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Diversity, distribution and physiology of the aerobic phototrophic bacteria in the mixolimnion of a meromictic lake

Natalia Yurkova, Christopher Rathgeber, Jolantha Swiderski, Erko Stackebrandt, J. Thomas Beatty, Ken J. Hall, Vladimir Yurkov
DOI: http://dx.doi.org/10.1111/j.1574-6941.2002.tb00952.x 191-204 First published online: 1 June 2002


The population of anoxygenic phototrophic bacteria in the aerobic zone of the meromictic Mahoney Lake was investigated using classical microbiological methods. This bacterial community was found to be very rich and diverse. Thirty-one new strains of the obligately aerobic phototrophic bacteria, and two new purple nonsulfur strains, were isolated in pure cultures and preliminarily characterized. The isolates contain a variety of carotenoids, bacteriochlorophyll a incorporated into pigment protein complexes, and are morphologically and physiologically diverse. These properties indicate a diversity of adaptations to the stratified environments of this meromictic lake. Phylogenetically all isolated strains belong to the α subclass of Proteobacteria.

1 Introduction

Mahoney Lake is a meromictic saline lake without an outflow, located near Penticton in the dry region of south central British Columbia. Meromictic lakes contain bottom waters (the monimolimnion) that do not mix with the surface waters (the mixolimnion), separated by a chemocline [1]. There is little seasonal change in the location of the chemocline in terms of salinity. However, the seasonal depth of the chemocline can vary by 0.5 to 0.75 m depending on the level of evaporation. Usually in Mahoney Lake, the top of the chemocline and the loss of oxygen in the vertical profile is defined in the 8.0 to 8.5 m zone. Meromixis is maintained by a sharp chemical discontinuity at the chemocline. Through the water column, Na+, Ca2+, Mg2+ and SO42− are the major ions in the lake water [2], attributed to the composition of lavas of the Marron formation in the watershed [3], and so Mahoney Lake has been classified as a mainly sodium sulfate lake [4].

Previous microbiological investigations of Mahoney Lake focused on anaerobic anoxygenic phototrophs and revealed an extremely dense population of the purple sulfur bacterium Amoebobacter purpureus in the chemocline of the lake [5,6]. The enormous A. purpureus population was proposed to be the major route for transfer of phosphorus, by periodic upwelling events, from the monimolimnion for the growth of heterotrophic bacteria in the mixolimnion of Mahoney Lake [5]. Strains of Rhodobacter capsulatus (a purple nonsulfur bacterium), Thiocapsa roseopersicina (a purple sulfur bacterium), Chloroherpeton thalassium and Prosthecochloris aestuarii (green sulfur bacteria) were also isolated from the chemocline at about 7 m depth [6]. Additionally, the cyanobacterium Gloeocapsa sp. has been found at depths up to 5 m [3].

Our goal was to investigate the microbial population of the Mahoney Lake mixolimnion with focus on a relatively newly described physiological group of aerobic bacteria, the aerobic phototrophic bacteria. Aerobic phototrophic bacteria have been found in different locations, including the extreme environments of acidic mine drainage waters, hot springs and deep ocean hydrothermal vent plumes [7]. The novel aspect of this increasingly large group of bacteria that contain bacteriochlorophyll (BChl) a and carotenoid pigments is the inability to use BChl for anaerobic photosynthetic growth. The aerobic phototrophic bacteria carry out limited anoxygenic photosynthesis under aerobic conditions, but light cannot be used as the sole source of energy and none of the aerobic phototrophic species has yet been shown to grow autotrophically. Most species are strict aerobes, and growth is dependent on organic substrates as the main source of carbon and energy [7]. Aerobic phototrophic bacteria are taxonomically classified into 15 different genera that include marine, freshwater and soil bacteria. A better understanding of the evolutionary origin and diversity of the physiological properties of this group depends on continuing studies of the existing species and isolation of new strains. In this paper we describe an investigation of new phototrophic bacteria isolated from different depths of the Mahoney Lake mixolimnion.

2 Materials and methods

2.1 Collection of samples

Samples were collected in October 1997 from the surface, 3 m and 5 m depths using a 3-l van Dorn bottle over the deepest site of the lake. Samples were immediately transferred into sterile 500-ml screw-cap bottles and placed in the dark at 4°C.

2.2 Enumeration of bacteria

After return to the laboratory, 10-fold dilutions of the samples were prepared and 0.1 ml of each dilution was spread in duplicate on 2% agar plates of the following media.

Medium N1 contained (in g l−1): KH2PO4, 0.3; MgSO4, 2.0; NH4Cl, 0.3; KCl, 0.3; CaCl2·2H2O, 0.05; Na2SO4, 15.0; NaHCO3, 0.5; Na-acetate, 1.0; Na-malate, 1.0; yeast extract, 1.0; peptone, 0.5.

Medium N2: same composition as medium N1 except that NaCl (15.0 g l−1) was substituted for Na2SO4, and Na-succinate (1.0 g l−1) for Na-malate.

Medium N3: same composition as medium N1, but without Na2SO4.

Medium N4: same composition as medium N1, except that the concentration of Na2SO4 was increased to 50.0 g l−1.

All the media were adjusted to pH 7.8–8.0 and supplemented with a mixture of vitamins (per liter of medium): 20 μg of vitamin B12; 200 μg of nicotinic acid; 80 μg of biotin; and 400 μg of thiamine, and with 2.0 ml of a trace element solution [8].

Plates were incubated aerobically at 30°C in the dark, and colonies were counted after 8 days. Pigmented colonies were resuspended in liquid media of identical composition and pure cultures were obtained by repeated plating.

The ability for anaerobic photosynthetic growth was tested in screw-cap test tubes completely filled with media for purple sulfur or nonsulfur bacteria [9], as well as with the medium of isolation.

2.3 Spectral analysis

Strains that formed pigmented colonies were grown under aerobic conditions at 32°C in rotating test tubes. After centrifugation (1–2 min at 15 000 rpm), pellets were resuspended in 125 μl of 10 mM Tris–HCl buffer (pH 7.8) and mixed with 375 μl of 30% bovine serum albumin solution to reduce light-scattering. Absorbance spectra were recorded between 350 and 1100 nm at room temperature with a Hitachi U2000 spectrophotometer (Hitachi, Tokyo, Japan).

2.4 Electron microscopy

Cells from aerobically grown exponential phase cultures in liquid medium were negatively stained with 1.0% aqueous uranyl acetate. For thin sections, the bacteria were embedded in Epon after fixation with 1.0% glutaraldehyde and 1.0% osmium tetroxide as described [10].

2.5 Physiology

Physiological and biochemical tests were performed as previously described [11,12].

2.6 16S rDNA-based phylogenetic analysis

Extraction of genomic DNA, PCR amplification of the 16S rDNA and direct sequencing of the purified PCR products were carried out according to Rainey et al. [13]. The sequencing reaction products were electrophoresed using a model 373A automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA). The partial 16S rDNA sequences were aligned with published sequences obtained from the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk) (Cambridge, UK) and the Ribosomal Database Project (http://rdp.cme.msu.edu/html) (RDP) using the ae2 editor [14] and similarity values were determined.

2.7 Nucleotide sequence accession numbers

The 16S rDNA sequences analyzed in this study were deposited in the EMBL database under the following accession numbers: ML1, AJ318407; ML10, AJ315686; ML14, AJ315689; ML19, AJ315688; ML20, AJ315694; ML21, AJ315695; ML30, AJ315684; ML35, AJ315693; ML36, AJ315692; ML45, AJ315685; ML3 (repr.), AJ315691; ML42 (repr.), AJ315683; ML4 (repr.), AJ315687; ML22 (repr.), AJ315690; ML37 (repr.), AJ315697; ML6 (repr.), AJ315682.

3 Results and discussion

3.1 Enumeration and isolation

Previous study has found that biomass of heterotrophic bacteria remained almost constant around 100 μg of C l−1 until July. However, it increased exponentially during late summer and autumn. This was caused by an increase in both bacterial cell numbers and bacterial cell volumes [5]. Integrated bacterial production reached a transient peak in May and increased steeply between July and November, parallel to the increase in bacterial biomass [5]. Therefore, in October 1997, when our samples were collected, the heterotrophic (facultative and obligate) bacterial community was near its maximum. Dissolved oxygen (DO) profiles of Mahoney Lake showed that the lake is oxic to ∼7 m depth, below which oxygen decreases rapidly to zero at 8 m [3,6]. On the sampling day in October 1997, the chemocline occurred at 8.35 m depth and extended down to 9.0 m, and the temperature and salinity were 18°C and 22 ppT at 8.5 m depth, respectively. At the surface, the temperature was 13.8°C, DO 8.6 mg l−1, salinity 5 ppT. At 3 m, the temperature was 25.3°C, DO 12.8 mg l−1, salinity 20 ppT, and at 5 m, the temperature was 28°C, DO 2.6 mg l−1, and salinity 22 ppT. Therefore the 3- and 5-m depth samples that we used were aerobic.

Aerobic phototrophic bacteria require oxygen and organic carbon. Thus, the reason why no selective medium has been developed to isolate aerobic phototrophic bacteria is because many nonphototrophic microorganisms grow well on aerobic organic media. We designed media that should satisfy bacterial needs for most growth factors and be close to the natural parameters of Mahoney Lake at various depths. Rich media have been used previously for the enumeration of aerobic phototrophs [7]. Since Mahoney Lake is mainly a sodium sulfate dominated lake with a mixolimnion SO42− content ranging from 5 g l−1 at the surface to 14 g l−1 at 5 m depth [4], medium N1, which has a high content of Na2SO4, was used to enumerate bacteria that require high amounts of sulfates. Medium N2 was devised to isolate and enumerate strains that prefer a NaCl-enriched medium for growth. Medium N4, which contains a very high concentration of Na2SO4 (50 g l−1), was used to detect bacteria that either require or are resistant to such high sulfate concentrations.

Data were collected on the abundance of the following groups of microorganisms that were capable of forming colonies on the employed media: aerobic noncolored bacteria, aerobic pigmented bacteria, obligately aerobic phototrophic bacteria, and facultatively aerobic purple nonsulfur bacteria.

The results are summarized in Tables 1 and 2. All samples gave rise to many nonpigmented colonies (representative of 2513 to 31 500 cells ml−1). The color of bacterial colonies (indicating of the presence of carotenoids) was used to identify presumptive phototrophic bacteria, which were subsequently screened for the presence of BChl in absorption spectra. Most of the pigmented cells contained carotenoids but not BChl. BChl a was detected in 31% of pigmented strains isolated from surface samples, in 28% of pigmented strains from 3 m and in 22% of pigmented strains recovered from 5-m depth. These data are similar to those reported for freshwater and seawater microbial mats, and for pelagic marine environments including deep ocean hydrothermal vent samples where BChl-containing strains comprised 10–30% of pigmented bacteria [12,1517]. However, the presence of BChl in different Mahoney Lake pigmented isolates did not correlate with the depth of sample, color of the colony or media used (Table 1). Thus, BChl-containing species seem dispersed throughout the mixolimnion.

View this table:

Distribution and enumeration of pigmented cells isolated from Mahoney Lake

Cell colorMediumAbundance (CFU ml−1)% of cells containing BChl
Surface3 m5 mSurface3 m5 m
NoncoloredN17 8502 5139 400NANANA
N23 2403 4833 785NANANA
N48 47528 80031 500NANANA
Pink–purpleN101204 555NA600
N204751 850NA36.40
N47902 7853 00028.611.158.4
Orange–redN14 23303 9506.7NA0
N21 360502 0005000
YellowN11 205000NANA
Brown–redN12 60022068679.462.575
N42401 3301 60010053.3100
  • ND, not determined; NA, not applicable.

View this table:

Some determinative characteristics of BChl a containing strains isolated from Mahoney Lake

StrainIsolated fromColorIn vivo carotenoid peaks (nm)In vivo BChl peaks (nm)MorphologyMotilityAnaerobic phototrophic growthMedium of isolation
ML14SurfaceBrown–orange465804, 870Short ovoid rods, chains of 2–3 cellsN1
ML15SurfaceBrown–orange420, 464802, 861Ovoid, thick rods, many long chains+N1
ML17SurfacePink–purple408, 484805, 866Ovoid rods+N1
ML19SurfaceBrown–orange462, 488807, 867Small, short ovoid or almost coccoid cells forming very long curved motile chains, connect by bubble-like structures. Produce prosthecae+N1
ML20SurfaceYellow427, 458, 488803, 868Thick ovoid rods, forming small chains+N1
ML21SurfaceBrown–red461, 488, 528805, 866Small, slightly curved rods, form short curved chains+N1
ML22SurfaceBrown–red464, 487755, 805, 864Extremely long thin rods, form chains. Cells often develop a small bubble like structure at one end+N1
ML23SurfaceBrown–red464, 488806, 864Long rods, forming long straight chainsN1
ML30SurfacePink460873Ovoid short rods, some chains of 2–3 cells+N4
ML32aSurfaceBrown–red468, 489749, 806, 867Ovoid short rods, some chains of 2–6 cells+N4
ML34SurfaceBrown–orange464, 487804, 869Ovoid short rods, some chains of 2–6 cells+N4
ML35bSurfaceYellow430, 459, 488805, 870Small ovoid rods, forming short chains+N4
ML13 mBrown–red462, 488762, 809, 869Ovoid cells+N1
ML33 mOrange–brown468804, 860Ovoid rods, forming chains. Production of buds and tendency for branchingN1
ML43 mBrown–red466, 491808, 866Rods. Aggregates of cells in presumably polysaccharide matrix. Bead-like formationsN1
ML6c3 mPurple408, 484805, 870Two types of cells, elongated rods, and bean shaped cells+N1
ML103 mPink475, 505866Large ovoid rods or elongated cells. Produce gas vacuoles+N2
ML37d3 mPink480859Long thin cells+N4
ML295 mPink409802, 861Pleomorphic+N2
ML42e5 mPink451, 479, 510803, 848, 880Short ovoid cells, forms long chains+++N4
ML455 mGrayish-yellow409806, 870Small short ovoid cells, forms short chains.+N4
  • +, positive; −, negative; RO, rich organic medium; Non-s, medium for purple nonsulfur bacteria.

  • a Represents group including ML31.

  • b Represents group including ML36.

  • c Represents group including ML16, ML18, ML33, ML38, ML39, ML40, ML44.

  • d Represents group including ML46, ML47.

  • e Represents group including ML43.

3.2 Morphology and cytology

Thirty-three strains that produce BChl a and carotenoids were isolated. Based on color, colony and cell morphology, cytology, absorption spectra and major physiological aspects, these 33 isolates were grouped as shown in Table 2. All purified strains form regular circular colonies on the surface of agar media, varying in size from 1–2 mm to 6–7 mm. Some isolates were nonmotile (Table 2), although the majority was motile having one long (Fig. 1A, strain ML42) or several wavy flagella (Fig. 2A, strain ML10). All isolates stained Gram-negative and this cell wall organization was confirmed by electron microscopy of thin sections (Figs. 1B,2B,C,3D,E,4C and 5C,D; strains ML42, ML10, ML19, ML4 and ML6, respectively). Most of the strains reproduced by binary division. Strains ML1, ML3, ML6, ML20, ML21, ML22, ML23, ML35 and ML40 divided by both symmetric and asymmetric constrictions. Production of buds and a tendency for branching was found in strains ML3 and ML4.


Strain ML42. A: Rod cell with one long flagellum. Negative staining. B: Ultra-thin section of cells showing Gram-negative cell wall and poorly developed photosynthetic intracytoplasmic membranes of thylakoid type (indicated by arrows). Bars: 0.5 μm (A), 0.25 μm (B).


Strain ML10. A: Negatively stained cells with possible multiple wavy flagellation. B,C: Ultra-thin sections of cells with extensive accumulation of presumed polyhydroxyalkanoate granules (indicated by arrow). Bars: 1 μm (A), 2 μm (B), 0.5 μm (C).


Strain ML19. A,D: Unusual form of cell connection. Bubble-like structures with micro-tubular ‘bridges’ in the middle are indicated by arrows. B,C: Prostheca formation is indicated by arrows. E: Initiation of prostheca formation. A–C: Negatively stained cells. D,E: Ultra-thin sections of cells. Bars: 1 μm (A), 0.5 μm (B,C,E), 0.25 μm (D).


Strain ML4. A: Rod cells incorporated into presumably polysaccharide matrix, and chains of bead-like formations (indicated by arrows). B: Ovoid cell and a chain of large ‘beads’. C: Elongated cell of irregular shape with prostheca-like formation (indicated by arrow). A,B: Negative staining. C: Ultra-thin section. Bars: 1 μm (A), 0.25 μm (B), 0.5 μm (C).


Strain ML6. A,B: Negatively stained bean-shaped and elongated cells with polar dark coloration of pointed ends. C,D: Ultra-thin sections of cells show polar periplasmic modifications (confirming polar staining in A and B; indicated by arrows). Bars: 1 μm (A), 0.5 μm (B), 0.25 μm (C,D).

Electron microscopy of thin sections of cells revealed that some strains accumulated storage materials. Strains ML1, ML3, ML4, ML6, ML10 and ML42 accumulated electron-clear granules attributed to polyhydroxyalkanoate compounds. Strains ML1 and ML3 accumulated electron-dense granules thought to be polyphosphate, whereas strain ML4 produced capsule material, presumed to be composed of polysaccharides, that formed a matrix with embedded cells (Fig. 4A, strain ML4). Accumulation of storage materials such as polysaccharides, polyphosphates and polyhydroxyalkanoates by aerobic phototrophic bacteria is common and has been discussed in a recent review [7].

A plethora of different cell shapes was observed among the new isolates under phase contrast light and electron microscopy (Table 2). The most interesting or unusual morphologies are presented in Figs. 1 2 3 4 5 6.


A: Strain ML3, which forms long chains of cells. B,C: Long cells of strain ML22 producing small bubble-like structures of unknown function at end of cell (indicated by arrows). Bars: 2 μm (A,B), 1 μm (C).

The pink-colored strain ML10 was found to be motile and formed ovoid rods or elongated cells of irregular shape (Fig. 2A–C). Elongated cells were often organized in rosettes of three to six cells (not shown). Massive formation of presumably polyhydroxyalkanoate granules was a specific feature of this strain (Fig. 2B,C).

The brown–orange strain ML19 had ovoid or almost coccoid cells forming long curved motile chains (Fig. 3A,D). Electron micrographs showed that cells in chains were connected by unknown bubble-like structures which had tubular connections in the middle. This type of connection has not been shown for previously described aerobic phototrophic species and calls for special study. Perhaps cells of strain ML19 exchange metabolites via these unusual structures. Another peculiarity of ML19 was the formation of prosthecae (Fig. 3B,C,E). Prosthecae increase significantly the surface-to-volume ratio of a cell, which may confer an increased ability to take up nutrients and expel wastes, and may function to provide buoyancy [18].

Ovoid rods of the brown–red strain ML4 excreted large amounts of capsular material, which holds cells together in small aggregates. Cells also produced unusual bead-like formations (Fig. 4A–C). Often these ‘beads’ were branched (Fig. 4A), and variable in size and shape. The functional role of these ‘beads’ as well as possible life cycle changes in ML4 requires further investigation.

A group of pink-purple strains, ML6, ML16, ML18, ML23, ML38, ML39, ML40 and ML44, showed interesting morphology. In liquid and especially on solid media two types of cells were produced: elongated rods and bean-shaped (vibroid) cells (Fig. 5A–D); both types of cells had pointed ends. Sometimes cells formed a helical shape. This morphology is similar to that of the purple nonsulfur bacterium Rhodocyclus purpureus[19]. Electron microscopy of negatively-stained cells revealed electron-dense areas at their poles (Fig. 5A,B), and thin sections indicated polar zones of significantly enlarged periplasmic space in both vibrioid cells and rods. The significance of this enlarged periplasm at the cell ends is unclear.

Strain ML22 had extremely long, thin cells (Fig. 6A,B), and many cells developed a small bubble-like formation at one end. Although this structure has not been investigated further, a possible function could be similar to that of a gas vacuole in regulating cell buoyancy.

3.3 Photosynthetic apparatus

The capability of the BChl-containing isolates for anaerobic photosynthetic growth (with tungsten filament lamp illumination level of about 30 microeinstein m−2 s−1) was tested in completely filled screw-cap test tubes and in agar (1%) deeps by using media (containing H2S, or Na2S2O3, and CO2 with or without acetate) for purple sulfur bacteria, or media (containing acetate, malate or succinate as sole carbon source) for nonsulfur bacteria. Anaerobic photosynthetic growth was also tested in the original liquid medium (N1, N2 or N4) used for isolation (see Table 2). Almost none of the isolates grew anaerobically in the light, which leads us to designate them as obligately aerobic phototrophic bacteria [7].

The isolates ML42 and ML43 grew photosynthetically (anaerobically) in purple nonsulfur bacterial media, whereas they did not grow in the purple sulfur bacterial medium that contained sodium sulfide (0.3 g l−1). This suggests that this concentration of sulfide was toxic for ML42 and ML43, and so we concluded that these two isolates are members of the purple nonsulfur bacteria. Anaerobic photosynthetic growth (although slow) yielded the highest cell densities in the liquid medium N4, whereas weaker growth occurred in the medium designed for purple nonsulfur bacteria. In all media, aerobically grown cultures were pink, whereas cultures grown anaerobically with illumination were light-brown, indicating a change in pigment composition. Such color difference between aerobically and anaerobically grown cultures is common for photosynthetic bacteria, and is due to a change in carotenoid composition and enhanced production of BChl [20]. Transfer of cells from aerobic plates to anaerobic photosynthetic conditions resulted in long lag phases of at least 48 h, whereas cells previously cultured under anaerobic photosynthetic conditions transferred to similar anaerobic photosynthetic conditions did not show such a long lag phase.

Cells of ML42 and ML43 grown photosynthetically (anaerobically) contained small amounts of intracytoplasmic membranes of thylakoid type, which we assume contained the photosynthetic apparatus (Fig. 1B). The absorption spectra of strains ML42 and ML43 grown under different oxygen conditions and media were significantly different (Fig. 7), although still indicative of reaction center and light-harvesting (LH) complexes. As can be seen from the in vivo absorption spectra, the LH apparatus consists of two types of antenna: an absorption peak at 880 nm that we attribute to an unusually red-shifted LH complex I; and absorption peaks at 804 and 848 nm that we assign to a peripheral LH complex II (Fig. 7A–C). The long wavelength absorption peak of LHI indicates an unusual protein environment of BChl in this complex.


Absorption spectra of the photosynthetic apparatus of strain ML42 synthesized under different growth conditions. Cells were grown in: (A) liquid medium for purple nonsulfur bacteria, anaerobically; (B) medium N4, aerobically on agar surface; (C) liquid medium N4, anaerobically.

The relative amounts of the LH complexes in ML42 and ML43 seemed to depend on the oxygen concentration and medium composition. This conclusion was reached because in aerobically grown cells the LHI (880 nm) peak amplitude was much greater than that of the LHII (804 and 848 nm) peaks, such that the LHII 848 nm peak was a barely detectable shoulder on the LHI 880 nm peak (Fig. 7B). In contrast, under anaerobic photosynthetic conditions in medium N4, cells produced both complexes at about similar levels, as indicated by the equal amplitudes of the LHII 848 nm and the LHI 880 nm peaks (Fig. 7C). After anaerobic photosynthetic growth to the stationary phase in the medium for purple nonsulfur bacteria the LHII peaks (at 804 nm and 848 nm) predominated, with the LHI absorption peak at 880 nm present as a small shoulder (Fig. 7A). Although the influence of oxygen and light intensities on LH complexes in purple photosynthetic bacteria is well known, we do not know whether the ML42 and ML43 isolates produced different forms of LHI and LHII complexes dependent on salinity, medium composition, temperature or light intensity, as reported for Rhodopseudomonas acidophila and Rhodopseudomonas palustris [2124]. The carotenoid composition (absorption peaks between 400 and 550 nm) as well as the amount of a presumed cytochrome (peak at 414 nm) also depended on the oxygen availability (Fig. 7A–C).

The absorption characteristics of obligately aerobic phototrophic strains are summarized in Table 2. The differences in carotenoid composition (absorption peaks in blue and green regions of the light spectrum between 400 and 550 nm) and BChl-protein complexes (absorption peaks in the infrared region of the light spectrum between 800 and 880 nm) are consistent with the colorful variety of isolates (from yellow to brown–red). We have detected at least eight variants of the LH complexes among these isolates (Fig. 8). The most interesting is the LH complex found in the eight strains that produced vibroid cells. These strains seem to have an unusual LHII complex that has one absorption peak at ∼805 nm (Fig. 8G, strain ML6). This absorption profile has been found only in the aerobic phototrophic genera Roseobacter and Rubrimonas [2527]. However, Roseobacter and Rubrimonas species have morphological, cytological and major physiological properties that are different from our isolates.


Diversity of photosynthetic light-harvesting complexes and carotenoid compositions found in new strains, as shown by absorption spectra. A: ML40. B: ML31. C: ML23. D: ML20. E: ML19. F: ML15. G: ML6. H: ML4.

3.4 Nutritional and other properties

A variety of diagnostic growth and physiological properties of the Mahoney Lake isolates are presented in Table 3.

View this table:

Comparative physiological characteristics of the aerobic phototrophic strains isolated from Mahoney Lake

Growth at pH
Utilization of
Yeast extract++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Hydrolysis of
Tween 60++++++++++++++++++++++++++++++++++NG+++++
Vitamin requirement
Antibiotic sensitivity
Penicillin G++++++++++++
Polymyxin B+++++++++++++
Nalidixic acid+++
  • +, substrate is utilized, substrate is hydrolyzed, vitamin required or antibiotic sensitive; ++, substrate is utilized for very good growth; −, substrate is not utilized, substrate is not hydrolyzed, vitamin is not required or antibiotic resistance; W, very weak growth; ND, not determined.

All strains were catalase and oxidase positive. Many strains hydrolyzed gelatin, Tween 60 and starch, indicating the presence of gelatinase, lipolytic and amylolytic activities (Table 3). Only three strains, ML10, ML45 and ML46, did not hydrolyze gelatin, Tween 60 or starch. Isolates from Mahoney Lake were able to grow over a wide range of temperatures, with all strains exhibiting growth at 5°C, and optimal growth at 28°C. All strains except ML16, ML17, ML18 and ML21 were able to grow at 37°C, and strain ML10 was capable of growth at as high as 45°C.

The pH of the Mahoney Lake mixolimnion showed a vertical variation from around pH 9 at the surface to about pH 8 near the chemocline, and all of the isolates tolerated a wide range of pH for growth. Most strains grew optimally without any difference in the range of pH 5.5–10. Strains ML6, ML16, ML17, ML18, ML30, ML33, ML38, ML39, ML40 and ML44 preferred a slightly higher pH with a minimum value of pH 6. All of these isolates were able to grow at a pH level as high as 11, and some of them grew equally well at pH 11 as at a lower pH. The strains ML34, ML35 and ML36 tolerated the broadest range of pH from 5 to 11.

The isolates were screened for their ability to utilize various organic compounds as sole carbon sources. In general, they can be divided into three groups. The first and largest group of strains (ML1, ML3, ML4, ML6, ML10, ML14, ML16, ML18, ML19, ML20, ML21, ML22, ML23, ML29, ML31, Ml32, ML34, ML38, ML40, ML43, ML44 and ML45) utilizes a wide variety of organic compounds as sole carbon source. This is typical for aerobic phototrophs associated with environments rich in organic matter [7]. The second group contains strains ML15, ML17, ML30, ML35, ML36, ML39, ML42 and ML46 that have very restricted metabolic abilities. These isolates utilize a limited number of organic compounds as a sole carbon source with best growth occurring when yeast extract, casamino acids or bactopeptone were provided. The third and smallest group of strains (ML33, ML37 and ML47) did not grow in media that contained only single organic compounds (organic acids or sugars), whereas growth was obtained in media supplemented with yeast extract or casamino acids.

Surprisingly, many of the aerobic phototrophic isolates demonstrated a requirement for biotin and/or vitamin B12. This requirement is unusual for the aerobic phototrophic bacteria, as only one genus Roseobacter has been shown to be vitamin dependent [26], although the growth of Citromicrobium bathyomarinum is stimulated by the addition of biotin to a minimal medium [28].

As is common for the aerobic phototrophic bacteria, there was variable resistance and sensitivity to several antibiotics (Table 3).

3.5 Effect of salinity

Because Mahoney Lake shows considerable vertical variation in salinity (4–40%o) [4], this could be an important environmental parameter that influences the growth and survival of microorganisms in this econiche. For this reason it was of interest to study the influence of salinity on the growth of the newly isolated strains of aerobic phototrophs. The salt requirement and tolerance of strains isolated from different depths were tested in aerated liquid media (N1, N2, N3 and N4) containing concentrations of NaCl or Na2SO4 from 0.5% to 15% (Table 4).

View this table:

The Mahoney Lake isolates exhibited a wide salt tolerance. Our experiments revealed that many strains grew in media with no NaCl or Na2SO4 added; however, none of these strains were obligately freshwater (Table 4; Fig. 9). Only one strain (ML3) appeared well suited for growth without added salts, showing little difference in growth as compared to saline media (Fig. 9B). Most of the strains able to grow in the absence of a salt supplement showed much weaker growth than on the same medium supplemented with a salt. Several strains (ML6, ML16, ML18, ML29, ML33, ML37, ML38, ML39, ML40, ML42, ML43, ML44, ML45 and ML46) could be described as obligately halophilic, because they did not grow in nonsupplemented media and very slow growth occurred at NaCl or Na2SO4 concentrations not lower than 0.5% (Table 4,Fig. 9A). Strains ML15, ML19, ML21, ML22 and ML23 grew in all media tested with slight preference for additional sulfate, whereas strains ML4, ML32, ML35, ML38, ML42 and ML45 grew best in the highly halophilic (supplemented with Na2SO4) medium N4 (Fig. 9C).


Growth curves measured in four different media types indicate salinity adaptation among investigated aerobic phototrophic strains isolated from Mahoney Lake. A: Obligately halophilic strain ML6. B: Strain ML3 is well adapted to fluctuating salinity, able to grow in both freshwater and saline media with small difference. C: Halophilic strain ML35 with preference for high sulfate concentrations. (♦), medium N1; (▪), medium N2; (▲), medium N3; (×), medium N4.

Compared to e.g. freshwater Sandaracinobacter sibiricus [17,29,30], which does not grow in media supplemented with 20 g NaCl l−1, the newly isolated strains of aerobic phototrophic bacteria clearly demonstrated broad salt tolerance. This ability may reflect adaptation to an environment with a steep gradient of salinity. A similar tolerance of a range of salt concentrations was noted for other aerobic phototrophic bacteria isolated from environments with variable salinity. Strains of Erythrobacter litoralis isolated from laminated marine mats on Texel island in the Netherlands [12] and strains of C. bathyomarinum recovered from a deep ocean black smoker plume waters [28] showed broad salt tolerance, indicating an adaptation to fluctuating salinities that may exist in a supralittoral zone and near deep ocean hydrothermal vents, respectively.

3.6 Phylogenetic analysis

Based upon partial 16S rDNA sequences (>400 bases) all studied isolates belong to the α subclass of Proteobacteria. With similarity values ranging between 97 and 100% most isolates are closely related to species of the genera Agrobacterium, Erythrobacter, Erythromicrobium, Porphyrobacter and Sphingomonas (Table 5). Whether these isolates should be regarded strains of described species or whether they represent new species should not be decided solely on the basis of molecular sequences. Other isolates are more distantly related (93–96% similarity) to described species of the genera Paracoccus, Bosea, Sulfitobacter, Roseobacter and Maricaulis and these isolates are more likely to represent novel species and, eventually, even novel genera if supported by other taxonomic criteria. The relationship of the lake isolates to members of the latter three genera is unexpected as they are of marine origin.

View this table:

Phylogenetic relatedness of new isolates to described species of the α subclass of Proteobacteria

Strain(s)Similarity (%) among each otherSimilarity (%) to nearest neighborNearest neighbor
ML1NA97.3Agrobacterium sanguineum
ML10NA93.6Bosea thiooxidans
ML14NA100Erythromicrobium ramosum
ML19NA98.7Genus Porphyrobacter
ML20NA97.2Genus Sphingomonas
ML21NA97.6Erythromicrobium ramosum
ML30NA95Genus Sulfitobacter
ML35NA98.9Genus Sphingomonas
ML36NA97.8Agrobacterium sanguineum
ML45NA94Genus Roseobacter
ML3, ML1510098.4Agrobacterium sanguineum
ML42, ML4310094.5Genus Paracoccus
ML4, ML31, ML3210098.8Porphyrobacter
98.4Erythrobacter litoralis
ML22, ML23, ML3410099.8Agrobacterium sanguineum
ML37, ML46, ML4710095Maricaulis maris
ML6, ML16, ML17, ML18, ML29, ML33, ML38, ML40, ML4410095.6Genus Paracoccus
  • NA, not applicable.

4 Concluding remarks

The mixolimnion of the meromictic, saline Mahoney Lake harbors a diverse population of anoxygenic phototrophic bacteria. Aerobic phototrophic bacteria are distributed in all oxygenated zones to at least a depth of 5 m, and are present in high numbers (Tables 1 and 2). Two purple nonsulfur photosynthetic isolates were also obtained from 5 m depth.

The aerobic phototrophic bacterial population of Mahoney Lake consists of a wide variety of morphological forms (Table 2; Figs. 1 2 3 4 5 6), and the cytology of these cells revealed a Gram-negative cell wall organization. Aerobic phototrophic bacteria isolated from Mahoney Lake do not develop extensive photosynthetic intracytoplasmic membranes characteristic of this group of bacteria [7,31].

Different types of LH complexes were found in the new isolates, as indicated by absorption spectra (Fig. 8), including several presumably new types of LH complexes. Further investigation of the photosynthetic apparatus in these isolates may reveal how the protein environment around BChl a affects its absorption properties.

The majority of strains isolated from Mahoney Lake were shown to utilize a variety of organic substrates, similar to other aerobic phototrophs previously described. However, some strains were unable to grow on any single organic compound tested, whereas a complex organic nutrient such as yeast extract supported growth (Table 3). This is unusual for aerobic phototrophic bacteria, and may indicate dependence on the organic nutrients (thought to be mainly complex, humic-like materials) available in the oligotrophic Mahoney Lake [5].

The majority of isolated strains have vitamin requirements or require as yet undetermined growth factors, and grow at high pH values found in Mahoney Lake (Table 3).

Mahoney Lake has vertical zones of variable salinity from the surface to the bottom. Different salt tolerances were established in strains isolated from different salinity concentrations. The majority of strains are adapted to the lake's salinity fluctuation, being able to grow in freshwater and in highly saline media (Fig. 9; Table 4). Perhaps because Mahoney Lake is a sulfate rich lake, most of the isolates grew best in sulfate enriched media (Table 4). The strains isolated from higher salt concentrations (5 m depth) grew best in highly halophilic medium N4 and did not grow without a salt supplement.

Similar to previously described species of the aerobic phototrophic and purple nonsulfur bacteria [7], new isolates belong to the α subclass of Proteobacteria closely related to each other and to nonphototrophic representatives of this subclass. An interesting observation of our phylogenetic study is a demonstration of the close relation of some strains purified from a meromictic lake to species obtained from different marine environments. Mahoney Lake is a relatively small lake, naturally and distantly isolated from the world oceans. Therefore, it is of interest to investigate the origin and evolution of these new strains in the context of the history and evolution of Mahoney Lake.


This work was supported by grants from NSERC (Canada) to V.Y., K.J.H. and J.T.B. We thank Elaine Humphrey (UBC, Vancouver) for generous help with electron microscopy preparations and Ina Kramer (DSMZ, Braunschweig) for participation in sequencing.


  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
  14. [14]
  15. [15]
  16. [16]
  17. [17]
  18. [18]
  19. [19]
  20. [20]
  21. [21]
  22. [22]
  23. [23]
  24. [24]
  25. [25]
  26. [26]
  27. [27]
  28. [28]
  29. [29]
  30. [30]
  31. [31]
View Abstract