Doc 0000173568

__ --------------!!!Piiiiili _____ .:::::::::;;: - 3 -~r.;~~:.:.:.: -:-:-:.~=:;·~!1!..•! -!.'.- ~-::--::- ---""!111---~::;;iilloliiiiili:=:;:::::..---------- ~~-.;,:-"';.;~~~~;.;.:.,..;~;a"i~i;.r/-2t' .. : x;~~~2 . / . i . ~ ;z.. - ; ~- : . e;:..s:ii.::u.!~;;;.~~;:.. ..;=~~.:~.~~;.;.;.:.;.," ,.,;_;.;,~~-·,~-· ·'- :...·-. .. ...: .{~~;:.~~;~.'-" ~ ' ~·~. :> '"';"-:. 3 ~~ .. .. .-.. ! I. ELECTRCCHEHICAL OBSERVATIONS Dl NICROBIOLCGICAL PROCESSES. GR01·iTH OF THIOBACILLUS THIOOXIDANS / ___.-.-_ /._ __ __ ...... . . . . ., I -~i .. ·- - -- , - .. ~ ~:.:_-, .. · 1. ) . ; . ; ' ~(~ t r;;. '' 1 lij ·-~-J· .. ~~~:-c· ~~:· ~-~ ~ ' , : : _ : ·: . , - .. ·--~ i .~•. ~~ -~"-: ~i- ' ·". '+ .,-;; l .:~~ -~"' <~: ·~ i .~f;~ .._ .oi;. SU!·lHARY The growth of Thiobacillus thiooxidans utilizing sulfur in three media was studied by observing changes in half-cell emf, bac- terial cell count and production of acid as·a of time. A functio~ comparison of the biological half-cell emf with comparable control !• half cells reveals that thiooxidans makes an electrochemical con- tribution to half-cell voltage. A change f'rom the more complex medium of Skerman's mineral salts to A.T.C.C. allowed a clearer delineation of' T. thiooxidans' ability to make an electrochemical contribution. Reproducible biological half-cell emf's obtained when ~.;ere the ferrous sulfate was removed from the A.T.C.C. medium. One half cell comprising !· thiooxidans utilizing sulfur in A.T.C.C. was ob served over a lll-day period. During this time the initial half cell voltage of -0.35 volts, decreased to a negative.value of -0.64 volts !• (hydrogen emf series). thiooxidans in utilizing sulfur produces only sulfate ion, thereby simplifying the identification of an -elec- trochemical contribution during growth. - 1- .. .- ... ~;:'%t"'F.z~~s ~, I;,~·il I. IIITRODUCTION The concept of converting chemical energy from natural occurring fuels into electrical energy by biochemical reaction has in 1 trigued mao for many years. Potter in 19ll"was the first to conduct experiments with biochemical galvanic cells. He observed that "the disintegration of organic compounds by microorganisms is accompanied by the liberation of electrical energy." His experiments were conducted primarily with the yeast-glucose system which gave open circuit voltages between 0.3 and 0.5 V. These exploratory experiments led to investi 2 gations in 1931 by Cohen, Who studied several bacterial cultures as electrical half cells. More recently, Bean,a Canfield,b Bitterleyc and their co-workers have been working on The image displays a stylized graphic of a bank vault door on the left side, with a blue neon glow emanating from the side. On the right, white text is overlaid on a dark background, stating that the document was obtained from "The Black Vault," an online database of declassified government documents. It further specifies that this particular record belongs to the MKULTRA/Mind Control Collection, containing over 20,000 declassified CIA pages, and provides a URL for downloading the collection. The text is presented in a clear, sans-serif font, with the URL highlighted in yellow. There are no photographs of people, locations, equipment, or subjects, nor are there any handwritten annotations, signatures, official stamps, forms, diagrams, tables, or redactions visible on this page. The visual elements primarily serve as a title card and source attribution, rather than conveying information about the document's content. The document is a typewritten page with several lines of text. The top line reads "ELECTROCHEMICAL OBSERVATIONS IN MICROBIOLOGICAL PROCESSES. I." Below that, in all capital letters and underlined, is "GROWTH OF THIOBACILLUS THIOOXIDANS." There are also some faint lines and dots that suggest a drawing or graph, but it is incomplete and unclear. In the lower right corner, the number "178" is circled in black ink. The page appears to be from a scientific report or thesis, and the circled number might be a page number or a reference. There are no photographs, stamps, forms, or evidence of experimental procedures visible. were conducted primarily with the yeast-glucose system which gave open circuit voltages between 0.3 and 0.5 V. These exploratory experiments led to investi 2 gations in 1931 by Cohen, Who studied several bacterial cultures as electrical half cells. More recently, Bean,a Canfield,b Bitterleyc and their co-workers have been working on various aspects of bioelec-. tricity for the National Aeronautics and Space Administration. Empha- sis in their investigations was placed on the utilization of organic foodstuffs as an energy source. In order to gain a better understanding of voltages developed in biological oxidations, a decision was made by the authors to investi- gate some of the autotrophic bacteria. Autotrophic bacteria, because of their ability to utilize inorganic substrates as an energy source a Philco Corporation, Newport Beach, California. b Magno Corporation, Anaheim, California. c The Marquardt Corporation, Van Nuys California. 1 . . .. . . . •'"/· - _~~ ~: ;:.~~i-~)~~-;;-~~~:~;;;~ ~·~.;]2~i:£.~~~S)~~_:::~?.·1-~~~~±~~~-t0;i;.~~1.£~~~;~ ~-~-·4:-~:~~~-~:~ -~------ -.: •• and carbon dioxide for their carbon requirements, offered a different and perhaps a simpler approach to associating electrochemical poten- tials with metabolic activity of bacteria. The sulfur oxidizing bacteria, Thiobacillus, were chosen since they were among the most I metabolically active autotrophs. In these studies, primary emphasis was placed on !· thiooA~dans. The electrochemical investigations reported in this paper assume that sulfate ion is the only metabolic product associated with !• the oxidation of sulfur by thiooxidans. This assumption has the 3 4 support of earlier workers such as Starkey, ' Starkey, Jones and 5 6 . 7 Fredrick, Vogler and Umbreit, and Parker and Prisk. -~ ;"~. ·.•r{j II. ELECTROCHEMICAL ACCESSORIES Carbon (UP-62-R) from the United Carbcn Company, Bay City,. Hichigan, •ras cut into electrodes. The ends of the electrodes were ·i -~- plated ;.lith copper from a CuS0 solution. Copper leads were soldered ~lLI 4 to the plated surfaces. The leads and their contact the carbon :~.~ ~.,ith ~j were treated with paraffin, to eliminate ~etting and direct contact between the copper and nutrient. Platinum electrodes were prepared from platinum gauze (45 mesh, 0.0078 in diameter) obtained from J. Bishop and Company. This ,,; a-~ gauze was cut into 2-in lengths, approximately 5/16 in wide.· Copper t'I leads were soldered to one end of the gauze. To prevent possible ~:~-~ -~ i - t~;~ 3 - ~i'o"~''i"~~1:fii~?'c'£~:E.:r:f'; S'"~"'f'''"'"1TI~C:Z'C'f"~~r;,:~/~0;'"'1"''2'09'i"!"f'ffi '"' oxidation o£ the copper, the lead wires were covered with plastic tubing. This This ,,; a-~ gauze was cut into 2-in lengths, approximately 5/16 in wide.· Copper t'I leads were soldered to one end of the gauze. To prevent possible ~:~-~ -~ i - t~;~ 3 - ~i'o"~''i"~~1:fii~?'c'£~:E.:r:f'; S'"~"'f'''"'"1TI~C:Z'C'f"~~r;,:~/~0;'"'1"''2'09'i"!"f'ffi '"' oxidation o£ the copper, the lead wires were covered with plastic tubing. This tubing 1-1as then anchored to the copper-platinum solder joint by coating the end of the tubing and the junction with an epoxy resin. To further prevent any possible diffusion of water to and through the plastic tubing and the epoxy-covered junction, the lead wires were kept above the--biological half-cell liquid level. . -- The thermocouple ef£ect for these copper-platinum electrodes was found to be negligible (5 ~V/°C). These biological half-cell investigations involved maintain- ing an air atmosphere above the media in the cells. The electrochemi- cal effect of supplying fresh air above versus bubbling it directly into the stirred media was-negligible as long as gaseous concentration gradients did not .exist within .the.media.~Table.IJ.---·--·- - -- --·-- TABLE I DYNAHIC AND STATIC AEROBIC CONDITIONS FOR STIRRED EIECTROCHEHICAL HALF CELLS Aerobic Potential, V Medium Atmosphere Static Dynamic (Bubbling) Sterile distilled water Air -0.323 -0.329 0.006 o.ooo Skerman's Air -0.365 -0.365 o.ooo A.T.c.c. Air -0.390 -0.390 o.ooo A.T.C.C. (minus FeS0 ) Air -0.420 -0.420 4 - 4 - -"' ~\rr~~ii]~;L ~:: '~;~c~·?·~ < ·.. :::(':·,,:..::~ ;:· :...: . :~_.; Since composition gradients were known to be generated through utilization of substrate by the uneven suspensions of bacteria, it was considered necessary to stir the half cells. The electro- uniform~ chemical effect of turning off the stirrer was checked for the unin- 1 oculated media in which such composition gradients were absent. Table II reveals that the effect of not stirring was appreciable in the sterile distilled It became negligible when conducting nutrients were ~ter. added to the water. In all large control and biological hali' cell experiments reported in this paper, the cell constituents were stirred and atmospheric air with its carbon dioxide available to the media ~s through sterile cotton plugs. TABLE II EFFECT OF NOT STIR.~IUG IARGE ET.ECTRCCHEHICA L HA IF CELIS (AIR BUBBUNG INTO CELL) Potential, V Medium On -Off Sterile distilled ~ter -0.329 -0.240 0.089 Skerman's -0.365 -0.363 0.002 A.T.C. C. -0.390 -0.388 0.002 A.T.c.c. (minus Feso ) -0.420 -0.418 0.002 4 '~~ - 5 - -(~;:;;:'}:~{0:)W'~{'.'3 ,,/-iS'~-!' ':7EC2~ ::;;gc:<1!ffC:':T"7~"'''{'' ""''':'~'17'''"'::'{1!'\',';':0"'C'T': ''":t:','CYT' iC7'.':T "":7:''·' ·::. An agar plug in the The page is a document with a title "SUMMARY" and contains a single block of text. There are no photographs, handwritten annotations, signatures, official stamps, forms, diagrams, schematics, organizational charts, tables, or visual evidence of experimental procedures, equipment, or facilities. The content is purely textual, detailing an experiment involving *Thiobacillus thiooxidans*. The bottom of the page contains a page number "-1-". The document is a scanned page of a declassified government report, featuring typed text and a footer indicating it is page "2-". There is no visual content such as photographs, diagrams, or stamps. The text discusses the concept of converting chemical energy into electrical energy using biochemical reactions and references various researchers and their experiments. There are a few superscript numbers and letters indicating footnotes and citations, but no handwritten annotations, signatures, or redactions are visible. (AIR BUBBUNG INTO CELL) Potential, V Medium On -Off Sterile distilled ~ter -0.329 -0.240 0.089 Skerman's -0.365 -0.363 0.002 A.T.C. C. -0.390 -0.388 0.002 A.T.c.c. (minus Feso ) -0.420 -0.418 0.002 4 '~~ - 5 - -(~;:;;:'}:~{0:)W'~{'.'3 ,,/-iS'~-!' ':7EC2~ ::;;gc:<1!ffC:':T"7~"'''{'' ""''':'~'17'''"'::'{1!'\',';':0"'C'T': ''":t:','CYT' iC7'.':T "":7:''·' ·::. An agar plug in the bend of the tube the tw-o half cells. separat~d - Although positive results for associating emf's rlth gro"t·rth ofT. thio- . oxidans were obtained vi th this initial design, a modification (Fig. 1) 1 which made a separate opening to the agar bridge for connecting a ref- erence calomel cell, was desired. Each of the original half cells, the biological and control, could now be monitored individually with the calomel half cell. Experiments w-ith this modified U-tube w-ere sat- isfactory, ho,.rever it was deficient in size and allowed concentration gradients to form so a new large biological half cell was designed. used to about 20 ml of nutrient. Furthermore, the long tubes narr~·t presented little opportunity for changes in electrode design, stirring, and continuous tneasurement of pH. Therefore, the new design used a large three- and, later, a five-necked 1 000 ml round-bottom flask (Fig. 2). A Teflon stirrer was suspended through the center neck sur- 1·ounded with a glass bearing. Its action diminished acid and mineral concentration gradients and insured uniform suspension of bacteria for population density determinations. An agar salt bridge and calomel cell were mounted in one opening and a glass and a platinum electrode were placed in the third neck. The calomel and glass electrodes w-ere - 6 - ---------- used for recording the pH values. This entire biological half cell was mounted in a constant temperature bath {29°). Prior to use of' this bath the electrochemical voltages 1¥ere observed to fluctuate in a cyclic manner with the room tem.n erature whenever -T. thiooxidans was present. This behavior was especially evident when pLitinum elec- trodes were used. The agar salt bridge, mounted in one opening of' the round- bottom flask, was led to a test tube containing saturated KCl main- tained at the same temperature as the biological half cell. A standard calomel electrode was mounted in the test tube as a rel'erence half' cell. Leads from the complete cell were connected to a K-3 potentiom- eter and a pH meter. All parts !:lf .the ab.o:ve .cells whiclLc.ould..ldthstand high_ .. _ .... __ _ temperatures -vrere the biological half cell. A standard calomel electrode was mounted in the test tube as a rel'erence half' cell. Leads from the complete cell were connected to a K-3 potentiom- eter and a pH meter. All parts !:lf .the ab.o:ve .cells whiclLc.ould..ldthstand high_ .. _ .... __ _ temperatures -vrere sterilized by autoclaving. The other parts \¥ere sterilized by rinsing in ethanol followed by three rinses with sterile distilled water. Before sterilization the electrodes were cleaned , with concentrated sulfuric acid and then washed with distilled water. All experimentation was oriented towards obtaining zero- current potentials of' complete as ••ell as half'-cell reactions. The initial electrochemical measurements were made with a Model K-3 Leeds- Northrup potentiometer. This instrument gave accurate voltage deter- minations when zero current conditions were established. However, 1-rhile balancing the galvanometer to obtain zero current conditions, - 7 - ~~r~:{XjS2.2;;;Ll; __ -_ ___ ._:_:__.L,,~lb{~E;;i'"iit'~~'L' -;a;.";..·z._i~~"""""""'"'''c: __ l:;:,~:-~~--~~;~ power was drawn initially from the cell causing, in some cases, a loss 11 of voltage. Since it was desirable to eliminate this probable contri- ..... I to variable results, a specially designed vacuum tube v.oltmeter butio~ was obtained for use with a recorder. Open conditions were 11 ~ircuit mair.tained by use of this vacuum tube voltmeter and continuous emf ~;,,:.·~ measurements could be taken with the recorder. III. MICROEIOLCGICAL TECHNIQUES AND OBSERVATIONS The application of microbiological techniques to support the electrochemical investigations was focused on two areas of study. The first involved obtaining reproducible bacterial growth. After such growth established, less complex media were sought by removing •~as area of study involved developing techniques for determining bacterial counts in the uniform biological half cell suspensions. A. of Thiobacillus thiooxidans Gr~~~h Successful growth of !· thiooxidans lias studied primarily in three media. At first, reproducible growth of!· thiooxidans was ob- 8 tained with shaker cultures using Skerman's basic mineral salts (17 salts). One per cent of sterile powdered sulfur was suspended in this 9 medium. High yields of 10 organisms/ml were obtained with mature cultu=es (maximtlin population density) after five days incubation. - 8 - - Since the interpretation of the emf measurements in the Skerman's medium vas exceedingly difficult, a simpler medium was sought. A.T.C.C. medium,d containing five salts plus 1 per cent sulfur, vas studied as a growth nutrient. This medium gave mature cultures with 8 populations of 10 bacteria/ml. A further consideratihn of reducing the salts, comprising This page is a scanned document containing only text. There are no photographs, handwritten annotations, official stamps, forms with filled fields, diagrams, schematics, organizational charts, tables, structured data, or redactions. The visual content is limited to the typed text of the document itself, which appears to be a scientific or technical paper, indicated by the presence of footnotes and a section heading like "II. ELECTROCHEMICAL ACCESSORIES". The document page displays text describing oxidation of copper and lead wires, anchored with epoxy resin, and maintaining an air atmosphere above media in electrochemical half-cells. It includes a table titled "TABLE I" that presents "DYNAMIC AND STATIC AEROBIC CONDITIONS FOR STIRRED ELECTROCHEMICAL HALF CELLS." This table lists different media (Sterile distilled water, Skerman's, A.T.C.C., and A.T.C.C. (minus FeSO4)) and their corresponding aerobic atmosphere, static potential, dynamic (bubbling) potential, and an additional column labeled "δ". There are no photographs, handwritten annotations, or official stamps visible. the emf measurements in the Skerman's medium vas exceedingly difficult, a simpler medium was sought. A.T.C.C. medium,d containing five salts plus 1 per cent sulfur, vas studied as a growth nutrient. This medium gave mature cultures with 8 populations of 10 bacteria/ml. A further consideratihn of reducing the salts, comprising A.T.c.c., brought about the removal of ferrous sulfate. The concentration of T. thiooxidans in mature cultures in 7 this medium, A.T.C.C~(-)1 was approximately 1 x 10 organisms/ml. The bacteria to be used with the biocells usually were taken from five-day-old mature shaker cultures. These cultures were grmm in 250 ml Erlenmeyer flasks with 30 ml of medium in each flask. Inctiba- tion was either at 29° or at room tem~erature. The mature cultures were harvested from the shaker· flasks by centrifuging the organisms at 9 000 rpm for 5 min in a Lourdes centri- 1 fuge. After decanting the supernatant, the cel+s were then washed twice with sterile medium and were suspended in the various volumes of the sterile medium for the particular bacterial concentration to be used for biological half-cell studies. 1 - ~ 9- J<~~ZJiii~~iif!lf.fi;T~~~:;i;~~;_;m,~'f&~'Ns:t;~f~\f~~]ftf;i;J';~c;:;~g,f(fB•{~'~(':~;~;;;;:f":~--w~-s-cs;~F:~'I? - ·•;f!!f2}!§~,,~":_ ..,-. . / )' :._1;::;.-,?-~ -~--:~---.?~-~ B. Bacterial Counts Two methods were applied for obtaining the bacterial popula- tions of the cultures in the preparation and operation of the biocells. These methods were the Petroff-Hausser chamber count and the micro- 9 Kjeldahl analysis for total nitrogen content of the bacteria. T. Thiooxidans was removed from the medium by filtration before a modified micro-Kjeldahl analysis was applied. Turbidity determina- tions for bacterial counts were not practical because of the presence of powdered sulfur. Pour and spread plate counts were discarded after obtaining irregular and time consuming results. The Petroff-Hausser chamber counts were used to calibrate the nitrogen content from the bacterial with their concentration in the medium. . The micro-Kjeldahl technique was only used when appreciable volumes of samples were available and concentrations of bacteria were 7 approximately 1 x 10 /ml or greater. Use of the Petroff-Hausser count- ing chamber technique was preferred for lower concentrations of bacteria and experiments where less than 1 ml of sample was available. The latter technique was adopted completely after the earlier of ~bases investigation in order to minimize disturbing the biological half-cell ecology. The total amount of liquid required for the samples by this technique 'rras negligible compared to the large biocell volume. - less than 1 ml of sample was available. The latter technique was adopted completely after the earlier of ~bases investigation in order to minimize disturbing the biological half-cell ecology. The total amount of liquid required for the samples by this technique 'rras negligible compared to the large biocell volume. - 10 - -.--.·--·-· - i}~~ C. General Microbiolo~ical Observations ···_ _·, : ~ :.- : ,· : _. ~ : - --L- + ~~ ; -- · ·· ~~ture populations obtained in the large biological half· cell ·,. :J ·~ (Table III) idth Skerma.n' s medium were consistently less than observed ··<t {\ ·: ; 1 > . : in the shaker cultures. Subsequent experimep.tation vit h grOi-rth of ..:':.: ~.~ · : - ~ ~ !· thiooxidans in less complex media showed that this d ~ ifference in -~}; :& population density decreased with A.T.C.C. and disappeared when ~t }~1 -.-_ .. : '- ~ ' A.T.c.c.(-) was used. ,{~ TABLE III .· ,._.. -~~ :~,-- ~$. -~ ~~- _-:~ TYPICAL MATURE POPU!ATION DENSITIES -'"~:,·~ ~ )~ ' Large Biological • ~ Shaker Culture Half Cell Number of -~ -!-;}·' 1>1edium ( organisms/ml) ( organisms/ml) Salts :~ ·. :.~ -~ -' " ~ ' Skerman's l X 10 9 l X 10 8 17 ·c. ti 8 7 ~ A.T.c.c. 1 X 10 l to 5 X 10 5 :...;. ·--~ 7 ~\ -~- -ii A.T.c.c.(-) 1 to 5 x 107 l to 5 x 10 4 t _-.. ~ -~;/~ '~ , ~ ~ ~ ~; · ~: - .: ~ .. . . 1 :it , Experiments with growth of !• thiooxidans in both shaker and ~1.1.: biocell cultures using Skerman's and A.T.C.C. as growth media showed that the reduction in the number of mineral salts caused a decrease in - --~-::.. the mature population density. Two typical biocells (Fig. 5) were started with similar inoculi and similar volumes of medium. The tem- perature for both biocells was held at 29°. Similar lag periods were :;,:;:.' ~ -.-. 1; observed during the first day, followed by a rise in bacterial cqncen- :.;..; ;.....&i ; ~.~-~- -~ ,_- :-. !~ ].:'~. trations during the second day. During the third day of incubation, ·-t~ --~ ~·t. ~?;: l-.~ y~') - ll - 1;. . ":"· ·;• <~~-{j -- .!;.·:~5}~:%~~-~-~~}rr~::~_ .;{~\}}:;\;;_~/~~;~;TJ~~-B~?:1--:~·-~?~;:~:~;-~ff:';;;'~;~;:~~,,~;f~(;::cr-;~;:'?f~;0;~~,?-;'~;:-~~':~~r;·?c~·~~,~,?~~,~~?~:~·-:?_ the bacteria concentrations were observed to rise more rapidly This page contains a typed document with a table presenting data from an experiment. The table is titled "EFFECT OF NOT STIRRING LARGE ELECTROCHEMICAL HALF CELLS (AIR BUBBLING INTO CELL)" and includes columns for "Medium," "On," "Off," and a delta value. The data shows potential measurements in Volts for different mediums like "Sterile distilled water," "Skerman's," and "A.T.C.C." There are no photographs, handwritten annotations, or stamps visible on this page. The bottom of the page features a page number "-5-". This page contains a typed document, numbered "6" at the bottom, discussing experimental designs for biological cells. There is no visible imagery, handwritten annotations, official stamps, forms, diagrams, tables, or redactions on this page. The content is solely textual, detailing modifications and limitations of laboratory equipment used in the experiments. rise in bacterial cqncen- :.;..; ;.....&i ; ~.~-~- -~ ,_- :-. !~ ].:'~. trations during the second day. During the third day of incubation, ·-t~ --~ ~·t. ~?;: l-.~ y~') - ll - 1;. . ":"· ·;• <~~-{j -- .!;.·:~5}~:%~~-~-~~}rr~::~_ .;{~\}}:;\;;_~/~~;~;TJ~~-B~?:1--:~·-~?~;:~:~;-~ff:';;;'~;~;:~~,,~;f~(;::cr-;~;:'?f~;0;~~,?-;'~;:-~~':~~r;·?c~·~~,~,?~~,~~?~:~·-:?_ the bacteria concentrations were observed to rise more rapidly in Skerrnan's than in A.T.c.c. After this period the population density 8 7 appeared.to stabilize at 1 x 10 for Skerman's and 4 x 10 bacteria/ml for the A.T.C.C. media. Dl. EIECTROCHEMICAL OBSERVATIONS HITH T. THIOOXIDANS The initial investigations were concerned with establishing that an electrochemical potential, different from that of a control !• cell, exists when thiooxidans utilizes sulfur. The U-ttibe was chosen for these studies. Each side of the U-tube comprised a half cell one 1 biological and the other an oxygen-carbon reference electrode. Dupli- cate U-tubes were prepared lnth only one difference. T. thiooxidans · was present .in. . the arm_of.o.ne .. of_ th_em •. ~The Qther half cell had only sulfur suspended in Skerman's medium. Figure 4 shows that a significant ~:.~ difference exists the complete cell emfls of the inoculated and :~:~r- b~tween .... ·.-ot.· • .:'~ : . .,.:i.·· :::~~~ control cell. Such results were typical both when carbon and ''hen ~J platinum electrodes were used in the cells. The increase in the dif- ference with time suggests that after acclimation to the cell, !· ~~t ~ ~- .:,.·..;'-""-"·~~ oxidans became active and started to utilize the sulfur which in turn ~.t·t·f.· ~i. fif.. J changed the electrochemical nature of the half cell. Biological and control half cell emf'~ using platinum elec- trodes,were measured as a function of .time in the modified U-tube design (Fig. 1). The biological half·cells consistently gave voltages k~~ ;I ~~ -~- lii5:fifi\'ll~ifl&:':1Wi~;~T~\W0T},'%7fe0Tf;B'Kf'?W\~'l}'':~~r:-;;;;;:~;;;'·r~;;;;:~~~'!J':i0?;0fS?%::tj7f0(i~0',~;:; - vhi ch '.rere lover than their contra ls however leaks through the agar 1 1 plug at various times after cell preparation encouraged pursuing experi- mentation with a better cell design. The remaining experiments were congucted with large biological half cells using platinum in preference to the slower responding carbon electrodes. Since quantitative data were expected from the use of this new cell design, a check was made on how increases in hydrogen and sulfate ion vould affect the half cell electrochemical emf as measured by this electrode. Sulfuric acid was added separately in approximately 20 incre- ments to 1 per cent suspensions of powdered sulfur from the use of this new cell design, a check was made on how increases in hydrogen and sulfate ion vould affect the half cell electrochemical emf as measured by this electrode. Sulfuric acid was added separately in approximately 20 incre- ments to 1 per cent suspensions of powdered sulfur in each of the three media under consideration for studying growth of!· thiooxidans. The initial pH values of' approximately 5.0 gave •ray upon additions of the acid to values of' ·1-.-o. ~·Each~ addi-~on Q~3tC_M_ _~ i~ted p__r_e>!i_y.ction of . •-·-- -- T" .. --- sulfuric acid by!· thiooxidans. The maximum variation in half cell emf1s •nth Skerman1s medium experiencing these changes ~n pH was 0.012 V. Subsequent experimentation with A.T.C.C. and A.T.C.C.{-) media gave a smaller maximum variation for the same total change in pH. Thus, the electrochemical background fluctuations to be expected when sulfate ion is produced by !· thiooxidans were identified. A. Medium Effect on Biocell Activity Shaker cultures ofT. thiooxidans were grown in Skerman1s 1 A.T.c.c., ar~ A.T.C.C.(-) media under similar conditions. The prep- arations for centrifuging, washing, and resuspension in fresh sterile - 1.3 - - media, were planned to give populations having a concentration of 7 l x 10 bacteria/ml. However, the indetermir~te losses in the trans- fers gave rise to a slight variance in initial bacterial suspensions in the large biological half cells. The sus~ension in A.T.C.C.{-) was 7 7 1.2 X 10 o The value for Skerman's was 1 x 10 and for A.T.C.c., 2 x 6 10 bacteria/ml. After inoculation, a lag phase was observed to take place during growth in each medium (Fig. 5). The values for bacterial popu- lation counts under the dotted line in Fig. 5 were below the micro- Kjeldahl analysis so they were estimated {Petroff-Hausser Count). After 70 hr the population in the Skerman' s sa 1t s which was ini- 1 1 tially similar to the other two media, was nOiv greater. Subsequent bacterial counts showed that the populations stabilized and after eight days the Skerman•s-medium had a population of 1 x 108 while both A.T.C.C. 7 media had approximately 5 x 10 bacteria/ml. These saturation popula- tions were typical of large biocell experiments with these three media (Table III). If the variation in the initial concentration in bacteria can be ignored the amount of total acid produced by !· thiooxidans seemed to depend upon the The document page contains typewritten text, with some faint horizontal lines and speckling that suggest age and texture of the paper. There are no photographs, handwritten annotations, signatures, stamps, forms, diagrams, tables, or redactions visible. The page appears to be a section of a scientific or technical report detailing experimental procedures likely related to electrochemical measurements, involving components such as a temperature bath, agar salt bridge, calomel electrode, and a potentiometer. The text describes the setup and measurements for a biological half cell. Finally, a page number "-7-" is visible at the bottom center. This is a scanned document page containing typed text and a page number at the bottom. There are no photographs, handwritten annotations, signatures, or official stamps visible. The page is mostly clear, with some faint vertical lines on the left margin and a blurred area at the top possibly indicating a water stain or part of the scanning process. There are no diagrams, schematics, forms, tables, or visual evidence of experimental procedures. The text focuses on microbiological techniques and observations related to bacterial growth, specifically mentioning "Thiobacillus thiooxidans" and experimental conditions. 7 media had approximately 5 x 10 bacteria/ml. These saturation popula- tions were typical of large biocell experiments with these three media (Table III). If the variation in the initial concentration in bacteria can be ignored the amount of total acid produced by !· thiooxidans seemed to depend upon the medium in which it gre-.;-1. Since it was not possible to wash residual amounts of acid from the centrifuged cells, the initial pH values differed. Subsequent accumulation of acid is shown in Fig. 6. Of particular interest was the fact that T. thiocxidans produced less - l4 - . ~-:-; . _:, . . . :.. .. ~ ,~· . . .:- .. -.~· .-... -~~-:...;:~~: -· ~ i ·; · : •:. ~ J. iJ . ~ - ~~=::,;-£·~\;~i~:- • i: • ·:.-::~i-:i<: ~~:.:.:·_ _: :.:~. - :~ - 2 -~ : : : . ;.--. >·."" ~~.~~;:...;.;..::. :.-,:_-; --~-- acid (0.73 mmole) in the Skerman's medium in attaining a greater cell 7 population 7 x 10 bacteria/ml than in the other two media (3.66 mmole 1 for A.T.C.C.(-) a