Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs

ELSEVIER

FEMS Microbiology Ecology 20 (1996) 69-77

Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs Niels-Ulrik

Frigaard, Kim L. Larsen ‘, Raymond P. Cox

*

Institute of Biochemistry, Odense University, Campusuej 55, 5230 Odense M, Denmark

Received 11 August 1995; revised 4 January 1996; accepted 8 January 1996

Abstract The combination of chromatographic separation using reverse-phase high-performance liquid chromatography and continuous monitoring of the column eluate using a diode-array spectrophotometer allowed qualitative and quantitative pigment profiling of extracts of photosynthetic material in a single run. Carotenoids and the spectrally distinct types of chlorophyll and bacteriochlorophyll can be unambiguously identified even when imperfectly separated on the column. The resulting spectrochromatogram is a fingerprint useful for the rapid characterization of pure cultures or mixed populations. We have developed software to allow recording, manipulation, and presentation of the resulting spectrochromatograms and present results from photosynthetic microbes in pure and mixed cultures. We describe a number of approaches to the presentation of the resulting data in a readily comprehensible form. Keywords: High-performance liquid chromatography; Diode-array spectrophotometry; Fingerprint; Pigment; Microbial phototroph

1. Introduction Phototrophic most other pigmentation.

microorganisms

are

distinct

from

microbes in their possession of strong These pigments are chlorophylls (Chl),

(BChl), carotenoids and phycobilins, which are involved in light energy capture in photosynthesis. With the exception of the phycobilins of cyanobacteria, cryptophytes and red algae (Rhodophyta), which are covalently attached to proteins [I], these pigments can be extracted by organic

bacteriochlorophylls

* Corresponding author. Tel: +45 66 15 86 00; Fax: +45 65 93 03 52, E-mail: [email protected]

’ Present address: Biotechnology Laboratory, Department of Civil Engineering, Aalborg University, Sohngaardholmsvej 57, 9000 Aalborg, Denmark. Federation of European Microbiological PII SO168-6496(96)00005-O

Societies

solvents. An extract of a pure or mixed culture in an organic solvent will therefore contain a complex mixture of porphyrins (chlorophylls, bacteriochlorophylls and derivatives) and carotenoids. The pigment composition is dependent on the type of phototroph present and is important information when characterizing pure cultures or the microbial distribution in natural environments [2-41. The informational value of absorption spectra is usually limited to determining the type and concentration of the major spectral species of (bacterio)chlorophylls and estimating the level of total carotenoid. Complete analysis of the mixture requires a separation technique, usually HPLC. However, recording the absorbance of the column eluate at a single wavelength provides no information about the chemical identities of the individual peaks. For well defined samples such as extracts from higher

70

N. 4.

Frigaard et al. / FEMS Microbiology

plant tissues this may be acceptable, but with previously unexamined source material there is a potential problem in assigning all the peaks and the risk that important components will be overlooked because the detection wavelength was poorly chosen. The solution to this problem is to combine spectrophotometry with chromatography. Diode-array spectrometers allow complete absorption spectra to be obtained within much less than a second, allowing the spectra of individual peaks in the chromatogram to be determined as they pass through the detector. Spectrochromatography is a two-dimensional technique, producing a data set containing absorbance values as a function of both wavelength and time. It contains in principle all the information needed for a total quantitative pigment analysis, leading to an enormous saving of time and material compared to the one-dimensional approaches of conventional HPLC or spectrophotometry. However, processing the large amount of data contained in a spectrochromatogram and converting it to useful information is a considerable problem. We describe here the approach we have taken in using a commercially-available high-performance diode-array spectrophotometer as an HPLC detector, which involved the development of special computer programs to collect the data and manipulate the results. We also describe methods for presenting the resulting spectrochromatograms on a monochrome output device such as a printer, and present representative results for a variety of types of microbial phototrophs, and some examples of the analysis of the mixed populations found in natural samples, all using the same HPLC analysis protocol.

2. Materials and methods 2. I. Organisms The pure cultures investigated were: Chlorojlexus aurantiacus Ok-70-fl, Chlorobium tepidum ATCC 49652, Chlorobium phaeobacteroides 1549, Chlorobium limicola 6230, Chlorobium vibrioforme NCIB 8327, Chromatium vinosum DSM 180, Gloeobacter violaceus PCC 7421, Synechococcus sp. OD 24, Chlamydomonas reinhardtii SAG 11-32, Rhodomonns sp. and an isolate of a purple non-sulfur

Ecology 20 (1996) 69-77

bacterium identified as Rhodobacter sphaeroides. All pure cultures were grown in batch culture under light-limiting conditions, except for Chlorobium tepidum which was grown in continuous culture. Natural sediment samples for preparation of Winogradsky columns were collected in the brackish Hofmannsgave bay, North Funen, Denmark. 2.2. Sample preparation Samples of 20-50 ml cell culture or 5-50 g sediment sample were centrifuged at 10000 X g for 10 min. The pellet was extracted with acetone:methanol (7:2 v/v) and centrifuged again. This was repeated until no more pigment could be extracted. If necessary, sonication was used to facilitate extraction of the pigments. The extracts were dried under N, or vacuum in the dark and redissolved in 0.5- 1.O ml solvent B (see HPLC procedure below). Particulate material was removed by centrifugation at 20000 X g for 5 min and passage through a 0.45 pm pore-size filter. 1.0 M ammonium acetate (10% v/v) was added to the sample as a buffer before injection onto the column. The amount of BChl or Chl in each injection was in the range 5-50 pg. Extracts were run immediately as the apolar carotenes tend to precipitate on standing in the solvent and BChl a, b and e are readily oxidized. For storage the extracts were dried under N, in the dark and kept at - 50°C. 2.3. HPLC procedure A Kontron HPLC system was used (Kontron Instruments AG, Zurich, Switzerland). A 3.9 X 300 mm column packed with octadec$silyl-bonded amorphous silica (Nova-Pak C18, 60 A pore size, 4 pm particles) was used with a pre-column (Nova-Pak C181, (Waters, Millipore Corporation, Milford, MA, USA). The method used was a modification of that used by Borrego and Garcia-Gil [5]. The gradient was composed of solvent A (methanol:acetonitrile:water, 42:33:25 by volume) and solvent B (methanol:acetonitrile:ethyl acetate, 39:31:30 by volume) as follows: at the time of injection 40% B, a linear increase to 100% B at time 60 min and back to 40% B in 3 min. The system was equilibrated for 5 min before injection. The flow rate was held constant

N.-U. Frigaard et al./ FEMS Microbiology Ecology 20 (1996) 69-77

at 1.0 ml mm’. Solvents were of HPLC grade (Rathburn, Walkerburn, UK). The column temperature was 30°C. 2.4. Data acquisition The eluent from the HPLC column was passed through a single wavelength detector and then through a flow cell with 10 mm path length in a Specord S 10 diode-array spectrophotometer (Carl Zeiss, Jena, Germany). This contains 1024 diodes and is capable of recording a spectrum from 190 nm to 1020 nm with a resolution between 0.81 and 0.83 nm depending on the wavelength. Data from the spectrophotometer was transferred to a personal computer with an Intel 486 processor. Software provided by the manufacturer allowed raw spectral data to be read into a function in the C programming language. A specially-written program in Turbo C (Borland International Inc., Scotts Valley, Calif., USA) was used to accumulate 8 spectra at the maximum rate possible and store the average on a RAM disk with 4 Mbyte capacity. Averaged spectra were stored at 3 s intervals for 1024 wavelength points at

Table 1 Spectrochromatographic

71

absorbance values in principle from - 3.0 to 3.0 at a nominal accuracy of 0.0001 A. A spectrochromatogram lasting 60 min contains 1200 spectra and occupied 2.3 Mbyte of binary data. The program displays the captured spectrum on the screen (1024 X 768 pixels) as a vertical line of pixels whose colour depends on the absorbance values. A maximum of 899 spectra corresponding to 45 min can be simultaneously shown on the screen. As the spectrochromatogram advances, the coloured display provides a visual picture of the results and it is possible to display either a chromatogram at any selected wavelength, or any previously recorded spectrum, in the intervals between data capture. A second program was used to display previously recorded spectrochromatograms and view and save individual spectra, chromatograms, or selected wavelength X time blocks as ASCII files for analysis and display by other software. 2.5. Data manipulation

and presentation

The data from a particular wavelength X time block was compressed and presented as contour plots

data for selected pigments

Retention time (min)

Pigment

Absorption

maxima (nm)

3.7-4.7 6.0-8.0 6.4 7.3 11.3 12-35 13-17 13-17 13.9 14.4 29.0 30.9 32.5 35-44 38.5 39.7 43-50 44-51 44-54 46-50 53.0 53.9

Oscillaxanthins Myxoxanthophylls Neoxanthin Violaxanthin Alloxanthin Major BChl c homologs Major BChl d homologs Major BChl e homologs Lutein Zeaxanthin BChl b BChl a Chl b Spheroidene-chromophores Spheroidenone Chl a Isorenieratene-chromophores cis-Isorenieratene-chromophores Chlorobactene-chromophores cis-Chlorobactene-chromophores a-carotene /3-Carotene

469,496,53 1 448,475,506 413,437,465 417,440,469 429,452,48 1 434,666 427,655 469,654 423,445,474 452,478 373, 795 364,770 463,648 428,453,484 482 430,663 427,452,479 343,422,447,473 436,461,490 352,432,456,485 422,446,474 449,473

72

300

400

500 Wavelength

Fig. 1, Absorption from the column.

spectra of the 5 spectral types of bacteriochlorophyll

600

700

600

900

(nm) (BChl) taken by the diode-array

spectrophotometer

as they elute

and ‘cross plots’ using specially-written software. The contour plots were generated in Windows Metafile format from blocks of resolution 4.9 nm X 12 s by a program written in Mathematics (Wolfram Research Inc., Champaign, IL, USA). The cross plots were generated in Hewlett-Packard Graphics Language format from blocks of resolution 1.6 nm X 6 s by a program written in Microsoft QuickBasic.

tivity at the wavelength of maximum absorbance comparable (around lo5 M- ’ cm-‘) [6,7,9].

2.6. Pigment analysis

A sequence of spectra as a function of time is frequently displayed in such a way as to give the impression of a perspective view of a three-dimensional landscape. While such plots are aesthetically appealing, we found that it was not possible to display spectrochromatograms of complex mixtures in this way without important features being obscured. To improve interpretation of the spectrochromatograms and provide documentation on a monochrome output device, we investigated the possibility of displaying the results as two-dimensional contour plots. For reasons of clarity, the number of contour levels should not exceed 10. However, if this number of contour levels is used with a linear scale, relatively small peaks will not be shown. One way to enhance to appearance of small peaks in the contour plot is to place the contour levels on a logarithmic

Chlorophylls, bacteriochlorophylls and their derivatives were identified from the red (Q,) and blue (Soret) band absorbance maxima listed in Table 1 and elsewhere [6,7]. Carotenoids were identified from their absorption maxima [8,9], and reported pigment compositions of the microorganisms [&lo]. The absorption spectra of the five spectrally distinct types of bacteriochlorophyll as they elute from the column are shown in Fig. 1. The absorption spectrum of Chl a resembles the spectrum of BChl d, and the spectrum of Chl b that of BChl e, apart from the maxima shifts indicated in Table 1. The relative molar amounts of chlorophylls, bacteriochlorophylls and carotenoids can be estimated from the absorption spectra since the molar absorp-

is

3. Results 3.1. Spectrochromatographic

contour plots

13

N.-U. Frigaard et al./ FEMS Microbiology Ecology 20 (1996) 69-77

a

b

C

-

-

-

-

-

-

-

-

z

Fig. 2. Illustration of 10 contour levels that are equidistant on (a) a linear scale and on a logarithmic scale with (b) c = 10 and (cl c = 50, (see Eq. (1) in the text for explanation). The large peak is cut by all levels. The small peak (10% of large peak) is not cut by any level above the baseline on the linear scale (a), but is cut by 2 and 4 levels in (b) and (c) respectively.

absorbance scale. In order to obtain a contour plot with logarithmically spaced levels which are independent of the amount of sample and to avoid problems with zero and negative absorbances due to changes in the background absorbance, the data were transformed by adding a constant, b, to all absorbance values such that the transformed data ranged over a desired factor c given by (A,,, + b)/( Amin + b), where A,,, and Amin are the maximum and minimum values in the original data. The contour levels were then placed equidistantly on a logarithmic absorbance scale going from Amin + b to A,,, + b. Increasing the value of c contracts the lower levels and widens the upper. The effect of this in enhancing small peaks is illustrated in Fig. 2. The equation for nlogarithmically equidistant contour levels in a spectrochromatogram is contour level i -i-l =

Cn-‘(Amax-Amin)

-Amax+CAmin

c-1

(1)

where i varies from 1 to IZ. Note that b does not appear in this expression. The first (i = 1) and last (i = n) contour levels are equal to the minimum and

maximum recorded absorbance values respectively and only occur as points in the spectrochromatogram. In a spectrochromatographic contour plot, the relative height of a peak may be estimated by counting the number of visible contour levels i’. By using Eq. the (1) with A,,, = 1, Amin=O, and i=i’+l, height of the highest contour level of the peak is (P’(“_‘) - l)/(c - 1). Fig. 3 shows a spectrochromatogram obtained from an extract of a pure culture of the green filamentous bacterium Chloroflexus aurantiacus depicted as a contour plot where c = 10 and 12= 10. Four BChl c homologs (discussed below) eluted after 22.1, 29.0, 32.6, and 34.9 min and several kinds of carotenoid eluted after 37.6, 37.7, 52.0, 54.5, and 54.9 min. Monochrome contour plots of this type are not always readily interpretable, especially to the inexperienced eye. We have therefore investigated altemative approaches to data reduction which allow the information to be presented in a more readily understandable manner. 3.2. Maximum

absorbance

chromatogram

Fig. 3 also shows a maximum absorbance chromatogram (MAC) of the same sample. Rather than showing the absorbance at a single wavelength as in a traditional chromatogram, this depiction shows the maximum absorbance at any wavelength in a given range (in this case 300-850 nm). In this way all pigments contribute to the chromatogram. 3.3. Spectrochromatographic

cross plots

A ‘cross plot’ is superimposable on the corresponding contour plot. Only pigments with a maximum absorbance above a certain threshold are included. The spectrum of a given peak is indicated by vertical lines through the points at which the absorbance is above a certain fraction of the maximum absorbance in that spectrum. For example, with an arbitrary threshold of 20%, the blue (Soret) band of BChl c is indicated by a line from around 309 to 453 nm and the red (Q,) band by a line from around 644 to 682 nm (Figs. 1 and 3). These lines are independent of the absolute absorbance values, so spectra of

74

N.-U. Frigaard et al./ FEMS Microbiology

Ecology 20 (1996) 69-77

spectral type (BChl c) because the peak positions are at similar wavelengths, and that the latest-eluting BChl c at 34.9 min was present in the largest amount because the horizontal peak lines are widest. It can also be seen that at least 4 of the 5 carotenoids which eluted after the bacteriochlorophylls were spectrally distinct. 3.4. Measurements

0

700 -

t

ttt

500 -

(

300 0

,I,

t,;tl,

20

, 40

,‘1

j~ 60’

Time (min) Fig. 3. Three different depictions of a spectrochromatogram obtained from an extract of Chlorojkxus aurantiacus. The absorbance range in the original data is - 0.03 to 1.85. Upper panel: contour plot where c = 10 and n = 10. Center panel: maximum absorbance chromatogram (MAC) using the wavelength range 300-850 nm. Lower panel: cross plot. The criterion for the appearance of a spectrum is a minimum peak height of 10% of the highest peak in the spectrochromatogram. Vertical lines correspond to absorbance values above 20% of the maximum in the spectrum; horizontal lines at peak maxima are scaled so that the highest peak in the spectrochromatogram cooresponds to 5% of the total length of the abscissa.

a particular type appear similar. The positions of the absorbance maxima are indicated by horizontal lines, whose length is proportional to the absolute absorbance at the peak maximum. In the case of BChl c the peak maxima are at 338, 434, and 666 nm (Figs. 1 and 3). From the cross plot in Fig. 3 it is apparent that the 4 porphyrins were of the same

on extracts from single strains

Fig. 4 shows spectrochromatograms (displayed as cross plots) for a number of representative phototrophs. The panels on the left of the figure show oxygenic phototrophs containing Chl a. The green alga Chlamydomonas reinhardtii contains Chl b eluting at 32.5 min and Chl a at 3.97 min; p-carotene eluted at 53.9 min and a number of xanthophylls eluted before Chl b. A tetrapyrrole was also seen which eluted within the first 5 min; this is probably a chlorophyllide degradation product. For the cyanobacterium Gloeobacter uiolaceus Chl a and p-carotene are seen as in the case of the green alga, together with a smaller component with a Chl a-like spectrum which eluted slightly later, and the sugarconjugated xanthophyll oscilloxanthin which eluted within 5 min as two isomers (the all-trans form and a form in which one of the double bonds is in the cis position). The cryptomonad alga Rhodomonas sp. contains Chl c (which has no esterifying alcohol and was not retarded under our chromatographic conditions) and alloxanthin which eluted after 11.3 min. In place of the expected Chl a there was a pigment with the same spectral properties but a slightly lower elution time (37.5 min); this may be an isomerisation product formed during sample preparation. Chromatium and Rhodobacter are purple bacteria, containing BChl a which eluted at 29-31 min. Rhodobacter sphaeroides is an isolate identified on the basis of morphology and the presence of spheroidenone (38.5 min) and 2 carotenoids of the spheroidene series (35.6 and 44.0 min) [l 11. The examples of green sulfur bacteria show a green-coloured strain containing BChl d (Chlorobium uibrioforme) and a brown-coloured strain containing BChl e (Chlorobium phaeobacteroides). As was the case for Chloroflexus aurantiacus (Fig. 3) there are a number of peaks with the same bacteriochlorophyll spectrum, corresponding to a series of

N.-U. Frigaard et al. / FEMS Microbiology

homologs. In Chlorojlexus these homologs differ in the esterifying alcohol. In the green sulfur bacteria one alcohol is predominant but there are several homologs with different aliphatic side chains attached to the tetrapyrrole (for example methyl, ethyl, n-propyl or isobutyl) [12]. Both these changes effect the polarity of the molecule and hence its retention time, but in different ways. The changes in the esterifying alcohol in Chlorojlexus (for example octadecanol to geranylgeraniol) can have large effects of the polarity, giving rise to peaks with considerably different retention times. The differences between the homologs in the green sulfur bacteria are relatively minor. The tetrapyrrole pigments from Chlorobium vibrioforme which eluted in the first 10 min are probably bacteriochlorophyllides of the corresponding BChl d homologs. The pigment compo-

Ecology 20 (1996) 69-77

15

sition of green sulfur bacteria can be extremely complex because minor amounts of bacteriochlorophyll with a different esterifying alcohol are also found. Up to 15 different bacteriochlorophyll peaks have been reported in a single strain [5]. The spectrochromatogram of ChEorobium phaeobacteroides also shows the carotenoid isorenieratene (49.4 min) which is typical of green sulfur bacteria containing BChl e. In the BChl c-containing green sulfur bacterium Chlorobium tepidum there were 10 closely-assocated peaks is the chlorobactene region (results not shown). In general, cis-isomers of chlorobactene and isorenieratene (characterised by an additional peak around 350 nm and a 5nm blueshift in the other peak maxima compared to the all-truns form [9]) were separated from the all-truns form by 0.7-0.8 min (results not shown).

Green zone

‘hodomonas

t

t1

/II,,!I,, i ,

iii. 40

60 0

20 40 Time (min)

60 0

20

40

I

60;

Fig. 4. Spectrochromatographic cross plots of extracts of Chlamydomonas reinhardtii, Gloeobacter uiolaceus, Rhodomonas sp., Chromatium uinosum, Rhodobacter sphaeroides, a sample from an anoxic, red zone of a Winogradsky column, Chlorobium uibriofonne, Chlorobium phaeobacteroides and a sample from an anoxic, green zone of a Winogradsky column. The parameters used in drawing the cross plots are as described in the legend to Fig. 3.

16

3.5. Measurements tions

N.-U. Frigaard et al./FEMS

Microbiology

on extracts from mixed popula-

In order to investigate the possible value of spectrochromatograms of samples derived from natural environments containg a variety of types of phototrophs, we made extracts from coloured areas of Winogradsky columns derived from a brackish water sediment. Fig. 4 shows the spectrochromatogram of a purple-red zone; BChl a is observed in the same position as for Rhodobacter, indicating the presence of purple bacteria. There is also Chl a indicating the presence of algae, cyanobacteria, or both. Fig. 4 also shows a spectrochromatogram of a green-coloured zone well below the surface of the column; the major features are the presence of a series of BChl c homologs and chlorobactene (45.7 min), indicating the predominance of green sulfur bacteria.

4. Discussion The combination of HPLC with diode-array spectrophotometry allows us to obtain a pigment profile of the sample in a single measurement. This is a considerable saving of time and sample compared to the alternative approaches of repeating the chromatography at a series of wavelengths, or collecting samples manually for separate determinations of the spectra. It appears that most of the commonly observed pigments in the various groups of phototrophs have distinct combinations of spectrum and polarity so that they can be unambiguously detected. There are several potential sources of error which need to be considered when making measurements of this type. One in the presence of degradation products, such as chlorophyllides, pheophytins and carotenoid isomers; these may be either genuinely present in the sample or artifacts produced during extraction, and they can greatly complicate the resulting spectrochromatogram and cause difficulties in its interpretation. A second type of problem is that the exact retention times obtained will inevitably depend on the column used and its age, and the exact spectra obtained are those in the particular solvent composition in the detector at the moment of elution, which is neither perfectly reproducible nor identical with the spectra given in the literature, which are

Ecology 20 (1996) 69-77

usually in pure solvents. In Table 1 we have collected typical values for retention times and wavelengths of peak maxima as the compounds pass through the detector, for a variety of pigments found in phototrophs. The values are derived both from the spectrochromatograms in Fig. 4 and from other measurements. Spectrochromatograms can be valuable at several levels of detail. The major algal classes and types of photosynthetic bacterium generally contain characteristic types of chlorophyll or bacteriochlorophyll. Information about this is immediately available from a spectrochromatogram, and will allow an unknown isolate to be placed in a general taxonomic grouping [8,10,12]. For example, the presence of BChl e is characteristic of brown-coloured green bacteria; the presence of chlorophyll b is characteristic of green algae or euglenoids. A spectrochromatogram of a mixed culture or natural sample will allow a quantitative determination of the relative contributions of the different classes of chlorophylls and bacteriochlorophylls to the total pigment pool and thus provide information about the relative importance of the different cell types of which they are characteristic. More detailed information about pigment content, especially the carotenoid composition, can allow taxonomic discrimation within the larger groupings defined by bacteriochlorophyll or chlorophyll compositions. For example, the characteristic series of related carotenoids observed in different types of purple and green bacteria [9- 11,131 provides information useful for identification at the level of the genus or species. Unfortunately it is not possible to identify carotenoids unambiguously from their absorption spectra. Nevertheless, valuable information is provided by a spectrochromatogram which gives both the peak position, which provides an indication of the number of conjugated double bonds in the chromophore, and the elution time, which provides an indication of the polarity of the molecule and hence an indication of the presence of oxygen atoms (xanthophylls) or their absence. (An exception to this rule is the esters of xanthophylls and long chain fatty acids which elute more slowly than carotenes on reverse-phase HPLC). Even if a spectrochromatogram contain pigments which can neither be specifically identified nor as-

N.-U. Frigaard et al. / FEMS Microbiology Ecology 20 (1996) 69-77

signed to a general class such as ‘carotenoids with very high polarities, possibly due to conjugation with sugars’ ) the patterns obtained may be useful as a characteristic fingerprint of a particular strain. Pigment profiles have considerable potential value in the taxonomy and ecology of microbial phototrophs. Their value at the present is limited by the scarcity of information about the biological significance of phenomena such as carotenoid composition or bacteriochlorophyll homolog distribution, particularly knowledge about the relative importance of genotype and environmental factors. The approach described here provides a powerful technique for investigations of such questions.

Acknowledgements We thank Prof. M.T. Madigan for providing a culture of Chlorobium tepidum, Prof. J. Ormerod for the cultures of Chlorobium phaeobacteroides and Chlorobium uibrioforme, N.T. Eriksen for providing cells of Rhodomonas sp. and Dr. M. Miller for valuable and encouraging discussions. This work was supported by the Danish Natural Sciences Research Council and the Carlsberg Foundation.

References [ll Grossman, A.R., Schaefer, M.R., Chiang, G.G. and Collier, J.L. (1993) The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbial. Rev. 57, 725-749. [2] Korthals, H.J. and Steenbergen, C.L.M. (1985) Separation and quantification of pigments from natural phototrophic microbial populations. FEMS Microbial. Ecol. 31, 177-185.

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[3] Palmisano, A.C., Cronin, S.E., D’Amelio, ED., Munoz, E. and Des Marais, D.J. (1989) Distribution and survival of lipophilic pigments in a laminated microbial mat community near Guerrero Negro, Mexico. In: Microbial Mats: Physiological Ecology of Benthic Microbial Communities (Cohen, Y. and Rosenberg, E., Eds.), pp. 138-152. American Society for Microbiology, Washington, D.C. [4] Repeta, D.J., Simpson, D.J., Jorgensen, B.B. and Jannasch, H.W. (1989) Evidence for anoxygenic photosynthesis from the distribution of bacteriochlorophylls in the Black Sea. Nature 342, 69-72. 151 Borrego, C.M. and Garcia-Gil, L.J. (1994) Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC. Photosynth. Res. 41, 157-163. [6] Oelze, J. (1985) Analysis of bacteriochlorophylls. Methods Microbial. 18, 257-284. [7] Hoff, A.J. and Amesz, J. (1991) Visible absorption spectroscopy of chlorophylls. In: Chlorophylls @cheer, H., Ed.), pp. 723-738. CRC Press, Boca Raton. [8] Rowan, K.S. (1989) Photosynthetic Pigments of Algae, 334 pp. Cambridge University Press, Cambridge. [9] Schmidt, K., Connor, A. and Britton, G. (1994) Analysis of pigments: carotenoids and related polyenes. In: Chemical Methods in Prokaryotic Systematics (Goodfellow, M. and O’Donnell, A.G., Eds.), pp. 403-461. Wiley, Chichester. [lo] Imhoff, J.F. (1992) Taxonomy, phylogeny, and general ecology of anoxygenic phototrophic bacteria. In: Photosynthetic Prokatyotes (Mann, N.H. and Carr, N.G., Eds.), pp. 53-92. Plenum Press, New York. [ 111 Imhoff, J.F. and Truper, H.G. (1991) The genus Rhodospirillum and related genera. In: The Prokaryotes, a Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications (Balows, A., Truper, H.G., Dworkin, M., Harder, W. and Schleifer, K.-H., Eds.), pp. 2142-2155. Springer, New York. 1121 Scheer, H. (1991) Structure and occurrence of chlorophylls. In: Chlorophylls @cheer, H., Ed.), pp. 3-30. CRC Press, Boca Raton. [13] Bobe, F.W., Pfennig, N., Swanson, K.L. and Smith, K.M. (1990) Red shift of absorption maxima in Chlorobiineae through enzymatic methylation of their antenna bacteriochlorophylls. Biochem. 29, 4340-4348.