Antarctic bacteria strain with organic matter decomposition and nitrogen removal function and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to an Antarctic strain with organic matter decomposition and metabolism and nitrogen removal functions and application thereof. Serratia proteamaculans The Antarctic strain is classified and named as CLB_ANTARCTIC_006 PLUS, and has an organic matter decomposition and metabolism and nitrogen removal activity in a 4-30 DEG C saline water and / or fresh water environment, is beneficial to degradation and utilization of organic matters in a low-temperature environment and nitrogen pollution removal, realizes collaborative treatment of solid organic waste and nitrogen-containing wastewater, and has application prospects in resource utilization of agricultural and forestry and breeding waste and biological remediation in a low-temperature environment in cold regions.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Antarctic strains with organic matter decomposition and metabolism and nitrogen removal functions and their applications. Background Technology

[0002] With global population growth and the intensification of agricultural development, the annual output of agricultural solid waste (such as crop straw and sawdust) is constantly increasing. These wastes are rich in recalcitrant components such as lignocellulose, which decompose slowly in the natural environment. Long-term accumulation not only occupies land resources but can also cause soil and air pollution through infiltration and volatilization. Simultaneously, in agricultural production and aquaculture activities, the degradation process of straw is often accompanied by the release and transformation of nitrogen, leading to the accumulation of nitrogenous pollutants such as ammonia nitrogen, nitrates, and nitrites in water bodies. This further exacerbates ecological risks such as eutrophication, hypoxia, and biotoxicity, threatening the balance of aquatic ecosystems and human water security.

[0003] Microbial degradation technology, due to its low cost and environmental friendliness, has become an important means to promote the resource utilization of agricultural waste and the treatment of water pollution. Through metabolism, microorganisms can not only degrade complex organic matter such as lignin, but also participate in nitrogen conversion processes, achieving the synergistic treatment of complex organic waste and nitrogen-containing pollutants. However, in high-latitude or low-temperature winter environments, the metabolic activity of microorganisms decreases significantly, and the enzyme catalytic efficiency decreases, leading to a slowdown in lignin degradation and hindering nitrogen conversion processes, thus severely limiting its practical application in cold environments. These effects result in a prolonged degradation cycle for complex polymers such as lignin and a significant decrease in the removal efficiency of nitrogen-containing pollutants (ammonia nitrogen, nitrate, and nitrite) in wastewater, leading to practical problems such as insufficient composting maturity of agricultural solid waste in winter and low operating efficiency of wastewater treatment plants in winter (e.g., in Northeast China). Summary of the Invention

[0004] This study isolated a cold-resistant strain of Serratia crus-galli, CLB_ANTARCTIC_006 PLUS, from the intestine of Trematomus bernacchii. This strain grows well in saline and / or freshwater environments at temperatures ranging from 4 ℃ to 30 ℃ and exhibits multi-type organic matter decomposition and nitrogen removal activities. Its cold-resistant characteristics, such as below 15 ℃, especially not higher than 10 ℃, and even as low as 4 ℃, provide feasibility for the degradation and utilization of various organic matter in low-temperature environments and the removal of nitrogen pollution, demonstrating great application potential in the resource utilization of organic waste and environmental bioremediation.

[0005] This invention provides an Antarctic strain with functions of organic matter decomposition and nitrogen removal, and the Antarctic strain is classified and named as follows: Serratia proteamaculansCLB_ANTARCTIC_006 PLUS, with accession number CCTCC NO: M20261129; the Antarctic strain described therein exhibits organic matter decomposition and metabolism and nitrogen removal activities in saline and / or freshwater environments at 4 ℃-30 ℃, and also has high lignin peroxidase activity.

[0006] The organic compound is selected from at least one of lignin, benzoic acid, peptone, alcohols, acetic acid, D-amino acids, vegetable oil, and diesel oil; wherein, The benzoic acid is selected from at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, 4-hydroxybenzoate, 3-hydroxybenzoic acid, and 3-hydroxybenzoate, preferably at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, and 4-hydroxybenzoate; The peptone is trypsin; The alcohol is glycerol; The acetic acid compounds are selected from acetic acid and / or acetates; The D-amino acid is selected from at least one of D-phenylalanine, D-alanine, D-serine, and D-glutamine, preferably at least one of D-alanine, D-serine, and D-glutamine; The vegetable oil is selected from at least one of soybean oil, flaxseed oil, and sunflower seed oil, preferably soybean oil and / or sunflower seed oil.

[0007] The nitrogen source is inorganic nitrogen, selected from at least one of ammonia nitrogen, nitrate, and nitrite.

[0008] This invention provides a product of an Antarctic strain, which is a culture or fermentation product of the Antarctic strain with organic matter decomposition and metabolism and nitrogen removal functions described above.

[0009] The present invention provides a microbial product containing the Antarctic strain with organic matter decomposition and metabolism and nitrogen removal functions described above and / or the culture or fermentation product of the Antarctic strain.

[0010] The present invention relates to the application of the Antarctic strain, or the product of the Antarctic strain, or the microbial product having the functions of organic matter decomposition and metabolism and nitrogen removal in the degradation of organic matter and / or nitrogen removal.

[0011] The organic compound is selected from at least one of lignin, benzoic acid, peptone, alcohols, acetic acid, D-amino acids, vegetable oil, and diesel oil; wherein, The benzoic acid is selected from at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, 4-hydroxybenzoate, 3-hydroxybenzoic acid, and 3-hydroxybenzoate, preferably at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, and 4-hydroxybenzoate; The peptone is trypsin; The alcohol is glycerol; The acetic acid compounds are selected from acetic acid and / or acetates; The D-amino acid is selected from at least one of D-phenylalanine, D-alanine, D-serine, and D-glutamine, preferably at least one of D-alanine, D-serine, and D-glutamine; The vegetable oil is selected from at least one of soybean oil, flaxseed oil, and sunflower seed oil, preferably soybean oil and / or sunflower seed oil.

[0012] The nitrogen source is inorganic nitrogen, selected from at least one of ammonia nitrogen, nitrate, and nitrite.

[0013] The present invention relates to the application of the Antarctic strains, or products of Antarctic strains, or microbial products having the functions of organic matter decomposition and nitrogen removal in sewage or wastewater treatment, and / or agricultural and forestry waste degradation, and / or vegetable oil or organic hydrocarbon pollution treatment.

[0014] The sewage or wastewater includes, but is not limited to, domestic sewage or wastewater, industrial sewage or wastewater, and aquaculture tailwater or wastewater.

[0015] The agricultural and forestry wastes include, but are not limited to, straw, vines, fruit pomace, seeds, peanut shells, bean pods, bran (wheat bran or rice husks, etc.), edible mushroom substrate, firewood, bark, branches, tree branches, rolled bark, wood shavings, sawdust, and other waste wood.

[0016] The vegetable oils include, but are not limited to, soybean oil, flaxseed oil, sunflower oil, etc., and the organic hydrocarbons include, but are not limited to, diesel oil, etc.

[0017] The present invention provides a method for degrading agricultural and forestry waste, the steps of which include: using the Antarctic strain with organic matter decomposition and metabolism and nitrogen removal functions described above, or the products of the Antarctic strain, or microbial products to ferment and degrade the agricultural and forestry waste.

[0018] The agricultural and forestry wastes include, but are not limited to, straw, vines, fruit pomace, seeds, peanut shells, bean pods, bran (wheat bran or rice husks, etc.), edible mushroom substrate, firewood, bark, branches, tree branches, rolled bark, wood shavings, sawdust, and other waste wood.

[0019] The present invention provides a method for treating sewage or wastewater, comprising the steps of: adding the Antarctic strain, or the product of the Antarctic strain, or the microbial product having the functions of organic matter decomposition and metabolism and nitrogen removal as described above, to the sewage or wastewater to degrade and remove organic matter and / or nitrogen in the sewage or wastewater.

[0020] The organic compound is selected from at least one of lignin, benzoic acid derivatives, peptone, alcohols, acetic acid derivatives, D-amino acids, vegetable oils, and diesel oil; wherein, the benzoic acid derivatives are selected from at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, 4-hydroxybenzoate, 3-hydroxybenzoic acid, and 3-hydroxybenzoate; the alcohol is glycerol; the acetic acid derivatives are selected from acetic acid and / or acetates; the D-amino acids are selected from at least one of D-phenylalanine, D-alanine, D-serine, and D-glutamine; and the vegetable oil is selected from at least one of soybean oil, flaxseed oil, and sunflower oil. The nitrogen is derived from inorganic nitrogen and is selected from at least one of ammonia nitrogen, nitrate, and nitrite.

[0021] The sewage or wastewater includes, but is not limited to, domestic sewage or wastewater, industrial sewage or wastewater, and aquaculture tailwater or wastewater.

[0022] The present invention provides a method for treating vegetable oil or organic hydrocarbon pollution, the steps of which include: using the Antarctic strain with organic matter decomposition and metabolism and nitrogen removal functions described above, or products of the Antarctic strain, or microbial products to degrade and remove vegetable oil or organic hydrocarbons.

[0023] The vegetable oils include, but are not limited to, soybean oil, flaxseed oil, sunflower oil, etc., and the organic hydrocarbons include, but are not limited to, diesel oil, etc.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The CLB_ANTARCTIC_006 PLUS strain of the present invention can grow continuously in the range of 4 ℃-30 ℃, especially under the condition of 4 ℃, and has a strong temperature adaptability, especially a significant cold adaptability.

[0025] (2) The CLB_ANTARCTIC_006 PLUS strain of the present invention continuously degrades lignin in the range of 4 ℃-30 ℃. It achieves a lignin degradation rate of about 24% after 5 days of cultivation in a 4 ℃ seawater environment and about 15% after 5 days of cultivation in a 4 ℃ freshwater environment, showing excellent low-temperature lignin degradation ability.

[0026] (3) The CLB_ANTARCTIC_006 PLUS strain of this invention exhibited peak LiP activity of approximately 1250 U / L and 1500 U / L at 4 ℃ and 10 ℃, respectively, peak MnP activity of 14 U / L and 36 U / L, and peak Lac activity of approximately 2 U / L. The LiP activity was more than 40 times that of MnP and more than 700 times that of Lac, respectively, demonstrating high LiP activity under low temperature conditions. CLB_ANTARCTIC_006 PLUS maintained high lignin degradation efficiency and high LiP activity even at extremely low temperatures, showing a significant advantage in cold adaptation.

[0027] (4) The CLB_ANTARCTIC_006 PLUS strain of this invention can not only complete lignin depolymerization, but also further utilize aromatic compounds from lignin degradation products. The combination of "high LiP activity + complete aromatic compound metabolic pathway" may be an important mechanism for CLB_ANTARCTIC_006 PLUS to achieve efficient low-temperature lignin degradation. The CLB_ANTARCTIC_006 PLUS strain of this invention can also utilize complex organic substrates such as peptone, glycerol, acetic acid, vegetable oil, D-amino acids, and diesel oil, and has a broad metabolic spectrum and environmental adaptability, which is conducive to maintaining continuous metabolic and degradation functions in complex environments.

[0028] (5) The CLB_ANTARCTIC_006 PLUS strain of this invention also has a strong denitrification capacity. In actual wastewater sample treatment experiments, the ammonia nitrogen removal rate was close to 80% within 96 hours.

[0029] The CLB_ANTARCTIC_006 PLUS strain of this invention has the characteristics of both lignin degradation and nitrogen removal and conversion functions, which makes it promising for the synergistic treatment of solid organic waste and nitrogen-containing wastewater.

[0030] The CLB_ANTARCTIC_006 PLUS strain of this invention can not only promote the degradation and resource utilization of lignocellulose in agricultural and forestry waste, but also reduce the nitrogen pollution load in the environment. It has good application prospects in the fields of resource utilization of agricultural and forestry and livestock waste in cold regions (such as straw treatment and purification of livestock wastewater) and low-temperature bioremediation. Attached Figure Description

[0031] Figure 1 Colony morphology (A) and growth characteristics at different temperatures (B) of strain CLB_ANTARCTIC_006 PLUS.

[0032] Figure 2 The lignin degradation capacity of strain CLB_ANTARCTIC_006 PLUS under different temperature conditions (A) and seawater or freshwater conditions (B) was investigated.

[0033] Figure 3 The activities of lignin peroxidase LiP (A) and manganese peroxidase MnP (B) in strain CLB_ANTARCTIC_006 PLUS under low temperature conditions were studied.

[0034] Figure 4 The degradation rate of corn stalks by CLB_ANTARCTIC_006 PLUS at different inoculation concentrations.

[0035] Figure 5 The utilization capacity of strain CLB_ANTARCTIC_006 PLUS of different organic substrates was investigated. The substrates used were: A: diesel oil; B: soybean oil; C: D-glutamine; D: tryptone; E: glycerol; F: sodium p-hydroxybenzoate; G: sodium acetate; H: sodium benzoate; I: D-serine; J: LB medium; K: sunflower oil; L: D-alanine; M: D-phenylalanine; N: sodium 3-hydroxybenzoate; O: linseed oil; and P: seawater.

[0036] Figure 6 The ammonia nitrogen removal efficiency of CLB_ANTARCTIC_006 PLUS under conditions of 10 ℃ and 25 ℃.

[0037] Figure 7 The nitrite removal efficiency of CLB_ANTARCTIC_006 PLUS under conditions of 10 ℃ and 25 ℃ is given.

[0038] Figure 8 The nitrate removal efficiency of CLB_ANTARCTIC_006 PLUS under conditions of 10 ℃ and 25 ℃.

[0039] Figure 9 The efficiency of CLB_ANTARCTIC_006 PLUS in removing ammonia nitrogen from wastewater at 4 ℃ is given. Detailed Implementation

[0040] Example 1: Strain Acquisition Antarctic fish with burr-shaped shoulder ( Trematomus bernacchii The intestinal contents of the bacteria were used as the bacterial source sample and enriched in an enrichment medium (pH 7.0-7.5, KH2PO4 1g / L, K2HPO4 1g / L, NaNO3 2g / L, MgSO4·7H2O 0.5g / L, CaCl2 0.02 g / L, alkali lignin 2 g / L, agar 15 g / L, and sterile artificial seawater as the solvent) at 4°C to obtain the target strain CLB_ANTARCTIC_006 PLUS.

[0041] CLB_ANTARCTIC_006 PLUS bacterial suspension with an OD600 of approximately 1.0 was inoculated onto the surface of LB solid medium and cultured at 4 ℃. CLB_ANTARCTIC_006 PLUS formed white, round colonies with regular edges and smooth surfaces on the LB medium. Figure 1 (A)

[0042] Genomic DNA was extracted from the bacterial strain using a rapid bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). The 16S rRNA gene was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO:1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO:2). The PCR reaction system (20 μL) included: 10 μL PCR premix, 1 μL each primer, 1 μL template DNA, and 7 μL ddH2O. The amplification program was: 95 ℃ pre-denaturation for 10 min; 29 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 90 s; and extension at 72 ℃ for 10 min. The PCR products were detected by 1% agarose gel electrophoresis and then sequenced. BLAST alignment analysis was performed using the NCBI database. Preliminary classification and identification were performed based on the 16S rRNA gene sequence, followed by phylogenetic analysis using GTDB-Tk based on conserved single-copy marker genes. Average nucleotide identity (ANI) analysis was used to compare the genomic similarity between CLB_006 and closely related strains to aid in determining its taxonomic position. Phylogenetic analysis of the 16S rRNA gene showed that strain CLB_ANTARCTIC_006 PLUS clustered with *Serratia proteamaculans* in the same clade.

[0043] The target strain CLB_ANTARCTIC_006 PLUS was deposited on May 29, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. It was classified and named... Serratia proteamaculans CLB_ANTARCTIC_006 PLUS, with accession number CCTCC NO: M 20261129, is considered viable after accession.

[0044] Example 2: Determination of bacterial growth characteristics CLB_ANTARCTIC_006 PLUS bacterial culture with an OD600 of approximately 1.0 was inoculated into LB liquid medium at a 1% (v / v) inoculation rate and cultured with shaking (200 rpm) at 4, 10, 15, 20, 25, and 30 °C, respectively. OD600 values ​​were measured every 6 h to plot growth curves. All experiments were performed in triplicate.

[0045] like Figure 1As shown in Figure B, the CLB_ANTARCTIC_006 PLUS strain can grow in the range of 4 ℃ to 30 ℃, with faster growth and higher final biomass at 20 ℃ to 25 ℃. It can also continue to grow at 4 ℃, indicating that it has a wide temperature adaptability range and a certain low temperature adaptability.

[0046] Genomic DNA was extracted from the bacterial strain using a rapid bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). After passing quality testing, the DNA was used for whole-genome sequencing. Sequencing data underwent quality control before genome assembly, and the assembly results were analyzed for basic characteristics such as integrity, GC content, gene number, and genome structure. Gene prediction and functional annotation were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The predicted protein-coding sequences were further compared with the COG database to analyze the functional gene composition and distribution of major functional categories of the strain. Simultaneously, metabolic pathways were annotated using the KEGG database, focusing on pathways related to carbon source utilization, aromatic compound degradation, lignin-related intermediate metabolism, nitrogen metabolism, and low-temperature adaptation.

[0047] Functional annotation and analysis revealed eight cold shock protein (CSP) genes in the CLB_ANTARCTIC_006 PLUS genome, a significantly higher number than in most mesophilic bacteria, indicating that this strain possesses good potential for low-temperature stress response. Previous studies have shown that CSPs are widely involved in RNA metabolism regulation and protein synthesis maintenance under low-temperature conditions, representing a crucial mechanism for polar microorganisms to adapt to cold environments. The CLB_ANTARCTIC_006 PLUS genome also detected multiple glycine-betaine, proline, and trehalose transport systems, including opuABCD, betT, proVWX, treBC, and thuGFE. These compatible solutes not only maintain cellular osmotic pressure balance but also enhance cell survival under low-temperature conditions by inhibiting ice crystal formation and stabilizing protein structure. Simultaneously, the strain contains multiple fatty acid desaturase encoding genes, participating in the synthesis and regulation of unsaturated fatty acids, increasing cell membrane fluidity, and thus maintaining the normal function of membrane proteins and transport systems under low-temperature conditions. Research indicates that increasing membrane lipid unsaturation is one of the most common cold adaptation mechanisms in psychrophilic and psychrophilic microorganisms. Furthermore, the CLB_ANTARCTIC_006 PLUS genome contains a variety of antioxidant enzyme systems, including SOD, KatE, KatG, GST, and glutathione peroxidase. Under low-temperature conditions, reactive oxygen species (ROS) easily accumulate in cells, leading to protein, lipid, and DNA damage. A robust antioxidant defense system can effectively scavenge ROS and reduce the damage caused by oxidative stress to cells. The aforementioned cold shock proteins, compatible solute transport systems, membrane lipid regulation mechanisms, and antioxidant enzyme systems collectively constitute the low-temperature adaptation network of CLB_ANTARCTIC_006 PLUS, providing crucial protection for maintaining high metabolic activity at 4 °C.

[0048] Example 3: Determination of Lignin Degradation Rate Take 1g of KH2PO4, 1g of K2HPO4, 2g of NaNO3, 0.5g of MgSO4•7H2O, 0.02g of CaCl2, and 2g of alkali lignin. Add sterile artificial seawater or distilled water to a final volume of 1 liter, with a pH of 7.0-7.5, to obtain seawater culture medium and freshwater culture medium with lignin as the sole carbon source.

[0049] CLB_ANTARCTIC_006 PLUS bacterial culture with an OD600 of approximately 1.0 was inoculated into seawater culture medium at a 1% (v / v) inoculation rate and cultured at 4, 10, 15, 20, 25 and 30 °C, respectively. Samples were taken every 24 h, centrifuged at 8000 rpm for 15 min and the OD280 value of the supernatant was measured. The residual lignin concentration was calculated based on the OD280 and lignin concentration standard curve.

[0050] CLB_ANTARCTIC_006 PLUS bacterial culture with an OD600 of approximately 1.0 was inoculated at a rate of 1% (v / v) into both seawater and freshwater culture media and cultured at 4 °C. Samples were taken every 24 h, centrifuged at 8000 rpm for 15 min, and the OD280 value of the supernatant was measured. The residual lignin concentration was calculated based on the OD280 versus lignin concentration standard curve.

[0051] The lignin degradation rate is calculated using the following formula: Degradation rate (%) = (C0) / (C0) C t ) / C0×100%, where C0 is the initial lignin concentration, C t Let t be the residual lignin concentration at time t.

[0052] like Figure 2 As shown in Figure A, the lignin degradation rate in the culture system gradually increased under different temperature conditions, indicating that the strain can utilize lignin as a carbon source for growth. Comparison under different temperature conditions revealed that CLB_ANTARCTIC_006PLUS exhibited the highest lignin degradation efficiency at 15 ℃, and maintained good degradation activity at 4 ℃, indicating that this strain possesses significant low-temperature lignin degradation capabilities.

[0053] like Figure 2 As shown in Figure B, after culturing at 4 ℃ for 5 days, the lignin degradation rate in the seawater system reached approximately 24%, while the degradation rate in the freshwater system reached approximately 15%. With prolonged culturing time, the lignin degradation rate further increased.

[0054] Example 4: Determination of Lignin-Degrading Enzyme Activity CLB_ANTARCTIC_006 PLUS bacterial culture with an OD600 of approximately 1.0 was inoculated into freshwater culture medium at a rate of 1% (v / v) and cultured at 4 ℃ and 10 ℃, respectively. Samples were taken every 24 h, centrifuged at 10000 rpm for 10 min, and the supernatant was measured.

[0055] Lac (laccase) activity was measured by the ABTS oxidation method, with absorbance changes at 420 nm; LiP (lignin peroxidase) activity was measured by the azuril demethylation method at 651 nm; and MnP (manganese peroxidase) activity was measured by the guaiacol oxidation method, with absorbance changes at 465 nm. All experiments were performed in triplicate.

[0056] like Figure 3As shown in Figure A, LiP activity rapidly increased in the early stages of cultivation, reaching a peak on days 3-4. At 10 °C and 4 °C, the highest activities reached approximately 1516 U / L and 1258 U / L, respectively. Subsequently, enzyme activity gradually decreased, but remained at a high level throughout the entire cultivation cycle. The LiP activity at 4 °C still maintained more than 80% of the level at 10 °C, demonstrating the good low-temperature catalytic stability of LiP.

[0057] like Figure 3 As shown in Figure B, MnP activity remained at a low level during the first 4 days of culture, then gradually increased, reaching a peak on day 8. The highest activity was approximately 36 U / L at 10 °C, while it was only approximately 14 U / L at 4 °C, after which it gradually decreased. MnP activity was significantly lower than that of LiP.

[0058] At both 10 ℃ and 4 ℃, the activity of Lac showed a slow increasing trend and tended to stabilize in the later stage of cultivation. The highest activity was approximately 2.0 U / L at 10 ℃ and approximately 1.7 U / L at 4 ℃.

[0059] Among lignin degradation-related enzymes, LiP exhibited the highest enzyme activity, with activities more than 40 times that of MnP and more than 700 times that of Lac. LiP is likely the most important functional enzyme in the low-temperature lignin degradation process of CLB_ANTARCTIC_006 PLUS. LiP can directly attack the non-phenolic aromatic structures in lignin, achieving the initial depolymerization of high-molecular-weight lignin, and is therefore considered a key oxidase in the lignin degradation system.

[0060] Example 5: Determination of Straw Degradation Effect Corn stalks were cut into segments approximately 3 cm long and longitudinally split to increase the contact area between the stalk substrate and microorganisms. The treated stalks were dried at 80 °C to constant weight and then weighed, denoted as W0. Subsequently, they were added to an inorganic salt culture medium (KH₂PO₄ 1.0 g / L, NaNO₃ 2.5 g / L, MgSO₄•7H₂O 0.3 g / L, CaCl₂ 0.1 g / L, NaCl 0.1 g / L, FeCl₃ 0.01 g / L, distilled water as solvent) at a concentration of approximately 10 g / L, with the corn stalks as the sole carbon source. After the strain CLB_ANTARCTIC_006 PLUS was cultured to the logarithmic growth phase, the initial bacterial concentration in the culture system was adjusted to 10... 5 10 6 and 10 7 CFU / mL, with an equal volume of sterile water used as a blank control instead of the bacterial culture. The culture was incubated at 4 ℃ and 200 r / min with shaking for 15 days.

[0061] After cultivation, collect the straw residue, gently rinse with sterile water to remove attached bacteria and culture medium residue, dry at 80 ℃ to constant weight, and record the weight as W. Calculate the straw degradation rate using the mass loss method: Degradation rate (%) = [(W0)] W) / W0]×100% Where W0 is the initial dry weight of straw (g) and W is the residual dry weight of straw after cultivation (g).

[0062] The validity of the experiment was determined by a change of no more than 5% in the dry weight of straw in the blank control group. All experiments were performed in triplicate.

[0063] like Figure 4 As shown, CLB_ANTARCTIC_006 PLUS can grow using corn straw as a carbon source at 4 ℃ and achieve significant straw degradation. After 7 days of cultivation, when the initial inoculum concentration was 10... 5 10 6 and 10 7 At CFU / mL, the straw degradation rates reached 12.35%, 15.40%, and 16.24%, respectively.

[0064] Example 6 Determination of utilization capacity of different organic substrates To verify the ability of strain CLB_ANTARCTIC_006 PLUS to further metabolize lignin degradation products and to evaluate its utilization of different types of organic substrates, a single carbon source culture method was used to determine its carbon source utilization profile. Inorganic salt medium (same as in Example 5) was used as the basal medium, with different types of carbon sources added as the sole carbon source. Since lignin can produce aromatic intermediates such as benzoic acid and hydroxybenzoic acid during microbial degradation, sodium benzoate, 3-hydroxybenzoate (3-HB), and 4-hydroxybenzoate (4-HB) were selected as representative lignin degradation intermediates. Meanwhile, to evaluate the strain's ability to utilize other organic substrates, peptone (Tryptone), glycerol, sodium ethanoate, D-phenylalanine, D-alanine, D-serine, D-glutamine, and vegetable oils [soybean oil, flaxseed oil, and sunflower oil] and diesel fuel were selected as representative substrates.

[0065] The final concentration of peptone was 1.3 g / L, and the final concentrations of sodium acetate, D-amino acids, and aromatic compounds were all set at 5 mmol / L. Glycerol, diesel oil, and vegetable oil were added to the culture medium at 2.5% (v / v) of the total culture volume. The strain CLB_ANTARCTIC_006 PLUS was first cultured in LB liquid medium to the logarithmic growth phase (OD600≈0.4), then centrifuged to collect the cells, and washed three times with sterile artificial seawater to remove residual nutrients. The washed cells were then resuspended in sterile artificial seawater and inoculated into culture systems containing different single carbon sources at the same inoculum size, and cultured at 4 ℃ for 7 days.

[0066] Samples were taken before incubation (Day 0) and after 7 days of incubation, serially diluted, and spread onto LB agar. Colony counts were performed after incubation at 4 °C for 7 days. Changes in colony counts before and after incubation were used to evaluate the strain's ability to utilize different carbon sources. All experiments were performed in triplicate, and a blank control group (P) was included, consisting of an equal volume of sterile artificial seawater instead of any carbon source.

[0067] The results are as follows Figure 5 As shown, CLB_ANTARCTIC_006 PLUS can grow using benzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid—typical aromatic intermediates produced during lignin degradation—as the sole carbon source, and forms distinct colonies. It exhibits good growth ability in all three aromatic compound media, indicating its ability to further metabolize small-molecule aromatic intermediates produced during lignin degradation, demonstrating a relatively complete lignin degradation metabolic potential. It also maintains stable growth on the two hydroxybenzoic acid media, indicating that this strain has the ability to utilize multiple lignin degradation intermediates. Furthermore, CLB_ANTARCTIC_006 PLUS can also grow using peptone, glycerol, sodium acetate, D-amino acids, diesel oil, and vegetable oils (peanut oil and sunflower oil) as the sole carbon source.

[0068] Genome functional annotation and analysis revealed that CLB_ANTARCTIC_006 PLUS contains 472 genes for amino acid transport and metabolism (COG-E) and 460 genes for carbohydrate transport and metabolism (COG-G), indicating its extensive basal metabolic capacity. Further analysis showed that CLB_ANTARCTIC_006 PLUS possesses a rich organic substrate uptake and utilization system, including 154 amino acid and peptide transport-related genes and various extracellular peptidases, suggesting its ability to utilize proteins and peptides as potential nutrient sources. This strain also showed 129 genes related to carbohydrate and organic acid transport, mainly including the ABC transport system, the phosphotransferase system (PTS), and various organic acid transport proteins, which can mediate the transmembrane transport of various monosaccharides and oligosaccharides such as glucose, fructose, arabinose, ribose, cellobiose, and trehalose. Furthermore, multiple glycoside hydrolase family members (including GH3, GH13, GH18, GH20, and GH39) were identified in the CLB_ANTARCTIC_006 PLUS genome, indicating its potential to degrade complex polysaccharides and plant-derived polycarbohydrates. These enzyme systems provide an important enzymatic basis for the initial degradation of hemicellulose and cellulose in lignocellulose components. Regarding lipid utilization, CLB_ANTARCTIC_006 PLUS also encodes a long-chain fatty acid transporter (FadL), extracellular lipase, and glycerol metabolism-related systems, suggesting its ability to utilize plant oils and lipids as carbon sources, thereby enhancing its adaptability to complex organic environments.

[0069] Further analysis revealed that CLB_ANTARCTIC_006 PLUS possesses a relatively complete gene system related to lignin and aromatic compound degradation. Regarding lignin degradation, two Dyp-type peroxidase encoding genes and one multi-copper oxidase encoding gene (Chromosome_2756) were detected in the CLB_ANTARCTIC_006 PLUS genome. One of the Dyp genes (Chromosome_2960) is located near the benzoic acid degradation gene cluster, indicating its potential involvement in lignin depolymerization and subsequent aromatic compound metabolism. The multi-copper oxidase encoding gene is presumed to possess laccase activity. CLB_ANTARCTIC_006 PLUS also contains multiple aromatic compound transporter genes, including benzoic acid transporter (benE), 4-hydroxybenzoic acid transporter (pcaK), 4-hydroxyphenylacetic acid transporter (hpaX), and 3-phenylpropionic acid transporter (hcaT). The CLB_ANTARCTIC_006 PLUS genome also contains several complete aromatic compound degradation gene clusters. Among them, the hpa gene cluster is involved in the degradation of 4-hydroxyphenylacetic acid; the pca gene cluster is involved in the degradation of 4-hydroxybenzoic acid; the ben-cat gene cluster is responsible for the degradation of benzoic acid via the catechol pathway; and the paa gene cluster is involved in the metabolism of phenylacetic acid. These metabolic pathways ultimately convert aromatic compounds into central metabolic intermediates such as pyruvate, acetyl-CoA, and succinyl-CoA, which then enter the tricarboxylic acid cycle. These results indicate that CLB_ANTARCTIC_006 PLUS not only possesses the initial depolymerization ability of lignin but also has the genetic basis for further utilization of lignin degradation products.

[0070] Example 7 Nitrogen Conversion Capacity Determination After activation in TSB medium, strain CLB_ANTARCTIC_006 PLUS was cultured to the logarithmic growth phase. The cells were collected by centrifugation and washed twice with sterile physiological saline. The cells were then resuspended to OD600=0.5 as the inoculum suspension.

[0071] Under aseptic conditions, 0.5 mL of bacterial suspension was inoculated into 100 mL of nitrification medium ((NH4)2SO4 0.28 g / L, sodium succinate 3.4 g / L, MgSO4·7H2O 0.2 g / L, KH2PO4 1.5 g / L, Na2HPO4 4 g / L, trace element solution 2 mL, pH 7.2), or 100 mL of denitrification medium (NaNO3 0.38 g / L, sodium succinate 3.4 g / L, MgSO4·7H2O 0.2 g / L, KH2PO4 1.5 g / L, Na2HPO4 4 g / L, trace element solution 2 mL, pH 7.2), or 100 mL of nitrite denitrification medium (NaNO2 0.05 g / L, sodium succinate 0.56 g / L, MgSO4·7H2O 0.2 g / L, KH2PO4 1.5 g / L). The solution was prepared in 250 mL Erlenmeyer flasks containing 4 g / L Na₂HPO₄, 2 mL of trace element solution, and pH 7.2, and then cultured with shaking at 10 °C and 25 °C (200 r / min). During the culture, samples were taken every 24 h for the HNM system and every 6 h for the DM and NDM systems.

[0072] Under aseptic conditions, a bacterial suspension was inoculated into 200 mL of wastewater sample (collected from a wastewater treatment plant in Northeast China during winter) at a 5% (v / v) inoculation rate. The sample was then incubated at 4 °C with shaking (200 r / min), and ammonia nitrogen (NH4) was measured periodically. + -N concentration.

[0073] NO2 was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry. NO3- concentration was determined by ultraviolet spectrophotometry. -N concentration was determined using Nessler's reagent method for NH4+. + -N concentration. All experiments were performed in triplicate, with uninoculated culture medium serving as a blank control.

[0074] Figure 6 In the nitrification culture system shown, at 10 ℃, in HNM medium with an initial ammonia nitrogen concentration of 54.59 mg / L, the ammonia nitrogen removal rate reached 95.99% after 72 h of culture; when the temperature was increased to 25 ℃, the degradation rate reached 98.65% after 72 h of culture.

[0075] Figure 7 In the nitrite denitrification culture system shown, the strain was able to rapidly utilize NO2 under both 10 ℃ and 25 ℃ conditions. - -N, after 24 h of cultivation, the nitrite removal rates reached 96.60% and 94.77%, respectively.

[0076] Figure 8 In the denitrification culture system shown, for a high nitrate concentration of approximately 127 mg / L, the nitrate removal rate reached 57.20% within 96 h, indicating that this strain has a strong nitrate utilization capacity. In a low nitrate concentration (75.62 ± 6.58 mg / L) culture system, NO3- removal can be achieved within 48 h. - Complete removal of -N.

[0077] CLB_ANTARCTIC_006 PLUS exhibited good inorganic nitrogen removal capabilities under various temperature conditions. At 10 ℃, the strain showed high removal efficiency for both ammonia nitrogen and nitrite nitrogen.

[0078] like Figure 9 As shown, at 4 ℃, the CLB_ANTARCTIC_006 PLUS strain was able to reduce approximately 65 mg / L of NH4 in the wastewater sample within 96 h. + -N was reduced to around 15 mg / L, and the ammonia nitrogen removal rate was close to 80%, demonstrating good low-temperature denitrification performance.

[0079] Genome functional annotation and analysis revealed that CLB_ANTARCTIC_006 PLUS possesses a relatively complete inorganic and organic nitrogen utilization system. Regarding inorganic nitrogen metabolism, the genome detected NarK family nitrate / nitrite transporters, the nitrate ABC transport system (NrtABC), nitrite transporter (FocA), and ammonium transporter (AmtB). Simultaneously, CLB_ANTARCTIC_006 PLUS exhibits both assimilated and dissimilated nitrate reduction pathways, including key genes such as narGHI, napAB, nirBD, nasAB, and nasDE, indicating its ability to utilize nitrate, nitrite, and ammonium as nitrogen sources. Regarding organic nitrogen metabolism, the CLB_ANTARCTIC_006 PLUS genome detected the putrescine transport system (PotE, PuuP), spermidine / putrescine ABC transport system, urea transport system (UrtABCDE), and cyanate transport system (CynX). Furthermore, key metabolic enzymes such as urea carboxylase, allantoic acid hydrolase, and cyanate lyase are present, indicating its ability to utilize a variety of organic nitrogen compounds. Regarding phosphorus metabolism, CLB_ANTARCTIC_006 PLUS possesses a low-affinity phosphate transport system (Pit), a high-affinity phosphate transport system (PstABCS-PhoU), and phosphonate transport and degradation systems (PhnCDE and PhnJHIJKLM). These genes endow it with the ability to utilize inorganic phosphorus, organic phosphorus, and phosphonates.

[0080] Example 8 Biosafety Evaluation To assess the safety of strain CLB_ANTARCTIC_006 PLUS in environmental remediation and aquaculture applications, hemolytic activity assays, zebrafish challenge experiments, and antibiotic susceptibility assays were conducted.

[0081] 1. Hemolytic activity assay The potential hemolytic activity of strain CLB_ANTARCTIC_006 PLUS was evaluated using the blood agar plate method. A 1000-fold dilution of CLB_ANTARCTIC_006 PLUS bacterial suspension in the logarithmic growth phase was used, and 2 μL of the suspension was evenly spread onto blood agar plates. The plates were incubated upside down at 25 °C for 24–48 h. The presence of a clear ring (β-hemolysis), a greenish ring (α-hemolysis), or no change (γ-hemolysis) around the colonies was observed. The potential pathogenicity of strain CLB_ANTARCTIC_006 PLUS was assessed by comparing the hemolytic activity around the colonies.

[0082] Blood agar plate culture results showed that no obvious transparent hemolytic zone (β-hemolysis) or grass-green hemolytic zone (α-hemolysis) was observed around the CLB_ANTARCTIC_006 PLUS colonies, exhibiting γ-hemolytic characteristics. This result indicates that CLB_ANTARCTIC_006 PLUS does not possess significant hemolytic activity.

[0083] 2. Zebrafish challenge experiment Healthy two-month-old zebrafish were selected as experimental subjects. After anesthetizing with 10 mg / L eugenol, a wound approximately 2-3 mm long and reaching the muscle layer was made in the dorsal muscle above the middle of the lateral line using a sterile dissecting needle. Immediately after treatment, the zebrafish were transferred to a fully aerated and stable culture water body for 24 hours to recover.

[0084] The experimental group placed the injured zebrafish in a solution containing 3×10 6 In aquaculture water containing CFU / mL CLB_ANTARCTIC_006 PLUS bacterial solution, the control group underwent only scratch treatment without contact with the bacterial solution. During the experiment, continuous oxygenation was maintained, and behavioral changes, wound infection status, and mortality rates of zebrafish were continuously observed over 48 hours to evaluate the potential biosafety of strain CLB_ANTARCTIC_006 PLUS.

[0085] Both the experimental and control groups of zebrafish maintained normal swimming and feeding behavior. During the observation period, no obvious redness, swelling, ulceration, or secondary infection was observed at the wound site on the experimental group, nor were any abnormal behaviors or deaths observed. At the end of the experiment, there was no significant difference in survival rate between the experimental and control groups, indicating that CLB_ANTARCTIC_006 PLUS did not exhibit significant pathogenicity to the damaged fish.

[0086] 3. Antibiotic susceptibility testing The bacterial suspension in the logarithmic growth phase was evenly spread on the surface of LB solid medium. Antibiotic susceptibility testing discs of different types, including commonly used antibacterial drugs such as β-lactams, aminoglycosides, tetracyclines, macrolides, and quinolones, were placed on the plates. After incubation at 25 °C for 24 h, the diameter of the inhibition zone was measured.

[0087] According to the Clinical and Laboratory Standards Institute (CLSI) standards, the response of strains to various antibiotics is classified into three levels: Sensitive (S), Intermediate (I), and Resistant (R) to assess their potential antibiotic resistance risk.

[0088] Antibiotic susceptibility testing results showed that CLB_ANTARCTIC_006 PLUS was sensitive to two antibiotics, moderately sensitive to five antibiotics, and resistant to the remaining antibiotics. The antibiotics that inhibited the growth of CLB_ANTARCTIC_006 PLUS mainly included β-lactams, fluoroquinolones, amyl alcohols, and polymyxins, indicating that this strain maintains good sensitivity to some commonly used clinical antibiotics and possesses a certain degree of overall antibiotic susceptibility.

[0089] Table 1. Results of susceptibility testing of CLB_ANTARCTIC_006 PLUS against 25 antibiotics. Hemolysis assays, zebrafish challenge assays, and antibiotic susceptibility analysis results showed that CLB_ANTARCTIC_006 PLUS exhibited good biocompatibility. Combined with its excellent low-temperature lignin degradation ability, high LiP expression level, and good nitrogen conversion and removal capacity, this strain holds promise as a novel functional strain for the resource utilization of agricultural waste and the remediation of low-temperature polluted environments in cold regions.

[0090] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. An Antarctic strain with functions of organic matter decomposition and nitrogen removal, characterized in that, The Antarctic strain was classified and named Serratia proteamaculans CLB_ANTARCTIC_006 PLUS, with accession number CCTCC NO: M20261129; the Antarctic strain described therein exhibits organic matter decomposition and metabolism and nitrogen removal activity in saline and / or freshwater environments at 4 ℃-30 ℃.

2. The Antarctic strain according to claim 1, characterized in that, The organic compound is selected from at least one of lignin, benzoic acid, peptone, alcohol, acetic acid, D-amino acid, vegetable oil, and diesel oil; the nitrogen is selected from at least one of ammonia nitrogen, nitrate, and nitrite.

3. The Antarctic strain according to claim 2, characterized in that, The benzoic acid is selected from at least one of benzoic acid, benzoate, 4-hydroxybenzoic acid, 4-hydroxybenzoate, 3-hydroxybenzoic acid, and 3-hydroxybenzoate. The alcohol is glycerol; The acetic acid compounds are selected from acetic acid and / or acetates; The D-amino acids are selected from at least one of D-phenylalanine, D-alanine, D-serine, and D-glutamine; The vegetable oil is selected from at least one of soybean oil, flaxseed oil, and sunflower seed oil.

4. A product of an Antarctic strain, characterized in that, It refers to the culture or fermentation product of the Antarctic strain described in any one of claims 1-3.

5. A microbial product, characterized in that, Contains the Antarctic strain according to any one of claims 1-3 and / or the product according to claim 4.

6. The use of the Antarctic strain according to any one of claims 1-3, or the product according to claim 4, or the microbial product according to claim 5 in the degradation of organic matter and / or removal of nitrogen.

7. The use of the Antarctic strain according to any one of claims 1-3, or the product according to claim 4, or the microbial product according to claim 5 in sewage or wastewater treatment, and / or degradation of agricultural and forestry waste, and / or treatment of vegetable oil or organic hydrocarbon pollution.

8. A method for degrading agricultural and forestry waste, characterized in that the steps include... include: Agricultural and forestry waste is fermented and degraded using the Antarctic strain described in any one of claims 1-3, the product described in claim 4, or a microbial product described in claim 5.

9. A method for treating sewage or wastewater, characterized in that the steps include... include: Adding the Antarctic strain according to any one of claims 1-3, the product according to claim 4, or a microbial product according to claim 5 to sewage or wastewater degrades and removes organic matter and / or nitrogen from the sewage or wastewater.

10. A method for treating vegetable oil or organic hydrocarbon pollution, characterized in that the steps include... include: The Antarctic strain according to any one of claims 1-3, the product according to claim 4, or a microbial product according to claim 5 is used to degrade and remove vegetable oil or organic hydrocarbons.

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