Biodegradation of pyraclostrobin by two microbial communities from Hawaiian soils and metabolic mechanism

Highlights

• Two microfloras capable of catabolizing pyraclostrobin were obtained from Hawaiian soils.

• More than 99% of pyraclostrobin (100 mg L⁻¹) was degraded by the microfloras in 5 days.

• A metabolic pathway involving carbamate hydrolysis was proposed.

• Pseudomonas expressing carboxylesterase might be able to degrade carbamate chemicals.

Abstract

Pyraclostrobin has been widely and long-termly applicated to agricultural fields. The removal of pyraclostrobin from ecological environment has received wide attention. In this study, using sequential enrichments with pyraclostrobin as a sole carbon source, two microbial communities (HI2 and HI6) capable of catabolizing pyraclostrobin were obtained from Hawaiian soils. The microfloras analysis indicated that only Proteobacteria and Bacteroides could survive in HI2-soil after acclimatization, whereas the number of Proteobacteria in HI6-soil accounted for more than 99%. The percentages of Pseudomonas in the HI2 and HI6 microfloras were 69.3% and 59.3%, respectively. More than 99% of pyraclostrobin (C₀ = 100 mg L⁻¹) was degraded by the HI2 and HI6 microorganisms within five days. A unique metabolite was identified by high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS/MS). A metabolic pathway involving carbamate hydrolysis was proposed. The tertiary amine group of pyraclostrobin was hydrolyzed to primary amine group with the decarboxylation, which facilitated pyraclostrobin detoxification because carboxylester was an important functional group. The metabolic mechanism suggested that Pseudomonas expressing carboxylesterase might be able to degrade carbamate chemicals. Therefore, Pseudomonas might be an ideal candidate for expression and cloning of carbamate-degrading gene in genomics studies. The current study would have important implications in detoxification and bioremediation of carbamates through the CN bond cleavage of methyl carbamate.

Introduction

Pyraclostrobin, a class of broad-spectrum strobilurins fungicide, was first announced by Syngenta in 2000, and then was ratified in the European Union in 2003 and enrolled at the U.S. Environmental Protection Agency in 2012 [1]. The strobilurin fungicide acts through inhibition of mitochondrial respiration by blocking electron transfer within the respiratory chain, which in turn causes severe disruption of important biochemical processes, and results in cessation of fungal growth. Pyraclostrobin has been widely used for chemical control of various fungi in many countries owing to the low mammalian toxicity, and the broad scope and high potency [[1], [2], [3]]. Although pyraclostrobin has low acute toxicity to mammals, its transformation products (TPs) may produce environmental risks due to the wide usage. Therefore, removal of pyraclostrobin from ecological system has attracted public attention.

It is known that, the conventional chemical or physical technologies have inherent drawbacks due to high operating cost, difficulty in operation and production of secondary pollutants [4]. Instead, bioremediation techniques have been recognized as powerful alternatives to conventional methods for decontaminating soil or water [5,6]. In ecosystem, bacteria play an important role in detoxification of xenobiotics because of their short life cycle, inexpensive cost, eco-friendly process, less secondary pollution and feasibly growth on various substrates [[7], [8], [9], [10], [11], [12]]. Therefore, the microbial influence on pollution control of organic pollutants for the environmental remediation has been received increasing attention by the scientific community. Recent, studies have been focusing on the bioremediation potential of pre-adapted microbes and improvement of biodegradation efficiency by bioaugmentation and biostimulation [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]].

Although many bacterial strains or microfloras has been isolated for biodegradation of pesticides and their transformation products (TPs), only few studies on biodegradation of strobilurins were reported in the literature. For instance, Lopes et al. isolated and obtained a pyraclostrobin-degrader Klebsiella sp. from soybean-grown soil after long-term use of this pesticide [25]. Howell and colleagues found that Cupriavidus sp. and Rhodanobacter sp. exhibited degrading activity against azoxystrobin [26]. Two Pseudomonas strains (Burkholderia and Pseudomonas aeruginosa) were isolated from pyraclostrobin-contaminated natural environment, which were capable of degrading pyraclostrobin and azoxystrobin etc [27,28]. As is known, the microbial metabolism is a major pathway responsible for pyraclostrobin degradation in natural soils. However, the biodegradation mechanism of pyraclostrobin remains unclear by now. It is essential to investigate the degradation products and metabolic mechanism of pyraclostrobin in molecular and biochemical levels, which would provide rationale and guidance for the construction of pyraclostrobin-degrading genes. On the other hand, because bioremediation in nature relies on cooperative metabolic activities of complex microbial populations, biodegradation by a pure strain does not represent the actual behaviors of environmental microorganisms in natural pyraclostrobin-contaminated soils [4]. Therefore, it is necessary to study microorganisms in the pyraclostrobin-contaminated environment.

The main objectives in this study were (1) to isolate and analyze a better-tolerability microbial community capable of catabolizing pyraclostrobin; (2) to investigate the biodegradability of pyraclostrobin by the microbial communities; (3) to elucidate the metabolic pathway and degradation mechanism on basis of the metabolite analyses. The current study would have important implications in detoxification and bioremediation of carbamate chemicals.