PHB production by Bacillus megaterium strain MNSH1-9K-1 using low-cost media

Abstract

Plastics are widely used for diverse applications due to their versatility. However, their negative impact on ecosystems is undeniable due to their long-term degradation. Thus, there is a rising need for developing eco-friendlier alternatives to substitute fossil-based plastics, like biopolymers. PHA are synthesized intracellularly by microorganisms under stressful conditions of growth and have similar characteristics to conventional polymers, like their melting point, transition temperatures, crystallinity, and flexibility. Although it is feasible to use biopolymers for diverse industrial applications, their elevated production cost due to the supplies needed for microbiological procedures and the low productivity yields obtained have been the main limiting factors for their commercial success. The present study assessed the ability of Bacillus megaterium strain MNSH1-9K-1 to produce biopolymers using low-cost media from different kinds of fruit-peel residues. The results show that MNSH1-9K-1 can produce up to 58 g/L of PHB when grown in a medium prepared from orange-peel residues. The data obtained provide information to enhance the scalability of these kinds of biotechnological processes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01232-7.

Introduction

Synthetic plastics are used in many different applications, as they are a family of versatile materials. Because of their desirable characteristics for industrial purposes, such as high stability and malleability, their use may be seen as unavoidable [1]. However, there is a global awareness of the environmental impact of these fossil-based polymers due to the major problems caused by their long-term degradation. Thus, the rising need to find eco-friendlier alternatives to substitute conventional plastics has led to the exploration of products of biological origin, like polyhydroxyalkanoates (PHA) [2].

PHA are industrially compostable bio-based polyesters [3] produced by a wide variety of microorganisms (i.e., bacteria, and microalgae) and transgenic plants [4–6], and efforts have been made for their study, due to PHA potential uses [7]. These compounds have similar characteristics to their petrochemical-derived counterparts, like their melting point, transition temperatures, crystallinity, and flexibility [8]. In general terms, PHA are thermoplastic or elastomeric, and their sufficiently high molecular mass provides them with properties similar to those of conventional polymers, such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), or polystyrene (PS) [9].

The macromolecules of microbial biopolymers are synthesized intracellularly from fatty acids and other aliphatic carbon sources and accumulated within the cytoplasm as carbon and energy reserve when the cells are grown under unbalanced conditions; as a precaution against nutrient starvation and stressful environments [3]. Specifically, it has been reported that PHA accumulation often occurs when microorganisms are subjected to an excessive amount of a carbon source, while other nutrients like N, P, Fe, S, Mg, or K are limited [10], as well as oxygen and pH considerations [11, 12].

Diverse Gram-positive and Gram-negative bacteria possess the ability to produce PHA [2, 13], among which the genus Bacillus is distinguished [14–16]. Even when the use of PHA seems to be a promising alternative to substitute traditional polymers, the elevated cost of culture media and the low bacterial productivity yields are the main limiting factors to its commercial success. In this respect, some viable options for inexpensive carbon sources to produce PHA, such as food and industrial waste, have been previously reported [17], like wafer residues, cane molasses, hydrolyzed citrus pulp [18], and papaya waste [19]. Some studies also reported the feasibility of the use of orange juicing waste and fruit-peel residues as low-cost sources to prepare inexpensive culture media for bacterial PHA obtention [13, 20], Specifically, orange peels have been previously identified as a viable carbon source for PHA production, using strains of Bacillus spp. and Pseudomonas sp. [21, 22].

In the present work, the ability of the strain Bacillus megaterium MNSH1-9K-1 to produce PHA was assessed using different kinds of fruit-peel residues as sources for the elaboration of low-cost culture media. Due to the results obtained, the production of the biopolymer using the medium from orange-peel residues was studied in further detail. Also, the PHA obtained was characterized to corroborate its nature. Undoubtedly, the world is heading toward the need for circular and sustainable approaches, so a strong emphasis needs to be given to the research and establishment of methodologies regarding the efficient and long-term use of bioresources.

Materials and methods

The strain used and general growth conditions

The bacterial strain used for this study is B. megaterium MNSH1-9K-1 (GenBank accession number KM654562.1), which was isolated from a high-metal content site in Guanajuato, Mexico [23]. Pre-inoculums were grown overnight in 125-mL flasks containing 10 mL of Nutrient Broth (NB), and a total of 1×10⁶ cells were further used to inoculate 125-mL flasks with 50 mL of medium. Microbial growth was performed at 150 rpm and 30 °C, and determinations of total cell counts were done using a Neubauer chamber. Fruit peel residues of Musa acuminata (banana; Ban), Citrus sinensis (orange; Ora), Carica papaya (papaya; Pap), Citrullus lanatus (watermelon; Wat), and Cucumis melo var. cantaloupensis (cantaloupe; Can) were used to prepare low-cost media, following the protocol described by Figueroa-Ocampo [24] based on extraction by infusion [25]. To this end, peels were manually separated from fruits followed by a wash with tap water. Once washed, the peels were cut into pieces of ~ 3 cm and dried at 75 °C for 24 h. Subsequently, 10 g of peels were added per 200 mL of boiling water and, without further heating, let them rest in the hot water for 120 min. The infusions obtained were filtered to eliminate solids, sterilized by autoclave, and stored at 4 °C. The resultant sterile infusions were diluted in half with sterile distilled water before being used for experimentation. NB medium was used as a control since it is commonly employed for bacterial growth.

Afterward, this protocol was improved to enhance PHA production in Ora medium [26, 27], as follows: once washed, the peels were cut into pieces of ~ 1 cm, which were dried at 65 °C for 24 h. Then, 10 g of peels were added per 100 mL of boiling water, letting them rest in the hot water for 30 min. Then, the infusion obtained was filtered, pH adjusted to 7 using NaHCO₃ 1M, and processed as previously explained. Improved PHA obtention is expressed in Table 1 as N_x/N_A ratio, where N_x is the PHA in g/L obtained in each condition divided by N_A, which is the g/L obtained with the first protocol of Ora medium preparation.

Table 1.

Improvement of PHA production yield in the medium produced from orange-peel residues

Condition	PHA production NX/NA
A (initial)	1.00 ± 0.15a*
B (final)	4.29 ± 0.66b

*Lowercase letters indicate groups of data that were significantly different by ANOVA (P ≤ 0.05)

Quantification of metabolites in culture media

Glucose and protein contents were determined at the beginning of the experimentation. The dinitrosalicylic acid (DNS) method was used with glucose at 0.1% (v/v) as standard. To this end, samples were first diluted 1:4 or 1:8, and 1.5 mL of DNS reagent was added; mixtures were heated (water bath) at 90 °C for 5 min to develop the red-brown color and 10 mL of distilled water was added for color stabilization. Finally, samples were cooled at room temperature in a water bath, and absorbance was measured at 540 nm [13, 28].

For the case of protein assessment, the Lowry method was followed, where a 2 mg/mL BSA solution was used to prepare the standard curve. Five milliliters of the Lowry reagent was added to each sample and incubated for 10 min at room temperature. Afterward, 0.5 mL of Folin-Ciocalteau Reagent was also added followed by a 30-min incubation period. Then, absorbance was measured at 750 nm. Based on the protein content, nitrogen (N) was calculated by its equivalents [N_eq], in which 16% of proteins were considered as N content [13, 29].

Optic microscopy

To observe the bacterial morphology and PHA production after 2 days of growth in Ora medium, a Nikon ECLIPSE Ni-U microscope was used, which allowed a 400- to 1200-fold magnification, as specified for each case. To identify the bacillary cell morphology, simple staining with safranin was performed, for which a drop of the sample was placed on a slide, and 95–100% (w/v) aqueous safranin was added, covering the entire sample for 30 min (until complete dryness). Subsequently, the sample was rinsed with distilled water until all residual safranin was removed; the sample was allowed to dry again and was then observed under the microscope.

To detect cell differentiation, that is, the presence of spores, Schaeffer-Fulton staining was performed [30, 31] according to the broadly known methodology but with some modifications. After the sample was placed on a clean slide and allowed to dry at room temperature (~ 27 °C) for 10–15 min, a piece of filter paper was placed on the sample, and malachite green 5% (w/v) was added until the entire sample was covered. The sample was then heated for 8 min, the filter paper was removed, and the sample was washed with distilled water. Finally, safranin at 95–100% (w/v) was added.

For PHA observation, Sudan Black staining was performed [13, 32], for which a drop of the sample was placed on a slide and fixed under the flame. Sudan Black B 0.02% (w/v) was added and allowed to react for 7 min, and the sample was rinsed with ethyl alcohol at 97% (v/v) until the dye residues were completely removed, then the sample was allowed to dry at room temperature (~ 27 °C) and observed.

PHA extraction, quantification, and characterization

PHA extraction was performed following the protocol previously described by Valdez-Calderón et al. [13]. Briefly, 50-mL samples were centrifuged at 8000 rpm for 15 min after culture growth. Once the supernatant was discarded, biomass was treated with 10 mL of chloroform 99% (v/v) (Meyer) and 10 mL of sodium hypochlorite 10–15% (v/v) (Sigma-Aldrich) per g of biomass; this suspension was incubated at 30 °C for 1.5 h at 120 rpm and then centrifuged at 4000 rpm for 30 min. The lower phase was recovered and 1:1 methanol was added for PHA precipitation, letting it stand for 24 h at 4 °C. Finally, the precipitate obtained was dried at 65 °C and stored at room temperature until further analyses.

For PHA quantification, gravimetric determinations were done in each case using a semi-analytical balance (VELAB®). For the characterization of the biopolymer obtained, UV-Vis spectrometry, Fourier transform infrared spectroscope (FTIR), and proton nuclear magnetic resonance (¹H-NMR) were performed. Additionally, the differential scanning calorimetry (DSC) curve was measured on a differential scanning calorimeter (DSC Discovery 2500, TA Instruments) under a nitrogen flow of 50 mL/min, in the temperature range of 25–300 °C with heating and cooling rates of 10 °C/min on samples in 40 μL aluminum crucibles sealed with pierced lids.

Statistical analyses

Basic statistical parameters and analyses of variance (ANOVA) were performed using the commercial statistical software OriginPro 8.0. Differences in P values ≤ 0.05 were considered statistically significant. To evaluate the effect of carbohydrate and protein content in the production of PHA, a factorial regression analysis of two factors was performed using the software Minitab 19, considering a confidence level of 95%.

Results and discussion

Bacterial growth and PHA production in media elaborated from fruit peel residues

It has been previously reported the suitability of using agro-industrial residues to sustain bacterial growth and PHA production [13, 17]. Thus, to assess the capability of strain MNSH1-9K-1 to grow in low-cost media, the bacterium was grown for 72 h at 30 °C and 150 rpm in media produced from different fruit peel residues, namely: banana (Ban), cantaloupe (Can), orange (Ora), papaya (Pap), and watermelon (Wat) [13], using NB as a laboratory medium control. The results obtained are shown in Fig. 1a, where it can be observed that the strain was able to grow under all media tested, being this capability Can > NB > Ora > Ban = Wat > Pap, in a range of 1.56×10⁷ to 1.00×10⁶ total cells/mL. It is interesting to note that, compared to the laboratory control medium used (NB), the microorganism presented higher viability in Can than in NB, and showed lower growth yields in all the rest of the low-cost media tested, compared to the one observed in NB, which may be reflecting some growth disadvantage or stress during culture growth in Ora, Ban, Pap, and Wat.

Fig. 1.

Bacillus megaterium MNSH1-9K-1 growth (a) and PHA production (b) in media prepared from different fruit-peel residues. Lowercase letters indicate groups of data that were significantly different by ANOVA (P ≤ 0.05)

Regarding the microbial biopolymer production, PHA was obtained between 1.63 and 9.79 g/L (Fig. 1b); the highest productions were observed in Ora, Pap, and Ban, and were similar to the one shown in the laboratory control medium (NB). The lowest productions were obtained in Can and Wat. The differences observed in cellular growth and PHA production could be due to the differential content of nutrients in the media since diverse nutrient composition affects bacterial growth [33] and PHA production [34]. These results seem to agree with previous reports, which stated that PHA production is enhanced during stressful growth conditions [3].

Thus, to explore these facts in further detail, the content of glucose and proteins (and from protein content, the theoretical calculation of N content expressed as [N_eq]) was determined (Online Resource 1); accounting that glucose could be considered as a metabolic marker for carbohydrate use since bacterial species predominantly prefer glucose when other carbohydrates or nutrients are available [35] because it can directly enter the glycolytic pathway [36], and it has been demonstrated that glucose promotes PHA production over other carbohydrate sources [37].

Subsequently, a factorial regression analysis of two factors was performed, to evaluate the effect of glucose and protein contents in the production of PHA (Fig. 2a). The Pareto diagram obtained shows that PHA production, under the conditions tested, is influenced by both protein and glucose presence, although this production seems to be more sensitive to the protein content, and thus to the N content [N_eq], than to the interaction of the concentrations of proteins and glucose, or to glucose content. Additionally, significant differences between the nutrient content can be observed when performing a graphical analysis of the protein and the glucose content of the different media (Fig. 2b). While Pap and Ban present at least 2.5 times more glucose than Ora and NB, Pap presents 4.5 to 7.0 times more proteins (and [N_eq]) than the other media where the highest PHB production was observed (Ban, Ora, and NB). It is also important to mention that, while glucose may be considered in excess in these four media [10], being at least three times higher in Ban, Ora, and Pap than in Wat, the protein content was registered between 1.07 and 7.40 g/L in these media, in contrast to Can and Wat, in which less than 1 g/L of proteins (≤ 0.15 of [N_eq]) were detected and are the media that presented the lowest PHA production. Since PHA production yield was similarly obtained in the low-cost medium Ora than in Pap and Ban, but with lower requirements of proteins and glucose (considering that lower nutrient requirements may benefit further experimentation), Ora was used for the subsequent studies.

Fig. 2.

Comparison of the carbohydrate and protein content in the different low-cost media tested to determine the degree of significance of each factor (a) and to graphically analyze the differences in the nutrient content (b)

PHA production in the medium elaborated from orange-peel residues

A more extensive analysis was performed to study the PHA production of B. megaterium MNSH1-9K-1 using Ora medium. First, it was considered to explore if modifications in the PHA production protocol could enhance the obtention yield of the biopolymer. Thus, diverse trials were made to upgrade the elaboration of Ora medium to increase PHA production, by varying some particular conditions, like the drying process of the peels, the proportion of the compounds used to prepare the medium, the infusion time used to produce the extract for the medium, and the pH of the medium at the beginning of experimentation [26, 27]. It has been reported that over-drying processes of citrus peels cause the loss of nutrients, vitamins, and carotenoids [38], and that the quantity of the materials used and the extraction times in water infusions modify the compounds that can be recovered from organic precursors [39, 40]. In this case, the improved condition was achieved when the peels were dried at a lower temperature and the double quantity of peels was used for the infusion process. Studying these facts in further detail, there were found previous reports showing that the extraction yields of components using water as the solvent of infusions reach more than 90% of their maximum after 4 min of rest in hot water and that these yields are not significantly different after 5 [41], 15, 30, or 45 min of extraction times [42]. Even more, these reports suggest that the size of the biomass used may influence the extraction yields in the first 3 min of the infusion, but not afterward [41]. Then, discarding the minor differences that may have occurred in the degradation of peel components in the drying processes at 65 and 75 °C [43–45], it can be inferred that the amounts of glucose and proteins were approximately doubled in the improved Ora medium compared to the original one (Online Resource 1) since half the volume of boiling water was used to prepare the infusion. Besides, as could be expected, the regulation of a neutral pH at the beginning of the experimentation seemed to benefit the process, as it may have promoted better cellular performance [46]. Once the protocol for the elaboration of Ora medium for PHA production was improved (moving from obtaining yield A to B, where B was achieved by optimizing the Ora medium production methodology), a fourfold increase in the PHA obtention was attained, as shown in Table 1.

Subsequently, PHA production was assessed at the different phases of growth of the microorganism in Ora medium. To this end, B. megaterium MNSH1-9K-1 was grown in this medium for 7 days at 30 °C and 150 rpm, and samples were periodically taken to determine culture growth and PHA production yield. As can be observed (Fig. 3a), the stationary phase of the culture began after 1 day of growth in Ora medium and presumably remained until day 7. However, spore-like structures were detected when performing the total cell count using the Neubauer Chamber to determine bacterial growth. Thus, morphological diversity (presence of vegetative cells and spores) was also determined by the same cell counting process, observing that spores appeared liberated in the medium after 1 day of growth, representing 80.7% of the cell population on this day. From day 2 to day 7, mainly spores could be observed under the microscope (≥ 90%).

Fig. 3.

Growth (a) and PHA production (b) kinetics of Bacillus megaterium MNSH1-9K-1 grown in the medium prepared from orange fruit-peel residues (Ora). Lowercase letters indicate groups of data that were significantly different by ANOVA (P ≤ 0.05). T, total cells; V, vegetative cells; S, spores

Regarding PHA production during the different times of growth, it was found that the culture began this production since the beginning of the experiment and was increased during the exponential growth (Fig. 3b); even more, the PHA production was enhanced when the stationary phase was reached (day 2) and was detectable until day 7. To study, in more detail, the production of the biopolymer during the cell differentiation circumstances, analyses using optic microscopy were performed to observe the bacterial morphology, and the PHA produced by the culture at the time in which the highest production was achieved (after 2 days of growth; Fig. 3b). As shown in Fig. 4, after simple staining with safranin, both bacilli (stained) and non-stained spore-like structures can be observed (Fig. 4a), typical of Bacillus spp., corroborating what was seen during the total cell counting procedure. Besides, a PHA granule covered by a safranin-stained layer is also clearly shown, which are structures known to be formed during the PHA production process [47]. After Schaeffer-Fulton staining, green-stained spores were obtained (Fig. 4b), showing that mature spores are produced, reflecting that the sporulation process is correctly developed in Ora medium and discarding the immaturity of spores [31]. Finally, culture sample staining with Sudan B, and the observation of different fields under the microscope (Fig. 4c and d), showed that some of the PHA produced seems to be contained in granules, and other is being liberated in the culture medium, as aggregations of the biopolymer can also be seen.

Fig. 4.

Micrographs of Safranin (a), Schaeffer-Fulton (b), and Sudan Black B (c, d) stainings of Bacillus megaterium MNSH1-9K-1 culture after 2 days of growth in the medium prepared from orange fruit-peel residues (Ora), to observe cell morphology and PHA production

Interestingly, the highest peak of PHA obtention was observed after 2 days of growth where 91% of the cells were spores. Also, it is important to consider that it seems that once the culture reached its maximum production, the PHA remained in the system and was continuously detected until the end of the experimentation. It has been previously stated that the sporulation process of Bacillus spp., caused by the stressful conditions of growth that can promote biopolymer production, may also diminish these productivities [48], so repression of this differentiation process has been addressed to enhance obtention yields [49]. Contrastingly, it has also been observed that successful sporulation and PHB production may be tightly linked in some Bacillus strains [50].

PHB is synthesized from the central metabolite acetyl-CoA [51]. Although acetyl-CoA is produced by oxidative decarboxylation of pyruvate from glycolysis, it can be also produced by the oxidation of fatty acids or by the oxidative degradation of certain amino acids, as they can also participate in the metabolic pathways that produce acetyl-CoA. Regarding glycolysis, the major carbohydrate entering the pathway is glucose, although other multiple simple sugars can be used, including galactose and fructose [52]. Thus, diverse carbon sources may be used by bacteria for PHA production. In addition, previous reports have shown that the total sugar content (glucose, fructose, and sucrose) in orange peels accounts, on average, for 34% of their dry weight, of which glucose represents around 10% [53], proteins 7%, and lipids 2.5% [54]. Based on this information, it was theoretically calculated that carbohydrate concentration in the improved Ora medium is  ~  83 g/L, plus 3 g/L, and 2 g/L of proteins and lipids, respectively. Thus, there are ~ 89 g/L of total carbon in this medium (already considering the carbon added by the sodium bicarbonate to adjust pH to 7). According to this total carbon content, the production yield (g PHB/g carbon source) is  ~ 0.64 [55, 56]. Hence, the PHB production yield achieved using strain MNSH1-9K-1 in Ora low-cost medium is comparable to those obtained with high-producing bacteria [13, 17–22, 55–58]. Besides, no PHA degradation in the stationary/sporulation phase was observed, which has been reported to be caused by nutrient depletion or stressful growth conditions [50]. Our results suggest that, once the vegetative cells lyse and mature spores are liberated to the medium (Fig. 4a and b), the PHA is also released from these vegetative cells, likely contained in the characteristic water-insoluble granules (Fig. 4c), and liberated to the environment, as observed in the agglutination of biopolymer in the medium (Fig. 4d), which is probably due to the degradation of the proteinaceous layer that covers the PHA granules [59].

Characterization of the PHA obtained

To determine the nature of the PHA obtained from the microorganism in Ora medium, a sample of the biopolymer extracted was analyzed at the highest time-point of production detected (after 48 h of growth) through spectroscopic methods (Online Resource 2). The techniques used included UV-visible, FTIR, and NMR-¹H, which confirmed a PHB structure by identifying its characteristic signals [13, 60]. Additionally, the DSC thermogram of the PHB obtained presented a melting temperature (Tm) of 172 °C, similar to the ones previously determined for this kind of microbial biopolymer [61], suggesting that the obtained PHB may be processed by injection molding or extrusion [62]. Also, it has been reported that PHA may be suitable candidates for applications where biocompatibility and/or biodegradability are required, like for the production of new materials for medical purposes [3, 36, 60, 62, 63].

The potential of B. megaterium strains for industrial and environmental applications has been widely stated [64, 65]. Specifically, the potential of this species for PHB production has been previously demonstrated [66]. However, as mentioned before, industrial exploitation of PHA production has been difficult due to production costs and low obtention yields. In the present study, it was shown that B. megaterium strain MSH1-9K-1 is able to produce PHB in a low-cost medium elaborated from orange-peel residues and that this capability is at least two times more elevated than the ones reported to date, including those grown in low-cost media [13, 17]. Further studies are now being performed to optimize the physicochemical parameters of this production and also to enhance the productivity of this strain using molecular approaches.

Author contribution

AMRC co-directed the study and wrote the manuscript; AVC performed the structural elucidation of the biopolymer obtained; AFAP determined the growth and PHA production kinetics in the medium prepared using orange-peel residues and improved the PHA production protocol; CBFO determined the bacterial culture growth and PHA production in the different low-cost culture media; SCI quantified the secondary metabolites in the different culture media; MQC performed the microscopic analyses; AER obtained DSC results for PHB at different temperatures; BDPG made the factorial regression analysis; NGRA received the funds, co-directed the study, and reviewed the manuscript.

Funding

This project was supported by the funds granted to Project SIP No. 20201305 of the Instituto Politécnico Nacional of Mexico.

Data availability

The datasets generated during and/or analyzed during the current study are available from the authors on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.