Bioremediation of Fungicides by Spent Mushroom Substrate and Its Associated Microflora (2024)

In Vitro Studies

Spent substrate of button mushroom was used for the isolation of dominant fungi and bacteria with their ability to biodegrade the two fungicides (carbendazim and mancozeb). The dominating fungi and bacteria were isolated by the method of serial dilution and plating on malt extract agar (MEA) and nutrient agar (NA) media, and incubation at 30±2°C. The dominating micro flora was selected based upon their colony forming units on respective media. The purified fungi were evaluated for their fungicide degradation potential by growing in ME broth with 100μgml⁻¹ concentration of the respective fungicide at 30±2°C for 6days. The degradation of fungicides was measured by determining the changes in concentration of two fungicides by the method mentioned under the heading residue assay.

In second stage experiment, fungi (3) and bacterial isolates (2) exhibiting higher fungicide degradation potential under broth culture conditions were inoculated in different permutations and combinations in sterilized button mushroom spent substrate premixed with respective fungicide at100μgg⁻¹ of SMS. The inoculated SMS was incubated at 30±2°C for next 15days and the fungicide degradation was recorded at an interval of 3days each by measuring the changes in fungicides residue by the method mentioned under the heading residue assay.

In Vivo Studies

The study was carried out by mixing different proportions of the recomposted button mushroom spent substrate in soil pre-mixed with 100ppm concentration of the respective fungicide. The crops of tomato and pea were also raised on fungicide mixed and spent mushroom substrate amended soil to study the biodegradation of fungicide under natural field conditions.

Residue Assay

1. Reagents Reagents of sodium sulphate (anhydrous), sodium bicarbonate, ammonia, ethyl acetate, hydrochloric acid, potassium hydroxide, carbon disulfide, lead (II) acetate (trihydrate), stannous chloride, sulphuric acid, methanol, carbendazim (50% WP, BASF) and mancozeb (75%, Indofil) were prepared with analytical grade chemicals.

2. Preparation of standards and working solutions

   1. Potassium hydroxide (0.5M) 7.0g of potassium hydroxide (KOH) was dissolved in methanol in 250ml volumetric flask and allowed to cool down at room temperature and made up to the mark with methanol. Potassium hydroxide (secondary standard) was standardized by titrating it with 0.5M oxalic acid (primary standard) to make it exactly 0.5M.

   2. Carbon disulfide (10μgml⁻¹) 0.5mg of carbon disulfide was taken in 50ml volumetric flask and made up to the mark with methanol.

   3. Lead acetate (30%) 30gm lead acetate (trihydrate) was taken in 100ml volumetric flask and final volume was made with distilled water.

   4. Stannous chloride (40% in HCl) 40g of stannous chloride was taken in 100ml volumetric flask and final volume was made with concentrated hydrochloric acid.

3. Mancozeb residue assay Mancozeb residue was assayed as per the method of Dubey and Stan [12]. The apparatus was set up as per the protocol described in the manual of pesticide residue analysis [13] with modified adsorption tubes. The first absorption tube was poured with 25ml of 30% lead acetate, while the second absorption tube with 15ml of concentrated sulphuric acid. The third tube was kept in an ice-water bath (0–4°C) and 15ml of 0.5M methanolic KOH was added in it. A known weight (50g) of sample was poured into the flask and 150ml water was added followed by heating for 15min. To this 15ml concentrated HCl+20ml stannous chloride was added and the sample was digested for 30min. The decomposition was carried out following DFGSIS method of CS₂ determination. The third tube containing methanolic KOH along with xanthate was allowed to stand for 20min at room temp after removal from ice-water bath. Residue assay was carried out by recording the absorbance at 302nm using UV–Visible spectrophotometer (Chemito 1500).

4. Apparatus Modified dithiocarbamate decomposition and distillation apparatus was used for hot acid decomposition of the sample.

5. Carbendazim residue assay Carbendazim residue was estimated as per the method of Nath et al. [14]. A 50g sample with equal amount of anhydrous sodium sulphate and 2.5ml of concentrated ammonia were blended at a high speed for 3min with 100ml of ethyl acetate. Sodium sulphate was used as an anti-emulsifying agent during blending. The upper organic layer was decanted and filtered over anhydrous sodium sulphate. The left over residue was again extracted with 100ml ethyl acetate and filtered in a Buchner funnel under vacuum. The filtrates were mixed together and final volume was reduced to about 80ml under vacuum at 40–45°C. 20ml filtrate was taken in a separating funnel and was partitioned twice with 10ml of 0.1N HCl.

The lower aqueous phase was pooled in another separatory funnel followed by addition of 40ml of ethyl acetate. To this about 30ml of saturated sodium bicarbonate solution (80%) was added with vigorous shaking for 2min. The lower aqueous phase was rejected. Organic phase was washed with water and then partitioned with 10ml of 0.1N freshly saturated HCl. Absorbance of 0.1N HCL was recorded at 280nm using UV–Visible spectrophotometer (Chemito 1500).

In Vitro Study

The data depicted in Figs.1 and ​and22 reveal the fungicides biodegradation potential of 6 SMS dominating fungi and bacteria under in vitro conditions. Out of six dominating microbes, highest degradation of carbendazim was recorded with B–I bacterial isolate during 6days (100–82.55μgml⁻¹) of incubation at 30±2°C, whereas highest degradation of mancozeb (100–81.95μgml⁻¹) was recorded with Trichoderma sp. The degradation of two fungicides in control was gradual and far less as compared to dominating fungi and bacterial isolates. In control, the degradation of mancozeb was almost negligible (100–93.75μgml⁻¹). Very scanty information is available on fungicide biodegradation potential of SMS and microbes inhabiting in it. However, role of white rot fungi particularly, Phanerochaete chrysosporium and a putative peroxidase mutant has been studied for co-mineralization of a mixture of pesticides including 2,4-D and 2,4,5-T under nutrient rich broth conditions, wherein the mycelial fractions were recorded to contain only 5% of the pesticides [3, 4].

The fungicide degradation potential of SMS dominating fungi and bacterial isolates was also studied for 15days at 30±2°C by inoculating them in different permutations and combinations in sterilized fungicide mixed button mushroom SMS (at100μgg⁻¹ of SMS). Carbendazim was degraded from lowest of 11.90% (100–88.10μgg⁻¹) in control to highest of 33.50% (100–66.50μgg⁻¹) in mixed inoculum of Trichoderma sp. and Aspergillus sp. It was followed by 33.00% degradation in mixed inoculum of Trichoderma sp., Aspergillus sp. and B-IV bacterial isolate (Table1). The degradation of mancozeb was higher than carbendazim and after 15days of incubation, it was highest of 49.50% (100–50.50μgg⁻¹) in mixed inoculum of Trichoderma sp., Aspergillus sp. and B–I bacterial isolate, followed by 41.60% in mixed inoculum of Trichoderma sp. and Aspergillus sp. (Table2). In control, the degradation of mancozeb was lower than carbendazim and it was only 10.00% in comparison to 11.90% for carbendazim. In earlier studies, the SMS has been recorded to stimulate the microbial population (10⁸–10⁹) in soil, which it maintains for 28days. The stimulated microbial population of Aspergillus sp., Penicillium sp., Trichoderma sp., Aeromonas sp. and Bacillus sp., has been reported to degrade alachlor within 7days of incubation under in vitro conditions [15, 16].

In Situ Study

The fungicide degradation potential of button mushroom spent substrate was also evaluated under in situ conditions by mixing different proportions of SMS in fungicide mixed soil and the residue level of two fungicides was assayed at a regular interval of 1month for next 6months. Among different proportions of SMS, the 20% (w/w) and 30% (w/w) SMS gave results at par with each other and very low level of carbendazim residue was detected after 6months of SMS mixing. At different intervals of residue assay, the level of carbendazim degradation in 30% SMS was 2–3 folds higher than the control (Fig.3). In soil carbendazim gets degraded at varied rates depending upon the nutrient status of the soil and the type of vegetation growing on it. On bare soil its DT₅₀ is 6–12months [17]. In line with the in vitro studies, the degradation of mancozeb under in situ conditions was also higher than carbendazim. After second and third month of SMS mixing, the degradation of mancozeb in 20 and 30% SMS treatments was 3–5 folds higher than the control (Fig.4). The mixing of SMS at 20 and 30% (w/w) has been found to stimulate the degradation of two fungicides and 6months time was recorded to be sufficient for complete degradation of the fungicides. As the previous studies have clearly revealed the growth and yield stimulatory role of SMS in different vegetables [7–10], the present findings further support the all-round role of SMS in raising organic field crops. In several earlier studies, the spent substrate from different mushrooms have been used to decontaminate the soils contaminated with PCP [4], 3-ring and 4-ring compounds [18], hazardous industrial wastes [19], and chlorophenols (PCP), polycyclic aromatic hydrocarbons (PAHS) and aromatic monomers [20].

Earlier in a similar study, Busswel [19] studied the bioremediation stimulating catabolic activities of indigenous microflora by optimizing their in situ growth conditions. However, Tekere et al. [21] reported very high rate of (about 82%) degradation of the pesticide, lindane by P. chrysosporium with very low initial concentrations of the pesticide (5 and 10mgl⁻¹). Faster degradation of fungicides in microflora inoculated treatments is attributed to the involvement of lignin degrading enzymes system. Some other findings have also revealed the role of laccase, Mn-dependant peroxidase and high carbon content in SMS in breakdown of organopollutants [22–24]. The SMS from Pleurotus pulmonarius has also been tried earlier to treat a PCP contaminated water system and after 2days of incubation, 89% removal of PCP has been reported with 5% SMS [24]. This has been attributed to biodegradation through lignolytic enzymes (70%) and bio-sorption by the SMS (19%).

The present study provides sufficient evidences that SMS has great potential in bioremediation of contaminated sites. Results from field study are of great importance, as the contamination of soil from agricultural chemicals is continuously increasing and plenty of untimely deaths had been reported time to time from the affected areas of the state of Punjab. The multi-facet utilities of SMS on using as manure in field crops will not only curtail the additional bourdon of chemical fertilizers on poor farmers and will also improve the affected soil for field crop raising.

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