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 .
Election/Restrictions
Applicant’s election of the species Gracilaria spp. (claims 48 and 49) and Haloferax spp. (claims 46, 50, 51, and 56) in the reply filed on 03/29/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Priority
The instant application filed on 01/08/2024 is a 371 of PCT/AU2022/050706 filed on 07/06/2022, which claims priority to AU2021902068 filed on 07/07/2021 and AU2022901400 filed on 05/24/2022. AU2021902068 finds support for the instantly claimed invention; therefore, the effective filing date for the instant application is 07/07/2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/17/2025, 11/26/2025, and 02/26/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawing Objections
The drawings are objected to because the Examiner cannot distinguish between the different temperatures in the bar graph of Figure 4. A higher resolution photo is requested for Figure 11, as the differences between the freshwater and seawater cannot be ascertained. A higher resolution graph for Figure 16 is requested, as the legend cannot be read. A higher resolution photo for Figure 19 is requested, as differences between the test tubes cannot be ascertained. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 112(b), Indefiniteness
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.
Claim 49 recites “selected from the group comprising”; however, Markush
language should be closed language, not open, and, based on this open claim language, it
is unclear what other alternatives are intended to be encompassed by the claim (see, e.g.,
MPEP 2111.03).
Claim Rejections - 35 USC § 103, Obviousness
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.
Claims 46-47, 50-51, 56, and 61-65 are rejected under 35 U.S.C. 103 as being unpatentable over Ghosh (Polyhydroxyalkanoates and biochar from green macroalgal Ulva sp. biomass subcritical hydrolysates: Process optimization and a prior economic greenhouse emissions break-even analysis; 2021 – cited in the IDS filed on 07/17/2025) in view of Vadlamani (US 2016/0222421; Date of Publication: August 4, 2016) and Munoz (EP 0622462; Date of Publication: November 2, 1994) as evidenced by Oren (Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei; 2014).
Ghosh’s general disclosure relates to “the simultaneous production of polyhydroxyalkanoates (PHA) and biochar from green macroalgae Ulva sp. is examined, applying subcritical water hydrolysis and Haloferax mediterranei fermentation” (see, e.g., Ghosh, abstract). Moreover, Ghosh discloses that “PHA production from seaweed hydrolysate using extreme halophiles coupled to biochar production could become a benign and promising step in a marine biorefinery” (see, e.g., Ghosh, abstract).
Regarding claim 46 pertaining to a process of producing PHAs from macroalgae, Ghosh teaches “The seaweed was cultivated in artificial seawater (ASW) cultivation medium which was prepared using distilled water containing dissolved dried Red Sea Salt (Red Sea Inc., IS) which had a total salinity of 37‰. Ammonium nitrate (NH4NO3, Haifa Chemicals Ltd., IS) and phosphoric acid (H3PO4, Haifa Chemicals Ltd., IS) were added to the ASW medium to maintain a concentration of 6.4 g m−3 of nitrogen (N2) and 0.97 g m−3of phosphorus (P) respectively” (see, e.g., Ghosh, Section 2.1, pg. 2). Furthermore, Ghosh teaches “Following hydrolysis, the liquid phase (hydrolysate) was utilized as a substrate for fermentation using Haloferax mediterranei ATCC 33500 (NCIMB2177) as a fermentative organism. The seaweed hydrolysates for PHA production were used at a concentration of 25% v/v of medium as observed in previous studies (Ghosh et al., 2019). The salinity of the medium was adjusted to the Hv-YPC medium (25% w/v) (Allers et al., 2010) and peptone was utilized as the nitrogen source. The experiments were performed in 100mL bottles (Schott Duran, USA) with 50mL working volume (Fig.1b). The initial pH of the medium was adjusted to pH7.2. The fermentation was performed at 42°C in a temperature controlled shaking incubator at 100rpm (Benchmark Scientific, USA)” (see, e.g., Ghosh, Section 2.4, pgs. 3-4).
Regarding claim 47 pertaining to the saltwater, Ghosh teaches “The seaweed was cultivated in artificial seawater (ASW) cultivation medium which was prepared using distilled water containing dissolved dried Red Sea Salt (Red Sea Inc., IS) which had a total salinity of 37‰. Ammonium nitrate (NH4NO3, Haifa Chemicals Ltd., IS) and phosphoric acid (H3PO4, Haifa Chemicals Ltd., IS) were added to the ASW medium to maintain a concentration of 6.4 g m−3 of nitrogen (N2) and 0.97 g m−3 of phosphorus (P) respectively” (see, e.g., Ghosh, Section 2.1, pg. 2).
Regarding claims 50-51 pertaining to the halophilic microbe, Ghosh teaches Haloferax mediterranei ATCC 33500 (NCIMB2177) as a fermentative organism (see, e.g., Ghosh, Section 2.4, pgs. 3-4). Haloferax mediterranei is inherently a microbe that grows in salt concentrations of at least 150 g/L (see, e.g., Oren, “Window for salt tolerance”, pg. 135).
Regarding claim 56 pertaining to fermenting the growth medium with a halophilic microbe, Ghosh teaches “Following hydrolysis, the liquid phase (hydrolysate) was utilized as a substrate for fermentation using Haloferax mediterranei ATCC 33500 (NCIMB2177) as a fermentative organism. The seaweed hydrolysates for PHA production were used at a concentration of 25% v/v of medium as observed in previous studies (Ghosh et al., 2019). The salinity of the medium was adjusted to the Hv-YPC medium (25% w/v) (Allers et al., 2010) and peptone was utilized as the nitrogen source. The experiments were performed in 100mL bottles (Schott Duran, USA) with 50mL working volume (Fig.1b). The initial pH of the medium was adjusted to pH7.2. The fermentation was performed at 42°C in a temperature controlled shaking incubator at 100rpm (Benchmark Scientific, USA)” (see, e.g., Ghosh, Section 2.4, pgs. 3-4). Therefore, Ghosh teaches fermenting the growth medium comprising the seaweed hydrolysate using Haloferax mediterranei ATCC 33500 in order to produce PHAs. Moreover, Ghosh does not teach sterilization of the growth medium or the fermentation system; therefore, one of ordinary skill in the art would readily understand that the medium and the fermentation system is not sterile.
Regarding claim 61 pertaining to the pH and temperature of the fermentation culture, Ghosh teaches that the pH of the fermentation medium was adjusted to a pH of 7.2 and the fermentation was performed at 42oC in a temperature-controlled shaking incubator at 100 rpm (see, e.g., Ghosh, Section 2.4, pg. 4).
However, Ghosh does not teach: enzymatically hydrolysing the macroalgae mixture to form a macroalgae hydrolysate (claim 46); or extracting the polyhydroxyalkanoates from within the halophilic cells using a salt-water based lysis process (claim 46); or wherein the step of extracting the polyhydroxyalkanoates from within the halophilic cells using a saltwater- based lysis process, comprises harvesting the cell biomass by separating it from the fermentation culture by centrifugation or filtration (claim 62); or wherein the separated halophilic cells are submersed in a saltwater-based solution to lyse them, and wherein the saltwater-based solution comprises seawater or water with added salts (claim 63); or wherein the saltwater-based solution comprises additional chemical or enzymatic agents, and wherein the additional chemical or enzymatic agents comprises sodium dodecyl sulfate (SDS) (claim 64); or wherein polyhydroxyalkanoates are recovered from the halophilic cells by submersing them in, followed by centrifuging or filtering them from, the saltwater-based solution one or more times and removing the aqueous phase (claim 65).
Vadlamani’s general disclosure relates to methods for the recovery of lipids, sugars, and proteins from microbial biomass, wherein the methods involve treating microalgae with a fungal acid protease or with a mixture of at least one protease and at least amylase (see, e.g., Vadlamani, abstract).
Regarding claim 46 pertaining to enzymatic hydrolysing the macroalgae, Vadlamani teaches two strains of algae, wherein “Biomass digestions were performed at a solid concentration of 10% (w/v) in 50 mM citrate buffer adjusted to a pH of 4.5. Protease alone or a mixture of protease with α-amylase and amyloglucosidase (all purchased from Sigma) were added to the biomass slurries” (see, e.g., Vadlamani, [0069]-[0070]).
Munoz’s general disclosure relates to “A procedure for the extraction of polyhydroxyalkanoates from halophilic bacteria which contain them, using lysis or rupture of halophilic cells (for example, of the halobacteria type) which develop in media with high salt concentrations, by concentration by centrifugation, and then dilution-resuspension in a medium with low salt concentration, for example, fresh or distilled water, and then centrifugation, sedimentation, or filtration of the suspension obtained” (see, e.g., Munoz, abstract). Furthermore, Munoz discloses “The present procedure is applicable to the extraction of granules of PHA produced by halobacteria and other halophilic bacteria. It is based on the weakness of the cell envelopes of these microorganisms when they are exposed to low concentrations of salts, for example, in fresh water; under these conditions the cells of halophilic bacteria lyse (rupture), releasing all the cell components into the medium. Since the granules of PHA are of considerable size and density, they can be recovered from the suspension of cells once lysed, by centrifugation at low speed, sedimentation, filtration, etc.” (see, e.g., Munoz, col 2, lines 6-18).
Regarding claims 46 and 62-65 pertaining to extracting PHAs using a salt-water based lysis process, Munoz teaches “To cause lysis of the cells, it is necessary to reduce the concentration of these salts in the medium which surrounds the cells to less than 0.5% for the NaCl and to less than 0.1% for the magnesium; otherwise, the lysis would not be effective. For this, once the medium has been eliminated, it is necessary to resuspend the cells in a quantity of water large enough that the concentration of salts reaches the level indicated” (see, e.g., Munoz, col 2, lines 50-58 & col 3, line 1). Moreover, Munoz teaches “A procedure for the extraction of polyhydroxyalkanoates from halophilic bacteria which contain them, characterized by lysis or rupture of halophilic cells (for example, of the halobacteria type) which develop in media with high salt concentrations, by concentration, centrifugation, and then dilution-resuspension in a medium with low salt concentration” (see, e.g., Munoz, claim 1). Therefore, for halophiles, Munoz teaches resuspension of the microbe in saltwater, wherein the concentration of salt is low.
Regarding claims 62 and 65 pertaining to harvesting the cell biomass and separating it using centrifugation, Munoz teaches “After obtaining the suspension of granules free of whole cells and cell residues of significant size, sedimentation or low-speed centrifugation is begun, recovering the PHA granules with a high level of purity. For this step, it is important that the starting culture medium be as free as possible of particulate matter, because if present it would be collected with the granules, contaminating the final product” (see, e.g., Munoz, col 3, lines 20-28).
Regarding claim 64 pertaining to the addition of SDS, Munoz teaches “The sediment containing the granules usually presents lipid and protein contamination. This contamination can be reduced to traces with one or a plurality of washing operations using water and a detergent which dissolves proteins, such as SDS” (see, e.g., Munoz, col 3, lines 29-33).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Ghosh’s macroalgae hydrolysate, wherein the macroalgae is undergoes enzymatic hydrolysis to form the hydrolysate, as taught by Vadlamani. One would have been motivated to so do because Vadlamani teaches that “Biological pretreatment methods, such as enzyme hydrolysis, have replaced the traditional acid hydrolysis due to the mild conditions, fewer by-products, and lack of corrosion issues” (see, e.g., Vadlamani, [0003]). Moreover, Ghosh teaches hydrolysis of macroalgae in order to ferment the sugars present in the macroalgal biomass to produce biofuels, such as bioethanol or biomethane, and polymers, such as polyhydroxyalkanoates (see, e.g., Ghosh, Introduction, pg. 2). Additionally, Ghosh teaches “One of the key steps for seaweed biomass fermentation includes the fragmentation of complex carbohydrates into their fermentable ingredients. Also, fermentability and PHA yield depend on the composition of the hydrolysates” (see, e.g., Ghosh, Introduction, pg. 2). Therefore, based on the teachings of Ghosh and Vadlamani, it would have been obvious to produce a macroalgal hydrolysate via enzymatic hydrolysis in order to obtain fragmented complex carbohydrates that can be used for fermentation to produce PHA. Also, it would have been obvious to utilize enzymatic hydrolysis due to the mild conditions, fewer by-products, and lack of corrosion issues of the method compared to other hydrolysis methods. One would have expected success because Ghosh and Vadlamani both teach enzymatic digestion and carbohydrate recovery of algal biomass.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce PHA by fermentation of H. mediterrani with the macroalgal biomass, as taught by Ghosh, wherein the PHA is obtained from H. mediterrani by a saltwater-based lysis process, as taught by Munoz. One would have been motivated to do so because Munoz teaches “A procedure for the extraction of polyhydroxyalkanoates from halophilic bacteria which contain them, using lysis or rupture of halophilic cells (for example, of the halobacteria type) which develop in media with high salt concentrations, by concentration by centrifugation, and then dilution-resuspension in a medium with low salt concentration, for example, fresh or distilled water, and then centrifugation, sedimentation, or filtration of the suspension obtained” (see, e.g., Munoz, abstract). Additionally, Munoz teaches that lysis using water allows for one to obtain a polymer with high yields and levels of purity (see, e.g., Munoz, col 2, lines 18-23). Moreover, Ghosh teaches lysis followed by centrifugation of the H. mediterrani cells allows for PHA produced in these cells to be extracted and further quantified (see, e.g., Ghosh, Section 2.5, pg. 4). Therefore, based on the teachings of Ghosh and Munoz, it would have been obvious to lyse H. mediterrani using a saltwater-based lysis process to obtain PHA from H. mediterrani because a saltwater-based lysis process would allow for one to obtain PHA with high yields and levels of purity.
It would have been thirdly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce PHA by fermentation of H. mediterrani with the macroalgal biomass, as taught by Ghosh, wherein the PHA is obtained from H. mediterrani by a saltwater-based lysis process that comprises the addition of SDS, as taught by Munoz. One would have been motivated to do so because Munoz teaches that after lysis and centrifugation of the cells containing the PHA granules, “The sediment containing the granules usually presents lipid and protein contamination. This contamination can be reduced to traces with one or a plurality of washing operations using water and a detergent which dissolves proteins, such as SDS” (see, e.g., Munoz, col 3, lines 29-33). Moreover, Ghosh teaches lysis followed by centrifugation of the H. mediterrani cells allows for PHA produced in these cells to be extracted and further quantified (see, e.g., Ghosh, Section 2.5, pg. 4). Therefore, based on the teachings of Ghosh and Munoz, it would have been obvious to add SDS to the saltwater-based lysis process in order to reduce lipid and protein contamination. One would have expected success because Ghosh and Munoz both teach processes for obtaining PHA from H. mediterrani.
Claims 48-49 are rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz and as evidenced by Oren as applied to claims 46-47, 50-51, 56, are 62-65 above, and further in view of Tuma (Upgrading end-of-line residues of the red seaweed Gelidium sesquipedale to polyhydroxyalkanotes using Halomonas boliviensis; 2020 – cited in the IDS filed on 07/17/2025).
The combined prior art of Ghosh, Vadlamani, and Munoz, herein referred to as modified-Ghosh-Vadlamani-Munoz, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
However, modified-Ghosh-Vadlamani-Munoz does not teach: wherein the macroalgae comprises cultivated Gracilariaceae (claim 48); or wherein the macroalgae comprises Gracilaria spp. (claim 49).
Tuma’s general disclosure relates to “Agar extraction from Gelidium and Gracilaria red seaweed species produces hundred thousand ton of carbohydrate-rich residues annually. Gelidium sesquipedale waste biomass obtained after agar extraction, still contained 44.2 % w/w total carbohydrates (dry-weight basis). These residues were biologically up-graded to poly-3-hydroxybutyrate (P3HB) after saccharification of their carbohydrate fraction to simple sugars. A combined hydrolysis treatment using sulfamic acid followed by enzymatic hydrolysis with cellulases produced a glucose-rich hydrolysate with a negligible content of inhibitors. With this treatment a sugar yield of circa 30 % (g glucose/g biomass) was attained. The algal hydrolysates were assessed as carbon source for the production of P3HB by the halotolerant bacteria Halomonas boliviensis. A cell concentration of 8.3 g L−1 containing 41 % (w/w) of polymer and a yield (YP/S) of 0.16 gpolymer/gglucose were attained in shake flask assays. In this work, cellulose-rich seaweed waste was shown to be an upgradable, sustainable source of carbohydrates” (see, e.g., Tuma, abstract).
Regarding claims 48-49 pertaining to Gracilaria spp., Tuma teaches that Gracilaria spp., can be hydrolyzed to produce major monosaccharides such as glucose, D-galactose, and anhydro-D-galatose (see, e.g., Tuma, Table 1). Moreover, Tuma teaches “Phosphoric acid was also successfully used in the saccharification of the red macroalga Gracilaria verrucosa [37] at a concentration of 1.5 % (w/v), 140 °C and 60 min reaction time” (see, e.g., Tuma, Section 3.2, pg. 7). Furthermore, Tuma teaches “Sulfamic acid (H3NSO3), also known as amidosulfonic acid, is considered a green catalyst [38]. It is a moderately strong acid (pKa = 1.01), non-corrosive and has a dual active site. It was tested as catalyst in the hydrothermal hydrolysis of the red alga Gracilaria verrucosa at a concentration of approximately 1 % (w/v) (130 °C and 90 min reaction time), resulting in a total reducing sugar yield of 39.9 % [29]” (see, e.g., Tuma, Section 3.2, pg. 7). Therefore, based on the teachings of Tuma, Gracilaria spp., can be hydrolyzed to produce monosaccharides.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce polyhydroxyalkanoates from macroalgae, as taught by modified-Ghosh-Vadlamani-Munoz, wherein the macroalgae is Gracilaria spp., as taught by Tuma. One would have been motivated to do so because Tuma teaches that Gracilaria spp., can be hydrolyzed to produce major monosaccharides such as glucose, D-galactose, and anhydro-D-galatose (see, e.g., Tuma, Table 1). Moreover, modified-Ghosh-Vadlamani-Munoz teaches that monosaccharides can be utilized in a fermentation process to produce PHA (see, e.g., Ghosh, Section 3.1, pg. 6). Therefore, based on the teachings of modified-Ghosh-Vadlamani-Munoz and Tuma, it would have been obvious to produce a macroalgae hydrolysate from Gracilaria spp., because this would result in production of major monosaccharides such as glucose, D-galactose, and anhydro-D-galatose, which can be used to produce PHA. One would have expected success because modified-Ghosh-Vadlamani-Munoz and Tuma teach production of PHA from macroalgae.
Claim 52 is rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz and as evidenced by Oren as applied to claims 46-47, 50-51, 56, are 62-65 above, and further in view of Azizi (Acid pretreatment and enzymatic saccharification of brown seaweed for polyhydroxybutyrate (PHB) production using Cupriavidus necator; 2017 – cited in the IDS filed on 07/17/2025).
The combined prior art of Ghosh, Vadlamani, and Munoz,herein referred to as modified-Ghosh-Vadlamani-Munoz, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
Regarding claim 52 pertaining to the macroalgal mixture being formed from wet macroalgae and saltwater, modified-Ghosh-Vadlamani-Munoz teaches “The seaweed was cultivated in artificial seawater (ASW) cultivation medium which was prepared using distilled water containing dissolved dried Red Sea Salt (Red Sea Inc., IS) which had a total salinity of 37‰. Ammonium nitrate (NH4NO3, Haifa Chemicals Ltd., IS) and phosphoric acid (H3PO4, Haifa Chemicals Ltd., IS) were added to the ASW medium to maintain a concentration of 6.4 g m−3 of nitrogen (N2) and 0.97 g m−3of phosphorus (P) respectively” (see, e.g., Ghosh, Section 2.1, pg. 2).
However, modified-Ghosh-Vadlamani-Munoz does not teach: wherein the macroalgae is broken down into smaller portions prior to forming the macroalgal mixture, and wherein the smaller portions of macroalgae comprise particles of less than approximately 2 mm in diameter (claim 52).
Azizi’s general disclosure relates to “The brown seaweed Sargassum sp. was used as a feedstock to produce polyhydroxybutyarte (PHB) using Cupriavidus necator PTCC 1615. In order to release monomeric sugars, dilute acid hydrolysis of Sargassum sp. biomass was followed by enzymatic saccharification (see, e.g., Azizi, abstract).
Regarding claim 52 pertaining to the particle size of the macroalgae, Azizi teaches grinding brown macroalgae Sargassum sp., biomass using a coffee grinder to obtain 50 mesh size particles (see, e.g., Azizi, Section 2.1). One of ordinary skill in the art would readily understand that 50 mesh size particles is less than 2 mm in diameter.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a macroalgal and saltwater mixture, as taught by modified-Ghosh-Vadlamani-Munoz, wherein the macroalgae is broken down to a particle size less than 2 mm in diameter, as taught by Azizi. One would have been motivated to do so because Azizi teaches that the seaweed biomass was washed several times to remove impurities, dried at room temperature, and then ground using a coffee grinder to obtain 50 mesh size particles (see, e.g., Azizi, Section 2.1, pg. 3). Furthermore, Azizi teaches that these ground seaweed particles were used to characterize the seaweed biomass and perform hydrolysis using a dilute acid (see, e.g., Azizi, Sections 3.1-3.2, pgs. 7-8). Therefore, the ground seaweed is used to further characterize the biomass and perform acid hydrolysis. Moreover, modified-Ghosh-Vadlamani-Munoz teaches cultivation of seaweed and hydrolysis of seaweed in order to produce PHAs (see, e.g., Ghosh, abstract) (see, e.g., Vadlamani, [0069]-[0070]). Therefore, based on the teachings of modified-Ghosh-Vadlamani-Munoz and Azizi, it would have been obvious to grind the seaweed into particles after drying out the seaweed in order to perform further analyses and experiments on. One would have expected success because modified-Ghosh-Vadlamani-Munoz and Azizi both teach production of PHA and/or PHB from seaweed using microbes.
Claim 53 is rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz, as evidenced by Oren, and Azizi as applied to claims 46-47, 50-52, 56, and 62-65 above, and further in view of Khalil (A review of extractions of seaweed hydrocolloids: Properties and application; 2018).
The combined prior art of Ghosh, Vadlamani, Munoz, and Azizi, herein referred to as modified-Ghosh-Vadlamani-Munoz-Azizi, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
Regarding claim 53 pertaining to the macroalgal mixture, modified-Ghosh-Vadlamani-Munoz-Azizi teaches “The seaweed was cultivated in artificial seawater (ASW) cultivation medium which was prepared using distilled water containing dissolved dried Red Sea Salt (Red Sea Inc., IS) which had a total salinity of 37‰. Ammonium nitrate (NH4NO3, Haifa Chemicals Ltd., IS) and phosphoric acid (H3PO4, Haifa Chemicals Ltd., IS) were added to the ASW medium to maintain a concentration of 6.4 g m−3 of nitrogen (N2) and 0.97 g m−3of phosphorus (P) respectively” (see, e.g., Ghosh, Section 2.1, pg. 2).
However, modified-Ghosh-Vadlamani-Munoz-Azizi does not teach: wherein hydrocolloids are extracted and removed (claim 53).
Khalil’s general disclosure relates to a review highlighting “Various applications of these seaweed hydrocolloids as thickeners, stabilizers, coagulants and salves (in the wound and burn dressings) and materials to produce bio-medical impressions in the food, pharmaceutical, and biotechnology industries” (see, e.g., Khalil, abstract).
Regarding claim 53 pertaining to removal of hydrocolloids, Khalil teaches “Extraction processes of seaweed hydrocolloids are slow and complicated processes, involving multiple steps of processing. Typically, hot water is the most popular solvent for the extraction of plain seaweed hydrocolloids due to their water soluble properties except for alginate/alginic acid extractions; which required hot alkali as major solvents” (see, e.g., Khalil, Section 3, pg. 302).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Ghosh-Vadlamani-Munoz-Azizi’s macroalgal mixture, wherein hydrocolloids are extracted and removed from the mixture, as taught by Khalil. One would have been motivated to do so because Khalil teaches “hydrocolloids components, namely alginate, agar and carrageenan tend to provide numerous ingredients to the food, pharmaceutical, cosmetic, textiles, paper and biotechnology industries as stabilizers, thickeners, emulsifier and fillers” (see, e.g., Khalil, Introduction, pg. 297). Therefore, one of ordinary skill in the art would readily understand that the hydrocolloids extracted from the macroalgal can be used in other food, pharmaceutical, cosmetic, and biotechnology processes. Moreover, modified-Ghosh-Vadlamani-Munoz-Azizi teaches production of a macroalgal-saltwater mixture (see, e.g., Ghosh, Section 2.1, pg. 2), that can undergo hydrolysis and microbial fermentation to produce PHAs (see, e.g., Vadlamani, [0003] & Ghosh, Introduction, pg. 2). Therefore, one of ordinary skill in the art would readily understand that removal of hydrocolloids for other food, pharmaceutical, cosmetic, and biotechnology processes would result in a more purified macroalgal product for hydrolysis and microbial fermentation to produce PHAs. One would have expected success because modified-Ghosh-Vadlamani-Munoz-Azizi and Khalil both teach processes involving macroalgal compositions.
Claim 54 is rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz, as evidenced by Oren, Azizi and Khalil as applied to claims 46-47, 50-53, 56, and 62-65 above, and further in view of Kwon (Production of sugars from macro-algae Gracilaria verrucosa using combined process of citric acid-catalyzed pretreatment and enzymatic hydrolysis; 2015).
The combined prior art of Ghosh, Vadlamani, Munoz, Azizi, and Khalil herein referred to as modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
However, modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil does not teach: wherein the macroalgal mixture is treated with a weak acid, and wherein the weak acid comprises citric acid (claim 54).
Kwon’s general disclosure relates to “a combined process of citric acid-catalyzed pretreatment and enzymatic hydrolysis was applied for production of total reducing sugar (TRS) from marine macro red-algae Gracilaria verrucosa as a potential re source. By citric acid-catalyzed pretreatment under the 1:10 solid-to-liquid ratio, 0.1 M citric acid, 150 °C, 60 min condition, a 50.9% TRS yield was obtained. By subsequent enzymatic hydrolysis using enzyme mixture (20 FBG/g-biomass and 28 FPU/g-biomass), a 57.8% TRS yield was obtained. Because citric acid-catalyzed pre-treatment is simple, cheap, and environmentally friendly (do not use hazardous chemicals), these results served to demonstrate the significant potential of citric acid-catalyzed pretreatment to produce TRS for biofuels and bio-chemicals refinement purposes” (see, e.g., Kwon, abstract).
Regarding claim 54 pertaining to treatment with citric acid, Kwon teaches treatment of G. verrucosa with citric acid for hydrolysis (see, e.g., Kwon, Section 2.2, pg. 294).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a macroalgal mixture, as taught by modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil, wherein the mixture is treated with citric acid, as taught by Kwon. One would have been motivated to do so because Kwon teaches that “Pretreatment is most commonly performed on lignocellulosic bio mass to obtain efficient enzymatic hydrolysis” (see, e.g., Kwon, Introduction, pg. 293) and “citric acid-catalyzed hydrothermal pretreatment of macro-algae is simple, cheap, and environmentally friendly. It does not use hazardous chemicals and generates low amounts of by-products than strong acids. Also, citric acid and its salts in hydrolysate, notably, can be used as a pH buffer system without further treatment during subsequent enzymatic hydrolysis” (see, e.g., Kwon, Introduction, pg. 293). Moreover, modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil teaches enzymatic hydrolysis of macroalgae in order to obtain PHAs (see, e.g., Vadlamani, [0003] & Ghosh, Introduction, pg. 2). Therefore, based on the teachings of modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil and Kwon, it would have been obvious to perform a citric acid pretreatment before enzymatic hydrolysis in order to obtain efficient enzymatic hydrolysis. One would have expected success because modified-Ghosh-Vadlamani-Munoz-Azizi-Khalil and Kwon both teach enzymatic hydrolysis of macroalgae to produce sugars.
Claim 55 is rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz and as evidenced by Oren as applied to claims 46-47, 50-51, 56, are 62-65 above, and further in view of Mohan (Conversion of Pine Sawdust into Polyhydroxyalkanoate Bioplastics; 2021 – cited in the IDS filed on 07/17/2025).
The combined prior art of Ghosh, Vadlamani, and Munoz, herein referred to as modified-Ghosh-Vadlamani-Munoz, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
Regarding claim 55 pertaining to enzymatically lysing the macroalgae to form a macroalgal hydrolysate, modified-Ghosh-Vadlamani-Munoz teaches two strains of algae, wherein “Biomass digestions were performed at a solid concentration of 10% (w/v) in 50 mM citrate buffer adjusted to a pH of 4.5. Protease alone or a mixture of protease with α-amylase and amyloglucosidase (all purchased from Sigma) were added to the biomass slurries” (see, e.g., Vadlamani, [0069]-[0070]). Moreover, following enzymatic hydrolysis, modified-Ghosh-Vadlamani-Munoz teaches “After obtaining the suspension of granules free of whole cells and cell residues of significant size, sedimentation or low-speed centrifugation is begun, recovering the PHA granules with a high level of purity. For this step, it is important that the starting culture medium be as free as possible of particulate matter, because if present it would be collected with the granules, contaminating the final product” (see, e.g., Munoz, col 3, lines 20-28). Therefore, the combined prior art of modified-Ghosh-Vadlamani-Munoz teaches the method of obtaining a hydrolysate after the macroalgal mixture is enzymatically hydrolysed.
However, modified-Ghosh-Vadlamani-Munoz does not teach: wherein the macroalgal hydrolysate is detoxicated (claim 55).
Mohan’s general disclosure relates to “the potential of producing polyhydroxyalkanoates (PHAs) from pine dust through chemical and microbial conversions” (see, e.g., Mohan, abstract). Moreover, Mohan discloses “To overcome microbial inhibition by the hydrolysates, we systematically investigated the role of different potential inhibitors in defined media and tested the effects of different detoxification treatments on microbial activity” (see, e.g., Mohan, abstract).
Regarding claim 55 pertaining to detoxification of the hydrolysate, Mohan teaches detoxification post hydrolysis reaction (see, e.g., Mohan, pg. 8385, “Post Reaction Detoxification”). Moreover, Mohan teaches that the crude hydrolysate was run through ion exchange chromatography, followed by an activated carbon treatment, followed by calcium hydroxide neutralization and overliming (see, e.g., Mohan, pg. 8385, “Post Reaction Detoxification”).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Ghosh-Vadlamani-Munoz’s macroalgal hydrolysate, wherein the hydrolysate is detoxicated post hydrolysis, as taught by Mohan. One would have been motivated to do so because Mohan teaches “Crude biomass hydrolysates contain numerous components that are quite toxic to microbial growth including furanics and organic acids from sugar breakdown, phenolics from lignin hydrolysis, and large molecular weight humic materials at various levels. Several detoxification methods generally remove inhibitors by their adsorption on various sorbents” (see, e.g., Mohan, Introduction, pg. 8384). Moreover, modified-Ghosh-Vadlamani-Munoz teaches enzymatic hydrolysis of macroalgae in order to obtain PHAs (see, e.g., Vadlamani, [0003] & Ghosh, Introduction, pg. 2). Therefore, based on the teachings of modified-Ghosh-Vadlamani-Munoz and Mohan, it would have been obvious to detoxicate the macroalgae post hydrolysis (i.e., macroalgal hydrolysate) in order to remove components that are toxic to microbial growth since the macroalgal hydrolysate is further fermented with microbes to produce PHAs. One would have expected success because modified-Ghosh-Vadlamani-Munoz and Mohan both teach production of PHAs using hydrolysis and bacterial fermentation steps.
Claims 57-60 are rejected under 35 U.S.C. 103 as being unpatentable over Ghosh, Vadlamani, Munoz and as evidenced by Oren as applied to claims 46-47, 50-51, 56, are 62-65 above, and further in view of Bedade (Emergent Approaches to Efficient and Sustainable Polyhydroxyalkanoate Production; 2021 – cited in the IDS filed on 07/17/2025).
The combined prior art of Ghosh, Vadlamani, and Munoz, herein referred to as modified-Ghosh-Vadlamani-Munoz, is discussed above as it pertains to a method of producing polyhydroxyalkanoates from macroalgae.
However, modified-Ghosh-Vadlamani-Munoz does not teach: wherein the fermentation culture is grown in a continuous or semi-continuous fermentation system (claim 57); or wherein the continuous fermentation system comprises two or more fermentation reactors operating sequentially or in a series or cascade, and wherein additional growth media is added to the fermentation culture during fermentation (claim 58); or wherein the carbon-nitrogen ratio of the fermentation culture inside the reactors increases from the first fermentation reactor to a last fermentation reactor (claim 59); or wherein the carbon-nitrogen ratio of the fermentation culture inside each reactor is kept at a constant ratio via feeding of additional growth media into each reactor, wherein, the continuous fermentation system comprises inlet feeds providing growth media to the fermentation reactors and outlet feeds, pumping fermentation culture from the first reactor, through each subsequent reactor, to a last reactor, and wherein an outlet feed from the last reactor is set at a flow rate equal to the sum of all inlet feed flow rates in the continuous fermentation system (claim 60).
Bedade’s general disclosure relates to a review that “describes various process innovations that build on fed-batch and semi-continuous modes of operation as well as methods that lead to high cell density cultivations” as well as “work to move from costly to lower cost substrates such as lignocellulose-derived hydrolysates, metabolic engineering of organisms that provide higher substrate conversion rates, the potential of halophiles to provide low-cost platforms in non-sterile environments for PHA formation, and work that uses mixed culture strategies to overcome obstacles of using waste substrates” (see, e.g., Bedade, abstract).
Regarding claim 57 pertaining to the fermentation culture, Bedade teaches that continuous and semi-continuous processes can be implemented for the fermentation culture (see, e.g., Bedade, Section 2.3, pg. 10).
Regarding claim 58 pertaining to continuous fermentation, Bedade teaches continuous fermentation comprising more than two fermentation reactors operating sequentially, wherein each fermentation reactor has a feed stream that supplies nutritional medium to the bioreactions (see, e.g., Bedade, Figure 4 & Section 2, pg. 4).
Regarding claim 59 pertaining to the carbon-nitrogen ratio and fermentation reactor, Bedade teaches two reactors in series, wherein the first bioreactor cultivated R. eutropha cells under limiting glucose conditions, and the second bioreactor had P3HB accumulation due to excess carbon source and limiting nitrogen conditions (see, e.g., Bedade, Section 2.3, pg. 10). Moreover, Bedade teaches that “The specific P3HB production rate was dependent on the C/N molar ratio such that, the C/N ratio of 30 in PHB production phase gave optimal results” (see, e.g., Bedade, Section 2.3, pg. 10). Therefore, Bedade teaches a continuous culture system wherein the carbon-nitrogen ratio increases from the first reactor to the second, or last, reactor.
Regarding claim 60 pertaining to the continuous fermentation system, Bedade teaches a continuous fermentation process “characterized by continuous addition at a constant flow rate of fresh media to the bioreaction which provides cells with fresh nutrients. To keep the bioreactor working volume constant, products and effluents are continuously removed” (see, e.g., Bedade, Section 2.3, pg. 10). From these teachings by Bedade, one of ordinary skill in the art would readily understand that the flow rate of the inlet and outlets are the same, or constant, for a continuous fermentation system. Bedade teaches in Figure 4 a multi-stage fermentation system that comprises stirred tank reactors: F1, F3, F5, F7, and F9; and feed streams that supply nutritional medium to the bioreactors R1, R2, R3, R4, and R5. F2, F4, F6, and F8 represent continuous transfer of the fermentation broth from one bioreactor to the next. Therefore, there are inlet and outlet feeds for the system (see, e.g., Bedade, Figure 4). Additionally, since Bedade teaches a continuous fermentation process “characterized by continuous addition at a constant flow rate of fresh media to the bioreaction which provides cells with fresh nutrients. To keep the bioreactor working volume constant, products and effluents are continuously removed” (see, e.g., Bedade, Section 2.3, pg. 10), one of ordinary skill in the art would understand that the carbon-nitrogen ratio is kept constant since growth media is added to each reactor, which is also represented in Figure 4 of Bedade.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce PHAs via microbial fermentation, as taught by modified-Ghosh-Vadlamani-Munoz, wherein the fermentation process is a continuous fermentation process, as taught by Bedade. One would have been motivated to do so because Bedade teaches “the rate of microbial growth is constant under steady-state conditions. A continuous cultivation process that runs at high specific growth rates can provide high productivities. Furthermore, continuous cultivations are desirable since they substantially decrease the frequency of bioreactor shutdown and cleaning operations. Also, continuous cultivation processes circumvent wash-out even at high dilution rates. This can lead to high productivity and concentrations of the product” (see, e.g., Bedade, Section 2.3, pg. 10). Moreover, modified-Ghosh-Vadlamani-Munoz teaches the production of PHAs using microbial fermentation with H. mediterranei (see, e.g., Ghosh, Section 2.4, pgs. 3-4). Therefore, based on the teachings of modified-Ghosh-Vadlamani-Munoz and Bedade, it would have been obvious to produce PHAs from H. mediterranei using continuous fermentation because the continuous fermentation process provides high productivity and concentration of the product. One would have expected success because modified-Ghosh-Vadlamani-Munoz and Bedade both teach microbial fermentation methods for PHA production.
Conclusion
Claims 46-65 are rejected.
No claims are allowed.
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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