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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/23/2026 has been entered.
DETAILED ACTION
Claims 2, 13-16, 18, 20, 23-24 and 31 are canceled. Claims 1, 3-12, 17, 19, 21-22, 25-30, and 32-34 are pending and under consideration in this action.
Priority
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, 25-29, and 32-34 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 reasonablyClaim 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 *** 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 *** has introduced new matter into the claims.
Claim *** as filed on *** recites a *ENTIRE CLAIM, NEW MATTER underlined
Applicant’s amendment, filed ***, directs to support to paragraphs *** of the specification filed **, and asserts that no new matter has been added. However, the specification as filed does not provide sufficient written description of the above underlined limitations.
Claims ** contain(s) new matter because of the limitation ___. Claim ** contains new matter because of the limitation of ___
The specification as filed and the original claims do not provide support for these limitations in claims **.
Such limitations recited in the instant claims ** (and 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.
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 03/23/2026 has introduced new matter into the claims.
Claim 1, as filed on 03/23/2026, recites a multi-stage, multi-strain fermentation method, comprising: a first fermentation process, comprising contacting one or more strains of microorganism(s) selected from Pyrococcus, Metallosphaera, Rhodospirillum, Chloroflexus, Cyanobacteria, Chlorella, Dunaliella, Nannochloropsis, Scenedesmus, and Botryococcus, using mineral medium, aeration of 0.01-0.4 vvm, and either light or no light conditions, and with a first primary feed stock wherein carbon dioxide is a primary carbon source, which first fermentation process produces a first fermentation broth; a second fermentation process, comprising contacting one or more strains of microorganism(s) selected from Aurantiacus, 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 with 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; and initiating a third fermentation process, comprising contacting a culture medium comprising the 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 03/23/2026, recites a multi-stage, multi-strain fermentation method, comprising: a first fermentation process, comprising contacting 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 a first primary feed stock comprising carbon dioxide, which first fermentation process produces a first fermentation broth; a second fermentation process, comprising contacting one or more strains of microorganism(s) selected from Aurantiacus, Sordaria, Trametes, Irpex, Lenzite, Phanerochaete, Klebsiella, Ochrobactrum, Dysgonomonas, Sphinpobacterium, Bacteroides, Parabacteroides, Flavobacterium, polymorphonuclear Aeromonas, Pleomorphomonas, Arcticibacter, Elizabethkingia, Neisseria, Mycobacterium, Trichoderma, Zymomonas, Stenotrophomonas, Paenibacillus, Nocardia, Nocardiopsis, Bacillus, Rhizobium, Cellulomonas, Vibrio, Cellvibrio, Cytophaca, Alistipes, Aspergillus, Ruminofilibacter, and Clostridium with 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 with an initial mineral medium pH of 7-8.5, and 2-10% lignocellulosic material by volume as a 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; and initiating a third fermentation process, comprising contacting a culture medium comprising the 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.
Applicant’s amendment, filed 03/23/2026, asserts that no new matter has been added. See p. 8 paragraph 1 of the remarks. However, Applicant has not pointed out where the amended claims are supported, nor does there appear to be a written description of the claim limitations ‘a first primary feed stock wherein carbon dioxide is a primary carbon source’ and ‘2-10% lignocellulosic material by volume as a primary carbon source’ in the application as filed. The original claims and specification do not provide sufficient written description of the above underlined limitations.
Claim 1 and dependent claims 3-12, 17, 19, 21, 26-29 and 32-34 contain new matter because of the limitation that requires a first primary feedstock wherein carbon dioxide is a primary carbon source.
Claim 22 and dependent claim 25 contain new matter because of the limitation that requires 2-10% lignocellulosic material by volume as a primary carbon source in the second fermentation process.
The specification as filed and the original claims do not provide support for the broader limitation in the amended claim 1. Original claim 18 (filed 05/12/2022) recites a first culture condition comprising: culturing the microorganism(s) using mineral medium, carbon dioxide as the main carbon source. Paragraph [0007] of the instant specification teaches a medium for fermentation in which carbon dioxide serves as the main carbon source. In fermentation A of examples 1-6, the specification teaches a first fermentation medium prepared with carbon dioxide as the main carbon source. See ex.1 [0051], ex.2 [0060], ex.3 [0069], ex.4 [0078], ex.5 [0087] and ex.6 [0096]. Under the broadest reasonable interpretation, the scope of the amended claim limitation encompasses a first primary feed stock wherein carbon dioxide is one primary carbon source. However, the original disclosure supports one medium species in which carbon dioxide is the main carbon source. Thus, the original disclosure does not reasonably convey possession of the subject matter of the amendment.
The specification as filed and the original claims do not provide support for the broader limitation in instant claim 22. Original claim 22 (filed 05/12/2022) recites: the microorganism(s) of the second fermentation are cultured using mineral medium, with 2-10% lignocellulose as the main carbon source. Paragraph [0009] of the instant specification discloses that lignocellulosic material serves as the main carbon source. Thus, the original disclosure does not reasonably convey possession of the broad scope encompassed by the amended claim limitation, which requires 2-10% lignocellulose material by volume as a primary carbon source.
Such limitations recited in the instant claims 1 and 22 (and 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.
Claim 1, 3-12, 17, 19, 21-22, 25-30, and 32-34 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: a first fermentation process comprising contacting one or more strains of microorganism(s)…using mineral medium, aeration of 0.01-04 vvm, and either light or no light conditions, and with a first primary feed stock wherein carbon dioxide is a primary carbon source. The term “contacting” renders the claim indefinite because the way in which the mineral medium, aeration and light affect the contact step is unclear. In one interpretation, the first fermentation process comprises culturing one or more strains of microorganism(s) in a mineral medium, with aeration of 0.01-0.4vvm and either light or no light conditions, and wherein carbon dioxide is the primary carbon source. In an alternative interpretation, the first fermentation process comprises contacting one or more strains of microorganism(s) with a primary feed stock wherein carbon dioxide is a primary carbon source of the primary feed stock; and during this contact step a mineral medium is required to be used in anyway, the contact step is required to occur under aeration of 0.01-0.4 vvm, and under either light or no light conditions. As such, one of ordinary skill in the art cannot ascertain the metes and bounds of the required contact step within the first fermentation process.
Claims 1 and 22 recite “mineral medium” (line 5 of claim 1 and line 14 of claim 22), which renders the claims indefinite because, in one interpretation, the mineral medium encompasses any medium containing a mineral, and under an alternative interpretation, the medium is limited to a specific composition. The instant specification teaches the composition of a first fermentation medium in paragraph [0052], the instant specification is however silent regarding the composition of the mineral medium. Therefore, the scope of the claimed mineral medium is unclear. It is unclear what ingredients are required in the claimed medium other than a mineral.
Claims 1, 22 and 30 recite “microorganism(s) selected from Aurantiacus” in line 8, 9 or 10, which render the claims indefinite because it is unclear whether the claim intends to reference the bacterium Chloroflexus aurantiacus, the bacterium Salipaludibacillus aurantiacus, or the flower Diplacus aurantiacus; however, flower D. aurantiacus is not a microorganism.
Claims 3-12, 17, 19, 21, 25-29, and 32-34 depend from claim 1 or 22 and are rejected for the reason set forth above.
Claim 12 recites “the at least one glycolipid or lipopeptide biosurfactant aqueous solution”, which renders the claim indefinite because in one interpretation claim 12 is referencing the “second aqueous solution containing the at least one glycolipid or lipopeptide biosurfactant” recited in claim 7 (last 2 lines), and under an alternative interpretation claim 12 is referencing at least aqueous solution containing glycolipid or lipopeptide.
Claims 26 and 30 recite “inorganic salt medium”. See claim 26 line 2, and claim 30 line 26. The instant specification is silent regarding the composition of the mineral medium. Therefore, it is unclear what ingredients are required in the claimed inorganic salt medium other than an inorganic salt.
Claim 30 recites “the medium” in the second to last line, which render the claim indefinite because it is unclear whether “the medium” intends to reference the inorganic salt medium, or the culture medium in the third fermentation process. Claim 30 recites “further comprises trace elements: Zn, Mn, Ca.” in the last two lines, which renders the claim indefinite because there is no conjunction at the end of the list. Therefore, it is unclear whether the claim is complete. To obviate this rejection, “Mn, Ca” can be amended to “Mn, and/or Ca”.
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 26-29 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1).
Regarding claims 1, Koskinen teaches cultivating autotrophic microorganisms in closed bioreactors. Gases containing concentrated CO2 (i.e. first primary feed stock) 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. 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 culture medium E05, which includes MgSO4 (i.e. Mg mineral). See [0203]. 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. However, Koskinen teaches an E05 medium that includes a mineral.
Koskinen does not teach combining a first fermentation broth and a second fermentation broth to form a first mixture. However, Koskinen teaches cell waste from previous fermentation.
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 use the E05 mineral containing medium of Koskinen for the autotrophic Cyanobacteria cultivation, and to further substitute cells including Cyanobacteria and the Bacillus cells for the cell waste in the Streptomyces fermentation. One of ordinary skill in the art would have been motivated to use the E05 mineral containing medium for the autotrophic Cyanobacteria cultivation, because Koskinen suggests that the medium contains pure components. See [0168]. There would have been a reasonable expectation of success because Koskinen demonstrates a fermentation using the E05 culture medium in example 2 (see [0202]). One of ordinary skill in the art would have been further motivated to substitute the cells for the cell waste in the Streptomyces fermentation, because Koskinen suggests that cell waste can be used to enhance the maximum growth in economical and low-cost cultivation conditions See [0208]. 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 lips. See [0052]. Lipids include glycolipids (i.e. a biosurfactant). See [0043].
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 26, Koskinen teaches a culture medium E05, which includes MgSO4 (i.e. an inorganic salt). See [0203]. In example 5, Koskinen teaches cultivating Streptomyces (i.e. a third fermentation strain) G009 in cell waste (i.e. carbon source). Streptomyces cells from previous fermentations are harvested by filtration, and oil-extracted cell residue is supplied to the fermentation broth. See [0217]. Koskinen teaches using cell waste as a nutrient source. See [0208].
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 7-12 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1), as applied to claims 1, 3-6, 17, 19 and 21 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 does 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 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 Farmer (US 2021/0267214, filed 10/08/2019), 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 glucose, CaCl2 · 2H2O and trace elements including ZnSO4 ·7H2O and MnSO4 ·H2O (i.e. Zn, Mn, Ca elements). 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 contacting 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 a first primary feed stock comprising carbon dioxide, which first fermentation process produces a first fermentation broth.
Ramírez does not teach a second fermentation process comprising an initial mineral medium pH of 7-8.5.
Farmer teaches reducing deleterious atmospheric gas comprising applying a composition to a source of the deleterious atmospheric gas. See claim 26 of Farmer. The deleterious gas includes carbon dioxide. See claim 33 of Farmer. The composition comprises a bacterium selected from a group that includes Pseudomonas chlororaphis. See claim 31 of Farmer. Furthermore, Farmer teaches P. aeruginosa. See [0194]. Farmer suggests that such application can have an effect on the site, for example by action of biosurfactant. See [0084]. Farmer discloses that the pH of the microbe-based composition should be suitable for the microorganism. The pH of the composition is about 3.5 to 7. See [0158].
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 the carbon dioxide deleterious atmospheric gas of Farmer to the Pseudomonas inoculated seed culture 1 of Ramírez, to further modify the pH of the B. subtilis batch culture of Ramírez in view of the 3.5 to 7 pH taught by Farmer. One of ordinary skill in the art would have been motivated to apply the carbon dioxide deleterious atmospheric gas of Farmer because Farmer suggests that there exists a need to reduce greenhouse gases, especially CO-2 to slow the detrimental effects of global warming. See [0016]. There would have been a reasonable expectation of success because Farmer suggests that P. aeruginosa may be useful in a composition for reducing carbon dioxide atmospheric gas. One of ordinary skill in the art would have been further motivated to apply the pH of Farmer to the B. subtilis batch culture of Ramírez, because Farmer teaches B. subtilis, and Farmer further suggests that a pH of 7 may be suitable for the microorganism. There would have been a reasonable expectation of success because Ramírez demonstrates a B. subtilis batch culture (i.e. a second fermentation process) in which 2%, 5% or 10% w/v of lignocellulosic OMW is the sole carbon source, and the batch culture is maintained at 37˚C and 160 rpm.
Ramírez and Farmer do not teach sterilizing a first mixture formed by the combination of a first fermentation broth and a second fermentation broth.
Ramírez and Farmer 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 and 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 32-34 are rejected under 35 U.S.C. 103 as being unpatentable over Koskinen (EP 2 546 352 A1), as applied to claims 1, 3-6, 17, 19, 21 and 26-29 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 does 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 33, Farmer teaches biosurfactants that are rhamnolipids. See claim 9.
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 03/23/2026 have been fully considered but they do not apply to the new grounds for rejection discussed above.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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