A Performance Study on 3D-Printed Bioplastic Pots from Soybean By-Products

Abstract

Sustainability is a key factor in the development of new materials for plant pots, given the significant environmental impact of traditional plastic-based pots. Researchers have paid attention to developing biodegradable and sustainable alternatives to petroleum-based pots. In this study, two novel bioplastic formulations are developed, which incorporated soy-based by-product fractions to produce plant pots with self-fertilizing capability while also being cost-competitive. A 3D-printing process, fused filament fabrication, is used to produce plant containers from the filaments of soy-based new materials. Further, a small-scale greenhouse experiment is conducted to compare the performance of the soy-based 3D-printed bioplastic pots with pure polylactic acid (PLA) 3D-printed pots and traditional plastic pots, by growing a fruit-bearing plant (tomato) and a flowering plant (zinnia). Plant growth properties and root circling are analyzed, and the results show that the soy-based pots performed comparably to traditional plastic pots, especially in dry conditions, and also reduced root circling. While a more in-depth analysis is necessary, these initial findings suggest that using soy-based fractions and 3D-printing technology could provide a sustainable approach to developing plant pots, which could reduce the environmental impact of plastic-based containers and improve plant health.

1. Introduction

It is widely known that petroleum-based plastics have a negative impact on the environment due to their long decomposition period and their contribution to greenhouse gas emissions during production [1]. In agriculture and horticultural applications, petroleum-based plastic pots have several negative environmental impacts. Petroleum-based plastic is made from oil, which is a non-renewable resource [2]. The extraction and processing of oil contribute to air and water pollution and greenhouse gas emissions. In terms of decomposition, plastic pots can take hundreds of years to decompose in landfills, leading to long-term environmental pollution [3]. Additionally, the production and improper disposal of plastic pots involve the use of chemicals, some of which can be toxic and harmful to human health and the environment [4].

As an alternative to traditional petroleum-based plastics, bioplastics have the potential to reduce the carbon footprint and offer a more sustainable alternative to traditional petroleum-based plastics [5,6]. Bioplastic pots, such as those made from soy-based bioplastics [7], offer several sustainability benefits, such as self-fertilization and biodegradability. Bioplastic pots made from renewable resources, such as plant-based or plant-waste materials, can be replenished. Depending on the specific materials and manufacturing process, they can also offer self-fertilizing properties. For example, bioplastics made from polyhydroxyalkanoate (PHA) polymer can be biodegraded by micro-organisms in the soil, releasing nutrients that can be taken up by plants [8,9]. In addition, bioplastic pots can degrade in a shorter period compared to petroleum-based plastics, reducing environmental pollution and waste [7,10]. The production of bioplastics generally results in lower greenhouse gas emissions than petroleum-based plastics [11]. Bioplastics are typically less harmful to the environment when disposed of properly. These sustainability benefits make bioplastic pots an eco-friendly alternative to traditional petroleum-based pots.

Recent research has focused on developing various bioplastic pots as an alternative to petroleum-based plastic pots. Bioplastic pots are usually made from biodegradable polymers such as polylactic acid (PLA) and fillers from plant waste and animal fibers, which make them plantable, compostable, and biodegradable [12]. Plantable bioplastic pots can be directly planted and biodegraded directly in the soil, whereas compostable bioplastic pots typically provide a more sturdy structure and have to be removed before planting and placed in a compost pile to biodegrade [12]. Sun et al. [13] evaluated the biodegradability of bioplastic pots produced from straw fiber with hydrolyzed soybean protein isolate/urea/formaldehyde copolymer-based adhesive. They observed that, during the degradation process, a number of bacteria and fungi selectively accumulated on the surface of the pot to accelerate its degradation [13]. Liew and Khor [14] analyzed the weight loss of bioplastic pots incorporated with newspaper pulp fibers. Their results showed that bioplastic pots tested below ground had a higher degradation rate than those planted above ground [14].

Bioplastic pots are also reported to provide plants with natural nutrients and improve their growth. Castronuovo et al. [15] assessed the performance of three different kinds of biodegradable pots (biodegradable polyester, plain, or added with plant fibers). Their investigation results showed that plants grown in pots with added plant fibers have good values of agronomical qualitative and quantitative indices. Schettini et al. [16] developed biodegradable pots with micrometric fibers derived from tomato peel and seed, as well as hemp strands. Their experimental results concluded that the developed biodegradable containers enhance the root development and plant growth, avoiding transplant shock and root deformation [16]. Rahman et al. [7] conducted a greenhouse study for soybean bioplastic-based pots and found that their biocomposite formulations outperformed traditional plastic pots for certain plant growth and biodegradability. However, bioplastic pot costs are currently higher than petroleum-based plastic pots [17], as the production process of bioplastics requires several additional pre-and post-processing steps compared to the straightforward injection molding of traditional plastic pots [7].

To minimize the processing steps involved while maintaining the sustainability benefits of bioplastic pots, this paper proposes an innovative 3D-printed (fused filament fabrication) bioplastic pot from soybean by-products. Soybean by-products are the leftovers from processing soybeans into various food and industrial products [18]. These by-products can include soybean hulls (SH) and soy protein isolate (SPI), among others [6,19]. Soybean by-products are often used in animal feed and in the production of biodiesel, industrial chemicals, and bioplastics [19,20,21]. In recent years, there has been a growing interest in finding novel ways to use soybean by-products, particularly in the development of sustainable materials and products [22].

Now, 3D-printing technology is advancing, with ongoing research and development efforts exploring new materials, improved printing techniques, and enhanced capabilities. It opens up possibilities for future innovations in plant pot manufacturing, including the incorporation of smart materials, sensors, and even bioengineered elements to promote plant health and growth. There are several advantages of using the fused filament fabrication (FFF) process for pot fabrication. For example, the FFF machines are inexpensive and the operation of an FFF machine is easy, and it can even be controlled remotely [23]. An FFF machine requires limited space for installation and operation. Moreover, costly tooling is not required to produce pots by the FFF process [24]. Also, FFF enables the creation of complex geometries and intricate designs that may not be easily achievable with traditional manufacturing methods like granulation and pressing molding. More importantly, the waste of materials or scrap is low for the FFF process as the material usage is optimized, which might lead to cost savings in the long term. In summary, pot fabrication through the FFF process provides unique advantages in terms of customization, design complexity, material efficiency, prototyping, manufacturing complexity, and future potential [25].

As a compliment to the traditional manufacturing processes, 3D-printing processes have several applications in agriculture, including the production of customized tools and equipment, replacement parts, and even foods [26,27]. One particular area where 3D printing has shown promise is in the creation of planters and containers for crops [28]. By using bioplastics and other sustainable materials, 3D-printing processes can help reduce the environmental impact of traditional agriculture practices while also improving plant health and growth. Thus, this paper proposes an alternative bioplastic pot production method with a 3D-printing approach.

In short, the contributions of this research are to (1) develop bioplastic pot formulations from soybean by-products by mixing PLA with SH and SPI, (2) access 3D printing as an alternative bioplastic pot production process, and (3) evaluate the performance of the 3D-printed bioplastic pots. The combination of using sustainable materials and state-of-the-art technology, such as 3D printing, has the potential to revolutionize the way crops are grown and food is produced, making agriculture more sustainable and efficient.

The rest of this paper is organized as follows: Section 2 includes information on the bioplastic pot formulations and 3D-printing process. In Section 3, the greenhouse experiments and plant growth measurement study are elaborately explained. Section 4 follows up with the data analysis and discussion towards sustainable development. Finally, concluding remarks are made in Section 5.

2. Materials and Manufacturing Methods

This research studied the performance of four types of pots. The summary of the material composition and manufacturing process of the four types of pots is given in

Table 1. Summary of formulations and manufacturing process.

Pot ID	Formulations *	Type of Pots	Manufacturing Methods
Pot 1 **	90% PLA + 7.5% SH + 2.5% PEOX	Biodegradable	3D-printed (soy-based filament)
Pot 2 **	87.5% PLA + 7.5% SH + 2.5% SPI + 2.5% PEOX	Biodegradable	3D-printed (soy-based filament)
Pot 3 **	100% PLA	Biodegradable	3D-printed (PLA filament)
Pot 4 (control) ***	Polyethylene	Non-biodegradable	Injection molding

* Polylactic acid (PLA), soy hulls (SH), Poly-2-ethyl-2-oxazoline (PEOX), and soy protein isolate (SPI). ** Produced in-house in the laboratory setting. *** Purchased from a commercial source.

. Three 3D-printed biodegradable pots are compared with a non-3D-printed, non-biodegradable polyethylene (commercial plastic) pot. Among the three 3D-printed biodegradable pots, two types of pots (Pot 1 and Pot 2) are printed from soy-based filaments, and one type of pot (Pot 3) is printed from pure PLA.

2.1. Materials and Procurement for the 3D-Printed Pots

In this research, the soy-based filaments are prepared from PLA, SH, SPI, and PEOX in different proportions. A filament is a form of material that is used to fabricate parts by the FFF process. PLA is a polymer matrix, whereas SH and SPI act as the fillers. PLA is a biodegradable and biocompatible polymer that is derived from renewable resources such as corn starch or sugarcane [29]. PLA is suitable as the matrix material for bioplastic pots due to its mechanical properties, biodegradability, and processability [25]. SH and SPI are two different by-products derived from soybeans [30,31]. SH is the outer covering of soybeans and is often considered a waste product. SH is low-protein and high-fiber. SPI is a highly refined protein-packed product that is made by removing the fat and carbohydrates from soybeans [32]. SH and SPI are selected as potential fertilizers to enhance plant health and speed up the pots’ biodegradability [30]. PEOX is a synthetic water-soluble polymer known for its biocompatibility and biodegradability. The application of PEOX in biocomposite filament formulation is to increase the bonding between the PLA matrix and the bio-based fillers [33].

After considering the filament production constraints, several smaller-batch formulations are experimented on with respect to maximizing the proportion of filler(s) with PLA, and we ended up with Pot 1 and Pot 2 ratios instead of other mass ratios. During pre-experimentation, it is found that SH exceeding 7.5% caused the filament to become excessively brittle and prone to breakage, leading to nozzle blockages and improper plastic extrusion through a standard 0.4 mm FFF machine nozzle.

The final compositions obtained are as follows: Pot 1 is produced from a soy-filament made up of 90% PLA + 7.5% SH + 2.5% PEOX, and Pot 2 is printed from a soy-filament that consisted of 87.5% PLA + 7.5% SH + 2.5% SPI + 2.5% PEOX. These two pot formulations are compared with Pot 3, which is 3D-printed from 100% PLA filament. PLA (3001D) is obtained from Nature Works, LLC, Minnetonka, MN, USA, while SH pellets are collected from NCI (Northern Crops Institute) Feed Production Center, Fargo, ND, USA. SPI is procured from Honeyville Inc. and PEOX is purchased from Sigma-Aldrich (St. Louis, MO, USA). Additionally, all three biodegradable 3D-printed pots are compared with commercial non-biodegradable regular plastic plant pots of similar size purchased from a commercial source.

2.2. Methods for Producing the 3D-Printed Pots

As the FFF process uses the filament as material, prior to the printing process, soy-based and pure PLA filaments are produced beforehand. There are three major steps in producing the 3D-printed pots, as demonstrated in Figure 1. The first step is material processing, the second step is filament production, and the third step is the 3D-printing process. All steps are carried out at the different laboratory facilities at North Dakota State University in Fargo (ND, USA).

In Step 1, the SH pellets are milled using the Retsch SK 100-mill machine and sieved to obtain a granular size of less than 100 μm. The SH particle size is reduced to avoid nozzle clogging during the FFF process. Meanwhile, SPI, which is already in powdered form, does not require preprocessing. The PLA pellets and SH particles underwent a drying process in a vacuum oven at 80 °C for 6 and 4 h, respectively, to remove any moisture content. The PLA, SH, SPI, and PEOX are mixed thoroughly using a mixer for 0.15 h to produce the two 3D-printing filament formulations for Pot 1 and Pot 2 based on prespecified proportions. The mixing is not required for the Pot 3 formulation as it has a single constituent, PLA.

Moving on to Step 2, the mixed constituents of each filament formulation are poured into the barrel of the Leistritz twin-screw extruder, which had three heating zones: feed zone, transition zone, and metering zone. The temperature at different zones varied from 135 °C to 165 °C for all three filament formulations. The output of the extrusion process is strands, which then passed through a water channel through the die of the extruder for cooling and solidification to obtain filament with 1.75 ± 0.1 mm diameter. These strands are then coiled onto a 3D-printing filament spool.

In Step 3, a retrofitted MakerBot Replicator Z18, an FFF machine, is used to fabricate the three biodegradable pots (Pots 1–3). The 3D-printed pots’ design and dimensions were obtained from the control Pot 4. The 3D-printing parameters employed are: layer thickness 0.34 mm, print temperature 210 °C, and infill density 100%. The produced Pots 1–3, along with the control Pot 4, are shown in Figure 2.

About 8 pots are 3D-printed for each type of biodegradable formulation, which included Pot 1, Pot 2, and Pot 3. These formulations are chosen based on their potential to be environmentally friendly and sustainable. After printing, a visual inspection was conducted to check for the print quality, including uniformity of the layers and the absence of warping or cracks. The defective rate is relatively high for printing due to low pot thickness. If no defects are found, the pots are sent to the greenhouse to be used for plant growth which will be detailed in the following section.

3. Greenhouse Performance Study

The greenhouse field experiment is a multi-stage process aimed towards optimizing plant growth and quality, as outlined in chronological order in Figure 3. To support plant growth, a commercial growing mix and water-soluble fertilizer (Jacks 20-20-20 General Purpose) are used for all plants. The growing medium used is Pro-Mix BX with microbial fungicide by Sun Gro^® Horticulture. A 200 ppm (parts per million) solution of the fertilizer is regularly applied to the growing medium during the seedling and transplantation processes and continued up until harvesting time to provide essential plant nutrients. After harvest, the plants are measured for their growth in fresh and dry conditions, and root circling is also observed. The collected data are further analyzed to evaluate the effectiveness of biodegradable formulations in promoting plant growth and health.

3.1. Overview of Greenhouse Experiment

Step 1: Seedlings. The first step is the seedling preparation, where the seeds are sown in the seed starter trays filled with a commercial growing medium. For horticultural plants, one fruit-bearing plant (tomato) and one flowering plant (zinnia) are selected based on the availability of seeds in the greenhouse during the experiment. Sheyenne tomato and zinnia seeds are planted in four-cell plastic seed starter trays filled with Pro-Mix BX growing medium in the North Dakota State University Plant Science Horticulture Greenhouse (Fargo, ND, USA). The conditions of the growing medium and seedlings’ growth are routinely monitored. Once the seedlings have germinated and grown to a sufficient height, they are then transplanted into the experimental pots with the same commercial growing medium.

Step 2: Transplantation. After approximately four weeks of seedling growth, the seedlings are transplanted into the four types of experimental pots (shown in Figure 2) on 11 October 2021. During the transplantation process, the seedlings are carefully removed from the plastic trays and replanted into the experimental pots to ensure adequate root establishment and space for growth. Transplantation is a critical step in the growth cycle of many plants, promoting better growth, improved yields, and environmental stress protection. Four samples (seedlings) are employed for each pot and plant type, totaling 32 pots in the greenhouse experiment (16 pots each for tomato and zinnia).

Step 3: Visual inspection. To ensure a healthy growth of the plants, a regular visual inspection is conducted at least once a week to check for any abnormalities in their growth or appearance. The inspection included checking for signs of disease or pests, nutrient deficiency, soil conditions, and any other environmental stressors. As shown in Figure 4, the tomato and zinnia plants are inspected during both the rooting and growth phases.

After four weeks of transplantation, the tomato and zinnia plants grow sufficiently tall and require support to prevent them from falling due to the weight of their stalks and stems in the pots. Therefore, fiberglass tree stakes are used to provide structural support for climbing. During this phase, a visual inspection is also carried out. All the tomato and zinnia samples displayed good growth without noticeable plant growth differences among the different pot types for each species. Moreover, no signs of plant diseases are observed during the inspection. However, one tomato plant grown in Pot 1 and one zinnia plant grown in the plastic pot (Pot 4) do not thrive past this stage and eventually died.

Step 4: Harvest. After two months of growth, the remaining 30 plants are harvested for plant growth measurement and root circling analysis. These measurements and analyses are critical for ensuring the success of the greenhouse experiment and obtaining accurate data for the future design of experiments. During harvesting, the plants are removed from their respective pots. Comparison photos of each plant from all four pots are taken for a qualitative analysis of plant growth, as shown in Figure 5.

3.2. Post-Harvest Processing

Step 5: Root circling observation. Observation of root circling is important because it indicates the potential for root-bound plants, which can lead to stunted growth and reduced yields. When roots are restricted to the shape of the container, they can begin to grow in circles instead of spreading out, which can limit their ability to take up water and nutrients [34]. To observe any instances of root circling, close-up photographs are taken from different angles after the harvest (before separating the root and shoot) of each plant for plant growth measurement. By observing root circling, a better understanding of the effect of container size and shape on plant growth can be obtained. These findings can be further used to make recommendations for optimal growing conditions.

Step 6: Plant growth measurement (fresh condition). Plant growth and health depend on two essential components of the plant: the shoot and the root. The shoot is the visible above-ground part of the plant, including the stem, leaves, flowers, and fruits. In contrast, the root is the hidden below-ground part that provides support and absorbs water and nutrients from the growing medium. To measure plant growth, the root is separated from the shoot, and the growing medium is washed away from the root. Four fresh condition parameters are measured immediately after harvesting: plant height, plant width, fresh shoot weight, and fresh root weight. Figure 6 illustrates different measured parameters.

The fresh weight measurements are taken immediately after the plants are harvested. This measurement includes all the water in the plant and reflects the plant’s current true state. Fresh weight is often used to determine the water content of the plant tissue and to calculate other growth parameters. Both fresh and dry weight measurements are important in plant growth studies, as they provide different types of information about the plant’s growth and overall health [35]. Additional drying processes are carried out before measuring the dry weight of the shoot and root.

Step 7: Drying. Once the fresh plant measurements have been completed, the roots and shoots of all plants are separated and placed in different paper bags. These bags, containing the different plant parts, are then subjected to a temperature of 176 °F (or approximately 80 °C) in a dryer for seven days prior to the measurement of dry condition parameters.

Step 8: Plant growth measurement (dry condition). Dry weight measurements are taken after the plant has been dried to remove all moisture. This measurement provides the information on the plant’s total biomass, as it represents the weight of the plant material that remains after all the water content has been removed. Dry weight is often used to assess the plant’s nutrient content, as well as the plant’s resistance to drought stress. After seven days in the dryer, plant parts are taken out of the paper bags and weighed to obtain the dry shoot weight and dry root weight.

In the next section, the statistical methods applied to analyze collected fresh and dry condition data are introduced.

3.3. Overview of Statistical Analysis

Statistical analysis is a subset of data analysis focusing on using statistical methods to analyze and interpret data [36]. Statistical analysis is employed in this research to identify relationships between study variables and make inferences based on the collected data from the greenhouse experiment described in the previous section. Two statistical methods employed are the one-way analysis of variance (ANOVA) and the Tukey pairwise comparison. The statistical analyses are conducted for tomato and zinnia plants, and all six plant growth measurement variables are collected from the greenhouse experiment.

ANOVA is a statistical method used in hypothesis testing to draw valid conclusions from collected greenhouse experiment data by testing differences in plant growth measurement means (μ) between two or more groups of pots (K). In this research, the groups are four types of pots labeled as Pots 1–4, with K = Pot 1, Pot 2, Pot 3, and Pot 4. Since the variation between the pots is analyzed based on plant growth measurement variables, which are assumed to be independent, a one-way ANOVA is employed. This allows for an analysis of the differences in means between the groups of pots (${\mu }_K$) and provides insights into which types of pots are more effective for plant growth in the given conditions.

In statistical analysis, the assumption of independence means that the values or measurements being analyzed are not influenced by each other. In the case of plant growth measurements in the greenhouse settings, the assumption of independence means that the growth of one plant in one pot does not influence the growth of other plants in other pots. This assumption is reasonable in most experimental settings [36], where the plants are treated similarly and are grown in separate pots or containers. If the plants are grown in the same pot or container, or if there are factors that could influence the growth of multiple plants, such as temperature or light, then the assumption of independence would not hold, and a different statistical analysis may be needed.

ANOVA involves defining null and alternative hypotheses (Equation (1)), choosing a significance level, calculating a test statistic, and making a decision based on the p-value. In this study, the null hypothesis ($H_o$) states that there is no significant difference in the means of plant growth measurements between Pots 1–4, while the alternative hypothesis (H_a) states that there is a difference in the means.

$$\begin{gathered} H_o: {\mu }_{Pot1}={\mu }_{Pot2}={\mu }_{Pot3}={\mu }_{Pot4} \\ {H}_a:At least one pair of means is different from each other \end{gathered}$$

(1)

A significance level (α) is pre-determined as a probability threshold for rejecting the null hypothesis, and, in this study, it is set to 0.05. This means that, if the calculated probability value (p-value) is less than or equal to 0.05, the null hypothesis is rejected. The p-value represents the strength of evidence against the null hypothesis. In one-way ANOVA, the p-value can be obtained from the F-test statistic by dividing the between-group mean square by the within-group mean square to obtain the F-value (Equation (2)). The p-value can then be found by looking up the F-value and the associated degrees of freedom in the F-distribution table.

$$\begin{gathered} F-value=\frac{explained variance}{unexplained variance}=\frac{between group mean square}{within group mean square} \\ =\frac{\sum _{i=1}^K{n_i\left({\overline{Y}}_i-\overline{Y}\right)}^2/(K-1)}{\sum _{i=1}^K\sum _{j=1}^{n_i}{\left({\overline{Y}}_{ij}-{\overline{Y}}_i\right)}^2/(N-K)} \end{gathered}$$

(2)

For the F-test numerator to obtain the between-group mean square, ${\overline{Y}}_i$ is the sample mean in the i-th group of pots, $n_i$ is the number of observations in the i-th group, $\overline{Y}$ is the overall mean of the collected data, and K is the number of group of pots (Pots 1–4). For the F-test denominator to obtain the within-group mean square, ${\overline{Y}}_{ij}$ is the j-th observation in the i-th out of K groups, and N is the overall sample size. The F-test statistics follow F-degrees of freedom of $d_1=K-1$ and $d_2=N-K.$

Hypothesis testing has two possible outcomes: accepting or rejecting the null hypothesis ($H_o$). The $H_o$ is accepted when the p-value is greater than the significance level (p-value > α), indicating that the difference between means is not statistically significant. On the other hand, the $H_o$ is rejected when the p-value is less than or equal to the significance level (p-value ≤ α), indicating that there is a statistically significant difference between at least two group means.

However, one-way ANOVA cannot determine which specific group means exhibit significant differences from each other when $H_o$ is rejected. Therefore, a post hoc analysis, Tukey pairwise comparison, is conducted [7]. This analysis involves comparing all possible pairs of group means using a confidence interval approach. Confidence intervals are calculated for each group mean, and then the intervals between pairs of groups are compared to determine if they overlap or not. If the intervals overlap, there is no significant difference between the means; if they do not overlap, the means are significantly different. This helps identify which specific groups have significant differences in their means.

3.4. Greenhouse Experiment Results

3.4.1. Plant Growth Measurements: Tomato

The size of the test Pots 1–4 is about 4 inch in diameter. When grown in small containers, horticultural plants with limited growing medium and space can become root-bound, resulting in root circling and potential girdling problems. When a tree’s roots are too large for a small container and kept for a long time, the roots hit the edge of the container and circle due to space constraints. Root circling has negative impacts on plant health, growth, and stability [37,38]. This experiment observed root circling of tomato plants grown in Pots 1–4, as shown in Figure 7. The results for the root circling observation reveal that tomato grown in soy-based bioplastic containers (Pot 1 and Pot 2) showed fewer root circles compared to the control Pot 4. Based on the experimental investigation, it can be concluded that bioplastic Pots 1–3 may result in less root circling compared to plastic Pot 4.

The fresh- and dry-condition plant growth measurements of tomato are shown in

Table 2. Plant growth measurement data for tomato.

Pot ID	Samples	Plant Height (cm)	Plant Width (cm)	Shoot Fresh Weight (gm)	Root Fresh Weight (gm)	Shoot Dry Weight (gm)	Root Dry Weight (gm)
Pot 1	S1	44.00	21.00	22.00	4.00	3.15	0.66
	S2	32.00	17.00	15.00	3.00	2.06	0.43
	S3	30.00	22.00	16.00	3.00	2.83	0.33
	S4 *	-	-	-	-	-	-
	Avg.	35.33	20.00	17.67	3.33	2.68	0.47
Pot 2	S1	32.00	25.00	18.00	6.00	3.33	0.73
	S2	29.00	27.00	19.00	7.00	3.27	0.73
	S3	27.50	12.00	6.00	1.00	0.57	0.09
	S4	37.00	35.00	24.00	7.00	5.48	0.93
	Avg.	31.38	24.75	16.75	5.25	3.16	0.62
Pot 3	S1	38.00	20.00	12.00	4.00	1.42	0.33
	S2	27.00	26.00	18.00	5.00	3.19	0.62
	S3	24.00	11.60	6.00	2.00	0.85	0.17
	S4	36.00	21.00	16.00	3.00	1.14	0.33
	Avg.	31.25	19.65	13.00	3.50	1.65	0.36
Pot 4 (Control)	S1	42.00	25.00	22.00	6.00	3.06	0.66
	S2	35.00	22.00	18.00	4.00	2.30	0.58
	S3	35.00	23.00	14.00	3.00	2.21	0.35
	S4	37.00	28.00	20.00	7.00	2.68	0.66
	Avg.	37.25	24.50	18.50	5.00	2.56	0.56

* Sample lost during the greenhouse experiment.

. There are four samples (S1–S4) observed for each pot type (Pots 1–4). The average (Avg.) growth measurements are taken from these four individual samples, and the comparison are shown in Figure 8. The error bar of a plant growth parameter in Figure 8 demonstrates the variation among samples for the growth parameter. Unfortunately, one tomato sample in Pot 1 died during the experiment process and, hence, is excluded from the data analysis.

Based on the data presented in Table 2 and Figure 8, the following observations and conclusions can be drawn from the tomato greenhouse experiments:

• In terms of plant height, tomato plants grown in non-biodegradable plastic Pot 4 outperformed those grown in 3D-printed bioplastic pots. However, for plant width, tomato plants grown in soy-based bioplastic Pot 2 showed comparable results to those grown in the control Pot 4.
• For fresh weight, the tomato shoot performed better when grown in Pot 4, while the root weight is comparable to the control Pot 4 when grown in soy-based bioplastic Pot 2. The higher fresh weight of a plant indicates that the plant has a greater amount of water or other fluids in its tissues, which can be an indication of greater nutrient (e.g., nitrogen) uptake or efficient photosynthesis [39].
• In terms of dry weight, tomato plants grown in soy-based bioplastic Pots 1 and 2 showed better shoot dry weight compared to the other pots. The root dry weight of tomato plants grown in bioplastic Pot 2 is also comparable to that of the control Pot 4. A higher dry weight of a plant can indicate that the plant has accumulated more nitrogen from pots [39,40].

Based on the greenhouse experiment results for tomato plants, the bioplastic pots only performed better in dry conditions. This may be due to the porosity of the biodegradable materials used in the bioplastic pots, which could result in these pots retaining less moisture than the control plastic pot. Plants may also absorb more nitrogen from soy hulls and soy protein isolate of pot materials. However, further analysis is required to determine the exact reasons for this observation, such as the impact of bioplastic degradation on soil moisture retention, nutrient availability, and microbial activity. In the next section, the results obtained for the zinnia plant will be detailed.

3.4.2. Plant Growth Measurements: Zinnia

In addition to plant growth measurements, the greenhouse experiment also aimed to assess the occurrence of root circling and potential girdling problems in horticultural plants grown in small containers. The root circling study for zinnia in Pots 1–4 is observed and documented, as shown in Figure 9. The observation results indicate that zinnia grown in soy-based bioplastic containers (Pots 2 and 3) have fewer root circles than those grown in Pot 4. This is likely because, when the roots of a plant come into contact with the bioplastic material, they are more likely to grow through and around the material rather than circling around the pot. This can lead to healthier root growth and, ultimately, healthier plants.

The fresh- and dry-condition plant growth measurements of zinnia are recorded in

Table 3. Plant growth measurement data for zinnia.

Pot ID	Samples	Plant Height (cm)	Plant Width (cm)	Shoot Fresh Weight (gm)	Root Fresh Weight (gm)	Shoot Dry Weight (gm)	Root Dry Weight (gm)
Pot 1	S1	67.00	17.00	36.00	26.00	6.50	3.38
	S2	65.00	16.00	29.00	16.00	4.60	2.08
	S3	51.00	15.00	22.00	13.00	3.54	1.71
	S4	52.00	15.00	22.00	13.00	5.51	2.87
	Avg.	58.75	15.75	27.25	17.00	5.04	2.51
Pot 2	S1	53.00	13.00	29.00	13.00	5.29	1.84
	S2	35.00	16.00	28.00	15.00	4.42	1.96
	S3	36.00	21.00	24.00	8.00	3.19	1.21
	S4	48.00	20.00	25.00	11.00	4.38	1.79
	Avg.	43.00	17.50	26.50	11.75	4.32	1.70
Pot 3	S1	44.00	18.00	31.00	16.00	4.77	1.90
	S2	38.00	12.00	17.00	7.00	2.38	0.96
	S3	36.50	14.00	21.00	14.00	3.81	2.05
	S4	29.00	18.00	14.00	10.00	1.94	1.08
	Avg.	36.88	15.50	20.75	11.75	3.23	1.50
Pot 4 (Control)	S1	46.50	16.00	24.00	10.00	3.96	1.08
	S2	40.50	15.00	21.00	14.00	3.31	1.95
	S3	73.50	20.00	36.00	24.00	6.47	3.51
	S4 *	-	-	-	-	-	-
	Avg.	53.50	17.00	27.00	16.00	4.58	2.18

* Sample lost during the greenhouse experiment.

. There are four samples (S1–S4) for each container and plant type. The average (Avg.) growth measurements are taken from these four individual samples. Unfortunately, one sample is lost during the growth process, and the zinnia plant that did not survive in the plastic pot (Pot 4) is excluded from the data analysis.

The average (Avg.) growth measurements are taken from these four individual samples shown in Figure 10 for all growth measurement parameters. Variations among samples for a plant growth measurement parameter are represented by an error bar in Figure 10. Based on the data presented in Table 3 and Figure 10, the following observations and conclusions can be drawn from the zinnia greenhouse experiments:

• Zinnia plants grown in soy-based biodegradable plastic Pot 1 showed superior plant height compared to other bioplastic pots (Pot 2 and Pot 3) and non-biodegradable control Pot 4. Soy-based bioplastic Pot 2 exhibited plant width results comparable to the control Pot 4, which is also observed for the tomato plant width detailed in the previous section.
• For fresh weight, the shoot of zinnia plants performed better when grown in soy-based bioplastic Pots 1 and 2, which is comparable to the control Pot 4. However, zinnia root performed better in soy-based Pot 1 and Pot 4 than in Pot 2 or Pot 3.
• Regarding dry weight, zinnia plants grown in soy-based bioplastic Pot 1 showed better shoot dry weight compared to the other pots. Moreover, the root dry weight of zinnia plants grown in bioplastic Pot 1 is comparable to that of the control Pot 4.

Unlike the results for tomato, where the bioplastic pots only performed better mainly in dry conditions, the zinnia experiments showed that the soy-based bioplastic Pot 1 outperformed the non-biodegradable plastic Pot 4 in all plant growth measurement categories, as well as the root circling observation. This could be due to the fact that soy-based bioplastic, being a biodegradable material, may allow for better air and water permeability, promoting healthier root growth and overall plant development. Additionally, the absence of harmful chemicals in the bioplastics may also contribute to better plant growth and health. Although more in-depth analyses are necessary, these initial findings suggest that the use of soy-based bioplastics could be a viable alternative to non-biodegradable plastic in horticultural applications.

4. Data Analysis and Discussion

4.1. Statistical Analysis Results

The cumulative ANOVA test results for tomato and zinnia based on the collected data from Table 2 and Table 3 are presented in

Table 4. ANOVA results for tomato and zinnia with 95% significance.

Plant	ANOVA	Plant Height (cm)	Plant Width (cm)	Shoot Fresh Weight (gm)	Root Fresh Weight (gm)	Shoot Dry Weight (gm)	Root Dry Weight (gm)
Tomato	p-value *	0.267	0.493	0.406	0.424	0.352	0.474
	Results	Accept Ho	Accept Ho	Accept Ho	Accept Ho	Accept Ho	Accept Ho
Zinnia	p-value *	0.045	0.703	0.445	0.445	0.260	0.308
	Results	Reject Ho **	Accept Ho	Accept Ho	Accept Ho	Accept Ho	Accept Ho

* p-value is compared to α = 0.05. ** Followed up with Tukey pairwise analysis.

. Based on the ANOVA results, it can be concluded that there is no significant difference between the plant growth measurements for tomato and zinnia when grown in different pots for most of the plant health parameters analyzed. This suggests that the choice of pot may not have a significant impact on the growth and health of these plants, which is a positive finding for growers and other stakeholders who are looking to reduce their environmental impact.

However, the ANOVA results also showed that the plant height of zinnia exhibited a significant difference, which indicates that the choice of pot may have an impact on the growth and health of certain plant species. This finding could be useful for growers and gardeners who are looking to optimize their plant growth and health by choosing the most appropriate pot for each plant species. Thus, a Tukey pairwise comparison is conducted only for zinnia plant height. The results are shown in

Table 5. Grouping information with Tukey pairwise comparison and 95% confidence.

Plant/Variable	Pot ID	Description	Mean (cm)	Grouping
Zinnia/ plant height	Pot 1	3D-printed (soy-based)	58.75	A	-
	Pot 2	3D-printed (soy-based)	43.00	A	B
	Pot 3	3D-printed (PLA)	36.88	-	B
	Pot 4 (control)	Injection molding	53.30	A	B

.

Tukey grouping provides a useful way to further explore the differences between group means after an ANOVA test has shown that there are significant differences between at least two groups. From the results in Table 5, it is shown that, for zinnia plant height, Pots 1, 2, and 4 belong to Group A, whereas Pots 2, 3, and 4 belong to Group B. For Pot 2 and Pot 4, they can belong to both Group A and B, which means that their plant height measurement is not significantly different from either group. Based on these results, it can be concluded that 3D-printed soy-based pots (Pot 1 and Pot 2) are comparable with traditional plastic pots (Pot 4) for zinnia plant health measurement. This can further suggest that soy-based pots are a viable and sustainable alternative to traditional plastic pots. By identifying which groups are significantly different from each other and which ones are not, Tukey grouping can help researchers understand the specific factors contributing to the differences in plant growth and health. This information can be valuable for optimizing growing conditions and selecting the most appropriate plants and growing methods for specific purposes, which can contribute to sustainable agriculture and horticulture practices.

4.2. Discussion and Future Work towards Sustainable Development

Developing sustainable materials for plant pots is crucial for achieving environmentally friendly agriculture and horticulture. This study presents two novel bioplastic formulations incorporating soy-based fractions with a 3D-printing process to fabricate the pots. As the 3D-printing process is a layer-by-layer process, gaps between two consecutive layers are observed due to low pot thickness and weak bonding between layers. These gaps may be reduced by analyzing the impact of the 3D-printing process parameters and making adjustments in the parameters accordingly [41]. It is observed that, as a result of the low water retention of 3D-printed pots due to gaps between layers, plants grown in these pots require more frequent watering.

While bioplastic pots are known to possess self-fertilizing capabilities [7,31,42], this is not always apparent in the 3D-printed pots, possibly due to their porosity [6]. The porosity of the 3D-printed structure may lead to water and nutrient loss, which can reduce the ability of the pots to provide self-fertilizing capabilities. The small gaps between the printed layers of the 3D-printed pots can create channels for the loss of water and nutrients, reducing the overall efficiency of the pots in providing self-fertilizing capabilities. Therefore, the porosity of the 3D-printed structure may affect the ability of the pots to retain water and nutrients and, thus, impact their self-fertilizing capabilities.

However, the 3D-printed pots could be optimized in their structure to possess better self-fertilizing and self-watering capabilities [41]. For example, increasing the thickness of the walls to reduce porosity can improve the water retention capacity of the pots. Moreover, incorporating additives such as hydrogels or other water-absorbing materials into the bioplastic formulation can increase the water-holding capacity of the pots [43,44]. Additionally, incorporating nutrients or fertilizers into the bioplastic formulation can help to release them slowly over time and provide a steady supply of nutrients to the plants [31,45]. Finally, the design of the pot can also be optimized to ensure that water and nutrients are distributed evenly throughout the soil and root system, promoting healthy plant growth [28].

Although this pilot study has limitations in terms of the limited sample size and variety of plants experimented in the greenhouse, it is a first step in determining whether 3D printing could complement traditional manufacturing processes to produce cost-competitive bioplastic pots with a customizable formulation for a specific plant species. To ensure the reliability of the results, the experiment is needed to be replicated with a wider variety of plants and pot samples to account for some plants that did not survive the greenhouse experiment. Investigating the long-term effects of using bioplastic pots on plant growth and soil health would provide valuable insights. Moreover, future work will also focus on the basic performance of plant pots, such as water/gas permeability, strength, additives concentration, etc. The plant nutrient (e.g., nitrogen) absorption from pot materials by plant roots will be experimentally investigated for different types of plants. In addition, other agriculture by-products, such as wood flour, rice husks, and corn flour, will be explored for biocomposite pot development.

5. Conclusions

This study presents a promising approach to developing sustainable bioplastic pots using soy-based fractions in novel bioplastic formulations coupled with 3D-printing technology. The results of the small-scale greenhouse experiment demonstrated that the soy-based 3D-printed bioplastic pots performed comparably to traditional plastic pots, particularly in dry conditions, and reduced the problem of root circling. These findings offer a feasible solution to developing environmentally friendly plant pots that promote healthy plant growth. Further studies are necessary to examine the long-term effects of the bioplastic pots and assess the economic feasibility and environmental impact of large-scale bioplastic production, recycling procedures, and waste management. However, the initial results presented here are encouraging for the sustainable development of agriculture and horticulture applications.