A type of *Protozoa zonatifida* HY-4 that degrades n-tetradecane and its application in environmental remediation.

Abstract

This invention discloses a strain of Protozoa zonae with the ability to degrade n-tetradecane (Tetrazane). Prototheca zopfii HY-4 and its applications. The algal strain has been deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252337. Single-factor degradation experiments showed that the algal strain could degrade n-tetradecane in an initial volume concentration range of 0.1%–5% (v / v); it exhibited a high degradation rate at an initial volume concentration of 0.5% (v / v) and an inoculum size of 2% (v / v); and it maintained a certain degradation activity within a pH range of 5–10. This invention can be used to prepare microbial agents for degrading n-tetradecane and is suitable for the remediation of wastewater or soil contaminated with n-tetradecane.

Description

Technical Field

[0001] This invention relates to the fields of environmental bioengineering, microbiology, and pollution ecological remediation technology. Specifically, it relates to a highly efficient n-tetradecane-degrading algal strain isolated from polluted environments—*Protocera zonata*. Prototheca zopfii HY-4 and its applications. Background Technology

[0002] n-Tetradecane ( n Tetradecane (n-tetradecane) is a typical medium- to long-chain straight-chain alkane, widely found in industrial solvents, chemical raw materials, lubricating oils, and fossil fuels. During industrial production, chemical product storage and transportation, and oily wastewater discharge, n-tetradecane may leak, be discharged, or migrate into aquatic and soil environments. Due to its low hydrophilicity and relatively stable chemical properties, n-tetradecane does not readily volatilize or degrade in the natural environment, easily accumulating in soil pores or oily wastewater systems. This can negatively impact soil structure, pore aeration, and the aquatic ecosystem, placing pressure on the ecological restoration of polluted areas.

[0003] Traditional physical and chemical remediation technologies for treating alkane pollution typically include thermal desorption, chemical oxidation, solvent elution, and adsorption separation. While these technologies can reduce pollutant concentrations under certain conditions, they often suffer from high engineering costs, significant disturbance during the remediation process, substantial impact on the native microbial ecosystem, and potential secondary pollution in practical applications. In contrast, microbial remediation technology utilizes the metabolic activity of functional microorganisms to transform or mineralize alkane pollutants such as n-tetradecane into low-toxicity products, cell biomass, carbon dioxide, and water. It offers advantages such as better environmental compatibility, milder operating conditions, and suitability for in-situ remediation, thus showing promising application prospects in the treatment of alkane pollutants.

[0004] However, in actual site remediation processes, conventional degrading microorganisms still face certain limitations when dealing with hydrophobic alkane pollutants such as n-tetradecane.

[0005] First, there is insufficient tolerance to higher concentrations of substrates. Alkane contaminants such as n-tetradecane may exist at high concentrations around oil storage facilities, in refining wastewater treatment systems, or in areas of localized leaks. High concentrations of alkanes can adversely affect the cell membrane structure and metabolic activity of microorganisms, leading to a decline in the activity of functional microorganisms and impacting the system's ability to sustain degradation.

[0006] Second, the bioavailability of pollutants is low. n-Tetradecane has low hydrophilicity and limited solubility in aqueous systems, readily existing in the oil phase or adsorbed form. Microorganisms have difficulty fully contacting the hydrophobic substrate, leading to limited substrate mass transfer and thus affecting the efficiency of alkane uptake, transformation, and degradation.

[0007] Third, changes in environmental pH conditions can affect degradation efficiency. The pH conditions at actual remediation sites are often influenced by factors such as industrial wastewater, soil buffering capacity, and pollutant composition, potentially resulting in an acidic or alkaline environment. Some conventional degrading bacteria are quite sensitive to pH changes; when deviating from their optimal range, their growth activity and degradation capacity may significantly decrease, limiting their application in complex polluted environments.

[0008] Protozoanthellae ( Prototheca zopfii As a chlorophyll-free unicellular algae, its cell structure and environmental adaptation characteristics differ from those of conventional bacteria, potentially giving it a certain ability to adapt to polluted environments. Therefore, screening protozoan algal strains capable of utilizing n-tetradecane and maintaining degradation activity within a certain concentration and pH range from polluted environments has practical significance and application value for enriching alkane-degrading microbial resources and expanding bioremediation technology pathways for n-tetradecane-contaminated wastewater and soil. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing bioremediation technologies, such as the poor tolerance of conventional bacteria to high concentrations of n-tetradecane toxicity, low utilization of hydrophobic substrates, and narrow adaptability to environmental pH, and to provide a *Protozoa zonata* (… Prototheca zopfii HY-4 and its applications.

[0010] This algal strain was able to grow in a culture system with n-tetradecane as the main carbon source and exhibited n-tetradecane degradation capabilities. The oil expulsion zone test was positive, suggesting that it may produce extracellular active substances that facilitate the emulsification or dispersion of hydrophobic substrates. A capsule-like coating structure was observed around the cell periphery of the algal strain, and it maintained its degradation activity within a certain range of n-tetradecane concentration and pH.

[0011] Another objective of this invention is to investigate the degradation patterns of the HY-4 algal strain under different initial substrate concentrations, different inoculum amounts, and different initial environmental pH values, so as to provide scientific and reliable process parameter guidance for its large-scale application in actual environmental engineering remediation.

[0012] To achieve the above objectives, the present invention employs the following detailed technical solution: (a) Classification, nomenclature and preservation information of algal strains This invention discloses a strain of *Protozoa zonata* HY-4 with the ability to degrade n-tetradecane. *Protozoa zonata* HY-4 is named... Prototheca zopfii HY-4 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, 430072, China. Accession number: CCTCC NO: M 20252337. Deposit date: October 24, 2025.

[0013] The 18S rDNA nucleotide sequence of the strain described in this invention is shown in SEQ ID NO.1: The *Protozoa HY-4* was separated from activated sludge collected from refinery wastewater.

[0014] (II) Morphological and physiological-biochemical characteristics of algal strains The present invention Prototheca zopfii The morphological and physiological-biochemical characteristics of HY-4 algal strain are as follows: Cellular morphology characteristics: Under an optical microscope, the cells of the HY-4 algal strain appeared as regular spherical shapes; after standard Gram staining, the result was a false positive (G+). Further observation confirmed that the algal strain lacked flagella and the ability to produce spores, but the cells were surrounded by a distinct capsule structure.

[0015] Physiological and biochemical metabolic characteristics: Biochemical reaction tests showed that the algal strain was positive for catalase, indicating that it has a certain ability to decompose hydrogen peroxide; the oil excretion zone test was positive, suggesting that it may produce extracellular active substances that are beneficial to the emulsification or dispersion of hydrophobic substrates. Among other routine biochemical indicators, the gelatin liquefaction test was negative, the methyl red (MR) test was inapplicable or negative, the VP test was negative, and the starch hydrolysis test was also negative.

[0016] (III) Degradation characteristics and optimal application parameters of HY-4 algal strain This invention systematically investigated the degradation performance of the HY-4 algal strain under different initial concentrations of n-tetradecane, different inoculum amounts, and different initial pH conditions through single-factor variable control experiments, clarifying the degradation pattern and suitable application parameters of this algal strain in the treatment of n-tetradecane pollutants: (1) Effect of initial substrate concentration (n-tetradecane): Under low concentration (0.1% v / v) of n-tetradecane, the algal strain exhibited high degradation capacity, with a degradation rate of 96% to 97%. When the initial n-tetradecane concentration increased to 0.5% v / v, the degradation rate remained within the range of 78% to 82%, indicating that the HY-4 algal strain had good substrate utilization capacity under low to medium concentrations of n-tetradecane. As the n-tetradecane concentration further increased to 1% to 5% v / v, the degradation rate of the algal strain changed, but remained within the range of 64% to 82%, with a degradation rate of 64% to 67% maintained under 5% v / v n-tetradecane conditions. These results indicate that the HY-4 algal strain can not only effectively utilize low concentrations of n-tetradecane, but also maintain a certain degradation activity under higher concentrations of n-tetradecane, demonstrating its good adaptability to different n-tetradecane concentrations.

[0017] (2) Effect of Initial Inoculation Amount: With the increase of the inoculation amount of HY-4 algae, the degradation rate of n-tetradecane generally showed an upward trend, indicating that appropriately increasing the initial inoculation amount helps to increase the effective biomass in the system, thereby improving the degradation efficiency of n-tetradecane. When the inoculation amount reached 2% (v / v), the degradation rate of the system reached 86.37% to 88.92%, which has entered a relatively high degradation level. When the inoculation amount continued to increase to 3% and 5% (v / v), the degradation rate further slowly increased to about 90%-92%, but the increase was relatively limited. Considering the degradation effect, the amount of bacterial agent added, and the cost of subsequent engineering applications, under the conditions of this experiment, an inoculation amount of 2% (v / v) can be regarded as a more suitable initial inoculation ratio for HY-4 algae to treat n-tetradecane pollutants, providing a reference for subsequent optimization of bacterial agent addition and setting of application parameters.

[0018] (3) The influence of initial environmental pH: The HY-4 algal strain exhibited a certain ability to degrade n-tetradecane within the pH range of 5 to 10, indicating that the algal strain has a certain adaptability to different acid and alkaline conditions. In a slightly acidic environment, the algal strain maintained high degradation activity at pH=5 and pH=6, with the highest degradation rate reaching 95%-96%, indicating that slightly acidic conditions did not completely inhibit its n-tetradecane utilization ability. Meanwhile, under slightly alkaline and strongly alkaline conditions, i.e., within the pH range of 8 to 10, the HY-4 algal strain maintained relatively stable degradation performance, with degradation rates remaining above 83%. Among them, the degradation rate was higher at pH=9, reaching 93.85%-98.49%; the degradation rate at pH=10 was 92.16%. The above results indicate that the HY-4 algal strain exhibits certain degradation activity under different initial pH conditions, and can maintain good n-tetradecane degradation capacity under slightly acidic and slightly alkaline conditions. It can provide candidate algal strains and parameter basis for the bioremediation of n-tetradecane-contaminated wastewater or soil under complex pH conditions.

[0019] (iv) A microbial agent, wherein the microbial agent uses the aforementioned Protozoa HY-4 as the active ingredient.

[0020] (v) The application of the aforementioned Protozoan HY-4 or the aforementioned microbial agent in the degradation of n-tetradecane.

[0021] The specific application involves adjusting the *Protozoa HY-4* algal suspension to... FROM 600 With a value of 0.95-1.05, it is inoculated into an environment containing n-tetradecane with an initial volume concentration not exceeding 5%, and degrades n-tetradecane in the environment under conditions of pH 5-10 and temperature of 28-32℃.

[0022] The inoculum size of the *Protozoa hy-4* is 1%-5% (v / v).

[0023] The environment is wastewater and / or soil.

[0024] The beneficial effects of this invention are as follows: 1. The HY-4 algal strain provided by this invention can grow in a culture system with n-tetradecane as the main carbon source and exhibits degradation ability within a certain range of n-tetradecane concentration. Experimental results show that this algal strain has a high degradation rate under low concentration of n-tetradecane and can be used as a candidate functional microbial resource for the remediation of n-tetradecane-contaminated environments.

[0025] 2. The HY-4 algal strain showed a positive oil film diffusion test, indicating that it possesses a certain oil film diffusion capacity, suggesting that this algal strain may produce extracellular active substances that are beneficial for the emulsification or dispersion of hydrophobic substrates. This characteristic helps to enhance the contact between the algal strain and n-tetradecane, thereby alleviating the problem of limited mass transfer of hydrophobic alkanes in aqueous systems.

[0026] 3. The HY-4 algal strain exhibited degradation ability within an initial volume concentration range of 0.1%-5% (v / v) of n-tetradecane. Notably, it maintained some degradation activity even at 5% (v / v) n-tetradecane concentrations, indicating its adaptability to higher concentrations of n-tetradecane.

[0027] 4. The HY-4 algal strain maintained its n-tetradecane degradation activity under pH conditions of 5-10, indicating that it has a certain adaptability to different acid and alkaline environments and can provide a candidate algal strain for the bioremediation of n-tetradecane-contaminated wastewater or soil under complex pH conditions.

[0028] 5. Under the condition of an initial volume concentration of n-tetradecane of 0.5% (v / v), the degradation effect of HY-4 algal suspension was better when the inoculum amount was 2% (v / v). This result can provide a reference for subsequent optimization of microbial agent dosage and setting of engineering application parameters. Attached Figure Description

[0029] Figure 1 For the present invention Prototheca zopfii SEM image of HY-4 algal strain.

[0030] Figure 2 A trend graph showing the effect of different initial n-tetradecane concentrations as a single-factor variable on the degradation rate of HY-4 algae.

[0031] Figure 3 A trend graph showing the effect of different initial inoculum amounts as a single-factor variable on the degradation efficiency of the HY-4 algae strain.

[0032] Figure 4A trend graph showing the effect of different initial environmental pH values ​​as a single-factor variable on the n-tetradecane degradation performance of the HY-4 algal strain. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto: The culture medium formulation used in the specific embodiments of the present invention is as follows: LB medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, pH 7.0, autoclaved at 121℃ for 20 min.

[0034] Inorganic salt culture medium (g / L): K2HPO4 1.0, KH2PO4 1.0, NH4NO3 1.0, MgSO4·7H2O 0.3, CaCl2 0.03, FeSO4 0.005, ZnSO4 0.002, MnSO4 0.0002, autoclaved at 121 ℃ for 20 min.

[0035] Example 1: Isolation, purification, microbiological morphology, physiological and biochemical characteristics, and molecular identification of HY-4 algal strain 1. Enrichment, separation, and purification of algal strains: Activated sludge was collected from wastewater at an oil refinery in Jiangsu Province, China. After settling for 24 hours, 5 mL of the supernatant was inoculated into 100 mL of inorganic salt medium with n-tetradecane as the sole carbon source. After incubation at 30 °C and 160 rpm for 7 days, 5 mL of the suspension was inoculated into 100 mL of fresh inorganic salt medium and incubated at 30 °C and 160 rpm for 7 days. This incubation process was repeated 3–5 times. A certain amount of the culture medium was then serially diluted (range set at 10⁻⁶). -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 The obtained 100 μL solutions of different concentration gradients were purified by repeated streak plating on an inorganic salt solid selective medium with n-tetradecane as the sole carbon source, ultimately yielding morphologically uniform single bacteria / algal colonies, which are the [cells / algae]. Prototheca zopfii HY-4 algal strain.

[0036] 2. Morphological and microscopic observation: Fresh, isolated, and purified algal colonies were selected, smeared, dried, and fixed, then Gram-stained and observed under an optical microscope. The results confirmed that HY-4 cells under the microscope exhibited a typical spherical structure; the cells showed false-positive staining (G+). Further observation confirmed that this algal strain lacked flagella and spore production, but had a distinct capsule covering the outer surface of the cells. Prototheca zopfii Electron microscopy results of HY-4 algal strain are as follows: Figure 1 As shown.

[0037] 3. Determination of key physiological and biochemical indicators: The metabolic capacity of the purified HY-4 algal strain was analyzed using conventional bacterial physiological and biochemical identification methods. The tests included gelatin liquefaction test, methyl red test, VP test, starch hydrolysis test, catalase test, and oil expulsion ring test.

[0038] The test results are shown in Table 1.

[0039] Table 1 Algal strains Prototheca zopfii Summary table of physiological and biochemical characteristics of HY-4. Table 1 shows the results: Gelatin liquefaction test: no liquefaction was observed, the result was negative. Methyl red (MR) test: the result was not applicable or negative. VP test: no red reaction was observed, the result was negative. Hydrolyzed starch test: no transparent hydrolysis zone appeared around the colony, the result was negative. Catalase test: a large number of bubbles were produced after adding hydrogen peroxide solution, the result was positive. Oil expulsion zone test: HY-4 culture medium was inoculated on a plate covered with a hydrophobic oil film. After incubation, a clear transparent "oil expulsion zone" appeared around the colony, pushing the oil film outward, the result was positive.

[0040] 4. Molecular identification and preservation of algal strain HY-4: The purified HY-4 isolate was sent to Zhejiang Tianke High-Tech Development Co., Ltd. in fresh culture medium for molecular identification. The testing unit performed nucleic acid extraction, PCR amplification, product purification, and Sanger sequencing according to the sample type. Given that HY-4 exhibited characteristics of eukaryotic algae isolates in colony morphology, cell morphology, and subsequent sequence alignment, molecular identification employed amplification and sequencing of the 18S rDNA region, which is suitable for eukaryotic algae. The sequenced sequences were then compared with [other sequences] using BLAST. Prototheca zopfii The related sequences showed high similarity. Based on morphological characteristics and sequence alignment results, HY-4 was identified as... Prototheca zopfii .

[0041] The 18S rDNA sequence of algal strain HY-4 is shown in SEQ ID NO.1: Homology comparison of this sequence with gene sequences in GenBank revealed that it belongs to... Prototheca zopfii The similarity is extremely high. Based on sequencing results and physiological and biochemical test results, the algal strain HY-4 was determined to belong to... Prototheca zopfii Therefore, this strain was named *Protozoanus zonifolius* (…). Prototheca zopfii HY-4, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252337, deposited on October 24, 2025, at Wuhan University, Wuhan, China, 430072, China.

[0042] Example 2: Effect of initial n-tetradecane concentration on the degradation performance of HY-4 algal strain Standard inorganic salt liquid culture medium was prepared, and different volume percentages of n-tetradecane were added as the sole carbon source to achieve initial concentrations of 0.1%, 0.5%, 1%, 2%, 3%, and 5%, respectively. The HY-4 algal strain culture was centrifuged to collect the bacterial cells, washed twice with sterile inorganic salt culture medium, and resuspended. The algal suspension concentration was adjusted to OD0.05. 600 = 1.0 ± 0.05. Subsequently, algal suspension was added at an inoculum rate of 1% (v / v), i.e., 1 mL of algal suspension was added to each bottle. Each treatment group was cultured at 30 ℃ and 160 rpm with constant temperature shaking for 15 days. After culture, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - Ct) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, and Ct is the residual concentration of n-tetradecane after the culture ends.

[0043] The results are as follows Figure 2 As shown in the figure. Experimental results indicate that algal strain HY-4 exhibits high degradation ability under low concentrations of n-tetradecane (0.1% v / v), with a degradation rate of 96%-97%. When the initial concentration of n-tetradecane is increased to 0.5% (v / v) and 1% (v / v), the degradation rate remains within the range of 78%-82%. With further increases in n-tetradecane concentration, the degradation rate of HY-4 changes, but it still maintains a degradation rate of 64%-67% under 5% (v / v) n-tetradecane conditions. These results demonstrate that algal strain HY-4 can utilize not only low concentrations of n-tetradecane but also maintain a certain level of degradation activity under higher concentrations, indicating its adaptability to different n-tetradecane concentrations.

[0044] Example 3: Effect of inoculum ratio on the n-tetradecane degradation efficiency of HY-4 algal strain In an inorganic salt liquid culture medium containing a uniform initial concentration of n-tetradecane, the effect of different inoculum sizes on the degradation of n-tetradecane was investigated using HY-4 algae as the inoculum. The HY-4 algae culture was centrifuged to collect the cells, washed twice with sterile inorganic salt medium, and resuspended. The concentration of the algal suspension was adjusted to... FROM 600 = 1.0 ± 0.05. The initial addition of n-tetradecane to each treatment group was 0.5 mL, i.e., the initial pollutant concentration was 0.5% (v / v). Subsequently, the inoculum was added to the culture system at inoculum rates of 0.1%, 0.5%, 1%, 1.5%, 2%, 3%, and 5% (v / v), respectively. Each treatment group was cultured at 30 ℃ and 160 rpm for 15 days with constant temperature shaking. After the culture was completed, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C) t ) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, C t This represents the residual concentration of n-tetradecane after the culture period.

[0045] Experimental results are as follows Figure 3 As shown, the degradation rate of n-tetradecane by the HY-4 algae strain generally increased with the increase of the initial inoculum size. At an inoculum size of 1.5% (v / v), the degradation rate reached approximately 80%; when the inoculum size reached 2% (v / v), the degradation rate reached 86.37%-88.92%, which is already at a relatively high level. Further increasing the inoculum size to 3% (v / v) and 5% (v / v) only slightly increased the degradation rate to around 90%-92%, indicating that further increasing the inoculum size had limited effect on improving degradation efficiency. Considering both the degradation effect and the amount of inoculant added, under the conditions of this experiment, a 2% (v / v) inoculum size can be considered a suitable initial inoculum size for the HY-4 algae strain to degrade n-tetradecane.

[0046] Example 4: Effects of environmental pH stress on the n-tetradecane degradation performance of HY-4 algal strain The initial pH values ​​of multiple groups of culture media containing equal amounts of n-tetradecane (each group with an initial n-tetradecane concentration of 0.5%) were adjusted to 5, 6, 7, 8, 9, and 10, respectively. The HY-4 algal strain culture media were centrifuged to collect the bacterial cells, washed twice with sterile inorganic salt medium, and resuspended. The algal suspension concentration was adjusted to... FROM 600= 1.0 ± 0.05. Subsequently, 2.0 mL of HY-4 algal suspension was added to each treatment group, representing an inoculum size of 2% (v / v). Each group was cultured at 30 ℃ and 160 rpm for 15 days with constant temperature shaking. After culture, n-tetradecane in the culture system was extracted with the organic solvent n-hexane, and the residual amount of n-tetradecane was determined by gas chromatography. The degradation rate was calculated based on the initial concentration. The n-tetradecane degradation rate was calculated using the following formula: Degradation rate (%) = (C0 - C) t ) / C0 × 100% Where C0 is the initial concentration of n-tetradecane, C t This represents the residual concentration of n-tetradecane after the culture period.

[0047] The results are as follows Figure 4 As shown, the HY-4 algal strain maintained a certain level of n-tetradecane degradation activity within a pH range of 5-10, indicating that the strain has a certain adaptability to different initial pH conditions. Under slightly alkaline conditions, its degradation efficiency was relatively stable, with the pH 9 treatment group showing a higher degradation rate. These results suggest that the HY-4 algal strain can serve as a candidate functional algal strain for the remediation of n-tetradecane-contaminated environments under complex pH conditions.

[0048] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any equivalent substitutions, modifications, or alterations made by those skilled in the art based on the technical content disclosed in the present invention, as long as they do not depart from the technical solution and spirit of the present invention, should fall within the protection scope of the present invention.

Claims

1. A *Protozoa zonae* HY-4 that degrades n-tetradecane, characterized in that, The *Protozoa zonata* HY-4 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252337, deposit date: October 24, 2025; named... Protothecazopfii HY-4.

2. The *Protozoa HY-4* according to claim 1, characterized in that, The 18S rDNA nucleotide sequence of the *Protozoa HY-4* is shown in SEQ ID NO.

1.

3. The *Protozoa HY-4* according to claim 1, characterized in that, The *Protozoa HY-4* was separated from activated sludge collected from refinery wastewater.

4. The *Protozoa HY-4* according to claim 1, characterized in that, Its cells are spherical, show false positive results in Gram staining, have a capsule structure, and are without flagella or spores.

5. A microbial inoculant, characterized in that, The microbial agent uses Protozoa HY-4 as the active ingredient, as described in any one of claims 1 to 4.

6. The use of Protozoania HY-4 as described in any one of claims 1 to 4 or the microbial agent as described in claim 5 in the degradation of n-tetradecane.

7. The application according to claim 6, characterized in that, The specific application involves adjusting the *Protozoa HY-4* algal suspension to... OD 600 With a value of 0.95-1.05, the inoculation is carried out in an environment where the initial volume concentration of n-tetradecane does not exceed 5% (v / v), and the n-tetradecane is degraded under conditions of pH 5-10 and temperature of 28-32 ℃.

8. The application according to claim 7, characterized in that, The inoculum size of the *Protozoa hy-4* is 1%-5% (v / v).

9. The application according to claim 7, characterized in that, The environment is wastewater and / or soil.

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