Response of ammonia oxidizing microbes to the stresses of arsenic and copper in two acidic alfisols
Introduction
Ammonia oxidation (AO) is the first and rate limiting step in nitrification (Di et al., 2009), and thus plays a decisive role in the global nitrogen cycle. For more than a century, it has been believed that AO is exclusively mediated by chemolitho-autotrophic ammonia oxidizing bacteria (AOB) (De Boer and Kowalchuk, 2001). The recent discovery of homologs of ammonia monooxygenase gene (amoA) in archaea and subsequent cultivation of autotrophic ammonia oxidizing archaea (AOA) affiliates to phylum Thaumarchaeota has shown the importance of AOA in the global nitrogen cycle (Könneke et al., 2005). Nevertheless, the key factors driving the abundance, diversity and activity of ammonia oxidizers are still unclear. Studies have demonstrated the changes in abundance and diversity of AOB and AOA in response to different abiotic stresses like temperature, pH, soil type, organic and inorganic fertilization (Chan et al., 2013, Chen et al., 2013, He et al., 2007, Nicol et al., 2008, Nyberg et al., 2012). However, the response of AOA to heavy metals in acidic soils remains poorly characterized. It has been estimated that over 50% of the world's potential arable lands are acidic (von Uexkull and Mutert, 1995). Currently about 12% of world land area in crop production is acidic in nature. Asian continent accounts for approximately 1044 million ha (26.4%) of the world's acidic soils. It is of paramount importance to know the effect of anthropogenic perturbations on nutrient cycling processes of acidic soils. For example, soil contamination by heavy metals is known to be one of the most important anthropogenic perturbations and is of great worldwide concern. Heavy metals adversely affect the activity and diversity of microbial community associating with soil elemental cycling (Dai et al., 2004, Khan et al., 2010). Among all metals, copper (Cu) and arsenic (As) have received much attention because of their high toxicity and solubility in agricultural soils (Sheldon and Menzies, 2005, Smedley et al., 1996). In addition to this, As has received a lot of attention in East and Southeast Asia because of the well-publicized catastrophes in India (West Bengal), Bangladesh, and Vietnam (Meharg and Hartley-Whitaker, 2002, Singh and Ma, 2007). Cu enters the soils due to several sources, e.g. mining, smelting, electroplating, biosolids, agricultural application of sludge, copper-containing pesticides and fertilizers (Lone et al., 2008). Elevated levels of As in soils on the other hand originate from the widespread use of arsenicals as pesticides, primary and secondary industrial inputs (Adriano, 2001). Numerous field and microcosm studies have demonstrated the adverse effects of As and Cu on soil microbial communities (Brandt et al., 2006, Chodak et al., 2013, Wightwick et al., 2013). However, little information is available on the effects of Cu and As on soil ammonia oxidizers (especially in acidic soils) which are an important functional groups within the N cycle.
Nitrification is an important soil function that could be reduced by heavy metals and therefore considered as sensitive microbial process with regards to heavy metal stress (Smolders et al., 2001). Studies have particularly emphasized the response of ammonia oxidizing bacteria to heavy metals such as Zn, Cu and Hg (Lee et al., 2011, Liu et al., 2010, Mertens et al., 2009, Vasileiadis et al., 2012). Some of these studies reported a sensitive response of the AOB community to heavy metals (Frey et al., 2008, Lee et al., 2011, Vasileiadis et al., 2012); AOA, on the other hand, appear to be tolerant to the stress of heavy metal such as Hg and Zn (Liu et al., 2010, Vasileiadis et al., 2012). Previously, AOA-amoA gene diversity profile in heavy metal polluted soils has been established by DGGE and T-RFLP based community shifts (Mertens et al., 2009, Ollivier et al., 2012, Vasileiadis et al., 2012). However, these studies could not offer detailed AOA phylogenetic information such as what are the AOA in heavy metal polluted soils. Moreover, little information is available on the responses of ammonia oxidizers to As and Cu in acidic soils, where AOA are considered to be a main contributor to ammonia oxidization (Lee et al., 2011, Zhang et al., 2012).
The objective of the present study was to assess the responses of ammonia oxidizing microbes (AOA and AOB) to the Cu and As stresses particularly in acidic soils after 10 weeks of incubation. The effects of heavy metal on abundance and community diversity were studied based on the q-PCR, DGGE and clone library sequencing analysis. The results may help to improve our understanding of the ecological effects of heavy metals on the abundance and diversity of ammonia oxidizers in acidic alfisols.
Section snippets
Soil samples and experimental setup
Two different types of pristine alfisols (from both India and China) were collected for short-term incubation experiments. Triplicate composite sampling was performed for soil collection. In brief, each top soil sample (10 cm in depth) was composed by pooling three subsamples obtained in a 1 × 1-m area. Alfisol-RA was collected from Rayka, a site located in semiarid region near the Mahi River basin, Gujarat, western India. ‘RA’ is an oligotrophic silty-loam soil, with low organic matter (0.86%),
Effects of As and Cu on gene abundance in RA alfisols
Among all the genes quantified, AOB-amoA and bacterial-16S rRNA genes were highly sensitive to both metals (As and Cu) in all treatments. The bacterial-16S rRNA gene copy numbers were lower in the As-amended soils than those in the Cu-amended soils (Fig. 1A). Copy number of bacterial-16S rRNA gene in CK soil, was 4.3 × 1010 copies g−1 dry soil, whereas the number significantly reduced in As-70 (7%), Cu-500 (5.03%) and As-70 + Cu-500 (6.9%) soils (Table 1). AOA-amoA gene abundance ranged from 1.5 × 108
Discussion
Soil ammonia oxidizing archaea (AOA) have been suggested to be more stable and abundant than ammonia-oxidizing bacteria (AOB) in acidic soils (He et al., 2012, Zhang et al., 2012). However, their relative distribution and potential contribution under contrasting environmental conditions are unclear. In the present study response of ammonia oxidizing microbes to the stresses of As and Cu were investigated in acidic soils. The findings demonstrated that As and Cu adversely affected the abundance
Conclusions
The present study shows a direct toxic effect of As and Cu on microbial ammonia oxidizing activity in acidic alfisols. Soil ammonia oxidizing microbes seems much vulnerable to stress of combined As and Cu. The effect of As and Cu on AOA community structure was not significantly different from the control soil. Sequencing and phylogenetic analysis of amoA gene revealed that the Thaumarchaeal-AOA group 1.1b was the only AOA in oligotrophic acidic soils and provide further importance of this group
Acknowledgements
This work was supported by the National Natural Science Foundation of China (41201523) and the Academy of Sciences for the Developing World (TWAS), Trieste, Italy and Chinese Academy of Sciences (CAS), Beijing, China under the scheme “TWAS-CAS fellowship programme for postgraduate research” to GS for the year 2010.
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