General Microbiology - Schlegel, H. 1987

Phototrophic bacteria and photosynthesis
Purple and green bacteria

The ability to use light as an energy source for growth is characteristic of two fundamentally different groups of Bacteria.

Purple and green bacteria, grouped into the order Rhodospirillales¹, can be regarded as relict organisms that have survived from the early Stages of Photosynthesis evolution. Unlike green plants, they are unable to use Water as a hydrogen donor; instead, they require Donors with a higher degree of reduction (H₂S, H₂, or Organic compounds). Consequently, photosynthesis in these bacteria occurs without the evolution of O₂. Such a process is referred to as anoxygenic photosynthesis. Bacteria of this group are typically aquatic organisms found in both fresh and marine waters. Their red, orange, or green coloration is due to the presence of bacteriochlorophylls and carotenoids.

Cyanobacteria use water as a hydrogen donor and release oxygen in the light, thereby performing oxygenic photosynthesis. Their pigment system includes chlorophyll a, carotenoids, and phycobilins. Since the photosynthetic process in cyanobacteria is fundamentally similar to that of green plants, this group of bacteria was until recently classified alongside photosynthetic eukaryotes and referred to as blue-green Algae. However, in terms of their cellular Structure, they are typical prokaryotes. Cyanobacteria have already been described in detail in Section 3.21 and will not be discussed here².

¹ Currently, green bacteria are classified into a separate order, Chlorobiales, and together with purple bacteria, they are grouped into the Class Anoxyphotobacteria. — Ed. note.

² In addition to cyanobacteria, prokaryotic organisms belonging to the order Prochlorales are also capable of oxygenic photosynthesis. Together with cyanobacteria, they are classified in the class Oxyphotobacteria. — Ed. note.

Phototrophic bacteria that perform anoxygenic photosynthesis are divided into two major groups: purple bacteria (Rhodospirillales) and green bacteria (Chlorobiales). Representatives of these two orders differ significantly in their cytological and physiological characteristics, as well as in their characteristic pigments (Table 12.1; Figs. 12.1, 12.6, and 12.10).

Fig. 12.1. Some representatives of purple sulfur bacteria (Chromatiaceae) and purple nonsulfur bacteria (Rhodospirillaceae).

Purple bacteria (Rhodospirillales)

A common feature of all members of the order Rhodospirillales is that their photosynthetic apparatus (light-harvesting systems and reaction centers) is located on internal membranes (thylakoids) formed by invaginations of Cell/30.html">The Plasma Membrane (see Figs. 2.23 and 2.24).

Table 12.1. Families of anaerobic phototrophic bacteria

¹ Bchl — bacteriochlorophyll.

² p-ABA — p-aminobenzoic acid.

³ PM — plasma membrane.

⁴ Certain species grow in the dark under aerobic or microaerophilic conditions. — Ed. note.

⁵ Almost all species grow in the dark under aerobic or microaerophilic conditions. — Ed. note.

The Morphology of thylakoid structures can vary greatly among different species, including vesicular, tubular, and lamellar forms (see Fig. 2.24). With few exceptions, the characteristic chlorophyll of this group of bacteria is bacteriochlorophyll a. All these bacteria are capable of fixing CO₂ via the ribulose bisphosphate cycle and utilizing Organic compounds as hydrogen donors and/or carbon sources.

Based on their ability to use elemental sulfur as an electron donor, the purple bacteria are divided into two families: the purple sulfur bacteria, or Chromatiaceae (formerly Thiorhodaceae), and the purple nonsulfur bacteria, or Rhodospirillaceae (formerly Athiorhodaceae)¹.

Purple sulfur bacteria (Chromatiaceae). Most purple sulfur bacteria can be easily recognized by intracellular sulfur inclusions visible as highly refractive globules (see Figs. 2.43, 12.2, and 12.3). Chromatium okenii (5 µm thick and 20 µm long) and Thiospirillum jenense (3.5 µm thick and 50 µm long) are giants among bacteria (Figs. 12.1, 12.2, and 12.3). They have long attracted the attention of microscopists and serve as model organisms for studying flagellar motility and tactic responses to various stimuli. Chromatium warmingii differs from C. okenii by its slightly smaller Cell size and polar positioning of sulfur inclusions. Large representatives of this genus are Kidney-shaped, whereas smaller ones are short rods. These include Chromatium vinosum strain D, which has been used in important studies on Bacterial photosynthesis. Thiocystis species (T. violacea, T. gelatinosa) are characterized by spherical, motile Cells. In Thiocapsa roseopersicina and T. pfennigii, the cells are also spherical, but nonmotile. Many species of purple sulfur bacteria possess gas vacuoles. Examples include the spherical motile bacteria Lamprocystis roseopersicina, as well as nonmotile forms such as Amoebobacter (spherical cells), Thiopedia (ellipsoidal cells), and Thiodictyon (rod-shaped cells).

¹ Currently, another family, Ectothiorhodaceae, is distinguished; previously, the corresponding species were included in the family Chromatiaceae. — Ed. note.

⁶ Recently, an anaerobic phototrophic bacterium, Heliobacterium chlorum, which carries out anoxygenic photosynthesis, has been discovered. It contains a unique chlorophyll (bacteriochlorophyll g) and, unlike other phototrophic bacteria, is Gram-positive. It is hypothesized to represent the most ancient form of phototrophs. — Ed. note.

A characteristic feature of Chromatiaceae is that during The oxidation of H₂S, sulfur is deposited within their cells as an intermediate product. Representatives of the genus Ectothiorhodospira (E. mobilis, E. halophila) deposit sulfur extracellularly rather than in the Cytoplasm, and further oxidize it to sulfate outside The Cell.

Fig. 12.2. Chromatium okenii (A) and Ch. warmingii (B). Bright-field micrographs; X ~ 800. (Photo by N. Pfennig.)

Chromatiaceae possess vesicular thylakoids (chromatophores) that typically fill the entire cell. Currently, only two exceptions are known: in Ectothiorhodospira species, the thylakoids form stacks of lamellae, whereas in Thiocapsa pfennigii they are tubular in shape.

Fig. 12.3. Thiospirillum jenense with monopolar polytrichous flagella and sulfur inclusions. Bright-field micrograph; X ~ 700. (Photo by N. Pfennig.)

Fig. 12.4. Rhodospirillum rubrum (A) and Rh. fulvum (B). Bright-field micrographs; x ~ 1200 and ~ 1800, respectively.

Purple non-sulfur bacteria (Rhodospirillaceae). Most known bacteria in this group can be assigned to two genera. Rod-shaped species are grouped into the genus Rhodopseudomonas; their species names largely reflect their physiological characteristics: R. palustris, R. viridis, R. acidophila, R. blastica, and R. sulfidophila. Spirilloid forms belong to the genus Rhodospirillum; differentiated by size and pigmentation are such species as R. rubrum (Fig. 12.4), R. salexigenes, R. fulvum, R. molischianum, and R. photometricum. The thylakoid structures in purple non-sulfur bacteria vary considerably in shape.

Fig. 12.5. Rhodomicrobium vannielii, a non-sulfur purple bacterium that reproduces by budding and forms hypha-like filaments. Bright-field micrograph; x ~ 1200. (Photo by N. Pfennig.)

A distinct position among the Rhodospirillaceae is held by Rhodomicrobium vannielii and Rhodocyclus purpureus. The former species (Fig. 12.5) reproduces by budding; the daughter cells remain attached to the mother cell via hypha-like filaments or detach to form cells with peritrichous flagella. Rhodocyclus purpureus is the only non-motile form in this family; its cells are semi-ring-shaped, and the photosynthetic apparatus is presumably located in the plasma membrane, which exhibits only very small and sparse invaginations.

Hydrogen sulfide inhibits the growth of many purple non-sulfur bacteria; however, some species are resistant to H₂S or even utilize it as a hydrogen donor during CO₂ fixation. Rhodopseudomonas sulfidophila and R. palustris oxidize hydrogen sulfide to sulfate without forming elemental sulfur as an intermediate product.

Green bacteria (Chlorobiales)

Representatives of the Chlorobiales are characterized by chlorosomes—Organelles containing pigments and appressed to the plasma membrane. These chlorosomes house bacteriochlorophyll (Bchl c, d, or e), which is the characteristic light-harvesting pigment of this group. In addition, these bacteria contain small amounts of bacteriochlorophyll a, which is directly linked to photosynthetic Reaction Centers and localized in the plasma membrane. Green bacteria differ from purple bacteria in lacking ribulose-1,5-bisphosphate carboxylase; consequently, they are unable to fix CO₂ via the Calvin-Benson cycle.

Fig. 12.6. Phototrophic green sulfur bacteria (Chlorobiaceae).

Fig. 12.7. Chlorobium limicola. Young culture with extracellular sulfur deposits. Bright-field micrograph; x ~ 1000. (Photo by N. Pfennig.)

Fig. 12.8. The green sulfur bacterium Pelodictyon clathratiforme (A), with cells connected in a network, and the purple sulfur bacterium Thiodictyon elegans (B), forming a looser network. Bright-field micrographs; x 1500 and 400, respectively. (Photo by N. Pfennig.)

Fig. 12.9. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF Chlorobium limicola. A. Ultrathin section. B. Chlorosomes visualized by freeze-etching. CM — chlorosomes; PM — plasma membrane; CW — Cell wall. Scale bar length approx. 100 nm. (Staehelin L. A. et al., Biochim. Biophys. Acta, 589 [1980], 30.)

The family Chlorobiaceae (Figs. 12.6–12.9) includes green (Chlorobium vibrioforme and Ch. limicola) and brown-colored (Ch. phaeobacteroides) species, as well as forms that aggregate in stellate clusters (Prosthecochloris) or networks (Pelodictyon clathratiforme).

Chlorochromatium aggregatum represents a symbiotic association of two bacterial types: colorless chemoorganotrophic rods motile by means of long polar flagella, and their ectosymbionts, green phototrophic bacteria. Pelochromatium roseum has a similar Organization, with the distinction that its ectosymbiont is brown. The marine bacterium Chloroherpeton thalassium differs from Chlorobium species in its filamentous cell shape and gliding motility.

Chloroflexaceae. The phototrophic green bacterium Chloroflexus resembles filamentous gliding bacteria in its morphology and mode of movement, yet it contains bacteriochlorophylls c and a and possesses chlorosomes of the same type as those found in green sulfur bacteria (Chlorobiaceae). At the same time, Chloroflexus differs from Chlorobium species in its capacity for aerobic heterotrophic growth on complex media both in the light and in the dark. Unlike Chlorobiaceae, Chloroflexaceae are virtually incapable of photoautotrophic growth on CO₂ and H₂; thus, these bacteria are more accurately classified as photoheterotrophs. C. aurantiacus is a globally widespread bacterium and constitutes the primary component of green or orange mats found at the bottom of thermal spring effluent streams.

12.1.1 Pigments of the Photosynthetic Apparatus

Due to Photosynthetic Pigments, sufficiently dense Suspensions of phototrophic bacteria display a green, blue-green, purple-violet, red, brown, or pink coloration. The color depends on The Nature and quantitative ratio of the pigments. Individual pigments can even be identified from the absorption spectra of intact cells (Fig. 12.10). Chlorophylls, for example, are responsible for the absorption maxima in the blue (< 450 nm) as well as the red and infrared (650–1100 nm) Regions of the spectrum. Absorption in the 400–550 nm range is primarily due to carotenoids, whereas in cyanobacteria, the 550–650 nm range is attributed to phycobiliproteins.

Various chlorophylls of phototrophic microorganisms differ mainly in the presence or absence of a double bond between carbon atoms 3 and 4 and in the substituents on the porphyrin Skeleton (Fig. 12.11). These modifications determine the absorption maxima in the near-infrared region. These differences are also clearly visible in the spectra of intact cells or isolated photosynthetic membranes, where pigments form specific complexes with Proteins (Fig. 12.10). The main maximum of chlorophyll a in green algae and cyanobacteria lies in the 680–685 nm region; that of bacteriochlorophylls c, d, and e in green sulfur bacteria and Chloroflexus is found in the 715–755 nm region; and that of bacteriochlorophyll a in most purple bacteria is located at 850–890 nm. Bacteriochlorophyll b absorbs in the range from 1020 to 1035 nm and has so far been found only in the cells of Rhodopseudomonas viridis, Ectothiorhodospira halochloris, Thiocapsa pfennigii, and certain other purple bacteria. Bacteriochlorophyll a in purple bacteria is represented by four spectral forms: B800, B820, B850, and B870–890. In this case, the differences in absorption depend on The Nature of the bond and THE POSITION OF the Bchla molecule within the pigment-protein complex of the photosynthetic apparatus. The number of peaks and their relative height vary from species to species, and in many organisms, they are also influenced by cultivation conditions.

Along with their characteristic bacteriochlorophylls c, d, or e, Chlorobiaceae also contain small amounts of Bchla with a single absorption peak at 810 nm, whereas in Chloroflexus cells, Bchla exhibits two distinct peaks at 808 and 868 nm.

Fig. 12.10. Absorption spectra of intact cells of phototrophic bacteria, cyanobacteria, and green algae. Types of bacteriochlorophylls are indicated in parentheses. Also shown are curves characterizing the infrared absorption capacity of filters used to obtain elective enrichment cultures of green and purple bacteria (A), purple bacteria containing bacteriochlorophyll a (B), and purple bacteria containing bacteriochlorophyll b (C). (Pfennig N., Ann. Rev. Microbiol. 21 [1967], 285.)

Fig. 12.11. Structural differences between chlorophyll a and bacteriochlorophylls a, b, c, d, and e.

*The bond between C-3 and C-4 is saturated

Carotenoids are so-called accessory pigments of phototrophic organisms. They absorb light in the spectral range from 400 to 550 nm. In purple bacteria, these are predominantly aliphatic C₄₀ compounds (tetraterpenoids) containing tertiary hydroxy and methoxy groups (lycopene, rhodopin, spirilloxanthin, spheroidene). Oxo and aldehyde groups can impart a dark red color to them (spheroidenone, okenone, rhodopinal). Carotenoids with aromatic rings (arylcarotenoids) — derivatives of γ- or β-carotene — have also been discovered. These carotenoids are found in representatives of only a few genera (okenone in certain Chromatiaceae; chlorobactam is a typical carotenoid of green species, and isorenieratene of brown species of Chlorobium).

Carotenoids perform two Functions: on the one hand, they participate in photosynthesis as light-harvesting pigments, i.e., they absorb light energy and transfer it to chlorophyll; on the other hand, they protect chlorophyll against photooxidation. Blue-green mutant strains of purple bacteria lacking carotenoids are capable of growing only under low light and die at high light intensities.

From the differences in the absorption spectra of green algae, cyanobacteria, purple bacteria, and green bacteria, it can be concluded that various groups of phototrophic organisms utilize light from different spectral regions for photosynthesis. This is related to the light conditions in the natural habitats of various phototrophic organisms. The observed differences are utilized when obtaining enrichment cultures of certain phototrophic bacteria (see Fig. 12.13).

Pigment localization. Photosynthetic pigments in purple bacteria are associated with internal membranes — vesicular or tubular outgrowths of the plasma membrane that remain connected to it while extending into the cytoplasm. In different bacterial species, these membranes have varying shapes. They can be tubules, vesicles, or aggregates of lamellae (arranged either concentrically or in stacks); sometimes they fill the entire cell interior (see Fig. 2.23). Membrane fragments released during Cell Disruption as vesicles and separated by centrifugation are referred to as "chromatophores." In the cells of green bacteria, pigments are associated with different structures: light-harvesting pigments are mainly associated with chlorosomes, whereas reaction center pigments are associated with the plasma membrane (see Figs. 2.4 and 12.9).

Regulation of pigment and thylakoid synthesis. The synthesis of photopigments depends on growth conditions, primarily illumination and (in facultative anaerobes) the presence of oxygen. The pigment content in cells is higher the lower the illumination was during their growth. Oxygen also affects pigment formation: much like bright light, it suppresses The formation of membrane structures containing pigments, and consequently the synthesis of bacteriochlorophylls and carotenoids. The amounts of photosynthetic pigments vary in parallel with Changes in the number of vesicles and tubules (intracellular membranes) visualized in cells using an Electron microscope. Furthermore, oxygen inhibits certain enzymatic steps of bacteriochlorophyll synthesis. The highest content of photopigments, as well as pigment-bearing vesicles and tubules, can be found in cells grown under anaerobic conditions at low illumination.

12.1.2 METABOLISM

Investigating the metabolism of phototrophic bacteria involves A number of difficulties. These bacteria are often characterized by great versatility. For example, many nonsulfur purple bacteria are capable of both anaerobic growth in the light and aerobic growth in the dark; other groups are represented by strict anaerobes and obligate phototrophs. Many species utilize molecular hydrogen as an electron donor, and some use hydrogen sulfide or sulfur. The assimilation of CO₂ and organic compounds is observed. Under anaerobic conditions in the dark, a small amount of energy can be generated via Fermentation, though it is insufficient for the growth of most species. In short, phototrophic bacteria are among the most metabolically versatile organisms. Therefore, we can only provide a General Overview of their physiological capabilities here.

CO₂ fixation. Almost all phototrophic bacteria studied to date are capable of fixing CO₂ via the ribulose bisphosphate cycle. To reduce 3-phosphoglycerate, purple and green bacteria utilize NADH₂ rather than NADPH₂ (unlike green plants). In addition, ferredoxin- or NAD(P)-dependent reductive carboxylation reactions participate in CO₂ assimilation. CO₂ fixation either enables completely autotrophic growth or serves to maintain a specific cellular redox level during the simultaneous assimilation of highly reduced organic compounds (e.g., Fatty acids). Chlorobium species assimilate CO₂ via the reductive Tricarboxylic Acid Cycle.

Hydrogen donors. Anaerobic phototrophic bacteria require an exogenous hydrogen donor. They are capable of utilizing H₂, hydrogen sulfide, elemental sulfur, thiosulfate, organic acids, alcohols, sugars, and even certain Aromatic Compounds. Molecular hydrogen is utilized by very many, but by no means all, phototrophic bacteria. Small species of Chromatium, some Rhodobacter species (e.g., R. capsulata), rhodospirilla, and Chlorobium grow in the light using H₂ and CO₂. The quantum yield in this process is roughly as high as in oxygenic photosynthesis. In both Rhodopseudomonas acidophila and the cyanobacterium Anabaena cylindrica, it amounts to 8 moles of quanta per 1 mole of fixed carbon dioxide.

Some purple nonsulfur bacteria, purple sulfur bacteria, and green sulfur bacteria oxidize hydrogen sulfide to sulfate. In most purple sulfur bacteria, sulfur is temporarily deposited intracellularly as an intermediate product. Apparently, the quantitative dominance of large Chromatium representatives in natural habitats (ponds) is related to their ability to rapidly oxidize H₂S in the light and accumulate sulfur; meanwhile, intracellular sulfur serves as a reserve of reducing power and allows CO₂ to be assimilated in the light without the uptake of external hydrogen donors. When utilizing thiosulfate, a distinction must be made between sulfane and sulfonic sulfur; only sulfane sulfur can be deposited in cells, whereas sulfonic sulfur is released into the environment as sulfate.

Some Chlorobium species primarily oxidize hydrogen sulfide only to sulfur, which is excreted into the medium. Such bacteria grow particularly well in co-culture with Desulfuromonas acetoxidans: this species, via "Anaerobic Respiration," reduces sulfur to H₂S while oxidizing ethanol or acetate. Chlorobium and Desulfuromonas represent a typical example of a functional association, or syntrophy, between two microorganisms (see Section 9.3).

Dark metabolism. Many purple nonsulfur bacteria and Chloroflexus are capable of aerobic growth in the dark provided that organic substrates are available. Consequently, they possess the components of respiratory metabolism, including The Tricarboxylic Acid Cycle. The latter also participates in metabolic transformations under anaerobic conditions in the light. The utilization of a wide range of organic acids and sugars by certain representatives of Chromatiaceae and Rhodospirillaceae indicates that although the metabolism of these phototrophic bacteria varies, it generally proceeds via well-known pathways (such as the fructose bisphosphate pathway, the Entner–Doudoroff pathway, the tricarboxylic acid cycle, etc.).

Since phototrophic bacteria must survive through the night, it is not surprising that energy-yielding processes still occur under anaerobic conditions in the dark. The energy source is fermentation, in which reserve substances serve as hydrogen donors and sulfur (where available) acts as the hydrogen acceptor. The end Products of Anaerobic dark metabolism are CO₂, acetate, propionate, and hydrogen sulfide. However, only isolated species of phototrophic bacteria are able to grow under anaerobic conditions in the dark.

H₂ production in the light. Some representatives of purple bacteria are capable of evolving molecular hydrogen in the light in the presence of suitable organic or inorganic hydrogen donors. H₂ production depends on the C/N ratio of the substrates present in the medium and is inhibited by free ammonium ions. N₂ also reversibly inhibits the evolution of molecular hydrogen by cells. Photoproduction of H₂ is associated with a side function of Nitrogenase; along with N₂, this enzyme is also capable of reducing protons, leading to the formation of molecular hydrogen. Therefore, an excess of energy and reducing power results in H₂ evolution.

Fixation of molecular nitrogen. The vast majority of investigated purple and green bacteria are capable of fixing nitrogen. However, their growth rate utilizing N₂ is generally lower than in the presence of ammonium ions.

Reserve Materials. Characteristic reserve materials for phototrophic bacteria include poly-β-hydroxybutyric acid, Polysaccharides, and polyphosphates. Depending on growth conditions, Chromatiaceae representatives may also contain sulfur inclusions, which exist within the cells as orthorhombic sulfur.

12.1.3 Distribution of Phototrophic Bacteria

Phototrophic purple and green bacteria inhabit the anaerobic zones of various aquatic environments, including shallow ponds, slow-flowing waters, lakes, and marine bays. Purple sulfur bacteria often form films On the surface of mud or decaying plant material, displaying a wide range of red hues from pale pink to deep red. Sometimes they swarm above the mud surface, forming a layer about a decimeter thick. Such water "blooms" in shallow water bodies (Fig. 12.12) are primarily caused by large purple bacteria such as *Chromatium okenii*, *C. warmingii*, *C. weissei*, and *Thiospirillum jenense*, although smaller representatives of Chromatiaceae and Chlorobiaceae are also found. Massive proliferation of purple sulfur bacteria can likewise be observed in shallow ponds whose surfaces are covered with a dense mat of duckweed (*Lemna*) or water lily leaves. This unique biological filter absorbs the spectral components of light that would otherwise be utilized by green algae and cyanobacteria, while transmitting the light absorbed by bacteriochlorophylls and dark-red carotenoids. Consequently, anaerobic phototrophic bacteria thrive beneath the duckweed cover, whereas green algae and cyanobacteria fail to develop (Fig. 12.12). In some natural habitats, certain species of phototrophic sulfur bacteria exist as almost pure cultures. *Rhodospirillaceae* can also be found consistently, though rarely in such massive quantities.

Fig. 12.12. Profile of a shallow water body showing massive development of purple sulfur bacteria.

Seasonal mass development of purple sulfur bacteria is also observed in the anaerobic zones of lakes below the thermocline (see Fig. 17.1). Here, the bacteria find the essential substances they require: hydrogen sulfide, CO₂, and organic compounds. Infrared solar radiation naturally does not penetrate to depths of 10–30 meters; at such depths, the energy maximum shifts to the blue and blue-green regions of the spectrum (450–500 nm), which corresponds precisely to the absorption range of carotenoids. This explains the relatively high carotenoid content in purple bacteria, which dictates their coloration: much like phycobiliproteins in red algae and cyanobacteria, carotenoids enable photosynthetic metabolism in purple bacteria at considerable depths. Accordingly, at such depths among green sulfur bacteria, brown forms rich in carotenoids predominate (*Chlorobium phaeobacteroides*, *C. phaeovibrioides*, *Pelochromatium*).

12.1.4 Enrichment Cultures

The method for isolating anaerobic phototrophic bacteria is based on the observation (described above) that purple sulfur bacteria sometimes achieve massive growth even in shallow water bodies when the entire water surface is blanketed with a dense layer of duckweed. By employing filters that absorb the short-wavelength portion of the spectrum and transmit infrared rays—which are utilized exclusively by specific groups of phototrophic bacteria—one can establish selective growth conditions for both green sulfur bacteria and purple bacteria containing bacteriochlorophyll *a* or *b*.

Whether purple or green bacteria will predominate in an enrichment culture (the "Winogradsky Column," Fig. 12.13) depends on the available hydrogen donors and the concentration of H₂S. If a cylinder containing sand, soil, and egg albumin is filled with water and then inoculated with a sample from a natural habitat of phototrophic bacteria, *Rhodospirillaceae* species will develop in the light. If, however, calcium sulfate is added to ensure the continuous generation of H₂S via sulfate reduction, the growth of purple non-sulfur bacteria will be suppressed, allowing the sulfur bacteria to dominate. In synthetic nutrient media supplemented with vitamin B₁₂, enrichment cultures of various green and purple bacterial species can be obtained by fine-tuning such parameters as hydrogen sulfide and nutrient salt concentrations, pH, Temperature, and light intensity. Forms that are particularly sensitive to environmental conditions—such as bacteria possessing gas vesicles—typically require lower H₂S concentrations, lower temperatures, and reduced light intensities. When isolating specific *Rhodospirillaceae* species, the choice of hydrogen donor and the presence of certain Vitamins (biotin, p-aminobenzoic acid, thiamine, nicotinic acid) play a decisive role.

Fig. 12.13. Enrichment cultures of phototrophic bacteria in "Winogradsky columns." The numbers above the columns indicate the spectral CHARACTERISTICS OF THE light filters beneath which preferential growth of the specified species and groups of bacteria is observed. A few days after Setting up the columns and inoculating the medium with pond mud and water, distinct red or green "bacterial plates" form within the water column.