ELR-CTRCCHE.IICAL OBSERVATIOLTS III @IICROBIOLX"TCAL PROCESSES. 1. GR(XJTH OF THIOBACILLUS THIOOXIDATIS 7777777 SLT141,!ARY 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 fundtiog of time. A comparison of the biological half-cell emf with comparable c2ontrol half cells reveals that T. thiooxidans makes an electrochemical con- tribution to half-cell voltage. A change from 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 contribation. Reproducible biological half-cell emfl2e were obtained when the ferrous sulfate was removed from the A.T.C.C. medium. One half cell comprising T. thiooxidans utilizing sulfur in A.T.C.C. was ob- served over a 111-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). T. thiooxidans in utilizing sulfur produces only sulfate ion,, thereby simplifying the identification of an-elec- trochemical contribution during growth. 777. J. I,.=ODUCTION The concept of converting chemical energy from natura occurring fuels into electrical energy by biochemical reaction has in- trigued man for many years. Potter' in 1911-was the first to conduct "the experiments with biochem2ical galvanic cells. He observed that disintegration of organic compounds by microorganisms is acc=panied by the libe gy ration of electrical ener His experiments were conduc primarily with the yeast-glucose system which gave open circuit voltages between 0.3 and 0.5 V. These exploratory experiments led to i=ves2ti- gations in 1931 by Cohen,2who studied several bacterial cultures as electrical half cells. More recently, Beana Canfieldb Bitterl and their c6-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 2 foodstuffs as an energy source, It 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. Autotraphic bacteria because of their ability to utilize inorganic substrates as an energy source a Philco Corporation, Newport Beach, California. 8 b Magna Corporation, Anaheim, California. C The llarq@iardt Corporation, Van Nuys, California. 2 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 lot bacteria., Thiobacillus, were chosen since they were among the most metabolically active autotrophs. In these studies, primary emphasis 2 was placed on T. thioo.%-idans. The electrochemical investigations reported in this paper assume that sulfate ion is the only metabolic product associated with the oxidation of sulfur by Z. thiooxidans. This assumption has the 3 4 su2pport of earlier workers such as Starkey, Starkey, Jones and 5 6 7 Fredrick, Vogler and Umbreit: and Parker and Prisk. 11. ELECTROC=CAL ACCESSORIES Carbon (T)P-62-R) from the United Carbcn Company, Bay City, 1-iichigan, was cut2 into electrodes. The ends of the electrodes were plated @rith copper from a CuSO4 solution. Copper leads were soldered to the plated surfaces. The leads and their contact with the carbon were treated with pataffin'to eliminate Wetting and direct contact between the copper and nutrient* Platinum electrodes were prepared from platinum ga0uze (45 mesh, 0.0078 in diameter) obtained from J. Bishop and Company. This -in gauze was cut into 2 lengths, approximately 5/16 in wide., Copper leads were soldered to one end of the gauze. To prevent:possible 3 oxidation of the copper, the lead wires were covered with plastic tubing. This tubing was then anchored to the copper-platinum solder joint by coating the end of the tubing and the junction with an To further prevent any possible diffusion of water to epoxy resin. -2covered junction, the and through the plastic tubing and the epoxy lead wires were kept above the-.biological half-cell liq@iid,level.,-, The thermocouple effect for these dopper-platinun electrodes was found to. be negligible (5 pv/"c). These biological half-cell investigations involved maintain- 2 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 vas-negligible as long as gaseous concentra on gradients did not -exist.within.th me Table-l)-..--.--,- 2 e- dia-@ TABLE I DYIIA14IC AND STATIC AEROBIC COITDITIONS FOR STIF@RED E=-@CTRO -M@IICAL HALF CELTS Aerobic Potential, V Medium Atmosphere Static Dymmic (Bubbling) 8 2 Sterile distilled water Air -0.323 -0.329 0.006 Sl,,erman's Air -0.365 -0.365 0.000 A.T.C.C. Air -0.390 -0.390 0.000 A.T.C.C. (minus FESO Air -0.420 -0.420 0.000 4) 4 Since composition gradients were known to be generated thr ough utilization of substrate by the uneven suspensions of bacteria, it was considered necessary to uniformly stir the half cells. The electro- chemical effect of turning off the stirrer was checked for the unin 2 oculated media in which such composition gradients were abse nt. Table II reveals that the effect of not stirring was appreciable in the sterile distilled water. It became negligible when conducting nutrients were added to the water. In all large control and biological half cell e2xper iments reported in this paper, the cell constituents were stirred and atmospheric air with its carbon dioxide was available to the media through sterile cotton plugs. TABLE II EFFECT OF NOT STIR'.RIITG 'LARGE ELE-CTROCHF14ICAL HATY CELLS (AIR BTJBBTING llITO CELL) Potential, V Medium On Off 6 Sterile distilled water -0.329 -0.240 0.089 Skerman's -0.365 -0.363 0.002 A.T.C.C. 9 -0.390 -0.388 0.002 A.T.C.C. (minus FeSO4) -0.420 -0.418 0.002 5 tion three basic biocell de- During the course of experimenta -volved combining a biolo The first design in signs were implemented. e half cell in a U-tube. 2 eal half cell with either a control or a referenc e tube separated the two half cells. An agar plug in the bend of th Although positive results for associating emfls with gro,,rth of T. thio- oxidans were obtained with this initial design, @L modification (Fig'. 1), which made aseparate opening to the2 agar bridge for connecting a ref- .erence calomel cell, was desired. Each of the original half cel3.s, the biological and control, could now be monitored individually with the calomel half cell. Experiments with this modified U-tube were sat- isfactory, however it was deficient in size and allowed concentration gradients to form so a new large biolo ical half cell w2as designed. 9 c -31-- The abo-Ve@-. e -@designs--l-@'Lmi-tt-d -th-z amount@--of--@.Uquid to- used to about 20 ml of nutrient. Furthermore, the long narrcnr tubes presented little oppor2tunity for changes in electrode design, stirr ngj, and continuous measurement 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- rounded with a glass bearing. Its action diminished adid and mineral concentration gradients and insure8d 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 were 6 .777-@ us ording the pH values. This entire biological half cell ed for rec was mounted in a constant temperature bath (29*). Prior to use of. this bath the electrochemical voltages were observed to fluctuate in a cyclic manner with the room temperature whenever T. thiooxidans was present. This behavior was especially2 evident when platinum elec trodes were used. The agar salt-bridge,, mounted in one opening of the round- bottcm flask, was led to a test tube containing saturated KCI main- tained at the same temperature as the biological half cell. A standard calomel electrode was mounted in the test tube as a reference half 2 -3 potentiom- cell. Leads from the complete cell were connected to a K eter and a pH meter. All parts -of th.e -.abDve- -cells whi-ch-.could- withstand -high- temperatures were sterilized by autoclaving. The other parts were sterilized by rinsing in 2ethanol followed by three rinses with sterile distilled water. Before sterilizat4-on the electrodes were cleaned with concentrated sulfuric acid and then washed with distilled water. All experimentation was oriented towards obtaining zero- -cell reactions. The current potentials of complete as wel2l as half T@- initial electrochemical measurements were made with a Iliodel X-3 eds Northrup potentiometer. This instrument gave accurate voltage deter- minations when zero current conditions were established. However.% while balancing the galvanometer to obtain zero cu4rrent coaditions 7 77-,- power was drawn initially from the cell causing, in some cases, a loss Of voltage. Since it was desirable to eliminate this probable contri- bution to variable results, a specially designed vacuum tube voltmeter' was obtained for use with a recorder. Open circuit conditions were maintained by use of this vacuum tube voltme2ter and continuous emf measurements could be taken with the recorder. III. l@aCROBIOU)GICAL TECMTIQUES 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 2 growth ,ras established, less complex media were sought by removing i ndividual-- cons=uent-&-&x-cm-th4-,--@@more L!L'LvfJfit@r area of study involved developing techniques for determining bacterial counts in the uniform biological half cell suspensions. 2 -.,th of I-rhiobacillus thiooxidans A. Gro Successful growth of T. thiooxidans was studied primarily in three media. At first, reproducible growth of T. thiooxidans was ob- tained with shaker cultures using Skerman'se basic mineral salts (17 salts). One per cent of sterile powdered sulfur was suspended in th8is medium. High yields of 109 organisms/ml were obtained with mature cultuz-es (maximum population density) after five days incubation. 8 Since the interpretation of the emf measurements in the Skerman's medium was exceedingly difficult., a simpler medium was sought. d A.T.C.C. medium, containing five salts plus 1 per cent sulfur, was studied as a growth nutrient. This medium gave mature cultures with 8 2 populations of 10 bacteria/ml. A further consideration of reducing the salts, comprising A.T.C.C., bmughtabout the removal of ferrous sulfite. The concentration of T. thiooxidans in mature cultures in 7 this medium, A.T6C.C.(-), was approximately 1 x 10 organism2s/ml. The bacteria to be used with the biocells usually were taken from five-day-old mature shaker cultures. These cultures were grown in 250 ml Erlemeyer f2.asks with 30 ml of medium in each flask. Incuba- tion was either at 29* or at room temperature. The mature cultures were harvested from the shaker flasks by 2 centrifuging the organisms at 9,000 rpm for 5 min in a Lourdes centri- fuge. After decanting the supernatant, the cells 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. d 7 -7 H20: 50.0; CaCl2, 25.0; Idilligrams/100 cc:. (ITH4)2SO4. 20.0; MgSO4 FeSO4, 5-0; ICi2PO4, 300.0. 9 B. Bacterial Counts la Two methods were applied for obtaining the bacterial popu tions of the cultures in the preparation and operation of the biocells. These methods were the Petroff-Hausser chamber count and the micro- Kjeldahl analysis for total nitrogen conten2t of the bacteria. T. Thiooxidans was removed from the medium by filtration before a modified micro-Kjeldahl analysis was applied. Turbidity dete=ina- 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 2 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 approximately 1 x 107/m, or greater. Use of the Petroff-Hausser count- ing chamber techniqu2e 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 phases of investi gation in order to minimize disturbing the biological half-cell ecology. The total amount of liq7aid required for the samples by this t9echnique ,7as negligible compared to the large biocell volume. -f, 10 C. General Microbiological Observations @lature populations obtained in the large biological half cell (Table III) with Skerman's medium were consistently less than observed in the shaker cultures. Subsequent experimentation with growth of difference in T2. thiooxidans in less complex media showed that this population density decreased with A.T.C.C. and disappeared when A.T.C.C.(-) was used. TABLE III TYPICAL MATURE POPUTATION DENSITIES Large Biological Shaker Culture 2 Half Cell Number of MediL= (organisms /ml) (organisms/ml) Salts 8 Skerman's I x 10 1 x 10 17 08 7 A.2T.C.C. 1 x I 1 to 5 x 10 5 A.T.C.C.(-) 1 to 5 x 107 1 to 5 x 107 4 Experiments with growth of T. thiooxidans in both shaker and biocell cultures using Skerman's and A.T.C.C. as growth media showed that the reduction in the number of 2mineral salts caused a decrease in the mature population density. Two typical biocells (Fig. 3) were The tem- started with similar inoculi and similar volumes of medium. perature for both biocells was held at 290. Similar lag periods were observed during the first day, followed by a rise in 1 bacterial cqncen- trations during the second day. During the third day of incubation, entrations were observed to rise more rapidly in the bacteria conc Skerman's than in A.T.C.C. After this period the population density 8 7 appeared.to stabilize at 1 x 10 for Skerman't and 4 x 10 bacteria/ml for the A.T.C.2C. media. :[V. EIECT.ROCHMIICAL OBSERVATIOI-TS I-RITH T. THIOOXIDALIS The initial investigations were concerned with establishing that an electrochemical potential, different from that of a control cell, exists when T. thiooxidans utilizes sulfur. The U-tube was chosen for these s2tudies. Each side of the U-tube comprised a half ce one gen-carban reference electrode. Dupli- biological and the other an oxy cate U-tubes were prepared with only one difference. T. thiooxidans was present i a-the.- qrm-of-.-O-ne..-Of-. them.. -,-The other half cell had only2 sulfur suspended in Skerman's medium. Figure 4 shows that a significant difference exists between the complete cell emfls of the inoculated and I cell. Such results were typical both wh contro en carbon and when platinum electrodes were used in the cells. The increase in the dif- ference wit2h time suggests that after acclimation to the cell, T. thio- oxidans became active and started to utilize the sulfur which in turn changed the electrochemical nature of the half cell. Biological and control half cell emflr-j using platinum elec- trodes ed as a function of time in the modified U-tube ,were measur 5 design (Fig. 1). The biological ha3.f.cells consistently gave voltages 12 which were lower than their controls, however, leaks through the agar plug at various times after cell preparation encourayed pursuing experi- mentation with a better cell desi-n. The remaining experiments were conducted with large biological half cells using platinum in preference to the slower responding carbon elec2trodes. 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 would 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 con2sideration for studying growth of T. thiooxidans. The initial pH values of approximately 5.0 gave way upon additions of the acid to values of -1-.0. --Each-add;Lttoa,-qf--Itc--kd -ted p3@ i Aimula -941@ct on of Sulfuric acid by T. thioo2xiclans. The maximum variation in half cell emf's with Skerman's medium experiencing these changes in pH was O.OIP V. Subsequent exoerimentation 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 e.-cpected wh7en sulfate ion is produced by T. thiooxidans were identified. A. Medium Effect on Biocell Activity Shaker cultures of T. thiooxidans were grown in Skerman's, A.T.C.C., arA A.T.C.C.(-) media under similar conditions. The prep- arations for centrifuging, washing, and resuspension in fresh sterile media, were planned to give populations having a concentration of 7 bacteria/ml. Howeverj the indeterminate losses in the trans- I x 10 fers gave rise to a slight variance in initial bacterial suspensions in the large biological half cells. The suspension in A.T.C.C.(-) was 2 7 1.2 x 107. The value for Skerman's was I x 10 and for A.T.C.C., 2 x 6 10 bacteria/ml. After i s observed to take place noculation, a lag phase wa during growth in each medium (Fig. 5). The v2alues 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 salts, which was ini- tially similar to the other two media, was noii greater. Subsequent 2i bacterial counts showed that the populations2 stabilized and after eight days the Skerman's-medi= had a population of 1 x 108 while both A.T.C.C. x 1 7 1. These saturation popula- media had approximately 5 0 bacteria/M tions were typical of large biocell experiments with these three media (Table III). If2 the variation in the initial concentration in bacteria can be ignored the amount of total acid produced by T. t'.Iiiooxidans seemed to depend upon the medium in which it grew. Since it was not possible to wash residual amounts of acid from the centrifuged cellsj, the initial pH values differed. Subsequent accumulation of acid is shown in Fig, 6. Of parti5cular interest was the fact that T. thiooxidans produced less cid (0.73 =ole) in the Skerman's medium in attaining a greater cell a 7 7 x 10 bacteria/ml than in the other two media (3.66 mmole population: for A.T.C.C.(-) and 1.89 mmole for A.T.C.C.) for the initial 70-hr rotrth interval2. 9 The growth of T. thiooxidans was followed by electrochemical measurements. The fact that-the control half cells (Fig. 7) started at exactly the same emf was coincidental. They usually differed by small amounts. T. thiooxidans was observed as a contaminant (Fig. 7) in the control half cell for the Skerman's medium after the 502-hr measure- ments. The visual presence of this microorganism was supported by a corresponding change in pH due to acid production. This microbiologi- cal activity caused a decrease in half-cell voltage as indicated by the values at the 70-hr interval. The change from the initial emfls for the Ske=an's and A.T.C.C2. half cell controls were typical for these media. Since their half-cell voltages usually stabilized after 20 hr, subsequent experi- mentation involved preparation and operation of two control half cells u=tilvoltage stability was observed. Then., one of the half cells was inoculated for comparison of their behavior as a function of time. These complications were5 minimzed when working with the A.T.C.C.(-) medium. Its half-cell emf was less erratic and stabilized quite readily. 15 7. ;,@ 77, The inoculated A.T.C.C.(-) (FiG. 8) half cell had the same initial voltage as its control half cello whereas the other inoculated half cells were higher than their controls. After 33 hr each of the inoculated half cells had voltages more negative than their control cells. The behavior of the inoculated A.T.C.C. and 2A.T.C.C.(-) half cells was comparable after 29 hr of operation. These cells gave lower voltages than the inoculated Skerman's half cell. The relatively small difference between the inoculated and control half cells comprising Skerman's medium pointed out the need to have a less complex medium intimately following growth of T.,thiooxidans electrochemica2lly. Thus: subsequent studies were conducted with A.T.C.C. and A.T.C.C.(-) media. The biological half cell emfls with these media were found to be more stable and further-remov,@d,@r;@@Aheir control half-ceu values. The removal of the ferrous sulfate from the A.T.C.C. medium offered addi-- tional improv2ement in control and biological half-cell stability And reproducibility. B. Long Term Biological Half-Cell Activ it Spedial precautions were taken with the preparation of one experiment which was allowed to run for an extended period of time. Emphasis was placed on minimizing external contact with the medium to eliminate the possibility of contamination and3 to increase the probabil- ity of long life. The supply of oxygen and car'@.-on dioxide for this 16 77@@- 777777@- ton If cell came from the atmosphere through sterile cot biological ha e wires leading into the p lu,,- c ,s pla ed in the small air gaps around th ogical half cel2l was cell through the rubber stoppers* The large biol ectrode at- ed with its Teflon stirrers pH meter, and the platinum el us It was filled with sterile A.T.C.C. medium' erized tachments. charadt 2 oxidand to give an (-pH, emf, sterility) and then inoculated with T. thio initial concentration of approximately 7 x 107 bacteria/ml. Sterile sulfur was used as the energy source in this medium. The cell counts pH and biological half-cell behavior for a 16-day period are shown in Fig. 9. Table IV describe2s the behavior of this cell approaching the lllth day. Between these times the emf and the bacterial counts fluc- tuated slightly with a fairly uniform accumulation of acid.- The general trend of the half cell potential was to become more negative. After the 111 days, the biological half cell was observed to be contaminated wi bacteria other2 than T. thiooxidans. The half-cell emf was observed to become more positive after b This observation was ec=ing contaminated. consistent with that obtained from other inoculated biological half cells which became contaminated with foreign bacteria. 17 TABLF, IV SUPPUVC,L'TARY DATA FO',R LOrk@j TERII BIOLCGICAL HALF CELL thiooxidans Utilizing Sulfur) Time Period Half-Cell emf (clays) (v) pH BacteriaLml 2 x 107 72 -0.61 1.18 5 x 107 76 -0.62 1.09 5 7 79 -0.61 2 1.05 3 x 10 7 83 -0.62 0.97 4 x 10 90 -0.63 0.92 4 x 107 7 99 -0.63 0.921 6 x 10 7 106 -0.64 0.95 4 x 10 ill -0.64 0.92 3 x 107 Several interesting features were worth noting in this long term experiment. lihe initial inocul2ation gave a population of 7 x 10 7 T. thiooxidans/ml. Death occurred lowering the population beloer the - level of detection by the end of thg- initial day of operation, the population was observed to increase to 1.5 x 107. Fluctuations between this value and 3 k 10 7 were observed 2 throughout the 17-day interval shown in Fig. 9. Between the end of this 7 period and the lllth day, a maximum count of 5 x 10 was obtained. The 7 Population dropped to 3 x 10 bacteria/ml towards the end of the experi- ment. The ecological factors 6 effecting bacterial growth kept the cell population in this range. The pH of the medium changed from an initial value of 4.85 to about 1..7 units after 15 days. Changes in its value after this time were small since appreciable amounts of acids had to be produced relative to the total amount present in order to bring about a change in pH. The fact that T. -thiooxidans was not increasing its popu- lation suggests inactivity but the increase in sulfuric acid concentra- tion indicates that a constant cell division and death rate existed. The general trend in the half-cell emf throughout this time period was t2o become more negative. The consistent trend in the curves obtained in this experi- ment indicated that many of the past variations in hilf cell potentials were no longer present and that reproducibility of subsequent biologi- cal half cells should be expected. One exception to this consistent trend toward a lower half cell potential may be significant. A notice- 2 able decrease in cell population appeared to take place after the eighth day. Thj,,s d4p,. recovery seemed to cause a simultaneous change in the electrochemical potential. C. ReDroducibi2.ity of Biological Half Cell Several biological half cells were prepared with T. thio- oxidans utilizing sulfur in the A.T.C;C.(-) medium to determine repro- ducibi2.ity of results. Figure 10 records their biological half-cell emf for a five-day period. Initially, the cells started at approxi- mately the same voltage followed by a slight rise and then a decrease. A slight divergence in values then takes place for one and one-half days. After this interv5al they gave equivalent results. The effect 19 er three days shown in Series XLVI when aft of hie.,er teuperature bath his time the thermostat for its the emf decreases markedly. At t 2 enced malfunctioned and temperatures exceeding 60* were probably experi during the evening period. This irreparable damage caused formation o a lower ha3-f-cell emf, measured upon return and subsequent control at 290. Figure 11 records the change in pH of each of these half cells.2 One can readiiy see that after the initial inoculation, a slight divergence takes place. This divergence disappears after about one day, giving rise to similar amounts of acid formed in each of the cells. The divergence in the microorganisms' contribution to emf and lp acid formation between the one-fourth and one and one-half day period 2 p g phase pi-sm- x eriencing a la appears to@-be,. c=3@,st-- in each of the biological half cells throughout this time interval. Figure 1P shows the bacterial cell count per milliliter as a function of time. A lag phase existed in each ce5U with a slight inconsistency in the recovery time. In general, the cell populations duplicated themselves in each of the cells. The last two values for the cell count in Series XLVI reflect the damage caused by the temperature of 7@ the bath exceeding the control value. 20 V. DISCUSSION The existence of an electrochemical contribution from T. thio- oxidans was established during the initial investigations with combined half cells comprising biological and contrar half cells with either a calomel or oxygen-carbon electrode. The cell comprising T. thiooxidane utilizing sulfur made contributio2ns to an electrochemical potential which reflected microbiological activity. Subsequent experiments with well defined biological half cells, comprising emf, pH, and bacterial count determinations, supported the initial observations. The inter- action of T. thiooxidans with the various nutrients as well as sulfur gave half cell emfls which were distinctly different t2han control half cells operated for comparable periods of time under identical experi-. mental conditions* The reduction in -the-n=berof.nutrient ions for growth of T. thiooxidans improved one's ability to measure its more intimate contributions to an electrochemical emf. However, since the measured half cell emf is an algebraic resultant of each ele2ctrochemi- cal contribution in the half cell it is not possible at.this time to say how T. thiooxidans is imp3-icatede If sufficient activity coeffi- -cients at these ionic concentrations would be available, the theoreti- cal contributions of the constituents of the media could be calculated 3 so the remaining biological contribution could be identified. This calculation is much too complicated at present. However,, as further 21 77 experimentation progresses towards finding the minim= n=ber of miner- als necessary for growth of T. thiooxidans utilizing sulfur the Dossi- bility of calculating the actual contribution of each constituent and thereby the specific contribution of T. thiooxidans becomes much greater. The premise that the study of electrochemical behavior!of autotrophs.,, especially Thiobacillus Sp., may be simpler than heterotrophs has not been resolved in thete-investigations. Additional study with both-types of species will be needed before a conclusion can be made that one or the other will offer the simpler approach to gaining a better under- standing of electrochemical processes that take place during micro- 2 biological growth. Ackno,@iled_7me,.it -The authors -t@who acted as consultants for obtaining optimum conditions for electrochemical measurements and microbiological growth. 22 3 7 7@ REFERRICES i. M. C. Potter. Proc. ROY. SOc. (T-ondon), 84B, 260 (1911). J. Bact.., 21, 18 (1931). 2. B. Cohn) 3. E. L. Starkey, J. Bact..* ioy 135-163 (1925). 4. R. L. Starkeyy J. 2Bact.-t 28jp 387-399 (1934). n. MicrobiOl. nes 5. E. L. Starkey, G. E. Jo and L. E. Fredrick, J. Ge 15., 329-334 (1956). it. Soil Sciencej, 51, 331-339 (1941). 2 6. K. G. Vogler and W. W. Umbre Gen. Microbio]L., 8 344-346 (@1953). 7. C. D. Parker and J. PriSk, J S. V. B. D. Skerman., A Guide t Identification of the Genera of Bacteria, Williams and Wilkins Company, 143-144 (1959). 9. E Kabat and M. Mayer, 5Experimental Imm'UnOcllemistry, Second Edition, s C-. Themas- Cnarle 71 23 Ili tLi ti O' bd> P. td )-d Ld H. ct la, @ii 0 0 ci- aq (D ra En (D ct 0) ci- (D (D PV (D fA OZI (D :i 0 M 4 0 2 (D0 (D Cf- FAm (D0 0(D ci C+ r) (+ r) 0II (D CL0 5 mP. ct (D ct $.It ci bJ @l- 1-4 bJ 2 tzi CY' ct- 0 H ro ID FJ 4D Fl 2 t-i t-i Fi o(D 0 cn ct0 Fl ct En ci- 3 c+ F- C+ P, C+ C+ ct Bacteria/ml td I 0 0 2 ell. C> 0 6 9--= C= I:d 0 > 0 > cn CD cn CD 0.12 0 0.08 0 0 0 0.04 0.00 28 20 24 24 O' 8 12 16 Time (days) -Tubes Voltage Difference Between Control. and Biocell, U Fig. 4 (D 0 I 0 ct- :k: ot4 0 cn bJ ct CD c-t FJ C-L0 ct 4 En 1>o 0 ct @:i H @-3 (n Fi (n ci- CD 0-0 V-17 VTPD;I ;UaaDjlTc UT poznzcza PTOV 9 gTa 09 Os ot or, OT 0 100-0 2 1010 eo I-o ti . . . .............. 0.3 A-A 0-0 rA ft 0 0- ap"2, 0 oi@-C 0.4 0 0.---'. 0 0.5 2 0 10 20 30 4 0 5 0 6 0 7 0 Time (hours) Fig. 7 Control flalf Cells with Different Media 2 (sulfur suspensions) C) A.T.C.C.(-) A.T.C.C. Skerma4n's -0.3 )t O.O.-O O O.... 0 -O 0-0. o@O 0 0 4 0. 2 0-0 r4 Ca bo 0 - 0.!5 0 0.61 0 1.0 20 30 4 0 50 60 7 0 2 Tizae (hours) -Lv>- 8 Inoculated Ifalf Cel.l.s wi.tli Different Media (T4 thiooxidans utilizing sulfur) A.T.C.C.(-) 8 A.T.C.C. O@@ 0 Skcritian's k lift -0.40 -0.45 to 0 Id o 0 0 0 A A 2 0 AC==.3 A A,'-c=:30A A A 0 2 4 8 10 12 14 16 6 Time (days) Fig. 9 Long T'erm Biological lialf Cell Behavior (T. thi.ooxi.dins, Siilfxir, A.T.C.C.) 0 .4 00 Cb C@ 0 0 1 2 3 4 5 Time (days) Fig. 10 Rep9roducibility of Biologic,-.1 Half Cel-1, e,-,,f (T. thiooxidans, A.T.C.'C.(-), sulfur) 0-0 Series XLVI Series XLVII 0-0 Series IZT o 10 I 2 3 5 6 Ti!rie (days) Fig. 11 Reproducibility of Biological Half Cell, pH (T. thiooxidans, A.T.C.C.(-)5, sulfur) 0-0 Ser-ies XLVI Series XLVII 0 0 Series LII 0 0 _.Z 0 0 0 7 10 2 4 5 6 2 Time (days) Fig. 12 Reproducibility of Bacterial Growth in Biological Ifalf Cell (T. thiooxidans, A.T.C.C.(-), sulfur) C)@@o Series XLVI Series XLVII3 Series LII July 1, 1965 Dea-- e Encrosed are two copies of our paper '?El ctrochemical Observations in Microbiological Processes. II. Gro,4th o-l' Th-iob@x@lu@-, Th ans. 2 'or release of this I would appreciate your making arrangements paper for publication. Any co=ents thel, you or yocx --s.sociates -proving the paper may have for im would be well received. Thar-k you for your kind assistance in getting this paper released. 9 Very truly yours, low -RVATIONS IN ICCriOBIOLOGICAL PROCESSES. II- ELEC-LROCHM.EECAL OBSZ GRO14TH OF T.TIIOBACILLUS THICOXIDANS SUI-24ARY The electrochemical activity of the individual chemicals in the nutrient medium for growth of Thiobacillus thiooxidans was studied along with the effect of the gases in7equilibri@= with their solutions. Several chemicals were active individually, howe2ver the magnitudes, as measured by changes in half-cell potential, were less than that observed when T. thicoxidans was present in their com- posite mixture. Sterilized Skerman's mineral salts and the American Type Culture Collection (without ferrous sulfate) media were not @74 sensitive electrochemically to changes from pure oxygen to nitrogen 2 atmospheres. When T. thiooxidans was present in these media,the biological half-cell emf became sensitive to changes in the oxygen content of the atmosphere in equilibri= with the organism and nutrient. The ability of T. thiooxidans to make an electrochemical contribution, as registered by a platinum electrode, is substantiated further by those investigations.1 I. Introduction The existence of an electrochemical contribution by Thiobacillus thiooxidans utilizing sulfur during growth was previously established. The fact, that this electro'chemical behavior existed, raised several questions about its origin. A review of the possible 2 2 half-ce 11 emfls which may exist when sulfur is oxidized in this system to sulfite thiosulfate, polythionate, or sulfate, suggests that the previously measured emf may be the resultant of several potentials due to the steady state concentrations of such intermediates. Even though Starkey and subsequent workers fo=d 2that only sulfate was formed by T. thiobxidans utilizing sulfur, the magnitude of the observed biological half-cell emf was such that it could rise from the presence of small amounts of intermediate products in steady state ratios. Therefore, microtechniques of analysis were employed to check whether or not any heretofore undetected oxidation-reductio2n reactions contributed to the observed biological half-cell emfls. Since T. thiooxidans is an aerobe, its growth depends upon the availability of o.-cygen. The effect of oxygen or the lack of it on the electrochemical character of control and inoculated half cells, was investigated also to isolate T. thiooxidans'con- 3 tribution to a half cell potential. II. Electrochemical Accessories The electrochemical apparatus used in these studies was equivalent to that described in the previous paper except for some minor additions. Of necessity, the vacuum @ube voltmpter and re- corder combination were used to measure the immediate electrochemical response to 2changes from aerobic to anaerob c atmospheres above the half cells. Figure 1 shows the essential elements of the circuitry ccmprising the electrochemical measurements. The voltage output on the output resistor was fed as an input signal to a Daystrom- Weston Model 6701, Type 1 continuous chart recorder. This recorder--- (an automat2ic recording potentiometer) remained continually bal- anced with the potential input from the voltmeter. The voltmeter output current was the direct result of the difference between the biological half-cell and the calomel reference,half-cell potentials. Open circuit conditions were maintained by use of this vacuum tube voltmeter. Calibrations with a stand2ard cell via a Leeds-Northrup K-3 potentiometer before and after experimentation insured correct recorder measurements of half-cell potentials. All half-cell emfls quoted in this paper are based on the standard hydrogen electrode. The large volume biological half cell, utilizing the platinum, glass, and calomel electrodes,agar-KC1 salt bridg9e, and Teflon stirrer, was used for these investigations. The preparation, 2 inoculation, and oueratio,-l of this cell duplicated our earlier in- vestigations except for one additioa to the cell design. A gas sparger was inserted into one of the five necks of the one liter flask. One of the rubber stoppers in an adjacent neck was perfo- rated to p;ovide an exhaust vent for the gases bubbled into the half 2 cell. This vent insured that the total pressure of the gases above the nutrient would not exceed that of the outside atmosphere. A Precision Scientific 'llet Test Meter" was used to measure the amount and flow of various gases into the half cells. This meter also saturated each gas with water vapor, thus reducing losses by evaporatio2n from the biocell. The effects of stirring the biocell and bubbling gases into. the media;-iveT@eL-d-L-teT-m±net@,,Pm-,,ftftl,,A'n-tt?-trgeir,'"-,=,d,,,o@c@.@ MV@-that,&s in magnitude of the half-2cell voltage for pure nitrogen and oxygen for each of the media were equivalent to those reported for air in the earlier paper, i.e. static atmosphere versus sparger bubbling, less thaa one millivolt; cn-off stirring action, less than two millivolts for each of the media considered in these studies. III. Microbiological Preparations and Observations5 T. thiooxidans for these biocell investigations were taken from five-day-old mature shaker cultures grown at room temperatures. 77777777--, a These cultures, comprising separately Skerrmn's' ATCC and ATCC (_)b mineral salts, were groiin in 250 ml Erldnmeyer flasks with ldp sulfur for an energy source. The mature cultures were har- vested by centrifuging the organisms at 9 000 rpm for 5 min. The2 supernatant was then decanted, the cells washed with fresh sterile medium, resuspended and recentrifuged. After another washing, they resuspended in the chosen sterile medium controlled at 29"C were for the biological half-cell investigations@ IV. Analysis for Sulfur Intermediates 2 The desire to relate emf changes to specific oxidation- reduction states within the sulfur system, made it mecessary to investigate the--possible--exis-tence-.nL-Rnlfur-intex=edi-a-tes-and-thejx__ changes with time. The approach to the analysis of the biocells was initially divided into two2 phases. Phase one was concerned with the gross description of thle cell medium at increasing time intervals during the experiments. Phase two concerned itself with the measurement of the amount of intermediate products formed during biocell operation. a. American Type Culture 1-tedium, mg/100 cc: (NH4)2SO4, 20.0; 1 50.0; CaCI2, 25.0; FeSO4, 5.0; KH 14gSO 4 - 7 HL 2@04, 300-0. b. Same as (a) without FeSO4. 4 :77 During the invettigations, the biocell wA@-- by titrating with standard bases and oxidizing agents. was i Sex:ples of the medium after different periods of growth showed that the presence of strong acid (sulfurid) could be assayed by titration with alkali to a pH o2f 5.0. An extension of the titration to a pH of 8.5 was employed to detect the presence of weak acids. These pH values were chosen frcm inflection points observed during ti- trating samples from the bi6cell. Since several of the possible sulfur intermediates that could be formed react with iodine, in- dependent and simul-taneous iodine tit.-ations were also conducted. Phase two concerned the development and application of a collirm chromatographic separation for the potential metabolic intermediates. This method was derived from' an adaption of 5 6 Trudinger's2 work on the medium of Thiobacillus X, and Iguchils methods of anion separation with anion-exchange resins. Approximately 10 =1 samples of cultures were removed from the biological half cell at prescribed intervals, and after fil- tration to remove all sulfur and bacteria, ala exact volume of 10 ml was eluted through a c8olumn of 3 g washed Dowex 1 x 4 anion exchange resin contained in an 8 = O.D. glass tube fitted with a glass stop- cock for flow control. The various fractions were eluted as follows: 5 1. The sulfite and sulfide fractions were eluted with 20 ml of 0.5 M a=onium acetate, pH S. 2. The thiosulfate was eluted with 10 ml of 2 M a=onium acetate, pH 5. 3. The polythionate fraction was removed in the following manner. A mixture of. -2l.ml bf I per -cent--qodi=- cyanide and .2 -ml of 1:10 annoni= hydroxide was added to the column and allowed to react with the remaining constitutents for 5 min. The solution was eluted and the resin was washed thoroughly with 20 ml distilled water. Two milliliters of 1:10 nitric acid was added to t2he column and allowed to react for 5 min. The eluate was then washed through with 1 ml of distilled water and collected in a graduated tube. The eluate, and-washings -to-tal--vol-ume-of S-m-2', with distilled water. The amounts of sulfite and thiosul..'Late eluted were deter- mined by iodine titration. The polythionates were assayed after the addition of two drops of 10 per cent ferric a=oni= sulfate to the prepared nitric acid eluates. The amount of color developed was determined by absorbance measurements of 460 nip with a Beckman LU spectrophotometer. 4These procedures were checked with known mixtures of the sulfur intermediates in cell media with and without T. thiooxidans before being applied to the analysis of biological half cells. 6 7777 The results from the analysis for the gross desdriptica of the shaker and biocell cultures are stn-..p ized in Tables I and 11 for typical changes in compositions as a function of time. Table III represents typical analyses for the specific sulfur intermediates. earlier observations 2with more These results are consistent with rapid accumulatioa of acid in the shaker culture as compared to the large biocells. Specific observations gove rning the data in these tables are as follows: 1. The accumulation of strong acid, indicated by alkaline titraticn to pH 5, was shown by subsequent precipitation with bar2i= chloride to be a direct result of the formation of sulfate ions. It was not unusual to obtain a pH of 1.0 within several weeks of operat -d "giver a total of 60--iM ion., - -For a,600 ml --bi@ocel-l-, th:ts-,woul of hydrogen ion or 30 mli of sul2fate ion. 2. The concentration of the weak acids, indicated by the second titration to pH 8.5, was very small, below 0.04 meq/ml. A significant increase during growth of T. thiooxidans was not ob- served. 3. The total oxidizable intermediates of the inoculated cells (Tablesl and II) was in9itially very small, below O-OC02 meq/ml. This concentration did not increase as a function of the time. The unimoculated controls showed essentially the same concentrations of these products as did the biocells. 7 TABLE I CROSS DESCRIPTION OF ACIDS MID OXIDIZABI@L" IIM-V-RMEDI-017LIS FORI@IED DURING LARGE- SEA=,-R CULTURE C-ROI-ITH (Medi=, ATCC) Time Titrati2ons (millieguivalents) For 10 ml Sample (days) pH NAOH to pH 5 NAOH to pH 8.5 Iodine 0 2.20 0.27 0.25 0.002 1 2.16 0.30 0.26 0.003 4 1.52 1.41 0.27 2 0.003 5 1.40 1.71 0.27 0.004 6 1.38 2.19 0.28 0.004 a 1.50 2.97 0.31 0.002 10 1.07 3.85 0.29 0.004 3 IP 1.04 4.40 0.25 0.003 8 TABLE II GROSS rr-.ccRIPTION OF ACIDS AIID OXIDIZABLE Illrrr-I.RIIEDIATES FCRIE-D RURING BIOLOGICAL F-us-CELL 001-ITH (Medi=, ATCC) Titrati2olis ('Lliillieguivalents) For 10 ml Samples Time pH NAOH to pH 5 NAOH to pH 8.5 Ic,.I-ine 0 4.45 0.01 0.24 0.002 4 hr 4.00 0.01 0.30 0.002 7 hr 2 3.68 0.01 0.36 0.002 1 day 3.00 0.05 0.30 0.002 2 days 2.42 0.21 0.26 0.002 3 days 2.13 0.37 0.26 0.0015 5 2dajs 1.95 0.49 0.26 0.002. 7 days 1.94 0.62 0.24 b.002 9 days 1.94 0.68 0.23 0.002 13 days 1.84 0.65 0.26 0.001 3 9 TABLE- III AITALYSIS FOR OXIDIZ-ABLE INTERIC-DIATES FORIC-D DURING BIOLOGICAL HALF-CELL GROWTH Titraticns (14illiequivalents) @or 10 ml Sa=les Time pH Sulfite Thio2sulfate PolythionatEs- (days) C*l B*2 c B c B c B 0 4.90 4.80 0.0012 O.CO12 0.0022 0.0022 0.022 0.022 1 4.90 2.72 0.0012 0.0017 0.0005 0.0007 0.0190 0.026 2 2 4.90 2.35 0.0010 O.CO10 0.0007 0.0007 0.0190 0.026 4.go 2.2o 0.0010 0.0010 0.0007 0.0007 0.0190 0.029 3 6 5.00 1.90 0.0012 0.0015 0.0007 0.0007 0.0190 0.019 a 9 '5. 00 1.87 0.0012 0.0015 0.0007 0.0007 0.0190 0.019 9 5.00 1450 0.0012 0.0015 0.0007 0.0007 0.0190 o.oi9 *1 Control half cell *2 Biological half cell 10 r sulfite or 4 for the accumulation of eithe Evidence thiosulfate ions over and above the control concentration which was below 0.00015 meq/ml (Table III) was not obtained. 5. There may have been a slight increase in the poly- 2 thionate concentration in one biocell (Table III) during the second -ntration day of incubation. The coace. went from 0.0022 meq/ml to 0. 00,'z.G me q/ml- on this day. This change may be a'signi.'icant in- crease. The control did not show a similar gain. This polyth2ionate concentration in the biocell subsequently rose to a high of 0.0029 meq/ml by the fourth day and then returned to control levels of O-CO19 meq/ml. These analytical results show that T. thiooxidans utili- zati= of sulfur gave rise primarily to accumulation of sulfate ion. If other intermediate2s are formed, their concentration levels are essentially below the limit of these methods of.inorganic analyses. Future analyses should be made for these intermediates on the sur- face or inside of the cell 3c wall of these bacteria since they do not appear to accumulate as metabolic products in the nutrient medium. 5 V. Electrochemical Observations Skerman's medium with 1 per cent sulfur was placed in the biological half cell which in turn was set in a constant temperature bath. The Daystrom-Weston recorder was connected to determine in- stantaneous27 changes in the half-cell emf. After an initial ob- servation period which showed the half-cell emf to be constant(the air bubbling into the cell), the air was replaced with pure nitro- geii. The top cu2rve in Fig. 2 shows that the presence Iof pure nitro- gen bubbling into the cell had little effect on the half-cell potential during this time period. This curve as well as those that follow are direct tracings from the recorder paper. A similar ex- periment in which the air in 'the bubbler was switched to pure oxygen indicated a slight, negligible reduction in 2the half-cell voltage. After the Skerman's medium had been inoculated with T. thioo,.Kidans and equilibrated with the air bubbling into the cell a change from this air to pure oxygen gave an i=ediate change in the half-cell emf (third graph, Fig. 2). The remaining curves on this page show that a change to air an2d nitrogen after the pure oxygen has Ibeen bubbled into inoculated medium for a time, also gave changes in the biological half-cell emf. Figure 3 shows the re- producibility of the oxygen effect in the inoculated Skermanla medium. These two curves were taken after the biocell's voltage had been stabilized =der pure nitrogen app6roximately 3 hr. Pure nitrogen raised and oxygen lowered the inoculated half-cell emf. The ex- tent of the gas effect under prolonged experimentation was observed 12 to depend udon the concentration of the inocul= as well as the time period after inoculation. Figure 4 gives an example of initial and subsequent oxygen effects c7oserved in an inoculated Skerman's medium. The majority of the effects, obtained from using the pure gases, took, place- --over- -a@ periiod@ of -several -minutes -i7a--thL- i=cul-ated- Skerman's medium. Appreciable oxygen, nitrogen, and carbon dioxide effects on the biological half-cell potential were observed only after the centrifuged, washed T. thiooxidans were placed in the medium. The latter effect, carbon dioxide, was not shown in2 the graphs; however, it essentially duplicates the effect observed with nitrogen. The ef f ex-t@ o@t- X-h=lgim. atmosphere in equilibrium with the biological half cell was found to be somewhat more complex when Skernnn's was replaced with ATCC medium. Switching f2rom pure oxygen to nitrogen caused changes in the emf of both control and biological half cells. The changes in emf of the biological half cell were much greater than that ob- servea with the control half cell. The following series'of experi- ments were initiated to individually, and then collectively, con- sider the degree of the gas effect for e7ach constituent in the ATCC medium. 13 The large biological half cell with the platinum electrcde was filled with approximately 600 ml of sterile distilled water. High voltages of approximately -0.24 v were obtained for a pure nitrogen atmosphere and a low of -0-30 v for pure oxygen atmosphere were obtained with this electrode. The half-cell emf'was allowed to s2tabilize with pi)re oxygen bubbling into it before the individual constituents of ATCC were added. After the addition of an individ- ual compound, the effects of the pure gases were determined by allowing the voltage to stabilize. This stability was identified by recording voltages until no change in half-cell emf was observed over a 10 min period. The2n the biocell was emptied and filled with fresh sterile distilled water for the next constituent Table IV silTr,.-narizes in part the results obtained from these investigations. These individual constituents make electrochemical con- tributions which in an oxygen atmosphere may bL- as high as -0.13 and as low as -0.426 v depending upon which one is selected. Except for calcium chloride and ferrous sulfate, the nitrogen and carbon dioxide effects almost duplicate themselves. Furthermore, it is of interest to note that there was relatively little difference in the nitrogen and oxygen effects except for KH2PO4. To silr.Tnn ize, varia- tions in partial p3ressures of carbon dioxide may affect the CaCl2 and Fel-:@04 contributions to emf, and variations in the nitrogen-oxygen 14 TABLR, IV C-ASI-:OUS EFF-NTS ON ATCC COI.TSTITURLLTS AS I-T-ASL-RFD BY HALF-CELL DiF Stabilized Half Cell E4F Pure Gases (2v Coacentration Oxygea tituent Cons (gm/l) Carbon Dio-.ide Nitro 0.5 -0.25 -0.20 -0.23 J-'!gSq4'1.H 20 2 CaCl 0.25 -0.19 -0.10 -0.13 2 F e l,'O 0.03 -0.72 -0.41 -0.42 4 KH2PO4 3.0 -0.32 -0.38 -0.46 (NH4:)2SO4 0.2 -0.23 -0.22 -0.24 pressures may affect the KH2PO4 contribution to an emf. Each of these ccmpouads and pure water, except for FESO., contribute electro- chemical voltages which were found to be significantly higher in the hydrogen scale series for half-cell potentials than have been observed with T. thiooxidans oxidizing 2 sulfur in thelpresence of air, i.e. -0.6 to -0.64 v. The half-cell.sensitivity to the FESO and 4 carbon dioxide interaction were part of the reason for eliminating FESO from the ATCC medium in subsequent studies. The individual behavior of the 2constituents in ATCC toward the various gases does not mean that such behavior is carried over in the complex medium. In order to further obtain data on how the half-cell emf may be aa'fected by these constituents, an experi-. ment.,was conducted in which the half-cell emf was recorded during an arbitrary sequence of adding each constituent2 to sterile distilled water while bubbling air into the mixture. Table V shows the stabi- lized voltages that were obtained after the sequential additions, starting with FESO During the experiment, the time for the half- cell voltage to stabilize was observed to vary with the individual components. Approximately 47 min was necessary for 1the FESO and 4 approximately 3-1/2 hr for the KH2po4 addition to stabilize. These variations in voltage suggest that the initial electrochemical be- havior of an ATCC medium may change if the time factor is not ;7 7 TABLL@ V ,TAU-Cr.LL Elg.,,S ruRII-IG SL,=TIAL ADDITIONS OF ATCC CONSTITU,-@i':iTS (AIR A7LI@OSPKr---Rl',' Sequential Additions to Sequential Stabilized Half- 2 Cell F,1,7's Distilled Wate:@ FESO -0.36 4 -0.35 4 -0.34 (NH 4 4)2SO4 -0.29 CaCI2 -0.42 KH2po4 17 sufficient for obtaining stability. This fact may explain some Of the variations that have been experienced in the past except for one additi=al step in the procedure which should contribute towards stability. After the media were prepared and thoroughly mixed., they were autoclaved for sterilization. This step should!act as a stabi- 2 lizing or uniform aging factor. In general, a review of the stabilized voltages obtained in this sequence shows that the resulting voltages are close together having a low of -0.42 and a high of -0.29 v. The addition of sterile powdered sulfur to the final mixture and subsequent sterilized media, did not change the electrochemical potential-of2 the media in the half cell$. An assessment of the previous experimental results di rected a study of changes in atmospheric composition in equilibrium with the ATCC medium without its ferrous sulfate constituent, ATCC(-). Figure 5 (top) shows that the short and long term operation of the sterile ATCC(-) medium under an atmosphere of pure nitrogen resulted in a relatively small and negligible change in half-cell emf as compared to air. After this determination, the nitrogen in the sparger was switched to oxygen (Fig. 5, bottom) and again a negligible change in half-cell emf was observed for the short and long term treatment. Thus, this medium responded to these two pure gases like the Skerman's and not like the original ATCC medium. 18 The next experimental sequence involved studying the specific gas effects an the inoculated ATCC(-) medium. Figure 6 4 shows that the presence of T. thiooxidans utilizing sulfur causes an instantaneous decrease in the half-cell emf when the air s effect in equilibrium with the cell was changed to pure oxygen. Thi was similar to those observed in both the Skermnn's and ATCC media. The ha lf-cell voltage, after 1. 2, and 4 hr, was higher than its original value and appeared to stabilize at -0.30 v. Similar treat- ment with the inoculated Skerman's medium reduced th half-cell 2 e voltage and caused it to remain below its original value. In another inoculated biological half cell (Series Lll) that had been operating under a normal air atmosphere, the air was changed to pure nitrogen (Fig. 7). The short term effect of this change was no'%, noticeable on the half-cell emf; howe2ver, the longer ter= effect, 1 and 5 hr, was appreciable. After this half-cell emf had stabilized in the presence of pure nitrogen, a change to pure oxygen was made (Fig. 8). In a relatively short period of tire, the half-cell voltage dropped to a value slightly below that experienced in Series '.KLVII (Fig. 6). Afte9r 2 hr this stabilized half-cell emf obtained from this switch to oxygen (i.e.p after a long term treatment with pure nitrogen) appeared to be different than 19 n7 77 the oxygen treatment of the air stabilized biological half cell (Series LXVII). The latter gave a value of -0.35 v which was above its initial cell emf while the former (Series Lll) gave a value which was lower, -0-55 v. Thus, the prolonged treatment wlth nitro- -"A gen on this biological half cell (Series LII2) suggests that a significant change from its-original character took-place. VI. Discussion The analysis for the existence and accumulation of partially oxidized sulfur intermediates did not reveal any concrete evidence for explaining the electrochemical contribution that T. thiooxidans appears to ma2ke when added to sterile media.- If such intetmediate .s exist, their concentratiQx3.g glre at or below the limit of analyses developed in this study. Other sources, such as the surface or inside of the T. thiooxidans' cell wall may make electrochemical contri- 2 butions. Justification or encouragement to explore such possibilities exist from this study. The investigations on individual and co=bined electrochemical investigations on the compounds comprising the ATCC and ATCC(-) identify their electrochemical activity. The activity of these ions reflected by their half-cell emfls especially with the6 ferrous sulfate eliminated from the nutrient, is higher in the hydro- gea electromotive series scale than experimentally observed during 20 idans. Thus active bibicaical half-cell operation with T. thioox sm by its presence makes an electro- the fact that this microorgani chemical contribution to the half cell is further substantiate through these investigations. 2 The greater sensitivity of the biological than the control half-cell t of the atmosphere potential to changes in the oxygen-ccnten and nutrient further suggests that viable T. thio@xidans is electro- chemically active. The actual individual or group of oxidized and reduced comPoundScontributing 2to this electrochemical. activity must still be iso lated. Aclmowledgmeg@@ The auth=e art-i-adebted.:L,,- who acted as consultants for identifying the electrochemical chax8,cte,--istic1s associated with T. thiooxidans utilizing sulfur. 21 REFERENCES 2. Latimer, W. M., The Oxidation States of the Elements and Their Potentials in Aqueous Solutions, Preatice-Hall,, Inc., p. 81 T1952).@ 3. Starkey., R. L., J. Bacteriology, 10, 135-163 (1925). 4. Skermpn, V. B. D.) A Guide7 to Identification of the Genera of Bacteria, Williams and Wilkins Company, p. 143-144 (1959). 5. Trudinger, P. A., Aust. J. Biol. Sci., 17, p. 446-458 (1964). 6. Iguchi$ A., Bull. Chem. Soc. Japan, 31, 597-600 (1958). @ll'[:P%@IATIOt.IAL CELL CALOi@,,]El- P O'I'E i\1'1- I 0 iN.,'] E T E R AGAR 2 E @ ICE I % L BRIDGE ELEC'F 'IODE -F U 0 VACIJUM 0 U'I' f) u C E L- I.- 0. I 2 VOLI-i@/IE-I'Et@ RESIS'fOl@ -F I i\l (J @A PL-A c 0 1) ELECI-,'IODE 2 -rci -!'A @l -F VOLTAGE COI\IS T 11 A t@l S t-- OR \!I E R CELL- @10.2 I-J L AT I @,l U t!ll -1@ODE EL.-ECI 120 V A.C. 6 0 1 FJ.g. I - 131.ock 43 41 2 4-1 '-tj 2 Pt C 141 4 L3 tv L) -0 3 uliteil (",,cr,tiian's Fig. Consistency of tli(.- OxY l,'c n L" f Ce c LI no 2 I lll.'L f -Cell -0 ini-,o Cell 0,@yffen Btibbled u 1 c lir Ati,.iorl)licr 9 4 6 lojr , OC ,IC-cc,-.,L pototiL.i.al- ACL 0..,CYGCI2I 1!"f.rect Oil li@ .I 1. .. VIC,. 4 Sulf Ur) Llii Lll-)bled -0.3 oxygen B 2 inlo cells Air Atinospl-iere o -0.4 d iie Media Ster Ill),-nedia-tel'Y After InoculatiOll -0.5 Several Days f,-fter Inoculation 6 7 5 4 2 3 0 1 Lnutes) Tiine (M A i BLII)].).I.eft i.nio Cel.1 -o.:50 Oyygen ]3ttl)bled into Cell. -0-35 2 o3eii ALitiosplici-c N:L Lr -0-40 -ibilizc(-l V,.tltte 5 (-I aiitt 2 hi- ItLLr) -0.45 0 2 3 4 5 6 7 -0 .4 Air ALjiiospliere 0;,yl-.,,Cn BLtbbled iiito Co!'I-l 14inut-,c,.) CA t;i C7, ri i,1.1. Li, o'i)ti A I-IlitL),;Vtit-,.L-c 0 ii 13Ltbbl.t-LI itilo Ct@] -0.4 U) 2 S-Labilized Value (2 hr later) Fig. 8 Oxygeh Effect in Itioctil.a-ted AII'CC(-) 14oditun (Series III, Age Six Days) 6 0 2 3 4 5 6 (TJ.me, A'-Ii.iiutes)