BACKGROUND:

Plastic pollution stands among climate change as one of the biggest threats to biodiversity and human health (Scott, 2000). On a larger scale, plastics directly affect wild animals through ingestion. Animals such as sea turtles and birds often confuse wayward plastic bags for a food source, leading to poisoning, choking and eventually death (Moser and Lee, 1992). Specifically, more than 180 animal species are known to accidentally ingest plastic fragments (Derraik 2002). Other animals have their growth impeded by plastic through constriction, where plastic items such as six-pack rings (used to hold canned beverages together) find their way around an animal's neck and cause growth deformities (Derraik 2002). 

Plastics spread through our environment through repeated fragmentation, where microscopic plastic particles break off from larger plastic objects after prolonged time in water. These fragments have been accumulating for about a century in marine water spreading worldwide, and represent approximately 85% of stranded plastic debris on shorelines (Brown et al 2007). These smaller plastics can be consumed by organisms much lower on the food chain, severely increasing the rate of plastic spread. Because of the complexity of the food web, these plastics are inevitably passed from aquatic to terrestrial animals (Moser and Lee, 1992). Plastic damage is not limited to just animals however, because many of these animals will eventually become our food, passing microplastics into our systems. Additionally, dissolved microplastics can find their way into human drinking water, and they are confirmed to cause obesity, cardiovascular disease, reproductive disorder and breast cancer among others (Campanale et al., 2020). Finally, because of its extremely long decomposition time, conventional plastic landfill is taking up a lot of valuable space. 79% of plastic waste is sent to landfill rather than recycling (representing a mere 9%), and it is estimated that, with poor waste management conditions continuing, 12,000 metric tons of plastic will be in landfill by the year 2050 (Wojnowska-Baryła et al., 2022). Luckily, the fairly recent development of biodegradable plastics can mitigate this issue by directly replacing conventional plastics.  

Biodegradable plastics differ from conventional ones in that they are able to degrade without any additional processing often in large composting sites (Wood et al., 2014). There is no one, definitive type of biodegradable plastic, and it is common to see new developments every year. However, Wood et al., mentions common formulations of biodegradables including plastics made from starch, bacteria, and cellulose (Wood et al., 2014). These plastics are able to decompose because of their specific, often plant-based material makeup which means they are able to return to the soil as CO2, methane, water, and edible biomass or compost. (Fillicioto et al., 2020). Detritivores (organisms whose primary role is to break down plant and animal matter to provide nutrients for the surrounding environment) like fungal colonies and bacteria in the soil are able to consume bioplastics as a food source and are the primary reason why they are able to degrade in as little as 3 to 6 months, whereas conventional plastic lasts for years (Wojnowska-Baryła et al., 2022). One of the best biodegradable plastics to study for this comparison are starch based plastics, which are highly varied and readily available.

Starch based plastics are one of the most common types of biodegradable plastic. They are derived from plant starches and are a component of many widely known biodegradable plastic items such as BioBag compostable garbage bags (BioBag, 2022). These items typically have fewer uses than conventional plastic equivalents because of how much faster they decompose. While conventional plastics should last for years in a compost heap before they fully break down, especially in the landfills where they most often pile up, biodegradables will last for mere months (Wojnowska-Baryła et al, 2022). A study on a smaller time scale by Gordon J. supports this claim (2022). Gordon tested the “biodegradability” of two different biodegradable plastics by burying them about 10 cm deep in organic compost along with conventional plastic, represented by a plastic bag. The plastics were allowed to sit for 60 days, after which they were removed to undergo a biodegradability test. Gordon’s methods, biodegradability test and biodegradable plastic recipes were adapted from a lengthy study by Orenia et al. (2018). The biodegradability test was mostly visual and measured on a 5 point scale, with a 1 indicating little to no visible degradation and 5 indicating heavy degradation (ie. holes and warping of the original structure). Unsurprisingly, the plastic bag received a score of 1 with almost no changes to shape or structure; the only differences were soil stains. On the other hand, Gordon’s experimental plastic, a cellulose based plastic, received a score of 4.75, losing most of its structural integrity and sporting countless holes on the surface (Gordon, 2022). Studies like Gordon’s support the idea that conventional plastics may be more chemically resistant and have a longer shelf life when compared to biodegradable ones, but biodegradables break down easily when placed in compost heaps. To combat this, stronger biodegradables that are as chemically resistant as conventional plastics but degrade just as well as common biodegradable plastics are being developed, such as in the case of Saranti et al.’s experiments (2021). In this investigation, they discovered that adding clay and black pepper essential oils to biodegradable films actually increase the plastic’s tensile strength (the ability of an object to resist pressure or stress ) by around 150% while still retaining high biodegradability (Saranti et al., 2021). Fortunately, the rarity of chemically resistant biodegradables is not a downside. It is because of their low chemical resistance and organic components that the environmental benefits of biodegradables far outweigh their structural faults. 

In many cases, chemical resistance and structural integrity does not matter as most conventional plastics appear in forms that are not reused, such as shopping bags and pet waste bags. Both of these items have commercially available biodegradable equivalents, with BioBag again being an extremely popular pet waste bag brand (BioBag, 2022). BioBag mostly uses starch plastics for their bags (BioBag, 2022), mainly because of their reliability, strength, and abundance. Certain starch based plastics are even more resistant to water than other biodegradables, making them more viable in the food industry while still retaining their biodegradability in compost heaps because starches are a natural part of the diet of several detritivores (Rushton, P. and Hassall, M., 1983). One such detritivore is the terrestrial isopod, which suggests the use of these organisms in compost heaps could further accelerate their rate of material degradation (Wood et al., 2014).

Terrestrial isopods (Isopoda oniscidea) are small land crustaceans which serve as detritivores in their natural environment. While they are terrestrial animals, terrestrial isopods still possess gill pads from their aquatic ancestors, which need to be kept damp for oxygen exchange (Ziegler et al., 2005). Like the majority of invertebrate decomposers, they will eat almost any organic matter they can access. This includes both plant and animal matter, but the majority of the terrestrial isopod’s diet consists of plant waste; leaf litter and decomposing logs provide them with most of the food they require (Rushton, P. and Hassall, M., 1983). Occasionally, if the opportunity presents itself, they will also feed on decomposing animal tissue, bones or waste, which provide calcium and other minerals needed to grow their shells (Ziegler et al, 2005). This omnivorous behavior makes them key players at breaking down large waste deposits in their natural environment. Among other well studied decomposers, terrestrial isopods provide countless modern benefits to humanity which makes them a very likely organism to assist in the degradation of biodegradable plastics. Their feeding behavior helps enrich our soil as the isopod’s feces, referred to as frass, serves as a natural fertilizer for plants (Rushton, P. and Hassall, M., 1983). They are helpful in gardens, compost heaps and farms alike, serving as one of the larger members of the cleanup crew. Unlike other well known garden insects, most species will not go for live plants, meaning that there is no harm in their presence. This ecological service makes them a keystone species in that countless organisms benefit from their presence. Isopods have evolved to consume decaying matter quickly and, as a result, have a high reproductive rate. This provides hundreds of other organisms with a steady and dependable food source. Additionally, through consuming decaying matter, isopods help maintain microbial and fungal populations (Rushton, P. and Hassall, M., 1983). 

Furthermore, complex adaptations in isopod feeding behavior and chemical tolerance make them more than suited to consume biodegradable plastics. In order to tolerate the toxic chemicals in the plant foods they eat, isopods make use of several soil bacteria species present in their digestive tract, two of the most common being Candidatus heptoplasma and Candidatus hepatincolla porcellionum. These species of bacteria engage in a symbiotic relationship with the isopods in which the bacteria break down and feed on toxic molecules found in plant matter (aside from safer components like cellulose and starch), while the isopods are in turn protected from toxicity (Zimmer, M. and Topp, W., 1998). Because cellulose and starch are the primary ingredients in the aforementioned biodegradables, these specific bacteria give isopods a serious advantage in breaking them down. As observed by Zimmer, M. and Topp, W., young isopods will obtain these beneficial bacteria through copophagic behavior, consuming their parent’s feces (Zimmer, M. and Topp, W., 1997). For this reason, isolating newborn isopods from adults or any sort of substrate could be life threatening, as they would not get the beneficial bacteria required to safely digest much of their food. 

To break up and consume their foods, isopods make use of modified limbs called mandibles, a pair of horizontal, jaw-like appendages used for ripping and tearing up their food matter. A high calcium content in the mandibles makes them sturdy, and a structure referred to as the corpus serves a function similar to the mammalian tooth (Ziegler et al, 2005). These work to rip and chew both meat and plant matter, but crush together horizontally instead of vertically like vertebrate jaws (Ziegler et al., 2021). Signs of isopod feeding are indicated by small holes or “bite marks'' found on possible food sources such as leaf litter. The efficiency of isopod consumption is well displayed in an experiment by Hattenschwiller et al., where groups of 9 isopods were fed about 300 mg of Ash tree leaf litter. About 20 days later, the animals had consumed about 150 mg of litter, meaning that the total amount of mass loss was about 50% (Hattenschwiller et al., 1999). At even higher concentrations these animals could be significantly more efficient. Fortunately, this is part of their nature; conspecific aggregation is a survival mechanism among terrestrial isopods.

Conspecific aggregation is a trait that makes terrestrial isopods an excellent candidate for the acceleration of biodegradable degradation. High concentrations of isopods on a food medium means it can be broken down significantly faster than most solitary decomposers. Akin to their photophobic traits, terrestrial isopods exhibit aggregation behavior for their own survival. They inhabit environments where their predators are diverse, widespread, and have numerous different methods of catching them (Ďurajková B, 2022). To combat this, isopods have evolved a mechanism similar to countless other prey animals: staying in large groups. Remaining in a group reduces the probability that each individual would get picked off by a predator. This is because when a predator grabs one of them and is occupied, others in the group have a chance to escape. Certain species can alert other members of a group of danger even before an attack, thus promoting survivability (Ďurajková B, 2022). In addition, living close to one another eliminates the need to search for a mate, eliminating another possible predation opportunity. This naturally evolved behavior combined with their reasonable size makes them one of the best choices for biodegradable decomposition and the best possible choice for this specific study.

Because of their general abundance, isopods have also been used as a model organism in soil ecotoxicology studies, as well as the effect of human activity on soil health. Countless companies, such as Carolina Biological, produce isopods in captivity to be purchased for experimental reasons because of their variety of applications and fairly straightforward care requirements (Carolina, 2022). A study by Van Oemen Cloke et al. used captive-bred isopods to study the negative effects of a soil pollutant, glucosinolates, on soil organisms. They compared two factors: death rates when isopods consumed foods containing pollutant chemicals vs. when isopods consumed soil containing pollutant chemicals. Glucosinates enter the soil as agricultural runoff from commercial crops. The glucosinolate compounds were spiked into the soil and food using acetone as a solvent (1 ml of acetone for each dry gram of soil or food medium). Finally, the isopods were able to eat either the soil or food medium for 30 days and it was found that, compared to a negative control with no chemicals added, exposure to the chemicals through food had almost no effect on isopod survivability percentage, while exposure to chemicals through soil resulted in survivability levels as low as 30% (Van Oemen Cloke et al., 2012). This indicated that, while soil organisms such as isopods could detect toxins in food, it was highly unlikely for them to avoid them when they were present in their environment, shining light on the detriment of soil pollution (Van Getsel Cam et al., 2018).

 In addition to their dependable role in environmental health studies, isopods are purchased to use as feed for domestic reptiles or amphibians, or to keep vivariums and terrariums clean (Neherp, 2022 and Sans Vertigo, 2022). Their adaptability also means they will thrive in most man made environments so long as there is some sort of organic matter available. Being hardy generalists makes them an excellent candidate for the consumption of biodegradables. 

The function of organisms such as isopods is not limited to their ability to consume biodegradable substances but instead with the waste that they produce. The aforementioned frass produced by isopods provides a high nutritional value for plants, but is often moist and difficult to collect en masse (Rushton, P. and Hassall, M., 1983). Here, another model organism is required: Tenebrio Molitor, the mealworm. Mealworms are the larval stage of the yellow mealworm beetle, an insect belonging to the darkling beetle family (Carolina, 2023). They fulfill a similar niche as terrestrial isopods, being detritivores in their natural environment. Burrowing and feeding are the two main priorities of larval mealworms, as they spend most of their time beneath the substrate. Like isopods, they have a widely varied diet, consuming both plant and animal matter and obtaining all of their moisture from their food. This includes starch and cellulose, key components of biodegradable plastics. This means that mealworms can also feed on biodegradable mediums (Kröncke et al., 2022). Their strong mandibles allow them to tear into a variety of different surfaces, making feeding possible. Unlike isopods, they breathe air through a set of laterally lined spiracles, a form of passive respiration where air is constantly flowing in and out of their bodies. Mealworms do eventually pupate and morph into adult beetles, where they will continue to accept almost any organic matter as a food source. They will breed quite readily so long as food is available, making them easy to propagate. They are most at home in a substrate they can burrow in, such as wheat bran, which also serves as a food source. Wheat bran is the most commonly used substrate for raising mealworms (Kröncke et al., 2022).

Farmers often raise mealworms in wheat bran as a source of feed for domestic animals such as poultry, given the high protein value the insects provide (Kröncke et al., 2022). According to a study by Zhao et al., freeze dried mealworms contain 15% fat and 20% protein, and given their abundance, can provide high nutritional value in relatively low amounts of mass. In addition, their carbon footprint is insignificant when compared to other protein sources such as cattle (Zhao et al., 2016). This is valuable considering another function of mealworms: the ability to consume styrofoam (Yang et al., 2015).

 This quality allows them to shine as a model organism, digestion of conventional plastics is extremely rare among animals. Polystyrene, the main component of styrofoam, is quite durable and does not typically degrade naturally. Mealworms are able to break it down thanks to their gut bacteria Exiguobacterium sp. strain YT2 (Yang et al., 2015). In accordance with a Stanford study by Yang et al., this entire process occurs in about 24 hours, after which the styrofoam is effectively degraded in the mealworm’s gut. Larvae fed on a styrofoam diet lived as well as those fed with a normal diet of wheat bran over a 1 month period. Additionally, during the 16 day test period, 48% of the ingested styrofoam carbon was converted into CO2 and the residue was expelled as waste with a limited fraction incorporated into the worm’s biomass. Tests confirmed that the polystyrene was mineralized to CO2 and incorporated into lipids, providing energy for the mealworms. No styrofoam product remained within the tested mealworms, making them completely viable for consumption and maintaining their position as a safe and nutritious animal feed (Yang et al., 2015). This makes them a viable option for human consumption in the future given their aforementioned low carbon footprint and high nutritional value (Zhao et al., 2016). However, mealworm frass serves as one of the main benefits of using these organisms instead of terrestrial isopods.

As mealworms eat they regularly produce pellets of waste, called frass. Frass comes in the form of small, dry grains which, when seen in high quantities, shares a similar appearance to a pile of sand (Nogalska et al., 2022). Through the use of a sieve, frass can be easily separated from a wheat bran substrate and stored in large quantities. When mixed into soil, insect frass provides outstanding results for plant growth (Kataga et al., 2012). Because of this ease of transport, mealworm frass could easily become a highly marketable fertilizer option. This is especially relevant when mealworms are fed biodegradable plastics or styrofoam. Theoretically, a company could create biodegradable plastics while also breeding mealworm beetles on site. The mealworms would feed on used biodegradable products the company receives through a compost program as well as shipments of styrofoam waste received from packing companies, producing high quantities of frass as a result. These companies could then package and sell frass to agriculturists to make a profit, which would help offset the cost of creating the biodegradable products in the first place while providing an alternative to high emission chemical fertilizers. In addition, extra mealworms could be sold as a feed option for insectivorous pets, as they already serve as one of the primary feeders in the exotic pet trade (Carolina, 2023).

The present study aims to build upon the understanding of styrofoam consumption in mealworms from Yang et al. in combination with a separate study by Kataga et al. on the ability of insect frass to provide nutrition for plants (Kataga et al., 2012). Kataga et al. did not use mealworms but instead the larvae of Mamestra brassicae, more commonly referred to as the cabbage moth, a common cause of crop damage in commercial farms (Jiang Long et al., 2020). Individuals were obtained from captive bred populations at the Center for Ecological Research, Kyoto University. In the study, the M. brassicae larvae were fed leaves from Brassica rapa L. var. Perviridis, the Japanese mustard spinach, which was grown in a nutrient rich compost. Frass from the larvae was then collected and oven dried at 60°C to prepare for experimental use. Frass was then mixed into soil mediums containing minimal fertilizer and the growth of plants was monitored from within a greenhouse. 2 grams of frass were scattered over the surface of the 110 g of soil in certain pots containing B. rapa seedlings two weeks of age. Other pots were left alone to serve as control groups. Aboveground biomass, referred to as Leaf N, was recorded for all of the plants at the end of the experimental period. This was done by removing, drying and weighing leaves. It was found that Leaf N increased significantly in pots sprinkled with the frass of M. brassicae, indicating the positive effects of insect frass on soil nutritional value  (Kataga et al., 2012).

To expand upon this research within the available classroom environment, a hybrid setup and experiment were formulated. Mealworms were purchased from Carolina Biological in bulk (1,500 individuals total) and housed in 16 quart plastic containers with ventilation holes, as they require regular airflow (Carolina, 2023). About 500 mealworms were placed in each of the 3 containers, which each had its own substrate: styrofoam packing peanuts, wheat bran, or BioBag waste bags (BioBag, 2022). Two other 16 quart containers were reserved for collecting adult beetles and pupa so that the mealworms can be bred effectively. Over the experimental period, frass produced from each of the containers will be collected every 4 days and stored in individual containers for later use. The current goal is to collect enough frass to use as a fertilizer in a similar manner as shown in the study by Kataga et al., where it will be sprinkled on the surface of the soil and absorbed by the plants (Kataga et al., 2012). Frass produced in all three bins will be tested as options alongside a control plant group. The plants being tested in this study will be a variant of Brassica Rapa, (a direct relative of the plant tested in Kataga et al. 's study) referred to as the Wisconsin Fast Plant . These plants are sourced from Carolina Biological and known for their hardy nature and rapid growth (Carolina, 2023). This as well as the appearance of their relatives in countless other studies on plant growth make them an excellent option for this research  (Kataga et al., 2012). Factors such as plant appearance, height and biomass will be recorded to test the effectiveness of different frass mediums. It is predicted that while the frass produced by mealworms feeding upon the three different mediums (BioBag, Styrofoam and wheat bran) will influence plant growth, frass from the BioBag and wheat bran groups will display the best growth given their components being more naturally occurring than the polystyrene found within the styrofoam. Whatever the results, this data will provide new insights on the benefits and limitations of frass based fertilizers, especially those produced by organisms fed on plastics.

In addition, combining isopod or mealworm behavior with other decomposers could prove even more effective in biodegradation efforts and significantly aid our global plastic pollution crisis. For example, the cohabitation of terrestrial isopods with springtails is advised given their mutual benefits (Rushton, P. and Hassall, M., 1983), but research into cohabitation with other decomposer organisms such as earthworms, beetle larvae, and even specific fungi species could prove effective at further accelerating decomposition of biodegradables. Finally, implementation of all these organisms into both private and commercial compost heaps could allow biodegradable plastics to be more widespread as there would be significantly more invertebrate volume to help break them down. This is a realistic goal and one that needs to be continually researched given how widespread, easy to reproduce, and generally effective these organisms are at breaking down both organic and inorganic waste. In addition, the packaging of frass based fertilizers could not only provide methods of recycling plastic material but also raising money which could be put towards environmental solutions. The impact on our global plastic crisis would be massive; not only would we see a reduction in the aforementioned volume of plastic pollution, but we would also receive new options for fertilizer and abundant food sources (processed mealworms). All of these benefits could stem from animals that are thriving off of our garbage, protecting biodiversity for years to come.

METHODS & PROCEDURE:

Safety and Precautions:

While working with mealworms, their food mediums, or preparing substrate mixtures, Personal Protective Equipment (including nitrile gloves, safety goggles and a lab coat) was worn. Goggles and a lab coat were less important given the lack of lethal chemicals in the experiment, however they were often worn for the sake of general cleanliness. 

In addition, microplastic frass was handled and often went airborne as a result of its low mass. To prevent inhalation or entry through the eyes, a KN-95 mask (YOTU, Product # B0971PBMVG) and lab safety goggles were always worn in addition to the standard Personal Protective Equipment. Work with microplastics was also mostly done under a LabAire Systems DynamicFLO Fume Hood.

General Study Overview

This study examined the effects of different kinds of mealworm frass (waste) on height, leaf count and general health of the Wisconsin Fast Plant, Brassica rapa.  Mealworms, Tenebrio molitor, were used as a replacement for the M. brassicae from Kataga et al.’s study given their easier care requirements, wide availability given their role as feeder insects and, most notably, their unique ability to consume polystyrene demonstrated in Yang et al.’s study (2012 ; 2015). M. brassicae was also not chosen given their frequency to fly and greater space requirements (Jiang Long et al., 2020). 

Fast plant seeds were separated into four groups, each of which contained 12 plants. The groups were based on the type of substrate medium the plants would grow in. The first group, representing a negative control, was planted in plain coco fiber, a substrate with almost no nutrient levels that would allow for an understanding of how fast plants would grow without the presence of fertilizer. This fits the known definition of a negative control, which is defined as a replicate that receives no additional treatment or independent variable, which in the case of this experiment is the presence of frass (Lipsitch et al., 2010). Coconut fiber is successful in this manner given that it has been confirmed to lack most nutrients (Balawejder et al., 2022). This group was compared with three other groups planted in substrates created by mixing mealworm frass with a coco fiber substrate. These represented experimental arms which would yield novel data. The frass mixtures tested included BioBag, a biodegradable pet waste bag made mostly of starch components, styrofoam packing peanuts, and the mealworm’s commercial food source, wheat bran, which technically served as a positive control since frass is known to influence plant growth (BioBag, 2021; Kataga et. al. 2012). Mealworms produced frass while feeding on these, which was then mixed into the coco fiber.

Plant growth was monitored for approximately 3 weeks after the initial sowing of Fast Plant seeds in the substrate. This allowed sufficient time for the plants to mature, as it states in Carolina’s care guide for fast plants that the plants flower and reach sexual maturity after 14 days (Carolina, 2023). After this period, they are less dependent on energy stored within the original seed and instead soak more nutrients from their environment (Carolina, 2023). Consequently, allowing growth for an additional week will allow for greater variation between the group’s growth rates.

Plants were monitored every two days and images were recorded and stored in Lab Notebooks, where trends in plant health and growth rate were compared. Leaf size/count as well as any plant death or wilting were recorded for later use (For more information on care requirements, see section Brassica rapa in Model Organisms and Care Requirements). Mealworm cohorts also received regular maintenance, however the health of the colonies was a minor concern given that the primary goal was to collect their waste product. The full maintenance of these colonies, including separating adults/pupa and hydration of the cultures, is all included in the following section: Tenebrio molitor in Model Organisms and Care Requirements.

After the end of the 3 week period, plants were removed from the substrate and quantitative data such as plant mass (dehydrated), root length, and leaf count were recorded and analyzed. The experimental question “How will different types of mealworm frass impact the height, leaf count and general health of Wisconsin Fast Plants?” was used as a guide for the study. It was hypothesized that while the wheat and BioBag frass may increase plant height and leaf count due to high nutrient content, styrofoam frass may stunt the growth of the plants because of the microplastics present in the waste. 

Model Organisms and Care Requirements

-Tenebrio molitor

Mealworms were purchased from Carolina Biological in bulk (1,500 individuals total, Product # 144264) and housed in 16 quart plastic containers (Rubbermaid, Product # RMCC160001) with ventilation holes, as they require regular airflow for effective respiration (Carolina, 2023). Holes were created using a heated glue gun (Assark, Product # ‎HL-E 20W) to bore into the plastic, and about 8 holes were created in each 16 quart container (4 on the top lid and 2 on either side of the container. About 500 mealworms were placed in each of the 3 containers, which each had its own substrate: styrofoam packing peanuts (Uboxes, Product # PEANUTS3CUFT), wheat bran (Josh’s Frogs, Product # B00KSJISFW), or BioBag waste bags (BioBag, Product # B01MQTTXJ3) (BioBag, 2022). The containers were filled about halfway deep (approximately 3 cm) with each substrate medium. Two other 16 quart containers were reserved for collecting adult beetles and pupa. Pupa, once fully matured into adult beetles, were transferred into the adult container. To identify the physical appearance of the different life stages, see the image below:                         

Because of their high breeding rate, the original intent was to collect eggs from the adult container to produce new larvae (Zhao et al., 2016). However, the initial amount of larvae used produced enough frass for the experiment, so breeding was not required.

To care for mealworm larvae and adults, the only additional maintenance required was spraying the containers with fresh water every three days to keep the mealworms hydrated. No additional feeding was needed given that all three substrates provided excess food for the mealworms, however beetles in the adult bin were fed apple slices every two weeks for additional nutrition. It is recommended to remove these after about 3 days as they will start to grow mold, which can cause health problems for a mealworm beetle culture.

Mealworm housing containers were stored by a window which received indirect sunlight, which did not have a negative impact on these insects considering their tendency to burrow (Kroncke et al., 2022). Keeping the housing containers in an area that receives partial shade maintains moisture and prevents the containers from overheating. The ideal temperature for mealworm growth is approximately 20 to 22 °C (68 to 72 °F), so keeping them anywhere around this range is helpful (Carolina, 2023). A storage cabinet is an easily accessible storage location that provides all of these benefits, so long as at least four ventilation holes are not obstructed.

-Brassica rapa

 The plants being tested in this study are a variant of Brassica rapa, (a direct relative of the plant tested in Kataga et al. 's study) referred to as the Wisconsin Fast Plant (2012). This choice was made with the intent of producing similar results. They were purchased as seeds from Carolina Biological and are known for their hardy nature and rapid growth (Carolina, 2023). Care for B. rapa is relatively simple; once seeds have been planted (see the Planting of B. rappa (Wisconsin Fast Plants) section) in soil substrate as prepared in the Preparation of Frass Substrate Blends section, regular daily spraying and constant access to light should be sufficient for growth. Starter pots (Bonviee, Product # B086X2Z8P8) were used to grow plants and include a water tray which, when filled, hydrates plants via capillary action. This was done when the laboratory was inaccessible on weekends to keep plants healthy, and 65 mL of water was measured with a 100 mL graduated cylinder and poured into the trays. In addition, the lids included were used to cover the plants and keep the environment damp by reducing evaporation. When not in use, seeds of B. rapa can be stored in the bag provided by Carolina, which prevents the entry of any unwanted light and moisture which may start plant growth prematurely. 

Collection of Mealworm Frass

Every 3 - 4 days, mealworm containers containing either Wheat Bran, BioBag or Styrofoam media were checked for frass, which could be identified as a small, powder-like substance akin to sand. Frass color was influenced by the properties of the food medium fed to the mealworms. Frass produced by mealworms feeding on styrofoam, for example, was a pure white color and particles were extremely light (For safe handling of this microplastic, please revisit the first section, Safety Precautions). Frass produced by worms feeding on wheat bran was heavier and took on a brown color almost identical to the color of the wheat bran. Unfortunately, BioBag frass was not produced to the same extent and very minimal feeding was observed. Mealworms in this group seemed to instead seem to prefer feeding on the sheddings of other mealworms. For this reason, the planned Biobag trial was completely removed from the experiment. See the image below, Frass ID: 3 Variants for specific frass identification, which includes an image of the frass produced by the BioBag group.

To separate the frass from the substrate medium, an AKAKD three sieve sifting pan (AKAKD, Product # B09MJ99PG5) was used. The smallest mesh had gaps of about 1 mm, which allowed nothing but frass particles to fall through. The sieve was placed on top of a 3 quart container (Anbers, Product # B083PT4BM1) where the frass was collected. The substrate was scooped from the different containers using the scooper tool that came with the AKAKD sifting pan, then dumped onto the sieve above the container, which was shaken rapidly to separate the frass from the substrate medium. This was repeated for each container until no frass remained. In the case of the styrofoam container, however, the procedure was altered slightly. As mealworms often preferred to burrow into the packing peanuts and eat them from the inside out, cracking open the peanuts was necessary to release the frass inside. Additionally, large, untouched packing peanuts were removed from the housing containers when frass was being collected so smaller particles collecting on the bottom of the bin could be easily scooped with the scooper tool. Frass containers were labeled with the corresponding food medium and stored near the mealworm containers.

Preparation of Frass Substrate Blends:

“Frass Substrate Blend” refers to the product created when mixing coconut fiber substrate with each type of mealworm frass. In the case of this experiment, two different types of frass substrate blends were created: Styrofoam and Wheat Bran Frass. As mentioned in the previous paragraphs, BioBag was not included given the mealworms' unwillingness to feed on it and produce frass. All were mixed using an identical procedure and frass to coconut fiber ratio. Approximately 84 grams of coconut fiber were weighed in a bowl on a Flinn Scientific Electronic Balance (Product # OB2143), then about 5 grams of the specific type of frass was measured and mixed evenly throughout the coconut fiber using a scoopula. To help bind the frass to the coconut fiber substrate, the mixture was sprayed anywhere from 5 to 9 times with water from the spray bottle. This was especially effective for the styrofoam frass blend, as the styrofoam particles tended to be disturbed easily when dry. Frass Substrate Blends were usually mixed just before potting and sowing of the plants, however they can be stored in zip-lock bags for later use. Ideally, if the ratio is followed and the same starter pots mentioned in the following section, Planting of Brassica rapa (Wisconsin Fast Plants) are used, there should be no remaining frass substrate blend after potting is complete as the amount of substrate created is based on the carrying capacity of these starter pots.

Planting of Brassica rapa (Wisconsin Fast Plants)

Planting fast plant seeds was relatively straightforward given their general hardiness. Starter pots (Bonviee, Product # B086X2Z8P8) came in three parts: water tray (bottom), plant slots (middle) and plastic lid (top). Four starter pots were set aside, labeled for each of the three different growth groups: Control, Wheat Frass and Styrofoam. Substrate blends prepared in the previous section were poured to fill each of the 12 slots in their respective pot (eg. the frass substrate blend created with wheat frass was poured into the starter pot labeled “Wheat Frass.”). For the starter pot labeled control, 89 grams of pure coco fiber substrate (lacking any mealworm frass) was poured into the plant slots. This would be used to judge the “normal” growth of Fast Plants in the prepared environment. Carolina Biological provides a care sheet which claims that, in soil with sufficient fertilizer, the plants will reach flowering age within 14 days of planting, however coconut frass lacks fertilizer, which means the plants may reached this stage at a slower rate (Carolina, 2023). Once all of the plant slots were filled, a small hole about 2-3 mm deep was poked into each plant slot using a glass stirring rod. After this was complete, a single fast plant seed was placed in each of these holes, and the substrate was pushed back over the seeds so they could germinate properly.

Seeded starter pots were placed alongside one another in a Johnny’s seeds seedling grow cart (Product # 7026) kept damp via plastic lids included with the starter pots and received lighting about 30cm above them controlled by a BN-LINK mechanical outlet timer (Product #  B00MVFF59S)  for approximately 12 hours per day, allowing them all to receive the same conditions for growth. They were regularly monitored using practices from the previous Model Organisms and Care Requirements section, under Brassica rapa. 

Data Collection:

After around 3 weeks of undisturbed growth and consistent conditions, mature plants were removed from the substrate, delicately as to preserve their roots and stems. Substrate was washed off as much as possible without causing any damage. Each plant was placed in an individual weigh boat, and these weigh boats were grouped based on what starter pot the plant originated. Shortly after, plant height, from start of stem (not including roots) to the top of the plant was recorded. Following this, plants were dehydrated in a Hamilton Beach Digital Food Dehydrator (Product # B012CG8N26) at 37.7° C for about 3 hours to remove excess water. After this, any remaining coco fiber substrate not removed by washing was easily removed from the roots and discarded. Dehydrated plants were weighed on a Flinn Scientific Electronic Balance (Product # OB2143) calculate “dry mass.” After the first trial Plants were discarded after this was complete, and starter pots were washed out.

Data Analysis:

Data values were stored within a Google Sheets file, and organized based on cohort and type of data. The first goal was the identification and handling of outlier data. Outliers were identified as plants that never sprouted from their seeds or never emerged from the soil. These would be represented with a “/” mark on the table, indicating that they would not be compiled with data. Conversely, plants that died during trials were counted as this was a trend amongst one specific experimental group. Plants that died were represented by an x in their data slot.

To compile this data, the negative control plant height and leaf count were averaged, then compared with average data points from the experimental groups. This excludes the styrofoam group given the fact that it caused plant death in every scenario. With the averages, two tailed independent T Tests were conducted. “Independent” was chosen given the fact that the plants in the different groups are individual biological replicates and unrelated. “Two tailed” refers to the fact that the values within each data set can be greater or less than one another. These test produced a P value via an equation setup in Google Sheets, and data was determined to be statistically significant if P values were less than or equal to 0.05, which would support the part of the original hypothesis that wheat bran frass would positively impact plant growth.

MATERIALS LIST AND PROCEDURE:

RESULTS AND DISCUSSION:

All figures displayed here are a result of research conducted in the 2023-2024 research period, and are not directly related to data from the preliminary research in 11th grade on the terrestrial isopod Porcellio scaber. Of course, it’s important to acknowledge that there are various similarities between these two experiments. Both Tenibrio molitor and Porcellio scaber were used to degrade plastic waste, whether commercial or biodegradable and both species were also cultured in similar setups incorporating all of their life stages (See Fig 1.) The main difference between the two experiments is that while P. scaber were fed plastics to see how fast they were able to consume them, T. molitor were fed plastics (among other food items) for the purpose of collecting their frass (see Fig 2.) for use as a fertilizer. Nonetheless, data collected while experimenting with P. scaber had a positive influence on this experimental setup and allowed for a well informed data analysis.

Figure 1 displays the life stages of the model organism, Tenebrio molitor and serves as a helpful way to identify each life stage for the culturing organization described in the methods section. During the majority of the frass collection period, beetles were in their larval stage (see image labeled “larva” in the the figure above), as this is how they were purchased from Carolina Biological. However, it is important to note that this is not the only stage in which they were able to produce frass. T. molitor continue to consume the same food and produce the same waste products in their adult stages (see images labeled “Adult (Hardening): and “Adult (Hardened”). Only as eggs or pupae are they unable to produce frass. A visual understanding of the actual process and timeline in which this frass is produced is outlined by Fig 2. 

Fig. 2 depicts how the styrofoam and wheat bran fed to mealworms breaks down over a 2-3 week period. It is important to understand that this period depends on the amount of food medium or amount of worms present. The period of time in this figure is solely based on a quantity of about 500 mealworms and in a 16 quart Rubbermaid bin filled about 4 cm deep with the respective food/plastic. It’s also important to note that this rate was achieved while constantly sorting out  adults and pupae from the bins, and removed individuals were not replaced. However the amount of larval mealworms per bin remained consistent during this period given the fact that most do not pupate until 8-10 weeks have passed (As described in Fig. 1) and the trial began shortly after shipping. As shown in Fig. 2, the way in which mealworms degrade styrofoam and wheat bran differs greatly. Wheat bran is already composed of small pieces, so frass is often difficult to identify until 2-3 weeks, when there is plenty of it. Sifting and separating frass from wheat bran with a sieve was used as an additional method of identification and quantification. In the case of styrofoam frass, degradation is visibly obvious, as holes begin to form in the packing peanuts which grow larger as time passes. Eventually, large masses of white frass appear around and within the degraded packing peanut, which can be easily collected without the need for a sieve. These different frass types were then mixed in with a soil called Black Gold, which was used to grow Wisconsin Fast Plant (B. rapa) seeds. This process and its associated data is outlined in Fig. 3.

Fig 3 displays data collected during the Black Gold soil trial. An outlier in this experiment would be classified as a plant that failed to sprout, however all plants successfully germinated so there was no need to account for outliers. Originally, Black gold soil was used with the intention that it lacked unnatural fertilizers and would allow for the observation of how mealworm frass enhances growth. However after data collection and closer observation of the ingredients, it was made quite clear that Black Gold contained a plethora of natural fertilizer additives. This list included canadian sphagnum peat moss, compost, earthworm castings, horticultural grade perlite and processed bark, which are all high nutrient materials that can overpower the effects of a small quantity of mealworm frass (Black Gold, 2022). They evidently have a large impact on plant growth, which can be seen by the plant’s masses, especially those represented by the highest margins of error. As a result, the differences between each group of plants were not at all significant, as indicated by the p values of 0.2 (control vs. wheat) and 0.5 (control vs. styrofoam). Furthermore, the error bars and margins of error were quite large, further supporting the fact that the spread of data was very random. Variables such as positioning in the dehydrator, amount of soil removed from plant roots and dried pieces breaking off are key contributors to this skewed data. Fig 3 showed that a secondary experiment needed to be conducted with a substrate that lacked any present nutrients so the effects of the frass alone could be studied. Coconut fiber stood out as the best possible choice because it is inert (lacking any nutrients), widely available and low in cost. This prompted a new trial which is represented by the data in the following figure, Fig 4. Instead of mass, height was measured due to the aforementioned cause of error. This allowed for the measuring of plants without needing to change their structure (via dehydration) or worry about removing soil from fragile roots. Most importantly, height is quick to measure.

Fig 4 allows for interesting conclusions regarding the effects of various types of frass on plant growth in coconut fiber. Similarly to Fig. 3, an outlier in this experiment would be classified as a plant that failed to sprout, however all plants successfully germinated so there was no need to account for outliers. Primarily, the fact that mealworm frass produced while feeding on their standard food mediums increases plant growth (in this case, quantified by height after a set period of time) is supported by this data. The mean value for plant height (in cm) for the group grown in wheat frass mixed with coconut fiber is 7.1 cm, which is greater than the value of 5.6 cm for those grown in the coconut fiber with no additives. Supporting this fact further is the p value produced when comparing the wheat group to the control, which is 2.0 x 10-5, a number significantly less than 0.05. This shows extreme statistical significance between the ranges of heights in the two groups, not just the averages. This suggests that the experimental setup was successful and did not hinder the plants ability to extract the nutrients from the frass in question.

Most interesting and novel, however, is the way in which the group grown in styrofoam behaved. The skull icon representing what would’ve been the styrofoam data indicates that these plants died approximately 3 days after germination. This could be for a variety of reasons, but the most logical is the presence of toxic styrofoam chemicals within the frass even after it is digested by the mealworms. As mentioned in the Background section, the flame retardant Hexabromocyclododecane (HBCD), which is a key component of styrofoam, still remains in mealworm frass after digestion (Yang et. al., 2015). After several watering sessions, it is possible that this chemical leaches out of the frass, mixes with  the soil, and is absorbed by the plants, acting as a pollutant which eventually kills them. This of course poses the question: why weren’t styrofoam-fertilized plants killed in the black gold trial? It’s possible that the abundance of fertilizer within the black gold soil enriched the plants first, allowing them to germinate and grow healthily before accessing the styrofoam frass. In short, the presence of the other fertilizers diluted the styrofoam toxins. In this trial, there was no additional fertilizer, forcing the plants to consume the styrofoam toxins first which eventually killed them.These results provide interesting prompts for further study. Are there plants more resistant to this chemical than B. rapa? Are there ways to further nullify the negative effects this flame retardant has on plants, or eliminate it entirely after it is processed by the mealworms? A proposed new experiment would include testing a variety of different widely available plant species and how they’re affected by the presence of frass within the soil. Testing different concentrations of frass would also prove useful. Plant height would still be collected as the main data point. A separate but related experiment would also focus on the analysis of the flame retardant’s chemical composition and its effects on certain organisms. This will help to provide a greater understanding as to how we can eventually work around this toxic component.

While the hypothesis about plain styrofoam frass being a viable option as fertilizer for B. rapa may have been refuted, data from this experiment is still quite useful and with no shortage of applications. Frass produced by the wheat group clearly boosts plant growth, and must be used as an alternative to chemical fertilizers whenever possible to protect our future environment. Better still is the fact that T. molitor are now one of many insect species being farmed en masse for human and livestock consumption, meaning their frass can be easily distributed as fertilizer as sustainable insect cuisine continues to grow in popularity. This is an exciting prospect but also one that will depend on a better educated public on why insect farming is so beneficial for the environment. Insect farming takes up significantly less space, as most can be raised in tiered bin systems, insects have insignificant nitrate emissions in comparison to commercial livestock, and lastly, are packed with a high amount of protein for their small size. Our future depends on more people having access to this information.

In addition, this experiment displayed that mealworms are very apt at breaking down polystyrene plastics relatively quickly as displayed by Fig 2, while still continuing their life cycle as usual. Most notable is the fact that they completely remove any traces of the styrofoam from their own bodies during their digestion, which means they still serve as a viable protein source/animal feed when fed on styrofoam. The only condition would be that they’re starved for about a week before harvest to make sure all of the excess, including the flame retardant, passes through their gut. With this knowledge, mealworms could reduce the mass of our landfills significantly as we continue to work towards a solution for the remaining flame retardant toxin. The results gathered here are a step forward in the right direction, however better research on flame retardants and other plastic components as well as the potential of plastic eating organisms will, again, allow for more impactful experiments in the future.

Lastly, the degradation behavior exhibited by T. Molitor in this research period will make them a good degredation companion for the P. scaber studied in the 2022-2023 research period. Both efficiently consume high quantities of organic matter, some of which makes up key components of biodegradable plastics. With both living together in compost heaps, we can effectively accelerate the rate at which our biodegradables break down while also producing a valuable source of fertilizer from both the waste the animals leave behind. And since both species are highly prolific, populations can always be harvested for use as animal feed. Both of these species provide an endless amount of benefit–and paint a better picture for the future of our ever-changing planet.

SUPPLEMENTAL INFO AND FIGURES:

Raw Data (Google Sheets File)

Frass Effects on Plant Growth - JG 

Link to Lab Notebook:

Lab Notebooks Collection JG 

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