Problem and Purpose

• Fritz Haber and Carl Bosch's collaboration revolutionized agriculture via liquid ammonia synthesis, paving the way for synthetic fertilizers. This was crucial for feeding a rapidly growing global population.
• Synthetic nitrogen fertilizer: Widespread use doubled human-made nitrogen compounds, posing severe pollution threats to water, soil, and the atmosphere.
• Nitrogen surplus: Vital for life but now a hazardous pollutant, contaminating water sources, endangering wildlife, and impacting human health. Contributes significantly to climate change through nitrous oxide emissions.
• Biological nitrogen fixation (BNF): Sustainable solution utilizing nitrogen-fixing microorganisms. However, BNF is energy-intensive, hindering atmospheric nitrogen conversion to plant-usable forms.
• Project objective: Enhance Azotobacter's biological nitrogen fixation efficiency by modifying nitrogenase enzyme activity through exposure to varied iron (II) ion and salinity concentrations.

Hypothesis 

Our hypothesis suggests that the addition of ferrous sulphate to the growth medium will augment the efficiency of nitrogen fixation by Azotobacter strains, owing to its provision of essential iron cofactors crucial for nitrogenase enzyme activity. Conversely, it also suggests that excessive concentrations of sodium chloride may impede nitrogen fixation efficiency due to potential osmotic stress and disruption of cellular processes, despite its necessity for bacterial growth. Thus, the hypothesis anticipates that optimizing ferrous sulphate concentration while maintaining low sodium chloride levels will yield maximal nitrogen fixation efficiency in the Azotobacter strains.

Characteristics of Nitrogenase

• Nitrogenase: Key enzymes in nitrogen fixation, converting atmospheric nitrogen to ammonia.
• Azotobacter's nitrogenase variations: Azotobacter species possess a diverse set of nitrogenases, with the molybdenum-iron nitrogenase being the most commonly used. The atlernative contains vanadium and is more active in low temperatures.
• Adaptability: Different nitrogenase types allow efficient nitrogen fixation in diverse environments. For instance, VFe nitrogenase excels at lower temperatures, aiding nitrogen fixation in colder settings. 
• Nitrogenase components: Component I (Dinitrogenase) and Component II (Dinitrogenase reductase). In the different types of nitrogenases, Component I consists of several homologous metalloproteins, specifically the molybdenum-iron (MoFe) protein in the Mo nitrogenase, vanadium-iron (VFe) protein in the V nitrogenase, and an iron (Fe) protein in the iron only nitrogenase.
  • Component I is further categorized into a P-cluster and a FeMo-cofactor (alternatively VFe or FeFe), the specific site of nitrogen fixation. Component II is an iron protein with the function of transferring electrons to Component I to reduce atmospheric nitrogen into ammonia.

Process of Biological Nitrogen Fixation

• Biological nitrogen fixation (BNF): Process that converts atmospheric nitrogen to ammonia via nitrogenase.
• Reaction: 
• Nif genes: Genes responsible for the coding of proteins associated with nitrogen fixation. Widely found among nitrogen fixing bacteria across diverse environments.
• Sensitivity to oxygen: Nitrogenases degrade in oxygen-rich conditions, causing many bacteria to cease enzyme production. Biological nitrogen fixation is therefore essentially a anaerobic process. Some nitrogen-fixing organisms use oxygen-binding proteins like leghemoglobin to facilitate oxygen supply and protect nitrogenase from being inactivated by oxygen.

Characteristics of Azotobacter

• Azotobacter: A genus of bacteria crucial in the nitrogen cycle that converts atmospheric nitrogen into ammonium ions.
• Interaction with plants: While some Azotobacter species engage in plant interactions, most perform independent nitrogen fixation without symbiotic relationships.
• Characteristics: Motile, oval or spherical diazotrophs forming resilient cysts, thriving as soil microbes.
• Function: Primarily involved in nitrogen fixation, thriving in diverse environments and occasionally in symbiotic relationships with plants.
• Biofertilizer significance: Globally acknowledged as an effective biofertilizer, Azotobacter is a pivotal model organism for diazotroph studies.
• Additional roles: Azotobacter serves multiple purposes, contributing to the production of food additives and specific biopolymers.

Azotobacter Nitrogen Fixation

• Azotobacter strains utilizes nitrogenase enzymes to convert atmospheric nitrogen into absorbable ammonium for plant roots.

• Enzymatic diversity: Azotobacter species host many types of nitrogenase, the most basic and commonly used is the molybdenum-iron nitrogenase. Vanadium-iron nitrogenase may also be present and is more effective in lower temperatures.

• Oxygen sensitivity: Due to the seneitivity of the nitrogen fixation process to oxygen, azotobacter hosts defense mechanisms that involve intensifying metabolism and reducing cellular oxygen levels to counter the detrimental impact of oxygen on nitrogen fixation.

Independent Variables:

• Concentration of Iron (II) Sulfate: The different concentrations of iron (II) sulfate (500 ppm, 1000 ppm, and 1500 ppm) represent the independent variable as they are deliberately manipulated to assess their effects on nitrogen fixation.
• Concentration of Sodium Chloride: Similarly, the different concentrations of sodium chloride (1%, 2%, and 3%) represent another independent variable that is manipulated to observe its impact on nitrogen fixation.

Dependent Variable:

• Ammonium Ion Concentration: The concentration of ammonium ions in the testing solutions derived from the agar samples serves as the dependent variable. This variable is measured to assess the effectiveness of nitrogen fixation by the Azotobacter strains under different nutrient compositions.

Controlled variables: 

• Strain of Azotobacter: The azotobacter strains were collected from the same bottle of biofertilizer. This ensures that the strain of bacteria growing on each plate will be similar in that they are derived from the same source.
• Temperature of the room: By maintaining a constant temperature in the experimental environment, we ensure that external fluctuations do not affect bacterial growth. Temperature variations can profoundly impact microbial metabolism and growth rates. Consistency in temperature provides a stable and controlled setting for observing the effects of the manipulated variables, such as iron (II) sulfate and sodium chloride concentrations, on nitrogen fixation efficiency.
• pH of growth medium: The pH level of the growth medium plays a crucial role in bacterial metabolism and nutrient uptake. Fluctuations in pH can influence enzyme activity, microbial growth rates, and nutrient availability. By keeping the pH of the growth medium constant, we eliminate potential pH-related variations that could confound the results. This ensures that any observed differences in nitrogen fixation efficiency are attributable to the manipulated variables rather than pH fluctuations.
• Time allowed for bacterial growth: Providing a consistent duration for bacterial growth allows for uniformity in observing nitrogen fixation efficiency across all experimental samples. By maintaining a fixed incubation period of 6 days, we ensure that all bacterial cultures have sufficient time to reach comparable growth stages, facilitating accurate comparisons of nitrogen fixation efficacy.
• Nutrient composition of the growth medium: Apart from the manipulated variables, such as iron (II) sulfate and sodium chloride concentrations, the nutrient composition of the growth medium remains constant as the same type of agar was used. Nutrients other than the ones under investigation can also influence bacterial growth and nitrogen fixation. Therefore, keeping the composition of other nutrients consistent ensures that any observed effects on nitrogen fixation are solely attributable to the manipulated variables and not to variations in other nutrients.
• Stirring/agitation of the growth medium: Uniform mixing or agitation of the growth medium is essential for ensuring equal distribution of nutrients and ions throughout the agar plates. Inadequate mixing can lead to uneven distribution of nutrients, resulting in variations in bacterial growth and nitrogen fixation within and across experimental samples. By maintaining consistent stirring or agitation techniques across experiments, we minimize the potential for localized nutrient gradients and ensure homogeneous growth conditions for all bacterial cultures.

Step 1: Preparation - Agar Plate Setup 

• Ferrous Sulphate Purification:
  • 120 g of ferrous sulphate fertilizer was dissolved in 160 mL of distilled water.
  • The solution was heated to 80° C and filtered two times to remove any insoluble impurities.
  • The solution was allowed to cool down to room temperature (around 20° C) overnight and was then left outside at 4° C for two days to allow crystallization.
  • After two days, the crystals of pure ferrous sulphate were harvested and dried.
• Addition of Substances:
  • Dissolve 50 mg of the iron (II) sulfate crystals in 100 mL of water to make a 500 ppm solution. Add 2.3 g of nutrient agar powder to make the 500 ppm iron growth medium.
  • Dissolve 100 mg of iron (II) sulfate to 100 mL of water to make a 1000 ppm solution. Add 2.3 g of nutrient agar powder to make the 1000 ppm iron growth medium.
  • Dissolve 150 mg of iron (II) sulfate to 100 mL of water to make a 1500 ppm solution. Add 2.3 g of nutrient agar powder to make the 1500 ppm iron growth medium.
  • Dissolve 1g of sodium chloride to 100 mL of water to make a 1% solution. Add 2.3 g of nutrient agar powder to make the 1% sodium chloride growth medium.
  • Dissolve 2g of sodium chloride to 100 mL of water to make a 2% solution. Add 2.3 g of nutrient agar powder to make the 2% sodium chloride growth medium.
  • Dissolve 3g of sodium chloride to 100 mL of water to make a 3% solution. Add 2.3 g of nutrient agar powder to make the 3% sodium chloride growth medium.
  • Dissolve the nutrient agar powder in each solution and heat each solution to 85° C, making sure all the agar has dissolved.
• Plate Pouring:
  • Allow each solution to cool down to 60° C and pour each solution into 3 separate petri dishes
  • Allow the poured agar to solidify in the Petri dishes and cool down to room temperature forming a nutrient-rich medium suitable for bacterial growth.

Step 2: Culture System Setup - Initial Phase

• Environmental Conditions:
  • Place the prepared plates in a controlled environment set to a constant temperature of 18 degrees Celsius.
  • Properly label each plate to ensure accurate tracking and observation throughout the experiment.
• Incubation Period:
  • Allow sufficient time, precisely 6 days, for the bacterial colonies, particularly Azotobacter strains, to grow and establish themselves on the nutrient-enriched agar.

Step 3: Assessment of Ammonium Ion Concentration

• Sample Collection for Testing:
  • Following the growth period, collect small samples of agar with a volume of approximately 2.5 mm^3 using the tip of a pipette from each plate.
• Preparation of Solutions for Testing:
  • Dissolve the collected agar samples from each plate in 10 mL of distilled water. Ensure thorough mixing to create consistent solutions.
• Ammonium Ion Concentration Test:
  • Utilize a specialized ammonia test kit calibrated to measure ammonium ion concentration accurately.
  • Adhere strictly to the provided instructions within the kit to execute the test protocol for each solution derived from the agar samples.
• Interpretation of Results:
  • Record and compare the ammonium ion concentration levels obtained from the testing solutions derived from the modified Azotobacter strains.

Step 4: Assessment of Ammonium Ion Concentration

• Analyze the results obtained from the initial phase to determine the optimal combination and quantities of iron (II) sulfate and sodium chloride that produced the maximum nitrogen fixation efficacy.

Graph 1: 

According to the first chart and table, the control group, devoid of added iron (II) ions, consistently displayed moderate levels of nitrogen fixation across all trials, with values ranging from 0.25 to 0.5. Interestingly, the introduction of ferrous sulfate at concentrations of 500 ppm resulted in an increase in nitrogen fixation, whereas the introduction of ferrous sulfate at 1000 ppm appeared to have no impact on nitrogen fixation, as the observed values remained relatively consistent with those of the control group. However, at the highest concentration tested, 1500 ppm of ferrous sulfate, a slight decrease in nitrogen fixation was observed across all trials compared to lower concentrations. 

Graph 2:

The data regarding ammonia concentrations across different concentrations of sodium chloride suggests that impact of salinity concentrations from 2% to 3% on nitrogen fixation is insignificant, while a salinity of 1% resulted in a slight increase in ammonia concentration. The control group, 2% sodium chloride, and 3% sodium chloride concentrations all displayed similar ammonia concentrations, indicating minimal influence of sodium chloride levels on nitrogen fixation at these concentrations. However, the 1% sodium chloride trials on average demonstrated higher nitrogen fixation. 

Graph 3:

The observed data on average ammonia concentrations across varying concentrations of ferrous sulfate reveals notable trends in nitrogen fixation activity. The introduction of ferrous sulfate at concentrations of 500 ppm resulted in the highest average ammonia concentration among the tested groups, indicating a potential enhancement of nitrogen fixation compared to the control. However, as the concentration of ferrous sulfate increased to 1000 ppm and 1500 ppm, the average ammonia concentrations decreased gradually. This trend suggests a possible toxic effect on Azotobacter at higher concentrations of ferrous sulfate

Table 4:

The data regarding average ammonia concentrations across different concentrations of sodium chloride suggests intriguing insights into its impact on nitrogen fixation. With an ammonia concentration at 0.58 ppm, the trials with a sodium chloride concentration of 1% performed the best. This may be attributed to the fact that sodium and chlorine are also neccesary macronutrient to many bacteria, including Azotobacter. However, the trials with a sodium chloride concentration of 2% and 3% performed very similarly to the control trials, only with slight variations in ammonia concentrations. This may be due to inhibitory effect and stress on the bacteria caused by high concentrations of sodium chloride. However, the Azotobacter was still able to maintain a nitrogen fixation efficiency comparable to the control trials. It is likely that due to the diverse arange of soil conditions on earth, it is probable that Azotobacter species have adapted to live and fix nitrogen in such concentrations of salinity. 

Table 5:

At 500 ppm of ferrous sulphate, average percent change in efficiency reaches 60%, indicating an optimal range for nitrogen fixation by Azotobacter. However, at 1000 ppm, average percent change in efficiency drops to 20%, suggesting reduced effectiveness. The anomalous -20% average percent change in efficiency at 1500 ppm indicates a potential toxic effect of high iron concentrations on Azotobacter. These findings underscore the importance of optimizing nutrient concentrations for bacterial activity, with higher concentrations potentially inhibiting nitrogen fixation.

Table 6: 

In trials containing a sodium chloride concentration of 1%, the average change in ammonia concentration is 40%. In trials containing sodium chloride concentrations of 2% and 3%, the average change in ammonia concentration is 0% and 20% respectively. This trend suggests that as the sodium chloride concentration reaches 1%, the nitrogen fixing efficiency in Azotobacter increases. However, the data also suggest that the nitrogen fixing efficiency more or less returns to the levels in the control tials as the sodium chloride concentrations increases to 2% and 3%.

Ferrous Sulfate:

The observed trend in the efficiency of iron (II) ions in promoting nitrogen fixation aligns with established principles of microbial ecology and nutrient dynamics.

• Initially, at lower concentrations of ferrous sulphate (500 ppm), the efficiency of iron (II) ions is notably higher, indicating an optimal range where the availability of this micronutrient facilitates nitrogen fixation by Azotobacter strains. This heightened efficiency is likely attributable to the essential role of iron in various enzymatic reactions involved in nitrogen metabolism within bacterial cells.
• As the concentration of ferrous sulphate increases to 1000 ppm and 1500 ppm, the efficiency of iron (II) ions in promoting nitrogen fixation decreases. This decline suggests that while iron is crucial for Azotobacter growth and nitrogen fixation, there exists a threshold beyond which excessive iron becomes toxic.
• The reduction in efficiency at higher concentrations could be attributed to the toxic effects of high iron levels on microbial cells. Iron toxicity disrupts cellular functions, inhibiting metabolic processes vital for nitrogen fixation and overall bacterial growth.
• The negative efficiency recorded at 1500 ppm underscores the severity of this toxicity, where the adverse effects on Azotobacter strains outweigh any potential benefits from increased iron availability.
• While higher concentrations of ferrous sulphate initially correlate with increased nitrogen fixation efficiency, the subsequent decline in efficiency coincides with alterations in ammonia concentration. This suggests a complex feedback mechanism wherein nutrient availability influences microbial processes, ultimately affecting the biogeochemical cycling of nitrogen in the ecosystem.

Sodium Chloride:

The data illustrates a clear relationship between sodium chloride concentration and its efficacy in promoting the studied process.

• The control group, devoid of sodium chloride, establishes a baseline efficiency of approximately 41.67%. This serves as a reference point against which the effects of varying sodium chloride concentrations are measured.
• Experimental groups treated with sodium chloride concentrations of 1%, 2%, and 3% demonstrate average efficiency changes of 40%, 0%, and 20% respectively. This observed trend suggests increased nitrogen fixing efficiency as the sodium chloride concentration reaches 1%. However, as the sodium chloride concentration reaches 2% and 3%, the nitrogen fixing efficiency returns to the levels in the control tials.
• At lower concentrations of sodium chloride (1%), the efficiency increases slightly when compared to the control group. This may indicate that much like most other bacteria, sodium and chlorine are also essential macronutrients for Azotobacter.
• As sodium chloride concentration escalates to 2% and 3%, efficiency declines to levels comparable to that of control trials, indicative of inhibitory effects at higher concentrations. The reduced efficiencies observed at 2% and 3% sodium chloride imply a pronounced inhibition.
• This inhibition may stem from osmotic stress induced by high salt concentrations, resulting in the dehydration of bacterial cells. By reducing water activity via osmotic pressure, sodium chloride limits the availability of water necessary for bacterial metabolism and proliferation.
• Consequently, this constraint on water availability could impose stress on the growth and activity of Azotobacter strains.
• However, the fact that the Azotobacter was still able to maintain a level of nitrogen fixation comparable to that of the control trials indicates that Azotobacter species may have evolved to tolerate heightened levels of salinity due to the wide range of soil conditions on earth.

The experimental data highlights the intricate relationship between nutrient concentrations and their impact on biological processes, specifically nitrogen fixation by Azotobacter strains. Analysis of the efficiency of iron (II) ions and sodium chloride revealed distinct trends with varying concentrations.

For iron (II) ions, an optimal concentration range was observed, with a peak efficiency at 500 ppm ferrous sulphate, followed by a decline at higher concentrations. This decline in efficiency suggests a threshold beyond which excessive iron levels may induce toxicity effects, inhibiting bacterial activity and nitrogen fixation. The negative efficiency recorded at 1500 ppm ferrous sulphate underscores the severity of this toxicity.

Similarly, low concentrations sodium chloride resulted in a brief increase in the nitrogen fixing efficiency of Azotobacter. However, the increase in sodium chloride concentrations also resulted in a decline in the nitrogen fixing efficiency at higher concentrations, albeit not as significant when compared to increases in ferrous sulphate concentrations. This suggests that sudium chloride also functions as a important macronutrient for Azotobacter but can also impose stress on the bacteria at higher concentrations due to osmotic stress. However, at these higher salinity concentrations, Azotobacter strains were still able to maintain a decent nitrogen fixing efficiency. This is most likely due to Azotobacter’s relative tolerace to these salinity levels, stemming from its ability to adapt to diverse soil environments on earth.

In the face of escalating concerns regarding environmental pollution and the sustainability of current agricultural practices, there has been a growing interest in exploring alternative approaches to nitrogen fertilization. One prominent solution is Biological Nitrogen Fixation (BNF), which offers a sustainable means of enriching soils with nitrogen through the activity of nitrogen-fixing microorganisms, most notably Azotobacter strains.

Many soils are naturally rich in iron, with adequate levels typically present despite low crop availability. Iron plays a crucial role in various physiological processes in plants, particularly for crops that thrive in acidic soils such as blueberries, strawberries, grain, soybeans, and cole crops like cabbage and broccoli. Optimal iron levels in soil are essential for supporting crop growth and maximizing yields.

The data from our study indicate that the efficiency of Azotobacter nitrogen-fixing activity can be significantly enhanced by optimizing iron concentrations in the soil. Specifically, the application of ferrous sulfate at a concentration of 500 ppm, which falls within the optimal range for iron availability, resulted in the highest nitrogen fixation efficiency. This suggests that by ensuring iron levels in the soil are within the optimal range, Azotobacter nitrogen-fixing activity can be effectively boosted.

Furthermore, it is important to consider optimal salinity levels in soil for maximizing nitrogen fixation efficiency. Our data suggest that low concentrations of sodium chloride (1%), naturally present in the soil, may slightly enhance nitrogen fixation. However, higher concentrations (2% and 3%) of sodium chloride may exhibit inhibitory effects and impose stress on the bacteria. Therefore, maintaining optimal salinity levels in soil, compatible with microbial activity, is essential for promoting effective nitrogen fixation.

Under optimal conditions, the application of this knowledge presents a feasible and sustainable approach to enhancing nitrogen fixation in agricultural systems. By leveraging the natural nitrogen-fixing capabilities of Azotobacter strains, optimizing soil iron levels, and ensuring appropriate salinity levels, farmers can reduce their reliance on synthetic nitrogen fertilizers, mitigate environmental pollution, and promote soil health and fertility in a sustainable manner.

Moreover, due to the molar mass of ferrous sulfate heptahydrate, a concentration of 500 ppm of ferrous sulfate heptahydrate correlates to a 100 ppm concentration of Fe2+ ions.  A concentration of 1000 ppm of ferrous sulfate heptahydrate correlates to a 200 ppm concentration of Fe2+ ions and A concentration of 1500 ppm of ferrous sulfate heptahydrate correlates to a 300 ppm concentration of Fe2+ ions.

Additionally, a concentration of 1% sodium chloride correlates to a soil salinity level of 15.6 dS/m, indicating slightly above moderately saline soil. A concentration of 2% sodium chloride correlates to a soil salinity level of 31.2 dS/m and a concentration of 3% sodium chloride correlates to a soil salinity level of 48.6 dS/m, both of which indicating highly saline soil.

During the dissolution process of ferrous sulfate into water and subsequent heating of the solution following the addition of agar powder, it is highly probable that some iron (II) ions underwent oxidation to iron (III) ions upon exposure to atmospheric oxygen gas. This oxidation occurred due to the stirring of the mixture, which facilitated contact with oxygen. Consequently, the total concentration of iron (II) ions in the agar growing medium might have decreased below the initially added quantities of 50 mg, 100 mg, or 150 mg.

This transformation from iron (II) to iron (III) ions is particularly significant because iron (III) ions are biologically unavaliable to Azotobacter.

Thus, there could have been a deficiency of available iron for the Azotobacter within the growth medium compared to the originally added amount. This phenomenon underscores the paramount importance of meticulously controlling the dissolution and heating processes, as well as minimizing exposure to atmospheric oxygen during the preparation of agar growing mediums, to uphold the desired concentration of bioavailable iron for microbial growth and experimentation. Additional precautionary measures, such as utilizing reducing agents or conducting reactions under inert gas atmospheres, may also be considered to mitigate the oxidation of ferrous ions during preparation.

Moreover, the experiment was conducted in a basement with an inconsistent heating schedule and during times of varying weather conditions. Consequently, the temperatures during the experiment may have exhibited slight fluctuations. Given that Azotobacter, like many other bacteria, may demonstrate different rates of growth and reproduction under varying temperature conditions, these fluctuations could have impacted the activation and efficiency of the nitrogen fixation function of Azotobacter.

Furthermore, during the measurement and determination of the ammonium ion concentration, it is crucial to acknowledge that other impurities may be present in the mixture, such as agar. This may potentially interfere with the testing solution and introduce errors in the final data.

Additionally, subjectivity in identifying the colours of the testing solutions may have further introduced potential errors. Therefore, rigorous attention to detail and adherence to standardized protocols are essential to minimize such sources of error and enhance the accuracy and reliability of the experimental results.

• Haber-Bosch process. Haber-Bosch Process - an overview | ScienceDirect Topics. (n.d.). https://www.sciencedirect.com/topics/engineering/haber-bosch-process#:~:text=The%20Haber%2DBosch%20process%20is,The%20Royal%20Society%2C%202020).
• Nutrients and eutrophication active. Nutrients and Eutrophication | U.S. Geological Survey. (n.d.). https://www.usgs.gov/mission-areas/water-resources/science/nutrients-and-eutrophication#:~:text=Eutrophication%20is%20a%20natural%20process,and%20clogging%20water%2Dintake%20pipes.
• Nitrogenase. Nitrogenase - an overview | ScienceDirect Topics. (n.d.). https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/nitrogenase#:~:text=Nitrogenase%20is%20a%20metalloenzyme%20system,an%20active%20area%20of%20interest.
• Einsle, O., & Rees, D. C. (2020). Structural Enzymology of Nitrogenase Enzymes. Chemical reviews, 120(12), 4969–5004. https://doi.org/10.1021/acs.chemrev.0c00067
• Seefeldt, L. C., Hoffman, B. M., & Dean, D. R. (2009). Mechanism of Mo-dependent nitrogenase. Annual review of biochemistry, 78, 701–722. https://doi.org/10.1146/annurev.biochem.78.070907.103812
• Wagner, S. (n.d.). Nature news. Biological Nitrogen Fixation. https://www.nature.com/scitable/knowledge/library/biological-nitrogen-fixation 23570419/#:~:text=Biological%20nitrogen%20fixation%20(BNF)%2C,to%20ammonia%20(NH3).
• Soumare, A., Diedhiou, A. G., Thuita, M., Hafidi, M., Ouhdouch, Y., Gopalakrishnan, S., & Kouisni, L. (2020). Exploiting Biological Nitrogen Fixation: A Route Towards a Sustainable Agriculture. Plants (Basel, Switzerland), 9(8), 1011. https://doi.org/10.3390/plants9081011
• Azotobacter. Azotobacter - an overview | ScienceDirect Topics. (n.d.). https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/azotobacter
• Sumbul, A., Ansari, R. A., Rizvi, R., & Mahmood, I. (2020). Azotobacter: A potential bio-fertilizer for soil and plant health management. Saudi journal of biological sciences, 27(12), 3634–3640. https://doi.org/10.1016/j.sjbs.2020.08.004
• Aasfar, A., Bargaz, A., Yaakoubi, K., Hilali, A., Bennis, I., Zeroual, Y., & Meftah Kadmiri, I. (2021). Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability. Frontiers in microbiology, 12, 628379. https://doi.org/10.3389/fmicb.2021.628379
• Bernhard, A. (n.d.). The Nitrogen Cycle: Processes, Players, and Human Impact. Nature news. https://www.nature.com/scitable/knowledge/library/the-nitrogen-cycle-processes-players-and-human-15644632/
• Encyclopædia Britannica, inc. (n.d.). Nitrogen cycle. Encyclopædia Britannica. https://www.britannica.com/science/nitrogen-cycle
• Soto-Urzúa, L., & Baca, B. E. (2001). [mechanisms for protecting nitrogenase from inactivation by oxygen]. Revista latinoamericana de microbiologia. https://pubmed.ncbi.nlm.nih.gov/17061570/#:~:text=The%20Nitrogenase%20enzyme%20complex%20(the,the%20deleterious%20effect%20of%20O2.
• Bradley, J. M. Et al. (2020, December 18). Bacterial iron detoxification at the molecular level. Journal of Biological Chemistry. https://www.sciencedirect.com/science/article/pii/S0021925817506432#:~:text=The%20toxicity%20of%20iron%20stems,formation%20of%20reactive%20nitrogen%20species.
• Larson, C. A., Mirza, B., Rodrigues, J. L. M., & Passy, S. I. (2018). Iron limitation effects on nitrogen-fixing organisms with possible implications for cyanobacterial blooms. FEMS microbiology ecology, 94(5), 10.1093/femsec/fiy046. https://doi.org/10.1093/femsec/fiy046
• Gregory, G. J. Et al. (2021, February 1). Stressed out: Bacterial response to high salinity using compatible solute biosynthesis and uptake systems, lessons from Vibrionaceae. Computational and Structural Biotechnology Journal. https://www.sciencedirect.com/science/article/pii/S2001037021000349#:~:text=Most%20bacteria%20that%20live%20in,heterotrophic%20bacteria%20are%20poorly%20understood.
• Korkeala, H., Alanko, T., & Tiusanen, T. (1992). Effect of sodium nitrite and sodium chloride on growth of lactic acid bacteria. Acta veterinaria Scandinavica, 33(1), 27–32. https://doi.org/10.1186/BF03546933
• Abdel Latef, A. A. H., Omer, A. M., Badawy, A. A., Osman, M. S., & Ragaey, M. M. (2021). Strategy of Salt Tolerance and Interactive Impact of Azotobacter chroococcum and/or Alcaligenes faecalis Inoculation on Canola (Brassica napus L.) Plants Grown in Saline Soil. Plants (Basel, Switzerland), 10(1), 110. https://doi.org/10.3390/plants10010110
• Ecological soil screening level for Iron. Oak Ridge National Laboratory. (2003, November). https://rais.ornl.gov/documents/eco-ssl_iron.pdf
• Global Map of Salt-affected Soils (GSASmap). GSASmap | Global Soil Partnership | Food and Agriculture Organization of the United Nations. (n.d.). https://www.fao.org/global-soil-partnership/gsasmap/en/

• We would like to sincerely thank our parents for their unwavering support, both emotionally and financially, throughout the duration of this project. Their encouragement and provision of a dedicated workspace have been invaluable.
• Furthermore, we extend our heartfelt gratitude to the teachers and student leaders in the STEM club of Dr. E.P. Scarlett High School. Their guidance, assistance, and invaluable advice have played a significant role in the success of this experiment.