Abstract

Background: Commercial paint pigments contain toxic heavy metals that harm humans and pollute the environment. To mitigate these harms, ecologically safe pigments are necessary. Objective: This experiment aims to create a biopaint de-novo using transformed Escherichia coli bacteria and compare it to commercial paint. Methods: Genetically engineered E. coli bacteria producing magenta pigment were grown in petri dishes. The pigment protein was extracted, filtered, and dehydrated into a crystalline powder. This was mixed with acrylic medium to make biopaint. The biopaint and commercial paint were applied on acrylic paper; red, green, blue, and total spectral intensities were measured daily under different testing conditions. Spectral intensity variability was measured and compared using the Coefficient of Variation (CV). Trends in spectral intensity were analyzed using regression analysis. Results: The differences in the CV of biopaint to commercial paint were less than 20% under all testing conditions. Spectral intensities for both biopaint and commercial paint did not show any significant change during the testing period under the conditions of room temperature, heat, and humidity. However, under the cold testing condition, biopaint showed a slight but statistically significant (p-value < 0.05) decline in total, red, and blue spectral intensities. Conclusion: This experiment proves that E. coli-derived pigments can be used to make biopaint which has a similar durability to commercial paint as measured by the spectral intensities.

Keywords

Synthetic Biology, E. coli Protein Pigments, Sustainable Paint, Biomaterials, Microbial Pigments, Biogenic Paint Development

Share and Cite:

1. Introduction

The utilization of mineral pigments across various industrial sectors has been standard practice due to their appealing characteristics of vibrant colors, stability, and consistency [1]. Paint use has been increasing annually with the global pigment consumption reaching approximately 9.7 million metric tons in 2014 [2] [3]. In addition, around 700,000 to 800,000 tons of pigment are used in the dye industry and a significant amount is used in the cosmetics industry [4]-[8]. Pigment compounds are typically comprised of inorganic materials and are largely used in paints and coatings [5] [9]. Inorganic dyes derived from coal-tar derivatives have applications in textiles, food and beverages, paints and coatings, cosmetics, and personal care [5].

The adverse health and environmental ramifications of these inorganic colorants have raised significant concerns [10] [11]. Toxic heavy metals, including lead, titanium, arsenic, cadmium, cobalt, nickel, zinc, and manganese, are integral components of inorganic pigments and have been associated with considerable risks to both human and environmental health [12]-[16]. The detrimental effects of heavy metal pigments on human health are profound [16]-[20]. Research indicates that exposure to heavy metals in paint correlates with a significantly higher risk of developing certain types of cancers [21] [22]. The National Institute of Health (NIH) has classified several of these metals as carcinogens and a Chinese study concluded that the total carcinogenic risk posed by heavy metals in paints can reach close to 1%, highlighting the significant threat they represent [21]-[23]. Exposure to these metals, whether through skin absorption, ingestion, or inhalation, can lead to severe outcomes, including organ damage, neurological disorders, and death [20] [24]-[26]. Children are notably at risk from lead exposure from paints, which is linked to developmental delays, behavioral issues, and learning disabilities [20] [27]. There is a 1.16% increase in the incidence of childhood developmental disorders attributable to lead exposure [20] [28]-[30].

The environmental ramifications of inorganic pigments are equally concerning [10] [31]. The pigment production cycle, encompassing mining, milling, extracting, and grinding, contributes to environmental degradation, releasing toxic substances into the air, water, and soil [32]. This pollution adversely affects ecosystems and wildlife, with aquatic life being particularly vulnerable to the toxic effluents from pigment production processes, leading to significant water pollution and habitat destruction [10] [31] [32]. The bioaccumulation of heavy metals through the food chain can result in devastating impacts on biodiversity and ecosystem health [33]. Processes and procedures to maximize the utilization of mineral resources and minimize the environmental, social, and economic impacts associated have shown promise [34]. Studies have been done to guide urban planning taking into account the influence of emissions on weather conditions [35].

Considering these adverse effects of inorganic pigments, a pursuit of sustainable and non-toxic alternatives is emerging. Sajjad et al. have suggested pigment production by cold-adapted bacteria and fungi can have wide-ranging applications and can represent a promising solution [36]. These organisms can produce a broad spectrum of colors without the negative environmental and health impacts associated with inorganic pigments [37] [38]. Non-toxic and sustainably sourced biopigments reduce the dependence on hazardous chemicals and processes. The potential applications of bacterial pigments are extensive and diverse [36] [39]-[41]. In the textile industry, which accounts for significant global water pollution, biopigments could offer an eco-friendly alternative to these inorganic dyes [42]-[44]. In the food industry, where consumers increasingly demand natural and safe products, natural colorants from bacteria could replace artificial additives [44]-[46]. Moreover, in cosmetics, bacterial pigments could provide safer, more sustainable coloring options, minimizing the risk of skin irritations and allergic reactions linked to inorganic colorants [44] [47] [48]. This research addresses the feasibility of utilizing Escherichia coli bacteria to produce sustainable pigments for paint applications. By leveraging specific bacterial strains known for their pigment-producing capabilities, this experiment aims to develop a viable method for generating safer and more environmentally friendly alternatives to commercial paint pigments. This innovation could pave the way to biopaint production and significantly reduce the environmental footprint of the paint, dye, and cosmetic industries.

2. Methods

Genetically engineered Escherichia coli bacteria capable of expressing a magenta pigment were cultured in petri dishes (plasmid vectors sourced from Amino Labs) (Figure 1). The bacterial cells were subjected to a lysis process to release the pigment. This was achieved through the application of Triton X-100, a nonionic surfactant, alongside Hen Egg White Lysozyme (HEWL), an enzyme that breaks down bacterial cell walls. The contents were allowed to settle for 24 hours at room temperature, to facilitate complete cell lysis and pigment release. The resultant solution underwent centrifugation at 15,000 revolutions per minute (RPM). This step facilitates the separation of the lysed cellular components from the soluble pigment. Subsequently, the supernatant was filtered through a 0.22 µm pore-size membrane filter, ensuring the removal of all cellular debris and leaving behind a pigment solution. This filtrate was then subjected to a dehydration process to harvest crystalline pigment powder (Figure 2). The pigment was then formulated into a biobased paint by amalgamating with an acrylic medium, creating a homogeneous mixture suitable for application (Figure 3).

For the experimental evaluation, eight one-centimeter by one-centimeter squares of acrylic painting paper served as a testing surface; four were coated with the biopaint and four with a commercially available paint (Liquitex Acrylic Magenta). The testing conditions were exposure to room temperature (20˚C), heat (40˚C), cold (−10˚C), and humidity. The assessment of the paint’s durability and color stability was conducted over a 10-day period. Spectral intensities (red, green, blue, total) of the painted squares were measured daily using the ColorPicker software application, which provided a quantitative measure of color retention and fading.

The variability in spectral intensity was quantified through the calculation of the coefficient of variation for both the biopaint and the commercial paint under each testing condition. A linear regression analysis was performed and analyzed to identify any significant trends over time. This approach allowed for a visualization of the biopaint’s performance against that of commercial paint, aiming to highlight the potential benefits and limitations of biobased pigments in commercial applications.

Figure 1. Escherichia coli bacterial colonies expressing pigment protein coded by jellyfish gene.

Figure 2. Extracted bacterial pigment.

Figure 3. Amalgamate bacterial pigment with acrylic binder.

3. Results

The baseline spectral intensity characteristics (Mean ± SD) for biopaint and commercial paint along with the CV under the testing conditions are shown in Table 1. The total spectral intensity Mean with Standard Deviation (SD) is 164 ± 3.5 for the biopaint with a CV of 2.1 and 184.6 ± 6.1 for the commercial paint with a CV of 3.3. A determination was made a priori, for the purposes of this study, a difference in CV% of ≥20% is considered significant. As shown in Table 1, the CV variance between the biopaint and commercial paint ranged from 0.0% to 14.5% across all tested conditions.

Table 1. Baseline spectral intensity comparison.

The changes in spectral intensity values, including Total, Red, Green, and Blue, over time for each testing condition, are presented in Figures 4-7. These figures indicate the durability of the paints under different testing scenarios.

Figure 4. Testing condition: Room temperature.

Figure 5. Testing condition: Heat.

Figure 6. Testing condition: Cold.

Figure 7. Testing condition: Humidity.

Table 2 shows the mean estimates of spectral intensity alongside their 95% confidence intervals (CI) and associated p-values. The mean estimate for biopaint’s total spectral intensity at room temperature is 165.73 with a 95% CI 160.06 - 171.40 (p = 0.45). The mean estimate for commercial paint’s total spectral intensity at room temperature is 187.33 with a 95% CI 177.43 - 209.50 (p = 0.52). As noted in Table 2, spectral intensity trends showed significant p-values for total, red, and blue spectral intensities for biopaint under the cold testing condition.

Table 2. Spectral intensity estimates, 95% confidence interval, and p-values testing for trends of significance.

4. Discussion

The key advantage of biopigments lies in their minimal environmental and health impact. Unlike synthetic pigments, biopigments are typically biodegradable, non-toxic, and sustainable, making them an eco-friendly alternative. Their production and use do not involve harmful chemicals, ensuring that they are safer for both human health and the environment. As a result, they are gaining traction as a viable replacement in industries seeking to reduce their ecological footprint while maintaining vibrant and stable color properties [1].

This experiment has demonstrated that protein pigments synthesized by genetically modified Escherichia coli bacteria can be extracted, dehydrated, and subsequently formulated into a functional biogenic paint. Testing conditions in this experiment were chosen to reflect real-world environmental conditions. In the comparative analysis using the Coefficient of Variation to evaluate the distribution of spectral intensity across both biopaint and commercial paints, we observed that all resultant values were below the 20% threshold. This suggests consistency in spectral behavior, underscoring the biopaint’s potential as a viable alternative to its commercial counterparts. In this experiment, a p-value <0.05 indicates the paint did lose or gain spectral intensity of statistical significance over the testing period. Conversely, a p-value >0.05 indicates the paint did not lose or gain spectral intensity of statistical significance over the testing period. The spectral intensity profiles of commercial paint remained largely unaltered across a spectrum of testing conditions over the ten-day period, as evidenced by p-values exceeding the 0.05 significance level. Notably, the biopaint exhibited similar stable spectral intensities under diverse environmental conditions, including Room Temperature, Heat, and Humidity. However, the biopaint’s spectral intensities for total, red, and blue under cold conditions exhibited statistically significant deviations, with p-values below 0.05. Despite this statistical significance, the actual magnitude of these differences was relatively minor (3.79 for total spectral intensity, 1.13 for red, and 1.74 for blue), likely imperceptible to the human eye. The significant p-values observed are potentially attributable to multiple comparisons. The family-wise error rate indicates an 81% chance of finding at least one false positive p-value.

We would like to acknowledge several study limitations. One limitation is the small sample size, necessitating future experimentation with a large sample size to validate these findings. Additionally, during the application, biopaint exhibited a more globular texture in comparison to commercial paint, potentially attributable to insufficient biopigment drying and grinding. It needs to be seen whether longer drying and finer grinding of the biopigment will enhance usability characteristics. The drying time for biopaint was observed to be longer than that of commercial paint, indicating potential practical implications. The extraction of pigments from bacterial sources also presents logistical and economic hurdles, given the labor-intensive process and the relatively low yield of pigment, raising questions about its scalability and commercial viability. Furthermore, the paint’s durability over extended periods remains unexamined, as does its performance under a wider array of environmental conditions. This study also focused exclusively on magenta pigments, limiting the generalizability of these findings to other pigmentary formulations.

Despite these challenges, the potential applications for biogenic pigments in areas where commercial pigments pose environmental, or health risks, are promising. The production of pigments through bacterial synthesis can be a costly endeavor due to the relatively low yield associated with microbial processes. However, this method holds significant promise in niche applications, particularly in the generation of unique or exotic colors that are difficult to achieve through conventional methods. Moreover, bacterial pigments offer a sustainable alternative in cases where synthetic pigments pose substantial environmental risks. The field of cosmetology may benefit from the development of biopigments as safer alternatives to inorganic pigments. Additionally, advances in synthetic biology, including the utilization of fungal systems for pigment production, offer exciting avenues for overcoming current limitations and enhancing the economic feasibility of biogenic pigment production.

One limitation affecting the usability of biopaint is its extended drying time. Several biologically derived drying agents, such as eucalyptus oil, chitosan, starch, and fruit stone powders, are available on the market and have shown potential in accelerating the drying process in various applications. However, further research is necessary to evaluate whether the incorporation of these agents can enhance the performance characteristics of biopaints, particularly with respect to drying efficiency, surface finish, and durability. Exploring these additives could improve the practicality of biopaints for broader commercial use.

In conclusion, this exploratory study substantiates the feasibility of creating a biopaint from bacterial pigments, which closely mirrors the spectral characteristics of traditional commercial paints under various conditions over 10 days. Despite its preliminary nature and the obstacles identified, this research paves the way for further innovations in the development of sustainable and environmentally friendly pigment sources.

Financial Disclosure: This work was funded in part by the Mayo Clinic Department of Medicine, Division of General Internal Medicine.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References