A strain of acidothermophilic sulphur-reducing archaea strain e233cd1 and uses thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a strain of acidophilic and thermophilic S. islandicus E233CD1 and use thereof. Saccharolobus islandicus The application is preserved in the China General Microbiological Culture Collection Center on July 31, 2023, and the preservation number is CGMCC NO. 26354. The application obtains a strain of acidophilic and thermophilic S. islandicus E233CD1 through domestication. Compared with a wild strain, the strain can grow normally in a high-concentration cellulose environment and has improved cellulose utilization capacity. In a 1.1% sodium carboxymethyl cellulose culture medium, the growth rate is obviously improved, and the maximum biomass that can be reached by the bacterial liquid is also significantly increased.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of acidophilic thermophilic Icelandic sulfur leaf mold E233CD1 and its uses. Background Technology

[0002] As a carbohydrate widely distributed in nature, lignocellulose is the world's largest renewable biomass resource. [1] The fermentation of lignocellulose to produce second-generation bioethanol (cellulosic ethanol) and other products can not only reduce agricultural waste pollution but also alleviate the energy crisis caused by the depletion of fossil fuels, thus possessing significant application and economic value. [2] .

[0003] Lignocellulose is an inexpensive and readily available renewable biomass resource from plant materials. It consists of 40-60% cellulose, 10-40% hemicellulose, 15-30% lignin, and minor components. [3] The specific components are as follows Figure 1 As shown, cellulose is composed of numerous D-glucopyranose monomers linked by β-1,4-glycosidic bonds. These chain-like molecules are arranged in parallel and form crystalline microfibrils through hydrogen bonds. [4] As the most common organic polymer on Earth, cellulose has an annual production of 1.5 × 10⁻⁶. 12 Tons of this material can be used as a base for the preparation of synthetic cellulose in coating materials, paper, textiles, optical films, and other applications. [5] Compared to cellulose, hemicellulose is amorphous and more hydrophilic, therefore, it is easily degraded and transformed by acids, alkalis, heat, and biological treatments. [6] Lignin has a relatively complex structure, with its basic structure consisting of aromatic compounds and a C3 chain, composed of phenylpropane linked by C-C bonds and ether bonds. [7] Lignin, as an amorphous multiphase polymer insoluble in water, effectively prevents structural damage from hydrolytic enzymes. It is precisely because of its complex and stable structure, composed of macromolecules such as cellulose, hemicellulose, and lignin, and supported by various intermolecular forces, that lignocellulose exhibits its strong resistance to the decomposition of foreign substances. [8] In industrial applications of lignocellulose, pretreatment is usually required to remove surface barriers and initially degrade lignocellulose into cellulose, hemicellulose, and lignin before utilization.

[0004] Pretreatment technologies for lignocellulose can be categorized into physical methods (microwave, radiation, and pulverization), chemical methods (acids, alkalis, oxidants, and organic solvents), physicochemical methods (acid heat treatment, steam explosion, and electrocatalysis), and biological methods (enzymes produced by microorganisms decompose lignocellulose). [8]Currently, the main method used in industry is chemical-physical (acid-thermal) decomposition, which is recognized as the pretreatment technology closest to commercialization.

[10] The acid and heat pretreatment process involves adding an appropriate amount of acid solution to lignocellulose and then subjecting it to high-pressure steam treatment. The acid treatment in this step effectively depolymerizes hemicellulose.

[11] High-temperature hydrothermal treatment further promotes the removal of the cellulose barrier.

[0005] While pretreated lignocellulose can be utilized more effectively, the pretreatment process generates a large amount of waste liquid. The acidic and hot environment in the waste liquid limits the utilization and fermentation of conventional microorganisms. Cellulase at room temperature usually cannot function under these harsh conditions, and restoring the waste liquid to a milder environment, such as neutral pH and room temperature, would significantly increase production costs.

[0006] *Icelandine sulfidea*, belonging to the Archaea domain and the Tack superphylum, is a type of extremophile microorganism capable of growing in high-temperature and highly acidic environments. Its acidophilic physiological characteristics give it excellent potential for industrial applications; for example, high temperatures can increase reaction rates while preventing contamination, facilitating the separation and recovery of volatile products, and reducing processing costs. *Icelandine sulfidea* S. islandicus As a model sulfur fungus species, *Sulphurella azedarach* has an optimal growth temperature of 75-80 °C and an optimal growth pH of 2.0-3.0. A well-established genetic system for *Sulphurella azedarach* has been established, including a marker-free gene knockout system and a shuttle vector expression system. This gives *Sulphurella azedarach* the potential to become a good chassis microorganism for cellulose degradation. However, currently, no *Sulphurella azedarach* strains that can grow normally under high concentrations of sodium carboxymethyl cellulose have been reported, and there has been no progress in research on the application of *Sulphurella azedarach* in cellulose degradation. Summary of the Invention

[0007] The purpose of this invention is to provide an acid-thermophilic Icelandic sulfur leaf strain E233CD1, which has a high efficiency in cellulose degradation and can withstand conditions such as acid heat and high concentrations of sodium carboxymethyl cellulose.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is: a strain of *Sulphurophytum latipes* E233CD1, which is *Sulphurophytum latipes*. Saccharolobus islandicus It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 31, 2023, with accession number CGMCC NO.26354.

[0009] Preferably, the present invention also provides a method for obtaining the acidophilic thermophyte Icelandic sulfur leaf strain E233CD1, which uses sodium carboxymethyl cellulose as the sole carbon source and employs a method of gradually increasing the concentration of sodium carboxymethyl cellulose to conduct subculture and domestication culture of strain E233.

[0010] Preferably, the concentration of sodium carboxymethyl cellulose is in the range of 0.4%-1.1%.

[0011] The present invention also provides the use of the strain E233CD1 in the degradation of cellulose.

[0012] Preferably, the strain can be used to prepare a cellulose degrading agent.

[0013] Preferably, the strain provided by the present invention can be used to treat waste liquid from lignocellulose pretreatment.

[0014] Preferably, the present invention also provides a cellulose degrading agent, wherein the cellulose degrading agent contains the strain E233CD1.

[0015] The beneficial effects of this invention are as follows: Through domestication, this invention obtains a strain of *Isochrysis galbana* E233CD1, which, compared to the wild strain, can grow normally in a high-concentration cellulose environment and exhibits enhanced cellulose utilization. In a 1.1% sodium methylcellulose medium, not only is the growth rate significantly increased, but the maximum biomass achievable by the bacterial culture also shows a significant increase. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the domestication and cultivation process of strain E233CD1 in an embodiment of the present invention; Figure 2 Comparison of the growth of wild-type strain and domesticated strain E233CD1 in MTVU-1.1% CMC medium; Figure 3 Micrographs of strain E233CD1: (a) Scanning electron microscope image; (b) Transmission electron microscope image; Figure 4 The following is a test of the carbon source utilization ability of strain E233CD1 in the embodiments of the present invention: (a) shows the growth of E233CD1 in a medium without added carbon source; (b) shows the growth of E233CD1 in a medium containing 1.1% CMC as a carbon source. Detailed Implementation

[0017] To facilitate understanding of this research, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. However, this research can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of this research.

[0018] The initial strain for domestication was *Sulphurella apiacea* strain E233, preserved in our laboratory, originating from Professor Shen Yulong's laboratory at the Institute of Microbial Technology, Shandong University. *Sulphurella apiacea* is an extreme acidophilic, thermophilic, aerobic heterotrophic microorganism, spherical in shape, lacking organelles. Its optimal growth temperature is 75-80 °C, and its optimal growth pH is 2.0-3.0. Its genome is 2.52 Mb in length, containing 2806 genes and expressing approximately 2644 proteins. Currently, a complete genetic operating system has been established for *Sulphurella apiacea*, including a marker-free gene knockout system and a shuttle vector expression system.

[0019] I. The *Icelandine Sulfophyllum* strain provided by this invention is derived from *Icelandine Sulfophyllum* strain E233 using the following methods and steps for domestication. The specific process is as follows: Figure 1 As shown: First, the growth of strain E233 was measured under low CMC concentrations. It was found that the growth of the strain was gradually inhibited when the concentration increased to 0.4%. Therefore, a CMC concentration of 0.4% was used as the initial concentration for acclimatization. The wild-type strain E233 was then used with an initial OD... 600 Inoculate at a ratio of 0.1 and grow to OD. 600 When the OD value is between 0.8 and 1.0, transfer is performed. The initial transfer takes a relatively long time, but this time gradually decreases with each subsequent transfer. At this point, the concentration of the inoculum is reduced, and the initial inoculum OD is lowered. 600 The value was reduced to 0.05, and the transfer continued. When the growth rate of the strain to be acclimatized in 0.4% CMC medium reached the same rate as that of the wild-type strain in ordinary medium, it was considered that the acclimatized strain had adapted to the 0.4% CMC concentration and reached stable growth at this concentration, and the CMC concentration in the medium was then considered to be increased. After 15 transfers at the 0.4% concentration, stable growth was achieved.

[0020] The CMC concentration was increased to 0.7%, and the acclimatized strains that had already grown stably at a concentration of 0.4% were used with the initial OD... 600 Inoculate at a ratio of 0.1 and grow to OD. 600 When the value is between 0.8 and 1.0, perform the transfer. When the growth rate improves to a certain extent, reduce the initial inoculation OD. 600 The value was lowered to 0.05, and the strain was continuously subcultured. After 28 subcultures, the acclimatized strain reached stable growth in 0.7% CMC medium.

[0021] The CMC concentration was increased to 0.9%, and the domesticated strains that had already grown stably at a concentration of 0.7% were used with the initial OD... 600 Inoculate at a ratio of 0.1 and grow to OD. 600 When the value is between 0.8 and 1.0, perform the transfer. When the growth rate improves to a certain extent, reduce the initial inoculation OD. 600 The value was lowered to 0.05, and the strain was continuously subcultured. After 52 subcultures, the acclimatized strain reached stable growth in 0.9% CMC medium.

[0022] The CMC concentration was increased to 1.1%, and the acclimatized strains that had already grown stably at a concentration of 0.9% were used with an initial OD... 600 Inoculate at a ratio of 0.1 and grow to OD. 600 When the value is between 0.8 and 1.0, perform the transfer. When the growth rate improves to a certain extent, reduce the initial inoculation OD. 600 The value was lowered to 0.05, and the strain was continuously subcultured. After 68 subcultures, the acclimatized strain reached stable growth in 1.1% CMC medium.

[0023] At this point, the CMC concentration was 1.1%, which was already a relatively high value for CMC in the culture medium environment. Continuing to increase the CMC concentration would make the medium too viscous; therefore, the acclimatization culture was terminated at a concentration of 1.1%. At this concentration, 52 more subcultures were performed to stabilize the acclimatization state. The acclimatization was thus completed, yielding an acidophilic thermophilic Iceland sulphurella strain capable of tolerating high concentrations of sodium carboxymethyl cellulose and growing normally under these conditions. This strain was named E233CD1, and its 16S rDNA sequence is shown in SEQ ID NO.1. This strain was deposited on July 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.26354. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] The genome of strain E233CD1 was sequenced and compared with that of the wild-type strain to explore the differences. Through whole-genome sequence comparison and SNP detection, mutations were found at multiple gene loci in strain E233CD1. The differences between strain E233CD1 and the wild-type strain E233 are shown in Tables 1 and 2, respectively.

[0025] Table 1. Indel comparison results of strain E233CD1 and wild-type strain E233 332229 SiRe_0370 331875-332336 A AT Asn Hypothetical protein 462561 SIRE_RS02555 462435-462635 TG T Pro Hypothetical protein 1010068 The intergenic region is 40 bp away from the next gene, SIRE_1060. 1010109-1010657 T TA — SIRE_1060: Hypothetical protein 1166198 SiRe_1215 1166100-1166471 C CT Val Hypothetical protein 1261560 The intergenic region is 182 bp away from the next gene, SiRe_1337. 1261743-1262327 G GA — Molybdenum cofactor guanylate transferase 1645656 The intergenic region is 11 bp away from the next gene, SiRe_1777. 1645675-1645983 A AT — 30S ribosomal protein S10 2035538 SiRe_2195 2034631-2035743 TG T Leu ABC transporter permease 2252896 The intergenic region is 31 bp away from the next gene SiRe_414 in the transcriptional direction. 2252926-2253861 T TA — Hypothetical protein Table 2. SNP comparison between strain E233CD1 and wild-type strain E233 nonsyn IEQ43_RS01415 SiRe_1776 NZ_JADAML010000001.1_234641 CG Translational extension factor EF-1 α-subunit nonsyn IEQ43_RS02170 SiRe_1199 NZ_JADAML010000002.1_96976 GA Pyruvate hydrated kinase syn IEQ43_RS04315 SiRe_0245 NZ_JADAML010000005.1_9865 CT Metallophosphatase nonsyn IEQ43_RS06525 SiRe_0996 NZ_JADAML010000008.1_55956 CT Glycine cleavage system H protein nonsyn IEQ43_RS07675 SiRe_2345 NZ_JADAML010000011.1_42394 CT MFS transporter nonsyn IEQ43_RS08165 SiRe_0739 NZ_JADAML010000013.1_45069 TC AMP-binding protein nonsyn IEQ43_RS08500 SiRe_2192 NZ_JADAML010000015.1_27129 CA Glycoside hydrolase 31 family proteins nonsyn IEQ43_RS10680 SiRe_0684 NZ_JADAML010000034.1_6596 AT MFS transporter nonsyn IEQ43_RS11075 SiRe_0262 NZ_JADAML010000039.1_7258 GA Glycoside hydrolase 15 family proteins nonsyn IEQ43_RS12300 SiRe_0799 NZ_JADAML010000060.1_35 AG The DUF1286 domain contains protein nonsyn IEQ43_RS12380 SiRe_0533 NZ_JADAML010000062.1_2234 GT Molybdenum-dependent oxidoreductases nonsyn IEQ43_RS12535 SiRe_0610 NZ_JADAML010000066.1_4140 GT Glycosyltransferase 2 family proteins nonsyn IEQ43_RS12865 SiRe_0525 NZ_JADAML010000077.1_626 GA The DUF1648 domain contains protein nonsyn IEQ43_RS12990 SiRe_0692 NZ_JADAML010000082.1_3166 TA RNA-guided endonucleases, TnpB family proteins NZ_JADAML010000082.1_3183 CT nonsyn IEQ43_RS13015 SiRe_0582 NZ_JADAML010000084.1_1497 TC DNA double-strand break repair nuclease NurA nonsyn IEQ43_RS13170 SiRe_0531 NZ_JADAML010000092.1_24 AT DUF973 family proteins nonsyn IEQ43_RS13200 SiRe_0575 NZ_JADAML010000094.1_1107 AT Helix-turn-helix domains contain proteins nonsyn IEQ43_RS13285 SiRe_0548 NZ_JADAML010000102.1_1137 TA S9 family peptidase nonsyn IEQ43_RS13315 SiRe_0799 NZ_JADAML010000104.1_1407 CT The DUF1286 domain contains protein nonsyn IEQ43_RS13545 SiRe_0581 NZ_JADAML010000126.1_676 AT ATP-binding protein NZ_JADAML010000126.1_683 AT II. Growth Characteristics Test of Strain E233CD1 1. Determination of bacterial growth rate: The growth rate was measured using two different media: conventional media (using sucrose as the carbon source) and media using CMC as the carbon source. Since the media used for the growth of wild-type strains and domesticated strains are different, the influence of residual carbon sources in the original culture medium of the strains must be excluded during the measurement. The cells were washed and then inoculated to obtain the growth curve.

[0026] First, wild-type strain E233 and strain E233CD1 were co-cultured until the bacterial culture reached OD. 600 The value was approximately 0.8. 20 ml of bacterial suspension was taken and centrifuged at 7000 rpm for 10 min to obtain bacterial cells. The bacterial cell precipitate was resuspended with an inorganic salt solution, centrifuged again, and the precipitate was resuspended again with an inorganic salt solution. At this point, the bacterial suspension had removed the residual carbon source from the original culture medium between the bacterial cells. Subsequently, growth curve inoculation was performed.

[0027] Determination of OD in bacterial suspension 600 The value, after calculation, was inoculated into 30 ml of MTSVU or MTVU-1.1% CMC medium to achieve the initial OD value of the bacterial culture. 600 The value was 0.04. The culture was carried out at 75 °C. The growth of the bacterial culture was measured at fixed time points, and growth curves were plotted to compare the growth of the two strains in the two culture media.

[0028] 2. Analysis of growth curve results: First: The domesticated strain E233CD1 grows significantly faster than the wild-type strain. It can quickly utilize CMC as a carbon source for growth, while the wild-type strain needs a longer adaptation period to grow. Second: The maximum biomass of the domesticated strain E233CD1 was significantly higher than that of the wild-type strain, and its maximum concentration in bacterial solution was also significantly higher than that of the wild-type strain. Third: Compared with the wild-type strain, the domesticated strain E233CD1 exhibits extremely high growth stability. Even after about 300 hours of growth, the bacterial concentration did not decrease.

[0029] 3. Results of cellulose utilization test: like Figure 4 As shown, (a) shows the growth of strain E233CD1 in a medium without a carbon source, and (b) shows the growth of strain E233CD1 in a medium containing 1.1% CMC as a carbon source. The comparison of the two growth curves reveals its maximum biomass (i.e., the maximum OD reached during growth). 600The concentration of the bacterial culture solution (Cc) was significantly increased. Based on this result, it can be confirmed that strain E233CD1 utilized the carbon source in the culture medium during its growth process, thereby increasing the bacterial concentration.

[0030] Because wild-type strain E233 utilizes reducing sugars in the bacterial culture as a carbon source for growth, the reducing sugar content in MTVU-1.1% CMC medium was measured. It was found that high temperatures hardly degraded CMC, directly producing reducing sugars. Therefore, strain E233CD1 needs to degrade CMC to generate reducing sugars before it can be utilized. This suggests that strain E233CD1 has an improved CMC degradation ability compared to the wild-type strain, meaning its cellulose degradation ability is also improved to some extent.

[0031] Compared to the wild-type strain, strain E233CD1 exhibits enhanced cellulose utilization, making it suitable for further applications in cellulose utilization scenarios. Furthermore, strain E233CD1 thrives in a warm, acidic environment, providing excellent conditions for cellulose utilization. The optimal growth temperature for strain E233CD1 is around 75 ℃, and this high-temperature environment is more conducive to the recovery of some fermentation products (such as ethanol fermentation). Therefore, this strain can serve as an excellent fermentation cell for some products.

[0032] 4. Culture method of Icelandic sulfur leaf fungus strain: Because strain E233 is a uracil-deficient strain, the normal wild-type strain was cultured on MTSVU medium; during the acclimatization process, different concentrations of MTVU-CMC medium with sodium carboxymethyl cellulose as the sole carbon source were used. The specific preparation method for the culture media is as follows: MTSVU medium (1 L): 2 g tryptone, 3 g ammonium sulfate, 10 mL potassium salt, 1 mL calcium and magnesium ions, 1 mL trace elements, and distilled water are added to a final volume of 800 mL. The mixture is stirred thoroughly to dissolve. The pH is adjusted to 3.1-3.3 with concentrated sulfuric acid. The final volume is brought to 1 L with distilled water. The mixture is autoclaved at 121 °C for 20 min. After cooling, 1 mL of vitamin mixture is added. Different carbon sources can be added as needed, with a concentration of 20% (w / v). 10 mL of 20% (w / v) sucrose (MTSV medium) is added. For uracil auxotrophic strains, uracil can be added to a final concentration of 10 μg / mL.

[0033] MTVU-CMC medium (1 L): First, prepare a 2% CMC stock solution by dissolving 20 g of CMC in 1 L of distilled water. Because the 2% concentration is high, heating is required during the dissolution process. After preparation, autoclave at 121 ℃ for 20 min. After cooling, the 2% CMC stock solution is obtained and used to prepare subsequent CMC culture media.

[0034] Dissolve 2 g tryptone, 3 g ammonium sulfate, 10 mL potassium salt, 1 mL calcium magnesium ion, and 1 mL trace element in a small amount of distilled water. Then, add the required amount of 2% CMC stock solution calculated according to the CMC concentration required for the culture medium. Adjust the pH to 3.1-3.3 with concentrated sulfuric acid, and bring the volume to 1 L with distilled water. Autoclave at 121 ℃ for 20 min. After cooling, add 1 mL vitamin mixture and 10 mL 20% (w / v) uracil to a final concentration of 10 μg / mL.

[0035] Potassium salt formula: 25 g K2SO4, 5 g KCl, 35 g g glycine.

[0036] Trace element formula: Add 0.8 g of MnCl2·4H2O, 2.1 g of Na2B4O7·10H2O, 0.11 g of ZnSO4·7H2O, 0.025 g of CuSO4·5H2O, 0.015 g of Na2MoO4·2H2O, 0.015 g of VOSO4·5H2O, 0.005 g of CoSO4·7H2O, 0.005 g of NiSO4·6H2O, and 2 g of FeSO4·7H2O. Dissolve in distilled water, then filter and sterilize using a 0.22 μm filter membrane.

[0037] Calcium and magnesium ion formula: Ca(NO3)2·4H2O 10.9 g, MgCl2·6H2O 203.3 g.

[0038] Vitamin Mixture Formula: Niacin 10 mg, pantothenic acid 10 mg.

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Claims

1. A strain of *Sulphurella asiatica* E233CD1, characterized by: This strain is *Sulfophyllum ibex*, a type of *Acidophilus thermophilus*. Saccharolobus islandicus It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 31, 2023, with accession number CGMCC NO.26354.

2. Use of the acidophilic thermophilic Iceland sulfide strain E233CD1 as described in claim 1 in the degradation of sodium carboxymethyl cellulose.

3. A sodium carboxymethyl cellulose degrading agent, characterized in that: It contains the strain E233CD1 as described in claim 1.

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