Determination. of Cellulose in Agricultural Residues Modi$cations of the Norman-Jenkins .Method ELISHA B. LEWIS' Chemistry Department, Kentucky Agricultural Experiment Station, Lexington, K y . The proposed method of cellulose isolation is based on the >-orman-Jenkins method but has seleral modifications. It is continuous, thus aioiding transference of samples, it requires much less Lime, and it is not susceptible to variations in chlorine conceutration within the limits giFen. The crude celluloses by both methods and their a- and y-cellulose and pentosan fractions compare very well but show marked differences in ash. The much lower ash in crude celluloses b j the proposed method is a function of the boiling water and 5 % sulfuric acid washes.
58' C. and 50 mm. of Tercury vacuum) for 40 minutes and cool under room conditions; add 10 and 20 ml. of 727, sulfuric acid (660 ml. of concentrated acid to 411 ml. of distilled water) a t about 4' C. to the samples in 50-nil. beakers, place in a refrigerator a t about 4" C., and let stand, with occasional stirring, to ensure complete removal of soluble material (at least 24 hours); dilute the suspensions to 5% acid concentration (add 219 ml. of water to the 10-ml. suspension and 438 ml. of water to the 20-ml. suspension); reflux 2 hours; filter; wash with boiling water until acid-free; dry a t 105" C.; cool in desiccator; weigh residues; then ash the residues and weigh again and calculate the lignin weights by difference. Pentosans viere determined by the A.O.A.C. method ( 2 ) except that constant volume TTas maintained by dropwise addition of 12% hydrochloric acid. Total cellulose, which is the equivalent of crude cellulose less ash, and the a- and y-cellulose fractions were determined in the crude cellulose by the TAPPI method (II), but substituting 0.1 4sodium thiosulfate, 10% potassium iodide, and 17, starch indicator, and using the factor: 1 ml. of 1 N potassium dichromate = 0.0068 gram of cellulose. The "pure" cellulose is the crude cellulose less ash, lignin, and pentosan as defined by Reid et al. ( 7 ) but may contain some hexosans, as in the case of woods. The per cent of available chlorine in sodium hypochlorite was determined as follows: Dilute 10 ml. of sample to 400 ml., shake, and take 50-ml. aliquot; add 10 ml. of 10% potassium iodide, 1 ml. of 20% sulfuric acid, and about 25 ml. of distilled water, and titrate with 0.1 N sodium thiosulfate, using 1% starch indicator.
I T H the ever-increasing demand for paper products and the time elemcnt involved in the replacement, of forests t o provide pulp products, the pulp and paper industries are supplementing their forest resources with agricultural residues. This practice seems destined t o become more important in t,he future, since it provides a supply produced annually in most cases and utilizes materials which are considered as wastes or are used to coniparatively small advantage on the farm. Because some of the standard methods for cellulose isolation and determination in evaluating agricultural residues for pulping are excessively t,ime-consuming and tedious there is a distinct need for a method which is rapid, simple, and accurat,e. This work brings out, such a method, in which handling of the.samples is reduced t o a minimum. It is based on the Xorman-Jenkins method (4),but incorporates several modificat.ions. In t,his paper, the Sornian-Jenkins method is used as a standard of comparison. &proximately 150 quantitative cellulose isolations and 10 batch isolations were made by the Kornian-Jenkins and proposed methods. The resulting crude celluloses n-ere analyzed for total celluloses a- and y-celluloses, pentosans, lignin, and ash. PREPARATION OF SAMPLES
APPARATUS
The three materials used in this study (tobacco stalks, hemp b u d s , and sorghum bagasse) are fairly representative Of most agricultural residues. The tobacco stalks and hurds were received air-dry and the sorghum b a g a m moist, fresh from the mill. The bagasse was dried for about 30 hours a t about 85" C. ______. in a forced draft dryer. All samples were ground to pass a 60mesh sieve and thoroughly stirred so as to be homogeneous.
porbsityto30-ml. sintered-glass crucibles 2 liter Pyrex Pyrex filter flasks forfilter suction. For areMedium used, attached stirring, a glass rod with rubber policeman is desirable.
~
REAGENTS
Sulfite-bicarbonate solution: 31.0 grams of anhydrous sodium sulfite and 10.3 grams of U.S.P. sodium bicarbonate per liter. Alkaline salt solution: 20 grams of sodium sulfite, 11 grams of sodium sulfate, 9 grams of sodium chloride, 9 grams of sodium bicarbonate, and 250 ml. of 0.5 N sodium hydroxide, and diluted to 1040 ml. with distilled water. Acid hypochlorite: 30 ml. of sodium hypochlorite (4.07, available chlorine) and 20 ml. of 20% sulfuric acid added to 1 liter of distilled water. Sulfuric acid 20%: 124 ml. of concentrated sulfuric acid, and 904 ml. of distilled water. Sulfuric acid 5%: 29 nil. of concentrated sulfuric acid, and 978 ml. of distilled lvater. Ammonium hydroxide 1 t o 20: 50 nil. of concentrated ammonium hydroxide, diluted to 1 liter with distilled water. Ethyl, alcohol, 957& Commercial ethyl ether.
ANALYTICAL PROCEDURE
for 3 Moisture was determined by drying 2 grams of hours a t 100" C. Ether-alcohol solubility (10)was detemnined on 5-gram samples by extracting 24 hours with ethyl ether and 50 hours with 95Yc ethyl alcohol. rllcohol-benzene soIubiIity (9) was determined by extracting 2 grams of sample with a sdution of 3 3 5 ethyl alcohol (95%) and 67% benzene by volume for the t h e necessary for good checks on dupllcate samples. Crude ceu&se, which is cellulose plus pentosans, hexosans, lignin, and ash, was determined by the Korman-Jenkins ( 4 ) and Proposed $sh was determined in the original material and in crude cellulose using 2- and 4-gram samples, respectively, by the &O.A.C. method ( 1 ) . Lignin was determined on 1- and >gram samples, respectively, by the method of Hessler ( 5 ) , which colnbines the methods of Korman-Jenkins (51, Ritter (8), and Peterson et al. ( 6 ) . The combined met,hod of lignin analysis using the 1- and 2-gram samples is essentially as follows: flux 1 hour in 100 and 200 ml. of 570 sulfuric acid (29 ml. of concentrated sulfuric acid to 978 ml. of distilled water) ; filter; wash, first with boiling distilled water until acid-free, then with 95% alcohol, and then with ether; dry in a Vacuum Oven ( a b u t
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present address, Projects Supervisor, Commonwealth Engineering co.of Ohio, 1771 Springfield St., Dayton, Ohio.
METHOD
Weigh accurately 0.59 to 0.61 gram of ground sample into the filtering crucible and attach to filter flask. I n all washes fill the crucible almost to the top, stir, let settle for 3 to 4 minutes, and filter using suction. Wash first with boiling alkaline salt solution, and repeat this wash until the filtrate is clear and colorless. Eight to ten washes are usually required.
910
.
911
NOVEMBER 1947 Then wash with boiling water, cool acid hypochlorite, and boiling sulfite bicarbonate solution, a t which point the residue is usually a dark purple. Repeat the washing series until no lignin color test is obtained as indicated by the white residue after the final sulfite-bicarbonate wash. All cool washes are a t room temperature (about 27" (3.). Wash once with boiling water, twice with cool 57, sulfuric acid, once with cool and four times with boiling 1 t o 20 ammonium hydroxide, once with alcohol, and once with ether. Place crucible with residue in a weighing bottle and dry in a vacuum oven (about 50" C. and 50 mm. of mercury) to constant weight. Usually 2 hours are sufficient. Cool in desiccator and weigh. Then remove residue (crude cellulose) mechanically, quantitatively, and weigh the weighing bottle with crucible. The difference in the weights is the weight of the crude cellulose. It is important that no weighings be made until this time because the crucible loses weight from the alkaline washes. FACTORS INFLUENCING METHOD
Liquid-Solid Ratio. The use of approximately 0.6-gram sam-' ples for quantitative isolat,ion of crude cellulose in 30-ml. crucibles is recommended, since this gives the same liquid-solid ratio as, in the Norman-Jenkins method (4),and the crude cellulose can be analyzed directly by the T A P P I method (11) volumetrically for total cellulose and the a- and y-cellulose fract.ions. Transfer of the samples during processing is avoided. Samples of approximately 0.6 gram are shown t o be representative in this work by the comparable results obtained from a considerble number of analyses of identical samples. Filtration. The medium-porosity Pyrex sintered-glass crucibles were used for representative retention of the residues along wit,h good filtration and minimum loss in crucible n-eight by the alkaline washes. These crucibles did not have equal rates of filtration, but this was remedied b y pouring hot. 17.57, sodium hydroxidc through them under suction until they gave about equal rates of filtration with cold water. In the first expcriments on cellulose isolation using t,he crucibles, filtration \\-as very- difieult on pretreating the samples by washing alternatc~ly with boiling 37, sodium sulfite and cool "neutral" bodiuni hypochlorite as in thc Sorman-Jenkins method very difficult on pretreating the samples s ng 37, sodium sulfite ~ a s l i e s . Additioii of sodium hypochlorite (4.0 to 5.0ccavailable chloliiie) to thc. sdfitebic:irboiiate solution in the volume ratio of 14 to 90 greatly d up tht, filtratioii, esprciall>- for tlie sorghum bagasse 1 3 ~ ~ 1 the ~ 1 , sodium hypochlorite hreaks down t o form sodium chloride and oxidizes an equivalcnt portion of sodium sulfit(. to sodium sulfate. I n this ivaj-. thc, composition of th(. allraline salt 7,)lution \vas cst~ablished. Tho presence of sodium bicarbonatc>:ipparently does not affect the ratc of filtrat,ion. Washes. The alkaline salt washes are used for easier filtration, as shown above, and removal of resinous materials. Thc hoiling n-at,er washes are used in the proposrd method bocause in preliminary experiments it was noted that this greatly intensified the lignin test color, especially at, thc start, and also becauw this, along with the final extractions with 5 5 , sdfuric acid, is instrumental in reducing the ash in the crude cellulose. The hoiling n-atcr is possibly effective in lignin removal. but t h e results are not conclusive, probably because of the slightly inhibitory effect of the sodium bicarbonate. The intensification of the sulfite lignin test color is probably due t,o removal of residual sulfite by the boiling water from the'residue, which in turn is actcd on more completely by the acid hypochlorite. Following the tioiling water washes with the cool acid hypochlorite raises the temperature of the acid hypochlorit,e \\-ashes only t o about 35" ('., which is not enough t,o cause ap'preciable cellulose break-
'
1 experiments were made in cellulose determination by the proposed method, using hypochlorites varying from 5.0 t o 4.0y0 available chlorine. The hypochlorites were used in t,he same volume ratio as specified for hypochlorite of 4.0yo available
chlorine. The resulting variations in salt and chlorine concent,rations had litt,le or no effect on the method as t o bleaching stages required, time, crude cellulose, or analysis of the crude cellulose. The use of sodium hypochlorit,e of 4.0% available chlorine is recommended simply for saving in reagent. The sodium bicarbonate is added to the 3% sodium sulfite solJtion, as heretofore given, because it has an inhibitory effect on t,he action of the sodium sulfite on the cellulose without seriously affecting t,he removal of lignin. This has been shown by Aronovsky ( l e )in fiber cooks under conditions of elevated temperature and pressure and is further evidenced by the stability of tke proposed method over a range of chlorine concentrat,ions. The t,wo extract,ions 1vit.h cool 5% sulfuric acid remove any residual sodium sulfite in the crude cellulose and reduce the ash. The cool and boiling 1 t o 20 ammonium hydroxide washes are used in the order recommended first to neutralize the residual acid in the crude cellulose and then t o remove the residual salts and any traces of acid. If the 57, sulfuric acid washes were followed by the boiling 1 to 20 ammonium hydroxide, danger of cellulose breakdon-n would arise, particular1)- in batch runs, due t o increased acid activity through heating before the ammoriium hydroxide completely penetrated the fibers. For the most part, the 1 t o 20 ammonium hydroxide was brought to boiling in the open and used quickly enough t o prevent excessive loss of ammonia. For a part of the work, it was brought t o boiling under a reflux condenser xvhich helped prevent ammonia loss. Drying. The vacuum oven is preferred t o the usual drying a t 100" t,o 105' C. in air ovens in order t,o avoid danger of cellJlose breakdoivn. SODIUhI HYPOCHLORITE CONCENTRATIOS S
I n analyzing a series of commercial sodium hypochlorite solutions labeled "5.25v0 sodium hypochlorite by \wight," it was found that the available chlorine ranged from 3.96 t o 4.0cC. The volumes of the hypochlorites used as reported in this paper w r c varied t o give the chlorine and sulfate conccntrirrions spccified for the Sorman-Jenkins and proposed mt:thods-for inst :tncc, 3.8 nil. of sodiuni hypochlorite (3.9BCc available ch11)riiic~i= 3 ml. of sodium hj-pochlorit,e ( 5 . 0 5 available ehloriiicx I . I n t,he acid hypochlorites, the volume riitio of 1iyp:)chloritc to acid ivas always 3 t,o 2 . The theoreticnl value for solutions of 5 , 2 5 5 sotii,ini hypochlorite by \wight is 5.0% available chlorine. IIon-cvt.r, on standing, the hypuchlorites gradually lose their ai-:dnl)lc ciilorinc. Since none of the sodium hypochlorite solutions in this work ran as high as 5.0vo availablc chlorine, the term "hypochlorite of 5.0% available chlorine" means that sufficient, voluiii(~sof hypochlorites of lesser available chlorine m r e used t,o givc tlie same chlorine and sulfate concentrations as 3, 15, and ki nil. of hypochlorite of 5.OCO available chlorine, respect acid hypochlorite, 105 ml. of "neutral" sodium h~pochloritc( d ) , and 104 nil. of alkaline salt solut,ion. COMPARISON O F METHODS
Cellulose determinations were made by the Sornim-Jonkins and proposed methods for comparison as t o time, hle tching stages, crude cellulose, total cellulose, a-cellulose, -pxJll dose, ash, lignin, and pentosan. For the first five, sodium hypochlorites of 4.0 and 5.0cC nvailable chlorine were used. For the subsequent comparison>, sodium hypochlorite of 5.0qo available chlorine was used f,)r the Norman-Jenkins method, which gives the chlorine concentrations specified for the original method. I n the Yornian-Jenkins method, the sodium hypochlorites of 4.0 and 5.0% available chlorine were used as follows: neutral hypochlorite, 15 ml of sodium hypochlorite t o 90 ml. of distilled water; acid hypochlorite, 3 ml. of sodium hypochlorite and 2 ml. of 20Vc sulfuric acid in 100 ml. of distilled water.
'
912
V O L U M E 19, NO. 1 1 Table I.
Analyses of Original Materials
Hemp Hurds Duplicate Moisture Ash Ether-alcohol extraction Alcohol-benzene
ash-free) Total cellulose (NormanJenkins)= Crude cellulose (~roDosedmethod. ash-free) Total cellulose (proposed method) u-Cellulose (Norman-Jenkins) a u-Cellulose (proposed method) Pure cellulose (Norman-Jenkins).’ Pure cellulose (proposed method) Lignin Pentosan 0
1
2
6.00
1.21 4,14
6.20 1.19 2.97
4 38
3.c
Sample Sorghum Bagasse Tobacco 1 2 1 6.11 2.80 15.66
6.07 2.79 13.13
6.42 8.90 13,22
Stalk 2 6.43 8.93 15.61
13.81
13.44
11.88
12.55
50.26
50.95
41.55
41.49
52.70
52.94
48.85
48.48
40.23
39.86
54.78
54.86
52,03
51.29
42.38
‘43.26
54.40
54.49
50.86
51.02
42.93
43.27
37.38
37.61
33.93
33,36
28,29
28,99
38.97
39.04
35.05
34.95
31.24
31.20
41.79
41.90
34.47
34.61
31.80
31.64
40.93
41.08
34.01
33.97
31.89
32.29
21.71 24.20
22.15 24.20
11.81 27.41
11.83 27.41
12.96 20.70
13.02 20.36
Sodium hypochlorite of 5.0% available C1 used.
tion levels is probably due to differences in ash and pentosan removal. The total cellulose values by the proposed method using sodium hypochlorites of 5.0 and 4.0% available chlorine are in good agreement between the two chlorination levels and with the crude cellulose values. The use of sodium hypochlorites of 5.0 and 4.0% available chlorine influences the Norman-Jenkins method also as to bleaching stages and time required. The time required by the proposed method is one half to one third that required by the Norman-Jenkins method. The crude celluloses are higher in a-cellulose by the proposed method than by the Norman-Jenkins method, but this is pretty well accounted for by the differences in ash and pentosan contents. In examining the pure and a-cellulose values, it is seen that the pure celluloses are somewhat higher than the a-celluloses in the tobacco stalks and hemp hurds. The same holds true for the Norman-Jenkins values in the sorghum bagasse, but the converse is true in the proposed method values. Fibrous residues from annual crops usually contain about 8% more or-cellulose than pure cellulose, whereas wood fibers usually contain considerably more pure cellulose than a-cellulose. This is probably due to higher percentages of hexosans in the crude wood celluloses than in the crude celluloses from the residues (11). The tobacco stalks and hemp hurds, though annual crop residues, are of a woody nature, and in the light of their pure and or-cellulose values may be considered borderline cases. The anomalous values obtained for the sorghum bagasse may be due to characteristic differences in the methods for bringing down the hexosan fractions.
Samples of 0.6 gram in 30-ml. medium-porosity Pyrex sinteredglass crucibles were used’ for the Norman-Jenkins method, since this gives the same liquid-solid ratio but avoids the transference of samples necessary in the original method. Batches of crude cellulose of 29 t o 35 grams each were made up by the Norman-Jenkins and proposed methods using No. 4 Buchner Coors porcelain funnels with No. 10 canvas filter circles, ACKNOWLEDGMENT preshrunk, proportionately increasing the weight of sample and The author wishes to express his appreciation to J. S. Mcvolume of solution. The ash, lignin, and pentosan analyses were Hargue, Karl E. Rapp, and Martha Koppius for their helpful critirun on these batches. cisms and assistance in preparingthe manuscript. I n running Norman-Jenkins cellulose determinations and batch isolations, the cooling of the sulfite washes can be retarded by placing watch glasses over the crucibles and funnels. Table 11. Comparison of Korman-Jenkins and Proposed blethods as to Time, Bleaching Stages, and Cellulose Yield -RESULTS
All results in Tables I to I11 are given in per cent and on oven-dry basis, except moisture. I n Table 11, a “bleaching stage” is a series of extractions with boiling water, acid hypochlorite, and boiling sulfite-bicarbonate for the proposed method and a series of extractions with acid hypochlorite-boiling sulfite for the NormanJenkins metliod. DISCUSSION
The proposed method of cellulose isolation and determination is based on the Norman-Jenkins method, modified by alkaline salt pretreatment, use of the boiling water wash, acid hypochlorite of lower chlorine concentration, and sodium bicarbonate in the sodium sulfite and the dilute acid and ammonia washes. I n the cellulose determination using sodium hypochlorites of 5.0 and 4.0% available chlorine, the crude cellulose values check very favorably. IIowever, the Norman-Jenkins total cellulose values using sodium hypochlorite of 5.0% available chlorine are somewhat lower than those using sodium hypochlorite of 4.0% available chlorine. The variations in the Norman-Jenkins total cellulose contents a t the two chlorina-
(Using sodium hypochlorite 5.0 and 4.0% available chlorine)
%
Check
Method
Bleaching stages
NormanJenkins Proposed
Time, hours
NormanJenkins Proposed
Crude cellulose (with ash)
NormanJenkins Proposed
Total cellulose
NormanJenkins Proposed
Available Chlorine in Hypo- Hemp chlorite 1 5.0 4.0 5.0 4.0 5.0 4.0 5.0 4.0 5.0 4.0 5.0 4.0 5.0 4.0 5.0 4.0
9 14 7 8 7.5 10.4 3.1 2.9 65.56 55.15 54.87 54.86 52.70 54.47 53.85 54.40
Hurd
Sorghum
9 14 7 8 7.5 10.4 3.1 2.9 55.87 55.29 55.55 54.95 52.94 54.90 54.16 54.49
15 23 15 14 11.1 15.9 4.35 3.7 50.79 50.27 51.42 52.12 48.85 50.34 50.59 50.86
2
1
Bagasse Tobacco 2
1
15 23 15 14 11.1 15.9 4.35 3.7 51.46 51.07 50.72 51.38 48.48 50.02 50.11 51.02
7 10 7 9 6.3 8.1 3.1 3.1 42.02 42.13 43.17 42.42 40.23 41.92 42.83 42.93
Stalk 2
7 10 7 9 6.3 8.1 3.1 3.1 41.96 41.99 43.26 43.30 39.86 42.20 43.32 43.27
Table 111. Analyses of Crude Celluloses Isolated by Norman-Jenkins and Proposed Methods Hemp
Hurd
Sorghum
Bagasse
Tobacco
Cheok
Method
1
2
1
2
1
2
a-Cellulose
Norman-Jenkina Proposed Norman-Jenkins Proposed Norman-Jenkina Propoded Norman-Jenkins Proposed Norman-Jenkins Proposed
67.57 71.35 16.02‘ 14.40 1.22 0.18 0.20 0.52 23.82 24.58
67.03 71.43 17.32 15.43 1.26 0.16 0.18 0.52 23.88 24.29
66.05 68.56 8.07 9.54 0.94 0.16 0.32 0.44 31.26 34.15
65.05 68.12 7.41 9.53 0.97 0.19 0.28 0.41 32.40 33.36
69.03 71.98 13 90 13.60 1.13 0.12 0.16 0.33 23.32 22.43
69.29 71.69 13.21 14.07 1.07 0.12 0.11 0.38 23.56 24.99
-,-Cellulose Ash Lignin Pentosan
Stalk
N O V E M B E R 1947
913
LITERATURE CITED
(l) Assoc. OfficialAgrs
Official and Tentative Methods Of Analysis, 5th ed., p. 354 (1940). (2) Zbid., 6th ed., p. 413 (1945). (3) Hessler, L. E., U. S. Department of Agriculture, unpublished report. (4) Norman, A. G., and Jenkins, s. H., BiOchem. J., 27, g18 (l933). ( 5 ) Zbid., 28,2147-68 (1934). (6) Peterson, C. J., Walde, A. W., and Hixon, R. M., IXD.Eh‘O. CHEM.,ANIL. ED.,4, 216 (1932). Chem.y
(7) Reid, J. D., Nelson, G. H., and Aronovsky, S. I., Zbid., 12, 255 (1940). (8) Ritter, G. J., Seborg, R. M.,and Mitchell, R. L., Ibid., 4, 202 (1932). (9) Tech., .4ssoc. Pulp and Paper Industries, Method T6m-41. zbid,, 31ethod T2,34m-35, (11) Ibid., Method T429m-42. (12) myse, L. E., “Wood Chemistry,” A. C. S. Monograph No, 97, p. 773, New York, Reinhold Publishing Corp., 1944. RECEIVEDDecember 5 , 1945.
Aerobic Fermentor with Good Foam-Control Properties JEROME F. SAEIZAN Forest Products Laboratory, Forest Service, U . S. D e p a r t m e n t of .4griculture, Madison, Wis.
I
which successfully and economically produced food yeast for human or animal consumption from sulfite waste liquor. The Waldhof fermentor appears t o be a solution t o a problem novel fermentor had been developed by Zellstofffabrik Waldhof, in the proposed production of food yeast from carbohydrate wastes, including those from dairies and canneries and in fruit production and processing. This proposal for yeast production has been receiving increasing attention in the United States (3, 4, 6, 8 ) , both from the standpoint of economical and sanitary waste disposal and as a means of obtaining a high-quality protein food product. T h e difficulty caused by foaming has been one of the main obstacles t o successful food-yeast production. When sulfite waste liquor and other wood sugars were used, foamingpreviously could be controlled only by the use of such large amounts of defoaming agent as t o constitute a major expense for the product. The German development seems t o have solved this problem, and gives reason t o believe that its principles may prove useful in other aerobic fermentations, such as the production of baker’s yeast, penicillin, SCALE 0 I 2 3 INCHES and inoculum for alcoholic fermentation, and in the actiVULCAN/ rE vated-sludge method for the treatment of certain concentrated wastes. The Waldhof process for fodder-yeast production permits fermentations t o be carried out continuously at a high rate of speed and maintains the yeast AIR CAP BO SHAFT in vigorous condition. It apSCALE BEA /?IN G BUSHING pears t o be suitable for use SCALE SCALE # I INCH 0 /INCH with any of the wood sugars thus far tested, in spite of the fact that wood sugar is Figure 1. Laboratory-Scale Experimental Fermentor with Mechanical Aerator
N T H E course of technical investigations during 1945 of the German wood-using industries (5, 7’), i t was found that a
w
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rroM,
