United tages Patent Office
1 2
BR*paRarro" or'*Xl8fff rAL rNsEcrrcr,E
trohn G Megna Bakerdield, Cultf., asdgnor o Blofern
Corporrtion, Wsco, Callt, s corpoftotr of Nevrh
No-Drawkgl Filod Jus 9, 1959' Se* I{o. tI8'999 o filtration or ccparation more diffisuh. For instance, in
2t Clalnts. (Cl. 19--96)
My iuvaation is dirccted to a Dw and uscul proass
for preparing a rnicrobial insecticidc and ls espesielly
soccrned with improvemeats in the inductio of spuula- l0 sharp contrast th*eto, when follwing the teachings of
tioa and e.rys{el formation of thc active insccticidal mate-
ial, all as is hereafter descrlbd i detail.
The micmbiological control ol lnscct has lon ago
bcen uggested end thc potentiai advantoges utliizing ,
sucl control mothods have becn well rccognizod. In
brel thcy may be 8mmed up as follows:
( I ) Thc harmless and non-toxic uature of irpet
pathogenr fcr othor forms of life and, theroforg the
ab'sens of toxis rssidue*.
(2) The uuurually higk dcgroo of speeicity of disdase 20 mentioned siltefful. It has bxn found that au utsually
organismr for their iusect hosts.
(3) The possiblo combincd npplication with organic
rssticides so es t iucreee protccon in an rea, media (oil-frss cot(onsocd cndospcrm ffour), l.67%o carn
(4) Thc easc and inexpensiveness with rrhieh insect _ _ stcep sotidn, with o bufier to maintain thc pfi vithin
patLogena can be producod.
(5) The apparent lack of rcsistanc.c cxhibited by issects.
In thr praotica of rny inventisn, it ls particularly
advantag*oue o use tho rpor+forming bacillu identified
as Bacilht thurlngiewis. Thie spore-forming bacillur u,a - - medium cotaiaing 1.4% beEt nelasser e& 1,2o c,arn
icolate i n. glinsr from disised larvae*of tbe Medi. s0 ipp solids, nd (2) . aqusous'iiquid medium conrain-
tsrransa flour srslh (Ztschr, f. das Gesam, Getreide-
3, 63J0, 191 1). Berlinor dcserlbed the organism
as a grsm positive, psritrichously flagellated spore"forming
rod. On sporulalion, tbe cells of the incct pathogen oo
contain at onE ond a spsre aRd at the other end a diamond-
thnped crystal or asporal boily, A proteinaceous toxin
identical in composition to tho crystalline inelusiu has
boen solated from sporulatcd cultures of Bdcillus
thurlnglensls, (C. L. Hannay and P. Fitu-Iatres, C4D. ,. satl'sfactory. imarly, althoug orn stefl! solids arc
J. Llicrub,, vol. I, pp. 694-710, 19J5.) Other rpore- *" speslIly desirable, in place therecf other materials can
forming bacilli vhich develo, an espor*I body or a
crysfallinc insect pathogeil in th spores Eatr elso be
utilized, ngteworthy among therr being Bacill,w sotto.
(Nature, vol, 173, pp. 545, 546, 1954; Can, I. Microb',
val, 2, pp,111*1U1, 1956.)
Whilc rporuhtion and crystsl formation occur,i.rrcident
to tte growtb o. Baciltttt thttringten$s sn fraclllat sotto
*ud similar insect pathogcn produclng hasilli under cer-
tain sonditions in certaiu fermonfation rnedia, ilero are
rumbers oi difrculties which are encountered in attcn:pt- g6 phases of my inveution, such meals or flours cari bE
ing to utilizg such known procedtrrce fir comrnercial
practice. Th,esc involve, arnong olher, unduly l,ong
Iermentation periods, nadcquatc or unduly alow sporula-
tion cnd cryctal formation, aud related problems in con-
nection with harveting of fbe actirt nect pathoeenic gg incorporated. My paticularly preforred media for the
nratcrial.
\a o
I bave dircovered, aoflg othor tlrings, in accor"Jance
wth my Frssent inventicn, tht, whcn tho fermentson
a
is efrected by meaus of e aforesaid nricroorganisme in
an aquous liquid nutrient medium undcr subnrerged
aerated conditi0ns, iffiporta[t improverrent result whsn
the raid nurricnt medium posossss wlmt may be char-
acteried a* a cortain balance. ,That bsianco consists, in
tlro pa$icularly preferred aspects of rny invention, in the
fermentable corbohydr*te ar<1 tbe nitrogen available fcr
growth of tbe eaid microorg*nismu boing so related to
oach other that thcy aro sxhausted or depleted at approxi"
matcly th $emi timo ulter rporulatian has commenced.
If the nforesald coadition is not eatisfisd, thpn sporula-
tion bagins very slowly and proceeds dt a slow rate rpith ?0 [icnter coataining the thus prepared nutrigrt medium is
lfit result tbaf, beforo an aplreciable fraction of the cell
parentedr*if*::
). 867" YnqL Ye. do (6Yr^ots"J^^
population has sporulatcd, thoe cells which initially
sportrlateil fracture, free rpores and crytals are fororcd,
and omo sporor begin to germinale thereby decreasiug .+tH
o, scaGd Jurywlc')
the total viable opore couut in addition to making tb
).q'{o
ertain unbalaaced conditios of fermenteble carbohy.
c.ot{
0
dratc and aitrogon available for growth in.a giveii autrisnt
rnediurn, not Gven 10% sporulation of the cullure can
bs obtinad wlthin practical fermentation rycle, In
G?'/, slnoP <'d d=*Qtd^')
tho prescrt luventiou, percmlags populalion porulition
ol '15Vo t 959h arr readily cibtained. Moreover, not
infrogueny, llrger crystale are obtined. $tiII anothef /1 , C-,,(n
adventago of tho proset lnvertion reidcs ia thc fait
ls that, bpcsuc tbo fermenttiori period ir rolatively ehort,
CC%
tho poseibiltticr of contamimtiol try ofh+r microorgeinirri
aro pprecibly lcssoned..
With thc fo,eorqS guiding priciplrs in vic'w, numrous
nutrient rneie an readily bc eireIved to mset thd Ior-
satisfactory nutrient thdiurii is a tiquid aqueous mediun'l
0,1"/o e{l
cottaining 1.87o carie or beet molases, 1.49o Pharma-
25 f$o preferrcd rarges s hereafter set forth. Calcium
carboate in au amouilt of 0.19 of tha mcdlum Is cape-
cialty sa{idactory as bufier. Good, bet ot as setis-
do rcYYLoutl^,o'
],\'lo H. c(o' -
f*ctory, resalts are obtaircd with (1) an aqueous liquid L^
iug 0.6% bct rnlassos sud 0,5% coru step solids, in
i,2" /o Corn [*{
each case with 0.17a calcium arbotrate B the buffcr.
Th,o latter two nutrient media gjve excllent sporulatiou s'UdS
but devclorent s apprcciably lcss than in tho cass
-. of tlrecetlaforesaid
particularly preferred nutritnt moum.
Wliile nurnbers of assimilablc carbohydratee can be
ucd su m varioug sugerg, s&ne solasser, bcct rnoln$es
and the likg I havo found trcst molasree to be particularly
$,t
olo
Goa b'r+cc
bs used such as soj'saucc, Sauce Sas* 2C (4. E. Staley
Mfe. eo.) whhh is a hydrolyzed corn p.mduct, arid
atrtolyeed j,eagts, althotrgh witb less satirfactnry retults.
Althug it 'has been found that the inclusiffi in rhe
45
medie of oil frce cottonseed eadaepe.tm our, extmplified
-- by the Phannanedh pmduct, is otstandi[gly effigacioue,
ia placc thercof olher ol free cereal ours or meal
such as eorn oll rneal, sa$wer oil meaf,'soya bean oil
meal and the like can bc used. Also, it thc broader
t.t,,-t
G'c'R
mitted, Io-wever, the utiliztion of the Pharmamedia
or tho like results i increase of sereral fold, for'istance,
frorn 2 to i timer and more, in the cell counte over the t t - O. L/o rnc(,o, >d"e
ernploymeat of mEdia in wnicn said mterial is not *{
oLiS*
O -5"/o c'e(*gt(c?
practco of tliie ivitioq are-tfos aqueous r:oedia con- ff?. \
tainiug from about 0,6{a to atg:ttt 2?o ana especiallv \ J
from i.4tfu to L,9Vo rrrolaces, sdrrentdgeolsly beet mola- \-/
}
.4"( c C*CP
scd; up to about 2?a afld advariageously from 1, r.o l.fo
?barmamedia or imilar oil froc coreal mel or flour;
rom rbout 0.5fo to about l,9%o aud espccially 0.8 to
1,77a aorfi steop sods; aud a cmall amount of a bufier,
most desirably calcium csrboatc, a suitable' auotmt
being about 0.17a.
In carrying out &e ferasotetdn proEodure, the pI{ of
thc outricnt medlum ls qitsted with sodium hydroxidc,
potassirm hydroxide orotlor uitable base to a pH n tho
rangc of 7.2-7.6. Tho rutriont nedhm is then batcb
elerjlized for 20-3S minutes at 121 degreos'Cl Thc ftr-
uol'rmGe\ ]Pt[
c
ioculated with about 59ft of ifs volumc of vegctative
,i
f noonl-v S% dd
InoorrrL
8tt
i.
s\
t
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 s,o?3r?49
78
of thc microorganlsg Bacilius tharngiensit, wbcrcby the 19. The metbod af claim 9, rrhich include the teps
fermentabte crbohydrate and nitrogen arailblc for of addirg a lter aid to -lhe fsmsntation at the cad o{
grorrth pre$eat n iaid ferrrsqtatioa mediuo are e& said fermentatiot operation' cfiecting rapid ffItratiol to
austed it *pprexirtately the same time after the coa- reduce fracturiog of it1cf ce1ls, and dryig tbe solids.
firetrssrpent f sporulation. 5 20. The mcthod of claim 14 which iaeludt thc steps
!4. A fermeda$on methd of preparing microbial i- cf adding a filter aid to tbe femeatation aJ the end of
secticides xhich comprises fermentilg ar aqueous liguid said fermentatiol operaon, e-ffcctiag rapid ltralioa- 1o
ieisrcntauon mcdiuri, nadcr submerged aerated codi- reduce fracturing of iatact cellq and dying tte solid-
Uo"i, iootrioing from about 0.6 to aiout 2? ,molacses, 21. In a fernrentatioa metho4,o! prcparilS oicrobial
qI to about 2?l o.an sil &ee cereal meal or f,our, hon lo insecticiilcs in an aqueous liquid fermetatisn medium
a"bout 0.5 to bout 1.9% com steep solitla ad a bu$cr, containiag fe$Icntable carbohydrate and assimilabtc
rrreaas oI an iseeticidal.prodrrcing spore-formiog uitroqen by neans of an insmticidal-produciag rporc-
microorganism of the,type whieb both sporulate and also forming microorgaaism of tlre type which botb _eporu-
forme ciystals. lates and also forms crystals, lbe ixxovement rrbjch cot-
15. A feraentaoa aethod of preparirg mierobisl iB- 1 sists in carryhg oul the fernettson under submerged
ecticides *hich comprises fermerticg au aqueous liquid aerated csrditiqns t Producs mi{ sporc: aad crystals,
fermeutation medium, under subsrergcd aerated cotrdi- adng a filter aid to the fermeutation at the end o'f said
t;;;rt"i"tg from about S.6 to abot 2$6 molarses, feffflenttia operatioo, efiectirg rapid filtration to re'
frsrr about L?o lo abolrt |J% of an oil &ce cottoa sead duce frac{uriog of intact ccl gernioation of pro$,
endosperrr meal or flour, from about 0.8 to abort Ii?o $) a*d/q autolys and ben dryiug said solids.
coru steep solid, ad a buffer in mannt to instease the 22. Is lerpefialion ssthsd of preparbg naicrobial
pH tCI 7-.2:1.5, by rneans of an inseccidatrpmduciag insccticides by tfiecting sggruleiion and crystal forra-
ipore-formiag microorganism of *be type xtich both tion itr an qx,eous li+gid-fermcntatio mcdum contaiu-
ior,late
- afo ako form crysal. ing ferurentable carbohydrate an assioilablc itrogc
16. A fermentatior Bctbod of prepariag oicrobial in- * by means of tbe ricroorgaism Bacllx thwingensis,
secticic wbich compriscs fcrmmtirg aa agueou* liguid the improvemert which cotri$ in csryiug out the fer-
fermetation medium, uader submorged eerated coldi- rrstatiron for a perod of absuf 14 io about 32 hous
tiots, containirg frorr about 1.4 to about 19% molasse, under submergrd aerated condilioss to pmducc taid
fromabout 19 to about LSqo of sn oil frr cottoased spores and crystl, arldi*g a lter aful to the fermenta"
endosperm meal or flou, from about 0.E to about fifa a0 on at the snd ef said fermentaioo oeration, efecti*g
corn iteep solids, and abou 0.I% calciun carbouate, by rapid Itraon is reduce fratturi?e ofinl?ct coll, euri-
meass of the rnicrocrganissr .Bcllfus thwrngiensl*. nation of sporc& sd/or autolysis, eod ther dryins seid
17. The method of claim 1, which iaelude the teps eolids.
of adding fllter aid to the fermentatioa *!-:S 9l Rs,f{rcnccs c*d is rhe file o[ this pateut
said fermetatioa operation, cffoctirg "t
repid filtration to ""
3
;;ce iriturng *f iotact Ua aaa**,ing tbe soli&. teinbaus: Annual Revie, of MicrobioloSy, vol. II'
18. Thc mstttod of claim 6, whicb iucludc ths slgX,a 1957, pp. 165-168.
of adding a tter aid to the fermert*,tion et" rk end af Faster et a1.: I. Bacl 1949, p9. 6.X946.
said fernrentatho oBeraiion, e,ffcc[iog rapid llration to - - Kyaysi; BacL Reve. 1948, pp, 53, 63 ad 66.
rpduc fracturing o{ ittact cells, ard ilrying the solids'
4