By ultrasonically lysing the engineered E. coli Rosetta that overexpressed Alginate lyase 2 (AL2), a crude enzyme solution was obtained and mixed with the alginate solution. The 3,5-dinitrosalicylic acid (DNS) method was used to test the concentration of reducing sugar and absorbance at 540 nm, together with the Bradford assay for protein concentration determination (The specific activity was defined as U/mg). Graph a shows the specific activity of the untransformed E.coli Rosetta(as control, the blue column) is 5U/mg, which may be caused by the subtle effect of other components of the cell or the experimental uncertainty. By contrast, the specific activity of alginate lyase (the red column) is 323U/mg, seeing a significant promotion of efficiency. For graphs B and C, different temperatures (25°C, 37°C, 45°C) and pH (pH2.5, pH6.1, pH7.4, and pH8.5) was used for testing, then we found that 37°C and pH7.4 is the optimum condition for alginate degradation. This result significantly proves the efficiency of alginate lyase in our recombinant bacteria, and provides a firm support for the further applications.

Cellulase Bgls was synthesized and cloned to the pET23b vector, the transformed to E.coli Rosetta. After culturing, the lysate of engineered E. coli was mixed with carboxymethylcellulose (CMC). The concentration of reducing sugar and the absorbance of 540nm was recorded by DNS method, as well as the concentration of protein by Bradford method. By using the untransformed E.coli Rosetta as control, graph a shows the activity of 0.5U/mg, which may results from the experimental uncertainty. By contrast, the crude enzyme shows a specific activity of 11.45U/mg. In graphs b and c, we then used different temperatures (25°C, 37°C, and 55°C) and different pH (pH5.8, pH6.5, pH7.4) for the same experiment, obtaining the result that 37°C and pH6.5 is the optimum condition for higher efficiency. Graph d shows the results of combining alginate lyase and cellulase together to digest the kelp, we can see the degradation rate increased as crude cellulase solution volume increased. The results proved the effects of cellulase, which can be used to boost efficiency with alginate lyase.

The SRRz suicide gene was synthesized and cloned downstream of the arabinose promoter in the pSB1A3 vector, then transformed to E.coli Rosetta. Different concentration of arabinose was used to induce the expression of SRRz. As shown in graph a, after monitoring the growth of bacteria for 28 hours, we found that the absorbance of bacteria culture at OD600 is inversely proportional to the concentration of arabinose, which indicates the suppressing effect of SRRz gene to the growth of bacteria. For graph B, we used One-way ANOVA to analyze the significance of induced culturing and non-induced culturing, p < 0.05 was considered significant.

When engineered bacteria are exposed to an environment with 1 mM of arabinose, the expression of SRRz is activated, leading to the complete lysis of the bacteria. The results are meaningful for preventing engineering bacteria to be released to the natural environment, which may cause biological contamination. However, under conditions of 0.1 mM arabinose, the expression level of SRRz cannot fully suppress bacterial proliferation. In such conditions, engineered bacteria can release alginate lyase and cellulase, which decompose kelp's intricate polysaccharides, providing invaluable resources for both industrial and agricultural sectors. In summary, by fine-tuning the arabinose concentration, the SRRz cleavage gene can be harnessed either to bolster biosafety or to break barriers in engineered bacteria.

To evaluate the effectiveness of engineered strains in kelp fermentation, we will apply the engineered strains to actual fermentation. We added the SRRz lysozyme gene to the aforementioned two engineered strains, controlled by an Arabinose promoter. To assess the performance of strains Bgls/SRRz and AL2/SRRz in kelp fermentation, we set up two groups. The CK group was not inoculated with any strains, while the Bgls/SRRz and AL2/SRRz group was co-inoculated with strains Bgls/SRRz and AL2/SRRz. The kelp was thoroughly cleaned with purified water to remove any accumulated dirt and dried at 65°C for 2 hours. Then, 1,000 g of dried kelp was soaked in water for 2 hours, chopped, and added to a 20 L fermentation bottle. Subsequently, 5 L of LB medium (with 100 μg/mL ampicillin) and 10% (v/v) of the engineered strains (OD600 = 0.6) were added. Based on the in vitro testing results of the SRRz lysozyme strain, 0.1 mM of L-arabinose was added to induce bacterial lysis and allow the engineered strains to continue growing. Fermentation was carried out at 37°C and 150 rpm. Every 48 hours, 50% of fresh LB medium (supplemented with 100 μg/mL ampicillin) was replaced in a laminar flow hood. After two weeks, the kelp in the fermentation bottle was filtered, washed, and dried at 65°C to a constant weight. The degradation rate was calculated by comparing the weight of the kelp before and after treatment. All experiments were conducted in triplicate, and the data will be expressed as mean±SD. It can be observed that the degradation rate of 1 kg of kelp by the mixed engineered strains Bgls/SRRz and AL2/SRRz was approximately 60% in the second week, as shown in Figure 4. These preliminary results suggest that our engineered bacteria can be applied in actual fermentation processes. In the future, we plan to use medium-sized fermenters equipped with stirring mechanisms, temperature and pH sensors, and flow meters to investigate the optimal fermentation conditions.

Alginate oligosaccharides have been shown to boost seed vitality and accelerate germination by promoting water uptake. Beside, the cellulase can hydrolyzes cellulose into monosaccharides, which serve as a source of energy for plants. To explore the potential of engineered bacterial degradation products as fertilizer, the kelp fermentation solution were collected and filtered using a 0.22-micron membrane to remove any bacteria present. The filtrate was collected and diluted to 10% with clean water to prevent adverse effects of high osmotic pressure on the seeds. Seeds in the test group were soaked in the filtered fermentation product for 24 hours. In contrast, the control group (CK) seeds were soaked in clean water. The treated seeds were then sown in seed cultivation trays for growth. On the third day, the germination status of each group of seeds was observed and recorded. Based on these observations, the germination rate for each group was calculated. We conducted a series of experiments to explore the impact of fermentation products on the germination rates of various seeds. The results revealed a significant increase in the germination rate of pine willow seeds after treatment with the fermentation products. In CK group, these seeds had a germination rate of 74%, but after the treatment, it rose to 90% (Table 1). Similarly, soybean seeds also showed a comparable trend, with their germination rate increasing from 76% to 84% (Table 1). In addition to these, we also tested the seeds of wheat, barley, and triticale. However, these seeds did not exhibit favorable germination in our experiments. Regardless of whether they were in the control group (CK) or the test group treated with fermentation products, the germination rates for these seeds were exceedingly low. We suspect that this might be due to issues related to our cultivation techniques or conditions. To address this issue, we plan to optimize and adjust our cultivation conditions in the future. We hope that by improving these conditions, we can enhance the germination rates of these seeds and further investigate the potential effects of the fermentation products on them.

Table 1. Germination rate of different treatment groups

In conclusion, the experiment data proves the signinficant effects of combining alginate lyase gene and bacterial cellulase together to decompose alginate polysaccharide to oligosaccharide, as well as the SRRz suicide gene, which enables engineered bacteria to be killed to prevent biological contamination. In order of the environmentally friendly and sustainable advantages, our system will be appropriate to be applied in fertilization fields, as the alginate oligosaccharides benefit the plants.