Method for improving stress tolerance of microalgae endocrine disrupter by utilizing phycobacterium

Abstract

The invention relates to the field of microorganisms, and discloses a method for improving the stress tolerance of microalgae endocrine disruptors by utilizing phycobacterium, which comprises the following steps: co-culturing phycobacterium and microalgae; the endocrine disrupter is nonyl phenol; the phycobacterium is rosette-shaped rhizobium and / or brevundimonas vesicula. According to the invention, the effect of improving the stress tolerance of the endocrine disrupter (nonyl phenol) of the microalgae by part of interalgal bacteria of the microalgae is found for the first time. By co-culturing the algae and the microalgae, the survival and growth capabilities of the microalgae under the stress of nonyl phenol can be effectively improved, so that the tolerance and removal capability of the microalgae to high-concentration endocrine disrupter polluted wastewater are expected to be improved in an algae-bacteria co-culture manner.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a method for improving the stress tolerance of microalgae to endocrine disruptors by utilizing algal bacteria. Background Art

[0002] Nonylphenol (NP) is the primary degradation product of nonylphenol ethoxylates (NPEOs), a nonionic surfactant widely used in the production of detergents, emulsifiers, and pesticides. In recent years, the discharge of large amounts of NP-containing wastewater and the degradation of NPEOs in the environment have led to increasingly prominent water pollution problems in rivers, lakes, and coastal seawater worldwide. Concentrations of NP in natural waters and wastewater have been reported to reach as high as 644 μg / L and 1350 μg / L, respectively. As a typical environmental endocrine disruptor, NP exhibits teratogenic, carcinogenic, and mutagenic effects, posing a serious threat to biological health and ecosystem stability. Currently, NP has been listed by the United Nations Environment Protection Agency (UNEP) as one of 27 priority persistent toxic pollutants. Traditional wastewater treatment technologies primarily include physical / chemical methods (which are costly) and microbial methods (which require significant energy input and are unsuitable for treating wastewater containing high concentrations of nutrients). These technologies generate large amounts of sludge, which creates an environmental burden.

[0003] Microalgae are the oldest photosynthetic autotrophs on Earth, contributing nearly 50% of global photosynthesis. They are primary producers in aquatic ecosystems, playing a crucial role in regulating ecological stability and biogeochemical cycles. Microalgae can utilize a large number of nutrients in wastewater for growth and have the ability to adsorb and degrade pollutants such as endocrine disruptors. Consequently, they have been used in the treatment of industrial, agricultural, and municipal wastewater. While treating wastewater, microalgae can also produce high-value-added products such as bioenergy, animal feed, and biofertilizer, achieving resource recovery. However, the effectiveness of microalgae in biologically removing pollutants such as endocrine disruptors varies depending on the algal species. Due to their small size and relatively simple structure, microalgae are limited in their growth and metabolism when exposed to the stresses of highly contaminated wastewater, resulting in a reduction in biomass and reduced wastewater treatment effectiveness. Therefore, it is necessary to improve the tolerance of microalgae to adverse environments to meet the needs of large-scale, efficient, and rapid wastewater treatment.

[0004] A limited number of reports indicate that exogenous addition of NaHCO₃ can help increase microalgae biomass, thereby enhancing their ability to remove endocrine disruptors from wastewater. Notably, bacteria also play a crucial role in microalgae's growth, development, adaptation, and tolerance to pollutants such as heavy metals, pesticides, and benzene. Co-cultivation with bacteria to enhance microalgae photosynthesis, thereby increasing their biomass and their ability to remove nitrogen and phosphorus from wastewater, has been widely reported. However, there are fewer reports on using bacteria to enhance microalgae's tolerance to stress, particularly to endocrine disruptors.

[0005] In summary, in order to meet the growing demand for wastewater treatment, if some bacteria that can improve the tolerance of microalgae to endocrine disruptor stress can be discovered, it is hoped that the tolerance and removal ability of microalgae to high-concentration endocrine disruptor-contaminated wastewater can be improved through algae-bacteria co-cultivation. Summary of the Invention

[0006] To address the above technical problems, the present invention provides a method for improving microalgae's tolerance to endocrine disruptor stress using phycobiotic bacteria. This invention, for the first time, discovers that some phycobiotic bacteria from microalgae have the ability to improve microalgae's tolerance to endocrine disruptor stress (nonylphenol). Co-culturing these bacteria with microalgae can effectively enhance the microalgae's ability to survive and grow under nonylphenol stress. Therefore, it is expected that co-culturing microalgae with bacteria could improve microalgae's tolerance to and removal of high-concentration endocrine disruptor-contaminated wastewater.

[0007] The specific technical solutions of the present invention are: In a first aspect, the present invention provides a method for improving microalgae's tolerance to endocrine disruptor stress using phycozobacteria, comprising: co-culturing the phycozobacteria with microalgae. The endocrine disruptor is nonylphenol, and the phycozobacteria are Rhizobium rosetteiformis and / or Brevundimonas vesicularis.

[0008] This study, published in the journal Nature Communications, demonstrates that some microalgae-derived bacteria can enhance their tolerance to endocrine disruptors (nonylphenols). Co-culturing these bacteria with microalgae effectively enhances their survival and growth under nonylphenol stress. This suggests that co-culturing microalgae with bacteria could potentially improve their tolerance to and removal of high-concentration endocrine disruptor wastewater. Specifically: (1) The present invention found that some phycobiotic bacteria can increase the cell density of microalgae under nonylphenol stress and significantly increase the chlorophyll and carotenoid content of cells. In addition, they can also reduce the oxidative damage to algal cells caused by nonylphenol exposure. The above results indicate that some phycobiotic bacteria can promote the photosynthetic growth and pollution tolerance and adaptability of microalgae under stress by reducing the damage to the photosynthetic organs of microalgae caused by nonylphenol stress and increasing the content of cellular photosynthetic pigments.

[0009] (2) The present invention found that some algal bacteria can significantly increase the total amount of EPS and PS content in the culture system. EPS is an important stress-resistant substance in microalgae and plays a key role in cell tolerance to pollution stress.

[0010] (3) The present invention found that nonylphenol stress significantly inhibited microalgae photosynthesis, energy metabolism (glycolysis, TCA cycle, oxidative phosphorylation), protein synthesis, amino acid metabolism (Arg, Pro, Ala, Tyr, etc.), vitamin synthesis and metabolism (VB1, VB2, VB5, VB6), and antioxidant capacity. Some algal bacteria can reduce the inhibitory effect of nonylphenol on gene expression in the corresponding metabolic pathways of algal cells and stimulate microalgae to fix CO2 and the synthesis and metabolism of intracellular substances.

[0011] Preferably, the rosette rhizobium is named ZJ2401-1, and was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33764, and the microbial classification is named Rhizobium rosettiformans.

[0012] Preferably, the Brevundimonas bulatal is named ZJ2403-1, and was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33765, and the microbial classification is named Brevundimonas bulatal. Preferably, the microalgae is Diplococtilla fasciata.

[0013] In a second aspect, the present invention provides a rosette-shaped rhizobium, named ZJ2401-1, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33764, and the microbial classification was named Rhizobium rosettiformans.

[0014] The rosette rhizobium selected by the present invention is particularly effective in improving the tolerance of microalgae to endocrine disruptor stress.

[0015] In a third aspect, the present invention provides the use of rosette rhizobium for improving the tolerance of microalgae to endocrine disruptor stress, wherein the endocrine disruptor is nonylphenol.

[0016] Preferably, the microalgae is Diplococtilla fasciata.

[0017] In a fourth aspect, the present invention provides a strain of Brevundimonas bulatal, named ZJ2403-1, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33765, and the microbial classification was named Brevundimonas bulatal.

[0018] The Brevundimonas vesicularis screened out in the present invention is particularly effective in improving the tolerance of microalgae to endocrine disruptor stress.

[0019] In a fifth aspect, the present invention provides the use of Brevundimonas vesicularis for improving the tolerance of microalgae to endocrine disruptor stress, wherein the endocrine disruptor is nonylphenol.

[0020] Preferably, the microalgae is Diplococtilla fasciata.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This study first discovered that some phycobiotic bacteria in microalgae can improve the microalgae's tolerance to endocrine disruptor (nonylphenol) stress. Co-culturing these bacteria with microalgae can effectively enhance the microalgae's survival and growth ability under nonylphenol stress. Therefore, it is expected that co-culturing microalgae with bacteria can improve the microalgae's tolerance to and removal of high-concentration endocrine disruptor wastewater.

[0022] (2) The present invention screened and obtained rosette rhizobium ZJ2401-1 and vesicular Brevundimonas ZJ2403-1, which are particularly effective in improving the tolerance of microalgae to endocrine disruptor stress and therefore have great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of bacteria on the growth of Dictyolithiasis under nonylphenol stress; Figure 2 This is a diagram showing the effects of different bacteria on the tolerance of Glechoma tenuifolia to nonylphenol stress; Figure 3 This is a diagram showing the effects of Brevundimonas on the photosynthetic pigments and cell structure of Dictyolithiasis under nonylphenol stress; Figure 4 The figure shows the effect of Brevundimonas on the secretion of extracellular polymers by Dictyolithiasis under nonylphenol stress; Figure 5 This is a diagram showing the effect of Brevundimonas on the gene expression of Dictyolithiasis under nonylphenol stress; Figure 6 This is a diagram of the bacterial species composition of the algal microbiome in Example 1. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the embodiments.

[0025] Example 1: Experiment on the effect of bacteria on the growth of Dictyolithiasis under nonylphenol stress To investigate the effects of bacteria on the growth of NP-stressed Diplodocella, normal algae (microbiome composition, bacterial species, and microbial composition) were grown in 50 mL of sterile BG11 (sodium nitrate: 1.5 g / L; potassium phosphate dibasic: 40 mg / L; magnesium sulfate heptahydrate: 75 mg / L; calcium chloride: 36 mg / L; citric acid: 6 mg / L; ammonium ferric citrate: 6 mg / L; disodium ethylenediaminetetraacetic acid: 1 mg / L; A5 trace metal solution: 1 mL) medium for 8 days. Figure 6 As shown), sterile algae NP exposure culture test. Microalgae were inoculated to the initial OD 680 The value was 0.06, and the concentration of nonylphenol NP was 0, 0.01, 0.1, 0.3, and 0.9 mg / L. The microalgae culture conditions were light intensity 60 μmol m -2 s -1 , temperature 28℃, relative humidity 60%, light / dark time ratio 14h / 10h.

[0026] The results are as follows Figure 1 As shown in the figure, ab is the cell density of normal algae and sterile algae when exposed to 0, 0.01, 0.1, 0.3, and 0.9 mg / L NP for 2, 4, and 8 days; cd is the growth inhibition rate of normal algae and sterile algae under different concentrations of NP exposure; ef is the actual growth picture of normal algae and sterile algae after exposure to different concentrations of NP for 8 days.

[0027] The results showed that the cell density of normal Reticulum spp. with or without NP exposure was higher than that of sterile algae ( Figure 1 NP's effect on normal algal growth exhibited a "low-promoting, high-inhibiting" effect: NP concentrations ≤ 0.3 mg / L significantly promoted normal algal growth, while high NP concentrations (0.9 mg / L) significantly inhibited microalgal growth. In contrast, low-concentration NP treatment (0.1 mg / L) significantly inhibited the growth of sterile algae (P < 0.05). Furthermore, in the presence of the phycosphere microbiome, the inhibition rate of 0.9 mg / L NP on microalgal growth decreased from 75.8%, 50.6%, and 31.9% to 53.7%, 25.0%, and 15.7% at 2, 4, and 8 days, respectively. After 8 days of culture, the darker, greener color of the normal algae culture medium compared to the sterile algae further demonstrated that the phycosphere microbiome enhanced the tolerance and growth of NP-exposed microalgae.

[0028] Example 2: Experiment on improving the tolerance of Reticulum glutinosinum to nonylphenol stress by different bacteria To investigate the effects of different bacteria on the tolerance of G. reticulum to NP (nonylphenol) stress, different bacteria were cultured in dual cultures with sterile G. reticulum. Sixteen bacterial strains, including five Rhizobium, five Pseudomonas, two Brevundimonas, two Blastomonas, and two Sphingomonas, were isolated from the periphyton of G. reticulum after 8 days of NP exposure by the spread and streak method. All bacteria were grown in LB medium (10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl). G. reticulum was grown in BG11 medium. All culture media were autoclaved at 121°C for 15 minutes before use.

[0029] A 4-day sterile algae and bacterial co-culture experiment was conducted in 50 mL of sterile BG11 medium. The concentration of the endocrine disruptor nonylphenol NP was 0.9 mg / L. The microalgae were inoculated to an initial OD 680 The value was about 0.06, and the bacteria (OD 600 ≈1) The inoculation amount was 0.05%, 0.5%, and 1% (v / v). The algae-bacteria co-culture conditions were: light intensity 60 μmol m -2 s -1 , temperature 28℃, relative humidity 60%, light / dark time ratio 14h / 10h.

[0030] The results are as follows Figure 2 As shown, ae is Rhizobium, f and h are Blastomonas, g is Brevundimonas, ik is Pseudomonas, and l is Sphingomonas. The cell density of sterile Glechoma subtilis exposed to 0 mg / L (CK) and 0.9 mg / L NP for 2 and 4 days with and without bacteria addition.

[0031] The results showed that 12 bacterial strains had the ability to promote the growth and resistance of microalgae. Among them, Rhizobium bacteria are typical growth-promoting bacteria in the algae. All five isolated bacteria could significantly (P < 0.05) promote the growth of Glechoma spp. under both NP stress and non-NP stress, and the algal cell concentration increased with the increase of bacterial addition ( Figure 2 ae); further, Rhizobium 2( Figure 2 b, CGMCC No.33764) had the best effect among the five Rhizobium bacteria, Brevundimonas 1( Figure 2g, CGMCC No.33765) had the best effect among all the isolated and screened strains. In addition, it is worth noting that the stress environment stimulated the bacteria (Brevundimonas sp.1, Blastomonas sp.1, Pseudomonas sp.1, 2, 4, Sphingomonas sp.1) to promote the growth of microalgae. For example, in the NP-free treatment group, the addition of Brevundimonas sp.1 had no significant effect on the growth of sterile algae (P>0.05); however, under 0.9 mg / L NP stress, the addition of 0.05%, 0.1%, and 0.5% Brevundimonas could significantly (P<0.01) increase the algal cell concentration; especially under 0.5% bacterial addition, the cell concentration of NP-stressed microalgae could increase by 39.3% ( Figure 2 g). The above results demonstrate that bacteria can significantly improve the tolerance of Dictyophora to NP stress.

[0032] Example 3: Effects of bacteria on the photosynthetic pigments and cell structure of Dictyota glutinosae under nonylphenol stress In order to clarify the specific role of bacteria in improving the tolerance of Dictyota glutinosae under nonylphenol stress, Brevundimonas vesicularis (CGMCC No. 33765), which has the most significant effect on promoting the growth of stressed microalgae, was selected for sterile algae-bacteria binary culture. The culture experiment was carried out for 8 days in 50 mL of sterile BG11 medium with a nonylphenol NP concentration of 0.9 mg / L. The microalgae were inoculated to an initial OD 680 The value was about 0.06, and the bacteria (OD 600 ≈1) The inoculum size was 0.05%, 0.5%, and 1% (v / v). The culture conditions were: light intensity 60 μmol m -2 s -1 , temperature 28°C, relative humidity 60%, light / dark ratio 14h / 10h. On the 8th day, the culture medium was collected, the cells were obtained by centrifugation, and after freezing and thawing three times, they were placed at -20°C overnight. The photosynthetic pigments of the microalgae were extracted using the 95% ethanol-water bath heating method, and the chlorophyll a, b and carotenoid contents were measured and calculated by spectrophotometry. At the same time, 2.5% glutaraldehyde solution was immediately added to the algal cells collected by centrifugation to fix the cells overnight. They were rinsed with 0.1mol / L phosphate buffered saline (pH 7.0) and fixed twice with 1% OsO4 solution (1-2h). After dehydration with a series of gradient ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, 100%), the algal cells were sectioned, stained, and the ultrastructure of the microalgae cells was observed using a projection microscope.

[0033] The results are as follows Figure 3Figure 2 shows the chlorophyll (a, b) and carotenoid contents of Diploctatoides glutinosae after 8 days of culture under NP stress with 0, 0.05%, 0.5%, and 1% bacterial addition. Figure 2 shows the ultrastructure of the microalgae cells with and without bacterial addition.

[0034] The results showed that Brevundimonas could significantly (P<0.05) increase the chlorophyll and carotenoid contents of microalgae cells while increasing the cell density of NP-stressed microalgae. Figure 3 In particular, at a 0.5% addition rate, chlorophyll a, b, and carotenoid contents increased by 39.8%, 43.6%, and 30.7%, respectively, compared to sterile algae exposed to 0.9 mg / L NP. Furthermore, unlike the plasmolysis and chloroplast / thylakoid degradation that occurred in algal cells exposed to 0.9 mg / L NP, bacterial addition mitigated the oxidative damage induced by NP exposure. These results suggest that bacterial addition can promote photosynthetic growth, pollution tolerance, and adaptability of stress-induced microalgae by mitigating NP damage to their photosynthetic apparatus and increasing cellular photosynthetic pigment content.

[0035] Example 4: Effects of bacteria on the secretion of extracellular polymers by Dictyolithiasis under nonylphenol stress The effect of Brevundimonas bacteria (CGMCC No. 33765) on the tolerance of Glechoma viridis to nonylphenol stress was further analyzed based on the secretion characteristics of microalgae extracellular polymers (EPS). The specific steps are as follows: after 8 days of culture, the culture medium was collected, the culture medium was removed by centrifugation, and the cells were resuspended in 0.6% NaCl solution. The microalgae EPS was extracted by high-speed centrifugation, and after passing through a 0.22μm filter membrane, the EPS and extracellular protein content were determined using total organic carbon analysis and BCA protein detection kits. The fluorescence spectral characteristics of the EPS solution were analyzed using a fluorescence spectrophotometer under the conditions of excitation wavelength of 220-400nm, emission wavelength of 250-500nm, increment of 5nm, and scanning rate of 1200nm / min.

[0036] The results are as follows Figure 4 As shown, ac is sterile algae (AA), 0.9 mg / L NP exposed algae (NP 0.9) under different proportions of bacteria added per unit cell (10 6 Figures 5 and 6 show the total amount of EPS, exopolysaccharide (PS), and protein (PN) contents in the cultured microalgae. Figures 5 and 6 show the three-dimensional fluorescence spectra of EPS solutions in the co-culture system of sterile algae with 0%, 0.05%, 0.5%, and 1% bacteria, respectively, without and with NP treatment. A and B represent tyrosine and tryptophan, respectively.

[0037] The results showed that the EPS of Glechoma microalgae was mainly composed of PS; NP stress significantly (P<0.05) stimulated the secretion of EPS in microalgae cells and increased the PS content ( Figure 4 When the addition of Brevundimonas bacteria was ≥ v.5%, the effect of NP exposure on EPS and PS secretion per unit cell was significantly weakened, indicating that the presence of bacteria alleviated the stress of NP on algal cells. At the same time, under NP exposure, compared with microalgae culture alone, the significant increase in microalgae cell density, especially at a 0.5% addition ratio, also led to a significant (P < 0.05) increase in the total EPS and PS content in the algae-bacteria co-culture system ( Figure 4 EPS is an important stress-resistant substance in microalgae and plays a crucial role in cellular tolerance to pollution stress. These results indicate that the addition of bacteria (0.5%) to the co-culture system enhances algal cell tolerance to NP stress by promoting microalgal growth and increasing EPS, particularly PS, content. Furthermore, although bacterial addition had no significant effect on total NP content per cell or in the system (P>0.05), a decrease in the abundance of tyrosine and tryptophan aromatic proteins further suggests that bacterial addition mitigates NP stress on microalgae.

[0038] Example 5: Effects of bacteria on gene expression in Dictyota glutinosae under nonylphenol stress To further analyze the underlying mechanisms by which algae-bacteria co-culture enhances NP tolerance in Glechoma reticulata, we compared gene expression in Glechoma reticulata with and without the addition of 0.5% Brevundimonas bacteria (CGMCC No. 33765) after 8 days of NP exposure. The following steps were performed: Algal broths were collected from the different treatments after 8 days of NP exposure at 0 and 0.9 mg / L. Total DNA was extracted using a Trizol kit and deoxyribonuclease was added to remove DNA. The resulting RNA was detected and purified, and then AT bases were ligated with polyA using Oligo(dT)-labeled magnetic beads to enrich for mRNA, which was then fragmented. cDNA was synthesized from the mRNA template, and a cDNA library was constructed for Illumina HiSeq sequencing. Sequencing data were filtered, assembled using Trinity sequencing, and then subjected to Corset hierarchical clustering analysis. Differentially expressed genes (DEGs) were identified based on a |log2FC| > 1 and an FDR < 0.05. At the same time, KEGG was used to functionally annotate DEGs, and the molecular mechanism by which bacteria enhance the tolerance of microalgae to NP stress was analyzed at the gene expression level.

[0039] The number of DEGs in different treatments Figure 5As shown in the results, after 8 days of 0.9 mg / L NP exposure, the number of genes significantly upregulated and downregulated was 305 and 1026, respectively, compared to the no NP treatment. In the presence of 0.5% Brevundimonas bacteria, the number of DEGs decreased by 70.5%, and the corresponding number of upregulated and downregulated genes was 165 and 215, respectively, indicating that bacterial addition alleviated the stress effect of NP on algal cells. Further analysis revealed that NP stress significantly inhibited microalgal photosynthesis, energy metabolism (glycolysis, TCA cycle, oxidative phosphorylation), protein synthesis, amino acid metabolism (Arg, Pro, Ala, Tyr, etc.), vitamin synthesis and metabolism (VB1, VB2, VB5, VB6), and antioxidant capacity. However, the addition of bacteria reduced the inhibitory effect of NP on gene expression in the corresponding metabolic pathways of algal cells and stimulated CO2 fixation and intracellular substance synthesis and metabolism, thereby improving the growth level and stress tolerance of microalgae under NP stress.

Claims

1. A method for improving the tolerance of microalgae to endocrine disruptor stress using algal bacteria, characterized in that: Co-cultivation of algal bacteria with microalgae; The endocrine disruptor is nonylphenol; The algal bacteria are rosette rhizobia and / or vesicular Brevundimonas.

2. The method according to claim 1, wherein: The rosette rhizobium is named ZJ2401-1, and was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33764, and the microbial classification is named Rhizobium rosettiformans.

3. The method according to claim 1, wherein: The brevundimonas bulatal was named ZJ2403-1, and was deposited in the General Microbiology Center of the China Culture Collection Administration on March 10, 2025, with a deposit number of CGMCC No. 33765, and the microbial classification was named Brevundimonas bulatal.

4. The method according to claim 1, wherein The microalgae is Diplococephala.

5. A rosette-shaped Rhizobium strain, characterized by: It was named ZJ2401-1 and deposited in the General Microbiology Center of China Culture Collection Administration on March 10, 2025, with the deposit number CGMCC No.33764. The microbial classification was named Rhizobium rosettiformans.

6. The use of the rosette rhizobium according to claim 5 for improving the tolerance of microalgae to endocrine disruptor stress, characterized in that: The endocrine disruptor is nonylphenol.

7. The method according to claim 6, wherein The microalgae is Diplococephala.

8. A strain of Brevundimonas albosum, characterized by: It was named ZJ2403-1 and deposited in the General Microbiology Center of China Culture Collection Administration on March 10, 2025, with the deposit number CGMCC No.33765. The microbial classification was named Brevundimonas bulatal.

9. The use of Brevundimonas vesicularis for improving the tolerance of microalgae to endocrine disruptor stress according to claim 8, characterized in that: The endocrine disruptor is nonylphenol.

10. The method according to claim 9, wherein The microalgae is Diplococephala.

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