Comparison of Two Lipases in the Hydrolysis of Oil and Grease in

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Comparison of Two Lipases in the Hydrolysis of Oil and Grease in Wastewater of the Swine Meat Industry Elisandra Rigo,† Roberta Eletı´zia Rigoni,† Patrı´cia Lodea,† De´ bora De Oliveira,† Denise M. G. Freire,‡ Helen Treichel,*,† and Marco Di Luccio† Department of Food Engineering, URI, Campus de Erechim, AVenida Sete de Setembro, 1621, Erechim, RS, 99700-000, Brazil, and Department of Biochemistry, Instituto de Quı´mica, UFRJ, CT, Bloco A, Lab 641, Rio de Janeiro, RJ, 21945-970, Brazil

Wastewaters of the meat industry usually present high contents of oil and fat, which present low biodegradability. Enzymatic hydrolysis may contribute to increase the biodegradation of fatty wastewaters, accelerating the treatment process. In this context, this work aimed to evaluate the enzymatic hydrolysis of wastewater of the swine meat industry. The effects of process variables on the hydrolysis were investigated. The performance of a commercial lipase (Lipolase 100T) was compared to a noncommercial lipase produced by solid-state fermentation (lipase SEP). Process kinetics showed that maximum hydrolysis was reached between 9 and 15 h of reaction. The conditions that maximize hydrolysis for each enzyme were established through statistical analysis of the results. The maximum conversion condition for lipase SEP yielded 100.1 µmol of free acid/mL using 5.0% (w/v) enzyme at 45 °C, while Lipolase 100T yielded 52.1 µmol of free acid/mL using the same enzyme amount but at 37.5 °C. Introduction Water is essential for activities of the meat industry. After use, it turns into a carrier of organic matter that is produced in the process.1 If not treated, it causes large-scale pollution of land and water with its high biochemical oxygen demand (BOD) and chemical oxygen demand (COD). Actually, it has become increasingly difficult to fulfill the disposal requirements.2 Primary processes, involving addition of chemicals (as polyelectrolytes), followed by dissolved air flotation, are often employed to reduce oil and grease concentrations prior to the usual biological treatment. However, the costs of chemicals can be high and some pretreatment systems may not be able to reduce the oil and grease (O&G) concentration to subinhibitory levels.2-4 The complexity of the interactions of microorganisms and the wastewater may complicate the operational control of conventional wastewater treatment plants, which may cause several problems in both aerobic and anaerobic processes.3,5 Fat and proteins present in meat industry wastewaters are usually poorly biodegradable, causing the generation of unpleasant odors, foam, solidification at low temperatures, poor flocculation and sedimentation, and damaging the operation and efficiency of biological processes. Oil and grease may adsorb on microorganism surfaces, hindering cell aqueous phase transfer rates, thus lowering conversion.2,3,5 Enzymes have potential applications in processes for the treatment of wastewaters, solid wastes, hazardous wastes, and soils. The use of hydrolytic enzymes may be of great interest to solve problems in biological wastewater treatment processes caused by high fat content and suspended solids. This kind of treatment has been investigated and presents some advantages such as control of products, nongeneration of toxic byproducts, mild operating conditions, reduction of energy costs, more * To whom correspondence should be addressed. Tel.: +55 21 35209000. Fax: +55 21 3520-9090. E-mail: [email protected]. † URI. ‡ UFRJ.

efficient utilization of raw materials, processing of waste streams to produce marketable byproducts, processing of raw materials, and decontamination of food waste byproduct streams.2,6-8 The employment of microbial enzymatic preparations in the pretreatment of dairy industry wastewaters with high O&G content has been extensively studied.2-5 Few studies involving enzymatic treatment of slaughterhouse wastewaters and restaurant wastewaters can be found.2,6 Unfortunately, the utilization of commercial enzymes to perform the hydrolysis of fats is still very expensive; thus the viability of this technique is strongly dependent on the amount of enzyme necessary for the hydrolysis step.4 The production of low-cost lipases, using alternative substrates as agroindustry residues in solid-state fermentation systems (SEPs), as well as the screening and isolation of new producer strains may increase the process economics and environmental attractiveness.9 Solid-state fermentation has been intensively studied due to the great diversity of extracellular enzymes produced by the process, low possibility of contamination, and low investment and energy costs.9,10 Related to these aspects, this work aimed to study the application of two different lipases on the hydrolysis of wastewater of the swine meat processing industry. The effect of process variables on enzymatic hydrolysis was investigated, and the maximization of hydrolysis was carried out using an experimental design technique. The production of free acids was monitored as a response in each experimental condition. Two different lipases were used: Lipolase 100T, a commercial enzyme produced by Novozymes, and an enzymatic complex obtained by solid-state fermentation (SEP) of babassu cake by Penicillium restrictum.11 After maximization, the effect of substrate (oil and grease) concentration was also investigated. Experimental Section Wastewater Sampling and Preparation. Wastewater samples used in all experiments were collected at a local swine meat processing plant (Erechim, RS, Brazil), which produces swine cuts, sausages, hams, and other industrialized meat products.

10.1021/ie0708834 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/13/2008

Ind. Eng. Chem. Res., Vol. 47, No. 6, 2008 1761 Table 1. Physical-Chemical Characterization of Wastewaters of Swine Meat Industry parameter

AFWW

PFWW

chemical oxygen demand (COD) (g/L) volatile suspended solids (VSS) (mg/L) total solids (TS) (mg/L) oil and grease (O&G) (g/L) protein (P) (%) average flow rate (AFR) (L/h)

2000 90 370 107 927 31.08 0.09

32.00 3912 8462 3.08 0.03 70 000

Composite samples were collected at two different points of the wastewater treatment plant (WTP). One sample was always collected after sieving and before the addition of chemicals for flotation and was denominated PFWW. The other sample was collected on the top waste of the flotation unit, rich in fat, and was denominated AFWW. Sample collecting was carried out during normal operation of the plant. The temperature and pH of the wastewater were monitored at the moment of sampling and ranged from 35 to 40 °C and from 5 to 6, respectively. The samples were transferred to a refrigerated container (4 °C) during collecting. The content of the container was homogenized and stored at 4 °C until use. Wastewater Characterization. The wastewater was characterized in terms of total solids (TS), oil and grease (O&G), total Kjeldahl nitrogen (TKN), and chemical oxygen demand (COD) according to the procedures described in Standard Methods for the Examination of Water and Wastewater.12 Table 1 presents the results of physical and chemical characterization of the wastewater used in all experiments in this work. Analysis of PFWW shows a high concentration of organic matter, expressed as COD, which is usually observed in wastewaters with a high content of oil and grease and suspended solids.13,14 Enzymes. Two lipases were used in this work: a nonimmobilized commercial lipase, Lipolase 100T, which is a fungal lipase, produced by a genetically modified organism (GMO) by Novozymes, for the detergent industry, and a solid enzymatic preparation obtained by solid-state fermentation of babassu cake (lipase SEP) by Penicillium restrictum12 and produced by the Biotechnology Group at the Federal University of Rio de Janeiro (UFRJ), which was kept at -10 °C and used without any purification. Lipase activity was determined by titrimetric method. An emulsion of olive oil (10% w/v) and arabic gum (5.0% w/v) in 0.1 mol‚L-1 sodium phosphate buffer, pH 7.0, was incubated with a sample of the enzyme extract at 37 °C and 160 rpm for 15 min. The reaction was stopped, and the fatty acids were extracted with a solution of acetone and ethanol (1:1). The fatty acids produced were titrated with 0.05 mol‚L-1 NaOH.15 One unit of lipase activity was defined as the amount of enzyme that produces 1 µmol of fatty acids/min, under the assay conditions. Enzymatic Treatment of Wastewater. A wastewater sample with 10 g/L O&G was transferred to a stoppered Erlenmeyer flask. Standardization of oil and grease content was carried out mixing PFWW and AFWW of known oil and grease content. Each reaction was carried out for 24 h, and the kinetics of hydrolysis was followed by titration of free acids formed during the reaction. An experimental run was carried out without the addition of the enzyme for use as a control. Samples (2 mL) of the reaction medium were collected and added to 20 mL of a solution of acetone:ethanol (1:1) to stop the reaction and extract the free acids (FA). This mixture was then titrated with 0.02 N NaOH until pH 11.15 The assessment of the effect of factors on the hydrolysis of oil and grease of the wastewater was carried out by an

Table 2. Matrix of Fractional Factorial Design 24-1 (Actual and Coded Factors) and Responses in Terms of FA for the Two Enzyme Preparations experimental conditions run

T (°C)

pH

1 2 3 4 5 6 7 8 9a 9b 9c

27 (-1) 27 (-1) 27 (-1) 27 (-1) 45 (+1) 45 (+1) 45 (+1) 45 (+1) 36 (0) 36 (0) 36 (0)

6 (-1) 6 (-1) 11 (+1) 11 (+1) 6 (-1) 6 (-1) 11 (+1) 11 (+1) 8.5 (0) 8.5 (0) 8.5 (0)

a

FAa (µmol/mL)

SR (rpm) E (% m/v) lipase SEP Lipolase 100T 0 (-1) 200 (+1) 0 (-1) 200 (+1) 0 (-1) 200 (+1) 0 (-1) 200 (+1) 100 (0) 100 (0) 100 (0)

0.1 (-1) 5.0 (+1) 5.0 (+1) 0.1 (-1) 5.0 (+1) 0.1 (-1) 0.1 (-1) 5.0 (+1) 2.55 (0) 2.55 (0) 2.55 (0)

6.2 47.2 85.1 6.2 98.1 16.7 6.4 76.0 71.2 71.2 72.9

17.6 13.7 20.8 7.7 35.0 24.4 7.0 40.9 39.9 39.1 40.2

Free acids in 15 h of reaction.

experimental design that allowed the obtaining of results with a reduced number of experimental runs.16 The ranges of the studied factors were as follows: amount of enzyme, 0.10-5.0% (w/v); stirring rate, no stirring to 200 rpm; temperature, 27-45 °C; and pH 6-11. The ranges were defined based on the literature.4 The matrix of the 24-1 fractional factorial design and the results obtained are presented in Table 2. All runs were carried out in duplicate and reproducibility was evaluated repeating some experimental runs in random order. After maximization of hydrolysis conditions, the effect of substrate (O&G) concentration on hydrolysis performance was evaluated with fixed enzyme amount, temperature, and pH. Results and Discussion Enzymatic Hydrolysis of the O&G Present in the Meat Industry Wastewater. Lipase activities for lipase SEP and Lipolase 100T were 43 U per gram of dry substrate (gds) and 2800 U per gram of the commercial enzyme preparation, respectively. Although these results are poorly comparable since the purities of the enzyme preparations are different, they suggest that the commercial enzyme is much more active in the standard substrate tested (olive oil) than the enzyme extract obtained by solid-state fermentation using Penicillium restrictum. The matrix of the experimental design and the results in terms of FA produced in the reaction for the two enzymes are presented in Table 2. The data shown in this table refer to the amount of FA formed after 15 h of reaction. The results show that the highest yield in FA was obtained with the lipase SEP, at 45 °C, with 5.0% (w/v) of enzyme preparation, at the lower level of pH (6.0), and with no stirring. For the experiments with Lipolase 100T the best results were obtained with conditions of run 8 and the central point. The experimental condition of the central point is probably the most attractive, since it uses a lower level of temperature (36 °C) and enzyme (2.55% w/v) than run 8 and obtains a similar amount of FA. Figures 1 and 2 present the hydrolysis kinetics for all the experimental conditions preestablished in the experimental design for both enzymes. It is possible to observe that the temperature and the amount of enzyme are relevant factors for hydrolysis using Lipolase 100T. It may also be noted that the maximum of FA is reached between 9 and 15 h of reaction, in general. Similar behavior is observed for lipase SEP, shown in Figure 2. Experimental error was evaluated through triplicate runs of central points and at the extreme points of the experimental design, at random. The results showed a good reproducibility of the enzymatic reactions, yielding variations lower than 5%.

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Figure 1. Kinetics of enzymatic hydrolysis of O&G of swine meat industry wastewater using Lipolase 100T at (a) 27 and (b) 45 °C.

Consumption of free acids by microorganisms present in the wastewater was assessed by experimental runs adding 50 mg‚L-1 sodium azide, to inhibit microbial growth. The results of enzymatic hydrolysis with and without the inhibitor did not differ, showing that microbial activity during the assays was negligible. These results show that the hydrolysis catalyzed by lipase SEP resulted in higher conversions when compared to Lipolase 100T. This behavior is peculiar, since the hydrolytic activity of Lipolase 100T in olive oil seemed higher than the activity of lipase SEP. The assay of enzymatic activity was carried out under standard conditions after the experiment using the wastewater, but no expressive change in activity before the run was observed. This result may be due to the different resistances to denaturation or to inhibitory effects caused by components present in the meat industry wastewater. Other studies report that the high concentration of free enzyme in the reaction medium may cause a decrease in the hydrolysis rate by adsorption of fat particles in the surface of enzyme, causing this inhibition. This limit generally varies according to the characteristic of each lipase.13 Another issue worth mentioning is the fact that the lipase SEP is a nonpurified enzyme extract and contains other hydrolases such as proteases, amylases, esterases, etc.2,4 Lipolase 100T is a commercial semipurified lipase, and no other hydrolase activity is reported for this enzyme preparation. Thus, the activity of Lipolase 100T over olive oil is much higher compared to lipase SEP, because the activity is determined per gram of practically pure lipase, while for lipase SEP the activity is determined per gram of fermented dry matter, which is not pure and contains other proteins. In wastewater

Figure 2. Kinetics of enzymatic hydrolysis of O&G of swine meat industry wastewater using lipase SSF at (a) 27 and (b) 45 °C.

Figure 3. Variation of initial reaction rate as a function of substrate concentration.

treatment many substrates are present and pools of hydrolases such as lipase SEP are usually more effective than pure enzymes such as Lipolase 100T.2-5 The matrix of results of the experimental design was statistically analyzed in terms of FA produced in the reaction for the two enzymes (Table 2). The effect of the factors on the enzymatic hydrolysis using lipase SEP and Lipolase 100T can be observed in the Pareto charts presented in Figures 4 and 5. The temperature of the reaction and the amount of enzyme were the variables that most influenced the hydrolysis. The highest

Ind. Eng. Chem. Res., Vol. 47, No. 6, 2008 1763

Figure 4. Pareto chart of standardized effects of factors on hydrolysis using lipase SEP (15 h of reaction).

Figure 5. Pareto chart of standardized effects of factors on hydrolysis using Lipolase 100 T (15 h of reaction).

hydrolysis rate occurred at the higher level of temperature (45 °C) and enzyme amount (5% w/v). This result is of relevance to an industrial application, since after scaling up the use of the enzymatic treatment would be just after the primary treatment (usually dissolved air flotation), for which common temperatures range from 27 to 40 °C. Leal et al.10 found the best conversions at 45 °C after 24 h (14 µmol/mL FA), using the same lipase of Penicillium restrictum for the hydrolysis of dairy wastewater, although the condition selected for hydrolysis was 35 °C for 12 h for the sake of economic feasibility. Cammarota et al.3 studied the effect of the concentration of lipase SEP on the hydrolysis of dairy wastewater and showed that the condition that yielded the highest conversion in FA (29 µmol/mL) was 1.0% (w/v) for 24 h, 35 °C, and 120 rpm. Jung et al.5 found similar results (23 µmol/mL) for hydrolysis of dairy wastewater using lipase SEP at 30 °C, 0.2% (w/v) for 8 h. Figure 4 shows that the stirring rate has a negative effect on hydrolysis while the pH presents a positive effect (p < 0.05). Since the best result of hydrolysis using lipase SEP (Table 2) was obtained at the lower level of pH, new experiments were

Table 3. Effect of pH and Stirring Rate (SR) on Hydrolysis Using Lipase SEP (Univariable Tests, T ) 45 °C, E ) 5% w/v) experimental conditions run

pH

SR (rpm)

FAa (mmol/mL)

1 2 3 4

6 (-1) 6 (-1) 6 (-1) 11 (+1)

0 (-1) 100 (0) 200 (+1) 200 (+1)

100.1b ( 5.7 92.8b ( 12.1 85.6b ( 2.3 85.9b ( 0.1

a Free acids in 15 h of reaction. b Results with the same superscripts do not significantly differ (Tukey test, p < 0.05).

proposed to find a condition that maximizes the FA production. The stirring rate did not influence the hydrolysis using Lipolase 100T, presented in the diagram of effects in Figure 5. In this case, pH showed a negative effect on the hydrolysis of the effluent. Table 3 presents the experiments carried out with lipase SEP after statistical analysis, at 45 °C and 5% (w/v) enzyme. The results at different pHs and stirring rates were analyzed by the Tukey test, and it was observed that both pH and stirring rate did not differ significantly (p < 0.05) in the range investigated. These results are interesting from the economics point of view,

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Table 4. Effect of pH and Stirring Rate (SR) on Hydrolysis Using Lipolase 100T (Univariable Tests, T ) 45 °C, E ) 5% w/v) experimental conditions run

pH

A (rpm)

1 2 3

11 (+1) 6 (-1) 11 (+1)

100 (0) 100 (0) 200 (+1)

FAa

(mmol/mL)

43.2b ( 0.6 39.5b ( 2.0 51.1b ( 3.2

a Free acids in 15 h of reaction. b Results with the same superscripts do not significantly differ (Tukey test, p < 0.05).

since the enzymatic treatment could be done without adjustment of the effluent pH, which is between 5 and 6, and without adjustment of the stirring rate. Table 4 shows the experiments carried out with Lipolase 100T after the statistical analysis. These results also did not differ significantly (p < 0.05). Thus, it is also possible to work with Lipolase 100T at lower levels of stirring rate and at the pH of the raw effluent. It is possible to note that lipase SEP presents a higher rate of hydrolysis in comparison to Lipolase 100T under similar operating conditions. This effect could be possible related to the higher stability of lipase SEP in the effluent, or to a higher affinity for the substrates present in the effluent. Therefore lipase SEP could find a promising application in the prehydrolysis of lipids of meat industry wastewater. Effect of Substrate Concentration. The evaluation of substrate concentration is important for kinetic parameter estimation in enzymatic systems, which is useful for scaling up the process. A series of batch experiments was carried out with different initial concentrations of O&G, fixing the enzyme concentration, pH, and stirring rate at previously optimized conditions for both enzymes. Analysis was based on FA production after 4 h of reaction. Experimental data was used to build a kinetic curve of initial reaction rate (Vo, µmol‚mL-1‚h-1) versus concentration of substrate ([S], g‚L-1), presented in Figure 3. This figure shows that the enzymatic hydrolysis is in the region of a first-order reaction. The Michalis-Menten equation (eq 1) can be used to obtain the maximum rate (Vmax) and Michalis-Menten constant (Km) only when the reaction is in the region of mixed order (0 and 1). In the cases where the reaction is of first-order kinetics ([S] < Km), the Michaelis-Menten equation is reduced to the expression in eq 2, which defines a linear relation between

V)

Vmax[S] Km + [S]

V)

Vmax[S] Km

(1)

(2)

reaction rate, V, and substrate concentration, [S]. In this case the kinetic parameter K1 ) Vmax/Km is the slope of the saturation curve. The comparison of the kinetic parameters for both enzymes shows that reactions catalyzed by lipase SEP are up to 3.5 times higher than the ones catalyzed by Lipolase 100T. These results agree with the ones observed in experimental designs, which showed that higher conversions might be obtained using lipase SEP at the same operating conditions. As discussed earlier, this behavior may be related to inhibition or inactivation of Lipolase 100T by compounds present in the wastewater.

Conclusions Maximum yield in free acids for both enzymes was obtained at a high amount of enzyme (5.0% m/v), at 45 °C, pH 6, and 100 rpm (FA ) 39.5 µmol/mL for Lipolase 100T and 100.1 µmol/mL for lipase SEP). For both enzymes pH could be altered according to the application since it did not affect significantly (p < 0.05) the hydrolysis of the effluent. Lipolase 100T presented a lower reaction rate than lipase SEP, which yielded the highest rates for conversion in FA for the latter when compared to the commercial lipase at the range investigated in this work. This behavior is possibly related to the effects of the adverse medium on Lipolase 100T, suggesting that lipase SEP is more indicated for hydrolysis of meat industry effluents, and more advantageous, since it is a nonpurified enzymatic preparation, produced with agroindustry residues. Acknowledgment The authors would like to acknowledge Allimentus Engenharia e Tecnologia for a scholarship and PIT/SCT/RS, FAPERGS, FAPERJ, and URIsCampus de Erechim for financial support. Literature Cited (1) Cavalcanti, J. E. W. A.; Braile, P. M. Manual de tratamento de a´ guas residua´ rias industriais; CETESB: Sa˜o Paulo, Brazil, 1993. (2) Cammarota, M. C.; Freire, D. M. G. A review on hydrolytic enzymes in the treatment of wastewater with high oil and grease content. Bioresour. Technol. 2006, 97, 2195. (3) Cammarota, M. C.; Teixeira, G. A.; Freire, D. M. G. Enzymatic pre-hydrolysis and anaerobic degradation of wastewaters with high fat contents. Biotechnol. Lett. 2001, 23, 1591. (4) Leal, M. C. M. R.; Freire, D. M. G.; Cammarota, M. C.; Sant’Anna, G. L., Jr. Effect of enzymatic hydrolysis on anaerobic treatment of dairy wastewater. Process Biochem. 2006, 41, 1173. (5) Jung, F.; Cammarota, M. C.; Freire, D. M. G. Impact of enzymatic pre-hydrolysis on batch activated sludge systems dealing with oily wastewaters. Biotechnol. Lett. 2002, 24, 1797. (6) Masse, L.; Kennedy, K. J.; Chou, S. The effect of an enzymatic pre-treatment on the hydrolysis and size reduction on fat particles in slaughterhouse wastewater. J. Chem. Technol. Biotechnol. 2001, 76, 629. (7) Nicell, J. A. Chemical Degradation Methods for Wastes and Pollutants: EnVironmental and Industrial Applications; Marcel Dekker Inc.: New York, 2003. (8) Ensuncho, L.; Cuenca, M. A.; Legge, R. L. Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale threephase fluidized bed reactor. Bioprocess Biosyst. Eng. 2005, 27, 185. (9) Pandey, A.; Soccol, C. R.; Rodriguez-Leon, J. A.; Nigam, P. SolidState Fermentation in Biotechnology; Asiatech Publishers Inc.: New Delhi, India, 2001. (10) Leal, M. C. M. R.; Cammarota, M. C. M.; Freire, D. M. G.; Sant’Anna, G. L., Jr. Hydrolytic enzymes as coadjuvants in the anaerobic treatment of dairy wastewaters. Braz. J. Chem. Eng. 2002, 19, 175. (11) Cammarota, M. C.; Freire, D. M. G.; Sant’Anna, G. L., Jr.; Russo, C.; Freire, D. D. C.; Castilho, L. R. Processo de preparac¸ a˜o e composic¸ a˜o de preparado enzima´tico para tratamento de efluentes dome´sticos e industriais com elevado teor de gorduras, proteı´nas e/ou carboidratos e processo para tratamento de efluentes dome´sticos e industriais com elevado teor de gorduras, proteı´nas e/ou carboidratos. (Production process and composition of an enzymatic preparation and its use for the treatment of domestic and industrial effluents of high fat, protein and/or carbohydrate content.) Brazilian Patent 0007101-3, 2000. (12) APHA, AWWA, WEF. Standard methods for the examination of water and wastewater, 19th ed.; Publication Office APHA: Washington, DC, 1998. (13) Masse, L.; Kennedy, K. J.; Chou, S. Testing of alkaline and enzymatic hydrolysis pre-treatments for fat particles in slaughterhouse wastewater. Bioresour. Technol. 2001, 77, 155.

Ind. Eng. Chem. Res., Vol. 47, No. 6, 2008 1765 (14) Masse, L.; Masse, D. I.; Kennedy, K. J. Effect of hydrolysis pretreatment on fat degradation during anaerobic digestion of slaughterhouse wastewater. Process Biochem. 2003, 38, 1365. (15) Freire, D. M. G.; Gomes, P. M.; Bon, E. P. S.; Sant’Anna, G. L., Jr. Lipase production by a new promising strain of Penicillium restrictum. J. Braz. Soc. Microbiol. 1997, 28, 6.

(16) Montgomery, D. C. Design and analysis of experiments, 5th ed.; New York, 2001.

ReceiVed for reView June 27, 2007 ReVised manuscript receiVed December 5, 2007 Accepted December 24, 2007 IE0708834