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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/23/2025 has been entered.
DETAILED ACTION
Claims 2, 6-10, 15, 18, 22, and 25-46 are cancelled. Claims 47-51 are new. Claims 1, 3-5, 11-14, 16-17, 19-21, 23-24 and 47-51 are pending. Claims 4-5, 17, and 19-21 withdrawn from consideration as being drawn to a non-elected invention.
Accordingly, claims 1, 3, 11-14, 16, 23-24 and 47-51 are under consideration in this action.
Response to Amendment
The amendment filed 03/27/2025 is entered.
The § 112(b) rejection of claim 11 for lack of antecedent basis is withdrawn in light of the amendment.
The § 103 rejection of claims 1, 3, 11-14, 16 and 23-24 over Haas with evidence from Yasin and Kato is withdrawn. However, a new § 103 rejection is discussed below.
Applicants' amendments and arguments filed on 03/27/2025 have been fully considered. Rejections and/or objections not reiterated from previous office actions are hereby withdrawn. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Priority
The instant claims are entitled to an effective filing date of 04/12/2021.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
New Matter
Claims 1, 3, 11-14, 16, 23-24 and 47-51 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. The amendment filed on 03/27/2025 has introduced new matter into the claims. The underlined limitations below constitute as new matter.
Claim 1 as filed on 03/27/2025 recites a method of culturing an anaerobic bacterium and an aerobic microorganism, comprising culturing in a microaerobic environment the anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; the anaerobic bacterium increases in cell density and produces an increased amount of acetate or ethanol compared to the anaerobic bacterium cultured without the aerobic microorganism; and the anaerobic bacterium is of the genus Clostridium, Eubacterium, or Sporomusa.
Claim 23 as filed on 03/27/2025 recites a method of producing a product, comprising culturing in a microaerobic environment anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2;the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; the anaerobic bacterium increases in cell density and produces an increased amount of acetate or ethanol compared to the anaerobic bacterium cultured without the aerobic microorganism; the aerobic microorganism produces the product; and the anaerobic bacterium is of the genus Clostridium, Eubacterium, or Sporomusa.
Claim 24 as filed on 03/27/2025 recites a method of syngas fermentation, comprising culturing in a microaerobic environment anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; the anaerobic bacterium increases in cell density and produces an increased amount of acetate or ethanol compared to the anaerobic bacterium cultured without the aerobic microorganism; the aerobic microorganism increases in cell density compared to the aerobic microorganism cultured without the anaerobic bacterium; the aerobic microorganism produces the product; and the anaerobic bacterium are of the genus Clostridium, Eubacterium, or Sporomusa.
Applicant asserts that support for the claim amendment can be found throughout the specification and in the claims as originally filed, for example. Applicant asserts that no new matter has been added. See the first paragraph on page 6 of the remarks filed 03/27/2025. However, the specification and claims filed 04/12/2022 do not provide sufficient written description of the above underlined limitations.
Claims 1, and 23 contain new matter because of the limitation requiring the anaerobic bacterium to increase in cell density and produce an increased amount of acetate or ethanol compared to the anaerobic bacterium culture without the aerobic microorganism. Claims 3, 11-14, 16 and 47-50 contain new matter because the claims depend from claim 1 and require the same limitation. Claim 24 contains new matter because the claim requires the anaerobic bacterium to increase in cell density and produce an increased amount of acetate or ethanol compared to the anaerobic bacterium culture without the aerobic microorganism; and because the claim requires the aerobic microorganism to increase in cell density compared to the aerobic microorganism cultured without the anaerobic bacterium. Claim 51 contains new matter because the claim depends from claim 24 and requires the same two limitations.
The specification as filed and the original claims do not provide support for these limitations in claims 1, 23 and 24. In example 2, the specification demonstrates a successful co-cultivation of Clostridium ljungdahlii and Escherichia coli in bioreactors sparged with 1% O2. The total growth was monitored by OD600. Product formation is determined by HPLC-RID. As shown in figure 4, C. ljungdahlii grows rapidly even with continual sparging, reaching a final OD of ~5. Within approximately 10 hours, C. ljungdahlii becomes the dominant microbe in the reactor. Additionally, acetate and ethanol products are detected in the broth. See the first paragraph on page 16. Figure 4A shows the co-culture growth (dark= E. coli, light= C. ljungdahlii, Black=redox) in a rich medium sparged with 1% O2. See lines 22-23 on page 4 and figure 4A below. Furthermore, in example 2, the specification teaches examining whether the co-culture can be established in a minimal medium. Simultaneous growth of both microbes (Figure 5) is observed but the relative abundances are different, with E. coli accounting for most of the biomass under these conditions. See figure 5 and the paragraph spanning pages 16-17. Figure 5 shows the successful co-culture growth in minimal medium sparged with 2% O2. See page 4 lines 25-26. Although figures 4A and 5 show an increase in co-culture cell density, the figures and specification do not compare the observed cell densities of the co-cultures to the cell densities of C. ljungdahlii and E. coli individually in monocultures. Moreover, figures 4A and 5 are silent regarding the acetate or ethanol production of C. ljungdahlii in the co-culture with aerobic E. coli as compared to the
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production of acetate or ethanol by C. ljungdahlii without E. coli.
The specification teaches a reverse order inoculation in which the reactor is initiated with C. ljungdahlii and a gas feed containing no oxygen. Then, oxygen is added to the feed and E. coli is inoculated after 24 hours of growth (Figure 6). A concurrent growth of both microorganisms is observed as evidenced by the density increases. The “short window of co-culture growth [is] due to exhaustion of xylose by C. ljungdahlii”. See the first full paragraph on page 17 and figure 6. The reverse order inoculation method described in example 2 does not constitute as support for the newly added limitations because figure 6 indicates that cell density of C. ljungdahlii decreases in the presence of both E. coli and oxygen. Figure 6, as shown below includes two vertical dotted lines to indicate either E. coli inoculation or oxygen introduction. There is a short increase in the cell density of C. ljungdahlii between the two vertical dotted lines. However, the cell density decreases after the second vertical dotted line. Therefore, the anaerobic C. ljungdahlii bacterium has a decreased cell density when cultured with the aerobic E. coli microorganism in a microaerobic environment continually sparged with
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oxygen.
Such limitations recited in the instant claims 1, 23 and 24 and required by the dependent claims, which did not appear in the specification or original claims, as filed, introduce new concepts and violate the description requirement of the first paragraph of 35 U.S.C 112. Applicant is required to provide sufficient written support for the limitations recited in the instant claims. Applicant can remove the new matter limitations from the claims to obviate this rejection.
Enablement
Claim 1, 3, 11-14, 16, 23-24 and 47-51 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention:
Under the broadest reasonable interpretation, claims 1, 3, 11-14, 16, 23-24 and 47-50 imply that culturing any anaerobic strain of Clostridium, Eubacterium, or Sporomusa with any aerobic microorganism in the instantly claimed microaerobic environment under continuous O2 sparging can increase the cell density of the anerobic bacterium and can increase the anaerobic bacterium’s production of acetate or ethanol, as compared to a culture of the anaerobic bacterium without the aerobic microorganism. Furthermore, claims 24, and 51 indicate that a syngas fermentation of any anaerobic Clostridium, Eubacterium, or Sporomusa bacterium and any aerobic microorganism in the instantly claimed microaerobic environment under continuous O2 sparging can further increase the cell density of the aerobic microorganism, as compared to the aerobic bacterium cultured without the anaerobic bacterium.
The state of the prior art and the level of predictability in the art:
With respect to the state of the art on anaerobic and aerobic co-cultures, Haas teaches culturing anaerobic C. ljungdahlii with aerobic E. coli in an environment with a gas mixture composed of 65% H2, 33% CO2, and 2% O2 at 37ºC (e.g. a microaerobic environment) in example 3. See paragraphs [0089] and [0092]. Haas indicates that C. ljungdahlii grows in this co-culture because Haas discloses that there was an increase in acetate from 330 mg/l to 378 mg/l. See paragraph [0094]. Thus, Haas indicates that the anaerobic bacterium cell density and acetate production increase throughout the co-culture with E. coli. However, in example 1, Haas teaches a C. ljungdahlii monoculture capable of producing more acetate, as compared to the co-culture of example 3. In example 1, Haas teaches culturing C. ljungdahlii in the presence of 0.15% O2--. See [0080]-[0080]. The wild strain C. ljungdahlii is cultured autotrophically in a complex medium. The gas trapped in the medium is removed by a frit. The reactors are purged with pre-mixed gas mixture comprising in 0.15% O2--. See [0081]. As disclosed in table 1, the experiment 1 shows the following: an increase in the OD600 from 0.042 to 0.406, an increase in ethanol production from 9 mg/L to 362 mg/L, and an increase in acetate production from 296 mg/L to 4495 mg/L. However, Haas discloses that in repeated experiments the bacteria cells were unable to grow in the presence of 0.15% O2--. See [0083]-[0084]. Thus, Haas illustrates the unpredictability in the art because Haas teaches a monoculture of C. ljungdahlii that is capable of producing more acetate (4495 mg/l), as compared to the co-culture with E. coli (378 mg/l).
With respect to the state of the art of symbiotic co-culture systems for increased cell density and productivity, Wu (Microbial cell factories, 2016, 15, 1-11) discloses that in general, a symbiotic system or co-culture system appears to be advantageous over single culture because of the potential for cooperative utilization of the metabolic pathways of all involved organisms. Cell growth of one micro-organism may be enhanced by activities of other micro-organisms. For example, both of C. acetobutylicum TSH1 and B. cereus TSH2 display poor growth when cultured individually, however, promotion of cell growth occurs in a symbiotic system. In the symbiotic system, not only cell growth but also solvent producing ability is enhanced. See the first two paragraphs of the ‘results and discussion section’. However, Wu suggests that acetone, butanol and ethanol fermentation still has a number of challenges to face. See the sentence spanning pages 1-2. Wu indicates that some reports confirm that aerobic bacteria, such as B. subtilis and B. cereus, have a positive interaction with anaerobic Clostridium, but the role of aerobic bacteria is not clearly established. Moreover, different microorganisms in a co-culture system may compete for substrates, therefore, the product yield and productivity are influenced compared with single cultures. The complicated community interactions between different microorganisms in co-culture systems are still unclear, so Wu suggests that further study is necessary. See the first full paragraph in the right column on page 2. Furthermore, in table 4, Wu teaches an example of a C. acetobutylicum mono-culture that produces 2.3 ± 0.04 g/L ethanol, as compared to a symbiotic system with B. cereus that produced 2.2± 0.1 g/L ethanol. This result indicates that a monoculture of an anaerobic Clostridium bacterium may produce more ethanol alone as compared to the Clostridium bacterium cultured in a symbiotic system with an aerobic B. cereus microorganism. In conclusion, Wu illustrates the unpredictability in the art of symbiotic systems for increased cell density and productivity because Wu suggests that not every aerobic microorganism and anaerobic Clostridium bacterium may form a symbiotic due to complicated community interactions.
The amount of direction provided by the inventor and the existence of working examples:
The specification does not provide a working example of anaerobic bacterium and aerobic microorganism co-culture in which the cell-density of the anerobic bacterium is increased and the acetate or ethanol productivity is increased, as compared to a monoculture of the anaerobic bacterium without an aerobic bacterium. In example 2, the specification teaches a reverse order inoculation in which the reactor is initiated with C. ljungdahlii and a gas feed containing no oxygen. Then, oxygen is added to the feed and E. coli is inoculated after 24 hours of growth. See page 17 and figure 6. Figure 6 includes two vertical dotted lines that indicate the time at which the E. coli is inoculated and the time at which oxygen is introduced. Between the two vertical dotted lines the cell density of the C. ljungdahlii increases. However, after the second dotted line the cell density of the C. ljungdahlii decreases. The specification discloses that this “short window of co-culture growth [is] due to exhaustion of xylose by C. ljungdahlii”. See the first full paragraph on page 17 and figure 6. Therefore, the reverse order inoculation method described in example 2 indicates that the anaerobic C. ljungdahlii bacterium has a decreased cell density when cultured with the aerobic E. coli microorganism in a microaerobic environment continually sparged with oxygen, as compared to a culture in which C. ljungdahlii is cultured without the E. coli, i.e. the time prior to the inoculation of E. coli in which C. ljungdahlii is cultured alone (see figure 6).
The specification provides a working example of a co-culture in which the cell density of the aerobic microorganism increases; however, the specification does not provide a comparison to a culture of the aerobic microorganism without the anaerobic bacterium. In example 2, the specification teaches examining whether the co-culture can be established in a minimal medium. Simultaneous growth of both microbes (Figure 5) is observed but the relative abundances are different, with E. coli accounting for most of the biomass under these conditions. See figure 5 and the paragraph spanning pages 16-17. Figure 5 shows the successful co-culture growth in minimal medium sparged with 2% O2. See page 4 lines 25-26. Although figure 5 shows the cell density of the E. coli increasing, the figures and specification do not compare the observed cell density to the cell density of an E. coli monoculture.
The quantity of experimentation needed to make or use the invention:
In view of the nature of the invention, the breadth of the claims, the guidance and
working examples in the specification, and the level of predictability within the art, as
evidenced above, one skilled in the art could not use the instantly claimed culture method to increase the cell density and acetate or ethanol production of an anaerobic Clostridium, Eubacterium or Sporomusa bacterium without undue experimentation. Prior to the effective filing date of the instantly claimed invention, it was well known that an anaerobic bacterium such as C. ljungdahlii can grow and produce acetate in a co-culture with an anerobic microorganism such as E. coli , as evidenced by Haas. However, Haas indicates that such co-culture may not consistently produce more cell density and acetate or ethanol production, as compared to a monoculture of the anaerobic C. ljungdahlii bacterium alone. The instantly disclosed working example indicates that the cell density of the anaerobic C. ljungdahlii bacterium may decrease in the presence of an aerobic E. coli bacterium and oxygen (figure 6). Although Wu suggests that symbiotic co-cultures can increase cell density and the production of products, Wu also indicates that not every aerobic and anaerobic microorganism combination can yield that intended result. The working examples in the specification focus on the cell density, acetate production and ethanol production of a C. ljungdahlii and E. coli co-culture. However, the specification does not compare the observed results to mono-cultures of C. ljungdahlii and E. coli. Consequently, there is no indication that the instantly claimed method can increase the cell density and acetate or ethanol production of any anaerobic Clostridium, Eubacterium or Sporomusa bacterial strain in a co-culture with any aerobic microorganism, which is commensurate with the scope of the claims; and, there is no indication that the instantly claimed syngas fermentation method can increase the cell density of the aerobic microorganism compared to the aerobic microorganism cultured without the anaerobic bacterium.
Accordingly claim 1 and dependent claims are enabled for a method of culturing an anaerobic bacterium and an aerobic microorganism, comprising culturing in a microaerobic environment the anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; and the anaerobic bacterium is of the genus Clostridium, Eubacterium, or Sporomusa. Claim 23 is enabled for a method of producing a product, comprising culturing in a microaerobic environment anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2;the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; the aerobic microorganism produces the product; and the anaerobic bacterium is of the genus Clostridium, Eubacterium, or Sporomusa. Claim 24 and dependent claim 51 are enabled for a method of syngas fermentation, comprising culturing in a microaerobic environment anaerobic bacterium and an aerobic microorganism; wherein the microaerobic environment does not require gas pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2; the aerobic microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium; the aerobic microorganism produces the product; and the anaerobic bacterium are of the genus Clostridium, Eubacterium, or Sporomusa.
Claim Interpretation
Only the enabled limitations discussed above are addressed below.
Claim 1 requires culturing an anaerobic bacterium, such as Clostridium ljungdahlii, and an aerobic microorganism, such as E. coli. See instant claims 11 and 16. Furthermore, claim 1 requires culturing the anaerobic and aerobic microorganisms in a “microaerobic environment”, which is interpreted as an environment where the dissolved oxygen concentration in the growth media is substantially reduced compared to the equilibrium concentration in the normal atmosphere, e.g. an environment with 2% oxygen at 37ºC. See the instant specification page 13 lines 27-29 for the definition, and see page 15 line 4 and page 17 lines 16-17 for an exemplary microaerobic environment. The microaerobic environment does not require a gas pre-treatment, such that any absence of a gas pre-treatment meets the instant limitation. The microaerobic environment is continually sparged with O2. The aerobic microorganism is required to consume oxygen in the culture to allow growth of the anaerobic bacteria; and the anaerobic bacteria are of the genus Clostridium, Eubacterium, or Sporomusa.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 3, 23, and 49-50 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wu (Microbial cell factories, 2016, 15, 1-11) with evidence from Huang ( Bioprocessing for value-added products from renewable resources, 2007, 185-223) and NOAA. (The Atmosphere. National Oceanic and Atmospheric Administration, 2023).
Regarding claims 1 and 23, Wu teaches a symbiotic system of Clostridium acetobutylicum TSH1 (e.g. anaerobic) and Bacillus cereus TSH2 (e.g. aerobic). Fermentation is performed statically at 37˚C without anaerobic treatment (e.g. discharge/removal of O-2). Bioreactor fermentation is caried out in a 5L fermenter containing P-2 medium. Microaerobic fermentation is achieved by flushing with air (e.g. oxygen) at a rate of 0.15 L/min. See the ‘culture conditions’ section on page 9. Wu discloses that cells grow well and produce 17.7 g/L acetate, butanol and ethanol (ABE) in the symbiotic system with continuous air sparing (e.g. continuous O-2 sparging). See the first passage on page 6 and the abstract. Wu discloses that B. cereus TSH2 consumes dissolved oxygen in the culture and offers an anaerobic condition for C. acetobutylicum TSH1. See the first full paragraph in the left column on page 4. Wu discloses that in the symbiotic system TSH06, not only cell growth (e.g. cell density) but solvent producing (e.g. ethanol) ability is enhanced. See the last paragraph in the right column on page 3 and figure 1B. Thus, Wu teaches a method of culturing an anaerobic bacterium and aerobic microorganism (relevant to instant claim 1), and Wu teaches a method of producing ABE products (relevant to instant claim 23) comprising culturing in a microaerobic environment the anaerobic bacterium and aerobic microorganism; wherein the microaerobic environment does not require an anaerobic treatment to remove O2, as evidenced by Huang below; the microaerobic environment is continually sparged with the oxygen in the air, as evidenced by NOAA; and the aerobic B. cereus microorganism consumes oxygen in the culture to allow growth of the anaerobic bacterium, and the anaerobic bacterium is of the genus Clostridium.
The “anaerobic treatment” of Wu is interpreted as the removal of O2 because evidentiary reference Huang states that anaerobic fermentation occurs in the fermentation vessel once the oxygen is discharged and replaced with N2, CO2 or another by-product. See the first 3 lines on page 211.
Evidentiary reference NOAA discloses that the atmosphere includes O2. See the table. Therefore, Wu teaches a microaerobic environment that is continually sparged with O2.
Regarding claim 3, evidentiary reference NOAA discloses that the atmosphere comprises O2, CO, H2, and CO2. See the table. Therefore, the air continually sparged into the microaerobic environment of Wu inherently includes O2, CO, H2, and CO2. See the first passage on page 6 and the abstract.
Regarding claim 49, Wu teaches the relative abundance of C. acetobutylicum TSH1 and B. cereus TSH2 at different inoculation ratios. As shown in figure 4, the relative abundance of the aerobic B. cereus TSH2 increases from hour 0 to hour 4 at each inoculation ratio. See figure 4 and it’s caption. Thus, Wu indicates that the aerobic microorganism increases in cell density.
Regarding claim 50, Wu discloses that C. acetobutylicum TSH1 becomes the dominant species and accounts for 99.85% of the whole population in solventogenic phase. Increasing inoculation ratio of B cereus TSH2 affects butanol producing ability of the symbiotic system, but the relative abundance of each strain is not affected in the final broth, and C. acetobutylicum TSH1 is the dominant species no matter how the inoculation ratio is changed. See the last paragraph of the conclusion section on page 9
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Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 11-14, 16, 23-24, 47- 51 are rejected under 35 U.S.C. 103 as being unpatentable over Haas (US 2017/0260552), in view of Wu (Microbial cell factories, 2016, 15, 1-11), with evidence from Yasid (Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate. Royal Society open science 2016, 3(8), pp.160187) and Kato (Anaerobe tolerance to oxygen and the potentials of anaerobic and aerobic cocultures for wastewater treatment. Brazilian Journal of Chemical Engineering 1997, 14, pp.395-407).
Regarding claims 1, 11, 16 and 47, Haas teaches a mixed culture of a first and second microorganism in an aqueous medium, wherein the second microorganism is selected from a group that includes aerobic microorganisms and the first microorganism is selected from a group that includes Clostridium ljungdahlii. See claims 1, 2 and 5. example 3, Haas teaches a method of culturing Clostridium ljungdahlii (i.e. an anerobic bacteria) and Escherichia coli, (i.e. an aerobic microorganism). See example 3 in paragraph [0087]. For the joint production phase, E. coli and C. ljungdahlii are grown in an environment with a gas mixture composed of 65% H2, 33% CO2, and 2% O2 at 37ºC (e.g. a microaerobic environment). See paragraphs [0089] and [0092]. Gas is trapped in the culture medium using a frit, i.e. a porous ring, which is mounted in the center of the reactors of a sparger [0092]. Haas suggests that the aqueous culture medium may comprise oxygen that is dissolved by any means known in the art, such as a continuous gas flow (e.g. sparging) [0025-0026]. The environment of Haas is considered a microaerobic environment because the environment contains 2% oxygen at 37ºC. Haas implies that a gas pre-treatment to remove trace O2 is unnecessary, because Haas discloses that C. ljungdahlii can tolerate the presence of O2 [0092][0083][0032]. Evidentiary reference Yasid discloses that E. coli grows by aerobic respiration in the presence of oxygen (lines 1-2 of page 2). Evidentiary reference Kato adds that aerobic bacteria can protect anaerobes from O2 exposure, as oxygen can be rapidly consumed (see the first six lines on page 8). Thus, Yasid and Kato suggests that E. coli inherently consumes any present oxygen in a culture. The oxygen condition established in the E. coli and C. ljungdahlii culture is considered to be a low-oxygen condition because the anaerobic C. ljungdahlii grew. See [0092-0094]. For clarity, Haas indicates that C. ljungdahlii grew in the culture because Haas discloses that there was an increase in acetate, which is a product of C. ljungdahlii. Specifically, Haas discloses that after the cultivation period the results showed that there is an increase in acetate from 330 mg/l to 378 mg/l. See paragraph [0094].
In summation, Haas teaches a method for culturing anaerobic C. ljungdahlii, comprising culturing in a microaerobic environment the anaerobic bacteria C. ljungdahlii (relevant to instant claims 11 and 47) and an aerobic microorganism E. coli (relevant to instant claims 16 and 47); wherein the microaerobic environment may not require pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2; and the aerobic E. coli microorganism of Haas inherently consumes oxygen such that the low-oxygen condition established is suitable for C. ljungdahlii growth. Yasid and Kato provide evidence for the inherent ability of E. coli to consume oxygen and establish a low-oxygen condition for anaerobic bacteria.
Haas does not explicitly teach a microaerobic environment that does not require gas pre-treatment to remove trace O2 (relevant to instant claim 1).
Wu discloses that strict anaerobes need special equipment and complicated operation to eliminate oxygen in the culture medium, for example, adding reducing agents or flushing with N2 gas which increases the total cost of acetate, butanol and ethanol fermentation. See the first passage on page 2. Wu teaches a symbiotic system of Clostridium acetobutylicum TSH1 (e.g. anaerobic) and Bacillus cereus TSH2 (e.g. aerobic). Fermentation is performed statically at 37˚C without anaerobic treatment (e.g. treatment to remove O-2). See the ‘culture conditions’ section on page 9. Wu discloses that cells grow well and produce acetate, butanol and ethanol (ABE) in the symbiotic system with continuous air sparing (e.g. continuous O-2 sparging). See the first passage on page 6 and the abstract for the indication that the sparging was continuous. Wu discloses that B. cereus TSH2 consumes dissolved oxygen in the culture and offers an anaerobic condition for C. acetobutylicum TSH1. See the first full paragraph in the left column on page 4. Wu discloses that in the symbiotic system, not only cell growth (e.g. cell density) but solvent producing (e.g. ethanol) ability is enhanced. See the last paragraph in the right column on page 3 and figure 1B.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to recognize that the removal of trace O2 is not required in the fermentation of Haas, because Haas suggests that C. ljungdahlii can tolerate O2 to an extent, and Wu suggests that an aerobic microorganism may consume the dissolved oxygen in the culture to offer an anaerobic condition. One would be motivated to skip the unnecessary step to save money because Wu suggests complicated operations to eliminate oxygen in the culture medium can increase the total cost. There would be a reasonable expectation of success because Haas suggested culturing the two strains in the presence of O2 and explicitly discloses that C. ljungdahlii could grow in the presence of 0.15% O2 and form typical products, such as acetate (see paragraph [0083]). Moreover, there would be a reasonable expectation of success because Wu demonstrates a co-culture with an anaerobic Clostridium bacterium and an aerobic microorganism that does not require an anaerobic treatment
Regarding claim 3, Haas teaches culturing C. ljungdahlii and E. coli in an environment containing a gas mixture of H2, CO2, and O2 and suggests that CO and/or CO2 may be used as a carbon source. See paragraphs [0092] and claim 1 on page 11.
Wu discloses that cells grow well and produce acetate, butanol and ethanol (ABE) in the symbiotic system with continuous air sparing (e.g. gas containing O2, CO, H2, and CO2). See the first passage on page 6 and the abstract
Regarding claims 12-14, Haas teaches a second microorganism, such as E. coli, capable of metabolizing acetate (i.e. an acetotroph microorganism) to a substituted or unsubstituted organic compound, such as an amino acid (e.g. a product). See Haas pages 11-12, claim 8, claim 13, and claim 14.
Regarding claim 23, Haas teaches a method of producing at least one substituted or unsubstituted organic compound, the method comprising contacting a mixed culture of a first and second microorganism with oxygen and a carbon source comprising CO and/or CO2 , wherein the first microorganism is an acetogenic microorganism, such as Clostridium ljungdahlii, capable of converting the carbon source to acetate and/or ethanol; and the second microorganism, such as E. coli, is capable of metabolizing acetate and/or ethanol to the substituted or unsubstituted organic compound; wherein the substituted or unsubstituted organic compound may be selected from a list that includes amino acids. See claims 8, and 12-14 of Haas. Oxygen and the carbon source are provided to the aqueous medium in a continuous gas flow (e.g. continually sparged). See claim 9. The continuous gas flow has a concentration of oxygen at a range of 0.01 to 10% by weight. See claim 11. In example 3, Haas teaches a joint production phase in which both growth cultures, E. coli and C. ljungdahlii are grown in 2% O2 (e.g. a microaerobic environment). See paragraph [0092]. Haas implies that a gas pre-treatment to remove trace O2 is unnecessary, because Haas suggests that C. ljungdahlii can tolerate the presence of O2 [0092][0083][0032]. Yasid and Kato provide evidence for the inherent ability of E. coli to consume oxygen and establish a low-oxygen condition for anaerobic bacteria. See lines 1-2 of page 2 of Yasid and the first six lines on page 8 of Kato. Thus, Haas teaches a method of producing an amino acid product comprising culturing in a microaerobic environment anaerobic bacteria and an aerobic microorganism wherein the microaerobic environment does not require gas pre-treatment to remove trace O2; the microaerobic environment is continually sparged with O2 through continuous gas flow; the E. coli aerobic microorganism inherently consumes oxygen in the culture to allow growth of the anaerobic bacteria; the E. coli aerobic microorganism produces the product; and the aerobic bacteria are of the genus Clostridium.
Wu discloses that in the symbiotic system TSH06, not only cell growth (e.g. cell density) but solvent producing (e.g. ethanol) ability is enhanced. See the last paragraph in the right column on page 3 and figure 1B.
Regarding claim 24, Haas teaches a synthesis gas culture (i.e. syngas fermentation) method, comprising culturing in an environment containing 2% O2 (e.g. a microaerobic environment) anaerobic C. ljungdahlii and aerobic E. coli [0088][0095-0096][0100]. Haas implies that a gas pre-treatment to remove trace O2 is unnecessary, because Haas suggests that C. ljungdahlii can tolerate the presence of O2 [0092][0083][0032]. Haas suggests that O2 is sparged into the medium, because Haas teaches trapping gas in the culture medium via a frit on a sparger, and suggests that oxygen can be dissolved in the medium by any means known in the art, such as a continuous gas flow (i.e. continually sparging). See paragraphs [0092] and [0025-0026]. Moreover, the E. coli is considered to inherently consume the oxygen present in the culture to an extent that allows the anaerobic C. ljungdahlii to grow, because Haas teaches growing E. coli with C. ljungdahlii [0092-0094]. Haas discloses that the second microorganism, such as E. coli, is capable of metabolizing acetate and/or ethanol to the substituted or unsubstituted organic compound (e.g. product). See claims 8, 13-14. Thus, Haas teaches a syngas fermentation method.
Wu discloses that in the symbiotic system TSH06, not only cell growth (e.g. cell density) but solvent producing (e.g. ethanol) ability is enhanced. See the last paragraph in the right column on page 3 and figure 1B.
Regarding claim 48, Haas teaches a mixed culture of a first and second microorganism. See claim 1. The second microorganism is selected from a group that includes aerobic microorganisms. See claim 2. The first microorganism is selected from a group that includes C. ljungdahlii and Eubacterium limosum. See claim 5.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the C. ljungdahlii bacterium in example 3 of Haas discussed above with the Eubacterium limosum. A person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Haas indicates that C. ljungdahlii and Eubacterium limosum are interchangeable as a first microorganism in a mixed culture with an anaerobic microorganism.
Regarding claim 49, Wu teaches the relative abundance of C. acetobutylicum TSH1 and B. cereus TSH2 at different inoculation ratios. As shown in figure 4, the relative abundance of the aerobic B. cereus TSH2 increases from hour 0 to hour 4 at each inoculation ratio. See figure 4 and the caption below. Thus, Wu indicates that the aerobic microorganism increases in cell density.
Regarding claim 50, Wu discloses that C. acetobutylicum TSH1 becomes the dominant species and accounts for 99.85% of the whole population in solventogenic phase. Increasing inoculation ratio of B cereus TSH2 affects butanol producing ability of the symbiotic system, but the relative abundance of each strain is not affected in the final broth, and C. acetobutylicum TSH1 is the dominant species no matter how the inoculation ratio is changed. See the last paragraph of the conclusion section on page 9 and figure 4 above.
Regarding claim 51, Haas teaches a method of producing at least one substituted or unsubstituted organic compound, the method comprising contacting a mixed culture of a first and second microorganism with oxygen and a carbon source comprising CO and/or CO2 , wherein the first microorganism is an acetogenic microorganism, such as Clostridium ljungdahlii, capable of converting the carbon source to acetate and/or ethanol; and the second microorganism, such as E. coli, is capable of metabolizing acetate and/or ethanol to the substituted or unsubstituted organic compound; wherein the substituted or unsubstituted organic compound may be selected from a list that includes amino acids. See claims 8, and 12-14 of Haas.
Response to Arguments
Applicant's arguments filed 03/27/2025 have been fully considered to the extent that they might apply to the new grounds of rejection set forth above, but they are unpersuasive.
103 rejection over Haas with evidence from Yasid and Kato
Applicant suggests that the obviousness rejection uses hindsight reconstruction. See the first full paragraph on page 7 of the remarks.
This argument is unpersuasive because Applicant has not explicitly stated any limitation that was arrived at through hindsight reasoning; and the prior art teaches every active method step required within the instant claims, such that no knowledge was gleaned from the instant disclosure. Moreover, MPEP 2144 indicates that the rationale may be expressly or impliedly contained in the prior art or may be reasoned from knowledge generally available to one of ordinary skill in the art, or established scientific principles. The instant rejection is based on a teaching-suggestion-motivation rationale, because Haas and Wu either expressly or impliedly teaches every claimed component. Furthermore, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Applicant argues that Haas does not teach culturing the anaerobic bacterium and aerobic microorganism in a microaerobic environment that does not require gas pre-treatment to remove trace O-2; nor does Haas teach an aerobic microorganism that consumes oxygen in the culture to allow growth of the anaerobic bacteria. Applicant argues that Yasid and Kato do not cure the deficiencies of Haas. See the second paragraph on page 8.
This argument is unpersuasive because paragraph [0032] of Haas implies that aerotolerant microorganisms, such as Clostridium, are capable of living in the presence of oxygen, and Wu demonstrates a fermentation process in which an anaerobic treatment (e.g. removal of oxygen) is unnecessary for a Clostridium bacterium. In paragraph [0032], Haas states that “[t]hese bacteria [i.e. aerotolerant bacteria] live by fermentation alone, regardless of the presence of oxygen in their environment” [0032]. In paragraph [0075] Haas states that “Clostridium ljungdahlii is adapted to grow in an environment comprising oxygen”. Therefore, Haas implies that a gas pre-treatment to remove O2 is not necessary. Moreover, Wu suggests that eliminating oxygen in a culture medium for strict anaerobes can increase the total cost. See the first passage on page 2. Wu demonstrates a co-culture in which the aerobic microorganism consumes dissolved oxygen to sustain an anaerobic environment. See the first passage on page 4. Therefore, one of ordinary skill in the art could have reasonably recognized that the microaerobic environment of Haas does not require a gas pre-treatment to remove trace O2 in view of the teachings, motivation and suggestions of Haas and Wu.
Applicant argues that Haas fails to provide any evidence of anaerobic bacterial growth under the instantly claimed conditions. Applicant argues that the acetogens described in Haas are well-documented to engage in resting cell metabolism, where cells remain metabolically active and produce intermediate metabolites such as formate and end-products like acetate and ethanol but do not undergo cell division or generate new biomass. Applicant references Bertsch & Müller, 2015; Cotter et all., 2009; and Schwarz & Müller, 2020. See the third paragraph on page 8 of the remarks.
This is unpersuasive because a reference is presumed to be operable until applicant provides facts rebutting the presumption of operability. See MPEP 2121. In example 3, Haas teaches a joint production phase in which “both growth cultures (E. coli at OD600 of 1.0 and C. ljungdahlii at OD600 of 0.2) were grown in 200 ml of production buffer”. See [0092]. Since Haas indicates that the cultures are “grown”, cell growth is presumed. Applicant references Bertsch & Müller, 2015; Cotter et al., 2009; and Schwarz & Müller, 2020. These references are not cited in the IDS filed 04/23/2025 or provided. Therefore, the facts Applicant intends to rely on to rebut the presumption of operability are unclear. Bertsch, J., & Müller (Biotechnology for biofuels, 2015 8, 1-12; provided herein) does not teach acetogens engaging in a resting cell metabolism, as argued. Rather, Bertsch indicates that acetogens grow on syngas. See the right column on page 10. For example, Bertsch discloses that “[c]ells of C. autoethanogenum growing on H2 + CO-2 produce not only acetate but also ethanol as end product[s]”. See the first full paragraph on page 10. Therefore, Bertsch indicates that acetate and ethanol production may be the result growing acetogen cells on H2 + CO-2. As discussed above, example 3 of Haas teaches culturing E. coli and C. ljungdahlii in the presence of H2, and CO-2. See [0092]. Therefore, it is unclear how Bertsch provides evidence to rebut the presumed operability. Based on the citations provided in paragraph 3 on page 8 of the remarks, it is unclear which Cotter et al., 2009 and Schwarz & Müller, 2020 references are being relied upon.
Applicant argues that the synthesis of 48 mg/L of acetate in example 3 of Haas is a trivial amount within the error range of NMR quantification. E. coli is known to produce acetate from cysteine (Hayes et al., 2023), thus there is no guarantee that the acetate was produced by C. ljungdahlii. See the paragraph spanning pages 10 to 11 of the remarks.
This argument is unpersuasive because the instant claims do not require any specific amount of acetate to be produced by the anaerobic bacterium. Thus, the argument is not commensurate in scope with the instant claims.
Applicant argues that the presence of acetate does not necessarily indicate growth, as resting cells of C. ljungdahlii can produce acetate from CO-2/H2. See the paragraph spanning pages 10-11 of the remarks.
This argument is unpersuasive because arguments of counsel cannot take the place of factually supported objective evidence (MPEP 2145 or 716.01(c)). See, e.g., In re Huang, 100 F.3d 135, 139-40, 40 USPQ2d 1685, 1689 (Fed. Cir. 1996); In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Applicant has not provided evidence to support the assertion that “resting cells of C. ljungdahlii can produce acetate from CO-2/H2”. As discussed above, Bertsch & Müller, 2015 do not teach “resting cells” of C. ljungdahlii, and it is unclear which Cotter et al., 2009 and Schwarz & Müller, 2020 references Applicant intends to rely upon.
Applicant argues that even if it is assumed that the acetate production is solely by C. ljungdahlii and was coupled to growth, the amount of C. ljungdahlii that could have been produced in this experiment is 2.4 mg, a trivial amount. The increase in formate described in example 3 of Haas is non-trivial. However, formate is an intermediate metabolite of C. ljungdahlii and can inhibit the rest of Wood-Ljungdahl pathway (Schwarz & Müller, 2020). Applicant asserts that significant formate production by acetogens has only been accomplished in resting cell suspensions (Schwar