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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/29/2025 has been entered.
Claim Status
The amendment of 12/29/2025 has been entered. Claims 38, 41-43, 46-49, 51, 53-63 are pending (claim set as filed on 12/29/2025).
Applicant previously elected invention Group II, drawn to a method of decreasing and/or controlling the amount of inorganic nitrogen compounds in an aqueous system, and of the pathogen species Streptococcus agalactiae, inorganic nitrogen species nitrite, the Bacillus strain species DSM 33349, the 16S rDNA species SEQ ID NO: 1, the strain characteristics a) an ability to grow in 2 mM bile, b) an ability to grow anaerobically, c) an ability to grow in the presence of 1 wt. % of NaCl for at least one day in claim 48, and of the animal species Crustacean, in the reply filed on 01/06/2025.
New claim 58 defines a mutant that has not been previously elected. As such, new claims 58-63 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 38, 41-43, 46-49, 51, and 53-57 are currently under examination and were examined on their merits.
Claim Rejections - 35 USC § 112 (b)
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.
Claim 38, 41-43, 46-49, 51, and 53-57 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.
Claims 38 and 57 recite “wherein the strain or preparation […] inhibits the growth of at least one pathogen in the aqueous system, wherein: […] spores of the Bacillus strain germinate within not more than 30 hours in an environment in which the amount of carbon sources is below 50 g per kg”, which renders the claims indefinite, because it is unclear if “an environment” recited in line 13 of claim 38 and in line 10 of claim 57 is referring to the aqueous system or is broader to include any environment.
One of ordinary skill in the art would not be able to determine the metes and bounds of claims 38 and 57, and thus could not clearly determine how to avoid infringement of the claims.
In the interest of compact prosecution, claims 38 and 57 are interpreted to the broadest embodiment claimed.
Dependent claims 41-43, 46-49, 51, and 53-56 are rejected since they do not clarify the indefinite language of claim 38.
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.
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 38, 41-43, 46-49, 51, 53, 55-57 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (CN 106906169 A, published on 06/30/2017), hereinafter ‘Wang’, as evidenced by Liu et al. (“Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus”, published on 12/02/2016, Fish & Shellfish Immunology, Vol. 60 (2017), pages 326-333), hereinafter ‘Liu’, as evidenced by Setlow (“Germination of Spores of Bacillus Species: What We Know and Do Not Know”, published on 01/31/2014, Journal of Bacteriology, Vol. 196, No. 7, pages 1297-1305), hereinafter ‘Setlow’, as evidenced by as evidenced by Chandler et al. (“Pollution monitoring with total organic carbon analysis”, published in 1976, Journal (Water Pollution Control Federation), Vol. 48, No. 12, pages 2791-2803), hereinafter ‘Chandler’, as evidenced by Shifeng (“Bacillus sp. (in: firmicutes) 16S ribosomal RNA gene, partial sequence”, published on 12/04/2016, NCBI, pages 1-2), hereinafter ‘Shifeng’, as evidenced by Ye et al. (“Global Gene Expression Profiles of Bacillus subtilis Grown under Anaerobic Conditions”; published in Aug 2000, Journal of Bacteriology, Vol. 182, No. 16, pages 4458-4465), as evidenced by Han et al. (“Strain-Specific Benefits of Bacillus on Growth, Intestinal Health, Immune Modulation, and Ammonia-Nitrogen Stress Resilience in Hybrid Grouper”, published on 03/06/2024, Antioxidants, Vol. 13, 317, pages 1-20), as evidenced by Liu et al. (“Purification of aquaculture effluent by Bacillus subtilis HAINUP40”, published on 05/16/2018, Fisheries Science (Dalian), Vol. 37, No. 2, abstract, one page), hereinafter ‘Liu et al. 2018’, as evidenced by MacWilliams et al. (“Luria Broth (LB) and Luria Agar (LA) Media and Their Uses Protocol”, published in 2016, retrieved from https://asm.org/protocols/luria-broth-lb-and-luria-agar-la-media-and-their-u, pages 1-4), hereinafter ‘MacWilliams’, as evidenced by Bio Basic (“Product Specification”, published on 08/12/2024, retrieved from https://web.archive.org/web/20240812082551/https://biobasic-asia.com/wp-content/uploads/2023/01/Product-Spec-Marine-Broth-2216E.pdf, 2 pages), hereinafter ‘Bio Basic’, and as evidenced by Pelzer et al. (WO 2019/002471 A1, published on 01/03/2019), hereinafter ‘Pelzer’.
Wang’s general disclosure relates to “the technical field of fish breeding, and in particular relates to a Bacillus subtilis HAINUP40 and an application thereof” (description, paragraph [0002]).
Regarding claims 38, 41-43, 46-49, 51, and 53, 55-57, please note the 112b rejection above.
Regarding claims 38 and 57, please note the elected the Bacillus strain species DSM 33349 under Claim Status above.
Pertaining to a method of decreasing/or controlling the amount of inorganic nitrogen compounds in an aqueous system, Wang teaches a method of decreasing and/or controlling the amount of inorganic nitrogen compounds in an aqueous system (description, paragraphs [0002], [0017], [0031]-[0032]), the method comprising supplying the aqueous system with a Bacillus strain or a preparation thereof (description, paragraph [0008]; see abstract), wherein the strain degrades at least one inorganic nitrogen compound (description, paragraphs [0017], [0031]-[0032]; see abstract) and inhibits the growth of at least one pathogen in the aqueous system (description, paragraphs [0010], [0017]; see abstract); and wherein the aqueous system is rearing water (description, paragraphs [0016], [0032]). The ability of Wang’s Bacillus strain Hainup40 to inhibit S. agalactiae in rearing water is further evidenced by Liu (page 330, paragraph 4; see Fig. 4).
Pertaining to germination of spores of the Bacillus strain, Bacillus strains are known to germinate within minutes after exposure to specific nutrients, as evidenced by Setlow (see abstract), wherein nutrients triggering Bacillus subtilis germination include L-alanine, L-valine, and L-asparagine (Setlow, page 1298, right column, paragraph 1). Since Wang teaches wherein the Bacillus strain is a Bacillus subtilis strain (description, see paragraph [0002]), it is the Examiner’s position that Wang’s Bacillus strain germinates within not more than 30 hours in an environment in which the amount of carbon sources is below 50 g per kg.
Regarding claims 38 and 53, pertaining to the C:N ratio, Wang teaches degradation of ammonia while the aqueous system comprises a COD/N ratio of 469.33 mg/L / 21.71 nitrogen mg/L (description, paragraph [0031]; note, 26.4 mg/L ammonia corresponds to about 21.7 mg/L nitrogen), resulting in a COD/N ratio of 21.61. It is noted that the COD can be converted to C by dividing by the conversion factor of 2.67 based on the molecular ratio of oxygen to carbon, as evidenced by Chandler (page 2793, left column, paragraph 7), thereby resulting in a C:N ratio of about 8. Wang further teaches degradation of nitrite wherein the aqueous system comprises a COD:N ratio of 488 mg/L / 0.052 mg/L of nitrogen (description, paragraph [0031]; note, 0.17 mg/L nitrite corresponds to 0.052 mg/L of nitrogen) resulting in a COD:N ratio of about 9385 which corresponds to a C:N ratio of about 3515.
Regarding claim 41, pertaining to the at least one pathogen, Wang teaches wherein the at least one pathogen is a pathogen of an aquatic animal (“tilapia-derived Streptococcus agalactiae”; description, paragraphs [0010], [0017]).
Regarding claim 42, please note the elected pathogen species Streptococcus agalactiae under Claim Status above.
Pertaining to the pathogen, Wang teaches wherein the pathogen is the elected Streptococcus agalactiae (description, paragraphs [0010]).
Regarding claims 43 and 51, please note the elected nitrogen species nitrite under Claim Status above.
Pertaining to the elected inorganic nitrogen compound nitrite, Wang teaches wherein the at least one inorganic nitrogen compound is nitrite (description, paragraphs [0031]-[0032]; see abstract).
Regarding claims 46 and 47, please note the elected 16S rDNA species SEQ ID NO: 1 under Claim Status above.
Pertaining to the elected 16S rDNA sequence, Wang teaches wherein the Bacillus strain has a 16S rDNA sequence, wherein said sequence has a sequence identity of 99.8% to instant sequence SEQ ID NO: 1 (“KY244143.1”; description, paragraph [0023]), as evidenced by Shifeng (“Bacillus sp. (in: firmicutes) 16S ribosomal RNA gene, partial sequence”, published on 12/04/2016, NCBI, pages 1-2; see alignment of instant SEQ ID NO: 1 with sequence KY244143.1 below). The Examiner notes that the 16S rDNA sequence of Wang’s strain has 99.79 % identity with instant SEQ ID NO: 1, corresponding to 99.8 % identity recited in instant claim 47.
RID: X5XDHCEJ114
Job Title:SEQ ID NO 1
Program: BLASTN
Query: SEQ ID NO 1 ID: lcl|Query_551315(dna) Length: 1457
Subject:Bacillus sp. (in: Bacteria) 16S ribosomal RNA gene, partial sequence ID: KY244143.1(nucleic acid) Length: 1514
Sequences producing significant alignments:
Scientific Common Max Total Query E Per. Acc.
Description Name Name Taxid Score Score cover Value Ident Len Accession
Bacillus sp. (in: Bacteria) 16S ribosomal RNA gene, partial... Bacillus sp.... NA 1409 2610 2610 98% 0.0 99.79 1514 KY244143.1
Alignments:
>Bacillus sp. (in: Bacteria) 16S ribosomal RNA gene, partial sequence
Sequence ID: KY244143.1 Length: 1514
Range 1: 88 to 1509
Score:2610 bits(1413), Expect:0.0,
Identities:1419/1422(99%), Gaps:0/1422(0%), Strand: Plus/Plus
Query 1 GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 88 GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCC 147
Query 61 GGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTG 120
||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||
Sbjct 148 GGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTG 207
Query 121 GCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC 180
|||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||
Sbjct 208 GCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGC 267
Query 181 TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 268 TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA 327
Query 241 CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 328 CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT 387
Query 301 GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 388 GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGG 447
Query 361 AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 448 AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGG 507
Query 421 CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 508 CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG 567
Query 481 GGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 568 GGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGG 627
Query 541 GGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGT 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 628 GGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGT 687
Query 601 AGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 688 AGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTG 747
Query 661 TAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 748 TAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC 807
Query 721 ACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 808 ACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA 867
Query 781 CGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACG 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 868 CGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACG 927
Query 841 GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 928 GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC 987
Query 901 AGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGAC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 988 AGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGAC 1047
Query 961 AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1048 AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA 1107
Query 1021 GCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1108 GCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTG 1167
Query 1081 ACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1168 ACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC 1227
Query 1141 ACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCAC 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1228 ACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCAC 1287
Query 1201 AAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTA 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1288 AAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTA 1347
Query 1261 GTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1348 GTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT 1407
Query 1321 CACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGC 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1408 CACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGC 1467
Query 1381 CGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTA 1422
||||||||||||||||||||||||||||||||||| ||||||
Sbjct 1468 CGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAAAAAGGTA 1509
Regarding claims 48-49, please note the elected strain characteristics a), b) and c) under Claim Status above.
Pertaining to the elected ability of the Bacillus strain to grow anaerobically (reading on (a)), Wang teaches wherein the Bacillus strain is Bacillus subtilis (description, paragraph [0002]), which is known as a facultative anaerobe bacterium with the ability to grow anaerobically, as evidenced by Ye (page 4458, left column, paragraph 1).
Pertaining to wherein the Bacillus strain is characterized by an enzymatic activity (reading on (d)), Wang teaches wherein the Bacillus strain has protease activity (description, paragraphs [0008], [0017]; see abstract).
Regarding claims 48-49 and 56, pertaining to the elected ability of the Bacillus strain to grow in the presence of 1 wt-% of NaCl for at least one day (instant claims 48-49, reading on (b)), and to the ability of the Bacillus strain to grow in the presence of 1.5 wt-% of NaCl (instant claim 56), Wang teaches Bacillus subtilis HAINUP40 (description, paragraph [0002]), which has the ability to grow in Luriae Bertani (LB) medium for 24 h, as evidenced by Han (page 3, paragraph 4), and further grows in 2216E medium, as evidenced by Liu 2018 (see abstract). The Examiner notes that LB medium contains 1 wt -% of NaCl, as evidenced by MacWilliams (page 2, paragraph 1), and that 2216E medium contains about 1.9 wt-% of NaCl, as evidenced by Bio Basic (see components on page 1). Thus, the evidentiary references indicate that Wang’s Bacillus has the ability to grow in 1 wt.-% of NaCl for at least one day, and further has the ability to grow in the presence of 1.5 wt.-% of NaCl.
Regarding claims 48-49 and 55, pertaining to the elected ability of the Bacillus strain to grow in the presence of 0.3 wt.-% porcine bile and/or in the presence of 0.3 wt.-% chicken bile (instant claims 48-49, reading on (e), and instant claim 55), Wang teaches Bacillus subtilis strain HAINUP40 (description, paragraph [0002]), which is considered able to grow in the presence of 0.3 wt.-% porcine bile and/or in the presence of 0.3 wt.-% chicken bile, since Wang’s strain HAINUP40 grows in the presence of 0.3 % (w/v) Oxgall, as evidenced by Liu (page 327, left column, paragraph 3; see abstract and Table 1, note the slight increase of viable cell counts in simulated intestinal fluid (SIF) at pH 6.8 within 4 h), and since Bacillus subtilis has been shown to have the ability to grow in presence of 0.3 wt.-% porcine bile and in presence of 0.3 wt.-% chicken bile, as evidenced by Pelzer (page 19, lines 33-37).
Wang does not teach wherein the Bacillus strain degrades nitrate, nitrite and/or ammonia while the aqueous system has a carbon to nitrogen (C:N) ratio of between 30 and 90 (instant claims 38 and 57), wherein the C:N ratio is between 40 and 80 (instant claim 53).
While Wang does not teach wherein the Bacillus strain degrades nitrate, nitrite and/or ammonia while the aqueous system has a carbon to nitrogen (C:N) ratio of between 30 and 90 (instant claims 38 and 57), wherein the C:N ratio is between 40 and 80 (instant claim 53), the instantly recited C:N ratios would be within the realm of routine experimentation since Wang teaches C:N ratios of about 8 and 3515. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine the optimal C:N ratios for maximal degradation of inorganic nitrogen compounds. Further, one would expect success since Wang’s teachings are directed to different inorganic nitrogen compounds (description, paragraph [0031]), and therefore, manipulation of the C:N ratio based on the teachings of the reference would be within the purview of an artisan. Generally, differences in concentration will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA1955). See MPEP § 2144.05 part II A.
Wang does not expressly teach wherein the Bacillus strain is the elected DSM 33349 or a preparation thereof (instant claim 38), and wherein the Bacillus strain is a mutant of the elected strain DSM 33349 with a sequence identity of at least 95%, or a preparation thereof (instant claim 57).
However, based on Wang's teachings, it is highly likely that Wang's strain and Applicant's strain are the same strain since both strains are Bacillus subtilis species (specification; page 1, lines 28-30; Wang, description, paragraph [0004]), and both degrade nitrite and ammonia and inhibit the growth of Streptococcus agalactiae, as discussed above. Thus, it is highly likely that Wang’s and Applicant’s strain are the same.
Still, if there should be a slight variation between Wang's strain and the instantly recited
strain, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have used the instant strain in lieu of Wang’s strain since both strains belong to the species Bacillus subtilis, and share the ability to degrade nitrite and ammonia and inhibit Streptococcus agalactiae.
Pertaining to a preparation of the elected Bacillus, a mutant of the elected Bacillus and a preparation thereof, Wang’s strain reads on the embodiment ‘preparation thereof’ (instant claim 38), and on ‘mutant’ of the elected Bacillus strain and ‘preparation thereof’ (instant claim 57). ‘Preparation’ according to the specification encompasses “active compounds at least one metabolite, preferably a mixture of metabolites, as further described below, and/or at least one enzyme selected from proteases, in particular subtilisin, xylanases and/or cellulases, and/or at least one peptide, and/or combinations thereof” (specification, page 24, lines 27-30). It is highly likely that Wang’s Bacillus strain produces peptides, metabolites, or enzymes identical with peptides, metabolites, or enzymes comprised in the instant preparations derived from the instant strain or its mutant, since Wang’s strain and the instant strain are both able to degrade nitrite and ammonia and inhibit the growth of Streptococcus agalactiae, as discussed above, and Wang’s strain produces extracellular enzyme activities (description, paragraph [0017]; see abstract).
The claimed mutants of the instant application are described as having a sequence identity of 95% with the elected Bacillus strain DSM 33349. The specification teaches wherein the sequence identity of the mutant relates to the genomic sequence of the instant strain (page 6, lines 21-25). However, no genomic sequence and associated SEQ ID NO is provided in the instant application (see under Claim Rejections - 35 USC § 112). The specification describes: “In a preferred embodiment of the invention, the mutants and variants of the strains DSM 33349, …, have the same identifying characteristics like the parent strain” (page 10, lines 13-15). It is noted, that no specific mutants are described in the specification. Therefore, Wang’s Bacillus strain anticipates the claimed mutants and preparations since Wang’s strain and the instant strain are both Bacillus subtilis, share the ability to degrade nitrite and ammonia, and are able to inhibit the growth of the pathogen Streptococcus agalactiae, as discussed above.
Still, Wang does not specify the Bacillus strain is the claimed elected strain or its mutant. However, if there should be a slight variation between Wang’s strain and the mutant of the instant strain, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have used the instant mutant to degrade nitrite or ammonia in Wang’s method, since both, Wang’s strain and the instant strain degrade nitrite and ammonia and inhibit the growth of the pathogen Streptococcus agalactiae, as discussed above.
Claims 38 and 54 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (CN 106906169 A, published on 06/30/2017), hereinafter ‘Wang’, as evidenced by Liu et al. (“Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus”, published on 12/02/2016, Fish & Shellfish Immunology, Vol. 60 (2017), pages 326-333), hereinafter ‘Liu’, as evidenced by Setlow et al. (“Germination of Spores of Bacillus Species: What We Know and Do Not Know”, published on 01/31/2014, Journal of Bacteriology, Vol. 196, No. 7, pages 1297-1305), hereinafter ‘Setlow’, as evidenced by Chandler et al. (“Pollution monitoring with total organic carbon analysis”, published in 1976, Journal (Water Pollution Control Federation), Vol. 48, No. 12, pages 2791-2803), hereinafter ‘Chandler’, as evidenced by Shifeng et al. (“Bacillus sp. (in: firmicutes) 16S ribosomal RNA gene, partial sequence”, published on 12/04/2016, NCBI, pages 1-2), hereinafter ‘Shifeng’, as evidenced by Ye et al. (“Global Gene Expression Profiles of Bacillus subtilis Grown under Anaerobic Conditions”; published in Aug 2000, Journal of Bacteriology, Vol. 182, No. 16, pages 4458-4465), as evidenced by Han et al. (“Strain-Specific Benefits of Bacillus on Growth, Intestinal Health, Immune Modulation, and Ammonia-Nitrogen Stress Resilience in Hybrid Grouper”, published on 03/06/2024, Antioxidants, Vol. 13, 317, pages 1-20), as evidenced by Liu et al. (“Purification of aquaculture effluent by Bacillus subtilis HAINUP40”, published on 05/16/2018, Fisheries Science (Dalian), Vol. 37, No. 2, abstract, one page), hereinafter ‘Liu 2018’, as evidenced by MacWilliams et al. (“Luria Broth (LB) and Luria Agar (LA) Media and Their Uses Protocol”, published in 2016, retrieved from https://asm.org/protocols/luria-broth-lb-and-luria-agar-la-media-and-their-u, pages 1-4), hereinafter ‘MacWilliams’, as evidenced by Bio Basic (“Product Specification”, published on 08/12/2024, retrieved from https://web.archive.org/web/20240812082551/https://biobasic-asia.com/wp-content/uploads/2023/01/Product-Spec-Marine-Broth-2216E.pdf, 2 pages), hereinafter ‘Bio Basic’, and as evidenced by Pelzer et al. (WO 2019/002471 A1, published on 01/03/2019), hereinafter ‘Pelzer’, in view of Xia et al. (“Isolation and characterization of a Bacillus subtilis strain with aflatoxin B1 biodegradation capability”, published on 12/26/2016, Food Control, Vol. 75 (2017), pages 92-98), hereinafter ‘Xia’, and in view of Cho et al. (“In vitro degradation of zearalenone by Bacillus subtilis”, published on 08/10/2010, Biotechnol Lett, Vol. 32, pages 1921–1924), hereinafter ‘Cho’.
Modified Wang’s teachings have been set forth above.
In addition, Wang teaches wherein the Bacillus strain “has strong extracellular protease and extracellular amylase activity, which can significantly promote the growth of farmed animals” (description, paragraph [0032]).
Wang does not teach wherein the Bacillus strain degrades mycotoxins (instant claim 54).
Xia’s general disclosure relates to “a bacterial strain capable of degrading aflatoxin B1 (AFB1)” (see entire document, including abstract).
Regarding claim 54, please note the 112b rejection above.
Pertaining to Bacillus degrading mycotoxins, Xia teaches wherein a Bacillus subtilis strain degrades the mycotoxin aflatoxin B1 (see abstract), and wherein “[t]he aflatoxin B1-degrading activity of isolate JSW-1 was predominantly attributed to the cell-free supernatant and this activity was found to be heat stable but sensitive to proteinase K treatment, indicating that the extracellular proteins or enzymes are responsible for the AFB1 degradation” (see abstract).
In addition, Xia teaches wherein “Aflatoxins are type of mycotoxins mainly produced by Aspergillus flavus and a common contaminant of food and grain, posing a serious economic and health problem worldwide.” (see abstract).
Cho’s general disclosure relates to the degradation of the mycotoxin zearalenone by Bacillus subtilis (see entire document, including abstract).
Regarding claim 54, pertaining to the bacteria degrading mycotoxins, Cho teaches wherein B. subtilis completely degrades Zearalenone (ZEN) (page 1923, left column, paragraph 4), and that some degradation activity could be found in the supernatant of the culture (page 1923, left column, paragraph 5; see Fig. 3).
Cho further discloses wherein “Zearalenone (ZEN) is a non-steroidal estrogen produced by many Fusarium species in cereals and other plants, and is frequently implicated in safety of foods and feeds” (see abstract).
While Wang does not teach wherein the Bacillus degrades mycotoxins (instant claim 54), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined Wang’s teachings with Xia’s and Cho’s teachings, to have expected that Wang’s strain degrades mycotoxins. One would have been motivated to do so since Wang’s strain produces strong extracellular enzyme activity and promotes growth of farmed animals (see above), and Xia and Cho disclose mycotoxin degradation by B. subtilis that is linked to extracellular activity produced by the strain. Based on Wang’s, Xia’s and Cho’s teachings it is highly likely that Wang’s strain degrades mycotoxins since all references are directed to Bacillus subtilis and to extracellular activity produced by B. subtilis.
Response to Arguments
Applicant has traversed the previous rejections under 35 U.S.C. 102/103 and under 35 U.S.C. 103 in the reply filed on 12/29/2025 (remarks, pages 8-14). Wang, Xia, and Cho are still relied upon in the above rejections. Applicant's arguments have been fully considered but they are not persuasive.
In Applicant’s reply, Applicant states that “neither Wang nor Crab, alone or in combination, discloses or suggests degradation of inorganic nitrogen compounds under the claimed C:N conditions”, and states that “COD values reported in Wang are not equivalent to carbon concentration and cannot be used to infer a C:N ratio” (remarks, page 11).
The Examiner responds that C:N ratios can be estimated from the COD and inorganic nitrogen concentrations provided by Wang, as discussed above. Since Wang’s strain decreases inorganic nitrogen in the presence of a low C:N ratio (about 8) and in the presence of a high C:N ratio (about 3515), it is highly likely that Wang’s strain degrades inorganic nitrogen in presence of the claimed C:N ratios, even if there should be slight differences between the estimated and actual C:N ratios.
Applicant describes that “Wang does not describe spore germination behavior of HAINUP40, does not disclose germination timing, and does not describe germination under carbon-limited conditions or in rearing water” (remarks, page 12).
The Examiner responds that Bacillus strains are generally known to have the ability to germinate within minutes, and Bacillus subtilis germination is triggered by amino acids, as discussed above. As such, it is highly likely that Wang’s strain germinates within not more than 30 hours in an environment in which the amount of carbon is below 50 g per kg, wherein the aqueous system is rearing water.
Applicant states that Wang “does not disclose degradation of any mycotoxin and does not identify any mycotoxin-degrading enzyme or activity”, and that “the cited references do not establish a biochemical or functional relationship between the extracellular activities disclosed in Wang and the mycotoxin-specific degradation activities disclosed in Xia and Cho” (remarks, page 13).
The Examiner notes that the instant specification does not provide evidence that Applicant’s Bacillus degrades mycotoxins. Applicant describes that the instant Bacillus secretes metabolites, enzymes and/or peptides into the surrounding medium (specification, page 24, lines 9-11). As such, since Wang’s Bacillus produces extracellular activity which promotes growth of farmed animals (description, paragraph [0032]), and extracellular activity has been linked to mycotoxin degradation by Bacillus subtilis, as discussed above, it is highly likely that Wang’s strain degrades mycotoxin.
Conclusion
No claims are allowed.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANDRA ZINGARELLI whose telephone number is (703)756-1799. The examiner can normally be reached M-F 9-5.
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/SANDRA ZINGARELLI/ Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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