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Environmental Microbiology (EM) | Biodegradation and Bioremediation
Microbiol. Biotechnol. Lett. 2023; 51(3): 257-267
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, email@example.com
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
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 [1−4]. 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
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 . 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 .
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) . For biochemical identification, the VITEK2 BCL card (The VITEK 2 system, BioMérieux, France) provides a reliable identification of
Pair of specific primer was designed for amplifying Class IV
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) . 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.
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 . 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.
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 “
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].
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 .
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
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
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 . 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) . 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 (
In this study, the cell growth and PHA biosynthesis from the newly isolated
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) . 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
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
The monomer composition of the PHA produced by
The specific thermal characteristics of the PHB produced by
In this work, the PHA was successfully produced from the newly isolated PHA-producing bacterium,
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.
The authors have no financial conflicts of interest to declare.