gas flow. See [0387]. Gas flow ranges from 0.05 to 0.3 vvm which overlaps with the instantly claimed 0.01-0.4 vvm. See [0388]. Nutrient addition solutions may be added during the run. See [0390]. Reed teaches chemicals for the growth of microbial cultures including: nitrate, e.g., potassium nitrate, ferrous sulfate FeSO4 7H2O (i.e. ferrous sulfate heptahydrate), zinc sulfate. See [0249]. Reed further teaches a trace mineral solution including ZnSO4.7H2O (i.e. zinc sulfate heptahydrate). See [0355].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Reed’s Rhodospirillum chemoautotrophic first fermentation process where CO2 is the primary carbon source for Koskinen’s autotrophic first fermentation process. One of ordinary skill in the art would have been motivated to do so because Reed suggests that chemoautotrophic organisms are particularly suited for processes for the conversion of CO2-to-organic chemicals ([0019]). There would have been a reasonable expectation of success because Reed teaches chemoautotrophic microorganisms that accumulate lipid (see claim 60 of Reed), and lipids encompass sphingolipids (i.e. biosurfactant) (see [0132]).
Koskinen and Reed do not teach combining a first fermentation broth and a second fermentation broth to form a first mixture.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Reed’s first fermentation broth and Koskinen’s second fermentation broth comprising Bacillus cells prior to the sterilization taught by Koskinen. One of ordinary skill in the art would have been motivated to do so because Koskinen suggests using cell waste in cultivations (see [0167]), where cell waste refers to a broth such as a spent culture medium used in the cultivation of microorganisms (see [0174]). There would have been a reasonable expectation of success because Koskinen states that: cell waste may be any microbial cell residue or debris obtained after cultivation of microbial cells and isolation of any desired products from the culture broth (see [0057]).
Regarding claim 3, Koskinen teaches producing oil by microorganisms that are able to convert CO2 to lipids. See [0052]. Lipids include glycolipids (i.e. a biosurfactant). See [0043].
Reed suggests that carbon dioxide may be sequestered in the process steps. See [0275].
Regarding claim 4, Koskinen teaches utilizing microorganisms capable of producing exoenzyme that hydrolyse polymeric sugars in lignocellulosic materials. See [0092]. Koskinen teaches mixed microbial cultures containing at least one microorganism capable of hydrolyzing polymeric sugars, such as cellulose and/or hemicellulose, to sugar monomers. See [0104].
Regarding claim 5, Koskinen teaches organisms, including Bacillus, which are capable of utilizing polymeric hemicellulose due to exoenzymes. See [0105]. Exoenzyme that hydrolyze polymeric sugars in lignocellulosic materials. See [0092].
Regarding claim 6, Koskinen teaches recovering oil from cells by extraction. See [0137]. In example 5, Koskinen teaches a chloroform extract of culture broth. See [0219]. Koskinen discloses that the terms lipid and oil are used synonymously. Lipids include glycolipids. See [0043].
Regarding claim 17, Koskinen teaches hemicellulose or cellulose from industrial practices. Materials can be agricultural residues, such as corn stover (i.e. which includes corn stalks), wheat straw and rice straw. Koskinen teaches wood materials or residues including e.g. sawmill and pulp. See [0089]. Koskinen teaches oil palm fronds leaves. See [0045].
Regarding claim 19, Koskinen teaches inoculation from spore stock to media; a transferring rate of 1-10%; temperature of 28˚C, and agitation of 200-330 rpm. See [0197].
Regarding claim 21, Koskinen discloses that cultivation can be carried typically in 3 to 10 days, which overlaps with the instantly claimed 4-10 days. See [0177].
Regarding claim 35, Koskinen teaches that the cultivation medium comprises cell waste or residue. The amount of cell waste or residue is 1-300 g per liter cultivation medium, preferably 5-200 g/l. See [0022].
Reed teaches whole cell products included within a feed or nutrient supply provided to another organism. See [0105]. Reed teaches removing media and biomass (i.e. fermentation broth) from a previous batch via peristaltic pump except for a residual volume, typically less than one liter, which is used to inoculate the next batch. See [0387]. Furthermore, Reed suggests that after the cell culture leaves the bioreactors, the cell mass can be separated from the liquid medium. Solid - liquid separation can be accomplished using processes and equipment well known in the art such as but not limited to continuous centrifuges or flowing broth through membrane filters to separate the cell mass from the liquid. Cell mass needed to replenish the cell culture population at a desirable level can be recycled back into the bioreactor. Surplus cell mass can be dried to form a dry biomass product which can be further post - processed into various feed. See [0440].
Koskinen and Reed do not teach the first fermentation broth is concentrated by 50% prior to combination with the second fermentation broth.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to adjust the concentration of Reed’s first fermentation broth prior to combining it with Koskinen’s second fermentation broth. One of ordinary skill in the art would have been motivated to do so because Koskinen suggests adjusting the concentration of the cell waste prior to using it in a fermentation with Streptomyces (third fermentation). There would have been a reasonable expectation of success because Reed demonstrates processing fermentation broth prior to its use as a feed for other organisms. MPEP 2144.05(II)(A) indicates that differences in concentration generally amount to “routine optimization” and will not support patentability unless there is evidence indicating the claimed feature is critical. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Claims 7-12 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1) and Reed (US 2020/0165733), as applied to claim 1 above, and further in view of Invally, (Food and bioproducts processing, 2019 114, 122-131; as previously relied upon) and Sun (CN103059108A, published 04/24/2013; as previously relied upon).
Regarding claim 7, Koskinen teaches recovering oil from cell biomass or culture broth using any method known in the art. For example, microorganisms are separated from the medium using filtration. See [0113]. Koskinen discloses that oil and lipid are used synonymously and that lipids include glycolipids. See [0043].
Koskinen and Reed do not teach an extraction process comprising: fractionating the biosurfactant fermentation broth to produce an aqueous phase; acid precipitating a first aqueous liquid from the aqueous phase; filtering the acid precipitated aqueous liquid to obtain a precipitate; dissolving the precipitate with an aqueous liquid at pH>8 to produce a resuspended precipitate; and filtering the resuspended precipitate to obtain a second aqueous solution containing the at least one glycolipid or lipopeptide biosurfactant.
Invally teaches an aqueous solution is obtained from (i.e. fractionating) a cell-free broth. See figure 1 (IV). Invally teaches performing acid precipitation on an aqueous solution to obtain a supernatant and precipitate. See figure 1 (IV). Invally teaches collecting precipitate by centrifugation (i.e. filtration to obtain precipitate). See section 2.2. Invally suggests re-dissolving rhamnolipid precipitate in neutral aqueous solution (e.g. pH 7) and performing calcium precipitation. See section 4. For calcium precipitation, CaCl2 and deionized water are added to a rhamnolipid solution. See section 2.5. Invally indicates that an aqueous rhamnolipid product stream is filtered from impurities. See figure 1 (VI).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply Invally’s extraction to Koskinen’s fermentation broth. One of ordinary skill in the art would have been motivated to do so because Invally suggests that the downstream processing may help improve the overall economic feasibility of rhamnolipid manufacturing as an industrial product. See the last 3 lines before section 2 of Invally. There would have been a reasonable expectation of success because Invally demonstrates extracting biosurfactant from fermentation broth.
Koskinen, Reed and Invally do not teach dissolving the precipitate with an aqueous liquid at pH>8 to produce a resuspended precipitate.
Sun teaches a method for purifying sodium subtilisin Bacillus subtilis lipopeptide. See claim 1 of Sun. In example 7, Sun teaches obtaining a supernatant from a fermentation liquid of Bacillus. Sun teaches adding hydrochloric acid to a supernatant and collecting the precipitate (i.e. acid precipitation). Then, sodium hydroxide is added for neutralization reaction. See [0076]. Sun discloses that neutralization reaction refers to adding an alkaline solution until the solution is neutral, i.e. reacting until the pH value is 6.5-8.5. See [0039].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize Invally’s dissolving step by adjusting the pH of the neutral aqueous solution based on Sun’s suggestion. One of ordinary skill in the art would have been motivated to do so because Sun suggests following acid precipitation with a neutralization reaction to a pH of 6.5-8.5, which overlaps with the instantly required pH >8. There would have been a reasonable expectation of success because Sun demonstrates a method in which a fermentation broth is neutralized (see [0076]).
Claim Interpretation: Based on the claim language of claim 8, adjusting a biosurfactant fermentation broth to pH=8-10, and centrifuging the pH-adjusted broth at 1000 x g produces a microbial material pellet, a clear aqueous liquid layer and a lipid phase.
Regarding claim 8, Sun teaches adjusting the pH of diluted fermentation liquid of Bacillus to a pH of 7.0-8.5, which overlaps with the instantly claimed pH=8-10. Sun teaches centrifuging the liquid at 10000g. See [0068].
Regarding claim 9, Koskinen teaches microorganisms that produce lipids in temperatures ranging between 10 to 45˚C. See [0133]. Furthermore, Koskinen suggests a time of 24-96h. See [0204].
Koskinen does not teach acid precipitation that comprises adjusting the first aqueous liquid to pH to produce an acidic aqueous liquid, then chilling the aqueous liquid at 4-10˚C for 24 hours prior to filtering.
Invally teaches an acid precipitation in which the supernatant is acidified to a pH of 2. The wet precipitate is collected by centrifugation (i.e. filtering). See section 2.2.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to adjust the temperature and time between Invally’s acid precipitation and Invally’s filtering based on Koskinen’s suggestions. One of ordinary skill in the art would have been motivated to adjust the temperature because Koskinen suggests that 10˚C may be appropriate for some microorganisms. There would have been a reasonable expectation of success because Invally demonstrates an acid precipitation step. One of ordinary skill in the art would have been further motivated to adjust the timing of the steps, because a person of ordinary skill in the art has good reason to pursue the known options. There would have been a reasonable expectation of success because Koskinen teaches a 24h time period.
Regarding claim 10, Sun teaches acidification precipitation. See claim 9 of Sun. Furthermore, Sun teaches a membrane filtration step at 10-50˚C, which overlaps with the instantly claimed 4-10˚C. See [0016]. Sun teaches a ceramic membrane with a pore size of 20nm to 100nm (i.e. 0.1 µm). See [0037].
Regarding claim 11, Sun teaches a membrane filtration step. See [0016]. Sun teaches a ceramic membrane with a pore size of 20nm to 100nm (i.e. 0.1 µm). See [0037].
Regarding claim 12, Koskinen teaches the lipid concentration in the cultivation medium. See [0215].
Koskinen does not teach concentrating the at least one glycolipid biosurfactant aqueous solution under vacuum at 50˚C.
Sun teaches preforming a neutralization reaction solution at 50˚C and spray drying (i.e. concentrating) the reaction solution to obtained the finished product. See paragraph [0066]. Sun teaches spray drying, which includes discharging water vapor by a vacuum device. See paragraph [0042]. Sun does not teach changing the temperature between the neutralization reaction and the spray drying, so Sun implies that the spray drying occurs at 50˚C.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply Sun’s vacuum spray drying to the lipid containing solution of Koskinen. One would be motivated to apply Sun’s vacuum spray drying, because Sun suggests that it makes material easier to store, transport and use. See [0042]. There would have been a reasonable expectation of success because Sun demonstrates spray drying (i.e. concentrating). See [0076].
Claims 22, 25 and 30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramírez (Bioresource technology, 2015, 198, 231-236) in view of Reed (US 2020/0165733), and Jadav (Journal of Natural Gas Science and Engineering, 2017, 43, 156-166).
Regarding claims 22 and 30, Ramírez teaches growing Pseudomonas aeruginosa and Bacillus subtilis with olive mill waste (OMW) as the sole carbon source. See the abstract. OMW has a high content of lignocellulosic material. See p. 232 second paragraph. Ramírez teaches inoculating bacteria onto nutrient-agar and incubating. Ramírez teaches preparing two seed cultures before a batch culture. Seed culture 1 is a medium in distilled water. Twenty ml of this medium is inoculated with cells and maintained. Seed culture 2 is a mineral salt medium (MSM) that includes MgSO4 ·7H2O, CuSO4 · 5H2O and ZnSO4 ·7H2O (Zn element), MnSO4 ·H2O (Mn element) and CaCl2 · 2H2O (Ca element). Seed culture 2 is inoculated with of seed culture 1, and grown again, for adaptation of cells in MSM. See p. 232 section 2.3 first paragraph. For batch fermentation, the MSM medium is used but the glucose is replaced with 2%, 5% or 10% w/v of OMW as the carbon source. Cultures are inoculated with 5% v/v of seed culture 2 and maintained at 37˚C and 160 rpm. See p. 232 section 2.3 second paragraph. Ramírez indicates that the B. subtilis and P. aeruginosa are grown separately. See, for example, Fig. 3 caption. Ramírez teaches carrying out all experiments in triplicate. See p. 232 section 2.3 paragraph 2.
Ramírez does not teach first fermentation process comprising inoculating one or more strains of microorganism(s) selected from Pyrococcus, Pseudomonas, Metallococcus sp., Metallosphaera, Rhodospirillum, Chloroflexus, Aspergillis, Cyanobacteria, Chlorella, Dunaliella, Nannochloropsis, Scenedesmus, and Botryococcus with the first primary feed stock comprising carbon dioxide, which first fermentation process produces a first fermentation broth.
Ramírez does not teach a mineral medium comprising (NH4)2SO4 with an initial mineral medium pH of 7-8.5.
Reed teaches Pseudomonas sp., Bacillus sp., Rhodospirillum sp. and cyanobacteria including Scenedesmus sp. See [0196]. Reed teaches inoculating a bioreactor and turning on gas flow. See [0387]. The gas composition includes CO2 (i.e. first primary feed stock). See [0388]. Nutrient addition solutions may be added during the run. See [0390]. Reed teaches chemicals for the growth of microbial cultures including ammonium sulfate (NH4)2SO4. See [0249]. Reed suggests controlling the pH of the microbial culture. See [0260]. Reed teaches a pH to 6.5. See [0414].
Ramírez and Reed do not teach an initial mineral medium pH of 7-8.5.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply Reed’s first primary feed stock comprising carbon dioxide to Ramírez’s Pseudomonas, to add Reed’s (NH4)2SO4 to Ramírez’s mineral medium, and to further adjust the pH. One of ordinary skill in the art would have been motivated to apply Reed’s carbon dioxide to Ramírez’s Pseudomonas, because Reed suggests that Pseudomonas species may function as an oxyhydrogen or knallgas strain (see [0196]), which are capable of growing on gaseous carbon including CO2 (see [0031]). There would have been a reasonable expectation of success because Ramírez demonstrates growing Pseudomonas, and Reed further suggests that Pseudomonas may use carbon dioxide as a carbon source. One would be further motivated to add Reed’s (NH4)2SO4, because Reed suggests that the (NH4)2SO4 can be used for growing or maintaining microorganisms. There would have been a reasonable expectation of success because Ramírez demonstrates growing Bacillus (the second microorganism) in a mineral medium and Reed suggests that (NH4)2SO4 may be used for growing or maintaining microorganisms including Bacillus species. One of ordinary skill in the art would have been motivated to adjust the pH, because Reed suggests controlling the pH. There would have been a reasonable expectation of success because Reed teaches a 6.5 pH from which one could reasonably optimize. MPEP 2144.05(II)(A) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Ramírez and Reed do not teach sterilizing a first mixture formed by the combination of a first fermentation broth and a second fermentation broth. However, Reed suggests sterilizing media components (see [0385])
Ramírez and Reed do not teach a third fermentation process, comprising contacting a culture medium comprising the first mixture with one or more biosurfactant producing strains selected from Pseudomonas, Bacillus, Candida, Acinetobacter, Pantoea, Streptomyces, Rhodococcus, Pseudozyma, Ustilaginales, and Moeziomyces under conditions and for a time sufficient to produce a biosurfactant fermentation broth that comprises at least one glycolipid or lipopeptide.
Jadav teaches allowing B. subtilis and P. aeruginosa to grow separately in minimal salt medium (MSM), which is formed by adding the nutrients. The nutrients aid the growth of the microorganism for the production of biosurfactant. After sterilization of flasks containing MSM these two microorganisms are inoculated. The samples are collected to observe the biosurfactant production. Microorganisms are cultivated until enough biosurfactant is produced. See p. 158 first passage.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine the MSM containing batch cultures of B. subtilis and P. aeruginosa of Ramírez as modified by Reed and to further apply the sterilization of Jadava prior to [a re-inoculation] of the B. subtilis and P. aeruginosa. One of ordinary skill in the art would have been motivated to combine the B. subtilis and P. aeruginosa batch cultures of Ramírez because Jadav suggests that P. aeruginosa and B. subtilis can be combined for biosurfactant production. There would have been a reasonable expectation of success because Ramírez demonstrates separately fermenting B. subtilis and P. aeruginosa in batch cultures containing MSM, and Jadava demonstrates growing B. subtilis and P. aeruginosa separately before combining the two in MSM. One of ordinary skill in the art would have been further motivated to sterilize the combined batch culture of Ramírez and Jadava, because Jadava suggests sterilizing MSM before inoculation and Ramírez suggests performing experiments in triplicates. There would have been a reasonable expectation of success because Jadava demonstrates inoculating B. subtilis and P. aeruginosa in MSM after sterilization.
Regarding claim 25, Ramírez teaches a 7 day culture. See, for example the caption of figure 1.
Claims 26-29 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1) and Reed (US 2020/0165733), as applied to claim 1 above, and further in view of Desai (US 2019/0292513)
Regarding claim 26, Koskinen teaches cultivating Streptomyces (i.e. a third fermentation strain) G009 in cell waste (i.e. a carbon source). See [0217].
Koskinen does not teach the inorganic salt medium comprising NO3, FeCl2, NaH2PO4, K2HPO4, MgSO4, KCI, choline chloride, yeast extract, and trace elements Zn, Mn, and CI.
Reed teaches chemicals used for maintenance and growth of microbial cultures including: nitrate (i.e. NO3), ferrous chloride FeCl2 ·4H2O, potassium phosphate KH2PO4, magnesium sulfate MgSO4 ·7H2O, and yeast extract. See [0249]. Furthermore, Feed teaches zinc sulfate or zinc chloride (i.e. Zn element and Cl element), manganese sulfate or manganese chloride (i.e. Mn element and Cl element). See [0249].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to add the inorganic salt medium components taught by Reed to the medium of Koskinen. One of ordinary skill in the art would have been motivated to do so because Reed suggests that the nutrients may be used to maintain or grow microorganisms. There would have been a reasonable expectation of success because Reed teaches chemoautotrophic microorganisms including Streptomyces (see [0199]), which is the same genus Koskinen demonstrates fermenting.
Koskinen and Reed do not teach NaH2PO4, KCl, and choline chloride.
Desai teaches media and fermentation methods for producing polysaccharide in a bacterial cell culture. See the abstract. Desai teaches a media containing choline chloride, and inorganic salts NaH2PO4 ·H2O and KCl. See table 2.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to add the Desai’s NaH2PO4, KCl, and choline chloride to the medium of Reed and Koskinen. One of ordinary skill in the art would have been motivated to do so because Desai suggests that the NaH2PO4, KCl, and choline chloride can be combined with additional inorganic salts and vitamins. There would have been a reasonable expectation of success because Desai suggests that the NaH2PO4, KCl, and choline chloride can be used for culturing bacteria.
Regarding claim 27, Koskinen teaches using cell waste as a nutrient source. See [0208]. Koskinen teaches cultivating Streptomyces G009 in 20 g/l (i.e. 2%) cell waste. See [0212]. Furthermore, Koskinen teaches cultivation medium comprising 1-300 g per liter cell waste or residue. See [0022].
Regarding claim 28, Koskinen, in example 5, teaches cultivating Streptomyces G009 at a temperature of 28˚C, an aeration rate of 0.5-1vvm, and with a stirring intensity of 100-280. See [0218]. Koskinen also teaches an agitation of 0.1-1.5 vvm. See [0171].
Regarding claim 29, Koskinen teaches cultivations carried out in 3 to 10 days. See [0177].
Claims 32 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1) and Reed (US 2020/0165733), as applied to claim 1 above, and further in view of Farmer (WO 2020/257109, as referenced in the action 06/24/2024)
Regarding claims 32, Koskinen teaches bacteria including Bacillus and Pseudomonas. See [0102]. Koskinen suggests that the bacteria are capable of producing lipids. See [0099]. Lipids include glycolipids. See [0043].
Koskinen and Reed do not teach a sophorose glycolipid.
Farmer teaches a method for enhanced production of one or more microbial growth by-products. See claim 1 of Farmer. Farmer teaches biosurfactant producing microorganisms including Pseudomonas and Bacillus. See p. 2 lines 4-5. Farmer teaches glycolipid biosurfactants such as sophorolipids. See lines 15-16 on page 17.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to ferment Koskinen’s Pseudomonas and/or Bacillus (i.e. third fermentation strains), and in the process obtain sophorolipids based on Farmer’s suggestion. One of ordinary skill in the art would have been motivated to do so because Koskinen suggests that Pseudomonas and/or Bacillus produce lipids, which encompassed glycolipids. There would be a reasonable expectation of success because Farmer suggests that sophorolipids are by-products of Pseudomonas and/or Bacillus.
Regarding claim 34, Farmer teaches biosurfactants including mannosylerythritol lipids. See page 5 line 9 and page 17 lines 14-16.
Response to Arguments
Applicant's arguments filed 08/14/2026 have been fully considered to the extent that the apply to the new grounds of rejection set forth above but they are unpersuasive.
Applicant argues that Koskinen’s cell waste is not equivalent to the instantly required “combining the first fermentation broth and the second fermentation broth to form a first mixture”; [and] sterilizing the first mixture to form a sterilized first mixture. Koskinen defines cell waste as what remains after oil extraction. Applicant indicates that the claims require a broth, which is an aqueous mixture, a liquid, whereas Koskinen teaches sludge separated from waste water prior to oil recovery. See the arguments spanning pgs. 12-13 of the remarks.
This argument is not persuasive. The rejection discussed above does not indicate that Koskinen’s cell waste is equivalent to the instantly claimed first mixture. Reed is relied upon for teaching the first fermentation broth. As set forth in the rationale, it would have been obvious to combine Reed’s first fermentation broth with the Bacillus containing second fermentation broth of Koskinen. Koskinen meets the instantly required sterilization step because Koskinen, in example 5, suggests that the medium comprising cell waste is sterilized prior to its use for Streptomyces fermentation (i.e. third fermentation process). See [0218]. Although Applicant argues that Koskinen’s cell waste is sludge not broth, the instant claims do not exclude additional unrecited elements, because claim 1 recites the open-ended term “comprising” in line 1, so the claims do not exclude additional unrecited elements.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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