INDUSTRI-4L d S D ENGINEERING CHEMISTRY
October, 1929
981
Toxicity of Water-Soluble Extractives and Relative Durability of Water-Treated W o o d Flour of Western Red Cedar' A. M . Sowder FOREST R E S E A R C H L.ABOR.A+ORY, SCHOOL
OF F O R E S T R Y , U N I V E R S I T Y OF I D A H O , M O S C O W , IDAHO
In order to determine the toxicity of the water-soluble extractives and the relative durability of kiln-dried and unseasoned heartwood and sapwood of western red cedar ( Thuja plicutu), finely ground samples of the various woods were treated with like amounts of hot and cold distilled water by the laboratory process. The toxicity of the various extractives was tested by the Petri dish method, the extractives having been mixed with malt agar in various concentrations and inoculated with pieces of a vigorous growing culture of a wood-destroying fungus. Samples of the various water-treated wood flours were also inoculated to determine the extent the fungus could grow on them as compared with the growth made on the same wood flours without water treatment. Lentinus lepideus, a sapro-
phytic fungus causing a brown cubical rot, was used in all inoculations. That the volatile materials driven off by subjecting small western red cedar blocks of heartwood to kiln-drying temperatures were toxic was determined by collecting the volatile materials and condensing them, then mixing with malt agar and inoculating. The heartwood extracts and the hot-water extracts were found to be more toxic to the fungus used than the sapwood extracts and the cold-water extracts, respectively. Kiln-drying temperatures have some influence in releasing chemical deposits contained within the heartwood, but the effect is of no important consequence in destroying the toxic properties of the heartwood.
HE fact that western red cedar, Thuja plicata, is quite resistant to wood-decay organisms is well established. Its decay resistance in service (9) places it as the most durable of our native Idaho woods. Laboratory tests ( 7 ) with ten different fungi acting on white pine, yellow pine, Douglas fir, white fir, larch, spruce, and cedar show conclusively that the latter is by far most resistant to wood decay. Its use as fence posts, telephone poles, and shingles has given it an eminent position among our woods for durability in actual service. The resistance of western red cedar to wood-destroying fungi is attributed to the presence in considerable quantities of a peculiar kind of toxic material in the heartwood. Other woods, durable under actual service conditions ( I ) , are known to contain water extractives that are toxic to wood-destroying fungi. On the other hand, non-durable woods are, as a rule, low in such extractives. Laboratory tests ( 1 ) of many durable woods have proved that the hot-water extracts are all more toxic than the coldwater extracts of the same material; also that the heartwood extract is more toxic than the corresponding sapwood extract. A frequent explanation is that the sapwood contains starch, sugar, protoplasm. etc., which are absent in the heartwood, but there have been no reliable quantitative determinations of these substances in wood. Apparently, then, the difference of heartwood and sapwood is due not to t,he presence of substances in the sapwood which promote decay, especially when one considers that, of the non-durable species of wood, there is little or no difference in the durability of heartwood and sapwood, yet there is no reason for supposing that there is any less starch, sugar, protoplasm, etc., than in the durable species. Previous work with western red cedar (6) indicates the unusual resistance of this species to be due to the presence of a toxic resin or other substance, or to some mechanical effect produced by the cell walls themselves, or to the structure of the cellulose molecule itself. It was found that acetone and alcohol extracted the toxic principle the most completely of various solvents tried. S o mention has been made of any effect on the extractives
that may be produced by subjecting the woods to seasoning processes, and it is the purpose of this paper to ascertain if any serious result is evident when the wood has been kilndried. This study also treats of the extractive effect of water on the toxic properties of unseasoned and seasoned heartwood and sapwood of western red cedar wood flour. The seasoned wood used in this work was kiln-dried and as such was subjected to the usual kiln temperature for cedar, which is about 80" C. (175" F.) (8). This temperature is about 44°C. (80°F.) above that to which air-seasoned wood is subjected so it is reasonable to suppose that certain volatile materials are driven off during kiln-drying. These volatile materials undoubtedly contain toxic properties and if they are driven off it is quite apparent that the service life of kilndried wood is shortened. Western red cedar, being resistant to decay, must therefore possess a greater percentage of toxic material than other readily available species; it is the logical species of wood to use for this experimental work. Owing to the period of years necessary for relative durability studies to be carried on under actual decay conditions in the field, the laboratory method used is of value in checking the field tests and, instead of wood blocks, finely ground wood flour was used, in order to secure quicker solubility in water.
T
1
Received March 27 1929.
Methods
It was impossible to distinguish any color difference between the unseasoned and seasoned blocks of either heartwood or sapwood. Sawdust of unseasoned and seasoned heartwood and sapwood of the western red cedar blocks-a total of four wood-flour samples-was ground to a size that would pass through a 0.5-mm. mesh. Since the heartwood and sapwood ( I , 4 ) vary greatly in their decay resistance, care was taken to separate them when present in the same block. A previous investigator (IO) found that, within a species, specific gravity of wood has a direct relation to its decay resistance. The specific gravity of the western red cedar used in this work was determined by removing a section 2.5 cm. square from the center of each of the original blocks. Samples of 350 grams of each of the four wood-flour specimens based on oven-dried weight were prepared in dupli-
INDUSTRIAL AND ENGINEERING CHEMISTRY
982
cate-one for treatment with cold distilled water and the other with hot distilled water-and were placed in large Erlenmeyer flasks. T o the samples treated with cold distilled water 3500 cc. were added and allowed to stand for 48 hours a t room temperature with intermittent shaking; to the samples treated with hot distilled water 3500 cc. were added and the flasks placed in a boiling-water bath for 3 hours. The contents of each flask were then poured on filters. After filtering each sample was washed with 1000 cc. of distilled water, cold or hot according to whether the previous treatment was with cold or hot water. In each case the combined filtrate and wash water was evaporated down to 350 cc. a t a temperature not to exceed 74" C. (165" F.). By so doing each cubic centimeter represents the extract from 1 gram of wood flour. The treated wood flour was dried to room temperature and tested in the manner described later. T a b l e I-Specific G r a v i t y and R i n g s p e r I n c h (2.5 c m . ) of Unseasoned and Seasoned Heartwood and Sapwood of W e s t e r n Red C e d a r WOODFLOUR Av. RINGSPER INCH SP. GR. Unseasoned heartwood 13 0.332 Unseasoned sapwood 31 0.310 Seasoned heartwood 16 0.339 Seasoned sapwood 25 0.309
Results of Tests
To determine what percentage of wood flour substance was removed by the various treatments, 10 cc. of each of the various extracts were pipetted into watch glasses of known weights. The liquid was allowed to evaporate to dryness a t room temperature, which took about 24 hours, then the watch crystals were placed in a drying oven for hour a t 79°C. (175" F.) until continued heating caused no further loss in weight. The results are recorded in Table 11. E x t r a c t from W e s t e r n Red Cedar Wood F l o u r WOODFLOUR MATERIAL EXTRACT FROM 10 cc. SOLUBLE
T a b l e 11-Water-Soluble WATER
TREATMENT Cold Hot Cold Hot Cold Hot Cold Hot
TAKEN FROM
Unseasoned heartwood Unseasoned heartwood Unseasoned sapwood Unseasoned sapwood Seasoned heartwood Seasoned heartwood Seasoned sapwood Seasoned sapwood
EXTRACT MATERIAL Gram 0.1763 0,3270 0.1112 0.1715 0.3295 0.5580 0,1009 0,1820
Per cent 1.76 3.27 1.11 1.71 3.30 5.58 1.01 1.82
The seasoned heartwood gave a higher percentage of soluble material than the unseasoned, but the sapwoods yielded about the same amounts. The differences in the case of the sapwoods are practically negligible and may have been caused by differences in the specific gravities of the materials. Both unseasoned and seasoned wood doubtless did not come from the same tree. According to Table I, the specific gravities for the unseasoned and seasoned sapwood are about the same, but the heartwoods show greater differences. A substance having toxic properties may poison the food of a wood-destroying fungus so that it inhibits growth or kills the fungus. The toxicity of each of the various extractives was tested by the Petri dish method (5) using malt agar. Three concentrations of each extract were used-10, 50, and 100 per cent. The various extracts were placed in groundglass stoppered bottles, clamped in a metal frame to prevent volatilization, and sterilized a t 8 pounds (0.55 kg. per sq. cm.) pressure for 30 minutes. The malt agar used was that employed in the usual cultural work (.5), consisting of Trommer's malt extract, bacto-agar, and distilled water. The sterile extracts were placed in Petri dishes and when cool inoculated with a small piece of a vigorous growing culture of Lentinus lepideus, a saprophytic fungus ( 2 ) causing a brown cubical rot.
T'ol. 21, No, 10
As a basis for a comparison of the growth of the fungus, controls were prepared in which the extract was omitted. Pure malt agar medium was used in the Petri dishes and later inoculated with the fungus. All the cultures were then placed in an incubator and growth readings taken from time t o time by measuring the average diameter of each growth in each Petri dish. Where the growth was not circular, two dimensions were taken to secure the average diameter. Fourteen days were required for the growth of the fungus in the controls to reach the margins of the dishes and this period of time is used as a basis for comparing the toxic strength of the various extracts. The percentage retardation of growth was determined by comparing the radial growth of the fungus on the media containing the extracts with the radial growth of the fungus in the control. These data are shown in Table 111. T a b l e 111-Retardation of Lentinus lepideus on t h e Various C o n c e n trations of W e s t e r n Red C e d a r Extractives a f t e r 14 Days WATER WOODFLOUR EXTRACT 10% 50% 100% TREATMENT TAKENFROM CONCN. CONCN. CONCN. Per cent Per cent Per cent Cold Unsea%onedheartwood 37 99= 990 Hot Unseasoned heartwood 81 99a 99; Cold Unseasoned sapwood 00 34 99 Hot Unseasoned sapwood 00 48 994 Seasoned heartwood Cold 99" lOOb lOOb Seasoned heartwood Hot 99a lOOb lOOb Cold Seasoned sapwood 00 36 99' Hot Seasoned sapwood 17 59 990 99 per cent retardation means growth of fungus inhibited but not killed a Fungus transferred to malt agar slant recovered. b Fungus transferred to malt agar slant did not recover
Ten days after inoculation some of the cultures planted on the higher concentrations of heartwood extract showed no growth whatever. In order to ascertain if the fungus had been killed or growth merely inhibited, a small portion of each inoculum was transferred to test-tube slants containing malt agar. Each inoculum was washed in sterile distilled water and transferred to the malt agar in a test-tube slant. The designation (a) in Table 111indicates that such transfers recovered after a period of from 5 days to 3 weeks so growth was inhibited for the 14-day period, but that those marked (6) failed to recover. In these transfers the fungus had been killed by the toxic properties of the extract. Tests on Water-Treated Wood Flour
For the purpose of determining t o what extent L e n t i n u s lepideus can grow upon the treated wood flour, samples of approximately 5 grams each of wood flour were prepared in series of nine and placed separately in 250-cc. Erlenmeyer flasks of known weights. This required seventy-two such flasks, since there were eight wood flour samples-namely, cold- and hot-water extracts of unseasoned and seasoned heartwood and sapwood. Similar samples of each of the untreated wood flours in series of five were prepared in the same manner. Also two flasks were prepared for each sample but were not to be inoculated with the fungus. The flasks containing the wood flour were then placed in an electric oven and dried to constant weight. Each flask was then plugged with cotton and sterilized. Twenty-five cubic centimeters of distilled mater were added to each flask containing wood flour. The water was allowed to diffuse through the wood flour for 3 hours before inoculation with a vigorous growing culture of L e n t i n u s lepideus. The cotton plugs were then covered with waxed paper and wapped tightly to prevent evaporation of the water. The flasks were then placed in an incubator and allowed to grow for 6 months. Table IV shows the average loss of sawdust in the flasks for each series. It is seen from Table I V that enough of the toxic substance in the heartwood wood flour of both the unseasoned and seasoned material was extracted by treating with both hot and
INDUSTRIAL A N D ENGINEERING CHEMISTRY
October, 1929
Growth of Lentinus IeDidous on Controls a n d on Hot- and
cold water t o permit the fungus to make some growth. Table I1 shows that a greater percentage of soluble material was obtained from the seasoned heartwoods than from the unseasoned heartwoods, and the same relation in regard to the loss percentage due to decay would he expected from the wood flour. This held except in the case of the hot seasoned heartwood, which for some unhccountable reason gave a low loss percentage of 4.53. The treated samples of s a p wood wood flour each had a dense growth of the fungus. Table
Iv-Loss In Welghf
of WesfernRed Cedar Wood Flour tnoculafed w l f h Lennnus Iepideu, HE*RTWOOD
TRBATUENT
I
UNSEASONED SZASONHD Per c e l l Per Lent
Cold water Hot water
5.61 7 73
""treated0 Untreated, not
2.58
6.83 4.5s 0.90
0.00
0.00
inoculated'
SAPWOOD WTNSEISONBD SBASONBD
cent 20.47
PSI
16.40 19.55
Per cent 18.28 16.94
0.01
10.06 0.01
* Controls.
Volatile Constituents of Western Red Cedar
In order to determine whether any of the toxic material present in the wood of western red cedar is driven off with the application of heat a t temperatures used in kiln-drying, small samples of the unseasoned and seasoned wood blocks were subjected t,o various temperatures in an electric oven. The blocks were placed in 2-quart fruit jars and sealed with a cork stopper through which was tightly fitted a glass tube extending from the jar to a condensing apparatus outside
Cold-Wafer E x t r a c t i r e s w i t h
9x3
hear
the oven. Heat was applied for 48 hours and the volatile material thus condensed in each case was mixed with 25-cc. of malt agar and placed in Petri dishes. Upon cooling a small piece of vigorous growing culture of Lentinus lepideus was placed upon the medium in the center of each Petri dish. Two controls for each run, using 25 cc. of malt agar for each plate, were also made and inoculated in the same manner. . Growthreadingsweremadefrom time to time, andit wasevident that thevolatilematerial driven off fromthe higher temperature NUS of the heartwood blocks was more toxic to the fungus than that secured from the runs a t the lower temperatures. However, the lowest temperature used-80.3" C. (176' F.)-showed that enough toxic volatile material was driven off to retard the growth of the fungus. Other temperatures used were 85" C. (IXSOp.); 90.2" C. (194' p.); 94.7" C. (203' F.); and 100" C. (212" F.). There seemed to be only a slight difference in the amount of retardation between the unseasoned and seasoned heartwood volatile material for the various temperatures, though a greater amount of volatile material was driven off from the unseasoned blocks-possibly they contained a larger amount of moisture. No apparent differences were noticeable in the same tests made with the sapwood. Any ill effect that kiln-drying temperatures may have in driving off the toxic volatile materials present is more than offset hy the beneficial results secured (5) in effectively arresting any fungus growth that may be present in the wood. The kiln-drying temperatures serve to sterilize the wood and any toxic volatile material driven off appears to he negligible.
984
19DCSTRliiL A S D ENGI.VEERISG CHEMISTRY Conclusions
(1) The hot-water extracts of western red cedar heartwood are all more toxic to Lentinus Eepideus than the coldwater extracts. (2) The heartwood extracts of western red cedar are more toxic to Lentinus Eepideus than are the sapwood extracts. (3) The usual temperatures used to kiln-dry the wood of western red cedar have a slight effect upon releasing the chemical deposits of the heartwood upon treatment with water. (4) Seasoning of western red cedar by kiln-drying has
VOl. 21, No. 10
little or no effect in destroying the toxic properties which make it more durable in actual service. Literature Cited (1) Hawley, Fleck, and Richards, IND.E N G . C H B Y . , 16, 699 (1924). (2) Hubert, J . Agr. Research, 39, 523 (1924). (3) Hubert, U. S. Dept. Agr., Bull. 1363 11924). (4) Humphrey, Mycologia, 8, SO (1916). (5) Richards, Proc. A m . Wood-Preservers' Assocn., 19, 127 (1923). (6) Schmitz, School of Forestry, University of Idaho, Idaho Foreiler, p 6 (1922). (7) Schmitz and Daniels, School of Forestry, University of Idaho, Bull. 1 (192 1). ( 8 ) Thelen, U. S. Dept. Agr., Bull. 1136 (1923). (9) White, Timberman, 33, 126 (1922). (10) Zeller, Ann. M o . Botan. Gardens, 4, 93 (1917).
Monosodium Glutamate as a Chemical Condiment' John E. S. Han Y-1085~ NORTH
SZRCHUBN
ROAD,S H A N G H A I ,
CHINA
As the corresponding threshold values of cane sugar and table salt are 1:200 and 1:400, respectively, the flavoring LL proteins yield amino acids upon hydrolysis. About power of monosodium glutamate is a t e e n times stronger eighteen amino acids have been obtained from pro- than cane sugar or seven times stronger than table salt. teins. They are probably condensation products of The intensity of the meatlike taste in a solution of monoamino acids, the condensation being assumed to be between sodium glutamate increases with the concentration of the carboxyl group of one and the amino group of another. (C6H8N04)-ions, but not inexact proportion. Thus when the When any particular protein is hydrolyzed, whether by concentration of (CBH8?J04)-ions is doubled, the taste is acid, alkali,-or steam, ihe intensified, but not quite same amino acids are prodoubled. On the contrary, the salty taste is more than duced and in the same proGlutamic acid, a rare chemical found only in reportion. During the procd o u b l e d when we exactly search laboratories in America, in the form of monodouble the concentration of ess of digestion the digessodium salt is freely used like salt and sugar by housetive enzymes convert the sodium chloride in a soluwives, restaurants, Buddhists, vegetarians, etc., in the tion. Evidently, the taste food protein very largely Far East. The Chinese alone used $1,130,000 worth of i n t o t h e same substances of (CbH8hT04)- ions would this chemical condiment in 1928. I t will be interesting not be appreciable if large as those produced by boilfor the American chemist to know how the chemists in quantities of both glutamate ing acids. The final prodthe Orient have magically transformed this rare chemiand sodium chloride existed ucts of hydrolysis are concal into an everyday necessity. i n t h e same solution. If s e q u e n t l y the units with such a solution is greatly which the Drocess of assimidiluted. the saltv taste dislation chieky deals (3). d-Glutamic acid, CsH9N04, one of the common amino appears and that of glutamate becomes predominant. The acids, was discovered by Ritthausen ( 7 ) in 1866 among the highest flavoring efficiency is obtained when glutamate is products of hydrolysis of wheat gluten by sulfuric acid. used in soup and other dishes that contain comparatively It occurs in both animal and vegetable proteins and is one little salt. The meatlike taste diminishes when vinegar is added to a of the principal constituents of meat and vegetable extracts. Gliadin and glutenin, the two chief proteins that occur in solution of monosodium glutamate. Probably this is due approximately equal proportion in wheat gluten, were found to the fact that the free glutamic acid formed upon acidifyt o yield, upon hydrolysis, 37.33 and 23.42 per cent of glu- ing is not appreciably dissociated to furnish (CaH8N04)- ions. The taste of very dilute solutions, such as those used for tamic acid, respectively ( I ) . Glutamic acid is harmless. When administered as food, 96 per cent is absorbed, part the threshold value test, is not meatlike, but sweet. The being used up in protein synthesis and the rest being oxidized sweet taste of cane sugar is, however, different from that of monosodium glutamate; the sweet taste of cane sugar is to urea (3). greatly intensified, when the concentration of the solution Properties of Monosodium Glutamate is increased. I n China and Japan monosodium glutamate is manufacGlutamic acid is dibasic and forms both acid and normal salts. Ikeda, during the course of researches on the sea- tured on a commercial scale and consumed as a condiment. weed Laminaria japonica,2 discovered that the univalent It is perhaps the only metallic salt of glutamic acid that is - of glutamic acid possesses decided meat- non-poisonous and can crystallize well. The pure salt is ion (C5H8X04) like taste. The intensity of the taste can be judged from the perfectly white, nearly odorless, and non-deliquescent. The fact that 1 part of monosodium glutamate, NaC5H8N04, commercial product is usually somewhat hygroscopic when dissolved in 3000 parts of water is just perceptible to taste. the humidity is high, and has a faint odor resembling that of dry casein if kept in closed vessels. Other salts of glutamic 1 Received April 10, 1929. acid in which only one hydrogen is displaced by metal, such 3 The seaweed is used for its flavor and has the taste of monosodium as monopotassium glutamate, KC6H8N04,acid calcium gluglutamate. Glutamic Acid
A
