CONTINUATION OF MKULTRA, SUBPROJECT #133 (W/ATTACHMENTS)

• Document Type: SPECIALCOLLECTION
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): 00693683
• Release Decision: RIPPUB
• Original Classification: U
• Document Page Count: 12
• Document Creation Date: January 23, 2025
• Document Release Date: February 12, 2002
• Publication Date: July 29, 1963

� ...LA., �
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DRAFT
29 July 1963
MEMORANDUM FOR THE RECORD
SUBJECT : Continuation of MKULTRA, Subproject #133
1. The purpose of M1CULTRA, Subproject 133 is to enable 11SD. to
utilize the services ofillgoAssociatesPrifeetroi-vtiVIsBiologye?
a
2.: El continues to provide data and. materials which lend.
themselves to new and. unique approaches to energy production and trans-
formation (bio-batteries ) His studies on basic raechani sins of mineral
transformations also 'provide new potential paths for deterioration of
metals.
3.. IlidgiaitEetreTuliciVtorrMeditteisilesearthk will function as ccriero
.0 and l!Ctittgout during this fiscal year. The cost of this program for one
year will be $9,000.00 to which must be added $360.00 which represents a;
iitA service charge to .be retained by the aut*ou# The total cost of the
program will not. exceed $9,360.00.. Charges should be made against 'Allot-
. _
ment No. 4125-1390-3902.
li. It is not anticipated that permanent equipment other than that
listed in the bilaget will be required for thik program. Title to the
equipment listed will be retained by the ens, _:;,L7u.te in lieu of higher over-
head rates. �
"
APPROVED FOR RELEASE
DATE:JAN 2002
sF�
5.. Documentation and accounting for travel expenses which are re-
imbursable by,
accepted practice of that organization.
6. 11111111111has.teen Cleared COI= end is unwitting and yi13.
11 conform to the
remain unwitting of the true nature of the. sponsor-...
Attachment:
Proposal
Distribution:
Original only
'TPA t.4i�
,
APPROVED FOR RELEASE
CTJ17.4..V1k3te
DATE: JAN 2002
Proposal No. waiieROPPIA.
entitled
for renewal of Griffts2inzaitt
for the p riod
1 September 1963 31 August 1964
Submitted on behalf of
-3A.S SCieivanuratExu xeb bUrALTINEr10.
APPROVED FetiAlrit@EA�t963 -
DATE:JAN 2002
I 33 -42'7
PURPOSE OF STUDY
This proposal is a request for renewal for the period 1 S4tember 1963-
31 August 1964 of the grant-in-aid presently supporting research on VIRTEMPrilera
bienerner The field of investigation covered 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 the research carry increasing sig..
nificance 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 locationsl. 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 additional clues to the understanding of manganese bio-
chemistry in public water supplies, where manganese together with iron can cause
discoloration of water and water containers, and where manganese and iron are de-
posited biologically in water mains and pipes. In the area of conservation of
mineral resources, the work underweartairroarprovides a basis for assessing and con-
trolling any relative instability of mineral deposits in relation to microbes. It
_eems evident that uncontrolled bacterial leaching could conceivably lead to de-
� � � . -
--pIetion and eventual complete. translocation of some deposits, In mineral explora-
tion this work indicates that special groups of microorganisms known to be able to
live at the expense of a specific mineral may be of great assistance in discovering
� new sites of mineral deposits. Finally, this work lends additional support to the
practice of using microbes in mineral extraction.
APPROVED FOR RELEASE
DATE:JAN 2002
2
a
� SUMMARY OF WORK ACCOMPLISHED UNDER THE 1962-63 CRANbFROM illiEgG.ESCHICKTERAFUNDIv
(a) Microbial Action on Mineral Sulfides
Quantitative studies on the action of the Thiobacillus-Ferrobacillus
group of bacteria on the minerals orpiment (As2S3), arsenopyrite (FeS2FeAs2), and
enargite (3Cu2SAs2S5), were initiated. The bacteria achieved significant solubili-
�
zation of orpiment, releasing arsenic as arsenite and arsenate. Oxidation of the
sulfur portion of orpiment is probable, but remains to be experimentally 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 reflect-
ed 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 3.5 to 5.0 in that time.
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, contrary to a sustained release of soluble arsenic from
orpiment by bacteria, only a limited amount of soluble arsenic was 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 concehtration of
soluble iron and arsenite and arsenate has been reached. Although some oxidation
of arsenopyrite occurred in the absence of bacteria, its extent was less and its
chemistry different, because iron arsenates and arsenites were 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.
� .
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DATE:JAN 2002
...
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 bacterial phenomenon also poses a problem when arsenic
and iron are to be extracted together from a natural mineral deposit.
Quantitative work on bacterial oxidation ofenargite has given results
resembling those with arsenopyrite Although the bacteria 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 slight-
ly 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 recognizable 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 3.5 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 an cuprous sulfide by the Thiobacillus-Ferrobacillus group, in-
vestigated chiefly by a graduate student, is showing variable responses on different
synthetic preparations. Bacterial enrichment cultures derived from several mine
effluents during the past year have, however, given more consistent growth responses
4
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 accompalging the Thiobacillus-
Ferrobacillus group of bacteria in mine water from inspuutionwmArAmonma The proto-
zoa included an amoeba and a flagellate, which appeared to grow at the expense of
the bacteria and fungi. The amoeba were observed to ingest such organisms.
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DATE:JAN 2002
Both kinds of protozoa are unusual in their tolerance of 800 pmcopper 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 Augfist,
CL,
1962, as guest of the thirty-four manganifer-
ous samples and 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 ana-
lyzed for total iron and manganese content. The results showed that the various
manganiferous samples were by no means alike. In some cases this was obvious by
visual inspection. In other cases, however, the differences were not macroscopically
apparent. Instead, the iron-manganese content, or the bacterial content, or the Mn
adsorptive power of the samples, showed differences. Results of experiments, de-
signed 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, bacterial enhancement 6f 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
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DATE:JAN 2002
L�
samples it was noted that Mn uptake by manganiferous material 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 Mn02 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 chemical and physical properties of manganiferous materials heap to
determine the effectiveness of bacteria in enhancing Mn adsorption. Moreoveri since
all but one of the samples of the 1962 collection came from extinct submarine volca-
noes (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 between Mn2+
and Mn02 forming Mn" when nodules adsorb Mn24', on the condition that Mn34' is
� stabilized so as to prevent it from undergoing the 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 stabilizers. Nodule
breakdown can thus be accomplished biologically with sugar as the reducing agent of
Mh0212 or nonbiologically by a dismutation reaction between Mi12* and Mn02. Why the
-latter reaction:Occurs*with Only some nodules even though all have Ma02 and adsorb.-
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, aid in Mn addition, but not Achromobacter. All three
organisms have been found in nodules. Arthrobacter, at high peptone concentrations,
causes clumping and heaping of nodular material; not so at low peptone concentra-
tions. A similar phenomenon has been noted in soil . The clumping and heaping
APPROVEDFORRELEASE
DATEAAN2M12
seems to reduce surface area, and therefore slows adsorption d'Mn.
Initial experiments have revealed that Arthrobacter can aid in Mn ad-
sorption by "synthetic" Mn02. However, the adsorption and incorporation process of
Mn2* is not permanent. The 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" Mn02 is a much poorer adsorbent than nodule
neterial. 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, collected last summer, were examined
by enrichment for bacterial content. The distribution of bacteria in these cores
was not uniform nor were the bacteria necessarily of the same kind at different
depths in the same core. These findings are similar to those cited by Zo Bell. _
Work is presently 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, must reflect upon Mn distribution in the sea and upon nodule dis-
tribution and structure.
REFERENCES
1. de Grys , Ann. Econ, Geol, 57: 1031-1044 (1962)
3. Martin, J.P., and S.A. Waksman. Soil Science SO: 29-47 (1940).
4. Zo Bell, C.E. MARINE MICROBIOLOGY. Chronica Botanica Co. Publ. 1946, p. 91.
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DATE:JAN 2002
7
� mo....00,
/
PROGRAM OF PUT RE 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 atten-
tion 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 forms of a given element dissolved and reprecipitated after bactetial
action. Such information should provide clues to the mechanism of action by the
bacteria. The detailed information on the fate of arsenopyrite and enargite, when
available, will provide information concerning the effect 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
tell material for resting cell studies and enzymatic investigations.
(b) ManFanese Nodules
Two aims will be followed experimentally in this work. One is the further
elucidation of environmental relationshins to the process of nodule development or
degradation. The second is the elucidation of the biochemical mechanism whereby Ma
is added to, or released from, manganese nodules by bacteria. To attain the first
aim, samples from various known sources will be studied in respect to physical,
chemical, and biological properties, and attempts will be made to correlate the re-
sults with geographic 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 material and on synthetic Nn02 to compare and contrast behavior. Since pre-
vious experiments have already shown a difference in action by bacteria between
nodule material and Mn02, a condition is provided to compare the effect of iron on
the two systems. It is planned to develop more specific assay procedures for
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DATE:JAN 2002
differentiating the various oxidation states of Mn and thereby,tp follow the exact
fate of adsorbed or desorbed Mn, and to establish more clearly the role bacteria
play in this process. The ultimate goal is to study the bacterial action on manga-
nese .on an. enzymic. level.
PUBLICATIONS: ARISING OUT. OF THE WORK UNDER THIS
� 01131
-ucaniiircErcr to . naLa.--c-1-101 .1
. ' (2sc
con. Geol.)
-tablItOr-STOMITS-1-un-vu c
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(3)274
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1963,p.
161.
FOR 1962-63
(Ip,prepara-
IAbstract.
(4),1 111 (Invited
TIPTiTiAo,lbelniblishedAmthe Proceedings of the Rudolf's Research Con-
ferencep�gutgers-University, 5-7 June 1963).
�
(5)aQIV-1Fiiiifdipited,.thav,,aspapeewill-bewritten for submission to Appl.
Microbiol. on the work with manganese nodules during the last year.
PERSONNELFOR�196364
Principal Investigator:
Technician:
Graduate Student:
0,1
AswittnytProfosSOr*off4mww
nioaogy2.�
Hampirdrafgrzupported
APPROVED FOR RELEASE
DATE:JAN 2002
C-
for renewal o
for the period
1 September 1963 - 31 August 1964
'..Personnel
Principal Investigator
-1/8 time-academic year
1/4 time-summer months
Technician-full time
� Consumable Supplies
� Chemicals
Glassware �
� Permanent Equipment
Travel and Conmtunication
To scientific meetings
Telephone
Publication of-Reports
Indirect Cost
14% of Total Direct Cost
hk
Proposal No. 148404111M116
ent tl d.
COST ESTIMATE
471: � -
April 1963
$1,175
785
$1,960
4,330
Total Personnel Payments
���������������=.
$6,290
$ 400
200
600
520
$ 250
35
� 285
200
Total Direct Cost
� $7,895
TOTAL
Research Division
1111131
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DATE:JAN 2002