Candida palmiticola strain and application thereof

Abstract

A salt- and acid-tolerant Candida palmis strain and its application, relating to the biological treatment of high-salt, high-acid, low-nitrogen, low-phosphorus organic wastewater. The strain is Candida palmis (…). Candida palmioleophila The strain CP, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 67160), has the nucleotide sequence shown in SEQ ID NO. 1. This strain can grow and maintain high metabolic activity under conditions of salinity 2%–8%, pH 2–9, temperature 15–45℃, and a wide range of carbon-nitrogen-phosphorus ratios of 100:5:1–500:5:1. This strain can be applied to treat high-salt, high-acid organic wastewater such as candied fruit wastewater, achieving not only efficient COD removal but also significant pH self-raising capabilities. It requires no neutralization agents, exhibits strong resistance to shock loads, low operating costs, and is environmentally friendly, thus solving the problem of difficult biological treatment of high-salt, high-acid, low-nitrogen-phosphorus wastewater.

Description

Technical Field

[0001] This invention relates to the biological treatment of high-salt, high-acid, low-nitrogen, low-phosphorus organic wastewater, and in particular to a salt- and acid-tolerant Candida palmis strain and its application in organic wastewater treatment. Background Technology

[0002] Various industries, including textile printing and dyeing, food processing, petroleum, and leather tanning, discharge high-salinity industrial wastewater (Phong NHV, Hao HN, Wenshan G, et al. Microalgae for saline wastewater treatment: a critical review[J]. Critical Reviews in Environmental Science and Technology, 2020, 50(12): 1224-1265.). High-salinity organic wastewater typically contains high concentrations of salts (such as K+). ＋ Na ＋ CO3 2- SO4 2- Cl - Wastewater contains organic pollutants and other potentially harmful components, and generally exhibits characteristics such as high chemical oxygen demand, extreme pH levels, and poor biodegradability. In the food manufacturing sector, factories involved in vegetable pickling, dairy production, meat product canning, and seafood processing generate concentrated wastewater with high concentrations of cations (Chen G, Talebi S, Gras S, et al. A review of salty waste stream management in the Australian dairy industry [J]. Journal of Environmental Management, 2018, 224:406-413.).

[0003] The treatment of high-salt and high-acid wastewater is a highly challenging problem in current industrial wastewater treatment, mainly due to the bottlenecks of strong biological inhibition and high operating costs. Firstly, in terms of biological treatment, the enormous osmotic pressure generated by high salt concentrations leads to dehydration and plasmolysis of microbial cells, severely damaging cell structure. Meanwhile, the highly acidic environment alters the charge state of proteins and nucleic acids, causing inactivation of key metabolic enzymes and hindering transmembrane transport of nutrients. This dual extreme environment of "high salt + strong acid" causes conventional activated sludge systems to collapse; only a very few specially acclimated, tolerant strains can barely survive, and their degradation efficiency is often unstable, greatly limiting the application of low-cost biotechnology. Furthermore, traditional physicochemical neutralization methods not only consume large amounts of alkaline agents but also generate large quantities of difficult-to-treat saline sludge, causing serious secondary pollution and a heavy burden on subsequent disposal.

[0004] Despite its challenges, biological methods offer several unique advantages in treating high-salt, high-acid wastewater. Firstly, they utilize naturally occurring microorganisms, eliminating the need for large amounts of chemical reagents and thus reducing the risk of secondary environmental pollution. Secondly, by screening and training specific salt- and acid-tolerant microorganisms, effective removal of specific pollutants can be achieved. These microorganisms remain active even under extreme conditions and can naturally degrade organic pollutants in wastewater. Furthermore, biological methods are generally more economical than physicochemical methods, especially when treating industrial wastewater requiring continuous operation, where the operating and maintenance costs of biological treatment systems are often lower. Moreover, biological treatment processes are self-regulating, offering a buffer against fluctuations in load and pollutant concentrations. Therefore, screening for degrading bacteria adapted to high-salt, high-acid organic wastewater is crucial.

[0005] Candida palmioleophila is a common fungus. Rincón et al. isolated this strain from the oil separator in the palm oil refining process. This environment is usually characterized by high organic load, low oxygen, and often acidity. The fact that it can become the dominant bacterium in such an environment and maintain a high activity of lipid degradation demonstrates its natural tolerance to the harsh environment of industrial wastewater. Furthermore, its ability to rapidly adjust its protein expression profile under different carbon source conditions shows its strong metabolic flexibility (Rincón LJ, Agualimpia B, Zafra G. Differential Protein Profiles of the Lipolytic Yeast Candida palmioleophila under Different Growth Conditions. [J] Chemical Engineering Transactions, 2018, 64: 343-348.). In addition, Candida palmioleophila possesses a variety of oxidoreductases and antibiotic resistance genes, and also exhibits a strong ability to degrade tetracycline under high-salt conditions (Li YZ, Cui WY, Yan JH, et al. A salt-tolerant tetracycline degrading fungal strain of Candida palmioleophila: Characterization and degradation pathway. [J]. Journal of hazardous materials, 2025, 496: 139459.). Summary of the Invention

[0006] The purpose of this invention is to provide a salt- and acid-tolerant Candida palmis strain to address the problems of low treatment efficiency and poor microbial tolerance in traditional biological methods for treating high-salt and high-acid organic wastewater.

[0007] Another object of the present invention is to provide the application of the salt- and acid-tolerant Candida palmis in the treatment of high-salt, high-acid, low-nitrogen, low-phosphorus organic wastewater.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0009] A salt- and acid-tolerant Candida palmis, namely Candida palmis ( Candida palmioleophila)CP, deposited on October 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 67160, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, uses Candida palmis as a microbial agent to treat high-salt, strong-acid, low-nitrogen, and low-phosphorus organic wastewater, represented by candied fruit wastewater.

[0010] Among them, the palm oil Candida ( Candida palmioleophila The nucleotide sequence of CP is shown in SEQ ID NO. 1 of the sequence listing, as follows:

[0011] .

[0012] Candida palmis ( Candida palmioleophilaThe individual morphology of CP is as follows: the colonies are smooth, moist, milky white, and have neat edges; its salt tolerance range (based on NaCl mass) is 20~80 g / L; it can grow in pH 2~9, temperature 15~45℃ and carbon-nitrogen-phosphorus ratio of 100:5:1~500:5:1.

[0013] This invention also provides the above-mentioned Candida palmis ( Candida palmioleophila The application of CP in organic wastewater treatment is as follows:

[0014] (1) Candida palmis ( Candida palmioleophila CP was activated and expanded in culture; the activation medium was acidic high-salt YM liquid medium, and the culture temperature was 28 ℃, the rotation speed was 180 r / min, and the culture was carried out until the bacterial concentration reached 10. 8 CFU / mL;

[0015] (2) Inoculation and treatment: The inoculation method involves either biofilm treatment or direct addition of bacterial solution. The minimum inoculation ratio of bacterial solution volume to wastewater volume is 30% (v / v). Under aerobic conditions, the aforementioned *Candida palmis* (…) Candida palmioleophila CP undergoes biodegradation.

[0016] This invention involves inoculating a pure culture fermentation broth into a reactor packed with packing material, allowing biofilm formation to complete within 3 days. An intermittent treatment method is employed, connecting the biofilm-formed reactor to a high-salt, high-acid organic wastewater source. The influent salinity and pH are adjusted, and the effluent quality is monitored to ensure treatment effectiveness.

[0017] In the wastewater treatment process, the operating parameters of the reactor, including reaction time, aeration rate, and stirring speed, can be adjusted according to the influent COD concentration, salinity, pH value, and the required effluent quality to optimize the treatment effect.

[0018] During wastewater treatment, the effluent quality and COD removal rate can be monitored regularly, and treatment conditions can be adjusted based on the monitoring results to ensure that the effluent quality meets discharge standards or reuse requirements.

[0019] Furthermore, the palm oil Candida ( Candida palmioleophila CP can be applied in the treatment of high-salt, strong-acid, and low-nitrogen-phosphorus organic wastewater. High salinity refers to a salinity of 2%–8% based on sodium chloride; strong acidity refers to a pH of 2–6; and low-nitrogen-phosphorus refers to a carbon-nitrogen-phosphorus ratio (COD:N:P) of 100:5:1–500:5:1, where N is total nitrogen and P is total phosphorus. The strain can maintain growth activity within a pH range of 2–9, making it suitable for treating strong-acid wastewater.

[0020] Compared with the prior art, the outstanding technical effect of the present invention is as follows:

[0021] 1. Highly efficient degradation: The palm oil Candida of this invention ( Candida palmioleophila The CP strain exhibits excellent salt and acid tolerance, and can grow and stably degrade organic matter under conditions of 2%~8% salinity, pH 2~9, temperature 15~45 ℃ and C / N / P ratio of 100:5:1~500:5:1. The removal effect of organic substrate is best at pH=4. It is suitable for the direct treatment of organic wastewater with high salt, strong acid and low nitrogen and phosphorus content, achieving pH increase and COD removal.

[0022] 2. Wide applicability: The strains of this invention can solve the problems of difficult biochemical treatment and high treatment costs of high-salt and high-acid organic wastewater. It is suitable for treating various high-salt and high-acid organic wastewater, such as candied fruit wastewater.

[0023] 3. Environmentally friendly: Compared with traditional chemical treatment methods, biological treatment is more environmentally friendly and reduces the risk of secondary pollution.

[0024] 4. Economic benefits: It reduces treatment costs and improves the economic efficiency of wastewater treatment. It has good economic and environmental benefits. Attached Figure Description

[0025] Figure 1 Candida palmis ( Candida palmioleophila ) CP strain colony morphology characteristics diagram.

[0026] Figure 2 Candida palmis ( Candida palmioleophila ) Morphological characteristics of individual CP strains.

[0027] Figure 3 Candida palmis ( Candida palmioleophila Comparison of COD degradation effects between CP and control strains.

[0028] Figure 4 Candida palmis ( Candida palmioleophila Comparison of pH enhancement effects between CP and control strains.

[0029] Figure 5 pH for Candida palmis ( Candida palmioleophila The effect of CP degradation activity is shown in the curve.

[0030] Figure 6 Salinity for Candida palmis ( Candida palmioleophila The effect of CP degradation activity is shown in the curve.

[0031] Figure 7 Temperature effect on Candida palmis ( Candida palmioleophila The effect of CP degradation activity is shown in the curve.

[0032] Figure 8 The carbon-nitrogen-phosphorus ratio for Candida palmis ( Candida palmioleophilaThe effect of CP treatment on COD removal rate of wastewater is shown in the curve.

[0033] Figure 9 The carbon-nitrogen-phosphorus ratio for Candida palmis ( Candida palmioleophila The effect of CP treatment on pH improvement of wastewater is shown in the curve.

[0034] Figure 10 Candida palmis ( Candida palmioleophila )CP treatment of actual candied fruit wastewater influent and effluent COD and removal rate variation curves.

[0035] Figure 11 Candida palmis ( Candida palmioleophila )CP treatment of actual candied fruit wastewater influent and effluent pH and pH increase curves. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments will be used in conjunction with the accompanying drawings to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Rather, the invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined by the claims. Furthermore, to provide the public with a better understanding of the invention, some specific details are described in detail below. Those skilled in the art can fully understand the invention even without these detailed descriptions. Where not described in detail, existing methods can be used. The control strains are all publicly available laboratory strains, obtainable by those skilled in the art through conventional means.

[0037] This invention provides a salt- and acid-tolerant strain of Candida palmis, which is Candida palmis (… Candida palmioleophila The accession number is GDMCC No: 67160. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 24, 2025. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0038] Palm oil Candida ( Candida palmioleophila CP was isolated from the activated sludge of a candied fruit wastewater treatment unit. The colony morphology on the screening plate was milky white, smooth, moist, and with neat edges. ITS sequencing identified it as Candida palmis, and it was numbered CP.

[0039] Example 1: Candida palmis ( Candida palmioleophila Isolation and identification of CP strains

[0040] (1) Sample source: Activated sludge from the candied fruit wastewater treatment device.

[0041] (2) Formula and preparation of acidic high-salt YM solid culture medium: 1.2 g yeast extract, 1.2 g tryptone, 1.2 g malt extract powder, 4 g glucose, 30 g sodium chloride, 15 g gellan gum, 1000 mL water, adjust pH to 3 with HCl, autoclave at 121 °C for 20 min.

[0042] (3) Isolation and purification steps: Take 10 mL of activated sludge from the candied fruit wastewater treatment device and add it to an Erlenmeyer flask containing 90 mL of sterile culture medium. Incubate at 28 ℃ and 180 r / min for two days, then dilute and spread it on a solid culture medium. Then pick colonies and isolate them by streaking multiple times until single bacteria are obtained. Store the isolated strains in 50% glycerol at -80 ℃. Inoculate the isolated pure strains onto plates and incubate at 28 ℃. Observe and record the morphological characteristics of the colonies to confirm that it is *Candida palmis*. Figure 1 , Figure 2 As shown, the individual morphology of Candida palmis is as follows: the colonies are milky white, smooth, moist, and have neat edges.

[0043] (4) ITS sequencing and results: The pure strains obtained from the isolation were subjected to ITS sequencing. The sequencing results are as follows:

[0044] The strain showed a 99% similarity to the Candida palmis strain in the NCBI database, confirming it as this species.

[0045] Example 2: Candida palmis ( Candida palmioleophila Comparison of CP's degradation ability and pH-raising ability with other strains

[0046] The laboratory currently has four other strains of bacteria, namely Saturnispora silvae, Geotrichum pandrosion, Pichia dushanensis, and Crinitomyces flavificans, which are used to study the palm oil Candida of this invention. Candida palmioleophilaThe differences in organic matter degradation and pH-raising abilities between CP and these four strains were noted. The four control strains mentioned above are all commercially available strains and are used here only to compare the degradation abilities of the strains of this invention.

[0047] The specific steps are as follows:

[0048] a. Prepare simulated wastewater: use 2.5 g / L glucose and 3.5 g / L citric acid as a mixed carbon source, adjust the pH to 3 and the salinity to 3% (calculated as NaCl).

[0049] Trace element solution 1: Cobalt chloride 0.25 g / L, ferric chloride 5.0 g / L, manganese chloride 0.05 g / L, nickel chloride 0.025 g / L, copper chloride 0.007 g / L, zinc chloride 0.025 g / L, sodium molybdate 0.005 g / L, boric acid 0.025 g / L, sodium selenite 0.025 g / L;

[0050] Trace element solution 2: calcium chloride 27.7 g / L, magnesium chloride 101 g / L;

[0051] b. The above-mentioned palm oil Candida ( Candida palmioleophila Seed culture of CP and the other four strains was expanded into pure cultures. Activation and expansion culture conditions: Activation medium was acidic high-salt YM liquid medium, with the following formula: 1.2 g yeast extract, 1.2 g tryptone, 1.2 g malt extract, 4 g glucose, 30 g sodium chloride, and 1000 mL water, adjusted to pH 3 with HCl; culture temperature was 28 ℃, rotation speed was 180 r / min, and culture time was 24–48 h; expansion culture was carried out until the bacterial concentration reached 10-1. 8 CFU / mL. 30 ml of the expanded bacterial culture was added to 100 ml of simulated wastewater for degradation. The reaction was carried out for 3 days. Each experiment had 3 parallel samples. The aeration rate was controlled at 1.5 L / min and the reaction temperature was 28 ℃.

[0052] like Figure 3 , Figure 4 As shown, Candida palmis ( Candida palmioleophila CP strain significantly outperformed the other four control strains in both organic matter degradation and pH enhancement. Regarding organic matter degradation, the initial COD concentration of all five strains was 5324 mg / L, but as the reaction time progressed, *Candida palmis* (*Candida palmis*) showed superior performance. Candida palmioleophilaCP exhibited extremely high degradation efficiency, with its COD value plummeting to 1064 mg / L by day 3, a degradation rate as high as 80%. In contrast, the best-performing control strain, *Pichia dushanensis*, only decreased to 2415 mg / L, while the worst-performing strain, *Crinitomyces flavificans*, still retained 3642 mg / L. This indicates that *Candida palmatum* (Candida palmatum) exhibits superior degradation efficiency. Candida palmioleophila CP has a higher efficiency in organic matter metabolism and transformation, and can more thoroughly remove pollutants from wastewater. In the comparison of pH raising ability, the initial pH of each experimental group was 3. During the 3-day treatment process, Candida palmis (… Candida palmioleophila CP caused the pH of the system to rise rapidly, eventually reaching 6.84, almost neutral, while the pH increase of the other four strains was only between 3.62 and 4.65, remaining in an acidic state. In summary, *Candida palmatum* (… Candida palmioleophila CP has significant advantages in organic matter degradation and pH enhancement.

[0053] Example 3 pH effect on Candida albicans (Palm oil) Candida palmioleophila Effect of CP degradation activity

[0054] By plotting oxygen consumption curves at different pH levels, the study investigated Candida palmis (…). Candida palmioleophila )CP degradation activity.

[0055] The specific steps are as follows:

[0056] a. Prepare simulated wastewater: 0.35 g / L citric acid, 0.25 g / L glucose, add 5 ml / L trace element solutions 1 and 2, the initial COD is about 500 mg / L;

[0057] Trace element solution 1: Cobalt chloride 0.25 g / L, ferric chloride 5.0 g / L, manganese chloride 0.05 g / L, nickel chloride 0.025 g / L, copper chloride 0.007 g / L, zinc chloride 0.025 g / L, sodium molybdate 0.005 g / L, boric acid 0.025 g / L, sodium selenite 0.025 g / L;

[0058] Trace element solution 2: calcium chloride 27.7 g / L, magnesium chloride 101 g / L;

[0059] Nitrification inhibitor: Allyl thiourea 20 mg / L;

[0060] Carbon dioxide absorption solution: 40% sodium hydroxide solution.

[0061] b. Adjust the salinity of the simulated wastewater to 3% (calculated as NaCl). Add 350 ml of wastewater to each reaction flask, adjusting the pH to 2, 3, 4, 5, 6, 7, and 9 respectively. Add 10 ml of pure bacterial culture and 1.5 ml of nitrification inhibitor, and control the temperature at 25 ℃. Then, using a microbial respiration measurement system, add a measured volume of the wastewater to be tested along with a magnetic stir bar to the reaction flask. Turn on the magnetic stir system, and then suspend the absorption cup containing sodium hydroxide solution below the flask opening, ensuring it does not contact the liquid surface but can effectively capture carbon dioxide in the flask. Tighten the flask caps, place the assembled reaction flasks in a pre-set constant temperature water bath, and turn on the magnetic stir system. Then, insert the needle on the oxygen delivery tube into the corresponding reaction flask cap, check all oxygen delivery lines to ensure the system is strictly sealed, then turn on the oxygen cylinder to supply oxygen and start the measurement program. The system will automatically begin continuous monitoring, ultimately converting the raw signal into biological oxygen consumption.

[0062] Figure 5 For Candida palmis under different initial pH values ​​( Candida palmioleophila The oxygen consumption curves of CP show that pH significantly affects the degradation activity of this strain, exhibiting a clear acidophilic characteristic. The figure shows that at around pH 4, the strain has the fastest oxygen consumption rate and the highest final oxygen consumption, reaching approximately 280 mg / L. This means that about 60% of the substrate is completely oxidized, indicating that the strain's metabolism is most vigorous and its degradation activity is optimal at this level. The reaction rate is even faster in the pH 3 group initially, and the oxygen consumption increases rapidly over time, eventually reaching approximately 230 mg / L. In contrast, although the strain can still survive and metabolize at pH 2, the oxygen consumption curve is significantly lower than that of the pH 4 group, indicating that the excessively acidic environment has a certain inhibitory effect on its activity. At pH 5-8, although the reaction rate is relatively fast in the initial stage, the final oxygen consumption is lower. In conclusion, this strain has the best removal effect on organic substrates under weakly acidic conditions at around pH 4, which is consistent with the acidic water quality characteristics of candied fruit wastewater.

[0063] Example 4: The effect of salinity on Candida albicans (Palm oil) Candida palmioleophila Effect of CP degradation activity

[0064] By plotting oxygen consumption curves at different salinities, the study of Candida palmis ( ) was conducted. Candida palmioleophila )CP degradation activity.

[0065] The specific steps are as follows:

[0066] a. Prepare simulated wastewater: 0.35 g / L citric acid, 0.25 g / L glucose, add 5 ml / L trace element solutions 1 and 2, the initial COD is about 500 mg / L;

[0067] Trace element solution 1: Cobalt chloride 0.25 g / L, ferric chloride 5.0 g / L, manganese chloride 0.05 g / L, nickel chloride 0.025 g / L, copper chloride 0.007 g / L, zinc chloride 0.025 g / L, sodium molybdate 0.005 g / L, boric acid 0.025 g / L, sodium selenite 0.025 g / L;

[0068] Trace element solution 2: calcium chloride 27.7 g / L, magnesium chloride 101 g / L;

[0069] Nitrification inhibitor: Allyl thiourea 20 mg / L;

[0070] Carbon dioxide absorption solution: 40% sodium hydroxide solution.

[0071] b. Adjust the pH of the simulated wastewater to 3. Add 350 ml of wastewater to each reaction flask, and adjust the salinity (as NaCl) to 2%, 3%, 4%, 5%, 6%, 7%, and 8% respectively. Add 10 ml of pure cultured bacteria and 1.5 ml of nitrification inhibitor, and control the temperature at 25°C. Then, using a microbial respiration measurement system, add a measured volume of the wastewater to be tested along with a magnetic stir bar to the reaction flask. Turn on the magnetic stir system, and then suspend the absorption cup containing sodium hydroxide solution below the flask opening, ensuring it does not contact the liquid surface but can effectively capture carbon dioxide in the flask. Tighten the flask caps, place the assembled reaction flasks in a pre-set constant temperature water bath, and turn on the magnetic stir system. Then, insert the needle on the oxygen delivery tube into the corresponding reaction flask cap, check all oxygen delivery lines to ensure the system is strictly sealed, then turn on the oxygen cylinder to supply oxygen, and start the measurement program. The system will automatically begin continuous monitoring, ultimately converting the raw signal into biological oxygen consumption.

[0072] Figure 6 Candida palmis at different salinities (calculated as NaCl) Candida palmioleophila The CP oxygen consumption curve shows that Candida palmis (…) Candida palmioleophilaCP can grow in a salinity range of 2% to 8%, exhibiting extremely strong salt tolerance. The figure shows that the green curve representing 5% salinity remained at the top throughout the entire 72-hour reaction period, exhibiting the shortest lag phase, the steepest exponential growth slope, and the highest final cumulative oxygen consumption of approximately 260 mg / L. This indicates that at this salinity, the enzyme system activity within the microorganism is strongest, resulting in the highest efficiency in the oxidative decomposition of organic substrates. Within the salinity range of 2% to 4%, the curves highly overlapped and were slightly below the peak at 5%, indicating that this strain possesses very stable adaptability and high degradation efficiency within this moderate salinity range. However, when the salinity further increased above the 5% threshold, the inhibitory effect of high osmotic pressure began to appear: as the salinity increased from 6% to 8%, the curve gradually shifted downwards, with the purple curve representing 8% salinity showing the worst performance, exhibiting a significantly slower start-up speed and the lowest final oxygen consumption. This reveals that excessively high salinity forces microorganisms to consume more energy to regulate the osmotic pressure balance inside and outside the cell, leading to a decrease in degradation activity. It is noteworthy, however, that even at an extremely high salinity of 8%, the system maintained a considerable degradation capacity, demonstrating that this strain possesses strong salt tolerance and is highly suitable for treating high-salinity industrial wastewater. This characteristic of maintaining activity across a wide salinity range confirms that this strain possesses excellent salt-tolerant genes and osmotic pressure regulation mechanisms, making it highly suitable for treating candied fruit wastewater with large salinity fluctuations and high concentrations. In practical engineering, it eliminates the need for extensive dilution to reduce salinity, demonstrating significant application advantages.

[0073] Example 5: The effect of temperature on Candida albicans (Palm oil) Candida palmioleophila Effect of CP degradation activity

[0074] The study investigated Candida palmis (Candida palmis) by plotting oxygen consumption curves at different temperatures. Candida palmioleophila )CP degradation activity.

[0075] The specific steps are as follows:

[0076] a. Prepare simulated wastewater: 0.35 g / L citric acid, 0.25 g / L glucose, add 5 ml / L trace element solutions 1 and 2, the initial COD is about 500 mg / L;

[0077] Trace element solution 1: Cobalt chloride 0.25 g / L, ferric chloride 5.0 g / L, manganese chloride 0.05 g / L, nickel chloride 0.025 g / L, copper chloride 0.007 g / L, zinc chloride 0.025 g / L, sodium molybdate 0.005 g / L, boric acid 0.025 g / L, sodium selenite 0.025 g / L;

[0078] Trace element solution 2: calcium chloride 27.7 g / L, magnesium chloride 101 g / L;

[0079] Nitrification inhibitor: Allyl thiourea 20 mg / L;

[0080] Carbon dioxide absorption solution: 40% sodium hydroxide solution.

[0081] b. Adjust the pH of the simulated wastewater to 3 and the salinity to 3% (calculated as NaCl). Add 350 ml of wastewater, 10 ml of pure bacterial culture, and 1.5 ml of nitrification inhibitor to each reaction flask. Control the temperature at 15 ℃, 25 ℃, 35 ℃, and 45 ℃ respectively. Then, using a microbial respiration measurement system, add a fixed volume of the wastewater to be tested along with a magnetic stir bar to the reaction flask. Turn on the magnetic stir system, and then suspend the absorption cup containing sodium hydroxide solution below the flask opening, ensuring it does not contact the liquid surface but can effectively capture carbon dioxide in the flask. Tighten the flask caps, place the assembled reaction flasks in a constant temperature water bath with the magnetic stir system turned on, and then insert the needle on the oxygen delivery tube into the corresponding reaction flask cap. Check all oxygen delivery lines to ensure the system is strictly sealed. Then, turn on the oxygen cylinder to supply oxygen and start the measurement program. The system will automatically begin continuous monitoring and ultimately convert the raw signal into biological oxygen consumption.

[0082] Figure 7 The palm oil Candida of the present invention at different temperatures ( Candida palmioleophilaThe CP oxygen consumption curves show that the strain exhibits the highest metabolic activity at 35 ℃, with a cumulative oxygen consumption of approximately 300 mg / L within 72 hours. The slope of the oxygen consumption curve is also steepest at the initial stage of the reaction, indicating that the microorganism's oxidative decomposition rate of the substrate is fastest at this temperature, and the enzyme system is in optimal catalytic condition, effectively overcoming the environmental pressures posed by strong acids and high salts. At 25 ℃, the strain's metabolic effect follows closely, with a final oxygen consumption of approximately 275 mg / L after 72 hours. Although slightly lower than the oxygen consumption at 35 ℃, the overall trend of the oxygen consumption curve is very close to that of the 35 ℃ group, indicating that the strain can maintain efficient organic matter degradation capacity within the mesophilic range of 25 ℃ to 35 ℃, making it suitable for the conventional temperature range of industrial wastewater treatment. In contrast, excessively high or low temperatures significantly inhibit the strain's degradation activity, with higher temperatures exhibiting a more severe inhibitory effect. At a low temperature of 15 °C, although the metabolic rate of the strain slowed down due to reduced enzyme activity, the final oxygen consumption after 72 hours was approximately 192 mg / L. However, the oxygen consumption curve still showed an upward trend, indicating that the microorganisms did not stop their metabolic work, but only the degradation rate decreased. At a high temperature of 45 °C, the degradation activity of the strain was severely damaged, and the final oxygen consumption after 72 hours was only about 104 mg / L, which was the lowest level within the experimental temperature range. It is speculated that this phenomenon is caused by the synergistic toxicity of high temperature and high salt and strong acid environment, which exacerbates the damage to the cell membrane of the strain or leads to the denaturation and inactivation of key metabolic enzymes of the strain, making the microorganisms almost lose their ability to process pollutants.

[0083] Experiments show that the present invention contains Candida palmis (…). Candida palmioleophila CP exhibits excellent degradation performance in a weakly acidic environment (pH=4) and demonstrates exceptional stability across a wide salinity range of 2%–8%, with peak activity at 5% salinity. Even in a high-salinity environment (8%), the strain's metabolic system does not collapse, eliminating the need for significant alkaline neutralization agents and wastewater dilution costs in engineering applications. Furthermore, the strain's optimal metabolic temperature is 35°C, allowing it to directly utilize waste heat from wastewater to maintain peak efficiency, effectively overcoming the dual pressures of high salinity and strong acidity. The strain's characteristics of "strong acidophilicity, wide salt tolerance, and high efficiency at medium temperatures" make it a low-cost, high-efficiency bioaugmentation solution for addressing the challenges of biological treatment of high-acid and high-salinity wastewater.

[0084] Example 6: Effect of carbon-nitrogen-phosphorus ratio on Candida palmis (C. palmis) Candida palmioleophila The impact of CP processing

[0085] Study on the effects of different carbon-nitrogen-phosphorus ratios on Candida palmis ( ) Candida palmioleophila The effects of CP on the degradation of organic matter and pH self-raising ability are detailed in the following steps:

[0086] a. Prepare simulated wastewater using glucose 2.5 g / L and citric acid 3.5 g / L as a mixed carbon source. Adjust the initial COD value of the wastewater to 5000 mg / L, pH=3, and salinity to 3% (calculated as NaCl). Adjust the carbon-nitrogen-phosphorus ratio to 100:5:1, 200:5:1, 300:5:1, 400:5:1, and 500:5:1, respectively. Use ammonium chloride as the nitrogen source and potassium dihydrogen phosphate as the phosphorus source. Set up 3 parallel samples for each ratio. The reaction lasts for 3 days as one batch, and the process is carried out continuously for 30 days.

[0087] b. The above-mentioned palm oil Candida ( Candida palmioleophila CP seed culture was expanded into pure culture; activation and expansion culture conditions: the activation medium was acidic high-salt YM liquid medium, with the following formula: yeast extract 1.2g, tryptone 1.2g, malt extract powder 1.2g, glucose 4g, sodium chloride 30g, water 1000mL, and the pH was adjusted to 3 with HCl; the culture temperature was 28℃, the rotation speed was 180r / min, and the culture time was 24~48h; the expansion culture was carried out until the bacterial concentration reached 10. 8 CFU / mL; The expanded bacterial culture was added to a reactor containing packing material (polyurethane foam packing material), and aerated continuously for 3 days to complete biofilm formation. After biofilm formation, the prepared simulated wastewater was introduced, and the aeration rate was controlled at 1.5 L / min, and the reaction temperature was 28℃.

[0088] like Figure 8 As shown, regarding COD degradation rate, regardless of whether the ratio was 100:5:1 or 500:5:1, the COD removal rate of each experimental group quickly reached a high of around 70% in the first batch and remained stable above 75% for the next 27 days. The differences between groups were small, and no significant performance degradation due to an imbalance in the carbon, nitrogen, and phosphorus ratio was observed. Similarly, as... Figure 9 As shown, the effluent pH of all experimental groups steadily increased from the initial 3.0 to a near-neutral range of 6.2 to 6.8, with a high degree of pH concentration across groups. The experiment indicates that *Candida palmis* (…) Candida palmioleophila CP exhibits strong adaptability to changes in the carbon-nitrogen-phosphorus ratio. Even under extreme nutrient conditions with severe nitrogen scarcity, it maintains stable metabolic activity without significant nitrogen limitation, and does not significantly affect its ability to degrade organic matter or raise pH. Therefore, in practical engineering applications, it is not necessary to control the dosage of carbon or nitrogen sources to achieve the optimal carbon-nitrogen-phosphorus ratio. It is particularly suitable for treating industrial wastewater with large fluctuations in carbon sources or high carbon content and low nitrogen content (such as high-sugar wastewater and food processing wastewater), which can greatly reduce the cost of chemical reagents and has significant economic benefits.

[0089] Example 7 Candida albicans (Palm oil) Candida palmioleophila )CP treatment effect on actual candied fruit wastewater

[0090] Study of Candida palmis ( Candida palmioleophila The long-term treatment effect of CP on actual candied fruit wastewater (wastewater from bayberry processing) is shown in the following steps:

[0091] a. The above-mentioned Candida palm oil ( Candida palmioleophila CP seed culture was expanded into pure culture; activation and expansion culture conditions: the activation medium was acidic high-salt YM liquid medium, with the following formula: yeast extract 1.2 g, tryptone 1.2 g, malt extract powder 1.2 g, glucose 4 g, sodium chloride 30 g, water 1000 mL, and the pH was adjusted to 3 with HCl; the culture temperature was 28 ℃, the rotation speed was 180 r / min, and the culture time was 24~48 h; the expansion culture was carried out until the bacterial concentration reached 10. 8 CFU / mL;

[0092] b. The carbon-nitrogen-phosphorus ratio of the candied fruit wastewater is 425:4.5:1. No external nitrogen or phosphorus sources are added. The pH of the candied fruit wastewater is adjusted to approximately 3, the COD to approximately 10000 mg / L, and the salinity to 3% (calculated as NaCl). The expanded bacterial culture is added to a reactor containing packing material (polyurethane foam packing material), and continuous aeration is carried out for 3 days to complete biofilm formation. After biofilm formation, the above-mentioned bayberry wastewater is introduced, and the aeration rate is controlled at 1.5 L / min. The reaction adopts a sequencing batch reactor, with each batch lasting 3 days, for a total of 90 days.

[0093] Experimental results are as follows Figure 10 As shown, the influent COD concentration of the actual bayberry wastewater exhibited drastic fluctuations, ranging from approximately 9000 mg / L to nearly 15000 mg / L, classifying it as typical high-concentration, high-impact organic wastewater. The temperature curve shows that the system experienced a temperature range from approximately 10 ℃ to 30 ℃. *Candida palmis* (… Candida palmioleophila CP exhibited extremely strong resistance to shock loads and temperatures, with its effluent COD consistently maintained at a low level below 2000 mg / L. After overcoming the initial low-temperature adaptation phase, the overall COD degradation rate steadily increased and remained at a high level of 85% to 90% for an extended period, without experiencing a collapse in treatment performance due to a surge in influent concentration or early low temperatures. Figure 11 As shown, the influent of the candied fruit wastewater was in a strongly acidic state with a pH of 3.0 to 3.5 for a long period of time. However, during the continuous batch operation for three months, the pH value of the system effluent was steadily increased and maintained in the near-neutral to slightly alkaline range of 6.0 to 8.0, and the ΔpH was stably maintained between 3 and 4.5 units.

[0094] Experimental data show that Candida palmis ( Candida palmioleophilaCP exhibits excellent resistance to water quality load fluctuations and temperature shocks in harsh environments characterized by high salinity, strong acidity, and low nitrogen and phosphorus content, enabling stable and efficient degradation of organic matter and autonomous pH elevation. Compared to the conventional limitation of maintaining a carbon-nitrogen-phosphorus ratio of approximately 100:5:1 in traditional biological treatment processes, this strain maintains stable and efficient metabolic activity even after three months of continuous operation under oligotrophic conditions without additional nitrogen and phosphorus sources. This characteristic effectively overcomes the limitation of traditional processes that heavily rely on exogenous nutrient addition when treating high-concentration organic wastewater, significantly reducing reagent consumption and overall operating costs in practical engineering applications.

[0095] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A salt- and acid-tolerant Candida palmis, characterized in that... It was named Candida palmis ( Candida palmioleophila )CP was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 24, 2025, with accession number GDMCC No: 67160.

2. The salt- and acid-tolerant Candida palmis strain as described in claim 1, characterized in that... The nucleotide sequence of the strain is shown in SEQ ID NO. 1 of the sequence listing.

3. The salt- and acid-tolerant Candida palmis strain as described in claim 1, characterized in that... The strains were grown under conditions of 2%–8% salinity (based on NaCl), pH 2–9, and temperature 15–45 °C.

4. The application of the salt- and acid-tolerant Candida palmis strain as described in claim 1 in the treatment of high-salt, high-acid, low-nitrogen, low-phosphorus organic wastewater.

5. The application as described in claim 4, characterized in that... When the salinity of organic wastewater (calculated as NaCl) is in the range of 2% to 8%, the strains can maintain high metabolic activity and stably degrade organic matter, and no dilution of high-salt wastewater is required in engineering applications; when the carbon-nitrogen-phosphorus ratio (COD:N:P) of the wastewater is in the range of 100:5:1 to 500:5:1, no additional nitrogen or phosphorus source is required, and the strains can achieve COD degradation and increase the pH value of the effluent.

6. The application as described in claim 4, characterized in that... The specific method is as follows: (1) The palm oil Candida as described in claim 1 ( Candida palmioleophila CP was activated and expanded in culture; the activation medium was acidic high-salt YM liquid medium, and the culture temperature was 28 ℃, the rotation speed was 180 r / min, and the culture was carried out until the bacterial concentration reached 10. 8 CFU / mL; (2) Inoculation and treatment: The inoculation method involves either biofilm treatment or direct addition of bacterial solution. The minimum inoculation ratio of bacterial solution volume to wastewater volume is 30%. Under aerobic conditions, the aforementioned *Candida palmis* (…) Candida palmioleophila CP undergoes biodegradation.

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