BIOFILM COMPOSITIONS WITH IMPROVED STABILITY FOR NITROGEN FIXING MICROBIAL PRODUCTS

Application No. 17/287,377

• Filed: Apr 21, 2021
• Examiner: ESPINOSA, CLAUDIA EDILMA
• Art Unit: 1654
• Tech Center: 1600 — Biotechnology & Organic Chemistry
• Assignee: Pivot Bio Inc.
• OA Round: 5 (Non-Final)

Office Action

DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/11/2025 has been entered.
Election/Restriction
Applicant’s election without traverse of Group I (i.e., claims 1-7, 11, 16, 24, 26, 30, 40-41 and 87 drawn to a composition comprising one or more isolated bacteria and one or more biofilms or isolated biofilm compositions produced by one or more microbes) and for Species A: a specific bacteria (i.e., Klebsiella, claims 1-6, 11, 24, 26, 30, 40-41), a specific nucleic acid sequence (i.e., SEQ ID NO: 193 comprising a deletion of nifL , claim 41), non-intergeneric remodeled bacteria (i.e., claims 24 and 26) and biofilms produced by Kosakonia (i.e., claims 1 and 7) in the reply filed on February 7th, 2024 is acknowledged.
Claims 43-49, 53-54, 58, 66, 74, 84-85 and 90 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on February 7th 2024.

Status of Claims
Claims 1-92 were originally filed and amended on April 21st 2021. The amendment cancelled claims 8-10, 12-15, 17-23, 25, 27-29, 31-39, 42, 50-52, 55-57, 59-65, 67-73, 75-83, 86, 88-89 and 91-92, and amended claims 1-2, 16, 43-44, 58, 87 and 90.
The amendment filed on July 9th 2024, amended claims 1, 16 and 30; and added new claims 93-96.
The amendment filed on December 12th 2024, amended claims 1, 43 and 93.
The claim set filed on December 11, 2025 did not amend, delete or add any claims.
Claims 1-7, 11, 16, 24, 26, 30, 40-41, 43-49, 53-54, 58, 66, 74, 84-85, 87, 90 and 93-96 are currently pending and claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96 are under consideration.
Priority
The present application claims status as a 371 (National Stage) of PCT/US2019/059450 filed November 1st 2019, and claims the benefit under 35 U.S.C 119 (e) to U.S. Provisional Application No. 62/754,468 filed November 1st 2018. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C 119 (e) or under 35 U.S.C 120, 121, or 365 (c) is acknowledged.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 12/11/2025 has been considered by the Examiner.
Sequence Interpretation
Regarding claim 41, please note that the Examiner is interpreting the scope of the nucleic acid sequence as open-ended requiring at least about 90%, 95% or 99% identity to SEQ ID NO:193 (note: elected by Applicants as the isolated bacteria, and thus interpretation limited to Applicants’ elected species). Since SEQ ID NO: 193 is 613 nucleic acids in length, a sequence that is at least about 90% identical would encompass up to 61 modifications including any insertions, substitutions, deletions etc., a sequence that is at least about 95% identical would encompass up to 30 modifications including any insertions, substitutions, deletions etc., and a sequence that is at least about 99% identical would encompass up to 6 modifications including any insertions, substitutions, deletions etc.
Claim Interpretation
For purposes of applying prior art, the claim scope has been interpreted as set forth below per the guidance set forth at MPEP § 2111. If Applicant disputes any interpretation set forth below, Applicant is invited to unambiguously identify any alleged misinterpretations or specialized definitions in the subsequent response to the instant action. Applicant is advised that a specialized definition should be properly supported and specifically identified (see, e.g., MPEP § 2111.01(IV), describing how Applicant may act as their own lexicographer).
Regarding claim 1, regarding the scope of “composition”, it is noted that the instant specification does not define what constitutes “composition”. Pursuant to MPEP 2111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. Oxford English Dictionary defines “composition” as the forming (of anything) by combination of various elements, parts, or ingredients (see Oxford English Dictionary at https://www.oed.com/dictionary/composition_n?tab=meaning_and_use#8850634, accessed on 01/29/2026). As such, the Examiner is interpreting the scope of “composition” in relation to one or more isolated biofilm composition(s) as a combination of various elements, parts, or ingredients that form the biofilm, being one or more species of microbes/microorganisms, cells, extracellular polymeric substances and/or inorganic particles.
Response to Arguments
1. Applicant's arguments, see Response, filed 12/11/2025, with respect to 35 U.S.C. 103, as being unpatentable over WO2018/132774 A1 (Temme et al.), International Publication Date 19 July 2018 (herein after “774”)(cited in the IDS filed on 12/08/2021), WO2017/208237 A1 (Shemesh), International Publication Date 07 December 2017 (herein after “Shemesh”), and Balasundararajan et al., Journal of Basic Microbiology, 2019, vol. 59, pp. 1031-1039 (herein after “Balasundararajan”), as evidenced by Feoktistova et al., Cold Spring Harb Protoc. 2016, 2016(4), pp. 343-347 (herein after “Feoktistova”); have been fully considered but they are not persuasive. The 35 U.S.C. 103 rejection to claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96 has been maintained.
New Rejections
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.

The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.

1. Claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96 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, is drawn to “[a] composition to reduce bacterial cell viability loss during liquid or dry storage, comprising:
(i) one or more isolated bacteria, and
(ii) one or more isolated biofilm compositions produced by one or more microbes;
wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it, and are non-native to the one or more isolated bacteria, and wherein the one or more isolated bacteria exhibit less viability loss in liquid or dry storage for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm compositions stored under the same conditions.”
It is understood that the instantly claimed composition is formed by combining two different constituents. The first being one or more isolated bacteria, and the second being one or more isolated biofilm compositions produced by one or more microbes. It is also understood that the one or more microbes/microorganisms that produce the one or more isolated biofilm compositions are non-native to the one or more isolated bacteria (i.e., constituent (i)). However, the second constituent (i.e., one or more isolated biofilm compositions) has been separated from the microorganism that produced it. In other words, the second constituent is a biofilm that lacks the microorganisms or cells that produced it.
Nonetheless, it has not been clearly defined and/or established whether separating the microbes/microorganisms that produce the isolated biofilm composition disrupts the integrity of the biofilm composition, and if it so, would the isolated composition still be considered a biofilm. Since the term biofilm, as described in the instant specification, refers to associated and/or accumulated and/or aggregated microbial cells, their products (e.g., exopolymeric substances) and inorganic particles adherent to a living or inert surface (see instant specification, pg. 11, para[0081]), and since the term composition is being interpreted as a combination of various elements, parts, or ingredients that form something (i.e., the biofilm). Thus, separating the microorganisms/microbes that produce the one or more isolated biofilm compositions would result in a complex or matrix or just a film/structure. Therefore, the claims are indefinite because the wherein clause reciting the separation of the one or more isolated biofilm compositions from the microorganisms that produced is ambiguous.
An ordinary skilled artisan would not be able to ascertain the metes and bounds of the claimed composition to reduce bacterial cell viability loss during liquid or dry storage, because it is not clear whether the second constituent (i.e., one or more isolated biofilm compositions produced by one or more microbes), is a biofilm, a matrix, a complex, a film, or simply the exopolymeric substances (i.e., extracellular polymeric substances) produced by the separated microorganisms or by different/unseparated microbes/microorganisms that also form the isolated biofilm.

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.

2. Claims 1-7, 16, 24, 26, 30, 40-41, 87 and 93-96 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 claims contain 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 inventors, at the time the application was filed, had possession of the claimed invention.
Note: claim 11 is omitted from this rejection because it further narrows claim 1, wherein the one or more isolated bacteria is Klebsiella variicola 137-1036 strain and the one or more microbes is Kosakonia sacchari. Thus, claim 11 meets the written description requirement.
MPEP § 2163 states that 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, 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.
To satisfy the written-description requirement, the specification must describe every element of the claimed invention in sufficient detail so that one of ordinary skill in the art would recognize that the inventor possessed the claimed invention at the time of filing. Vas-Cath, 935 F.3d at 1563; see also Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572 (Fed. Cir. 1997) (patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that “the inventor invented the claimed invention’); In re Gosteli, 872 F.2d 1008, 1012 (Fed. Cir. 1989) (“the description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed’).
In the instant case, independent claim 1, is drawn to “[a] composition to reduce bacterial cell viability loss during liquid or dry storage, comprising:
(i) one or more isolated bacteria, and
(ii) one or more isolated biofilm compositions produced by one or more microbes;
wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it, and are non-native to the one or more isolated bacteria, and wherein the one or more isolated bacteria exhibit less viability loss in liquid or dry storage for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm compositions stored under the same conditions.”
Therefore, the scope of the instantly claimed composition encompasses (i) one or more isolated bacteria, and (ii) one or more isolated biofilm compositions produced by one or more microbes that when combined exhibit the function of reducing bacterial cell viability loss during liquid or dry storage for at least 30 days; with the caveat that the one or more isolated biofilm compositions has been separated from the microorganism (i.e., one or more microbes) that produced it.
In other words, a vast array of isolated bacteria and a vast array of isolated biofilm compositions produced by one or more microbes when put together or combined, reduce or diminish cell viability loss in liquid or dry storage for a determined period of time. As such, any isolated bacteria (regardless if it is one or more) when combined with any isolated biofilm composition (regardless if it is one or more), should exhibit the function as claimed (i.e., reduce bacterial cell viability loss during liquid or dry storage for at least 30 days). However, there is no indication as to what constitutes the structure of “a composition” that exhibits the claimed function.
The scope of instant claim 1 is broad, generic and is drawn to a genus of elements (i.e., one or more isolated bacteria and one or more isolated biofilm compositions produced by one or more microbes; wherein the biofilm composition has been separated from the one or more microorganism (i.e., microbes) that produced it ) that when combined to form a whole (i.e., a composition), reduce bacterial cell viability loss under certain conditions (i.e., liquid or dry storage for at least 30 days). The instant specification is silent about the structure (i.e., components) of a composition wherein the one or more isolated bacteria exhibit less viability loss in liquid or dry storage for at least 30 days. There is no indication of the defined structural limitations for a composition wherein one of its components (i.e., one or more isolated bacteria) exhibits less viability loss in liquid or dry storage for at least 30 days, when combined with one or more isolated biofilm compositions produced by one or more microbes, however the isolated biofilm compositions lack the one or more microorganisms/microbes that produced it.
Pursuant to MPEP 2163.05 (II) B, the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species. 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 instant specification is silent about a representative number of species which adequately describe the entire genus.
Rather, the specification only discloses examples wherein Kosakonia sacchari (i.e., a microbe/bacterium and biofilm former that also exhibits nitrogen fixation) is combined with Klebsiella variicola (i.e., a bacterium) (see instant specification, Examples 2-5, pp. 260-293). Starting with Example 2, the specification recites that a biofilm was used as a protective agent during liquid storage or dry storage of the bacteria, Klebsiella variicola (see pg. 260, para[0635]). In particular, Example 2, clearly describes that K sacchari was grown in a growth medium while shaking to produce a biofilm, which was isolated by filtration to collect the resulting microbial biofilm composition and subjected to one or more washes to remove effluent and loosely-attached K sacchari cells (see instant specification, pg. 260, para[0635]). The biofilm was then subjected to a heat shock sufficient to kill any remaining K. sacchari (see instant specification, pg. 260, para[0635]).
Accordingly, the specification is silent about an isolated biofilm composition that has been separated from the microorganism(s) that produced it. In Example 2, the “isolated biofilm composition” is being interpreted as isolating/separating the biofilm produced by K. sacchari from the growth medium and the inert surface where it was grown (i.e., plate/shaker). Additionally, the step of subjecting the isolated biofilm produced by K. sacchari to a heat shock, sufficient to kill any remaining K. sacchari implies that the isolated biofilm, after growing under dynamic conditions (i.e., growth medium while shaking), being subjected to filtration and subsequent washes with the intend of removing effluent and loosely attached K. sacchari cells, still comprised K. sacchari cells (i.e., the microorganism that produced it). Additionally, exposing an isolated biofilm composition to heat shock in order to kill any remaining organisms/cells that produced the biofilm, does not necessarily translate to separating the biofilm composition from the microorganism that produced it.
Furthermore, the instant specification recites that the term “biofilm” refers to associated and/or accumulated and/or aggregated microbial cells, their products (e.g. exopolymeric substances) and inorganic particles adherent to a living or inert surface (see instant specification, pg. 11, para[0081]). Thus, the isolated biofilm described in Example 2, still reads on this definition. Also, according to the definition, if the microbial cells that produce the biofilm are separated from the biofilm, then the biofilm would no longer be an association, accumulation or aggregation of microbial cells and their produces (i.e., biofilm composition). As such, the separation process described in Example 2, fails to teach wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it, as recited in instant claim 1.
Since the scope of the instantly claimed composition encompasses a large array of isolated bacteria and a large array of isolated biofilm compositions, since the specification fails to teach a representative number of species of isolated bacteria and isolated biofilm compositions which adequately represent the claimed genus, since there is no clear indication as to what is the necessary core structure or combination needed in order for the claimed composition to exhibit the desired function of reducing bacterial cell viability loss during liquid or dry storage of the one or more isolated bacteria that are part of the composition, then the instant specification does not reasonably convey to one skilled in the relevant art that Applicants had possession of the claimed invention.
The written description may be met by provided a representative number of species of the genus in the specification and/or in light of the state of the art. In the instant case, a representative number of the genus would be evidence of compositions comprising (i) one or more isolated bacteria (i.e., different from Klebsiella variicola) and (ii) one or more isolated biofilm compositions produced by one or more microbes (i.e., different from Kosakonia sacchari); wherein the one or more isolated biofilm compositions has been separated from the microorganisms that produced it. However the instant specification only teaches Examples 2-5 which are drawn to K. sacchari and Klebsiella variicola.
As discussed above, the scope of claims 1-7, 16, 24, 26, 30, 40-41, 87 and 93-96 encompass a composition, which comprises any isolated bacteria (regardless if it is one or more) and any isolated biofilm composition (regardless if it is one or more), wherein the isolated biofilm compositions has been separated from the microorganisms that produced it, and still exhibit the claimed function. Thus the claimed genus encompasses a vast array of isolated bacteria, and a vast array of isolated biofilm compositions. The instant specification does not constitute a representative number of species that fall within the claimed genus and still exhibit the function. The instant specification only teaches examples drawn to one species of composition, corresponding to isolated bacteria Klebsiella variicola and the isolated biofilm composition produced by Kosakonia sacchari. However the Kosakonia sacchari was not separated from the isolated biofilm composition. Therefore, the species of composition exhibiting the claimed function of less viability loss in liquid or dry storage for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm compositions stored under the same conditions is not representative of the claimed genus. Thus, the species exemplified in Examples 2-5 do not constitute a representative number of species that fall within the claimed genus, given that the claimed genus encompasses any one or more isolated bacteria and any one or more isolated biofilm compositions produced by any one or more microbes, wherein the any one or more isolated biofilm compositions has been separated from the any microorganism that produced it. Without a core structure/elements of the composition that correlate to the claimed function, the specification does not convey possession of the breath of the claimed genus.
Accordingly, claims 1-7, 16, 24, 26, 30, 40-41, 87 and 93-96 do not meet the written description requirement. As noted above, claim 11 was omitted from this rejection and meets the written description requirement.
Maintained Rejections/Modified Rejection
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.

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.
103 - KSR Examples of 'Rationales' Supporting a Conclusion of Obviousness
(Consistent with the "Functional Approach" of Graham)

Further regarding 35 USC 103(a) rejections, the Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S. 398, 127 S. Ct. 1727, 82 USPQ2d 1385, 1395-97 (2007) (KSR) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit.

Exemplary rationales that may support a conclusion of obviousness include:

(A) Combining prior art elements according to known methods to yield predictable results;

(B) Simple substitution of one known element for another to obtain predictable results;

(C) Use of known technique to improve similar devices (methods, or products) in the same way;

(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;

(E) "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;

(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;

(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.

Note that the list of rationales provided is not intended to be an all-inclusive list. Other rationales to support a conclusion of obviousness may be relied upon by Office personnel.
Also, a reference is good not only for what it teaches by direct anticipation but also for what one of ordinary skill in the art might reasonably infer from the teachings. (In re Opprecht 12 USPQ 2d 1235, 1236 (Fed Cir. 1989); In re Bode 193 USPQ 12 (CCPA) 1976).

2. Claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96 are rejected under 35 U.S.C. 103 as being unpatentable over WO2018/132774 A1 (Temme et al.) International Publication Date 19 July 2018 (herein after “774”)(cited in the IDS filed on 12/08/2021) as evidenced by Fagorzi et al., Microorganisms. 2022.,10, 1777, pp. 1-3 (herein after “Fagorzi”) and Lembre et al., 2012, Chapter 13: “Exopolysaccharides of the Biofilm Matrix: A Complex Biophysical World”, edited by Desiree Nedra Karunaratne, The Complex World of Polysaccharides, pp. 371-392, publisher IntechOpen (herein after “Lembre”); in view of WO2017/208237 A1 (Shemesh), International Publication Date 07 December 2017 (herein after “Shemesh”); and Balasundararajan et al., Journal of Basic Microbiology, 2019, vol. 59, pp. 1031-1039 (herein after “Balasundararajan”) as evidenced by Feoktistova et al., Cold Spring Harb Protoc. 2016, 2016(4), pp. 343-347 (herein after “Feoktistova”).
Regarding claims 1 and 93, ‘774 provides a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system, that has been bred to fix atmospheric nitrogen in an agricultural system that has been fertilized with more than 20lbs of Nitrogen per acre (see ‘774, pg. 2, para[0007]). Microbes useful in the methods and compositions of ‘774 may be obtained from any source (see ‘774, pg. 54, para[0204]). Microbes may be bacteria, archaea, protozoa or fungi (see ‘774, pg. 54, para[0204]), the microbes may be nitrogen fixing microbes, for example a nitrogen fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi, nitrogen fixing yeast, or nitrogen fixing protozoa (see ‘774, pg. 54, para[0204]). The microbes useful in the methods and compositions disclosed by ‘774 may be spore forming microbes, for example spore forming bacteria (see ‘774, pg. 54, para[0204]), thereby constituting a composition comprising: (i) one or more isolated bacteria as recited in instant claims 1 and 93. The bacteria may include nitrogen fixing bacterial consortia of two or more species (see ‘774, pg. 58, para[0213]). In some cases, one or more bacterial species of the bacterial consortia (i.e., biofilm) may be capable of fixing nitrogen (see ‘774, pg. 58, para[0213]). The bacteria which fix nitrogen and the bacteria which enhance the ability of other bacteria to fix nitrogen may be the same or different (see ‘774, pg. 58, para[0213]), thereby constituting a composition comprising: (ii) one or more isolated biofilm compositions produced by one or more microbes as recited in instant claims 1 and 93. ‘774 adds that selection of plant growth promoting microbes based on nitrogen fixation can be done by co-culturing the bacteria with competitive or beneficial microbes to elucidate cryptic pathways (see ‘774, pg. 35, para[0148]). ‘774 teaches that bacteria that can be produced by the methods disclosed include Klebsiella sp., (see ‘774, pg. 58, para[0214]); in some cases, a bacterial species selected from at the genera Klebsiella variicola are utilized (see ‘774, pg. 58, para[0214]), thereby constituting a composition comprising: (i) one or more isolated Klebsiella sp. bacterial cells as recited in instant claim 93. ‘774 also teaches that Examples of compositions may include seed coatings; the compositions comprising the bacterial populations may be coated onto the surface of a seed (see ‘774, pg. 67, para[0244]). Compositions may also be dehydrated in a suitable manner that maintains cell viability and the ability to artificially inoculate and colonize host plants (see ‘774, pg. 67, para[0244]). The compositions comprising bacterial populations may be coated on a surface of a seed, and may be in liquid form (see ‘774, pg. 66, para[0239]), thereby constituting a composition that reduces bacterial cell viability loss during dry or liquid storage as recited in instant claims 1 and 93.
With respect to reducing bacterial cell viability loss during liquid or dry storage, although ‘774 does not expressly teach that the composition reduces bacterial cell viability loss during liquid or dry storage, the Examiner would like to remind Applicants that the preambles of claims 1 and 93 recite a composition, and while the use of a descriptive clause, i.e. “to reduce …,” when referring to the contemplated use (i.e. “intended use”) of a claimed composition is proper, it is not a limitation and thus of no significance in determining the patentability thereof over the prior art. See, e.g., In re Otto, 312 F.2d 937, 938, 136 USPQ 458, 459 (CCPA 1963) (The claims were directed to a core member for hair curlers and a process of making a core member for hair curlers. The court held that the intended use of hair curling was of no significance to the structure and process of making.).Therefore, the ‘774 teachings satisfy the claimed intended use in the preambles of instant claims 1 and 93
‘774 teaches isolation of bacteria, in particular microbes useful in the method and compositions and adds that the microbes can be obtained by extraction from surfaces of tissues of native plants, and that non-limiting example of plant tissues may include a seed, seedling, leaf, cutting, plant, bulb or tuber (see ‘774, pg. 31, para[0131]). Also that microbes can be isolated from plant tissues to assess microbial traits and that the parameters for processing tissue samples may be varied to isolate different types of associative microbes, such as rhizopheric bacteria, epiphytes, or endophytes (see ‘744, pg. 31, para[0133]). Another method of isolating microbes may be through the isolation of bacteria from soils (see ‘774, pg. 31, para[0132]). Some examples of bacteria isolated according to ‘774’s methods can be a member of one or more of the following taxa: […] Klebsiella, Kosakonia … (see ‘774, pg. 58, para[0215]), and in some cases a bacterial species selected from at least one of the following genera are utilized: […] Klebsiella, Kosakonia …(see ‘774, pg. 59, para[0216]). ‘774 goes on to teach the isolation of diazotrophs from roots, which were harvested and washed with sterile deionized water to remove bulk soil (see ‘774, pg. 143, para[0346]). Root tissues were homogenized with 2mm stainless steel beads in a tissue lyser for three minutes at setting 30, and the samples were centrifuged for 1 minute at 13,000 rpm to separate tissue from root-associated bacteria (see ‘774, pg. 143, para[0346]). The supernatants were split into two fractions, one was used to characterize the microbiome through 16S rRNA amplicon sequencing and the remaining fraction was diluted and plated on nitrogen-free broth media(see ‘774, pg. 143, para[0346]).
As evidenced by Fagorzi, endophytes are studied for different purposes, spanning from the search for relevant activities (e.g., biocontrol, bioremediation, metabolite production, plant growth and resistance) to ecological investigation, including adaptation to harsh conditions (see Fagorzi, pg. 1, fourth paragraph) and for many endophytes, the soil is their original habitat and once the presence of the plant roots is perceived, many bacteria are attracted and proliferate around the roots, also forming biofilms over the root surface (see Fagorzi, pg. 1, paragraph 5). These new habitats are called rhizosphere and rhizoplane, meaning the soil environments close to the roots and the root surface, respectively (see Fagorzi, pg. 1, paragraph 5).
Additionally, Lembre provides evidence of various methods used to extract EPS (Extracellular Polymeric Substances) from biofilms, which include physical extraction, chemical extraction and enzymatic extraction (see Lembre, pg. 374, Figure 2). Among the physical extraction methods listed in Lembre’s Figure 2, are centrifugation, ultracentrifugation, sonication, filtration, heating, dialysis and lyophilization (see Lembre, pg. 374, Fig. 2). A biofilm is a complex structure made of aggregates of microbial cells within a matrix of extracellular polymeric substances (EPS) (see Lembre, pg. 371, first paragraph and Figure 1), and since some EPS are tightly associated to the biofilm structure, sometimes through covalent bounds to the cells surface and are not directly extracted (see Lembre, pg. 374, second paragraph). Others free EPS are directly released; the easily released EPS can be separated using physical methods such as high-speed centrifugation and ultrasonication (see Lembre, pg. 374, second paragraph).
Therefore, ‘774’s parameters for processing tissue samples (i.e., the roots) for the isolation of microbes in plant tissues, in particular the isolation of diazotrophs as described by ‘774 at pg. 143, para[0346], constitutes wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it as recited in instant claims 1 and 93. Since ‘774 teaches isolation of diazotrophs (i.e., root-associated bacteria that form a biofilm) from the plant tissue (i.e., harvested root) by centrifuging the homogenized tissue; and since centrifugation is one of the physical methods used to extract extracellular polymeric substances from a biofilm; it must follow that the root-associated bacteria (i.e., microorganism) is separated from the isolated biofilm composition (i.e., the harvested roots and the soil environments close to the roots and the root surface). As such, the teachings of ‘774 as evidenced by Fagorzi and Lembre are suggestive of the claim limitations recited in instant claim 1. Additionally, a person of ordinary skill in the art would have been motivated to separate the microorganisms/microbe/microbial cells from an isolated biofilm composition after reading the teachings of ‘774.
‘774 does not expressly teach wherein the one or more isolated biofilm compositions produced by one or more microbes are non-native to the one or more isolated bacteria; nor wherein the one or more isolated bacteria exhibits less viability loss in liquid or dry storage for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm composition stored under the same conditions, as recited in instant claims 1 and 93.
Shemesh is in the field of bacterial compositions and teaches a method of preparing a bacterial composition comprising: (a) in vitro co-culturing beneficial bacteria with biofilm-producing bacteria in a growth substrate under conditions that generate a biofilm which comprises the beneficial bacteria and the non-pathogenic bacteria; and (b) isolating the biofilm from the growth substrate, thereby preparing the bacterial composition (see pg. 2, lines 27-31). Shemesh hypothesized that extracellular matrix produced by robust biofilm former bacterium B. subtilis may provide increased protection to other species such as probiotic bacteria during their growth in co-culture biofilm system (see Shemesh, pg. 45, lines 1-4). As shown in Shemesh’s Figure 13, the co-culture cultivation had no effect on L. plantarum and B. subtilis growth ( compared to their growth in pure culture), indicating that there are no antagonistic interactions between these bacteria at given conditions (see Shemesh, pg. 45, lines 7-10), thereby constituting wherein the one or more isolated biofilm compositions produced by one or more microbes are non-native to the one or more isolated bacteria, as recited in instant claim 1. Shemesh adds that bacteria prefer to grow in complex community of multicellular cells called biofilm and not as free-living (planktonic) cells (see pg. 1, lines 28-29). Shemesh also teaches that once sufficient quantities of beneficial bacteria are propagated (and encapsulated in the biofilm), the biofilm is harvested (i.e., removed from the growth substrate) (see pg. 30, lines 24-26). Following isolation from the growth substrate, the biofilm (and/or bacteria incorporated therein) may be subject to drying (i.e., dehydrating), freezing, spray drying or freeze drying; preferably, the biofilm is treated in a way that preserves the viability of the bacteria (see pg. 30, lines 27-30). Shemesh also teaches that in some embodiments the bacterial compositions are formulated for use as an agricultural product, which further comprises an agent which promotes the growth of plants (see Shemesh, pg. 38, lines 5-6); and that the agricultural formulation comprises a fertilizer, preferably one that does not reduce the viability of the bacterial composition by more than 20 %, 30 %, 40 %, 50 % or more (see pg. 37, lines 1-5 and lines 16-18); and that at least 50% of the bacteria in the composition are viable (see pg. 53, claim 23). Interactions between the species embedded in the biofilm and their environment result in the formation of a complex structure, capable of resisting to environmental stress and exposure to antimicrobial agents; thus, biofilm formation represents a strategy for persistence under unfavorable conditions in diverse environments (see pg. 2, lines 1-5). Therefore, the teachings of ‘774 when combined with the teachings of Shemesh are suggestive of the claim limitations as recited in instant claims 1 and 93.
With respect to wherein the one or more isolated bacteria exhibits less viability loss in liquid or dry storage for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm composition stored under the same conditions, as recited in instant claims 1 and 93.
Balasundararajan teaches that Quorum sensing (QS)‐mediated biofilm‐forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere (see Balasundararajan, pg. 1031, Abstract). A total of 48 QS‐positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings (see Balasundararajan, pg. 1031, Abstract). The in vitro biofilm production and genetic diversity as revealed by BOX‐PCR fingerprinting showed high variability among the isolates (see Balasundararajan, pg. 1031, Abstract). The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7‐4; QS36‐2), A. enteropelongenes (QS20‐8), A. veronii (QS36‐3), Enterobacter sp. (QS20‐11), Klebsiella pneumoniae (QS24‐6), Kosakonia cowanii (QS24‐21), Providentia rettigeri (QS24‐2), Sphingomonas aquatilis (QS24‐17), and Pseudomonas sihuiensis (QS24‐20) (see Balasundararajan, pg. 1031, Abstract). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions (see Balasundararajan, pg. 1031, Abstract). The plant‐associated bacteria use QS signals for biofilm mediated colonization as well as for the production of antagonistic compounds to avoid competition from other microbial communities (see Balasundararajan, pg. 1036, right column, paragraph 1). QS increases the cell density of a strain and further tends to produce extracellular polymeric substances in which cells are embedded (see Balasundararajan, pg. 1036, right column, paragraph 1). This structure known as the biofilm is a microhabitat of that strain in which the cells live in with maximum protection (see Balasundararajan, pg. 1036, right column, paragraph 1). In Balasundararajan’s work, K. cowanii, a reclassified species of Enterobacter was also reported to be present in rice root (see Balasundararajan, pg. 1037, right column, paragraph 1). Kosakonia spp. found in clinical samples, soil and tree species and several species promotes plant growth by nitrogen fixation (see Balasundararajan, pg. 1037, right column, paragraph 1). Balasundararajan also teaches that diversified soil‐dwelling bacteria are capable of producing biofilms through N-acyl homoserine lactone‐mediated QS (see Balasundararajan, pg. 1037, right column, paragraph 2). These bacteria may be commensal, pathogenic, or beneficial to the rice crop (see Balasundararajan, pg. 1037, right column, paragraph 2); and that by careful examination of their plant-growth-promoting traits and virulence, it could be possible to develop a biofilm-forming inoculant for providing nutrients and for biotic and abiotic stress mitigation (see Balasundararajan, pg. 1037, right column, paragraph 2).
Furthermore, Balasundararajan teaches that all the bacterial isolates were assessed for biofilm formation in a 96-well titer plate and the biofilm production quantified with Crystal violet staining (see Balasundararajan, pg. 1032, right column, first paragraph). The time-course data (4, 6, 8, and 12 days) on biofilm population (optical density [OD] at 550nm) (see Balasundararajan, pg. 1032, right column, first paragraph). Balasundararajan also teaches that de-husked rice seeds were surface sterilized, transferred to a sterile petri plate and incubated at room temperature in the dark for 24 h (see Balasundararajan, pg. 1033, left column, last paragraph). All the elite biofilm-forming rhizobacterial strains were grown overnight in LB broth at 30°, the bacterial cells were harvested by centrifugation, washed twice in 10 mM MgCl2 and resuspended in the same; this suspension was inoculated (20µl per seed) at the hypocotyl region of 5 days old seedlings at OD660 of 0.05 (108 CFU/ml), whereas uninoculated controls were maintained simultaneously (see Balasundararajan, pg. 1033, right column, first paragraph). The colonization and biofilm formation on the surface of the root were visualized and photographed on the third day after inoculation (see Balasundararajan, pg. 1033, right column, first paragraph). Balasundararajan’s Figure 4, shows biofilm formation by elite quorum sensing bacteria (QS-bacteria) isolates on the root surface of rice seedlings (see Balasundararajan, pg. 1036, Figure 4); where QS24-6 corresponding to Klebsiella pneuminiae (i.e., isolated bacteria from the genera Klebsiella) and QS24-21 corresponding to Kosakonia cowanii (i.e., isolated bacteria from the genera Kosakonia) inoculated at the hypocotyl region of the 5 day old seedlings appear to have formed a biofilm growth on the root of the seedlings (see Balasundararajan, pg. 1036, Figure 4). It is also noted that the results depicted in Balasundararajan’s Figure 4 also include an uninoculated control (i.e., seedlings where no bacteria was applied).
As evidenced by Feoktistova, crystal violet staining is a quick and versatile assay for screening cell viability under diverse stimulation conditions (see Feoktistova, pg. 345, Discussion). One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA (see Feoktistova, pg. 343, Abstract). Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture (see Feoktistova, pg. 343, Abstract).
Since Balasundararajan teaches evaluating biofilm formation of the isolated strains, and since crystal violet staining is a method of evaluating cell viability, a person of ordinary skill in the art would have been motivated to determine the cell viability of a composition comprising one or more isolated bacteria, and one or more isolated biofilm compositions produced by one or more microbes; wherein the one or more isolated bacteria are stored in liquid or dry form for at least 30 days, and compare the cell viability loss to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm compositions stored under the same conditions.
Additionally and/or alternatively, the MPEP 2112-2112.02 states that when a reference discloses all the limitations of a claim except for a property or function, and the examiner cannot determine whether or not the reference inherently possesses properties which anticipate or render obvious the claimed invention but has basis for shifting the burden of proof to applicant as in In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980). In the instant case, ‘774 teaches a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen, where the bacterial species include Klebsiella variicola and one or more members of the taxa Kosakonia; and Balasundararajan teaches root colonization and biofilm formation of Klebsiella sp. and Kosakonia sp. on laboratory sprouted seed. The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether the composition as suggested by the combination of ‘774 and Balasundararajan would exhibit less viability loss when stored in liquid or dry form for at least 30 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm composition stored under the same conditions. The cited art taken as a whole demonstrates a reasonable probability that the suggested composition exhibits either identical or sufficiently similar viability loss when stored in liquid or dry form for at least 30 days when compared to a control and that whatever differences exists are not patentably significant. Therefore, with the showing of the reference, the burden of establishing non-obviousness by objective evidence is shifted to the Applicants.
From the teachings of the references, the Examiner recognizes that it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the teachings of Shemesh and Balasundararajan as part of the bacterial composition of ‘774 in order to arrive at the claimed invention.
One of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so because it was known that bacteria prefer to grow in complex community of multicellular cells called biofilms and not as free-living (planktonic) cells, therefore one would have been motivated to prepare a bacterial composition by in-vitro co-culturing beneficial bacteria with biofilm-producing bacteria in a growth substrate under conditions that generate a biofilm and using the composition as an agricultural product as taught by Shemesh. An ordinary skilled artisan would have been motivated because it was also known that the viability of the bacteria propagated and encapsulated in the biofilm can be preserved by drying (i.e., dehydrating), freezing, spray drying or freeze drying; because the QS-mediated biofilm-forming rhizobacteria such as Kosakonia cowanii was known to increase the cell density of a strain and further tends to produce extracellular polymeric substances in which cells are embedded, and because bacterial isolates (i.e., Kosakonia cowanii) were assessed for biofilm production and biofilm population for a time-course of 12 days, as taught by Balasundararajan.
One of ordinary skill in the art before the effective filing date of the claimed invention would have had a reasonable expectation of success given that the composition of ‘774 comprises one or more bacteria where the one or more bacteria include a combination of bacterial species such as Klebsiella and Kosakonia for use in fixing atmospheric nitrogen in an agricultural system. Given that ‘774 teaches parameters and methods to isolate microbes from plant tissue or soil by processing (i.e., homogenization in a tissue lyser) the harvested tissue comprising the microbe of interest (i.e., the roots) and separating the root-associated bacteria from the tissue by centrifuging, thereby resulting in an isolated biofilm composition separated from the microorganisms that produced it. And given that Shemesh bacterial composition for agricultural use comprised at least 50% viable bacteria.
Therefore, incorporating a cell density increasing Kosakonia bacterial strain as a (QS)-mediated biofilm-forming rhizobacteria as taught by Balasundarajan, and separating the isolated biofilm composition from the Kosakonia bacterial strain by following the tissue processing parameters and separation by centrifugation of the root-associated bacteria from the tissue as taught by of ‘774 in order incorporate the isolated biofilm composition as part of a bacterial composition for agricultural use as taught by Shemesh; thereby resulting in a biofilm-forming inoculant for providing nutrients and for biotic and abiotic stress mitigation (i.e., cell viability loss during liquid or dry storage) as taught by Balasundararajan, would support the increase in cell density of the strain and further production of extracellular polymeric substances in which one or more bacterial cells are embedded thereby fixing atmospheric nitrogen in an agricultural system by constituting some teaching, suggestion or motivation in the prior art that would have let one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention pursuant to KSR.
Regarding claim 2, ‘774 teaches that bacteria that can be produced by the methods disclosed include Klebsiella sp., (see ‘774, pg. 58, para[0214]); in some cases, a bacterial species selected from at the genera Klebsiella variicola are utilized (see ‘774, pg. 58, para[0214]), thereby constituting where the one or more isolated bacteria are selected from the genera Klebsiella as recited in instant claim 2. ‘774 also teaches that bacteria isolated according to methods of the disclosure can be a member of the Kosakonia taxa (see ‘774, pg. 59, para[0215]), thereby constituting where the one or more isolated bacteria are selected from the genera Kosakonia as recited in instant claim 2.
Regarding claims 3-5, ‘774 teaches that a combination of bacterial species from the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella (see ‘774, pg. 59, para[0216]). The species utilized can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis (see ‘774, pg. 59, para[0216]), thereby constituting wherein the one or more isolated bacteria are selected from Klebsiella variicola and Kosakonia sacchari as recited in instant claim 3; constituting wherein the one or more isolated bacteria is from the genus Klebsiella as recited in instant claim 4, and also constituting wherein the one or more isolated bacteria is a Klebsiella variicola as recited in instant claim 5.
Regarding claim 6, ‘774 also teaches Table A: Microorganisms Deposited under the Budapest Treaty where one of the entries corresponds to Depository NCMA with pivot strain designation number 137-1036, Taxonomy Klebsiella variicola, Accession Number 201712002 and deposit date of December 20, 2017 (see ‘774, Table A, pg. 63), thereby corresponding to wherein the one or more isolated bacteria is a Klebsiella variicola 137-1036 strain as recited in instant claim 6.
Regarding claim 7, ‘774 also teaches that bacteria isolated according to methods of the disclosure can be a member of the Kosakonia taxa (see ‘774, pg. 59, para[0215]). Additionally, ‘774 teaches the microbe "6-403" was deposited as NCMA 201708004 and can be found in Table A and is a mutant Kosakonia sacchari (also called C:M037) (see ‘774, pg. 149, para[0374] and Table A); thereby constituting where one or more microbes are selected from the Kosakonia species as recited in instant claim 7.
Regarding claim 11, ‘774 teaches the microbe "137-1036," which is a progeny mutant Klebsiella variicola strain from CI137 WT, was deposited as NCMA 201712002 and can be found in Table A (see ‘774, pg. 149, para[0375]). It is noted that entries 4-11 in Table A also displays microorganisms deposited under the Budapest Treaty which include Kosakania sacchari, Klebsiella variicola (see ‘774, pg. 64, Table A). Additionally, ‘774 teaches that in some cases, the species utilized can be one or more of Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis (see ‘774, pg. 59, para[0216]). As such the teachings of ‘774 satisfy the claim limitations as recited in instant claim 11.
Regarding claim 16, the MPEP 2112-2112.02 states that when a reference discloses all the limitations of a claim except for a property or function, and the examiner cannot determine whether or not the reference inherently possesses properties which anticipate or render obvious the claimed invention but has basis for shifting the burden of proof to applicant as in In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980). In the instant case, ‘774 teaches a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen, where the bacterial species include Klebsiella variicola and one or more members of the taxa Kosakonia. The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether the composition as suggested by the combination of ‘774 and Balasundararajan would exhibit less viability loss when stored in liquid culture for at least 90 days compared to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm composition stored under the same conditions. The cited art taken as a whole demonstrates a reasonable probability that the suggested composition exhibits either identical or sufficiently similar viability loss when stored in liquid culture for at least 90 days relative to a control and that whatever differences exists are not patentably significant. Therefore, with the showing of the reference, the burden of establishing non-obviousness by objective evidence is shifted to the Applicants.
Regarding claims 24 and 26, the microbes taught by ‘774 are “non-intergeneric” which mean that the microbes are not intergeneric (see ‘774, pg. 20, para[0092.25]). An “intergeneric microorganism” is a microorganism that is formed by the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera (see ‘774, pg. 20, para[0092.24]). An exemplary “intergeneric microorganism” includes a microorganism containing a mobile genetic element which was first identified in a microorganism in a genus different from the recipient microorganisms (see ‘774, pg. 20, para[0092.24]). Therefore, the teachings of ‘774 suggest the claim limitations recited in instant claim 24, wherein the one or more isolated bacteria are non-intergeneric remodeled bacteria, and methods for detecting the non-intergeneric engineered microbes derived from the WT strains (see ‘774, pg. 47, para[0178]).
‘774 also teaches that in order to utilize elemental nitrogen (N) for chemical synthesis, life forms combine nitrogen gas (N2) available in the atmosphere with hydrogen in a process known as nitrogen fixation (see ‘774, pg. 26, para[0118]). Because of the energy-intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen gas) have evolved sophisticated and tight regulation of the nif gene cluster in response to environmental oxygen and available nitrogen (see ‘774, pg. 27, para[0118]). Nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation (see ‘774, pg. 27, para[0118]). Although some endophytes have the ability to fix nitrogen in vitro, often the genetics are silenced in the field by high levels of exogenous chemical fertilizers (see ‘774, pg. 29, para[0123]). As such, the teachings of ‘774 satisfy the claim limitations as recited in instant claims 24 and 26.
Additionally, with respect wherein the non-intergeneric remodeled bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen as recited in claim 26, the discovery of a new use for an old structure based on unknown properties of the structure might be patentable to the discoverer as a process of using. In re Hack, 245 F.2d 246, 248, 114 USPQ 161, 163 (CCPA 1957). However, when the claim recites using an old composition or structure and the "use" is directed to a result or property of that composition or structure, then the claim is anticipated. In re May, 574 F.2d 1082, 1090, 197 USPQ 601, 607 (CCPA 1978) and In re Tomlinson, 363 F.2d 928, 150 USPQ 623 (CCPA 1966). See M.P.E.P. § 2112.02. Moreover, “[t]he discovery of a previously unappreciated property of a prior art composition, or of a scientific explanation for the prior art’s functioning, does not render the old composition patentably new to the discoverer.” Atlas Powder Co. v. Ireco Inc., 190 F.3d 1342, 1347, 51 USPQ2d 1943, 1947 (Fed. Cir. 1999). Thus the claiming of a new use, new function or unknown property which is necessarily present in the prior art does not necessarily make the claim patentable. As such, claim 26 can be deemed to recite an intended use of the claimed non-intergeneric remodeled bacteria capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. A recitation of an intended use must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. Accordingly, since ‘774 teaches a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen encompassed by the presently claimed invention, the intended use of the non-intergeneric remodeled bacteria being capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen is also obvious.
Regarding claim 30, ‘774 teaches that one can decouple the sensing of exogenous nitrogen from expression of the nitrogenase enzyme to facilitate field-based nitrogen fixation (see ‘774, pg. 29, para[0123]); and that the methods described also contemplate introducing genetic variation into the nifH, nifD, nifK and draT genes (see ‘774, pg. 28, para[122]). Additionally, that the genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase and glutaminase (see ‘774, pg. 38, para[0152]). As such, the teachings of ‘774 suggest the claim limitations as recited in instant claim 30.
Regarding claim 40, ‘774 teaches the microbe "137-1036," which is a progeny mutant Klebsiella variicola strain from CI137 WT, was deposited as NCMA 201712002 and can be found in Table A (see ‘774, pg. 149, para[0375]). It is noted that entries 4-11 in Table A also displays microorganisms deposited under the Budapest Treaty which include bacterium deposited as NCMA 201701003 (i.e., entry 4 in Table A), a bacterium deposited as NCMA 201701002 (i.e., entry 5 in Table A), a bacterium deposited as NCMA 201708004 (i.e., entry 6 in Table A), a bacterium deposited as NCMA 201708003 (i.e., entry 7 in Table A), a bacterium deposited as NCMA 201708002 (i.e., entry 8 in Table A), a bacterium deposited as NCMA 201708001 (i.e., entry 9 in Table A), a bacterium deposited as NCMA 201712001 (i.e., entry 10 in Table A), and a bacterium deposited as NCMA 201712002 (i.e., entry 11 in Table A). As such the teachings of ‘774 satisfy the claim limitations as recited in instant claim 40.
Regarding claim 41, ‘774 teaches Table G, which displays a list of engineered non-intergeneric microbes (see ‘774, pg. 178 to pg. 193). One of the entries in Table G, more specifically, the third entry on pg. 184, displays Strain ID 137-1036 with SEQ ID NO: 193 and genotype ΔnifL::PinfC, where starting at 24bp after the A of the ATG start codon, 1372bp of nifL have been deleted and replaced with the 137 PinfC promoted sequence (see ‘774, pg. 184, Table G). Note: Applicants elected instant SEQ ID NO: 193 as the isolated bacteria. Upon comparing instant SEQ ID NO: 193 with SEQ ID NO: 193 taught by ‘774, it was noticed that both sequences are 100% identical. Therefore, the teachings of ‘774 satisfy the claim limitations as recited in instant claim 41.
Regarding claim 87, MPEP 2112-2112.02 states that when a reference discloses all the limitations of a claim except for a property or function, and the examiner cannot determine whether or not the reference inherently possesses properties which anticipate or render obvious the claimed invention but has basis for shifting the burden of proof to applicant as in In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980). In the instant case, ‘774 teaches a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen, where the bacterial species include Klebsiella variicola and one or more members of the taxa Kosakonia. The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether the composition as suggested by the combination of ‘774 and Balasundararajan would exhibit less viability loss when stored at 37°C, compared to a control composition comprising the one or more isolated bacteria and lacking the one or more biofilms or isolated biofilm compositions stored under the same conditions. The cited art taken as a whole demonstrates a reasonable probability that the suggested composition exhibits either identical or sufficiently similar viability loss when at 37°C, and that whatever differences exists are not patentably significant. Therefore, with the showing of the reference, the burden of establishing non-obviousness by objective evidence is shifted to the Applicants.
Regarding claim 94, ‘774 does not expressly teach that the one or more isolated biofilm compositions has been processed to kill any remaining microbes that produce the biofilm as recited in instant claim 94, since ‘774 teaches bacterial compositions comprising biofilm-forming bacterial populations of Klebsiella variicola and Kosakonia sacchari to coat the surface of seeds thereby constituting a well-known composition comprising one or more isolated biofilm compositions, the functional property (i.e., killing any remaining microbes) of the isolated biofilm as claimed and the known composition comprising biofilm-forming bacterial populations of Klebsiella variicola and Kosakonia sacchari would necessarily read upon the same. The discovery of a previously unappreciated property of a prior art composition, or a scientific explanation for the prior art’s functioning, does not render the old composition patentably new to the discoverer. Atlas Powder Co. v. Ireco Inc., 190 F.3d 1342, 1347, 51 USPQ2d 1943, 1947 (Fed. Cir. 1999). Thus, the claiming of a new functional property (i.e., killing any remaining microbes) which would necessarily read upon the prior art does not necessarily make the claim patentable. In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 4333 (CCPA 1977).
Additionally and/or alternatively, it is unnecessary for a prior art reference to teach that the composition has been processed to kill any remaining microbes because a functional property/intended use of the isolated biofilm composition (i.e., has been processed to kill any remaining microbes) does not state a condition that is material to patentability or provide a structural limitation that would further limit the claimed peptide. The court has found that the determination of whether clauses such as “wherein” and “whereby" is a limitation in a claim is dependent on the specific facts of the case. If the “wherein" or “whereby” clause limits a process claim where the clause gives meaning and purpose to the manipulative steps, it should be given patentable weight. However, the court also found (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)) that a “‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’” In the instant case, the functional property/intended use of the isolated biofilm composition (i.e., has been processed to kill any remaining microbes) is an intended result of the claimed isolated biofilm composition that gives little meaning and purpose to the structure of the claimed isolated biofilm composition. Accordingly, claim 94 recites an intended result that does not render material to patentability.
Regarding claims 95-96, ‘774 teaches that one can decouple the sensing of exogenous nitrogen from expression of the nitrogenase enzyme to facilitate field-based nitrogen fixation (see ‘774, pg. 29, para[0123]). Specific targets for genetic variation to facilitate field-based nitrogen fixation using the methods described herein include one or more genes selected from the group consisting of nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifT, nifB, and nifQ (see ‘774, pg. 29, para[0123]). ‘774 teaches that the methods described also contemplate introducing genetic variation into the nifH, nifD, nifK and draT genes (see ‘774, pg. 28, para[122]). The activity of NifA is also regulated post-translationally in response to environmental nitrogen, most typically through NifL-mediated inhibition of NifA activity. (see ‘774, pg. 28, para[0121]). In general, the interaction of NifL and NifA is influenced by the PII protein signaling cascade via GlnK, although the nature of the interactions between GlnK and NifL/NifA varies significantly between diazotrophs; in Klebsiella pneumoniae, both forms of GlnK inhibit the NifL/NifA interaction, and the interaction between GlnK and NifL/NifA is determined by the overall level of free GlnK within the cell (see ‘774, pg. 28, para[0121]). Additionally, ‘774 teaches that the genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase and glutaminase (see ‘774, pg. 38, para[0152]). As such, the teachings of ‘774 suggest the claim limitations as recited in instant claims 95 and 96.
In light of the foregoing discussion, the Examiner concludes that the subject matter defined by the above claims would have been obvious to one of ordinary skill in the art within the meaning of 35 USC 103. Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, as evidenced by the references above.
Response to Arguments
Regarding Applicants’ arguments with respect to the 35 U.S.C 103 rejection to claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96, have been considered but are not persuasive.
As discussed in the rejection above, the combined teachings of ‘774 as evidenced by Fagorzi and Lembre, in view of Shemesh and Balasundararajan as evidenced by Feoktistova suggest a composition to reduce bacterial cell viability loss during liquid or dry storage, more specifically a composition comprising isolated Klebsiella sp. bacterial cells and one or more isolated biofilm compositions produced by one or more microbes such as Kosakonia sp. Thus, the 35 U.S.C 103 rejection above renders the amended claims obvious.
In response to Applicants’ argument, i.e., none of the cited art teaches or suggest that the biofilm was isolated and separated from the biofilm former Kosakonia sacchari, as explained in Example 2 of the as-filed specification (see Remarks, filed 12/11/2025, pg. 9 to pg. 10), it is found unpersuasive.
It is noted that the features upon which applicant relies (i.e., Example 2, K. sacchari was grown in a growth medium while shaking to produce a biofilm, which was isolated by filtration to collect the resulting microbial film composition and subjected to one or more washes to remove effluent and loosely-attached K. sacchari cells. The biofilm was subjected to a heat shock sufficient to kill any remaining K. sacchari.) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). As discussed in the “Claim Interpretation” section above, the scope of the instantly claimed isolated biofilm composition is being interpreted as encompassing a combination of various elements, parts, or ingredients that form the biofilm, and those elements, parts or ingredients are one or more species of microbes/microorganisms, cells, extracellular polymeric substances and/or inorganic particles. Therefore, the scope of claim 1 and dependent claims do not correlate that the isolated biofilm composition had been separated from the microorganism that produced it. Additionally, the scope of the instantly claimed composition is unclear and/or ambiguous, as discussed in the 35 U.S.C 112(b) rejection above, because an ordinary skilled artisan would understand that a biofilm comprises the microorganisms/microbes/cells embedded in a matrix of composed of extracellular polymeric substances, carbohydrates, proteins and particles. Thus, removing or separating the microorganisms from the biofilm would disrupt the biofilm composition.
Moreover, the fact that the inventor has further clarified that the Kosakonia sacchari biofilm was isolated and separated from the biofilm former by filtration, which would naturally flow from following the suggestion of the prior art, cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Even if the scope of the instant claim encompassed an isolated biofilm composition that has been separated from the microorganisms that produced it; and assuming arguendo that the prior art does not expressly suggest wherein one or more isolated biofilm compositions has been separated from the microorganism that produced it. Since the prior art teaches separation of root-associated bacteria from the tissue (i.e., root) by centrifugation it would naturally follow that that separation of the biofilm composition present in the tissue (i.e., the roots) is separated from the microorganism that produced it (i.e., root-associated bacteria) would necessarily be due to centrifugation.
As such, the reason (i.e., an isolated biofilm composition separated from the microorganisms that produced it) an ordinary skilled artisan would modify the teachings of ‘774 and separate tissue from root-associated bacteria does not preclude a finding of obviousness. In other words, it is not necessary for the prior art to expressly teach that the one or more isolated biofilm compositions has been separated from the microorganism that produced it, because the prior art teaches separation and isolation of tissue-associated bacteria from the tissue by physical extraction such as centrifugation and/or filtration and/or heating.
Furthermore, as discussed in the action dated 09/19/2025; the instant specification does not define what constitutes “separated”. Pursuant to MPEP 2111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. In the instant case, the term “separated” was given its plain meaning, i.e., to set or keep apart as defined by Merriam Webster Dictionary. As such, the Examiner interpreted that the biofilm composition was set apart or kept apart from the microorganism that produced it, and if the microorganism that produces the biofilm is found in a growth substrate, then it would naturally follow that the biofilm composition is separated from the microorganism that produces it. Additionally, it is noted that the instant specification refers to the terms "isolate," "isolated," and like terms, to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue, etc.) (see instant specification, pg. 19, para[0120]). Therefore, the phrase “has been separated from the microorganism that produced it” further emphasizes that the microorganisms that produces the biofilm are present in a growth substrate such as soil, water, plant tissue, etc. (i.e., a growth substrate), and thus once the biofilm compositions are separated from the microorganism that produced it , it is inherent that the biofilm compositions are set apart or kept apart from the microorganisms that produced it.
Accordingly, the Examiner maintains that the teachings of ‘774 as evidenced by Fagorzi and Lembre, in view of Shemesh and Balasundararajan as evidenced by Feoktistova read on the claim limitations recited in instant claim 1, wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it.
Additionally, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, as discussed above in the 103 rejection, the teachings of ‘774, Shemesh and Balasundararajan are suggestive of the claim limitations as recited in instant claim 1 and dependent claims 2-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96. In particular, ‘774 teaches bacterial compositions that comprise nitrogen fixing bacterial consortia of two or more species (i.e., microbes, bacteria, archaea, fungi, yeast or protozoa), wherein the compositions may be dehydrated in a suitable manner that maintains cell viability. As previously discussed, ‘774 also teaches parameters for processing and separating tissue from root-associated bacteria by tissue lysis and centrifugation. Fagorzi provides evidence that the root-associated bacteria (i.e., endophytes) are attracted to plant roots, proliferate and form biofilms over the root surface. As evidenced by Lembre, centrifugation is one of the physical methods of extracting microbial cells from the bacterial biofilm matrix. Therefore, an ordinary skilled artisan would have been motivated with a reasonable expectation of success after reading ‘774 to separate the one or more isolated biofilm composition from the microorganisms that produced it.
Furthermore, Shemesh also teaches bacterial compositions comprising: (a) in vitro co-culturing beneficial bacteria, with biofilm-producing bacteria in a growth substrate under conditions that generate a biofilm; and (b) isolating the biofilm from the growth substrate, thereby preparing the bacterial composition. Shemesh also teaches that the interactions between the species embedded in the biofilm and their environment results in formation of a complex structure capable of resisting to environmental stress and exposure to antibiotics; thus representing a strategy for persistence under unfavorable conditions (i.e., cell viability loss). Finally, the teachings of Balasundararajan demonstrate in-vitro biofilm production with strains of soil-dwelling bacteria capable of producing biofilms wherein cell-viability was evaluated with crystal violet staining. Therefore a person of ordinary skill in the art would have been motivated with reasonable expectation of success to incorporate the teachings of ‘774 with the teachings of Shemesh and Balasundararajan in order to arrive at the claimed composition. One of ordinary skill in the art would have been motivated with reasonable expectation of success to determine the cell viability of a composition comprising one or more isolated bacteria, and one or more isolated biofilm compositions produced by one or more microbes; wherein the one or more isolated biofilm compositions has been separated from the microorganism that produced it, and wherein the one or more isolated bacteria are stored in liquid or dry form for at least 30 days. One of ordinary skill in the art would have determined the cell viability of the isolated bacteria and isolated biofilm compositions and compared the cell viability loss to a control composition comprising the one or more isolated bacteria and lacking the one or more isolated biofilm compositions stored under the same conditions.
In response to Applicants’ argument (i.e., the proposed modifications cannot render the prior art unsatisfactory for its intended purpose (see Remarks, filed 12/11/2025, pg. 14), it is found unpersuasive.
MPEP 2143.01(V) states that [i]f a proposed modification would render the prior art invention being modified unsatisfactory for its intended purpose, there may be no suggestion or motivation to make the proposed modification. However, In re Urbanski, 809 F.3d 1237, 1244, 117 USPQ2d 1499, 1504 (Fed. Cir. 2016) (The patent claims were directed to a method of enzymatic hydrolysis of soy fiber to reduce water holding capacity, requiring reacting the soy fiber and enzyme in water for about 60-120 minutes. The claims were rejected over two prior art references, wherein the primary reference taught using a longer reaction time of 5 to 72 hours and the secondary reference taught using a reaction time of 100 to 240 minutes, preferably 120 minutes. The court held that both prior art references "suggest[ed] that hydrolysis time may be adjusted to achieve different fiber properties. Nothing in the prior art teaches that the proposed modification would have resulted in an ‘inoperable’ process or a dietary fiber product with undesirable properties." (emphasis in original)).
It is the Examiner’s understanding that Applicant is suggesting that modifying the teachings of the prior art in the manner suggested would forego the benefits taught by Shemesh (i.e., resisting to environmental stress and exposure to antimicrobial agents; thus, biofilm formation represents a strategy for persistence under unfavorable conditions (i.e., loss of cell viability) in diverse environments) and Balasundararajan (i.e., increasing the cell density of a strain and producing extracellular polymeric substances in which cells are embedded with maximum protection); thereby teaching away from the combination. However, the suggested combination of the teachings of ‘774 with Shemesh and Balasundararajan as discussed above, do not result in an inoperable method of biofilm formation with increased cell density, increased production of extracellular polymeric substances and increased resistance to environmental stress. Furthermore, a person of ordinary skill in the art would have been motivated with reasonable expectation of success to combine the teachings of ‘774, Shemesh and Balasundararajan in order to arrive at the claimed composition to reduce cell viability loss during liquid or dry storage.
Accordingly, the Examiner maintains that a case of obviousness has been established in light of the foregoing discussion and as evidenced by the references cited above. Therefore 35 U.S.C. 103 rejection to claims 1-7, 11, 16, 24, 26, 30, 40-41, 87 and 93-96 is maintained.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CLAUDIA E ESPINOSA whose telephone number is [phone]. The examiner can normally be reached Monday-Friday 9:30-5:30 EST.
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/CLAUDIA ESPINOSA/Patent Examiner, Art Unit 1654
/LIANKO G GARYU/Supervisory Patent Examiner, Art Unit 1654
OK, I

Prosecution Timeline

• Apr 21, 2021: Application Filed
• Apr 05, 2024: Non-Final Rejection — §103, §112
• Jul 09, 2024: Response Filed
• Oct 19, 2024: Final Rejection — §103, §112
• Dec 12, 2024: Response after Non-Final Action
• Dec 23, 2024: Response after Non-Final Action
• Jan 15, 2025: Request for Continued Examination
• Jan 17, 2025: Response after Non-Final Action
• May 13, 2025: Non-Final Rejection — §103, §112
• Aug 12, 2025: Response Filed
• Sep 13, 2025: Final Rejection — §103, §112
• Dec 11, 2025: Request for Continued Examination
• Dec 15, 2025: Response after Non-Final Action
• Jan 30, 2026: Non-Final Rejection — §103, §112 (current)