Service, Chattanooga, Tenn., August 1964. Greene, H. L., Lane, W. R., “Particulate Clouds: Dusts, Smokes, and Mists,” E. and F. N. Spon Ltd., London, 1964, p p 138-41. Interbranch Chemical Advisory Committee, “Selected Methods for the Measurement of Air Pollutants,” PHS Publication no. 999-AP-11, 1965. Lundgren, D. A., Cooper, D. W., “Effect of Humidity o n Light-Scattering Methods of Measuring Particle Concentration,’’ APCA Paper no. 68-107, 61st Annual Meeting of the Air Pollution Control Association, St. Paul, Minn., June 23-27, 1968. Martin, A., Barber, F. W., J. Inst. Fuel 39,294-307 (1966).
Montgomery, T. L., Corn, M., J . Air Pollut. Contr. Ass. 17 (S), 512-17 (1967). Smith, M. E., “Reduction of Ambient Air Concentrations of Pollutants by Dispersion From High Stacks,” Proceedings of the Third National Conference on Air Pollution, PHS Publication no. 1649, Dec. 12-14, 1966, p p 151-60. Stone, G. N., Clarke, A. J., Combustion 39, 41-9 (1967). Turner, D. B., “Workbook of Atmospheric Dispersion Estimates,’’ Public Health Service, Cincinnati, Ohio, National Air Pollution Control Administration, PHS Publication no. 999-AP-26,1969. Receiced for review May 21, 1970. Accepted December 28, 1970.
Effect of Peroxyacetyl Nitrate (PAN) in vivo on Tobacco Leaf Polysaccharide Synthetic Pathway Enzymes Lawrence Ordin, Morris J. Garber, Juanita I. Kindinger, Sherry A. Whitrnore, L. Carl Greve, and 0. Clifton Taylor Departments of Biochemistry, Statistics, Horticultural Science, and Statewide Air Pollution Research Center, University of California, Riverside, Calif. 92502
m Tobacco plants were exposed to 1 ppm of peroxyacetyl
nitrate (PAN) for 1 hr. Subsequent exposure of the plants to light caused leaf-cell collapse, which was noted after 24 hr. Assay of the leaves at that time showed a strong inactivation of alkali-soluble glucan, cellulose, and lipid synthetases, no effect o n phosphoglucomutase, and a stimulation of UDP glucose pyrophosphorylase. Assay of the leaves a t the end of the gassing period showed no effect of the PAN except for relatively decreased alkali-soluble glucan and cellulose synthetase activities. Plants placed in a dark chamber a t that time showed no subsequent lethal effects. It was concluded that PAN treatment of potentially sensitive green plant tissue can cause a nonlethal, and probably reversible, decrease of cellulose synthetase (and of growth).
G
rowth and cell-wall metabolism, particularly cellulose synthesis, measured as incorporation of 4C from 14C-glucose, was inhibited by peroxyacetyl nitrate (PAN) treatment of oat coleoptile sections (Ordin, 1962; Ordin et al., 1970). Wall metabolism and growth of oat coleoptile sections responded to auxin similarly, Even in nongrowing coleoptiles (auxin omitted), wall metabolism was decreased by a PAN pretreatment. Pretreatment of oat coleoptile sections with PAN inhibited P-glucan synthetases such as cellulose synthetase (Ordin and Hall, 1967). Phosphoglucomutase was also inhibited by PAN pretreatments; UDP glucose pyrophosphorylase, however, was not inhibited (Hall and Ordin, 1967). Glucan synthetase level corresponded with growth rate of coleoptiles (Hall and Ordin, 1968). The inhibitory pattern induced by PAN for P-glucan synthetase was similar to the pattern of growth rate reduction in oat coleoptiles as shown by unpublished work in this laboratory. The question arises: Will PAN treatment of growing green leaves lead to inhibition
of wall glucan synthetases and of other enzymes on the pathway of glucan synthesis without necessarily producing lethal consequences ? Ozonated hexenes suppressed linear growth, fresh and dry weight and leaf area of beans without symptoms of visible injury (Todd and Garber, 1958). Height increase of pea plants was temporarily arrested by such treatments. Low concentrations of NO? suppressed pinto beans and tomato growth without producing necrosis (Taylor and Eaton, 1966). As indicated in published work (Taylor, 1969), long-term plant growth effects by PAN (without lethal symptoms) have not been studied. Tobacco leaves displayed a sensitivity to Los Angeles ambient air, which varied according to the physiological stage of development of the leaf (Glater et al., 1962). The gradient from tip to base of the tobacco leaf is a gradient from senescent to young tissues with leaf expansion occurring primarily by cell expansion in the second and third leaves (Avery, 1933). Taylor (1967) has shown that tobacco leaves display a sensitivity to PAN which varies according t o physiological stage of development in the leaf itself-i.e., similar to Glater’s finding. This response was measured by cell collapse visible to the naked eye. Damage was found in leaves which were still expanding. Decreased photosynthesis could cause decreased growth. I t was reported, however, that PAN did not cause photosynthesis to decline until water-soaked areas became visible (Dugger et al., 1963; Taylor, 1969). We will show below that nonlethal inhibition of glucan synthetase can occur in sensitive tobacco leaves. Materials and Methods Plant Growth Condition and Selection. Tobacco plants, Nicotiana tabacuni L. var. Bel W-3, were grown in a mixture of peat moss and pearlite (1-to-1) in natural light in carbonfiltered air in a greenhouse. Temperatures were 27 ’ to 32 “C for day and 21 ‘C for night. Plants were watered once a day; Volume 5, Number 7, July 1971 621
Hoagland's solution was used on alternate days and deionized water o n the intervening days. At least three days before exposure to PAN,the plants were placed in a carbon-filtered air growth chamber with 21°C day and 16.7"C night temperatures. Light intensity was 2500f t candles from 90 fluorescent and 10 incandescent bulbs for 12 hr a day. Watering was continued as outlined above. Plants which received a post-light treatment and plants which were used to determine extent of damage were returned to this chamber after PAN treatment. Six plants were selected for treatment, three for control and three for PAN treatment. Each plant had a t least four expanded leaves. An upper limit of 60 cm (approximately eight expanded leaves) was imposed by the exposure chamber height. I n all cases, just prior to fumigation, a section from one half of the second expanded leaf counting from the apex, was removed from four plants for assay (Figure 1). Where enzymes were to be assayed immediately after fumigation, the remaining sections of the selected leaves from the plants were removed immediately after the exposure. In other experiments where enzymes were to be assayed the next day, the plants were returned to the growth chamber after fumigation. The next day, the remaining sections of the selected leaves were removed from the plants. In both types of experiments, leaf sections were removed, as described above, from the other two plants (one control and one PAN-treated) and the remaining leaf tissue was used to rate damage. Leaf sections were transported from the greenhouse to the laboratory in plastic bags. Gassing of Plants. The light intensity during exposure varied for these experiments as a result of the use of natural light. Light intensity readings were taken just before and immediately after fumigation. The range for the experiments was from 2600 to 6000-ft candles, determined by means of a Tipp Tronic Inc. Precision Light Meter, Model 0-1 MA. PAN was prepared by earlier published methods (Stephens
REMOVED PRIOR TO EXPOSURE
TO STEM
Figure 1. Diagram of second tobacco leaf, showing manner in which lamina was cut before gassing 622 Environmental Science & Technology
et al., 1965; Stephens et al., 1969). PAN, kept at 13"C, was fed through Teflon tubing from the cold room into a flow meter manifold, and from there to the exposure chamber, where it was mixed with filtered greenhouse air which was pulled through the chamber at 5 ft3/min. Control plants were subjected to the filtered greenhouse air only, a t the same flow rate. The chamber used was that described elsewhere (Heck et al., 1968), but with Teflon windows. Plants were placed in the chambers and gassed for 1 hr. During fumigation, the PAN level was monitored on a Mast ozone recorder. The exact PAN levels were determined by gas chromatography and electron capture detection (Darley et al., 1963). Preparation of Enzymes. Sections to be used as cellulose synthetase, phosphoglucomutase, and soluble UDPG pyrophosphorylase enzyme sources were divided into halves with the piece closest to the plant stem designated the base and the other piece the center. I n experiments where both soluble and particle UDPG pyrophosphorylase enzyme were to be assayed, sections to be used were left intact-Le., center and base were combined. Each section was weighed, minced with a razor blade and ground at 0°C in a modified Kontes-Dual1 tissue grinder, which contained 0.5 ml of buffer for divided sections and 1 ml of buffer for undivided sections. The buffer used (for grinding as well as resuspension and dilutions later) was 0.1M tris, 0.004M Na? EDTA,0.001M dithiothreitol (DTT) a t p H 8.0. The homogenates were centrifuged a t maximum speed (about 1000 X G) in an International clinical centrifuge at 0°C for 5 min. The supernatant solutions were transferred to 3.5-ml Sorvall tubes and recentrifuged a t 1000 X G (maximum), 0°C for 5 min. The resulting supernatant solutions were transferred again to 3.5-ml Sorvall tubes and centrifuged a t 25,000 X G (maximum), 0°C for 25 min. The supernatant solutions were removed and the volumes were noted. Since Day (1968) in this laboratory showed that cell sap concentration had to be nearly constant to obtain directly comparable pyrophosphorylase activities in crude extracts, these sample volumes were adjusted with buffer to the same weight to volume ratio as the weight to volume ratio which existed for the smallest (by weight) tissue, after corrections were made for any transfer loss during preparation of the enzyme. These solutions were assayed for phosphoglucomutase and for soluble UDP glucose pyrophosphorylase. The pellets were resuspended in a volume of buffer equal to the volume in which tissue was originally ground and recentrifuged at 25,000 X G (maximum), 0°C for 25 min. The resulting supernatant solution was discarded. For cellulose synthetase assays the pellet was resuspended in 0.15 ml of buffer. For particulate UDPG pyrophosphorylase assay, the sample which contained the smallest (by weight) tissue was resuspended in 0.2 ml of buffer. The remaining samples were resuspended with buffer on the same weight to volume basis after corrections were made for transfer losses during preparation of the enzyme. Enzyme Assays. SOLUBLE PHOSPHOGLUCOMUTASE. Reaction mixtures for the assay contained 50 pmoles of cysteine-HC1 buffer at p H 7.6, 12.5 pmoles of glucose-1-phosphate, 9.6 pmoles of MgCl?, 5 pmoles of Na2 EDTA at p H 8.0, 0.67 pmole of NADP,0.875 of a unit of glucose-6-phosphate dehydrogenase for a total volume of 2.8 ml. T o this was added 0.05 ml of sample enzyme containing 200 to 400 pg of protein. Reactions were carried out at 35°C. Reduction of NADP was followed at 340 m p in a Gilford Model 2000 multiple-sample absorbance recorder.
SOLUBLEAND PARTICLE UDPG PYROPHOSPHORYLASE ASSAY. Reaction mixtures for the assay contained 0.6 pmole of Na! UDP glucose, 1.2 pmoles of Na4P20,, 9.6 pmoles of MgC12, 5 pmoles of Na, EDTA a t p H 8.0,lOO pmoles of tris-HCI buffer a t p H 7.6, 0.67 pmole of NADP,0.875 of a unit of glucose-6phosphate dehydrogenase, 5 units of phosphoglucomutase for a total volume of 2.9 ml. To this mixture was added 0.05 ml of sample enzyme. After a rate was established for the particle enzyme, unless noted otherwise, 0.02 ml of 10% Triton X-100 was added to each reaction mixture, and the reaction was allowed to proceed until a second rate was established. Protein content of particulate enzyme aliquots was 300 to 550 pg. Reactions were carried out at 35 "C. Reduction of NADP was followed a t 340 m p in a Gilford Model 2000 multiple-sample absorbance recorder. CELLULOSE,ALKALI-SOLUBLE GLUCAN AND LIPID SYNTHETASES. The final incubation solution of 0.2 ml volume contained 4 pmoles of MgCl,, 0.4 pmole of EDTA, 10 pmoles of tris-HCI p H 8, 0.7 pmole of UDP-ghICOSe-"C (ca. 100,000 cpm), 0.1 pmole of DTT, 1 mg of cellobiose, and 0.05 ml of pellet containing 75 to 230 pg protein. Incubations were carried out in a shaking bath a t 25°C for 15 min. Reaction was stopped by heating the tubes in a boiling water bath for 5 min. One milligram of acid swollen cellulose and 0.5 ml of water were added to each tube, and the mixture was reheated. The solutions were centrifuged a t 25,000 X G (maximum) for 10 min and the extracts were siphoned off. The pellets were resuspended in sequence with various solvents, followed by centrifugation at 25,000 G, and each supernatant solution was siphoned off. Thus, the pellets were extracted, in sequence, twice more with 1-ml hot water (temperature of boiling water bath) for 5 min, once with 1 ml of chloroform-methanol (1 to 2), once with 1 ml of methanol (combined with chloroformmethanol extract), and twice with 1 ml of hot 1N NaOH (temperature of boiling water bath) for 5 min. The pellet was finally washed with 1 ml of cold water (combined with the alkali extract) and the residue was suspended in water for plating on planchets and subsequent radioassay. Aliquots of chloroform :methanol and methanol mixed extracts were radioassayed. The N a O H extracts were assayed for protein and for radioactivity. The chloroform: methanol-soluble is a measure of the enzyme activity referred t o as lipid synthetase which was not characterized further here, but which is probably a glycosyl transferase involved in glycolipid synthesis (Pinsky and Ordin, 1969; Ongun and Mudd, 1970). PROTEIN DETERMINATION AND STATISTICS. Proteins were determined by the modified method of Folin and Ciocalteu (Lowry et al., 1951). F o r the cellulose synthetase protein, a volume of 1 ml from the alkali-soluble extract was used. A 0.05-ml sample was used to determine protein content of phosphoglucomutase and soluble and insoluble UDPG pyrophorylase preparations. Particulate UDPG pyrophosphorylase samples were treated with 0.5 ml of hot 1 N N a O H for 10 min prior to the standard protein analysis. Analysis of variance was carried out by standard techniques, making use of the ratios of enzyme level. Each enzyme was analyzed separately for statistical significance. Product Hydrolysis. Alkali insoluble material was removed from the planchets with 0.2 ml 1N N a O H before the planchets were rinsed three times with 0.3 ml water. All of the samples were combined into one tube and centrifuged at 25,000 X G at 15°C for 10 min, and the supernatant solution was discarded. The pellet was then rinsed three times with 1 ml of water before being recentrifuged at 25,000 x G at 15°C for 10 min each time.
Pellets were hydrolyzed with 0.167 mg Streptomyces sp. B814 cellulase in 0.5 ml 2.5mM phosphate at p H 5.8 a t 50°C for 24 hr. Reaction was stopped by heating 10 niin in a boiling water bath. The solution was dried at room temperature in vacuo, spotted on S&S 589 White Ribbon paper and chromatographed three times in butanol, pyridine, water (30:10:15) for 10 h r each time. The paper was cut into I-cm pieces along its length and the activity on each piece was determined in a Nuclear Chicago, 720 Series, liquid scintillation counter. Samples of lipid-free, water-insoluble material were hydrolyzed with cellulase in the original incubation tube. QM
Results
Table I shows the levels of synthetases as a function of position in the expanding leaf. The amounts of UDP glucose converted to alkali-soluble and insoluble glucans were very small compared to conversion in oat coleoptile tissue. The leaf tip was not used in the investigation, since earlier experiments indicated that it was more resistant to PAN than was the center or base of the leaf. The data indicate that alkali-insoluble glucan (cellulose) synthesis was higher in the base, but the difference was significant only at the 10% level. Other synthetases (alkali-soluble glucan and lipid) were essentially not different between the two regions. At this stage, the basal part probably was growing faster than the center and the tip was probably not growing at all or very little. Growth was mainly cell expansion (Avery, 1933). The nature of the glucans produced by the reaction of UDP glucose is shown in Figures 2 and 3. An experiment was carried out as described and the alkali extraction was omitted. Water-insoluble, lipid-free material was hydrolyzed with cellulase, which contained P-1,4-glucanase and a trace of p1,3-glucanase (Reese and Mandels, 1963), and was chromatographed to yield the data shown in Figure 2. The @-1,3-dextrin series (laminaribiose and laminaritriose) as well as cellobiose and cellodextrin (P-1,4 series) were produced. This indicated the production of cellulose and of a minor amount of callose
Table I. Lipid and Glucan Synthetases in Tobacco as Function of Location in Leaf. Lipid NaOH-sol. Cellulose cpm/100 p g protein,/l5 min
Center Base Standard error of mean
2049 1885
232 243
65 86
95
28
7
Differences between center and base were not significant for a given synthetase at 0.1 level of probability except for cellulose synthetase, where the difference was significant at the 0.1 level but not at the 0.05 level. a
Table 11. UDP Glucose Pyrophosphorylase and Phosphoglucomutase in Tobacco as a Function of Location in the Leaf. UDPG PP PGM fimoles of substrate convertediminlmg protein
Center Base Standard error of mean
15.44 16.92
207.60 181.79
1.19
8.73
Differences between locations were not significant at 0.05 level of probability. UDPG PP = UDP glucose pyrophosphorylase; PGM = phosphoglucomutase. a
~
Volume 5, Number 7, July 1971 623
teo-
120-
E n 0
60
n
-
1
"01
5
20
IS
10
25
cm
Figure 2. Distribution of radioactivity in various carbohydrate components formed by cellulase digestion of water-insoluble product of incubation of U 3 P glucose with particulate enzyme from tobacco leaves. Products of several experiments with untreated tobacco plants were pooled to provide sufficient material for hydrolysis CD, cellodextrin; LT, laminaritriose; CB, cellobiose; LB, laminaribiose; G , glucose. See methods for details of incubation medium. Origin of chromatogram indicated by arrow
tw,
40
OO
5
IO
15
.20
G
25
30
cm Figure 3. Distribution of radioactivity in various carbohydrate components formed by cellulase digestion of alkali-insoluble product of incubation of UDP glucose with particulate enzyme for tobacco leaves. Products of several experiments with untreated tobacco plants were pooled to provide sufficient material for hydrolysis CD, cellodextrin; CB, cellobiose; LB, laminaribiose; G , glucose. See methods for details of incubation medium. Origin of chromatogram indicated by arrow 624
Environmental Science & Technology
(@-1,3-glucan). The alkali-insoluble material, which was hydrolyzed, yielded only cellobiose (Figure 3). Unhydrolyzed material was probably high molecular weight cellulose, since undissolved carrier cellulose remained in the hydrolysis tubes. Data for distribution of phosphoglucomutase and soluble UDPG pyrophosphorylase are given in Table 11. There was n o significant difference for amount of either enzyme as a function of position in the leaf. Preliminary experiments showed that the variation among selected leaves was too great to allow a direct comparison of enzyme level after gassing. Consequently; leaf halves were paired to provide internal controls. The ratio of enzyme activity after gassing compared to that before gassing was determined for control plants and for PAN-treated plants. Plants showed no visible symptoms for at least 4 to 5 hr after gassing. In fact, when such plants were placed in the dark within a n hour of termination of gassing, they were not damaged at all. The minimum light period after gassing needed to produce lethal effects was not determined here. The simple effect of treatment was statistically evaluated for center and for base. F o r lipid and for cellulose synthetases, the simple effect was not significant for center or for base as shown in Table 111. For alkali-soluble glucan synthetase, PAN treatment was highly significantly different from the control for the leaf base but not significant for the center. Interaction between treatment and location in leaf was highly significant for the alkali-soluble glucan synthetase and the main effect was therefore not examined statistically. Because there was no significant interaction between location in leaf and treatment for either lipid synthetase or cellulose synthetase, the main effects-Le., for center and base combined-were examined to provide better estimates of the treatment effect than could be obtained from either location separately. The PAN-treated plant mean was significantly lower than the control mean for cellulose synthetase but was not significantly different for the lipid synthetase. Phosphoglucomutase and soluble UDPG pyrophosphorylase from three experiments, where plants were sampled immediately after exposure to 850 to 986 ppb PAN, did not differ significantly by treatment. F o r pyrophosphorylase, the controltreated plant ratio was 1.08, while PAN-treated plant ratio was 1.17. For phosphoglucomutase, the control-treated plant ratio was 0.82, while PAN-treated plant ratio was 0.88. I n both cases, because of n o interaction between treatment and leaf area, the ratios were based on center and base together. If plants were assayed 24 h r after gassing, severe inhibition of all three synthetases was obvious. About 80% of the leaf area was damaged on both plants gassed with 840 ppb PAN in one experiment. The overall after-gassing/before-gassing ratios for control plants for lipid, alkali-soluble glucan, and cellulose synthetases in this case were: (center and base added together) 0.89, 0.88, and 0.74 while after-gassing/before-gassing ratios for PAN-treated plants were, respectively : 0.22, 0.44, and 0.06. Examination of phosphoglucomutase ratios (center and base added together) showed them to be 0.92 for control and 0.94 for PAN. The UDPG pyrophosphorylase ratios under similar circumstances were 1.09 for control and 2.46 for PAN. This increase in pyrophosphorylase activity suggested either a mechanism whereby leaf damage enhanced synthesis, caused a release of enzyme from a bound form, or activated the enzyme, As a first approximation, release of particle-bound enzyme was hypothesized since earlier unpublished studies showed sonication of oat coleoptile pyrophosphorylase increased activity. Consequently, plants were treated with PAN and the next day the leaves were ex-
Table 111. Effect of PAN Treatment of Tobacco Plants on Lipid and Glucan Synthetases Immediately after Gassing. Ratio: activity after gassing/activity before gassing Lipid NaOH-Sol Cellulose Base C B Center Center C B Center Base Base 1.25 1.11 1.70 1.61 1.17 0.98 1.08 1.26 1.13 0.87 1.32 0.72 1.11 1.01 1.28 0.94
+
+
Control PAN^ Significance Simple effects Interactions Main effects
NSC
NSc
NSc
NSc
NSC
____ C: B
+
1.66 1.22
NSc NSc
d
NAf
NSc
+
Six experimental runs. C B = average of center and base. 850 to 986 ppb PAN (V/V). c NS, difference between ratios not significant at 0.05 level of probability. d Difference between ratios significant at 0.05 level of probability. e Difference between ratios significant at 0.01 level of probability. f NA, not applicable. 4
amined for soluble and particulate enzyme. Particles were treated with detergent to get maximum bound activity released. PAN-treated leaves showed a striking rise in soluble enzyme ratio (Table IV) and a marked decrease in particlebound enzyme ratio. Discussion Sensitive tobacco leaves exposed to adequate light after PAN fumigation showed the expected cell collapse and eventual necrosis 24 hr later. In initial stages of cell death, cellulose and alkali-soluble glucan synthetases were strongly inhibited. This was not unexpected since recent findings (Pinsky and Ordin, 1969) showed that membrane integrity was important for cellulose synthetase functioning. Phosphoglucomutase, an enzyme which is sensitive to PAN,was unaffected and UDP glucose pyrophosphorylase, which is partially membrane bound, increased in activity. The increase was probably a result of release from degraded membranes. Breakdown in ultrastructure of beans following PAN gassing occurred when adequate light was provided afterward (Thomson et al., 1965). The most important finding, however, was that, if cell necrosis is prevented by transferring plants to decreased light early enough, a relative decrease of cellulose synthetase still occurred. Although synthetase apparently increased during the exposure period, it increased less in PAN-treated plants, indicating either interference with further formation and (or) inactivation of enzyme. Because of correlation between growth and P-glucan synthetases in oats (Hall and Ordin, 1968) and as shown by unpublished work in this laboratory, this finding is highly significant with respect to short-term growth effects in the field. If sensitive plants were exposed to PAN just before sunset-e.g., as is common in the eastern part of the Los Angeles basin-they might suffer a temporary setback in synthetase level and growth.
Table IV. Effect of PAN Treatment of Tobacco Plants on Soluble and Particle-Bound UDP Glucose Pyrophosphorylase 2 4 hr after Gassing Ratio : activity after gassing/activity before gassing Soluble Particulates” Control 0.48 0.69 PAN^ 1 ,72 0.25 Significance (three exp.). (two exp.)c a Particulate enzyme preparation was treated with 0.068 % Triton x-100. * 810 to 1400 ppb PAN (vlv). Difference between ratios significant at 0.01 level of probability.
Although evidence is strong that PAN directly inactivated the glucan synthetases (Ordin and Hall, 1967), interference with further formation of synthetase in oat coleoptiles in the absence of plant growth hormone was also reported (Ordin et al., 1969). Since pyrophosphorylase was readily released only by membrane degradation, and this degradation did not occur until several hours after gassing with PAN,it is unlikely that cellulose synthetase declined in the early period due to an effect on membranes to which synthetase is attached or of which it is an integral portion. However, if the effect on the membrane was subtle and reversible, inactivation of the enzymes by PAN-induced changes of plasma membrane is possible. The latter is yet to be shown however. Because of the high variability in tobacco plant leaves as well as the relative insensitivity of the plants to PAN, similar investigations on more uniform and more sensitive tissue in green plants are in progress. Acknowledgment
Streptomyces sp. QM B814 cellulase was a gift from E. T. Reese, U S . Army Natick Laboratories, Natick, Mass. Literature Cited Avery, G . S., Jr., Amer. J. Bot. 20, 565-92 (1933). Darley, E. F., Kettner, K. A., Stephens, E. R., Anal. Chem. 35, 589-91 (1963). Day, D., National Science Foundation Summer Student Report, 1968. Dugger, W. M., Jr., Koukol, J., Reed, W. D., Palmer, R. L., Plant Physiol. 38 (4), 468-72 (1963). Glater, R . B., Solberg, R . A., Scott, F. M., Amer. J . Bot. 49, 954-70 (1 962). Hall, M. A., Ordin, L., Physiolog. Plant. 20, 624-33 (1967). Hall, M. A., Ordin, L., “Biochemistry and Physiology of Plant Growth Substances.” Runne - Press, Ottawa, Canada, 1968, pp 659-71. Heck, W. W., Dunning, J. A., Johnson, H., U S . Public Health Service. National Center for Air Pollution Control Pub. APTD-68-6 (1968). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R . N., J . Biol. Chem. 193, 265-75 (1951). Ongun, A., Mudd, J. B., Plant Physiol. 45(3), 255-62 (1970). Ordin, L., Plant Physiol. 37(5), 603-8 (1962). Ordin, L., Hall, M. A., Plant Physiol. 42(2), 205-12 (1967). Ordin, L., Hall, M. A., Kindinger, J., Arch. Enciron. Health 18,623-6 (1969). Ordin, L., Garber, M. J., Kindinger, J., Physiolog. Plant. 23, 117-23 (1970). Pinsky, A,, Ordin, L., Plant Cell Physiol. 10, 771-85 (1969). Reese, E. T., Mandels, M., “Advances in Enzymic Hydrolysis Volume 5 , Number 7, July 1971 625
of Cellulose and Related Materials,” Pergamon Press, New York, 1963, pp 197-234. Stephens, E. R., Burleson, F. R., Cardiff, E. A., J . Air Poll. Contr. Ass. 15(3), 87-9 (1965). Stephens, R., Burleson, F., Holtzclaw, K., J . Air Poll. Contr. ASS.19(4), 261-4 (1969). Taylor, 0. C., 1967, unpublished. Taylor, 0. C., J . Air Poll. Contr. Ass. 19(5), 347-51 (1969). Taylor, 0. C., Eaton, F. M., Plant Physiol. 41(1), 132-5 (1966).
Thomson, W. W., Dugger, W. M., Jr., Palmer, R . L., Botan. GOZ.126, 66-72 (1965). Todd, G. W., Garber, M. J., Botan. Gaz. 120, 75-80 (1958). Receiced for reoiew June 17, 1970. Accepted Nocember 9, 1970. This incestigation was supported in part by research grant AP 00213 and contract no. PH86-68-71 ,from the National Air Pollution Control Administration OJ the U S . Public Health Sercice.
Removal of Sulfur Dioxide from Stack Gases by a Modified Claus Process Robert T. Struck, Metro D. Kulik, and Everett Gorin Research Division, Consolidation Coal Co., Library, Pa. 15129
The results of a bench-scale study on the potential application of a low-temperature Claus process to the treatment of dilute SO2-containingstack gases are presented here. Optimum results are obtained at a temperature of about 100°C. Under these conditions, the sulfur formed quantitatively condenses on the catalyst and the product gas contains less than 50 ppm of sulfur compounds. Data are given for a two-stage regeneration process which eliminates catalyst poisoning caused by sulfate formation. Of the sulfur recovered, 93 % is elemental sulfur, the rest is ammonium sulfate. These results were obtained with a synthetic flue gas having the same composition as a power-plant stack gas, with the exception that NO, was absent. Preliminary results showed that the presence of NO, accelerates poisoning of the catalyst, but that the above twostage regeneration procedure is still effective for reactivating the catalyst. H
R
esearch and development is being conducted by government and industry to develop processes for removing SO, from stack gases. The removal of SO? from power-plant stack gases is, of course, receiving the major attention, but removal of SO, and H?S-SO> mixtures from other process tail gases is also important. The latter, for example, includes off gases from roasting of sulfide ores as well as tail gases from sulfuric acid manufacturing plants and from conventional Claus plants. A method for accomplishing this objective has been proposed based on the use of a “modified”-type Claus process. The process operates by injecting H2Sinto the gas to provide an HyS,’SO,mol ratio of 2, as required for the catalytic reaction, 2 H,S
+ SO? = X3 S, + 2 H?O
(1)
I t differs from the normal Claus process in that the proc:ss is carried out at a much lower temperature and the sulfur product is largely condensed on the catalyst. The conventional Claus process operates at a higher temperature which is always above the dew point of sulfur vapor. The conversions that can be achieved at the higher tempera626
Environmental Science & Technolog)
ture are limited by the thermodynamic equilibria, as has been previously discussed by Gamson and Elkins (1953). Generally, the conversion is below 95 %, even when more than one catalytic stage is used. One main potential advantage of a Claus-type process is that the amount of reductant required to produce elementary sulfur from SO? is reduced to very nearly the minimum of 2 mol/mol of sulfur as dictated by the overall process,
Other regenerable SOuremoval processes now under development require a minimum of 3, and as many as 4 mol of reductant, expressed as hydrogen, per mole of sulfur recovered (Bienstock et al., 1965; Oldenkamp and Margolin, 1969). The first attempt to apply the present type of process to gas purification was made to coke oven gas by Audas (1951). In this case, the process was applied in reverse-Le., SO? was added to the H?S-containing gas, and the modified low-temperature Claus process was conducted with condensation of sulfur on the alumina catalyst with subsequent regeneration. Application of the concept to flue gas treating was proposed by Kerr (1968). In both the Audas and Kerr processes, the sulfur-fouled catalyst is cycled through a thermal regeneration step, where sulfur is removed by distillation at about 500°C. Princeton Research has undertaken more recent work to develop this type of process (Chem. Eng. News, 1968) under the auspices of the National Air Pollution Control Administration. Little information is available, however, about the results of their work at this time. One aspect of this type of process is that the hydrogen sulfide content of the treated gas must be maintained at a very low level, and thus the efficiencyof conversion must be very high. Consolidation Coal Co. undertook evaluation of the modified Claus process in its laboratories because, economically, it seems to be potentially one of the most attractive processes for treating flue gas. The present paper deals with a study of the major features of the process and the limitations in its potential application to particular gases. The following subjects are covered: thermodynamic limitations of the process, experimental determination of the effects of the major flue gas components and process variables, and a study of methods of catalyst regeneration. A two-step re-
