Effects of copper on methane emission, methanogens and methanotrophs in the rhizosphere and bulk soil of rice paddy

doi:10.1016/j.catena.2015.05.024

CATENA

Volume 133, October 2015, Pages 233-240

https://doi.org/10.1016/j.catena.2015.05.024 Get rights and content

Highlights

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Cu^2 + decreased diversity and abundance of methanogens and methanotrophs.
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Methanotrophs suffer more from Cu^2 + addition (200 to 800 mg kg^− 1) than methanogens.
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Rhizosphere environment alleviated Cu^2 + stress on methanogens and methanotrophs.
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Methylosarcina-related methanotrophs are resistant to 800 mg kg^− 1 Cu^2 + application.

Abstract

Copper contamination is common in paddy fields due to wastewater irrigation and application of sludge and Cu-containing fungicides. We aimed to study the effects of copper (Cu^2 +) application on methane emission, methanogens and methanotrophs in both the rhizosphere and bulk soil. The study was conducted in rhizobox in which flooded soil was applied with different Cu^2 + concentrations. Methane emission was collected with static chamber method and determined by gas chromatography. The diversity and composition of methanogens and methanotrophs were studied using PCR–DGGE and sequencing analysis of methanogenic 16S rRNA and pmoA genes. The abundance of methanogens and methanotrophs was determined using quantitative real-time PCR of mcrA and pmoA genes, respectively. The results showed that Cu^2 + application decreased methane emission along with the diversity and abundance of methanogens and methanotrophs, although the application of 200 mg kg^− 1 Cu^2 + did not significantly decrease the diversity of methanogens and methanotrophs in the rhizosphere. In addition, Cu^2 + decreased methanotrophs diversity more profoundly than methanogens diversity. Methanogens in both the rhizosphere and bulk soil were closely related to Methanosaeta, Methanosarcina, Methanobacterium and Methanomicrobia archaeon. Methanotrophs in rhizosphere soil were clustered into four groups (type I methanotrophs, Methylobacter, Methylomonas and Methylosarcina) while those in bulk soil were much less diverse. The addition of 200 to 800 mg kg^− 1 Cu^2 + did not dramatically change the composition of methanogens; however, for methanotrophs, only one DGGE band belonging to Methylosarcina was present after the addition of 800 mg kg^− 1 Cu^2 +. We conclude that methanotrophs were more sensitive to Cu^2 + addition than methanogens, and that the rhizosphere environment alleviated Cu^2 + stress on methanogens and methanotrophs.

Introduction

Methane is the one of the most important greenhouse gases (Thielemann et al., 2000). Its annual increase rate in the atmosphere was estimated to be the highest among all greenhouse gases (Khalil, 1999). Anthropogenic activities including rice cultivation, domestic animal grazing, landfillings, coal mining, and oil and gas extraction, contribute more than 60% of global methane emission (IPCC, 2014). Of these anthropogenic activities, rice cultivation is the most significant source. Paddy fields are responsible for approximately 15–20% of global total anthropogenic methane emission (Li et al., 2011).

Copper contamination is common in rice field soil, mainly due to wastewater irrigation, sludge treatment and application of Cu-containing fungicides (Komárek et al., 2010). One study has shown the effect of Cu^2 + and other heavy metals on soil methane emission (Jiao et al., 2005). However, knowledge about the effect of Cu^2 + on methanogens and methanotrophs in paddy soil is still limited. Methane emission from paddy fields is the net result of processes including methane production, oxidation and transportation (Cai et al., 2007). More than 80% of total emissions are transported through the aerenchyma system of rice plants (Cheng et al., 2006). A detailed investigation into the influence of Cu^2 + on methanogens and methanotrophs along with on plant growth might clarify the effects of Cu^2 + on methane emission. Aquatic plants such as rice plants, survive anoxic conditions by supplying their root system with oxygen from the atmosphere (Chen et al., 2008). The root exudates with released oxygen result in different microbial communities and methane emission flux between rhizosphere and bulk soil (Aulakh et al., 2001). We therefore hypothesized that the effects of Cu^2 + on methane emission, methanogens and methanotrophs might vary between the rhizosphere and bulk soil.

Methanogens and methanotrophs can be characterized by the 16S rRNA gene or functional genes including mcrA and pmoA. The mcrA gene encodes for the K-subunit of methyl coenzyme M reductase (MCR), the key catabolic enzyme of methanogens (Ramakrishnan et al., 2001). The pmoA gene encodes an active subunit of the particulate methane monoxygenase (pMMO). All known methanotrophs possess a pmoA gene except Methylocella and Methyloferula (Dedysh et al., 2000, Vorobev et al., 2011). The mcrA and pmoA genes have been widely used as phylogenetic markers for methanogens and methanotrophs, respectively. The phylogeny of methanogens based on mcrA gene is consistent with that based on 16S rRNA gene (Luton et al., 2002). As a result, 16S rRNA is an alternative gene to mcrA for the study of methanogens (Watanabe et al., 2009). Phylogenetic studies of methanogens based on methanogenic 16S rRNA or mcrA gene and methanotrophs based on pmoA gene have revealed community composition of flooded rice soil (Watanabe et al., 2006, Zheng et al., 2008). In the present study, soil methanogens were studied using the 16S rRNA and mcrA genes and methanotrophs were studied using the pmoA gene. To test our hypothesis, we focused on the effect of Cu^2 + application on methane emission along with the diversity and abundance of methanogens and methanotrophs in the rhizosphere and bulk soil.

Section snippets

Soil sampling and physiochemical properties

One soil sample was collected from plowed layer at a depth of 15 cm in a farmland at Wujiang County, Jiangsu Province of China (30°38′N, 120°41′E). Soil sampling was carried out in June 2007, after rice was harvested. The climate is subtropical with an average annual precipitation of 1000 mm and an average temperature of 16 °C. After sampling, the fresh soil was immediately sieved through a 2-mm mesh. Soil aliquots were subjected to measurements of physiochemical properties using routine methods (

Methane emission

Methane emission from both the rhizosphere and bulk soil increased during the 49-day experiment, especially in the tillering and jointing stage which started approximately 26 days after rice planting (Fig. 1). The magnitude of increase was much higher in the rhizosphere than in the bulk soil. For Ct, methane emission from rhizosphere increased from 0.10 to 45.93 mg m² h^− 1, from day 0 to day 49, while that from the bulk soil increased from 0.12 to 2.46 mg m² h^− 1. Copper exhibited a significant

Discussion

The present study clearly showed methane emission, diversity and abundance of methanogens and methanotrophs in the rhizosphere and bulk soil, along with their responses to Cu^2 + additions. Most DGGE bands of methanogens were closely related to three genera including Methanosaeta, Methanosarcina and Methanobacterium. Methanosaeta exclusively use acetate as a substrate; Methanobacterium use H₂/CO₂ and formate, and Methanosarcina generate methane from acetate, H₂/CO₂, and methyl compounds (Conrad,

Conclusions

This study clearly demonstrated that Cu^2 + application decreased methane emission, along with the biodiversity and abundance of methanogens and methanotrophs. The decreased methane emission can be attributed to the toxic effect of Cu^2 + addition on methanogens and rice growth. Nevertheless, the rhizosphere environment alleviated Cu^2 + stress on methanogens and methanotrophs. In addition, methanotrophs were much more sensitive to Cu^2 + addition than methanogens. Only the genus Methylosarcina was

Acknowledgments

This study is financially supported by the Natural Science Foundation of China (40671101).

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¹: The two authors contributed the same.

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Effects of copper on methane emission, methanogens and methanotrophs in the rhizosphere and bulk soil of rice paddy

Highlights

Abstract

Introduction

Section snippets

Soil sampling and physiochemical properties

Methane emission

Discussion

Conclusions

Acknowledgments

Adv. Agron.

J. Microbiol. Methods

Soil Biol. Biochem.

FEMS Microbiol. Lett.

Chemosphere

Soil Biol. Biochem.

Environ. Int.

Soil Tillage Res.

Soil Biol. Biochem.

FEMS Microbiol. Lett.

Soil Biol. Biochem.

FEMS Microbiol. Ecol.

Anal. Biochem.

Org. Geochem.

Soil Biol. Biochem.

Soil Biol. Biochem.

Soil Biol. Biochem.

FEMS Microbiol. Ecol.

Syst. Appl. Microbiol.

Appl. Soil Ecol.

Impact of root exudates of different cultivars and plant development stages of rice (Oryza sativa L.) on methane production in a paddy soil

Plant Soil

Methane flux and high-affinity methanotrophic diversity along the chronosequence of a receding glacier in Greenland

Ann. Glaciol.

Effects of nitrogen fertilization on CH4 emissions from rice fields

Soil Sci. Plant Nutr.

Ammonia-oxidizing archaea: important players in paddy rhizosphere soil?

Environ. Microbiol.

Effects of nitrogen fertilization on CH₄ emissions from rice fields