Use of spent substrate after Pleurotus pulmonarius cultivation for the treatment of chlorothalonil containing wastewater

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Abstract

Lignocellulosic materials are used as substrate for the cultivation of the edible mushroom Pleurotus pulmonarius. After two or three flushes of mushrooms, the spent substrate is discarded although it still has an important enzymatic activity that can be used for several purposes. In this study, we sought to determine the technical feasibility of using spent substrate from P. pulmonarius to degrade chlorothalonil. Reaction mixture was prepared with 6 ml of pesticide aqueous solution (2 mg active ingredient/l) and 3 ml of enzymatic extract obtained from spent P. pulmonarius substrate. The enzymatic reaction (27 °C, pH 7.4) was conducted for 1 h with sampling at 15 min intervals. The effect of storage time and temperature (freezing or refrigerating) of spent substrate and enzymatic extract, respectively, on the activity over chlorothalonil was determined. Freshly obtained spent substrate extract was able to reduce 100% of the initial concentration of chlorothalonil (2 mg/l) after 45 min of reaction. Storage time had a negative effect on the stability of the enzymatic activity: with spent substrate stored for a week, chlorothalonil concentration was reduced in 49.5% after 1 h reaction and with substrate stored for two and three weeks, the degradation efficiency decreased to 9.15% and 0%, respectively. Cooling and freezing the spent substrate extract also had a negative effect on chlorothalonil degradation.

Introduction

Oyster mushrooms are a group of several edible Pleurotus species cultivated worldwide. In the production of oyster mushrooms, every tonne of mushroom produced results in one or two tonnes of dry spent residual material. This spent mushroom substrate (SMS) may be used in land filling, crop production or for many other purposes (Rinker, 2002). However, in this time of limited natural resources and great concerns over human health and the environment, additional and alternative uses are under investigation, among them, those involving the use of the fungal enzymatic capability for bioremediation (Martirani et al., 1996, Cerniglia and Sutherland, 2001, Ward et al., 2004, Mougin et al., 2009).

Oyster mushrooms are classified as white rot fungi because they are able to degrade lignin, cellulose and hemicellulose. They produce several kinds of phenol oxidases and some enzymes of this group are considered as responsible for the degradation of lignin. The use of white rot fungi for bioremediation purposes is of great interest because of the ligninolytic system they have which makes them able to degrade a wide group of environmental pollutants (Canales et al., 2007).

In Mexico, 3.5 × 106 t pesticides are used annually (INSP, 2005). Among those pesticides, chlorothalonil (CTN, tetrachloroisophthalonitrile, a broad spectrum, non-systemic, organochlorine fungicide) has an elevated use, mainly in the production of tomatoes, almonds, onions, and tropical crops like banana and papaya (Hernández-Hernández et al., 2007, Kegley et al., 2009). CTN is used as a foliage and soil protectant fungicide (Carlo-Rojas et al., 2004). Cox (1997) mentioned that CTN is the second most widely used agricultural fungicide in the US with applications totaling 6000 t annually. Pesticides spills are common at the sites, within the farms, where activities of mixing/loading and machinery washing (post application) are conducted. This can lead to severe contamination of soil and surface waters because of improper handling of wastes such as equipment rinsates. It has been estimated that these effluents contain about 10% of the recommended dose of pesticide. In the case of chlorothalonil this would be 16 mg/l (Salgado-Cárdenas and Bello-Mendoza, submitted for publication). CTN is considered a genotoxic substance with low level of bioaccumulation and a questioned high degradability in water when compared to other organic pollutants. CTN is classified as moderately toxic in mammals, however, it is considered as toxic to humans, including carcinogenicity, reproductive and developmental toxicity, neurotoxicity, and acute toxicity (Kegley et al., 2009, PANNA, 2007). It is highly toxic for fish and aquatic invertebrates (EPA, 1999). Because of this, we investigated the use of SMS on the degradation of CTN in agricultural effluents with the aim of determining a new option for using this biomass by-product from the mushroom industry.

Section snippets

Strain and culture media

The strain Pleurotus pulmonarius ECS-0190 from the mycological collection of El Colegio de la Frontera Sur (ECOSUR) was used. For maintenance and culture of the strain, the media potato dextrose (Bioxon) and yeast (0.5%, BBL) were used. The spawn was prepared with sorghum grains sterilized for 30 min at 121 °C (Quimio, 2002) and Pangola grass (Digitaria decumbens) was used as substrate for production of mushrooms.

Cultivation of mushrooms and spent mushrooms substrate

Substrate preparation and cultivation of oyster mushrooms were made by following

Results

The variation with time of chlorothalonil content in the reaction mixture is shown in Fig. 1. After 45 min reaction, the chlorothalonil content was reduced to undetectable levels suggesting 100% degradation. When extract from P. pulmonarius carpophores was used, the residual content of chlorothalonil was 10.1% of initial value after 60 min contact. In the control, the residual chlorothalonil was 98.8% after 60 min.

The efficiency of chlorothalonil removal obtained with SMS at different weeks of

Discussion

The ligninolytic ability of the genus Pleurotus has been demonstrated by several authors and it has been considered capable of degrading many phenolic related pollutant compounds (Martirani et al., 1996), among them are some chlorinated pesticides like endosulfan (Goswami et al., 2009, Sánchez et al., 2007).

The fact that syringaldazyne, ABTS, phenol red and catechol were oxidized by the enzymatic extract indicates the presence of laccase, MnP and phenol oxidase activities in the SMS and

Conclusion

Freshly obtained spent substrate extract from P. pulmonarius cultivation was able to reduce 100% of the initial concentration of chlorothalonil (2 mg/l) after 45 min of reaction at 27 °C and pH 7.4. This opens the possibility of using such an abundant by-product from the mushroom industry for cleaning effluents polluted by organochlorine pesticides. However, more research must be done in order to optimize the use of SMS to degrade chlorothalonil, since our results indicate a high sensibility of

Acknowledgements

The authors wish to thank Lilia Moreno and Gerardo Hernández for their technical support and Javier Valle for his help in the statistical analysis of the data.

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