Determination of the efficiency of moving bed biofilm reactors for the treatment of cianurated waters Fernando Antonio Chaves Vallejos Químico, estudiante de Maestría Director: Julio Andrés Cardona, Magister en Química Codirector: Luz Adriana Betancur Jaramillo, Doctora en Ciencias Químicas Programa de maestría en Química Universidad de Caldas Facultad de Ciencias Exactas y Naturales Manizales Caldas, June 2020

1. Problem statement and justification 2. Conceptual framework 3. Background 4. Research question 5. Objectives 6. Methodology 7. Abstract 8. References C ontent

3 U.S. Geological Survey, Mineral commodity summaries 2019: U.S. Geological Survey, 200 p., (2019). available: C olombia exported 36.6 tons of gold in Problem statement and justification

3 Department Gold (Oz Troy) Antioquia Chocó Bolívar Nariño85787 Cauca78557 Caldas77803 Tolima26050 Córdoba21957 Risaralda6592 Huila5605 Santander Problem statement and justification I n Caldas the production of gold focuses on Marmato U.S. Geological Survey, Mineral commodity summaries 2019: U.S. Geological Survey, 200 p., (2019). available:

Mining Legal Illegal 4 1. Problem statement and justification M ining produces effluents with high concentrations of cyanide compounds Lara-Rodríguez, J. S. How institutions foster the informal side of the economy: Gold and platinum mining in Chocó, Colombia. Resour. Policy (2020)

5 Jaszczak, E., Polkowska, Ż., Narkowicz, S., & Namieśnik, J. Cyanides in the environment—analysis—problems and challenges. Environmental Science and Pollution Research, 24(19), 15929– (2017). C yanide affects cytochrome c causing cell death Cyanide inhibition 1. Problem statement and justification

T here are several methods to decrease the concentration of cyanide compounds in mining effluents 6 Cyanide treatment methods Alkaline chlorination Hydrogen peroxide Biological oxidation Phytoremediation Microorganisms Activated carbon Catalytic oxidation Kuyucak, N. & Akcil, A. Cyanide and removal options from effluents in gold mining and metallurgical processes. Minerals Engineering 50–51, 13–29 (2013). Mosa A., Saadoun l, Kumar K.,, Dhankher O. Potential Biotechnological Strategies for the Cleanup of Heavy Metals and Metalloids. Frontiers in Plant Science (2016). absorption volatilization 1. Problem statement and justification

2. Conceptual framework 7 1. Yazici, E. Y., Ahlatci, F., Yilmaz, E., Celep, O. & Deveci, H. Precipitation of zinc from cyanide leach solutions using Trimercapto-s-triazine (TMT). Hydrometallurgy 191, (2020). Extraction Trituration Restoration Leaching Debris Treatment Foundry Final product Extractiasofhashfio sahfoashfioashfaifh asifhiafhiiahisahfio ashfon Gold recovery A n extraction and leaching process is necessary to obtain the gold Gold mining

8 Razanamahandry, L. C. et al. Performance of various cyanide degrading bacteria on the biodegradation of free cyanide in water. J. Hazard. Mater. 380, (2019). A bioreactor is a biologically active system where chemical reactions occur 2. Conceptual framework General bioreactor scheme

9 Sustrat e B atch reactors are used for preliminary testing Works in batches Constant nutrients Suspended biomass Easy to do Control variables Low cost 2. Conceptual framework Characteristics batch reactor Burneo, B. S., Juárez, A. S. & Nieto-Monteros, D. A. Un-steady state modeling for free cyanide removal and biofilm growth in a batch process. J. Hazard. Mater. 388, (2020).

10 Sinnott, R. & Towler, G. Chapter 15 - Design of Reactors and Mixers. in Chemical Engineering Series (eds. Sinnott, R. & Towler, G. B. T.-C. E. D. (Sixth E.) 1039–1146 (Butterworth-Heinemann, 2020). M icroorganisms inhabit the bed and the reaction occurs here Fixed bed More expensive Little versatility Multi-stage Fixed biomass Works continuously 2. Conceptual framework Fixed bed bioreactors

11 Di Biase, A., Kowalski, M. S., Devlin, T. R., & Oleszkiewicz, J. A. Moving bed biofilm reactor technology in municipal wastewater treatment: A review. Journal of Environmental Management, 247, 849–866. (2019). 2. Conceptual framework I n a moving bed biofilm reactor the biomass is added to a support making the reaction more effective MBBR general scheme Influent Support Aeration system Enfluent Biomass Support tank

11 Di Biase, A., Kowalski, M. S., Devlin, T. R., & Oleszkiewicz, J. A. Moving bed biofilm reactor technology in municipal wastewater treatment: A review. Journal of Environmental Management, 247, 849–866.(2019). 2. Conceptual framework I n a moving bed biofilm reactor the biomass is added to a support making the reaction more effective MBBR general scheme Support Aeration system Biomass Support tank

12 Calero Díaz, G. Biorreactores de lecho móvil y de membrana para el tratamiento de aguas residuales a bajo tiempo de retención hidráulico: estudio de la influencia en la eliminación de contaminantes. (2019). 2. Conceptual framework E ffluents reach the tank and the aeration system keeps them moving Possible configuration of the tank and aeration system Aeration system Support tank Sensors

12 Calero Díaz, G. Biorreactores de lecho móvil y de membrana para el tratamiento de aguas residuales a bajo tiempo de retención hidráulico: estudio de la influencia en la eliminación de contaminantes. (2019). 2. Conceptual framework E ffluents reach the tank and the aeration system keeps them moving Possible configuration of the tank and aeration system Aeration system Support tank

13 Liu, J. et al. Performance evaluation of a lab-scale moving bed biofilm reactor (MBBR) using polyethylene as support material in the treatment of wastewater contaminated with terephthalic acid. Chemosphere 227, 117–123 (2019). 2. Conceptual framework M icroorganisms are added to the supports, forming a biofilm Made with polyurethane Hgh area-surface ratio Density close to water Have channels Can be ceramic Supports types

17 Microorganisms used Mushrooms Trichoderma harzianum, Fusarium solani, Trichoderma polysporum Bacteria Pseudomonas sp, Serratia spp, Bacillus magaterium They have specific enzymes Dash, R. R., Gaur, A. & Balomajumder, C. Cyanide in industrial wastewaters and its removal: A review on biotreatment. J. Hazard. Mater. 163, 1–11 (2009) Conceptual framework

15 Chen, G. et al. Biofilm as a live and in-situ formed membrane for solids separation in bioreactors: Biofilm succession governs resistance variation demonstrated during the start-up period. J. Memb. Sci. 608, (2020). 2. Conceptual framework I n the biofilm the transformation of the substrate into less toxic products occurs Biofilm formed Perform bioconversion Grow in support Formed biofilm Are specific Low cost Constant movement

16 Singh, U., Arora, N. K. & Sachan, P. Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri. Brazilian J. Microbiol. 49, 38–44 (2018).. 2. Conceptual framework P seudomonas synthesize an exopolysaccharide that facilitates cell adhesion and biofilm formation Pseudomonas Biofilm Bacilli Exopolysaccharide Electron micrograph

17 Web of science 764 results Scopus 1021 results S earch carried out on June 16 using the keywords moving bed biofilm reactor, showed the following results 3. Background

T he word cyanide was added to the previous search showed the following results, adding the word Pseudomona no results were found 17 Web of science 5 results Scopus 8 results 3. Background

18 Mekuto, L., Ntwampe, S. K. O., Utomi, C. E., Mobo, M., Performance of a continuously stirred tank bioreactor system connected in series for the biodegradation of thiocyanate and free cyanide. Journal of Environmental Chemical Engineering, 5(2), 1936–1945. (2017). 3. Background P erformance of a continuously stirred tank bioreactor system connected in series for the biodegradation of thiocyanate and free cyanide Reactor scheme and results

19 Cabello, P., Luque, V. M., Olaya, A., Sáez, L. P., Moreno-Vivián, C., & Dolores Roldán, Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: From omic approaches to biotechnological applications. FEMS Microbiology Letters, 365(6), 1–7. M. (2018). 3. Background A ssimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344 Nitrilase Cyanase PII–type nitrogen regulator Alkyl–hydroperoxide reductase Dihydropicolinate synthase

20 3. Background M oving bed biofilm reactor to treat wastewater Leyva-Díaz, J.C., Martín-Pascual, J. & Poyatos, J.M. Moving bed biofilm reactor to treat wastewater. Int. J. Environ. Sci. Technol. 14, 881–910 (2017) Scheme and variables of a bioreactor Q = Volumetric flow S = Substrate X = Biomass

21 Knowledge discovered Microorganisms can degrade cyanide substances Pseudomonas can tolerate cyanide compounds MBBR reactor designs exist The behavior of polyurethane supports is known Gaps in knowledge Here are no reports of the use of pseudomonas in an MBBR reactor Little information on the ceramic supports used in MBBR reactors The behavior of pseudomonas in ceramic supports is unknown In Colombia the MBBR technology for the treatment of mining effluents is null ¿What is the efficiency of moving bed biofilm reactors designed in this investigation, for the degradation of cyanide compounds present in synthetic water? R esearch question Determination of the efficiency of moving bed biofilm reactors for the treatment of cianurated waters

22 Establish chromatographic parameters for the analysis of cyanidated species and their degradation products Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions Determination of the efficiency of moving bed biofilm reactors for the treatment of cianurated waters Determine the efficiency of moving bed biofilm reactor for the treatment of cyanide waters, using Pseudomona putida Assess degradation of cyanide species in a moving bed biofilm reactor 5. Objectives G eneral objective S pecific objectives

23 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products 6. Methodology Ion chromatography analysis Simulated conditions Eluent isocratic, 27 mM de KOH Flow 1 mL/min Runing time 20 min Temperature 30°C Optimization Mix with 30 ppm concentration of NO 3 -, NO 2 -, SO 4 -2, CN -, SCN - Ivanova, V., Surleva, A., & Koleva, B. Validation of Ion Chromatographic Method for Determination of Standard Inorganic Anions in Treated and Untreated Drinking Water. IOP Conference Series: Materials Science and Engineering, 374(1). (2018). E xperimental design for optimization Dionex ICS 5000

6. Methodology From the solid reagents Prepare stock solutions 1000 ppm Make 30 ppm dilutions of nitrate, nitrite, sulfate, thiocyanate and cyanide ions Analyze on the ion chromatograph 24 E xperimental design for optimization Ivanova, V., Surleva, A., & Koleva, B. Validation of Ion Chromatographic Method for Determination of Standard Inorganic Anions in Treated and Untreated Drinking Water. IOP Conference Series: Materials Science and Engineering, 374(1). (2018). 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products

6. Methodology 25 NameSymbolAnalyseObjective Capacity factorKHalfObjective reached ResolutionRHalfObjective reached SelectivityaHalfMaximize EfficiencyNHalfMaximize Response variable E xperimental design for optimization NameUnitsTipePapelLowHigh A:FlowmL/minContinuouosControllable B:EluentmMContinuouosControllable C:Temperature°CContinuouosControllable Controllable factors Type of factorsType of design Central pointsDesign is Randomized? Number of Replications Total Executions Process, main effects Factorial 2^3 2, RandomYes230 Design 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products

26 6. Methodology D etermination of some validation parameters Linearity Experimental process Prepare solutions of each ion in a concentration range of 10 to 100 ppm and analyze in triplicate Statistical process Test f Confidence limits Detection limit Quantification limit Sensitivity Živojinović, D. Z., & Rajaković, L. V. Application and validation of ion chromatography for the analysis of power plants water: Analysis of corrosive anions in conditioned water–steam cycles. Desalination, 275(1), 17–25. (2011). 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products

27 6. Methodology Precision Instrumental repeatability Standard solutions of 20, 50 and 80 ppm, perform 5 repetitions Intermediate precision On different days inject standard solutions of 20, 50 and 80 ppm, perform 5 repetitions Živojinović, D. Z., & Rajaković, L. V. Application and validation of ion chromatography for the analysis of power plants water: Analysis of corrosive anions in conditioned water–steam cycles. Desalination, 275(1), 17–25. (2011). 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products Determine mean, standard deviation, coefficient of variation. D etermination of some validation parameters

28 6. Methodology Accuracy Analysis of solutions known concentration Prepare solutions 10.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 70.0 ppm inject 5 repetitions Adding standard to a known sample To a 20 ppm synthetic water sample add standard of 50 ppm and perform 5 repetitions Adding standard to a problem sample To a sample of mining effluent water, add a standard of 50 ppm and perform 5 repetitions Živojinović, D. Z., & Rajaković, L. V. Application and validation of ion chromatography for the analysis of power plants water: Analysis of corrosive anions in conditioned water–steam cycles. Desalination, 275(1), 17–25. (2011). 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products Error percentage Recovery percentage D etermination of some validation parameters

29 6. Methodology S ome preliminary chromatograms Flow: 0.5 mL/min Eluent: 22mM Fluoride Chloride Nitrite Sulfat e Bromide Nitrat e Phosphate Thiocyanate

30 6. Methodology S ome preliminary chromatograms Fluoride Chloride Nitrite Sulfat e Bromide Nitrat e Phosphate Thiocyanate Flow: 1 mL/min Eluent: 22mM

31 6. Methodology S ome preliminary chromatograms Fluoride Chloride Sulfat e Bromide Nitrat e Phosphate Thiocyanate Flow: 1 mL/min Eluent: 27mM

32 6. Methodology C alibration preliminary curve for thiocyante DataValue Slope0.096 Standard deviation of the slope0.001 Determination coefficient R intercept1.229 uS Standard deviation of the intercept0.046 Calibration curve deviation0.044 LOQ ppm LOD6.278 ppm 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products

Reactivation of microorganisms Preparation Rehydration of vials 1.5 mL BHI broth for thirty minutes Espitia and Díaz scheme 0.5 ml of the suspension in 5 ml of BHI broth incubate at 37 ° C for 24 h Incubation Peals of the pure culture on BHI agar Incubate at 37°C for 24h Characterize microscopically and macroscopically Characterizatio n Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

Reactivation of microorganisms Preparation Rehydration of vials 1.5 mL BHI broth for thirty minutes Espitia and Díaz scheme 0.5 mL of the suspension in 5.0 mL of BHI broth and incubate at 37 ° C for 24 h Incubation Peals of the pure culture on BHI agar Incubate at 37°C for 24h Characterize microscopically and macroscopically Characterizatio n Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

Reactivation of microorganisms Preparation Rehydration of vials 1.5 mL BHI broth for thirty minutes Espitia and Díaz scheme 0.5 mL of the suspension in 5.0 mL of BHI broth and incubate at 37 ° C for 24 h Incubation Peals of the pure culture on BHI and cyanide agar Incubate at 37 °C for 24 h Characterize microscopically and macroscopically Characterizatio n Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

Pseudomonas spp, «Pathogen Safety data sheet-Infectious Substances», Public Health Agency of Canada, available in Microscopic Macroscopic Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

From concentrated solution Conservation Shake at 200 rpm Adjust to optical density Kirsop protocol Take 2 ml vials Add to each 15 discs of Whatman No. 4, 5 mm filter paper From BHI broth Take 0.1ml Add to the vials and homogenize Store at 4°C Storage Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

From concentrated solution Conservation Shake at 200 rpm Adjust to optical density Kirsop protocol Take 2 mL vials Add to each 15 discs of Whatman No. 4, 5 mm filter paper From BHI broth Take 0.1ml Add to the vials and homogenize Store at 4°C Storage Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

From concentrated solution Conservation Shake at 200 rpm Adjust to optical density Kirsop protocol Take 2 mL vials Add to each 15 discs of Whatman No. 4, 5 mm filter paper From BHI broth Take 0.1 mL Add to the vials and homogenize Store at 4 °C Storage Parra Huertas, S. L., Pérez Casas, M. M., Bernal Morales, M., Suárez Moreno, Z. & Montoya Castaño, D. Implementación y evaluación de dos métodos deconservación y generación de la base de datos del banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). Nova 4, 39 (2006) Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions

NameUnitsAnalyseObjetive Degradation%HalfMaximize NameUnitsTipePapelLowHigh A:pH ContinuouosControllable B:Substratemg/LContinuouosControllable C:TimehContinuouosControllable Type of factorsType of design Central pointsDesign is Randomized? Number of Replications Total Executions Process, main effects Factorial 2^3 2, RandomYes Response variable Controllable factors Experimental design 6. Methodology 2. Assess the effect of Pseudomona putida on the degradation of cyanidated species under in vitro conditions D etermination of degradation conditions of thiocyanate and cyanide in vitro

J. P. McQuarrie and J. P. Boltz, “Moving Bed Biofilm Reactor Technology: Process Applications, Design, and Performance,” Water Environ. Res., vol. 83, no. 6, pp. 560–575, (2011) Methodology 3. Assess degradation of cyanide species in a moving bed biofilm D esign, start-up and optimization of the bioreactor

38 6. Methodology 3. Assess degradation of cyanide species in a moving bed biofilm V= 4 L Acrylic Cylindrical 12w pump Synthetic water NutrientsConcentration (g/L) NaH 2 PO 4· 2H 2 O K 2 HPO 4 0,400 MgSO 4 ·7H 2 O MnCl 2 ·4H 2 O CaCl 2 ·2H 2 O0.500 ZnCl CoCl 2 ·6H 2 O1.200 NiCl 2 ·6H 2 O1.200 FeCl Cyanide compounds Li, C., Liang, J., Lin, X., Xu, H., Tadda, M. A., Lan, L., & Liu, D. Fast start-up strategies of MBBR for mariculture wastewater treatment. Journal of Environmental Management, 248, (2019). R eactor design and effluent preparation

J. P. McQuarrie and J. P. Boltz, “Moving Bed Biofilm Reactor Technology: Process Applications, Design, and Performance,” Water Environ. Res., vol. 83, no. 6, pp. 560–575, (2011) Methodology 3. Assess degradation of cyanide species in a moving bed biofilm Selec carrier Add to the broth Make biofilm Quantify biomass S upport configuration and biofilm formation

J. P. McQuarrie and J. P. Boltz, “Moving Bed Biofilm Reactor Technology: Process Applications, Design, and Performance,” Water Environ. Res., vol. 83, no. 6, pp. 560–575, (2011) Methodology 3. Assess degradation of cyanide species in a moving bed biofilm 99 mL 9 mL99 mL 9 mL 1 mL Dilution 1: Dilution 1:10000 Dilution 1: Dilution 1:100 S upport configuration and biofilm formation Many colonies Isolated colonies

41 6. Methodology 3. Assess degradation of cyanide species in a moving bed biofilm Li, C., Liang, J., Lin, X., Xu, H., Tadda, M. A., Lan, L., & Liu, D. Fast start-up strategies of MBBR for mariculture wastewater treatment. Journal of Environmental Management, 248, (2019). R eactor starup and monitoring

42 Response variable Controllable factors NameUnitsTipePapelLowHigh A: Support type CategoricalControllablePolyure -thane ceramic B: TRHhContinuouosControllable648 C: FR%ContinuouosControllable630 Experimental design Type of factorsType of design Central pointsDesign is Randomized? Number of Replications Total Executions Process, main effects Factorial 2^3 2, RandomYes Methodology NameUnitsAnalyseObjetive Degradation%HalfMaximize D etermination of degradation conditions in the reactor

43 Abstract The leaching process for gold extraction releases effluents with a high load of cyanide compounds that are toxic and degrade ecosystems, various treatments for wastewater of mining origin exist, but are expensive and generators of harmful by-products, as an alternative there are reactors of biofilm, that are economical, specific and generate non-toxic waste products. In this project bioreactors will be designed and optimized for the preliminary treatment of synthetic water, physicochemical parameters will also be determined to assess the viability of the reactors and propose their use in systems real. Douglas Gould, W. et al. A critical review on destruction of thiocyanate in mining effluents. Minerals Engineering 34, 38–47 (2012). Sotomayor Burneo, B., Sánchez Juárez, A. & Nieto Monteros, D. A. Un-steady state modeling for free cyanide removal and biofilm growth in a RBC batch process. J. Hazard. Mater. (2019).

References CORPOCALDAS. Plan de acción institucional , (2016). Guamán Guadalima, M. P. & Nieto Monteros, D. A. Evaluation of the rotational speed and carbon source on the biological removal of free cyanide present on gold mine wastewater, using a rotating biological contactor. J. Water Process Eng. 23, 84–90 (2018). Hendry-Hofer, T. B. et al. A Review on Ingested Cyanide: Risks, Clinical Presentation, Diagnostics, and Treatment Challenges. Journal of Medical Toxicology 15, 128–133 (2019). Douglas Gould, W. et al. A critical review on destruction of thiocyanate in mining effluents. Minerals Engineering 34, 38–47 (2012). Sotomayor Burneo, B., Sánchez Juárez, A. & Nieto Monteros, D. A. Un-steady state modeling for free cyanide removal and biofilm growth in a RBC batch process. J. Hazard. Mater. (2019). doi: /j.jhazmat Connolly, D., Barron, L. & Paull, B. Determination of urinary thiocyanate and nitrate using fast ion-interaction chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 767, 175–180 (2002). Demkowska, I., Polkowska, Z. & Namiesnik, J. Application of ion chromatography for the determination of inorganic ions, especially thiocyanates in human saliva samples as biomarkers of environmental tobacco smoke exposure. J. Chromatogr. B 875, 419–426 (2008). Ragusa, A. et al. HPLC Analysis of Phenols in Negroamaro and Primitivo Red Wines from Salento. Foods 8, 45 (2019). Gillio Meina, E., Raes, K. & Liber, K. Models for the acute and chronic aqueous toxicity of vanadium to Daphnia pulex under a range of surface water chemistry conditions. Ecotoxicol. Environ. Saf. 179, 301–309 (2019). Nava-Alonso, F., Elorza-Rodríguez, E., Uribe-Salas, A. & Pérez-Garibay, R. Análisis químico de cianuro en el proceso de cianuración revisión de los principales métodos. Rev. Metal. 43, 20–28 (2007). Dash, R. R., Gaur, A. & Balomajumder, C. Cyanide in industrial wastewaters and its removal: A review on biotreatment. J. Hazard. Mater. 163, 1– 11 (2009). Restrepo, O., Montoya, C. & Muñoz, N. Microbial degradation of cyanide from gold metallurgical plants utilizing P.fluorecens. DYNA 73, 45–51 (2006). Huertas, M. J. et al. Alkaline cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Influence of pH. J. Hazard. Mater. 179, 72–78 (2010). Salinas, E. et al. Mejora del proceso de cianuración de oro y plata, mediante la preoxidación de minerales sulfurosos con ozono. Rev. la Soc. Química México 48, 315–320 (2004). 44

Thank you 45

5. Methodology E xperimental design for optimization Experiment Flow (mL/min) Eluent (mM) Temperature (°C)KR αN 10, , , , , , , , , , , , , , , , , ,

5. Methodology E xperimental design for in vitro condition Experiment pH Substrate (ppm)Time (h) Degradation (%) 1 7, , , , , , , , , , , , , , , , , ,

5. Methodology E xperimental design for reactor Experiment Suppor TRH (h)FR (%) Degradation (%) 1 polyurethane66 2 ceramic486 3 polyurethane486 4 polyurethane ceramic ceramic630 8 polyurethane630 9 ceramic polyurethane polyurethane66 12 ceramic polyurethane polyurethane ceramic66 16 ceramic ceramic polyurethane ceramic polyurethane polyurethane66 22 ceramic polyurethane polyurethane ceramic66 26 ceramic ceramic polyurethane ceramic polyurethane2718

6. Methodology NameSymbolAnalyseObjective Capacity factorKHalfObjective reached ResolutionRHalfObjective reached SelectivityaHalfMaximize EfficiencyNHalfMaximize Response variable E xperimental design for sturdiness NameUnitsTipePapelLowHigh A:FlowmL/minContinuouosControllable X-0.1X+0.2 B:EluentmMContinuouosControllableX-1.0 X+1.0 C:Temperature°CContinuouosControllableX-1.0 X+1.0 Controllable factors Type of factorsType of design Central pointsDesign is Randomized? Number of Replications Total Executions Process, main effects Factorial 2^3 2, RandomYes212 Design 1. Establish chromatographic parameters for the analysis of cyanidated species and their degradation products.

RECUPERACIÓN: Corresponde a la diferencia (en porcentaje)entre la concentración med ida de un analito en una muestra fortificada (a la que se le ha agregado una cantidad conocida de estándar) y la concentración medida en la misma muestra sin fortificar, dividido por la concentración de sustancia agregada Analizar una muestra real y en paralelo analizar la misma muestra a la que se ha agregado una concentración dada de estándar. La concentración final debe estar dentro del rango de determinación del método. Calcular la recuperación de acuerdoa la siguiente expresión

Volumen del reactor 59

16 Perspective Design and optimize bioreactors for the treatment of cyanurated compounds Design bed Determine the best conditions for the development of the microorganism and biofilm formation Evaluate which cyanide compounds the microorganism can treat Compare the results and show them in scientific events

16 we can do it because the research group has the experience: Cardona, J., Taborda, O., (2016). Estudio de la degradación de fenol sobre catalizador de arcilla pilarizada. Revista Científica, 25, Jaramillo, M., Marin, Y., Ocampo, D., (2018). Effects on the photosynthetic level in three species of aquatic plants treated with waste water of mining origin. Boletin Cientifico Del Centro de Museos, 22(1), 43–57. Jaramillo, M., Buitrago, D., Galvis, J., (2016). Manejo demacrófitas acuáticas en la acumulacióny transformación de cianuroproducto del beneficio del oro en lamina la coqueta. Boletin Cientifico Del Centro de Museos, 20(1), 63–77. Perspective

Pseudomonas 14 Kang, M. H., & Park, J. M. (1997). Sequential Degradation of Phenol and Cyanide by a Commensal Interaction Between Two Microorganisms. Journal of Chemical Technology & Biotechnology, 69(2), 226–230 RESTREPO, O., MONTOYA, C., & MUÑOZ, N. (2006). MICROBIAL DEGRADATION OF CYANIDE FROM GOLD METALLURGICAL PLANTS UTILIZING P.fluorecens. Dyna, 73(149), 45–51. Pseudomona

Condiciones equipo Para el análisis de aniones y cationes se utilizó un cromatógrafo de intercambio iónico Thermo Scientific con una bomba dionex IC 5000, un generador de eluente dionex IC 5000, un automuestreador Dionex y un detector de conductividad dionex IC 5000 columna Dionex IonPac ® AS20 63

13 Cyanide metabolic pathways Neha Gupta, Chandrajit Balomajumder, V.K. Agarwal, Enzymatic mechanism and biochemistry for cyanide degradation: A review, Journal of Hazardous Materials, Volume 176, Issues 1–3, 2010, Pages 1-13 Douglas Gould, W., King, M., Mohapatra, B. R., Cameron, R. A., Kapoor, A., & Koren, D. W. (2012). A critical review on destruction of thiocyanate in mining effluents. Minerals Engineering, 34, 38–47. Nitriles:

13 Neha Gupta, Chandrajit Balomajumder, V.K. Agarwal, Enzymatic mechanism and biochemistry for cyanide degradation: A review, Journal of Hazardous Materials, Volume 176, Issues 1–3, 2010, Pages 1-13 Douglas Gould, W., King, M., Mohapatra, B. R., Cameron, R. A., Kapoor, A., & Koren, D. W. (2012). A critical review on destruction of thiocyanate in mining effluents. Minerals Engineering, 34, 38–47. Cyanide metabolic pathways Cyanide:Thiocyanate:

17 Cabello, P., Luque, V. M., Olaya, A., Sáez, L. P., Moreno-Vivián, C., & Dolores Roldán, M. (2018). Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: From omic approaches to biotechnological applications. FEMS Microbiology Letters, 365(6), 1–7. 4. Background A ssimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344

Balances reactor 67

Cyanide species in gold extraction processes Type of cyanideFeaturesExample Cyanide ionFree anionCN - Molecular cyanide Neutral molecule HCN Simple Cyanide CompoundsIonic compoundsNaCN Cyanide (WAD)Dissociates to pH 4.5Complexes of Cu, Cd, Ni, Zn, Ag Cyanide (SAD)dissociates to pH 2Complexes of Fe, Co, Au Cyanide derivativesCome from the transformations of cyanide compounds. SCN -, OCN - 6 Álvarez García, R. Aplicación de sistemas pasivos para el tratamiento de soluciones residuales de procesos de cianuración en minería de oro. TDR (Tesis Doctorales en Red) (2007). Jaszczak, E., Polkowska, Ż., Narkowicz, S., & Namieśnik, J. (2017). Cyanides in the environment—analysis—problems and challenges. Environmental Science and Pollution Research, 24(19), 15929–

Cyanide treatment methods Removal methodsAdvantagesDisadvantages Reaction Alkaline chlorinationTechnology well established. The cyanate is relatively less toxic and further oxidized to carbon, dioxide and nitrogen at lower pH Adds potentially objectionable cations/anions to water. Excess hypo-chlorite is toxic. Chlorine can react with organics to form chlorinated compounds. NaCN + Cl 2 CNCl + NaCl CNCl + 2NaOH NaCNO + NaCl + H 2 O 2NaCNO + 3NaOCl 2CO 2 + N 2 + 3NaCl + 2NaOH Hydrogen peroxideExcess reagent decomposes to water and oxygen Relatively simple to operate. Reagent costly Requires accurate measurement of chemical dose CN - + H 2 O 2 OCN - + H 2 O Activated carbonEffective method Used as a polishing process Cost is more Used only for low concentrations of cyanide Active site: CN- + * * -CN Catalytic oxidationEffective in presence of copper or other catalysts Not a full treatment process System TiO 2 /uv: CN - + 2OH OCN - + H 2 O 8 Kuyucak, N. & Akcil, A. Cyanide and removal options from effluents in gold mining and metallurgical processes. Minerals Engineering 50–51, 13–29 (2013).

Removal methodsAdvantagesDisadvantages Biological oxidation/biodegradationNatural approach, received well publicity, and by regulators. Use heaps as reactors reducing total washed volume, and possibly reach low flow areas of the heap more effectively. Relatively inexpensive. Biomass can be activated by aeration Can treat cyanides without generating another waste stream. No chemical handling equipment or expensive control needed. Superior resistance to shock and upsets No toxic byproducts, hence environmental friendly. Technology is not well established. Requires combination of metallurgy, biology and process engineering. Tends to be very site specific with specific evaluation and study require for each type. Can not treat high concentrations. 8 Kuyucak, N. & Akcil, A. Cyanide and removal options from effluents in gold mining and metallurgical processes. Minerals Engineering 50–51, 13–29 (2013). Cyanide treatment methods

9 Kuyucak, N. & Akcil, A. Cyanide and removal options from effluents in gold mining and metallurgical processes. Minerals Engineering 50–51, 13–29 (2013). Mosa A., Saadoun l, Kumar K.,, Dhankher O. Potential Biotechnological Strategies for the Cleanup of Heavy Metals and Metalloids. Frontiers in Plant Science (2016). Phytoremediation: Tolerant to high concentrations Be accumulators Rapid growth rate Easy harvested Cyanide treatment methods

Microorganisms used in cyanide elimination 12 Dash, R. R., Gaur, A. & Balomajumder, C. Cyanide in industrial wastewaters and its removal: A review on biotreatment. J. Hazard. Mater. 163, 1–11 (2009). Mushrooms: Trichoderma harzianum Fusarium solani F. Oxysporum Trichoderma polysporum Scytalidiym thermophilum Penicilium miczynski they have: Cyanases that produce ammonium and carbon dioxide Cyanide hydrates and amidases that convert cyanide to formic acid Rhodanases for cyanide detoxification Acremonium strictum, thiocyanate removal

Microorganisms used in cyanide elimination Compound removedMicroorganismReactorSource Cyanide WADPseudomonas spBatchActive sludge Potassium cyanideBacillus magateriumBatchOil waste Potassium cyanideBacillus pumilisFixed bedActive sludge Zinc complexesCitrobacter sp.,BatchMining sand Cyanide and iron complexes(II)Pseudomona fluorescensReactore fixed bedMining sand Sodium cyanatePseudomona putidaBatchBeef, ThiocyanatePseudomona putidaBatch Phenol and CyanidePseudomona putidaReactor fixed bed Cyanide and zinc complexesPseudomona spBatch Active sludge Mining sand Cyanide, niquel complexes(II), Phenol, Ni(CN) 4 -2 Pseudomona fluorescensBatch CyanideMethylobacterium sp.BatchActive sludge Sodium cyanatePseudomona pseudoalcaligenesBatchOil waste Sodium cyanate, cyanates and thiocyanate Pseudomona putidaFixed bedActive sludge 12 Dash, R. R., Gaur, A. & Balomajumder, C. Cyanide in industrial wastewaters and its removal: A review on biotreatment. J. Hazard. Mater. 163, 1–11 (2009). Bacteria:

Tratamientos cianuro 74

Complejos de cianuro 75

Degradación natural de cianuro 76

Reacciones en microorganismos 77

Reacciones en microorganismos 78

Filogenia pseudomona 79

Formación de biopelicula 80

81 Formación de biopelicula

82 Formación de biopelicula

83

84

Especies precipitación oro 85

Fosforilacion 86

Respiración aeróbica 87

Respiración anaerobia 88

Nitrificación - des nitrificación 89

Cloruro de cianogeno 90

Citocromo c 91