Fungal bioaugmentation of two rice husk-based biomixtures for the removal of carbofuran in on-farm biopurification systems
The ligninolytic fungus Trametes versicolor was employed in the bioaugmentation of compost- (GCS) and peat-based (GTS) biomixtures for the removal of the insecticide-nematicide carbofuran (CFN). Among several lignocellulosic substrates, fungal colonization was best supported in rice husk, and this pre-colonized substrate was used to prepare the biomixtures. Estimated half-lives for CFN were 3.4 and 8.1 days in the GTS and GCS biomixtures, respectively. The CFN transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at the moment of CFN application, but their concentration continuously decreased to complete removal in both biomixtures. Mineralization of 14C-radiolabeled CFN was faster in GTS (k = 0.00248 day−1) than in GCS (k = 0.00188 day−1). Complete elimination of the toxicity in the matrices was demonstrated after 48 days. Overall data suggest that the bioaugmentation improved the performance of the GTS rather than the GCS biomixture.
KeywordsBiopurification system Bioaugmentation Degradation Pesticides Fungi Toxicity
On-farm biopurification systems represent a biotechnological approach for the mitigation of point source contamination by pesticides, for example with wastewater derived from agricultural activities (Castillo et al. 2008). The main component of the biopurification systems is the biomixture, which acts as the biologically active core that accelerates the degradation of pesticides. The traditional biomixture developed in Sweden (Torstensson and Castillo 1997) consisted of straw, peat, and soil at a volumetric ratio of 2:1:1.
Components of the biomixture include soil, a humic material, and a lignocellulosic substrate. Soil should be preferably pre-exposed to the target pesticide as it contains important degrading microbiota. Peat is the most used humic component, employed to enhance the retention of the pesticides in the matrix; it can be substituted by compost, usually resulting in good removal performances (Coppola et al. 2007; Kravvariti et al. 2010). The lignocellulosic substrate promotes the colonization by ligninolytic fungi, which are involved in the degradation of diverse organic pollutants, thanks to their enzymatic systems including lignin-modifying enzymes (laccases and peroxidases) and intracellular complexes (cytochrome P450) (Doddapaneni and Yadav 2004; Pointing 2001). The organic contaminants degraded by ligninolytic fungi comprise polycyclic aromatic hydrocarbons, textile dyes, pharmaceuticals, brominated flame retardants and pesticides (Asgher et al. 2008; Cruz-Morató et al. 2013; Rodríguez-Rodríguez et al. 2012a), and their ability to remove pesticides in biomixture-like systems and other solid-matrices has been reviewed (Rodríguez-Rodríguez et al. 2013).
In this work, carbofuran (CFN) was employed as the model pesticide. It is a carbamate commonly used as a broad spectrum systemic nematicide, insecticide, and acaricide. It is highly toxic for mammals and aquatic life (Gupta 1994), and for this reason, it has been banned in agricultural activities in USA, EU, and in other countries; nonetheless, developing countries still use this pesticide and unregulated applications result in its presence in different environmental compartments (Otieno et al. 2010).
The aim of this work was to evaluate the performance of bioaugmented biomixtures in the removal of CFN, employing the ligninolytic fungus Trametes versicolor as the bioaugmentation agent. T. versicolor is a white rot fungus, capable of degrading diverse organic pollutants in several matrices (Tuomela et al. 1998; Rodríguez-Rodríguez et al. 2014). Biomixtures prepared using either compost or peat, soil, and rice husk as the lignocellulosic substrate were used to eliminate CFN and two of its transformation products; for this reason, we also monitored the mineralization of radiolabeled CFN and the toxicity reduction in the matrix.
Materials and methods
Chemicals and reagents
Commercial CFN formulation (Furadan® 48SC, 48 % w/v) was acquired from a local store. Analytical standards CFN (2,2-dimethyl-2,3-dihydro-1-benzofuran-7-ylmethylcarbamate, >99 % purity), 3-hydroxycarbofuran (99.5 %), and 3-ketocarbofuran (99.5 %) were obtained from Chemservice (West Chester, Pennsylvania, USA). Radio-labeled CFN (14C-CFN; [Ring-U-14C]-Carbofuran; 2.89 × 109 Bq g−1; radiochemical purity 100 %; chemical purity 99.5 %) was obtained from Izotop (Institute of Isotopes Co., Budapest, Hungary). Carbendazim-d3 (surrogate standard, 99.0 %) and carbofuran-d4 (internal standard, 99.5 %) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Potassium hydroxide analytical grade was purchased from Merck (Darmstadt, Germany). Ultima Gold cocktail for Liquid Scintillation Counting was purchased from PerkinElmer (Waltham, Massachusetts, USA). Solvents and extraction chemicals are those reported by Ruiz-Hidalgo et al. (2014).
Fungal strain and biomixture components
T. versicolor (ATCC 42530) was obtained from the American Type Culture Collection and maintained by subculturing every 30 days on potato dextrose agar slants (pH 4.5) at 25 °C in the dark. T. versicolor-blended mycelial suspension was prepared according to a procedure by Font Segura et al. (1993), modified by using Sabouraud dextrose broth as culture medium. Clay loam soil (sand 40 %, silt 27 %, clay 33 %; 2.71 % C; 0.29 % N; pH 5.9) was collected from the upper soil layer (0–20 cm) of an onion field with history of CFN application, in Tierra Blanca, Cartago, Costa Rica. Soil was air-dried and sieved (<2 mm). Rice husk obtained from an agricultural input supplier from Tierra Blanca, Cartago, Costa Rica, was employed as the lignocellulosic substrate. Garden compost, employed as the humic component, was collected from a composting station located at Universidad de Costa Rica, dried and sieved (<2 mm). Peat moss (Berger, Saint-Modeste, Québec, Canada) was acquired from a local store.
Screening of fungal colonization on lignocellulosic substrates
Colonization of five lignocellulosic substrates by T. versicolor was evaluated before choosing the substrate to be used in the biomixtures. These substrates were newsprint paper, coconut fiber, rice husk, wood chips, and cane bagasse. The screening was performed in triplicate flasks (250 mL) containing the autoclaved lignocellulosic substrate (ratio 1:2, dry substrate/water, w/v), which was then inoculated with blended mycelial suspension (0.8 mL g−1 of dry substrate). Flasks were incubated at 25 °C (±1) for 24 days; colonization was visually monitored, and samples were periodically taken for extraction and determination of laccase activity.
CFN degradation assays were performed with 5 g of biomixture. Plastic tubes (12 cm × 3.5 cm) containing the proper amount of humidified rice husk were sterilized at 121 °C for 15 min prior to the inoculation of blended T. versicolor mycelial suspension (0.35 mL g-1 of dry rice husk). After fungal colonization for 10 days at 25 °C (±1), biomixtures were prepared by adding pre-exposed soil and either peat or compost at a ratio colonized-rice husk/soil/peat (or compost) of 50:25:25 % (v/v), to obtain the biomixtures GTS (peat-based biomixture) and GCS (compost-based biomixture). The biomixtures were then spiked with Furadan® 48SC to a CFN final concentration of ∼10 mg kg−1 and incubated in static conditions at 25 °C (±1) until the end of the assay. Water was periodically added in order to keep a constant water content in the matrices. Two sets of triplicate biomixture samples were periodically sacrificed over a 62-day period to determine the concentration of CFN and its transformation products, ecotoxicological tests, and laccase activity.
The mineralization of 14C-CFN was determined by trapping the evolved 14CO2 in 0.1 M KOH in flasks containing 50 g of the biomixtures and spiked with commercial CFN (50 mg kg−1) and 14C-CFN (3000 dpm g−1) (Ruiz-Hidalgo et al. 2014). Each treatment was replicated three times. The flasks were incubated in the dark at 25 °C (±1) for 64 days; water content was kept constant as mentioned above. KOH solutions in the flasks were withdrawn at selected times and replaced with the same amount of fresh KOH. Flasks containing 50 g of non-bioaugmented soil were used as controls. 14C activity from the mineralized 14CO2 was determined in the KOH samples as described below.
Extraction and quantification of CFN and transformation products
Selected transitions and other parameters in the detection of CFN and its transformation products using the dynamic multiple reaction monitoring (MRM) method
Retention time (min)
Collision energy (V)
Type of transition
238 → 163
238 → 107
236 → 161
236 → 179
222 → 165
222 → 123
196 → 164
196 → 136
225 → 165
225 → 123
Determination of 14CO2
Scintillant liquid (8 mL) was added to 2-mL aliquots taken from the KOH solution, and the 14C activity was measured by liquid scintillation using a Beckman LS6000SC counter. The total cumulative 14CO2 activity and the initially added 14C-CFN activity were used to calculate the percentage of 14C-pesticide mineralized. Pesticide mineralization was modeled by a first-order model.
Laccase was extracted from solid samples according to Rodríguez-Rodríguez et al. (2010); briefly, 30-mL acetate buffer (pH 5) was added to 3 g of homogenized biomixture, shaken for 30 min and centrifuged at 2862g for 15 min at 4 °C. Laccase activity of the supernatant was measured by a modified version of the method for the determination of manganese peroxidase activity (Kaal et al. 1993), using 2,6-dimethoxyphenol (DMP) as the substrate (extinction coefficient 24 800 M−1cm−1; λ = 468 nm) (Wariishi et al. 1992). Enzymatic activity was expressed as activity units (U) per kilogram of biomixture. One unit was defined as the number of micromoles of DMP oxidized per minute.
Acute toxicity: Daphnia magna immobilization test
An acute test based on D. magna immobilization was employed to estimate the residual toxicity in the biomixtures. The test was conducted following the U.S. EPA guidelines (EPA 2002) modified as described by Ruiz-Hidalgo et al. (2014), using aqueous extracts from unitary biomixtures sampled during a 62-day period as analytical matrix. Aqueous extracts were obtained according to the protocol EPA-823-B-01-002 (2001). EC50, the concentration producing 50 % of immobilization in the daphnids, was determined using the TOXCALC (Toxicity Data Analysis Software from Tidepool Scientific Software). Toxicity was expressed as toxicity units (TU), calculated according to the expression: TU = (EC50)−1 × 100.
Results and discussion
The degradation of CFN was assayed in two fungal bioaugmented rice husk-based biomixtures employing T. versicolor as the bioaugmentation agent. The efficiency of the biomixtures was evaluated by considering the decrease in concentration of the parent compound, the production of transformation metabolites, the mineralization of 14C-CFN, and the decrease in the toxicity during the removal treatment.
Removal and mineralization of CFN and transformation products in different matrices related to the design of bioaugmented biomixtures
CFN estimated half-life (day)
Mineralization rate constant, k (day−1)
Transformation products from CFN
3-Hydroxycarbofuran (initial concentration of 0.244 mg kg−1 to <LOD after 13 days); 3-ketocarbofuran (initial concentration of 0.163 mg kg−1 to <LOD after 34 days)
3-Hydroxycarbofuran (initial concentration of 0.193 mg kg−1 to <LOD after 6 days); 3-ketocarbofuran (initial concentration of 0.105 mg kg−1 to <LOD after 20 days)
Chin-Pampillo et al. (2015a)/This work
3-Hydroxycarbofuran (<LOQ, 0.021 mg kg−1); 3-ketocarbofuran (<LOQ, 0.164 mg kg−1)
Chin-Pampillo et al. (2015b)
3-Hydroxycarbofuran (peak of 0.0569 mg kg−1 after 4 days); 3-ketocarbofuran (peak of 0.805 mg kg−1 after 8 days)
Rice husk + T. versicolor
3-Hydroxycarbofuran (peak of 1.56 mg kg−1 after 15 days)
Ruiz-Hidalgo et al. (2014)
Typically biomixtures used in biopurification systems have employed peat as the humic component; nonetheless, given that peat is not easily available, it can be replaced by compost (or even spent mushroom substrate, Gao et al. (2015)). These compost-based biomixtures can be more efficient in degrading pesticides than peat-based biomixtures (Coppola et al. 2007, 2011; Vischetti et al. 2008). These reports contrast with the findings of the present work, since the bioaugmentation only enhanced the performance of the peat-based biomixture.
The elimination of CFN by T. versicolor in rice husk was slow, with a half-life of 29.9 days (Ruiz-Hidalgo et al. 2014), almost ninefold higher than that obtained with the bioaugmented GTS; in addition, the CFN removal was only 55 % after 60 days. Therefore, the use of this lignocellulosic substrate within a biomixture can significantly increase the removal performance.
The transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at concentrations below 0.25 mg kg−1 in the bioaugmented biomixtures at the moment of spiking with the commercial formulation of CFN. Concentration profiles revealed a continuous removal of both metabolites in the biomixtures to levels below detection limits (Fig. 2b) suggesting that their removal was faster than their potential production in these matrices. A less efficient performance was achieved in the non-bioaugmented GTS, where some accumulation of these products (particularly 3-ketocarbofuran, up to 0.8 mg kg−1) occurred within the first days (Chin-Pampillo et al. 2015b). The product 3-hydroxycarbofuran accumulated during the first 15 days of CFN removal (peaks of around 1.5 mg kg−1) in the rice husk bioaugmented with T. versicolor, whereas 3-ketocarbofuran was not detected (Ruiz-Hidalgo et al. 2014).
Although laccase activity is not involved in the initial degradation of CFN by T. versicolor (Mir-Tutusaus et al. 2014), it has been related to the degradation of a broad spectrum of organic pollutants (Asgher et al. 2008). Initial laccase activities were 117.8 and 79.6 U kg−1 in GTS and GCS biomixtures, respectively. Nonetheless, this enzymatic activity was negligible during the rest of the incubation period, which may depend on either the competition effect of the microbial communities present in the soil and the compost (McErlean et al. 2006) or laccase inactivation in the matrix (Botterweck et al. 2014). The removal of CFN by T. versicolor in rice husk alone produced a maximum in laccase activity of 62.9 U kg−1 (Ruiz-Hidalgo et al. 2014). Biopiles containing T. versicolor showed laccase activity during the removal of pharmaceuticals in sewage sludge in non-sterile conditions (Rodríguez-Rodríguez et al. 2012b). Nonetheless, the conditions of humidity and the amount of indigenous microbiota were presumably lower in the sludge systems with respect to the biomixtures, thus promoting the better fungal colonization and activity in the former. Laccase activity also plays a role in the formation of non-extractable residues of pesticides, by favoring the formation of strong covalent bonds between these molecules and the soil humic substances; this phenomenon has been described for metalaxyl and its major transformation product, metalaxyl acid, in the presence of immobilized laccase (Botterweck et al. 2014). This indicates that if this enzyme is active in the matrix, it may contribute to the apparent elimination of CFN in the biomixtures, rather than to produce derivative metabolites.
Reports on the bioaugmentation of biomixtures for biopurification systems are scarce. Biomixtures containing T. versicolor and barley straw removed 36–58 % of pesticides such as metalaxyl, terbuthylazine, atrazine, diuron, iprodione, and chlorpyrifos (Bending et al. 2002); however, these biomixtures were previously sterilized, and the effect of indigenous microbial communities was suppressed. Similarly, isoproturon was completely removed after 100 days in biomixtures containing straw when they were bioaugmented with Phanerochaete chrysosporium, another white rot fungus, whereas the removal accounted for around 80 % without bioaugmentation (von Wirén-Lehr et al. 2001). In the case of bacterial bioaugmentation, Verhagen et al. (2013) could not find significant differences in the removal rate of chloropropham between the non-bioaugmented and the bioaugmented biomixtures with a chloropropham-degrading strain of Delftia acidovorans or with a chloropropham-degrading enrichment culture.
The fungal bioaugmentation of rice husk-based biomixtures represents a suitable biotechnological option for the removal of CFN. A clear enhancement in the performance of the biomixtures occurred when T. versicolor was employed in the bioaugmentation of GTS, the biomixture employing peat as a humic component; in this case, the removal of CFN and its transformation products was faster than in the non-bioaugmented biomixtures or in the pre-exposed soil. A complete removal of toxicity was achieved in the biomixtures after 48 days of treatment, which supports the ecological relevance of the biomixture. The fact that the bioaugmentation as a strategy for the enhancement of biomixture efficiency was effective for only one of the two tested biomixtures highlights the importance of a careful design and evaluation of biomixtures before their large-scale use.
The authors acknowledge Vicerrectoría de Investigación, Universidad de Costa Rica (projects 802-B2-046, 802-B4-503 and 802-B4-609), the Costa Rican Ministry of Science, Technology and Telecommunications, MICITT (project FI-093-13), and the Joint FAO/IAEA project TC COS5/029 for supporting this work.
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