Polyhydroxyalkanoate production in Priestia megaterium strains from glycerol feedstock

Andrew J. Cal; Victor J. Chan; Winston K. Luo; Charles C. Lee

doi:10.1371/journal.pone.0322838

Abstract

Our society relies heavily on plastic, but most plastic is petroleum-based and non-biodegradable resulting in major negative environmental impacts. Polyhydroxyalkanoates (PHAs) are a type of biopolymer that can be produced from microorganisms cultured on renewable feedstocks, such as glycerol. PHAs are biodegradable and have properties similar to petroleum-based plastics. Several Priestia megaterium isolates have been demonstrated in previous studies to produce PHA when cultured on glycerol, but there has been no comparison of strains available in public repositories. Such comparison would be useful to identify the most promising strains for further development. In this study, we screened a number of P. megaterium strains from readily accessible repositories for their ability to produce PHA from glycerol. The strains had a wide range of growth and PHA production characteristics on glycerol: cell dry weight (0.5–5.7 g/L), percent PHA (4–42%), PHA titer (0.1–1.9 g/L), and yield (26–303 mg/g). The time course of PHA production varied widely among the different strains. There was also a dramatic difference in molecular weights which ranged from 119 kD to 402 kD. This information will be valuable to groups in selecting a PHA strain to develop based on their specific requirements.

Figures

Citation: Cal AJ, Chan VJ, Luo WK, Lee CC (2025) Polyhydroxyalkanoate production in Priestia megaterium strains from glycerol feedstock. PLoS One 20(4): e0322838. https://doi.org/10.1371/journal.pone.0322838

Editor: Ranjit Gurav, UPES: University of Petroleum and Energy Studies, INDIA

Received: October 4, 2024; Accepted: March 28, 2025; Published: April 30, 2025

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Modern society is dependent on the benefits of petroleum-based plastics in all aspects of our economy; however, there are significant negative environmental consequences of this lifestyle. Traditional plastic production results in the release of harmful greenhouse gases into the atmosphere, and the plastic itself is generally not biodegradable thus polluting the land and water for many years. Therefore, there are intense efforts to develop sustainable alternatives. One promising approach is the use of polyhydroxyalkanoates (PHAs) which are biodegradable polyesters produced by certain microorganisms [1,2]. The characteristics of PHAs vary widely based on their specific subunit composition, and these polymers can emulate the properties of many different traditional plastics [3,4]. The most commonly produced PHA is poly-3-hydroxybutyrate (PHB) which is composed of 3-hydroxybutyrate subunits. PHB has properties (tensile strength [~30 MPa] and modulus [~3.25 GPa]) that are similar to polypropylene [5].

A major barrier to broad adoption of PHA polymer is the higher cost compared to traditional plastics. One of the major expenses of PHA production is the feedstock used to culture the microorganisms. To alleviate this problem, waste streams from different processes have been explored as feedstocks for PHA production [6–10]. Recently, glycerol has emerged as a promising substrate because it is a major byproduct of biodiesel manufacturing [11–13]. The production of biodiesel has been increasingly yearly and is predicted to reach 50 billion liters by 2030 [14,15]. Approximately, 1 g of glycerol is generated for every 10 g of biodiesel. Although there are currently applications for glycerol that do not involve PHA production, the amount of glycerol made available through the biodiesel industry far exceeds the current market.

The bacterium Priestia megaterium (formerly known as Bacillus megaterium) is an excellent candidate to produce economical levels of PHA. P. megaterium was the first microorganism discovered to produce PHA, specifically PHB [16]. The bacterium has been widely used in biotechnological applications to produce industrial biochemicals [17]. The fact that P. megaterium is a Gram-positive bacterium is an advantage over Gram-negative bacteria containing contaminating lipopolysaccharide endotoxins. There are many reagents available to genetically engineer P. megaterium [18,19]. P. megaterium is capable of growing on a wide variety of feedstocks, and economic modeling has demonstrated that it is feasible to commercially manufacture PHB from glycerol [20]. To produce PHB, P. megaterium uses a heteromeric PHB synthase enzyme composed of PhaC and PhaR subunits [21].

Multiple research groups have demonstrated PHA production from P. megaterium using glycerol as a feedstock; however, the majority of these studies utilize isolates not available in public strain repositories [20,22–30]. Additionally, experimental culturing conditions vary greatly between these studies, making direct comparisons impossible.

In order to facilitate further development of P. megaterium for PHA production and select appropriate strains for functional modification by genetic engineering [31], we have conducted a side-by-side comparison of 12 publicly-available strains in this study. P. megaterium strains were compared for their ability to produce PHB from glycerol feedstock under identical experimental conditions. Cell biomass, polymer percent, titer, and yield were monitored over the course of time. Polymers were analyzed for monomer composition, molecular weight, and thermal properties. Knowledge of the PHB production characteristics will allow groups to make informed decisions about which strain to utilize and further develop based on their industrial priorities. All the strains in this study are easily obtainable from cell repositories available to the public.

Materials and methods

Strains and media

Twelve P. megaterium strains were acquired from readily available public sources: ATCC (American Type Culture Collection; Virginia, USA), BGSC (Bacillus Genetic Stock Center, Ohio, USA), Boca Scientific Inc. (Massachusetts, USA), DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), and NRRL (ARS Culture Collection, Illinois, USA). All chemicals and reagents were obtained from Sigma-Aldrich (MO, USA) unless otherwise specified. Growth media was adapted from Cardozo et al. [31] and was composed of the following (per L): 1.0 g yeast extract, 1.0–3.0 g (NH₄)₂SO₄, 0.2 g MgSO₄·7H₂O, 20.0 g carbon substrate (glycerol or glucose), 1.5 g KH₂PO₄, and 3.6 g Na₂HPO₄, and 1.0 mL of trace element solution. The trace element solution was adapted from Kim et al. [32] and was composed of the following (per L): 10.0 g FeSO₄·7H2O, 2.25 g ZnSO₄·7H2O, 1.0 g CuSO₄·5H₂O, 0.364 g MnSO₄·H₂O, 2.0 g CaCl₂·2H₂O, 0.121 g Na₂B₄O₇, 0.11 g (NH₄)₆Mo₇O₂₄·4H₂O, and 10.0 mL 35% HCl. For solid media, recipe was supplemented with 3 g/L NH₄SO₄ and 1.5% agar.

16S rRNA sequencing

Cultures were grown in media supplemented with 3 g/L NH₄SO₄. Genomic DNA was isolated from the cells using PureLink Genomic DNA Minikit (ThermoFisher Scientific, MA, USA). Genomic DNA was used as template in a PCR reaction using Herculase II Fusion DNA polymerase as described by the manufacturer (Agilent, CA, USA). The PCR primers used to amplify the 16S rRNA gene are listed below [33]:

27f: 5’- agagtttgatcmtggctcag-3’

1525r: 5’- aaggaggtgwtccarcc-3’

The resulting PCR product was processed with the DNA Clean & Concentrator-5 kit (Zymo Research, CA, USA) and sequenced with the 27f and 1525r primers. DNA Sanger sequencing was conducted by Elim Biopharmaceuticals (CA, USA). All sequences were deposited into the NCBI database (NIH, MD, USA).

PHB production

Liquid cultures were shaken at 225 rpm at 30°C. Starter cultures were grown by inoculating 50 ml media (supplemented with 3 g/L NH₄SO₄) in 250 ml flasks using a colony picked from a freshly streaked agar plate. After overnight growth, the starter cultures were used to inoculate 200 ml media (supplemented with 1 g/L NH₄SO₄) in 1 L baffled flasks to OD₆₀₀ = 0.2. Cultures were grown for 96 h, and time points were collected periodically. Cultures were spun down, and cell pellets were washed with 1% NaCl. This wash procedure was repeated, and the cell pellets were frozen or lyophilized. All strains were cultured in triplicate.

Polymer analysis: gas chromatography and mass spectrometry

To 10 mg of lyophilized bacterial sample, 1 ml of methanolysis reagent (15% sulfuric acid and 85% methanol) and 1 ml chloroform were added to a 12-ml glass tube and heated at 105°C for 2 hours. After cooling on ice, 1.5 ml water was added, and the mixture was vortexed then centrifuged 10 min at 500 x g. The chloroform layer was collected and dehydrated with 50 mg anhydrous magnesium sulfate. The samples were diluted into chloroform and analyzed as previously described [34]. In brief, 2 μl were injected onto ThermoScientific Trace 1310 gas chromatograph) equipped with a TG-5MS column (ThermoFisher Scientific). Products were analyzed using a coupled ThermoScientific IQS LT Single Quadrupole Mass Spectrometer. Quantification and peak identification were obtained using the ThermoScientific Chromeleon software. Besides 3-hydroxybutyrate, no signals for other 3-hydroxy fatty acids (3-hydroxyvalerate/3-hydroxyhexanoate) were detected. A standard curve was made with pure PHB (TianAn, Zhejiang, China) ranging from 1 to 10 mg of polymer.

Polymer analysis: gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA)

Wet cell pellets were lysed by resuspension in lysozyme solution (27.5 mg/ml lysozyme in 50 mM potassium phosphate and 16.7 mM magnesium chloride). The suspension was incubated at 37°C for 30 min, sonicated, and centrifuged at 6,000 X g for 20 min. The PHB pellet was collected and lyophilized. Aliquots containing approximately 6 mg of PHB were solubilized in 1.8 ml chloroform at 100°C for 1 h. The solution was cooled, and 1.8 ml water was added. The solution was vortexed and centrifuged at 500 X g for 10 min. The chloroform fraction was collected and twice the volume of heptane was added to precipitate the PHB. The solution was centrifuged at 16,000 X g for 15 min, and the pellet was collected and dried.

For GPC analysis to calculate molecular weight, polymers were resuspended in chloroform to a final concentration of 1 mg/ml and injected onto an Agilent 1260 Infinity II HPLC equipped with a PLgel 10 µm MiniMIX-B column (Agilent, CA, USA). A calibration curve was developed with EasiVial GPC/SEC polystyrene size standards (Agilent) [35].

For FTIR analysis, polymers were loaded on a Nicolet iS10 instrument equipped with a diamond ATR. For each sample, 16 scans were averaged from 650 to 4000 cm⁻¹ at a resolution of 4 cm⁻¹. Spectra were normalized using OMNIC Spectra software (ThermoFisher Scientific).

For DSC analysis, polymers were characterized on a PerkinElmer DSC 8000 instrument calibrated for melting and enthalpy with indium (PerkinElmer, MA, USA). Approximately 10 mg of polymer sample was sealed in an aluminum DSC pan and subjected to a basic heat scan. Samples were held at 40°C for 1 min, then heated to 200°C at a rate of 10°C min⁻¹. Samples were held at 200°C for 0.5 min, cooled to -20°C at a rate of 10°C min⁻¹, and finally heated to 200°C at the same rate. Percent crystallinity (X_c) was determined according to the formula X_c = (ΔH_m/ ΔH⁰_PHB), where ΔH_m is the enthalpy of fusion and ΔH⁰_PHB is the theoretical enthalpy of melting a pure crystal of PHB (146 J/g) [36].

For TGA analysis, polymers were characterized on a Mettler Toledo TGA-DSC 3 + instrument (Mettler-Toledo, OH, USA). Approximately 10 mg of samples were loaded into alumina crucibles and heated from 25–350°C at a rate of 10°C min⁻¹ with a nitrogen flow of 60 mL min^-1.

Glycerol and ammonia analysis

Culture supernatant samples (200 μl) were applied to a 96-well filter plate (FiltrEX #3510; Corning, CA, USA). The filtered media samples were analyzed for glycerol on an Agilent 1100 HPLC equipped with an Aminex HPX-87H column (Biorad, CA, USA) in 0.01 N sulfuric acid in water (pH 2.4) at a flow rate of 1 ml/min. Standard curves were made from 12.5 to 200 g/L glycerol.

Ammonium measurements were made with the API Ammonia Test kit following manufacturer’s instructions (API Aquarium Pharmaceuticals, PA, USA). Absorbance at 595 nm was read on a SpectraMax M3 plate reader (Molecular Devices, CA, USA). A standard curve was made from 0.015 to 0.1 g/L ammonium sulfate.

Results and discussion

Twelve Priestia megaterium strains in our collection that were obtained from publicly accessible repositories, including two common industrial strains [37], QM B1551 and DSM319, were studied (Table 1). The majority of the strains were acquired from the ARS Culture Collection (IL, USA) and were all deposited by different researchers. The strains were assayed for growth and PHB production on defined media containing glycerol as carbon substrate, and timepoints were collected for up to 96 hours (S1 Fig). Interestingly, although QM B1551 grew on solid media with glycerol substrate, this strain did not have significant growth in liquid culture. PV 586 is a plasmidless derivative of QM B1551 and likewise did not grow in liquid culture.

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Table 1. P. megaterium strains examined in this study.

https://doi.org/10.1371/journal.pone.0322838.t001

The ten P. megaterium strains that did grow in liquid culture demonstrated a wide variety of growth and PHB production profiles (Table 2 and S1 Fig). To illustrate these differences, three strains with distinct PHB production profiles were compared (Fig 1). YYBm1 has the fastest time of maximum PHB titer at 8 h whereas NRRL B-3254 and NRRL B-350 do not accumulate significant levels of polymer until 24 h (Fig 1A). At its peak PHB titer at 48 h, NRRL B-350 produced more PHB that YYBm1 (1.9 g/L vs 1.2 g/L). NRRL B-3254 had the lowest PHB titer of the three strains at 0.8 g/L.

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Table 2. Growth and PHB production.

https://doi.org/10.1371/journal.pone.0322838.t002

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Fig 1. Time course of growth and PHB production of three P. megaterium strains.

(A) PHB titer, (B) cell dry weight, (C) residual glycerol, and (D) residual ammonium. All data is the average of three replicates.

https://doi.org/10.1371/journal.pone.0322838.g001

NRRL B-3254 had the longest period of stable PHB titer with little change from 24 h to 96 h (Fig 1A). On the other hand, after the NRRL B-350 PHB titer peaks at 48 h, the amount drops steadily from 1.9 g/L to 0.8 g/L at 96 h. YYBm1 showed the greatest change in PHB titer where the titer rapidly dropped to very low levels from 8 h to 12 h.

The biomass levels of NRRL B-3254 and NRRL B-350 trend proportionally with their PHB titers with NRRL B-350 biomass decreasing after 48 h and the NRRL B-3254 biomass not showing a dramatic decrease from 24 h to 96 h (Fig 1B). On the other hand, the biomass of YYBm1 does not parallel the PHB titer. Although the PHB titer drops almost completely after 8 h, the YYBm1 biomass increases up to 24 h before gradually decreasing up to 96 h.

The consumption rates of glycerol and ammonium were initially much greater for YYBm1 compared to NRRL B-3254 and NRRL B-350 which is consistent with the more rapid initial growth rate of YYBm1 (Fig 1C and 1D). Only NRRL B-350 consumed all the available glycerol which is reflected in the highest PHB titer among the three strains at 48 h.

YYBm1 is a mutant of DSM 319 in which the nprM and xylA genes are deleted. Similar to its mutant derivative, DSM 319 also had a maximum PHB titer at 8 h (Table 2 and S1 Fig). Although the accumulated biomass (cell dry weight) of both DSM319 and YYBm1 were similar, the YYBm1 strain had a greater %PHB (42% vs 28%) and thus a higher PHB titer (1.2 g/L vs 0.7 g/L). It is unclear why the YYBm1 strain had a higher PHB titer relative to the DSM319 parent strain. It is possible that the deletion of the nprM and xylA genes allows more resources to be directed towards PHB synthesis. In addition, compared to the other P. megaterium strains, both the DSM 319 and YYBm1 strains had relatively narrow windows of time in which PHB was rapidly produced and then broken down after 12 h. Thus, it is possible that both strains had similar maximum PHB titers which occurred at slightly different times that were not captured in our sample collection regimen.

The sizes of the polymers were analyzed by GPC (Table 3). Among the strains that were grown on glycerol, DSM 319 had the highest molecular weight (402 kD). The other strains all had molecular weights that ranged from 119 kD to 319 kD. It is known that polymers from bacteria cultured on glycerol are typically smaller than those cultured on glucose [12]. Glycerol is known to serve as a chain terminator in PHB polymerization and can attach to the end of the polymer via the primary or secondary hydroxyl of the molecule. Previous studies have demonstrated that using glycerol as feedstock decreased the molecular weight of the polymer, and this effect was correlated with the concentration of glycerol [38,39].

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Table 3. Gel permeation chromatography of PHB from different P. megaterium strains.

https://doi.org/10.1371/journal.pone.0322838.t003

All polymers were isolated from cultures grown with glycerol substrate unless otherwise specified. Mw (weight averaged MW), Mn (number averaged MW), Mp (peak MW), Mv (viscosity average MW), Mz (third moment), and PD (polydispersity). All MW are expressed in daltons.

There is likely an additional mechanism responsible for the lower molecular weights. P. megaterium PHB polymerase is a Class IV synthase composed of two subunits: the main PhaC synthase and a smaller PhaR accessory [21]. The PhaRC synthase complex has been demonstrated to also have an alcoholysis scission activity [40]. The enzyme can use an alcohol to cleave the polymer internally thereby capping the smaller polymer with a new molecule [41]. When recombinant E. coli strains transformed with Class IV synthases were cultured on Luria Bertani (LB) media, the initial molecular weight of the polymer decreased from 10⁵ to 10⁴ Da over the course of 12 h to 60 h [42]. The GPC chromatogram of the intermediate timepoints showed the initial polymer gradually reducing in size to develop a bimodal distribution that eventually resolved to a single peak of lower molecular weight polymer. This reduction in molecular weight is believed to be caused by the alcoholysis scission activity using endogenously produced ethanol [40]. When the NRRL B-349 strain was grown in media containing glucose instead of glycerol, the intermediate time point that was analyzed showed the polymer had a higher molecular weight than that of the polymer from the culture grown in glycerol (Table 3). The GPC chromatogram of the two polymer samples showed that the polymer from the glucose culture had a bimodal distribution similar to that seen in the previous study [42] (Fig 2). It is likely that the polymer from the glucose culture would have eventually resolved into a single peak of lower molecular weight at a later time point.

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Fig 2. Molecular weight distributions of polymer from NRRL B-349.

Polymers were extracted from cells grown in cultures supplemented with glycerol (solid) or glucose (dashed).

https://doi.org/10.1371/journal.pone.0322838.g002

The polymers produced by the NRRL B-349 cultured on either glycerol or glucose were also compared by FT-IR (Fig 3). Both polymers had very similar spectra with multiple bands (1720, 1452, 1379, and 1277 cm^-1) that are characteristic of PHB [25,31,43]. The strongest band at 1720 cm^-1 corresponds to the ester carbonyl group. The bands at 1452 cm^-1 and 1379 cm^-1 are attributed to -CH₂ and -CH₃ groups, respectively. The thermal properties of both polymers were analyzed by differential scanning calorimetry (T_m, T_c, ΔH_m, and X_c) and thermogravimetric analysis (T_10% and T_max) (Table 4). These values were similar between both polymers indicating that the thermal properties were not dramatically impacted by the difference in polymer molecular weight distribution.

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Table 4. Thermal properties of polymers extracted from strain NRRL B-349 grown in cultures supplemented with glycerol or glucose.

https://doi.org/10.1371/journal.pone.0322838.t004

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Fig 3. FT-IR of polymers from NRRL B-349.

Polymers were extracted from cells grown in cultures supplemented with glycerol (solid) or glucose (dashed).

https://doi.org/10.1371/journal.pone.0322838.g003

There is a great deal of variation in the PHA production profile and molecular weights among the multiple strains analyzed (Tables 2 and 3) although the reason for these differences is currently unknown. The strains may have different tolerances to glycerol or metabolize the feedstock with varying efficiencies. Another possibility is that the PHB polymerases might have different activities. Thus, a more efficient PHB polymerase could lead to more and/or higher molecular weight biopolymer. The PHB polymerases might also have different alcoholysis scission activities which would also modulate the final molecular weight of the biopolymer.

Table 5 summarizes results from previous work producing polyhydroxyalkanoates from P. megaterium strains cultured on glycerol feedstock including strain NRRL B-1851 from this study. Many of the strains from the past studies have not been deposited in publicly accessible repositories. It is difficult to compare the different strains due to the variety of growth conditions. For instance, the culture temperatures ranged from 25°C-37°C while the timepoints analyzed varied from 12 h to 84 h. Even the glycerol in the media varied with some groups using pure lab grade glycerol while others used crude preparations of glycerol from biodiesel waste streams. In general, the highest PHB titers were derived from bioreactors employing batch and fed-batch feed strategies compared to flasks. A direct comparison was made when Moreno et al. grew P. megaterium strain B2 in both flask and bioreactor and obtained approximately three times more PHB in the latter [27].

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Table 5. PHB production from P. megaterium cultured on glycerol feedstock.

https://doi.org/10.1371/journal.pone.0322838.t005

When compared to other strains that were previously grown in flasks, several of the strains in this study had higher PHB titers (1.8–1.9 g/L) than all of the previously studied strains except for P. megaterium strain MK386891 (2.73 g/L).

Conclusions

This study provides comparison of microbial PHB production using glycerol feedstock from different P. megaterium strains that are available from readily accessible repositories. Based on this information, strains can be selected for further production optimizations depending on user priorities. For instance, if a longer window of stable PHA titer were essential, one could select a strain such as NRRL B-350 which maintains a constant titer from 24–96 hours. Alternatively, the common industrial strain DSM 319 could be selected if production speed were most critical since PHB rapidly accumulated to the maximum titer at only 8 h. Although the PHB titer does rapidly decrease after 12 hours, the DSM 319 strain could serve as a target for genetic modification to maintain higher levels of polymer over time.

Besides the different PHB production profiles, the P. megaterium strains were also demonstrated to produce biopolymers in a range of molecular weights. While high molecular weight polymers are valued for their higher tensile strength, lower molecular weight PHB could also have utility if greater flexibility is required. Thus, different P. megaterium strains could be selected for PHB production depending on the end applications.

Regardless of the specific strain that is chosen for PHB production from glycerol feedstock, optimization of growth conditions would also be expected to increase PHB levels. Simply growing the bacteria in bioreactors should yield large improvements compared to the flask conditions used in this study. Furthermore, modifications of the culture media (temperature, pH, ammonium level, glycerol source and concentration, etc.) can all impact PHB production in P. megaterium.

Supporting information

S1 Fig. Growth and polyhydroxyalkanoate production time courses.

https://doi.org/10.1371/journal.pone.0322838.s001

(PDF)

Acknowledgments

We thank Rena Kibblewhite, Joel Martinez, and Zach McCaffrey for technical assistance. Mention of trade names or commercial products in this report is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer.

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Correction

Abstract

Figures

Introduction

Materials and methods

Strains and media

16S rRNA sequencing

PHB production

Polymer analysis: gas chromatography and mass spectrometry

Polymer analysis: gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA)

Glycerol and ammonia analysis

Results and discussion

Conclusions

Supporting information

S1 Fig. Growth and polyhydroxyalkanoate production time courses.

Acknowledgments

References

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