Study of The Biodegradation of Poly (3-Hydroxybutyrate) (PHB) and High-Density Polyethylene (HDPE) by Microorganisms from the Sea waters of the Atlantic Coast of Brazil

The objective of this study was to evaluate the biodegradation of Poly (hydroxybutyrate) (PHB) and high-density polyethylene (HDPE) in static systems, using as uid the seawater of the Coastal Region of the State of Pernambuco (Brazil). The physical and chemical modications of the polymers, as a function of biodegradation, were evaluated by Fourier transform infrared spectroscopy (FTIR), mechanical tensile assay, differential scanning calorimetry (DSC), gravimetric test, and microbiological analysis. Through the FTIR, it was possible to observe in the PHB a decrease of 23.22% in the carbonyl index for the crystalline phase and 32.30% in the amorphous phase after 180 days, which evidences the effect of the biodegradation present. The mechanical properties of PHB were altered with biodegradation, but the thermal properties remained. During the gravimetric tests, there was a reduction in mass and consequently higher degradation rates for PHB, which is corroborated by the microbiological tests of the system. All characterizations demonstrated that the surface of the HDPE is less susceptible to biolm formation and, consequently, to the enzymatic action of microorganisms. After 180 days of immersion, no signicant microbiological degradation was observed in the HDPE, except for some abiotic alterations.


MATERIALS
The investigation was carried out with PHB -poly (hydroxybutyrate), and HDPE -high-density poly (ethylene). The analysis uid used in the experiment was collected at a point in the Region of Marco Zero, Recife-PE, speci cally at latitude 8º3'15" and longitude 34º52'53".
The PHB is naturally produced by a fermentative process of some bacteria, and depending on the species, there may be characteristics that in uence properties from one batch to another. The PHB used in this study was produced by the bacterium Burkholderia sacchari. The material was supplied in powder form by PHB Industrial S/A (Serrana, SP). And high-density polyethylene (HDPE) was obtained in the form of small grains (pellets), and supplied by the thermoplastic company of Nordeste Eteno LTDA. Table  1 presents the main properties of these polymers. The experiments were conducted in static systems using glass bioreactors. The specimens were suspended by nylon wires and attached to the bioreactor lid ( Figure 1), in order for the test bodies to be exposed at the same depth in the uid, for a period of up to 180 days.
For the collection of samples, intervals ranging from 7, 14, 30, 60, 90, 120, and 180 days of experiments were used, counted from the date of assembly of the bioreactors. For each analysis in the aforementioned interval, ten previously numbered and weighed specimens were removed.

Extruder processing
The polymers were processed in SENAI -CIMATEC, in modular IMACON extruder, with double thread, corotating, with ratio L/D = 30 and thread speed equal to 250 RPM. These materials were processed in the form of threads that passed through a stainless-steel vat, containing water at room temperature, in order to reduce the temperature gradient. Table 2 shows the processing temperature conditions for the polymers processed in the extruder.  PHB  145  145  145  150  155  155  155  160  175   HDPE 165  165  170  170  175  175  180  185  185 Source: Author, 2021 The next step was pre-drying by a compressed air collimator jet. Following, the formation of pellets the SAGEC equipment, model SG70, 220 v, with rotating knives, at a speed adjusted with the production of the thread.

Injection
After granulation and pellet formation, the materials were placed in trays in an air-circulation oven (PALLEY) at a temperature of 100 °C and kept for a period of 4 hours, in order to improve drying and thus mitigate the electrolytic degradation of these materials during processing [21]. The specimens were molded by the injection process in SENAI -CIMATEC, according to ISO 527 standards for tensile testing [22]. For this stage, a Primax ROMI injector with a capacity of 100 tons of closing force was used. Table 3 shows the injection conditions of the polymers under study.

Analyses of sessile-organisms
The cell concentrations of the microbial groups present in the bio lms were determined according to the periods of removal of the specimens to evaluate the biodegradation. The water inside the bioreactors was replaced every fourteen days, which is the time to exhaust the nutrients present in the seawater [23]. To quantify the sessile microorganisms, the specimens removed were placed in containers with 30 ml of saline solution and/or 30 ml of the reducing solution for the analysis of aerobic and anaerobic heterotrophic microorganisms, respectively. Then, these samples were submitted to the ultrasonic bath for 15 seconds to ensure the removal and dispersion of sessile microorganisms. The bio lms formed on the surface of the polymeric test bodies were removed by scraping the surface with a sterile spatula in the appropriate solutions. All procedures were performed in aseptic conditions.
Microorganisms were quanti ed using the following methods: a) aerobic heterotrophic bacteria (AHB) and Pseudomonas spp.: according to the methodology described by Silva

Gravimetric Assay (Biodegradation Rate)
In the mass loss tests, the specimens were weighed before the start of the process and at the end of the process. Mass loss was quanti ed by the difference between its original weight and its weight after the test. For the calculation of the biodegradation rate of polymers, the average of 5 specimens was considered. When removed from the systems, the bio lms were removed from the specimens, then washed with distilled water, ethyl alcohol, and acetone, and were subsequently dried with warm air.

Mechanical tensile test
The tensile tests, before and after the biodegradation process, were performed at SENAI -CIMATEC, in a universal MACHINE EMIC, model DL 2000 with a load cell of 20 kN. Each test used 5 test bodies, with procedures and dimensions speci ed by ISO 527. The data of the mechanical test were analyzed through analysis of variance (ANOVA) using the software Statistica, version 10.0.228.8.   In the intensity of absorbance at 1748 cm -1 , corresponding to the carbonyl group (C=O), there was a difference when the specimens were submitted to seawater, similar to that occurred in the literature [28], which reports the decrease in the intensity of the carbonyl band with the test time in the water of the Atibaia River.

FTIR
In general, the spectra found are similar ( Figure 2), but the form used to quantify the degradation of polyesters is the determination of the presence of terminal carboxylic groups in the polymer. This can be used to quantify degradation because the formation of these groups is due to the degradation of polyester by the microorganism producers of extracellular esterase. For a better resolution of the bands, a deconvolution was performed, through the Lorentzian function, of the PHB spectra ( Figure 3) for the normalization of the absorbance intensity of a band considered as an internal standard at 1380 cm -1 , attributed to the deformation of CH groups [29,30].
From the values obtained with deconvolution performed on the graphs in Figure 2, it was possible to calculate (Equation 1), analyze, and compare the carbonyl indices of the amorphous phase and the crystalline phase of PHB initial and after biodegradation ( It was observed that the indexes of terminal carboxylic groups for all samples showed a decrease in this functional group during the degradation time. The results showed a decrease of 23.22% in the carbonyl index for the crystalline phase and 32.30% in the amorphous phase after 180 days. The decrease in absorbances related to carbonyl indices, both in the amorphous and crystalline phase, evidences the effect of present biodegradation, resulting from the action of esterase that probably occurred in carbonyl carbon of the ester bond.
Thus, PHB samples submitted to microbial treatment in seawater were biodegraded by the action of microorganisms in this biome, occurring hydrolytic reactions of ester groups and oxidative breaks of the polymer chain.
Observing Figure 4, we notice the presence of three absorption bands in the HDPE, these three bands refer to the different vibrations of the C-H bond.
The presence of a band at 1464 cm -1 , can be attributed to angular deformation outside the plane of CH 2 , which does not vary depending on the degradation time. Figure 5 shows the expanded FTIR spectra in the region from 1800 to 1400 cm -1 , which is the region of interest.
Comparing Figures 4 and 5, it is possible to observe that the periods of immersion in seawater did not cause changes in the formation of carbonyl groups or double bonds that indicate the process of biodegradation of the HDPE [31].
One of the characteristics of this polymer is hydrophobicity, which can hinder enzymatic attack, evidencing a great resistance to biodegradation with microorganisms present in the studied uid. But Liyoshi et al. (1998) [32] submitted three species of lignin-degrading fungi, including P. chrysosporium, to degradation tests with the HDPE and concluded that manganese peroxidase enzymes caused signi cant degradation of this polymer, suggesting that this enzymatic complex may be the key to polyethylene degradation. This is because the biodegradation of the material is linked to the type of enzyme, microorganism, temperature, and exposure time [18].

Differential Scanning Calorimetry (DSC)
DSC analyses were used to investigate the crystallization and fusion events of PHB and HDPE before and after exposure to the marine environment for periods of 7, 14, 30, 60, 90, 120, and 180 days. The samples were initially heated to erase the thermal history, and then cooled quickly and heated again. Table 6 presents the parameters of crystallization temperature (Tc), melting (Tm), and the most endothermal heat (ΔH) and crystallinity (ΔX).
For the PHB sample ( Figure 6), during cooling, a peak of cold crystallization (C1) was detected for the PHB/0 days. From 7 days of exposure to the marine environment, a very subtle peak appears around 49 ºC and remains with more advanced exposure times, resulting from a possible reordering of the crystalline structure of the PHB due to the attack of microorganisms. Some authors raise the hypothesis of inter-spherulitic segregation [33].
Then, during the second PHB/0 day heating, two thermal events are observed: (1) an exothermic peak associated with a crystalline reordering of PHB or secondary crystallization (C2) [33]; and, (2) the fusion of crystals, which occurs in the range of (159 °C). The fusion of these structures is characterized by a double peak, which could be explained in terms of fusion/recrystallization processes [33]: peak I is characterized by a shoulder around 159°C, corresponds to fusion of imperfect crystals, while peak II, peak itself, around 166.3°C, corresponds to the fusion of perfect crystals produced by crystallization during cooling.
A subtle decrease in crystallinity was observed in the rst 60 days, and an increase in the crystallinity of the samples from 90 days on. Due to the hydrolytic scissions of the chain segments, increases in the degree of crystallinity and melting temperature indicate reorganization of the remaining polymer chains after the consumption of the amorphous phase and increased thickness of the lamellae.
With regard to crystallization in cooling, it can be seen in Figure 7 that the HDPE presents a narrow and well-de ned peak around 118, characteristic of the formation of well-uniform crystals. Table 4 shows that crystallization temperatures did not change with increased exposure to the marine environment, and only a discrete shift of Tc after 180 days of exposure can be seen.
In reheating, only a single endothermic peak is detected, close to 130 ºC, the melting temperatures of the polymers after exposure remained almost equal to the melting temperatures of the HDPE without exposure. For the HDPE, there was an increase in its crystallinity of 81.6%, with 60 days of exposure to the marine environment. The crystallinity of polymeric materials is often one of the factors that hinder the access of microorganisms to biodegradation, that is, amorphous parts are preferably attacked, consequently the crystallinity of the HDPE increases by the division of its chains, followed by subsequent recrystallization of smaller chains.
It can also be observed a decrease in crystallinity after 60 days of exposure to the marine environment, the reason for this could be the lack of homogeneity introduced from the formation of the bio lm during exposure that can act as obstacles during the growth of crystalline lamellae. Crystalline lamellae have to overcome the obstacle that slows the general crystallization process [34].
Bio lms are materials composed of extracellular polymeric substances excreted by microorganisms, favoring the adhesion of cells on the surface of the material and on other cells [35]. During the stages of biodegradation evaluation, it is common to observe the formation of a bio lm on the surface of the exposed materials. With the formation of bio lm, microorganisms, the metabolism of the beings involved, produce several enzymes and substances such as acids that interact with the surface of the polymer promoting chemical reactions responsible for degradation [36,37].

Seawater Plankton Bacteria
The cellular concentrations of aerobic heterotrophic plankton bacteria (AHB), iron precipitants (IPB), anaerobic heterotrophic bacteria (AnHB), and sulfate reducers (SRB) quanti ed in seawater, studied in this study, were quanti ed and presented in Figure 8. mentioned, the concentrations obtained were lower than those found in the present study for all plankton groups, except for IPB that presented lower concentrations.
The presence of these bacteria in seawater evidences a medium with potential for the occurrence of biodegradation. These analyses can generate different results, as it is in uenced by the location, time, climatic conditions, and other factors [40]. An analysis in the graphs allows us to observe that, at 7, 14 and 30 days of the experiment (Figures 9, 10, and 11) the aerobic microbiota (AHB, IPB, and lamentous fungi) had higher sessile concentrations for the PHB polymer. Only at 7 days, the lamentous fungi had similar sessile concentrations for the two polymers. Pseudomonas spp. were only quanti ed at 7 days for PHB polymer. The sessile concentrations of anaerobic microorganisms (AnHB and SRB) were also bene ted on the surfaces of PHB polymers in these experiment times. With 14 days, the AnHB had similar concentrations for the two polymers. These higher concentrations of the microbiota for PHB demonstrate that this polymer is more susceptible to the adherence of microorganisms, with biodegradation initiated within fewer days than the HDPE, as shown in the graph in Figure 11.

Sessile Bacteria in Different Systems
It is also important to highlight that aerobic bacteria (AHB and IPB) showed a reduction in their growth from 7 to 14 days, probably due to nutrient de ciency and/or oxygenation of the medium. This reduction in the concentration of these microbial groups may also be related to the increase in sessile exopolysaccharides (SEP), because the IPB aggregate iron precipitates in the bio lm, decreasing oxygenation and leading to the growth of anaerobic heterotrophic bacteria, as shown in Figures 9, 10 and 11 [41]. From 30 days of the experiment, the concentrations of anaerobic microorganisms increased continuously up to 180 days. In the nal times analyzed, the aerobic microbiota (AHB and IPB) also had high concentrations, Pseudomonas spp. were again quanti ed in the systems and the lament fungi maintained a low concentration in the two polymers ( Figure 12, 13, and 14).
As already discussed in the literature, PHB is a natural polyester, that is, it is produced by bacteria as an intracellular energy reserve. Thus, this material is compatible with the mechanisms of bio assimilation of smaller chains, generated from hydrolysis and biological oxidation promoted by enzymes produced by bacteria and fungi [42].
However, for the HDPE, its hydrophobicity limits the interaction between the polymer and enzymes, hindering their attack and, consequently, biodegradation (Figure 4) [43]. Therefore, the count in the initial periods, 7, 14, and 30 days of the experiment were lower in the HDPE, since this polymer did not serve as a source of carbon to microorganisms. Over time, microorganisms start to develop new enzymes, which facilitate biodegradation, and the microbial population increases concentration [28].

GRAVIMETRIC BIODEGRADATION ASSAYS
The gravimetric assay is the most commonly applied and relatively sensitive method for determining changes caused by microbial attack in polymers [44]. Figure 15 shows the pro le of the degradation rate of PHB and HDPE over 180 days of the experiment.
Two different degradation pro les are observed, comparing polymers, higher degradation rates for PHB are shown. This is corroborated by the microbiota of the systems, shown in Figures 9, 10, and 11, which demonstrated that the surface of the HDPE is less susceptible to the formation of bio lms and, consequently, to the enzymatic action of microorganisms.
This can be explained by the chemical composition of polymers, PHB contains in its structure carboxylic groups, which are hydrophilic. The HDPE, because it is formed by hydrophobic chemical groups, hinders the enzymatic action of microorganisms on the polymer surface [45] con rming what was presented by the analysis of the chemical structure of polymers by FTIR.
Through mass loss analyses, it was observed that biodegradation can be favored, depending on the microbiota of the uid, exposure to abiotic agents, and the structure of polymers. Table 5 presents the results of the stress at rupture, modulus of elasticity and speci c strain for the PHB before and after the inoculation process. Table 5 shows that after the periods of exposure of the specimens to the marine environment, there were changes in all mechanical properties analyzed as a function of time. However, from the exposure time of 30 days to 90 days, the rupture stress, the elastic modulus, and the speci c strain did not undergo signi cant changes in their values. During the biodegradation process, the surface and interior of the specimens are lled with voids, facilitating the propagation of cracks during the tensile test, resulting in the decrease of mechanical properties [46]. For this reason, it was not possible to perform the tensile tests of the specimens of 120 and 180 days. PHB is a very hard and fragile material, has a low viscosity, and is a polymer with a high modulus of elasticity [47]. Evaluating the modulus of elasticity in traction of the initial PHB and after the biodegradation process, it is observed, in general, that with the time of exposure to the marine environment this property is decreased.

Traction
The modulus of elasticity is a measure of the stiffness of the material, that is, it is directly related to the material's ability to resist deformations [48]. For better visualization of the behavior of the samples, the elastic module graphs ( Figure 16a) and Rupture Stress (Figure 16b) were plotted: Analyzing the modulus of elasticity (Figure 16a), there was a decrease of 26.9% after 7 days. In the 14day period, the modulus of elasticity showed no signi cant difference in relation to the previous point.
Behavior that corroborates the results of mass loss ( Figure 15) and counting of microorganisms ( Figure   10), which did not present alterations at these times. In the times of 30, 60, and 90 days, the elasticity modules showed non-signi cant decreases over time. Figure 16b indicates the behavior of the maximum tensile stress found for the studied times. It can be observed that the maximum tensile stress values gradually decreased over time. This property corresponds to the greatest tension that the material can resist, if this tension is applied and maintained, the result will be the fracture.
Through the data obtained in the tensile tests, it is veri ed that the PHB presents fragile behavior. Elastic deformation in the polymer occurs due to its crystalline part, which provides resistance to the material and still provides elasticity due to the relative mobility of crystalline chains [49]. As most of the PHB structure is amorphous, it breaks before the crystalline portion of the material, causing irreversible deformations, which lead to decreased material resistance and consequently rupture of polymer chains.
It is noted that the mechanical properties of PHB samples were in uenced by the conditions of the biodegradation tests. It can be explained by a possible decrease in the molecular mass of PHB. According to Montoro (2010) [50], molar mass is an extremely important factor, because it directly affects the mechanical resistance of the polymer, the ability to swell, the ease of being hydrolyzed, and, consequently, the rate of biodegradation, which is related to its crystallinity. Table 6 and Figure 17 show the results of the stress at rupture, modulus of elasticity, and speci c deformation obtained for the HDPE before and after the biodegradation process. It is observed that the HDPE is highly tenacious, that is, it is capable of suffering a high degree of deformation before breaking, and presents low tensile strength and low modulus of elasticity ( Figure   17a).
The modulus of elasticity and maximum tensile stress did not undergo signi cant changes after 7 and 14 days. The elasticity modules for the HDPE after 30, 60, and 90 days immersed in seawater differed by 3.5%, 3.54, and 4.2%, respectively, when compared to HDPE before the experiment. A fact probably resulting from the increase in population density of microorganisms present on the surface of the material, since the compositions are practically similar to those of the pure material. The results show that the HDPE presented behavior similar to those reported in the literature by Elleuch and Taktak (2006) [51].

Conclusion
For the period of time studied, the microorganisms present in the water collected in the Marco Zero region in Recife-PE were able to biodegrade the PHB. The HDPE remained resistant to microbial action. PHB biodegradation occurs proportionally in the amorphic and crystalline phases and for the HDPE there were no changes in the bands that indicate the biodegradation process. The PHB showed higher rates of degradation when compared to HDPE, demonstrating that the surface of PHB is more susceptible to the formation of bio lms and, consequently, to enzymatic actions of microorganisms. Analyzing the results obtained and comparing with the HDPE, it was observed that after the inoculation periods there were changes in all mechanical properties analyzed for PHB. The HDPE, on the other hand, did not undergo signi cant changes. Impacted environments such as the Marco Zero region of Recife-PE are signi cant in prospecting microorganisms capable of biodegrading polymers.
Bioreactor of static systems Values for elastic modulus (a) and rupture stress (b) obtained before and after HDPE biodegradation