*Article* **Fungi and Circular Economy:** *Pleurotus ostreatus* **Grown on a Substrate with Agricultural Waste of Lavender, and Its Promising Biochemical Profile**

**Simone Di Piazza 1,\* , Mirko Benvenuti <sup>2</sup> , Gianluca Damonte <sup>2</sup> , Grazia Cecchi <sup>1</sup> , Mauro Giorgio Mariotti <sup>1</sup> and Mirca Zotti <sup>1</sup>**


**Abstract:** The increasing production of essential oils has generated a significant amount of vegetal waste that must be discarded, increasing costs for farmers. In this context, fungi, due to their ability to recycle lignocellulosic matter, may be used to turn this waste into new products, thus generating additional income for essential oil producers. The objectives of our work, within the framework of the European ALCOTRA project FINNOVER, were two-fold. The first was to cultivate *Pleurotus ostreatus* on solid waste of lavender used for essential oil production. The second was to provide, at the same time, new products that can increase the income of small and medium farms in the Ligurian Italian Riviera. This paper presents two pilot tests in which *P. ostreatus* was grown on substrates with five different concentrations of lavender waste, ranging from 0 to 100% (*w/w*). Basidiomata grown on all the substrates and their biochemical profiles were characterized using high-performance liquid chromatography coupled to mass spectrometry. The biochemical analysis of mushrooms proved the presence of molecules with antioxidant and potential pharmacological properties, in particular in mushrooms grown on lavender-enriched substrates. The results open the possibility of producing mushrooms classified as a novel food. Furthermore, the results encourage further experiments aimed at investigating how different substrates positively affect the metabolomics of mushrooms.

**Keywords:** essential oil production; agro-waste recycling; mushroom cultivation; closing the loop; HPLC-MS analysis

#### **1. Introduction**

Due to environmental changes and the high degree of competitiveness of national and international markets, the agri-food industry faces numerous economic challenges; hence, the industry is constantly looking for economically sustainable solutions. These difficulties are manifested in rural areas where small- and medium-sized farms face challenges to market their products and to make them competitive with multinational corporations or foreign products. These difficulties may be caused by the poor efficiency of the production cycles and/or by the lack of competitiveness of the products themselves. A circular economy could be a valid alternative to the habitual "take-make-waste" approach, providing a concrete solution to transform the system into a more efficient, sustainable, and eco-friendly approach. Many studies have highlighted that a circular approach, through closing the production loop, minimizes external inputs and the production of additional waste, making the processes both economically and environmentally sustainable [1]. A circular approach, if properly applied, could allow the survival of many local farm businesses that would

**Citation:** Di Piazza, S.; Benvenuti, M.; Damonte, G.; Cecchi, G.; Mariotti, M.G.; Zotti, M. Fungi and Circular Economy: *Pleurotus ostreatus* Grown on a Substrate with Agricultural Waste of Lavender, and Its Promising Biochemical Profile. *Recycling* **2021**, *6*, 40. https://doi.org/10.3390/ recycling6020040

Academic Editor: Leonel Jorge Ribeiro Nunes

Received: 3 May 2021 Accepted: 8 June 2021 Published: 11 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

otherwise not be economically sustainable. In recent years, the scientific community has worked to optimize production processes from a circular economy perspective.

Concerning bioresources, research has focused on the isolation and selection of particular organisms that are exploitable in circular processes [2]. In this context, fungi—due to their natural roles in ecological cycles—are good candidates to be exploited to recycle and to transform vegetal by-products and waste from agriculture into more valuable products. To date, there are more than 100,000 known species of fungi, but it is estimated that there are over 3,000,000 species that are still unknown [3]. Due to their biological characteristics, fungi are able to colonize almost every environment on Earth, and thus they play a key role in nutrient cycling in trophic chains. For example, lignicolous fungi (that is, wood-decaying fungi) play a crucial role in recycling lignin and cellulose in forest ecosystems [4]. On this basis, it is clear that several species of lignicolous fungi can also be applied fruitfully from a circular economy perspective and can be exploited to turn woody by-products and waste from agriculture into food or other valuable products. Mushrooms, in particular, are a group of fungi characterized as having sporomata (fruiting bodies) visible to the naked eye; they have been collected and cultivated by humans for thousands of years both for food and for medicinal purposes [5]. Almost all traded mushroom species today are saprotrophic or lignicolous fungi cultivated on substrates based on different decaying organic matter, most frequently wood. Recently, the increasing use of mushroom species such as *Pleurotus ostreatus* (Jacq.) P. Kumm., *Lentinula edodes* (Berk.) Pegler, *Ganoderma lucidum* (Curtis) P. Karst. and *Grifola frondosa* (Dicks.) Gray for food [6,7], nutraceutical products, and cosmetics [8,9], has motivated scientists to undertake new research regarding the applications of mushroom-forming fungi. Some examples are enzyme production through solid or liquid fermentation [10,11], bioremediation of polluted environments [12], new composite biomaterial for green building [13], and medical and nutraceutical applications [5,9]. In a recent paper, an international group of researchers [14] further underlined how, due to the research carried out in recent years, fungi can play a central role in our lives and, if properly exploited, they can help address many of the challenges humans are likely to face in the future.

*P. ostreatus*, also known as the oyster mushroom, is one of the most used and affordable species of cultivated mushrooms globally [6]. Due to its economic, ecological, and medicinal values, *P. ostreatus* is widely cultivated. Several authors have highlighted its usefulness in bioremediation of petroleum and aromatic polycyclic hydrocarbons [15–17]. Furthermore, from a biochemical perspective, *P. ostreatus* is an excellent supplier of different nutrients and well-known molecules with beneficial effects, such as vitamins, amino acids, and essential fatty acids. The nutritional value of these compounds, and their numerous effects, such as anti-inflammatory and antioxidant properties, are well known [18–20]. Within the extensive literature on *P. ostreatus*, research about cultivation techniques and the effects of different substrates on the production of sporomata has shown how different agricultural waste can be used for sporomata production [21–26]. These findings confirmed the versatility of this mushroom and suggest the possibility of cultivating it on other types of agricultural waste from a circular perspective.

True lavender (*Lavandula angustifolia* Miller) and its cultivars are widely utilized in the essential oil industry. The natural range of *L. angustifolia* lies in the south of France, the Pyrenees (Spain) and, partially, the north-western Italian Riviera [27]. Today, in addition to France and Spain, Bulgaria, the United Kingdom, China, Ukraine, and Morocco are among the biggest global producers. Bulgaria, in particular, has recently become the world's leading producer, producing 100 tons of lavender oil per year. During the extraction process, the relatively low quantity of essential oil in fresh lavender (0.8–1.3%) results in enormous quantities of solid residues (tens of thousands of tons globally) that still have a high content of useful substances. These wastes are usually discarded directly in nearby locations or disposed of as special waste, leading to potential environmental issues.

In this work, within the framework of the European project ALCOTRA 1198 FINNOVER (http://www.interreg-finnover.com accessed on 3 May 2021), we hypothesized that *P. os-*

*treatus* can be cultivated on substrates enriched with waste derived from the extraction of lavender essential oils. The pilot tests were established at the Stalla Company, a small agricultural business in Liguria (north-western Italy). The company was founded in 1900 and in the past 20 years has cultivated and hybridized several species of flowers, including the *L. angustifolia* cultivar *imperia* used in this work. The activities were carried out in two pilot tests with the main goal of confirming the feasibility of cultivating *P. ostreatus* mushrooms on waste of the *L. angustifolia* cultivar *imperia* in a small local and rural business. The biochemical profile of sporomata grown on lavender-based substrates was analyzed through high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) to evaluate the variation due to the lavender enrichment. The biochemical data showed that the mushrooms grown on the enriched substrates have a high content of useful substances that can add value to the final product. The results of this work highlight the possibility of using lavender residues according to the circular economy principle for the production of mushrooms.

#### **2. Materials and Methods**

#### *2.1. Pilot Tests*

*P. ostreatus* was used in the tests because it has several interesting characteristics: it grows quickly, is relatively easy to manage, has good organoleptic characteristics, and has interesting, well-known medicinal properties. Moreover, it also grows spontaneously in many areas of Liguria and northern Italy.

In 2017, two strains of *P. ostreatus* called POA (autochthonous wild strain, isolated from a Ligurian locality in the province of Savona) and POC (domesticated strain) were isolated and tested for the pilot plant cultivation. The two strains were isolated under a laminar flow on Malt Extract Agar (MEA) and Potato Dextrose Agar (PDA) at 24 ◦C for 15 days. Once purified, the two strains were preserved on PDA slants at 6 ◦C in the ColD collection of the Laboratory of Mycology DiSTAV (University of Genoa). The two tests described started in December (2018 and 2019, respectively) with spawn production and incubation, and the cultivation phases were carried out in spring at the Stalla Company.

Substrates were prepared according to Yang et al. [23] and modified as follows. In December, plant residues of the *L. angustifolia* cultivar *imperia* derived from the extraction of lavender essential oil were properly shredded and mixed with barley straw for the preparation of growth substrates with different lavender/straw ratios (Table 1). The water content of the final mixed substrates was adjusted to 70%.


**Table 1.** Details of the different percentages of lavender present in the substrates.

The prepared substrates were placed in 25 × 45 cm thermoresistant polypropylene bags (600–700 g per bag), sealed, and autoclaved at 125 ◦C for 30 min. Once cooled, the substrates were inoculated under a laminar flow hood using 30 g of 7 day old cultures of the POA and POC strains grown on PDA. The spawn was incubated in the dark at 24 ◦C and 65–70% relative humidity (RH) for 50–60 days to allow the mycelium to colonize the entire substrate.

The pilot cultivation test was carried out in April at the Stalla Company. The pilot plant was set up in a company greenhouse adapted for cultivation. The 25 m<sup>2</sup> pilot area was set up using special sterilized plastic tarps. Cultivation benches (90 cm high) were set up in the pilot area, adjacent to a fog irrigation system to manage the RH during the cultivation phase. In addition, to prevent contamination, a small anteroom was set up to provide double protection against contaminants to the pilot area. Before starting cultivation, all surfaces were sanitized using a 20% hypochlorite solution. The spawn was placed in the pilot area: all bags were placed on the benches, spaced 20 cm apart from each other. To enable the formation of primordia, six holes of 2 cm in diameter were made using a sterile scalpel on each spawn bag. During the cultivation period, the fog irrigation system was used to keep the RH at about 80%; moreover, the irrigation contributed to control the temperature at around 22 ± 3 ◦C during the entire period of cultivation.

The basidiomata were harvested when the pileus margins were flat to slightly rolled upwards. They were counted, weighed, and preserved at −20 ◦C for the subsequent biochemical analysis.

The weight data of the collected basidiomata were processed using Microsoft Excel to calculate the mean, perform one-way analysis of variance (ANOVA), and create charts of production on the different substrates. In addition, the percentage yield on the different substrates (Y) was calculated using the following equation:

$$\Upsilon = \frac{\text{basidiomata fresh weight}}{\text{weight of substrate}} \times 100$$

#### *2.2. Sample Preparation and Biochemical Analysis*

The basidiomata were divided into aliquots of 2 g fresh weight each, taking care to eliminate growth substrate usually attached to hyphae. Each aliquot was placed within an Eppendorf tube. The samples obtained were dried using a Speed-Vac freeze dryer slightly heated to 35 ◦C to facilitate the evaporation of the water in the vegetation. The samples were finely pulverized in a mortar to obtain a greater contact surface with the solvent, placed inside heat-resistant glass vials with 2 mL of anhydrous ethanol, methanol, or acetonitrile next to a magnetic anchor, and the vials were hermetically closed. Water was not used as an extraction solvent because in samples rich in polymeric sugars such as those examined, there is a rehydration of the sample itself, which is not desirable because it prevents effective filtration and centrifugation. The sample was left for 2 h on a heating plate at 55 ◦C and then filtered through filter paper and transferred to settle in a test tube in a refrigerator for 30 min. This allowed agglutination of the residual excess carbohydrates, causing it to precipitate. A final centrifugation at 13,000 rpm for 10 min was used to obtain clear extract without solid residue.

The HPLC-ESI-MS analysis was performed using an Agilent 1100 chromatograph directly coupled with an MSD ion trap mass spectrometer. Chromatographic separation was conducted using a C-18 Symmetry column (Waters Corporations). The column was chosen considering the complexity of the matrix to be analyzed and the reproducibility of the method.

For the mobile phase, HPLC-grade acetonitrile (Merck, Darmstadt, Germany) and Milli-Q water (Millipore Corp., Bedford, Italy) were used, and were both filtered, degassed, and added, respectively, to 0.5 and 1% formic acid (Carlo Erba, Sabadell, Spain) to facilitate and improve ionization. SIAD (Bergamo, Italy) supplied the research-grade nitrogen (>99.995%). Absolute ethanol (Carlo Erba) was used for the extraction. The characterization of a given signal can be undertaken by analyzing the "full scan" and tandem spectra with Massbank EU, an exhaustive "open access" tool available on the Internet. The m/z ratio of the parent ion was entered and, to focus the search and exclude some substances, the m/z ratios of one or more fragment ions were also entered. For each search, a relative intensity normalized to 100 and an adequate tolerance regarding the accuracy and resolution of the instrument used was set. In our case, although the instrument used has an accuracy of 0.05 UMA when considering the m/z ratios, the tolerance was set at 0.3 UMA.

The main issue with this kind of sample is the dominant presence of long-chained polysaccharides, such as chitin and related polymers, whose behavior as "chemical sponges" can interfere with the optimal separation in HPLC and ionization in the mass spectrometer. To overcome these problems, great care was applied in the filtration and centrifugation

processes; these steps are essential to ensure the quality of the samples that are injected in the HPLC-MS system.

"

#### **3. Results**

#### *3.1. Pilot Tests*

"

The entire mushroom production process on substrates enriched with lavender waste was established to be cost effective and well integrated, with the lowest impact on the usual activities of the company. As shown in Figure 1, the mushroom production process began with the waste derived from essential oil extraction (center of Figure 1). After the mushroom production process, the exhausted substrate consisting of vegetable material partially degraded by the biological activity of the mushrooms was used within the company as a soil conditioner or to produce biocompost.

**Figure 1.** The entire circular process for mushroom cultivation. The yellow area concerns the cultivation of lavender and the essential oil production process. The green area represents the mushroom production process and the valorization of the spent substrates for biocompost production.

In 2018 (the first pilot test), during the incubation phase, 97% of the inoculated bags completed the incubation phase, whereas 3% were contaminated by a common microfungus of the genus *Trichoderma*. In 2019 (the second pilot test), all of the incubated bags reached the optimal colonization. The incubation time required to achieve complete colonization of the substrates was 40 ± 5 days in 2018 and 47 ± 7 in 2019. The substrates consisting of 100% lavender residues needed 12 ± 8 days longer in 2018 and 15 ± 7 days longer in 2019 to complete colonization compared with the control substrate. There were no significant differences between the two strains used regarding growth. During the cultivation phase, 90 and 95% of the bags in 2018 and 2019, respectively, reached the production of primordia and developed fruit bodies.

The weight of the basidiomata grown on the different substrates in 2018 and 2019 is shown in Table 2. The one-way ANOVA confirmed that the substrate composition affected the weight (*p* < 0.01), but there was no difference in the growth between the two strains tested.


**Table 2.** Mushroom production (expressed in grams) on the different substrates tested.

As shown in Figure 2, the percentage yield (Y) for both pilot tests showed a lower production efficiency for the substrate containing high lavender waste concentrations compared with the control (100% straw), which had a Y between 27.4 and 29.8. The substrate with 100% lavender had a Y between 15.2 and 16.9, the substrate with 50% lavender had a Y between 17.9 and 22.6, and the substrate with 40% lavender had a Y between 18.2 and 24.2. The Y for the substrate containing 30% lavender—between 24.4 and 26.4—was closest to the control substrate. — —

**Figure 2.** Production yield expressed as the percentage of the two strains grown on different substrates (each color refers to different straw/lavender ratios: blue = 100/0; red = 0/100; green = 50/50; violet = 60/40; light blue = 70/30) containing different concentrations of lavender waste in 2018 and 2019.

#### *3.2. Biochemical Analysis*

The analysis of sporomata extracts grown on the substrates with lavender showed that an average mass of extract between 1.6- and 2.1-fold higher in weight was produced compared with the sporomata grown on the control substrate. In addition, domesticated sporomata grown on the control substrate tended to produce fewer metabolites than the wild strain. The analysis was performed using multiple samples of mushrooms grown under the same conditions. This examination demonstrated the repeatability of the extraction and analytical methods. As shown in Figures 3 and 4, the chromatograms of the extracted samples overlap perfectly.

**Figure 3.** A comparison between samples of the same strain grown under the same conditions and extracted in ethanol. The perfect overlap of the different extracts confirms the reliability and reproducibility of the extraction and analytical methods.

**Figure 4.** A comparison between samples of the same strain grown under the same conditions and extracted in acetonitrile.

This method isolated and identified >50 molecules belonging to numerous chemical families, such as di-tripeptides, fatty acids, and their epoxides. There were no major differences between the domesticated and wild strains regarding production of biomass and metabolites, although there were differences in the metabolite profile of *P. ostreatus* harvested on only straw compared with the enriched substrates. As shown in Table 3, the

HPLC-MS method allowed dividing the various classes of compounds into five groups relative to their retention time within the chromatographic analysis. The obtained separation could be very useful because it is predictive of the biological activities that each fraction, or specific compounds, from *P. ostreatus* extracts may have for different applications, including for human health.

**Table 3.** Classification of the isolated and identified compounds relative to the percentage of eluent B and their retention time in high-performance liquid chromatography.


#### **4. Discussion**

The two pilot tests carried out demonstrated the application of mushroom cultivation on a lavender farm following the circular economy principle. This process, as already proposed by other authors, enables farmers to reuse the agricultural waste produced during the main production cycle to obtain a new product [11,20,26]. The particular substrate leads to distinct metabolomic profiles, a phenomenon that improves the commercial value compared with mushrooms grown on traditional substrates. This difference compensates for the lower yield of mushrooms. These data are consistent with the feasibility study carried out as part of the FINNOVER project (www.interreg-finnover.com accessed on 3 May 2021). The vegetal waste partially inhibited the mycelial development, increasing the incubation time when grown on the substate with lavender, and, later, the development of basidiomata (lowering the production yield compared with the control). Based on the average yields reported in Table 1, there were significant differences in the weight of basidiomata produced on the substrates tested (*p* < 0.001), but there were no differences in the biomass of the produced sporomata between the two strains tested. This result was further confirmed by calculating the production efficiency index Y, as shown in Figure 2. These calculations revealed that the yields on a substrate composed of 100% of lavender residue were lower than those on the control substrate composed exclusively of straw. These differences in biomass production suggest the use of lavender residues to grow mushrooms may be unsuitable because, as shown in Figure 2, the Y value is inversely proportional to the lavender waste content in the substrate. However, as discussed below, the metabolomic characteristics and added nutraceutical value provided by the lavender to the cultivated basidiomata could compensate for a small drop in production. Because the substrate containing 30% lavender residue had slightly lower yields (ranging from 1 to −5.4%) than those observed in samples without lavender residue, this substrate may provide an appropriate balance between biomass production and improved metabolomic profile.

*P. ostreatus* is a well-known food that is rich in essential dietary elements [28,29]. Specifically, we found that the wild strain is a slightly better producer of metabolites in response to the presence of an environmental substrate than the domesticated strain, if dried extracts and just secondary metabolites are considered. This variable production probably depends on the fact that the domesticated strain, which has been cultivated for

generations in a protected and controlled environment through cloning of the mycelium—a type of asexual reproduction that does not allow genetic mixing—depresses or silences in successive generations various secondary metabolic pathways that are no longer useful for maintaining physiological activity. These metabolic pathways are not silenced in the wild strain, because in nature it is in direct contact with environmental stressors, and direct competitors and pathogens. The substrate enriched with lavender, a plant particularly rich in terpenes and other molecules it produces as antibacterial and antifungal agents, stimulates the fungus to activate its secondary metabolism, as evidenced by the presence of compounds that are not part of the fungus's usual primary metabolomic pattern. Although weight data from the different growth phases of the fungus showed that the fungus grown on straw generally produced greater biomass, HPLC-MS and tandem mass analysis showed that the levels of *n*-acetylglucosamine and its precursors remained constant in each sample, independently of the type of substrate used for growth.

Of the many molecules extracted and characterized, significant interest exists in the amino acids, glucosides, and small dipeptides, that are particularly known in the fungi kingdom. There are myriad roles for these peptides and their derivatives; it has been shown that these peptides have antifungal, antimicrobial, immunostimulant, and growthpromoting properties [30]. The evolution of these peptides can be seen in the large cyclized peptide molecules produced by some fungi, such as amatoxins of the *Amanita* genus, a clear evolutionary trend that leads from simple molecules to complex cycled peptides with defensive functions. Amanitin toxins, in general, are among the most effective toxins produced by superior fungi, with a clear defensive role. Therefore, the condition by which peptides and dipeptides are produced in *P. ostreatus* appears to be archaic or, at least, less evolved [30]. Of significant interest is the notable amount of glutathione in the samples. Its action is highly relevant regarding free radicals and peroxide ions, and largely justifies the powerful antioxidant action of *P. ostreatus* extracts analyzed on cell cultures treated with oxidizing agents (personal data).

Of note is the presence of fatty acid epoxides, particularly those of myristoleic, linoleic, and linolenic acids produced by the fungus grown on lavender-enriched substrates. Fungi are an excellent source of polyunsaturated fatty acids, whose role has long been recognized in the prevention of inflammatory heart and other diseases [31]. Moreover, it is evident that a balanced intake of polyunsaturated fatty acids is fundamental for human health. We also found a notable share of palmitic acid, a saturated fatty acid. The samples of *P. ostreatus* grown on substrate enriched with lavender also showed the presence of numerous molecules derived from fatty acids, in particular a series of fatty acid epoxides. These molecules are produced by the fungus in response to environmental stresses through the activation of the monooxygenase domain of cytochrome P450, which is common to all fungi and has been highly preserved during evolution [32]. In particular for linoleic and linolenic acid, in the samples cultivated on substrates enriched with lavender, we noticed a partial decrease in the content of polyunsaturated fatty acids in favor of the presence of fatty acid epoxides. This trend was also confirmed by the presence of cholanic acid in the samples of fungi cultivated on substrates enriched with lavender. These molecules of steroid origin are similar to those found in human bile salts and mammals in general. These molecules are probably involved in the fatty acid mobilization inside fungal cells, consistent with the presence of lipid drops in the fungal cells. The presence of fatty acid epoxides in fungi is interesting. It is known that fatty acids often have cytotoxic activity or otherwise are harmful to health [33]. It should be noted that epoxides of fatty acids were only present in fungi cultivated on enriched substrates, and were absent or present in trace amounts in fungi grown on only straw, confirming that lavender acts as a stressor, and suggesting the role of fatty acids epoxides as biomarkers of stress in Basidiomycota. In contrast to plants, in which the role of peroxygenases in the production of fatty acid epoxides is known, in fungi it appears that only the monooxygenase domain of cytochrome P450 is involved in their synthesis [34]. Nevertheless, the role of fatty acid epoxides in the regulation and suppression of inflammatory processes has been recognized [35]. Several

toxic and tumor growth-promoting activities are known [36], so further investigation to verify the activity of fatty acid epoxides produced by *P. ostreatus* would be useful.

#### **5. Practical Implications of This Study**

The pilot tests conducted allowed us to evaluate the technical feasibility of exploiting the cultivation of mushrooms to recycle agricultural residues in small rural farms. The results confirmed the feasibility of this approach, but showed a lower production yield compared with the standard substrate in optimal conditions. The reasons for this reduced efficiency are due to the different substrate and to the fact that the Stalla Company, which produces lavender essential oil, is not specialized in the production of mushrooms. Although the yields were low, a positive aspect that emerged from these tests is the presence of interesting substances within the basidiomata. This positive effect, due to the different substrates, should be investigated because it could add value to the product and make it extremely profitable for the farmers. Future tests will contribute to optimizing and improving the efficiency of the process, and to better understand how different residues can influence the biochemical composition of the fungi produced.

#### **6. Conclusions**

The pilot tests carried out in this work showed that mushrooms can be fruitfully exploited in the agricultural circular economy. In particular, we found that a lavenderbased waste product could be recycled, resulting in interesting characteristics from a food that is currently commercially available, making it a niche product and a potential novel food. The same model could be tested using different residues and fungi. By evaluating different combinations, other interesting products could emerge that may be exploited in different rural contexts. The results obtained in this work should spur further research on this topic.

**Author Contributions:** Conceptualization, S.D.P., M.B., G.D., G.C., M.G.M., M.Z.; methodology, S.D.P., M.B., G.D., M.Z.; validation M.Z. and G.D.; formal analysis; investigation S.D.P., M.B.; data curation, S.D.P., M.B., G.C.; writing—original draft preparation, S.D.P., M.B., writing—review and editing, S.D.P., M.B., G.D., G.C., M.G.M., M.Z.; supervision, G.D. and M.Z.; project administration, M.Z.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a Interreg-Alcotra European Union project called FINNOVER (n◦ 1198), http://www.interreg-finnover.com/ accessed on 3 May 2021.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge Franco Stalla for the technical work during the pilot tests.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Biodegradation of Hemicellulose-Cellulose-Starch-Based Bioplastics and Microbial Polyesters**

**Mateus Manabu Abe <sup>1</sup> , Marcia Cristina Branciforti <sup>2</sup> and Michel Brienzo 1,\***


**\*** Correspondence: michel.brienzo@unesp.br

**Abstract:** The volume of discarded solid wastes, especially plastic, which accumulates in large quantities in different environments, has substantially increased. Population growth and the consumption pattern of societies associated with unsustainable production routes have caused the pollution level to increase. Therefore, the development of materials that help mitigate the impacts of plastics is fundamental. However, bioplastics can result in a misunderstanding about their properties and environmental impacts, as well as incorrect management of their final disposition, from misidentifications and classifications. This chapter addresses the aspects and factors surrounding the biodegradation of bioplastics from natural (plant biomass (starch, lignin, cellulose, hemicellulose, and starch) and bacterial polyester polymers. Therefore, the biodegradation of bioplastics is a factor that must be studied, because due to the increase in the production of different bioplastics, they may present differences in the decomposition rates.

**Keywords:** biodegradation; bioplastics; lignocellulosic fibers; microbial polyesters

#### **1. Introduction**

Consumption demands for industrialized materials such as plastics in their various applications have increased over the past years. This consumption is generating residues, which require alternatives for their proper disposal and recycling. Disposal, recycling, and plastic substitution are potential research areas towards urgent and necessary solutions. Most commercial plastics come from the petrochemical industry, which uses natural gas and fossil hydrocarbons as feedstock. Such synthetic plastics are biodegradable and degradable only for a long period. Therefore, they are considered neither biodegradable nor renewable [1]. Synthetic polymers, such as polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), nylon, polyester (PS), and epoxy are examples of plastic components of high resistivity, chemical and biological inertness, resistance, flexibility, and other interesting properties [2–5].

At the beginning of the large-scale production of synthetic plastic materials, their properties seemed adequate for good quality development. However, such materials are non-biodegradable, thus generating large accumulations of residues in different landscapes. Thus, they have been a cause of growing concerns due to environmental problems. New materials based on biological sources have been developed towards solving or reducing the above-mentioned problems. However, in addition to be renewable and biodegradable, bioplastics must have vapors barrier properties and mechanical properties that meet the different applications of this material, and the attention has now evolved towards the possible ecotoxic effects of bioplastics and active properties for a cover of food.

The names of biodegradable and/or bioplastic products given by companies and reported in the literature, when drawn up wrongly, can lead to misunderstandings by the general public due to incorrect classifications of the polymeric materials [6–9]. A bioplastic

**Citation:** Abe, M.M.; Branciforti, M.C.; Brienzo, M. Biodegradation of Hemicellulose-Cellulose-Starch-Based Bioplastics and Microbial Polyesters. *Recycling* **2021**, *6*, 22. https://doi.org/10.3390/ recycling6010022

Received: 28 January 2021 Accepted: 11 March 2021 Published: 22 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

can be biodegradable or not. However, a biodegradable material does not necessarily come from a biological source. Towards the avoidance of errors, the following definitions, reported in this article, must be clarified:


Different biotic and abiotic factors contribute to the different degradation processes [11]. Thermal, mechanical, and chemical degradation, as well as photodegradation, are examples of abiotic degradation. A degradation process is related to the fragmentation of material into small elements or molecules, or just physical and chemical changes in a polymer. Due to high temperatures, polymers can be thermally degraded. The chemical bonds in their chains are broken by a thermo-degradation effect [12].

Mechanical degradation is an abiotic degradation mechanism that occurs through shear forces (due to aging, turbulence in water and air, snow pressure, and other factors), tension and/or compression. Under environmental conditions, it acts synergistically with different abiotic factors [13].

Abiotic chemical degradation occurs by the degradative effect of chemicals substances, and represent one of the most important mechanisms of abiotic degradation, since the polymer matrix is affected by atmospheric or agrochemical pollutants, such as oxygen (i.e., O<sup>2</sup> or O3), which produce free radicals through oxidation, attacking covalent bonds [13]. Abiotic chemical degradation differs from biotic chemical degradation, mainly regarding the origin of the chemical with a degrading effect.

Photodegradation is the process of degradation of polymers by the action of light, resulting in the oxidation of the material. UV rays interact with chromophores groups of polymers (carbonyl, hydroxyls, and aldehydes), which are degraded by chain fission, photoionization, crosslinking, and oxidation reaction [11,13–15].

Microbial biodegradation is a degradation process of polymers and other materials through the action of microorganisms [11] resulting in CO<sup>2</sup> and/or methane, water, cell biomass, and energy. However, in the natural environment and even in the process of controlled biodegradation, abiotic effects help or even occur synergistically with biodegradation. This consideration of synergism is important for the elaboration of biodegradation procedures.

With environmental concern, this review evaluated the biodegradation process (considering the synergistic action of biotic and abiotic agents) of bioplastics elaborated with polysaccharides from plant biomass and microbial polyesters. Moreover, addressing the definitions, biodegradation mechanism, and factors that affect the biodegradative process of bioplastics. The scope of this review does not address the biodegradation of bioplastics produced from polymers of animal origin (natural polymer), and bioplastics derived from petroleum (PBAT, PBS, PVA, PCL, and PGA [16]. However, the definitions presented in this review do not exclude these types of bioplastics.

#### **2. Problems Related to Plastics**

The current geological era, the so-called Anthropocene, is exposed to the influence of human actions in different environments. Indicators from such anthropic actions are biodiversity reduction, deforestation, climate, and other environmental changes [17]. However, materials produced by human society, like plastics, are also indicators of the Anthropocene. Plastics directly (i.e., environmental impacts from the plastic production chain) affect different environments (e.g., terrestrial and marine).

An environment in which plastic waste currently generates several problems, is the oceans, due to the large accumulation of these materials. The plastic that reaches the oceans mostly is generated in coastal population regions, where the disposal and management of this waste is destined for uncontrolled landfills [18]. Due to urban runoff and inland waterways, such plastics reach oceans and are transported via tide and winds. It is estimated that between 4.8 and 12.7 million metric tons of plastics produced in the continent (distribution varies according to the analyzed location) reached the marine environment in 2010 [18].

In recent years, environmental concerns (e.g., harmful effects of plastics on the environment, since they are not biodegradable [19], or slowly degraded) have been intensified. Large accumulations of floating plastics in the oceans have been reported- approximately 1.8 trillion pieces of plastic have been quantified in the Great Pacific Garbage Patch (GPGP) [20]. The ingestion of plastic fragments by the marine fauna is a major concern due to their small size [18,21,22], the so-called microplastics, which are smaller than 5 mm [21]. Besides, since plastic fragments are present on the surface and floor of oceans, as well as in several maritime regions (coastal areas) and the Arctic sea ice, strategies, as a reduction in inputs [18] and the elaboration/utilization of biodegradable materials, would be adequate measures to reduce the impacts of plastics.

Even with the area of studies on the impacts of plastics on fauna and for the various organisms still under development, some studies point to the occurrence of toxicological effects of this synthetic waste [23,24]. A plastic intake and entanglement can lead to the lower life quality of organisms, loss of mobility, external and internal injuries, blockage of digestion, and other harms [25]. Goldstein and Goodwin [22] identified the presence of microplastics (mainly PE, PS, and PP) in the digestive tract of 33.5% of *Gooseneck crustaceans* (*Lepas* spp.).

The development of innovative technologies represents a means for both sustainable development and the growth of emerging countries, such as Brazil, whose sustainable energy has been highlighted by innovation technologies. Thus, even with bioplastic not representing a material for total replacement of non-biodegradable plastic, researches and production of bioplastics are a technological alternative for the development of a more sustainable and balanced society. Therefore, aligning with the current trend (socio-political and environmental), in which concerns with the environment is growing.

#### **3. Biodegradation Process**

The microbial biodegradation of materials occurs by the action of microorganisms, such as fungi and bacteria [26], and is classified as physical, chemical, and enzymatic according to modifications in the materials. Biodegradation is a natural process of vital importance for nutrients and energy recycling [27]. Microorganisms use organic material as a source of nutrition for their metabolism; except for the substances used in metabolic incorporation, the rest is oxidized by cellular respiration, thus leading to the formation of simple and small submetabolites, released in the environment [28,29].

Biodegradation due to physical degradation occurs from the adhesion of microorganism species to the surface of organic materials through the secretion of a gum [30] produced by microorganisms. This gum represents a complex matrix made of natural polymers (e.g., polysaccharides and proteins). Such a thick complex, together with microorganisms, infiltrates the material and changes its volume, size, pores distribution, moisture content, and thermal transfers. A few microorganisms (e.g., filamentous fungi) lead to cracks in the materials due to mycelial growth, i.e., both their durability and resistance properties are reduced [13,31]. Microorganism biofilms are a matrix that protects microorganisms from different environmental conditions and results in a major change in materials [13,32].

Biodegradation by chemical degradation refers to the production of chemical substances by living organisms, which facilitate and increase the speed of the process. Emulsifying substances produced by microorganisms help the exchange between hydrophobic and hydrophilic phases, which are important interactions for the penetration of microorganisms in the polymeric material [33]. Such a lime formation (polymers secreted by microorganisms mixed with different microbial species) improves the material deterioration. It represents a point of accumulation of polluting and chemical substances (abiotic chemical degradation), thus benefitting microbial proliferation [34].

Examples of chemical substances released into the environment by microorganisms, which play an important role in chemical biodegradation, are nitrous acid, nitric acid, and sulfuric acid. All of these compounds are produced by chemolithotrophic bacteria, such as *Nitrosomonas* spp., *Nitrobacter* spp., and *Thiobacillus* spp., respectively [29,33,35]. Apart from the action of chemical substances generated by those organisms, chemoorganotrophic microorganisms generate organic acids with potential for chemical degradation (e.g., oxalic, citric, gluconic, glutaric, glyoxalic, oxaloacetic, and fumaric acids).

The action mechanisms of such acids (organic or inorganic) are diverse and include an increase in surface erosion when adhering to the material surface [36]. The use of those acids as nutrients benefits the growth of filamentous fungi and bacteria [13]. Another action mechanism of biotic chemical degradation is the oxidation of organic material. Certain fungi and bacteria have specific proteins in their membrane that capture iron-chelating compounds (siderophores) [37]. With this mechanism microorganisms capture cations from a matrix.

Biodegradation by enzymatic degradation occurs due to the depolymerization of polymeric chains of a matrix through the action of hydrolase enzymes that catalyze the reactions of chemical bonds breakage adding a water molecule. These bonds are ether, peptide-like, and ester, present in biodegradable bioplastics. The main enzymes are amylases and cellulases, which cleave starch and cellulose polymers, respectively. However, other enzymes (breakage of ester bonds), such as esterases and lipases, can degrade co-polyesters.

A mechanism that explains the action of hydrolases (e.g., depolymerase) in polyesters hydrolysis (synthetic and natural) through biodegradation is related to three amino acids, namely serine, histidine, and aspartate. A hydrogen bond is formed when a component reacts with the histidine ring, thus guiding interaction between histidine and serine, and forming an alcohol group of high nucleophilic character (-O). Histidine plays a deprotonating role for serine, i.e., as a base. The alkoxide group includes an ester bond and generates an acyl-enzyme and an alcohol group. Finally, a free enzyme and a terminal carboxyl group are generated by the action of the water molecule under an acyl-enzyme. This entire enzymatic degradation process is termed catalytic triad [30,38,39], and the products generated are metabolized or not by microorganisms that have depolymerizing enzymes. Therefore, a consortium of microorganisms is important for complete biodegradation [13]. Figure 1 depicts the mechanism of action of depolymerizes and the catalytic triad.

Apart from the biodegradation of cellulose, starch, and polyesters, hemicellulose is another polymer that can be degraded by microbial enzymes. A catalytic action of hemicellulases (enzymatic pool) on different types of hemicellulose polysaccharides produces monomeric sugars, acetic acids [40]. For example, enzymes that degrade xylan (hemicellulose from grasses) are endo-1,4-β-xylanase (cleavage results in oligosaccharides), xylan 1,4-β-xylosidase (cleavage of oligosaccharides generate by xylan, which forms xylose monomers), and accessory enzymes, such as xylan-esterases, ferulic and p-coumaricesterases, α-Ï-arabinofuranosidases, and α-4-*O*-methyl glucuronidase [41]. Both enzymes act synergistically so that xylans and hemicellulose mannans of some types of plant cell walls are depolymerized [42]. Nevertheless, some polymers are not biodegraded by common enzymatic hydrolysis, i.e., polymers can be oxidized by enzymes such as laccase, dioxygenase, peroxides, monooxygenase, and oxidases [43]. Thus, such enzymes are not hydrolases and influence the cleavage process of polymers differently from hydrolases (oxygen insertion, hydroxylation, oxidation, and free radical formation lead to polymer cleavage) [43]. Figure 2 depicts the enzymatic biodegradation process.

**Figure 1.** Enzymatic hydrolysis of polymers and catalytic site of depolymerase enzymes [13].

β β α α The result of biodegradation, for example of bioplastic from natural polymers (e.g., polysaccharides) is the generation of small molecules from a polymer. Microorganisms cannot employ large substances insoluble in water for obtaining organic or inorganic nutrients for their metabolism. They produce enzymes and chemicals used in extracellular environments and, therefore, depolymerize the materials. After hydrolysis and/or oxidative action of microorganism enzymes on different polymers, which results in monomers, metabolism oxidation occurs. In this system, organic compounds lead to a loss of electrons and the consequent production of ATP molecule (adenosine triphosphate). This is the last biodegradation stage, in which organic matter is mineralized. The microorganisms use smaller and simple organic molecules, such as oligomers and monomers, for their metabolic activities. However, byproducts are generated from microbial metabolism (e.g., carbon dioxide—aerobic degradation), water, biomass, methane, and hydrogen sulfide (anaerobic degradation) [45,46]. Figure 3 displays the biotic and abiotic degradation of plastic.

**Figure 2.** Enzymatic biodegradation process [44].

**Figure 3.** Disintegration, biodegradation, and mineralization process of plastic polymeric materials. Adapted [47].

#### *3.1. Factors That Influence Biodegradation*

The microbial population available is a key factor for biodegradation in an environment (soil, air, and water), and several properties (e.g., the chemical constitution of materials) affects the efficiency of the biodegradation process. Chemical composition influences the biodegradation of plastics through different patterns of crystallinity, hydrophilic and hydrophobic character, conformational flexibility, polymer accessibility, surface area, molecular weight, melting temperature, hydrolyzable and oxidizable bonds in polymer chains, morphology, and stereoconfiguration [27,48,49].

Crystallinity influences biodegradability because it affects the accessibility of the enzyme to the material polymer. More organized regions of polymers (crystalline) tend to hinder enzymatic hydrolysis since catalytic proteins diffuse with greater difficulty. On the other hand, water molecules diffuse more easily between amorphous (less organized) regions, and enzymes can easily access the material polymers in such regions [28].

The polarity of bioplastics directly influences biodegradation, since materials developed with hydrophobic polymers are less susceptible to enzymatic attack. Degrading microorganisms depend on a hydrophilic surface to adhere to and catalyze the depolymerization reaction by means of hydrolytic enzymes. However, this enzymatic accessibility to the material is reduced on hydrophobic polymeric surfaces. This impediment occurs not only because the microorganisms and enzymes are more hydrophilic, but also due to the aqueous medium (usual water), in which the enzyme is contained, to have their contact with the material (bioplastic) reduced. For example, glycolic polyacids (PGA) are more easily biodegraded than poly (lactic acids) (PLA), since PLA is more hydrophobic) [50].

Blends in a polymeric bioplastic matrix are common when it is desired to obtain materials with certain characteristics, and also interfere with biodegradation (increase or reduce biodegradation), since the different components of biocomposites can influence the accessibility of the enzyme to the polymeric material in different ways.

The molecular weight of polymers affects the biodegradability of plastics, since the heavier the molecular weight, the greater the difficulty for microorganisms to break it down and assimilate. Therefore, the lower the molecular weight of the polymer, the easier the biodegradation, since the need for extracorporeal digestion is reduced. Aliphatic polyester is one of the few biodegradable polymers of high molecular weight [13]. However, it is worth mentioning that in addition to the molecular weight, the types of bonds in the polymeric chain (considering that bioplastic, like plastic, is formed by a polymeric matrix), and different chemical groups in polymers influence the biodegradation process.

Although the term "bio" degradation is directly correlated with the fragmentation of a polymer by the action of microorganisms, these microorganisms do not act in isolation on the polymeric material, since abiotic agents influence the fragmentation efficiency. The abiotic degradation of organic matter such as thermal, mechanical, chemical, and by the action of light are examples of degradative processes. These processes work synergistically with biodegradation, reducing the material to dimensions that allow microbial assimilation [13,51].

#### *3.2. Assessment and Biodegradation Quantification*

Biodegradation can be measured through metabolic products, physical and chemical properties of plastics/bioplastic, acidification of the medium, and other ways. CO<sup>2</sup> is a product of biodegradation, more specifically, of the oxidation of organic matter, and can be used for direct or indirect measurement of material biodegradation over a period of time. Its content released in a degradation process is quantified by the respirometry technique, which can use a closed CO<sup>2</sup> production and a capture system. International methods, such as ASTM D5338-15 [52] and ISO 14855-2: 2018 [53] are applied for the quantification of the CO<sup>2</sup> produced in a microbial degradation process.

The measurement of consumed oxygen (ISO 17556: 2003) [54] is another method of quantifying biodegradation by respirometry. Respirometry involves techniques that measure parameters indicative of cellular respiration. The higher the consumption of oxygen and the release of CO<sup>2</sup> by microorganisms, the better the biodegradation indicator. For details and examples of other standard methods of respirometry analysis (ASTM, EN, and ISO), see specialized literature [55].

Methane molecules can also be used for measurements of materials biodegradation. However, unlike the above-mentioned respirometry techniques, CH4, CO2, and other gases quantification is generally conducted under anaerobic conditions. Analysis methods such as ASTM D5511-02 [56], is used for this purpose.

Apart from microbial proliferation in plastic/bioplastics materials, analyses of color change, surface roughness, cracks, and holes are also alternatives for checking the deterioration of materials [13,36]. Analysis parameters can be used especially for materials of difficult biodegradation and low CO<sup>2</sup> release. However, the results of such analyses (e.g., microbial growth in the polymeric matrix) are not recommended for the conclusion of biodegradation or abiotic degradation directly [13]. Additional techniques, such as electron microscopy, photon microscopy, microscopy of polarization, and atomic force microscopy reinforce the results [13,57,58].

The physical properties of plastics/bioplastics (e.g., tensile strength, elongation at break, modulus of elasticity, crystallinity, cold crystallization temperature, and glass transition temperature) can be measured as biodegradation indicators. The weight loss of a sample determined by the burial method can be used in plastic/bioplastic biodegradation analyses, although it may result from the solubility and volatility of certain substances [13]. The analysis of weight loss of bioplastics by burying in soil, or composting systems, may result in conclusion errors, since in addition to the mass of the soil or compost account for the variation in the bioplastic mass, in bioplastics washing processes (a step which precedes weighing procedures), can cause fragmentation and loss of material derived from bioplastic. Thus, even though the method of analyzing mass loss is frequently reported in the literature, as is usual in determining the biodegradation of bioplastics, this technique ends up being difficult to perform [59]. Recent articles evaluating the biodegradation of bioplastics by burying in soil and compost has used image evaluation as a tool for analysis, that is, the reduction of the area of bioplastics, detected by image registration (from the insertion of the bioplastic in a mold/grid with known dimensions) [60,61].

The indication of biodegradation through products generated by microorganisms is another way of measuring the process. For example, the biodegradation of polymeric cellulose materials can be measured according to the release of glucose [62], or the quantification of 1,4-butanediol as an indicator of the biodegradation of PBA and PBS polymers [63].

The increase in microbial biomass (weight or number of cells) is indicative of a biodegradation process since a single source of carbon (plastic or bioplastic material) in a closed environment can point out the occurrence of biodegradation and/or surface changes and molecular rearrangements. However, conclusive statements about the amount of mineralized material cannot be directly made.

The evaluation and quantification of bioplastic and/or plastic biodegradation by the above-mentioned methods can be conducted in an aqueous medium and soil. However, each condition of analysis imposes different requirements, which leads to different responses from different methods.

#### *3.3. Biodegradation of Bio-Based Polymers Bioplastics*

In this topic, biodegradation of bioplastics developed with polysaccharides from plant biomass/lignocellulose and microbial polyesters was followed as the scope of this review. It was exemplified the biodegradation of a category of bioplastics, those developed with natural polymers (vegetable and microbial). Therefore, this review does not intend to address issues related to the development of bioplastics of vegetable and microbial origin, advantages and disadvantages in addition to the viability of this material (related to the economic aspects and properties of bioplastics). To obtain this information, it is recommended reading of the specialized literature [6,64–66].

#### 3.3.1. Biodegradation of Plant-Based Polymers Bioplastics

The mass loss of bioplastics from rice straw showed complete degradation after 105 days [67]. Rice straw bioplastics were composed mainly of cellulose and trifluoroacetic acid. On the first day of contact with the soil, the bioplastic showed an increase in mass, due to the phenomenon of water absorption by the material. According to the authors, its mechanical properties are similar to those of polystyrene (bioplastic in the dry state).

The mass loss of bioplastics consisting of acetylated starch and acetylated sugarcane fibers (lignin, hemicellulose, and cellulose) resulted in 24.2 to 39.3% degradation after 5 weeks [68]. The acetyl group may have created stable biodegradable sites; however, an increasing effect on the crystallinity of the bioplastic with the addition of cellulose may have contributed to the low biodegradation rate due to the restriction effect of the microbial enzyme's activity. In addition to the crystallinity and chemical structure of cellulose, microbial diversity, carbon availability and the period of biodegradation considered can influence its depolymerization.

Bioplastics (glycerol, acetylated starch, and acetylated nanocellulose composition) subjected to biodegradation in a petri dish with *Trametes versicolor* were completely degraded in 60 days, and after 40 days with starch and non-acetylated reinforcement. The starch bioplastics were completely biodegraded after 30 days, and the addition of cellulose to the formulation of bio-based plastics resulted in a longer biodegradation time [69]. Water and moisture absorption is important in the biodegradation process of bioplastics [70]. The starch-based bioplastics investigated in this study were composed of different concentrations of oxidation starch (20, 40, and 60%). Oxidation decreases biodegradation due to reduced swelling and water absorption from the soil by bioplastic.

Hemicellulose is another natural plant-derived polymer of potential application for the development of bioplastics. However, in addition to the elaboration that biomaterial, the study of the biodegradation of these carbohydrates in bioplastics have not received attention, as the area of use of hemicellulose for bioplastic focus on physicochemical properties and modifications of this macromolecule. The bioplastic based on xylan (of the hemicellulose type of grasses) and blended with gelatine was completely biodegraded after 15 days of conditioning (determined by the burial procedure) [71]. This bioplastic was considered 100% biodegradable since the sample could not be recovered for weighing. A bioplastic made with 50% xylan (from beechwood) and PVA (polyvinyl alcohol) was 56% biodegraded after 30 days by burial in soil [72]. PVA reduced the biodegradation of the bioplastic produced by the PVA/xylan mixture. The sample with 25% xylan was 42.2% biodegraded after 30 days of burial in soil.

Xylan was grafted with poly-(ε-caprolactone) (PCL), and biodegradation was evaluated by BOD (biological oxygen demand). The biodegradation (aerobic and activated sludge) kinetics of bioplastics with high concentrations of PCL was delayed in comparison to materials made with pure hemicellulose or with lower graft concentrations [66]. Despite changes in the kinetics, the biodegradation property of the bioplastic was not altered and ranged between 95.3 and 99.7%.

Recalcitrant substances also influence the biodegradation of natural polymers. Lignin is a constituent of lignocellulosic fibers, shows the highest degree of recalcitrance in the plant cell wall [73,74]. This polymeric complex of phenylpropane units hinders the biodegradation of the material or products that contain it, such as bioplastics, and reduces the contact surface of lignocellulosic fibers with degrading enzymes [74]. Lignin requires different enzymes to degrade due to the different units that comprise its polymeric complex [75]. In anaerobic environments lignin may persist biodegradation for a longer time, with this process is primarily more efficient in aerobic environments [76], due to the catalytic action involved in oxygen.

Starch and lignin (lignosulfonate) bioplastics were completely biodegraded after 4-month burial [77]. Biodegradation was measured through the analysis of CO<sup>2</sup> and morphological characteristics. The samples with lignin analyzed after 5 weeks of biodegradation tests were fragmented, however, small residues of the bioplastic were identified. After the 2-month burial, the samples with lignin showed a significant biodegradation effect, with small fragments of the material still observed. After 4 months of testing, residues of bioplastic fragments were no longer detected. A bioplastic made from the addition of lignin (1.2% *w*/*t*) to the bio-PTT matrix (Bio-poly (trimethylene terephthalate)) increased its weight loss through biodegradation in soil [78]. In 140-day burial, bio-PTT/lignin bioplastic showed more than 50% mass weight loss.

A higher CO<sup>2</sup> emission was reported from films with lignin in comparison to the bioplastic composed only of starch, due to the greater amount of carbon atoms in its formulation [77]. However, such CO<sup>2</sup> may have originated from the metabolism of soil organic compounds, i.e., the bioplastic may have stimulated the microbial degradation of stable organic compounds in the soil through the priming effect. A strategy for the biodegradation of bioplastics composed of lignin, due to the recalcitrance of this phenolic complex, is the application of UV radiation prior to chemical, microbiological and/or enzymatic treatments. Lignin is susceptible to photodegradation due to the UV effect [79]. After photodegradation, other treatment combinations can be applied for the degradation or biodegradation of lignocellulosic fibers, such as enzymatic or oxidative treatments. One of the advantages of using lignin in the development of thermoplastic formulations is its processing at high temperatures [80]. However, studies using lignin in the bioplastic formulation, have not received much attention.

3.3.2. Biodegradation/Enzymatic Degradation of Plant-Based Polymers Bioplastics in Relation to Derivatization

The assessment of biodegradation, disintegration, and enzymatic degradation of bioplastics made with natural polymers (such as proteins, starch, cellulose, and hemicellulose) is not recurrent in the literature. Biodegradation has received lower attention when compared to the objective of most studies, which is to evaluate the physicochemical and mechanical properties of the materials. However, this limitation in the studies is even greater when compared to the biodegradation of bioplastics made with modified polymers.

The comparison between bioplastics developed from unmodified and modified hemicellulose presents few studies intending to analyze the enzymatic degradation [81], and biodegradation. This low number of studies with hemicellulose could be related to the difficulties in obtaining a plastic polymer matrix from this heterogeneous vegetable polysaccharide. However, in addition to the analysis of the physicochemical properties of modified bioplastics, the effects of chemical, physical, and biological (and enzymatic) modifications of polymers on biodegradation must be considered. The enzymes involved in the enzymatic degradation of unmodified and modified polysaccharides may be different. Moreover, a more complex enzymatic pool will be required for modified polysaccharides.

As pending groups are attached to the polysaccharides chain, new enzymes will be required for further hydrolysis. According to a recent review article, physical modifications of polysaccharides hardly result in a change in the biodegradation process [82]. However, chemical changes result in different degradation mechanisms. Considering a chemical similarity, the enzymatic degradation of cellulose acetate can be catalyzed by acetyl esterases, an enzyme common for xylan deacetylation. The modification or functionalization of polysaccharides may result in a reduction in biodegradation since modified bioplastics (acetylated cellulose, acetylated xylan, acetylated starch, starch propionate, starch butyrate, starch valerate, and starch hexanoate) showed a reduction in anaerobic biodegradation [83]. For example, the degree substitution (DS) > 1.5, 1.5, 1.2 for starch, cellulose, and modified xylan (acetylated) respectively, represented the minimum modification necessary to delay the biodegradation of bioplastics.

The chemical modifications of the polysaccharides that make up bioplastics, such as acetylation, increase the degree of hydrophobicity of the polymers and the plastic matrix. This has the advantage of reducing the solubilization of the polymers in polar solutions. However, resulted in a decrease the enzymatic degradation. It was observed a reduction in two mannases of *Cellvibrio japonicus* (CjMan5A and CjMan26A), with reduced catalytic activities on galactoglucomannan substrates (hemicellulose) due to the decrease of the solubility of the polymers [81]. Other studies in the literature showed the influence of chemical modification of hemicellulose in relation to solubility, thermal resistance, crystallinity [84], and biodegradation rate [85]. Therefore, the diffusion of water by the composite and biodegradation is a parameter affected by chemical derivatization.

Modified xylans with an increase in the DS reduced enzymatic degradation by xylanolitic enzyme [86]. However, a rapid biodegradation rate (80%) on the first day of the evaluation was achieved for (hydroxypropyl)xylan. Substitutions above 1.5 reduced enzymatic degradability by 10%. However, the modification of cellulose with hydroxypropyl led to a reduction in biodegradation (20% in 18 days). Regarding the DS and the enzymatic activity, the article justifies the limitation of the recognition of the xylanolitic enzyme to the substrate due to chemical modification. In addition to the sterile impediment, when it changes the polysaccharide polarity through modification, it may be another explanation for the degradability reduction [87].

Modifications of polysaccharides may result in a less hydrophobic bioplastic, favoring the process of biological and abiotic degradation. Xylan carboxymethylation for bioplastic production showed an increase in water absorption at high relative humidity, demonstrating, therefore, the hydrophilic character of the carboxymethyl groups [88]. Carboxymethylation is a procedure for the production of hemicellulose-based bioplastics with increases in hydrophilic characteristics [89]. This procedure results in the development of environmentally favorable materials considering biodegradation.

A modification of hemicellulose by subtraction of chemical constituents may result in a different biodegradation process. An enzymatic modification of arabinoxylan resulted in an increase in the bioplastic crystallinity as the arabinose content was reduced [90,91]. In both of these studies, the effects of enzymatic modification of hemicellulose in relation to biodegradation were not evaluated. However, the increase in crystallinity may be a retarding factor in the bioplastic biodegradation due to the degree of organization of the molecules limiting enzymatic action, probably reducing the water absorption effect and reducing microbial growth.

The different modifications in natural bio-based polymers (for example, polysaccharides) may result in a difficulty in biodegradation or enzymatic degradation. The rate of degradation of these materials can reduce in a given period. However, the material can still be metabolized or degraded using enzymes. For example, acetylated xylan is the form found in natural lignocellulosic materials, therefore, although acetyl groups result in a delay in biodegradation, these polysaccharides are biodegradable by microbial enzymes, such as xylanases and esterases, whereas the acetylated xylan form is predominant in the environment.

#### 3.3.3. Biodegradation of Microbe-Based Polymers Bioplastics

Under the nutritional abundance of carbon and nitrogen, some bacteria can synthesize energy reserve polymer (inclusions). Polymers like polyhydroxyalkanoate (PHA) (intracellular granules), can be produced via microbial fermentation of biomass (animal or vegetable). Regarding applications, these natural polymers are an important alternative for the manufacture of bioplastic materials since they are biodegradable and biocompatible, and used in the medical field [92]. With the 41% increase in world production of PHAs between 2010–2017, this polyester has become a polymer of significant interest in the development of bioplastics. The properties of this microbial polyester can contribute to a reduction of environmental impacts due to the closed carbon cycle generated by biodegradation [93].

There is a growing interest in the development of materials formulated with PHAs, the study of the biodegradability of these materials. However, factors that influence the degradation of composites and bioplastics are necessary. Some of the marine microorganisms that are known to degrade PHAs [94] are *Aestuariibacter halophilus* S23; *Alcanivorax* sp. 24; *Alcanivorax dieselolei* B-5; *Pseudoalteromonas haloplanktis*; *Alteromonas sp*. MH53; *Bacillus* sp.; *Bacillus* sp. strain NRRL B-14911; *Bacillus* sp. MH10; *Comamonas testosteroni* YM1004;

*Enterobacter* sp.; *Aliiglaciecola lipolytica*; *Gracilibacillus* sp.; *Marinobacter* sp. NK-1; *Nocardiopsis aegyptia*; *Pseudoalteromonas* sp. NRRL B-30083; *Pseudoalteromonas gelatinilytica* NH153; *Pseudoalteromonas shioyasakiensis* S35; *Pseudomonas stutzeri* YM1006; Psychrobacillus sp. PL87; *Rheinheimera* sp. PL100; *Shewanella* sp. JKCM-AJ-6,1α; *Streptomyces* sp. SNG9. Terrestrial microbial representatives degraders PHAs [95] are *Alcaligenes faecalis*; *Pseudomonas lemoignei*; *Acientobacter* sp.; *Acientobacter schindleri*; *Bacillus* sp.; *Pseudomonas* sp.; *Stenotrophomonas maltophilia*; *Variovorax paradoxus*; *Stenotrophomonas rhizophilia*; *Penicillium* sp.; *Purpureocillium lilacinum*; *Verticillium lateritium*; *Burkholderia* sp.; *Nocardiopsis* sp.; *Streptomyces* sp.; *Bacillus cereus*; *Burkholderia* sp.; *Cupriavidus* sp.; *Gongronella butleri*; *Penicillium oxalicum*.

As in polysaccharide-based bioplastics, crystallinity in polyester bioplastics from microbial synthesis plays an important role in the biodegradation process. In bioplastics with higher proportions of amorphous regions, depolymerization occurs more quickly through abiotic or biotic action. For example, higher biodegradation was obtained with hydroxybutyrate (PHB), hydroxybutyrate-co-hydroxyvalerate (PHBV-40), PHBV-20, and P (3HB, 4HB) (10% mol of 4HB) and PHBV-3 [96]. According to the quantification of CO<sup>2</sup> in a composting vessel, PHBV-40 and P (3HB, 4HB) (10% mol 4HB) showed the highest degrees of biodegradation, due to a reduction in crystallinity with the addition of higher percentages of HV (valerate hydroxide—indicated by the numbering in front of the acronym) and 4HB. Biodegradation was 90.5%, 89.3%, 80.2%, 90.3% and 79.7% in 110 days of analysis for bioplastics formulated by PHBV-40, PHBV-20, PHBV-3, P (3HB, 4HB) and PHB, respectively.

The advantage of using PHBV in comparison to PHB is the ease of processing and good toughness. Certain PHBV disadvantages such as low thermal stability and a high degree of crystallinity must be overcome [96]. Improvements, mediated by chemical changes, must be performed together with the preservation of the material's biodegradation property, which, depending on the HV percentage, maybe rigidity or flexibility, similarly to commercial synthetic plastics (polyethylene, polypropylene, and polyvinylchloride), and assurance of biodegradation of the formulated bioplastic [96].

A commercial Ecoflex bioplastic (commercial product of BASF) was compared to PHB and poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in activated sludge for 18 days. Bioplastics composed of PHBHHx showed a higher degree of biodegradability than Ecoflex and PHB, with weight losses of 40, 20, and 5%, respectively [97]. The low crystallinity and morphology of the surface of the bioplastic proved a determining factor in the biodegradation process, observed mainly in bioplastics with 12% HHx (hydroxyhexanoate), which displayed a rough and porous surface before and after undergoing activated exposure to sludge and lipases (Figure 4).

**Figure 4.** Surface morphology of bioplastic made with PHB (12% HHx) before (**left**) and after degradation (**right**) [97].

Besides surface morphology and crystallinity of the bioplastics, other factors, such as mixing components, depth of burial (due to environmental and/or microbial differences), and time of exposure to the soil also determine the biodegradability degree. In the study

performed by Weng et al. [98], evaluating through appearance and fragmentation, the following results were achieved for the biodegradation of polymeric blends (poly (3 hydroxybutyrate-co-4-hydroxybutyrate and poly (lactic acid)—(P (3HB, 4HB)/PLA)): In the first month of testing, blends composed of 100% P (3HB, 4HB) and those with 25% PLA showed loss of integrity (appearance), whereas in the second month, both bioplastics had been almost completely biodegraded. This behavior was similar for the different depths of burial used (20 and 40 cm); however, at 20 cm and 2 months of testing, a greater difficulty was observed in the collection of fragments of blends with 75% of P (3HB, 4HB). For both depths of burial, the higher the concentration of PLA in the blends, the longer the biodegradation time. However, the biodegradation behavior was the opposite for higher concentrations of P (3HB, 4HB).

Polymer blends with 100% and 75% P (3HB, 4HB) were degraded more easily at 20 cm depth, although the presence of PLA in the bioplastics represented a delay in biodegradability at both depths tested. At 40 cm, PLA suffered greater disintegration, due to the anaerobic conditions, providing better conditions for degradation of the PLA, as reported by the authors [98].

In addition to temperature, bioplastic composition, crystallinity, degree of hydrophilicity and environmental conditions in relation to oxygen concentration, another factor that must be taken into account is the abundance of microbial biomass and the efficiency of fungi and bacteria biodegradation in different environments. The biodegradation of microbial polyesters by fungi was reported as dominant in soil [95]. However, the biodegradation of polymers in aquatic (marine) medium was faster with the use of bacteria [99,100].

PHA bioplastics as well as other bioplastics have limitations in their applications and achievements due to the high cost, low mechanical resistance, and impairment of biodegradation in functionalization processes and mixtures with other polymers [93]. In addition to the low ductility property, one of the main disadvantages of using PHAs in the production of bioplastics is the formation of brittle bioplastics, properties that can be improved by mixing biodegradable polymers from oil. However, the sustainability of bioplastic manufacturing is affected since the use of oil in the extraction and refinement stage generates the carbon dioxide production [101]. Another impact that should be considered in the production of PHA bioplastics with synthetic polymers such as the use of PCL is the reduction in the rate of biodegradation [102]. However, the development of polyester bioplastics with bio-based fibers origin (blends development), such as lignocellulosic fibers, can assist in overcoming the low ductility property of bioplastics [93]. This blend reduces costs, ensuring a biodegradable and renewable product.

The application of polyhydroxyalkanoate as a bioplastic has major limitations (e.g., its production costs for replacing conventional plastics [103]). A potential alternative for the optimization of PHA production technologies is the use of organic residues, such as lignocellulosics [104]. As an example, hemicellulose [105–107], cellulose [108] and a mixture of hemicellulose and cellulose hydrolyzate [109] have been used for the production of microbial polyesters, such as PHA and P3HB (PHB). However, the use of lignocellulosic fibers poses limitations mainly related to the production yield and generation of inhibitory substances for PHA-producing microorganisms [104]. Table 1 shows some properties and biodegradation times of different biodegradable bioplastics produced from biopolymers.

Poly(lactic acid) (PLA) is another polyester that may be partially derived from the microbial fermentation of biopolymers. The lactic acid produced by the bacteria is polymerized by a chemical route, thus forming PLA, which offers several advantages, such as rigidity and miscibility with other biodegradable plastics. However, in several application areas (e.g., manufacture of 3D printers), its fibers have been used by Brazilian companies due to the PLA lower heat loss in comparison to oil-derived plastics [110]. Nevertheless, bioplastics from bacterial polyesters should be considered, since PHAs like PBH present several advantages in comparison to PLA [111]. Table 1 shows some properties and biodegradation times of different biodegradable bioplastics produced from biopolymers.


**Table 1.** Properties and biodegradation time of different biodegradable bioplastics produced from biopolymers.

CONB = Biodegradation conditions; BPR = Biodegradation period and rate; PAB = Biodegradation analysis procedure; TS = Tensile strength; E = elongation; . . . = not reported; \* = Newton (N).

Bioplastics are renewable and/or biodegradable and display good mechanical properties such as tensile strength similar to certain synthetic plastics in common use (Table 1). Polypropylene and polystyrene, fossil-based synthetic plastics, show TS between 25–40 MPa and 30–55 MPa, respectively [68], whereas it ranges between 55–124 and 9–17 for CellophaneTM and low-density polyethylene (LDPE), respectively [123,124]. This similarity of mechanical properties of bioplastics and plastics was also reported by Hansen et al. [125]. Therefore, even if at present and in the future, the total replacement of non-biodegradable plastic from petroleum, is something unlikely, for some applications, such as bioplastics for use in agriculture (mulch) and packaging (short lifetime), this technology can represent one of the alternatives (along with other actions and technologies) to mitigate the environmental impacts related to plastics.

#### *3.4. Effect of the Bio-Based Polymer Addition on the Biodegradation Rate of PHAs Bioplastics*

The addition of bio-based polymers in polyester microbial biocomposites is vast in the literature [93,126,127]. The bio-based polymers application, mainly lignocellulosic fibers, is due to the improvement in the biodegradation rate of the formulated bioplastic [93]. This improvement in the biodegradation of PHAs biocomposite is related to the increase in hydrophilicity and water absorption by bioplastics. A mixture of 30% Sisal fibers (wt) and PHBV resulted in increased water absorption of 14% compared to pure PHBV (0.8%) [128]. The use of Kenaf fibers (main cellulose) in the blend with poly(3-hydroxybutyrate-co-3 hydroxyhexanoate) [P(3HB-co-3HHx)], resulted in a greater loss of mass in biodegradation in soil due to greater water absorption and microbial binding sites in the microbial polyester from binding with Kenaf fiber [129]. This study suggested that the accelerated deterioration of the blend (reduction of mechanical properties), after 6 burial weeks (soil) was due to the weakening of the adhesion between the fiber/[P(3HB-co-3HHx )], with the access of water to the internal hydrophobic regions of the polymer.

The use of hemicellulose with PHAs is also an alternative to increase the blends biodegradation rate in relation to pure microbial polyesters [93]. Bioplastics from PHBV/ Peach Palm Particles (lignocellulosic fiber with considerable hemicellulose content) were biodegraded faster than pure PHBV in soil [130]. The authors reported cracks, corrosion, and discoloration after 2 months of biodegradation. The poor adhesion between the fiber/PHBV interface, which resulted in greater water absorption and accessibility of soil microorganisms, was suggested as contributed to the deterioration.

Starch, another polysaccharide from vegetable biomass, can also be used in the production of bioplastics with reduced biodegradation time. The mixture of starch and PHBV (50/50% wt) was fully biodegraded in the soil after 33 days, i.e., there was a 50% reduction in biodegradation time compared to pure PHBV [131]. The addition of starch reduced the crystallinity of the blend, facilitating the absorption of water by the matrix and increased the enzymatic activity on the surface and in the inner region of the blend. There was an increase in the biodegradation of PHA/starch blends as the starch content increased in the formulation, with biodegradation of PHA/30% starch (wt) corresponding to 44% in 6 months of burial in soil [132].

Chemical modifications of polysaccharides, such as cellulose acetylation (cellulose acetate) can result in partial or total inhibition of blends biodegradation, due to reduced solubilization and hydrophobicity of the fibers. The acetyl and butyryl group in cellulose reduced the rate of biodegradation of the PHB blend/modified cellulose due to the impediment of the substituents and reduced the blends/water interactions [133]. However, in the same study, the mechanical properties were improved by increasing the concentration of cellulose acetate butyrate. Related to cellulose, the degree of substitution above 2.5 results in the inhibition of biodegradation [93]. However, some chemical modifications can positively influence the biodegradation of microbial and bio-based polymer blends. This improvement in biodegradation is due to the increase in the contact area surface of the fibers, resulting from the surface treatments of the fibers, such as, an increase in the

fiber rugosities with the application of NaOH, which removes the hemicellulose and lignin fibers [93].

Lignin is the most recalcitrant constituent of lignocellulosic fibers due to the complexity of the composition of this phenolic macromolecule [82,134]. The lignin enzymatic catalytic degradation needs different enzymes [75,82], or even the synergy of an enzyme complex. The inclusion of lignin in the blends of PHAs and PLA results in steric impediment of the enzyme and reduction of the degree of hydrophilicity, which is shown in the literature as a factor in reducing the biodegradation of polyester blends [93,132]. For example, the biodegradation in soil of the PHA/lignin blend was 4% after 24 weeks, which was lower than the rate of biodegradation of the PHA/10% starch, PHA/cellulose (11.1 and 100% respectively) [132]. An alternative for obtaining bioplastics from microbial polyesters, with guaranteed polymer biodegradability, is the use of enzymes and microorganisms capable of catalyzing the breakdown of lignin. The main enzymes involved in lignin oxidoreduction are laccases (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) [82], and the recently discovered enzymes dye-decolorizing peroxidases and unspecific peroxygenase [134,135].

The application of specific microorganisms that degrade lignin and/or PHAs can be an alternative to improve the biodegradation of the blends of these polymers. In nature, these enzymes act synergistically, and some microorganisms can produce the three enzymes, while others only produce a few of the necessary enzymes [82]. LiP has a key role in the degradation of lignin, due to the distinct characteristics of the active site of the enzyme. However, the catalytic action of LiP is mediated by H2O2, which is generated by Lac [135]. The effectiveness of the microorganisms is essential for the biodegradation of PHA/lignin blends, as some microorganisms such as *Phanerochaete chrysosporium* are considered excellent for the degradation of lignin [134,136]. However, Brown-rot fungi due to the degradation mechanisms of lignin not being oxidative, presents a reduced degradation process of lignin [82]. Another example is the case of the bacteria *Streptomyces viridosporus*, which can result in a reduced degradation process of lignin since this bacterium acts in the non-phenolic regions of lignin [82,134].

#### **4. Conclusions**

Advances in the development of materials and technologies with fewer environmental impacts are highly expected, mainly due to the progress in the area of biopolymers over the past two decades. However, biopolymers application and use in the various sectors of society is limited, i.e., the annual production of bioplastics compared to plastics is still low. In this way, the use of plant biomass and microbial polyesters can help the development of bioplastic feasible, due to the availability of resources, biocompatibility, biodegradability and generally does not result in ecotoxicity. However, the physicochemical and biodegradation properties must be considered for the study of the optimization of bioplastic from natural polymers. Several actions must be taken so that bioplastic can become a reality on a large scale. The state of São Paulo (Brazil) has established a law that prohibits the supply of disposable plastic products to commercial establishments, which may increase the production scale of some bioplastics, thus reducing costs. The approval of a law by the Chinese government that prohibits the import of international plastic wastes for recycling can also encourage the production of bioplastics. An increase in the production and distribution of bioplastics is not sufficient for the development of a more conscious and sustainable society, i.e., care must be taken for the identification of a bioplastic and/or biodegradable material towards no final consumers' mistakes and no unsuitable actions or disposal habits.

**Funding:** The authors acknowledge the São Paulo Research Foundation (FAPESP) for the financial support of this research project (process number 2019/16853-9; 2019/12997-6; and 2017/22401-8).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Characterization of Used Lubricant Oil in a Latin-American Medium-Size City and Analysis of Options for Its Regeneration**

**Carlos Sánchez-Alvarracín \*, Jessica Criollo-Bravo, Daniela Albuja-Arias, Fernando García-Ávila and M. Raúl Pelaez-Samaniego**

> Faculty of Chemical Sciences, University of Cuenca, Av. 12 de Abril and Av. Loja, Cuenca 10202, Ecuador; jessica.criollo@ucuenca.edu.ec (J.C.-B.); daniela.albuja@ucuenca.edu.ec (D.A.-A.); fernando.garcia@ucuenca.edu.ec (F.G.-Á.); manuel.pelaez@ucuenca.edu.ec (M.R.P.-S.)

**\*** Correspondence: carlos.sancheza@ucuenca.edu.ec; Tel.: +593-991-708-800

**Abstract:** Petroleum-derived products, such as lubricant oils, are non-renewable resources that, after use, must be collected and processed properly to avoid negative environmental impacts. A circular economy of used oils requires the re-refining and reuse of the same. Similar to most countries in Latin America, the management of used oils in Ecuador is still incipient and few cities collect and treat this material properly. In Cuenca, the ETAPA company collects ~1344 t/year of used oils, which are subjected to pretreatment operations prior to their use as fuel in a cement factory. However, combustion generates polluting gases and disallows the adding of value to the used oils. The lack of studies on the characterization and methods utilized for recovering used oils under the conditions found in medium-size Latin-American cities (e.g., Cuenca), alongside a lack of government policies, have hindered the adoption of re-refining operations. The objective of this work is to characterize the used lubricant oils in Cuenca, to compare them with the properties of used oils from other countries, and to suggest some re-refining technologies for oils with similar properties. Used oil samples were collected from mechanic shops and car-lubricating shops for characterization. Its physicochemical properties and metal contents are comparable to the used oils in other countries globally. Specifically, the flash point, kinematic viscosity, TBN, and concentrations of Zn, Cd, and Mg are similar to the properties of used oils in Iraq, Egypt, and the United Arab Emirates. Based on these results, the best re-refining option for used oils in Cuenca is extraction with solvents in which sedimentation and dehydration (already conducted in Cuenca) is followed by a solvent reaction process, a vacuum distillation process, a finishing process with bentonite, and a final filtration step.

**Keywords:** waste lubricating oil; characterization; used oil management; circular economy

#### **1. Introduction**

Lubricating oil (or engine lubricant oil), a product derived from petroleum refining, is a mixture of essential oils (either virgin or processed, mineral or synthetic) and additives. Lubricant oils are used in equipment with moving parts to reduce friction and surface wear off [1], which makes these oils widely employed for the operation of internal combustion engines (ICEs; both gasoline- and diesel-type engines). During the operation of ICEs, the additives in the oil are partially consumed and the oil quality decreases over time due to degradation by oxidation, decomposition of oil and its additives, and contamination with water, gasoline (or diesel), dirt, metals, and carbon particles [2]. The degradation of lubricant oil reduces its life, which severely limits its reusability. Thus, it needs to be replaced with new oil [3]. The used lubricant oil must be collected and stored adequately to avoid environmental pollution, such as from spillage. Often, used lubricant oils are burnt without any treatment, which emits harmful gases into the environment. Sometimes these materials are even spilled in rivers [4].

Used lubricating oils contain heavy metals (e.g., Cr, Cd, As, and Pb) and harmful chemical compounds, such as polynuclear aromatic hydrocarbons, benzene, chlorinated

**Citation:** Sánchez-Alvarracín, C.; Criollo-Bravo, J.; Albuja-Arias, D.; García-Ávila, F.; Pelaez-Samaniego, M.R. Characterization of Used Lubricant Oil in a Latin-American Medium-Size City and Analysis of Options for Its Regeneration. *Recycling* **2021**, *6*, 10. https:// doi.org/10.3390/recycling6010010

Received: 15 October 2020 Accepted: 12 January 2021 Published: 2 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

solvents, polychlorinated biphenyl (PCBs), and polycyclic aromatic hydrocarbons (PAHs). Therefore, proper management of used oils is necessary to avoid the negative effects inflicted on human health and nature [5]. From an environmental point of view, combustion of used oils is not recommended [5], since the improper incineration of 5 L of used oil could pollute an amount of air equivalent to that needed by a person to live over three years [6]. The adsorption of Cr and its compounds released by burning used oil can cause some types of cancer [5]. The negative impacts of the improper management of used oil on the environment and on human health [7] require the exploration of options for regenerating these oils with the intent of producing new lubricants and other petroleumderived products. The yield of lubricant oils through the re-refining of used oils is higher than the yield from virgin crude petroleum refining [2], promoting a reduction of about 90% of the environmental impacts that otherwise could result from the production of petroleumderived lubricant oils [7]. Some companies in the world, particularly large industries that consume lubricants, regenerate used oil in their own facilities by physicochemical processes to remove the contaminants. Then, the regenerated oil is added to new oils to operate ICEs or is used in other industrial applications, such as gear lubricants, cutting oil, and metal-rolling lubricants [8]. This option has the advantage of reducing losses in each processing step, although the regenerated oil cannot be used again in the engines or gearboxes in vehicles due to degradation. Thus, lubricating oils must be re-refined for further use as lubricants in ICEs [9].

Our literature review suggests that there is a limited amount of studies reporting on the collection methods, characterization, and reuse techniques for used lubricating oils in Latin-American countries. Mexico and Brazil are the leading countries in the region on the reuse of used lubricant oils. In Mexico, Bravo Energy recovers around 100 million L of used oil in the form of fuels from Mexico and other countries (the US, Argentina, and Chile) [8]. Brazil consumes around 1 billion L/year of lubricating oil, 60% of which corresponds to automobile ICEs. More than 36% of the collected used oil in the country is derived from cars' ICEs [10]. The Sindicato Nacional da Indústria do Rerrefino de Óleos Minerais, "Sindirrefino" [11], ensures that the oil-refining activities in Brazil follow the latest technology, obtaining products duly specified by the National Petroleum Agency, according to Portaria ANP-130/1999. The industrial park has three differentiated technologies: (a) an acid/clay system with a "cracking term", obtaining a neutral heavy base oil; (b) a flash distillation or skin evaporation system, which allows obtaining a light and medium neutral base oil; and (c) an extraction system selective of propane solvents to obtain a medium neutral base oil. Currently, there are several Latin-American cities that possess private re-refining plants. Some examples of re-refining plants include CILCA (Lima, Peru) [12], Biofactor (Duran, Ecuador) [13], Nueva Energía S.A. (Buenos Aires, Argentina) [14], and other plants in Medellin and Bogota (Colombia) [15]. In September 2019, the IDB (Inter-American Development Bank) approved a loan to co-finance an oil recovery plant in Central America in partnership with the Costa Rican company Metalub, which currently collects and ships used oils to the US for re-refinement [16]. However, in the region, re-refining is mostly solely conducted at the city scale; thus, countries as a whole are not involved in the efficient collection and re-refining systems of used oils.

In Ecuador, the State, through the Agency of Regulation and Control of Hydrocarbon Fuels (ARCH) [1], regulates and controls all activities related to the production, distribution, and use of petroleum and petroleum-derived fuels and lubricants. According to the Central Bank of Ecuador, from January to July 2018, the country imported 70,081 t of lubricants, corresponding to an FOB value above US\$ 100 million [17]. In May 2019, the Ministry of the Environment of Ecuador (Ministerio del Ambiente) promulgated Law 042 to regulate the management of used lubricant oils and the containers of these materials; it establishes general guidelines on the storage, collection, and final disposal of used lubricating oils, and the minimum collection goal corresponds to 20% calculated on the total tons of lubricating oil imported or manufactured annually [18]. However, the law does not make specific reuse operations or the re-refining of used oils mandatory. According to data from Arc

& Pieper S.A. (from Quito), around 84 million L/year of used oils are not recovered in the country. From it, 30% correspond to automotive and industrial used oils [19]. Few municipal governments (i.e., Cuenca, Ambato, Quito, Ibarra, and El Coca) and the Provincial Government of Tungurahua have protocols to collect and recycle used oils. In Quito, the BIOfactor company [13] collects all types of oils generated by the city and neighboring cities in the north of Ecuador. The company has a re-refining capacity of 9 million L/year (in the city of Duran), which corresponds to approximately 11% of the oil used in the country. Nevertheless, only the municipal government of Cuenca has an environmental license from the Ministry of Environment (MAE) to collect and treat used oils [19]. This city (a medium-size Latin-American city, with less than half a million inhabitants), through the ETAPA company (Municipal Company of Telecommunications, Potable Water, and Sewerage), has been carrying out a program called "Collection of Used Batteries and Used Oils" since 1998, aiming to generate environmental awareness among the population and promoting an adequate management of these wastes [20].

Currently, ETAPA collects 151,200 L/month (~1534 t/year) of used oil from "used oil generators", a group of approximately 1300 car-lubricating shops, mechanic shops, car wash shops, and tire repair shops, and some miscellaneous industrial companies in Cuenca. This amount of used oil collected is approximately 57% of the total consumed in the city [20]. The remaining is sold to an informal market that employs this oil as fuel in furnaces for bricks, tile manufactures, and, less frequently, for wood treatment, concrete formwork, spraying of cars' bottoms after car-washing, as a pesticide, to reduce dust on unpaved roads, or to protect cattle from subcutaneous parasites [19]. The practice of employing used oils as fuel is common in Latin-American countries (see, for example, [21]). Evidently, some of these uses are not environmentally friendly. It is expected that, in Ecuador, Law 042 could promote better management practices of used oils and reduce pollution.

The oil collected by ETAPA is subsequently treated in its own waste treatment facility through decantation, filtration, and clarification processes to separate the water and solid impurities. Following the Ecuadorian Technical Standard NTE-INEN 2266: 2017 for the transport, storage, and handling of hazardous materials, the treated oil is sent to a cement company in Guayaquil where it is burnt as fuel [20]. However, the management system of used oils in the city requires expenses that currently are paid by both the municipal government and the used oil generators. The sale of used oils to an informal market could be explained in part by the propensity of used oil generators to reduce such additional costs. Thus, adding value to used oils could help to reduce its utilization in informal markets. Despite these problems, the collection and treatment system have been effective at avoiding spillages and harmful discharges of used oil.

The implementation of Law 042 [18] would boost the required steps towards a circular economy that prioritizes regeneration and reuse of used lubricant oil. For this purpose, a deep understanding of the properties of used oil and the potential options for adding value to this material are critical [22]. In the case of Ecuador, information on the properties of used oils is scarce. Almeida et al. [4] characterized some properties of used oils in Quito. In Cuenca, Jaramillo et al. [23] characterized two types of oils commonly used in taxis (SAE 20W50 and SAE 10W30) to determine the most common causes of engine wear. Likewise, ETAPA has determined some properties of the used oils they collect (mixtures of automotive and industrial used oils) [24]. However, there are no current works showing a complete characterization of used oils or suggestions about possible routes for re-refining these materials as part of a circular economy strategy in the conditions of Cuenca and other cities in Ecuador. The objective of this work was twofold: (a) to characterize the used lubricant oils collected in Cuenca and compare the resulting properties with those of used oils elsewhere; and (b) to recommend regeneration options for used oils, considering the characteristics of the oils and the experiences reported in other places. The work hypothesizes that, if the characteristics of used oils in Cuenca are comparable to those in other places where used oil management is adequately performed, it is possible to adapt such proven processes to the conditions of our city. This paper summarizes the main

findings of the research and expects to contribute with ideas for a better management of used lubricating oils in medium- (e.g., Cuenca) and small-size cities. Figure 1 summarizes the process to be developed in this research in order to meet the proposed objective.

oils elsewhere; and (b) to recommend regeneration options for used oils, considering the characteristics of the oils and the experiences reported in other places. The work hypothesizes that, if the characteristics of used oils in Cuenca are comparable to those in other places where used oil management is adequately performed, it is possible to adapt such proven processes to the conditions of our city. This paper summarizes the main findings of the research and expects to contribute with ideas for a better management of used lu-

Figure 1 summarizes the process to be developed in this research in order to meet the proposed objective.

*Recycling* **2021**, *6*, x FOR PEER REVIEW 4 of 23

bricating oils in medium- (e.g., Cuenca) and small-size cities.

**Figure 1.** Process for the development of this research.

#### **Figure 1.** Process for the development of this research. **2. Materials and Methods**

#### *2.1. Materials*

**2. Materials and Methods** *2.1. Materials* Samples of used lubricant oils were obtained from mechanic car shops and other used oil generators in the city of Cuenca. A preliminary step consisted of surveying a group of 265 randomly selected (out of the 1300) used oil generators registered by ETAPA to identify the collection methods and storage conditions of the used oils [25]. Based on the preliminary results, it was determined that the number of storage containers in the city is 16, for which a sample was taken from each one and the four most frequently repeated were duplicated, taken from different establishments (Samples 4 and 7, 5 and 6, 8 and 9, 14 and 15). The collection of three extra samples directly from the engines of three gasoline vehicles (i.e., used oil without being mixed or exposed to contamination from external agents) also was conducted for a comparison of the properties. Therefore, twenty-three oil samples were collected in total. The twenty samples were mixtures of various types of used automotive oils (mostly engine and gearbox lubricant oils) that have been stored under different conditions (e.g., using plastic or metallic containers), as presented in Table 1. As seen in the table, in Cuenca, the used oils are stored in the used oil generators' facilities for up to three months prior to collection by ETAPA. Samples of 500 mL were extracted directly from the storage tanks. The collected samples were then sent to the Chemical Samples of used lubricant oils were obtained from mechanic car shops and other used oil generators in the city of Cuenca. A preliminary step consisted of surveying a group of 265 randomly selected (out of the 1300) used oil generators registered by ETAPA to identify the collection methods and storage conditions of the used oils [25]. Based on the preliminary results, it was determined that the number of storage containers in the city is 16, for which a sample was taken from each one and the four most frequently repeated were duplicated, taken from different establishments (Samples 4 and 7, 5 and 6, 8 and 9, 14 and 15). The collection of three extra samples directly from the engines of three gasoline vehicles (i.e., used oil without being mixed or exposed to contamination from external agents) also was conducted for a comparison of the properties. Therefore, twenty-three oil samples were collected in total. The twenty samples were mixtures of various types of used automotive oils (mostly engine and gearbox lubricant oils) that have been stored under different conditions (e.g., using plastic or metallic containers), as presented in Table 1. As seen in the table, in Cuenca, the used oils are stored in the used oil generators' facilities for up to three months prior to collection by ETAPA. Samples of ~500 mL were extracted directly from the storage tanks. The collected samples were then sent to the Chemical Control Laboratory of the Guangopolo Power Plant (Termopichincha Unit—CELEC EP) for characterization.

#### Control Laboratory of the Guangopolo Power Plant (Termopichincha Unit—CELEC EP) *2.2. Characterization of the Used Oil Mixtures*

for characterization. The twenty three samples were characterized with the purpose of determining the water content, density at 15 ◦C, viscosity index, base number, flash point, kinematic viscosity at 100 ◦C, and metals content (Al, Ba, B, Cd, Ca, Cu, Cr, Sn, P, Fe, Mg, Mn, Mo, Ni, Ag, Pb, Si, Na, Ti, V, and Zn). Water content was determined following ASTM D95 [26]. Density was determined as per ASTM D4052-11 [27]. The viscosity index was calculated from the kinematic viscosity at 40 and 100 ◦C and following the ASTM D 2270-93 procedure. The base number (TBN) was obtained by potentiometric titration, following ASTM D2896- 11 Procedure B [28]. The flashpoint was obtained in accordance with ASTM D92-12b [29]; i.e., using the Cleveland open cup method. The thermal bath method (ASTM D445-15 Procedure A) [30] was used for the kinematic viscosity index at 100 ◦C. The metal content was determined in accordance with ASTM 6595-00 (2011) [31]. All tests were conducted in

duplicate. Table 2 show the analytical instruments' specifications for the characterization of the used oil mixtures.


**Table 1.** Storage conditions of the used oils in mechanics and car lubricating shops in Cuenca.

<sup>a</sup> Refers to a screening process to remove large particles (e.g., wipe cloth) prior to storing in the tanks. <sup>b</sup> Indicates if the storage container has been under roof conditions or not. <sup>c</sup> Frequency of collection of the used oils from the used oil generators' facilities.

**Table 2.** The analytical instruments' specifications.


#### *2.3. Statistical Analysis of the Used Oil Properties*

The results obtained were statistically analyzed in three steps: The first step focused on identifying the similarities among the 23 oil samples, from a physicochemical perspective. For this purpose, a cluster analysis using the SPSS program was conducted and the samples with similar characteristics were grouped [32]. Then the agglomeration method (i.e., a hierarchical classification in which algorithms are used to group objects, using a measure of Euclidean squared distance between data and the Ward linkage that measures remoteness between groups) was employed. According to [32], this method is used to analyze quantitative variables when the sample size is relatively small (<50). With this analysis, a classification tree or dendrogram was prepared (see Figure 2). Since small distances

indicate homogeneous conglomerates and large distances show diversified conglomerates, it is convenient to stop the grouping process when the horizontal lines become long [32]; this procedure determines the number of clusters. In the second step of the analysis, the inference statistical technique "confidence interval" (with a 95% level of confidence) was employed [33]. In the third step, a comparison of the physicochemical characteristics of the used oils in Cuenca to the properties of used oils in other countries was conducted. Box and whisker plots were employed to summarize the data and to identify outliers (using the SPSS software). analysis, a classification tree or dendrogram was prepared (see Figure 2). Since small distances indicate homogeneous conglomerates and large distances show diversified conglomerates, it is convenient to stop the grouping process when the horizontal lines become long [32]; this procedure determines the number of clusters. In the second step of the analysis, the inference statistical technique "confidence interval" (with a 95% level of confidence) was employed [33]. In the third step, a comparison of the physicochemical characteristics of the used oils in Cuenca to the properties of used oils in other countries was conducted. Box and whisker plots were employed to summarize the data and to identify outliers (using the SPSS software).

The results obtained were statistically analyzed in three steps: The first step focused on identifying the similarities among the 23 oil samples, from a physicochemical perspective. For this purpose, a cluster analysis using the SPSS program was conducted and the samples with similar characteristics were grouped [32]. Then the agglomeration method (i.e., a hierarchical classification in which algorithms are used to group objects, using a measure of Euclidean squared distance between data and the Ward linkage that measures remoteness between groups) was employed. According to [32], this method is used to analyze quantitative variables when the sample size is relatively small (<50). With this

*Recycling* **2021**, *6*, x FOR PEER REVIEW 6 of 23

Flashpoint Equipment to flashpoint Fisher Tag Flash Tester 926 Metal content Atomic emission spectrometer Spectroil M/F-W 626

*2.3. Statistical Analysis of the Used Oil Properties*

**Figure 2.** Cluster analysis of the physicochemical properties of the mixtures of used oils (a dendrogram using the Ward link). **Figure 2.** Cluster analysis of the physicochemical properties of the mixtures of used oils (a dendrogram using the Ward link).

#### *2.4. Analysis of Options for Re-Refining Used Oils in Cuenca 2.4. Analysis of Options for Re-Refining Used Oils in Cuenca*

The results of the characterization and experiences reported in the literature served to propose options of technologies that could be employed to re-refine used oils in Cuenca. This stage sought to establish the requirements that could enable the processing and utilization of used oils as part of the circular economy strategy in the city. The options that were analyzed considered five factors: (1) the amount of available used oil collected in the city alongside the possibility of expanding used oil collection in neighboring cities; (2) the quality of the used oil; (3) the yield of byproducts that can be obtained from used lubricant oils through re-refining, based on the literature and the limitations of some re-refining The results of the characterization and experiences reported in the literature served to propose options of technologies that could be employed to re-refine used oils in Cuenca. This stage sought to establish the requirements that could enable the processing and utilization of used oils as part of the circular economy strategy in the city. The options that were analyzed considered five factors: (1) the amount of available used oil collected in the city alongside the possibility of expanding used oil collection in neighboring cities; (2) the quality of the used oil; (3) the yield of byproducts that can be obtained from used lubricant oils through re-refining, based on the literature and the limitations of some re-refining technologies; (4) the availability of materials for the treatment process close to the city; and (5) the costs of the regeneration technologies and the possible uses of the waste.

#### **3. Results and Discussion**

#### *3.1. Physical Characteristics of the Used Oils, and Their Metal Content, in Cuenca*

The initial test of normality of the properties of the used oils in Cuenca showed that the physical properties, such as water content, density, viscosity index, kinematic viscosity, and the presence of elements (Al, B, Cd, Cr, Fe, Mg, Mn, Mo, Ni, Ag, Na, Ti, and V), present a normal behavior. Thus, the central tendency measure is the mean. However, the TBN and the concentrations of Ba, Ca, Cu, Sn, P, Pb, Si, and Zn in the used oil did not show a normal distribution. Therefore, for these properties, we performed a data transformation through the Cox–Box method, using Minitab 19 software. After these considerations, the ranges of the values for each physical property and the metal content are shown in Table 3.


**Table 3.** Physical properties and metal content of the used oils in Cuenca city.

<sup>a</sup> 95% level of confidence.

#### *3.2. Influence of Storage Conditions on Used Oil Properties*

The result of the cluster analysis of the physicochemical properties of the twenty-three samples is presented in Figure 2. For the analysis, Euclidean squared distance and Ward's linkage were chosen to create homogeneous groups [32]. In the dendrogram (Figure 2), a distance of 5 was chosen, obtaining four clusters (according to the similarity of their physicochemical properties) called Clusters A, B, C, and D. Cluster A contains nine samples (i.e., Samples 8, 18, 13, 5, 7, 15, 3, 1, and 12), Cluster B also consists of nine samples (i.e., Samples 17, 23, 2, 9, 4, 19, 6, 16, and 20), Cluster C consists of two samples (i.e., Samples 14 and 21), and Cluster D consists of three samples (i.e., samples 10, 11, and 22). Sample 2, as seen in Figure 2, corresponds to the average values of the properties of the 23 oil samples. The corresponding properties moved away as the Euclidean distances of the other samples moved too. An ANOVA test was performed to compare the means of the groups (at the 95% confidence level). Figure 2 suggests that the storage conditions are not grouped but are different within the same cluster. Samples 1, 2, and 3, which were taken directly from vehicles' engines (Table 1), are located within Clusters A and B, with characteristics similar to the other stored samples, suggesting that the storage time (i.e., up to three months) does not affect the properties of the used oils. Clusters C and D, despite having fewer samples, also show different storage conditions. Therefore, this finding indicates that there is no correlation between the storage conditions (i.e., type of material of the containers and time) and the oil's characteristics. This can be confirmed because the properties of the oils extracted directly from the vehicle engines, which correspond to the zero storage date, were analyzed under the same environmental conditions as the other stored samples. Furthermore, due to the climatic conditions of Cuenca (which is more or less constant throughout the year), there are no temperature variations that could compromise the properties of the oil during storage.

#### *3.3. Physical Properties of the Used Lubricating Oils in Cuenca and Comparison with the Properties of the Used Oils in Various Countries*

Table 4 shows the values of the physical properties and metal content of the used oils in Cuenca (Column 2) and the new lubricant oils that are employed in the city (Column 3). The table also presents the typical values and expected limits of both the physical properties and metal contents reported in the literature, as well as the expected tendencies of each property or value compared to the new oils. It can be seen that, for used oils in Cuenca, most values are below the accepted limits, according to [34]. Moreover, the water content is below the limits reported by [34] and the rest of the results (except the concentration of some metals) are in the range of expected values [8,35]. This result suggests that the re-refining options for used oils in Cuenca should take advantage of the experiences in other places that process used oils with similar properties to guarantee the properties of the regenerated oil as required for further use.

**Table 4.** The physical properties and metal concentration of the used lubricant oils in Cuenca, in comparison with other oils.


Table 5 shows a comparison of the physical properties, presence of metals, and concentration of additives of the used oils in Cuenca with those of used oils in other countries. Some properties are difficult to compare due to a lack of data. However, it is seen that, as with other materials, the water content is below the values reported in other countries. Properties such as viscosity are of interest since they show that the oils have not degraded too much during their use, as well as indicating a lower water content.

Metals such as Al, Cd, Cr, Cu, Fe, Pb, Mn, Mg, Ni, Ag, Sn, Ti, and V (Table 6), known as wear metals [8], arise in the used oil due to the engines' high operating temperatures and pressures. Thus, the concentrations of metals found in the used oil vary depending on the brand and the type of used oil, the conditions of the engine that originated them, and the distance traveled by the vehicle until oil change is performed [36]. Ti is outside the range of typical values for used oils with a much higher value. Oil liquid Ti is widely used to reduce the friction and wear of engine components [37], which could explain such high values for Ti content. In the case of engine wear metals, all values are below the mean of the countries reported in the literature (except for S and V, from which there are not enough results to compare in Table 5); however, they are below the typical ranges as reported by Parry [8] and Widodo et al. [35], as seen in Table 4. The results indicate that the oils have a low contamination by wear metals, especially in the case of Si, Al, Cu, Cr, and Fe. Si is considered a highly abrasive material [38]. Its presence in the used oil is mainly due to contamination by sediments or some brake fluids, as well as by dust (dirt) entering the engine. The concentration of additives such as Ba, B, Ca, Mg, Mo, Na, P, and Zn (Table 7) is reduced with respect their initial concentrations. P and Zn are elements that are generally found in anti-wear additives and antioxidants used to protect metal segments under engines' extreme operating conditions. Ca, Mg, and Na sulfonate are mainly associated with the detergents/dispersants used for engine cleaning. Some engine oils contain MoS2 to reduce wear at high temperatures and pressures. B and Ba are used in some synthetic oil formulations and act as anti-wear additives [22,36,39]. Regarding B, there are not enough data to compare with other countries (Table 5), but it is within a typical range according to Widodo. et al. as seen in Table 4. Some metals that are components of the engines also serve as additives in lubricating oils. This is the case of Al, Cu, Mg, Ti, and Mo. If the concentration of an element in the used oil increases with respect to the concentration of the virgin oil, it is considered a wear metal, otherwise it is considered an additive. Ba and Mg content is below the mean of the typical values reported in the literature (Table 7), which suggests that part of these elements was lost during the operation of the engines. The Mo, Na, P, and Zn concentrations are also below the mean values (right column), suggesting loss of these metals, but they are still within the typical ranges (see Table 4). Ca has a higher value than the expected values [8,9]. Its content depends on the types or brands of oils present in the mixture.

In general, it is observed that the characteristics of the oils used in Cuenca are within the ranges of values obtained from other countries. The results of this research are close to those reported for used oils in other countries in South America (e.g., Colombia, Chile, and Venezuela), except for the higher water content of the used oils in Chile. This could possibly result from similar climatic conditions, age of the car fleet, and types of oils consumed. Likewise, in Peru, a higher presence of wear metals in used oils is seen, although those used oils retain more additives. The literature also shows that, in Australia, used oils are less contaminated by wear metals, but there is greater loss of additives. Works conducted in the US on the concentration of metals in used oils show values close to used oils in Cuenca. In the case of Spain and Portugal, there is less metal contamination in the used oil, except for Fe. Iraq, Nigeria, and Poland present approximately similar results, although those oils appear less contaminated by metals. The same happens in the case of used oils in the UAE, except for the high content of Pb. The physical characteristics of the used oils in Egypt, Pakistan, and Saudi Arabia show some similarities to the used oils analyzed in this work. However, the metals content cannot be compared due to a lack of information. A high water content and low viscosity index is evident in South Africa's used oil. Kazakhstan, Ukraine, and Ghana show the highest metal-contaminated automotive used oil.


**Table 5.** The physical properties, concentration of wear metals, and additives of the used lubricating oils in Cuenca, in comparison with the properties of the oils used in various countries.

#### *3.4. Analysis of Options for Re-Refining Used Oils in Cuenca*

The treatment of used lubricant oils involves three processes: recovery, reprocessing, and regeneration (or re-refining) [8]. Reprocessing is carried out by the elimination of contaminants from used oils and may include distillation and chemical treatment, as well as a combination of methods with those used for recovery. With the re-refining process, the highest degree of contaminant removal is reached, obtaining the base oil to manufacture new lubricating oils. Table 6 shows the methods commonly employed for re-refining of used oils at the laboratory scale and Figure 3 summarizes the sequence of these steps. Regardless of the re-refining process, it begins with a pretreatment step that depends on the characteristics of the oil. According to Fong et al. [5], good quality oils can be obtained from used oils and their production costs are relatively lower than the costs of producing oils through petroleum refining. A gallon of used oil (3.78 L) provides 2.5 L of lubricating oil, which, otherwise, should require 42 gallons (>158 L) of crude oil [57]. Besides, the byproducts of re-refining can be converted into valued end products for other manufacturing processes, such as asphalt. The yields of the products and byproducts can cover the cost of buying the chemicals needed to keep the operation of the re-refining plant and, therefore, to make the process profitable [5]. An oil of higher quality than the corresponding original oil cannot be obtained using these processes. For this reason, for cost-effective recycling of used oil, it is important to separate the different types of oils in the places used oils are generated because, from the mixture of high-quality and low-quality used oils, only low-quality oils are obtained [8]. Speight and Exall [58] (cited by [35]) indicated that, in the re-refinement process, solid particles and water are separated from the used oil by physical treatments to obtain an oil comparable to the original, but the contaminant metals could not be completely removed.


**Table 6.** Steps for re-refining used oils and the technologies employed at the laboratory scale.

**Table 6.** *Cont.*

**Figure 3.** Main stages of conventional technologies for the re-refining of used oils. Adapted from [35]. **Figure 3.** Main stages of conventional technologies for the re-refining of used oils. Adapted from [35].

**Acid clay** dehydration vacuum distillation acid wash adsorption with clay filtration Recovery (63%) filtration Recovery (63%) dehydration vacuum distillation clay filter Re-refining consists of four stages: pre-treatment (i.e., removal of water and solid particles), regeneration (i.e., elimination of degradation products), fractionation of the bases (i.e., separation of light hydrocarbons), and finishing (i.e., improvement of color and smell of the treated oil) [35,59,63]. Some of these processes still have some disadvantages, such as the poor performance of oil regeneration or the generation of other environmentally

> adsorption with clay

**Figure 4.** Summary of the technologies employed at the industrial level for re-refining used lubri-

hydro-reaction adsorption

**Acidactivated clay** 

adsorption

vacuum distillation

cant oil. Adapted from [66,67].

evaporation

reaction

distillation 

dehydration thin film

**Extraction** dehydration solvent

**treatment** dehydration vacuum

**Thin/clean film** 

**Solvent** 

**Hydro-**

(74%) 

(85%) 

filtration Recovery

with clay filtration Recovery

hazardous contaminants [35]. Following the logic of the circular economy, the ideal regeneration method for used lubricating oils should guarantee the function the product had at the beginning, as many times as possible after a treatment is conducted. **Regeneration** Acid treatment

Solid waste and water

*Recycling* **2021**, *6*, x FOR PEER REVIEW 14 of 23

**Used lubricating oil**

Storage (mixed oil)

**Pre-treatment** Filtration Centrifugation Magnetization Decanting Sedimentation Heating

For the finishing step, excellent results have been obtained with the use of bentonite [44,50,60,61]. This is the process that appears most suitable for used oil treatment in Cuenca, due to the large amounts of bentonite available in the province of Napo [64], located ~400 km from Cuenca. Bentonite can be used as bleaching land to clarify and reduce (by adsorption purification) the intense color of the oils [65]. It is expected that used oil generators (e.g., mechanic and car lubricating shops) in the city will play a vital role in the collection system as they are responsible for the handling, separating, and storing operations for different types of used oils before delivering them to authorized collectors to ensure proper treatment [22]. Caustic treatment Actived carbon / clay treatment Acid clay treatment Irradiation and ultrasonic adsorption Solvent extraction **Base fractionation** Vacuum distillation Consecutive vacuum and Sludge (heavy metals, chlorinated hydrocarbons, and polyaromatic compouunds) Solvent recovered

In Ecuador, in the city of Quito, the re-refining of used lubricating oils is carried out, in this process methods such as filtration, extraction by solvents, and vacuum distillation are applied, while in the city of Cuenca only filtration, decantation, and sedimentation is performed; in Figure 3 these processes are marked in red. atmospheric distillation **Finish** Adsorption Re-refined base oil Additive metals

At an industrial scale, the main technologies employed for re-refining used oils are (a) the acid/clay process; (b) activated clay process; (c) thin-film evaporation (TFE), based in vacuum distillation; (d) solvent extraction process; and (e) hydro-treatment process (hydrogen extraction) [58,65,66]. The steps for each re-refining technology and the yields that can be recovered from the process are presented in Figure 4. Neutralization Decanting Filtration + Additive New Lube oils **Figure 3.** Main stages of conventional technologies for the re-refining of used oils. Adapted from

[35].

**Figure 4.** Summary of the technologies employed at the industrial level for re-refining used lubri-**Figure 4.** Summary of the technologies employed at the industrial level for re-refining used lubricant oil. Adapted from [66,67].

cant oil. Adapted from [66,67].


**Table 7.** The main technologies employed at an industrial scale for re-refining used lubricating oils.

Details on each technology and the corresponding advantages and disadvantages are presented in Table 7. Other new technologies are currently available, especially in Europe and China, including the combined finishing of TFE and clay, TFE and solvent finishing, TFE and hydro-treatment, thermal deasphalting (TDA), clay finishing, and TDA and hydro-treatment. Kupareva et al. [66] presented an exhaustive review of the technologies employed at the industrial scale for re-refining used oils in Europe and CleanOil in China, as well as a list of the companies/countries using those technologies (see Table 8). Most of those commercial plants use the technologies described above, with small modifications in some cases.

The selection of a specific technology for re-refining used oil at an industrial level involves several criteria [67,68], including (a) technical and sustainability aspects; (b) health, safety, and environmental impacts; and (c) economic considerations. The thinfilm evaporation (TFE) technology appears to offer the best results (in terms of product quality), followed by the solvent extraction, the hydrotreatment, and the activated acid–clay and acid–clay technologies. However, the TFE technology is economically viable only at large scales. The minimum capacity needed for a plant with this technology, according to Kupareva, is around 40,000 t/year (see Table 8), which is several times higher than the ~1534 t/year of used oil collected in Cuenca. Therefore, TFE technology is not currently viable in Cuenca.

The hydrotreatment and the activated acid–clay and acid–clay technologies, however, are incapable of producing products with adequate quality while possibly creating environmental problems [67] (see Table 7). Thus, these technologies are not appropriate compared to the TFE or the solvent extraction technologies. For these reasons, in this study, we believe the solvent extraction technology is the best option for re-refining used oils in Cuenca. The solvent extraction process is more adequate for processing relatively low volumes of used oils and the quality of the product complies with engine requirements. The oil recovery rate is relatively high (around 74%) and the process accepts both synthetic and mineral oils used in vehicles [66]. The solvents can be recovered and reused in the process. Moreover, this technology is widely used in other countries [66]. Since the physical properties of the used oils in Cuenca resemble those of countries such as the UAE [44], Iraq [53], and Egypt [56], which recover oils through solvent extraction, the use of this technology is justified and currently feasible.


**Table 8.** The main re-refining processes currently used in European countries and China (adapted from [66,71]).


115

The pretreatment of used oil that ETAPA currently carries out (water and sludge removal through sedimentation, centrifugation, and filtration) is similar to that used in the UAE [44] and Egypt [56]. Thus, the preliminary steps required for the solvent extraction process are already being performed. In the re-refining process through solvent extraction, a 1:3 oil-to-solvent ratio is proposed (where the solvents are blends of butanol + toluene + methanol or butanol + KOH), following the processes used in the two aforementioned countries [44,56]. The solvent extraction process is followed by a vacuum distillation step for the recovery of solvents that will be reused in the process to remove metals (especially Pb and Fe) [44]. For the finishing step, different adsorbents can be used. However, better results are obtained with alumina, bentonite, and activated bentonite. The use of almond and palm kernel powders can also be effective [44]. At the moment, ETAPA's Used Oil Collection Program sends the filtered and ceded oil sludge to the ECOTECNO Foundation attached to HOLCIM, where a thermal destruction of the waste is carried out with the endorsement of the local and regional Environmental Control Entity. The waste generated by the re-refining plants in Europe and China are used as a component in asphalt preparation and as fuel in boilers [66]. These could be an option for re-using the wastes of the re-refining processes since the city has a small asphalt production plant that could process all this by-product. The development of a re-refining plant with a capacity to process higher amounts of used oils than that currently collected in the city is suggested. Our estimates indicate that there could be up to 7330 t/year of used oil to be processed in the proposed plant (which is around 10% of the total lubricant oil consumed in Ecuador; see Section 1). This amount would result from (a) a complete recovery of used oil in Cuenca; and (b) the contribution of neighboring cities in the South of Ecuador (e.g., Loja, Azogues, Machala, and other smaller cities) that are located at distances less than 220 km from Cuenca (Figure 5). According to [72], the added total number of cars in these cities is approximately double that in Cuenca. However, further work is necessary to better define the collection, transport, and storage logistics of used oil in those cities to comply with the requirements of a re-refining plant and Law 042. The benefit for Ecuador is enormous since the import of up to 7330 t/year of lubricant could be avoided in these conditions. In addition to environmental and social benefits, the country could save around US\$ 30 million/year, according to data from the Central Bank of Ecuador [73], due to avoiding the import of lubricant oils. *Recycling* **2021**, *6*, x FOR PEER REVIEW 19 of 23 The pretreatment of used oil that ETAPA currently carries out (water and sludge removal through sedimentation, centrifugation, and filtration) is similar to that used in the UAE [44] and Egypt [56]. Thus, the preliminary steps required for the solvent extraction process are already being performed. In the re-refining process through solvent extraction, a 1:3 oil-to-solvent ratio is proposed (where the solvents are blends of butanol + toluene + methanol or butanol + KOH), following the processes used in the two aforementioned countries [44,56]. The solvent extraction process is followed by a vacuum distillation step for the recovery of solvents that will be reused in the process to remove metals (especially Pb and Fe) [44]. For the finishing step, different adsorbents can be used. However, better results are obtained with alumina, bentonite, and activated bentonite. The use of almond and palm kernel powders can also be effective [44]. At the moment, ETAPA's Used Oil Collection Program sends the filtered and ceded oil sludge to the ECOTECNO Foundation attached to HOLCIM, where a thermal destruction of the waste is carried out with the endorsement of the local and regional Environmental Control Entity. The waste generated by the re-refining plants in Europe and China are used as a component in asphalt preparation and as fuel in boilers [66]. These could be an option for re-using the wastes of the re-refining processes since the city has a small asphalt production plant that could process all this by-product. The development of a re-refining plant with a capacity to process higher amounts of used oils than that currently collected in the city is suggested. Our estimates indicate that there could be up to 7330 t/year of used oil to be processed in the proposed plant (which is around 10% of the total lubricant oil consumed in Ecuador; see Section 1). This amount would result from (a) a complete recovery of used oil in Cuenca; and (b) the contribution of neighboring cities in the South of Ecuador (e.g., Loja, Azogues, Machala, and other smaller cities) that are located at distances less than 220 km from Cuenca (Figure 5). According to [72], the added total number of cars in these cities is approximately double that in Cuenca. However, further work is necessary to better define the collection, transport, and storage logistics of used oil in those cities to comply with the requirements of a re-refining plant and Law 042. The benefit for Ecuador is enormous since the import of up to 7330 t/year of lubricant could be avoided in these conditions. In addition to environmental and social benefits, the country could save around US\$ 30 million/year, according to data from the Central Bank of Ecuador [73], due to avoiding the import of lubricant oils.

**Figure 5.** Map of Ecuador showing Cuenca and some neighboring cities that could supply used lubricant oils for the operation of a re-refining plant.

#### **4. Conclusions**

Disposing of used lubricating oils by direct incineration causes damage to the environment while losing a non-renewable resource. A circular economy requires the reuse of used lubricating oils to reduce waste and environmental damage while promoting social benefits. While the current used oil collection system in Cuenca is primarily focused on avoiding spills and the collection of used oils, strategies to re-refine and therefore add value to these materials are important steps to consider. The work has found that the storage conditions of the used oil in the generators' premises do not significantly affect its properties. This result shows that the system currently used by used oil generators in Cuenca can be used for further processing and no changes to the system or logistics are required. The characterization of used oils in the city has shown that most of the properties are comparable to those of used oils in other countries (for example, the United Arab Emirates, Iraq, and Egypt). Some critical properties, such as the water content, are below those reported in the literature, which helps the re-refining process. Consequently, technologies to recover and add value to used oils in Cuenca must consider the experiences of other nations in the processing of similar used oils. European industries show great progress on the subject, as well as good recovery rates and quality of the regenerated product. The union of several methods improves the final product even more, but the economic investment increases. However, small- and medium-sized cities such as Cuenca do not generate large amounts of used oil in order to have plants with the same capacities as those in Europe. Therefore, the selection of treatment technology must also consider aspects such as the available market, operating costs, transportation, energy, and the quality of oil to be obtained. Due to the conditions in Cuenca, a good option is the solvent extraction process since it can be adapted by small-scale plants, providing adequate quality and performance of the products, as well as reducing negative environmental impacts. For the finish (improvement of color and odor), bentonite, a material that is available near the city, can be used. Furthermore, in Cuenca the initial step is already being carried out; that is, the filtration/dehydration and sedimentation process. Re-refining used oils in Cuenca and other medium-sized cities, whether in Latin America or elsewhere, is necessary to comply with the principles of a circular economy, taking advantage of non-renewable resources and contributing to the economy, society, and environment.

**Author Contributions:** Conceptualization, C.S.-A., J.C.-B. and D.A.-A.; methodology, C.S.-A., J.C.-B., M.R.P.-S. and D.A.-A.; sampling, J.C.-B. and D.A.-A.; software, C.S.-A. and F.G.-Á.; validation, C.S.-A., J.C.-B. and M.R.P.-S.; formal analysis C.S.-A., J.C.-B. and M.R.P.-S.; investigation, C.S.-A., J.C.-B., F.G.-Á. and D.A.-A.; data curation, C.S.-A. and F.G.-Á.; writing—original draft preparation, C.S.-A., J.C.-B. and F.G.-Á.; writing—review and editing, C.S.-A. and M.R.P.-S.; funding acquisition, J.C.-B. and D.A.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Research Department of the University of Cuenca (DIUC) through the project "Análisis y definición de estrategias y escenarios para el desarrollo de sistemas de mantenimiento industrial orientado a la eficiencia energética y amigable con el ambiente en la ciudad de Cuenca".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** The authors acknowledge Raul Pelaez-Garcia for English editing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

