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Substrate conversion by fungal spores entrapped in solid matrices
ARCHIVES
OF
Substrate
BIOCHEMISTRY
.ZND
Conversion
Northern
Received
130, 384-388 (1969)
BI0PHYeICS
by Fungal
Spores
D. E. JOHNSON
rlND
Regional
in Solid
Matrices
A. CIEGLER
Research Laboratory,
September
Entrapped
3, 1968; accepted
Peoria,
Illinois
December
61604l
16, 1968
Conditions for the operation of a fungal spore-continuous column process are described wherein sucrose inversion is used as a model detection system. ECTEOLA-cellulose, acting as asolid support, provided good retention and flow-rate characteristics in columns prepared with Aspergillus and Penicillium spores. Spore columns so prepared were stable and needed only a cursory washing before storage or re-use. Germination on the columnwas negligible. The interact,ious occurring between variations in spore concentration, substrate concentration, and flow rate are reported, including the effect each has upon product yield.
The use of enzymes in industry is limited because of their cost and the time involved in their purification and handling. To some extent, the attachment of enzymes to solid matrices would lower the costs involved because such preparations could be used repeatedly and are readily applicable to continuous operations. A variety of enzymes-for example, proteases (l-3), urease (4), ribonuclease (5), aminoacylase (6-g), and many others-have been been rendered insoluble by using solid supports. Enzymatically active insoluble preparations are best adapted to column operation. Unlike a batch fermentor, the continuous flow characteristics of a column make delays for substrate introduction and product recovery unnecessary. By entrapping fungal spores instead of enzymes on a column, the difficulties inherent with enzyme purification and stability are avoided. Though whole cells would be difficult to maintain in a sationary phase on a column, spores by definition are in a resting stage and remain so on a column. Spores are not physiologically inert, however, and do possess selective enzymic activity (10). Research at our Laboratory has shown
Spore production. Stock ctdtures of 8. oryzae NRRL 1989, Aspergillus we&ii NRRL 2001, and Penicillium ropueforti NRRL 3360 were maintained on potato-dextrose-agar slants. Spores of each were raised in 2.8-liter Fernbach flasks containing 300 g of coarsely cracked corn moistened with 150 ml of water. The sterile corn was inoculated with spores washed directly from a 7-day-old slant. Incubation at 28” for 10 days provided good sporulation. Spores were harvested according to the procedure previously described (12). Adsorbents. Insoluble carriers employed to trap the spores on columns included several ionexchange cellulose derivatives such as diethylaminoethyl (DEAE), mixed amines (ECTEOLA), carboxymethyl (CM), and phosphonic acid (P)all were obtained from Bio-Rad Laboratories,2 Richmond, California. A commercial polyacrylamide gel, Bio-Gel CM (Bio-Rad) and other assorted chromatographic materials (silica gel, Brinkmann Instruments Inc., Westbury, New York; Amerlite
1 This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.
2 The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
that spores of Aspergillus oryzae contain an active invertase which rapidly hydrolyzes sucrose to give equal molar amounts of glucose and fructose (11). This spore-substrate system was used as a model for invest’igating the techniques and operations involved in spore-column arrangements. MATERIALS
384
AND
METHODS
COLUMN
ENTRAPMENT
IH-120, Mdlinckrodt Chemical Works, St. Louis, Missouri; neutral cellulose, Carl Schleicher & Schuell Co., Keene, New Hampshire) were also examined for spore-column applications. L’olunlns. Glass columns, 9 mm i.d., 400 mm in length, and 15 mm i.d., 500 mm in length, were supplied by Fischer & Porter Co., Warminster, Pennsylvania. A 250-ml round-bottomed flask was fused to the top of each column to serve as a substrate reservoir. The columns were surrounded by water jackets, which provided a means of temperntllre control during operation. Spore-column formation. Since each resin was unique, a given set of conditions did not necessarily result in columns of the same dimensions and flow rate. For simplicity, column formation with ECTEOLA-cellulose is described. Depending upon the desired column height, a measured amouut of resin was washed with several volumes of 0.05 M KzHPOd-NaOH, pH 5.8. Spores were added directly to the aqueous resin from a stock solution t.o give a final concentration ranging from 5 X lo9 t,o 3 X 10’0 spores per column. The mixtllre was then poured into the column and allowed to settle undisturbed. Eight grams of ECTEOLA-cellulose, when packed, formed a column approximately 200 mm in height (wit,h a 15.mm i.d. column). Columns could be washed as often as needed with 200-300 ml of buffer and either stored at 4” or held by circulating cold water through t,he jackets when not in llse. Sucrose inversion. After sufficient equilibration with wash buffer. the spore column was charged with substrate. Initial dilution of substrate was avoided by a nearly complete removal of equilibration buffer at) the top of the column before substrate application. Sucrose solutions (ranging from 0.02 M to 0.2 M in 0.05 M phosphate buffer) were eluted from the column at rates controlled by variable-flow Teflon stopcocks. Flow rates were expressed as ml/hr/cms, or in some inst,ances, simply as ml/hr. Fractions were collected on an LKB Ultrorac fraction collector (LKB Instruments Lt,d., Surrey, England). Temperature was held constant at 30” during elution by circulating preheated water throllgh the column jackets. Individual fractions were assayed for reducing sltgar by the Nelson-Somoygi method (13, 14). Dtlplicate readings of each fraction were made with a Beckman Model B spectrophotometer and compared with a standard curve, previous11 plotted from a mixtllre containing equal amolmts of gIrlrose and fructose. RESULTS
The spore-column technique first necessitated a search for a suitable insoluble carrier, or resin, which would secure the spores
OF FUNGAL
SPORES
x.35
within a column matrix. Based on the relatively large size of mold spores, ranging from 2.8 to 8.0 ,J in diameter, a purely physical entrapment on a column appeared possible and column ndsorbents were selected accordingly. Several materials used for chromatographic separations, including silica gel (0.05-2-mm and 0.2-0.5-mm pore diamet,er), Amberlite IR-120, and neutral cellulose were used in forming columns with A. oryzae spores. These materials proved to be unsuit,able for spore-column applications because of poor spore-retention characteristics and in some instances, extreme flow-rate restrictions. Because flow-rate requirements demanded a more porous carrier, several ion-exchange cellulose resins were examined. In Table I, retention and flow-rate characteristics of three species of spores are compared on four representative resins, consisting of two cation exchangers (CM- and P-cellulose) and t13.0 anion exchangers (DEAF:and ECTEOLA-cellulose). Retention descriptions are arbitrary and can be used only as a means of comparison. Columns of spores and resin were poured at both an acid (5.0) and a basic (9.0) pH, and flow rates were measured from the beginning of column formation. A destruction of the ion-exchange capacity of a resin (pH 9.0 for CM- and Pcellulose, pH 5.0 for DEAFGcellulose) resulted in :t loss of P. roqueforti and ‘4. wentii spores from the column. 8. myzae spores were surprisingly resistant to pH changes. Since ECTEOLA-cellulose showed no loss of retention power at pH 5.0-9.0, this resin provided a fairly wide range of pH for operating conditions. The differences in retention characterist’ics of spores within the resin matrix indicate the existence of charge binding in addition to physical entrapment. The difference in retention bctween spores of A. wysae and the other two species might reflect either a varikon in their surface charges or merely a difference in size. An ion-exchange polyacrylamide gel, Rio-Gel CM, exhibited poor spore-retention characteristics and was generally unsuitable for spore-column techniques. Variations in the operation of the spore column \verc investigated by the use of the sucrose inversion system already men
386
JOHNSON
AND
CIEGLER
TABLE
I SPORES ON ION-EXCHANGE
ADSORPTION CHARACTERISTICS OF Fc-SGAL
Ion-exchange cellulose resins
PH
Column height (mm)
Aspergillus oryzae SRRL
Penicillium
RESINS=
roqueforli
NRRL 3360
1989
I
Retention
Retention
Aspergillus
w&ii
NRRL 2001 I
Retention
--
5.84
++ +
5.84 3.48
+++ +
5.06 4.27
++ +
4.29 3.82
2.29 2.23
+ ++
2.29 2.75
+ +++
2.16 2.75
3.15 3.25
+++ +++
4.19 4.19
+++ +++
3.98 3.36
5.62 4.49
++
140 140
5.39 5.17
Diethylaminoethyl (DEW
240 240
Mixed amines (ECTEOLA)
150 150
Carboxymethyl
Phosphonic
(CM)
acid (P)
-I
-
0 Columns were 9 mm id.. with 2 g of resin poured into each as a slurry. recorded during the first hour of column operation. b +++ = Excellent, ++ = good, + = poor, - = no retention.
tioned. Since the reaction is rapid, it can be easily followed by the measurement of reducing power and so it is well suited to the spore-column arrangement. An elution diagram (Fig. 1) of a column packed with 3 X lOlo spores reveals that 50 % conversion of 0.02 M sucrose is attained within an hour and that the same rate of conversion is maintained throughout the elution period, covering a 7-hr interval. When a run is finished, the column can be washed with a dilute buffer solution to remove all the substrate and then refrigerated until its next use; or if tap water is cold enough, it can be circulated through the water jackets to provide sufficient refrigeration without moving the entire column to a cold room. Germination was negligible in columns that were properly washed after each run. However, continuous application of 0.2 M sucrose on columns for 8-10 hr or more usually caused signs of germination to appear if column washing was not initiated by that time. Iodoacetic acid, 1 mM, (Sigma Chemical Co., St. Louis, Missouri), when incorporated with the substrate solution, seemed to inhibit germination without af-
Retention
and flow rate were
60 -
t
s 40 ; -z z 20 0.
I 1
I 2
415
9'0
I 3
I
135 1 Hours180 : ml.
I 5
I
215
260 f
7 315
FIG. 1. Elution diagram of sucrose inversion by column-bound spores of Aspergillus oryzae. Column dimensions: 3 X lOi spores, 7.5 g ECTEOLA-cellulose, 180 mm height, 15 mm i.d., 0.02 M sucrose, flow rate 1.10 ml/hr/cm3, temperature 30”. The arrow indicates the time at which column washing was started.
fecting invertase activity, although additional evidence must be obtained on the effects of iodoacetate in the column before a concise evaluation can be made about its usefulness. Results from a series of columns arranged to provide data on the interactions that occur between spore concentration, substrate concentration, and flow rate are plotted in Fig. 2. TWO values (high and low) for each
COLI’MN
ENTItAl’?rlENT
factor were assigned, and the eight possible combinations of columns and conditions were run simultaneously. Analysis of the data plotted in graphic form reveals a greater efficiency of substrate conversion at the low sucrose concentration (0.02 M), even though greater absolute amounts of substrate were converted at a concentration of 0.2 M sucrose. A significant increase in product yield was apparent at’ 3 X lOlo spores per column, 0.02 M sucrose, and the slow (0.65 ml/hr/cm3) flow rate. A decrease in flow rate was insignificant in determining yield at either substrate concentration with 1 X lOlo spores per column, but it did co11tribute to an increase in yield at 3 X 10”’ spores per column. Columns of equal height and flow rate but differing in spore concentration did not invert sucrose o11 a linear basis corresponding t’o an increase in spore count. Although the yield increased with rising spore concentrations, it appeared to approach a maximum at the high (3 X lOlo) concentration. Counts surpassing 3 X 1O’O spores per column (200-mm height, 15mm i.d.) tended to restrict, the flow rate and so could not be compared wit’h the results in Fig. 3. Variations in column height failed to provide definitive data for improved substrate conversion. Columns of equal spore concent’ration and flow rate, but unequal in height, convert,ed sucrose at a fairly uniform rate, though conversion decreased in the tallest (400-mm) column (Fig. 4).
0
* I
I
0.2 0.02 Sucrose, M
0.2
FIG. 2. Three-factor column interactions and their relationship to product yield. In A, 1 X 10’0 spores per column; B, 3 X 1O’Ospores per column; flow rates, 0.65 ml/hr/cm3 (p) and 1.28 ml/hr/ cm3 (- - -). Columns: 8 g of ECTEOLA-cellulose, 15-mm i .d., 200-mm height, 30”.
OF FI:N(;AI,
SI’OI~ES
3s7
601 50 40 s .E 30F2 E 20lo-
0.5 1.0 2.0 3.0 Spore Concentration [XIO’O) FIG. 3. Effect of spore concentration on sticrose inversion. All columns were poured with 8 g of ECTEOLA-cell&se but with different, amolints of spores. Flow rate in all colr~~nns-1.28 ml/hr/ cm3,
I * 40 z ‘5 20 I -w
100 200 300 400 Columnlength, mm.
FIG. 4. Effect of column height on sucrose inversion. All columns contained 1 X lOI spores, but were pollred with varying amotints of ECTEOLA-cellulose-4, 7, 12. and 16 g. Flow rate in all columns-45 ml/hr.
Eluent from one column could be routed through a second to provide increased COIIversion. Product yield from 0.2 M sucrose, normally only 15-20%, was increased to 30-35% by passage through a second column. Rerout’ing of eluent through the same column produced a similar increase. Other conversion systems lvere applied to the spore-column technique but, with less success than t’he sucrose. Fatty acid oxidation by spores of P. roqueforti on a column created collection difhcuhies because of the extreme volatility of the products. Another system, involving starch hydrolysis by spores of A. wentii (12), was not rapid enough to be effective on n column and so yields were low.
388
JOHNSON DISCUSSION
The entrapment of micro-organisms in columns offers potential for a continuous conversion fermentation process. Spores, though less active than vegetative cells, are more stable on a column and can be used as enzymically active catalysts in much the same way as purified “insoluble” enzymes. The spore-column technique is limited to solutions of low viscosity. Sucrose solutions greater than 0.2 M restricted flow rate. The selection of certain ion-exchange cellulose resins as insoluble carriers, which provided superior retention and flow-rate characteristics, is in general agreement with the literature. Cellulose (6-8, 15), Dowex-50 and -2 (5) and derivatives of Sephadex (9, 16) have all been used successfully to form insoluble enzyme complexes. Polyacrylamide gel has been used to entrap various enzymes and lichens (17), and the preparation subsequently appled in active form to columns. However, the commercially polymerized gel we used was inadequate for spore-column operation. Column height did not appreciably affect maximum sucrose conversion within a range from 100-300 mm. The slight decrease in inversion with a 400-mm column could be the result of on-column metabolism of inversion products by the spores. Substrate diffusion through an extended bed could also be responsible for diminished activity, since the spores were separated by a proportionately greater distance than in the shorter columns. In addition to the obvious advantages of a continuous conversion system as we stated in the beginning, a useful application of the spore-column technique might lie in the areas plagued by feedback inhibition. A biochemical reaction that has been inhibited by the
AND
CIEGLER
formation of an end product could be controlled effectively by removing the end product as it is formed. A continuously flowing column process accomplishes this task simply by eluting the product. Consequently, a concentrated product solution should not arise, and any feedback inhibition mechanism that is involved would be alleviated. REFERENCES 1. BAIL-ELI, A., AND KATCHALSKI, E., Xature 188, 856 (1960). 2. CEBR.Z, J. J., GIVOL, D., SILMAN, H. I., AND K9TCH24LS~I, E., J. Biol. Chem. 236, 1720 (1961). 3. BAR-ELI, A., AND KATCHALSKI, E., J. Biol. Chem. 238, 1690 (1963). 4. RIESEL, E., AND KATCHALSKI, E., J. Biol. Chem. 239, 1521 (1964). 5. BARNETT, L. B., AND BULL, H. B., Biochim. Biophys. Acta 36, 244 (1959). 6. TOSA, T., MORI, T., FUSE, N., AND CHIBATA, I., Enzymologia 31, 214 (1966). 7. Tom, T., MORI, T., FUSE, N., AND CHIISATA, I., Enzymologia 31, 225 (1966). 8. TOSA, T., MORI, T., FUSE, N., AND CHIBAT.~, I., Enzymologia 32, 153 (1966). 9. TOSA, T., MORI, T., FUSE, N., AND CHIUATA, I., Biotechnol. Bioeng. 9, 603 (1967). 10. KNIGHT, S. G., Ann. N. Y. Acad. Sci. 139, 8 (1966). 11. NELSON, G. E. N., JOHNSON, D. E., AND CIEGLER, A., Develop. Ind. Microbial., in press (1968 Ed.). 12. JOHNSON, D. E., NELSON, G. E. N., AND CIEGLER,A., AppZ. Microbial. 16, 1678 (1968). 13. NELSON, N., J. Biol. Chem. 163, 375 (1944). 14. SOMOGYI, M., J. BioZ. Chem. 196, 19 (1952). 15. KAY, G., .~ND CROOI~, E. M., AVature 216, 514 (1967). 16. AX&N, R., AND POR~TH, J., Natwe 210, 367 (1966). 17. MOSBACH, K., AND MOSBXH, R., Acta Chem. &and. 20, 2807 (1966).
OF
Substrate
BIOCHEMISTRY
.ZND
Conversion
Northern
Received
130, 384-388 (1969)
BI0PHYeICS
by Fungal
Spores
D. E. JOHNSON
rlND
Regional
in Solid
Matrices
A. CIEGLER
Research Laboratory,
September
Entrapped
3, 1968; accepted
Peoria,
Illinois
December
61604l
16, 1968
Conditions for the operation of a fungal spore-continuous column process are described wherein sucrose inversion is used as a model detection system. ECTEOLA-cellulose, acting as asolid support, provided good retention and flow-rate characteristics in columns prepared with Aspergillus and Penicillium spores. Spore columns so prepared were stable and needed only a cursory washing before storage or re-use. Germination on the columnwas negligible. The interact,ious occurring between variations in spore concentration, substrate concentration, and flow rate are reported, including the effect each has upon product yield.
The use of enzymes in industry is limited because of their cost and the time involved in their purification and handling. To some extent, the attachment of enzymes to solid matrices would lower the costs involved because such preparations could be used repeatedly and are readily applicable to continuous operations. A variety of enzymes-for example, proteases (l-3), urease (4), ribonuclease (5), aminoacylase (6-g), and many others-have been been rendered insoluble by using solid supports. Enzymatically active insoluble preparations are best adapted to column operation. Unlike a batch fermentor, the continuous flow characteristics of a column make delays for substrate introduction and product recovery unnecessary. By entrapping fungal spores instead of enzymes on a column, the difficulties inherent with enzyme purification and stability are avoided. Though whole cells would be difficult to maintain in a sationary phase on a column, spores by definition are in a resting stage and remain so on a column. Spores are not physiologically inert, however, and do possess selective enzymic activity (10). Research at our Laboratory has shown
Spore production. Stock ctdtures of 8. oryzae NRRL 1989, Aspergillus we&ii NRRL 2001, and Penicillium ropueforti NRRL 3360 were maintained on potato-dextrose-agar slants. Spores of each were raised in 2.8-liter Fernbach flasks containing 300 g of coarsely cracked corn moistened with 150 ml of water. The sterile corn was inoculated with spores washed directly from a 7-day-old slant. Incubation at 28” for 10 days provided good sporulation. Spores were harvested according to the procedure previously described (12). Adsorbents. Insoluble carriers employed to trap the spores on columns included several ionexchange cellulose derivatives such as diethylaminoethyl (DEAE), mixed amines (ECTEOLA), carboxymethyl (CM), and phosphonic acid (P)all were obtained from Bio-Rad Laboratories,2 Richmond, California. A commercial polyacrylamide gel, Bio-Gel CM (Bio-Rad) and other assorted chromatographic materials (silica gel, Brinkmann Instruments Inc., Westbury, New York; Amerlite
1 This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.
2 The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
that spores of Aspergillus oryzae contain an active invertase which rapidly hydrolyzes sucrose to give equal molar amounts of glucose and fructose (11). This spore-substrate system was used as a model for invest’igating the techniques and operations involved in spore-column arrangements. MATERIALS
384
AND
METHODS
COLUMN
ENTRAPMENT
IH-120, Mdlinckrodt Chemical Works, St. Louis, Missouri; neutral cellulose, Carl Schleicher & Schuell Co., Keene, New Hampshire) were also examined for spore-column applications. L’olunlns. Glass columns, 9 mm i.d., 400 mm in length, and 15 mm i.d., 500 mm in length, were supplied by Fischer & Porter Co., Warminster, Pennsylvania. A 250-ml round-bottomed flask was fused to the top of each column to serve as a substrate reservoir. The columns were surrounded by water jackets, which provided a means of temperntllre control during operation. Spore-column formation. Since each resin was unique, a given set of conditions did not necessarily result in columns of the same dimensions and flow rate. For simplicity, column formation with ECTEOLA-cellulose is described. Depending upon the desired column height, a measured amouut of resin was washed with several volumes of 0.05 M KzHPOd-NaOH, pH 5.8. Spores were added directly to the aqueous resin from a stock solution t.o give a final concentration ranging from 5 X lo9 t,o 3 X 10’0 spores per column. The mixtllre was then poured into the column and allowed to settle undisturbed. Eight grams of ECTEOLA-cellulose, when packed, formed a column approximately 200 mm in height (wit,h a 15.mm i.d. column). Columns could be washed as often as needed with 200-300 ml of buffer and either stored at 4” or held by circulating cold water through t,he jackets when not in llse. Sucrose inversion. After sufficient equilibration with wash buffer. the spore column was charged with substrate. Initial dilution of substrate was avoided by a nearly complete removal of equilibration buffer at) the top of the column before substrate application. Sucrose solutions (ranging from 0.02 M to 0.2 M in 0.05 M phosphate buffer) were eluted from the column at rates controlled by variable-flow Teflon stopcocks. Flow rates were expressed as ml/hr/cms, or in some inst,ances, simply as ml/hr. Fractions were collected on an LKB Ultrorac fraction collector (LKB Instruments Lt,d., Surrey, England). Temperature was held constant at 30” during elution by circulating preheated water throllgh the column jackets. Individual fractions were assayed for reducing sltgar by the Nelson-Somoygi method (13, 14). Dtlplicate readings of each fraction were made with a Beckman Model B spectrophotometer and compared with a standard curve, previous11 plotted from a mixtllre containing equal amolmts of gIrlrose and fructose. RESULTS
The spore-column technique first necessitated a search for a suitable insoluble carrier, or resin, which would secure the spores
OF FUNGAL
SPORES
x.35
within a column matrix. Based on the relatively large size of mold spores, ranging from 2.8 to 8.0 ,J in diameter, a purely physical entrapment on a column appeared possible and column ndsorbents were selected accordingly. Several materials used for chromatographic separations, including silica gel (0.05-2-mm and 0.2-0.5-mm pore diamet,er), Amberlite IR-120, and neutral cellulose were used in forming columns with A. oryzae spores. These materials proved to be unsuit,able for spore-column applications because of poor spore-retention characteristics and in some instances, extreme flow-rate restrictions. Because flow-rate requirements demanded a more porous carrier, several ion-exchange cellulose resins were examined. In Table I, retention and flow-rate characteristics of three species of spores are compared on four representative resins, consisting of two cation exchangers (CM- and P-cellulose) and t13.0 anion exchangers (DEAF:and ECTEOLA-cellulose). Retention descriptions are arbitrary and can be used only as a means of comparison. Columns of spores and resin were poured at both an acid (5.0) and a basic (9.0) pH, and flow rates were measured from the beginning of column formation. A destruction of the ion-exchange capacity of a resin (pH 9.0 for CM- and Pcellulose, pH 5.0 for DEAFGcellulose) resulted in :t loss of P. roqueforti and ‘4. wentii spores from the column. 8. myzae spores were surprisingly resistant to pH changes. Since ECTEOLA-cellulose showed no loss of retention power at pH 5.0-9.0, this resin provided a fairly wide range of pH for operating conditions. The differences in retention characterist’ics of spores within the resin matrix indicate the existence of charge binding in addition to physical entrapment. The difference in retention bctween spores of A. wysae and the other two species might reflect either a varikon in their surface charges or merely a difference in size. An ion-exchange polyacrylamide gel, Rio-Gel CM, exhibited poor spore-retention characteristics and was generally unsuitable for spore-column techniques. Variations in the operation of the spore column \verc investigated by the use of the sucrose inversion system already men
386
JOHNSON
AND
CIEGLER
TABLE
I SPORES ON ION-EXCHANGE
ADSORPTION CHARACTERISTICS OF Fc-SGAL
Ion-exchange cellulose resins
PH
Column height (mm)
Aspergillus oryzae SRRL
Penicillium
RESINS=
roqueforli
NRRL 3360
1989
I
Retention
Retention
Aspergillus
w&ii
NRRL 2001 I
Retention
--
5.84
++ +
5.84 3.48
+++ +
5.06 4.27
++ +
4.29 3.82
2.29 2.23
+ ++
2.29 2.75
+ +++
2.16 2.75
3.15 3.25
+++ +++
4.19 4.19
+++ +++
3.98 3.36
5.62 4.49
++
140 140
5.39 5.17
Diethylaminoethyl (DEW
240 240
Mixed amines (ECTEOLA)
150 150
Carboxymethyl
Phosphonic
(CM)
acid (P)
-I
-
0 Columns were 9 mm id.. with 2 g of resin poured into each as a slurry. recorded during the first hour of column operation. b +++ = Excellent, ++ = good, + = poor, - = no retention.
tioned. Since the reaction is rapid, it can be easily followed by the measurement of reducing power and so it is well suited to the spore-column arrangement. An elution diagram (Fig. 1) of a column packed with 3 X lOlo spores reveals that 50 % conversion of 0.02 M sucrose is attained within an hour and that the same rate of conversion is maintained throughout the elution period, covering a 7-hr interval. When a run is finished, the column can be washed with a dilute buffer solution to remove all the substrate and then refrigerated until its next use; or if tap water is cold enough, it can be circulated through the water jackets to provide sufficient refrigeration without moving the entire column to a cold room. Germination was negligible in columns that were properly washed after each run. However, continuous application of 0.2 M sucrose on columns for 8-10 hr or more usually caused signs of germination to appear if column washing was not initiated by that time. Iodoacetic acid, 1 mM, (Sigma Chemical Co., St. Louis, Missouri), when incorporated with the substrate solution, seemed to inhibit germination without af-
Retention
and flow rate were
60 -
t
s 40 ; -z z 20 0.
I 1
I 2
415
9'0
I 3
I
135 1 Hours180 : ml.
I 5
I
215
260 f
7 315
FIG. 1. Elution diagram of sucrose inversion by column-bound spores of Aspergillus oryzae. Column dimensions: 3 X lOi spores, 7.5 g ECTEOLA-cellulose, 180 mm height, 15 mm i.d., 0.02 M sucrose, flow rate 1.10 ml/hr/cm3, temperature 30”. The arrow indicates the time at which column washing was started.
fecting invertase activity, although additional evidence must be obtained on the effects of iodoacetate in the column before a concise evaluation can be made about its usefulness. Results from a series of columns arranged to provide data on the interactions that occur between spore concentration, substrate concentration, and flow rate are plotted in Fig. 2. TWO values (high and low) for each
COLI’MN
ENTItAl’?rlENT
factor were assigned, and the eight possible combinations of columns and conditions were run simultaneously. Analysis of the data plotted in graphic form reveals a greater efficiency of substrate conversion at the low sucrose concentration (0.02 M), even though greater absolute amounts of substrate were converted at a concentration of 0.2 M sucrose. A significant increase in product yield was apparent at’ 3 X lOlo spores per column, 0.02 M sucrose, and the slow (0.65 ml/hr/cm3) flow rate. A decrease in flow rate was insignificant in determining yield at either substrate concentration with 1 X lOlo spores per column, but it did co11tribute to an increase in yield at 3 X 10”’ spores per column. Columns of equal height and flow rate but differing in spore concentration did not invert sucrose o11 a linear basis corresponding t’o an increase in spore count. Although the yield increased with rising spore concentrations, it appeared to approach a maximum at the high (3 X lOlo) concentration. Counts surpassing 3 X 1O’O spores per column (200-mm height, 15mm i.d.) tended to restrict, the flow rate and so could not be compared wit’h the results in Fig. 3. Variations in column height failed to provide definitive data for improved substrate conversion. Columns of equal spore concent’ration and flow rate, but unequal in height, convert,ed sucrose at a fairly uniform rate, though conversion decreased in the tallest (400-mm) column (Fig. 4).
0
* I
I
0.2 0.02 Sucrose, M
0.2
FIG. 2. Three-factor column interactions and their relationship to product yield. In A, 1 X 10’0 spores per column; B, 3 X 1O’Ospores per column; flow rates, 0.65 ml/hr/cm3 (p) and 1.28 ml/hr/ cm3 (- - -). Columns: 8 g of ECTEOLA-cellulose, 15-mm i .d., 200-mm height, 30”.
OF FI:N(;AI,
SI’OI~ES
3s7
601 50 40 s .E 30F2 E 20lo-
0.5 1.0 2.0 3.0 Spore Concentration [XIO’O) FIG. 3. Effect of spore concentration on sticrose inversion. All columns were poured with 8 g of ECTEOLA-cell&se but with different, amolints of spores. Flow rate in all colr~~nns-1.28 ml/hr/ cm3,
I * 40 z ‘5 20 I -w
100 200 300 400 Columnlength, mm.
FIG. 4. Effect of column height on sucrose inversion. All columns contained 1 X lOI spores, but were pollred with varying amotints of ECTEOLA-cellulose-4, 7, 12. and 16 g. Flow rate in all columns-45 ml/hr.
Eluent from one column could be routed through a second to provide increased COIIversion. Product yield from 0.2 M sucrose, normally only 15-20%, was increased to 30-35% by passage through a second column. Rerout’ing of eluent through the same column produced a similar increase. Other conversion systems lvere applied to the spore-column technique but, with less success than t’he sucrose. Fatty acid oxidation by spores of P. roqueforti on a column created collection difhcuhies because of the extreme volatility of the products. Another system, involving starch hydrolysis by spores of A. wentii (12), was not rapid enough to be effective on n column and so yields were low.
388
JOHNSON DISCUSSION
The entrapment of micro-organisms in columns offers potential for a continuous conversion fermentation process. Spores, though less active than vegetative cells, are more stable on a column and can be used as enzymically active catalysts in much the same way as purified “insoluble” enzymes. The spore-column technique is limited to solutions of low viscosity. Sucrose solutions greater than 0.2 M restricted flow rate. The selection of certain ion-exchange cellulose resins as insoluble carriers, which provided superior retention and flow-rate characteristics, is in general agreement with the literature. Cellulose (6-8, 15), Dowex-50 and -2 (5) and derivatives of Sephadex (9, 16) have all been used successfully to form insoluble enzyme complexes. Polyacrylamide gel has been used to entrap various enzymes and lichens (17), and the preparation subsequently appled in active form to columns. However, the commercially polymerized gel we used was inadequate for spore-column operation. Column height did not appreciably affect maximum sucrose conversion within a range from 100-300 mm. The slight decrease in inversion with a 400-mm column could be the result of on-column metabolism of inversion products by the spores. Substrate diffusion through an extended bed could also be responsible for diminished activity, since the spores were separated by a proportionately greater distance than in the shorter columns. In addition to the obvious advantages of a continuous conversion system as we stated in the beginning, a useful application of the spore-column technique might lie in the areas plagued by feedback inhibition. A biochemical reaction that has been inhibited by the
AND
CIEGLER
formation of an end product could be controlled effectively by removing the end product as it is formed. A continuously flowing column process accomplishes this task simply by eluting the product. Consequently, a concentrated product solution should not arise, and any feedback inhibition mechanism that is involved would be alleviated. REFERENCES 1. BAIL-ELI, A., AND KATCHALSKI, E., Xature 188, 856 (1960). 2. CEBR.Z, J. J., GIVOL, D., SILMAN, H. I., AND K9TCH24LS~I, E., J. Biol. Chem. 236, 1720 (1961). 3. BAR-ELI, A., AND KATCHALSKI, E., J. Biol. Chem. 238, 1690 (1963). 4. RIESEL, E., AND KATCHALSKI, E., J. Biol. Chem. 239, 1521 (1964). 5. BARNETT, L. B., AND BULL, H. B., Biochim. Biophys. Acta 36, 244 (1959). 6. TOSA, T., MORI, T., FUSE, N., AND CHIBATA, I., Enzymologia 31, 214 (1966). 7. Tom, T., MORI, T., FUSE, N., AND CHIISATA, I., Enzymologia 31, 225 (1966). 8. TOSA, T., MORI, T., FUSE, N., AND CHIBAT.~, I., Enzymologia 32, 153 (1966). 9. TOSA, T., MORI, T., FUSE, N., AND CHIUATA, I., Biotechnol. Bioeng. 9, 603 (1967). 10. KNIGHT, S. G., Ann. N. Y. Acad. Sci. 139, 8 (1966). 11. NELSON, G. E. N., JOHNSON, D. E., AND CIEGLER, A., Develop. Ind. Microbial., in press (1968 Ed.). 12. JOHNSON, D. E., NELSON, G. E. N., AND CIEGLER,A., AppZ. Microbial. 16, 1678 (1968). 13. NELSON, N., J. Biol. Chem. 163, 375 (1944). 14. SOMOGYI, M., J. BioZ. Chem. 196, 19 (1952). 15. KAY, G., .~ND CROOI~, E. M., AVature 216, 514 (1967). 16. AX&N, R., AND POR~TH, J., Natwe 210, 367 (1966). 17. MOSBACH, K., AND MOSBXH, R., Acta Chem. &and. 20, 2807 (1966).