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Environmental Microbiology (EM)  |  Biodegradation and Bioremediation

Microbiol. Biotechnol. Lett. 2023; 51(3): 257-267

Received: May 30, 2023; Revised: July 23, 2023; Accepted: July 31, 2023

Valorization of Pineapple Peel Waste for Sustainable Polyhydroxyalkanoates Production

Kannika Bunkaew1, Kittiya Khongkool1, Monthon Lertworapreecha1, Kamontam Umsakul2, Kumar Sudesh3, and Wankuson Chanasit1*

1Microbial Technology for Agriculture, Food and Environment Research Center, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand
2Division of Biological Sciences, Faculty of Science, Prince of Songkla University, Songkhla 90110, Thailand
3Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia

Correspondence to :
Wankuson Chanasit,

The potential polyhydroxyalkanoates (PHA)-producing bacteria, Bacillus megaterium PP-10, was successfully isolated and studied its feasibility for utilization of pineapple peel waste (PPW) as a cheap carbon substrate. The PPW was pretreated with 1% (v/v) H2SO4 under steam sterilization and about 26.4 g/l of total reducing sugar (TRS) in pineapple peel hydrolysate (PPH) was generated and main fermentable sugars were glucose and fructose. A maximum cell growth and PHA concentration of 3.63 ± 0.07 g/l and 1.98 ± 0.09 g/l (about 54.58 ± 2.39%DCW) were received in only 12 h when grown in PPH. Interestingly, PHA productivity and biomass yield (Yx/s) in PPH was about 4 times and 1.5 times higher than in glucose. To achieve the highest DCW and PHA production, the optimal culture conditions e.g. carbon to nitrogen ratios of 40 mole/mole, incubation temperature at 35℃ and shaking speed of 200 rpm were performed and a maximum DCW up to 4.24 ± 0.04 g/l and PHA concentration of 2.68 ± 0.02 g/l (61% DCW) were obtained. The produced PHA was further examined its monomer composition and found to contain only 3-hydroxybutyrate (3HB). This finding corresponded with the presence of class IV PHA synthase gene. Finally, certain thermal properties of the produced PHA i.e. the melting temperature (Tm) and the glass transition temperature (Tg) were about 176℃ and -4℃, respectively whereas the Mw was about 1.07 KDa ; therefore, the newly isolated B. megaterium PP-10 is a promising bacterial candidate for the efficient conversion of low-cost PPH to PHA.

Keywords: Polyhydroxyalkanoates (PHAs), 3-hydroxybutyrate (3HB), pineapple peel hydrolysate (PPH), Bacillus megaterium, low-cost carbon source

Graphical Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible microbial polyesters synthesized and accumulated by various microorganisms as energy and carbon storage compounds. The accumulation of PHA normally occurs when nitrogen sources are limited, but carbon sources are in excess in the culture medium [14]. Due to their unique properties, PHAs have attracted increasing attention as an eco-friendly alternative to petroleum-based plastics. On the other hand, PHAs are environmental-friendly bioplastics because they can be degraded by microorganisms resulting in water and carbon dioxide. Moreover, PHAs are nowadays utilized in a wide range of medical and agricultural applications due to their biocompatibility and biodegradability characteristics [3, 5, 6]. PHAs are commonly classified into two major categories: short-chain-length (SCL-) and medium-chain-length (MCL-) PHAs. The repeat units of SCL-PHAs are composed of C3−C5, whereas MCL-PHAs contain C6−C14. The monomer compositions of the synthesized PHAs are influenced by the bacterial strain, type and quantity of carbon sources supplied to the culture medium [3, 4, 7]. Gram-positive bacteria such as Bacillus megaterium was reported as the first poly-3-hydroxybutyrate (PHB) producing bacteria. It has been extensively known as a suitable microorganism for PHB production because of high PHB yields and its cell walls are deficient in immunogenic lipopolysaccharides making them an ideal source of PHA for implantable polymers [6, 8]. For example, some strain of B. megaterium accumulated up to 70% PHB content per dry cell weight under optimal conditions [9, 10]. However, the major obstacle of commercial PHAs is their high production cost, which is from $5.0 to 6.1/kg and the substrate cost accounts for over 40% of the total operating cost of PHA production [1, 2, 5, 11]. To solve this problem, inexpensive and renewable substrates or waste feedstocks are used as the carbon source for PHA production. Among these, pineapple peel residue has become an attractive low-cost substrate owing to its high sugar content, e.g. sucrose, glucose, fructose, and other carbohydrates, which provide the necessary nutrients for bacterial growth and PHA biosynthesis [1, 2, 12]. Valorization of this lignocellulosic waste could be a sustainable and eco-friendly solution, in addition to non-competition with food values [5, 13, 14]. In this study, we aim to investigate the feasibility of pineapple peel waste for PHAs production. Specifically, the potential PHAaccumulating bacterium was isolated and then the culture conditions were optimized to enhance the bacterial growth and PHAs accumulation.

Isolation and screening of PHA-producing bacteria

The pineapple peel waste (PPW) samples were collected from various sites in the pineapple plantation area, Pa Bon District, Phatthalung, Thailand [7°16′12″N 100°10′12″E]. Then, the bacteria were isolated by adding 10 g of soil into 90 ml of nitrogen-limiting mineral salt medium (MSM) consisting in g/l: glucose, 20; (NH4)2SO4, 1.0; KH2PO4, 2.0; Na2HPO4, 0.6; MgSO4·7H2O, 1.0; 1 ml trace element [15]. The initial pH was adjusted to 7.0. After 3 days of incubation at 30℃ with shaking at 200 rpm, 0.1 ml aliquots from each enrichment culture were plated onto MSM agar. For the rapid detection of PHA-producing bacteria, 0.02% of Sudan black B, a preliminary screening agent for lipophilic compounds, including PHA granules, was applied to stain bacterial colonies. The colonies that are able to incorporate the Sudan black B appeared bluish black color [7, 16]. The positive colonies were then further confirmed the PHA granules biosynthesis inside the bacterial cells by Sudan black B staining under microscope observation. In addition, the positive colonies were grown on MSM containing Nile red fluorescence dye for quantitative estimation of PHA production under UV light. The strong bright orange fluorescent colonies indicate high PHA content [1, 3, 16]. Finally, the bacterial isolates that showed the maximum PHA content were further confirmed for PHA accumulation by transmission electron microscope (TEM) analysis. Briefly, the cells were separated by centrifugation, washed with a saline buffer (10% NaCl, 0.1M sodium phosphate buffer, pH 7.2), and were then re-suspended in a 2.5% (v/v) glutaraldehyde solution for overnight at 4℃ before fixed with 1.0% osmium tetroxide. The ultrathin sections were then stained with uranyl acetate followed by lead citrate before viewing with JEOL JEM 2010F TEM (Jeol, Japan), with an accelerating voltage of 150−200 kV [17].

Identification of the bacterial isolate

The genomic DNA of the PHA-accumulating bacterial isolate PP-10 was extracted from the cell pellet using a PureLink™ genomic DNA extraction kit (Invitrogen, Thermo Fisher Scientific, USA). The 16S rRNA gene amplification was performed by PCR using Taq polymerase (Invitrogen, Thermo Fisher Scientific) with the universal primers 20F (5′-GAG TTT GAT CCT GGC TCA G-3′) and 1500R (5′-GTT ACC TTG TTA CGA CTT-3′). The amplification program was comprised of 1 cycle at 94℃ for 3 min, 25 cycles of denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min and elongation at 72℃ for 2 min, followed by a final amplification step at 72℃ for 3 min [1, 16]. The amplified PCR products were purified using a Qiagen PCR purification kit (Qiagen, USA) and sent for DNA sequencing at First BASE Laboratories, Malaysia. The 16S rRNA gene sequence analysis was carried out using the NCBI BLAST program. A phylogenetic tree of the 16S rRNA genes was constructed using EzBioCloud by maximum parsimony method (the robustness for individual branches was estimated by 1,000 replication bootstrap) [18]. For biochemical identification, the VITEK2 BCL card (The VITEK 2 system, BioMérieux, France) provides a reliable identification of Bacillus species and members of related genera [19].

Analysis of phaC synthase gene

Pair of specific primer was designed for amplifying Class IV phaC gene by aligning and analyzing Class IV PHA biosynthesis operon sequences belonging to the Bacillus species as described by Berekaa (2012). The primers designs were; P1:5'-GAT GTG TAT TTG CTT GAC TGG GG-3' and P2: 5'-AGC CAA TCG CCG ATT GAA GGA TA-3') with an annealing temperature of 64℃ [20].

Pretreatment and hydrolysis of Pineapple peel waste (PPW)

The PPW was dried in a hot air oven at 65℃ followed by grinding using a blender and sieved to the particle sizes between 0.841−0.420 mm (-20/+40 mesh). Then, 10 g of samples were pretreated by using H2SO4 (varying 1% (v/v) to 3% (v/v)) and autoclaved at 121℃, 15 psi for 30 min (method modified from Sukruansuwan and Napathorn 2018) [2]. The pretreated samples were filtered through Whatman filter paper No. 1, and the filtrate was collected and finally adjusted pH to 7.0 to obtain pineapple peel hydrolysate (PPH) containing fermentable sugars. The compositions and concentration of the sugars in PPH were analyzed using highperformance liquid chromatography (HPLC) with refractive index detector (RID), Zorbax NH2 column, mobile phase: acetonitrile/water (75−25% v/v) and flow rate of 1.5 ml/min (high-performance liquid chromatograph, Agilent Technologies, 1200 series, USA) [2, 12]. Additionally, the presence of amino acids and essential minerals in PPH were analyzed by HPLC and Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES), respectively as described by standard procedures.

Biosynthesis of PHA by the newly bacterial isolate

The bacterial isolate PP-10 was pre-cultured in basal culture medium (BCM) consisting of (g/l): yeast extract, 10; peptone, 10; beef extract, 5; NaCl, 5; and glucose, 10 with an initial pH of 7.0 [15]. The culture was then incubated at 30℃ and 200 rpm until the mid-log phase was reached. Then, 5% (v/v) inoculum suspension (OD600 = 0.5) was transferred into 250 ml Erlenmeyer flask containing 50 ml of MSM supplemented with 2% (w/v) glucose or 2% (v/v) of total reducing sugar (TRS) in PPH for enhancing PHA production under the culture conditions as described above. The samples were then taken every 12 h for 3 days for further cell growth and PHA analysis.

PHA biosynthesis under statistical optimal condition

To achieve the maximum cell growth and PHA production, the experimental design and statistical analysis were optimized using response surface methodology (RSM) with Box-Behnken design (BBD) (Design Expert ver. 12 software, Stat-Ease Inc., USA). Three key factors such as carbon to nitrogen ratios (C/N): (20−60 mole/mole), incubation temperature: (30−40℃) and shaking speed: (100−300 rpm) and two variable responses such as DCW and PHA concentration were used to fit a second-order response surface (Table S1). The analysis of variance (ANOVA) was performed and the model terms was deemed significant when “Prob > F” values were less than 0.05. The non-significance of lack-of-fit F-value is considered a good model. The model was validated with respect to the DCW and PHA concentration under the predicted conditions from the significant model [1, 2123].

Thermal properties of PHA

The thermal properties of the PHA sample were characterized by a differential scanning calorimeter (DSC) thermal analysis system (Perkin Elmer Pyris 1) in the range of -50 to 250℃ at a heating rate of 20℃/min. The glass transition temperature (Tg) and melting point temperature (Tm) were determined from the second scan of DSC thermogram [1, 24, 25].

Determination of molecular weight

The molecular weight was determined at 40℃ using a gel permeation chromatography (Agilent Technologies, 1260GPC/SEC MDS, USA) equipped with a refractive index detector and SHODEX K-802 and K-806M columns. Chloroform was used as the eluent at a flow rate of 0.8 ml/min. The samples were prepared by dissolving the extracted PHA in chloroform at a concentration of 1 mg/ml [24].

Analytical Procedures

Dry cell weight. The cells were harvested by centrifugation (5,000 rpm, 20 min at 4℃) followed by freeze-drying of the cell pellets until constant cell weights were obtained. The dry cell weight (DCW) was calculated in g/l [15, 21].

PHA quantification and monomer characterization. Approximately 20 mg sample of freeze-dried cells of the bacterial isolate PP-10 was added into 2 ml each of chloroform and acidified methanol [15% (v/v) H2SO4]. The mixture was then heated at 100℃ for 3 h. After cooling to room temperature, 2 ml of distilled water was added, followed by vigorous shaking, and then the reaction was left overnight for phase separation. The chloroform portion containing the PHA methyl ester was then analyzed by a gas chromatography-flame ionization detector (GC-FID) for PHA quantitation and gas chromatography-mass spectrometry (GC-MS) in the totalion scan mode at a mass-to-charge ratio (m/z) = 45−600 for detection of monomer compositions. Monomers of methyl hydroxyalkanoates (Larodan, Sweden) and benzoic acid methyl ester (Sigma-Aldrich, USA) were used as an external standard and internal standard, respectively [21, 22, 26]. The compound identifications were achieved by matching query spectra to spectra present in a reference library (NIST 2020/2017/EPA/NIH).

Total reducing sugar (TRS). The total reducing sugar concentration was measured by 3,5-dinitrosalicylic acid (DNS). Briefly, 500 μl of cell-free supernatant was added to 500 μl of the color reagent. These solutions were heated in boiling water for 10 min and immediately transferred on ice, and the absorbance was measured at 540 nm when the calibration curve was glucose at 0 to 1.0 g/l [2, 12].

Fermentation kinetics. Production kinetics of cell growth and PHA amount were calculated in maximum specific growth rate μmax (h-1), the product yield of PHA concerning sugar consumption YP/S (g/g), the product yield of PHA with respect to biomass YP/X (g/g), biomass yield related to sugar consumption YX/S (g/g) and volumetric productivity of PHA (g/l/h) of the culture media [11, 12, 15, 22, 26].

Statistical analysis. All the data presented were representative of the results of three independent experiments and were expressed as the mean values ± standard deviations (S.D.). Analysis of variance (one-way ANOVA) followed by Post hoc: Tukey’s test for testing differences among means was performed using SPSS. Differences were considered significant at p < 0.05 (IBM® SPSS® Statistics) [22].

Isolation of potential PHA-producing bacteria

The isolation and screening of new bacterial species from the lignocellulosic habitats are efficient methods to obtain the PHA-producing bacteria that are able to utilize agricultural wastes, including pineapple peel waste (PPW), as a carbon source [1, 5, 12, 27]. A total of 56 bacterial isolates were obtained from soil samples around the pineapple plantation area in Pa Bon District, Phatthalung, Thailand. Among these, bacterial isolate PP-10 exhibited a maximum intensity of fluorescence under UV light after staining with Nile red. Additionally, the PHA granules were detected by Sudan black B staining under the light microscope as well as under a transmission electron microscope (Fig. 1). In addition, the biochemical characteristics were examined using VITEK® 2 system with Bacillus card as described by Halket et al. (2010) [19]. The result revealed that the bacterial isolate PP-10 was identified as B. megaterium with 93% probability (Table S1). This biochemical identification corresponded with the 16S rDNA analysis presented in the phylogenetic tree (Fig. 2). The 16S rDNA sequence of the isolate PP-10 showed a 100% sequence homology to the type strain of B. megaterium IAM 13418T. It was then deposited in GenBank with an accession number OQ859945. Moreover, to confirm the biosynthesis of PHA, the PHA synthase gene e.g. phaC in B. megaterium PP-10 was analyzed by using Class IV PHA biosynthesis operon sequences belonging to the Bacillus spp. and the result of PCR product is demonstrated in Fig. 3. The results proved the presence of phaC as an expected size about 760 bp that similar to positive control of Bacillus sp. ST1C [28]. The sequence of phaC in B. megaterium PP-10 was further compared with phaC gene among Bacillus species and exhibited 98.2% identity to phaC of B. megaterium strain ST41 based on NCBI databases. B. megaterium was the first discovered bacteria to be studied for PHB production. It possesses class IV PHA synthase gene that is generally favor short-chain-length (SCL-) monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization [6, 7, 20]. Notably, they are able to utilize a wide range of carbon substrate and have high resistance to osmotic pressure and temperature as well as producing relatively high PHB yields and need few fermentation factors [8, 9]. Many studies have reported the use of lignocellulosic waste including corn straw, sugarcane bagasse, banana pseudo-steam, as an alternative sources of substrate for PHB production by B. megaterium [5, 6, 14, 25].

Figure 1.PHA accumulation in bacterial isolate PP-10: Colony morphology (A) Detection of PHA production by Nile red fluorescence (B) PHA granule stained with Sudan black B under a microscope (C) and TEM micrograph of PHA granules (D) when cells were grown in 2%(v/v) PPH.
Figure 2.Phylogenetic tree based on the 16S rDNA gene sequence showing a comparison of Bacillus species. Lactobacillus arizonensis NRRL B-14768T was used as an out-group. Values at nodes indicate bootstrap values for 1,000 replicates.
Figure 3.Detection of phaC in Bacillus megaterium PP-10 using specific primer of Class IV phaC gene among Bacillus species [lane 1(left); 100 bp DNA ladder, lane 2; positive control: amplified phaC in Bacillus sp. ST1C [26], lane 3; amplified phaC in B. megaterium PP-10, and lane 4; negative control].

Acid pretreatment of pineapple peel waste

The pineapple peel waste (PPW) was pre-treated with acid, e.g. 1%(v/v) to 3%(v/v) H2SO4, under steam heat to generate fermentable sugars for supporting growth and PHA biosynthesis. The sugar compositions and concentrations in PPH were analyzed by HPLC and it found that glucose was a major component which reached about 1.51 ± 0.02%(w/v) followed by fructose (1.30 ± 0.01%w/v) and minor of sucrose and xylose (Table 1). In general, the composition of plant cell wall varies in cellulose (40−80%), hemicellulose (10−40%), and lignin (5− 25%) content depending on the type of biomass [5, 13]. In this study, the lignocellulosic biomass compositions in PPW were determined and found that it contained about 23%(w/w) of cellulose, 14%(w/w) of hemicellulose, 18%(w/w) of total lignin whereas Sukruansuwan and Napathorn (2018) reported about 36.8% and 5.12% of cellulose and lignin, respectively in pineapple waste obtained from the canned pineapple industry [2]. However, these PPW have to be pretreated to remove lignin and reduce the crystallinity of cellulose by acid hydrolysis at high temperatures to breakdown of long chains carbohydrate to sugar monomers. The disadvantage of this method was the generation of toxic compounds such as furfural and 5-Hydroxymethylfurfural (5-HMF) which further decreases the yields of fermentable sugars and inhibit the bacterial growth by decreasing the intracellular pH resulted in cell death [13, 14]. Xylose is known to dehydrate to furfural under acidic conditions while glucose dehydrate to 5-HMF, which can be further hydrolyzed to levulinic acid (LA) [29]. In this study, three major microbial inhibitors e.g. furfural, 5-HMF and LA were examined in various percentages of diluted sulfuric acid concentration and the results in Table 1 showed that at 1% of H2SO4 the lowest amount of furfural and 5-HMF were produced while no LA detection. Similar to the previous study, 1%(v/v) of H2SO4 was used to pretreat PPH producing mainly glucose with a maximum content up to 35 g/l. On the other hand, the use of alkaline i.e. Ca2OH or NaOH for PPW pretreatment resulted in a reduction of TRS while a using of H2SO4 from 1%(v/v) to 3%(v/v) concentration was found to produce the highest TRS mainly glucose in the range of 20 to 35 g/l [2, 30]. While in this work, the total reducing sugar (TRS) concentration in PPH was analyzed by DNS method and showed the highest TRS concentration of 26.4 ± 0.02 g/l at 1%(v/v) acid pretreatment condition, which was significantly higher than the other treatments (p < 0.05). These results suggested that the type and concentration of acid strongly affect the types includes the amount of sugar released from the lignocellulosic biomass; however, a commonly used concentration range of sulfuric acid pretreatment for lignocellulosic biomass is between 1% (v/v) to 5% (v/v) to maximize sugar release and/or minimize the formation of inhibitory compounds. This phenomenon can be explained that acid pretreatment can result in higher delignification and solubilization of hemicellulose, thus increasing the accessibility of cellulose for enzymatic hydrolysis. The sugars released during acid pretreatment are generally more fermentable compared to those produced through alkaline pretreatment [2, 5, 13]. Moreover, Castro et al. (2016) studied the pineapple peel physicochemical properties and revealed that it contained total carbohydrate and total protein about 27.08 and 0.63 g/100 g of pineapple peel including trace of Mg, K and Zn [27]. These nutrients are able to support the microbial growth and their metabolic activities [1, 2, 12, 30].

Table 1 . Total reducing sugar, sugar composition and microbial inhibitors in pineapple peel hydrolysate (PPH) after acid hydrolysis pretreatment.

%(v/v) H2SO4Total reducing sugar (g/l)Sugar content [%(w/v) ±SD]Microbial inhibitors (ppm)
1%26.40 ± 0.77a1.30 ± 0.011.51 ± 0.02<0.25<0.2519.60 ± 0.149.47 ± 0.46N.D.
2%21.16 ± 0.95b1.27 ± 0.051.41 ± 0.01<0.25<0.2521.34 ± 0.2519.60 ± 0.35N.D.
3%21.16 ± 0.24b1.24 ± 0.041.43 ± 0.01<0.25<0.2555.30 ± 0.1711.29 ± 0.17N.D.

a,bDifferent letters above bars indicate significant differences (p < 0.05; post-hoc Tukey’s test-one way ANOVA analysis), N.D., not detected.

Comparison of PHA biosynthesis between pure sugar and PPH

In this study, the cell growth and PHA biosynthesis from the newly isolated B. megaterium PP-10 were evaluated and compared the efficiency of using glucose, a pure sugar and the PPH which contains mixed sugars as carbon sources. The initial substrate concentration in the MSM production medium was set at 20 g/l. The results clearly demonstrated that the maximum cell growth of 3.63 ± 0.07 g/l of DCW was obtained when the cells were grown in 2%(v/v) TRS in PPH at 12 h of cultivation (Fig. S1A) which accounted for almost threetimes of a maximum specific growth rate higher than in 2%(w/v) glucose (Table 2). Correspondingly, the highest PHA content of 54.58 ± 2.39 %DCW was produced when cultivated B. megaterium PP-10 in PPH for 12 h, which was higher than in pure sugar e.g. glucose. Remarkably, after the cells reached maximum growth, the PHA content was likely to decrease along the cultivation time (Fig. S1B). These results can be explained that the significant reduction of PHA content after 12 h was provided for maintaining the bacterial growth by utilization of this storage PHA as a carbon and energy source. On the other hand, some sporulation was observed but only after 12 h (Fig. S2). Some types of PHAs were also served as a carbon source for biosynthesis of spore components. This PHA production in Bacillus species is produced during the early stages of sporulation therefore the sporulation can suppress the formation of PHA [68, 27]. In addition, the fermentation kinetic parameters e.g. YP/X, YP/S, YX/S, and PHA productivity between the cultivation in 2%(v/v) TRS in PPH and 2%(w/v) glucose were determined. All those fermentation kinetics presented in Table 2, in 2%(v/v) TRS in PPH had higher than in 2%(w/v) glucose particularly the productivity of PHA in PPH was about four times higher than in glucose. These results also corresponded to the consumption of sugar by B. megaterium PP-10. A similar observation was found in Bacillus sp. SV13 that the conversion rate of DCW to PHA in pineapple waste (Yp/x, 0.15) was more significant than in glucose and sucrose [12]. Moreover, a maximum PHAs yield of about 3.44 g/l was also produced when grown Bacillus drentensis BP17 in an extracted pineapple peel juice, followed by sucrose (2.77 g/l) and glucose (2.42 g/l) [1]. It implies that mixed sugar in pineapple waste could promote the growth and PHA production of bacteria, whereas pure sugars, e.g. glucose and sucrose could not. These may be due to amino acids and some minerals, as well as essential vitamin in pineapple juice and/or PPH are necessary for bacterial growth [1, 12, 31]. In this study, the amino acid compositions and certain minerals contained in PPH were further analyzed. The results strongly confirmed that the PPH consisted of the following minerals (in mg/l): Ca (149.60), Mg (87.64), Na (11.48), Fe (8.29), Zn (2.50), and K (2.32). The major amino acids in PPH were threonine (1.14 μmol/ml), valine (0.69 μmol/ml), and aspartic acid (0.61 μmol/ml). Threonine plays a role in the formation of the murein cell wall of bacteria, which is essential for maintaining cell structure and integrity [30, 31]. The amino acid profile in the PPH was shown in Table S2.

Table 2 . Fermentation kinetic parameters when grown B. megaterium PP-10 in MSM containing 2% (v/v) TRS in PPH or 2% (w/v) glucose as the sole carbon source.

Carbon sourceResidual substrate concentration (g/l)Fermentation kinetic parameters
T (h)μmax (h-1)YP/X (g/g)YP/S (g/g)YX/S (g/g)PHA productivity (g/l/h)
2% (v/v) TRS in PPH8.70 ± 0.09120.3030.550.230.420.17
2% (w/v) Glucose12.25 ± 0.02360.0910.430.120.270.04

Cultivation time, T (h), maximum specific growth rate, μmax (h-1), the product yield of PHA with respect to sugar consumption YP/S (g/g), the product yield of PHA with respect to biomass YP/X (g/g), biomass yield related to sugar consumption YX/S (g/g) and volumetric productivity of PHA (g/l/h).

Statistical optimization of microbial growth and PHA biosynthesis from PPH

RSM, a powerful optimization method, can enhance DCW and PHA yield by optimizing the independent factors of the experiment, namely X1 (A-C/N), X2 (Bincubation temperature), and X3 (C-shaking speed) [23]. This approach proves valuable in optimizing media components and critical variables that influence biomolecule production. The BBD experiments (Table S3) were specifically designed to determine the optimal conditions for maximizing cell growth and PHA production in B. megaterium PP-10. The equations below represent the quadratic regression model used to evaluate the highest DCW and PHA concentration:

DCW (g/l) = +3.9-0.41X1-0.19X2+0.089X3-0.068X1X2+0.41X1X3+0.039X2X3-0.95X12-0.26X22-0.55X32

PHA concentration (g/l) =+2.38-0.30X1-0.11X2-0.030X3+0.077X1X2+0.20X1X3+0.021X2X3-0.92X12-0.31X22-0.56X32

Where X1, X2, X3 are the linear; X12, X22, X32 are the squared; and X1X2, X1X3, X2X3 are interaction coefficients.

Both DCW and PHA concentration were well-fitted into the quadratic model with high reliability ratings (R2 = 0.9465 and R2 = 0.9539, respectively, at p < 0.0001) [1, 23]. The lack of fit in the model was considered insignificant, indicating a good fit. The response surface plot displayed the optimal values for DCW and PHA concentration, and the contours of the surface plots revealed the interaction between the variables and the correlation between predicted and actual values for DCW and PHA concentration (Figs. S3A and B, and Fig. S4). Certain model terms, such as A, A2, and C2, were found to be significant for both DCW and PHA concentration. The interactive effect of variables AC (C/N and shaking speed) had the most significant impact on DCW, while A, A2, B2, and C2 had the most impact on PHB concentration (Table S4 and S5). The optimized culture conditions were found to be a C/N of 40 mole/mole, incubation temperature of 35℃, and shaking speed of 200 rpm, resulting in the highest DCW of 4.24 g/l and a maximum PHA concentration of 2.68 g/l after 12 h of cultivation (Table 3). The RSM model significantly improved microbial growth and PHA production in B. megaterium PP-10, with a 7% increase in DCW and an 11% increase in PHA production compared to the predicted optimal conditions. Table 4 summarized previous reports that utilized pineapple waste (e.g. juice, core, and peel) as a carbon source for PHA biosynthesis by different bacteria. The DCW ranged from 2.15 to 14.42 g/l, while the PHA concentration varied from about 0.4 to 7.7 g/l, with productivities between 0.008 to 0.2 g/l/h [1, 2, 12, 27, 30, 32].

Table 3 . Predicted and experimental values of the responses at optimal culture conditions.

ConditionC/N (mole/mole)Incubation temperature (℃)Shaking speed (rpm)DCW (g/l)PHA (g/l)
Modified40352004.24 ± 0.042.68 ± 0.02

Table 4 . Cell growth and PHA biosynthesis from pineapple residue by various bacteria.

Bacterial strainCarbon sourceDCW (g/l)PHA conc. (g/l)PHA content (%DCW)PHA productivity (g/l/h)YP/SReference
Bacillus sp. PP-10PPH4.24 ± 0.042.68 ± 0.0261.14 ± 1.020.2230.356This study
Bacillus drentensis BP17EPPJ145.6ni0.156ni[1]
Cupriavidus necator A-04PPH CAE5.3 13.60.7 7.712.7 600.010 0.1600.10 0.45[2]
Bacillus sp. SV13Pineapple waste4.00.40ni0.008ni[12]
Ralstonia eutropha ATCC 17697PPH2.15-3.26ni44.8nini[27]
Cupriavidus necator CCU 52338PWJ14.422.01ninini[30]
Bacillus cereusPPH4.71.7ni0.035ni[32]

PPH; pineapple peel hydrolysate, CAE; crude aqueous extract of pineapple waste products (peel and core), PWJ; pineapple waste juice, EPPJ; extracted pineapple peel juice; ni: not indicated.

Characterization of monomer composition in the produced PHA

The monomer composition of the PHA produced by B. megaterium PP-10 was investigated and characterized. The cells were grown in MSM with 2% (v/v) TRS in PPH as the sole carbon source. Through GC-MS chromatography (Fig. S5), the chromatogram was identified a retention time of 13.85 min, corresponding to 3-hydroxybutyrate (3HB) methyl ester, and 16.44 min, representing benzoic acid methyl ester (used as an internal standard). As a result, the PHA produced by B. megaterium PP-10 was determined as homopolymer consisting exclusively of 3-hydroxybutyrate (3HB) monomers. These 3HB monomers are short-chain length-PHA units containing four carbon atoms with a methyl group as a side chain. The presence of the phaC gene, belonging to class IV PHA synthase, in this strain supported these findings, as this gene preferentially polymerizes the 3HB monomer [3, 6, 20].

B. megaterium and similar microorganisms typically produce PHB homopolymer from sugars due to the organization of PHA biosynthetic genes and their substrate specificity [6, 7]. PHB materials are known for being stiff and brittle, making their properties quite similar to polypropylene [3, 7].

Thermal properties and molecular weight

The specific thermal characteristics of the PHB produced by B. megaterium PP-10 when cultivated in PPH were examined. The melting temperature (Tm) was approximately 176℃, and the glass transition temperature (Tg) was around -4℃. Additionally, the average molecular weight (Mw) to be 1.07 KDa, the number-average molecular weight (Mn) to be 0.73 KDa, and the polydispersity index (PDI) of 1.45, were shown in Table 5. These thermal properties of the PHB produced by B. megaterium PP-10 were comparable to those of commercial PHB (Sigma-Aldrich) and other research findings [4, 33, 34]. Previous research indicates that the molecular weights (Mw and Mn) of PHA can vary widely, ranging from 5.0 × 104 Da to 1.0 × 107 Da, and the PDI is within the range of 1.1 to 6.0 [1, 3, 4]. However, the PHB obtained from PPH in our current study exhibits a lower Mw and PDI compared to PHB produced from glucose, which is considered the standard PHB. Penkhrue et al. (2020) proposed that a low PDI polymer is potentially suitable for packaging and tissue engineering applications [1]. Additionally, the preference for low molecular weight PHA as a storage material in bacteria, which can be rapidly metabolized, suggests that the reduction in molecular weight may be related to providing a rapid energy supply for endospore development in Bacillus spp. [6].

Table 5 . Thermal properties and molecular weight of the produced PHA by B. megaterium PP-10 when grown the cell in PPH.

Bacterial strainPHAThermal propertiesMolecular weightReferences
Melting temp. (Tm, ℃)Glass transition temp. (Tg, ℃)Mn (g/mol)MW (g/mol)PDI
Bacillus megaterium PP-10PHB176.41- 473,475107,0961.45This study
Bacillus drentensis BP17PHB172-1172,600115,0001.59[1]
P(3HB) Sigma-AldrichPHB175-1804506,000 ± 900ni[34]

Weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (PDI = Mw/Mn) ; ni: not indicated.

In this work, the PHA was successfully produced from the newly isolated PHA-producing bacterium, B. megaterium PP-10 by using pineapple peel waste (PPW) as a lignocellulosic substrate. About 26.40 g/l of total reducing sugar (TRS) in pineapple peel hydrolysate (PPH) was produced after acid-pretreatment with 1%(v/v) H2SO4. The highest biomass of 3.63 g/l and a maximun PHB concentration of 1.98 g/l (54.58% DCW) were achieved when grown the cells in PPH. In addition, up to 4.24 g/l of DCW and a maximum PHA concentration of 2.68 g/l were obtained under optimal conditions based on RSM design model. Finally, the produced PHAs was characterized and found that only 3HB monomer was detected with a Mw of 1.07 KDa, Tm of 176℃ and Tg of -4℃.

This work was financially supported by National Higher Education, Science, Research and Innovation Policy Council, Thaksin University (Research project grant no. TSU-65A105000021) Fiscal Year 2022.

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