Background

The extensive use of chemical fertilizers in modern agriculture has caused a plethora of detrimental effects on the environment and human health. In the next 26 years, the population is projected to grow by approximately 2 billion people (United Nations, 2022). This, combined with the rising global per capita food demand, indicates that by 2050, a 50% increase in current food production levels will be required to uphold existing food security standards (Baulcombe et al., 2009; World Health Organization, 2019). Chemical fertilizer usage has more than quadrupled in the past 60 years alone, will continue to grow at an increasingly rapid rate, and exacerbate the significant problems they cause if mitigatory solutions aren’t found (Ritchie, 2021). In order to address these challenges while promoting food security, a new sustainable, effective, and economically viable method of agriculture that focuses on maintaining soil health is required. 

Soil health is paramount to food security and human health, as it directly influences the nutrient content and quality of crops (Das et al., 2022). Modern soil research has found that heterotrophic microorganisms capable of nitrogen fixation, mineral solubilization, and other beneficial chemical reactions play a crucial role in maintaining soil health and supporting robust plant growth (Cao et al., 2023). However, the specific bacteria required for these processes and the mixture of bacteria required for effective soil remediation are still under investigation. Although nascent, this research indicates the possibility of one day creating an artificial consortia of bacteria inspired by their unique interactions and soil remediation capabilities. 

Consortia, compared to a singular species that can only execute one unique chemical process, are both novel and revolutionary in the field of synthetic biology, as they can perform many complex functions (Sadvakasova et al., 2023). By harnessing the beneficial interactions between soil bacteria and crops, researchers aim to develop biofertilizers that enhance nutrient uptake, improve soil health, adapt to variable conditions, and increase crop yields while minimizing adverse environmental impacts (Cao et al., 2023; Devi et al., 2022; Ikhajiagbe et al., 2021; Sadvakasova et al., 2023). On a broader scale, researchers seek to develop economically viable and sustainable biofertilizers that can meet the demands of modern agriculture while protecting the environment and human health (Aqeel et al., 2023; Devi et al., 2022; Kumar & Shastri, 2017; Sadvakasova et al., 2023; Weerahewa & Dayananda, 2023). Hence, this review article explores the potential of biofertilizers, in the form of microbial consortiums, as a realistic, practical, and eco-friendly alternative to traditional synthetic fertilizers. 

As the global population continues to grow at a rapid pace and climate change creates new agricultural challenges, the issues that stem from the overuse of chemical fertilizers are exacerbated (Baulcombe et al., 2009; United Nations, 2022: World Health Organization, 2019). With the exponentially increasing global population exacerbating major issues like food insecurity and, therefore, widespread unsustainable agriculture, it is imperative that scientists research new, more efficient methods of farming (Baulcombe et al., 2009; Devi et al., 2022; Luqman et al., 2023; World Health Organization, 2019). Moreover, with climate change models predicting a 1.5 ℃ average increase in global temperature and more droughts in arable regions by 2030, farmers may be unable to grow traditional water-intensive crops (Garthwaite, 2023). In the past 40 years alone, a third of the Earth’s arable land has been lost to erosion, pollution, and climate change (Milman, 2015). In efforts to mitigate these issues and meet the ever expanding demand for food, each year 128,000 square kilometers of wooded area is destroyed (Greenpeace, n.d.). This destruction is a direct consequence of the need for more arable farmland and accounts for 80% of annual global deforestation (Greenpeace, n.d.). If scientists are to effectively address this dire situation, they must focus their research on gaining a stronger comprehension of crop biology. This understanding goes beyond just crop production; it encompasses the broader ecological context in which plants play pivotal roles.

From acting as animal habitats to carbon sinks, which are natural environments that absorb and store large amounts of carbon dioxide from the atmosphere, plants are extremely important to both micro and macro ecosystems around the world (Khan et al., 2014). When humans grow plants, though, it’s usually for agricultural purposes rather than to maintain the balance of native ecosystems. Monocropping, the act of planting one kind of plant in a large area of land repeatedly, is a common agricultural practice that is extremely harmful to the environment and widespread in traditional farming practices (Kogut, 2020). In clearing natural habitats to plant millions of the same crop, monocropping dismantles biodiversity, habitats, and natural carbon sinks, thereby destroying ecosystems and contributing to the climate crisis (Kogut, 2020). Additionally, monocropping reduces the availability of nutrients of which the grown crop needs to develop and causes increased spreading of diseases and pests since there isn’t enough biodiversity to mitigate the outbreaks (Balogh, 2021). In the United States alone, 442 million acres of natural ecosystems have been demolished to use for monocropping (Charski, 2020). It is therefore of utmost importance to protect areas with a bounty of biodiverse plants, like forests, from industrial agriculture. Also, if land could produce crops with higher yields per acre or farmers planted crops that have increased calories per gram of produce, the unrelenting expansion of agricultural enterprise could be significantly mitigated without affecting profits (Weerahewa et al., 2021). Instead of selecting crops that satisfy these requirements, agriculture has relied on different kinds of fertilizers to bolster crop yields per acre and extend the life of arable land (Kumar and Shastri, 2017). In the modern era, however, harmful synthetic fertilizers, manufactured fertilizer derived from chemicals like ammonia, atmospheric nitrogen, phosphate minerals, natural gas, and potassium, have dominated the agricultural industry (Ritchie, 2021). When it comes to the prices of synthetic fertilizers, periods of economic shock have a profound impact on fertilizer prices and usually lead to unpredictable fluctuations in their price, meaning that they can end up being exceedingly expensive (Lahmiri et al., 2017). This problem is only amplified by the fact that these fertilizers have temporary effects, meaning that farmers have to buy them year after year (Lahmiri et al., 2017). Basic science research focused on plant physiology and the intricate processes through which plants absorb vital soil nutrients is extremely important, not only because it plays a fundamental role in plant development, but also because it provides new insight into alternative methods of fertilization.

Plant roots don’t expand simply to find sources of water, but also to absorb and utilize minerals that reside within soil pores that are necessary for their growth such as nitrogen, phosphorus, and potassium (Şahin Bal et al., 2022). Almost all fertilizers, but especially synthetic ones, aid the plant by increasing the abundance of these necessary nutrients. With access to an abundance of nutrients, plants can experience accelerated growth, making the use of fertilizers a common approach to achieve this objective. For example, crops grown with 40 kg/acre of nitrogen fertilizer are twice the size compared to those without, illustrating the substantial impact of nutrient supplementation on plant development (Thunder Said Energy, n.d.). Because farmers are able to customize the ratio of nutrients, mainly nitrogen, phosphorus, and potassium, in their synthetic fertilizers, they are able to efficiently grow a wide variety of plants with differential nutrient requirements. Additionally, they are much more effective than traditional sustainable fertilizers, like manure, wood ash, and bone meal, due to the fact that they are almost entirely made up of nutrients usable by plants (Pokorny, 2015). In the past century, chemical fertilizers have massively increased agricultural yield per acre, and, without them, there would only be enough food to feed 50% of the world’s population (Walling and Vaneeckhaute, 2020). In addition to this, since 1961, the overall use of synthetic fertilizers has increased sixfold, from an average of 20 kg/acre to 120 kg/acre (Ritchie, 2021). This data perfectly displays our overreliance on synthetic fertilizers. Although revolutionary for agriculture, synthetic fertilizers cause major harm to surrounding ecosystems, human health, the microbiome of the soil, and contribute to greenhouse gas emissions (Wang et al., 2023). 

Overuse of chemical fertilizers creates contaminated runoff, which is excess water that flows on the surface of an area of land into other areas and carries contaminants like excess nutrients (United States Environmental Protection Agency, n.d). This runoff seeps into surrounding aquatic ecosystems, causing the rampant growth of detrimental algae, like cyanobacteria, leading to significant habitat loss (Washington State Department of Ecology, n.d.). This surplus water can also flow into and contaminate water supplies. Additional negative effects of synthetic fertilizers involve general soil health. In a study conducted by Jin et al., the scientists investigated the effects of synthetic fertilizers on pineapple yield, soil bacteria, and soil pH (2023). They found that chemical fertilizers significantly increase soil pH compared to untreated soil, thereby killing and decreasing the biodiversity of beneficial soil bacteria (Jin et al., 2023). Maintaining soil health is critical, as healthy soil promotes plant growth by retaining water, allowing air to reach the roots of the plant, and serving as a habitat for beneficial microorganisms (Heiskanen et al., 2022). The continued, widespread use of synthetic fertilizers continues to damage ecosystems around the world and decrease soil fertility, but our existing dependency and the lack of suitable replacements makes avoiding them impossible. Because our need to produce crops at high yields per acre won’t disappear, similarly effective yet sustainable alternatives must be found through extensive research (Wang et al., 2023).

Instead of adding easily accessible yet temporary nutrients to the soil, other methods focus on helping the plant utilize self-renewing but normally inaccessible forms of nutrients. An example of this approach is the addition of symbiotic nitrogen-fixing bacteria, microbes that latch onto the roots of plants and convert nitrogen gas (N2) into ammonia (NH3), to the soil (Johnson, 2005). Compared to plants grown with chemical fertilizer, plants grown with biofertilizer containing nitrogen fixing bacteria have an average root length that is 4 cm longer (Devi et al., 2022). Additionally, through the process of nitrogen-fixation, the microbes turn nitrogen gas, which is normally inaccessible to plants, into accessible ammonia (Johnson, 2005). This ammonia can later be turned into usable nitrogen, which helps growth regulation, nutrient uptake from the soil, and is a key component in chlorophyll (Buchholz, 2022). Nitrogen-fixing microbes aren’t the only type of beneficial soil bacteria, however. Different kinds of bacteria can have unique functionalities, one of the most important being mineral solubilization, the conversion of minerals that are inaccessible to plants into forms that plants can absorb (Rafique et al., 2022). While almost all soil bacteria are advantageous, some are parasitic and kill plants, which in turn promotes the growth of more beneficial bacteria (Mishustin et al., 1973). Biofertilizers composed of solely beneficial bacteria with functionalities like nitrogen fixation or mineral solubilization could act as a sustainable, effective, and profitable alternatives to traditional fertilization methods.

In their 2023 study, Rangasamy et al. grew Finger millet (Eleusine coracana) and Green gram (Vigna radiata) in the presence of the symbiotic nitrogen-fixing bacterium, Rhizobium mayense. The researchers grew all arms of their experiment at pH 7, 30 ℃, and utilized 1% glucose and 0.05% ammonium sulfate as the sole sources of nutrients, as they found those were the optimal parameters for Rhizobium mayense (Rangasamy et al., 2023). After 80 days, the researchers observed that the bacterium enhanced all growth metrics in both organisms. Most significantly for the Green gram, on average the root length increased from 7.2 cm to 9 cm, the fresh weight increased from 10.5 g to 15.6 g, and the chlorophyll levels increased from 15.1 mg/L to 19.5 mg/L (Rangasamy et al., 2023). This study’s results provide clear evidence that these microbes can significantly increase the growth of common agricultural crops. A limitation of this study, however, is that it only utilized one kind of bacteria. This begs the question: could an amalgam of many species of soil bacteria better facilitate increased growth in plants? Furthermore, could mixes of helpful bacteria be an eco-friendly and viable alternative to synthetic fertilizers?

Because of environmental damages caused by synthetic fertilizers and the results of newer bacteria studies, research on the effects of communities of helpful bacteria on crops has become more popular in the scientific community. In a groundbreaking study by Aqeel et al., in which researchers compared plants grown in sterilized soil to plants grown in soil with microbes, the researchers found that communities of soil microbes can act as nutrient recyclers, promoters of plant growth, and generally maintain soil health (Aqeel et al., 2023). In nature, monocultures, soils with a single type of microbe, do not exist. Thus, it is critical that we think about soil bacteria instead as a complex community made up of multiple species. Without these communities, it is unlikely that many plant ecosystems today would exist, so it’s crucial that researchers investigate them and consider ways we can replicate these communities in large-scale agricultural settings.

In a review article by Cao et al., the scientists examined several studies that conducted research on soil bacteria communities and concluded that these communities can undeniably and significantly promote plant growth (Cao et al., 2023). One of the studies they wrote about, by Liu et al., concluded that a community of plant-growth-promoting-bacteria (PGPB) comprised of Stenotrophomonas rhizophila, B. sphaericusand, and B. amyloliquefaciens increased total soluble protein and nitrogen content in rapeseed plants (B. napus) (Liu et al., 2021). As previously mentioned, different microbes serve different purposes in the process of promoting plant growth. A novel example of a potentially beneficial community could include multiple bacterial species that each offer unique nutrient services. For example, a mixture of bacteria that promotes nitrogen fixation, another that solubilizes minerals plants normally have a hard time utilizing for growth, and another that breaks down dead plant matter into nutrients could be extremely advantageous. One of the greatest knowledge gaps in this field is the lack of information about the unique functions offered by various species of soil bacteria. Because there haven’t been many studies published on soil bacteria, not many microbial services have been annotated. In other words, many of the effects of soil microbes on the growth and health of crops, especially in the presence of other bacteria, are simply unknown. In just the past few years however, many scientists have been researching the effects of consortiums, or communities, of soil bacteria on crops like wheat, rice, maize, and rye (Negi et al., 2022; Awasthi et al., 2022; Tyagi et al., 2023). Very recently, this list has expanded to include a drought-resistant grain named, amaranth. 

Along with its ability to survive in dry, hot environments, many plants in the genus amaranthus produce high-protein, high-calorie, healthy grain (Ikhajiagbe et al., 2021). Also, amaranth is also considered a ‘superfood,’ meaning that its grain contains many vital nutrients, lipids, and vitamins (Wang et al., 2023). With climate change a bigger threat than ever and its warming of areas of massive agricultural production, amaranth thriving in drought-like conditions is extremely important. Moving towards the future, a switch from conventional crops to amaranth could mitigate famines in a hotter and more densely populated world. The possibility of amaranth becoming a staple grain in the future and the need for a new kind of eco-friendly fertilizer makes amaranth a perfect model organism to investigate the benefits of soil bacteria (de la Rosa et al., 2009). Although microbial consortiums have gained increasing attention as a research topic, only a limited number of studies have explored their relationship with amaranth. However, there is no doubt that further examination of this topic will be pursued in the future.

One of these studies, conducted by Devi et al. in 2022, researched how a microbial consortium composed of nitrogen-fixating and mineral solubilizing bacteria strains affected the growth of amaranth (Amaranthus hypochondrius L.). The researchers isolated rhizospheric bacteria from the soil through serial dilution, a step-wise method of dilution in which the dilution factor stays constant. They proceeded to use primary and secondary screening to evaluate if the bacteria had plant growth promoting traits (Olicón-Hernández et al., 2022). They later created a consortium of bacteria with positive traits, the two main species having nitrogen-fixation and mineral solubilization capabilities respectively. In the two positive arms of their experiment, Devi et al. grew amaranth with the recommended dosage of nitrogen-phosphorus-potassium (NPK) synthetic fertilizer as well as a half dose. In their negative arm, they grew the amaranth with no fertilizer or bacteria present. Finally, in their experimental arm, the amaranth was grown in the presence of the microbial consortium. After 70 days, compared to plants grown with the 100% recommended dosage of NPK synthetic fertilizer plants grown in the presence of the microbial consortium had, on average, 7/6ths times longer shoot lengths, had roots 3.9 cm longer, were double the dry weight (5 g to 10 g), nearly double the fresh weight (15 g to 27 g), and had 50% more chlorophyll (37 ug/ml to 58.5 ug/ml) (Devi et al., 2022). These results are incredibly significant, as almost every measured aspect of amaranth growth with the microbial consortium was larger than those grown with no additives and, more importantly, those grown with the standard dose of synthetic fertilizer. This suggests that not only can bacterial consortiums be an equivalent and sustainable alternative to synthetic fertilizers, but also that they have the potential to help crops grow significantly larger (Devi et al., 2022). The results from the study are reason enough to pursue more research of bacterial consortiums and to view them as a realistic, alternative biofertilizer that could replace conventional fertilizers.

Because the specific strains of soil bacteria Devi et al. used in their study would be impossible to obtain in the classroom, my study seeks to replicate theirs with soil bacteria called Pseudomonas fluorescens, Bacillus subtilis, and Azotobacter chroococcum. They are common, easy to purchase, and, due to the fact that they are understudied as soil bacteria, are perfect candidates with which to conduct research. My hypothesis is that the amaranth in the presence of a consortium of Pseudomonas fluorescens, Bacillus subtilis, and Azotobacter chroococcum will grow significantly larger than the amaranth in the presence of no soil additives and the amaranth receiving NPK synthetic fertilizer. Additionally, amaranth in the presence of these three species of soil bacteria individually will grow to a size in-between the amaranth in the presence of no soil additives and the amaranth that got the 100% dosage of NPK synthetic fertilizer. Although the effects of these bacteria on other model plants are well documented, only Pseudomonas fluorescens’s effects on amaranth have been published, and only in one study (De Salamone et al., 2012; David et al., 2018). This study, conducted by Ikhajiagbe et al., grew amaranth in the presence of Pseudomonas fluorescens and found that the plants grew significantly more leaves compared to plants not in the presence of the bacteria (Ikhajiagbe et al., 2021). They also took data on shoot and root length but found inconclusive results, leaving a large knowledge gap about why the bacteria altered the number of leaves without affecting amaranth’s growth. I seek to help close the aforementioned knowledge gap by utilizing the effective setup and amaranth model organism published by Devi et al. as well as the soil bacterium, Pseudomonas fluorescens. My study hopes to combine the findings of Devi et al. and Ikhajiagbe et al. to find that a consortium of Pseudomonas fluorescens, Bacillus subtilis, and Azotobacter chroococcum stimulates growth of crops from the Amaranthus genus more than unfertilized soil and soil with the 100% recommended dosage of NPK synthetic fertilizer. In building upon the research of Devi et al. with the difference of the experimental bacteria, my study aspires to uncover the effects of an understudied soil bacteria on amaranth. As there is a lack of literature studying the effects of the Pseudomonas fluorescens, Bacillus subtilis, and Azotobacter chroococcum soil bacteria on amaranth, there is a specific yet important knowledge gap about how these bacteria affect the growth of amaranth. My research helps to close this knowledge gap and, therefore, is important to the scientific community.

The practical applications of the findings of Devi et al., and other research into microbial consortiums are numerous and exceptional. The move from conventional synthetic fertilizers towards biofertilizers has the potential to improve both soil and human health, mitigate droughts, prevent runoff, enhance the economic viability of farmers, and increase the worldwide production of crops. By implementing these consortiums as biofertilizers, major issues that have been exacerbated by rising global population, like the clearing of natural ecosystems, food insecurity, and climate change, will be mitigated. Because harmful runoff, destruction of ecosystems, degradation of soil health and fertility, and poisoning of water resources are directly caused by the use of synthetic fertilizers, it is clear that biofertilizers would diminish these effects. Embracing microbial consortiums as a possible alternative to synthetic fertilizers will help to address the challenges facing global food production and preserve the planet's ecosystems. Because of new developments in synthetic biology, such as functional genomics and metabolic profiling, if researchers were to have comprehensive knowledge of how different types of soil bacteria affected plants, they would be able to create biofertilizers that could promote greater levels of plant growth than synthetic fertilizers while also maintaining and creating healthier soil (Sadvakasova et al., 2023). Overall, these microbes are vital to the health of ecosystems around the world and research funding should be allocated to investigate how they can be utilized for widespread sustainable agriculture.