A method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae and its application

Abstract

This invention discloses a method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae, comprising the following steps: (1) culturing *W. asterioni* strains in BG-11 medium under continuous light for 24 hours until the logarithmic growth phase; (2) inoculating *Bacillus* strains from solid LB medium into liquid LB medium and activating them at 37°C for 24 hours, then transferring the activated bacterial solution in the liquid LB medium into a seed culture medium; (3) first inoculating the *W. asterioni* strain from step (1) into the molasses alcohol wastewater, and then adding the activated bacterial solution from step (2) on the 6th day of cultivation; the bacterial and algal biomass in the treated wastewater can be used as fertilizer. This invention is the first to utilize *W. asterioni* and *Bacillus* synergistically to treat molasses alcohol wastewater, and the synergistic treatment of wastewater by bacteria and algae has a significant effect on removing organic matter. Furthermore, the combined use of bacterial and algal biomass and purified wastewater for the application of biofertilizer can effectively promote plant growth.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment and microbial fertilizer technology, and specifically relates to a method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae and its application. Background Technology

[0002] Currently, the main methods for wastewater treatment include physical methods (sedimentation, filtration, membranes, etc.), chemical methods (adsorption, ion exchange, etc.), and biological methods (bioadsorption, bioprecipitation, etc.). These methods have the advantage of removing substances from different types of wastewater and using different methods to remove pollutants. However, they also have disadvantages such as uneven removal efficiency, high installation costs, and uneconomical practices. Furthermore, they may cause secondary pollution and increase the difficulty of subsequent treatment. Microalgae have broad application prospects in wastewater treatment. They can effectively treat wastewater, solve environmental problems, and generate valuable byproducts. It has been found that co-culturing microalgae and bacteria has a positive effect on the accumulation of useful substances by microalgae and can degrade complex organic matter in wastewater into simple molecules that microalgae can utilize, which is more beneficial for wastewater treatment. However, the key to improving the removal efficiency of the system is to select the optimal combination of bacteria and algae, which requires strict screening of microalgae and bacteria.

[0003] Biofertilizers, comprising microalgae and microorganisms, contribute to the synthesis of fertilizer forms and promote the delivery of essential nutrients to developing crops to aid growth. Biofertilizers play a crucial role in organic agriculture by increasing the supply or availability of essential nutrients to host plants. The purpose of using biofertilizers is to increase the number of microorganisms, thereby accelerating microbial processes and improving the availability of nutrients for plant use. Therefore, combining wastewater and biomass resource utilization offers multiple benefits, including wastewater purification and biofertilizer development. A low-cost, easy-to-operate method needs to be developed for practical application. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention relates to a method for the co-treatment of molasses alcohol wastewater by bacteria and algae, and its application. Different bacterial strains have their own characteristics and potential application value. This application is the first to utilize *Asteris cuspidata* and *Bacillus* to co-culture and degrade substances in molasses alcohol wastewater. Subsequently, the bacterial and algal biomass can be used for corn planting, which can promote corn growth, couple the resource utilization of wastewater, reduce cultivation costs, harvest biomass, and simultaneously benefit the development of high-value agricultural fertilizers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae, wherein the strain used in the method is Bacillus sp. GXU-B2, which was isolated, screened and purified from wild dragon blood tree stems collected in Chongzuo, Guangxi in the previous laboratory. It is now deposited at the China Center for Type Culture Collection, with accession number CCTCC M20231974.

[0007] Includes the following steps:

[0008] (1) The star-shaped Wortellella species were cultured in BG-11 medium under continuous light for 24 hours until the logarithmic growth phase was reached;

[0009] (2) The Bacillus strain was inoculated from solid LB medium into liquid LB medium and activated at 37°C for 24 h. Then the activated bacterial solution in liquid LB medium was transferred to seed medium.

[0010] (3) First, inoculate the star-shaped Wortleyia from step (1) into the molasses alcohol wastewater, and then add the activated bacterial solution from step (2) on the 6th day of cultivation.

[0011] Furthermore, in step (1), the initial light intensity during continuous light cultivation is 100 μmol·m⁻¹. -2 ·s -1 During the later stages of cultivation, the light intensity was gradually increased to 250 μmol·m⁻². -2 ·s -1 Providing light can enhance the photosynthetic capacity of algae.

[0012] Furthermore, in step (1), a mixture of air containing 1% CO2 is continuously supplied during the cultivation process, which can promote the growth of the strain.

[0013] Furthermore, in step (3), the inoculum size of *W. stellaria* is OD750 = 0.7-0.9, and the inoculum size of the bacterial solution is OD600 = 0.9-1.1. The mixing ratio of *W. stellaria* and the bacterial solution is conducive to their symbiosis.

[0014] Furthermore, in step (3), the concentration of the molasses alcohol wastewater is 500 mg / L to 4500 mg / L, with the optimal concentration being 3500 mg / L.

[0015] Furthermore, the algae and bacteria biomass cultivated by the method, along with the treated wastewater, can be used as a bio-organic fertilizer to promote the growth of corn.

[0016] The alcohol wastewater used in this invention is molasses alcohol wastewater collected from Guangxi Guitang Group Co., Ltd.; the algal species is *Vischeria stellata* SAG 887-2, which was purchased from the Algal Culture and Collection Center (SAG) of the University of Göttingen, Germany (https: / / www.uni-goettingen.de / en / 45175.html) and preserved in BG-11 medium. The algal species underwent aseptic preservation culture and is now preserved in the Laboratory of Environmental Microbiology Resources and Utilization, College of Life Sciences and Technology, Guangxi University, with accession number GXU-A13; the bacterial strain is *Bacillus sp.* GXU-B2, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 20231974 and deposit date of October 20, 2023.

[0017] The advantages of this invention are as follows: This invention is the first to utilize Algae wiltii and Bacillus spp. to synergistically treat molasses alcohol wastewater. The synergistic treatment of wastewater by bacteria and algae has a significant effect on removing organic matter. Furthermore, the combined use of bacterial and algal biomass and purified wastewater for the application of bio-fertilizer can effectively promote plant growth. By cultivating bacterial and algal biomass in wastewater at low cost, the effective utilization of wastewater resources and the sustainable development of bio-fertilizer can be achieved. Attached Figure Description

[0018] Figure 1 These are optical microscope images of the star-shaped Wortellella of the present invention;

[0019] Figure 2 This is an optical microscope image of the algal symbiosis of the present invention;

[0020] Figure 3 This is a diagram showing the growth of *W. sterigmata* in molasses alcohol wastewater of different concentrations according to the present invention.

[0021] Figure 4 This is a diagram showing the growth of the bacteria and algae symbiosis in molasses alcohol wastewater of different concentrations according to the present invention;

[0022] Figure 5 This is a comparison image of the fluorescence characteristics of organic matter in molasses alcohol wastewater before and after purification by *W. stellatus* according to the present invention;

[0023] Figure 6 This is a comparison of the fluorescence characteristics of organic matter before and after purification of molasses alcohol wastewater by the bacterio-algae symbiosis method of the present invention;

[0024] Figure 7 This is a comparison diagram of the morphological characteristics of seedlings after the addition of bacterial and algal fertilizer according to the present invention. Detailed Implementation

[0025] The essential features of the present invention can be seen from the following embodiments, but these embodiments are for illustrative purposes only and are not intended to limit this aspect.

[0026] Example 1

[0027] A method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae includes the following steps:

[0028] (1) After the star-shaped *Westphalia sterigmata* strain was cultured in BG-11 medium with an air mixture containing 1% CO2 under continuous light for 24 h until it reached the logarithmic growth phase, the initial light intensity for continuous light culture was 100 μmol·m⁻¹. -2 ·s -1 During the later stages of cultivation, the light intensity was gradually increased to 250 μmol·m⁻². -2 ·s -1 .

[0029] (2) The Bacillus strain was inoculated from solid LB medium into liquid LB medium and activated at 37°C for 24 hours. Then the activated bacterial solution in liquid LB medium was transferred to seed medium, and the absorbance of the bacterial solution at OD=600nm was measured at regular intervals.

[0030] (3) First, inoculate the star-shaped Weissella in step (1) into the molasses alcohol wastewater. On the 6th day of cultivation, add the activated bacterial solution in step (2). Measure the biomass biomass every three days. Use three-dimensional fluorescence to measure the wastewater purification effect of the initial and final wastewater.

[0031] In step (3), the inoculum size of *Westphalia stellaria* is OD750 = 0.7–0.9, and the inoculum size of the bacterial solution is OD600 = 0.9–1.1.

[0032] In this embodiment, the alcohol wastewater was collected from the molasses alcohol wastewater of Guangxi Guitang Group Co., Ltd.; the algal species was Vischeria stellata SAG 887-2, which was purchased from the Algal Culture and Collection Center (SAG) of the University of Göttingen, Germany (https: / / www.uni-goettingen.de / en / 45175.html) and preserved in BG-11 medium. The algal species underwent aseptic preservation culture and was preserved in the Laboratory of Environmental Microbiology Resources and Utilization, College of Life Sciences and Technology, Guangxi University, with accession number GXU-A13; the bacterial strain was Bacillus sp. GXU-B2, which was deposited in the China Center for Type Culture Collection, with accession number CCTCC M 20231974 and deposit date of October 20, 2023.

[0033] In the treatment of the aforementioned molasses alcohol wastewater, a grouping model was adopted, dividing the treatment into two groups: a single treatment group for *W. werendrum erinaceus* and a symbiotic treatment group for bacteria and algae. The concentrations of the molasses alcohol wastewater used were 500 mg / L, 1500 mg / L, 2500 mg / L, 3500 mg / L, and 4500 mg / L, respectively. Each group had three control cases. During cultivation, the morphology of the algae and the symbiotic bacteria and algae was observed using optical microscopy. Figure 1 It can be seen that the cell membrane of *W. astrosa* in molasses alcohol wastewater is smooth, while under normal, non-stress conditions, the cell membrane is wrinkled, indicating that the wastewater environment affects the growth and metabolic processes of *W. astrosa*. Figure 2 It can be seen that in the symbiotic relationship between bacteria and algae in molasses alcohol wastewater, bacteria surround and attach near the algae, and the cell walls of the algae exhibit hollow regions. This indicates that there are structural connections and interactions between bacteria and algae, suggesting that bacterial-algae interaction is beneficial for survival in the molasses alcohol wastewater environment. Figure 3 and Figure 4 It can be seen that regardless of whether it is the single treatment group of *W. wilfordii* or the symbiotic treatment group of bacteria and algae, the biomass grows proportionally with the increase of wastewater concentration. Moreover, under the same wastewater concentration, the growth of bacteria and algae symbiosis is better than that of *W. wilfordii* alone, indicating that the interaction between bacteria and algae plays a positive role in growth. Therefore, this bacteria-algae combination has the potential economic value of producing high material energy.

[0034] Figure 5 and Figure 6 The images show a comparison of the fluorescence characteristics of organic matter in molasses alcohol wastewater before and after purification, diluted 10 times. The images show that microalgae can degrade, assimilate, and absorb organic matter in the wastewater, reducing its toxicity. Microorganisms and their enzymes mainly participate in the decomposition of organic matter in the wastewater. Both groups effectively reduced the concentration of various organic substances, and the higher the wastewater concentration, the more significant the organic matter removal effect. However, under the same wastewater concentration conditions, the fluorescence intensity removal of organic matter in molasses alcohol wastewater treated by the synergistic treatment of microorganisms and algae was superior to that of *W. w.* alone, indicating that the synergistic treatment capacity of microorganisms and algae is stronger than that of *W. w.* alone.

[0035] The application of the molasses alcohol wastewater after co-treatment of bacteria and algae as a biological organic fertilizer specifically involves using the bacterial and algal biomass after co-treatment of the molasses alcohol wastewater as fertilizer for crop growth, and using the algal and bacterial biomass together with the treated wastewater to irrigate the roots of plants as fertilizer.

[0036] When using bacterial and algal biomass as corn fertilizer, the corn seed used was Zhengdan 958. The experiment was conducted in a greenhouse, and the soil was sterilized at 121 degrees Celsius. Seedlings were grown in pots. Each pot contained an equal amount of finely ground, sieved, and air-dried soil. Corn seedlings of uniform size (4-5 cm in bud length and 3-4 cm in root length) were selected and planted, with three seedlings per pot and three parallel plants per group.

[0037] The algae-bacteria synergistic treatment group with a wastewater COD concentration of 3500 mg / L exhibited the highest biomass and best purification effect. Therefore, this group was used as the cultivation condition for bio-fertilizer. The cultured algae-bacteria biomass, along with the treated wastewater, served as bio-organic fertilizer. The application amounts (one-time) of the algae-bacteria bio-fertilizer from left to right were 0 ml, 50 ml, 100 ml, 150 ml, 200 ml, and 250 ml. The growth and development of the corn plants were observed. Plant height and stem diameter were measured on day 21. The plants were then removed from the pots. Figure 7 Wash with deionized water, separate the stems, leaves and roots, and measure the length and weight of the stems, leaves and roots respectively.

[0038] Table 1. Effects of different bacterial and algal concentrations on maize growth

[0039] Fertilizer application rate Plant height (cm) Stem diameter (cm) Root length (cm) Fresh root weight (g) Whole fresh weight (g) 0ml 23.52±0.65 0.31±0.01 18±0.43 0.16±0.02 1.06±0.12 50ml 26.13±0.43 0.42±0.02 15±0.23 0.13±0.06 1.13±0.32 100ml 43.01±1.32 0.63±0.01 53±0.97 1.22±0.02 6.27±0.45 150ml 41.55±1.1 0.51±0 45±1.23 1.12±0.01 4.84±0.33 200ml 35.52±0.32 0.54±0.02 29±0.76 0.25±0.04 2.87±0.56 250ml 30.17±0.54 0.41±0.01 36.3±0.31 0.35±0.02 2.89±0.46

[0040] Table 1 shows the effects of different fertilizer application rates on maize growth. With increasing fertilizer application rate, maize plant height followed a normal distribution, with the best growth observed at a 100ml fertilizer application rate, reaching plant height, stem diameter, and root length of 43.01cm, 0.63cm, and 53cm, respectively. This significantly promoted growth compared to the control group without fertilizer. This indicates that the purified wastewater can still be used for soil irrigation, achieving zero wastewater discharge, and that the wastewater can be used to cultivate bacteria and algae at low cost for use as bio-fertilizer. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 7 It was found that corn plants treated with microbial-algae fertilizer had more developed root systems. With 100ml of fertilizer added, the root length and fresh root weight reached 53cm and 1.22g respectively, and the corresponding above-ground plant height was also the highest. This indicates that applying microbial-algae fertilizer can promote root growth, increase root hair density, increase root volume, root absorption surface area, and root density, which is beneficial for expanding the soil space for crop water and nutrient absorption, and promoting the growth of the corn's above-ground parts. Therefore, the combination of microbial-algae organic fertilizer aligns with the principles of green and sustainable agricultural development and has enormous potential application prospects.

[0041] Although the specific embodiments of the present invention have been described and illustrated in detail above, it should be noted that various changes and modifications can be made to the above embodiments without departing from the spirit of the present invention and the scope set forth in the appended claims.

Claims

1. A method for the synergistic treatment of molasses alcohol wastewater by bacteria and algae, characterized in that, The strain used in the method is Bacillus sp. GXU-B2, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC M20231974. Includes the following steps: (1) The star-shaped Wortellella species were cultured in BG-11 medium under continuous light for 24 hours until the logarithmic growth phase was reached; (2) The Bacillus strain was inoculated from solid LB medium into liquid LB medium and activated at 37°C for 24 h. Then the activated bacterial solution in liquid LB medium was transferred to seed medium. (3) First, inoculate the star-shaped Wortleyia from step (1) into the molasses alcohol wastewater, and then add the activated bacterial solution from step (2) on the 6th day of cultivation.

2. The method for synergistic treatment of molasses alcohol wastewater by bacteria and algae according to claim 1, characterized in that, In step (1), the initial light intensity during continuous light cultivation is 100 μmol·m⁻¹. -2 ·s -1 During the later stages of cultivation, the light intensity was gradually increased to 250 μmol·m⁻². -2 ·s -1 .

3. The method for synergistic treatment of molasses alcohol wastewater by bacteria and algae according to claim 1, characterized in that, In step (1), an air mixture containing 1% CO2 is continuously supplied during the cultivation process.

4. The method for synergistic treatment of molasses alcohol wastewater by bacteria and algae according to claim 1, characterized in that, In step (3), the inoculum size of *Westphalia astericae* is OD750 = 0.7–0.9, and the inoculum size of the bacterial solution is OD600 = 0.9–1.

1.

5. The method for synergistic treatment of molasses alcohol wastewater by bacteria and algae according to claim 1, characterized in that, The concentration of the molasses alcohol wastewater in step (3) is 500 mg / L to 4500 mg / L.

6. The application of the algal and fungal biomass cultured by the method described in claim 1, together with the treated molasses alcohol wastewater, as a bio-organic fertilizer.

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