Influence of sucrosis on the degradation of pesticide by white rot fungus Influência da sacarose na degradação de pesticida por fungo da podridão branca Influencia de la sacarosa en la degradación de plaguicidas por el hongo de la putrefacción blanca

The influence of sucrose on the removal of Paraquat (PQT) in synthetic aqueous medium was evaluated by Phanerochaete chrysosporium. Initially, a toxicity test was performed on plates containing paraquat at concentrations of 1, 5, 10, 20 and 30 mg.L. Then, they were carried out in batches agitated batch (RBA) and sequential batch (RBS). Four reactors were submitted, containing medium with 30 mg.L of paraquat, under a reaction time of 144 h, the reactors being RBA-2 and RBS-2 with the addition of 2 gL of sucrose, and without the adding sucrose to the RBA-0 and RBS-0 reactors. In all reactors, paraquat was removed, but in RBS-0, the best mean removal efficiency was obtained (41.1 ± 0.89%). The best values of apparent speed of degradation (k) were found in reactors with sucrose RBA-2 and RBS-2, 0.015 ± 0.002 h and 0.018 ± 0.002 h, respectively, indicating that the addition of sucrose influenced the speed removal of paraquat. It was also verified that the pollutant was not completely removed by adsorption to fungal biomass, which microorganisms predominated in the medium at the end of the treatment, demonstrating their role in the paraquat bioremediation process. Therefore, the addition of sucrose influenced the removal speed of the PQT and COD, but not the removal efficiency.


Introduction
Environmental pollution from pesticide use is seen as a serious problem due to its adverse effect on human health, plants and animals (Galic et al., 2018;Pandiselvam et al., 2020). Pesticides are chemical compounds that are highly required in agricultural activities, as they are efficient in controlling and eliminating pests, contributing to the maintenance of the quality and yield of agricultural products (Hyland & Laribi, 2017). However, the careless use of these chemicals can contribute negatively on the environment and, thus, consequently, on human health (Abhilash & Singh, 2009;Han et al., 2015;Rowland et al., 2011).
The negative effects of pesticide exposure on human health depend mainly on the toxicological profile of the product, as well as on the intensity and exposure, which can occur through inhalation, ingestion and skin contact, resulting in acute and chronic health problems (Pereira, 2011, Lopes & Albuquerque, 2018. Exposure increases the risk of the emergence of several pathologies, especially cancerous tumors, in addition to hormonal disorders and congenital malformation (Vasconcelos, 2018;Lemarchand et al. 2017;Lerro et al. 2019).
Paraquat is a non-selective contact herbicide used and banned in over 50 countries due to its high chemical toxicity (Wesseling et al., 2001). It is known and marketed as gramoxone, gramocil, agroquat, gramuron and paraquol, or also as a constituent in mixtures with other active ingredients, as in secamate (Serra, Domingos & Prata, 2003). Its classification regarding the potential environmental hazard is II (Very Dangerous) and its toxicological class is type I (Highly Toxic) (Anvisa, 2005). In regions of agricultural development, with high plantations, paraquat is the main source of pollution of water and soil resources (Sorolla et al., 2012). Exposure to this pollutant can cause serious side effects (Dong et al., 2013;Fukushima et al., 2002;Song et al., 2020) and has been used as a method of suicide (Wu et al., 2014). This active ingredient is banned in Because of its polluting potential, it is necessary to seek ways to eliminate it from the environment. In the literature one can find works that perform the degradation of Paraquat from photocatalysts (WONGCHAROEN & PANOMSUWAN, 2018), by adsorption (Suo et al., 2019), zeolite (Keawkumay et al., 2019) and by bioremediation (Wongputtisin et al., 2021).
Bioremediation or biological remediation being a technique in which natural microorganisms such as bacteria, fungi, yeast and enzymes (present or added to the site) are used in the biochemical degradation of organic and inorganic contaminants present in soils, waters and various other environments (Bernoth et al., 2000;Sanchéz et al., 2013).
According to Jou & Huang (2002) bioremediation is an economical and efficient technique for the elimination of pollutants in aqueous media. Fungi have been showing great versatility in remediating polluted environments, because they are able to grow and survive in high concentrations of contaminating compounds, use them as a source for obtaining energy, in addition to what Research, Society andDevelopment, v. 10, n. 15, e344101522790, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.22790 3 are shown to be skillful organisms in the synthesis of enzymes that act in the safe removal of contaminants from the environment (Das & Dash, 2014).
On an industrial scale it is of utmost importance the reaction time in which the synthetic water will be treated, i.e. the yield is one of the important factors to determine the operation cost. Therefore, the faster the degradation reaction occurs, the lower the cost. Hence the need for a study on the addition of a carbon source that can accelerate the reaction time.
From the bioremediation with fungi, this research seeks to evaluate the kinetics and the influence of sucrose, as a carbon source, in the efficiency of removal of pesticide Paraquat in stirred batch and sequential reactors, inoculated with Phanerochaete chrysosporium (PHC) using synthetic effluent doped with 30 mg.L -1 of paraquat.

Methodology
The research is of the quantitative experimental type carried out on a laboratory scale (Pereira et al., 2018), in which mathematical analyzes were carried out based on the results obtained, such as means, standard deviations, percentages and statistics. The work was carried out at the Environmental Technology Laboratory (LATAM) of the Federal Institute of Education, Science and Technology of Ceará (IFCE). The present research was divided into 5 steps: (I) plate toxicity test, (II) cultivation and counting of fungal spores, (III) assembly and operation of the reactors, (IV) adsorption test and (V) contamination evaluation.

(I) Plaque Toxicity Test
This step consisted in evaluating the tolerance capacity of the fungus Phanerochaete chrysosporium to the pollutant under study. Petri dishes containing Martin culture medium were prepared and different concentrations of Paraquat (1 mg.L -1 , 5 mg.L -1 , 10 mg.L -1 , 20 mg.L -1 and 30 mg.L -1 ) were used. The fungus was inoculated using the 6 mm diameter mycelium plugs methodology (OTTONI, 2012). The growth of the fungal species was observed for 7 days by photographic recording and measurement of colony diameters at the times of 0 h, 24 h, 72 h, 120 h and 168 h. The measurement was performed with the help of a pachymeter.
The culture was kept in a microbiological oven under 28°C for seven days. Afterwards, the spores were removed with the help of isotonic Tween 80 solution and were stored in a sterile vial for later microscopic counting in a Neubauer chamber. A concentration of 2 x 10 6 spores/mL was used for the reactor inoculum.

(III) Assembly and operation of reactors
The highest paraquat concentration tested at which fungus growth occurred in the plate test was chosen to be studied in the batch reactors. The synthetic water was prepared using a concentration of 30 mg.L -1 of PQT added to the water supply.

• Stirred batch reactors with dispersed biomass
The stirred batches were performed in a bench-top incubator with rotary shakers, at 150 rpm and 30°C ± 2. The reactors were erlenmeyer flasks with a reaction volume of 200 mL (useful volume of 250 mL), previously sterilized in autoclaves, and sealed with aluminum foil and Nonwoven Fabric to prevent contamination. The reactors were packed with newspaper to avoid photodegradation of the medium. The experiment took place in duplicate.
Fourteen RBA-0 (30 mg. L -1 of PQT and without sucrose) and 14 RBA-2 (30 mg. L -1 of PQT and 2 g.L -1 of sucrose) reactors were operated. Every 24 h, 2 flasks of each reactor (RBA-0 and RBA-2) were removed for the analyses, so that there was no interference in the useful volume of the reactor, totaling 7 days. Control reactors (RC) were also operated as an abiotic factor (without addition of fungus). The RC-0 being a control reactor without addition of sucrose and RC-2 a control reactor with 2 g.L -1 of sucrose.
• Sequential batch reactors with immobilized biomass Duran flasks with useful of 4.5 L were used as reactors. The reactor RBS-2 received the synthetic wastewater doped with paraquat and with 2 g.L -1 of sucrose as a co-substrate, and RBS-0, control reactor, received the same amount of synthetic wastewater, but without the addition of co-substrate. The reactors were operated in 5 cycles with 7 days duration each.
For the adherent growth of the biomass the support material used was polyurethane foam, wrapped with polyethylene nets to group the foams and improve immobilization. The material was previously washed with alcohol and distilled water and sterilized in an autoclave at 121°C for 20 minutes to remove possible impurities.
The total reaction time for each cycle of the reactors was 144 h, and the reactors were a after 6 days, and the reaction times of 12, 24, 48, 72, 96, 120, and 144 h were studied.

Analyzes performed
Analyses of pH, temperature, Chemical Oxygen Demand (COD), sucrose concentration and Paraquat (PQP) were performed, as well as reaction kinetics.
The pH and temperature analyses were checked in order to observe the metabolic activity of the fungus, following the APHA (2005) methodology.
The COD analyses were performed with filtered samples, so that there was no influence of the biomass present in the reactor, also following the procedures described by APHA (2005).
The determination of paraquat was performed according to AOAC Method 969.09 (2000), which comprises the reaction between Na2S2O4 -sodium dithionite 1% (diluted with NaOH 0.1 mol. L -1 ) and samples containing the herbicide, obtaining a blue coloration. Readings were taken in a spectrophotometer at a wavelength of 600 nm. The statistical evaluation of each parameter studied was determined by analysis of variance (ANOVA) and comparison by Tukey's Test (95% confidence level, p ≤ 0.05). The programs used were Microsoft Excel, Minitab 17 Statistical Software and OriginLab.
(IV) Adsorption test The adsorption test was performed to evaluate the adsorption capacity of PQT by the polyurethane foam used as support medium in the reactors and by the fungal biomass.
The adsorption capacity of PQT on the support medium was evaluated from the initial and final concentration of the pesticide (SILVA, 2015). The same synthetic effluent used in the sequential batch reactors was used. To determine the concentration of PQT during the test, Equation 2 was used.
Being, mg: absorbed PQT mass C 0: Initial concentration of PQT (mg.L -1 ) The adsorption capacity of the fungal biomass was performed from the methodology of Barbosa (2016). Initially, an aliquot of fungal biomass was removed from the sequential batch reactors. Subsequently, the PQT present in the sample was removed with methanol addition and followed by centrifugation. At the end, the separation was done with a vacuum membrane. The solid part was characterized from the analysis of volatile suspended solids and the liquid part read in the spectrophotometer.

(V) Contamination test
At the end of each reaction time in the reactors, aliquots were submitted to the contamination test, which aims to verify the existence of other microorganisms that had not been inoculated, in quantitative terms, which may have arisen during the process. The methodology used was according to APHA (2005).

Plaque Toxicity Test
The toxicity step in plates aimed to evaluate the growth of fungal colonies for 7 days, with different concentrations of pesticide ( Figure 1) and define the concentration to be used in sequential batch reactors. of the fungus. Therefore, PHC is also tolerant to these heavy metals.
From this perspective, Wongputtisin et al. (2021) observed that the fungal species Aspergillus tamarii was able to degrade 80% of PQT in contaminated soil in 15 days, indicating the capacity of these microorganisms to remediate contaminated areas.

pH and temperature
Regarding pH and temperature, the temperature of the reactors averaged 28 ± 2 °C. The reactors started with an average pH of 6.5 ± 0.5. Only the RBA-2 reactor presented an average pH equal to the initial range (6.28 ± 0.34). The RBS-2 reactor presented the lowest final average pH (2.83 ± 0.11) which may be related to the production of by-products formed by According to Han et al. (2014) and Reyna et al. (2017), the best pesticide removals occur at acidic pH, as it enhances the enzymatic activity of the fungi and favors the degradation process. The pH being close to 5, the best for the growth of most fungal species.

Influence of sucrose on paraquat degradation
The results of the removal efficiencies of PQT, consumption of sucrose and degradation kinetics constants from the stirred-batch reactors with dispersed biomass and in sequencing with immobilized biomass, inoculated with Phanerochaete chrysosporium are presented in Table 1.  In all reactors there was removal of PQT. Being that the sequential batch reactor without addition of sucrose, RBS-0, obtained efficiency of 41.1 ± 0.89% and with sucrose, RBS-2, obtained 34.3 ± 2.31%. Therefore, the addition of the cossubstrate did not influence positively for the removal of PQT in the sequencing reactors. However, in the stirred batch reactors, the addition of sucrose improved the efficiency of PQT removal. RBA-2 obtained 21.1 ± 0.52 % and RBA-0 only 3.1 ± 0.02.
It is noteworthy that greater removal of PQT was observed in reactors with sucrose in sequential batch (RBS-2) when compared to reactors with stirred batch also containing an external source of carbon (RBA -2), which may indicate that for the consumption of PQT, the form of fixation of the microorganism on the support medium may influence the achievement of better removal percentages, as already reported by Bouabidi, El-Naas and Zhang (2019).
Statistically, the means of PQT removal of the 4 reactors are significantly different from each other. However, although fungal metabolism and growth are positively influenced by the presence of glucose (YAO et al., 2013), the noninfluence of sucrose, in this research, is due to the fact that the microorganisms used the pesticide as a carbon source. The addition of a cossubstrate also influenced negatively in the study of the authors Silva et al. (2013), when testing the ability Cunninghamella elegans to degrade phenanthrene in aqueous medium with the addition of glucose, the highest concentration Research, Society andDevelopment, v. 10, n. 15, e344101522790, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.22790 8 added (1 g.L -1 ) inhibited the cell growth of the fungus. In contrast, the authors Silva et al. (2018) had better paraquat removal results (70%) when added 1 g.L -1 of glucose and 1 g.L -1 of cassava peel in batch reactors inoculated with Aspergillus niger AN 400, with reaction time of 168 h with initial concentration of 30 mg.L -1 of PQT. The removal efficiency was higher than in this work, but two carbon sources were used, which may have been more easily assimilated by the fungus than sucrose. As well, the cassava husk may have been a source of extra fiber and micronutrients for the environment.
From the kinetic study, it was possible to evaluate the influence of the cosubstrate on the degradation of PQT. The degradation curve profiles of the reactors (Figure 3) followed the first order reaction and the constant k was calculated in OriginPro 6 software. RBA-0 = stirred batch reactor without addition of sucrose; RBA-2 = stirred batch reactor with 2 g.L -1 sucrose; RBS-0 = sequential batch reactor without addition of sucrose; RBS-2 = sequential batch reactor with 2 g.L -1 sucrose. Source: Authors (2021).
In this present study, it was observed that the decay curve of the reactor RBA-0 did not fit as degradation. It was also found that the reactors with sucrose addition, RBA-2 and RBS-2, obtained better average kinetic rates, 0.015 h -1 and 0.018 h -1 , respectively. Therefore, the addition of sucrose influenced the rate of degradation of the pollutant. When statically evaluated, the two reactors showed no significant differences between them with respect to the kinetic constants. The authors Silva et al. (2018), also found higher removal speed of PQT (k = 0.2051 h -1 ), but were added 3 gL -1 of glucose, a higher concentration than the sucrose concentration used, in this work.
The difference in sucrose consumption may be due to the type of biomass used and consequently may also have influenced the removal of PQT. The use of dispersed biomass with stirring, the pH and temperature range in the stirred batch reactors resulted in a pellet-like fungal growth (Figure 3). These pellets are small spheres of hyphae (Papagianni, 2004) and this format makes it difficult for nutrients to reach the interior of the pellets when they are in larger sizes, so the pellets must have small sizes to avoid the emergence of nutritional deficiency (GIBBS et al., 2000;KRULL et al., 2013). Therefore, the formation of these pellets in the RBA-0 and RBA-2 reactors cannot have influenced the low removal of the PQT since the sucrose consumption was only 56.5 ± 0.05% and a consumption rate of 0 .0166 h -1 .
The highest PQT removal means occurred in sequential batch reactors with immobilized biomass (RBS-0 =41.1 ± 0.89 %; RBS-2 = 34.3 ± 2.31%). According to the authors De Filippi and Lewandowski (1998) and Jou and Huang (2003), the use of immobilized biomass has four main advantages, namely: simplicity of operation, ability to withstand shocks from organic loads, low solids production, and beyond Furthermore, little energy is needed. Furthermore, the use of immobilized biomass reduces the adaptation time (Sharma & Gupta, 2012) and offers great efficiency and stability to the process, especially when a high degradation rate is needed (Alves, 1999). Therefore, these statements corroborate the results of this study, as the sequential batch reactors with immobilized biomass favored the removal of the pollutant and were the ones with the best removals of PQT (RBS-0 = 41.1 ± 0.89%) and best mean sucrose consumption and kinetic constant, 92.4 ± 0.864% and 0.147 h -1 for the RBS-2 reactor. Figure 4a shows the sequential reactor with the support material and Figure 4b shows the reactor 15 days after fungus inoculation and the biomass already formed.

Influence of sucrose on organic matter degradation in terms of COD
The influence of sucrose on COD degradation was evaluated only in reactors RBS-0 and RBS-2, with mean initial concentrations of 959.13 ± 53.1 mg. L -1 and 2424.58 ± 236.7 mg. L -1 , respectively. Even without sucrose addition, the RBS-0 reactor still presents organic matter because of the synthetic effluent. The highest removals in RBS-2 occurred in the fourth and fifth cycle, presenting 80.8% and 80.4%, respectively. The average COD removal efficiency of all cycles of RBS-2 was 79.9% ± 0.95, as shown in Table 5. In RBS-0, the highest average removals of COD occurred in the fourth and second cycles, presenting 79.6% and 77.2%, respectively. The average COD removal efficiency in all cycles of RBS-0 was 75.4% ± 2.8. Statistically, the means of COD removal are not significantly different. Therefore, the addition of sucrose did not influence positively on the removal of organic matter, however, as mentioned earlier, the addition of sucrose favored the removal speed of the PQT. Figure 5 shows the decay profiles of COD. The COD removal reaction followed first order kinetics. The speed in which the COD was removed, during the final reaction time of 144 h, in RBS-2 presented an average of the kinetic constant of 0.014 ± 0.001 h -1 , being the fourth cycle the fastest in the removal (0.016 h -1 ). As for RBS-0, the average kinetic constant was 0.006 ± 0.002 h -1 , with the first and fifth cycles being the fastest in removal (0.008 h -1 ). However, the fifth cycle besides being faster, presented a higher efficiency in the removal of COD compared to the first cycle. Therefore, the addition of sucrose negatively influenced the rate at which COD was removed, since the reactor without sucrose (RBS-0) had a rate 2 times faster than the reactor with sucrose (RBS-2).
According to Singh (2006), the optimal concentration of the bio-substrate to be used depends on the microbial species, the type of pollutant and the type of reactor.
When adding 3 mgL -1 of glucose in the sequential batch reactor inoculated with Aspergillus niger AN 400, the authors Marinho et al. (2017) obtained better COD removal (50 ± 3%) when compared to the reactor without glucose (25 ± 4%), these values are lower than those found in this study with the addition of sucrose, which may be associated with co-substrate used.

Adsorption test
Initially, the adsorption test was performed on the support material used in the sequential batch reactors to verify the amount of adsorbed paraquat. The saturation point reached by the pollutant was 2 minutes with a maximum capacity of 0.0001 g of PQT per gram of support material. The total mass balance of pesticide removed by reactors RBS-0 and RBS-2 was averaged and an average removal of 0.00259 g of PQT was verified. Therefore, the reactor with 15g of support material has 0.000173 g of PQT for each gram of support material. Since the maximum adsorption capacity of the foam was 0.0001 g, the value found in the degradation of MDP in the reactors was higher than the maximum capacity, meaning that adsorption was not the main removal mechanism. Therefore, the fungus Phanerochaete chrysosporium collaborated with the degradation.
The adsorption test was also performed on the fungal biomass. 0.00090 g of COD was absorbed for 15.2 g of total biomass. Therefore, the removal via adsorption in the biomass was only 1.7%. According to Pinto et al. (2012), that there are few studies on fungal degradation that perform the adsorption test and that each fungal species has different adsorption processes.

Contamination assessment in reactors
The contamination evaluation was performed at the end of each cycle in each reactor. It consists in verifying the predominance of the fungus at the end of each treatment. In all reactors the presence of fungus was higher than that of bacteria (Table 3). Therefore, the degradation of the PQT and COD was predominantly done by fungal action.

Conclusion
From the toxicity test it was verified that the microorganism Phanerochaete chrysosporium under different concentrations of paraquat, was resistant to all concentrations tested.
In relation to the addition of sucrose in the reactors, this did not influence positively in the efficiency of pesticide and COD removal, however, the addition of the co-substrate influenced the speed of degradation of the PQT and COD, since the reactors with the addition of sucrose were the ones that presented the highest kinetic constants.
It was possible to observe that the reactors with immobilized biomass showed better removal of PQT when compared to the reactors with dispersed biomass.
In both types of reactors the presence of fungi was higher than that of bacteria, therefore the removal of paraquat and COD was predominantly done by fungal action.
As a suggestion for future work, evaluate the performance of other fungal species in the biological treatment of pesticides, as well as other possibilities for using a co-substrate that has a lower cost.