ENDOGENOUS FERMENTATION IN THIORHODACEAE'

DANIEL D. HENDLEY

Department of Biochemistry (Fels Fund), University of Chicago, Chicago, Ilinoi8

Received for publication April 2, 1955

The photosynthetic bacteria grow wellanaerobically in the light, and many membersof the group are strict anaerobes, but theirabilities to ferment added substrates appearlimited and no one has succeeded in growingthese organisms anaerobically in the dark onany substrate (Gest, 1951). On the other hand,it has been known since the work of Gaffron(1934) and Roelofsen (1935) that a strain ofpurple sulfur bacteria (Chromatium strain D),when grown on a medium containing thiosulfateor H2S, may exhibit an active endogenous fer-mentation over a period of many hours.

Gaffron (1934, 1935) believed he had shownthat the photochemical oxidation of H,S tosulfate by this organism is a reversible reaction,and that the endogenous fermentation representsan oxidation of reserve food materials coupledwith the reduction of sulfate to H25.The evidence for this was that the addition

of sulfate stimulated the endogenous fermenta-tion and apparently was necessary for thephotoassimilation of malate and butyrate. Whenincubated in the dark, the bacteria were alsofound to produce large quantities of an iodine-reducing material which gave a qualitative testfor H2S.

Roelofsen (1935), however, was unable todemonstrate any influence of sulfate on gas andacid production in the dark and Van Niel (1936)showed that the effect of sulfate on the lightmetabolism of organic substrates was a non-specific salt effect.Van Niel did, however, confirm the production

of an iodine-reducing material at a rate com-parable to that reported by Gaifron, but thereductant appeared to be largely nonvolatile inacid solution and therefore not H2S. The actualrate of production of H2S, specifically determinedby trapping it in acidified CdSO4 solution, provedto be barely 10 per cent of rates reported by

1 Part of this paper has been abstracted from athesis submitted by the author in partial fulfill-ment of the requirements for the Ph.D. degree.

Gaffron for the production of iodine-reducingmaterial. If the cells were devoid of storedelemental sulfur, the rate of H2S production waseven lower and could not be increased by theaddition of sulfate.Van Niel concluded that H2S production was

not an important metabolic activity of thepurple sulfur bacteria, and that the traces whichwere formed arose from reduction of sulfurrather than sulfate.

In the following study the endogenous fer-mentation of Chromatium strain D has beenreinvestigated. The results support Gaffron'sbelief that the iodine-reducing material producedin the dark is indeed H2$. However, it appearsthat only the first step of the photochemicaloxidation of H2S is readily reversible, sincecolloidal sulfur and not sulfate proved to be thesource of the H2S, in accord with the results ofVan Niel (1936).

Other products of the endogenous fermentationby Chromatium and the reduction of colloidalsulfur by baker's yeast were also investigated.

MATERIALS AND METHODS

The purple sulfur bacterium used for most ofthis work was "strain D," a strictly anaerobicorganism which has been variously called aThiocystis or Chromatium species. A culture waskindly supplied by Dr. Wilson of the Universityof Wisconsin, who had been maintaining it oninorganic medium. According to the account ofRoelofsen, strain D was isolated by him from anenrichment culture used by Gaffron and wassubsequently used by both Roelofsen (1935) andVan Niel (1936). Its favorable characteristics formetabolic studies are rapid growth on inorganicmedium with negligible formation of mucoidclumps and films.

Culture medium. The composition of theculture medium is shown in table 1. An essentiallyinorganic medium was used so as to approximatethe cultural conditions in the experiments ofGaifron (1934, 1935) and Roelofsen (1935).

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TABLE 1Composition of medium

Solution A1.0%o NaCl0.2%/o NH4Cl0.1% KH2PO40.1% MgClg26H200.02% CaCi2 anhyd.trace elements (Larsen)1.0 mg % indigo carmine0.90% v/v conc. HCl

Solution B1.26% Na2COs anhyd.0.6% Na2SOs205H200.02% disodium ethylene diamine tetraacetate(0.01% Na2S.9H20 added just before auto-claving)

In pyrex-distilled water

Parts A and B are autoclaved separately andmixed in equal volume after cooling. The result-ing pH should be near 8.0. The final concentra-tions of NH4Cl, KHI2P4, MgCl2*6H20 and traceelements are those of Larsen's medium (Larsen,1952) with the exception that the iron concentra-tion is increased 50 per cent. Indigo carmine is

used as an indicator for absence of oxygen(Hutner, 1950). With the addition of 2 to 2.5per cent agar this medium was used for main-taming pure cultures for inoculation.The bacteria were grown in a continuous cul-

ture apparatus shown in figure 1. In order tomaintain a relatively constant pH and pCO2, a

stream of 4 per cent CO2 in nitrogen was bubbledthrough the culture flak. Traces of oxygen inthe nitrogen (prepurified grade, Matheson Co.)were removed by washing the gas with a solu-tion of chromous sulfate. Hydrogen sulfide can

be admitted to the gas stream to cause re-

duction of dissolved oxygen initially present inthe culture medium or to supply an additionalfeeding after the thiosulfate has been consumed.After harvesting about 90 per cent of the bac-teria, new culture solution is injected by gravity-feed from sterile reservoirs of solutions A andB. Injections are made simultaneously fromthe two reservoirs through 20-gage stainless steelsyringe needles thrust, with sterile precautions,through the thick rubber of the sampling outlet.

Centrifugations of the bacterial suspensions

,'=78! / ~~~~2-CO2

CrSO4 SOON.

MEDIA INJECTEDHERE

Figure 1. Anaerobic culture apparatus

were made in completely filled and stopperedtubes. Dissolved oxygen was removed by theaddition of 0.01 per cent Na2S *9H20.Advantages of this continuous culture method

over the filled bottle technique of van Niel(1931) may be summarized as follows: (1) a largeinoculum of cells from the previous culture isalways available; (2) a pure culture can bemaintained with little effort over a period ofmonths; (3) pH fluctuations are minimized sincethe PCO2 is held constant; (4) temperaturefluctuations do not cause entrance of air; and (5)additions and samplings can be readily madewith little fear of contamination. With a 10 percent inoculum full growth is achieved withintwo days at 30 C.

Samples from continuous cultures were checkedfrom time to time for purity by both microscopicexamination and incubation in the dark bothaerobically and anaerobically on "A C medium"(Difco), with the addition of 0.3 per cent sodiumsulfate to detect sulfate reducers. In only oneinstance was a continuous culture vessel found tobe contaminated.For some of the work described here bacteria

were cultured in a stream of 4 per cent CO2 inhydrogen freed of oxygen by passage througha palladium catalyst (Baker and Co., Inc.). Themedium used was that shown in table 1 with theexception that thiosulfate was eliminated,(NH4)2S04 was substituted for NThCl, and thefinal concentration of NaHCOs was reduced from0.5 per cent to 0.25 per cent. Growth of thebacteria in this medium to a density of 13-mlcells per liter of medium has proved possible.

Cell volume determinations were made bysedimenting cells to a constant volume at 2,000

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x G. One ml of packed cells of sulfur-free bacteriahas a dry weight of approximately 0.34 g. In someinstances in this paper the amount of H2S evolvedby the bacteria is expressed in terms of "cellvolumes." This means the volume of H2S evolved,corrected to standard conditions, divided by thevolume of cells.

Determinations of CO2 and total acid produc-tion were made by the Warburg manometricmethod. Hydrogen production was usuallymeasured by the difference between two vessels,one containing palladium black to absorb hydro-gen (Roelofsen, 1935). Hydrogen sulfide wascontinuously removed by precipitation of ZnSon a large roll of filter paper in the center wellsaturated with a solution 0.25 M in ZnSO4 and0.5 M in malic acid adjusted to pH 5.1 with NaOH.This mixture quantitatively trapped the H2Swithout interfering with the CO2 measurements.At the end of the incubation period H2SO4 wastipped into the suspension to bring the pH tobelow 2. After 20 min the vessel was opened andthe filter paper was removed and submerged inexcess acidified iodine solution. The remainingiodine was back-titrated with thiosulfate. Con-trol experiments indicated that 95 per cent ormore of 4 gM H2S added could be recovered bythis method.When only H2S was to be measured, the bac-

terial suspensions were incubated in 5- to 30-mlsyringes. Advantages of syringe methods foranaerobic studies have been noted by Kirk andHansen (1951). Samples were withdrawn, freefrom contact with air, with the aid of a 0.25- or1.0-ml syringe. Each sample was then injectedthrough a rubber seal into the center compart-ment of a Conway diffusion dish where the H2Swas liberated by contact with acid. Furthersteps in the analysis followed the method ofArchibald and Gordon (1953).

Total fatty acids were determined by steamdistillation of the supernatant from the acidifiedbacterial suspension after centrifugation. Thedistillates were titrated with 0.1 N Ba(OH)2 ina stream of C02-free air. Excess standard acidifiediodine was added to an aliquot of the resultingsolution and back-titrated with thiosulfate togive a measure of the traces of SO2 usuallypresent. Excess Ba(OH)2 was added to theremainder of the distillate and the solutionreduced to a volume of 0.2 to 0.4 ml. The bariumwas precipitated as the sulfate by the addition of

excess (NH4)2SO4. Aliquots of the supernatantwere used for paper chromatography on WhatmanNo. 43 paper with a solvent consisting of 100 ml2-butanol + 25 ml 1 M NH40H aq. (Kennedy,1953, personal communication). After solventdevelopment the chromatograms were air-driedand sprayed with 0.08 per cent phenol blue in25 per cent aq. ethanol 0.005 N in H2S04.

Colloidal sulfur was prepared by the methodsof Od6n (1913). The final fraction used flocculatedin 0.2 M NaCl. This fraction contained relativelylittle adsorbed pentathionate (Freundlich andScholz, 1922). Kurtenacker's (1938) HgC12method was used for the determination ofadsorbed pentathionate and tetrathionate. In theexperiments to be reported, the amount ofpentathionate present never amounted to morethan 15 per cent of the H2S produced.

In the analysis for the radioactive isotope S35,carrier sulfate was added to samples containingorganic sulfur which were then dried and fusedin a platinum crucible with 4 g NaOH and 0.5 gKNO3 to effect oxidation of the sulfur to sulfate(Treadwell and Hall, 1942). Precipitations ofBaSO4 were made in dilute solution 0.01 N inHCl at 90 C. After being dried and weighed theBaSO4 was pressed into a circular depression in acounting planchet to make an "infinite thickness"layer and the radioactivity measured with aflow counter.

RESULTS

Identity of the reducing msbstance. When washedsuspensions of bacteria grown in thiosulfatemedium were directly titrated with iodinesolution before and after incubation in the dark,an increase in iodine titer always occurred.During illumination the titer decreased. Theseresults confirmed earlier observations by Gaffron(1935) and Van Niel (1936). The suspensions,taken from Warburg vessels or syringes, weretreated with an excess of iodine at a pH of ap-proximately 2 and immediately back-titratedwith standard thiosulfate solution.

Contrary to the results of Van Niel, however,the reducing substance was found to be largelyvolatile in acid solution. When the cells wereseparated from the supernatant, most of thereducing substance remained in the supernatant.The reducing agent in the supernatant wasfound to be acid labile, and precipitation withzinc and lead salts showed it to be H2S. A reduc-

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6~~~

z

L&i

0-i

I~~~~~~0 _

5 6 7 20HOURS

Figure S. Change of iodine-reducing titer duringillumination. A, Titer of entire suspension. B,Titer of supernatant. C, Sulfide titer in super-natant. Each Warburg vessel contained 0.052 mlbacteria grown in thiosulfate medium, 9 pM bicar-bonate, 500 p NaCl, in a total volume of 2.8 ml.The gas phase was 2 per cent C02 in purified ni-trogen.

ing capacity of 13.05 to 13.23 jsEq I2 couldbe recovered as ZnS or PbS from a superna-tant with a total iodine titer of 13.60 ;uEq.ZnS was precipitated in the presence of ammoniaand ammonium ion, and PbS in excess NaOH.Control experiments showed that under theseconditions thiosulfate, sulfite, cysteine, andmethyl mercaptan stay in solution.

In another experiment the H,S production inthe dark from two mucus-forming strains ofThiorhodaceae was measured. In each case atleast 80 per cent of the iodine-reducing titer ofthe supernatant could be precipitated as ZnS.Although H2: does not account for the entire

iodine titer of a whole cell suspension, it is ap-

parently responsible for the changes in titerduring light and dark periods. Figure 2 showschanges in light and dark periods of the iodinetiter of a suspension, the supernatant titer, andthe HES titer. Each set of points was determinedfrom three samples withdrawn by syringe froman alkalinized suspension in a Warburg vessel,avoiding contact with air. The titer of thesuspension was obtained by adding one sampleto an excess of acid iodine solution and im-mediately filtering out the cells under pressure

through asbestos. The titer of the supernatant

TABLE 2Rate of H2S production

In each experiment, washed cells of Chromatiumstrain D, grown in a medium containing the speci-fied H-donor, were suspended in a bicarbonatesolution under a gas phase containing 2-5 per centC02 in purified nitrogen and incubated in the darkat 34-35 C, pH 7-8. In the experiments of Gaffronthe change in iodine titer of the suspension isassumed to represent H2S production.

Time of Rate iExperimnent Cultumrl Conditions Incuba- Celi Vol-

tion umes HiSper Hour

1 S2,,-2 medium 2.5 0.462 S20-2 medium 4.4 0.333 H2 medium 5.6 0.084 H2 medium* 4.9 0.002

Gaffron lt S%00' medium 1.5 0.42Gaffron 2 S,O2-' medium 24 0.33van Niel§ malate medium + 24 0.045

H2S feeding

* Cysteine, 0.01 per cent used instead ofNa2S-9H20, 0.01 per cent in harvesting and wash-ing procedures.

t Gaffron, 1935, table VI, 4.t Ibid., table VIII, b), 1.§ van Niel, 1936, p. 329, expt. 3, No. 2.

was determined by rapid filtration of anotheraliquot directly into acidified iodine solution.HIS was determined by filtration of the thirdaliquot into ammoniacal ZnSO4 solution, theZnS being then washed and allowed to react withacid iodine solution. In each case, back titrationwith standard thiosulfate determined the amountof iodine reduced.The results indicate no significant decrease in

the cell titer, as measured by the difference be-tween the whole suspension and supernatanttiters, during the light period, or increase duringthe dark period. The differences between thetotal supernatant and H,S titers are probablylargely due to losses of ZnS during the washingoperation. In other experiments, better recoveriesof ZnS were obtained.

Rate of production of H2S. Table 2 presentsa few representative rates of H2S formation col-lected from various experiments. Two ratesfrom Gaffron's experiments (1935) and one fromVan Niel's (1936) are included for comparison. Itmay be observed that the rates obtained with thio-sulfate-grown celLs in the present experiments are

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in the same range as those reported by Gaffron.Since the rate of H2S production falls with time,van Niel would doubtless have observedsomewhathigher rates had he used shorter incubation times.

Influence of pH. H2S production increasesabout 25 per cent as the pH is raised from 7.5to 9.5, but drops rapidly to zero in weakly acid(pH 4.5) solution.

Effect of oxygen. It is important to maintainstrictly anaerobic conditions when measuringH2S production, since the bacteria catalyze a

very rapid oxidation of H2S by even traces ofoxygen. For this reason 0.005 to 0.01 per centNavS *9H20 was initially added to suspensionscontained in syringes to reduce the dissolvedoxygen present. In manometric experiments thegas mixtures used for flushing were freed fromtraces of oxygen by washing with a chromoussulfate solution or, in the case of hydrogenmixtures, by pasage through a palladiumcatalyst.Experiment with radioactive sulfate. The con-

firmation of H2S production again raised thepossibility of sulfate reduction. This possibilityhad been strengthened by a report of Nakamura(1939) that a species of Thiorhodaceae absorbedhydrogen in the dark when sulfate was added.The availability of the radioactive isotope S35suggested an experiment which could reveal even

traces of net sulfate reduction or exchange withreduced sulfur compounds in the cell.The general plan of the experiment was to

compare S3'04-2 reduction in a bacterial sus-

pension incubated in the dark with one incubatedin the light. The soluble and insoluble fractions(the solvents being 0.01 N H2SO4 and boiling 80per cent ethanol) were freed of unreacted sulfateand analyzed to find the percentage of the addedcounts that had been fixed. The incorporationof S35 into the H2S formed in the suspensionincubated in the dark was determined separatelyafter the addition of Na2S .9H20 carrier. TheH2S was trapped in a solution of 3 per centH202 made 0.5 M in NH40H, where the sulfidewas oxidized to sulfate (Treadwell and Hall,1942).A summary of the results is shown in table 3.

Only a very small percentage of the S5 addedcould be recovered in the bacterial fractions.The total sulfate actually fixed is not known sincethe amount remaining in the washed cells at thestart of the incubation was not determined.However, even if we assume a generous cellular

TABLE 3Failure to reduce S5g0i-2

Each vessel contained 0.13 ml washed cells ofChromatium strain D grown on thiosulfatemedium, 0.011 M NaHCOs, 0.5 per cent NaCl,and 0.2 pM of sodium sulfate and sulfuric acidcontaining 6.1 X 106 counts/min of S86. Liquidvolume = 25 ml. Gas phase was 2 per cent CO2in nitrogen. Vessels were incubated in either lightor darkness for 4 hours at 33-35 C. During incuba-tion 12.5 um H2S were produced in the dark vessel.Approximately 10 per cent of the cells containedsulfur inclusions.

Per CentCounts/Min of AddedCounpts/Mm

Added asS310i-2............. 6.1 X 106Recovered as S-2 (dark ves-

sel) ...................... 670 0.01Recovered in insol. fraction,dark vessel................ 390 0.007

Recovered in insol. fraction,light vessel................ 640 0.01

Recovered in soluble frac-tion,* dark vessel.......... 2,250 0.04

Recovered in soluble frac-tion,* light vessel......... 2,120 0.04

* Freed of S31047- by two precipitations withBaCl2 of added carrier sulfate.

concentration of 0.1 M for sulfate, the proportionof H2S arising from sulfate could not have beengreater than about 0.01 per cent assuming freeexchange of intracellular and extracellular sulfate.There was no measurement made of the

permeability of these cells to sulfate but it wouldappear to be high, at least during illumination,since sulfate is an end product of the photo-metabolism of reduced sulfur compounds andmust escape rapidly from the cell.One type of sulfur intermediate which would

not be detected by the procedures used is a non-volatile compound which is oxidized quantativelyback to sulfate by air in strongly acid solutionin the presence of bacteria. With this possibleexception we may conclude from this experimentthat strain D, when grown on thiosulfate mediumand still containing some elemental sulfur,reduces sulfate to a negligible extent in bothlight and dark compared to the production ofH2S in the dark.The reduction of S504-2 was not studied in

cells entirely devoid of stored sulfur by growth

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oS / ~ADDED.2 _ MORE BOILED BACTERIA-ADDED (ACID EXTRACTED)

0' I.0 20 40 60 80 100 120 140

MINUTES

Figure S. Stimulation of H2S production bycolloidal sulfur. 0 = H2-grown bacteria alone,colloidal sulfur added at 40 mins. 0 = H2-grownbacteria + boiled thiosulfate-grown cells, addi-tional boiled cells, acid extracted, added at 112min. Each syringe contained 0.013 ml H2-grownbacteria per ml of 0.02 M phosphate buffer pH 7.4containing 0.01 per cent Na2S-9H20. Temp = 34 C.

in a medium containing hydrogen as H-donor.It was found, however, that the addition ofsulfate to such cells did not stimulate H3S produc-tion. Van Niel (1936) found the same result usingcelLs grown on a malate medium.

Desulfhydration. Many species of bacteriahave been found to catalyze a desulfhydration ofadded cysteine and homocysteine (Fromageot,1951). The over-all equation for the process maybe written:

cysteine (or homocysteine) -+ a-keto acid +NHs + H2S

If the H2S produced by strain D were comingfrom intracellular cysteine, one might thenexpect to find a concomitant production of NHi.However, in several experiments where NH$production in the supernatant was measured bydirect nesslerization, it was found to be of theorder of only H0 the H2S production. It isconclu&dd that the production of H2S cannot beaccounted for by this mechanism.

Effect of boiled bacteria and colloidal &fur.Purple sulfur bacteria grown in an atmosphereof hydrogen and C02, with sulfate as the solesulfur source, exhibit very low rates of H2Sproduction. One example of this is given inexperiment 3 of table 2. When, however, boiledcells of thiosulfate-grown bacteria were added to

the hydrogen-grown bacteria a rapid productionof H2S ensued. Boiled cells of hydrogen-grownbacteria had no stimulatory effect. The factor inthe boiled cells of thiosulfate-grown bacteriawhich caused the stimulation was not extractedfrom the insoluble matter by boiling in 0.1 NHCl followed by boiling in 0.5 M NH4OH, andresisted oxidation by air. Colloidal sulfur wasfound to duplicate the effect of boiled cellsof thiosulfate-grown bacteria. The effects ofadded sulfur and boiled celLs on H2S productionin hydrogen-grown bacteria are compared infigure 3. Note the similarity in rates after theaddition of sulfur and the second addition ofboiled bacteria (this time acid-washed). The lagafter the addition of the sulfur or boiled cellsuntil the new rate is established can be shownto be due to oxidation of some H,S by oxygendissolved in the injected liquid, and does notrepresent a delay in the reduction of added sulfur.

It follows from these experiments that thedifference in rates of H2S production betweenthiosulfate-grown and hydrogen-grown bacteriais due to the accumulation of sulfur in the former.The source of sulfur for the much lower produc-tion of H2S from hydrogen-grown bacteria provedto be traces of colloidal sulfur arising from oxida-tion by air of the 0.01 per cent NaiSS9H20 addedroutinely during the harvesting procedure andto the wash water. If 0.01 per cent cysteine issubstituted for the sulfide as reductant, the H2Sproduction falls to the extremely low level indi-cated by experiment 4, table 2.An interesting aspect of the H2S production

induced by added sulfur is that the rate is higherper cell volume than that observed in cellsnaturally filled with sulfur granules by growthin thiosulfate medium. The initial rate of reactionafter the addition of colloidal sulfur in theexperiment was 2.0 cell volumes H2S per hour,more than three times the highest rates observedin the absence of added sulfur. The effect of sul-fur can always be duplicated by boiled, acid-extracted, sulfur-filled bacteria.

This result indicates that the availability ofsulfur normally limits the rate of H2S productioneven in thiosulfate-grown cells. It suggests thatthe intracellular sulfur is not as available forreduction as the extracellular sulfur, or that thesurface area of one or two intracellular sulfurgranules is simply not sufficient for maximumrates of reaction. The particle size of the added

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sulfur proved to be important since a hydrophilicsuspension of sulfur particles above colloidal sizewas found to give a lower rate of H2S productionthan colloidal sulfur. The frequency of bombard-ment of the cell surface by the sulfur particlesmay be a factor in determining the rate of reac-tion.Given an excess of colloidal sulfur, the bacteria

may produce up to three cell volumes of H2S in a4-hour period. To reduce this amount of sulfursome reductant must be initially present in thecells at a concentration of at least 0.27 N. Thereductant was not identified but the evidenceindicates that only a small part of it could besulfhydryl compounds. If this were not the casethen one would expect the iodine titer of the cellsto show a large decrease during incubation inthe dark, equivalent to the H25 production.Figure 2, however, shows that the changes in celltiter are small compared to the H2S changes.In some experiments the equivalent H2S produc-tion was found to be four times larger than theinitial acid iodine titer of the celLs.The utilization of added pyruvate or malate

for sulfur reduction is suggested by the fact thatthe addition of a mixture of these substances wasfound to increase H2S production from bacteriaplus colloidal sulfur by 25 per cent. The experi-ments of Gaffron (1935) and Van Niel (1936)also indicate a stimulation of H2S production byadded malate.

Growth experiment. Although the coupleH2S/S has a low potential at pH 7 (Latimer,1938), near that of DPNH/DPN+ (Rodkey,1954), it is energetically possible that the oxida-tion of such a substrate as pyruvate by sulfurmight furnish enough energy for coupled forma-tion of high energy phosphate bonds and growth.It was decided to test the possibility of anaerobicgrowth in the dark with sulfur as oxidant althoughKohlmiller and Gest (1951) had failed to demon-strate growthof a relatedorganism, Rhodospirillumrubrum, using KaFe(CN)e as oxidant. Sulfur is amuch poorer oxidant than KaFe(CN)6 but isa more natural substrate for purple bacteria.Accordingly, strain D was inoculated into ananaerobic medium containing malate, pyruvate,yeast extract and 0.5 per cent colloidal sulfur.The H2S formed was continuously removed.No growth occurred, however, until the culturewas exposed to light after a week of incubationin the dark.

Identification of fermentation acis. Gaffron

(1934) and Roelofsen (1935) observed theproduction of considerable and varying amountsof unidentified acids, which decreased thebicarbonate concentration during endogenousfermentation in the dark. In the present experi-ments, 70 to 100 per cent of the acid producedby both hydrogen-grown and thiosulfate-grownbacteria could be recovered as steam-volatileacid.Paper chromatography and a Duclaux distilla-

tion indicated that more than 90 per cent of thesteam-volatile acid is acetic acid. Further con-firmation came from the fact that treatment ofthe volatile acids with dichromate-H2S04 oxi-dizing mixture did not decrease the volatile acidtiter. Kohlmiller and Gest (1951) have reportedendogenous production of acetic acid at anunspecified rate from cells of R. rubrum, grownon organic medium. They found the acetic acidto be accompanied by varying proportions ofC02, hydrogen, propionate, and higher fattyacids, depending on the pCO2 and culturalconditions.

Production of hydrogen. No evidence could beobtained for the production of any gas in thedark other than CO2 from cells grown on thio-sulfate medium. From hydrogen-grown cells,however, which had been harvested usingcysteine instead of Na2S as reductant, theproduction of another gas at a rate of up to 0.11cell volumes per hour could be observed. Sincethis gas was absorbed by palladium black butnot by alkali, it was presumed to be hydrogen.These observations confirm those of Roelofsen(1935) who found hydrogen production from cellsof strain D grown on peptone medium but notfrom cells grown on thiosulfate medium. Whencolloidal sulfur is added to hydrogen-grown cells,the production of hydrogen is inhibited (table 4,expt. 1) and any hydrogen in the gas phase willbe actively absorbed down to a partial pressureas low as 0.1 to 0.2 per cent. Thus it againappears that the difference in endogenousmetabolism between the thiosulfate- and hydro-gen-grown bacteria may be explained by thepresence of sulfur in the former.

It should be noted that hydrogen-grownbacteria carry on an endogenous fermentation inthe absence of elemental sulfur but the rate ofproduction of C02 and acetic acid, especiallyC02, is greatly stimulated by the addition ofcolloidal sulfur (table 4, expt. 1).

Other products of the endogenous fermentation.

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TABLE 4Endogenous fermentation products of Chromatium strain D

In each experiment, washed cells of Chromatium strain D, grown in inorganic medium containingeither molecular hydrogen ("IH2-grown") or thiosulfate ("S-grown") as H donor, were suspended in amedium containing NaHCO,, 0.006-0.008 m, and NaCl, 0.02-0.03 M and incubated in the dark at 34 C.The gas phase was 2 per cent CO, in nitrogen unless CO is indicated, in which case 2 per cent CO2 inCO was used.

Experi- Time of Tol1 O H1| Tment Incubation Cniis 0 H2 iS Acid (Acid

kr PikpM DIR pZq

1 1.0 H2-grown 0.4 0.4 <0.01 2.0H2-grown + S* 2.8 0.0 4.1 3.6

2 4.7 S-grown 3.5 0.0 5.1 2.1 2.0

3 2.5 S-grown 21 22 17 12

4 2.7 H2-grown 1.0 1.3 4.7H2-grown in CO 0.5 0.0 6.1

5 4.4 S-grown 6.6 7.2 5.2 5.5S-grown, in CO 4.2 8.0 6.5 6.7

6 1.9 S-grown 3.5 4.7 1.6S-grown+ DNPt 4.9 6.2 4.5

* Colloidal sulfur added at the beginning of the incubation period.t 2,4-dinitrophenol 3 X 104 M.

Besides C02, acetic acid, H,S, or, in the case ofhydrogen-grown bacteria, hydrogen, no otherproducts of the endogenous fermentation were

detected in quantities equivalent to more thanhO of the acetic acid production. Analyses weremade for the following substances: neutralvolatile material oxidizable by a dichromate-HS104 mixture, lactate, glycerol, 2,3-butyleneglycol, and acetoin. The analyses were made on

bacterial suspensions cleared by Zn(OH)g pre-cipitation, according to methods 1, 8, 17, 18, and23, collected and modified by Neish (1952).Other acids besides the fatty acids were notlooked for since in most experiments more than

90 per cent of the acid produced was steam-volatile.Endogenous photoreduction. The HIS and

acetic acid produced by endogenous fermentationin the dark can be reutilized as hydrogen donorsby the bacteria during a subsequent light period.This is evidently the cause of the phenomenon ofendogenous photoreduction or "autoassiailation"observed by Gaffron (1934, 1935) and Roelofsen(1935) using strain D. Gaffron (1935) proposed

HoS as the substrate for the endogenous photo-reduction, whereas Roelofsen (1935) implicatedorganic acids.

Effect of inhibitors. The rate of production ofH,S from colloidal sulfur is 90 per cent inhibitedby heating anaerobically for 3 min at 70 C andmore than 98 per cent inhibited by heating at90 C, suggesting that enzyme-catalyzed reactionsare involved.

Gaffron (1935) found that the presence of COinhibits acid formation during the photometabo-lism of thiosulfate. The effect of CO on theendogenous fermentation, on the other hand, is toincrease acid production and decrease CO),production from cells grown on either thiosulfateor hydrogen media (table 4, expts. 4 and 5).The production of hydrogen is completely in-hibited by CO, but HiS production is not.Another experiment demonstrated that thesubstitution of helium for nitrogen in the gasphase does not alter the proportions of fermenta-tion products. These results suggt that heavy-metal enzymes are involved in the productionof H2 and C00 but not Hvg or acetic acid.

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ENDOGENOUS FERMENTATION IN THIORHODACEAE

At certain concentrations 2 ,4-dinitrophenolsignificantly increases the rate of production ofall fermentation products, especially fixed acid(table 4, expt. 6). According to prevailing theoriesof the mode of action of dinitrophenol (Simon,1953) this suggests that the endogenous fer-mentation is normally coupled to phosphateesterification.H2S production from yeast. It has been known

since the work of Dumas (1874) that yeast cellscan reduce sulfur, but a search of the literaturefailed to reveal data useful for comparison withthe results found here for Thiorhodaceae. Someexperimental work was therefore necessary.

When colloidal sulfur was added to washedcells of fresh baker's yeast (Fleischmann), initialrates of H2S production of up to 1.5 cell volumesper hour, and a total production up to 2.7 cellvolumes, could be observed. In rate and mag-

nitude these values fall in the range of thosefound previously for the purple sulfur bacteria.As with the bacteria, the H2S production may

greatly exceed the initial iodine titer of the cells.Direct titration of live or boiled yeast cells withiodine at pH 1 gave values between 0.25 and0.30 ,uEq per 10 AL cells, which is about } ofthe equivalents of H2S that may be evolved.The production of H2S is accompanied by the

evolution of C02 at approximately the same

rate. This rate of COM production is about 10times that of the endogenous rate without addedsulfur. Tetrathionate reduction proceeds at ap-

proximately the same rate as sulfur reduction.The addition of H2S, thiosulfate, sulfite, dithio-nate, or oxidized glutathione caused no detectablestimulation of the endogenous fermentation.As with Chromatium, 112S production from

yeast cells is retarded more than 90 per cent byheat denaturation.

DISCUSSION

The reduction of sulfur by yeast would appear

to be a rather unusual reaction for this organism,but the reduction of sulfur by Thiorhodaceaemust be considered a normal physiological proc-

ess, since these organisms are usually found innature containing intracellular S droplets. Therelatively high rate at which H2S may be pro-

duced, up to 12 cell volume per hour for a periodof several hours, is suggestive of the endogenousrespiration of green plants, with S instead of02 serving as oxidant. Although it apparently

does not allow growth, such a "sulfur respira-tion" might nevertheless provide useful energyfor flagellar movements and synthetic reactionsrequired for survival in the dark.While this paper was in preparation it was

learned that Larsen (1953) observed the evolu-tion of considerable amounts of a gas absorbableby a HgC12 solution, presumably H2S, fromsuspensions of Chlorobium thiosulfatophilumincubated anaerobically in the dark. It thusseems probable that H2S production is an im-portant feature of the metabolic pattern in allphotosynthetic bacteria capable of utilizing H2Sfor growth in the light.

ACKNOWLEDGMENTS

The author is indebted to Dr. Hans Gaffronfor suggestions and criticism of this work.

SUMMARY

The endogenous fermentation of washed cellsof a Chromatium species strain D, grown on aninorganic medium containing thiosulfate, resultsin the formation of varying proportions of H2S,CO2 and acetic acid. The formation of significantamounts of nonvolatile iodine-reducing materialas previously reported (Van Niel, 1936) could notbe confirmed. The evidence indicates that thesource of the H2S is elemental sulfur which isenzymatically reduced in a reaction coupledwith the oxidation of reserve organic materials.Reduction of sulfate, measured with the radio-active isotope S", was found to be negligible,at least with cells containing traces of storedsulfur.When the cells are grown on an inorganic

medium containing molecular hydrogen asH-donor, the endogenous fermentation results inthe production of C02, acetic acid, and hydrogen,but not H2S unless colloidal sulfur is present.The addition of colloidal sulfur to cells devoidof stored sulfur was also found to stimulate theendogenous production of CO2 and acetic acid,but anaerobic growth in the dark on an organicmedium with sulfur as oxidant could not bedemonstrated.The endogenous production of CO2 by baker's

yeast is stimulated about tenfold by the additionof colloidal sulfur. For equal cell volumes, therate and magnitude of the production of H2S ap-proximates that observable in Chromatiumstrain D, with added colloidal sulfur.

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HENDLEY

A continuous culture method has been de-scribed which presents advantages over previousmethods for cultivation of the Thiorhodaceae.

REFERENCESARcHIBALD, R. M. AND GORDON, H. 1953 Rho-

danese and desulfhydrase. Federation Proc.,12, 170.

DUMAS, M. 1874 Recherches sur la fermenta-tion alcoblique. Ann. Chim. et Phys., ser.5, 3, 57-108.

FREUNDLICII, H. AND SCHOLZ, P. 1922 Ueberhydrophobe und hydrophile Sole des Schwe-fels. Kolloidehem. Beih., 16, 234-66.

FROMAGEOT, C. 1951 Desulfhydrases, pp. 1237-43 in The enzymes, Volume I, part 2. Aca-demic Press Inc., New York.

GAFFRON, H. 1934 tJber die Kohlensiiure-assimilation der roten Schwefelbakterien.I. Biochem. Z., 269, 447-53.

GAFnRON, H. 1935 tlber die Kohlensiiureassi-milation der roten Schwefelbakterien. II.Biochem. Z., 279, 1-33.

GEST, H. 1951 Metabolic patterns in photosyn-thetic bacteria. Bacteriol. Revs., 15, 183-210.

HENDLEY, D. D. 1954 Metabolism of purplesulfur bacteria. Ph.D. thesis, University ofChicago.

HUTNER, S. H. 1950 Anaerobic and aerobicgrowth of purple bacteria (Athiorhodaceae)in chemically defined media. J. Gen. Micro-biol., 4, 286-93.

KIRK, J. E. AND HANSEN, P. F. 1951 A newprocedure for determination of respiration oftissue homogenates. Federation Proc., 10,208-9.

KOHLMILLER, E. F., JR. AND GEST, H. 1951 Acomparative study of the light and darkfermentations of organic acids by Rhodo-spirillum rubrum. J. Bacteriol., 61, 269-82.

KURTENACKER, A. 1938 Analytische Chemie derSauerstoffsduren des Schwefels. F. Enke,Stuttgart.

LARSEN, H. 1952 On the culture and generalphysiology of the green sulfur bacteria. J.Bacteriol., 64, 187-96.

LARSEN, H. 1953 On the microbiology and bio-chemistry of the photosynthetic green sulfurbacteria. Kgl. Norske Videnskab. Selskabs,Skrifter, NR 1.

LATIMER, W. M. 1938 The oxidation states ofthe elements and their potentials in aqueoussolutions. Prentice-Hall, Inc., New York.

NAKAMURA, H. 1939 Beitriige zur Stoffwechsel-physiologie der Purpurbakterien. V. ActaPhytochim. (Japan), 11, 109-25.

NEISH, A. C. 1952 Analytical methods for bac-terial fermentations. Report No. 46-8-3 (2ndrevision), National Research Council ofCanada.

ODAtN, S. 1913 Der Kolloid Schwefel. Akade-mische Buchdruckerei, Upsala.

RODKEY, F. L. 1954 Potentiometric studies onthe diphosphopyridine nucleotide system.Federation Proc., 13, 282.

ROELOFSEN, P. A. 1935 On photosynthesis ofthe Thiorhodaceae. Ph.D. thesis, Universityof Utrecht.

SIMON, E. W. 1953 Mechanisms of dinitrophenoltoxicity. Biol. Revs. Cambridge Phil. Soc.,28, 453-79.

TREADWELL, F. P. AND HALL, W. T. 1942 Ana-lytical chemistry, 9th Ed., Vol. II. John Wileyand Sons, Inc., New York.

VAN NIEL, C. B. 1931 On the morphology andphysiology of the purple and green sulfurbacteria. Arch. Microbiol., 3, 1-112.

VAN NIEL, C. B. 1936 On the metabolism of theThiorhodaceae. Arch. Microbiol., 7, 323-58.

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