DOI: 10.3303/CET2293026 Paper Received: 24 January 2022; Revised: 13 March 2022; Accepted: 13 May 2022 Please cite this article as: Mayorca Clemente S., Castaneda-Olivera C.A., Benites Alfaro E.G., 2022, Microbial Biofilm as a Methodology for Treatment of Cyanide-contaminated Water, Chemical Engineering Transactions, 93, 151-156 DOI:10.3303/CET2293026 CHEMICAL ENGINEERING TRANSACTIONS VOL. 93, 2022 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Marco Bravi, Alberto Brucato, Antonio Marzocchella Copyright © 2022, AIDIC Servizi S.r.l. ISBN 978-88-95608-91-4; ISSN 2283-9216 Microbial Biofilm as a Methodology for Treatment of Cyanide- Contaminated Water Sadit Mayorca Clemente, Carlos Castañeda Olivera, Elmer Benites-Alfaro Universidad César Vallejo, Campus LN, Alfredo Mendiola 6232 Lima 15314, Perú ebenitesa@ucv.edu.pe Cyanide-contaminated wastewater is a very important problem of negative environmental impact. The objective of the research was the reduction of cyanide in the contaminated water emitted in a gold recovery process in the district of Chala-Arequipa after being used in mining processes, the method used was the formation of a microbial biofilm on suitable supports for its growth. The presence of cyanide in two water samples before treatment was 1,352 mg/L respectively. The treatment was performed in bioreactors with biofilms of Pseudomona aeruginosa and Escherichia coli supported on polypropylene and polyurethane for biofilm formation and growth. The tests were conducted over a period of 5, 6 and 7 days, obtaining the following results: in test 1, with Pseudomona aeruginosa biofilms, the cyanide concentration was reduced to 38.98 mg/L (97.11 %), with a pH of 10.77, total suspended solids at 10.79 mg/L, electrical conductivity at 10. 63 mg/L and turbidity at 7.46 NTU; test 2, in bioreactors with Escherichia coli biofilms was also favourable, decreasing cyanide to 325.1 mg/L (75.95 %), with a pH of 8.7, total suspended solids at 1.43 mg/L, dissolved oxygen at 6.70 mg/L, electrical conductivity at 51.13 µS/cm and turbidity at 7.13 NTU. Thus, it was established that the biofilms of Pseudomona aeruginosa and Escherichia coli constitute an environmentally sustainable methodology for the reduction of cyanide in water, which can be complemented with other technology to culminate with the elimination or removal of cyanide to levels below the maximum permissible limits of the environmental quality standards, avoiding the negative impacts that these discharges produce to ecosystems and human beings. 1. Introduction Presence of cyanide in wastewater has become a crucial problem to solve in the world, as is the case of the population of Chala - Arequipa (Peru) where the Organismo de Evaluación y Fiscalización-OEFA found cyanide concentrations above the environmental quality standards; Therefore, the need to remediate the water resource has made it possible to seek new technologies in the detoxification and destruction of contaminants such as cyanide, lead, chromium, etc., replacing the traditional approach of disposal and confinement (Garzón et al., 2017), methods such as electrochemical precipitation (Li et al., 2022), photoelectrocatalytic oxidation (Wei et al., 2022), using quaternary polyethylenimine - cellulose fibers (Lin et al., 2022), combined methods of electrocoagulation, adsorption and wetlands (Pratiwi et al., 2021) and natural adsorbents (up to 89 %) (Sain et al., 2021). The forms of environmentally friendly technologies are bioremediation using microorganisms, fungi, plants enzymes produced by them (Muñoz and Guillén, 2015). Bioremediation of water using microorganisms is a method that is being investigated more frequently in order to find the best efficiency of their metabolization to degrade pollutants such as cyanide. This biological process carried out by certain microorganisms with the capacity to use cyanide to obtain carbon or nitrogen results in a less toxic by-product (Restrepo et al., 2006). For cyanide-contaminated wastewater contaminated with cyanide have used as bioremediation medium Pseudomonas pseudoalcaligenes which is able to grow in cyanide medium as the only source of nitrogen, they synthesize by metabolism the enzymes nitrilases and oxidases that provide the carbon to transform cyanide, at alkaline pH, it was possible to biodegrade up to 90 % in 8 days (Carmona, 2016); something similar was done with the biological detoxification of water from a reactor, with the same bacterial strain Pseudomonas pseudoalcaligenes in alkaline medium, controlling the pH (at 10) to avoid volatilizing hydrogen cyanide (HCN) 151 and dissolved oxygen (10%) decreasing cyanide by 2. 8 mg CN/L (Huertas et al., 2010). In gold mining waste with in vitro assays and using the bacteria pseudomonas fluorescens, pseudomonas alcaligenes, acinetobacter, enterobacter and Bacillus, reduced cyanide up to 2.8 mg CN/L (Huertas et al., 2010). Bacillus, reduced up to 96 % of cyanide in 9 days (Cardona, 2015). Another bacterium used was Chromobacterium violaceum together with activated sludge that reduced from 50 mg/L cyanide to the amount of 7.5 mg/L (Shin et al., 2019); in a study using a biofilm-forming bacterium to act a degrading bacterium Rhodococcus rhodochrous BX2 reduced organic cyanide from groundwater (An et al., 2018). In other cases, microorganisms in the form of cells reduce pollution and can also be a source of energy (Sawasdee V. and Pisutpaisal N., 2018). In another research Pb was reduced with a consortium of microbial (Peens J. et al., 2018). Bioremediation provides a solution to the problem of contamination by chemical compounds in water and soil, being a viable and promising alternative (Garzón et al., 2017); therefore, this study provides new basic knowledge about this biotechnological treatment using a microbial biofilm with microorganisms for remediation of cyanide contaminated wastewater, verifying the feasibility and efficiency of the method to recover cyanide wastewater, avoiding negative impacts on the environment. 2. Methodology The experimental process of the research followed the following phases: • Obtaining 60 liters of contaminated water sample from a gold processing plant in the town of Chala- Arequipa, following the normative protocol RS 004-94-EM-DGAA and subsequent evaluation to determine the level of cyanide compared with the Maximum Permissible Limits stipulated in the DS. 010-2010-MINAN. • Isolation of microorganisms from the water sample in the laboratory, which were subjected to adaptation and purification in high cyanide medium. • Identification of Pseudomona aeruginosa and Escherichia coli microorganisms by biochemical Gram stain test. Figure 1: Microorganisms identified, Pseudomona aeruginosa y Escherichia coli Figure 2: Biofilm supports • Design and construction of 0.30 m long, 0.25 m wide and 0.35 m deep bioreactor cells (Figure 3). Figure 3: Bioreactor in operation • Preparation of supports for the microbial biofilm of polypropylene and polyurethane material in cubic form of 1 cm side (Figure 2). • Treatment of the cyanide contaminated water samples in the bioreactor cells coded and numbered as indicated in the matrix presented in Table 1. • Finally, the characterization of the treated water samples was carried out. 152 Table 1: Treatment factor matrix Type of microorganism for the biofilm Amount of inoculum (mg/L) Treatment time (days) Amount of carriers (units) Repeat code Pseudomona aeruginosa 1500 5 100 R1 (3 repetitions in reactor 1) 6 100 R2 (3 repetitions in reactor 2) 7 100 R3 (3 repetitions in reactor 3) Escherichia coli 1500 5 100 R1 (3 repetitions in reactor 1) 6 100 R2 (3 repetitions in reactor 2) 7 100 R3 (3 repetitions in reactor 3) 3. Resulted and discussion 3.1 Initial characteristics of the water sample for treatment Table 2 shows that the industrial wastewater sample contained 1,352 mg/L of cyanide; this value exceeds the maximum permissible limit (MPL) for discharges of liquid effluents from mining-metallurgical activities, established by Peruvian regulations (MINAM, 2010), whose values are 1 mg/L at any time and 0.8 mg/L for the annual average. Likewise, the pH exceeds the MPL, which establishes that it must be between 6 and 9, as well as total suspended solids, which is set at 50 mg/L (limit at any given time). Table 2: Initial characteristics of contaminated water Parameters Valour units Total cyanide 1,352 mg/L pH 13.4 pH units Temperature 24.5 °C DBO 70 mg/L Dissolved Oxygen 1 mg/L Electrical conductivity 60 µS/cm Total suspended solids 70 mg/L Turbidity 90 NTU 3.2 Kinetics of biofilm inoculum growth with Pseudomonas aeruginosa and Escherichia coli The growth of the biofilm of the bacteria Pseudomonas aeruginosa and Escherichia coli was verified as shown in Table 3, observing an increase in the volume of the inoculum due to the kinetics of growth over time due to the generation or reproduction of the bacteria. bacteria, having favourable temperature and pH for it (Ramírez et al., 2006) Table 3: Initial characteristic of contaminated water Biofilm Time (h) 0 1 2 3 4 5 6 7 8 9 N° of Pseudomonas aeruginosa (CFU/mg/L) 0.001 0.016 0.024 0.033 0.039 0.043 0.048 0.039 0.055 0.059 N° of Escherichia coli (CFU/mg/L) 0.001 0.019 0.03 0.038 0.043 0.048 0.038 0.039 0.048 0.069 3.3 Results of growth kinetics in the treatments With the result of the verification of bacterial growth, the contaminated water samples were treated. Figure 4 shows the growth kinetics of the Pseudomonas aeruginosa biofilm in three bioreactors, it is observed that there is an increase in the bacterial population due to the favourable conditions presented such as alkaline pH (Carmona, 2016), the temperature also favoured bacterial growth although the most optimal is around 35 °C (PAHO/WHO, 2015). 153 Figure 4: Growth kinetics of Pseudomas aeruginosa biofilm. Figure 5 shows the growth kinetics for the Escherichia coli biofilm, tested in three bioreactors, also verifying the adaptability and growth of the bacteria. Figure 4: Kinetics of Escherichia coli biofilm growth Table 4 shows the quantification of the biofilm population of Pseudomona aeruginosa and Escherichia coli at the beginning and end of the treatment of the cyanide-contaminated water samples. Similarly, Table 5 shows the quantification for Escherichia coli. In both cases, the increase in the microbial population was determined, which will intervene in the reduction of the cyanide present in the water samples. Table 4: Initial and final population of Pseudomona aeruginosa in bioreactors during treatment Reactor Initial Population (CFU/mL) Final Population (CFU/mL) R1 550 x 106 1.48 x 1012 R2 385 x 106 1.44 x 1012 R3 522.5 x106 1.5 x 1012 Table 5: Initial and final population of Escherichia coli microorganisms in the bioreactors during treatment Reactor Initial Population (CFU/mL) Final (CFU/mL) R1 6.05 x 1010 1.65 x 1013 R2 4.29 x 1010 1.83 x 1013 R3 3.465 x1010 1.72 x 1013 3.4 Characteristics of contaminated water samples after treatment Figure 6 shows the reduction of cyanide in water after treatment with a biofilm of Pseudomonas aeruginosa microorganisms. It is observed that after 5 days cyanide was reduced from 1,352 mg/L to 38.98 mg/L (97.11%) 0.005 0.045 0.048 0.062 0.072 0.109 0.011 0.035 0.066 0.075 0.08 0.096 0.117 0.015 0.04 0.059 0.058 0.065 0.066 0.07 0.11 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0 20 40 60 80 100 120 140 160 180 CF U / (m g/ L) Time (h) Bioreactor - 1 Bioreactor- 2 Bioreactor - 3 0.009 0.035 0.046 0.06 0.073 0.125 0.031 0.049 0.05 0.054 0.067 0.078 0.109 0.007 0.039 0.045 0.07 0.064 0.071 0.083 0.134 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 20 40 60 80 100 120 140 160 180 CF U /m g/ L Time (days) Bioreator - 1 Bioreator - 2 Bioreator - 3 154 in reactor R1, while in the other reactors R2 and R3 the reduction percentage was lower.This possibly happens due to the operating conditions, mainly due to the activity of the microbial population as established by the scientific literature, which indicates that it can reach between 90% and 96% (Carmona, 2016) and Cardona, 2015). Using immobilized Psudomona fluorescens, cyanide was reduced by 99.12% in 48 hours with a degradation of 1.0445 mg/L-h (Naranjo and Manuel, 2006).Figure 7 shows the reduction of cyanide in water after biofilm treatment of Escherichia coli microorganisms. It is established that after 6 days the cyanide was reduced from 1,352 mg/L to 325.1 mg/L (75.95%) in the R2 reactor, while in the other R1 and R3 reactors the reduction percentage was lower. This biofilm was less efficient than the Pseudomonas aeruginosa biofilm in reducing cyanide; however, these reductions do not reach the level of the maximum permissible limits of the Peruvian standard, which is 1 mg/L (MINAM, 2010), taking into account that cyanide is lethal with an intake of 2-5 mg/kg of body weight (Agudelo C. et al., 2010) Figure 6: Reduction of cyanide with Pseudomona aeruginosa biofilm Figure 7: Cyanide reduction with Escherichia coli biofilm The physicochemical properties of the wastewater after being treated with the biofilms are presented in Table 6, the pH decreased due to the acids generated by the bacteria but remains in the optimal range for bacterial population growth. It was found that the pH and total suspended solids in the wastewater were within the maximum permissible limits to be disposed of in the receiving body, in accordance with Peruvian environmental regulations (MINAM, 2010). The pH is very important in the recovery of aqueous cyanide compounds since at pH greater than 10.2 more than 90% of the total cyanide is as CN- ion and its recovery is more feasible, when the pH is less than 8.4 it is as HCN with a high volatility due to its high vapor pressure (100 kPa at 26°C), causing a decrease in the concentration of cyanide in the solution (Pérez and Higuera, 2008); consequently, this could have been reflected in the result of the investigation, since in the reactor with Psudomona aeruginosa the pH was 10.77. Table 6: physicochemical characteristics of treated water Treatment pH Temperature(°C) Electrical conductivity (µS/cm) DBO5 (mg/L) Dissolved Oxygen (mg/L) Turbidity (NTU) Total Suspended Solids (mg/L) No treatment 13.4 24.5 60 70 1 90 70 With Pseudomonas aeruginosa 10.77 25.97 10.63 10.10 5.76 7.46 10.79 With Escherichia coli 8.70 23.10 51.13 38.20 6.70 7.13 13.43 4. Conclusion With microbial biofilms of Pseudomonas aeruginosa and Escherichia coli, supported on polypropylene and polyurethane material, the concentration of cyanide in contaminated industrial wastewater was reduced by 97.11% and 75.95% for each biofilm in 5 and 7 days of treatment ; Therefore, biotechnology with microorganisms is an environmentally advantageous alternative to other methods that use chemicals and more research is needed to scale the methodology to larger volumes of effluents of this type of pollutant, which is very dangerous, lethal to life and the environment. 1,352 38.98 316.00 320.00 0 200 400 600 800 1,000 1,200 1,400 1,600 Initial (0 days) R1 5 days R2 6 days R3 (7 days) Cy an id e (m g/ L) Tests 1,352 418.8 325.1 285 0 200 400 600 800 1,000 1,200 1,400 1,600 Initial (0 days) R1 5 days R2 6 days R3 (7 days) Cy an id e (m g/ L) Tests 155 Acknowledgments To Universidad César Vallejo, program “Research UCV”, for the academic and financial support for the dissemination of this research. References Agudelo C, R.M., Betancur U, J., Jaramillo C, C.L., 2010. Biotratamiento de residuos cianurados y su relación con la salud pública. Revista Facultad Nacional de Salud Pública 28, 7–20. An X., Cheng Y., Huang M., Sun Y., Wang H., Chen X., Wang J., Li D., Li C., 2018. Treating organic cyanide- containing groundwater by immobilization of a nitrile-degrading bacterium with a biofilm-forming bacterium using fluidized bed reactors. Environmental Pollution 237, 908–916. https://doi.org/10.1016/j.envpol.2018.01.087 Cardona E., 2015. Microorganismos Potenciales degradadores de Cianuro en residuos de minería de oro (Tesis de Título). Universidad de Colombia, Medellin. Carmona M.I., 2016. Estudio del papel de los genes cio en la resistencia al cianuro de “Pseudomonas pseudoalcaligenes” CECT5344. Universidad de Extremadura, Extremadura, España. Garzón J.M., Rodríguez Miranda J.P., Hernández Gómez C., 2017. Aporte de la biorremediación para solucionar problemas de contaminación y su relación con el desarrollo sostenible. Rev. Univ. salud 19, 309. https://doi.org/10.22267/rus.171902.93 Huertas M.J., Sáez L.P., Roldán M.D., Luque-Almagro V.M., Martínez-Luque M., Blasco R., Castillo F., Moreno- Vivián C., García-García I., 2010. Alkaline cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Influence of pH. Journal of Hazardous Materials 179, 72–78. https://doi.org/10.1016/j.jhazmat.2010.02.059 Li M., Li B., Chen J., Cui S., Yang Y., Shen X., Liu K., Han Q., 2022. Purifying cyanide-bearing wastewaters by electrochemical precipitate process using sacrificial Zn anode. Separation and Purification Technology 284. https://doi.org/10.1016/j.seppur.2021.120250 Lin X., Tran D.T., Song M.-H., Yun Y.S., 2022. Development of quaternized polyethylenimine-cellulose fibers for fast recovery of Au(CN)2- in alkaline wastewater: Kinetics, isotherm, and thermodynamic study. Journal of Hazardous Materials 422. https://doi.org/10.1016/j.jhazmat.2021.126940 MINAM, 2010. D.S. 010-2010-Minam: Apreuban Límites Máximos Permisibles para la descarga de efluentes líquidos de actividad Minero - Metalúrgicas. Naranjo F.J., Samta-María M., 2006. Evaluación de la inmovilización de Pseudomonas fluorescens para la biodegradación de cianuro en solución acuosa 109. Muñoz A., Guillén G., 2015. Sistema de Biorremediación Para La Regeneración De Suelos Hidromórficos Del Estero Chicharrón Y Río Cucaracha De La Comuna De Montañita, Provincia De Santa Elena. (Tesis de Título). Politécnica del Litoral, Guayaquil. OPS/OMS, 2015. Peligros biológicos [WWW Document]. Pan American Health Organization / World Health Organization. Peens J., Wu Y., Brink H., 2018. Microbial pb(ii) precipitation: the influence of elevated pb(ii) concentrations. Chemical Engineering Transactions 64, 583–588. https://doi.org/10.3303/CET1864098 Pérez, J., Higuera, O., 2008. Comportamiento electroquímico del cianuro. Ingeniería y Desarrollo 24 (14).. Pratiwi I., Mukimin A., Zen N., Septarina I., 2021. Integration of electrocoagulation, adsorption and wetland technology for jewelry industry wastewater treatment. Separation and Purification Technology 279. https://doi.org/10.1016/j.seppur.2021.119690 Ramírez N., Serrano J., Sandoval H., 2006. Microorganismos extremófilos. Actinomicetos halófilos en México. Revista Mexicana de las ciencias farmacéuticas 37, 17. Restrepo O.J., Montoya C.A., Muñoz N.A., 2006. Degradación microbiana de cianuro procedente de plantas de beneficio de oro mediante una cepa nativa de p. flurrecens. DYNA 73, 45–51. Sain M., Pramanik S., Baltrenaite-Gediene E., Chandra Ghanta K., Dutta, S., 2021. Abatement of cyanide and ammoniacal-N from coke-oven wastewater using natural adsorbents: A comparative study. Journal of the Indian Chemical Society 98. https://doi.org/10.1016/j.jics.2021.100230 Sawasdee V., Pisutpaisal N., 2018. Microbial community from tannery wastewater in microbial fuel cell. Chemical Engineering Transactions 64, 397–402. https://doi.org/10.3303/CET1864067 Shin D., Park H., Lee J.C., Kim M., Lee J., 2019. Microbial community responses to cyanide in a biological treatment reactor for cyanide containing wastewater from gold processing plant. Presented at the IMPC 2018 - 29th International Mineral Processing Congress, pp. 3222–3230. Wei P., Zhang Y., Huang Y., Jia Y., Chen L., Wang M., 2022. Effect and mechanism of cyanide degradation and Cu/Zn recovery by photoelectro-catalytic oxidation. Separation and Purification Technology 282. https://doi.org/10.1016/j.seppur.2021.120050 156 49mayorcaclemente.pdf Microbial Biofilm as a Methodology for Treatment of Cyanide-Contaminated Water