DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 01/20/2026 has been entered.
Status of claim rejections
The rejection of record under 35 USC 112(a) is maintained/modified in view of Applicant’s arguments in the response filed 01/20/26.
The rejection of record under 35 USC 112(b) is maintained/modified in view of Applicant’s arguments in the response filed 01/20/26.
The rejections of record under 35 USC 103 are maintained/modified in view of Applicant’s arguments in the response filed 01/20/26.
Claim Objections
Claim 95 objected to because of the following informalities: Claim 95 recite “a method of claim 95.” It is suggested for Applicant to amend the claim to recite “The method” for consistency with the other claims. Appropriate correction is required.
Maintained/Modified 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 1-2, 4-13, 17-18, 21-23, 85-91, and 94 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.
The term “at or below a threshold concentration” in claim 1 and 94 is a relative term which renders the claim indefinite. The term “threshold concentration” 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.
Here, the instant claims require “generating NH3 with the one or more microorganisms in the bioreactor; and maintaining a concentration of the NH3 at or below a threshold concentration.” It is unclear what this “threshold concentration” of NH3 would be in the bioreactor, such that one of ordinary skill would not understand the concentration of NH3 that must be maintained inside of the bioreactor. The specification only speculates that “production of a desired end product by bacteria located within the solution may be controlled by limiting a concentration of bioavailable nitrogen, such as in the form of ammonia, amino acids, or any other appropriate source of nitrogen useable by the bacteria within the solution to below a threshold nitrogen concentration. However, and without wishing to be bound by theory, the concentration threshold may be different for different bacteria and/or for different concentrations of bacteria.” (see paragraph 100 of the specification as instantly filed). As such, the claim is indefinite. For the purposes of compact patent prosecution, the examiner has interpreted the limitation of “maintaining a concentration of the NH3 at or below a threshold concentration” to encompass maintenance of the concentration of NH3 to any concentration (including any de minimis amount of NH3). Note that the other dependent claims are also indefinite for dependency on indefinite claim 1.
Claim 91 recites, inter alia, “wherein the one or more microorganisms comprises a nucleotide sequence as set forth in NCBI reference sequence NC_009720.1.” It is unclear how one or more microorganisms can comprise a nucleotide sequence of a single species (e.g., a species of X. versatilis). Furthermore, it is unclear how a microorganism can comprise a nucleotide sequence, unless the microorganism contains that nucleotide sequence. Thus, the claim is indefinite. For the purposes of compact prosecution, the examiner is interpreting the microorganism to contain the nucleotide sequence.
Response to Arguments
Applicant’s arguments filed 01/20/26 have been fully considered but they are not persuasive.
On pg. 8-9 of the remarks, Applicant argues that the Specification provides sufficient examples for characterizing and determining suitable threshold concentrations. Applicant argues “paragraph [0100] of the instant Application provides an example of threshold concentrations of bio-available nitrogen at a variety of optical densities of microorganisms. In view of this, a person of ordinary skill in the art would be capable of ascertaining whether a threshold concentration is met for the claimed species of microorganism and other microorganisms”, such that a specific threshold concentration is not required.
In response, the examiner disagrees. As set forth in the rejection above, the specification only speculates what the threshold concentration could be based on an exemplary embodiment using a single microorganism species. This is not enough to provide one of ordinary skill enough information to deduce what the threshold concentration of NH3 would be as it relates to the claimed invention, as well as what would be considered “at or below” the threshold. As such, the rejection is maintained as set forth above.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 95 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 95 recites, inter alia, “A method of claim 95, wherein the one or more microorganisms comprise Xanthobacter autotrophicus.” As written, claim 95 is dependent on itself. A claim dependent on itself cannot further limit the subject matter previously recited in the claims. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements. For the purposes of compact patent prosecution, the examiner is interpreting the claim to be dependent on claim 92.
Modified 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 1-2, 4-13, 17-18, 21-23 and 85-100 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 written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the inventor was in possession of the claimed genus. A “representative number of species” means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014). The issue is whether the skilled artisan would understand inventor to have invented, and been in possession of, the invention as claimed.
Claim interpretation: The independent claims (and thus the dependent claims) requires “a method of producing a biofertilizer in a bioreactor, comprising: a) generating H2 in a bioreactor comprising one or more microorganisms which express a hydrogenase and a nitrogenase, wherein the bioreactor further comprises a source of N2 and CO2; and (b) growing the one or more microorganisms in the bioreactor in culture media in the presence of the H2 to produce a biofertilizer, wherein glutamine synthetase, in the one or more microorganisms, is inhibited by an inhibitor comprising phosphinothricin, wherein the one or more microorganisms comprise Xanthobacter autotrophicus and wherein the one or more microorganisms exhibit a resistance to reactive oxygen species (ROS); generating NH3 with the one or more microorganisms in the bioreactor; and maintaining a concentration of the NH3 at or below a threshold concentration.”
Instant claim 21-23 recites a massive genus of microorganisms (e.g., bacteria, archaea, fungi, Acidiphilum species, Athrobacter species, etc.) in any combination within the bioreactor.
The instant specification as published describes one or more mutant microorganisms, which may comprise one or more beneficial phenotypes or traits, e.g., resistance to reactive oxygen species (RORs). The term “mutant” refers to a microorganism obtained by direct mutant selection but also includes microorganisms that have been further mutagenized or otherwise manipulated (e.g., via the introduction of a plasmid). Accordingly, embodiments include mutants, variants, and or derivatives of the respective microorganism, both naturally occurring and artificially induced mutants. For example, mutants may be induced by subjecting the microorganism to known mutagens, such as N-methyl-nitrosoguanidine, using conventional methods. Conventional methods are available for obtaining or otherwise constructing desirable mutants of any bacteria or microorganism (see para 0131). Based on the specification, the examiner has interpreted the “microorganism exhibits a resistance to reactive oxygen species” to encompass any microorganism (either naturally occurring or microorganisms containing nebulous mutations) capable of exhibiting resistance to ROS as a beneficial phenotype (including the claimed X. autotrophicus). The question at issue is whether the skilled artisan would have understood Applicant to have been in possession of the massive genus of microorganisms as encompassed by the instant claims.
Reduction to practice and disclosure of drawings or structural chemical formulas: Applicant discloses in the specification the cultivation and use of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus in a bio-electrochemical reactor to make biofertilizer for growth of radish seeds (see Examples, para 00170-00207; 00213-14).
Sufficient, relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure: Applicant has not provided information as to the defining structural characteristics that would lead one of ordinary skill in the art to the identity of a microorganism capable of exhibiting a resistance to reactive oxygen species as claimed. Applicant’s claims encompass a massive genus of microorganisms (bacteria, archaea, algae, fungi, etc.) including microorganisms resistant to ROS naturally or via induction of artificial mutations. However, Applicants have not disclosed a single embodiment of a mutant capable of such an ability other than speculative embodiments in the specification. None of the instant claims further characterize such a microorganism, and as such Applicant does not have requisite support in the specification for such microorganisms. Applicant has claimed the microorganisms present in the bioreactor based on ability to resist ROS, however this indicates what the microorganism(s) are able to do (function), not what it is (relevant structure or identification). Even with knowledge in the art regarding the mutation of microorganisms generally, one of ordinary skill would not know what structural features of the microorganisms are required for the outcome of “exhibiting a resistance to reactive oxygen species” without a recognized correlation between structure and function. This is not sufficient to meet the written description requirement. The specification does not provide adequate disclosure for microorganisms with that much variation for use in a bioreactor.
Lack of support for massive genus of microorganisms resistant to ROS: The specification fails to teach and/or provide support for possession of the microorganisms (mutant or otherwise), as there is no correlation or defining characteristic that would convey the identity of them with the ability to perform the function as broadly claimed.
Applicant has provided no support of the claimed genus of ROS-resistant microorganisms within the specification. Applicant has only demonstrated possession of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus, and more specifically X. autotrophicus for use in a bioreactor for making biofertilizer (see Examples as well as Figs. 5-11). However, there is no support in the specification for the use of any microorganism that is resistant to ROS. Applicant’s claim broadly encompasses any microorganism (including X. autotrophicus) having some nebulous natural or artificial mutations that would somehow exhibit ROS resistance, however the specification only provides support for the use of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus. Furthermore, even though there is support for these specific microorganisms in the specification, Applicant has not identified or provided any data that shows whether any of the microorganisms utilized are capable of the ROS-resistant function (including X. autotrophicus) as instantly claimed. Thus, the data generated for the use of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus disclosed in the specification cannot reasonably be extrapolated to and applied to support possession of an entire claimed genus of microorganisms resistant to ROS as claimed, because no one species, combination, or variant accounts for the variability amongst the claimed genus. As in Ariad, merely drawing a fence around the outer limits of a purported genus is not an adequate substitute for describing a variety of materials constituting the genus and showing that one has invented a genus and not just a species. “A patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.” Brenner v. Manson, 383 U.S. 519, 536 (1966).
Lack of possession of one or more microorganisms comprising a nucleotide sequence as set forth in NCBI reference sequence NC_009720.1:
Instant claim 91 also recites “wherein the one or more microorganisms comprises a nucleotide sequence as set forth in NCBI reference sequence NC_009720.1.” This is extremely problematic because the specification does not disclose the requisite possession of sequence(s) that comprise the accession number. The material identified by the NCBI reference sequence number is subject to change. This means that the number indicated within the disclosure is arbitrary and does not establish possession of the claimed nucleotide sequence. To this point, reference sequence NC_009720.1 has been removed/suppressed from the NCBI website by RefSeq staff and the only record available is of the obsolete version (see below).
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MPEP 608.01(p) states, in part: "Essential material" may be incorporated by reference, but only by way of an incorporation by reference to a U.S. patent or U.S. patent application publication, which patent or patent application publication does not itself incorporate such essential material by reference. "Essential material" is material that is necessary to: (1) Provide a written description of the claimed 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 set forth the best mode contemplated by the inventor of carrying out the invention as required by 35 U.S.C. 112(a); (2) Describe the claimed invention in terms that particularly point out and distinctly claim the invention as required by 35 U.S.C. 112(b); or (3) Describe the structure, material, or acts that correspond to a claimed means or step for performing a specified function as required by 35 U.S.C. 112(f). Merely including reference to a nucleotide sequence from an accession number in a claim is not sufficient to meet the written description requirement.
The specification, then, is considered devoid of sufficiently detailed, relevant, identifying characteristics demonstrating that Applicant was in possession of the claimed genus of subject(s) in need thereof, i.e., additional complete or partial structures, other physical and/or chemical properties, functional characteristics coupled with a known or disclosed correlation between function and structure, or some combination thereof demonstrating possession of the claimed genus.
Response to Arguments
Applicant’s arguments filed 01/20/26 have been fully considered but they are not persuasive.
On pg. 9-10 of the remarks, Applicant argues that the Office has ignored the description of the instant specification related to ROS resistance. Applicant argues that paragraph 0131 and response to the previous Office Action state that the microorganisms described may include “one or more microorganisms whihch comprise one or more beneficial phenotypes or traits”, goes on to state “mutants may be induced by subjecting the microorganism to known mutagens . . . using conventional methods”, and that microorganisms that exhibit ROS resistance may be capable of producing PHB. From this, Applicant urges that they have sufficient disclosure on how to develop bacteria that exhibit resistance to reactive oxygen species.
In response, the examiner disagrees. As set forth above, Applicant has provided no support of the claimed genus of ROS-resistant microorganisms within the specification. Applicant has only demonstrated possession of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus, and more specifically X. autotrophicus for use in a bioreactor for making biofertilizer (see Examples as well as Figs. 5-11). However, there is no support in the specification for the use of any microorganism that is resistant to ROS. Applicant’s claim broadly encompasses any microorganism (including X. autotrophicus) having some nebulous natural or artificial mutations that would somehow exhibit ROS resistance, however the specification only provides support for the use of X. autotrophicus 7CT, B. japonicum, R. eutropha, and V. paradoxus. Furthermore, even though there is support for these specific microorganisms in the specification, Applicant has not identified or provided any data that shows whether any of the microorganisms utilized are capable of the ROS-resistant function (including X. autotrophicus) as instantly claimed. Mere cursory statements regarding general, conventional mutagenesis techniques are not enough to provide adequate support for the genus of bacteria, archaea, and fungi containing this ROS resistant function as instantly claimed. Thus, the rejection is maintained as set forth above.
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 (i.e., changing from AIA to pre-AIA ) 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.
First rejection
Claims 1-2, 4-13, 17-18, 21-23, 85-86, 92-100 are rejected under 35 U.S.C. 103 as being unpatentable over Reed et al. (US 2020/0165733, prior art of record, hereinafter “Reed”) in view of Torella et al. (PNAS, 112(8):2337-2342 (2015), IDS-NPL, hereinafter “Torella”; prior art of record), Colnaghi et al. (Plant Soil, 194:145-154 (1997), IDS-NPL, hereinafter “Colnaghi”; prior art of record), Berlicki et al. (Bioorg. Medic Chem., 14:4578-4585 (2006), hereinafter “Berlicki”; prior art of record) as evidenced by Schink (FEMS Microbiol. Let., 13(3):289-293 (1982), prior art of record) and Al Rowaihi et al. (Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions. PloS One. 2018 Apr 26;13(4):e0196079; hereinafter “Al Rowaihi”; prior art of record).
Reed teaches a method of producing a biofertilizer in a bioreactor comprising growing one or more microorganisms in the bioreactor in a culture media to produce the biofertilizer (para 0044-0045, 0060, 0062, 105-106). Reed further teaches that the bioreactor comprises a source of nitrogen gas (N2), hydrogen gas (H2), and CO2 (para 0236-0238, 0249).
Reed teaches that the biofertilizer is a microbial biomass, e.g., a biomass and culture media (paras. 0002-0003, 0200, 0220). Reed also teaches that the biofertilizer is a protein-rich biomass, i.e., enriched with a carbon energy source (para 0045). Reed teaches liquid or solid culture medium to produce biomass as well as drying biomass (solid) and liquid suspensions of biomass (paras 0123, 0149, 0190, 0220, 0315-0136).
Instant claim 1 further recites that the one or more microorganisms express a hydrogenase and a nitrogenase. Reed teaches identical genera and species of microorganisms as recited by instant claims 1 and 23, including Xanthobacter autotrophicus, Alcaligenes species, Alcaligenes paradoxus, Arthrobacter species, Azospirillum species, Azospirillum lipoferum, Beggiatoa species, Cupriavidus necator, Derxia species, Hydrogenophaga species, Hydrogenophaga pseudoflava, Microcyclus species, Microcyclus aquaticus, Nitrosococcus species, Nocardia species, Nocardia autotrophica, Nocardia opaca, Paracoccus species, Paracoccus denitrificans, Pseudomonas species, Pseudomonas facilis, Ralstonia species, Ralstonia eutropha, Renobacter species, Renobacter vacuolatum, Rhizobium species, Rhizobium japonicum, Rhodobacter species, Rhodobacter sphaeroides, Rhodomicrobium species, Variovorax species, and Variovorax paradoxus (see e.g., para 0047, 0190-0192, 0195-0200). Reed also teaches that the microorganisms comprise bacteria, archaea, or fungi, and can naturally accumulate polyhydroxybutyrate (PHB) (para 0175, 0194, 0200, 0218, 0262).
As evidenced by Schink, Xanthobacter autotrophicus (claims 19 and 23) expresses both hydrogenase and nitrogenase (pg. 289, Introduction). Schink further evidences that X. autotrophicus can grow autotrophically with hydrogen, oxygen, carbon dioxide, and molecular nitrogen as sole sources of electrons, energy, carbon, and nitrogen; fixes carbon dioxide via the Calvin cycle; and utilizes hydrogenase to supply electrons for reduction of nitrogen by nitrogenase (pg. 289, Introduction). As evidenced by Colnaghi, diazotrophs—i.e., nitrogen fixing bacteria—convert N2 to NH3 by electron reduction and protonation of gaseous dinitrogen, which is further assimilated by glutamine synthetase (pgs. 145-146, Introduction).
As such, absent evidence to the contrary, the microorganisms taught by Reed, which are identical to those recited by the instant claims, would inherently express hydrogenase and nitrogenase, and exhibit the same properties as those recited by instant claims 1-3 and 6. Moreover, use of more than one of the microorganisms taught by Reed would reasonably encompass hydrogenase and nitrogenase expression from different microorganisms since each microorganism would individually express species-specific versions of the enzymes.
Reed does not explicitly teach that one or more of the microorganisms present in the bioreactor exhibits a resistance to reactive oxygen species. However, Al Rowaihi further evidences that PHB is produced in native and engineered microorganisms by accumulating as granules in the cytoplasm in response to conditions of physiological stress, and cells with high PHB content have enhanced survival and tolerance toward oxidative stress/damage caused by reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anion, and hydrogen peroxide (pg. 2, para 2-3). Al Rowaihi further evidences that microorganisms such as Methylobacterium extorquens and Cupriavidus necator (previously known as Ralstonia eutropha) are well-studied strains of microorganisms that produce PHB (pg. 2, para 2-3).
As such, absent evidence to the contrary, at least one of the microorganisms taught by Reed (i.e., R. eutrophica as instantly claimed) would inherently be resistant to reactive oxygen species (the property required of instant claim 1) as Al Rowaihi evidences that the production of PHB by R. eutrophica confers increased ROS resistance and enhanced survival and tolerance toward oxidative stress/damage.
Reed teaches that “[c]ertain embodiments of the present invention leverage intermittent renewable sources of power, such as solar and wind, to produce the H2 required for carbon fixation” (para 0449). More particularly, Reed teaches use of an electrolyzer to produce H2 from water for use in the bioreactor (para 0449). The reference does not explicitly teach generating H2 in the bioreactor. However, it would have been prima facie obvious at the time of filing to generate H2 in the bioreactor taught by Reed because Torella teaches H2 generation in a bioreactor, using cathode and anode water-splitting catalysts, which can be utilized by microorganisms to produce, e.g., biomass (Abstract; FIG. 1, produced below).
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One of ordinary skill in the art would have been motivated to modify Reed in view of Torella in order to advantageously produce a renewable supply of H2 in the bioreactor taught by Reed with a reasonable expectation of success.
Reed and Torella do not explicitly teach a step of inhibiting glutamine synthetase, thereby inhibiting assimilation of NH3 into intracellular biomass in favor of extracellular NH3 accumulation in the bioreactor culture media.
However, Colnaghi teaches diazotrophs—i.e., nitrogen fixing bacteria—convert N2 to NH3 by electron reduction and protonation of gaseous dinitrogen, which is further assimilated by glutamine synthetase (GS) (pages 145-146, Introduction). Colnaghi further teaches that genetic or biochemical interference with GS activity results in extracellular NH3 accumulation (page 147, first column). Colnaghi also teaches that GS activity can be prevented by specific chemical inhibitors such as methionine sulfoximine (MSX) (page 146, second column). Berlicki teaches that MSX and phosphinothricin (PPT) were well-known, potent inhibitors of GS (page 4578, second column).
Therefore, one of ordinary skill in the art would have been motivated to modify Reed and Torella to include an inhibitor of GS, including PPT, which Berlicki teaches is a potent GS inhibitor, in order to advantageously enrich the biofertilizer taught by Reed and Torella with ammonia (NH3) with a reasonable expectation of success.
Furthermore, the maintenance of a concentration of NH3 in the bioreactor (as instantly claimed) would have been the result of routine optimization using standard laboratory techniques available at the time of filing, as Reed teaches that the production and distribution of molecules produced by the microorganisms can be optimized by control of bioreactor conditions, control of nutrient levels, genetic modifications of the cells by maintaining specific growth conditions (e.g. levels of nitrogen, oxygen, phosphorous, sulfur, trace micronutrients such as inorganic ions) (see paragraph 262-263) (see MPEP 2144.05).
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Second rejection
Claims 87-89 are rejected under 35 U.S.C. 103 as being unpatentable over Reed, Torella, Colnaghi, and Berlicki as applied to claims 1-2, 4-13, 17-18, 21-23, 85-86, and 92-100 above, and further in view of Jiang et al ((2015), Electrodeposited Cobalt-Phosphorous-Derived Films as Competent Bifunctional Catalysts for Overall Water Splitting. Angew. Chem. Int. Ed., 54: 6251-6254; hereinafter “Jiang”).
As discussed above, the claims were rendered prima facie obvious by the combined teachings of Reed, Torella, Colnaghi, and Berlicki.
As further discussed above, Torella teaches a bioreactor that has a Co-Pi water-splitting anode, with NiMoZn and stainless steel (SS) cathodes (see Fig. 1 of Torella reproduced below; and pg. 2338, col 1, para 2).
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The difference between the references and the instant claims is that none of the references explicitly teach that the cathode comprises a cobalt-phosphate (Co-P) alloy catalyst (as in claim 87).
However, Jiang teaches cobalt-phosphorus derived films as competent bifunctional catalysts for water splitting (title, abstract). Jiang specifically teaches Co-P films can be directly utilized as electrocatalysts for both HER and OER (H2 and O2 evolution reactions, respectively) in strong alkaline electrolytes and alkaline medium solutions and can be employed as a catalyst on both anode and cathodes for overall water splitting with 100% Faradaic efficiency, rivalling the integrated performance of Pt and IrO2 cathode/anode configurations (see abstract, pg. 6253, col 1-2).
Therefore, one of ordinary skill would have been motivated to modify the bioreactor of Reed, Torella, Colnaghi, and Berlicki with the Co-P alloy catalyst cathode of Jiang in order to advantageously effectuate water splitting for H2 and O2 evolution reactions with 100% Faradaic efficiency that performs better than known cathode/anode configurations.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Third rejection
Claim 91 is rejected under 35 U.S.C. 103 as being unpatentable over Reed, Torella, Colnaghi, and Berlicki as applied to claims 1-2, 4-13, 17-18, 21-23, 85-86, and 92-100 above, and further in view of Xanthobacter autotrophicus Py2, complete genome GenBank: CP000781.1; available online 28 Jan 2014; retrieved Jan 29, 2026 from https://www.ncbi.nlm.nih.gov/nuccore/CP000781.1).
As discussed above, the claims were rendered prima facie obvious by the combined teachings of Reed, Torella, Colnaghi, and Berlicki.
None of the references explicitly teach the microorganism comprises NCBI Reference Sequence: NC_009720.1.
However, CP000781.1 teaches a nucleotide sequence of Xanthobacter autotrophicus Py2, which is identical to NCBI Reference Sequence: NC_009720.1 (whose record is suppressed; see 112(a) rejection above). It would have been prima facie obvious to one of ordinary skill at the time of filing to modify the method of over Reed, Torella, Colnaghi, and Berlicki by using the nucleotide sequence of X. autotrophicus as disclosed by CP000781.1 to advantageously produce a biofertilizer in a bioreactor (such as the one disclosed by Reed) using a known bacterial species.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments filed 01/20/26 have been fully considered but they are not persuasive.
On pg. 10-14, Applicant argues that the motivation to combine Torella with Reed is based on improper hindsight. Applicant argues that the references are directed to different applications with different goals and mechanisms of operation that cannot be combined because Reed describes biosynthetic production of amino acids, proteins, and other biological nutrients while Torella describes development of an integrated bioelectrochemical system where R. eutropha is used to convert hydrogen and oxygen produced from water splitting into biomass and fusel alcohols. Applicant concedes that both references describe using C1 substrates but argues that the purpose of Reed is to consume H2 and hydrogen and is only generated by Reed as a separate feedstock, nor does it disclose needing to do so within a bioreactor. Applicant argues Fig. 32 of Reed shows that the generation of hydrogen is separate from the microorganisms while the microorganisms of Torella are in the same container as an opposite arrangement from Reed. Applicant argues Reed intentionally and precisely controls the amount of oxygen but that because oxygen production is inherently tied to hydrogen production via water splitting, precise control of amounts of oxygen would not be possible if it was generated in the bioreactor of Reed using the electrolyzer of Torella. Applicant urges that Torella describes alcohol and fuel production such that one of ordinary skill would not look to the method of Torella, such that the rejection is based on cherry-picking from the references. Finally, Applicant argues the combination of Reed and Torella would leave Reed unsatisfactory for its intended purpose because the microorganisms of Reed are chemoautotrophic (such that the level of oxygen must be controlled otherwise the organisms would die) versus the bioreactor of Torella that does not control amounts of oxygen.
In response, the examiner disagrees. First, in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). As outlined above, Reed specifically teaches the use of at least one microorganism resistant to ROS (see rejection above), as Al Rowaihi evidences that C. necator (i.e., R. eutropha, one of Applicant’s claimed species) is capable of producing PHB, which confers enhanced survival and tolerance toward oxidative stress/damage caused by reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anion, and hydrogen peroxide (pg. 2, para 2-3). Reed also explicitly teaches that the production of the biomass occurs in a bioreactor, and Torella provides one of ordinary skill the teaching, suggestion, and motivation to the use the water-splitting bioreactor to advantageously produce a renewable supply of H2 in the bioreactor taught by Reed. Furthermore, the use of the bioreactor of Torella would not render the teachings of Reed unfit for its intended purpose because at least one of the microorganisms (as required by the instant claims) has, absent evidence to the contrary, resistance to ROS. Second, Reed only discloses that the amount of oxygen can be controlled as part of the preferred embodiments, not that it must be controlled. One of ordinary skill would have readily understood the disclosure of Reed to disclose that the control of oxygen is but one of many options as to maintenance or production of biomass in the method. Furthermore, the prior art teachings of Reed and Torella in combination provides one of ordinary skill the teaching, suggestion, and motivation to create biofertilizer in a bioreactor with specific microorganisms, such as the ones claimed, without Applicant’s disclosure. As such, the rejections are maintained as set forth above.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Gómez, M.A., Rodelas, B., Sáez, F. et al. Denitrifying activity of Xanthobacter autotrophicus strains isolated from a submerged fixed-film reactor. Appl Microbiol Biotechnol 68, 680–685 (2005).
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/G.C.R./Examiner, Art Unit 1651
/THOMAS J. VISONE/Supervisory Patent Examiner, Art Unit 1672