[PAGE 1]
NKULaRA Subprojsct

At Date of Criginal Period Bne Extended: Allobent Ascunt or Authorization Covered To Ruobes Czigetion

XNVOTCE CHOK LISP

[PAGE 2]
Cost Account h125-1390-3902

Object Class

[PAGE 3]
at29 July 1963

MEMORANDUM FOR: C/TSD/FASS

SUBJECT

; MKULTRA,Subpcojest\_No.133

Under the authority granted ia the meoiorandem from the DCI to thè DD/A and the extension of this authorty memoranda, Subproject133 has een approved, and\$9,360.00 of the over-all Project MKULTRA funds have been oliated to subproject's expenses and should be charged to cost cntor 4125-1390-3902

oAsst, Chiet, TSDforND

Concur:

APPROVED FOR OBLIGATION OF FUNDS:

Chief, DD/P/TSD

Date:

Distribution: Orig & 1 - Addreosee 2 TSDI BF

Chief, Sci. TSD/R&D

Ceayy14, 680 XD-4,680

CETEY THT FNOG ARS ATARARLE 9
4125-1390-3902 二 AVinONIRss OrNC...

133-2

[PAGE 4]
P E A   /     E  t t  dn A

[PAGE 5]
3125-1390-3902 Cost Account

Object Class

133-4

[PAGE 6]
Decot à. Jaly jve

ERASDON FOR: The Cerptrol1er

ATTENTICA :Plamee Divietda.

SUHECT 1 EULTrA, Suopoje:t 133

3 Vade qo auteeri 3. grested iu rhe carrd 13 i 2
frco the to the Dof. and the extenoico cY rhãs MAECIA in sub-31 Y \$9.0s.00
:f :20 cr-al 2 K7WRA fuds …1 aka ExT2249 sul aheend te 2 eect

5 Sramnb

22 i Griginel signed by Sidney Cottieh Acdon

2510

Wtarieuetcp

V1. ISD FASS BBCalega IVC 2--TSD.BB

133-5

CEOAR 040176 103125-1390-3909

[PAGE 8]
Cost Account2115-590-1902

Object Class

133-7

[PAGE 9]
DATS: 25 Mey 161

MEORANDUM FOR A EE COFIROLLER

ATIENTTON

Finacce Division

SUBJSOT

: ULA, Subprojact133

Uader the euthority grented in the pezorardoa dated 13 Aycil 195

frca tha DOI to the-Do/A, eni the extension of this euthority 1n sub-

eequent czsoronde, Subgrojeot 133has been approved, and\$2,36o.00..

of the ovor-all Project-lUJtA fuds bave beei obligated to cover tha

aubproject's expeoces sod should-be\_chargetto\_ccst contor\_2l25-130:36e.

Chte? TSD/Reacerch Branch

APPRCVAD FOR ORLIGATICK

OF FUSDS:

CERTIFY THAT FUNDS ARE AVARLASLE CSUGATION RELRENCE 010 235139396

AUüKORZING OFFCER

Date:

12350

Distrlbutioo; Ocig. & 2 - Addrecsee XTSD/FAES 2-TSD/RB (1 chron, 1 file(finance)

133-8

G

[PAGE 10]
12 July 1961

MEMORANDUM FOR: Chief, FInance Divlsion

VIA

: \_TSD/Budget Olllcer

SUBJECT : MKULTRA, Subprofect 133, Involce # Allotmeat 2125-1390-3902

1. Invoice No. l covering the above subproject is attached. Payment should be mada as followe:

Cashler's check lu the amount of \$9, 000.00 drawa on a payable to B

Cashlorie chock in the amount of \$360. 00 drawn on a payable to

2. Please forward the checks to Chief, TSD/Research Dranch, through TSD/Budget Officor, no later than 24 July 1961.

3. Thle In a final involce. However, since it ls anticipatod that additional funds will be obligated for this project, the Cles should not be cloaed,

8033

133-9

[PAGE 11]
13--10

[PAGE 12]
71.3 S0OTLIOS
7050
282
4--5

[PAGE 13]
CERTIFICATION

This is to certiry that I have received an aceunting from MULIRA Sub-project f 133 for the period 1 September 1964 - 30 Apr11 1965. The accouating reflecte expezses in the aoount of \$307.88 for the resaining grant balance.

The accouating statomcat and paymont certification vill bo retained by TsD and will te nade avallable for review in TSD, if necessary.

I certiry that satiefactory aervices represeated by the accouating havs been received and that to the bost of zy kaowledge and belier the fuads expended were for tha puposes authorized by the project approval.

133-12

7

[PAGE 14]
I dertify that services or rateriala havo teen: satisfactorily received e oxpenditures Fere incurred on officialnsg

[PAGE 15]
The current balance on account 440.11 i6 \$307,88. Thie sum will oe used for psyment of puolioation coste of a paper in CONOMIC GEOL GY ana the remainder for the puronese of expendable laboratory items.

[PAGE 16]
October 12,1964

In response to your letter of October 8, attached please find an accounting ofe funds provided for rasearch performed under the dlrection of for the strid beglaning Septenber 1, 1962 and ending C

We trust that the report Is sufflciently detalled for your purposes.

Please advise what dispositlon should be made of the unexpended balance.of \$307.88.

4 Assistant to the Comptroller

133-15

[PAGE 18]
12 0ctober 1964

12 Thie ie to soknowledge tne extenaion of the expiration taccount no.) fron the date of Wora of the exteneion wae tranamitted to me via teleohone by on.12 0ctober 1964.

The ourrent balance on account 440.11. is \$307.88. Thie eum will be-used for payment of publication costs or a paper in CONOMIC GEOLOGY ena the remainder for the purchase or expendable\_laboratory iteme. A

Thank yor for permitting this d@ extension.

cc Research Division Comptroller

[PAGE 19]
MOMORANDUH POR : Chief, Covert Claine Branch

SUBJECTMULTRA SubOJect 133

Atteobed aro certifications from Chief, Biologtcal Brench,

for HULIRA Oubproject 133 Invofces 2 and 3 ant sbould be recor-

and in accout 760.

10130 C/TSD/SS

Distribution: Original & 1 - Addressce

133-17

19 October 1964

[PAGE 20]
October 12,1964

In response to your letter of October 8. attached please find an accounting of ws funds provided for cesearch performed under. the direction of for August 31,1964

We trust that the report Is sufficlently detalled for your purposes

Please advise what disposition should be mado of the unexpended balance of \$307.88.

Very truly yours

133-18

enclosure

[PAGE 21]
Supplies & Expenses

I certify that servire ar atoclals have been satisfactorily receivel… the exponditures vere incurred cn offioial h,siress.

Hicroblology of Kinerals

[PAGE 23]
DATE TRANSMITTAL SLIP TO: TSD SS ROOM NO. BUILDING

KEMAKAS: ORIGINAL AUTHORIZATION FOR SUB 133 WAS FOR \$9,000. HOWEVER, ONLY 48,50O PASSED BY H(THE REMAINING FSCO IS PART OF \$2914.93 STILL TO BE RETURNED BY THIS ACCOUN TING WITH \$16O.39:ADDED FOR PUBLICATON AND REPORT CHARGES CLEARS OP THE \$850O
UNDER INVOICE 1.

133-20

[PAGE 25]
Salerles & Yagus 3u2e11es \$.Exjsn70s traveli ether Costs  Roprints Indlrect Coats 14%

Interla Report 41
210410/1/61-11/30/62
\$4,673.85
2.435.12
194.48
12.00
37,315.45
1.024.1€
\$8,339.61
160.39
8,500.00

I have exaninecaltted expenditure.

T Datea 2/1/63 1

44006

133-21

[PAGE 26]
During the previous year concentrated his efforts on.the elucidation of mechanisms ofimicrobialaction on mineralsulfides and manganese nodules.

Bacterial dissolution of As2S3, FeS2FeAs2,\_and 3Cu2SAs2S5 was demonstrated.The end products wore shpwn to-be arsenite, arsenate, Fe, Ferr, and Cu", with the fate, of the\_sulfur stiil undetermined.

Studies on CuS indicated that Thiobacillus sp. were unable to solubilize this mineral in pure culture. They found that dissolution of this mineral under natural conditions necessitated the presence of protozoa and fungi. These organisms were all active under-conditions of copper concentration which would normally be lethal. 10

Samples-of manganoso nodules collected by in B the Atlantic.Occan (as a guest of a were studied todetermine the nature of possible bacterial transformations. A-preliminary scheme for this activity was determined to be a non-biological dismutation Mn and MnO2 forming Mn// This latter.ion must be stabilized and it is likely that organic by-products\_of bacterial metabolism play this-role.

has furnished three cultures with interesting characteristics for application to the power. source. studies. under MKULTRA # 78.

During the present year methods for mass culture of mineral oxidizers-have-been-developed which will\_furnish cell material for enzymatic and resting cell studies..Continued work on manganese\_transformations is cmphasizing environ mental effects.

133-22

PROJECT CRYPTO

MKULTRA

[PAGE 27]
Date 5 Septomber 1963

BranchBB Category NEW MATERIALS & CONCEPTS (X) (formerlyIVc)

Microbial transfor-Project Title mations of Minerals Item Classifcation Unclassified (Reports)

Unclassified. Project Crypto. MKULTRA 133 Crypto.Classifcation

Branch Project No N.A.Project Engineer

Contract No... N.A. Task No N.A..

Type of ContractMKULTRADate-Initiated 12 July 1963

Cost \$9,360.00 Comopletion Date Continuing

Purpose: To provide the services of  and to support research on fundamental-mechaaisms-o mineral transformation fy microorganisns.\_in\_addition\_to the direct potontial for new energy sourcc systems, the, contract may provido byproducts.useful for material ceterioration application.

Status: Current and satisfactory.

Requirement: Intornally generated in support of tho roquironents to seek new and better sources of energy for battery application.

133-22 REVISED

[PAGE 28]
Receiptis:hereby acknowledged\_of Check No. 0102970, dated 23 August 1963, drawnion the

Receipt is hereby acknowledged of Check No. 0102971, dated 23 August 1963, drawn: on the

DATE Syd 10 196 3

11V 3

133-23

RECEIPT

[PAGE 30]
29 July 1963

\$9,360.00

4125-1390-3902

Chief, Sei. TSD/R&D

133-25

[PAGE 31]
BranchBB Category\_New Materiale\_ &\_Concepts (X)

Microbial transformations Project Title of Minerals Item Classification Unclassified (Reports)

Project CryptoMULTRA #133 Crypto ClassificationVnelassified

A Branch Project No. N.A. Project Engineer.

Contractor.

Contract NoN.A. Task NoN.A.

Date Initiated 12 July 1963 Type of Contract MKULTRA

9360.00 Continuing Cost Completion Date

Purpose: To provide the services of and to support research on fundamental mechaniams of mineral transformatión-by microorganisms. In addition to the direct potential for new energy source systems, the contract may provide by-products useful for material deterioration application.

Status: Current and satisfactory.

REQUIREMENT SOURCE: Intornally generated in support of the requirements to seek now and better sources of energy for battery application.

133-26

Date 29 July 1963

[PAGE 32]
MEMOKANDUM FOR:THE RECORD

: SUBJECT : Continuation of MKULTRA, Subproject-f133

2. continues to provide data and materials which lend themselves to nev and unique approaches to energy production and transformation (bio=batteries). His studies on basic mechanisms of mineral transformations also provide new potential paths for deterioration of metale.n

3 will function as

and  during this fiscal year. The cost of this program for one year wi11 be=\$9,000.00 to which must be added \$360.00 which represents a h% service-charge to be retained by the The total cost of the program wi11 not exceed \$9,360.00. Charges should be\_made against Allotment No. 4125-1390-3902.

4. It is-not anticipated that pernarent equipment other than that 1 listed in the budget will be required for this program.Title to the equipment listed will be retained by the  in lieu of higher overhead rates.

133-27

DRAFT

29 July 1963

[PAGE 33]
5. Documentation and eccounting for travel expenses which are rewili\_conform to the imbursable by accepted practice of that organization.

6. has been cleared COVERTLY and:is unwitting and will

remain unwitting of the true nature of the sponsor.

Attachment: Proposal

Distribution: Original\_only

133-27

2

[PAGE 34]
Submitted on bchalf of

April\_1963

[PAGE 35]
This proposal is a request for renewal for the period 1\_Septepber 19o3-
31 August 1964 of the grant-in-aid presently supporting research on The field of investigation covored by the present grant is so broad and
relatively unexplored that several additional years of research can be profitably
spent in unraveling the problem, The results of tho research carry increasing sig
nificence not alone academically but also practically, as in public health, conser
vation of mineral resources, mineral exploration, and mineral extraction. In public
health, these investigations are providing clues to the possible origin and control
of acid ferruginous, and/or acid cupriferous, and/or acid arsenical-stream pollu
tion, deriving from bacterial action on natural mineral deposits in certain geo
graphical locations,With continued population increase, maintenance of all water
resources in a condition fit for human consumption becomes, more and more important.
The work is also providing additionel clues to the undorstanding of manganese bio
chemistry in publić water supplies, where manganese together with iron can ceuse
discoloration of wáter and water containers, and whore manganese and iron are de
posited biologically in water mains and pipes. In the area of conservation of provides a básis for assessing and con
mineral resources, the work under trolling any relative instability of minerel deposits in relation to microbes. It seems evident that uncontrolled bacterial leaching could conceivably lead to depletion and eventual complete translocation of some deposits, In mineral nloration, this work indicatos that special groups of microorganisms known to be able to livo at the expense of a specific mineral may bo of great assistance in discovering new sites of minoral deposits. Finally, this work lends additional-support to the practice of using microbes in mineral extraction

133-27

PURPOSE OF STUDY

[PAGE 36]
SUMMARY OF WORK ACCOMPLISHED UNDER THE 1962-63 FROM (a) Microbial Action on Mineral Sulfides

Quantitative studies on the action of the Thiobacillus-Ferrobacillus group of bacteria on-the minerals orpiment (As,S,), arsenopyrite (FeS,FeAs,), and enargite (3Cu SAsS.). were initiated.The bacteria achieved significant solubilization of orpiment.:releasing arsenic as arsenite and arsenate. Oxidation of the sulfur portion of orpiment is probable, but remains to be experimentaliy verified. Some spontaneous, nonbiological oxidation of orpiment occurred, but it was only about one-third as extensive as with bacteria, The chemistry of bacterial oxidation of orpiment appears.to differ significantly from nonbiological oxidation as reflected by pH changes during the processes, With bacteria the pH fell from 3.5 to 2.0 in thirty-five days. but without bacteria it rose from 3.5ito 5.0 in that time. The precise chemical mechanism of orpiment\_ oxidation remains to be worked out.

Quantitative work on bacterial oxidation of arsenopyrite showed release of iron, arsenic, and probably sulfur, from the mineral. Most of the analyses in this study were carried out by an undergraduate student for his senior thesis. The results have shown that, contrery to a sustained release of soluble arsenic from oroiment by bacteria, only a limited amount of solublc arsenic\_wes released\_ by\_them from arsenopyrite, : This happened in spite of a pronounced, release of iron.  The reason for limited release of soluble arsenic by bacteria, from arsenopyrite is the precipitation of iron\_arsenites and arsenates after a critical concentration of soluble iron and arsenite\_and\_arsenate\_has been\_reached, Although some oxidation of arsenopyrite occurred in the absence of bacteria, its extont was less and its chemistry different, because iron arsenates and arsenites wore-not precipitated without bacteria, and the pH of the medium dropped from 3.5-to 2,5 in thirty-six days with bacteria and rose from\_3.5\_to 4.0 in that time without bacteria,

133-27

[PAGE 37]
The extensive reprecipitation of iron and arsenic through bacterial action has direct implications with respect to possible translocation of the constituents of arsenopyrites in nature. The bactorial phenomenon also poses a problem when arsenic and iron are to be extracted togethor from a natural mineral deposit.

Quantitative work on bacterial oxidation of enargite\_ has given-results resembling those with arsenopyrite. Although the bacterie are evidently acting on the mineral, a sustained solubilization of the component arsenic is not noted. Indeed, in the presence of bacteria the dissolved arsenic concentration drops after an initial rise. In the absence of bacteria the arsenic concentration rises slightly but continually. -It is not clear from the results so far what the fate of the arsenic or copper is after bacterial action because no reçognizable precipitate was formed. Contaminating iron is released extensively by bacteria from the mineral but not without them. . The released iron appears to remain in the-ferrous state. The pH changes are from 3.5 to 2.5 with bacteria, and from 3.\$ to 4.5 without bacteria, in thirty-six days.A precise description of the chemical changes that enargite undergoes remains to be worked out.

Growth on cuprous sulfide by the Thiobacillus-Ferrobacillus group, investigated chiefly by a graduate student, is showing variable responses on different synthetic preparations,Bacterial enrichment cultures derived from sevoral mine effluents during the.past year have, howover, given more consistent growth responses on these preparations,The possibility arises that consistent action on cuprous sulfide requires the participation of more than one organism.In this connection, the principal investigator found protozoa and fungi acccapanying the Thiobacillus-Ferrobacillus group of bacteria in mine water from The protozoa included an amoeba and a flagellate, which appoared to grow at the expense of the bacteria and fungi. The amoeba wero observed to ingest such organisms,

133-27

[PAGE 38]
Both kinds of protozoa are umusual in their tolerance of 800 ppm copper and upwards of 2000 ppm iron, Ordinarily\_these\_metal concentrations would be expected to be lethal. The flagellate has been repeatedly subcultured in an iron-salts medium in mixed culture with the Thiobacillus-Ferrobacillus group of bacteria.

(b) Manganese Nodules

During three cruises in the Atlantic Ocean in June, July, and August, 1962, as guest of the thirty-four manganiferous samples end twenty-four cores of bottom deposits were taken.About two-thirds of the manganiferous samples have now been tested for their bacterial content and for the ability of these bacteria in aiding manganese addition to the respective samples, as previously described by Most of these samples were also analyzed for total iron and manganese content. The results showed that the various manganiferous samples were\_by no\_means alikc. In some\_ cases this was obvious by visual inspection. In other cases, however, the differences were. not macroscopically apparent, Instead, the iron-menganose content, or the bacterial content, or the Mn adsorptive power of the samples, showed diffcrences. Results of experiments, designed to test for the enhancement of Mn adsorption by manganiferous material with the help of the native bacterial flora, could be divided into four major categories. In one, bacterial enhancement\_of Mn adsorption occurred with or without prior surface-sterilization.In a second, it occurred only with surface sterilization, and in a third only without it, In a fourth, bacteria1 enhancement of Mn adsorption was not noted under either condition, Since not all bacteria from Mn nodules can enhance Mn adsorption, inclusion of surface-sterilization in the procedure causes selection of different types of organisms from\_the\_nodule\_flora than omission of surface-sterilization.However, the full explanation for the variability of bacte. rial enhancement of Mn adsorption is more complicated than that. With a number of

133-27

[PAGE 39]
samples it was noted that Mn uptake by manganiferous materlal resulted in a net loss of manganese from the adsorber. The reaction accounting for this phenomenon is the result of a dismutation between Mn²+ and Mn0, and will be discussed further below. This reaction was exhibited by about one-sixth of the samples tested and shows that the properties of these samples are different from the rest. It can therefore be concluded that chenical and physical properties of manganiferous meterials help to determine the effectiveness of bacteria in enhancing Mn adsorption. Moreover, since all but one of the samples of the 1962 collection came from extinct submarine volcances (sea mounts),and since-quite a few of these samples behaved differently from earlier samples from other sources, it is postulated that geographic location of manganiferous samples may affect their chemical, physical, and biological properties.

A clue to the nature of nonbiological Mn release from manganiferous samples is provided by an experiment which shows a dismutation reaction botween Mn²+ and MnO2 forming Mn3+ when nodules adsorb Mn², on the condition that Mn³+ is stabilized so as to-prevent it from undergoing thc reverse-reaction.Pyrophosphate was found to be an effective stabilizer, Since pyrophosphate does\_ not occur in a marine environment, protein or amino acids may serve instead as-stabilizors. Nodule breakdown can thus be accomplished biologically with sugar as \_the reducing agont of MnO, 2 or nonbiologically by a dismitation reaction botween Mn²+ and MnO,. Why the 2\* latter reaction occurs with only some nodules, even though all have Mn0, and adsorb 2 Mn²+, remains to be explained.

The ability of Arthrobacter to aid in Mn addition to nodular substance was shown in pure culture experiments, From similar tests it could be concluded that Vibrio can similarly aild in Mn eddition, but not Achromobacter, Al1 three organisms have been-found in nodules. Arthrobacter, at high peptone concentrations, causes clumping and heaping of nodular matorial; not so at low peptone concentrations. A similar phenomenon has been notod in soil . The clumping and heaping

133-27

[PAGE 40]
seems to reduce surface area. and therefore slows adsorption of Mn.

Initial experiments have rcvealed that Arthrobacter can aid in Mh adsorption by "synthetic" Mno,. However, the adsorption and incorporation process of M²+ is not permancnt. Ths previously cited dismutation reaction seems to come into play eventually, probably when the bacteria become physiologically inactive\_at the end of their growth cycle. "Synthetic" Mn0, is a much poorer adsorbent than nodule matorial, It is postulated that the iron in nodule material helps to stabilize the manganese in it and, for as yet unknown reasons, increases its adsorptive power.

The core samples of bottom deposits, collccted last summer, were examined by enrichment for bacterial content. The distribution of bacteria in these cores was not uniform, nor were tho bacteria necessarily of the same kind at different depths in the same core. Theso findings are similar to those cited by Zo Bel1". Work is presontly under way to test the ability of the core samples to adsorb Mn with and without bacterial growth. Differences. in Mn adsorptive capacity seem to exist among cores on the basis of tests so far, Such differences, if corroborated by further tests, rust reflect upon Mn distribution in the sea and upon nodule distribution and structure.

REFERENCES

1. de Grys, Ann. Econ, Geol. 57: 1031-1044 (1962), C

3. Martin, J.P., and S.A, Waksman. Soi1 Scienco 50: 29-47 (1940).

4. Zo Be11, C.E. MARINE MICROBIOLOGY. Chronica Botanica Co., Pub1. 1946, p. 91.

133-27

[PAGE 41]
PROGRAM OF FUTURE WORK

(a) Mineral Sulfides

Although it is the intention to continue testing other\_mineral sulfides for susceptibility to\_attack by the Thiobacillus-Ferrobacillus group, special attention will be paid to the details of bacterial action on orpiment, arsenopyrite, and enargite. Quantitative methods have to\_be\_developed for determining the proportions of various forns of a given element dissolved and reprecipitated after bacterial action. Such information should provide clues to the mechanism of action by the becteria. The detailed information on the fate of arsenopyrite and enargite, when available, will provide information concoring the\_effcct of-side\_reactions\_on the overall process of bacterial mineral oxidation.. Ultimately; it is hoped\_to\_develop methods of mass culture of a desired\_organism on a particular mineral\_to get\_enough cell material for resting cell studies and cnzymatic investigations.

(b) Manganese Nodules

Two aims will be followed experimentally in this work. One is the further elucidation of environmental relationships to\_the\_process of nodule development or degradation. The second is the\_elucidation of the biochemical mechanism whoreby Mm is added to, or released from, manganese nodules by bacterie. To attain the first aim, samples from various known scurces will be studied in respect to physical, chemical, and biological properties, and attempts will be made to corrclate the results with geographie location. In studying the manganese fixing capacity of bottom muds, possible correlation with nodule development will be sought. To attain the second aim, further studies with purified cultures from nodules will be made on nodule matorial and on synthetic Mn0, to compare and contrast behavior. Since pre-2 vious exporirents have already shown a difference in action by bacteria bctweon nodule material and Mn0,, a condition is provided to compare the effect of iron on the two systems. It is planned to develop nore specific assay procedures for

133-27

[PAGE 42]
differentiating the various oxidation statos of Mn and thereby to follow the exact fate of adsorbed or desorbed Mn, and to establish more clearly the role bacteria play in this process. -The ultimate gool is to study the bacterial action\_on manpanese on an enzymic level.
B
FUBLICATIONS ARISING OUT OF THE WORK UNDER THIS FOR 1962-63

(5) It is enticipated that a paper will bo written for submission to Appl. Microbiol. on the work with manganese nodules during the last year.

PERSONNEL FOR 1963-64

Principal Investigator:  Associate Professor of Biology

133-27

[PAGE 43]
Personnel

Apr11 1963

133-217

[PAGE 44]
MEMORANDUM FOR: C/TSD/FASS

SUBJECT MKUL.TRA, Subpcoject No.133.

Under the authority-granted in ths menorandeo sate"

from the DCI to the. DD/A,. and the axtsasion ci. this utac. .y

memoranda, Subproject133has en proved \$9,360.00

of the over-all Project MKULTRA funds ha  teea oali at::t.

subproject's axpenses-and should be charged to co4125-1390-3902

Concur:

Asst, Chief, TSDfer R&D

Concur:

APPROVED FOR OBLIGATION OF FUNDS:

Chief.D0/P/TSD

Oate:

Distation: Or: 1: 1- Addressee 2. IS0/

133-28

29 July 1963

[PAGE 45]
Date 18 February 1963

Branch CategoryBiological Energy Systems (IVc)

Project Title..of Minerals Item Classification Unclassified (Reports)

Project Crypto KULTRA Sub. # 133 Crypto Classification Unclassified

Branch Project No.N.A. Project Engineer

Contractor. C

Contract No.N.A. Task No. N.A.

Type of Contract\_\_ MKULTRADate Initiated12 July 19€1

Cost \$9,000.00 Completion Date Continuing

Purpose: To provide the services of and to support research on fundanental mechanisms of mineral transformation by microorganisms. addition to the direct\_potential for new energy source systems, the contract-may-provide-by-products-useful for material-deterioration application.

Status: Current and satisfactory.

SOURCE: Internally generated in support of the requirement to seek new eni better.sources-of enorgy for battery epplication.

REVISED 133-29

[PAGE 46]
I hereby acknowledge receipt\_of the following:

Check No. 0069284, dated Oct 11, 1962, drawn on the

in the amount of \$9, 000.00.

Check No. 0069286, dated Oct 11, 1962. drawn

in the amount of \$360.00

133-30

[PAGE 48]
3.kRASUM FOR: The.Ccptrollar

ATCTETION Ficepea Divisien

SUNECT ■1 LUL FA. Sobprojeet 2 133 一

Uacer .tos suthority greared in the wasócaedaa cared 13 Aail
froa tão tc 4 ad C 1113 3 六
aeqent cedcrasd 2 5 133 PAS bEER aporqvad a04 \$9,360.00 cvat-ail r E0LTA Porde 1 odeo à te SCET 3125-1390-3902

AP2R0E1OR O00ATT02

xxxxcxxxxxxxxx Acting Chief, TSD ule2519

Praarre

133-32

Daàs: 24 July 1962

[PAGE 49]
SUBJECT :Continuation of MKULTRA; Subproject 133

1. The purpose of MKULTRA, Subproject 133is to enable TSD/BB B to utilize the\_services-of

2. During the first year of the program has made significant contributions to the all too scanty knowledge of the mechanisms of mineral transformátions. A technical discussion of these accomplishments is attached hereto, with an outline of proposed work for the coming year. It is possible that these investigations may well lead to new approaches for energy transfer systems (bio-batteries) and deterioration of metals.

functioned as and during the first year of this project. This service will heretofore The cost of be furnished by the this program-for the second year will be \$9, 000.00 to which must be added \$360.00 which represents a 4% service charge to be retained by the The total cost of the program; therefore, will not exceed \$9, 360.00. Charges should be made against Allotment No.3125-1390-3902.

4. It is not anticipated that permanent equipment other than that listed in the budget will be required for this program Title to the

133-33

DRAFT

24 JUL 1962

MEMORANDUM FOR: THE RECORD

[PAGE 50]
-2- equipment listed will be retained by the lieu of higher overhead rates.

5. Documentation and acconnting for travel expenses which

the accepted practice-of that organization.

6.has been cleared COVERTLY and is unwitting

and will remáin unwitting of the true nature of the-sponsor. a

TSD/BIOLOGICAL BRANCH

APPROVED FOR OBLIGATION OF FUNDS:

AC/TSD

DATE:\_

Attached: Budget Proiect Summary and Proposal

Distribution: Original only

133-33

[PAGE 51]
Attenticn: Subject: Preposal-entitled

Please find enclosed one copy of the subject proposal submitted on behalf of

The proposal is not being submitted elsewhere for possible supporti

Your-consideration of our proposal will\_be appreciated and we-look forward to hearing from you.

133-33

Enclosure

[PAGE 52]
Proposal entitled

133-33

Submitted\_on\_behalf.of Associate Professor\_of Biology

June 1962

[PAGE 53]
Purpose of Study:

The purpose of this proposal is a request for financial support to continue an-investigation of microbial action on marine manganese nodules and terrigenous mineral sulfides, which the principal investigator has been-pursuing since 1958. Very intensive work on these materials is being carried on-by him, with fruitful results, during the current year, 1961-62, under a  from the

Sunmary of Past Work:

a. Bacteriology of mineral sulfides.

Attempts were made-to evaluate the microbial flora isolablefrom unsterilized, crushed sulfide minerals by enrichment in mineral solütion. The folloving minerals were studied:alabandite, arsenopyrite, bornite, chalcocite, chalcopyrite, cinnabar, cobaltite, covellite, enargite,-galena, marcasite, orpiment, pyrite, pyrrhotite, realgar, and sphalerite. Of these minerals, only cobaltite, enargite, galeta, pyrite, pyrrhotite, realgar, and sphalerite yielded microorganisms. For the most part these organisms were heterotrophic and probably represented contaminants. However; Hyphomicrobium, isolated from realgar, a pink yeast repeatediy isolated

133-33.

DEPARTMENT OF BIOLOGY

June-14, 1962

[PAGE 54]
from sphalerite, and Arthrobicter, isolated from cobaltite, galena, pyrrhotite, realgar, and sphalerite may constitute part of a normal flora. The action of any of these organisms with respect to the mineral with which they were found associated remains to be established.

After surface-sterilization, some of the above mineral sulfides, when enriched in mineral solution, have yielded iron-oxidizing autotrophs. These minerals include arsenopyrite, pyrite, pyrrhotite, chalcopyrite, enargite, galena, marcasite, and sphalerite.-At least some of the isolated bacterial strains are not restricted to a diet of iron for energy, but can use sulfur or, probably, some other oxidizable metals.

The ability\_to.grow on any of the above sulfide minerals was tested by inoculatiag surface-sterile\_ samples in oxidizing.columns with Ferrobacillus ferrooxidans,-and-attempting to recover the organism from effluent fceding solution over a period of two months or more. So far, positive results have been obtained with arsenopyrite, enargite, chalcopyrite, marcasite, galena, pyrite, pyrrhotite, and sphalerite.Negative results have been obtained with alabandite, bornite, cobaltite, covellite, chalcocite, and onc sample of galena. Cinnabar, orpiment; and realgar are being currently investigated.

In addition to the foregoing qualitative work, quantitative studics on the rates of oxidation of synthetic CuS \_and natural arsenopyrite are presently being undertaken. From these studies it has become clear that synthetic CuS can be oxidized at least 4x as fast by bacteria than by autoxidation, and-that arsenopyrite can be more rapidly oxidized by bacteria than-by autoxidation. Results with the latter material are not yet sufficient to establish an exact rate comparison. The precise mech-

-2-

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[PAGE 55]
anism of bacterial oxidation remains. to be established.The work with synthetic Cu,S proves, what some other workers seem to doubt, that F ferrooxidans can-oxidize metals other\_than iron.

b. Manganese Nodules

Oceanographers have-felt pretty strongly in the past that the origin and development of manganese nodules in the oceans is attributable to purely physicochemical processes.However,

on finding organic nitrogen in nodules, concluded that biological agents were involved In nodule genesis. At his suggestion, the principal investigator attempted to find out if bacterla might play a role- 1n-this. He found that bacteria were-indeed present in the nodular-substance after surface-sterilization-(a rough estimate at present is 104 per gram). They included Achromobacter. Arthrobacter, Bacillus, Brevibacterium, Staphylococcus, Vibrio, an unidentified rod, and an unidentified coccus. The-principal investigator:showed in\_quantitative experiments that nodular substance can adsorb manganous ion from sea water, and that this adsorptton is accelerated by bacteria:that grow from the nodular material.The acceleration of manganous ion-adsorption is explainable on the basis-that the bacteria oxidize the adsorbed manganese, which facilitates further adsorption of manganous manganese. The acceleration requires the presence-of peptone, to pcrmit bacterial development.If peptone and glucose are present, manganese is-released from the nodular substance rather than adsorbed, at least in a net effcct.Since some nodules were apparently initiatcd around shark's teeth, ear bones of whales pumice, etc., in the sea, attempts were made to see. if oyster shells can adsorb manganous manganese and thus serve as possible foci of nodules.

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[PAGE 56]
It was found that they do adsorb it and that peptone did not stimulate this adsorption (no\_bacteria were present!). As far as a survey of the lterature has gone, these-observations with respect to manganese nodules have not been reported before.

Pertinent literature:

The early. literature dealing with microbial action \_on minerals has
been covered by Alexander (1).A review by Lyalikova suomarizes much-of
the past important work on Thiobacillus ferrooxidans and-Ferrobaciilus
ferrooxidans(2) An intimate association of-iron-oxidizing autotrophs
with natural mineral sulfides has been indicated by the -work of and by that of Lyalikova (5). Differences of optnion-exist between
Bryner and Anderson-(6), Malouf and Prater (7), and-Ivanov, Nargirvyak, (8) on the other
and Stepanov on the- one:hand, and
about a mechanism of mineral sulfide oxidation of chalcopyrite, molybdenite
chalcocite, and sphalerite, for instance. No previous studies on bacteria
in menganese nodules has been reported. However, bacterial manganese oxidation and reduction by-soil bacteria has been known for-some-time.An important quantitative study on large-scale bäcterial-manganese metabolism is that of Mann and Quastel-(9). Descriptions-of manganese nodules are-given by Murray (10) and Dietz (11). A chemical and physical study of nodules
was made by Buser and Gruetter (12). The fioding of organic nitrogen in nodules was first. reported byGraham (13) and Graham and Cooper (14), who also suggested a biölogical origin-of the nodules on this basis.

7

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[PAGE 57]
1. Alexander, M., INTRODUCTION TO SOIL MICROBIOLOGY, John Wiley & Sons-Co., 1961.

2. Lyalikova, N.N., Mikrobiologiya 29: 773-779 (1960).

5. Lyalikova,N. N., Mikrobiōlogiya, 30: 135-139(1961).

6. Bryner, L. C., and R. Anderson, Ind. Eng. Chem. 49: 1721-1724 (1957).

7. Malouf, E.E., and J. D.Prater, J. Metals 13: 353-356 (1959).

8. Razzell, W. E., Annual Western Meeting, Vancouver, Oct. 1960 Transactions, LXV, 1962 pp.-135-136.

9. Mann, G., and-J. H. Quaste1, Nature 158: 154-156 (1946).

10. MUrray,J VOYAGE OF H.M.S. CHALLENGER. DEEP SEA DEPOSITS. Her Majesty's Stationary Office. 1891.

11. Dietz, R. S., J. Calif. Mines and Geol. 51: 209-220, (1955).

12. Buser, W. and A. Gruetter, Schweiz. mineralog. .petrogr. Mitt. 36:-49-62,(1956).

13. Graham,J.W., Science 129: 1428-1429 (1959).

14. Graham, J. W. and Susan Cooper, Nature 183: 1050-1051,

Pertinent Publications by Principal Investigator:

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References:

[PAGE 58]
Continuation of present lines of investigation:

a. Mineral-Sulfides:

Continuation of survey of natural sulfide minerals for a normal flora, with particular emphasis on-large-scale microbial-action on minerals.

2. Characterization of isolates (physiologlcal and morphological).

3. Examination of isolates for specific mineralizing activities.

4. Elucidation of blochemical mechanisms of mineral transformation.

b. Manganese Nodules:

1. Qualitative and quantitative bacteriological comparison of manganese nodulesfrom different oceans.

2. Study of the blochemical mechanism of manganous oxidation and MnO reduction in the various bacteria-isolated.

3.-Deteraination of the mechanism of iron-incorporation into manganege nodules.

The methods to be used in these studies will be adaptations of standard procedures of\_bacteriology, physiology, and biochemistry.

Personnel:

Principal Investigator: Assoc. Prof. of Blology.

Technician:

Graduate Student:

(not presently supported)

Undergraduate student: ndergraduate research fellow)

(smmer-1962) (summer1963)

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Proposed New Work:

[PAGE 59]
CONSUMABLE SUPPLIES Chemicals S 300 Glassware 340

OTHER EXPENSES Publication \$--180 Telephone 20

[PAGE 60]
Date 24 July 1962

BranchBBCategory Biological Energy Systems (IVC)

Project Title Microbial Transformation Item Classification. Unclassified (Reports) of Minerals

Project Crypto\_ MKULTRA, Sub #133 Crypto ClassificationUnclassified

Branch Project No. N. A. Project Engineer. A

Contractor blc

Contract No. N.A. Task NoN.Ai

12July1961 Type of Contract MKULTRA Date Initiated.

Cost\$9000.00 Completion Date Continuing

Purpose: To provide\_the services of and to support research on fundamental mechanisms of mineral transformation by microorganisms.

[PAGE 61]
Gantlemeo:

We are ploasod to be ablo to transmit to you the following

E No, 268654 drawn cn tho for \$360.00.

Cashler'g Check No. 2-064674 drawn on the 1oz-\$9,000.00

These funds ropresent a contributicn for the use of your
directore in carrying out the very worthwhile rosearch goals
of your organization. Yours truly,

Enclosures (2)

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14 Augunt 1961

[PAGE 62]
12 July 1961

MEMORANDUM FOR: Chiol, Finance Divislon

VIA 1. TSD/Budget Oflleer

SUBJECT t MKULTRA, Subproject 133, Iavoiee #1 Allotmept 2125-1390-3902

L Invoice No. l covering the above subproject is attached. Payment should be made as follows:

essr payablo toy

Cashlor's check In tho amount of \$360. 00 draya on e B payablo tol

2. Plesse forward tho checka to Chlef, TSD/Rosearch Branch, through TSD/Budgot Offcer, no later than 24 July 1961.

3. This is a fnal lnvoice, However, eince it ia anticlpated that additional funde will be obligated for this projact, the fles should not ba closed.

TSD/Regearch Branch

Attached:

Invoice & Certilficatioos

Diotributlon:

Orig u 2- Addrsasee

1-TSD/FASS

2--TSD/RB

TSD/RBA

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[PAGE 63]
(1) It is hereby certiried that this is Xuvoice 1 apply ng to sub-projeet No. j3 of MuLTRA, that perforuence is satiofactory, that servicen aro being accouplished in accordance vith mutual agreezents, that a detailed agenda of the payuenta and receipts is on file in TSD/RB, that thia bill 1s just aul correct and that payment thereof hae not yet teen male

(2) It is hereby.certiried that this invoice applles to SubProject 133 of HKulTRA which was duly approved, end that the project ia being carrled out in accordance with the menorandum of 13 April 1953 from the DCI to the DU/A, end the extencicn of this authority in subsequent tevoranda.

[PAGE 65]
SUBJECT :-Initiation of MKULTRA, Subproject 133

1. The purpose of MKULTRA, Subproject 133 is to enable TSD/RA to utilize the services of

will conduct a program of research on mineral transforwing microorganisms. The work vill consider both ecological and physiological aspects of the problem. Ways mey also be shown for growing food from minerals by a non-photosyuthetic process because-some bacteria, already known to attack certain minerals, can change carbon-dioxide into organic mattēr with the energy that they deriye from the oxidation of minerals.

shall function 3. as and  for this project. The cost of this program for the first year wi11 be \$9,000.00 to which must be added \$360.00 which represents-a 4% service charge to be retained by the  The total cost-of the program, therefore w11l not exceed\_ \$9,360.00. Charges.should be made against Allotment No. 2125-1390-3902.

4. It is not anticipated that any permapent equipuent will be required for this program.

5. Docugentation and açogunting for travel\_expenses which are will conforu to the reimbursable by accepted practice of that organization.

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MEMORANDUM. FOR:The RecOrd

[PAGE 66]
has been cleared COVERTLY and is unwitting and 6

will remain unwitting of the true nature of the sponsor.

APPROVED FOR OBLIGATION OF FUNDS:

Chief TSD/Research Branch

Date:

Attached; Budget Proposal & Literature Reviev

Distributiou: Original only

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[PAGE 67]
1. Salaries \$4,500.00 Principal Investigator \$4,000.00 Assistant

\$ 200:00 2. Travel

3. Supplies

133-3P

[PAGE 68]
A STUDY OF MINEHAL=TRANSFORMING MICRCORGANISMS

Purpose:

The project proposed herein is antended to provide information on the microbiological participation in mineral transformation. Only scanty knowledge-is:available in this field at the present time. The work will coneider ooth ecologicel and physiological aspectg of the proolem, The findings should be a valuable-contribution to fundamental knowledge in microbiology, geology, and allied fields. They should also be a valuaole contrioution to practical knowledge in these areas because ways may be shown to harness some or these microorganisms to do useful work for Han, such as aiding in the conversion of minerals of low economic value into substancee-of high economic valueWeys may also be shown for-growing food from minerals-by-a-non-photosynthetic process,-oecause some bacteria, already known to attack certain-minerals, can dharge=CO2\_into organic matter-with the energy which they derive from the oxidation of the minerele, (1). This organic matter may then in turn serve as food for other microorganismg whicn may oe used directly or indirectlv es food for Man.

From preliminary-investigations oy the seniorinvestigator. (2), it has become spparent that mineral-transforming miuroorganiems are intinately associated with various types of minerals. The proposed research is designed to follow up the leads from this earlier work to delineate clearly the envronmental end functional

00

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interrelationships-between mineral habitatg endthe-microorganisms found therein.

Objectives:

a) Igolation-of repregentative organisms by enrichment and pure culture methods: . 1. from ores and other minerals 2.from drainage weters of mineral deposits 3.from soils

b) A study of the over-all chemical activity on varlous mineral suostances by enrichment cultires and pure-cultures.The mineral suostances will include

1. natural minerals

2.synthetic minerals

c) A study of the mechanisms of action on various mineral substances by microbial isolates.

plan of Research:

a) Isolation of representative cultures-by enrichment and pure culture methods.

Miorobial enrichments will be derived fron crushed ores,-other mineral substances, and- soilg by percolating nutrlent salt solutions-through percolation columns charged with these materials.The effluent solutions will 5e subcultred in eppropriateselective media, such-es-iron broth (3), sulrur oroth (4); thiosulfate broth (4), nutrient broth, etc.

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[PAGE 70]
Microbial enrichments will also oe derived from flask cultüres prepared by overlaying crushed ore, other mineral eubstancess and soils with a nutrient salt solution.Microbial enrichments of-drainage-waters from mineral deposits will oe made oy inoculating suitable volumes into the various, selective media already oited. Other enrichment methods:may-be used as they become appropriate. The ores and other mineral substences will be used before and after surface sterilization.

Pure cultures fromtthe varicusenrichments will be prepared oy streking on soliairied inorganic and organic\_neaiawhenever possible. In cases-where neither highly-purified agar or silica gels work, a dilution technique ueing selective liduid media may be employed. Culture purity will oe eetablished by aporopriate morphological and cultural obserrations Attempts at establishing the taxonomic identity of the-1solates-will be mede.

o) A study of the over-all chenical activity on various mineral substances oy enrichment cultures and pure cultures. The-over-all chenical activity will be studied with enrichment-cultures and pure cultures-acting on: mineral substances or-known-oompogition.In these-gtudies, qualitative and ouantitative measurements will be made on gubstrate decomposition,-produot accumuletion, change: in tituatable acidity, pH, redox potential, consumption of CO2, 02, and organic matter, if any.A comnarieon

.3.

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[PAGE 71]
of-activitles in enrichment cultures with activities in pure oultures on identical media will reveal whether the cooperation of more than one micpoorganiem is required for a given transformation.

c) A study of the-mechanismsof action on various minerel substances=by micpobial isolates.

The-mechanism of action on various mineral substrates by pure-cultures wlll oe investigated by the use of standard chemical,biochemical, and physiological techriques. The exoerimental approach will include-a quantitative analysis of the-action on pure mineral compounds,=on eoluole forms of the cations and-anions or which the-respective minerals are composed,-and-on the theoretically possiole intermediates of metabolism:Such information will be-required for the formulation ofa scheme that woild explain the possible over-all chemical reaction ooserved.:

Referencest

1. Silvermen and Lundgren, J. dacteriol. 78:326-(1959).

3. Silverman and-Lundgren,J.3acterio1.77:-642-(1959).

4. MANUALOF-MICRO3IOLOGICAL HETHODS, MOGraW-H111 300k 0o., Inc., New York, 1957, p. 114.

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[PAGE 73]
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[PAGE 74]
A STIDY OF MINERAL-TRANSFORMG MICROORGANISHS133-3 Literature Review:

A number of reports exist in the literature\_about microorganieme which seem to be associated with processes of mineral transformation. In many instances, the function which thege microorganisms play is not known or nas been misinterpreted. The following is scdiscussion of some reports on microoial action on minerale common in nature. Sulfur:

An inspection of-tne ?th edition of Bergey's Manual of Determinative Bacteriology reveals that bacteria have been associated:in the past with sulrur oxidations and reductione (1,2). The Thiobacteriacese include members that have oeen snown to oxidize various formé of sulrur more reduced than sulfate,  These organisms are all ooligately or fecultatively chemoautotrophic. Most are strictly aerobic, except for Thiobpciilus denitrificane, which is facultative. Photosynthetic bacteria, belonging to the Onlorobacteriaceae and tne Thiorhodaceae, nave been shown to use inorganic eulfur compounda other than sulrate as-reductants in photosynthesis. More questionablé is the assooiation of nigher bacteria, the Beggiatoaceae and Acnromataceae, witn sulfur ozidatio Their association witn sulfur is largely based on the ooeervetion or intracellular depoeitton of sulfur granules. Fauet and Wolre have reoently shown tnat Beggiatoa alba's activity on sulfur is restricted ir nov absent, at least in laboratory culture (3). The nabivats-of all tnese oacteria seem to.oe soil, fresn and marine waters, and-sulfur deposite (4). In at leest one instance a sulfur oxidizer nas oeen isolated from coprolite-rock (fossil dung) or tne Triassio period (l). A fairly recent review of the sulfur bacteria-is contained in (5);-

Sulfir reduotion has been nost extensively studied in relation to the activities of Desulfoviorio desulruricans of

[PAGE 75]
the Spirillaceae. Members of this genus reduce\_eulrates, thiosulfates,-sulfites, eto. to hydrogen sulfide under anaerobio conditione. The habitats or theee bacteria include goil, sewage; water,oil-bearing strata, etc. Uptodate discussions of the phyeiology. or these bacteria are contained in (6,7).

The interaction or bacteria with iron compounds was observed as early as-l888 (8). In tnis work, a gheathed bacterium, Leptothrix, was ooserved to deposit ferrichydroxide in its sheath. It wegbelieved that the organiem was chemoautotrophic, getting its energy for-carbon dioxide aseimilation from the oxidation or ferroue ipon. However,\_the cnemoautotrophic nature=or this-organiem and many other iron bacteria has not been satisfactorily proven in the light:of our present understanding of chemoautotrophy. At present it\_seems more-likely tnat the sheethed bacteria (Chlamydobaoteriaceae, Crenotnficaceae) deposit ferric-hydroxide from ferruginous waters in their sheaths without metebolic mediation. The same kind of ferric hydroxide deposition is believed to take place in the\_case of the stalked bacteria (Caulobacteriaceae). In their case, too, it is at present questioned whether ohemoautotrophy accoants for the ferric hydroxide deposition. he one exception in this instance may be Gallionella ferruginea (9). Among encapsulated baoteria (Siderocapsaceae), tne majority of the bacteria seem to deposit ferric hycroxide-in tneir capsules or outer cell=surfaces by non-chemoautotrophic.means.Only the genus Ferrobacillus in this family oxidizes-ferrous-iron by strictly onemoautotrophic means, at a very acid pH (1o).This genus is probably related to the Thiobacteriaceae (1). The

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[PAGE 76]
reported habitat for all these bacteria is iron-beering water.
Certain iron ores-also seem to cantain them;(ll).

Various common bacteria ars able to precipitate iron from organic salt\_combination (12). Such genera as Escherichia Aerobacter, Pseudomonas, Bacillus, etc. are!able to bring this about oy digesting away the organic portion of the-ealt. Aotinomycetes or tne-genera Nocardia and Streptomyces can also carry out such activity.\_

Ferric iron reduction may be promoted airectly or indirectly oy microbial agente. The indirect effect ia-probably tne more common, taking place in a reducing environment at\_acid pH created by barious bacteria. However, Bacillus ciroulans can reduce ferric iron tnrough direct metabolic, interaction (13). Iron sulfides-may-be precipitated tnrough tne formation or hydrogen silfide by sulrate reducers or by release or hydrogen sulfide from organic-sulrur compounds by various common bacteria. Manganese compounds:

aepos1ugun Manganese/has peen asgociated with many or the Siderocapsaceae
which are also associated with iron deposition.-In general, evidence
is lacking for metabolio dependence of this manganese precipitation
(1,2), although Beijerinck reported long ago that some true
pacteria anà fungi from soil could oxidige,manganese gulfate or
manganese carbonate (heterotrophically) (14,15). Some bacteria
seem to be-able to reduce manganese dioxide under conditions where
this compound can act as an electron acceptor in place of oxygen.
Quastel and Scholefiela (16) reported on such a-process.Chémo
autouroohic\_manganese\_oxidation has been reported by Prave(17)
and by Sartory and Meyer (18), but confirmation or tnese observations is required.

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[PAGE 77]
Transformation or copper compounde nas been reported by Ciferri and Scaramuzzi (19),\_Hurwitz (20), and Bryner and Jamegon (21). Only in the last or these tnree. reports doeg evidence exiat that enzymatic action on copper compounds-is involved: in fact, tne bacteria in that report are described as onemoautotrophs-allied to tne cnemoautotrophic iron oxidizers. In tne first two reporte, metabolic endeproducts are held responsible-for tne transformation of insoluble copper compounds. Molybdenum compounda:

An organiem\_related to tne cnemoautotrophic iron-oxidizers has been discovered to be aole to oxidize the-mineral molybdenite (MoS) (22). The produots or its action on this mineral are suifate and nolybdic aoid.Notning furtner-seems-to be known about thigttrensrormation. Silicates:

Bacillus siliceus has been reported to release potassium from a ooind etate in aluminum eilicates wnen tne organism is growing in a potassium dericient environment (23).-Aspergillus niger has also been reported to decompose clay minerals to satisfy its potassium requirements (24). Acid formation and its subsequent action on tne minerals probably: accounts for tne potassium release. The acios may include capbonic,\_organic, nitric, and suifuric acids.

Conolusion:

This:literature review is intended to bring out tne fact that microorganhsms may oring about mineral traneformation in two ways:.by direct enzymatic interaction,:ueually causing oxidation or reduction: or by-indireot aotion through tne production of environmental conditione wnicn promote traneformations\_tnrough

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Copper compounds:

[PAGE 78]
the production or environmental conaitions wnich promote transformatione tnrough chemical-reactions or a purely non-biological nature. It is the first type-or transrormation which will pe-svressed-in the proposed work.

In soite-or the imoression tnat certain problems\_or microoial mineral-transfcrmation have been:resolved. more extensiveinspeotion ofthe pertinent literature reveals contradictione.Theee examples bearing onitne problem or tcrobial action in mineral sulfide transformation will be cited for illustration.

Leathen et al.(25) claimed that ehey:could\_observe Ferrobacillus ferrooxidans to oxicize marcaeite but not pyrite. Both these minerals have the same composttion (FeS2). They attributed tnis-dirrerence in action or the baateria\_ to a dirference in crystal stricture between marcasite and: pyrite, Yet,Bryner et al. (21) and could ootain growth on pyrite by F. ferrooxidane or a closely related organism.

A second unresolved problem concerns tne oxidation of molybdenite\_(2l). in tnis work, an organism related-to F. ferrooxidans oxidized tne mineral to sulrate and molyodic acid. ine workers assumed tnat oxidation or the sulfur components of the mineral accounted for this reaction. They overlooked completely the fact that molyodenum had itselr undergone an oxidation. The-question thus arises whether the bacteria were directly responsibae for molybdenum oxiaation.

A tnird item or conflioting intormation concerns tne reoorted ability or F. ferrooxidans to grow on-media\_goliaified with agar (26). dryner-and Jameson could nou confirm tnie observation (21).

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[PAGE 79]
It is thue clear that even on tnoge mioroorganiems for whicn extensive information is available concerning tneir role in mineral tremsformation. furtner wrk is needed to clarify various points or conflicting inrormation.

References:

1. Bergey'e Manual or Determinative Bacteriology, 7th. ed.. The Will1ams and Wilkins Co., Daltimore 195?

2. Skerman.V. B. D., A GUIDE TO THE IDENTIF1CATION OF THE GENsRA OF BACTERIA. The Williams and Wilkins Co., Baltimore,1959

3. Faust and-Wolre, J. Bacteriol. 81: 99 (196l)

4. Kusnetzov, Bacteriol. Proc., ly6l, p.36;

5. Vishniac and Santer, Bacteriol. Rev. 21:.195=(1957)

6. Postgatee Ann. Rev. Microbiol. 13: 505 (1959)

7. Mecnalas-and Rittenberg, J. Bacteriol. 0: 501-(1960)

8. Winogradeky,\_Botan. Zeitung 46: 261 (1888)

9. Vatter: ana Wolre, Bacterlol. Proc. 1955,-p.35

10. S11verman ana Lundgren, J. Bacteriol. 78: 326 (1959)

C 11.

12. Halvoreon, So11 Sc1. 32: 141 (1931) 2.

13. 8romrield, J. Gen. Microbiol. 11: 1 (1954)

14. Beijerinck. Fo11a Microbiol. 2:123 (1913)

15. Be1jerinok, Verslag Akad. Wetensonappen, 22: 415 (1913)

16. Quastel and Scholefield, So1l Sci.75: 279 (1953)

17. Prave, Arcn Mikrobiol. 27:33 (1957)

18. Sartory and Meyer, Comot. Rend. Acad. Sci. 225: 541 (1947)

19. Cirerri\_and Scaramuzzi.\_Atti Ist, Bot. Univ. Lab. Crittog. Pav1a=Ser.5.3:-233-(1937)

-6一 2

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20. Hurwitz, So11 Sc1.Soc. Am., Proc. 12: 195 (1947)

21. Bryner and Jameson, Appl. Micro0iol. 6: 281 (1958)

22. Bryner, Loren, and Anderson, Ind. Eng. Chem. 49! 721(1957)

23. Aleksandrov,and Zak, Mikrooioloiya 19: 9y (1950)

24. Eno and Heugzer, So11 Sc1. 80: L99 (1955)

26. Leatnen, Braley, and McIntyre, Appl Microoiol. 1: 65 (1953)

27. Temple and Colmer, J. Bacteriol. 62: 605 (1951)

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