Polyacrylamide-degrading bacterium

Abstract

A highly efficient polyacrylamide-degrading bacterium, belonging to *Tistrella bauzanensis* (accession number CCTCM20221847), was isolated from fracturing fluid flowback fluid in an oilfield. After 7 days of cultivation in a selective liquid medium with polyacrylamide as the sole nutrient source, the strain significantly degraded HPAM. After optimizing the degradation conditions, under the following conditions: 175 rpm, 37°C, 3% (v / v) inoculum, pH 3.0, urea as the nitrogen source, glucose as the carbon source, and the addition of 0.05 g / L CuCl2, 0.1 g / L CaCl2, and 0.1 g / L ZnSO4, a degradation efficiency of 72.7% was achieved, exceeding the approximately 40% degradation rate reported in other literature, thus realizing highly efficient HPAM degradation. GPC and FIRT results confirmed the significant degradation of HPAM. This strain enables low-cost, pollution-free, and highly efficient degradation of HPAM.

Description

Technical Field

[0001] This invention relates to a strain of Tistrella bauzanensis that is highly efficient at degrading polyacrylamide, belonging to the field of industrial microbial technology. Background Technology

[0002] Polyacrylamide (PAM) is a general term for polymers obtained by copolymerizing acrylamide homopolymer with other monomers. It is a linear, water-soluble polymer that can be classified into nonionic, cationic, and anionic types. Water-soluble anionic polyacrylamide (HPAM) is widely used in oilfield enhanced oil recovery as an additive in drilling and fracturing fluids. By injecting an aqueous solution of polyacrylamide, the oil-water mobility ratio is improved, thereby increasing oil recovery. HPAM can also be used as a water-blocking agent and profile control agent to effectively seal high water-cut formations.

[0003] With the increasing use of HPAM, its residues have caused environmental pollution problems, such as landfill blockage and water pollution. Although polyacrylamide itself is non-toxic, its monomer, acrylamide, is a water-soluble toxic substance. Once it enters surface water and groundwater, it can be absorbed through the skin, mucous membranes, respiratory tract, and mouth, causing neurological poisoning and leading to sensory and motor disorders. Long-term exposure to acrylamide can lead to acute, subacute, and chronic poisoning. Under natural conditions, polyacrylamide undergoes very slow degradation, producing toxic acrylamide monomers, which cause long-term environmental harm.

[0004] Biodegradation of polyacrylamide is an important degradation method due to its advantages of low cost, no pollution, and high efficiency. However, due to the complexity of the strain's growth environment and the recalcitrant nature of polymers, the degradation efficiency of currently screened degrading bacteria in high-concentration polymer systems is generally low, mostly ranging from 10% to 40%. Highly efficient degrading strains are key to establishing efficient polyacrylamide biodegradation technology. Summary of the Invention

[0005] One object of the present invention is to provide a novel polyacrylamide-efficient degrading bacterium.

[0006] Another objective of this invention is to provide an optimized degradation process for degrading bacteria, which can significantly improve the degradation efficiency of polyacrylamide by Tistrella bauzanensis.

[0007] The present invention discloses a highly efficient polyacrylamide-degrading bacterium, characterized in that the strain is *Tistrella bauzanensis*, which was deposited at the China Center for Type Culture Collection (Wuhan University) on November 30, 2022, with accession number CCTCC M 20221847.

[0008] The degrading bacterium Tistrella bauzanensis provided by this invention was isolated from oilfield fracturing fluid flowback fluid. After being cultured on tryptic soybean peptone agar (TSB) plates at 37 °C for 48 h, the colony morphology was milky white, oval, with a raised center, viscous, opaque, with neat edges and a smooth surface.

[0009] The morphological characteristics of the Tistrella bauzanensis strain provided by this invention are: Gram-negative, short rod-shaped cells, no flagella, and no spores.

[0010] The physiological and biochemical characteristics of the *Tistrella bauzanensis* strain provided by this invention are as follows: growth temperature is 20-45 ℃, growth pH is 3-9, and it is positive for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H2S production, urease, and gelatin hydrolysis tests, while it is negative for oxidase, VP, indole production, β-galactosidase, and citrate utilization tests.

[0011] The 16S rRNA gene sequence characteristics of *Tistrella bauzanensis* provided by this invention are as follows: *Tistrella bauzanensis* was inoculated into TSB medium and cultured at 37 ℃ and 175 rpm for 36–40 h. Genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). PCR amplification was performed using the upstream primer 5'-AGAGTTTGATCMTGGCTCAG-3' and the downstream primer 5'-TACGGYTACCTTGTTACGACTT-3' to obtain the 16S rRNA gene fragment. The PCR conditions were as follows: 94 ℃ for 5 min; 94 ℃ for 45 s, 56 ℃ for 45 s, 72 ℃ for 90 s, 30 cycles; 72 ℃ for 10 min. The amplified product was sequenced by Shanghai Sangon Biotech Co., Ltd. The obtained homologous sequences were analyzed by BLAST on the NCBI website. A phylogenetic tree of the strain was constructed using MEGA 7.0 software and ClustalX 2.0. Sequencing results showed that the 16S rRNA gene sequence of this strain was 1402 bp in length, and had the highest similarity (99.5%) to Tistrella bauzanensis (accession number: NR_117256.1). Its 16S rRNA gene sequence is shown in the sequence listing.

[0012] The screening process for Tistrella bauzanensis provided by this invention includes the following steps:

[0013] Enrichment: To maximize the selection of the target bacteria, the bacteria were enriched twice in the beef extract peptone enrichment medium to obtain an enriched mixed bacteria.

[0014] Acclimation: The enriched mixed bacteria were transferred to a selective liquid medium with polyacrylamide as the sole nutrient source and gradually acclimated in selective liquid medium with increasing polyacrylamide concentrations of 0.3 g / L, 0.5 g / L, and 1.0 g / L for a total of three acclimation cycles, each lasting 7 days.

[0015] Isolation: Under aseptic conditions, use a pipette to aspirate the acclimated culture medium, serially dilute it with sterile water, and spread it onto selective solid culture medium. Incubate at 37 °C for an appropriate time. After colonies have grown on the plates, select colonies with good growth and different morphologies.

[0016] Purification: The obtained colonies were repeatedly streaked across four zones of plates until a single strain was obtained. The degradation rate of polyacrylamide was determined using the starch-chromium iodide method, and the change in polyacrylamide concentration was used as the evaluation index to screen for highly efficient polyacrylamide-degrading bacteria, *Tistrella bauzanensis*.

[0017] The optimized degradation process of Tistrella bauzanensis strain provided by this invention includes the following steps:

[0018] Based on the selective liquid culture medium, the inoculation time, shaking speed, culture temperature, inoculum size, initial pH of the culture medium, nitrogen source composition, carbon source composition, and metal ion composition of the culture medium were optimized. Except for the process conditions to be optimized, the other conditions were as determined after optimization in the previous experiment.

[0019] Growth curves were plotted based on the biomass of the strains, and strains in the logarithmic growth phase were selected for inoculation and subsequent measurements.

[0020] Different shaking speeds were set, and after culturing in selective liquid medium for 7 days, the bacterial concentration and HPAM degradation rate were measured to determine the optimal shaking speed.

[0021] By setting different culture temperatures and culturing in selective liquid medium for 7 days, the optimal culture temperature was determined by measuring bacterial concentration and HPAM degradation rate.

[0022] Different inoculum sizes were set, and after culturing in selective liquid medium for 7 days, the optimal inoculum size was determined by measuring bacterial concentration and HPAM degradation rate.

[0023] Different initial pH values ​​were set for different culture media. After culturing in selective liquid medium for 7 days, the optimal pH was determined by measuring bacterial concentration and HPAM degradation rate.

[0024] Different nitrogen sources, including peptone, ammonium sulfate, yeast extract, urea, ammonium nitrate, corn syrup, ammonium chloride, and ammonium bicarbonate, were used for optimization screening. After culturing in selective liquid medium for 7 days, the optimal nitrogen source was determined by measuring bacterial concentration and HPAM degradation rate.

[0025] Different carbon sources, including glucose, sucrose, fructose, D-mannose, maltose, D-galactose, soluble starch, lactose, D-cellobiose, cellulose, and liquid paraffin, were used for optimization screening. After culturing in selective liquid medium for 7 days, the optimal carbon source was determined by measuring bacterial concentration and HPAM degradation rate.

[0026] Different concentrations of copper chloride, calcium chloride, and zinc sulfate were used for optimization screening. After culturing in selective liquid culture medium for 7 days, the optimal metal ion species were determined by measuring bacterial concentration and HPAM degradation rate.

[0027] Advantages of this invention:

[0028] The *Tistrella bauzanensis* strain provided by this invention was isolated from fracturing fluid flowback fluid in oil fields and exhibits good tolerance to high concentrations of polyacrylamide.

[0029] By employing effective fermentation control methods to provide suitable dissolved oxygen, pH, temperature, biomass, carbon source, nitrogen source, and trace elements for cell growth, Tistrella bauzanensis achieved a degradation rate of 72.7% for HPAM, thus realizing highly efficient degradation of HPAM.

[0030] The degrading bacteria significantly reduced the molecular weight of polyacrylamide from 3.7 × 10⁻⁶ before degradation. 6 Da decreased to 1.87 × 10 5 Da. FTIR results showed that the amide group of HPAM was converted into a carboxyl group during biodegradation. Attached Figure Description

[0031] Figure 1 This is a diagram of Tistrella bauzanensis coated on a TSB plate;

[0032] Figure 2 This is a growth curve of the Tistrella bauzanensis strain;

[0033] Figure 3The graph shows the biomass and polyacrylamide removal rate of Tistrella bauzanensis strain under different (a) rotation speed (b) inoculum size (c) temperature (d) pH conditions.

[0034] Figure 4 The graph shows the biomass and polyacrylamide removal rate of Tistrella bauzanensis strain under different (a) nitrogen source (b) carbon source (c) metal ion culture conditions.

[0035] Figure 5 This is a comparison of the infrared spectra of Tistrella bauzanensis strain before and after degradation. Detailed Implementation

[0036] Example 1 Screening of highly efficient polyacrylamide-degrading bacteria

[0037] Enrichment medium: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1000 mL distilled water, pH 7.0 ± 0.2.

[0038] Selective liquid culture medium: 1.0 g polyacrylamide, 1.6 g K2HPO4·3H2O, 0.4 g KH2PO4, 0.06 g MgSO4, 0.01 g CaCl2, 0.5 g NaCl, 0.005 mg CuSO4, 0.005 mg ZnSO4, 0.003 mg Na2BO4, 1000 mL distilled water, pH 7.0 ± 0.2.

[0039] Selective solid culture medium: 1.0 g polyacrylamide, 1.6 g K2HPO4·3H2O, 0.4 g KH2PO4, 0.06 g MgSO4, 0.01 g CaCl2, 0.5 g NaCl, 0.005 mg CuSO4, 0.005 mg ZnSO4, 0.003 mg Na2BO4, 20 g agar powder, 1000 mL distilled water, pH 7.0 ± 0.2.

[0040] Tryptic soybean peptone liquid medium (TSB): 17.0 g tryptic peptone, 3.0 g plant peptone, 2.5 g K2HPO4·3H2O, 5.0 g NaCl, 2.5 g glucose, 1000 mL distilled water, pH 7.0 ± 0.2.

[0041] The upper water sample of the fracturing fluid flowback fluid was first cultured in beef extract peptone enrichment medium at 37°C and 175 rpm for 24 h. Then, 2 mL of the enrichment culture was added to fresh enrichment medium and cultured under the same conditions for 24 h to obtain the enriched mixed bacteria, thus completing the enrichment process.

[0042] Acclimation: The enriched mixed bacteria were transferred to a selective liquid medium with polyacrylamide as the sole nutrient source and gradually acclimated in selective liquid medium with increasing polyacrylamide concentrations (0.3 g / L, 0.5 g / L, and 1.0 g / L, respectively), with each acclimation cycle lasting 7 days.

[0043] Isolation: Under aseptic conditions, 0.1 mL of the acclimated culture medium was pipetted and serially diluted with sterile water, then spread onto selective solid plates and incubated at 37 °C for an appropriate time. After colonies grew on the plates, colonies with good growth and different morphologies were selected.

[0044] Purification: The obtained colonies were repeatedly streaked across four zones on plates until a single strain was obtained. The degradation rate of polyacrylamide was determined using the starch-cadmium iodide method, the specific method of which is as follows:

[0045] Preparation of HPAM standard solution: Accurately weigh 500 mg of HPAM solid, add ultrapure water to make up to 1000 mL, and gradually dilute to HPAM solutions with concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L.

[0046] Accurately pipette 5 mL of sodium acetate buffer solution into a 50 mL volumetric flask. Then, pipette 2 mL of polyacrylamide solution of different concentrations and 20 mL of distilled water into the volumetric flask and mix well. Next, add 2 mL of saturated bromine water to the volumetric flask, let it stand for ten minutes, and observe the color of the solution. The amount of bromine water added should be such that the solution turns yellow or light yellow after the reaction (ensuring excess bromine water). Prepare 5 mL of 1% sodium formate solution, add it to the mixed solution, shake well, and let it stand for 5 minutes. Prepare 5 mL of starch-cadmium iodide reagent, add it to the mixed solution, and dilute to 50 mL with distilled water. Shake well and let it stand for 10 minutes. Prepare a separate set of solutions without polyacrylamide as a reference. Then, measure the absorbance of different concentration solutions at wavelengths of 400–800 nm to determine the wavelength corresponding to the maximum absorption peak. The measurement is performed using a UV spectrophotometer. Finally, based on the measured relationship between polyacrylamide concentration and absorbance, plot a standard curve.

[0047] The degradation rate of polyacrylamide is calculated using the following formula:

[0048]

[0049] In the formula: C0 represents the mass concentration of polyacrylamide before degradation (mg / L);

[0050] C1 represents the mass concentration (mg / L) of the degraded polyacrylamide.

[0051] Using the change in polyacrylamide concentration as an evaluation index, the highly efficient polyacrylamide-degrading bacterium, Tistrella bauzanensis, was finally screened.

[0052] Example 2: Morphological and physiological-biochemical identification of the *Tistrella bauzanensis* strain provided by this invention.

[0053] The *Tistrella bauzanensis* strain provided by this invention was isolated from fracturing fluid flowback fluid and purified by repeated dilutions and plate-coating. The strain's morphology and physiological and biochemical reactions were identified using Gram staining, morphological observation, and a Gram-negative bacterial identification system.

[0054] Tistrella bauzanensis can grow single colonies on TSB plates at 37°C for 48–72 h. These colonies are milky white, oval, with a raised center, viscous, opaque, with neat edges and a smooth surface (see attached image). Figure 1 This strain is Gram-negative, with short rod-shaped cells, no flagella, and no spores. Its growth temperature is 20-45 ℃, and its growth pH is 3-9. It is positive for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H2S production test, urease, and gelatin hydrolysis test, and negative for oxidase, VP, indole production test, β-galactosidase, and citrate utilization test.

[0055] Example 3 Cloning and sequencing of the 16S rRNA gene of *Tistrella bauzanensis* provided by the present invention

[0056] The 16S rRNA gene sequence characteristics of *Tistrella bauzanensis* provided by this invention were as follows: The strain was inoculated into TSB medium and cultured at 37 ℃ and 175 rpm for 48–72 h. Genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). PCR amplification was performed using the upstream primer 5'-AGAGTTTGATCMTGGCTCAG-3' and the downstream primer 5'-TACGGYTACCTTGTTACGACTT-3' to obtain the 16S rRNA gene fragment. The PCR conditions were as follows: 94 ℃ for 5 min; 94 ℃ for 50 s, 56 ℃ for 45 s, 72 ℃ for 90 s, 30 cycles; 72 ℃ for 10 min. The PCR-amplified 16S rRNA gene fragment was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results showed that the 16S rRNA gene sequence of this strain was 1402 bp in length, and had the highest similarity (99.5%) to Tistrella bauzanensis (accession number: NR_117256.1). Its 16S rRNA gene sequence is shown in the sequence listing.

[0057] Example 4: Preparation of Seed Liquid

[0058] The *Tistrella bauzanensis* strain was streaked on TSB solid medium and incubated at 37 °C for 4 days to activate it. Once yellow colonies appeared, the culture was stored at 2–4 °C for a short period before use. A single activated colony was inoculated into a 50 mL Erlenmeyer flask containing 50 mL of TSB liquid medium and incubated at 37 °C with a shaker at 175 rpm for 36 hours to obtain a primary seed culture. This primary seed culture was then inoculated at a 2% inoculum into a 250 mL Erlenmeyer flask containing 50 mL of selective liquid medium and incubated at 37 °C with a shaker at 175 rpm for 7 days. The degradation efficiency of polyacrylamide was then determined.

[0059] Example 5: Optimization of degradation conditions by degrading bacteria

[0060] A cell growth curve was plotted based on the biomass of the strain, and the OD was measured every two hours using a UV spectrophotometer. 600 The absorbance value at nm was used to determine that 36 h was the optimal inoculation time for the bacterial strain (see Appendix). Figure 2 ).

[0061] The oscillation speeds were set to 145 rpm, 155 rpm, 165 rpm, 175 rpm, and 185 rpm, respectively. By measuring the bacterial concentration and HPAM degradation rate, 175 rpm was determined to be the optimal oscillation speed.

[0062] The temperature was controlled at 20 °C, 25 °C, 30 °C, 37 °C, and 40 °C at 175 rpm. The optimal culture temperature was determined to be 37 °C by measuring bacterial concentration and HPAM degradation rate.

[0063] Under conditions of 175 rpm and 37 ℃, different inoculum amounts (v / v) of 1%, 2%, 3%, 4%, 5%, and 6% were set. By measuring bacterial concentration and HPAM degradation rate, 3% (v / v) was determined to be the optimal inoculum amount.

[0064] The initial pH of the culture medium was adjusted to 3, 4, 5, 6, 7, 8, and 9 under conditions of 175 rpm, 37 ℃, and an inoculum size of 3% (v / v). By measuring bacterial concentration and HPAM degradation rate, pH 3.0 was determined to be the optimal pH (see Appendix). Figure 3 ).

[0065] Different nitrogen sources, including peptone, ammonium sulfate, yeast extract, urea, ammonium nitrate, corn syrup, ammonium chloride, and ammonium bicarbonate, were used for optimization screening. Urea was determined to be the optimal nitrogen source by measuring bacterial concentration and HPAM degradation rate.

[0066] Different carbon sources, including glucose, sucrose, fructose, D-mannose, maltose, D-galactose, soluble starch, lactose, D-cellobiose, cellulose, and liquid paraffin, were used for optimization screening. Glucose was determined to be the optimal carbon source by measuring bacterial concentration and HPAM degradation rate.

[0067] Different concentrations of CuCl2, CaCl2, and ZnSO4 were used for optimization screening. By measuring bacterial concentration and HPAM degradation rate, 0.05 g / L CuCl2, 0.1 g / L CaCl2, and 0.1 g / L ZnSO4 were determined to be the optimal metal ion composition (see Appendix). Figure 4 ).

[0068] Example 6 Analysis of Degradation Products

[0069] Based on the characteristic that HPAM is easily soluble in water but insoluble in most organic solvents, the degradation products of polyacrylamide were extracted with methanol. The culture medium after degradation was centrifuged at 6000 r / min for 5 min, the precipitate was discarded and the supernatant was retained. Two volumes of anhydrous ethanol were added and the mixture was shaken and mixed evenly. After standing for 5 min, the precipitate was collected by centrifugation at 3000 r / min for 5 min. The precipitate was then vacuum dried to obtain the sample to be tested.

[0070] The changes in HPAM functional groups were analyzed using a Fourier transform infrared spectroscopy (Nicolet 6700, Thermo Scientific). The FTIR spectral acquisition range was 4000 cm⁻¹. -1 ~500 cm -1 The obtained precipitate was washed three times with methanol solution and dried in a vacuum drying oven at 50 °C. The dried sample was then pressed into a KBr pellet and subjected to infrared spectroscopy analysis. The sample degraded by *Tistrella bauzanensis* showed an infrared spectrum at 3300 cm⁻¹. -1 The characteristic peaks on both sides are broader and shift to lower wavenumbers compared to the original sample, indicating amide hydrolysis during biodegradation. After biodegradation, the peak at 1666 cm⁻¹... -1 A new characteristic peak appears, which is the characteristic absorption peak of the carbonyl group. It was originally located at 1610 cm⁻¹. -1 The stretching absorption peak representing the CO bond disappears after biodegradation, at 1074 cm⁻¹. -1 A new absorption peak appeared at the point, indicating that the amide group has been degraded (see Appendix). Figure 5 FTIR results showed that the amide group of HPAM was converted into a carboxyl group during the biodegradation process.

[0071] The molecular weight of HPAM samples before and after biodegradation was determined by gel permeation chromatography (Waters 2695 HPGFC). Samples were filtered through a 0.22 μm filter before the experiment. Water was used as the mobile phase, dextran as the standard, and the flow rate was 0.5 mL / min, with a column temperature of 40 ℃. The molecular weight of HPAM before degradation was 3.7 × 10⁻⁶. 6 Da, after degradation by Tistrellabauzanensis, has a molecular weight reduced to 1.87 × 10⁻⁶. 5 Da.

Claims

1. A polyacrylamide-degrading bacterium ( Tistrella bauzanensis (), deposited at the China Center for Type Culture Collection, accession number CCTCC M 20221847.

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