Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a divisional application of PCT/EP2020/0661 98, filed June 11,
2020. Applicant's amendments filed on March 20, 2026 is acknowledged. Claims 2, 5-12, and 22 are canceled, and claims 23-25 are newly added. Claims 1 and 4 are amended. Currently claims 1, 3-4, 13-21, and 23-25 are pending and under examination.
Claim Objections
Claim 24 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 23. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
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.
Claim 25 is 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 25 recites the limitation "the ammonium" in line 1. There is insufficient antecedent basis for this limitation in the claim.
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 non-obviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3-4, 13, 19-21, and 23-25 are rejected under 35 U.S.C. 103 as being unpatentable over Furigo et al. (Bioprocess Engineering, 1993, vol. 9, pgs. 119-127, cited in PTO-892 mailed 9/20/24, hereinafter “Furigo”) in view of Larsen (US 2011/0244543A1, cited in PTO-892 mailed 04/02/2025).
Regarding claims 1, 3, 13, 21, and 25, Furigo teaches nitrogen limited growth of a methanotrophic culture with Methanococcus capsulatus as the main species in a mixed culture, in a 5 liter fermenter equipped with devices for control of pH and temperature (title, pg. 119, col. 1, para 2; sec. 2.1). Furigo teaches a liquid substrate composition (fermentation broth), natural gas as the carbon-substrate, and ammonia as the nitrogen-substrate in the broth (pg. 119, col. 2, para 1, pg. 120, sec. 2.2; Fig. 1). Furigo teaches the flow of gas to the fermenter is kept at approximately 200 liter/day and the air is supplied at a rate corresponding to a methane/oxygen ratio of 0.7, which is adequate for an excess in order to maintain the nitrogen limitation, a form of regulating the nitrate concentration (pg. 120, sec. 2.2). Furigo teaches vigorous stirring yielding an approximate transfer coefficient kLa of 700/h ensures that neither oxygen nor natural gas becomes limiting, wherein an oxygen probe in the fermenter confirms this during fermentations, which meets the limitations of claims 1 and 13 (pg. 119, sec. 2.1).
Furigo teaches nitrate concentration in the liquid phase is determined by the Brucine method, wherein samples are added to Brucine solution and measured by spectrophotometer (pg. 120, sec. 2.5). Furigo teaches the nitrogen concentration in the broth is near 0 mM (below 0.5 mM) at dilution rates up to 0.22 /h, and is about 4 mM (0.248 g/l) at a dilution rate of 0.22/ h, wherein the fermentations carried out at dilution rates of 0.01-0.22/h, the nitrogen concentration (which comprises nitrate) is significantly below 0.5 mM (0.031 g/l) approaching 0.0 g/l (pg. 121, Fig. 5). Furigo teaches when there is an excess of ammonia, the consumption of nitrate becomes maximum as soon as ammonia falls back to the steady state level and has been observed that ammonia is the preferred nitrogen source and leaves nitrate if present together with ammonia in the broth, thus it is reasonable to assume that ammonia exerts an inhibitory effect on the reduction of nitrate and nitrite and creates pools of these two nitrogen compounds, particularly if they are formed because of excess of ammonia (pg. 122, col. 2, para 2). Although Furigo does not explicitly disclose the nitrate levels are kept below 0.01 g/L, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Furigo teaches a nitrogen control process that keeps nitrate levels significantly low, close to the claimed nitrate concentration, thus is obvious. Furigo also teaches ammonium pulse experiments, wherein 15 mmol pals of ammonium as sulphate are pulsed into the broth, and discloses the observation that consumption of the nitrate formed from the ammonia impulse will not commence until excess ammonia has disappeared (pg. 125, col.1, para 2, Fig 9). In each experiment pulsing 15 mmol and 25 mmol ammonium, nitrate concentration peaks at 2-4 hours at concentrations of 0.124 g/L, then reduces to 0.006 g/L after 6 hours (Fig. 9-10), thus further underlining the disclosure of regulating a nitrogen substrate to maintain nitrate concentration below 0.01 g/L.
Furigo teaches the fermentation reactor comprises a gas inlet that is controlled by a wet precision gasmeter, that enables a fine balance of volume of gases (pg. 119, sec. 2.1, Fig. 1).
Furigo does not teach a fermentation reactor comprising a sensor for determining the concentration of nitrate in the fermentation broth by in-line analysis. Furigo also does not teach the gas inlet is controlled by a computer based on data from the nitrate sensor, as part of an ‘in-line analysis’.
However, Larsen teaches at least one sensor or analyzer for determining the concentration of nitrate in the fermentation broth (fermenter includes one sensor configured to measure concentration of nitrate in the broth; [0094]); and wherein the fermentation reactor comprises at least one supply pump configured and/or controlled to automatically regulate the nitrate concentration in the fermentation broth (FIG. 4: a pump (111) coupled to the injection points (110) for introducing gases; [0086]; the addition of gases into the fermenter is controlled by a control system based on data (nitrate concentration) obtained by the at least sensor; [0097], [0103], [0111]; thus, the pump is configured and/or controlled automatically regulate the nitrate concentration in the fermentation both). Larsen teaches addition of substrate components in liquid solution, additional water, recirculation of supernatant as make-up for the withdrawn broth and substrate gases are controlled by a computer receiving data from the gas sensors in the headspace, ion sensors or analyzers, thermo sensors, pressure sensors, etc. and calculating the necessary amounts of each component for obtaining optimized growth of the microorganisms, thus meeting the limitation of an in-line analysis [0097].
Therefore 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 modify the fermentation process wherein a fermentation reactor supplies a carbon substrate, a nitrogen substrate (i.e. ammonia), and maintains the nitrate concentration in the fermentation broth significantly below 0.035 g/L by regulating the flow of carbon-substrate, nitrogen-substrate, and oxygen, and combine the reactor configuration of a nitrate sensor that sends data to a computer that regulates inlet of gases for maintaining the nitrate concentration as taught by Larsen with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to include a sensor for determining nitrate in the fermentation broth that would allow real-time regulation of nitrate concentrations in the fermentation, wherein appropriate amounts of gas fed to the culture would result in optimized fermentation conditions, since excess ammonia exerts an inhibitory effect on the reduction of nitrate and nitrite, thereby slowing the fermentation process, as taught by Furigo.
Regarding claim 4, modified Furigo further teaches the experiments were carried out in a steady state, which deduced the important parameters as yield on carbon, nitrogen, and maximum growth rate (pg. 120, sec. 3). As evidenced by the Specification (pg. 20, lines 9-10), a “steady state fermentation” is also known as a continuous phase fermentation.
Regarding claim 19, Furigo further teaches the substrate being gaseous, a very large mass transfer number from gas phase to liquid phase is necessary; this latter condition can only be met in nozzle fermenters which can produce volumetric mass transfer coefficients far beyond those in stirred tank fermenters (pg. 119, col. 1, para 1). Furigo does not explicitly teach the circulating fermentation liquid is affected by alternating pressure, which increases the mass transfer and solubility of the substrate gases.
However, Larsen teaches the productivity of the fermentation process is further optimized in that the circulating fermentation liquid experiences an alternating pressure during circulation in the fermenter and has an increased mass transfer and solubility of substrate gases into the liquid phase in the zone having an increased pressure [0045].
Therefore, 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 optimize by modifying the fermentation process taught by Furigo by alternating pressure on the circulating fermentation liquid which increases mass transfer and solubility of the substrate gases as taught by Larsen with a reasonable expectation of success. Both Furigo and Larsen acknowledge reactor tanks with only vigorous mixing requires the use of excessive energy and additional tank configurations (i.e. nozzle fermenters) for efficient mass transfer to the liquid, thus it would be obvious to one of ordinary skill to alternate pressures in the tank for enhancing the mass transfer and solubility of the substrate gases for optimal fermentation production, as taught by Larsen.
Regarding claim 20, Furigo does not teach the use of heterotrophic bacteria that are co-fermented with the methanotrophic microorganisms. However, Larsen teaches one or more heterotrophic bacteria is added to the methanotrophic fermentation liquid [0050]. Larsen teaches M. capsulatus (the same methanotrophic organism taught by Furigo) culture will accumulate acetic acid and other carboxylic acids due to the content of higher hydrocarbons in the natural gas, which will inhibit growth of M. capsulatus [0059]. Therefore, Larsen teaches it may be useful to co-ferment one or more strains of heterotrophic bacteria with the methanotrophic bacteria for digesting higher hydrocarbons (alcohols, carboxylic acids, etc.) e.g. ethanol, acetate, citrate, etc. or degradation products of partially digested dead or decaying biomass [0059]. Thus, 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 co-ferment the methanotrophic microbe with heterotrophic bacteria to reduce inhibition of toxic byproducts (carboxylic acids) and enhance the fermentation process, as disclosed by Larsen.
Regarding claims 23-24, Furigo teaches in the steady state condition of the dilution rates of 0.01/h-0.10/h, cell density is maintained around 6.3 g/l to 4.5 g/L dry cell weight, wherein at those dilution rates, the total nitrogen concentration is below 0.5mmol/L (pg. 121, Figs. 2 & 4), which would suggest to one of ordinary skill in the art that maintaining a low nitrogen concentration would effectively result in at least 4.6 g/L of biomass in the fermentation process, thus would be obvious to one of ordinary skill in the art at the time of filing the claimed method.
Claims 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Furigo and Larsen as applied to claims 1, 3-4, 13, 19-21, and 23-25 above, and further in view of Vigreux (EP 0057152-B2, cited in PTO-892 mailed 04/02/2025).
Regarding claims 14 and 15, Larsen teaches a fermentation reactor comprising a loop part (FIG. 2: U-shaped fermenter {100) having a loop part; [0073], [0076]) and a top tank (FIG. 2: U-shaped fermenter (100) having a top tank (top part 104); [0073], [0076]), said loop-part comprising a downflow part, connected to an upflow part via a U-part (FIG. 2: U-shaped fermenter includes a down-flow leg (101) and an up-flow leg (102); [0073], [0076]), wherein the top tank comprises: (i) a first outlet connecting the top tank to the downflow part of the loop-part and allowing a fermentation liquid present in the top tank to flow from the top tank into the loop-part (FIGS. 2-3: opening of the top tank (top part 104) connected to the down-flow leg (101); [0073], [0076]); (ii) a first inlet connecting the top tank to the upflow part of the loop-part, allowing fermentation liquid present in the loop-part to flow from the loop part into the top tank (FIGS. 2-3: opening of the top tank (top part 104) connected to the up-flow leg (102); [0073], [0076]); wherein the fermentation reactor further comprises: (v) at least one inlet for supplying a nitrogen-source comprising ammonia, an ammonium compound and/or molecular nitrogen (injection points 110 or through other nozzles (not shown) placed in the down-flow leg or up-flow leg or the U-bend of the fermenter; [0049], [0063], [0086], [0110]; Claim 46); and (vi) at least one sensor or analyzer for determining the concentration of nitrate in the fermentation broth (fermenter includes one sensor configured to measure concentration of nitrate in the broth; [0094]); and wherein the fermentation reactor comprises at least one supply pump configured and/or controlled to automatically regulate the nitrate concentration in the fermentation broth (FIG. 4: a pump (111) coupled to the injection points (110) for introducing gases; [0086]; the addition of
gases into the fermenter is controlled by a control system based on data (nitrate concentration) obtained by the at least sensor; [0097], [0103], [0111]; thus, the pump is configured and/or controlled automatically regulate the nitrate concentration in the fermentation broth).
Furigo teaches the fermentation tank comprises a gas outlet, but neither Furigo nor Larsen explicitly teach a vent tube for discharging effluent gas from the top tank. However, Larsen does disclose wherein the gas from a headspace (117) of the top tank is release via a valve (120) [0089]). Further, Larsen discloses wherein the vent tube is well-known in the art for venting gas or gases separated in the headspace of a top tank (FIG. 1: venting tube 6; [0070]). In view of Larsen, it would have been obvious to one of ordinary skill in the art to have incorporated a venting tube into the top tank of Larsen for the purpose of venting gas or gases separated in the headspace of a top tank, as disclosed by Larsen (FIG. 1: venting tube 6; [0070]). Further, one of ordinary skill in the art would have made said modification since such component is well-known in the art as disclosed by Larsen ([0070]).
Neither Furigo nor Larsen explicitly teach the fermentation reactor comprises a visual inspection means, such as an inspection hole.
However, Vigreux teaches a fermentation reactor comprising an inspection hole for periodic maintenance operations (see FIG. 1 and page 4, para 9 of the English translation).
Therefore 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 modify the fermentation process taught by Furigo, with utilizing a fermentation reactor comprising a loop-part and top tank, connected to via a U-part, wherein the top tank comprises a vent tube for discharging effluent gases as taught by Larsen, and including a visual inspection hole in the top tank taught by Vigreux with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to incorporate an inspection hole into the top tank of Larsen for the purpose of facilitating periodic maintenance operations of the top tank as disclosed by Vigreux.
Regarding claim 16, modified Larsen teaches the fermentation reactor comprises a flow reducing device (e.g., valve and narrowing of diameter/cross section of a portion of the fermentation reactor; [0039]-[0040] and [0081]).
Regarding claim 17, modified Larsen further discloses wherein the fermentation reactor further comprises a first pressure controlling device and a second pressure controlling device and optionally, a third pressure controlling device ([0038]-[0040]).
Regarding claim 18, modified Larsen further discloses wherein the first pressure controlling device is selected from a valve, a pump such as a propeller pump, a lobe pump, a turbine pump or nozzles or jets, and wherein the second and/or optionally third pressure controlling device is selected from a valve, a static mixer, a hydrocyclone, a pump such as a propeller pump, a lobe pump, a turbine pump, a pressure controlled valve, a plate with holes, nozzles or jets or a narrowing of the diameter or cross-section of the fermentation reactor part in which it is placed ([0038]-[0040] and [0081]).
Response to Arguments
Applicant's arguments filed March 20, 2026 have been fully considered but they are not persuasive.
Regarding the 103 rejections, Applicant argues the phrase of "regulating flow of the nitrogen-substrate to the fermentation reactor to maintain a nitrate concentration of the fermentation broth below 0.01g/" as recited in amended claim 1 is a technical solution on the premise that ammonia is sufficiently supplied to meet the growth needs of microorganisms, but not so much that it inhibits the production of biomass. The threshold limitation specifically of nitrate has inherent differences in technical scenarios and purposes from the nitrate concentration of 0 caused by insufficient ammonia for research purposes disclosed in Furigo. A core purpose is to avoid nitrate excretion and inhibition of biomass production caused by excessive ammonia on the premise of ensuring the sufficient supply of nitrogen source required for microbial growth, rather than making the nitrate concentration approach 0 by "not supplying sufficient ammonia." This is an important feature the present invention and a necessary condition for achieving efficient biomass production in industrial fermentation. Efficient production of biomass is neither explicitly or inherently disclosed in Furigo. Applicant argues Furigo merely describes the interplay between total broth nitrogen and biomass content in Figures 2-5. Starting from ammonium (N1/N2 in Figure 8), the pathway initially leads to either enzymatic formation of nitrite (N3/N3e in figure 8) or biomass (dXW). The produced nitrite may either be converted (reduced) back into ammonium (N2 in figure 8) or may be converted into nitrate by oxidation (N4/N4e in figure 8). Furigo fails to teach or suggest any negative correlation between biomass and nitrate content, on the contrary Furigo specifically states that the excreted levels of nitrate up to 0.31g/l are well tolerated (cf. page 123, left column, 3rd paragraph lines 9-13, page 126, right paragraph, line 9-15). Thus, Furigo neither recognizes the nitrate concentration as limiting biomass production nor suggests that further reduction would provide any benefit. Moreover, Furigo certainly does not explicitly or inherently discuss any threshold level of nitrate concentration that impacts biomass production. Applicant argues in the presently claimed invention, the "nitrate concentration exceeding 0.01 g/L leads to a significant decrease in biomass" and "regulating flow of the nitrogen-substrate to the fermentation reactor to maintain a nitrate concentration of the fermentation broth below 0.01g/l, while maintaining a level of nitrogen-substrate in the fermentation broth above 0 g/l to support growth of the microorganism" are both realized in the industrial scenario where ammonia supply meets the basic growth needs of microorganisms and ensures biomass production. Even the lowest ammonia pulse (0.01 g/L) in the experiments always ensures sufficient ammonia supply, and there is never a situation of insufficient nitrogen source, which is inherently different from the scenario of "nitrate concentration of 0 caused by insufficient ammonia." The examples demonstrate that reducing nitrate to <0.01 g/L results in a disproportionately higher biomass relative to cultures grown at higher nitrate concentrations (cf. Table 2 of the present application), an effect that would not have been expected in view of Furigo's teaching that 0.31 g/L already provides satisfactory growth. This demonstrates the criticality of the claimed upper limit. Applicant argues in Figure 5 of Furigo, when D < 0.22/h, the total nitrogen concentration in the culture medium approaches 0 mmol/L, which is a state of insufficient ammonia in continuous chemostat culture where the nitrogen source supply rate is lower than the microbial consumption rate. It is not an industrial fermentation process for achieving efficient biomass production, nor is it an operating condition adopted in actual production. The description of the process in Furigo belongs to a completely different scenario from the nitrate concentration control achieved by the present invention in the industrial fermentation scenario of sufficient ammonia supply and oriented to biomass production. A person of ordinary skill in the art would not have been taught, suggested or motivated to rely on Furigo as a comparable fermentation process in any attempt to achieve the presently claimed invention related to nitrate monitoring and control.
In response to applicant's argument that Furigo did not explicitly teach regulating nitrate concentration below 0.01 g/L in the fermentation broth to achieve maximum biomass production as presently claimed, the fact that the inventor has recognized another advantage which would flow naturally 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). Furigo’s method teaches nitrogen-substrate supply limitation, and describes a model wherein excess ammonium in the broth inhibits exert an inhibitory effect on the reduction of nitrate and nitrite thus creating pools of these two nitrogen compounds, and discloses the assumption that nitrate is not consumed until ammonia has disappeared is confirmed and in accordance with the statement that ammonia and nitrate exhibit diauxi (pg. 126, col. 2, para 1). Although Furigo does not explicitly disclose nitrate concentrations below 0.1 g/L would enable efficient biomass production, Furigo does disclose the observation that ammonia is the preferred nitrogen source and leaves nitrate if present together with ammonia in the broth, thus it is reasonable to assume that ammonia exert an inhibitory effect on the reduction of nitrate and nitrite thus creating pools of these two nitrogen compounds particularly if they are formed because of excess of ammonia. Furthermore, Fig. 6 shows high g dry cell to mol N ratio when dilution rates are kept below 0.1/h, but above 0.4/h, which further suggests keeping nitrogen concentrations low for efficient biomass production (pg. 122). Thus, Furigo teaches regulation of the nitrogen-substrate supply with a methanotroph culture under ammonia limiting conditions and describes the inhibitory effect of ammonia on biomass production, as well as nitrate concentrations in the broth, therefore the claimed method is prima facie obvious over the prior art.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA EDWARDS whose telephone number is (571)270-0938. The examiner can normally be reached M-F 8am-5pm EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Louise Humphrey can be reached at (571) 272-5543. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
Prosecution Insights