Potential of Aspergillus niger Tiegh 8285 in the bioremediation of water contaminated with benzonitrile

Benzonitrile is a compound found in pesticides. The use of these pesticides can cause environmental contamination, and the search for non-aggressive methods to eliminate these residues is necessary. In this study, fungi Aspergillus isolated from cocoa were investigated for their benzonitrile bioremediation potential. The fungi were cultured in a solid medium supplemented with nitrile and glucose ( a ), nitrile ( b ), and glucose ( c ). Independent variables: time, inoculum, and nitrile were optimized using a central composite design to determine the best microbial growth and wet biomass (dependent variable) as a response in the bioremediation process. A. niger Tiegh 8285 showed good adaptation, especially in situation b in nitrile 5 days, 3 mycelial inoculums and 54 μ L of benzonitrile for microbial growth, resulting in 1.83 ± 0.03 g of wet biomass, confirming the efficiency of the selected mathematical model. A. niger Tiegh 8285 proved to be a promising bioremediation agent for benzonitrile. siendo necesaria la búsqueda de métodos no agresivos para la eliminación de estos resíduos. En este estudio, los hongos Aspergillus aislados del cacao fueron investigados por su potencial para la biorremediación del benzonitrilo. Los hongos se cultivaron en medio sólido suplementado con nitrilo y glucosa (a) , nitrilo (b) y glucosa (c) . Variables independientes: tiempo, inóculo y nitrilo fueron optimizaron mediante un diseño de Central Composite para determinar el mejor crecimiento microbiano y biomasa húmeda (variable dependiente) como respuesta en el proceso de biorremediación. A. niger Tiegh 8285 mostró buena adaptación, principalmente en situación b en 5 días, 3 inóculo micelial y 54 μ L de benzonitrilo para crecimiento microbiano, resultando 1.83 ± 0.03 g de biomasa húmeda, confirmando la eficiencia del modelo matemático seleccionado. A. niger Tiegh 8285 demostró ser un agente de biorremediación prometedor para el benzonitrilo.

Any chemical contamination in a natural environment, such as rivers and groundwater, is undesirable in any circumstance. Therefore, the environmental pollution problems associated with social and technological development have spurred the search for different methods of water treatment, the most promising of which is bioremediation (Fu, et al., 2020).
Bioremediation is a branch of biotechnology in which microorganisms such as bacteria and fungi (Quintella, et al., 2019) are used to transform contaminating and toxic compounds, such as dioxins (Dao, et al., 2019), agricultural effluents (Neoh, et al., 2016), soil contaminated with oil (Chaudhary, et al., 2019;Li, et al., 2020) or heavy metals and pesticides , and wastewater from the pharmaceutical industry (Shah, et al., 2020) into compounds of low or zero toxicity, while obtaining water and carbon dioxide during the process (Liu, et al., 2017). Moreover, bioremediation has been extensively explored in wastewater treatment (Lu, et al., 2014;Catania, et al., 2020;Hubabillah, et al., 2020;Zhou, et al., 2020) and can be characterized as a renewable process since it uses living organisms. Another advantage is that this process does not require the use of chemical catalysts and the reactions can be carried out at approximate room temperature (Zhang, et al., 2013).
Studies have been carried out involving the bioremediation of several compounds such as Myceliophthora thermophile (Salami, et al., 2018) and Coriolopsis gallica (Vidal-Limon, et al., 2012) in the bioremediation of environmental contaminants, Sinanaonta woodiana, a freshwater mollusk, in the bioremediation of aquaculture effluents (reservoirs for breeding marine species, such as fish and shellfish) (Sicuro, et al., 2020), and Bacillus velezensis in the bioremediation of textile dyeing residues (Gowri, et al., 2020).
In bioremediation, each microorganism reacts differently depending on the conditions. Therefore, it is important to provide ideal conditions for the microorganism to increase its bioremediation potential. For this purpose, chemometric techniques are considered useful alternatives. Thus, the aim of this study was to investigate the bioremediation potential of fungi of the genus Aspergillus, isolated from the stem and leaves of cocoa in southern Bahia, in the presence of benzonitrile and to optimize the conditions of bioremediation using central composite design (CCD) using the variables time, inoculum, and volume of nitrile.

Central composite design for optimization of bioremediation of water contaminated with benzonitrile
Aspergillus niger Tiegh 8285, previously grown in a minimum solid mineral medium a, was inoculated in a minimum liquid mineral medium (De Oliveira, et al., 2013), situation a. An Erlenmeyer flask (250 mL) containing 100 mL of minimal liquid mineral medium supplemented with glucose (1.5 g) was autoclaved (CS Prismatec, Itu, Brazil) at 121°C for 15 min.
When it reached room temperature, benzonitrile was added. In parallel, small slices of the minimum solid medium containing the fungus mycelia were cut from the stock culture and inoculated. Then, the experiment was incubated in an orbital shaker (Tecnal, Piracicaba, São Paulo, Brazil) at 30°C at 120 rpm. Finally, the reaction was filtered using a vacuum pump (Prismatec, Itu, Brazil), and the wet biomass was weighed.
The procedure was optimized using a central composite design (CCD) (Dos Santos et al., 2016;Marques et al., 2018) with the independent variables reaction time (t), quantity of inoculum (In), and volume of nitrile (Nit) ( Table 1) and the dependent variable wet biomass. The experimental project was modeled and analyzed using Statistica v.12.0 software (Statsoft, USA). The CCD obtained for the maximum biomass production was quadratic, as suggested by the software, and presented in equation 1. In the experimental matrix, the coded values of all parameters vary in five levels (-α, -1, 0, +1, α) (Table 1), totaling 17 experiments (Table 2). At the beginning and end of each reaction, pH was measured only as a reaction indicator.

Model adequacy and validation
The adjustment quality of the statistical model was verified by the coefficient of determination (R 2 ), and the significance of the model was tested by Fisher's test (F-value) through analysis of variance (ANOVA), according to Ferreira et al 2007. Model adequacy was analyzed using the observed vs predicted graph. The interactions between the variables and their influence on the response obtained were analyzed using a Pareto chart, and the optimal point of maximum bioremediation was obtained using equation two and the response surface graph. The selected statistical model was validated by running the experiment under optimized conditions and comparing it with the expected response (Ferreira, et al., 2007;Bezerra, et al., 2020).

Screening in a minimal solid medium in the presence of benzonitrile
Fungi of the genus Aspergillus from different biomes in the state of Bahia with biocatalytic potential in the presence of aromatic nitriles were selected through screening in a minimal solid mineral medium supplemented with glucose and benzonitrile, according to situations a, b, and c, using quantitative evaluation through the number of spores (Table 3). As observed in Table 3, the fungi showed microbial growth in situations a, b, and c and a better adaptation in media supplemented with benzonitrile (a and b), indicating the inducing role of nitrile since it is the only source of nitrogen, as well as the possible biocatalytic potential of the respective fungi. These results corroborate data from the literature that stress the importance of nitriles as an inducer in microbial growth (Coady, et al., 2013;De Oliveira, et al., 2013;De Oliveira, et al., 2014), in particular, benzonitrile (Agarwal, et al., 2017;Serra, et al., 2019). Source: Authors.
According to Table 3, A. niger Thiegh 8285 showed a significant increase in spores in the situation that only contained benzonitrile b (63.2 x 10 4 spores x mL -1 ), which represents an increase of approximately 35 times compared to situation c. The good adaptation of A. niger Thiegh 8285 revealed that the addition of benzonitrile alone in a medium with minimal amounts of nutrients was sufficient to supply the needs of the microorganism.

Optimization of bioremediation
From the results obtained from screening in minimal solid mineral medium (Table 3), A. niger Tiegh 8285 was selected to evaluate its potential in the benzonitrile bioremediation process through the central compound design with 17 experiments (Table 2). Research, Society andDevelopment, v. 11, n. 8, e42711831078, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i8.31078 6 The analysis of variance (ANOVA) calculated model efficiency and adequacy for the experimental design used, as shown in Table 4. The computed F value (22.80) for the model was considerably higher than in the Table 4 (3.67), showing that the model was significant. The model's capacity was assessed using the R 2 determination coefficient, which was calculated to be 1.0. The value found for R 2 (0.97) indicates that the model reported 97% of the experimental data, and there were only 3% of errors, to which noise can be attributed. The observed vs. predicted graph (Figure 1)   Research, Society andDevelopment, v. 11, n. 8, e42711831078, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i8.31078 7 indicating that an increase in time reacts negatively to the response and results in a decrease in wet biomass, revealing that all the nutrient is bioconsumed in a short time; therefore, A. niger Thieg 8285, in addition to being resistant, needs little time to bioremediate nitrile. The quadratic variable had a positive correlation, indicating that, as the volume of nitrile increased, the expected response (biomass) also increased since the culture medium is deficient in nutrients and the only source of nitrogen is the benzonitrile itself. This result shows that A. niger Thieg 8285 was induced to consume nitrile to survive, thus proving it adapted to the established conditions. The analysis of the Pareto chart allowed selection of statistically significant terms at 95% significance and removed the non-significant terms (P >0.50) from the mathematical model. After being the adequacy of the model approved using ANOVA, response surfaces (Figure 3a, b and c) were obtained, making it possible to observe the influence of combinations between the independent variables. Polynomial Eq. 2 was used to express the relationship between the coded independent variables and to predict a response: Y = 1.81 -0.067X1 + 0.034X2 + 0.066X3 -0.146X1 2 -0.066X2 2 + 0.134X3 2 + 0.01X1X2 + 0.005X1X3 -0.02X2X3 (2) Based on equation 2 and the response surface, the maximum biomass production point of reaction was 5 days and 12 hours, inoculum 3.38 mm and 54.31 μL of benzonitrile with theoretical production of 1.82 g of wet biomass. The reaction time obtained (5 days and 12 hours) is within the average time interval for several species of A. niger, which is between 5 (Sattar, et al., 2019;Papadaki, et al., 2020;Putri, et al., 2020) and 7 days (Khan, et al., 2019;Aboyeji, et al., 2020). The volume of nitrile obtained from the statistical design used (CCD) revealed that the microorganism studied has resistance to nitrile since microorganisms generally have limited tolerance to high concentrations of nitriles due to their toxicity (Sattar, et al., 2019).

Statistical validation of bioremediation
For model validation, experiments at the optimum point were performed in triplicate using the ideal conditions provided by equation 2 and the response surface ( Figure 3). The wet biomass value predicted by the model was 1.82 g. By performing the optimal point experiment in triplicate, it was possible to obtain an average of 1.83 ± 0.03 g and a recovery of approximately 100% between the theoretical and the experimental value. These results corroborate those obtained in the central composite planning and validate the use of the proposed method for the bioremediation of water contaminated with benzonitrile by A. niger Tiegh 8285.

Conclusion
The role of benzonitrile as an inducer was confirmed with fungi of the genus Aspergillus of the state of Bahia, particularly Aspergillus niger Tiegh 8285, isolated from cocoa beans. This study has a high scientific value since a chemometric tool (central compound design) was used for the statistical optimization of bioremediation of water contaminated with benzonitrile by A. niger Thiegh 8285. Moreover, this study is relevant in terms of environmental preservation concerns by presenting a promising method for the treatment of water contaminated with residual nitriles. The result is encouraging for future studies on the bioremediation of aromatic nitriles.