0099-2240/78/0035-0038$02.00/0APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1978, p. 38-44Copyright © 1978 American Society for Microbiology

Vol. 35, No. 1Printed in U.S.A.

Phototrophic Purple and Green Bacteria in a SewageTreatment Plant

E. SIEFERT,* R. L. IRGENS,t AND N. PFENNIG

Institut fiir Mikrobiologie der Gesellschaft fir Strahlen- und Umweltforschung mbH, D-3400 Gottingen,Federal Republic of Germany

Received for publication 22 July 1977

In all purification stages of a biological sewage treatment plant, phototrophicbacteria were detected by the method of viable cell counts. The predominantspecies identified belonged to the genus Rhodopseudomonas of purple nonsulfurbacteria. The number of phototrophic bacteria was highest in wastewater con-taining sludge. In activated sludge, an average of 105 viable cells/ml was found;the number depended upon concentration of sludge rather than on seasonalchanges in light conditions in the course of a year. Bacteriochlorophyll a wasextracted from activated sludge. Relative to the viable counts of phototrophicbacteria, the content of bacteriochlorophyll a was 5- to 10-fold higher than thatof three representative pure cultures. By incubation of activated and digestersludge under different environmental conditions, it was shown that phototrophicbacteria can compete with other bacteria only under anaerobic conditions in thelight.

Phototrophic purple and green bacteria arefound in nearly all aquatic environments. Purpleor green sulfur bacteria can be easily recognizedwhen they form water blooms. Purple nonsulfurbacteria, however, rarely appear in visible con-centrations (19). Their distribution in nature,therefore, can only be evaluated from resultsobtained by enrichment techniques (8) or themembrane filter method (1, 17).The presence of purple bacteria is particularly

dependent upon the degree to which water ispolluted by organic matter. Their growth con-tributes to the purification of heavily pollutedwater exposed to sunlight, as, for example, insewage lagoons (4, 7). In Japan, phototrophicbacteria are used in the main purification stageof organic wastewater treatment (9-11).The occurrence of phototrophic bacteria in

conventional organic wastewater purificationplants has not been previously studied. Thispaper presents viable cell counts and identifica-tion of phototrophic purple and green bacteriain all aerobic and anaerobic purification stagesat the sewage treatment plant in Gottingen(West Germany). Additionally, experimentswere carried out to determine which type ofmetabolism (phototrophic, respiratory, or fer-mentative) is used by the phototrophic bacteriato compete with other bacteria in the differentparts of the sewage plant.

t Present address: Department of Life Sciences, SouthwestMissouri State University, Springfield, MO 65802.

MATERIALS AND METHODS

Agar media. The medium for purple nonsulfurbacteria contained, per liter: 2.0 g of disodium succi-nate (anhydrous); 0.5 g of KH2PO4; 0.4 g ofMgSO4 7H20; 0.4 g of NH4Cl; 0.5 g of CaCl2 2H20;2.0 g of yeast extract (Difco); 0.5 ml of ethanol; 5.0ml of iron(III)-citrate solution (100 mg/100 ml); 10 mlof trace element solution (14); 12 g of agar (Difco).The pH was adjusted before autoclaving to 6.9.The medium for purple and green sulfur bacteria

was prepared as described by Pfennig and Lippert(14).Method for viable cell counts. (i) Purple non-

sulfur bacteria. Sludge samples were first agitatedfor 10 min by a Vibromixer (Fa. Sartorius) to obtainhomogeneous suspensions. Serial dilutions (1:10) weremade in sterile 0.2% NaCl. From each of three suitabledilution steps, 1 ml was pipetted into 100 ml of lique-fied agar medium at 400C. After the agar was mixed itwas distributed into four petri dishes. The plates wereincubated in tall glass jars under an atmosphere of95% H2 and 5% CO2 (28 to 30°C, 2,000 to 3,000 lx).Colonies were counted after 10 to 14 days of incuba-tion.

(ii) Green and purple sulfur bacteria and pur-ple nonsulfur bacteria. Serial dilutions (1:10) ofhomogeneous sludge suspension were prepared in agarshake tubes according to the method of Pfennig (13)and were sealed with paraffin-paraffin oil (1:3). Theagar tubes were incubated for 14 days at 280C and2,000 lx of incandescent light. Phototrophic sulfur andnonsulfur bacteria in digester sludge were countedusing agar shake tubes with the medium of Pfennig(13), supplemented by 0.05% acetate, and the vitaminsolution of Pfennig and Lippert (14).

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Samples. Samples were obtained at the Gottingensewage treatment plant between 8:00 and 9:00 a.m. insterile 250-ml bottles. The analysis was started within1 h after collection. In the case of activated sludgesamples, the bottles were not completely filled to avoidanaerobic conditions.Test for decomposition of gelatin. Gelatin was

added to the agar medium for purple nonsulfur bac-teria to a final concentration of 0.4%. When coloniesof the test strains had developed, the plates werecovered with a solution containing 1.5 g of HgCl2 and2 ml of 12 M HCI in 10 ml of distilled water. Theappearance of a clear zone around the colonies indi-cated hydrolysis of gelatin.Experiments with sludge samples under dif-

ferent culture conditions. (i) Activated sludge.Wastewater from the primary sedimentation tank wasadded to an equal volume of activated sludge. Threehundred-milliliter amounts of this mixture were incu-bated in 1,000-ml Erlenmeyer flasks on a rotary shakerat 150 rpm for 2 weeks at 280C. Anaerobic flasks wereflushed with N2 and closed with rubber stoppers. Foraerobic cultivation, fluted Erlenmeyer flasks cappedwith felt cloth were used. Illuminated cultures wereincubated at 2,000 lx of incandescent light.

(ii) Digester sludge. Digester sludge or digestersludge-raw sludge (1:1) was diluted with distilled water(1:1). Four 200-ml portions of diluted sludge wereincubated in 1,000-ml Erlenmeyer flasks. The flaskswere flushed with N2 and closed with rubber stoppers,which contained an air seal filled with par-affin oil to compensate for gas pressure. The flaskswere incubated at 280C for 4 weeks on a rotary shakerat 150 rpm. For illuminated flasks, a light intensity of2,000 lx was used.

Extraction of bacteriochlorophyll a from acti-vated sludge. Two liters of activated sluge was cen-trifuged, and the pellet was resuspended in 100 ml ofdistilled water. To extract bacteriochlorophyll a, 900ml of acetone-methanol (7:2) was added and stirredfor 1 h in the dark at +40C. Before centrifugation, 25ml of 12 M HCI was added to convert bacteriochloro-phyll into stable magnesium-free bacteriopheophytin.

After centrifugation, 100 ml of carbon tetrachloridewas added to the decanted supernatant. By additionof 1,000 ml of water, an acetone-methanol-water phaseand a carbon tetrachloride phase were obtained. Thelatter phase, containing bacteriopheophytin and otherlipid-soluble pigments, was separated and washed sev-eral times with distilled water. Remaining water wasbound by the addition of anhydrous Na2SO4.Chromatography. Bacteriopheophytin was sepa-

rated from other pigments by column chromatographyon silica gel (Mallinckrodt, 100 mesh) at 40C. Assolvent, carbon tetrachloride-acetone (85:15) was used.

The bacteriopheophytin fraction was further puri-fied by repeated chromatography on silica gel plates(Merck, 5553). Mixtures of carbon tetrachloride andacetone in ratios between 85:11 and 97:3 were used assolvents. Bacteriopheophytin a was recovered fromthe plates by extraction of the silica gel fraction withcarbon tetrachloride. Purified bacteriopheophytin a,isolated from Rhodospirillum rubrum, served as areference.

Spectrophotometric determinations. Absorp-

tion spectra of pigments dissolved in carbon tetrachlo-ride were measured with a Zeiss spectrophotometer(DMR 21).The concentration of bacteriopheophytin a was es-

timated from the height of the peak at 760 nm. Theextinction coefficient of bacteriopheophytin a in car-bon tetrachloride was calculated from a calibrationcurve according to Thiele (thesis, University of Got-tingen, Gottingen, West Germany, 1966): ae76 = 37liters/mmol cm. The value for bacteriopheophytin awas multiplied by the factor 1.025 to obtain the cor-responding bacteriochlorophyll a concentration.

Determination of bacteriochlorophyll a. Bac-teriochlorophyll a was extracted from pure culturesof purple nonsulfur bacteria with acetone-methanol(7:2). Ten milliliters of cell suspension was centrifuged,the pellet was resuspended in 1 ml of distilled water,and 9 ml of solvent mixture was added. After 30 minat 40C, the mixture was centrifuged. The absorbanceof the supernatant was measured at 775 nm. Thebacteriochlorophyll a concentration was calculated us-ing the extinction coefficient E75 = 75 liters/mmol cmgiven by Clayton (2).

RESULTSViable cell counts of phototrophic bacte-

ria in different parts of the sewage treat-ment plant in Gottingen. Phototrophic bac-teria were found in all purification stages of thebiological sewage treatment plant in Gottingen,which was constructed by Loos and Preuss (12).Most of the species identified belonged to thepurple nonsulfur bacteria (Table 1). A smallpercentage of purple and green sulfur bacteriawas found in samples containing either activatedsludge or sludge from the primary settling tank.The total number of phototrophic bacteria inthese samples was high.

Phototrophic bacteria were found to be al-ready present in the raw sewage of the inlet.During the mean residence time of about 4 h inthe primary settling tank, the viable cell countincreased, especially in the sedimented rawsludge. This sludge is subsequently digested inthe methane digester. The mean residence timeof the sludge in this digester is about 20 days.Table 1 shows that the number of phototrophicbacteria in the digester sludge was less thanthat in the raw sludge, indicating that the deathrate ofphototrophic bacteria under digester con-ditions cannot be compensated for by an as-sumed growth rate of any of the species.The wastewater leaving the primary settling

tank is purified in an aerobic activated sludgeprocess. In this purification step the number ofphototrophic bacteria increased about 10-fold(Table 1).Phototrophicbacteriainactivated sludge.

To establish by which type of metabolism thephototrophic bacteria grow in activated sludge,the following experiments were performed.

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TABLE 1. Numbers ofpurple and green bacteria in the different tanks of the sewage treatment plant inGottingen

Colony counts/ml

No. of Purple nonsulfur bacteriasamples Purple sulfur bacteria Green sulfur bacteria

Mean Rangevalue

Raw sewage 1 1,700 0 (in 10 ml) 0 (in 10 ml)

Supernatant of the primary set- 2 8,100 7,350-8,910 50 0 (in 0.1 ml)tling tank

Settled raw sludge of the primary 5 116,000 4,100-550,000 0 (in 0.1 ml) 800settling tank

Digester sludge 5 16,000 100-80,000 0 (in 0.1 ml)

Activated sludge 15 100,000 2,900-650,000 1,000 1,300

(i) Determination of illumination condi-tions in the activated sludge tank. Anaerobictube cultures, freshly inoculated with Rhodo-pseudomonas capsulata, were incubated for 3weeks in different depths of the tank. Duringthis period the weather was sunny. Light-de-pendent growth occurred only to a depth ofabout 10 cm below the surface of the activatedsludge tank. It is apparent that anaerobic light-dependent growth of phototrophic bacteria mustbe strongly light-limited in the activated sludgetank, which has a depth of 3.8 m. We expected,therefore, that the number of phototrophic bac-teria would vary with the different light condi-tions of the seasons.

(ii) Effect of seasonally changing lightconditions. Phototrophic bacteria were countedmonthly over 1 year. The numbers of photo-trophic bacteria fluctuated during the year from103 to more than 105 per ml, but the fluctuationswere not in accordance with the changing lightconditions throughout the year. Rather, it wasfound that the cell number increased with thesludge concentration: a linear relationship wasobtained when the square root of the cell num-ber was plotted against the dry-weight concen-tration of the activated sludge (Fig. lb). Thesame correlation was observed for those che-moorganotrophic bacteria that grew under an-aerobic conditions on the agar plates simulta-neously with the phototrophic bacteria (Fig. la).The numbers of these chemoorganotrophic bac-teria correlated strictly to the numbers of thephototrophic bacteria. It is reasonable to con-clude, therefore, that growth of both photo- andchemoorganotrophic bacteria is favored by themicroaerobic conditions at high sludge concen-trations.

(iii) Detection of bacteriochlorophyll a inactivated sludge and its concentration re-

a62000

.0

840000 OE z

a r-' ' 20000 D0

IV &u 0.

c! <2 20

0

. 800-6._o s-

o 'o 6000 aL-

a Q- 400,) a

O0 200D

1 L5 2 25Dry weight of activated sludge (mg/rl)

FIG. 1. Dependence of colony counts of anaerobicheterotrophic (a) and phototrophic (b) bacteria on

the varying concentrations ofactivated sludge within1 year.

lated to the viable counts of phototrophicbacteria. When pure cultures of purple nonsul-fur bacteria grow aerobically as chemoorgano-trophs, their bacteriochlorophyll synthesis be-comes repressed by oxygen (3). To find outwhether the purple nonsulfur bacteria in acti-vated sludge contain bacteriochlorophyll or

whether their bacteriochlorophyll formation isrepressed by oxygen, the content of bacterio-chlorophyll a of activated sludge was determinedand related to the viable cell counts. The ab-sorption spectra of the acid extract (Fig. 2a)show that activated sludge contains detectableamounts ofbacteriochlorophyll a. Successive pu-

a

b

DI

0[

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PHOTOTROPHIC BACTERIA IN A SEWAGE PLANT

400 500 600 700 800nm365 392 530 690 760

Wave Length

FIG. 2. Spectrophotometric detection of bacterio-pheophytin a in activated sludge extract and its chro-matographic separation from other pigments. (a) Ac-tivated sludge extract; (b) after separation on a silicagel column; (c) after thin-layer chromatography; (d)after a second thin-layer chromatography; (e) absorp-tion spectrum ofpure bacteriopheophytin a (Thiele,thesis).

rification of bacteriopheophytin a from otherpigments, e.g., pheophytin of algae and carote-noids, provided evidence that the shoulder at760 nm of spectrum a (Fig. 2) was caused bythe absorption of bacteriopheophytin a.When the bacteriopheophytin content of the

activated sludge was related to the number ofviable cells of phototrophic bacteria in thesludge, a theoretical bacteriochlorophyll contentof these cells was obtained that was 5- to 10-fold higher than that of viable cells from purecultures of Rhodopseudomonas sphaeroides, R.capsulata, and R. gelatinosa, which were grownanaerobically under light limitation (Table 2).

(iv) Determination ofenvironmental con-ditions under which phototrophic bacteriamultiply in activated sludge. Activatedsludge was incubated aerobically or anaerobi-cally, both in the light or in the dark. Photo-trophic bacteria were found to grow only underanaerobic light conditions (Table 3). Both theviable cell count and the dry weight increasedunder such conditions, indicating photoassimi-lation of soluble organic compounds present inthe wastewater by phototrophic bacteria. Theviable cell counts decreased in anaerobicallydark- and aerobically light- and dark-incubated

TABLE 2. Bacteriochlorophyll a content of activatedsludge and ofpure cultures (isolated from activated

sludge)Bacteriochloro-

Source of bacteriochlorophyll a phyll a(pg/105 viable cells)

Activated sludge (29 Aug. 1972) ... 2.5 x 10-3Activated sludge (22 Sept. 1972) ... 1.9 x 10-3Activated sludge (4 Oct. 1972) .... 1.8 x 10-3

R. capsulataa 0.62 x 10-3R. sphaeroides ... 0.48 x 10-3R. gelatinosa . ...0.15 x 10-3

aPure cultures were grown anaerobically underlight limitation at 500 lx from a tungsten lamp.

TABLE 3. Survival and growth ofphototrophicbacteria in activated sludge incubated under

different conditionsAt the beginning After 7 days

Photo- Drywt Photo- Drywtof acti- of acti-Incubation trophic bac- vated trophic bac- vated(colon sludge (colon sludge(colon(mg/ colony (mg/counts/mi) (mg/ counts/ml) MI)

LightAerobic 8.6 x 104 0.89 4.2 x 103 0.60Anaerobic 8.6 x 104 0.89 1.8 x 107 0.99

DarkAerobic 8.6 x 104 0.89 2.3 x 103 0.59Anaerobic 8.6 x 10' 0.89 4.4 x 103 0.77

activated sludge. The decrease was largest underaerobic dark conditions and least under anaero-bic conditions. Also, a loss of activated sludgedry weight took place (Table 3). The decreasein the viable cell count was proportionally largerthan could be expected from the mineralizationof the sludge alone.Genera and species of phototrophic bac-

teria in activated sludge. Several genera andspecies of phototrophic bacteria can be recog-nized by the morphology of their cells (15) andthe color and shape of their colonies on agar.The percentage of the different species was es-timated using the same agar plates for the viablecounts (Table 4). The identification of Rhodo-spirillum tenue, Rhodopseudomonas viridisand R. palustris, Rhodomicrobium vanielii,Chromatium vinosum, Thiocapsa roseopersi-cina, and Chlorobium limicola was quite easy.In contrast it was sometimes difficult to distin-guish R. sphaeroides, R. capsulata, and R. ge-latinosa. The three latter species formed thelargest number of colonies on the agar plates.Three representative strains were isolated andidentified unequivocally by growth tests on 37different substrates. For one strain, however,

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TABLE 4. Distribution of different species ofphototrophic bacteria in activated sludge

Species DistributionSpecles ~~~~~(%)

Rhodopseudomonas sphaeroides 5175Rhodopseudomonas gelatinosa JRhodopseudomonas palustris 6-25Rhodopseudomonas capsulata 6-25Rhodospirillum tenue 1-5Rhodopseudomonas viridis 1-5Chromatium vinosum <1Thiocapsa roseopersicina <1Chlorobium limicola <1

these growth tests did not give sufficient infor-mation to identify it as R. gelatinosa or R.sphaeroides.

Gelatin hydrolysis was tested to identify R.gelatinosa, because this character has been de-scribed as typical for this species. Half of thestrains that were identified as R. capsulata,however, also hydrolyzed gelatin. Other strainshaving the cell morphology and colony color ofR. gelatinosa did not hydrolyze gelatin. Gelatinhydrolysis, therefore, did not prove to be conclu-sive for the identification of R. gelatinosa.The strictly anaerobic brown-colored species

of the genus Rhodospirillum, R. fulvum, R. mol-ischianum, and R. photometricum, were not de-tected on plates used to determine viable cellcounts. It was possible, however, to enrich R.photometricum repeatedly in activated sludgeby incubating samples in test tubes at 1,000 lx

and 30°C. Growth spots of accumulated R. pho-tometricum appeared after a few days on theilluminated side of the tube. From such enrich-ments, one strain was isolated in pure culture.Some phototrophic bacteria, picked from a

single colony, failed to grow on second plates ofthe same medium. Among these, R. tenue strainsproved to have a requirement for vitamin B12,which was not added to the original agar me-

dium.Culture experiments withdigester sludge.

The numbers ofphototrophic bacteria decreasedin the methane digester (Table 1). To determinewhether this was due to the conditions in thedigester, e.g., any inhibitory effects of its micro-bial population on the phototrophic bacteria or

the complete absence of light, the following ex-

periments were done. Diluted digester sludgewith and without the addition of raw sludge wasincubated anaerobically in the dark and in thelight. The illuminated flasks showed a large in-crease in the numbers of phototrophic bacteriaafter 2 and 4 weeks. In flasks kept in the dark,neither growth of the whole population of pho-totrophic bacteria (Table 5) nor growth of indi-vidual species could be observed, judging from

TABLE 5. Survival and growth ofphototrophicbacteria in digester sludge incubated anaerobically

in the dark or in the lightPhototrophic bacteria (colony counts/ml)

Diluted digester Diluted digesterTime sludge mixed with ilutddgeseraw sludge sludge

Light Dark Light Dark

At the beginning 1 x 105 1 X 105 2.8 x 10i 2.8 x 10:'After 2 weeks 8.3 x 108 2 x 104 2.9 x 10 1.6 x 10:After 4 weeks 3.0 x 109 8 x 103 8.0 x 108 9.0 x 10:'

the appearance of the different types of colonies.Thus, phototrophic bacteria in digester sludgehave a selective advantage only in the presenceof light.

DISCUSSIONPhototrophic bacteria are present in all puri-

fication stages of the municipal sewage treat-ment plant in Gottingen; their contribution tothe purification process, however, is negligible,as shown by the viable cell counts presented inthis paper. Even the unexpectedly high colonycounts of phototrophic bacteria in the activatedsludge tank, 5 x 104/mg (dry weight) of activatedsludge, is small in comparison to total viablecounts of aerobic bacteria amounting to 107/ml(dry weight) (16). In heavily polluted waters orhighly eutrophic ponds, the number of photo-trophic bacteria is in the same range as thatfound in activated sludge: 104 to 105 cells/ml asreported by Biebl and Drews (1) and Kaiser (8).The presence of phototrophic bacteria in acti-vated sludge was demonstrable both by the platecount method and by detection of bacteriochlo-rophyll a in activated sludge. Quantitative de-terminations of bacteriochlorophyll a showedthat activated sludge contained much more bac-teriochlorophyll a than would be expected fromthe colony counts (Table 2). There are severalpossible explanations for this finding. First, theplate count medium used did not support growthof all phototrophic cells present in the sludge;second, due to cell aggregates, one phototrophiccolony from an activated sludge sample repre-sented more than one single cell; and third, theactivated sludge contained bacteriochlorophylla or bacteriopheophytin a in dead cells of pho-totrophic bacteria which could not be recordedby the viable count method.

In spite of these uncertainties, one can con-clude from the relatively high content of bacter-iochlorophyll a in activated sludge that photo-trophic bacteria grow under the reduced oxygentension in the sludge flocs, because bacterio-chlorophyll synthesis would be repressed if the

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PHOTOTROPHIC BACTERIA IN A SEWAGE PLANT 43

bacteria were exposed to the oxygen concentra-tion measured in the free water phase of theaeration tank (2 to 4 mg of 02 per liter).

Full pigmentation usually indicates that thebacteria were grown anaerobically by photosyn-thetic energy conversion, but they might alsohave grown by respiratory energy conversion atlow oxygen partial pressure (5). It is likely thatphototrophic bacteria take advantage of bothtypes of metabolism. In activated sludge, thephotosynthetic metabolism appears to be lesssignificant, since only the uppermost 10 cm ofthe total depth of 3.8 m receives sufficient lightto allow phototrophic growth, as we have found.Due to the mixing procedure, phototrophic bac-teria stay, intermittently at best, for 2.6% of theday in that layer, where they can grow photo-synthetically.Although this quantity of light in our experi-

ence is rather low for phototrophic growth, lightdoes not appear to be the growth-limiting factor,since no correlation was observed between col-ony counts and seasonal light conditions. Thisapparent contradiction is solved if one assumesthat phototrophic bacteria obtain energy by res-piration during their stay in the dark. This as-sumption would also explain why the facultativeaerobic species R. gelatinosa, R. capsulata, andR. sphaeroides predominated in activatedsludge.The assumption that phototrophic bacteria

grow using mainly respiratory energy appliesonly to microaerobic conditions. Two facts in-dicate that too much oxygen is unfavorable.First, with decreasing sludge concentration inthe aeration tank aerobic conditions become im-proved, but the colony counts of both photo-trophic and anaerobic heterotrophic bacteria arediminished quadratically (Fig. la and b). Sec-ond, phototrophic bacteria in activated sludgedecreased in number when cultivated both inthe light and in the dark under strict aerobicconditions in fluted Erlenmeyer flasks on a ro-tary shaker (Table 3).Low oxygen concentrations at which photo-

trophic bacteria can grow fully pigmented inactivated sludge are probably present in theactivated sludge flocs. About 95% of the totalnumber of phototrophic bacteria are attachedto the sludge flocs (Siefert, thesis, University ofGottingen, Gottingen, West Germany, 1972).As reported by Uffen and Wolfe (18), photo-

trophic bacteria might also grow under stronglyreducing conditions anaerobically in the darkby a fermentative metabolism. If this metabo-lism is of ecological significance, phototrophicbacteria should increase in viable cell counts inthe strictly anaerobic environment of the meth-ane digester. Colony counts of digester sludge,

however, were much lower than those of theraw sludge entering the digester. Also, it wasimpossible to obtain an increase in viable cellcounts of phototrophic bacteria when digestersludge, enriched with raw sludge, was incubatedanaerobically in the dark (Table 5). In illumi-nated control vessels, colony counts greatly in-creased. It is, therefore, conclusive that underanaerobic conditions in the absence of light pho-totrophic bacteria cannot compete with chemo-trophic anaerobes, because their fermentativeenergy metabolism is not efficient enough (6).Our results show that in conventional sewage

treatment plants under normal operating con-ditions, phototrophic bacteria are not capableof competing with chemotrophic bacteria be-cause of either the limited availability of lightor the presence of too much oxygen, or both.Nevertheless, these bacteria are permanentlypresent in all stages of the plants and can beeasily enriched, if samples are incubated underappropriate conditions. If light and oxygen sup-ply are adjusted to the requirements of the pho-totrophic bacteria, they should become the pre-dominant bacteria involved in the purificationprocess. Sewage treatment plants utilizing pho-totrophic bacteria for the main purification stepalready are in operation in Japan (11). The useof phototrophic bacteria has some advantagesover the conventional biological sewage treat-ment. First, the major organic and inorganicconstituents of sewage are assimilated ratherthan dissimilated, resulting in an augmenteduptake of minerals. Thus, a third purificationstep for removal of inorganic nutrients couldpossibly be saved. Second, the cell protein ofthe phototrophic bacteria is a useful by-product,which can be utilized as a supplement in animalnutrition.

ACKNOWLEDGMENTSWe express gratitude to H. Ahlborn, K.-H. Pelzer, and W.Backhaus, who helped to take samples in the sewage purifi-cation plant in Gottingen.

LITERATURE CiTED1. Biebl, H., and G. Drews. 1969. Das in-vivo-Spektrum

als taxonomisches Merkmal bei Untersuchungen zurVerbreitung von Athiorhodaceae. Zentralbl. Bakteriol.Parasitenkd. Infektionskr. Hyg. Abt. 2 123:425-452.

2. Clayton, R. K. 1963. Toward the isolation of a photo-chemical reaction center in Rhodopseudomonas sphe-roides. Biochim. Biophys. Acta 75:312-323.

3. Cohen-Bazire, G., W. R. Sistrom, and R. Y. Stanier.1957. Kinetic studies of pigment synthesis by nonsulfurpurple bacteria J. Cell. Comp. Physiol. 49:25-68.

4. Cooper, P. E., M. B. Rands, and C.-P. Woo. 1975.Sulfide reduction in felmongery effluent by red sulfurbacteria. J. Water Pollut. Control Fed. 47:2088-2099.

5. Drews, G., H.-H. Lampe, and R. Ladwig. 1969. DieEntwicklung des Photosyntheseapparates in Dunkelk-ulturen von Rhodopseudomonas capsulata. Arch. Mi-krobiol. 65:12-28.

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6. Gurgun, V., G. Kirchner, and N. Pfennig. 1976.Vergarung von Pyruvat durch sieben Arten phototro-pher Purpurbakterien. Z. Allg. Mikrobiol. 16:573-586.

7. Holm, H. W., and I. W. Vennes. 1970. Occurrence ofpurple sulfur bacteria in sewage treatment lagoon. Appl.Microbiol. 19:988-996.

8. Kaiser, D. 1966. Contribution a l'tude de l'ecologie desbacteries photosynth6tiques. Ann. Inst. Pasteur Paris111:732-749.

9. Kobayashi, M. 1975. Role of photosynthetic bacteria infoul water purification. Prog. Water Technol. 7:309-315.

10. Kobayashi, M., M. Kobayashi, and H. Nakamishi.1971. Construction of a purification plant of pollutedwater, using photosynthetic bacteria. J. Ferment. Tech-nol. 49:817-825.

11. Kobayashi, M., and Y.-T. Tchan. 1973. Treatment ofindustrial waste solutions and production of useful by-products using a photosynthetic bacterial method. Wa-ter Res. 7:1219-1224.

12. Loos, K., and F. Preuss. 1970. Die Erweiterung dervollbiologischen Abwasserreinigungsanlagen in Gottin-gen. Wasser Boden 9:243-245.

13. Pfennig, N. 1965. Anreicherungskulturen fur rote undgrine Schwefelbakterien. In Anreicherungpkultur und

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Mutantenauslese. Zentralbl. Bakteriol. Parasitenkd. In-fektionskr. Hyg. Abt. 1 Orig. Suppl. 1, p. 179-189,503-505.

14. Pfennig, N., and K. D. Lippert. 1966. Uber das VitaminB12-Bediirfnis phototropher Schewfelbakterien. Arch.Mikrobiol. 55:245-256.

15. Pfennig, N., and H. G. Triiper. 1974. The photosyn-thetic bacteria, p. 24-25. In R. E. Buchanan and N. E.Gibbons (ed.), Bergey's manual of determinative bac-teriology, 8th ed. The Williams & Wilkins Co., Balti-more.

16. Pike, E. B., and C. R. Curds. 1971. The microbialecology of the activated sludge process, p. 123-147. InG. Sykes and F. A. Skinner (ed.), Microbial aspects ofpollution, Society for Applied Bacteriology Symposium,Ser. no. 1. Academic Press Inc., London.

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18. Uffen, R. L., and R. S. Wolfe. 1970. Anaerobic growthof purple nonsulfur bacteria under dark conditions. J.Bacteriol. 104:462-472.

19. Van Niel, C. B. 1971. Techniques for the enrichment,isolation and maintenance of the photosynthetic bac-teria. Methods Enzymol. 23A:3-28.

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