enzyme concentration, response surface method- ology. INTRODUCTION. Cellulase is an enzymatic complex which associates oxidative and hydrolytic ...
Bioresource Technology 43 (1993) 155-160
MEMBRANE CONCENTRATION OF F U N G A L CELLULASES J. Carlos Roseiro, Alexandra C. Concei~fio & M. T. Amaral-Colla~o* Laboratorio Nacional de Engenharia e Tecnoiogia Industrial, Departamento de Tecnologia das Industrias Quimicas, Unidade de Microbiologia Industrial-Biotecnologia, Azinhaga dos Lameiros, 1699 Lisboa Codex, Portugal
(Received 20 January 1992; revised version received 5 May 1992; accepted 18 May 1992)
of the remaining solution. The concentration technique normally in use is ultrafiltration by the utilization of membranes with a cut-off (solute molecular weight at which 90% macromolecules are retained by the membranes) not larger than 10000 Da. In addition, ultrafiltration can be part of a more elaborate downstream scheme preceding salt precipitation or energy-intensive processes, such as spray-drying or lyophilization, to obtain a solid cellulase enzyme (Esterbauer et al., 1991). The main feature of ultrafiltration lies in the anisotropic structure of the membrane which, associated with cross-flow of the fluid, avoids irreversible blocking. Anisotropic membranes are usually made from polysulfones and are available in a separation range between 500 and 1000000 Da, from bench to industrial scale. The concentration procedure takes place with a constant pressure providing the ultrafiltration driving force (Flaschel et al., 1983). In this paper the importance of pressure inside the fiber during the concentration procedure is assessed and compared with the cross-flow rate. A statistical experimental design according to the Box-Hunter distribution was used for carrying out the experimental work. The use of this method allowed the quantification of the relative importance of the factors on the ultrafiltration output (Box & Wilson, 1951; Deming & Morgan, 1987).
Abstract A 10000 Da hollow-fiber membrane characterized by an average hydraulic resistance of 9.5 x 106 Pa s m - i and an average transmembrane pressure of 1"5 x 106 Pa was used to concentrate a commercial fungal cellulase solution. The level of enzymatic activity increased slowly for the first 3 h before it started rising steeply in an exponential concentration effect due to solute polarization at the inner layer of the membrane, producing a flow rate decrease of approximately 20%. Enzymatic activity yield and permeate flow were studied in a range between 940 and 1300 ml rain- i for cross-flow rate, 0.68 x 106 and 1"22 x 106 Pa for outlet pressure, and ultrafiltration time varying between 90 and 300 min. Pressure was revealed to be more important than cross-flow rate in the efficiency of the concentration. Also, a long lasting operation produced better results in terms of the studied responses than any combination of the other factors. The values of the specific activity of the cellulase components for an 8-h ultrafiltration period remained constant, indicating that no shear inactivation occurred. Key words: Cellulase, Trichoderma sp., ultrafiltration, enzyme concentration, response surface methodology.
INTRODUCTION Cellulase is an enzymatic complex which associates oxidative and hydrolytic enzymes to degrade cellulose interactively. Although bacterial cellulases have been widely studied (Gilkes et al., 1991), those produced by fungi are more effective (Goyal et al., 1991), Trichoderma sp. being the basic microorganism for the production of commercial preparations (Esterbauer et al., 1991 ). Following fermentation of cellulose, the downstream processing entails two steps, the separation of insoluble matter and mycelium and the concentration
METHODS Process material Process fluid was a cellulase solution of CeUuclast 1.5 L (enzyme complex containing 1,4-(1,3;1,4)-fl-Dglucan 4-glucanohydrolase, EC 3.2.1.4; 1,4-fl-oglucan cellobiohydrolase, EC 3.2.1.91; and fl-glucosidase, EC 3.2.1.21), 5 NCU in citrate buffer 0.01 M, pH 4-8. The enzyme complex was obtained from Novo Industries A/S, Copenhagen, Denmark. Analytical methods Enzymatic activity of the cellulase complex was measured using filter paper as substrate. Endoglucanase, cellobiohydrolase and fl-glucosidase activities were assessed by using carboximethylcellulose, cotton wool and p-nitrophenyl-fl-o-glucopyranoside
*To whom correspondence should be addressed. Bioresource Technology 0960-8524[92/S05.00 O 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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as substrates, respectively (Mandels et al., 1976). Glucose was measured by the dinitrosalicilic acid technique. Dinitrosalicilic acid was purchased from Merck, Darmstadt, Germany, and o-glucose was obtained from BDH Chemicals Ltd, Poole, England. Filter paper used was Whatman 1. One enzyme unit (U) was defined as the amount of enzyme producing 1 /~mole of glucose min- ~in 1 ml of sample. Ultrafiltration system Ultrafiltration was carried out using a polysulfone hollow-fiber unit, cartridge HIP10-20, cut-off 10 000 Da, obtained from Amicon Co., Danvers, Massachusetts, USA, operated under specific conditions according to the experimental design. Cross-flow was set by the working flow rate of a Cole Parmer peristaltic pump (Chicago, Illinois, USA), which varied between 0 and 1550 ml min- ~. Vibration due to fluid peristaltic flow was avoided by means of a Cole Parmer flow integrator placed immediately before the ultrafiltration cartridge inlet port. Pressure drop and transmembrane pressure were calculated from the readings of two pressure meters at both ends of the cartridge and controlled by an outlet valve. Permeate was collected through a downstream port where flow measurements took place periodically to assay flow alteration. Fiber cleaning was a major step in ensuring that all the runs had similar starting conditions. A flow of a 0.5-r~ sodium hydroxide solution inside the fibers at the maximum pump speed rate and minimum outlet pressure (valve fully open) was kept until a 10 ml min-L permeate flow was obtained. The system was then flushed with distilled water under the same conditions until a 12 ml min-1 permeate flow was reached. Periodically, a more extensive cleaning was performed involving circulation of 1% pepsin solution followed by several minutes of backflushing. After cleaning, the cartridge was stored in a 0"2% sodium azide solution at 4°C before the next use. Experimental design Experimental distribution for three factors, according to the Box-Hunter design (Box & Wilson, 1951; Deming & Morgan, 1987), was used to produce response surfaces. Eighteen experiments (including four replicates at the center of the experimental domain) were carried out using an initial 2-liter volume of the enzyme solution within an experimental domain with cross-flow rate (X~), between 940 and 1300 ml min-l, outlet pressure (X2), varying between 0-68x 106 and 1.22× 106 Pa, and ultra-
filtration time (X3), between 90 and 300 min. Coded representation of the factors was used for calculation purposes. The responses studied in the design were the permeate flow drop (determined at the permeate outlet) and enzymatic activity yield (ratio of the final activity to the number of units at the beginning of the operation). The model used to express the responses was a second order polynomial equation (eqn ( 1)). ei = flo + Eflixi + Eflijxix i + E flii X2
(1)
where Yi=response from experiment i; fl=parameters of the polynomial model; x= experimental factor level (coded units). Simple matrix algebra was computed in the professional spreadsheet Quattro (Borland, Scotts Valley, California, USA) and used to calculate the equation parameters fl, along with the analysis of variance for the effectiveness of the factors and for the lack of fit. Isoresponse contours were obtained by using an in-house BASIC program. RESULTS AND DISCUSSION The membrane was characterized by determination of the hydraulic resistance ( W ). The hydraulic resistance is the description of permeability of the membrane for pure solvent, and increases significantly in the presence of solute due to the attachment of molecules to the membrane. It is obtained graphically from the equation: j=I
Ap
(2)
W where J=permeate flow, W= hydraulic resistance and A P = transmembrane pressure. Table 1 shows that the hydraulic resistance did not vary with the cross-flow rate in the range of the experimental domain. Efficiency of ultrafiltration for this membrane is thus dependent solely on the nature and concentration of the solute. The transmembrane pressure acts as the driving force and controls the permeate flux, achieving a maximum of 1.56 x 106 Pa. Ceilulase concentration The main factors affecting ultrafiltration are the membrane stability and the hydrodynamic conditions which are dependent on the pressure of the fluid inside the fiber and the cross-flow rate of the fluid. Table 2 shows the results obtained during the progress of the experimental design for different cross-flow rates, pressure at the fiber outlet and ultra-
Table 1. Hydraulic resistance of the membrane within the experimental domains
Cross-flow rate (ml s- t) Hydraulic resistance (Pa s m-~ x 10") Maximum transmembrane pressure (Pax 10n)
5.6 10.0 1.46
No variation of the resistance was detected for distilled water operation.
11.5 9.0 1.49
18.7 9.9 1'53
24.3 8.9 1.56
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Membrane concentration offungal cellulases Table 2. Box & Hunter experimental design, test conditions and studied responses
Test
Cross-flow rate ml min- 2
Outlet pressure Pax 104
Ultrafiltration time min
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 300 1 300 1 300 1 300 940 940 940 940 1 415 830 1 120 1 120 1 120 1 120 1 120 1 120 1 120 1 120
1"22 1-22 0"68 0"68 1"22 1"22 0"68 0"68 0"95 0"95 1"4 0"49 0"95 0"95 0"95 0"95 0"95 0"95
300"0 90"0 300"0 90"0 300-0 90"0 300"0 90"0 195"0 195"0 195"0 195"0 371"6 18"4 195"0 195"0 195-0 195"0
Permeate flow drop ml rain- 2 3"9(8"4 - 4"5 ) 2"5(7"5 - 5) 2-3(5"3 - 3) 1-3(4"5 - 3"2) 3-8(8 - 4"2) 1"5(7 - 5"5) 2-0(5 - 3) 1"0(4"5 - 3"5) 3"6(6 - 2"4) 1"8(5"5 - 3"7) 3"0(7"5 -4"5) 1"5(4"3 - 2"8) 3"8(7"2- 3"5) 1"0(7 - 6) 2"3(5"8 - 3-5) 2"9(5-7 - 2"8) 2-9(5"8 - 2"9) 2-8(5"8 - 2"8)
Enzyme yield
Ut/Uo 4"3 1"7 2-2 1-4 3"8 1"3 2"6 1"0 1"8 1"4 2"5 1"6 4-1 1"0 2-4 1-9 1"9 2"0
Four replicates at the center of the experimental domain provide the degrees of freedom required for the analyses of variance.
filtration time (factors X~, X 2 and X 3, respectively). Maximum enzymatic activity yield was obtained in tests 1, 5 and 13 where the operational factors (particularly pressure) were at their maximal for long periods of operation. For the conditions in these runs the volumetric concentration factor, given by the ratio of initial to final volume of concentrate, was 6.7, 5.4 and 5.7, respectively. For reduced periods of operations, even when the operational factors were at high levels (tests 2, 9 and 11 ), the enzyme yield obtained was particularly low, corresponding to volumetric concentration factors of 1"4, 2" 1 and 2"3, respectively. Therefore, it is apparent that the longer the operation lasts the higher is the level attained by the responses. This is due to the gel formation at the membrane which decreases the permeate flow and also to a maximum volume reduction obtained after a long period, resulting in the increase of the enzyme activity yield. Consequently, the complete processing of the fluid produces higher efficiency in the use of ultrafiltration as a concentration technique. This general analysis is further developed by comparison of the parameters of the linear models. Table 3 shows the levels of the parameters affecting the two responses studied in the system, fl0 expresses the response at the center of the experimental domain (cross-flow rate, 1120 ml min-2; pressure, 0"95 x 106 Pa; process time, 195 min). Pressure strongly alters both the permeate flow drop and the enzymatic activity yield. According to the parameters fit and f12, pressure is a more important factor than cross-flow rate in the concentration (ill
