Enhanced Degradation of Polycyclic Aromatic Hydrocarbons (Pahs) in the Rhizosphere of Sudangrass (Sorghum � Drummondii)

Chemosphere 234 (2019) 789e795

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Enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) in the rhizosphere of sudangrass (Sorghum drummondii)

* John Jewish A. Dominguez a, , Hernando P. Bacosa a, b, Mei-Fang Chien a, Chihiro Inoue a a Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai, 980-8579, Japan b Department of Marine Sciences, Texas A&M University at Galveston, Galveston, TX, United States highlights graphical abstract

Sudangrass emerged superior in removing PAHs in soil than other previously reported grasses. Sudangrass specially enriched Sphingomonadales, a potent PAHs degrader, in its rhizosphere. The abundance of PAHs degradation biomarker genes, PAH-RHDa and nidA, was highest in soil planted with sudangrass.

article info abstract

Article history: Grasses are advantageous in the removal of polycyclic aromatic hydrocarbons (PAHs) in soil because of Received 30 January 2019 their ﬁbrous root, high tolerance to environmental stress, and low nutritional requirements. In this study, Received in revised form a pot experiment was conducted to test the ability of four grasses to remove PAHs in the soil, and to 21 May 2019 investigate the corresponding bacterial community shift in the rhizosphere of each. Sudangrass achieved Accepted 30 May 2019 the maximum removal of PAHs at 98% dissipation rate after 20 days. Polymerase chain reaction- Available online 3 June 2019 denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing revealed that su- Handling Editor: T. Cutright dangrass specially enriched the growth of a known PAHs degrader, Sphingomonadales, regardless of the presence or absence of PAHs in the soil. Moreover, the gene copy numbers of PAHs catabolic genes, PAH- Keywords: RHDa and nidA, as measured by real time-PCR (RT-PCR) were highest in the soil planted with sudangrass. Polycyclic aromatic hydrocarbons Overall, this study suggested that sudangrass further enhanced the dissipation of PAHs by enriching Phytoremediation Sphingomonadales in its rhizosphere. Rhizodegradation © 2019 Elsevier Ltd. All rights reserved. Sphingomonadales Rhizosphere effect

1. Introduction carcinogenic, teratogenic, mutagenic, and eco-toxic (Abdel-shafy and Mansour, 2016; Brown et al., 2017; Samanta et al., 2002; Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants Wang et al., 2018). Once released into the environment, PAHs tend known to pose risks to both ecological and human health. It is to adsorb in soil humic contents in the topsoil where they tend to concentrate (Kanu and Anyanwu, 2005; Okere and Semple, 2012). In order to remove PAHs with low cost and environmental burden, one method is to use living organisms such as plant, bacteria or * Corresponding author. Inoue Laboratory, Graduate School of Environmental both (Abdel Ghany et al., 2015; Gan et al., 2009; Shukla et al., 2013). Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai, 980-8579, In particular, rhizoremediation is proposed to be the approach with Japan. E-mail address: [email protected] (J.J.A. Dominguez). most potential in remediating PAHs in soil (Shukla et al., 2013). https://doi.org/10.1016/j.chemosphere.2019.05.290 0045-6535/© 2019 Elsevier Ltd. All rights reserved. 790 J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795

Rhizoremediation is the process in which plants indirectly 30 seeds of Italian ryegrass, tall fescue, and perennial ryegrass were degrade PAHs by stimulating the microbial community in its root accordingly dispersed in each pot and covered with 3 cm of soil. (Dzantor, 2007). This can be achieved in multiple ways, namely: 1) Sudangrass seeds are bigger than the other grasses. The average plant provides aeration to the soil and microbes thereby enhancing germination rate for sudangrass was 86%, 85% for tall fescue, 88% aerobic reactions (Anderson et al., 1993; Bisht et al., 2015); 2) plants for perennial rye grass, and 90% for Italian ryegrass. The soil secrete organic compounds (i.e. sugar, organic acids, secondary moisture content was maintained to 20% throughout the plant metabolites) that can stimulate microbial growth, select experiment. PAHs-degrading bacteria, and induce PAHs catabolic reactions Depending on the plant used, five experimental treatments (Anderson et al., 1993; Balasubramaniyam, 2015; Rohrbacher and were prepared with (1) or without (0) the addition of PAHs solution St-Arnaud, 2016); and finally, plants can increase the bioavail- (Table 1). The treatments include the following: unplanted control ability of PAHs through physical and chemical means (Lefevre et al., (H1, H0); tall fescue (T1, T0); sudangrass (S1, S0); perennial ryegrass 2013; Rohrbacher and St-Arnaud, 2016; Zhu et al., 2009). (P1, P0); lastly, Italian ryegrass (I1, I0). Each treatment was prepared Plants under the family Poaceae, collectively known as grasses, in triplicate. After 20 days of cultivation at room temperature, soil are often employed in rhizoremediation studies due to their fast samples were stored in 4 C until extraction. growth, tolerance to PAHs, deep and fibrous roots, resistance to environmental stress, and low nutritional requirements 2.3. Analytical procedure (Balasubramaniyam, 2015; Sivaram et al., 2018a). Furthermore, in a study conducted by Olson et al. (2007), grasses emerged as the The remaining PAHs in the soil were extracted using methods most effective in dissipating PAHs in soil among the eight families from Lee et al. (2008). Briefly, 10 g fresh weight of soil were ho- tested. Thus, it is not surprising that numerous PAHs rhizor- mogenized and transferred to 50 mL centrifuge tube and shaken emediation studies have been conducted using grasses (Fu et al., vertically with 25 mL of dichloromethane at 300 rpm for 1 h. The 2012; Gaskin and Bentham, 2010; Khan et al., 2009; Kuiper et al., tubes were then centrifuged at 5000 rpm and the supernatant was 2001). decanted and stored in 4 C until analysis. PAHs were analyzed Of particular interest in this study is sudangrass (Sorghum x using GC-4000 gas chromatography (GL Sciences Inc., Tokyo, Japan) drummondii). Despite reports of superior PAHs dissipation equipped with flame ionization detector (FID) and an InertCap 17 enhancing ability (Reilley et al., 1996; Sivaram et al., 2018b; Su et al., MS column. The column temperature was held at 80 C for 2 min, 2008), sudangrass remained relatively not studied and the infor- then increased to a maximum temperature of 300 C at the rate of mation about how the plant influences its rhizosphere during rhi- 10 C/min. The injector and detector temperatures were main- zoremediation is lacking. Among the four plants tested in a study tained at 350 C. Helium was used as a carrier gas. The concentra- conducted by Su et al. (2008), sudangrass dissipated most of pyrene tion of PAHs was calculated based on a five-point standard curve. but enriched the soil microbial population the least. Hence, it is worth investigating how sudangrass possibly achieve dissipation of 2.4. Microbial community analyses PAHs with respect to its rhizosphere microbial community. fl In this study, the ability of sudangrass to dissipate PAHs ( uo- Total DNA was extracted from the soil using Powersoil DNA fl rene, phenanthrene, uoranthene, and anthracene) in soil and the extraction Kit (Qiagen, Inc., Hilde, Germany) based on the manu- corresponding changes in the bacterial community of the rhizo- facturer's protocol. The extracted DNA was used as template for sphere soil was investigated. Furthermore, known PAHs degrada- polymerase chain reaction-denaturing gel gradient electrophoresis a tion biomarkers such as PAH-RHD and nidA genes were analyzed (PCR-DGGE). Briefly, the V3 region of 16s rRNA gene was amplified in parallel. These were conducted together with other grasses by PCR using universal primers 341F-GC (50-CGC CCG CCG CGC GCG previously reported to enhance PAHs degradation, namely: tall GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG fescue (Festuca arundinacea), perennial ryegrass (Lolium perenne) CAG-30) and 518R (50-ATT ACC GCG GCT GCT GG-30). PCR was per- fl and Italian ryegrass (Lolium multi orum). formed in reaction mixtures composed of 25 ml of Promega Master Mix (Promega Corp., WI, USA), 2 mL(10mM) each of forward and 2. Materials and methods reverse primers, approximately 10 ng of DNA extract and filled with DNAse free water to a volume of 50 mL. The amplification conditions 2.1. Chemicals and plant samples in the 2720 Thermal Cycler (Applied Biosystems, Foster, CA, USA) were as follows: initial denaturation at 94 C for 5 min, followed by fl fl Analytical grade uorene, phenanthrene, uoranthene, and 30 cycles of 94 C for 30 s, 55 C for 30s and 72 C for 30s, and a final pyrene were procured from Wako Chemicals, Ltd. (Osaka, Japan). extension step at 72 C for 7 min. The presence of PCR products was Four grasses were used in the study, namely: tall fescue (Festuca confirmed by gel electrophoresis. arundinacea), sudangrass (Sorghum x drummondii), perennial DGGE was performed using the DCode Universal Mutation fl ryegrass (Lolium perenne), and Italian ryegrass (Lolium multi orum). Detection System (Bio-Rad, California, USA). An 8% polyacrylamide & The seeds were purchased from Takii Co., Ltd (Miyagi, Japan). gel with a 30e70% gradient was prepared using urea and form- amide as denaturants. The samples were then loaded and run at a 2.2. Biodegradation experiment constant voltage of 70V and constant temperature of 60 C for 16 h.

For the soil, four bags of lawn soil with unknown composition (Daisin Co., Ltd., Miyagi, Japan) were thoroughly mixed to achieve a Table 1 homogenous soil composition. The PAHs in the lawn soil were Experimental set-up employed in the biodegradation experiment. below detection limit. Four hundred grams of the soil was weighed, Soil with 400 ppm PAHs No PAHs added fl sprayed with PAHs solution (1 mg/mL each of uorene, phenan- Unplanted control H1 H0 threne, fluoranthene, and pyrene) to a final concentration of Tall fescue T1 T0 400 mg/kg, and mixed thoroughly using a hand mixer. The solvent, Sudan grass S1 S0 acetone, was allowed to evaporate for five days. The soil was then Perennial ryegrass P1 P0 Italian ryegrass I1 I0 transferred to a 0.55 L vinyl pots. Twenty seeds of sudangrass and J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795 791

After the run, the gel was unloaded, stained with ethidium bromide PAH is still limited and sometimes conflicting. In this study, all and viewed under UV illumination for documentation. plants enhanced the dissipation of phenanthrene and pyrene which The bands of interest were excised from the gel and centrifuged is consistent with previous reports (Binet et al., 2000; Chouychai briefly. PCR reamplification using 341F and 518R primers was per- et al., 2009; Reilley et al., 1996). However, statistically significant formed as previously mentioned. The PCR products were subjected removal of fluoranthene was only achieved in sudangrass and to gel electrophoresis and were excised and purified using QIAquick ryegrasses but not in tall fescue. In contrast, Robinson et al. (2003) PCR purification kit (Qiagen, Hilden, Germany) according to man- reported tall fescue to enhance dissipation of fluoranthene but not ufacturer's instructions. After purification, the products were used phenanthrene in an aged creosote-contaminated soil. Thus, plant as template for sequencing using BigDye Terminator v3.1 chemistry information alone does not suffice in predicting the outcome of in 3130 Genetic Analyzer (Applied Biosystems Inc., USA). All se- PAHs dissipation. Discrepancies can be attributed to other factors quences obtained were compared to available sequences in the such as aging and soil properties (Sivaram et al., 2018a). In this GenBank database using the Basic Local Alignment Search Tool study, freshly spiked lawn soil was used and the results may not be (BLAST). the same in other soil with different properties and PAHs content as Soil samples planted with sudangrass were submitted for demonstrated by Smith et al. (2011). According to a meta-analysis pyrotag sequencing using Roche 454 pyrosequencer (Chunlab Inc., conducted by Ma et al. (2010), plants have more apparent effect South Korea). on freshly spiked soil than in long-term contaminated soil. Never- theless, as with past studies, this study provided strong evidence 2.5. Real-time PCR that given specific conditions, plants can further the rate of PAHs dissipation in soil. Quantitative PCR was performed to estimate the PAH-ring hy- In terms of the mechanism of dissipation, both abiotic (e.g. droxylating dioxygenase (PAH-RHDa) and pyrene dioxygenase volatilization and chemical degradation) and biotic (e.g. microbial (nidA) genes using the primers or probe designed by Ding et al. degradation and plant uptake) factors can be accounted for the loss (2010) and Debruyn et al. (2007), respectively. Real-time PCR as- of PAHs (Ghosal et al., 2016; Reilley et al., 1996). In a phytor- says were performed using a 7500 Real-Time PCR System (Applied emediation system, plants and bacteria can both contribute to the Biosystems Inc., USA) based on the procedure described by Bacosa degradation of PAHs. Specifically, PAHs can be adsorbed, absorbed, and Inoue (2015). Briefly, the 20-ml reaction mixture containing 5 ml accumulated, and degraded by some plants (Alagic et al., 2015; Gao template DNA, 10 ml Power SYBR Green PCR Master Mix (Applied and Zhu, 2004; Liu et al., 2015). However, more evidence points Biosystems, USA), 1 ml (10 pmol/ml) each of forward and reverse toward enhanced microbial degradation as the main mechanism of primers, and 2 ml of DNAse free water was prepared. Then, the run biotic PAHs dissipation (Alagic et al., 2015). was conducted using the following cycling conditions: 2 min at 50 C for carryover prevention; 10 min at 95 C for enzyme acti- 3.2. Enhanced PAHs degradation by sudangrass vation; and 40 cycles of 30 s at 95 C, 1 min at 57 C, and 30 s at 72 C. All samples and standards were run in triplicates with Among the grasses tested, sudangrass (S1) exhibited the most negative controls included in all quantifications. Standards were remarkable decrease in total PAHs concentration equivalent to 98% prepared by cloning target genes in pCR2.1 vector using the TOPO dissipation of the initial concentration. Compared to other grasses, TA cloning kit (Invitrogen, California, USA) according to the man- PAHs rhizodegradation studies focusing on sudangrass are few but ufacturer's protocol. Once the loaded vectors are transformed into the experimental results obtained in this study is parallel with TOP10 Escherichia coli cells, eight-point standard curves were made previous accounts in terms of its positive effect on PAHs degrada- using 16S rDNA and nidA genes from Mycobacterium vanbaalenii tion (Reilley et al., 1996; Sivaram et al., 2018a). This superior per- PYR-1, and PAH-RHDa genes from a bacterial consortium in the formance by sudangrass can be attributed to its faster growth. As a laboratory. C4 plant, sudangrass has faster photosynthesis rate that could result to more rhizodeposits which in turn stimulate more bacteria 2.6. Statistical methods to degrade PAHs (Sivaram et al., 2018b). Aside from this, according to the results of Sivaram et al. (2018a), sudangrass has higher PAHs All statistical analyses have been conducted using Paleontolog- bioaccumulation potential than tall fescue and ryegrass though ical Statistics (PAST) software (Hammer et al., 2001). minimal in concentration. While the results are limited only to up to 20 days post-germination period, the time frame was long 3. Results and discussion enough to differentiate the grasses based on their ability to enhance PAHs dissipation. Further, the results suggest that sudangrass is 3.1. Dissipation of PAHs more practical to use since the removal of PAHs in soil can be achieved faster than the other grasses. The dissipation of PAHs in lawn soil after 20 days is shown in Fig. 1. Even without plants (H1), the PAHs concentration decreased 3.3. Bacterial community shift four times the initial concentration (from 400 mg/kg to 105 mg/kg) implicating degradation by either biotic or abiotic means. Among The change in bacterial community before and after the exper- the four PAHs, only fluoranthene and pyrene remained after 20 iment is presented in Fig. 2. Selected bands were excised and days in the planted treatment. In general, the degradation of PAHs sequenced as shown in Table 2. Two general trends can be seen: 1) decreases with increasing molecular weight (Bacosa and Inoue, the lawn soil was dominated by Fulvivirga kasyanovii (band A in 2015). Hence, low molecular weight PAHs such as fluorene and Fig. 1) that remained abundant even after the biodegradation phenanthrene might have been degraded first. Also, it is apparent experiment; 2) the treatment indiscriminately enriched Massilia that all planted treatments exhibited significant reduction of niabensis and Novosphingobium indicum (band c and d in Fig. 2, overall PAHs concentration. The ability of plants to enhance dissi- respectively). Fulvivirga kasyanovii is a gram-negative bacteria pation of PAHs in soil is a widely reported phenomenon (Fu et al., originally isolated from the sea water (Nedashkovskaya et al., 2012; Gao et al., 2010; Lee et al., 2008; Sivaram, 2014). However, 2007). This might hint where the lawn soil is extracted. On the information regarding what plant can enhance degradation of what other hand, some strains of M. niabensis and N. indicum are 792 J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795

Fig. 1. Residual PAHs in lawn soil after 20 days. One-way ANOVA analysis revealed that treatments in ﬂuoranthene and pyrene vary signiﬁcantly, p < 0.05 and p < 0.01, respectively. Post-hoc analysis is based on Tukey's HSD.

2016; Liu et al., 2014). N. indicum is also known as a PAHs degrader (Yuan et al., 2009). The presence of these bacteria can account for the degradation even in unplanted soil.

3.4. Enrichment of Sphingomonadales by sudangrass

One specific band of interest corresponding to Sphingomona- daceae bacterium (band g), appears to be specially enriched in planted treatments with PAHs (lane H1, T1, S1, P1 and I1). Sphin- gomonadaceae is a genus known for its ability to degrade PAHs (Bacosa and Inoue, 2015; Leys et al., 2004; Luo et al., 2012). Based on this result, Sphingomonadaceae can be implied to play an important role in plant-induced enhancement of PAHs degradation. Interestingly, sudangrass without PAHs (lane S0) also enriched the growth of this bacterium. Thus, sudangrass may be capable in shaping the rhizosphere bacterial community competent for PAHs degradation by enriching Sphingomodaceae. While the specific mechanism by which sudangrass enrich Sphingomonadaceae is unknown, it is possible that sudangrass exude compounds that structurally resemble PAHs (Rohrbacher and St-Arnaud, 2016; Singer, 2006). Soil samples planted with sudangrass were further analyzed by pyrosequencing. Fig. 3 showed that Sphingomonodales dominated Fig. 2. DGGE profiles of the 16S rRNA gene amplified from lawn soil before cultivation both samples but is further enriched in soil with PAHs. It can be said and after 20 days of biodegradation experiment. with confidence that the degradation of PAHs can be attributed to this order. Together with Sphingomonodales, other bacteria that implicated in PAHs degradation. While M. niabensis is originally are enriched in the presence of PAHs include Burkholderiales, isolated in air samples (Weon et al., 2008), the genus Massilia was Alteromonodales, Methylophilales, Xanthomonodales, Pseudomo- found to be a predominant genus in some grasses (Wemheuer et al., nodales and Legionellales. In contrast, those that are reduced, if not 2017) and reclaimed grasslands (Chaudhary and Kim, 2017). Some eliminated, include Myxococcales, Chromatiales, Anaerolinaeles, Massilia species were reported to degrade phenanthrene (Gu et al., Cytophagales, Sphingobacteriales and Steroidobacter. The families that were enriched in the presence of PAHs have also been reported J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795 793

Table 2 Identiﬁcation of the bands excised from PCR-DGGE.

Band Phylogenetic Group Closest relative in the GenBank (Accession No.) Similarity

a Bacteroidetes Fulvivirga kasyanovii KMM 6220168 (NR043975) 178/190 (94%) b Gammaproteobacteria Steroidobacter denitriﬁcans FS (NR044309) 185/194 (95%) c Betaproteobacteria Massilia niabensis 5420S-26 (NR044571) 189/193 (98%) d Alphaproteobacteria Novosphingobium indicum H25 (NR044277) 189/192 (98%) e Bacteroidetes Fulvivirga kasyanovii KMM 6220168 (NR043975) 172/192 (93%) f Gammaproteobacteria Steroidobacter denitriﬁcans FS (NR044309) 185/193 (96%) g Alphaproteobacteria Sphingomonadaceae bacterium E4A9 (NR044320) 169/169 (100%) h Alphaproteobacteria Sphingomonadaceae bacterium E4A9 (NR044320) 182/182 (100%) i Bacteroidetes Fulvivirga kasyanovii KMM 6220168 (NR043975) 173/185 (93%) j Alphaproteobacteria Novosphingobium indicum H25 (NR044277) 185/189 (98%) k Alphaproteobacteria Sphingomonadaceae bacterium E4A9 (NR044320) 191/191 (100%)

to be enriched in other petroleum hydrocarbon- or PAHs-polluted environments (Atlas et al., 2015; Chemerys et al., 2014; Ding et al., 2012; Dong et al., 2015; Gutierrez et al., 2013; Zhang et al., 2011). Taking all of these together, despite the presence of potential PAHs degraders across treatments, only sudangrass enriched the bacterium under the phylogenetic group Sphingomonadales even without PAHs. Thus, sudangrass might have contributed to the increase of this potent PAHs degrader in the soil resulting to an enhanced PAHs degradation superior to other grasses. This results provide insights by which difference in plants and their ability to enhance PAHs dissipation can be conﬁrmed using a microbial community perspective.

3.5. Abundance of PAHs catabolic genes

While PAHs degradation analysis and DGGE data showed enhanced degradation of PAHs in the presence of plants and spe- ciﬁc bacteria, it does not provide direct evidence that plants stimulate bacteria to degrade PAHs. Thus, quantitative analysis of the bacterial PAH catabolic genes, PAH-RHDa and nidA, in the presence or absence of plants has been conducted (Bacosa and Inoue, 2015; Debruyn et al., 2007; Ding et al., 2010). As shown in Fig. 4, increased copy numbers of these genes were observed in treatments spiked with PAHs. Interestingly, the genes were also present in soil planted Fig. 3. Bacterial community composition of sudangrass with (S1) and without PAH with sudangrass even without PAHs. This reinforced the hypothesis (S0) at day 20 based on pyrosequencing. that sudangrass might be releasing compounds that is structurally similar to PAHs resulting to the increase of these catabolic genes. Furthermore, the quantity of these genes coincides with the

Fig. 4. Total abundance of 16s rRNA, PAH-ring hydroxylating dehydrogenase alpha (PAH-RHD) and pyrene dioxygenase (nidA) genes at day 20. 794 J.J.A. Dominguez et al. / Chemosphere 234 (2019) 789e795 degradation rate obtained in each treatment. Specifically, soil Chouychai, W., Thongkukiatkul, A., Upatham, S., Lee, H., Pokethitiyook, P., planted with sudangrass that exhibited largest decrease in PAHs Kruatrachue, M., 2009. Plant-enhanced phenanthrene and pyrene biodegradation in acidic soil. J. Environ. Biol. 30, 139e144. concentration also displayed highest copy number of the catabolic Debruyn, J.M., Chewning, C.S., Sayler, G.S., 2007. Comparative quantitative preva- genes. While the mere presence of these genes do not necessarily lence of mycobacteria and functionally abundant nidA , nahAc , and nagAc equate to activity, it provides strong evidence that sudangrass dioxygenase genes in coal tar contaminated sediments. Environ. Sci. Technol. 41, 5426e5432. cultivate bacteria capable of degrading PAHs. Surprisingly, nidA, Ding, G., Heuer, H., Smalla, K., 2012. Dynamics of bacterial communities in two which was specifically designed for mycobacteria solicited same unpolluted soils after spiking with phenanthrene : soil type specific and com- response as the more general PAH-RHDa gene despite the absence mon responders. Front. Microbiol. 3, 1e16. https://doi.org/10.3389/fmicb.2012. 00290. of Mycobacteria (Fig. 3). It is possible that other bacterium that Ding, G., Heuer, H., Zu, S., Spiteller, M., Pronk, G.J., Heister, K., et al., 2010. Soil type- possess similar conserved region have reacted to the test. Never- dependent responses to phenanthrene as revealed by determining the diversity theless, the use of these biomarkers were confirmed to be a good and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system. Appl. Environ. 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