APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1989, p. 639-644 Vol. 55, No. 3 0099-2240/89/030639-06$02.00/0 Copyright © 1989, American Society for Microbiology Carbon Isotope Fractionation by Thermophilic Phototrophic Sulfur : Evidence for Autotrophic Growth in Natural Populations MICHAEL T. MADIGAN,'* RAY TAKIGIKU,2 ROBERT G. LEE,2 HOWARD GEST,3 AND J. M. HAYES2 Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901,1 and Biogeochemical Laboratories, Departments of Chemistry and Geology,2 and Department ofBiology,3 Indiana University, Bloomington, Indiana 47405 Received 17 October 1988/Accepted 20 December 1988

Purple phototrophic bacteria of the Chromatium can grow as either photoautotrophs or photohetero- trophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotroph- ically grown cultures of C. tepidum yielded '3C fractionation factors near -20%o. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7%o being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20 %c, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.

Anoxygenic phototrophic bacteria play important roles in specific biomarkers of C. tepidum. In this paper, we present the anaerobic cycling of organic matter through their activ- the results of these experiments and show that carbon- ities as primary producers and as consumers of both organic isotopic analyses can be used to assess the nutritional status compounds and hydrogen sulfide (4, 21, 26). Thus, mass of natural populations of phototrophic bacteria. developments of purple and green sulfur bacteria are quite common in illuminated anaerobic habitats in which sulfide is present (6, 21, 26, 27). Sulfide is produced biogenically in MATERIALS AND METHODS such habitats by metabolic activities of the obligately anaer- obic sulfate- and sulfur-reducing bacteria (21, 26, 41), whose Organism, media, and culture conditions. Pure cultures of development is ordinarily dependent on the presence of C. tepidum MC (20) were grown either photoautotrophically low-molecular-weight organic compounds generated from or photoheterotrophically in the medium previously de- fermentation processes or present as pollutants (6, 21, 26, scribed (20). Photoautotrophic growth was achieved in me- 27). The availability of such organic compounds offers the dium containing 4 g of NaHCO3 per liter as the sole carbon potential for accompanying blooms of purple and green source, whereas for photoheterotrophic growth the medium bacteria to develop photoheterotrophically. However, de- contained 0.8 g of NaHCO3 per liter and 10 mM sodium spite the fact that organic substances are present in natural acetate. All media contained 4 mM sulfide (pH 7). Cultures ecosystems of this type, it is generally considered that of C. tepidum were grown for 24 h at 49 to 50°C at a light phototrophic sulfur bacteria develop autotrophically (4, 36). intensity of ca. 2,000 lux (incandescent illumination). Cells, Recent studies that point to the importance of acetate as a harvested by centrifugation, were washed once in 50 mM photoheterotrophic substrate for development of mass accu- potassium phosphate (pH 7) and stored under N2 at -80°C. mulations of phototrophic bacteria, however, indicate that Field sampling. The springs sampled lie in the upper photoautotrophy may not be the norm in all natural situa- terraces of Mammoth Hot Springs, Yellowstone National tions (1, 15, 37). Park, Wyoming, and have been given the unofficial names In sulfide springs, phototrophic sulfur bacteria usually Roland's Well, New Pit, and New Spring by D. M. Ward develop in response to sulfide of geochemical origin. For (see reference 38) and R. W. Castenholz (5). These relatively example, in the Mammoth area of Yellowstone National small springs (Roland's Well is ca. 0.5 m in diameter) are Park, thin mats of the thermophilic purple sulfur bacterium, located southeast of Painted Pool and Bath Lake as shown in Chromatium tepidum, develop in thermal sulfide springs at the map of Castenholz (5) and are described in more detail in temperatures between 40 and 60°C (19, 20). Recent microbi- the review by Ward et al. (38). Samples of thin red mats of C. ological studies of these mats have shown that strains of the tepidum and thick mats of green Chloroflexus strains (11) green phototrophic bacterium Chloroflexus and various het- were collected with spatulas and placed in 50-ml sterile erotrophic bacteria, including sulfate-reducing bacteria, co- plastic tubes. Mat material was kept at 0°C for 48 h before exist with C. tepidum (38). Consequently, it was of interest being frozen at -80°C and shipped to Carbondale, Illinois, or to determine the carbon metabolism pattern of C. tepidum in Bloomington, Indiana, for analyses. such circumstances. This was done by measuring the car- Pigment processing. Photosynthetic pigments were ex- bon-isotopic composition of natural populations of cells and tracted from pure cultures or natural material with methanol or acetone. Crude bacteriochlorophyll a and carotenoid pigments were obtained from rotary evaporates of solvent * Corresponding author. extractions (25) and further purified by silica-gel thin-layer 639 640 MADIGAN ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 1. Characteristics of pure cultures of C. tepidum grown photoautotrophically and photoheterotrophically at 50°C "3C fractionation factors (%O)b RuBP Growth conditions carboxylase £p (biomass/ Ebchl (bacterio- Ephy (phytol/ ancr (spirillox- 5car (rhodovibrin/ activitya carbon source) chlorophyllide a! carbon source) anthin/carbon carbon source) carbon source) source) Photoautotrophic 33 -20.5 -20.0 -23.6 -24.1 ND" Photoheterotrophic 0.1 -7.6 -7.6 -12.4 -12.9 -13.1

" In nanomoles - minute-l * milligram of protein-'; assayed as described in reference 14. b Calculated by using equation 3 (for photoautotrophic growth) or equation 2 (for photoheterotrophic growth) as described in Materials and Methods. ND, Not determined.

chromatography (developed with hexane-n-propanol [9:1]). isotopic composition was therefore essentially constant. Preparation and isolation of bacteriochlorophyllide a and Accordingly, values were calculated by using equation 2. phytol was done by the procedure of Ellsworth et al. (8). Consumption of C02 was not negligible under conditions of Carbon isotopic analyses and calculations. Spring water autotrophic growth. In this case, sp was determined from the collected by syringe was immediately transferred to evacu- following equation (22): ated serum vials and analyzed for total dissolved carbon within 6 h. After acidification of spring water samples with t:p = (adf 8d0)/ln (1 - f) (3) H3PO4, the total amount of dissolved inorganic carbon (CO2, where 5,lf is the isotopic composition of dissolved CO2 at a H2CO3, and HC03-) was measured manometrically, and the fractional consumption f (f -, 1 as growth of the culture C02 was trapped for isotopic analyses. Cells or isolated progresses) and 5d, is the isotopic composition of dissolved pigment fractions were placed in quartz combustion tubes C02 atf = 0. A plot of (85,f, - ado) as a function of ln (1 - f) and dried by rotary evaporation. After addition of 0.3 g of yields a straight line with slope = Ep. Values of (8df' - ado) cupric oxide to each sample, the tubes were sealed under vacuum and combusted at 850°C overnight (40). Carbon dioxide from combusted samples and from spring water samples was purified by cryogenic vacuum distillation and TABLE 2. Field measurements and isotopic characteristics of natural populations of C. analyzed in either a Finnigan MAT Delta E or a Nuclide 6-60 tepidum mass spectrometer. Isotopic compositions are reported in Source terms of the conventional delta notation from the following Parameter Roland's New New equation and are referred to the PeeDee Belemnite standard Well Pit Spring (PDB [7]): Spring water chemistry all = [(R,, - Rr)lRr]103 (1) pH 6.7 6.5 6.5 Temperature (°C) 52 55 53 where R = (13C/'2C) and the subscripts u and r refer to the H2S (>M)a 41 34 NDb unknown and reference samples, respectively. Total dissolved carbonate (mM) 21.7 15.2 10.7 Isotopic compositions of biosynthetic products are always &, (%c vs PDB) -2.1 -2.0 -4.1 dependent on the isotopic composition of the carbon source ti (%C vs PDB)' -7.2 -5.8 -7.8 as well as on fractionations caused by isotope effects asso- ciated with the biosynthetic reactions. It is the latter frac- Isotopic compositions (%G vs PDB) tionations which are characteristic of a particular organism Biomass (mostly C. tepidum) -28.7 ND ND or process, whereas isotopic compositions of carbon sources Bacteriochlorophyllide a -27.7 -26.4 -28.8 Related phytol -31.1 -29.7 may vary due to -32.4 unrelated environmental factors. It follows Bacteriochlorophyllide c -28.8 -28.9 ND that meaningful biogeochemical interpretation of isotopic Related alcohol -31.3 -31.7 ND compositions of natural products must be based on observed Spirilloxanthin ND -27.6 -28.4 or inferred fractionation factors rather than on absolute Rhodovibrin ND -27.9 -28.8 isotopic compositions. Accordingly, Tables 1 and 2 are constructed to emphasize the former values and to suppress Fractionation factors vs dissolved irrelevant information such as the absolute isotopic compo- CO°, (%G) sitions of the reagents employed as carbon sources for the Materials related to C. tepidum laboratory cultures. ,p (total biomass) -21.6 ND ND In open systems, such as the continuously flowing hot E5chl (bacteriochlorophyllide a) -20.5 -20.8 -21.1 Ephy (phytol) -24.0 -24.0 -24.8 springs studied here, the isotope fractionation factor, s, is scar (spirilloxanthin) ND -22.0 -20.8 calculated by using the equation: scar (rhodovibrin) ND -22.3 -21.2 Materials related to Chloroflexus spp. {x= + + - 1}1,000 (2) {[(8x 1,000)/(bS 1,000)] Ebchl (bacteriochlorophyllide c) -21.7 -23.3 ND where the subscript x designates a particular organic carbon Erelated alcohol -24.3 -26.1 ND phase (P for primary biomass, bchl for bacteriochlorophyll, "Data from reference 38. phy for phytol, and car for carotenoid pigments) and the b ND, Not determined. subscript s designates the carbon source (d for dissolved Calculated by using equations 4 and 5 as indicated (see Isotopic analyses C02, a for acetate). Laboratory culture systems were and calculations). Because the pH is near pK, for carbonic acid, the isotopic difference between 5,, and &, varied significantly. closed. case However, in the of photoheterotrophic growth d Calculated by using equation 2, with b,d as specified for each location (row on acetate, only 5% of the acetate was utilized and its 6 of this table). VOL. 55, 1989 CARBON ISOTOPE FRACTIONATION BY 641 from two different cultures were in good agreement and were of carbon flow. The lipid-related values of -12.4 to -13.1 combined to yield the best estimate of Ep. Fractionation cannot be interpreted without knowing the pattern of isoto- factors for specific compounds were determined by measur- pic ordering within the acetate carbon source. The 5.4%o ing the isotopic difference between them and total biomass in enrichment of 13C in biomass and bacteriochlorophyllide is harvested cells. probably related to assimilation of dissolved inorganic car- Because carbon dioxide is the substrate for photosynthetic bon at C2 -_ C3 and C3 -> C4 steps in anabolic reactions (see carbon fixation, Cp is correctly defined (equation 3) in terms later discussion and note that in these experiments, the of 5d' the isotopic composition of dissolved CO2. Isotopic isotopic composition of the acetate carbon source was analysis of total dissolved carbonate does not directly pro- -11.6%o versus PDB, while that of the total carbonate vide a value for bd. In the pH range of interest, the isotopic present in the medium was -5.2%o). composition of total dissolved carbonate is given by Isotopic composition of natural populations of C. tepidum. 2 + Table presents carbon isotopic data for natural populations .= Xdad Xb5b (4) of C. tepidum. Material was collected from three small hot where X is the mole fraction of total dissolved carbonate and springs in the Mammoth region of Yellowstone. Acetate was the subscripts E and b refer to total and bicarbonate carbon, undetectable in spring water (data not shown), but dissolved respectively (the concentration of H2CO3 is so low that inorganic carbon was high and sulfide was readily detectable neglecting it is not consequential isotopically). Values of X (Table 2). Only in Roland's Well was it possible to obtain were determined by reference to the pH and to the known samples of C. tepidum essentially free of Chloroflexus temperature dependence (12) of carbonic acid dissociation strains, and hence isotopic analyses of total cell carbon were constants. The isotopic fractionation between bicarbonate performed only from this site. However, the isotopic com- and dissolved CO2 was calculated from the following equa- position of bacteriochlorophyllide a isolated from the three tion (24): different springs and used as a biomarker for C. tepidum differed by only 2.4%o (-26.4 to -28.8%o versus PDB; Table {[(5d + 1000)I(5b + 1,000)] - 1}1,000 = 24.12 - 9.866/T (5) 2). Moreover, when the results were recast in terms where T is the absolute temperature (e.g., of the hot spring) of fractionation factors (i.e., when differences in the iso- in degrees Kelvin. Rearrangement of equation 5, and substi- topic composition of dissolved CO2 were taken into tution in equation 4, allows calculation of ad from observed account), the range of variation was reduced to 0.6%o (-20.5 values of 85. to -21.1%o; Table 2). The average value Of Ebchl -20.8%o, is in reasonable agreement with the value observed for C. tepidum grown autotrophically in pure cultures in the labo- ratory (-20.0; Table 1). This result is, therefore, consistent RESULTS with autotrophic growth in the natural system. A comparison Isotopic composition of pure cultures of C. tepidum. The with the photoheterotrophic value for Ebchl (-7.6%o; Table 1) thermophilic purple sulfur bacterium C. tepidum grows cannot be made because no acetate (or other volatile car- photoautotrophically in mineral media containing CO2 as the boxylic acid) could be detected and isotopically analyzed in sole carbon source and sulfide as the electron donor (19, 20). the natural system. Under such conditions, a thermostable ribulose-1,5-bisphos- Supporting the conclusion that photoautotrophy is a dom- phate (RuBP) carboxylase/oxygenase (ribulose bisphosphate inant process in C. tepidum mats was the fact that RuBP carboxylase) is present (14). C. tepidum can also assimilate carboxylase was detectable in crude extracts of natural acetate and pyruvate (20), and availability of these sub- populations of C. tepidum (data not shown). However, strates severely represses synthesis of Calvin cycle en- specific activities of this Calvin cycle enzyme were highly zymes. This is illustrated in Table 1, in which levels of RuBP variable in different samples and in general were lower than carboxylase in photoautotrophic and photoheterotrophically those of photoautotrophically-grown pure cultures, probably grown cells are compared. because of the considerable time interval between collection Reflecting these differences in RuBP carboxylase-specific and the time of enzyme assay (1 month). Thus, the accumu- activities, major differences in the carbon isotopic composi- lated evidence strongly indicates that the natural populations tion of photoautotrophically and photoheterotrophically of C. tepidum were growing photoautotrophically. grown cells were observed. Total cellular carbon as well as Isotopic compositions of polyisoprenoid lipids related to purified bacteriochlorophyll a and carotenoid pigments from C. tepidum and obtained from the natural populations are autotrophically grown C. tepidum were more highly depleted consistent with biosynthesis via photoautotrophy but indi- in "3C (Table 1). Fractionation factors summarized in Table cate some differences between laboratory and natural popu- 1 reflect widely observed intermolecular contrasts, appar- lations. A key point of consistency was observed when ently of biosynthetic origin. The "3C content of bacteriochlo- values Of Cbchl and Ephy were compared. The Ebchl - Ephy rophyllide carbon is near that of total biomass, or slightly difference found in nature, 3.5%o (Table 2), is almost pre- enriched (13). Lipids are depleted in 13C relative to total cisely equal to that observed in the photoautotrophically biomass. Monson and Hayes (23) related this contrast to grown laboratory culture (3.6%o; Table 1). It is assumed that depletion of 13C at carbon positions derived from the car- bacteriochlorophyllides and phytol are synthesized simulta- boxyl carbon of acetyl coenzyme A (acetyl-CoA). Bearing in neously and thus share a common biosynthetic feedstock. mind that polyisoprenoid lipids contain acetyl-CoA methyl/ Accordingly, the isotopic difference between them should be acetyl-CoA carboxyl carbon in the ratio 3/2, the 3.6 to 4.1%o controlled only by isotope effects in their respective biosyn- difference between lipids and biomass observed for photoau- thetic pathways and should be the same in nature and in totrophically grown cells suggests an isotopic difference of 9 vitro. Like phytol, the carotenoid pigments are also polyiso- to 101%oo between methyl and carboxyl positions in acetyl- prenoid lipids. In the laboratory cultures, the isotopic com- CoA used by C. tepidum for lipid biosynthesis. The even positions of the carotenoids and of phytol were similar (to larger lipid-biomass difference found in the photohetero- whatever extent they differed, the carotenoids were more trophically grown cells is linked to a much different pattern depleted in 13C). In nature, however, the carotenoids were 642 MADIGAN ET AL. APPL. ENVIRON. MICROBIOL. enriched in '3C relative to phytol, and even approached the higher cell densities as photoheterotrophs. Accordingly, it is isotopic composition of bacteriochlorophyllide. No defini- likely that the isotopic composition of purple sulfur bacteria tive explanation for this difference can be put forward, but it in most natural ecosystems would be (isotopically) heavier may be that in these natural populations, phytol and caro- than of cells growing strictly autotrophically. tenoids were not synthesized at the same time. By contrast, the carbon-isotopic composition of cells and Although the focus of the present study was on the purple pigment fractions of the thermophilic purple bacterium C. bacterium C. tepidum, a few isotopic analyses were per- tepidum growing in Yellowstone sulfide springs clearly re- formed on cells and pigment fractions from the obligately flects autotrophic processes and suggests that C. tepidum is phototrophic green Chloroflexus filaments (11) that form a true primary producer in its natural habitat. Although C. thick mats in many of the same springs in which C. tepidum tepidum readily assimilates acetate when grown in pure is found. Interestingly, fractionation factors obtained from culture (20), photoheterotrophy is apparently not a signifi- analyses of pigment fractions of bacteriochlorophyll c, used cant metabolic process in natural populations of this pho- as a biomarker for green Chloroflexus strains (11), showed totroph. Perhaps the continuous supply of sulfide from dramatic depletion in 13C, even more so than for bacterio- geothermal sources favors maintenance of the photoau- chlorophyll a obtained from C. tepidum (Table 2). However, totrophic state. Indeed, sulfide limitation, a condition which unlike the results with C. tepidum, the enzyme RuBP probably rarely exists for natural populations of C. tepidum, carboxylase was totally undetectable in cell extracts of has been identified as a major trigger of photoheterotrophy in natural populations of green Chloroflexus strains (data not other phototrophic sulfur bacteria (15, 37). shown). Studies of carbon isotope fractionation by cultures of Chloroflexus aurantiacus OK-70 fl (grown photoautotrophi- cally on H2 plus C02) have shown this organism to discrim- DISCUSSION inate less against "3CO2 than known Calvin cycle organisms Purple sulfur bacteria regularly coexist in nature with (16) (although the pathway of autotrophy in Chloroflexus heterotrophic organisms, in particular with sulfate- and aurantiacus is not known, it is not the Calvin cycle [16]). sulfur-reducing bacteria (6, 21, 26, 27, 35, 41). Biogenic Assuming that green Chloroflexus strains and Chloroflexus sulfide production is largely dependent on the availability of aurantiacus share the same autotrophic CO2 fixation path- organic matter (41), and the presence of soluble organic way, we were surprised to find bacteriochlorophyllide c compounds may then affect the nutrition of any phototrophic isolated from green Chloroflexus strains to be isotopically so population that develops. A number of organic compounds light. This finding raises the possibility that in nature green can be assimilated by phototrophic bacteria, and acetate in Chloroflexus cells assimilate isotopically light organic com- particular seems to be well metabolized (27, 32). Acetate is a pounds excreted by C. tepidum. The latter situation would major product of many different fermentations and in most be analogous to the assimilation by Chloroflexus aurantiacus anaerobic environments is one of the most abundant organic of organic compounds excreted by the cyanobacterium compounds (17). In green bacteria (29) and in the nutrition- Synechococcus lividus (2, 3) and of fermentation products ally restricted Group I members of the family Chromati- generated from decompositional processes in the mat (1). On aceae (32), which frequently predominate in blooms (34), the other hand, thick stands of green Chloroflexus strains do acetate incorporation is strictly sulfide dependent (15, 20, 32, occur in springs too hot to support mats of C. tepidum (11, 37). The oxidation of sulfide by these phototrophs in nature 20), which indicates that green Chloroflexus strains can also could therefore signal either photoautotrophic or photohet- grow autotrophically. Further isotopic studies will be neces- erotrophic reactions. Indeed, in chemostat studies of purple sary to determine the metabolic patterns of green Chloro- and green bacteria it has been shown that environmental flexus strains in situ. factors, in particular light and sulfide limitations, greatly A major technical problem in carbon isotopic studies of stimulate the incorporation of acetate; when light and sulfide complex microbial ecosystems has been the difficulty of are limiting, the bulk of cell material may actually be derived obtaining data on a specific organism in mixtures of different from acetate rather than from CO2 (15, 37). organisms, such as occur in a microbial mat (9). Our ap- The presence of assimilable organic compounds may proach of measuring specific biomarkers serves to overcome significantly affect the amount and route of CO2 fixation by this problem. When a particular biomarker can be clearly anoxyphototrophs. For example, in Rhodospirillum rubrum associated with a particular organism and can be obtained in (10) and in Chromatium vinosum (18), acetate severely pure form, the complexity of the ecosystem should be of represses synthesis of RuBP carboxylase (note that this was little consequence in assessing the carbon-isotopic composi- also true of C. tepidum [Table 1]). However, because acetate tion of that member of the community. Bacteriochlorophylls is assimilated by many purple and green sulfur bacteria via and specific carotenoids are ideal biomarkers because they the activity of pyruvate synthase (acetyl-CoA + CO2 + 2H can be obtained from mixed cultures in pure form and are -> pyruvate) (10, 21) and since the pyruvate formed is further frequently highly specific for particular organisms (33). In carboxylated to yield C4 carbon skeletons (30), a limited view of this, it is hoped that the methods developed for use amount of CO2 fixation always occurs during photohetero- on hot spring phototrophs can be used to determine the trophic growth on acetate. Fixation of CO2 by these path- isotopic composition of phototrophic bacteria developing in ways, however, is not associated with the significant carbon response to biogenic sulfide. Such analyses would be useful isotope fractionation observed in Calvin cycle reactions (28, for testing our hypothesis that photoheterotrophy is a major 31). Thus, we assume that purple sulfur bacteria growing at metabolic pattern in such blooms of phototrophic bacteria. the expense of biogenic sulfide probably assimilate organic compounds, such as acetate, in addition to small amounts of CO2. Indeed, this conclusion is supported by the early ACKNOWLEDGMENTS studies of Wassink and Manten (39), who showed that pure This work was supported by grants PCM-8415291 (to H.G.), cultures of purple sulfur bacteria enriched and isolated under PCM-8404996 (to J.M.H. and H.G.), and PCM-8505492 (to M.T.M.) strictly photoautotrophic conditions grew faster and to from the U.S. National Science Foundation and by grant NGR- VOL. 55, 1989 CARBON ISOTOPE FRACTIONATION BY PURPLE SULFUR BACTERIA 643

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