Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4

Polymer Degradation and Stability 95 (2010) 1335e1339

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Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4 Kouhei Mizuno a, *, Aya Ohta a, Manami Hyakutake b, Yousuke Ichinomiya b, Takeharu Tsuge b a

Division of Biochemical Engineering, Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 802-0985, Japan b Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 December 2009 Received in revised form 23 January 2010 Accepted 27 January 2010 Available online 6 February 2010

We describe the characterization of polyhydroxyalkanoate (PHA)-producing bacteria isolated from an ammunition-polluted soil in Kitakyushu City, Japan. Over 270 strains were evaluated for PHA accumulation based on a colony staining method using Nile red. Of these, nine strains were selected based on the intensity of Nile red fluorescence and the cells were quantitatively analyzed for PHA by gas chromatography. PHA accumulation was observed in five strains, all of which are inferred to be close to the Bacillus cereus group according to 16S rDNA sequence analysis. Interestingly, these strains produced a PHA copolymer, poly(3-hydroxybutyrae-co-3-hydroxyvalerate) [P(3HB-co-3HV)], with a 3HV fraction up to 2 mol% with glucose as a carbon source. Further characterization was performed on one isolate, B. cereus YB-4. Gel permeation chromatography analysis revealed that the number of average molecular weights of PHA accumulated in B. cereus YB-4 drastically changed from 722,000 to 85,000 over a 72-h cultivation period. Furthermore, the PHA synthase genes were cloned and the deduced amino acid sequences were determined. This study provides new insights into PHA biosynthesis by members of the B. cereus group. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Polyhydroxyalkanoate Bacillus Molecular weight change Polluted soil Isolation

1. Introduction Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by bacteria as storage compounds for energy and carbon, normally under nutrient-limiting conditions with excess carbon. PHAs are biodegradable thermoplastics that can be obtained from renewable resources such as sugars and vegetable oils [1,2]. They are water insoluble, non-toxic, biocompatible, and have recently received attention because of their applications in the packaging industry, medicine, agriculture, and food industry. Currently, there are plans to produce some PHAs on an industrial scale employing gram-negative bacteria such as Ralstonia eutropha and recombinant Escherichia coli [3,4] because these bacteria show better growth and higher accumulation of PHAs than others, including gram-positive bacteria. However, PHAs isolated from gram-negative bacteria contain outer membrane lipopolysaccharide (LPS) endotoxins, which induce a strong immunogenic reaction in humans. Hence, these PHAs are undesirable, particularly for biomedical applications. Gram-positive bacteria have thick

* Corresponding author. Tel./fax: þ81 93 964 7303. E-mail address: [email protected] (K. Mizuno). 0141-3910/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2010.01.033

outer membranes that do not contain LPS, and therefore, are potentially better sources of PHAs for use in biomedical applications [5]. In addition, some studies have addressed gram-positive bacteria as preferable candidates for host cells of PHA production. For example, members of the genus Rhodococcus which frequently resides in arid sites like deserts permits the cells to survive in stressful environments [30], and may therefore adapt to the case of PHA production under stressful conditions. Corynebacterium glutamicum, which has long been used for fermentation production of amino acids, has been investigated as a candidate for a safe and established platform for industrial PHA production [31]. Gram-positive bacteria have another potential advantage in terms of raw materials for PHA production. The gram-positive genera Corynebacterium, Nocardia, and Rhodococcus are capable of naturally synthesizing the commercially important copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from abundant and inexpensive carbon sources such as glucose [4,6]. In contrast, gram-negative bacteria need expensive structurally related substrates such as propionic acid, valeric acid, or other fatty acids with an odd number of carbon atoms to produce 3HV units [4]. The relatively high expenditure involved is a major hindrance in PHA copolymer production. Hence, gram-positive producers could considerably reduce the production cost.

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The best characterized gram-positive bacterial group and the first PHA producer from the genus Bacillus was identified as Bacillus megaterium in 1926 [7]. Till date, many species of PHA-producing bacilli have been isolated from various environments and characterized. Some of these are able to produce PHA copolymers from inexpensive and structurally unrelated carbon sources. Bacillus sp. 88D isolated from a municipal sewage treatment plant is able to produce P(3HB-co-3HV) from glucose as a sole carbon source [8]. Bacillus cereus SPV is able to use fructose, sucrose, and gluconate to produce P(3HB-co-4-hydroxybutyrate) [P(3HB-co-4HB)] and P (3HB-co-3HV-co-4HB) [9]. Bacillus sp. INT005 isolated from a gas field soil produces P(3HB-co-3-hydroxyhexanoate) [P(3HB-co3HHx)] from glucose [10]. The diversity of PHA products in the genus Bacillus are presumed to be due to class IV PHA synthase, which was recently identified as a new class and has relatively broad monomer specificity [4]. This indicates that the genus Bacillus could be used for the industrial production of PHA copolymer, which prompted us to isolate new copolymer producers with excellent cell growth and polymer accumulation from a unique environment in Kitakyushu City, Japan. Distinctly evolved bacteria such as toluene-degrading bacteria in an ammunition-polluted soil from this area have been reported [11]. Thus, this is a source of novel bacterial strains. In this study, hundreds of bacteria isolated from a polluted soil in Kitakyushu City, Japan, were examined for their ability to produce PHAs with glucose as a carbon source. Nine PHAproducing strains were selected, and their products were further characterized. The strain YB-4 that showed the best production of PHA copolymer among the isolates, was further investigated with respect to the molecular weight of the PHA and its time-dependent change during cultivation. Furthermore, genetic analysis of the PHA synthase genes of the strain YB-4 was performed. This study provides a basis for further investigation of PHA biosynthesis by members of the B. cereus group. 2. Materials and methods 2.1. Samples Samples were collected from an ammunition-polluted area located in Fukuoka Prefecture, Japan, in 2008. This area had been used as an ammunition bunker until 1972, and reportedly deformed frogs [12] and 2,4,6,-trinitrotoluene (TNT)-metabolizing bacteria [11], the likely cause for which was residual pollution, were found here. All samples used for this study were transferred to the laboratory and stored at 4  C. 2.2. Isolation of PHA-producing bacteria

washed with distilled water to remove the remaining carbon sources, and then lyophilized. 2.4. Polymer characterization The PHA content of the lyophilized cells was determined by gas chromatography (GC) after methanolysis of the lyophilized cells in the presence of 15% sulfuric acid [14]. Methanolyzed 3HA was also analyzed by electron impact ionization gas chromatographyemass spectrometry (GCeMS) on a GCMS-QC2010 system (Shimadzu Co., Ltd., Kyoto, Japan) using an Inter Cap 1 column (GL Science Co., Ltd., Tokyo, Japan). The polymers accumulated in the cells were extracted with chloroform for 72 h at room temperature and purified by precipitation with methanol. Molecular weight data were obtained by gel permeation chromatography (GPC) as described previously [15]. 2.5. PCR amplification, sequencing, and similarity analysis of 16S rDNA sequences Eubacterial 16S rDNA was amplified using the following primer sets: 341F (50 -CCT ACG GGA GGC AGC AG-30 ) and 907R (50 -CCG TCA ATT CCT TT[A/G] AGT TT-30 ), corresponding to positions 341e357 and 907e926 in E. coli, respectively, and 27F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1525R (50 -AAA GGA GGT GAT CCA GCC-30 ), corresponding to positions 8e27 and 1543e1525 in E. coli, respectively. Bacterial colonies were picked using a sterile pipette tip and dipped into a PCR reaction mixture consisting of 5 U/mL Ex-Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), 10 mM primers, 10 mM MgCl2, and 10 mM reaction buffer (Takara Bio) in a final reaction volume of 50 mL. PCR was performed in a thermal cycler (MIR-D40, Sanyo Co., Osaka, Japan) using the following reaction conditions: 94  C for 5 min, followed by 30 cycles at 94  C for 30 s, 55  C for 30 s and 74  C for 1 min. Then, the PCR products were cloned into E. coli using a TOPO TA Cloning Kit (Invitrogen, Hilden, Germany). The cloned plasmids were extracted and purified using a Miniprep Plasmid Extraction Kit (Qiagen Co., Hilden, Germany). The DNA sequences of the plasmids were obtained using a DNA sequencing service (Hokkaido System Science Co., Ltd., Sapporo, Japan). All highly accurate sequences were compared to sequences deposited in the Genbank DNA database using the BLAST algorithm [16]. The full sequence of 16S rDNA (1544 bp) from the strain YB-4 has been deposited in DDBJ database (accession number AB535529). The formation of cereulide, the emetic toxin of B. cereus, was determined using a PCR-based assay in which the cereulide synthetase (ces) gene was targeted [17]. 2.6. Biochemical characterization of the strain YB-4

In order to isolate bacteria, a 10 g portion of each sample was homogenized in 30-mL autoclaved distilled water, and serial dilutions were plated onto agar medium (0.5 g yeast extract, 1 g peptone, 10 g glucose, 15 g agar per liter of deionized water) that contained Nile red (Sigma, St. Louis, MO, USA) at 0.5 mg per liter of medium [13]. The plates were incubated aerobically at 30  C for 2 days, and the colonies were then exposed to UV-light to visualize the strains capable of producing PHA.

The strain YB-4 was tested using an API 50 CH system (bioMérieux Co., Marcy l'Etoile, France). The API 50 CH strips were inoculated, incubated, and interpreted according to the manufacturer's instructions. Then, the data were analyzed with API labo software (bioMérieux). To analyze growth temperature, the strain YB-4 was cultured at 30e50  C with constant shaking at 120 rpm for 72 h. Growth was monitored by measuring turbidity using a spectrophotometer (Gene quant proS; GE Healthcare UK Ltd., Buckinghamshire, England) at 600 nm.

2.3. Polymer production

2.7. Cloning of PHA synthase genes from the strain YB-4

PHA production was carried out in LB medium (5 g yeast extract, 10 g tryptone, 10 g NaCl per liter of deionized water) plus 20 g/L glucose in a 500-mL shaking flask containing 100-mL medium at 30  C for 72 h or less. After cultivation, the cells collected were

The chromosomal DNA of the YB-4 strain was isolated and used as a template for PCR amplification of the PHA biosynthesis operon involving PHA synthase genes (phaR and phaC) and the 3ketoacyl-CoA reductase gene (phaB). Oligonucleotide primers for

K. Mizuno et al. / Polymer Degradation and Stability 95 (2010) 1335e1339

PCR amplification of the PHA operon were designed based on the nucleotide sequences from B. cereus E33L (accession number CP000001) and B. cereus subsp. cytotoxis NVH391-98 (CP000764) as follows: 50 -TCA TTA TGT AGC ATG ATT GTG CAT CAC CTC-30 and 50 -AAG CTC CCG TAT CCC AAA ACA ACT TAC CAG-30 . The PCR product was cloned into the TA-cloning vector pT7-Blue (Merck & Co., Inc., NJ, USA). DNA sequencing was performed with several PCR products to obtain error-free sequence data. The sequence data have been deposited in DDBJ database (accession number AB525763). 3. Results and discussion 3.1. Screening of PHA-producing strains We attempted to isolate PHA-producing bacteria from the ammunition-polluted area where the mutated frogs and toluenemetabolizing bacteria had been reported [11,12]. The textures of the soil samples used in this study were classified into two groups: moist brown and sandy. The rates of the PHA-producing isolates were 69% and 41% from moist brown and sandy soils, respectively. Hundreds of isolates were scanned to reveal a count of 65 positive for Nile red, from which nine exhibiting a significantly high intensity of fluorescence were selected, as listed in Table 1. Based on 16S rDNA analysis, seven strains were identified as members of the genus Bacillus, five of which belonged to the B. cereus group (including B. cereus, Bacillus anthracis, Bacillus mycoides, Bacillus mycoidespseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis). It is well known that members of this group are able to produce PHA, consisting mainly of shortchain-length monomers [4]. The other two strains were identified as Paenibacillus and Streptomyces. To date, no studies have reported on PHA accumulation by Paenibacillus, whereas more than ten strains belonging to the Streptomyces genus have been reported to produce PHA. The 16S rDNA sequence of YR-1 shows high similarity to that of Streptomyces griseus (99%), which is reported to produce P(3HB) at 3.5 wt% from glucose [4,18].

Table 1 16S rDNA homologies and PHA accumulation levels of the isolated strains. Strain 16S rDNA homology from BLAST match

YB-1 YB-4 SA-2 SA-3 SA-4 SA-5 SA-6 SA-7 YR-1

PHA accumulation

Closest strain (Accession number)

Identity (%)

Dry cell PHA PHA weight content composition (g/L) (wt%) 3HB 3HV (mol%) (mol%)

Paenibacillus sp. (EU497636) Bacillus cereus group (CP001177) Bacillus sp. (DQ079001) Bacillus sp. (EF625229) Bacillus cereus group (Z84594) Bacillus cereus group (DQ234844) Bacillus cereus group (Z84594) Bacillus cereus group (Z84594) Streptomyces griseus (FJ405912)

99.4 (584/587)

0.9

ND

e 98

e

99.9 (1543/1544) 6.1

35

2

99.2 (555/559)

1.5

ND

e

e

99.8 (558/559)

5.8

ND

e

e

100 (559/559)

6.7

29

100

Trace

100 (559/559)

4.3

30

100

Trace

100 (559/559)

6.1

15

98

99.8 (560/561)

4.7

44

100

Trace

99.6 (567/569)

4.3

ND

e

e

2

Cells were cultured in 500-mL shaking flasks containing 100-mL LB medium plus glucose (20 g/L) at 30  C for 72 h. 3HB: 3-hydroxybutyrate, 3HV: 3-hydroxyvalerate. ND: not detected. Trace: less than 1 mol%.

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3.2. PHA production by isolated strains The nine isolated strains were examined for PHA production from glucose in shaking cultures at 30  C for 72 h. The results are listed in Table 1. The strains SA-4, YB-4, SA-6 showed good cell growth exceeding 6 g/L, whereas the cell growth of YB-1 and SA-2 was as low as 0.9e1.5 g/L. PHA accumulation was observed in the range of 15e44 wt% with five strains, YB-4, SA-4, SA-5, SA-6, and SA-7, all of which are members of the B. cereus group, as inferred from 16S rDNA sequence analysis. Other strains did not produce PHA under these culture conditions or may have initially produced PHA that degraded within the 72-h cultivation period. In terms of PHA composition, the major component of the polymers produced here was the 3HB unit. As a minor component, 3HV units up to 2 mol% were detected by GC analysis, particularly in the PHAs produced by strains YB-4 and SA-6. The presence of the 3HV unit was confirmed by GC/MS (data not shown). As mentioned earlier, P(3HB-co-3HV) is a commercially important polymer, hence, the strains YB-4 and SA-6 may be potential candidates for industrial P(3HB-co-3HV) production. However, 2 mol% of the 3HV unit is too low for practical use. As demonstrated with Alcaligenes sp. SH-69 [19], a chemical mutagenesis approach might be effective to enhance 3HV incorporation of these strains on glucose. On comparison of the strains YB-4 and SA-6, the former was able to produce PHA more than the latter. Thus, we chose the strain YB-4 for further characterization. 3.3. Taxonomy of the strain YB-4 We taxonomically analyzed the YB-4 strain, which was found to be a gram-positive, rod-shaped bacterium of length 2.0e4.0 mm. It grew well within a temperature range of 30e45  C (optimum ¼ 35  C) and exhibited motility. Analysis of the full sequence of the 16S rDNA (1544 bp) indicated that the strain YB-4 was closely related to B. cereus AH187 (99%). The profile from the API 50 CH system for the strain YB-4 is shown in Table 2, which shows 95.2% identity to B. cereus [20], as well as the results of genetic identification. From these results, we concluded that the strain YB-4 is B. cereus. The formation of cereulide, the emetic toxin of B. cereus, was examined using PCR-based detection [17], and it was confirmed that the strain YB-4 is unable to produce cereulide. This result suggests that strain YB-4 is a non-emetic B. cereus [21]. Among the PHA-producing Bacillus strains previously reported, Bacillus sp. INT005 demonstrates most similarity to the YB-4 strain in several characteristics, including its growth temperature range (30e45  C) and cell size (1.0  2.0e4.0 mm). The two strains differed in their nutritional characteristics in that YB-4 utilizes glycerol, salicin, cellobiose, starch, and glycogen, which are not utilized by the Bacillus sp. INT005, whereas YB-4 is not capable of utilizing sucrose, which is utilized by Bacillus sp. INT005 [10]. Starch may be an appropriate substrate for the industrial PHA production by B. cereus YB-4. 3.4. Molecular weight of PHA synthesized by B. cereus YB-4 The molecular weight of PHA extracted from 72-h cultured B. cereus YB-4 was determined by GPC as shown in Table 3, and the molecular weight distribution is shown in Fig. 1. The number average molecular weight (Mn) and the weight average molecular weight (Mw) were 85,000 and 146,000, respectively. The molecular weight (Mn) is quite low when compared to those of B. cereus SPV (339,000) [22] and Bacillus sp. INT005 (281,000) [10], but similar to that of Bacillus thuringinesis R-510 (53,000e65,000) [23].

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Table 2 Taxonomic characteristics of B. cereus YB-4. Morphological characteristics Shape Size (mm) Motility Gram staining Spore formation Physiological characteristics Cereulide formation Growth condition Temperature Anaerobic condition Aerobic condition

Rod 1.0  2.0e4.0 þ þ þ e 30e45  C þ þ

Nutritional characteristics þ e   þ      þ þ          þ þ

Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol b-Methyl-D-xylose Galactose Glucose Fructose Mannose Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol a-Methyl-D-mannose a-Methyl-L-glucose N-Acetylglucosemine Salicin

     e     þ þ   e   þ    þ þ

D-Tagatose D-Fucose L-Fucose Lyxose Xylitol D-Arabitol L-Arabitol Gluconate 2-Keto-gluconic acid 5-Keto-gluconic acid Cellobiose Maltose Lactose Melibiose Sucrose Gentiobiose D-Turanose Trehalose Melezitose Raffinose Inulin Starch Glycogen

To study the time-dependent change of molecular weight, the polymers extracted from 14-h to 24-h cultured cells were analyzed (Table 3 and Fig. 1). GPC analysis showed that these polymers were of relatively high molecular weights, and their Mn and Mw were in the range of 697,000e722,000 and 1,930,000e2,030,000, respectively. The molecular weight drastically decreased between 24 and 72 h of cultivation, which may be explained by the intracellular mobilization of the accumulated polymers. Previous reports on B. thuringiensis ATCC35646 indicate that intracellular PHA depolymerase functioned at the late stationary growth phase even in a nutrient-rich medium such as LB medium [24]. The changes in P (3HB) accumulation levels during mobilization by several Bacillus strains have been studied; however, there are no reliable reports on the molecular weights of PHAs undergoing mobilization. The low molecular weight exhibited by B. thuringiensis R-510 might be the value after intracellular mobilization of PHA. This study also shows the accumulation of low molecular weight PHA at the late culture phase. Thus, the remarkable reduction in the molecular weight at Table 3 Molecular weight of PHA accumulated in B. cereus YB-4. Culture time (h)

14 24 72

Dry cell weight (g/L)

4.2 6.1 6.1

PHA content (wt%)

PHA composition

Molecular weight

3HB (mol%)

3HV (mol%)

Mn (103)

Mw (103)

Mw/Mn

12 23 35

98 98 98

2 2 2

722 697 85

1930 2030 146

2.68 2.91 1.73

Cells were cultured in 500-mL shaking flasks containing 100-mL LB medium plus glucose (20 g/L) at 30  C.

Fig. 1. Molecular weight distribution of PHAs that were isolated from 14-, 24- and 72-h cultured B. cereus YB-4.

the stationary growth phase might be a common feature of PHAproducing Bacillus strains associated with spore and septum development to provide a rapid energy supply by polymer degradation, as observed by Slepecky and Law [25]. Indeed, we observed spore formation in B. cereus YB-4 (Table 2) and the number of spores formed increased at late culture time (data not shown). Since high molecular weight polymers are potentially useful as plastic materials, avoiding the reduction of PHA molecular weight is an important issue for industrial production using Bacillus strains. 3.5. Cloning of PHA synthases from B. cereus YB-4 PHA synthases have been classified into four classes, based on their substrate specificities and subunit composition of enzymes [26]. PHA-producing Bacillus strains possess class IV synthase that comprises PhaC and its subunit PhaR. Class IV synthases preferentially polymerize short-chain-length monomers. At the pha locus of B. cereus YB-4, there are three genes similar in organization to the pha loci of class IV synthase possessing Bacillus [9,27,28]. The PHA synthase gene and its subunit gene, referred to as phaCYB4 (1086 bp) and phaRYB4 (483 bp), respectively, are represented in Fig. 2. The deduced amino acid sequence of phaCYB4 exhibited higher identity to the PHA synthases of B. cereus SPV (99%), Bacillus sp. INT005 (99%), B. anthracis str. “Ames Ancestor” (99%), B. thuringiensis serovar konkukian str. (99%), B. cereus E33L (98%) than that of B. megaterium ATCC11561 (71%). The Cys-151 of PhaCYB4 in the lipase box-like sequence (G-X-C151-X-G), which is highly conserved in all known polyester synthases, is proposed to be involved in the transesterification reaction, as well as Cys-149 in the PHA synthase from Allochromatium vinosum [29]. Also, the deduced amino acid sequence of phaRYB4 exhibited higher identity to the PHA synthase subunits of B. cereus E33L (99%), Bacillus sp. INT005 (99%), B. cereus SPV (98%), B. anthracis str. “Ames Ancestor” (98%), B. thuringiensis serovar konkukian str. 97e27 (98%) than that of B. megaterium ATCC11561 (46%). A putative 3-ketoacyl-CoA reductase is encoded by phaBYB4 (744 bp), which is located between phaRYB4 and phaCYB4 genes

Fig. 2. Genetic organization of PHA biosynthesis genes of B. cereus YB-4 (accession number AB525763). phaCYB4 gene encoding PHA synthase (PhaCYB4); phaBYB4 encoding 3-ketoacyl-CoA reductase (PhaBYB4); phaRYB4 encoding a subunit of the PHA synthase (PhaRYB4).

K. Mizuno et al. / Polymer Degradation and Stability 95 (2010) 1335e1339

(Fig. 2), and is presumed to provide (R)-3HA-CoA as the substrate of PHA synthase from 3-ketoacyl-CoA. 4. Conclusions In this study, some PHA-producing bacteria were isolated from ammunition-polluted soil. We demonstrated that bacteria belonging to B. cereus group are capable of producing P(3HB-co3HV) copolymer from glucose as a carbon source. Notably, the isolated strains YB-4 and SA-6 were capable of incorporating a higher fraction of the 3HV unit (2 mol%) than others. On the basis of taxonomic characterization, the strain YB-4 was identified as non-emetic B. cereus. The molecular weight of PHA accumulated in B. cereus YB-4 decreased drastically from 722,000 to 85,000 during the 72-h cultivation period, which is thought to be common in PHA-producing Bacillus strains. This study provides new insights into PHA biosynthesis in terms of copolymer composition and molecular weight changes by the members of B. cereus group. References [1] [2] [3] [4]

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