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Full text of "A chemical study on mature, cured and fermented Pennsylvania cigar-leaf tobacco [microform]"

Author: Royo, Renato Marino 

Title: A chemical study on mature, cured and fermented 

Pennsylvania cigar-leaf tobacco 

Place of Publication: 



Copyright Date: 1942 

Master Negative Storage Number: MNS# PSt SNPaAg031 .1 3 



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100 1 Royo, Renato Marino. 

245 12 A chemical study on mature, cured and fermented Pennsylvania cigar- 
leaf tobacco $ca thesis by Renato Marino Royo. 
260 $c1942. 

61 leaves $bill. $c29 cm. 

Thesis (M.S.)-Pennsylvania State College. 

Bibliography: leaves 59-61. 

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300 
502 
504 



590 



590 



(^ ^ 



tiffi PHHEYLVAKU ST/^E COLLBOS 

Ti-o Gradurrts Sohool 

D©i>art«wt of Agricultural and BiologloaX. Chealatary 



fUSSTLTiUnA ClOAR-LEAi' TOMCCO 



A ThMla 

by 

itenaio i!arino Hoyo 



Sutaittod in mrtinl f-af ilL-ujnt 
of Uie roquirotxntB 

for tho degree of 

MALTHR OF ilCimiCU 

August, 1942 



Approved » 



.^^4MlscL 



..-« *-»-..«. ,«» , ii< ll« W !■» Ill Wl i n 







Head of tneI^eSrte©at 



4MMilPMWMMa*«« 



A::a:o7»-LEuaEMa;:T 






The author withos to ex^rees his appreciation 
Dr. D.E. Haley for his eTer-willinc cooperation 



and encouragement durin^- the course of this etudy. 

And to Ur. It^idoro vol6n, lIonoraLle CorjiiiBEion- 
cr of Agriculture of Puerto Rico for his personal 
intereet in the furtherance of ny education • 



^r 






i 



fABL. ^;F C013TEKTS 

Pa:j0 

Iritrodviction * * ' ^ ^ 

Review of tho Literature • ' 

toalytical x roceoures •....•• • *.**.. 

Detericdnatioii of Uoi^ turc • • • • • • ^"^ 

D-3terr.inatiori of Ash 

beterrdnation of tl.e Total Allcalinity of tUc Crude 

Ash • . • • ^^ 

Detonrdnation of the Total Alkalinity of the Soluble 

Asl ••......» • ^"^ 

Determination of the Alkalii:ity of the Insoluble Ash 14 

Preparr.cion of the Or.jAnic Acid Fraction 1* 

Determination of Oxalic Aoid 1* 

Detemination of Citric Acid - • • ^^ 

Determination of L^^xc Acid ^' 

Brterr-inntion of Potaeciun; ^^ 

Determination of Total iUtror,en ^^^ 

Determination of Protein lUtro^-en ♦ • • 21 

Detorirxination of Hon-Protein llitrogen • . . . 21 

Dotermination of Calciura • 

Determination of the Volatile Organic Acidity ...... 21 

Purpose and Plan of tl^e Experiments 23 

Tahlc I - libcperiment T .•....-... • 

27 
Table II - Sxporintf^nt I ••• 

Discussion - ExperL-nont I ■> ^^ 



Table of Contents Cont*d. 



Tables 111 anu lY * Kxr;erli-cnt I — 



« • • • • • 



• •• •••••«•••• 



Tables V and VI - Sxjjeriuient 1 



• ••«!•*•••• 



Table VII - Experimert II •.. 



•••••«• 



»••••••••• 



••«••••••* 



Tables VIII and IX ir-iixp®* iiat^xit II ..*.« 



Disc ass ioii - 3y;peri^'ient II 



Tables X. and X I - Iijcper i ..^.ent II ,..,♦.... 



•••••• 



•* T 



Table ^.11 - Kxpcrxinen'^ i±- 



»••••••••••• 



Discasr.ion - HUperkej^t III 



Tabic Illll - Sxpcrix^ent IV 



• »•»««'•••**# 



Dl8c^&sior. - ExpcrirTiOnt IV 



»»*#•■•«•••••••• 



«••»••••»••• 



<^i^. - 



bu:::i^ry 



Biblio; raphy 



k'B.'Q 



«. )' 



32 

35 

41 

42 

43 

46 

50 

51 

54 

55 



57 



69 



WTRCWJCTIO!} 



Althou-Ji tobacco is tropica in origin, its Culture is world wiao. 
Tiie plairt in rov:n as far i:orth as central oweden and a.z far south ac 
Southarn Australia and Kew ::e?.lc>j:id. Thuc, this riant can be -rov-n 
succesefull; under a very v^ide ran^ie of cliratic conditio gj but on 
the ot'.er naiid, the co: . ercial valuo of t:.e product dspends largely 



on the onvir 



To'.-acco i a; 



nt in which it ia produced, 
been U.e eubjcct of irvef;ti^;ation for aUty years, but 



much of the earlier uc-^a can no longer Id re;^arded a6 reliable. TLe 
dearth of e.mlyticol procedures adequate to cope v.-ith the problems ore- 



se 



nted by the c.^oruic".! cor.nlexity of the tobacco leaf lias been a ser- 



ious 



obetaclo in the pathway of progrees along this line. 

A marked procrcEs has been jiiade rosardin^ the different cheitical 
clian-es which the tobacco leaf undersoes in the field, the curing ehed 
and the factory. Wny inveetijators are trying to elucidate the com- 
plex nature of ti»e different cheaical chan:-ee that are constantly go- 
ing on in the tobacco ,.lant especially during its grovrth jeriod. Oth- 
ers are endeavorin- to advance our chemicd laiowlod,;e in regard to the 



►■»/^/> tTJ C f 



c e t; 3 



of curing. A knowledi;;e of thoee chemical clian^ee tliat take 
place in the leaves of the tobacco plants when these are detached and 
allowed to dry with maximl exposure to air is very important, eince 
it represents those changes wiiich aflect the physical and chemical 
propertlee of the leaf - ir^ore particularly those properties; whicb in- 



clucio bi.e o'^pacity to hold firo and tl^e color, texture, and elasticity. 

A nur:ber o.' v/orkerts are trying to solve the ajfiter- which iii- 
▼oItcs tlie fermentation of tobr.cco. Different theorioc Lave been ad- 



vat 



iced to accoant for the cherical changes ocaurring during the fer- 



mentation process by different workers on the cubjectt ^^ile certain 
progress has been rnade in this particular I^cld, a ceneidcrable ainount 
of inforriation rcLiains to be obtained* 

More research v^orh along these different lines of investi^'fttioni 
and lore attention -ivcn to the cultural ana fcrbili^cr praotices a« 
they arc related to quality of the leaf .ill srcally increase our laiow- 
l#dg« of tobacco cherdstry and add to the success of the tobaceo in- 
dustry • 



REVIEW OF TIO:; LITERATURE 



In snite of tae alTnoct universal occarrence of or^;anic acids in 
-)lant£-, surprisingly, little tru3t\^orthy irirori..atioix i« available on 
the exact identity of the e.oid* fnurd ir a given species. Most of 
the available inforraatior. about t!.e -etabolisi- of tie organic acids 
has been obtai)iea froi:. ttuciies of t.ie iov/er fomis of plant life, es- 
ptcially i-oldc and yeasts. 

It had been pointed ont in the rtvievvs ox rranzen ^iO, ii, it., xc , 
14,15) and his co-workers thie.t the older dv.ta etbablishing the iden- 
tity of the or--arac acids of the tobacco leaf are very unBatisfac tory. 



Ti.e exter!sii7e invostifabions of the Rustuiaii ii^orkers on cired and fer- 
mented tobaccos of the oriental types had recently been sumuarized on 



the v/ork of Shuiuck (38). These v/orkert on t>ieir qualitative invo 



4- 1 



gationc identified citric, mlic, jxmUc, succinic, and funiaric acids 
on cured and fermented tobaccoe. A more rucent publication by Viclc- 
ery, and i^ioher (51) contains a very corr.prohensive review of tie lit- 
erature relating to the organic acids of the tobacco plant • 

Pennsylvania cigar-leaf tobacco contains appreciable quantities 
of inalic, citric and oxalic acidsi in fact, these actuolly constitute 



more 



than four-fifths of the total ether-soluble acidity of the loaf 






tissue. 



fh# Whole subject of the metabolism of organie acids of ci'^eJ^ 
plants has been recently reviewed by Bennett-CIur.. (7) and rcforG..co 



should be r-Ade to his paper t^ for a coi-prohtneire statement oi the pre- 
sent position of our knovledcc oI the or-anic acids in plant piiy.iolocy. 
The work of Viokery and Puchor (40) on the i:.cta:.oliSTi of tlie orcanic 
ids of the tobacco plant durin,- culture reve. ic that t^e three chief 



ac 



n 



The aut/iors 



W&aiic acids of the tobacco leaf, rralio, citric, and oxalic ucide, un- 
deri.o vovy litUe ohMge in ahtola oo as^ount durlnr culture of the loavei 
in the li •,l.t, the total or-anic acidity remains osceutiall/ couBtuut. 
Durir.: culture in the dark, nov-evgr, quantity of lialic acid diniiniahec 
and the ciuuntity of citric acid increases considerably, the oxalic 
acid and the total orr^anio acidity both rcxi:. uno^.a^xged. A consider- 
at on of the nossible sources iror: which the acwly for-ed citric acid 
y bo derived indicates thet ,.mlic acid is the r,o«t probable co-,.rce. 
ertain suggections ae to the poss.tlo chemical r.echa..iu..s to accotM* 
for the intercoiiversion I»ad bean advanced by tlie authors. 
clso found that oxalic acid deoreaseB, but does not entirely disappear 
during the develo^-ment of the plant. Smirnov also found it to be the 
case with Oriental tobaccos. Malic acid attains its raxii^ruir. oon©«n- 
tration in the young plant and tVie concentration do*i not olAn^e appre- 
ciably in the nature leaf. Concentration of citric acid is alco . axi- 

1 h. the young plant, decreasing ii*rkedly in the mature iresh leaf 
and asain increasing durin-^ the ouriag pro«*«», Malic acid v.-ac, at all 
tta<'e6 of f,ro*'th tl.e prodoiuinatln.i acid of the leaves. oxaUc acid be- 
ing n»xt in order, with citric acid in sniallest quantities. Th« a- 
mour.t of unkriowi acids ««• interraediate between the oxalic a-A citric 



acius* 



G 



In 1914 Garner, Bucoii, u..u r^Jubert (lo) reported duUi on the 
mlio, citric and oxalic acid couteut of smr^ples of dried ,-reen leaves 






cured leaves of several vari«tiea of -ouii<.cticut tobacco. 



Tx.e 



work of Pickery und Pucher {b^) on tl.« turiug of »eversil varieties of 
ewmecticut tobacco shmm that th» quantity of rr^lic acid did not c)...anf:8 
dMrlac the first twelve cays li' curir.- but subsequently di:...iiiisiuid by 
about 1G;jJ. Ti.c quantity of citric acid in tho leaf Lncrrased enorr.ou«- 
ly. at U.e oxpiratioii of txvelvo days nearly five tl-ues --is mch of this 
acid was proseul, ir. u.e louf j and at tnc end of the curing period, tho 
quantity had further iucrcaGod to £>.3 thes the cri-inal r.-...oarit. The 
flynthesis of citric acid -A-as ono oi the r-ajor ohan-es occaring during 
cur in:-. They present data in this bullotiii Luplyiac tlxat oxalic acid 



is a relatively un 



constituent of the tobfcoco lc(-i at a-.y 



sta'-e of curing* Ruhland rer^arde oxalic acid ab oI ra;.u:-u.er/uai si*^- 
nif ictmca in the •Wieral s.etubolisu of mny plants being called forth 
in r«»foase to t}.e formtion of Manonia as a part of the inechanism to 
provide for the raiuteimnce of the reaction of the tiaaue within the 

proper lir.iits. 

Hook (2) r.akin- a study of the non-volatile organic acids of 
Pennsylvania ci-ar-loaf tobacco found tl:^t during tho ouring period 
the oxalic uciu and tiie citric acida increased in quaritity whereas 
the mile acid decreased. He concluded that the better (iuali*l». of 
cured tobacco y:nn found to I^ave a greater iu.ount of citric acid whereas 
Uio quaiaitie. of »alic and oxalic acids were appro, irately tf^e .a^.c. 
B« found timt thcro v.'as no influence on the citric, ,^li^, tffd o:calic 
acid content of the tobacco leaf by varying the fertiliser treateent 
or the ayatom of rotation* 



• 



Duriix: tUe .Tocessin^; of tobacco of the ci^avletvi type certain 
-periods of ferrientatior. arc involved* Althou -h tl.e f erx! entation of 
tobacco has hecr. tLe subject of st>idy for .icre thai; fifty years, little 
rt knowledge exietc ar to the different factors involred* To Jen- 
kins (^2) belonrs the cri?dit for ti.^ first deter:i).in-tion of soTie of tlia 



cl 



lomical ohan-ee occ\iTix.r, durin;; f^nuentation of ci^ar-leaf tobacco. 



■hV^r* -r^r- 



tadrnov (40) studied the boha-rior of the orcanic acidG auriiu. cno xe 
Iitatio5o of tobacco ard reached the followinr conclusions t- 

(1) Under anaerobic eruditions nalic aiid citric acid practically 
di«appear* in presence of oxygen there is scfireel^' any chonr© at all. 

(2) FfiriLentation decrear>06 the percentage of organic acids but 
ti'ie change hi th^. citric acid content is greater than that of mlic 

acid* 

The work ^f ..c.KListry (27) shoy.s that the noWPil fermentation is 
al.mye *ocornijanied by a marked progressivo decrease in the total etner- 
soluble orf.anic acidity and a corresponding; rise in the allmlinity #r 

dirrdnution of the -alic aoid and citric acid 



the tissue ♦ A ^ro 



A 



content of the leaf is asuociated with & satisfactory fementation, 
the oxalic acid present in the tissue is not apprcciall^ altered. 

The earXioet reference to the addition of y»a«t to fermenting 
tobaeoo was by Koller in 1068 (24). Koller inoculated tobacco with 
a yeast infusion in an effort to imsten the fermentation. Sumr-artes 
•f the work of several Genrian inTesti^^atore aro to be found in the 
publication of Wagner (5G) and Kisrlinr, (23). 

BwtnilE are given for the use of yeaet in nujneroue myt* Street 
(48) v.as the firct to report on the use of yeaat in femientntion of 






■X 

■A 



tobacco in this country. The addition of livijaf, yaast cells to to- 
bacco ray be iMt..rpretec ar. either a biological or biocher.ical rethod. 
TMst. are simpl. Tors of fun,;i iu the classification of plants and 
parallel reactions are coiia:.oii. 

The early work on tlic chendcal factors influencing the quality and 
burn of the tobacco leaf ims concerned minly with the mineral con- 
stituents and the toVa organic acidity. ;>chloesin5 (37) may be call- 
ed tho pioneer in the scientific inveetijation of the cheii.ic.l lactors 
affectinc the burning qualities of tobacco. He m. tho oriijina.or of 
the theory o. combtittlbility of to-oacco that attributes to pota.sium 
•alts of organic acids a decidedly beneficial influence to the burn- 
ing qualitieo, ana to the potassiu- calte of sulfuric and ruuriatic 
acids a very hamiful effect inhibitins the burn. He oxplaiiieu t .. 
favorable action being due to the .clliag of the potaeciu,-. s^t. of 
oxalic, citric and i,«alic acie,6. which, ^^hen heated nelt before they 
burn and by further heating yield an inflated and highly porous coal 
favorable for holding fire He clair.ed that there i^ in eeneral a 
a direct relationship between the alkalinity of the v^ater-soluble cor.- 
Btituents of a tobacco ash and its fire holding capacity. 
Unity of the ash solution is due alir^ost entirely to the potassium car- 
bonate which it contains. The loaf doesn't contain potassium carbon- 
ate. but wi.en it is burned, the potas.iun salts of tho organic acid. 
are oxidized to carbonates. So the alkalinity of the soluble aeh ap- 
pears to be a good ii-.dcx to fire holdinc capacity. 

l,^ analysinc the ash of -jood burning, tobacco, van Bei:r-.elen (47) ul- 
^ys found a greater quantity of potassiurr. present as the carbonate and 
lesser quantities as the chloride and sulpliate. 



X'H.^ cJ.Xi£&''."" 



8 



« 



B#hre:as (C) ac a result of his IrveEtl-.atiov.al work on the burn- 
ing quaUties of toboc.o. i^ate. that a high coutora of potaseiux. in 
eo^.bination with citric. r.alic and oxalic acids largely deterr.ii:e. th« 

burnini; qualities. 

Gar-er (17) ruide extrftetfi from both cood burning; and poor burning 
leaves and conluded that both contained about the sar.e q-anntities of 
pota..iu». but tie inferior leaf contained a moh higher percontace of 
3.ineral acida. Hence, ho concluded that the potas.iuB. ealts of or- 
ganic acids such a« mlic an6 citric are the chief factora c«trollins 

the burn# 

Haley. Kasset, and Olson on a study of certain constituents of tl« 

leaf and their relation to the burning, qualities of tobacco (1^) ar- 
rived at t!;e follov-ine couclu.ionc .- (I) The ether-.olu.le organic acids 
of the plant q)?car to occur aL.ost wholly in combination ..Ith the al- 
kali earth ;.etais as masurec by tho almlinity of the aoluble ash and 
insoluble ash. In .ra.tically all oases they found a par.llelisn. be- 
tween the burnin;: qualities and the alkalinity of the soluble a.T. 
They concluded that the season and the fom of potash supplied as fer- 
tilizer affected alkaliiaity of the soluble ash in practically all ca.es. 
Field fertilizers experiments on tobacco has been done by many in- 

-1 i • ..^ fhf> wrtrk of f'rear (1^) Olson (29,30. 
veeti-ators since early tx3..eB, Tue worK oi * reui v , 

SI. i:). Haley (20,30.4li) and Thorpes (45) on Pennsylvania oigar-leaf 
tobacco should be mentioned. 

The mrk done of t:.e effect of fertilization on the organic acids 
of the tobacco-loaf is very ..ea^er. Siuuuck (5Q) presented evidence to 
.how that the citric acid content increased with an Lnoreaso in ferti- 






i 

I 



lii;ation. KaM«t (23) ctvidiod tho effect of fort llization on the or- 
ganic acid oojabent at; -wBli as tLe effect of tVe orcanic acidity on 
burninr- quality* Block (O) fomid xic influence on tho citric, malic, 
mad oxalic acid content of the tobacco leaf by varying the fortili£:©r 

treatment or b' e K.rste^ of rotation* 

Kuroimtoir and co-v:or:ierG (2G) and Ps^re- (31) working with high 
citric acid Rueeiaii tobacco, foiond timt the nmkii-iun accu^Tiulahion of 






rio acid vjas obtained by the uso of R potash fertiliser. Aiiobl'.er 



au£i;ian v--jrLcr, VladimirDv (S'l.Su) v.'or';ing with the saiiie t/po oi 



.'^ ^~ 



O" 



liacc 



fouiid timt en i-crc^ice in tl-iS potassiuji content of tiie nutrient 
nedinTi dccrc. .:..ed the citrio aoid content and the total acidity of fue 
tobacco leaves. lie also found tliat araaoniuti caltt: jis a Gource of 
nitrofen decreased the total acidity and «te •etltent of citrio acid, 
*hil© the use of nitrate as a source of nitrogen resulted in increas- 
ed acidity and citric acid content. 

The literature of plant composition, as influenced by the form in 
*hiok nitrogen is supplied, ie very liinited. Most investigators have 
worked fro.r. the agricultural point of view and Imve recorded tiieir re- 
sults in tonriB of 6ij;c, crop yield, luxuriance of foliage, or at best 
in terms of dry weight of the plants. 

Haley and Thomas (45) workin- on the absorption of plant nutrients 
in relation to tho production of Pennsylvania ci^ar-leaf tobacco found 
that the effect of nitroren on growth and development of tobacco plant 
depends on the balance of nutrient material i^resont but likewise on the 
fonr. of nitroeen used. They found that cottonseed ir^eal fro-i standpoint 
of yield, is r.oro satisfactory ae a carrier of nitrogen than HallOj. 



i 



10 
ftl0y also fouyid t!.ai the a1.:corption of nitro'-on occai-L. curing the grov.- 
inc scuEon, altiiou^ih tic poroantace of uitrocen falls at maturity. Tf.e 
cloYO-r and alfalfa, T.'hRn -ro%wi in rT'.:.-tio-.i, adds co' .sidurably to fcao 
quantity of nitro;-en uv« llaM-^ for aLeor^.:biOii. 

The uso of aE2.io/-iw:. aalts as tho source of nitrogen for t3ie .-row- 
ing of tobacco has been exteuaively studied by Anderso:. and his v-jollab- 

oratoro (1,44). 

Clarh (9) working with toniato plants found an outnbn-.'Jing diffnr- 
ence in the relative asiounts of organic acide in the tisG..o3 when nit- 
ro-en was supplied as ari:uoniu« aitro^en and wl;en it was cupplied as 
nitrate nitroeen. Fxirthenrjore, the distribution of this organic ocid- 
ity with respect to the relative proportions of oxalic, malic, citric 

The 



^•^ f^ -.^ . , 



and ur:^.o..u «»f«anic acids was widely different In the two c 
leaves and stalks of plants crovm on anmonium nitrogen contained re»- 
pectively 71 and 66 raillx-equivalents of organic acids per 100 gna» 
of dry tissue, tliose of the plants ,^rown on nitrate nitrogen contained 
153 and 147 i.iilli-9quival>.'it6. 

Vickery, Pucher and V.'akeBian (S3) very recently worked von the ef- 
fect upon the corposition of the tobacco plant of the form in whioh 
nitrocen U supplied. They presented results leading to tl^e conclusion 
that tho tobacco plant is extremely responsive to the nature of the ni- 
troj'enous nutriment with T«hich it is supplied. The plants grown with 
the higher i-roportions of uia;.onia in the culture solution contained 
very little organic acid. Malic acid is the doii^inant acid in the 
"nitrate plants", but it appears to be tho acid jsost extensively af- 
fected by a change in tho nitrogen supply Citric acid appeared to be 
also extensively affected especially v.t.en the amount of airoaoniuia nit- 



w 



11 



1 



fiegen v^g i :cr ^aifdt The acids of the unlmoTOi £:ron,; in the pruso 
MM chBiWM ro-arkabl:- littlo in ab .solute quantity ova 



■ii 4-- 






range di 



nitrogen nutrition (0 to 60;.- 



), also the oxalic acid v.^g only 



modwrately affected. 

The behavior of the a] callnlty of tho ash is particularly sbrik- 
ins, Th« altrate plants yielded a highly alkaline ash, the "aic.oniiur; 
nitroson pUnts" a ir.oderately alkaline one. The quantity of ash par 
plant was greatly affected* 

Tte •aloiurn in the ash of ttio leaT»B followK % cur-re th^t closely 
resemblee that for the orjanic solids. The variation of calciua as 
the range of nitrogen nutrition varies fror. to 80,'J aamonium nitrojen 
nas not nearly so striking ac vims the variation in the organic acids. 
It can bo concluded fror. tho dita tiiat the calciun rtiorption at a 
constant level of calcium mm^V "^^ 1^*^^® affected by the ch.anc,o in 
the font: of nitrogen in the BOlutioni such differences as were noted 



can 



be in'.erpreted only as aecondarj* results of the change in the 



nitrogen nutrition. Riosphate absorption, like that of ealclum, ims 
not sreatly affected by an increase in the proportion of auSioniua ion 
available to tho plants when ti^e caloiuri and phosphate were provided 
e.t a constant concentration in the several culture solutions. 

Records of the analysis of cured tobacco grora in 1927 at the 
Windsor Tobacco Substation (50) showed that a sample prepared from 
plants fertilized ivith llaHOg contained 3.5;.' of nitrate nitrogen, while 
one from plants fertilised with awaoniun sulphate contai ed 19.5°i 



,0< 



&air.ples from plants that Iiad received various oreanio nitroseneous 
fertilizers lay between these extremes. The explanation of the low 



% 



nitrate nitrogen content of tlie sodium nitrat« plants Ig doubtless the 
leaching of the soluble s»lt froir, the unusually iicr.v-y rains of that 
particular growinc. Airjnonitun ealts, on the co--trary are not readily 
reEioved in thi^ v.r.y and it is obviouc that an exUn.ive transfonration 
to nitrates it the coil must htve occurred. 



j 






1 



,3 



mMJTI 1 CM. PROG SDURSS 



> 



All sarirlcc were anal,;:ied in duplicate and exprei-sed on a nolBturo 



free basis 



Dctenoiimtion of Moisture 



\0m\i t i0i' ■ Mi^i'o 



lioisturo ^.L detcrnired by dryin^^, a ono r^ram saB^ple to constant 
)^i,ht ill a vacuui. dosGicutcr containixig sulfuric acid^ according to the 
l^thods of the As?;ociation of Official A^-ricultural Chemists {b). 
Detorr^ination cf_j^^ 

.^.sh vjab deteriaiiiod by heatinp; the Samples in porcelain dishes 
for al;out 16 hours in an oloctrio 3:.uf i Ic furnace at 450-500 C 
Deterrinabion of the Total /.Ikaliuiy o£ t he Crude Ash 



Transfer tlio ash to a beaker and add exactly 50cc. or v.i: ex- 
ccr,: of ll/lO Sixlinrlc acid, heat to boilteg and Xiltor. ..a:,h the 
precipitate thorou-hly and titrate with h/10 KaOH solution usinc methyl 
orance or xylene cyanide -no thyl orange as aii indioator. Tho results 
are expressed as the total aij;ount of normal acid rocjuirec to neutralize 
^e total ulLalinity of the ash in 100 craras of the original plant 

natorinl - ^U.O froe. 

Pete r^nination of the Total ; aLi.llnity of the Solu bleAdj. 

Add about 150cc, of hot water to the crude ash, filter and wash 
v.'Ell .vith hot water. Titrate the filtrate Y^ith l^/lO EzSO^, using 



thyl 



bjf Jb^PHPp 



The 



results are expressed as the nuiuber of cc. of normal aoid required t» 
BeulraliEo the alkalinity of iiie sollAle ash contained in 100 erane 
•f the orisinal material - K-O free. 



u 



De 



'^ 
M 

■^ 



t e rrnina t ion oil J^j f . . Alj £)_ lj]iitx. p_r,t: . o Jn6_o lubU_A8h 

IkalLnity ox' tho ir;iiolublc ash «M ccilculatod by Sv.btract- 



The a 



ins tLe alkaliy.it7 of tJ.e soluble ach fro- t-.o totel alkalr-iity of tne 
«tll» TLe reculLt arc exproa. ed «.« the quentity of nowal f^cid re- 
quired to neutralUe the alL-aliiiity of tho Insoluble ash in 100 .-rars 
of the ori-inal pleuib inaterial - IIoO free. 

ration of tue Orgfflaic Acid Fr^^o bljW 



Tue or. a:.ic acids of tri© toU^cco leaf were extracted according 
to the technique of I-ucLcr. Yiolxry and Wakeman (S2). Two graauB of 
the dried ar,d powdered tic cue «• aoldlfied to pH I.O with sulfuric 
acid, mi:vGd with 3.5 sraniB of Rcbeutos, and extracted with the anhy- 
drous ethyl ether in a Goxhlet extraction apparatus for a period of 
24 hours. At the end of the extraction period, 25inl. of ciiEtilied 
water MM added to the ether -extract, the Eycter. was thoroughly ag- 
itated to transfer the acide to tho aqueous phase, and the ether v^as 
carefully distilled. The aqueous solution was cooled in the refrig- 
err-.tor in order to solidify the resinous rater .al which was then re- 
moved by filtration throuch a Gooch crucible, 
tract was Eade to a definite voluaue. 

Detenaination of the Tofl fc:ther - Soluble O rganj^^Ao^d .Content, 
The ordinary titration procedure was employed in determininc 
the total etlier soluble organic acidity of the sample. A 25ml. 
ali-iot of the organic acid fraction was diluted with 10ml. of dis- 
tilled water and titrated with .0411 NaOH using phenolphthalein as an 

indicator. 

Pete miinat ion of Oxalic Acid 

Oxalic acid was determined by the method of Pucher, Viokery 



The or";anic acid ex- 



15 



^ 












I 



'1 



1 



and V.aV.orxm (S!^) as iollo-st 

A 25iiil. aliq.uot of the organic acid oxtruot -<huii acidifiot to 
Congo Red with 0.5 ?! IICI. The precipitate tixat fora^d Was allov--6d to 
spttle, filtered off on Mtett«« in a Goooh crucible, and washed with 
«ftt«;r. A crop of aethyl-re-i was added to the clear filtrat* and air.- 
nonium hydroxide ^s added to a fain* alkaline reaction. Two to 
three j:-.l, of i^lacial acetic acid wore added, followed by 5 ml. of 10?{ 
oaloiuir. c:nlorido ijolution. After Btanding at least two hoara, th« 



calcii.m oxalate 



iltcred on asbsstoe in a Goooh cruciblo a d -was 



iMiBlied with a little very dilute aimTioniUEi hydroxid"-. Crucible and 
contents v.ero then transferred to a 100 ml. beaker. Five tl. of 50fc 
H2SO4 and 20 lul. of mter wre addec, and the solution vme heated to 
boilinc and titrated while very hot with 0.02 K potacBiup. petwanganate , 

Golutioii. 

Dcte rraiimiion of Cit ric_ Acid 

Citric acid, when treated with potaseiuiu perwanganate and potassium 
broirdde under U;e proper conditiWW. is couTertec Lnto the insoluble 
•ubstance pentabronoacetone . Thiu reaction was originally employed by 
Stahre (2) for the qualitative recognition of citric acid, but was pla- 
ced upoa a (juantitacivc basis by the work of Kunz (25) and of llartinan 
and 'lilliG (21) and it is now widely used for the deteniiination of cit- 
ric acid, Pucher, Violory said Uaker&n (34) taking advanta,',e of this 
and other ideas of sore Russian workers have developed an accurate 
voltUTictric i;.ethod to deterriine citric acid in small '^uantlti^fl. 
this method, citric acid and i:*lic acids are oxidized si^nultaneously, 
and pentabroMoacetone which is formed from the citric acid is :,c,arated 
fror. the malic acid oxidation product by extraction with petroloun 



In 



16 
tthor, after Wiiioli the acide 9Ct% ditsnd&ed aermratcly, TI»c prococlure 
that nafi used imc as follov;s: 

A 5 ml. aliquot of the organic acid solution war, dilated to 20 rlt 
with water avid 3 ir.l* of 50JJ 112^0,;^ wnro addedt The mixture ime boiled 
gently for ei,jvt to ten ininutes to expel traces of ether, coolcv , a :d 
1 ml* ol saturated brenlM Wtter Mls added* After five ralaittee the so- 
lution imt filtered with gentle suction through a Gooch cricil le into 



I 



f 



ft suction flask Liarhed at 55 nil., and 2 ml*, of li. potassiuip. brordde 



or^O 



v/erc mUM# The temperature of the solution mis then adjusted at 20 
to 22^ for 10 ninutes with occasional s'^irring, and ivae then ohilled to 
5^ to lO^C euid deooloriaed ^ith Z% Er,0-, added dropwise with vigorous 

£mlt 



stirri.-i •• 



The oxidation mixture was transferred to a 125 ml* pear-shaped 



separatory fla^k, and the suction flask "was riMtd into the 



ratory 



flaek with 25 ral* of petrolouxa other used in spaII quantities* The 
flask was shaken vigorously and the aqueous layer v/ae darawn off* The 
petroleum ether layer v»tj.s transferred to a second flask* The aqueous 
solution was shaken ar.ain v/ith 20 !• of petroleum other and was then 



put atiide for the detemdnation of malic acid* The tv:o petroleun ei- 
ther extracts v/er© combined and washed four ti^es with 5 ml. of water 
and the vmehin^^s wore added to the aqueous extract for the malio acid 
detonuinatiout A S ml* quantity of 4kfu sodium sulfide solution was 
added to the separatory flask, and the mixture was sliaken vigorously* 
The aqueous layer, \rhich turned reddish^ was dravm off into a 50 ml* 
Erlenmeyer flack, the petroleum either was then treated a second tir.>e 
with 3 ml* of sodium sulfide, and was washed three tiyr^es with 3 ml* to 
4 ml* of water^ the aqueous solution all bcinj; combined* To this ^o- 



,* 



D ■# 



17 



I 



lution wae added 2 ml. of 2 H sulfuric acid and a few quartz psbbles} 
and the solution was boiled gently for three Eienutec to expel hydrogen 
sulfide. It ifcTit then cooled to roor temperature and sufficient 1.5 K 
potassium pennan<;;anato added to produce a red color reraanent for 20 
seconds. This usually took about 0.8 to 1.0 ml. of perrransanate. The 
color vrtiE discharged by the addition of a ssall excess of a specially 
prepared halocen-free h(|dro^:en peroxide (34,35), followed by the addition 
of 2 El. of concentrated nitric acid, S ir.l. of standard silver nitrate, 
1 1. of ferric alus'. indicator solution, and 3 to 4 t^I. of ethyl •Mwr. 
The flask was siiaken vi-'oroucly to coa-ulato the silver brordde, and 
the solution WM titrated vith aamonium thiocyaiiate from a micro-burette 
to a faint salmon-pink color. In as riuoh as the conversion ol citric 
acid to pentabroiuoacetone is not strictly quantitative, it was neoeeaary 
to multiply the q lantity of citric acid determined by a factor of 1.12. 

Determination of Ifelic Acid 

Malic acid wac determined by the method of Pucher, Viokery and 
Wakeman (54). The rethod described in the present paper depends on the 
observation that malic acid, when treated vdth potassium pennancanate 
in the presence o£ potassium bromide under the proper conditio s, i« 
converted into a bror.ine compound t'uat is volatile v.-ith steam. This 
substance yields an extremely insoluble condensation product when heat- 
ed with dinitrophenylhydrasine in acid solution. The ratio between 
tlie amo-ont of this product and of the .nAlic acid submitted to oxidation 
is constant, and the quantity can be easily estimtod by dissolvin?; the 
product in pyridine, dilutin,:; with water, and .aking the solution alka- 
line with sodium hydroxide. The i..tense blue color formed can be meas- 
ured ViTith a colorir.'.eter. 



y 



18 



■-J5 



l'^ 



Tho method ie ae follov/et- Tne aqueous solution, together with the 
waeh fluid from the petroloum ether (obtained rs described under the de- 
tennination of oltric ccid), ^^-as diluted to a 100 ml, A 26 ei1. ulicaot 
was transferred to a 300 ml. Kjeldahl flask to which a 25 ml. of mter 
aind a few quartz pebbles vrere adfcd. To insure the destruction of the 
hydrogen peroxide, about 0.5 al. oi" potassium permnganate was added 
and tho solution decolorized by the addition of 2 ml. of sodium sulfite 
collation. This step has been eliy^inatod in the ir.odifications of tho 
methods recently publishoo (2£) by the authors. 

The flask was fitted v/itb a bent distillation tube which extended 
into a 250 rrl. wide-nouth Erleniueyer flask charged with 10 ml. of a 
freshly filtered 2, 4-dii.itropaenylhydraeinc solution and 20 ml. of 
imter. The end of the distillation tube vjas dipped beneath tlie sur- 
face of the reareut in the receiving flask, and the flame of a micro- 
burner was applied to t]ie Kjeldahl flask. The receivinc flask was 
iirunersed in a cold water b^U- r.n«i the rate of distillation was adjusted 
60 tliat the receiving solution did not boil vigorously durin^s *"« <iis- 
tillation. This last procedure is contrafcy to the directions as ^iven 
in the original paper (34), but ^ms advised by the authors in later 
reodificatious (35). The distillation me coiitinued until the voluira 
of the solution rer.-^inins in the Kjeldahl flack v/as reduced to so..:ewhat 
less than 10 .1., usually taking fro. 12 to 15 minutes. The roceiv- 
inr flask vms then rer.oved and the end of the distillation tube rinsed 



into it. 



Th© orange precipitate wai? transferred £s ooir.pletoly as possible 
with mtor to a sirixll No. 2 Gooch crucible furnished with an asbestos 
jnat. and ms dried for a short tiine at 100 to 110*^0. Meanwiiile the 



19 



4 



a 



-■■ 




receiver was thoroughly drained, and the last tr.uos of prMlpi%it» 



urere 



taken up la hot .^yridliMl used in several succecsivo portions, but 



not more than 5 to v rl. in all. Th© p;y'ridine m«hings vmre then 
trancforred to a 25 inlt voluiiiatric flfttk, the orucibld was then fitted 
into a rubber stopper, carried on a cylindrical funnel — which is at- 
tached to a test tube equipped ^rlth a eide arm ~ and marked at 20 iil* 
Boiling pyridine was ad ied in snail proportions to the crucibloj and 
the contents Vv'ere gently titruated witli a rlastJ rod, after which suct- 
ion Twas applied and the puridine was drawn through each tLrie# Tliree 
or four wEihinge usually were sufficient to dissolve and transfer all 
the precipitate to the £5 rrJ. flask used for t!ie rinsin- of the receiver. 
After beinc cooled to roOi:i temperature, the solution was nAde to voluao 
with pyridine and mixed. It was then allowed to settle or ^vas filter- 
ed, if necessary, to remove shreas of asbestos* 

Either a 2 ial# of a 6 nl. aliquot of the pyridine solution was 
transferred to a 100 ml. flask and 50 ml. of .mter v/ere added, followed 
by a 5 nl* of 5N KaOii solution. The solution was tiien diluted to the 
Eiark, and the blue color v/as reo-d in a colorL-ieter by coLiparison with a 
standard mlic acid solution. This standard ynalic acid solution con- 
taining 10 nillisrar;ic per 5 ml# was treated ir. exactly the saiTie manner 
as described for the aliquot of the organic acid fraction. 

Deterrination of Potassium 



Mikrtl*Ma«M«ri»M 



Potassiuj;! wa 



£3 detcrrdned by the aithod outlined by Thoiras (46 )i 



A 2.5 .;^ran sarxple was v/eiched into a porcelain crucible and placed in 
a muffle furnace, rejulatod so as not to exceed a temperature of 400 C., 
for 24 hours. The ash was taken up with a 5 ril. of dilute nitric acid 



ana 



5 rl. of dilute hydrochloric acid, filtered, and washed with hot 



^0 



one percent 



nitrio acid. The ahlose filter paper and residue were rc- 



pUe 



ed in the crciLlc and burned i-i the muffle furnace at a full red heat. 
This ash ms then taken up with an equal fixture of concentrated li.dro- 
chloric and nitric aoidB, boiled, diluted, filtered, and washed with hot 
1% nitric acid. The filtrate and waehinre were niade up to a voluiae of 
250 ml. To a 50 r:J. aliquot in a porcelain evaporating dleh was added 
1 Kl. of W^ sulfuric acid. The solution was evaporated to drynesc on 
the steam bath, and the dish vms then heated over a Meekor burner until 
the residue was white. The residue* was taken up with 1 ml. of concen- 
trated KCl and hot water added with stlrrinc until solution was com- 
plete. Then 5 rrJ. of 2,t platinic chloride were added and the solution 
evaporated on the etear. bath just to dryP.08S. The residue was taken 
up with 25 ml. of acidulatec alcohol {?.,2h M Hydrochloric acid in 30^ 
alcohol) and the mixture wat thoroughly stirred. After ctandinc for 
a half an hoar, the contcntt; of the dish were filtered through a wwlgh- 
ed Gooch crucible, wathed with 30f4 alcohol, dried at 100*^0. for half 

an hour, and •*rei^;,hed. 

De ter mination of Total Eitrocen 

Total nitrogen wae dotorm.ned by the official i.iethod of tli« 
Association of Official ivgricultural Cheiaists. Tl^e Gunning ir.odifi- 
oation of the Kjeldahl method to include nitrate nitrogen (2) was fol- 
lowed up to the point where the raterial was ready for dieostion. 
After this the Kjeldahl Gunr-in-rArnold (3) method war, followed, with the 
exception tV^t copper sulfate and a selenized granule v»re used instead 
of Eicrcuric oxide or i:iercury as catalysts. 



rf 



tl 



DrtomlJiation o f Pre be in Kitroge n 



.» !»■ Ill III ■ " ■ 



H 



■I 



i 



0\vs : 



Protein nitrogen '^^ detenained by the method employed by a Re- 
search Departiuent of one oi' the large tobacco maaufacturerE, ae foil- 
One rroia of tobacco nAC plaoeu in a 400 ml, beakor and 100 ml. 
of 0,5% acetic acid t.ue added. The contents were boiled for ttf 
minutes on a hot plate and allowed to cool. The eolation me filter- 
ed, the filtrate discs-rded, and the filter washed with hot acetic acid 
mter. The entire rats of tobacco, including the filter paper, was 
tranBierred to a 000 ml. fgeldahl flask and the Kjeldahl Gunr, in^-Arno Id 
procedure, as jr.entioned above, vus followed. 
p»^-«,Y^mi nation of Mon-Protein lUtro-en 

The non-protein nitrogen content of the leaf tissue ms calcu- 
lated by substractins the percentf-GC of protein nitrogen froir, the total 

nitro:en of the leaf. 

Detenrdnatior. of Calcium 

Calciur,; wae determined by the official riethod of the AsEociation 

of Agricultural Chei:dst8» 

Dcten-^dnati on of th.o_ volatile organi c aeiditjr 

The standard jnethou employed by tlxe Rcsef.rch Departnent of on. 
of tiie larger eaetorn tobacco rimufacturin;. companies was used to de- 
terraine volatilo or-anic acids. 

Five ml. of tartaric acid and 100 ml. of carbon-dioxide free water 
W.S added to 5 grains of pov.dsred tobacco in a 500 ml. Kjeldahl flaak. 
The etill ^-au connected and heat applied to the flask re^ulatin^ the 
flaE'.e 60 tliat the boiling ocoured in 5 to 8 minutes. At the expir- 
ation of 10 minutes from the time of applying, the flaiao. sbear. was 
passed in to the flask and 500 ml. of distillate was collected at the 



22 



« 



rate of 10 -1. per ?iinut". The original volur..c ::f the solution in 
tUo reaction flask waa mrked avA maintained durinj tl.c distillation. 
The Dietillato was transferred to a 600 r.l. bealcor and heated to 95<»0. 
6 drops of phenol red Eolution vrets addea, and titrated with 0.021! 
NaOII, us in- a dayli-lit lair.p for illumination. After aubetractins 
the titration value of the blank, the total volatile acids mte cal- 



cu 



lated as acetic acid on a aio is tare -free basis. 



1 



'I 



2*^ 



PURPOSE AlID nj£ OF TlIC EXPERIMSKTS 



Table I gives definite infonnation as to how the ei^;ht outlylBg 
Dlots for the season of 1^41 were treated^ These plots v/ere local- 
ised in different parts of Lancaster County, the center of the to- 
bacco industry in Pennsylvania* 

An application of 1000 pounds per acre of a 4-8-12 fortilizer 
ims riven to tlie olots froi- which samples A and B were taken. The 
experiment mis planned making nitrogen t}is inofit important variable. 
Tiie nitrogen «M applied to samplee _A. iu a more readily available 
form (75-;? NaHOs and 25;i oottonoeed neal) and to sai.iples B^ in a less 
available i'orm (75,' cottonseed raoal and 25:?o HalfOj) 

A study of the moisture, nitrogen (total, protein, and non- 
protein) potassiusi and calciun content was made first on saEplee of 
the mture tobacco. This analyeis ^me followed by a study of the 
soluble and insoluble ash constituents of both ether soluble organic 
acidity, ae u-ell as the three most ir-portant non-volatile organic 
acids found in the tobacco loaf, oxalic, citric and i^lio, were r.ade 



The 



of both the imture and cured sanples. 

The anal G is, of saniples A were compared to sar^jles B_. 
differences in tixe ohendcal conposition of the leaf, when the two 
different applications of nitrogen nfide, were studied. The pocsi- 
bility of usin^ this fertilizer as a coneral treatment of cigar-leaf 
tobacco is discuEced. A study of the organic acid metabolisr-. of tiie 



«4 



iMtturo and cured i;a,.plec is presented* 



Exrinm^sin) i 



Table YII --ives definite inI"ori:iation as to how certain cured and 
femiented Sta^Xtt picked from the 1341 crop were treated. 

Appar^ntl/^ samples v/Viich had been yeast treated durin;; feraen- 
tation BG^.i to be rore coT^'nletoly ferx^iented tlian the untreated sam- 
ples. Tlie ieniientation of the cured samples vmB carried in two waysj 
some v/ere yeast treated, while others received no yeast treatment* 
A comparison of tlie cherr.ical co. .position of the yeast treated and un- 
treated samples vrais i^iade. A study of ti^e alkalinity of the soluble 
and insoluble ash of the cured and ionaei^ted saraples (untreated and 
yeast treated) was litoiiriso L.ade# This was followed by a stady of 
the ether Goluble organic acidity, the oxalic, citric and malic acid 
content of the cured and fexnented samples. 

Special attention was fjiven to the organic acid xnatabolisni of 
the cured and fermented samples end a comparison was made between the 
untreated and /east treated sarirles to see if the yeast treatment af- 
fected the cheraical coiajpositior, of the leaf. 



Eiir^iiLiiisn: iii 

The ohe^nical analysis of two cured saiaples from the crop of 1941 
is sliovm. The "S ilvcstrius saiiiplc'' represents a special caiy.ple of 
the 1941 crop mA is characterized by a low nicotine content. T! e 
usual 4-8-12 fertilizer treatrr^ent had been / iven to both sampler. The 
*2phrata sDxiple" is ciJixractcrized by bein;; grovm under drought oon- 
ditiors. ▲ oor.parison of the chemical conposition of the leaf of 



i 



s» 



thoso trv»c. samnlee ro'AH under e^!'circlv dlffere.it ivi-tor conditions is 
inado* 



EXPliiRIMMT I? 

Table XIII shows the analysis of the soluble and iiisolublo a^/h 
of fermented samples of tobacco of tlio l;:-":^! croi>« Aii ctra sairrnle 
ta!:eB froni the market in cigar fom, X ic analyzed for £olul Ic and 



insoluble ash and compared to tlie other fermented saniploc. of the 



1941 crop. Attllysis of the 
presented. 



volatile or£:anic acide it aluo 



TABLE I 



EXPEillKiiNT I 
Data showing how tiie sar.ples for Experiment I wore treated. 



i ■ * i ^^ii— I'iMi' r^* ^ 



iK^ Al^ 1. ^'- X C f 
y^xriLur \ ITnrio of Grower 



lOlA 

lOlD 



Denlinger 
Koah E# 
Donlin^er 



102 J 



Ciiarles Vu 
Leferer 

Ciiarles .V, 
Loferer 



103A 

103fl 



104A 
1 1043 



Geor:*e Craj:ier 



George Cramer 



Longenecker 
Longenecker 



Location 



Treatirtent 



4-8-12 



Paradise 
Paradise I 4-8-12 



Dramore 
DruTTiOre 



1 



4-C-12 
4-3/12 



Pequea 



4-S-12 



Mt. Joy 



AiTiOiirit 



Planted 



1000^ 

.^ -»j^i'-«ir* ■»«<»! -j.i — ■■■a* ■«» 

lOOOj 



5/2S-&/24 7/18 
6/2-6/3 



1000 ■' 6/2-r/z 
1000. e/5-c/ii 



Topped 



7/13 






8/4 
3/4 

0/11 



^2 1000 ■■ J:/9-S/K ■ S/11 , 3^5 S/gS-gZ^ 



uuckered 



r 



8/2 
8/2 



;i.it 



8/3-B/ll 
8/3-5/11 



8/23 8/25 
8/23 1 3/2 i 
S/26 



5 



4-8-12 



1 



lit. Joy I 4-8-12 

iM Mill - -M Lfrf—m\ -^^.— — -....^^.^■J— *— i^ 



lOOOz 



6/14 8/12 
U ' g/14 ' 8/1 



9/u 



O /o O Q /•? <H 

I 



»/• 



.£ZS L-iZ5 



8 



A - Ready available nitro-en (75/o GaUOj and 25;:^ cottonseed r;e:.a) 
B - Cottonseed Meal (75;t cottonseed meal and 25;J Kal^O^) 



r3 



TABLE II 



nxj^:^:iBiSHT i 



Cor.position of Mature Tobacc 






from Outlyir.^: Plots for tho EoaL-oi oi' 1941 



27 



{iouaple 

iu::;ber 



.OU 



OIB 



iloicture 



LOSA 



.023 



^03A 



.03B 



.(Ak 



||04B 



5.38 
6.30 



7.20 



6,74 



5,66 



5.98 



,77 



5.32 



Nitro?-Gn 



rotul j Protein 



7' 

3.33 
3.66 



4»24 






2.29 



..Oil- 
Protein 

1.16 

1.37 



Potaesium 



2.44 



'1.17 ! 2.33 



4.63 



4.06 



4.04 



4.23 



2.94 



2.50 



2.2G 



1.30 



l.:i 



— —'—«.. 



7 ^■ 



1«J30 



1.78 



3*0 



2 



5.41 



2^6S 



^ R/!. 



C»D^x 



3*09 



.Gl 






2.37 I 1.8« I 2.75 

■ ■ I .■■» ■ I. .-.»—.■■ IM ■ Ml III I >■ .1. » ■ II I II I. 



Cal- 
c iu' i 



' A.r 






\ 



L m Xi-* 



\ 2.90 



U^.22^_ 



J. . t^' c 






3.53 1.41 



3.09 l.i 



o5 



2.92 !1*07 
2.30 l.Gl 

2.93 l.e4 



28 



E^p'raMECT I 



DiGciiSsioni 

Due to ti.e liriited number oi soxiplcs studied in all the exper- 
iiaentsi it &#•• Hot seeni proper to dra.. ...any definite conclusions. 
It can be *jaid in general, however, tliat alfchoufh the number of sairi- 
plcii ct -".died was srall, thoy sho>v a roneral trend of LoLavior under 
tlie epecified coadltions oi the experiment* 
liltro ;cn, Potassiixi, Bnd Calciivni 

Ta:;le I -ives definite infowmtiwi as to how the inature soli- 
ploc fror: the outlying pious of 1941 were treated. Table II pre- 



sents tie analysis of these 6aj:.plcs» 



1^0 aDpreciable difference 



¥/aG foxmd between sanplee A and B^ in the total, protein and non- 
protein nitrogen. Any difference noticeable is of a minor nature. 
Although saiiiplec A }.ave access to a greater proportion of nitrogen 



in a Lioro readily available foriii them sainples B^ no real diffe 

on the availability of nitroren, as chovm by the ohendcal analysis 

of the leaf is demonstrated. 

Hie know that the tobucco plant, as nany other plants, take noet 
of its nitroy,'en froiri the soil in the form of nitrates. The nitro- 



gen 



in cottonseed real and most of the orgcoiic nitro^_^eneous ferti- 



lizers is supposed to be in an unavailable form and can not le read- 
ily utilized by the plant. It is only by conplicated decor!>rosition 
process carried on by soil micro or^^anisms tliat the nitro-en in this 
Eiaterial is finally broken dovm to the nitrate fonr. and becomes a- 
va liable to the plant. 



2d 



The nitrogen Eupplied to saniples A ic in a readily availaxl 



e 



fori, and ehould «'^ive eno\i "h supply of nitrogen for U:o carl./ titages 
of ^rowth* Kitroren la absorbed and utillzod normally by the 
growin?;; tobacco plejit in the eeurly stages of growth* After the 
plant is "topped* the absor tio.a and utilij^.ation of nitrogen apr-ear 
to bo r.oro Iianaful than beneficial. The nitrogen aiqpli4l4 to cam- 
ple s B roved to be as effective in regard to absorption of nitro- 
gen by the plant, as for saniple A* 

It is safe to say that the amount of available nitrogen in fer- 
tilizer B seerae to be sufficient to furnish enough nitrogen for tie 
earliest stages of ^o^h, the £;lant utilizing the nitrogen fror; the 
cottonseed meal as it becomes slowly available due to the action of 
microor^;anisms# Also, at the earliest staMB of growth the plant 
could Iiave riade use of the nitrates usually present, althou^:,h in 
eiDall amoimts, in the soil. 

It will not make any difference fro;: wl'iat source the nitrogen 
coiries as long as the "organic nitroj^en** is given enough time to un- 
dergo decomposition to the '^nitrate fom" and becone available to 
tiie plant* Of course, nitrogen hi so:ae readily available fonu should 
be applied at planting so that it can be utilized for the earliest 
sta-es of r;rowth and avoid nitrogen shorta^^e. 

Samples B gave the same nitrogen analysis ac samples A duo to 
tiie fact that there was a general decomposition of the *'or;;anic nit- 
roren** froii the cottonseed meal into nitrate form and iii this v/ey it 



became available to tlie plant* In samples A althou^^h a r;reater pro- 
portion of nitrate nitrogen "v^us applied still the amount of protein 



30 



nitrogen xmj r^reatcr than the non-pro tciii -nitro.jeru iiitrmte re- 
duction occurs vvithin the plant to ''tiaino nitrogen" ViiiicI: consti- 
tute the nitrogen part of the protein noleoule in stoiples J3, there 
are probably three ;;^.roc6Cscs occurring siniultaiioouslyt A decoxa-* 



pos 



it ion of the ''or-anic nitrogen" to '^nitrate nitrogen** in the coil. 



followed by the absorption of "nitrate nitrogen** by the nlant, and 
finall^'- the reduction of this **nitrate nitrogen** to the "amino ni- 
tro^^en" v:lthin the plant ♦ 

Tlie aiiount of potassiuri and calcium absorbed by the t?/o plants, 
as shovm by the annlysis of the leaf^ does not indicate any differ- 
once under these two different conditions of nitro:;on nutrition. 

Potash is very essential where quality production of cigar-loaf 
tobacco is desirable* Under nonnal conditions, nitrocen is absorb- 



ed by the plant r.ore readily than potash* An !J/k ratio o. I, or- 
dinarily is desirable for the c^rowin,:-; of the best quality of cigar- 
leaf tobacco* With the exception of sanples lOlA and 161B1 the N/K 
ratios are far froir. unity 8ho'.>fin|v; that those applications ol nitro- 
gen fertilizer will :;:iv0 an oxcciSi; o.: nitrogen in tiie leaf as comparGd 
to potash under the conditions of tlieso cx?«riiaents* Usually, a 
concentration of Z.Syt total nitroc^n in the mature leaf will suffice 
if the concentration of potassium is In excess of this figure* 
Sonie stable forms of nitrogen, J,uoh as proteins, should B^lvmys bo 
present in the leaf since they contribute to certain qualities with- 
in the leaf itself. If too high a concentration of proteins ie 
present a satisfactory fenr.entation i:;ay be prevented* 



31 



We should oapliRsisc again that no single factor contributes nore 
toward desirable burning; qualiticG of tobacco Vab.ii tuc jr^otassltcn. con- 
tents The faTorablo influence of potaseium on the burning; qualities 
of tobacco ie obtained only when irieasurable quantities of thit: ele- 
ment are in combination v/ith other-soluble organic aeidc, especially 
hydroxy acidi: such as citric and nr.lic aoids5« 



Alkalinity of tljjp Ash 

Tables III and IV nVioy: the analysis of the alkalinity o£ the 
ach of the raturo and cured sanplas* Tha alkalinity of tho sol- 
uble ash of the mature staples did not show any considerable dif- 
ference betv;eon the san.ples v^nioh \7ore treated with the available 
forni of nitrof.en and those which were treated with the uawailable 
foiTTi. Samples 102A a::/ 1021^ were subjected to a drouj:ht condition 
and this probably caused the low value • Ko consistent results 
could be obtained for the values of the alkalinity of tlia soluble 
ash» Apparently, the fom of nitrogen applied on a fertilizer 
does not seem to have an effect on the allcalinity of the soltftla 
asht The samples, to *^ioh nitro,j3n is applied in an available 
forrti, care a hi j. her value for the total alkalinity tlian the yiuio- 
les to which nitrocen is applied ii' an unavailable fonnt This is 
naturally due to the greater ano^ont of IJallOj applied to sanples A# 
This will provide for a p;reator amount of sodium ions iWiiich will 
f;:ive an increase in the total alhallnity of the ash# The insol- 
uble alkalinity for sanples A wic also of a higher value than for 
sejuploo 3. This is to be expected since 3a;;.plv3s A javo a higher 



TABLK III 



m:PE2mmT i 



52 



Alkalinities of tho A^h 
of the ilatoi'C ..arple 



Sair.ple 



lOU 

101 .J 

102A 
102B 



Ash 

/a 



17*b7 

IC.hQ 



^al 

Soluble 



r* 



ish 



Insoluble 
M»o./lOO 







49,98 

5G.49 



j 10 oA 
1033 



2S,58 
9.97 



40.58 



41.79 

4o.'i-i; 



, — 



199.5 

18y.43 



179.59 

171.D3 



Total 



814.67 

13G.75 



258.97 



.^ —. .*^.^ ■■*■■»■! 



-4 



TAiiLE IV 



EX:n:itiIMLl,T I 






4U.51 
244.92 



808.19 
201.5 



cy r^ ty r\ f^ 

227.G3 



500*7C 
2G6.70 



Sample 
Im:mber 



lOU 



i:2A 

102B 



lO*^-'- 



OJX 



104A 
104B 



Ash 
/J 



21«96 



4U C. • <^ 1 
«^1 • \J J 



v." • 



18.93 

20.36 



Alkalinitiee of the Ash 
of the Cured oaniple* 



Allca linity of Ash 
Soluble '^ IrisolubTe 



M.e./lOO £';ms» 



20.36 



! A^, .fj 



12.86 

il;.36 



Total 






13.93 



im w' I »i «iP ■ " ■ '■ ^ 



19.28 

19.28 



M.e ,/l 00 r^tas. M.e./lQO gms. 



292,47 



286,39 
272.7 



312.83 



■I III ■ ii » 



299,75 
235.58 



202.46 



«74.37 
242.31 



29(5,39 



194.15 
261.59 






value for tLe total alkaliiiity of tlie ash tl.an samples 3. Tho 
sa^rlee v/lIo! v/ore supplied ^vith nitro -6.11 in an available foni, 
gavo a hi;^hei'- percerita-e of ash* This is due to the greater a- 
mount of mineral ratter isod in HiIb fertilizer as cor^^arod to 
fertilizer B. 

Duriiif^ tliC curiti^; procosti^ tho ash content incroa:;iod in all 
•Itces* Tliic ic accompanied by an increase in the total alkalir.itjr 
of tlie aeh* Samples 104A and 104B did not show any conciderable 
chan^-!;e in the total alkalinity durin;^; curi:xg« Thie is prohaoly 
due to the fact that in thece two saiiples we have a higher percent- 
age of calciun -^reBent, contrihutin;" to tho greater ar.ount of tlie 
total alkali..ity» Probably most of thie calciu:. is ^recent in an 
insoluble form* Galcium i\. this ion. is quite inactive and v;lll 
not under -o any considerable ohan-^e durin^; the curing procosc* 
That the calcium Is h an insoluble and inactive form can be »hr n 
by the hi-h yercrMta-e of oxalic acid ;iven by these tv/o samples ♦ 
W© knov/ thAt the lar^-er the aL.ount 01 nitrogen present, the ir.ore 
proteins are forined and of course the more oxalic, acetic, succinic 
and formic acids are produced in the protein nictaholism^ Oxalic 
acid is ali:o a product of carbohydrate metabolism but to a far great- 
er extent a product of protein roete.bolien. One of the phyeiolo ical 
rolcc of calcium in jlant netabolism is to precipitate or neutralixe 
oxalic acid as v/ell as many of the other acids produced witliin the 
plant or taken from the eoil in surplus quantity* If the calciun 
is present in an insoluble foriri it will not be effective in neutra- 
lizing other or^^onic acids x->roduced by tlie protein metal: olisn, and it 



i 



3^ 



will not take part in t\\e !iOi7::ial reactions of r^lant netabolisn* 



In ali.iofct every ca^o a direct correlation v/ac found to e?List 
b#tiN»en tte ittount of potacsiuic. present ajflul th« allralinity of th© 



•o 



luble ach# It wac i'oand by Ilaley (1Q) that ^lot only the amount 



of rotat;h nreecnt out alco the form of potaeh affects tue alkalinity 
of the ash« He conclidec that vilien tibL# plants received iniiriate of 
potacn, the alhalinity cf the soluble aeh wan less, at: a n.:le, than 
When the sulphate was usedt A profound decrease took place in tht 
alhallnlty of the soluble ash duriiig the curing process* 



Or-anic Acid ia;tabolis::is 



«M«aMft*~ 



Tablet V and YI shov' the analysis of tlie organic acids of th© 
Kiature and cured soaqplat froir the plotG of 1341» 

Api)arently, differcivt applicationc of fonis of nitro^on does 
not seor to affect th.e tot^l eti:er Bcluble organic acidity of the 
xaature saiuplcs. Ko j^reat difference ifi noticeable bet-Aecn sanploc 
A and B. In the other cate, the data presented show that the na- 
t^ire sauples B, which iiad been subjected to an application of an 
unavailable form of nitroi^en (75/^ cottoneccd xeal and 25>i Ka])JO.J, 



igave a hi^^her percunta^^e of oxalic acid in all caces ti.on sarrplec 
A| v/hioh were subject to an application of an availakla form of 
nitrogen (75/^ KaKOj and 25>o cottovicieed neal). 

SairylcG lOlA and 104B are very interesting and unusual sojiiples 
duo b» '^t3 hi^h percentage of oxalic acid. As we knovr, the larcer 
onount of nitrog n presant, the r.ore roteins are formed. Those 
•ampl s cs^ve a hi/j-h value for the nitro^;cn content* Oxalic, acetic, 
succinic a. A fomic acide are products of protein and carbohydrata 
inctr.bolisTn« 



TABLE Y 



BUPKHWBIf 1 



Analysis of t]:e non-'volatile orgifftic acidd 
oxalic, cii^rlc, ana itialic-of the -mture sariplcs» 



•3" r* 



e 



NuT^ber 



T* Total ...tlier l>ol- 
! ublo Or:*aiiic 



lOlA 

loi: 



Acidity 






^__J____j_. 



177,77 

J. J t ' # ^ D 



Oxalic 
Ac id 



„•' 






— 



102A 
"102B 



103A 
103B 



« 



104A 



::ii.24 

207.94 



20C.15 



190.05 



11043 15G.35 



.73 
1.27 



3.42 



\ 



Citric 



Acid 

„ * 









.6i 






5.89 



'^ r, n 



2*99 



"^1 



7,04 j 3.D3 



Malio 
Acid 



of 



-»-i» ,»-■ . I fc ♦ . 



10.10 
10 ♦G3 



v/ tX 



3 



7 






7,98 



MB>* «**-*• l». » i i i w -tfco n wt , |i1i#»ar— 



0.07 
7.37 



' /5 J-' -^ 



'^.^'■el 






■m-^ 



m 



TABLE VI 



Analysis of the non-volatile or^janic acids- 
oxalic, citric and nalic - of the cured sairiples. 



Total Ether 
Sample ! Colulle Cr- 
Number I p'anic Acidity 



— — «ii*> ■ »*. ' 



-> «!■ m% wm I 



X '. X^ i. 



102A 
102B 



\ 

V 



103A 

— »«— I. I 

104A 
1043 



/■ 



.0 ./ 100 frms, 



lD3t99 



Oxalic 
Acid 

OS' 



3,29 



/ 0«^X 

167a3 



■.^ -^-^fc..i I— - ■li«i».iw» n ^» W iii^iaii I— i « 



2C-7.42 



Wfc MWli^W »!• Mil 



164. 2G 
181,45 



5.97- 
5.01 



.m.-^ , 



r 



.02 



-»■- I ■ I 11 !■ Mil »■ 






• 33 
6*90 



Citric 

Ac id 

% 



4*07 



Malio 

Acid 



»■■ v mm I m^* 



3.74 
3,86 



4,64 



7,45 



5.40 
6,32 






•^••45 



4.93 

4,72 



S.7 



r 



5.G7 






- 



36 

Apparently^ t/ie calciiu-i is in fin iueoluble foiTA and is not able 
to neutralize all the acids produced by tho plant ...etabolisru Prob- 
ably tlierc arc li^any other factors tliat should be taken into account* 
We probably have a very intcnsivo carbohydrate and protein metabo- 
lism in these two lanvS, in whioh case nfli Aight have a ^reater a* 
mount of oxalic acid than is nori.-ially produced* Perhaps, the a- 
mount of calciijiri preuont ic not sifficiontly available to be able to 
neutralise t:.o c.-cess of oxalio acid* Of course, v.^e should reir^n- 
ber that the eletj^ont calcror. has other physiolo-i^^al roles* CAL- 
ciuni is supposed to influence the translocation of carbohydrates and 
is also active in influencing, bh© physiolo-ical availability of other 
ions* Calci'xn is also a cor.iponent part of plant structures and com- 
pounds • Oxalio ucid is produced in rreater 'paantitioc tl^an the oth- 
er acids tliat are norr.ially produced in protein and carbohydrate ne- 
tabolism in these tvTO samples* This explains to a certain extent 
the excess of oxalic acid in the plant but ur ought conditions should 
bo coneiderede Due to the intensive metabolism precont in these t\^/o 
plantG an excess of calciuri inay be necessary to fully eutrnlize the 
effect of the de-radation products of protein and carbohydrate ne- 
tabolism as nv-ell as to liave enough calcium available to assune its 
other duties in the plant* Anyway, the case is abnorr'/il* 



Malic acid v-uc produced in greater a:r.ounts in samples B^* 



Ap- 



pture 



ntly, the form of nitrosett •pplied seems to affect the quantity 
of malic acid produced in the leaf* ilow tlie form and amount of a- 



va 



liable nitrogen affect the quantity of malic acid produced is not 



known, probably in an indirect %vuy by influencinp, the rate of for- 



37 



mat ion or tlie malic acid by affecti);;;, one or nioro of the .Any 
factors involved in the cor.iplcx malic acid rotabolisLi of t:..o to- 
bacco ,lant. The citric Mcid did not aaov; a..y consistent olmxisee 
by tlio applloatio.. of the tv^o forms of nitroi;;en» 

thm non-volatile organic acids - pjaiio, citric, anc oxalic - 



cons 



titutc about SO^!! of th« ttrtal other soluble acidity (2?) and 
ly about 20;! of the total ortr;anic solids of the tobacco leaf 
tissue • Tlie bo called "unknoim acls" constitute about 20;! of ti.o 
total ether solu.lo acidity*, ©f coarse, this will vary and will 
dopcnd on th# axao^xnt of oxalic, citric and malic acid formed* Our 
idea is to tr;; to reduce the oxalic acid content, and increase tlio 



citric and rmlic acid in the leaf. In trying to do this the a- 
mwxkt of unhnonn acids vull be reduced. 7/e want to increase the 



a:; 



.ount of citric and nalic acids, expoc tally a combination of po- 



tacsium with the tvvo acids, since it is tliis corabination n'Jiich is 

so conducive to an even and conplete coxabustion of the tobacco leaf 

accou^anied by a li-^ht and coherent coirtposition of tiie ash. 

In General, it ahould be said that although there is a decided 

difference in the cher.iical coinposition of the or,^anic acids when 

the two different amounts of available nitro^jen are added, the dif- 

forences are not of such a jreat nature as to allow for Yory defi- 

•• 

nite ot«elusior:S. 

Gh a^ijos of the organic acids durinr cur in ^ t 

During the curing process, apparently the total other soluble 
orranic acidity underwent no sicnifioant ohanre. The oxalic acid 



8i 



-1 * ."» 



d 



TiOi seem to U^/e a r/jB-jor art ir, the "curing procccc" 



Th© 



paraiiount changes taking place Curing curing arc the increase in 
citric acid and the corresponding; decrease in malic acid* These 
olian--e6 confim the finc.'.nrs of Vickcjry and Pucher (5C) and other 
Saf^ttiga tor c (S). Three poGcibilitiea art given in the litera- 
ture for the pronour-nccd iucrease in citric acid durin- c-.ringt (l) 
it riav hcYc ari&en ac a by-nroduct of the dcairdnation of Mdno a- 
olds I it rr-ay have ooir.e fror;i the deoonpoeition of carbohydrates | or 
it Liay liave been co.iverted f roia uxi\TL0^»\r. acids ^rocursorG. btill, 
thore liat; been nroposed a reasonable hypct].oci3 for tl.e fortaation 
of citric aciC frora malic acid by the mechaniem of Knoop and Lartius 
(23a) • Vickcry and I\i-her (49), -^/lile studyin- the chemical chan^OG 
that occur in tobacco leaves during culture in li^ht and in darkness, 
showed that there T.-as a considerable decrease in the nalic acid con- 
tent and an increase in the citric acid content \\hen the loaves were 
cultured in v/ater solution in the darL» 

It ooems improbable tlmt tiie deoonposition products of the pro- 
tein xuetabolisTTi played an iiiiportant jjart in the fonaation of citrio 



ac 



id. That the citric acid rfxlght ]kave been foniod fron the decon- 



posttion product of carbohydrate rietabolish. ^eons reasonable fror.. a 
chemical point oi view. It hat. lon^, been loiown tl»t oarbohydrat#« 



can 



be converted into citric acid by nolds and the recent experiiients 



of Clirzaszcz and Turkow (Ga) are o: significance in this connection. 
Althou,:;h the view that citric acid forrued during curing; originates 
from carbohydrates Ijblq irtuch to rocorsnend it, an equally strong argu- 
ment can be advan^^ed for au entirely different origin of this acid. 



r 

i 



39 






\.orI: of Vickcry and Pucher (52) Jsho^cC tLai tl-e ajiantiby of 



citric ticld v/hioh apncarea durinr curii;r-^ is the 2:>av.:e order oi' niar: 



nitttidi us th© uiiimo'wn acids tl^t disap^. cared. Ko evidence of this 
WIS found in this i^ vostigaticiu In fact, there was no consistent 
change w^ith rospcct to tlie ijinlmov/a acids durin- the curing process. 
In some cases there was an incrof^M of thjtM acids during curing 
and in other cases there rmc a decrease. If it coulc! be eliovm that 
these ^'unknown acics" could be converted into citric acid by natural 
proO«»6eL, this t1«w of the origin of the citric acid will receive 
Miterial support. If h.ov;e/or, theee acids turn out to be lar :oly 
substances of a totally different t;y^ tiie idea of carbohydrate origin 
for the citric acid will becoiTie r.ioro probabl . 

No £;rcat and no consistent difference was found bet-^^.TDon the 
cher.ical Go^/:'03iton of citric, oxalic fiiid umlic acid between the 
cured samples A and 2\s . Apparently, the amount of available ni- 
trof.en appled as a fcrt.Lll.;e2- does not influence the organic acid 
Kietabolism during the curing procesa. 

By the application of tliis particular fortlliser in which ni- 
troj-^en Vi?as applied in two different proportions, vm Were able to ob- 
tain a higher peroonox^e of citric acid in tlio cured leaf than in 
preceding years* The inalic acid content Vw^at^ lower, while the oxalic 
acid content for the 1041 crop vvac much higher than for the preced- 
ing ye: r:;. (Block (8) found that the better quality cured tobacco 



WBl 



8 found to have a , roater amount of citric acid whereas t^e quan- 



tities of Sialic and citric acide tf«p© approxir,iately th« Murie# So 

v/e chould try by different fertiliser practices to increase the citric 



40 



Itfid »alic acidc contents, aiid reduce the anoujrb of oxalic acid in 
the leaf* With rs-ard to the ^mt^-irc leaves Block (o) foioiid that 



the bettor :rade o. 



tobacco v;aE charaoterizod by a lower content 



of citric and oxalic acids than tols found in the poorer ^Tades, 
In the mature saniplcs for the 1941 crop a higher parcentare of cit- 
ric and oxalic acid vmr, fov.aid ai3 compai^ed to previous years, and a 
lnjfmr percentage of ralio acid. 

The problem of dctrrrd.nin;- the effect of a definite application 
of a fertilisior o;i the cho;..ical co-ipocition of the loaf of a ^lant 
is cor.plicatcd b; the exlstonce of corcr(\l r^odifyini": factors. One 
of the rr.ost important factors to be conridered in a stucy of this 
ca^e is the clmractcr of the season, particularly the amount and 
distribution of rainfall. The cliaracter of the vfoathor during the 
growing and curin^^ season, ha& an ii^portai t bearing on the results 
obtained for any particular year* Thli is particularly true in 
case of fcfctilizor experi>aents, although it applies sonewhat to all 



field experiments* In case of the trtacco plant the character 



of 



the weather Liodifies not only the physical cluaracterictics of the 
tobacco leaf, but also affects the avi il^bilit;/ of the fertilizer 
which in turn affect the composition of the leaf. 



fkBlE VII 



EXPEIlUffiKT II 



Data shov/ing how the saraples 
'or Experiment II wBre treated 



41 






Sample 1 - Bo:c 1- 



Muixoa Tobacco 



Troatnent 4-3-12 



Kreidor Seed 






II n I > I ■Mimi 



Pemonted SamDlcs 



<— 111 If II 1—1 >.iiwin^imw 



Sample 14 - Bex S 

Liurmria Tobacco 
Troatment 4-o-12 
Krelder and Se"warr Seed 



• Ml 



Sample 8 " 3ox 4-5 
Street Tobacco 

Treatment 3-6-12 



I M 



ml II !■ !■ 



SaL-plo 1- Bo:: 1 (untreated) 
MmnLja ToLacco 
Troa-tanent 4-3-12 
Kroider i>oed 



S^.plc 2 - Box 2 (Yeai)t 

treated) 

IfumKia Tobacco 

Treatnent 4-3-12 
Kroider Leed 

Sannle 14 - Box 3 (veast 

treated) 
Mumrna Tobacco 
Treatment 4-3-12 
ICreider and Sewarr Seed 



Sample 8 - Box 4 (untreated) 

Street Tobacco 
Treatment; 3-6-12 

Sarriple 9 - box 5 (yeast 

treated) 
Street Tobacco 
Treatrr.ent S-6-12 



««bi««*M><«»>rtM» «•»•< 



mtmmmm''mmmmmmmm^ 



TibU: VIII 



42 



Nunibor 



EXPliJRIIO^T II 

Analysis of tlie al-alinitic b of the at;h 

of the currd saiiiplos 



14 



8 &.. 9 



'* ' * 4- 



8#3G 






7.o0 






Alka linity of Ash 
3 o 1 ab Ic In d^olubXe ^ 






• 44 
19»99 



LI*e,/lOO i^e./lOO 



18»9fi 



54.70 



I ! 



Total 
M*e./lOO 

-J^}:^* ....J.. J2i?i:L„ 

337.82 









321. DC 






I 

1:^0 J« v>o i <::^ I oO 



II PI* HI— ^ m I iMi -t .«>.'-- 



I (J w 



TA.3LE IX 



Sample 
Nuinber 



14 



EXPBRIIEKT II 

Anf.lysis of the alkaliidtiec of the Asl; 
of trio femexite . Liv.rlcs 



iNMfc «ri»MM*Ma 



Moisture Ash 



% 



9.16 



% 



I * m M>pji» » i j « wr ^iti^ pi^^ 



25.95 



0.15 



9.14 



8 



9.12 



■^ ■ ■ » iw - 



9.17 



25.51 



22,52 



23.01 



20. CG 



Alkalinity of Aeh 
Insoluble 1 



i-oluble 

M*c,/lOO b.o./lOO 



61.02 



82.5 



o7.5 



25.07 



21.43 



305^.22 
279. S8 



Total 

:.e./ioo 



366.24 



.- —- —- * 



274.76 



313.92 



261.97 






3C1.88 



342.26 



;'^,n on 



n o '7 / 



43 



EX]'i"iIIvni"T II 



Alkalinities oi' tlie Ashi 



mimm " >m \\wm\f < m 



Table VII ahowe Lov: the fiain;>lus used in the second eicperiiaont 
wore treated* The yeast treatij^xib produced tobacco wiiicli seeined 






snore conpletely formented, ai; j id^od by appoarance and snokiii 
qualities bLam the untreatoc G<'.r.plos» 

Tables YIII and IX siioi/ ti:e aiicil/ais of the soluble and in- 
soluble ash Ox the curcu saBipl.e. boecialiy striking' is the hij^h 
value of the solubl at.!: of 8anpl#i I and XJY. This ie sonething 
wx) dave do '.ook forward cinco there Locr.s -o e.'rii^t a correlation 
betv.oon the soluble alLalinity of the ash of the soiuples and their 
burninr qualities* The quantity of alhalinity of the soluble ash 
is affected by t!ie fom and the amount of potash applied as a fer- 
tilizer and to a laarked e:cfcont by the soason# Ihe usual rai^-^^e ot 
the alkalinity of the soluble ash of Pennsylvariia d^iar-lcaf to- 
bacco is aro:;.nd 40-dO . .illi-equiv^lents per 100 r^rar.s of sarr.le. 
A& v^-o see there is a marked difference between the value obtained 
for saiTpJec I and the usual ron^e of the soluble aldalirity of the 
ash of Peniisylvania ci[^ar-loaf tobacco* 

The value obtained for the total alkalinity of the ash for the 
flifferent saxriples is about normal; v;ith the exception of sample VIII 
lAich cave a lonrer value* The soluble alkalinity of the ash vdiich 
gave a lower value* The soluble alkalinity of tho ach for tiiii 
soEiple v/as lov/* 



u 



On* to the uaucually hifjh vtxlue of t:.c soluble allialinivV oi 



i. 



tho asl: of samples I and XIV, a lovrer value tl-an normal ^»m£ 



OD 



tained lor tde insoluble clLnliiiity of tl.e aslu Of course, tliis 
is (Icsirowble to a certain extent* 

D-arin- tr.e ferr.entation process an incr^ate in the ash con- 
t«nt tm» found in all cauec* This increase in ash ehoitld produce 
a correEpondinc; increase in tlie alkalinity of the tissue • An in- 
oreace in ths alkalinity of the tissue is usually acooirpanied by a 



cjrroepondiiig decr«a«d in the ethsr soluble organic acidity i: 



"1 or 



dor to inaintain the ncrjjxl conetaiit level of pll necestuiry for all 
livin;;:: tissues* This v.ue found to be tho case* McPIinstry (27) 



in his work oonoludsd thitt th 



e norrrial form^ntation is ?hmys i^c- 



coTT.panied by a narked pro^reosive doorcase in the total ether sol- 
uble or.'^anic acidity and a corresponding rlco in the alkalinity 
of the tiesuc* 

Apparently, tho yeaet treated saijplee gave a lo'v/er ash oon- 
tent than the corrc si or.din^ untroated sanplcs* Of course, tlie 
yeact treated eaixiples gave a lower value for the total alkalinity 
of tho ash# Ho ooncistent reculbs vroro fou3id in regard iNI tht 
soluble al -ilLnity of the ash* In two cases, (noiiiplos I and XIV) 
tOki increase in the soluble aUoiliiiity of the aeh vv.g found when 
the sa^^ploc wore yeast treated* The untreated sanplcs showed a 
dooroase in the value of the solubel alkalinity from the corres- 
ponding cured saraplec. The untreated sample 7III bohlvcd tlie 
sajTie way, but the corresponding, yeast treated sample IX failed 
to give an increase in the soluble alkalinity of the ash* Also^ 



45 



tlx total allrialinity of the yoaet treated sairiDle 1\ fallo 






an increase in tho total alkalinity* Tiiic iij rather unusual sine 







a "nonml** fermentation ;.roccsG is alv.u:c .-ceo -^nniccl hv 



an increase 



in the total alkalinity* Still, tho untreated sample YI"^! bokavod 
norr-filly as well as the otherc untreated and yeat^t treated E£L->lc:i. 



The insoluble alkalinity increar:©c! uuriu;- fernontatl 



on m every 



ca::c, the i:xreat;e sho^Oi by the untreated ca^iples boin^_; of a higher 
value than tiie corresponding yeast treated eanples. This is to be 



ox^.ecued since the yeast troatrient apparently caused a greater in- 
crease in the coluhle alkalinity which means less inGoluble alkalin- 
ity of tixe ash* 



Or;;;anic Acids Metabolicrit 

Tables X and XI chow tho analysis of the total other soluble 
organic acidity and of the non- volatile or'TOnic acids - oxalic. 
citric and rialie - of the cured and ferr.iented samplec. In the 
first case, a pronounce.^ decrease in tlie total ether 6ol Ic or- 
ganic acidity mo found to accompany trie fermentation of the son- 
plec stuc'led. Apparently, the yeast treated saraplee cliowed a 
greater decrease in tho ether soluble orp-onic acidity. The de- 
crease in the ether soluble organic acidity is in accordance vrith 
the total alkalinit;' increase chewed by all tho couples* 

HcKinetry (27) arrived at the sajLae concluelon* Hie data and 
the data precentod in this invoctigation in ro3ard to the change in 



the total ether soluble organic acids durinf; forraentation are at 
iance v/itli the cone lue ions drawn by Pucher^ Vickery and Yl 



(3C). 



TABLE X 



Rf.oermont II 



AttftlysiE of the non-volatile organic acids- 
oxalic, ciLrio and malic- of the cured samples 



46 



Sar.ple 
Nujuber 






14 



8 (S: » 



Total Ether 
Soluble Or- 
ganic Acidity 
2»ev/lOO :;ia£# 

I 1 -r . -^.-^^^ — r J» » II ill* 



Oxalic 

Acid 
-^ 

/o 



Citric 
Acid 

% 






9*07 



mm»iB<yj<',«w>M--«»Pi«whiMMwi— ■^'Wf^' 



277.56 



li<ii H «. I.. 



269.75 



2,G1 



2.96 



. ,*-.* -■t.wi* ' ■ >#i, . i m i i iMiiiii«i>w ^1 






*7 



.43 



2,61 



—l 



CO 



Ilhtlio 
Acid 






2.81 



4.45 



TA3LE XI 



KKPERB-'HI^T II 



.lysis Ox the non-vola^r.ile organic acids- 
oxalic, citric and loalic- of the f^riaented eamples 



S«apl© 
Uumber 


Total Ether 
Soluble Or- 
gi^nic Acidity 
M#e,/lOC gmSt 


Oxalic 
Acid 


Citric 
Acid 


Malic 
Acid 

c/ 


1 


225.17 


3.37 


0.81 


1.53 1 


t 


230.51 


3.61 


0.51 


1,73 


14 


214.52 


4,03 


0,21 


1,67 


8 


223.30 


4.02 
4.12 


1.57 


0.64 1 


t 


213,18 


1.64 


0.93 










! 



47 



Those v/orl:ers re^ortou tliat the changes in the cor.cG.ntrat.lon of tlie 



organic acid:: of thit leaf duriii'; fo 



tion are of a xr.inor nature • 



Ac McKinstry ©xplaine in his work (27), the conclusions of the Conn- 
••tittUt wwrkers L^ay be applied with propiety to tlie caLe-formentod, 
Connocticut tobacco; this type of leaf is usually subjeot#d to a 
Tery mild and very slow fomentation. But o\ir data dononstrates 
iimirial'ly tfeMi fact tliat tho noafwal bulk fomentation of Pennsyl- 
vania cigar-leaf fcobacco is always accompanied by a promwnced de- 
crease in the orr.anic acl<!lty of the tissue • 

The data obtained in this investigation revealed tho fact that 
cibric and F^lic acid, present irainly in the leaf as the potasaium 
•Altft, Bxo dissinilated to a marked ; ogreo clurin the proce^u? of 
ferinentation. Tliis is in accordance Ydth the work of pWVtiMa in- 
vesti^;ators (27). This is vjiiat to be exDocted since the citric 
and malic acids present in tlio foru* of potassium salts will satis- 
factorily serve as an energy source for the mioroorEanisms 
dated wi;.:i the :iorn)al fomentation process. This lias been proved 
by experiment with pure culture studiasi in vMoL it has been dem- 

* 

onstrated that the potass tur. salts or the ainmonium salts of citric 
and Hialic acids are in a fom readily available as ai: enery source 
for the rpaid proliforatinG mlcifoflora. To this, oxalic acid is 
•n exception since it exists usually in the xon.i of tho insoluble 
calciuiri 8alt# Tlie changes shovired by tho oxalic acid content of 
the leaf tissue durinr the fermentation process is not of a con- 
sistent nature. in general, there is no appreciable chan e in the 



oxalic acid content. The relative inertness of oxalic acid during 



48 



f^nintetioii ic not surprising# It had bMn showi preTiouoly (43) 

tJmt thlB acid occurE rjainly in the fonr. of insoluble oxalatec in 



the tobacco leaf» Furthermoro, 



culture studies uul demon- 



stimted that the oxalate ion -- La eny chemical combination - will 
not serve as an enerr^^y source for any of the microorganisme assoc- 
iated with the noruml fenuentation of oi^ar-leaf tobacco. 

The decrerse in citric acid curinr fenuentation is of a high- 
er value than rralic acid. Smirnov (40) •todying the bc,.avior of 
the non«-volatilo organic acids of sono oriental types of tobacco 
durin : fenrientation reaci ed the sane conclusion# 

Appar#ntly9 there v/ac no great difference in the chemical 00:2- 
position of the non-volatilo orj^a^iic acids betv/een the untreated 
and yeast treated ■•■ipl0«» 

Durinr^ tlxe fermentation it appears as if the yeast treated 
samples give a higher percentage of oxalic acid than the untreated* 
The 8a»i thing appears to be true for liialic acid. The decreawi, 
or rather w.e dissi^nilation of rmlic acid ie riorc on the untreated 
sanplos during the fermentation process. The citric acid did not 
show any coi-sictont results j the ycact treated sanples showing a 
greater decrease in the citric acid content in one case, and a 
SLialler decrease tlian the untreatea in the other case. 

The so-called •'unlmown acids" present in the tobacco loaf ara 
still a niattor oi speculation. They can be calculated by pubrtract- 
ing the sum of the percenta>;;es of tae oxalic, citric, and njilia a^ 
oids from the percentages of the total etlxr soluble organic acidity. 
Ho consistent results could be found in regard to these acids. 



49 



Apparently there is no i^reat change on the value of these aclde 
during feraeutation* Pro- ably t) cce acids can not be utilised 
as a source of energy hy the luiordor^aniems preeent in tlxe fer- 
mentation proceec* Any <tha n^e they undergo durinfj feriaentation 
is probably of a small nature • More work done on these acids 
might reveal very illuninatin^ and intereGtio^ infommtion^ 



TABLE XII 



EXPERILISirr III 



Analysis of two cured samples frora the 1941 crop, 
Silvostrius and Drhrata, ^ro\m under entirely different moisture conditions 



Sample Moisture 

% 



I 

Silvos- 

rius Q«50 



I o- 



i:L,p:urata 



1 5.18 



Total 

Ash 






«**••« 



20.75 



20.80 



Soluble 



Aljaliriity of Ach 

> I IS club le [ Total 



M.e.AoO ' I^.e./lOO 



ns 






29.38 



18.36 



272.39 



.- 



'otal 



l:.c*/ioo ? 



/*» 



Hitro:ren 

p?^t^r 









-♦-^ 



I.IQ 



Protoin 
7^ 



T 






/ 



235.09 1303.4 5 { 2.21 j 1.59 [ 0_ 



1.72 11.47 ..34 
•62 ll.GO 1.13 



o 



51 



EXI^EKI:.':viIT III 



Table XII s:.Dwr> two sa-ples fror tlie crop of 1941 ♦ Tliclr 
only difference uvue that the Ephrat>a sanple ims groYm on a decided- 
ly drought condition. Their fcrtilii^er treatment uus ti;e usual 



4-8-12 • The .SilveBtriuG cample was characterized by its 1 



o\v nxc- 



#tiw content. The analyeii; is prcBented to show how a dr-^ ceas- 
on miglit affect the chemical corr.position of the tobacco leaf. 

The irioisture recuircnontc of bhe tobacco :>lant arc hi£;h be- 
cause of its rroat leaf area and the desirability of rapid -rorth. 
Any factor tending to lov/cr the noieture requirement of the to- 
bacco riant affects ti^e yield and quality of the tobacco loaf. 



From the analysis of the two samplec it appears that th 



e one 



which Iiad been subjected to a dry spell £;avo a lovrer value for t 



.0 



soluble alkalinity of the ash. Tiie form and aiTiount of potash, and 
to a certain extent the season, have a jreat deal to do with the 
total quantity of the soluble alkalinity of the ach. Of course, 
the rioisture content of both samples wtrt quite different. This 
is to be expected for the differences in nioisture conditions upon 
which both plants 2;^0"*^« 

The total ash content apparently does not seem to be affected 
by a dry season* The same appears to be true for the total alka- 
linity of the ash. The total nitrogen is lov^ior in the Epiirata sai..- 
pie, but the protein nitro£;en value is x.i^her in this sample. The 
non-protein nitro-en is lowier in the Ephrata sample, that is the 
eaL.plo subjectec to a dry spell. This is probably due to the smll 



52 



I 



I 



teiount of v;l^t0r of tra iclocation in this plar^t^ There ic robably 
»ot enourh vmtor L\ Vr.lz tobacco loiit to accovtiplish a conpleto trans- 
location of the nitratec fro:.: the coll to the , la.it and ri:rially to he 
deposited in the leaf. The lotif whan analysed Imi: to givo a higher 
percentage of protein nitro<^en sir>ce it is lov/ in non-protein nitro- 
gen« 

Tho tobacco plant is one of the plantc w;iich ic aifectod most by 
•aj" vairation in clisaate and weather. Seasonal variations in weather 
will nat irally affect the quality of tLc leaf, just as re, :lonal dif- 
ferences in oliriate affect both tjrpe and qu«ility* Uaually, too low 
tcnperaturds laay prevent tho full ripeness of the loaf neccssai-y for 
the beet quality. Hi|:h teraperaturer, reduce tlie water content of the 
plant and favor etron^, aroina and thickenin- of the leaf. Optiiiun 
moisture conditions, partial shade, and absence of v/ind favorc thi 
production of a large, broad, thin fine-veined, open textured, elas- 
tic, li-ht colored, bright leaf with lov; nicotine content. Lece 
favorable !.ioisturo conditions mean a simllcr, narrower, denser, heav- 
ier, thicker-Yoined, darker colored, slower bumin- leaf v/ith more 
nicotine, ctronger aro.na - cualitios desired in different dec^rees in 
cigar filler, oi^parottes, pipe smokini^, chowin<:: and snuff tobaccos. 
Hoeearch in recent years lias Qhox^-n how to L.odify noistnre con- 
ditions to oouQ extent by fertilizer practices and the u«e of the 

right kind of organic niattcr in tlie soil. The methods of topping 

• > 
has also been found to have Gome effect on tiie moisture relauxons. 

It hac been found in research v/ork in recent years tiiat a heavy po- 
tash fertilization and the use of very lirrrited quantities ol c],lor- 



S3 



ides in the rertillzcr are both effoctivc i::. increasing drought re- 
sistance, Certax.-L weeds and crop ^lantc :>recedi:i tobac -o ii; the 
rotation are decidedly beneficial, vdxile others arc deiinitcly in- 
iurious in increasing drought resistance. 

So far a^ our experience shows^ froi.i the standpoint of quality 
of -roduot, touacoo i^ rci-rkahly seniitivo to its ouvironinent| and 
iOm Mftjor problcre of the -ro^:or is to control the environ: cnt of the 
plant; sonethin^ wo can do only to a very limited extent. 



mm 



TAIiLL XIII 



EXPERIMENT IV 



Analysis of tie solTible, insolublo, and total allailinity 

of fenaente:. samples of the 1941 croo 
Analysis of the stoam volatile orp:anic acids is also sxiown 
The X saiuple is taken frox:. the comn.ercial narket. 




A represents a sar.ple taken fror.; the Pennsylvania liiarket. 






65 



KXPERIMEIiT JV 



Table XIII •hows the analyiio of the soluble and ineoluble 
alkalinity of the ash of forzr.ented aaiapUB of toLaoco of the 1341 
crop. The £ saiuple ws talcen fro-a the coimr.eroial jirket anJ con- 
pared to the Bajtploe froir the 1941 crop. 

The ralue obtained for the alkalLnity of the solullu adi rep- 
resents about the averaf* value for Pennsylvania fcnucnted cir;ar- 

The oiear SMpltt represented is an excaptionally 
tiood £;rade of tobacco fror, the Pennsylmnia market. Its hi h 
value for the alkalinity of the soluble ash indicator- itt -ood 
bumin-;: ytuilitios. 



leaf tobacco. 



The 



lysis for the steaii volatile organic acids for these 



3ar;:ples is alco shovm. The extreme high value of the cigar sarr^ple 
as corapared to tho other sarcples should be noticed, JicKinstry (27) 
showed that during the early sta-et- of the fernientation proc.cs 
there is a pronounced increase in the volatile organic acidity. 
This initial increase is followed by a significant decrease during 
the reminder of the process. The origin of tho volatile or-anic 
acids formed during the initial period? of fermentation, us well a* 
the underlying factors involved in their subsequent disappearance 
are still lur^-ely matters of speculation, i^any of the volatile or- 
Caiiic acids furnish ideal energy sources for the ...icroorganisn. as- 
sociated with the ferr;..entatior. of tobacco. The possibility that 
this volatile organic acids, produced as the result of trancfoniuitions 
of the more comple.. leaf constituents, are in turn utilized in t:.e 






energy me-Uibolisr. of the ruioroor^txnisins is an attractive hyootiiesis. 



Today the subject is a r^tic^r ox £>puoala oioxi. 



57 



BVUi'.ktti 



1. Under conditlone of Experiment I, in which nitro en 



was 



made tlxe only variable, there was no appreciable difieroucc iii. the 
amount of nitrogen (total, protein, non-proteiiO absorbed r^^ tiLowii 



by the chemical cori:position of the leaf bety/een sav:^)leL 



s A ana ij# 



2. The amount of potaseimu and calcium absorbed by the two 
plants under the two different applic /.tions of nitro^^on ferbili- 
•ation (75;;J mm^^ and 25% cottonseod liieal for sainples j. and 75^ 
cottonseed neal and 25;^ Na^Oj for samples J]) mxs apparently the 
same* 

5. samples A, to ^whioh nit o-en was applied in a roro readily 
available form, gave a hi.^her peroentar;e of ash, a hij^her value for 



the total alkalinity and the altatlinity of the incoluble ach tl. 



an 



the correspondinr; ^ sanples, to which nitrosen was applied in a lets 
a'fmilAble foriii. The alkalinity of the tolable ash was found to be 
about the same in both cases* 

During the curing process we have €tn apparent increase in 
the ash content, and increase in the total alkalinity of the ash \vlth 
a correepondini; decrease in the ether-soluble ore;anio acidity; and 
an increase in the alkalinity of the insoluble ash. 

4. With the use of these fertilizers, a higher value lor oxo.lic 
and citric acid and a lower value for rrialic acid was obtained than 
for preceding years. 

Application 6f fertilizer B^ {^ave a hip;her percentap;e of 
oxalic and raalic acid, v/hile no great difference for tho values for 






citric acid was obtained as compared to tlie application of fort ill- 



t#r A» 



During the curing process the ojcalio acid did not undergo 
Miy ^reat chan -e, the citric acid increased considerably while the 
malic acid underwent a decrease in value ♦ 

6. The fermented aaiiples are characterized by a hi-her per- 
centa(5,e of ash content axid by an increase in the total, ana the al- 
kalinity of the eolublo and inroluble ash. 



The yeaet ferii.ented sanplac did not show as hi'-h incre 



as# 



in the total and inBoluble alkalinity of the ash as the untreated 
samples* 

6# The foniientation was accompanied by a decrease in the total 
ether soluble or-onic acidity* A profound diminution of iiAlic and 
citric acid accor. panied the ferr.ientation, wl.ile the oxalic acid did 
not under-o any appreciable alteration. The yeast ferruented samp- 
les gave a higher value for oxalic and malic acid, wliile the citric 
acid did not show any great difference butween the untreated and 
yeast treated samples* 

7, ,vhen a tobacco plant grows on drought conditions, the mois- 
ture content ol the leaf, the value lor the soluble alkalinity of 
the ash and the total and non-protein nitro-en are lower than wyien 
the plant is -rown under optinuiti moisture conditions. The potassium 
content was found hi-herj while the asli content and the total alka- 
linity of the ash did not show any great difference* 



69 



BIBI^IOOHAPHy 



1. Anderson, P.J., Swanback, T.?\., and Street, O.K., Conn, A-r* 
Expt* Sta., Bull. 3B0 (1996). 

^* Aito c iatlon of Offioi al Agricultural Cheinistg t .Methods of An* 
alyt:!^^ ItK edition, Ci;;3lTT)ae 25 • ^ " ' 

8. Ibidt pa-^e 25 

4. Ibidt pa£;e 125 

5. Ibidt .pa^o 336. 

6m Behrens, J., I^indw. Ver. Sta., 431, 27-30 (1304). 



?• Bennot and Clark, T«A», 



phi'tolor.ist, 32, 37,121,1^7, (1933) 



8# Block, S.S., M.S. Thee is. The Pennsylvania State Collo-e (1941) 
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9* Clark, 11. E., Plant Hiysiol., £l, 5 (195G) 

10. Franzen, lU, ar.d Ltern, E.Z., Physiol. Che^ . li:^^, 270,(1921) 

11. Franaen, i:., and ilelwert, F.L., PliyBiol. Chom. , 122, 46, (1922) 

12. Franzen, !!., and Keyssnor, E., Biochein. Z., IS., 1 r, (1923) 

— ■■ 11^ a m 

13. PYanzen, H#, and lielv/ert, F., Biochem. Z., 131 , 3S4, (1923) 

14. Franzen, !!•, and Ilelwert, ?., Biochem. Z., 13G , 291, (1923) 

15. Franzen, il., and Oeterta^-, R., Biochem. Z., 13'^, 327, (1923) 

16. Frear, ,*illiai:., Pennsylvania wts.te Collo^^e Agr. Expt. bta.. 
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17. Garner, iAi.V»., U.S. Dept. Agr. Bur. Plant ind.. Bull. 106, (1907) 

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20. Haley, D.E., Olson, Otto^ Plant Pliysiolocy, ^, 177-132, (1D31) 



60 



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23. Kisslin^, iU, Ilandbuch der TaMlcundec, des Tabakbaues, ujkI do 

. TaLalcfacricatio/, i-.-aitioa 7, xcrlin, [iDc^) 

23a. Kaoop, F., and ^lartiiu, 0., Z. Phyeiol. Chci^. 242, 1, (l^3u) 

24* Kollor, J.r;,c., L^nJY/irtscLartlicLer, U. Tccchniecher, r^ezie- 
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25. 
26. 



Trudy 



27, 



2c;. 

29. 

30. 



Kmu, *:U, Arch. Chc3rn>, L-ikrpekop, 7^, 235, (l.ld) 

Kurchato-v, P.W., Lupinovich, T.S., and Bu«yuk, I!.Y., 
Veloness ^el'^Lo Klioz Lir.t,, 5, Gl, (1936) 

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NaSMt, K.*,, W.L,, T;io8is, The fennBylvania iitatc Colle -e, (1;!27) 

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Olson, Otto, litley, D.2., Pcnn. State College Apr. Expt. ^te , 
Bull. 240, (1J29) ' 



31. isurcY, G.M,, Tabak, 8_, 36, (1938) 



32, 



I-uchcT, G.U., Vickery, II.B., and w'akeman, A. J., j. ird. iJri". 
Oh©i.i. (anal, ed.), 6, 140, (1934) 



33. Ib.idt £, 190, (1934) 

34. Ibidt _6, 238, (1954) 

35. Ibidt 13, 244. (1941) 

36. Ibid: riant Physiol. _13, 621, (1930) 

IT. Schloosinc, Th., Compt. Rend. Acad. Sci. (Pr.ria) 50, lo;;y-lO30, 

33. Sotouok, iwA., Chemistry oi' Tobaooo and Manufactured Products 
(in liuBBian) Krasnodar (1930) 

39. Schmuck, A., iiull. Acad. w,ci. U.i..S.a. i>Gr. loi., 05r, (1330) 

40. Sirirnov.A., U.S.S.R. State Inat. Tobacco Investigation. Bull.. 34, 
(1927) , — 



61 



41, Snirnov, A., I,, "The Physiological i:.ioche' ical Pri:ciplec oi 

Tobacco ^urin^: and Feriient^ tio.:" (in distlar.) Kraenodar (lb?33) 

4t# Stahre, L», Nord. Farmazeutik Tids,, £, 141, (1895) 

4S» Street, E»C*, Report for 3.y'3? of the Tobacco Substation at 
Iftindeor, bull, ^.10, (l£37) 

44. SwTBJibe.ck, T.R,, and Anclereon, r»J., Conn. A^^^r. Expt. Sta. Bull., 
355, 359, (1934) 

— H— III! Ill IJ I 

45. ThorAfi, JmJ.g Haley, D.L%, Olson, -tto. P«nn* ^tate College A^r» 
and Bxpt* i;ta# Lull. 3^,1, (1L/3C) 

46. Thomas, J»J,, ?h*D» Ibesis The Penney Ireiiia ^tato Coll.^-;e (1935) 

47. van iienur^len, J«>A», L©.nQf^» Vcr. :.t^« 37^, 409-43C, (1099) 

48t Vickery, K.B., and l\icher, d.f*.. Jour, Biol. Cheni., 90, 637, (1j31) 



^ta*, iiuli. 511, 



49* Ibid: 119, 523, (1931) 

50. Vickery, II. B#^ and Pucher, G.'»U, Conn. Arr. 
(1930) 

51. Ibidi Bull, 528, (1931) 

52. Ibid I Bull. 352 , (1932) 

53. Viokery, H.B., Pucher, d«W«, and Viiakeman, J.A*, Gonii. Agr. 
Focpt. ;:>ta. Bull, 44?., (1940) 

54. VlaAteiirov, A.V., Chemisation i^ocialistic ^^-^^ U.^,S.H. 8, 36, 
(1939) 

55. Ibidt Conpt. Rerid. Aoad. ii^ci. U.S.o.R., 699, (1939) 

56. Ufasner, L.V., Tabak Kultur, Tabak und Zisarrenfabrikation 248-49, 
••einar (1884)