Rice has been suggested as a major route for Sb exposure, especially in mining areas [14, 36], and is reported to be more efficient in taking up Sb(III) than Sb(V) [14]. SbO will generate the less mobile Sb(V) and thus reduce the uptake of Sb by the rice. Therefore, SbO may be beneficial to attenuate the consequences Sb contamination in rice paddies. The anoxic conditions and the high levels of nitrate in rice paddies may facilitate nitrate-dependent SbO, which, however, has never been reported in rice paddies. Therefore, the current study tackles this important but less understood environmental issue to investigate the potential of nitrate-dependent SbO in the Sb-contaminated rice paddies.
Our current understanding of anaerobic Sb(III) oxidizers is mainly based on three isolates [13, 18, 27]. Culture-independent tools such as DNA-SIP may be available to expand the list of nitrate-dependent SbOB. However, clear Sb-dependent growth is difficult to observe because SbOB require high concentrations (millimolar range) of Sb(III) to prompt significant increases in biomass [26]. The slow growing nature of SbOB may increase the required duration of 13C incorporation and thus incur cross-feeding, which complicates the interpretation of SIP data. The current study aims to examine the proof of concept of using DNA-SIP combined with 16S rRNA gene amplicon sequencing to reveal nitrate-dependent SbOB. Given that the long incubation time for DNA-SIP may cause cross-feeding among microorganisms, shotgun metagenomic sequencing was further performed on the heavy DNA fractions of 13CSbN to examine whether some key genes responsible for nitrate-dependent SbO (i.e., Sb(III) oxidation, nitrate reduction, and carbon fixation) were present in the putative nitrate-dependent SbOB to confirm their role in nitrate-dependent SbO.
Nitrate-dependent SbO potential of the Sb-contaminated rice paddy soil
Anaerobic Sb(III) oxidation to Sb(V) was clearly demonstrated in the anoxic rice-paddy cultures amended with Sb(III) and NO3−, but not in the cultures amended with NO3− or Sb(III) only (Fig. 1), suggesting that the addition of nitrate may facilitate SbO. Nitrate reduction with concomitant SbO is further supported by the conversion of nitrate to nitrite (Fig. 1c and d). Sterile controls showed no formation of Sb(V) supporting that nitrate-dependent SbO is a biotic process. Overall, this observation confirms that bacteria can mediate nitrate-dependent SbO in rice paddy soils.
aioA may be the key gene for nitrate-dependent SbO
Given similar chemical properties shared by Sb and As elements, it has been proposed that microbes may drive Sb transformation by using similar metabolic pathways with As. Previous studies suggest that arsenite oxidase (encoded by the aioA gene) may be responsible for SbO. For example, the aioA gene has been detected in some known nitrate-dependent SbOB including Hydrogenophaga taeniospiralis strain IDSBO-1 and Sinorhizobium sp. GW3 [13, 27]. In addition, the transcription level of the aioA gene in Sb(III)-oxidizing Sinorhizobium sp. GW3 significantly increased upon Sb(III) addition under anaerobic conditions and a mutation in the aioA gene reduced the anaerobic SbO rate by over 70% [13]. Consistently, several observations in this study also support that the aioA gene is involved in anaerobic SbO: (i) the copy numbers of the aioA gene increased only in the cultures with nitrate-dependent SbO (Fig. 2a). The abundance of the aioA gene showed significant positive correlations with the concentration of Sb(V) produced over the course of nitrate-dependent SbO in the treatment amended with both Sb(III) and NO3− (R = 0.88, P < 0.05) (Fig. 2b), whereas such correlation was not detected in two other treatments where nitrate-dependent SbO was not observed (data not shown); (ii) following a 60-day incubation period, the highest relative abundance of the aioA gene was observed to gradually shift to the heavier DNA fractions only in the 13CSbN treatment where nitrate-dependent SbO occurred, while no obvious shifts to the heavier fractions were found in other treatments (i.e., 13CSbN, 13CN and 12CN). These observations collectively support that the aioA gene is responsible for nitrate-dependent SbO. A Sb(III) oxidase, encoded by anoA gene, belonging to the short-chain dehydrogenase/reductase family was recently identified and proposed to be responsible for aerobic SbO [20]. In this study, the anoA gene, however, was neither successfully amplified from any of the treatments nor observed in the metagenome, implying that anoA may not be responsible for nitrate-dependent SbO in this rice paddy soil.
Putative nitrate-dependent SbOB identified by DNA-SIP
A number of genera, such as Azoarcus, Azospira and Chelativorans, were proposed as putative nitrate-dependent SbOB in the current study. The relative abundance of Azoarcus spp. increased from undetectable in the original rice paddy soil inoculum to 78% at day 60 in the cultures amended with Sb(III) and NO3− (Fig. 3). Since Azoarcus was not enriched in the cultures amended with NO3− only, it suggests that SbO likely supported its growth. Furthermore, as seen in the DNA-SIP result (Fig. 5), Azoarcus dominated (close to 50%) in the heavy DNA fractions of the 13CSbN treatment, but was not found in the 13CN treatments, thus demonstrating that Azoarcus incorporated 13C-NaHCO3 only during nitrate-dependent SbO. Azoarcus spp. are well known for their capability to mediate nitrate-dependent As(III) oxidation via AioA in paddy soils and other environments [34, 38]. Consistently, aioA genes were observed in the bin associated with Azoarcus (bin9), supporting their role also in SbO. In addition, genes for denitrification and carbon fixation were observed in the Azoarcus-associated bin9, suggesting its capability for denitrification and autotrophy (Fig. 6). Collectively, these results support that Azoarcus-associated bacteria are responsible for the autotrophic oxidation of Sb(III) linked to nitrate reduction in the paddy soil. Bacteria associated with Azospira dominated (42 ± 6%) the bacterial communities in the treatment amended with Sb(III) and NO3− at day 30 (Fig. 3) and was observed to be significantly enriched in the heavy DNA fractions of 13CSbN treatment compared to 13CN (Fig. 5). In addition, a bin associated with Azospira containing the aioA gene was detected in the 13C-heavy-fraction metagenome (Fig. 6). These observations suggest that Azospira may be a putative nitrate-dependent SbOB. The detection of genes for denitrification and carbon fixation in the Azospira-associated bin also supported its capability for nitrate-dependent SbO. Nitrogen cycling by the genus Azospira has been previously described, including nitrogen fixation and denitrification [39, 40]. Although Azospira spp. has not previously been shown to oxidize Sb(III) under either aerobic or denitrifying conditions, autotrophic Azospira sp. strain ECC1-pb2 isolated from sludge and sediment samples was capable of As(III) oxidation linked to chlorate reduction [41]. Our current study identified Azospira spp. as putative nitrate-dependent SbOB. Chelativorans-affiliated bacteria were identified as putative nitrate-dependent SbOB in this study because of two reasons: (i) they were significantly enriched in the heavy fractions of 13CSbN than their counterparts in 13CN (Fig. 5); (ii) aioAB genes and denitrifying genes were observed in the Chelativorans-associated bin (Fig. 6), supporting their potential ability for nitrate-dependent SbO. Members of Chelativorans have been extensively identified as heterotrophic denitrifiers and have been enriched in uranium-contaminated soil [42–44]. However, the detection of genes for carbon fixation suggested that they hole the potential to oxidize Sb(III) autotrophically.
Metagenomic-binning of the 13C-heavy-fraction metagenome provides an additional method to examine the physiological traits of the nitrate-dependent SbOB community. Many bins, such as those associated with Thauera, Ramlibacter and Anaeromyxobacter, contained an aioA gene. Although Thauera, Ramlibacter and Anaeromyxobacter have been detected in As-contaminated sites previously [45–47], their role in either As(III) or Sb(III) oxidation has not been reported. The presence of aioA and the genes responsible for denitrification and carbon fixation in the bins related with these genera (Fig. 6), suggests that they have the potential for nitrate-dependent SbO.
Relatively higher abundance of Gemmatimonas was observed in the heavy DNA fractions of both 13CSbN and 13CN than those in corresponding light fractions. Neither aioA nor aioB, however, was detected while genes involved in denitrification and carbon fixation were observed in the Gemmatimonas-associated bins (bin8). These observations suggested that Gemmatimonas may be more likely autotrophic denitrifier without the capability to oxidize Sb(III). In addition, bacteria associated with Halomonas, Geobacter and Pelagibacterium were significantly enriched in the heavy fractions in the 13CSbN treatment than that of 13CN (Fig. 5). Although Halomonas was identified as As(III) oxidizers [48], Geobacter spp. are notable for their capability for metal reduction [49] and Pelagibacterium has never been associated with As or Sb transformation. Unfortunately, bins associated with these three genera were not detected by the metagenomic-binning, thus we cannot determine whether they are potentially nitrate-dependent SbOB. Further investigation, such as isolation of members of these genera, are necessary to reveal their role in nitrate-dependent SbO.
The current study provided a proof of concept of using DNA-SIP to identify nitrate-dependent SbOB. The long incubation time (60 day) are necessary to observe obvious shift of 13C-incorporating microbial communities. Because long incubation time may incur cross-feeding [50], shotgun metagenomics followed by DNA-SIP is suggested to provide the physiological traits of the putative nitrate-dependent SbOB and identify the scavenging denitrifies or other microorganisms incorporating 13C from cross-feeding.