Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1312-4
ORIGINAL PAPER
New strategy to apply perfluorodecalin as an oxygen carrier in lipase production: minimisation and reuse Erika Souza Vieira • Taˆmara Karoline de Oliveira Fontes • Matheus Mendonc¸a Pereira • Hofsky Vieira Alexandre • Daniel Pereira da Silva ´ lvaro Silva Lima Cleide Mara Faria Soares • A
•
Received: 6 March 2014 / Accepted: 15 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A novel strategy for the production of lipase by Bacillus sp. ITP-001 in a stirred tank fermenter using perfluorodecalin (PFD) was studied. Firstly, a response surface methodology 22 with three central points was employed to optimise the effect of agitation speed and aeration rate in lipase production. According to the response from the experimental designs, 300 rpm (revolutions per minute) and 0.5 vvm (air volume/liquid volume per minute) were found to provide the best condition (lipolytic activity: LA = 3,140.76 U mL-1). Then, the influence of PFD concentration on the fermentation process was evaluated. Incorporation of PFD at all concentrations above 1 % had no statistically significant influence on lipase production, that is, the previous optimisation allowed the reduction of the amount of PFD added besides increasing lipase production. Furthermore, PFD could be used in three sequential fermentations without altering the statistical production of lipase, reducing by 67 % the cost of PFD addition.
E. S. Vieira T. K. de Oliveira Fontes ´ . S. Lima (&) H. V. Alexandre C. M. F. Soares A Programa de Po´s-Graduac¸a˜o em Engenharia de Processos, Universidade Tiradentes, Av. Murilo Dantas 300, Farolaˆndia, Aracaju 49032-490, Brazil e-mail: [email protected] ´ . S. Lima M. M. Pereira H. V. Alexandre C. M. F. Soares A Instituto de Tecnologia e Pesquisa, Av. Murilo Dantas 300, Farolaˆndia, Aracaju 49032-490, Brazil D. P. da Silva Nu´cleo de Engenharia de Produc¸a˜o, Universidade Federal de Sergipe, Av. Marechal Rondon s/n, Jardim Rosa Elze, Sao Cristovao 49100-000, Brazil
Keywords Lipase Oxygen carrier Perfluorodecalin Experimental design Fermentation
Introduction Lipases (triacylglycerol acylhydrolase, E.C.3.1.1.3) constitute an important group of enzymes associated with lipid metabolism, and may be obtained from animal, plant, and microbial sources [1]. These enzymes are hydrolases, which act under aqueous conditions on the carboxyl ester bonds present in triacylglycerides to liberate fatty acids and glycerol [2, 3]. Microbial lipases are immensely applied in various facets of industrial biotechnology such as in fat and oil hydrolysis [4], food industries [5], detergent industries [6], pharmaceutical industries [7], and biodiesel synthesis [8] due to their stability, selectivity, and broad substrate specificity [9]. A considerable number of microorganisms have been used to produce lipases [10–12], especially the genus Bacillus [13, 14]. Solid-state fermentation, SSF [15], and submerged fermentation, SmF0 [16], are effective techniques for microbial lipase production. However, SmF has been used traditionally because the recovery of extracellular enzymes and determination of biomass can be performed by simple filtration or centrifugation [17]. For SmF, the literature presents several studies to optimize and devise strategies of lipase production in terms of medium composition such as carbon and nitrogen source [18, 19], and operational conditions such as temperature, pH value, aeration rate (concentration of dissolved oxygen), and agitation speed [20–23]. In this context, the literature reports that oxygen is required as a final electron acceptor to reoxidise nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2) and generate ATP for
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metabolism [24]. Therefore, low oxygen levels induce or repress transcription, synthesis of different enzymes, or both, thereby affecting all metabolism and, consequently, product yield and productivity [25]. Moreover, the solubility of oxygen in water is low (approximately 8.82 mg L-1 at 20 °C) [26] and, consequently, the oxygen mass transfer is also low. Therefore, oxygen is an indispensable raw material that must be supplied in large amounts during industrial aerobic fermentation [27]. A way to minimise the low oxygen mass transfer in SmF is by the use of a water-immiscible liquid, i.e., a nonconventional organic solvent [28], which presents high oxygen solubility. This strategy is called a two-liquidphase system, in spite of four phases are present in the system (hydrophobic, aqueous, gas phases and cells) [29]. According to Villemur et al. [30], this approach can be divided into two categories; in the first one, the waterimmiscible liquid is the substrate for the microorganisms (petroleum hydrocarbon, n-alkanes, or vegetable oil), and in the second, the non-aqueous liquid is added to the bioreactor to enhance oxygen mass transfer (silicone oil, hexadecane, or perfluorochemicals (PFCs)]. PFCs are derivatives of aliphatic, cyclic, or polycyclic hydrocarbons in which most or all of the hydrogen atoms have been replaced by fluorine, increasing the molecular mass [31]. Furthermore, PFCs have an exceptional thermal stability; they are chemically inert, non-toxic towards cells, and have specific densities of about twice that of water and 10–20 times higher oxygen solubility than water [32, 33]. One commonly used PFC is perfluorodecalin, PFD (C10F18), a bicyclic perfluorinated alkane [34]. As previously mentioned, the use of a second liquid phase, namely PFD, an oxygen-carrying component, in the culture media increases the availability of oxygen to the microorganism [35]. Coelho and co-workers reported that PFC addition (20 %, v/v) benefited lipase production that increased 11-fold [36], corroborating the results from Elibol and Ozer [37], which found the highest lipase production with 10 % (v/v) PFC. The highest PFD concentration has the disadvantage the increasing the cost of enzyme production. For this reason, the goal of this work was to search for a new approach to minimise the use of PFD in multiphase fermentation, by optimising the agitation speed of parameters and aeration rate, then checking the effect of the addition of perfluorodecalin and their potential for reuse. Approach has not yet reported in the literature.
Materials and methods Microorganism and chemicals Bacillus sp. ITP-001 strain, isolated from a petroleumcontaminated soil and belonging to a culture collection of
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Bioprocess Engineering Laboratory, Research and Technology Institute, ITP/Sergipe, Brazil, was used throughout this study. The bacterial strain was maintained in nutrient agar slant at 4 °C. Crude coconut oil was used as inducers of lipase production and carbon sources, and was purchased at a local market. PFD, the common brand name of which is Flutec PP6, was purchased from F2 Chemicals Ltd. (Preston, Lancs, UK). The relevant physical properties of PFD at 25 °C and 1.01 9 105 Pa are as follows: density 1.917 g mL-1, vapour pressure 810 Pa, boiling point 142 °C, and oxygen solubility 127.8 mg L-1 [38]. All other chemicals used were of analytical grade and commercially available. Experimental system Experiments were performed in an aerated 4.5 L baffled bioreactor TEC-Bio-V (Tecnal, Piracicaba-Sa˜o Paulo, Brazil) with 3 L working volume. The system geometry is shown in Fig. 1. Agitation was provided by two impellers, the first of which is a six-flat-bladed Rushton turbine, and the other is a six-curve-bladed Smith turbine. Aeration between 0.5 and 1.5 vvm (air volume/liquid volume per minute) was provided by means of a ring sparger situated directly below the lower Smith turbine. The temperature was maintained at 37 ± 0.1 °C by means of a heating jacket and coiling coils. Submerged fermentation conditions Lipase production was carried out with a 10 % (v/v) inoculum of Bacillus sp. ITP-001 grown in a medium containing (%, w/v): KH2PO4 (0.1); MgSO4.7H2O (0.05); NaNO3 (0.3); yeast extract (0.6); peptone (0.13), and starch (2.0). The medium was sterilised at 121 °C for 20 min. The 4.5 L bioreactor with 3.0 L of culture medium was incubated at 37 °C and pH 7.0. The agitation speed and aeration rate were 100–300 rpm (revolutions per minute) and 0.5–1.5 vvm, respectively. After 48 h of fermentation, crude coconut oil was added (2 % v/v) into the medium to induce lipase production [10]. Experimental design and statistical analysis Response surface methodology (RSM) was used to optimise lipase production, using two independent variables (agitation speed and aeration rate) with a significant influence on the response variable (lipolytic activity). The process involved three important steps: performing the statistically designed experiments, estimating the coefficients in the mathematical model, and predicting the response and checking the adequacy of the model.
Bioprocess Biosyst Eng Fig. 1 Schematic diagram of the bioreactor
Each parameter had three levels as shown in Table 1. A first-order equation (Eq. 1), which included all interaction terms, was used to calculate the predictive response: X X Y ¼ bo þ bi xi þ bij xi xj ð1Þ where Y represents the predictive response (lipase activity expressed in U mL-1), bo is the intercept, bi the first-order coefficient, bij the coefficient interaction effect, and xi and xj the coded levels of variables Xi and Xj investigated in experiments. The variable Xi was coded as xi according the equation: xi = (Xi -Xo)/DXi, where xi is the coded value of the variable Xi (dimensionless), Xo is the value Xi at the central point level, and the step change value DXi = (high level - low level)/2. Coding was required since the factors were expressed in different units. The central composite design (CCD) was used to acquire data to fit the above equation. A 22 full factorial design, with three replicates at the central point resulting in seven experiments, was used to investigate the three Table 1 Codes and actual levels of independent variables for the design of experiments Variables
Agitation speed (rpm) Aeration rate (vvm)
Symbols
X1 X2
Coded levels -1
0
?1
100 0.5
200 1.0
300 1.5
selected variables. The levels of variables were chosen after a series of preliminary experiments based on literature [39–41]. Influence of perfluorodecalin on lipase production Initially, PFD was filter sterilised through 0.20 lm pore size filter paper and was aseptically saturated with oxygen by bubbling gas. Different concentrations of PFD (1, 5, and 10 % v/v) were added to the medium; and lipase activity, biomass, and volumetric coefficient of oxygen transfer (KLa) were observed. After the fermentation process, PFD was separated from aqueous phase by centrifugation at 10,000 rpm for 10 min. The organic phase was filtered to remove the biomass, and then distilled. Finally, the PFD was reused in the process. Statistical analysis The experimental data were subjected to multiple regression analysis using the STATISTICA Program (StatSoft, Inc., Tulsa, OK, USA) to obtain the coefficients. The F test was performed to evaluate if the model was significant. The multiple coefficients of correlation R and the determination coefficient R2 were calculated to evaluate the performance of the regression equation. Statistical testing of the model was done in the form of analysis of variance (ANOVA), which was required to test the significance and
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adequacy of the model. An effect that exceeded the vertical line (p = 0.05) was considered significant. The mean values were significantly separated using Tukey’s test [least significant difference (LSD)]. Analytical assay The KLa was determined by the dynamic gassing-out method [42], in which oxygen concentration was measured during gas saturation of the liquid layer. This method involves stopping the supply of air to the fermenter at the start of the experiments, which results in a linear decline in the concentration of dissolved oxygen due to the respiration of the microorganisms. The concentration of dissolved oxygen was followed with a polarographic oxygen electrode (Metler Toledo oxygen meter, USA) fitted with Teflon membrane and with an electrolytic solution of Na3PO4 in the cell. The balance of mass transfer leads to the following equation: C ln ð2Þ ¼KL a t C C The mass transfer coefficient KLa values were determined from the measured concentration (C) of oxygen dissolved in the culture medium aerated by circulated PFD and the known saturated oxygen concentration (C*). Ten millilitre samples from the fermentation broth were centrifuged at 3,000 rpm for 15 min. The supernatant was used to analyse starch, glucose, fat, oil and grease (FOG), and lipolytic activity. The harvested biomass was washed once with petroleum ether to remove possible traces of residual oil, and twice with distilled water, and the cells were finally dried at 105 °C for 24 h to constant weight, cooled in a desisccator and weighed. The concentration of starch was determined by Soccol’s method [43]. Assay reaction was initiated by adding 7.2 mL of iodine solution (KI 12 g L-1 and I2 0.12 g L-1) in sample of fermented broth free cell. The absorbance was determined in spectrophotometer at 620 nm. Soluble starch was used as standard. Glucose was analysed using the glucose-oxidase method (Glicose Liquiform Assay Kit, Labtest, Lagoa Santa-Minas Gerais, Brazil). FOG were analysed by infrared determination, using chlorotrifluoroethylene (S-316) as a solvent. The solvent was added at a ratio 1:1 (sample:solvent). After shaking for 2 min, the system was separated, and the organic phase (contenting the oil and grease) was used to determine the FOG using an infrared spectrophotometer (TOG/TPH analyser—Model CVR, Infracal Wilks Enterprise, USA). Lipolytic activities were assayed using the oil emulsion method according to a modification used by Zanin et al. [44]. The substrate was prepared by mixing 50 mL of oil
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olive with 50 mL of Arabic gum solution (7 %, w/v). The reaction mixture containing 5 mL of the emulsion, 2 mL of 100 mM sodium phosphate buffer (pH 7.0) and enzyme extract (1 mL) was incubated in a thermostated batch reactor for 5 min at 37 °C. A blank titration was done on a sample where the enzyme was replaced with distilled water. The reaction was stopped by the addition of approximately 0.33 g of sample in 2 mL of acetone–ethanol–water solution (1:1:1). The liberated fatty acids were titrated with 40 mM potassium hydroxide solution in the presence of phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme that liberated 1 lmol of free fatty acid per min (lmol min-1) under the assay conditions (37 °C, pH 7.0).
Results and discussion Optimisation of lipase production PFD has been applied to enhance the mass oxygen coefficient in the fermentation process to produce lipase [33, 37]. In all cases, the highest lipase production has been obtained using a high concentration of PFD. In this work, a new approach to improve oxygen transfer in the fermentation process for the production of lipase, to minimise the addition of PFD as an oxygen carrier was studied. For this reason, initially, an optimisation of agitation speed and aeration rate was performed, and then the influence of addition of PFD as an oxygen carrier was carried out. A complete factorial design and response surface methodology analysis 22 with three central points was adopted to optimise the agitation speed and aeration rate for lipase production by Bacillus sp. ITP-001. Minimum and maximum levels of each parameter were determined according to the literature [40, 41]. As shown in Table 2, the highest and lowest lipase production was detected in experiments 02 [lipolytic Table 2 Experimental design of lipase production by Bacillus sp. ITP-001 at pH 7.0 and 37 °C Run
Agitation speed (rpm)
Aeration rate (vvm)
Lipolytic activity (U mL-1) Experimental
Predicted
01
100
0.5
3,026.14
2,993.87
02
300
0.5
3,140.76
3,108.49
03
100
1.5
2,713.64
2,681.37
04
300
1.5
1,495.81
1,463.54
05
200
1.0
2,462.23
2,561.82
06 07
200 200
1.0 1.0
2,549.39 2,544.79
2,561.82 2,561.82
Bioprocess Biosyst Eng Table 3 Analysis of variance (ANOVA) for the second factorial design
R = 99.16, R2 = 98.31, F1;5;0.01 = 16.26
Source
Degrees of freedom
Sum of square
Mean square
Fcal
p Value
Aeration rate (X2)
1
957,902.6
957,902.6
398.18
0.0025
Agitation speed (X1)
1
304,268.1
304,268.1
126.48
0.0078
(1) 9 (2)
1
443,855.8
443,855.8
184.50
0.0054
Lack of fit
1
9,716.1
9.716.1
4.04
0.1822
Pure error
2
4,811.0
2.405.7
–
4
Total
6
1,720,554.0
–
–
–
activity (LA) 3,140.76 U mL-1) and 04 (LA 1,495.81 U mL-1), respectively. The independent and dependent variables were fitted by the following linear equation: LA (U mL1 Þ ¼ 2561:82 þ 275:80X1 489:36X2 333:11X1 X2
ð3Þ
where LA is the lipase activity as a function of agitation speed (X1) and aeration rate (X2). The deviation between experimental and predicted values was between 8.79 and 70.42 U mL-1, which corresponds to coefficients of variation between 0.34 and 2.80 %. The goodness of fit was evaluated by the R and R2. The above model is reliable and predictive for the experimental results, as the value of R (0.9916) indicated a good agreement between the experimental and predicted values. R2 (0.9831) was close to 1, indicating the efficacy of the model, i.e., the response model can explain 98.31 % of the total variations. ANOVA was used to check the adequacy of the fitted model (Table 3). The p values were used as a tool to check the significance of each coefficient; all term coefficients were found to be significant (p \ 0.01). The pure error calculated from the central points was very low, about 0.14 % of the total sum of squares, indicating good reproducibility of the experimental data. Based on the F test, the model is highly predictive, since the calculated F value (587.19) was 36 times higher than the listed F (16.26). The Pareto diagram shown in Fig. 2 presents the standardised effects of the independent variables and their interactions on the dependent variables. All variables and interactions were significant; however, the negative coefficient for the model components X1, X2, and X1X2 demonstrated an unfavourable effect on lipase production by Bacillus sp. ITP-001. Pandit and co-workers [22] reported that increasing the aeration rate (gas-flow rate from 19.45 to 50.34 cc/s] resulted in the best lipase production by Candida rugosa. The production of lipase by Candida cylindracea in stirred tank reactor achieved the best value (41.035 U mL-1) at 30 °C, 400 rpm and 1 vvm, which corresponds to the central point of the factorial design [45].
Fig. 2 Pareto diagram with the values for the effects
The contour and three-dimensional plot between the independent variables showed an increase in lipase production with a decrease in agitation speed and aeration rate (Fig. 3). The evaluation of the operating conditions using complete factorial design showed that for low aeration rate, the agitation speed has a positive effect in lipase production. However, for high aeration rate, the agitation speed has a negative effect, possibly due to the high turbulence caused by the combination of variables. On the other hand, the best value was found using high agitation speed (300 rpm) and low aeration rate (0.5 vvm). Based on this, the interaction of agitation speed and aeration rate presents results with distinct profile of literature, nevertheless it has important role in reducing the concentration of the PFC in subsequent experiments. The fermentation process was monitored by the time course up to 144 h (Fig. 4 and Table S1 of Supporting Information), to observe the profile of lipase production, biomass, starch consumption, glucose concentration, and TOG in the best condition found in the complete factorial design and response surface methodology analysis (300 rpm and 0.5 vvm) at 37 °C and pH 7.0. Before the addition of coconut oil (48 h), 81 % of the starch had been consumed and the microorganism was in the exponential growth phase. The high consumption of starch is associated with the hydrolytic action of cellulolytic enzyme [46], which was confirmed by varying the glucose concentration.
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Fig. 3 Response surface (a) and contour diagrams (b) for lipase activity as a function of agitation speed and aeration rate
Gupta et al. [47] also reported the highest consumption of sugar at 24 h of fermentation using Aspergillus terreus. It should be noted that after addition of coconut oil occurred the highest biomass production (7.24 g L-1 at 72 h—stationary growth phase) and lipase production increased periodically, up to 120 h (LA -1 3,140.76 U mL —death phase), and then the lipolytic activity decreased, probably due to depletion of easily assimilated substrate (90 % consumption) and complex substrate (70 % of coconut oil in terms of TOG). In accordance with the lipase production by Burkholderia sp. C20 [40], Pseudomonas aeruginosa PseA [48], and Aspergillus niger [49], the process using Bacillus sp. ITP001 was also associated with the growth of the microorganism. In this way, the KLa decreased with the increase of biomass, reaching the lowest value (19.95 h-1 or 46 % of the initial value) from the beginning of the stationary growth phase to the end of the death phase. This observation allows us to infer that oxygen is still a limiting factor of cell growth. Influence of PFD addition on lipase production It is known that PFD plays an important role in oxygen transference in submerged fermentation [31]. For this reason, the effect of adding PFD (1, 5, and 10 % v/v) as an oxygen carrier was studied for lipase production using Bacillus sp. ITP-001 (Fig. 5 and Table S2 of Supporting Information). Addition of PFD enhanced oxygen solubility in the fermentation medium and allowed the highest lipase production (3,509.46 ± 44.75 U mL-1; 11.74 % increase compared with without PFD) at the highest PFD
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concentration (10 %), corroborating with the results of Coelho et al. [35] in lipase production using Y. lipolitica. The addition of PFD decreased the biomass. At 120 h, the biomass concentration was 3.18 ± 0.51 g L-1 (control—no PFD), 2.64 ± 0.20 g L-1 (1 % PFD), 2.02 ± 0.03 g L-1 (5 % PFD) and 2.52 ± 0.2 g L-1 (10 % PFD). Elibol and Ozer [37] observed that the inclusion of 10 % PFD in the fermentation medium results in higher lipase production, however, the biomass concentration did not change significantly. However, a previous optimisation of fermentation conditions (aeration rate and agitation speed) resulted in a greater efficiency of oxygen transfer. In this way, lipase production with PFD concentrations of above 1 % (v/v) was statistically similar when the Tukey’s test was applied, meaning that an excess of PFD did not result in a higher lipase production in this case. The commercial value of PFD is approximately 300.00€/kg PFD, that is, a 3 L fermentation with the addition of 1 % PFD corresponds to an increase of 17.25€ to process costs, while an incorporation of 10 % corresponds to an increase of 172.53€. A new approach to the use of PFD, starting with optimisation in terms of aeration and agitation followed by the study of the influence of PFD addition allowed an economy of 90 % of the process costs. Reuse of PFD in lipase production Normally, the addition of perfluorocarbon, like PFD, in the fermentation process is accompanied by a surfactant such as Pluronic F-68 and Pluronic F-127 [32]. According to Marrucho and co-workers [50], the stability of
Bioprocess Biosyst Eng
Fig. 5 Effect of PFD concentration (0 % unfilled circle, 1 % filled circle, 5 % filled square, and 10 % unfilled square) on lipase production. Same letters in the same columns do not differ significantly (p B 0.05)
Fig. 4 Time course of lipase production in submerged fermentation using Bacillus sp. ITP-001 at 37 °C, pH 7.0, 300 rpm, and 0.5 vvm. a Profile of starch consumption (filled square) and glucose concentration (unfilled square); b profile of KLa (unfilled square) and biomass (unfilled circle); c profile of TOG (filled diamond) and enzymatic activity (unfilled diamond)
perfluorocarbon in a water emulsion depends on the temperature, perfluorocarbon type, composition of the aqueous phase, and the surfactant used. In our case, after the fermentation process, the broth was brought to repose and the phase was gently separated because the composition of medium was surfactant free. In a similar fermentation process, Bacillus sp. ITP-001 [51] and B. subtilis [52] produced surfactin; however, we infer that the surfactant production or the temperature of the process (37 °C) was not enough to stabilise the emulsion.
Fig. 6 Assessment of reuse of perfluorodecalin (1 % v/v) in lipase production. Same letters in the same columns do not differ significantly (p B 0.05)
The PFD was filtered and reused in a new fermentation, and the influence of reuse on lipase production is shown in Fig. 6 (Table S3 of Supporting Information). It was observed that there was no statistically significant difference between the initial fermentation and the first reuse, or between the two subsequent reuses. Therefore, it is understood that PFD can be reused twice without affecting lipase production. A simplified economic analysis of the fermentation process with 1 % PFD allows us to infer that
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the use of PFC in three fermentation processes represents 67 % savings in the cost of production.
Conclusions A new approach to implementation of PFD as a carrier of oxygen to improve the production of lipase from Bacillus was initially performed by optimisation of agitation speed and aeration rate. The highest lipase activity was 3,140.76 U mL-1 at 300 rpm and 0.5 vvm. Then, the influence of PFD on lipase production was studied, and no statistically significant difference was observed using PFD between 1 and 10 %, because the parameters that promoted the transfer of oxygen had already been optimised, in other words, an economy of 90 % compared with the processes applying 10 % PFC reported in the literature. The PFC was distilled and used three times without a statistically significant difference in lipase production, corresponding to an economy of 67 % compared with systems with single use of 1 % PFC.
References 1. Treichel H, Oliveira D, Mazutti MA, Di Luccio M, Oliveira JV (2010) Food Bioprocess Tech 3:182–196 2. Gupta R, Gupta N, Rathi P (2004) Appl Microbiol Biotechnol 64:763–781 3. Kamini NR, Fujii T, Kurosu T, Iefuji H (2000) Process Biochem 36:317–324 4. Ramani K, John KL, Ramakrishnan M, Sekaran G (2010) Process Biochem 45:1683–1691 5. Aravindan R, Anbumathi P, Viruthagiri T (2007) Indian J Biotechnol 6:141–158 6. Liu R, Jiang X, Mou H, Guan H, Hwang HM, Li X (2009) Biochem Eng J 46:265–270 7. Gotor-Ferna´ndez V, Brieva R, Gotor V (2006) J Mol Catal B Enzym 40:111–120 8. Tongboriboon K, Cheirsilp B, H-Kittikun A (2010) J Mol Catal B Enzym 67:52–59 9. Romdhane I, Fendri A, Gargouri Y, Gargouri A, Belghith H (2010) Biochem Eng J 53:112–120 10. Feitosa IC, Barbosa JMP, Orellana SC, Lima AS, Soares CMF (2010) Acta Sci-Technol 32:27–31 11. Fang HHP, Liang DW, Zhang T, Liu Y (2006) Water Res 40:427–434 12. Lima VMG, Krieger N, Mitchell DA, Fontana LD (2004) Biochem Eng J 18:65–71 13. Guncheva M, Zhiryakova D (2011) J Mol Catal B Enzym 68:1–21 14. Sifour M, Zaghloul TI, Saeed HM, Bereka MM, Abdel-Fattah YR (2010) New Biotechnol 27:330–336 15. Gutarra MLE, Godoy MG, Maugeri F, Rodrigues MI, Freire DMG, Castilho LR (2009) Bioresource Technol 100:5249–5254 16. Teng Y, Xua Y, Wang D (2009) J Mol Catal B Enzym 57:292–298 17. Coradi GV, Visitac¸a˜o VL, Lima EA, Saito LYT, Palmieri DA, Takita MA, Oliva-Neto P, Lima VMG (2013) Ann Microbiol 63:533–540
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18. Gupta N, Sahai V, Gupta R (2007) Process Biochem 42:518–526 19. Burkert JFM, Maugeri F, Rodrigues MI (2004) Bioresource Technol 91:77–84 20. Liu CH, Chen CY, Wang YW, Chang JS (2011) Biochem Eng J 58–59:96–102 21. Potumarthi R, Subhakar C, Vanajakshi J, Jetty A (2008) Appl Biochem Biotechnol 151:700–710 22. Puthli MS, Rathod VK, Pandit AB (2006) Biochem Eng J 27:287–294 23. Essamri M, Deyris V, Comeau L (1998) J Biotechnol 60:97–103 24. Xu H, Dou W, Xu H, Zhang X, Rao Z, Shi Z, Xu Z (2009) Biochem Eng J 43:41–51 25. Martı´nez I, Bennett GN, San KY (2010) Metab Eng 12:499–509 26. Helm I, Jalukse L, Vilbaste M, Leito I (2009) Anal Chim Acta 648:167–173 27. Song P, Chen C, Tian Q, Lin M, Huang H, Li S (2013) Biochem Eng J 71:1–10 28. van Sonsbeek HM, Beeftink HH, Tramper J (1993) Enzyme Microb Tech 15:722–729 29. van der Meer AB, Beenackers AACM, Burghard R, Mulder NH, Fok JJ (1992) Chem Eng Sci 47:2369–2374 30. De´zil E, Comeau Y, Villemur R (1999) Biodegradation 10:219–233 31. Pilarek M, Szewczyk KW (2008) Biochem Eng J 41:38–42 32. Fraker CA, Mendez AJ, Inverardi L, Ricordi C, Stabler CL (2012) Colloid Surf B 98:26–35 33. Amaral PFF, Freire MG, Rocha-Lea˜o MHM, Marrucho IM, Coutinho JAP, Coelho MAZ (2008) Biotechnol Bioeng 99:588–598 34. Lowe KC (2002) J Fluorine Chem 118:19–26 35. Amaral PFF, Almeida APR, Peixoto T, Rocha-Lea˜o MHM, Coutinho JAP, Coelho MAZ (2007) World J Microb Biot 23:339–344 36. Amaral PFF, Rocha-Lea˜o MH, Marrucho IM, Coutinho JAP, Coelho MA (2006) J Chem Technol Biotechnol 81:1368–1374 37. Elibol M, Ozer D (2000) Process Biochem 36:325–329 38. Dias AMA, Cac¸o AI, Coutinho JAP, Santos LMNBF, Pin˜eiro MM, Vega LF, Gomes MFC, Marrucho IM (2004) Fluid Phase Equilib 225:39–47 39. Freire DM, Teles EMF, Bon EPS, Sant’Anna GL Jr (1997) Appl Biochem Biotechnol 63–65:409–421 40. Liu CH, Huang CC, Wanga YW, Chang JS (2012) J Taiwan Inst Chem E 43:511–516 41. Burkert JFM, Maldonado RR, Maugieri F, Rodrigues MI (2005) J Chem Technol Biot 80:61–67 42. Ni X, Gao S, Cumming RH, Pritchard DW (1995) Chem Eng Sci 50:2127–2136 43. Soccol CR, Iloku I, Marin B, Roussos S, Raimbault M (1994) Food Sci Technol 3:320–323 44. Soares CMF, Castro HF, Moraes FF, Zanin GM (1999) Appl Biochem Biotech 79:745–758 45. Salihu A, Alam MZ, Abdulkarim MI, Salleh HM (2011) J Mol Catal B Enzym 73:187–192 46. Carvalho NB, Souza RL, Castro HF, Zanin GM, Lima AS, Soares CMF (2008) Appl Biochem Biotech 150:25–32 47. Gulati R, Saxena RK, Gupta R (2000) Process Biochem 36:149–155 48. Mahanta N, Gupta A, Khare SH (2008) Bioresour Technol 99:1729–1735 49. Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV (2002) Process Biochem 38:715–721 50. Freire MG, Dias AMA, Coelho MAZ, Coutinho JAP, Marrucho IM (2005) J Colloid Interf Sci 286:224–232 51. Silva MTS (2013) MSc thesis, Tiradentes University, AracajuSergipe, Brazil 52. Wang SL, Yeh PY (2006) Process Biochem 41:1545–1552
ORIGINAL PAPER
New strategy to apply perfluorodecalin as an oxygen carrier in lipase production: minimisation and reuse Erika Souza Vieira • Taˆmara Karoline de Oliveira Fontes • Matheus Mendonc¸a Pereira • Hofsky Vieira Alexandre • Daniel Pereira da Silva ´ lvaro Silva Lima Cleide Mara Faria Soares • A
•
Received: 6 March 2014 / Accepted: 15 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract A novel strategy for the production of lipase by Bacillus sp. ITP-001 in a stirred tank fermenter using perfluorodecalin (PFD) was studied. Firstly, a response surface methodology 22 with three central points was employed to optimise the effect of agitation speed and aeration rate in lipase production. According to the response from the experimental designs, 300 rpm (revolutions per minute) and 0.5 vvm (air volume/liquid volume per minute) were found to provide the best condition (lipolytic activity: LA = 3,140.76 U mL-1). Then, the influence of PFD concentration on the fermentation process was evaluated. Incorporation of PFD at all concentrations above 1 % had no statistically significant influence on lipase production, that is, the previous optimisation allowed the reduction of the amount of PFD added besides increasing lipase production. Furthermore, PFD could be used in three sequential fermentations without altering the statistical production of lipase, reducing by 67 % the cost of PFD addition.
E. S. Vieira T. K. de Oliveira Fontes ´ . S. Lima (&) H. V. Alexandre C. M. F. Soares A Programa de Po´s-Graduac¸a˜o em Engenharia de Processos, Universidade Tiradentes, Av. Murilo Dantas 300, Farolaˆndia, Aracaju 49032-490, Brazil e-mail: [email protected] ´ . S. Lima M. M. Pereira H. V. Alexandre C. M. F. Soares A Instituto de Tecnologia e Pesquisa, Av. Murilo Dantas 300, Farolaˆndia, Aracaju 49032-490, Brazil D. P. da Silva Nu´cleo de Engenharia de Produc¸a˜o, Universidade Federal de Sergipe, Av. Marechal Rondon s/n, Jardim Rosa Elze, Sao Cristovao 49100-000, Brazil
Keywords Lipase Oxygen carrier Perfluorodecalin Experimental design Fermentation
Introduction Lipases (triacylglycerol acylhydrolase, E.C.3.1.1.3) constitute an important group of enzymes associated with lipid metabolism, and may be obtained from animal, plant, and microbial sources [1]. These enzymes are hydrolases, which act under aqueous conditions on the carboxyl ester bonds present in triacylglycerides to liberate fatty acids and glycerol [2, 3]. Microbial lipases are immensely applied in various facets of industrial biotechnology such as in fat and oil hydrolysis [4], food industries [5], detergent industries [6], pharmaceutical industries [7], and biodiesel synthesis [8] due to their stability, selectivity, and broad substrate specificity [9]. A considerable number of microorganisms have been used to produce lipases [10–12], especially the genus Bacillus [13, 14]. Solid-state fermentation, SSF [15], and submerged fermentation, SmF0 [16], are effective techniques for microbial lipase production. However, SmF has been used traditionally because the recovery of extracellular enzymes and determination of biomass can be performed by simple filtration or centrifugation [17]. For SmF, the literature presents several studies to optimize and devise strategies of lipase production in terms of medium composition such as carbon and nitrogen source [18, 19], and operational conditions such as temperature, pH value, aeration rate (concentration of dissolved oxygen), and agitation speed [20–23]. In this context, the literature reports that oxygen is required as a final electron acceptor to reoxidise nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADH2) and generate ATP for
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metabolism [24]. Therefore, low oxygen levels induce or repress transcription, synthesis of different enzymes, or both, thereby affecting all metabolism and, consequently, product yield and productivity [25]. Moreover, the solubility of oxygen in water is low (approximately 8.82 mg L-1 at 20 °C) [26] and, consequently, the oxygen mass transfer is also low. Therefore, oxygen is an indispensable raw material that must be supplied in large amounts during industrial aerobic fermentation [27]. A way to minimise the low oxygen mass transfer in SmF is by the use of a water-immiscible liquid, i.e., a nonconventional organic solvent [28], which presents high oxygen solubility. This strategy is called a two-liquidphase system, in spite of four phases are present in the system (hydrophobic, aqueous, gas phases and cells) [29]. According to Villemur et al. [30], this approach can be divided into two categories; in the first one, the waterimmiscible liquid is the substrate for the microorganisms (petroleum hydrocarbon, n-alkanes, or vegetable oil), and in the second, the non-aqueous liquid is added to the bioreactor to enhance oxygen mass transfer (silicone oil, hexadecane, or perfluorochemicals (PFCs)]. PFCs are derivatives of aliphatic, cyclic, or polycyclic hydrocarbons in which most or all of the hydrogen atoms have been replaced by fluorine, increasing the molecular mass [31]. Furthermore, PFCs have an exceptional thermal stability; they are chemically inert, non-toxic towards cells, and have specific densities of about twice that of water and 10–20 times higher oxygen solubility than water [32, 33]. One commonly used PFC is perfluorodecalin, PFD (C10F18), a bicyclic perfluorinated alkane [34]. As previously mentioned, the use of a second liquid phase, namely PFD, an oxygen-carrying component, in the culture media increases the availability of oxygen to the microorganism [35]. Coelho and co-workers reported that PFC addition (20 %, v/v) benefited lipase production that increased 11-fold [36], corroborating the results from Elibol and Ozer [37], which found the highest lipase production with 10 % (v/v) PFC. The highest PFD concentration has the disadvantage the increasing the cost of enzyme production. For this reason, the goal of this work was to search for a new approach to minimise the use of PFD in multiphase fermentation, by optimising the agitation speed of parameters and aeration rate, then checking the effect of the addition of perfluorodecalin and their potential for reuse. Approach has not yet reported in the literature.
Materials and methods Microorganism and chemicals Bacillus sp. ITP-001 strain, isolated from a petroleumcontaminated soil and belonging to a culture collection of
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Bioprocess Engineering Laboratory, Research and Technology Institute, ITP/Sergipe, Brazil, was used throughout this study. The bacterial strain was maintained in nutrient agar slant at 4 °C. Crude coconut oil was used as inducers of lipase production and carbon sources, and was purchased at a local market. PFD, the common brand name of which is Flutec PP6, was purchased from F2 Chemicals Ltd. (Preston, Lancs, UK). The relevant physical properties of PFD at 25 °C and 1.01 9 105 Pa are as follows: density 1.917 g mL-1, vapour pressure 810 Pa, boiling point 142 °C, and oxygen solubility 127.8 mg L-1 [38]. All other chemicals used were of analytical grade and commercially available. Experimental system Experiments were performed in an aerated 4.5 L baffled bioreactor TEC-Bio-V (Tecnal, Piracicaba-Sa˜o Paulo, Brazil) with 3 L working volume. The system geometry is shown in Fig. 1. Agitation was provided by two impellers, the first of which is a six-flat-bladed Rushton turbine, and the other is a six-curve-bladed Smith turbine. Aeration between 0.5 and 1.5 vvm (air volume/liquid volume per minute) was provided by means of a ring sparger situated directly below the lower Smith turbine. The temperature was maintained at 37 ± 0.1 °C by means of a heating jacket and coiling coils. Submerged fermentation conditions Lipase production was carried out with a 10 % (v/v) inoculum of Bacillus sp. ITP-001 grown in a medium containing (%, w/v): KH2PO4 (0.1); MgSO4.7H2O (0.05); NaNO3 (0.3); yeast extract (0.6); peptone (0.13), and starch (2.0). The medium was sterilised at 121 °C for 20 min. The 4.5 L bioreactor with 3.0 L of culture medium was incubated at 37 °C and pH 7.0. The agitation speed and aeration rate were 100–300 rpm (revolutions per minute) and 0.5–1.5 vvm, respectively. After 48 h of fermentation, crude coconut oil was added (2 % v/v) into the medium to induce lipase production [10]. Experimental design and statistical analysis Response surface methodology (RSM) was used to optimise lipase production, using two independent variables (agitation speed and aeration rate) with a significant influence on the response variable (lipolytic activity). The process involved three important steps: performing the statistically designed experiments, estimating the coefficients in the mathematical model, and predicting the response and checking the adequacy of the model.
Bioprocess Biosyst Eng Fig. 1 Schematic diagram of the bioreactor
Each parameter had three levels as shown in Table 1. A first-order equation (Eq. 1), which included all interaction terms, was used to calculate the predictive response: X X Y ¼ bo þ bi xi þ bij xi xj ð1Þ where Y represents the predictive response (lipase activity expressed in U mL-1), bo is the intercept, bi the first-order coefficient, bij the coefficient interaction effect, and xi and xj the coded levels of variables Xi and Xj investigated in experiments. The variable Xi was coded as xi according the equation: xi = (Xi -Xo)/DXi, where xi is the coded value of the variable Xi (dimensionless), Xo is the value Xi at the central point level, and the step change value DXi = (high level - low level)/2. Coding was required since the factors were expressed in different units. The central composite design (CCD) was used to acquire data to fit the above equation. A 22 full factorial design, with three replicates at the central point resulting in seven experiments, was used to investigate the three Table 1 Codes and actual levels of independent variables for the design of experiments Variables
Agitation speed (rpm) Aeration rate (vvm)
Symbols
X1 X2
Coded levels -1
0
?1
100 0.5
200 1.0
300 1.5
selected variables. The levels of variables were chosen after a series of preliminary experiments based on literature [39–41]. Influence of perfluorodecalin on lipase production Initially, PFD was filter sterilised through 0.20 lm pore size filter paper and was aseptically saturated with oxygen by bubbling gas. Different concentrations of PFD (1, 5, and 10 % v/v) were added to the medium; and lipase activity, biomass, and volumetric coefficient of oxygen transfer (KLa) were observed. After the fermentation process, PFD was separated from aqueous phase by centrifugation at 10,000 rpm for 10 min. The organic phase was filtered to remove the biomass, and then distilled. Finally, the PFD was reused in the process. Statistical analysis The experimental data were subjected to multiple regression analysis using the STATISTICA Program (StatSoft, Inc., Tulsa, OK, USA) to obtain the coefficients. The F test was performed to evaluate if the model was significant. The multiple coefficients of correlation R and the determination coefficient R2 were calculated to evaluate the performance of the regression equation. Statistical testing of the model was done in the form of analysis of variance (ANOVA), which was required to test the significance and
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adequacy of the model. An effect that exceeded the vertical line (p = 0.05) was considered significant. The mean values were significantly separated using Tukey’s test [least significant difference (LSD)]. Analytical assay The KLa was determined by the dynamic gassing-out method [42], in which oxygen concentration was measured during gas saturation of the liquid layer. This method involves stopping the supply of air to the fermenter at the start of the experiments, which results in a linear decline in the concentration of dissolved oxygen due to the respiration of the microorganisms. The concentration of dissolved oxygen was followed with a polarographic oxygen electrode (Metler Toledo oxygen meter, USA) fitted with Teflon membrane and with an electrolytic solution of Na3PO4 in the cell. The balance of mass transfer leads to the following equation: C ln ð2Þ ¼KL a t C C The mass transfer coefficient KLa values were determined from the measured concentration (C) of oxygen dissolved in the culture medium aerated by circulated PFD and the known saturated oxygen concentration (C*). Ten millilitre samples from the fermentation broth were centrifuged at 3,000 rpm for 15 min. The supernatant was used to analyse starch, glucose, fat, oil and grease (FOG), and lipolytic activity. The harvested biomass was washed once with petroleum ether to remove possible traces of residual oil, and twice with distilled water, and the cells were finally dried at 105 °C for 24 h to constant weight, cooled in a desisccator and weighed. The concentration of starch was determined by Soccol’s method [43]. Assay reaction was initiated by adding 7.2 mL of iodine solution (KI 12 g L-1 and I2 0.12 g L-1) in sample of fermented broth free cell. The absorbance was determined in spectrophotometer at 620 nm. Soluble starch was used as standard. Glucose was analysed using the glucose-oxidase method (Glicose Liquiform Assay Kit, Labtest, Lagoa Santa-Minas Gerais, Brazil). FOG were analysed by infrared determination, using chlorotrifluoroethylene (S-316) as a solvent. The solvent was added at a ratio 1:1 (sample:solvent). After shaking for 2 min, the system was separated, and the organic phase (contenting the oil and grease) was used to determine the FOG using an infrared spectrophotometer (TOG/TPH analyser—Model CVR, Infracal Wilks Enterprise, USA). Lipolytic activities were assayed using the oil emulsion method according to a modification used by Zanin et al. [44]. The substrate was prepared by mixing 50 mL of oil
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olive with 50 mL of Arabic gum solution (7 %, w/v). The reaction mixture containing 5 mL of the emulsion, 2 mL of 100 mM sodium phosphate buffer (pH 7.0) and enzyme extract (1 mL) was incubated in a thermostated batch reactor for 5 min at 37 °C. A blank titration was done on a sample where the enzyme was replaced with distilled water. The reaction was stopped by the addition of approximately 0.33 g of sample in 2 mL of acetone–ethanol–water solution (1:1:1). The liberated fatty acids were titrated with 40 mM potassium hydroxide solution in the presence of phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme that liberated 1 lmol of free fatty acid per min (lmol min-1) under the assay conditions (37 °C, pH 7.0).
Results and discussion Optimisation of lipase production PFD has been applied to enhance the mass oxygen coefficient in the fermentation process to produce lipase [33, 37]. In all cases, the highest lipase production has been obtained using a high concentration of PFD. In this work, a new approach to improve oxygen transfer in the fermentation process for the production of lipase, to minimise the addition of PFD as an oxygen carrier was studied. For this reason, initially, an optimisation of agitation speed and aeration rate was performed, and then the influence of addition of PFD as an oxygen carrier was carried out. A complete factorial design and response surface methodology analysis 22 with three central points was adopted to optimise the agitation speed and aeration rate for lipase production by Bacillus sp. ITP-001. Minimum and maximum levels of each parameter were determined according to the literature [40, 41]. As shown in Table 2, the highest and lowest lipase production was detected in experiments 02 [lipolytic Table 2 Experimental design of lipase production by Bacillus sp. ITP-001 at pH 7.0 and 37 °C Run
Agitation speed (rpm)
Aeration rate (vvm)
Lipolytic activity (U mL-1) Experimental
Predicted
01
100
0.5
3,026.14
2,993.87
02
300
0.5
3,140.76
3,108.49
03
100
1.5
2,713.64
2,681.37
04
300
1.5
1,495.81
1,463.54
05
200
1.0
2,462.23
2,561.82
06 07
200 200
1.0 1.0
2,549.39 2,544.79
2,561.82 2,561.82
Bioprocess Biosyst Eng Table 3 Analysis of variance (ANOVA) for the second factorial design
R = 99.16, R2 = 98.31, F1;5;0.01 = 16.26
Source
Degrees of freedom
Sum of square
Mean square
Fcal
p Value
Aeration rate (X2)
1
957,902.6
957,902.6
398.18
0.0025
Agitation speed (X1)
1
304,268.1
304,268.1
126.48
0.0078
(1) 9 (2)
1
443,855.8
443,855.8
184.50
0.0054
Lack of fit
1
9,716.1
9.716.1
4.04
0.1822
Pure error
2
4,811.0
2.405.7
–
4
Total
6
1,720,554.0
–
–
–
activity (LA) 3,140.76 U mL-1) and 04 (LA 1,495.81 U mL-1), respectively. The independent and dependent variables were fitted by the following linear equation: LA (U mL1 Þ ¼ 2561:82 þ 275:80X1 489:36X2 333:11X1 X2
ð3Þ
where LA is the lipase activity as a function of agitation speed (X1) and aeration rate (X2). The deviation between experimental and predicted values was between 8.79 and 70.42 U mL-1, which corresponds to coefficients of variation between 0.34 and 2.80 %. The goodness of fit was evaluated by the R and R2. The above model is reliable and predictive for the experimental results, as the value of R (0.9916) indicated a good agreement between the experimental and predicted values. R2 (0.9831) was close to 1, indicating the efficacy of the model, i.e., the response model can explain 98.31 % of the total variations. ANOVA was used to check the adequacy of the fitted model (Table 3). The p values were used as a tool to check the significance of each coefficient; all term coefficients were found to be significant (p \ 0.01). The pure error calculated from the central points was very low, about 0.14 % of the total sum of squares, indicating good reproducibility of the experimental data. Based on the F test, the model is highly predictive, since the calculated F value (587.19) was 36 times higher than the listed F (16.26). The Pareto diagram shown in Fig. 2 presents the standardised effects of the independent variables and their interactions on the dependent variables. All variables and interactions were significant; however, the negative coefficient for the model components X1, X2, and X1X2 demonstrated an unfavourable effect on lipase production by Bacillus sp. ITP-001. Pandit and co-workers [22] reported that increasing the aeration rate (gas-flow rate from 19.45 to 50.34 cc/s] resulted in the best lipase production by Candida rugosa. The production of lipase by Candida cylindracea in stirred tank reactor achieved the best value (41.035 U mL-1) at 30 °C, 400 rpm and 1 vvm, which corresponds to the central point of the factorial design [45].
Fig. 2 Pareto diagram with the values for the effects
The contour and three-dimensional plot between the independent variables showed an increase in lipase production with a decrease in agitation speed and aeration rate (Fig. 3). The evaluation of the operating conditions using complete factorial design showed that for low aeration rate, the agitation speed has a positive effect in lipase production. However, for high aeration rate, the agitation speed has a negative effect, possibly due to the high turbulence caused by the combination of variables. On the other hand, the best value was found using high agitation speed (300 rpm) and low aeration rate (0.5 vvm). Based on this, the interaction of agitation speed and aeration rate presents results with distinct profile of literature, nevertheless it has important role in reducing the concentration of the PFC in subsequent experiments. The fermentation process was monitored by the time course up to 144 h (Fig. 4 and Table S1 of Supporting Information), to observe the profile of lipase production, biomass, starch consumption, glucose concentration, and TOG in the best condition found in the complete factorial design and response surface methodology analysis (300 rpm and 0.5 vvm) at 37 °C and pH 7.0. Before the addition of coconut oil (48 h), 81 % of the starch had been consumed and the microorganism was in the exponential growth phase. The high consumption of starch is associated with the hydrolytic action of cellulolytic enzyme [46], which was confirmed by varying the glucose concentration.
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Fig. 3 Response surface (a) and contour diagrams (b) for lipase activity as a function of agitation speed and aeration rate
Gupta et al. [47] also reported the highest consumption of sugar at 24 h of fermentation using Aspergillus terreus. It should be noted that after addition of coconut oil occurred the highest biomass production (7.24 g L-1 at 72 h—stationary growth phase) and lipase production increased periodically, up to 120 h (LA -1 3,140.76 U mL —death phase), and then the lipolytic activity decreased, probably due to depletion of easily assimilated substrate (90 % consumption) and complex substrate (70 % of coconut oil in terms of TOG). In accordance with the lipase production by Burkholderia sp. C20 [40], Pseudomonas aeruginosa PseA [48], and Aspergillus niger [49], the process using Bacillus sp. ITP001 was also associated with the growth of the microorganism. In this way, the KLa decreased with the increase of biomass, reaching the lowest value (19.95 h-1 or 46 % of the initial value) from the beginning of the stationary growth phase to the end of the death phase. This observation allows us to infer that oxygen is still a limiting factor of cell growth. Influence of PFD addition on lipase production It is known that PFD plays an important role in oxygen transference in submerged fermentation [31]. For this reason, the effect of adding PFD (1, 5, and 10 % v/v) as an oxygen carrier was studied for lipase production using Bacillus sp. ITP-001 (Fig. 5 and Table S2 of Supporting Information). Addition of PFD enhanced oxygen solubility in the fermentation medium and allowed the highest lipase production (3,509.46 ± 44.75 U mL-1; 11.74 % increase compared with without PFD) at the highest PFD
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concentration (10 %), corroborating with the results of Coelho et al. [35] in lipase production using Y. lipolitica. The addition of PFD decreased the biomass. At 120 h, the biomass concentration was 3.18 ± 0.51 g L-1 (control—no PFD), 2.64 ± 0.20 g L-1 (1 % PFD), 2.02 ± 0.03 g L-1 (5 % PFD) and 2.52 ± 0.2 g L-1 (10 % PFD). Elibol and Ozer [37] observed that the inclusion of 10 % PFD in the fermentation medium results in higher lipase production, however, the biomass concentration did not change significantly. However, a previous optimisation of fermentation conditions (aeration rate and agitation speed) resulted in a greater efficiency of oxygen transfer. In this way, lipase production with PFD concentrations of above 1 % (v/v) was statistically similar when the Tukey’s test was applied, meaning that an excess of PFD did not result in a higher lipase production in this case. The commercial value of PFD is approximately 300.00€/kg PFD, that is, a 3 L fermentation with the addition of 1 % PFD corresponds to an increase of 17.25€ to process costs, while an incorporation of 10 % corresponds to an increase of 172.53€. A new approach to the use of PFD, starting with optimisation in terms of aeration and agitation followed by the study of the influence of PFD addition allowed an economy of 90 % of the process costs. Reuse of PFD in lipase production Normally, the addition of perfluorocarbon, like PFD, in the fermentation process is accompanied by a surfactant such as Pluronic F-68 and Pluronic F-127 [32]. According to Marrucho and co-workers [50], the stability of
Bioprocess Biosyst Eng
Fig. 5 Effect of PFD concentration (0 % unfilled circle, 1 % filled circle, 5 % filled square, and 10 % unfilled square) on lipase production. Same letters in the same columns do not differ significantly (p B 0.05)
Fig. 4 Time course of lipase production in submerged fermentation using Bacillus sp. ITP-001 at 37 °C, pH 7.0, 300 rpm, and 0.5 vvm. a Profile of starch consumption (filled square) and glucose concentration (unfilled square); b profile of KLa (unfilled square) and biomass (unfilled circle); c profile of TOG (filled diamond) and enzymatic activity (unfilled diamond)
perfluorocarbon in a water emulsion depends on the temperature, perfluorocarbon type, composition of the aqueous phase, and the surfactant used. In our case, after the fermentation process, the broth was brought to repose and the phase was gently separated because the composition of medium was surfactant free. In a similar fermentation process, Bacillus sp. ITP-001 [51] and B. subtilis [52] produced surfactin; however, we infer that the surfactant production or the temperature of the process (37 °C) was not enough to stabilise the emulsion.
Fig. 6 Assessment of reuse of perfluorodecalin (1 % v/v) in lipase production. Same letters in the same columns do not differ significantly (p B 0.05)
The PFD was filtered and reused in a new fermentation, and the influence of reuse on lipase production is shown in Fig. 6 (Table S3 of Supporting Information). It was observed that there was no statistically significant difference between the initial fermentation and the first reuse, or between the two subsequent reuses. Therefore, it is understood that PFD can be reused twice without affecting lipase production. A simplified economic analysis of the fermentation process with 1 % PFD allows us to infer that
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Bioprocess Biosyst Eng
the use of PFC in three fermentation processes represents 67 % savings in the cost of production.
Conclusions A new approach to implementation of PFD as a carrier of oxygen to improve the production of lipase from Bacillus was initially performed by optimisation of agitation speed and aeration rate. The highest lipase activity was 3,140.76 U mL-1 at 300 rpm and 0.5 vvm. Then, the influence of PFD on lipase production was studied, and no statistically significant difference was observed using PFD between 1 and 10 %, because the parameters that promoted the transfer of oxygen had already been optimised, in other words, an economy of 90 % compared with the processes applying 10 % PFC reported in the literature. The PFC was distilled and used three times without a statistically significant difference in lipase production, corresponding to an economy of 67 % compared with systems with single use of 1 % PFC.
References 1. Treichel H, Oliveira D, Mazutti MA, Di Luccio M, Oliveira JV (2010) Food Bioprocess Tech 3:182–196 2. Gupta R, Gupta N, Rathi P (2004) Appl Microbiol Biotechnol 64:763–781 3. Kamini NR, Fujii T, Kurosu T, Iefuji H (2000) Process Biochem 36:317–324 4. Ramani K, John KL, Ramakrishnan M, Sekaran G (2010) Process Biochem 45:1683–1691 5. Aravindan R, Anbumathi P, Viruthagiri T (2007) Indian J Biotechnol 6:141–158 6. Liu R, Jiang X, Mou H, Guan H, Hwang HM, Li X (2009) Biochem Eng J 46:265–270 7. Gotor-Ferna´ndez V, Brieva R, Gotor V (2006) J Mol Catal B Enzym 40:111–120 8. Tongboriboon K, Cheirsilp B, H-Kittikun A (2010) J Mol Catal B Enzym 67:52–59 9. Romdhane I, Fendri A, Gargouri Y, Gargouri A, Belghith H (2010) Biochem Eng J 53:112–120 10. Feitosa IC, Barbosa JMP, Orellana SC, Lima AS, Soares CMF (2010) Acta Sci-Technol 32:27–31 11. Fang HHP, Liang DW, Zhang T, Liu Y (2006) Water Res 40:427–434 12. Lima VMG, Krieger N, Mitchell DA, Fontana LD (2004) Biochem Eng J 18:65–71 13. Guncheva M, Zhiryakova D (2011) J Mol Catal B Enzym 68:1–21 14. Sifour M, Zaghloul TI, Saeed HM, Bereka MM, Abdel-Fattah YR (2010) New Biotechnol 27:330–336 15. Gutarra MLE, Godoy MG, Maugeri F, Rodrigues MI, Freire DMG, Castilho LR (2009) Bioresource Technol 100:5249–5254 16. Teng Y, Xua Y, Wang D (2009) J Mol Catal B Enzym 57:292–298 17. Coradi GV, Visitac¸a˜o VL, Lima EA, Saito LYT, Palmieri DA, Takita MA, Oliva-Neto P, Lima VMG (2013) Ann Microbiol 63:533–540
123
18. Gupta N, Sahai V, Gupta R (2007) Process Biochem 42:518–526 19. Burkert JFM, Maugeri F, Rodrigues MI (2004) Bioresource Technol 91:77–84 20. Liu CH, Chen CY, Wang YW, Chang JS (2011) Biochem Eng J 58–59:96–102 21. Potumarthi R, Subhakar C, Vanajakshi J, Jetty A (2008) Appl Biochem Biotechnol 151:700–710 22. Puthli MS, Rathod VK, Pandit AB (2006) Biochem Eng J 27:287–294 23. Essamri M, Deyris V, Comeau L (1998) J Biotechnol 60:97–103 24. Xu H, Dou W, Xu H, Zhang X, Rao Z, Shi Z, Xu Z (2009) Biochem Eng J 43:41–51 25. Martı´nez I, Bennett GN, San KY (2010) Metab Eng 12:499–509 26. Helm I, Jalukse L, Vilbaste M, Leito I (2009) Anal Chim Acta 648:167–173 27. Song P, Chen C, Tian Q, Lin M, Huang H, Li S (2013) Biochem Eng J 71:1–10 28. van Sonsbeek HM, Beeftink HH, Tramper J (1993) Enzyme Microb Tech 15:722–729 29. van der Meer AB, Beenackers AACM, Burghard R, Mulder NH, Fok JJ (1992) Chem Eng Sci 47:2369–2374 30. De´zil E, Comeau Y, Villemur R (1999) Biodegradation 10:219–233 31. Pilarek M, Szewczyk KW (2008) Biochem Eng J 41:38–42 32. Fraker CA, Mendez AJ, Inverardi L, Ricordi C, Stabler CL (2012) Colloid Surf B 98:26–35 33. Amaral PFF, Freire MG, Rocha-Lea˜o MHM, Marrucho IM, Coutinho JAP, Coelho MAZ (2008) Biotechnol Bioeng 99:588–598 34. Lowe KC (2002) J Fluorine Chem 118:19–26 35. Amaral PFF, Almeida APR, Peixoto T, Rocha-Lea˜o MHM, Coutinho JAP, Coelho MAZ (2007) World J Microb Biot 23:339–344 36. Amaral PFF, Rocha-Lea˜o MH, Marrucho IM, Coutinho JAP, Coelho MA (2006) J Chem Technol Biotechnol 81:1368–1374 37. Elibol M, Ozer D (2000) Process Biochem 36:325–329 38. Dias AMA, Cac¸o AI, Coutinho JAP, Santos LMNBF, Pin˜eiro MM, Vega LF, Gomes MFC, Marrucho IM (2004) Fluid Phase Equilib 225:39–47 39. Freire DM, Teles EMF, Bon EPS, Sant’Anna GL Jr (1997) Appl Biochem Biotechnol 63–65:409–421 40. Liu CH, Huang CC, Wanga YW, Chang JS (2012) J Taiwan Inst Chem E 43:511–516 41. Burkert JFM, Maldonado RR, Maugieri F, Rodrigues MI (2005) J Chem Technol Biot 80:61–67 42. Ni X, Gao S, Cumming RH, Pritchard DW (1995) Chem Eng Sci 50:2127–2136 43. Soccol CR, Iloku I, Marin B, Roussos S, Raimbault M (1994) Food Sci Technol 3:320–323 44. Soares CMF, Castro HF, Moraes FF, Zanin GM (1999) Appl Biochem Biotech 79:745–758 45. Salihu A, Alam MZ, Abdulkarim MI, Salleh HM (2011) J Mol Catal B Enzym 73:187–192 46. Carvalho NB, Souza RL, Castro HF, Zanin GM, Lima AS, Soares CMF (2008) Appl Biochem Biotech 150:25–32 47. Gulati R, Saxena RK, Gupta R (2000) Process Biochem 36:149–155 48. Mahanta N, Gupta A, Khare SH (2008) Bioresour Technol 99:1729–1735 49. Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV (2002) Process Biochem 38:715–721 50. Freire MG, Dias AMA, Coelho MAZ, Coutinho JAP, Marrucho IM (2005) J Colloid Interf Sci 286:224–232 51. Silva MTS (2013) MSc thesis, Tiradentes University, AracajuSergipe, Brazil 52. Wang SL, Yeh PY (2006) Process Biochem 41:1545–1552
