Results & Discussion

Results/Discussion and Final Figures

 Plants gather nutrients for their growth through trading ions with particles in the soil, thus understanding how different soil additives affect this process is imperative for the future of agriculture. Figure 1 can be used as a tool to visualize how amaranth uptakes nutrients and the differences between the experimental arms. With no changes, Figure 1 can be interpreted as the no additives arm. With more available nutrients in the soil, it better represents the fertilizer arm. Lastly, with rhizobacteria on the roots that make this trading process easier for the plant, it can be seen as the P.f. arm. Preliminary research in 11th grade was based on Devi et al.’s 2022 study on the plant growth promoting abilities of a consortium of mineral solubilizing and nitrogen fixing bacteria on amaranth and Ikhajiagbe et al.’s 2021 research on the growth responses of amaranth when grown with the Pseudomonas fluorescens (P.f.) rhizobacteria. To gather the results of each arm of the experiment, each plant in each arm had its height in centimeters and number of leaves measured every 10 days for 40 days. The number of leaves/plant was recorded as it is a good overall indicator of plant health. The plants receiving fertilizer were watered with the recommended dosage of 20-20-20 NPK chemical fertilizer every 3 days, and the plants receiving P.f. had their soil inoculated with 1 mL of a confluent P.f. solution.

During their development, plants take up nutrients from the soil as a growth mechanism. Traditional fertilization methods seek to infuse soil with an abundance of nutrients to facilitate the growth of plants to a larger size. In this case, the recommended dosage of 20-20-20 NPK chemical fertilizer acted as a positive control, as amaranth are grown using this method in industrial agriculture around the world and how this fertilizer impacts growth is already well understood (Kumar and Shastri, 2017). On the other hand, rather than seeking to artificially put nutrients into the soil through harmful chemical fertilizer, studies like Devi et al’s (2022) are trying to harness symbiotic relationships between plants and rhizobacteria to facilitate the uptake of nutrients that are harder for plants to utilize. This is a newly emerging idea and it is not well understood, thus the 1 mL dosage of a confluent Pseudomonas fluorescens solution acted as the unknown arm.

Like in Devi et al.’s 2022 study, a significant increase in height was observed in the P.f. arm when compared to the no additives group (p = 0.01; p < 0.05). The mean height after 40 days for the no additives group was 6.3 cm (+/- 1.2 cm) while, for the P.f. group, it was 8.1 cm (+/- 1.4 cm) (Figure 2A). Additionally, it was observed that the fertilizer arm and the P.f. arm had no significant difference, with the mean height after 40 days for the fertilizer arm being 8.4 cm (p = 0.77; p > 0.05) (Figure 2A). 

A significant difference in number of leaves per plant was also observed between the no additives arm and the P.f. arm (p = 0.02; p < 0.05). The mean number of leaves after 40 days for the no additives arm was 4.9 (+/- 1.4 leaves/plant) while it was 6.2 (+/- 0.7 leaves/plant) for the P.f. group (Figure 2B). It was also observed that the P.f. arm had significantly more leaves per plant on average than the fertilizer arm (p = 0.002; p < 0.05). The mean number of leaves after 40 days for the fertilizer arm was 4.4 (+/- 1.1 leaves/plant) (Figure 2B).

This result was unexpected, but demonstrated the potential of rhizobacteria to grow plants larger and healthier. These results suggest that a single species of nitrogen-fixating rhizobacteria could act as a sustainable alternative to the standard 20-20-20 NPK chemical fertilizer as they both grew plants significantly larger than plants in the no additives group. However, if a single species has such a large effect, this data suggests that a consortium of rhizobacteria could improve amaranth growth. The P.f. culturing process was the biggest challenge while conducting this preliminary study. In addition to the large amount of time each culture would take to start, there were inconsistencies in the incubation process that compromised the reliability of the cultures. This led to a search for a more efficient and reliable method to make the cultures to remove this bottleneck from future experimentation. Instead of creating new cultures from scratch each time one was required, glycerol stocks were created from fully confluent bacteria solution, making the process quicker and more reliable. Thus, adapting this project to study the effects of a consortium of beneficial rhizobacteria on amaranth was much easier. Wisconsin Fast Plants were chosen to take preliminary data primarily for their fast growth and widespread use as a model organism (Kelly, 2004).

Based on junior year’s preliminary results, the senior year study focused on the effects of a novel consortium of beneficial rhizobacteria on amaranth height and number of leaves grown . Figures 3A and 3B show data on average fast plant height (cm) and number of leaves respectively after 30 days. This data was collected to see whether a consortium of P.f. and B.s. would improve plant growth better than either of them individually. The hypothesis was that the plants inoculated with a 50-50 consortium of Pseudomonas fluorescens and Bacillus subtilis would grow significantly taller and have more leaves than their counterparts in the no additives arm due to the nitrogen fixating and mineral solubilizing properties of the bacteria. This hypothesis also asserts that plants inoculated with one type of bacteria, Pseudomonas fluorescens or Bacillus subtilis respectively, would grow significantly taller and have significantly more leaves than their counterparts grown with no additives for the same reason.

After performing two-tailed independent t-tests, significant differences in height were observed between the no additives arm (10.6 cm +/- 2.2 cm) & the P.f. arm (17.1 cm +/- 2.8 cm) (p = 0.000002; p < 0.05), between the no additives arm (10.6 cm +/- 2.2 cm) & the B.s. arm (17.2 cm +/- 2.4 cm) (p = 0.00; p < 0.05), and between the B.s. arm (17.2 cm +/- 2.4 cm) & the 50-50: P.f.:B.s. arm (19.4 cm +/- 2.5 cm) (p = 0.04; 0 < 0.05) (Figure 3A). Significant differences in number of leaves were observed between the no additives arm (5.4 leaves/plant +/- 1.4 leaves/plant) & the P.f. arm (6.9 leaves/plant +/- 0.9 leaves/plant) (p = 0.005; p < 0.05), between the no additives arm (5.4 leaves/plant +/- 1.4 leaves/plant) & the B.s. arm (7.0 leaves/plant +/- 1.5 leaves/plant) (p = 0.01; p < 0.05), and between the B.s. arm (7.0 leaves/plant +/- 1.5 leaves/plant) & the 50-50: P.f.:B.s. arm (8.5 leaves/plant +/- 1.7 leaves/plant) (p = 0.03; 0 < 0.05) (Figure 3B). No other t-tests were conducted, as these results are sufficient substantial evidence to support the experimental hypothesis.

Because the preliminary data taken on Fast Plants confirmed that, when compared to individual strains of rhizobacteria, a consortium of P.f. and B.s. helps Wisconsin Fast Plants grow taller and healthier, the experiment was continued using that consortium on amaranth. The plants grown with no additives and those grown with the 50-50 mix of P.f. & B.s. are being compared to plants grown with fertilizer to answer if consortia of rhizobacteria could act as suitable replacements for chemical fertilizers. The hypothesis was that plants inoculated with a 50-50 consortium of P.f. and B.s. would grow significantly taller and have more foliage than plants grown with no additives, similarly high to plants in the fertilizer arm, and significantly more leaves than plants in the fertilizer arm. It also predicts that plants grown with fertilizer would grow significantly taller than their no additive counterparts. Figures 4A and 4B show data on average fast plant height (cm) and number of leaves respectively after 40 days. This data was taken to see whether a consortium of Pseudomonas fluorescens and Bacillus subtilis would grow the amaranth better than the recommended dosage of 20-20-20 NPK chemical fertilizer.

After performing two-tailed independent t-tests, significant differences in height and number of leaves were found (Figure 4A & 4B). Significant differences in height were observed between the no additives arm (10.9 cm +/- 1.1 cm) & the 50-50: P.f.:B.s. arm (14.5 cm +/- 1.4 cm) (p = 0.00001; p < 0.05), between the no additives arm (10.9 cm +/- 1.1 cm) & the fertilizer arm (12.7 cm +/- 1.3 cm) (p = 0.0035; p < 0.05), and between the fertilizer arm (12.7 cm +/- 1.3 cm) & the 50-50: P.f.:B.s. arm (14.5 cm +/- 1.4 cm) (p = 0.01; 0 < 0.05) (Figure 4A). Significant differences in number of leaves were observed between the no additives arm (5.4 leaves/plant +/- 1.3 leaves/plant) & the 50-50: P.f.:B.s. arm (8.8 leaves/plant +/- 1.8 leaves/plant) (p = 0.0002; p < 0.05), between the no additives arm (5.4 leaves/plant +/- 1.3 leaves/plant) & the fertilizer arm (7.0 leaves/plant +/- 0.9 leaves/plant) (p = 0.006; p < 0.05), and between the fertilizer arm (7.0 leaves/plant +/- 0.9 leaves/plant) & the 50-50: P.f.:B.s. arm (8.8 leaves/plant +/- 1.8 leaves/plant) (p = 0.01; 0 < 0.05) (Figure 4B). These results are substantial evidence to support most aspects of the experimental hypothesis. For example, the hypothesis stated that plants in the 50-50: P.f.:B.s. arm would grow similarly to plants in the fertilizer arm, yet the plants in the 50-50: P.f.:B.s. arm grew significantly higher (p = 0.01; 0 < 0.05). Devi et al’s 2022 research on the effects of a consortium of rhizobacteria on amaranth also found that a consortium grew plants significantly more than fertilizer, suggesting that this result wasn’t caused by experimental error. The hypothesis also stated that plants with no additives wouldn’t grow significantly less foliage than plants in the fertilizer arm, which was disproven (p = 0.006; 0 < 0.05). Although junior year preliminary data conflicts with this, it is very plausible that increased nutrient availability in non-degraded soil could cause plants to be healthier.

Although the hypothesis that a consortium of rhizobacteria could grow plants similarly to or more than fertilizer was supported during this experiment, there were some experimental issues and confounding variables that led to the death of some plants, which impacted the length of the study. One issue was a fungus gnat infestation, stemming from the high humidity environment that the plants were kept in. Even though it may seem like there is a possibility that the bacteria killed the plants, it would be very unlikely considering only one to two plants from each arm of the experiment died and there was no similar problem for the fast plants grown with the same bacteria just weeks prior.

The results of this study suggest that the possibility that consortia of beneficial rhizobacteria could act as sustainable replacements for harmful chemical fertilizers is rising. The potential of biofertilizers created with consortia of rhizobacteria in the field of sustainable agriculture is extensive. Implementing these types of biofertilizers in agricultural settings would completely eliminate the destructive downsides of chemical fertilizers, leading to increased soil health and fertility, decreased runoff, and decreased levels of ecosystem destruction. Future studies should focus on better understanding the widespread agricultural implementation of consortia of rhizobacteria and using new advancements in the field of synthetic biology to find the most efficient community of these bacteria for the growth and productivity of common crops like wheat or rice. This information could change the face of industrial agriculture, eliminate a large percentage of food insecurity through increased levels of crop production, and preserve millions of hectares of ecosystems around the world.