Optimization of lipids enzymatic hydrolysis present in swine slaughterhouse effluents Aniela Pinto Kempka

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1 Optimization of lipids enzymatic hydrolysis present in swine slaughterhouse effluents Aniela Pinto Kempka ABSTRACT Santa Catarina State University, Food Engineering Department, Pinhalzinho, SC, Brazil Effluents from slaughterhouses are characterized by a high concentration of oils and greases that can cause problems in conventional treatments. The fats and proteins present in effluents have low biodegradability, which represents serious problems for the biological degradation processes. The degradation of difficult pollutants in more easily degradable products can be mediated by enzymes. Enzymatic hydrolysis facilitates the biodegradation because it makes the organic material is made available as compounds of lower molecular weight, the lipids being made available in the form of fatty acids. The application of enzymes as processing aids in effluent treatment has some advantages, such as the specificity that allows control of the products, which leads to increased income generation for the non-toxic byproducts, moderate conditions of operation, cost reduction in terms and energy and equipment, making this process attractive from the point of view of environment. The objective of this work was optimizing the conditions for lipids enzymatic hydrolysis present in swine slaughterhouse effluents using lipolytic enzymes. Temperature, ph and enzyme concentration were the variables tested. The enzymes showed good activity and could therefore be used for the hydrolysis process proposed here. The optimized conditions to maximize the release of fatty acids were: temperature of 3 C, ph.5 and enzyme concentration of 1.1 %, yielding fatty acids in the order of 31.5 µmol/ml for lipase and 31.5µmol/mL for phospholipase. All variables influenced the release of fatty acids, but the most significant variables were ph and enzyme concentration. Keywords: effluents; hydrolysis; lipids; lipase; phospholipase. INTRODUCTION The effluents from slaughterhouses contain high levels of lipids and proteins that cause low biodegradability [1] impairing the process of biodegradation. Besides the decrease of biodegradability, fats and proteins cause serious problems even in aerobic and anaerobic biological processes as the development of odors, solidification of fats at low temperatures [] and still foaming, preferential pathways in sludge bed and drag the biomass, leading to loss of efficiency and even the collapse of the reactor [3]. Among the alternatives to minimize the problems occurring in biological reactors for effluent from slaughterhouses, the use of enzymes has been extensively studied, because they allow the development of technological processes very close to efficient processes executed by nature [] with the advantage of reducing the effluent residence time in the reactors and still having several potential advantages such as simplicity and ease of process control, without the need for acclimatization of biomass, not suffering the effects of shock loading of pollutants. Enzymes can be applied in cases with low or high concentrations of pollutants, with wide ranges of ph, temperature and salinity [5]. Lipases have the feature to hydrolyze phospholipids and lipid molecules, the latter being characterized by lipid molecules containing phosphoric acid as mono or diester []. They are widely found in nature and from animals such as lipases from the pancreas of pigs and also in human beings, the last being the most known and investigated among all lipases [7]. They are particularly important due to the fact that specifically hydrolyze oils and greases, and this is of great interest in the treatment of industrial effluents that are high in fat [] as is the case for the slaughterhouse industry. Phospholipases are a group of enzymes that catalyze the hydrolysis of esters, linked to glycerophospholipids. Phospholipids are major constituents of biological membranes and in vivo, its hydrolysis is regulated by phospholipases with an actual physiological role [9]. This study describes two enzymes, lipase and phospholipase hydrolysis of an effluent from the slaughterhouse industry, being analyzed the main variables that take part of the process, performing an optimization of the same from the use of statistical tools and techniques of planning experimental.

2 MATERIALS & METHODS Materials On completion of laboratory tests were used two commercial enzymes (fungal lipase and a phospholipase porcine origin). The effluent used in hydrolysis reactions was collected from a wastewater treatment station from a swine slaughterhouse located in the city of Chapecó SC. The effluent being used now only by the process of railing. The effluent was stored in plastic containers and stored at freezing temperature. Characterization of the effluent With the aim to characterize the effluent, aliquots of 5mL were sampled and immediately transported to the laboratory for determining the physical chemical characteristics. Content in oils, greases and solids, Chemical Demand of Oxygen (COD), total alkalinity and dissolved oxygen were determined according to standards recommended by official methods [1]. Planning of experiments and optimization of hydrolysis conditions The effects of ph, temperature and enzyme concentration on the hydrolysis of oils and greases from wastewater were investigated by using techniques of experimental design in order to determine the influence of variables in generating the maximum concentration of free fatty acids. Table 1 shows the levels and values of variables. All experiments were performed in duplicates. Table 1. The levels and values of variables Variables Temperature (ºC) ph Enzyme concentration % (w/v) Hydrolysis of the effluent The hydrolysis reactions of the effluent were performed in 15 ml Erlenmeyer flasks placed in a water bath. The temperature was maintained according to the experimental design. Each reaction was conducted during 1 hours and the hydrolysis was followed by measurement of the releasing of fatty acids. Aliquots of ml hydrolyzed effluent were withdrawn every two hours and were diluted in ml of a solution of acetone: ethanol (1:1), whose goal is to stop the reaction. This mixture was then titrated with a solution of.n NaOH until ph 11. [11]. The beginning of each reaction was done the procedure with a rate of not hydrolyzed effluent, measuring the fatty acids present in the same initials. Statistical analysis of results was performed using the Statistica. sotware (STATSOFT, INC). RESULTS & DISCUSSION Characterization of the effluent The characterization of the effluent resulted a content in oil and grease of 5. mg/l, ph.55, total solids of 13. mg/l and COD of 315. mg/l. Other studies show similar values to those found in this study for the COD, 393. and 7. mg/l [1, 13]. The ph value obtained in this work is similar to values obtained by the works cited above, being. and.5 respectively. Optimization of hydrolysis conditions for enzymes Figures 1 and show the final concentrations of fatty acids in µmol/ml for the tests performed with the lipase and phospholipase, respectively. For both enzymes, the best results were obtained in experiments 15 and 1, which correspond to the midpoint, whose hydrolysis conditions were: temperature 3 ºC, ph.5 and enzyme concentration of 1.1 %. The average fatty acid using lipase obtained for the experiments cited above was 31.5 µmol/ml. As for the experiments using phospholipase, the average fatty acid obtained was 31.5 µmol/ml Hydrolysis curves of the effluent using lipase Figure 3 shows the hydrolysis curves of the effluent to the experimental conditions with a temperature of 7 ºC and central point. It is observed that the best response is presented in the experiment whose conditions are ph. and enzyme concentration. % (w/v), but this condition presents much less the condition of the

3 central point in terms of release fatty acids. It was also noted that the temperature of 7 C significantly reduces the release of fatty acids in relation to the central point condition. Studies on the influence of operating conditions in the synthesis of fatty acids using immobilized lipase produced by Candida antarctica at temperatures of 5 ºC, 35 C, 5 C, 55 ºC and 5 ºC and 1 % of enzyme in all experiments, found an increase in the conversion of fat fatty acids from 9. % to. % when increasing the temperature of 5 ºC to 5 ºC and 5 C operating condition which released the lowest percentage of fatty acids [1]. Fatty acids (µmol/ml) Experiment 1 Experiment Experiment 3 Experiment Experiment 5 Experiment Experiment 7 Experiment Experiment 9 Experiment 1 Experiment 11 Experiment 1 Experiment 13 Experiment 1 Experiment 15 Experiment 1 Fatty acids (umol/ml) Experiment 1 Experiment Experiment 3 Experiment Experiment 5 Experiment Experiment 7 Experiment Experiment 9 Experiment 1 Experiment 11 Experiment 1 Experiment 13 Experiment 1 Experiment 15 Experiment 1 Figure 1.Generation of fatty acids by lipase. Figure. Generation of fatty acids by phospholipase. Figure shows the hydrolysis curves of the effluent to the experimental conditions with a temperature of 5 ºC and central point. It appears that the central point condition were better than the others, which have a temperature of 5 ºC in all stages of the experiment. The conditions were ph. with enzyme concentration of. and. % (w/v), respectively, were immediately following the conditions regarding the release of fatty acids //, 7// 7/11/, 7/11/ 3/,5/1, Figure 3. Hydrolysis to the effluent at 7ºC and the //, 5// 5/11/, 5/11/ 3/,5/1, Figure. Hydrolysis to the effluent at 5ºC and the Figure 5 shows the hydrolysis curves of the effluent to the experimental conditions with a temperature of 3 ºC (including the midpoint). Analyzing the experimental temperature of 3 ºC, it is observed that the interaction between the variables is presented visually significant, since we obtained results are much poorer in terms of release of fatty acids to keep the temperature constant and varying the concentration of enzyme and ph. In the experiment whose initially the ph was 13., did not become a low release of fatty acid over time. This can be explained by the high alkalinity of the medium, due to the methodology applied in the quantification of fatty acids, we did not allow the determination of fatty acids in media where the ph is above 11.. It should be noted that even considering the context of the experiment was conducted to obtain the experimental design in a comprehensive manner. Figure presents the extreme conditions of the experimental design, which changed the temperature of the effluent, and remained constant ph conditions and enzyme concentration. The temperature of 51 C was obtained by an increased release of fatty acids in relation to temperature of 1 C, maintaining the ph and enzyme concentration constant (ph.5 and enzyme concentration 1.1 %). However, the condition of the central point remains as the best experimental design for

4 the enzyme lipase, which shows that for lipase study in the higher temperature and higher concentration of enzyme does not lead to an increased release of fatty acids //1,1 3/13/1,1 3/,5/, 3/,5/, 3/,5/1, Figure 5. Hydrolysis to the effluent in the temperature of 3ºC /,5/1,1 51/,5/1,1 3/,5/1, Figure. Hydrolysis to the effluent at temperatures of 51ºC, 1 C and the Hydrolysis curves of the effluent using phospholipase Figure 7 shows the hydrolysis curves of the effluent to the experimental conditions at 7 C and the central point, using the enzyme phospholipase. As for the lipase to phospholipase at 7 C did not show a good performance in the hydrolysis process, but in terms of ph, is observed in the curves immediately below the central point that at the end of the experiment we obtained values release of fatty acids very close to ph. and 11.. Figure shows the hydrolysis curves of the effluent to the experimental conditions where the temperature was 5 ºC and central point. It is observed that for phospholipase temperature of 5 C, the conditions were the best that the enzyme concentration was. %, and the release of fatty acids into the condition of initial ph. was higher than release fatty acids of the condition of initial ph 11. unlike the lipase, the ph had the greatest influence being that for a temperature of 5ºC the best conditions of release for initial ph, with. % and. % concentration of enzyme, respectively //, 7// 7/11/, 7/11/ 3/,5/1, Figure 7. Hydrolysis to the effluent at 7ºC and the //, 5// 5/11/, 5/11/ 3/,5/1, Figure. Hydrolysis to the effluent at 5ºC and the Figure 9 shows the hydrolysis curves of the effluent to the experimental conditions at a temperature of 3 ºC. Keeping the temperature constant and varying the enzyme concentration and ph levels to a minimum and maximum perceives the relationship between the variables mentioned and the generation of fatty acids. The experiment where the enzyme concentration increased to. % as well as for lipase, results were not superior to the midpoint. Figure 1 shows the curves of hydrolysis of the effluent by phospholipase to the extreme experimental conditions of the experimental design. It is observed that the condition has a higher temperature showed better performance in the release of fatty acids in relation to the lowest temperature. This result is analyzed without taking into account the experiments presented here confirms that at higher temperatures the enzymes have a better activity, however, taking into account the central point, it appears that the result obtained for the temperature of 51 ºC corresponds to approximately 5 % of the value obtained at the

5 midpoint. Therefore, the enzyme phospholipase the best condition for obtaining a greater release of fatty acids is the condition for obtaining a greater release of fatty acids is the condition of central point //1,1 3/13/1,1 3/,5/, 3/,5/, 3/,5/1, Figure 9. Hydrolysis to the effluent in the temperature of 3 C /,5/1,1 51/,5/1,1 3/,5/1, Figure 1. Hydrolysis to the effluent at temperatures of 51 ºC, 1 C and the Statistical Analysis of Experimental Results The effects of the variables can be seen in Figures 11 and 1, which presents the Pareto chart for the system catalyzed by lipase and phospholipase, respectively. It is observed that the effect was more pronounced the negative effect attributed to the ph followed by the enzyme concentration for the two enzymes studied. ph (Q) Enzyme concentration (Q) -7.1 Temperature (Q) -5.3 ()ph (L) -.5 (3)Enzyme concentration (L) 3.73 (1)Temperature (L).7 1 by by 3.33 Bloc(1). by 3. p=.5 Effect Estimate (Absolute Value) Figure 11. Pareto chart - lipase. -.5 ph(q) Enzyme concentration(q) -7.5 Temperature(Q) -5. (3)Enzyme concentration(l).7 ()ph(l) -.9 1Lby3L 1.5 (1)Temperature (L) 1. 1LbyL -. Lby3L.5 Bloc(1).13 p=,5 Effect Estimate (Absolute Value) Figure 1. Pareto chart - phospholipase. Figures 13 and 1 contain the response surfaces obtained for the lipase and phospholipase, respectively. Figure 13. Response surface for lipase. Figure 1. Response surface for phospholipase.

6 Observe that the region with maximum release of fatty acids corresponds to the central point, whose operating conditions are maintained at 3 ºC, ph.5 and enzyme concentration of 1.1 % (w/v). Equations 1 and present the mathematical models of simple statistical system catalyzed by lipase (R =.) and phospholipase (R =.), respectively, through empirical model according to the coded variables adjusted for fatty acids (µmol/ml). FA = 15, +,7.T,75.pH +,3.E 1,.T 1,.pH 15,.E (1) FA = 31, 3,5.pH + 7,.E 11,5.T 1,7.pH 15,5.E () Where: FA = fatty acids (µmol/ml), T = temperature (ºC) e E = enzyme concentration % (w/v). CONCLUSIONS The enzymes showed good activity and could therefore be used for the hydrolysis process proposed here. The results were satisfactory, where the maximum conversion of fatty acids for both enzymes was the temperature of 3 ºC, ph.5 and enzyme concentration of 1.1% (w/v), which is the central point. We obtained a value of 31.5 µmol/ml for lipase and 31.5 µmol/ml for phospholipase and all variables influenced the release of fatty acids, but the most significant variables were ph and enzyme concentration. To check the efficiency of the process, the use of effluent in a reactor containing hydrolyzed sludge should be tested in order to obtain a better rate of biodegradation. REFERENCES [1] Cammarota, M.C..; Freire, D.M.G.. A review on hydrolytic enzymes in the treatment of effluent with high oil and grease content. Bioresearches Technology, 97,,195-,1. [] Cammarota, M.C.; Teixeira, G.A.; Freire, M.D.G. 1. Enzymatic pre-hydrolysis and anaerobic Degradation of effluents with higt fat contents. Biotecnology Letters, 3 (19), 1,591-1,595. [3] Castro, H.F., Mendes, A. A., Pereira, E. B., Furigo Júnior, A. 5. Aplicação de lipases no tratamento de águas residuárias com elevados teores de lipídios. Química Nova, (), [] Hasan, F., Shah, A. A., Hameed, A.. Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, [5] Karam, J.; Nicell, J. A Potential applications of enzymes in waste treatment. Journal of Chemical Technology & Biotechnology, 9, [] Fennema, O.R.. Química de los alimentos. Zaragoza: Acribia. 1 p. [7] Pandey, A.; Soccol, C.R. and Mitchell, D.. New Developments in solid state fermentation: bioprocesses and products. Process Biochemistry, 35, 1,153-1,19. [] Leal, M. C.M.R., Freire D. M.G., Cammarota, M.C., Sant Anna Jr, G.. Effect of enzymatic hydrolysis on anaerobic treatment of dairy effluent. Process Biochemistry, 1, 1,173-1,17. [9] Siloto, A.M.P. 1. Seleção de microrganismos produtores de fosfolipase, otimização da produção e caracterização da enzima bruta. Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos. Programa de Pós-Graduação em Ciência de Alimentos Dissertação de Mestrado. [1] APHA American Public Health Association, American Water Works Association, Water Federation Environment Standard Methods for the examination of water and effluent. Washington: APHA. [11] FREIRE, D.M.G., GOMES, P.M., BON, E.P.S, SANT ANNA JR., G.L Lipase production by a new promising strain of Penicillium restrictum. Journal of the Brazilian Society for Microbiology,, -1. [1] Pereira, E.B. 5. Tratamento enzimático para remoção de gorduras dos resíduos gerados por indústrias de produtos avícolas. Universidade Federal de Santa Catarina, Departamento de Engenharia Química e Engenharia de Alimentos Programa de Pós-Graduação em Engenharia Química - Tese de Doutorado. [13] Dors, G.. Hidrólise enzimática e biodigestão de efluentes da indústria de produtos avícolas. Universidade Federal de Santa Catarina, Departamento de Engenharia Química e Engenharia de Alimentos Programa de Pós-Graduação em Engenharia Química Dissertação de Mestrado. [1] Trubiano, G.; Bori, D.; Errazu, A. 7. Influence of the operating conditions and the external mass transfer limitations on the synthesis of fatty acid esters using Candida antarctica lipase. Enzyme and Microbial Technology,, 71-7.