Guayule Rubber J
MICROBIOLOGICAL IMPROVEMENT BY SHRUB RETTING PAUL J . .ALLEN1 AKD RALPH EMERSON2 Guayule Rubber Extraction Research Unit, C'nited States Depurtnaeni of Agriculture, Salinas, Cultf.
T h e quality of rubber from guayule shrub may be markedly and consistently improved by retting the shrub prior to milling. The resinous contaminants of the crude are reduced to about one half the quantity present i n unretted guayule rubber, tensile strength is increased about 5070, and some other phSsica1 properties are improved. Advantages may also be gained in handling and milling the shrub, and the recovery of rubber hjdrocarbon is excellent. The improvement is caused by microorganisms, and its success therefore depends upon the provision of conditions favoring the rapid development of an active microbial flora, which may include molds, bacteria, or actinomycetes. Access of oxygen to the shrub is essential, but must
UAYULE rubber obtained by conventional means is admittedly inferior to Hevea rubber both h chemical composition and in physical properties An improvement in the quality of guayule would therefore be a material contribution toward competitive production of this native rubber. The Continental Mexican Rubber Cornpany a t Torreon, Mexico, found it possible under war conditions to improve the quality of a small proportion of its production by chemical means, but a t high cost. The possibility existed of markedly improving tne quality of the entire production a t low cost simply by storing the harvested shrub in the proper manner prior to milling. When the Government of the United States undertook the job of producing rubber for the war emergenev, this possibility urgently needrd investigation. Spence (15)showedin 1930and 1933that storageofmoist guayule shrub in various nays resulted in the production of a rubber n ith reduced acetone extract and improved physical properties. Appreciable losses in dry weight of shrub were recorded, and the resulting greater throughput t o the mill vias emphasized. Spence also indicated that this moist storage hastened coagulation of the latex, and that improvement in the qualitv of the rubber took place more rapidly with cut or crushed than x i t h n-hole shrub. This treatment of the shrub Spence termed a retting process, in loose analogy with the retting of flax, thereby suggesting t h a t the observed iniprovement might result from microbial action. The specific agents responsible for retting were not, however, clearly defined, the precise conditions requisite fo? uniformly good retting were not known, and the fundamental nature of the process \va3 not understood. Past failure t o achieve unifoi nily successful rets on a large scale niuit be ascrihci t o this ldck of knowledge concerning the conditions and agents involved in retting. In 1942, as a part of the goveinment-sponsored guayule research project, preliminary efforts a t the Eastein Regional Research Laboratories were therefore directed toward solution of some of the fundamental problems involved in shrub retting. Following indications in the records of the Intercontinental Rubber Company that uniform aeration of the shrub is e,SriitiaI 1 Present address, Department of Botany, University of M lsconcln, Madison, WIS. 2 Present address, Department of Botany, University of California, Berkeley, Cal1f.
be accompanied b.i temperatures and moistures within a suitable range, which will vary somewhat depending upon the degree of mechanical reduction of the shrub and the frequency of mixing. Practical methods for attaining favorable conditions have been devised and tested, and their applicability to commercial operation is indicated. Outstanding are the methods of bale retting, floor retting, and retling in continuously rotating drumfi. With the latter method optimum improvement is obtained in about 72 hours. Evidence is presented indicating that microorganisms bring about improved physical properties through selective removal of deleterious resinous contaminants.
for good retting but difficult to achieve on a large scale, White and his eo-workers (23) investigated the use of forced aeration in pilot scale tests. Appreciable, progressive improvement in rubber quality was obtained in their experiments, and a close interrelation among retting time, resin content, tensile strrngth, and the content of rubber hydrocarbon wa? noted. Other concurrent studies ( I j deinonetrated that certain molds and bacteria are capable of decomposing one or inore fractions of the acetonrsoluble contaminants of guayule rubber, and clearly indicated that these resin-decomposing microorganisms might be the agents responsible for retting. Beginning late in 1943, investigations of shrub retting were resumed a t the Guayule Rubber Extraction Research Unit in Salinas, Calif., and extensively developed during the following 2 years. The results obtained make it posaible (1) to present c-onclusive proof of the microbial nature of guayule retting, (2) to identify the active microorganisms, (3) to relate their growth requirements and metabolism to the retting proceas, (4)t o formulate with precision the complex of conditions requisite for optimal retting, (5) to evaluate on a sound basis the relative merits of some eight different methods of retting and their application to commercial practice, and (6) t o gain some insight into the relation between the improvement in chemical composition and the improvement in physical properties of rubber from rrtted shrub The first three aspects are discussed in ail account ( 6 ) concerned primarily with the purely biological phases of the problem I n the present discussion, covering the last three aapectq, attention is focused primarily on the practical application of i etting as a means of improving the qualitv of guayule rubber. This 16 a highly arbitrary division dictated by the extensive nature of thp material covered and the widely diver gent fields of interest c7oncerned. Had it been possible, integration of all phases in a single monograph vould have permitted a more effective prescntation of the \\-hole subjert, since only by correlhting the microbiology with the rubber technologv can one expect to reach a rrally sound understanding of guayule retting. This relation between the microorganisms and the processing of guayule holds not only for retting but also for other phases of processing between harvesting and milling. Like every plant or animal product, guayule shrub is subject to microbial decomposi-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
tion, which can be entirely prevented only by extreme environmental conditions impossible to attain in ordinary practice. If completely uncontrolled, these virtually unavoidable deconipositions may actually be detrimental to rubber quality, rubber recovery, or both. If controlled and properly directed they can be utilized to achieve improvements that would not otherwise be realized. The controlled use of microorganisms in industrial processes dates back to man’s early history. The production of wines, beers, and other alcoholic beverages, the making of cheeses, sauerkraut, and silage, and the retting of flax to prepare linen fibers all depend on the action of certain microorganisms, the development of which is favored by the control over conditions of storage of a wide variety of raw materials of plant or animal origin. With the growth of the sciences of bacteriology and mycology during the past few decades, industrial processes utilizing microorganisms under controlled conditions have come into greater prominence. The present methods for large scale production of alcohol and glycerol by yeast fermentations, citric and gluconic acid by mold fermentations, and a variety of important chemicals, including butanol, acetone, and propanol by bacterial fermentations, all eloquently testify to the feasibility of such applications. The idea of developing to a commercial scale a process utilizing microorganisms for the improvement in quality of guayule rubber is therefore backed by a long history of precedent. MATERIALS AND STANDARD PROCEDURES Guayule (Parthenium argentatum Gray) is a perennial, woody shrub of the temperate zone, native to semiarid regions of northern Mexico and the southwestern United States. It is one of many rubber-bearing plants in the family Compositae, but has been the focus of particular attention because of its outstandingly high content of rubber. Shrub that is 4 years old may contain as much as 20% rubber hydrocarbon on a dry-weight basis. I n the early years of the rubber boom, guayule rose to a place of prominence, a t one time providing nearly one tenth of the entire, annual world production of crude rubber. As in Hevea and other rubber trees, the rubber occurs in the plant as a latex, but in contrast with Hevea and certain other commercial rubber plants, this latex is confined within discrete cells, rather than being present in continuous ducts. Most of the rubber-bearing cells occur in the outer or cortical tissues of stem and root, a fact of significance in the retting of whole shrub.
Most of the shrub used in the present investigation was 2 t o 3 years old, and was grown in the Salinas area, partly on irrigated fields, partly on dry land. In ordinary practice the plants are harvested by cutting the roots approximately 6 inches below the ground. Following digging, shrub for the present investigation was either brought in for immediate processing (lush shrub), or spread out to dry in the fields, usually for 5 days (field-conditioned shrub). On arrival a t the pilot plant or factory, plants were (1) stored without defoliation, ( 2 ) parboiled, defoliated, and used immediately, or (3) parboiled, defoliated, and dried rapidly to approximately 15% moisture in a current of warm air, then stored in a bodega (open shed or loft) until used. The terms “foliate” and “defoliated” are used here to designate, respectively, shrub with the leaves on and shrub with leaves removed after parboiling for approximately 10 minutes. I n general, the term “conditioning” has been rather loosely applied to various treatments of the shrub between harvesting and milling, but as used here applies only t o those treatments, such as field conditioning or bodega conditioning, in which microbial activity is minimized. Other types of treatment, in which a greater or smaller amount of microbial activity is involved, are included in the category of retting. Unless otherwise specified, all shrub used in these experiments was parboiled and defoliated immediately on arrival from the field. Three alternative procedures were employed to prepare shrub for retting: whole shrub was used directly, or it was chopped in blade cutters equipped with screens to give particle sizes ranging from several inches down to 0.125 inch, or it was cut t o the desired size and then further broken up by passing through corrugated crushing rolls set with approximately 0,004-inch clearance.
347
Where necessary and feasible, rcpresentative samples for control and test millings were separated out with a Jones binary divider, a process generally called riffling. When this was unnecessary or impossible, other methods of sampling were employed. Samples of shrub for chemical analysis were obtained by riffling a 1-quart aliquot out of the milling sample. The adequacy of this procedure for obtaining truly representative aliquot,s is shown by the data presented in Table I X . Moisture, resins, and rubber hydrocarbon were determined by standard procedures (16, 24). D a t a from the analytical laboratories early indicated that analyses for rubber hydrocarbon in crushed shrub are erratic and not always indicative of the true rubber content, owing t o excessive agglomeration of the rubber as a result of crushing. For this reason, only uncrushed, cut shrub was used for analyses in these investigations. Rough estimates of the extent of coagulation of the latex were made microscopically, and measurements of the p H of shrub samples were taken with a Beckman p H meter equipped with a glass electrode. I n order to estimate yields and determine the quality of ,millable rubber, shrub was milled in 27-inch pebble mills following a standard assay procedure. Unless otherwise specified, a charge of 4 kg. dry weight was crushed twice (at 0.004-inch setting) arid milled for 90 minutes with a water-solids ratio of 5 t o 1. The resulting rubber was then decorked, scrubbed to reduce dirt contaminants (acetone- and benzene-insolubles) and dried to. less than 1% moisture in a vacuum oven at 1 2 e C. A 0.25% acetone solution of p,p’-diphenylphenylenediamine(JZF) Tas sprayed on the wet rubber to minimize oxidative deteriorat,ion during drying. Chemical analyses of the crude rubber were carried out in t h e usual manner (24). Depending on the information needed, analyses were made for moisture, acetone- and benzene-insoluble materials, rubber hydrocarbon (by bromination), and resins (by difference), or for resins only by direct acetone extraction. Shrub moistures are expressed on a wet-weight basis; all other analytical data are on a dry-weight basis. Physical tests included (I) occasional determinations of t h e molecular weight and plasticity of the crude and (2) tensile strength, modulus at 500, 600, and 700% elongation, ultimate elongation, Shore hardness, and permanent set of the vulcanixate. Rubber was cured for 30, 60, and 90 minutes at 127” C. after compounding with the following proportions of ingredients: crude rubber, 100; mercaptobenzothiazole, 1.0; zinc oxide 6.0; stearic acid, 4.0; sulfur 3.5. Unless otherwise specifically stated, all physical tests reported in the present paper were made with rubber compounded on this formula. Clark and Place (E) concluded that such a formula effectively brings out differences in quality between different samples of guayule rubber, although considerably more rapid cure and higher tensile strength can often be obtained a t optimum cure by the use of such aromatic amines as p p’-diaminodiphenylmethane (Tonox) and/or diphenylguanidfue (DPG). Tonox or similar compounds have been applied in variable amounts to much commercial guayule rubber, but to obviate the disturbing effectsof such activators in comparative studies, the authors have always employed the nonactivating antioxidant, p,p’-diphenylphenylenediamine (JZF), and have used diphenylguanidine or Tonox only in particular instances. All other procedures in the compounding and testing of rubber conformed with A.S.T.M. specifications. Two of the most striking features of the inferiority of rubber from young unretted guayule are its high content of resinous contaminants and its relatively low tensile strength. As a consequence of shrub retting, rubber is consistently obtained with a reduced content of resins and with improved physical properties (14, 16, 2 3 ) . hlthough other beneficial changes likewise occur, the reduction in resins and the increase in tensile strength appear. to reflect most accurately the degree of improvement obtained. These changes were therefore adopted ’as the main criteria by which to judge the efficacy of any particular ret. Of these two. criteria the resin content of the crude rubber is less influenced by extraneous factors such as the acetone- and benzene-insolubles. Furthermore, a quantitative evaluation of the resin content involves fewer operations following the termination of the retting process itself. For these reasons, data on changes in resin content of the crude rubber are more precise, and are drawn upon in the following discussion more frequently than data on tensile strength. A detailed discussion of the relation between physical: properties of the vulcanixate and chemical composition of t h e crude is given herewith.
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
348 TABLEI.
CHAXGES I N CHEMICAL COMPOSITION AND TENSILE STREXGTH OF RCBBER OBTAINED B Y RETTING S H R U B O F DIFFERENT ORIGINS AND LTARIETIES"
Variety 593
Field Treatment Nonirrigated b
593
Iionirrigated 6
406
Irrigatedb
406
Nonirrigated
...
Irrigatedc
Day8 Conditioned 5 5
17 45
b
"Id
Days Retted 0 7 0 7 0 7 0 7 0 7
composition of Dry Crude Rerins, Insoliihlrs. c/,
56
22.8 11.4 22.7 12.1 18.0 8.9 21.3 13.8 24.5 14.6
5.8 8.7 6.0 7.7 14.4 13.2 14.1 13.0 9.5 8.8
a All rets mere carried out with 0.5 inch cut shrub at 50% moisture, spread turned 6 times daily. b 2.5-year-old shrub. C 1-year-old seedlings.
FACTORS I N F L U E S C I N G RETTIA-G I n order to understand the principles and practice of suceeseful retting, it is necessary to realize that the factors of the external environment have a direct influence in determining which microorganisms develop, and that the improvement in rubber quality is in turn dependent on the type and number of microorganisms which do develop. This three-wav relationship can be simply pictured as follomrs: LIicroorganisms
F"1
A+C Environment provided in retting shrub
Amount of resin reduction in crude rubber
~~2$ip~f
Vnlcliniaate. Lb./Sq. I n c h 2520 3400 25G0 3210 2440 2855 2420 2860 1930 3150
Vol. 41, No. 2 We can now, therefore, take C diup the relation A+ rectly, arid show how the factors of the external environment influence the rate, uniformity, and quality of rets carried out in a variety of ways with the natural microflora. ORIGIN AND PRETREATMENT O F SHRUB
It seems probable that a n improvement in rubber quality on floor in 10-inch deer, beds, and can be achieved by retting regardless of the age, variety, or conditions of growth of guayule shrub. At least, the authors have obtained a marked decrease in the resin content of the crude and an increase in the tensile strength of the vulcanized rubber from shrub of varieties 593 and 406, both irrigated and nonirrigated, varying in age from 1 t o 4.5 years (cf. Table I and other data belon-). Within limits, the treatment of the shrub after harvesting likewise has no basic effect on the retting process, provided the plants are always parboiled and defoliated prior t o retting. Equally good yields and improvement in the rubber have been obtained from rets of lush shrub, shrub kept onlv long enough to effect partial coagulation of the latex, and shrub stored for considerable periods. I n certain instances storing the shrub after harvesring will have some influence on the degree of improvement possible. First, it is generally recognized, as shown in Table 11, that proper conditioning-i.e., rapid drying of the shrub at low temperature (about 25' to 30" C.)-results in a small but tiistinct reduction in resins, often accompanied bv a slight improvement in physical properties, and thus give? the unrettcd control rubber n slightly improved quality. During such treatment there is little or no development of niicroorganisins on the shrub, and it is therefore probable that these small improvements are a result of physiological changes occurring \Tithin the cells of the guayule plant itself. At present the nature of these changes is almost entirely unknown, but it is probablv significant that they take place during the period %hen the maior part of the latex is coagulating into solid rubber particles. TJ7hatever their cltiise, sucn changes contribute t o differences in quality of unretted controls. Secondly, stoiage prior to retting frequentlv leads to one or more of the following deleterious changes: (1) The molecular weight and plasticity of guayule rubber undergo a marked decrease during shrub storage. ( 2 ) The acetone- and benzene-
A fundamental part of the investigation of retting has been a Well over a hundred distinct species study of the relation A+B. of microorganisms have been isolat'ed from retting guayule shrub and identified. With few exceptions they fall int,o t x o main natural groups, the filamentous molds and the bacteria. Estensive studies of these organisms in culture, singly and in various combinations, have shown that the factors of their environment determine not only the kinds of organisms which can occur but also the extent of their growth and activity in natural rets. The relation B-+- C has also been extensively studied, and has been found t o be of equally crucial importance. I t has been clearly demonstrated (1, 6 ) that microorganisms are the agents responsible for the improvements resulting from shrub retting; only some of the organisms occurring on retting shrub are capable of attacking the resins; only those organisms which can decompose the resins will, when BETWEEN SHRUB CONDITIOSIXG AND QUALITY O F RUBBER FROM grown in pure culture on the TABLE11. RELATION UNRETTED SHRUB shrub, effect a reduction in Shrub Crude Rubber Days Conditioned RHC Tensile the resin content of the, crude; Strength, Moisture, Resins, Insolubles, Reoovery, PrePostand a close correlation exists defoliation defoliation 70 % 70 Lb./Sq. I n c h Shruh Source between the occurrence of 9.2 92 2595 P u r a field. 2 31.5 24.1 7 11.3 102 2495 Sept. 1944 4 20.1 20.7 8 members of this resin-decom12.1 103 2705 1 17 . Y 19.5 12 2620 15 1 16.3 20.4 11.8 100 posing group and the over-all 35.2 21.8 8.2 100 2625 Wilmot field auccess of a natural ret. 3 0 20.6 10.2 104 2815 Dee. 1914 15.7 9 3 Through their influence on the 2585 18.1 11.2 102 16.7 16 3 2450 3 63 1 1 . 5 17.6 1 3 . 2 100 minroflora, the various factors Guidotti field, 28.4 24.3 5.8 105 2760 3 3 of the environment exert proFeb. 1945 11 20.7 22.6 9.1 100 2320 3 2 2 . 9 9 . 3 101 2520 nounced effects on the re1 8 . 8 18 3 2500 3 70 10.0 21.5 11.4 . . . duction of resins in the crude a For E discussion of recoveries over 100% see section o n relation between retting and mill recovery. rubber and on other features of improvement in quality.
349
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February 1949
r
'*
I
\
\
I
8
4
lo
I
,
I
I
4
8
12
a0
16 DAYS STORED
t
24
28
I
IO
I
BO
30
40
DAYS STORED
Figure 1. Comparison of Decline in Resin Content of Crude Rubber Shrub stored under anaerobic (-)
t
8 '
and aerobic (-
- -)
conditions
insolubles in the crude rubber and the permanent set of the vulcanized rubber increase progressively as a result of shrub storage at moistures below 25y0. (3) The recovery of rubber may be seriously impaired, particularly if the shrub undergoes prolonged or variable exposure to sun, rain, high temperatures, or the uncontrolled activity of microorganisms. As the small changes resulting from conditioning give no advantage in the ultimate quality of the ret,ted rubber, storage of shrub for any period prior to retting should be avoided.
Figure 2.
Interruption of Decline in Resin Content of Crude Rubber
Following transfer of shrub t o anaerobic storage (-) aerobic retting (-)
-
a t intervals during
oxygen have been demonstrated. (1) Following a period of anaerobic storage, the resulting ensilage may be satisfactorily retted aerobically, as shown in Figure 1. (2) At any stage of aerobic retting, the retted shrub may be stored anaerobically without further change in the quality of the rubber finally milled out, as shown in Figure 2. The possible application of these findings to the storage of cut shrub before or after retting is evident. MOISTURE
AERATION
A positive relation between a n adequate supply of oxygen and
Spence ( 1 4 ) rightly emphasized the importance of keeping the shrub "in a moist condition" to obtain good retting. The simple reason for this is now evident. Just as foods can be preserved from the decomposing action of microorganisms by dehydration, so the microbial retting of guayule is inhibited by drving the shrub. I n the course of this investigation this necessity of adequate moisture for successful retting has been repeatedly observed. It is, nevertheless, difficult to reduce the moisture content rapidly enough to prevent microbial action completely without subjecting the rubber in the shrub to the deleterious effects of exposure t o oxygen a t high temperatures. If. however, whole shrub is loosely piled in a strong current of air, a t room temperature, immediately after parboiling and defoliation, the moisture content can be brought below 15% within 1 t o 2 weeks. The control shrub in retting experiments was often treated in this manner, and under such conditions never showed any visible evidence of mold growth or other microbial activity.
good retting was suggested in the experiments of Spence ( 1 4 ) . Acting on this suggestion, White et al. (23) obtained further evidence indicating that access of air to all parts of the shrub mass was a primary requisite for good retting. To verify this relation further and establish definitely the necessity of a good supply of oxygen for successful natural retting, many experiments have been done comparing the effectiveness of retting under aerqbic a n d anaerobic conditions. Anaerobic conditions were obtained by packing or sealing cut shrub in containers without access of .air. The natural microflora exhausted the small amount of oxygen initially present in the shrub within 18 hours, and typical ensilage formation ensued, with a heavy development of lactic acid bacteria and a drop in p H t o about 5.0 or below (see also discussion of acidity and alkalinity). Aerobic conditions were provided in a variety of ways discussed in &ore detail below. T h e differences in the uuality of the rubber from these two types of treatment were TABLE 111. COMPARISONS OF CHEMICAL COMPOSITION AND PHYSICAL PROPERTIES OF RUBBER strikingly pronounced and FROM AEROBICALLY RETTEDSHRUB( t)AND ENSILED SHRUB(-) consistent. As shown in FigModulus a t Tensile Ultimate Rubber ures 1, 2, and 6 and Table Strength, Elongation, 500% Elongation, Hydrocarbon, Expt. No. Aeration Resins. yo % Lb./Sq. Inch % Lb./Sq. Inch 111, virtually no change in -
I
t h e quality of the rubber occurred as a result of anaerobic storage for varying intervals, whereas marked improvement resulted from aerobic retting. Two additional points confirming the prime importance of
+++ -
12.4 74.6 2800 760 560 24.2 67.0 2260 855 185 13.6 72.6 2860 770 565 3A; 15.4 75.2 2780 750 480 3B 24.6 68.9 1920 825 180 Shrub 2 years old cut 0.25 inch held 4 weeks without control of moisture or temperature 8 f e e t deep in small tanks. A, aerobio to6 portions, 30 i o 50% moisture and 20° t o 60° C.; B, anaerobic bottom'portiona, 40 t o 50% moisture and loo to 20" C. b P a r t of 1B aerobically retted for 1 week. C Shrub 3 years old cut 0.5 inch crushed once, held 3 days a t 5 2'% moisture a n d 2 5 O t o 3 5 O C. in rotating drums, A, aerated; B, not aArated-Le., skaled. 1Aa
1Ba 2b
@
INDUSTRIAL AND ENGINEERING CHEMISTRY
350
Reference to Table 11 and Figure 7 \Till indicate the minor estent of changes in rubber quality occurring under these conditions. The striking changes in qualit,y which occur, on the other hand, when uncut siirub is stored wet (25 t o 509; moisture) for relat,ive!y short periods, arc sh0n.n in Figures 7 and 10 Kithin t,his range, likevise, retting is niorc rapid a t higher moistures (Figure 10) than a t lower nioistures (Figure 7). The active retting of whole shrub occurring at, such moistures is correlated with a profuse developnient of microorganisms, most conspicuously filamentous molds. The probability existed t,hat nioistures in a range above those completely limiting niicrobial activity might play a more suhtle role in determining the rate, extent, and nature of the retting process. A more careful st,udy \vas therefore made of the effect of moisture in the range from 35 to 70(r,. These experiments were performed using the rotating drums described below, because they provided the best means for achieving EL maximum of uniformity and cont,rol over temperature, moisture, and other variables. All rhrub was prepared and treated folloving the standard practice described in connection v-it11 the rotating drums. Retting, as judged by decrease in the percentage of resins in the crude, did not occur at all at, 70Yc moist,ure, but was almost equally effective between 35 and 58% (Figure 3). The failure to achieve any retting a t 70% rcsulted, not from the moisture itself but from t,he inadequate aerat,ion of such a wet mash and the consequent formation of typical ensilage. The appreciably more rapid rate of retting during the first 2 days a t 58 and 5270 moisture as compared with that. a t 35 and 43y0 can probably be ascribed to the difference in the microfloras and their relative rates of growth. At the higher levels of moisture there is a rapid developnient of resin-decomposing bacteria and no growth of filamentous molds; a t the lower levels, the development of a bacterial population is slower and is accompanied by the growth of filamentous molds which, however, become a well established part of the resin-decomposing flora only aft'er 2 days.
Vol. 41, No. 2
imperative that temperatures above GO" C. be avoided if niierobial action and consequent retting are to be encouraged. In a study of the effects of temperature on retting, White et al. ( 2 3 ) attempted t o control the temperature a t a uniform level throughout a deep stationary inass of shrub by periodically wetting with thermostatically controlled water sprays. Their re. d t s showed that good retting could be achieved a t tmnperatures in the range of 42' to 50" C., but the method was not entirely satisfactory for demonstrating the relation of tempera-, turc t o retting. Maintenance of a temperature in the noighborhood of 37" C. required such frequent spraying that the shrub became water-logged, and retting failed t o occur because the shrub was anaerobic, not because the temperature was unsuitable. A logical and simple niet,hod of prevent,ing the development of high temperatures in decompositing shrub is to increase the surface-volume ratio of the mass until a balance is reached bctween heat evolution and heat, loss. By spreading the shrub out on the floor in layers 1 or 2 feet deep, temperatures can be maintained below 60" C. Retting in such shallow piles of shrub m-as found by Spenee ( 1 4 ) to be superior to that obtained in bins or silos. I n the authors' experience this method has likewise given very satisfactory retting in many small scale experiments (see section on floor retting), but the factors of degree of mixing, particle sizc, and depth of shrub have been found to exert effects of critical importance. These factors must therefore be talcen int,o consideration if the maximum advantages are to be rcalized with this method of retting. If there is no mixing of the shrub during such rets, they are consistently satisfactory, although subject to a certain degree of heterogeneity. Shrub covering a !Tide range of particle sizes can be used, and temperatures can be mainOained below 60" C. by keeping the beds sufficiently shallow. Periodic mixing, even hourly, does not give control of temperature in appreciably deeper beds, but does provide greater uniformity. I n this case only the finer cuts of shrub can be used (see discussion of mechanical reduction in particle
re
TEMPERATURE
Whenever moist, plant materials are stored with access of oxygen, a n evolution of heat occurs as a result of strongly esot,hermie decompositions caused by microorganisms. If cut shrub is stored in sufficiently large masses, thc insulating effect of the outer portions of the pile prevents dibsipat,ion of the heat evolved, and a rapid rise in temperature to a high level occurs. h striking demonstration of this heating x a s encountcrcd during the storage of 23 tons of cut shrub in a cubical bin provided . with means for forced aeration. After an init,ial lag, the temperaturc of the shrub, particularly in the upper portions, rose rapidly until it reached 65' to 70" C. Once active thermogenesis had started, the heat produced could not be removed by forced aeration without excessive desiccation. Thc accompanying figures show that n o retting occurred a t the excessively high temperatures that prevailed in a largc port,ioii of the shruli mass.
Shrub S ~ n i p l e
To
1 control 2: kept 14 days in bin, temperature GO' to 70' C . for most of period
23.2
Tensile Strenzth ofVulcanizate, Lh./Sq. I n c h 2370
22.7
2313
Reiin Content of Crude,
Cultural tests of sample 2 revealed that there m r e no living organisms in the shrub a t the time of milling and showed that failure t o achieve any retting mas a direct result of sterilization from prolonged exposure to high tempciatures. A similar selfsterilization of hay resulting from microbial thermogenesis was described by Miehe ( 1 1 ) , who attributed it to the high temperatures, possibly in conjunction with bactericidal subqiances. At any rate, in rets where reinoculation is not achieved, it is
0
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zro W
6
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12
14
16
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35
40
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45 50 55 S H R U B MOISTURE, %
I
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60
65
70
Figure 3. Effect of Shrub Moisture during Retting on Hato and Extent of Change i n Resin Content of Crude Rubber D a t a averaoed from three series of rets of crushed shrub a t 30' C . iinder c o n t h e d conditions i n rotating drums. Each curve shows total change in resins resulting from retting for number of days indicated
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1949
351
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12
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20
-I
25
30
35
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TEMPERATURE,
45
50
55
'C
Figure 5. Effect of Shrub Temperature during Retting on Rate and Extent of Change of Tensile Strength of Vulcanized Rubber
D a t a averaged from four series of rets of crushed shrub a t 5 2 % moisture under controlled conditions in rotating drums. E a a h curve shows t o t a l change i n resins resulting from retting for number of days indicated
D a t a from s a m e series of experiments as Figure 4
size), however, and the frequency of mixing becomes a factor of critical importance intricately related t o the temperature and microflora. With shrub cut to 0.5 inch, spread in beds 10 inches deep, and held at 50 to 52% moisture, prevailing temperatures are between 45' and 60' C., but successful retting is obtained only when mixing is limited t o a few times during the day shift. Similar rets mixed hourly are completely unsuccessful. An understanding of their failure required more exact data on the temperature relations of retting with continuously mixed shrub. The rotating drums described below provided a means of conducting such experiments at controlled temperatures while at the same time maintaining uniform moisture and aeration. Using crushed shrub at 52y0moisture, several series of rets were carried out at six levels of temperature in the range from 24" to 54" C. Figure 4, showing the relation of temperature to the drop in resin content of the crude rubber obtained, brings out two important points: (1) good resin reduction was obtained at all temperatures from 24"to about 40" C., but (2) there was a sharp decrease in improvement above 40" C., and little or no resin reduction occurred a t levels above 45" C. A similar relation between temperature and improvement in tensile ,strength of the vulcanized rubber is shown in Figure 5. Another series a t 35% moisture gave essentially the same results, but the drop in improvement between 40' and 45" C. was not so abrupt. An explanation of the success or failure of the various rets which have been discussed in connection with temperature requires an understanding of the effects which temperature and mixing have on the resin-decomposing microflora (Or, 6). The fundamental relations arc as follows:
'
TEMPFRITIIRE. *C
Figure 4. Effect of Shrub Temperature during Retting on Rate and Extent of Change i n Resin Content of Crude Rubber
I n rets where the shrub is not mixed a t all, filamentous molds can develop. They arc all capable of decomposing resins, a n i have temperature limits for active growth extending up to 55 and 60" C. Any ret of unmixed shrub in which the prevailing temperatures do not exceed 60' C. is therefore successful if other conditions are satisfactory. If the shrub is turned only a few times during the day shift, filamentous molds do not develop, but a heavy growth of resindecomposing actinomycetrs appears after 3 to 4 days. These organisms, which are capable of growing a t temperatures up to 60" C., are probably the agents responsible for the success of infrequently turned rets. In shrub which is continuously mixed at 52% moisture, neither filamentous molds nor actinomycetes develop appreciably, and
bacteria are the retting agents. Since resin-decomposing bacteria have temperature limits for active growth extending only to 45' C., retting of continuously mixed shrub of high inoistur: content is successful only if the temperature is kept below 45 . At lower moistures, some development of filamentous molds occurs in continuously mixed shrub, but appears to be insufficient to produce appreciable retting at temperatures above 45' C. MECHANICAL REDUCTION IN PARTICLE SIZE
The breakage of intact plants during harvesting is responsible for the initial development of microorganisms on the shrub. Mechanical reduction of the harvested shrub either by cutting or crushing, greatly accelerates the rate of development of this microflora, and hence, as Spence (14) observed, hastens the retting process. The effect of widely different degrees of mechanical reduction was studied with a view to determining whether any particular type of treatment was to be preferred Thoroughly satisfactory retting has been achieved with degrees of mechanical reduction ranging all the way from whole shrub down to shrub cut t o 0.125-inch pieces or finely crushed. The cutting operation itself appears to have no direct bearing on the quality of the resulting rubber. The finer the cut, the more rapid is the development of a microflora, but the greater also is the rate of heating and of depletion of oxygen in the shrub mass. If whole or coarsely cut shrub is used, molds will be the primary retting agents, probably because of their capacity to penetrate the shrub tissues more extensively than do the bacteria Such material must not be mixed, as mechanical action generally inhibits the dcvclopment of filamentous molds. The broad temperature tolerance of these organisms, however, renders control of temperature less critical. If finely cut shrub is used, the more rapid development and intensive activity of the bacteria can be exploited, but rigid control of aeration and temperature IS essential if thc advantages of fine reduction are to be realized. These problems and their relation to the practical aspects of commercial retting are discussed below. ACIDITY AND ALKALINITY
The changes in p H which occur during a natural ret can in most instances serve as a useful criterion by which t o judge whether or not retting is progressing satisfactorily. Ordinarily the initial pH of cut shrub which has been parboiled and defoliated is slightly below neutral, between 6.0 and 7.0. The p H of lush
INDUSTRIAL AND ENGINEERING CHEMISTRY
352
TABLEI\'+ AND
Vol. 41, No. 2
RELATIONS BETWEES p H O F SHRUB, A E R A T I O N , REDUCTION OF RESINS IX CRUDERUBBER
Description PH Decrease of Shrub Initial Final i n Resins Aeration Freshly c u t 8.9a 7.5 so K O Freshly cut 8.9a 8.3 Yes Yes Ensiled 7.5a 8.9 Yes Yes Freshly c u t 6.3 5.3 NO P-0 Freshly c u t 6.6 7.6 Yes Yes Ensiled 5.3 7.7 Yes Yes a p H adjusted t o these values a t s t a r t of experiment by mixing in a b o u t 10% CaCOa a n d 1.0% KazCOa, based on d r y weight of shrub.
4 1
0
I
I
1
20
40
60
I I 80 100
The retting of ensilage is therefore feasible, although the Iow initial pH may a t first reduce the rapidity of retting by temporarily interfering with the rapid development of retting bacteria. INOCULATION
5 $ 8 U U I
10
t 14
'
0
I
I
20
40
I
60 HOURS IN R O T A T I N G D R U M
I
I
80
100
Figure 6. Relation between pH of Shrub during Retting and Change in Resin Content of Crude Rubber All experiments with crushed shrub a t 30° C. under controlled conditions in rotating drums 6 . Markedly low pH a n d negligible amount of retting occurring under anaerobic conditions with shrub a t 52% moisture 0. Conspicuous rise i n p H and drop in resins characteristic of good aerobic bacterial ret,of shrub at,52% m o i s t y e X . Relatively slight change in pH associated with good aerobic retting a t 35% moisture, bacterial action accompanied b y extensive activity of molds
shrub is closer to neutrality than that of shrub conditioned for some time. During the progress of a good natural ret, there may a t first be a temporary slight drop in pH, but this is soon followed, under most conditions, by a rise to a final level between 7.5 and 8.5, frequently accompanied by an active production of ammonia. Under certain exceptional but well-defined conditions, a microflora can develop which is capable of effecting good retting without raising the p H of the shrub above neutrality, but it has never been possible to achieve good retting when the p H dropped much below 6.0 and remained there. A summary of these relations between p H and resin reduction is presented in Figure 6. When it is realized that the strongly acid reaction (solid circles) was a result of anaerobic storage, it becomes clear that the lack of oxygen (Figure 1) and not the low p H was the direct cause of unsatisfactory retting. That the actual level of p H or the direction of change in p H itself is not consistently related to resin reduction is shown in Table IV. The important relation proves t o be one already discussed-viz., the relation between aeration, independently of pH, and resin reduction. Even if the p H is initially low, good retting may be obtained if the shrub is supplied with oxygen.
The recognition of guayule retting as a microbial process led to a study of inoculation as a potential means of accelerating the process or making it more effective. Shrub to be retted in the rotating drums was artificially inoculated with well-retted shrub or pure cultures of resin-decomposing bacteria or molds isolated from natural rets. The results of these shrub-inoculation tests 3hO~iedno clear-cut advantage of inoculated shrub over the controls, as regards either rate of retting or final quality of rubber. Apparently, therefore, it is difficult to impiove by artificial inoculation upon the natural inoculation which the shrub receives during handling and wetting after parboiling. Despite the negative results of these preliminary experiments, however, it would he premature t o discard the idea of inoculation as unprofitable without a great deal of further study. Such investigations should include the use of inoculum derived from rets a t various stages of completion, the addition of inoculum a t various periods during the ret, and experiments with pureculture inoculation using other species of organisms than those already tried. If any method of inoculation should prove successful, it could readily be incorporated into a scientifically controllable process such as drum retting. OPTIMAL COMBINATION OF FACTORS
The basic conclusions drawn in the foregoing discussion of factors influencing retting can be summarized as follows: The quality of rubber from virtually any type of guayule shrub can be improved by rett,ing. In order t,o realize the ma,ximum advantages of retting, lush shrub s h d d be used. If operating considerations require storage or conditioning prior to processing, this period should be reduced t o a minimum. I n order to obt'ain rapid and uniform retting, a plentiful supply of oxygen must, be maintained throughout the shrub mass. Methods of retting that will ensure adequate aeration are discumed below. In general, the best retting will occur in a range of shrub moistures between 35 and 55% on a wet-m.eight basis. h considerable degree of retting may also occur above and below this range, but the extreme limits (20 and 70%) should be approached wit,h caution and with the realization that detrimental factors are likely to become operative. Retting with continuous mixing of shrub must be carried out in t,he relatively narrow range of 25 ' to 35' C. For all other types of retting herein discusted, temperature limits are not so precise, but a maximum of 60 should never be exceeded, and a safer upper limit is 55". The more the shrub is reduced in particle size (by cutting or crushing) the more rapidly and uniformly it will ret, provided aeration and temperature are controlled. Coarsely cut and whole shrub can be very effectivelv retted, but the process goes more slowly. The natural pH of guayule shrub is satisfactory for initiating the retting Drocess and does not require further adjustment. Successful-r&ting a t higher moistures is accompanied by an increase in alkalinity of the shrub, but may occur a t lower moistures with little change in pH.
February 1949 The potentialities of artificial shrub inoculation have not been sufficiently explored t o warrant any recommendation, but should be further investigated in any future study of the retting of guayule shrub.
INDUSTRIAL AND ENGINEERING CHEMISTRY TABLE V.
IMPROVEMENT IN QUALITYOF RUBBER FROM CUT SHRUBRETTED WITHOUT MIXING AERATION,IN RELATION TO SIZE AND DIMENSIONS OF SHRUBM a s s
BUT WITH
Scale of Experiment
Origin of Sample Tested
EVALUATION O F Laboratory Composite Surface Pilot plant METHODS Subsurface Pilot plant Surface Factory Spence's discovery of shrub Factory Subsurface retting and his perception of its value as a method for facilitating the recoverv and improving the quality of rubber from guayule shrub led him t o an extensive investigation of methods of carrying out the process. His consistent success in small scale experiments provided grounds for believing that retting should be capable of translation to large scale operation. The realization of this belief, which the present investigations have proved t o be fully justified, would have been difficult without an understanding of the microbiological nature of the process. Having recognized the fundamental role of microorganisms, however, and having shown how the factors of their environment influence their activity and thus determine the progress of a ret, we can proceed t o a n evaluation of methods of shrub retting. This evaluation is based primarily on the degree of improvement in rubber quality obtained in extensive tests of each method, and it is shown that the suocess or failure of a method is directly related to its adequacy in maintaining a t an optimal level the environmental factors discussed above. It is often difficult or impossible to translate from small scale to commercial operation without introducing new and sometimes prohibitive limitations. This fact has been recognized, and in so far as possible promising methods have been developed from laboratory to pilot plant t o factory scale operation.
-
RETTING
353
WITHOUT MIXING SHRUB
Pit Retting. One of the methods of retting suggested in the early stages of this investigation consisted of burying the shrub in shallow outdoor pits and leaving it covered with earth for a period of weeks. I n a small number of experiments, successful retting was obtained after several weeks of storage, but for the most part the shrub became waterlogged, with resulting production of ensilage and no improvement in rubber quality. (This work was done under the direction of Kenneth Taylor, of the Forest Service, who kindly allowed the authors to include the experiments in their discussion.) This method has little to recommend it, as the satisfaction of temperature and aeration requirements, poor at best, is subject to the vagaries of the weather. For large scale aerobic retting the method is entirely unsuitable, while for small scale operation one of the other methods discussed below would be more satisfactory. Crib Retting. I n another method tried in the early stages of this investigation, attempts were made to provide automatic aeration by placing shrub cut to 1-inch pieces in cribs with slatted sides. Tests on a pilot plant scale (about 1 ton of shrub) showed little or no improvement in rubber quality even in the narrowest (1.5-foot) cribs. An adequate supply of oxygen throughout the shrub was not obtained, and in all cases deleteriously high temperatures (above 60 C.) were reached. The development of a retting flora was therefore spotty, and largely restricted to peripheral portions of the shrub mass. Hence this method is inadequate for the retting of finely cut shrub, and would find its only possible application in the retting of very coarsely cut or whole shrub. Tank or Bin Retting. I n view of the failure to achieve spontaneous aeration even in relatively small masses of the finer cuts of shrub, it appeared necessary to investigate the practicality and advantages of forced aeration, espccially with the ultimate
Approx. Size of Shrub Mass Horiz. diam., Depth,
ft.
1.5-2.5 6 6 12 12
.
ft.
3 10 10 12 12
Days Retted 15 12 12
14 14
Change in % Resins of Crude 10 -5
-
+4
-6 0
Change in Teniile Strength of Vulcanizate, Lb./Sq. Inch 4- 600 700 0 300 -250
+ +
aim of translating the process to large scale operation. The experiments of White et al. (23) had indicated the feasibility of supplying oxygen by means of forced aeration t o masses of shrub cut to 2-inch pieces, cracked to 0.25- to 0.5-inch thickness, and piled 5 feet deep, and it seemed likely that a modification of their method could be successfully applied to even greater depths of shrub. The authors' experiments were carried out in tanks or bins aerated by blowing air through a false bottom on which the 0.25- to 1-inch cut shrub rested. If aeration was sufficiently rapid (more than 0.5 cubic foot of air per cubic foot of shrub per hour) t o carry away a major part of the carbon dioxide produced, good retting was always obtained in the peripheral zones of shrub, but these zones represented an increasingly smaller proportion of the total mass as the scale of experiments was increased from laboratorv to pilot plant to factory. consequently, the over-all improvement in numerous laboratory experiments was good, but in pilot plant experiments was poorer, and in a factory scale experiment was negligible (see Table V). The failure to obtain any retting except in the peripheral zones of the larger masses of shrub, even though aeration was fully adequate, can be attributed primarily t o excessive overheating which quickly resulted in the death of most or all of the resin-decomposing organisms. Only by allowing large masses of shrub to pass through the period of active thermogenesis and become gradually reinoculated over a period of weeks, could a measure of resin reduction, chiefly by molds, be obtained in the crude rubber, and the long periods of storage required introduced deleterious changes more than counterbalancing the advantages of reduced resins. The utter failure of these efforts to translate the process t o a large scale demonstrates the serious and fundamental nature of the shortcomings inherently operative in unmixed, aerated, bin rets. Clear evidence of the factors responsible and their opposing effects was afforded by a critical analysis of these experiments, and was later substantiated by the results of carefully controlled laboratory experiments. A brief summary of the interplay of the factors involved will emphasize their importance. The heat evolved by microbial thermogenesis results in a rise in temperature which may be as much as 6" C. per hour. Consequently it is essential to provide means for artificially cooling the mass, if sterilization is to be prevented and a suitable temperature for the growth of retting organisms is to be maintained. This was attempted in two ways: by direct water sprays and by increased flow of air through the shrub. The former method proved inapplicable because it waterlogged the shrub and hence interfered with the uniform access of oxygen. The failure of the second method arises primarily from the low heat capacity of air. Adequate cooling of a n actively retting mass of shrub would require approximately 100 cubic feet of air per cubic foot of shrub per hour, as compared to 0.5 cubic foot of air per cubic foot of shrub per hour needed to satisfy the oxygen requirements. Although a supply of air of this magnitude is not in itself a serious problem, it does prevent the maintenance of a uniformly optimum moisture in the shrub. Drying of the shrub is rapid and unequal and can not be prevented by the use of air saturated with mois-
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354
ture. Temperatures in the shrub above that of the entering air decrease the relative humidity of the air and permit it, to pick up additional moisture which may later be deposited in the cooler outerzones or carried through and lost in the atmosphere above the shrub. In actual practice other more, complex but similar effects undoubtedly contribut,e to the heterogeneity and over-all drying of the shrub. The authors’ experience with large masses of cut shrub piled in deep bins and subjected to forced aeration has shown that very pronounced gradients of temperature and moisture develop and are impossible to avoid, with the result that retting is spotty, slow, and generally inferior. [The progressive improvement in quality up to 3 weeks found by White et al. (23) may possibly be ascribed to the gradual incorporation of previously unretted port,ions of t,he shrub mass, rather t,han to a continuous improvemcnt in the quality of rubber in any particular region. A similar progressive improvement has been not,ed in certain pit, rets, where the central portions of thc mass were at first, anaerobic but were later gradually retted as drying of the outer layers permitted deeper penetration of oxygen.] Floor Retting. The disadvantages inherent in bin retting can be largely obviated by floor ret,ting. in which the moist shrub is spread out in a shallow bed. By a proper combination of shrub particle size and depth of bed, aeration becomes spontaneous, and the dissipation of heat is sufficiently rapid to prevent a rise in temperature above 60” C. I n practice, the requirements for floor space, which will in any event be large, must be kept a t a minimum. Conscrvat,ion of space can be achieved in two ways: by reducing particle size and thereby incrtasing densit,? and rat,e of retting, and by increasing the depth of shrub. Either change, however, alone or in combination with the other, will ultimately lead to conditions of inadequak aeration and excessive heating. There is a pract,ical limit, therefore, belo\
