Supporting hydroponic farming using engineered bacteria: hormone-targeting and pH-adjusting systems

Introduction

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The food issue has been existing and escalating in recent years due to global warming and climate change, both of which are having an enormous impact on plant growth. The Special Report on Climate Change published by the Intergovernmental Panel on Climate Change indicates that around 46.2% of the global land area is either covered by drylands or facing desertification, which is reducing agricultural productivity.[1] In addition to natural factors, improper agricultural practices and food trade restrictions also intensify the food problem, and thereby affect the national economy, trade, and tourism. The wide variety of research programs focusing on safe food supplies or food security is now recognized as an important element in underpinning sustainable development. As we were looking into these matters, we found out developed countries are facing challenges in food security as well. For example, the UK has had a massive increase in food bank users from 61468 in 2010 to over 2 million in 2022. Moreover, a significant portion of these food bank users is minors. Food Banks Since 2010 - YouTube (from Trades Union Congress (TUC))

The children’s chances of graduating from high school are reduced by food insecurity. This concerns us very much, as we are a high school team.

Insufficient Food Supply and Urban Condition

According to the World Food Programme, 135 million people suffered from acute hunger in 2020, largely due to man-made conflicts, climate change, and economic downturns. The hunger problem is an intensifying emergent problem, and it is particularly related to climate change.

The Special Report on Climate Change and Land indicates that a half-billion people are living in places that are turning into deserts, and soil is being lost between 10 and 100 times faster than it is forming. In addition, climate change makes growing plants more difficult, as floods, drought, storms and other types of extreme weather threaten to disrupt, and over time shrink, the global food supply. [2] This issue greatly impacts food production around the world. In August 2022, the droughts in Southwest China caused huge damage to crops in regions near the Yangtze River, which provides the water needed in croplands nearby. The water availability dropped dramatically in this crucial farming season, so their local food supply is facing a huge challenge. In Argentina, where 12 to 13 million tonnes of wheat used to be exported, a historically recorded drought reduced the soil moisture rate to the lowest since 2010, cutting down wheat production.[13] Both the drought cases in China and Argentina show the consequences of droughts and how extreme weather is now more frequent and severe. Notably, these disasters contribute to hunger, especially when resources are not distributed fairly.

Hunger and malnutrition are often associated with the rural and remote, yet for the four billion of us who live in urban areas, these problems are progressing increasingly close to home. Due to globalization, urban regions rely heavily on the import of food from other places. Our food supply might be affected by extreme weather around the world, thus increasing food prices, and forcing people who live under the poverty line to suffer from hunger. The COVID-19 pandemic may contribute to the deterioration, causing over 130 million people to suffer acute hunger by the end of 2020.[4] Experts warn that food shortages could lead to an increase in cross-border migration[2], and conflicts and protests may happen more frequently, eventually leading to a turbulent society.

Hunger leads to undernutrition which causes weight loss, failure to grow, losing body fat and muscle, mental problems, and a vulnerable immune system to defend against disease. It also causes the low birth weight of infants, as well as inadequate development and growth in children and teenagers who require more energy and nutrients to develop their bodies. [14]

Therefore, we will need a more sustainable system to nourish more than 7.7 billion people on Earth currently, to 9.7 billion in 2050. [5] The system should be able to support food production to deal with emergent cases.

Potential Solution: Hydroponics in Urbanized Region

Hydroponics seems to be a promising solution. Hydroponics, a soil-free farming method that uses a culture medium, can be designed vertically. It has high energy and water efficiency since it does not require energy consumption for large-scale farming machines, and its recycling system reuses water and nutrition. Since the crops are grown indoors, there is no longer a need for hazardous pesticides because of the well-controlled environment.

Harvesting and distributing products within the same urban area shortens the supply chain. Selling to grocery stores or direct to growers eliminating transportation and/or packaging needs.

Enhancing agricultural productivity and sustainable food production is crucial to alleviating the perils of hunger. The implementation of a hydroponic system would therefore have multiple advantages in supporting the urban regions and contributing to a more sustainable society. First, hydroponics saves space. Many cases use abandoned buildings to run vertical farms, which is sustainable and operational. In “The Plant” vertical farm in Chicago, they use abandoned buildings to grow plants on a commercial scale.[3] Second, there is a low possibility of pest problems, so pesticides can be eliminated. In indoor or half-indoor conditions (greenhouses or balconies), most factors are well-controlled to prevent pathogens. Third, hydroponics also produces fewer carbon emissions than great areas of soil farming. Large farming equipment that is commonly used generates much more carbon than hydroponics due to burning fossil fuel. Also, hydroponics reduces the carbon footprint produced by transportation. Fourth, due to water conservation and recycling, hydroponics uses less water than soil agriculture. [5]

Hydroponics provides an opportunity for developing the urban agricultural industry.[5] In urban areas, there are no large areas for soil farming as it is expensive and scarce. This global issue can also be applied to Macau, where the population density, 21645 per Km^2 (56,059 people per m^2)[2], is the 7th highest in the world. To develop a more suitable farming method that is productive, sustainable, and space-saving, after our research, hydroponics has great potential to be put into practice in Macau.

Furthermore, over 99% of Macau’s daily consumption is imported from other places (mainly Mainland China). The carbon footprint of food imports, together with Hong Kong, is three times larger than the direct carbon emissions in Mainland China in the last two decades.[4] The carbon emissions are contributed by fossil-fuel transportation. As a densely-packed city, we the citizens are hoping to find a more sustainable way for self-farming in urban places. These issues draw our attention to food production and possible solutions.

Using synthetic biology to overcome limitation in hydroponics

However, hydroponics has its limitations. First of all, the autonomy level of current hydroponics is low due to the strict requirements for nutrient and pH level monitoring. The nutrient solution is cycled, therefore the secretion of roots will accumulate, and the nutrient level decreases over time. If the pH drifts too high (past 7), the plant's uptake of some nutrients becomes less efficient. For example, plants can become iron deficient, even if sufficient iron ions are present in the nutrient solution.[15] These affect the toxicity, the amount of nutrients, the pH level, and their stability, which are all crucial for plant growth. Particularly in pH maintenance, through modelling the growing conditions of different plants in different pH environments, the result showed that range pH 6.5-7 is most beneficial for most plants. To maintain the pH value in water culture, we propose an algorithm that will automate the farming process and reduce the needed manpower. [16]

To optimize hydroponics, higher autonomy and efficiency are required to develop a more sustainable urban farming method in Macau.

pH value cannot be fixed since the plants and the nutrient solution both affect the pH level. About one week after initial germination, the pH will reduce from 7 to 5 approximately. To further validate whether the pH affects the plant growth, plants (spinach and soybeans) were emerged in solutions with different pH. We observed that the health condition of the plant grows in pH 5 was worst, and the height was the lowest among pH 5, 6, 7 and 8. We have then investigate the consequences of low pH in our modeling, using data to analysis the best growth pH value for seven kinds of plants. As we tested the pH level in the hydroponic machine, the pH decrease steadyily in days. Since low pH environment is not an ideal growth environment for plants because the nutrients will be too mobile and be abosorbed excessively by the plants, which eventually leads to toxicity, pH level needs to be maintained. This requires manual interference to add chemicals, which reduces the autonomy level. To deal with the pH-maintaining problem, we used ldhA and glsA genes in our plasmid design to control the pH level, which is called the genetic pH shooting system. This system includes pH-sensing promoters that can respond to high or low pH levels and generate acidic or alkaline substances in self-responsive pH adjustments. The asr promoter is the promoter of the acid shooting circuit, while the P-atp2 promoter is the promoter of the base shooting circuit. The Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. [12] (Figure 2.)

The next step in improving productivity

To monitor and enhance plant growth, hormone control is a crucial part. To deal with severe natural disasters and abiotic stress like draughts, extreme temperatures, etc, plants evolved sophisticated mechanisms to pass on the stress signal to stop different growing phases.[7] Some of the phytohormones inhibit plant growth, which proves that the hormones produced by plants aren't always helping plants to grow faster. For example, Abscisic acid (ABA) is a kind of hormone produced by plants such as soybeans which stops or delays the seed germination phase in order to protect the plant from extreme weather. In contrast, in hydroponic conditions, the environment is well controlled, so the ABA is excessive and might increase the time required for planting. Instead of involving the use of chemical fertilizers/plant hormones or generating transgenic plant seeds, which is both harmful to the human body and the balance of the ecosystem, we chose to use synthetic biology to solve this problem. We engineered different recombinant E.coli strains that would have the ability to produce and secrete plant hormone-binding domains. These different classes of plant hormones binding domains in our engineered E. coli strains mediates the function of the plant hormones expressed by the plant, and thereby promotes the growth of the plant. Furthermore, our design could complement the current plant hormone-based agriculture work, providing a terminator for the use of plant hormones and benefiting future agricultural works. Additionally, we can sustain the pH in our hydroponic system at a slightly acidic environment of around pH 6-6.5 to help plants absorb enough nutrients from the water culture. We also engineered the recombinant E.coli strains that would have the ability to produce chemicals to control the system.

On one hand, specifically, we used the E. coli transformed with a green fluorescent protein to test the growth curve of E. coli in a hydroponic setting. After that, we used NSP4-T2R4, the ABA binding protein with added NSP4 peptides to regulate ABA. Once T2R4 binds to ABA, it will stop ABA signaling and promote plant growth.[8][9]

On the other hand, we have used PYL8 in our project, which is a hypersensitive ABA regulating protein and could bind to the ABA. Its function has been tested in soil-grown plants, so we will test both constructs to select the best ABA binding protein.[10][11] Via competitive inhibition, the signaling of ABA will be reduced and therefore enhance plant growth. (Figure 3.) We designed the plasmid using the pET11a (T7 locking system) vector. With the genetic pH shooting system and glsA (acid shooting circuit), we also used K2762014 (asr-sfGFP) to test fluorescence in an acid environment.

Aim and Target

Enhanced efficiency is necessary to increase the possibility of applying hydroponics as a support system and therefore ameliorate food supply problems. Through the proposed solution, we are aiming to optimize productivity by implementing our system in the new industrial district in the future; in the case of Macau, where our teams is from, our system would compensate 2% of the imported produce, which is 150,206 tons of vegetables and fruits (1,334,881,000 MOP; $165,324,000 USD) in 2021. [6]