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Article

Microbial Consortia in the Remediation of Single-Use Waste: The Case of Face Masks

by
María del Refugio Castañeda Chávez
,
Luz María Campos García
,
Christian Reyes Velázquez
,
Fabiola Lango Reynoso
,
David Reynier Valdés
,
Isabel Araceli Amaro Espejo
and
Gabycarmen Navarrete Rodríguez
*
Tecnológico Nacional de México/Instituto Tecnológico de Boca del Río. Km. 12 Carretera Veracruz-Córdoba, Boca del Río CP 94290, Ver, Mexico
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(4), 2070-2084; https://doi.org/10.3390/microbiolres15040139
Submission received: 27 August 2024 / Revised: 30 September 2024 / Accepted: 3 October 2024 / Published: 7 October 2024
Browse Figures
Versions Notes

Abstract

:
This study presents the results of evaluating hydrocarbonoclastic consortia in the biodegradation of microplastics derived from single-use, triple-layered polypropylene face masks. The choice of this carbon source was driven by the need to address the increase in single-use waste generated during the recent SARS-CoV-2 pandemic, as the use of face masks was a mandatory protective measure. Two bubble column bioreactors were used, each containing hydrocarbonoclastic consortia sourced from the Port of Veracruz and the Gulf of Mexico. The biodegradation activity of these consortia was assessed by observing the physical appearance of microplastic samples under a stereoscope and a microscope, as well as by calculating the weight loss of polypropylene after 15 days. The results revealed that the consortium from the Gulf of Mexico, with a maturity of 1 year, showed a higher capacity for polypropylene biodegradation, achieving a 19.98% degradation rate. This consortium also demonstrated more stable kinetics during the experimentation period. In contrast, the younger consortium from the Port of Veracruz exhibited a lower biodegradation rate of 3.77% and variable growth kinetics. Hydrocarbonoclastic bacteria identified within the consortia included Pseudomonas aeruginosa, Enterococcus faecalis, and Vibrio parahaemolyticus, among others. The hydrocarbonoclastic consortia have the potential to biodegrade from various forms of plastic waste, including single-use face masks.

1. Introduction

The term “plastic”, derived from the Greek word for malleability under heat, also known as “polymer”, describes carbon-based compounds with long molecular chains derived from hydrocarbons. Due to its remarkable flexibility, plastic can be integrated into various stages of production and final products, underpinning its economic relevance [1,2]. Plastics are categorized by size, with microplastics (MPs) typically defined as plastic fragments measuring less than 5 mm in any dimension, though the lower limit is not precisely established. In contrast, “nanoplastic” refers to plastic particles that are smaller than 100 nm [3]. The ongoing COVID-19 pandemic, caused by the SARS-CoV-2 virus, led the World Health Organization to implement stricter protective measures, including lockdowns, social distancing, and the use of personal protective equipment (PPE) to prevent the spread of the virus through droplets [4,5].
Face masks, a form of PPE, have been used since early pandemics and are now a form of single-use hydrocarbon-derived waste that are intended to reduce the spread of droplets or aerosols between individuals and their surroundings [5,6].
Single-use face masks are made from durable and resistant materials such as polyethylene and polypropylene. However, poor waste management can lead to their dispersion into marine ecosystems, where they fragment into microplastics, becoming persistent pollutants that disrupt marine ecology [1,7,8,9].
Since these products retain their resistance characteristics as hydrocarbon derivatives, plastic biodegradation in the environment can be very slow due to their complex composition, depending on the proportion of their fractions [1,10].
Nonetheless, biological treatment technologies aim to remediate such situations using the metabolic activities of specific organisms, such as plants, fungi, and bacteria, to degrade, transform, or eliminate contaminants, converting them into harmless metabolic products. These technologies have a more positive environmental impact as contaminants are eliminated or degraded [10,11,12].
In the biodegradation process of plastics, there are two different situations—a direct action, where the transformation of plastics provides nutrients for microbial growth, and an indirect action, where microbial metabolic products affect the plastic’s structure [13].
The use of diesel as a substrate in bubble column bioreactors and the consortium characterized by García et al. [14] comprising Phylum Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Firmicutes, and Acidobacteria in a mineral medium over 14 days resulted in diesel biodegradation from 13.0 to 0.00 g L−1 in just 12 days [15]. Currently, it is imperative to address the biodegradation of single-use waste using hydrocarbonoclastic microbial consortia and to generate scientific knowledge. These consortia have the potential to demonstrate their ability to biodegrade plastic polymers such as polypropylene, which is present in products like face masks derived from hydrocarbons.
Therefore, this study aimed to evaluate the efficiency of hydrocarbonoclastic microbial consortia obtained from the Port of Veracruz and the Gulf of Mexico in the biodegradation of single-use face masks as a carbon source in bubble column bioreactors (BCBs) as a remediation method.

2. Materials and Methods

The research was conducted at the Aquatic Resources Research Laboratory (LIRA) of the Technological Institute of Boca del Río of the National Technology of Mexico (ITBOCA/TECNM), located in the municipality of Boca del Río Veracruz, Mexico.
Two groups of microbial consortia obtained through composite samples were used. One was obtained in the Gulf of Mexico by the Universidad Autónoma Metropolitana (UAM) through its isolation from the rhizosphere of Cyperus laxus, a native plant of the southeastern Mexican wetlands capable of growing in petroleum-contaminated soils; this consortium was identified through 16S ribosomal gene analysis by Lizardi and Gutiérrez [16]. The second consortium corresponded to a new microbial group also isolated from a composite sample of sediments extracted from the Port of Veracruz area. The reactors with the seed consortia used in this research were maintained mainly following the methodology of Lizardi and Gutiérrez [16].

2.1. Sediment Sample Collection

The sampling stations were selected in the Port of Veracruz to isolate the microbial consortium according to Pucci et al. [17]. These stations were chosen considering a 1.5 Km radius in the port area, taking into account interactions related to ship traffic and docking, as well as potential hydrocarbon exposure. The geographic coordinates of the stations were as follows: Station 1 (19°11′52.07″ N–96°7′47.42″ O) and Station 2 (19°11′51.15″ N–96°7′46.75″ O). At each station, two 100 g sediment samples were collected at a depth of 20 cm in the low tide zone using a stainless-steel shovel. Each sediment sample was placed in a container to remove stones, sticks, and other unwanted elements. The samples were then homogenized with a spatula. A 30 g portion of each sample was combined to form a composite sample, which was placed in sterile plastic bags and immediately sealed. These composite samples were stored at 4 °C until laboratory use.

2.2. Microcosm Generation Prior to Isolation and Scaling

The sample was placed in two glass containers and incubated for 30 days at 40 revolutions per minute, following a microcosm system that controls factors influencing process efficacy [17]. A 90% mineral medium (g/L) composed of 6.75 g NaNO3 (J.T. Baker; 99.9%), 2.15 g K2HPO4 (J.T. Baker; 99.3%), 1.13 g KCl (J.T. Baker; 99.9%), and 0.54 g MgSO4 ∙ 5H2O (J.T. Baker; 100.1%) was added. Additionally, 10% of the collected sediment sample, 1 mL of sterilized diesel for the first microcosm, and 1 mL of petroleum for the second were included. The pH was adjusted to 6.5 with 1.0 N HCl and 99% BaCl2 [18].

2.3. Maintenance and Scaling of Parent Bioreactors

After forming the microcosm over 30 days, the Port consortium was scaled. This process was conducted under sterile conditions using 90% mineral medium and 10% of the microbial consortium sample, with a diesel concentration of 13 g L−1 as the carbon source. The Port consortium obtained from the microcosm was processed in a 250 mL Erlenmeyer flask. Meanwhile, the hydrocarbonoclastic consortium obtained by Lizardi and Gutiérrez [16] from the Gulf of Mexico, maintained and matured for a year under the same conditions, was processed in a 500 mL Erlenmeyer flask [18]. The medium was adjusted to a pH of 6.5 using 1.0 N HCl and 99% BaCl2, and aeration was provided using Elite 800 oxygen compressors for aquariums and tanks up to 37.8 L, with a flow rate of 1.5 L m−1, over a 15-day period. This aimed to maintain the consortium in a stationary phase and prevent the growth curve from declining towards cell death before inoculation in larger volume bioreactors [19].

2.4. Selection of Carbon Source

During the COVID-19 pandemic, face masks became essential in containing the virus spread, surpassing other alternatives in efficacy, and becoming the predominant global choice. Despite their public health effectiveness, the widespread use of face masks has had an adverse environmental impact, generating significant single-use plastic waste. Given this scenario, finding solutions to reduce the environmental impact of masks has become urgent. This context prompted us to choose face masks as the carbon source for the study with the objective of taking advantage of and transforming these materials into resources that have less impact on the environment.

2.5. Inoculation

Two glass bioreactors were used for each bubble column due to the resistant properties of the microbial consortium, with a height-to-diameter (H/D) ratio of 4, obtaining a total volume of 4 L for each bioreactor, 50 cm in height, 10 cm in diameter, and 2 mm in thickness. A working volume of 3 L was used, and an L-shaped diffuser with seven 1 mm holes was adapted and connected to silicone hoses in each bioreactor, which were secured with clamps to keep them in place. These hoses were connected to Elite 800 oxygen compressor pumps designed for aquariums up to 37.8 L, with a flow rate of 1.5 L min−1 [20]. Each bioreactor was inoculated with 10% of the total volume of the corresponding microbial consortium. The microbial consortium from the Gulf of Mexico, with a maturation period of 1 year, was inoculated into one bioreactor, while the microbial consortium from the Port of Veracruz, with a maturation period of 30 days, was inoculated into the other bioreactor. Both consortia were maintained under sterile conditions during the inoculation process to prevent external contamination.
The mineral medium used (a total of 2850 L) consisted of a mixture (g L−1) of 6.75 g of NaNO3 (J.T. Baker; 99.9%), 2.15 g of K2HPO4 (J.T. Baker; 99.3%), 1.13 g of KCl (J.T. Baker; 99.9%), and 0.54 g of MgSO4 ∙ 5H2O (J.T. Baker; 100.1%) dissolved in distilled water [18]. Each bioreactor was inoculated with 150 mL of the microbial consortium and a carbon source (polypropylene coverslip) was added at a concentration of 13 g L−1 [21]. The pH of the medium was adjusted to 6.5 using 1.0 N HCl and BaCl2 at 99% through a Consort C6010 multiparameter. To avoid external contamination, cork caps that were adapted for the diffuser inlet were placed on each bioreactor. The bioreactors were operated at room temperature and pressure for a period of 15 days, with continuous aeration provided by the oxygen compressor pumps to ensure an adequate supply of oxygen to the microbial consortia, thus favoring the biodegradation process [18].

2.6. Evaluation of Substrate Biodegradation

After 15 days of bioreactor operation, the substrate was extracted to determine the amount that had been degraded by the microbial consortium. The contents of each bioreactor were transferred to 1 L glass containers and sterilized in an autoclave at a temperature of 121 °C (15 lb) for 20 min. After reaching room temperature, the contents were filtered using a strainer and were washed with distilled water for 3 min. Then, organic matter was removed by adding 30% hydrogen peroxide (H2O2) and letting it act for approximately 60 min until the substrate was completely covered.
Once the organic matter removal time had elapsed, the amount of hydrogen peroxide absorbed by the substrate was partially reduced. The substrate was then placed in porcelain capsules for drying in an oven at 125 °C for 24 h until it reached a constant weight. This was conducted to obtain the biodegradation percentage.
The formulas used to calculate biodegradation are presented below. To obtain a value by weight difference expressed in grams, the following Equations (1) and (2) were used to determine the biodegradation percentage according to Quiroga [19]. Here, P0 is the initial weight; P1 is the final weight; and PP is the weight lost.
P0 − P1 = PP
% of biodegradation (PP) (100)/P0

2.7. Physical Evaluation of Substrate Biodegradation

A qualitative analysis of the substrate obtained after being in the bioreactors for 15 days was conducted using a light-colored MICAPSA-brand binocular stereoscopic microscope with a 4× resolution. Additionally, a detailed observation was carried out using an OPTIKA-brand binocular microscope model B-600B with a 40× objective.

2.8. Growth Kinetics of the Microbial Consortium Using McFarland Scale and Spectrophotometry

An evaluation of bacterial growth in the medium was conducted using the turbidity scale known as the McFarland scale. This scale is used in microbiology to assess the concentration of cells in microbial suspensions by visually evaluating turbidity, providing numerical values that indicate cell density. The scale, with typical values ranging from 0.5 to 4 corresponding to various levels of turbidity and cell concentrations, is used to standardize the concentration of microorganisms in microbiological preparations, such as adjusting cell density in experiments.
The visual comparison involves matching the turbidity of the suspension with standard turbidity patterns on the scale, and when it visually matches a specific value, the cell concentration is assumed to be approximately equivalent [22,23]. A calibration curve was created using linear regression with the McFarland scale after measuring the absorbance of prepared barium sulfate (BaSO4) solutions.
The values were obtained using a Thermo Scientific UV-VIS 300 spectrophotometer. The absorbance results obtained from the spectrophotometer (experimental optical density) were positive according to the coefficient of determination and were graphically represented. Notably, the values are significantly close to the trend line, resulting in an R² value of 0.99, indicating higher precision in determining the concentration of microorganisms in suspension. Subsequently, to measure the growth of microorganisms from the consortium present in the Gulf of Mexico and the Port, tests were conducted using a Thermo Scientific UV-VIS 300 spectrophotometer. Samples were taken every 24 h for 15 days. These samples were placed in a quartz cell, and the equipment was set to a wavelength of 625 nm to measure absorbance through spectrophotometry. The absorbance data obtained were used in the following Equation (3), according to Escobar et al. [24].
log [CFU/mL] = 3 × 1010 [Abs] + 0.0236
These absorbance data were captured in Excel every 24 h for 15 days. The absorbances obtained for each inoculated medium sample in the bioreactors were then incorporated into the resulting data from the application of the formula for CFU/mL. From these data, two microbial growth kinetics graphs were generated, one for each consortium. These graphical representations were useful in showing variations in absorbances in relation to the growth of microorganisms over time. This approach allowed for an evaluation of the stability and a comparison of the growth of each consortium during the kinetics.

2.9. Identification of Microorganisms

To identify the hydrocarbon-degrading microorganisms present in the microbial consortia from the Gulf of Mexico and the Port in larger experimental bioreactors that are responsible for the biodegradation of polypropylene in face masks, samples from both consortia were inoculated into selective media. These media included Pseudomonas F Agar (BD Bioxon), Pseudomonas P Agar (BD Bioxon), Bile Esculin Agar (CONDA), MacConkey Agar (BD Bioxon), TCBS Agar, and Endo Agar (BD Bioxon), and were incubated for 24 h at a temperature of 35 °C.
After the incubation period, isolated colonies from the selective media were cultured on Tryptic Soy Agar (TSA) (MCD LAB) and incubated at 35 °C for 24 h. Subsequently, isolated colonies were selected for Gram staining to observe the morphological characteristics of the bacteria using an OPTIKA-brand binocular microscope model B600B with a 40× objective. Oxidase and catalase tests were also performed on the selected colonies.

3. Results and Discussion

3.1. Biodegradation of Disposable Face Masks

The biodegradation percentages of the face masks were calculated from the initial substrate amounts introduced into each bioreactor, which was 3.822 g and rounded to 4 g for a working volume of 3 L, considering a density of 0.098 g/mL for the three-layer face mask (Table 1). These calculations, as specified in the methodology, were employed to determine the percentages. It is noteworthy that the Gulf of Mexico consortium achieved a 19.98% biodegradation of polypropylene, while the Puerto consortium showed the lowest biodegradation percentage at 3.77%. According to Fadare and Okoffo [25] and Wu et al. [26], the release of microfibers from surgical masks is influenced by the brand used, the time, and the environmental conditions to which they are subjected during degradation. Oliviera et al. [27] indicated that there is a limited number of studies on the biodegradation and ecotoxicity of masks due to the pollutants generated by them; furthermore, they highlighted that the uncertainties and uncontrolled variables present during experimental procedures complicate the final comparison of the behavior of masks with that of plastic waste.
The biodegradation percentages of face masks in bioreactors with microbial consortia are shown in Table 1. The initial substrate weight was standardized to 4 g for a working volume of 3 L, based on a density of 0.098 g mL−1 for the three-layer face mask. The Gulf of Mexico consortium achieved a biodegradation percentage of 19.98%, while the Puerto consortium showed a biodegradation percentage of 3.77%.

3.2. Morphological Changes in Face Mask Microfibers

A separation between the fibers of the substrate subjected to biodegradation by the microbial consortia is observed compared to the intact substrate (Figure 1). The openings in the fibers of the substrate exposed to the Puerto consortium are smaller than those in the substrate treated by the Gulf of Mexico consortium. These structural changes were more evident in the microplastics treated with the Gulf of Mexico consortium (Figure 1). Cai et al. [28] reported that several pretreatments, using physical and chemical methods, of plastics can improve the efficiency of their biodegradation; this would contribute to a significant reduction in toxic contamination by plastics.
However, Wu et al. [26] stated that the abundance, size distribution, and morphology of microfibers released after mechanical abrasion by sediments are related to the type of mask used. They indicated that the abundance of microfibers was highest with surgical masks > common masks > facial filter masks (FFPs). Saliu et al. [29] reported that a single surgical mask subjected to artificial weathering with ultraviolet light irradiation and vigorous shaking in artificial seawater for 180 h could release up to 173,000 fibers per day. Wu et al. [26] also indicated that for surgical masks, the differences in the length of the fibers released ranged from 47.78 μm to 3.93 mm, while 72.41% to 89.58% of the total number of microfibers released had a length of 0.1 to 1 mm. The structural changes in the substrate obtained in this research were also related to the exposure time, due to variations in the biodegradation kinetics over 15 days in both bioreactors.
In Figure 2, we observe that in the untreated samples, the edges are uniform and the fibers maintain a compact structure (Figure 2). In contrast, in the samples subjected to biodegradation by the Puerto and Gulf of Mexico consortia, the edges appear irregular, and the fibers constituting the layers show greater opening in the structure, as well as irregularities in their edges. This change is even more evident in the substrate subjected to the Gulf of Mexico consortium. It is relevant to highlight that throughout the 15-day kinetic observation period, a change in the color saturation of the substrate exposed to the Puerto consortium was observed, as the polypropylene microplastics took on a slight yellowish hue, possibly related to biomass development. On the other hand, the substrate treated with the Gulf of Mexico consortium experienced a slight decrease in saturation, adopting a softer tone of the initial color.
The exposure time and type of plastic influence the biodegradation process of face masks; the release of microfibers from surgical masks is affected by the brand used, degradation time, and environmental conditions [26]. In contrast, Zhao et al. [30] indicated that environmental factors indirectly affect microplastics in the process of interaction and the transport of pathogens by altering their surface properties. Saliu et al. [29] reported that submerging the masks in seawater after ten days of exposure did not generate fragments or fiber aggregates in the sieved fractions, and only after filtration was a significant number of microfibers collected.
The findings of this study gain relevance by presenting similarities with the research conducted by Hermoza [31], who used airlift bioreactors for 7 days under controlled conditions and inoculated bacteria of the species Pseudomonas aeruginosa with a low-density polyethylene substrate, achieving a 2% biodegradation of the substrate. Additionally, with Aspergillus brazilensis, a 7% biodegradation was achieved. This confirms that this method is a developing alternative for the treatment and mitigation of low-density polyethylene, a hydrocarbon derivative. Thakur et al. [32] emphasized the importance of considering that biological degradation depends on various factors, including chemical structure, functional groups, molecular weight, crystallinity, and additives.
Cai et al. [28] indicated that additives, dyes, and organic contaminants in plastics can have negative effects both on public health and on the environment. In addition, they reported that plastic biodegradation is also particularly affected by plastic additives and the plasticizer contained in these plastics, which are capable of causing harmful impacts on biota. Maddela et al. [33] indicated that plastic additives not being able to add covalent bonds freely in the environment contributes to their presence in different environmental matrices, which can be associated with their significant toxicity. The impact on the environment has been reported, and only 25% of these have been characterized for their potential possibility for public health and the middle environment.
Meanwhile, in their study, Narciso et al. [34] demonstrated that bacterial strains isolated from a forest showed the ability to biodegrade polyethylene terephthalate, a component of microplastics collected from sediments in the state of Veracruz. The microplastics exhibited physical modifications such as cracks, cavities, erosion, and holes on the surface, attributed to the biodegradation process carried out by the microorganisms. This aligns with our study’s observations on polypropylene substrate, where the edges became irregular and the fibers showed separation.
Additionally, the microbial consortium composed of Xanthomonas sp., Acinetobacter bouvetii, Shewanella sp., and Aquamicrobium lusatiense, previously isolated from an oil-contaminated area in Veracruz according to the research of Tzintzun et al. [35], was evaluated to determine its capacity to degrade diesel at a concentration of 20 g L−1, achieving this process in 10 days using an airlift bioreactor.
Similarly, the study conducted by Valdivia [36] evaluated the capacity of an aerobic bacterial consortium to degrade polypropylene (PP) and polyethylene (PE) in both liquid and solid environments. The results revealed that the consortium managed to degrade approximately 8.15% of PP and 10.12% of PE. These findings confirm that the consortium exclusively uses plastic polymers as a carbon source for its metabolic activities. This suggests that the consortia investigated in our study also have the ability to metabolize polypropylene substrate as a carbon source, in addition to being aerobic. Therefore, they achieved a biodegradation percentage, with the Puerto consortium reaching 3.77% and the Gulf of Mexico consortium achieving 19.98%, all within a kinetic period of 15 days.

3.3. Growth Kinetics of the Gulf of Mexico Consortium (BR1)

The absorbance values corresponding to bioreactor 1 show that on day 5, a significant increase in bacterial growth was observed, reaching 3.26 × 1010 CFU mL−1 during the exponential phase (Figure 3a). Subsequently, on day 6, bacterial growth decreased to 2.75 × 1010 CFU/mL, followed by an increase on day 7 to 3.14 × 1010 CFU mL−1, considering these values within the stationary phase. On day 11, another decrease was observed to 2.25 × 1010 CFU mL−1, with a slight increase in bacterial growth on days 14 and 15.

3.4. Growth Kinetics of the Puerto Consortium (BR2)

In bioreactor 2, on day 1, prior to 24 h of inoculation, the bacterial density was 4.350 × 109 CFU mL−1, which was the highest result in the kinetics, indicating the start of the exponential phase after the lag phase (Figure 3b). Days 2 to 5, 7, 13, and 14 show low and similar values in bacterial density. The increase recorded on day 6 is not linked to the exponential phase; however, an increase to 3.120 × 109 CFU mL−1 was observed. Days 5, 8, 9, 10, 11, 12, and 15 show the lowest values. The growth kinetics of the Puerto consortium reveal variability, possibly attributable to its status as a young consortium, suggesting the need for a longer maturation and maintenance period.
The consortia were worked on following the methodology and recommendations of Medina et al. [18], which involved maintaining the medium at a pH of 6.5. However, the temperature was kept at ambient level and was variable, which could have interfered with the bacterial growth of each consortium. Additionally, the Gulf of Mexico consortium was maintained for a year before being subjected to the biodegradation test, while the Puerto consortium was in adaptation in microcosms for 30 days and underwent scaling with maintenance for 15 days prior to the biodegradation test. Therefore, it can be inferred that the Gulf of Mexico consortium had a greater maturity and adaptation to the medium compared to the Puerto consortium.
These findings are related to the conclusions of Gutierrez [37], who highlights the importance of factors such as medium pH and temperature in microbial growth and in the biodegradation process of low-density polyethylene by the bacterium Pseudomonas aeruginosa. It is worth noting that this bacterium is also present in the consortia analyzed in our research. Furthermore, Tirado et al. [38] indicated that the biodegradation of waste can be delayed or even not occur when microbial populations have low densities. This aligns with the biodegradation results, where the Puerto consortium showed a lower percentage of biodegradation and unstable microbial growth kinetics with lower values compared to the Gulf of Mexico consortium. The latter obtained a higher percentage of biodegradation and more stable microbial growth kinetics with higher density values.

3.5. Identification of Bacterial Consortia from Puerto and Gulf of Mexico

Microbial consortia samples were inoculated into selective culture media to promote the growth of specific bacteria while suppressing others. This selective cultivation resulted in pure colonies of targeted microorganisms, allowing for subsequent identification and analysis. Castañeda et al. [39] highlighted the importance of identifying the dominant bacterial species within a consortium through various biochemical and phenotypic tests, which aids in determining optimal growth conditions. Thus, utilizing selective culture media enabled the isolation of pure bacterial colonies, facilitating the identification of their potential to biodegrade microplastics.
Holt et al. [40] emphasized that following incubation in these media, isolated pure colonies allow for more precise morphological and biochemical characterization. Castañeda et al. [39] further noted that accurate microbial identification often requires additional biochemical analyses such as oxidase and catalase tests, along with Gram staining. In the process of microorganism isolation, selecting the appropriate growth medium and maintaining optimal incubation conditions are crucial for successful cultivation [41]. Castañeda et al. [39] demonstrated that a series of biochemical and phenotypic tests allowed for the characterization of microbial consortia, determining their potential use in the degradation of hydrocarbons like diesel. This underscores the promising role of microorganisms in degrading a variety of compounds. Similarly, Kotova et al. [42] noted that microbial plastic degradation research has explored a broad spectrum of plastics, including polyethylene, polystyrene, and polyethylene terephthalate (PET). As such, the screening of plastic-degrading microorganisms through a selective approach enabled a more thorough and precise analysis of the bacterial composition within specific environments.
Upon performing Gram staining, the identified microorganisms displayed a Gram-negative structure in both analyzed consortia. Additionally, the catalase test was positive for all the genera found. On the other hand, the oxidase test was positive for most, except for Enterococcus faecalis, which tested negative. This is significant because most isolated microorganisms with a better adaptation capacity were Gram-negative [43,44,45,46,47]. These are characterized by their hydrocarbon-degrading capabilities, attributed to the presence of lipopolysaccharides in their membranes, which facilitates the formation of hydrocarbon emulsions in aqueous environments.
The bacteria identified in the consortia are considered facultative hydrocarbonoclastic bacteria, as they adapted to the microcosms prior to scaling, as well as to the mineral medium in which they were inoculated, demonstrating their degradative capacity (Table 2). The biotechnological application of hydrocarbonoclastic microbial consortia has been implemented in the degradation of various carbon sources. Castañeda et al. [39] reported a 98.47 ± 0.38% degradation of diesel in an airlift bioreactor with an initial concentration of 13 g L−1. Additionally, Crisafi et al. [48] demonstrated that hydrocarbon-degrading bacteria are capable of colonizing virgin mask material and inducing morphological changes in the components of the masks’ surfaces.
Six bacterial genera were identified—Enterobacteriales (Escherichia coli, Shigella flexneri, and Salmonella typhimurium), Vibrionales (Vibrio spp., V. parahaemolyticus), Lactobacillales (Enterococcus faecalis and Streptococcus pyogenes), Pseudomonadales (Pseudomonas aeruginosa), Burkholderiale (Burkholderia cepacia), and Stenotrophomonas (Stenotrophomonas maltophilia). In contrast, Castañeda et al. [39] and Medina-Moreno et al. [49] identified only three genera of hydrocarbonoclastic bacteria (Pseudomonas, Vibrio, and Diplococcus) during the hydrocarbon biodegradation process.
Various studies have reported the capacity of both axenic microorganisms and mixed microbial consortia isolated from contaminated sites to biodegrade polypropylene (PP) and microplastics generated from COVID-19 sanitary waste [50]. This biodegradation ability has also been reported in bacterial consortia composed This biodegradation ability has also been described in bacterial consortia capable of utilizing and degrading PP as their sole carbon source including a large group composed of Pseudomonas stutzeri, P. chlororaphis, Bacillus subtilis, B. flexus, and Vibrio sp [51,52]. Additionally, bacteria such as Pseudomonas and Burkholderia are known to thrive in various environments and are capable of degrading hydrocarbons and their derivatives in soil and water [53,54]. Moreover, Vijaya-Lakshmi et al. [55] reported that P. aeruginosa VJ 1 can biodegrade untreated PP masks, demonstrating a versatile biological process for the managing PPE used during the SARS-CoV-2 pandemic, achieving a 5.37% weight reduction in PP film over 30 days.
Microorganisms belonging to various genera, such as Bacillus, Actinobacteria, Pseudomonas, Aspergillus, Penicillium, Cyanobacteria, and several species of microalgae, have shown a remarkable ability to degrade the microplastics present in the environment [28,56]. In line with these findings, the group of microorganisms with potential for biodegradation is broad and diverse. Among them is Stenotrophomonas panacihumi, which exhibited a degradation rate of 20.3 ± 1.39% of PP over a 90-day period [57]. Another species, Rhodococcus rhodochrous, demonstrated the ability to biodegrade PP films containing metal stearates, which act as pro-oxidants due to the enzymes involved in plastic biodegradation [50,58].
According to Ballesté et al. [59], microorganisms, including those considered potentially pathogenic, can colonize plastic surfaces in aquatic environments. Castañeda et al. [39] noted that Escherichia coli, while typically considered a pathogenic bacterium, has also been observed to tolerate the presence of hydrocarbon contaminants in its environment. Ballesté et al. [59] reported a close relationship between plastics and pathogenic bacteria, as plastic biofilms act as reservoirs for E. coli, thus contributing to the survival and persistence of fecal bacteria in aquatic systems.
Hernández-Sánchez et al. [60] indicated that microplastics act as a reservoirs for microorganisms by increasing the presence of fecal and pathogenic bacteria indicators in beach areas. Microplastics serve as substrates for growth by altering their properties and becoming transport mediums for the dispersion of pathogenic organisms, constituting a new route of exposure with implications for public health [60,61]. Thangaraj-Uthra et al. [62] suggested that microplastics are multi-faceted stressors in ecosystems, ranging from carriers of toxic chemicals and pathogen vectors that can alter microbial diversity to contributing to antibiotic resistance and impact on diseases such as diarrheal disease. These findings emphasize the broad implications of emerging contaminants for public health. Zhao et al. [30] underscored the importance of increasing the knowledge of the ecological behavior of microplastics and pathogens, as well as the potential hazards associated with their inherent exposure.
Understanding the risks associated with fecal indicator behavior and plastic pollution has significant implications on water quality assessments in marine environments [59]. Kotova et al. [42] highlighted that the lack of standardized protocols for microbial degradation of plastics complicates the comparison of results between different authors. Current research trends focus on accelerating the microbial degradation of plastics. However, it has been demonstrated that the degradation of PET by recombinant hydrolases from thermophilic actinobacteria has proven to be the most efficient process for plastic degradation known to date [42].
Meanwhile, Stoeck et al. [63] emphasize that sediment plays a crucial role as it acts as a support that promotes the growth of microorganisms and facilitates the mineralization of hydrocarbons. This implies that these microorganisms can efficiently metabolize the carbon source provided by hydrocarbons. According to Pucci et al. [17], the constant exposure of microorganisms in sediments or liquids to hydrocarbons due to maritime traffic leads to adaptability and an increase in microbial density with hydrocarbon-degrading abilities. It is important to highlight that the Puerto and Gulf of Mexico consortia were obtained from sediments in areas exposed to hydrocarbon spills or maritime traffic, as mentioned previously. These consortia are composed of microorganisms that have adapted to the environment in which they grew, demonstrating their ability to metabolize the hydrocarbons present in their surroundings.

4. Conclusions

The Gulf of Mexico bacterial consortium demonstrated a higher capacity for the biodegradation of microplastics present in polypropylene three-layer masks, achieving 19.98% biodegradation compared to the 3.77% achieved by the Puerto consortium. The bacterial growth kinetics of the Gulf of Mexico consortium, evaluated through the McFarland scale and spectrophotometry, show more stable absorbance values. In contrast, the bacterial growth kinetics of the Puerto consortium display notable instability over the 15-day evaluation period.
The aerobic bacteria present in the consortia are classified as facultative hydrocarbon degraders, as they demonstrated their ability to adapt both in the microcosm before scaling and in the mineral medium in which they were inoculated, thus evidencing their degradative capabilities. Among the most abundant bacteria are Pseudomonas aeruginosa, Burkholderia cepacia, Escherichia coli, Stenotrophomonas maltophilia, Streptococcus pyogenes, Salmonella Typhimurium, Shigella flexneri, Vibrio parahaemolyticus, and Enterococcus faecalis. Although significant progress has been made in the biodegradation of single-use masks through microbial consortia under controlled conditions, challenges remain in achieving complete biodegradation within a short period. Therefore, it is crucial to explore additional improvements in the project for future applications. These improvements could include optimizing the bioreactor used, adjusting the concentration of microbial consortia for inoculation, and implementing a metagenomic analysis. The latter approach would provide a more concrete and detailed understanding of the diversity and functioning of the microbial communities as a whole. Although significant progress has been made in the biodegradation of single-use face masks using microbial consortia under controlled conditions, challenges remain in achieving complete biodegradation in a short period of time.

Author Contributions

Conceptualization, M.d.R.C.C., C.R.V. and L.M.C.G.; methodology, C.R.V., L.M.C.G. and M.d.R.C.C.; validation, M.d.R.C.C., I.A.A.E. and C.R.V.; investigation, M.d.R.C.C., C.R.V., G.N.R. and L.M.C.G.; resources, D.R.V., I.A.A.E. and F.L.R.; writing—original draft preparation, M.d.R.C.C., L.M.C.G. and G.N.R.; writing—review and editing, M.d.R.C.C. and G.N.R.; supervision, F.L.R., D.R.V. and I.A.A.E.; project administration, M.d.R.C.C., I.A.A.E. and F.L.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

This research was carried out with the financial support of the Instituto Tecnológico Nacional de México/Instituto Tecnológico de Boca del Río (TecNM/ITBOCA) and of the Consejo Nacional de Ciencia y Tecnología (CONAHCYT).

Conflicts of Interest

The authors declare no conflicts of interest.

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Microbiolres 15 00139 g001
Figure 1. Morphology of the integrity of the fibers of the three layers in the mask after exposure to bacterial consortia. The surface of the original face mask fiber remains relatively intact compared to the consortia (microscope with a 40× objective).
Figure 1. Morphology of the integrity of the fibers of the three layers in the mask after exposure to bacterial consortia. The surface of the original face mask fiber remains relatively intact compared to the consortia (microscope with a 40× objective).
Microbiolres 15 00139 g001
Microbiolres 15 00139 g002
Figure 2. Morphology of the integrity of the fibers of the three layers in the mask after exposure to bacterial consortiums. The fiber surface of the original face mask is shown relatively intact with respect to the consortia (microscope with a 4× objective).
Figure 2. Morphology of the integrity of the fibers of the three layers in the mask after exposure to bacterial consortiums. The fiber surface of the original face mask is shown relatively intact with respect to the consortia (microscope with a 4× objective).
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Microbiolres 15 00139 g003
Figure 3. (a) Microbial growth kinetics of bioreactor 1. (b) Microbial growth kinetics of bioreactor 2.
Figure 3. (a) Microbial growth kinetics of bioreactor 1. (b) Microbial growth kinetics of bioreactor 2.
Microbiolres 15 00139 g003
Table 1. Biodegradation of face masks in bioreactors with microbial consortium.
Table 1. Biodegradation of face masks in bioreactors with microbial consortium.
ConsortiumFace MasksValue% Biodegradation
P0 (g)P1 (g)PP (g)
PuertoThree-layer4.03.88410.1513.77
Gulf of MexicoThree-layer4.03.18840.799219.98
Abbreviations: P0—initial weight; P1—final weight; PP—weight lost.
Table 2. Identification of bacteria consortia isolated in the study area.
Table 2. Identification of bacteria consortia isolated in the study area.
Puerto Consortium
BacteriaCatalaseOxidaseGram
Streptococcus pyogenesPositivePositiveNegative (Diplococci-coccobacilli)
Pseudomonas aeruginosa,
Burkholderia cepacia,
Escherichia coli,
Stenotrophomonas maltophilia		PositivePositiveNegative (Diplococci-coccobacilli)
Salmonella
Typhimurium
Shigella flexneri		PositivePositiveNegative (Diplococci-bacilli-coccobacilli)
Vibrio parahaemolyticusPositivePositiveNegative (Diplococci-bacilli-coccobacilli)
Gulf of Mexico consortium
Enterococcus faecalisPositiveNegativeNegative (Diplococci-bacilli-coccobacilli)
Pseudomonas aeruginosa
Burkholderia cepacia
Escherichia coli
Stenotrophomonas maltophilia.		PositivePositiveNegative (Diplococci-bacilli- curved bacilli-Vibrio)
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Castañeda Chávez, M.d.R.; Campos García, L.M.; Reyes Velázquez, C.; Lango Reynoso, F.; Reynier Valdés, D.; Amaro Espejo, I.A.; Navarrete Rodríguez, G. Microbial Consortia in the Remediation of Single-Use Waste: The Case of Face Masks. Microbiol. Res. 2024, 15, 2070-2084. https://doi.org/10.3390/microbiolres15040139

AMA Style

Castañeda Chávez MdR, Campos García LM, Reyes Velázquez C, Lango Reynoso F, Reynier Valdés D, Amaro Espejo IA, Navarrete Rodríguez G. Microbial Consortia in the Remediation of Single-Use Waste: The Case of Face Masks. Microbiology Research. 2024; 15(4):2070-2084. https://doi.org/10.3390/microbiolres15040139

Chicago/Turabian Style

Castañeda Chávez, María del Refugio, Luz María Campos García, Christian Reyes Velázquez, Fabiola Lango Reynoso, David Reynier Valdés, Isabel Araceli Amaro Espejo, and Gabycarmen Navarrete Rodríguez. 2024. "Microbial Consortia in the Remediation of Single-Use Waste: The Case of Face Masks" Microbiology Research 15, no. 4: 2070-2084. https://doi.org/10.3390/microbiolres15040139

APA Style

Castañeda Chávez, M. d. R., Campos García, L. M., Reyes Velázquez, C., Lango Reynoso, F., Reynier Valdés, D., Amaro Espejo, I. A., & Navarrete Rodríguez, G. (2024). Microbial Consortia in the Remediation of Single-Use Waste: The Case of Face Masks. Microbiology Research, 15(4), 2070-2084. https://doi.org/10.3390/microbiolres15040139

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