Method for promoting growth and carbon sequestration of scenedesmus by using polyacrylamide in oilfield wastewater

Abstract

The invention relates to a biomass energy utilization technology, and aims to provide a method for promoting scenedesmus growth and carbon sequestration by using polyacrylamide in oilfield wastewater. Comprising the following steps: inoculating an original scenedesmus strain into a photobioreactor filled with a liquid culture medium, providing a proper illumination condition, and introducing CO2 / N2 mixed gas into the reactor; the concentration of polyacrylamide in the liquid culture medium is increased in a gradient increasing mode, and inoculation is repeated multiple times till domestication is completed; scenedesmus strains capable of stably inheriting are selected for enlarged culture, oilfield wastewater containing polyacrylamide is used for preparing a liquid culture medium, and decomposition of polyacrylamide is promoted while carbon sequestration growth of scenedesmus is achieved. The polyacrylamide in the oilfield wastewater is utilized to promote the growth and carbon sequestration of the scenedesmus, the polyacrylamide can be degraded while the carbon sequestration growth is realized, the double results of carbon sequestration and waste removal are realized, and the comprehensive benefit of the method is far better than that of a fungus waste removal scheme in the traditional technology.

Description

Technical Field

[0001] This invention relates to biomass energy utilization technology, and in particular to a method for promoting the growth and carbon fixation of Scenedesmus using polyacrylamide in oilfield wastewater. Background Technology

[0002] Polyacrylamide (PAM) is an extremely important oilfield chemical, widely used as a lubricant, suspending agent, oil displacement agent, flocculant, and thickener. However, polyacrylamide cannot be effectively recycled, leading to serious environmental pollution problems. Residual polyacrylamide in wastewater can naturally and slowly degrade into toxic acrylamide monomers, damaging the nervous system of humans and animals and exhibiting toxicity to organisms. Therefore, research on the degradation and utilization mechanisms of polyacrylamide in oilfield wastewater is of great practical significance.

[0003] Currently, there are various technologies and methods for treating polyacrylamide-containing wastewater, each with its own advantages and disadvantages. Biological methods primarily utilize microbial communities that can use polyacrylamide as a nutrient source, gradually degrading it into small-molecule organic and inorganic substances through metabolic activity. Compared with traditional physical and chemical treatment technologies, biological methods have significant advantages such as no secondary pollution and low operating costs. Furthermore, they can efficiently remove polymers from effluent through specific metabolic pathways of microorganisms, demonstrating good application potential in the field of organic wastewater treatment. Multiple studies have reported that the amide groups in polyacrylamide are easily degraded by microorganisms, providing nitrogen and carbon sources for microbial growth and metabolism. This physiological process is mainly catalyzed by key enzymes in microbial metabolism. Urease is an extracellular hydrolytic enzyme secreted by organisms that can degrade the amide groups in polyacrylamide. Numerous studies have confirmed that urease activity is directly related to changes during the polyacrylamide degradation process. Research indicates that denitrification in polyacrylamide solutions is mainly catalyzed by urease; after the polyacrylamide molecules break off from the amide groups, they are decomposed into long-chain polymers.

[0004] However, existing research on microorganisms used for polyacrylamide degradation typically focuses on fungi, failing to cover a wider range of biological species. For example, patent publication CN120173750A proposes a polyacrylamide-degrading fungus, its screening and domestication methods, and its applications; patent publication CN119776219A proposes a strain for degrading polyacrylamide and its applications. The drawback of using fungal products is that the fungi are continuously consumed during the degradation process due to their limited tolerance, thus requiring significant investment in the continuous production of the fungi themselves. Furthermore, the effectiveness of using fungi to degrade polyacrylamide is limited, resulting in low overall benefits. Summary of the Invention

[0005] The main objective of this invention is to overcome the shortcomings of the prior art and provide a method for promoting the growth and carbon fixation of Scenedesmus using polyacrylamide in oilfield wastewater.

[0006] To solve the above-mentioned technical problems, the solution of the present invention is:

[0007] A method for promoting the growth and carbon fixation of Scenedesmus using polyacrylamide in oilfield wastewater is provided, comprising the following steps:

[0008] (1) The original strain of Scenedesmus was inoculated into a photobioreactor containing a liquid culture medium containing a preset concentration of polyacrylamide; appropriate light conditions were provided, and a CO2 / N2 mixture was introduced into the reactor to provide a carbon source for the growth and carbon fixation of Scenedesmus.

[0009] (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium; repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner.

[0010] (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture.

[0011] (4) Prepare a liquid culture medium using oilfield wastewater containing polyacrylamide and add it to a photobioreactor; introduce an appropriate amount of improved Scenedesmus strain into the liquid culture medium for inoculation and introduce a CO2 / N2 mixed gas; promote the decomposition of polyacrylamide while the Scenedesmus is growing and fixing carbon.

[0012] As a preferred embodiment of the present invention, the *Scenedesmus* strain is from the freshwater algae strain bank of the National Aquatic Biological Germplasm Resource Bank, with the strain number FACHB-44.

[0013] As a preferred embodiment of the present invention, the illumination conditions are 70–90 μmol·m⁻². -2 ·s -1 The incubation temperature is 23–26℃.

[0014] As a preferred embodiment of the present invention, the CO2 / N2 mixture has a CO2 volume concentration of 6% and a flow rate of 8-12 mL / min.

[0015] As a preferred embodiment of the present invention, the liquid culture medium adopts the typical formulation of Mineral Medium numbered BG11 in the ATCC culture medium database.

[0016] As a preferred embodiment of the present invention, the gradient increase of polyacrylamide in the liquid culture medium during the gradient acclimatization process is as follows: the initial concentration is set to 10 mg / L, the concentration at the second inoculation is 100 mg / L, and the concentration is increased by 100 mg / L for each subsequent inoculation.

[0017] As a preferred embodiment of the present invention, during the gradient acclimatization process, 3 to 5 parallel groups are set up, and algal liquid samples are collected after each culture for biomass determination; after intergroup analysis with the original strain, the group with the highest microalgal growth rate and microalgal CO2 fixation rate is selected for the next acclimatization step.

[0018] As a preferred embodiment of the present invention, when using the improved strain of Scenedesmus for large-scale culture, the concentration of polyacrylamide in the liquid culture medium is controlled to be 400-500 mg / L.

[0019] Description of the invention principle:

[0020] Microalgae, as the microorganisms with the highest photosynthetic efficiency and the fastest carbon fixation growth, have unique advantages in treating and purifying waste gas and wastewater. Due to its thick cell wall and strong stress resistance, *Scenedesmus* is often used in industrial wastewater treatment. However, existing research has not given sufficient attention to the applications of *Scenedesmus* beyond industrial wastewater treatment, and related research results are rarely reported.

[0021] Through long-term research, the inventors' team discovered that *Scenedesmus*, after gradient domestication, can grow normally in the presence of polyacrylamide and simultaneously secrete urease to promote the degradation of polyacrylamide into polyacrylic acid and ammonia. The ammonia decomposition product can be used by *Scenedesmus* to enhance its amino acid synthesis and energy supply, thereby further improving its carbon fixation efficiency. Based on this discovery, this invention innovatively proposes using oilfield wastewater rich in polyacrylamide for *Scenedesmus* growth and carbon fixation. This achieves both carbon fixation and waste removal by utilizing urease to degrade the amide groups in polyacrylamide simultaneously.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. This invention reveals the mechanism by which Scenedesmus utilizes polyacrylamide in oilfield wastewater to promote growth and carbon fixation, providing a new pathway for microalgae waste removal applications.

[0024] 2. In a growth environment containing polyacrylamide, Scenedesmus can secrete urease to promote the degradation of polyacrylamide into polyacrylic acid and ammonia. The ammonia in the decomposition products can enhance reaction pathways such as amino acid synthesis and energy supply, thereby improving carbon fixation efficiency.

[0025] 3. Using polyacrylamide in oilfield wastewater to promote the growth and carbon fixation of Scenedesmus can simultaneously degrade polyacrylamide, achieving both carbon fixation and waste removal. Its comprehensive benefits far exceed those of traditional fungal waste removal solutions. Attached Figure Description

[0026] Figure 1 This diagram illustrates the degradation effect of the original *Scenedesmus* strain and the improved strains in each embodiment on polyacrylamide.

[0027] Figure 2 This is a comparison chart showing the carbon fixation capacity of the original *Scenedesmus* strain and the improved strains from each embodiment. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] I. Implementation Scheme of the Invention

[0030] The method for promoting the growth and carbon fixation of Scenedesmus using polyacrylamide in oilfield wastewater, provided by this invention, is characterized by comprising the following steps:

[0031] (1) The original strain of Scenedesmus was inoculated into a photobioreactor containing liquid culture medium containing 10 mg / L of polyacrylamide; appropriate light conditions were provided, and a CO2 / N2 mixture was introduced into the reactor to provide a carbon source for the growth and carbon fixation of Scenedesmus.

[0032] As an optional example, the *Scenedesmus* strain was obtained from the Freshwater Algae Genetic Resources Bank of the National Aquatic Organism Germplasm Bank, with the strain number FACHB-44. The light conditions were 70–90 μmol·m⁻². -2 ·s -1 The incubation temperature was 23–26℃. The CO2 volume concentration in the CO2 / N2 mixture was 6%, and the flow rate of the mixture was 8–12 mL / min. The liquid culture medium used was the typical formulation of Mineral Medium (BG11) from the ATCC culture medium database.

[0033] (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium. Repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner as follows: the concentration at the second inoculation is 100 mg / L, and the concentration of each subsequent inoculation is increased by 100 mg / L.

[0034] During the gradient acclimatization process, 3 to 5 parallel groups were set up. After each culture, algal liquid samples were collected for biomass determination. After intergroup analysis with the original strain, the group with the highest microalgal growth rate and the highest microalgal CO2 fixation rate was selected for the next acclimatization step.

[0035] (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture.

[0036] (4) Prepare a liquid culture medium using oilfield wastewater containing polyacrylamide, add it to the photobioreactor, and control the concentration of polyacrylamide in the liquid culture medium to be 400-500 mg / L. Introduce an appropriate amount of improved Scenedesmus strain into the liquid culture medium for inoculation, and introduce a CO2 / N2 mixed gas; while the Scenedesmus is growing and fixing carbon, it promotes the decomposition of polyacrylamide.

[0037] II. Examples and Control Groups

[0038] Example 1

[0039] (1) The original *Scenedesmus* strain FACHB-44 was inoculated into a 300 mL photobioreactor containing liquid culture medium. The liquid culture medium was a typical formulation of Mineral Medium (BG11) from the ATCC culture medium database, containing 10 mg / L polyacrylamide. The light conditions were controlled at 80 μmol·m⁻². -2 ·s -1 The culture temperature was 25℃. A CO2 / N2 mixed gas was continuously introduced into the reactor at a rate of 10 mL / min to provide a carbon source for the growth and carbon fixation of Scenedesmus; the volume concentration of CO2 in the CO2 / N2 mixed gas was 6%.

[0040] (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium. Repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner as follows: based on the initial concentration, the concentration of the second inoculation is 100 mg / L, and the concentration of each subsequent inoculation is increased by 100 mg / L.

[0041] During the gradient acclimatization process, five parallel groups were set up. After each culture, algal liquid samples were collected for biomass determination. After intergroup analysis with the original strain, the group with the highest microalgal growth rate and the highest microalgal CO2 fixation rate was selected for the next acclimatization step.

[0042] Test method description: During the domestication and cultivation process, 10 mL algal solution samples were collected periodically for biomass determination.

[0043] First, the absorbance (OD) of *Scenedesmus* at a characteristic wavelength of 680 nm was measured using an ultraviolet spectrophotometer. 680 The sample was then centrifuged at 5000 rpm for 10 min, followed by washing the algal sludge three times with deionized water. The sludge was then transferred to a vacuum drying oven at 80℃ and dried for 24 hours until the weight remained constant. The final dry weight of the biomass was then determined. The dry weight and OD of the Scenedesmus biomass were obtained through formula fitting. 680 The linear regression equation is as follows. Microalgae samples were taken every 24 hours in the experiment to test and calculate the daily biomass dry weight.

[0044] Biomass dry weight (g / L) = 0.521 × OD 680 (1)

[0045] The absorbance of the microalgae was controlled at around 0.5 during inoculation and cultivation. The dry weight of the biomass at this time is recorded as m1, and the dry weight at harvest is recorded as m2. The growth time is t days, and α represents the carbon content in the algal cells. The measured value of α in *Scenedesmus* cells is 50%. Then, what are the microalgal growth rate λ and the microalgal CO2 fixation rate V? CO2-fixation The calculation formulas are as follows:

[0046] λ (g / L / d)=(m2-m1) / t (2)

[0047] V CO2-fixation (g / L / d) = (λ×α×44) / 12 (3)

[0048] (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture.

[0049] (4) Prepare a liquid culture medium with oilfield wastewater containing polyacrylamide, with a polyacrylamide concentration of 450 mg / L, and then add it to the photobioreactor; introduce an appropriate amount of improved Scenedesmus strain into the liquid culture medium for inoculation, and introduce CO2 / N2 mixed gas; promote the decomposition of polyacrylamide while the Scenedesmus fixes carbon and grows.

[0050] Example 2

[0051] (1) The original *Scenedesmus* strain FACHB-44 was inoculated into a 300 mL photobioreactor containing liquid culture medium. The liquid culture medium was a typical formulation of Mineral Medium (BG11) from the ATCC culture medium database, containing 10 mg / L polyacrylamide. The light conditions were controlled at 70 μmol·m⁻². -2 ·s -1 The culture temperature was 23℃. A CO2 / N2 mixture was continuously introduced into the reactor at a rate of 8 mL / min to provide a carbon source for the growth and carbon fixation of Scenedesmus; the volume concentration of CO2 in the CO2 / N2 mixture was 6%.

[0052] (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium. Repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner as follows: based on the initial concentration, the concentration of the second inoculation is 100 mg / L, and the concentration of each subsequent inoculation is increased by 100 mg / L.

[0053] During the gradient acclimatization process, three parallel groups were set up. After each culture, algal liquid samples were collected for biomass determination. After intergroup analysis with the original strain, the group with the highest microalgal growth rate and microalgal CO2 fixation rate was selected for the next acclimatization step.

[0054] (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture.

[0055] (4) Prepare a liquid culture medium with oilfield wastewater containing polyacrylamide, with a polyacrylamide concentration of 400 mg / L, and then add it to the photobioreactor; add it to the photobioreactor; introduce an appropriate amount of improved Scenedesmus strain into the liquid culture medium for inoculation, and introduce CO2 / N2 mixed gas; promote the decomposition of polyacrylamide while the Scenedesmus fixes carbon and grows.

[0056] Example 3

[0057] (1) The original *Scenedesmus* strain FACHB-44 was inoculated into a 300 mL photobioreactor containing liquid culture medium. The liquid culture medium was a typical formulation of Mineral Medium (BG11) from the ATCC culture medium database, containing 10 mg / L polyacrylamide. The light conditions were controlled at 90 μmol·m⁻². -2 ·s -1 The culture temperature was 26℃. A CO2 / N2 mixed gas was continuously introduced into the reactor at a rate of 12 mL / min to provide a carbon source for the growth and carbon fixation of Scenedesmus; the volume concentration of CO2 in the CO2 / N2 mixed gas was 6%.

[0058] (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium. Repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner as follows: based on the initial concentration, the concentration of the second inoculation is 100 mg / L, and the concentration of each subsequent inoculation is increased by 100 mg / L.

[0059] During the gradient acclimatization process, four parallel groups were set up. After each culture, algal liquid samples were collected for biomass determination. After intergroup analysis with the original strain, the group with the highest microalgal growth rate and the highest microalgal CO2 fixation rate was selected for the next acclimatization step.

[0060] (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture.

[0061] (4) Prepare a liquid culture medium with oilfield wastewater containing polyacrylamide, with a polyacrylamide concentration of 500 mg / L, and then add it to the photobioreactor; add it to the photobioreactor; introduce an appropriate amount of improved Scenedesmus strain into the liquid culture medium for inoculation, and introduce CO2 / N2 mixed gas; promote the decomposition of polyacrylamide while the Scenedesmus fixes carbon and grows.

[0062] Control group

[0063] Perform the procedure as described in Example 1, except that the original strain of Scenedesmus, FACHB-44, is introduced into the liquid culture medium for inoculation.

[0064] In steps (4) of Examples 1-3 and the control example, except that the inoculated algal strain and polyacrylamide concentration were performed according to their respective determined schemes, other experimental conditions should be kept the same to avoid deviations in test results. Specifically, the inoculation OD value was 0.8; the flow rate of the CO2 / N2 mixture was 10 mL / min, and the CO2 volume concentration was 6%; the light condition was 80 μmol·m⁻¹. -2 ·s -1 The incubation temperature is 25℃.

[0065] During the algal culture process, the algal solution was sampled and tested periodically according to the method described in step (2) of Example 1.

[0066] III. Test Results and Analysis

[0067] from Figure 1 The comparison of polyacrylamide degradation rates between the original and improved algal strains shows that the gradient domestication and improvement of *Scenedesmus* in this invention significantly improves its efficiency in degrading and utilizing polyacrylamide in oilfield wastewater. Specifically, the polyacrylamide degradation rate of *Scenedesmus* in the control group was 44%; the polyacrylamide degradation rates of the improved *Scenedesmus* strains in Examples 1-3 were 71%, 55%, and 64%, respectively, all significantly higher than the control group. Furthermore, *Scenedesmus* domesticated under the conditions of Example 1 was more conducive to the degradation and utilization of polyacrylamide.

[0068] from Figure 2The comparison of carbon fixation capacity between the original and improved algal strains shows that gradient domestication and improvement of *Scenedesmus* can significantly enhance its carbon fixation capacity. Specifically, the peak carbon fixation rate of *Scenedesmus* under the control group was 0.45 g / L / d; the peak carbon fixation rates of the improved strains in Examples 1-3 were 0.92, 0.75, and 0.81 g / L / d, respectively, all significantly higher than the control group. Furthermore, *Scenedesmus* domesticated under the conditions of Example 1 was most capable of utilizing the degradation products of polyacrylamide for carbon fixation and growth.

[0069] Extensive cultivation experiments and data analysis conducted by the applicant show that a polyacrylamide concentration of 400–500 mg / L in the culture medium is most favorable for the growth and carbon fixation of the improved *Scenedesmus* strain. Specifically, the dry weight of *Scenedesmus* biomass reached 2.4 g / L on the seventh day of harvest, which is 115% of the control group; the peak carbon fixation rate reached 0.92 g / L / d, which is 124% of the control group. Excessively high concentrations of polyacrylamide (≥500 mg / L) have a delayed effect on the growth and carbon fixation of *Scenedesmus*. These experimental results can serve as an important reference when designing industrial production plans.

[0070] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A method for promoting the growth and carbon fixation of Scenedesmus using polyacrylamide in oilfield wastewater, characterized in that, Includes the following steps: (1) The original strain of Scenedesmus was inoculated into a photobioreactor containing a liquid culture medium containing a preset concentration of polyacrylamide; appropriate light conditions were provided, and a CO2 / N2 mixture was introduced into the reactor to provide a carbon source for the growth and carbon fixation of Scenedesmus. (2) After the culture is completed, take out an appropriate amount of algal strain and inoculate it into a new photobioreactor to increase the concentration of polyacrylamide in the liquid culture medium; repeat the inoculation in this way multiple times, and increase the concentration of polyacrylamide in the liquid culture medium in a gradient manner. (3) When Scenedesmus can grow stably and continuously in a liquid culture medium containing 1000 mg / L polyacrylamide, it is considered to have completed domestication; the genetic stability test of the domesticated Scenedesmus improved strains is carried out according to the conventional procedure, and the strains that can stably inherit are selected for large-scale culture. (4) Prepare a liquid culture medium using oilfield wastewater containing polyacrylamide and add it to the photobioreactor; An appropriate amount of the improved Scenedesmus strain was introduced into the liquid culture medium for inoculation, and a CO2 / N2 mixture was introduced; this promoted the decomposition of polyacrylamide while the Scenedesmus was growing with carbon fixation.

2. The method according to claim 1, characterized in that, The *Scenedesmus* strain was obtained from the freshwater algae strain bank of the National Aquatic Biological Germplasm Resource Bank, with the strain number FACHB-44.

3. The method according to claim 1, characterized in that, The illumination conditions were 70–90 μmol·m⁻². -2 ·s -1 The incubation temperature is 23–26℃.

4. The method according to claim 1, characterized in that, The CO2 / N2 mixture has a CO2 volume concentration of 6% and a flow rate of 8–12 mL / min.

5. The method according to claim 1, characterized in that, The liquid culture medium used is a typical formulation of Mineral Medium numbered BG11 in the ATCC culture medium database.

6. The method according to claim 1, characterized in that, During the gradient acclimatization process, the polyacrylamide in the liquid culture medium was increased in the following manner: the initial concentration was set at 10 mg / L, the concentration at the second inoculation was 100 mg / L, and the concentration was increased by 100 mg / L for each subsequent inoculation.

7. The method according to claim 1, characterized in that, During the gradient acclimatization process, 3 to 5 parallel groups were set up. After each culture, algal liquid samples were collected for biomass determination. After intergroup analysis with the original strain, the group with the highest microalgal growth rate and the highest microalgal CO2 fixation rate was selected for the next acclimatization step.

8. The method according to claim 1, characterized in that, When using an improved strain of Scenedesmus for large-scale culture, the concentration of polyacrylamide in the liquid culture medium should be controlled at 400–500 mg / L.

Images