Biodegradation of profenofos, an acetylcholine esterase inhibitor by a psychrotolerant strain Rahnella sp. PFF2 and degradation pathway analysis

Highlights

• A psychrotolerant Rahnella sp. was isolated from the apple orchard of HP, India.

• The strain was efficiently degraded 50µg/ml profenofos at 20 °C, at 28 °C and at 15 °C.

• 4-Bromo-2-chlorophenol was detected as one of the major pathway metabolites.

• A probable profenofos biodegradation pathway has been proposed.

• Enzyme organophosphorus hydrolase might be responsible for initial degradation.

Abstract

Profenofos (PFF) is an organophosphate insecticide and used worldwide to control harmful pests and insect populations. Removal of PFF from the environment is important because of its high mammalian acute toxicity due to acetylcholinesterase enzyme inhibition. Biodegradation in this context is beneficial as it is a cost-effective and sustainable process. In the present study, microbial degradation of PFF was investigated by using a psychrotolerant bacterium Rahnella sp. PFF2. The bacterial strain was isolated from the soil samples of apple orchards situated in Kufri, Himachal Pradesh, India. Quantitative analysis through High-performance liquid chromatography revealed that the bacterium PFF2 was able to degrade 100% PFF (50 μg/ml) within 14 days at 20 °C, within 16 days at 28 °C, and within 20 days at 15 °C. GC/MS and HPLC studies showed the presence of pathway metabolites 4-Bromo-2-chlorophenol, phosphoric acid, and 3, 4 – dimethyl benzoic acid. Based on these data a probable PFF degradation pathway has been proposed. An inducible and intracellular organophosphorus hydrolase enzyme might responsible for the initial degradation process. To the best of our knowledge, the current finding is the first report of PFF degradation at both the psychrophilic and mesophilic temperature conditions by any psychrotolerant Rahnella sp. isolated from Western Himalayan regions.

Introduction

With the increasing population, the global grain output has increased significantly, and simultaneously the risk factor for storage and protection of crops from pests and insects has also increased. India produces on an average 250 million tons of total grains per year but losses 11–15% of the total output due to harmful pest and insect population (Walter et al., 2016). To avoid such losses, a large number of pesticides are used worldwide and nowadays they have become indispensable components of modern agriculture. Profenofos (O-(4-Bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate; PFF) (Fig. S1), an organophosphate (OP) insecticide is vastly used for this purpose on field crops, vegetables, and fruit crops. According to WHO, PFF is reported as moderately hazardous and toxicity class II pesticide (WHO, 2010). Every year, nearly 775,000 pounds (lbs.) active ingredient (ai) of profenofos pesticide is applied to cotton, and about 85% of it is used for the control of lepidopteran species. PFF is broadly used in many countries situated in north, south and central America, the Indian subcontinent, Africa, southeast and east Asia (Dogheim et al., 2002; Mansour, 2004; Radwan et al., 2005; Loutfy et al., 2008; PAN UK, 2008; Li et al., 2010; Swarnam and Velmurugan, 2013; Van Toan et al., 2013) for controlling pests in various crops, paddy, and vegetable cultivation (Talebi and Ghassami, 2004; He et al., 2010; Ooraikul, 2011; Eijaza et al., 2015; Kelageri et al., 2015; Kushwaha et al., 2016). In India, this “restricted use” pesticide is available in 50% EC (Emulsifiable Concentrate) formulation. Himachal Pradesh, a northern state of India is famous for the production of Apple crops, and to protect the harm caused by pests and insects, PFF is used extensively in this region (Sharma and Nath, 2003). Extensive use of this pesticide in both open field and poly house conditions results in its (or its residues) presence in the samples of market crops and vegetables (Kelageri et al., 2015) as well as in different parts of the environment viz. soil, surface water, and ambient air (Anwar et al., 2012; Harnpicharnchai et al., 2013).

Due to their ample application, PFF is responsible for multifaceted environmental and human health problems (Kushwaha et al., 2016), and even at low concentrations, PFF is highly persistent and toxic (Zhao et al., 2008). In comparison to other OP insecticides, the PFF parent form is reported as a significant inhibitor of acetylcholine esterase (AChE) (Das et al., 2006; Nillos et al., 2007). Blood cholinesterase inhibition in rats has been reported due to exposure to PFF (McDaniel and Moser, 2004). The widespread application of this pesticide is the main factor for the loss of honey bee colonies (Abaga et al., 2011) and it also affects the soil microflora, especially bacterial population and di-nitrogen fixers (Martínez Toledo et al., 1992). Apart from its developmental and reproductive toxicity, PFF was found to be genotoxic (i.e., DNA-damaging) to non-target aquatic and terrestrial organisms such as mice, fish, insects, and earthworms (Kushwaha et al., 2016 and references therein). Profenofos is mutagenic (Fahmy and Abdalla, 1998) and also reported as a potent endocrine-disrupting chemical (Moustafa et al., 2007).

To search for its removal from the environment, different conventional methods (e.g. chemical treatment, incineration, and landfills) were adopted but found less promising due to the secondary risk of exposure and economically restricted use. In this context, microbial degradation technology was found more effective because of its cost-effective and sustainable application. In the environment, different classes of microorganisms can inhabit one niche. Among them, coexisting bacterial communities are well reported for their quick adaptation through genetic changes to biodegrade ranges of xenobiotics compounds including pesticides, polyaromatic hydrocarbons, and other POPs (Singh 2009). The symbiotic microbial population was also studied for their unique strategy of nutrient recycling in the bioremediation process in the municipal wastewater treatment plants where C/N (organic carbon to nitrogen loading rates) ratio was found as the controlling factor for the microbial population (Sepehri et al. 2018, 2020). Among the bacterial classes reported to date, Proteobacteria (gamma-proteobacteria, beta-proteobacteria, and alpha-proteobacteria), Actinobacteria, and Flavobacteria are the dominating phyla reported for pesticide biodegradation (Doolotkeldieva et al., 2018).

To date, very limited information has been published related to PFF biodegradation and detailed pathway analysis. Apart from that, no psychrotolerant bacterium has been reported yet which can efficiently degrade PFF. Few bacterial genera including Pseudomonas, Burkholderia (Malghani et al., 2009a; Siripattanakul-Ratpukdi et al., 2014), Bacillus (Salunkhe et al., 2013), Stenotrophomonas (Deng et al., 2015), Pseudoxanthomonas (Talwar and Ninnekar, 2015), Achromobacter Citrobacter (Jabeen et al., 2015), was reported for PFF degradation whereas, only two fungal sp., viz., Aspergillus sydowii CBMAI 935 and Pencillium raistrickii CBMAI 931 were found efficient to degrade PFF pesticide (Da Silva et al., 2013). It is important to mention here that due to its toxicity and structural constrain, degradation of PFF by soil microbes is not a straightforward process and requires multiple enzymatic systems for its complete disintegration. The objective of the present study was to investigate the biodegradation of pesticide PFF by the indigenous bacterium and the determination of the degradation pathway. Pathway analysis will help us to get an idea about the enzyme(s) responsible for the degradation of PFF which may use in the future for other biotechnology applications. Isolation and identification of psychrotolerant bacterium in this context not only showed the presence of this genus in this region of Himalayas but also showed its potentiality for pollution abatement. To the best of our knowledge, this is the first report of PFF degradation by any psychrotolerant Rahnella sp. isolated from the western Himalayas.