439
remediation of PHE-copper co-contaminated soils
440
The inoculation of Sphingobium PHE-1 significantly improved the growth of 441
ryegrass (Figure 5). Interestingly, the growth of ryegrass was also found to be greatly 442
stimulated by Cu(II), with plants grown in PHE-copper co-contaminated soils having 443
higher biomass than those grown in soils with PHE contamination only (Figure 5), 444
which was consistent with the trends of ahdA1b-1 and xyLE gene transcription in 445
MSM (Figure 2B and Figure 4B). This improvement in ryegrass growth was 446
attributed to the increasing activities of Sphingobium PHE-1 in the presence of Cu(II). 447
It was reported that some heavy metal-resistant bacteria could promote the growth of 448
host plants. Examples included a copper-resistant plant growth-promoting bacterial 449
(PGPB) strain, Ax10, which was isolated from a copper mine soil and facilitated 450
Brassica juncea growth and Cu(II) uptake (Ma et al., 2009). Besides, the 451
nickel-resistant strain PGPB SRS8 was also found to be capable of stimulating plant 452
growth and nickel accumulation in the crops Ricinus communis and Helianthus
453
annuus (Ma et al., 2011). 454
The residual PHE concentrations in soils subjected to different treatments on day 455
56 were presented in Figure 6. The results clearly illustrated that more PHE was 456
removed from soils inoculated with Sphingobium PHE-1, compared to the 457
uninoculated treatments, and ryegrass planting also resulted in lower residual PHE. 458
The best performance in terms of PHE reduction occurred in the treatment with both 459
ryegrass and Sphingobium PHE-1. This optimal PHE removal was explained by 460
microbial degradation and, to some extent, by enhanced adsorption to roots and 461
accumulation in ryegrass shoots, as found in our previous study (Wang et al., 2012). 462
Besides, the bioavailability of PAHs, which often limits the biodegradation of these 463
compounds, may be increased with the aid of some components in the root exudation 464
(An et al., 2010; Cerniglia, 1993; Gao et al., 2010). 465
The abundance of xylE genes in soils was also studied to monitor the effect of 466
ryegrass on the growth and degrading activities of Sphingobium PHE-1, attributing to 467
its higher specificity than ahdA1b-1 and 16s rRNA genes. A higher abundance of xylE
468
gene in soils with ryegrass was found than that in soils without ryegrass (Figure 7, 469
p<0.05), which proved that ryegrass could encourage the abundance of Sphingobium
470
PHE-1. Additionally, the copy numbersof xylE gene in soil was also enhanced by the 471
Cu(II) addition, which was in agreement with the transcription of xylE in 472
Sphingobium PHE-1 (Figure 2B). The increase of xylE abundance in soils planting 473
with ryegrass was attributed to the positive influence of rhizospheric effect on 474
microbes. The fibrous root of ryegrass observed in this work provided microbial 475
attachment sites and enhanced soil aeration, and the vegetation cover created 476
favourable environmental conditions such as temperature and soil moisture. More 477
importantly, the root exudates as the ecological driver in the rhizosphere supply
478
microorganisms with a relatively constant source of nutrients, such as water soluble
479
carbon, nitrogen and phosphorus, especially in poor soils. The rhizosphere therefore
480
improves the microbial growth, activities and the abundance of functional genes
481
(Haritash and Kaushik, 2009). Besides, many secondary plant metabolites in root
482
exudation with similar structure to aromatic hydrocarbons may stimulate the
483
metabolic pathways of PAHs degraders (Martin et al., 2014).It was observed that
484
more microbes, enhanced activities and increased abundance of PAHs-degrading 485
genes in planted versus unplanted soils occurred in PAHs contaminated soils (Haritash
486
and Kaushik, 2009; Thomas and Cebron, 2016). 487
In the present study, the growth of ryegrass was improved by Sphingobium 488
PHE-1. In turn, the ryegrass provided abundant nutrients to Sphingobium PHE-1 and 489
promoted its colonisation in the rhizosphere, which enhanced the dissipation of PHE. 490
It was proved that PHE and pyrene (PYR) dissipation with the aid of microbes in soils 491
was improved by planting ryegrass and the levels of PHE and PYR increased with the 492
distance from the root surface (Gao et al., 2013). A similar phenomenon was also 493
observed in another study, in which the growth of ryegrass significantly increased soil 494
peroxidase activities, leading to enhanced dissipation of PHE and PYR in, and 495
additionally, the interactions of ryegrass with the two microbes further improved the 496
dissipation of PHE and PYR (Yu et al., 2011). In the present study, the growth of 497
ryegrass was improved by inoculation with Sphingobium PHE-1, further benefiting 498
bacterial growth by rhizospheric effect and improving PHE dissipation as well. The 499
capacity of strain Sphingobium PHE-1 to exhibit Cu(II) tolerance and degrade PHE 500
suggests its feasibility in the remediation of PAHs-heavy metal co-contaminated soils 501 and sediments. 502 503 Acknowledgments 504
This study was supported by the National Natural Science Foundation of China 505
(Nos. 41322008 & 41673111), the Joint Funds of the National Natural Science 506
Foundation of China and the Natural Science Foundation of Guangdong Province, 507
China (U15012345), and the Doctoral Scientific Research Foundation of Guangdong 508
Province (No. 2016A030310118). 509
510
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