Results

Our project aims to investigate the potential of utilizing engineered microalgae strains and microalgae-bacteria interaction systems for water remediation. Firstly, we optimized the efficiency of the microalgae-bacteria interaction system by screening different ratios of P. tricornutum and Q. aquimaris. Subsequently, we compared various immobilization methods to enhance the stability and repeatability of the microalgae-bacteria system. Furthermore, through synthetic biology techniques, we constructed engineered microalgae strain ——FUCO3 with enhanced nitrogen absorption, lipid accumulation, and fucoxanthin accumulation, which were then screened and filtrated. Finally, we evaluated the bioremediation effectiveness of these engineered strains under varying water quality conditions and analyzed their impact on aquatic environments. This study provides a new bioremediation idea and method for solving water pollution problems using engineered microalgae strains and microalgae-bacteria interaction systems.

Phaeodactylum tricornutum is an economically significant microalga in marine ecosystems. It serves as both aquaculture feed and a source of valuable bioactive compounds such as EPA, DHA, and fucoxanthin. Qipengyuania aquimaris, a widely distributed bacterium found in marine environments, exhibits functions including organic matter degradation and heavy metals detoxification. The interaction between P. tricornutum and Q. aquimaris holds great potential for bioremediation strategies aimed at effectively addressing issues related to nitrogen-phosphorus elements, heavy metals, and organic pollutants in marine ecosystems. However, the molecular mechanisms and regulatory factors governing this interaction remain unclear; furthermore, the physiological effects of different bacteria-to-microalgae ratios on microalgae require further research and exploration.

In order to explore the optimal ratio of microalgae-bacteria interaction between P. tricornutum and Q. aquimaris, we used the cell density ratio of the two as the basis, and set up four treatment groups with microalgae-bacteria ratios of 1:1, 1:3, 1:5, and 1:7, respectively, to monitor physiological indicators. The results showed that in the interaction system with Q. aquimaris, the cell growth rate increased significantly with the increase of bacteria concentration, and the biomass increased significantly in the 1:3 and 1:5 treatment groups (Figure 1 A,B), but the photosynthetic efficiency Fv/Fm decreased significantly (Figure 1 C). In addition, combined with the analysis of chlorophyll and fucoxanthin relative content (Figure 1 C,D), it was found that the accumulation of microalgal pigments had a significant positive effect within a certain range with the increase of bacteria concentration.

Additionally, by conducting visual analysis of the microalgae oil plasts and analyzing the average fluorescence intensity of the oil plasts using flow cytometry (Figure 2), it was observed that the inclusion of Q. aquimaris in the culture enhanced lipid accumulation in microalgae to a certain extent when compared to the control.

Figure 1 Effects of different microalgae-bacteria ratios on microalgae related indicators
(A, cell growth curve; B) biomass; C, photosynthetic efficiency Fv/Fm; D, relative chlorophyll content; E, relative content of fucoxanthin; microalgae: bacteria is cell density ratio.)

Figure 2 Visualization and mean fluorescence intensity analysis of microalgal oil plasts

Conclusions

By culturing P.tricornutum with Q. aquimaris at various density ratios, we assessed the growth rate, biomass, oil accumulation, photosynthetic efficiency, and pigment accumulation of P.tricornutum. The findings revealed that:

·The addition of Q. aquimaris has a more obvious improvement on the growth rate of P. tricornutum, and has a significant promotion effect on biomass.

·As the concentration of Q. aquimaris increased in the co-culture system, the photosynthetic efficiency of P.tricornutum initially increased rapidly and then gradually .The treatment groups with the ratios of 1:1 and 1:3 exhibited significant improvement in Fv/Fm on D1, while the group with a ratio of 1:3 maintained good photosynthetic efficiency until D5.

·Relative pigment content demonstrated that treatment groups with ratios of 1:1 and 1:3 promoted pigment accumulation in P.tricornutum approximately 1.3-fold respectively; However, treatment group with a ratio of 1:7 exerted a significant inhibition on chlorophyll content consistent with poor photosynthetic efficiency.

Regarding lipid accumulation in the interaction system between P.tricornutum and Q. aquimaris , there was a substantial elevation in lipid accumulation within the groups with ratios of 1:1 and 1:3. Specifically, lipid accumulation increased by 80.08% compared to the control for the 1:3 treatment group.

Based on these findings, we primarily analyzed photosthetic efficiency and lipid accumulation as key factors to evaluate bioremediation performance in co-cultivation systems. Consequently, we selected a co-cultivation ratio of P. tricornutum to Q. aquimaris at of 1:3 for further experiments.

Prospect

These findings suggest a mutualistic relationship between P. tricornutum and Q. aquimaris, potentially involving the exchange of nutrients, signaling molecules, and antioxidants. The results of this study offer an effective co-culturing method for marine microalgae and bacteria with significant implications for the development of microalgae-based biofixation, biodiesel and functional pigments synthesis

Comparing different immobilization schemes for microalgae-bacteria interaction system

Introduce

Results

In order to further analyze the interaction mechanism of P. tricornutum and Q. aquimaris and screen out a relatively optimal immobilization cultivation mode, we designed five interaction cultivation forms under the premise of the selected microalgae-bacteria ratio.

By monitoring the nitrogen consumption rate in the medium (Figure 3 A), we found that the ABO treatment group had the fastest nitrogen absorption rate compared with the conventional interaction form of AB control group, and AO and ABS had relatively slower nitrogen absorption rates. According to the above phenomenon, it can be seen that when microalgae are isolated by embedding materials, although they are still in the same medium with bacteria, their influence by the substances produced by bacteria is significantly enhanced, but when microalgae and bacteria are mixed and co-embedded (ABS), this phenomenon does not occur.

Then, by comparing the fucoxanthin (Figure 3 B) and chlorophyll (Figure 3 C) of each treatment group, it was found that when bacteria were immobilized in one of the forms in the treatment group, they had a positive effect on the accumulation of chlorophyll and fucoxanthin. In addition, by oil plasts visualization analysis (Figure 4), it was found that when bacteria were separately embedded and immobilized, the lipid accumulation of microalgae in the same environment was significantly reduced, but the volume and fluorescence intensity of microalgal chloroplasts increased.

Figure 3 The effect of different embedding conditions on microalgae
(A, the change of total nitrogen content in the medium; B, the relative chlorophyll content of different treatment groups; C, the relative fucoxanthin content of different treatment groups. microalgae-bacteria liquid co-culture (AB), microalgae (AO) or bacteria (BO) separately immobilized and co-cultured with bacteria or microalgae, microalgae and bacteria separately immobilized and co-cultured (ABO), microalgae and bacteria co-immobilized (ABS); chlorophyll and fucoxanthin detection for D7)

Figure 4 TAGs accumulation in microalgae
（TAGs content was determined by BODIPY 505/515 Laser scanning confocal microscope LSM (Leica),with 488 nm excitation wavelength and 530 nm emission wavelength）

Conclusions

Filtrating of the engineered microalgae strains

By designing corresponding primers, we successfully amplified and cloned BBa_K4688002 to BBa_K4688004 from the P. tricornutum genome. Then, we used the infusion method to connect the obtained fragments to the expression vector provided by the 2022 CHINA-FAFU team, and successfully identified them (Engineering-Design Cycle 3: Test).

We transformed the constructed expression vector into P. tricornutum by electroporation, and obtained potential engineered microalgae strain monoclonal clones on the selection plate with zeocin resistance after 15 days (Figure 5 A); then, we randomly picked monoclonal microalgae strains and cultured them in seawater with resistant medium, and verified the picked monoclonal clones at DNA, RNA, and Protein levels respectively (Figure 5 B,C,D).

At the Protein level verification (Figure 5 B), we took the monoclonal microalgae strains that had been enlarged and cultured, and used the tag in the expression vector to perform Western Blot verification. The results showed that only FUCO3 engineered microalgae strain had a protein size that matched the expectation (FUCO3: 84.15kDa), while FUCO1 (81.51 kDa) and FUCO2 (84.16 kDa) results were much larger than the expected protein band size.

In order to further confirm the correctness of the engineered microalgae strain, we verified it at the DNA level (Figure 5 C), using the positive control of the expression vector that was verified correctly in the previous stage as a reference, and using the universal identification primers designed with 50bp before and after the gene insertion site in the expression vector to perform PCR amplification on DNA; by comparison, we successfully identified FUCO1 and FUCO3 engineered microalgae strains. However, since FUCO1 and FUCO2 had similar results that were too large at the protein level, and FUCO2 microalgae strain did not pass the identification at the DNA level, we chose to perform a new round of microalgae transformation on FUCO1 and FUCO2, and temporarily continued with FUCO3 microalgae strain for subsequent identification.

In addition, in order to analyze whether the expression vector successfully overexpressed the target gene in microalgae, we performed RNA level identification on FUCO3 (Figure 5 D), and obtained positive results. The expression level of PtFUCO3 gene in FUCO3 microalgae strain was about 8-fold higher than that in WT at the same period.

Finally, after multiple identifications, we selected FUCO3 engineered microalgae strain to carry out subsequent experimental plans.

Figure 5 Identification of the engineered microalgae strain
(A, monoclonal transgenic microalgae strain; B, protein level identification; C, DNA level identification; D, RNA transcription level identification)

Evaluating the bioremediation potential of genetically modified microalgae strain

Introduce

Results

In terms of cell growth rate and water nitrogen utilization, we found that FUCO3 microalgae strain had a higher growth rate in the early stage of the growth cycle (Figure 6 A), and reached a similar level to the control in the later stage; in terms of nitrogen utilization efficiency, FUCO3 microalgae strain had a faster nitrogen utilization efficiency than the control (Figure 6 B), and achieved a more efficient nitrogen removal after forming an microalgae-bacteria interaction system with Q. aquimaris.

Figure 6 The monitoring of microalgae growth and nitrogen use efficiency
（WT，wild type；FUCO3，Overexpressing strain of PtFUCO3；WT-Qa：Bacteria and microalgae interactions of wild type microalgae strains；FUCO3-Qa，Bacteria and microalgae interactions in microalgae strains overexpressing PtFUCO3）

In terms of photosynthetic efficiency and pigment accumulation, analysis of the Fv/Fm measurement results revealed that the engineered microalgae strain FUCO3 exhibited significantly higher photosynthetic efficiency during the D1 adaptation period of the growth cycle (Figure 7 A). This enhancement could potentially be attributed to increased levels of antioxidant substances such as fucoidan within its microalgae, thereby considerably shortening the adaptation period for this engineered strain. Furthermore, examination of NPQ results pertaining to light energy dissipation capacity demonstrated that FUCO3 displayed superior photosynthetic resistance under both conventional cultivation and microalgae-bacteria interaction cultivation conditions. Compared to WT, FUCO3 exhibited an approximate 35% increase in both photosynthetic resistance and high light (Figure 7 B). In terms of microalgal pigment accumulation, FUCO3 exhibits a significant improvement compared to the wild type, with an approximately 1.75-fold increase in fucoxanthin content (Figure 7 C).

Figure 7 Measurement of microalgal photosynthetic capacity and pigment accumulation
(A, photosynthetic efficiency Fv/Fm; B, relative content of fucoxanthin; C, photosynthetic NPQ level)

Regarding lipid accumulation, through visualization of microalgal oil bodies and analysis of average fluorescence intensity during the D7 cultivation cycle, we observed that the FUCO3 microalgae strain exhibited a 40% increase in lipid accumulation under conventional cultivation conditions and a 22% increase under microalgae-bacteria interaction mode compared to the control (Figure 8).

Figure 8 Visualization analysis and mean fluorescence intensity analysis of microalgal oil plasts

In addition, in order to further test the ability of engineered microalgae and the designed bacteria-microalgae co-culture system in wastewater bioremediation, we used simulated artificial wastewater for testing in the laboratory. Since the expectation for wastewater bioremediation in the real society is fast enough and takes into account the effect, we focus on the early data of the engineered microalgae in the test phase. Through analysis, it can be found that in the artificial wastewater environment, the cell growth rate, biomass and photosynthetic efficiency of FUCO3 microalgae are significantly higher than those of the control (Figure 9 A, B,C). Moreover, analysis of cell growth rates indicated that incorporation of the bacteria-microalgae co-culture system conferred a certain level of resistance to wild-type P. tricornutum in simulated artificial wastewater conditions (Figure 9 A).

Figure 9 Physiological monitoring of microalgae under artificial wastewater conditions
(A, cell growth curve; B, Biomass; C, photosynthetic efficiency Fv/Fm; WT: wild type; FUCO3, PtFUCO3 overexpressing engineered microalgae; WT-W & FUCO3-W, two microalgae cultured under simulated wastewater conditions; WT-W-Qa & FUCO3-W-Qa: two microalgae co-cultured with bacteria under simulated wastewater conditions)

Finally, we evaluated the expression levels of key genes, fucoxanthin synthesis, and nitrogen and phosphorus utilization pathways in FUCO3 microalgae, which possess superior characteristics such as enhanced lipid accumulation, removal of eutrophication-causing elements, and improved photosynthetic efficiency. Encouragingly, a significant up-regulation trend was observed for most of the key genes in these pathways (Figure 10), providing robust molecular evidence to support our subsequent analysis on the role of PtFUCO3 in related metabolic processes.

Figure 10 Analysis of the key gene expression in different metabolic pathways

Conclusions

Summary

In our project, we introduced a study on the co-culture of P. tricornutum and Q. aquimaris, and an experiment of overexpressing fucoidan synthesis related genes in P. tricornutum by genetic engineering. The results showed that the co-culture of bacteria and microalgae can improve the growth rate, biomass, lipid accumulation, photosynthetic efficiency and pigment accumulation of microalgae, and FUCO3 engineered microalgae showed better performance than wild-type P. tricornutum in these aspects, and also showed great potential in wastewater bioremediation. These findings revealed the important role of bacterial-algal interaction and fucoidan synthesis genes in microalgal metabolism, and provided new ideas and methods for the application of microalgae and bacterial-algal co-culture systems.

Additional Product

The compilation of the "Bacteria and Algae manual"

Bacteria-microalgae symbiosis is a mutually beneficial relationship that holds significant potential for applications in bioenergy, biopharmaceuticals, and other fields. However, the knowledge and technical applications of bacteria-microalgae symbiosis are not yet widespread or comprehensive enough. Many beginners who wish to enter this field face the challenge of lacking systematic learning materials and guidance. To address this issue, we have authored a handbook on bacteria-microalgae symbiosis that aims to summarize the fundamental knowledge and practical methods of this topic while facilitating quick initiation into related research for beginners. In writing this handbook, we drew upon relevant literature from both domestic and international sources as well as our own research experience in the field of bacteria-microalgae symbiosis. Our goal was to provide comprehensive yet concise content with clear structure and rigorous logic. We hope that our handbook will serve as a valuable resource for those new to the field while also providing reference material for researchers already working in it.

The manual serves as a comprehensive guide to the applications of fungal-microalgae interactions, specifically designed to facilitate beginners in swiftly initiating their research endeavors within this field.

The manual covers the following content:

·The definition, characteristics and classification of microalgae and bacteria, as well as the morphological structure and physiological features of common microalgae and bacteria;

·The application of microalgae and bacteria in various fields, such as water purification, bioenergy, biopharmaceuticals, etc.;

·The cultivation methods of microalgae and bacteria and the protection and management measures;

·The principle, mechanism, effect and advantage of microalgae-bacteria interaction.

The manual has the following characteristics

·The content is comprehensive, encompassing the fundamental knowledge, technical methodologies, and practical applications of microalgae-bacteria interaction.

·The language is concise yet informative, accompanied by abundant illustrations to facilitate comprehension and implementation.

·The structure exhibits clarity and the logical flow maintains rigor, rendering it convenient for both reference and learning purposes.

The manual holds the following significance

·It offers a comprehensive learning resource for novices in the field of microalgae-bacteria interaction, enabling them to acquire essential knowledge and skills.

·It functions as an informative guidebook for promoters involved in the field of microalgae-bacteria interaction, effectively showcasing their accomplishments and demonstrating the significance of this research area.