Impact of Plant Species on the Microbial Community of an Aquaponic System
Aquaculture, Hydroponics, and Aquaponics Aquaculture is the term used to describe the breeding, raising, and harvesting of living organisms in an aquatic environment (US Department of Commerce, 2019). “Fish farming” is often a common term. A common example of this is recirculating aquaculture system (RAS). While systems may vary, the concept remains the same. The water that holds the fish circulates out along with any waste or leftover food. It is then brought through a filtration, then an aeration unit that will return oxygen to the water and remove carbon dioxide (Losordo, et. al., 1999). Fish are not the only organism that can be raised as algae, seaweed, and other aquatic plants can also be grown. Hydroponics is a similar concept, however terrestrial plants are produced. It includes growing crops in water rather than soil (Tripp, 2014). Fertilizers, including vitamin and minerals are added to the water to ensure the plants have what is needed for growth. While both hydroponics and aquaponics provide significant advantages, specifically when addressing overconsumption of our food supply, recent studies have shown that a combination of the two could provide an even greater benefit (Junge et al., 2017). Aquaponics, not to be confused with aquaculture, integrates the recirculation of water and aeration of a RAS but uses the terrestrial plants and nitrifying bacteria from hydroponics to rid the system of waste products harmful to the fish. The fish will produce nitrogen waste in the form of ammonia. This ammonia is then broken down into nitrite and then nitrate by the microbes. A buildup of ammonia or nitrite can be harmful to the fish however nitrate is relatively harmless. Nitrate is also the preferred source of nitrogen for plants (Rakocy, et al., n.d). In the end the plant, fish, and bacteria all benefit. Microbes in the System While all components of the system are important, this study will look specifically at the microorganisms. Hydroponics, like aquaponics, often use nitrifying bacteria to produce nitrate, so it can be assumed that the microbiota will be similar, however it is unclear on how the aquaculture aspect will affect these microbes. It is not unlikely to find plant growth-promoting rhizobacteria (PGPR) in a hydroponic system. Rhizobacteria are the bacteria that grow directly on the edges of the roots. This means that they have direct contact with the roots. They maintain a symbiotic relationship and help promote plant growth accomplishing one of the following: reducing nitrogen into nitrate, enhancing stress resistance, stabilizing phosphate and potassium intake (Cakmakci, Donmez, and Erdoan, 2007). While they are extremely common in soil, PGPRs are also used as a biofertilizer to help plant growth in both hydroponics and soil grown crops. It was specifically found that Bacillus spp. Gliocladium spp., Trichoderma spp., and Pseudomonas spp. were common bacteria and can be found in a healthy hydroponic system (Lee, and Lee, 2015). All the previous species are also considered Rhizobacteria. Each species benefits the plant differently. For example, one distinction of Bacillus spp is that some species lowered the infection rate of some pathogens (Nihorimbere et al., 2011). In addition, specific species were reported to increase water quality and growth rate (Nihorimbere et al., 2011). Some species of Pseudomonas spp. were shown to decrease root rot and have some antifungal properties on certain species (McCullagh, et al., 1996). Now turning the attention to the aquaculture side, one study found that two main types of bacteria were found: autotrophic and heterotrophic bacteria. Many autotrophic bacteria were ammonia oxidizers such as Nitrosococcus, Nitrosospira, and Nitrosomonas or nitrate oxidizers such as Nitrobacter and Nitrospira (Rurangwa and Verdegem, 2015). These oxidizers help reduce the amount of ammonia and nitrite (that are harmful to the fish) into nitrate or nitrogen gas that are much less toxic. The heterotrophic bacteria were found to be different depending on the species of fish. For example one study using carp found Alphaproteobacteria and Betaproteobacteria. Later the same group used goldfish instead and found a larger variety of microbes that included Actinobacteria, Bacilli, Gammaproteobacteria, Planctomycetacia, Sphingobacteria, Hyphomicrobium denitrificans, Rhodovulum euryhalinum and Nitrospira moscoviensis (Sugita, et al., 2005). Advantages of Aquaponics/Implications and Relevance Food security is becoming an increasingly relevant topic globally. The United Nations Food and Agriculture Organization (FAO) estimated that by 2050, the global population will reach 9.1 billion and our agricultural system allone will not be able to supply enough food (Sohngen, 2017). In addition, studies have shown a great decline in labor in agriculture (Christiaensen, Rutledge, and Taylor, 2020). Finally, as the amount of people going into agriculture decreases, it was also found that those going into the agricultural sector have less knowledge than previous generations (Kuiper, Shutes, Van Mejil, et al., 2019). Without the proper knowledge and understanding, a series of trial and errors will occur that could have been prevented. Now that we are aware of the problem, it is now time to come up with a solution. Aquaponics, as previously mentioned, recirculation of water along with the aeration of a RAS but uses the terrestrial plants and nitrifying bacteria from hydroponics to rid the system waste products harmful to the fish. The end result would be both a supply of fish and produce grown from the plants. Aquaponics also has a multitude of other benefits. Some agricultural policies such as pesticides, fertilizers, and over use of soil have shown negative effects on the environment (Goudie and Viles, 2003). Aquaponics recirculates the water and only produces organic waste that is easily broken down (Konig, et al., 2016). This makes aquaponics an eco-friendly solution. As well as being an environmentally friendly solution, it is also economically friendly. The largest cost of maintaining an aquaponic system in a commercial style system was fish food (Konig, et al., 2016). The aeration unit, bacteria, and plants contribute to make the water reusable for the fish meaning the cost of water significantly decreases. Finally, aquaponic brings forth the idea of having a local food source rather than having dependence on food being brought in. This dependence can be seen currently in the pandemic. Studies have shown that because of travel restrictions, some consumers are no longer able to visit their typical spot nor were their typical products available (Laguna, et. al, 2020). It is also predicted to see an 17 million increase in food insecure Americans in 2020 compared to 2018 (Gundersen, et al., 2020). If used accordingly aquaponics could help reduce the dependence on transported goods. Aquaponics could be one part of solving the problem of food insecurity. It has shown to have great promise in multiple aspects economically, environmentally, and even more so in food sustainability. However, its full potential cannot be reached without a thorough understanding of the system. Abundant research has been done on the microbial ecosystem of hydroponics and aquaculture, but little has been done in aquaponics. Traditional aquaculture does not typically look into the microbes of the system. One of the main reasons being that DNA sequencing is very expensive to be proforme. In 2015, it was estimated that a whole human genome sequence would cost approximately $4,000 (Wetterstrand, 2020). This study will contribute to the knowledge on the microbial ecosystem. The objective of this study is to identify common microbes of an aquaponic system and to see if the type of plant has any effect on the microbes within the systems. The hypothesis is that while there are some microbes that are predicted to be the same, there will be a significant difference between the systems with different types of plants. Methods: Overview of setup Seven (7) five-gallon tanks will be used in this study. 3 tanks will have plant A (most likely basil), and 3 tanks will have plant B (most likely swiss chard). The remaining 2 tanks will be a control and hold additional fish respectively. These fish will be used as a replacement if one of the fish in the trial system passes on. All tanks will be cleaned with 20% bleach and thoroughly washed out with deionized water. They will then be air dried for 7 days to ensure that any left over chlorine is degraded. The figure below depicts a larger version of the system that will be implemented in this study. It will use a cylindrical tubing cut to look like a “C” to hold the plants grown in rock wool. A submersible water pump will be used to force water into the tubing. The tubing will be held at an angle to allow water to flow back into the tank. To speed up the growth of bacteria, nitrifying bacteria will be added as an additive (this is common in aquaculture). Three goldfish will be placed in each trial tank along with the control. The fish will be fed 1.2 grams of food. Measurements of temperature, ammonia, nitrite, nitrite, pH, chlorine, water hardness, and dissolved oxygen will be taken every Monday, Wednesday, and Friday before the addition of food to the system. This will be done to record and understand the health of the system while the microbiota is being established. This system will be allowed to run for 45-60 days. After the allotted time, microbial samples will be collected, and analyzed. This system was designed by a group of New Jersey students for the Lunar Plant Growth Challenge put on by NASA. Image Credit: Atlantic County Institute of Technology (Smith, 2009) Microbial Analysis In order to identify the microbes, the following procedure will be performed. A sample of water from each tank will be collected in a sterilized beaker. During transportation a clean tin foil cover will be used to prevent contamination. The samples will then be immediately filtered using a filter membrane with .45 micrometer pores. This is large enough to catch any organism, yet still be able to filter out any waste. The DNA will then be extracted from the filter by using an Illustra Bacteria GenomicPrep Mini Spin Kit. Once extracted, PCR will then be used along with a 16s microbiome kit that will help enrich and amplify the bacterial DNA. After this, DNA Clean & Concentrator™ Kits, Zymo Research will then be used to purify the bacteria and ensure that any leftover material from the PCR. Finally, DNA sequencing will be able to be performed and the data will then be analyzed to identify the species of microbes Data Analysis Once the microbes are identified, a series of two group t-tests will be performed. Since the data will not be numbers, some modifications will have to be made. To begin, each system with plant A will be paired with a system form with plant B. In all there will be 3 pairs. Then, all the found microbes will be placed on a spreadsheet. At this point, the microbes will be translated into 1 or 0 depending if the microbe was found in the system (1), or not found in the system (0). Each pair will then have a two group T-test run on them to see if there is any significant difference between the two. After the calculations are completed, confidence intervals will be used and a conclusion will be made. Timeline: Fall 2020: (current) prepare proposal, research, trial systems, present proposal Spring 2021: continue trial research (determining if this is the setup I want to us), practice jjjjjjjjjjjjjjjjjjj maintaining system (to ensure less problem during experimentation) Summer 2021: Work and earn money for project Fall 2021: Perform project, gather and organize data, begin analyzing results Spring 2022: take Bio 499, begin final report, senior presentation Budget: Fish: .25$ each $2 6 in one test strips: 100 for 15$ $15 Submersible water pumps with tubing $84 5-gallon tank 8 x 10 $80 DNA Clean & Concentrator™ Kits, Zymo Research present in lab 16s Microbial DNA prep kit present in lab PCR reagents $~50 ZymoBiomics DNA miniprep kit $274 Extraction filter present in lab Various glassware present in lab PCR machine present in lab Plastic petri dishes (40) $22 Nutrient Agar powder $46.30 Flow cell-NGS Nanopore $100 Overall price: $673.30 Any suggestions would be extremely beneficial, and I thank you for your time. 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