JOURNAL OF BACrERIOLOGY, Oct. 1970, p. 462-472Copyright 0 1970 American Society for Microbiology

Vol. 104, No. 1Printed in U.S.A.

Anaerobic Growth of Purple Nonsulfur BacteriaUnder Dark ConditionsROBERT L. UFFEN AND R. S. WOLFE

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received for publication 3 April 1970

Purple nonsulfur photosynthetic bacteria were cultured anaerobically in the ab-sence of light by a modification of the Hungate technique. Growth was slow and re-

sembled that of fastidious anaerobes; on yeast extract-peptone-agar medium, eachcell produced about 16 descendants in 15 to 20 days. Growth was stimulated byaddition of ethyl alcohol, acetate and H2, or pyruvate and H2. Cells grown in the pres-ence of pyruvate and H2 produced acetate and C02; each cell produced approxi-mately 10 descendants in 24 hr under anaerobic, dark conditions. Spectrophotomet-ric evidence obtained from cells which were the product of five generations suggestsno difference between the bacteriochlorophyll and carotenoids synthesized by cellsgrown anaerobically under dark or light conditions. Likewise, the ultrastructure ofthe photosynthetic apparatus in cells grown anaerobically in the dark and in thelight appears similar.

The ability of photosynthetic bacteria to growin an anaerobic environment with dependenceupon radiant energy was documented in a reviewby van Niel in 1944 (37). In the studies described,growth did not occur under identical anaerobicconditions in the dark. Recently, Pfennig (25)summarized knowledge of the photosyntheticbacteria and emphasized the anaerobic nature oftheir ecological niche. Yet, it is not unusual tofind photosynthetic bacteria in anaerobic en-vironments which are inaccessible to radiantenergy, such as sewage sludge or layers of blackmud from streams, as noted by Kandratieva (17);little is known of their persistence under suchconditions. Certain nonsulfur purple bacteriahave been shown to ferment substrates anaero-bically in the dark (16, 21-23), and fructose wasobserved to stimulate the formation of coloniesof Rhodospirillum rubrum on the surface of asolid growth medium (29). Nevertheless, thegrowth of photosynthetic bacteria in the darkunder controlled anaerobic conditions has neverbeen demonstrated.

In the present study, a pink-red colony wasobserved during the isolation of methane bacteriafrom anaerobic mud by the Hungate technique(4, 14). This organism proved to be a nonsulfurpurple photosynthetic organism which grewslowly in the dark under conditions being usedfor the cultivation of methane bacteria. We havefound that other representatives of the purplenonsulfur bacteria do indeed grow slowly under

strictly anaerobic conditions in the absence ofradiant energy. In this communication, we definethe conditions under which growth occurs andpresent evidence that the membrane system ofdark-grown cells is similar to that of light-growncells.

MATERIALS AND METHODS

Bacteria. R. rubrum strain Si was obtained from H.Gest. R. rubrum strain 4 (Giesbrecht) and Rhodo-pseudomonas viridis NHTC 133 were obtained fromC. Sybesma. Rhodopseudomonas spheroides strain2.4.1 was obtained from S. Kaplan, and Rhodopseu-domonas palustris was isolated in this laboratory froman anaerobic enrichment; N. Pfennig kindly identifiedthis isolate.

Growth media. Cells were grown on a solid mediumconsisting of (grams per 100 ml distilled water): yeastextract (Difco), 0.3; Bacto-peptone (Difco), 0.3; agar,2.0; and resazurin (Allied Chemicals, New York,N.Y.), as a redox indicator, 0.01. The mineral solu-tion of Pfennig and Lippert (27) and a vitamin solu-tion (39) also were added in 0.1- and 0.8-ml amounts,respectively, to 100 ml of growth medium. The cys-teine-sulfide reducing agent of Bryant and Robinson(4) was sterilized and added separately to the growthmedium. Liquid media of the same composition butwithout agar and supplemented with ethyl alcohol,sodium acetate, or sodium pyruvate (final concentra-tion, 0.3 g per 100 ml of medium) were prepared.Ethyl alcohol and sodium pyruvate were filter-ster-ilized and added separately to the growth medium.Growth media were prepared and sterilized under astrictly anaerobic argon atmosphere by a modificationof the Hungate technique (14) as described by Bryant

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and Robinson (4). Sodium carbonate (0.2 g per 100ml of growth medium) was added to media when gasmixtures which contained 20 or 50% CO2 were used,and sodium phosphate buffer (pH 7.0) at a final con-centration of 0.01 M was added to liquid growth me-dium which contained sodium pyruvate. The final pHof the sterile media was 6.9 to 7.1. For aerobic growthof cultures in the dark, liquid media of the same com-position were prepared by omitting agar, resazurin,and cysteine-sulfide reducing agent.

Growth conditions. A simple modification of theHungate technique was developed to maintain andgrow cells under strictly anaerobic conditions. Sterileagar medium (25 ml) was placed into a 150-ml pre-scription bottle under a stream of argon. The bottlewas aseptically sealed with a solid black-rubber stop-per, and the agar was allowed to solidify on the flatside of the bottle. Cells were transferred asepticallywith a platinum loop into the anaerobic bottle, andwere spread over the agar by means of a glass rod (Fig.IA) while a stream of 02-free, sterile gas was passedinto the bottle. Finally, the bottle was tightly sealedwith a solid black-rubber stopper (Fig. 1B), and theneck was dipped into melted paraffin. Aseptic tech-nique and manipulation of gas probes were performedas described previously (3,4). When gas mixtures whichcontained CO2 were used, the growth medium was al-lowed to equilibrate with CO2 before cells were spreadonto the agar surface. Liquid media used in studies ofgrowth under either anaerobic, light or anaerobic,dark conditions were prepared in a similar mannerand dispensed into sterile test tubes as described byBryant and Burkey (2). For certain purposes, cellswere grown anaerobically under low light conditions(55 to 60 ft-c) obtained with a 60-w incandescent lightbulb placed approximately 45 cm from the growth bot-tles or tubes. Dark-growth conditions were maintainedby placing anaerobic bottles or test tubes in fiberboard(3 mm thick) cylinders with light-proof closures. Simi-larly, flasks of liquid media for aerobic, dark growth ofcells were placed in a fiberboard cylinder which wasfitted onto the table of a rotary shaker (New BrunswickScientific Co. model VS-100). Each Erlenmeyer flaskwas filled to 30% capacity and was shaken at about 300rev/min. Unless stated otherwise, cells to be used forinocula were serially subcultured at least twice underthe specific conditions of the proposed experiment be-fore an inoculum was taken for a growth experimentunder the same conditions. The growth temperature inexperiments was 34 to 37 C.

Preparation of gases. Gases used to maintain anaer-obic environments were vigorously scrubbed to re-move all traces of oxygen. Argon-CO2 gas mixtureswere prepared by means of a flow meter (SHO-RATE"dual-flow," Brooks Instrument Division, Hatfield,Pa.) with a common outlet. High-purity Ar, H2, andH2-CO2 (80:20 or 50:50 mixture) gases were pur-chased from Matheson Gas Products, Inc. (Joliet,Ill.). Carbon dioxide was obtained from Simpson Dis-tributing Co., Urbana, Ill. Trace amounts of 02 wereremoved by passing the gases over hot reduced copperfilings as described by Bryant, McBride, and Wolfe (3).In these experiments, two furnaces were connected inseries. In addition, for certain experiments the gases

rubber stopper

Aagar

gas

FIG. 1. Schematic represenitation of technique usedfor growth of bacteria on an agar surface under strictanaerobic conditionts: (A) inoculation of growth me-dium under continuous gassing; (B) rubber-stopperedgrowth bottle.

were bubbled through a photochemically reducedmethylviologen solution at pH 10 (31) as modified byYu and Wolin (40). This additional precaution wastaken to detect as well as to remove trace amounts ofoxygen.

Determination of cell numbers. Cell growth waswashed from the surface of solid growth media andcollected into a known volume. Samples of these cellsand cells grown in liquid growth medium were counteddirectly by means of a Petroff-Hausser bacteria-count-ing chamber.

Protein. Samples were dried in vacuo and digested at37 C for 1.5 hr in 1 N NaOH. Protein content was esti-mated by the method of Lowry et al. (19). Crystallinebovine serum albumin was used as a protein standard.

Bacteriochlorophyll. An acetone-methanol (7:2,v/v) solvent system was used to extract bacterio-chlorophyll from washed cells (7), and the optical den-sity was measured at 775 nm with a Beckman modelDU spectrophotometer. The bacteriochlorophyll con-tent in solvent extracts was estimated according to themethod of Cohen-Bazire, Sistrom, and Stanier (7).

Gas chromatography. Carbon dioxide was detectedwith a Packard gas chromatograph equipped with asilica gel column connected to an argon ionization de-tector. Volatile fatty acids were resolved by placingacidified samples of culture supernatant liquid onto aChromosorb W (60/80 mesh), acid-washed, 20%Tween 80 and 2% H3PO4 gas chromatographic columnas described by Erwin et al. (11).

Spectroscopy. The detection and characterization ofbacteriochlorophyll and carotenoids were accom-plished spectrophotometrically with whole-cell sus-pensions in 60% sucrose solution as described byPfennig (26). Spectra were obtained by use of a Cary14 recording spectrophotometer.

Measurements of light-induced absorbancy changesin washed whole cells were obtained with a split-beamabsorption difference spectrophotometer as described

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by Sybesma and Fowler (33). Cells were suspended inthe salts solution used by these investigators.

Photomicroscopy. Photomicrographs were takenwith a flash attachment to a Zeiss photomicroscope.

Electron microscopy. Cells were fixed by the pro-cedure of Kellenberger, Ryter, and S&chaud (15).Cells grown anaerobically in the dark were fixed witha 1% (w/v) solution of osmium tetroxide for 3 hr.Agar blocks were dehydrated in aqueous ethyl alcoholas described by Holt and Canale-Parola (13), andwere embedded in Epon 812 by the method of Luft(20). Sections were cut on a Reichert ultramicrotomewith a diamond knife and were mounted on uncoated300- or 400-mesh copper grids. Sections were stainedwith a 1% solution of uranyl acetate for 45 min at 45 C(J. Murphy, personal communication) and with Pb-citrate, prepared according to the method of Venableand Goggeshall (38), for 6 min. All sections wereexamined with a Siemens Elmiskop I electron micro-scope operating at 60 kv, equipped with a 50-Mm ob-jective aperture.

RESULTS AND DISCUSSION

Rhodopseudomonas spheroides, R. palustris, R.viridis, and two strains of Rhodospirillum rubrumwere grown under strictly anaerobic conditionsin the dark. A typical culture of R. rubrumgrown anaerobically in the dark, on the surfaceof the unsupplemented agar medium, is presentedin Fig. 2. The organism was grown under a 20%C02-80% H2 atmosphere; however, similargrowth was readily obtained under an argon at-

FIG. 2. Photograph of anaerobic bottle showinganaerobic, dark growth of R. rubrum Si. Cells weregrown under a CO2 (20%)-H2 (80%) atmosphete at37 C for 30 days.

mosphere. The use of bottle cultures (Fig. 1 and2) has the advantages of the streak-plate forpure culture isolation, as well as the advantagesof cell yields obtained through heavy inoculationand subsequent confluent growth. Furthermore,additional redox indicators may be included withinthe bottle but not in contact with the medium,as described below.To establish that an anaerobic environment

was maintained throughout our growth studies,the gas used during preparation of media andinoculation (Fig. 1) of the anaerobic bottles wasextensively cleansed of oxygen, and resazurinwas placed in the growth medium. Throughoutthe medium preparation and bacterial growthperiod, the medium remained colorless. Withinseconds after exposure to air, the resazurin be-came oxidized, as indicated by a color change topink.

Additional evidence was sought to demonstratethat anaerobiosis was obtained and maintainedby this technique. An alkaline solution of methyl-ene blue (0.1%, w/v) in 0.5% glucose (w/v) wasboiled under a stream of 02-free gas until color-less, and 10 ml of the reduced solution wasadded anaerobically to a growth bottle of R.palustris which had been placed on its side, agarside up. In this manner, the reduced methyleneblue solution had a large surface exposed to thegas atmosphere but did not contact the agarmedium. After 10 days of incubation in the dark,bacterial growth was apparent on the agar; theindicators remained colorless. Upon exposure toair, the methylene blue solution turned blue andthe resazurin in the growth medium turned pink.R. spheroides and R. palustris grew under identicaldark conditions when the methylene blue indicatorsolution was replaced with 10 ml of a slightlyturbid culture of Escherichia coli in NutrientBroth (Difco). After 10 days, typical growth ofthe photosynthetic organisms had developed onthe surface of the solid medium. Upon exposureto air, the resazurin contained in the solid yeastextract-peptone medium turned pink, and E.coli in the broth grew to a high density (109cells/ml). It was concluded from these experi-ments that strictly anaerobic conditions weremaintained in the culture bottles during thegrowth of purple nonsulfur bacteria. Moreover,the same anaerobic bottle technique supportedgrowth of the strictly anaerobic, methane-pro-ducing bacteria Methanosarcina and Methanobac-terium strain M.o.H. Growth media have beenmaintained in a reduced state under these condi-tions for several months, and Methanosarcinaremained viable for 7 months in an unopenedbottle with methanol as substrate, indicating theabsolute anaerobic state of the culture conditions.

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To show that no light was available duringgrowth, photographic plates were attached to theinside of the fiberboard cylinders; these platesfailed to become exposed. Dark growth of thepurple nonsulfur bacteria was unaffected wheninfrared radiation was excluded by wrapping thegrowth bottles or the fiberboard cylinder, orboth, with heavy duty aluminum foil.On the unsupplemented agar medium, R.

rubrum, R. palustris, and R. spheroides showedgrowth in heavily inoculated areas 10 days afterinoculation, and individual colonies about 1 mmin diameter developed by 20 days. Growth wasnot stimulated on this medium by replacing theAr atmosphere with H2, C02, or gas mixturescontaining 20% CO2 and 80% H2 or Ar. Growthand motility could be maintained by repeatedtransfer of each organism under dark, anaerobicconditions. Cells maintained under dark condi-tions were transferred quickly in a semidark room.The growth of R. rubrum Si and R. rubrum 4

under anaerobic, dark conditions was determinedby direct cell count. A cell suspension of youngcells grown anaerobically in the dark was pre-pared, and a 0.1-ml sample was spread onto thesurface of agar growth medium with a glassspreader. The argon-filled bottles were incubated,agar side up, in the dark. After 23 days, cellgrowth was determined (Table 1). Motile cellswere observed under phase-contrast microscopy(Fig. 9). Both strains of R. rubrum grew at a veryslow rate, requiring approximately 5 days for onegeneration. When experiments were conductedwith R. palustris, similar growth occurred, but anaccurate cell count was not obtained as the cellsformed clumps which could not be dispersed.Cells of the purple nonsulfur bacteria investigatedpossessed good pigmentation after growth underanaerobic, dark conditions.

Kohlmiller and Gest (16) and Nakamura (21-23) reported that under dark, anaerobic conditionsR. rubrum Si and R. palustris fermented certainorganic acids. It is conceivable that the samefermentative pathways are functional in cellsgrowing anaerobically in the dark. Cells of R.rubrum SI were placed into the liquid yeast ex-tract-peptone growth medium to which variousshort-chain fatty acids, alcohols, and dicarboxylicacids were added singly or in combination. It wasfound that growth under anaerobic, dark condi-tions could be significantly stimulated by theaddition of ethyl alcohol under an Ar atmosphereand acetate or pyruvate under an H2 atmosphere(Table 2). The best cell growth obtained after10 days at 37 C with ethyl alcohol or acetatewas 2.30 X 108 and 2.24 X 108 cells/ml, respec-tively. Pyruvate stimulated the growth of R.rubrum which, after 36 hr at 37 C, reached ap-

TABLE 1. Growth of Rhodospirillum rubrum underanaerobic, dark conditions on yeast extract-

peptone-agar medium"

No. of cellsStrain generationo b

Initial (No) Final (Ng)

SI 4.2 X 107 107 X 107 4.74 17.6 X 107 345 X 107 4.3

aCells grown under Argon atmosphere for 23days at 37 C.

b Number of generations = log2N, - log2N0where N represents the number of cells.

TABLE 2. Growth of Rhodospirillum rubrum Si onliquid medium under anaerobic, dark conditions

Period No. of cellsGs of No. of

Substrate Ghase incu- -genera-bation Initial (No) Final (Ni) tions

Ethyl al- Argon 240 0.68 X 107 23.0 X 107 4.75cohol

Acetate Hy- 240 0.48 X 107 22.4 X 10' 5.54dro-gen

Pyruvate Hy- 36 1.30 X 107 23.4 X 107 4.17dro-gen

proximately 2 X 108 cells/ml. The addition ofpyruvate reduced the time required for onegeneration from approximately 5 days on yeastextract-peptone-agar growth medium to 8.6 hr inliquid medium (Tables 1 and 2). The growth ofR. rubrum Si under anaerobic, dark conditions inbroth with pyruvate is shown as an increase inprotein and bacteriochlorophyll in Fig. 3 and 4.Figure 3 presents the protein increase of cellsgrown for 72 hr. In this experiment, R. rubrumSt cells were grown anaerobically in the light inthe presence of pyruvate for 24 hr and were thenplaced into fresh medium with pyruvate underanaerobic conditions in the dark. There was nolag in protein synthesis when the cells weretransferred from light to dark growth conditions.During the first 6 hr after transfer to a dark en-vironment, the bacteriochlorophyll content de-creased, and then it increased parallel with pro-tein synthesis. Protein synthesis by cells subcul-tured serially twice in a dark environment ispresented in Fig. 4. Again, no lag in protein syn-thesis was observed, and bacteriochlorophyll in-creased proportionately. The growth rate of cellspassed from anaerobic, light to anaerobic, darkconditions (Fig. 3) was greater than that of cellspassed from anaerobic, dark to anaerobic, darkgrowth conditions. The rate of growth was sig-

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tot\h 30

'1320UN 10_K O

cLE0

th

-

;Vt

rteI mg IBChlI IL9Aprotein mmgJ

-N,

1-

Zr:

Vt0,k-,

¼~KzVt'

24 48 72TIME (hours)

FIG. 3. Increase of protein (0) and the bacterio-chlorophyll (A) content in R. rubrum Si cells grownon pyruvate in the light anaerobically and transferredinto dark, anaerobic conditions. Bacteriochlorophyllconcentration as a function ofprotein synthesis is pre-sented above (@).

nificantly increased under an atmosphere of H2by the addition of pyruvate (Tables 1 and 2).Schoen (29) observed that R. rubrum formedcolonies in the dark under anaerobic conditionsif fructose was present in the growth medium. Weobserved no stimulation of the anaerobic, darkgrowth of R. rubrum Si in the presence of glucose,fructose, or glycerol.

Kohlmiller and Gest (16) reported that R.rubrum Si cells grown under both anaerobic,light and aerobic, dark conditions fermentedpyruvate anaerobically in the dark and producedC02, acetate, and propionate. In our experiments,R. rubrum Si cells grown under dark, anaerobicconditions (5 to 15 generations) produced onlyCO2 and acetate. In our system, anaerobic, darkgrowth of R. rubrum on pyruvate under a H2-CO2 (50:50) gas phase failed to form propionate.Trace amounts of propionate were detected onlyin the supernatant solutions of cultures grownunder light, anaerobic conditions. The fermenta-tion of pyruvate during dark, anaerobic growthresulted in a drop in the pH value of the mediumfrom 7.0 to 5.7 within 48 hr after inoculation.

This might account for the observation that,after anaerobic, dark growth on pyruvate for 48hr at 37 C, R. rubrum Si cells rapidly becamenonphototactic and lost viability. Consequently,to maintain cells under anaerobic, dark growthconditions they were transferred every 36 hr intofresh growth medium.

Bacteriochlorophyll-a was extracted from cellsof R. rubrum S1 described in Table 1. The bac-teriochlorophyll-a content in the solvent extract(7) and the protein content remaining in the celldebris were determined (Table 3). The bacterio-chlorophyll-protein ratios of strains SI and 4 aredifferent; the significance of this difference is notknown. The bacteriochlorophyll-protein ratio for

\s 30< 20

10

I',th8P0~

K.0i:

-N3

N-4

-4

it

(Z30~

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TIME (hours)

FiG. 4. Increase ofprotein (0) and bacteriochloro-phyll (A) in R. rubrum S1 cells grown on pyruvate,under dark, anaerobic conditions. Bacteriochlorophyllsynthesis as a function ofprotein increase is presentedabove (0).

TABLE 3. Bacteriochlorophyll produced by R. rubrumgrown under anaerobic, dark conditionsa

Bacterio- Bacterio-Strain chlorophyllb Proteinc chlorophyll!

mg of protein

pg mg Ag

SI 1.28 0.256 14.04 8.00 0.573 5.00

a Cells grown for 23 days at 37 C.b Corrected for bacteriochlorophyll content in

inoculum. (Si = 2.75,g; strain 4 = 2.26,g underanaerobic, light conditions)

c Contained in cell residue after extraction ofbacteriochlorophyll.

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R. rubrum Si is similar to that obtained by Schickand Drews (28) for cells placed under semi-aerobic, dark conditions, and is significantly lessthan that obtained with R. rubrum grown anaer-obically under low-intensity light (6, 18, 30).The ratio of bacteriochlorophyll to protein in

R. rubrum Si cells grown anaerobically under low-intensity light immediately decreased in the ab-sence of light and stabilized at a new lower value(Fig. 3 and 4).Whole cells of R. palustris which had been sub-

cultured at least once under anaerobic, darkconditions were suspended in a 60% sucrosesolution (w/w) and were observed spectro-photometrically. Figure 5 presents a comparisonof the light absorption spectra of 5-day-old cellsgrown aerobically in the dark, anaerobically in thedark, and anaerobically in the light. As notedby previous investigators (5-6, 8, 18, 35), absorp-tion maxima typical of bacteriochlorophyll orcarotenoids were not observed in cells grownaerobically in the dark. The spectra obtainedfrom cells of R. palustris grown anaerobicallyunder dark or light conditions were identical,with bacteriochlorophyll-a maxima at 375 (notpictured), 597, 803, 854, and 890 (shoulder) nm;characteristic carotenoid absorption maxima wereobserved at 469, 499, and 532 nm, as noted byPfennig (25, 26; personal communication) andCohen-Bazire et al. (7). Figure 6 shows theresults of a spectrophotometric study of R.rubrum Si grown under similar conditions. Cellsgrown aerobically in the dark show no charac-teristic absorption maxima, and cells grown an-aerobically under both light and dark conditionsshow identical absorption maxima for bacterio-chlorophyll-a at 588, 801, and 882 nm and forcarotenoids at 484, 511, and 548 nm (7).

R. viridis (10, 24) was grown anaerobicallyunder light and dark conditions on the yeastextract-peptone-agar medium previously describedto which sodium malate (0.3 g per 100 ml ofmedium) was added. The spectra obtained fromwhole cells grown under both conditions weresimilar (Fig. 7), with absorption maxima at 397(not pictured), 605, 825, and 1,015 nm, whichare typical for bacteriochlorophyll-b (9, 12, 24).The remaining absorption maxima are attributedto carotenoids, with the exception of the bac-teriochlorophyll-b maximum at 684 nm in cellsgrown anaerobically in the light; the nature ofthis maximum is not understood (36).

Separate cultures of R. rubrum Si, R. palustris,and R. spheroides 2.4.1 were allowed to growanaerobically under dark conditions for ap-proximately five generations before preparationfor electron microscopy. The cells of each strainused for the inoculum had been maintained under

FIG. 5. Absorption spectra of R. palustris. Cellswere suspended in 60% (w/w) sucrose solution.

-'-aerobic, dark

0.5

--\-a erobic, light0.4-

0.3

X.naerobic, dark0.2-

0. I

500 600 700 800 900WAVELENGTH (nm)

FIG. 6. Absorption spectra of R. rubrum S,. Cellswere suspended in 60% (w/w) sucrose solution.

anaerobic, dark growth conditions. Figure 9 is aphase-contrast photomicrograph of motile photo-tactic cells of R. rubrum Si which had been grownunder anaerobic, dark conditions. For compara-

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FIG. 7. Absorption spectra of R. viridis NHTC 133.Cells were suspended in 60% (w/w) sucrose.

tive purposes, cells grown anaerobically in thelight also were prepared for cytological studies.

Electron micrographs of thin sections of R.rubrum Si cells grown under anaerobic, darkconditions (Fig. 8) appeared identical with thoseof cells grown under anaerobic, light conditions(Fig. 10). From our observations, "chromato-phore" structures are distributed through thecell cytoplasm of cells grown anaerobicallyunder dark conditions, as is also the case in cellsgrown under light conditions (Fig. 10). Bieder-mann et al. (1) and Drews and Giesbrecht (8)also found extensive "chromatophore" formationin cells of R. rubrum placed under semiaerobic,dark conditions.

Electron micrographs of sectioned cells of R.spheroides 2.4.1 grown anaerobically underdark and light conditions are presented in Fig. 11and 12, respectively. Cells grown under anaerobic,dark conditions (Fig. 11) contained a membranesystem of characteristic "chromatophore" struc-tures. Membrane content was, however, lessextensive than in cells grown under anaerobic,light conditions (Fig. 12). Figure 14 is an electronmicrograph of a cross section through the lamel-lar membrane system of R. palustris formed incells grown under anaerobic, dark conditions.The membrane network in these cells was lessextensive than that in cells grown under anaerobic,light conditions (Fig. 13). Numerous cellsgrown anaerobically in the dark were fixed whilein the process of cell membrane invagination, a

process leading to the formation of the lamellar-type membrane apparatus (34) shown in Fig. 13

and 14. An example of this process is presentedin Fig. 15.No cytological distinctions were observed be-

tween fifth or sixth generation cells grown anaer-obically under dark and light conditions. Thedimensions of the unit membrane networks werenot significantly different. It was not possible toascertain this for R. spheroides because of poorfixation of the light-grown cells (Fig. 12).Biedermann et al. (1) found that purple non-

sulfur bacteria, which can respire aerobically,formed an apparently functional photoreceptivemembrane network in the absence of, or withrestricted amounts of, oxygen. Moreover, carote-noid- and bacteriochlorophyll-containing mem-brane structures also were synthesized in an en-vironment where they serve no photoreceptivefunction. Results of chemical and cytologicalstudies, as well as observations of phototaxis,indicate that these photoreceptive membranesare functional.

R. rubrum Si cells were grown for approximately20 generations under dark, anaerobic conditions,and the light reactivity of the photosynthetic ap-paratus was observed spectrophotometrically.Figure 16 is a light minus dark difference spec-trum of the photoactive P890 and P800 center(32). Likewise, the light minus dark differencespectrum of R. rubrum cytochromes C422 andC428 is presented in Fig. 17 (33). When cellsgrown anaerobically in the dark were exposed toan actinic light beam at 874 nm, the cytochromesbecame oxidized, as characterized by a decreasein absorption; when the actinic light was shutoff, the absorption increased as the cytochromesbecame reduced. The "fast" decrease in absorp-tion of cytochrome C428 oxidation and the "fast"increase in absorption of cytochrome C422reduction is presented in Fig. 17. The same cellsexhibited a rapid light-induced nicotinamideadenine dinucleotide reduction when observed byfluorometric techniques. These data indicate thatcells of R. rubrum Si growing under anaerobic,dark conditions continued to synthesize a com-plete light-responsive photosynthetic apparatus.So far, we have examined only members of

the Athiorhodaceae, but there appears to be noreason why members of the Thiorhodaceae andChlorobacteriaceae should not grow anaerobicallyin the dark. Since the photosynthetic mode oflife for bacteria in nature is located in anaerobicenvironments (25), it could be of considerableadvantage for a cell to have a preformed photo-receptive apparatus while in a transitory anaer-obic, dark environment. Continued study ofanaerobic, dark growth of photosynthetic bac-teria may provide information helpful in under-

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..oI.

FIG. 8. Electron micrograph of longitudinal thin section of R. rubrum Si grown under anaerobic, darkconditions. Unless otherwise noted, for electron micrographs the main fixation time was 3 hr and each sectioni waspoststained with uranyl acetate and lead citrate. X 112,000.

FIG. 9. Phase-contrast photomicrograph of motile R. rubrum Si cells grown anaerobically in the dark.X 1,900.

FIG. 10. Electron micrograph of thin transverse section of R. rubrum SI grown under anaerobic, light con-ditions. X 104,000.

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FIG. 11. Electron micrograph of R. spheroides 2.4.1. Longitudinal thin section. Cell growii underanaerobic, dark conditions. X 56,000.

FIG. 12. Electron micrograph of thin section of R. spheroides 2.4.1. Cell grown under anaerobic, lightconditions. X 52,000.

FIG. 13. Electron micrograph of thin transverse section of R. palustris. Cell grown under anaerobic, lightconditions. Main fixation time was 11 hr; poststained with uranyl acetate and lead citrate. X 71,400.

FIG. 14. Electron micrograph of thin transverse section of R. palustris grown under anaerobic, dark condi-tions. X 115,000.

FIG. 15. Electron micrograph of thin section of R. palustris grown under anaerobic, dark conditions. Cellmembrane (CM) in process of invagination to form "photosynthetic" apparatus. X 207,000.

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1.5

1.0

0.5

-0.5

-1.01

-1.5

780 800 820 840 860 880 900WAVELENGTH (nm)

FIG. 16. Light minus dark difference spectrum inthe near infrared spectral region of R. rubrum Si cellsgrown anaerobically in the dark and induced by 586-nmactinic light.

420 440WAVELENGTH (nm)

FIG. 17. Light minus dark difference spectra ofcyto-chromes in R. rubrum Si cells grown under anaerobic,dark conditions. Cells were suspended in mineral-saltssolution. Cytochrome C428 lighlt-induced absorbancydecrease at low (0) and medium (A) light intensities;0.2 and 2.8 nE/cm2 sec, respectively. Light inducedresponse of cytochrome C422 (0) is presented as thefast absorbancy increase after shutting off the light.(1874 = actinic light beam at 874 nm.)

standing the development of the photosyntheticapparatus and its functioning.

ACKNOWLEDGMENTSWe thank H. Gest and S. Kaplan for providing cultures of

photosynthetic bacteria and N. Pfennig for kindly identifying ourisolate. We are grateful to C. Sybesma and C. F. Fowler for help-ful discussions and assistance in spectrophotometric techniquesand to M. P. Bryant for advice and for cultures of methanogenicbacteria.

This investigation was supported by Public Health Servicefellowship At-33148 to R. L. U.

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