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 .
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.
Claims 7-16, 21-22, and 24 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 7 recites “The bio-enhanced remediation model system of claim 1 with the further element of a filamentous microbe.”; however, the “filamentous microbe” is not provided a recitation so as to structurally and/or functionally relate the filamentous microbe to the operative embodiment of the model system of Claim 1.
Similarly, Claim 9 recites “The bio-enhanced remediation model system of claim 1, further comprising a mixture of microbes, at least one of which is a filamentous microbe.”; however, the “mixture of microbes” is not provided a recitation so as to structurally and/or functionally relate the mixture of microbes to the model system of Claim 1.
Applicant is suggested to provide the particular structure the microbes are associated with/within or recite their function within the system.
In Claim 10, the claim further recites “observing hyphae in the secondary channels…” however the presence of hyphae to be afforded to be observed in the first place is not established in the claims. By this, the origin of the hyphae is unclear. Does Applicant intend an incubation step to allow the hyphae to grow? Does Applicant intend to provide a further step to the colonization of the microbial composition that produces or correlates with hyphae? Is the incubated microbial composition assessed in some manner so as to define a presence/absence or quantifiable amount of hyphae therein?
Further, the “identifying” step is broadly interpreted herein to mean any form of identifying the filamentous microbe as a bio-enhanced remediation microbe, even mere selection of the microbe for study as discussed by the research publication references discussed below.
As recited herein, the step also appears as a given conclusion, and is absent any particular evaluation and/or satisfaction of a criterion(ia) the correlate to being drawn to qualifying the microbe as a “bio-enhance remediation microbe.” Does Applicant intend a qualitative or quantitative comparison with the prior observed hyphae to inform if the microbe is a bio-enhanced remediation microbe or not?
Claim 11 recites “wherein the NAPL comprises a compound present at a bio- enhanced remediation site” wherein this recitation is contradictory as the compound of the NAPL within the microfluidic device could not possibly also simultaneously be present as a bio-enhanced remediation site.
Further, a bio-enhanced remediation site is not a positive element of the claims as the claims are drawn to the applied steps given to the microfluidic device of claim 1.
Applicant may wish to amend the claim to recite something on the order of “wherein the NAPL comprises a compound relevant to a bio-enhanced remediation site”. Further, the orientation of the “site” within the metes and bounds of the method and device thereof is unclear. The site appears to amount to mere prospective intended use; however, does Applicant intend the site to be an additional element of the device, such as an additional channel?
Claim 13 recites “and selecting for bio-enhanced remediation of the NAPL the microbial composition with greater hyphae infiltration into the NAPL” wherein the metes and bounds of the criteria guiding the “selecting” step are unclear. It appears Applicant intends selecting the microbial composition with greater hyphae infiltration into the NAPL for directed evolution or for tailoring to a respective remediation site, however, the relative term “greater hyphae infiltration” does not have a comparative measurement thereto so as to assess what meets this desired parameter.
This is likewise seen in claim 15 to the “greater NAPL removal”.
Does Applicant intend to provide a set of measurements of the removal of NAPL and assign a value therewith as it pertains to showing its “removal” (i.e. percentage volume removed versus original volume), and provide a comparison between the two for which has a greater percentage volume removed?
Claim 21 recites “wherein at least one species of microbe is genetically modified” wherein it is unclear to what microbes the genetic modification refers. Applicant may wish to amend the claim to recite on the order of “wherein at least one of the filamentous microbe or the second species of microbe is genetically modified”. Or does applicant intend to refer to a different or additional microbe than those claimed? Further,
Claim 24 recites “wherein the filamentous microbe is genetically modified to metabolize at least one contaminant of the bio-enhanced remediation site” wherein the metes and bounds of the term “contaminant” are unclear. One of ordinary skill has no way to discern from a soil mixture what the instant claim encompasses as a “contaminant” and what is not a contaminant. The instant specification further does not provide guidance to what compounds/species are particularly encompassed by the term “contaminant”. Applicant is suggested to provide the particular chemical classifications/groups instead of reciting “contaminant”. Further, Applicant is suggested to correlate the capabilities of the microbe to the particular chemicals species/groups being targeted rather than general discussion to a remediation site which has no basis in the confines of the utilized device and which indefinitely provides for the target chemicals intended herein.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 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 1-6 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gao et al. (Gao, Beibei; et al.; “Chemotaxis to Local Chemical Gradients Enhanced Bacteria Dispersion and Bioavailability of PAH in a Heterogenous Porous Medium”, SSRN preprint, 9 Sep 2022.), hereinafter “Gao”.
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Regarding Claim 1, Gao teaches a model system (See the “Introduction” section: “pore-scale model”. See also: Abstract.) comprising:
a microfluidic device (Fig. 1a) comprising:
a main flow channel comprising an inlet, an outlet, and a main flow channel width (See the annotated Fig. 1 above. Further note that the main flow channel comprises a branching and unbranching segment formed by the columnar structures 1.4 mm in diameter as seen through Fig. 1.);
one or more microfluidic beds in fluid communication with the main flow channel (See the annotated Fig. 1 above showing the microfluidic bed between the segments of the main flow channel.), at least one microfluidic bed comprising a plurality of structures (See the annotated Fig. 1 above showing the columnar structures comprised in the microfluidic bed. See also section 2.2: “cylindrical obstacles that were periodically arranged with a distance between pore walls of...0.05 mm in low permeable areas (sides), as shown in Fig. 1a.”) that define secondary channels (The narrower branching pore spaces shown in the annotated Fig. 1a surrounding the structures.), at least a portion of the secondary channels having a width narrower than the main flow channel width (See Fig. 1a showing the secondary channels 0.05 mm across (See section 2.2.) as significantly narrower in diameter (multiple of the secondary channels fitting within the confines of the main channel portion(s)) compared to the main channel portion(s) being 0.36 mm across.);
an aqueous phase dispersed within at least a portion of the main flow channel (See section 2.2: “To mimic PAH contamination in subsurface environment under hydraulic flow, deionized water was flushed at a pore velocity of 5 m/d (within typical range of groundwater velocity) for 30 min.” See also section 2.3 discussing the NAPL-water interface. See also Fig. 1b showing the main flow channel being flushed with water while the brown-colored NAPL remains in the secondary channels.); and
a non-aqueous phase liquid (NAPL) dispersed within at least a portion of the secondary channels (See Abstract: “we conducted transport experiments and numerical simulations with chemotactic bacteria and naphthalene trapped within a non-aqueous phase liquid (NAPL) mainly in low permeable areas [the secondary channels] of a dual-permeability microfluidic device.” See also section 2.2: “Prior to introducing bacteria into the chamber, the micromodel was saturated by a NAPL mixture of chemoeffector naphthalene and 2,2,4,4,6,8,8-Heptamethylnonane (or HMN). HMN is a model NAPL used in many studies, which is known not to elicit chemotactic response in PpG7, sustain growth of bacteria (Ghoshal et al., 1996), or be toxic to bacteria (Marx and Aitken, 2000).”);
a fluid source in fluid communication with the inlet (See the annotated Fig. 1 above showing the syringe fluid source.); and
an imager in visual communication with at least a portion of the microfluidic device (See section 2.3: “A wide-field microscope (Olympus IX-70, FL) with a phase ring NO.1, 20×/0.40 lens and a CCD camera was used to record bacterial density in pores, especially at the junctures of different permeabilities.”),
as in Claim 1.
Regarding Claim 2, the prior art meets the limitations of Claim 1 as discussed above. Further, Gao teaches the model system discussed above further comprising a computer in electronic communication with the imager (See section 2.3: “A temporal series of 100 images was taken at each NAPL-water interface consecutively at an interval of 50 ms, and imported as a stack into ImageJ.” – As the images are imported into ImageJ, an image analysis software, the images in Gao must have been taken electronically, and thereby, a computer must necessarily be in electronic communication with the imager.), as in Claim 2.
Regarding Claim 3, the prior art meets the limitations of Claim 1 as discussed above. Further, Gao teaches the model system discussed above wherein the microfluidic device comprises two microfluidic beds in fluid communication with the main flow channel (See the annotated Fig. 1a above showing a first and a second microfluidic bed, the two microfluidic beds being in fluid communication with the main flow channel.), as in Claim 3.
Regarding Claim 4, the prior art meets the limitations of Claim 1 as discussed above. Further, Gao teaches the model system discussed above wherein the fluid source comprises a syringe (See the annotated Fig. 1a above showing the fluid source as being a syringe.), as in Claim 4.
Regarding Claim 5, the prior art meets the limitations of Claim 4 as discussed above. Further, Gao teaches the model system discussed above wherein the syringe is controlled by a pump (Gao section 2.2 requires a specific flow pressure of DI water be maintained for 30 minutes to flush out the NAPL mixture: “To mimic PAH contamination in subsurface environment under hydraulic flow, deionized water was flushed at a pore velocity of 5 m/d (within typical range of groundwater velocity) for 30 min.” Thereby, Gao reasonably suggests the syringe is actuated by a syringe pump, rather than by hand, so as to achieve a steady flow state necessary for accuracy of the groundwater flow, as well as replicability between experimental trials where by-hand pumping would introduce significant variability confounding overall results.), as in Claim 5.
Regarding Claim 6, the prior art meets the limitations of Claim 4 as discussed above. Further, Gao teaches the model system discussed above wherein the main flow channel has a width of from 1 μm to 10 cm (See the annotated Fig. 1a above showing a width of the main flow channel as being 0.36 mm, falling within the claimed range of 1 μm to 10 cm.), as in Claim 6.
Claims 17, 19-20, 23, and 25-30 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ghanem et al. (Ghanem, Khaled; et al.; “Bioremediation of Diesel Fuel by Fungal Consortium Using Statistical Experimental Designs”; Pol. J. Environ. Stud. 2016; 25(1): 97-106.), hereinafter “Ghanem”.
Regarding Claim 17, Ghanem teaches a bio-enhanced remediation composition comprising a filamentous microbe (See page 2, first column: “The aim of this study is to degrade diesel fuel by locally isolated fungi under the influence of some cultural conditions of incubation period and different levels of both diesel and surfactant. To enhance the biodegradation process a fungal consortium of A. alternata and A. ustus was used.” -- A. alternata and A. ustus are classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). – Regarding the composition being a “a bio-enhanced remediation composition”, see page 2, first column: “The biodegradation of diesel polluted soil was also done by the fungal consortium.”.), as in Claim 17.
Regarding Claim 19, the prior art meets the limitations of Claim 17 as discussed above. Further, Ghanem teaches the composition discussed above further comprising a second species of microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].”), as in Claim 19.
Regarding Claim 20, the prior art meets the limitations of Claim 19 as discussed above. Further, Ghanem teaches the composition discussed above wherein the second species of microbe is a filamentous microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].” Further note that both A. alternata and A. ustus disclosed by Ghanem are classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). As such, a combination of those two species of filamentous microbes is reasonably suggested by Ghanem.), as in Claim 19.
Regarding Claim 23, Ghanem teaches a method of bio-enhanced remediation, the method comprising introducing into a bio-enhanced remediation site the bio-enhanced remediation composition of claim 17 (See page 2, first column: “The biodegradation of diesel polluted soil was also done by the fungal consortium.” – Therein, such polluted soil represents that of a remediation site, and such remediation at genuinely polluted sites is the clear aim of Ghanem, wherein Ghanem further provides for the composition of claim 17.), as in Claim 23.
Regarding Claim 25, the prior art meets the limitations of Claim 23 as discussed above. Further, Ghanem teaches the method discussed above wherein the composition comprises a plurality of different microbes, at least one of which is a filamentous microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].” Further note that both A. alternata and A. ustus disclosed by Ghanem are classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). As such, a combination of those two species of filamentous microbes is reasonably suggested by Ghanem.), as in Claim 25.
Regarding Claim 26, Ghanem teaches a bio-enhanced oil recovery composition comprising a filamentous microbe (See page 2, first column: “The aim of this study is to degrade diesel fuel by locally isolated fungi under the influence of some cultural conditions of incubation period and different levels of both diesel and surfactant. To enhance the biodegradation process a fungal consortium of A. alternata and A. ustus was used.” -- A. ustus is classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). – Regarding the composition being a “a bio-enhanced remediation composition”, see page 2, first column: “The biodegradation of diesel polluted soil was also done by the fungal consortium.”.), as in Claims 26.
Regarding Claim 27, the prior art meets the limitations of Claim 26 as discussed above. Further, Ghanem teaches the composition discussed above further comprising a second species of microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].”), as in Claim 27.
Regarding Claim 28, the prior art meets the limitations of Claim 27 as discussed above. Further, Ghanem teaches the composition discussed above wherein the second species of microbe is a filamentous microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].” Further note that both A. alternata and A. ustus disclosed by Ghanem are classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). As such, a combination of those two species of filamentous microbes is reasonably suggested by Ghanem.), as in Claim 28.
Regarding Claim 29, Ghanem teaches a method of recovering oil from an oil recovery site, the method comprising introducing into the oil recovery site the bio-enhanced oil recovery composition of claim 26 (See page 2, first column: “The biodegradation of diesel polluted soil was also done by the fungal consortium.” – Therein, such polluted soil represents that of a remediation site, and such remediation at genuinely polluted sites by introducing into the site the bio-enhanced oil recovery composition is the clear aim of Ghanem, wherein Ghanem further provides for the composition of claim 26.), as in Claim 29.
Regarding Claim 30, the prior art meets the limitations of Claim 29 as discussed above. Further, Ghanem teaches the method discussed above wherein the composition comprises a plurality of different microbes, at least one of which is a filamentous microbe (See page 2, first column: “Some recent studies have been reported to use a mixed population of fungal strains that could enhance biodegradation efficiency, especially on high concentrations of oil [2, 14].” Further note that both A. alternata and A. ustus disclosed by Ghanem are classified under the filamentous hyalohyphomycetes/mould group (Aspergillus section Usti). As such, a combination of those two species of filamentous microbes is reasonably suggested by Ghanem.), as in Claim 30.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 7-12 are rejected under 35 U.S.C. 103 as being unpatentable over Gao in view of Baranger et al. (Baranger, Clair; et al.; “A compartmentalized microsystem helps understanding the uptake of benzo[a]pyrene by fungi during soil bioremediation processes”; Science of The Total Environment, Volume 784, 17 April 2021, 147151, ISSN 0048-9697.), hereinafter “Baranger”. Gao has been discussed above.
Regarding Claims 7 and 8, the prior art meets the limitations of Claim 1 as discussed above. Further, Gao does not specifically teach the model system discussed above further comprising a filamentous microbe, nor wherein the filamentous microbe is a hyphal fungus, as in Claims 7 and 8.
However, Baranger teaches a respective model microfluidic system wherein Talaromyces helicus (See Abstract.) is the pollutant-remediating microbe, and wherein Talaromyces helicus is a filamentous microbe (See section 1: “Talaromyces helicus is a filamentous soil ascomycete [a type of fungus] with a cosmopolitan distribution, found on several continents in temperate to tropical climates...”) and a hyphal fungus (See section 1: “It can grow at moderate temperatures (20 °C) and presents relatively fine hyphae of 1 to 3 μm in diameter...”). Therein, Baranger thereby represents a similar arrangement as in Gao where a microfluidic device is utilized to assess the efficacy of a pollutant-remediating microbe.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the model system of Gao further comprising a filamentous microbe, and wherein the filamentous microbe is a hyphal fungus, such as suggested by Baranger, as the hyphal fungus of Baranger is a known alternative pollutant-remediating model organism wherein one would be motivated to implement said fungus in Gao so as to evaluate fungal rather than bacterial remediation in Gao’s device. The fungus would predictably perform the identical relevant function of serving as a pollutant-remediating microorganism in a microfluidic model environment. The fact that the fungus reaches the pollutan5t through hyphal extension rather than chemotaxis represents a difference in biological mechanism-not a difference in the claimed function. Further, the hyphae of Baranger are only 1-3 μm in diameter (See section 1.) and would thus easily reach into the secondary channels of Gao, thereby representing a reasonable expectation of success.
Regarding Claim 9, the prior art meets the limitations of Claim 1 as discussed above. Further, Gao does not specifically teach the model system discussed above further comprising a mixture of microbes, at least one of which is a filamentous microbe, as in Claim 9.
However, as discussed above regarding Claim 7, one of ordinary skill in the art would find it obvious to provide the fungus of Baranger to the device of Gao so as to merely investigate a different mechanism of pollutant-remediation with a different model organism. Further, Baranger discusses “The elucidation of the mechanisms by which filamentous fungi influence PAH bioavailability in soils (Posada-Baquero et al., 2019) and transport these molecules through the hyphal network (Harms et al., 2011) are also important to understand how they can positively interact with other types of organisms within combined bioremediation strategies (Baranger et al., 2021). Notably positive results in bioremediation of PAH impacted soils have been obtained through combination of mycoremediation and phytoremediation (Ma et al., 2021).” (See section 1.). Thus, Baranger reasonably suggests combining other microbes with the filamentous microbe so as to examine the effects of their interaction.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the model system of Gao further comprising a mixture of microbes, at least one of which is a filamentous microbe, such as suggested by Baranger, so as to examine the effects of microbial-interaction on the efficacy of the intended pollutant remediation. Therein, Examiner further notes that given this language in Baranger, one would find it obvious to add the filamentous microbe as an additional microbe to the bacterial microbe of Gao so as to better simulate a soil environment and its effects on the bacterial remediation achieved by Gao.
Regarding Claim 10, Gao teaches a method for identifying a filamentous microbe as a bio-enhanced remediation microbe, the method comprising:
providing the bio-enhanced remediation model system of claim 1 (See Claim 1 above.);
introducing a microbial composition into the aqueous phase (See section 2.1: “...bacteria were harvested via centrifugation at 2680g for 1 min 30 s (Thermo Scientific, PA) and resuspended in 10 % random motility buffer (RMB, containing 11.2 g/L K2HPO4 (Fisher Scientific, NY), 4.8 g/L KH2PO4 (Amresco), and 0.029 g/L EDTA (Sigma-Aldrich, MO)) to an optical density of 1.5 at 590 nm.” – See further the rejection of Claim 1 above, and the results and discussion section of Gao, which discuss the microbes remediating the NAPL by traveling to the aqueous/organic interface.),
incubating the bio-enhanced remediation model system under conditions effective to allow members of microbial composition to colonize the main flow channel (See section 3.1 and Fig. 2 where the bacterial distribution is measured after 50 min of incubation time and colonized the channels of the device.);
Further regarding Claim 10, Gao does not specifically teach the method discussed above wherein the microbial composition comprises at least one filamentous microbe; and further comprising observing hyphae in the secondary channels of at least one microfluidic bed; and identifying the filamentous microbe as a bio-enhanced remediation microbe, as in Claim 10.
However, as discussed above regarding Claim 7, one of ordinary skill in the art would find it obvious to provide the fungus (filamentous microbe) of Baranger to the device of Gao so as to merely investigate a different mechanism of pollutant-remediation with a different model organism. Further, Baranger discusses observing the fungal hyphae in various areas of the compartmentalized model including secondary channels (microchannels) (See Figs. 4a and 4b.) and utilize such observation to determine if the microbe is a suitable remediation microbe (See sections 4 and 5.). Therein, such methodology is useful in determining suitability of a microbial remediator via determination of the mechanisms thereto the remediation processes.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao wherein the microbial composition comprises at least one filamentous microbe; and further comprising observing hyphae in the secondary channels of at least one microfluidic bed; and identifying the filamentous microbe as a bio-enhanced remediation microbe, such as suggested by Baranger, so as to determine the suitability for and mechanism of action of a microbial bioremediation organism.
Regarding Claim 11, the prior art meets the limitations of Claim 10 as discussed above. Further, Gao teaches the method discussed above wherein the NAPL comprises a compound present at a bio- enhanced remediation site (See Abstract: “Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, EPA-designated priority pollutants for soil and groundwater, remaining recalcitrant to bioremediation because of limited bioavailability.”), as in Claim 11.
Regarding Claim 12, the prior art meets the limitations of Claim 10 as discussed above. Further, Gao does not specifically teach the model system discussed above wherein the microbial composition comprises a plurality of microbes, at least one of which is a filamentous microbe, as in Claim 9.
However, as discussed above regarding Claim 7, one of ordinary skill in the art would find it obvious to provide the fungus of Baranger to the device of Gao so as to merely investigate a different mechanism of pollutant-remediation with a different model organism. Further, Baranger discusses “The elucidation of the mechanisms by which filamentous fungi influence PAH bioavailability in soils (Posada-Baquero et al., 2019) and transport these molecules through the hyphal network (Harms et al., 2011) are also important to understand how they can positively interact with other types of organisms within combined bioremediation strategies (Baranger et al., 2021). Notably positive results in bioremediation of PAH impacted soils have been obtained through combination of mycoremediation and phytoremediation (Ma et al., 2021).” (See section 1.). Thus, Baranger reasonably suggests combining other microbes with the filamentous microbe so as to examine the effects of their interaction.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao wherein the microbial composition comprises a plurality of microbes, at least one of which is a filamentous microbe, such as suggested by Baranger, so as to examine the effects of microbial-interaction on the efficacy of the intended pollutant remediation. Therein, Examiner further notes that given this language in Baranger, one would find it obvious to add the filamentous microbe as an additional microbe to the bacterial microbe of Gao so as to better simulate a soil environment and its effects on the bacterial remediation achieved by Gao.
Claims 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Gao in view of Baranger and Hopke et al. (Hopke, Alex; et al.; “Crowdsourced analysis of fungal growth and branching on microfluidic platforms”; PLoS ONE 16(9): e0257823; September 29, 2021.), hereinafter “Hopke”. Gao and Baranger have been discussed above.
Regarding Claim 13, Gao teaches a method of comparing bio-enhanced remediation of a non-aqueous phase liquid (NAPL) by different bio-enhanced remediation microbial compositions, the method comprising:
providing a first bio-enhanced remediation model system of claim 1 (See Claim 1 above.), comprising the NAPL in the microfluidic bed (See the annotated Fig. 1b above.); introducing a first microbial composition into the aqueous phase of the first bio-enhanced remediation model system (See section 2.1: “...bacteria were harvested via centrifugation at 2680g for 1 min 30 s (Thermo Scientific, PA) and resuspended in 10 % random motility buffer (RMB, containing 11.2 g/L K2HPO4 (Fisher Scientific, NY), 4.8 g/L KH2PO4 (Amresco), and 0.029 g/L EDTA (Sigma-Aldrich, MO)) to an optical density of 1.5 at 590 nm.” – See further the rejection of Claim 1 above, and the results and discussion section of Gao, which discuss the microbes remediating the NAPL by traveling to the aqueous/organic interface.); incubating the first bio-enhanced remediation model system under conditions effective to allow members of first microbial composition to colonize the main flow channel of the first bio-enhanced remediation model system (See section 3.1 and Fig. 2 where the bacterial distribution is measured after 50 min of incubation time and colonized the channels of the device.); measuring infiltration into the NAPL of at least one microfluidic bed of the first bio-enhanced remediation model system (See section 3.1 and Fig. 2a showing fluorescent measurements of the microbes infiltration of the “low-permeability” microfluidic beds, wherein the NAPL-water interface is represented by a dashed line.); as in Claim 13.
Further as in Claim 13, Gao does not specifically teach the method discussed above wherein the microbial compositions comprise a filamentous microbe and the hyphal extension thereof being the measured infiltration, nor further comprising providing a second bio-enhanced remediation model system of claim 1, comprising the NAPL in the microfluidic bed; introducing a second microbial composition into the aqueous phase of the second bio-enhanced remediation model system, incubating the second bio-enhanced remediation model system under conditions effective to allow members of second microbial composition to colonize the main flow channel of the second bio-enhanced remediation model system; measuring infiltration into the NAPL of at least one microfluidic bed of the second bio-enhanced remediation model system; and selecting for bio-enhanced remediation of the NAPL the microbial composition with greater hyphae infiltration into the NAPL, as in Claim 13.
However, Baranger teaches a respective model microfluidic system wherein Talaromyces helicus (See Abstract.) is the pollutant-remediating microbe, and wherein Talaromyces helicus is a filamentous microbe (See section 1: “Talaromyces helicus is a filamentous soil ascomycete [a type of fungus] with a cosmopolitan distribution, found on several continents in temperate to tropical climates...”) and a hyphal fungus (See section 1: “It can grow at moderate temperatures (20 °C) and presents relatively fine hyphae of 1 to 3 μm in diameter...”). Therein, Baranger thereby represents a similar arrangement as in Gao where a microfluidic device is utilized to assess the efficacy of a pollutant-remediating microbe. Further, Baranger discusses observing the fungal hyphae in various areas of the compartmentalized model including secondary channels (microchannels) (See Figs. 4a and 4b.) and utilize such observation to determine if the microbe is a suitable remediation microbe (See sections 4 and 5.). Therein, such methodology is useful in determining suitability of a microbial remediator via determination of the mechanisms thereto the remediation processes.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao wherein the microbial compositions comprise a filamentous microbe and the hyphal extension thereof is the measured infiltration, such as suggested by Baranger, so as to provide a means for determining suitability of a microbial remediator via determination of the mechanisms thereto the remediation processes.
Further, regarding the claimed providing of a second microfluidic device of Claim 1 and selecting for the fungus having the greatest hyphal infiltration into the NAPL, Hopke teaches a respective method comprising incubating different fungal (both mutant and WT) strains across plural identical microfluidic chips (as well as different chips) so as to identify the best performers in terms of growth velocity (infiltration of the fungus across the various channels of the chip) (See the “Discussion” section.), thereby optimizing the fungus for a particular purpose (See the “Introduction” section.).
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao/Baranger further comprising providing a second bio-enhanced remediation model system of claim 1, comprising the NAPL in the microfluidic bed; introducing a second microbial composition into the aqueous phase of the second bio-enhanced remediation model system, incubating the second bio-enhanced remediation model system under conditions effective to allow members of second microbial composition to colonize the main flow channel of the second bio-enhanced remediation model system; measuring infiltration into the NAPL of at least one microfluidic bed of the second bio-enhanced remediation model system; and selecting for bio-enhanced remediation of the NAPL the microbial composition with greater hyphae infiltration into the NAPL, such as suggested by Hopke, so as to identify best performers of fungal species in terms of infiltration into a NAPL so as to discover a most efficient/efficacious fungus for bio-remediation.
Regarding Claim 14, the prior art meets the limitations of Claim 13 as discussed above. Further, Gao does not specifically teach the model system discussed above wherein the first microbial composition or the second microbial composition comprises a plurality of different microbes, at least one of which is a filamentous microbe, as in Claim 14.
However, as discussed above regarding Claim 7, one of ordinary skill in the art would find it obvious to provide the fungus of Baranger to the device of Gao so as to merely investigate a different mechanism of pollutant-remediation with a different model organism. Further, Baranger discusses “The elucidation of the mechanisms by which filamentous fungi influence PAH bioavailability in soils (Posada-Baquero et al., 2019) and transport these molecules through the hyphal network (Harms et al., 2011) are also important to understand how they can positively interact with other types of organisms within combined bioremediation strategies (Baranger et al., 2021). Notably positive results in bioremediation of PAH impacted soils have been obtained through combination of mycoremediation and phytoremediation (Ma et al., 2021).” (See section 1.). Thus, Baranger reasonably suggests combining other microbes with the filamentous microbe so as to examine the effects of their interaction.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao wherein the first microbial composition or the second microbial composition comprises a plurality of different microbes, at least one of which is a filamentous microbe, such as suggested by Baranger, so as to examine the effects of microbial-interaction on the efficacy of the intended pollutant remediation. Therein, Examiner further notes that given this language in Baranger, one would find it obvious to add the filamentous microbe as an additional microbe to the bacterial microbe of Gao so as to better simulate a soil environment and its effects on the bacterial remediation achieved by Gao.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Gao in view of Baranger, Hopke, and Mohsenzadeh et al. (Mohsenzadeh, Fariba; et al.; “Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils”; Iranian J. Environ. Health Sci. Eng.; 2012 Dec 15; 9(1): 26.), hereinafter “Mohsenzadeh”. Gao, Baranger, and Hopke have been discussed above.
Regarding Claim 15, Gao teaches a method of comparing bio-enhanced remediation of a non-aqueous phase liquid (NAPL) by different bio-enhanced remediation microbial compositions, the method comprising:
providing a first bio-enhanced remediation model system of claim 1 (See Claim 1 above.), comprising the NAPL in the microfluidic bed (See the annotated Fig. 1b above.); introducing a first microbial composition into the aqueous phase of the first bio-enhanced remediation model system (See section 2.1: “...bacteria were harvested via centrifugation at 2680g for 1 min 30 s (Thermo Scientific, PA) and resuspended in 10 % random motility buffer (RMB, containing 11.2 g/L K2HPO4 (Fisher Scientific, NY), 4.8 g/L KH2PO4 (Amresco), and 0.029 g/L EDTA (Sigma-Aldrich, MO)) to an optical density of 1.5 at 590 nm.” – See further the rejection of Claim 1 above, and the results and discussion section of Gao, which discuss the microbes remediating the NAPL by traveling to the aqueous/organic interface.); incubating the first bio-enhanced remediation model system under conditions effective to allow members of first microbial composition to colonize the main flow channel of the first bio-enhanced remediation model system (See section 3.1 and Fig. 2 where the bacterial distribution is measured after 50 min of incubation time and colonized the channels of the device.); as in Claim 15.
Further as in Claim 15, Gao does not specifically teach the method discussed above wherein the microbial compositions comprise a filamentous microbe, nor further comprising providing a second bio-enhanced remediation model system of claim 1, comprising the NAPL in the microfluidic bed; introducing a second microbial composition into the aqueous phase of the second bio-enhanced remediation model system, incubating the second bio-enhanced remediation model system under conditions effective to allow members of second microbial composition to colonize the main flow channel of the second bio-enhanced remediation model system; measuring relative NAPL removal between the model systems, and selecting for bio-enhanced remediation of the NAPL the microbial composition with greater NAPL removal, as in Claim 13.
However, Baranger teaches a respective model microfluidic system wherein Talaromyces helicus (See Abstract.) is the pollutant-remediating microbe, and wherein Talaromyces helicus is a filamentous microbe (See section 1: “Talaromyces helicus is a filamentous soil ascomycete [a type of fungus] with a cosmopolitan distribution, found on several continents in temperate to tropical climates...”) and a hyphal fungus (See section 1: “It can grow at moderate temperatures (20 °C) and presents relatively fine hyphae of 1 to 3 μm in diameter...”). Therein, Baranger thereby represents a similar arrangement as in Gao where a microfluidic device is utilized to assess the efficacy of a pollutant-remediating microbe. Further, Baranger discusses observing the fungal hyphae in various areas of the compartmentalized model including secondary channels (microchannels) (See Figs. 4a and 4b.) and utilize such observation to determine if the microbe is a suitable remediation microbe (See sections 4 and 5.). Therein, such methodology is useful in determining suitability of a microbial remediator via determination of the mechanisms thereto the remediation processes.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao wherein the microbial compositions comprise a filamentous microbe and the hyphal extension thereof is the measured infiltration, such as suggested by Baranger, so as to provide a means for determining suitability of a microbial remediator via determination of the mechanisms thereto the remediation processes.
Further, regarding the claimed providing of a second microfluidic device of Claim 1 and selecting for the fungus having the greatest removal of the NAPL, Hopke teaches a respective method comprising incubating different fungal (both mutant and WT) strains across plural identical microfluidic chips (as well as different chips) so as to identify the best performers in terms of growth velocity (infiltration of the fungus across the various channels of the chip) (See the “Discussion” section.), thereby optimizing the fungus for a particular purpose (See the “Introduction” section.). Further, Mohsenzadeh teaches comparison of removed model pollutant across fungal strains to determine efficiency (See the “Evaluation of petroleum removing” section.), thereby optimizing the fungus for pollutant remediation.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the method of Gao/Baranger further comprising providing a second bio-enhanced remediation model system of claim 1, comprising the NAPL in the microfluidic bed; introducing a second microbial composition into the aqueous phase of the second bio-enhanced remediation model system, incubating the second bio-enhanced remediation model system under conditions effective to allow members of second microbial composition to colonize the main flow channel of the second bio-enhanced remediation model system, such as suggested by Hopke; and selecting for bio-enhanced remediation of the NAPL the microbial composition with greater NAPL removal, such as suggested by Mohsenzadeh, so as to identify best performers of fungal species in terms of model pollutant removal so as to discover a most efficient/efficacious fungus for bio-remediation.
Regarding Claim 16, the prior art meets the limitations of Claim 15 as discussed above. Further, Gao does not specifically teach the model system discussed above wherein the first microbial composition or the second microbial composition comprises a plurality of different microbes, at least one of which is a filamentous microbe, as in Claim 9.
However, as discussed above regarding Claim 7, one of ordinary skill in the art would find it obvious to provide the fungus of Baranger to the device of Gao so as to merely investigate a different mechanism of pollutant-remediation with a different model organism. Further, Baranger discusses “The elucidation of the mechanisms by which filamentous fungi influence PAH bioavailability in soils (Posada-Baquero et al., 2019) and transport these molecules through the hyphal network (Harms et al., 2011) are also important to understand how they can positively interact with other types of organisms within combined bioremediation strategies (Baranger et al., 2021). Notably positive results in bioremediation of PAH impacted soils have been obtained through combination of mycoremediation and phytoremediation (Ma et al., 2021).” (See section 1.). Thus, Baranger reasonably suggests combining other microbes with the filamentous microbe so as to examine the effects of their interaction.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the model system of Gao wherein the first microbial composition or the second microbial composition comprises a plurality of different microbes, at least one of which is a filamentous microbe, such as suggested by Baranger, so as to examine the effects of microbial-interaction on the efficacy of the intended pollutant remediation. Therein, Examiner further notes that given this language in Baranger, one would find it obvious to add the filamentous microbe as an additional microbe to the bacterial microbe of Gao so as to better simulate a soil environment and its effects on the bacterial remediation achieved by Gao.
Claims 18, 21-22, and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Ghanem in view of Cortes et al. (Cortés-Espinosa, Diana; et al.; “Heterologous expression of manganese peroxidase in Aspergillus niger and its effect on phenanthrene removal from soil.”; J. Mol. Microbiol. Biotechnol.; 2011; 21(3-4): 120-9.), hereinafter “Cortes”. Ghanem has been discussed above.
Regarding Claims 18 and 21, the prior art meets the limitations of Claim 17 as discussed above. Further, Ghanem does not specifically teach the composition discussed above wherein the first and/or second filamentous microbe is genetically modified, as in Claims 18 and 21 respectively.
However, Cortes teaches use of a genetically modified filamentous microbe for soil remediation (See Abstract: “A strain of Aspergillus niger, previously isolated from sugarcane bagasse because of its capacity to degrade phenanthrene in soil by solid culture, was used to express a manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium, aiming at increasing its polycyclic aromatic hydrocarbons degradation capacity...Transformation success was confirmed by PCR amplification using gene-specific primers, and a single fragment (1,348 bp long, as expected) of the recombinant mnp1 was amplified in the DNA from transformants, which was absent from the parental strain.”). Therein, Cortes thereby represents a similar composition as in Ghanem but where the effect under study is genetic enhancement rather than the effect being between wild type species.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the composition of Ghanem wherein the first and/or second filamentous microbe is genetically modified, such as suggested by Cortes, so as to merely investigate another promising pathway for enhancing bio-remediation, wherein both the recombinant and WT fungi serve the identical functions of remediating contaminated soil.
Regarding Claim 22, the prior art meets the limitations of Claim 21 as discussed above. Further, Ghanem is modified in view of Cortes as discussed above regarding Claims 18 and 21 so as to provide a genetically modified filamentous microbe for soil pollutant remediation. Therein, the genetic modification of Cortes increases consumption of a non-aqueous phase liquid (NAPL) (See Abstract: “The recombinant A. niger SBC2-T3 strain developed MnP activity and was able to remove 95% of the initial phenanthrene (400 ppm) from a microcosm soil system after 17 days, whereas the wild strain removed 72% under the same conditions.”), as in Claim 22.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious that, when providing the genetically modified fungi of Cortes into the composition of Ghanem, to provide the genetic modification to increase consumption of a non-aqueous phase liquid (NAPL), so as to provide a most efficient microbe for fully rather than partially remediating polluted soil.
Regarding Claim 24, the prior art meets the limitations of Claim 23 as discussed above. Further, Ghanem does not specifically teach the method discussed above wherein the filamentous microbe is genetically modified to metabolize at least one contaminant of the bio-enhanced remediation site, as in Claim 23.
However, Cortes teaches use of a genetically modified filamentous microbe for soil remediation (See Abstract: “A strain of Aspergillus niger, previously isolated from sugarcane bagasse because of its capacity to degrade phenanthrene in soil by solid culture, was used to express a manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium, aiming at increasing its polycyclic aromatic hydrocarbons degradation capacity...Transformation success was confirmed by PCR amplification using gene-specific primers, and a single fragment (1,348 bp long, as expected) of the recombinant mnp1 was amplified in the DNA from transformants, which was absent from the parental strain.”), wherein the genetic modification is to metabolize at least one contaminant of the bio-enhanced remediation site (See page 6, column 2: “This result can be attributed to mnp1 expression because this is the only difference between the two strains, which were grown under the same culture conditions. These results allow us to infer that the observed differences in metabolism were caused by the presence of MnP recombinant enzyme.”). Therein, Cortes thereby represents a similar composition as in Ghanem but where the effect under study is genetic enhancement rather than the effect being between wild type species.
Thus, one of ordinary skill in the art before the effective filing date of the claimed invention would have found it obvious to modify the composition of Ghanem wherein the filamentous microbe is genetically modified to metabolize at least one contaminant of the bio-enhanced remediation site, such as suggested by Cortes, so as to merely investigate another promising pathway for enhancing bio-remediation, wherein both the recombinant and WT fungi serve the identical functions of remediating contaminated soil.
Conclusion
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/B.J.K./Examiner, Art Unit 1798
/NEIL N TURK/Primary Examiner, Art Unit 1798
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