OW 89 NO. 4US-139Oo3902 Cost Acvmt 2 Ob*t- Cli@4 Obliga:ljons Obligations Unliquidated Date Remarks and Imuried Liquidated Balance 9 36(@oo- 9t366.00 1 ?01963 MTLTU Sub Pi@6J*-133 7/29/63 15AUG 2 MYl.@t(,)rtA,NI)UM FOI- C/TSD/FASS ..SIIBJI--CT MKUL'FRA, @@thority granted I -oll@i: -Li Under the at frorn the DCI to the DD/A, and ttie extenrion of -Zliis -tL" I "I Olen-iori)ida, Subl.,r $9,,360 00 of ttio over-all Project L@IKULI'RA biiids 'iave b--czi,. alubprojectla expe-tises a-id Should 2 4125-1390-3502 9 ief, r-. Tgi-eT)- DC i TM/ Corcur., APP!IC;VZD FOR C)BLICTATION OF X-UNDS: I C-@ Chie D SD Ot2@ig I Addronsee 2 T D/ (When F ed Zn) NAME OF ADVANCEE DATE AMOI!!.klC).C( VOUCHER NO. (Fino"ce use on REQUEST FOR ADVANCE A L @q 3 Atl,: @t).s t 1,)Cl co@;. o") OF FUNDS PAYAOLE TO C#A Du' Lo, I Nr. w,-.LTF L S-,c 2 cr- PURPOSE Advance 1,0 MMLT.@A Subprojec"v@ J:l 133, invo:Lc-- 3 for ac-livltzl ",.Iy C/T-.Z) on July 19f-3. AccQunt'A.@-,6 for 'rbl-st adiarce w'i2.11 be in eLceordw,---,, wi4,ii &tAacki-,vrt,/Ieof Fiscoa Amir;,-x. PIS Corwar@,l 'r oorf-e tipat I wjjj fully accou2nt for this advowe f wouci@ere mw re do/ snip unexpended balance #.W oubminsion 0 STATUS OF OUTSTAND I A@VANCES: to the POP*ftioid 2PoAnt stated and A@v the due detel c d be4ow. in ithe event ofmy lei Ufa to a* account and refund any r@x@dod balance'. Zouthoorios ddoeta*n lary to DATE AMOUNT Uft4ccou"TEDOAL&Mf @R PORT N POIN C AT I @, i F t4A CE 2 ON A -ST l@A7110fhi ION 0 MONTH@Y ON THE AST 2 WORKDAY @F EACH T" 77 REOUESTlf4G OFFICER -APPROV 0 FUNDS ADVANR@2D SIONAruptiE DATJE SIGNATURE OF APPROVING OFFICER edge re ot oh* t stated here- under o be used a di to accounted fe as show" above. -1 CIERTIFY FUNDS ARE AVAILABLE AMOUNT OOLISATION REVE 2 PENCE NO. CHARGE ALLOTMENT 40. @E-RTIFIED FOR PAYMENT OR CRE61* 4125-@1390-39W DATE 'AUTHORI@ZED CEPTIFYINO SIGNATURE OF El! VA-.E 0 S@GNATURE AVG 1'4.1 Of, 3 SPACE BELOW FOR EXCLUSIVE USE OF FINANCE DOVISION %RIED OY 7"EVI-Eweo ay VOUCHER NO. 7-12 DESCRIPTION-ALL OT04ER ACCOUNTS 213-33 34-3* 43 5. 47-SZ 58.67 66-70 7'1-80 STA'I ION 40-42 l@r 4PA PIL . 53 54-97 ALLOY. Olt COST AMOUNT 2 Rer@ No. GEN .0, c - u DESCRIPTION- co E OERI ERAL 2 Acc-r. P40. N CA ADVANCE ACCOUNTS 13-27 A, 'r. NO. 33 no. 9 TOTALS !1-60 281 use POEVIOUS EDITIO 0* (3$-A#) VI' Loo oooo9cg6 -ooso9c'6 YUIM $at-I-VES9 2 I-W-unavI palvpln rl aiaa Meplnbliva, suot .lv.VT ,Iqo- @--suo!ivBqqo MID 12@arqo junooov isoo ON 9LI Sidney, A< TS D- F -s -TSD. BB C@v T- 2 3 NA 470UMT OUCHER NO. (Finance use only) 5 October 1962 2 9. R ADVANCE -'e e) PAYABLE TO OF FUNDS REQUEST FO SFF attached. !PURPOSE To 2 r nding MKULTRA Subproject 133 Invoice #2 which activity was approved by C/TSD on 25 July 1962. Accounting to be ln""ac@cordance with the Fiscal Annex Attachrnent C. I agree that I will fu2lly account for i a advance by mu anion of wouchero and ro fund of any unexp dad belong* STATUS OF OUTSTANDING ADVANCES to the reporting pean't,atated and by the &a dot checked 6* *v. In the 0 t of @,w ta4@@wre to oo2 accoup d rofuod Any @RopexpencF@d bjoJ@nce'@ I motho@in. d@duc@@on f@om my amiar t* erfe.@ wattloment. DATE AMOUt4y 2 UNACCOUNTEDDAIANCE REPOtt@IN pboni DUE DATE IERS FINA CF DIVISION HEAD U ON ARRIVAL AT 0 ON OR ABOUT 2 MONTHLY @ ON THE LAST WORK@DAV @OF EACH MONTH 7--7 2 REQUESTING OFFIC-IF? @APPR ED RECEIPT F FUNDS ADVANCED DATE IDA,T E SIGMA PROVIN43 OFFICGR Z acknowledole F* !!a mhownlmbove. I ney tt 0 TE I MOUNT I CERTIFY FUNDS 2ARE AVAILABLE 0 C TSD i C 04AC15ALJIT :FOP PAYMENT CREDIT OBLIGATION REFERE CERTIFIED @w 2 AUTHOR12,Eo CERTIFYING OFFICER sichl ATURE OFi ADVAf4cEE OAT§. ,I SIGNATURE OCT 1962 2 SPACE BELOW FOR EXCLUSIVE USE OF FINANCE DIVISION '!ARED BY REVIEWED by VOUCHER NO. 7-12 .............. DESCRIPTION-ALT. OTHER ACCOUNTS 13-33 34-39 43 432.46 A7-SZ 66-70 71-to -33 OPLIG. SA-57 ALLOY. 00,4:OST our, AMOUNT 28 ST Ar , ON AO-42 F PAY! E 'W/A no. CODE EXPEO90 U P r. NO. GENERAL Acc-r. NO. ..... . ............... PER 6vimoct CA 2 P. P.@o," CODIE i4 0. A ....... @IEDR.ER @2, ON- a r No PRO @1 I $LCCT.@ OPT 13,27 ........ .... r-r 2ID 'yp ACCT. No. ADVA@COFE4'A:CRCOUMTS coo emo@@ x an P. 0. IC L ok s 9 ivy 2 C ltrk,@ T;i= t I i-7 i-i II IL 01 01 9 ALS i CiSs. A*) @5-60 281 use POCYOOUS toolloms. NO. OV Cost-Ac-le@ount Object Clii@@- 8 Obligatiora Obl;g4Cions Urdlquidated Balance Date Remarks-and--Rel4@@@- Incurred Liquidated 24 JLIL S961 2 AUG 13 t@-v, l@@- fr= the DCT tn I)Q of t'n-e 2 zelte,,l to eul)proj,e;ut i; I CEKTIFY- THAT FLINDS APE AVAItAgLk 2 @t-Y.AYCA 10 A@-Iii(DK,ZING D I s t @.-:Vi ,it. C. a; 2 WD/FAES 2 -ISD@/RZ-(l chron, 1-file(finance) X,7 -I--! 12- "'r lgiu MEMORANDUM MRt@: Cbltto ViiiU=-* Division VIA TSD/budget 0ifte" AU6tiii@ZUS-1390039oz 2 et to- attuh*4. Invvico e Paymed should b4 C4L&hi*X!A-Clt U-- 4-4ii@of$9,1000.00- @kk I th, to 2e-e- k-thikk lim th4 $360 00 drawn on a r Branch# @h TSDIZWgot ter-- Y196lik 3. that aaatima tiA@lyu"ui*W for this p2riOI44@6--Ahi iiiij@ should acot b4 Chief T /Res-"Ych Branch Fi F Cli Aftacht4,.. R- EC; Invoice c@@r-tin" I- Distributiow. Orig & a -Addit*#44- Zq mob, CONFIDENTIAL FUNDS POSTING VOUCHER VOUCHER NO. 7-i2 DATE 2. o UCHER NO. 7-12 DESCRIPTION-ALL OTHER ACCOUNTS 13-33 342-39 43 145'. 4 47-52 50-67 66-70 71-60 STATION 40-42 F p OBLOG. 53 S4-57 ALLOT. OR COST DUE .A 2 7@ 303. CODE EXPEND u ky REF. NO. GENERAL ACCT. NO. DATE AMOUNT PE JR f. 2 .1 DESCRIPTION- ........ .......... CODE CA LEDGER 62.67 ..... ADVANCE ACCOUNTS 13-27 F-.O. NO. Poor. no. LIO. CK . two. DEJECT @2t&b' ... YR ACCT. NO. BIT CREDIT ;;;;EC;, ry s CODE ;;;:,No. 'x' CLASS Div. 2 -47 ?14 &4 ZZ& 4 4 EXPLANATION OF ENTRY 2 I TPTA@S Ilse DATE PREPARED V DATE REVNE*ED BY CERTIFIED FOR PAYMENT OR C@IEDIT 5 DATE Si G@T60C OF CCOTTFYING OFrICEft@ FORM 10- 59 606 use PREVIOUS goty....sw (Whofn FilJed In) SUBMITTED By DATE - VOUCHER NO. 2-12 ACCOUNTING BY INDIVIDUAL FOR ADVANCE 2 PERIOD OF ACCOUNTING f? ri OK4 TO 2.965 N07'E: Follow ZD*tfuctiOve O" X&Vorv@ 2 ftn -V Ari-I -- 1. CASH ON HAND BEGINNI2NG OF PERIOD 3C XPENSES THIS PERIODS '10 1, R E DESCRIPTION AMOUNT .UV 2 tE 2. OUTSTANDING ADVANCES BEGINNING OF PERIOD DAT 3. RECEIPTS THIS PEROODI NY-venom - fte -S 2 ECEIPT 'r DESCRIPTION 307-tZ NUMBER DW F TOTAL EXPENSES 2 !EfrV@@ED HEREWITH ICA$" I ICHECK-J- @M*Nev ONO$'.* A VA4CES END OF E 2 vTs7 No N,.7 AD R109 (Attech ]Amtin S. :ASH 0 @#4@000 END! OF PER100 jOR BALANCE 06C AOVANCEE i 4. TOTAL TO ACCOUNT FOR 2 S TOTAL! OLINTED FOR @;i@APPROVEO I & expen4fitures listed hereon and I CERTIFY FUNBS ARE A@AILABLE 2 Cor@ify t a th attee to were inegirted for official p POE PAN ACCOUNT NO@ DAT SIGNAT@ 2 PROVING FFICQ i OBLIG.RC NO. CHA f fidetitioa! @nature,@, that payinent or 2@o . e i I been 1 fec*4v&d@ and t A4 co r 2 go not!, root t trve and S GNAT OF A%)TI4,()RIZONG OFFICER CERTIFIED FO2R PAYMENT OR!CREDIT @IPAYEE DATE OF DATE SIGN TUR9 Of CERTIFYING OFfrl JR 2 himime SPACE BELOW FOR EXCLUSIVE USE OF OFFICE OF FINANCE CSCRIP'rlOI4-ALL OTHER ACCOUNTS 13-33 34-39 40. 42-47 46. so 5i-s4 55-60 67-70 71-80 F-0 - STATION 41 00LIG. Ag CA COST - FAN OBJECT I AMOUNT 2 CLASS 28 ERAL ACCOUNT SYMIOO@ CODE REF, 0 PAY OR GEN ....... C . . . . . . . PER C LEDGER 2 ' : I : 61-66 :60 ----DESCRIPTIO - p CCT. NO ! - 70 C'J 0 A 2 CKI POO. DUE LIC: , CREDIT 13-27 me. Accy ... UN ........ DEONT ADVANCE ACCOUNTS 2 ..................... F ..... CODIE 'r ow:iccy 0 Per. we . DATE 7 2 . . . . . . . . . . DATE REPAREO REVIEWED SY DATE TOTALS: FOR 9 282 1;33 for -tbo-okloa I ftptmber- 19" '30-AV@4 -1965-- - !U ren"u7*xpna"-ja the ammt of-43W.68 for tht gm+. be] 2 eatim-,dm be reuij" by TSD &SI ViU be Me& sy&U&Xa for rrdav In TSDI bY 'as* lotow%OgCoWbolltt tho b&" bass --pavoe"-authoriz- by.tm f Wb" O)TODU44 ad 'rot* I 6-t - Fravicut lkeport 20636.81 2 0636 59.48 132.11- 2 sa @w__ (9 (:50 so Patty cash ..6 T210@ ExPenles----- 2-9636.81 -- ---- 2 Total of-ts 15-519.40 269 77 15 739 17 lndlroct Costs 14'% of 0 1 rect CO@M77--- 2-172.71 Total T@. Fundi frovids.4 18,0002.00 6*1&-nce (writte@n-Off) .)5 I dOr lf-y that servicec, oi- teeil satief"t6riii-rece-lv-pd oxponditures I-OrQ-Iiieurred--or, off 77777777 2 00%obej@@ 1964-- extenstori o T410 ie/@o aok I o,.tnt 'no. )from_ia 2 Word of. 'ov I t t e d7--V4Y -on 12 Oetooer-1964t, e oarr n _on-aoo n TKV# yrie.,@t Tof O St' ,er OL-@)OY in 200,\ ..... . exoendable I*boratory,-It-ems tene@o to r-_ 1) no v a OrL-- oo ase,9ro'q comio October-12,1964 att*ched-please -in retponse7tocyour I f Octaor, f Ind an account-IK"f performed under@--t@,e dir the peridd beginn2in"o@@@t, 1961 and.endin Augus t 3 1, we trust -that the-repor't I"fficti4ii-@@alted for ------ your purposes Please advis@w@ @ts osltlon should be ffade of the p unexpwded-be I*hce -of $307-88. verv bpvtro I I or A" stant to t e@ losure $3,703-27 Assoc.Prof6ss r---- i,i4o.1 i ab. Assistant -J#958.72 i4,362;60 -88321031 ob. Tec2hnician 8. TT2 $up .gilts L .$1,055.11 $729-057--- Phot raphic Servlco--- -47-024 2 09 7 50 -30. -34 Institute Stores 17-56 12-78 2 -25 Tole ho6c Expense II-.50- 7:731 1> 19 75 1.75. Central Duplicating 2 -1.75 Petty Cash 3 60@ 15 1 .40 -31-70 ----- --- '117a- r ry c is 44 4-6@@ .73 2 13:05 -05 13. 5100 9.5 14-55 LL 4.3 2 4.30 0 5.75 -15@ 222.21 -- ----- 121.06 101. 2 z6.89 -26.89 7.40-- 5-. 7 1.70 - 07 239 33---- 100 0 249.4o Fonic"torees -6 '46.3 .36 2 36.85 360 21047 21'.47 14,.50 l4.t5O co 2 2 38 Total Expenses ---sT7"ML-L:!--$1#253.0 4-- Total Direct Costs .$7-090,63 $8,428.77 Indirect Cost 14% of- Direct2- Cost $992-.@69 $2#172-72 --$l$180,03 Total $8,003-32 #692 12 $91000-00, Fu6nds Provided $91000600 0 ----($608-80) $307 (Qvertxpended) Underexpended $916 -68-- .98 -- - -- ---- -- '64 r 19 1 2Ootobe titension T@h col t no. 2 T, 2,,&LA ord the tengro7 'w@a a ttt7e37 o me via teleohone OY 2Octobev.-:@-1964. on I 0- The 6 ent b&lstn,@o 0o@n -t26e-mn t 4.4 II: is $307-88-- Thi a be-used.-fo7-- ent of P-11-blicsitio-h-:coets or a-T)aDer in &GO!.'OMIC GEOL,,-AY-ena---the-rem-a-inde-r e.,idabl@-lqboratory lteme. exp 8 8 Thank Y@"- f or- oermittlng this 4ppow e?Ltension, co Resenr@oh Di-vision Comptroller 13--3 19 Oct4ober ILGA XDIMAN" FM I Chief, 004vrCM*li6i@ Br&Aoh SL4L7= WILTRA -SkAp*ojoot- 133- Attacbed we -.-"titioatiom frcs Mief2 Bi*108104Ll 3ftWb- rer xctnm abtOO404- 133 @im 2 3 ll!M iiii,@4 'lt4 r-ee- -or_-... &A in accoot T604 OCT 7964 6f Distribution.- Origiml 1 4- -Addrois L3 Octobee 12 219( In respcnse--to your 16tt@@r-of-october 80-attoched please find an accountlhg bf@ fund search performed under-tht ditec2i@ion 0 for the period beiinnlng Septerber August 31,1964, We trust-that-the repdr tI ssf f I c Fe-n-t u y purposes Please advise what disoo@tt.ion-s-hould be made -of-the- 4 unexpended bat4h@@@$307.88, Ve ry truly_y tta t to liir enc o'su 9/i/62 salaries UlIL64 Totel@ @@@ASSOC.Professor--- $1.748 1 v9S5 $3,703.27 2 -958- 72 --4t362.60 $11321.3-2 Lab, Assistant --3# Lob. Technl-cl*K 8s8,oa7 858 Tota I T5-.-n T3' $12,882.5T es Expenses 1, 055-92 $1*785.49' 7 ------54- 74- phic Service -47.24 .50 2 -56 Institute Stores 17 -75- -31--- Telephone Expense 196 )if .25 Central Ouplicating 1.75 77- 2 -- - 1.75-- --3,60 15418-0 ig.40 ---- - ---- !I-.70- 31-.70 es2 rar char 114.46 -13-05 -t- 1.05@ 9.55 5.00 -4.30 4.3Q @5-75 54 75 101- 5@@----222@-V 26.89 -26.89 Le ar- i @RE 1 70 7.40 2 -104-07-- -@----239033 ---- 49.40 2 otro c Sto es --.84 Economic Ge,@Ol@Pu 46,36 -46-36 -4 2 2lo47 2 7-- .14.50-- 14 50 Xerox Service 2. 8 Total Expentev777---- 192 J.01 $-2,7362.81 Total Oirect Costs $7.090-63 -428.77 $15 519.40 Indirect Cost 14% of oirect cost i8o- -- $2,172-72 $992.69- $1 3 2 ---$9-000 @oo funds Provided $9,000.00@ 000.00 (Overexpended) Underexpended- $307-88- I certi-fi,- thl-'Ll se-,-,v r, cn VOUCHER NO. (Financt use Only) suam.$TTED By ACCOUNTING BY INDIVIDUAL PERIOD OF ACCOUNTING FOR ADVANCE 2 FROM --7@o- 3. Oot*bw 30 Sovonbw I-W I N079: Follow Yn#tfuctiO"e DISBURSEMENTS 2 RECEIPTS DESCRIPTION AMOUNT 1. CASH ON HAND BEGINNING OF PERIOD s oesc"%PTI2ON 2. DATE TOTAL EXPENSES 3a. F 4. REFUNAEO-MEREW!TN F-T-. A.. cite[* S . cokSI4 ON NO IE@D@OF PRIOD : : @OTA@ C UNTED FOR 2 TOTAL TO ACCOUNT FOR I @@@ he a ondit@r*A iigi#d hereon and I CCRTIFY:FU-4DS ARE AVAILABLE APPROVED t 0 to 0 inc@rr*d for of2ficiati pur'. SIG I CRI fid iai thot paymenlt Or OBLIGATION FTEFERENI-.E 0. C04.APGE TME"T NO. DATE 2 A tuo'a' N he tl;bo n flocei,,Od. and that 2 r AO and co,, f-Pet- uo s I 04ATURIE OF AUTHOR I 2 I MG OFF IOR CIERTIO F I ED FOR PAYOENT OR C ED IT OAT 2 I - I SIGNATURE F CER'TiIF@il$iG OFFICER l@ 10 A 7'E@ SPACO BELOW FOR EXCLUSIVEIUSE OF FINAN E DIVI2SION VOUCHER No. 7.12 REVIE@ED BY PARED BY T77 2 770 71.6* 0 DESCRIPTION - ALL OTHER ACCOUNT 9 40 42 413 45.AS $4.s7 2LLOT OR co AMOUIMY TATION X;,@. F OPLIG 53 ACC@. NO. DATE 13.33 A II.A;L E 2 s @PAY RE t4 GENERAL ...... C.... U PC!. CA LEDGER 3A73..3. coo CODE " .0. I D 2 YR ATCT CK. NO- OXJZCT riEscRIPTION ADVANCE ACCOUNTS 13. 27 , f. . ... too, 0"i ',' ' ''I2@, c f. A 9 s s t D ... iv@33 @ooitct $-D. coo @NO @477770T@LS FORM Is- 61 282 USE PREVIOUS EDITION$. C)ATE TRANSMITTAL SLIP To ROOM NO- :4 2 CPLI A) -r/ ROOM NO. EKION BUILD114G IE?LACES FORM 36 i 3 'o IFE VKHICH kAY BC V-ED. V6"rob e r 1,8 Dear f f i-iid :-our,: irst@ scal@@repor ease f f En2clo5@ p Derio r@ 'the d v y4w ery: ru y. Enclos4ure 10/l/61 14-673 '!ravel i Total CO. 61 -L t P, d TOS/Ch.@iniloal D Datwll@ OIWMKULTRA PROJECT CRYPTO @IKULTRA--# 133----- PRINCIPAL INVESTIGATOR-@.7 n '29 JiiIY71963 DATE RENEW'G During the-gl@@olis ye_ar e.neen rated his efforts 2on the elii@i-d@itioll of f'microbial-actiw mineral sulf-idb@@@@ganese-nodul@@s.----- Bacterial-diss6liiti6n of --As - S,3,- FeS-2-Fe-As - and 3CuoSAS- B - 2- 2 5 was demonstrated-----Tl2i@@ -products were shown to-be-arsenite arsenate, F&t7Fe-fff;76:@ Cu-t@-*ith th7e-fate, of the@Lsulfu"till undetermined-.------ Studiea::@@S2--indicated that Thiob@dillus-sp. w&r@@ble to solubilit"hi@g@if"a2 n pure cultutb@@i Thia@fouft -"at dis-@ solution of-tti'l@i---mindral-@V@er ffktu"1-66nditions.necessitated the presence@@of:protozoa@and futigi.- TFies-e prganisms-were all--- active under-conditions-of copg"_-concentra-tioii-which-vo@uld 2 mally be lethal-@---7 Samples of@l"ng"69t--@dules-"ll-e-cted b the Atlanti-c@@@Cas@a studied to'determine -the -nature of 1:@a-ct-it@-i-ial:Ltransfor- mations. A:@:@@@"ch-e-me f6f--this--adt.iv@2Lt"as determined to be a LfOt!Ug M This latter--io-n--m-u-st b-e----stabilived'and it--i_s-liki@l)@:that--Org"ic by-products-of:Lbacterial-metabolism play tbis-t:@role.. -dUl-t-u--res2 with interest ng ha urnished three charac-tc4r!st or application to the powe r,source-studies- under NIKULTRA-". During--the present year_methods foi@7-tMss culture of min- eral oxidizers@::have::@b-een-dd-V-el@$@@@117ftitnish cell 2 material for-enzymatic-and restiiig--cell-7@@tudies.- Continued work on manganese:Aransformati6n8 is empha--,izing-7environ men------ tal effects.---- KULTRA REVISED s@s e @Rteri@211 D@@t 2 7C E @@T Brarich__P.3 Category----;@.- (foriperly IVC) @ii-crobial transfor- nerc,tis --T Project Title_..,@a2tions of Ili tem Clawfce,,ion.Une 1 p@ l@4E@z,)4ar t S) MKLU.,TRA 133 Cryp+uo Classi -@ica+@,.on--- Uneliss lieci Project Crypto--@@- Bra,ich Proiect No.-------AT 2 P.roj,-,^t Encpinee Contractor. Contract No,- N.A. Task- No.- Type of Contract- @,,I-KUL@fRA 12 Jlijy $9,360.00 Coil t i 2I-lil i 11 @l' Cost- CorrPl!@tio-m. Dat Purpose: To provi-do t--n-e sorvice-S of i@ C', t 0s I'@ lp l@') 11) il L 11 search o----4 2 r;iiiier.,il, transfoi,,,ii.-LL,ioi fy microorganisi-is.-Iii-adli-,-,"-toil--@-0 tiie diree-L ot(-,-itial -"or new oner,,-,y sotirce-systeirs, -'Eh,- 2i,.,,-:ty provicle I)y- produe+.s.useful for waterii-,- k.'@@@ter.-Loratioii sfal.-US: Ctirrent and satislactory. Requireii-ient: Intornall-i., generated in support o@IL 'tio t 2 seek new and be@tter sources of ener-y 'or 'ca@-ory RECEI Receipt is-herebyiacknowled Check 9970 clated 21,37)AUgust 1963,. drawn on the 10 woom, n the amount-6f-$360-.0o.2- Receipt is hereby ackhbwle-dged of _4___-ak I dgte-dl--2 drawn on - th o 3 Aif@-It- pay-----7 able- the amount-of $9!,000.00.1 -- ---- .@N 5 DATF. NA OF ADVANCEE DATE AMOUP VOUCHER NO. (IFiDOnCO U90 Only) ME ICEOUEST FOR ADVANCE PAYABLE TO 2 ROOM BUILDING TELEPHONE EXT. OF FUi4DS puptpo$E Ml 3 Corwaret CZ--!!SAO Mrs that I w2ill full aoc f *him ad@o&Ac by OubmiAgO an of woo-chers md refund of my oo.xpondod bel4wes STATUS OF OUTSTANDING ADVANCES g,*-the Foportint pcoant eta ted And 0,4$te cateked be 2 ret*@DACCOunt modiralund ,one any xp@Oded bolooes, I outh*rixo 'frow my N;ior t DATE AMOUNT ~COLWTIEDB ;tFP*R7i riopost4 CA@f 2 i i I row"-N@E 11 Is O. MCIADOU; 0 F-Rs 2 ON ARR$,VAL AT 0 ST T'lloo O" op''@BOUT MONTHLY - ON TM LAST WOITKOAY 0 @EACH MONTH F UNDS2 REOUESTING OFFICER APPROVED I CCE@ OVANCEP 2 A DATE _ISIISPIATIJFIF ID rE SIONATORE OF O@,'P 0 OIOFICER I ACKnowso re 0 n *Novi$ stAt;wh-ero. 2 rder io i,atoool ami c4wnted far ho," 2 PAIIE A I CERTIFY FUNDS ARE AVAILABLE @Mou 1NY1 OBLIGATION REFERENCE 2 NO. CHARGE ALLOTMENT NO. CERTIFO D FOR PAYMENT OR@CF.EDirr 412 DATE AUTHORIZED CIEFT ifyiNG p7FIC R sirNATUItE Of ADVA DATE SIGNATURE 2 SPACE BELOW FOR EXCLUSIVE USE OF FINANCE DIVISION APED By REVIEWED By VOUCHER NO. 7-12 3A.39 2 47-52 58-67 60-70 04SCRIPTION-ALL OTH@L!R ACCOUNRS 13- 33 STATION 40.4; 43 4S-46 08 54.@7 ALLOT. 00 COST IDUE AMOUNT ,25; 13 F PAY Cl 1110 9 no Eft, GENERAL ACCT., "20. DATE CODE DESCRIPTION. N ADV CA Le R i L I O@- AC)VA E ACCOUNTS 13-Z7 ......... ;'to --- C'T'o,o.T777;;7 1 i t v* AECT 0991 IT CODE' 2 x,oxv I 1 1 t I I I I To,ALs ro 2 4$4-49) 281 maywous EDITION$. -9-.. -SIE P 29 July 1963 133 4125@139Q@3902 4 JUL 0 L I CEPTil-Y TVAT FLINE'@5 AR@, AV,@ILAaLE, Bran t mierobialAranef6rmations Project Title--Ot MA sis---I-- - -- -- ---.- - --- ---Uiielaasified LRe or@lsa iteh-CIA@iificatio ..ProjectCrypto----1294L'M"133- -t--Clas@itlcatiorL --- v a_ua fLi-e-4. ryp 0 -A Branch Project No- @@-Project Fngin Contractor.. NLTEA &ntract No Task of Contract TPA Date Initiat6d--@@L@@ 2 12 July 63 9 0.6o- Continuing cog Completion Date---; PutYm: To providCtli6 sorvic and to supportl"search on - fundamlital:bttbmii transformati6n-by microorganisms. 2 In addition--Wtht-dtro@4@@tial-fd-r--hW-tnergy source_systems, the cotltract-may p;pyw by-li@ts %mful for material.det@erioratiori applications- status: Current ari& s MUIREHENT saing 1 in support of -the requirements to took rAw and-Utttr-sources of energy, fdirvattery application. DRAFT--.- 29-JuV@ M&40RAMM FOR THE REC r*Ject-il33 SUBJF.CT _Continuation of @O@@ubP 1. The- 2se of-n,,ULTRA,,_goproject 133-i;Btto enable-TSD/BB-to------------ utilize the services o Now ____Wcontinues to provide data and@@rials which lend themselve.4Ao-new and-uniqpe-approaches to energy roduction- formation 2(blo-batt@eries). His studit@@--6Wb&Rc mechantOO"f miderai transformations also@- rovide tev--potential:@@s for deterioration of- metals 4@- mompr_wvill function as AM08 The cost of this program2 for one year wi-11 W$9.,000.00 to which must be added-$360-00 which represents a 4$ service-ebarg"ol7oO@retained The -total Cos the ,-360.00@@---che.iges should-be made against A program will@"t-exceed-$9 2 llot- ment No. 4125-139Q@!39p@@- 4. It-is-not antici ted that t4@nt equipment other tban-that --Y6V!red fo- - thi 0 ram. listed in the budget will be r o pr Title to -the eovipment list#d-vill be retained by the i0n lieu of higher over- head rates-.- f6ii--tr"el expenses-vbich-ar-e---re----- 5. ting ill-coELform, to the imbursable ace4apted pi@actitt-Of that organitatio2n.- been cl@@d-CovERTLY andi is unititting and vill-- 6. remain unvittit4@:oUthe ttud--nature of -tbe--tj@onsor. TSD/BiolOgical Brane 5 sD i-.T Attachment: - Distribution: original:bnly Fropo"v ertt itlt@@ I-S 6i@h@963-0 31-Augu3t 1964-.! Submitted -on behdl f -@6f April-1963--- PURPOSE OF STUDY This pro-oosel is a request -for renewal for- the period I -September 1963. 31 August 1964 of tbe-grant-in-aid presently supporting research -of investigation-covored by the ptesent-grant is so-brotd "a The fitild relatively unexplorbd that several additional years of @ieso&rch can -be@ Prof ItOl y spent in unraveling the probltm, -The----ftsul-t"f tho -research ttm-incr,-asing sig* nificance not alone academically but also practically@-"-iti@public health- co2nsor. vation of mineral rosour@x@mineral--oi@pl6Mion, and mineT&174Utution, -Iii-pblic healtbj theso investigations are-providing clues to-tlfe-7Wsiblo--orii[ih@@ control- of acid forruginoug,, and/or acid--cupiifoi@6%is- and/or acid arsenical-ttr2eam poll-u--- tion, deriving from bitt4 ial-tetion on-natural--mineral d@@@ @@rtain geo-- rephical locationst With continued population increaso,.maintenance-of 411-"ter resources in a condition--fwf(@f n cons@u*tion-becomos. more- a2nd more important. The work. Is also providiti"di-tibha,l-clM-t-o, the understanding-of manganese chemistry In public--water s lies,, 01@@ganoo togethe"ith -iron -can cause discoloration of wiltor W@@ manganose-and- iron--aro--de- posited biologically in water mains-an 2s In the mineral resources, the work-und r provides a basis for 6s!ossihg_and ton- trolling any relative lnstability-of@-tiiieral-deposits In relation to-microbes.-- It seems evident that urt-c trolled battor'lal--Icaching could--ConcoivablyF-lead to de. 2 pletion and eventual cooV@@@@@@of-idr,"cpbtits@-71n minCTSI-(;@@10ra- tion,, this work indicates--that sgp@@grbvs of microorganisms known t-o-be-'a.bi-e-to--- livo at the expense-of7t-tp ine-ral may be of- grea-t@ assistance -in -ditt6o-ting 7 tow sites of minoTal-deposits..- Finally@-7tbis work lends additional--suppoi-t-to the practice of using microbes in-mineral-6Aract-ion-i------ 2 SUMMY OF WORK ACCOMPLISHED:UNDER THE-1962-.63fAM-F- (a) Microbia!:Action-on-Wineral Siilfides QUantitatiV* 3tudi"-on the action of the Thiobtcillus,-Forrobatillut@-- group of ba2cteria oii-the-minerals o*@@(At-2S-)---aiiibnopyritd (FtS Ft@@)-t --- and 2 enargite (3Cu SAs S were initttttd,---The t@@@ri"hie-v-ed significkit-solubili- 2 22 s zation of orpiment,,ireleasing arsenic as arsenite and-arsenate,,-- Oxidation of the sulfur portion of V4t-@ia-to be experimentally verifi4-d,- Some spontaneous, nonbiol4@-gital oxidati@6h@6rpiment oceuvrod but- i-t--w-a-s- -only 2 about one-third as extensive--$S-Wllth-b-acteria, The-chemistry of bacterial oxidation of orplunt appears@to-diff,6-r---@ik@@@y fr-o--m-nonbiological-oxidation--as@---ttfl@-@------@- ed by pH changes durih"he- --recesses, With l2i@id- in thirty-five days -but without bacteria it rose-from 3.S',to--S-O-in--that time precise chemical mechanitti@bf-o iron-t-oxi-da-tion -ftmainsLO be worked Quantitat nopyrito showed-roloasd@7- of iron, arsenic, and probably sulf@@2fr---o -m- the Most - of -th4@@Mat@@@- this study were hi"niO"h-esis.- The - ------ results have shown that cbhtrary--to-&-t"tained release orpiment by bacteria-only a limited -iin@ of solb4l6--"nic was from arsenopyrite, , Wis h2a --a Pronounced, releasol@of AtO. The------- reason for limited reltn"f froarsonopyrite is the precipitation of ir on-aTSenitts-@oAd-arsehates after-a-criticali@concontratiort soluble iron and a isenate rsenit2e and a -has been reached, Although some oxidation Of 8TSOnOpyritO occurred In th6-abidnce-of bacteria -its extont was less and its-- chemistry different, because iron artenates and arsenitos were-not precipitated without bacteria, md-tho@@ of th@--aditi6-dtopped fitt3-3-,-S-to-2-,S-ih th-itty@.tik days with bacteria and7id@@-fitd@3 S@@-0 in that time without bacteria. The extensive reprdeipitation-of it6"d arsonic-through @atteiial -action has direct implications with respect to possiblb--@locatioA of-the constituents of arsonopyrit e, The ba torial7@h"oronon-also pc@ses@a problem when arsenic es iii r.Itur --2 - --c and iron are to be extracted togethor-from a natural mihOal--depoSit. Quantitative work@@@terial--bxidation of enarlite-has-given-result@------ resembling those witk-arsenopyritoo- Although the bacteria tire-evide-ntly acting on the mi2neral, a sustained -tolubiliioib"f th@@@@iit- arsenic- is-wot, noted,,- Indeed, in the preseric of b-act6rie-the dfWlVtd arsente,concontration droo after an- initial, rise, the arstnic concentration rise"lig@t---- ly but continually It is- not clear fr66-the----r-c-sult-s- so- fi@t@@wbat- -the f6t"f arsenio or copper action becauso no-reco izabl@--@ipitate was formed. Contaminati" by -bac,,teria-@@@@but not without them. -Thd released iron appears to remain in e 2 to-2-5 with b;ictoria and-ifot@-34,S7!",S without bacteria, pH changes are frowE@3@ In thirty-six days, A@@ise-descri@ti6"f tho 4zhemical-ch4Aos- that undergoes remains to b4@@ked out. Growth on-Leuprpps sulfide 2bj@l@ Thiobacillus.Forrob&di-ius in-.- vestigated chiefl by7"raduate student,-is_shdwing variable-aspq@@@ on different synthetic preparations.-BtdMidl enritht@6@ItUt6" ived from-sev&ral mine 07 2 during the past have h&"er,, effluents __y"r ivon more on those preparations-,-7@@lbe p@qoibility-ari-ses- that consistent action on cuprous sulfide requires the articipation of@@ than one-organism.-----In---this connection, the principal in2vestigator found-p-r-oto--zot@@ funpi accompanying-the Thiobacillus Ferrobacillus it-roup of-bactipria in m-ine-water f The proto- zoa included an a@ooba-and a f lagellato@@ which -dpoara to grow at- the egoose of the bacteria and fuhgi@---The-AWeba worc observed to ingest-3uch t@"isms-, 4 Both kinds of protozoa are tMUSUal-in their tolerance of 800 pP;!@ or and Wwaards of 2000 ppm iron. Ordindiily these-metal concentrations would be expected to be lethal. The flagellate has been repeatedly-subcultured in an-iron:#salts mediiiat In mix2ed culture with the Thiobacill4@@@i6Vk,itlii@tOW::§"actoria. (b) Manganese Nodu -o Puring three-cruises in@the--AQantic cean in June, July, and Augu-stp thitty 2 -four mantmifef 1962i as guest of tfie ous samples and twenty-fd s-of bottom depo-sits--were takeni-@About two-thirds of the manganiferous-samplotaave now--b6en- tested--fV"heir bacterial content -and for the ability of th2ese addition-to the re.spect -ve samples, as previously ddsctibed-by Most of those-samt)los were also anao-- lyzed for total iron and manganese content, showed--tnat manganiferous samples-were byLno-mearis-olikb@@In some cases-ithitDWtt::@iW@@Lby visual ifispection. I"ht@"atts.-however, the differences wete-not macroscopically a parent. Instead,.tho iron-man--g--ane-so-,content, or the bacterial content or the Mn--- p I 2 adsorptive power of th4-ib# -,--sh-ow-ed diff6i6ht@ds-Results-of:@6)(Poiti@Ms--de. signed to test for the tnt@antbi@ent-bf Mn Adsorption by P-antanifetdus material with-- the help of the native bacterial--flora,-could bo--divided-2into four-major-categories. In one, bacterial enhancement-of -M:"sotp:t@@@ with or without prior surface-sterilizationo--In-a--sec--o-nd,--i-t--oc--cu-rred only with surfaco-sterilitation.- and in a third only without it, of Mn-adsorption 2 was not noted under cith-@i-t6hditidn.---Since not all Vttt4tia from-Itn-nodules can-- enhance Mn adsorption--inclusion of -surface-sterilliatibn in-the procedure causes selection of different.-types of--o-r-g--a--u]'LS--M-S--fro-m-th@@--nod7ule-flora-than omission of surface-storilizations -However.-the full explanation fc@r-the variability of bacte. rial enhancement of Mn adsorption is more complicated than that, With a:numbi@i--bf s samples it was noted-that-,Mn-uptako-:@@@fftbqt@inatoTial rosmitod--in a not lots of mwganese from tW-adsorbero--- Th6-@ction accounting for-this P4"p"non-is @the result of a disou2tation-between Mn2l and @tnoi@od will be discussed further below, Ihis reaction was exhibited by about one-.six-th--of tho-samples tested and shows that the properties of these tAtT@"t 6to diffb@@from the- rest, - _it-tidi--thereforo be concluded that chemical and ph sical Mrop2erties of-td6WifttWt--matorialt@ho to p determine the offe@ bacteria in enhancing Mn adsotg@ etivenet"f since all but one of the sampl6"f the 1962-tblltttion came from extinct subma2rine volta. noes (sea mounts),: and- sinco-quite- a few of th@O-@Wles b-ebav"ifferently@@_ earlier samles from other sources-@-it--is-- ted that goograohic-ldtation of manganiferous s&M@et:tayAkff0t--their chemitdls- physicali and biological- -properties. A clue2 to-the-nature-of-hdaiol@kical Mn rolo"e-from manganiferous samples is provided by Aii@exporiii@6nt-which shows adismutatiort-@@on-b6tween M2* and MnO forming MA3, d rb Mn2* on the condition that Mn3+ is 2 SAon-n-c@dules a so 2 0 - stabilized so as to-ptevent it-frdii-undergoing tho reverse-roaction- Pyto@hosrhate was found to be an off&aiV4--stabilizer, Since p@@spbate-does-not occur-in marine environment, protein or amih"tids may serve instead-as-stabili-zors, Nlodule,----------- 2 breakdown can thus be-accomplish@@@@ll"th-sugar-as-the reducing agent of mno .2 or nonbiologicallyll@@a dishitation reaction botwoon-Mn2"' and MnO---- Why--the 2 latter reaction oteur"ith@thly somo-nodules, even though all@htva 2 2 and adsorb Mn2+,, remains to be explained*-- ne abilit"f-Atthrobactor to aid in Mn-addit.ion to nodular@tubstance was shown in pure culture experiments,, FrW similar tests it could be concluded that Vibrio can siudlarly aid-in ktn additiibii but-not Achromobacter. All three org6anisms have boon-f6Wid in-hodul6t,---Arthro-bacter, at h.i"WW-concentrations, causes clumping ai'id heaping of nodular matc-,,rial-, not so at low poptone concentra- tions. A similar phenomenon has been notod in soil . lbo clwping and heaping 6 ------ seems to reduce surfacelar-ea, and-therefore slows Adsorption of Mn. Initial experiments have revealed that ArthTobatter can aid-in gi-ed- sorption by "synthetic"tng the adsorption and.incorporation p@@oss of mn2+ is not permanent.- Ilia Drevi6Utl--y cithd dismutation reaction seemtito come into play eventually, prbbably:Aen-the-bacteria become physiologically inactive at the and of their growth,,cyclo. 114@ohe-tic"-Mn it--@h--06ter-ads2orbent -than nodule-- material, It is postul"7thtt the --i-ron- In- nodule--m-a-t-ori-41 helps - to stab-ili-ze -the- manganese in it and, for as yet unknown to6t6ns, increases-its-@odsorp!.iyo-po@or6- The tore samples of bot2tom d6@Oit-1--collectod lost summer, were examined is-t-Tibutio-n 7@df-bacteria -in as by enrichment for bicterial-@@onto@@. --The d' --tho-3-0---cor-- was not uniform. not-weroitho-btj2@tj@tia ily of t -@ame-kind at different -wessar depths in the same findings are similar to th-ose7cited- WZo Be Work is presently undi@"it:l(@@ the ability of tWC-OT-0@sawles- to adsorb Mn with and without bacterial-Fr2owth,-- Differences In Mn tLdso-rptive,capec@ty seem to f exist among cores or&--tht---b-4ti tests so--fa-r. Such-differences--if'corroborated t by further tests, must rofi@@@Mn--dis-t2-ribution in tht@sea::atrdLtTq"odule di3- tribution and structure,-- REFERENCES 1. de Grys, Ann, Econ. Ce--ol,- 7,---1031-1044-(1962). 31 Martin, JaPes ;@nd-SI-A-, Sci6htt@-SO.- 29@@.47 (1940)5. 4. Zo Bell, C.Eo MARINE-7MrCROBIOLOCT@:@@tca B6tmica Co.g Publ.--1946 91, 7 PROGRAM OF FUTURE WORK (fLa) Mneral Sulftdes Althoue.h it-is the Intention-toLcontinue testi g other mineral Sulfi4ts-- n for susceptibility to-attack ial atten@-- tion will be paid to tho-detalls of iment. arsonop"o, -_and oiti enargite. Quantitative via-thods-have-to-be-developed for determinin@g-th6@pr6portibiis---.----- Of Various forms of a-giv6 element-dissolved and repr@@ated afte-r bi4@terial action. Such inform'ation should-provide clue-"o--the-L@nchanism of 7MiWby::the- bacteria. I'he detailed informati@* on-the- ato of arsonopyiite and onargites when available, will provide information coneetnia"he effect overall proc2ess of bacterial mineral-oxidation,.LUItimately., it is tiopod-to develop- methods of mass culttiro of7"esired-oiganism on-a-particular mineral to-got onouph- cell material for resting cell studies-and eniyma-tic-investivations. (b) "40dulos---, Two aims will-be 2 C@@6 i-@-the further elucidation of onvironmental:@"lAti6Mhi4-t6 tho-p- ro-c-oss -of or degradation. The socond is the-eiii@idi-ti6h-6f-7ih@i bio6h6acql7oi@anitii-Uhoroby is adde2d to, or roleased-from,-manganese-nodule-s-by- bacteria. T6@Attain@@the-fitst aim, samples from various-khdwr"c-u-rces W I 0 3t n-tespect-to physical, ehomical, and biological -properties, _and attempt -s will be mad,.% -tz@-cortolato the to- sults with 2 b- tt- 0 Om aWs, possible corroldtid"ilh@:WU-16Ae--V--Olopm-ent:@iill be- sought. To attain the second aim, further studi"-wlth-purified cultures from nodule2"ill-b6-madd--o-n--------------- nodule miterial and on synthetic @InOi:lo compare and contrast tehavior. Since pre- vious experiments havo-already-3hdidi- A-di f ft@-fCntd-iii --ittioii by bacteria -botw-c-e---n--- nodule 2material and MnO 0 a condition It provided to compare the eff@tt--6f-iioh-on, 2 the two Systems, It is planned to develop more specific assay procedures for differentiating the various-oxidAtibh-"ates of @In and thereby to follow the exact- fate of adsoibed or desorbod @Int and to establish@@e el6arly tho role bgetetia play in this process. --li@"Itimato goil is to study tho bacterial--actio-n-on mopa- nose on an enzymic level, 2 T,UBLICATIO@14S ARISING-OUT-OVTHE klORK LNDER THIS goooo"962.63 (2) (5) It is antlelpat d 'th-at-7&= or-will bo written fo"ubmis,@@@Q@l. - -Microbiol. on the work 'th-mqneanese n@Mb@"u-Yinp the-l-t--- 0 WI as year. PERSONNEL FOR 1963-64 p-, Ptbf6t@@f rincipal Investigator: AssociAte Bio ogy - Technician: Graduate Student:- April 1963----- Proposdl Nd ti. for-the 'no-Tiod 31 August 1964 I_Septembt@t 2 CL)ST-ESTIMATE Personnel Principal InVjstipator 1 7 5 1/8 78S 1/4 tire-suwer- mntbs Technician-full time_ $6s290 2 Tottl-Pei-sbnnel "ents- Cons=able pmlies 400 Chemicals -@200 Glassware 520 Permanent E2uipment - Travel.and Communicatto T2o scientific in6otin73:::@@ 2so--- 35 --- 28 Telephone rts Publication of PERL s----_- T6-ttkl-Direct Cost -$7 89S indirect Cost 2 11105 14% of Total Direct Co-s-t-- TOTAL,---,- -$9 000 Research D hk July @963 2 'N4KLIL.TRA-, itv Una@Sr the a,@thor -or--i :i he ri""I to the 2 froin 4.@ D D t,k, arici LI',e a t!@t $9,360 aw O-V --ubl>roject h, of the ol;,e-r-all Projecz 2 4125-1390-3902 @,jbp r oj a c t! a axp 4n s e .3 a-id shoule, be clill Concur- Concur! AP-PR(@V',-:'D ;;,OR OF FU@l 1)" 'ST) 9 /T. D -18 Fe'brtiai-y 1963 Dioloeical-Energy Sys te= Branch- Category...----- Mierdoial-Tr@-angfdtmatioh Project Title 2 -ciis it6iw s-iricatior,- Project Cryp 1,,rKULTRA -80@@@@crypto.-C-Ikssjl@C-8-tio Unclsssified --- --- ----- Branch Project No.----A*A 2 et Eog@h6e Contractor. N.A.' N.A. Contract No,- @@--T"k -NO.- 12 jufy 1961 Type of Contract-- 14MTIL --Date- In2itiated r)letion Dat Continu n;i@ -Com e- Cost . .. .......... ftrp-ose- To providi@-th@@:sb@tvices-of to sapport-reeearch on -------- 2 -by fundar.-L-htalAfidtht@nit@M 0 n@@ rens ion midrootp-shism@@@@ additiorl@ to the dl i@@dt-lotential for AOW-e@y sOurCe7sy§teM$ , the, contract-1may-proYI44tby-product,s-u"ful for--materie-l-deterioration application.-- Status: Current andltatisfactor SOURCE: Intemally-&nerated t@tt. of the requirement to-seek new ar.,! better- so-iirces-of I hereby acknowl d of e ge receipt- -_the-followi g:, 00 94, dated Oct llp 1962,--drawn--- yabf@-. in the amount of $9, 000. 00. @-ec k N"Q69286, rawil y7ab in the amount of 3 0.-00-- DATE 5 2 AMOUNT 0, ge-s@q VOUCHER NO. (Finance use only) NAN%4 October 196 REQUEST FOR ADVANCE 9 PAYABLE TO2 7 T OF FUNDS See attached. PURPOSE JIMfor funding MKULT RA Subproject 233 Invoice l@Z v,,hicii activity wac approved T - 0-1!' iii by C/TSD on2 25 July 196Z. Accottntinp, to be In accordance with the Fi,,,cal Am-iex Attaclirn,5@.-,it C. z y account for th Josion of vouchers and refund of anyunexpended balance 2 t:groo that Jr wlyl full a advance by subm 7' the foportjng point sits@ed and by the d data @liecked @nt *for f$JJure to so 2 and re fund STATUS OF OVTSTANOING ADVANCIES In the ovd any unexpe@dd balance I X Mthoi@xo d.4@@ ion f@om my oafs offleat Set2tlement. DATE AMOUNT tftqlccoojl4lrgo "A@AKE DUE DATE IME@@A HANC2E D@IVIS@ION 000ARTERS -STINAT,$ON ON 'AR7tVAL %-r OIL 2 ON PABOUT ot LAST m 2 EJ 'ro4@ wRK Or A(:" MONTH T7--l REOUESTlf4G OFFICER ARPROV@D 2 k R15CEI@T FOR i UNDS ADVANCED DATE SIGNATURE DATY. SIGNATT) ' 'r. acknowledge re lot a n at ted OFF2ICgk. I6@d., to bl used accou7t*d fair 77 h@W. above. I CERTIFYIFUNDS ARE AVAILABLE 5 ild 3D@ TSD@ 'I 2 Amo T OBLIGATION REFIER CEPTIF )R PAYME@T OR CR@EDIT 'T3V Fmcf7V) OZ DATE AUTIOltlZF-D CIERT,L@2YING O@ ICEFI SIPNATURE OF Ao@@ C@E DATE SIGNATURE SPACE BELOW FOR EXCLUSIVE USE OF FINANCE DIVIS 4 PREPARED BY REVIEWED ay VOUC54ER no.,2 7-12 DESCRIPTION-ALL OTHER ACCOUNTS 13-33 34-39 3 AS.46 A7-S2 58-67 60-70 71-60 STATION 40-42 IUF 53 5A-57 ALLOT. OR COST DUE AMOUNT 2 26- 33 C*01 EXPEND 0" E PAY IIEF' GENERAL ACCT. 40. DATE -0;: CODE PER, A LEOGElt 2 DESCRIPTION- P. N P;. IN Lio@ ......... cetoot ADVANCE ACCOUNTS. 13-27 .... 0 E A el. No. x Piet'. no 2 r-Y IS cr 0,1 III D@LLLL TOTALS@ FORM AJONW,1 S. 60 281 use PRKVIOUS EDITIONS. 24 July 196? 133 1-33 $9, 360@LW- 3125-tl Agt 2 4 JUL 1962 D R@WFT MEMORAND,-UM THE RECOR SUBJECT co-ntin-u-ationof-MKULT-R-A, Subproject_133_ 2 -Subproject 133 iV-t6-6ftabli@TS 1. The"*o-se-of MKULTRK@ D/B@ to utilize the-s@@@ces of 2. During the first year of-th6-p" made significant contributions to th-e-all7t&6-@@ty knowle'age--bf the mechanisms - it-@f6n of -these acco of mineral transf6kiiiAtions Aiechnical d' mplishmen-ts- is attached hereto, with an outline of proposed work f6j@(@@g year. It is possible- that the@@inves-zigations may well-l- ad-to new approaches-2- -for energy trarLsf6f:syotems- (bio-batteries) and deterioration of metals 3. ctioned aspilliwlj un and during the first-year of this projec Th s service-will-her_etofd"-.-------- be furnished by @the@@@ e cost of this program -for the second-yi67@- will W$9, 000-. 00 to-which must be added $ 360. 00 which repre sents a 47o k@@,rvice charge to be retained by the 4X$IW The total cost of the-p----r-ogram, therefdres exceed $9, 360. 00. Charges 4 should be made against Allotment N6-.-- 3125-1390-3902-.--- 4. It is not anticipated that permanent ecuipment other than that listed in the budget will Vib@@ired for this program;- -Title to the -2 equipm t listed will be retained b@( lieu of,highor en overhead rates -and ac 'ng for travel which 5.: Dbt@@tioa coanti -eNp@q@s 2 are reimbursable b will conform to -of that orktaization. the accepted ractice 6. nas been clea-r-a-C-(@V-9-PTLY@@d'is-unwitting- and will remiin unwittiniz,of the- true n-a-ture-of-the@sponsora---2----@@ TSD/BIOL( TSD/BIOLOGICAL-BRANCH APPROVED-FOR OBL-IG-ATION OF FUNDS@--@ AC/TSD DATE: Attached: Budget Project Summary and Pr@6@al Distribution,. Original only 25-june 1962 tt nti Subject: P@rep-osal-@-0i ed 2.e o -subje, ro- q-t- -P Pli@@f2 ini ene- behalf 6 posal submitted-on-- -is- The proposal not being submitted elsewhere for possible support. Y6utf@@bn4ideration of our proposal will-be ap re- 3 ciated andn@@look forward to bear@@fr-o-m-yo-u-4, yourso sistant-Dirt"Or L Enclosure ed Submitted on-behalf-of Assoc ate Viofes-s6-r-of-Bl@6 Jufte 1962 OF--BIOLWY 1962---- Purpose of -Study: The purpose offthi"roposal is--a request-for financial support to continue an-investigation-o"icr-obial-4@etio6@6h marine manganese nodules and terrige-nou&@@mi@hdral-sulfidk"hich the@ p-cincipal-invelsti--- gator has been-pursuiA@@@1958,-Ver.v iWt6"ive wor'k-on-tlves materials is results, during the current.yiar,-1961-62,-_un-tier-a- from the 2 nce relatively little is kdown aoo-t m i u on, And in view-of current acadd&i@@@practical iii@ @f microbiologists,- geologists,, mining engineers, soil-461thtists, oc"h6gt&Aers. etci@v--in-the subj4ct, this research- shoul"kd:@tuabl6 -conttibution-to-- science,----L:L@:!=-- Sum-mary-of-@ast 1?6rk: a. Ba.@teriology=of -7tih@@l-@dif ides-& e2- lable--from Att mpts were madefto@tvalua-te the m-tcrobia-l-flora iso unsterilized, crushed--giilfidA--tiherals by enrichment in mineral solutim@@ The follo,,,iing-mintral6-vtre-studied-.---alabandite---4rsenopyrite, bornite, 2 chalcocite, c-halc6pytitd,,-cinnabar, cobaltit6, dovellitei@te64tgite,-galena, mareasite, orpiment, pyrite pyrrhotite -realgar and sphtlerit6-,- Of-these minerals, only-cobaltit-4-,---e-na-rgite-, galeta, pyrite; pyrrhotite, realgar.- 3and sphalerite yielded microorganisms, For-the most-part these-organisms were heterotrophic and probably reotettnted-tbntaminants. H6w4vet@- Hy-phomicrobiums---isolated ftd@i-t4AlgAr, a--pink-ye-ast repeatedly -2 from sphalerite, and Arthrdb@ctdr, idblaftd from cobaltite galdna pyrrhotite, r4alg4j@;--and sohalbtit@i tAy--donstitiite-part of 7i--ft6rmal flora. The action..of any@@.of -these-organis66172@@tth@@spect-to the mineral- with which they were f64hd-associated-f4@i@--Wb6-establf-ahed-,------------- After surface.-isterilitA-tioni some of the above mineral-sulfides-,---- when enriched-ih-&ineral::ji@@dtibn, hAVi@--yielded iror,,-oxidizitig auto- trophs. Thete-mi- le@-incWddl:arsenopyri@"yrite@ chat----- n6-ra copyrite, enargite, _galena,_ marcasite,- and--"hal-eritei@@-At - 10@"t7@d2ome of the isolated bacterial----tt-rains are not--rest-rieted to a diet of-iron for energ)r, but can useE::sulfut@@i, probably, some other oxidizable metali-;-7777 The abiLity--t6- grow on any of-th"b@-ove-sulfide' minerals was tes e by inocul2ati-,tg-surfAte-tterile-sampl67@@idizi4t@columns-with Fettd.-- bacillus ferroo@@__ nd it-tet6o-tin8@-tb te4boVer-the-organism feedin& solution overi"eriod-of-t@i@@h-9 or more-.-So-far. -positive results have beentbbtain2ed:@4ith- itsi@hopfttt@@@@@te, -chalcopyrite,- marcasite, ga-lena,--pyrite pyrrhotit@i-,-a-n-d --s---ph-a-leritei7:Ntptivd- results have been obtaid@@d:@iith-alabandite-,- bornite, cobalttte, covellite,,-chaltb@@ 2 cit6, and one sampl"_f gl-Yl@@@ihh"@i@-6YPimdiht-,. and rea1ttf7d@being- c rrently investigate In addition-to the foregoing--- litAtiV6-iWdr% quantitative studies- -q.ua--- on the ra2tes of -oxidati6"fi@@)@@@S-@-64tural Artenopyrite are presently being-und6ttAken-0----Ffdta7@hese studi6s-it has become clear that synthetic Cu2S can b-6 oxidized at lea@gt 4y--as--fast by-bacteria than by-- autoxidation, and-that Arsdhopyrite can be more rapidly oxidized,by--- bacteria than-by autoxidati6n, Rd-6ults with the-latter material are not yet sufficiont to establish 4ii-exAct-rate-comvarison. The precise mech-@--- anism of bacterial oxidation re-mains to be established.@@The-work synthetic Cu S proves-what-some-otber-workers -seem to doubt, that F----- 2 ferrooxidans can-oxi8ize-metalt other than iron. b. Manganese Ndd4les Oceanographers-have-felt vrett2y strongly in the past@that the-origiii------ and development-of-manzanese-nod@j es-iii7Ah-e-dceans-is-attributable to purely physitochemidAl@@oroc--e-s-s--es-&---- Nowe@ver@ on finding organic'nitrogth-in nodules--,- 2 concluded that bidlogt!@@gents-were involved-iti@dul4-gen4sis-@- At his suggestion, the ovineWl-investig4Ui@-attempted to-@.find@:@f@ll@a-c-te--rici----.1 'might play a role-in this-- Rt@found-that bacteria were-indeod--@pr-esent In the nodular-substance-aft@@r-surft@@O@tiliza-tio-n- (a rough estimate at present is 10@- er gram) @They included-Atii@bacter"rthrobacter. p 0 -- - - --- - - -- - -- Bacillus. Br6vibacterium. St@@1-0-co2-ccus, Vibti6, an unidentified7tO, an unidentified -ebetui.----The-pri.neipal-investiga-tor-@showed-irt quantitative experiments that nodular substance can ad-so-rb- mankanous ion--from sea water,-, and that this adsorption is-accelerated@W bacteria that grow from the 2 nodular mate rial,--Thd@acttleration of manganous ion-adsorptio-n-is-explAin---- able on the basis-that-the b@icteria-o-x-idiie--the adsorbed mang:anese.-which facilitates futthtt-adsorption-of mangaftquv@@ganese- The acceleration requires t2he presente--of p4ptone-;@to permit bacterial development. If peptone and glucose are present mangar,@ese is-releared ftd6@the-tibdulaf--- substance rather than adsorbed, at-least in a net effect. -- Since som-e---- nodules were apparently initiated around shark's teeth, ear bones of whales, pumice, etc.$ in the sea, atteml)ts were made to see if oyster shells can adsorb mang.-inous manganese and thus serve-as possible-foci-of nodules. It was found that they,,do adsorb it and that peptonq did not stimulate tl-.is adsorption (no-batterta, uere-present.1). As far as a-survey-o-f the-Iterature has gone, th@ese-observations with respect to mangangse-nodules--have not been reported before@ttt Pertinent li terature-2---- The early literature-dealing with microbial action on-m-in-orals-has---- been covered by Alexanddir- (l)-o----A -review by Lyaliko:va summarize-a-much@of the past important work 0 ferLooxildoans-(2).----An-intimate ass"iation of-itbA-oxidizihg-autotrophs---- 2 with natural mineral sqlfidd@@@hAs been-inditated by the vor,t o-f and by thii"f f4@7@@f b@gtii6t-exist between Bryner and And6taw(6) Mal6@if-@dn-d-PtAter-(7) and lvanov@-Nargirvyak- 2 31 e--r- and StepAnov,on-the--one@hand-;@@ h( about a mechanism of@bift@@sulfide oxidatidtc-of chaloopyrite, molybdenite- chalcocite,; anho- n to cal). 3, Examination of it@-olA-@for "ecif i-e- mineralizing-activi-t-ies-,- 4. Elucid@ition of bio-chemittil mechanisms -of 7tDiffdrtl- trans- b. Manganese Nodules-.- dt riologitalft rison --Qualitative -Aiid-444@@titb:tiVe ba e ompa n -Uro-m different oceans 2 of ma aOst@@nodul" 26---- Study of-tWbi@@ical mech4hist7--Qf man&anous-6xidation and-Mhai:@@ett@@n-in the various bacteria-iOlatedo-- 3,-@@Detemitidtion of-thi-@anidt--@f 1-ton2-incorp6ration into- manganese-7nodult§7;@ The methods to be used-in these studids-will t@@ptations-of procedures of @@tt@riolo@gy@@-thy@@@d biochemistry. Personnel: Principal-IhV6stigator-, Ass2oc. Prof. of Biold&. Technician: craduati@, Student: C,_qoftftWnot presently supported)-- te student: U'@iiWndergraduate -Ado- I'dohim e -1962) research fellow) m 1 (s,@om r (s, xmer -1963) -7 Proposed Budgqt: ---- PERSONNEL Principal Investigator-- 1/8 time-academic year 130.50 I./4 tim4-surnmer-months - - 700 - Technic2ian@i ifull-tift& 4-.-IS5 5;905- PER14A.NENT EQUIPMENT @luorimeter@@@ 900-- CONSUMABLE SUPPLIES-- --7:@@300 C2hemical@ Glassvare------- -. ------ 640- TRAVEL To scientif ic@meetings 250- OTHER EXPENSES7::::@@@- Publica:tion-- 1820 Telephone 20 200-- T6"1 -Dii@@ct- -Cost 7,895 INDIRECT COST @ 14% of Total Direct-C@6@ --- 2 Total Cd@@t-7@7 9,000 Date 24july- 1962 Br --C fied-(Reports Project Tit emicrobi aa@s-f6kiiiAti6n I n - al Ti tern Classificatio Uncla" of M MKULTRA Sub LL3 3 Project Cryp Crypto CMifi Branch Project No--N Project Conttacto N N. A Cont2ract No- Jill MKULTRA----- Type of, ContracL- Date C6htinuiniz CosL $9000600- ---Completioa_Da'te 2 m m Purpose: To pyovid6Lthe-tervices 03 ME&nd. to support research on fundamental mechanism3 of -miiia -ral tion by microorganisx'h,s.- t 7 y Cd Status: The first year of thit"jec actoi '-in t a sumrnary zeport has 14 AUV$T 1961 W*arcpl"iodi@beabl@@-ti*-ttM$MittOYOU Z68 54 360.00. tilc -o 2 2 -4 on the W74 rawn t6ri$90 000. 0 -t" of VOW The$* &nds regreseiv for th@ 6th diractotgjr -OAz evorywortkwhilores"rchgoals------- Of your-organizauou. Yo%lrf-tr4ly, Enclosures (Z) U My 1961 Divition VIA SVWEGT UKULT" -$"Ptoj*@e-t zu -1 co L int@,tc* NO@ hod4-wlr4" " P"eAt 0 2 w payame to CA h6ck liA the PIS",$ fo@rward tilb: eblea# -to CbA#(#,TSDIP-iiitarch wr-&-"h-s "ac4t la* th" wiuly 1941, wo" Ts am-- It to anticip-ate4 be Proi4kCtt tho-til*# t,W should tA b4b @4@@ TS D/RostAVCIL BtOAth JX#tributtaW. t4 l@@]@SD FASS 5 z---- TSD/RB Ts@D/-R-ffi ce ce to are 'O:d jr,@a'ctordanct vitb@@iWt, 196 2 tsv/@Ba that this bill -to iuiit@- aA Pt tbo reof -h6e-- not @'Cei@aft lr& 4--@ correct ard that payment 0 thi oiiti' t4 6 323 tt fied d- I ----d d: that@'the pro,,@eat ng: rarr@ LM vhi4h wai; u y.-approve A;ccr"nee:viW-.Alie -fteso 2 1953 from @@@t to! 1,, e :.and VW au nt Dates. DA'Z'I this" AU%" -6u ritg, ta be- been. -,rlp"vbd--2, ttnd me"r. en7@dbliZa Ate be @,tb4 _ted, tc . 2 Qi39d coal @mom Fca @CW04TI64 cr 'Dig Addit lg @ & -. TW/FAW 5 i tiie(fir*ance (I chron., D R A P T40w-- Sub@io@ SUBJECT :--initiation of MMTRA@ ect 133:::::7@7 1. The se of KCLJLTRA,-SubproJect 133-i$-!tO-enable TSD/FS t2o utilize:@-@6 services of 2 co n du a t a program of research on mineral transforming microorganisms.- The work will consider-both ecological and ybysiological@:@t"f-the probltm.- Ways may-al-s--O--be-shown for groving food from min2eral@-ttA@@ @tosynthetic-process because:@@soawj*@teriai@A@ @nown to attack certain minera a can chsngp@l@@dioxide.-into organic-@dAtter.vith the@--energy that they deriwfrom the 3. sball function 2 as for this project. The cott@77of-tbis program for the firavyear will be $9-000-00-to which must be added $36o.oo which represents a 4% aervice dh&tg6--to t@6--tdtained b)r@th_e The total cost-of therefore vilr-hot exceed-$9)360.00 Charges-should be--made against Allotment N6-@-Zl25-1390.3902.-- 4. it7it that any perma@nent-ec -riot antio@@ Luippl@@ll- be required-fo"is program. 5- D00umentat8io--n-and a ting for travel expens reimbureabitil@y, will tonform to the Moo- accepted practice -of that orgppi-z-ation.- bas been cleared COVFMLY and is unvitting -an4- viU remaim@U@@ Of the true natum-of AppROVED FOR OBLIGATION OF -Resear Dates.;. Attached:--- Bu4O t- - Proj2@i" Litei6tVi4@Ri@-viev Distributio6o Original-06IY777-- BUDGET- 1. Salaries-- $4 500-00 Principal Iiiijit-i-&tor Assistant 200-400 2. Tra)rel:@@ 4 30O.W 3 - Supplies $92000.00-------- TORAL@@ A STUDYLIO"lNr-,KAL---TRAtISF(,'RI41@44 1-.47r CROOK(-sr kNT C-,14S Purpose: The projot@t-@propd'--ed herein Is ttitended toidrovide inforMAtion--Qn the-microbiological in mineral transformation. Onl -e2danty know'IiOdg6-is-availab e--in y this field-at the present time. The-ivork will consider both ecol-o-gleal-i@n-d @-,hyelo@[@ioal-ACP#6t-g@ of the problem.- e --con r Th findit@@shoild be A i'oiition--to funda@@-- mental knowl--e@,d------In microoiblogy@@@@-, and allied ge-- fields. T@@c,;hbiAld-also be a valiiabIO-@n'triolition---- to practlbal l@@@@htse area2s bee@-Rijae ways-may be shown to@harnpas some-of ti@f@ microorgani6mg-to do useful work::@t:@K@, b@tah@@iding ift-th,e7oonver-clon-of------------- econom m.i.neralo- 6f@I td-@lue Into siibqt-q2ncee--of7high economic vali@@ys--May al--@@@be-,ohou,,n-for- growing-food from minera hotosyhthetic- process@@@ecalise - some bacteria, ai:ii-@y--kri-oW-n to-attack-oertAn-mine.-als, 2 I to M-itter-iqith-the-energy which can dba,noe-C,02-1-@@ they derivief om the-oxidattdn of the miner,-olc, (l)-;@ This orc-,ania-m then lft--t,,.i-rn---.qerve as food for-- other microorg-inisn-g- W2'ni:-Ch MqT7t@@@d directly -or In--- M, d-irectl' as-7fdod'@fot an. From-prelitAh-@@r,v-iftliectigAtions 6y the senior-invee-- tia:cttor (2) It h4is 'oecome L-,p,2,oq.rent th-at mi.,ieral-trais- foruilng microorea.ni,:ms--"e-inti-ia-@pely as.socia%@,ed with variol-te types--of min@rala-.--The proposed reaearch is--- designed t-o-follovi.iip the lead#-fic@@ia e@n,.rlier wor9k to delineate el-6" y tii@@onment,*.l-pnd functiong interrelationships-betw6en mitieral h;kbitsLt@@:,-,ndithe mioroor-o@@@--- gani,pme found Objectives: a) lool-F-ttld"-reort@@entptive organiamL*-by--enri-ohment and pur"lil-tii2r@e 1. fii@@a --and -other mineral6@7 -d"inage 'ti;FLters 0- m 2. -- f j@ft J6 ---Ine-ral-deposits- 3. f rom -- s oil Is b) A atudY70f:AW ove-r-&1177b2htmical aotivity on vari-oa-is wil inclade 2. -,,qynthet-ic-mineral@9----- c) A study--of of .9.ction7:@on--various mineral -st-ib-s-tkff6"-by microble-1-1-lqolates. Plan of Research: P) IS-01@@@--reoresen-tativ-e,--c)-xl-tiires-by-enriolr@ient-.qnd -&6thod ,)ure-cult4i,e -s. 2 Hier6'aial776""hm8nts will:@bt derived-from- cruehed-ores@@other@minerpl-@§@ibbtAnces, qnd=-coil-4-by peroolating-nlitrient colunins charged with these mAteri@nig@@tffThe@efflu-ent- 2 .mol-,Ations will b@ Aqbc,,iltired In- appropriqte selective -media, such eq-iron broth b roth (4),-n@ttrient broth, etc. Mierobtal@ enrioments will derived frm.flask cultures-7pre,,oared by overlAying@7-crushed-orei-other mineral-eubs'tanoesi; qnd ,-oils with a--nlj-trlent solu-tibh-.=Microbta.1-6"lchments of--draina,ge waters from-mine@""eDosits will-o-e-ffgde-oy inooilgting -suitg-ble vol4k#s---t-nto--the-various. eelec-tiVt:7t-t@41A alre,"y=oited. Other enrichment metho2ds may7be used as they beconie7-approoriate*- The --ores --and -other=----- mineral@fOqbsts-hoe-a will-'&"ed-befori@@,qnd,After- surf ao e- t e ri 1-.I-z-a-ti on. OM---th(e---v-a-ricus-enrichment<-, will be@@Drenared-W-ttrVki@ft@@@if led@:tftoX@@c -Rnd or-ge,nie riediaiilwnenever possible, 1 e heith6t-highly@-Durif agar 11- --A ---- -4-@k. 2 -0" 0 ge a dtlqli@6h@:@niqiAe acirso, sel-edtive-114AId---mOd a may be@:7empl'6@@Culture- .o)-irity b"p ,propriate morpholb,2--leal obser-,rq tons.- Attem@,ots-@@t@@@@-t,qxonomic- identity of --- m4de. b) A study-o"he ov-6r--all miner,ql@ftb-e-t&ft"-t7-o"!@rich@ment cultures P-nd2 pure oult@@6tF;7=-- The-ovex@@l@@6h@@Mic-,ql-a-c-tlvi-ty i%Till b#---*-tiidied with enrichme.nt-cl-ilttir-es-e.nd-,.ol-ire cult,-irea--Reting on- iaineral tq2nocal@@f@known-oomlbOgitl b -a - -on.-: I ff- the,,2e@@Vtl-ldies, ($,,Ie.litatiTe-Ftnd iiiin-ntitative ineasiirements will7b-6@de on eiibstrate deoo,.,--qDocition,9 product qccl-imtilpti.on, chqngep- In acidity-*- pH, redox-Dotentip.1, cono-tim,)-tion ,in . oro,.qn c ni ,a er any A - c ori:n Of CO@4w';-# -- 02 -cl- of-aetivitteg- in enrich-raent oult.@ire-9 with activities in oure --(jri identioal@@i8 )Fill @"v"l -whether the-cooderation of@7M-dre--than--one-mievoorganism -is- req-iired-for A criven tr--qnsformpttion.------- c) A study 6f--the-medha,nitms of "tion on variouc minerp-I stibe tanes.-a:;:by - midbo'@,3i6.r7t"la tOd. The@@@@-of- actiori- on veriou,.z -mineral@. sii'o2strat-@Lel-,-- by pure7@@:cul III b--e--i-nv-e-etig@@ed7@by the-use-of -;tandard@@hemical@tbloch6midal@-, P-n(i ph -101-ogical 'Uechl.l uess The exoerimental--a-D-y-)j@@ch wi 11-21"lude-,q-,iliantitp--tlve analysis of the@@ao@ti@dh on-DiAre mineral coriipound#7;@@bneoltibl76Tform.0-- of tl.-ie-cRtions And anions of Vqich the@;;@@res-oective minerplq 9-re comoo,.;ed@@@h"q-e-oretically Doc-sibl@@7i@@t-6@diate,-* of metaboll--cm-.--646h@:Thf6tmation will be@@reiiiired--for the form4l:;.7tibh-of7k::@"hemt-thp,t-wo,ild7@lal"h!5-@ossible 2 over-all@@!dhe-m,-16417ii-@tibn Ob-cerved. Ref erene es,. 1. Silitermpn P-nd7Ui@4dgrtn, J.-3"ter.tol@ ?87f@26 (1959)7@@--@@ 3 Si ren, 7-@@2 (i8g verm,qn 9.nd Liind@g 4. 14.kNUAL;;OF-MICR0310LO(,'T-CAL-I,!E@THoDS, Oo.,, Trio., New-Y6tX7;@19,5?- T). 114. MINIRAL-THANSFORK'-4 $14-S sa> A,- "F-- G 1141CROORGANT Literature Review, A number of @@orts -exist in the literatire -aidoiit microorganisms which seem to b"ssoclat-ed-with 2-orooesse,,o of mineral-transf6rmatioh. In many instanoes---the function which those mid@roorganiame Dlay-is not known dr has been-misintery,)reted.- - ThOC@X'oll*Ving-is - &-,'-discussion of some redorts o microoial--O:@ion on minera 8-200mmon 1-n@"t).tre Sulfur: An indoection of;thd ?th---i@ditio6-of iiergey@s Maniial- -of Determi.nAtiVe BAc-teriol- ale-that bacteria-have been- associated'in-the@Lbast- with e-.ilfii@idationg - and r-e@6tione (1 02).__ The Thloba6tertacelg@e in-clalt--@mber-s-'Chkt-hav"ee"nown-to oxi- dize varioiis -Tiiese--- organisms Are:::@l so,-iltativel" mba@it6troohie. Most are 2 which Is f.@oultatiVt-o P-h-bt oq y -bacteria belonging to -uhe _Ozhttic Onlorobacteri&ceae--p-nu tne Thi-drtiodAceae@l"ve-beerv::shown--t-o--i-ia-e------------,- inorganic sulfur comoo2i-i-n-d-e-----ol@@-tnan--sulfate se-red@.-ictqnts--in photosynthesis. M@6@ @tionabl4--i-- the assoolation of nigher bacteria, @t-ne@LBOgglAtonceae and Aciitft&tAdt&pF@witti oqlfuii--@aidAtion. 2 Their associatidh witn sulfur--I-s-lergel"sed7on-tnt-7-ooservp--tio-n of intracellial-O@r--- -6 itton of--Bu-lf4ii--Vahules-----Faus-t and-Wolfe- dCD b have reoently-ghowa@-tn2at-B-----I-toR-albals activity on sul*L'ur Is restriatec'L lf@@-@@bsent, at le"t iti@labdratory culture-:@(3), ,i:ne nabli;ats--of:::7All:@Z,6nese oaoteria seem co oe soil, fresh and--- narine waters-and-six-If@i"tt@"Its (4)6-- In at le net-qnce, oxidi"r7@"a -d from -o a sulfur --oeen itolat@e o;prolite--rook (fossil d(ing)-of rne Trlq,*,sio Y)eriod (1). A fairlyLLrecent-review of The -q,,i'-f iir bacteria- is -contained In (5) Siilfltr=:-redi,iotion hqs Oeen mogt extensiV*ly-@@atiidled In relation l.'#o the activiti-e@-df Dti@difoviori-O des@AlfArie@khs---of5- the Spirillaceae. Members of this gonlis @re.dace-eulfAtes,--- thio-oulfates--sulfites, etc. to hyd,rd"A@gulfide undor anaerobic conditions, TWhabitate of, theae bacteria Include soil, sewagei walter oil-be$Lring strata, ece. Up@odate disou2ssione-or the physiology. ot,--thes-e-b"T."Ware o-ontained in (6,7). Tron comdo unds: The lnte act on of bacteria with i-L-on compotinde-wac; observed as eariy as-1888--(8)-. In tni--- work a sheathed7bacterilim--- LeRtothr2iX. wAg-o@)eerved7Wd""-It--f,e-r-rio--hyAroxi-de----I-n--its sheath.-- It wa elieve"hit the organism was-onemoautotroohio,,-gettitig IU--- energy for-carbon dl"id-e ac-,-*-i-milation from-the oxidation-of--ferrous- lvon, However7i-@@tne-onemoa@,itotrophio and many other lr baoteria-hp.9-not-been satisfact -ar-ily- proven in--- the ligiig : ot::d@ptesent under-qtandi@*--of - ohemoautotropqy At-- Dresent2 1"1@i-seeme- more -lAkelY---V"t--the sheathe-d-bp.-cL-ter a"C'nlamy!4,@ baoteriaceae-i@ Gr-e-no-Vn-:kloeLoiae_) deposit--f6kt-id@@hydii"ide--from ferruginous-waters-in thei-ii--sheathe-withotit-m2etab-o-lic--mO-d-iAtion@@-- The se-me kind--o"-6@o l@@@ide-d#ojb@alti.on-is bell-6v-e-d-t,--O----take---------, place in the case of--thi6--stLqlXtUb@acteria-.(Caiilobacteriaceae). 2 In their ea-se, too, it IC@4@@,qe.-nt--qxe--s@ioned@Vhether-ohemoaiito- troohy acooints for the ftrrio-hydr6xide 4epositi-on. -4he one rcui R&il n ee ad - exception In thli@7IMte-noe--may be-Gsill6i2i@@lla- f -e-L- Among enea-osul&td -babte-ria (SideroosLngaceae), tne mqjori-i@y of tne ba-ateria--,seem-to-d- t ferric hydroxide--in tneir capsales or outer :cell@@aiirf Ae-e-t@ by non-cliemoalitotroohic means @@- Only t1he gen@,is Ferrob"illue In- this f "ily--6XIdi ze a - f errolie - iron- by - -- - - strictly 'enentoaiAtotrot)hi@6-fftAns-- 9-t-a very aold pH-(10). -This genus Is probAbl"16@ed-to--the-ThlobALdti@rlaceae (1). The reported habitat f r all th@Wbaot$ria It iron-bearing voters Certain Iron ores-al" -6-@-to "ntain tnem;(11)8 Variol@e-oommon--baoteria are able-to precipitate Iron from organic ealt-oombination 112). Siich genera Aerobacter, Pgtudomonas t2his about by digeating-away-The--organic portioniof the salt@-@otino- myeetes of tne-genern -- ----- -- out such activity. Ferrio@:lr6h reduction may be-70oin-o-ted--dii@@c-tly--or- lncLireotlf--- 2 by microbla a.- Th@@7indiiiect-effeot la- rababl--t--n--e--more common, 'caking pl"e-iri a rediid!Lno environment a-t acid pli created by tarloi-te-LibA-ot"ia. kna can rediioe f ertio- iror2@7tnrough direct -metaboli-e-, Inter-a-et-lon (13).--- Iron ailfidea-mq 7 reelpitated-tnroli@zli t.ne-f@@@tion or hydrogen s:ulfi-(i:PLby@:-t;tilfate rediiarT-or@by@release of hydrogen===@=--2--- sul-fide from--organto-sulf4iti-7doi6,oounds by various common bacteria-- Manganese oomdounds*, Matigane has oeen a.asoolated-with many-of--the-Sid"ocaosaceae which are also-assoc-late"lth iron-d"@"Ition.--In gener@@@evidence Is lacking for metabo o epenctence bf--thig manganese-preelpitation (1,2)1, althotigh-B#ij"t"r-"'O@6rted-l@@-@&go-7that some- t-rae oaoteria aAd:7t4ngi f rotd -coll-6bAld -oxidi%e m2angqnese- s!,ilfatt or.- manganese carbonate (he-terotroohically)'(14,15)i -Som6 bacteria seem to be-a:ble to@redi-ioe mqngAnels-e--dioxide@@@nde"nnditi-6ns--where this comnound-@@n &-c-t as An elf6tti-@ @tor-in idlace-of oxy2gen, quastel and-SchoWl-e-ld (16) re,,oorted on such-a-,-orocese-i7--Chemo- autottroohic-manganese oxidsltion has--been r@ported@bt Prs@@(17)-- and by Sartory-and oonfirmati.on-oi' -@ne-ce observations to recluired.-6------- COIDDer com-ooundg:- Transformati.on--or copper com ol-inds nas been re,,oorted-by-- Ciferri and-Sdkt&da@Mi--(19) Hurwitz (20), and Bryner--and-@=-- Jameson (21)4 -Only--in the-lqs ree-reports does eviclence epast tiiat enzymatic action on copi.Der-compounds-is- involved; in-faot,-'tne b#@d@@IA--that redort-are deserl'ued@@ as ciienmautotroohs-all!tA@:l@@nemoautotroohlo-iron oxiditttt@-@1--l--- In tne first2-two reports, metabolic end@prddaote akie@-held-- responsibl e-fb@@the-transf6@idn of tnsolut)le---obt@ter@@@ounds. Molybden'UM An or abitt re e -to-- ne-onemoautotro-.ohic Iron-oxidizers- has been di-seov-ere t a2di-d to oxidlie ;iietmineral molybdenite-- (MO92) (22).- Th"rodiiets:@bt, - it-g---Aotion on - thi"ittttal-6,re- a-,ilfate ar@d mol@y-bdit@l"id@:@@ng-furtner-seeme- to be known about thietl-tranef6Mati62ft-I Silicates,. BP-oill4" s- as been reported -t--o releas-@-tac-aiiim-- from a ooind et-kte-in al461ta"iIT6A-@@nen-ta"rgantem is growing ln-&::-@asaiiim-defiolOht-t-hvirohm6at (23),AmLrgilliis ol&er has a go@been-ii-@t@@@deootiioose olay.-minerals to satisfy ite@@potaesium requirdments-(24) Aold formation and Its subsequen@ action on tne -minerals-prob-ably accoiints--ror Tne@: po--- tassium release.- may Include -carbonic, -organto, nitric, ahd-s@Alfuric@:"Ide,-- Conllil4.sloqa. This.literkt4i@@View 1"-htended to bring out tne fact--- t.qat microorganlt&i:b"-'2orl6"boqt mineral transformation In two ways;: by -direct enzymatic-- I"erEcotion,, iieuRIly CAU-Sing o./,Idation or reductlon, or b3i-indireot action throi-igh trie- Of production environmental conditions wnion promote TirA3na'L'or,,nAti6ne ttirough- the prodiiction-of environmental-conclitionalwnioh promote transformations tnrough-onemioal7::r"otions o -" DiArely non-biological nat It 16 ttie first tyde of tranarormation ,i-tr e which will oe-e-6ressed-2in tht-prdposed In "it"r-the--Imnression--tnat certain-problems-or microbial mineral@iti@&nafe,@rmqtion have been-resolved, more- extensive:inspeotioi,i or t-,qe--pertinent 11-te-rat,,xre reveals contradiotiont@@@ r 2 lea bearing on.tne-problft-6i@-&&b-r@dbial--7- ee exa action in'mineral sti-if lde--t-rangf 6i@@tion will::I@"it-ed-f dii--Illlis-- tration, LeatherL-et,--a-l@@A25) -claimed -that they; oould -buse ve Ferrobaoillue--feti@IdAns to oxici.ize mareaei-tO@-@@pyrite. tsoth these@--min6ralA have tkie same comiDosttion-(M-)-f- Ttiey attri-- 2 -2 - @, bated t.,ii'@@@:-differenoe in aetion--of-The bp-.eteria-to a difference In crystal stritatum- be-Lween maroasite anck@ pyrite-. xe ryner et al,, (21) 00,Ild ootain growth on I)yrite by F. fer.-oox-ld@48 or a-ologe y re@lated 6rRanism, A seoofid iinre-qo-lve4::@"b -em--nonceic@ne.-IoxidAtion of molybdenite (21). in @nia work- an organism related-to F, OXidans oxidutd-tne and-malyodic acid, ::-a2 that oxidation of th ine workers- asi-imed- e et-tlfdi@::domponents of the minerRI aoooan-@4"or- thi-a -reactit-)n. _ -If hey overlookt-d- - COMDletely-tfte-fact that molt&d@@@hkd-itselfAn(tergone- an-..2---- oxidation. The ciiestion thiiq arises whetber-the brcteria were directly responalbtt-"jti-molybdenum-oxiaation. A rnird-1,@"--oi,-oonflAotih@7information ooncerna--tne renorted aoility-o"@@@"ooxidans to grcfw-2on-media-q-*o@lictifted--- with agar (26). 3ryner-and Jameson coixld noti confift --@hle observation (21). It Is tnue-olear i;nat even--on--tnose-mloroorganitims-for whion extensive information-is qvailabl-6--concerning-,cneir-role-in mineral transfomation, fiirt"i@--Wrk7 is- needed@@t@-Olarify various Doints of oonflioting Referenoes2, 1. 8ergey's Manial--or D6ttrmihAtive Bacteriology,- 'ith@@77e-do, The 1957- 20 Skerman GUIDE-TO-,rR& lDt;NTIelOATION Ok'T- GENh;RA@6F!@BACTF,RrA, Th@6-Will!Lbi6ii-=i@h@Wilkir@t@.@, 6altimore-1959 3. Faust snd Wolf",-Bk6t"iol-- 61 -9"1961) 4o Kiienetzov--Baoteriol, Prooo, 5. VishniaeL@an@L-SanteV,,-!3"t"iol@-Rev# -21i*b 195-(195?) 2 6. Pdatgi ktb Anne R6v. Mic-r-obiol-,- 120.- 505 (1959) - 't. Meenal&s an@cv--R JO-B 1-. -tO -3 0 1 8 Winogradek@(- Bot-an Zel-t-ung-46: 261 (218e8)- 9. 10,, Silverman ama-- ndgren, (1959) 12, lialvorson@-Soil S--c-i. @32*.-. 14@@@ 13. l4i6jti-"Iol -(19511-) 14. Beijerinok F-oliA 1.-Iidr6biol@-20123 (1913) 15. BeijezLlnok,-Veralk"kAd, WttOhsdhApp6h, 22. 41-5--(1913-)- 16. @Zaastel and S6!1 5 i _5,.--279 (1953)-,---- 17. Prave, Aroh-Mikt@@ol",?.'-733-,(193'?)-----@ 18. Sartory-anck Meyer----Cbmot-;-Rend, Aca-d- Sci. 225 19. Ciferri and-ScattWUI, Attl-ls-t7@ dot. Unive L&b@-7Cfittdgo- Pavia-Ser,@--5,- (1937) 20. tiiirwlzz,- $oil-Sol, Soce ',4,m. Proo, 12: l95-(l94?) 21. Bryner ancL@-a@6h@- -6:- 281--(1958) 22. -bryner, Lofeif; end Anderson, Ind 6ng. Chem. 4 "721 (195?) 223. Aleksandrov,- i-9 4.@ 91) (1950) 24. Eno and lieiiszer Soll@@@t--L99 11 0 - -(-1955) 26. Leatne)n, tiralty ,And M616tyee, Apol 141o.roDiol-;:I@@@195 27. Temple and-Cd mer, a o t 5 (I 9 51