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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.
Remarks
The amendments and remarks filed on 02/25/2026 have been entered and considered. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action. The rejections and/or objections presented herein are the only rejections and/or objections currently outstanding. Any previously presented objections or rejections that are not presented in this Office Action are withdrawn.
Claims 1-19 are pending.
Claims 1 and 3 are amended.
Claims 11 and 13 are withdrawn.
Claims 1-10, 12, and 14-19 have been examined on the merits.
Priority
This application, U.S. Application No. 19202839, is a continuation in part of U.S. Application No. 18532043, filed on 12/07/2023, now issued as U.S. Patent No. 12297423, which claims benefit under 35 U.S.C. 119(e) to a provisional application No. 63534123 filed on 08/23/2023.
Claim Rejections - 35 USC § 112(b), or 112, Second Paragraph
Claims 1-10, 12, and 14-19 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 pre-AIA the applicant regards as the invention.
Claim 1 is indefinite due to the recitation of “said urea is the primary nitrogen source for said yeasts”. The claim specifically defines that urea is not added until more than 90% of free amino nitrogen (10-400 mg/liter) is consumed by yeasts, which indicates that the fermentation broth does not contain urea until <10% of free amino nitrogen is left in the broth. It is unclear how the urea can be a primary nitrogen source for yeasts while the fermentation broth does not contain urea and the yeasts use the free amino nitrogen (FAN) as the nitrogen source, before the urea is added to the broth when >90% of FAN is consumed.
The remaining claims are rejected for depending from an indefinite claim.
Claim Rejections - 35 USC § 112, First Paragraph
The following is a quotation 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 35 U.S.C. 112 (pre-AIA ), first paragraph:
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 1-3, 5-10, 12, and 14-19 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling a method for controlling bacterial contamination while growing yeasts possessing a nickel-independent urea-utilizing enzyme, does not reasonably provide enablement for a method for controlling bacterial contamination while growing yeasts not possessing a nickel-independent urea-utilizing enzyme. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention the invention commensurate in the entire scope with these claims.
In making a determination as to whether an application has met the requirements for enablement under 35 U.S.C. 112 ¶ 1, the following factors enumerated In re Wands, 8 USPQ2d 1400, at 1404 (CAFC 1988) are considered: (1) the breadth of the claims, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the quantity of experimentation necessary. While it is not essential that every factor be examined in detail, those factors deemed most relevant should be considered.
The breadth of the claims. Claims 1-3, 5-10, 12, and 14-19 are directed to a method for controlling bacterial contamination while growing yeasts, wherein urea is a primary nitrogen source for the yeasts after >90% of free amino nitrogen is consumed by yeasts, wherein an amount of nickel in the fermentation broth during the entire fermentation time is less than 1 mg nickel per kg of the broth. It is noted that the scope of “less than 1 mg nickel per kg of said fermentation broth” recited in the claim 1 encompasses a zero amount of nickel in the broth. It is also noted that the yeasts recited in the claims can be any yeasts, which include yeasts possessing a nickel- independent urea-utilizing enzyme as well as those yeasts not possessing any nickel-independent urea-utilizing enzyme, but only possessing a nickel-dependent enzyme for utilizing urea to support yeast growth. For example, Schizosaccharomyces pombe encompassed by the yeasts of the claims is a yeast containing only a nickel-dependent urease enzyme whose activity relies on the Ni2+ ion, and this yeast cannot utilize urea when nickel is absent or blocked from entering the cells of the yeast; and on the other hand Saccharomyces cerevisiae encompassed by the yeasts of the claims does not express any nickel-dependent enzyme, as evidenced by Milne et al. (Metab. Eng., 2015, 30:130–140, see abstract/lines 7-10) and Eitinger et al. (J. BIOL. CHEM., 2000, 275(24): 18029–18033, see abstract/lines 12-15).
The amount of direction or guidance presented and the existence of working examples. Examiner notes that the specification discloses that the claimed method for controlling bacterial contamination by using urea as a nitrogen source and limiting nickel is based on the assumption that bacteria require nickel as a cofactor for their urease enzymes in order to use urea for growth, while yeasts do not require nickel as a cofactor for their enzymes that utilize urease for growth. As such, the presence of a nickel-independent urea-utilizing enzymes in the yeasts is essential for the claimed invention. In the specification there is a working example (pages 28-29), which uses Candida utilis yeast (i.e. Cyberlindnera jadiniimore) containing a nickel-independent enzyme that utilizes urea, as well as a contaminated Lactobacillus fermentumnd bacterium that does not contain nickel-independent urease enzymes. In this example, the Candida utilis yeast is cultured in a broth containing urea, but not nickel, in the presence of the contaminated Lactobacillus fermentumnd. The results demonstrate that when urea is the nitrogen source and no nickel is present during fermentation, yeasts having nickel-independent urea-utilizing enzymes (e.g. Candida utilis) grow faster than contaminating bacteria not having nickel-independent urea-utilizing enzymes (e.g. Lactobacillus fermentuma), thereby controlling bacterial contamination. However, the specification fails to provide any information regarding how to reach the goal of controlling bacterial contamination when a yeast (e.g. Schizosaccharomyces pombe) not containing any nickel-independent urea-utilizing enzymes is cultured in a broth with urea as a main nitrogen source in the absence of nickel in the claimed method.
The state of prior art, and the predictability or unpredictability of the art. The prior art, as evidenced by Milne et al. and Eitinger et al. described above, teaches that when yeasts (such as S. pombe) contain only a nickel-dependent urease enzyme, the yeasts cannot utilize urea when nickel is absent or blocked from entering yeast cells. As such, yeasts not having nickel-independent urea-utilizing enzymes cannot utilize urea in the absence of nickel, thus not growing faster than contaminating bacteria not having nickel-independent urea-utilizing enzymes, therefor not being able to reaching the goal of controlling bacterial contamination in the claimed method.
The quantity of experimentation necessary. It is not routine in the art to grow yeasts not having nickel-independent urea-utilizing enzymes in a broth comprising urea as a main nitrogen source and contaminating bacteria in the absence of nickel for reaching the goal of controlling bacterial contamination. Neither the prior art nor the specification supports or shows that bacterial contamination can be prevented or controlled when yeasts not having any nickel-independent urea-utilizing enzymes is used in the claimed method. Therefore, in absence of some guidance as to how to control bacterial contamination when yeasts cannot utilize urea, one of skill in the art would have to carry out a large amount of experimentation to find which additional steps or additional compounds need to be included in the disclosed method, or how to modify the disclosed method, to reach the goal of controlling bacterial contamination.
Therefore, Claims 1-3, 5-10, 12, and 14-19 are not enabled, and neither the specification nor the prior art enable the entire scope of the claimed invention.
Claim Rejections - 35 USC § 103
Claims 1-5, 7, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Narendranath et al. (Applied and Environmental Microbiology, 1997, 63(11): 4158-4163, of record in parent application #18532043) in view of Yue et al. (Biomass and Bioenergy, 2012, 39:48-52, of record in parent application), Thomas et al. (Applied and Environmental Microbiology, 1990, 56(7): 2046-2050, of record), Kakimoto et al. (Appl Microbiol Biotechnol, 1990, 32:538-543, of record), Strope et al. (BMC Evolutionary Biology, 2011, 11:80, pages 1-15, cited in IDS of parent application), and Whiting et al. (Applied and Environmental Microbiology, 1992, 58(2):713-716, cited in IDS of parent application), as evidenced by Gramss (Advances in Nutrition and Food Science, 2020, 2020(5): 1-9; of record in parent application). This rejection is maintained.
Regarding Claims 1 and 4-5, Narendranath et al. teach a method for yeast-catalyzed fermentations for ethanol production in the presence of lactobacilli bacteria including Lactobacillus fermentum that compete with yeast cells for nutrients (see abstract) (Note: these bacteria read on the “contaminating bacteria” recited in the instant claims 1 and 5), the method comprising a step of growing saccharomyces cerevisiae yeast (reading on the “yeasts” recited in the instant claims 1 and 4), at a starting pH in a fermentation broth comprising: a wheat mash (reading on the “sugar solution” in the instant claim 1), urea as a primary nitrogen source, the yeast, and the contaminating bacteria (abstract; page 4159, “Materials and methods” section, left col./para 3 – right col./para 3), wherein the wheat mash contains glucose, which is released from wheat starch enzymatically hydrolyzed by a-amylase and glucoamylase (page 4159, right col: para 2/lines 4-6 and 9-10, para 3/lines 4-5). Narendranath et al. also teach a concentration of yeast at 106 CFU/mL (page 4159, right col, line 2) and multiple concentrations of contaminating lactobacilli bacteria, including 105 CFU/mL and 106 CFU/mL (see table 1), which read on the claimed range of “a starting concentration of less than 107 CFU/mL” in the instant claim 1; and that the presence of contaminating bacteria inhibits ethanol production, wherein when yeasts are inoculated at 106 CFU/mL a 2% decrease in ethanol production is observed, and when up to 109 CFU/mL of contaminating bacteria are present a 3.8 – 7.6% reduction in ethanol production is observed (abstract, lines 9-10 and 15-16). Narendranath et al. further teach that bacterial contamination is a major cause of reduction in ethanol production in fermentation by Saccharomyces cerevisiae, and the lactic acid bacteria of genus Lactobacillus are the major concern to distilleries and fuel alcohol plants (page 4158, first para).
Regarding the limitation “a mineral source” in the fermentation broth of the claim 1, the wheat mash in the fermentation broth of Narendranath et al. is a known source of minerals as evidenced by Gramss, who teaches that wheat wort (wheat mash) contains minerals such as Cu, Mn, Ni, and Zn (see Table 2 in page 5). Thus, the fermentation broth of Narendranath meets the limitation about the mineral source.
Regarding the limitation about an initial yeast concentration “at least 50 g/L” of wet weight of yeast, it is equivalent to about 109 CFUs/mL, as evidenced by the disclosure of the specification of the instant application (see page 18, para [0076]). Narendranath et al. teach the fermentation broth contains yeast at a single initial yeast concentration of about 106 CFUs/mL (page 4159/right col/line 2), along with the contaminating bacteria with a variable concentration at about 105, 106, 107, 108, or 109 CFUs/mL (page 4159/left col/last full para). Narendranath et al. do not teach an initial concentration of about 109 CFUs/mL for yeasts. However, Narendranath et al. demonstrated that the more outnumbered the contaminating bacteria are, the more reduced ethanol production is observed (see abstract). One of ordinary of skill in the art would have recognized that increasing an initial concentration of the yeast would facilitate controlling of contaminating bacteria. It is noted that the yeast concentration of Narendranath et al. is readily modified to a higher concentration by routine optimization for controlling of contaminating bacteria. It is well settled that routine optimization is not patentable, even though it results in significant improvement over the prior art (see MPEP 2144.05). Thus, the limitation would be obvious over the cited prior art.
Regarding the limitation “free amino nitrogen” in the fermentation broth of the claim 1, it is noted that the wheat mash in the fermentation broth of Narendranath et al. comprises free amino nitrogen (FAN), which is released from wheat during the process of wheat mashing (see Narendranath et al., page 4159/right col/para 2), as evidenced by the specification of the instant application (page 16, para 0071) and as supported by Thomas et al. (page 2046, right col, para 2, lines 9-11). Thus, the fermentation broth of Narendranath comprises FAN. Regarding the further limitation about the starting concentration 10 – 400 mg/L of free amino nitrogen in the claim 1, Narendranath et al. are silent about the specific concentration. However, it would have been obvious to include FAN of wheat mash at the claimed concentration range in the fermentation broth in the method of Narendranath et al. for ethanol production, because it is well known in the art that wheat mash contains FAN at a concentration of 10 – 400 mg/L, as supported by Thomas et al., who teach that wheat mashes have FAN in a range of 54 mg to 58 mg per liter or at a concentration of 74.4 mg per liter (page 2047: right col, para 3, lines 4-5; page 2048: right col, para 2, lines 8-10), which read on the claimed concentration range. Thomas et al. also teach that brewery wort/mash (prepared from other grains and used for alcohol fermentation) contains FAN in a range of 150 – 300 mg/L (page 2047: right col, para 3, lines 3-4), which reads on the claimed concentration range too.
Narendranath does not teach adding urea to the fermentation broth via fed-batch feeding during fermentation over time, as required by the claim 1.
Yue teaches a method for producing ethanol through yeast fermentation and studying the effect of different nitrogen sources, comprising a step of growing yeast, Saccharomyces cerevisiae, within a fermentation broth comprising: sweet sorghum juice containing glucose, sucrose, and fructose; Urea CO(NH2)2 or ammonium (NH4)2SO4 as a primary nitrogen source; and the yeast (abstract; page 49: left column/paras 2-4), wherein urea was supplemented during the fermentation time and was added initially at the start of fermentation; wherein urea is then supplemented at 3 hours and 8 hours of fermentation time, i.e. via fed-batch feeding; wherein urea or ammonium was added directly to the concentrated sweet sorghum juice at a concentration of 0.8 g nitrogen/L, and the control without adding nitrogen source has an initial nitrogen concentration of 0.07 g/L (page 49/right col/para 3; page 50/right column/section 3.3). Yue et al. further teach that nitrogen source is completely consumed during the first 12 hours, and due to the limitation of nitrogen source the formation of organic acids occurs, consequently pH decreases to 4.6 after the 12 hours’ fermentation; and higher initial concentrations of urea resulted even more ethanol yields (page 50, section 3.3/lines 1-6). Yue et al. continue to teach that adding supplemental urea over the fermentation process (i.e. a fed-batch feeding of urea, such as 3 h and 8 h) to maintain the nitrogen source at a constant level (page 50, section 3.3/lines 8-11), thus preventing the formation of organic acids and maintaining the pH of the fermentation broth. Yue et al. further teach that compared to the free ammonium nitrogen source, the use of urea as a nitrogen source not only promotes the specific growth rate and ability of ethanol tolerance, but also increases the ethanol yield and reduced the formation of byproducts (e.g. glycerol and acetic acid); and Yue et al. concluded that urea is a preferred nitrogen source during ethanol fermentation (abstract, conclusion in page 51).
It would have been obvious to one of ordinary skills in the art to modify the method of Narendranath by supplementing urea via fed-batch feeding over the fermentation time at an appropriate amount for inhibiting formation of organic acid by-products, maintaining pH, and improving ethanol production, as taught by Yue. One of ordinary skills in the art would have been motivated to do so, because Yue et al. teach that adding fed-batch urea prevents the limitation of nitrogen source, which is the cause of forming organic acid by-products, reducing pH and affecting ethanol production. In addition, Yue et al. teach that addition of supplemental urea helps maintaining a viable yeast cell number (see sections 3.1 and 3.3.). Furthermore, the adding fed-batch urea has an additional advantage to the growth of yeasts, in view of that Narendranath et al. teach that production of organic acid by-products (lactic acid) leads to a growing amount of contaminating bacteria, contributing to a decrease of yeast’s growth (see the para spanning pages 4160 and 4161). One of ordinary skill in the art has a reasonable expectation of success at applying the teachings of Yue et al. to modify the method of Narendranath et al., because both Yue et al. and Narendranath et al. are directed to method of alcohol fermentation through Saccharomyces cerevisiae with urea as a primary nitrogen source, and the fed-batch feeding of urea is readily applicable to the method of Narendranath et al.
Regarding the limitation of “a starting concentration of sugars greater than 50 g/L” in the claim 1, Yue et al. teach a starting sugar concentration 300 g/L (page 49/left col/last full para/last 4 lines), reading on the claimed range. Thus, the limitation is obvious over the cited prior art.
Regarding the limitation about a nickel amount “less than 1 mg/kg” recited in the claim 1, Narendranath et al. do not require adding any exogenous nickel to the fermentation broth in their method. Even considering an amount of Nickel unintentionally added to the fermentation broth, nickel is still at a level much less than 1 mg/kg, as evidenced by Gramss, who further teaches that the nickel amount is about 0.013 to 0.27 mg/kg in the wheat wort (i.e. wheat mash). Thus, the fermentation broth using mashed wheat in the method of Narendranath is lower than 1mg/kg, meeting the limitation. Furthermore, it would have been obvious not to add any nickel agent to the fermentation broth and to specifically limit nickel in the fermentation broth to a level of substantially zero or below 1mg/kg in the method of Narendranath for inhibiting growth of contaminating bacteria, thus promoting growth of yeast and increasing ethanol production. This is because Narendranath et al. do not require adding any exogenous nickel to the fermentation broth in their method for growing yeasts for ethanol production; and it is well known in the that the enzyme, urea amidolyase, used by the yeast (S. cerevisiae) for breaking down urea does not need nickel for its catalytic activity, while the urease enzyme used by the contaminating bacteria for breaking down urea needs nickel for its catalytic activity. In support, Kakimoto et al. isolated and examined the urease enzyme of Lactobacillus fermentum (a contaminating bacterium in the method of Narendranath), and teach that this urease enzyme contains nickel and is a nickel metallo-protein (for its catalytic function) and production of the urease enzyme increases in the presence of nickel (abstract, the section “Analysis of nickel” in pages 540-541). Further in support, Strope et al. teach that urea amidolyase in fungus/yeast (including Saccharomyces species, S. cerevisiae) breaks down urea into consumable ammonia as nitrogen source, and urease enzymes are not present in those of the subphylum Saccharomycotina (including Saccharomyces species, S. cerevisiae) (abstract, Table 2), and Strope et al. further teach that there is a selective advantage of using urea amidolyase of Saccharomyces species over using nickel-containing urease (of bacteria), because this allows the Saccharomycetes species to jettison all Ni2+ dependent metabolisms, thus no nickel being needed (page 4, right col, lines 11-17). Furthermore, Narendranath et al. teach that contaminating bacteria of genus Lactobacillus is a major cause of reduction in ethanol production in fermentation by S. cerevisiae, and they are the major concern to distilleries and fuel alcohol plants. As such, one of ordinary skill in the art would expect that a combination of using urea as primary nitrogen source and limiting nickel leads to the inhibition of growth of contaminating Lactobacillus bacteria, and improving ethanol production.
Regarding the recited fermentation time “between two hours and 18 hours” recited in the claim 1, Narendranath et al. does not teach the claimed time range for the fermentation. However, adjustment of the fermentation time is deemed merely a matter of design choice, judicious selection, and routine optimization, which is well within the purview of the skilled artisan having the cited reference before him/her as a guide. The claimed fermentation time range would have been obvious over the cited prior art in the absence of any showing of unexpected results or criticality.
Regarding the limitation of “fed-batch feeding commencing after said yeasts consume more than 90% of said free amino nitrogen” in claim 1, the combined teachings of Narendranath et al. and other cited prior art. suggest a starting concentration 10-400 mg/L of FAN (free amino nitrogen) as indicated above, and they are silent about a specific level of FAN when the fed-batch feeding of urea commences. However, it would have been obvious to start the fed-batch feeding after FAN is completely consumed or almost depleted (e.g. more than 90% is consumed) in the modified method of Narendranath et al. for facilitating control of the bacterial contamination, because urea can only be consumed by the yeast, leading to inhibiting growth of contaminating bacteria, while FAN is a universal nitrogen source for both yeast and contaminating bacteria, as supported by the cited prior art described above. Regarding the further limitation “the pH of said fermentation broth does not rise, due to addition of said urea, during said entirety of said fermentation time” recited in the claim 1, the cited prior art does not teach this limitation. However, the recited limitation is directed to the outcome of the fed-batch feeding under the claimed conditions, i.e. it is directed to what the fed-batch feeding does to the pH of fermentation broth, not to what the step of fed-batch feeding is. The teachings of the cited prior art suggest a step of fed-batch feeding having all the claimed limitations. In the absence of evidence to the contrary, it is presumed that a substantially same step is capable of performing substantially the same function and generating the same outcome.
Narendranath et al. do not teach separating and recycling the yeasts from the fermentation broth, wherein recycled yeasts are not washed with acid and a portion of the recycled yeasts is used in subsequent fermentation. However, it would have been obvious to further modify the method suggested by Narendranath et al. and other cited prior art for separating and recycling yeasts from the fermentation broth and reusing a portion of recycled yeasts in subsequent fermentations for ethanol production, because it is a universal practice in the ethanol industry to recycle yeasts and re-use them in fermentations, and techniques are well established in the art for separating yeasts from contaminating bacteria. In support, Whiting et al. teach that yeast recycling is universally practiced in brewery industries (page 713, left col, para 3, lines 1-2). Whiting et al. further teach centrifuging fermentation broth at different gravities for effectively separating the yeasts (S. cerevisiae) from contaminating bacteria, specifically: first conducting a low-speed centrifugation at 11 x g over the fermentation broth to recover the yeasts and then a high-speed centrifugation at 5100 x g over the yeast-depleted supernatant to recover the contaminating bacteria in pellets (abstract/lines 3-4; page 714/left col/last full para; page 715: left col/para 3, and right col/lines 1-3). Given the process of Whiting et al. do not require washing the separated/recycled yeasts with acid for decontamination, the teachings of the cited prior art meet the requirement of the claimed limitation about not washing the recycled yeasts.
Regarding the additional limitation “the remaining fraction of said recycled yeasts is used as a yeast cream co-product” recited at the end of the claim 1, it is noted that this limitation is very broad because the claim does not recite any limitation to define how this yeast co-product is used. It would have been obvious to one of ordinary skill in the art to use the remaining recycled yeasts as a feedstock and subjecting it to a drying process in the modified method of Narendranath et al. for preparing active dry yeasts for future applications, because it is well known in the art that alive yeasts can be effectively dried and stored in a dried form to be used in fermentation process, as supported by Narendranath et al. (page 4159, left col/last para) and Thomas et al. (page 2046, right col/last para/lines 1-3), who teach using active dry yeasts for starting their fermentation cultures.
Regarding Claims 2-3 and 10, these claims recite product by process limitations because they describes a step(s) involved in producing the sugar solution. The recited sugar solution is not limited to the manipulations of the recited step(s), only to the structure implied by the step(s). See MPEP 2113. Narendranath et al. teach a wheat mash (sugar solution) prepared from wheat by a process comprising grinding, digestion with alpha-amylases and glucoamylases, and filtering (page 4159, right col, paras 2-3).
The wheat mash of Narendranath et al. appears to be structurally the same as that of the claims 2 and 10, so it anticipates the claimed sugar solution. However, even if the wheat mash of Narendranath et al. is not the exact same as the claimed sugar solution, the difference would be obvious because processes of preparing wheat mash is well known in the art and they are readily modified for preparing the claimed sugar solution. Regarding the claim 3, the wheat mash of Narendranath et al. comprises glucose and the sweet sorghum juice of Yue et al. comprises glucose, sucrose, and fructose, which appear to be structurally the same as the claimed sugar solution. Thus, the claims would be obvious over the cited prior art.
Regarding Claim 7, Whiting et al. teach using centrifugation for separating the yeasts from the broth. The claim would be obvious over the cited prior art.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-10 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Narendranath et al. (Applied and Environmental Microbiology, 1997, 63(11): 4158-4163, of record in parent application # 18532043) in view of Yue et al. (Biomass and Bioenergy, 2012, 39:48-52, of record in parent application), Thomas et al. (Applied and Environmental Microbiology, 1990, 56(7): 2046-2050, of record), Kakimoto et al. (Appl Microbiol Biotechnol, 1990, 32:538-543, of record), Strope et al. (BMC Evolutionary Biology, 2011, 11:80, pages 1-15, cited in IDS of parent application), and Whiting et al. (Applied and Environmental Microbiology, 1992, 58(2):713-716, cited in IDS of parent application), as applied to Claims 1-5, 7, and 10, further in view of Burlew et al. (US 2014/0024064, 2014, of record), as evidenced by Gramss (Advances in Nutrition and Food Science, 2020, 2020(5): 1-9; of record in parent application). This rejection is maintained.
The teachings of Narendranath et al. modified by Yue et al., Thomas et al., Kakimoto et al., Strope et al., and Whiting et al. are described above.
Regarding Claim 6, the modified Narendranath et al. do not teach a distillation step for separating ethanol from the fermentation broth.
It would have been obvious to one with ordinary skills in the art to include a distillation step in the modified method of Narendranath for separating ethanol from the fermentation broth and producing ethanol products, because it is common practice in the art to apply distillation for separating ethanol and removing impurities for generating ethanol products in the ethanol fermentation industry, as supported by Burlew et al., who teach a process for production of fermentative ethanol/alcohol in a fermentation broth by using a microorganism such as S. cerevisiae yeast (abstract, paras 0088, 0105, 0213-0214), wherein ethanol/alcohol is separated from the fermentation broth through distillation (para 0105/lines 10-12).
Regarding Claim 8, Narendranath et al. teach separating the sugar solution from solid fraction through filtering a saccharified solution, do not teach subjecting it to centrifugation to produce the sugar solution/supernatant and a co-product cake which is then produced as a dried animal feed product.
It would have been obvious to replace the filtration step with a centrifugation step in the modified method of Narendranath et al. for separating the saccharified solution into the sugar solution and a co-product cake, wherein the co-product cake is further processed into a dried animal feed product, because centrifugation is an art-recognized alternative process of filtration for performing liquid-solid separation, and it is well-known in the art to apply centrifugation to saccharified solutions in fermentation industry for producing a liquid fraction of fermentable sugars and a solid co-product cake, which can be processed into a dried animal feed product. In support, Burlew et al. further teach before fermentation, solids are removed from a mash feedstock by centrifugation, wherein the solids are referred as wet cake (i.e. co-product cake) and it can be further dried to be a dry cake and can be used as animal feed (paras 0073: page 5/last 6 lines and page 6/first line; paras 0113-0114; paras 0200-201, Fig. 4). Regarding the limitation “dried to less than 12 wt%” recited in the claim, the cited art does not teach the claimed range. However, a moisture level in the dried animal feed product in the modified method of Narendranath et al. is readily mortified through routine optimization for increasing stability of the feed product. It is noted that the techniques for drying the co-product cake into animal feeds are well established in the art, as supported by Burlew et al., who teach drying wet cakes through evaporation and remove any free moistures from the cake by using a vacuum source (paras 0549/lines 6-8 from bottom of left col, and 0115/last 8 lines). It would have been obvious to remove any free moisture from co-product cake for producing an animal feed, thus arriving at the claimed moisture range.
Regarding Claim 9, Burlew et al. further teach that solids from whole stillage include crude protein, fatty acids, and fatty acid esters, which may be used, wet or dry, as an animal feed (para 0202), and that thin stillage separated from whole stillage may be combined with cook water for preparing a new batch of fermentable mash (para 0177, lines 10-15 from bottom). In view of Burlew et al., it would have been obvious to mix a portion (thin stillage) of the whole distillation stillage with water to form slurry for preparing a new batch of fermentable mash and using the remainder (concentrated solid) as an animal feed in the modified method of Narendranath et al.
Regarding Claim 17, Yue et al. teach controlling the amounts of carbon source and nitrogen source; and Yue et al. teach that after fermentation, essentially no nitrogen derived from urea is in the fermentation broth (level between 0 and 0.05g/L), and that essentially no urea is left after a 60-hour fermentation time (see figure 3; page 50, right column, section 3.3, lines 1-2). It would have been obvious to control a ratio of carbon source vs. nitrogen source in the modified method of Narendranath et al. for essentially consume all the urea in the fermentation broth, thus making the process of ethanol fermentation cost-effective.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-5, 7, 10, 12 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Narendranath et al. (Applied and Environmental Microbiology, 1997, 63(11): 4158-4163, of record in parent application # 18532043) in view of Yue et al. (Biomass and Bioenergy, 2012, 39:48-52, of record in parent application), Thomas et al. (Applied and Environmental Microbiology, 1990, 56(7): 2046-2050, of record), Kakimoto et al. (Appl Microbiol Biotechnol, 1990, 32:538-543, of record), Strope et al. (BMC Evolutionary Biology, 2011, 11:80, pages 1-15, cited in IDS of parent application), and Whiting et al. (Applied and Environmental Microbiology, 1992, 58(2):713-716, cited in IDS of parent application), as applied to Claims 1-5, 7, and 10, further in view of Wu et al. (US Patent No, 6252016, 2001, of record), as evidenced by Gramss (Advances in Nutrition and Food Science, 2020, 2020(5): 1-9; of record in parent application). This rejection is maintained.
The teachings of Narendranath et al. modified by Yue et al., Thomas et al., Kakimoto et al., Strope et al., and Whiting et al. are described above.
Regarding Claims 12 and 14, the modified Narendranath et al. do not teach a plate heat exchanger comprising stainless grade 316, or a spiral plate heat exchanger comprising stainless grade 316.
It would have been obvious to incorporate a plate heat exchanger or a spiral plate heat exchanger comprising stainless grade 316 plates into the apparatus system in the modified method of Narendranath et al. for performing heat exchange, thus providing a heat source for maintaining a temperature of fermentation broth (i.e. in operable communication with fermentation broth) at a desired level, because it is well known in the art to use these plate heat exchangers for controlling temperatures of a reaction broth in a reactor, as supported by Wu et al., who teach a method of using a plate heat exchanger for performing a polymerization reaction in a reactor, wherein the plate heat exchanger exposes to a temperature control medium and then controls temperatures of reaction broth/mixture in the reactor (abstract; column 3/lines 30-43), wherein the plate heat exchanger comprises heat exchanger plates which serve as heat exchange surfaces and can be made of stainless steel type 316, and the plate heat exchanger is a spiral plate heat exchanger (column 3/lines 52-55 and 57; column 5/lines 3-4, 8-10, and 28-30).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-5, 7, 10 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Narendranath et al. (Applied and Environmental Microbiology, 1997, 63(11): 4158-4163, of record in parent application # 18532043) in view of Yue et al. (Biomass and Bioenergy, 2012, 39:48-52, of record in parent application), Thomas et al. (Applied and Environmental Microbiology, 1990, 56(7): 2046-2050, of record), Kakimoto et al. (Appl Microbiol Biotechnol, 1990, 32:538-543, of record), Strope et al. (BMC Evolutionary Biology, 2011, 11:80, pages 1-15, cited in IDS of parent application), and Whiting et al. (Applied and Environmental Microbiology, 1992, 58(2):713-716, cited in IDS of parent application), as applied to Claims 1-5, 7, and 10, further in view of Verbelen et al. (Appl Microbiol Biotechnol, 2009, 82:1143–1156, of record), as evidenced by Gramss (Advances in Nutrition and Food Science, 2020, 2020(5): 1-9; of record in parent application). This rejection is maintained.
The teachings of Narendranath et al. modified by Yue et al., Thomas et al., Kakimoto et al., Strope et al., and Whiting et al. are described above.
Regarding Claims 18 and 19, the modified Narendranath et al. do not teach that the fermentation broth is oxygenated, wherein the oxygenation is carried out by air sparging.
Verbelen et al. teach that volumetric productivity of ethanol fermentation
process can be increased during high cell density (HCD) brewery fermentation by using a higher inoculum size of yeast (abstract/lines 1-3). Verbelen et al. also teach a process of performing HCD fermentation in combination with oxygenation of wort (i.e. fermentation broth) and yeasts for improving growth yield of yeast (Saccharomyces cerevisiae) during the HCD fermentation (abstract, page 1144/right col/para 3 - page 1145/para 2), wherein the wort/fermentation broth is oxygenated by sparging with air or oxygen (page 1145/left col/lines 4-5, Table 1) and the yeasts are oxygenated with oxygen (the para spanning pages 1144 and 1145). Verbelen et al. further teach that optimization of the oxygen conditions prior to HCD fermentation in their process can efficiently increase the performance and stability of the fermentation, and the wort oxygenation either alone or in combination with yeast preoxygenation gives a high net growth, thus ensuring sustainability of HCD fermentations (page 1156, left col, last para, lines 1-6).
It would have been obvious to modify the method suggested by Narendranath et al. other cited prior art by oxygenating the fermentation broth with air sparging along with HCD fermentations of the yeast for increasing productivity of ethanol fermentation and efficiently increasing the performance and stability of the fermentation, as taught by Verbelen et al. One of ordinary skill in the art would have been motivated to do so, because Verbelen et al. teach that HCD fermentations increase productivity of ethanol fermentation process and oxygenation of fermentation broth efficiently increases the performance and stability of the HCD fermentation. One of ordinary skill in the art has a reasonable expectation of success at applying the teachings of Verbelen et al. to modify the method suggested by Narendranath et al. and other cited prior art, because both are directed to a method of ethanol fermentation by using S. cerevisiae yeast.
Regarding the limitation “sufficient to support aerobic growth of said yeasts” in claim 18, the claim does not define any specific concentration of oxygen in the broth or a specific extend of aerobic growth of yeasts. Given Verbelen et al. teach oxygenating both yeasts and fermentation broth, and their oxygenated conditions meet all the limitations recited in the claim, it is assumed that substantially the same step in the method suggested by the cited prior art is capable of generating the same outcome, i.e. aerobic growth of yeasts.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-5, 7, 10 and 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Narendranath et al. (Applied and Environmental Microbiology, 1997, 63(11): 4158-4163, of record in parent application # 18532043) in view of Yue et al. (Biomass and Bioenergy, 2012, 39:48-52, of record in parent application), Thomas et al. (Applied and Environmental Microbiology, 1990, 56(7): 2046-2050, of record), Kakimoto et al. (Appl Microbiol Biotechnol, 1990, 32:538-543, of record), Strope et al. (BMC Evolutionary Biology, 2011, 11:80, pages 1-15, cited in IDS of parent application), and Whiting et al. (Applied and Environmental Microbiology, 1992, 58(2):713-716, cited in IDS of parent application), as applied to Claims 1-5, 7, and 10, further in view of Suryanarayan et al. (US 6197573, 2001, of record), as evidenced by Gramss (Advances in Nutrition and Food Science, 2020, 2020(5): 1-9; of record in parent application). This rejection is maintained.
The teachings of Narendranath et al. modified by Yue et al., Thomas et al., Kakimoto et al., Strope et al., and Whiting et al. are described above.
Regarding Claims 15 and 16, the modified Narendranath et al. do not teach that the fermentation broth is cooled by evaporative cooling, wherein the air is circulated and evaporates water and reduces temperatures of the broth is sprayed and the air evaporates water from the spray. However, it would have been obvious to cool the fermentation broth by evaporative cooling coupled with water evaporation in the modified method of Narendranath et al. for reducing temperature of the fermentation broth and facilitating the fermentation process, because it is well known in the art that fermenting microorganisms generates heat, which builds up heat inside the bioreactor and is problematic to the fermentation process, and the evaporative cooling is effective at removing heat and reducing temperatures of the broth. In support, Suryanarayan et al. teach that the process of fermenting microorganisms generates heat, the buildup of heat inside the bioreactor is problematic to the fermentation process; and a method of removing heat is evaporative cooling combined with removing water from fermentation broth, which may be achieved by sending air through bioreactor/fermentation broth and venting the air out (i.e. circulating air for evaporating water) the collector plates 24 (col. 12, lines 51-60; Fig. 5).
It is noted that Suryanarayan et al. does not teach spraying the broth. However, it is a routine practice in the art for evaporating water, which would have been obvious to one of ordinary skill in the art.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments
Applicant's arguments about the claim rejections under 35 U.S.C. 103 in the response filed on 02/25/2026 (pages 8-14) have been fully considered but they are not persuasive for the following reasons.
In response to Applicant’s arguments based on urea hydrogen peroxide (UHP) and antibacterial agent H2O2 of Narendranath in page 9/para 3 of the 02/25/2026 response, Examiner notes that Applicant repeats the same arguments as those in Applicant’s previous response filed on 10/29/2025 (in page 10/para 2). These arguments are moot because Applicant mistakenly referred to a reference of Narendranath which is completely different from the prior art of Narendranath et al. cited in the 103 rejections, as explained in the previous office action dated 12/29/2025 (see details in Examiner’s response to arguments in pages 22/para 3 of the previous office action).
Applicant’s arguments based on teachings of Yue in page 9/para 4 – page 10/para 1 of the 02/25/2026 response are not persuasive for the reasons of record (see Examiner’s response to arguments in the paragraph spanning pages 22 and 23 of the previous office action dated 12/29/2025).
Applicant’s arguments based on nickel amounts in fermentation broth in the 02/25/2026 response (page 10, para 2) are not persuasive for the reasons of record (see Examiner’s response to arguments in the paragraph spanning pages 23 and 24 of the previous office action dated 12/29/2025).
Applicant's further arguments based on that the examiner's conclusion of obviousness is based upon improper hindsight reasoning in the 02/25/2026 response (page 10/last para) are not persuasive for the reasons of record (see Examiner’s response to arguments in the paragraph spanning pages 24 and 25 of the previous office action dated 12/29/2025). In view of the combined teachings of Narendranath et al., Kakimoto et al. and Strope et al., the claimed limitations about supplementing urea as a primary nitrogen source and limiting nickel for controlling bacterial contamination during the fermentation time would have been obvious to one of ordinary skill in the art for all the reasons indicated above.
Applicant’s arguments in first para of page 11 in the 02/25/2026 response are based on Declaration under 37 CFR 1.132 by Inventor Hamrick filed on the same day. However, the Hamrick Declaration is insufficient to overcome the 103 rejections of record. It is noted that the declaration provides only arguments, no objective evidence relevant to non-obviousness of the claimed invention. The arguments in Hamrick Declaration (pages 3 and 4) have been fully considered, but they are unpersuasive. Examiner notes that the conclusion of obviousness is established based on whether the combined teachings of prior art references would have suggested to one of ordinary skill in the art the claimed invention before the effective filing date of the claimed invention, not based on whether a single prior art reference (such as Narendranath and Wongsurakul) teaches the claimed invention. Although no single prior art reference teaches the claimed method for controlling bacterial contamination in yeast culture by using urea as a primary nitrogen source and simultaneously limiting nickel in the fermentation broth, the combined teachings of Narendranath, Yue, Thoms, Kakimoto, Strope, and Whiting render the claimed method (claims 1-5, 7 and 10) to be obvious to one of ordinary skill in the art for the reasons described above in the 103 rejection (pages 7-17). Examiner further notes that Inventor Hamrick’s arguments about “whole paper is about peroxides” in page 4/para 1 of the declaration are moot because none of the cited prior art references is related to using peroxidases for reducing bacterial contaminations. The declaration referred to a wrong prior art reference. In response to Applicant’s arguments based on a “long-felt need” in page 11/para 1 of the response, MPEP 716.04 provides guidance on Long-Felt Need: “Establishing long-felt need requires objective evidence that an art recognized problem existed in the art for a long period of time without solution. The relevance of long-felt need and the failure of others to the issue of...”. Industrial fermentation companies currently use antibacterial agents such as antibiotics and modified urea (UHP) to boost specific growth rates of yeasts and inhibit growth of contaminated bacteria, and also use filtration methods to get rid of contamination. There is no objective evidence to support the bacterial contamination problem is only solved by the claimed invention, but not solved by others. In view of the foregoing, when all of the evidence is considered, the totality of the rebuttal evidence of nonobviousness fails to outweigh the evidence of obviousness.
With regard to Applicant’s arguments in paras 2-3 of page 11 of the response, these arguments are not persuasive for the reasons of record (see Examiner’s response to arguments in page 25/para 2 of the previous office action dated 12/29/2025). Examiner further notes that the technical features listed under (a), (b), and (c) in page 11 of the response are not Applicant’s invention or findings, rather these features are well known in the art, as supported by the prior art references of Kakimoto et al. and Strope et al. cited in the 103 rejections above, who teach: (a) yeasts such as S. cerevisiae contain Ni-independent urea amidolyase enzyme; (b) contaminating bacteria such as L. fermentum contain Ni-dependent urease enzyme, not the urea amidolyase enzyme, and (c) bacteria that can grow on urea have the Ni-dependent urease enzyme, which requires nickel as the cofactor. The combined teachings of Narendranath et al., Yue, Thomas et al., Kakimoto et al. and Strope et al. suggests the claimed technical features including: controlling bacterial contamination in yeast culture by limiting nickel below 1 mg/kg and using urea as primary nitrogen source which is added to fermentation broth via fed-batch feeding without increasing the pH of the broth during the entire fermentation, for the reasons indicated in the 103 rejections above.
With regard to Applicant’s arguments in the paragraph spanning pages 11 and 12 of the response, these arguments are not persuasive for the reasons of record (see Examiner’s response to arguments in the paragraph spanning pages 25 and 26 of the previous office action dated 12/29/2025). Examiner further notes that it is known in the art that pH of a culture medium can be prevented from rising and maintained to be pH-neutral by controlling an amount of urea added to the medium such that ammonia released from urea is exactly balanced by its assimilation into yeast cells, as evidenced by Hensing et al. (Appl. Microbiol. Biotechnol, 1995, 43:58-64), see page 63/right col/para 3/last 5 lines. Applicant failed to provide any factual evidence (experimental data) to support the arguments based on the “critical feature” in the claimed method. There is no factual evidence to support that the claimed limitations (adding urea via fed-batch feeding and controlling the fed-batch feeding such that pH is not increased due to urea addition over entire fermentation) are critical for controlling bacterial contamination in the claimed method.
With regard to Applicant’s arguments in page 12, para 2 of the response, these arguments are not persuasive for the reasons of record (see Examiner’s response to arguments in page 26 of the previous office action dated 12/29/2025). Examiner notes that the prior art cited in the 103 rejections suggest a method for controlling bacterial contamination by supplying urea to yeast as a nitrogen source in the absence of nickel, as indicated above. The absence of nickel suggested by the cited prior art reads on the claimed nickel amount “less than 1 mg/kg”. Thus, the claimed range of less than 1 mg/kg has no novelty.
In response to Applicant’ arguments based on the reference of Wongsurakul et al. in the 02/25/2026 response (the para spanning pages 12 and 13), it is noted that no teachings about the claimed invention in Wongsurakul et al. does not change the fact that the claimed invention would have been obvious to one of ordinary skill in the art over the combined teachings of the cited prior art. With regard to the use of nickel at a concentration higher than 185 ppm for inhibiting yeast taught by Wongsurakul et al., this use does not support the novelty of the claimed invention for the reasons of record (see Examiner’s response to arguments in the paragraph spanning pages 26 and 27 of the previous office action dated 12/29/2025).
In response to Applicant’ argument that the examiner has combined a large number of references in the 103 rejections in the 02/25/2026 response (page 14, first para), reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991). MPEP 2145(V).
Overall, The conclusion of the obviousness of the claims 1-10, 12, and 14-19 has been established for all the reasons indicated above.
Conclusion
No claim is in condition for allowance.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Qing Xu, Ph.D., whose telephone number is (571) 272-3076. The examiner can normally be reached on Monday-Friday from 9:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao, can be reached at (571) 272-0939. Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to the receptionist whose telephone number is (571) 272-1600.
/Qing Xu/
Patent Examiner
Art Unit 1656