Alginate is a high molecular weight polysaccharide extracted from seaweed, widely used in various fields such as food, medicine, cosmetics, and agriculture. However, due to its non-degradable nature, its production and use lead to significant waste and environmental pollution. Therefore, finding an efficient method for alginate degradation is crucial. Kelp is a typical brown algae rich in alginate and cellulose polysaccharides. We employed microbial alginate lyase and cellulase enzymes to hydrolyze kelp, aiming to enhance its degradation efficiency and product quality. The microbial alginate lyase can break down alginate into smaller oligosaccharides and monosaccharides, while cellulase can decompose cellulose into smaller cellulose molecules. Additionally, cellulase helps increase the solubility of hydrolysis products. We overexpressed alginate lyase (AL2) and bacterial cellulase bgls separately and verified their functions. To enhance the safety of engineered bacteria and release alginate lyase and bacterial cellulase, we also studied the lysis effect of SRRz under the control of the arabinose promoter. Moreover, the oligosaccharides produced by alginate lyase can promote plant growth and enhance their stress resistance, while the sugars produced by cellulase can provide energy for plants. To verify the benefits of the fermentation products from the engineered bacteria on plants, we applied these recombinant engineered bacteria in kelp fermentation, treated seeds with the fermentation liquid, and then tested their germination rate.

In order to maximize the utility of alginate oligosaccharides, we first need to produce them. Alginate oligosaccharides are formed through the hydrolysis of alginate. After reviewing the literature, we discovered that alginate lyase can perfectly accomplish this task. As a biological enzyme, it offers advantages such as mild reaction conditions, ease of operation, low energy consumption, high yield, high specificity, and environmental friendliness. After a thorough comparison, we found that enzymatic degradation has significant advantages over chemical and physical degradation methods. Therefore, we have decided to use it as our method for hydrolyzing alginate.

The recombinant vector was transformed into E. coli Rosetta cells ，the cell pellet was collected by centrifugation and resuspended in Tris-HCl (pH 7.4). The cells were then lysed through ultrasonication, yielding the cell lysate. The total protein concentration in the lysate was determined using the Bradford assay kit . To validate the function of the alginate lyase, 0.9 mL of substrate solution alginate , 50 mM Tris-HCI buffer, 200 mM NaCl, was mixed with 0.1 mL of cell lysate and incubated at 37°C for 30 minutes before sampling. The concentration of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) method. The absorbance at 540nm was recorded to calculate the activity. The activity in the control group was measured at 5 U/mg. Under our experimental conditions, the specific activity of the alginate lyase crude extract was determined to be 323 U/mg in figure4A. This means that each milligram of crude extract can release 323 micromoles of reducing sugar per minute. This result demonstrates the high efficiency of the alginate lyase in our engineered bacteria, providing strong support for further applications.

To determine the optimal reaction temperature for alginate lyase, we diluted the cell lysate with Tris-HCl buffer at pH 7.4 and mixed it with a 0.5% (w/v) solution of alginate. After incubating at 25°C, 37°C, and 45°C for 30 minutes, we measured the concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method. To determine the optimal reaction pH for alginate lyase, we mixed the cell lysate with a 0.5% (w/v) solution of alginate using different buffer solutions. We used a 10 mM citrate buffer at pH 6.1, as well as Tris-HCl buffers at pH 7.4, 8.5, and 10.2. After incubating at 37°C for 30 minutes, we measured the concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method.The result is shown in Figure 4B and 4C. The optimal reaction temperature for AL2 is 37°C, and the optimal pH condition is 7.4.

The results have shown that alginate lyase is capable of hydrolyzing alginate efficiently and in a sustainable way. However, since algae in seaweed, not only do they have large amounts of alginate, but they also contain tons of cellulose that might surround the alginate inside the cells of algae and seaweed, preventing the alginate lyase from hydrolyzing as much alginate as possible. Therefore, we want to see if there is a way to hydrolyze the cellulose effectively.

After brainstorming and literature collection within the group, we found that bacterial cellulase is very suitable for the task of degrading cellulose. Bacterial Cellulase, an enzyme produced by bacteria, can break down cellulose into a usable carbon source. Bacterial Cellulase belongs to a complex enzyme system, including Endoglucanase (EC 3.2.1.4), Exoglucanase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21), all of which play roles in cellulose degradation. With the three enzymes working together, the end product would be glucose.

To evaluate the expression and activity of Bacillus subtilis cellulase Bgls in E. coli Rosetta, we first synthesized the bgls gene and cloned it into the pET23b vector. Engineered E. coli Rosetta was cultured in LB broth medium for 3 days at 37°C, and then centrifuged at 13,000 rpm for 5 minutes. Cell pellet was resuspended in PBS buffer (10 mM, pH 7.4) and then lysed through ultrasonication (150 W, 1s sonication with 3s intervals, for a total of 20 minutes). 1 ml of the supernatant (crude enzyme solution) was mixed with one ml of 1% carboxymethylcellulose (CMC, Sigma-Aldrich) solubilized in PBS buffer (10 mM, pH 7.4) and incubated at 37°C for 30 minutes under shaking (120 rpm). One ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the mixture was boiled for 5 minutes, then the absorbance was measured at 540 nm.

Under our experimental conditions, the specific activity of bgls was determined to be 11.45 U/mg in figure8A. Translation: To determine the optimal reaction temperature of cellulase, we mixed crude enzyme solution with a 1% CMC solution and incubated it at 25°C, 37°C, and 55°C for 30 minutes. After that, we used the 3,5-dinitrosalicylic acid (DNS) method to measure the concentration of reducing sugars. We found that the optimal reaction temperature for cellulase was 37°C, as shown in Figure 9B.

Furthermore, to determine the optimal reaction pH of cellulase, we mixed the crude enzyme solution with a 1% CMC solution under 37°C conditions. After incubation for 30 minutes at pH 5.8, 6.5, and 7.4 (in PBS buffer), we measured the concentration of reducing sugars using the DNS method. The optimal pH for Bgls was found to be 6.5, as shown in Figure 9C.

To verify the digestion efficiency of AL2 and bgls, the kelp was thoroughly washed with purified water to remove any accumulated dirt and dried at 65°C for 2 hours. 1g of dried kelp was soaked in water for 2 hours and then cut into 1cm2 pieces. The crude enzyme solution of cellulase and brown algae enzyme was prepared using Tris-HCI (pH 7.4). 1g of dried kelp and 20 mL reaction mixture containing 10 mM Tris-HCI buffer, 10 mL brown algae enzyme solution, and varying volumes of cellulase solution were prepared. After incubation at 37°C and 180 rpm for 24 hours, samples were taken. The kelp was filtered, washed, and dried at 65°C until a constant weight was achieved. Degradation rates were calculated by comparing the weight of the kelp before and after treatment. As shown in Figure 10, we found that the degradation rate of the kelp increased progressively with increasing volume of the crude cellulase solution. When the volume of crude cellulase solution reached 10 mL, the degradation rate of the kelp reached 59.11%.

By the results given above, we have proven that bacterial cellulase is capable of breaking down cellulose and forming glucose in a sustainable and highly efficient way. However, as the bacterial cellulase and alginate lyase are expressed through genetic engineering in Escherichia coli and our substrates are located outside the bacterial cells, we need to lyse the bacteria to allow the expressed enzymes to come into contact with the substrates and facilitate the reaction. Additionally, as we are incorporating Escherichia coli in our fertilizer production process, ensuring biosafety is of utmost importance. Therefore, we would need to find a way to make sure all E. coli die before the alginate oligosaccharide (as an end product) gets used as a fertilizer.

The recombined E.coli left will cause contaminations to the environment, we decided to insert SRRz lytic gene, which are linked genes consist of perforin gene S, phage lysosome(transglycosidase) gene R, and gene RZ, into the plasmid to control bacteria to decompose by itself.

We used linked genes composed of the perforin gene S, the phage lysozyme (transglycosidase) gene R, and the gene RZ. Arabinose promoter, BBa B0015 terminator is used. The constructed plasmid was introduced into Escherichia coli Rosetta receptor cells.

The figure 13A shows the growth of E.coli Rosetta in 28 hours with different concentration of arabinose. It is obvious that the concentration of arabinose is inversely proportional to the absorption value, in which when there is 0 mM arabinose, the absorption value at OD600 is about 2.25, but when there is 1 mM arabinose, the absorption value is about 0.2. The results show the significant decrease of the amount of bacteria as arbinose concentration increases, which proves the obvious effect of arabinose and SRRz genes.

The figure 13B shows the rate at which the bacteria proliferate under different concentrations of arabinose. The horizontal coordinate indicates different concentrations of arabinose, and the vertical coordinate indicates the absorption value of 600nm wavelength detected by the bacteria over a 28-hour period. The higher the absorption value, the more quantity. The results showed that with the increase of arabinose concentration, the light absorption value decreased and the number of bacteria decreased. We use One-way ANOVA to determine the statistical significance between induced and non-induced culture at the 28th hour, and p < 0.05 was considered statistically significant. The results showed that bacterial growth inhibition was enhanced with the increase of induction intensity.

The application of the SRRz cleavage gene in the field of biotechnology has garnered widespread attention. Especially in terms of the biosafety of engineered bacteria, this gene has demonstrated significant potential. 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. This means that at this concentration of arabinose, engineered bacteria cannot survive, ensuring they do not inadvertently leak into the external environment, posing a potential threat to the ecosystem. However, under conditions of 0.1 mM arabinose, the expression level of SRRz is not sufficient to fully inhibit bacterial growth. This offers us a unique opportunity where, under these conditions, bacteria can release alginate lyase and cellulase. These enzymes are crucial for the degradation of kelp, as they can break down the complex polysaccharides in kelp, providing valuable raw materials for industry and agriculture. In summary, by precisely controlling the concentration of arabinose, we can utilize the SRRz cleavage gene to achieve biosafety and barrier disruption in engineered bacteria, offering a new strategy for biotechnological applications.

To evaluate the effectiveness of engineered strains in kelp fermentation, we will apply the engineered strains to actual fermentation in the future. We added the SRRz lysozyme gene to the aforementioned two engineered strains, controlled by an Arabidopsis sugar 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. Tissue culture membrane sealing was used to ensure oxygen supply. 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 14.

We designed an experiment to explore the potential of fermentation products from engineered bacteria on kelp as fertilizers. Several types of seeds were chosen as test subjects to evaluate the impact of the fermentation products on their germination rates.

To obtain the fermentation products, we fermented kelp using engineered bacteria. After fermentation, we filtered the products using a 0.22-micron membrane to remove any potential bacteria and then diluted it to a concentration of 10%.

Seeds were divided into two groups: the test group seeds were soaked in the filtered fermentation product, while the control group seeds were soaked in clean water. After a 24-hour soak, they were sown in cultivation trays. On the third day, we observed and recorded the germination status of each seed group. Notably, pine willow seeds treated with fermentation products showed a germination boost from 74% to 90%. Similarly, soybean seeds exhibited an increase from 76% to 84%. However, wheat, barley and triticale seeds did not perform well in our tests, with low germination rates in both the control and treatment groups. This could be attributed to our cultivation methods or conditions.

Table 1. Germination rate of different treatment groups

The fermentation products of engineered bacteria indeed promote germination in certain seeds, providing preliminary evidence for their potential application in agriculture. Additionally, experimental conditions, such as the concentration of fermentation products and soaking duration, may significantly influence the results, suggesting that we need to be more meticulous in selecting and optimizing these conditions in future design and build phases. Given the low germination rate of some seeds, a more comprehensive cultivation condition should be considered in future testing stages. Lastly, we need to further investigate the long-term effects of fermentation products when applied in agriculture.

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.