Patent Application 17918666 - METHOD OF CULTURING ALGAE

Title: METHOD OF CULTURING ALGAE

Application Information

Invention Title: METHOD OF CULTURING ALGAE
Application Number: 17918666
Submission Date: 2025-05-19T00:00:00.000Z
Effective Filing Date: 2022-10-13T00:00:00.000Z
Filing Date: 2022-10-13T00:00:00.000Z
National Class: 435
National Sub-Class: 257100
Examiner Employee Number: 72793
Art Unit: 1645
Tech Center: 1600

Rejection Summary

102 Rejections: 0
103 Rejections: 1

Cited Patents

The following patents were cited in the rejection:

Office Action Text

DETAILED ACTION
Claims 1-5, 7, 13-15 and 20-30 are currently pending.

Claim Rejections - 35 USC § 112-2nd paragraph-2nd paragraph
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 1-5, 7, 13-15 and 20-30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 and dependent claims thereof are vague and indefinite because the oxygen saturation of the medium is an unclear parameter as it depends on different other parameter such as for examples the pressure of the air/gas present as well as the temperature of the media. Hence these results of different concentration of oxygen present in the media having great influence of the growth of microalgae accordingly the method is unclear and adequate method steps and ingredients are not recited in the claims. The method claim is incomplete for omitting essential steps without the active step for maintaining 75% oxygen saturation and the specific ingredients and conditions. See MPEP § 2172.01. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed. Appropriate clarification and/or correction is required.
Claims 3, 7, 20 and 21 and dependent claims thereof are vague and indefinite due to the use of the term “optionally”. All of the features listed after the word "optionally", "preferably" and the like are purely optional. The list of potential alternatives in the claims can vary and ambiguity arises. The claims are indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. Accordingly, the metes and bounds of the invention cannot be understood. Further, claim 20 recites several different ‘optionally’ clauses making the claim incredibly confusing. Appropriate clarification and/or correction is required. See MPEP § 2173.05(h).
Claim 14 is vague and indefinite because it recites “optionally wherein a majority of the carbon source is glycerol, optionally wherein the carbon sources consists of glycerol”. It is unclear what is considered a ‘majority”. The sentences preceding this phrase recite at least, 50, 60, 65, 70, 75, 80, 85, 90, 95%. Are all of these percentages a majority? The claim wording and arrangement is very confusing. . Appropriate clarification and/or correction is required.
Claim Rejections - 35 USC § 112-Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.

The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.

Claims 1-5, 7, 13-15 and 20-30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The instant claims are broadly drawn, for instance, to:
1. (CURRENTLY AMENDED) A heterotrophic method of culturing algae of the class Cyanidiophyceae, comprising: (a) Growing algae in a medium comprising a carbon source and a nitrogen source, wherein the oxygen saturation of the medium is above 75%.
The dependent claims provide a laundry list of potential and optional ingredients in so many different combinations which result in completely different media and culture conditions. They also allow for the use of many different algae species from the broad class of Cyanidiophyceae. The instant specification teaches a method of culturing algae, specifically Galdieria sulphuraria, under heterotrophic conditions to produce good amounts of photosynthetic pigments, e.g., phycocyanin. The present inventors have discovered that, as well as typical carbon sources, the alternative carbon source of glycerol, can be used as a basis for such heterotrophic culture and still provide high productivity as long as high oxygen saturation is maintained in the culture medium. The inventors teach that an oxygen saturation of at least 75% effectively compensates for the lack of light in heterotrophic culture by providing an alternative source of reactive oxygen species (ROS) to the algae. Such ROS are usually generated as a by-product of photosynthesis under light and stimulate the algae to produce protective pigments such as phycocyanin. In the absence of light, photosynthesis doesn’t take place and the pigment production typically falls due to inactivation. However, the specification teaches that the provision of high oxygen saturation can mimic the stimulation light usually provides. Furthermore, the high oxygen concentration means that the key enzyme, CPO, is retained within the algal cells and can function effectively to produce these pigments. The inventors have further found that the effect of high oxygen saturation persists with alternative carbon sources such as glycerol and does not require use of glucose, although glucose can be used to achieve similarly good results. The method of the invention has a phycocyanin productivity which is 567 times higher compared to existing Spirulina autotrophic cultures, and at least 20 times higher phycocyanin productivity compared to existing mixotrophic reactor based-Spirulina cultivation. The method of the invention further improves upon the closest heterotrophic cultures of Galdieria sulphuraria known in prior the art. The method of the present invention is able to achieve phycocyanin productivity of over 1.7 g.Lt.day, compared with a previously reported highest level of 0.86 g.L.day* using glucose as a carbon source (Graverholt and Eriksen 2007). Surprisingly this level of productivity has been achieved by the inventors using a carbon source of glycerol which has often been regarded as a poor substrate compared with glucose. When using glucose, the method of the present invention is able to achieve an even higher phycocyanin productivity of over 1.75 g.L4.day. The invention provides an alternative novel method of culturing algae, e.g., Galdieria sulphuraria, under heterotrophic conditions to produce good amounts of valuable chemicals, especially photosynthetic pigments such as phycocyanin. The inventors have discovered that the alternative carbon source of glycerol can be used as a basis for such heterotrophic culture and still provide high productivity as long as the nitrogen source is added into the culture when required and is not pre-mixed with the glycerol before entry into the bioreactor.
The present invention found that oxygen saturation during semi- and continuous heterotrophic cultivations is identified as a key driver for phycocyanin productivity, and a series of specially adapted bioreactors are developed in order to achieve this parameter.
The specification teaches that suitable oxygen or air is bubbled through the medium at a pressure of between 0.5 to 5 bar, suitably between 1 to 3 bar, suitably between 1 to 1.5 bar. Suitably the oxygen or air is bubbled through the medium at a pressure of around 1.5 bar.
See TABLE 1 and TABLE 2 on page 34 of the instant specification.
It was not possible to maintain high oxygen concentration (by mixing) whilst also maintaining high productivity of biomass and phycocyanin. In order to solve this problem, we developed a low-shear airlift bioreactor design specifically adapted to achieve high oxygen concentrations for high density cultivation of G. sulphuraria.
However, these results and working examples do not enable the breadth of the current claim of ‘culturing algae of the class Cyanidophyceae, as the prior art teaches culturing different algae under different conditions is highly unpredictable and often requires different parameters and/or ingredients. The instant claims also fail to provide an active step for keeping the oxygen saturation of the medium of above 75% in a heterotrophic method. There are no active steps in the heterotrophic cultivation method claims provide an alternative source of reactive oxygen species (ROS) to the algae to keep the oxygen saturation of the medium above 75% which is an essential step in the claimed heterotrophic method. The specification teaches that suitable oxygen or air is bubbled through the medium at a pressure of between 0.5 to 5 bar, suitably between 1 to 3 bar, suitably between 1 to 1.5 bar. Suitably the oxygen or air is bubbled through the medium at a pressure of around 1.5 bar. This appears to be an essential step in the method of heterotrophic culture of the G. sulphuraria. The claimed method also does not provide the conditions and specific media ingredients that allow for the unexpected results achieved in the instant specification.
Galdieria sulphuraria species is able to utilize various organic carbon substrates, either heterotrophically or mixotrophically. The polyextremophilic nature of G. sulphuraria allows for the use of cultivation conditions that are unsuitable for many other microorganisms, reducing the risk of contamination. Previous findings indicate that this species contains a high protein content when growing mixotrophically, including the blue phycobiliprotein C-phycocyanin, a high-value pigment.
The working examples in the instant specification are with Galdieria sulphuraria. Taking these attributes into account, G. sulphuraria is considered a unique species for the claimed methods and it would be unpredictable to extrapolate results across the entire class of Cyanidophyceae class of algae as the prior art as evidenced above teaches it is unpredictable to culture different types and strains of alga and different combinations of ingredients and culture conditions come into play which are not predictable.
Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001 clearly states: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 148 USPQ 689, 696 (1966) (stating, in context of the utility requirement, that "a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.") Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.”
Accordingly, the broadly recited instant claims are not enabled and do not contain the features which provided the unexpected results in Galdieria sulphuraria phycocyanin cultivation.
Claim Rejections - 35 USC § 112-Deposit
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.

The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.

Claim 21 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claim 21 recites:
21. (CURRENTLY AMENDED) [[A]] The method according to claim 10, wherein the algae is Galdieria sulphuraria, preferably—optionally selected from the following strains: ACUF141, SAG 108.79, 074G, 074G-G1, 074G-G2, CCMEE 5587.1, SAG 108.71, and UTEX#2919, preferably—optionally wherein the algae is Galdieria sulphuraria strain ACUF141
The specification lacks complete deposit information for the deposit all of these strains. Because it is not clear that the properties of these strains are known and publicly available or can be reproducibly isolated from nature without undue experimentation and because the best mode disclosed by the specification requires the use of the strains, a suitable deposit for patent purposes is required.
If the deposit has been made under the provisions of the Budapest Treaty, filing of an affidavit or declaration by applicant or assignees or a statement by an attorney of record who has authority and control over the conditions of the deposit over his or her signature and registration number stating that the deposit has been accepted by an International Depository Authority under the provisions of the Budapest Treaty, that all restrictions upon public access to the deposit will be replaced if viable samples cannot be dispensed by the depository is required. This requirement is necessary when deposits are made under the provisions of the Budapest Treaty as the Treaty leaves this specific matter to the discretion of each State. Amendment of the specification to recite the date of the deposit and the complete name and full street address of the depository is required.
If the deposits have not been made under the provisions of the Budapest Treaty, then in order to certify that the deposits comply with the criteria set forth in 37 CFR §1.801-1.809, assurances regarding availability and permanency of deposits are required. Such assurance may be in the form of an affidavit or declaration by applicants or assignees or in the form of a statement by an attorney of record who has the authority and control over the conditions of deposit over his or her signature and registration number averring:
(a) during the pendency of this application, access to the deposits will be afforded to the Commissioner upon request;

(b) all restrictions upon the availability to the public of the deposited biological material will be irrevocably removed upon the granting of a patent on this application;

© the deposits will be maintained in a public depository for a period of at least thirty years from the date of the deposit or for the enforceable life of the patent or for a period of five years after the date of the most recent request for the furnishing of a sample of the deposited biological material, whichever is longest; and

(d) the deposits will be replaced if they should become non-viable or non-replicable.

In addition, a deposit of the biological material that is capable of self-replication either directly or indirectly must be viable at the time of the deposit and during the term of deposit. Viability may be tested by the depository. The test must conclude only that the deposited material is capable of reproduction. A viability statement for each deposit of a biological material not made under the Budapest Treaty must be filed in the application and must contain:
1)The name and address of the depository;
2)The name and address of the depositor;
3)The date of deposit;
4)The identity of the deposit and the accession number given by the depository;
5)The date of the viability test;
6)The procedures used to obtain a sample if the test is not done by the depository; and
7)A statement that the deposit is capable of reproduction.

If the deposit was made under the provisions of the Budapest Treaty, filing of an affidavit or declaration by Applicants, assignees or a statement by an attorney of record over his or her signature and registration number stating that deposit has been accepted by an International Depository Authority under the provisions of the Budapest Treaty, that all restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this application and that the deposit will be replaced if viable samples cannot be dispensed by the depository is required. This requirement is necessary when a deposit is made under the provisions of the Budapest Treaty as the Treaty leaves this specific matter to the discretion of each State. Amendment of the specification to recite the date of the deposit and the complete name and address of the depository is required.
As a possible means for completing the record, applicant may submit a copy of the contract with the depository for deposit and maintenance of each deposit.
If the deposit was made after the effective filing date of the application for patent in the United States, a verified statement is required from a person in a position to corroborate that the cell line described in the specification as filed is the same as that deposited in the depository. Corroboration may take the form of a showing of a chain of custody from applicant to the depository coupled with corroboration that the deposit is identical to the biological material described in the specification and in the applicant's possession at the time the application was filed.
Applicant's attention is directed to In re Lundak, 773 F.2d. 1216, 227 USPQ 90 (CAFC 1985) and 37 CFR §1.801-1.809 for further information concerning deposit practice.

Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.

Claim(s) 1-5, 7, 13-15 and 20-30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sloth et al (Enzyme and Microbial Technology Volume 38, Issues 1–2, 3 January 2006, Pages 168-175), Schmidt et al (Biotech. Bioengineering. 2005. 90(1): 77-84) and Gross (Plant and Cell Physiology, Volume 36, Issue 4, June 1995, Pages 633–638) in view of Grama et al (ACS Sustainable Chemistry & Engineering. 2016. 4 (3), 1611-1618) and further in view of Perez Garcia et al (“Microalgal heterotrophic and mixotrophic culturing for bio-refining: from metabolic routes to techno-economics." Algal biorefineries (2015): 84-131; provided by Applicants).
Sloth et al teach the cultivation and growth of Galdieria sulphuraria and growth media for its cultivation in Section 2.1 of the reference it is taught that G. sulphuraria strain 074G was kindly supplied by Dr. Wolfgang Gross, Freie Universität Berlin. Stock cultures were maintained under constant light (30–0 μmol photons m−2 s−1) by sequential transfer into photoautotrophic batch cultures and grown at room temperature, pH 2 in a defined growth medium with no organic carbon substrates. The same growth medium, but with various concentrations of the nitrogen source, (NH4)2SO4, and supplemented with organic carbon sources (glucose, fructose or glycerol) was used in heterotrophic and mixotrophic growth experiments. In section 2.3 it is taught that heterotrophic and mixotrophic continuous flow cultures were grown in a 3 L Applikon BTS 05 bioreactor (Applikon, The Netherlands) containing 2.5 L culture. The bioreactor consisted of a cylindrical glass jar with a diameter of 13 cm fitted with a top plate of stainless steel. The bioreactor was placed in a cabinet equipped with eight fluorescent tubes (Osram 15W/31-830) and equipped with a Pt100 temperature sensor and autoclavable pH and oxygen electrodes (Mettler Toledo). The temperature was maintained at 42 °C and the culture was stirred at 500 rpm and aerated with 3 L min−1 air. The exit gas was passed through a condenser at 4 °C to reduce evaporation. Depending on the number of fluorescent tubes turned on, the average light intensity on the reactor wall was between 0 and 395 μmol photons m−2 s−1. When changing the light intensity, it was changed in the opposite direction of the previous change in order to avoid a continuous selection pressure for mutants with a different tolerance towards light compared to the original strain. The flow rate of growth medium through the culture was 1.58 L day−1 giving a dilution rate of 0.63 day−1. Cultures were grown at pH 2 with 1.0 g L−1 glucose or glycerol as carbon source and 0.55 g L−1 (NH4)2SO4 as nitrogen source. Sloth teaches that phycoyanin (PC) is found in the unicellular, acidophilic red alga Galdieria sulphuraria, which has been shown to be a candidate for a PC production process. G. sulphuraria contains PC and minor amounts of a second phycobiliprotein, allophycocyanin. Sloth teaches that Compared to the established PC production processes using S. platensis, a production process involving G. sulphuraria may offer several advantages. It is taught that G.suphuraria also produces a second phycobiliprotein, allophycocyanin. Some strains of G. sulphuraria grow well heterotrophically and at least partly retain their photosynthetic apparatus, including PC, when grown in darkness. Production of PC in these strains can therefore be carried out heterotrophically without the need for external light sources, or mixotrophically, in which case only low light intensities would be needed. The natural habitat of G. sulphuraria is hot, acidic springs, so the optimal growth conditions are found at temperatures above 40 °C and at pH 1–3. In addition, this alga is able to utilize many different carbon sources. In the ‘Discussion’ section it is taught that carbon limitation in the presence of excess nitrogen resulted in accumulation of relatively high cellular phycocyanin content 20 mg g-1 DW. It is also taught that glycerol gave higher biomass yield than glucose. Page 175 teaches that G. sulphuraria accumulates enough PC (10–30 mg g−1 DW dry weight) in carbon-limited conditions to make this organism a suitable alternative host for production of PC, particularly because G. Sulphuraria can be grown to very high cell densities (>100gL-1 biomass dry weight) without the need for external light sources and still product considerable amounts of PC.
Schmidt et al teach growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry wt. on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, resp. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry wt. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry wt. concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.
Gross et al teach the acido-and thermophilic red alga Galdieria sulphuraria (Galdieri) Merola grows under mixo- and heterotrophic conditions on 27 different sugars and sugar alcohols as sole carbon source. They separated two strains from an isolate originally collected at Mt. Lawu (Indonesia). These strains are indistinguishable in growth and pigmentation under autotrophic conditions. However, under heterotrophic conditions, strain 074 W lost most of its pigments whereas strain 074 G stayed green on all substrates tested. Strain 074 G had the highest pigment content when grown on sugar alcohols. Usually, the alga exhibited a short lag-phase followed by logarithmic growth. However, when transferred from auto- to heterotrophic conditions a lag-period of about 45 days was observed with the sugar alcohol dulcitol. Similarly, long lag-periods were also noticed for strain 074 G grown on D-mannitol and for strain 074 W grown on D-ribose. The length of the lag-phase is a function of the length of the previous culture under autotrophic conditions. This enormous versatility in the heterotrophic growth of Galdieria sulphuraria presents an ideal system to study the metabolism of rare sugars and sugar alcohols. See abstract.
Grama et al teach that glycerol was used as an organic substrate to enhance the biomass production rate of a Dactylococcus microalga during photoheterotrophy while simultaneously reducing the need for cell culture gas exchange. Photoheterotrophic cultivations were carried out at concentrations of 6-, 30-, and 150-mM glycerol in parallel with photoautotrophic and heterotrophic cultivations. The highest biomass productivity was with 30 mM glycerol, the concentration where the net oxygen exchange between the cells and the culture medium was minimized, thus implying a balance between respiration and photosynthesis and internal recycling of O2 and CO2. The max. specific growth rate and biomass productivity were subsequently increased by 43 ± 9% and 108 ± 16%, resp., compared to the photoautotrophic cultivations. The net oxygen production rate could be modeled as a function of the light intensity, chlorophyll content, the photosynthetic efficiency as measured by PAM fluorometry, and the respiration rate. Glycerol addition decreased the cellular chlorophyll a content and the photosynthetic efficiency, but increased carbon fixation by respiration. These results show the addition of a waste product, e.g., glycerol from biodiesel, could be used as an algal bioprocess substrate to reduce or eliminate the aeration energy demand while simultaneously increasing biomass production.
Perez-Garcia et al discusses different variables of culture conditions of microalgae for the production of chemical of interest (fig.1) as well cultivation methods (5 Cultivation Methods) disclosing different parameters bioreactor, mode of culture, culture media, oxygen supply, temperature, etc...
The skilled person is well aware of the parameters, bioreactor, mode of culture, culture media, oxygen supply, temperature, etc. as evidenced in the teachings of Perez-Garcia and other cited references above, which influence the growth of microalgae for the production of chemicals of interest. The cited prior art teaches that culturing Cyanidiophyceae, a class of extremophilic red algae, requires specific conditions to support their growth and metabolism. These conditions include providing adequate carbon, nitrogen, and oxygen sources, as well as maintaining optimal temperature and pH levels. While Cyanidiophyceae, which include Galdieria sulphuaria, primarily thrive in acidic and high-temperature environments, they still require oxygen for respiration. Adequate aeration or oxygen transfer during cultivation is necessary to support their metabolism. Hence, the skilled person carrying out routine methods would have arrived to the claimed subject matter without inventive skills and with reasonable expectation of success. Although the prior art references do not specifically recti the oxygen saturation levels of the medium as ‘above 75%’, the references teach the importance of maintaining proper oxygen levels. Sloth, in particular, recites that the bioreactor was placed in a cabinet equipped with eight fluorescent tubes (Osram 15W/31-830) and equipped with a Pt100 temperature sensor and autoclavable pH and oxygen electrodes (Mettler Toledo). The temperature was maintained at 42 °C and the culture was stirred at 500 rpm and aerated with 3 L min−1 air. Grama et al teach that glycerol was used as an organic substrate to enhance the biomass production rate of a Dactylococcus microalga during photoheterotrophy while simultaneously reducing the need for cell culture gas exchange. Photoheterotrophic cultivations were carried out at concentrations of 6-, 30-, and 150-mM glycerol in parallel with photoautotrophic and heterotrophic cultivations. The highest biomass productivity was with 30 mM glycerol, the concentration where the net oxygen exchange between the cells and the culture medium was minimized, thus implying a balance between respiration and photosynthesis and internal recycling of O2 and CO2. The max. specific growth rate and biomass productivity were subsequently increased by 43 ± 9% and 108 ± 16%, resp., compared to the photoautotrophic cultivations. The net oxygen production rate could be modeled as a function of the light intensity, chlorophyll content, the photosynthetic efficiency as measured by PAM fluorometry, and the respiration rate. Glycerol addition decreased the cellular chlorophyll a content and the photosynthetic efficiency, but increased carbon fixation by respiration. Accordingly, keeping oxygen saturation in the medium at appropriate levels, including above 75%, would have been an obvious parameter to be optimized and, if not inherent in the methods recited above, it would be particularly obvious by the teachings of Sloth.
Moreover, the particular growing conditions claimed do not show an unexpected/ surprising effect for the skilled artisan on which inventive step could be based. It has long been settled to be no more than routine experimentation for one of ordinary skill in the art to discover an optimum value of a result effective variable. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation." Application of Aller, 220 F.2d 454, 456, 105 USPQ 233, 235-236 (C.C.P.A. 1955). "No invention is involved in discovering optimum ranges of a process by routine experimentation." Id. at 458, 105 USPQ at 236-237. The "discovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219 (C.C.P.A. 1980). Since the cultures and specific limitations recited in instant claims are not specific for any particular purpose or solve any stated problem and the prior art teaches that methods and culture media can often vary according to the particular Cyanidiophyceae and, solutions and parameters appear to work equally as well, absent unexpected results, it would have been obvious for one of ordinary skill to discover the optimum workable ranges of the methods disclosed by the prior art by normal optimization procedures known in the heterotrophic art. It is noted that the carbon sources, nitrogen sources, pH, temperature, media, etc. are all taught by the prior art references cited above. As stated in the 112, first paragraph rejection above, there is no active step that provides an alternative source of reactive oxygen species (ROS) to the algae to keep the oxygen saturation of the medium above 75% which is an essential step in the claimed heterotrophic method. The specification teaches that suitable oxygen or air is bubbled through the medium at a pressure of between 0.5 to 5 bar, suitably between 1 to 3 bar, suitably between 1 to 1.5 bar. Suitably the oxygen or air is bubbled through the medium at a pressure of around 1.5 bar. This appears to be an essential step in the method of heterotrophic culture of the G. sulphuraria for the unexpected result shown in the instant specification of phycocyanin productivity of over 1.7 g.Lt.day, compared with a previously reported highest level of 0.86 g.L.day*. With respect to instant claims 28 and 29, they are product by process claims. The claim from which they currently depend recites a method which is taught by the prior art references, as there is no step of providing oxygen, etc. Accordingly, the Cyanidiophyceae produced by the cited methods, would inherently comprise the same/similar intracellular concentrations of phycocyanin and allophycocyanin. The patentability of a product does not depend upon its method of production. If the product in [a] product-by-process claim is the same as or obvious from a product of the prior art, [then] the claim is unpatentable even though the prior [art] product was made by a different process.” In re Thorpe, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). Once the examiner provides a rationale tending to show that the claimed product appears to be the same or similar to that of the prior art, although produced by a different process, the burden shifts to applicant to come forward with evidence establishing an unobvious difference between the claimed product and the prior art product. In re Marosi, 218 USPQ 289, 292 (Fed. Cir. 1983).

Pertinent art, not prior to effective filing date:

Fernandez et al (ACS Sustainable Chem. Eng. 2025, 13, 5, 2132–2140).

Oxygen-balanced mixotrophy (OBM) is a particular type of microalgae mixotrophic cultivation, where the supply of an organic carbon substrate is adjusted to match heterotrophic oxygen consumption with photosynthetic production. In this way, the need for aeration is eliminated due to intracellular gas recycling during daytime. After implementing this process at lab scale, we sought to explore its scalability in a tubular photobioreactor (TPBR). In this study, OBM was implemented in a two-phase tubular photobioreactor of 1700 L placed in a greenhouse and exposed to sunlight. The process was run with the polyextremophilic species Galdieria sulphuraria, using glucose as a carbon source. The gas phase was continuously recirculated, and the oxygen concentration was monitored and utilized to manage the glucose supply through a proportional-integral controller. An excessive rate of night aeration, however, resulted in CO2 limitation issues. Subsequent tuning and optimization of controller settings and the nighttime aeration rate effectively addressed the problem. The average biomass productivity reached 0.81 g·L–1·day–1, a significant improvement over autotrophic productivity in the same pilot system. On the other hand, the biomass yield on the substrate was 0.68 C-molx·C-mols–1, indicating that considerable carbon recycling took place but to a lower extent than at lab scale. These results provide a solid foundation for the large-scale industrial implementation of OBM.

Prior art, not presently relied upon:

Sun et al (Bioresource Technology. 2018 250: 868-876).
Influence of oxygen on the biosynthesis of polyunsaturated fatty acids in microalgae
As one of the most important environmental factors, oxygen is particularly important for synthesis of n-3 polyunsaturated fatty acids (n-3 PUFA) in microalgae. In general, a higher oxygen supply is beneficial for cell growth but obstructs PUFA synthesis. The generation of reactive oxygen species (ROS) under aerobic conditions, which leads to the peroxidation of lipids and especially PUFA, is an inevitable aspect of life, but is often ignored in fermentation processes. Irritability, microalgal cells are able to activate a number of anti-oxidative defenses, and the lipid profile of many species is reported to be altered under oxidative stress. In this review, the effects of oxygen on the PUFA synthesis, sources of oxidative damage, and anti-oxidative defense systems of microalgae were summarized and discussed. Moreover, this review summarizes the published reports on microalgal biotechnology involving direct/indirect oxygen regulation and new bioreactor designs that enable the improved production of PUFA.

Lopez et al (Scientific Reports volume 9, Article number: 10791 (2019)
A search for extremophile organisms producing bioactive compounds led us to isolate a microalga identified as Galdieria sp. USBA-GBX-832 from acidic thermal springs. We have cultured Galdieria sp. USBA-GBX-832 under autotrophic, mixotrophic and heterotrophic conditions and determined variations of its production of biomass, lipids and PUFAs. Greatest biomass and PUFA production occurred under mixotrophic and heterotrophic conditions, but the highest concentration of lipids occurred under autotrophic conditions. Effects of variations of carbon sources and temperature on biomass and lipid production were evaluated and factorial experiments were used to analyze the effects of substrate concentration, temperature, pH, and organic and inorganic nitrogen on biomass production, lipids and PUFAs. Production of biomass and lipids was significantly dependent on temperature and substrate concentration. Greatest accumulation of PUFAs occurred at the lowest temperature tested. PUFA profiles showed trace concentrations of arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5). This is the first time synthesis of these acids has been reported in Galdieria. These findings demonstrate that under heterotrophic conditions this microalga’s lipid profile is significantly different from those observed in other species of this genus which indicates that the culture conditions evaluated are key determinants of these organisms’ responses to stress conditions and accumulation of these metabolites.

Bozidar et al (Engineering in Life Sciences, Wiley, Weinheim, DE 10(2): 165-170, April 1, 2010; provided by Applicants).
Bozidar et al disclose the cultivation of a Euglena gracilis algae in a media as follows "the content of dry matter in potato liquor was 32.8 g/L containing 5% nitrogen and 31.8% carbon" (Materials and methods) and glucose “Corn steep liquor was obtained as a solution with a dry mass content of 50% from Sigma-Aldrich" (Materials and methods). The culture conditions were as follows "Cultivations in the presence of potato liquor were carried out without pH control starting with an initial pH of about 5. During cultivations on synthetic medium the pH was maintained at 4.5 by the addition of 10% v/v phosphoric acid solution" (2.3 Operating conditions for stirred-tank cultivation). The results were analyzed as follows "For bioprocess monitoring samples were taken under sterile conditions. All samples were analyzed in duplicate. Cell number density (N) was measured by microscopic cell counting in a Thoma chamber or by automatic counting in a flow counter [...] or the analysis of the paramylon concentration, the cell suspension obtained after centrifugation was resuspended in distilled water and then treated by ultrasound (Branson, USA) for cell disruption. [...] Total carbon and nitrogen contents in media and in biomass samples were determined by Elementar analysator vario EL (Elementar Analysensysteme, Germany).” (2.4 Analytical procedures). Bozidar discloses also a culture condition with O2 saturation of at least 30% by control of air flow rate and stirrer speed, if required.

Li et al.
PATENT NO. KIND DATE APPLICATION NO. DATE
--------------- ---- -------- --------------------- --------
CN 102021208 A 20110420 CN 2010-10545871 20101116
WO 2012065545 A1 20120524 WO 2011-CN82261 20111116
PRIORITY APPLN. INFO.: CN 2010-10545871 A 20101116
CN 2010-10567920 A 20101201
PATENT STATUS PATENT INFORMATION:
PATENT NO. KIND STATUS STATUS DATE
--------------- ---- ------------- -----------
CN 102021208 A Dead 20201121
WO 2012065545 A1 Dead 20201201
ED Entered STN: 26 Apr 2011
AB The title method comprises inoculating microalgae into heterotrophic
culture medium (its pH 4.0-10.0) in a bioreactor, carrying out
heterotrophic culture at 10-50° under controlling pH < 10.0
and dissolved oxygen > 1% to obtain microalgae culture liq., dilg.
with org. carbon source-free liq. culture medium (its pH 4.0-10.0)
to cell d. 0.1-50 g/L, introducing into a photoinduced device, inducing
with photoinduced culture medium at 5-50° and illumination
intensity 0.1-150 klx for 1-150 h under natural light or artificial light
irradn., extg., and sepg. to obtain lipid product. The method can be used
for industrial prodn. of biofuel (such as diesel oil or aviation kerosene)
with low cost.

Graverholt et al (Applied Microbiology and Biotechnology, (NOV 2007) Vol. 77, No. 1, pp. 69-75).
Production of biomass and phycocyanin (PC) were investigated in highly
pigmented variants of the unicellular rhodophyte Galdieria
sulphuraria, which maintained high specific pigment concentrations when
grown heterotrophically in darkness. The parental culture, G.
sulphuraria 074G was grown on solidified growth media, and intensely
coloured colonies were isolated and grown in high-cell-density fed-batch
and continuous-flow cultures. These cultures contained 80-110 g L-1
biomass and 1.4-2.9 g L-1 PC. The volumetric PC production rates were
0.5-0.9 g L-1 day(-1). The PC production rates were 11-21 times higher
than previously reported for heterotrophic G. sulphuraria 074G
grown on glucose and 20-287 times higher than found in phototrophic
cultures of Spirulina platensis, the organism presently used for
commercial production of PC.

Ahthane et al:
PATENT NO KIND DATE WEEK LA PG
---------------------------------------------------------
FR 3081880 A1 20191206 (201998) * FR 20[1]
WO 2019228947 A1 20191205 (201998) FR
EP 3802782 A1 20210414 (2021032) FR
US 20210230534 A1 20210729 (2021062) EN
US 11560542 B2 20230124 (2023008) EN
FR 3081880 B1 20241220 (2025003) FR
NOVELTY - Method for cultivating unicellular red algae for producing a
biomass rich in phycocyanins involves (i) mixing in mixotrophic or
heterotrophic mode of unicellular red algae on a culture medium
comprising carbon source comprising glucose and (ii) recovering
biomass by adding sufficient amount of glycerol to the culture medium
to increase production of phycocyanin as compared to culture without
glycerol.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the
following:
(1) method for preparing phycocyanins involves producing a biomass
and extracting phycocyanins from recovered biomass; and
(2) food product comprising the biomass or phycocyanin obtained by
the method.
USE - The method is useful for cultivating unicellular red algae
to produce biomass, which is used in food product. The unicellular red
algae is chosen from Cyanidioschyzon , Cyanidium or Galdieria ,
preferably C.merolae 10D, C.merolae DBV201, C.caldarium ,
C.daedalum , C.maximum , C.partitum , C.rumpens ,
G.daedala , G.maxima , G.partita or G.sulphuraria (all
claimed). The biomass includes food pigments or coloring.
ADVANTAGE - The method is suitable for industrial production of
biomass enriched with phycocyanins in a simple and cost-effective manner
with high yield and productivity.
TI Cultivating unicellular red algae used for producing biomass enriched
with phycocyanins used in food product by mixing in mixotrophic or
heterotrophic mode of unicellular red algae on culture medium and
recovering biomass

Gilmour et al (Current Topics in Biotechnology (2012), 7, 1-12).
A review. An airlift loop bioreactor (ALB) with microbubble dosing was
used to grow microalgae on high CO2 content steel plant exhaust gas,
generated from the combustion of off gases from steel processing. The gas
anal. of CO2 uptake in the 2200 L bioreactor showed a specific uptake rate
of 0.1 g l-1 h-1, an av. 14% of the CO2 available in the exhaust gas with
a 23% compn. of CO2. This uptake led to a steady prodn. of chlorophyll,
biomass and total lipid content in the bioreactor, with a best doubling
time of 1.8 days. The gas anal. also showed anti-correlation of CO2
uptake and O2 prodn., which along with the apparent stripping of the O2 to
the equil. level by the microbubbles, strongly suggests that the
bioreactor is not mass-transfer limited, nor O2 inhibited. Subsequently,
an array of 3 L lab. bench ALBs have been developed for screening
purposes, with the notion that conventional shake flask incubation for
screening is oxygen inhibited. The small ALBs achieve accelerating
exponential growth, resulting in the desired levels of algae d. an
order of magnitude faster than the undosed control. Large-scale screening
time in industrial labs. can thus be decreased significantly while using
environmental conditions appropriate for full scale prodn., including
stack gas as part of the medium. Finally, microbubble gas exchange with
an airlift loop effect is not limited to photobioreactors. The
circulation and mixing benefits can be replicated by engineering algal
ponds, as the baffles and diffusers needed to direct the airlift loop
effect are inexpensive.

Correspondence regarding this application should be directed to Group Art Unit 1645. Papers related to this application may be submitted to Group 1600 by facsimile transmission. Papers should be faxed to Group 1600 via the PTO Fax Center located in Remsen. The faxing of such papers must conform with the notice published in the Official Gazette, 1096 OG 30 (November 15,1989). The Group 1645 Fax number is 571-273-8300 which is able to receive transmissions 24 hours/day, 7 days/week.

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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jennifer E. Graser whose telephone number is (571) 272-0858. The examiner can normally be reached on Monday-Thursday from 8:00 AM-6:30 PM.

If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Gary Nickol, can be reached on (571) 272-0835.

Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-0500.

/JENNIFER E GRASER/Primary Examiner, Art Unit 1645 5/14/25

Patent Application 17918666 - METHOD OF CULTURING ALGAE - Rejection

Patent Application 17918666 - METHOD OF CULTURING ALGAE

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