Responses of soil ammonia-oxidizing microorganisms to repeated exposure of single-walled and multi-walled carbon nanotubes

Highlights

• The impact of repeated exposure of carbon nanotubes on the microbial communities was investigated.

• The first exposure caused stronger effects on microbial biomass compared with the second exposure.

• SWNTs and MWNTs produced different effects on the net N nitrification.

• The abundance and diversity of AOA were higher than that of AOB under the exposure of carbon nanotubes.

Abstract

The impacts of carbon nanotubes (CNTs) including single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) on soil microbial biomass and microbial community composition (especially on ammonium oxidizing microorganisms) have been evaluated. The first exposure of CNTs lowered the microbial biomass immediately, but the values recovered to the level of the control at the end of the experiment despite the repeated addition of CNTs. The abundance and diversity of ammonium-oxidizing archaea (AOA) were higher than that of ammonium-oxidizing bacteria (AOB) under the exposure of CNTs. The addition of CNTs decreased Shannon–Wiener diversity index of AOB and AOA. Two-way ANOVA analysis showed that CNTs had significant effects on the abundance and diversity of AOB and AOA. Dominant terminal restriction fragments (TRFs) of AOB exhibited a positive relationship with NH₄⁺, while AOA was on the contrary. It implied that AOB prefer for high-NH₄⁺ soils whereas AOA is favored in low NH₄⁺ soils in the CNT-contaminated soil.

Introduction

Nanotechnology has provided a basis for innovation in a wide range of fields and resulted in an exponential increase in products of novel materials (Gao et al., 2013, Maurer-Jones et al., 2013). Carbon nanotubes (CNTs) are one of the most promising nanomaterials and have become the subject of numerous investigations in chemical, physical and material science research areas since its discovery in 1991 (Iijima, 1991, Odom et al., 1998). Based on their outstanding physicochemical and mechanical properties, CNTs could be potentially applied in consumer electronics, building materials, agricultural smart delivery and so on (Rodrigues and Elimelech, 2010, Tong et al., 2012, Li et al., 2013a). CNTs can be classified as single-walled carbon nanotubes (SWNTs), which are hollow tubes of carbon capped at either end with a hemi-fullerene (Iijima and Ichihashi, 1993), and multi-walled carbon nanotubes (MWNTs) consisting of concentric layers of graphene sheets, where smaller diameter tubes are encased in larger diameter tubes (Ebbesen et al., 1993).

With their expanding role of CNTs in manufacturing, some environmental exposure is raising concerns about their potential environmental risk (Helland et al., 2007). The knowledge on the impacts of CNTs on the environment microbes is currently limited and most researches have been limited to pure culture studies (Ahmed et al., 2012, Rodrigues and Elimelech, 2010), which were carried out in less complex and more controlled environment than in the soil environment (Jin et al., 2013). Toxicity studies taking Escherichia coli (E. coli) as a model microorganism showed that over 80% of the cells attached to non-functionalized SWNTs aggregates died after an incubation of 60 min (Kang et al., 2008). Gene analysis of the E. coli which was exposed to CNTs showed an efficient expression of stress-related genes, corroborating the cytotoxic effects of CNTs on E. coli (Rodrigues and Elimelech, 2010). There are a handful of studies focused on the toxic effects of CNTs on microbial communities (Rodrigues et al., 2013). However, the majority of the studies addressed the bacteria in activated sludge (Goyal et al., 2010, Luongo and Zhang, 2010), wastewater (Kang et al., 2009), animals (Lam et al., 2004, Roberts et al., 2007, Zhang et al., 2008) and plants (Caňas-Carrell et al., 2008, Stampoulis et al., 2009). Although some recent studies have addressed the fate of CNTs in soil, few studies have investigated their toxicity on microbial communities in soil environment (Li et al., 2013b, Li et al., 2013c). Jin et al. found that SWNTs can reduce the soil enzyme activity and alter the microbial community structures (Jin et al., 2013, Jin et al., 2014). Shrestha et al. observed that there was no effect of MWNTs on soil respiration, enzymatic activities, and microbial community at 10, 100 and 1000 mg kg^− 1 soil (Shrestha et al., 2013), while a study indicated that most enzyme activity was repressed under MWNTs of 1000 mg kg^− 1 soil (Chung et al., 2011). These different results are divergent. Furthermore, the effects of repeated exposure of CNTs on the soil microorganisms are still not clear.

Nitrification is a key process in the soil nitrogen (N) cycle (Tan et al., 2013). Ammonium-oxidizing microorganisms (AOM) including AOB (Rousidou et al., 2013) and AOA (Konneke et al., 2005) have been identified as the key group that controls the rate-limiting step of nitrification i.e. the oxidation of ammonium to hydroxylamine (Prosser and Nicol, 2008). A previous study reported that the abundance of AOB and AOA could serve as a relevant and cost-effective bio-indicator for soil monitoring (Wessén and Hallin, 2011). Meanwhile, Pereira-Silva et al. suggested that ammonium-oxidation represents a well measurable disturbance-sensitive microbial process and proposed that analysis of the abundance and diversity of AOM could provide a good estimation of soil health (Pereira-Silva et al., 2013). Studies on the pollutants that affect AOM have been mostly focused on fertilization (Zhou et al., 2014), pesticides (Li et al., 2008, Wan et al., 2014), and nitrification inhibitor (Kleineidam et al., 2011). To our knowledge, the effects of nanomaterials on AOM, especially CNTs, have still not been explored.

In this study, the impacts of repeated exposure of SWNTs or MWNTs on soil microbial activity were investigated. The potential effects on AOB and AOA were examined using quantitative real-time PCR (qPCR) analysis and terminal restriction fragments length polymorphism (T-RFLP) during a short term laboratory incubation experiment. The results will provide a wide range of data to characterize the communities of AOM and stimulate a subsequent research aiming at the risk assessment of CNTs that were repeatedly accumulated in soil environment.